ISCHEMIA/REPERFUSION INJURY IN KIDNEY TRANSPLANTATION

MECHANISMS AND PREVENTION

Maciej Kosieradzki 1) , Wojciech Rowiński 2)

1) Department of General & Transplantation Surgery, Transplantation Institute,

Medical University of Warsaw, Poland

2) Department of Medical Sciences, University of Warmia & Mazury, Olsztyn, Poland

Address correspondence to: Maciej Kosieradzki M.D., PhD. Department of General &

Transplantation Surgery, 59 Nowogrodzka St, 02-006 Warsaw, Poland. E-mail:

mkosieradzki@wp.pl

Abstract

Ischemia has been an inevitable event accompanying the procedure of kidney

transplantation. Ischemic changes start with brain death which is associated with severe

homodynamic disturbances: increasing intracranial pressure results in bradycardia and

decrease in cardiac output; Cushing reflex causes tachycardia and increase of blood pressure,

and after short period of stabilization, systemic vascular resistance declines with hypotension

leading to cardiac arrest. Free radical mediated injury occurs and release of pro-inflammatory

cytokines and activation of the innate immunity develops. It has been is suggested that all of

these changes - the early innate response and the ischemic tissue damage play a role in

development of adaptive response which in turn may lead to acute kidney rejection,

Hypothermic kidney storage of various duration before transplantation adds to ischemic tissue

damage. The final stage of ischemic injury occurs during reperfusion. Reperfusion injury, as

an effector phase of ischemic injury, develops hours or days after initial insult. Repair and

regeneration process occur along with cellular apoptosis, autophagy and necrosis, and the fate

of the organ depends on whether cell death or regeneration prevails. The whole process has

been described as the ischemia/reperfusion injury. It has profound influence not only on the

early, but also late function of the transplanted kidney. Prevention of I/R injury should be

started before recovery of the organs (donor pretreatment).

Organ shortage has become one of the most important factors limiting extension of the

deceased donor kidney transplantation worldwide. This has caused an increasing use of

suboptimal deceased donors („high risk‟, „extended criteria‟, „marginal‟ donors) and

uncontrolled non-heart beating donors. Kidneys from such donors are exposed to much

higher ischemic damage before recovery, and have lower chances for a proper, early, as well

as long term function. Storage of kidneys, especially those recovered from ECD (or NHBD )

donors should be carried out using machine perfusion.

Mechanisms of ischemic injury:

Most of the data on ischemic injury mechanisms in eucariotic cells come from

cardiomyocyte studies, the heart can however reveal many stages of ischemia-reperfusion

injury as stunning, hibernation, arrhythmias and infarction. Such stages are not well-defined

in other tissues and organs. Transplant ischemia is somehow unique as complete cessation of

blood supply, observed in removed organ, and hardly ever occurs in ischemic organs (heart,

kidneys, brain, liver, skeletal muscle) left in situ after arterial/portal flow occlusion. Herein,

we discuss cellular mechanisms of ischemia-reperfusion injury of transplanted organ and

methods to prevent this injury with available organ preservation methods.

Cellular and molecular mechanisms of ischemia

Deprivation of oxygen. Cessation of arterial blood flow with immediate oxygen deprivation

of the cell, i.e. hypoxia with accumulation of metabolic products is what defines ischemic

injury. A switch to anaerobic glucose metabolism pathway, most likely driven by change of

oxyredox state with activation of glycolytic enzymes and their genes, occurs within minutes

and depends on metabolic demand of the tissue. Although net synthesis of 36 molecules of

ATP for each and every glucose particle in oxidative phosphorylation is not exactly true,1

anaerobic metabolism generates only minimal amount of high energy phosphates, definitely

not sufficient to meet the demand of aerobic tissues. In hypoxic muscle cells glucose

consumption was found to be 12-fold increased.2 However, apart from oxygen provision,

ischemia results also in metabolic substrates starvation: fewer glycogen granules were

observed in ischemic tissue. Although it‟s not the most important contributing factor, had

cellular glycogen been used up, anaerobic glycolysis arrested immediately. More

importantly, even if glycogen is still present but ATP was already used up, phosphorylation of

fructose-6-phosphate cannot occur and glycolysis pathway is not „ignited‟. Toxicity of

metabolic products, which are not washed out or eliminated, increases in paralel with osmolar

load. Tissues accumulate inorganic phosphate, protons, creatine, and glycolysis products.

Increased H+, lactates and NADH inhibit glycolytic enzymes, namely glycerylaldehyde

phosphate dehydrogenase. What‟s more, ATP synthase now becomes a hydrolase and swiftly

dephosphorylates ATP to ADP. Existing stores of phosphocreatine are lost within seconds of

ischemia to rephosphorylate ADP at least temporarily. ADP is then gradually catabolized to

inorganic phosphate, adenosine and inosine, which are permeable to the cellular membrane

and can escape to extracellular compartment. This results in deprivation of substrate for the

synthesis of high-energy phosphates even if subsequent restitution of blood flow occurs. This

also decreases so called ATP phosphorylation potential, a determinant of cellular function at

the end of ischemia. Although activity of ATP synthase is diminished already after 1 hour of

cold ischemia, it can be restored after reoxygenation. However, when ischemia lasts too long

(>24 hours) and energy substrates are lost, synthase activity does not recover after

reperfusion,3 meaning lethal cell injury.

Oedema. High-energy phosphates are vital in most of cellular function: from maintaining

homeostasis, signal integration and transduction, cell proliferation and differentiation to

execution of apoptotic death cycle. Although membrane phosphatases run slow during

ischemia,4 ATP depletion inhibits NA

+/K

+ membrane phosphatase and ability to maintain

membrane potential and cell excitability is lost due to gradient-driven K+ and Na

+ ions

trafficking. Sodium however enters cytoplasm accompanied by large amount of water,

causing oedema dependent on the extent and duration of ischemia.

An arrest of glycolysis due to NAD+-deficiency driven glycerylaldehyde phosphate

dehydrogenase inhibition results in various intermediates (glucose, glucose-6 and -1-

phosphates, α-glycerol phosphate) and products (lactate, NADH, H+) accumulation, and

strongly increases osmolar load of ischemic cells. Also, when ATP is gradually

dephosphorylated to soluble adenosine and 3 particles of inorganic phosphate, intracellular

compartment osmolarity rises greatly. Hyperosmolarity also attracts water into the cell

through simple diffusion, aquaporin and chloride channels as well as glucose transporter –

actually this mechanism, not Na+/K

+ pump insufficiency is thought responsible for ischemic

oedema. Ischemic injury to the phospholipid bilayer can also be of importance. Naturally, in

no flow ischemia metabolites leak out and accumulate in extracellular space unless some

balance between intra- and extracellular osmolarity and membrane permeability is achieved

and progression of oedema is slowed down. The phenomenon becomes a major issue when

extracellular space is washed out with machine perfusion graft preservation, and, had the

osmolarity of the perfusion solution not been enhanced, extracellular space easily could

become hypotonic and accelerated oedema formation could be seen.

Oedema results in disruption of cellular membranes: not only of the outer cellular

membrane (strech-activated channels are opened to counteract for the volume increase,

however membrane semi-conductance dissipates), but of endoplasmic reticulum, Golgi

apparatus, mitochondrial membranes and microtubules of cytoskeleton, which are ischemia

time-dependent.5 ER enables execution of hundreds of biochemical reactions, requiring

different milieu (pH, ions concentration, redox state, catalysers) at the same time due to

compartmentalization of cytoplasm.6 When this feature is lost, uniform conditions don‟t allow

for most of activity. Oedema to mitochondrium elongates the path energy-rich compounds

(phosphocreatine) need to cover from inner mitochondrial membrane to the cytoplasm.

Similarly, 3C-acids and glucose must travel the same distance. Mitochondrion looses

coordination of metabolic cycle and phosphorylation, transition pore opens. If ATP stores

were lost, necrosis occurs. When ATP is at least partly preserved, cytochrom c is released and

caspases are activated, resulting in subsequent cell death.7 When swollen mitochondria lose

their cristae and show matrix densities, irreversible phase of injury is set.8

. Acidosis is a an immediate result of anaerobic glycolysis – as energy demand

is high, reaction turnover must follow to generate enough ATP. However, NAD+ is lost in the

process, and it can only be regenerated with fermentation of pyruvate to lactic acid, which

strongly acidifies the cytoplasm. In acidosis, NAD+ decomposition occurs with formation of

glycolysis-inhibiting metabolites. To prevent excessive acidosis, phosphofructokinase, a key

enzyme in glycolysis pathway is inhibited by low pH. Altogether, with no blood flow to

remove lactate, energy production is halted. Decreased pH was uniformly found in ischemic

tissues.9,10

Although it may be involved in protection on reperfusion,11

acidosis during

coronary artery occlusion correlated well with tissue injury measured with myoglobin

release.12

Noteworthy, acidosis caused impaired recovery of contractile and microvascular

endothelial functions in hypothermia-arrested hearts not exposed to ischemia.13

Opening of

acid-sensing ion channels with calcium influx could be one of universal pathophysiologic

mechanisms, though these channels have not been studied in liver ischemia. However,

interstitial lactic acidosis in the liver strongly correlated with reperfusion injury with 24-hour

AST levels >2000 IU/L.14

Calcium overload. Mitochondrial dysfunction is a critical event during ischemia, as it begins

both necrosis and apoptosis cascades during reperfusion. The organelle are both the site,

where noxious particles are produced and the preferred target of injury. On ischemia,

Na+/Ca

2+ antiporter stops pumping calcium out of the cell, as sodium accumulated within the

cell cannot be removed by ineffective Na/K-ATPase and Na/Ca exchanger starts to work in

reverse direction. However so called intracellular calcium overload early in ischemia occurs

mostly due to redistribution of calcium from endoplasmic reticulum stores.15

Influx of

extracellular calcium occurs only in prolonged ischemia and during reperfusion.16

Increased

intracellular calcium causes calpain activation and translocation of Na/K ATP-hydrolase to

the cytoplasm, progressing its deficit.17

To maintain mitochondrial membrane potential with

increased intracellular sodium and calcium, Ca2+

retention in the mitochondrial matrix is

necessary. In fact, mitochondrial calcium at the end of ischemia was linked to the extent of

injury to the cardiomyocytes.18

Calcium overload in mitochondria causes early NADH-

coenzyme Q oxidoreductase (complex I) inhibition and resultant cytochrome aa3 oxidation.

With progression of ischemia, complexes III and IV are affected. Cytochrome c looses its

anchoring in cardiolipin elements of the inner mitochondrial membrane and permeates to the

cytosol and activates caspase 3 on reperfusion. Cytochrome release occurs via mitochondrial

permeability transition pore, pore-related miochondrial matrix swelling with unfolding inner

membrane cristae and braking of the outer membrane or other mechanism. Mitochondrial

permeability transition (mPT) is a non-selective pore in the inner mitochondrial membrane,

almost impermeable in physiological conditions, which opens due to high mitochondrial

calcium. Equilibration of H+ concentration on both sides of membrane abolishes ATP-syntase

driving force. Opening of mPT was observed in post-ischemic cardiac muscle reperfusion and

in fact, prolonged opening differentiates between reversible and irreversible reperfusion

injury.19,20

Inhibition of mPT pore at reperfusion with so cold „postconditioning‟ (brief

periods of ischemia and reperfusion after ischemia) or pharmacological intervention increases

trigger concentration of calcium necessary for mPT opening and can attenuate ischemia-

reperfusion injury. Noteworthy, cyclosporine A is a potent mitochondrial transition pore

inhibitor.21

Proteases, phospholipases, and free radicals. Increased cytosolic calcium at neutral pH

can activate some cytoplasmic phospholipases and proteases. Two important groups of

proteases activated in response to increased intracellular calcium are calpains (calcium-

dependent cysteine proteases) and caspases. The former cleave variety of proteins, including

protein kinase C, fodrin and cytoskeleton and has substrates within plasma membrane,

lysosomes, mitochondria and nucleus. The latter, aspartate-specific cysteine proteases, are

involved in execution of apoptosis and cell death. A clinical trial on efficacy of pan-caspase

inhibitor added to preservation medium and to flush solution in attenuation of liver injury

confirms caspase activation during ischemia or very early at reperfusion.22

Phospholipase A2

plays an important role in ischemic injury, as knockout mice lacking this enzyme activity

showed smaller infarct size, decreased post-reperfusional myeloperoxidase activity and

released less arachidonic acid.23

Free radicals are formed even during global ischemia, when very small amount of

oxygen is available. Deamination of adenosine provides substrate for xantine oxidase – as the

enzyme xantine dehydrogenase has changed its conformation due to ischemia – and is

believed to result in free radical formation. However more important source of free radicals in

ischemia is respiratory complex III itself.24

Thus, superoxide anion radical can be generated

by almost every oxidase. Substrate deficiency for nitric oxide synthase turnover can also

become a source of peroxynitrite, which additionally inhibits NOS and augments endothelial

disfunction.25

Whatever the source, production of radicals is controlled during warm

ischemia for some 25 minutes and increases rapidly afterwards.26

Warm vs cold ischemia

Cold itself is detrimental to the tissues and can cause changes similar to those

observed in warm ischemia even when the blood flow continues. Mitochondrial swelling with

rounding of their shape, extra- and intracellular oedema and margination of chromatin were

seen in proximal tubules hypothermia.27

This might reflect inability to maintain oxidative

phosphorylation and membrane transport in non-physiologic temperature. Although oxidative

phosphorylation is halted, energy stores are lost slowly in the cold, according to van‟t Hoff‟s

rule. Complete ATP loss was observed after 40 minutes of cardiac warm ischemia,28

which

never happened within clinically-justified time range of cold ischemia, although even 50%

decrease in ATP/ADP ratio was noted.29,30

As a remnant of millions of years of anaerobic evolution, hypoxia is able to switch on

substantial number of genes. However, as transcription and translation processes are time-

consuming and eucaryotic, aerobic cell subjected to oxygen deprivation needs to respond

quickly to energy demand, all of the enzymes needed for glycolysis are expressed

constitutively. To boost the response, CsrA/B system differentially regulates mRNA stability

of genes involved in glycogen synthesis, gluconeogenesis and glycolysis. On the other hand,

redundant genes of respiratory complex II are shut off. Protein content of enzymes involved in

glycolysis may be quickly enhanced with biding of HIF1α to the genome and de novo protein

synthesis. Most of glycolysis enzymes have HIF1α binding sites. Although there are some

notions on gene expression during hypothermia, we rather believe these authors observed

increased mRNA from RNA stabilization which occurs in the cold. There is little or no

evidence that double-stranded DNA unfolding, nucleotide transport and enzyme-dependent,

energy consuming mRNA synthesis can occur in warm-blooded animals since even in

bacteria temperature downshift results in arrest of transcription, which can only be restored

with prolonged adaptation to cold.31

Different types of ischemia generate different free radicals: peroxynitrite anions,

important in WI, do not take part in cold ischemic injury to the kidneys.32

In cardiac muscle,

CI injury is rendered by superoxide and hydroxyl radicals as well as hydrogen peroxide.33

Resistance of various cell populations to different types of ischemia is difficult to

assess, as major morphologic changes observable with light microscopy occur only after long

periods of time – changes can be seen sooner when reperfusion with blood is allowed. Cardiac

endothelial cells appear quite resistant to warm ischemia, as over 50% of them seemed viable

after 12 hours of WI as assessed with trypan blue exclusion, acetylcholine relaxation and

transmission electron microscopy.34

Major endothelial injury developed only when ischemia

was followed by reperfusion. Thus, warm ischemic injury in the heart is mostly to

cardiomyocytes and free radicals and inhibition of nitric oxide synthase from substrate

deprivation are probably primary noxious factor influencing post-reperfusion coronary flow

and contractile function, which is not true for prolonged cold ischemia.35

WI rendered also

prominent injury to hepatocytes and reduced the number of viable Kupffer cells.36

On the

other hand, cold ischemia followed by reperfusion showed marked changes in sinusoidal

endothelial cells with early necrosis, without influencing hepatocytes.37

In the kidney

proximal tubular cells are primary target of injury by warm ischemia. Although they can

tolerate short-time exposure, changes in transepitelial potential and cell-to cell junctions

appear very early, increasing leakage and slowing down active transport.38

Organs can tolerate prolonged cold ischemia and some warm ischemia without

significant deterioration of function. However, if both factors act in the same tissue, they can

easily cause profound injury with marked cellular death.39, 40

Brain death and donor pretreatment

Brain death results from rapid increase in intracranial pressure due to haemorrhage or

brain oedema. Altered intracranial volume affects venous outflow and speeds up increase in

pressure until structures of the brain are pushed towards foramen magnum and arterial blood

flow is completely halted. Ischemia of the pons drives autonomic reaction with bradycardia,

increased blood pressure and respiratory irregularity. With distal progression of ischemia,

necrosis of the vagal and cardiomotoric and respiratory nuclei occurs and uncontrolled

sympathetic reaction can be seen, unless total sympathetic de-nervation due to death of

oblongate medulla manifests. Thus, brain death causes two distinct haemodynamical phases:

uncontrolled sympathetic activation with arterial blood pressure well above 200 mmHg and

tachyarrythmia, followed by much longer hypotension period resulting from sympathetic

insufficiency, loss of vascular tonus and decrease in peripheral resistance.41

Early-phase

catecholamine storm depends on the velocity of intracranial pressure increase and increase in

serum adrenaline concentration can be as high as 200-1000 fold.42

Increased hydrostatic

pressure causes immediate tissue oedema, often haemorrhagic.43

On the other hand, necrosis of the hypothalamus and pituitary gland dissipates

thermoregulation44

and hormonal homeostasis: serum thyroid hormones do not meet

metabolic demand,45

diabetes insipidus is not controlled by vasopressin,46

production of

cortisol is halted47

and insulin release is inadequate.48

Free radicals and proteolytic enzymes

are released and slow, however progresive and inevitable death of cells and tissues occurs in

necrotic and apoptotic mechanism.49

These changes result in deterioration of brain-dead donor

organ function impairment, including impairment of kidney function in both optimal and

marginal donors.50

Hence, even if effect of cold ischemia was eliminated in experimental

setting, influence of brain death on renal transplant function remains significant,51

including

more frequent acute rejection episodes52

and decreased long-term survival.53

54

Brain death

was found to increase tissue CD68,55

CD4550

and CD8 infiltrates,49

ICAM1, VCAM156

and E-

selectin expression,57

as well as serum pro-inflammatory cytokines.58

To avoid or at least minimize consequences of brain death, various methods of

treatment applied to the brain-dead donor had been tried, both experimentally and clinically.

Early aggressive donor management with stabilization of donor haemodynamics has been

shown to increase the number of donations and organ yield.59

60

There‟s good and increasing

evidence, that treatment with small-dose catecholamines, especially dopamine, may improve

renal graft function and survival.61

62

Despite doubts if it has any benefit over agressive fluid

management with haemodynamic stabilization, Papworth protocol consisting of

methylprednisolone bolus, vasopressin, thriiodothyronine and insulin infusions, with its

numerous modifications, facilitated control of heamodynamic disturbances and seemed to

successfully increase the number of transplanted hearts and many large-volume cardiac

transplant centers have adopted it.63

Kidney storage

Kidney storage in hypothermia, which is necessary for logistic reasons, has to

maintain the organ viability during the time between recovery and transplantation. The

importance of ensuring successful preservation of kidneys between retrieval and implantation

has been recognized since renal transplantation started. Two approaches have developed to

limit ischemic damage, cold static storage (CS) and machine pulsatile perfusion (MP), both

technologies have persisted and continued evolving since their early development. In 1967

successful organ perfusion preservation was introduced by Belzer. Two years later Geoffrey

Collins described preservation solution with intracellular ionic content which allowed for

successful storage in simple hypothermia for up to18-24 hours. Soon, Belzer and Southard

developed different type of solution for hypothermic storage, so called UW solution, which

has become a gold standard since.

Simple static cold storage is the most widely used form of preservation in every day

clinical practice. It is simple and effective when the kidney from the standard deceased donor

is stored up to 24 hors. In last 10 years a number of different solutions have been introduced

into market which are successfully used for kidney and other organs hypothermic storage.64

However for longer storage and for preservation of kidneys recovered from extended criteria

(or non-heart beating) donors another approach is needed.

Hypothermic pulsatile perfusion kidney storage before transplantation was introduced

by Belzer more than 30 years ago. This method of preservation was not widely used, because

of the simplicity of cold storage, especially since the UW solution came to the market which

allowed for successful preservation for up to 24-30 hours. In the last 10 years an interest in

machine perfusion has grown due to the use of organs recovered from extended criteria, and

NHB donors. Machine perfusate solutions often consist of dialyzed hydroxyethyl starch to

prevent interstitial edema.. Adenosine is added as an ATP synthesis stimulator, phosphate as

an H+ ion buffer and ATP production stimulator, gluconate for cellular swelling suppression,

glutathione as an antioxidant, as well as other agents. Numerous studies confirmed the

usefulness of MP preservation of kidneys over 30 hours.

Wight et al65

conducted a systematic review and metaanalysis of the effectiveness of

machine perfusion and cold storage techniques in reducing delayed graft function (DGF) and

improving graft survival in recipients of kidneys from beating and non-heart-beating donors.

They documented that studies of high methodological quality and sufficient size are required

to determine whether machine preservation leads to reduce rates of DGF. Predicted impact on

graft survival implies that direct evidence would require a large population followed up over a

long period of time. Registry database analysis supported by validation of the link between

DGF and graft survival may be preferable and more feasible than randomized controlled

trials. Several studies have shown beneficial effects of Machine Perfusion (MP) on early

graft function and survival. Kwiatkowski et al in their prospective and retrospective studies

have shown beneficial effects of machine perfusion (MP) on early and long-term graft

survival.66

What‟s more, significantly lower incidence of chronic rejection, interstitial

fibrosis/tubular atrophy in kidneys preserved by MP was found.67

. Recent prospective, randomized Eurotransplant study comparing cold storage with

machine perfusion preservation documented the value of MP. This method decreased the

incidence of delayed graft function not only in standard, but also in extended criteria donors.

MP also reduced the incidence, the duration and the severity of DGF and ameliorates graft

function of NHBD kidneys after kidney transplantation.68

Also, as shown recently, machine

perfusion in a long term observation is economically superior to CS.69

Hypothermic machine perfusion not only facilitates longer storage, but also allows for

the assessment of the extent of ischemic damage of the organ. Machine perfusion

preservation allows also on the assessment of the quality of kidneys before transplantation .

Measuring flow, resistance, lactates excretion and alpha-GST in the perfusate it is possible

to predict whether the kidney will have immediate of delayed function after

transplantation.70

71

72

73

74

75

Reperfusion injury

Reperfusion injury, as an effector phase of ischemic injury, develops during hours or

days after initial insult. Repair and regeneration process occurs along with cellular apoptosis,

autophagy and necrosis, and the fate of the organ depends on whether cell death or

regeneration prevails. As apoptosis needs energy and protein synthesis, it occurs mostly at

reperfusion – cytochrome C release and caspase activation was noted as early as 5 minutes

after reperfusion, while it was virtually absent during prolonged ischemia of

cardiomyocytes.76

If cytosolic calcium, increased during ischemia, returns to normal values,

cells are able to recover from the injury. However progressive increase in cytosolic calcium

marks irreversible phase of injury.77

Rapid burst of free radicals shortly following reperfusion

is well-confirmed phenomenon. Most likely, discordant respiratory chain enzymes are the

main source of radicals on reperfusion, reduced ubiquinone reacts with free oxygen to form

superoxide, metabolized to hydrogen peroxide with catalse or froming extremely reactive

hydroxyl radical in the presence of ferrous/ copper ions. Radicals peroxidate lipids of cellular

and mitochondrial membranes at their double bonds, aromatic rings and thiol groups. This

includes cardiolipin and other phospholipids. Free radicals probably trigger endothelial injury,

as only after reperfusion endothelia, which seemed fairly well preserved after ischemia phase,

become oedematous and leaky to the proteins and small particles. As a source of both free

radicals (with potent radical-scavenging potential on the other hand) and cytochrome c,

mitochondria seem to be a gatekeeper to cellular injury during reperfusion. Mitochondrial

permeability transition pore remains closed during ischemia (it is inhibited by acidosis) and

opens on reperfusion.78

Although Na/K-ATPase inhibition did not play a major role in

oedema formation during ischemic phase, on reperfusion it is inhibited by free radicals and

intensifies the oedema.

Damaged endoplasmic reticulum fragments proliferate and concentrate in the

reperfused tissue, leading to formation of autophagosomes and indicating upcoming

disintegration of the cells.79

Soon, lysosomal activity of proteases and cathepsins increases

leading to necrotic or apoptotic cell death. Apoptosis can occur due to reduced mitochondrial

activity and loss of electric potential, increased Ca2+

with calpaine activation, free radical

generation, NO, decreased BCL-2 and increased BAX protein,80

overexpression of FAS

ligand, disintegration of cytoskeleton, and other triggers. Nitric oxide is extremely interesting

molecule, capable of both protection of microcirculation from the injury and tissue injury via

peroxynitrite formation, depending on whether it was fromed by constitutive or inducible

isoforms of NOS.81

Neutrophils are attracted by a set of chemokines including CXCL10, RANTES, IL17,

MCP1 and recruited by endothelial cells expressing ICAM1, E and P selectin which cross-talk

with integrins and L-selectin on polymorphonuclear cells. Matrix proteins, MMP-9,

fibronectin and laminin also play important role, facilitating leukocyte migration across the

vessel. Expression of these proteins significantly increases after reperfusion.82

83

Neutophils

may cause direct cytotoxicity via generation of oxygen free radicals and generate cytokines.

They control perivascular tissue oedema, damage endothelial cells directly and promote

platelets aggregation. Extent of infiltration with these cells is proportional to the magnitude of

injury to the reperfused organ. Although reperfusion begins with relative hyperaemia, often

exceeding normal blood flow rate, then followed by much longer phase of diminished blood

flow. Although mostly driven by decreased metabolic demand and extracellular oedema, it

partly depends on neutrophils and platlets accumulation within blood vessels, attenuating flow

and causing “no reflow” phenomenon,84

however negative role of flow reduction is disputed.

Prevention of reperfusion injury

Normothermic reperfusion

In spite of the latest developments of new preservation solution formulas (Vasosol,

ET-Kyoto, SCOT, IGL-1), major progress can be expected with normothermic preservation or

resuscitation. Kootstra group presented spectacular results of resuscitation of kidneys

severely damaged with 2 hours of warm ischemia. When reperfused in near-normothermia

with their EMS medium, kidneys were successfully rescued from lethal injury.85

These

experiments were followed lately by a Leicester group, where renal blood flow, high energy

stores and renal function after warm ischemia and 16 hours of cold storage were nicely

restored with 2 hours of normothermic resuscitation with donor‟s blood.86

However most

impressive clinical progress was achieved with TransMedics Organ Care System which

allows for extracorporeal normothermic preservation in donor bood-based solution. Leicester

group proved superiority of prolonged warm preservation of controlled and uncontrolled

NHBD kidneys,87

but technology seems promising in preservation, viability assessment and

expansion of utilized NHBD livers88

and improvement of cardiac allograft quality.89

. Recently

some groups have pointed at the beneficial effects of normothermic machine perfusion as a

new perspective in organ preservation and transplantation.90

Treatment of the recipient

To successfully prevent reperfusion injury, actions must be applied early, at stage of

the donor hemodynamic disturbances, organ retrieval and during preservation. Some

interventions however, may prove productive when used at graft reperfusion in the recipient.

Although free radicals play an undoubted role in pathology of ischemia-reperfusion

injury, administration of free radical scavengers at reperfusion used to give questionable

results until the Land‟s study, which confirmed significant late-term protection.106

Lately,

pyruvate has been shown to directly detoxify peroxynitrite and H2O2 and protect against

myocardial reperfusion injury.91

As described above, cyclosporines are able to inhibit permeability transition and, when

administered on reperfusion, they have been shown to decrease creatine kinase release and

infarct size in humans undergoing PCI intervention for acute cardiac ischemia.92

This needs

thorough evaluation in transplant models and remains in contrast with current policy to delay

CNI administration in renal ischemia-reperfusion injury.

Intermittent ischemia and reperfusion prior to prolonged ischemia, called ischemic

preconditioning, has been shown to salvage the heart and some other tissues from prolonged

ischemia. Similar phenomenon that can be applied at the procedure of transplant implantation,

called ischemic postconditioning, has been described lately. If gradually elongated kidney

reperfusion periods were intermitted with short-time ischemia, serum BUN and creatinine

decreased faster than in non-postconditioned animals exposed to renal warm ischemia. The

functional improvement was accompanied by more effective complex II mitochondrial

respiration and lesser peroxide production and protein oxidation93

as well as inhibition of

apoptosis in reperfused tissue.94

Although the phenomenon was confirmed in the heart, brain

and skin flap in some models, some doubts exist95

and call for further studies. Interestingly,

postconditioning protection had been induced pharmacologically with administration of 3%

v/v sevoflurane from the beginning of reperfusion the heart, which decreased infarct size and

mitochondrial injury,96

although hyperglycemia abolished this protection.97

The role of hemoxigenase 1, enzyme which converts heme into biliverdin, carbon

monoxide and free Fe, in protection from ischemia-reperfusion injury has been extensively

studied. Exposition of the liver transplant recipient animals to inhaled CO decreased serum

ALT, hepatocyte necrosis and neutrophil infiltrates dose-dependently.98

Noteworthy, HO-1

can be induced by simvastatin precoditioning.99

Interestingly, nicotine has been shown to reduce tubular damage in experimental model when

administered before reperfusion during warm ischemia through its strong anti-inflammatory

effect. It prevented neutrophil infiltration, decreased CXC, KC, TNF-alpha and high-mobility

group box-1 protein release. Less tubular apoptosis and proliferation was observed in

nicotine-treated mice.100

Ischemia reperfusion and immune injury

Ischemic injury to an allograft significantly increases risk of poor initial function. This

in turn was associated with increased rate of acute rejection episodes. Although there was no

satisfactory supporting hypothesis, many researchers thought an association between ischemia

and graft immunogenity to be reasonable. Elegant explanation came with Matzinger‟s injury

theory which explained the link between damage to the tissue, innate immunity response

differentiating „self‟ from „non-self‟ or „damaged-self‟ and communicating the danger signal

to adaptive immunity via Toll-like receptors and antigen presenting cells.101

„Danger signals‟

or „alarmins‟ released during ischemia and reperfusion could be graft-derived DNA and RNA,

oxidized proteins and lipids, high mobility group box 1 (HMGB1) and uric acid and calcium

pyrophosphate crystals.102

Lately, HMGB1 has grown much interest. It was shown to be

actively secreted from cells at risk via free-radicals dependent pathway during hepatocyte

ischemia/reperfusion. This process required intact TLR4 signaling and calcium-dependent

kinases.103

104

TLR4 expression increases in tubular epithelial cells following

ischemia/reperfusion. When TLR4 or its signaling pathway protein, MyD88 were absent,

kidneys were protected from I/R.105

According to injury theory, the lesser initial injury, the smaller agitation of adaptive immunity

and chances for early and late response to the allograft. Land immediately noticed, how

beautifully this theory explains results of their experiment of delayed kidney graft protection

with recombinant superoxide dismutase.106

107

Prolonged ischemia (>15 hours) can indeed

increase graft immunity, as it was found to be an independent risk factor for post-rejection de

novo production of high-titer anti-HLA class I PRAs.108

Circulating anti-donor antibodies as

well as IgM and C4d deposits in renal glomeruli accompanied severe (72 hours of cold

storage) ischemic injury 7 days after engraftment. When ischemia was limited to 24 hours, no

signs of damage and antibody-mediated immunity were present at day 7.109

Lately, we have

shown that although machine perfusion is not an effective method to decrease immediate

effects of ischemic injury, it significantly ameliorates late histological picture of renal

allograft. Tubular atrophy and interstitial fibrosis (former CAN) were observed in 90% of

biopsies from cold-stored and 64% machine perfused kidneys and chronic rejection

(according to Banff 2005 consensus), respectively in 9% and 3% of renal allografts.110

In an

exquisite study involving 600 protocol biopsies, Yilmaz has linked risk of chronic allograft

changes, including interstitial inflammation and fibrosis, tubular atrophy, vascular wall and

tubular basal membrane thickening, with cold ischemia duration.111

Clinical relevance of I/R injury

Organ shortage is a universal problem. The waiting lists are growing in all countries

and the gap between demand and number of available organs is increasing.. For this reason

extended criteria (ECD) and non heart beating donors (NHBD) are widely used. Such

kidneys are more susceptible to ischemic damage which leads to delayed graft function or

primary non-function, as well as worse graft function and survival. Kidneys recovered from

such donors should be stored using pulsatile perfusion, which allows for better protection

during preservation related ischemia, but also allow to measure several parameters (flow,

resistance, lactates excretion, alfa GST) which might be useful for the assessment of the

extent of ischemic injury.

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