Acute Kidney Injury: Pathogenesis, Diagnosis, and Management

Charles L. Edelstein

Acute kidney injury (AKI) (defined as an increase in serum creatinine >0.5 mg/dL) occurs in 1% of hospital admissions (1), and up to 7% of hospitalized patients develop AKI (1). Twenty-five percent of patients in the intensive care unit (ICU) develop AKI as defined by oliguria or a serum creatinine >3.5 mg/dL (1). Five percent of patients in the ICU will need renal replacement therapy (RRT) (1,2). Dialysis is the only Federal Drug Administration (FDA)-approved treatment for AKI (3). Even though both intermittent hemodialysis (IHD) and continuous RRT (CRRT) are widely used, the reported mortality rates of AKI are between 30% and 80% (4,5). In spite of an increase in the degree of comorbidity of patients with AKI, the in-hospital mortality rate has declined over the period 1988 to 2002 (6).

AKI is defined as a sudden decrease in the glomerular filtration rate (GFR) occurring over a period of hours to days. The Acute Dialysis Quality Initiative (ADQI) has developed the RIFLE (Risk Injury, Failure, Loss of kidney function, and End-stage kidney disease) classification of AKI that divides AKI into the following stages: (a) risk, (b) injury, (c) failure, (d) loss of function, (e) and end-stage kidney disease (Fig. 10-1) (7–9). The term “acute kidney injury” replaces the term “acute renal failure” (ARF), and ARF is restricted to patients who have AKI and need RRT. The RIFLE criteria have been validated in multiple studies, that is, as the RIFLE class increases, so does mortality (7–9).

The Acute Kidney Injury Network (AKIN) has also developed a classification of AKI (8–10) (Table 10-1). The AKIN group recommends a smaller change in serum creatinine (0.3 mg/dL) be used as a threshold to define the presence of AKI and identify patients with Stage 1 AKI (analogous to RIFLE-Risk). In the AKIN classification of AKI, a time period of 48 hours over which AKI occurs (compared to 1–7 days for the RIFLE criteria) is given. Patients receiving RRT are classified as Stage 3 AKI (RIFLE-Failure). In addition, the AKIN criteria differ from the RIFLE criteria as follows: (a) The AKIN classification includes less severe injury in the criteria. (b) AKIN avoids using the GFR as a marker in AKI, as there is no dependable way to measure GFR in AKI and equations to measure GFR in AKI are not reliable if the serum creatinine change is not in a steady state. (c) AKIN suggests that volume status should be optimized and urinary tract obstructions be excluded when using oliguria as a diagnostic criterion. The Kidney Disease Improving Global Outcomes (KDIGO) classification of AKI builds on the RIFLE and AKIN classifications. The KDIGO classification has both the increase in serum creatinine (0.3 mg/dL) over 48 hours and the 1.5- to 1.9-fold increase in serum creatinine known or presumed to have occurred over 1 to 7 days.

When AKI is not the result of primary vascular, glomerular, or interstitial disorders, it is referred to as acute tubular necrosis (ATN). In fact, in the clinical setting, the terms “acute renal failure” and “acute tubular necrosis” have become synonymous (11). However, ATN is a renal histologic finding and may not be consistently detectable in patients with AKI, despite profound kidney dysfunction (12–15). Thus, in the strictest sense, the terms AKI and ATN should not be used interchangeably (16). ATN has recently been defined as a syndrome of physiologic and pathologic dissociation (16).

After prerenal and postrenal azotemia have been excluded, the diagnosis of intrarenal or intrinsic AKI can be entertained. These problems may be renal vascular (large or small vessel), tubular, interstitial, or glomerular (Table 10-2). The Madrid AKI Study Group reported that the commonest cause of AKI was ATN accounting for 38% of hospitalized patients with AKI and 76% of ICU patients with AKI (4). The second and third leading causes of AKI were prerenal azotemia and urinary tract obstruction. Sepsis was the leading cause of AKI and more common than ischemic causes in the ICU (4,17–19). The diseases may be primary renal or part of a systemic disease. The diseases of vessels and glomeruli will be dealt with in Chapter 15. This chapter therefore will focus primarily on the ischemic and nephrotoxic causes of AKI and acute interstitial nephritis (AIN).

Pathogenesis of AKI

The nature of proximal tubular injury in ischemic AKI (20,21) includes reversible sublethal dysfunction (loss of polarity, swelling, loss of the apical brush border), lethal injury (necrosis necroptosis and apoptosis) (13,20) and autophagy, a normal physiologic process that tries to rescue the destruction of cells in the body. Autophagy maintains homeostasis or normal functioning by protein degradation and turnover of the destroyed cell organelles for new cell formation.

In rat models of ischemic AKI and in posttransplant AKI in humans, there is reversible sublethal injury during the first 6 hours of reperfusion followed by necrosis at 24 hours of reperfusion (22–24). Proximal tubular cell death due to ischemic AKI in vivo in rodents and hypoxia in vitro results predominantly in necrosis, hence the term “acute tubular necrosis,” or ATN (25). Apoptotic cell death in ischemic renal injury in vivo has been demonstrated (26,27). When apoptosis has been demonstrated in early ischemic AKI, it is often present in the distal tubules (28–30). The significance of apoptosis in distal tubules is uncertain. Apoptosis in proximal tubules may play a role in tubular regeneration and was demonstrated to occur later at 3 days after ischemic injury in regenerating proximal tubules (31).

Dissociation of spectrin and other basolateral cytoskeletal proteins plays a major role in the well-documented sublethal injury and loss of polarity, which leads to proximal tubule dysfunction during renal ischemia (22,32,33). Spectrin is the major component of the membrane-associated cytoskeleton and is also important in the maintenance of cell membrane structural integrity. In the cytoskeleton of the proximal tubule, Na⁺/K⁺ ATPase is linked to the cytoskeleton/membrane complex by a variety of cytoskeletal proteins including spectrin (32,34). ATP depletion and renal ischemia cause dissociation of the basolateral cytoskeleton in rat kidneys (33,35) and in human transplanted kidneys (22). Na⁺/K⁺ ATPase and spectrin dissociate from the cytoskeleton during ischemic AKI (22).

A complete redistribution of Na⁺/K⁺ ATPase from the basolateral to the apical membrane, that is, total loss of polarity, is not necessary to decrease sodium reabsorption. It has been demonstrated that (a) translocation of Na⁺/K⁺ ATPase to the cytoplasm results in depolarization confined to the proximal tubule; (b) fractional excretion of lithium, a surrogate measure for the fraction of filtered sodium that is delivered to the macula densa, the site of tubuloglomerular feedback, is massively increased; and (c) these abnormalities persist for the duration of the maintenance phase of postischemic AKI (22,23). These results provide evidence for decreased proximal reabsorption of sodium, resultant increased sodium delivery to the macula densa, tubuloglomerular feedback (“tubular communication with the glomerulus”), and resultant filtration failure that accompanies ischemic AKI.

The loss of polarity is also associated with redistribution of integrins. Tubular cells detach from their matrix, which results in increased cast formation and provides an experimental mechanism for the back-leak of glomerular filtrate. The consequences of loss of polarity, that is, tubuloglomerular feedback, cast formation with tubular obstruction, and back-leak of glomerular filtrate, are major factors in the pathogenesis of experimental ischemic AKI (26).

Necroptosis is a form of programmed or regulated necrosis or inflammatory cell death. Conventionally, necrosis is associated with unprogrammed cell death. Necroptosis shows that cells can execute necrosis in a programmed fashion and that apoptosis is not the only form of programmed cell death. Necroptosis is seen in human kidney cells subjected to ATP depletion (36). Necroptosis is mediated by receptor-interacting protein 1 (RIP1) and RIP3. Necroptosis was investigated in a mouse model of renal ischemia/reperfusion (I/R) injury. Treatment with necrostatin-1, an inhibitor of necroptosis, reduced organ damage and renal failure, even when administered after reperfusion (37,38). Inhibition of the core components of the necroptosis pathway RIP1 or RIP3 by gene knockout or a chemical inhibitor results in decreased cisplatin-induced proximal tubule damage in mice (39). Necroptosis is thought to contribute to AKI in kidney transplantation (40). The cytotoxicity of crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent necroptosis in five different cell types (41). These studies demonstrated that necroptosis is a major mechanism of proximal tubular cell death in AKI.

Autophagy is a process that takes place in all eukaryotic cells that keeps cells alive under stressful conditions (42). In autophagy, there is the sequestration of damaged organelles into double-membraned autophagosomes that subsequently fuse with lysosomes where their cargoes are delivered for degradation and recycling. In the healthy kidney, autophagy plays an important role in the homeostasis and viability of renal tubular epithelial cells.

Inhibition of autophagy using an ATG5 siRNA increases apoptosis during rewarming after cold storage in renal tubular epithelial cells (43). Autophagy occurs prior to apoptosis in renal tubular cells during AKI suggesting that autophagy is an early response of the cells to stress and not a result of apoptosis (44,45). Together, these studies suggest that autophagy is a renoprotective mechanism that protects against apoptosis to enable cell survival (42). Autophagy may be a protective mechanism to decrease apoptosis through the degradation of mitochondria (46,47). Removal of mitochondria by autophagy can increase the threshold for induction of apoptosis (47). Depolarized mitochondria that are not cleared by autophagy release caspase activators (cytochrome c and Smac) into the cytoplasm to induce apoptosis.

In cisplatin-treated proximal tubule cells, inhibition of autophagy by pharmacological inhibitors or genetic knockdown increases apoptosis (44). In vitro, pharmacological or genetic suppression of autophagy sensitizes tubular cells to apoptosis induced by hypoxia (48). Mice with kidney-specific knockout of autophagy (ATG5 or ATG7) are viable but develop worse ischemic or cisplatin-induced AKI demonstrating the renoprotective role of autophagy in the kidney (44,45). Together, these studies suggest that autophagy is a renoprotective mechanism against apoptosis for cell survival (42).

Telomerase deficiency delays renal recovery in mice after I/R injury by impairing autophagy (49). Telomerase reverse transcriptase (TerT) and RNA (TerC) are essential to maintain telomere length. TerC or TerT knockout significantly delayed recovery in ischemic AKI. Electron microscopy and LC3-II showed a significant delay of autophagosome formation in TerC and TerT knockout mice. The mTORC1 inhibitor, rapamycin, partially restored the I/R-induced autophagy response.

In summary, basal autophagy in the kidney is vital for the normal homeostasis of the proximal tubules (50). There is a complex connection between autophagy, apoptosis, and regulated necrosis in AKI that merits further study (50).

Potential mediators/mechanisms of AKI cause tubular injury, inflammation, or vascular injury (Table 10-3). These mediators of tubular injury, inflammation, or vascular injury will now be discussed in more detail.

Tubular Injury

Ca²⁺ ACCUMULATION AND CELL INJURY

Ca²⁺ overload is characteristic of tissues with lethally injured cells, since the breakdown of the plasma membrane barrier to Ca²⁺ causes a large increase in cytosolic Ca²⁺, which is sequestered in part by the mitochondria. Specifically, building on the hypothesis that homeostatic mechanisms controlling cellular Ca²⁺ are disturbed in AKI, it has been shown that radiocontrast-induced AKI (51,52) and cadaveric kidney transplant dysfunction (53,54), for example, can be attenuated by administration of chemically dissimilar Ca²⁺ channel blockers. These are two clinical conditions in which intense renal vasoconstriction is demonstrable, a situation where delivery of oxygen and nutrients to renal tubules is compromised. The administration of Ca²⁺ channel blockers reduces the intensity of renal vasoconstriction and provides better delivery of nutrients to renal tissues. With ischemia, the poor nutrient flow to renal tubules also results in tubule Ca²⁺ overload, which can be lessened by the Ca²⁺ channel blockers. Although Ca²⁺ channel blockers have been shown to be efficacious in these two aforementioned clinical conditions, a full understanding of the mechanisms by which cytosolic or tissue Ca²⁺ increases in underperfused situations and how this increase may contribute to organ injury is the focus of much recent research. It is important, therefore, to understand the normal cellular Ca²⁺ regulation before discussing the newer insights that have been gained using experimental approaches to further improve our understanding of the pathogenesis of AKI.

Normal Regulation of Cell Ca²⁺

Three major cellular Ca²⁺ pools exist: (a) a pool bound to plasma membranes, (b) a pool bound to or sequestered within intracellular organelles, and (c) a pool both free and bound within the cytoplasm (55).

Although 60% to 70% of all Ca²⁺ in renal epithelial cells is located in the mitochondria, cytosolic free ionized Ca²⁺ is the most critical with regard to regulation of intracellular events. Cytosolic free Ca²⁺ (Ca²⁺)i is normally kept at about 100 nM, which is 1/10,000 of the extracellular level (56). Ca²⁺ efflux is mediated on basolateral membranes by both Ca²⁺ ATPase, which is adenosine triphosphate (ATP) dependent, and a Na⁺/Ca²⁺ exchanger on the basolateral membrane, which is ATP independent (57). Normally, the cell membrane is impermeable to Ca²⁺ and maintains a steep Ca²⁺ gradient between the cytosol and the extracellular space. However, when cytosolic Ca²⁺ increases in response to increased cellular membrane permeability or decreased Ca²⁺ efflux or both, the mitochondria and endoplasmic reticulum actively increase their Ca²⁺ uptake. Mitochondrial uptake and retention of Ca²⁺ become substantial only when cytosolic levels exceed 400 to 500 nM, as occurs with cell injury (56). Mitochondrial uptake is regulated by a Ca²⁺ uniporter in the mitochondrial inner membrane. During cell injury, active mitochondrial sequestration appears to be quantitatively the most important process for buffering elevations in cytosolic Ca²⁺.

Tubular Effects of Ca²⁺ Accumulation

In vivo studies of intact kidney cannot discriminate between protective effects at the vascular sites compared to tubular sites or a combination thereof. As the proximal tubule is the main site of injury in I/R models in vivo and the human allograft with AKI (58), the study of isolated proximal tubules during conditions of oxygen deprivation either in suspension or in primary culture has provided insight into the pathophysiology of proximal tubular injury. Numerous studies in both freshly isolated rabbit and rat proximal tubules as well as various models of proximal and distal tubules in culture have demonstrated an increase in cytosolic Ca²⁺ in these renal epithelial cells during chemical anoxia, hypoxia, and Ca²⁺ ionophore treatment (59–67). When exposed to anoxia in vitro, proximal and distal tubules in culture rapidly exhibit cell death after reoxygenation (68). However, if Ca²⁺ is removed from the bathing medium during the first 2 hours of reoxygenation and then replaced, cell viability is greatly enhanced (68). Ca²⁺ channel blockers have also been shown to delay the onset of anoxic cell death in primary cultures of rabbit proximal tubules and cortical collecting tubules, suggesting that Ca²⁺-mediated hypoxic cell death is not limited to the proximal tubules (69).

Ca²⁺ channel blockers have no effects on the rate of Ca²⁺ influx into normoxic proximal tubules. However, during hypoxia or anoxia in vitro, Ca²⁺ influx rate into tubules is increased above normal levels, and Ca²⁺ channel blockers reduce this rate to or toward normal (70). This is an important observation because (Ca²⁺)i could increase as the result of normal influx rates in the presence of reduced efflux rates secondary to decreased ATP-dependent Ca²⁺ ATPase or decreased Na⁺/Ca²⁺ antiporter activity. The efficacy of Ca²⁺ channel blockers to prevent the increased Ca²⁺ influx rate during hypoxia and not during normoxia suggests a hypoxia-induced alteration in membrane permeability to Ca²⁺ that is sensitive to Ca²⁺ channel blockers. This permeability pathway appears to be sensitive, in part, to the decrease in ATP that occurs during hypoxia. For example, reduced ATP levels in rat proximal tubules with a phosphate-free incubation medium result in increased Ca²⁺ influx rate (71). This ATP-dependent change in Ca²⁺ permeability has not been examined in detail; however, acidosis prevents the increased Ca²⁺ influx rate in tubules and delays the onset of cell injury, as assessed by lactate dehydrogenase (LDH) release even though ATP remains at low levels (72). Cellular protection is also observed with an acidotic perfusate in the isolated perfused kidney (73). Intracellular acidosis is more likely to develop in complete anoxia than in hypoxia, and this may explain the only very short-lived increase in Ca²⁺ influx rate (70) as well as the absence of appreciable tissue Ca²⁺ overload during anoxia, as assessed by atomic absorption spectroscopy (74).

On the basis of these observations, the role of Ca²⁺ influx rate in mediating proximal tubule hypoxic injury was examined. By employing a combination of ethylene glycol tetraacetic acid (EGTA) and various Ca²⁺ concentrations in the tubule bathing medium (Ca²⁺-modified Krebs buffer), a delay in the onset of cell injury during hypoxia was seen when extracellular Ca²⁺ concentration was <10⁻⁵ M (64).

Thus, Ca²⁺ ions enter renal proximal tubules at a faster rate than normal during oxygen deprivation. The removal of extracellular Ca²⁺ ions or administration of Ca²⁺ channel buffers reduces the injury associated with this increased influx rate of Ca²⁺. Acidosis also reduces Ca²⁺ influx rate (72) and exerts cytoprotective effects (71–74). Finally, if Ca²⁺ ions do enter hypoxic or anoxic cells, their deleterious effects can be mitigated by calmodulin inhibitors (69). Together, these data strongly suggest that it is the increased cytosolic or intracellular burden of Ca²⁺ that initiates the development of cell injury.

The level of the free cytosolic Ca²⁺ increase during ATP depletion in proximal tubules has been studied. Previously it was difficult to determine peak cytosolic Ca²⁺ levels using the high-affinity Ca²⁺ fluorophore Fura-2. The (Ca²⁺)i increases to >100 μM in ATP-depleted proximal tubules using the low-affinity Ca²⁺ fluorophore Mag-Fura-2 (75). Experiments were done in the presence of 2 mM glycine, which approximates the physiologic concentration in vivo. Ninety-one percent of the tubules studied in an individual experiment had a free cytosolic Ca²⁺ that exceeded 10 μM. Thirty-five percent had levels >500 μM with no cell membrane damage. In this study, proximal tubules had a remarkable resistance to the deleterious effects of increased Ca²⁺ during ATP depletion in the presence of glycine. In the isolated perfused rat kidney, intracellular Ca²⁺ increases have also been measured using 19^F NMR and 5^F BAPTA. In these studies, there was a partially reversible increase from 256 to 660 nM of Ca²⁺ (76,77).

The level of oxygen deprivation that is required to increase cytosolic Ca²⁺ has also been studied. A rise in cytosolic Ca²⁺ in anoxic but not hypoxic tubules was demonstrated (78). In hypoxic perfusion, oxygen tension measured with a very sensitive electrode was 5 to 6 mm Hg. Complete anoxia was achieved with oxyrase in a nonperfused system. Ca²⁺ did not increase during hypoxia, but there was an increase in Ca²⁺ during anoxia. This increase paralleled the collapse in mitochondrial membrane potential as measured by rhodamine fluorescence. Because cell membrane damage occurred during both anoxia and hypoxia, it was concluded that an increase in cell Ca²⁺ is not always necessary for cell injury.

However, despite these studies, a crucial question remained to implicate Ca²⁺ as the primary factor in cell injury. Does the increase in cytosolic Ca²⁺ precede the injury, or is it a postlethal event? To answer this question, a video imaging system was designed in which the rise in cytosolic Ca²⁺ as well as cell membrane injury could be simultaneously measured in freshly isolated proximal tubules (79). (Ca²⁺)i in freshly isolated proximal tubules, as assessed with Fura-2, increased significantly after 2 minutes of hypoxia and continued to increase progressively with continued hypoxia (67). This increase in (Ca²⁺)i precedes the uptake by nuclei of the membrane-impermeable dye propidium iodide (PI) (67). PI staining is reduced when hypoxic rat proximal tubules are incubated either in a Ca²⁺-free medium or with the intracellular Ca²⁺ chelator BAPTA (67). This study strongly supports the hypothesis that a cause-and-effect relationship exists between the elevation in (Ca²⁺)i and the development of hypoxic membrane damage. Furthermore, this early rise in (Ca²⁺)i after 5 to 10 minutes of hypoxia is reversible, since return to a well-oxygenated medium results in a prompt (1 minute) return of (Ca²⁺)i to baseline level. If membrane injury had been the cause of the increase in (Ca²⁺)i, a return to basal levels would not have occurred with reoxygenation.

In support of a pathogenic role of Ca²⁺ in cell injury, it has been demonstrated that voltage-dependent Ca²⁺ channels are involved in cellular and mitochondrial accumulation of Ca²⁺ that follows ATP depletion and that voltage-dependent Ca²⁺ channels play an important role in regulating mitochondrial permeability transition, cytochrome c release, caspase activation, and apoptosis (80). In this study, in a rat renal proximal tubular cell line treated with antimycin A, ATP depletion-induced apoptosis was preceded by increased [Ca(²⁺)]i and mitochondrial Ca²⁺ before activation of mitochondrial signaling. Antagonizing L-type Ca(²⁺) channels with azelnidipine administration ameliorated cellular and mitochondrial Ca(²⁺) accumulation, mitochondrial permeability transition, cytochrome c release, caspase-9 activation, and resultant apoptosis.

MECHANISMS OF Ca²⁺-INDUCED PROXIMAL TUBULAR INJURY

There is now compelling evidence that hypoxia-induced rise in (Ca²⁺)i activates Ca²⁺-dependent intracellular events that mediate membrane injury. These potential Ca²⁺-dependent mechanisms include changes in the actin cytoskeleton of proximal tubule microvilli, activation of phospholipase A₂ (PLA₂), and activation of the calcium-dependent cysteine protease, calpain.

Ca²⁺-Dependent Changes in the Actin Cytoskeleton

In the presence of ATP depletion, both Ca²⁺-independent as well as Ca²⁺-mediated processes can disrupt the actin cytoskeleton during acute hypoxic proximal tubule cell injury (81,82). To better define the role of Ca²⁺ in pathophysiologic alterations of the proximal tubule microvillus actin cytoskeleton, freshly isolated tubules were studied. The intracellular free Ca²⁺ was equilibrated with highly buffered, precisely defined medium Ca²⁺ levels using a combination of the metabolic inhibitor, antimycin, and the ionophore, ionomycin, in the presence of glycine, to prevent lethal membrane damage (83). Increases in Ca²⁺ to ≥10 µM were sufficient to initiate concurrent actin depolymerization, fragmentation of F-actin into forms requiring high-speed centrifugation for recovery, redistribution of villin to sedimentable fractions, and structural microvillar damage consisting of severe swelling and fragmentation of actin cores. These observations implicate Ca²⁺-dependent, villin-mediated actin cytoskeletal disruption in hypoxic tubule cell microvillar damage.

Ca²⁺-Dependent Activation of PLA₂

PLA₂ hydrolyzes the acyl bond at the sn-2 position of phospholipids to generate free fatty acids and lysophospholipids. Free fatty acid release has been well documented in rat proximal tubules (84). This release is thought to be mediated to a large extent by activation of intracellular PLA₂ during hypoxia (85). It has been shown that both the messenger RNA (mRNA) for PLA₂ and the PLA₂ enzyme activity are increased in hypoxic rabbit tubules (86).

The mechanism of PLA₂-induced cell membrane damage is controversial. In proximal tubules, hypoxia has been shown to cause an increase in free fatty acids, which was initially believed to contribute to cell injury (84). However, a recent study has shown that unsaturated free fatty acids protect against hypoxic injury in proximal tubules and that this protection may be mediated by negative feedback inhibition of PLA₂ activity (87).

There are various isoforms of PLA₂, and most isoforms of PLA₂ require Ca²⁺ for catalytic activity (88). The cytosolic form, cPLA₂, preferentially releases arachidonic acid from phospholipids and is regulated by changes in intracellular Ca²⁺ concentration (88).

PLA₂ enzymatic activity was measured in cell-free extracts prepared from rat renal proximal tubules (85). Both soluble and membrane-associated PLA₂ activity was detected. All PLA₂ activity detected during normoxia was Ca²⁺ dependent. Fractionation of cytosolic extracts by gel filtration revealed three peaks of PLA₂ activity. Exposure of tubules to hypoxia resulted in stable activation of soluble PLA₂ activity, which correlated with disappearance of the highest molecular mass form (>100 kDa) and appearance of a low-molecular-mass form (approximately 15 kDa) of PLA₂. Hypoxia also resulted in the release of a low-molecular-mass form of PLA₂ into the extracellular medium. This study provides direct evidence for Ca²⁺-dependent PLA₂ activation during hypoxia. However, Ca²⁺-independent forms of PLA₂ have also been found to play a role in hypoxic proximal tubular injury (89).

cPLA₂-deficient mice have been developed. The cPLA₂ knockout mice have smaller infarcts and develop less brain edema and fewer neurologic deficits after transient middle cerebral artery ischemia (90,91).

There is evidence of an increased macula densa cell calcium concentration with a reduction in fluid load to the macula densa (92). An increase in macula densa cell calcium activates PLA₂ to release arachidonic acid, the rate-limiting step in the formation of prostaglandins like PGE₂. Adenosine also has an important function in the juxtaglomerular apparatus. It stimulates calcium release in afferent arteriolar smooth muscle cells, leading to contraction of the afferent arteriole as part of the tubuloglomerular feedback mechanism.

CYSTEINE PROTEASES

The cysteine proteases are a group of intracellular proteases that have a cysteine residue at their active site. The cysteine proteases consist of three major groups: cathepsins, calpains, and caspases. The cathepsins are non–Ca²⁺-dependent lysosomal proteases that do not appear to play a role in the initiation of lethal cell injury (93–95). Calpain is a cytosolic Ca²⁺-activated neutral protease. The caspases are a family of intracellular cysteine proteases. The term “caspase” embodies two properties of these proteases in which “c” refers to “cysteine” and “aspase” refers to their specific ability to cleave substrates after an aspartate residue. Caspases play a crucial role in inflammation and apoptotic cell death.

Calpain

Calpain is a cytosolic neutral cysteine protease that has an absolute dependence on Ca²⁺ for its activation (96). There are two major ubiquitous or conventional isoforms of calpain, the low Ca²⁺-sensitive μ-calpain and the high Ca²⁺-sensitive μ-calpain (97,98). The isoenzymes have the same substrate specificity but differ in affinity for Ca²⁺. Procalpain exists in the cytoplasm as an inactive proenzyme and becomes active proteolytically at the cell membrane only after it has become autolyzed (99,100). The autolyzed calpain is released either into the cytoplasm, where it hydrolyses substrate proteins, or it remains associated with the cell membrane and degrades cytoskeletal proteins involved in the interaction between the cell cytoskeleton and the plasma membrane. Activity of the autolyzed calpain is subject to a final regulation by a specific endogenous inhibitor called calpastatin (99,100). Calpain plays a role in platelet activation and aggregation (101), cytoskeleton and cell membrane organization (102,103), regulation of cell growth, differentiation, and development (104–106), and pathologic states, including Alzheimer disease, aging, cataract, muscular dystrophy, sepsis, Wiskott–Aldrich syndrome, Chédiak–Higashi syndrome, inflammation, arthritis, and malaria (107). Calpain 10 is a recently discovered mitochondrial calpain that plays a role in calcium-induced mitochondrial dysfunction (108).

The Ca²⁺-dependent calpains have been shown to be mediators of hypoxic/ischemic injury to brain, liver, and heart (109–112). Calpain plays a role in hypoxic injury to rat renal proximal tubules (113–115). This role of calpain in proximal tubule injury has been confirmed in subsequent studies (116,117). The calpain inhibitors PD150606 and E-64 ameliorated the functional and histologic parameters in a rat model of ischemic AKI (118). Injection of a fragment of calpastatin, which inhibits calpain, protects against the functional and histologic changes in the kidney in a mouse model of AKI (119). In recent studies, it has been demonstrated that calpains increase epithelial cell mobility and play a critical role in tubule repair. In vitro, exposure of human tubular epithelial cells (HK-2 cells) to μ-calpain reduced adhesion of HK-2 cells to extracellular matrix and increased their mobility. In a murine model of ischemic AKI, injection of a fragment of calpastatin, which specifically blocked calpain activity, delayed tubule repair and increased the worsening of kidney function and histologic lesions after 24 and 48 hours of reperfusion.

Caspases

Caspases are Ca²⁺-independent cysteine proteases. There are 14 members of the caspase family, caspases 1 to 14. Caspase-14 has been characterized and found to be present in embryonic tissues but absent from adult tissues (120). Caspases share a predilection for cleavage of their substrates after an aspartate residue at P1 (121,122). The members of the caspase family can be divided into three subfamilies on the basis of substrate specificity and function (123). The peptide preferences and function within each group are remarkably similar (123). Members of group 1 (of which caspase-1 is the most important) prefer the tetrapeptide sequences ­Trp-Glu(OMe)-His-Asp(OMe) (WEHD) and YVAD =Ac-Tyr-Val-Ala-Asp (YVAD). This specificity is similar to the activation sequence of caspase-1, suggesting that caspase-1 may employ an autocatalytic mechanism of activation. Caspase-1 (previously known as interleukin-1 [IL-1] converting enzyme, or ICE) plays a major role in the activation of proinflammatory cytokines. Caspase-1 is remarkably specific for the precursors of IL-1 and IL-18 (interferon-γ-inducing factor), making a single initial cut in each procytokine that activates them and allows exit from the cytosol (124,125). Group III “initiator” caspase-8 and caspase-9 prefer the sequence (L/V)EXD. This recognition motif resembles activation sites within the “executioner” caspase proenzymes, implicating this group as upstream components in the proteolytic cascade that serve to amplify the death signal. These “initiator” caspases pronounce the death sentence. They are activated in response to signals indicating that the cell has been stressed or damaged or has received an order to die. They clip and activate another family of caspases, the “executioners.” The optimal peptide sequence motif for group II, or “executioner caspases” (of which caspase-3 is the most important), is DEXD (123,126,127). This optimal recognition motif is identical to proteins that are cleaved during cell death.

There are two major pathways of caspase-mediated apoptosis (128). In the mitochondrial or “intrinsic” pathway, stress-induced signals affect the balance between pro- and antiapoptotic Bcl-2 family proteins to cause cytochrome c release from mitochondria. Caspase-2 is a recently discovered caspase that is a crucial initiator of the mitochondrial apoptosis pathway (129). Activation and increased activity of caspase-2 is required for the permeabilization of mitochondria and release of cytochrome c (129). Cytochrome c binds to the cytosolic protein, apoptosis protease-activating factor-1 (APAF-1), which recruits and activates caspase-9. Active caspase-9 in turn recruits and activates the “executioners” procaspase-3 and procaspase-7. In the “extrinsic” pathway, the binding of a ligand to its death receptor recruits an adaptor protein that in turn recruits and activates procaspase-8. For example, Fas ligand (FasL) binds to its death receptor Fas that recruits an adaptor protein called Fas-associated death domain (FADD). FADD in turn recruits and activates procaspase-8.

The caspase pathways that are centrally important in cell death involve the “initiator” caspase-8 and caspase-9 and the “executioner” caspase-3 (130). The central role of these caspases is supported by caspase-8, caspase-9, and caspase-3 (−/−) mice that have strong phenotypes based on cell death defects, developmental defects, and usually fetal/perinatal mortality. The critical role of “initiator” caspases is illustrated in caspase-9 (−/−) mice that demonstrate the absence of downstream caspase-3 activation (131). Activation of caspase-1, caspase-8, caspase-9, and caspase-3 has been widely described in hypoxic renal epithelial cells and cerebral ischemia (28,132,133). Caspase-1 may also cause cell injury by activation of the proinflammatory cytokines IL-1 and IL-18 (125). To establish a direct pathogenic role of specific caspases in this well-established cascade, knockout mice have been used. Caspase-1 (−/−) mice are protected against cerebral ischemia (134). Caspase-3 (−/−) mice are protected against Fas-mediated fulminant hepatitis (135).

For many years it was not known how caspase-1 was activated. It has recently been discovered that procaspase-1 is activated in a complex called the inflammasome (136,137). The inflammasome is a protein scaffold that contains pyrin domain-containing protein (NALP) proteins, an adaptor protein apoptosis-associated speck-like protein containing a caspase-recruiting domain (CARD) (ASC), procaspase-1, and caspase-5. The interaction of the CARD of procaspase-1 is mediated by the CARD of ASC and the CARD present in the C-terminus of NALP-1. Active caspase-1 in the inflammasome is a regulator of the “unconventional” protein secretion of “leaderless” proteins like IL-33, IL-1α, and fibroblast growth factor (FGF)-2 (138). IL-33 is an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type-2 associated cytokines like IL-4, IL-5, and IL-13 that can lead to pathologic changes in mucosal organs (139). IL-1α is increased in the kidney in mice in endotoxemic AKI (140) and cisplatin-induced AKI (141).

As caspase-1 is activated in the inflammasome, we investigated the inflammasome in cisplatin-induced and ischemic AKI (142). To determine whether the NACHT, LRR and PYD domains (NLRP3) inflammasome plays an injurious role in cisplatin-induced AKI, we studied NLRP knockout NLRP3(−/−) mice. In cisplatin-induced AKI, the blood urea nitrogen (BUN), serum creatinine, ATN score, and tubular apoptosis score were not significantly decreased in NALP3(−/−) mice compared with wild-type mice. NLRP3(−/−) mice with ischemic AKI had significantly lower BUN, serum creatinine, and ATN and apoptosis scores than the wild-type controls. The difference in protection against cisplatin-induced AKI compared with ischemic AKI in NLRP3(−/−) mice was not explained by the differences in proinflammatory cytokines IL-1β, IL-6, chemokine (C-X-C motif) ligand 1, or tumor necrosis factor-α (TNF-α). Thus the NLRP3 inflammasome is a mediator of ischemic AKI but not cisplatin-induced AKI (142).

Caspases participate in two distinct signaling pathways, (a) activation of proinflammatory cytokines and (b) promotion of apoptotic cell death (121,127,143,144). While caspases play a crucial and extensively studied role in apoptosis, there is now considerable evidence that the caspase pathway may also be involved in necrotic cell death (145). Caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis (146). Caspase inhibition has been demonstrated to reduce ischemic and excitotoxic neuronal damage (134,147,148). Moreover, mice deficient in caspase-1 demonstrate reduced ischemic brain injury produced by occlusion of the middle cerebral artery (133,134,149). Inhibition of caspases also protects against necrotic cell death induced by the mitochondrial inhibitor antimycin A in PC12 cells, Hep G2 cells, and renal tubules in culture (150,151). Caspases are also involved in hypoxic and reperfusion injury in cultured endothelial cells (152). Rat kidneys subjected to ischemia demonstrate an increase in both caspase-1 and caspase-3 mRNA and protein expression (25).

An assay for caspases in freshly isolated rat proximal tubules using the fluorescent substrate Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl coumarin (Ac-YVAD-AMC) was developed (153). Freshly isolated proximal tubules were preincubated with the caspase inhibitor Z-Asp-2, 6-dichlorobenzoyloxymethylketone (Z-D-DCB) for 10 minutes before being exposed to hypoxia. Tubular caspase activity was increased after 15-minute hypoxia in association with increased cell membrane damage as assessed by LDH release. Z-D-DCB attenuated the increase in caspase activity during 15-minute hypoxia and markedly decreased LDH release in a dose-dependent fashion. The fluorescent substrate Ac-DEVD-AMC, which is cleaved by caspase-3, was also used. Caspase activity was measured in normoxic and hypoxic tubules with both caspase-1 and caspase-3 substrates. Significant fluorescent activity was detected with Ac-YVAD-AMC (caspase-1 substrate) compared with Ac-DEVD-AMC (caspase-3 substrate), suggesting that caspase-1 is predominantly involved in hypoxic injury. In another study, the deleterious effect of caspase-1 on proximal tubules in vitro in the absence of inflammatory cells and vascular effects was demonstrated (154).

Caspase-1-Mediated Production of Interleukin-18

To establish a pathogenic role of caspase-1 in cell injury, caspase-1-deficient (−/−) mice have been used. These caspase-1 (−/−) mice have a defect in the production of mature IL-1β and IL-18 and are protected against lethal endotoxemia (149,155). The fact that IL-1β (−/−) mice are not protected against endotoxemia (156) suggests a potential role of IL-18 in the lethal outcome during sepsis. Moreover, in ischemic AKI, IL-1 receptor knockout mice or mice treated with IL-1 receptor antagonist (IL-1Ra) are not protected against ischemic AKI (157). Taken together, therefore, these previous studies suggest that IL-18 may be a potential mediator of ischemic AKI.

Since caspase-1 activates IL-18, lack of mature IL-18 might protect these caspase-1 (−/−) mice from AKI. Thus it was determined whether mice deficient in the proinflammatory caspase-1, which cleaves precursors of IL-1β and IL-18, were protected against ischemic AKI (158). Caspase-1 (−/−) mice developed less ischemic AKI as judged by renal function and renal histology. These animals had significantly reduced BUN and serum creatinine levels and a lower morphologic tubular necrosis score than did wild-type mice with ischemic AKI. In wild-type animals with ischemic AKI, kidney IL-18 levels more than double and there is a conversion of the IL-18 precursor to the mature form. This conversion was not observed in caspase-1 (−/−) AKI mice or sham-operated controls. Wild-type mice were then injected with IL-18-neutralizing antiserum before the ischemic insult, and there was a similar degree of protection from AKI as seen in caspase-1 (−/−) mice. In addition, there was a fivefold increase in myeloperoxidase (MPO) activity, as an index of leukocyte infiltration, in control mice with AKI but no such increase in caspase-1 (−/−) or IL-18 antiserum-treated mice. Caspase-1 (−/−) mice also show decreased neutrophil infiltration, suggesting that the deleterious role of IL-18 in ischemic AKI may be due to increased neutrophil infiltration.

IL-18 function is neutralized in IL-18-binding protein transgenic (IL-18BP Tg) mice. It was determined whether IL-18BP Tg mice are protected against ischemic AKI (159). IL-18BP Tg mice were functionally and histologically protected against ischemic AKI, as determined by the BUN, serum creatinine, and ATN score. The number of macrophages was significantly reduced in IL-18BP Tg compared with wild-type kidneys. Multiple chemokines/cytokines were measured using flow cytometry-based assays. Only CXCL1 (also known as KC or IL-8) was significantly increased in AKI versus sham kidneys and significantly reduced in IL-18BP Tg AKI versus wild-type AKI kidneys. This study demonstrates that protection against ischemic AKI in IL-18BP Tg mice is associated with less macrophage infiltration and less production of CXCL1 in the kidney.

It was determined whether macrophages are a source of injurious IL-18 in ischemic AKI in mice (160). On immunofluorescence staining of the outer strip of the outer medulla, the number of macrophages staining for IL-18 was significantly increased in AKI and significantly decreased by macrophage depletion using tail vein injection of liposomal-encapsulated clodronate (LEC). Adoptive transfer of 264.7 cells, a mouse macrophage line that constitutively expresses IL-18 mRNA, or mouse peritoneal macrophages deficient in IL-18 reversed the functional protection against AKI in LEC-treated mice. In summary, adoptive transfer of RAW cells, that constitutively express IL-18, reverses the functional protection in macrophage-depleted wild-type mice with AKI. In addition, adoptive transfer of peritoneal macrophages in which IL-18 function was inhibited also reverses the functional protection in macrophage-depleted mice, suggesting that IL-18 from adoptive transfer of macrophages is not sufficient to cause ischemic AKI. Possible sources of injurious IL-18 in AKI include the proximal tubule and lymphocytes. In this regard, freshly isolated proximal tubules from mice release IL-18 into the medium when exposed to hypoxia, and proximal tubules from caspase-1-deficient mice are protected against hypoxic injury (154).

Caspase-1-deficient (−/−) mice are protected against sepsis-induced hypotension and mortality. The role of caspase-1 and its associated cytokines was investigated in a nonhypotensive model of endotoxemic AKI. In mice with endotoxemic AKI, the GFR was significantly higher in caspase-1 (−/−) versus wild-type mice at 16 and 36 hours. IL-1β and IL-18 protein were significantly increased in the kidneys of mice with endotoxemic AKI versus vehicle-treated mice. However, inhibition of IL-1β with IL-1Ra, or inhibition of IL-18 with IL-18-neutralizing antiserum-treated or combination therapy with IL-1Ra plus IL-18-neutralizing antiserum did not improve the GFR in mice with endotoxemic AKI, suggesting that neither IL-1β nor IL-18 is the mediator on endotoxemic AKI (140).

The role of IL-18 was investigated in cisplatin-induced AKI. In IL-18Rα knockout vs. wild-type mice with cisplatin-induced AKI, there was worse kidney function, tubular damage, increased accumulation of CD4⁺ and CD8⁺ T cells, macrophages, and neutrophils, upregulation of early kidney injury biomarkers (serum TNF, urinary IL-18, and KIM-1 levels), and increased expression of proinflammatory molecules downstream of IL-18 (161). Anti-IL-18Rα and anti-IL-18Rβ antibody treatment increased cisplatin nephrotoxicity in wild-type mice. Thus, signaling through the IL-18 receptor α attenuates inflammation in cisplatin-induced AKI (161). Cisplatin-induced AKI is associated with an increase in cytokines including IL-18 in the kidney (141). However, IL-18 antiserum or transgenic mice that overexpress IL-18 binding protein, a natural inhibitor of IL-18, were not protected against cisplatin-induced AKI (141). Thus, unlike ischemic AKI where IL-18 is a mediator of injury, IL-18 is not a mediator of cisplatin-induced AKI.

Interaction between Calpain and Caspases in Hypoxic/Ischemic Proximal Tubular Injury

Studies suggest that both calpain and caspases play a role in hypoxia-induced cell membrane damage in proximal tubules (25,113,115,150,153). A prelethal increase in cytosolic Ca²⁺ is a cardinal feature of the hypoxic proximal tubule model (67). How are the non–Ca²⁺-dependent caspases activated during hypoxia? There are two possibilities. Caspase activation may be downstream of Ca²⁺-mediated activation of calpain, or caspases may be activated in a separate pathway independent of Ca²⁺. Since an interaction between caspases and calpains during cell injury has been suggested (149), the effect of the specific calpain inhibitor (2)-3-(4-iodophenyl)-2-mercapto-2-propenoic acid (PD150606) on the hypoxia-induced increase in caspase activity in proximal tubules was studied (153). PD150606 inhibited calpain activity and protects against hypoxic injury in rat proximal tubules (114). PD150606 also attenuated the hypoxia-induced increase in caspase activity. However, PD150606 did not inhibit the activity of purified caspase-1 in vitro, suggesting that calpain may be upstream of caspases during hypoxic proximal tubular injury. Next, the effect of caspase inhibition on calpain activity was determined (153). The specific caspase inhibitor Z-D-DCB attenuated the hypoxia-induced increase in calpain activity in proximal tubules. However, Z-D-DCB did not inhibit the activity of purified calpain in vitro.

In summary, these data suggest that both caspase-mediated activation of calpain and calpain-mediated activation of caspases occur during hypoxic proximal tubular injury. These data are supported by other studies that demonstrate simultaneous activation of both calpain and caspases during cell death (162). Thus, it is possible that during hypoxic proximal tubule injury, there are different proteolytic pathways involving different caspases and calpains.

Figure 10–2 Calpains and caspases in proximal tubular necrosis. Hypoxic/ischemic proximal tubular necrosis results in activation of cysteine protease pathways involving calpains and both caspase-1 and caspase-3 (164). There is increased activity of calpain (113–115) and caspase-1 (153) in hypoxic proximal tubular injury. During ischemic AKI, there is early calpain activation associated with downregulation of calpastatin protein, decreased calpastatin activity, and activation of caspase-3 (163). Also, impaired IL-18 processing protects caspase-1-deficient mice from ischemic AKI (158).

The interaction between calpain and caspases during ischemic AKI in vivo was investigated (163). An increase in the activity of calpain, as determined by (a) the appearance of calpain-mediated spectrin breakdown products and (b) the conversion of procalpain to active calpain, was demonstrated. Since intracellular calpain activity is regulated by its endogenous inhibitor, calpastatin, the effect of ischemia on calpastatin was determined. On immunoblot of renal cortex, there was a decrease of a low-molecular-weight form of calpastatin during ischemic AKI compared to sham-operated controls. Calpastatin activity was also significantly decreased compared to sham-operated rats, indicating that the decreased protein expression had functional significance. In rats treated with the caspase inhibitor Z-D-DCB, the decrease in both calpastatin activity and protein expression was normalized, suggesting that caspases may be proteolyzing calpastatin. Caspase-3 activity increased significantly after I/R compared to sham-operated rats and was attenuated in ischemic kidneys from rats treated with the caspase inhibitor. In summary, during ischemic AKI there is (a) calpain activation associated with downregulation of calpastatin protein and decreased calpastatin activity and (b) activation of caspase-3. In addition, in vivo caspase inhibition reverses the decrease in calpastatin activity. The proposed relationship between calpain and caspases in hypoxic/ischemic injury is shown in Figure 10-2 (153,158,164).

ROLE OF NITRIC OXIDE IN HYPOXIA/ISCHEMIA-INDUCED PROXIMAL TUBULE INJURY

Nitric oxide (NO) is a messenger molecule mediating diverse functions, including vasodilatation, neurotransmission, and antimicrobial and antitumor activities (165). A variety of cells produce NO via oxidation of L-arginine by the enzyme nitric oxide synthase (NOS) (166). Thus far, four distinct NOS isoforms have been isolated, purified, and cloned: neuronal, endothelial, macrophage, and vascular smooth muscle cell (VSMC)/hepatocyte (167,168). Identification of the specific isoform of NOS is important because the four isoforms vary in subcellular location, amino acid sequence, regulation, and hence functional roles. Neuronal and endothelial NOS (eNOS) are continuously present and thus are termed constitutive NOS (cNOS) (168). NO is produced by these enzymes when Ca²⁺/calmodulin interaction permits electron transfer from nicotinamide adenine dinucleotide phosphate (NADPH) via flavin groups within the enzyme to a heme-containing active site (169). This activation is very short lived. In contrast, VSMC/hepatocyte and macrophage isoforms are only expressed when the cells have been induced by certain cytokines, microbes, and microbial products and are therefore called inducible NOS (iNOS) (170). iNOS expression results in sustained production of NO. Unlike cNOS, iNOS activity is believed to be insensitive to changes in intracellular Ca²⁺, since calmodulin is tightly bound to the molecule. Once synthesized, iNOS remains tonically activated, producing NO continuously for the life of the enzyme (171).

Both cNOS and iNOS isoforms have been identified in the kidney, specifically in macula densa cells (cNOS), inner medullary collecting ducts (cNOS and iNOS), and proximal tubules (cNOS and iNOS) (168,172). In the kidney, physiologic amounts of NO play an important role in hemodynamic regulation and salt and water excretion (173).

It has been demonstrated that NOS activity is increased during hypoxia in freshly isolated rat proximal tubules. In this study, membrane damage, as assessed by LDH release into the medium, was prevented by both a nonselective NOS inhibitor (L-NAME) and a NO scavenger (hemoglobin) (174). In a separate study, hypoxia stimulated prompt and sustained NO release in the proximal tubule suspension as assessed by a NO-selective sensing electrode (175). NO concentration remained unmeasurable during normoxia. L-NAME completely inhibited hypoxia-induced NO release in parallel with marked cytoprotection. Further studies in freshly isolated proximal tubules from knockout mice have also been revealing about the role of NO in ­hypoxic/ischemic tubular injury. Hypoxia-induced proximal tubule damage, as assessed by LDH release, was no different between wild-type mice in which eNOS and nNOS were “knocked out.” However, proximal tubules from the iNOS knockout mice demonstrated resistance to the same degree of hypoxia (176).

In vivo, targeting of iNOS with oligodeoxynucleotides protects the rat kidney against ischemic AKI (177). This study provided direct evidence for the cytotoxic effects of NO produced via iNOS in the course of ischemic AKI. Augmented expression of iNOS and the prevalence of nitrotyrosine residues in kidneys have been demonstrated in osteopontin-deficient mice versus wild-type counterparts (178). Animals with the disrupted osteopontin gene exhibited ischemia-induced renal dysfunction and structural damage, which was twice as pronounced as that observed in mice with the intact osteopontin response to stress, also suggesting a role of iNOS in ischemic AKI. iNOS-deficient mice also have less renal failure and better survival than the wild-type mice after renal artery clamping (179). An induction of heat shock protein (HSP) was also observed in the iNOS knockout mice as a potential contributor to the protection.

In a renal artery clamp model in mice in which alpha-melanocyte-stimulating hormone (ά-MSH) was shown to block the induction of iNOS, there was decreased neutrophil infiltration and functional and histologic protection (180). A subsequent study examined the relative importance of ά-MSH on the neutrophil pathway by examining the effects of ά-MSH in ICAM-1 knockout mice and the neutrophil-independent isolated perfused kidneys (181,182). In this study, it was found that ά-MSH decreases renal injury when neutrophil effects are minimal or absent, indicating that ά-MSH inhibits neutrophil-independent pathways of renal injury.

Interestingly, however, L-NAME administration to the rat kidney clamp model actually worsens ischemic and endotoxemic AKI (177). This result was interpreted as an overriding blocking effect of eNOS activity with the nonspecific effects of L-NAME (26). This would worsen the renal vasoconstriction and resultant injury, thus obscuring any salutary effect at the level of the proximal tubule (183). Thus, opposing abnormalities in NO production within the endothelial and tubular compartments of the kidney may contribute to renal injury (26) (Fig. 10-3). Reduced eNOS-derived NO production causes vasoconstriction and worsens ischemia; increased iNOS-derived NO production by tubular cells adds to the injurious effects of ischemia on these cells. Therapeutic interventions to modulate NO production in ischemic AKI may require selective modulation of different NOS isoforms in the tubular and vascular compartments of the kidney (184).

MATRIX METALLOPROTEINASES

Matrix metalloproteinases (MMP) play a crucial role in remodeling of the extracellular matrix, which is an important physiologic feature of normal growth and development. In the kidney, interstitial sclerosis and glomerulosclerosis have been associated with an imbalance of extracellular matrix synthesis and degradation (185). Alterations in renal tubular basement membrane matrix proteins, laminin and fibronectin, occur after renal I/R injury (186).

Meprin A is a zinc-dependent metalloendopeptidase that is present in the brush border membrane of renal proximal tubular epithelial cells. The redistribution of this metalloendopeptidase to the basolateral membrane domain during AKI results in degradation of the extracellular matrix and damage to adjacent peritubular structures. The effect of meprin A, the major matrix-degrading metalloproteinase in rat kidney, on the ­laminin–nidogen complex was examined. Following ischemic injury, meprin A undergoes redistribution and/or adherence to the tubular basement membrane. Nidogen-1 (entactin), which acts as a bridge between the extracellular matrix molecules, laminin-1 and type IV collagen breakdown products, is produced as the result of partial degradation of tubular basement membrane by meprin A following renal tubular I/R injury (187).

Figure 10–3 Proposed imbalance of NO production in ischemic/septic AKI. In ischemic AKI, increased NO derived from iNOS is damaging to proximal tubules (176,177,179). In ischemic AKI, renal endothelial damage results in decreased NO derived from eNOS (26). In endotoxemic AKI, increased iNOS activity decreases eNOS activity possibly via NO autoinhibition (387). The nonselective NOS inhibitor, L-NAME, worsens ischemic and endotoxemic AKI due to an overriding blocking effect on eNOS.

Inbred strains of mice with normal and low meprin A activity have been studied (188). The strains of mice with normal meprin A developed more severe renal functional and structural injury following renal ischemia or the injection of hypertonic glycerol compared with the two low-meprin A strains. These findings suggest that meprin A plays a role in the pathophysiology of AKI following ischemic and nephrotoxic AKI (188).

The disruption of cadherin/catenin complexes in AKI may be associated with the transtubular back-leak of glomerular filtrate. In endothelial cells isolated from ischemic kidneys, the proteolytic activity of proMMP-2, proMMP-9, and MMP-9 was increased. Occludin, an in vivo MMP-9 substrate, was partly degraded in the endothelial fractions during ischemia, suggesting that the upregulation of MMP-9 was functional. These data suggest that AKI leads to the degradation of the vascular basement membrane and to increased permeability related to the increase in MMP-9 (189). In renal cells, in vitro cleavage of cadherins in normal rat kidney (NRK) cells requires active membrane-type (MT)1-MMP (MT1-MMP), also known as MMP-14 (190). In contrast to the potential injurious role of some MMPs, MMP9 protects the S3 segment of the proximal tubule and the intercalated cells of the collecting duct from apoptosis in AKI, most likely by releasing soluble stem cell factor, an MMP9 substrate (191).

HEAT SHOCK PROTEINS

HSPs protect cells from environmental stress damage by binding to partially denatured proteins and dissociating protein aggregates to regulate the correct folding and to cooperate in transporting newly synthesized polypeptides to the target organelles (192). Stresses that trigger the heat shock response include hyperthermia, hypothermia, generation of oxygen radicals, hypoxia/ischemia, and toxins (193).

HSPs are identified by their molecular weight. The most important families include proteins of 90, 70, 60, and 27 kDa (193). The HSP70 family includes proteins that are both constitutively expressed and induced by stress. They are the most highly induced proteins by stress and function as chaperones binding to unfolded or misfolded proteins.

Renal ischemia results in both a profound fall in cellular ATP and a rapid induction of HSP70 (194,195). It has been demonstrated that a 50% reduction in cellular ATP in the renal cortex during ischemia must occur before the stress response is detectable. Reduction in ATP below 25% control levels produces a more vigorous response. Reperfusion is not required for initiation of a heat shock response in the kidney (196).

In vitro studies have demonstrated that HSP induction protects cultured renal epithelial cells from injury. It has been determined that prior heat stress protects cultured opossum kidney (OK) cells from injury mediated by ATP depletion (197). Also HSP70 overexpression is sufficient to protect LLC-PK1 proximal tubular cells from hyperthermia but is not sufficient for protection from hypoxia (198).

The effect of HSP induction by hyperthermia on ischemic AKI has been studied. One study found that prior heat shock protected kidneys against warm ischemia (199). In another study, prior induction of HSP by hyperthermia was not protective against the functional and morphologic parameters of ischemic AKI in ischemia reflow in intact rats or medullary hypoxic injury (200). These variable results may be explained by the complexity of the intact animal compared with cultured cells; the degree, duration, and timing of the hyperthermic stimulus; and the differential response of mature and immature kidneys (201,202).

The mechanism of HSP protection against ischemic AKI is evolving. It has been demonstrated that HSPs participate in the postischemic restructuring of the cytoskeleton of proximal tubules (203). HSP72 complexes with aggregated cellular proteins in an ATP-dependent manner, suggesting that enhancing HSP72 function after ischemic renal injury assists refolding and stabilization of Na⁺/K⁺ ATPase or aggregated elements of the cytoskeleton, allowing reassembly into a more organized state (204). Another study suggested that there are specific interactions between HSP25 and actin during the early postischemic reorganization of the cytoskeleton (205).

Another potential mechanism of HSP protection against proximal tubular injury is the inhibition of apoptosis. OK proximal tubule cells exposed to ATP depletion develop apoptosis, and prior heat stress reduced the number of apoptotic cells and improved cell survival compared with controls (206).

APOPTOSIS

Apoptosis is a physiologic form of cell death that occurs in a programmed pattern and can be triggered by external stimuli (207). The triggers of apoptosis include (a) cell injury, for example, ischemia, hypoxia, oxidant injury, NO, and cisplatinum; (b) loss of survival factors, for example, deficiency of renal growth factors, impaired cell-to-cell or cell-to-matrix adhesion; and (c) receptor-mediated apoptosis, for example, Fas (CD 95) and transforming growth factor-β (TGF-β) (208).

Apoptosis has been demonstrated in cultured proximal and distal tubules exposed to hypoxia and chemical ATP depletion (132,206,207–211). A feature of these in vitro studies is that severe or prolonged ATP depletion leads to necrosis, while milder and shorter ATP depletion leads to apoptotic cell death (132). Apoptosis has been demonstrated in distal and proximal tubules during both the early phase and the recovery phase of ischemic AKI in rats and mice (28–30,212–222). The role that apoptosis of proximal and distal tubular cells plays in the loss of renal function and the recovery phase of ischemic AKI, as well as the relationship between apoptosis and necrosis in ischemic AKI, still needs to be elucidated (208,223,224).

Cisplatin is a commonly used chemotherapeutic agent that causes apoptosis or necrosis of renal tubular epithelial cells in vitro. After cisplatin injection in mice, renal apoptosis peaks on day 2, which precedes the peak in serum creatinine, ATN scores, and neutrophil counts, which peak on day 3. Renal dysfunction, apoptosis, ATN scores, and neutrophil infiltration were all reduced in caspase-1 (−/−) mice treated with cisplatin. Active caspase-3 was also reduced in caspase-1 (−/−) mice (225). This study confirms the injurious role of caspases and apoptosis in cisplatin-induced AKI.

Erythropoietin (EPO) is upregulated by hypoxia. EPO receptors are expressed in many tissues, including renal tubules. Multiple animal studies have shown that EPO is protective against AKI, and the protective effect may be related to inhibition of caspases and apoptosis (Table 10-4). In a cisplatin-induced AKI model in the rat, functional recovery was significantly improved in animals that received EPO compared with controls, and the enhanced recovery was secondary to increased regeneration of tubules, as shown by increased uptake of radioactive thymidine (226). In another study, rats that were pretreated with EPO before induction of ischemic AKI had a lower serum creatinine and decreased apoptosis compared with controls (227). In both in vivo and in vitro models of tubular injury, EPO provided protection from I/R injury by inhibiting apoptosis and increasing tubular cell regeneration (228). EPO was shown to be protective against interstitial fibrosis and inflammation in a rat model of cyclosporine nephrotoxicity (229). EPO prevents the decrease in the GFR in a rat model of contrast nephropathy (230). Kolyada et al. demonstrated that EPO decreased iohexol-induced activation of caspase-3 and caspase-8 and subsequent apoptosis in renal tubular epithelial cells (231). EPO and/or α-MSH treatment significantly prevented urinary-concentrating defects and downregulation of renal aquaporins (AQP) and sodium transporters in ischemic AKI in rats (232). EPO (300 units/kg) reduced tubular injury, prevented caspase-3, caspase-8, and caspase-9 activation, and reduced apoptotic cell death in vivo in mice (233). In human proximal tubule epithelial cells in vitro, EPO reduced DNA fragmentation, prevented caspase-3 activation, and attenuated cell death in response to oxidative stress (233). In a rat model of hemorrhagic shock, administration of EPO before resuscitation reduced the increase in the activities of caspase-3, caspase-8, and caspase-9, and prevented renal dysfunction and liver injury (234). In a model of endotoxemia-induced AKI in mice, EPO significantly decreased renal superoxide dismutase and attenuated the renal dysfunction as assessed by insulin-GFR (235).

The β-common receptor (βcR) plays an important role in the nonhematopoietic tissue-protective effects of EPO. In a mouse model of lipopolysaccharide (LPS)-induced AKI, the AKI was attenuated by EPO given 1 hour after LPS in wild-type but not in βcR knockout mice (236). In a cecal ligation model of AKI in older mice, AKI was attenuated by EPO treatment in wild-type mice but not in βcR knockout mice. Thus, activation of the βcR by EPO is essential for the protection against AKI in either endotoxemic young mice or older mice with polymicrobial sepsis, and for the activation of well-known signaling pathways by EPO (236).

Elimination of the mitochondrial fusion protein mitofusin 2 (Mfn2) sensitizes proximal tubular cells to apoptosis in vitro (237). The role of proximal tubular Mfn2 in ischemic AKI in vivo was investigated in ischemic AKI. Mice with a conditional knock out of proximal tubular Mfn2 (cKO-PT-Mfn2) had much less survival than wild-type mice with ischemic AKI. Increased cell proliferation, but no significant differences in ATN score, apoptosis, or necrosis were detected between genotypes. In ATP depletion in vitro, Mfn2 deficiency significantly increased proximal tubular proliferation and persistently activated extracellular signal-regulated kinase 1/2 (ERK1/2). Ischemic AKI reduced the Mfn2-Retrovirus-associated DNA sequences (RAS) interaction and increased both RAS and p-ERK1/2 activity in the renal cortical homogenates of cKO-PT-Mfn2 mice. These results suggest that, in contrast to its proapoptotic effects in vitro, selective PT Mfn2 deficiency accelerates recovery of renal function and enhances animal survival after ischemic AKI in vivo, in part by increasing Ras-ERK-mediated cell proliferation.

Conformational change in transfer RNA is an early indicator of acute cellular damage before the detection of apoptosis (238) Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, it was demonstrated that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased early in ischemic AKI before other known tissue injury biomarkers. It was concluded that tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage (238).

ALTERED GENE EXPRESSION

Immediate early genes and protooncogenes are induced during the early reperfusion period after renal ischemia (239). There is c-fos and c-jun activation as well as an increase in DNA synthesis (240). There is accumulation of early growth response factor-1 (Egr-1) and c-fos mRNAs in the mouse kidney after occlusion of the renal artery and reperfusion (241,242). Transient expression of the genes c-fos and Egr-1 may code for DNA-binding transcription factors and initiate the transcription of other genes necessary for cell division (243). JE and KC, growth factor-responsive genes with cytokine-like properties that play a role in inflammation, are also expressed during early renal ischemia (244). These genes may code for proteins with chemotactic effects that can attract monocytes and neutrophils into areas of injury (242). Studies demonstrate that c-fos and c-jun are expressed following renal ischemia as a typical immediate early gene response, but they are expressed in cells that do not enter the cell cycle (245,246). The failure of the cells to enter the cell cycle may depend on the co-expression of other genes.

The pathways that lead to the early gene response are interesting. At least two quite different pathways lead to the activation of c-jun (247–249). Growth factors activate c-jun via the mitogen-activated protein kinases (MAPKs), which include extracellular regulated kinases (ERKs) 1 and 2. This pathway is proliferative in nature. In contrast, the stress-activated protein kinase (SAPK) pathway is separate from the MAPK pathway. These kinases include c-Jun N-terminal kinase (JNK) 1 and 2. Activation and the effect on cell fate of the SAPK pathway are very different from the MAPK pathway. The SAPK pathway is essentially antiproliferative and can lead to either cell survival or cell death. During renal ischemia, SAPKs are activated, and inhibition of SAPKs after ischemia protects against renal failure (250,251). Thus, it is possible that manipulation of this pathway could lead to therapies that may ameliorate AKI. Also, exploration of the early gene response in renal ischemia using DNA microarrays and other genome-scale technologies should extend our knowledge of gene function and molecular biology (252).

Microarray analysis of kidney has given clues to the pathogenesis of AKI (252,253). There was an increase in genes involved in cell structure, extracellular matrix, intracellular calcium binding, and cell division/differentiation in kidneys from mice with AKI (254). In another study in mice with AKI, transcription factors, growth factors, signal transduction molecules, and apoptotic factors demonstrated consistent patterns of altered gene expression in the first 24 hours of postischemic reperfusion (255). In rats with AKI, microarray analysis demonstrated that nine genes were upregulated in the early phase (ADAM2, HO-1, UCP-2, and thymosin β4) and established phase (clusterin, vanin1, fibronectin, heat-responsive protein 12, and FK506) (256). Nine genes were downregulated in the early phase (glutamine synthetase, cytochrome p450 IId6, and cyp 2d9) and established phase (cyp 4a14, Xist gene, PPARγ, α-albumin, uromodulin, and ADH B2) Laser capture microdissection of immunofluorescently defined cells (IF-LCM) can isolate pure populations of targeted cells from the kidney for microarray analysis (257). This technique has been used to label and isolate thick ascending limb cells in the kidney for mRNA analysis (257).

Two genes that have been discovered to be activated in the kidney in AKI are kidney injury molecule-1 (KIM-1) in the proximal tubule (258) and neutrophil gelatinase-associated lipocalin (NGAL) in the distal tubules (259–261). KIM-1 is a phosphatidylserine receptor that recognizes and directs apoptotic cells to lysosomes in proximal tubular cells. KIM-1 also mediates phagocytosis of necrotic cells and oxidized lipoproteins by renal proximal tubular cells and increases clearance of the apoptotic debris from the tubular lumen (262). KIM-1 may play an important role in limiting the immune response to injury (262). In early ischemic AKI, KIM-1 expression is antiinflammatory by causing phagocytosis in tubular cells (263). KIM-1-mediated epithelial cell phagocytosis of apoptotic cells protects the kidney after acute injury by downregulating innate immunity and inflammation (263). NGAL is an iron-transporting protein. Purified recombinant NGAL inhibits apoptosis, enhances proliferation, and results in significant functional and pathological protection against AKI in murine models (261). NGAL forms a complex with iron-binding siderophores and stops inappropriately liganded iron from producing damaging oxygen radicals (264).

HYPOXIA-INDUCIBLE FACTOR-1α

Hypoxia-inducible factor-1 (HIF-1)α is an important molecule for the adaptation of cells to low oxygen or hypoxia. Systemic hypoxia, anemia, renal ischemia, or cobalt chloride results in an increase in HIF-1α in renal tubules (265). HIF-1α activation with carbon monoxide protects against ischemic (266) and cisplatin-induced (267) AKI. HIF-1α heterozygous deficient mice have worse AKI compared with control mice (268). Treatment of mice with l-mimosine and dimethyloxalylglycine, agents that activate HIF-1α by inhibiting HIF hydroxylases, protects against ischemic AKI in mice (268). Pharmacologic agents that induce HIF-1α may in the future be a potential therapy for AKI.

CELL CYCLE

Most normal tubular cells are quiescent at the G0 phase of the cell cycle. In AKI, there is cell proliferation in the damaged renal tubules (269). Death or loss of tubular cells may result in the neighboring cells stretching to cover the denuded area. These neighboring cells become dedifferentiated, and activate the cell cycle, driven by cyclins and cyclin-dependent kinases (CDKs) (262). The newly generated cells can develop into polarized, functional tubular cells for kidney repair (262). Cell cycle inhibitors like p21 are also induced during AKI resulting in G1 phase cell cycle arrest (269–272). P53 is another major cell cycle inhibitor that is rapidly induced in AKI is p53 (271,272). Transient cell cycle activation followed by cell cycle arrest may contribute to the development of fibrosis and loss of kidney function after AKI (262). Some tubular cells may become arrested at the G2/M phase, acquire a senescence-like phenotype and produce factors that promote fibrosis. The G1 cell cycle arrest factors, insulin-like growth factor-binding protein-7 (IGFBP7) and tissue inhibitor of metalloproteinase-2 (TIMP-2), in the urine are biomarkers of AKI (see later section on biomarkers of AKI).

The CDK inhibitor p21 was investigated in ischemic AKI and ischemic preconditioning (273). Ischemic AKI and renal histology was worse in the p21 knockout than in wild-type mice. Ischemic preconditioning attenuated I/R injury in wild-type but not p21-knockout mice. Ischemic preconditioning increased renal p21 expression and the number of cells in the G1 phase of the cell cycle before ischemic AKI demonstrating that renal p21 is essential for the beneficial effects of renal ischemic preconditioning. Transient cell cycle arrest induced by ischemic preconditioning by a p21-dependent pathway is important for subsequent tubular cell proliferation after I/R (273).

MITOCHONDRIA

Mitochondrial dynamics are markedly altered in ischemic and nephrotoxic AKI (269). Mitochondrial fragmentation arises before overt renal tubular injury or cell death (274). There is rapid fragmentation of mitochondria by a dynamic process termed fission regulated by proteins such as dynamin-related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1). Mitofusin 1 (Mfn1) and Mfn2 play a role in mitochondrial fusion. Fragmented mitochondria are a less efficient source of ATP and can undergo the mitochondrial permeability transition which results in influx of water and mitochondrial swelling, cell death through the release of calcium, cytochrome c, proapoptotic proteins, and reactive oxygen species (ROS) (269). Mitophagy results in recycling of damaged mitochondria. AKI is associated with an excess of mitochondrial fission compared with fusion. Pharmacologic inhibition of DRP1 improves mitochondrial morphology and protects against ischemic AKI and improved mitochondrial morphology (275). There is increased mitophagy in AKI to repair or clear fragmented mitochondria. In this regard, the autophagy molecules sestrin-2 and BNIP-3 are upregulated as seen on immunohistochemistry and immunoblot analysis in the ischemic AKI suggesting that autophagy is induced in renal tubules by at least two independent pathways involving p53-sestrin-2 and HIF-1α-BNIP3 (276).

In cisplatin-induced AKI, both oxidative stress and mitochondrial damage are associated with reduced levels of renal sirtuin 3 (SIRT3) (277). Treatment with the AMP-activated protein kinase (AMPK) agonist AICAR or the antioxidant agent acetyl-L-carnitine (ALCAR) restored SIRT3 expression and activity, improved renal function, and decreased tubular injury in wild-type mice but had no effect in Sirt3(−/−) mice (277). Sirt3-deficient mice had worse AKI. In cultured human tubular cells, cisplatin reduced SIRT3, resulting in mitochondrial fragmentation, while restoration of SIRT3 with 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and ALCAR improved cisplatin-induced mitochondrial dysfunction. This study suggests that SIRT3 improves mitochondrial dynamics in AKI (277).

TUBULAR OBSTRUCTION IN RENAL CELL INJURY

Increased excretion of tubular epithelial casts is a hallmark of recovery from AKI (201). The presence of tubular casts on renal biopsy as well as urinary casts has provided morphologic support for a role of tubular obstruction due to intraluminal cast formation in the pathogenesis of ischemic AKI (278). Finn and Gottschalk using micropuncture techniques during saline loading demonstrated clear evidence of increased tubular pressures in postischemic compared with normal kidneys (279). Renal vasodilation to restore renal blood flow also demonstrated increased tubular pressures in ischemic AKI in the rat. Tanner and Steinhausen (280) found that perfusing the proximal tubule with artificial tubular fluid at a rate that did not increase tubule pressure in normal animals increased tubule pressures in animals after a renal ischemic insult. Moreover, venting those obstructed tubules led to improved nephron filtration rates. Burke et al. also demonstrated that prevention of ischemic AKI in dogs with mannitol led to a decrease in intratubular pressures, suggesting that the induced-solute diuresis led to relief of cast-mediated tubular obstruction (281).

While it is clear that brush border membranes, necrotic cells, viable cells, and perhaps apoptotic tubular epithelial cells enter tubular fluid after an acute renal ischemic insult, the actual process and predominant location of the cast formation is, however, less clear. In AKI, distal tubules are obstructed by casts formed by tubular debris, cells, and Tamm–Horsfall protein (THP) (278). Since there are arginine–glycine–aspartic acid (RGD) adhesive sequences in human THP, there may be direct integrin-mediated binding of tubular cells to THP. Alternatively, polymerization of THP may result in entrapment of the cells in its gel. Adhesion of LLC-PK(1) cells to THP-coated wells was directly measured, and THP concentrate was dissolved in solutions of high electrolyte concentration that mimic urine from AKI and collecting ducts (282). LLC-PK(1) cells did not directly adhere to THP, a finding against integrin-mediated binding as a mechanism for in vivo tubular cell/THP cast formation. The high electrolyte concentration of AKI solutions was associated with THP gel formation. Thus, with renal ischemia and proximal tubule cell shedding, AKI and collecting duct fluid composition enhance THP gel formation and thus favor tubular cast formation and obstruction.

Integrins also play a role in cast formation. They recognize the most common universal tripeptide sequence, RGD, which is present in a variety of matrix proteins (283). These integrins can mediate cell-to-cell adhesion via an RGD-inhibitable mechanism (284). Experimental results support a role for adhesion molecules in the formation of casts. It has been shown that a translocation of integrins to the apical membrane of tubular epithelial cells may occur with ischemia (284–286). Possible mechanisms for the loss of the polarized distribution of integrins include cytoskeletal disruption, state of phosphorylation, activation of proteases, and production of NO (287,288). These integrins are known to recognize RGD tripeptide sequences (289,290). Thus, viable intraluminal cells could adhere to other luminal or paraluminal cells. There is experimental evidence for this cell-to-cell adhesion process as a contributor to tubule obstruction in ischemic AKI. Synthetic cyclical RGD peptides were infused before the renal ischemic insult in order to block cell-to-cell adhesion as a component of tubule obstruction (291–295). Using micropuncture techniques the cyclic RGD tripeptides blocked the rise in tubular pressure postischemic insult (289). An in vivo study of RGD peptides in ischemic AKI in rats demonstrated attenuation of renal injury and accelerated recovery of renal function (291). Systemic administration of fluorescent derivatives of two different cyclic RGD peptides, a cyclic Bt-RGD peptide and a linear RhoG-RGD peptide, infused after the release of renal artery clamp ameliorated ischemic AKI in rats (292,294). The staining of these peptides suggests that cyclic RGD peptides inhibited tubular obstruction by predominantly preventing cell-to-cell adhesion rather than cell-to-matrix adhesion (290).

ROLE OF ABNORMAL VASCULAR FUNCTION IN AKI

In organ ischemia, the restoration of perfusion may add to the problem of organ injury. Organ dysfunction attributable to reperfusion has been demonstrated in the heart, lung, brain, intestine, liver, and other organs. The importance of these findings is in their probable contribution to clinical features of myocardial infarction, AKI, and stroke. The implications of reperfusion injury are important in the clinical settings of flow diversion in surgical bypass and for function of transplanted heart, lung, kidney, and other organs.

Injury induced by I/R leads to organ dysfunction, in part by direct injury of parenchymal cells. Vascular dysfunction is an early and prominent aspect of I/R injury, with consequent impairment of blood flow and its regulation. For instance, there may be a progressive loss of regional organ blood flow following I/R. There also may be an exaggerated constriction to neurohumoral agonists, failure to respond to physiologic and pharmacologic vasodilators, and paradoxical vasoconstrictor responses to changes in arterial pressure and blood flow following a period of transient organ ischemia and reperfusion. Evidence suggests that disordered vascular function subsequent to I/R injury may itself have a substantial impact on organ recovery, since normalization of blood flow influences the rate of parenchymal cell restoration.

Basal vascular tone is essential for perfusion of complex and distinct vascular beds and is dictated in large part by metabolic requirements of individual organs. It is clear that both transmural pressure and shear stress from blood flow contribute to basal arterial vascular tone. The predominant effect of vessel wall pressure is to increase tone; that of flow is to reduce tone. The mechanisms mediating the tonal response to these physical forces are only partially understood. Ca²⁺ entry, at least in part, through unique stretch-operated channels is important in pressure-induced vasoconstriction. VSMC transmembrane Na⁺ concentrations are a factor in flow-related vasodilation. In addition, endothelial factors (NO, prostaglandins) are involved in flow-related vasodilation. Aside from its role in mediating shear-induced vasodilation, evidence indicates that endothelial-generated NO independently contributes to normal vascular tone. Other neurohumoral factors that contribute to changes in arterial tone dictated by metabolic demand are adenosine, oxygen, and carbon dioxide (296). Factors that modify vascular tone are listed in Table 10-5.

Vascular Dysfunction due to I/R Injury

The kidney model that exemplifies I/R injury is ischemia-induced AKI. A severe form of this disorder in which the renal artery is clamped for 40 to 70 minutes followed by immediate reflow (297,298) and a less severe form in which high-dose norepinephrine (NE) is infused into the renal artery for 90 minutes with slow spontaneous return of blood flow (297,299) have been studied extensively in rats. In the clamp model, there is a brief postocclusion hyperemia, then a sustained small reduction in renal blood flow, and an attenuated response to endothelium-dependent dilators (299). In the first few hours after reflow, in the NE model there is a modest reduction in renal blood flow compared with the preischemia level without hyperemia, a decreased response to endothelium-dependent vasodilators, and a small but significant reduction in the constrictor response to the NOS inhibitor L-NAME (296). There is partial endothelial cell detachment without ultrastructural changes in individual endothelial cells at 6 hours in both the renal artery clamp and NE AKI models. By 48 hours of reperfusion, the basal renal blood flow remains 20% reduced in the renal artery clamp model, and there is a reduced vasoreactive response to changes in renal perfusion pressure to constrictor agonists and to endothelium-dependent and endothelium-independent dilators (296). The predominant histologic finding at this time in the small resistance arteries and arterioles is VSMC necrosis, present in 55% to 60% of the vessels (300,301). It is assumed that the lack of response to vasoactive stimuli is due to the diffuse VSMC injury related to both the relative severity of ischemia and the rapidity of reperfusion. In the NE AKI model, at 48 hours, the basal renal blood flow also is approximately 20% less than normal (296,297). However, vascular reactivity is strikingly different from that in the renal artery clamp AKI model. The difference likely is due to less severe ischemia and a slower rate of reperfusion. There is an exaggerated renal vasoconstrictor response to angiotensin II and endothelin-1 (ET-1) both in vivo and in arterioles isolated from these kidneys (296,302). The response to endothelium-dependent vasodilators is reduced, but the constrictor response to L-NAME is actually increased (296). cNOS can be identified as at least as strongly reactive or more reactive than normal, as determined with cNOS monoclonal antibody in the resistance arterial vessels (303). While there is a dilator response to cyclic adenosine monophosphate-dependent PGI₂ in the 48-hour postischemic renal vasculature, there is no increase in renal blood flow to the NO donor sodium nitroprusside. Taken together, these data indicate that at 48 hours after ischemia in NE AKI in the rat kidney, vascular cNOS activity is not diminished but rather is maximal such that it cannot be stimulated further by endothelium-dependent vasodilators. The available NO under basal conditions has fully activated VSMC-soluble cyclic guanosine monophosphate such that there is no additional response to an exogenous NO donor.

In examining the mechanism for the constrictor hypersensitivity in the 48-hour postischemic vasculature in NE AKI, measurements of VSMC cytosolic Ca²⁺ have been made in the isolated arterioles from these kidneys perfused at physiologic pressures (302). Compared to similar vessels from sham AKI kidneys, there is a significantly higher baseline and an earlier and greater increase in VSMC Ca²⁺ in response to a normal half-maximal constricting concentration (EC₅₀) of angiotensin II, which correlates with the initially lower and more intense reduction in lumen diameter in the postischemic AKI vessels.

Another novel observation regarding VSMC Ca²⁺ in 48-hour postischemic renal arterioles in vitro is a paradoxical change in VSMC cell Ca²⁺ in response to changes in lumen pressure. In normal afferent and efferent arterioles, increasing lumen pressure (stretch) within an autoregulatory range for these vessels results in an increase in VSMC Ca²⁺. Conversely, decreasing lumen pressure is associated with a decrease in VSMC Ca²⁺. In the NE AKI vessels, the reverse relationships are observed. There are also corresponding paradoxical changes in lumen diameter, representing, at least, a loss of the myogenic response and, at most, a “reverse” myogenic response. This abnormal VSMC Ca²⁺ and myogenic response to pressure is suggested to be the basis of the markedly abnormal in vivo autoregulatory response between 48 hours and 1 week after AKI induction that is likely the most significant and clinically relevant I/R disorder of vasoreactivity in the kidney.

It was at first thought that Ca²⁺ channel blockers might be exerting their protective effects entirely at the vascular level by promoting the enhancement of renal blood flow. There are unquestioned renal vascular effects of Ca²⁺ channel blockers, with renal blood flow improving more rapidly after ischemia with Ca²⁺ channel blocker treatment (304). Renal blood flow and glomerular filtration will not decrease as severely during radiocontrast administration in dogs when Ca²⁺ channel blockers are coadministered (305). Ischemic AKI is characterized by a loss of autoregulatory ability, an enhanced sensitivity of renal blood flow to renal nerve stimulation, and injury to the endothelial lining of renal vessels (304). Much of this injury may be related to Ca²⁺ overload in VSMCs and/or endothelial cells, since verapamil and diltiazem partially obviate the loss of autoregulatory capacity and hypersensitivity to renal nerve stimulation (304).

Warm and cold ischemia during transplantation surgery may also contribute to vascular injury, and Ca²⁺ channel blockers are protective in experimental models of these clinical entities (306,307). However, other renal vasodilators such as prostacyclin do not restore autoregulatory integrity or reverse the increased sensitivity to renal nerve stimulation (304). Thus, it also seems that a unique effect of Ca²⁺ channel blockers is exerted at the vascular level.

At 1 week after ischemic injury, the endothelium appears normal, smooth muscle necrosis is less evident, but perivascular fibrosis is marked in the mid- to small-sized arterial vessels (297). Functionally, the response to endothelium-dependent dilators is reduced, L-NAME constrictor response is increased, and immunologically detectable NOS is present (303). There is a decreased dilator response to sodium nitroprusside but a measurable, albeit slightly reduced, dilator response to PGI₂ (303). These findings suggest maximal endothelial cNOS activity similar to that at 48 hours. Unlike 48-hour vessels, the vasoconstrictor response to angiotensin II was markedly attenuated both in vivo and in vitro at 1 week (296,308). On the other hand, as previously alluded, a paradoxical vasoconstriction to a reduction in perfusion pressure in the autoregulatory range could be demonstrated in vivo. It is difficult to suggest a single mechanism that explains this series of functional aberrations at 1 week. It is likely that more than one pathophysiologic process is operating to produce these complex responses.

Intravital two-photon microscopy has been used to study the microvascular events within the functioning kidney in vivo (309–312). Intravital two-photon microscopy enables investigators to follow functional and structural alterations with subcellular resolution within the same field of view over a short period of time. Endothelial cell dysfunction within the microvasculature was observed and quantified using the infusion of variously sized, differently colored dextrans or proteins. Movement of these molecules out of the microvasculature and accumulation within the interstitial compartment are readily observed during AKI. The FVB-TIE2/GFP mouse, in which the endothelium is fluorescent, has been used to study morphologic changes in the renal microvascular endothelium during I/R injury in the kidney (313). Alterations in the cytoskeleton of renal microvascular endothelial cells correlated with a permeability defect in the renal microvasculature as identified using fluorescent dextrans and two-photon intravital imaging. This study demonstrates that renal vascular endothelial injury occurs in ischemic AKI and may play an important role in the pathophysiology of ischemic AKI.

In patients with AKI, it has been demonstrated that diminished NO generation by injured endothelium and loss of macula densa neuronal NOS may impair the vasodilatory ability of the renal vasculature and contribute to the reduction in the GFR (314). Fifty patients who had a cadaveric renal transplant were studied: urinary nitrite and nitrate levels were determined, and intraoperative allograft biopsies were performed. In patients with sustained AKI, urinary nitrite and nitrate excretion was lower than in patients without AKI. In the kidney biopsies, eNOS expression diminished from the peritubular capillaries of 6 of 7 subjects in the sustained AKI group but from only 6 of 16 subjects in the recovery group.

Endothelial Injury

Normal epithelium and endothelium are separated by a small interstitial compartment. The endothelium is coated by a glycocalyx. In I/R injury there is swelling of endothelial cells, disruption of the glycocalyx and endothelial monolayer, and upregulation of adhesion molecules such as ICAMs, VCAMs, and selectins, resulting in increased leukocyte–endothelium interactions (262). There is formation of microthrombi in blood vessels and leukocytes migrate through the endothelial cells into the interstitial compartment (262). There are inflammatory cells and interstitial edema in the interstitial compartment. In ATN, the peritubular capillaries have vacuolar degeneration of the endothelial cell, thickening and multilayer basement membrane formation and attachment and penetration of monocytes (262).

Microparticles are cell membrane-derived particles that can promote coagulation, inflammation, and angiogenesis, and play a role in cell-to-cell communication (315). Microparticles are released by endothelial and circulating cells after sepsis-induced microvascular injury and contribute to endothelial dysfunction, immunosuppression, and multiorgan dysfunction—including sepsis-AKI (315). Glomerular endothelial injury, possibly mediated by a decreased vascular endothelial growth factor (VEGF) level, plays a role in the development and progression of AKI and albuminuria in the LPS-induced sepsis in the mouse (316). In AKI, impaired endothelial proliferation and mesenchymal transition contribute to vascular rarefaction and may contribute to the development of chronic kidney disease (CKD) (317).

The role of caspases and calpain in cisplatin-induced endothelial cell death was investigated (146). Cultured pancreatic microvascular endothelial (MS1) cells were exposed to 10 and 50 µM cisplatin. Cells treated with 50 µM cisplatin had severe ATP depletion, increased caspase-3-like activity, and displayed extensive PI staining indicative of necrosis at 24 hours. The increase in LDH release and the nuclear PI staining with 50 µM cisplatin at 24 hours was reduced by either the pan-caspase inhibitor, Q-VD-OPH, or the calpain inhibitor, PD-150606. Thus, in cisplatin-treated endothelial cells, caspases, the major mediators of apoptosis, can also cause necrosis. A calpain inhibitor protects against necrosis without affecting caspase-3-like activity suggesting that calpain-mediated necrosis is independent of caspase-3.

The causes of endothelial injury in AKI were investigated. Toll-like receptor 4 (TLR4) regulates early endothelial activation in ischemic AKI (318). There was increased TLR4 expression on endothelial cells of the vasa recta of the inner stripe of the outer medulla of the kidney in ischemic AKI in mice (318). Adhesion molecule (CD54 and CD62E) expression was increased on endothelia of wild-type but not TLR4 knockout mice in vivo. Further, the addition of high-mobility group protein B1, a TLR4 ligand released by injured cells, increased adhesion molecule expression on endothelia isolated from wild-type but not TLR4 knockout mice. TLR4 was localized to proximal tubules in the cortex and outer medulla after 24 hours of reperfusion. Thus, both endothelial and epithelial cells express TLR4, each of which contributes to renal injury by temporally different mechanisms during ischemic AKI (318).

In summary, I/R injury is accompanied by dramatic changes in basal and reactive vascular function of the organ involved. Endothelial injury also occurs in ischemic AKI in mice. There are similarities in altered organ vascular function, particularly in the early reperfusion period of 24 to 48 hours, including changes in permeability, decreased basal organ blood flow, hypersensitivity to vasoconstrictor stimuli, and attenuated response to vasodilators. The reduced responsiveness to endothelium-dependent vasodilators may be due to an actual reduction in eNOS activity or to an actual spontaneous maximal NOS/NO activity that cannot be stimulated further by endothelium-dependent agents.

When renal perfusion pressure is reduced, preglomerular arterial resistance decreases and efferent arteriolar resistance increases to maintain glomerular capillary hydraulic pressure and single-nephron GFR relatively constant. The efferent arteriolar constriction is mediated, in large part, through the local renin–angiotensin system (RAS) (319). Activation of the RAS stimulates synthesis of cyclooxygenase products, including the vasodilator prostaglandins PGI₂ and PGE₂ (320). PGI₂ and PGE₂ oppose the constrictor effects of angiotensin II, thereby attenuating the reduction in renal blood flow as renal perfusion pressure declines. The modulating vasodilator effect of prostaglandins in the setting of reduced renal perfusion appears to be greater in afferent than efferent arterioles. When PGI₂ and PGE₂ were administered exogenously during reduced renal perfusion, filtration fraction increased, with better preservation of the GFR than renal blood flow (321,322), suggesting that vasodilator prostaglandins preferentially caused preglomerular vasorelaxation under these conditions.

Prostaglandin synthesis was found to be increased in animal models of ischemic AKI (321,323), aminoglycoside nephrotoxicity (324), sepsis, and endotoxic shock (325,326). The indication that an increase in prostaglandin activity was renoprotective by maintaining glomerular hemodynamics showed that cyclooxygenase inhibitors in these disorders augmented the reduction in renal blood flow and the GFR (327,328).

Other evidence of protection in AKI was the finding that infusion of biologic prostaglandins and their analogs in ischemic (321,322), mercuric chloride (329), and glycerol-induced AKI (330) results in protection against AKI. The prostaglandin E₁ analog, misoprostol, was found to provide significant protection against ischemia-induced renal dysfunction in rats subjected to renal artery occlusion (331). Misoprostol-treated rats had GFRs almost threefold greater than control animals, although renal blood flow and renal vascular resistance were not significantly different. Misoprostol also protected against renal dysfunction in a model of toxic renal injury produced by mercuric chloride. In an in vitro model employing primary cultures of proximal tubule epithelial cells subjected to hypoxia and reoxygenation, misoprostol, prostaglandin E₂, and prostacyclin limited cell death. This study demonstrated that prostaglandins protect renal tubule epithelial cells from hypoxic injury at the cellular level independent of hemodynamic factors. Another study demonstrated that inhibitors of cyclooxygenase and lipoxygenase pathways exert a direct protective effect against the hypoxia–­reoxygenation-induced cell injury in renal tubules, a model independent of vascular and inflammatory factors (332).

The PGE₁ study group has performed a pilot study with intravenous PGE₁ administered before radiocontrast media in patients with renal impairment (333). Results from this pilot study suggest that intravenous PGE₁ may be used efficaciously and safely to prevent radiocontrast medium-induced renal dysfunction in patients with preexisting impaired renal function.

Natriuretic Peptides

In 1981, the natriuretic effects of an extract of mammalian atrial myocytes were first reported (334). Subsequently, this substance has been characterized as a polypeptide. The primary stimulus to atrial natriuretic peptide (ANP) synthesis and release is distention of the atria, where storage granules have been identified. Infusion of normal saline into human volunteers increases plasma ANP (335), and plasma ANP levels were elevated in edematous states that involved increased intravascular volume and atrial enlargement such as congestive heart failure.

Natural and synthetic ANPs cause dose-dependent reductions in systemic arterial pressure. The mechanism involves both peripheral vasorelaxation and a reduction in cardiac output (336,337). The magnitude of arterial pressure reduction is dependent on the state of basal vascular tone. ANP has been shown to inhibit both secretion and activity of the renin–angiotensin–aldosterone (338) and adrenergic nervous systems (339), as well as that of vasopressin (340) and ET-1 (341).

ANP has an important effect on the kidney. In vivo infusion of ANPs, both synthetic and naturally occurring from a variety of species, markedly increased the GFR while having a proportionately smaller effect on renal blood flow (342). Studies suggest that ANP-induced renal vasorelaxation was specific for the preglomerular arterioles (343,344). Other studies examining the rat renal microvasculature in vitro indicated that ANP not only directly vasodilated the afferent arteriole but also constricted the efferent arteriole (345,346). The tubular natriuretic effects of ANP involve inhibition of sodium and water transport in the loop of Henle, connecting tubules, and collecting ducts. Among other possible mechanisms, ANP has been shown to interfere with vasopressin effect and alter adenylate cyclase activity.

Other natriuretic peptides have been discovered. Another class of natriuretic peptides is referred to as brain natriuretic peptide (BNP). It has been isolated from both brain and heart (347,348). BNP, which contains 32 amino acids, has diuretic and natriuretic effects similar to ANP, while the hypotensive effect is not as potent. BNP is now FDA approved for clinical use in congestive heart failure (349).

Numerous animal studies have demonstrated a protective effect of ANPs on ischemic and nephrotoxic in models of AKI (345,350–353). ANP is effective in AKI models even when given after the initiating insult.

On the basis of the encouraging animal experimental results and the unique combination of pharmacologic properties, clinical studies were performed. A multicenter, randomized, double-blind, placebo-controlled clinical trial of ANP in 504 critically ill patients with AKI was performed. The patients received a 24-hour intravenous infusion of either ANP or placebo. The primary end point was dialysis-free survival for 21 days after treatment. The administration of ANP did not improve the overall rate of dialysis-free survival in critically ill patients with ATN. However, ANP decreased the need for dialysis in patients with oliguria (354). In a subsequent study, 222 patients with oliguric AKI were enrolled in a multicenter, randomized, double-blind, placebo-controlled trial. There was no statistically significant beneficial effect of ANP in dialysis-free survival or reduction in dialysis in these subjects with oliguric AKI (355). Mortality rates through day 60 were 60% versus 56% in the ANP and placebo groups, respectively. However, 95% of the ANP-treated patients versus 55% of the placebo-treated patients had systolic blood pressures <90 mm Hg during the study drug infusion (P < 0.001). The hypotensive effect of ANP in these recent trials no doubt obscured any intrarenal beneficial effect of the compound.

Calcium Channel Blockers

Calcium channel blockers (CCBs), which inhibit voltage-gated Ca²⁺ entry, have been shown to protect against ischemic and nephrotoxic (cisplatinum, gentamicin) AKI in various animal models (356–362). The protective effect involves less renal vasoconstriction and improved renal blood flow. At a tubular level, there is less AKI and improved mitochondrial function. More recently, it has been demonstrated that the CCB benidipine can ameliorate the ischemic AKI in rats and that the renoprotective effect was associated with the reduction in apoptosis in tubular epithelial cells (363). Diltiazem also improves renal function in endotoxin-induced AKI in the rat (364).

CCBs have also been examined in clinical studies. Gallopamil resulted in more rapid recovery of renal function in five patients with malaria- or leptospirosis-related AKI (365). Other human experience with CCBs has largely been in the setting of renal transplantation. Verapamil improved early graft function when administered to donors before harvesting the kidneys (54). Diltiazem administered to transplant patients immediately after graft placement resulted in better graft function and a lower incidence of posttransplant AKI (366). More recently, it was demonstrated that isradipine results in a better renal function after kidney transplantation (367). However, the protective effect was independent of delayed graft function or acute rejection.

Fractalkine

Proinflammatory cytokines increase expression of the CX₃C chemokine, fractalkine, on injured endothelial cells. The fractalkine receptor (CX₃CR1) is expressed on natural killer (NK) cells, monocytes, and some CD8⁺ T cells (368). Fractalkine has a mucinlike stalk that extends the chemokine domain away from the endothelial cell surface, enabling presentation of the CX₃C-chemokine domain to leukocytes. Expression of fractalkine enables bypassing of the first two steps of the adhesion cascade (i.e., rolling and triggering) and mediates cell adhesion between circulating leukocytes and endothelial cells as well as extravasation of these cells. Thus, fractalkine serves the dual function of an adhesion molecule and a chemoattractant (368). Fractalkine is a major chemoattractant for NK cells and monocytes but not neutrophils (369). Fractalkine expression is increased in patients with renal tubulointerstitial inflammation, with the strongest expression localized to vascular sites near to macrophage inflammation (370). Fractalkine is a strong candidate for directing mononuclear cell infiltration induced by vascular injury (370). Fractalkine expression is increased in the endothelium of large blood vessels, capillaries, and glomeruli in ischemic AKI (371). Fractalkine receptor inhibition is protective against ischemic AKI (371). Fractalkine expression is also increased in the blood vessels in mouse kidneys exposed to cisplatin (372). However, fractalkine inhibition did not protect against the functional and histologic abnormalities in cisplatin-induced AKI in mice (372).

Clinical Relevance of I/R Vascular Injury

The course of human ischemia-induced AKI is highly variable. An important and relevant observation regarding the variable duration and, in particular, the prolonged course in AKI patients was made by Solez et al. (14). In individuals with AKI duration of longer than 3 weeks, a prominent finding in biopsy or autopsy specimens was fresh tubular renal ischemic lesions that could not be related to the remote initial ischemic insult. A possible explanation for the fresh ischemic lesions was altered reactivity of the renal vasculature. Abnormal vascular reactivity in established ischemic AKI animal models includes loss of renal blood flow autoregulation. A number of investigators (300,308,373) have found an attenuated autoregulatory response from 2 to 7 days after AKI induction in the renal artery clamp model in rats.

Against the background of these postischemic vascular perturbations is the observation that a decrease in renal perfusion pressure is not associated with autoregulation of either the GFR or renal blood flow (296,303,304,308,374,375). In fact, rather than renal vasodilation, renal vasoconstriction occurs with a fall in renal perfusion pressure in the postischemic kidney. Thus, a degree of hypotension, which is of no clinical significance in the normal kidney, may cause renal damage in the kidney during the recovery phase of AKI. The same increased sensitivity in the postischemic kidney has also been shown to occur with nephrotoxic agents such as aminoglycosides.

These data have important clinical implications, as a modest arterial pressure reduction during the course of this disease, such as frequently occurs with hemodialysis treatment, can actually result in recurrent ischemic injury and prolongation of AKI (376).

VASOACTIVE RESPONSE TO SEPSIS

Sepsis is the most frequent cause of AKI in ICUs (4,17). AKI occurs in approximately 19% of patients with moderate sepsis, 23% of patients with severe sepsis, and 51% of patients with septic shock (19). The combination of AKI and sepsis is associated with a >80% mortality (4).

Complex vasoactive responses occur in septic AKI. Over the past three decades, sepsis has been studied in various species, including rats, dogs, pigs, primates, and humans. Recently a mouse model of septic AKI has been developed; this model allows the use of newer molecular techniques, including knockout and transgenic mice, to study the pathogenesis of AKI associated with sepsis.

The initial effects of sepsis in causing AKI primarily involve renal vasoconstriction (377). This renal vasoconstriction can be demonstrated in the absence of sepsis-mediated hypotension (377) as well as in the absence of later events, including apoptosis, leukocyte infiltration, and morphologic evidence of coagulation (e.g., glomerular fibrin) (378–381).

There is evidence that several vasoconstrictor and vasodilator pathways are activated during sepsis in various experimental models. During sepsis, the cytokine-mediated induction of NO results in a hyperdynamic state in which systemic vasodilation is associated with a secondary increase in cardiac output (19). The rise in cardiac output, however, may not be maximal for the degree of afterload reduction because of the myocardial depressant effect of cytokines such as TNF-α. The arterial underfilling associated with systemic arterial vasodilation is known to activate the RAS and the sympathetic nervous system (SNS) (382–385). While these events attenuate or abolish any systemic hypotension, they also lead to renal vasoconstriction. The vasoactive events of sepsis are, however, more complex than those initiated by arterial underfilling. The endotoxin-mediated increase in TNF-α is associated with an increase in iNOS (378,386). There is evidence in the endotoxemic rat that the increased NO that results from the upregulation of iNOS exerts a negative feedback on the eNOS in the kidney (387). Moreover, the secondary messenger of NO, cyclic guanosine 5′-monophosphate (GMP) has been shown to increase in the renal cortex during the initial 16 hours of sepsis but then at 24 hours to be downregulated in spite of continued high plasma levels of NO (388). Both of these events, namely, NO-mediated decreased eNOS and downregulation of cyclic GMP, would impair the normal counterregulatory vasodilator pathways that attenuate the renal vasoconstriction associated with activation of the RAS and SNS. ET-1 has been shown to be increased during endotoxemia in several species (389–393). The capillary leak that leads to interstitial edema and decreased plasma volume during endotoxemia has been reversed with ET receptor blockade in the rat, albeit with a decrease in blood pressure (389).

INFLAMMATION

The inflammatory response may play a major role in the pathogenesis of ischemic ARF (394,395). Both the innate and adaptive immune response is important in the pathophysiology of ischemic injury (262). The innate occurs early, is non–antigen-specific and is composed of neutrophils, monocytes/macrophages, dendritic cells, NK cells, and natural killer T cells (NKT cells) (262). The adaptive response occurs within hours, lasts a few days, and is activated by specific antigens. The adaptive response includes dendritic cell maturation and antigen presentation, T lymphocyte proliferation and activation, and T to B lymphocyte interactions (262).

The role of neutrophils, lymphocytes, macrophages, and NK cells has been studied in AKI and will be discussed in the next section.

Neutrophils

The role of neutrophils in AKI has been addressed in many studies and remains controversial (396). There is evidence that leukocytes, particularly neutrophils, mediate tubular injury in AKI derived from studies that show an accumulation of neutrophils in ischemic AKI and studies demonstrating a beneficial role of anti-ICAM-1 therapy in AKI (397). Rats depleted of peripheral neutrophils by antineutrophil serum were not protected against ischemic AKI (398). In another study, mice depleted of peripheral neutrophils by antineutrophil serum were protected against ischemic AKI (397).

The adherence of neutrophils to the vascular endothelium is an essential step in the extravasation of these cells into ischemic tissue (399). After adherence and chemotaxis, infiltrating leukocytes release ROS and enzymes that damage the cells (399). Activated neutrophils have been shown to enhance the decrease in the GFR in response to renal ischemia, at least in part due to release of oxygen radicals (400–403). In contrast, infusion of oxygen radical-deficient neutrophils from patients with chronic granulomatous disease did not worsen the course of ischemic injury (402). The mechanism by which adherent leukocytes cause ischemic injury is unclear but likely involves both the release of potent vasoconstrictors including prostaglandins, leukotrienes, and thromboxanes (404) as well as direct endothelial injury via release of endothelin and a decrease in NO (26,405).

Increased systemic levels of the cytokines, TNF-α and IL-1, may upregulate ICAM-1 after ischemia and reperfusion in the kidney (397). The administration of a monoclonal antibody against ICAM-1 protected against ischemic AKI in rats (402,406). ICAM-1-deficient mice are protected against renal ischemia (397). Thus, ICAM-1 is a mediator of ischemic AKI probably by potentiating neutrophil–endothelial interactions. There is also evidence that upregulation of adhesion molecules may contribute to this impaired medullary blood flow postischemic injury (407–409).

P-selectin is another important molecule involved in adherence of circulating leukocytes to tissue in inflammatory states. Renal ischemia has also been shown to be associated with upregulation of endothelial P-selectin with enhanced adhesion of neutrophils (410). A soluble P-selectin glycoprotein ligand prevents infiltration of leukocytes and protects functionally against ischemic AKI (288).

The role of neutrophils in AKI has been explored in many studies and remains controversial (396,411). There is evidence that neutrophils mediate renal tubular injury in AKI. This evidence is derived from studies that show an accumulation of neutrophils in ischemic AKI and studies demonstrating a beneficial role of anti-ICAM-1 therapy in AKI (397). In another study, mice depleted of peripheral neutrophils by antineutrophil serum were protected against ischemic AKI (397). However, rats depleted of peripheral neutrophils by antineutrophil serum were not protected against ischemic AKI (398).

Mice with ischemic AKI were treated with the pan-caspase inhibitor Quinoline-val-asp(Ome)-CH₂-OPH (OPH-001) (412). OPH-001 induced a marked (100%) reduction in BUN and serum creatinine and a highly significant reduction in the ATN score compared to vehicle-treated mice. OPH-001 significantly reduced the increase in caspase-1 activity and IL-18, and prevented neutrophil infiltration in the kidney during ischemic AKI. To further investigate whether the lack of neutrophil infiltration was contributing to the protection against ischemic AKI, a model of neutrophil depletion was developed. Mice were injected with 0.1 mg of the rat IgG2b monoclonal antibody RB6-8C5 intraperitoneally 24 hours before renal pedicle clamp (413). This resulted in depletion of neutrophils in the peripheral blood and in the kidney during ischemic AKI. Neutrophil-depleted mice had a small (18%) reduction in serum creatinine during ischemic AKI but no reduction in the ATN score despite a lack of neutrophil infiltration in the kidney. Remarkably, caspase-1 activity and IL-18 were still significantly increased in the kidney in neutrophil-depleted mice with AKI. Thus, to investigate the role of IL-18 in the absence of neutrophils, neutrophil-depleted mice with ischemic AKI were treated with IL-18-neutralizing antiserum. IL-18-antiserum-treated neutrophil-depleted mice with ischemic AKI had a significant (75%) reduction in serum creatinine and a significant reduction in the ATN score compared with vehicle-treated neutrophil-depleted mice. These results suggested a novel neutrophil-independent mechanism of IL-18-mediated ischemic AKI (412).

The IL-23/IL-17 and IL-12/IFN-γ cytokine pathways plays a role in abnormal adaptive immunity. The hypothesis was tested that early production of IL-23 and IL-12 following ischemia reperfusion injury (IRI) activates downstream IL-17 and IFN-γ signaling pathways and promotes kidney inflammation in a mouse model of ischemic AKI (414). Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and monoclonal antibody neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in AKI. IL-17A produced by neutrophils was critical for AKI. IFN-γ administration reversed the protection seen in IL-17A (−/−) mice subjected to IRI, whereas IL-17A failed to reverse protection in IFN-γ (−/−) mice. These results demonstrate that the innate immune component of AKI requires dual activation of the IL-12/IFN-γ and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-γ (414).

Lymphocytes

The role of other leukocytes, for example lymphocytes, has recently been reported. Mice with genetically engineered deficiency of both CD4⁺ and CD8⁺ T cells demonstrate a marked improvement in renal function and less neutrophil infiltration in the ischemic kidney compared with control mice. Also mice deficient in CD4 T cells, not CD8 T cells, are significantly protected from AKI (415). Direct evidence for a pathophysiologic role of the CD4 T cell was obtained when reconstitution of CD4-deficient mice with wild-type CD4 T cells restored postischemic injury.

However, there is also a study that CD4 T-cell depletion is not sufficient to protect against ischemic AKI (416). Mice were injected with 10 mg/kg of the rat IgG monoclonal antibody GK1.5 IP or vehicle. Complete CD4⁺ T-cell depletion with GK1.5 was confirmed by flow cytometry of lymph nodes before induction of AKI and at 24 hours of postischemic reperfusion. Serum creatinine and the ATN score were not different in vehicle-treated and CD4 T–cell-depleted mice with ischemic AKI. These results suggest that CD4⁺ T cells are not required for the development of ischemic AKI. Therefore, the hypothesis was tested that more than one subset of lymphocyte may need to be depleted for protection against ischemic AKI. T-cell receptor α chain (TCRα) (−/−) mice lack conventional αβ T cells and are deficient in both CD4⁺ and CD8⁺ T cells. TCRα (−/−) mice were not protected against ischemic AKI.

IL-33 is a recently discovered member of the IL-1 family of cytokines. IL-33 is a nuclear protein that is also released into the extracellular space (417). IL-33 is released as an early response to tissue injury (418). Full-length (active) IL-33 specifically binds the ST2R on CD4 T cells and increases secretion of proinflammatory cytokines (419). Thus, IL-33 is a chemoattractant for CD4 T cells. We have observed increased protein expression of full-length IL-33 in the kidney following induction of AKI with cisplatin (420). Compared with cisplatin-induced AKI in untreated mice, mice treated with a soluble ST2 fusion protein that binds IL-33 had fewer CD4 T cells infiltrate the kidney, lower serum creatinine, and reduced ATN and apoptosis. In contrast, administration of recombinant IL-33 (rIL-33) exacerbated cisplatin-induced AKI, measured by an increase in CD4 T-cell infiltration, serum creatinine, ATN, and apoptosis; this did not occur in CD4-deficient mice, suggesting that CD4 T cells mediate the injurious effect of IL-33. Wild-type mice that received cisplatin and rIL-33 also had higher levels of the proinflammatory chemokine CXCL1, which CD T cells produce, in the kidney compared with CD4-deficient mice. Mice deficient in the CXCL1 receptor also had lower serum creatinine, ATN, and apoptosis than wild-type mice following cisplatin-induced AKI. Taken together, IL-33 promotes AKI through CD4 T cell-mediated production of CXCL1. These data suggest that inhibiting IL-33 or CXCL1 may have therapeutic potential in AKI (420).

Studies in the acute high-dose cisplatin model of AKI (25 mg/kg/d for 3 days) demonstrate that an increase in neutrophils (225) and macrophages (372) occurs late in the course of cisplatin-induced AKI and that neither neutrophil (141) nor macrophage depletion (372) is protective. In an acute model of cisplatin-induced AKI in mice without cancer, data demonstrate that CD4 (−/−) mice are protected against AKI (420). A pathophysiologic role for CD4 T cells in an acute model of cisplatin-induced AKI has been also been demonstrated in two other studies (421,422). It was determined whether CD4 (−/−) mice are protected against AKI in a more clinically relevant 4-week chronic model of cisplatin-induced AKI in mice with lung cancer (423). Kidney function, serum NGAL, ATN, and tubular apoptosis score was the same in wild-type mice and CD4 (−/−) mice with AKI. The lack of protection against AKI in CD4 (−/−) mice was associated with an increase in extracellular signal-regulated kinase (ERK), p38, CXCL1 and TNF-α, mediators of AKI and fibrosis, in both cisplatin-treated CD4 (−/−) mice and wild-type mice. The lack of protection was independent of the presence of cancer or not. Tumor size was double and cisplatin had an impaired therapeutic effect on the tumors in CD4 (−/−) versus wild-type mice. These data warn against the use of CD4 T-cell inhibition to attenuate cisplatin-induced AKI in patients with cancer.

It was determined whether HSP exerts a renoprotective effects through regulatory T cells (Tregs) (424). T cells from heat-preconditioned mice failed to reconstitute ischemic AKI when adoptively transferred to T–cell-­deficient nu/nu mice in contrast to T cells from control mice. Tregs were also increased in heat-preconditioned AKI kidneys. Depleting Tregs before heat preconditioning abolished the renoprotective effect, while adoptive transfer of these cells back into Treg-depleted mice partially restored the beneficial effect of heat preconditioning. The renoprotective effect of HSP70 may be partially mediated by a direct immunomodulatory effect through Tregs (424).

Pharmacologic recruitment of Tregs is a potential therapy for ischemic AKI (425). Pretreatment of mice with the naturally occurring sphingosine N,N-dimethylsphingosine (DMS) was found to increase both tissue-infiltrating T effectors (Teffs, CD4(+)Foxp3(−)) and Tregs (CD4(+)Foxp3(+)) in the early phase of ischemic AKI. Renoprotection was abolished by administration of the Treg-suppressing agents, anti-CTLA-4 or anti-CD25 monoclonal antibodies, suggesting that Tregs play a key role in DMS-induced renoprotection. Thus, Tregs recruited to the kidney by DMS ameliorate AKI (425).

Endogenous Toll-like receptor 9 (TLR9) regulates AKI by promoting Treg recruitment (426). In cisplatin-induced AKI, Tlr9 (−/−) mice developed worse renal injury and exhibited fewer intrarenal Tregs compared with controls. A series of reconstitution and depletion studies defined a role for TLR9 in maintaining Treg-mediated homeostasis in cisplatin-induced AKI. This study identified a pathway by which TLR9 promotes renal Treg accumulation in AKI (426).

The IL-2/anti-IL-2 complex attenuates renal I/R injury through expansion of Treg cells (427). IL-2C administered before ischemic AKI induced Treg expansion in both spleen and kidney, improved renal function, and attenuated histologic renal injury and apoptosis after IRI. Also, IL-2C administration reduced the expression of inflammatory cytokines and attenuated the infiltration of neutrophils and macrophages in renal tissue. Depletion of Tregs with anti-CD25 antibodies abrogated the beneficial effects of IL-2C. IL-2C-administered ischemic AKI also enhanced Treg expansion in spleen and kidney, increased tubular cell proliferation, improved renal function, and reduced renal fibrosis (427).

Macrophages

Both monocyte/macrophages and NK cells are well-known sources and targets of injurious cytokines and chemokines (428–431). In a model of macrophage depletion using liposomal clodronate, it was demonstrated that macrophages contribute to tissue damage during acute renal allograft rejection (432) and ischemic AKI (371,433,434). Gene therapy in rats expressing an amino-terminal truncated monocyte chemoattractant protein-1 (MCP-1) reduced macrophage infiltration and ATN (435).

It was determined whether macrophages are a source of IL-18 in ischemic AKI in mice (160). On immunofluorescence staining of the outer stripe of the outer medulla, the number of macrophages double-stained for CD11b and IL-18 was significantly increased in AKI and significantly decreased by macrophage depletion using clodronate. Adoptive transfer of RAW 264.7 cells, a mouse macrophage line that constitutively expresses IL-18 mRNA, reversed the functional protection against AKI in both macrophage-depleted wild-type and caspase-1 (−/−) mice. To test whether IL-18 in macrophages is necessary to cause AKI, macrophages in which IL-18 was inhibited were adoptively transferred. Peritoneal macrophages isolated from wild-type mice, IL-18 binding protein transgenic (IL-18 BP Tg) mice, and IL-18 (−/−) mice were used. IL-18 BP Tg mice overexpress human IL-18 BP and exhibit decreased biologic activity of IL-18. Adoptive transfer of peritoneal macrophages from wild-type as well as IL-18 BP Tg and IL-18 (−/−) mice reversed the functional protection against AKI in LEC-treated mice. In summary, adoptive transfer of peritoneal macrophages in which IL-18 function was inhibited reverses the functional protection in macrophage-depleted mice, suggesting that IL-18 from adoptive transfer of macrophages is not sufficient to cause ischemic AKI.

IL-34 and CSF-1 mediate macrophage survival and proliferation. In ischemic AKI in mice, the time-related magnitude of macrophage-mediated AKI and subsequent CKD were markedly reduced in IL-34-deficient mice (436). IL-34 was generated by tubular epithelial cells and promoted macrophage-mediated tubular epithelial destruction during AKI that worsened subsequent CKD via two mechanisms: enhanced intrarenal macrophage proliferation and elevated bone marrow myeloid cell proliferation. Kidney transplant patients with AKI expressed IL-34, c-FMS, and PTP-ζ in tubular epithelial cells and IL-34 expression increased with worse donor kidney ischemia. The study concluded that IL-34-dependent, macrophage-mediated, CSF-1 nonredundant mechanisms promote persistent ischemic AKI that worsens subsequent CKD (436).

However, another study demonstrated that CSF-1-mediated expansion and polarization of resident renal macrophages/dendritic cells is an important mechanism promoting renal tubule epithelial regeneration after AKI (437). Macrophage colony-stimulating factor (CSF-1 or M-CSF) is important for kidney repair after ischemic AKI (438). CSF-1 is upregulated in tubule epithelial cells in response to AKI. CSF-1 binds to its receptor, CSF1R, in an autocrine and paracrine manner (438). Proximal tubule production of CSF-1 is important for polarizing and skewing macrophages toward an M2 phenotype, and for recovery from AKI (439).

After kidney I/R injury, monocytes home to the kidney and differentiate into activated macrophages. Proinflammatory macrophages contribute to the initial kidney damage. However, an alternatively activated macrophage reparative phenotype may promote normal renal repair (440). Macrophages isolated from murine kidneys during the tubular repair phase after AKI exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4-stimulated STAT6 signaling. Tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and in ischemic AKI in vivo, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. These data demonstrate that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phenotype that supports tubular proliferation in ischemic AKI (440).

Dendritic Cells

In mice, depletion of kidney and spleen macrophages using liposomal clodronic acid prevented AKI, while adoptive transfer of macrophages restored the AKI response (433). To determine whether macrophages or dendritic cells or both play a role in ischemic AKI, we performed ischemic AKI in CD11b-DTR mice that have a diphtheria toxin (DT)-induced depletion of CD11b cells (macrophages) and CD11c-DTR mice that have a DT-induced depletion of CD11c cells (dendritic cells) (441). While liposomal clodronic acid (that depletes both macrophages and dendritic cells)-treated animals had a significant functional protection from AKI, CD11b-DTR and CD11c-DTR mice were not protected against AKI despite a similar degree of renal macrophage and dendritic cell depletion. Protection against AKI in LEC-treated compared to CD11b-DTR or CD11c-DTR mice was partially explained by differences in proinflammatory cytokine profiles like CXCL1 and MCP-1. These findings suggested that simple depletion of renal macrophage or dendritic cell subpopulations is not protective against ischemic AKI in mice. Another study also demonstrated that macrophage/monocyte depletion by clodronate, but not DT, improves renal I/R injury in mice (442). In this study, clodronate did not deplete CD206-positive renal macrophages and, unlike DT, left resident CD11c-positive cells unchanged while inducing dramatic apoptosis in hepatic and splenic mononuclear phagocyte populations. Abolition of the protection against AKI by administration of DT to clodronate-treated mice suggested that the protective effect of clodronate resulted from the presence of a cytoprotective intrarenal population of mononuclear phagocytes sensitive to DT-mediated ablation (442).

Mice depleted of dendritic cells before or at the time of cisplatin treatment but not at later stages experienced more severe renal dysfunction, tubular injury, neutrophil infiltration, and greater mortality than mice not depleted of dendritic cells (443). This study demonstrates that resident DCs reduce cisplatin nephrotoxicity and its associated inflammation. The role of endogenous IL-10 and dendritic cell IL-10 in cisplatin-mediated kidney injury was investigated (444). Cisplatin treatment caused an increase in renal IL-10R1 expression and STAT3 phosphorylation. IL-10 knockout mice had worse cisplatin-induced AKI, indicating that endogenous IL-10 ameliorates kidney injury in cisplatin nephrotoxicity. Mixed bone marrow chimeric mice lacking IL-10 in dendritic cells showed moderately greater renal dysfunction than chimeric mice positive for IL-10 in dendritic cells. This study demonstrated that endogenous IL-10 reduces cisplatin-induced AKI and associated inflammation and that IL-10 produced by dendritic cells themselves account for the protective effect of dendritic cells in cisplatin-induced AKI (444).

Delivery of IL-10 via injectable hydrogels improves renal outcomes and reduces systemic inflammation following ischemic AKI in mice (445). Injectable hydrogels can be used to deliver drugs in situ over a sustained period of time. An injectable hydrogel with or without IL-10, or IL-10 was injected under the capsule of the left kidney. At 28 days after ischemic AKI, treatment with IL-10 reduced renal and systemic inflammation, serum IL-6, lung inflammation, urine NGAL, renal histology for fibroblast activity, collagen type III deposition, and fibrosis. Thus injectable hydrogels are suitable for local drug delivery following renal injury, are biocompatible, and help mitigate local and systemic inflammation (445).

Dendritic cell-mediated NKT cell activation is critical in initiating the immune response following ischemic AKI. Adenosine is an important antiinflammatory molecule. Mice with adenosine 2A receptor-deficient dendritic cells are more susceptible to ischemic AKI (446). Administration of dendritic cells treated ex vivo with an A₂AR agonist protected against ischemic AKI I by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. The study concluded that ex vivo A₂AR-induced tolerized dendritic cells suppress NKT cell activation in vivo and may be a potential cell-based strategy to attenuate ischemic AKI (446).

NK Cells

NK cells are a type of lymphocyte that mediate innate immunity against pathogens and tumors via their ability to secrete cytokines (447). NK cells are unique in their constitutive expression of receptors for cytokines, for example, IL-18, that are produced by activated macrophages (448). NK cells are activated by IL-18 independently of IL-12 (449). NK cells in mice express mostly the same receptors as humans, including NK 1.1. A model of NK cell activation in injured tissues has been proposed (450). In this model, it is hypothesized that NK cells are recruited to sites of injury from the bloodstream. Once in the tissue, NK cells become activated and release cytokines like IL-18 (450). In support of this hypothesis, it is known that NK cells play a role in numerous disease processes (451).

NK cell depletion in wild-type C57BL/6 mice is protective against ischemic AKI (452). Adoptive transfer of NK cells worsened injury in NK, T, and B cell-null Rag2(−/−) γ(c)(−/−) mice with ischemic AKI. NK cell-mediated kidney injury was perforin (PFN) dependent as PFN(−/−) NK cells had minimal capacity to kill tubular epithelial cells in vitro compared with NK cells from wild-type mice.

Mast Cells

Mast cells are innate immune cells that are involved in immunoglobulin E (IgE)-mediated hypersensitivity, asthma, and host defense against parasites (453). Mast cells are multifunctional pluripotent cells that migrate through vascularized tissues, completing their maturation in the end organs. Mast cells are often located in vascular beds and epithelial surfaces where they play key roles as sentinels and first responders in host defense. Mast cells contain mediators that are released upon degranulation: cytokines, chemokines, growth factors, leukotrienes, proteases and preformed TNF which can be released immediately after degranulation.

Mast cells mediate AKI through the production of TNF (453). Mast cell-deficient mice with cisplatin-induced AKI had attenuated renal injury, reduced serum levels of TNF, and reduced recruitment of leukocytes to the inflamed kidney. Mast cell-deficient mice also exhibited significantly lower intrarenal expression of leukocyte chemoattractants. Mast cell-deficient mice reconstituted with mast cells from wild-type mice exhibited similar cisplatin-induced renal damage and the same serum levels of TNF as wild-type mice. In contrast, cisplatin-induced AKI was attenuated in mast cell-deficient mice reconstituted with mast cells from TNF-­deficient mice The mast cell stabilizer sodium cromoglycate significantly abrogated cisplatin-induced AKI (453).

Renal tubular epithelial cells express increased amounts of the TLRs, TLR2 and TLR4, resulting in increased release of cytokines and chemokines which attract inflammatory cells and modulate the degree of injury (262). The proximal tubular epithelium expresses major histocompatibility complex II resulting in the presentation of antigen to T cells and the expression of costimulatory molecules (262). There is increased expression of B7-1 and B7-2, costimulatory tubule cell molecules on tubular cells that interact with CD28 on T lymphocytes and facilitate cytokine production (262).

The activation of innate immunity involves the release of pathogen-associated molecular patterns (PAMPs) and their binding to pattern recognition receptors (e.g., TLR4). Damage-associated molecular patterns (DAMPs) are molecules that are released dying cells activate cellular receptors resulting in inflammation (454). Proximal tubular cells are sensors of both DAMPs and PAMPs using pattern recognition receptors like TLR4 (455,456). Sepsis induces changes in the expression and distribution of TLR4 in the rat kidney (457). TLRs also play a critical role in ischemic injury, because loss of epithelial TLR4 and MyD88 (458) results in decreased cytokine and chemokine production, decreased inflammation in the kidney, and improved kidney function in ischemic AKI. Thus, TLR-mediated LPS signaling in proximal tubular injury results in the early initiation of damage-associated signaling cascades involving DAMPs and PAMPs. The best characterized DAMP is high-mobility group box 1 (HMGB1) protein which is a ubiquitously expressed nonhistone DNA-binding protein that regulates transcription and is a proinflammatory mediator (454). HMGB1 is chemotactic for immune cells and inflammatory cells. HMGB1 binds to the receptor for advanced glycation end products (RAGE) and causes NF-κB-induced transcription through interactions with TLRs and RAGE. Other DAMPs include S100 protein, uric acid, galectins, ATP and adenosine, thioredoxin, the intranuclear cytokine, IL-33 and THP (or uromodulin) (454). A neutralizing anti-HMGB1 antibody is functionally and histologically protective against ischemic AKI in mice associated with less tubulointerstitial infiltration by neutrophils (day 1) and macrophages (day 5) and markedly reduced apoptosis of tubular epithelial cells (459). Anti-HMGB1 antibody-treated IRI kidneys had significantly lower levels of IL-6, TNF-α, and monocyte chemoattractant protein 1 (MCP1). Administration of recombinant HMGB1 worsened ischemic AKI. Protection against AKI in TLR4-deficient mice was not affected by administration of either anti-HMGB1 antibody nor recombinant HMGB1 demonstrating that endogenous HMGB1 promotes ischemic AKI possibly through the TLR4 pathway (459). In another study, wild-type mice pretreated with recombinant HMGB1 before ischemia were functionally and histologically protected against ischemic AKI associated with less tubulointerstitial neutrophil and macrophage infiltration, and less tubular epithelial cell apoptosis (460). Gene expression of TLR downstream cytokines and chemokines were also significantly reduced. HMGB1 preconditioning provided additional protection to TLR2 but not TLR4 knockout mice. The protective effect of rHMGB1 preconditioning involved Siglec-G upregulation, a negative regulator of HMGB1-mediated TLR4 pathway activation.

NF-κB

NF-κB is a transcription factor that regulates the expression of many genes involved in immune and inflammatory processes and cell survival (461,462). The effect of direct inhibition of NF-κB transcriptional activity on kidney function, kidney inflammation, tubular apoptosis, and necrosis following the administration of cisplatin was determined (463). Mice were treated with JSH-23 (20 or 40 mg/kg) which directly affects nuclear factor-κB (NF-κB) transcriptional activity. Kidney function, tubular necrosis, but not tubular apoptosis and MPO activity were significantly improved by JSH-23 (40 mg/kg). Sixty-one NF-κB-responsive genes were increased by cisplatin of which 21 genes were decreased by JSH-23. Genes that were decreased by JSH-23 that are known to play a role in cisplatin-induced AKI were IL-10, IFN-γ, chemokine [C-C motif] ligand 2 (CCL2), and caspase-1. Another gene, caspase recruitment domain family, member 11 (CARD11), not previously known to play a role in AKI, was increased more than 20-fold and completely inhibited by JSH-23. CXCL1 and TNF-α, known mediators of cisplatin-induced AKI, were decreased by JSH-23. RIPK1 and 3, receptor-interacting serine/threonine-protein kinases, that play an important role in necroptosis, were decreased by JSH-23. Thus, NF-κB transcriptional inhibition in cisplatin-induced AKI ameliorates kidney function and ATN without a significant effect on apoptosis and is associated with a decrease in proinflammatory mediators and CARD11.

Tubular epithelial NF-κB activity regulates ischemic AKI (464). There was widespread NF-κB activation in renal tubular epithelia and in interstitial cells that peaked 2 to 3 days after ischemic AKI. Mice expressing the human NF-κB super-repressor IκBαΔN in renal proximal, distal, and collecting duct epithelial cells were protected against AKI tubular apoptosis, and neutrophil and macrophage infiltration. Primary proximal tubular cells isolated from IκBαΔN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice. The results indicate that in ischemic AKI NF-κB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response (464).

Adiponectin is a multifunctional cytokine that has a role in regulating inflammation. Adiponectin knockout mice were functionally and histologically protected against ischemic AKI (465). Knockout of adiponectin was found to inhibit the infiltration of neutrophils, macrophages, and T cells into the injured kidneys. This was associated with inhibition of NF-κB activation and reduced expression of the proinflammatory molecules IL-6, TNF-α, MCP-1, and MIP-2 in the kidney. Wild-type mice engrafted with adiponectin-null bone marrow had less AKI than adiponectin-null mice engrafted with wild-type bone marrow. Conversely, adiponectin-null mice engrafted with wild-type bone marrow had similar renal dysfunction and tubular damage compared with wild-type mice engrafted with wild-type bone marrow. These results demonstrate that adiponectin plays a role in the pathogenesis of AKI perhaps via NF-κB activation and that adiponectin may be a potential therapeutic target (465).

Uric Acid

The hypothesis has been presented that uric acid, at levels that do not cause tubular obstruction, may contribute to AKI (466). There are a number of mechanisms by which uric acid may contribute to AKI. Uric acid induces inflammation. Uric acid increases production of the chemotactic factor MCP-1 in VSMCs and C reactive protein synthesis in human vascular endothelial and smooth muscle cells (467). Hyperuricemic rats have a significant increase in macrophage infiltration in their kidneys independent of crystal deposition (468). Renal vasoconstriction also occurs in rats with experimentally induced hyperuricemia. The vasoconstriction is caused by an increase in resistance of the afferent (and, to a lesser extent, efferent) arterioles and a reduction in the single-nephron GFR, which can be attenuated by lowering the uric acid with allopurinol (469). The vasoconstriction is reversed by L-arginine, suggesting that a loss of NO in endothelial cells may be the cause of the vasoconstriction (470). In summary, uric acid may have vasoconstrictive, proinflammatory and pro-oxidative properties that could promote the development of AKI.

Adenosine

Extracellular adenosine is derived mainly via phosphohydrolysis of adenosine 5′-monophosphate (AMP) by ecto-5′-nucleotidase (CD73). Extracellular adenosine plays an antiinflammatory role, especially during conditions of limited oxygen availability. The four known adenosine receptor (AR) subtypes (A₁, A_2a, A_2b, A₃) are expressed in the kidney (471). CD73-dependent adenosine production plays a crucial role in the regulation of the tubuloglomerular feedback (472). It has been demonstrated that protection from ischemic AKI in mice by adenosine A2A agonists or endogenous adenosine requires activation of receptors expressed on bone marrow-derived cells (473). In addition, adenosine A2A agonists mediate protection against ischemic AKI by an action on CD4 T cells (474). Activation of the adenosine A1A receptor plays a protective role in ischemic AKI. Adenosine A1 receptor knockout mice demonstrate increased ischemic AKI (471), and adenosine A1 receptor activation inhibits inflammation, necrosis, and apoptosis in ischemic AKI in mice (475). The adenosine A2B receptor antagonist PSB1115 blocks renal protection induced by ischemic preconditioning, whereas treatment with the selective adenosine A2B receptor agonist BAY 60-6583 dramatically improves renal function and histology following ischemia alone (476). Adenosine A2B receptors were exclusively expressed in the renal vasculature (476). Studies using A2BAR bone marrow chimera conferred kidney protection selectively to renal A2BARs. These results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia. Pharmacologic or gene-targeted inhibition of CD 73 abolishes renal protection induced by ischemic preconditioning, and treatment of mice with soluble 5′-nucleotidase restores the renal protection induced by ischemic preconditioning (477). In summary, ARs are novel therapeutic targets in ischemic AKI.

The regulatory function of extracellular adenosine on renal perfusion was investigated (478). Equilibrative nucleoside transporters (ENTs) terminate adenosine signaling and it was observed that ENT inhibition in mice elevated renal adenosine levels and protected against ischemic AKI. ENT1 knockout mice were protected against AKI. Crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented the postischemic no-reflow phenomenon seen in AKI. These studies identified ENT1 and ARs as important in reestablishing renal perfusion following ischemic AKI (478). These studies provide novel insight into the preservation of postischemic renal perfusion (479). Endothelial cell adenosine A2B receptors are antagonized by adenosine reuptake into proximal tubule cells by equilibrative nucleotide transporter 1 that can be inhibited by dipyridamole (479). Adenosine A2B receptor agonists and inhibition of ENTs by dipyridamole may offer therapeutic avenues in ischemic AKI (479).

THERAPEUTIC ROLE OF GROWTH FACTORS

The growth factors insulin-like growth factor (IGF-1), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) are known to bind specific receptors in the proximal tubule and regulate metabolic, transport, and proliferative responses in these cells (480). Studies in this area have fallen into two broad categories: (a) those that have examined the renal expression of genes encoding growth factors or transcriptional factors associated with the growth response that is induced after AKI and (b) those that have examined the efficacy of exogenously administered growth factors in accelerating recovery of renal function in experimental models of AKI (481). EGF, HGF, and IGF-1 accelerate the recovery of renal function and regeneration of damaged proximal tubular epithelium and improve mortality in postischemic rat tubular injury (482–484). IGF-1 attenuates delayed graft function in a canine renal autotransplantation model (485). A relationship between expression of antiapoptotic Bcl-2 genes and growth factors in ischemic AKI in the rat has recently been described (222). It has been demonstrated that antiapoptotic Bcl-2 genes as well as both EGF and IGF-1 are upregulated in the surviving distal tubules and are detected in the surviving proximal tubules, where these growth factors are not usually synthesized (222,486).

The role of epidermal growth factor receptor (EGFR) activation in the recovery from acute ischemic AKI was investigated. Mice with a specific EGFR deletion in the renal proximal tubule—EGFR(ptKO)—were generated (487). Renal function recovery was markedly slowed in EGFR(ptKO) knockout mice. At day 6 after ischemic AKI, there was minimal evidence of injury in the control mice while both EGFR(ptKO) and erlotinib, an EGFR inhibitor, -treated mice still had persistent proximal tubule dilation, epithelial simplification, and cast formation. This study provides both genetic and pharmacologic evidence that proximal tubule EGFR activation plays an important role in the recovery phase after ischemic AKI (487).

A clinical study on IGF-1 in AKI has been performed. The study was designed as a randomized, double-blind, placebo-controlled trial in ICUs in 20 teaching hospitals (488). Seventy-two patients with AKI were randomized to receive recombinant IGF-1 (rhIGF-I) (35 patients) or placebo (37 patients). In this study, rhIGF-I did not accelerate the recovery of renal function in severely ill AKI patients.

HGF is a growth factor that promotes repair and regeneration. Mice with a knockout of the HGF receptor, c-met, had worse cisplatin or ischemic-induced AKI (489). c-met knockout mice had higher serum creatinine, more severe ATN, and increased apoptosis, associated with increased expression of Bax and FasL and decreased phosphorylation/activation of Akt and increased chemokine expression and renal inflammation. Overexpression of HGF in vivo protected against AKI in control mice, but not in Ksp-met(−/−) mice (489).

THERAPEUTIC ROLE OF MESENCHYMAL STEM CELLS

Mesenchymal stem cells (MSCs) have a well-known role in regeneration and immunomodulation. A search of clinicaltrials.gov revealed 40 clinical trials of MSCs in patients with Crohn disease, multiple sclerosis, graft versus host disease, ischemic stroke, organ rejection, cartilage repair, lupus nephritis, and heart disease. Administration of MSCs protects against ischemic AKI in rats (490). In this study, the expression of IL-1β, TNF-α, IFN-γ, and iNOS was significantly reduced by intravenous administration of MSCs. In addition, the beneficial effects of MSCs were found to be mediated by paracrine actions and not by their differentiation into target cells. Human MSCs improve renal function and survival in mice with cisplatin-induced AKI (491). Treatment of mice with autologous and allogeneic MSCs after AKI was safe and reduced renal fibrosis in mice that survived AKI (492). A phase 1 study of MSCs in patients at risk for AKI after cardiac surgery is underway.

The mechanism of the protective effect of MSC therapy in AKI remains unclear. Studies have indicated that MSCs could attenuate inflammation-related organ injury by induction of Tregs. MSCs protected against functional and histologic changes in AKI and downregulated IFN-γ production of T cells in the AKI kidney (493). MSCs increased the percentage of Tregs in the spleen and the ischemic kidney. Antibody-dependent depletion of Tregs blunted the therapeutic effect of MSCs. Coculture of splenocytes with MSCs caused an increase in the percentage of Tregs. Splenectomy abolished attenuation of ischemic injury, and downregulated IFN-γ production and the induction of Tregs by MSCs. Thus, MSCs ameliorate ischemic AKI by inducing Tregs through interactions with splenocytes (493).

Another study investigated the mechanism of the protective effect in AKI of MSCs. Mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs (miRNAs) play a role in protection against AKI (494). Phenotypic changes induced by extracellular vesicles have been implicated in mesenchymal stromal cell-promoted recovery of AKI. miRNAs are potential candidates for cell reprogramming toward a pro-regenerative phenotype. miRNA depletion in mesenchymal stromal cells and extracellular vesicles significantly reduced their intrinsic regenerative potential in AKI, suggesting a critical role of miRNAs in recovery after AKI (494).

COMPLEMENT SYSTEM

There is increased deposition of C3 along the tubular basement membrane in rat and mouse models of ischemic AKI (495). Extrahepatic production of complement proteins, especially by renal tubular epithelial cells, can promote local complement activation and injury. Preclinical studies demonstrate that activation of the complement system is a critical cause of AKI (495). ATN is characterized by activation of the alternative pathway of complement (496). Lack of a functional alternative complement pathway ameliorates ischemic ARF in mice (497). Complement activation within the injured kidney is a proximal trigger of many downstream inflammatory events in ischemic AKI (495). Complement activation may also account for the systemic inflammatory events that contribute to remote organ injury and patient mortality (495). Complement inhibitory drugs that have entered clinical studies may provide a therapeutic or preventive approach for patients with AKI.

MICRORNAS

miRNAs are short RNAs that regulate gene expression. miRNAs suppress the expression of target genes by binding to the 3′ untranslated regions and inducing repression or degradation of target mRNAs, resulting in reduced protein expression (498). miRNAs play a role in homeostasis as well as causing certain pathological processes. Dicer is a key enzyme in miRNA biogenesis. Mice with a proximal tubular cells knockout of Dicer were markedly protected against ischemic AKI with better renal function, less tubular apoptosis, and better survival compared with wild-type littermates (498). Microarray analysis showed demonstrated that some miRNAs were induced and others were downregulated. Notably, miRNA-132, -362, and -379 showed a continuous change during 12 to 48 hours of reperfusion. miRNA 687 (miR-687) as a key regulator and therapeutic target in ischemic AKI (499). miR-687 is markedly upregulated by HIF-1 in the kidney in ischemic AKI in mice and in hypoxic cultured kidney cells. miR-687 repressed the expression of phosphatase and tensin homolog (PTEN) and facilitated cell cycle progression and apoptosis. Inhibition of miR-687 preserved PTEN expression and attenuated cell cycle activation and renal apoptosis, resulting in functional and histologic protection against ischemic AKI.

An miR integrative network regulating toxicant-induced AKI has been discovered (500). miR-122 was mostly downregulated by cisplatin, whereas miR-34a was upregulated. Foxo3 was identified as a core protein to activate p53. The miR-122 inhibited Foxo3 translation as assessed using an miR mimic, an inhibitor, and a Foxo3 3′-UTR reporter. The role of decreased miR-122 in inducing Foxo3 was confirmed by the ability of the miR-122 mimic or inhibitor to replicate results. Increase in miR-34a also promoted the acetylation of Foxo3 by repressing Sirt1. Cisplatin facilitated the binding of Foxo3 and p53 for activation, which depended not only on decreased miR-122 but also on increased miR-34a. These studies also identified Foxo3 as a bridge molecule to the p53 pathway.

p53 is renoprotective in ischemic AKI by reducing inflammation (501). p53-knockout mice (p53(−/−)) had worse kidney injury, compared with wild-type mice, and had increased and prolonged infiltration of leukocytes. Acute inhibition of p53 with pifithrin-α in wild-type mice mimicked the observations in p53(−/−) mice. Chimeric mice that lacked p53 in leukocytes sustained injury similar to p53(−/−) mice, suggesting an important role for leukocyte p53 in ischemic AKI. A smaller proportion of macrophages in the kidneys of p53(−/−) and pifithrin-α-treated mice were the antiinflammatory M2 phenotype. Leukocyte p53 is protective by reducing the extent and duration of this inflammation and by promoting the antiinflammatory M2 macrophage phenotype (501).

miRNA-489 induction by HIF-1α protects against ischemic AKI (502). There is miRNA-489 induction in ischemic AKI kidneys. HIF-1α deficiency was associated with decreased miRNA-489 induction in cultured rat proximal tubular cells subjected to hypoxia and kidney tissues of mice after AKI. Inhibition of miRNA-489 increased apoptosis in renal tubular cells after ATP depletion injury in vitro. In mice, inhibition of miRNA-489 enhanced tubular cell death and ischemic AKI. On deep sequencing analysis, there were 417 mRNAs recruited to the RNA-induced silencing complex by miRNA-489, of which 127 contained miRNA-489 targeting sites. Sequence analysis and in vitro studies validated poly(ADP-ribose) polymerase 1 as a miRNA-489 target. These results demonstrate that miRNA-489 is induced via HIF-1α during ischemic AKI (502).

miRNA-24 inhibition reduces ischemic AKI (503). miR-24 was upregulated in the kidney in mice in ischemic AKI and in patients after kidney transplantation. There was specific miR-24 enrichment in renal endothelial and tubular epithelial cells in AKI. Transient overexpression of miR-24 alone in hypoxic cells induced apoptosis whereas silencing of miR-24 ameliorated apoptotic responses. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites rescued functional parameters in endothelial and tubular epithelial cells. In vivo, silencing of miR-24 in mice before ischemic AKI resulted in a significant improvement in survival and kidney function, a reduction in apoptosis, improved ATN scores, and less inflammatory cell infiltration in the kidney. Overall, these results indicate miR-24 promotes ischemic AKI by stimulating apoptosis in endothelial and tubular epithelial cells (503).

Hematopoietic miRNA-126 protects against renal I/R injury by promoting vascular integrity (504). Hematopoietic overexpression of miR-126 increased neovascularization of subcutaneously implanted Matrigel plugs in mice. In ischemic AKI in mice, overexpressing miR-126 resulted in a marked decrease in urea levels, fibrotic markers, and injury markers (KIM-1 and NGAL). The protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. These results demonstrate that overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotion of vascular integrity that supports recovery of the kidney after IRI (504).

ABNORMAL REPAIR OF INJURED PROXIMAL TUBULES AND THE PROGRESSION FROM AKI TO CKD

Epidemiologic and mechanistic studies suggest that the AKI and CKD are not distinct entities but rather are closely interconnected—CKD is a risk factor for AKI, AKI is a risk factor for the development of CKD, and both AKI and CKD are risk factors for cardiovascular disease (505). In a large, community-based cohort of 556,090 adult patients with preexisting normal or near-normal kidney function, an episode of dialysis-requiring ARF was a strong independent risk factor for a long-term risk of progressive CKD and mortality (506). There was a 28-fold increase in the risk of developing Stage 4 or 5 CKD and more than a twofold increased risk of death after dialysis-requiring AKI (506).

The potential mechanisms of the transition from AKI to CKD have been studied in rodent models of AKI. Tubular epithelial cells undergo apoptosis or necrosis or sloughing from the basement membrane in AKI. The surviving cells dedifferentiate, migrate along the basement membrane, proliferate to restore cell number, and then differentiate, in order to restore the functional integrity of the nephron (262). Loss of peritubular microvessels and the chronic activation of macrophages may contribute to interstitial fibrosis (262). The molecular switch that determines whether injured tubular cells undergo repair or a fibrotic response is not known. Epithelial-mesenchymal transition (EMT) may be a major pathways toward fibrosis in other organs (507). However, unequivocal evidence that EMT is a pathway toward renal fibrosis is lacking (508). The injured epithelial cell can produce profibrotic cytokines like TGF-β. In ischemic, toxic, and obstructive models of AKI, a causal association between epithelial cell cycle G2/M arrest and a fibrotic outcome has been demonstrated (509). G2/M-arrested proximal tubular cells activate c-jun NH(2)-terminal kinase (JNK) signaling, which acts to upregulate profibrotic cytokine production and treatment with a JNK inhibitor, or bypassing the G2/M arrest by administration of a p53 inhibitor or the removal of the contralateral kidney, rescues fibrosis in the unilateral ischemic injured kidney (509). Atrophic tubules that are not recovering have increased signaling of PI3K-Akt-mTOR, ERK-MAPK, JNK-MAPK, and TGF-β pathways, with markedly increased expression of profibrotic peptides PDGF-B, CTGF, and TGF-β (510). In vitro and in vivo studies demonstrate that increased profibrotic TGF-β signaling in tubules recovering from AKI is, in part, attributable to autocrine signaling by lysophosphatidic acid (510). Lysophosphatidic acid signaling acts through separate G protein-coupled receptors triggering αvβ6 integrin-dependent activation of latent TGF-β as well as transactivation of EGFR and ERK-MAPK (510).

Endothelin plays a role in AKI to CKD transition. Unilateral ischemia caused progressive renal ET-1 protein/mRNA increases with concomitant endothelin A (ETA), but not endothelin B (ETB), mRNA elevations (511). Unilateral ischemia produced progressive renal injury as indicated by severe histologic injury and a 40% loss of renal mass. Pre- or postischemic treatment with the ETA receptor antagonist atrasentan produced protective effects such as decreased tubule/microvascular injury, normalized tissue lactate, and total preservation of renal mass. On the other hand, ETB blockade had no protective effect. These findings provide the first evidence that ET-1 operating through ETA can have a critical role in progression of ischemic AKI to CKD (511).

It is not known whether injury of epithelial cells, endothelial cells, or inflammatory cells plays a role in the AKI to CKD transition. A mouse model of kidney injury using the Six2-Cre-LoxP technology to selectively activate expression of the simian DT receptor in renal epithelia was developed (512). By adjusting the timing and dose of DT, a highly selective model of tubular injury was studied. The DT-induced sublethal tubular epithelial injury was confined to the S1 and S2 segments of the proximal tubule. Acute injury was promptly followed by inflammatory cell infiltration and robust tubular cell proliferation, leading to complete recovery after a single toxin insult. In striking contrast, three insults to renal epithelial cells at 1-week intervals resulted in maladaptive repair with interstitial capillary loss, fibrosis, and glomerulosclerosis, which was highly correlated with the degree of interstitial fibrosis. The study concluded that selective epithelial injury can drive the formation of interstitial fibrosis, capillary rarefaction, and glomerulosclerosis, confirming a direct role for damaged tubule epithelium in the pathogenesis of CKD (512).

The presence of CKD makes AKI worse in mice (513). Sepsis-induced AKI by cecal ligation was induced in mice with a 5/6 nephrectomy mouse model of progressive CKD. The presence of CKD intensified the severity of kidney and liver injury, cytokine release, and splenic apoptosis. Accumulation of HMGB1, VEGF, TNF-α, IL-6, or IL-10 was increased in CKD or sepsis alone and to a greater extent in CKD-sepsis. Although VEGF neutralization with soluble fms-like tyrosine kinase 1 (sFLT-1) (a soluble VEGF receptor) effectively treated sepsis, it was ineffective against CKD-sepsis. A single dose of HMGB1-neutralizing antiserum did not protect against sepsis alone but attenuated CKD-sepsis. Splenectomy transiently decreased circulating HMGB1 levels, reversing the effectiveness of anti-HMGB1 treatment on CKD-sepsis. The study concluded that CKD increases the severity of sepsis, in part, by reducing the renal clearance of several cytokines. CKD-induced splenic apoptosis and HMGB1 release were found to be mediators for both CKD and sepsis (513).

Blockade of cysteine-rich protein 61 attenuates renal inflammation and fibrosis after ischemic kidney injury (514). In unilateral renal ischemia, increased expression of Cyr61 was detected, predominately in the proximal tubular epithelium. Treatment of mice with an anti-Cyr61 antibody attenuated the upregulation of kidney MCP-1, IL-6, IL-1β, and macrophage inflammatory protein-2, and reduced the infiltration of F4/80-positive macrophages on days 7 and 14 after IRI and reduced expression of collagen, TGF-β, and plasminogen activator inhibitor-I as well as the degree of collagen fibril accumulation, as evaluated by picrosirius red staining, and levels of α-smooth muscle actin proteins by day 14. Treatment of mice with an anti-Cyr61 antibody preserved peritubular microvascular density on day 14. It was concluded that Cyr61 blockade inhibits the triad of inflammation, interstitial fibrosis, and capillary rarefaction after severe ischemic AKI, mechanisms that underlie the AKI to CKD transition (514).

Diagnosis of AKI

THE BIOLOGY BEHIND BIOMARKERS FOR THE DIAGNOSIS AND PROGNOSIS OF AKI

BUN and serum creatinine that are currently used for the diagnosis of AKI are not very sensitive and specific markers of kidney function in AKI as they are influenced by many renal and nonrenal factors independent of kidney function. Biomarkers that are released into the blood or urine by the injured tubular cells and are analogous to the troponin release by injured myocardial cells after myocardial ischemia or infarction have been studied in detail for the early and more specific diagnosis of AKI. Biomarkers of AKI that have been detected in the urine or serum of patients with AKI and that increase before serum creatinine in AKI include urine IL-18, urine NGAL, urine KIM-1 and urine liver-type fatty acid-binding protein (L-FABP), urinary TIMP2 and IGFBP7 (which is known as NephroCheck—the first FDA-approved biomarker of AKI).

IL-18 is a proinflammatory cytokine that plays a role in both the innate and acquired immune response (515,516). Immunohistochemistry of mouse kidneys demonstrated an increase in IL-18 protein in injured tubular epithelial cells in AKI kidneys compared to normal controls. It was also determined that hypoxic proximal tubules had high levels of IL-18 (154). Urine IL-18 was increased in mice with ischemic AKI compared to sham-operated mice (158).

NGAL is a 21-kDa protein of the lipocalin superfamily. NGAL is a critical component of innate immunity to bacterial infection and is expressed by immune cells, hepatocytes, and renal tubular cells in various disease states (517). NGAL is a small secreted polypeptide that is protease resistant and thus may be easily detected in the urine. NGAL protein increases massively in the renal tubules and in the first urine output after ischemic AKI in rats and mice (518).

KIM-1 is a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain. KIM-1 mRNA and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney (258). Urinary KIM-1 is a noninvasive, rapid, sensitive, and reproducible biomarker for the early detection of both cisplatin-induced AKI and ischemic AKI in rats (519).

Cystatin C is a 13-kDa protein produced by all nucleated cells. It is freely filtered by the glomerulus, completely reabsorbed by the proximal tubules and is not secreted by the renal tubules (520). Thus some of the limitations of serum creatinine, for example, effect of muscle mass, diet, gender, and tubular secretion may not be a problem with cystatin C. Cystatin C is best measured by an immunonephelometric assay. Cystatin C is a better marker of GFR than serum creatinine as demonstrated in the following studies (521,522–524).

L-FABPs are a family of carrier proteins for fatty acids and other lipophilic substances such as eicosanoids and retinoids. FABPs facilitate the transfer of fatty acids between extra- and intracellular membranes. Urinary L-FABP was increased in mice with ischemic AKI and cisplatin-induced AKI (525). L-FABP was evaluated as a biomarker of renal ischemia in both human kidney transplant patients and a mouse model of AKI (526).

TIMP2 is a human gene. TIMP2 is a member of a gene family that encodes proteins that are natural inhibitors of the MMP. Metalloproteinases are peptidases that play a role in degradation of the extracellular matrix. IGFBP7 regulates the availability of insulin-like growth factors in tissues and modulates IGF binding to its receptors. IGFBP7 stimulates cell adhesion and cancer growth. TIMP2 and IGFBP7 are also markers of cell cycle arrest. Renal tubular cells enter a period of G1 cell cycle arrest after ischemia or sepsis (527). It is proposed TIMP2 and IGFBP7 are expressed in the tubular cells in response to DNA damage and other forms of injury. TIMP2 and IGFBP7 block the effect of the cyclin-dependent protein kinase complexes on cell cycle promotion which results in G1 cell cycle arrest for short periods of time to prevent damaged cells from dividing (528).

BIOMARKERS FOR THE EARLY DIAGNOSIS OF AKI

See Table 10-6.

Urine IL-18

Studies in humans demonstrated that urine IL-18 is an early predictive biomarker of AKI (529). The TRIBE-AKI (Translational Research Investigating Biomarkers in Early Acute Kidney Injury) Clinical Consortium was established to hasten the development of biomarkers. In the TRIBE-AKI study, urine IL-18 and NGAL were studied as early biomarkers of AKI in a prospective multicenter observational cohort study of 1,219 patients receiving cardiac surgery (530). It was demonstrated that urine IL-18, urine NGAL, and plasma NGAL associate with subsequent AKI and poor outcomes. Urine IL-18 and urine and plasma NGAL levels peaked within 6 hours after surgery. After multivariable adjustment, the highest quintiles of urine IL-18 and plasma NGAL associated with 6.8-fold and fivefold higher odds of AKI, respectively, compared with the lowest quintiles. Elevated urine IL-18 and urine and plasma NGAL levels associated with longer length of hospital stay, longer ICU stay, and higher risk for dialysis or death. Urine IL-18 and plasma NGAL significantly improved the area under the receiver operating characteristic (ROC) curve to 0.76 and 0.75, respectively.

Urine NGAL

The usefulness of NGAL as an early biomarker of AKI was reviewed in a meta-analysis. Fifty-eight manuscripts reporting on NGAL as a biomarker of AKI in more than 16,500 patients were analyzed (531). Following cardiac surgery, NGAL measurement in over 7,000 patients was predictive of AKI and its severity, with an overall area under the ROC curve of 0.82 to 0.83. Similar results were obtained in over 8,500 critically ill patients. In over 1,000 patients undergoing kidney transplantation, NGAL measurements predicted delayed graft function with an overall area under the curve (AUC) of 0.87. In all three settings, NGAL significantly improved the prediction of AKI risk over the clinical model alone.

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