Kidney Preservation: Review of Present and Future Perspective

F. Catena, F. Coccolini, G. Montori, C. Vallicelli, A. Amaduzzi, G. Ercolani, M. Ravaioli, M. Del Gaudio,R. Schiavina, E. Brunocilla, G. Liviano, G. Feliciangeli, and A.D. Pinna

0041-1345/1http://dx.doi

3170

ABSTRACT

One of the main problems in transplant surgery is the preservation of the organ during thecold ischemic time. The interrupted blood supply triggers a cascade of biological modifi-cations resulting in cell death, which predisposes to discharge of a large quantity of toxicmetabolites at the moment of organ reperfusion. Many approaches have been studied toprevent the toxic processes. Immediately after procurement, kidneys are flushed with thesesolutions. Two main: techniques of organ preservation are cold static storage and hypo-thermic machine perfusion (HMP). Based on age and comorbidities, individuals can begenerally divided into 2 groups: ideal and marginal donors. Characteristics of organs frommarginal donors are associated with an increased rate of delayed graft function andprimary graft nonfunction (PNF), which reduce transplant survival and increase the acuterejection risk. In the last 20 years, the United Network of Organ Sharing has reporteda 170% increase in deceased donors older than 50 years of age. Techniques of perfusionhave been demonstrated to play a pivotal role in graft function after transplantation. Somestudies suggest that HMP may improve outcomes after transplantation.

From theGeneral and TraumaSurgery Department (F.C.), ParmaHospital, Parma, Italy; General, EmergencyandTransplantSurgeryDepartment (F.Co.), Sant’Orsola-Malpighi University Hospital,Bologna, Italy;General andEmergencySurgeryDepartment (G.M.),Ospedali Riuniti of Bergamo, Bergamo, Italy; Unit of Urology (C.V.,A.A., G.E., M.R., M.D.G., A.D.P., R.S., E.B.), Sant’Orsola-MalpighiUniversity Hospital, Bologna, Italy; and Internal Medicineand Nephrology Department (G.L., G.F.), Sant’Orsola-MalpighiUniversity Hospital, Bologna, Italy.

Address reprint requests to Fausto Catena, MD, PhD, Generaland Trauma Surgery Department, Parma Hospital, Parma, Italy.

KIDNEY TRANSPLANTATION is universally consid-ered to be the treatment choice for established renal

failure or end-stage renal disease. Successful transplantationdepends on a sequence of events related to donor selection,graft preservation, surgical implantation, and recipienttreatment. During this progression of events the kidney isexposed to a series of extreme nonphysiologic insults. At thetime of organ retrieval from the donor, the blood supply isinterrupted, leading to ischemic insults to renal function.The ischemic period needs to be as short as possible,because damage resulting there from is extremely detri-mental to the organ.The ischemic period is divided into warm ischemic time

(WIT) and cold ischemic time (CIT). The former is definedas the period when the organ is bereft of blood, oxygen,and nutrients while cellular metabolism continues at fullstrength, whereas the latter is the moment during whichkidney is conserved in a hypothermic solution to slowcellular activity and reduce the production of toxicmetabolites.1

Removal of the kidney from the circulatory system leadsto an absence of oxygen and to anaerobic metabolism. Thefirst biochemical manifestation of ischemia is a rapiddecrease in intracellular adenosine triphosphate (ATP)levels. Impaired Na/K ATPase activity results in passiveentrance of Na into the cell with subsequent inflow of water.

3/$esee front matter.org/10.1016/j.transproceed.2013.02.145

Cells swell and form protruding pockets. Within 4 hoursnearly 95% of ATP has disappeared; energetic aerobicmetabolism is replaced by anaerobic glycolysis, productionof lactic acid, and consequently, cellular acidification.Severe acidosis activates phospholipases and proteasescausing lysosomal damage and eventually cell death.Furthermore, cold preservation activates matrix metal-loproteinases leading to endothelial cell detachment fromthe underlying matrix. Reduced metabolic activity becomescrucial to prevent irreparable organ damage. Reducing thecore temperature of the kidney to 4�C reduces enzymeactivity and metabolism by 58% in most cells.2 During thecold ischemic period it is possible to accomplish tissue

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Transplantation Proceedings, 45, 3170e3177 (2013)

KIDNEY PRESERVATION 3171

typing and crossmatching as well as organ allocation andtransportation and preparation of the recipient.Another crucial phase occurs at the time of reperfusion,

which is not devoid of side effects. The graft switches froma cold ischemic profile in which metabolism is minimalto restoration of blood flow with resumption of cellularactivity. The damage that incurs at this time is defined as theischemic-reperfusion injury. This complex cascade of eventsproduces further damage with consequent activation ofinflammatory mediators, damage to endothelium as well asactivation, and infiltration of neutrophils with generation ofreactive oxygen species.3 Cellular edema compresses thecapillary network limiting blood flow, elevates calciumconcentrations, and impairs contractile structures, oxygendelivery, and leukocyte, funoteres dependent upon thenewly restored blood flow. Moreover, it releases inflam-matory factors that result in the production of superoxideanions and other reactive oxygen species, which causes

Table 1. Composition of the M

Component UW HTK IG

Collides (g/L)HES (g/L) 50 d

PEG-35 (mmol/L) d d 0Impermeants (mmol/L)

Glucose d d

Citrate d d

Mannitol d 38Trehalose d d

Lactobionate 100 d 1Sucrose d d

Raffinose 30 d

Buffers (mmol/L)K2HPO4 d d

KH2PO4 25 d

NaHCO3 d d

NaH2P04 d d

Na2HP04 d d

Histidine d 180Electrolytes (mmol/L)

Sodium 25 15 1Potassium 120 9Chloride 20 32Calcium d 0.0015Magnesium d 4Magnesium sulphate (MgSO4) 5 d

ROS scavengers (mmol/L)Glutathione 3 d

Allopurinol 1 d

Tryptophan d 2Additives (mmol/L)

Adenosine 5 d

Ketoglutarate d 1Aminoglutaminic acid (mmol/L) d d

OH (mmol/L) d 310Osmolarity (mOsm/kg) 320 310 3pH 7.2e7.4 7.02e7.2 7.

UW, University of Winsconsin solution; HTK, histidine-tryptophan-ketoglutarate sphosphate-buffered sucrose (experimental trial only); HOC, hypertonic citrate/Marpolyethylene glycol; ROS, reactive oxygen species.

severe damage to cellular membranes phospholipids,nucleic acids and proteins. Because chemical injury linkedto preservation strongly correlates with immediate and long-term kidney function,4 preservation methods and solutionsmust seek to counteract the ischemic injury.Static cold storage (CS) and hypothermic machine

perfusion (HMP) are the most commonly used techniquesfor kidney preservation. This essay reviews the current useof preservation techniques, solutions, and methods inkidney transplantation.

MAIN PRESERVATION SOLUTIONS

After the introduction of static CS by Collins in 1969,prolonged kidney preservation became feasible. Nowadays,the main solutions for kidney preservation1,5 are Universityof Wisconsin (UW), histidineetryptophaneketoglutarate(HTK), Celsior, Institute George-Lopez (IGL-1), Polysol,

ain Preservation Solutions

L-1 Celsior Polysol EC PBS HOC

d d d d d d

.03 d 20 d d d

d d d 195 d d

d d d d d 80d 60 d d d 185d d 5.3 d d d

00 80 d d d d

d d d d 140 d

30 d 3.2 d d d

d d 15 d d

25 d d 42.5 d d

d d d 10 d d

d d d d 13 d

d d d d 56 d

d 30 6.3 d d d

20 100 120 10 125 8425 15 15 115 d 79d 42 d 15 d d

0.5 0.25 d d d d

d 13 d d d d

5 d d d d 40

3 3 5.6 d d d

1 d d d d d

d d d d d d

5 d 5 d d d

d d d d d d

d 20 d d d d

d 242e368 d d d d

20 320 d d d d

2e7.4 7.3 d d d 7.1

olution; IGL-1, institute-George-Lopez solution; EC, EuroCollins solution; PBS,shalls solution (experimental trial only);48e50 HES, hydroxyethyl starch; PEG,

3172 CATENA, COCCOLINI, MONTORI ET AL

EuroCollins, hypertonic citrate or Marshall solution(HOC), and phosphate-buffered sucrose (PBS) (Table 1).These solutions are intracellular-type or extracellular-type,considering their electrolyte composition.6 Intracellularsolutions are characterized by the high potassium and lowsodium concentrations necessary to prevent cell swelling.During hypothermia Naþ/Kþ-ATPase is inactive. There-fore, a solution in the extracellular space with an Naþ/Kþ

balance equal to the intracellular compartment reachesDonnan equilibrium preventing sodium and chloride fromentering the cell.7 Intracellular-type solutions such UW arecurrently considered to be important to preserve cellviability.7,8 The extracellular-type, such HTK, has lowpotassium and high sodium concentrations. Recent studieshave suggested that this composition is equal to, or evenbetter than, the intracellular type, as potassium can inducevasospasm.9,10

University of Winsconsin Solution

This medium is considered to be the gold standard amongorgan preservation solutions.11 UW combines metabolicallyinert substrates, such as lactobionate and raffinose, withhydroxy-ethyl starch (HES) acting as a colloid as well as anATP precursor (adenosine) and oxygen radical scavengers(glutathione and allopurinol).5 The principal benefit of thissolution is that it shows a good outcomes of short- and longterm kidney preservation. Its disadvantages are due to thehigh viscosity that compromises the microcirculation, andthe high potassium concentration that case produce vaso-constriction with hyperaggregation of HES.5,12 Two kinds ofUW solution have been utilized for HMP and for CS. In CS,UW-CSS is the main solution, while UW-G is used forHMP. Lactobionate is replaced by gluconate and thepotassium level is reduced in UW-G, achieving a consis-tency move like an extracellular solution.5

Histidine-Tryptophan-Ketoglutarate Solution

HTK introduced in 1980 by Bretschneider, was originally asa cardioplegic.5 HTK contains histidine and tryptophan asmembrane stabilizators as well as antioxidants and keto-glutarate for anerobic metabolism. HTK studies have shownconflicting results.14,36a,35 However, its low viscosity mayimprove microperfusion. Because of this characteristic,HTK is recommended in HMP at low pressures and highperfusion volumes.5

Celsior Solution

Celsior solution is a colloid developed in 1994 and originallyused in heart transplantation, before being extended toother abdominal organs. It combines the osmotic efficacy ofUW (lactobionate and mannitol) with the potent bufferingability of HTK (histidine).5 Colloid solutions contain addedmacromolecules that 20 not pass the cellular membrane andthus prevent tissue edema caused by the hydrostatic pres-sure of HMP.

Institute-George-Lopez

IGL-1 a rather new preservation solution developed inFrance5 combines the advantages of UW and Celsior.Polyethylene glycol is bound to cell and tissue surfaces, thusstabilizing them from adverse interactions. Studies suggestthat this feature modifies the inherent immunogenicity ofdonor tissue as a consequence of ischemia-reperfusioninjury.13 Badet et al14 demonstrated a reduced incidenceof delayed graft function (DGF) compared with kidneyspreserved with UW. However, a recently published multi-center study showed no significant difference in DGFcomparing IGL-1 with UW.5,15

Polysol

Polysol has been recently introduced with the goal of facil-itating transplantation of ischemically damaged organs.5

Therefore, amino acids, vitamins, potent buffers, and anti-oxidants have been added to support metabolism underhypothermic conditions.16 The solution has also been testedduring HMP.16,17 A case study by Schreinemachers et al18

demonstrated a superior graft survival with Polysol com-pared with UW in a porcine renal autotransplant model.Further reports by the same group have shown beneficialeffects of Polysol also for kidneys damaged by warmischemia.19 Polysol is currently used in experimental trialsonly.16,17 While recent reports on Polysol seem promising,more clinical data are necessary to evaluate its efficacy andbenefits.

Hypertonic Citrate

HOC, or Marshall’s solution, an intracellular-mimickingmedium, is extensively used for clinical transplantation inthe United Kingdom and Australia.51,52 HOC uses mannitolas the impermeant and citrate as its buffer to aid calciumextension from the cell while maintaining a physiologic pH.As a hypertonic solution, HOC prevents the entry of fluidinto cells. Experimental trials using canine kidneys havedemonstrated it to be effective for preservation periods upto 72 hours.52,58

Phosphate-Buffered Sucrose

As shown in Fig 1, nowadays, in the United States the mostcommonly used solution is UW, while in Europe the mainsolutions are UW and HTK.

STATIC COLD STORAGE

Since the 1980s, the majority of organs have been preservedby static CS, which is still considered the gold standard inkidney preservation. CS is the technique according to whichthe kidney is flushed with a sterile preservation solutionusing hydrostatic pressure immediately after procurement.A catheter is inserted in the renal artery and the blood iscompletely washed out from the kidney. Thereafter, theorgan is left in cold perfusion solution in a disposable plasticbag, which is then covered with ice to maintain a tempera-ture between 4�C and 0�C.

Fig 1. Comparison among different types of perfusion solutions used in Europe and in the United States.20

KIDNEY PRESERVATION 3173

In 1963, Calne et al21 showed that simple cooling ofkidneys in iced water preserved renal function for 12 hours;because the reduced temperature showed cellular metabo-lism. Subsequent studies demonstrated the effectiveness ofpreservation solutions to prolong CIT significantly andimprove organ quality.22 In fact, hypothermia reduces of themetabolic activity, which finally leads to cellular degrada-tion. As a consequence of circulatory interruption, oxygen isgradually exhausted, shifting metabolism from aerobic toanerobic. Up to now the principal advantages23e25 of CShave been its simplicity and low cost allowing kidney pres-ervation for CITs longer than 35 hours before trans-plantation. As a result, CS can be applied in any hospital byany surgeon without complicated or long training, withoutspecific, expensive materials that might be difficult to diffusethroughout the community.Themain disadvantage of CS is the intrinsic impossibility to

apply a standardized parameter to evaluate the health statusof the organ and to measure ischemic damage in order topredict graft viability as an index of post-transplant outcome.

HYPOTHERMIC MACHINE PERFUSION

A machine perfusion system is a pump continuouslyperfusing the kidney with cold preservation solution.Immediately after procurement, the organ is attached to themachine via the renal artery, after surgical preparation tomake the seal airtight. Circulation of the perfusate is ach-ieved through a device that generates either continuous orpulsatile flow by a peristaltic pump. The kidney is perfused

with hypothermic preservation solution through the arteryusing a temperature between 0�C and 4�C. It providesa continuous supply of nutrients with or without oxygen aswell as eliminates toxic substrates and free radicalsproduced during the ischemic period.In 1967 Belzer developed a machine equipped with

a pulsatile pump to perfuse and preserve kidneys for 72hours, employing cryoprecipitate and oxygenated plasma atlow pressure and at a temperature of 6�C to 10�C. After theintroduction of CS by Collins, in 1969, interest in HMPdecreased, because of CS was more simple and economicand allowed preservation up to 30 hours.12

There are 2 main HMP devices. In the Europe LifePortkidney transporter (Organ Recovery System, from Chicagoand Brussel) and in America The Waters Medical RM3(from Rochester, Minn, USA). The former portablemachine perfusion system perfuses a single kidney withoutsupervision. It uses the KPS-1 preservation solution, a UW-type medium.34 The latter renal preservation system isnonportable that under supervision perfuses 2 kidneyssimultaneously.Numerous advantages of HMP have been reported in the

literature. This method may decrease vasospasm, whileproviding additional parameters, such as flow, resistance,temperature, and pressure, that allow an evaluate ofischemic damage and organ viability as predictors of post-transplant outcome.26e29 HMP offers the possibilities toimprove organ quality by pharmacologic and gene transfertherapies.26,30,31 Moreover, HMP maintains the hemody-namic stimulation of the vasculature, which plays a critical

Table 2. Maastricht Categories of Donors After Cardiac Death

Masstricht category Description

I Dead on arrival (uncontrolled)II Unsuccessful resuscitation (uncontrolled)III Awaiting cardiac arrest (controlled)IV Cardiac arrest while brain dead (controlled)

3174 CATENA, COCCOLINI, MONTORI ET AL

role under normal physiologic conditions. Notably, thispotential benefit of HMP remains poorly understood.5

In recent years the interest in HMP has increased dueto the greater number of organs from marginal donors,which are predisposed to primary graft nonfunction (PNF)and DGF.26,27,32 In fact, marginal grafts not only showcompromised quality, but also are more prone to ischemicinsults. Recent data suggest superior outcomes, when theseorgans undergo HMP.4,33,59,60 There are 2 main disadvan-tages of HMP: the great cost of perfusion devices and thedifficulty to provide adequate training for personnel.

MARGINAL DONORS

Due to the increased waiting lists for kidney transplantationbecause of modified qualifications and criteria for kidneydonation, it has been important to expand the donors poolduring the last decades. During the 1980s kidneys wereobtained only from healthy young individuals who weredeceased due to brain death, who were defined as “idealdonors.” Currently, after the reduced number of Idealdonors and the lengthened waiting lists, kidneys are alsoretained from older donors, defined as “marginal donors”.This donor category increased by 170% between 1988 and2005, as reported in The United Network of Organ Sharingdatabase,35,36,48 but this rate was not accompanied bya similar number of renal transplantations. Marginal donorsshow a 1.70-fold increased relative risk of graft loss. Thedefinition includes all donors aged at least 60 years evenwith no comorbidities, but also all 50 to 59 years with atleast 2 of these 3 conditions: cerebrovascular cause of death,renal insufficiency with terminal creatinine levels greaterthan 133 mmol/L,34 and/or history of hypertension.37

Marginal kidneys show an increased risk of DGF andPNF, which reduce graft survival and augment rejectionrisk. In fact, older donors display a physiologic loss ofnephrons worsening renal viability. Furthermore, the pres-ence of comorbidities as hypertension and cardiovasculardisorders significantly irrepair renal function. Finally, variouscauses of deathdcardiac death donors (DCD) or brain deathdonors (DBD)dtrigger different physiopathologic mecha-nisms. Kidneys from DCD may not be exposed to the samesevere hormonal disturbances that accompany braindeath.41,61

DCD are categorized according to the Maastricht criteria(Table 2) which contrast controlled (cardiac death expected,but criteria for brain stem death not fulfilled) with uncon-trolled (cardiac death unexpected) circumstances.34 Esti-mates for the pool of donors in categories II and III rangefrom 20% to 40% of the whole donor pool. The reason thatthese organ donors are underutilized is because CS is anineffective preservation method when WIT exceeds 20minutes.Kidneys from DCD, particularly those categorized as

uncontrolled, may experience long WIT. As a result, kidneysfrom DCD can show higher rates of DGF, wherein the graftdoes not function immediately or PNF, the graft never

functions, than those from DBD. In addition, PNF and earlygraft loss are associated with increased risk of death overthe ensuing months.34 While the severity of early graftdysfunction depends on many factors, it is predominatelyinfluenced by WIT- and CIT-related injuries in DCDdonors.38,39

ISCHEMIC CHANGES RELATED TO BRAIN DEATH

Many randomized trials have investigated the role ofpharmacologic preconditioning of DBD donors with diltia-zem, epoprostenol, dopamine, and other vasodilators. Forexample, diltiazem has been administered either to thedonor or in the preservation solution to improve graftfunction after kidney transplantation with disappointingresults. Evidence in experimental animals suggesting thatbrief ischemic periods protect the organ against longerischemia times was the foundation for randomized trials ofischemic preconditioning in liver transplantation. Thesetrials have shown disappointing outcomes. Remote ischemicpreconditioning offers several advantages: a brief period oftissue ischemia many protect distant organs. Remoteischemic preconditioning is feasible before organ recoveryand a randomized trial is ongoing. Although clinical inves-tigations are difficult, other drugs tested in this settinginclude steroids, thyroxine, insulin, volatile anesthetics, andopioids.

DISCUSSION

Some studies consider that HMP improves outcomes aftertransplantation, but the majority of there have not applied,a randomization scheme and the patient numbers havenot been large enough to extrapolate of the results. Wightet al32 performed meta-analysis of the current literatureconcerning HMP versus CS. They showed a 20% reductionin DGF among the HMP group. An European multicenter,prospective, randomized clinical trial by the EurotransplantGroup, including Netherlands, Belgium, and Germany,compared HMP and CS.40 The results suggested beneficialeffects of HMP over CS both in terms of fewer cases ofDGF and of graft failures in the first 6 months aftertransplantation.After this clinical international trial, Moers et al27 in

2009, compared out comes of 336 kidneys preserved withHMP versus 336 with CS. Their data showed a significantreduced risk of DGF (20.8 vs 26.5, P ¼ .05) and increased1-year allograft survival (94% vs 90%, P ¼ .04) amongkidneys perfused with HMP. However, subgroup analysis

KIDNEY PRESERVATION 3175

did not find any significant difference between standard-criteria vs expanded criteria donors (P ¼ .75) or betweenDCD vs DBD (P ¼ .42).In 2010, however, Watson et al41 published a multicenter,

randomised, controlled trial comparing HMP with CS forkidneys from DCD. The trial was stopped when there wereavailable data for 45 pairs of kidney. There was no signifi-cant differences between kidneys assigned to HMP or CS inDGF incidence (58% vs 66% respectively), or graft (93.3%vs 97.8%, P ¼ .3) and patient survival at 1 year (93% vs100%, P ¼ .08). The authors state that the differences inkidneys from DBD and DCD may relate to the nature of theischemic injury: kidneys from DCD may not be exposed tothe severe hormonal disturbances accompanying braindeath, but are affected instead by a significant period ofwarm ischemic injury. These differences could explain whyHMP is superior to CS for DCD.In 2010, Jochmans et al53 published a randomized

controlled trial comparing HMP and CS in 82 kidney pairsfrom DCD donors. They observed a reduced incidence ofDGF from 69.5% to 53.7% (P ¼ .025) among HMP kidneysand better early graft function. One-year graft and patientsurvivals were similar between groups (93.9% vs 95.1%).

FUTURE PERSPECTIVE

The cutting edge preservation method currently is normo-thermic machine perfusion (NMP), which representsa transition from the usual practice. This technique has thepotential to provide metabolic support during the period oforgan storage.5,42 Normothermia with continuous or pulsa-tile flow offers the opportunity to restore normal aerobicmetabolism at a physiologic temperature. During NMP, theorgan is perfused and preserved at, or close to, the bodytemperature of 37�C. In theory, NMP at body temperatureallows the organ to maintain physiologic metabolism, thusminimizing accumulation of toxic substrates and formationof free radicals. It has been suggested that NMP may beparticularly advantageous for organs with extensive warmischemic injuries.5 Kidneys from marginal or DCD areparticularly susceptible to injury during hypothermic pres-ervation. Experimentally, extracorporeal membrane supportto maintain circulation before cooling and organ retrievalhas been used to improve the condition of DCD kidneys,showing lower rates of DGF compared with standardretrieval conditions. Normothermic preservation techniqueshave the potential to be adapted to improved methods to ofretain tissue viability compared with hypothermic tech-niques and may be used to enhance and assess the conditionof the organ, which may be particularly beneficial for kidneysfrom DCD donors. For the time being, this type of perfusioncannot be performed like HMP, that is to say immediatelyafter procurement, but only in a transplant center. Thereforethe organ is procured and immediately placed in cold staticstorage to be transported to the transplant center. Becauseof the great necessity of economic, scientific, and logisticefforts to eventually introduce this kind of perfusion on

a large scale, it is necessary to have a deeper understandingand scientific proof that it is safer and more effective thanstandard procedures. However, a few experimental studiesusing rabbit, canine, and porcine kidneys and livers havesuggested that organ preservation with NMP offersadvantages over traditional hypothermic techniques.5,42e45

These benefits demonstrated in preclinical studies seem tojustify an eventual clinical trial of this method,54e57 evenwhen considering its higher cost of compared with CS orHMP. To be introduced into common practice, NMP mustbe demonstrated to be not only as effective as other tech-niques, but also to have the potential to enlarge the donorpool. Indeed, future studies must also explore protectivemechanisms in HMP with pulsatile versus continuousperfusion.46,47

Protective mechanisms associated with pulsatile perfusionremain largely obscure. It can be speculated that pulsatileperfusion may not only provide nutrition and elimination oftoxins, but also sustain physiologic flow-mediated endothe-lial vasoprotective mechanisms, which may significantlyimpact ischemia-reperfusion injury and early innate immuneresponse events.In conclusion, every innovation regarding organ preser-

vation must face the wide scatter of perspective donorsthroughout the community, which poses barriers that aredifficult to overcome. This difficultly poses the need forgreat effort to improve the quality of the scientifically basedevidence regarding to preservation of organs destined fortransplantation.

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