Fisiopatologia de IRA Isquemica

8/3/2019 Fisiopatologia de IRA Isquemica

1/12NATURE REVIEWS|NEPHROLOGY VOLUME 7 | APRIL 2011 | 189

Division of Nephrology,Department ofMedicine, IndianaUniversity School ofMedicine, 950 WestWalnut Street, R2202,Indianapolis, IN 46202,USA (A. A. Sharfuddin,B. A. Molitoris).

Correspondence to:B. A. [email protected]

Pathophysiology of ischemic acute kidney

injuryAsif A. Sharfuddin and Bruce A. Molitoris

Abstract | Acute kidney injury (AKI) as a consequence of ischemia is a common clinical event leading to

unacceptably high morbidity and mortality, development of chronic kidney disease (CKD), and transition

from pre-existing CKD to end-stage renal disease. Data indicate a close interaction between the many cell

types involved in the pathophysiology of ischemic AKI, which has critical implications for the treatment of

this condition. Inflammation seems to be the common factor that links the various cell types involved in this

process. In this Review, we describe the interactions between these cells and their response to injury following

ischemia. We relate these events to patients who are at high risk of AKI, and highlight the characteristics

that might predispose these patients to injury. We also discuss how therapy targeting specific cell types can

minimize the initial and subsequent injury following ischemia, thereby limiting the extent of acute changes and,hopefully, long-term structural and functional alterations to the kidney.

Sharfuddin, A. A. & Molitoris, B. A. Nat. Rev. Nephrol.7, 189200 (2011); published online 1 March 2011; doi:10.1038/nrneph.2011.16

Introduction

The kidney is comprised of heterogeneous ce popua tions that function together to perform a number oftighty controed and compex processes. Acute kidneyinjury (AKI) is a common cinica event that disruptsthis homeostasis, eading to unacceptaby high morbid ity and mortaity. One cause of AKI is ischemia, whichcan occur for a number of reasons, for exampe, withthe use of vasoconstrictive drugs or radiocontrast agents;

hypotension inked to sepsis or bood oss after surgeryand trauma is aso a known cause of ischemia. The bodyis abe to adapt to a reduction in bood fow to a certaineve, but when deivery of oxygen and metaboic sub strates becomes inadequate, ceuar injury eads to organdysfunction. In this Review, we describe the interactionsbetween the various ce types and processes invoved inthe pathophysioogy of ischemic AKI. We aso outine anapproach that wi faciitate patient care and deveopmentof therapies for ischemic AKI in the future.

Patients at high risk of ischemic AKI

A cardiovascuar event can ead to ischemic AKI in

any patient, but there are certain individuas who are atinherenty high risk of deveoping AKI foowing mid tomoderate reductions in kidney perfusion. Thakar et al.1were the first to emphasize the importance of under standing and quantifying this increased risk. Subsequentstudies have identified and vaidated cinica variabes,such as age, existing chronic kidney disease (CKD), and

proteinuria, that contribute to this increased risk,25 andcinica staging systems, incuding the RIFLE system,have been deveoped to cassify patients according totheir risk or injury.6,7

These parameters shoud be used together with tradi tiona biomarkers when evauating risk in a patientundergoing eective procedures or receiving therapiesthat potentiay reduce rena bood fow.8 Rather than

a singe characteristic, patients often have mutipeassociated risk factors that resut in a cumuative risk ofdeveoping ischemic AKI (Box 1).1 Identifying patientsat risk is an essentia component of the medica workup,invoving both history taking and a physica examina tion, as this wi maximize care and reduce risk. Forexampe, improving the hemodynamic status of a patientis important to minimize AKI from nephrotoxic drugssuch as cispatin and aminogycosides, or with the useof radiocontrast agents that reduce kidney perfusion.9Additiona factors, such as high eves of oxidativestress or infammation, aso increase a patients risk ofdeveoping AKI.10

Prerena azotemia is the most common cause of AKIand accounts for 4055% of a cases.1113 This condi tion resuts from kidney hypoperfusion owing to areduced effective arteria voume, that is, the voumeof bood effectivey perf using the organs. Prerenaazotemia is divided into voume responsive and voumenonresponsive forms. In patients with the voume non responsive form, additiona intravascuar voume doesnot restore kidney perfusion and function. For exampe,patients with conditions such as congestive heart faiureand sepsis might not respond to intravenous fuid therapy,as decreased cardiac output and tota vascuar resistanceprevent kidney perfusion.1416 Compensation for prerena

Competing interests

B. A. Molitoris declares associations with the followingcompanies: Eli Lilly and Quark Pharmaceuticals. See the articleonline for full details of the relationships. A. A. Sharfuddindeclares no competing interests.

FOCUS ON AKI IN CRIT ICAL CARE

2011 Macmillan Publishers Limited. All rights reserved
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2/12190 | APRIL 2011 | VOLUME 7 www.nature.com/nrneph

azotemia incudes activation of baroreceptors, which ini tiates a cascade of neura and humora responses, activa tion of the sympathetic nervous system, and an increasein the production of catechoamines, especiay norepi nephrine.17 Increased reease of vasopressin is mediatedby hypovoemia and a rise in extraceuar osmoa ity, resuting in vasoconstriction, water retention, and

Key points

During ischemic acute kidney injury (AKI), ATP depletion results in cytoskeletal

changes in epithelial and endothelial cells, causing disruption of function, and a

decrease in glomerular filtration rate

Apoptosis and necrosis are major mechanisms of cell death that have

important roles in ischemia, with the contribution of each pathway depending

on the extent of the injury

Under physiological conditions, endothelial cells regulate permeabili ty, vascular

tone, coagulation, and inflammation; endothelial cells that are dysfunctionalsubstantially contribute to the extension phase of AKI

Inflammation and its mediators orchestrate the extension phase of ischemicAKI, and limit injury to tubular epithelial cells and vascular endothelial cells,

thereby promoting repair

Complex interactions between epithelial cells, endothelial cells, inflammatory

mediators, and cytokines can result in persistent injury during acute tubular

necrosis

Stem cells, mesenchymal cells, and endothelial progenitor cells contribute to

the repair and regeneration of tubular cells and endothelial cells following injury,

and could provide attractive targets for therapeutic intervention

back diffusion of urea into the papiary inter stitium.Activation of the reni nangiotensin adosteronesystem eads to increased production of the potent vaso constrictor angiotensin II, which preferentiay increasesefferent arterioar resistance.18 Gomeruar fitration rate(GFR) is preserved by the resutant increase in gomeru ar hydrostatic pressure. Angiotensin II activity is asoincreased during severe voume depetion, which eadsto afferent arterioar constriction, reducing rena pasmafow, GFR, and the fitration fraction.19

Concomitanty, compensatory mechanisms of kidneyautoreguation preserve gomeruar perfusion.20 Underphysioogica conditions, autoreguation of rena boodfow works above a mean systemic arteria bood pres sure of 7580 mmHg. Beow this pressure, the gomeru ar utrafitration pressure and GFR decine abrupty.21Rena production of prostagandins, kaikrein, kinins,and nitric oxide (NO) are increased and contribute tovasodiation.22,23 Nonsteroida anti infammatory drugsinhibit the production of prostagandins and reducekidney perfusion. Angiotensin converting enzyme

inhibitors bock the synthesis of angiotensin II anddisturb the deicate baance between afferent and effer ent arterioar tone in patients who have severe reductionsin effective arteria voume, such as those with congestiveheart faiure or biatera rena artery stenosis, thus wors ening prerena azotemia. Conversey, very high eves ofangiotensin II, as seen in circuatory shock, cause con striction of both afferent and efferent arterioes, whichnegates its protective effect.24

Taken together, these data provide strong evidence thatcertain identifiabe patients are at high risk of deveopingAKI. Increased attention to maximizing conditions, suchas hemodynamic status, drug dosing based on GFR, and

voume status is essentia to prevent or ameiorate AKIin these patients.

Cellular changes during ischemic AKI

Acute epithelial cell injury

Foowing a reduction in effective kidney perfusion,epitheia ces are unabe to maintain adequate intra ceuar ATP for essentia processes. This depetion ofATP eads to ce injury and, if severe enough, ce deathby necrosis or apoptosis. A segments of the nephroncan be affected during an ischemic insut, but the mostcommony injured epitheia ce is the proxima tubuarce. These ces are particuary susceptibe for a number

of reasons. First, this ce type has a high metaboic raterequired for mediating ion transport and a imited cap acity to undergo anaerobic gycoysis. Second, owingto the unique bood fow in the outer stripe of the S3segment of the nephron, there is marked microvascuarhypoperfusion and congestion in this region after injury,which persists and mediates continued ischemia evenwhen cortica bood fow might have returned to near norma eves. Endotheia ce injury and dysfunctionare primariy responsibe for this phenomenon, knownas the extension phase of AKI.25 Understanding that isch emic injury can be ocaized to specific microvascuardomains rather than throughout the kidney is important,

Box 1 | Causes of reduced effective ar terial volume and kidney hypoperfusion

Intravascular volume depletion

Hemorrhage (e.g. following trauma, surgery, postpartum)

Gastrointestinal losses (e.g. from diarrhea, vomiting, nasogastric loss)

Renal losses (e.g. diuretics, osmotic diuresis, diabetes insipidus)

Skin and mucous membrane losses (e.g. burns, hyperthermia)

Nephrotic syndrome

Cirrhosis

Capillary leak

Reduced cardiac output

Cardiogenic shock

Pericardial disease (e.g. restrictive, constrictive, tamponade)

Congestive heart failure

Valvular heart disease

Pulmonary disease (e.g. pulmonary hypertension, pulmonary embolism)

Sepsis

Systemic vasodilation

Cirrhosis

Anaphylaxis

Sepsis

Renal vasoconstriction

Early sepsis

Hepatorenal syndrome

Acute hypercalcemia

Molecules and drugs (e.g. norepinephrine, vasopressin, nonsteroidal anti-

inflammatory drugs, angiotensin-converting enzyme, calcineurin inhibitors)

Radiocontrast agents

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as quantifying tota rena bood fow as a measure ofeffective bood fow coud be miseading.

The other major epitheia ces of the nephron invovedin the pathophysioogy of ischemic AKI are those ofthe meduary thick ascending imb ocated distay.Apoptotic changes have been detected in human AKI,as shown in dista nephron segments during nephro toxic acute tubuar necrosis. Apoptosis of dista tubuarces aso occurs in donor biopsies before engraftment,which was predictive in one study of deayed graft func tion.26 In an ex vivo mode of hypoxic AKI, administra tion of FG 4497 (a specific proy hydroxyase inhibitorthat activates hypoxia inducibe factor [FibroGen, SanFrancisco, CA, USA]) in the isoated perfused kidneyed to decreased seective outer meduary dista tubuarinjury.27 Proxima tubuar ce injury and dysfunctionduring ischemia or sepsis eads to afferent arterioarvasoconstriction mediated by tubuogomeruar feed back, umina obstruction, and backeak of fitrateacross injured proxima tubuar ces, resuting inineffective gomeruar fitration and a profound drop

in GFR (Figure 1).28,29

Morphological changes

A hamark of ischemic injury is oss of the apica brushborder of proxima tubuar ces. Disruption of microviiand their detachment from the apica ce surface eadsto formation of membrane bound bebs eary foow ing ischemia that are reeased into the tubuar umen.Detachment and oss of tubuar ces exposes areasof denuded basement membrane, resuting in foca areas ofproxima tubuar diatation, as we as formation of distatubue casts.30 The soughed tubuar ces, brush bordervesice remnants, and ceuar debris in combination with

uromoduin form these granuar casts, which have thepotentia to obstruct the tubue umen, eading to noGFR in that functiona unit.31 Necrotic ce death is rareand restricted to the highy susceptibe outer meduaryregions, whereas features of apoptosis are commony seenin both proxima and dista tubuar ces (see beow).32

Injured gomeruar epitheia ces foowing isch emic or septic injury are not usuay seen on histoogicastains, athough studies have shown reversibe podocyte specific moecuar and ceuar changes. Wagner et al.33demonstrated in a rat mode that rena ischemia inducedpodocyte effacement with oss of sit diaphragm integ rity and proteinuria owing to rapid oss of interactions

between the tight junction proteins Neph1 and ZO 1.Ce cuture modes using human podocytes showed thatATP depetion resuted in rapid oss of Neph1 and ZO 1binding, and redistribution of Neph1 and ZO 1 from thece membrane to cytopasm; ATP recovery increasedphosphoryation of Neph1 and restored Neph1 and ZO 1binding and their ocaization at the ce membrane.33

Cytoskeletal and structural changes

The actin cytoskeeton has an integra roe in maintain ing ce structure and function, poarity, endocytosis,signa transduction, motiity, movement of organees,exocytosis, ce division, migration, barrier function of

the junctiona compexes, and cematrix adhesion.34Maintaining the integrity of the cytoskeeton is especiay

important for proxima tubuar ces in which ampifica tion of the apica membrane by microvii is essentia fornorma ce function. Depetion of ceuar ATP eadsto rapid disruption of apica F actin by depoymeriza tion mediated in part by cofiin, and redistribution ofthe cytoskeeta F actin core. This disruption causesinstabiity of the surface membrane and formation ofmembrane bound extraceuar vesices or bebs that areeither exfoiated into the tubuar umen or internaizedto potentiay be recyced.3539 Other proteins invoved inthe depoymerization process are tropomyosin and ezrin.During ischemia, ezrin becomes dephosphoryated andthe attachment between the microviar F actin core

and pasma membrane is ost.40 Simiary, tropomyosinsbind to and stabiize the F actin microfiament core in thetermina web by preventing access to cofiin. Foowingischemia, there is dissociation of tropomyosins from themicrofiament core, which enabes access of the micro fiaments in the termina web to the binding, severing,and depoymerizing actions of cofiin.41

Another important consequence of disruption ofthe actin cytoskeeton is the oss of tight junctions andadherens junctions. These junctiona compexes activeyparticipate in numerous functions, such as paraceuartransport, ce poarity, and ce morphoogy. Eary isch emic injury resuts in opening of tight junctions, eading

Endothelial injury

ActivationDysfunctionDetachmentApoptosisNecrosis

Inammation

WBC recruitmentNeutrophils

MacrophagesLymphocytesDendritic cell

activation

Vasoconstriction

Cytokine releasePermeability

Leukocyte adhesionmolecules

Rouleaux formationReduced ow

Leukocyte activation

Cytokine releaseMargination

Tissue migrationReduced ow

Cellular sheddingCellular debrisLoss of polarity

Loss of tight junctionsCytokine release

Epithelial cell injury

Sub-lethal injury Cytoskeleton

disruption

Lethal injury Necrosis Apoptosis

TubularobstructionBackleak

TGF-

Defective functionReduced GFR

High FENaConcentrating defect

Figure 1 | Pathogenesis of ischemic AKI. The major pathways of GFR impairmentin ischemic acute tubular injury are caused by ATP depletion in vascular andtubular cells. Numerous interactions exist between endothelial cells, WBCs,and epithelial cells in the pathophysiology of ischemic AKI. These interactions arebidirectional between the cells involved, and result in specific functional and

structural alterations. Inflammatory mediators released from proximal tubular cellsinfluence endothelial cell processes (e.g. increase vasoconstriction andexpression of cell adhesion molecules) that in turn influence the interactionsbetween WBCs and endothelial cells, leading to reduced microvascular flow andcontinued hypoxia within the local environment. Additional functional changesoccur, such as a marked reduction in production of erythropoietin and25-hydroxylation of vitamin D. Electrolyte accumulation can rapidly lead torequirement of renal replacement therapy. Metabolic acidosis as a consequence ofAKI must also be carefully monitored. Abbreviations: AKI, acute kidney injury;FENa, fractional excretion of sodium; GFR, glomerular filtration rate; TGF-,transforming growth factor ; WBC, white blood cell.





to increased paraceuar permeabiity and backeak ofthe gomeruar fitrate into the interstitium.42 Duringischemia, epitheia ces aso ose their attachment tothe underying extraceuar matrix owing to disruptionof integrins. Depetion of ATP resuts in reocaization of integrins from the basa membrane to the apica mem brane, with subsequent detachment of viabe ces fromthe tubuar basement membrane.43 The exfoiated cesthen bind to each other and form ceuar casts withinthe tubuar umen.

Aterations to the actin cytoskeeton during ischemiaresut in changes in ce poarity and function (Figure 2).Basoatera Na+/K+ATPase pumps redistribute to theapica membrane as eary as within 10 min foowingdisruption of the spectrinactin cytoskeeton, which isresponsibe for attaching the pumps to the membrane.44Redistribution of the pumps resuts in bidirectionatransport of sodium and water across the apica, as weas the basoatera, epitheia ce membrane, with ceuarsodium being transported back into the tubuar umen.This process is one the major mechanisms of the highfractiona excretion of sodium seen in patients with acutetubuar necrosis, and the inefficient use of ceuar ATP,

as ischemic conditions uncoupe ATP use and effec tive transceuar sodium transport.45 A high sodiumconcentration in the fitrate reaching the dista tubueeads to a reduction in GFR by activation of tubuargomeruar feedback, with stimuation of the macuadensa mediating afferent arterioar vasoconstriction.

Apoptosis and necrosis

The fate of the epitheia ce foowing an ischemicevent utimatey depends on the extent of injury. Cesundergoing sub etha or ess severe injury have the cap abiity of functiona and structura recovery if the insutis interrupted. Ces that suffer a more severe or ethainjury undergo apoptosis or necrosis, eading to cedeath. Apoptosis is an energy dependent, programmedce death that resuts in condensation of nucear andcytopasmic materia to form apoptotic bodies. Thesemembrane bound apoptotic bodies are rapidy phago cytosed by macrophages and neighboring epitheia ces.During necrosis, there is ceuar and organee sweing,oss of pasma membrane integrity, and rapid reease of

cytopasmic and nucear materia into the umen or inter stitium.46 Secondary necrosis occurs when ces under going apoptosis do not have adequate ceuar eves ofATP to support the staged demise of the ce.

Apoptotic mechanisms are compex with factorsaffecting a number of pathways. The caspase famiy ofproteases is an important initiator and effector of apop tosis.47,48 Both intrinsic (mitochondria) and extrinsic(death receptor) apoptotic pathways are activated inAKI. Specificay, activation of procaspase 9 primariydepends on intrinsic mitochondria pathways reguatedby the Bc 2 famiy of proteins, whereas pro caspase 8activation resuts from extrinsic signaing via ce surface

death receptors, such as FAS and FADD.47,48 Considerabecrosstak aso exists between the intrinsic and extrinsicpathways. Caspase 3, caspase 6, and caspase 7 are effec tor caspases that are abundant and catayticay robust,ceaving many ceuar proteins, which resuts in thecassic apoptotic phenotype. Inhibition of caspase activ ity has been shown to be protective against injuryin vitroand in AKI in vivo.49,50

Severa apoptotic pathways, incuding the intrinsic(Bc 2 famiy, cytochrome c, caspase 9), extrinsic (FAS,FADD, caspase 8) and reguatory (p53 and nucearfactor B) pathways, seem to be activated during isch emic rena tubuar ce injury. The baance between

ce surviva and apoptotic ce death aso depends onthe reative concentrations of the proapoptotic (BAX,BAD, and BID) and antiapoptotic (Bc 2 and B c 2 ike protein 1) members of the Bc 2 famiy of proteins.Overexpression of proapoptotic or reative deficiencyof antiapoptotic proteins can ead to formation of mito chondria pores. Other proteins that have an importantroe in apoptotic pathways incude nucear factor Band p53.51,52 The centra proapoptotic transcriptionfactor p53 can be activated by hypoxia, via hypoxia inducibe factor, as we as other noxious stimui suchas certain drugs. Kinases are responsibe for mediat ing ceuar responses invoved in apoptosis, surviva,

Brush border

Blebbing of microvilli

Injury Recovery

Tight junctions

Basement membrane

Loss of actin

cytoskeletal structure

Cast formation

Loss of cellcell contacts

Redistribution of

Na+/K+-ATPases

and integrins

to apical location

Epithelial

cell swelling

Backleak

Actin cytoskeleton

Cell adhesionmolecules

Integrins

Na+/K+-ATPase

Figure 2 | Effects of sub-lethal injury to tubular cells and their recovery. Damage toepithelial cells occurs early during ischemia and involves alterations to thecytoskeleton and in surface membrane polarity. ATP depletion induces rapiddisorganization of the actin cytoskeleton structure, which disrupts tight junctionsand in turn leads to backleak of tubular filtrate. Loss of cellcell contacts and celladhesion molecules results in flattened nonpolarized epithelial cells, denudedbasement membranes, and expression of mesenchymal markers. Na+/K+-ATPasepumps normally located at the basolateral membrane and tethered by the actinspectrin cytoskeleton, redistribute to the apical membrane of the proximal tubularcell and are internalized into the cytosol during ischemic injury. Morphologically,proximal tubular cells lose their brush borders, undergo swelling, and blebbing ofmicrovilli during injury, leading to cast formation. Severely injured proximal tubularcells undergo mesenchymal differentiation and subsequent re-epithelization.Recovery of proximal tubular cells begins with integrin reattachment, reassembly ofthe actin cytoskeleton, repolarization of the surface membranes, and redistribution

of the sodium pumps back to their basolateral location.

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and repair through their interaction with signas fromgrowth factors, incuding hepatocyte growth factor,insuin ike growth factor I, epiderma growth factor, andvascuar endotheia growth factor (VEGF).53,54 Thesemechanisms, which can be activated independenty vianonischemic pathways in other types of injury, inhibitproapoptotic proteins and activate the antiapoptotictranscription of cycic AMP response eement bindingfactors. Knock down of p53 in proxima tubuar cesusing short interfering RNA ed to a dose dependentattenuation of apoptotic signaing and kidney injury incamp ischemia and transpant modes, indicating poten tia therapeutic benefit for ischemic and nephrotoxickidney injury.55,56

Necrosis of epitheia ces resuts from increasedintraceuar cacium and the activation of membranephosphoipases and capain.57,58 Necrotic ces do not,therefore, exhibit the nucear fragmentation or chro matin condensation seen in apoptosis, and neither dothey form apoptotic bodies. Functionay, severe ATPdepetion resuts first in mitochondria injury with sub

sequent arrest of oxidative phosphoryation, causingfurther depetion of energy stores and robust forma tion of reactive oxygen species. Reactive oxygen species,such as the hydroxy radica peroxynitrite and hyper chorous acid, are generated by cataytic conversion inepitheia ces during ischemic injury. These moecuesdamage ces in a variety of ways, incuding peroxidationof ipids in the pasma membrane and intraceuar mem branes, and destabiization of cytoskeeta proteins andintegrins required to maintain cece adhesion as weas interactions between ces and the extraceuar matrix.Reactive oxygen species can aso have vasoconstrictiveeffects by scavenging NO.59

Autophagy, which is the process invoved in degrada tion of a ces own components through the ysosomamachinery, is now increasingy recognized as perhaps themost frequent ce death pathway for injured epitheium.Li et al.60 demonstrated the important roe of autophagyin rena epitheia ces in obstructive uropathy modes,whie Koesters et al.61 showed that expression of trans forming growth factor eads to excessive autophagy ininjured tubues.

Endothelial dysfunction

Endotheia ces contribute to vascuar tone, reguationof bood fow to oca tissue beds, moduation of coagua

tion and infammation, and vascuar permeabiity. Bothischemia and sepsis have profound effects on the renaendotheium, resuting in microvascuar dysreguationand continued ischemia and further injury, especiay inthe outer stripe of the kidney. Histopathoogica examina tion shows vascuar congestion, formation of edema,diminished microvascuar bood fow, and marginationand adhesion of infammatory ces to the endotheium,eading to the extension phase of AKI.34 Athough amarked decrease in tota kidney perfusion resuts ingoba ischemia, decreased regiona perfusion can extendischemic injury ocay without affecting goba perfu sion. The compexity of the vascuar beds within the

kidney makes interpretation of tota kidney bood fowchaenging foowing ischemic or septic injury.

Vascular tone

Conger et al.62,63 were among the first to demonstratethat postischemic rat kidneys dispayed vasoconstrictionin response to decreased rena perfusion pressure and,therefore, coud not autoreguate bood fow, even whentota rena bood fow had returned to baseine vaues upto 1 week after injury. This response can be bocked byCa2+ antagonists, and oss of endotheia NO synthase(eNOS) function is due to a reduced vasodiator responseto acetychoine and bradykinin.6466 Seective inhibition,depetion, or deetion of inducibe NOS (iNOS) showsrenoprotective effects during ischemia.64,65 Overa, thereis an imbaance of eNOS and iNOS in ischemic AKI, andit has been proposed that owing to a reative decreasein eNOS, secondary to endotheia dysfunction anddamage, there is a oss of antithrombogenic propertiesof the endotheium eading to increased susceptibiity tomicrovascuar thrombosis.66

Administration of l arginine, the NO donor mo sidomine, or the eNOS co factor sapropterin can pre serve meduary perfusion and attenuate AKI induced byischemiareperfusion injury; conversey the administra tion ofNnitro l arginine methy ester, an NO bocker,has been reported to aggravate the course of AKI.67 Severapharmacoogica studies have assessed the contribution ofeNOS impairment to the overa course of reduced renafunction foowing ischemiareperfusion injury.68,69

Permeability

The endotheia barrier separates the umen of the boodvesse from the surrounding tissue, and contros the

exchange of ces and fuid between these two compart ments. The endotheium is defined by transceuar andparaceuar pathways, the atter being a major contribu tor to endotheia dysfunction induced by infammation.Sutton et al.70 studied the roe of endotheia ces in AKIby utiizing fuorescent dextrans and two photon intra vita imaging in a series of experiments. The increasedmicrovascuar permeabiity observed in AKI is ikey tobe caused by a combination of factors, such as disrup tion of the endotheia monoayer and actin cytoskeeton,breakdown of perivascuar matrix, aterations in contactsbetween endotheia ces, upreguated eukocyteendo theia interactions, and severe aterations in the integrity

of the adherens junctions of the rena microvascuature.In vivo two photon imaging demonstrated a oss ofcapiary barrier function within 24 h of reperfusion,with maxima effects seen at 24 h after injury.70

Breakdown of the barrier function provided by theendotheium might aso be due to activation of matrixmetaoproteinase 2 or matrix metao proteinase 9,which temporay correates with an increase in micro vascuar permeabiity.25,71 Minocycine, a broad spectruminhibitor of matrix metaoproteinases, and the geatinaseinhibitor ABT 518 (Abbott, Abbott Park, IL, USA), bothameiorated the increase in microvascuar permeabiityin a rat mode of ischemic rena injury.71





Coagulation

Endotheia ces have a centra roe in coaguation

through their interaction with protein C and thrombo moduin. Protein C is activated by thrombin mediatedceavage and the rate of this reaction is augmented1,000 fod when thrombin binds to the endotheiace surface receptor thrombomoduin.72 The activa tion rate of protein C is further increased by approxi matey 10 fod when endo theia protein C receptor(EPCR) binds protein C and presents it to the thrombinthrombomoduin compex. Activated protein C acquiresantithrombotic and profibrinoytic properties, and partici pates in numerous anti infammatory and cytoprotectivepathways to restore norma homeostasis.73 Activatedprotein C is aso an agonist of proteinase activated

receptor 1.74During an infammatory response, many of the

natura anticoaguants, incuding protein C, aredegraded, or their production is decreased togetherwith downreguation of EPCR and thrombomoduinexpression, which decreases the anticoaguant and anti infammatory effects of the protein C pathway. Damagedendotheia ces undergo apoptosis, which ampifiesthe coaguation cascade further by providing a pro coaguant surface.75 Continued activation of infamma tion and the coaguation pathway eads to increasedmicrovascuar coaguation and further endotheiace dysfunction. Utimatey, microvascuar function is

compromised, resuting in disseminated intravascuarcoaguation and thrombosis, decreased oca tissue per

fusion, and organ dysfunction or faiure. Treatment withthrombomoduin both before and after injury attenuatesdamage, with minimization of vascuar permeabiitydefects, and improved rena bood fow.76

Activation of eukocytes and their reease of c yto kines requires signas from chemokines circuatingin the boodstream or through direct contact with theendotheium (Figure 3). Roing eukocytes can be acti vated by chemoattractants, such as compement C5a andpateet activating factor. Once activated, integrins on theeukocytes bind to endotheia igands to promote firmadhesion, with integrin 2 being the most important.77These interactions with the endotheium are medi

ated through endotheia adhesion moecues that areupreguated during ischemic conditions.

Singbart et al.78 found that P seectin on pateets,but not on endotheia ces, was the main determi nant in neutrophi mediated ischemic AKI. Bockadeof the common igand for E seectin, P seectin, andL seectin provided protection from both ischemicinjury and mortaity, which seemed to depend onthe presence of a key fucosy sugar on the s eectinigand.79,80 In a ceca igation and puncture (CLP) modeof septic peritonitis, mice engineered to be deficientfor E seectin, P seectin, or both, were competeyprotected against injury.81,82

a

Loss of endothelial

cellcell contacts

Coagulation

Impaired ow

Rouleauxformation

Endothelial cellswelling

Expression of

adhesion molecules

Transendothelial migration

Interstitium

Proximal tubuleCapillary

Cytokinerelease

Cytokines

Chemokines

ROS

ECMbreakdown

Permeability

Cytokines

DCLeukocyteendothelial

cell adhesion and

interaction

Lumen

b

Figure 3 | Events in endothelial cell activation, injury, and reduced microvascular flow. a | Ischemia causes upregulation andexpression of genes encoding various cell surface proteins, such as E-selectin, P-selectin, vascular cell adhesion protein 1,

and intercellular adhesion molecule 1, and downregulation of thrombomodulin. Activated leukocytes bind to endothelialcells through these adhesion molecules. Endothelial injury increases the production of endothelin-1 and decreasesendothelium-derived nitric oxide synthase, which induces vasoconstriction and platelet aggregation, promoting ahypercoagulable environment. The combination of leukocyte adhesion and activation, platelet aggregation, and endothelialinjury serves as the basis for vascular congestion of the cortical and medullary microvasculature. Permeability defectsbetween endothelial cells occur as a result of alterations in tight junctions and adherens junctions. The close proximity andcrosstalk between the epithelial proximal tubular cells and microvascular endothelial cells, as well as release of cytokinesand chemokines, further increase inflammation. Dendritic cells also have a role in this inflammatory cascade, and amplifyinflammatory signals between endothelial cells and epithelial cells. b | Hematoxylin and eosin stain of a human kidneybiopsy from a patient with AKI following ischemic injur y. Abbreviations: AKI, acute kidney injury; DC, dendritic cell; ECM,extracellular matrix; ROS, reactive oxygen species.

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Long-term effects of endothelial injury

Injury to endotheia ces coud contribute to chronicdisease. Basie et al.83 documented a considerabedecrease in the density of bood vesses foowing acuteischemic injury, which ed to the phenomenon of vascu ar dropout. This phenomenon was verified by Hrbetet al.84 who found that vascuar density was reduced byamost 45% at 4 weeks after an ischemic insut. Thisobservation indicates that, unike rena epitheia tubuarces, the rena vascuar system acks comparabe regener ative potentia. Whether apoptosis and necrosis contrib ute to vascuar dropout is not yet cear. Ischemia has beenshown to inhibit VEGF, whie inducing the VEGF inhibi tor ADAMTS1.85 The ack of vascuar repair was postu ated to be due to the reduction in VEGF expression, asadministration of VEGF to postischemic rats preservedmicrovascuar density.86 Vascuar dropout might mediateincreases in the expression of hypoxia inducibe factorand fibrosis, and ater proper hemodynamics, eading tohypertension. Basie and co workers have shown thatthe poor regenerative potentia of endotheia ces and

transformation into fibrobasts is in arge part owingto the ack of VEGF expression,87 which coud acceeratethe progression of CKD foowing initia recovery fromischemiareperfusion injury induced AKI. 83,88 Vascuardropout coud predispose individuas to recurrentischemic events and AKI.9

Leukocytes and inflammation

Infammation and recruitment of eukocytes during epi theia injury are now recognized as major mediators ofa phases of endotheia and tubuar ce injury (Figure 3).Eary infammation is characterized by margination ofeukocytes to the activated vascuar endotheium via

interactions between seectins and igands that enabefirm adhesion, foowed by transmigration into the inter stitium. A number of potent mediators are generatedby the injured epitheia proxima tubuar ce, incud ing proinfammatory cytokines, such as tumor necrosisfactor (TNF), intereukin (IL) 6, IL 1, IL 8, C C motifchemokine 2, transforming growth factor , and C Cmotif chemokine 5.89 To ike receptor (TLR) 2 is animportant mediator of endotheia ischemic injury, whieTLR4 has been shown in anima modes of both ischemicand septic injury to aso have a roe in injury,90 especiayin proxima tubuar ces.91

Neutrophis are the first ces to accumuate at the site

of ischemic injury.91 Bockade of neutrophi functionor neutrophi depetion provides ony partia protec tion against injury, indicating that other eukocytes asomediate injury. These infammatory mediators incudemacrophages, B ces, and T ces.92 Seective deetionof these ces in knockout mouse modes, and throughantibody mediated bockade, shows that these cesmediate tubuar injury at various phases of the process,and there are synergistic interactions between differ ent ce types.93 Expression of the compement compo nent C5a is markedy upreguated on proxima tubuarepitheia ces as we as interstitia macrophages, andis a powerfu chemoattractant with procoaguant

properties.94 Compement cascades are activated duringsepsis, and C5a has been found to be eevated in rodentmodes of sepsis.95 Bocking C5a or its receptor coudimprove surviva in sepsis.95,96

Thurman et al.97,98 showed that C3a was requiredfor CXC chemokine production by epitheia ces, andCrry (a compement inhibitor ocaized to the basoateramembrane of epitheia ces) was decreased foowingischemic injury. Knock down of C3a and caspase 3 usingRNA interference protected rena function in a transpantmode.99 Numerous cytokines, whether reeased from theendotheium or epitheia ces, work together to augmentthe infammatory response foowing ischemic or septicinjury.100 Cutured mouse tubuar ces stimuated withipopoysaccharide ed to an upreguation of TLR2, TLR3,and TLR4 and secretion of C C chemokines, such as C Cmotif chemokine 2 and C C motif chemokine 5. Thesedata indicate that expression of tubuar TLRs might beinvoved in mediating interstitia eukocyte infitrationand tubuar injury during bacteria sepsis.101

TLR2 and TLR4 are constitutivey expressed on

rena epitheium, and their expression is increased fo owing rena ischemiareperfusion injury. E Achkaret al.102 showed in a CLP rat mode of sepsis that Tr4expression was increased markedy in a tubues (proxi ma and dista), gomerui, and the rena vascuature.Furthermore, this group demonstrated that sepsis edto a Tr4 dependent increase in the expression of theproinfammatory mediator Cox 2; this protein wasmosty restricted to cortica and meduary thick ascend ing oops of Hene, which characteristicay express andsecrete uromoduin.103 Uromoduin coud stabiize theouter medua during injury by decreasing infammation,possiby through an effect on TLR4.104 Genetic deetion

of either TLR2 or TLR4 protects against rena ischemiareperfusion injury,91,105 thus indicating the prominentroe of TLRs in AKI.

Macrophages produce proinfammatory cytokines thatcan stimuate the activity of other eukocytes. Dayet al.106showed that depetion of macrophages in the kidney andspeen using iposoma codronate before rena ischemiareperfusion injury prevented AKI, whereas adoptivetransfer of macrophages reconstituted AKI. This groupaso demonstrated that agonists of the sphingosine 1 phosphate receptor (S1PR) induced ymphopenia, whichhad a protective effect.106,107 However, studies have asoshown a ymphocyte independent roe of S1PR in main

taining structura integrity after AKI, as S1PRs in theproxima tubue are necessary for stress induced cesurviva, and agonists of this receptor are renoprotectivevia direct effects on tubuar ces.108 Dendritic ces areaso thought to have a roe in AKI. Dong et al.109 demon strated that after AKI, rena dendritic ces producethe proinfammatory cytokines TNF, IL 6, C C motifchemokine 2, and C C motif chemokine 5, and thatdepetion of dendritic ces before ischemia substantiayreduced the eves of TNF produced by the kidney.

T reguatory (TREG

) ces aso have a roe in ischemicAKI. Gandofo et al.110 showed in a murine mode of isch emic AKI that T

REGce trafficking into the kidneys was





increased after 3 days and 10 days. Postischemic kidneyshad increased numbers of T ce receptor (TCR) +CD4+and TCR+CD8+ T ces, with increased production ofproinfammatory cytokines. The researchers noted thatdepetion of T

REGces using anti CD25 anti body 1 day

after ischemic injury increased rena tubuar damage,reduced tubuar proiferation, increased the productionof cytokines from infitrating T ces at 3 days, and TNFgeneration by TCR+CD4+ T ces at 10 days. In a separatestudy, infusion of CD4+CD25+ T

REGces 1 day after initia

injury reduced production of interferon by TCR+CD4+T ces at 3 days, improved repair, and reduced cytokinegeneration at 10 days.111 These studies demonstrate thatT

REGces infitrate reperfused kidneys during the heaing

process, promoting repair, ikey through moduation ofproinfammatory cytokine production of other T cesubsets. Partia depetion of T

REGces with an anti

CD25 monocona antibody potentiated kidney damageinduced by ischemiareperfusion injury, and resutedin more neutrophis, macrophages, and innate cytokinetranscription in the kidney.111 Furthermore, mice

deficient in Foxp3+ TREG ces had a greater accumua tion of infammatory eukocytes after rena ischemiareperfusion injury than mice containing T

REGces;

co transfer of isoated TREGS

ces and Scurfy ymph nodeces attenuated ischemiareperfusion injury inducedrena injury and eukocyte accumuation.112

Natura kier ces have been reported to infitrate thepostischemic kidney by 4 h after reperfusion. Li et al.113demonstrated the essentia roe of natura kier cesand neutrophis in the innate immune response to renaischemiareperfusion injury by mediating neutrophiinfitration and production of interferon . Furthermore,considerabe protection from kidney ischemia

reperfusion injury was evident in mice deficient in naturakier ces and mice administered an anti CD1d mono cona antibody, which bocked the interaction betweenantigen presenting ces and natura kier ces. 113

The anticoaguant function of antigen presentingces is responsibe for suppressing ipopoysaccharide induced stimuation of the proinfammatory media tors angiotensin converting enzyme 1, IL 6, and IL 18,perhaps accounting for its abiity to moduate renahemodynamics and protecting against septic AKI.114Taken together, these findings show that suppressionof infammation is a key target towards preventing andimiting AKI.

Investigators have shown that T ces aso have a majorroe in vascuar permeabiity during ischemic injury.Gene microarray anaysis showed that the productionof TNF and interferon was increased in CD3 andCD4 T ces from the bood and kidney after ischemia.Furthermore, it has aso been demonstrated that, in micedeficient in T ces expressing CD3, CD4, and CD8, thereis an attenuated increase in rena vascuar permeabiityafter ischemic injury. In this way, T ces directy con tribute to the increased vascuar permeabiity, potentiaythrough production of cytokines.115,116

Another feature noted during infammation andendotheia ce injury is the phenomenon of erythrocyte

trapping with Roueaux formation, causing obstructionand proonging the reduction in microvascuar boodfow and exacerbating tubuar injury.35

Distant organ effects of AKI

Ischemic AKI can have distant effects that potentiayater the function of other organs. Keyet al.117 demon strated the effects of rena ischemia on cardiac tissuesas shown by induction of IL 1, TNF, and interceuaradhesion moecue 1 mRNA expression as eary as 6 hafter ischemia. Kramer et al.118 showed that rena isch emic injury ed to an increase in pumonary vascuarpermeabiity defects, which were mediated throughmacrophages. Furthermore, this group showed in a ratmode of biatera rena ischemic injury or nephrectomythat expression of ung epitheia sodium channe, Na+/K+ATPase, and aquaporin 5 were downreguated, whichwas not the case in uniatera ischemic modes, indicat ing a roe for uremic toxins in moduating these effectsin the ung.119 Functiona changes in the brain have asobeen shown in the setting of AKI as noted in mice that

had increased neurona pyknosis and microgiosis inthe brain.120 In addition, extravasation of Evans bue dyeinto the brain indicated that the boodbrain barrier wasdisrupted in mice with AKI.120

Extrarena organs might conversey reguate isch emic AKI. Traumatic brain injury eicits a cytokine andinfammatory response that eads to rena infammationin transpants from brain dead, but not iving donors. 121The fact that AKI is associated with high morbid ity and mortaity indicates that mutiorgan crosstakis ikey to be a major contributor to dysfunction ofnonrena organs.

Molecules that protect against injuryMuch of the discussion above has focused on proteins orevents that promote injury. However, there are protec tive mechanisms that provide a defense against numer ous stresses. The heat shock protein system is inducedduring stress conditions; for exampe, overexpressionof heat shock proteins 25, 90, and 72 before injury havebeen found to have protective effects.122124 These pro teins are beieved to hep restore norma ce function byassisting in the refoding of denatured proteins, as weas aiding the appropriate foding of newy synthesizedproteins. Heat shock proteins aso degrade irreparabeproteins and toxins to imit their accumuation.

The enzyme heme oxygenase 1 has anti infammatory,vasodiatory, cytoprotective, antiapoptotic, and anti proiferative effects.125127 Mice deficient in this enzymewere shown to have marked exacerbation of gycero induced AKI, whereas overexpression of heme oxy genase 1 in cutured rena epitheia ces inducedupreguation of the ce cyce inhibitory protein p21,which conferred resistance to apoptosis.125127 Therefore,the actions of heme oxygenase 1 make it a potentiaytherapeutic enzyme in the prevention and reductionof AKI. More importanty, the upreguation or over expression of heme oxygenase 1 might aso be beneficiain the repair and regeneration of tubuar ces.

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where they ameiorate AKI through both paracrineeffects as we as repair of the injured microvascuature.146Human HSCs administered systemicay 24 h after kidneyinjury were seectivey recruited to injured kidneys ofimmunodeficient mice and ocaized prominenty inand around the vascuature.147 This recruitment wasassociated with repair of the kidney microvascuatureand tubuar epitheia ces, improved functiona recov ery, and increased surviva. HSCs recruited to the kidneyexpressed markers consistent with circuating endotheiaprogenitor ces and synthesized high eves of proangio genic cytokines, which promoted proiferation of bothendotheia and epitheia ces. Athough purified HSCsacquired endotheia progenitor markers once recruitedto the kidney, engraftment of human endotheia ces inthe mouse capiary was was rare, indicating that renarepair by human stem ces is mediated by paracrinemechanisms rather than repacement of the vascua ture.147 Targeting mechanisms of injury in this way tobock dysfunctiona intraceuar processes coud be ofkey therapeutic vaue.

Conclusions

A number of processes resuting in AKI invove isch emia foowed by a compex interaction of various cetypes within the kidney. Epitheia ce injury mediatesfunctiona aterations through direct faiure of the ces totransport ions and moecues, or indirecty by mediating

a decrease in GFR. Epitheia ces aso infuence the func tion of endotheia ces by reeasing chemokines, cyto kines, and other soube mediators. Interactions betweenendotheia ces and eukocytes contribute to continuedhypoxia, infammation, and further epitheia ce injuryand dysfunction. Numerous therapeutic targets havebeen identified that prevent or imit ongoing injury.Additiona approaches to improve repair and minimizefibrosis and vascuar dropout wi aso be critica in imit ing the deveopment of CKD and transition from CKDto end stage rena disease as a consequence of ischemicAKI in patients at high risk.

Review criteria

This Review was based on a search of the PubMed and

OVID databases using a combination of search terms that

included acute kidney injury, acute tubular necrosis,apoptosis, endothelial cell injury, inflammation,

leukocytes, cytokines, coagulation, stem cells,

endothelial progenitor cells, prerenal azotemia,

cytoskeletal alterations, renal blood flow, glomerularfiltration rate, actin, oxidative stress, reactive

oxygen species, heme oxygenase, heat shock

proteins, repair, vascular permeability, neutrophils,

T cells, and macrophages. Reference lists of selectedarticles were searched for further material. Articles were

chosen based on their originality and relevance to this

Review, and only English-language articles were selected.

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Author contributions

A. A. Sharfuddin and B. A. Molitoris contributedequally to researching data for the article, discussionof the content, writing and reviewing/editing of themanuscript before submission.

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