Prasad Devarajan

Acute renal failure (ARF) secondary to ischemic injury remains a common and potentially devastating problem. A transcriptome-wide interrogation strategy was used to identify renal genes that are induced very early after renal ischemia, whose protein products might serve as novel biomarkers for ARF. Seven genes that are upregulated >10-fold were identified, one of which (Cyr61) has recently been reported to be induced after renal ischemia. Unexpectedly, the induction of the other six transcripts was novel to the ARF field. In this study, one of these previously unrecognized genes was further characterized, namely neutrophil gelatinase-associated lipocalin (NGAL), because it is a small secreted polypeptide that is protease resistant and consequently might be readily detected in the urine. The marked upregulation of NGAL mRNA and protein levels in the early postischemic mouse kidney was confirmed. NGAL protein expression was detected predominantly in proliferating cell nuclear antigen-positive proximal tubule cells, in a punctate cytoplasmic distribution that co-localized with markers of late endosomes. NGAL was easily detected in the urine in the very first urine output after ischemia in both mouse and rat models of ARF. The appearance of NGAL in the urine was related to the dose and duration of renal ischemia and preceded the appearance of other urinary markers such as N-acetyl-beta-D-glucosaminidase and beta2-microglobulin. The origin of NGAL from tubule cells was confirmed in cultured human proximal tubule cells subjected to in vitro ischemic injury, where NGAL mRNA was rapidly induced in the cells and NGAL protein was readily detectable in the culture medium within 1 h of mild ATP depletion. NGAL was also easily detectable in the urine of mice with cisplatin-induced nephrotoxicity, again preceding the appearance of N-acetyl-beta-D-glucosaminidase and beta2-microglobulin. The results indicate that NGAL may represent an early, sensitive, noninvasive urinary biomarker for ischemic and nephrotoxic renal 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 of ischemic AKI and of medullary blood but have been in these fully for the profound loss of renal and human of vasodilators such as have to in in ARF of renal blood flow However, alterations are to a major as Alterations in in tubule include and of The consistent histologic findings of proximal tubular dilation and distal tubular in human biopsies that to tubular flow occurs in ischemic AKI. The for which is by the thick ascending limb as a an is enhanced by the that is encountered in the distal tubule in AKI This an for with tubule and brush However, it is that can for the intense because human that used with or have an on the and renal rate of patients with ARF. 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Neutrophil gelatinase-associated lipocalin (Ngal), also known as siderocalin, forms a complex with iron-binding siderophores (Ngal:siderophore:Fe). This complex converts renal progenitors into epithelial tubules. In this study, we tested the hypothesis that Ngal:siderophore:Fe protects adult kidney epithelial cells or accelerates their recovery from damage. Using a mouse model of severe renal failure, ischemia-reperfusion injury, we show that a single dose of Ngal (10 microg), introduced during the initial phase of the disease, dramatically protects the kidney and mitigates azotemia. Ngal activity depends on delivery of the protein and its siderophore to the proximal tubule. Iron must also be delivered, since blockade of the siderophore with gallium inhibits the rescue from ischemia. The Ngal:siderophore:Fe complex upregulates heme oxygenase-1, a protective enzyme, preserves proximal tubule N-cadherin, and inhibits cell death. Because mouse urine contains an Ngal-dependent siderophore-like activity, endogenous Ngal might also play a protective role. Indeed, Ngal is highly accumulated in the human kidney cortical tubules and in the blood and urine after nephrotoxic and ischemic injury. We reveal what we believe to be a novel pathway of iron traffic that is activated in human and mouse renal diseases, and it provides a unique method for their treatment.