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CAVEAT LECTOR
Alberto Ortiz, Silvia González Cuadrado, Corina Lorz, Jesús Egido.
Division of Nephrology. Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain.
Received 12/01/95; Accepted 01/26/96; On-line 03/01/96
The occurrence of apoptosis has been demonstrated in several renal diseases. However, there are numerous unanswered questions regarding the precise role of apoptosis in renal damage and the extracellular and intracellular factors that induce and prevent apoptosis in the kidney. The possible therapeutic value of the modulation of renal cell apoptosis in kidney diseases has not been adequately investigated yet.
4.1 ROLE OF APOPTOSIS IN RENAL DISEASE
Both apoptosis of intrinsic renal cells and of infiltrating leukocytes may contribute to the pathogenesis of renal disease.
4.1.1. Apoptosis as a mechanism of depletion of intrinsic renal cells.
Apoptosis may play a role in the loss of parenchymal cells at several stages of renal damage. The correct knowledge of the contribution of apoptosis to each of these stages in different renal pathologies is required for the design of therapeutic strategies.
Apoptosis triggered by ischemia, exogenous toxins or endogenous mediators of damage may be the initial insult capable of causing renal disease. Apoptosis may also contribute to the persistence of renal injury. Thus, foci of inflammation in response to other stimuli may render the microenvironment inappropriate to cell survival. Resolution of hypercellularity in proliferative glomerulonephritis or during the recovery phase of acute renal failure may result from apoptosis of the redundant cells. Progression of renal disease may be a consequence of a persistently high apoptotic rate of renal parenchymal cells leading to glomerular or tubular atrophy. Alternatively, a low rate of fibroblast apoptosis may promote renal fibrosis.
4.1.2. Apoptosis in the regulation of inflammation in kidney diseases
Many renal diseases are characterized by a mononuclear cell infiltrate composed mainly of monocytes/macrophages and T cells (138,139). Less frequently, a full blown inflammatory response that includes other leukocytes such as neutrophils and eosinophils is observed (140). Inflammatory cells may provide factors that cause parenchymal cell apoptosis. Monocytes/macrophages release TNF-alpha, Fas ligand, oxygen radical species, and nitric oxide (29,141). In contrast, the combination of perforin/granzyme B and Fas ligand accounts for most of the cytotoxicity of T lymphocytes (55).
In addition, apoptosis may stimulate or quench the inflammatory response. In the following sections the reciprocal interactions of apoptosis and inflammation are discussed.
4.1.2.1. Can apoptosis cause inflammation?
There is a harbored notion that apoptosis does not generate inflammation. However, generation of apoptosis and inflammation may be associated. Some factors, such as TNF-alpha, induce both apoptosis and chemotactic factors that lead to the recruitment of leukocytes (20,21,139,142). For example, in tubular cells the dose response curve for TNF-alpha-induced rantes mRNA expression and apoptosis are similar (21,142). Apoptosis itself might promote inflammation through two mechanisms: 1) Lysis of the apoptotic cells. Disintegration of apoptotic cells with release of non-specific pro-inflammatory factors may be a consequence of a failure of the recognition/engulfing mechanism. The presence of a low pH, cationic molecules and fragments of extracellular matrix proteins interfere with the uptake of apoptotic cells by phagocytes (143-145). The reactivity of antiphospholipid antibodies with the phosphatidylserine exposed on the surface of apoptotic cells might also interfere with their clearance (146). Massive apoptosis occurring in an organ not physiologically prepared for such an event may also lead to failure of apoptotic cell clearance. For example, apoptotic cell lysis has been observed after Fas activation in the liver (52). 2) Active release of proinflammatory cytokines. The genetic program activated during apoptosis may provide specific chemotactic substances for phagocyte recruitment, as it does provide new surface determinants for recognition and phagocytosis. There is scattered evidence for this notion. ICE is a component of the apoptotic machinery and it also activates the proinflammatory cytokine IL-1ß (96,106). In fact, macrophages undergoing apoptosis, but not those undergoing necrosis, process IL-1ß (147). Cell recruitment may also depend on binding of apoptotic bodies to specific receptors on the surface of monocytes with resulting cell activation and cytokine release (148).
4.1.2.2. Regulation of inflammation by apoptosis.
Clearance of inflammatory cells by apoptosis may contribute to resolution of inflammation and failure of this clearance may contribute to the persistence of the inflammatory process. Leukocytes, including neutrophils, macrophages and lymphocytes undergo apoptosis if there is not an adequate amount of survival factors (149-152). This phenomenon has been observed in vivo in the resolution of renal inflammation (153,154). Apoptotic leukocytes may be engulfed by local cells, like mesangial cells (153). Survival factors for leukocytes may differ from those for renal cells. TNF-alpha, for example, prevents apoptosis in monocytes, but kills renal cells (20,21,151). Cytokines expressed by renal cells, such as TNF-alpha and Fas ligand (27,29), may contribute to the progression of renal disease through prolonged survival or activation of macrophages and T lymphocytes (58,151,152).
4.1.3 Apoptosis and the immune response
Apoptosis plays a fundamental role in the control of the immune response in the thymus and the periphery (12). While the details of this involvement are beyond the scope of this review, it should be noted that alterations in apoptosis-related genes such as bcl-2 and fas result in autoimmune diseases and renal damage (53,60-63,121). Apoptosis by itself may generate autoimmunity. In effect, autoreactivity has been recognized against antigens present in apoptotic cells (155). If apoptotic cells are not adequately cleared, their contents might be released and further stimulate this autoimmune response (156). It has been suggested that this may help explain the relationship between infection and the initiation/exacerbation of autoimmunity, as infection can trigger apoptosis (4).
4.2. EXPRESSION OF APOPTOSIS GENES IN RENAL DISEASE
In this section we will review information regarding the role of apoptosis and apoptosis genes in different forms of renal damage.
Cell turnover in the healthy glomerulus is low. Apoptotic cells represent about 0.01% of rat glomerular cells (22) and 0.03/10 glomerular cross-sections in humans (157). During renal injury apoptosis may contribute to the clearance of excessive intrinsic glomerular cells and leukocytes (158). The resolution of the mesangial proliferation characteristic of anti-Thy-1 nephritis depends on apoptosis of excessive mesangial cells, which peaks at 0.25% of glomerular cells (22). Apoptosis of neutrophils is prominent in nephrotoxic nephritis in the rat (140,153) and in acute postinfectious glomerulonephritis in humans (157). More recently, apoptosis has been noted in the first hours (<12h) after induction of anti-Thy1 nephritis (159,160). Together with the ability of anti-Thy1 to induce apoptosis of cultured mesangial cells (31,159), this suggests that apoptosis may also cause glomerular injury. An increased occurrence of apoptosis is also present in several experimental models of progressive glomerular scarring that include the nephropathy seen in growth hormone transgenic mice, adriamycin nephropathy, 5/6 nephrectomy and crescentic anti-glomerular basement membrane antibody-induced nephropathy (161,162). A 50 to 100-fold increase in apoptosis was observed in human proliferative nephritis such as IgA nephropathy, lupus nephritis and anti-neutrophil cytoplasm antibody (ANCA)-positive vasculitis (157,163). The role of apoptosis in these diseases is unclear. By contrast the rate of apoptosis was much lower in non-proliferative glomerulonephritis such as membranous nephropathy (159,163). Apoptosis of glomerular epithelial and endothelial cells has also been noted during glomerular injury.
Glomerular apoptosis appears to be related to changes in the local expression of apoptosis regulatory genes. Expression of lethal factors such as TNF-alpha and fas ligand is increased in several types of glomerular injury (29 and reviewed in 164). Increased expression of fas, bcl-2 and bax has been observed in mesangial cells in proliferative glomerulonephritis (65,165,166). An association was found between glomerular Fas expression and glomerular cell death (166). The Bcl-2/bax ratio decreased in glomeruli showing matrix expansion with decreased cellularity (165). Bcl-2 deficient mice display marked glomerular abnormalities (122) that resemble the epithelial crescents typical of rapidly progressive glomerulonephritis. Bcl-2 and bax expression was not different from controls in patients with diseases characterized by glomerular normocellularity such as minimal change nephrotic syndrome and membranous nephropathy (165). High ICE mRNA levels are present in the experimental models of progressive glomerular scarring mentioned above (161).
4.2.2 Acute renal failure
Tubular cell apoptosis has been observed during ischemic, toxic and obstructive acute renal failure (15,17,167-171). In these conditions there is also an on-going necrosis and the relative contribution of the two mechanisms of death to the initial cell loss is uncertain. Apoptosis may also occur in cells proliferating in a compensatory fashion after renal injury. These cells may be more sensitive to absolute or relative deficits in survival factors. In this setting apoptosis might contribute to the persistence or delayed recovery from acute renal failure. Apoptosis may also contribute to an adequate resolution of damage (168,170,171). In this case it may represent a physiologic balance to check an exaggerated compensatory proliferative response. Shimizu et al. observed, after 60 minutes of ischemia, an early peak of necrosis and apoptosis in the first 48-72h of acute renal failure, and a second, bigger peak of apoptosis after 7-14 days, when the necrotic tubules had been completely reconstituted by a hyperplastic epithelium (168).
Changes in the expression of both extracellular and intracellular apoptosis regulatory factors occur during acute renal failure. A cytokine microenvironment permissive for cell death includes decreased renal levels of pre-pro-EGF, IGF-1 and TGF-alpha mRNA (169,172-174), and increased systemic TNF-alpha and local TGF-ß1 and fas ligand (174,175 and unpublished observation). Among the receptors, renal Fas is increased in experimental septic and toxic acute renal failure (65,176), as well as in human acute tubular necrosis (176). Expression of transcription factors involved in apoptosis is also high. c-myc and c-fos are increased in the early stages of several models of acute renal failure (177,178). c-myc overexpression confers a dependence on external survival factors (75), and it may promote cell death in the adverse cytokine microenvironment found in acute renal failure. c-myc activates p53 transcription (78). More recently, increased p53 expression has also been noted in obstructive nephropathy (179). This finding is in agreement with our prior observation that bcl-2 is decreased and bax is increased during toxic acute renal failure in mice (37). Another apoptosis regulatory gene, bcl-xL is also increased in this model (37). Bcl-2 is decreased in obstructive nephropathy (180).
4.2.3 Chronic renal atrophy and renal fibrosis
Chronic renal atrophy is characterized by a progressive loss of renal parenchymal cells (181). Apoptosis of tubular epithelial cells has been observed in chronic tubular atrophy induced by chronic ischemia, papillary necrosis, subtotal nephrectomy and HIV nephropathy (15,17,182,183). The interstitial infiltration by macrophages and T cells in progressive kidney diseases may provide cytokines and inflammatory mediators that induce apoptosis (138,139).
Parenchymal atrophy and interstitial fibrosis are almost invariably associated. However, the relationship between these two phenomena is poorly understood. Transdifferentiation of parenchymal cells (such as tubular epithelial cells) into fibroblasts may explain their association (184). Alternatively, fibrosis may promote atrophy by providing an adverse microenvironment for epithelial cell survival. Both an abnormal extracellular matrix (25,26) and the release of cytokines that induce apoptosis, such as TNF-alpha and Fas ligand, by fibroblasts (29) may contribute to this microenvironment.
Accumulation of interstitial fibroblasts may also be caused by altered regulation of cell survival. Fibroblasts obtained from fibrotic kidneys accumulate more rapidly in culture and survive longer than those obtained from healthy kidneys (185). In this sense, fibroblasts involved in skin wound repair are eliminated by apoptosis (186).
4.2.4 Polycystic renal disease
Both the occurrence of apoptosis and abnormalities of apoptosis-related genes have been reported in polycystic kidneys (187-189). Transgenic mice overexpressing c-myc suffer from polycystic kidneys (188), and overexpression of c-myc has been detected in the kidneys of cpk/cpk mice (189). The lack of functional Bcl-2 results in murine neonatal polycystic renal disease (122). It is theoretically possible that overexpression of bax, the endogenous antagonist of bcl-2, may result in the development of renal cysts. However, levels of bax mRNA in kidneys or tubular cells from cpk/cpk mice were normal (unpublished observation).
The PKD1 gene, responsible for most of the cases of human autosomal dominant polycystic kidney disease has recently been cloned (190). The encoded protein may participate in interactions with extracellular ligands. The intriguing possibility that it may mediate cell survival or death has not been addressed yet.
Apoptosis decreases the mass of unneeded metanephric mesenchyme following induction by the ureteric bud (14,191). NGF, EGF, IGF-I, IGF-II, TGFalpha and HGF can rescue metanephric cells from apoptosis (14,192,193). Metanephric stem cells also possess receptors for lethal factors such as Fas and TNF-alpha (unpublished observation).
Developing kidneys express high levels of bcl-2, bak, FADD, bcl-xL, ich-1L and p53 (74,102,119,126,133,194). Our own data indicate that Bcl-2, bax and bcl-xL are expressed by metanephric stem cells in culture (195 and unpublished). However, as already suggested by the normal renal phenotype of ICE-deficient mice (98), ICE mRNA was not detected in the developing kidney (161).
Altered apoptosis during kidney development can result in renal dysplasia or agenesis. Mice carrying targeted mutations of bcl-2 have small neonatal polycystic kidneys with persistence of immature cells (122,196). Mice lacking functional WT-1 suffer from renal agenesis as a result of massive apoptosis of the metanephric blastema (197).