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, 8, 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, 8, 9).
The Acute Kidney Injury Network (AKIN) has also developed a classification of AKI (8, 9, 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, 13, 14, 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).
 Figure 10-1 RIFLE criteria for the classification of AKI. RIFLE includes three grades of severity of AKI (risk, injury, and failure) and two outcome variables (loss of function and end-stage kidney disease). The RIFLE criteria attempt to convey the notion that kidney injury occurs before kidney failure. Studies have demonstrated that as the RIFLE class goes up, so does mortality. RIFLE, Risk, Injury, Failure, Loss of Kidney Function, and End-stage kidney disease; SCr, serum creatinine; AKI, acute kidney injury. |
Table 10-1 AKIN and KDIGO Classification of AKI | ||||||||||||||||||
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Causes of AKI
INTRARENAL OR INTRINSIC AKI
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, 18, 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).
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, 18, 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).
Table 10-2 Conditions That Cause “Intrinsic” or Parenchymal AKI | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Pathogenesis of AKI
THE NATURE OF PROXIMAL TUBULAR INJURY
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, 23, 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, 29, 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.
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).
Table 10-3 Mediators/Mechanisms of Ischemic AKI | |||||||||||
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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, 60, 61, 62, 63, 64, 65, 66, 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, 72, 73, 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 19F NMR and 5F 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, 94, 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, 105, 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, 110, 111, 112). Calpain plays a role in hypoxic injury to rat renal proximal tubules (113, 114, 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).
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.
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, 114, 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).
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 (&aacgr;-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 &aacgr;-MSH on the neutrophil pathway by examining the effects of &aacgr;-MSH in ICAM-1 knockout mice and the neutrophil-independent isolated perfused kidneys (181, 182). In this study, it was found that &aacgr;-MSH decreases renal injury when neutrophil effects are minimal or absent, indicating that &aacgr;-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).
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, 208, 209, 210, 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, 29, 30, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 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).
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).
Table 10-4 Erythropoietin Protects against AKI | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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, 248, 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, 260, 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).
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, 270, 271, 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).
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, 285, 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, 292, 293, 294, 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.
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.
Table 10-5 Factors That Modify Vascular Tone | ||||
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Normal Vascular Tone and Reactivity
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.
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, 310, 311, 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.
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.
an actual reduction in eNOS activity or to an actual spontaneous maximal NOS/NO activity that cannot be stimulated further by endothelium-dependent agents.
ROLE OF VASODILATORY SUBSTANCES
Endogenous vasodilators are involved in the hemodynamic changes that both initiate and maintain AKI.
In this section, the roles of endogenously generated vasodilators in the pathophysiology of ischemic, septic, and nephrotoxic AKI will be considered, as well as the therapeutic use of vasorelaxing substances in animal models and in clinical AKI.
Prostaglandins
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.
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