Acute renal failure (ARF), classically defined as an abruptdecrease in kidney function that leads to accumulation of nitrogenouswastes such as blood urea nitrogen and creatinine, is a commonclinical problem with increasing incidence, serious consequences,unsatisfactory therapeutic options, and an enormous financialburden to society (1–5). ARF may be classified as prerenal(functional response of structurally normal kidneys to hypoperfusion),intrinsic renal (involving structural damage to the renal parenchyma),and postrenal (urinary tract obstruction). This review focuseson intrinsic ARF, which has emerged as the most common and serioussubtype in hospitalized patients and can be associated pathologicallywith acute tubular necrosis (ATN). Consequently, it still iscommon clinical practice to use the terms intrinsic ARF andATN interchangeably. Despite decades of pioneering basic researchand important technical advances in clinical care, the prognosisfor patients with intrinsic ARF remains poor, with a mortalityrate of 40 to 80% in the intensive care setting. Two major problemshave plagued the field and hindered progress. First, well over20 definitions for ARF have been used in published studies,ranging from dialysis requirement to subtle increases in serumcreatinine (6). In an attempt to standardize the definitionand reflect the entire spectrum of the condition, the term acutekidney injury (AKI) has been proposed (4). AKI refers to a complexdisorder that comprises multiple causative factors and occursin a variety of settings with varied clinical manifestationsthat range from a minimal but sustained elevation in serum creatinineto anuric renal failure. Prerenal azotemia and other fully reversiblecauses of acute renal insufficiency are specifically excludedfrom the spectrum of AKI. An inherent shortcoming of this termis the continued reliance on serum creatinine measurements,and the definition of AKI undoubtedly will undergo enhancementsas novel early biomarkers for the identification of ARF beforethe rise in serum creatinine come to light (7). This reviewavoids the term ATN and uses the expressions AKI and intrinsicARF transposably. The second problem is an incomplete understandingof the cellular and molecular mechanisms that underlie AKI.This review updates the reader on current advances in basicand translational research that hold promise in human ischemicAKI. Classic concepts are mentioned briefly as founding principlesbut expanded on only if contemporary findings substantiate orrefute them. The reader is referred to recent publications thataddress the mechanisms that underlie other causes of intrinsicAKI, such as sepsis (8) and nephrotoxins (9). However, fromthe clinical viewpoint, it is acknowledged that AKI is frequentlymultifactorial, with concomitant ischemic, nephrotoxic, andseptic components and with overlapping pathogenetic mechanisms.
If function depends on form, then it follows that mechanismsthat are invoked to elucidate kidney dysfunction in AKI alsomust explain the morphologic alterations. In this regard, theterm ATN is a misnomer, because frank tubule cell necrosis israrely encountered in human ARF. This fact has once again beendriven home by careful examination of protocol kidney biopsiesthat are obtained soon after deceased-donor transplantation(10), which represents a predictable model for ischemic AKI(11). Prominent morphologic features of ischemic AKI in humansinclude effacement and loss of proximal tubule brush border,patchy loss of tubule cells, focal areas of proximal tubulardilation and distal tubular casts, and areas of cellular regeneration(12). Necrosis is inconspicuous and restricted to the highlysusceptible outer medullary regions. The glomeruli are usuallyunimpressive, unless a primary glomerular disease had causedthe ARF. This apparent disparity between the severe impairmentof renal function and the relatively subtle histologic changesin AKI traditionally has been bothersome. More recently, however,reconciliation has been forthcoming from a consistent findingof apoptotic cell death in both distal and proximal tubulesin both ischemic and nephrotoxic forms of human AKI (9,10).In addition, a great deal of attention has been directed towardthe peritubular capillaries, which display a striking vascularcongestion, endothelial damage, and leukocyte accumulation (13–15).The mechanisms that underlie these newly emphasized morphologicfindings and their implications for the ensuing profound renaldysfunction are detailed herein.
An intense and persistent renal vasoconstriction that reducesoverall kidney blood flow to approximately 50% of normal haslong been considered a hallmark of intrinsic ARF, promptingthe previous designation of "vasomotor nephropathy" (1). Asif to add insult to injury, the postischemic kidney also displayspeculiar regional alterations in blood flow patterns. Notably,there is marked congestion and hypoperfusion of the outer medullathat persist even though cortical blood flow improves duringreperfusion after an ischemic insult (13–15). Even undernormal conditions, the medullary region subsists on a hypoxicprecipice as a result of low blood flow and countercurrent exchangeof oxygen, although paradoxically housing nephron segments withvery high energy requirements (e.g., the S3 segment of the proximaltubule and the medullary thick ascending limb of Henle’sloop). The characteristic postischemic congestion worsens therelative hypoxia, leading to prolonged cellular injury and celldeath in these predisposed tubule segments. Sophisticated imagingtechniques have documented these changes in regional renal bloodflow in animals and validated them in human AKI (16). Mechanismsthat underlie these hemodynamic alterations have begun to surface,and they relate primarily to endothelial cell injury (13–17).This results in a local imbalance of vasoactive substances,with enhanced release of vasoconstrictors such as endothelinand decreased abundance of vasodilators such as endothelium-derivednitric oxide (NO) (2). Endothelin receptor antagonists ameliorateischemic AKI in animals (18), but human data are lacking. Similarly,both carbon monoxide and carbon monoxide–releasing compoundsare protective in animal models of ischemic AKI (19,20), likelythrough vasodilation and preservation of medullary blood flow,but have not been tested in humans. All things considered, thesemacrohemodynamic abnormalities cannot account fully for theprofound loss of renal function, and several human trials ofvasodilators such as dopamine have failed to demonstrate improvementin GFR in established ARF despite augmentation of total renalblood flow (21). However, microvascular alterations now arerecognized to play a major role, as discussed next.
Documented derangements in tubule dynamics include obstruction,backleak, and activation of tubuloglomerular feedback. The consistenthistologic findings of proximal tubular dilation and distaltubular casts in human biopsies indicate that obstruction totubular fluid flow certainly occurs in ischemic AKI. The intraluminalcasts stain strongly for Tamm-Horsfall protein, which normallyis secreted by the thick ascending limb as a monomer. Conversioninto an obstructing gel-like polymer is enhanced by the increasedluminal sodium concentration that typically is encountered inthe distal tubule in AKI (22). This provides an ideal environmentfor cast formation along with desquamated tubule cells and brushborder membranes. However, it is unlikely that obstruction alonecan account for the intense dysfunction, because human studiesthat used forced diuresis with furosemide (23) or mannitol (24)did not have an impact on the survival and renal recovery rateof patients with established ARF. Similarly, although movementof the glomerular filtrate back into the circulation has beenshown to occur, this accounts for only a very minor componentof the decrease in GFR in human ARF (2). Finally, a role foractivation of tubuloglomerular feedback has been proposed onthe basis of human studies (25). The increased delivery of sodiumchloride to the macula densa as a result of cellular abnormalitiesin the ischemic proximal tubule would be expected to induceafferent arteriolar constriction via A1 adenosine receptor (A1AR)activation and thereby decrease GFR (26). However, recent studieshave shown that a knockout of the A1AR resulted in a paradoxicworsening of ischemic renal injury, and exogenous activationof A1AR was protective (27). Therefore, tubuloglomerular feedbackactivation after ischemic injury indeed may represent a beneficialphenomenon that limits wasteful delivery of ions and solutesto the damaged proximal tubules, thereby reducing the demandfor ATP-dependent reabsorptive processes. Any salutary effectof exogenous A1AR activation in human AKI remains to be determined.
A profound reduction in intracellular ATP content invariablyoccurs early after ischemic renal injury, which sets in motiona number of critical metabolic consequences in tubule cells(28). These events are detailed next, and their interrelationshipis illustrated in Figure 1.
Figure 1. Alterations in tubule cell metabolism after ischemic acute kidney injury (AKI). The initiation phase is typified by profound ATP depletion, which primes the cell by activating a number of oxidative and cell death mechanisms. During the extension phase, prolonged ischemia followed by reperfusion propels these pathways to completion, resulting primarily in apoptosis and oxidant injury. Inhibition of these pathways may provide novel therapeutic approaches, as recently demonstrated with caspase inhibitors, iron chelators, and antioxidants. Illustration by Josh Gramling—Gramling Medical Illustration.
Alterations in Adenine Nucleotide Metabolism
Oxygen deprivation leads to a rapid degradation of ATP to ADPand AMP. With prolonged ischemia, AMP is metabolized furtherto adenine nucleotides and to hypoxanthine. Hypoxanthine accumulationcontributes to generation of reactive oxygen molecules by mechanismsexplained below. Adenine nucleotides freely diffuse out of cells,and their depletion precludes re-synthesis of intracellularATP during reperfusion. However, although provision of exogenousadenine nucleotides or thyroxine (which stimulates mitochondrialATP regeneration) can mitigate ischemic AKI in animal models,this approach has yielded disappointing results in human ARF(29,30).
Alterations in Intracellular Calcium
ATP depletion leads to impaired calcium sequestration withinthe endoplasmic reticulum as well as diminished extrusion ofcytosolic calcium into the extracellular space, resulting inthe well-documented rise in free intracellular calcium afterAKI. Potential downstream complications include activation ofproteases and phospholipases and cytoskeletal degradation (28).However, the increased cytosolic calcium dramatically inducescalcium-binding proteins such as annexin A2 and S100A6, whichplay an important role in the cell proliferation during recoveryfrom AKI in animal models (31). Reflective of this contradiction,a recent meta-analysis showed that calcium channel blockersmay provide some protection from renal injury in the transplantsetting (32), but evidence for their efficacy in other formsof human AKI is lacking.
Generation of Reactive Oxygen Molecules
There now is substantial evidence for the role of reactive oxygenspecies in the pathogenesis of AKI. During reperfusion, theconversion of accumulated hypoxanthine to xanthine (catalyzedby xanthine oxidase formed from xanthine dehydrogenase eitheras a result of proteolytic conversion or as a result of oxidationof sulfhydryl residues) generates hydrogen peroxide and superoxide.In the presence of iron, hydrogen peroxide forms the highlyreactive hydroxyl radical. Concomitantly, ischemia induces NOsynthase in tubule cells, and the NO that is generated interactswith superoxide to form peroxynitrate, which results in celldamage via oxidant injury as well as protein nitrosylation (28).Collectively, reactive oxygen species cause renal tubule cellinjury by oxidation of proteins, peroxidation of lipids, damageto DNA, and induction of apoptosis. A recent study documenteda dramatic increase in oxidative stress in humans with ARF,as evidenced by depletion of plasma protein thiols and increasedcarbonyl formation (33). Disappointing, intermittent hemodialysisresulted in only a limited and transient beneficial effect onthe redox status of plasma protein thiol groups and no effecton protein carbonyl content (33). Several scavengers of reactiveoxygen molecules (e.g., superoxide dismutase, catalase, N-acetylcysteine)protect against ischemic AKI in animals, but human studies havebeen inconclusive. A promising new advance in the field is theprotective effect of edaravone, a potent scavenger of free radicalsand inhibitor of lipid peroxidation when administered at thetime of reperfusion in a rat model of ischemic AKI (34). Edaravonehas been approved for human use in the treatment of cerebralischemia, and results of its use in human AKI are awaited withanticipation. Also, free iron that is derived from red cellsor other injured cells now is recognized as one of the mostpotent factors in the generation of reactive oxygen species,and the iron scavenger deferoxamine does alleviate ischemia-reperfusioninjury in animal models. However, the associated systemic toxicity(primarily hypotension) precludes its routine clinical use inhuman AKI (35). Two major advances have come to light in thearea of iron chelation. The first is the availability of humanapotransferrin, an iron-binding protein that protects againstrenal ischemia-reperfusion injury in animals by abrogating renalsuperoxide formation (36). Apotransferrin has been used successfullyfor the reduction of redox-active iron in patients who haveundergone hematologic stem cell transplantation without anyadverse effects (37). The second is the discovery of neutrophilgelatinase–associated lipocalin (NGAL), a major iron-transportingprotein complementary to transferrin, as one of the most highlyinduced genes and proteins in the kidney after ischemic injury(38,39). The biology of NGAL in AKI is detailed further next.Administration of NGAL provides remarkable structural and functionalprotection in animal models (40,41). The potential use of bothof these endogenous agents (apotransferrin and NGAL) in humanAKI currently is under investigation.
Contemporary techniques have provided novel insights into thecell biology of the proximal tubule in ischemic AKI. The structuralresponse of the tubule cell to ischemic injury is multifacetedand includes loss of cell polarity and brush borders, cell death,dedifferentiation of viable cells, proliferation, and restitutionof a normal epithelium, as illustrated in Figure 2. The mechanismsthat underlie this morphologic sequence of events are complexand examined next.
Figure 2. Alterations in tubule cell structure after ischemic AKI. The initiation phase leads to sublethal injury, with loss of brush borders and disruption of cell polarity and the cytoskeleton. If the injury is alleviated at this stage, then complete recovery ensues. If not, then the injury flows into the extension phase, with cell death, desquamation, luminal obstruction, and an inflammatory response (shown in Figure 4). Inhibition of apoptosis and inflammation at this stage may represent a powerful therapeutic approach. The maintenance phase is characterized by a balance between tubule cell death and restoration (which occurs largely as a result of dedifferentiation of viable tubule cells, although stem cells and progenitor cells also may play a role). Measures to accelerate the endogenous regenerative process may hasten recovery. Illustration by Josh Gramling—Gramling Medical Illustration.
Alterations in the Apical Cytoskeleton
Cellular ATP depletion leads to a rapid disruption of the apicalactin cytoskeleton and redistribution of actin from the apicaldomain and microvilli into the cytoplasm (42). The ensuing alterationsin microvillar structure lead to formation of membrane-bound,free-floating extracellular vesicles, or "blebs," that are eitherinternalized or lost into the tubular lumen. Brush border membranecomponents that are released into the lumen contribute to castformation and obstruction. These casts and vesicles that containactin and actin depolymerizing factor (ADF; also known as cofilin)have been detected in the urine in animal as well as human AKI(42). The role of ADF/cofilin in the apical microvillar breakdowncurrently is under active investigation. ADF/cofilin is a cytosolicprotein that normally is maintained in the inactive phosphorylatedform by Rho GTPases. In cultured renal tubule cells, ATP depletionleads to Rho GTPase inactivation, with resultant activationand relocalization of ADF/cofilin to the surface membrane andmembrane-bound vesicles (43,44). Concomitant, ATP depletiondissociates the actin-stabilizing proteins tropomyosin and ezrin(45), allowing the activated ADF/cofilin to bind and consequentlysever actin, which in turn leads to microvillar breakdown. Anotherwell known mechanism for ADF/cofilin activation involves familiesof phosphatases such as Slingshot and Chronophin (46). Activationof ADF/cofilin also can induce apoptosis by triggering cytochromec release, which may contribute further to its deleterious effects(47). Therefore, inactivation of ADF/cofilin, perhaps via transientinhibition of Slingshot, may represent a promising but unexploreddirection in AKI.
Disruption of the apical cytoskeleton by ATP depletion alsoresults in loss of tight junctions and adherens junctions. Reducedexpression, redistribution, and abnormal aggregation of a numberof key proteins that constitute the tight and adherens junctionshave been documented after ischemic injury in cell culture,animal models, and human studies (48). The consequent loss oftight junction barrier function potentially can magnify thetranstubular backleak of glomerular filtrate that is inducedby obstruction (49).
Alterations in the Basolateral Cytoskeleton
Ischemia results in the early disruption of at least two basolaterallypolarized proteins, namely Na,K-ATPase and integrins. The Na,K-ATPaseis normally tethered to the spectrin-based cytoskeleton at thebasolateral domain via the adapter protein ankyrin. In cellculture, animal models, and human studies, ischemia leads toa reversible cytoplasmic accumulation of Na,K-ATPase, ankyrin,and spectrin in viable cells (50). The mislocated Na,K-ATPaseremains bound to ankyrin but is devoid of spectrin. Postulatedmechanisms that lead to loss of Na,K-ATPase polarity includehyperphosphorylation of ankyrin with consequent loss of spectrinbinding and cleavage of spectrin by ischemia-induced activationof proteases such as calpain (2,50). A physiologic consequenceof the loss of basolateral Na,K-ATPase is an impairment in proximaltubular sodium reabsorption and a consequent increase in fractionalexcretion of sodium, which are diagnostic signatures of intrinsicARF.
The 1 integrins are normally polarized to the basal domain,where they mediate cell-substratum adhesions. Ischemic injuryleads to a redistribution of integrins to the apical membrane,with consequential detachment of viable cells from the basementmembrane. There is good evidence for abnormal adhesion betweenthese exfoliated cells within the tubular lumen, mediated byan interaction between apical integrin and the Arg-Gly-Asp (RGD)motif of integrin receptors. Administration of synthetic RGDcompounds attenuates tubular obstruction and renal impairmentin animal models, and the recent development of orally activeintegrin antagonists holds promise for human AKI (51). A recentanimal study has shown that the state of 1 integrin activationalso is critical for maintenance of tubule epithelial integrity(52). Preischemic intravenous administration of anti-activated1 integrin therapy (via mAb named HUTS-21 that selectively recognizethe active form) resulted in preservation of renal histopathologyand function, maintenance of cell-substratum interactions, andamelioration of the inflammatory response (52). The multifacetedprotective effect renders HUTS-21 a potentially attractive therapeuticcandidate for human AKI, but the need for pretreatment is anobvious disadvantage.
Alterations in Cell Viability
Both experimental and human studies indicate that tubule epithelialcells can suffer one of three distinct fates after ischemicAKI. The majority of cells remain viable, suggesting that theyeither entirely escape injury or are only sublethally injuredand undergo recovery. A subset of tubule cells display patchycell death that results from at least two pathophysiologic mechanisms.Necrosis is an explosive, chaotic process that is characterizedby loss of membrane integrity, cytoplasmic swelling, and cellularfragmentation. Apoptosis is a quiet, orderly demise that istypified by cytoplasmic and nuclear shrinkage, DNA fragmentation,and breakdown of the cell into membrane-bound apoptotic bodiesthat are rapidly cleared by phagocytosis. These two forms ofcell death can coexist and are considered two extremes of aspectrum. After ischemic renal injury, the mode of cell deathdepends primarily on the severity of the insult and the resistanceof the cell type. Necrosis usually occurs after more severeinjury and in the more susceptible nephron segments, whereasapoptosis predominates after less severe injury and especiallyin the ischemia-resistant distal nephron segments. Apoptosiscan be followed by "secondary necrosis," especially if the apoptoticcells are not rapidly removed. The commonly used assays forapoptosis (terminal deoxynucleotidyltransferase-mediated dUTPnick-end labeling assay and DNA laddering) do not adequatelydistinguish between apoptosis and necrosis. Strict morphologiccriteria are desirable for the detection and quantificationof apoptosis, including nuclear (condensation and fragmentation)and cytoplasmic (cell shrinkage and blebbing) changes.
Mounting evidence now indicates that apoptosis is the majormechanism of early tubule cell death in contemporary clinicalARF (53–55). During recent years, several animal modelsof ischemic AKI consistently and unequivocally demonstratedthe presence of apoptotic tubule cells using a variety of sensitiveassays (56–79). Importantly, this now has been confirmedby several investigators in human models of AKI (10,80–85).Nevertheless, controversies still exist regarding the contributionof apoptosis to the syndrome of ARF. First, most estimates placethe peak incidence of apoptosis at only approximately 3 to 5%of tubule cells after ischemic injury, which arguably is insufficientto explain the profound renal dysfunction. In response to thisskepticism is that the degree of apoptosis is vastly underestimatedbecause it is a rapidly occurring heterogeneously distributedevent that is notoriously difficult to identify and quantifyin tissues. Second, apoptosis is more commonly encountered inthe distal tubule, whereas loss of viable cells occurs predominantlyin proximal segments. Reconciliation of this argument is providedby the demonstration of both necrosis and apoptosis in the proximaltubule, where these processes may represent a continuum. Third,apoptosis generally is regarded as a physiologic process thatremoves damaged cells and therefore may be beneficial to theorgan and the organism. The counterpoint to this suppositionis that apoptosis after ischemic AKI is a double-edged swordthat occurs in two waves, at least in animal models. The firstwave is detectable within 6 to 12 h of the insult, peaks atapproximately 3 d, and rapidly diminishes. This phase deletespreviously healthy tubule cells, thereby contributing to theensuing dysfunction. The second wave becomes apparent approximately1 wk later, removes hyperplastic and unwanted cells, and thereforemay play a role in the remodeling of injured tubules.
Because the evidence is overwhelmingly in favor of apoptosisas a pathogenetic mechanism, considerable attention has beendirected toward dissecting out the molecular pathways involved.A multitude of pathways, including the intrinsic (Bcl-2 family,cytochrome c, and caspase 9), extrinsic (Fas, FADD, and caspase8), and regulatory (p53 and NF-B) factors, seem to be activatedby ischemic AKI, as illustrated in Figure 3. A leading contenderfor many years, the role of the Fas-FADD pathway in animal models,was reaffirmed recently by demonstration of upregulation ofthese proteins in apoptotic tubule cells after ischemia (58)and the functional protection that is afforded by small interferingRNA duplexes that target the Fas gene (61). However, convincinghuman data are lacking, because the induction of the Fas genethat was shown in one study of human cadaveric kidney transplants(83) was not reproduced in two subsequent publications (10,85).However, there is growing evidence implicating an imbalancebetween the proapoptotic (Bax and Bid) and antiapoptotic (Bcl-2and Bcl-xL) members of the Bcl-2 family in both animal (63,79)and human (10,82–84) situations. Of the regulatory factors,the proapoptotic transcription factor p53 has been shown tobe induced at the mRNA (56) and protein (55) levels, and inhibitionof p53 by pifithrin- suppresses ischemia-induced apoptosis byinhibiting transcriptional activation of Bax and mitochondrialtranslocation of p53 (57). However, pifithrin- is an unlikelycandidate for therapeutic consideration in humans because generalizedinhibition of p53-dependent apoptosis likely will promote survivalof damaged or mutation-bearing cells in other organ systems.
Figure 3. Major apoptotic pathways in human ischemic AKI. The extrinsic pathway requires activation of plasma membrane Fas receptor, with signal transduction via FADD resulting in activation of caspase 8. The intrinsic pathway requires translocation of Bax to the mitochondria, thereby forming pores for the release of cytochrome c and activation of caspase 9. Cross-talk between these pathways is provided by Bid activation. Bax also is activated by p53-dependent pathways. Bax activation is prevented in normal cells by Bcl2 and Bcl-xL. Both caspases 8 and 9 activate caspase 3, which initiates the final morphologic cascades of apoptosis. Inhibition of these pathways holds significant therapeutic promise in human acute renal failure. Many additional molecules that have been implicated in animal models (but not in human studies) are not shown. Illustration by Josh Gramling—Gramling Medical Illustration.
Inhibition of apoptosis does hold promise in ischemic AKI (86).Caspase activation is by and large the final common "execution"step in apoptosis (although caspase-independent apoptosis alsohas been described), and cell-permeant caspase inhibitors haveprovided particularly attractive targets for study (73). Currentlyavailable inhibitors largely have been investigated only inanimals, provide only partial protection, and are most effectivewhen administered before the injury. However, these characteristicsrender caspase inhibition as a potentially attractive approachto reducing apoptotic damage during cold storage of deceased-donorkidneys before transplantation, as has been demonstrated inanimal models (87). In this regard, an orally active pan-caspaseinhibitor (IDN-6556) was developed recently (88) and shown tobe effective in preventing injury after lung and liver transplantationin animals (88,89). IDN-6556 is currently undergoing evaluationin Phase II clinical trials for injury prevention in human livertransplantation.
Several other modalities ameliorate apoptosis and AKI in experimentalsituations. Pretreatment with erythropoietin conferred structuraland functional protection, inhibition of apoptosis, and upregulationof the antiapoptotic transcription factor NF-B (65,77). However,inhibition of NF-B by intrarenal transfection of decoy oligonucleotidesresulted in a paradoxic attenuation of ischemic AKI, perhapsby inhibiting transcription of proinflammatory factors (90).-1-Acid glycoprotein (an acute-phase protein of unknown function[59]), minocycline (62), A1 adenosine receptor agonists (64),peroxisome proliferator–activated receptor ligands (66),geranylgeranylacetone (an inducer of heat-shock proteins [HSP][67]), and poly(ADP-ribose) polymerase inhibitors (91) all haveprovided encouraging functional protection from ischemic AKIwith inhibition of apoptosis and inflammation, but the underlyingmechanisms have not been fully elucidated. Some of these agentsare already widely available and safely used in other humanconditions, and results of their use in AKI should be forthcoming.Challenges for the future clinical use of apoptosis inhibitionin AKI include determining the best timing of therapy, optimizingthe specificity of inhibitor, minimizing the extrarenal adverseeffects, and tubule-specific targeting of the apoptosis modulatorymaneuvers.
The mechanisms whereby the majority of tubule cells escape celldeath and either emerge unscathed or recover completely afterischemic AKI remain under active investigation. HSP have surfacedas prime arbitrators of this cytoprotection (92–94). Inductionof HSP is part of a highly conserved innate cellular responsethat is activated swiftly and robustly after ischemic AKI. HSPpromote cell survival by inhibiting apoptosis (95), and liposomaldelivery of HSP-72 into cultured renal tubule cells blocks ischemia-inducedapoptosis (96). HSP also facilitate the restoration of normalcellular function by acting as molecular chaperones that assistin the refolding of denatured proteins as well as proper foldingof nascent polypeptides. For instance, there now is broad evidencefor the role of HSP in the restoration of cytoskeletal integrityand Na,K-ATPase polarity after ischemic AKI in animal models(92–94). In cultured tubule cells, inhibition of the HSPresponse by gene-silencing techniques produced profound impairmentof cellular integrity and Na,K-ATPase polarity (97), and overexpressionof HSP-70 mitigated the loss of Na,K-ATPase polarity after ATPdepletion (98). Collectively, these findings suggest that maneuversthat enhance the innate HSP response have potential benefitin human AKI but that transition of therapy from bench to bedsidehas not been achieved yet. However, that this also may activateundesirable cellular processes such as increased immunogenicity(99) serves to caution us against leaping from the frying paninto the fire when contemplating HSP therapies.
Mechanisms of Repair
Surviving renal tubule cells possess a remarkable ability toregenerate and proliferate after ischemic AKI (100). Morphologically,repair is heralded by the appearance of dedifferentiated epithelialcells that express vimentin, a marker for multipotent mesenchymalcells (101). The origin of these cells remains contentious (andis addressed next), but they most likely represent survivingtubule cells that have dedifferentiated. In the next phase,the cells upregulate genes that encode for a variety of growthfactors, such as IGF-1, hepatocyte growth factor (HGF), andfibroblast growth factor, and undergo marked proliferation.In the final phase, cells express differentiation factors suchas neural cell adhesion molecule (NCAM) and osteopontin andundergo re-differentiation until the normal fully polarizedepithelium is restored. Therefore, during recovery from ischemia,renal tubule cells recapitulate phases and processes that arevery similar to those during normal kidney development (100–102).Recently identified examples of genes that lend support to theconcept of "recapitulation of phylogeny by ontogeny" includeNGAL (38–41), leukemia inhibitory factor (103), transcriptionfactor Ets-1 (104), and Wnt-4 (105). All of these transcriptsnot only are critical to early kidney development but also aremarkedly induced in the mature kidney after ischemic injury,where they seem to play a crucial role in the regeneration andrepair processes.
Understanding the molecular mechanisms of repair may provideclues toward accelerating recovery from ARF. For example, HGFhas been documented extensively to be renoprotective and renotrophicin animal models of AKI, mediated by its proliferative, antiapoptotic,and anti-inflammatory actions (68). However, the use of HGFin humans has been hampered at least in part by the widespreadexpression of its receptor, raising the possibility of seriousextrarenal adverse effects (106). Indeed, transgenic mice withgeneralized overexpression of HGF display diverse tumors, polycystickidney disease, and inflammatory bowel disease (106). Encouraging,selective overexpression of the HGF transgene in the proximaltubule via the -glutamyl transpeptidase-1 promoter bestowedremarkable functional protection from ischemic AKI, with enhancedtubule cell proliferation and decreased apoptosis (107). Selecteddelivery of HGF to the human proximal tubule, however, remainsa challenge. In the case of IGF-1, enthusiasm for its renoprotectiveeffects has been dampened by its exacerbation of inflammationand neutrophilic infiltration in the kidney after ischemia inanimals (108), and clinical trials with recombinant human IGF-1have not proved to be beneficial (109). Interest in IGF-1 therapyhas been rekindled by the recent discovery of ghrelin, a naturallyoccurring growth hormone secretagogue that markedly increasesserum IGF-1 levels and protects the kidney from ischemic injuryby improving endothelial function and vasodilation in animals(78).
Identification of the source of multipotent mesenchymal cellsthat are involved in the regeneration and repair processes mayhave important therapeutic implications and has been a matterof intense contemporary research. Evidence for an extrarenalprogenitor, namely bone marrow–derived mesenchymal stemcells (MSC), stems from detection of tagged MSC in the recipientkidney after cross-gender transplantation or systemic infusion(111–115). Administered MSC clearly enhance recovery fromischemic AKI in animals, but several controversies exist. First,the number of bone marrow–derived MSC that finally appearin the postischemic kidney is very small, and their detectionhas been fraught with technical false-positive results (116,117).Second, the majority of these bone marrow–derived cellswere detected in the interstitial compartment and not in nephronsegments, raising concerns for accentuation of renal fibrosisafter AKI (118,119). Third, the overall balance of evidencenow is in favor of the notion that restoration of the tubuleepithelium after ischemic AKI occurs predominantly via proliferationof endogenous renal cells (116–120). Although residentstem cells with tubulogenic capacity have been described inthe kidney (121–123), their contribution to the repairprocess is unclear, and the native tubule epithelial cells seemto represent the primary healing source. Fourth, the mode ofprotection by exogenously administered MSC may be related notto transdifferentiation processes but rather to powerful anti-inflammatoryand antiapoptotic mechanisms that are being uncovered (117,124,125).Because bone marrow–derived MSC are easily accessible,this therapeutic approach certainly holds promise in human AKI.
In recent years, the concept of the vascular endothelium asan organ that is both a source of and a target for inflammatoryinjury has become widely appreciated, and the role of endothelialalterations in the initiation and extension of AKI has receivedincreasing attention (13–17). Morphologically, disruptionof the actin cytoskeleton and junctional complexes, similarto those previously described in tubule epithelial cells, nowhas been documented in endothelial cells in experimental AKI(126,127). Consequent endothelial cell swelling, blebbing, death,and detachment of viable cells occur, and circulating endothelialcells have been demonstrated in humans with septic shock (128).Sites of endothelial denudation may be prone to prolonged vasoconstriction,and minimally invasive intravital microscopy has revealed sporadiccessation and even reversal of blood flow in peritubular capillariesduring reperfusion (129,130). Systemic or intrarenal administrationof fully differentiated endothelial cells into postischemicrat kidneys resulted in a significant functional protection(130). A similar, albeit less impressive, amelioration was achievedusing surrogate cells that expressed endothelial NO synthase.Furthermore, ischemic injury leads to a marked upregulationof angiostatin, a widely known antiangiogenic factor that inducesapoptosis of endothelial cells (131). Collectively, these findingsprovide a rationale for the use of proangiogenic agents thatcan increase the pool or mobilization of endothelial progenitorcells, such as erythropoietin, bone morphogenic protein, vascularendothelial growth factor, and statins (17).
Ischemic AKI also leads to increased endothelial expressionof a variety of adhesion molecules that promote endothelial–leukocyteinteractions. These include intercellular adhesion molecule-1(ICAM-1), P-selectin, and E-selectin (14,132). Although ablationof the ICAM-1 gene and pretreatment with ICAM-1 antibody renderedmice resistant to ischemic AKI, human trials with anti–ICAM-1mAb administered after ischemia did not prevent AKI in cadaverictransplant recipients (14). Similarly, gene knockouts, mAb,and pharmacologic inhibitor studies have suggested a role forE- and P-selectins (132–134). However, subsequent studieshave shown that it is platelet P-selectin, not endothelial P-selectin,that is the key component leading to AKI (135). Possible mechanismsinclude (1) adhesion of platelets to the endothelium, with subsequentleukocyte adhesion, and (2) adhesion of platelets to neutrophils,with consequent aggregate formation and trapping in narrow peritubularcapillaries (132). These abnormalities, combined with otherderangements in the coagulation cascade such as alterationsin tissue-type plasminogen activator and plasminogen activatorinhibitor-1 in the kidney (136), may account for the fibrindeposits that characteristically are found in the renal microvasculatureafter ischemic injury.
A growing body of evidence indicates that the inflammatory responseplays a major role in ischemic AKI. Inflammatory cascades thatare initiated by endothelial dysfunction can be augmented dramaticallyby the generation of a number of potent mediators by the ischemicproximal tubule, which is thought to represent a "maladaptiveresponse" (13,14,137–139). These include proinflammatorycytokines (e.g., TNF-, IL-6, IL-1, TGF-) and chemotactic cytokines(e.g., monocyte chemoattractant protein-1 [MCP-1], IL-8, RANTES).Elegant human studies demonstrated recently that the levelsof the proinflammatory cytokines IL-6 and IL-8 in the plasmapredict mortality in patients with AKI (139), and the levelsof CXCR3-binding chemokines in the urine predict AKI after kidneytransplantation (140), attesting to the clinical significanceof these mechanisms. Toll-like receptor 2 (TLR2) may representa major component of this proinflammatory response (70). Renaltubular expression of TLR2 is enhanced upon ischemic AKI, andTLR2 gene silencing by knockout and antisense treatment preventsischemia-induced renal dysfunction; neutrophil influx; tubuleapoptosis; and induction of MCP-1, TNF-, IL-6, and IL-1. Bonemorphogenic protein-7 also is induced in postischemic tubules(75) and protects against ischemic AKI by decreasing the levelsof MCP-1, IL-8, IL-6, and IL-1 in cultured proximal tubule cells(141).
Morphologically, several leukocyte subtypes have been shownto aggregate in peritubular capillaries, interstitial space,and even within tubules after ischemic AKI (Figure 4), and theirrelative roles remain under investigation (14). Neutrophilsare the earliest leukocytes to accumulate in the postischemickidney. However, neutrophil depletion or blockade of neutrophilfunction provides partial functional protection in some butnot all animal models. Furthermore, neutrophils are not a prominentfeature of ischemic AKI in humans, casting doubts about theclinical significance of neutrophil infiltration. Macrophagesare the next to accumulate in animal models (90), in responseto upregulation of MCP-1 in tubule cells (142) and inductionof its cognate receptor CCR2 on macrophages (143). Selectivemacrophage depletion ameliorates ischemic AKI, but the inductionof tissue injury by macrophages seems additionally to requirethe coordinated action of T cells and neutrophils (144). However,T cells have been identified in animal as well as human modelsof ischemic AKI (14), and T cell depletion is protective inexperimental AKI (145–147). Double CD4/CD8 knockout miceare protected from ischemic AKI (148), and adoptive transferof wild-type T cells into the null mice abrogates this protectiveeffect (149). However, inconsistencies exist, and recent datasuggest that the role of T cells in ischemic AKI may be complex,with the identification of both protective (Th2 phenotype) anddeleterious (Th1 phenotype) subtypes of T cells (150). Moreover,animals that are deficient in both T and B cells are not protectedfrom ischemic AKI (151), and depletion of peripheral CD4 T cellsfailed to bestow protection from ischemic AKI (152). The potentialrole of B cells in ischemic AKI is intriguing. Compared withwild-type animals, B cell–deficient mice are partiallyprotected from structural and functional ischemic renal injury,despite comparable neutrophil and T cell infiltrations (153).Wild-type serum transfer but not B cell transfer into B cell–deficientmice restored susceptibility to ischemic AKI, implicating asoluble serum factor as a mechanism by which B cell deficiencyconfers renal protection (153).
Figure 4. Alterations in the microvasculature and inflammation in ischemic AKI. During the extension phase, endothelial injury leads to intense vasoconstriction, microvascular sludging, and microvascular congestion with leukocytes. Activated leukocytes produce a number of inflammatory mediators and reactive oxygen species that potentiate tubule cell damage. In addition, tubule cells exhibit a maladaptive response by generating cytokines and chemokines that further amplify the inflammation. PMN, polymorphonuclear leukocyte; Th1, T-helper 1 cell. Strategies that modulate the inflammatory response may provide significant beneficial effects in ischemic AKI. Illustration by Josh Gramling—Gramling Medical Illustration.
Activation of the complement system in ischemic AKI, with aresultant amplification of the inflammatory response in thekidney, has received widespread attention in recent years (154–157).Whereas ischemia-reperfusion injury in most organs activatesthe complement cascade via classical pathways, studies in animals(158) and humans (159) have implicated a predominant role forthe alternative pathway in ischemic AKI (160). However, thisremains debatable, because other reports have identified a rolefor the mannose-binding lectin pathway as well in complementactivation after animal and human ischemic AKI (161,162). Equallycontroversial is the identification of the final active complementcomponent. Whereas earlier studies pointed to the C5b-directedformation of a membrane attack complex (163,164), several recentobservations have challenged this view and have identified apredominant role for C5a in ischemic AKI (154–157). C5ais a powerful chemoattractant that recruits inflammatory cellssuch as neutrophils, monocytes, and T cells. The kidney is oneof the few organs in which the C5a receptor is normally expressed,in proximal tubule epithelial cells as well as interstitialmacrophages (165). C5a receptor expression in tubule epithelialcells is markedly upregulated after ischemia-reperfusion injury(166) and sepsis (167). Inhibition of C5a generation using mAbprotected against renal dysfunction that was induced by ischemiaand inhibited neutrophil and macrophage influx in experimentalmodels (168). Importantly, pretreatment with orally active small-moleculeC5a receptor antagonists substantially reduced the histologicand functional impairment that was induced by ischemic AKI inanimal models (166,169). Small-molecule antagonists for C5areceptor are currently undergoing a Phase II clinical trialin rheumatoid arthritis and represent promising agents for thetreatment or prevention of ischemic AKI.
Other strategies that modulate the inflammatory response alsomay provide significant beneficial effects in human AKI, andseveral have already been tried in experimental situations.For example, IL-10 is a potent anti-inflammatory cytokine thathas been shown to provide functional protection against ischemicAKI by inhibiting maladaptive cytokine production by Th1 cells(170). Administration of a mAb against the proinflammatory cytokineIL-6 ameliorated structural and functional consequences of ischemicAKI, decreased neutrophil infiltration, and reduced proinflammatorycytokine production (171). Bimosiamose, a novel pan-selectininhibitor, protected from ischemic AKI in a kidney transplantmodel by reducing infiltration of macrophages and T cells andinhibiting intragraft expression of chemokines and cytokines(172). In addition to cholesterol lowering in humans, widelyused statins possess several properties that may be beneficialin ischemic AKI, including profound anti-inflammatory effects,inhibition of reactive oxygen species, and stimulation of endothelialNO production (173–175). Not surprising, several investigatorshave reported impressive structural and functional protectionfrom ischemic AKI by short-term pretreatment with statins (176–179).Similarly, erythropoietin, used extensively in humans for stimulatingerythropoiesis, also has prominent antiapoptotic and anti-inflammatoryactions (179) and has been used successfully for functionalamelioration of ischemic AKI in animals (65,77,180). Finally,-melanocyte stimulating hormone, an anti-inflammatory cytokine,protects against ischemic AKI by inhibiting the maladaptiveactivation of genes that cause inflammatory and cytotoxic renalinjury (180,181). Of interest, -melanocyte stimulating hormonepotentiates the beneficial effect of erythropoietin (180), remainseffective even when administered after the renal ischemia (180),and also protects against the distant lung injury that occursafter ischemic AKI (181). The widespread clinical experiencewith some of these interventions and their overall safety recordrender them highly promising candidates for the prevention andtreatment of ischemic AKI.
Attempts at unraveling the molecular basis of the myriad pathwaysthat are activated by complex nephrologic processes such asischemic AKI have been facilitated by recent advances in functionalgenomics and cDNA microarray-based technologies (100,182–185).Several investigators have used these techniques in human andanimal models of ischemic AKI to obtain expression profilesof thousands of genes. When combined with bioinformatic tools,these studies have identified novel genes with altered expression,new signal transduction pathways that are activated, and evennew drug targets and biomarkers in AKI. One of the first inducedmolecules to be identified in the postischemic kidney usinggenomic approaches was kidney injury molecule 1 (KIM-1) (186).KIM-1 protein subsequently was demonstrated to be upregulatedin postischemic animal and human kidney tubules, predominantlyon the apical membranes of proximal tubule epithelial cells,where it may play a role in renal regeneration (187,188). Anectodomain is shed into the urine, making KIM-1 a promisingnoninvasive urinary biomarker of ischemic human AKI (187–189).
Another recent example is NGAL, one of the most highly inducedgenes in the early postischemic kidney (56,100). NGAL proteinis markedly upregulated in kidney tubules very early after ischemicAKI in animals (38) and humans (190) and is rapidly excretedin the urine, where it represents a novel, sensitive, earlybiomarker of ischemic AKI (38,191). In the postischemic kidneytubules, NGAL protein accumulates in a punctate cytoplasmicdistribution that co-localizes with endosomal markers (38).Importantly, NGAL is highly expressed in tubule cells that areundergoing proliferation, suggesting a protective or regenerativerole after AKI. Further support for this notion derives fromthe critical role played by NGAL in kidney development, duringconversion of kidney progenitors into epithelia and tubules(192). Indeed, administration of NGAL in experimental modelsbefore, during, or even shortly after ischemic injury providesremarkable protection at the functional and structural levels,with an induction of proliferation and striking inhibition ofapoptosis of tubule epithelial cells (40,41). In this context,NGAL mitigates iron-mediated toxicity by providing a reservoirfor excess iron and may provide a regulated source of intracellulariron to promote regeneration and repair. Exogenously administeredNGAL also markedly upregulates heme oxygenase-1, a proven multifunctionalprotective agent in experimental AKI that works by limitingiron uptake, promoting intracellular iron release, enhancingproduction of antioxidants such as biliverdin and carbon monoxide,and inducing the cell cycle regulatory protein p21 (193–195).Because of its multifaceted protective action, NGAL has emergedas a prime therapeutic target in ischemic AKI.
Another maximally induced gene that is identified very earlyafter ischemic injury is Zf9, a Kruppel-like transcription factorthat is involved in the regulation of a number of downstreamtargets (196). Zf9 protein is strongly expressed during kidneydevelopment and is markedly upregulated in the postischemictubule cells, along with its major transactivating factor, TGF-1.Gene silencing of Zf9 abrogated TGF-1 overexpression and mitigatedthe apoptotic response to ATP depletion in vitro (196). Thesestudies thus have identified a hitherto unrecognized pathwaythat may play a critical role in the early tubule cell deaththat accompanies ischemic renal injury.
Transcriptome profiling in rat models recently has identifiedthrombospondin 1 (TSP-1), a previously known p53-dependent proapoptoticand antiangiogenic molecule in malignant cells (197), as anothermaximally induced gene early after ischemic AKI (74). The TSP-1protein product is upregulated in the postischemic proximaltubule cells, where it co-localizes with activated caspase-3.TSP-1 null mice were partially protected from ischemic injury,with striking structural preservation of kidney tissue (74).These results thus have identified yet another previously unknownapoptotic pathway that is activated in proximal tubule cellsearly after ischemic AKI.
It is an underappreciated fact that after an initial "recovery"from an episode of clinical ARF, a significant proportion ofpatients exhibit persistent or progressive deterioration inrenal function. It now is clear that the postischemic kidneyis not fully restored to its immaculate preinjury state, andanimal models display a reduction in renal microvasculature,interstitial fibrosis, tubular hypercellularity and atrophy,and persistent inflammation when closely examined in the recoveryperiod (195,198–202). Microarray analysis has been usedto identify persistently altered gene expression in the kidneyduring an intermediate stage, after functional "recovery" fromischemic AKI but before the onset of chronic fibrotic or scleroticchanges (203). Markedly induced transcripts that were novelto the ARF field included C4 complement (inflammation associated),calcium-binding protein S100A4 (fibrosis inducer), and matrixGla protein (calcification related). Future downstream studiesshould shed light on the role of these and other gene productsin the long-term consequences of ischemic AKI.
Temporal Patterns of Structural and Functional Alterations
The clinical course of ischemic AKI has been divided classicallyinto three phases: Initiation, maintenance, and recovery (204).To this paradigm, the addition of an "extension" phase afterthe initiation phase has been proposed, primarily to reflectpreviously underestimated amplification processes (15,205).Recent advances in the pathogenesis of ischemic AKI allow usto postulate temporal relationships between the clinical phasesand the cellular alterations detailed in this review. The initiationphase is the period during which initial exposure to the ischemicinsult occurs, kidney function begins to fall, and parenchymalinjury is sprouting but not fully entrenched. IntracellularATP depletion is profound, sublethal injury to the tubule epithelialand endothelial cells predominates, generation of reactive oxygenmolecules is initiated, and activation of inflammatory mechanismscommences. Intrarenal protective mechanisms, such as inductionof HSP in tubule cells, also are brought to play during theinitiation phase. If the injury is alleviated at this stage,then complete restitution and recovery is the rule. Prolongationof ischemia followed by reperfusion ushers in the extensionphase. Blood flow returns to the cortex, and tubules undergoreperfusion-dependent cell death but also commence the regenerationprocess. In contrast, medullary blood flow remains severelyreduced, resulting in more widespread tubule cell death, desquamation,and luminal obstruction. Injured endothelial and epithelialcells amplify the raging inflammatory cascades, and the endothelialdenudation potentiates the intense vasoconstriction. The GFRcontinues to decline. This phase probably represents the mostoptimal window of opportunity for early diagnosis and activetherapeutic intervention. During the maintenance phase, theparenchymal injury is established, and the GFR is maintainedat its nadir even though renal blood flow begins to normalize.Both cell injury and regeneration occur simultaneously, andthe duration and severity of this phase may be determined bythe balance between cell survival and death. Repair of bothepithelial and endothelial cells seems to be critical to overallrecovery. Measures to accelerate the endogenous regenerationprocesses may be effective during this phase. The recovery phaseis characterized functionally by improvement in GFR and structurallyby reestablishment of tubule integrity, with fully differentiatedand polarized epithelial cells. However, the repair processmay be incomplete, and both microvascular and tubular dropouthas been demonstrated in animal studies.
According to Greek mythology, the Hydra was a dreaded multitaskingmonster with several heads, a serpent body, and deadly breath.One of its heads was invincible to weapons, and if any of theother heads were severed, then another would burgeon in itsplace. Annihilating the Hydra required a diversified, multifunctionalapproach, including finding its hiding place, cutting off eachhead, cauterizing the stumps, burying the invincible head undera boulder, and systematically chopping up the serpentine body.A modern-day Hydra, ischemic AKI, is a potentially lethal conditionwith multiple pathophysiologic mechanisms that interplay withand amplify each other. Just like Heracles was summoned to vanquishthe Hydra, a call to arms has been issued to the nephrologycommunity to intervene in AKI (206,207). Conquering AKI alsowill require a comprehensive approach, including finding thebeast (making an early diagnosis) and executing a multifacetedtherapeutic approach that is based on a better understandingof the multiheaded pathophysiology. The remarkable progressin basic and translational AKI research that has been made sincethis topic last was reviewed in JASN less than 3 yr ago (208)is chronicled in this update. The cellular and molecular toolsof modern science undeniably have provided critical new insightsinto the roles of apoptosis, oxidant and iron-mediated injury,endothelial changes, regeneration and repair, and the inflammatoryresponse in the pathogenesis of ischemic AKI. Novel strategiesthat have emerged from these recent findings (Table 1) holdtremendous promise for the proactive treatment of human AKIand currently are under intense investigation.
Table 1. Emerging therapies for AKI on the basis of recent mechanistic insightsa
Acknowledgments
Studies that are cited in this review and were performed inthe author’s laboratory were supported by grants fromthe National Institutes of Health/National Institute of Diabetesand Digestive and Kidney Diseases (R01 DK53289, P50 DK52612,and R21 DK070163), a Grant-in-Aid from the American Heart AssociationOhio Valley Affiliate, and a Translational Research InitiativeGrant from Cincinnati Children’s Hospital Medical Center.The author is indebted to Dr. Norman J. Siegel (1943–2006)for his critical comments, encouragement, and guidance, whichhave shaped not only this manuscript but also the author’sentire career.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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1 integrins are normally polarized to the basal domain, where they mediate cell-substratum adhesions. Ischemic injury leads to a redistribution of integrins to the apical membrane, with consequential detachment of viable cells from the basement membrane. There is good evidence for abnormal adhesion between these exfoliated cells within the tubular lumen, mediated by an interaction between apical integrin and the Arg-Gly-Asp (RGD) motif of integrin receptors. Administration of synthetic RGD compounds attenuates tubular obstruction and renal impairment in animal models, and the recent development of orally active integrin antagonists holds promise for human AKI (51). A recent animal study has shown that the state of 
B) factors, seem to be activated by ischemic AKI, as illustrated in Figure 3. A leading contender for many years, the role of the Fas-FADD pathway in animal models, was reaffirmed recently by demonstration of upregulation of these proteins in apoptotic tubule cells after ischemia (58) and the functional protection that is afforded by small interfering RNA duplexes that target the Fas gene (61). However, convincing human data are lacking, because the induction of the Fas gene that was shown in one study of human cadaveric kidney transplants (83) was not reproduced in two subsequent publications (10,85). However, there is growing evidence implicating an imbalance between the proapoptotic (Bax and Bid) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family in both animal (63,79) and human (10,82–84) situations. Of the regulatory factors, the proapoptotic transcription factor p53 has been shown to be induced at the mRNA (56) and protein (55) levels, and inhibition of p53 by pifithrin-
suppresses ischemia-induced apoptosis by inhibiting transcriptional activation of Bax and mitochondrial translocation of p53 (57). However, pifithrin-
-glutamyl transpeptidase-1 promoter bestowed remarkable functional protection from ischemic AKI, with enhanced tubule cell proliferation and decreased apoptosis (107). Selected delivery of HGF to the human proximal tubule, however, remains a challenge. In the case of IGF-1, enthusiasm for its renoprotective effects has been dampened by its exacerbation of inflammation and neutrophilic infiltration in the kidney after ischemia in animals (108), and clinical trials with recombinant human IGF-1 have not proved to be beneficial (109). Interest in IGF-1 therapy has been rekindled by the recent discovery of ghrelin, a naturally occurring growth hormone secretagogue that markedly increases serum IGF-1 levels and protects the kidney from ischemic injury by improving endothelial function and vasodilation in animals (78). 
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Introduction
Alterations in Morphology
