Update on Mechanisms of Ischemic Acute Kidney Injury

Acute renal failure (ARF), classically defined as an abrupt decrease in kidney function that leads to accumulation of nitrogenous wastes such as blood urea nitrogen and creatinine, is a common clinical problem with increasing incidence, serious consequences, unsatisfactory therapeutic options, and an enormous financial burden 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 focuses on intrinsic ARF, which has emerged as the most common and serious subtype in hospitalized patients and can be associated pathologically with acute tubular necrosis (ATN). Consequently, it still is common clinical practice to use the terms intrinsic ARF and ATN interchangeably. Despite decades of pioneering basic research and important technical advances in clinical care, the prognosis for patients with intrinsic ARF remains poor, with a mortality rate of 40 to 80% in the intensive care setting. Two major problems have plagued the field and hindered progress. First, well over 20 definitions for ARF have been used in published studies, ranging from dialysis requirement to subtle increases in serum creatinine (6). In an attempt to standardize the definition and reflect the entire spectrum of the condition, the term acute kidney injury (AKI) has been proposed (4). AKI refers to a complex disorder that comprises multiple causative factors and occurs in a variety of settings with varied clinical manifestations that range from a minimal but sustained elevation in serum creatinine to anuric renal failure. Prerenal azotemia and other fully reversible causes of acute renal insufficiency are specifically excluded from the spectrum of AKI. An inherent shortcoming of this term is the continued reliance on serum creatinine measurements, and the definition of AKI undoubtedly will undergo enhancements as novel early biomarkers for the identification of ARF before the rise in serum creatinine come to light (7). This review avoids the term ATN and uses the expressions AKI and intrinsic ARF transposably. The second problem is an incomplete understanding of the cellular and molecular mechanisms that underlie AKI. This review updates the reader on current advances in basic and translational research that hold promise in human ischemic AKI. Classic concepts are mentioned briefly as founding principles but expanded on only if contemporary findings substantiate or refute them. The reader is referred to recent publications that address the mechanisms that underlie other causes of intrinsic AKI, such as sepsis (8) and nephrotoxins (9). However, from the clinical viewpoint, it is acknowledged that AKI is frequently multifactorial, with concomitant ischemic, nephrotoxic, and septic components and with overlapping pathogenetic mechanisms. Alterations in Morphology If function depends on form, then it follows that mechanisms that are invoked to elucidate kidney dysfunction in AKI also must explain the morphologic alterations. In this regard, the term ATN is a misnomer, because frank tubule cell necrosis is rarely encountered in human ARF. This fact has once again been driven home by careful examination of protocol kidney biopsies that are obtained soon after deceased-donor transplantation (10), which represents a predictable model for ischemic AKI (11). Prominent morphologic features of ischemic AKI in humans include effacement and loss of proximal tubule brush border, patchy loss of tubule cells, focal areas of proximal tubular dilation and distal tubular casts, and areas of cellular regeneration (12). Necrosis is inconspicuous and restricted to the highly susceptible outer medullary regions. The glomeruli are usually unimpressive, unless a primary glomerular disease had caused the ARF. This apparent disparity between the severe impairment of renal function and the relatively subtle histologic changes in AKI traditionally has been bothersome. More recently, however, reconciliation has been forthcoming from a consistent finding of apoptotic cell death in both distal and proximal tubules in both ischemic and nephrotoxic forms of human AKI (9,10). In addition, a great deal of attention has been directed toward the peritubular capillaries, which display a striking vascular congestion, endothelial damage, and leukocyte accumulation (13–15). The mechanisms that underlie these newly emphasized morphologic findings and their implications for the ensuing profound renal dysfunction are detailed herein. Alterations in Hemodynamics An intense and persistent renal vasoconstriction that reduces overall kidney blood flow to approximately 50% of normal has long been considered a hallmark of intrinsic ARF, prompting the previous designation of “vasomotor nephropathy” (1). As if to add insult to injury, the postischemic kidney also displays peculiar regional alterations in blood flow patterns. Notably, there is marked congestion and hypoperfusion of the outer medulla that persist even though cortical blood flow improves during reperfusion after an ischemic insult (13–15). Even under normal conditions, the medullary region subsists on a hypoxic precipice as a result of low blood flow and countercurrent exchange of oxygen, although paradoxically housing nephron segments with very high energy requirements (e.g., the S3 segment of the proximal tubule and the medullary thick ascending limb of Henle’s loop). The characteristic postischemic congestion worsens the relative hypoxia, leading to prolonged cellular injury and cell death in these predisposed tubule segments. Sophisticated imaging techniques have documented these changes in regional renal blood flow in animals and validated them in human AKI (16). Mechanisms that 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 endothelin and decreased abundance of vasodilators such as endothelium-derived nitric oxide (NO) (2). Endothelin receptor antagonists ameliorate ischemic AKI in animals (18), but human data are lacking. Similarly, both carbon monoxide and carbon monoxide–releasing compounds are protective in animal models of ischemic AKI (19,20), likely through vasodilation and preservation of medullary blood flow, but have not been tested in humans. All things considered, these macrohemodynamic abnormalities cannot account fully for the profound loss of renal function, and several human trials of vasodilators such as dopamine have failed to demonstrate improvement in GFR in established ARF despite augmentation of total renal blood flow (21). However, microvascular alterations now are recognized to play a major role, as discussed next. Alterations in Tubule Dynamics Documented derangements in tubule dynamics include obstruction, backleak, and activation of tubuloglomerular feedback. The consistent histologic findings of proximal tubular dilation and distal tubular casts in human biopsies indicate that obstruction to tubular fluid flow certainly occurs in ischemic AKI. The intraluminal casts stain strongly for Tamm-Horsfall protein, which normally is secreted by the thick ascending limb as a monomer. Conversion into an obstructing gel-like polymer is enhanced by the increased luminal sodium concentration that typically is encountered in the distal tubule in AKI (22). This provides an ideal environment for cast formation along with desquamated tubule cells and brush border membranes. However, it is unlikely that obstruction alone can account for the intense dysfunction, because human studies that used forced diuresis with furosemide (23) or mannitol (24) did not have an impact on the survival and renal recovery rate of patients with established ARF. Similarly, although movement of the glomerular filtrate back into the circulation has been shown to occur, this accounts for only a very minor component of the decrease in GFR in human ARF (2). Finally, a role for activation of tubuloglomerular feedback has been proposed on the basis of human studies (25). The increased delivery of sodium chloride to the macula densa as a result of cellular abnormalities in the ischemic proximal tubule would be expected to induce afferent arteriolar constriction via A1 adenosine receptor (A1AR) activation and thereby decrease GFR (26). However, recent studies have shown that a knockout of the A1AR resulted in a paradoxic worsening of ischemic renal injury, and exogenous activation of A1AR was protective (27). Therefore, tubuloglomerular feedback activation after ischemic injury indeed may represent a beneficial phenomenon that limits wasteful delivery of ions and solutes to the damaged proximal tubules, thereby reducing the demand for ATP-dependent reabsorptive processes. Any salutary effect of exogenous A1AR activation in human AKI remains to be determined. Alterations in Tubule Cell Metabolism A profound reduction in intracellular ATP content invariably occurs early after ischemic renal injury, which sets in motion a number of critical metabolic consequences in tubule cells (28). These events are detailed next, and their interrelationship is illustrated in Figure 1. Alterations in Adenine Nucleotide Metabolism Oxygen deprivation leads to a rapid degradation of ATP to ADP and AMP. With prolonged ischemia, AMP is metabolized further to adenine nucleotides and to hypoxanthine. Hypoxanthine accumulation contributes to generation of reactive oxygen molecules by mechanisms explained below. Adenine nucleotides freely diffuse out of cells, and their depletion precludes re-synthesis of intracellular ATP during reperfusion. However, although provision of exogenous adenine nucleotides or thyroxine (which stimulates mitochondrial ATP 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 within the endoplasmic reticulum as well as diminished extrusion of cytosolic calcium into the extracellular space, resulting in the well-documented rise in free intracellular calcium after AKI. Potential downstream complications include activation of proteases and phospholipases and cytoskeletal degradation (28). However, the increased cytosolic calcium dramatically induces calcium-binding proteins such as annexin A2 and S100A6, which play an important role in the cell proliferation during recovery from AKI in animal models (31). Reflective of this contradiction, a recent meta-analysis showed that calcium channel blockers may provide some protection from renal injury in the transplant setting (32), but evidence for their efficacy in other forms of human AKI is lacking. Generation of Reactive Oxygen Molecules There now is substantial evidence for the role of reactive oxygen species in the pathogenesis of AKI. During reperfusion, the conversion of accumulated hypoxanthine to xanthine (catalyzed by xanthine oxidase formed from xanthine dehydrogenase either as a result of proteolytic conversion or as a result of oxidation of sulfhydryl residues) generates hydrogen peroxide and superoxide. In the presence of iron, hydrogen peroxide forms the highly reactive hydroxyl radical. Concomitantly, ischemia induces NO synthase in tubule cells, and the NO that is generated interacts with superoxide to form peroxynitrate, which results in cell damage via oxidant injury as well as protein nitrosylation (28). Collectively, reactive oxygen species cause renal tubule cell injury by oxidation of proteins, peroxidation of lipids, damage to DNA, and induction of apoptosis. A recent study documented a dramatic increase in oxidative stress in humans with ARF, as evidenced by depletion of plasma protein thiols and increased carbonyl formation (33). Disappointing, intermittent hemodialysis resulted in only a limited and transient beneficial effect on the redox status of plasma protein thiol groups and no effect on protein carbonyl content (33). Several scavengers of reactive oxygen molecules (e.g., superoxide dismutase, catalase, N-acetylcysteine) protect against ischemic AKI in animals, but human studies have been inconclusive. A promising new advance in the field is the protective effect of edaravone, a potent scavenger of free radicals and inhibitor of lipid peroxidation when administered at the time of reperfusion in a rat model of ischemic AKI (34). Edaravone has been approved for human use in the treatment of cerebral ischemia, and results of its use in human AKI are awaited with anticipation. Also, free iron that is derived from red cells or other injured cells now is recognized as one of the most potent factors in the generation of reactive oxygen species, and the iron scavenger deferoxamine does alleviate ischemia-reperfusion injury in animal models. However, the associated systemic toxicity (primarily hypotension) precludes its routine clinical use in human AKI (35). Two major advances have come to light in the area of iron chelation. The first is the availability of human apotransferrin, an iron-binding protein that protects against renal ischemia-reperfusion injury in animals by abrogating renal superoxide formation (36). Apotransferrin has been used successfully for the reduction of redox-active iron in patients who have undergone hematologic stem cell transplantation without any adverse effects (37). The second is the discovery of neutrophil gelatinase–associated lipocalin (NGAL), a major iron-transporting protein complementary to transferrin, as one of the most highly induced 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 functional protection in animal models (40,41). The potential use of both of these endogenous agents (apotransferrin and NGAL) in human AKI currently is under investigation. Alterations in Tubule Cell Structure Contemporary techniques have provided novel insights into the cell biology of the proximal tubule in ischemic AKI. The structural response of the tubule cell to ischemic injury is multifaceted and includes loss of cell polarity and brush borders, cell death, dedifferentiation of viable cells, proliferation, and restitution of a normal epithelium, as illustrated in Figure 2. The mechanisms that underlie this morphologic sequence of events are complex and examined next. Alterations in the Apical Cytoskeleton Cellular ATP depletion leads to a rapid disruption of the apical actin cytoskeleton and redistribution of actin from the apical domain and microvilli into the cytoplasm (42). The ensuing alterations in microvillar structure lead to formation of membrane-bound, free-floating extracellular vesicles, or “blebs,” that are either internalized or lost into the tubular lumen. Brush border membrane components that are released into the lumen contribute to cast formation and obstruction. These casts and vesicles that contain actin 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 breakdown currently is under active investigation. ADF/cofilin is a cytosolic protein that normally is maintained in the inactive phosphorylated form by Rho GTPases. In cultured renal tubule cells, ATP depletion leads to Rho GTPase inactivation, with resultant activation and relocalization of ADF/cofilin to the surface membrane and membrane-bound vesicles (43,44). Concomitant, ATP depletion dissociates the actin-stabilizing proteins tropomyosin and ezrin (45), allowing the activated ADF/cofilin to bind and consequently sever actin, which in turn leads to microvillar breakdown. Another well known mechanism for ADF/cofilin activation involves families of phosphatases such as Slingshot and Chronophin (46). Activation of ADF/cofilin also can induce apoptosis by triggering cytochrome c release, which may contribute further to its deleterious effects (47). Therefore, inactivation of ADF/cofilin, perhaps via transient inhibition of Slingshot, may represent a promising but unexplored direction in AKI. Disruption of the apical cytoskeleton by ATP depletion also results in loss of tight junctions and adherens junctions. Reduced expression, redistribution, and abnormal aggregation of a number of key proteins that constitute the tight and adherens junctions have been documented after ischemic injury in cell culture, animal models, and human studies (48). The consequent loss of tight junction barrier function potentially can magnify the transtubular backleak of glomerular filtrate that is induced by obstruction (49). Alterations in the Basolateral Cytoskeleton Ischemia results in the early disruption of at least two basolaterally polarized proteins, namely Na,K-ATPase and integrins. The Na,K-ATPase is normally tethered to the spectrin-based cytoskeleton at the basolateral domain via the adapter protein ankyrin. In cell culture, animal models, and human studies, ischemia leads to a reversible cytoplasmic accumulation of Na,K-ATPase, ankyrin, and spectrin in viable cells (50). The mislocated Na,K-ATPase remains bound to ankyrin but is devoid of spectrin. Postulated mechanisms that lead to loss of Na,K-ATPase polarity include hyperphosphorylation of ankyrin with consequent loss of spectrin binding and cleavage of spectrin by ischemia-induced activation of proteases such as calpain (2,50). A physiologic consequence of the loss of basolateral Na,K-ATPase is an impairment in proximal tubular sodium reabsorption and a consequent increase in fractional excretion of sodium, which are diagnostic signatures of intrinsic ARF. The β1 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 β1 integrin activation also is critical for maintenance of tubule epithelial integrity (52). Preischemic intravenous administration of anti-activated β1 integrin therapy (via mAb named HUTS-21 that selectively recognize the active form) resulted in preservation of renal histopathology and function, maintenance of cell-substratum interactions, and amelioration of the inflammatory response (52). The multifaceted protective effect renders HUTS-21 a potentially attractive therapeutic candidate for human AKI, but the need for pretreatment is an obvious disadvantage. Alterations in Cell Viability Both experimental and human studies indicate that tubule epithelial cells can suffer one of three distinct fates after ischemic AKI. The majority of cells remain viable, suggesting that they either entirely escape injury or are only sublethally injured and undergo recovery. A subset of tubule cells display patchy cell death that results from at least two pathophysiologic mechanisms. Necrosis is an explosive, chaotic process that is characterized by loss of membrane integrity, cytoplasmic swelling, and cellular fragmentation. Apoptosis is a quiet, orderly demise that is typified by cytoplasmic and nuclear shrinkage, DNA fragmentation, and breakdown of the cell into membrane-bound apoptotic bodies that are rapidly cleared by phagocytosis. These two forms of cell death can coexist and are considered two extremes of a spectrum. After ischemic renal injury, the mode of cell death depends primarily on the severity of the insult and the resistance of the cell type. Necrosis usually occurs after more severe injury and in the more susceptible nephron segments, whereas apoptosis predominates after less severe injury and especially in the ischemia-resistant distal nephron segments. Apoptosis can be followed by “secondary necrosis,” especially if the apoptotic cells are not rapidly removed. The commonly used assays for apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay and DNA laddering) do not adequately distinguish between apoptosis and necrosis. Strict morphologic criteria are desirable for the detection and quantification of apoptosis, including nuclear (condensation and fragmentation) and cytoplasmic (cell shrinkage and blebbing) changes. Mounting evidence now indicates that apoptosis is the major mechanism of early tubule cell death in contemporary clinical ARF (53–55). During recent years, several animal models of ischemic AKI consistently and unequivocally demonstrated the presence of apoptotic tubule cells using a variety of sensitive assays (56–79). Importantly, this now has been confirmed by several investigators in human models of AKI (10,80–85). Nevertheless, controversies still exist regarding the contribution of apoptosis to the syndrome of ARF. First, most estimates place the peak incidence of apoptosis at only approximately 3 to 5% of tubule cells after ischemic injury, which arguably is insufficient to explain the profound renal dysfunction. In response to this skepticism is that the degree of apoptosis is vastly underestimated because it is a rapidly occurring heterogeneously distributed event that is notoriously difficult to identify and quantify in tissues. Second, apoptosis is more commonly encountered in the distal tubule, whereas loss of viable cells occurs predominantly in proximal segments. Reconciliation of this argument is provided by the demonstration of both necrosis and apoptosis in the proximal tubule, where these processes may represent a continuum. Third, apoptosis generally is regarded as a physiologic process that removes damaged cells and therefore may be beneficial to the organ and the organism. The counterpoint to this supposition is that apoptosis after ischemic AKI is a double-edged sword that occurs in two waves, at least in animal models. The first wave is detectable within 6 to 12 h of the insult, peaks at approximately 3 d, and rapidly diminishes. This phase deletes previously healthy tubule cells, thereby contributing to the ensuing dysfunction. The second wave becomes apparent approximately 1 wk later, removes hyperplastic and unwanted cells, and therefore may play a role in the remodeling of injured tubules. Because the evidence is overwhelmingly in favor of apoptosis as a pathogenetic mechanism, considerable attention has been directed 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 caspase 8), and regulatory (p53 and NF-κB) 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-α suppresses ischemia-induced apoptosis by inhibiting transcriptional activation of Bax and mitochondrial translocation of p53 (57). However, pifithrin-α is an unlikely candidate for therapeutic consideration in humans because generalized inhibition of p53-dependent apoptosis likely will promote survival of damaged or mutation-bearing cells in other organ systems. 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 also has been described), and cell-permeant caspase inhibitors have provided particularly attractive targets for study (73). Currently available inhibitors largely have been investigated only in animals, provide only partial protection, and are most effective when administered before the injury. However, these characteristics render caspase inhibition as a potentially attractive approach to reducing apoptotic damage during cold storage of deceased-donor kidneys before transplantation, as has been demonstrated in animal models (87). In this regard, an orally active pan-caspase inhibitor (IDN-6556) was developed recently (88) and shown to be effective in preventing injury after lung and liver transplantation in animals (88,89). IDN-6556 is currently undergoing evaluation in Phase II clinical trials for injury prevention in human liver transplantation. Several other modalities ameliorate apoptosis and AKI in experimental situations. Pretreatment with erythropoietin conferred structural and functional protection, inhibition of apoptosis, and upregulation of the antiapoptotic transcription factor NF-κB (65,77). However, inhibition of NF-κB by intrarenal transfection of decoy oligonucleotides resulted in a paradoxic attenuation of ischemic AKI, perhaps by inhibiting transcription of proinflammatory factors (90). α-1-Acid glycoprotein (an acute-phase protein of unknown function [59]), minocycline (62), A1 adenosine receptor agonists (64), peroxisome proliferator–activated receptor β ligands (66), geranylgeranylacetone (an inducer of heat-shock proteins [HSP] [67]), and poly(ADP-ribose) polymerase inhibitors (91) all have provided encouraging functional protection from ischemic AKI with inhibition of apoptosis and inflammation, but the underlying mechanisms have not been fully elucidated. Some of these agents are already widely available and safely used in other human conditions, and results of their use in AKI should be forthcoming. Challenges for the future clinical use of apoptosis inhibition in AKI include determining the best timing of therapy, optimizing the specificity of inhibitor, minimizing the extrarenal adverse effects, and tubule-specific targeting of the apoptosis modulatory maneuvers. The mechanisms whereby the majority of tubule cells escape cell death and either emerge unscathed or recover completely after ischemic AKI remain under active investigation. HSP have surfaced as prime arbitrators of this cytoprotection (92–94). Induction of HSP is part of a highly conserved innate cellular response that is activated swiftly and robustly after ischemic AKI. HSP promote cell survival by inhibiting apoptosis (95), and liposomal delivery of HSP-72 into cultured renal tubule cells blocks ischemia-induced apoptosis (96). HSP also facilitate the restoration of normal cellular function by acting as molecular chaperones that assist in the refolding of denatured proteins as well as proper folding of nascent polypeptides. For instance, there now is broad evidence for the role of HSP in the restoration of cytoskeletal integrity and Na,K-ATPase polarity after ischemic AKI in animal models (92–94). In cultured tubule cells, inhibition of the HSP response by gene-silencing techniques produced profound impairment of cellular integrity and Na,K-ATPase polarity (97), and overexpression of HSP-70 mitigated the loss of Na,K-ATPase polarity after ATP depletion (98). Collectively, these findings suggest that maneuvers that enhance the innate HSP response have potential benefit in human AKI but that transition of therapy from bench to bedside has not been achieved yet. However, that this also may activate undesirable cellular processes such as increased immunogenicity (99) serves to caution us against leaping from the frying pan into the fire when contemplating HSP therapies. Mechanisms of Repair Surviving renal tubule cells possess a remarkable ability to regenerate and proliferate after ischemic AKI (100). Morphologically, repair is heralded by the appearance of dedifferentiated epithelial cells that express vimentin, a marker for multipotent mesenchymal cells (101). The origin of these cells remains contentious (and is addressed next), but they most likely represent surviving tubule cells that have dedifferentiated. In the next phase, the cells upregulate genes that encode for a variety of growth factors, such as IGF-1, hepatocyte growth factor (HGF), and fibroblast growth factor, and undergo marked proliferation. In the final phase, cells express differentiation factors such as neural cell adhesion molecule (NCAM) and osteopontin and undergo re-differentiation until the normal fully polarized epithelium is restored. Therefore, during recovery from ischemia, renal tubule cells recapitulate phases and processes that are very similar to those during normal kidney development (100–102). Recently identified examples of genes that lend support to the concept of “recapitulation of phylogeny by ontogeny” include NGAL (38–41), leukemia inhibitory factor (103), transcription factor Ets-1 (104), and Wnt-4 (105). All of these transcripts not only are critical to early kidney development but also are marked