ARTICLE IN PRESS

EXPERIMENTAL

ANDTOXICOLOGIC

PA THOLOGY

0940-2993/$ - se

doi:10.1016/j.et

�CorrespondGuadalajara, Ja

E-mail addr1Both author

Experimental and Toxicologic Pathology 58 (2006) 185–195

www.elsevier.de/etp

Renal dysfunction as a consequence of acute liver damage

by bile duct ligation in cirrhotic rats

Sandra Rivera-Huizara,1, Ana Rosa Rincon-Sancheza,b,�,1, Amador Covarrubias-Pinedoc,Marıa Cristina Islas-Carbajala, Genaro Gabriel-Ortızd, Jose Pedraza-Chaverrıe,Adriana Alvarez-Rodrıgueza, Eduardo Meza-Garcıaa, Juan Armendariz-Borundaa,f

aInstitute for Molecular Biology in Medicine and Gene Therapy, CUCS, University of Guadalajara, Apdo. Postal 2-123,

Guadalajara, Jalisco 44281, MexicobInstitute for Chronic-Degenerative Diseases, CUCS, University of Guadalajara, MexicocUnit of Clinical Investigation OPD Civil Hospital Juan I Menchaca, MexicodCentro de Investigacion Biomedica de Occidente, IMSS Guadalajara, Jalisco, MexicoeDepartment of Biology, Faculty of Chemistry, UNAM, 04510 D.F., MexicofOPD Civil Hospital of Guadalajara, Mexico

Received 3 May 2005; accepted 2 May 2006

Abstract

Renal failure is a common complication in patients with alcohol-induced cirrhosis who undergo a superimposedsevere alcoholic hepatitis.

Aim: Our aim was to evaluate renal dysfunction established as a consequence of acute liver damage (ALD) inducedby bile duct ligation (BDL) in cirrhotic rats. Hepatic and renal functional assays were performed.

Results: Hiperbilirubinemia and increased alanine aminotransferase and aspartate aminotransferase (po0:05) inrats with BDL were observed since the first day of bile obstruction in cirrhotic rats. Urinary volume and urinarysodium concentration showed a significant reduction (po0:05) on days 3 and 5 after BDL. Plasma renin activity,plasma renin concentration, serum creatinine, and BUN values increased (po0:05) from day 1 to day 7 after BDL.Glomerular filtration rate was substantially decreased from day 1 to day 7. Histological changes became apparent sinceday 3 after BDL in which glomeruli with mesangial hipercellularity took place in the absence of tubular necrosis; withportal inflammation and proliferation of biliar conduits. Results of the present work demonstrate that ALD inducedby BDL in cirrhotic rats produces changes in renal function. In conclusion, this experimental model demonstrates thatan ALD of variable etiology, either surgical or induced by CCl4, can cause important damage that eventually results inrenal function deterioration. This experimental model may be suitable, to study the physiopathology of this syndrome,as well as for the evaluation of different pharmacological therapies.r 2006 Elsevier GmbH. All rights reserved.

Keywords: Cirrhosis; Bile duct ligation; Renal damage; Hepato-renal syndrome

e front matter r 2006 Elsevier GmbH. All rights reserved.

p.2006.05.001

ing author. Institute for Molecular Biology in Medicine and Gene Therapy, CUCS, University of Guadalajara, PO Box 2-123,

lisco, C.P. 44281, Mexico. Tel./Fax: +52 3617 4159.

esses: arrincon@cencar.udg.mx, rincon_ana@hotmail.com (A.R. Rincon-Sanchez).

s participated and contributed equally to this paper.

ARTICLE IN PRESSS. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195186

Introduction

Renal failure is a common complication in patientswith alcohol-induced cirrhosis whom undergo a super-imposed severe alcoholic hepatitis resulting in a rapiddeterioration of liver function (Monasterolo et al.,2002). Clinical characteristics of patients with decom-pensated cirrhosis and ascites are arterial hypotensiondue to a decrease in peripheral vascular resistance, highcardiac output, and hypovolemia. The degree of portalhypertension in decompensated cirrhosis closely corre-lates with activity of the rennin–angiotensin system(RAS). Patients with high risk to develop hepato-renalsyndrome (HRS) have either a marked sodium andwater retention or present a paramount alteration ofsystemic circulation (low arterial pressure) and intenseactivation of vasoconstrictor systems (RAS and sympa-tic nervous system (SNS). The etiology of the hyperdy-namic circulation is still controversial, yet most likely, isinitiated by vasodilatation induced by an increase inactivity of endothelial-dependent and independentvasodilators, like nitric oxide (NO) and prostaglandins(PG) (Tokuyama et al., 2002). Consequently, a hallmarkof HRS is a progressive renal cortical vasoconstriction(Cardenas et al., 2003) and the circulatory dysfunction isa main precursor of severe complications in cirrhosis.Thus, HRS is associated with bad prognosis (Arroyoet al., 2002). Given the fact that patients with type-2HRS have a small probability of survival (months), butpatients with type-1 have an even slighter probability(days or weeks) (Gines et al., 2003); appropriate clinicalhandling of HRS patients becomes paramount andrequires the use of parameters with prognostic factor topredict the evolution to decrease complications (Brigliaand Anania, 2002; Barada, 2004). Nonetheless, themechanisms of renal vasoconstriction taking place inHRS remain to be elucidated, and it may possibly be ofmultifactorial origin (Lancestremere et al., 1962).Although different experimental models have been usedto explain and study the pathogenesis of renal altera-tions associated with hepatic damage, they make use ofonly some of the biochemical and hemodynamicalterations commonly found in HRS patients (Allisonet al., 1978; Better and Massry, 1972; Kountouras et al.,1984; Poo et al., 1997; Jeyarajah et al., 2003).

Recently, an experimental animal model with highsimilarity to human HRS characteristics was developedin our laboratory. Such a model is raised by theadministration of an acute CCl4 dose to alreadycirrhotic rats (Rincon et al., 1997). In this experimentalmodel, a significant decrease of mean arterial pressure(MAP) was observed, followed by renal dysfunctioncharacterized by a decrease in the glomerular filtrationrate (GFR) and sodium urinary concentration withrenin activity induction (Rincon et al., 1999). In thatmodel, data produced suggested that renal damage was

not directly produced by CCl4. However, in order to ruleout the possibility that CCl4 causes part of the renaldamage, a surgical alternative to induce acute hepaticdamage was devised in rats with established cirrhosis.

Thus, cirrhotic rats were subjected to bile ductligation (BDL) as an alternative experimental model toproduce a superimposed acute liver damage (ALD) inwhich hepatic biochemical and histopathologic altera-tions have been observed in rats (Schaffner et al., 1971).Depending on the obstruction time, an acute or chronichepatic damage can take place.

This experimental model of decompensated cirrhosisis important because a hallmark of HRS is a progressiverenal cortical vasoconstriction and the circulatorydysfunction is a main precursor of severe complicationsin cirrhosis and HRS is associated with a bad prognosis.

Materials and methods

Induction of liver cirrhosis

One hundred and fifty male Wistar rats (Animalfacilities, CUCS, U. de G.) weighing 60–80 g, were used(Fig. 1A). Liver cirrhosis was induced by intraperitoneal(I.P.) injection of 0.20ml of a mixture of CCl4 (MerckCompany, Darmstadt, FRG) and mineral oil (SigmaChemical Company, St. Louis, MO, USA) three timesper week during 8 weeks. The percentages of CCl4 inmineral oil (v/v) were increased progressively as follows:week 1, 13%; week 2, 16%; week 3, 20%; and weeks4–8, 25% (Ehrinpreis et al., 1980). Rats were fed withstandard rat chow (Purina), and water ad libitum andthey were subjected to 12 h light–dark cycles. Animalswere housed in the animal facility of University ofGuadalajara and all animal studies were conducted inaccordance with the principles and procedures outlinedin the National Institutes of Health’s Guide for the Care

and Use of Laboratory Animals.

Animal groups

At the end of CCl4 chronic treatment, 135 cirrhoticanimals were randomly divided in two groups: sham-operated control (n ¼ 45) and BDL rats (n ¼ 90) and 15cirrhotic rats (Fig. 1A); this last group was used ascirrhotic control (no-sham, no BDL). In order to avoidexperimental caveats due to the use of ether, we dividedall animals in three different groups according to thetype of study to be carried out (Fig. 1B).

Bile duct ligation (BDL)

Rats were anesthetized with ethylic ether and, underaseptic conditions, subjected to either BDL as previously

ARTICLE IN PRESS

A

Weeks

60-80 g

1 2 3 4 5 6 7 8

CIRRHOTIC RATS

n=150

Initial body weight

SHAM (without BDL)

Bile Duct Ligated (BDL) n= 90

n= 45

n=15

n=135

BDL

SHAM

BDL

SHAM

BDL

SHAM

n=30

n=15

Hemodynamic studiesand plasma volume

Renin Hepatic and renal function

Cirrhotic (control)

Cirrhotic (control)

Cirrhotic (control)

n=5

n=30

n=15

n=5

n=30

n=15

n=5

B

C

(BDL)

0

n=6 in each time

1 x 3 5 7 x 9Days

n = 30 BDLn=5Cirrhotic

n = 15 SHAM n=3 in each time

CIRRHOTIC RATS n=15

x x

Fig. 1. Schematic representation of distribution groups and treatment. (A) represents chronic CCl4 treatment to induce cirrhotic rats

(n ¼ 150) (60–80 g initial body weight). After 8 weeks of treatment, 135 cirrhotic animals were randomly divided in two groups:

sham-operated control (n ¼ 45) and BDL rats (n ¼ 90) and 15 cirrhotic rats remained with no surgery. The last group was used as

cirrhotic control (day 0). (B) shows distribution between groups. All animals were divided in three groups according to the type of

study to be carried out: (1) hemodynamic and plasma volume; (2) plasma renin activity and concentration; (3) hepatic and renal

function. (C) represents sacrifice times in all groups at 1, 2, 3, 4, and 5 days after acute liver damage.

S. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195 187

described (Lee et al., 1986; Rodriguez-Garay, 2003) orsham surgery. After a midline abdominal incision close tothe sternum, the common bile duct was identified, a doubleligature was made with 3/0 silk and after that, a cut wasmade between both ligatures (Liu et al., 2003). In cirrhotic-sham-operated (CR-SHAM) rats, the bile duct wasexposed and gently mobilized with a sterile cotton-coveredapplicator before closure of the abdominal wound.

Renal function assays

At the end of chronic CCl4 treatment, cirrhotic ratswere placed in metabolic cages (Nalgene Company,Rochester, NY, USA) 3 days before surgery foracclimatization period. Thirty animals underwent BDLand 15 were used as control or Sham rats. Twenty-four-hour urine samples were collected from all experimentalanimals to determine total urinary volume, urinarysodium, creatinine, and osmolarity. Six animals of BDLgroup and three CR-SHAM-operated control rats werekilled at 1, 3, 5, 7, and 9 post-operative days (Fig. 1C).

Biochemical studies

For these determinations, another group of 30animals with BDL and 15 shams were used. Rats weredeprived of food, but not water overnight, and werekilled by decapitation to perform liver and renalfunction tests. Blood samples were immediately centri-fuged at 4 1C, and plasma was kept at �20 1C until reninactivity and plasma renin concentration (PRC) assays

were performed. Serum was used to measure totalproteins, albumin, and total bilirubin by colorimetricmethod (Merck Company, Darmstadt, FRG). Aspar-tate aminotransferase (AST), alanine aminotransferase(ALT), and alkaline phosphatase activities were deter-mined by colorimetric enzymatic method (MerckCompany, Darmstadt, FRG). Serum and urine sodiumwere measured by flame photometry (model PFP7C,Jenway LTD, England). Blood urea nitrogen (BUN)and serum creatinine were measured by colorimetricmethod using an autoanalyzer (Technicon RA-1000,Technicon Instruments Corporation, Tarrytown, NY,USA). Creatinine clearance was used to estimate GFR.Plasma renin activity (PRA) was measured by radio-immunoassay (RIA) of angiotensin I (AI) (Du PontNew England Nuclear, Boston, MA, USA) producedfor endogenous renin substrate after incubation ofplasma at pH 6.0 for 1 h at 37 1C in the presence ofangiotensinase inhibitors. PRC was measured by RIAof AI after incubation of plasma (1 h at 37 1C) with anexcess of renin substrate (Ibarra-Rubio et al., 1990).Urine and serum osmolarity were performed using amicro-osmometer (model 5004. Precision Systems Inc.,Sudbury, MA, USA); standards of calibration were usedto gauge the apparatus and osmolarity was measured bypoint of freezing after freezing of the samples.

Hemodynamics and plasma volume measurements

For hemodynamic studies, an additional group of 30animals with BDL and 15 sham rats were used (Fig. 1B).

ARTICLE IN PRESSS. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195188

These animals were conscious at all times during theprocedure. In other words, they were not anesthetized tocarry out these assays. MAP and heart rate weremeasured according to the indirect blood pressurerecording technique using an Electro-sphyngoman-ometer for rats (PE-300, Narco Bio-Systems, Austin,TX, USA) which involves the occlusion of circulation ofthe tail with an annular cuff and the detection of pulsewith a pneumatic pulse transducer as the cuff pressure islowered. At the end of the hemodynamic studies, plasmavolume was measured by the Evan’s Blue dilutionmethod to discard hypovolemia (Wang, 1959). Forplasma volume measurements rats were anesthetizedwith ethylic ether later, and a catheter was placed intothe femoral artery. Evan’s Blue dye solution 0.2ml (5%of Evan’s blue dye W/V in sterile isotonic saline) wasinjected. Before injecting the dye, a blood sample wasobtained as basal value and 15min after introducingEvan’s solution another sample was draw to quantifybody dye distribution.

Plasma dye concentration was read at 610 nm on aspectrophotometer (Beckman DU 650) and plasmavolume was determined using a curve of concentrationwith the dye as reported before (Lieberman et al., 1969).

Histopathological analysis

The liver and kidneys were obtained just from thegroup of animals that were used for renal function tests.Small portions of these organs were obtained forhistological evaluation and immediately were fixed inbuffered para-formaldehyde (0.1M, pH 7.4) at roomtemperature (Oberti et al., 1997). Representative frag-ments were washed in phosphate buffered and dehy-drated in graded concentrations of ethanol; thefragments were embedded in Paraplas-plus (Oxford,Labware, St. Louis MO, USA). From each liver andkidney, 4-mm-thick sections were obtained and slideswere stained with hematoxylin and eosin. Microscopyevaluation was performed by two pathologists blindedto the study.

Table 1. Hepatic function tests in cirrhotic plus BDL rats

Group Total protein (g/100ml)

CR-SHAM 5.7870.37

CR (day 0) 5.9470.44

1 day 5.8070.20

3 days 5.9070.30

5 days 5.8070.30

7 days 5.8070.21

9 days 5.2070.40

Hepatic function tests were performed in age-matched cirrhotic sham-ope

cirrhotic rats with acute liver damage 1, 3, 5, 7, and 9 days after bile duc

significant at po0:05 when compared with the cirrhotic-sham group.

Statistical analysis

Results are expressed as mean7S.D. of at least sixdeterminations. Data were analyzed for statisticalanalysis using ANOVA. Differences were consideredto be statistically significant when po0:05.

Results

Hepatic function tests

Hepatic function was evaluated at progressive timepoints (1, 3, 5, 7, and 9 post-operative days). For totalserum protein, albumin included, no significant differ-ences were observed between BDL group and CR-SHAM controls. However, a statistically significantincrease (po0:05) in total bilirubin concentration in ratswith BDL was observed since day 1 (Table 1), and thesevalues remained elevated during 7 subsequent days toBDL. AST, ALT, and alkaline phosphatase levels wereincreased (po0:05) in rats with BDL (Fig. 2). Theincrease in these enzyme levels were approximately four-fold during the first 5 days, returning to initial controlvalues 7 days after BDL.

Renal function tests

Kidney function tests showed a significant reduction(po0:05) in urinary volume and urinary sodiumconcentration 3 and 5 days after BDL (Fig. 3). Thesedrops in urinary volume and urinary sodium concentra-tion were statistically significant. Moreover, when weseparate dead of surviving animals values, we founda higher significant difference (po0:05) among them(Fig. 3). Serum creatinine and BUN values increased(po0:05) since the first days and remained elevated upto day 7 subsequent to BDL, compared with controlanimals and initial time (day 0). Then, these valuesreturned to normal levels on day 9 post-BDL. Mean-time, GFR was substantially decreased from day 1 to

Albumin (g/100ml) Total bilirubin (g/100ml)

3.1870.19 0.370.2

3.1870.21 0.570.2

2.7070.23 5.270.6*

2.7070.25 7.371.4*

2.8070.15 7.271.3*

2.9070.16 7.171.8*

2.9070.15 2.570.4*

rated rats (control) (CR-SHAM), cirrhotic rats (CR) (day 0) and in

t ligation (BDL). Results are expressed as mean7S.D. *Statistically

ARTICLE IN PRESSA

LAN

INE

AM

INO

TR

AN

SF

ER

AS

E

(IU

/L)

AS

PA

RT

AT

E

AM

INO

TR

AN

SF

ER

AS

E

(IU

/L)

ALK

ALI

NE

PH

OS

PH

AT

AS

E

(IU

/L)

DAYS AFTER BILE DUCT LIGATION

0

100

200

300

400

0

100

200

300

400

500

*

* *

* *

*

*

0

50

100

150

200

CR-SH 0 1 3 5 7 9

*

* *

*

*p < 0.05

Fig. 2. Levels of ALT, AST, and AP in cirrhotic sham-operated

rats (CR-SH) (n ¼ 3) empty circle, cirrhotic rats (day 0) (n ¼ 5),

and cirrhotic rats with acute liver damage 1, 3, 5, 7, and 9 days

after bile duct ligation (BDL) (n ¼ 6) in full circles. ALT, AST,

and AP levels were assessed by conventional serum biochemical

determinations. IU/L ¼ International Units/ Liter. Results are

expressed as mean7S.D. *po0:05 vs. cirrhotic-sham group.

0

0.5

1

1.5

2

2.5

3

3.5

4

Urin

ary

Vol

ume

(ml/k

g/h)

*

**

******

*

Urin

ary

Sod

ium

(m

Eq/

Kg/

h)

0

0.1

0.2

0.3

0.4

0.5

0.6

*

**

*

****

*

*

CR-SH 975310

DAYS AFTER BILE DUCT LIGATION*p < 0.05

**p < 0.05

vs.

vs.

Fig. 3. Urinary volume and urinary sodium excretion in

cirrhotic sham-operated rats (CR-SH) (n ¼ 3) empty circle,

cirrhotic rats (day 0) (n ¼ 5) and cirrhotic rats with acute liver

damage 1, 3, 5, 7, and 9 days after bile duct ligation (BDL)

(n ¼ 6) in full circles. Twenty-four hours urine samples were

collected during 9 days. Total urinary volume of each rat was

measured and tested for sodium. The gray circles represent the

values of the animals that died before being sacrificed. Results

are expressed as mean7S.D. *po0:05 vs. cirrhotic-sham

group. **po0:05 when we compared survived vs. died animals

values at the same experimental time.

S. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195 189

day 7; returning to normal values on day 9 post-BDL(Fig. 4). PRA and PRC (Fig. 5) were increased (po0:05)after BDL peaking at 3–5 days after BDL, returning tocontrol levels at 9 days post-surgical procedure.Proteinuria values were not statistically significantamong groups. Urinary osmolarity increased (po0:05),while serum osmolarity decreased (po0:05) in the BDLanimals (Table 2).

Hemodynamic studies

MAP was significantly lower 48 h after BDL com-pared to CR-SHAM group at the same time(64.772.3mmHg vs. 83.374.5mmHg) (po0:05) andpersisted down 72 h after. However, 5 days post-BDL,arterial pressure recovered to values of 80.074.8mmHg(po0:05). These animals did not show significantchanges in plasma volume after BDL, 39.075.3ml/kg

compared with control group values of 40.074.4ml/kg.These evidences suggest that renal alterations observedwere not due to hypovolemic state.

Histopathologic studies

Morphologic abnormalities were observed in livers ofcontrol cirrhotic rats (Fig. 6a), where liver tissue showedfibrosis, inflammation and steatosis, bridging fibrosis,and bands of connective tissue (Fig. 6a). No morpho-logic abnormalities were observed in livers of normalrats (data not shown). Fibrosis was confirmed by twoindependent board certificated pathologist. Rat liverwith cirrhosis 1 day after BDL (Fig. 6b), exhibited aturbid swelling (pale cytoplasmic and fine granulations)and diffuse vacuolization of the hepatocytes (Fig. 6c).Three days after BDL, livers exhibited fibrosis, cloudyswelling, hiperchromatic nuclei, and cytoplasmic vacuo-lization (Fig. 6d). Five days after BDL, livers showeddiffuse vacuolization, cloudy swelling, and perinuclear

ARTICLE IN PRESS

0

0.3

0.6

0.9

1.2

1.5

1.8

0

20

40

60

Ser

um C

reat

inin

e (m

g/dl

)B

UN

(m

g/dl

)G

FR

(m

l/min

)

* **

*

*

**

*

DAYS AFTER BILE DUCT LIGATION

CR-SH 9753100

0.2

0.4

0.6

0.8

1

*

**

*

*p < 0.05

Fig. 4. Serum creatinine, blood urea nitrogen (BUN) and

glomerular filtration rate (GFR) in cirrhotic sham-operated

rats (CR-SH) (n ¼ 3) empty circle, cirrhotic rats (day 0)

(n ¼ 5), and cirrhotic rats with acute liver damage 1, 3, 5, 7,

and 9 days after bile duct ligation (BDL) (n ¼ 6) in full circles.

Creatinine clearance was used to estimate GFR. Results are

expressed as mean7S.D. *po0:05 vs. cirrhotic-sham group.

P R

A(n

g A

1/m

l/h)

P R

C (

ng A

1/m

l/h)

DAYS AFTER BILE DUCT LIGATION

0

5

10

15

20

25

30

35

40

** *

*

CR-SH 0 1 3 5 7 9

20

40

60

80

100

*

* *

*

*

*p < 0.05

Fig. 5. Plasma renin activity (PRA) and plasma renin

concentration (PRC) in cirrhotic sham-operated rats (CR-

SH) (n ¼ 3) empty circle, cirrhotic rats (day 0) (n ¼ 5), and

cirrhotic rats with acute liver damage 1, 3, 5, 7, and 9 days

after bile duct ligation (BDL) (n ¼ 6) in full circles. Plasma

renin activity (PRA) was measured by determining angiotensin

I as indicated in Material and Methods. Results are expressed

as mean7S.D. *po0:05 vs. cirrhotic-sham group.

Table 2. Proteinuria and urinary and plasma osmolarity in

cirrhotic plus BDL rats

Group Proteinuria

(g/100ml)

U Osm

(mOsm/kg)

P Osm

(mOsm/kg)

CR-SHAM 770781 1053739 352721

CR (day 0) 790779 1074740 349730

1 day 920742 1775792* 237721*

3 days 847775 18197134* 238711*

5 days 886778 1752739* 238722*

7 days 896745 1736753* 239721*

9 days 810759 1765755* 236724*

Urine osmolality (U Osm) and plasma osmolality (P Osm) in cirrhotic-

sham (CR-SHAM), cirrhotic (CR) (day 0) and in cirrhotic rats with

acute liver damage 1, 3, 5, 7, and 9 days after bile duct ligation (BDL).

Results are expressed as mean7S.D. *Statistically significant at

po0:05 when compared with the cirrhotic-sham group.

S. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195190

halos (Fig. 6e); 7 days after BDL rat livers exhibitedfibrosis, hiperchromatic nuclei, and diffuse vacuoliza-tion (Fig. 6f) (Table 3).

Kidneys of control rats and cirrhotic-sham showed nosignificant macroscopic differences (data not shown).Cirrhotic kidneys showed minimal change comparedwith normal kidney (Fig. 7a). Kidneys of cirrhotic rats1, 3, and 5 days after BDL, showed glomerularmesangial hipercellularity (Figs. 7b–d), with glomerularcells displaying hiperchromatic nuclei and cloudyswelling. Hydropic degeneration was seen in the con-voluted tubules 7 and 9 days after BDL (both, proximal,and distal), and perinuclear halos were observed (Fig. 7eand f) (Table 4).

Discussion

The pathogenesis of renal function alteration asso-ciated to hepatic damage commonly found in HRSpatients remains to be completely unraveled. That is the

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Fig. 6. Histopathological liver analysis. No morphologic abnormalities were observed in livers of normal rats (without liver

damage) (data not shown). Morphologic abnormalities were observed in livers of control cirrhotic rats (day 0) (Fig. 6a), where liver

tissue showed fibrosis, inflammation, and steatosis, bridging fibrosis and bands of connective tissue. In order to show fibrosis and

collagen bands, the picture was taken at lower magnification (Fig. 6a) (225� ). Fibrosis was confirmed in all cirrhotic rats. Rat liver

with cirrhosis after 1 day of BDL (Fig. 6b) exhibited a turbid swelling (cytoplasmic pale and fine granulations) and diffuse

vacuolization of the hepatocytes. At 3 days after BDL liver exhibited fibrosis, cloudy swelling, hiperchromatic nuclei, and

cytoplasmic vacuolization (Fig. 6c). At 5 days after BDL liver showed diffuse vacuolization, cloudy swelling, and perinuclear halos

(Fig. 6d). At 7 days after BDL rat livers exhibited fibrosis, hiperchromatic nuclei, and diffuse vacuolization (Fig. 6e). Nine days after

BDL livers had steatosis; less inflammatory cells and collagen deposits are visible (Fig. 6f). Pictures (a–e) were taken at 225� .

S. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195 191

main reason why its accurate diagnosis is cumbersomeand represents a challenge to achieve. The naturalhistory of HRS onset in cirrhotic patients with asciteshas been described recently (Gines et al., 1993). Patientswith high risk to develop HRS have either a markedsodium and water retention or present a paramountalteration of systemic circulation (low arterial pressure)

and intense activation of vasoconstrictor systems(rennin–angiotensin and SNS).

Unfortunately, no specific test can be used in thediagnosis of HRS. The International Club of ascites hasrecently proposed a series of approaches that should betaken into account for HRS diagnosis (Arroyo et al.,1996). Therefore, an experimental model suitable to

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Table 3. Degree of histopathological liver changes in cirrhotic plus BDL rats

Group Clowdy swelling Vacuolar degeneration Hiperchromatic nuclei Necrosis

CR-SHAM 0 0 0 0

CR (day 0) 0 0 0 0

1 day 100 100 66.6 16.7

3 days 66.6 100 100 33.3

5 days 33.3 100 100 66.3

7 days 33.3 100 33.3 16.7

Cirrhotic-sham (CR-SHAM), cirrhotic (CR) (day 0) and in cirrhotic rats with acute liver damage 1, 3, 5, 7, and 9 days after bile duct ligation (BDL).

Values are expressed as percentage of rats with change.

Fig. 7. Histopathological kidney analysis. Kidneys of control rats and cirrhotic-sham were indistinguishable between them (data not

shown). Microscopic examination exhibited light changes in the kidney of cirrhotic rats (Fig. 7a). Pictures seem of normal kidneys.

Kidneys of cirrhotic rats 1, 3, and 5 days after BDL, showed glomerular mesangial hipercellularity (Figs. 7b–d), glomerular cells

exhibited hiperchromatic nuclei and cloudy swelling. Hydropic degeneration was seen in the convoluted tubules (both, proximal and

distal), and perinuclear halos were observed (Figs. 7e and f). Pictures (a–e) were taken at 225� .

S. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195192

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Table 4. Degree of histopathological kidney changes in

cirrhotic plus BDL rats

Group Hiperchromatic

nuclei

Clowdy

swelling

Hydropic

degeneration

CR-SHAM 0 0 0

CR (day 0) 0 0 0

1 day 100 100 33.3

3 days 100 100 33.3

5 days 66.3 66.3 16.3

7 days 66.3 66.3 16.3

Cirrhotic-sham (CR-SHAM), cirrhotic (CR) (day 0) and in cirrhotic

rats with acute liver damage 1, 3, 5, 7, and 9 days after bile duct

ligation (BDL). Values are expressed as percentage of rats with change.

S. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195 193

analyze the sequence of physiopathologic events beforeand during the acute phase of this syndrome becomeshandy.

The results of the present work show that ALDinduced by BDL in cirrhotic rats produces importantchanges in the renal function, similar to those observedin cirrhotic patients with renal function alteration(decompensated cirrhosis). In agreement with previousobservations (Mayoral et al., 1999), association betweenhemodynamic and renal function alterations was de-tected in all bile-duct ligated rats. Besides, our studyreveals that cirrhotic rats with biliar obstructionpresented significant decrease in MAP, which wasaccompanied of a statistically significant decrease inGFR, similar to observed by other investigators (Shashaet al., 1976; Bosch et al., 1983) using dogs with BDLand, with our previous studies with cirrhotic rats plusALD induced with CCl4 (Rincon et al., 1999; Islas-Carbajal et al., 2005).

In this study, sodium concentration in animalsdisplayed a sharp decrease in urinary excretion sincethe third day of biliary obstruction. Furthermore, asignificant decrease in the urinary and sodium excretionand oliguria at 72 h after obstruction were observed, andmaintained up to the fifth day. Thus, our results aresuggestive that an ALD of different etiology (surgical)or induced by CCl4 can cause an important damage thateventually results in renal function deterioration.Histopathological changes became apparent at the thirdday of biliary obstruction in which glomeruli withmesangial hipercellularity took place in the absence oftubular necrosis; with portal inflammation and prolif-eration of biliary ductules. Morphological examinationsrevealed that BDL rats underwent a loss of hepaticstructure. Proliferation of portal and periportal biliaryductules with disorganization of the hepatocytes platesdilated portal spaces and areas of inflammatoryinfiltrate were observed (Jin et al., 2005).

Hiperbilirubinemia and increases in ALT and ASTactivity were observed 1 day after BDL in cirrhotic rats.

Hemodynamic studies showed alterations in splenic andsystemic circulations, changes that have been describedin different animal species before (Allison et al., 1978;Better and Massry, 1972; Kountouras et al., 1984; Pooet al., 1997; Jeyarajah et al., 2003).

In agreement with previous observations, (Mayoralet al., 1999) association between hemodynamic andrenal function alterations was detected in all bile-ductligated rats. In this study, sodium concentration inanimals displayed a sharp decrease in urinary excretionsince day 3 of biliary obstruction. Histopathologicalchanges became apparent at the third day of biliaryobstruction in which glomeruli with mesangial hipercel-lularity took place in the absence of tubular necrosis;with portal inflammation and proliferation of biliaryductules.

No nephrotoxic effects in rats made cirrhotic after 12weeks of treatment with repeated small doses of CCl4were detected (Wensing et al., 1990), but in our previouswork with ALD induced by a single intragastric dose ofCCl4, decompensated cirrhosis was observed withhemodynamic and renal function alteration similar tothose observed in patients with HRS (Rincon et al.,1999). In the present work, serum bilirubin and liverenzymes were also significantly increased, similar tothose reported (Schaffner et al., 1971; Lee et al., 1986;Rodriguez-Garay, 2003; Wensing et al., 1990) for thesame BDL model.

It has been reported that BDL reduces antioxidantcell defenses, diminishes liver concentrations of glu-tathione and increases free-radical formation (Mayoralet al., 1999; Singh et al., 1992). Additionally, interac-tions between reactive oxygen species and reactivenitrogen species, mainly NO, could mediate some ofthe pathological effects associated with chronic inflam-mation. Mayoral et al. (1999) showed in BDL animalsthat there was a clear increase in liver inducible nitricoxide synthase (iNOS) expression, with no changes inconstitutive NOS (cNOS) levels (Mayoral et al., 1999).In our previous work, we evaluated NO role in renalfailure induced during decompensated cirrhosis bymeans of NOS expression (Islas-Carbajal et al., 2005).We found that renal endothelial nitric oxide synthase(eNOS) expression diminished and renal iNOS expres-sion increased during ALD without use of bothinhibitors (L-NAME, and aminoguanidine). Theseresults suggest that iNOS isoform is participating inthe kidney as an alternative pathway with an increasedNO production in kidney dysfunction as a consequenceof decompensated cirrhosis (Islas-Carbajal et al., 2005).Our former studies concurred with the present workusing a cirrhosis-induced kidney failure model andsuggest that iNOS expression might be participatinginducing NO production).

Although ALD caused by CCl4 is transient, it occursdifferently with BDL. In this study we could show

ARTICLE IN PRESSS. Rivera-Huizar et al. / Experimental and Toxicologic Pathology 58 (2006) 185–195194

that when liver damage is established (cirrhosis) andadditional ALD is superimpose in the first daysresembles in a similar way what happens in patientswith hepatorenal syndrome.

In conclusion, cirrhotic rats subjected to BDL showedlow arterial pressure and the intense arterial vasodilata-tion originates a filled vascular markedly insufficientproducing further sodium and water retention. After-wards, a compensated mechanism of vasoconstrictionincreasing low pressure take place before renal damageis produced. This model can be useful to understandmechanism of pathogenesis of HRS and to design newand specific pharmacological therapy.

Acknowledgments

The authors greatly appreciate the support ofpersonnel at CUCS Animal Facilities, especially thehelp of Dr. Pedro Dıaz. The authors are also indebted toMario Cardenas and Rosa Lina Torres-Rodrıguez fortheir invaluable technical help.

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