Ischemia/Reperfusion Injury in Kidney Transplantation: Mechanisms and Prevention

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schemia/Reperfusion Injury in Kidney Transplantation:echanisms and Prevention

. Kosieradzki and W. Rowinski

ABSTRACT

Ischemia has been an inevitable event accompanying kidney transplantation. Ischemicchanges start with brain death, which is associated with severe hemodynamic disturbances:increasing intracranial pressure results in bradycardia and decreased cardiac output; theCushing reflex causes tachycardia and increased blood pressure; and after a short periodof stabilization, systemic vascular resistance declines with hypotension leading to cardiacarrest. Free radical-mediated injury releases proinflammatory cytokines and activatesinnate immunity. It has been suggested that all of these changes—the early innate responseand the ischemic tissue damage—play roles in the development of adaptive responses,which in turn may lead to an acute font of kidney rejection. Hypothermic kidney storageof various durations before transplantation add to ischemic tissue damage. The final stageof ischemic injury occurs during reperfusion. Reperfusion injury, the effector phase ofischemic injury, develops hours or days after the initial insult. Repair and regenerationprocesses occur together with cellular apoptosis, autophagy, and necrosis; the fate of theorgan depends on whether cell death or regeneration prevails. The whole process has beendescribed as the ischemia-reperfusion (I-R) injury. It has a profound influence on not onlythe early but also the late function of a transplanted kidney. Prevention of I-R injuryshould be started before organ recovery by donor pretreatment. The organ shortage hasbecome one of the most important factors limiting extension of deceased donor kidneytransplantation worldwide. It has caused increasing use of suboptimal deceased donors(high risk, extended criteria [ECD], marginal donors) and uncontrolled non–heart-beating(NHBD) donors. Kidneys from such donors are exposed to much greater ischemic damagebefore recovery and show reduced chances for proper early as well as long-term function.Storage of kidneys, especially those recovered from ECD (or NHBD) donors, should use
machine perfusion.
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OST OF THE DATA on mechanisms of ischemicinjury in eukaryotic cells are derived from cardiac-

yocyte studies. The heart can reveal many stages ofschemia-reperfusion injury, including stunning, hiberna-ion, arrhythmias, and infarction. These stages are not asell defined in other tissues and organs. Ischemia of a

ransplant is somehow unique as complete cessation of thelood supply as is observed in a removed organ and hardlyver occurs in ischemic tissues—heart, kidneys, brain, liver,keletal muscle—which are left in situ after arterial/portalow occlusion. Herein, we discuss cellular mechanisms of

schemia-reperfusion injury of transplanted organs andethods to prevent this injury with available organ preser-

ation methods. S

2008 Published by Elsevier Inc.60 Park Avenue South, New York, NY 10010-1710

ransplantation Proceedings, 40, 3279–3288 (2008)

ELLULAR AND MOLECULARECHANISMS OF ISCHEMIAeprivation of Oxygen

essation of arterial blood flow with immediate oxygeneprivation of cells (ie, hypoxia with accumulation ofetabolic products) is defined as ischemic injury. A switch

From the Department of General & Transplantation SurgeryM.K.), Transplantation Institute, Medical University of Warsaw,oland, and Department of Medical Sciences (W.R.), Universityf Warmia & Mazury, Olsztyn, Poland.Address reprint requests to Maciej Kosieradzki, MD, PhD, De-

artment of General & Transplantation Surgery, 59 Nowogrodzka
t, 02-006 Warsaw, Poland. E-mail: [email protected]
0041-1345/08/$–see front matterdoi:10.1016/j.transproceed.2008.10.004

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3280 KOSIERADZKI AND ROWINSKI

o the anaerobic glucose metabolism pathway, most likelyriven by a change in the oxeredox state with activation oflycolytic enzymes and their genes, occurs within minutes;ts severity depends on metabolic demand of the tissue.lthough a net synthesis of 36 molecules of adenosine

riphosphate (ATP) for each glucose particle undergoingxidative phosphorylation is not exactly true,1 anaerobicetabolism generates only a minimal amount of high-

nergy phosphates, which is definitely insufficient to meethe demands of aerobic tissues. In hypoxic muscle cells,lucose consumption was observed to be increased 12-fold.2

owever, apart from provision of oxygen, ischemia alsoesults in metabolic substrate starvation: fewer glycogenranules are observed in ischemic tissue. Although not theost important contributing factor, exhaustion of cellular

f glycogen immediately arrests anaerobic glycolysis. Moremportantly, even if glycogen is still present but ATP haslready been exhausted, phosphorylation of fructose-6-hosphate cannot occur, the glycolysis pathway is notignited.” The toxicity of metabolic products, which are notashed out or eliminated, increases in parallel with thesmolar load. Tissues accumulate inorganic phosphate,rotons, creatine, and glycolysis products. Increased H�,

actates, and NADH inhibit glycolytic enzymes, namelylycerylaldehyde phosphate dehydrogenase. In addition,TP synthase now becomes a hydrolase, swiftly dephospho-

ylating ATP to adenosine diphosphate (ADP). Existingtores of phosphocreatine are lost within seconds of isch-mia due to at least temporary rephosphorylating of ADP,hich is then gradually catabolized to inorganic phosphate,denosine, and inosine, which being permeable to the cellularembrane escape to the extracellular compartment. This

vent results in deprivation of substrate for the synthesis ofigh-energy phosphates even when there is subsequent resti-ution of blood flow. The decreased ATP phosphorylationotential determines cellular function at the end of the

schemia. Although ATP synthase activity is diminished after 1our of cold ischemia, it can be restored after reoxygenation.owever, when ischemia lasts more than 24 hours and energy

ubstrates are lost, synthase activity does not recover aftereperfusion,3 leading to lethal cell injury.

dema

igh-energy phosphates are vital for most cellular func-ions: from maintaining homeostasis, signal integrationransduction, cell proliferation and differentiation to execu-ion of the apoptotic death cycle. Although membranehosphatases act slowly during ischemia,4 ATP depletion

nhibits NA�/K� membrane phosphatase, thereby impair-ng the ability to maintain membrane potential and cellxcitability, which require protection from gradient-driven� and Na� ion trafficking. Sodium, however, enters the

ytoplasm accompanied by large amount of water, produc-ng edema, the degree of which is dependent on the extentnd duration of ischemia.

An arrest of glycolysis due to NAD�-deficiency-driven,
lycerylaldehyde phosphate dehydrogenase inhibition re- t
ults in accumulation of various intermediates—glucose,lucose-6 and -1-phosphates, �-glycerol phosphate—androducts—lactate, NADH, H�. This greatly increases thesmolar load of ischemic cells. Also when ATP is graduallyephosphorylated to soluble adenosine and three particlesf inorganic phosphate, the osmolarity rises greatly in the

ntracellular compartment. Hyperosmolarity also attractsater into the cell through simple diffusion, aquaporin, andhloride channels as well as the glucose transporter. Actu-lly this mechanism, not Na�/K� pump insufficiency, iselieved to be responsible for ischemic edema. Ischemic

njury to the phospholipid bilayer can also be of importance.aturally, in a no-flow situations ischemia metabolites leakut, accumulating in extracellular space unless there is aalance between intra- and extracellular osmolarity andembrane permeability, which slows the progression of

dema. The phenomenon becomes a major issue when thextracellular space is washed out with machine perfusionMP) for graft preservation, particularly if the osmolarity ofhe perfusion solution was not augmented. The extracellu-ar space can easily become hypotonic with accelerateddema formation.

Edema results in disruption of cellular membranes: notnly the outer cellular membrane by opening of stretch-ctivated channels that counteract the volume increase,ith dissipation of the semiconductance of the membrane,ut also of endoplasmic reticulum (ER), Golgi apparatus,itochondrial membranes, and cytoskeletal microtubules,hich are ischemia time-dependent.5 The ER performsundreds of biochemical reactions, which require a specialompartmentalized cytoplasic milieu based upon pH, iononcentrations, redox state, and catalysts.6 When this fea-ure is lost, uniform conditions do not permit most of thectivities. Edema of mitochondria elongates the path way ofnergy-rich compounds (phosphocreatine), which is neededo be covered from the inner mitochondrial membrane tohe cytoplasm. Similarly, 3C-acids and glucose must travelhe same distance. The mitochondrion loses coordination ofhe metabolic cycle and phosphorylation, leading to open-ng of the transition pore. Necrosis occurs when ATP storesre lost. When ATP is at least partially preserved, cyto-hrome c is released with caspase activation, resulting inell death.7 When swollen mitochondria lose their cristaend show matrix densities, an irreversible phase of injury iset.8

Acidosis is an immediate result of anaerobic glycolysis: asnergy demand is high, reaction turnover must follow toenerate sufficient ATP. However, NAD�, lost in therocess, can only be regenerated with fermentation ofyruvate to lactic acid, which strongly acidifies the cyto-lasm. In acidosis, NAD� decomposition occurs with for-ation of glycolysis-inhibiting metabolites. To prevent ex-

essive acidosis, phosphofructokinase, a key enzyme in thelycolysis pathway, is inhibited by the low pH. With nolood flow to remove the lactate, energy production isalted. A decreased pH is uniformly observed in ischemic
issues.9,10 Although it may be involved in protection on

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MECHANISMS AND PREVENTION OF I-R INJURY 3281

eperfusion,11 acidosis during coronary artery occlusionorrelates well with tissue injury as measured with myoglo-in release.12 Noteworthy, acidosis impairs recovery ofontractile and microvascular endothelial functions inypothermia-arrested hearts not exposed to ischemia.13

pening of acid-sensing ion channels with calcium influxay be a universal pathophysiologic mechanism, though

hese channels have not been studied in liver ischemia.owever, interstitial lactic acidosis in the liver strongly

orrelates with reperfusion injury as evidenced by 24-hourspartate transferase (AST) levels �2000 IU/L.14

alcium Overload

itochondrial dysfunction is a critical event during isch-mia, as it initiates both necrosis and apoptosis cascadesuring reperfusion. The organelle is both the site whereinoxious particles are produced and the preferred target ofhe injury. During ischemia, the Na�/Ca2� antiporter stopsumping calcium out of the cell, since sodium accumulatingithin the cell cannot be removed by the ineffective Na/K-TPases, leading the Na/Ca exchanger to start to work in

he reverse direction. However, so-called intracellular cal-ium overload early in ischemia occurs mostly due toedistribution of calcium from ER stores.15 Influx of extra-ellular calcium occurs only during prolonged ischemia andeperfusion.16 Increased intracellular calcium activates cal-ain and causes translocation of Na/K ATP-hydrolase tohe cytoplasm, increasing its deficit.17 To maintain mito-hondrial membrane potential with increased intracellularodium and calcium, Ca2� retention in the mitochondrialatrix is necessary. In fact, mitochondrial calcium at the

nd of ischemia has been linked to the extent of injury toardiacmyocytes.18 Calcium overload in mitochondriaauses early inhibition of NADH-coenzyme Q oxidoreduc-ase (complex I) with resultant cytochrome aa3 oxidation.pon progression of ischemia, complexes III and IV are

ffected. Cytochrome c loses its anchoring within cardio-ipin elements of the inner mitochondrial membrane andermeates to the cytosol, activating caspase 3 on reperfu-ion. Cytochrome release occurs via the mitochondrialermeability transition pore, Pore-related miochondrialatrix swelling with unfolding of the inner membrane

ristae and breaks in the outer membrane. Mitochondrialermeability transition (mPT) is a nonselective pore in the

nner mitochondrial membrane, which is almost imperme-ble under physiological conditions, but opens due to a highitochondrial content of calcium. Equilibration of H�

oncentrations on both sides of membrane abolishes theTP-synthase driving force. Opening of mPT was observed

n postischemic cardiac muscle reperfusion; in fact, pro-onged opening differentiates reversible from irreversibleeperfusion injury.19,20 Inhibition of mPT pores at reperfu-ion by so-called postconditioning namely, brief periods ofschemia and reperfusion after ischemia, or by pharmaco-ogical intervention, increases the trigger concentration of
alcium necessary for mPT opening, thereby attenuating t
schemia-reperfusion injury. Noteworthy, cyclosporine is aotent mitochondrial transition pore inhibitor.21

roteases, Phospholipases, and Free Radicals

ncreased cytosolic calcium at neutral pH activates someytoplasmic phospholipases and proteases. Two importantroups of proteases that are activated in response toncreased intracellular calcium are calpains (calcium-ependent cysteine proteases) and caspases. The formerleave a variety of proteins, including protein kinase C,odrin, and cytoskeleton while capases have substratesithin plasma membranes, lysosomes, mitochondria, anducleus. Caspases aspartate-specific cysteine proteases) are

nvolved in the execution of apoptosis and cell death. Alinical trial on the efficacy of a pan-caspase inhibitor addedo the preservation medium and to the flush solutioneeking to attenuate liver injury confirmed caspase activa-ion during ischemia or early after reperfusion.22 Phospho-ipase A2 plays an important role in ischemic injury;nockout mice lacking this enzyme showed smaller infarctizes, decreased postreperfusional myeloperoxidase activi-ies, and less release of arachidonic acid.23

Free radicals are formed even during global ischemia,hen small amounts of oxygen are available. Deaminationf adenosine provides, a substrate for xanthine oxidase. Thenzyme xanthine dehydrogenase changes its conformationn response to ischemia. It is believed to result in freeadical formation. However, a more important source ofree radicals in ischemia is the respiratory complex IIItself.24 Superoxide anion radicals can be generated bylmost every oxidase in this complex. Substrate deficiencyor nitric oxide synthase (NOS) turnover can also become aource of peroxynitrite, which additionally inhibits NOS,ugmenting endothelial dysfunction.25 Whatever theource, radical production is controlled for about 25 min-tes during warm ischemia, increasing rapidly afterwards.26

ARM VERSUS COLD ISCHEMIA

old itself is detrimental to tissues. It can cause changesimilar to those observed in warm ischemia even withontinued blood flow. Mitochondrial swelling with roundingf their shape, extra- and intracellular edema, and margin-tion of chromatin have been seen in proximal tubulesuring hypothermia.27 This observation may reflect the

nability to maintain oxidative phosphorylation and mem-rane transport at nonphysiological temperatures. Al-hough oxidative phosphorylation is halted, energy storesre slowly lost in the cold, according to van’t Hoff’s rule.omplete ATP loss has been observed after 40 minutes of

ardiac warm ischemia,28 which never happens within thelinically justified time range of cold ischemia, although a0% decrease in ATP/ADP ratio has been noted.29,30

As a remnant of millions of years of anaerobic evolution,ypoxia is able to switch on a substantial number of genes.owever, as transcription and translation processes are

ime-consuming and as eukaryotic cells subjected to oxygen

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eprivation need to respond quickly to energy demands, allnzymes needed for glycolysis are expressed constitutively.o boost the response, the CsrA/B system differentially regu-

ates mRNA stability of genes involved in glycogen synthesis,luconeogenesis, and glycolysis. In contrast, redundant genesf the respiratory complex II are shut off. The protein contentf enzymes involved in glycolysis may be quickly enhanced byinding of HIF1� to the genome and by de novo proteinynthesis. Most glycolytic enzymes have HIF1� binding sites.lthough there are varying opinions on gene expressionuring hypothermia, we believe that the increased mRNAesults from RNA stabilization in the cold. There is little or novidence that double-stranded DNA unfolding, nucleotideransport, and enzyme-dependent, energy-consuming mRNAynthesis occur in warm-blooded animals, since even in bac-eria, a temperature downshift results in transcription arrest,hich can only be restored with prolonged adaptation toold.31

Various types of ischemia generate different free radicals:eroxynitrite anions, important in warm ischemia, do notake part in cold ischemic injury to the kidneys.32 In cardiacuscle, cold ischemic injury is rendered by superoxide and

ydroxyl radicals as well as by hydrogen peroxide.33

The resistance of various cell populations to differentypes of ischemia is difficult to assess, since major morpho-ogical changes observable with light microscopy occur onlyfter long periods of time; changes can be seen sooner wheneperfusion with blood is allowed. Cardiac endothelial cellsppear quite resistant to warm ischemia, as over 50% ofhem seemed viable after 12 hours of warm ischemia, asssessed with trypan blue exclusion, acetylcholine relax-tion, and transmission electron microscopy.34 Major endo-helial injury develops only when ischemia was followed byeperfusion. Thus, warm ischemic injury to cardiacmyocytess mostly due to free radicals and inhibited NOS fromubstrate deprivation, which are probably the primary nox-ous factors influencing postreperfusion coronary flow andontractile functions, phenomena that are not true forrolonged cold ischemia.35 Warm ischemia also rendersrominent injury to hepatocytes, and reduction of theumber of viable Kupffer cells.36 In contrast, cold ischemiaollowed by reperfusion causes marked changes in sinusoi-al endothelial cells with early necrosis without influencingepatocytes.37 In the kidney the primary target of injury byarm ischemia is proximal tubular cells. Although they can

olerate a short exposure, changes in transepithelial poten-ial and cell-to-cell junctions appear early, increasing leak-ge and slowing active transport.38

Organs can tolerate prolonged cold ischemia or somearm ischemia without significant deterioration of function.owever, when both factors act in the same tissue, they

asily produce profound injury with marked cell death.39,40

RAIN DEATH AND DONOR PRETREATMENT

rain death results from a rapid increase in intracranial
ressure due to hemorrhage or brain edema. Altered n
ntracranial volume affects venous outflow, speeding up thencreased pressure until brain structures are pushed towardo the foramen magnum, completely halting arterial bloodow. Ischemia of the pons drives the autonomic reactionsf bradycardia, increased blood pressure, and respiratory

rregularity. With distal progression of ischemia, necrosisf the vagal, cardiacmotoric, and respiratory nuclei oc-urs with uncontrolled sympathetic reactions, unlesshere has been total sympathetic denervation due toeath of the medulla oblongata. Thus, brain death causeswo distinct hemodynamic phases: uncontrolled sympa-hetic activation with arterial blood pressures well above00 mm Hg and tachyarrythmia, followed by a muchonger hypotensive period resulting from sympatheticnsufficiency, loss of vascular tonus, and decreased pe-ipheral resistance.41 Early-phase catecholamine stormepends on the velocity of the intracranial pressure

ncrease with heightened serum adrenaline concentra-ions by as much as 200- to 1000-fold.42 Increasedydrostatic pressure causes immediate tissue edema,hich is often hemorrhagic.43

Necrosis of the hypothalamus and pituitary gland dissi-ates thermoregulation44 and hormonal homeostasis: se-um thyroid hormones do not meet metabolic demand,45

iabetes insipidus is not controlled by vasopressin,46 pro-uction of cortisol is halted,47 and insulin release is inade-uate.48 Free radicals and proteolytic enzymes are released,

eading to slow, progressive, and inevitable death of cellsnd tissues via necrotic and apoptotic mechanisms.49 Thesehanges result in deterioration of organ function in brain-ead donors, including impaired kidney function in bothptimal and marginal donors,50 hence, even if the effect ofold ischemia was eliminated in an experimental setting.here is as significant influence of brain death on renal

ransplant function,51 including more frequent acute rejec-ion episodes52 and decreased long-term survival.53,54 Braineath has been observed to increase tissue CD68,55

D45,50 and CD8 infiltrates;49 ICAM, VCAM,56 and E-electin expression57; as well as serum content of proinflam-atory cytokines.58

To avoid or at least minimize the consequences of braineath, various experimental and clinical treatment methodsave been applied to brain-dead donors. Early aggressiveonor management with stabilization of donor hemody-amics has been shown to increase the number of donationsnd the organ yield.59,60 Interestingly the evidence suggestshat treatment with small doses of catecholamines, espe-ially dopamine, may improve renal graft function andurvival.61,62 Despite doubts whether it has any benefiteyond that of agressive fluid management with hemody-amic stabilization, the Papworth protocol of methylpred-isolone bolus, vasopressin, triodothyronine and insulin

nfusions and its numerous modifications seems to facilitateontrol of hemodynamic disturbances and increase the
umber of transplanted hearts.63

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IDNEY STORAGE

idney storage in hypothermia, which is necessary forogistical reasons, must maintain organ viability betweenecovery and transplantation. The importance of ensuringuccessful preservation of kidneys between retrieval andmplantation has been long recognized. Two approachesave been developed to limit ischemic damage: cold statictorage (CS) and machine pulsatile perfusion. Both tech-ologies have continued to evolve since their early devel-pment. In 1967, successful organ perfusion preservationas introduced by Belzer. Two years later, Geoffrey Collinsescribed a preservation solution with intracellular ionicontents, which allowed successful storage in simple hypo-hermia for up to 18 to 24 hours. Soon, Belzer and Southardeveloped a different type of medium for hypothermictorage, so-called University of Wisconsin (UW) solution,hich has become a gold standard.Simple static cold storage is the most widely used form of

reservation in everyday clinical practice. It is simple andffective when a kidney from a standard deceased donor istored for up to 24 hours. In last 10 years, a variety ofolutions have been introduced into market for successfulypothermic storage of kidneys and other organs.64 How-ver, another approach is needed for longer storage timesnd for preservation of kidneys recovered from extendedriteria (or non–heart-beating) donors.

Hypothermic pulsatile perfusion for kidney storage be-ore transplantation was introduced by Belzer more than 30ears ago. This preservation method was not widely used,ecause of the simplicity of cold storage, especially since theW solution came to the market since it allowed successful

reservation for up to 24 to 30 hours. In the last 10 years,ncreased interest in MP has grown due to the use of organsecovered from extended criteria and nonheart-beatingonors. MP solutions often contain dialyzed hydroxyethyltarch to prevent interstitial edema, with adenosine addeds a stimulator of ATP synthesis; phosphate as an H� ionuffer and ATP production stimulator; gluconate to sup-ress for cellular swelling; glutathione as an antioxidant; asell as other agents. Numerous studies have confirmed thesefulness of (MP) preservation of kidneys stores for over0 hours.Wight et al65 performed a systematic review and meta-

nalysis of the effectiveness of MP and CS techniques toeduce delayed graft function (DGF) and improve grafturvival among recipients of kidneys from heart-beating andon–heart-beating donors. They documented that studiesf high methodological quality and sufficient size showedP led to reduce DGF rates. The predicted impact of this

rocedure on graft survival requires a large patient popu-ation followed for a long period. Registry database analysisupports the link between DGF and graft survival, suggest-ng that evaluation of this complication may be moreeasible than randomized controlled trials. Several studiesave shown beneficial effects of MP on early graft function
nd survival. In prospective and retrospective studies, Kwi- e
tkowski et al showed beneficial effects of MP on early andong-term graft survival.66 Furthermore, the incidences ofhronic rejection and, interstitial fibrosis/tubular atrophyere significantly lower among MP-preserved kidneys.67

A recent prospective, randomized Eurotransplant studyhat compared CS with MP for preservation documentedhe value of MP: this method decreased the incidence ofGF not only among standard, but also extended criteria,

onors. MP also reduced the incidence, the duration, andhe severity of DGF and ameliorated graft function ofon–heart-beating donor kidneys after transplantation.68

s shown recently using long-term observation, MP wasconomically superior to CS.69

Hypothermic MP not only facilitates longer storage, butlso allows assessment of the extent of ischemic damage tohe organ. MP preservation also allows an assessment ofidney quality before transplantation. Measurements ofow, resistance, lactate excretion, and alpha-GST in theerfusate yields possible predictors of whether the kidneyill display immediate or delayed function after transplan-

ation.70–75

EPERFUSION INJURY

eperfusion injury, as an effector phase of ischemic injury,evelops during hours or days after the initial insult. Repairnd regeneration processes occur together with cellularpoptosis, autophagy, and necrosis; the fate of an organepends on whether cell death or regeneration prevails. Aspoptosis needs energy and protein synthesis, it occursostly upon reperfusion. Cytochrome c release and caspase

ctivation has been noted as early as 5 minutes aftereperfusion, while it was virtually absent during prolongedschemia of cardiacmyocytes.76 When cytosolic calcium thatad increased during ischemia returned to normal values,he cells were able to recover from injury. However, arogressive increase in cytosolic calcium marked an irre-ersible phase of injury.77 The rapid burst of free radicalshortly following reperfusion is a well-documented phe-omenon. Most likely, discordant respiratory chain en-ymes are the main source of radicals on reperfusion.educed ubiquinone reacts with free oxygen to form super-xide, which is metabolized to hydrogen peroxide by cata-

ase, or extremely reactive hydroxyl radicals in the presencef ferrous or copper ions. Radical-mediated peroxidation of

ipids in cellular and mitochondrial membranes occurs inouble bonds, aromatic rings, and thiol groups, which areresent in cardiolipin and other phospholipids. Free radi-als probably trigger endothelial injury, since only aftereperfusion do endothelial cells, which seem fairly wellreserved after the ischemic phase, become edematous and

eaky to proteins and small particles. As a source of bothree radicals and potent radical-scavenging potential viaytochrome c, mitochondria seem to be a gatekeeper toellular injury during reperfusion. The mitochondrial per-eability transition pore which remains closed during isch-

mia due to inhibition by acidosis, opens upon reperfu-

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ion.78 Although Na/K-ATPase inhibition does not play aajor role in edema formation during the ischemic phase,

pon reperfusion it is inhibited by free radicals, intensifyinghe edema.

Damaged endoplasmic reticulum fragments increase ineperfused tissues, leading to formation of autophago-omes, which signal upcoming cellular disintegration.79

oon, lysosomal activity of proteases and cathepsins in-reases, leading to necrotic or apoptotic cell death. Apo-tosis may occur due to reduced mitochondrial activity and

oss of electric potential, to increased Ca2� with calpainctivation, to free radical generation, to nitric oxide toecreased BCL-2 and increased BAX proteins,80 to over-xpression of FAS ligand, to cytoskeletal disintegration, ando other triggers. Nitric oxide is capable of protection bothf the microcirculation and of tissue injury via peroxynitriteormation, depending on whether it was formed by consti-utive or inducible isoforms of NOS.81

Neutrophils attracted by a set of chemokines, includingXCL10, RANTES, Interleukin-17, MCP-I, are recruitedy endothelial cells expressing ICAM1, E and P selectin,hich cross-talk with integrins and L-selectin on polymor-honuclear cells. Matrix proteins, MMP-9, fibronectin, and

aminin also play important roles to facilitate leukocyteigration across vessels. Expression of these proteins sig-

ificantly increases after reperfusion.82,83 Neutophils mayause direct cytotoxicity via generation of oxygen freeadicals and release of cytokines. They control perivascularissue edema, damage endothelial cells directly, and pro-ote platelet aggregation. The extent of their infiltration is

roportional to the magnitude of injury to the reperfusedrgan. Although reperfusion begins with relative hyper-mia, often exceeding the normal blood flow rate, it isollowed by much longer phase of diminished blood flow.lthough mostly driven by decreased metabolic demand

nd extracellular edema, it depends in part on neutrophilnd platelet accumulation within blood vessels, which at-enuate flow and cause the “no-reflow” phenomenon84;owever, there is dispute concerning the negative role ofow reduction.

REVENTION OF REPERFUSION INJURYormothermic Reperfusion

arallel to the development of new preservation solutionsor CS (Vasosol, T-Kyoto, SCOT, IGL), major progress inormothermic preservation or resuscitation of organs haseen made. The Kootstra group presented spectacularesults of resuscitation of kidneys that had been severelyamaged by 2 hours of warm ischemia. When reperfused atear-normothermia with their own exsanguinous metabolicupport medium, the kidneys were successfully rescuedrom lethal injury.85 These experiments have been latelyollowed by the Leicester group: renal blood flow, highnergy stores, and renal function after warm ischemia and6 hours of cold storage were nicely restored by 2 hours oformothermic resuscitation with donor blood.86 However,
he most impressive clinical progress was reported with the n
ransMedics Organ Care System, which allows for extra-orporeal normothermic preservation in a donor blood-ased solution. The Leicester group showed the superiorityf prolonged warm preservation of controlled and uncon-rolled non–heart-beating kidneys.87 This technology alsoeems promising for preservation, viability assessment, andxpansion of utilized non–heart-beating livers88 and cardiacllografts.89 Recently some groups have suggested benefi-ial effects of normothermic MP as a new approach in organreservation and transplantation.90

reatment of the Recipient

o successfully prevent reperfusion injury, actions must bepplied early; at the stages of donor hemodynamic distur-ances, organ retrieval, and preservation. Some interven-ions, however, may prove productive when used duringraft reperfusion in the recipient.

Although free radicals play an undoubted role in theathology of the ischemia-reperfusion injury, administra-ion of free radical scavengers at reperfusion seemed toield questionable results until Land confirmed a significantate-term protection.91 Lately, pyruvate has been shown toirectly detoxify peroxynitrite and H2O2, protecting againstyocardial reperfusion injury.92

As described above, cyclosporine inhibits permeabilityransition and, when administered on reperfusion, de-reases creatine kinase release and infarct size in humansndergoing percutaneous coronary intervention for acuteardiac ischemia.93 This needs thorough evaluation in trans-lant models; it remains in contrast with the current policy toelay calcineurin inhibitor administration in renal ischemia-eperfusion injury.

Intermittent ischemia and reperfusion prior to prolongedschemia, called ischemic preconditioning, has been showno salvage the heart and some other tissues from prolongedamage, a similar phenomenon can be applied at the timef transplant implantation. If gradually elongated kidneyeperfusion periods were interrelated with short-time isch-mia, serum blood urea nitrogen and creatinine decreasedaster than among non-postconditioned animals exposed toarm ischemia. The functional improvement was accompa-ied by more effective complex II mitochondrial respirationnd lesser peroxide production and protein oxidation,94 asell as inhibition of apoptosis in the reperfused tissue.95

lthough the phenomenon was confirmed in hearts, brains,nd skin flaps, some doubts call for further studies.96

nterestingly, postconditioning protection had been in-uced pharmacologically with administration of 3% v/vevoflurane from the beginning of reperfusion of the heart,hich decreased infarct size and mitochondrial injury,97 al-

hough hyperglycemia abolished this protection.98

The role of hemoxygenase 1, the enzyme that convertseme into biliverdin, carbon monoxide, and free Fe, has beenxtensively studied in protection from ischemia-reperfusionnjury. Exposure of liver transplant recipient animals to in-aled CO decreased serum alanine transferase, hepatocyte
ecrosis, and neutrophil infiltrates in dose-dependent fash-

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on.99 Noteworthy, HO-1 can be induced by simvastatinrecoditioning.100 Interestingly, through its strong anti-

nflammatory effect, nicotine has been shown to reduceubular damage in experimental models of warm ischemiahen administered before reperfusion. It prevented neu-

rophil infiltration, decreased CXC, KC, tumor necrosisactor-alpha, and high-mobility group box-1 (HMGB1)rotein release. Less tubular apoptosis and proliferationere observed among nicotine-treated mice.101

SCHEMIA-REPERFUSION AND IMMUNE INJURY

schemic injury to an allograft significantly increases theisk of poor initial function, which in turn has been associ-ted with an increased rate of acute rejection episodes.lthough there was no satisfactory supporting hypothe-

is, many researchers thought the association betweenschemia and graft immunogenity to be reasonable. Anlegant explanation came from Matzinger’s injury theoryo explain the links between tissue damage, innate im-une responses differentiating “self” from “non-self” or

damaged-self,” and communication of danger signals todaptive immunity via Toll-like receptors and antigen-resenting cells.102 “Danger signals” or “alarmins” re-

eased during ischemia and reperfusion may includeraft-derived DNA and RNA, oxidized proteins andipids, HMGB1, uric acid, and calcium pyrophosphaterystals.103 Lately, HMGB1 has been shown to be activelyecreted from at-risk cells via a free-radicals-dependentathway during hepatocyte ischemia-reperfusion. Thisrocess requires intact TLR4 signaling and calcium-ependent kinases.104,105 TLR4 expression is increased

n tubular epithelial cells following ischemia-reperfusionnjuries. When TLR4 or its signaling pathway protein,

yD88, were absent, kidneys were protected fromschemia-reperfusion.106

According to the injury theory, the less the initial insult,he smaller the agitation of adaptive immunity and theower the chances for early and late responses to thellograft. Land immediately noticed how beautifully thisheory explained his results of delayed kidney graft protec-ion after administration of recombinant superoxide dys-utase.91,107 Prolonged ischemia (�15 hours) indeed in-

reases graft immunity; it was shown to be an independentisk factor for postrejection de novo production of high-iter anti-HLA class I panel-reactive antibodies.108 Circu-ating anti-donor antibodies as well as immunoglobulin and4d deposits in renal glomeruli accompanied severe (72ours of CS) ischemic injury occurring 7 days after engraft-ent. When ischemia was limited to 24 hours, no signs of

amage or antibody-mediated immunity were present atay 7.109 Lately, we have shown that although MP is not anffective method to decrease immediate effects of ischemicnjury, it significantly ameliorated the late histological inju-ies in renal allografts. Tubular atrophy and interstitialbrosis (formerly known as CAN were observed in 90% of
iopsies from cold-stored and among 64% of machine- m
erfused kidneys with evidences of chronic rejection (ac-ording to Banff 2005 consensus) among 9% and 3% ofenal allografts, respectively.110 In an exquisite study involv-ng 600 protocol biopsies, Yilmaz et al linked the risk ofhronic allograft changes, including interstitial inflamma-ion and fibrosis, tubular atrophy, vascular wall and tubularasal membrane thickening, with the duration of cold

schemia.111

LINICAL RELEVANCE OFSCHEMIA-REPERFUSION INJURY

rgan shortage is a universal problem. The waiting lists arerowing in all countries with an increased gap betweenemand for and number of available organs. For thiseason, extended criteria and non–heart-beating donors areidely used. These kidneys are more susceptible to ischemicamage, leading to DGF or primary nonfunction, as well asorse graft function and survival. Kidneys recovered from

uch donors should be stored using pulsatile perfusion,hich allows better protection during preservation-related

schemia, as well as allows measurement of several param-ters—flow, resistance, lactate excretion, alfa GST—whichay be useful to assess the extent of ischemic injury.

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Ischemia/Reperfusion Injury in Kidney Transplantation: Mechanisms and Prevention

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