prevented experimental AKI, decreasing apoptosis and improving renal function.

Attachment to a normal basement membrane will prevent anoikis in podocytes and tubular cells. Some disease processes are characterized by hereditary (e.g., Alport syndrome caused by mutations in type IV collagen genes) or acquired (e.g., diabetic nephropathy) alterations of basement membrane matrix proteins. An altered basement membrane may contribute to reduced podocyte survival, and similar processes may be operative for tubular cells. The recovery of live podocytes from urine suggests that detached podocytes may be rescued from anoikis if an appropriate microenvironment is restored. Extracellular matrix proteins activate survival mechanism dependent on focal adhesion kinase and Ras-ERK signaling pathway. In addition to basement membrane changes, podocyte or tubular cell injury may lead to impaired function of receptors for extracellular matrix proteins. In podocytes, α3β1 integrin, α-actinin-4, and the dystroglycan complex are required for podocyte survival by facilitating adhesion to the glomerular basement membrane. α3β1 integrin is decreased in podocytes from humans and rats with diabetes, and high glucose media decreases the expression of α3β1 integrin via TGFβ in cultured podocytes. During AKI injured tubular cells lose polarity, dedifferentiate, and detach. The requirement for a normal extracellular matrix is also observed in mesangial cells. Laminin protects rat mesangial cells from apoptosis induced by serum starvation and DNA damage by a β1 integrin– mediated mechanism.

Novel relevant antiapoptotic molecules for renal cells have been recently identified. Cyclin I has a survival role in podocytes. By binding to CDK5, cyclin I increases BclxL and Bcl2 expression and decreases BAD expression. Cyclin I knockout podocytes were more susceptible to apoptosis both in vitro and in vivo through stabilization of p21. In addition, the CDK2 inhibitor p27kip1 has been related to apoptosis. Indeed, p27kip1–/– mice develop more intense tubular apoptosis after ureteral obstruction as well as more severe glomerulonephritis. Survivin, an inhibitor of apoptosis protein, has recently been identified as a constitutive prosurvival molecule in tubular cells that protects from experimental AKI.

4.2. Lethal factors

Cytokines, ischemia, endogenous toxic metabolites, or exogenous toxins may cause renal cell death in the complex environment of the injured kidney. Cytokines and hyperglycemia may induce apoptosis of glomerular and tubular cells. However, the main target of

ischemia-reperfusion injury and xenobiotics are tubular cells, especially proximal tubular cells, as a result of the presence of transporters that favor the intracellular accumulation of toxins and the high number of mitochondria to fuel molecular transport. Endothelial cells have been less studied, but they may also succumb to cytokines, ischemia/reperfusion toxic metabolites, and xenobiotics.

4.2.1. TNF superfamily cytokines

TNFα, FasL, TRAIL, and TNF-like weak inducer of apoptosis (TWEAK) can induce, depending on the microenvironment, apoptosis of mesangial cells, tubular epithelial cells, podocytes, and renal endothelial cells. The importance of cooperation between lethal factors has been underscored by the analysis of complex biological systems. Changes in the level of expression or activation of apoptosis regulatory molecules may explain the cooperation of cytokines in inducing cell death. As an example, TNFα increases the expression of TWEAK receptor, Fas, Bax, and Smac/DIABLO while decreasing that of BclxL in tubular epithelium. In tubular cells, TNFα-induced apoptosis is facilitated by deprivation of survival factors. FasL requires the upregulation of Fas receptor expression by survival factor deprivation or by the presence of an inflammatory milieu. By contrast, Fas activation induces death in nonstimulated mesangial cells in vitro and in vivo. TWEAK alone induces mesangial cell apoptosis, but not tubular cell death, that requires the concomitant presence of TNFα and interferon-γ (IFNγ). TRAIL is the most upregulated TNF superfamily gene in diabetic nephropathy tubulointerstitium. TRAIL is more lethal for tubular cells in a high-glucose inflammatory milieu. However, the most studied lethal cytokine is FasL. A number of apoptotic factors or settings involved in the pathogenesis of renal injury upregulate Fas expression in renal cells, and at least some of them render the cells more susceptible to FasL-induced apoptosis: cytokines (TNFγ, IFNγ, interleukin [IL] 1β, IL-1α), bacterial lipopolysaccharide (LPS), nephrotoxins, HIV infection, and deprivation of survival factors (Figure 22-2). Plasma from patients with thrombotic microangiopathy induces apoptosis and Fas expression in renal microvascular endothelial cells. In mesangial and tubular epithelial cells, protein synthesis inhibitors induce apoptosis and also sensitize to apoptosis mediated by the death receptors TNFR and Fas; these finding suggest that ongoing synthesis of protective proteins is required to prevent programmed cell death.

Tubular Fas-associated death domain protein (FADD) is upregulated in experimental AKI. FADD-DD is a

Bacterial products

Viral infection

Cytokines

(TNFγ, IFNγ, Fas

IL-1β, IL-1α)

Deprivation

of survival

factors

Nephrotoxins

Figure 22-2. The microenvironment modulates the sensitivity of renal cells to lethal stimuli. The influence of microenvironmental factors on the expression of Fas and the sensitivity to FasL-induced death has been extensively studied in renal cells. Inflammation (cytokines), viral (HIV) or bacterial (LPS) infection, nephrotoxins (cyclosporin A, acetaminophen), and deprivation of survival factors increase Fas expression in renal cells, and, with the exception of nephrotoxins, sensitivity to FasL.

truncated molecule corresponding to the death domain (DD) of FADD that behaves as a FADD antagonist in some cell systems. Surprisingly, in tubular cells, FADDDD is sufficient to promote a caspase-independent form of cell death. This is consistent with a role for FADD in death receptor–independent events.

4.2.2. Other cytokines

Several cytokines may induce apoptosis by triggering the intrinsic pathway of apoptosis independently of death receptors. Two key mediators of renal injury, TGFβ1 and angiotensin II, may induce apoptosis in renal tubular epithelial cells and podocytes.

The expression of TGFβ1 and its receptors is increased in a variety of glomerular diseases characterized by podocyte injury and proteinuria, including membranous nephropathy, diabetic nephropathy, and focal segmental glomerulosclerosis. Podocytes secrete TGFβ1 in response to several agents, such as high glucose, lowdensity lipoprotein (LDL), or thrombin. TGFβ1-induced apoptosis requires activation of p38 MAP kinase and engages several downstream mediators. SMAD-7, Bax synthesis, and caspase-3 activity are increased in TGFβ- induced apoptosis. The proapoptotic effects of Smad7 over-expression and of TGFβ1 are additive. However, unlike TGFβ1, Smad7 inhibits the nuclear translocation and transcriptional activity of the cell survival factor nuclear factor kappa B (NF-κB). The cyclin-dependent kinase inhibitor p21 is also increased in podocytes in experimental membranous nephropathy and diabetic nephropathy models. TGFβ1 increases p21 levels in

cultured podocytes and, in turn, p21 prevents the compensatory upregulation of antiapoptotic Bcl-2 that takes place under disease conditions to improve the chances of survival. The fact that p21-null podocytes were protected from TGFβ1-induced apoptosis supports a critical role for p21 in TGFβ1-induced apoptosis. In addition, TGFβ1 impairs the adhesion to the glomerular basement membrane by downregulating the expression of α3β1 integrin. TGFβ1 may also induce tubular cell apoptosis and epithelial-mesenchymal transition.

Angiotensin II is a mediator of stress tension (induced by mechanical stretch)–induced podocyte apoptosis and directly causes podocyte apoptosis through activation of the AT1 receptor.

4.2.3. Glucose

Besides the proapoptotic actions of cytokines expressed in diabetic tissues, hyperglycemia directly induces apoptosis in cultured podocytes and tubular cells (Figure 22-1). Glucose may also sensitize to cell death induced by other stimuli by upregulating Bax and Basp1 and downregulating BclxL in tubular cells. A further mechanism of podocyte loss in diabetes may relate to the detachment of podocytes from an abnormal glomerular basement membrane. Activation of poly (ADP ribose) polymerase (PARP) plays an important role in the pathophysiology of various diseases associated with oxidative stress, such as diabetes. PARP inhibitors blocked hyperglycemia-induced podocyte apoptosis in vitro. In addition, glucose degradation products, such as 3,4-dideoxyglucosone-3-en (3,4-DGE), induce Baxdependent apoptosis in tubular cells and podocytes.

Other agents whose role in glomerular injury is less well characterized also promote renal cell apoptosis. Oxidized LDL induced apoptosis in human cultured podocytes by reducing Akt activity. Reactive oxygen species themselves promote apoptosis of renal cells.

4.2.4. Drugs and xenobiotics

There are multiple nephrotoxic drugs. However, for some of them, nephrotoxicity is the dose-limiting side effect. Examples include the immunosuppressant CsA, the aminoglycoside antibiotics, the antifungal amphotericin B, the antiviral cidofovir, and the antineoplastic cisplatin. All of them may cause AKI and CKD. In addition, acetaminophen overdoses may cause AKI. The study of the molecular mechanisms engaged by nephrotoxins that induce AKI and cultured tubular cell apoptosis has disclosed stimulus-specific pathways that may lead to specific interventions (Figure 22-3).

Lysosomes

Nucleus

ER

Acetaminophen

Cisplatin

CsA increases Fas expression in tubular cells in culture and in vivo. However, neither neutralizing anti-FasL antibodies nor caspase-8 inhibitors decreased apoptosis induced by CsA. Similar observations were made for acetaminophen. This suggests that some changes in apoptosis-related molecules are epiphenomena not directly related to cell death. By contrast, Bax-mediated mitochondrial injury and caspase activation are key events in CsA-induced apoptosis of tubular cells. CsA induces Bax aggregation and translocation to mitochondria, causing mitochondrial outer membrane permeabilization, release of cytochrome c and Smac/DIABLO, and activation of caspases-9 and -3. Initiator caspase-2 is also activated and may lead to mitochondrial injury. In a positive feedback loop, caspases further damage the mitochondria, leading to loss of mitochondrial transmembrane potential. The feedback loop is essential for apoptosis and cell death to proceed because caspase inhibitors prevented both. This is one of several models for the participation of mitochondrial injury in apoptosis. Bax antisense oligodeoxynucleotides prevent CsAinduced apoptosis. Bax is also required for apoptosis and cell death induced by 3,4-DGE, a toxic glucose metabolite. CsA is a potent inhibitor of macrophage apoptosis through the inhibition of inducible nitric oxide synthase, illustrating cell-specific pathways.

Acetaminophen induces caspase-dependant apoptosis of tubular cells without characteristic mitochondrial alterations or Bax involvement. Acetaminophen

not from eventual cell death. By contrast, BcxL protected tubular cells from death. BclxL interacts with several ER proteins. CsA increased CHOP/GADD153 expression but failed to activate caspase-12, suggesting that CHOP upregulation may be induced by non-ER stressors. The ER stressor tunicamycin induced severe histological tubular injury, which was decreased both in CHOP/GADD153 and caspase-12 knockout mice. Although these studies serve as a proof of concept for the relevance of ER stress in tubular injury, tunicamycin has no direct clinical relevance. In a more clinically relevant model, ischemia/reperfusion, ORP150 (150-kDa oxygen-regulated protein), an inducible ER chaperone, was upregulated in tubular epithelium and shown to protect from ischemia/reperfusion or hypoxia.

Aminoglycoside nephrotoxicity is an example of lysosomal participation in apoptosis. Lysosomal accumulation of gentamicin may initially prevent its more toxic cytosolic localization. Eventually, lysosomal membrane permeabilization releases free gentamicin to the cytosol and/or releases other lysosomal components that trigger a Bax-mediated mitochondrial pathway of apoptosis.

The proapoptotic role of p53 has been characterized in cisplatin nephrotoxicity. Cisplatin damages genomic DNA and markedly induces p53 expression and phosphorylation. Pifithrin-α inhibits transcriptional and nontranscriptional activities of p53 and protects tubular cells in culture and in vivo. p53