Altered bile composition after liver transplantation is associated with the development of...

Molecular and biochemical mechanisms of bile duct injury after liver transplantation

Carlijn I. Buis

This thesis is funded by: . Different parts of this thesis were funded by grants

from the Jan Kornelis de Cock Foundation and the Groningen Graduate School for Drug

Exploration GUIDE.

The financial support of the following institutions and companies in the publication of this

thesis is highly appreciated:

Buis, C.I.

Molecular and biochemical mechanisms of bile duct injury after liver transplantation.

Thesis, University of Groningen, The Netherlands

ISBN: 978-90-367-3639-8

© Copyright 2008 Carlijn I. Buis, The Netherlands

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or

transmitted in any form or by any means, without prior permission of the author.

Cover: ICO-Communucations & Carlijn Buis

Lay-out: Gildeprint drukkerijen, Enschede, the Netherlands

Printed by: Gildeprint drukkerijen, Enschede, the Netherlands

Molecular and biochemical mechanisms of bile duct injury after liver transplantation

Proefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. F. Zwarts,

in het openbaar te verdedigen op

maandag 8 december 2008

om 13:15 uur

door

Carlijn Ineke Buis

geboren op 29 december 1978

te Vught

Promotor: Prof. dr. R. J. Porte

Beoordelingscommissie: Prof. dr. H.J. Metselaar

Prof. dr. M.J.H. Slooff

Prof. dr. H.J. Verkade

Paranimfen: Marieke de Boer

Mark-Hugo Maathuis

The cover shows an old advertisement of ‘ossegalzeep’ by Jawson Wood, dated in

1915. This soap made from ox bile was especially used in the twentieth century to

clean clothes with fatty stains. The bile salts acts as detergents and thereby enables

fatty stains to dissolve in water by formation of micelles.

A similar pattern can be found in human bile after transplantation. Bile salts form

micelles with phospholipids in the bile. In case phospholipids are relatively reduced

compared to bile salts, in other words if there is a low biliary phospholipids-to-bile

salt ratio, bile can act as a detergent for the bile ducts by recruiting phospholipids

from the membrane. After liver transplantation the bile formation is altered and in

some patients, a detergent – toxic – bile composition, with a low phospholipids-to-

bile salt ratio, is observed. This toxic bile is found to contribute to the development of

bile duct injury after liver transplantation.

Contents

Chapter 1 Introduction and outline of this thesis 9

Chapter 2 Causes and consequences of ischemic type biliary 15

lesions after liver transplantation.

Journal of HPB surgery 2006; 13:517–24.

Part I. Non-anastomotic biliary complications after liver transplantation

Chapter 3 Non-anastomotic biliary strictures after adult liver 37

transplantation part I: radiological features and risk factors

for early versus late presentation

Liver Transpl 2007; 13:708-718.

Chapter 4 Non-anastomotic biliary strictures after adult liver 61

transplantation part 2: Management, outcome and

risk factors for disease progression

Liver Transpl 2007; 13:725-732.

Part II. Bile physiology after liver transplantation

Chapter 5 The role of bile salt toxicity in the pathogenesis of bile duct 81

injury after non heart-beating porcine liver transplantation

Transplantation 2008; 85:1625–1631.

Chapter 6

Altered bile composition after liver transplantation is associated

with the development of Nonanastomotic biliary strictures

J of Hepatol, in press.

99

Chapter 7 Polymorphisms of hepatobiliary phospholipid transporter 123

MDR-3 associated with non anastomotic strictures after

human liver transplantation

submitted

Part III. HO-1 and hepatobiliary injury after liver transplantation

Chapter 8 Expression of Heme oxygenase -1 in human livers before 139

transplantation correlates with graft injury and function

after transplantation

Am J Transplant. 2005; 5:1875–1885.

Chapter 9 Heme oxygenase-1 genotype of the donor is associated 167

with graft survival after liver transplantation.

Am J Transplant. 2008; 8:377–385.

Chapter 10 Summary, discussion and future perspectives 191

Nederlandse samenvatting 203

List of contributing authors 211

List of publications 217

Dankwoord 221

Curriculum Vitae 229

List of abbreviations

Introduction and outline of this thesis

1

Introduction and outline

10

Chapter 1

Introduction and outline of this thesis

Liver transplantation is the ultimate treatment for end-stage liver disease. Survival following

liver transplantation has improved substantially over the years due to better pre-transplant

care, improved anesthesia and surgical techniques, enhanced intensive care medicine, and

more effective immunosuppressant medications. Currently, 1-year patient survival rate is

almost 90% and 5-year patient survival rate is 75% (1).

The first attempt to transplant a liver in a human was reported by Starzl in 1963 (2). In the

Netherlands, the first liver was transplanted in Groningen in 1979 (3). Nowadays, around 120

livers are transplanted annually in the Netherlands.

In the Netherlands, around 135 patients are currently on the waiting list for liver transplantation.

Although transplantation accounts for 77% of the outflow from the waiting list, unfortunately

still 12% of the patients die whilst on the waiting list. Worldwide, around 17000 patients are on

a waiting list for liver transplantation, while the estimated number of liver transplants performed

in 2008 will be less then 14000 (4). The focus on the recruitment of organ donors therefore

remains of vital importance in order to continue and improve the success of transplantation.

Posttransplant-related complications can grossly be classified into primary graft dysfunction,

vascular complications, graft rejection, recurrent disease, and biliary complications.

Reconstruction of biliary drainage is historically considered as the technical ‘Achilles heel’ of

liver transplantation (5). Although the surgical technique of biliary reconstruction has emerged

and is now a more or less standardized technique, complications arising from the bile duct

and its reconstruction remain a serious source of morbidity. The resulting biliary complications

comprise leakage and strictures. Depending on the localization, strictures are classified as

anastomotic or non-anastomotic. Non-anastomotic strictures (NAS) are considered to be

the most troublesome biliary complication after liver transplantation. NAS are defined as any

stricture, dilatation or irregularity of the intra- or extrahepatic bile ducts detected on imaging

studies of the biliary tree after liver transplantation. Approximately one in seven patients suffers

from NAS after liver transplantation. In patients with NAS graft loss is reported in up to 50% after

2 years (6). Accepted risk factors for NAS are hepatic artery thrombosis, chronic ductopenic

rejection, and ABO blood group incompatibility. In 1991 it was first described that NAS may

Chapter 1

11

also occur in the absence of these known risk factors (7). Because of the resemblance of

intrahepatic biliary strictures occurring after hepatic artery thrombosis, NAS that appeared

despite occlusion of the hepatic artery were also called ischemic type biliary lesions (ITBL).

The two names NAS and ITBL are still both used in the literature. A relationship between

NAS and the duration of cold ischemia time was discovered soon after. Ever since, research

in this area has focussed on identifying pathophysiological mechanisms and implementing

therapeutic strategies. Nevertheless, NAS still occur in many patients and in most cases no

apparent clinical risk factor can be identified. Therefore, the aim of this thesis was to perform a

more fundamental analysis, using genetic, molecular and biochemical methods in an attempt

to identify the underlying mechanisms of these biliary complications.

This thesis is divided in three parts, focusing on I) Clinical risk factors for the development and

progression of NAS, II) The role of bile salt toxicity in the development of bile duct injury and

NAS after liver transplantation, III) The role of heme oxygenase-1 (HO-1) in the protection of

liver grafts from ischemia / reperfusion (I/R) injury.

The three parts are preceded by a general overview of the causes and consequences of non-

anastomotic biliary strictures (chapter 2).

Part I. Non-anastomotic biliary complications after liver trans-

plantation.

The specific aims of this section were to describe the various forms of NAS and the

accompanying clinical risk factors as well as to study clinical risk factors for progression of

NAS. Chapter 3 describes the non-anastomotic biliary strictures in the Groningen cohort of

liver transplant recipients. All imaging studies of the biliary tree were reviewed. Localization and

severity of NAS at first presentation were categorized using a newly developed classification.

Time interval between transplantation and the initial presentation of NAS were recorded. The

purpose of this study was to identify risk factors for the clinical and radiological presentation of

NAS, as well as for the timing of NAS after liver transplantation. Chapter 4 concerns the cohort

of patients identified with NAS in chapter 3. This chapter focuses mostly on the consequences

of NAS. We defined a number of serious complications of NAS, studied their prevalence and

risk factors, and evaluated the effects of therapeutic measures.

Introduction and outline

12

Chapter 1

Part II. Bile physiology after liver transplantation.

The specific aims of this section were to evaluate the contribution of bile composition to the

development of bile duct injury. Bile salts have potent detergent properties and may damage

cells of the biliary tract by affecting the integrity of the membranes. The detergent properties

of bile salts are normally counteracted by phospholipids. By forming mixed micelles of bile

salts, phospholipids and cholesterol, phospholipids “neutralize” bile salts thereby protecting

against cellular injury. In a previous study our group has shown that bile produced early after

transplantation has an abnormal composition characterized by a low phospholipids-to-bile

salt ratio (8). Based on these findings we hypothesized that bile salt toxicity early after liver

transplantation contributes to the formation of NAS.

NAS are a frequently encountered complication after non-heart-beating (NHB) liver

transplantation. Aim of chapter 5 was to study the role of bile salt toxicity in the pathogenesis of

bile duct injury after NHB liver transplantation. We hypothesized that NHB liver transplantation

is associated with increased bile salt toxicity early after liver transplantation depending on the

length of the warm ischemia time in the donor. To test this hypothesis we studied bile composition,

graft survival and the degree of bile duct injury in a porcine liver transplant model.

Chapter 6 describes the role of altered bile composition in the development of NAS after

human liver transplantation. In a large clinical study in 111 patients bile composition and the

development of NAS were studied in a prospective fashion. The aim was to test whether bile

composition is involved in the pathogenesis of NAS.

Chapter 7 concerns the genetic variations in hepatobiliary transporters. These transporter

proteins are responsible for bile secretion. The bile salt export pump (BSEP, official name ATP

binding cassette, subfamily B, member 11. ABCB11) mediates ATP-dependent secretion of bile

salts across the canalicular membrane of hepatocytes. Multidrug resistant protein 3 (MDR3,

official name ATP binding cassette, subfamily B, member 4. ABCB4) acts as a primary active

phospholipid flippase and translocates phosphatidylcholine from the inner to the outer leaflet

of the canalicular membrane. Multidrug resistant related protein 2 (MRP-2, official name ATP

binding cassette, subfamily C, member 2. ABCC2 is a multispecific organic anion transporter

that mediates biliary excretion of a broad spectrum of divalent organic anions, including bilirubin

and glutathione. Via the subsequent passive diffusion of water into the bile, this process is

the most significant contributor to the bile salt–independent bile flow. Aim of this study was to

assess whether genetic variations in the above described transporters, present in the donor

liver, are associated with the occurrence of NAS in the recipient after transplantation.

Chapter 1

13

Part III. HO-1 and hepatobiliary injury after liver transplantation.

HO-1 has been proposed as a graft survival gene. Upregulation of HO-1 is considered to be

one of the most critical cellular protection mechanisms during cellular stress such as ischemia

and reperfusion occurring during a transplant procedure. The specific aim of this section was

to study the role of HO-1 expression in relation to postoperative hepatobiliary injury and graft

function.

Chapter 8 concerns endogenous HO-1 expression levels in human liver transplants. We

studied changes in HO-1 expression levels during liver transplantation and correlated this

with immediate postoperative hepatobiliary injury and graft function after transplantation.

Chapter 9 describes two genetic polymorphisms in the promoter influencing the inducebility

of HO-1: a (GT)n polymorphism and a single nucleotide polymorphism (SNP), A(-413)T. We

analyzed these two functional HO-1 promoter polymorphisms in donor genomic DNA in

relation to hepatobiliary injury and outcome after human liver transplantation. Furthermore,

we studied the functional relevance of these polymorphisms by measuring hepatic messenger

ribonucleic acid (mRNA) expression.

Finally, in Chapter 10 the results as described in this thesis are summarized and future

perspectives are discussed.

Referenceswww.unos.org; www.eurotransplant.nl1.

Starzl TE, Marchioro TL, Vonkaulla KN, Hermann G, Brittain RS, Waddell WR. Homotransplantation of the liver 2.

in humans. Surg Gynecol Obstet 1963; 117:659-76.

Krom RA, Gips CH, Houthoff HJ, Newton D, van der Waaij D, Beelen J, Haagsma EB, Slooff MJ. Orthotopic 3.

liver transplantation in Groningen, The Netherlands (1979-1983). Hepatology 1984; 4:61S-65S.

O’Leary JG, Lepe R, Davis GL. Indications for liver transplantation. Gastroenterology. 2008;134:1764-76. 4.

Calne RY. A new technique for biliary drainage in orthotopic liver transplantation utilizing the gall bladder as a 5.

pedicle graft conduit between the donor and recipient common bile ducts. Ann Surg 1976; 184:605-09.

Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical 6.

course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003;3:885-890.

Sanchez-Urdazpal L, Gores GJ, Ward EM, Maus TP, Wahlstrom HE, Moore SB, et al. Ischemic-type biliary 7.

complications after orthotopic liver transplantation. Hepatology 1992;16:49–53.

Geuken E, Visser D, Kuipers F, Blokzijl H, Leuvenink HG, de Jong KP, et al. Rapid increase of bile salt secretion 8.

is associated with bile duct injury after human liver transplantation. J Hepatol 2004;41:1017-25

Causes and Consequences of ischemic type biliary lesions after liver transplantation2

Jounal of HPB surgery 2006; 13:517-524

Carlijn I BuisHarm H HoekstraRobert C Verdonk

Robert J Porte

16

Causes and consequences of ITBL after liver transplantation Chapter 2

Abstract

Biliary complications are a major source of morbidity, graft loss and even mortality after liver

transplantation. The most troublesome are the so called ischemic type biliary lesions (ITBL),

with an incidence varying between 5-15%. ITBL is a radiological diagnosis, characterized

by intrahepatic strictures and dilatations on a cholangiogram in the absence of hepatic

artery thrombosis. Several risk factors of ITBL have been identified, strongly suggesting a

multifactorial origin. Main categories of risk factors for ITBL include ischemia related injury,

immunological induced injury and cytotoxic injury by bile salts. However, in many cases no

specific risk factor can be identified. Ischemia related injury comprises prolonged ischemic

times and disturbance in blood flow through the peribiliary vascular plexus. Immunological

injury is assumed as risk factor based on the relationship of ITBL with ABO incompatibility,

polymorphism in genes coding for chemokines, and pre-existing immunologically mediated

diseases as primary sclerosing cholangitis and autoimmune hepatitis. The clinical presentation

of patients with ITBL is often not specific, symptoms may include fever, abdominal complaints

and increased cholestatic liver function tests. Diagnosis is made by imaging studies of

the bile ducts. Treatment starts with relieving symptoms of cholestasis and dilatation by

endoscopic retrograde cholangiopancreaticography (ERCP) or percutaneous transhepatic

cholangiodrainage (PTCD) followed by stenting if possible. Eventually up to 50% of the patients

with ITBL will require a re-transplantation or may die. In selected cases, a re-transplantation

can be avoided or delayed by resection of the extra hepatic bile ducts and construction of a

hepatico-jejunostomy. More research on the pathogenesis of ITBL is needed before more

specific preventive or therapeutic strategies can be developed.

17

Chapter 2

Introduction

Biliary complications have since long been recognized as a major cause of morbidity and graft

failure in patients after orthotopic liver transplantation (OLT) (1-3). Bile leakage and bile duct

strictures are the most common complications. According to the localization, strictures can be

classified as anastomotic or non-anastomotic. Non-anastomotic intrahepatic strictures (NAS)

are considered to be the most troublesome biliary complication. NAS were first described

in OLT associated with hepatic artery thrombosis, where the biliary tree becomes ischemic

and eventually necrotic, resulting in a typical cholangiographic picture of biliary strictures,

dilatations and intraductal cast formation (4). However, these cholangiographic abnormalities

of strictures and dilatations can also be seen in patients who do not have an hepatic artery

thrombosis (5,6), so the term ischemic-type biliary lesions (ITBL) emerged (figure 1).

The reported incidence of ITBL differs greatly between different series, ranging from 1-19%

(7,8). Variations in the definitions of ITBL used in different studies as well as the reporting

of only symptomatic patients can at least partly explain these differences. In the majority of

series an incidence of 5 to 15% is reported (9-16).

A B

Figure 1. Cholangiogram 4 months after OLT. (A) normal, (B) ischemic type biliary lesions (ITBL).

18


Etiology and risk factors

The exact pathophysiological mechanism of ITBL is still unknown. However, several risk

factors of this often cumbersome complication have been identified, strongly suggesting a

multifactorial origin (Table 1). In general, risk factors of ITBL can be divided in three different

categories: ischemia related injury to the biliary epithelium, imunologically mediated injury and

cytotoxic injury induced by bile salts. These categories may point towards different etiological

mechanisms of ITBL, as will be described below.

Table 1. Risk factors for the development of ITBL

Ischemic injury

Warm ischemia in the donor

Prolonged cold ischemia

Reperfusion injury

Warm ischemia during implantation

Disturbed blood flow in the peribiliary plexus

Immunological injury

ABO incompatibility

Pre-existing disease with auto immune component

Auto-immune hepatitis

Primary sclerosing cholangitis

Cytomegalovirus infection

Chronic rejection

Chemokine polymorphism CCR5 delta 32

Bile salt induced injury

Hydrophilic bile salts are cytoprotective

Hydrophobic bile salts are cytotoxic

19

Chapter 2

A. Ischemic injury

The similarities between the radiological abnormalities of ITBL and the bile duct lesions seen

in the presence of hepatic artery thrombosis strongly suggest an ischemic factor in the origin

of ITBL. The quest for pathogenic mechanisms, therefore, started with factors associated with

ischemia.

A.1. Cold ischemic and reperfusion injury

Multiple studies have indicated that prolonged cold ischemia time (CIT) predisposes the graft to

the development of ITBL (6,15,17-20). In 1992, Sanchez-Urdazpal et al, reported an incidence

of ITBL of 2% in livers with a CIT < 11.5h, rising to 35% in livers with a CIT between 11.5h and

<13h and even up to 52% in grafts with a CIT > 13h (6). Nowadays many centers therefore

try to keep the CIT below 10h. However, even with a CIT shorter then 10h, Guichelaar et al

have shown that the duration of cold storage is still a risk factor for the development of ITBL

(17). The strong positive correlation between CIT and ITBL can be explained by either direct

ischemic injury of the biliary epithelium, increased susceptibility of the biliary epithelium for a

second factor such as reoxygenation injury, or secondary ischemia of the biliary epithelium

due to damage to the peribiliary arterial plexus (6).

The hypothesis that reperfusion injury during OLT contributes to bile duct injury is supported

by data provided by the experimental work of Noack et al (21). Using cell cultures, these

investigators have shown that biliary epithelial cells are more susceptible to reperfusion /

reoxygenation injury than hepatocytes. In an anoxic environment bile duct epithelial cells

and hepatocytes show equally reduced levels of ATP. However, the rate of cell death after

reoxygenation was significantly higher in the bile duct epithelial cells, compared to hepatocytes.

Increased production of reactive oxygen species by bile duct epithelial cells as well as a

lower intracellular concentration of glutathione as antioxidant, may explain this difference (21).

Clinical evidence for a contributing role of preservation injury is provided in a clinical study by

Li et al. These investigators have shown that the incidence of ITBL is significantly increased in

livers with increased preservation injury, as reflected postoperative peaks in serum aspartate

aminotransferase and alanine aminotransferase (20).

20


A.2. Injury of the peribiliary vascular plexus

Preservation injury results in increased arterial resistance and may cause circulatory disturbances

in small capillaries, such as the biliary plexus (20). Since the blood supply to the biliary tract is

solely dependant on arterial inflow, disturbances in the blood flow through the peribiliary plexus

may result in insufficient preservation and subsequent damage of the biliary epithelium.

Several studies have indicated that the viscosity of preservation solutions may play a

role in the development of ITBL (22,23). The highly viscous University of Wisconsin (UW)

preservation solution, now routinely used in most centers, might not completely flush out the

small donor peribiliary arterial plexus. Microcirculatory disturbances in the peribiliary plexus

may lead to obstruction and subsequently result in insufficient bile duct preservation (23).

Strengthening the evidence that insufficient perfusion of the peribiliary plexus might contribute

to the development of ITBL is provided in a study by Moench et al (24). These investigators

have shown that additional flushing of the peribiliary plexus by controlled arterial back-table

pressure perfusion is associated with a considerable reduction in ITBL after preservation with

UW solution (24). Apart from this, a proper harvesting technique of the liver and the extra

hepatic bile duct is critically important to preserve the viability and vasculature of the bile duct.

Although, never studied in a clinical trial, it is accepted by every surgeon that the extra hepatic

bile duct should be left covered with as much tissue as possible. Stripping of the bile duct

should be avoided in order not to injure the microcirculatory blood supply.

A.3. Warm ischemic Injury

Two periods of warm ischemia can be distinguished during the transplant procedure. The first

warm ischemia time (WIT), during harvesting and before cold preservation, and the second

WIT during graft implantation and before complete reperfusion. The first WIT is especially

a major concern in grafts from non heart-beating (NHB) donors. Several studies have

shown that liver grafts form NHB donors are at increased risk of developing ITBL (25-27).

Concern exists that harvesting time, extending the first WIT, in addition to subsequent CIT

and ischemia-reperfusion injury may result in damage to the biliary epithelium (25). Despite

plausible reasoning, no direct clinical evidence has directly linked prolonged harvesting time

with ITBL, and the literature concerning this item is not conclusive (25-29).

To reduce the incidence of ITBL, attempts have been made to reduce the second WIT.

During revascularization of the graft the most common technique is initial reperfusion via the

21

Chapter 2

portal vein with subsequent reconstruction and reperfusion of the hepatic artery. Bile ducts,

solely dependant on the hepatic artery for their blood supply, are exposed to warm ischemia

during reperfusion via the portal vein alone. This situation has been hypothesized to increase

damage of the biliary epithelium. To overcome this potential harmful situation, Sankary et al.

(18) have studied the impact of simultaneous versus sequential reperfusion of the portal vein

and hepatic artery on the incidence of ITBL. These investigators have observed a significant

reduction of ITBL when livers were reperfused simultaneously via the portal vein and hepatic

artery (18). However, in a more recent study, we were not able to demonstrate a favorable

effect of simultaneous arterial and portal reperfusion on the incidence of ITBL (30).

In an attempt to reduce the second WIT further, some investigators have introduced retrograde

perfusion of the liver graft via the inferior vena cava, after completing its anastomosis and

during construction of the portal vein anastomosis (31). Although this technique certainly

results in an earlier reperfusion of the graft, the central venous blood it is reperfused with

has a lower oxygen pressure than the portal or arterial blood. In a randomized controlled

clinical trial, Heidenhain et al. (32) have recently observed a higher incidence of ITBL in livers

that were reperfused in a retrograde fashion, compared to antegrade reperfusion via the

portal vein. The low perfusion pressure obtained during retrograde perfusion via the caval

anastomosis may be an explanation for this. This low venous pressure may result in poor flush

out and reperfusion of the peribiliary plexus, causing more ischemic biliary injury (J. Langrehr,

personal communication, 2005).

B. Immunological injury

Several papers have provided evidence for an immunological component in the pathogenesis of

ITBL (15,17,33). ITBL has been associated with various immunologically mediated processes,

such as ABO incompatible liver transplantation, pre-existing diseases with a presumed

autoimmune component (such as primary sclerosing cholangitis (PSC) and autoimmune

hepatitis (AIH)), cytomegalovirus (CMV) infection, chronic rejection, and finally with genetic

polymorphism of chemokines.

B.1. ABO incompatibility

ABO blood type mismatched liver transplantation has since long been recognized to give

rise to multiple complications (5,34). The incidence of ITBL in ABO-incompatible OLT varies

22


from 20-82% (15). An explanation for this could be the fact that the antigens of the blood type

system are not only expressed on the vascular endothelium, but also on the biliary epithelial

cells, making them a target for preformed ABO blood group antibodies (5,15). Because of this

high rate of complications and reduced graft survival rates, transplantation across the ABO

border is nowadays discouraged.

B.2. Association with pre-existing disease

It has been well described in several studies that patients who are transplanted for PSC have

a higher incidence of ITBL after transplantation (13,14,17,35,36). The association between

ITBL and AIH has only been described recently (17). PSC and AIH share a similar genetic

predisposition to autoimmunity (17). All together, these findings strengthen the hypothesis that

ITBL may have an underlying (auto) immune component.

B.3. Cytomegalovirus

In patients suffering from acquired immunodeficiency syndrome (AIDS), infection with CMV has

been shown to contribute to biliary problems, like cholangitis (37). After OLT, CMV infection

has been associated with an increased incidence of anastomotic strictures and biliary leaks

(38). CMV inclusions have been demonstrated histopathologically in the extra-hepatic bile duct

specimen in a liver transplant patient developing a biliary stricture during CMV infection (38,39).

A clear association between CMV and ITBL, however, has never been demonstrated (17). In

a recent large study of 1714 liver transplant recipients, Heidenhain et al. (40) could not find a

higher incidence of ITBL in patients who had suffered from CMV infection versus those who had

not. The role for CMV infection in the pathogenesis of ITBL, therefore, remains unclear.

B.4. Chronic rejection

Chronic rejection has been implicated as a potential cause of biliary strictures (12,41,42). This

effect is thought to be modulated not via direct injury to the biliary epithelium, but rather via the

arteriopathy accompanying chronic rejection, leading to narrowing of the medium-sized arteries.

The resulting ischemia of the bile duct wall seems to play an important role in the loss of small bile

ducts (15,43,44). Although chronic rejection has been identified as a risk factor for the development

of ITBL in several series (15,20,41,45), this could not always be confirmed by others (13,46).

Therefore the role of chronic rejection in the pathogenesis of ITBL remains to be elucidated.

23

Chapter 2

B.5. Chemokines

Chemokines play a key role in the postoperative immunomodulation, especially during rejection

as well as in post-ischemic injury. Evidence for a role of chemokines in the pathogenesis of

ITBL after OLT has been provided by a genetic association study focusing on CC-chemokine

receptor 5 (CCR5). CCR5 is a receptor for CC-chemokine ligand (CCL) 3 (macrophage

inflammatory protein 1 alpha) and CCL4 (macrophage inflammatory protein 1 beta), which

are over-expressed in infiltrating leukocytes (47). Biliary epithelial cells have been shown to

produce CC-chemokines that may bind specifically to CCR5 (48). CCR5∆32 polymorphism is

a nonfunctional mutant allele of CCR5 with an internal deletion of 32 base pairs. A study on

this polymorphism showed no differences in patient survival, rejection rates, re-transplantation

rates, and survival in OLT patients with CCR5∆32 compared with patients with wild-type CCR5

(49). Interestingly however, Moench et al recently found a very strong association between

the presence of the CCR5∆32 polymorphism in recipients and the development of ITBL after

OLT (33). These findings add to the existing evidence that immunological factors play a role

in the pathogenesis of ITBL.

C. Bile salt induced injury

Another potential factor in the pathogenesis of bile duct injury after liver transplantation

is bile salt toxicity. Bile salts have potent detergent properties towards cellular

membranes of hepatocytes and biliary epithelial cells. Normally, the toxic effects of

bile salts are prevented by complex (mixed micelle) formation with phospholipids.

Evidence for a pivotal role of bile salt-mediated hepatotoxicity in the pathogenesis of I/R injury

of liver grafts, has gradually emerged during the last decade. Using experiments in pigs, Hertl

et al. (50) have shown that bile salts can seriously amplify preservation injury of the biliary

epithelium. When porcine livers are flushed at the time of procurement with saline containing

hydrophobic bile salts, intrahepatic bile ducts are more seriously injured after even short

periods of ischemia, compared to control livers which are flushed with saline (50-52). Injury

of the biliary tree can be prevented when an infusion of hydrophilic, instead of hydrophobic,

bile salts are given to the donor animals prior to liver procurement (50). Moreover, it has been

demonstrated that morphological characteristics of human common bile ducts alter significantly

when livers are perfused with UW solution mixed with gallbladder bile, compared to livers which

are preserved with normal UW solution (53). Of interest, we recently found that microscopic

24


bile duct injury occurring early after human liver transplantation correlates with the formation

of toxic bile, characterized by a high bile salt / phospholipids ratio (54). Whether an increased

bile salt / phospholipids ratio contributes to hepatic injury or is an epiphenomenon, however,

could not be identified in this clinical study. Therefore, we recently initiated a study, using a

model of arterialized liver transplantation in mice that are heterozygous for the disruption of

the gene encoding for the transporter of phospholipids into the bile, the Mdr2 gene (Multidrug

resistance protein 2) (55). These mice disclose approximately half of the normal phospholipid

concentration in bile, leading to an abnormally high bile salt/phospholipid ratio, but have a

normal liver histology under normal conditions. When Mdr2+/- livers were transplanted after,

a short period of cold storage, into wild-type recipients serious biliary injury developed. These

findings provide evidence that endogenous bile salts act synergistically to I/R in the origin

of bile duct injury in vivo. In addition, these data indicate that intrahepatic cholestasis and

intracellular bile salt retention may be critical mechanisms triggering hepatobiliary injury after

liver transplantation. Even when the primary insult occurs to the bile ducts, hepatocellular

injury is an invariable feature of cholestasis, associated with accumulation of bile salts in the

liver and blood (56).

Current evidence indicates that bile salt retention is a key early event that contributes to

hepatocellular and biliary injury after OLT. Until more specific strategies become available, great

care should be taken to avoid exposure of bile duct epithelium to toxic bile salts during the

cold storage. Careful retrograde flushing of the bile ducts with preservation solution is therefore

considered to be critical to remove residual bile salts. Furthermore, the extra-hepatic bile duct

should not be ligated during organ procurement in order to ensure the flush out of bile and bile

salts during organ procurement and cold storage.

Clinical presentation

The clinical presentation of ITBL is often not specific; symptoms may include fever, abdominal

complaints and cholestatic liver function tests. In many patients, asymptomatic elevation of

serum gamma glytamyl transferase and/or alkaline phosphatase is the first sign of biliary

complications, prompting initiation of further examinations, such as cholangiography (16). Most

patients with ITBL present with symptoms within the first 6 months after OLT (7,12,13,17,57).

25

Chapter 2

Diagnostic work-up

The appropriate diagnostic workup has been discussed in several recent review papers (58-

60). Direct visualization of the bile ducts by endoscopic retrograde cholangiopancreaticography

(ERCP), percutaneous transhepatic cholangiodrainage (PTCD) or drain-cholangiography

remains the gold standard for making the diagnosis ITBL (7,12,13,17,24,61). Magnetic resonance

cholangiopancreaticography (MRCP) is becoming increasingly important as a diagnostic test,

with high positive and negative predictive values (62-64). Cholangiographic imaging can show

mucosal irregularities, narrowing of the lumen, and ductal dilatations (65). A classification of ITBL

has been proposed based on the localization of the abnormalities, distinguishing type I (extra-

hepatic lesions), type II (intrahepatic lesions), and type III (intra- and extra-hepatic alterations)

(66,67). However, this classification has not been widely accepted and used. In all cases of

non-anastomotic biliary strictures, patency of the hepatic artery should be carefully studied and

confirmed before the diagnosis of ITBL can be made. The presence of ITBL can be suggested

by biliary abnormalities in a liver biopsy, such as ductular proliferation and cholestasis (13).

However, ITBL remains a macroscopic and not a microscopic entity. No studies have been

conducted correlating histological abnormalities in liver biopsies and the presence of ITBL.

Treatment

More than in any other biliary complication, treatment of ITBL has to be individualized. Direct

treatment of strictures should be attempted via endoscopy or percutaneous dilatations and

stenting. With prolonged and intensive endoscopic or radiological treatment, over 50% of

patients can be treated successfully (7,12,17,20,68,69) some centers even reporting success

in over 70% (70). In many other cases, re-transplantation may at least be postponed by using

this strategy. Success will depend mainly on the severity of strictures and their localization, with

extra-hepatic strictures responding better to therapy. In patients with successful radiological

treatment, liver tests may improve, but often remain disturbed (14,69). Many physicians will

provide medical treatment with ursodeoxycholic to their patients in order improve bile flow and

to obtain a more favorable composition of the bile (68,71,72). However, the efficacy of this

strategy in influencing the incidence or outcome of ITBL has never been properly evaluated in

a randomized controlled clinical trial.

26


If non-operative techniques are unsuccessful, surgery may be appropriate in selected cases.

Especially when lesions are predominantly present at the level of the bile duct bifurcation,

resection of the extrahepatic bile ducts and Roux-en-Y hepatico-jejunostomy should be

considered. Schlitt et al. (73) have reported clinical and biochemical improvement in 14 out of

16 patients with hilar ITBL, who were treated by a hepatico-jejunostomy or portoenterostomy.

If all other treatment options have failed, retransplantation may be the only therapy left.

Especially in the presence of secondary biliary cirrhosis, recurrent cholangitis, or progressive

cholestasis due to extensive intrahepatic ITBL, retransplantation is mostly unavoidable.

The presence of ITBL is associated with a marked decrease in graft survival. Ultimately, up to

50% of patients with ITBL either die or need a re-transplantation, however mortality rates differ

markedly amongst studies (12,15,17).

Conclusion

Since the introduction of liver transplantation, biliary drainage has formed the so called ‘Achilles

heel’ of this procedure. Early studies have reported disabling complications of the biliary tract in

over 30% of the patients (74). Fortunately, much has changed during the last decades. Liver

transplantation is nowadays a standard treatment for patients with end stage liver disease and

survival is excellent, with one-year patient survival rates of 80 to 90%. Multiple improvements

in patient selection, perioperative management, as well as changes in surgical technique have

contributed to the success of OLT today. Unfortunately, despite these important improvements

and enormous gain in experience, biliary complications can still be regarded as the ‘Achilles

heel’. The most incomprehensible type of biliary complications is ITBL. Although several risk

factors for ITBL have been identified in recent years, the direct cause of ITBL can often not

be identified in an individual patient. Although it is most likely that the pathogenesis of ITBL is

multifactorial, several studies have strongly suggested a critical role for ischemic injury of the

peribiliary vascular plexus. In addition, studies have provided evidence for the involvement of

immunological processes, as well as bile salt induced injury of the biliary epithelium. Despite the

important progress that has been made in the understanding of the pathogenesis of ITBL, the

actual cause remains unidentified in many patients suffering from this troublesome complication

after OLT. Therefore, more research will be needed in this area to better identify and understand

the mechanism of ITBL. Only in this way, more specific preventive and therapeutic strategies

can developed, which may further improve patient and graft survival after OLT

27

Chapter 2

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Part I

Non-anastomotic biliary complications after liver transplantation

Non-anastomotic biliary strictures after adult liver transplantation: part I: radiological features and risk factors for early versus late presentation3

Liver Transpl 2007; 13:708-718

Carlijn I BuisRobert C Verdonk

Eric J Van der JagtChristian S van der Hilst

Maarten JH SlooffElizabeth B Haagsma

Robert J Porte

38

NAS after liver transplantation: risk factors for early versus late presentation Chapter 3

Abstract

Non-anastomotic biliary strictures (NAS) are a serious complication after orthotopic liver

transplantation (OLT). The exact pathogenesis is unclear. The purpose of this study was to

identify risk factors for the clinical and radiological presentation of NAS, as well as for the period

of presentation of NAS after OLT. A total of 487 adult liver transplants performed between 1986

and 2003 were studied. All imaging studies of the biliary tree were reviewed, cholangiography

was routinely performed between postoperative day 10-14 and later on demand. Localization

of NAS at first presentation was categorized into 4 anatomical zones of the biliary tree. Severity

of NAS was semi-quantified as mild, moderate or severe. A large number of donor, recipient

and surgical variables were analyzed to identify risk factors for NAS. NAS developed in 81

(16.6%) of the livers. Thirty-seven (7.3%) were graded as moderate to severe. In 85% of the

cases, anatomical localization of NAS was around or below the bifurcation of the common bile

duct. A large variation was observed in the time interval between OLT and first presentation of

NAS (median 4.1 months; range 0.3-155 months). NAS presenting early (≤ 1 year) after OLT

was strongly associated with preservation-related risk factors (Cold ischemia time Early NAS

694 min (501 - 797), Late NAS 490 min (394 - 650) (p=0.01)) and most frequently located in

the central bile ducts. NAS presenting late (> 1 year) after OLT was found more frequently in

the periphery of the liver and associated with immunological risk factors (PSC as indication for

OLT Early NAS n=12 (24%), Late NAS n=14 (45%) (p< 0.05)).

In conclusion, by separating cases of NAS based on the time of presentation after transplantation,

we were able to identify significant differences in risk factors, indicating different pathogenic

mechanisms depending on the time of initial presentation.

Introduction

Biliary complications are a major cause of morbidity and graft failure in patients after orthotopic

liver transplantation (OLT) (1-3). Non-anastomotic biliary strictures (NAS) are considered to be

the most troublesome biliary complication (4,5). NAS were first described in association with

bile duct ischemia due to hepatic artery thrombosis after OLT (6). However, intrahepatic biliary

lesions, such as strictures and dilatations, can also be seen in patients without hepatic artery

thrombosis (7,8). Another name that is frequently used to describe this type of complication is

39

Chapter 3

‘ischemic-type biliary lesions’ based on the radiological resemblance with biliary abnormalities

that can be seen after hepatic artery occlusion (8). The reported incidence of NAS varies

greatly between different series, ranging from 1-19% (9,10). This variation can, at least partly,

be explained by differences in the definition of NAS used in different studies, as well as the

reporting of only symptomatic patients and variations in the length of follow up after OLT. In the

majority of series an incidence between 5 to 15% has been reported for NAS (11-18).

The exact pathogenic mechanisms of NAS occurring in the absence of hepatic artery

thrombosis are still unknown. However, previous studies have strongly suggested two major

groups of risk factors: a) preservation (ischemia / reperfusion) injury-related factors and b)

variables related to immunological processes (4,19-21). In addition, recent studies have

indicated that hydrophobic bile salts are involved in the pathogenesis of biliary injury after

OLT (22-25).

In most previous studies, all patients with NAS were considered as one group, independent

from the time of occurrence after OLT and the anatomical localization (8,17,19,21,26-29), In

some studies only NAS occurring within 6 months after OLT were analyzed (20). However,

the time of presentation of NAS after OLT varies widely among different patients. In addition,

the severity and anatomical localization of biliary abnormalities at initial presentation may vary

considerably. We therefore performed a analysis of the anatomical localization and the severity

of NAS at the time of initial presentation in a large group of liver transplant recipients with long-

term follow-up. By separating cases based on the time of presentation after transplantation,

we were able to identify significant differences in risk factors for NAS, suggesting different

pathogenic mechanisms depending on the time of initial presentation. Progression of the

disease after initial presentation as well as long-term outcome of NAS in the same cohort of

liver transplants are presented separately (30).

Patients and Methods

Patients

Between January 1986 and May 2003 a total number of 717 liver transplants were performed

in 639 patients at the University Medical Center Groningen. After exclusion of children (<18

years), and patients with NAS based on hepatic artery thrombosis, 487 transplants in 428

adult patients were included in this study. Follow-up was until November 1, 2005 and median

40


follow-up was 7.9 years (interquartile range 4.2-12.6 years). Clinical information was obtained

from a prospectively collected database. If necessary the original patient notes were reviewed

for missing information. Retrospective studies were approved by the institutional ethical

committee.

Surgical Procedure

ABO blood group identical or compatible grafts from brain-death donors with normal or near

normal liver function tests were used for all patients. Organ procurement was performed

according to standard techniques, using either university of Wisconsin (UW) preservation

fluid, histidine-tryptophane-ketoglutarate (HTK) solution, or Euro-Collins (EC) solution (before

1989) (31). On the back table, bile ducts were thoroughly flushed with preservation solution. A

standardized technique was used for implantation, as has been described previously (32,33).

In our institution a duct-to-duct bile duct anastomosis is preferred, including in patients with

primary sclerosing cholangitis (PSC) if the recipient bile duct is suitable (34). A straight, open

tip silicon drain was placed transanastomotically in the bile duct, independent from the type of

bile duct anastomosis (duct-to-duct or Roux-en-Y hepatico-jejunostomy).

Postoperative Management

Two types of immunosuppressive scheme was used during the study period. For patients

with autoimmune diseases like autoimmune hepatitis, primary biliary cirrhosis, and primary

sclerosing cholangitis a triple immunosuppressive scheme [prednisolon, azathioprine and

cyclosporine A (CyA)]. All other patients received a double immunosuppressive scheme,

consisting of prednisolon together with either tacrolimus or CyA. In patients with compromised

renal function calcineurin inhibitors were withheld until creatinine clearance was over 50 mL/

min. If postoperative renal insufficiency was anticipated, induction therapy with basiliximab

was started. Biopsy-proven acute rejection was treated, when clinically indicated, with a

bolus of methylprednisolone on three consecutive days. Steroid-resistant rejections were

treated either by conversion to tacrolimus in patients on cyclosporine A, or by giving 5 doses

of antithymocyte globulin (4 mg/kg i.v.) on alternating days. When the cytomegalovirus

(CMV) status of the donor/recipient combination was positive/negative, prophylaxis with oral

ganciclovir was started at postoperative day 10 and continued for three months.

41

Chapter 3

Doppler ultrasound was performed routinely at postoperative days 1, 3, and 7 and on demand,

to rule out vascular or biliary complications or parenchymal lesions. Cholangiography via the

bile drain was routinely performed between postoperative day 10-14 and later on demand

(i.e. for rising cholestatic parameters or dilatation of bile ducts on ultrasound). The drain was

clamped when no anastomotic leakage or biliary complications were found at cholangiography.

The timing of bile drain removal has increased during the study period from one to currently

six months after transplantation. When a biliary complication was suspected after removal

of the bile drain, the preferred method for further imaging and or treatment was endoscopic

retrograde cholangiopancreaticography (ERCP). This technique has been available in our

center since the early 1980’s. In case of a hepatico-jejunostomy, percutaneous transhepatic

cholangiographic drainage (PTCD) was used to treat biliary complications. In recent years,

magnetic resonance cholangiopancreaticography (MRCP) has been used more frequently as

a diagnostic tool.

Diagnosis and Radiological Classification of NAS

For the purpose of this study, NAS were defined as any stricture, dilatation or irregularity of the

intra- or extrahepatic bile ducts of the liver graft, either with or without biliary sludge formation,

after exclusion of hepatic artery thrombosis by either Doppler ultrasound or conventional

angiography. Isolated strictures at the bile duct anastomosis were, by definition, excluded

from this analysis and have been described elsewhere (35). The time of first presentation of

NAS was recorded for all patients.

For the purpose of this study, all imaging studies of the biliary tree (cholangiography via the

biliary drain, PTCD, MRCP, or ERCP) of patients diagnosed with NAS were reviewed by a single

radiologist (EJ), who was blinded to the clinical information. The localization of biliary lesions

at the time of initial presentation was categorized according to predefined criteria, based on

the region and side of the liver. For this purpose we developed a schematic presentation of

the biliary tree in 4 different zones: the extrahepatic common bile duct (CBD) including the

hilar bifurcation (Zone A), the bile ducts between 1st and 2nd order branches (Zone B), the

bile ducts between 2nd and 3rd order branches (Zone C), and bile ducts in the periphery of

the liver (Zone D). In addition, the location of the stricture(s) was/were categorized as left or

right-sided, or bilateral (Figure 1).

42


Figure 1. Schematic presentation of the anatomical zones of biliary tree used to define the localization of NAS

after liver transplantation

The severity of biliary strictures was categorized based on an arbitrary severity index in which

strictures were scored per area as mild, moderate or severe. Severity scoring was based on

number of strictures in total, the severity according to the degree of narrowing, pre-stenotic dila-

tation and mucosal irregularity and finally the extensiveness of the strictures per area. (fig. 2)

Risk Factors for NAS

A large number of potential risk factors for NAS were studied by comparing the group of

patients with NAS with those who did not develop NAS. In addition, patients with NAS within

the first year after transplantation were compared with those who developed NAS after the first

year. Risk factors were grouped as donor-related variables (age, gender), recipient-related

variables (age, gender, indication for transplantation and Child-Pugh score), surgical variables

(preservation solution, cold ischemia time, warm ischemia time, revacularization time, type of

graft and bile duct reconstruction) and postoperative outcome variables (anastomotic leakage,

serum aspartate amino-transferase (AST), type of immunosuppression, length of stay in ICU,

CMV infection, and acute rejection).

43

Chapter 3

Figure 2. Cholangiography of patients presenting with different severities of NAS. (A) Example of mild NAS. Central

bile duct stenosis without more peripheral intrahepatic strictures and dilatations. (B) Example of moderate NAS.

Central stenosis and a stenosis in the left hepatic duct, with intrahepatic dilatations. (C) Example of severe NAS.

Diffuse strictures and irregularities of both the extra- and intrahepatic bile ducts on both sides of the liver.

44


Statistical Methods

Continuous variables were presented as medians with interquartile range (IQR) and categorical

variables as numbers with percentages. Time to occurrence of NAS was calculated according

to the Kaplan-Meier method. Categorical variables were compared using Pearson’s chi-

square test or Fisher exact test where appropriate. Comparison of continuous variables was

performed using the Mann-Whitney U test. The level of significance was set at 0.05. Statistical

analysis was performed using the SPSS/PC+ Advanced Statistics Package, Version 12.0.2

(SPSS, Chicago, IL).

Results

Initial Clinical and Radiological Presentation of NAS

Clinical characteristics of donor and recipients for the entire series are presented in Table 1.

Out of the total of 487 liver grafts, NAS was found in 81 (16.6%) livers, transplanted in 77

patients. Within the group with NAS, 71 were first transplants and 10 were retransplants. Four

patients developed NAS in both a first and a second graft.

The majority of patients with NAS presented with either elevated serum liver enzymes (n=49,

60%), and/or an episode of cholangitis (n=24, 30%). In 13 (16%) cases, the diagnosis of NAS

was based on coincidental findings on routine cholangiography in an otherwise asymptomatic

patient. The radiological modality, which led to the diagnosis of NAS, was cholangiography

via ERCP in 29, bile drain cholangiography in 24, MRCP in 23, and PTCD in 5 patients.

According to the inclusion criteria, all patients had a patent hepatic artery as confirmed by

Doppler ultrasonography or angiography.

The anatomical distribution of biliary lesions at the time of presentation is shown in Table 2.

Imaging studies for radiological evaluation was present in 78 of the 81 (96%) transplants.

45

Chapter 3

Table 1. Clinical Characteristics of Donor and Recipient for the Entire Series of Liver Transplants (n=487)*

Donor variables

Age (years) 40 (25 - 50)

Gender (male/female) 251 / 236 (52% / 48%)

Recipient variables

Age (Years) 45 (33 - 53)


Disease

PSC 82 (17%)

PBC + SBC 72 (15%)

Post viral cirrhosis 79 (16%)

Auto-immune hepatitis 47 (10%)

Alcoholic cirrhosis 38 (8%)

Cryptogenic cirrhosis 55 (11%)

Other 114 (23%)

Child Pugh Classification (A/ B/ C) 62 / 235 / 190 (13% / 48% / 39%)

Re-transplantation 60 (12%)

Surgical variables

Preservation solution

Low viscosity (EC or HTK) / High viscosity (UW) 44 / 443 (9% / 91%)

Cold ischemia time (minutes) 599 (440 - 760)

Warm ischemia time (minutes) 56 (47 - 65)

Bile duct reconstruction (dtd / Roux-Y) 410 / 71 (84% / 15%)

Type of graft (whole / reduced size) 472 / 15 (97% / 3%)

Postoperative variables

Anastomotic bile leakage 22 (5%)

serum AST postoperative day 2 (U/L) 351 (172 - 848)

Postoperative immunosuppressive treatment

Azathioprine / Tacrolimus / Cyclosporine 13 / 246 / 124 (3% / 50% / 25%)

ICU length of stay (days) 4 (2 - 8)

CMV infection 190 (49%)

Acute rejection 174 (36%)

* Continuous variables are presented as median and interquartile range, categorical variables as numbers with percentage.

Rejection: BANFF grade II - III or grade I and treated.

46


Biliary lesions were observed around or below the bifurcation of the CBD (Zone A) in 66 (85%)

cases. Biliary abnormalities became less frequent towards the periphery of the liver. The right

and left system, however, were equally affected in all zones of the biliary tree.

The severity of biliary strictures was classified as mild in 43 (55%) and as moderate to severe

in 35 (45%) of the cases. The cumulative incidence of moderate to severe NAS in the entire

population of liver transplant recipients was 7.3%.

Table 2. Anatomical Localization of NAS at Time of First Presentation.

Localization

Number (%) *

Extrahepatic or Bifurcation

Zone A 66 (81%)

left 9

right 8

both 46

CBD Only 3

Intrahepatic

Zone B 52 (67%)

left 8

right 9

both 35

Zone C 33 (42%)

left 6

right 6

both 21

Zone D 15 (19%) 1

left 2

right 12

both 1

*) More than one area could be involved in one patient.

47

Chapter 3

When Are NAS First Detected After Liver Transplantation?

A large variation was observed in the time interval between transplantation and the initial

presentation of NAS. The median time from transplantation to diagnosis of NAS was 4.1 months

(IQR 1.2-25.3 months). More than 50% of the cases of NAS presented within the first year after

transplantation (Figure 2). However, more long-term follow-up showed that the number of grafts

that develop NAS gradually continued to increase up to 12 years after transplantation. This

resulted in a sharp initial rise of the curve representing the cumulative incidence of NAS during

the first year after OLT, followed by a smaller increment beyond the first year (Figure 2). The

cumulative incidence was 14%, 15% and 16% at 3, 5 and 10 years after OLT, respectively.

Cum

ulat

ive

inci

denc

e of

NAS

(%)

5

10

15

20

Years after Transplantation

1 2 3 4 5 10 15

0

Numbers at risk 437 428 409 375 342 193 60

Figure 3. Cumulative incidence of NAS after liver transplantation in the time period 1986-2003.

48


Which Risk Factors Are Associated with NAS?

A comparison of demographic and clinical variables between grafts without NAS and the

entire group of livers that developed NAS, independent from the time of occurrence after

OLT, is presented in Table 3. The only significantly different variables between the two groups

were PSC as the indication for transplantation, type of preservation solution (high-viscosity

(UW-solution) versus low-viscosity solution (EC and HTK)), the type of bile duct reconstruction

(duct-to-duct versus Roux-Y hepatico-jejunostomy), and postoperative CMV infection.

However, standard testing whether a patient suffered from a CMV infection became only

routine clinical practice in our center around 1992, and was therefore available in only a subset

of 383 patients. In this first analysis, ischemia times did not emerge as a risk factor for NAS.

This was surprising, because cold and warm ischemia time have both been associated with

NAS in previous studies (8,20,26). However, as we have noted above, a change in the pattern

of the cumulative incidence could be observed after one year and about half of the cases of

NAS in our series were detected beyond the first year after transplantation. It is not likely that

the length of warm or cold ischemia still has an impact on the development of NAS at such

a long interval after transplantation. Therefore, we next examined radiological characteristics

and potential risk factors for early (< 1 year) versus late (> 1 year) initial presentation of NAS

after OLT.

49

Chapter 3

Tab

le 3

. Co

mp

aris

on

of

Do

no

r an

d R

ecip

ien

t C

har

acte

rist

ics

of

Liv

er G

raft

s W

ith

an

d W

ith

ou

t N

on

An

asto

mo

tic

Bili

ary

Str

ictu

res.

NAS

no N

AS

(n

= 8

1)

(n =

406

)

P-

valu

e

Don

or v

aria

bles

Age

(yea

rs)

41(3

2 - 5

0)39

(24

- 50)

0.16

Gen

der (

mal

e/fe

mal

e)40

/ 41

(49%

/ 51

%)

211

/ 195

(52%

/ 48

%)

0.67

Gen

der m

atch

(don

or/re

cipi

ent)

0.75

M

/M23

(28%

)10

4(2

7%)

F

/F21

(26%

)10

6(2

6%)

M

/F17

(21%

)10

7(2

6%)

F

/M20

(25%

)89

(21%

)

Rec

ipie

nt v

aria

bles

Age

(Yea

rs)

46(3

7 - 5

4)45

(32

- 53)

0.21

Gen

der (

mal

e/fe

mal

e)43

/ 38

(53%

/ 47

%)

193

/ 213

(48%

/ 52

%)

0.36

Dis

ease

PSC

26(3

2%)

56(1

4%)

<0.0

1

PBC

+ S

BC10

(12%

)62

(15%

)0.

50

Post

vira

l cirr

hosi

s9

(11%

)70

(17%

)0.

17

Auto

-imm

une

hepa

titis

7(9

%)

40(1

0%)

0.74

Alco

holic

cirr

hosi

s6

(7%

)32

(8%

)0.

88

Cry

ptog

enic

cirr

hosi

s8

(10%

)47

(12%

)0.

66

Oth

er15

(19%

)99

(24%

)0.

26

Chi

ld P

ugh

Cla

ssifi

catio

n (A

/ B/ C

)13

/ 39

/ 29

(16%

/ 48

% /

36%

)49

/ 19

6 / 1

61(1

2% /

48%

/ 40

%))

0.58

Re-

trans

plan

tatio

n 10

(12%

)49

(12%

)0.

94

50


NAS

no N

AS

(n

= 8

1)

(n =

406

)

P-

valu

e

Tim

e of

tran

spla

nt

Qua

rtile

(1st

/2nd

/3rd

/4th

)15

/ 21

/ 22

/ 23

(19%

/26%

/27%

/28%

)10

6 / 1

00 /

101

/ 99

(26%

/25%

/25%

/24%

)0.

53

Surg

ical

var

iabl

es

Pres

erva

tion

solu

tion

Low

vis

cosi

ty (E

C o

r HTK

) / H

igh

visc

osity

(UW

) 2

/ 79

(2%

/ 98

%)

42 /

359

(10%

/ 90

%)

0.02

Col

d is

chem

ia ti

me

(min

utes

)60

9(4

49 -

780)

594

(437

- 75

6)0.

54

War

m is

chem

ia ti

me

(min

utes

)55

(46

- 63)

56(4

7 - 6

5)0.

42

Bile

duc

t rec

onst

ruct

ion

(dtd

/ R

oux-

Y)62

/ 19

(77%

/ 23

%)

384

/ 52

(86%

/ 13

%)

0.02

Type

of g

raft

(who

le /

redu

ced

size

)80

/ 1

(99%

/ 1%

)32

9 / 1

4(9

7% /

3%)

0.29

Post

oper

ativ

e va

riabl

es

Anas

tom

otic

bile

leak

age

5(6

%)

17(5

%)

0.43

seru

m A

ST p

osto

pera

tive

day

2 (U

/L)

329

(163

- 63

0)37

0(1

73 -

885)

0.34

Post

oper

ativ

e im

mun

osup

pres

sive

trea

tmen

t

Azat

hiop

rine

/ Tac

rolim

us /

Cyc

losp

orin

e2

/ 54

/ 24

(3%

/66%

/30%

)11

/ 19

5 / 1

02(3

%/4

8%/2

5%)

0.81

ICU

leng

th o

f sta

y (d

ays)

4(2

- 7)

4(2

- 9)

0.23

CM

V in

fect

ion

44 /

29(6

0%)

146

/ 164

(47%

)0.

04

Acut

e re

ject

ion

23(2

8%)

151

(37%

)

0.13

Con

tinuo

us v

aria

bles

are

pre

sent

ed a

s m

edia

n an

d in

terq

uarti

le ra

nge,

cat

egor

ial v

aria

bles

as

num

bers

with

per

cent

age.

51

Chapter 3

NAS

no N

AS

(n

= 8

1)

(n =

406

)

P-

valu

e

Tim

e of

tran

spla

nt

Qua

rtile

(1st

/2nd

/3rd

/4th

)15

/ 21

/ 22

/ 23

(19%

/26%

/27%

/28%

)10

6 / 1

00 /

101

/ 99

(26%

/25%

/25%

/24%

)0.

53

Surg

ical

var

iabl

es

Pres

erva

tion

solu

tion

Low

vis

cosi

ty (E

C o

r HTK

) / H

igh

visc

osity

(UW

) 2

/ 79

(2%

/ 98

%)

42 /

359

(10%

/ 90

%)

0.02

Col

d is

chem

ia ti

me

(min

utes

)60

9(4

49 -

780)

594

(437

- 75

6)0.

54

War

m is

chem

ia ti

me

(min

utes

)55

(46

- 63)

56(4

7 - 6

5)0.

42

Bile

duc

t rec

onst

ruct

ion

(dtd

/ R

oux-

Y)62

/ 19

(77%

/ 23

%)

384

/ 52

(86%

/ 13

%)

0.02

Type

of g

raft

(who

le /

redu

ced

size

)80

/ 1

(99%

/ 1%

)32

9 / 1

4(9

7% /

3%)

0.29

Post

oper

ativ

e va

riabl

es

Anas

tom

otic

bile

leak

age

5(6

%)

17(5

%)

0.43

seru

m A

ST p

osto

pera

tive

day

2 (U

/L)

329

(163

- 63

0)37

0(1

73 -

885)

0.34

Post

oper

ativ

e im

mun

osup

pres

sive

trea

tmen

t

Azat

hiop

rine

/ Tac

rolim

us /

Cyc

losp

orin

e2

/ 54

/ 24

(3%

/66%

/30%

)11

/ 19

5 / 1

02(3

%/4

8%/2

5%)

0.81

ICU

leng

th o

f sta

y (d

ays)

4(2

- 7)

4(2

- 9)

0.23

CM

V in

fect

ion

44 /

29(6

0%)

146

/ 164

(47%

)0.

04

Acut

e re

ject

ion

23(2

8%)

151

(37%

)

0.13

Con

tinuo

us v

aria

bles

are

pre

sent

ed a

s m

edia

n an

d in

terq

uarti

le ra

nge,

cat

egor

ial v

aria

bles

as

num

bers

with

per

cent

age.

Is There a Difference in Radiological Presentation of Early Versus

Late NAS?

Differences in the anatomical localization of NAS presenting early (< 1 year) versus late (> 1

year) after transplantation are shown in Table 4. In contrast to the group with early presentation

of NAS in which the vast majority of lesions were found around the bifurcation and the CBD

(Zone A), biliary abnormalities in the group with late presentation of NAS were more frequently

identified in the periphery of the liver, at a level which reached statistical significance (table 4).

There were no significant differences in the severity of biliary strictures occurring early or late

after OLT. In the group of livers presenting with NAS early after OLT lesions were classified as

mild in 31 (64%) and as moderate to severe in 17 (36%) of the cases. In the group with late

presentation of NAS, lesions were classified as mild in 15 (50%) and moderate to severe in 15

(50%). Moreover, when severity of NAS at time of presentation was studied per zone of the

biliary tree, also no differences were found.

Table 4. Anatomical Localization of NAS Presentating Early (≤ 1year) Versus Late (> 1 year).

Localization Early NAS Late NAS

Number (%)* Number (%)* p-value

Extrahepatic or Bifurcation

Zone A

total 41 (85%) 25 (81%) 0.87

Intrahepatic

Zone B 30 (63%) 22 (73%) 0.72

bilateral 21 14

unilateral 9 8

Zone C 17 (35%) 16 (53%) 0.40

bilateral 10 11

unilateral 7 5

Zone D 4 (4%) 11 (37%) 0.04

bilateral 3 9

unilateral 1 2

*) More than one area could be involved in one patient.

52


Are Early and Late NAS Associated With Different Risk Factors?

When comparing all potential risk factors for NAS between the livers with early or late

presentation important differences were noted. Relevant variables with a p-value ≤ 0.1 are

presented in Table 5. The cold ischemia time was significantly longer for the group with early

NAS, compared to late NAS. In addition, the warm ischemia time was longer in the group

with early NAS, although this did not reach statistical significance. Furthermore, all cases of

anastomotic bile leakage, a condition generally associated with local bile duct ischemia, were

observed in the group with early NAS.

In contrast, significantly more patients transplanted for PSC, as well as more female/male

gender matches and Roux-Y bile duct reconstructions, were observed in the group with a

late presentation of NAS, compared to the group with early NAS. These findings indicate

that different mechanisms are involved in the pathogenesis of NAS depending on the time of

presentation after transplantation.

Table 5. Relevant Characteristics of Liver Grafts Presenting with NAS Early (≤ 1year) Versus Late (> 1 year) After OLT *

Early NAS Late NAS

(n = 50) (n = 31) P-value

Donor variables

Gender match (donor/recipient) <0.01

M/M 16 (32%) 7 (23%)

F/F 15 (30%) 6 (19%)

M/F 13 (26%) 4 (13%)

F/M 6 (12%) 14 (45%)

Recipient variables

Age 49 (37 - 57) 42 (36 - 51) 0.06

PSC as indication for OLT 12 (24%) 14 (45%) < 0.05

Surgical variables

Cold ischemia time (minutes) 694 (501 - 797) 490 (394 - 650) 0.01

Warm ischemia time (minutes) 57 (48 - 65) 53 (45 - 57) < 0.05

Bile duct reconstruction (dtd / Roux-Y) 43 / 7 (86% / 14%) 19 / 12 (61% / 39%) 0.01


Bile leakage 5 (10%) 0 0.07

* Only variables with p value ≤ 0.1 are presented in this table.

53

Chapter 3

Discussion

Strictures of the bile ducts are a serious complication after OLT, causing increased morbidity

and graft loss (9,20). Although the exact pathogenesis of this type of biliary complication

remains unknown, both preservation-related factors and immunological processes have

been suggested to play a role (4,19,20). Results from previous clinical studies focusing on

potential risk factors of NAS, however, are not unequivocal and conflicting data have been

found (20,36,37)

In this study we were able to identify differences in the anatomical localization as well as

differences in risk factors for NAS depending on the time of first presentation after OLT. While

ischemia and preservation-related variables were most prominent in the group with early

presentation, a late presentation of NAS was more frequently associated with immunological

factors. These findings provide new insights in the pathophysiological mechanisms of NAS.

In the current series, the cumulative incidence of NAS at 15 years after transplantation

was almost 17%, and 7% of all liver grafts were radiological graded as having moderate or

severe biliary strictures. These figures are in line with most previous studies (11,15,18,20).

However, lower percentages have also been reported in some series, which may be explained

by differences in the definition and diagnostics used, as well as differences in the duration

of follow up (17,38). The routine use of a biliary drain and postoperative cholangiography

allowed us not only to carefully identify and localize all biliary abnormalities, but also to include

minor or single strictures in otherwise asymptomatic patients. Routine cholangiography has

not always been used in previous studies. These factors may largely explain the differences in

the incidence of NAS reported in different series (9-18,20). In addition, we had a long follow-

up in our series with a median of almost 8 years. However, a limitation of this retrospective

single centre study could be different imaging modalities over the years and should be kept

in mind.

When all cases with NAS were studied as one group, independent from the severity and time

of occurrence after OLT, only PSC as the indication for transplantation, the type of preservation

solution and postoperative CMV infection could be identified as risk factors for NAS. The

higher incidence of NAS in patients transplanted for PSC (15,20,21) and patients who suffered

CMV infection postoperatively are in accordance with previous studies (19,36,37,39). Also the

54


more frequent occurrence of NAS in patients with a Roux-Y hepatico-jejunostomy has been

reported before and this can be explained by the more frequent use of this type of bile duct

reconstruction in patients transplanted for PSC, compared to patients transplanted for other

indications (20). The lower incidence of NAS in our series in livers which were preserved with

a low viscosity preservation solution is in agreement with previous reports (40). It has been

suggested that the peribiliary vascular plexus is better flushed out and better preserved when

low viscosity fluids are used compared with high viscosity fluids. These observations, however,

have not yet been confirmed to our knowledge in randomized controlled trials (41,42).

In contrast with previous studies, we were not able to identify an association between the

lengths of warm or cold ischemia time and the development of NAS when we analyzed all

grafts with NAS as one group, regardless the time of occurrence after OLT. Most previous

studies, however, had a short postoperative follow-up of less than one year, whereas in our

series the median follow-up was 7.9 years with an interquartile range of 4.2 to 12.6 years.

Although the cumulative incidence of NAS increased sharply within the first year, almost 50%

of all cases were detected beyond the first year after transplantation. Biologically it is not

plausible that preservation-related factors are still responsible for NAS that first present more

than one year after OLT. We therefore performed a second analysis comparing patients with

early (< 1 year after OLT) versus late (> 1 year after OLT) presentation of NAS. This analysis

showed significant associations between preservation-related risk factors, such as the length

of cold ischemia time and bile duct anastomotic leakage, and the occurrence of NAS early after

OLT. Ischemia reperfusion- and preservation injury-related variables are well described risk

factors for NAS, and include prolonged cold ischemia time (> 12 hours) or warm ischemia time

(> 60 min) and variables related to the efficacy of preservation of the peribiliary plexus, such

as viscosity and perfusion pressure of the preservation fluid (20,26,40). Moreover, the higher

incidence of NAS in liver transplantation from donors after cardiac death (non-heart-beating

donors) also strongly suggests an ischemia-related factor in the pathogenesis of NAS (43-45).

For many years, it has been policy in our center to keep the cold ischemia time as short as

possible and recipient operations usually start before the donor liver has arrived and as soon

as the liver has been judged transplantable by the surgical team performing the procurement

operation. In addition, the use of the piggyback technique has allowed us to shorten the warm

ischemia time during implantation, in comparison with conventional implantation (33,46). With

this policy we were able to keep the median cold and warm ischemia time below 12 hours

55

Chapter 3

and 60 min, respectively. Nevertheless, we could still identify cold ischemia time as one of the

most important discriminators of NAS occurring early after OLT.

Risk factors for the development of NAS late (> 1 year) after transplantation were a female to

male donor/recipient match, and PSC as the indication for transplantation. These variables

are not associated with preservation injury and suggest a more immunological pathogenesis

of NAS presenting late after OLT. An immunological origin of NAS has been suggested by

other investigators based on the relationship between NAS and ABO incompatibility, the

strong association with pre-existing diseases with a presumed autoimmune component (such

as PSC and autoimmune hepatitis), CMV infection, chronic rejection, but also with genetic

polymorphism of chemokines (17,20,28). It is very likely that recurrent PSC may have been

accountable for the occurrence of biliary lesions in some of the patients presenting with

NAS late after OLT. Based on radiological evaluation, however, recurrent PSC cannot be

distinguished from a late presentation of NAS. Although some of our patients fit well within the

definition of recurrent PSC as proposed by Graziadei et al. (47), more than half of our patients

who presented with NAS late after OLT were not transplanted for PSC.

Several studies have shown a lower survival rate for grafts from female donors transplanted in

male recipients. (48-51). Although some investigators have tried to explain this by differences

in estrogen receptor expression (49), reduced outcome for the female to male donor/

recipient match has also been described after OLT in children below 10 years of age (48).

This observation makes it less likely that a sex hormone-related pathogenesis is the only

explanation for the worse outcome of female livers into male recipients and immunological

processes have been suggested to play a role as well in other transplant settings (52).

Immunologically-mediated injury of the bile ducts resulting in NAS may be a direct result of

activated proinflammatory cytokines and influx of inflammatory cells. However, it cannot be

deducted from a clinical study like this whether this type of bile duct injury is (at least partially)

also caused by relative ischemia of the biliary epithelium due to immune-mediated obliterative

arteriopathy of the peribiliary vascular plexus (8,9,19). Further research in this area seems

warranted.

Very few studies to our knowledge have focused on the anatomical localization of NAS at the

time of first presentation. In the current series, over 80% of the NAS were localized around or

below the bifurcation of the CBD and less than 20% presented in the peripheral branches of

the biliary tree. Livers presenting with NAS more than one year after OLT had more frequently

56


involvement of the smaller and peripheral bile ducts of the liver. These differences in the

anatomical localization between NAS presenting early or late after OLT provide additional

support for differences in the pathogenesis of NAS depending on the time of presentation after

OLT. The critical relevance of arterial blood supply for the viability of the larger and extrahepatic

bile ducts is well described (53). This part of the biliary tree depends entirely on the arterial

peribiliary plexus which is perfused via the gastroduodenal artery and the hepatic artery.

During OLT, blood supply via the pancreatic head and the gastro-duodenal artery, supplying

the peribiliary plexus, is interrupted and the bile ducts become entirely depended on arterial

blood from the hepatic artery, making them more prone to hypoperfusion and ischemia. This

may explain the central localization of NAS presenting early after OLT. In addition, previous

studies have shown a large morphological and functional heterogeneity of different sized

intrahepatic bile ducts, providing an explanation why biliary lesions predominates in specific

sized bile ducts in various types of diseases affecting the biliary tree (54). This could also

be an explanation why immunologically-mediated NAS, presenting late after OLT, is more

pronounced at the level the smaller bile ducts.

In summary, by separating cases of NAS based on the time of presentation after transplantation,

we were able to identify significant differences in risk factors, indicating different pathogenic

mechanisms depending on the time of initial presentation. NAS presenting within the first

year after transplantation is strongly correlated with ischemia related risk factors, whereas

NAS presenting late, more than one year after OLT, is more associated with immunologically

related risk factors. These finding have implications for the development of new strategies to

prevent or treat NAS.

57

Chapter 3

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Non-anastomotic biliary strictures after liver transplantation part 2: Management, outcome and risk factors for disease progression4

Liver Transpl 2007; 13:725-732

Robert C VerdonkCarlijn I Buis

Eric J Van der JagtAnnette SH Gouw

Abraham J LimburgMaarten JH SlooffJan H Kleibeuker

Robert J PorteElizabeth B Haagsma

62

NAS after liver transplantation: risk factors for disease progression Chapter 4

Abstract

Non-anastomotic biliary strictures (NAS) after orthotopic liver transplantation (OLT) are

associated with high retransplant rates. The aim of the present study was to describe the

treatment, and identify risk factors for radiological progression of bile duct abnormalities,

recurrent cholangitis, biliary cirrhosis and retransplantation in patients with NAS. We

retrospectively studied 81 cases of NAS. Strictures were classified according to severity and

location. Management of strictures was recorded. Possible prognostic factors for bacterial

cholangitis, radiological progression of strictures, development of severe fibrosis/cirrhosis and

graft and patient survival were evaluated. Median follow up after OLT was 7.9 years. NAS

were most prevalent in the extrahepatic bile duct. Twenty-eight patients (35%) underwent

some kind of interventional treatment, leading to a significant improvement in biochemistry.

Progression of disease was noted in 68% of cases with radiological follow-up. Radiological

progression was more prevalent in patients with early NAS and one or more episodes of

bacterial cholangitis. Recurrent bacterial cholangitis (> 3 episodes) was more prevalent in

patients with a hepaticojejunostomy. Severe fibrosis or cirrhosis developed in 23 cases,

especially in cases with biliary abnormalities in the periphery of the liver. Graft but not patient

survival was influenced by the presence of NAS. Thirteen patients (16%) were re-transplanted

for NAS. In conclusion, especially patients with a hepatico-jejunostomy, those with an early

diagnosis of NAS, and those with NAS presenting at the level of the peripheral branches of

the biliary tree, are at risk for progressive disease with severe outcome.

63

Chapter 4

Introduction

Biliary complications are common after orthotopic liver transplantation (OLT). Biliary strictures

and leakage of bile are most frequently encountered. Strictures are often referred to as

anastomotic or non-anastomotic. Non-anastomotic strictures (NAS) are generally considered

to be the most troublesome type of biliary complications after liver transplantation, with a graft

loss rate of up to 46% after two years (1).

In a separate study we have analyzed the radiological characteristics of NAS at the time

of diagnosis and risk factors for the development of NAS (2). In this study, we were able to

identify significant differences in risk factors for the development of NAS depending on the

time of initial presentation. In addition, large variations in anatomical localization and severity

of NAS at the time of presentation were found, indicating that NAS is not a single disease,

but rather a group of biliary abnormalities with different pathogenesis. It is unknown whether

the different subtypes of NAS are also associated with difference in outcome and prognosis.

Previous studies concerning the treatment and outcome of NAS have not considered different

types of NAS as relevant subgroups and risk factors for radiological and clinical progression

once the diagnosis has been established have not been identified so far.

The aim of the present work was to study NAS in a large cohort of liver transplant recipients

with long-term follow up and to describe the results of treatment. In addition, we aimed to

identify risk factors for radiological progression of bile duct abnormalities, recurrent cholangitis,

biliary cirrhosis and re-transplantation.


Patients

Between January 1986 and May 2003 a total number of 717 liver transplants were performed

in 639 patients at the University Medical Center Groningen. After exclusion of children (<18

years), and patients with NAS caused by hepatic artery thrombosis, 487 transplants in 428 adult

patients were included in this study. Follow-up was until November 1st 2005, allowing a minimal

follow up time after transplantation of 2.5 years. Eighty-one grafts with NAS were identified in

77 patients as described previously (2). In short, all post-transplant radiological material of the

biliary tree was reviewed by a radiologist blinded to the clinical data (EJ). Anatomical extent

64


and severity of the biliary abnormalities were classified using a standardized scoring system.

The scheme used to classify anatomical localization and extension of NAS is depicted in

Figure 1. Severity was arbitrarily scored as mild, moderate or severe, according to the degree

of narrowing, pre-stenotic dilatation, and mucosal irregularity. Patient characteristics as well

as anatomical localization and severity of NAS are summarized in Table 1.

Figure 1. Schematic presentation of the anatomical zones of biliary tree used to define the localization of NAS

after liver transplantation

Study Endpoints

Clinical variables. Clinical information was obtained from the original patient notes, operation

notes and endoscopy reports. Records were reviewed for patient characteristics, indication for

liver transplantation, type of biliary reconstruction and outcome. Laboratory values of alkaline

phosphatase (APh), gamma glutamyltransferase (GGT), alanine-aminotransferase (ALT) and

total bilirubin (bili) were studied for the following time points: at the time of presentation, at the

beginning of treatment, and to study the effect of treatment, at a stable level within 3 months

after the last intervention.

65

Chapter 4

Management of NAS. Information about interventions was obtained from the patient notes.

Endoscopic retrograde cholangiopancreaticography (ERCP), percutaneous transhepatic

cholangiodrainage (PTCD), surgery, and medical therapies (ursodeoxycholic acid, antibiotics)

were noted. When ERCP or PTCD with interventions had been performed, the number of

sessions was registered, as well as technical details of the procedure. In case of surgical

treatment, the type of surgical procedure was recorded. Complications of treatment

were registered. Radiological progression. To study radiological progression of NAS all

cholangiograms (drain cholangiography, PTCD, MRCP, ERCP) that were performed after

transplantation were reviewed by a single radiologist (EJ), blinded to clinical information,

and using the same scoring system as described above. Bacterial cholangitis. Bacterial

cholangitis episodes were noted. Bacterial cholangitis was defined as an episode of liver

test abnormalities combined with fever for which antibiotic treatment was given. Recurrent

cholangitis was defined as three or more episodes of cholangitis.

Table 1. Patient Characteristics and Possible Prognostic Factors

Characteristic N (% or range)

Age at time of transplantation (median, range)Gender (M/F)Primary liver disease: PSC / OtherBiliary reconstruction: Duct-to-duct / Roux-en-YRe-transplant graftIBD before OLTIBD after OLTEarly NAS (<1 year after OLT)Extent of NAS at presentation* Zone AZone BZone CZone DSeverity of NAS at presentation* Mild / Moderate / SevereType of immunosuppresionPrednisone / azathioprine / cyclosporinePrednisone / tacrolimusPrednisone / tacrolimus / azathioprineOther

46 (18-66)40 / 3725 (31%) / 56 (69%)62 (77%) / 19 (23%)10 (12%)13 (16%)14 (17%)50 (62%)

66 (81%)52 (67%)33 (42%)15 (19%)

43 / 28 / 7

51 (63%)8 (10%)6 (7%)16 (20%)

* Data on patients with radiological material available (n=78)

66


Pathology. To see whether NAS led to biliary fibrosis of cirrhosis, the most recent available

pathology specimen of the liver of all patients was retrieved and scored by a liver pathologist

(AG) blinded to the clinical context. Liver fibrosis was scored as absent, minimal, moderate or

severe, with severe being either extensive bridging fibrosis or cirrhosis.

Survival. Graft and patient survival were analyzed by comparing patients with NAS to controls

matched for age and period of transplantation. Controls also had to be alive at the time of

diagnosis of NAS in the patients with NAS. Causes of death and graft failure were noted.

Prognostic factors. Possible prognostic factors for several outcome parameters are listed

in Table 1. The definition of inflammatory bowel disease (IBD) after liver transplantation was

an episode of abdominal pain and/or diarrhea, with inflammation seen during endoscopy,

confirmed pathologically and after exclusion of infectious causes. In addition the following

factors were included in the analysis: (type of) interventional treatment, the presence of

radiological progression, the occurrence of bacterial cholangitis, the maintenance use of

antibiotics, and the use of ursodeoxycholic acid.

Statistical Methods

Data were analyzed using SPSS 12.0 software. Comparison between groups was made using

the Chi-square test for categorical variables and the Mann-Whitney U test for continuous

variables. When indicated, a risk estimate was made calculating the relative risk (RR) and

confidence intervals using a Chi-Square test. Comparison of survival between groups

was made using Kaplan-Meier statistics with a log-rank test. A p-value of 0.05 or less was

considered to indicate statistical significance.

Ethical statement

Retrospective studies are approved by the institutional ethical committee.

Results

NAS were present in 81 grafts of 77 patients. In four patients NAS occurred in both a first and

second graft. Apart from NAS, a concomitant anastomotic stricture was diagnosed at some

point in the postoperative course in 21 patients. Median follow-up after the diagnosis of NAS

was 6.0 years (1.0-17.0). Median follow-up after OLT was 7.9 years (range 1.0-17.1). The

biliary reconstruction was duct-to-duct in 62 cases (77%), and a hepaticojejunostomy with

Roux-en-Y deviation in 19 cases (23%).

67

Chapter 4

Which Modalities Were Used for Treatment of NAS?

Interventions. Twenty eight patients (35%) were treated with ERCP, PTCD, surgery, or a

combination of these. Thirteen patients underwent one or more sessions of ERCP. Dilatation

was performed in all cases; in 12 also one or more stents were placed. Complications occurred

in 7, mostly cholangitis. No severe complications were observed. The median number of

therapeutic ERCP’s in these patients was 3 (range 1-11).

Seven patients underwent PTCD. Four patients underwent both ERCP and PTCD. In patients

treated with PTCD dilatation and stenting was performed in all cases. In two cases an

expandable metal stent was placed. The median number of therapeutic PTCD sessions in

these patients was 3 (range 1-6). A minor complication occurred in 2 cases.

In the end, eight patients underwent surgery for NAS, four after previous ERCP or PTCD. The

surgical procedure was conversion of duct-to-duct anastomosis to a hepatico-jejunostomy in

five patients, and revision of a previous hepatico-jejunostomy in three. Patients with a dilated

biliary tree were treated surgically more often than those without dilatation (20% vs. 2%,

p=0.01). All concomitant anastomotic strictures were successfully treated with success by

ERCP (n=13), PTCD (n=5), surgery (n=1) or a combination of these (n=2).

Ursodeoxycholic acid. Seventy-one patients (88%) were treated with long-term ursodeoxycholic

acid, mostly at a dose of 600 mg b.i.d.

Biochemical response to interventions. When the biochemical response within 3 months after

completion of interventional treatment was studied, significant improvements in serum ALT

(median 65 U/l vs. 36 U/l, p=0.015), bilirubin (median 46 µmol/l vs. 23 µmol/l, p<0.000) and

GGT (median 360 U/l vs. 125 U/l, p=0.014) was noted, compared to pretreatment values. No

significant improvement in APh was seen. In 8 of the 28 patients no biochemical response to

treatment was seen.

Is NAS a Progressive Disease?

Radiological progression. Material for retrospective radiological evaluation of NAS at

presentation was available in 78 of the 81 transplants (96%). In 59 cases (80%) follow-up

cholangiography was performed and available for determination of progression of the biliary

abnormalities. The median time between the diagnostic and last cholangiography was 1.7

years (range 0.1 – 11.7). Progression of the severity of biliary abnormalities was observed

in 28 (42%) of the 59 grafts with follow up cholangiography. At the time of diagnosis, the

68


severity of NAS was scored as mild in 32 (54%), moderate in 22 (37%) and severe in 5 (9%)

cases. At the end of follow up the severity of NAS was scored as mild in 17 (29%), moderate

in 22 (37%) and severe in 20 (34%) cases. Progression of the anatomical extent of the biliary

abnormalities was seen in 36 (61%) of the patients with follow up cholangiography. The details

are listed in Table 2. Progression was seen at all levels of the biliary tree.

Table 2. Radiological Progression of NAS in Patients With Follow-up Cholangiography (n=59)*

LocalizationPresentationN (%)

End of follow upN (%)

ExtrahepaticZone A all

IntrahepaticZone B left right both

Zone C left right both

Zone D left right both

50 (58)

5 (8.5)7 (11.9)27 (45.8)

5 (8.5)5 (8.5)15 (25.4)

1 (1.7)2 (3.4)7 (11.9)

56 (97)

4 (6.8)8 (13.6)40 (67.8)

4 (6.8)3 (5.1)25 (42.4)

4 (6.8)0 (0)13 (22)

* More than one area could be involved in one patient.

Casts and sludge were seen at some time point after transplantation in 21 (27%) and 18

(23%) patients respectively. Cholangitis episodes. Thirty-nine subjects (48%) had at least

one episode of cholangitis. Nineteen had to be admitted repeatedly for recurrent bacterial

cholangitis (defined as three or more episodes). The median number of cholangitis episodes

in these 19 was 5 (range 3-17). Thirty patients were put on maintenance use of antibiotics for

some time, mostly ciprofloxacin.

Liver pathology. Pathology specimens were available from 63 livers. The mean time from

transplantation to biopsy was 3.7 years (range 0.1-15.9). At the end of follow up, pathologically

proven biliary cirrhosis or severe bridging fibrosis had developed in 17 cases (25%). In an

additional six patients the diagnosis of cirrhosis was made on clinical grounds: these patients

were known with severe NAS, and developed ascites, abnormal coagulation or varices with

radiological evidence of cirrhosis while the portal vein was open. Thus, in the end severe

fibrosis or cirrhosis developed in 23 (28%) of the livers with NAS.

69

Chapter 4

Are Patient and Graft Survival Affected by NAS?

Graft survival. Graft survival of the patients with NAS after one, five and ten years was 91%

(3.1), 73% (5.0) and 63% (6.1) respectively (standard error in parentheses). Graft survival

was significantly lower in the patients with NAS, compared to matched controls without NAS

(p=0.001, fig. 3).

Thirteen patients (16%) underwent re-transplantation of the liver for NAS after a median of 0.9

years (mean 3.9 years, range 0.2 – 12.3). At the end of this study, two patients were awaiting

liver re-transplantation for NAS.

Patient survival. Compared to matched controls, patient survival was lower in patients with

NAS, although this did not reach statistical significance (fig. 3).

At the end of the study 17 patients had died. In 5 cases, the cause of death was related

to NAS. In four patients the cause of death was multi-organ failure after sepsis due to

cholangitis, in one case liver failure due to biliary cirrhosis. Two patients had been offered a

re-transplantation, but refused.

Which Factors Are Predictive for Progression of NAS?

An overview of the analyses of prognostic factors is presented in Table 3.

Table 3. Prognostic Factors for Progression and Outcome of NAS

Outcome parameter Prognostic factor RR (95% CI), p-value

Radiological progression

Recurrent cholangitis

Biliary cirrhosis/bridging fibrosis

Severe outcome **

Asymptomatic course***

Early NAS (< 1 year)One or more episodes of cholangitis

Roux-en-Y hepaticojejunostomy

Abnormalities at Zone B*Abnormalities at Zone C*

Abnormalities at Zone C*Radiological progression during follow up

Mild abnormalities*

1.9 (1.1-3.4), 0.0042.0 (1.0-4.2), 0.018

3.6 (1.7-7.6), 0.001

1.5 (1.1-1.9), 0.0211.8 (1.2-2.8), 0.022

1.7 (1.1-2.7), 0.0291.8 (1.1-3.0), 0.026

1.9 (1.3-2.7), 0.002

* at presentation

** defined as: death due to NAS, cirrhosis/fibrosis, retransplantation

*** defined as: no cholangitis, no fibrosis or cirrhosis, no re-transplantation, no need for treatment

70


Patients diagnosed with NAS

(N=81)

Treatment

N=28

ERCP: 11

PTCD: 5

ERCP+PTCD: 4

Surgery 4

ERCP+Surgery: 2

PTCD+Surgery: 2

No treatment

N=53

FOLLOW UP

Outcome

Rec. Cholangitis 5 (18%) *

Cirrhosis 8 (29%)


Death d/t NAS 2 (7%)

Outcome

Rec. Cholangitis 5 (10%)

Cirrhosis 13 (25%)



Figure 2. Clinical course and outcome in patients with NAS. * A total of 14 patients experienced recurrent

cholangitis (3 or more episodes). A total of 5 patients experienced recurrent cholangitis after treatment was

finished.

Predictors of radiological progression. When patients with progression of radiological

abnormalities were compared with patients in whom the severity and extent of abnormalities

was not progressive, two risk factors for progression were identified: early NAS presenting

within 1 year after transplantation and one or more episodes of cholangitis. Patients presenting

with early NAS were also at increased risk for both casts (RR 1.6, 95%CI 1.2-2.2, p=0.008)

and sludge (RR 1.8, 95%CI 1.4-2.4, p=0.001). Predictors of bacterial cholangitis. The only risk

factor for recurrent cholangitis, defined as 3 or more episodes, was a biliary reconstruction

with a Roux-en-Y hepatico-jejunostomy. Predictors of progression of fibrosis. Radiological

abnormalities at the intrahepatic level were identified as risk factors for development to severe

bridging fibrosis or cirrhosis. These concerned Zone B and Zone C.

71

Chapter 4

Patients diagnosed with NAS

(N=81)

Treatment

N=28

ERCP: 11

PTCD: 5

ERCP+PTCD: 4

Surgery 4

ERCP+Surgery: 2

PTCD+Surgery: 2

No treatment

N=53

FOLLOW UP

Outcome

Rec. Cholangitis 5 (18%) *

Cirrhosis 8 (29%)



Outcome

Rec. Cholangitis 5 (10%)

Cirrhosis 13 (25%)



Figure 2. Clinical course and outcome in patients with NAS. * A total of 14 patients experienced recurrent

cholangitis (3 or more episodes). A total of 5 patients experienced recurrent cholangitis after treatment was

finished.

Predictors of radiological progression. When patients with progression of radiological

abnormalities were compared with patients in whom the severity and extent of abnormalities

was not progressive, two risk factors for progression were identified: early NAS presenting

within 1 year after transplantation and one or more episodes of cholangitis. Patients presenting

with early NAS were also at increased risk for both casts (RR 1.6, 95%CI 1.2-2.2, p=0.008)

and sludge (RR 1.8, 95%CI 1.4-2.4, p=0.001). Predictors of bacterial cholangitis. The only risk

factor for recurrent cholangitis, defined as 3 or more episodes, was a biliary reconstruction

with a Roux-en-Y hepatico-jejunostomy. Predictors of progression of fibrosis. Radiological

abnormalities at the intrahepatic level were identified as risk factors for development to severe

bridging fibrosis or cirrhosis. These concerned Zone B and Zone C.

Figure 3. Patient and graft survival in patients with NAS (n=81) and matched controls (n=81). N.s.: not significant

Predictors of an asymptomatic course. In 23 cases (28%) the NAS were completely

asymptomatic, defined as no episodes of cholangitis, no biliary fibrosis or cirrhosis, and no

need for interventional treatment. When these patients were compared with the other 58

patients, the radiological findings at presentation were predictive of an asymptomatic course:

patients with abnormalities that were scored as mild at the time of diagnosis had a significantly

higher chance of an asymptomatic course (44% if mild vs. 11% not mild, p=0.002).

Predictors of severe outcome. To analyze risk factors for NAS with severe outcome, we

identified three markers of severe outcome: death due to NAS, re-transplantation, and biliary

cirrhosis or severe bridging fibrosis. Patients experiencing one or more of these were compared

with the remaining group of patients. Two risk factors for severe outcome were identified: NAS

presenting at the intrahepatic Zone C, and NAS that showed radiological progression during

follow up. With respect to severe outcome there was no difference between the 28 patients

who received interventional treatment (ERCP, PTCD, surgery) versus the 53 patients who did

not (severe outcome 46% versus 37%, p=0.389.

72


Discussion

NAS or intrahepatic biliary strictures are a common and often troublesome complication after

liver transplantation. Although previous studies on this subject differ markedly concerning

methodology and results, a high incidence of retransplantation has been reported almost

uniformly, as well as the need for frequent biliary interventions and admissions (3-5). In the

present study, we found a relatively high incidence of NAS compared to previous reports.

Whereas most large series report an incidence of NAS of 5-10% (6-8), we found that 17% of

patients were diagnosed with NAS at some point after OLT. Most likely, this difference is due

to the fact that we defined any type of biliary stricture other than anastomotic strictures as

NAS. In adition, postoperative cholangiography via the biliary drain has been routine practice

in our center. This allowed us to identify a large number of cases without any persisting clinical

signs of biliary disease (23 cases, 28%). This is also reflected by the rather low number of

retransplantations (16%) compared to previous reports from other centers. Another possible

explanation is the large number of patients transplanted for PSC (17%) and relatively low

number transplanted for viral disease (16%) in our center. PSC is a known risk factor for the

development of NAS (9-12).

From the present material it becomes clear that NAS after liver transplantation is not just one

disease, but a spectrum of abnormalities, ranging from slight, localized mucosal irregularity

to extensive and diffuse biliary strictures. Possibly, not all areas of non-anastomotic bile duct

narrowing are due to a fibrotic type of stricture (13). We thus aimed to identify from this diverse

group of NAS those cases that would progress to a clinically relevant or progressive disease.

We found radiological progression in 68% of our patients with cholangiographic follow-up.

Most likely, this number is lower for the entire group of patients with NAS, since those patients

without radiological follow-up probably did not have marked progression. Interestingly, NAS

presenting early after transplantation had a higher risk of progression. Previous investigators

have also mentioned a more severe course of disease in patients with NAS presenting

early after transplantation (14-16). Besides a higher risk of radiological progression, these

cases also showed a significantly higher risk for the development of casts and sludge. These

differences are probably due to a different pathogenesis of NAS presenting early or late after

transplantation, as has been described by Buis et al. earlier in this journal.

The most critical clinical consequences of NAS are recurrent cholangitis and biliary cirrhosis.

73

Chapter 4

Both may necessitate re-transplantation. We found recurrent cholangitis (arbitrarily defined as

three or more episodes) in 19 of our patients, despite treatment with maintenance therapy with

antibiotics in most patients. The only risk factor for recurrent cholangitis that was identified

was the presence of a hepaticojejunostomy instead of a duct-to-duct anastomosis. Most likely,

this type of biliary reconstruction leads to reflux of bacteria into the biliary tree, as has been

shown in animal models (17). In a patient that is immunosuppressed and has diminished flow

of bile due to NAS, it is foreseeable that this situation will lead to bacterial colonization of the

bile ducts and repeated episodes of cholangitis.

Biliary cirrhosis is the ‘end-point’ of long-standing NAS. We found biliary cirrhosis or severe fibrosis

in 23 of our cases (28%). At the end of follow up, nine of these 23 (39%) were re-transplanted.

Interestingly, the two risk factors for development of biliary cirrhosis were strictures at the level

of the segmental (Zone B) and sub-segmental (Zone C) branches. Apparently, these strictures

cause more long-term damage to the liver than more centrally located lesions. This may be due

to a number of factors. Perhaps, this type of NAS has a different pathogenesis than the more

‘proximal’ type of NAS, leading to ongoing biliary damage. Another possible explanation is that

these abnormalities are less amenable to treatment. Knowing that strictures at the site of the

segmental and sub-segmental branches are a risk factor for biliary cirrhosis, one can make an

estimate of the risk for progressive disease at the time of diagnosis.

When biliary cirrhosis, retransplantation and death due to NAS were combined to define serious

disease, strictures at the level of the subsegmental branches (Zone C) and radiological progression

of strictures were identified as significant risk factors. Thus, one can use these characteristics to

define patients with a higher risk of serious disease in the future.

Although we did see NAS-related mortality in our series, overall patient survival was not significantly

affected. This corresponds to previous studies on this subject (18-21). Graft survival however was

impaired compared to matched controls (73% vs. 94% after five years). This is not a surprising

finding. It is not possible from our results to conclude whether or not treatment for NAS prevented

re-transplantation in a number of cases. Although the number of re-transplantations was similar

in patients with and without endoscopic, percutaneous or surgical therapy; we do not know

what these numbers would have been like without treatment. Previously, others have described

successful treatment of NAS with a number of modalities (22-26).

We did not study the success of treatment in these patients, since the group of patients

treated with ERCP, PTCD or surgery is rather small (28 cases), heterogeneous concerning

74


location and severity of abnormalities, and several types of treatment modalities were used.

However, in the majority of patients an improvement in liver test was seen, although this is

not synonymous with uneventful long-term outcome. To date, practically all studies on the

treatment of NAS are retrospective and descriptive in nature. Definitive answer on the best

treatment modality for NAS should come from a multi-center, prospective, randomized study.

However, practical difficulties in such a study would be the large variability in the timing of

presentation and the progression of the biliary abnormalities. Our study on risk factors for

the occurrence of NAS (2), combined with the current study on outcome and prognostic risk

factors for disease progression, facilitates in the identification of important subgroups and

clinical variables that can be used for stratification in a prospective study (see also figure 4).

Roux- en-Y

anastomosis

Preservation injury

(Ischaemia)

Immunology

(PSC, Gender mismatch)

Intrahepatic NASEarly NAS Late NAS

Bacterial

Cholangitis

Radiological Progression of NAS

Severe Outcome Fibrosis/cirrhosis

Figure 4. Schematic representation of risk factors and prognostic factors for the development of early and

late NAS, bacterial cholangitis, progressive radiological abnormalities and severe outcome. Each connection

represents a statistical correlation (present study and work by Buis et al (2)). NAS: non-anastomotic strictures,

PSC: primary sclerosing cholangitis.

75

Chapter 4

In conclusion, non-anastomotic biliary strictures are a common complication after orthotopic

liver transplantation. The radiological and clinical picture of NAS shows a spectrum ranging

from minor abnormalities without any symptoms to severe strictures eventually leading to

re-transplantation. Graft survival is significantly reduced in patients suffering from NAS.

Especially patients with a hepatico-jejunostomy, those with an early diagnosis of NAS, and

those with NAS presenting at the level of the peripheral branches of the biliary tree, are at

risk for the development of recurrent cholangitis, radiological progression, development of

cirrhosis and eventually retransplantation.

76


References


course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003 Jul;3:885-90.

Buis CI, Verdonk RC, van der Jagt EJ, van der Hilst CS, Slooff MJH, Haagsma EB, Porte RJ. Non-anastomotic 2.

biliary strictures after adult liver liver transplantation part one: Radiological features and risk factors for early

versus late presentation. Liver Transpl 2007; 13:708-18.




complications after orthotopic liver transplantation. Hepatogastroenterology 1997 Jan;44:258-62.

Rull R, Garcia Valdecasas JC, Grande L, Fuster J, Lacy AM, Gonzalez FX, et al. Intrahepatic biliary lesions after 5.

orthotopic liver transplantation. Transpl Int 2001 Jun;14:129-34.


transplantation. Radiology 1994 Jun;191:735-40.




orthotopic liver transplantation. Transpl Int 2001 Jun;14:129-34.






associated with nonanastomotic biliary strictures in human hepatic allografts. Transplant Proc 1993

Apr;25:1964-7.


the introduction of transcystic stenting. Transplantation 1998 Nov 15;66:1201-7.



Kuo PC, Lewis WD, Stokes K, Pleskow D, Simpson MA, Jenkins RL. A comparison of operation, endoscopic 14.

retrograde cholangiopancreatography, and percutaneous transhepatic cholangiography in biliary complications

after hepatic transplantation. J Am Coll Surg 1994 Aug;179:177-81.

77

Chapter 4

Sanchez-Urdazpal L, Gores GJ, Ward EM, Maus TP, Buckel EG, Steers JL, et al. Diagnostic features and 15.

clinical outcome of ischemic-type biliary complications after liver transplantation. Hepatology 1993 Apr;17:

605-9.

Sanchez-Urdazpal L, Gores GJ, Ward EM, Hay E, Buckel EG, Wiesner RH, et al. Clinical outcome of ischemic-16.

type biliary complications after liver transplantation. Transplant Proc 1993 Feb;25(1 Pt 2):1107-9.

Chuang JH, Chen WJ, Lee SY, Chang NK. Prompt colonization of the hepaticojejunostomy and translocation of 17.

bacteria to liver after bile duct reconstruction. J Pediatr Surg 1998 Aug;33:1215-8.




ischemic-type biliary lesions following orthotopic liver transplantation. Liver Transpl 2004 Mar;10:434-9.

Otto G, Roeren T, Golling M, Datsis K, Hofmann WJ, Herfarth C, et al. [Ischemic type lesions of the bile ducts 20.

after liver transplantation: 2 years results]. Zentralbl Chir 1995;120:450-4.

Sanchez-Urdazpal L, Gores GJ, Ward EM, Hay E, Buckel EG, Wiesner RH, et al. Clinical outcome of ischemic-21.

type biliary complications after liver transplantation. Transplant Proc 1993 Feb;25(1 Pt 2):1107-9.


complications after orthotopic liver transplantation. Hepatogastroenterology 1997 Jan;44:258-62.

Schlitt HJ, Meier PN, Nashan B, Oldhafer KJ, Boeker K, Flemming P, et al. Reconstructive surgery for ischemic-23.

type lesions at the bile duct bifurcation after liver transplantation. Ann Surg 1999 Jan;229:137-45.

Sung RS, Campbell DA, Jr., Rudich SM, Punch JD, Shieck VL, Armstrong JM, et al. Long-term follow-up 24.

of percutaneous transhepatic balloon cholangioplasty in the management of biliary strictures after liver

transplantation. Transplantation 2004 Jan 15;77:110-5.

Theilmann L, Kuppers B, Kadmon M, Roeren T, Notheisen H, Stiehl A, et al. Biliary tract strictures after orthotopic 25.

liver transplantation: diagnosis and management. Endoscopy 1994 Aug;26:517-22.

Zajko AB, Sheng R, Zetti GM, Madariaga JR, Bron KM. Transhepatic balloon dilation of biliary strictures in liver 26.

transplant patients: a 10-year experience. J Vasc Interv Radiol 1995 Jan;6:79-83.

Part II

Bile physiology after

liver transplantation

The role of bile salt toxicity in the pathogenesis of bile duct injury after non heart-beating porcine liver transplantation5

Transplantation 2008; 85:1625-1631

Marit J YskaCarlijn I Buis

Diethard MonbaliuTheo A Schuurs

Annette SH GouwOlivier NH Kahman

Dorien S VisserJacques Pirenne

Robert J Porte

82

Bile salt toxicity and bile duct injury after NHB porcine liver transplantation Chapter 5

Abstract

Background. Intrahepatic bile duct strictures are a serious complication after non-heart-

beating (NHB) liver transplantation. Bile salt toxicity has been identified as an important factor

in the pathogenesis of bile duct injury and cholangiopathies. The role of bile salt toxicity in the

development of biliary strictures after NHB liver transplantation is unclear.

Methods. In a porcine model of NHB liver transplantation, we studied the effect of different

periods of warm ischemia in the donor on bile composition and subsequent bile duct injury

after transplantation. After induction of cardiac arrest in the donor, liver procurement was

delayed for 0 min (group A), 15 min (group B) or > 30 min (group C). Livers were subsequently

transplanted after four hours of cold preservation. In the recipients, bile flow was measured

and bile samples were collected daily to determine the bile salt / phospholipid ratio. Severity

of bile duct injury was semi-quantified by using a histological grading scale.

Results. Posttransplant survival was directly related to the duration of warm ischemia in

the donor. The bile salt / phospholipid ratio in bile produced early after transplantation was

significantly higher in group C, compared to group A and B. Histopathology showed the highest

degree of bile duct injury in group C.

Conclusion. Prolonged warm ischemia in NHB donors is associated with the formation of

toxic bile after transplantation, with a high biliary bile salt / phospholipid ratio. These data

suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury after NHB liver

transplantation.

83

Chapter 5

Introduction

The success of orthotopic liver transplantation as a therapy for patients with end-stage liver

disease has resulted in an increasing demand for donor livers. In many parts of the world this

created a growing shortage of organs from brain death or deceased donors. A possible solution

to reduce shortage of donor organs is expansion of the donor pool by accepting donation after

cardiac death (DCD) or non-heart-beating (NHB) donors. Patient survival after transplantation

of livers from NHB donors has shown to be comparable to survival after transplantation of

livers from brain death donors (1-4). Graft survival after NHB liver transplantation, however,

is about 10-15% lower due to a higher rate of primary non-function and other graft-related

complications. Intrahepatic bile duct strictures, also known as non-anastomotic strictures or

ischemic-type biliary lesions, are a serious cause of morbidity and a leading cause of graft

failure after NHB liver transplantation (5-7). Although the exact pathogenesis is unknown, it is

generally believed that warm ischemia in the NHB donor due to hypotension before cardiac

arrest, as well as during the time period between cardiac and organ procurement, is a critical

factor in the pathogenesis of these biliary strictures (5-7). Although hepatocytes may recover

from the warm ischemic insult in donor, bile duct epithelial cells have a poor tolerance towards

ischemia and regeneration of cellular ATP is much slower than in hepatocytes (8-12). Apart from

this direct detrimental effect of ischemia on the bile duct epithelium (13), there is accumulating

evidence that bile salt toxicity contributes to bile duct injury after liver transplantation (14-

16). Although secretion of bile salts by hepatocytes is the main driving force of bile flow, bile

salts can act as detergents injuring cellular phospholipid membranes. Under physiological

circumstances, bile salts are therefore neutralized in bile by phospholipids after formation of

mixed micelles (17).

Experimental studies in mice as well as clinical studies in humans have indicated that bile

formation early after liver transplantation may be disturbed, resulting in the formation of more

toxic bile with a relatively high bile salt / phospholipid ratio. A high bile salt / phospholipid ratio

has been associated with more severe bile duct injury after transplantation (14-16).

The role of bile salts in the pathogenesis of bile duct injury after NHB liver transplantation has not

been studied before. We hypothesized that bile salt toxicity acts in concert with warm ischemia

injury in the pathogenesis of intrahepatic bile duct injury after NHB liver transplantation. To study

the role of bile salts in the development of bile duct injury after NHB liver transplantation, we

84


have used a well established model of NHB liver transplantation in pigs (18,19). The specific

aim was to study whether increasing length of warm ischemia in NHB donors is associated with

more toxic bile formation after transplantation, as indicated by the bile salt / phospholipid ratio,

and subsequently more severe injury of the intrahepatic bile ducts.

Materials and Methods

Animals and NHB Liver Transplant Model

Inbred female Landrace pigs, weighing 18 to 37 kg, were used as donors and recipients.

In donors, cardiac arrest was induced by ventricular fibrillation, followed by standardized

periods of warm ischemia before cold preservation and procurement of the liver, to mimic

NHB donation. The pigs were divided in 5 groups (n=6 each) with different periods of donor

warm ischemia time (WIT): 0 min (controls; group A), 15 min (group B), and 30, 45 or 60

minutes. Because of a low rate of survivors in the latter three subgroups (2/6, 2/6, and 0/6

at postoperative day 4, respectively) we regarded these as one group for analysis (group C,

> 30 min WIT). In general, there were no major differences in outcome parameters between

these three groups.

After the period of warm ischemia, the liver was flushed with ice cold histidine tryptophan

ketoglutarate (HTK) preservation solution. During cold perfusion, a cholecystectomy was

performed and the common bile duct was transected and flushed out with cold saline solution.

Subsequently, livers were stored at 4˚C for four hours until transplantation.

In the recipients, a midline laparotomy was performed, the native liver was removed, and the

donor liver was implanted in an orthotopic position. No veno-venous bypass was used. After

completing the anastomosis between the suprahepatic inferior vena cava of the recipient

and donor, the portal vein was reconstructed and the liver was reperfused. Subsequently, the

infrahepatic vena cava was reconstructed. Arterial recirculation was established by an end-to-

side anastomosis between the donor aorta (left in continuity with the hepatic artery) and the

recipient aorta. There were no significant differences in the duration of the anhepatic time or

the time interval between portal reperfusion and restoration of arterial blood low among the

three groups. Mean (range) duration of the anhepatic phase in group A, B and C was 23 min

(20-26 min), 25 min (20-30 min), and 24 min (18-30 min), respectively. Time interval between

portal and arterial reperfusion was 38 min (30-45 min), 42 min (28-68 min), and 37 min (20-

85

Chapter 5

68 min), respectively. Flow probes (Transonic Systems, Ithaca, NY, USA) were implanted

around the hepatic artery and portal vein and connected to a dual channel ultrasonic transit-

time volume flow meter (T206, Transonic). Blood flow was measured continuously during the

first 3 hours after reperfusion and twice daily thereafter. In addition, patency of the vascular

anastomoses was macroscopically inspected during necropsy. All hepatic artery anastomoses

were found to be patent.

Perioperatively, arterial blood pressure was monitored via an arterial line in the left common

carotid artery. Central venous pressure was monitored via a catheter in the left external jugular

vein. Infusion of intravenous fluids was individually guided by clinical signs of hypovolemia,

hemodynamic parameters, and laboratory blood analysis. Although moderate hypotension up

to a period of 30 min was well tolerated during the anhepatic phase, 500 ml of oxyplatin was

administered IV during the anhepatic phase to avoid severe hypotension (20). In general,

there were no major differences in hemodynamics among the groups.

During transplantation a catheter was inserted in the common bile duct and externalized via

the abdominal wall. Daily bile production was completely diverted into a collecting bag. To

maintain the enterohepatic circulation of bile salts, bile was readministered via a jejunostomy

catheter. Antibiotic prophylaxis was provided by IV Ceftazidime, 500 mg.

Postoperatively, animals received tacrolimus (0.05 mg/kg bid) as immunosuppressant. All

animals had free access to water and food. All surviving animals were able to feed themselves

normally as of postoperative day 2 and there were no apparent differences between the groups.

The postoperative observation period was limited to four days to minimize confounding effects

caused by sepsis and other late-onset phenomena. After four days the pigs were killed and

animals surviving less than four days were autopsied to identify the cause of death (19).

Experiments were performed in accordance with the Belgian law regarding animal welfare.

Biochemical serum analyses

Serum levels of aspartate aminotransferase (AST) and bilirubin were determined using routine

chemical methods.

Collection of Bile and Determination of Bile Composition

Bile production was measured 3 hours after reperfusion and daily thereafter to calculate bile

flow (bile production / kg body weight of the donor). Bile samples were collected daily to

86


examine bile composition and determination of the biliary bile salt / phospholipid ratio. Total

biliary bile salt concentration was measured spectrophotometrically using 3α-hydroxysteroid

dehydrogenase (21). Biliary phospholipid concentration was analysed using a commercially

available enzymatic method (Wako Chemicals GmbH, Neuss, Germany).

Hepatic Gene Expression of Bile Transporters

In parallel with the measurement of bile composition, we measured hepatic mRNA expression

of the bile salt transporter (bile salt export pump; BSEP or Abcb11) and the phospholipid

translocator (multidrug resistance protein; MDR3 or Abcb4). The gene sequence of porcine

MDR3 was not known and, therefore, determined for this experiment (NCBI Accession #:

EF067318).

Wedge biopsies were taken one hour after reperfusion and on postoperative day (POD) four

in surviving animals. Biopsies were snap frozen and stored at -80° C until analysis. RNA

isolation from liver biopsies was performed using TRIzol (Invitrogen Life Technologies, Breda,

The Netherlands), Chloroform (Merck, Darmstadt, Germany) and the DNAse-kit from Sigma

(Sigma-Aldrich, Zwijndrecht, The Netherlands). RNA integrity was quantified by electrophoresis

using agarose-gel (Sphaero Q, Leiden, The Netherlands) and ethidiumbromide (Sigma-

Aldrich). The enzyme M-MLV reverse transcriptase (Sigma-Aldrich) was used to convert

RNA (1µg in a final volume of 21 µl) in copy-DNA (cDNA). Taq polymerase (Invitrogen Life

Technologies, Breda, The Netherlands) and conventional PCR were used to multiply the cDNA

and make it detectable with DNA electrophoresis by an UV-transilluminator. For quantitative

real-time detection, sense, anti-sense porcine primers (Invitrogen, Paisley, Scotland) and

fluorogenic probes (Eurogentec, Herstal, Belgium) were designed for the hepatobiliary

transporters BSEP and MDR3, using Primer Express software (PE Aplied Biosystems, Foster

City, CA, USA). All probes were 5’ labeled by a 6-carboxy-fluorescein (FAM) reporter and 3’

labeled with a 6-carboxy-tetramethyl-rhodamine (TAMRA) quencher (table 1). In each PCR

reaction duplicate samples of 5 µl cDNA (25x) (2 ng RNA / µl) were used in a final volume

of 20 µl (qPCR Core Kit Eurogentec, Seraing, Belgium). Every PCR sample was duplicated

in triplo, in a real-time RT PCR 384 wells plate (Applied Biosystems). mRNA copy numbers

of transporter genes were normalized to those of porcine β-actine mRNA. The ABI PRISM

7700 sequence detector (Applied Biosystems) was used for quantitative real-time RT PCR

according to the manufacturer’s instructions.

87

Chapter 5

Histopathological Grading of Bile Duct Injury

Bile duct injury in biopsies taken during and after transplantation was semiquantified by

calculating a modified bile duct injury severity score (BDISS) as described previously (21), and

based on the following two components: bile duct epithelial damage (graded as 0 = absent, 1

= mild, 2 = moderate, 3 = severe; modified from the Banff criteria for acute rejection (22)) and

ductular reaction (graded as 0 = absent, 1 = mild, 2 = moderate, 3 = severe). This resulted in

a minimal BDISS of 0 and a maximum score of 6 points. All histological assessments were

performed by a single pathologist (ASG) who was unaware of the study group of the animals

and of the other study data.

Statistics

Values are expressed as mean ± standard error of the mean (SEM). Data were analyzed

using SPSS software version 14.0 for Windows (SPSS Inc., Chicago, Il, USA). Differences

within and between groups were compared using a paired and non-paired Student-T test,

respectively. Total course of biochemical variables during the first week was compared by

calculating the area under the curve (AUC, using the trapezium rule). All p-values were two-

tailed and considered statistically significant at a level of less than 0.05.

Results

Survival Analysis

Postoperative survival of animals was directly related to the duration of warm ischemia in the

donor (figure 1). In the group A (0 min donor WIT), four days survival rate was 100%, compared

to 90% in the group B (15 min donor WIT) and 20% in group C (> 30 min donor WIT).

In group B, one recipient was found death on POD 1, despite a good initial recovery from the

transplant procedure. On necropsy, death was contributed to hypoxia resulting from severe

pulmonary edema. In group C, one animal was awake and recovering from the procedure,

but could not be weaned from the ventilator, due to lack of spontaneous respiratory activity,

possibly as a result of brain stem injury. In accordance with the international guidelines on

animal welfare, this animal was sacrificed 12 hours after surgery. One animal recovered

from the procedure, but was found death on POD 1. A subsequent necropsy did not reveal

a clear cause of death (normal aspect of all thoraco-abdominal organs, no ascites, and no

88


other indications of liver failure). The remaining animals that died were diagnosed with early

postoperative liver failure or primary graft non-function. Usually, these animals displayed an

incorrectable metabolic acidosis with increasing levels of lactate and severe coagulopathy

after reperfusion, and could not be weaned from the ventilator. On necropsy, typically, large

amounts of hemorrhagic ascites were found as a result of the severe coagulopathy and portal

hypertension due to congestion in failing liver.

In parallel with the clinical course, serum levels of AST at 3 hrs after reperfusion were

significantly higher in group C, compared to group B and A (1938+170, 1116+520 and 288+58

U/L, respectively; p<0.05). However, there were no significant differences in serum AST levels

among the three groups in the surviving animals at POD 4 (237+57, 327+157 and 190+56

U/L, respectively).

0

20

40

60

80

100

WI = 0min

WI = 15min

WI > 30min

an

imal su

rviv

al (%

)

30 21 4

0

0 1 2 3 4

postoperative days

Figure 1. Survival after porcine NHB liver transplantation in relation to various time periods of warm ischemia

in the donor.

89

Chapter 5

Early Recovery of Bile Flow and Bile Composition

Animals which died immediately postoperative from early graft failure (mainly in group C)

displayed very minimal or no bile production and, therefore, were not included in the bile

analyses. Bile flow at 3 hr and 24 hr after graft reperfusion was significantly lower in the

surviving animals in group C (> 30 min WIT) compared to the control group A, while there was

no significant difference in bile flow recovery between the group B (15 min WIT) and the control

group A (figure 2). The composition of bile produced by livers with a WIT > 30 min (group C)

was also more cytotoxic, as expressed by a significantly higher bile salt / phospholipid ratio

early after transplantation (figure 3). Serum bilirubin levels increased during the postoperative

course in all groups and there were no significant differences between the groups at POD 4.

P = 0.019*

P = 0.010*

3

0

2

4

6

8

48

bil

efl

ow

(m

l/kg

/day)

24

WI = 0 min

WI = 15 min

WI > 30 min

time after

transplantation (hours)

3 4824

Figure 2. Bile flow after NHB liver transplantation in relation to various time periods of warm ischemia in the donor.

90


0

10

20

30

40

50

1 2 3 4

WI = 0min

WI = 15min

WI > 30minP=0,008

P=0,012

postoperative days

bil

iary

BS

/ P

L r

ati

o

Figure 3. Mean biliary bile salts / phospholipid (BS / PL) ratio during four days after NHB liver transplantation.

AUC, area under the curve.

Histological Evaluation of Bile Duct Injury

In the surviving animals, histological analysis of postoperative liver biopsies showed a higher

degree of bile duct injury in livers with prolonged warm ischemia in the donor. There were no

differences in the mean BDISS in the group of livers with a WIT of 0 min (group A), compared

to the group with WIT of 15 min (group B). However, the BDISS was significantly higher in

the group with a WIT > 30 min (group C) compared to the groups A and B together (3.0 + 0.2

versus 2.2 + 0.2; p= 0.013). Representative examples of histology of liver biopsies with a low,

intermediate or high BDISS are presented in figure 4.

91

Chapter 5

A

C

B

Figure 4. Representative examples of histology of liver biopsies (Masson Trichrome staining). A) Low BDISS: a

portal tract showing a bile duct with mild epithelial damage, loss of nuclei and infiltration by a neutrophilic gran-

ulocyte. B) Intermediate BDISS: a portal tract containing inflammatory cells. A hepatic artery is shown on the

left and a bile duct on the right side. The damaged bile duct shows epithelial desquamation (lumen), infiltration

by inflammatory cells and disruption of the basement membrane (arrows). There is nuclear atypia, stratification

and loss of biliary epithelial cells. C) High BDISS: a portal tract showing severely damaged and malformed bile

ducts (arrows). There is loss of and diffuse damage to epithelial cells, disrupted basement membrane and heavy

infiltration by inflammatory cells.

creo

92


Gene Expression of Bile Transporters

Hepatic expression of BSEP and MDR3 mRNA decreased after transplantation in all three

groups (figure 5). However, there were no statistically significant differences between the

three groups.

BS

EP

mR

NA

levels

MD

R3 m

RN

A l

evels

A

0.00

0.50

1.00

1.50

2.00

WI = 0 min

WI = 15 min

WI > 30 min

0 60 96 B

0.00

0.50

1.00

1.50

2.00

60 960

time after transplantation (hours)

A

time after transplantation (hours)

B

Figure 5. Relative BSEP (A) and MDR3 (B) mRNA levels in porcine liver grafts after 0 min, 15 min or > 30 min WIT.

Biopsies were taken at 0 min, 60 min and 4 days after transplantation. Genes of interest were standardized for β-actin

mRNA.

Discussion

The aim of this study was to investigate whether prolonged warm ischemia in NHB donors

is associated with the production of more toxic bile early after transplantation, which may

subsequently contribute to the development of intrahepatic biliary strictures after NHB liver

transplantation. In a porcine model of NHB liver transplantation we have shown that livers

obtained from donors who suffered > 30 min of warm ischemia produced bile with a significantly

93

Chapter 5

higher bile salt / phospholipid ratio after transplantation than livers from donors with 0 or 15 min

warm ischemia in the donor. In addition, bile duct injury was more severe and the survival rate

was lower the group with > 30 min of warm ischemia in the donor. These findings indicate that

prolonged warm ischemia in the NHB donor is associated with the posttransplant production

of cytotoxic bile, characterized by a high biliary bile salt / phospholipid ratio, and suggest that

these changes in bile composition contribute to the pathogenesis of bile duct injury after NHB

liver transplantation.

The current findings are in accordance with previous experimental and clinical studies,

which indicated that bile salts contribute to the development of bile duct injury after liver

transplantation (15,23,24). Although the secretion of bile salts by hepatocytes into the bile

canaliculus is the main driving force behind the generation bile flow, bile salts are also

potentially cytotoxic due to their detergent activity (17). Under normal conditions, bile salts

form mixed micelles with phospholipids and cholesterol, which prevents bile salt toxicity. In

case of excess of bile salts, either due to increased bile salt secretion or reduced secretion

of phospholipids, free non-micellar bile salts may cause cholangiocyte injury, pericholangitis

and periductal fibrosis (17,25). In human liver transplantation, it has been shown that bile

salt secretion recovers more rapidly after liver transplantation than phospholipid secretion,

resulting in a cytotoxic bile composition (14). A high bile salt / phospholipid ratio early after

transplantation is correlated with the histological degree of bile duct injury. In an experimental

mouse model of liver transplantation it was recently shown that livers from Mdr2 +/- mice,

which secrete only 50% of the normal amount of phospholipids into the bile, develop severe

bile duct injury after transplantation, as reflected by enlarged portal tracts with cellular damage,

ductular proliferation, bile stasis and a dense inflammatory infiltrate (16). In contrast, no such

abnormalities were seen in transplanted wild-type mouse livers. In addition to these studies,

which focussed on the detrimental effects of endogenous bile salts, others have shown similar

deleterious effects of exogenous administered bile salts. Experimental studies in pigs have

shown that infusion of hydrophobic bile salts before liver procurement results in significantly

increased intrahepatic biliary injury after transplantation, compared to control livers flushed

with saline (15). The observed high biliary bile salt / phospholipid ratio early after NHB liver

transplantation in the current study, suggest that bile salt toxicity is a contributing factor in the

development of bile duct injury after NHB liver transplantation. The bile salt / phospholipid

ratio correlated well with the length of warm ischemia due to cardiac arrest in the donor. It

94


is likely that the ischemic insult to the biliary epithelium remains a key determinant in the

pathogenesis bile duct injury after NHB liver transplantation, however, bile salt toxicity could

aggravate the degree of injury.

Hepatobiliary secretion of bile salts and phospholipids is an active process which is determined

by the hepatic transporters BSEP and MDR3, respectively. Various molecular changes of

hepatocellular-transport systems have been described in patients with cholangiopathy or

cholestatic disorders (17), illustrating the importance of these transporter functions. Decreased

activity of the MDR3 (26-28) or BSEP (29) transporters, due to a gene mutation for example,

is associated with decreased bile formation and cholestasis. In our study we observed a

reduction in the expression of BSEP and MDR3 mRNA after transplantation. However, no

significant differences were noted between the three groups. These data are in accordance

with a recent study in human livers from heart-beating (deceased) donors where also no

differences were found in BSEP and MDR3 mRNA expression at three hours after graft

reperfusion (13). In this human study, however, a small, but significant, increase in BSEP

expression was found at one week after transplantation (14). Follow-up in our study was limited

to only four days and further studies will be needed to determine whether similar changes also

occur in this porcine model of NHB donor liver transplantation. In general, current findings

suggest that the observed differences in bile composition are caused by posttranscriptional

processes or changes in transporter activity rather than a direct effect on gene transcription.

It is increasingly recognized that changes in protein levels of BSEP and NTCP are largely

determined by the subapical storage or mobilization of these transporter proteins and to a

lesser degree by changes in gene expression (30). Unfortunately, we were unable to perform

immunohistochemistry or western blot analyses, due to the lack of adequate antibodies

against porcine BSEP and MDR3.

The accumulating evidence supporting the concept of bile salt toxicity as an important

determinant in the pathogenesis of bile duct injury after liver transplantation opens new

avenues for preventive and therapeutic measures. One obvious option would be the exogenous

administration of hydrophilic bile salts, such as ursodeoxycholic acid, which lack the detergent

properties of hydrophobic bile salts. Daily oral administration of ursodeoxycholic acid is a well

known therapy to reduce bile salt toxicity by replacement of the hydrophobic bile salts in the

bile salt pool (31,32). In addition, hydrophilic bile salts have been shown to possess more direct

cytoprotective properties which are independent from the reduction in hydrophobic bile salts,

95

Chapter 5

and involve inhibition of apoptotic pathways (15,31). Another interesting therapeutic target

could be MDR3, given the key role of biliary phospholipids in protecting bile duct epithelium

from potentially toxic, aggressive biliary content (30). Therapeutic strategies aimed at reducing

bile toxicity through stimulation of MDR3 expression and function may be an important future

therapeutic approach to prevent bile duct injury after liver transplantation. Administration of

fibrates, statins or peroxisome proliferators, have been shown to stimulate biliary phospholipid

secretion by the induction of MDR3 (or its rodent homolog mdr2), making bile less toxic (33-

35). However, more research in this area, including assessment of potential side effects of

these compounds will be needed before clinical application of these compounds to prevent

bile duct strictures can be advised.

Currently, there is not an established animal model of bile duct injury after NHB-donor liver

transplantation. Development of such a model, however, is of great relevance to facilitate

studies on the pathogenesis and development of biliary strictures in liver grafts from NHB

donors. In the current study we have focussed on injury of the small (microscopic) bile ducts

in the liver parenchyma. In clinical practice, bile duct lesions in livers from NHB donors are

typically found in the larger (macroscopic) bile ducts (9). More research using the current

porcine model with more longterm follow-up will be needed to determine whether bile salt

toxicity is also involved in the development of bilary strictures in the larger bile ducts. The ideal

model of NHB liver donation is one in which the time period of cardiac arrest results in a timely

recoverable hepatocellular injury (an thus animal and graft survival), but yet the developement

of enough biliary damage to develop biliary strictures more longterm after transplantation. In

this respect, 30 minutes of donor warm ischemia appeared to be a useful model for further

research.

In summary, we investigated the role of toxic bile composition in the pathogenesis of bile

duct injury after NHB liver transplantation, using a well established porcine model. Our data

indicate that the length of warm ischemia due to cardiac arrest in the NHB donor correlates

with the formation of toxic bile, characterized by a high biliary bile salt / phospholipid ratio, after

transplantation. These findings suggest that bile salt toxicity contributes to the pathogenesis

of bile duct injury after NHB liver transplantation.

96


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College of Critical Care Medicine, Society of Critical Care Medicine. Crit Care Med 2001; 29: 1826-1831.

Neuberger J. Developments in liver transplantation. Gut 2004; 53: 759-768.2.

Reddy S, Zilvetti M, Brockmann J, McLaren A, Friend P. Liver transplantation from non-heart-beating donors: 3.

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Zawistowski CA, Devita MA. Non-heartbeating organ donation: a review. J Intensive Care Med 2003; 18: 189-4.

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Abt PL, Desai NM, Crawford MD et al. Survival following liver transplantation from non-heart-beating donors. 6.

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Garcia-Valdecasas JC, Tabet J, Valero R et al. Evaluation of ischemic injury during liver procurement from non-7.

heart-beating donors. Eur Surg Res 1999; 31: 447-456.

Lewis WD, Jenkins RL. Biliary strictures after liver transplantation. Surg Clin North Am 1994; 74: 967-978.8.


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Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. J Clin Gastroenterol 2005; 39: 10.

S90-S102.

Cameron AM, Busuttil RW. Ischemic cholangiopathy after liver transplantation. Hepatobiliary Pancreat Dis Int 11.

2005; 4: 495-501.

Noack K, Bronk SF, Kato A, Gores GJ. The greater vulnerability of bile duct cells to reoxygenation injury than 12.

to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation. Transplantation 1993;

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Lazaridis KN, Strazzabosco M, Larusso NF. The cholangiopathies: disorders of biliary epithelia. Gastroenterology 13.

2004; 127: 1565-1577.

Geuken E, Visser D, Kuipers F et al. Rapid increase of bile salt secretion is associated with bile duct injury after 14.

human liver transplantation. J Hepatol 2004; 41: 1017-1025.

Hertl M, Harvey PR, Swanson PE et al. Evidence of preservation injury to bile ducts by bile salts in the pig and 15.

its prevention by infusions of hydrophilic bile salts. Hepatology 1995; 21: 1130-1137.

Hoekstra H, Porte RJ, Tian Y et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver 16.

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Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med 1998; 339: 1217-1227.17.

Monbaliu D, Crabbe T, Roskams T, Fevery J, Verwaest C, Pirenne J. Livers from non-heart-beating donors 18.

tolerate short periods of warm ischemia. Transplantation 2005; 79: 1226-1230.

Monbaliu D, van PJ, De VR et al. Primary graft nonfunction and Kupffer cell activation after liver transplantation 19.

from non-heart-beating donors in pigs. Liver Transpl 2007; 13: 239-247.

Oike F, Uryuhara K, Otsuka M et al. Simplified technique of orthotopic liver transplantation in pigs. Transplantation 20.

2001; 71: 328-331.

Turley SD, Dietschy JM. Re-evaluation of the 3 alpha-hydroxysteroid dehydrogenase assay for total bile acids 21.

in bile. J Lipid Res 1978; 19: 924-928.

Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997; 25: 22.

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Schmucker DL, Ohta M, Kanai S, Sato Y, Kitani K. Hepatic injury induced by bile salts: correlation between 23.

biochemical and morphological events. Hepatology 1990; 12: 1216-1221.


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Arrese M, Trauner M. Molecular aspects of bile formation and cholestasis. Trends Mol Med 2003; 9: 558-564.25.

de Vree JM, Jacquemin E, Sturm E et al. Mutations in the MDR3 gene cause progressive familial intrahepatic 26.

cholestasis. Proc Natl Acad Sci U S A 1998; 95: 282-287.

Deleuze JF, Jacquemin E, Dubuisson C et al. Defect of multidrug-resistance 3 gene expression in a subtype of 27.

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Jacquemin E, de Vree JM, Cresteil D et al. The wide spectrum of multidrug resistance 3 deficiency: from 28.

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Suchy FJ, Ananthanarayanan M. Bile salt excretory pump: biology and pathobiology. J Pediatr Gastroenterol 29.

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Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev 30.

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Fickert P, Zollner G, Fuchsbichler A et al. Effects of ursodeoxycholic and cholic acid feeding on hepatocellular 31.

transporter expression in mouse liver. Gastroenterology 2001; 121: 170-183.

Trauner M, Fickert P, Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic 32.

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peroxisome proliferators in the mouse liver. J Hepatol 1997; 26: 1331-1339.

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Hooiveld GJ, Vos TA, Scheffer GL et al. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) 34.

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Chianale J, Vollrath V, Wielandt AM et al. Fibrates induce mdr2 gene expression and biliary phospholipid 35.

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Altered bile composition after liver transplantation is associated with the development of Nonanastomotic biliary strictures6

J of Hepatol. In Press

Carlijn I BuisErwin Geuken

Dorien S VisserFolkert Kuipers

Elizabeth B HaagsmaHenkjan J Verkade

Robert J Porte

100

Bile composition after liver transplantation and NAS Chapter 6

Abstract

Nonanastomotic biliary strictures are troublesome complications after liver transplantation.

The pathogenesis of NAS is not completely clear, but experimental studies suggest that

bile salt toxicity is involved. In 111 adult liver transplant bile samples were collected daily

posttransplantation for determination of bile composition. Expression of bile transporters was

studied perioperativly. Nonanastomotic biliary strictures were detected in 14 patients (13%)

within one year after transplantation. Patient- and donor characteristics and postoperative

serum liver enzymes were similar between patients who developed nonanastomotic biliary

strictures and those who did not. Secretions of bile salts, phospholipids and cholesterol were

significantly lower in patients who developed strictures. In parallel, biliary phospholipids/bile

salt ratio was lower in patients developing strictures, suggestive for increased bile cytotoxicity.

There were no differences in bile salt pool composition or in hepatobiliary transporter

expression.

Conclusion. Although patients who develop nonanastomotic biliary strictures are initially

clinically indiscernible from patients who do not develop nonanastomotic biliary strictures, the

biliary bile salts and phospholipids secretion, as well as biliary phospholipids/bile salt ratio in

the first week after transplantation, was significantly lower in the former group. This supports

the concept that bile cytotoxicity is involved in the pathogenesis of nonanastomotic biliary

strictures.

101

Chapter 6

Introduction

Biliary complications are a major cause of morbidity and graft failure in patients after liver

transplantation (1-3). Nonanastomotic strictures (NAS) of the larger bile ducts are considered

to represent the most troublesome biliary complication as they are frequently resistant to

therapy (4). The reported incidence of NAS is 5-15% (5-11). The occurrence of NAS can be

partly attributed to thrombosis of the hepatic artery. The pathogenesis of NAS that develop in

the absence of hepatic artery thrombosis is less clear (1,12). In general, three mechanisms

contributing to bile duct injury after liver transplantation have been postulated: preservation or

ischemia-related injury (7,13-18), immunological processes (7,19,20) and injury induced by

cytotoxicity of biliary bile salts (21-24).

Bile salts have potent detergent properties and may damage cells by affecting the integrity of

cellular membranes (22,25). In the biliary tree, the toxic effects of bile salts are usually reduced

by the formation of mixed micelles with phospholipids (26,27). Studies in mice and pigs, as well

as clinical studies in humans, have indicated that bile formation early after liver transplantation

may be disturbed, resulting in more cytotoxic bile with a relatively low phospholipids / bile salt

ratio (1,21-24,28,29). We previously showed a strong relationship between this ratio early after

liver transplantation and injury of the small bile ducts in the liver (21,24,29). The small bile ducts,

however, are lined by distinct cholangiocytes, that have different characteristics compared with

cholangiocytes in larger bile ducts, i.e. the location of NAS (30-33). It is unknown whether bile

toxicity is also involved in the pathogenesis of transplantation-related injury of the large bile

ducts, which may lead to the development of NAS.

In contrast to the cytotoxic properties of bile salts, evidence has accumulated that bile salts

may also influence cholangiocyte proliferation and survival, especially in the larger bile

ducts (31,34-36). Some bile salts, including taurocholate and taurolithocholate stimulate

cholangiocyte proliferation in vitro and in vivo, and bile salts are considered a survival factor

for cholangiocytes in the larger bile ducts (34,35). Cholangiocytes of the large bile ducts

are able to take up bile salts from bile via the apical Na+-dependent bile acid transporter

(ASBT, gene symbol SLC10A2). After basolateral secretion, bile salts are transported back

to hepatocytes and resecreted into bile, thereby contributing to bile flow via the “cholehepatic

shunt pathway” (30,37). Bile production and composition, is therefore not exclusively

determined by hepatocytes.

102


It has remained unclear whether bile salts are detrimental or beneficial for cholangiocyte

function in large bile ducts after human liver transplantation, and whether or not bile production

and composition are involved in the pathogenesis of NAS. In contrast to the small bile ducts,

bile salts may not only have toxic effects but could also exert a proliferative restoration or

preservation of the biliary epithelial lining of large bile ducts after transplantation. If bile

composition is involved in the pathogenesis of NAS, one would expect that the bile composition

in the first week after liver transplantation is different in those patients who will develop NAS

as compared to patients who will not develop NAS. We tested this hypothesis by prospectively

assessing bile production and composition within one week after liver transplantation and the

subsequent development of NAS in a large cohort of adult liver transplant recipients.


Patients

Between August 2000 and December 2004 a total of 222 liver transplants were performed

at the University Medical Center Groningen. After excluding children (<18 years; n=70) and

non heart-beating donor liver transplants (n=5), 147 patients were potential candidates for

the study. Thirty six cases were excluded, because of graft loss within 90 days (n=22), initial

poor graft function (defined as in (38,39); n=12), or hepatic artery thrombosis (confirmed by

either Doppler ultrasound or angiography; n=2). This resulted in a study population of 111 liver

transplant procedures. Surgical technique and perioperative management were as previously

described by our group (5,40,41). Clinical variables and laboratory data were prospectively

collected in a computerized database. Tissue and data collection was performed according to

the guidelines of the medical ethical committee of our institution and the Dutch Federation of

Scientific Societies.

Diagnosis of NAS

NAS was defined as any stricture, dilatation, or irregularity of the intra- or extrahepatic bile ducts

of the liver graft, occurring within the first year after transplantation (Figure 1). The diagnosis

NAS was based on at least one adequate imaging study of the biliary tree, after exclusion of

hepatic artery thrombosis by either Doppler ultrasound or conventional angiography. Imaging

studies of the arterial vasculature were repeated over time if no other explanation for the

103

Chapter 6

NAS was found and to confirm patency of the hepatic artery (5). Severity of NAS was graded

according to a semi-quantitative scale, as described previously (5). Isolated strictures at the

bile duct anastomosis were not included in this analysis. The time of first presentation of NAS

was recorded for all patients.

A B

Figure 1. Postoperative cholangiography in liver transplant recipients. (A) Example of normal cholangiogram,

with smooth lining and equal filling of the biliary tree. (B) Example of non anastomotic biliary strictures (NAS),

characterized by diffuse strictures and irregularities of both the extra- and intrahepatic bile ducts on both sides

of the liver with intrahepatic dilatations.

Collection of Liver Biopsies

Specimens of liver tissue were obtained during routine diagnostic biopsies of the liver grafts.

According to our protocol, three consecutive needle biopsies were collected: at the end of

cold preservation, approximately 3 hours after reperfusion, and 1 week after transplantation.

An aliquot of the biopsy specimen was immediately snap-frozen for isolation of total RNA, the

remaining material was used for routine histological analysis. Pieces of normal liver tissue

from hepatic resections for colorectal metastasis were collected after obtaining informed

consent and served as controls (n=9). All liver biopsies were snap-frozen and stored at -80°C

until further processing.

104


Collection and Analysis of Bile Samples

Before transplantation, the gallbladder was removed and the bile ducts were flushed with

preservation fluid on the backtable during preparation for implantation. During the transplantation

an open tip silicon catheter was inserted in the recipient common bile duct and placed retrograde

through the anastomosis. Via this open biliary tube, bile flow was entirely diverted outside the

patient into a collection bag that was placed below the horizontal bed level (42). Interruption

of the enterohepatic circulation in the patient was prevented by re-administration of bile via a

percutaneous feeding jejunostomy catheter. Samples of bile were collected daily in the first

postoperative week between 8:00 and 9:00 am. Bile samples were frozen and stored at -80°C

until further processing. None of the patients received a statin or ursodeoxycholic acid during the

first week after transplantation. Bile samples were analyzed for total bile salts, phospholipids,

and cholesterol contents. Total bile salt concentrations were measured with fluorescent method

using 3α-hydroxysteroid dehydrogenase (43). Phospholipid and cholesterol concentrations in bile

were assayed spectrophotometrically, using commercially available enzymatic methods (Wako

Chemicals GmbH, Neuss, Germany; and Roche Diagnostics GmbH, Mannheim, Germany;

respectively). Postoperative secretion of bile components was defined as concentration

multiplied by daily bile production per kilogram body weight of the donor. Bile salt composition

of bile samples was determined by capillary gas chromatography in a 50µL bile sample on a

Hewlett-Packard gas chromatograph (HP 5880A) equipped with a 50 m x 0.32 mm CP-Sil-19

fused silica column (Chrompack B.V., Middelburg, The Netherlands) (44). Subsequently the

hydrophobicity of the bile salt pool was determined by the Heuman index (45).

RNA Extraction and Reverse Transcription Polymerase Chain

Reaction

Isolation and reverse transcription of RNA was performed as described previously (21). Messenger

RNA levels of following hepatobiliary transporters were analyzed: the most prominent bile salt

uptake system (NTCP, Na+-dependent taurocholate cotransporting polypeptide: gene symbol

SLC10A1) and secretion system (BSEP, bile salt export pump: gene symbol ABCB11), the

phospholipid translocator (MDR3, multidrug resistance protein 3: gene symbol ABCB4) and the

main canalicular organic anion transporter and driving force of the bile salt independent bile flow

(MRP2, multidrug resistant associated protein-2: gene symbol ABCC2). Additionally, cholesterol

7α-hydroxylase (gene symbol CYP7A1) was analyzed by real-time polymerase chain reaction

105

Chapter 6

(PCR), using the ABI PRISM 7900 HT Sequence detector (Applied Biosystems, Foster City, CA,

USA). Nucleotide sequences of Primers (Invitrogen, Paisly, Scotland) and Probes (Eurogentec,

Herstal, Belgium) were designed using Primes Express software (Applied Biosystems, Foster

City, CA, USA). Probes were 5’ labeled by a 6-carboxy-fluoresceine (FAM) reporter and 3’ labeled

with a 6-carboxy-tetra-methyl-rhodamin (TAMRA) quencher and are listed in Table 1. Messenger

RNA copy numbers of genes were normalized to those of 18S rRNA. Real time PCR data were

analyzed using the comparative cycle threshold (CT) method (46).

Table 1. Sequences of Primers and Probes Used for Real-Time PCR Analysis

Gene Alternative Name Primers and Probes PCR Product (bp)

SLC10A1 NTCP sense 5’ -TGA TAT CAC TGG TCC TGG TTC TCA -3’ 74

antisense 5’-GCA TGT ATT GTG GCC GTT TG -3’

probe5’ FAM-TCC TTG CAC CAT AGG GAT CGT CCT CA - TAMRA 3’

ABCB11 BSEP sense 5’ -ACA TGC TTG CGA GGA CCT TTA -3’ 105

antisense 5’ -GGA GGT TCG TGC ACC AGG TA -3’

probe 5’ FAM-CCA TCC GGC AAC GCT CCA AGT CT - TAMRA 3’

ABCB4 MDR3 sense 5’ -CTA TGG AAT TAC TTT TAG TAT CTC ACA AGC ATT -3’ 100

antisense 5’ -AGC GCA TAT GTC CAT TCA CAA T -3’

probe 5’ FAM-TTT TTC CTA TGC CGG TTG TTT - TAMRA 3’

ABCC2 MRP-2 sense 5’ -TGC AGC CTC CAT AAC CAT GAG -3’ 139

antisense 5’ -CTT CGT CTT CCT TCA GGC TAT TCA -3’

probe5’ FAM-CAG CTT TCG TCG AAC ACT TAG CCG CA - TAMRA 3’

CYP7A1 sense 5’-GAG AAG GCA AAC GGG TGA AC-3’ 181

antisense 5’-GGT ATG ACA AGG GAT TTG TGA TGA-3’

probe5’ FAM-TGG ATT AAT TCC ATA CCT GGG CTG TGC TCT-TAMRA 3’

18S sense 5’ -CGG CTA CCA CAT CCA AGG A -3’ 109

antisense 5’ -CCA ATT ACA GGG CCT CGA AA -3’

probe 5’ FAM-CGC GCA AAT TAC CCA CTC CCG A - TAMRA 3’

106


Statistical Analysis

Collection of laboratory values from the central laboratory database was conducted as

described previously (46). Continuous variables were presented as medians with interquartile

range (IQR) or means with standard error of the mean (SEM) when appropriate. Categorical

variables were presented as numbers with percentages and compared using Pearson’s chi-

square test. Comparison of continuous variables was performed using the Mann-Whitney

U test. Area under the curve (AUC) was analyzed by the trapezium method. The level of

significance was set at 0.05. Statistical analysis was performed using SPSS 14.0 (SPSS,

Chicago, IL, USA).

Results

Development of NAS

NAS was diagnosed in 14 of the 111 liver transplant recipients (13%) at a median time interval

of 2.4 months (IQR 1.3 - 4.0 months) after transplantation. Signs of NAS were mild/moderate

in 12 patients and severe in 2 patients. There were no significant differences in donor and

recipient characteristics or surgical variables in patients who developed NAS compared to

patients who did not develop NAS (Table 2).

Serum Markers of Hepatocellular Injury and Cholestasis

Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in

the first week after transplantation, as markers of ischemia reperfusion injury, were similar

in patients who did or did not develop NAS (Figure 2). Similarly, gamma glutamyltransferase

(γGT) and alkaline phosphatase (ALP), markers of cholestasis, were not different between the

two groups in the first postoperative week (Figure 2).

107

Chapter 6

Days after transplantation

Se

rum

AS

T (

U/L

)

AUC p=0.73

A 0 1 2 3 4 5 6 7 80

200

400

600

800

1000

1200

Se

rum

ALT

(U

/L)

AUC p=0.64

B 0 1 2 3 4 5 6 7 80

200

400

600

800

1000

1200


Se

rum

AL

P (

U/L

)

AUC p=0.37

C 0 1 2 3 4 5 6 7 80

100

200

300

400

Se

rum

G

T (

U/L

)

AUC p=0.36

D 0 1 2 3 4 5 6 7 80

100

200

300

400

500

Days after transplantation Days after transplantation

Figure 2. Comparison of median serum levels (IQR) of aspartate aminotransferase (AST; panel A), alanine ami-

notransferase (ALT; panel B), alkaline phosphatase (ALP; panel C), and gamma glutamyltransferase (γGT; panel

D) during the first 8 days after liver transplantation in patients who later developed non anastomotic biliary

strictures (NAS, closed squares) and patients who did not develop NAS (open triangles).

Biliary Secretion of Bile Salts, Phospholipids and Cholesterol

Bile production increased 7-fold during the first week after transplantation in both groups

(Figure 3). Biliary bile salt secretion increased after transplantation in both groups. Bile flow

increased in linear fashion with the higher bile salt secretion rate in both groups. The bile salt

independent bile flow (Y-intercept) and the bile salt dependent bile flow (slope) were similar in

both groups (NAS group: flow = 0.028 x BS-secretion + 1.12, r2=0.47; Controls: flow = 0.024 x

BS-secretion + 1.67, r2= 0.56). However, in patients who did not develop NAS, the increase in

bile salt secretion was over 1.5 fold higher compared to patients who did develop NAS (99 ±

23 µmol/day/kg versus 166 ± 27 µmol/day/kg at day 8) (Figure 3). In parallel with the relatively

reduced bile salt secretion, secretion of phospholipids and cholesterol was also significantly

lower in patients developing NAS (Figure 3). In patients who developed NAS, the secretion of

108


biliary phospholipids during the first week after transplantation was even more compromised

than the secretion of bile salts. This resulted in a significantly lower biliary phospholipid / bile

salt ratio in the patients developing NAS, compared to patients who did not develop NAS

(Figure 4).

BS

se

cre

tio

n (

um

ol/

da

y/k

g)

AUC p=0.02

B 0 1 2 3 4 5 6 7 80

50

100

150

200

Bil

e p

rod

uc

tio

n (

ml/

da

y)

AUC p=0.15

A0

100

200

300

400

500

0 1 2 3 4 5 6 7 8


AUC p=0.01 AUC p=0.01

PL

se

cre

tio

n (

um

ol/

da

y/k

g)

CH

se

cre

tio

n (

um

ol/

da

y/k

g)

DC0 1 2 3 4 5 6 7 8

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 80

1

2

3

4

5



Figure 3. Comparison of median (IQR) daily bile production (panel A), bile salt (BS; panel B) secretion, phos-

pholipid (PL; panel C) secretion, and cholesterol (CH; panel D) secretion during the first 8 days after liver trans-

plantation in patients who later developed non anastomotic biliary strictures (NAS, closed squares) and patients

who did not develop NAS (open triangles). Overall BS, PL and CH secretion, as determined by the area under the

curve (AUC), was significantly lower in the patients who developed NAS.

109

Chapter 6

0.05

0.10

0.15 AUC p=0.02

PL

/BS

ra

tio

0 2 4 6 8

0.00

0.05


PL

/BS

ra

tio

Figure 4. Comparison of the mean biliary phospholipid / bile salt (PL/BS) ratio in the first 8 days after liver trans-

plantation in patients who developed non anastomotic biliary strictures (NAS, closed squares) and patients who

did not develop NAS (open triangles). The PL/BS ratio was significantly lower in patients who developed NAS.

150

300

450

600

750

Ab

so

lute

am

ou

nt

(nm

ol)

DC C CDC UDC0

Ab

so

lute

am

ou

nt

(nm

ol)

Figure 5. Composition of bile salts in bile at day 3 after transplantation in patients who later developed non

anastomotic biliary strictures (NAS, dark bars) and patients who did not develop NAS (open bars). There were

no significant differences in the absolute amounts of the various bile salts between the two groups of patients.

Abbreviations: DC: deoxycholate, C: cholate, CDC: chenodeoxycholate, UDC: ursodeoxycholate.

110


Tab

le 2

. Co

mp

aris

on

of

Do

no

r, R

ecip

ien

t, S

urg

ical

an

d P

ost

op

erat

ive

Var

iab

les

of

Liv

er G

raft

s W

ith

or

Wit

ho

ut

No

n A

nas

tom

oti

c S

tric

ture

s (N

AS

).

NA

SC

ontr

ol O

LT

(n

= 1

4)

(n =

97)

P-

valu

e

Don

or v

aria

bles

Age

(yea

rs)

47(3

9 - 5

7)48

(37

- 58)

0.98

Gen

der (

mal

e/fe

mal

e)6

/ 8(4

3% /

57%

)44

/53

(45%

/ 55

%)

Gen

der m

atch

(don

or/re

cipi

ent)

0.42

M

/M2

(14%

)28

(29%

)

F

/F5

(35%

)24

(25%

)

M

/F4

(29%

)16

(17%

)

F

/M3

(21%

)29

(30%

)

Body

wei

ght d

onor

72.5

(65

- 86.

3)70

(65

- 80)

0.54

Labo

rato

ry v

aria

bles

*

Hem

oglo

bulin

(m

mol

/L)

7.1

(5.8

- 8.

4)7.

0(6

.2 -

7.8)

0.90

Tota

l bilir

ubin

(um

ol/L

)8.

4(5

.5 -

10.5

)10

.1(6

.0 -

16.0

)0.

31

Alan

ine

Amin

o tra

nsfe

rase

(U

/L)

20(1

3 - 2

5)23

(15

- 45)

0.17

gam

ma

Glu

tam

yl tr

ansf

eras

e (U

/L)

25(2

0 - 6

3)22

(14

- 38)

0.63

Alka

line

phos

phat

ase

(U/L

)55

(50

- 86)

53(3

9 - 6

6)0.

31

Cau

se o

f dea

th0.

89

Cer

ebra

l Vas

cula

r Acc

iden

t11

(79%

)72

(74%

)

Trau

ma

2(1

4%)

19(2

0%)

Mis

cella

neou

s1

(7%

)6

(6%

)

Rec

ipie

nt v

aria

bles

Age

(yea

rs)

54(4

4 - 5

8)50

(40

- 55)

0.31

111

Chapter 6

Gen

der (

mal

e/fe

mal

e)5

/ 9(3

6% /

64%

)57

/ 40

(59%

/ 41

%)

0.10

Dis

ease

0.

42

Prim

ary

Scle

rosi

ng C

hola

ngiti

s4

(30%

)20

(21%

)

Prim

ary

and

Seco

ndar

y Bi

liary

Cirr

hosi

s2

(14%

)9

(9%

)

Vira

l hep

atiti

s0

20(2

1%)

Auto

imm

une

hapa

titis

2(1

4%)

10(1

0%)

Alco

holic

cirr

hosi

s2

(14%

)10

(10%

)

Cry

ptog

enic

cirr

hosi

s2

(14%

)5

(5%

)

Oth

er2

(14%

)23

(24%

)

Chi

ld P

ugh

Cla

ssifi

catio

n (A

/ B/ C

)1

/ 7 /

6(7

% /

50%

/ 43

%)

19 /

38 /

37(2

0% /

40%

/ 40

%)

0.49

Re-

trans

plan

tatio

n 3

(21%

)15

(16%

)0.

57

Surg

ical

var

iabl

es

Pres

erva

tion

Solu

tion

0.23

Hig

h vi

scos

ity (U

W)

14(0

%)

88(9

1%)

Low

vis

cosi

ty (H

TK)

0(1

00%

)9

(9%

)

Col

d is

chem

ia ti

me

(min

utes

)50

0(4

06 -

595)

489

(409

- 58

7)0.

86

War

m is

chem

ia ti

me

(min

utes

)48

(42

- 54)

45(4

0 - 5

1)0.

26

Rev

ascu

lariz

atio

n tim

e (m

inut

es)

78(6

4 - 9

8)93

(80

- 109

)0.

21

Bile

duc

t rec

onst

ruct

ion

(duc

t to

duct

/ R

oux-

Y)11

/ 3

(79%

/ 21

%)

76 /

21(7

8% /

22%

)0.

99

Post

oper

ativ

e va

riabl

es

ICU

-leng

th o

f sta

y (d

ays)

2.5

(1.0

- 10

.8)

2(1

.0 -

6.3)

0.70

Acut

e re

ject

ion

5(3

6%)

35(3

6%)

0.

98

112


Before After 1 week0.0

1.0

2.0

3.0

4.0

AB

CB

11

F

old

In

du

cti

on

A

*

** **

Before After 1 week

0.0

1.0

2.0

3.0

4.0

SL

C1

0A

1

Fo

ld i

nd

uc

tio

n

B

** **

Before After 1 week

0.0

1.0

2.0

3.0

4.0

AB

CC

2

Fo

ld I

nd

uc

tio

n

DBefore After 1 week

0.0

1.0

2.0

3.0

4.0

AB

CB

4

Fo

ld I

nd

uc

tio

n

C

Figure 6. Relative gene expression of the bile transporters ABCB11 (panel A), SLC10A1 (panel B), ABCB4 (panel

C) and ABCC2 (panel D) in human liver grafts. A comparison was made between patients who developed non

anastomotic biliary strictures (NAS, dark bars) and patients who did not develop NAS (open bars). Genes of

interest were standardized for 18S rRNA. In livers that later developed NAS, a significant decrease in ABCB11

mRNA expression was found immediately after transplantation, compared to pretransplant values. This de-

crease was not observed in livers that did not develop NAS. In both groups mRNA expression of the bile salt

transporters ABCB11 and SLC10A1 increased significantly during the first week after transplantation. However,

there were no significant differences between the two groups. Before: before reperfusion. After: 3 hours after

reperfusion. One week: one week after liver transplantation. *) p<0.05, when compared to values before trans-

plantation. **) p<0.05, when compared to values after reperfusion.

113

Chapter 6

CY

P7

A1

Fo

ld I

nd

uc

tio

n

5

10

15

p=0.07

CY

P7

A1

Fo

ld I

nd

uc

tio

n

NAS Control

0

5

Figure 7. Relative CYP7A1 gene expression one week after transplantation in livers of patients who developed non

anastomotic biliary strictures (NAS) and patients who did not develop NAS. CYP7A1 catalyzes the conversion of

cholesterol into 7α-hydroxycholesterol and is considered to be the rate-controlling step in bile salt synthesis.

Bile Salt Pool Analysis

In a subset of 22 patients (9 NAS and 13 controls) bile salt pool composition at postoperative

day 1, 2, 3 and 7 was analyzed using gaschromatography. This analysis did not reveal

any significant differences between the two groups. Amounts of the various bile salts at

postoperative day 3, when the difference in phospholipids / bile salt ratio between the two

groups was most pronounced, are shown in Figure 5. In addition, no differences in biliary

hydrophibicity, as reflected by the Heuman index, were found at any time point between the

two groups.

Hepatic Expression of Bile Transporters and CYP7A1

Perioperative changes in the hepatic expression of hepatobiliary transporters are presented in

Figure 6. Compared to preoperative values, mRNA levels of the bile salt transporter ABCB11

were significantly decreased at 3 hrs after reperfusion in livers that developed NAS, whereas

this change was not observed livers that did not develop NAS. In both groups, mRNA levels

of the bile salt transporters ABCB11 and SLC10A1 increased significantly during the first

week after transplantation. In contrast, no significant changes were observed in the hepatic

expression of ABCB4, the phospholipid translocator, and ABCC2. There were no significant

differences in transporter expression between the two groups at any time point.

114


In parallel with the low bile salt secretion, expression of CYP7A1 (the rate-controlling enzyme

in de novo bile salt synthesis) at one week after transplantation was substantially lower in

patients who developed NAS, compared to those who did not (Figure 7).

Discussion

In a prospective clinical study, we evaluated the potential role of bile composition and especially

the relative contribution of bile salts and phospholipids in the development strictures of the

large bile ducts, or NAS, after otherwise successful liver transplantation. Interestingly, the

overall biliary secretion of bile salts, phospholipids and cholesterol during the first week after

transplantation was significantly lower in patients who later developed NAS, compared to

patients who did not develop NAS. The secretion of phospholipids was relatively more affected

than bile salt secretion, resulting in a lower biliary phospholipids / bile salt ratio in patients who

developed NAS. These findings indicate that the development of strictures of the large bile

ducts is preceded by abnormal bile composition early after transplantation, several weeks

before clinical symptoms of bile duct injury appear. This study supports the hypothesis that

early changes in bile composition contribute to the relatively late stricturing of the large bile

ducts, leading to the radiological diagnosis of NAS after transplantation.

In the current study the incidence of NAS up to one year after transplantation was 13%. This

rate is similar to data reported in most previous studies (6,7,10,46) but higher than reported in

some others (15,18,47). Variations in the reported incidence of NAS among different studies

can be explained by differences in study design (retrospective versus prospective) and

differences in the diagnostic criteria used.

Bile salts possess potent detergent properties and as such, are potentially cytotoxic

(48,49). In case of relative excess of bile salts, either due to increased bile salt secretion

or reduced secretion of phospholipids, micellar bile salts may cause cholangiocyte injury,

pericholangitis and periductal fibrosis (50,51). In previous studies we have shown that toxic

bile composition early after transplantation, characterized by a low biliary phospholipid / bile

salt ratio, is associated with histological signs of injury of the small bile ducts in the liver

(21,24,29). The role of bile salt toxicity in the pathogenesis of injury of the small intrahepatic

bile ducts was also demonstrated in an experimental study using a liver transplant model

in mice (24). Livers transplanted from Abcb4-/+ mice, which have only 50% expression of

115

Chapter 6

the phospholipids translocator Abcb4, into wild-type recipients developed signs of severe

injury of the small intrahepatic bile ducts within two weeks after transplantation (24). In the

current study we focused on the development of NAS, which is a disease of the large bile

ducts (5,52). Our results suggest for the first time that bile salt toxicity is also involved in the

development of large bile duct injury, leading to the clinical and radiological diagnosis of NAS.

Despite the observed low phospholipid / bile salt ratio in patients developing NAS, reflecting

bile toxicity, the overall biliary secretion of bile salts in these patients was lower than in

patients who did not develop NAS. This observation was not expected and introduces the

intriguing possibility that, apart from relative bile salt toxicity, relative bile salt deprivation could

(also) contribute to cholangiocyte injury and the development of NAS. There is substantial

evidence that bile salts are potent inducers of cholangiocyte proliferation and thus bile duct

repair (31,34-36). Uptake of bile salts by cholangiocytes is mediated by the transporter ASBT

(SLC10A2) at the ductular membrane of these cells (30,37). In contrast to cholangiocytes

of the small bile ducts, cholangiocytes in larger bile ducts do express ASBT and, therefore,

these cells can re-absorb bile salts from bile (30,37). This important difference between

cholangiocytes from small and large bile ducts may explain why a previous clinical study

focusing on posttransplant injury of the small bile ducts did not reveal a relationship between

small bile duct injury and reduced bile salt secretion. Collectively, these observations raise the

possibility that the pathogenesis of biliary injury after liver transplantation is different for small

and large bile ducts. In this respect it would have been interesting to study the expression of

ASBT (SLC10A2) in the large bile ducts in the current study. However, it is difficult to take serial

biopsies of the large bile ducts in patients and we were unable to detect ASBT (SLC10A2)

mRNA expression in liver biopsies, which mainly contain small bile ducts (data not shown).

Some bile salts have a more pronounced effect on cholangiocyte proliferation than others.

Taurocholate, for example, may enhance proliferation, while ursodeoxycholate may reduce

the proliferative effects of other bile salts (36,53). In the current study we found no differences

in the bile salt pool composition in patients who developed NAS, compared to those who did

not. Therefore, we have no evidence to suggest that differences in composition of the bile salt

pool are involved in the altered overall bile salt secretion or in the pathogenesis of NAS after

liver transplantation.

A key question that emerges from this study is: what determines the low bile salt secretion

in livers that are developing NAS? Theoretically, reduced biliary bile salt secretion can result

116


from a) decreased de novo synthesis, b) impaired hepatobiliary transport at the level of the

canalicular membrane (ABCB11), and/or c) impaired intestinal bile salt re-absorption and fecal

loss of bile salts leading to reduced bile salt pool size. In the classical pathway of de novo bile

salt biosynthesis, CYP7A1 catalyzes the conversion of cholesterol into 7α-hydroxycholesterol,

which is considered to be the rate-controlling step. In humans, the classical pathway accounts

for approximately 80% of total bile salt synthesis (54,55). We observed a lower hepatic

expression of CYP7A1 in patients who later developed NAS, compared to those who did not.

It is tempting to ascribe the reduced bile salt secretion in patients who developed NAS to the

lower expression of CYP7A1. Yet, three issues should be considered in this respect: a) the

difference in CYP7A1 expression was striking, but it did not reach statistical significance, in

contrast to the difference in bile secretion; b) no information is available on the correlation

between CYP7A1 mRNA levels and actual cholate synthesis in the early post-transplant period;

and c) it can be anticipated that the amount of bile salts secreted after liver transplantation is

increasingly derived from re-absorbed (“conserved”) bile salts from the intestine.

To demonstrate or refute increased intestinal loss of bile salts as an explanation for the

differences in biliary bile salt secretion we would have needed the collection of faeces. Although

this was not performed, we have other arguments to assume that the observed differences in

bile salt secretion are not caused by differences in intestinal bile salt loss. Reduced bile salt

pool size due to impaired intestinal reabsorption would be expected to lead to an increased

rather than a decreased hepatic CYP7A1 expression. In addition, a previous study from our

centre has shown that serum bile salt concentrations increase during the first week after

transplantation, which is not compatible with increased intestinal losses (56).

Hepatobiliary secretion of bile salts is an active process which, under normal circumstances,

is mainly influenced by the sinusoidal transporter SLC10A1 and the canalicular transporter

ABCB11. Theoretically, impaired hepatobiliary transport could have resulted form a reduced

expression of these transporter proteins. Compared to pretransplant values, ABCB11 mRNA

expression was decreased immediately after transplantation in livers that later developed

NAS. Although this decrease was not observed in livers that did not develop NAS, there

were no significant differences between the two groups either before or immediately after

transplantation. In accordance with previous observations by Geuken et al. (21), we observed

an increased mRNA expression of the bile salt transporters SLC10A1 and ABCB11 in both

groups after transplantation, while mRNA levels of the phospholipid translocator ABCB4 did

117

Chapter 6

not change. These findings are compatible with the relatively low biliary phospholipid / bile salt

ratio observed early after transplantation. However, there were no significant differences in

the expression of the bile transporters between the two groups, suggesting that the observed

differences in bile salt secretion cannot be explained by differences in gene transcription.

Based on the current study, we cannot exclude that posttranscriptional processes or changes

in transporter activity are involved. Unfortunately, we were unable to perform Western blot

analyses for quantification of transporter protein levels due to the small amount of liver tissue

obtained from needle biopsies.

We also examined whether differences in bile composition between patients who developed

NAS and those who did not could be explained by differences in phospholipids secreted per

bile salt. Therefore we additionally analyzed the biliary hydrophobicity index and the bile salt

independent bile flow. There were no significant differences in the hydrophobicity index or in

the bile salt independent bile flow, indicating that these factors cannot explain the observed

differences between the two groups (57,58).

Several other factors have been shown to contribute to the development of NAS after liver

transplantation, including long cold or warm ischemia times (7,9), inadequate washout and

perfusion of the peribiliary capillary plexus (16,17), and immunological injury (19,59). In the

current study we found no differences in the duration of cold and warm ischemia in livers with

or without NAS. These data support previous suggestions that the pathogenesis of NAS is not

only related to a direct ischemic injury of the biliary epithelium (1,12).

In summary, the results of this prospective clinical study strongly support the hypothesis

that bile composition is involved in the pathogenesis of NAS after liver transplantation.

Patients who developed NAS within one year after liver transplantation were initially clinically

indiscernible from patients who did not develop NAS. However, bile composition in this early

postoperative period was different in these two groups. Patients who developed NAS were

characterized by a reduced biliary secretion of bile salts and phospholipids and a decreased

biliary phospholipid / bile salt ratio. We speculate that those early defects in bile formation,

possibly genetically based, play a role in the injury of the biliary epithelium of large bile ducts

early after transplantation, subsequently leading to the formation of biliary strictures.

118


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after liver transplantation, part 2: Management, outcome, and risk factors for disease progression. Liver Transpl

2007;13:725-732.

Alvaro D, Gigliozzi A, Attili AF. Regulation and deregulation of cholangiocyte proliferation. J Hepatol 2000;33:333-53.

340.

Vlahcevic ZR, Stravitz RT, Heuman DM, Hylemon PB, Pandak WM. Quantitative estimations of the contribution 54.

of different bile acid pathways to total bile acid synthesis in the rat. Gastroenterology 1997;113:1949-1957.

Duane WC, Javitt NB. 27-hydroxycholesterol: production rates in normal human subjects. J Lipid Res 55.

1999;40:1194-1199.

Haagsma EB, Huizenga JR, Vonk RJ, Albers CJ, Grond J, Krom RA, et al. Composition of bile after orthotopic 56.

liver transplantation. Scand J Gastroenterol 1987;22:1049-1055.

Verkade HJ. Inhibition of biliary phospholipid and cholesterol secretion by organic anions affects bile canalicular 57.

membrane composition and fluidity. J Gastroenterol 2000;35:481-485.

Verkade HJ, Vonk RJ, Kuipers F. New insights into the mechanism of bile acid-induced biliary lipid secretion. 58.

Hepatology 1995;21:1174-1189.

Carrasco L. Effects of cold ischemia time on the graft after orthotopic liver. Transplantation 1996;61:393.59.

Polymorphisms of hepatobiliary phospholipid transporter MDR-3 associated with non anastomotic strictures after human liver transplantation7

Submitted

Carlijn I BuisGerrit van der Steege

Ilja M NolteDorien S VisserRobert J Porte

124

ABCB4 gene polymorphism and NAS Chapter 7

Abstract

Non-anastomotic biliary strictures (NAS) are an important and troublesome biliary

complication after liver transplantation. The pathogenesis of NAS is not completely clear,

but studies have suggested that bile salt toxicity, due to an imbalance between biliary bile

salts and phospholipids is involved. Hepatobiliary transporter proteins are responsible for bile

secretion and composition. Aim of this study was to assess whether genetic variations in these

transporters are associated with the development of NAS.

In 461 liver transplant procedures, we studied donor genotype of three of the most relevant

hepatobiliary transporters: the bile salt export pump (ABCB11), the transporter of phospholipids

(ABCB4) and the transporter of glutathione and bilirubin (ABCC2). Four to five tagging single

nucleotide polymorphisms (SNPs) with an equal physical distribution per gene were selected

using HapMap data. Haplotypes were constructed using an Expectation-Maximization

algorithm to estimate haplotype frequencies. The occurrence of NAS was determined for

livers with the various transporter genotypes.

NAS were detected in 77 patients (16.7%) after transplantation. Patients who received a donor

liver with ABCB4 haplotype AGGTA developed NAS almost twice as often as donor livers with

other haplotypes (28% versus 15%; p = 0.007). In a multivariate Cox regression analysis, the

AGGTA haplotype of the ABCB4 gene in the donor, was confirmed as an independent risk

factor for NAS (HR=2.23, 95% CI= 1.29 – 3.85; p = 0.004). Various haplotypes of the ABCB11

and the ABCC2 gene, or individual SNPs, were not associated with NAS.

Conclusion: A common haplotype in the transporter of phospholipids (ABCB4) in donor livers

is independently associated with a two-fold increased risk for NAS after liver transplantation.

Transport of phospholipids into the bile in livers with this risk haplotype might be altered after

liver transplantation, contributing to the development of NAS.

125

Chapter 7

Introduction

Biliary complications are reported in 10 to 30% of the patients after liver transplantation

representing a major cause of morbidity and mortality (1). Non-anastomotic biliary strictures

(NAS) are considered to be the most troublesome biliary complication, because they may

occur at multiple locations in the biliary tree and are frequently resistant to therapy (2,3). Graft

survival is markedly reduced in patients developing NAS; 16% of all patients with NAS need a

re-transplantation and 35% will require an interventional treatment (3).

The occurrence of NAS can partly be attributed to thrombosis of the hepatic artery. The

pathogenesis of NAS developing in the absence of hepatic artery thrombosis is less clear

(1,4). In general, three mechanisms contributing to bile duct injury after liver transplantation

have been postulated: preservation or ischemia-related injury (5-11), immunological processes

(7,12,13) and injury induced by cytotoxicity of biliary bile salts (14-17). Bile salts have potent

detergent properties and may damage cells in the absence of phospholipids by affecting the

integrity of cellular membranes (15,18). Damage to the canalicular membrane of the biliary

epithelial cells could result in progressive destruction of bile ducts (19). Normally, these toxic

effects of bile salts are prevented through neutralization by phospholipids. We have recently

shown that changes in bile formation, leading to cytotoxic bile with a relative low phospholipid-

to-bile salt ratio, are associated with bile duct injury and the development of NAS after liver

transplantation (14,17,20,21).

Bile production depends on an active process involving the transport of bile acids,

phospholipids and other osmotic compounds across a concentration gradient into the bile

canaliculus. Hepatobiliary transporter proteins play a rate-limiting role in this process. Genetic

variations in the phospholipid translocator, multiple drug resistance protein 3 (MDR3, official

name ATP binding cassette, subfamily B, member 4 or ABCB4) have been associated with

abnormal phenotypes, characterized by the production of bile with a low biliary phospholipid

content, leading to bile duct injury and intrahepatic cholestasis. A genetic variation inevitably

leading to disease is the mutation in the ABCB4 gene associated with progressive familial

intrahepatic cholestasis type III (PFIC III) (22). Characteristic clinical features of these patients

are jaundice, recurrent cholangitis and elevated serum γ-glutamyltransferase levels, reflecting

the destruction of cell membranes of the biliary epithelium. Other genetic variations of the

ABCB4 gene have a less stable phenotype and may lead to symptoms only under specific

126


circumstances. An example of this is intrahepatic cholestasis of pregnancy (ICP), which

may occur in women who were previously without symptoms, but develop jaundice during

pregnancy. (19,23-28).

SNPs in the bile salt exporter pump (BSEP, official name ABCB11) have been related with

a spectrum of clinical phenotypes such as the syndrome of benign recurrent intrahepatic

cholestasis (BRIC) (29), PFIC-2 (30), and ICP (28,31). Multidrug resistance related protein 2

(MRP-2, official name ABCC2), which is a transporter of bilirubin and glutathion (GSH) into

bile, is known from the benign human disorder Dubin Johnson, which is characterized by an

increase of conjugated bilirubin without elevation of liver enzymes (32).

It has remained unclear whether variations in these genes encoding for hepatobiliary

transporters might contribute to the pathogenesis of NAS after liver transplantation. We

hypothesized that there variations in these genes that do not result in an abnormal phenotype

under normal, physiological conditions, but that are associated with to the development of bile

duct injury under stressful conditions, such as liver transplantation. Such a mechanism would

be similar to the development of ICP in women with certain ABCB4 polymorphisms.

The aim of the present study was to determine whether genetic variations in the genes

encoding for hepatobiliary transporters are associated with the development of NAS in a large

prospective cohort of 461 adult liver transplant recipients.


Patients

Between January 1990 and January 2005, 720 liver transplantations were performed in 621

patients in our center. After exclusion of pediatric patients (n=160), 461 adult liver transplant

recipients remained. Cryopreserved splenocyts from the donors were used for the genotyping.

Recipient follow-up was until December 2007, resulting in a median follow-up of 8.2 years

(interquartile range 4.6–12.6 years). Surgical procedure and postoperative management

have been described previously (2). In short, ABO blood group-identical or compatible grafts

from brain-death donors with normal or near normal liver function tests were used for all

patients. Immunosuppressive protocols were based on a calcineurin inhibitor (tacrolimus or

cyclosporine A) either with or without azathioprine and a rapid taper of steroids. Biopsy-proven

acute rejection was treated when clinically indicated with a bolus of methylprednisolone on

127

Chapter 7

three consecutive days. Doppler ultrasound was performed routinely at postoperative days

1, 3, and 7 and later on demand to rule out vascular or biliary complications or parenchymal

lesions. Cholangiography via a biliary drain was routinely performed between postoperative

day 10 –14 and later on demand (i.e. for rising cholestatic laboratory parameters or dilatation

of bile ducts on ultrasound).

Donor data was collected from the donor forms and checked and completed with information

from the archives of the Eurotransplant Organization, Leiden, The Netherlands. Recipient

data were obtained from a prospectively collected computer database. If necessary the

original patient notes were reviewed for missing information. Tissue and data collection was

performed according to the guidelines of the medical ethical committee of our institution and

the Dutch Federation of Scientific Societies.

Diagnosis of NAS

Primary outcome parameter in this study was the development of NAS. For this study NAS

were defined as any stricture, dilatation, or irregularity of the intra- or extrahepatic bile ducts

of the liver graft, either with or without biliary sludge formation, after exclusion of hepatic artery

thrombosis by either Doppler ultrasound or conventional angiography. Isolated strictures at

the bile duct anastomosis were, by definition, excluded from this analysis and have been

described elsewhere (33).

Selection of Hepatobiliary Transporter SNPs and Genotyping

The following genes were studied: the phospholipid translocator (ABCB4), the most prominent

bile salt transporter (ABCB11), and the main canalicular organic anion transporter and driving

force of the bile salt independent bile flow (ABCC2). Genomic DNA was isolated from donor

splenocytes using a commercial kit (Gentra Systems, Minneapolis, MN, USA). SNPs were

selected based on data from the HapMap database (release #16, www.hapmap.org), using

the Haploview tagging tool. Per locus only those SNPs were selected that tagged haplotypes

with a frequency in the HapMap Caucasian dataset of more than 10% and with a minor allele

frequency around 20%. In addition, we tried to combine this selection criterion with an equal

physical distribution across the genes, preferably with exonic location. TaqMan assays were

used to genotype the ABC transporter SNPs. These were obtained from Applied Biosystems

(Foster City, CA, USA) by the assay-on-demand or the assay-by-design services. Details of

the various SNPs are given in Table 1.

128


Tab

le 1

. Mar

ker

dat

a

Gen

eA

pplie

d as

say

IDSN

PPo

sitio

n N

ucle

otid

e ch

ange

Am

ino

Aci

d ch

ange

MA

F *

ABC

B11

(BSE

P)hC

V319

7737

rs38

1438

15’

- U

TRG

-->

AA

(30%

)

chro

mos

ome

2hC

V203

4236

rs22

8761

8in

tron

8G

-->

AA

(30%

)

hCV2

0342

47rs

2058

996

intro

n 10

G --

> A

A (4

8%)

hCV8

8135

44rs

4733

513’

- U

TRG

-->

AA

(31%

)

ABC

B4 (M

DR

3)hC

V184

3476

rs23

0238

7ex

on 3

G --

> A

syno

nym

ous

Leu

--> L

euA

(11%

)

chro

mos

ome

7hC

V831

7490

rs12

0228

3ex

on 5

A -->

Gsy

nony

mou

sAs

n -->

Asn

G (3

3%)

desi

gnrs

1149

222

intro

n 9

T -->

GG

(17%

)

desi

gnrs

3167

4in

tron

13

C --

> T

T (1

8%)

hCV1

5780

446

rs23

7359

3in

tron

23A

--> C

C (1

1%)

ABC

C2

(MR

P2)

hCV1

6121

737

rs20

7333

6in

tron

3A

--> T

T (4

0%)

chro

mos

ome

10hC

V281

4669

rs27

5610

9in

tron

7T

--> G

G (4

5%)

desi

gnrs

2273

697

exon

10

G --

> A

nons

ynon

ymou

sIle

-->

Val

A (1

6%)

desi

gnrs

2002

042

intro

n 19

C --

> T

T (3

0%)

hCV2

8146

42rs

7176

203’

- U

TRG

-->

AA

(23%

)

* M

AF

: M

ino

r al

lele

fre

qu

ency

129

Chapter 7

Statistical Analysis

Descriptive, continuous variables are reported as median and interquartile ranges (IQR) and

categorical variables are reported as numbers with percentage. The power of this study was greater

than 80% to detect an odds ratio (HR) of 1.71 or larger for SNPs with a minor allele frequency

of at least 20% at the statistical significant level of 5%. Prior to all analyses, Hardy–Weinberg

equilibrium was confirmed for all genotypes using the chi-squared test. Single SNP analysis

was performed with chi-squared test. Haplotype analysis was performed using an Expectation-

Maximization algorithm to estimate haplotype frequencies (in-house software). The frequencies

were estimated on the total group of patients and these frequencies were used to estimate the

haplotype frequencies among patients with and patients without NAS. In addition for each specific

haplotype a log-likelihood ratio test was performed to test whether the frequencies of this haplotype

differed between patients with and without NAS. Incidence rate of NAS was estimated with the

Kaplan–Meier actuarial method and compared with the log-rank test. Furthermore, if the probability

of a specific haplotype combination (using frequencies of the total group) was higher than 90%, that

combination was assumed to the true combination. In this way, being carrier of specific haplotypes

could be determined for use as a covariate in a multivariate Cox regression model.

Statistical analyses were performed using SPSS Version 14.0 for Windows (SPSS Inc., Chicago,

IL, USA). All p-values were two-tailed and considered as statistically significant at levels < 0.05.

130


Results

Incidence NAS After Liver Transplantation

Clinical characteristics of donors and recipients, as well as perioperative variables of the entire

series are presented in Table 2. NAS were detected in 77 of the 461 (16.7%) liver grafts studied.

Among the liver grafts that developed NAS, 67 were first transplants and 10 were re-transplants.

1.0

0.8

0.6

Cu

mu

lati

ve

ris

k o

f N

AS Log Rank p=0.007

20151050

0.4

0.2

0.0

Years after transplantation

Cu

mu

lati

ve

ris

k o

f N

AS

yes

no

Carrier AGGTA

Figure1. Cumulative risk of NAS during the first 10 years after liver transplantation in carriers of the risk haplo-

type versus non-carriers of the risk haplotype AGGTA. The incidence of NAS was almost doubled in the group

of patients carrying the risk haplotype AGGTA (log-rank test p < 0.01)

Association between bile transporter haplotypes and NAS

In a univariate analysis, we found no relationship between any of the selected individual SNPs

in the ABCB11, ABCB4, or ABCC2 gene and the occurrence of NAS (data not shown). We

next constructed different haplotypes of these genes, as is presented in Table 3. In 33 patients

haplotypes could not be assigned, because the probability of a specific haplotype combination

was below 90%. Haplotype analysis of the phospholipid transporter ABCB4 showed a strong

correlation with NAS. The haplotype variant AGGTA was 2-times more frequent in patients who

developed NAS, compared to patients who did not develop NAS (13.2% versus 6.7%, p=0.01).

131

Chapter 7

Analyses of haplotype variations of the ABCB11 and ABCC2 genes were not associated with

the occurrence of NAS (data not shown).

As shown in figure 1, the cumulative incidence of NAS in livers with the AGGTA risk haplotype

was almost two-times higher than in livers that did not carry the AGGTA risk haplotype (28%

versus 15%; p < 0.01).

To determine whether the AGGTA haplotype of the ABCB4 gene is an independent risk factor

for NAS, we next performed a multivariate Cox regression analysis. In this analysis we included

all accepted clinical risk factors for NAS that have been described previously, including type

of perfusion solution, cold ischemia time, warm ischemia time, indication for transplantation,

gender match, as well as the risk haplotype AGGTA. In this multivariate Cox regression model,

the donor AGGTA haplotype of the ABCB4 gene was independently associated with NAS

(HR=2.23, 95% CI= 1.29 – 3.85; p=0.004).

132


Table 2. Donor, Recipient, Surgical and Postoperative Variables of Liver Grafts (n=461)*

Donor variables

Age (years) 43 (30 - 51)


Recipient variables

Age (years) 46 (35 - 54)


Disease

Cholestatic disease 142 (31)%

Parenchymal disease 205 (44)%

Metabolic liver disease 54 (11)%

Vascular liver disease 12 (3)%

Acute liver failure 32 (7)%

Liver tumor 5 (1)%

Other 11 (2)%

Child Pugh Classification (A / B / C) 72 / 189 / 200 (15% / 41% / 43%)

Retransplantation 62 (13%)

Surgical variables

Preservation solution

Low viscosity / High viscosity 23 / 438 (5% / 95%)

Cold ischemia time (minutes)** 564 (441 - 728)

Warm ischemia time (minutes)# 54 (45 - 63)

Revascularization time (minutes)## 96 (79 - 115)

Bile duct reconstruction (duct-to-duct / Roux-Y) 381 / 73 (82% / 16%)

Type of graft (whole / reduced size) 447 / 14 (97% / 3%)


ICU-length of stay (days) 4 (2 - 8)

Acute rejection 150 (33%)

* Continuous variables are presented as median and interquartile range, categorical variables as numbers

with percentage.

** Cold ischemia time; between start cold perfusion in the donor and end of cold preservation of the liver graft

# Warm ischemia time; between the end of cold ischemic preservation of the liver and portal vein reperfusion

## Revascularization time; between the end of cold ischemic preservation of the liver and arterial reperfusion

133

Chapter 7

Table 3. Haplotype frequencies among donor livers with and without NAS.

ABCB11 NAS no-NAS overall

SNP1 SNP2 SNP3 SNP4 n=77 n=384 LR p-value

A G A G 25,7% 23,9% 24,2% 0,20 0,66

G G A G 30,4% 31,1% 31,0% 0,01 0,91

A A A G 6,8% 8,8% 8,4% 0,65 0,42

G A A G 5,1% 6,7% 6,4% 0,52 0,47

A G G A 2,6% 1,2% 1,4% 1,39 0,24

A A G A 9,4% 11,9% 11,5% 0,71 0,40

G A G A 16,9% 13,7% 14,2% 0,86 0,35

Total * 97% 97% 97%

ABCB4 NAS no-NAS overall

SNP1 SNP2 SNP3 SNP4 SNP5 n=77 n=384 LR p-value

G A T C A 48,7% 48,8% 48,8% 0,14 0,71

G G T C A 19,3% 19,4% 19,4% 0,00 1,00

G A G C A 2,6% 3,2% 3,1% 0,09 0,76

A G T C A 1,5% 1,5% 1,5% -0,02 1,00

A G G T A 13,2% 6,7% 7,7% 6,46 0,01

A G G T C 4,6% 3,3% 3,5% 0,65 0,42

G A T C C 2,5% 5,0% 4,6% 1,83 0,18

G G G T A 3,3% 4,3% 4,1% 0,21 0,65

G G T T A 1,5% 1,9% 1,9% 0,04 0,84

Total * 97% 94% 95%

ABCC2 NAS no-NAS overall

SNP1 SNP2 SNP3 SNP4 SNP5 n=77 n=384 LR p-value

A T G C G 8,0% 13,7% 12,8% 3,58 0,06

A T G T G 30,0% 25,4% 26,2% 0,92 0,34

A T A C A 27,0% 19,1% 20,4% 3,49 0,06

T T G C G 5,0% 4,3% 4,4% 0,16 0,69

T T A C G 13,7% 15,3% 15,1% 0,24 0,63

T G A C G 16,0% 20,8% 20,0% 1,58 0,21

Total * 100% 99% 99%

* Only haplotypes with a frequency > 1% are shown

134


Discussion

We have tested the hypothesis that genetic variability in hepatobiliary transporters in donor

livers is associated with the development of NAS after transplantation. We have evaluated

this in a large cohort of 461 liver transplant recipients. The most important finding in this

study was a strong association between the phospholipid translocator ABCB4 genotype and

the development of NAS after liver transplantation. A common haplotype in the ABCB4 gene

was significantly more present in livers that developed NAS, compared to those that did not

develop NAS. A multivariate Cox regression analysis confirmed that the risk haplotype of

the ABCB4 gene is an independent risk factor for the development of NAS. We found no

association between haplotypes of the ABCB11 and ABCC2 gene and the occurrence of NAS

after liver transplantation.

There is accumulating evidence from both clinical and experimental studies that altered

bile composition with decreased phospholipid secretion may contribute to biliary injury and

subsequent intrahepatic biliary strictures after liver transplantation (14,17,20). Although previous

studies have suggested that changes in the expression of the phospholipids translocator ABCB4

may play a role in the reduced biliary excretion of biliary phospholipids after liver transplantation,

the impact of a genetic predisposition has not been reported before (14,21).

Although we did not perform bile analysis in this large cohort of patients, the observed

association between the AGGTA ABCB4 haplotype strongly suggests that biliary phospholipid

secretion in livers with this haplotype is reduced after liver transplantation. Reduced biliary

secretion of phospholipids results in a lower biliary phospholipids-to-bile salt ratio, which has

been associated with increased bile duct damage. Normally, the toxic effects of bile salts

are prevented by the neutralization of bile salts by phospholipids through the formation of

mixed micelles in bile. In case of relative excess of bile salts, either due to increased bile

salt secretion or reduced secretion of phospholipids, free non-micellar bile salts may cause

cholangiocyte injury, pericholangitis and periductal fibrosis (34,35). The role of diminished

biliary phospholipids secretion and increased bile salt toxicity in the pathogenesis of bile

duct injury has previously been demonstrated in an experimental study using a murine liver

transplant model (17). In this study livers from Abcb4-/+ mice, expressing only 50% of the

phospholipids translocator Abcb4, were transplanted into wild-type recipients. Although livers

and bile ducts from Abcb4-/+ mice are phenotypically normal under normal circumstances,

135

Chapter 7

these livers developed severe injury of the intrahepatic bile ducts after transplantation. This

finding indicates that, although a reduction of biliary phospholipid secretion of up to 50%

alone does not result in bile duct injury, this may result in overt bile duct injury when a second

insult is present, such as ischemia / reperfusion injury (17). This animal study suggest that

the impact of cold ischemia and reperfusion on ABCB4 function – specifically in genetically

susceptible individuals with an ABCB4 genetic variation – could contribute to bile duct injury

following liver transplantation (36).

Several genetic variations in hepatobiliary transporters have been linked to various types of

cholestatic disorders and cholangiopathies (19,36). A large number of clinicall relevant SNPs

of the ABCB4 gene have been reported in literature (22,24,26-28,37-39). For two reasons

we found it not rational to analyse these individual SNPs in the current study. First of all,

some of the reported gene variations have been linked to a known and permanent cholestatic

phenotype, such as PFIC-3, and it is unlikely that patients with such a phenotype were

selected as organ donor. Secondly, the prevelance of most of the individual SNPs reported in

the literature is low, and it is unlikely that very rare SNPs could accountable for a complication

such as NAS with an incidence of around 16%.

In conclusion, in this large series of 461 liver transplant recipients, we established a strong

association between donor ABCB4 haplotype and the development of NAS after liver

transplantation. Livers with the AGGTA haplotype of the ABCB4 gene were found to have a

two-times higher risk of developing NAS, compared to livers without this haplotype. These

data contribute to the accumulating evidence that a (relative) reduction in biliary phospholipid

secretion, resulting in the increased toxicity of bile salts, play an important role in the

development of bile duct injury and NAS after liver transplantation, and that alterations in bile

composition after transplantation may have part of its origin in the donor.

136


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van Mil SW, van der Woerd WL, van der Brugge G, Sturm E, Jansen PL, Bull LN, et al. Benign recurrent 29.

intrahepatic cholestasis type 2 is caused by mutations in ABCB11. Gastroenterology 2004;127:379-84.

Strautnieks SS, Bull LN, Knisely AS, Kocoshis SA, Dahl N, Arnell H, et al. A gene encoding a liver-specific ABC 30.

transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet 1998;20:233-8.

Noe J, Kullak-Ublick GA, Jochum W, Stieger B, Kerb R, Haberl M, et al. Impaired expression and function of the 31.

bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis. J Hepatol 2005;43:536-

43.

Toh S. Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and 32.

mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome. American journal of human genetics

1999;64:739-46.

Verdonk RC, Buis CI, Porte RJ, Van der Jagt EJ, Limburg AJ, van den Berg AP, et al. Anastomotic biliary 33.

strictures after liver transplantation: Causes and consequences. Liver Transpl 2006;12:726-35.

Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med 1998 22;339:1217-27.34.

Arrese M, Trauner M. Molecular aspects of bile formation and cholestasis. Trends Mol Med 2003;9:558-64.35.

Trauner M, Fickert P, Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic 36.

syndromes. Semin Liver Dis 2007;27:77-98.

Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defect in adults with symptomatic intrahepatic and 37.

gallbladder cholesterol cholelithiasis. Gastroenterology 2001;120:1459-67.

Eloranta ML. Association of single nucleotide polymorphisms of the bile salt export pump gene with intrahepatic 38.

cholestasis of pregnancy. Scandinavian journal of gastroenterology 2003;38:648-52.

Pauli-Magnus C, Kerb R, Fattinger K, Lang T, Anwald B, Kullak-Ublick GA, et al. BSEP and MDR3 haplotype 39.

structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology

2004;39:779-91.

Part III

HO-1 and hepatobiliary

injury after liver transplantation

Expression of Heme oxygenase -1 in human livers before transplantation correlates with graft injury and function after transplantation 8

Am J Transplant. 2005; 5:1875-1885

Erwin GeukenCarlijn I Buis

Dorien S VisserHans Blokzijl

Han MoshageBalazs Nemes

Henri GD LeuveninkKoert P de Jong

Paul MJG PeetersMaarten JH Slooff

Robert J Porte

144

HO-1 before transplantation and graft injury and function after transplantation Chapter 8

Abstract

Upregulation of heme oxygenase-1 (HO-1) has been proposed as an adaptive mechanism

protecting against ischemia/reperfusion (I/R) injury. We investigated HO-1 expression

in 38 human liver transplants and correlated this with I/R injury and graft function. Before

transplantation, median HO-1 mRNA levels were 3.4-fold higher (range 0.7-9.3) than in normal

controls. Based on the median value, livers were divided into two groups: low and high HO-1

expression. These groups had similar donor characteristics, donor serum transaminases,

cold ischemia time, HSP-70 expression, and distribution of HO-1 promoter polymorphism.

After reperfusion, HO-1 expression increased significantly further in the initial low HO-1

expression group, but not in the high HO-1 group. Postoperatively, serum transaminases were

significantly lower and bile salt secretion was higher in the initial low HO-1 group, compared

to high expression group. Immunofluorescence staining identified Kupffer cells as the main

localization of HO-1.

In conclusion, human livers with initial low HO-1 expression (< 3.4 times controls) are able

to induce HO-1 further during reperfusion and this is associated with less injury and better

function than initial high HO-1 expression (> 3.4 times controls). These data suggest that

increase in HO-1 during transplantation is more protective than a high HO-1 expression before

transplantation.

145

Chapter 8

Introduction

Orthotopic liver transplantation (OLT) is an effective treatment for end-stage liver diseases (1).

However, ischemia and subsequent reperfusion of the liver remain a major cause of graft injury,

causing liver dysfunction and even failure after transplantation (2). This is particularly true for

livers from older donors and steatotic livers, which have a higher susceptibility to ischemia/

reperfusion (I/R) injury (3,4). During organ procurement and transplantation, the liver is exposed

to oxidative stress. Besides the ischemia during cold storage, hypoxia may occur before or

during procurement due to hypotension or cardiac arrest in the donor. After graft reperfusion,

several cascades are triggered leading to the formation of reactive oxygen species (ROS), which

are well-known sources of oxidative stress. Methods to protect liver grafts against I/R injury have

considerable clinical consequences and are therefore of great interest.

It is increasingly recognized that cells respond to stressful events, such as ischemia, hypoxia

and ROS, by the activation of various cytoprotective genes and pathways. Heme oxygenase-1

(HO-1) has recently been proposed as a graft survival gene (5,6). Up-regulation of HO-1 is

considered to be one of the most critical cellular protection mechanisms (7,8). It is rapidly

induced under various conditions of oxidative stress, including hypoxia, hyperoxia and ROS

(9). HO-1 catalyzes the rate-limiting step in the oxidative detoxification of excess heme, by

cleaving the α-methene bridge into equimolar amounts of free iron, biliverdin and carbon

monoxide (CO) (9). Free iron, catalyzing oxidative reactions, is bound by iron regulatory

proteins that stimulate synthesis of ferritin, thereby preventing iron-dependent oxidative

stress (10,11). Biliverdin is subsequently converted into bilirubin and both have the ability

to scavenge ROS (12-15). CO has been shown to serve as an endogenous regulator for

maintaining microvascular blood flow of the liver (16,17).

Two- to three-fold induction of HO-1 by pharmacologic agents or genetic engineering has been

shown to reduce I/R injury in rat liver grafts after extended cold ischemia time (6). Moreover,

steatotic livers from genetically obese Zucker rats are markedly protected against I/R injury

after exogenous upregulation of HO-1 (5). Based on these observations, exogenous induction

of HO-1 prior to transplantation has been proposed as a potentially powerful therapeutic option

to protect liver grafts against I/R injury (5,6). Molecules such as HO-1, however, are probably

not exclusively cytoprotective and each of the products generated by the action of heme

oxygenase (Fe2+, bilirubin and CO) can cause injury under certain circumstances (18). Indeed,

146


several experimental studies have shown that excessive overexpression of HO-1 is directly

related with increased injury (19-21). Recently, also a (GT)n dinucleotide repeat polymorphism

that modulates the level of HO-1 inducibility was identified in the promoter region of the human

HO-1 gene. Short GT repeats (<25) are associated with highly significant upregulation of HO-1

in response to inflammatory stimuli (22,23). Therefore, it is critically important to understand the

endogenous changes in HO-1 expression under clinical conditions, such as transplantation,

before the exogenous induction of HO-1 can be safely attempted as a possible therapeutic or

prophylactic measure to reduce I/R injury.

We have therefore studied the changes in endogenous HO-1 expression in human liver

grafts before and after transplantation and correlated these with biochemical markers of graft

injury and hepatobiliary function. This study provides important new information on the role of

endogenous HO-1 expression during human liver transplantation


Patient and Donor Data

Thirty-eight patients undergoing OLT were included. All patients received livers from brain

death, multi-organ donors. In the control group (n=5), biopsies were collected in patients

undergoing partial hepatectomy for metastatic tumors. Tissue and data collection was

performed according to the guidelines of the medical ethical committee of our institution and

the Dutch Federation of Scientific Societies.

Collection of Liver Biopsies and Bile Samples from Recipients

Three sequential needle biopsies were taken from each liver graft: at the end of cold storage,

3 hours after reperfusion and 1 week after transplantation. Biopsies were immediately divided:

one part was snap-frozen in liquid nitrogen for RNA and protein isolation and one part was

frozen in isopentane at -80°C for histology studies. During transplantation a bile drain was

routinely placed into the common bile duct, allowing collection of bile (24). To avoid interruption

of the entero-hepatic circulation bile was daily readministered via a jejunostomy catheter. After

the transplantation, bile samples were collected daily between 8 and 9 am. Liver and bile

specimens were stored at -80°C.

147

Chapter 8

RNA Isolation and Reverse-Transcriptase Polymerase Chain

Reaction

Total RNA was isolated from liver biopsies using TRIzol (Invitrogen Life Technologies, Breda, the

Netherlands) and quantified using Ribogreen (Molecular Probes, Inc., Eugene, OR). Reverse

transcription was performed on 3.36 µg RNA using random primers in a final volume of 75 µl

(Reverse Transcription System, Promega, Madison, WI). For quantitative real-time detection

RT-PCR (25,26), sense and antisense primers (Invitrogen, Paisley, Scotland) and fluorogenic

probes (Eurogentec, Herstal, Belgium) for HO-1, HSP-70 and 18S were designed using Primer

Express software (PE Applied Biosystems, Foster City, CA). For HO-1, the primers and probe used

were 5’-GACTGCGTTCCTGCTCAACAT-3’ (sense), 5’-GCTCTGGTCCTTGGTGTCATG-3’

(antisense), and 5’-TCAGCAGCTCCTGCAACTCCTCAAAGAG-3’ (probe), generating a 75

base pair PCR product. For heat shock protein-70 (HSP-70), used as a molecular stress marker,

the following primers and probe were used: 5’-TCTTCTCGCGGATCCAGTCT-3’ (sense),

5’-GGTTCCCTGCTCTCTGTCG-3’ (antisense), and 5’-CCGTTTCCAGCCCCCAATCTCAG-3’

(probe), generating a 70 base pair PCR product. For 18S, the primers and probe used were

5’-CGGCTACCACATCCAAGG-3’ (sense), 5’-CCAATTACAGGGCCTCGAAA-3’ (antisense),

and 5’-CGCGCAAATTACCCACTCCCGA-3’ (probe), generating a 109-base pair PCR

fragment. The ABI PRISM 7700 (Applied Biosystems, Foster City, CA) was used for PCR.

Protein Isolation and Western Blot Analysis

Frozen liver tissue was homogenized in buffer containing protease inhibitors. Protein

concentrations were measured using a standard Lowry assay. Fifteen µg of protein was

fractioned on a 5% SDS-PAGE gel and transferred to PVDF membranes (Pall Life Sciences,

Ann Arbor, MI). The membranes were blocked with 1% SKIM milk (Fluka BioChemica, Buchs,

Switzerland) and labeled with the anti HO-1 polyclonal antibody (dilution, 1:5000, StressGen,

Victoria, British Columbia, Canada). After washing in PBS/0.05% Tween-20 (Sigma, Malden,

The Netherlands), blots were incubated with a horseradish peroxidase-labled goat anti-rabbit

IgG (dilution, 1:2000, DAKO, Glostrup, Denmark). Finally membranes were developed with

ECL (Amersham, Chalfont St Giles, UK). Five separate cases were examined in each group.

148


HO-1 Genotype assessment

Genomic DNA was isolated from donor splenocytes using a commercial kit (Gentra Systems,

Minneapolis, MN). PCR and genotyping procedures were similar as described by de Jong

et al. (27). The 5’-flanking region of the HO-1 gene containing the poly (GT)n repeat was

amplified by PCR using as forward primer 5’-CAGCTTTCTGGAACCTTCTGG-3’, carrying

a 6-FAM flourescent label (Sigma, Malden, the Netherlands), and as reversed primer

5’-GAAACAAAGTCTGGCCATAGGAC-3’. Sequence analysis of the amplification products of

individuals homozygous for the 222 and 229 basepairs alleles showed correspondence with

GT numbers 26 and 29, respectively (results not shown). We divided allelic repeats into two

subclasses using a classification as previously described in transfection studies (28). Short

repeats, with less than 25 GT repeats (amplicons of 220 basepairs and less), were designated

as allele class S (short), and long repeats with 25 or more GT repeats as allele class L (long).

Recipients of class S allele liver transplants (homozygous S/S and heterozygous S/L) were

compared with recipients of non-class S allele transplants (L/L).

Immunofluorescence Microscopy

Frozen liver sections were stained for HO-1 and the Kupffer cell marker CD68, using an anti-HO-1

polyclonal antibody (dilution, 1:100, StressGen) and an anti-human CD68 monoclonal antibody

KP-1 (dilution, 1:2000, DAKO). After washing, sections were subsequently incubated with a

goat anti-rabbit IgG with a red fluorescent label (Alexa Fluor 568, Molecular Probes, Leiden, the

Netherlands), and with a goat anti-mouse IgG with a green fluorescent label (Alexa Fluor 488,

Molecular Probes). Double-positive cells were identified as those stained yellow. Percentages

of HO-1-positive Kupffer cells were calculated by dividing the number of cells stained yellow by

the number of cells stained green (29). Five different high power fields (x400) were analyzed in

an individual liver sample, and five separate cases were examined in each group. Images were

taken with a Leica DM LB fluorescence microscope (Leica, Wetzlar, Germany).

Total Bile Salt Secretion and Serum Biochemistry

Postoperatively, bile flow was expressed as daily bile production in mL per kg body weight

of the donor. Total bile salt concentration was measured spectrophotometrically with

3α-hydroxysteroid dehydrogenase (30). Serum samples were analyzed for aspartate- and

alanine aminotransferase (AST and ALT) and gamma glutamyltransferase (GGT), by routine

clinical chemistry testing.

149

Chapter 8

Statistics

Statistical analyses were performed using SPSS Version 11.5 for Windows (SPSS Inc.,

Chicago, IL). All data are reported as median and interquartile ranges (IQR). Groups were

compared with the Mann-Whitney U-tests, Wilcoxon Signed Ranks-tests, Pearson X2-tests

and the Fisher’s Exact Test where appropriate. Postoperative biochemical variables were

compared using the daily values, but also the total course during the first week was compared

by calculating the area under the curve (AUC), using the trapezium rule. All P values were

2-tailed and considered as statistically significant at levels < 0.05.

Results

Effects of OLT on HO-1 Gene and Protein Expression.

Before transplantation, the median HO-1 mRNA level was 3.4-times higher in donor livers

than in normal control livers (P = 0.001; Figure 1), suggesting that HO-1 is already induced in

brain-death donors or during organ procurement.

Re

lative

HO

-1 m

RN

A le

ve

ls

P = 0.001 P = 0.002

P = 0.005

3

4

5

6

Re

lative

HO

control

livers

Before

OLT3 hours after

reperfusion1 week

after OLT

liver grafts

0

1

2

3

Figure 1. HO-1 mRNA levels in human liver grafts (n=38) and normal control livers (n=5). HO-1 mRNA was

standardized for 18S rRNA. HO-1 expression in control livers was set to 1.0. Values represent medians and

interquartile ranges.

150


At 3 hours after reperfusion, there was no significant overall change in HO-1 expression. One

week after transplantation, HO-1 gene expression decreased by 38% compared to the values

after reperfusion (P = 0.002; Figure 1). However, HO-1 expression remained strongly elevated

during the first postoperative week compared to normal control livers (Figure 1).

Initial low HO-1 expression

Initial high HO-1 expression

3

4

5

6

P = 0.04

- 1 m

RN

A le

ve

ls

P = 0.001

P = 0.003

P = 0.03

P = 0.001

P = 0.003

A

before

OLT

3 hours after

reperfusionbefore

OLT

3 hours after

reperfusioncontrol

livers

liver grafts

0

1

2

3

Re

lative

HO

-

HO-1 protein (32 kD)B

1 2 3 4 5

Figure 2. A) Course of HO-1 mRNA levels in human liver grafts with low or high HO-1 expression before trans-

plantation; initial low HO-1 expression group (n=19) and initial high HO-1 expression group (n=19), respectively.

HO-1 mRNA was standardized for 18S rRNA. HO-1 mRNA levels in normal control livers was set to 1.0. Values

represent medians and interquartile ranges. B) Western blot analysis of HO-1 protein expression in the initial

low and initial high HO-1 group.

151

Chapter 8

At 3 hours after reperfusion, there was no significant overall change in HO-1 expression. One

week after transplantation, HO-1 gene expression decreased by 38% compared to the values

after reperfusion (P = 0.002; Figure 1). However, HO-1 expression remained strongly elevated

during the first postoperative week compared to normal control livers (Figure 1).



3

4

5

6

P = 0.04

-1 m

RN

A le

ve

ls

P = 0.001

P = 0.003

P = 0.03

P = 0.001

P = 0.003

A

before

OLT

3 hours after

reperfusionbefore

OLT

3 hours after

reperfusioncontrol

livers

liver grafts

0

1

2

3

Re

lative

HO

-

HO-1 protein (32 kD)B

1 2 3 4 5

Figure 2. A) Course of HO-1 mRNA levels in human liver grafts with low or high HO-1 expression before trans-

plantation; initial low HO-1 expression group (n=19) and initial high HO-1 expression group (n=19), respectively.

HO-1 mRNA was standardized for 18S rRNA. HO-1 mRNA levels in normal control livers was set to 1.0. Values

represent medians and interquartile ranges. B) Western blot analysis of HO-1 protein expression in the initial

low and initial high HO-1 group.

A wide variation in HO-1 gene expression was detected in liver biopsies that were collected

before transplantation, ranging from 0.7- to 9.3-times the levels in normal control livers. To

be able to identify donor variables that are associated with HO-1 induction, and to study the

possible impact of HO-1 on I/R injury and graft viability after transplantation, we decided to

divide liver grafts into two groups based on the level of HO-1 expression before transplantation.

A low HO-1 expression group (n=19) was formed by livers with an initial HO-1 mRNA level

below the median value (< 3.4-times control levels) and a high HO-1 expression group (n=19)

was formed by livers with an initial HO-1 gene expression above the median value (> 3.4-times

control levels). Median HO-1 expression in the low and high expression group was 2.0- and

5.0-times higher than in control livers (Figure 2A). Interestingly, HO-1 mRNA level increased

significantly by 43% after reperfusion in the initial low expression group, whereas HO-1

expression decreased by 23% after reperfusion in the inital high expression group (Figure

2A). In both groups, HO-1 gene expression remained significantly elevated during the first

postoperative week, compared to controls (data not shown).

Changes in HO-1 protein concentrations, as detected by Western blot analysis, were similar to

the changes in HO-1 mRNA expression. HO-1 protein concentration was low in normal control

livers, compared to the donor livers. After reperfusion, HO-1 protein expression increased further

in the initial low HO-1 expression group, but not in the initial high HO-1 group (Figure 2B).

Comparison of Donor Data for Livers with Low and High HO-1

Expression

A large number of donor characteristics and laboratory values were investigated in an attempt

to explain the differences in HO-1 gene expression before transplantation. Several events

that are known to induce HO-1 expression in animal models, such as hypotension, cardiac

arrest, blood transfusions and ischemia, may also occur in brain-death donors or during organ

procurement. In addition to this, some drugs (i.e. dopamine) have been shown to induce HO-1

expression (31). We have compared all these donor-related events and variables in the two

groups, but were unable to find statistically significant differences (Table 1).

152


Table 1. Comparison of donor, recipient and surgical variables in initial low HO-1 expression group

and initial high HO-1 expression group.

Low HO-1 Expression High HO-1 Expression

Donor variables

Age (years; median [IQR]) 39 (25-60) 48 (41-58)

Gender (M/F) 7/12 8/11

ICU stay (days; median [IQR]) 2.5 (0.8-4.5) 1.2 (0.3-3.2)

Duration of liver procurement (minutes; median [IQR])

150 (51-177) 150 (67-195)

Hypotension (no. of donors)a 7/19 11/19

Cardiac arrest (no. of donors)b 2/19 3/19

Dopamine (no. of donors)c 8/19 11/19

Bloodtransfusion (no. of donors)c 5/19 7/19

Temperature (oC; median [IQR]) 36.1 (36.0-36.8) 36.5 (36.1-37.0)

Diuresis last hour (ml; median [IQR]) 220 (113-300) 200 (130-320)

Bloodpressure (mmHg; median [IQR]) 120/60 (110/60-124/73)

120/67 (110/65-137/78)

pO2 (kPa; median [IQR]) 16.5 (13.1-21.8) 13.6 (11.8-20.1)

FiO2 (%; median [IQR]) 40 (36-47) 40 (40-57)

AST (U.L-1; median [IQR]) 27 (15-93) 42 (19-67)

ALT (U.L-1; median [IQR]) 24 (18-61) 25 (14-45)

GGT (U.L-1; median [IQR]) 20 (15-29) 20 (13-63)

Total Bilirubin (U.L-1; median [IQR]) 4.0 (1.3-10.0) 10.0 (5.0-16.5)

Hemoglobin (mmol.L-1; median [IQR]) 7.6 (6.3-8.9) 7.0 (5.8-8.9)

Recipient and Surgical variables

Age (years; median [IQR]) 45 (28-58) 47 (35-54)

Gender (M/F) 9/10 13/6

ICU stay (days; median [IQR]) 3 (2-6) 2 (2-7)

Acute rejection of the graft (no. of recipients)d 11/19 4/19

1st Warm Ischemia Time, WIT (minutes; median [IQR])e

43 (36-57) 42 (28-49)

Cold Ischemia Time, CIT (minutes; median [IQR])

465 (415-567) 574 (457-620)

2nd WIT (minutes; median [IQR])f 43 (37-47) 48 (43-56)

a) Donors who suffered at least one episode of hypotension or b) cardiac arrest within 24 hrs prior to

procurement of the liver.

c) Number of donors who were administered dopamine or blood within 24 hrs before donor hepatectomy.

d) Number of recipients who suffered from rejection of the graft within the first week after transplantation.

e) 1st WIT: time between start cold perfusion in the donor and procurement of the liver graft.

f) 2nd WIT: time between the end of cold ischemic preservation of the liver and start of reperfusion in the

recipient.

There were no statistical significant differences for any variables between the two groups (Mann Whitney

U-test or Pearson Chi-Square-test).

153

Chapter 8

There were also no significant differences in the time between start of in situ cold perfusion

in the donor and actual hepatectomy (1st “relatively” warm ischemia) or in the duration of

cold storage (Table 1). Interestingly, there were also no differences in donor serum markers

of liver injury (AST, ALT and GGT) or liver function (bilirubin) between the two groups (Table

1). Moreover, there was no significant difference in pretransplant mRNA expression of the

stress protein HSP-70 in the low and high HO-1 group (1.18 [ IQR 0.30 – 3.76] versus 0.57

[IQR 0.22 – 2.27]; p = 0.44). These data suggest that differences in HO-1 expression in liver

grafts before transplantation cannot simply be explained by a higher number of compromised

donors in the high HO-1 expression group.

The Effect of HO-1 Donor Genotype

To examine whether the differences in initial HO-1 expression could be explained by the the

number of (GT)n repeats in the HO-1 promoter region, HO-1 donor genotypes were analyzed.

Allele class S/S was present in 8% of the donors, 35% of the donors were heterozygous for

class S alleles (S/L), and 57% of the donors were non-carriers of the class S allele (L/L).

Distribution of the numbers of (GT)n repeats was not different for donor livers in the initial low

and high HO-1 expression group (Figure 3). There were also no significant differences in the

distribution of class S allele donor livers (S/S and S/L) and non-class S donor livers (L/L) in

the two groups (Table 2).

Table 2. Distribution of HO-1 genotype in the livers with initial low or high HO-1 mRNA expression.

Initial HO-1 Expression

Genotype* Low High

p-value = 1

Short Allele (SS or SL) 8 (42%) 8 (44%)

Long Allele (LL) 11 (58%) 10 (56%)

19 (100%) 18** (100%)

a) Short allele (S) status defined as < 25 (GT) repeats in the HO-1 promoter region; Long allele (L) status

defined as > 25 (GT) repeats in the HO-1 promter region.

b) Genomic DNA for gene sequencing was not available in one donor.

154


40

50

60

70

Alle

le F

req

ue

ncy (

%)



0

10

20

30

40

19 21 23 25 27 29 31 33 35

Number of (GT)n Repeats

Alle

le F

req

ue

ncy (

%)

Figure 3. Allele frequencies of the HO-1 (GT)n repeat promoter polymorphism in liver grafts with initial low or

high HO-1 mRNA expression.

Post-transplant Outcome in Relation to HO-1 Expression

To examine whether the magnitude of HO-1 induction was associated with differences in

outcome after transplantation, laboratory values and recipient characteristics were analyzed.

Posttransplant serum levels of AST and ALT were used as well-accepted markers of I/R injury.

Although there were no differences in serum AST levels in the donors, we found a significant

positive correlation between serum AST levels in the recipient on postoperative day 1 and

HO-1 expression in the donor liver before transplantation (Figure 4). When comparing the

two groups, serum AST levels on postoperative days 1 through 3 were significantly higher

in recipients of livers with high HO-1 expression (Figure 5A). Also serum ALT levels were

significantly higher on postoperative day 1 in recipients of livers with high HO-1 expression

(Figure 5B). Hepatobiliary function, as reflected by biliary bile salt secretion, was significantly

worse in the group with high HO-1 expression, compared to the group with low expression

(Figure 5C). When groups were categorized based on the ability of increasing HO-1 expression

during reperfusion of the liver graft, serum AST levels in the induction group (n=15) were

significantly lower on postoperative days 2 and 3 than in the HO-1 reduction group (n=23).

Serum ALT levels and biliary bile salt secretion however, did nof differ between the groups in

the latter classification (data not shown).

155

Chapter 8

40

50

60

70

Alle

le F

req

ue

ncy (

%)



0

10

20

30

40

19 21 23 25 27 29 31 33 35

Number of (GT)n Repeats

Alle

le F

req

ue

ncy (

%)

Figure 3. Allele frequencies of the HO-1 (GT)n repeat promoter polymorphism in liver grafts with initial low or

high HO-1 mRNA expression.

Post-transplant Outcome in Relation to HO-1 Expression

To examine whether the magnitude of HO-1 induction was associated with differences in

outcome after transplantation, laboratory values and recipient characteristics were analyzed.

Posttransplant serum levels of AST and ALT were used as well-accepted markers of I/R injury.

Although there were no differences in serum AST levels in the donors, we found a significant

positive correlation between serum AST levels in the recipient on postoperative day 1 and

HO-1 expression in the donor liver before transplantation (Figure 4). When comparing the

two groups, serum AST levels on postoperative days 1 through 3 were significantly higher

in recipients of livers with high HO-1 expression (Figure 5A). Also serum ALT levels were

significantly higher on postoperative day 1 in recipients of livers with high HO-1 expression

(Figure 5B). Hepatobiliary function, as reflected by biliary bile salt secretion, was significantly

worse in the group with high HO-1 expression, compared to the group with low expression

(Figure 5C). When groups were categorized based on the ability of increasing HO-1 expression

during reperfusion of the liver graft, serum AST levels in the induction group (n=15) were

significantly lower on postoperative days 2 and 3 than in the HO-1 reduction group (n=23).

Serum ALT levels and biliary bile salt secretion however, did nof differ between the groups in

the latter classification (data not shown).

3000

4000

5000

6000

Seru

m A

ST

level on P

OD

1

R2 = 0.15

P = 0.017

0

1000

2000

0 0.1 1 10

mRNA HO-1 before transplantation

Seru

m A

ST

level on P

OD

1

Figure 4. Correlation between hepatic HO-1 mRNA expression before transplantation and serum AST level on

postoperative day 1 (POD 1) in all liver transplant recipients (n=38).

These findings indicate that liver grafts with an initial high (> 3.4-fold) HO-1 expression

before transplantation exhibited more I/R injury and have poorer hepatobiliary function after

transplantation than grafts with an initial low (< 3.4-fold) HO-1 expression, despite the fact

that there were no differences in biochemical or molecular markers of graft injury in the donor

before organ procurement.

156


AS

T (

U/L

)

Postoperative days

1 2 3 4 5 6 7

*

*

*

0

200

400

600

800

1000

1200

1400

AUC P < 0.05

ALT

(U

/L)

400

600

800

1000

1200

1400

*

AUC P < 0.05

A

B

Postoperative days

1 2 3 4 5 6 7

0

200

400

Bili

ary

Bile

Sa

lt O

utp

ut

(µm

ol·d

ay

-1·k

g-1

)

AUC P < 0.05

Postoperative days

1 2 3 4 5 6 7

0

50

100

150

200

250

300

350

400

C

Figure 5. Serum AST (panel A) and ALT (panel B) levels and biliary bile salt secretion (panel C) in the first week

after OLT in the initial low (open bars; n=19) and high HO-1 (closed bars; n=19) expression groups. Values

represent medians and interquartile ranges. The asterisks indicate significant differences between the groups

(p<0.05). Total course during the first week was calculated as the area under the curve (AUC), using the trape-

zium rule.

157

Chapter 8

Immunofluorescence Microscopy

Specific immunostaining showed that HO-1 was predominantly localized in irregular and

star-shaped cells. These characteristics suggested that HO-1 protein is mainly expressed in

Kupffer cells, which was confirmed by double-color immunofluorescence labeling, using the

anti-HO-1 and anti-human CD68 MoAb KP-1, a marker of Kupffer cells. As shown in Figure 6,

the distribution of anti-HO-1 positive (red) cells overlapped with that of KP-1-positive (green)

cells, resulting in a yellow staining. In control livers, a considerable proportion of Kupffer cells

did not express HO-1-associated immunoreactivity and displayed mainly a green staining

(Figure 6A). In contrast with this, almost all Kupffer cells in liver grafts demonstrated positive

staining for HO-1 (Figure 6B-E). Indeed, morphometrical analysis showed significantly higher

percentages of HO-1-positive Kupffer cells in liver grafts before transplantation, compared

to normal control livers (low and high HO-1 expression group 88% and 95%, respectively,

compared to 50% in normal control livers, P < 0.02 for both groups; Table 3). After reperfusion,

HO-1 expression in Kupffer cells increased further, resulting in a positive staining of all Kupffer

cells in both groups (Table 3).

Although, after reperfusion, all Kupffer cells in both groups stained positive for HO-1, the red

staining (HO-1) per cell was far more intense in the group with high HO-1 expression than

in the low expression group (Figure 6C and E). This indicates that not only the percentage

of Kupffer cells expressing HO-1 is increased in liver grafts, but that also the HO-1 protein

expression per Kupffer cell is enhanced, where the latter seems to discriminate the group with

high HO-1 expression from the livers with low HO-1 expression. This is in line with the higher

HO-1 mRNA and protein levels after reperfusion in the group with high HO-1 expression,

compared to the low expression group.

158


A. Normal control liver

B. Initial low HO-1 expression: before OLT C. Initial low HO-1 expression: after reperfusion

D. Initial high HO-1 expression: before OLT E. Initial high HO-1 expression: after reperfusion

Figure 6. Immunofluorescence double-staining of liver biopsies. Sections are stained for HO-1 (red) and the

Kupffer cell marker CD68 (green). Colocalization of these two colours can be recognized by the yellow colour.

Panel A; normal control liver. Panel B; pretransplant biopsy of a liver with low initial HO-1 mRNA expression.

Panel C; postreperfusion (3hrs) biopsy of a liver with low initial HO-1 mRNA expression. Panel D; pretransplant

biopsy of a liver with high initial HO-1 mRNA expression. Panel E; postreperfusion biopsy (3hrs) of a liver with

high initial HO-1 mRNA expression.

159

Chapter 8

Tab

le 3

. Mo

rph

om

etri

cal a

nal

ysis

of

cell

typ

e sp

ecifi

c ex

pre

ssio

n o

f H

O-1

in h

um

an li

ver

tran

spla

nts

wit

h lo

w o

r h

igh

HO

-1 e

xpre

ssio

n a

nd

co

ntr

ol l

iver

s.

Con

trol

In

itial

Low

HO

-1 E

xpre

ssio

n In

itial

Hig

h H

O-1

Exp

ress

ion

Bef

ore

OLT

Afte

r Rep

erfu

sion

Bef

ore

OLT

Afte

r Rep

erfu

sion

Sing

le im

mun

osta

inin

g

HO

-1(+

) (no

. of c

ells

; med

ian

[IQR

])a 20

[17-

23]

31 [2

7-47

]e 2

7 [1

7-36

]e 4

0 [3

7-44

]e,f

37

[27-

44]e

CD

68(+

) (no

. of c

ells

; med

ian

[IQR

])b37

[35-

43]

31 [2

5-40

] 3

0 [2

3-36

] 4

2 [4

0-44

]f 3

6 [3

3-43

]g

Dou

ble

imm

unos

tain

ing

HO

-1(+

) Kup

ffer c

ells

(no.

of c

ells

; med

ian

[IQR

])c20

[17-

23]

28 [2

3-35

] 2

3 [1

3-30

] 4

0 [3

7-43

]f 3

5 [3

0-43

]g

% H

O-1

(+) K

upffe

r cel

ls (%

; med

ian

[IQR

])d50

[45-

63]

88 [7

8-99

]e10

0 [4

0-10

0]e

95

[93-

100]

e10

0 [8

8-10

0]e

a) N

umbe

r of H

O-1

and

b) C

D68

pos

itive

cel

ls.

c) N

umbe

r and

d) p

erce

ntag

e of

HO

-1 p

ositi

ve K

upffe

r cel

ls.

Anal

yses

bas

ed o

n ob

serv

atio

ns in

five

diff

eren

t hig

h po

wer

fiel

ds w

ithin

one

live

r bio

psy

at 4

00X

e) P

< 0

.02,

com

pare

d w

ith th

e co

ntro

l gro

upf)

P <

0.03

, com

pare

d w

ith th

e va

lues

bef

ore

OLT

of t

he in

itial

low

exp

ress

ion

grou

pg)

P <

0.0

1, c

ompa

red

with

the

valu

es a

fter r

eper

fusi

on o

f the

initi

al lo

w e

xpre

ssio

n gr

oup

160


Discussion

We have investigated HO-1 expression in human liver allografts during transplantation and

correlated this with clinical signs of graft injury and hepatobiliary function. There are three

novel findings in this study. First, we have shown that, compared to normal control livers,

HO-1 gene and protein expression in human liver grafts from brain-death donors is induced

already prior to transplantation. After reperfusion, HO-1 expression increased further in livers

with relatively low initial HO-1 expression (< 3.4 times controls), but not in livers with initial

high HO-1 expression (> 3.4 times controls). Second, allografts with initial high expression of

HO-1 demonstrated significantly more I/R injury and had worse hepatobiliary function than

grafts with a low upregulation of HO-1. Finally, we were able to identify Kupffer cells as the

main site of HO-1 protein expression in human liver grafts. While about 50% of the Kupffer

cells in normal control liver expressed HO-1, positive staining for HO-1 was found in 100% of

the Kupffer cells of transplanted livers. These findings provide important new information on

the endogenous regulation of HO-1 during human liver transplantation.

There is accumulating evidence that the HO-1 system has important vasoregulatory properties

and actively maintains hepatic microperfusion and tissue oxygenation via the production of

CO (16). In addition to this, the HO-1 system has been shown to have anti-oxidant, anti-

inflammatory, anti-apoptotic and platelet aggregation-inhibiting properties and, therefore, it

has been proposed a graft survival gene. Animal studies have suggested that exogenous

induction of HO-1 before transplantation may confer cytoprotective and immune regulatory

functions (6,32-34) and could become a novel and potentially powerful strategy to protect

(marginal) liver grafts from I/R injury (5,8). Induction of HO-1 can be obtained by a variety of

methods, such as administration of HO-1 inducers (i.e. cobalt protoporphyrin) or adenoviral

HO-1 genetransfer (5,8). These methods generally lead to a 2 to 3-fold upregulation of

HO-1 activity (5). There is increasing evidence that overexpression of HO-1 higher than this

is not exclusively cytoprotective (19,21). In fibroblast cell cultures, low induction of HO-1

(less than 5-fold) was shown to be cytoprotective against hyperoxia, but excessive HO-1

activation resulted in the accumulation of free divalent iron and increased oxidative injury (19).

Moreover, it has been shown that highly increased (about eight- to nine-fold) activity of HO-1

contributes to endotoxin-induced shock in rats, due to the increased production of CO, a potent

vasorelaxant (21). Therefore, it is of paramount importance that the endogenous changes in

161

Chapter 8

HO-1 expression during transplantation, as well as the therapeutic window of protection, are

well defined before clinical application of HO-1 inducing protocols are attempted.

All donor livers in our study were obtained from brain-death multi-organ donors. The

increased HO-1 mRNA and protein expression observed in these livers before transplantation

suggests that HO-1 is induced in brain-death donors. This observation is in line with studies

in kidney allografts from brain-death donors (35). We have tried to identify variables which

could have contributed to the increased expression of HO-1 in the donor livers before

transplantation. Several factors have been shown to induce HO-1 gene expression in vivo,

including hypotension (36), hypoxia (37-39), hyperoxia (9,40), blood transfusions (41,42),

and inotropic drugs like dopamine (31). All of these factors may also occur in postmortem

organ donors. Comparison of these known inducers of HO-1 gene expression, as well as

several other donor and procurement related variables, however, did not show any statistically

significant differences between the two groups. Variations in initial HO-1 expression could

also not be explained by differences in the distribution of the (GT)n repeat polymorphism of

the HO-1 promoter. The functionally relevant short allele status (<25 repeats) was not found

more frequently in livers with initial low HO-1 expression. Further studies will be necessary to

elucidate the mechanisms of endogenous HO-1 induction in organs from brain death donors.

Although we did not find differences in biochemical (liver enzymes) or molecular (HSP-70)

markers of liver injury before transplantation between the liver grafts with low or high HO-1

expression, we did observe a significant correlation between postoperative serum AST in the

recipients and initial HO-1 expression. In parallel with this, serum AST levels were significantly

higher and biliary bile salt output significantly lower after transplantation in recipients of livers

with high HO-1 expression, compared to grafts with low HO-1 expression. Liberation of

divalent iron is one of the effects resulting from increased HO-1 activity (9). Iron is a mediator

of the generation of ROS and it has been shown to play an important role in I/R injury (43,44).

We, therefore, speculate that exaggerated HO-1 activity in liver grafts may cause increased

injury due to the liberation of iron, resulting in a pro-oxidant condition and higher susceptibility

to I/R injury. The apparent paradox of one molecule or pathway causing both cytoprotection

and cytotoxicicty has also been found in other systems, like the nitric oxide system (45). More

studies will be needed to clarify this issue.

Interestingly, a significant further increase in HO-1 expression was found after reperfusion of livers

with an initially low expression, whereas a small, but significant decrease in HO-1 expression was

162


observed in livers with initially high HO-1 expression. This data could imply that HO-1 mRNA

expression cannot be further upregulated upon reperfusion when levels are already high to start

with, whereas further upregulation can occur in livers with moderately elevated HO-1 expression

before reperfusion. Although we observed a better postoperative outcome in the initial low HO-1

expression group, it remains indefinite whether it is the initial low HO-1 expression or the ability

to increase HO-1 expression upon reperfusion that confers cytoprotection.

We identified Kupffer cells as the main site of HO-1 expression in human livers. Makino et al.

(29) have recently reported similar findings in human cirrhotic livers. These studies in human

liver are in contrast with data from rat livers, where considerable expression of HO-1 has also

been found in hepatocytes (46). While in our study about 50% of the Kupffer cells in the control

livers expressed HO-1, this was more than 80% in the liver grafts before transplantation and

even 100% after transplantation. These findings suggest that a subpopulation of Kupffer cells,

which does not express HO-1 under normal circumstances may induce HO-1 expression. It

has been suggested that Kupffer cells may serve as sensor cells detecting local hemodynamic

changes and mechanical forces in sinusoids (29,47). By increasing HO-1 activity and the

generation of the vasorelaxing gaseous CO, Kupffer cells are able to maintain microvascular

blood flow in the liver (29). On the other hand, it is well-known that Kuppfer cells play a

critical role in the pathogenesis of I/R injury of the cold preserved liver through the production

of ROS and cytokines, like tumor necrosis factor-α (48,49). Our data suggests that high

overexpression of HO-1 in Kupffer cells prior to transplantation contributes to the deleterious

effects of these cells in I/R injury.

Although there is a large body of evidence suggesting that exogenous up-regulation of HO-1

in transplant models in animals confers cytoprotective effects (5,32-34), our findings caution

against an uncontrolled application of non-cell specific methods to induce HO-1 expression in

human organ donors. Exogenous induction of HO-1 in postmortem organ donors could further

increase an already elevated HO-1 expression, resulting in potentially detrimental effects

instead of cytoprotection. The main difference between our study in patients undergoing

liver transplantation and studies in animal models of liver transplantation is that in the clinical

situation liver grafts are usually obtained from brain death organ donors, whereas healthy

animals are used as donors in experimental models. Moreover, cellular localization of HO-1

expression in human liver transplantation is predominantly restricted to the Kupffer cells,

whereas in stress-exposed rat livers, HO-1 is also upregulated in hepatocytes (46).

163

Chapter 8

Our data suggest a dual role for HO-1 in human liver transplants, with either cytoportection or

increased cytotoxicity, depending on the initial level of overexpression. New pharmacological

interventions should probably not focus on the induction of HO-1 prior to transplantation, but

rather aim for induction during transplantation.

164


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Carlijn I BuisGerrit van der Steege

Dorien S VisserIlja M Nolte

Bouke G HepkemaMaarten Nijsten

Maarten JH SlooffRobert J Porte

170

HO-1 genotype of the donor and graft survival Chapter 9

Abstract

Heme oxygenase 1 (HO-1) has been suggested as a cytoprotective gene during liver

transplantation. Inducibility of HO-1 is modulated by a (GT)n polymorphism and a single

nucleotide polymorphism (SNP) A(-413)T in the promoter. Both a short (GT)n allele and the

A-allele have been associated with increased HO-1 promoter activity.

In 308 liver transplantations, we assessed donor HO-1 genotype and correlated this with

outcome variables. For (GT)n genotype, livers were divided into two classes: short alleles

(<25 repeats; class-S) and long alleles (≥ 25 repeats; class-L). In a subset, hepatic mRNA

expression was correlated with genotypes.

Graft survival at 1 year was significantly better for A-allele genotype compared to TT-genotype

(84% versus 63%, p=0.004). Graft loss due to primary dysfunction occurred more frequently

in TT-genotype compared to A-receivers (p=0.03). Recipients of a liver with TT-genotype had

significantly higher serum transaminases after transplantation and hepatic HO-1 mRNA levels

were significantly lower compared to the A-allele livers (p=0.03). No differences were found for

any outcome variable between class S and LL-variant of the (GT)n polymorphism. Haplotype

analysis confirmed dominance of the A(-413)T SNP over the (GT)n polymorphism.

In conclusion, HO-1 genotype is associated with outcome after liver transplantation. These

findings suggest that HO-1 mediates graft survival after liver transplantation.

171

Chapter 9

Introduction

Orthotopic liver transplantation (OLT) is the best available treatment for patients with endstage

liver failure. It is well recognized that, during the transplant procedure, livers are exposed to

various stressful stimuli such as ischemia and reperfusion injury. Heme oxygenase 1 (HO-1)

has been shown to provide cytoprotection during liver ischemia and reperfusion. Moreover,

it has been suggested to have an immune modulating effect (1). In various experimental

OLT models, upregulation of HO-1 has been shown to protect livers from I/R injury and to

improve graft survival (2,3). HO-1 catalyzes the oxidative detoxification of excess heme

resulting in equimolar amounts of free iron (Fe2+), biliverdin and carbon monoxide. All products

formed in this process possess potential beneficial effects in the transplant setting. CO has

vasodilatating effects, thereby maintaining microvascular hepatic blood flow (4,5). Biliverdin

and the subsequently formed bilirubin possess potent anti-oxidant effects (6-9). Free iron is

highly reactive by itself, however cellular Fe2+ released via heme degradation up-regulates the

expression of the Fe2+ sequestrating protein ferritin as well as that of an Fe2+ pump, thereby

limiting the amount of free iron and preventing the generation of reactive oxygen species (10-

12).

We previously studied the endogenous regulation of HO-1 during human liver transplantation

and showed a dual role for HO-1, with either cytoprotection or increased cytotoxicity, depending

on the initial level of overexpression (13). However, none of the clinical variables analyzed in

this study could explain the variation in initial expression of HO-1 in the donor livers. We

therefore decided to study the impact of genetic differences on HO-1 expression and outcome

after OLT.

Expression of the HO-1 gene is modulated by two functional polymorphisms in the promoter:

a (GT)n polymorphism and a single nucleotide polymorphism (SNP) (14-19). (GT)

n is the most

frequent of the simple repeats scattered throughout the human genome and many of these

exhibit a length polymorphism (20). Most of these variable sites are not expected to have any

functional effect, since they are located in intragenic regions and introns. However, the HO-1

(GT)n repeat resides in a regulatory sequence and a short (GT)

n allele has been associated

with enhanced transcriptional activity of the gene (14,17-19). In kidney transplantation the

influence of HO-1 (GT)n polymorphism has recently been studied by Baan et. al. (21) and Exner

et. al. (22), who found a positive correlation between a short GT repeat and graft function and

172


survival after transplantation. In addition to the (GT)n polymorphism, the A(-413)T SNP has

been identified as a functionally relevant variation of the HO-1 gene (15,16). Using a transient

transfection assay of HO-1 promoter luciferase genes in bovine aortic endothelial cells Ono et

al have shown that the A-allele of this SNP is associated with a higher promoter activity than

the T-allele. Interestingly, the A(-413)T SNP appeared in vitro to be more important for HO-1

promoter activity than the (GT)n polymorphism (15,16). Only limited work has been conducted

evaluating the A(-413)T SNP in clinical research (15).

Based on the accumulating evidence that HO-1 is an important enzyme influencing graft survival

after transplantation, we hypothesized that these two functionally relevant polymorphisms

in the HO-1 promoter are associated with outcome after OLT. Therefore, we analyzed the

two functional HO-1 promoter polymorphisms in donor genomic DNA in relation to outcome

after human liver transplantation. Furthermore we studied the functional relevance of these

polymorphisms by measuring hepatic mRNA expression.

Patients and methods

Patients

Between January 1996 and January 2005, a total number of 465 consecutive OLT’s were

performed at the University Medical Center Groningen. After exclusion of children (<18

years), 320 transplants in 282 adult patients were included in this study. Of 308 donors (96%)

cryopreserved splenocytes were available for the HO-1 genotyping. Median follow up time for

this cohort was 4 years and 6 months (range 20-81 months).

ABO blood group-identical or compatible grafts from brain-death donors with normal or

near normal liver function tests were used for all patients. A standardized technique was

used for implantation, as has been described previously (23,24). During the study period,

immunosuppressive protocols were based on tacrolimus or cyclosporine A, either with or

without azathioprine and a rapid taper of steroids. Biopsy-proven acute rejection was treated

when clinically indicated, with a bolus of methylprednisolone on three consecutive days.

Steroid-resistant rejections were treated either by conversion to tacrolimus in patients on

cyclosporine A, or by giving 5 doses of antithymocyte globulin (4 mg/kg i.v.) on alternating days.

Doppler ultrasound was performed routinely at postoperative days 1, 3, and 7 and on demand

to rule out vascular or biliary complications or parenchymal lesions. Cholangiography via a

173

Chapter 9

biliary drain was routinely performed between postoperative day 10-14 and later on demand

(i.e. for rising cholestatic parameters or dilatation of bile ducts on ultrasound). Tissue and data

collection was performed according to the guidelines of the medical ethical committee of our

institution and the Dutch Federation of Scientific Societies.

HO-1 genotype assessment

Genomic DNA was isolated from donor splenocytes using a commercial kit (Gentra Systems,

Minneapolis, MN, USA). The 5’-flanking region of the HO-1 gene containing the (GT)n

polymorphism was amplified by polymerase chain reaction (PCR) using as forward primer

5’-CAG CTT TCT GGA ACC TTC TGG-3’ (sense), carrying a 6-FAM fluorescent label (Sigma,

Malden, the Netherlands), and as reversed primer 5’-GAA ACA AAG TCT GGC CAT AG

GAC-3’ (antisense). PCR and genotyping procedures were similar as described earlier (25).

Sequence analysis of the amplification products of individuals homozygous for the 222 and 229

base-pairs alleles showed correspondence with GT numbers 26 and 29, respectively (results

not shown). Allelic repeats were divided into two subclasses using a similar classification

based on transfection studies as described previously (26). A short allele, with less than 25 GT

repeats, were designated as class S, and long allele with 25 or more GT repeats (amplicons

of 220 base-pairs and more), as class L (26). Recipients of class S allele liver transplants

(homozygous SS and heterozygous SL) were compared with recipients of non-class S allele

transplants (LL).

The single nucleotide polymorphism A(-413)T (rs2071746) was analyzed using the ABI7900HT

TaqMan system (Applied Biosystems, Foster City, CA, USA) with a probe/primer assay

hCV15869717, developed by and purchased from Applied Biosytems (Assay-on-Demand).

Recipients of at least one A-allele liver transplants (homozygous AA and heterozygous AT)

were compared with recipients of heterozygous T-allele recipients (TT).

Collection of liver biopsies, RNA isolation and reverse-transcriptase

polymerase chain reaction

In a subset of 38 patients we collected liver biopsies at the end of cold storage to compare

HO-1 mRNA expression in the various genotype groups. RNA isolation and cDNA synthesis

were performed as described before (13). cDNA levels of HO-1 and 18S were measured

by Real Time Polymerase Chain Reaction (PCR) using the ABI PRISM 7900 HT Sequence

174


detector (Applied Biosystems). Nucleotide sequences of Primers (Invitrogen) and Probes

(Eurogentec) were designed using Primes Express software (PE Applied Biosystems). Probes

were 5’ labeled by a 6-carboxy-fluoresceine (FAM) reporter and 3’ labeled with a 6-carboxy-

tetra-methyl-rhodamin (TAMRA) quencher. Real time RT-PCR data were analyzed using the

comparative cycle threshold (CT) method. Briefly, the difference in cycle times, ∆CT, was

determined as the difference between the tested gene and the reference RNA, 18S. We then

obtained ∆∆CT by finding the difference compared to a control group of liver biopsies from

patients undergoing an hemihepatectomy for colorectal metastasis. The fold induction (FI)

was calculated as 2-∆∆CT.

Clinical Outcome parameters

Outcome parameters included serum concentrations of aspartate aminotransferase (AST) and

alanine aminotransferase (ALT), as marker for ischemia / reperfusion injury after OLT, graft

survival, incidence of acute rejection, and causes of graft loss. Recipient data were obtained

from a prospectively collected database. Donor data were extracted from the national and

hospital’s donor databases.

Acute rejection was suspected on the basis of daily liver function tests, fever and deterioration

of the clinical condition and proven by needle biopsy of the liver. The degree of acute rejection

was histologically graded according to the Banff classification (27). Only rejections within

the first three months with grade II and III, or grade I with a clinical indication for treatment,

were considered in this study. As individual causes of graft loss, five different etiologies were

identified: 1) Primary dysfunction (PDF), defined as either primary non function (PNF) or graft

loss due to initial poor function (IPF). PNF was defined as non life sustaining function of the liver

requiring retransplantation or leading to death within seven days after OLT. IPF was defined

as early graft dysfunction characterized by serum AST levels > 2000 U/l on any day between

postoperative day 2-7, and a prothrombine time (PT) >16 sec (modified according to Ploeg

et. al. (28)), which was not explained by biliary or vascular complications; 2) Hepatic artery

thrombosis, which was always confirmed by doppler ultrasonography and or angiography; 3)

Non-anastomotic biliary strictures, as detected on imaging studies of the biliary tree and in

the absence of arterial complications (29); 4) Recurrent disease, and 5) non-graft related graft

loss, including extrahepatic conditions that contributed to the loss of the donor liver, such as

postoperative sepsis and multi-organ failure.

175

Chapter 9

Statistics

All data are reported as median and interquartile ranges (IQR) or number with percentage.

Collection of laboratory values from the central hospital database was conducted as follows.

For every postoperative biochemical variable of each patient a time curve was constructed

before further analysis. In case multiple measurements of a parameter were performed on

one day, these values were averaged to a single value before further analysis. Likewise,

in case laboratory values were missing on certain days, these values were interpolated.

Extrapolations were not performed.

Groups were compared with the chi-square test or Mann Whitney U test, where appropriate.

Biochemical variables were compared using the daily values, but also the total course during

the first two post operative weeks was compared by calculating the area under the curve (AUC),

using the trapezium rule. Graft survival curves were calculated according to the Kaplan-Meier

method and compared using the log-rank test. A two-tailed p-value of < 0.05 was considered

statistically significant. Statistical analyses were performed using SPSS version 12.0.2 (SPSS

Inc., Chicago, IL, USA).

To study linkage disequilibrium between the two polymorphisms in the promoter of the HO-1

gene, the frequencies of the combined genotypes of the (GT)n polymorphism and A(-413)T

were counted. Linkage disequilibrium is the occurrence of two or more polymorphism variants

together on the chromosome more often than could be expected based on recombination

possibilities, most likely due to their close locations, but it may also arise when the combination

confers a selective advantage. A haplotype is a vector of polymorphisms. Haplotype frequencies

were estimated from the genotype counts using the expectation-maximization algorithm (own

software). Linkage disequilibrium is then determined from these haplotype frequencies by

means of D’ and the correlation coefficient R2, which both range from 0 to 1 with 1 implying the

strongest possible linkage disequilibrium and 0 as no linkage disequilibrium.

176


30

40

50

Alle

le f

req

uen

cy (

%)

19 20 21 22 23 24 25 26 27 28 29 30 31 32 34 35 36 38

Number of GT repeats of the HO-1 promoter in 308 liver donors

0

10

20

Alle

le f

req

uen

cy (

%)

Figure 1. Allele distribution of the (GT)n polymorphism in 308 liver donors.

Results

Distribution of HO-1 genotypes in the donor population.

The allelic distribution of the (GT)n polymorphism in the HO-1 promoter of liver donors is given

in figure 1. The distribution of (GT)n is bimodal, with a peak at 22 repeats (22%) and the other

at 29 repeats (45%). Forty two (14%) patients received a liver from a donor homozygous

for class S allele, 130 (42.2%) from a heterozygote (SL) and 136 (44.2%) from a donor

homozygous for the class L allele.

With respect to the T(-413)A SNP, the distribution of the genotypes was as follows: 92 (30%)

patients received a liver from an AA genotype donor, 153 (50%) from an AT genotype donor,

and 61 (20%) from a TT genotype donor, (in two samples genotyping of A(-413)T failed).

There were no significant differences in donor- and recipient characteristics of patients

receiving a liver from a donor with a class S allele (SS or SL) or from a donor without a

class S allele (LL). Also no significant differences were found between the group of patients

who received a liver from a donor with an A-allele (AA or AT) and the group of patients who

received a liver without an A-allele (TT genotype) (table 2).

177

Chapter 9

30

40

50

Alle

le f

req

uen

cy (

%)

19 20 21 22 23 24 25 26 27 28 29 30 31 32 34 35 36 38

Number of GT repeats of the HO-1 promoter in 308 liver donors

0

10

20

Alle

le f

req

uen

cy (

%)

Figure 1. Allele distribution of the (GT)n polymorphism in 308 liver donors.

Results

Distribution of HO-1 genotypes in the donor population.

The allelic distribution of the (GT)n polymorphism in the HO-1 promoter of liver donors is given

in figure 1. The distribution of (GT)n is bimodal, with a peak at 22 repeats (22%) and the other

at 29 repeats (45%). Forty two (14%) patients received a liver from a donor homozygous

for class S allele, 130 (42.2%) from a heterozygote (SL) and 136 (44.2%) from a donor

homozygous for the class L allele.

With respect to the T(-413)A SNP, the distribution of the genotypes was as follows: 92 (30%)

patients received a liver from an AA genotype donor, 153 (50%) from an AT genotype donor,

and 61 (20%) from a TT genotype donor, (in two samples genotyping of A(-413)T failed).

There were no significant differences in donor- and recipient characteristics of patients

receiving a liver from a donor with a class S allele (SS or SL) or from a donor without a

class S allele (LL). Also no significant differences were found between the group of patients

who received a liver from a donor with an A-allele (AA or AT) and the group of patients who

received a liver without an A-allele (TT genotype) (table 2).

Are the two HO-1 polymorphisms in linkage disequlibrium?Haplotype frequencies were estimated from data in table 1A and are presented in table 1B.

The two most prevalent haplotypes were A(-413)_(GT)29 and (-413) T_(GT)22 indicating that

the “favorable” A-allele is in linkage disequilibrium with the “unfavorable” class L genotype.

The linkage disequilibrium measures D’ and R2 were 0.87 and 0.50, respectively indicating

strong linkage disequilibrium between the two promoter polymorphisms. The class L genotype

cosegregates at -413 more often than expected with the A-allele, the same holds for the

combination of the class S genotype and T-allele at -413.

Table 1A. Number of A(-413)T genotypes in each genotype of the (GT)n polymorphism

(GT)n repeat length

polymorphism TT AT AA

(21, 29) 0 5 0

(22, 22) 10 2 0

(22, 23) 13 2 1

(22, 24) 5 0 0

(22, 29) 1 65 3

(22, 36) 5 0 0

(23, 29) 0 12 0

(23, 30) 0 4 0

(24, 29) 0 8 0

(25, 29) 0 4 2

(26, 29) 0 6 5

(28, 29) 0 0 4

(29, 29) 1 4 57

(29, 30) 0 1 7

(29, 36) 0 16 0

Combinations occurring less then 4 times are not shown in this table

178


Table 1B. Estimated haplotype frequencies from table 1A with the EM algorithm. Haplotypes with a frequency

of less than 1% are not shown.

(GT)n number of repeats

repeats -413 Estimated haplotype frequency (%)

21 T 1.3

22 A 1.4

22 T 20.3

23 T 7.2

24 T 3.0

25 T 1.8

26 A 1.2

26 T 1.9

27 A 1.1

29 A 43.2

29 T 1.2

30 A 4.3

36 T 5.4

Is there an association between HO-1 genotypes and mRNA

expression?

In a subgroup of 38 livers, material was available to measure hepatic HO-1 mRNA expression. The

fold induction of the HO-1 mRNA in liver biopsies, retrieved at the end of the cold storage period,

was significantly higher in A-receiver genotype livers compared to the TT-genotype (p=0.03) (figure

2). No difference in mRNA expression was found for the recipients of class S or LL- livers.

Although these findings provide support for a functional relevance of the A(-413)T SNP and

not for the (GT)n polymorphism, they do not demonstrate the dominance of one of these

polymorphisms. Therefore we next studied HO-1 mRNA expression in the various haplotype

combinations (figure3). Within the group of non class S allele transplants (LL), HO-1 mRNA

was higher in livers with an A allele (haplotype A-L, A-L) compared to livers with a T-allele

(haplotype T-L, T-L). A similar comparison of different haplotypes within the group of class S

livers was not possible due to the low frequency of the S-A, S-A haplotype (table 1B).

179

Chapter 9

1 F

old

In

du

cti

on

in

liv

er

bio

ps

y

2

3

P=0.03

(n=38)

HO

-1 F

old

In

du

cti

on

in

liv

er

bio

ps

y

TT genotype A-allele genotype

0

1

Figure 2. Fold induction of the HO-1 gene in biopsies taken at the end of cold storage in a group of 38 patients.

Liver grafts with at least one A-allele had a significantly higher expression of HO-1 mRNA compared to TT geno-

type liver grafts (p=0.03).

Are HO-1 polymorphisms associated with outcome after liver

transplantation?

Survival. In the entire cohort of 308 transplants, overall actuarial graft survival rate at 1 and 5

years was 80% and 71%, respectively. Graft survival rates were significantly better in recipients

of livers with at least one A-allele, compared to recipients of a TT genotype liver; log rank p=0.004

(Figure 4). In addition, within the group of livers with at least one A-allele, there were no differences

between AA and AT genotypes. No differences were found between recipients of class S or LL-

livers. Ischemia / reperfusion injury. Postoperative serum levels of AST and ALT as a marker of

ischemia / reperfusion injury, are presented in Figure 5 A and B. Recipients of a liver with TT

genotype had significantly higher serum transaminase levels, as expressed by the AUC for the

first two weeks (AST (p=0.01, ALT p=0.009)). There were no significant differences in serum AST

or ALT in recipients of a class S liver, compared to recipients of a non class S liver (LL).

180


Table 2. Comparison of donor and recipient characteristics in relation to donor HO-1 genotype.

(GT)n polymorphism A(-413)T SNP *

S-Receiver LL A-Receiver TT

(n = 172) (n = 136) P value (n=245) (n=61) P value

Donor

Donor age (years) 46 (35 - 55) 46 (38 - 53) 0.67 45 (37 - 54) 47 (37 - 55) 0.77

Gender (M/F) 76 / 96 (44% / 56%) 70 /64 (53% / 47%) 0.11 120 / 125 (49% / 51%) 34 / 27 (43% / 57%) 0.43

Laboratory variables**

Hemoglobulin (mmol/L) 7.1 (6.1 - 8.0) 7.3 (6.2 - 8.2) 0.46 7.1 (6.2 - 8.0) 7.2 (6.0 - 8.1) 0.97

Total bilirubin (umol/L) 10 (7 - 16) 11 (7 - 16) 0.90 11 (7 - 16) 12 (7 - 14) 0.45

AST (U/L) 28 (18 - 48) 27 (18 - 44) 0.60 27 (18 - 44) 32 (20 - 54) 0.23

ALT (U/L) 21 (14 - 34) 22 (14 - 42) 0.57 21 (13 - 36) 27 (18 - 46) 0.02

γ-GT (U/L) 20 (11 - 37) 24 (14 - 40) 0.13 21 (12 - 39) 24 (15 - 37) 0.66

AP (U/L) 53 (40 - 72) 55 (42 - 79) 0.33 54 (41 - 76) 49 (36 - 68) 0.25

Dopamine use (n=177) 100 (58%) 77 (57%) 0.92 137 (56%) 40 (66%) 0.56

Blood transfusion 76 (44%) 54 (40%) 0.43 108 (44%) 22 (36%) 0.25

Cause of death 0.07 0.94

Cerebral Vascular Accident 124 (73%) 98 (73%) 177 (73%) 44 (73%)

Trauma 44 (21%) 28 (26%) 57 (23%) 15 (25%)

Miscellaneous 4 (7%) 10 (2%) 11 (10%) 2 (3%)

Recipient

Recipient age (years) 49 (37 - 55) 46 (35-53) 0.08 46 (35 - 55) 49 (38 - 54) 0.33

Gender (M/F) 95 / 77 (55 % / 45%) 71 / 65 (52% / 48%) 0.60 131 / 114 (53% / 47%) 33 / 28 (54% / 46%) 0.93

Diagnosis 0.04 0.76

Cirrhosis 147 (85.5%) 102 (75%) 196 (80%) 51 (84%)

Acute Failure 12 (7%) 11 (8%) 19 (8.5%) 4 (6.5%)

Tumors 1 (.5%) 6 (4.5%) 5 (1.5%) 2 (3%)

Non-cirrhotic 12 (7%) 17 (12.5%) 25 (10%) 4 (6.5%)

MELD Score 15 (10-22) 14 (11-19) 0.52 14 (11-20) 16 (11-24) 0.31

Preservation Solution 0.80 0.39

High viscosity 157 (92%) 125 (93%) 225 (92%) 58 (95%)

Low viscosity 14 (8%) 10 (7%) 20 (8%) 3 (5%)

CIT (minutes) 515 (415 - 640) 526 (439 - 685) 0.13 519 (434 - 643) 531 (411 - 663) 0.99

WIT (minutes) 50 (43 - 62) 45 (45 - 60) 0.40 51 (44 - 60) 50 (42 - 63) 0.83

LOS at ICU 3 (2-10) 3 (2-7) 0.10 3 (2-7) 3 (2-12) 0.54

*) SNP analysis failed for two donors. **) At time of donor procedure

Continuous variables are presented as median and interquartile range, categorical variables as numbers with percentage.

181

Chapter 9

Table 2. Comparison of donor and recipient characteristics in relation to donor HO-1 genotype.

(GT)n polymorphism A(-413)T SNP *

S-Receiver LL A-Receiver TT

(n = 172) (n = 136) P value (n=245) (n=61) P value

Donor

Donor age (years) 46 (35 - 55) 46 (38 - 53) 0.67 45 (37 - 54) 47 (37 - 55) 0.77

Gender (M/F) 76 / 96 (44% / 56%) 70 /64 (53% / 47%) 0.11 120 / 125 (49% / 51%) 34 / 27 (43% / 57%) 0.43

Laboratory variables**

Hemoglobulin (mmol/L) 7.1 (6.1 - 8.0) 7.3 (6.2 - 8.2) 0.46 7.1 (6.2 - 8.0) 7.2 (6.0 - 8.1) 0.97

Total bilirubin (umol/L) 10 (7 - 16) 11 (7 - 16) 0.90 11 (7 - 16) 12 (7 - 14) 0.45

AST (U/L) 28 (18 - 48) 27 (18 - 44) 0.60 27 (18 - 44) 32 (20 - 54) 0.23

ALT (U/L) 21 (14 - 34) 22 (14 - 42) 0.57 21 (13 - 36) 27 (18 - 46) 0.02

γ-GT (U/L) 20 (11 - 37) 24 (14 - 40) 0.13 21 (12 - 39) 24 (15 - 37) 0.66

AP (U/L) 53 (40 - 72) 55 (42 - 79) 0.33 54 (41 - 76) 49 (36 - 68) 0.25

Dopamine use (n=177) 100 (58%) 77 (57%) 0.92 137 (56%) 40 (66%) 0.56

Blood transfusion 76 (44%) 54 (40%) 0.43 108 (44%) 22 (36%) 0.25

Cause of death 0.07 0.94

Cerebral Vascular Accident 124 (73%) 98 (73%) 177 (73%) 44 (73%)

Trauma 44 (21%) 28 (26%) 57 (23%) 15 (25%)

Miscellaneous 4 (7%) 10 (2%) 11 (10%) 2 (3%)

Recipient

Recipient age (years) 49 (37 - 55) 46 (35-53) 0.08 46 (35 - 55) 49 (38 - 54) 0.33

Gender (M/F) 95 / 77 (55 % / 45%) 71 / 65 (52% / 48%) 0.60 131 / 114 (53% / 47%) 33 / 28 (54% / 46%) 0.93

Diagnosis 0.04 0.76

Cirrhosis 147 (85.5%) 102 (75%) 196 (80%) 51 (84%)

Acute Failure 12 (7%) 11 (8%) 19 (8.5%) 4 (6.5%)

Tumors 1 (.5%) 6 (4.5%) 5 (1.5%) 2 (3%)

Non-cirrhotic 12 (7%) 17 (12.5%) 25 (10%) 4 (6.5%)

MELD Score 15 (10-22) 14 (11-19) 0.52 14 (11-20) 16 (11-24) 0.31

Preservation Solution 0.80 0.39

High viscosity 157 (92%) 125 (93%) 225 (92%) 58 (95%)

Low viscosity 14 (8%) 10 (7%) 20 (8%) 3 (5%)

CIT (minutes) 515 (415 - 640) 526 (439 - 685) 0.13 519 (434 - 643) 531 (411 - 663) 0.99

WIT (minutes) 50 (43 - 62) 45 (45 - 60) 0.40 51 (44 - 60) 50 (42 - 63) 0.83

LOS at ICU 3 (2-10) 3 (2-7) 0.10 3 (2-7) 3 (2-12) 0.54

*) SNP analysis failed for two donors. **) At time of donor procedure

Continuous variables are presented as median and interquartile range, categorical variables as numbers with percentage.

182


Acute rejection. The overall incidence of clinically relevant acute rejection within the first three

months after OLT was 34%. There were no statistically significant differences in the incidence of

acute rejection between any of the genotypes. Moreover, no differences were found in the severity

of rejection among the different genotype groups (Table 3).

1,5

2

2,5

3

1 F

old

In

du

cti

on

in

liv

er

bio

ps

y

Haplotype combinations

0

0,5

1

(T-L,T-L) (A-L,A-L)

HO

-1 F

old

In

du

cti

on

in

liv

er

bio

ps

y

Figure 3. HO-1 m RNA expression in relation to HO-1 haplotypes. Within the group of LL livers, HO-1 mRNA

expression was higher when the LL allele variant was combined with two A-alleles, compared to LL allele carriers

combined with two T-alleles.

183

Chapter 9

Acute rejection. The overall incidence of clinically relevant acute rejection within the first three

months after OLT was 34%. There were no statistically significant differences in the incidence of

acute rejection between any of the genotypes. Moreover, no differences were found in the severity

of rejection among the different genotype groups (Table 3).

1,5

2

2,5

3

1 F

old

In

du

cti

on

in

liv

er

bio

ps

y

Haplotype combinations

0

0,5

1

(T-L,T-L) (A-L,A-L)

HO

-1 F

old

In

du

cti

on

in

liv

er

bio

ps

y

Figure 3. HO-1 m RNA expression in relation to HO-1 haplotypes. Within the group of LL livers, HO-1 mRNA

expression was higher when the LL allele variant was combined with two A-alleles, compared to LL allele carriers

combined with two T-alleles.

Tab

le 3

. In

cid

ence

of

acu

te r

ejec

tio

n w

ith

in t

he

firs

t th

ree

mo

nth

s af

ter

OLT

in r

elat

ion

to

do

no

r H

O-1

gen

oty

pe.

(GT)

n pol

ymor

phis

mA(

-413

)T S

NP

*

S-R

ecei

ver

LL

A-

Rec

eive

r TT

(n =

172

)

(n

= 1

36)

P

valu

e

(n=2

45)

(n=6

1)

P va

lue

Acu

te R

ejec

tion

60(3

5%)

44(3

2%)

0.64

82(3

4%)

22(3

6%)

0.70

Gra

de**

0.55

0.26

I15

(25%

)15

(35%

)

24(3

0%)

6(2

9%)

II32

(55%

)20

(47%

)

44(5

5%)

8(3

8%)

III11

(19%

)

8(1

9%)

12(1

5%)

7

(33%

)

*) S

NP

anal

ysis

faile

d fo

r tw

o do

nors

.

**) G

rade

s of

reje

ctio

n ac

cord

ing

to th

e BA

NFF

cla

ssifi

catio

n. F

or th

ree

patie

nts

no h

isto

logi

cal d

ata

wer

e av

aila

ble.

Cat

egor

ical

var

iabl

es a

s nu

mbe

rs w

ith p

erce

ntag

e.

Tab

le 4

. Cau

ses

of

gra

ft lo

ss g

rou

ped

by

do

no

r H

O-1

gen

oty

pe

(GT)

n pol

ymor

phis

mA(

-413

)T S

NP

*

S-R

ecei

ver

LL

A-R

ecei

ver

TT

(n =

172

)

(n =

136

)

P va

lue

(n=2

45)

(n

=61)

P

valu

e

Prim

ary

dysf

unct

ion

6(3

%)

5(4

%)

0.84

5(2

%)

6(1

0%)

0.03

Hep

atic

arte

ry th

rom

bosi

s7

(4%

)5

(4%

)0.

948

(3%

)4

(7%

)0.

66

Non

ana

stom

otic

bila

ry s

trict

ures

4(2

%)

5(4

%)

0.41

7(3

%)

2(3

%)

0.68

Rec

urre

nt d

isea

se4

(2%

)5

(4%

)0.

418

(3%

)1

(2%

)0.

23

Not

gra

ft re

late

d25

(15%

)18

(13%

)0.

8833

(13%

)10

(16%

)0.

33

Mis

cella

neou

s 5

(3%

)0

0.

053

(3%

)2

(3%

)0.

54

*) S

NP

anal

ysis

faile

d fo

r tw

o do

nors

.

184


Causes of graft loss. The number of grafts lost in patients receiving a liver with a S-allele

was 50 (29%), the number of grafts lost in patients receiving a liver with LL genotype was 37

(27%). The number of grafts lost in patients receiving a liver with an A-allele was 62 (25%), the

number of grafts lost in patients receiving a liver with TT genotype was 25 (41%) (p=0.004).

To find an explanation for the observed differences in overall graft survival in relation to the

A(-413)T SNP, we next examined the individual causes of graft loss (Table 4). Primary graft

dysfunction was a significantly more frequent cause of graft loss in the group of TT-genotype

livers (10%) compared to livers with an A-allele (2%); odds ratio 3.73 (95% Confidence interval

1.02 to 13.60; p=0.03). For the other most common causes of graft loss, including hepatic

artery thrombosis, non anastomotic biliary strictures, recurrent disease and non graft related

causes, no significant differences were found in the distribution among the different genotypes

(Table 4).

100

80

60

Ac

tua

ria

l g

raft

su

rviv

al

(%)

A-allele genotype

4003002001000

Days post OLT

40

20

0

Ac

tua

ria

l g

raft

su

rviv

al

(%)

TT genotype

A-allele genotype

Log-rank p = 0.004

Figure 4. Kaplan Meier 1-year survival curve for liver grafts in relation to donor HO-1 A(-413)T SNP. Log-rank test

for livers with an A-allele (AA or AT genotype) versus no A-allele (TT genotype): p-value = 0.004.

185

Chapter 9

Causes of graft loss. The number of grafts lost in patients receiving a liver with a S-allele

was 50 (29%), the number of grafts lost in patients receiving a liver with LL genotype was 37

(27%). The number of grafts lost in patients receiving a liver with an A-allele was 62 (25%), the

number of grafts lost in patients receiving a liver with TT genotype was 25 (41%) (p=0.004).

To find an explanation for the observed differences in overall graft survival in relation to the

A(-413)T SNP, we next examined the individual causes of graft loss (Table 4). Primary graft

dysfunction was a significantly more frequent cause of graft loss in the group of TT-genotype

livers (10%) compared to livers with an A-allele (2%); odds ratio 3.73 (95% Confidence interval

1.02 to 13.60; p=0.03). For the other most common causes of graft loss, including hepatic

artery thrombosis, non anastomotic biliary strictures, recurrent disease and non graft related

causes, no significant differences were found in the distribution among the different genotypes

(Table 4).

100

80

60

Ac

tua

ria

l g

raft

su

rviv

al

(%)

A-allele genotype

4003002001000

Days post OLT

40

20

0

Ac

tua

ria

l g

raft

su

rviv

al

(%)

TT genotype

A-allele genotype

Log-rank p = 0.004

Figure 4. Kaplan Meier 1-year survival curve for liver grafts in relation to donor HO-1 A(-413)T SNP. Log-rank test

for livers with an A-allele (AA or AT genotype) versus no A-allele (TT genotype): p-value = 0.004.

TT genotype

A-allele genotype

Seru

m A

ST

level (U

/l)

Postoperative day

0

200

400

600

800

1000

1200

1400

1600

1 2 3 4 5 6 7 8 9 10 11 12 13 14

** ** * ** ** *

AUC p = 0.01A

* P < 0.05

** P < 0.01

Postoperative day

Seru

m A

LT

level (U

/l)

Postoperative day

AUC p = 0.009B

0

200

400

600

800

1000

1200

1400

1600

1 2 3 4 5 6 7 8 9 10 11 12 13 14

* * * * **

Figure 5.

A. Serum levels of AST in the first two weeks after OLT. On day 8,9,11- 14, recipients of a liver with at least one

A-allele had significant lower AST levels. Total course during the first two weeks, calculated by the area under

the curve, was significantly lower in liver grafts with at least one A-allele (p=0.01).

B. Serum levels ALT in the first two weeks after OLT. On day 9 – 14, recipients of a liver with at least one A-allele

had significant lower ALT levels. Total course during the first two weeks, as calculated by the area under the

curve, was significantly lower in liver grafts with at least one A-allele (p<0.01).

186


Discussion

In this study we have examined the relationship between two functionally relevant

polymorphisms in the promoter of the HO-1 gene in the donor and postoperative outcome

in a large cohort of 308 liver transplant recipients. There are three novel findings in this

study. Firstly we observed significantly worse outcome in patients receiving a liver from a TT

genotype donor, compared to recipients from donors with at least one A allele. Secondly, we

have shown that the A(-413)T SNP and the (GT)n polymorphism are in linkage disequilibrium

with each other in this predominantly Caucasian population. Finally, we have shown, for the

fist time in a human population, the differences in functional relevance of these two HO-1

promoter polymorphisms. Our association study of the various haplotypes and actual HO-1

mRNA expression suggests that the A(-413)T SNP is of greater functional relevance than the

(GT)n polymorphism. No differences were found in any outcome parameter between the class

S and the LL-receivers of the (GT)n polymorphism. The power of this study with an overall

sample size of 308 subjects was greater than 80% to detect a difference of 13% in graft

survival at the statistical significant lever of 5%.

An association with functional polymorphisms of the HO-1 gene and clinical outcome parameters

has also been found in other pathological conditions, such as pulmonary emphysema, certain

cardiovascular diseases and malignancies (14,19,30-35). With respect to transplantation, two

groups have previously reported an association between HO-1 polymorphism in the donor

and outcome after kidney transplantation (21,22). Baan et al and Exner et al have shown

a positive correlation between the presence of a short (GT)n allele in the HO-1 promoter

and a favorable outcome after kidney transplantation (21,22). Although our data and the

two studies in kidney transplant recipients all point towards a critical role for the HO-1 / CO

pathway in maintaining graft function after solid organ transplantation, in detail the studies

are different. The two studies in kidney transplantation revealed an association between the

(GT)n polymorphism and outcome after transplantation, whereas we found an association

with the A(-413)T SNP. Unfortunately, this SNP was not tested in the two previous studies in

kidney transplant recipients. Moreover, a third large genetic association study between the

(GT)n polymorphism and outcome after kidney transplantation did not provide evidence for

a protective effect of class S alleles on kidney graft survival (36). The linkage disequilibrium

between the short (GT)n variant and the T-allele at -413 in the current study, in combination

187

Chapter 9

with the known dominant effect of the A(-413)T SNP in relation to the (GT)n polymorphism

(16), could possibly explain the inconsistent results of studies in kidney transplant recipients

focusing on the (GT)n polymorphism only. It could well be that HO-1 expression has actually

been lower in kidney grafts with a short (GT)n allele. Unfortunately, tissue levels of HO-1

mRNA, as a marker of actual HO-1 gene expression, were not measured in the three studies

in kidney transplantation.

In our study population, which mainly consisted of Caucasians, we found the A(-413)T

SNP and the (GT)n polymorphism within the promoter of the HO-1 gene to be in linkage

disequilibrium. The two most frequent haplotypes were the A-allele at -413 in combination

with a long (29) (GT)n allele (43.2%) and the T-allele at -413 in combination with a short (22)

(GT)n allele (20.3%). This finding is in accordance with results from previous studies in

Japanese populations (15,16). Theoretically, these combinations are counterproductive, as

the A-allele at -413 and a short (GT)n allele are both associated with enhanced expression

of HO-1, whereas the T-allele and a long (GT)n allele are associated with reduced HO-1

expression. Our data, however, consistently point towards a dominant effect of the A(-413)

T SNP over the (GT)n polymorphism. Not only clinical outcome parameters but also hepatic

HO-1 mRNA correlated with the A(-413)T SNP, but not with the (GT)n polymorphism. To

our knowledge this is the first study in humans suggesting an association between mRNA

expression and the various HO-1 haplotypes. Similar observations have been made by Ono et

al. who have studied the functional role of the A(-413)T SNP and the (GT)n polymorphism in an

in vitro system of bovine aortic endothelial cells, using a luciferase reporter assay (15). These

investigators suggested that, with respect to HO-1 promoter activity, the A(-413)T SNP is

dominant over the (GT)n polymorphism. In a previous study we have shown the HO-1 mRNA

expression correlates well with protein expression in human livers (13).

The exact mechanisms explaining the clinical observations in this study are incompletely

understood. Experimental studies have previously shown that upregulation of HO-1 protects

liver grafts against ischemia / reperfusion injury and improves graft survival (2,3,37-39)

Especially, steatotic livers, which are highly sensitive to ischemic injury, seem effectively

protected against this type of injury by induction of HO-1 (37,40). The inferior outcomes of livers

with the unfavorable TT genotype (associated with a reduced HO-1 promoter activity) in the

current study supports these previous findings. The effect of HO-1 genotype on graft survival

could, at least partly, be explained by a higher incidence of PDF in livers with a TT-genotype,

188


compared to livers with an A-allele (p=0.03). However, when the Kaplan Meier curves are

carefully observed, the lines started to separate from day 25 and were further divergent later

after transplantation. In accordance with this, the differences in serum transaminases became

more pronounced in the second week after transplantation. These observations suggest that

the observed differences in graft survival are not only explained by differences in ischemia

/ reperfusion injury, but also result from other mechanisms. In fact, the absolute number

of grafts lost due to PDF is relatively small, again suggesting that other factors have also

contributed to the observed differences in graft survival. Apparently, the impact HO-1 is not

limited to the early postoperative period. We speculate that other, possibly immune-mediated

processes, could explain the more late effects of HO-1 on graft survival. Several studies have

shown that HO-1 is a key enzyme in certain immune processes. Nevertheless, we observed

no differences in the incidence or severity of acute rejection between the various genotypes.

However, it would be of interest to study HO-1 mRNA expression in donor livers more long-

term after transplantation and to see if differences in HO-1 expression persist. Unfortunately,

we had no access to repeated biopsies during long term follow-up after OLT. More studies

on the mechanisms underlying the more long-term effects of HO-1 on graft survival will be

needed.

In conclusion, in this large series of 308 liver transplant recipients, we found an association

between donor HO-1 genotype and outcome after liver transplantation. Livers with at least

one A-allele of the A(-413)T SNP had significantly better graft survival rate and a lower rate of

PDF than livers with the TT genotype. In addition, our data indicate a functional dominance

of the A(-413)T SNP over the (GT)n polymorphism. These data suggest that HO-1 is critically

involved in maintaining graft function during and after liver transplantation.

189

Chapter 9

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Summary, general discussion and future perspectives10

194

Summary, discussion and future perspectives Chapter 10

Summary

Chapter 1 provides a short introduction of the facts and figures in liver transplantation.

Furthermore, the aims of this thesis are discussed. These aims were to evaluate the molecular

and biochemical mechanisms of bile duct injury after liver transplantation.

In Chapter 2 the literature regarding the causes and consequences of non-anastomotic

strictures (NAS) is reviewed. The aim of this chapter was to describe the current knowledge

about the pathophysiological mechanisms, the clinical presentation, and the treatment of

NAS. NAS is a radiological diagnosis, characterized by intrahepatic strictures and dilatations

on a cholangiogram. NAS were first described after liver transplantation in association with

hepatic artery thrombosis (HAT). In case of early HAT after liver transplantation the biliary tree

becomes ischemic and eventually necrotic, resulting in a typical cholangiographic picture of

biliary strictures, dilatations and intraductal cast formation. However, these cholangiographic

abnormalities of strictures and dilatations can also be seen in patients who do not have a

hepatic artery thrombosis, so the term ischemic-type biliary lesions emerged. In this thesis the

term NAS was used to describe intrahepatic biliary strictures and dilatations in the confirmed

absence of HAT. The incidence of NAS varies around 15% in different series. Several risk

factors for NAS have been identified, strongly suggesting a multifactorial origin. Main categories

include ischemia related injury, immunological induced injury and cytotoxic injury by bile salts.

However, in many cases no specific risk factor can be identified. The clinical presentation of

patients with NAS is often not specific. Symptoms may include fever, abdominal complaints and

increased cholestatic liver function tests. The diagnosis is made by imaging studies of the bile

ducts. Treatment starts with relieving symptoms of cholestasis and dilatation of the stenosed

bile ducts by endoscopic retrograde cholangiopancreaticography (ERCP) or percutaneous

transhepatic cholangiodrainage (PTCD), if possible followed by stenting. Eventually up to

50% of the patients with NAS will require a re-transplantation or may die. In selected cases, a

re-transplantation can be avoided or delayed by surgical intervention.

In the clinical study described in Chapter 3 we aimed to identify clinical risk factors for the

development of NAS after liver transplantation. A total of 487 adult liver transplants with a

median follow-up of 7.9 years were studied. All imaging studies of the biliary tree were reviewed.

195

Chapter 10

Localization of NAS at first presentation was categorized into 4 anatomical zones of the biliary

tree. Severity of NAS was semiquantified as mild, moderate, or severe. NAS developed in 81

livers (16.6%). In 85% of the cases, anatomical localization of NAS was around or below the

bifurcation of the common bile duct. The severity of biliary strictures was classified as mild

in 43 (55%) and as moderate to severe in 35 (45%) of the cases. The cumulative incidence

of moderate to severe NAS in the entire population of liver transplant recipients was 7.3%.

A large variation was observed in the time interval between liver transplantation and first

presentation of NAS (median 4.1 months; range 0.3-155 months). NAS presenting early (<1

year) after liver transplantation were associated with preservation related risk factors. Cold and

warm ischemia times were significantly longer in patients with early NAS compared with NAS

presenting late (>1 year) after transplantation, and early NAS were more frequently located in

the central bile ducts. NAS presenting late after transplantation were more frequently found

in the periphery of the liver and were more frequently associated with immunological factors,

such as primary sclerosing cholangitis as the indication for liver transplantation. By separating

cases of NAS on the basis of the time of presentation after transplantation, we were able to

identify differences in risk factors, indicating different pathogenic mechanisms depending on

the time of initial presentation.

The population of patients suffering from NAS as described in Chapter 3 is further studied

in Chapter 4. The aim of this particular study was to describe the treatment, and identify risk

factors for radiological progression of bile duct abnormalities, recurrent cholangitis, biliary

cirrhosis and retransplantation in patients with NAS. Progression of disease was noted in

68% of cases in whom follow-up radiology was available. Radiological progression was more

common in patients with early NAS (≤ 1 year) and with one or more episodes of bacterial

cholangitis, and less prevalent in patients with extrahepatic biliary abnormalities. Recurrent

bacterial cholangitis (3 or more episodes) was more frequently seen in patients with a Roux-

en-Y anastomosis. Severe fibrosis or cirrhosis developed in 23 cases, especially in cases with

peripheral biliary abnormalities. Graft survival, but not patient survival, was influenced by the

presence of NAS. Thirteen patients (16%) were retransplanted for NAS. The conclusion of the

study is that especially patients with a hepatico-jejunostomy, those with an early diagnosis of

NAS, and those with NAS presenting at the level of the peripheral branches of the biliary tree,

are at risk for progressive disease with severe outcome.

196


In Chapter 5 we took a closer look at the role of bile composition in the development of bile duct

injury after liver transplantation, in a porcine model of non heart-beating liver transplantation.

After non-heart-beating (NHB) liver transplantation, the occurrence of NAS is a serious and

often encountered complication. Bile salt toxicity has been identified as an important factor

in the pathogenesis of bile duct injury and cholangiopathies in general. The role of bile salt

toxicity in the development of biliary strictures after NHB liver transplantation was, however,

unclear. In a porcine model of NHB liver transplantation, we studied the effect of different

periods of warm ischemia in the donor on bile composition and subsequent bile duct injury after

transplantation. After induction of cardiac arrest in the donor, liver procurement was delayed

for 0 min (group A), 15 min (group B), and 30 min or more (group C). Subsequently livers were

transplanted after 4 hr of cold preservation. In the recipients, bile flow was measured, and bile

samples were collected daily to determine the phospholipids-to-bile salt ratio. Severity of bile

duct injury was semi quantified by using a histological grading scale.

Survival after transplantation was directly related to the duration of warm ischemia in the

donor. The phospholipids-to-bile salt ratio in bile produced early after transplantation was

significantly higher in group C, compared with group A and B. Histopathologic examination

showed the highest degree of bile duct injury in group C. Based on these results, it was

concluded that prolonged warm ischemia in NHB donors is associated with the formation of

toxic bile after transplantation, characterized by a low biliary phospholipids-to-bile salt ratio.

These data suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury

after NHB liver transplantation.

The previous chapter, as well as other studies from our group, have indicated that bile

formation early after liver transplantation may be disturbed, resulting in more cytotoxic bile

with a relatively low phospholipids-to-bile salt ratio. It was unknown whether bile toxicity is

also involved in the pathogenesis of NAS, a disease of the larger bile ducts. If bile composition

is involved in the pathogenesis of NAS, one would expect that the bile composition in the

first week after liver transplantation is different in those patients who will develop NAS than

in patients who will not develop NAS. We tested this hypothesis in a prospective clinical

study, described in Chapter 6. In this study, bile production and composition within one week

after liver transplantation were correlated with the subsequent development of NAS in a large

cohort of adult liver transplant recipients. In 111 adult liver transplants bile samples were

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Chapter 10

collected daily after transplantation for determination of bile composition. Expression of bile

transporters was studied perioperatively. NAS were detected in 14 patients (13%) within

one year after transplantation. Patient and donor characteristics and postoperative serum

liver enzymes were similar between patients who developed NAS and those who did not.

Secretions of bile salts, phospholipids and cholesterol were significantly lower in patients

who developed NAS. In parallel, biliary phospholipids-to-bile salt ratio was lower in patients

developing NAS, suggestive for increased bile cytotoxicity. There were no differences in bile

salt pool composition or in hepatobiliary transporter mRNA expression.

Although patients who develop NAS were initially clinically indiscernible from patients who

did not develop NAS, the biliary bile salts and phospholipids secretion, as well as biliary

phospholipids-to-bile salt ratio in the first week after transplantation, was significantly lower in

the former group. This supports the concept that bile cytotoxicity is involved in the pathogenesis

of NAS.

In the previous chapter we have shown that altered bile composition, with a lower phospholipids-

to-bile salt ratio is associated with NAS after liver transplantation. Hepatobiliary transporter

proteins are responsible for the biliary secretion of phospholipids and bile salts. Aim of Chapter

7 was to assess whether variations in the genes in the donor encoding for these transporters

are associated with the occurrence of NAS in the recipient. Without transplantation, genetic

variations itself may not result in bile duct injury. However, early after transplantation, when

the graft is still recovering from I/R injury, these variations might be a critical second factor in

the sequence of events leading to bile duct injury. A similar phenomenon can be found in other

diseases, such as intrahepatic cholestasis of pregnancy (ICP), where patients with a genetic

variation in hepatobiliary transporters display an abnormal phenotype only during pregnancy.

Of 458 procedures in adults, cryopreserved splenocytes were available form the donors and

used for genotyping. The following genes were studied: bile salt export pump (ABCB11),

transporter of phospholipids (ABCB4) and transporter of glutathione and bilirubin (ABCC2).

Four to five tagging single nucleotide polymorphisms (SNPs) with an equal physical

distribution per gene were selected using HapMap data. Haplotypes were constructed using

an Expectation-Maximization algorithm to estimate haplotype frequencies. NAS was detected

in 77 patients (16%) after transplantation. Patients who received a donor liver with ABCB4

haplotype AGGTA developed NAS almost twice as often (28%) as donor livers with other

198


haplotypes (15%) (p=0.007). Analysis in a multivariate Cox regression model showed AGGTA

haplotype of ABCB4 from the donor to be an independent risk factor for NAS (p=0.004,

OR=2.23, 95% CI= 1.29 – 3.85). ABCB11 and ABCC2 haplotypes or single SNPs, were not

associated with NAS.

These data indicate that a common haplotype in the transporter of phospholipids (ABCB4)

in donor livers is independently associated with a two-fold increased risk for NAS after liver

transplantation. Transport of phospholipids into the bile in livers which are carriers of this risk

haplotype might be altered in the time period early after transplantation.

Upregulation of heme oxygenase-1 (HO-1) has been considered an adaptive and protective

mechanism against ischemia/reperfusion (I/R) injury. In Chapter 8 we studied the role of

endogenous HO-1 expression in human liver transplants in relation to early postoperative

hepatobiliary injury and dysfunction. Before transplantation, median HO-1 mRNA levels were

3.4-fold higher (range: 0.7–9.3) in donors than in normal controls. Based on the median value,

livers were divided into two groups: low and high HO-1 expression. There were no differences

in donor characteristics, donor serum transaminases or cold ischemia time between the two

groups. Postoperatively, however, serum transaminases were significantly lower and the

bile salt secretion was higher in the group with an initial low HO-1 expression, compared to

the high expression group. Immunofluorescence staining identified Kupffer cells as the main

localization of HO-1.

To study possible effects of HO-1 induction upon reperfusion, we categorized groups based

on the ability to increase HO-1 expression during reperfusion of the liver graft. In this analysis,

serum AST levels immediately after liver transplantation were significantly lower in the group

with an increase in HO-1 expression compared to livers without upregulation of HO-1 upon

reperfusion. These findings suggest that the ability to induce HO-1 expression at the time of

graft reperfusion may confer hepatobiliary protection. Further research will be necessary to

determine which is more important: a low expression of HO-1 before liver transplantation, or

the ability to induce HO-1 at the time of graft reperfusion.

In the previous chapter, the endogenous regulation of HO-1 during human liver transplantation

was studied. None of the clinical variables analyzed in this study could explain the variation in

initial expression of HO-1 in the donor livers. We therefore hypothesized that genetic variations

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Chapter 10

may be responsible for the differences in HO-1 expression and subsequent outcome after

liver transplantation. The inducibility of HO-1 is modulated by a (GT)n polymorphism and a

single nucleotide polymorphism (SNP) A(-413)T in the promoter. Both a short (GT)n allele and

the A-allele have been associated with increased HO-1 promoter activity. In Chapter 9, a study

is described in which HO-1 genotype in the donor was tested and correlated with outcome in

308 adult patients. For (GT)n genotype, livers were divided into two classes: short alleles (<25

repeats; class-S) and long alleles (≥ 25 repeats; class-L). For the A(-413)T SNP, livers were

grouped as A-carriers (AT or AA) versus TT-genotype livers. In a subset of each group, hepatic

mRNA expression was correlated with genotypes. Graft survival at 1 year was significantly

better for A-allele genotype compared to TT-genotype (84% versus 63%, p=0.004). Graft loss

due to primary dysfunction occurred more frequently in TT-genotype compared to A-receivers

(p=0.03). No differences were found for the occurrence of NAS in both groups. Recipients of

a liver with TT-genotype had significantly higher serum transaminases after transplantation.

Hepatic HO-1 mRNA levels were significantly lower in TT genotype livers compared to the

A-allele livers (p=0.03). No differences were found for any outcome variable between class S

and LL-variant of the (GT)n polymorphism. Haplotype analysis indicated the dominance of the

A(-413)T SNP over the (GT)n polymorphism.

The main conclusion of this study was that the HO-1 promoter polymorphism A(-413)T is

associated with outcome after liver transplantation. The TT variant is linked with worse graft

survival, more primary dysfunction, increased I/R injury and reduced HO-1 mRNA levels.

Furthermore we provided evidence for a greater functional relevance of the A(-413)T SNP

over the (GT)n polymorphism.

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General discussion and future perspectives

Part I: Non-anastomotic biliary complications after liver trans-

plantation

The specific aims of this section were to describe the various forms of NAS and the accompanying

clinical risk factors as well as to study the risk factors for progression of NAS. We found a

difference in risk factors for NAS presenting early (≤1 year) and NAS presenting late (>1 year)

after transplantation. NAS occurring early after transplantation were correlated with prolonged

ischemia times. NAS occurring late after transplantation were more strongly associated with

immunological risk factors. These data suggest that there are different subtypes of NAS that

have different etiologies. This aspect should be considered in future studies.

The following groups of patients were found to have an increased risk for disease progression:

patients with a hepaticojejunostomy, those with an early diagnosis of NAS, and those with

NAS presenting at the level of the peripheral branches of the biliary tree. In clinical practice it

is important to identify these patients for a close follow up and early intervention.

Our newly proposed classification system for NAS is a promising tool to better classify patients

with NAS. However, to become useable and successful in the currently expanding international

field of liver transplantation, our system should be validated. This would enable us to confirm

our findings on the relevance of the localization of NAS and subsequent consequences

for prognosis and management. Aligning many different international centres with different

protocols, facilities and expertise for a prospective study into this classification system might

be complex and time consuming. Therefore and second best, this validation could be achieved

by retrospectively studying other cohorts of liver transplant patients by reviewing the images

of the biliary tree and correlating the classification with risk factors and level of progression.

It is likely that recurrent PSC may have been accountable for the occurrence of late NAS in a

number of patients. On the basis of radiological evaluation, however, recurrent PSC cannot

be distinguished from a late presentation of NAS. Although some of our patients fit well within

the definition of recurrent PSC, more than half of our patients who presented with NAS late

after transplantation were not transplanted for PSC. In an attempt to reduce the occurrence of

early NAS, it remains important to focus on a further reduction of ischemic times, in particular

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Chapter 10

the cold ischemia time. However, many centres have already put a lot of effort in this, and

it is questionable whether a substantial further reduction of cold ischemic time is feasible.

New perspectives in preservation of the liver graft might realize these assiduously sought-

after improvements of graft quality. Maintaining organ viability via (normothermic) machine

perfusion during preservation might be effective in reducing postoperative bile duct injury.

The central concept behind (normothermic) perfusion is to maintain normal function of the

liver during the whole period of preservation and enable immediate graft function and protect

the vulnerable biliary epithelial cells from I/R injury. Currently great efforts are being taken to

better understand the concepts of machine perfusion, as well as to find the ideal preservation

fluid and to create possibilities to implement machine perfusion in daily practice (1).

Part II: Bile physiology after liver transplantation

The specific aims of this second section were to evaluate the contribution of bile composition to

the development of bile duct injury. We found supporting evidence that toxic bile, characterized

by a low phospholipids-to-bile salt ratio, contributes to the development of bile duct injury, not

only at a microscopic level but also at a macroscopic level, like NAS.

The questions whether bile duct injury and toxic bile composition are not just two consequences

of the same underlying factor, has been studied previously by our group. Using mice

heterozygous for disruption of the Mdr2 gene (equivalent of human MDR3), Hoekstra et al.

confirmed that there is indeed a cause-effect relationship between toxic bile formation and bile

duct injury after liver transplantation and ruled out the possibility that toxic bile composition

and bile duct injury both result from the same underlying factor (2). In this study it was

demonstrated that endogenous bile salts act synergistically with I/R in the origin of bile duct

injury in vivo.

The question of which role toxic bile plays in bile duct injury is of great interest. What is

exactly happening on a cellular level? What is happening on epithelial level? What is the

sequence of events before the epithelial cells are damaged so severely that we can detect

it by radiological examination? Fickert et al proposed a very appealing mechanism similar to

the pathogenesis of primary sclerosing cholangitis in humans. They stated that due to a lack

of phospholipids the nonmiccellar-bound, free bile acids might damage the tight junctions and

basement membranes of the epithelial lining, leading to leakage of potentially toxic bile acids

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into the periductal area. As a result the inflammatory response is induced, ultimately resulting

in fibrosis and narrowing of the biliary ducts (3).

Therapeutic strategies to modify intrahepatic cholestasis and to prevent bile duct injury after

OLT may include the administration of the hydrophilic bile salt ursodeoxycholic acid. Daily oral

administration of ursodeoxycholic acid is a well-known therapy to reduce bile salt toxicity by

replacement of the hydrophobic bile salts in the bile salt pool (4,5). Although the exact mechanisms

underlying its cytoprotective effect are not fully understood, it may reduce bile salt–induced injury

by replacing the toxic hydrophobic biliary bile salts. In addition, it has been shown to stimulate

cannalicular transport and biliary excretion, enhancing bile flow and reducing the exposure time

of biliary epithelium to toxic bile salts (4). The potentially beneficial effects of ursodeoxycholic

acid make this drug an interesting strategy to prevent NAS. Current experimental and clinical

research provides strong support for a prospective clinical trial focussing on the abilities of

ursodeoxycholic acid to prevent NAS early after liver transplantation.

Another interesting therapeutic target could be the MDR3 gene, given the key role of biliary

phospholipids in protecting bile duct epithelium from potentially toxic, aggressive biliary

content (5). Administration of fibrates, statins, or peroxisome proliferators in mice, have been

shown to stimulate biliary phospholipid secretion by the induction of MDR3 making bile less

toxic (7-9). Further research in this direction seems justified.

Part III: HO-1 and hepatobiliary injury after liver transplantation

The specific aim of the third section was to study the role of HO-1 in relation to postoperative

hepatobiliary injury and graft function. We showed that upregulation of HO-1 during liver

transplantation correlates with better hepatobiliary function after transplantation. Furthermore

we demonstrated that patients possessing a polymorphism that is associated with reduced

HO-1 expression on mRNA level have a worse hepatobiliary function after transplantation and

an increased risk of graft loss on the long run. The role of HO-1 as a cytoprotective protein

was confirmed by these studies.

It was noted that HO-1 is already upregulated in many livers form brain death donors. The

variations in the observed upregulation of HO-1 mRNA levels could not be explained by a

larger number of marginal donors in the group with high HO-1 expression. Moreover, factors

associated with major hemodynamic alterations in the donor and several surgical variables

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Chapter 10

were similarly distributed amongst the donor groups with initial low expression of HO-1,

compared to the donor group with initial high expression of HO-1. To find an explanation

for these differences we studied two functional polymorphisms in the promoter region of the

gene: a (GT)n polymorphism and the single nucleotide polymorphism A(-413)T SNP.

The finding that the A(-413)T SNP exerts its effect not only in the immediate moments after

the transplant procedure, but has also consequences in the longer term (figure 4 in chapter 9),

is interesting. This could indicate that not only attenuation of I/R injury by a favourable HO-1

phenotype is beneficial but that HO-1 mediated processes may also play a role in later phases

after the transplantation.

Clinical application of interventions in the HO-1 system should be considered. However, we

should bear in mind that the beneficial effects of HO-1 may have a narrow therapeutic window

as shown in chapter 8. Highly overexpressed HO-1 displays pro-oxidant properties secondary

to iron accumulation, and may therefore be harmful instead of cytoprotective.

It would be very interesting to focus this research on the specific effect of HO-1 on biliary

epithelial cells which are especially vulnerable for I/R injury. We know from the study

described in chapter 8 that human HO-1 in the liver is mainly located in the Kupffer cells, and

not abundantly present in biliary epithelial cells. Strategies to enter HO-1 in these cells might

be of great interest to study whether HO-1 over expression could protect the bile ducts from

injury resulting from I/R injury or bile toxicity.

In summary, new insights are provided into the molecular and biochemical mechanisms of bile

duct injury after liver transplantation. We have proposed a classification system of NAS based

on the localization and severity of the biliary abnormalities. This classification system appeared

valuable in identifying different etiologies of NAS and also allowed the identification of patients

with NAS who are more at risk for complications or disease progression. Toxic bile composition,

characterized by a low phospholipids-to-bile salt ratio was discovered as a contributing

mechanism in the development of bile duct injury and NAS after liver transplantation. Further

interventional studies aimed at prevention of NAS based on the principle of this altered bile

composition are warranted. Finally, we have demonstrated a cytoprotective role of HO-1 in

liver transplantation, opening new avenues for the development of novel preventive strategies

or therapies.

204

Summary, discussion and future perspectives

References

Maathuis MH, Leuvenink HG, Ploeg RJ. Perspectives in organ preservation. Transplantation 2007 27;83:1289-1.

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Fickert P, Fuchsbichler A, Wagner M, Zollner G, Kaser A, Tilg H et al. Regurgitation of bile acids from leaky bile 3.

ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice. Gastroenterology 2004;127:261-74.

Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Pojer C, Zenz R, et al. Effects of ursodeoxycholic and cholic 4.

acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 2001;121:170-183.

Trauner M,Fickert P,Wagner M. MDR3(ABCB4) defects: Aparadigm for the genetics of adult cholestatic 5.

syndromes. Semin Liver Dis 2007; 27: 77.

Trauner M, Boyer JL. Bile salt transporters: Molecular characterization, function, and regulation. Physiol Rev 6.

2003; 83: 633.

Miranda S, Vollrath V, Wielandt AM, et al. Overexpression of mdr2 gene by peroxisome proliferators in the 7.

mouse liver. J Hepatol 1997; 26: 1331.

Hooiveld GJ, Vos TA, Scheffer GL, et al. 3-Hydroxy-3-methylglutarylcoenzymeAreductase inhibitors (statins) 8.

induce hepatic expression of the phospholipid translocase mdr2 in rats. Gastroenterology 1999; 117: 678.

Chianale J, Vollrath V, Wielandt AM, et al. Fibrates induce mdr2 gene expression and biliary phospholipid 9.

secretion in the mouse. Biochem J 1996; 314: 781.

Nederlandse samenvatting

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Samenvatting

Levertransplantatie is de aangewezen behandeling voor patiënten met eindstadium leverfalen.

Het succespercentage van een transplantatie is groot, na 5 jaar is meer dan 75% van de

patiënten nog in leven. Er kunnen echter complicaties optreden, onder meer van de galwegen.

Complicaties van de galwegen betreffen lekkage, stricturen van de anastomose (AS) en

stricturen en verwijdingen van de galwegen in de lever, de non-anastomotische stricturen

(NAS).

In dit proefschrift zijn NAS nader onderzocht, we hebben gekeken naar moleculaire en

biochemische mechanismen van deze complicatie.

NAS is een diagnose die gesteld wordt door de radioloog op basis van afbeelding van de

galwegen, een cholangiogram. Het beeld wordt gekarakteriseerd door vernauwingen en

verwijdingen van de galwegen in de lever (intrahepatisch). NAS na levertransplantatie zijn

initieel veel beschreven in combinatie met een trombose van de arteria hepatica (HAT).

In het geval van een vroege HAT na levertransplantatie worden de galwegen ischemisch

en uiteindelijk necrotisch, hetgeen resulteert in een typisch beeld op het cholangiogram

met vernauwingen en verwijdingen. Deze typische cholangiografische afwijkingen van

vernauwingen en verwijdingen worden echter soms ook gezien in de afwezigheid van HAT,

vandaar dat de term ‘ischemic-type biliary lesions’ (ITBL) is ontstaan. In dit proefschrift is de

term NAS gebruikt om intrahepatische vernauwingen en verwijdingen te beschrijven in de

bevestigde afwezigheid van HAT. De incidentie van NAS varieert rond 15% in verschillende

onderzoeken. Er zijn meerdere risico factoren voor NAS geïdentificeerd wat sterk suggereert

dat er een multifactoriele origine is. De belangrijkste categorieën zijn ischemisch gerelateerde

schade, immunologisch geïnduceerde schade en schadelijke effecten door galzouten. In

sommige gevallen kan echter geen specifieke risicofactor worden aangewezen. De klinische

presentatie van patiënten met NAS is vaak niet specifiek. Symptomen als koorts, buikklachten

en afwijkende lever waarden in het bloed duidend op cholestase kunnen voorkomen. De

diagnose wordt gesteld met behulp van beeldvormende studies van de galwegen. De

behandeling begint met het verlichten van de klachten veroorzaakt door de cholestase en

verwijding van de eventueel gestenoseerde galwegen met behulp van ‘endoscopisch retrograde

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cholangiopancreaticography’ (ERCP) of ‘percutanes transhepatische cholangiodrainage

(PTCD) zo mogelijk gevolgd door het achterlaten van een stent in de galwegen. Uiteindelijk

moet er bij een deel van de patiënten een re-transplantatie plaatsvinden of komen ze te

overlijden. In geselecteerde gevallen kan een re-transplantatie worden vermeden of in ieder

geval uitgesteld door chirurgische interventie.

Part I: Non-anastomotische galwegstricturen na levertransplantatie

In dit eerste gedeelte beschrijven we een tweetal studies met als doel a) de verschillende

vormen van NAS en bijbehorende klinische risico factoren te beschrijven, b) te achterhalen

welke patiënten met NAS een risico lopen op het ontwikkelen van ernstige problemen en de

behandeling daarvan.

In hoofdstuk 3 presenteren we een nieuwe classificatie van NAS. Met deze methode hebben

we alle beeldvorming van de patiënten met NAS van de afgelopen jaren in het UMCG opnieuw

beoordeeld. Verder hebben we gekeken naar risicofactoren voor het ontstaan van NAS, het

moment na de transplantatie waarop NAS zich presenteren en de progressie van de ziekte in

de loop van de jaren na de transplantatie.

In een groep van 487 volwassen transplantatie patiënten met een mediane follow-up van bijna 8

jaar waren er 81 (16.6%) patiënten die NAS ontwikkelden. We hebben aanwijzingen gevonden

dat er 2 vormen van NAS bestaan. NAS welke zich vroeg, binnen 1 jaar na de transplantatie,

presenteren en NAS welke zich na meer dan 1 jaar presenteren. Vroege NAS zijn geassocieerd

met preservatie en ischemisch gerelateerde risocofactoren, zoals een langere koude en

warme ischemie tijd. Tevens presenteren de vroege NAS zich vaker centraal in de lever. Late

NAS daarentegen zijn meer geassocieerd met immunologische risico factoren, zoals primair

scleroserende cholangitis als indicatie voor de transplantatie. Deze late vorm van NAS werd

vaker gezien in de periferie van de galwegen. We kunnen dus zeggen dat er verschillende

pathogenetische processen een rol spelen bij het ontstaan van NAS.

Als we verder kijken naar de groep patiënten met diagnose NAS, in hoofdstuk 4, konden

we de volgende risicofactoren identificeren voor radiologische progressie, wat bij bijna

70% van de patiënten optrad: vroege NAS en één of meerdere episodes van bacteriële

cholangitis. Bij patiënten met NAS bleek een galwegreconstructie met een Roux-Y hepatico-

jejunostormie een risicofactor voor het ontstaan van bacteriële cholangitis. Ernstige fibrose

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of cirrose ontstond in 23 gevallen, vooral in gevallen waarbij de NAS perifeer in de lever

gelokaliseerd was. Transplantaatoverleving, maar niet patiëntenoverleving, werd beïnvloed

door de aanwezigheid van NAS. Dertien patiënten (16%) onderging een re-transplantatie

vanwege de NAS.

De conclusie van deze studie was dat vooral patiënten met een Roux-Y reconstructie,

patiënten met vroege NAS en patiënten met NAS in de periferie van de lever risico lopen op

voortschrijdende ziekte met ernstige uitkomsten.

Part II: Gal fysiologie na levertransplantatie

De specifieke doelen van dit tweede gedeelte waren te evalueren welke bijdrage de gal

samenstelling heeft op het ontstaan van galwegschade. Van galzouten is het bekend dat ze

een detergente, vetoplossende werking hebben (zie de titelpagina verklaring). Galzouten,

zonder beschermende fosfolipiden, hebben een schadelijke werking. Het is bekend dat

ze een rol spelen in het ontstaan van galwegschade en cholangiopathie bij vele andere

ziektebeelden. Galformatie direct na de transplantatie kan verstoord zijn hetgeen resulteert in

een dergelijke schadelijke samenstelling met een lage fosfolipiden – galzouten ratio. De rol van

schadelijke galsamenstelling in de ontwikkeling van galwegstricturen na levertransplantatie is

echter onduidelijk.

Na ‘non heart-beating’ (NHB) levertransplantatie zijn NAS een vaak voorkomende complicatie. In

hoofdstuk 5 wordt in een varkensmodel van NHB levertransplantatie het effect van verschillende

periodes van warme ischemie in de donor op galsamenstelling en daaropvolgende schade aan

de galwegen na transplantatie bestudeerd. In drie groepen werd een oplopende vertraging

voor uitname van de lever toegepast, waarna het orgaan wordt getransplanteerd na 4h koude

bewaartijd. De galflow werd gemeten en galmonsters werden dagelijks verzameld om de

fosfolipiden – galzout ratio te bepalen. De mate van schade aan de galwegen werd gescoord

door middel van een histologische scoringsschaal. De resultaten toonden dat de fosfolipiden –

galzouten ratio in de gal die vlak na de transplantatie werd geproduceerd significant lager is in

groep met de langste warme ischemietijd in de donor. In deze groep was ook de grootste mate

van galwegschade te zien. Op basis van de resultaten van deze studie werd geconcludeerd dat

langere warme ischemie in NHB donoren is geassocieerd met de vorming van schadelijke gal

na transplantatie, deze bevindingen suggereren dat schade door galzouten bijdraagt aan het

ontstaan van galwegschade na NHB levertransplantatie.

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In het voorgaande hoofdstuk en ook andere studies uit onze groep hebben aangetoond dat

galformatie direct na de transplantatie verstoord kan zijn wat resulteert in een schadelijke

samenstelling van de gal met een lage fosfolipiden - galzouten ratio. Het was onbekend

of deze veranderde samenstelling ook en rol speelt bij de ontwikkeling van NAS, een

aandoening van de grotere galwegen. Indien veranderingen in de galsamenstelling

een rol spelen bij het ontstaan van NAS na transplantatie dan is te verwachten dat de

galsamenstelling na transplantatie anders is bij patiënten die NAS ontwikkelen ten opzichte

van patiënten die geen NAS ontwikkelen. Deze hypothese werd getest in een prospectieve

klinische studie welke beschreven is in hoofdstuk 6. In deze grote cohort studie bij

volwassen levertransplantatiepatiënten werd galproductie en samenstelling in de eerste

week na transplantatie gecorreleerd aan het ontstaan van NAS in het verdere beloop na

transplantatie. In 111 levertransplantatiepatiënten werden dagelijks galmonsters verzameld

om de samenstelling te analyseren. NAS werden gediagnosticeerd in 14 patiënten (13%)

binnen 1 jaar na transplantatie. Patiënten die uiteindelijke NAS ontwikkelden bleken minder

galzouten, fosfolipiden en cholesterol uit te scheiden in de gal. Tegelijkertijd was de biliare

fosfolipiden - galzouten ratio lager in patiënten die NAS ontwikkelden, wat duidt op mogelijk

meer schadelijke samenstelling van de gal. Deze bevindingen passen in het concept dat

galzouten betrokken is bij het ontstaan van NAS.

In het voorgaande hoofdstuk hebben we laten zien dat schadelijke gal samenstelling met

een verlaagde fosfolipiden - galzouten ratio geassocieerd is met NAS na levertransplantatie.

Hepatobiliare transporteiwitten zijn verantwoordelijk voor de secretie van fosfolipiden en

galzouten vanuit de hepatocyten naar de gal. Omdat de verschillen in de galsamenstelling

niet te verklaren waren door klinische variaties bij de donor of ontvanger was het doel van

hoofdstuk 7 om te analyseren of variaties in de genen die coderen voor deze transporters in

de donor geassocieerd zijn met het ontstaan van NAS in de ontvanger. Zonder transplantatie

leiden deze variaties op zichzelf niet tot galwegschade. Echter, direct na transplantatie, op

het moment dat de lever nog herstellende is van de schade van de ischemie en reperfusie

(I/R), kunnen deze variaties juist een belangrijke factor zijn in de serie van gebeurtenissen die

leidt tot het ontstaan van galweschade. Een vergelijkbare situatie wordt gezien bij patiënten

met intrahepatische cholestase tijdens de zwangerschap. Wanneer deze patiënten niet

zwanger zijn hebben zij geen klachten, echter op het moment dat er iets bijzonders gebeurt,

een zwangerschap vertonen ze ziekte verschijnselen. Bij 458 levertransplantatie procedures

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konden we de verschillende hepatobiliare transport eiwitten genotyperen. Zevenenzeventig

patiënten (16%) ontwikkelden NAS na transplantatie. Patiënten die een donorlever ontvingen

met een genetische variatie in de fosfolipidentransporter ontwikkelde bijna 2 keer zo vaak

NAS (28%) als patiënten die een donorlever ontvingen zonder deze variatie (15%). Ook in een

multivariate analyse was deze variatie een onafhankelijke risicofactor voor het ontstaan van

NAS. Hoewel we dat in deze studie niet hebben onderzocht, zou het transport van fosfolipiden

naar de gal in levers welke drager zijn van het risico haplotype veranderd kunnen zijn in de

direct postoperatieve periode en op deze wijze een bijdrage kunnen leveren aan het ontstaan

van NAS.

Part III: HO-1 en hepatobiliaire schade na lever transplantatie

Het specifieke doel van het derde deel was om de rol van heme oxygenase-1 (HO-1) te

bestuderen in relatie tot hepatobiliaire schade en leverfunctie. Opregulatie van HO-1

wordt beschouwd als een belangrijk beschermingsmechanisme tegen I/R schade bij

levertransplantatie. In hoofdstuk 8 hebben we in 38 volwassen levertransplantatiepatiënten de

rol van endogene HO-1 expressie, voor tijdens en na transplantatie, bestudeerd in relatie tot

postoperatieve hepatobiliaire schade en functie direct na transplantatie. Voorafgaand aan de

operatie was de mediane HO-1 expressie reeds 3,4-keer verhoogd (spreiding 0,7 tot 9,3-keer

verhoogd). Deze spreiding was niet te verklaren door de klinische condities of behandelingen

van de donoren. We vonden dat in de groep van levers die het vermogen hadden om het

HO-1 tijdens de transplantatie verder op te reguleren, de schade aan de lever minder was dan

bij de patiënten waarbij de HO-1 expressie in de lever niet toenam. Dit suggereert dat levers

die tijdens reperfusie het HO-1 kunnen induceren beter beschermd zijn tegen I/R-schade

dan donorlevers die dit niet kunnen. Verder onderzoek zal nodig zijn om te achterhalen wat

belangrijker is: een lage HO-1 expressie voor aanvang van de transplantatie, of het vermogen

om HO-1 tijdens de reperfusie te induceren.

In het voorgaande hoofdstuk is de endogene regulatie van HO-1 tijdens levertransplantatie

onderzocht. Omdat de variatie die werd gevonden in de initiële HO-1 expressie in de donor

levers kon niet worden verklaard door klinische variabelen, werd de hypothese opgevat

dat genetische verschillen verantwoordelijke zouden kunnen zijn voor de variatie in HO-

1. De expressie van HO-1 wordt in belangrijke mate bepaald door 2 variaties in het gen,

zogenaamde polymorfismen. Eén daarvan is het ‘single nucleotide polymorfisme’ (SNP)

211

A(-413)T. Aangezien ieder mens 2 allelen heeft kunnen de volgende variaties ontstaan: AA,

AT en TT. De A-variant is geassocieerd met een verhoogde HO-1 activiteit. In hoofdstuk 9

beschrijven we een studie waarin de genetische variatie van de donor werd geanalyseerd en

gecorreleerd aan uitkomsten na transplantatie in een groep van 308 volwassen patiënten die

een levertransplantatie ondergingen. In een subgroep werd de HO-1 genexpressie in de lever

gecorreleerd aan de genotypen. Overleving van het transplantaat na 1 jaar was beter voor

A-varianten in vergelijking met de TT-genotypes. Verlies van het transplantaat als gevolg van

primaire disfunctie werd vaker waargenomen bij levers met het TT-genotype. Er werd geen

verschil gezien in de incidentie van NAS in beide groepen. Ontvangers van een TT-genotype

lever hadden meer schade aan de lever direct na transplantatie. HO-1 genexpressie in de

lever was lager in de levers met het TT-genotype, in vergelijking met levers met een A-allel.

De belangrijkste conclusie van deze studie was dat het A(-413)T polymorphisme in de HO-1

promoter geassocieerd is met uitkomsten na levertransplantatie.

Tot besluit kunnen we stellen dat onderzoek naar de moleculaire en biochemische mechanismen

van het ontstaan van galwegschade belangrijke nieuwe gezichtspunten hebben opgeleverd.

We hebben een nieuw classificatie systeem voor NAS voorgesteld, dat is gebaseerd op de

lokalisatie en ernst van de galwegafwijkingen. Dit classificatiesysteem bleek waardevol in het

identificeren van verschillende ontstaansmechanismen van NAS, tevens was het mogelijk

patiënten te identificeren die een groter risico liepen op complicaties en progressie van de

ziekte. Schadelijke samenstelling van de gal, gekarakteriseerd door een lage fosfolipiden -

galzouten ratio, werd geïdentificeerd als een belangrijk bijdragende factor aan het ontstaan

van galwegschade en NAS na levertransplantatie. Verder interventie onderzoeken gericht op

het voorkómen van NAS gebaseerd op de bevindingen van deze veranderde gal samenstelling

zijn nu het aangewezen vervolg. Tot slot hebben we een beschermende rol aangetoond voor

HO-1 in levertransplantatie, dit opent nieuwe wegen voor het ontwikkelen van preventieve

strategieën en therapieën.

List of contributing authors

214

List of Contributing Authors


Dr. H. Blokzijl

Department of Gastroenterology and Hepatology

University Medical Centre Groningen

Groningen, the Netherlands

Dr. W. Geuken

Surgical Research Laboratory, Department of Surgery.

Currently Department of Pathology



Dr. A.S.H. Gouw

Department of Pathology



Dr. E.B. Haagsma




Dr. B.G. Hepkema

Department of Laboratory medicine, Transplantation Immnology



Drs. C.S. van der Hilst

Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery



215

Drs. H.H. Hoekstra




Dr. E.J. Van der Jagt

Department of Radiology



Dr. K.P. de Jong




O.N.H. Kahmann

Surgical Research Laboratory



Prof. dr. H. Kleibeuker




Prof. dr. F. Kuipers

Pediatric Gastroenterology, Department of Pediatrics



216


Dr. H.G.D. Leuvenink




Dr. A.J. Limburg




Dr. D. Monbaliu

Department of Abdominal Transplant Surgery and coordination

University Hospitals Leuven

Leuven, Belgium

Prof. H. Moshage




Dr. B.A. Nemes




Dr. M. Nijsten

Surgical Intensive Care Unit



217

Dr. I.M. Nolte

Department of Epidemiology



Dr. P. M.J.G. Peeters




Prof. dr. J. Pirenne

Department of Abdominal Transplant Surgery and coordination

University Hospitals Leuven

Leuven, Belgium

Prof. dr. R.J. Porte




Dr. T.A. Schuurs




Prof. dr. M.J.H. Slooff




218


Dr. G. van der Steege

Department of Genetics



Dr. R.C. Verdonk




Prof. dr. H.J. Verkade

Pediatric Gastroenterology, Department of Pediatrics



Ing. D.S. Visser

Surgical Research Laboratory, Department of Surgery



Drs. M.J. Yska





List of publications

220

List of Publications


Buis CI, Bakker SJL. ACE- inhibitie niet effectief bij voorkómen van restenose na coronaire

stentimplantatie. Ned Tijdschr Geneeskd 2001; 145:2051. (short review paper).

Buis CI, Wijdicks EFM. Serial MR imaging in central pontine myelinolysis. Liver Transpl 2002;

8:643-5.

Buis CI, Wiesner RH, Krom RAF, Kremers WK, Wijdicks EFM. Acute confusional state following

liver transplantation for alcoholic liver disease. Neurology 2002; 59:601-5.

Geuken E, Buis CI, Visser DS, Blokzijl J, Moshage H, Nemes B, Leuvenink HGD, Jong de

KP,Peeters PMJG, Slooff MJH, Porte RJ. Expression of Heme oxygenese-1 in human livers

before transplantation correlates with graft injury and function after transplantation. Am J

Transplant 2005; 5:1875-85.

Buis CI, Porte RJ, Slooff MJH. Levertransplantatie. In: H.G. Smeenk, N.W.L. Schep, W.M.U.

van Grevenstein ed. Leidraad chirurgie. Houten: Bohn Stafleu van Loghum, 2005; 211.

Buis CI, Porte RJ, Slooff MJH. Niertransplantatie. In: H.G. Smeenk, N.W.L. Schep, W.M.U.

van Grevenstein ed. Leidraad chirurgie. Houten: Bohn Stafleu van Loghum, 2005; 219.

Verdonk RC, Buis CI, Porte RJ, Haagsma EB. Biliary complications after liver transplantation,

A review. Scand J Gastroenterol 2006; 243:89-101.

Buis CI, Hoekstra H, Verdonk RC, Porte RJ. Causes and Consequences of Ischemic Type

Biliary Lesions After Liver Transplantation. J Hepatobiliary Pancreat Surg 2006; 13:517-24.

Su Huawei, van Dam GM, Buis CI, Visser DS, Hesselink JW, Schuurs TA, Leuvenink HGD,

Contag CH, Porte RJ. Spatiotemporal Expression of heme oxygenase-1 Detected by in vivo

bioluminescence after hepatic ischemia in HO-1/Luc mice. Liver Transpl 2006; 12:1634-9.

221

Verdonk RC, Buis CI, Porte RJ, van der Jagt EJ, Limburg AJ, vd Berg AP, Slooff MJH, Peeters

PMJG, de Jong KP, Kleibeuker JH, Haagsma EB. Anastomotic biliary strictures after liver

transplantation: prevalence, presentation, management and outcome. Liver Transpl 2006;

12:726-35.

Mantel HTJ, Buis CI, Homan van der Heide JJ, van den Berg AP, Verkade HJ, Haagsma EB,

Peeters PMGJ, de Jong KP, Slooff MJH, Porte RJ. Gecombineerde lever-niertransplantaties:

Indicaties en resultaten in het UMC Groningen. Ned Tijdschr Geneeskd 2006; 150:2260-5.

Buis CI, Verdonk RC, van der Jagt EJ, van der Hilst CS, Slooff MJH, Haagsma EB, Porte

RJ. Non-anastomotic biliary strictures after adult liver transplantation part one: Radiological

features and risk factors for early versus late presentation. Liver Transpl 2007; 13:708-18.

Verdonk RC, Buis CI, van der Jagt EJ, Gouw ASH, Limburg AJ, Slooff MJH, Kleibeuker JH,

Porte RJ, Haagsma EB. Non-anastomotic biliary strictures after adult liver transplantation

part two: Management, outcome and risk factors for disease progression. Liver Transpl 2007;

13:725-32.

Buis CI, Steege vd G, Visser DS, Nolte IM, Hepkema BG, Nijsten M, Slooff MJH, Porte

RJ. Heme oxygenase-1 genotype of the donor is associated with graft survival after liver

transplantation. Am J Transplant 2008; 8:377-85.

Buis CI, Hofker HS, Nieuwenhuijs VB. Diverticulitis of the Jejunum, an uncommon diagnosis.

Dig Surg. 2008; 25:83-4.

Yska MJ, Buis CI, Monbaliu D, Schuurs TA, Gouw ASH, Kahmann ONH, Visser DS, Pirenne

J, Porte RJ. The role of bile salt toxicity in the pathogenesis of bile duct injury after non heart-

beating porcine liver transplantation. Transplantation 2008; 85:1625-31.

Buis CI, Geuken E, Visser DS, Kuipers F, Haagsma EB, Verkade HJ, Porte RJ. Altered

bile composition is associated with the development of nonanastomotic biliary strictures. J of

Hepatol, in press.

222

Buis CI, Steege vd G, Visser DS, Nolte IM, Porte RJ. Polymorphism of hepatobiliary

phospholipid transporter ABCB4 associated with nonanastomotic biliary strictures after human

liver transplantation. Submitted.

Hoekstra H, Buis CI, Verdonk RC, van der Hilst CS, van der Jagt EJ, Haagsma EB, Porte

RJ. Is Roux-Y choledochojejeunostomy an indipendant risk factor for non-anastomotic biliary

strictures after liver transplantation? Submitted.


Summary, discussion and future per-spectives

Dankwoord

Summary, discussion and future per-spectives

224

Dankwoord

Dankwoord

Het állerleukste van promoveren is dat je met zoveel verschillende mensen mag samenwerken

en van hen kan leren. Graag w il ik velen van hen in dit meest gelezen hoofdstuk noemen.

Professor dr. R.J. Porte, beste Robert, vanzelfsprekend ben jij de eerste, de allerbelangrijkste.

Jij hebt voor mij de mogelijkheid gecreëerd om AGIKO te worden. Jij hebt mij in staat gesteld

dit mooie (al zeg ik het zelf) proefschrift af te leveren. Het is fantastisch om met je te werken!

Je bezieling voor wetenschappelijk onderzoek is ongelooflijk groot en voortdurend aanstekelijk

omdat het is gebaseerd op een zeer scherpe en heldere analyse van de onderwerpen. Iedere

keer na overleg met jou had ik nóg meer inspiratie om aan de slag te gaan, jammer dat de

promotie nu klaar is. Gelukkig is er in ieder geval nog één artikel dat we samen verder mogen

polijsten, zodat de samenwerking op deze manier nog even door mag gaan.

Tot slot, je hebt een uitermate groot talent om een hele goede, leuke en bovenal gezellige groep

van onderzoekers om je heen te verzamelen, het is genieten om daar onderdeel van uit te

mogen maken.

Professor dr. M.J.H. Slooff, beste professor, u bent het boegbeeld van de hepatobiliare

chirurgie en levertransplantatie in Groningen. Op de achtergrond bent u voor mij heel belangrijk

geweest voor het welslagen van deze promotie. Uw wijsheid en warmte die ik heb leren kennen

voor het vak, maar zeker ook voor de ‘andere mooie dingen’ van het leven zal ik niet vergeten.

Dank voor het beoordelen van dit proefschrift.

Professor dr. H.J. Verkade, beste Henkjan, ik heb heel veel van je geleerd. Voor mij ben je

hét voorbeeld van combinatie van klinische top zorg met top wetenschappelijk onderzoek. Je

analyseert, denkt (en praat) zó snel dat het voor een gewone sterveling zoals ik vaak moeilijk

is bij te houden. Maar ik heb je altijd alle vragen mogen stellen totdat ik het begreep, dank voor

alle heldere uitleg. Dank voor het beoordelen van dit proefschrift.

Professor dr. H.J. Metselaar, beste Herold, ik heb je (en de onderzoeksgroep uit Rotterdam)

mogen ontmoeten op vele mooie plaatsten in de wereld. Dank voor het beoordelen van dit

proefschrift.

225

Professor dr. H.J. ten Duis, beste opleider, zonder uw steun was dit proefschrift nooit in deze

vorm tot stand gekomen. Graag wil ik u danken voor het vertrouwen dat u al heel vroeg in mij

heeft gesteld.

Dr. M. Eeftinck Schattenkerk, beste dr. Schattenkerk, graag wil ik ook u danken voor het

vertrouwen dat u reeds vroeg in mij gesteld heeft, dat ik mijn opleiding juist in Deventer mag

vervolgen. Nu dit proefschrift succesvol is afgerond kijk ik er erg naar uit onder uw leiding ook

mijn chirurgische vaardigheden verder te ontwikkelen.

Drs. M.T. de Boer, lieve Marieke, samen kunnen wij de wereld aan! Rio de Janiero, Milaan, San

Fransisco, Los Angeles, Amsterdam, Mumbai, (New York...?). Een voor een hoogtepunten. Kijk

uit naar (het feest van) jouw promotie. De manier waarop jij je vak bedrijft is een groot voorbeeld

voor me, ik ben apetrots dat jij mijn paranimf bent.

Dr. M.H.J. Maathuis, lieve Hugo, wat een feest was het om tegelijk met jou onderzoek te doen.

Het is fantastisch om met jou samen te werken, je bent ongelooflijk positief, organisatorisch de

beste, en een echte teamspeler, superlatieven te kort. Van het organiseren van het SEOHS heb

ik dan ook intens genoten. Ik weet zeker dat je succesvol en gelukkig zal worden in je nieuwe

functie. Ik ben waanzinnig vereerd dat je mijn paranimf bent.

Dr. W. Geuken, beste Erwin, dank voor het mede opzetten van de lijn galwegcomplicaties

na levertransplantatie in het lab. Dr. R.C. Verdonk, beste Robert, nu is het mijn beurt jou te

bedanken voor het samenwerken. Dank ook voor alle biopten waar ik een stukje van mocht

hebben. Ik kom graag op je oratie over een paar jaar! Drs. M.J. Yska, beste Marit, jij hebt als

student in het lab een uitzonderlijke prestatie geleverd! Dank voor het trekken van het ‘Leuven’

project, en veel succes in je verdere carrière.

Ing D.S. Visser, Beste Dorien, dank voor je grote hulp bij alle labbepalingen! We hebben

fantastisch samengewerkt, hetgeen ook blijkt uit het feit dat jullie zoon pas werd geboren toen al

het werk voor dit proefschrift klaar was! Veel geluk met het leven op Ameland!

Dr. G. van der Steege, beste Gerrit, jij nam alle tijd om mij in te wijden in de wereld van de

polymorfismen en haplotypes. Het was genieten om samen te puzzelen achter jouw computer.

226

Dankwoord

Hoofdstuk 9 is inmiddels heel mooi gepubliceerd, en ik weet zeker dat dit ook met Hoofdstuk 7

gaat lukken! Dr. E.J. van der Jagt, beste dr. van der Jagt, dank voor de vele uren dat u samen

met Robert Verdonk en mij alle beeldmaterieel van mogelijke NAS patiënten heeft gereviseerd

en gescoord. De uitkomsten van dit werk waren essentieel voor dit proefschrift. Professor dr.

F. Kuipers, beste Folkert, hoe fantastisch is het ergens te mogen werken waar je grootheden

als jij gewoon tegen het lijf loopt bij het koffieapparaat. Dank voor je warme betrokkenheid bij

mijn promotie onderzoek en in het bijzonder hoofdstuk 6. Dr. I.M. Nolte, beste Ilja, jouw inzicht

in statistiek is onnavolgbaar, toch slaagde je erin mij te laten begrijpen wat je deed. Dank voor

je hulp bij Hoofdstuk 7 en 9. Dr. E.B. Haagsma, beste dr. Haagsma, als begeleider van Robert

Verdonk bent u betrokken geweest bij het welslagen van Part I van mijn proefschrift, dank

daarvoor. Professor dr. J. Pirenne en dr. D. Monbaliu, beste professor, beste Diethard, dank

voor de succesvolle samenwerking en de warme ontvangst in Leuven, ook voor Marit.

De medeauteurs van de artikelen en nog niet eerder genoemd; Dr. A.S.H. Gouw, beste

Annette, dank voor uw hulp bij alle pathologische vraagstukken. Dr. B.G. Hepkema, beste

Bouke, dank voor de hulp vanuit de transplantatie immunologie bij hoofdstuk 7 en 9. Dr. M.

Nijsten, beste Maarten, dank voor de hulp bij het verkrijgen van alle labwaarden van de

levertransplantatiepatiënten. Dr. H. Blokzijl, beste Hans, alweer lang geleden heb jij mijn eerste

schreden in het lab begeleid, dank daarvoor en voor al het advies ‘along the way’. Drs. C.S. van

der Hilst, beste Christian, al mijn statistische kennis heb ik van jou! Dank voor je voortdurende

uitleg. Drs. H.H. Hoekstra, beste Harm, ik heb respect voor jouw eigen manier waarop je je

onderzoek bedrijft. Je muizen studie is mechanistisch gezien voor dit proefschrift van groot

belang geweest.

HPB en Levertransplantatie chirurgen en fellows uit Groningen dank ik voor alle uitleg en het

verzamelen van materiaal zodat er onderzoek gedaan kon worden; Dr. P.M.J.G. Peeters,

beste Paul, de precisie en toewijding waarmee jij opereert en klinische zorg verleent heb ik als

keuzeco leren kennen en zal voor altijd een voorbeeld blijven. Daarnaast heb ik de gesprekken

over zeilen, schilderen en het leven bijzonder gewaardeerd. Dr. K.P. de Jong, beste Koert, na

ons gestrande KOROCA project durfde ik bijna niet opnieuw bij de HPB club in Groningen aan

te kloppen, ik ben blij dat ik toch de stap heb genomen. Dr. I.Q. Molenaar, beste Q, hoe kan ik

jou bedanken? Het feestje had al ruim 2 jaar geleden kunnen zijn ;-) . Je bent een waanzinnig

227

groot voorbeeld van een ambitieuze chirurg met een prachtgezin! Dr. W.G. Polak, beste Wojtek,

dr. B.A. Nemes, dear Balasz, dr. S. Eguchi, dear Susumu, dr. A. Soyama, dear Aki en dr. E.

Sieders, beste Ger, thank you so much for all your explanations during the past years.

Professor dr. H. Kleibeuker, dr. A.J. Limburg, dank voor uw bijdrage aan hoofdstuk 4 van dit

proefschrift. Dr. A.P. van den Berg, beste Aad, dank voor al je uitleg en warme belangstelling

tijdens en na mijn keuzecoschap tijd.

Graag wil ik de volgende mensen danken voor hun bijdrage aan mijn proefschrift. Tina Crabbé

uit Leuven is onmisbaar geweest voor het uitvoeren van de experimenten uit Hoofdstuk 5,

zodat wij met de verkregen monsters onderzoek konden doen. Jan Bottema, dank voor het

verzamelen van gal voor Hoofdstuk 6, zodat ik ook eens een weekendje weg kon! Renze

Boverhof, dank voor het uitvoeren van de gaschromatografie van de galmonsters in hoofdstuk

6. Mariska Geuken, Fjodor van der Sluijs en Petra Suichies-Ottens, voor jullie technische

ondersteuning, en niet alleen voor hoofdstuk 8! Marcel Mulder, dank voor het uitvoeren van

de genotypering van Hoofdstuk 7 en 9. Tot slot Daniëlle Nijkamp, dank voor vele zaken, maar

ook voor de gezellige tijd in het oude archief van Eurotransplant in Leiden alwaar we alle oude

donordata hebben nagezocht!

De omgeving waar ik mijn onderzoek heb uitgevoerd is het Chirurgisch Onderzoekslaboratorium.

Vanuit de kliniek uiteraard professor dr. R.J. Ploeg, dank voor uw vele scherpe vragen tijdens

de labbesprekingen, en voor het meenemen van de jonge dokter op donor (vlucht (!) met

whisky). Dr. G.M. van Dam, je bent een waanzinnig enthousiaste wetenschapper en een super

begeleider van een jonge assistent.

Organisatorisch waren het soms roerige tijden, maar het chirurgisch lab is een fantastische

plek om te werken. Veel dank, dr. T. Lisman, beste Ton, voor je komst om te beginnen, je

wetenschappelijke inzicht en gezelligheid! Dr. H.G.D. Leuvenink, beste Henri, dank voor je

opvang van de jonge en onervaren onderzoeker in het lab die ik in het begin was! Dr. T.A

Schuurs, beste Theo, dank voor je wetenschappelijk inzicht en je begeleiding van Marit en het

totstandkomen van Hoofdstuk 5. Ing J.J Zwaagstra, beste Jacco, je weet dat ik groot respect

heb voor hoe jij het hoofdanalist-schap invult! Veel dank ook aan Ing A.van Dijk, beste Anthony,

dank voor het dierexperimentele werk, ik heb veel van je geleerd op microchirurgisch gebied,

228

Dankwoord

Ing J. Wiersema-Buist, beste Janneke, dank voor je algemene ondersteuning.

Het laboratorium kindergeneeskunde en MDL wil ik graag danken voor hun gastvrijheid en

voortdurende hulp tijdens mijn onderzoek. Beter een goede buur dan een verre vriend! Met

name dr. K.N. Faber en professor dr. A.J. Moshage. Drs. J. Mulder, beste Jaap, jammer

dat we (nog) geen artikel samen hebben geschreven, wel veel dank voor al je advies (op PCR

gebied).

Huawei Su, MD, dear Su, it was great to get to know and work with you! You published a

wonderful paper in the American Journal and I am pleased with my first citation in a Chinese

journal!

Collega onderzoekers en kamergenoten, Mijntje Nijboer (collega, we blijven elkaar zeker nog

tegenkomen!), Jayant Janandunsing (aan de andere kant van het doek), Cyril Moers (collega,

de lat is gelegd), Lyan koudstaal (succes met afronden van jouw proefschrift), Anne Margot

Roskott (dank voor de overheerlijke cappu’s!), Micheal Sutton (lang leve de galwegen), Tan

Hongtao (thank you so much for your contribution in the rapamycin project), Hugo Maathuis

(paranimf!) en Ilona Peereboom (dank voor het zijn van een hele hele leuke collega, dat dat nog

maar lang mag duren!), dank voor alle gezellige momenten en (wetenschappelijke) reflectie.

Graag wil ik ook op deze plaats de mensen bedanken die mijn eerste schreden op het

wetenschappelijke pad hebben begeleid en gestimuleerd. Professor dr. T.H. The, door mij te

selecteren voor de JSM en goede adviezen nadien. Professor dr. C.H. Gips, zoals gezegd, de

eerste basis is gelegd bij de GISH-T! Dankzij mijn succesvolle afstuderen in the Mayo Clinics

ben ik gestimuleerd door te gaan met wetenschap. Professor dr. R.A.F. Krom, icoon op het

gebied van levertransplantatie, dank voor uw begeleiding op en naast het wetenschappelijke

gebied en uw voortdurende belangstelling in mijn (helaas chirurgische) carrière. Professor dr.

E.F.M. Wijdicks, uw kunde om in een razend tempo een goed artikel te schrijven zal me altijd

bijblijven! Het was fantastisch met u samen te werken! Dr. C.B. Rosen, dear Chuck, our small

project together resulted in the first international oral presentation in my career, thanks for the

opportunities you created for me as a medical student to come to the OR and join in liver

transplant and donor procedures.

229

Het leverteam is een prachtige Groningse traditie! Dank aan alle studenten die in de loop van

de jaren hebben geholpen met het verzamelen van biopten tijdens de transplantaties! Verder wil

ik ook graag de andere studenten waar ik kort of lang mee heb mogen samenwerken bedanken:

Danka, Olivier (medeauteur H5!), Mohammed, Bakhtawar, Mickey, Henk-Jan (Mayo-collega

en auteur van CLKTx stuk), Maurits (weer terug!) en natuurlijk Fraukje en Heleen.

It has been a great honour to work with professor dr. G.J. Barrit, head of the department of

Medical Biochemistry, Flinders Medical School, Adelaide, Australia. Dear Greg and dear Yabin

Zhou, thank you for your hospitality in and outside the lab. Dr. V.B. Nieuwenhuijs, beste Vincent,

dankzij jouw contacten was het voor mij mogelijk naar Greg’s lab te gaan en een nieuw project,

dat we hier ook samen met Robert hebben geschreven, daar op te zetten. Dutchies! Marije,

Heleen, Fraukje, Claire, Meike, Claire en Judith, dank voor de ongelooflijke gezelligheid, op

het lab en daarbuiten en voor alle hele mooie tripjes die we hebben gemaakt!

Onderzoekers van het eerste uur uit het TRIADE gebouw Anne Brecht (dank voor het precedent

van een uitgebreid dankwoord), Tjeerd, Lucas, Annemarie, Kirsten, Marten, Eric, Esther en

Martin. In mijn eerste maanden werd ik meteen opgenomen in ‘de club’. Het was als jonge

onderzoeker fantastisch om ‘collegae’ te hebben!

SEOHS bestuur 2006, Hugo, Marcel, Justine (OH’s 4ever), Coralien, Hilke, Anton, Marinus

en Martin, het was een topcongres! Dank voor de heerlijk afwisseling in het wetenschappelijke

werk in de vorm van onze vele vergaderingen.

Onderzoek en congresbezoek-collegae; Quintus, Marieke, Nienke, Sander, Daantje, Christian,

Harm, Ilona, Margijske het is fantastisch met jullie de halve wereld over te vliegen!!

Collegae assistenten en chirurgen van het UMCG. Het voelde fantastisch om als jonkie

meteen opgenomen te worden in de club. Veel dank voor alle gezelligheid tijdens mijn tijd als

onderzoeker. Veel dank voor het wegwijs maken van datzelfde jonkie 2 jaar later in de kliniek

toen ik eindelijk ‘mocht’. En alvast voor de toekomst: veel dank voor alle gezelligheid en het

wegwijs blijven maken in de chirurgie!

230

Dankwoord

Ongelooflijk veel dank aan Linda, de regassen van C4 en alle andere secretaresses, voor het

bestellen van vele statussen en andere regeldingen! Veel dank ook aan de medewerkers van C4

waar ik niet alleen begon als keuzeco, maar ook nog heel vaak kwam om gal te verzamelen.

Veel mensen die niet direct bij het onderzoek betrokken zijn geweest, maar wel in mijn leven

een belangrijke rol spelen; Kickers (vanaf nu meer tijd om die ‘band voor het leven’ weer wat

meer aandacht te geven), GGGG (lieve oud-huisgenootjes: “wat wij hebben, heeft niemand!”),

Boswandeling (weekend van 22 januari 2009 staat geblokkeerd!), en alle verschillende

hockeyteams die mijn tijd in Groningen van de nodige inspanning en ontspanning hebben

voorzien. Een klein stukje gedicht voor jullie allen (naar Eric Brey):

Ik denk terug aan duizendtallen hapjes, nipjes, slokken

Maar ‘t was vooral m’n Ziel die daar zo gulzig zat te schrokken

Dat wij zo weer eens met elkaar langdurig haute-cuisine’den:

Kom, laat er altijd Eten zijn, en Drinken zijn, met Vrienden...

Lieve Pappa & Mamma en Hanne & Rikkert, Oom Wouter, dank voor alle 1001 leuke

momenten die wij met elkaar hebben! Mamma, dank voor alle wijze adviezen die ik gevraagd

en ongevraagd van je krijg op zoveel terreinen. Pappa, dank voor het inzicht dat ik van je heb

mogen leren (afkijken eigenlijk) terwijl jij alles maakte wat je bedacht (boot!). Wel spannend dat

ik straks als chirurg sta te knutselen zonder jou! Lieve Hanne, dank voor het zijn van mijn steun

en toeverlaat! Het is zo heerlijk om je te realiseren dat jullie voor altijd mijn Pappa, Mamma en

Zussie zijn!!

Mijn allerliefste Luitzen,

Je zoenen zijn zoeter dan

zoeter dan honing en ik vind je

mooier en liever, liever

en aardiger nog

dan de koning

Naar Judith Herzberg en Herman van Veen

Curriculum Vitae

232

Curriculum Vitae

Curriculum Vitae

Carlijn Buis was born on December 29th, 1978 in Vught, the Netherlands. After 6 years of

primary school at the ‘Rudolf Steiner Vrije School’ in ’s-Hertogenbosch she went to high

school (Gymnasium Beekvliet) in st. Michielsgestel where she graduated in 1997. In the

same year she started her medical study at the University of Groningen. During her study

at university Carlijn contributed as a board member to AIESEC (l’Association International

des Etudients en Science Economique et Commercial), a worldwide student organisation

focussing on international exchange. As a medical student Carlijn participated in the Junior

Scientific Master Class programme, under the guidance of professor dr. T.H. The. Carlijn

conducted her graduating scientific rotation via professor dr. C.H. Gips in Rochester, MN, USA

at the liver transplant unit in the Mayo Clinic under the guidance of professor dr. R.A.F. Krom

and professor dr. E.F.M. Wijdicks. In 2001 she passed her doctoral examination.

She followed her clinical rotations with great pleasure in the ‘Deventer Ziekenhuizen’ in

Deventer, the Netherlands. As a preparation for her MD-clinical trainee ship she conducted

her last clinical rotation at the Hepatobiliary Surgery and Liver Transplant unit at the University

Medical Center Groningen (UMCG). After graduating as a medical doctor, in January 2004,

Carlijn started her PhD at the UMCG, in the surgical research laboratory under the guidance

of professor dr. R.J. Porte. In May 2004 a MD-Clinical Research Traineeship (AGIKO) was

granted by the Netherlands Organization for Scientific Research (NWO). After conducting

2 years of research Carlijn accomplished the first year of her surgical training at the UMCG

under the guidance of professor dr. H.J. ten Duis. In 2008, a visiting research traineeship

was conducted at the Department of Medical Biochemistry and the Liver Transplant Unit at

Flinders Medical Center, Adelaide, in Australia under guidance of professor dr. G. Barritt, dr.

V.B. Nieuwenhuijs and professor R.J. Porte. Carlijn started a new project that is currently

being taken forward by various graduating students from the Netherlands. The upcoming year

Carlijn will continue her surgical training in the UMCG. In September 2009 and onwards she

will continue her surgical training in het ‘Deventer Ziekenhuis’ under guidance of dr. M Eeftinck

Schattenkerk.

Altered bile composition after liver transplantation is associated with the development of...

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