the Periphery of the Glomerular Capillary Wall Toward the Center of Disease
http://www.100md.com
糖尿病学杂志 2005年第6期
1 Department of Internal Medicine (Klinik fe Innere Medizin III), University Hospital, Jena, Germany
2 Department of Medicine, Renal-Electrolyte and Hypertension Division, Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT
Nephropathy is a major complication of diabetes. Alterations of mesangial cells have traditionally been the focus of research in deciphering molecular mechanisms of diabetic nephropathy. Injury of podocytes, if recognized at all, has been considered a late consequence caused by increasing proteinuria rather than an event inciting diabetic nephropathy. However, recent biopsy studies in humans have provided evidence that podocytes are functionally and structurally injured very early in the natural history of diabetic nephropathy. The diabetic milieu, represented by hyperglycemia, nonenzymatically glycated proteins, and mechanical stress associated with hypertension, causes downregulation of nephrin, an important protein of the slit diaphragm with antiapoptotic signaling properties. The loss of nephrin leads to foot process effacement of podocytes and increased proteinuria. A key mediator of nephrin suppression is angiotensin II (ANG II), which can activate other cytokine pathways such as transforming growth factor- (TGF-) and vascular endothelial growth factor (VEGF) systems. TGF-1 causes an increase in mesangial matrix deposition and glomerular basement membrane (GBM) thickening and may promote podocyte apoptosis or detachment. As a result, the denuded GBM adheres to Bowman’s capsule, initiating the development of glomerulosclerosis. VEGF is both produced by and acts upon the podocyte in an autocrine manner to modulate podocyte function, including the synthesis of GBM components. Through its effects on podocyte biology, glomerular hemodynamics, and capillary endothelial permeability, VEGF likely plays an important role in diabetic albuminuria. The mainstays of therapy, glycemic control and inhibition of ANG II, are key measures to prevent early podocyte injury and the subsequent development of diabetic nephropathy.
Diabetic nephropathy is the leading cause of end-stage renal disease and is clinically characterized by proteinuria and progressive renal insufficiency. Dating back to the first description by Kimmelstiel and Wilson (1), histological analyses have focused on the increase in mesangial matrix as the main lesion of diabetic glomerulopathy. In addition, glomerular basement membrane (GBM) thickening has been considered an important pathophysiological event in the disease. These pathological alterations have been linked to functional consequences, first described in landmark structure-function studies done in patients with type 1 as well as type 2 diabetes (2eC4). Specifically, mesangial matrix expansion correlates closely with both proteinuria and deterioration of renal function. It has been proposed that accumulation of matrix in the mesangial area reduces the capillary surface area available for filtration, thereby contributing to the progressive loss of renal function (2). Renal failure may also arise from nephron dropout due to tubulointerstitial fibrosis (5), and this process is aggravated by the harmful downstream effects of proteinuria on the tubules (6). However, the genesis of proteinuria in diabetes is not readily explained by the associated mesangial matrix expansion. Rather, consideration should be given to alterations of the glomerular filtration barrier, which is composed of the glomerular endothelium, the GBM, and the podocyte (glomerular visceral epithelial cell). Widespread endothelial dysfunction is believed to result in proteinuria (7), which is exacerbated by intraglomerular hemodynamic stress (8). Although it is highly fenestrated, the glomerular endothelium might pose some hindrance to protein permeability (9,10). As for the GBM, its conspicuous thickening in diabetes, perhaps due to accumulation of collagen IV and alterations in its architecture and composition (11), would seem to constitute a more effective barrier to the filtration of proteins but is in fact more porous to proteins (12). While loss of charge selectivity in the GBM has been proposed to partly explain the proteinuria (13), a decrease in negatively charged proteoglycans occurs late in the course of diabetic nephropathy, sometimes long after the appearance of microalbuminuria (14). This leads to the conclusion that the final barrier restricting plasma proteins to the vasculature is the slit diaphragm of the podocyte; little research, however, has been conducted on podocyte biology in diabetes until recently. The advent of improved cell culture techniques, the discovery of key podocyte-specific molecules, and advances in transgenic technology have revolutionized the study of podocytes in health and disease (15). The purpose of this review is to summarize the emerging evidence that podocytopathy plays a pivotal role in the manifestations of diabetic glomerulopathy, expanding upon the "mesangiocentric" dogma of diabetic glomerular disease.
PODOCYTE BIOLOGY
A comprehensive description of the biology of this highly differentiated cell is beyond the scope of this article, but a basic understanding is necessary to appreciate how podocyte malfunction contributes to diabetic nephropathy (15). Podocytes extend long processes toward the GBM to which they affix by cell surface adhesion proteins such as the 31 integrin and dystroglycan (16eC18). The foot processes of adjacent podocytes interdigitate and are separated by narrow spaces (30eC40 nm) that are bridged by a porous membrane called the slit diaphragm. These membranes contain pores that are freely permeable to water and small solutes but relatively impermeable to plasma proteins (19,20). Thus, the integrity of the slit diaphragm is one of the principal determinants of the permselective properties of the glomerular filtration barrier, and knowledge of its molecular architecture will help elucidate the role that the slit diaphragm plays in proteinuria (15). A major advance was the discovery of nephrin, whose gene is mutated in the congenital nephrotic syndrome of the Finnish type, a rare form of hereditary nephrosis characterized by diffuse foot process effacement of the podocytes (21). Nephrin, a transmembrane protein with a large extracellular portion, self-associates in a zipper-like arrangement through homophilic dimerization, forming the molecular substrate of the slit diaphragm (22). Other proteins such as podocin and CD2-associated protein (CD2AP) interact with nephrin in cholesterol-rich regions of the cytoplasm called lipid rafts and anchor nephrin to actin filaments of the podocyte cytoskeleton (15,23). CD2AP links nephrin and podocin to phosphoinositide 3-OH kinase, and this complex has cell-signaling properties that stimulate Akt, a serine-threonine kinase (24). One target of nephrin/CD2AP-induced phosphorylation is Bad, a proapoptotic protein of the Bcl-2 family (24). Upon phosphorylation, Bad is inactivated and apoptosis does not occur. Thus, afferent signaling coming from intact slit diaphragms prevents podocyte apoptosis (24,25), a beneficial effect given that terminally differentiated podocytes fail to proliferate and are not easily replaced (26). The inability of podocytes to proliferate may be secondary to upregulation of cell cycle inhibitory proteins, p57 and p27 (22). Expression of p27 in cultured podocytes can be stimulated by high glucose, and this further prevents cell replication (22).
Because it does not normally divide, the podocyte represents one of the last unexplored frontiers in renal cell culture research. Podocytes in primary culture can be coaxed into reentering the cell cycle, but they invariably lose their differentiated characteristics. Besides, the cell homogeneity in primary culture is questionable because glomerular parietal epithelial cells often contaminate and overgrow the podocytes (27). To circumvent these difficulties, "conditional immortalization" has been developed to obtain a pure population of podocytes that can either proliferate or differentiate (28). The technique involves the introduction of an oncogene whose gene product is temperature sensitive, such as the tsA58 variant of the SV40 large tumor antigen (TAg) (29). The TAg protein complexes with and disables p53 and retinoblastoma proteins, removing two major barriers to cell cycle progression and allowing the podocyte to be propagated in culture. When differentiated podocytes are needed for experimentation, the cells can be "thermoshifted" to a higher temperature that will inactivate tsA58, reverse the immortalization, and permit differentiation to occur.
PODOCYTOPATHY IN HUMAN DIABETIC KIDNEY DISEASE
Experiments from 25 years ago in diabetic rats described foot process widening of podocytes (30), a finding later confirmed in patients with relatively advanced nephropathy due to type 1 diabetes (31). More recent investigations also described an increase in foot process width in microalbuminuric type 1 diabetic subjects, and foot process width correlated directly with the urinary albumin excretion rate (32). In addition to foot process widening, the number and density of podocytes have been reported to be markedly reduced (podocytopenia) in diabetic patients, whether afflicted with type 1 or type 2 diabetes (33eC36). While an ideal protocol for counting podocytes in biopsy specimens has not been developed yet, it has been argued that modern morphometric techniques can yield reasonable approximations. For instance, it has been estimated that the podocyte number is significantly reduced, even in diabetes of short duration (33). In Pima Indians with type 2 diabetes, podocyte density is drastically reduced, and the remaining foot processes are widened to maintain GBM coverage (34,37). Among all glomerular morphological characteristics, the decreased number of podocytes per glomerulus was the strongest predictor of progressive renal disease, with fewer cells predicting more rapid progression (38). Additionally, a recent morphometric study (36) in European type 2 diabetic patients has shown that a diminution of podocyte density rather than absolute reduction in podocyte number is more predictive of the presence and magnitude of albuminuria. The numerical density of podocytes per glomerulus was reduced in patients with microalbuminuria and was decreased even further in patients with overt proteinuria (36). A cross-sectional study also reported a significant inverse correlation between proteinuria and both podocyte number and density per glomerulus (39). Consistent with the observed loss of podocytes in diabetes, podocytes were present in the urine in 53% of type 2 diabetic patients with microalbuminuria and in 80% with macroalbuminuria, while normoalbuminuric patients and healthy control subjects had undetectable levels of urinary podocytes (40). Interestingly, treatment with an ACE inhibitor reduced the number of urinary podocytes (40).
Podocytopenia may exacerbate the development of proteinuria because a denuded GBM can come into contact with Bowman’s capsule and promote synechiae formation, an initial step in the development of glomerulosclerosis (15). Morphologic changes in the podocyte are also believed to engender proteinuria. With foot process widening, the decrease in slit diaphragm length might impede the filtration of water and lower the glomerular filtration rate. However, if protein permeability remains intact, as postulated in an innovative theory (41), the amount of protein relative to water is increased in the urinary space, and the elevated protein concentration could exceed the tubule’s reabsorptive capacity and manifest as proteinuria. The merit of this theory is that it reconciles the paradox of increasing proteinuria in the face of declining renal function, a characteristic of diabetic nephropathy, but the theory remains unproven and controversial (41). Even so, the worsening proteinuria could induce tubular atrophy and interstitial fibrosis, and these irreversible tubulointerstitial changes, together with glomerulosclerosis, then lead to chronic renal insufficiency (6).
To detect earlier functional abnormalities in podocytes, several recent studies have focused on the expression of podocyte-specific proteins in diabetic patients. For instance in type 1 diabetes, nephrin excretion in the urine (nephrinuria), detected by Western blot analysis, was present in 30% of the patients with normoalbuminuria, 17% of those with microalbuminuria, and 28% of those with macroalbuminuria, whereas none of the nondiabetic subjects had nephrinuria (42). These findings were not correlated with biopsies, but they suggest that increased urinary nephrin equates with early podocyte injury, even before the onset of microalbuminuria. Another study (43) found that nephrin staining was extensively reduced in renal biopsy specimens from nephropathic patients with type 1 diabetes. Several studies have evaluated nephrin mRNA and protein expression in type 2 diabetes. In general, nephrin protein production was downregulated in the diabetic subjects compared with the nondiabetic control subjects (44eC46), and the decrease in nephrin correlated with the broadening of the foot process widths. In contrast, CD2AP expression was not reduced in podocytes from diabetic patients, suggesting that the reduction in nephrin was not due to widespread podocyte loss or injury (45); this underscores the importance of nephrin in maintaining podocyte integrity. Finally, type 2 diabetic patients who were treated with the ACE inhibitor perindopril for 2 years exhibited a nephrin mRNA expression that was close to normal when compared with nondiabetic control subjects; patients not treated with the ACE inhibitor had significantly reduced nephrin transcripts (46).
PODOCYTE DYSFUNCTION IN EXPERIMENTAL DIABETES
Animal experiments allow for the repeated sampling of kidney tissue over time, furnishing a longitudinal record of the development of diabetic nephropathy and the efficacy of pharmacological interventions. Serial investigations (47) on kidneys from obese Zucker fa/fa rats, a model of type 2 diabetes that develops segmental glomerulosclerosis, revealed that nephropathy started with damage to podocytes, manifesting as foot process effacement and cytoplasmic accumulation of lipid droplets. Early podocyte damage antedated the development of glomerulosclerosis and tubulointerstitial damage in this model (48). In the streptozotocin (STZ)-induced diabetic rat, a model of type 1 diabetes, several studies (49,50) have reported a decrease in podocyte number, broadening of the foot processes, and reduction in nephrin expression.
PODOCYTE APOPTOSIS AND/OR DETACHMENT IN HUMANS AND ANIMAL MODELS
The exact etiology for podocyte loss in diabetes remains speculative, but two mechanisms can be suggested: apoptosis and cell detachment. Evidence for a primary role of apoptosis in either human or experimental diabetic nephropathy is scant (51eC54). However, podocyte apoptosis can be demonstrated in cell culture. For instance, angiotensin II (ANG II) induces apoptosis in cultured rat glomerular epithelial cells, and this effect is mediated by the transforming growth factor- (TGF-) system since it is inhibited by an antieCTGF- antibody (55). In TGF-1eCoverexpressing transgenic mice, albeit nondiabetic, the podocyte undergoes apoptosis in situ shortly after the sclerotic lesion appears in the glomerulus (56). Thus, the heightened intraglomerular activity of TGF-1 that is characteristic of diabetes may theoretically be responsible for apoptosis (57), a process that can be mediated through Smad7, which inhibits the nuclear translocation of the cell survival factor nuclear factor-B (56,58).
The other mechanism of podocyte loss in diabetes may relate to the detachment of podocytes from the GBM. This scenario does not exclude a role for apoptosis, since cells may first detach and then undergo apoptosis because of interruption of viability cues deriving from cell-matrix interactions (17). Alternatively, detached cells can be shed in the urine as live podocytes (59). In fact, podocyturia worsens with the progression from normoalbuminuria to microalbuminuria to overt proteinuria (40). Loss of cell anchorage to the GBM may result from downregulation of the 31 integrin receptor, the principal adhesion complex that attaches the podocyte to the GBM (16,17,60,61). Several studies (62eC64) have shown that the 31 integrin is decreased in the podocytes of humans and rats with diabetes. Furthermore, high glucose media in cultured rat or human podocytes decreases the expression of 31 integrin (62,64); this downregulation is perhaps mediated by increased levels of TGF-1 (65,66).
ROLE OF ANG II IN NEPHRIN EXPRESSION AND PODOCYTE INJURY
Pharmacological interventions in animal models have supported the view that an increased ANG II activity is involved in podocyte injury in diabetes. ACE inhibition or AT1 receptor antagonism attenuated podocyte foot process broadening in rats with STZ-induced diabetes (49). In another study (67) involving the STZ-induced diabetic rat, therapy with an ACE inhibitor, but not an endothelin receptor antagonist, prevented loss of podocytes and podocyte injury. Similarly, an ACE inhibitor, but not aminoguanidine, an inhibitor of advanced glycation end product (AGE) formation, attenuated the diabetes-associated reduction in nephrin expression (50). Likewise, an AT1 receptor antagonist, but not the calcium channel blocker amlodipine, normalized the reduced nephrin expression in podocytes from spontaneously hypertensive rats with superimposed STZ-induced diabetes (68,69). Finally, the renal damage that occurs in obese Zucker rats was more effectively controlled by combined treatment with an ACE inhibitor and an AT1 receptor antagonist than either monotherapy alone (70). Thus, it may be concluded that suppression of nephrin expression in podocytes can be caused by increased renal ANG II activity in diabetes, but the molecular mechanisms are incompletely understood.
Podocytes express the AT1 and probably the AT2 receptors after injury and therefore could respond to stimulation with ANG II (15,71). Transgenic rats with targeted overexpression of the podocyte’s AT1 receptor showed pseudocysts in podocytes, followed by foot process effacement and local detachment, with subsequent progression to focal segmental glomerulosclerosis (72). High glucose concentrations induce ANG II formation in podocytes through upregulation of angiotensinogen expression (15). Furthermore, ANG II production in podocytes can be activated by proteinuria, likely by the transit of proteins through the filtration barrier (73) and by mechanical stretch, which mimics the hemodynamic effects of intraglomerular hypertension (74). Interestingly, ANG II formation as a consequence of mechanical stretch appears to be independent of ACE (74). As an alternative to ACE for the conversion of ANG I to ANG II, chymase has been shown to be upregulated in the glomeruli of patients with nephropathy due to type 2 diabetes (75). Because chymase is not blocked by ACE inhibitors, glomeruli in the diabetic state may still generate ANG II despite ACE inhibition. Some evidence indicates that the increase in angiotensinogen expression is signaled by intracellular reactive oxygen species (ROS) (74). Oxidative stress is a leading initiator of cellular dysfunction in diabetes complications (76,77), and increased ROS generation can induce podocyte dysfunction (78,79).
ROLE OF HEPARAN SULFATE PROTEOGLYCANS
A decrease in the GBM content of negatively charged HSPG contributes to the loss of charge selectivity in the glomerular filtration barrier and to the progression of proteinuria (80,81). One potential mechanism for decreased de novo synthesis of proteoglycans is the increased generation of ROS (81,82). Although the predominant proteoglycan in the GBM was thought to be perlecan, it has become more clear that agrin is the more abundant HSPG (83). Synthesis of proteoglycans occurs in all three glomerular cell types, but podocytes are an especially important source of these negatively charged molecules. Proteoglycan synthesis in podocytes is differentially influenced by high ambient glucose and ANG II (83,84); high glucose suppresses the production of agrin’s core protein, whereas ANG II decreases synthesis of the core protein and diminishes the sulfation of its side chains (83,84). Podocytes exposed to ANG II decrease the amount of HSPG on their cell surfaces and in the extracellular matrix (83); these results may partly explain the antiproteinuric effect of ACE inhibitors and angiotensin receptor blockers in diabetic nephropathy.
ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR
Losses of nephrin and proteoglycan are not the only pathological changes fostering proteinuria in diabetic nephropathy. Vascular endothelial growth factor (VEGF), a survival and angiogenic factor with strong microvascular permeabilizing properties (85), may increase the permeability of the glomerular filtration barrier to circulating proteins. The most convincing evidence that VEGF overexpression is involved in the proteinuria of diabetes comes from studies in type 1 diabetic (STZ-induced) rats and type 2 diabetic db/db mice. Neutralization of VEGF with a systemically administered anti-VEGF antibody reduced the urinary albumin excretion by at least 50% compared with the untreated diabetic controls (86,87). In addition, VEGF induces endothelial nitric oxide synthase, thereby promoting the vasodilation and hyperfiltration that are typical of early diabetic nephropathy (86,88).
The expression of VEGF in the glomerulus is most pronounced in the podocytes, and experimental diabetes increases VEGF mRNA and protein expression (89,90). In cultured podocytes, VEGF expression can be stimulated by high glucose concentrations (91), by TGF-1 (91,92), and by ANG II acting via AT1 and AT2 receptors (93,94). The gene expression of VEGF is also stimulated by a transcription factor called hypoxia-inducible factor-1 (95). We have demonstrated in PC12 cells that hypoxia-inducible factor-1 expression is stimulated by ANG II in a posttranscriptional mechanism involving AT2 receptors (96). This increase is caused by downregulation of a prolyl hydroxylase (SM-20/PHD3), the enzyme responsible for initiating hypoxia-inducible factor-1 degradation (96). Upregulation of podocyte-derived VEGF in diabetes may depend on signals downstream of the receptor for AGE, or RAGE (90). RAGE expression is increased in the podocytes of diabetic db/db mice (90), and inhibition of AGE-RAGE interactions by soluble RAGE treatment significantly depressed VEGF expression in the kidney and ameliorated albuminuria and glomerulosclerosis (90). Precursors of AGE such as Amadori-glycated serum albumin may mediate diabetic proteinuria (97) and promote glomerular production of hydrogen peroxide (98). Whether increased ROS production stimulates podocyte VEGF expression remains to be investigated.
An intriguing concept that has come to attention is that podocytes not only produce VEGF but are also acted upon by VEGF (92,99). This VEGF autocrine loop may play important roles in podocyte biology since VEGF decreases intracellular calcium concentrations and protects against cytotoxicity (99), possibly via the antiapoptotic actions of nephrin (25). These cytoprotective effects were reversed when endogenous VEGF was blocked by either a class III tyrosine kinase inhibitor PTK787/ZK222584 (99) or a monoclonal anti-VEGF antibody (25).
VEGF also stimulates the podocyte to produce the 3 chain of collagen IV, a principal ingredient of the GBM; this effect may be mediated by VEGFR-1 signaling (92). When endogenous VEGF secretion by podocytes was stimulated by TGF-1 treatment, the production of 3(IV) collagen increased (92). Blockade of endogenous VEGF action by a specific inhibitor of VEGF receptor kinases, SU5416, reduced the TGF-1eCinduced expression of 3(IV) collagen by 50%, establishing a pivotal role for the VEGF autocrine system in at least one aspect of the regulation of GBM composition by podocytes (92).
It may be speculated that VEGF-stimulated podocyte production of 3(IV) collagen contributes to diabetic thickening of the GBM and its altered permselectivity. Extrapolating from our in vitro work in cultured podocytes, we treated diabetic db/db mice with intraperitoneal injections of SU5416 at 2 mg/kg body wt, given twice a week for 8 weeks (100). SU5416 treatment, which had no adverse effects, significantly prevented GBM thickening and albuminuria without affecting blood glucose levels (100 and S.H. Sung, S.C., F.N.Z., unpublished data). These findings with SU5416 compare favorably with those of the anti-VEGF antibody in db/db mice (87); however, that study did not report the effect of anti-VEGF therapy on GBM thickening. That SU5416 particularly benefited the podocyte was also suggested by the preservation of nephrin protein, assayed by immunostaining (S.H. Sung, S.C., F.N.Z., unpublished data). Glomerular nephrin levels, which are typically reduced in the db/db mouse, were restored almost to normal by SU5416 treatment (S.H. Sung, S.C., F.N.Z., unpublished data), suggesting that nephrin preservation represents another mechanism whereby VEGF blockade might ameliorate diabetic albuminuria.
ROLE OF TGF-1
TGF- plays an integral role in the pathogenesis of diabetic nephropathy (101). This fibrogenic cytokine is stimulated by the diabetic state, is increased in the kidney during diabetes, and is able to recapitulate the hypertrophic and sclerotic features of diabetic renal disease (102). Elevated levels of TGF- have been measured in the glomeruli of STZ-induced diabetic rats by micropuncture techniques (103,104), and an intracellular transducer of TGF- signaling, Smad3, has been shown to translocate into the glomerular nuclei of diabetic db/db mice (105), attesting to the overactivity of the TGF- system in diabetic nephropathy. Inhibition of TGF- by a panselective neutralizing antibody in diabetic db/db mice prevented diabetic renal hypertrophy, mesangial matrix expansion, and the development of renal insufficiency (106). However, there was no significant effect on albuminuria in the diabetic mice (106).
A clear role for TGF- in the pathogenesis of diabetic albuminuria cannot be established at present because of conflicting studies. In nondiabetic models, overexpression of active TGF-1 in transgenic mice caused mesangial expansion, interstitial fibrosis, renal insufficiency, and progressive proteinuria (107). In addition, an increase in albumin permeability was observed when isolated glomeruli from normal rats were exposed ex vivo to TGF-1 (108). However, in our studies on the type 2 diabetic db/db mouse, we failed to see a significant reduction in albuminuria after 8 weeks of treatment with a neutralizing antieCTGF- antibody, perhaps because VEGF expression in the kidney cortex was kept elevated by other diabetic factors (106). Interestingly, the work of Benigni et al. (109) in uninephrectomized STZ-induced diabetic rats revealed that one but not another species of antieCTGF- antibody significantly reduced proteinuria when compared with control IgG. Nevertheless, either antibody could potentiate the antiproteinuric effect of an ACE inhibitor when given in combination (109). Other studies showed that when a portion of the TGF- signaling pathway was selectively inhibited by the creation of a Smad3 knockout mouse, the albuminuria persisted when the mice were rendered diabetic with STZ (100). However, another cohort of Smad3 knockout mice with a different strain background showed significant amelioration of albuminuria in STZ-induced diabetes (110). Nevertheless, both diabetic Smad3 knockout models displayed prevention of mesangial matrix expansion (110 and S.C., S.H. Sung, N.J. Laping, F.N.Z., unpublished data). Thus, it is possible to dissociate albuminuria from mesangial matrix expansion, and the pathogenesis of proteinuria may relate more to VEGF and nephrin while the pathogenesis of glomerulosclerosis may deal more with TGF- and other fibrogenic pathways.
The podocyte is also subject to the increased levels and overactivity of intraglomerular TGF- in diabetes. As discussed above, podocyte detachment and/or apoptosis can be caused by increased TGF- activity (55,58,62). Additionally, TGF-1 stimulates the podocyte expression of 3(IV) collagen (91), but surprisingly, TGF-1 suppresses the production of both 1(IV) and 5(IV) collagen (91), important exceptions to the dogma that TGF- is uniformly profibrotic. TGF-1 also stimulates the podocyte expression of VEGF (91) that in turn increases the activity of the VEGF autocrine loop, with all of its attendant effects (25,92,99).
Unlike other renal cells, podocytes may not respond to the diabetic state by increasing their expression of TGF-. The production of TGF-1 in cultured mouse podocytes was not significantly stimulated by high glucose (91) or by ANG II (S.C., F.N.Z., unpublished data). Instead, these metabolic stimuli significantly upregulated the expression of the TGF- type II receptor (91 and S.C., F.N.Z., unpublished data), which binds to the TGF- ligand and initiates the TGF- signaling cascade (111). In theory, the increase in type II receptor could make the podocyte more sensitive to the elevated levels of TGF- in the diabetic state, and it could enhance a paracrine interaction between the podocyte and the mesangial and/or glomerular endothelial cell.
CONCLUSIONS
Although the pathophysiology of mesangial cells has long been considered to be the major factor in the development of glomerulosclerosis in type 1 or type 2 diabetes, more recent evidence suggests that podocytes also play a critical role in the early functional and structural changes of diabetic kidney disease. On the one hand, podocytes are the victims of pathogenetic events surrounding the progression of diabetic nephropathy, including the harmful effects of high glucose, AGE, ANG II, ROS, TGF-, and mechanical stretch. On the other hand, podocytes are the culprits in diabetic nephropathy in that a reduced nephrin expression may lead to podocyte foot process broadening and effacement, while increased VEGF production may exacerbate GBM thickening and proteinuria, leading to tubular atrophy and interstitial fibrosis (Fig. 1). Many of these events are mediated by ANG II, whose local concentration is increased in diabetic kidney disease. ANG II in turn suppresses nephrin and induces TGF- type II receptor expression in podocytes, perhaps intensifying the paracrine effects of TGF- derived from the mesangium or glomerular endothelium. Increased TGF- activity may lead to podocyte apoptosis and/or detachment with podocytopenia and the development of progressive glomerulosclerosis. An additional sequela is VEGF overactivity that could increase glomerular hemodynamic stress, alter the production of GBM components, and repress nephrin expression, all maladaptive events that foster proteinuria (Fig. 1).
The ideal therapy for diabetic nephropathy remains elusive, but excellent glycemic control and ANG II antagonism have become the foundations of current therapy. Promising new treatments that target the roles of VEGF, TGF-, nephrin, HSPG, glycated proteins, and ROS are beginning to appear on the horizon. In this regard, protection of podocytes will be of paramount importance in preventing the development and progression of diabetic nephropathy.
ACKNOWLEDGMENTS
G.W. is supported by a grant from the Deutsche Forschungsgemeinschaft. S.C. is supported by a grant from the National Institutes of Health (DK061537). F.N.Z. is supported by grants from the American Diabetes Association and the National Institutes of Health (DK44513 and DK54608) and by the Department of Veterans Affairs.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
AGE, advanced glycation end product; ANG II, angiotensin II; CD2AP, CD2-associated protein; GBM, glomerular basement membrane; HSPG, heparan sulfate proteoglycans; RAGE, receptor for AGE; ROS, reactive oxygen species; STZ, streptozotocin; TGF-, transforming growth factor-; VEGF, vascular endothelial growth factor
REFERENCES
Kimmelstiel P, Wilson C: Intercapillary lesions in the glomeruli of the kidney. Am J Pathol 12: 82eC97, 1936
Mauer SM, Steffes MW, Ellis EN, Sutherland DER, Brown DM, Goetz FC: Structural-functional relationships in diabetic nephropathy. J Clin Invest 74: 1143eC1155, 1984
Osterby R, Gall MA, Schmitz A, Nielsen FS, Nyberg G, Parving HH: Glomerular structure and function in proteinuric type 2 (non-insulin-dependent) diabetic patients. Diabetologia 36: 1064eC1070, 1993
White KE, Bilous RW: Type 2 diabetic patients with nephropathy show structural-functional relationships that are similar to type 1 disease. J Am Soc Nephrol 11: 1667eC1673, 2000
Ziyadeh FN, Goldfarb S: The renal tubulointerstitium in diabetes mellitus. Kidney Int 39: 464eC475, 1991
Remuzzi G, Bertani T: Pathophysiology of progressive nephropathies. N Engl J Med 339: 1448eC1456, 1998
Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, Jensen T, Kofoed-Enevoldsen A: Albuminuria reflects widespread vascular damage: the Steno hypothesis. Diabetologia 32: 219eC226, 1989
Zatz R, Meyer TW, Rennke HG, Brenner BM: Predominance of hemodynamic rather than metabolic factors in the pathogenesis of diabetic glomerulopathy. Proc Natl Acad Sci U S A 82: 5963eC5967, 1985
Deen WM, Lazzara MJ, Myers BD: Structural determinants of glomerular permeability. Am J Physiol Renal Physiol 281: F579eCF596, 2001
Deen WM: What determines glomerular capillary permeability J Clin Invest 114: 1412eC1414, 2004
Kefalides NA: Basement membrane research in diabetes mellitus. Coll Relat Res 1: 295eC299, 1981
Isogai S, Mogami K, Shiina N, Yoshino G: Initial ultrastructural changes in pore size and anionic sites of the glomerular basement membrane in streptozotocin-induced diabetic rats and their prevention by insulin treatment. Nephron 83: 53eC58, 1999
Menne J, Park JK, Boehne M, Elger M, Lindschau C, Kirsch T, Meier M, Gueler F, Fiebeler A, Bahlmann FH, Leitges M, Haller H: Diminished loss of proteoglycans and lack of albuminuria in protein kinase C-eCdeficient diabetic mice. Diabetes 53: 2101eC2109, 2004
Vernier RL, Steffes MW, Sisson-Ross S, Mauer SM: Heparan sulfate proteoglycan in the glomerular basement membrane in type 1 diabetes mellitus. Kidney Int 41: 1070eC1080, 1992
Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 83: 253eC307, 2003
Tsilibary EC: Microvascular basement membranes in diabetes mellitus. J Pathol 200: 537eC546, 2003
Kretzler M: Regulation of adhesive interaction between podocytes and glomerular basement membrane. Microsc Res Tech 57: 247eC253, 2002
Kojima K, Kerjaschki D: Is podocyte shape controlled by the dystroglycan complex Nephrol Dial Transplant 17 (Suppl. 9): 23eC24, 2002
Tryggvason K: Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm. J Am Soc Nephrol 10: 2440eC2445, 1999
Wartiovaara J, Ofverstedt LG, Khoshnoodi J, Zhang J, Makela E, Sandin S, Ruotsalainen V, Cheng RH, Jalanko H, Skoglund U, Tryggvason K: Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography. J Clin Invest 114: 1475eC1483, 2004
Tryggvason K, Ruotsalainen V, Wartiovaara J: Discovery of the congenital nephrotic syndrome gene discloses the structure of the mysterious molecular sieve of the kidney. Int J Dev Biol 43: 445eC451, 1999
Khoshnoodi J, Sigmundsson K, Ofverstedt LG, Skoglund U, Obrink B, Wartiovaara J, Tryggvason K: Nephrin promotes cell-cell adhesion through homophilic interactions. Am J Pathol 163: 2337eC2346, 2003
Saleem MA, Ni L, Witherden I, Tryggvason K, Ruotsalainen V, Mundel P, Mathieson PW: Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation. Am J Pathol 161: 1459eC1466, 2002
Huber TB, Hartleben B, Kim J, Schmidts M, Schermer B, Keil A, Egger L, Lecha RL, Borner C, Pavenstadt H, Shaw AS, Walz G, Benzing T: Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol 23: 4917eC4928, 2003
Foster RR, Saleem MA, Mathieson PW, Bates DO, Harper SJ: Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol 288: F48eCF57, 2004
Griffin SV, Petermann AT, Durvasula RV, Shankland SJ: Podocyte proliferation and differentiation in glomerular disease: role of cell-cycle regulatory proteins (Review). Nephrol Dial Transplant 18 (Suppl. 6): vi8eCv13, 2003
Weinstein T, Cameron R, Katz A, Silverman M: Rat glomerular epithelial cells in culture express characteristics of parietal, not visceral, epithelium. J Am Soc Nephrol 3: 1279eC1287, 1992
Mundel P, Reiser J, Ze瞚ga-Meje-Borja A, Pavenstdt H, Davidson G, Kriz W, Zeller R: Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248eC258, 1997
Jat PS, Sharp PA: Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature. Mol Cell Biol 9: 1672eC1681, 1989
Steffes MW, Leffert JD, Basgen JM, Brown DM, Mauer SM: Epithelia cell foot process width in intact and uninephrectomized diabetic and nondiabetic rats. Lab Invest 43: 225eC230, 1980
Ellis EN, Steffes MW, Chavers B, Mauer SM: Observations of glomerular epithelial cell structure in patients wit type I diabetes mellitus. Kidney Int 32: 736eC741, 1987
Berg UB, Torbjornsdotter TB, Jaremko G, Thalme B: Kidney morphological changes in relation to long-term renal function and metabolic control in adolescents with IDDM. Diabetologia 41: 1047eC1056, 1998
Steffes MW, Schmidt D, McCrery R, Basgen JM: Glomerular cell number in normal subjects and in type 1 diabetic patients. Kidney Int 59: 2104eC2113, 2001
Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, Meyer TW: Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 99: 342eC348, 1997
White KE, Bilous RW, Marshall SM, El Nahas M, Remuzzi G, Piras G, De Cosmo S, Viberti G: Podocyte number in normotensive type 1 diabetic patients with albuminuria. Diabetes 51: 3083eC3089, 2002
Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, Fioretto P: Is podocyte injury relevant in diabetic nephropathy Studies in patients with type 2 diabetes. Diabetes 52: 1031eC1035, 2003
Nelson RG, Meyer TW, Myers BD, Bennett PH: Clinical and pathological course of renal disease in non-insulin-dependent diabetes mellitus: the Pima Indian experience. Semin Nephrol 17: 124eC131, 1997
Meyer TW, Bennett GL, Nelson RG: Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria. Diabetologia 42: 1341eC1344, 1999
White KE, Bilous RW: Structural alterations to the podocyte are related to proteinuria in type 2 diabetic patients. Nephrol Dial Transplant 19: 1437eC1440, 2004
Nakamura T, Ushiyama C, Suzuki S, Hara M, Shimada N, Ebihara I, Koide H: Urinary excretion of podocytes in patients with diabetic nephropathy. Nephrol Dial Transplant 15: 1379eC1383, 2000
Smithies O: Why the kidney glomerulus does not clog: a gel permeation/diffusion hypothesis of renal function. Proc Natl Acad Sci U S A 100: 4108eC4113, 2003
Patari A, Forsblom C, Havana M, Taipale H, Groop PH, Holthofer H: Nephrinuria in diabetic nephropathy of type 1 diabetes. Diabetes 52: 2969eC2974, 2003
Doublier S, Salvidio G, Lupia E, Ruotsalainen V, Verzola D, Deferrari G, Camussi G: Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II. Diabetes 52: 1023eC1030, 2003
Koop K, Eikmans M, Baelde HJ, Kawachi H, De Heer E, Paul LC, Bruijn JA: Expression of podocyte-associated molecules in acquired human kidney diseases. J Am Soc Nephrol 14: 2063eC2071, 2003
Benigni A, Gagliardini E, Tomasoni S, Abbate M, Ruggenenti P, Kalluri R, Remuzzi G: Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy. Kidney Int 65: 2193eC2200, 2004
Langham RG, Kelly DJ, Cox AJ, Thomson NM, Holthofer H, Zaoui P, Pinel N, Cordonnier DJ, Gilbert RE: Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition. Diabetologia 45: 1572eC1576, 2002
Gassler N, Elger M, Kranzlin B, Kriz W, Gretz N, Hahnel B, Hosser H, Hartmann I: Podocyte injury underlies the progression of focal segmental glomerulosclerosis in the fa/fa Zucker rat. Kidney Int 60: 106eC116, 2001
Coimbra TM, Janssen U, Grone HJ, Ostendorf T, Kunter U, Schmidt H, Brabant G, Floege J: Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int 57: 167eC182, 2000
Mifsud SA, Allen TJ, Bertram JF, Hulthen UL, Kelly DJ, Cooper ME, Wilkinson-Berka JL, Gilbert RE: Podocyte foot process broadening in experimental diabetic nephropathy: amelioration with renin-angiotensin blockade. Diabetologia 44: 878eC882, 2001
Kelly DJ, Aaltonen P, Cox AJ, Rumble JR, Langham R, Panagiotopoulos S, Jerums G, Holthofer H, Gilbert RE: Expression of the slit-diaphragm protein, nephrin, in experimental diabetic nephropathy: differing effects of anti-proteinuric therapies. Nephrol Dial Transplant 17: 1327eC1332, 2002
Ortiz A, Ziyadeh FN, Neilson EG: Expression of apoptosis-regulatory genes in renal proximal tubular epithelial cells exposed to high ambient glucose and in diabetic kidneys. J Investig Med 45: 50eC56, 1997
Murata I, Takemura G, Asano K, Sano H, Fujisawa K, Kagawa T, Baba K, Maruyama R, Minatoguchi S, Fujiwara T, Fujiwara H: Apoptotic cell loss following cell proliferation in renal glomeruli of Otsuka Long-Evans Tokushima Fatty rats, a model of human type 2 diabetes. Am J Nephrol 22: 587eC595, 2002
Kumar D, Zimpelmann J, Robertson S, Burns KD: Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney. Nephron Exp Nephrol 96: e77eCe88, 2004
Kumar D, Robertson S, Burns KD: Evidence of apoptosis in human diabetic kidney. Mol Cell Biochem 259: 67eC70, 2004
Ding G, Reddy K, Kapasi AA, Franki N, Gibbons N, Kasinath BS, Singhal PC: Angiotensin II induces apoptosis in rat glomerular epithelial cells. Am J Physiol Renal Physiol 283: F173eCF180, 2002
Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke P, Mundel P, Bottinger EP: Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 108: 807eC816, 2001
Yoo J, Ghiassi M, Jirmanova L, Balliet AG, Hoffman B, Fornace AJ Jr, Liebermann DA, Bottinger EP, Roberts AB: Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation. J Biol Chem 278: 43001eC43007, 2003
Schiffer M, Mundel P, Shaw AS, Bottinger EP: A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta-induced apoptosis. J Biol Chem 279: 37004eC37012, 2004
Vogelmann SU, Nelson WJ, Myers BD, Lemley KV: Urinary excretion of viable podocytes in health and renal disease. Am J Physiol Renal Physiol 285: F40eCF48, 2003
Korhonen M, Ylanne J, Laitinen L, Virtanen I: Distribution of beta 1 and beta 3 integrins in human fetal and adult kidney. Lab Invest 62: 616eC625, 1990
Adler S: Characterization of glomerular epithelial cell matrix receptors. Am J Pathol 141: 571eC578, 1992
Chen HC, Chen CA, Guh JY, Chang JM, Shin SJ, Lai YH: Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes. Life Sci 67: 2345eC2353, 2000
Regoli M, Bendayan M: Alterations in the expression of the alpha 3 beta 1 integrin in certain membrane domains of the glomerular epithelial cells (podocytes) in diabetes mellitus. Diabetologia 40: 15eC22, 1997
Kitsiou PV, Tzinia AK, Stetler-Stevenson WG, Michael AF, Fan WW, Zhou B, Tsilibary EC: Glucose-induced changes in integrins and matrix-related functions in cultured human glomerular epithelial cells. Am J Physiol Renal Physiol 284: F671eCF679, 2003
Kagami S, Border WA, Ruoslahti E, Noble NA: Coordinated expression of beta 1 integrins and transforming growth factor-beta-induced matrix proteins in glomerulonephritis. Lab Invest 69: 68eC76, 1993
Kumar NM, Sigurdson SL, Sheppard D, Lwebuga-Mukasa JS: Differential modulation of integrin receptors and extracellular matrix laminin by transforming growth factor-beta 1 in rat alveolar epithelial cells. Exp Cell Res 221: 385eC394, 1995
Gross ML, El-Shakmak A, Szabo A, Koch A, Kuhlmann A, Munter K, Ritz E, Amann K: ACE-inhibitors but not endothelin receptor blockers prevent podocyte loss in early diabetic nephropathy. Diabetologia 46: 856eC868, 2003
Bonnet F, Cooper ME, Kawachi H, Allen TJ, Boner G, Cao Z: Irbesartan normalises the deficiency in glomerular nephrin expression in a model of diabetes and hypertension. Diabetologia 44: 874eC877, 2001
Davis BJ, Cao Z, de Gasparo M, Kawachi H, Cooper ME, Allen TJ: Disparate effects of angiotensin II antagonists and calcium channel blockers on albuminuria in experimental diabetes and hypertension: potential role of nephrin. J Hypertens 21: 209eC216, 2003
Toblli JE, DeRosa G, Cao G, Piorno P, Pagano P: ACE inhibitor and angiotensin type I receptor antagonist in combination reduce renal damage in obese Zucker rats. Kidney Int 65: 2343eC2359, 2004
Gloy J, Henger A, Fischer KG, Nitschke R, Mundel P, Bleich M, Schollmeyer P, Greger R, Pavenstdt H: Angiotensin II depolarizes podocytes in the intact glomerulus of the rat. J Clin Invest 99: 2772eC2781, 1997
Hoffmann S, Podlich D, Hahnel B, Kriz W, Gretz N: Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats. J Am Soc Nephrol 15: 1475eC1487, 2004
Abbate M, Zoja C, Morigi M, Rottoli D, Angioletti S, Tomasoni S, Zanchi C, Longaretti L, Donadelli R, Remuzzi G: Transforming growth factor-beta1 is up-regulated by podocytes in response to excess intraglomerular passage of proteins: a central pathway in progressive glomerulosclerosis. Am J Pathol 161: 2179eC2193, 2002
Durvasula RV, Petermann AT, Hiromura K, Blonski M, Pippin J, Mundel P, Pichler R, Griffin S, Couser WG, Shankland SJ: Activation of a local tissue angiotensin system in podocytes by mechanical strain. Kidney Int 65: 30eC39, 2004
Huang XR, Chen WY, Truong LD, Lan HY: Chymase is upregulated in diabetic nephropathy: implications for an alternative pathway of angiotensin II-mediated diabetic renal and vascular disease. J Am Soc Nephrol 14: 1738eC1747, 2003
Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813eC820, 2001
Evans JL, Goldfine ID, Maddux BA, Grodsky GM: Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 23: 599eC622, 2002
Vega-Warner V, Ransom RF, Vincent AM, Brosius FC, Smoyer WE: Induction of antioxidant enzymes in murine podocytes precedes injury by puromycin aminonucleoside. Kidney Int 66: 1881eC1889, 2004
Kojima K, Matsui K, Nagase M: Protection of alpha(3) integrin-mediated podocyte shape by superoxide dismutase in the puromycin aminonucleoside nephrosis rat. Am J Kidney Dis 35: 1175eC1185, 2000
Adler S: Structure-function relationships associated with extracellular matrix alterations in diabetic glomerulopathy. J Am Soc Nephrol 5: 1165eC1172, 1994
Kanwar YS, Liu ZZ, Kashihara N, Wallner EI: Current status of the structural and functional basis of glomerular filtration and proteinuria. Semin Nephrol 11: 390eC413, 1991
Kashihara N, Watanabe Y, Makino H, Wallner EI, Kanwar YS: Selective decreased de novo synthesis of glomerular proteoglycans under the influence of reactive oxygen species. Proc Natl Acad Sci U S A 89: 6309eC6313, 1992
Brinkkoetter PT, Holtgrefe S, van der Woude FJ, Yard BA: Angiotensin II type 1-receptor mediated changes in heparan sulfate proteoglycans in human SV40 transformed podocytes. J Am Soc Nephrol 15: 33eC40, 2004
Yard BA, Kahlert S, Engelleiter R, Resch S, Waldherr R, Groffen AJ, van den Heuvel LP, van der Born J, Berden JH, Kroger S, Hafner M, van der Woude FJ: Decreased glomerular expression of agrin in diabetic nephropathy and podocytes, cultured in high glucose medium. Exp Nephrol 9: 214eC222, 2001
Neufeld G, Cohen T, Gengrinovitch S, Potorak Z: Vascular endothelial growth factor and its receptors. FASEB J 13: 9eC22, 1999
De Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH: Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. J Am Soc Nephrol 12: 993eC1000, 2001
Flyvbjerg A, Dagnaes-Hansen F, De Vriese AS, Schrijvers BF, Tilton RG, Rasch R: Amelioration of long-term renal changes in obese type 2 diabetic mice by a neutralizing vascular endothelial growth factor antibody. Diabetes 51: 3090eC3094, 2002
Schrijvers BF, Flyvbjerg A, De Vriese AS: The role of vascular endothelial growth factor (VEGF) in renal pathophysiology. Kidney Int 65: 2003eC2017, 2004
Cooper ME, Vranes D, Youssef S, Stacker SA, Cox AJ, Rizkalla B, Casley DJ, Bach LA, Kelly DJ, Gilbert RE: Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes 48: 2229eC2239, 1999
Wendt TM, Tanji N, Guo J, Kislinger TR, Qu W, Lu Y, Bucciarelli LG, Rong LL, Moser B, Markowitz GS, Stein G, Bierhaus A, Liliensiek B, Arnold B, Nawroth PP, Stern DM, D’Agati VD, Schmidt AM: RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy. Am J Pathol 162: 1123eC1137, 2003
Iglesias-de la Cruz MC, Ziyadeh FN, Isono M, Kouahou M, Han DC, Kalluri R, Mundel P, Chen S: Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes. Kidney Int 62: 901eC913, 2002
Chen S, Kasama Y, Lee JS, Jim B, Marin M, Ziyadeh FN: Podocyte-derived vascular endothelial growth factor mediates the stimulation of 3(IV) collagen production by transforming growth factor-1 in mouse podocytes. Diabetes 53: 2939eC2949, 2004
Rizkalla B, Forbes JM, Cooper ME, Cao Z: Increased renal vascular endothelial growth factor and angiopoietins by angiotensin II infusion is mediated by both AT1 and AT2 receptors. J Am Soc Nephrol 14: 3061eC3071, 2003
Kakizawa H, Itoh Y, Imamura S, Matsumoto T, Ishiwata Y, Ono Y, Yamamoto K, Kato T, Hayakawa N, Oda N, Goto Y, Nagasaka A, Senda T, Itoh M: Possible role of VEGF in the progression of kidney disease in streptozotocin (STZ)-induced diabetic rats: effects of an ACE inhibitor and an angiotensin II receptor antagonist. Horm Metab Res 36: 458eC464, 2004
Freeburg PB, Robert B, St. John PL, Abrahamson DR: Podocyte expression of hypoxia-inducible factor (HIF)-1 and HIF-2 during glomerular development. J Am Soc Nephrol 14: 927eC938, 2003
Wolf G, Schroeder R, Stahl RA: Angiotensin II induces hypoxia-inducible factor-1 alpha in PC 12 cells through a posttranscriptional mechanism: role of AT2 receptors. Am J Nephrol 24: 415eC421, 2004
Cohen MP, Sharma K, Jin Y, Hud E, Wu VY, Tomaszewski J, Ziyadeh FN: Prevention of diabetic nephropathy in db/db mice with glycated albumin antagonists: a novel treatment strategy. J Clin Invest 95: 2338eC2345, 1995
Daniels BS, Brandt C, Aggarwal M: Increased glomerular H202 production in diabetes: role of glycated proteins (Abstract). J Am Soc Nephrol 4: 791A, 1993
Foster RR, Hole R, Anderson K, Satchell SC, Coward RJ, Mathieson PW, Gillatt DA, Saleem MA, Bates DO, Harper SJ: Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes. Am J Physiol Renal Physiol 284: F1263eCF1273, 2003
Sung SH, Chen S, Laping NJ, Ziyadeh FN: Albuminuria is ameliorated by an inhibitor of VEGF receptors, SU5416, in diabetic db/db mice but is unaffected in Smad3-knockout mice rendered diabetic with streptozotocin (Abstract). J Am Soc Nephrol 15: 720AeC721A, 2004
Chen S, Jim B, Ziyadeh FN: Diabetic nephropathy and transforming growth factor-beta: transforming our view of glomerulosclerosis and fibrosis build-up. Semin Nephrol 23: 532eC543, 2003
Ziyadeh FN: Mediators of diabetic renal disease: the case for TGF- as the major mediator. J Am Soc Nephrol 15 (Suppl. 1): S55eCS57, 2004
Wang S-N, Hirschberg R: Growth factor ultrafiltration in experimental diabetic nephropathy contributes to interstitial fibrosis. Am J Physiol Renal Physiol 278: F554eCF560, 2000
Wang S-N, LaPage J, Hirschberg R: Role of glomerular ultrafiltration of growth factors in progressive interstitial fibrosis in diabetic nephropathy. Kidney Int 57: 1002eC1014, 2000
Hong SW, Isono M, Chen S, Iglesias-de la Cruz MC, Han DC, Ziyadeh FN: Increased glomerular and tubular expression of TGF-beta1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 158: 1653eC1663, 2001
Ziyadeh FN, Hoffman BB, Han DC, Iglesias-de la Cruz MC, Hong SW, Isono M, Chen S, McGowan TA, Sharma K: Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor- antibody in db/db diabetic mice. Proc Natl Acad Sci U S A 97: 8015eC8020, 2000
Kopp JB, Factor VM, Mozes M, Nagy P, Sanderson N, Bottinger EP, Klotman PE, Thorgeirsson SS: Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease. Lab Invest 74: 991eC1003, 1996
Sharma R, Khanna A, Sharma M, Savin VJ: Transforming growth factor-beta1 increases albumin permeability of isolated rat glomeruli via hydroxyl radicals. Kidney Int 58: 131eC136, 2000
Benigni A, Zoja C, Corna D, Zatelli C, Conti S, Campana M, Gagliardini E, Rottoli D, Zanchi C, Abbate M, Ledbetter S, Remuzzi G: Add-on anti-TGF-beta antibody to ACE inhibitor arrests progressive diabetic nephropathy in the rat. J Am Soc Nephrol 14: 1816eC1824, 2003
Fujimoto M, Maezawa Y, Yokote K, Joh K, Kobayashi K, Kawamura H, Nishimura M, Roberts AB, Saito Y, Mori S: Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy. Biochem Biophys Res Commun 305: 1002eC1007, 2003
Massaguee J: TGF- signal transduction. Annu Rev Biochem 67: 753eC791, 1998(Gunter Wolf, Sheldon Chen)
2 Department of Medicine, Renal-Electrolyte and Hypertension Division, Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT
Nephropathy is a major complication of diabetes. Alterations of mesangial cells have traditionally been the focus of research in deciphering molecular mechanisms of diabetic nephropathy. Injury of podocytes, if recognized at all, has been considered a late consequence caused by increasing proteinuria rather than an event inciting diabetic nephropathy. However, recent biopsy studies in humans have provided evidence that podocytes are functionally and structurally injured very early in the natural history of diabetic nephropathy. The diabetic milieu, represented by hyperglycemia, nonenzymatically glycated proteins, and mechanical stress associated with hypertension, causes downregulation of nephrin, an important protein of the slit diaphragm with antiapoptotic signaling properties. The loss of nephrin leads to foot process effacement of podocytes and increased proteinuria. A key mediator of nephrin suppression is angiotensin II (ANG II), which can activate other cytokine pathways such as transforming growth factor- (TGF-) and vascular endothelial growth factor (VEGF) systems. TGF-1 causes an increase in mesangial matrix deposition and glomerular basement membrane (GBM) thickening and may promote podocyte apoptosis or detachment. As a result, the denuded GBM adheres to Bowman’s capsule, initiating the development of glomerulosclerosis. VEGF is both produced by and acts upon the podocyte in an autocrine manner to modulate podocyte function, including the synthesis of GBM components. Through its effects on podocyte biology, glomerular hemodynamics, and capillary endothelial permeability, VEGF likely plays an important role in diabetic albuminuria. The mainstays of therapy, glycemic control and inhibition of ANG II, are key measures to prevent early podocyte injury and the subsequent development of diabetic nephropathy.
Diabetic nephropathy is the leading cause of end-stage renal disease and is clinically characterized by proteinuria and progressive renal insufficiency. Dating back to the first description by Kimmelstiel and Wilson (1), histological analyses have focused on the increase in mesangial matrix as the main lesion of diabetic glomerulopathy. In addition, glomerular basement membrane (GBM) thickening has been considered an important pathophysiological event in the disease. These pathological alterations have been linked to functional consequences, first described in landmark structure-function studies done in patients with type 1 as well as type 2 diabetes (2eC4). Specifically, mesangial matrix expansion correlates closely with both proteinuria and deterioration of renal function. It has been proposed that accumulation of matrix in the mesangial area reduces the capillary surface area available for filtration, thereby contributing to the progressive loss of renal function (2). Renal failure may also arise from nephron dropout due to tubulointerstitial fibrosis (5), and this process is aggravated by the harmful downstream effects of proteinuria on the tubules (6). However, the genesis of proteinuria in diabetes is not readily explained by the associated mesangial matrix expansion. Rather, consideration should be given to alterations of the glomerular filtration barrier, which is composed of the glomerular endothelium, the GBM, and the podocyte (glomerular visceral epithelial cell). Widespread endothelial dysfunction is believed to result in proteinuria (7), which is exacerbated by intraglomerular hemodynamic stress (8). Although it is highly fenestrated, the glomerular endothelium might pose some hindrance to protein permeability (9,10). As for the GBM, its conspicuous thickening in diabetes, perhaps due to accumulation of collagen IV and alterations in its architecture and composition (11), would seem to constitute a more effective barrier to the filtration of proteins but is in fact more porous to proteins (12). While loss of charge selectivity in the GBM has been proposed to partly explain the proteinuria (13), a decrease in negatively charged proteoglycans occurs late in the course of diabetic nephropathy, sometimes long after the appearance of microalbuminuria (14). This leads to the conclusion that the final barrier restricting plasma proteins to the vasculature is the slit diaphragm of the podocyte; little research, however, has been conducted on podocyte biology in diabetes until recently. The advent of improved cell culture techniques, the discovery of key podocyte-specific molecules, and advances in transgenic technology have revolutionized the study of podocytes in health and disease (15). The purpose of this review is to summarize the emerging evidence that podocytopathy plays a pivotal role in the manifestations of diabetic glomerulopathy, expanding upon the "mesangiocentric" dogma of diabetic glomerular disease.
PODOCYTE BIOLOGY
A comprehensive description of the biology of this highly differentiated cell is beyond the scope of this article, but a basic understanding is necessary to appreciate how podocyte malfunction contributes to diabetic nephropathy (15). Podocytes extend long processes toward the GBM to which they affix by cell surface adhesion proteins such as the 31 integrin and dystroglycan (16eC18). The foot processes of adjacent podocytes interdigitate and are separated by narrow spaces (30eC40 nm) that are bridged by a porous membrane called the slit diaphragm. These membranes contain pores that are freely permeable to water and small solutes but relatively impermeable to plasma proteins (19,20). Thus, the integrity of the slit diaphragm is one of the principal determinants of the permselective properties of the glomerular filtration barrier, and knowledge of its molecular architecture will help elucidate the role that the slit diaphragm plays in proteinuria (15). A major advance was the discovery of nephrin, whose gene is mutated in the congenital nephrotic syndrome of the Finnish type, a rare form of hereditary nephrosis characterized by diffuse foot process effacement of the podocytes (21). Nephrin, a transmembrane protein with a large extracellular portion, self-associates in a zipper-like arrangement through homophilic dimerization, forming the molecular substrate of the slit diaphragm (22). Other proteins such as podocin and CD2-associated protein (CD2AP) interact with nephrin in cholesterol-rich regions of the cytoplasm called lipid rafts and anchor nephrin to actin filaments of the podocyte cytoskeleton (15,23). CD2AP links nephrin and podocin to phosphoinositide 3-OH kinase, and this complex has cell-signaling properties that stimulate Akt, a serine-threonine kinase (24). One target of nephrin/CD2AP-induced phosphorylation is Bad, a proapoptotic protein of the Bcl-2 family (24). Upon phosphorylation, Bad is inactivated and apoptosis does not occur. Thus, afferent signaling coming from intact slit diaphragms prevents podocyte apoptosis (24,25), a beneficial effect given that terminally differentiated podocytes fail to proliferate and are not easily replaced (26). The inability of podocytes to proliferate may be secondary to upregulation of cell cycle inhibitory proteins, p57 and p27 (22). Expression of p27 in cultured podocytes can be stimulated by high glucose, and this further prevents cell replication (22).
Because it does not normally divide, the podocyte represents one of the last unexplored frontiers in renal cell culture research. Podocytes in primary culture can be coaxed into reentering the cell cycle, but they invariably lose their differentiated characteristics. Besides, the cell homogeneity in primary culture is questionable because glomerular parietal epithelial cells often contaminate and overgrow the podocytes (27). To circumvent these difficulties, "conditional immortalization" has been developed to obtain a pure population of podocytes that can either proliferate or differentiate (28). The technique involves the introduction of an oncogene whose gene product is temperature sensitive, such as the tsA58 variant of the SV40 large tumor antigen (TAg) (29). The TAg protein complexes with and disables p53 and retinoblastoma proteins, removing two major barriers to cell cycle progression and allowing the podocyte to be propagated in culture. When differentiated podocytes are needed for experimentation, the cells can be "thermoshifted" to a higher temperature that will inactivate tsA58, reverse the immortalization, and permit differentiation to occur.
PODOCYTOPATHY IN HUMAN DIABETIC KIDNEY DISEASE
Experiments from 25 years ago in diabetic rats described foot process widening of podocytes (30), a finding later confirmed in patients with relatively advanced nephropathy due to type 1 diabetes (31). More recent investigations also described an increase in foot process width in microalbuminuric type 1 diabetic subjects, and foot process width correlated directly with the urinary albumin excretion rate (32). In addition to foot process widening, the number and density of podocytes have been reported to be markedly reduced (podocytopenia) in diabetic patients, whether afflicted with type 1 or type 2 diabetes (33eC36). While an ideal protocol for counting podocytes in biopsy specimens has not been developed yet, it has been argued that modern morphometric techniques can yield reasonable approximations. For instance, it has been estimated that the podocyte number is significantly reduced, even in diabetes of short duration (33). In Pima Indians with type 2 diabetes, podocyte density is drastically reduced, and the remaining foot processes are widened to maintain GBM coverage (34,37). Among all glomerular morphological characteristics, the decreased number of podocytes per glomerulus was the strongest predictor of progressive renal disease, with fewer cells predicting more rapid progression (38). Additionally, a recent morphometric study (36) in European type 2 diabetic patients has shown that a diminution of podocyte density rather than absolute reduction in podocyte number is more predictive of the presence and magnitude of albuminuria. The numerical density of podocytes per glomerulus was reduced in patients with microalbuminuria and was decreased even further in patients with overt proteinuria (36). A cross-sectional study also reported a significant inverse correlation between proteinuria and both podocyte number and density per glomerulus (39). Consistent with the observed loss of podocytes in diabetes, podocytes were present in the urine in 53% of type 2 diabetic patients with microalbuminuria and in 80% with macroalbuminuria, while normoalbuminuric patients and healthy control subjects had undetectable levels of urinary podocytes (40). Interestingly, treatment with an ACE inhibitor reduced the number of urinary podocytes (40).
Podocytopenia may exacerbate the development of proteinuria because a denuded GBM can come into contact with Bowman’s capsule and promote synechiae formation, an initial step in the development of glomerulosclerosis (15). Morphologic changes in the podocyte are also believed to engender proteinuria. With foot process widening, the decrease in slit diaphragm length might impede the filtration of water and lower the glomerular filtration rate. However, if protein permeability remains intact, as postulated in an innovative theory (41), the amount of protein relative to water is increased in the urinary space, and the elevated protein concentration could exceed the tubule’s reabsorptive capacity and manifest as proteinuria. The merit of this theory is that it reconciles the paradox of increasing proteinuria in the face of declining renal function, a characteristic of diabetic nephropathy, but the theory remains unproven and controversial (41). Even so, the worsening proteinuria could induce tubular atrophy and interstitial fibrosis, and these irreversible tubulointerstitial changes, together with glomerulosclerosis, then lead to chronic renal insufficiency (6).
To detect earlier functional abnormalities in podocytes, several recent studies have focused on the expression of podocyte-specific proteins in diabetic patients. For instance in type 1 diabetes, nephrin excretion in the urine (nephrinuria), detected by Western blot analysis, was present in 30% of the patients with normoalbuminuria, 17% of those with microalbuminuria, and 28% of those with macroalbuminuria, whereas none of the nondiabetic subjects had nephrinuria (42). These findings were not correlated with biopsies, but they suggest that increased urinary nephrin equates with early podocyte injury, even before the onset of microalbuminuria. Another study (43) found that nephrin staining was extensively reduced in renal biopsy specimens from nephropathic patients with type 1 diabetes. Several studies have evaluated nephrin mRNA and protein expression in type 2 diabetes. In general, nephrin protein production was downregulated in the diabetic subjects compared with the nondiabetic control subjects (44eC46), and the decrease in nephrin correlated with the broadening of the foot process widths. In contrast, CD2AP expression was not reduced in podocytes from diabetic patients, suggesting that the reduction in nephrin was not due to widespread podocyte loss or injury (45); this underscores the importance of nephrin in maintaining podocyte integrity. Finally, type 2 diabetic patients who were treated with the ACE inhibitor perindopril for 2 years exhibited a nephrin mRNA expression that was close to normal when compared with nondiabetic control subjects; patients not treated with the ACE inhibitor had significantly reduced nephrin transcripts (46).
PODOCYTE DYSFUNCTION IN EXPERIMENTAL DIABETES
Animal experiments allow for the repeated sampling of kidney tissue over time, furnishing a longitudinal record of the development of diabetic nephropathy and the efficacy of pharmacological interventions. Serial investigations (47) on kidneys from obese Zucker fa/fa rats, a model of type 2 diabetes that develops segmental glomerulosclerosis, revealed that nephropathy started with damage to podocytes, manifesting as foot process effacement and cytoplasmic accumulation of lipid droplets. Early podocyte damage antedated the development of glomerulosclerosis and tubulointerstitial damage in this model (48). In the streptozotocin (STZ)-induced diabetic rat, a model of type 1 diabetes, several studies (49,50) have reported a decrease in podocyte number, broadening of the foot processes, and reduction in nephrin expression.
PODOCYTE APOPTOSIS AND/OR DETACHMENT IN HUMANS AND ANIMAL MODELS
The exact etiology for podocyte loss in diabetes remains speculative, but two mechanisms can be suggested: apoptosis and cell detachment. Evidence for a primary role of apoptosis in either human or experimental diabetic nephropathy is scant (51eC54). However, podocyte apoptosis can be demonstrated in cell culture. For instance, angiotensin II (ANG II) induces apoptosis in cultured rat glomerular epithelial cells, and this effect is mediated by the transforming growth factor- (TGF-) system since it is inhibited by an antieCTGF- antibody (55). In TGF-1eCoverexpressing transgenic mice, albeit nondiabetic, the podocyte undergoes apoptosis in situ shortly after the sclerotic lesion appears in the glomerulus (56). Thus, the heightened intraglomerular activity of TGF-1 that is characteristic of diabetes may theoretically be responsible for apoptosis (57), a process that can be mediated through Smad7, which inhibits the nuclear translocation of the cell survival factor nuclear factor-B (56,58).
The other mechanism of podocyte loss in diabetes may relate to the detachment of podocytes from the GBM. This scenario does not exclude a role for apoptosis, since cells may first detach and then undergo apoptosis because of interruption of viability cues deriving from cell-matrix interactions (17). Alternatively, detached cells can be shed in the urine as live podocytes (59). In fact, podocyturia worsens with the progression from normoalbuminuria to microalbuminuria to overt proteinuria (40). Loss of cell anchorage to the GBM may result from downregulation of the 31 integrin receptor, the principal adhesion complex that attaches the podocyte to the GBM (16,17,60,61). Several studies (62eC64) have shown that the 31 integrin is decreased in the podocytes of humans and rats with diabetes. Furthermore, high glucose media in cultured rat or human podocytes decreases the expression of 31 integrin (62,64); this downregulation is perhaps mediated by increased levels of TGF-1 (65,66).
ROLE OF ANG II IN NEPHRIN EXPRESSION AND PODOCYTE INJURY
Pharmacological interventions in animal models have supported the view that an increased ANG II activity is involved in podocyte injury in diabetes. ACE inhibition or AT1 receptor antagonism attenuated podocyte foot process broadening in rats with STZ-induced diabetes (49). In another study (67) involving the STZ-induced diabetic rat, therapy with an ACE inhibitor, but not an endothelin receptor antagonist, prevented loss of podocytes and podocyte injury. Similarly, an ACE inhibitor, but not aminoguanidine, an inhibitor of advanced glycation end product (AGE) formation, attenuated the diabetes-associated reduction in nephrin expression (50). Likewise, an AT1 receptor antagonist, but not the calcium channel blocker amlodipine, normalized the reduced nephrin expression in podocytes from spontaneously hypertensive rats with superimposed STZ-induced diabetes (68,69). Finally, the renal damage that occurs in obese Zucker rats was more effectively controlled by combined treatment with an ACE inhibitor and an AT1 receptor antagonist than either monotherapy alone (70). Thus, it may be concluded that suppression of nephrin expression in podocytes can be caused by increased renal ANG II activity in diabetes, but the molecular mechanisms are incompletely understood.
Podocytes express the AT1 and probably the AT2 receptors after injury and therefore could respond to stimulation with ANG II (15,71). Transgenic rats with targeted overexpression of the podocyte’s AT1 receptor showed pseudocysts in podocytes, followed by foot process effacement and local detachment, with subsequent progression to focal segmental glomerulosclerosis (72). High glucose concentrations induce ANG II formation in podocytes through upregulation of angiotensinogen expression (15). Furthermore, ANG II production in podocytes can be activated by proteinuria, likely by the transit of proteins through the filtration barrier (73) and by mechanical stretch, which mimics the hemodynamic effects of intraglomerular hypertension (74). Interestingly, ANG II formation as a consequence of mechanical stretch appears to be independent of ACE (74). As an alternative to ACE for the conversion of ANG I to ANG II, chymase has been shown to be upregulated in the glomeruli of patients with nephropathy due to type 2 diabetes (75). Because chymase is not blocked by ACE inhibitors, glomeruli in the diabetic state may still generate ANG II despite ACE inhibition. Some evidence indicates that the increase in angiotensinogen expression is signaled by intracellular reactive oxygen species (ROS) (74). Oxidative stress is a leading initiator of cellular dysfunction in diabetes complications (76,77), and increased ROS generation can induce podocyte dysfunction (78,79).
ROLE OF HEPARAN SULFATE PROTEOGLYCANS
A decrease in the GBM content of negatively charged HSPG contributes to the loss of charge selectivity in the glomerular filtration barrier and to the progression of proteinuria (80,81). One potential mechanism for decreased de novo synthesis of proteoglycans is the increased generation of ROS (81,82). Although the predominant proteoglycan in the GBM was thought to be perlecan, it has become more clear that agrin is the more abundant HSPG (83). Synthesis of proteoglycans occurs in all three glomerular cell types, but podocytes are an especially important source of these negatively charged molecules. Proteoglycan synthesis in podocytes is differentially influenced by high ambient glucose and ANG II (83,84); high glucose suppresses the production of agrin’s core protein, whereas ANG II decreases synthesis of the core protein and diminishes the sulfation of its side chains (83,84). Podocytes exposed to ANG II decrease the amount of HSPG on their cell surfaces and in the extracellular matrix (83); these results may partly explain the antiproteinuric effect of ACE inhibitors and angiotensin receptor blockers in diabetic nephropathy.
ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR
Losses of nephrin and proteoglycan are not the only pathological changes fostering proteinuria in diabetic nephropathy. Vascular endothelial growth factor (VEGF), a survival and angiogenic factor with strong microvascular permeabilizing properties (85), may increase the permeability of the glomerular filtration barrier to circulating proteins. The most convincing evidence that VEGF overexpression is involved in the proteinuria of diabetes comes from studies in type 1 diabetic (STZ-induced) rats and type 2 diabetic db/db mice. Neutralization of VEGF with a systemically administered anti-VEGF antibody reduced the urinary albumin excretion by at least 50% compared with the untreated diabetic controls (86,87). In addition, VEGF induces endothelial nitric oxide synthase, thereby promoting the vasodilation and hyperfiltration that are typical of early diabetic nephropathy (86,88).
The expression of VEGF in the glomerulus is most pronounced in the podocytes, and experimental diabetes increases VEGF mRNA and protein expression (89,90). In cultured podocytes, VEGF expression can be stimulated by high glucose concentrations (91), by TGF-1 (91,92), and by ANG II acting via AT1 and AT2 receptors (93,94). The gene expression of VEGF is also stimulated by a transcription factor called hypoxia-inducible factor-1 (95). We have demonstrated in PC12 cells that hypoxia-inducible factor-1 expression is stimulated by ANG II in a posttranscriptional mechanism involving AT2 receptors (96). This increase is caused by downregulation of a prolyl hydroxylase (SM-20/PHD3), the enzyme responsible for initiating hypoxia-inducible factor-1 degradation (96). Upregulation of podocyte-derived VEGF in diabetes may depend on signals downstream of the receptor for AGE, or RAGE (90). RAGE expression is increased in the podocytes of diabetic db/db mice (90), and inhibition of AGE-RAGE interactions by soluble RAGE treatment significantly depressed VEGF expression in the kidney and ameliorated albuminuria and glomerulosclerosis (90). Precursors of AGE such as Amadori-glycated serum albumin may mediate diabetic proteinuria (97) and promote glomerular production of hydrogen peroxide (98). Whether increased ROS production stimulates podocyte VEGF expression remains to be investigated.
An intriguing concept that has come to attention is that podocytes not only produce VEGF but are also acted upon by VEGF (92,99). This VEGF autocrine loop may play important roles in podocyte biology since VEGF decreases intracellular calcium concentrations and protects against cytotoxicity (99), possibly via the antiapoptotic actions of nephrin (25). These cytoprotective effects were reversed when endogenous VEGF was blocked by either a class III tyrosine kinase inhibitor PTK787/ZK222584 (99) or a monoclonal anti-VEGF antibody (25).
VEGF also stimulates the podocyte to produce the 3 chain of collagen IV, a principal ingredient of the GBM; this effect may be mediated by VEGFR-1 signaling (92). When endogenous VEGF secretion by podocytes was stimulated by TGF-1 treatment, the production of 3(IV) collagen increased (92). Blockade of endogenous VEGF action by a specific inhibitor of VEGF receptor kinases, SU5416, reduced the TGF-1eCinduced expression of 3(IV) collagen by 50%, establishing a pivotal role for the VEGF autocrine system in at least one aspect of the regulation of GBM composition by podocytes (92).
It may be speculated that VEGF-stimulated podocyte production of 3(IV) collagen contributes to diabetic thickening of the GBM and its altered permselectivity. Extrapolating from our in vitro work in cultured podocytes, we treated diabetic db/db mice with intraperitoneal injections of SU5416 at 2 mg/kg body wt, given twice a week for 8 weeks (100). SU5416 treatment, which had no adverse effects, significantly prevented GBM thickening and albuminuria without affecting blood glucose levels (100 and S.H. Sung, S.C., F.N.Z., unpublished data). These findings with SU5416 compare favorably with those of the anti-VEGF antibody in db/db mice (87); however, that study did not report the effect of anti-VEGF therapy on GBM thickening. That SU5416 particularly benefited the podocyte was also suggested by the preservation of nephrin protein, assayed by immunostaining (S.H. Sung, S.C., F.N.Z., unpublished data). Glomerular nephrin levels, which are typically reduced in the db/db mouse, were restored almost to normal by SU5416 treatment (S.H. Sung, S.C., F.N.Z., unpublished data), suggesting that nephrin preservation represents another mechanism whereby VEGF blockade might ameliorate diabetic albuminuria.
ROLE OF TGF-1
TGF- plays an integral role in the pathogenesis of diabetic nephropathy (101). This fibrogenic cytokine is stimulated by the diabetic state, is increased in the kidney during diabetes, and is able to recapitulate the hypertrophic and sclerotic features of diabetic renal disease (102). Elevated levels of TGF- have been measured in the glomeruli of STZ-induced diabetic rats by micropuncture techniques (103,104), and an intracellular transducer of TGF- signaling, Smad3, has been shown to translocate into the glomerular nuclei of diabetic db/db mice (105), attesting to the overactivity of the TGF- system in diabetic nephropathy. Inhibition of TGF- by a panselective neutralizing antibody in diabetic db/db mice prevented diabetic renal hypertrophy, mesangial matrix expansion, and the development of renal insufficiency (106). However, there was no significant effect on albuminuria in the diabetic mice (106).
A clear role for TGF- in the pathogenesis of diabetic albuminuria cannot be established at present because of conflicting studies. In nondiabetic models, overexpression of active TGF-1 in transgenic mice caused mesangial expansion, interstitial fibrosis, renal insufficiency, and progressive proteinuria (107). In addition, an increase in albumin permeability was observed when isolated glomeruli from normal rats were exposed ex vivo to TGF-1 (108). However, in our studies on the type 2 diabetic db/db mouse, we failed to see a significant reduction in albuminuria after 8 weeks of treatment with a neutralizing antieCTGF- antibody, perhaps because VEGF expression in the kidney cortex was kept elevated by other diabetic factors (106). Interestingly, the work of Benigni et al. (109) in uninephrectomized STZ-induced diabetic rats revealed that one but not another species of antieCTGF- antibody significantly reduced proteinuria when compared with control IgG. Nevertheless, either antibody could potentiate the antiproteinuric effect of an ACE inhibitor when given in combination (109). Other studies showed that when a portion of the TGF- signaling pathway was selectively inhibited by the creation of a Smad3 knockout mouse, the albuminuria persisted when the mice were rendered diabetic with STZ (100). However, another cohort of Smad3 knockout mice with a different strain background showed significant amelioration of albuminuria in STZ-induced diabetes (110). Nevertheless, both diabetic Smad3 knockout models displayed prevention of mesangial matrix expansion (110 and S.C., S.H. Sung, N.J. Laping, F.N.Z., unpublished data). Thus, it is possible to dissociate albuminuria from mesangial matrix expansion, and the pathogenesis of proteinuria may relate more to VEGF and nephrin while the pathogenesis of glomerulosclerosis may deal more with TGF- and other fibrogenic pathways.
The podocyte is also subject to the increased levels and overactivity of intraglomerular TGF- in diabetes. As discussed above, podocyte detachment and/or apoptosis can be caused by increased TGF- activity (55,58,62). Additionally, TGF-1 stimulates the podocyte expression of 3(IV) collagen (91), but surprisingly, TGF-1 suppresses the production of both 1(IV) and 5(IV) collagen (91), important exceptions to the dogma that TGF- is uniformly profibrotic. TGF-1 also stimulates the podocyte expression of VEGF (91) that in turn increases the activity of the VEGF autocrine loop, with all of its attendant effects (25,92,99).
Unlike other renal cells, podocytes may not respond to the diabetic state by increasing their expression of TGF-. The production of TGF-1 in cultured mouse podocytes was not significantly stimulated by high glucose (91) or by ANG II (S.C., F.N.Z., unpublished data). Instead, these metabolic stimuli significantly upregulated the expression of the TGF- type II receptor (91 and S.C., F.N.Z., unpublished data), which binds to the TGF- ligand and initiates the TGF- signaling cascade (111). In theory, the increase in type II receptor could make the podocyte more sensitive to the elevated levels of TGF- in the diabetic state, and it could enhance a paracrine interaction between the podocyte and the mesangial and/or glomerular endothelial cell.
CONCLUSIONS
Although the pathophysiology of mesangial cells has long been considered to be the major factor in the development of glomerulosclerosis in type 1 or type 2 diabetes, more recent evidence suggests that podocytes also play a critical role in the early functional and structural changes of diabetic kidney disease. On the one hand, podocytes are the victims of pathogenetic events surrounding the progression of diabetic nephropathy, including the harmful effects of high glucose, AGE, ANG II, ROS, TGF-, and mechanical stretch. On the other hand, podocytes are the culprits in diabetic nephropathy in that a reduced nephrin expression may lead to podocyte foot process broadening and effacement, while increased VEGF production may exacerbate GBM thickening and proteinuria, leading to tubular atrophy and interstitial fibrosis (Fig. 1). Many of these events are mediated by ANG II, whose local concentration is increased in diabetic kidney disease. ANG II in turn suppresses nephrin and induces TGF- type II receptor expression in podocytes, perhaps intensifying the paracrine effects of TGF- derived from the mesangium or glomerular endothelium. Increased TGF- activity may lead to podocyte apoptosis and/or detachment with podocytopenia and the development of progressive glomerulosclerosis. An additional sequela is VEGF overactivity that could increase glomerular hemodynamic stress, alter the production of GBM components, and repress nephrin expression, all maladaptive events that foster proteinuria (Fig. 1).
The ideal therapy for diabetic nephropathy remains elusive, but excellent glycemic control and ANG II antagonism have become the foundations of current therapy. Promising new treatments that target the roles of VEGF, TGF-, nephrin, HSPG, glycated proteins, and ROS are beginning to appear on the horizon. In this regard, protection of podocytes will be of paramount importance in preventing the development and progression of diabetic nephropathy.
ACKNOWLEDGMENTS
G.W. is supported by a grant from the Deutsche Forschungsgemeinschaft. S.C. is supported by a grant from the National Institutes of Health (DK061537). F.N.Z. is supported by grants from the American Diabetes Association and the National Institutes of Health (DK44513 and DK54608) and by the Department of Veterans Affairs.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
AGE, advanced glycation end product; ANG II, angiotensin II; CD2AP, CD2-associated protein; GBM, glomerular basement membrane; HSPG, heparan sulfate proteoglycans; RAGE, receptor for AGE; ROS, reactive oxygen species; STZ, streptozotocin; TGF-, transforming growth factor-; VEGF, vascular endothelial growth factor
REFERENCES
Kimmelstiel P, Wilson C: Intercapillary lesions in the glomeruli of the kidney. Am J Pathol 12: 82eC97, 1936
Mauer SM, Steffes MW, Ellis EN, Sutherland DER, Brown DM, Goetz FC: Structural-functional relationships in diabetic nephropathy. J Clin Invest 74: 1143eC1155, 1984
Osterby R, Gall MA, Schmitz A, Nielsen FS, Nyberg G, Parving HH: Glomerular structure and function in proteinuric type 2 (non-insulin-dependent) diabetic patients. Diabetologia 36: 1064eC1070, 1993
White KE, Bilous RW: Type 2 diabetic patients with nephropathy show structural-functional relationships that are similar to type 1 disease. J Am Soc Nephrol 11: 1667eC1673, 2000
Ziyadeh FN, Goldfarb S: The renal tubulointerstitium in diabetes mellitus. Kidney Int 39: 464eC475, 1991
Remuzzi G, Bertani T: Pathophysiology of progressive nephropathies. N Engl J Med 339: 1448eC1456, 1998
Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, Jensen T, Kofoed-Enevoldsen A: Albuminuria reflects widespread vascular damage: the Steno hypothesis. Diabetologia 32: 219eC226, 1989
Zatz R, Meyer TW, Rennke HG, Brenner BM: Predominance of hemodynamic rather than metabolic factors in the pathogenesis of diabetic glomerulopathy. Proc Natl Acad Sci U S A 82: 5963eC5967, 1985
Deen WM, Lazzara MJ, Myers BD: Structural determinants of glomerular permeability. Am J Physiol Renal Physiol 281: F579eCF596, 2001
Deen WM: What determines glomerular capillary permeability J Clin Invest 114: 1412eC1414, 2004
Kefalides NA: Basement membrane research in diabetes mellitus. Coll Relat Res 1: 295eC299, 1981
Isogai S, Mogami K, Shiina N, Yoshino G: Initial ultrastructural changes in pore size and anionic sites of the glomerular basement membrane in streptozotocin-induced diabetic rats and their prevention by insulin treatment. Nephron 83: 53eC58, 1999
Menne J, Park JK, Boehne M, Elger M, Lindschau C, Kirsch T, Meier M, Gueler F, Fiebeler A, Bahlmann FH, Leitges M, Haller H: Diminished loss of proteoglycans and lack of albuminuria in protein kinase C-eCdeficient diabetic mice. Diabetes 53: 2101eC2109, 2004
Vernier RL, Steffes MW, Sisson-Ross S, Mauer SM: Heparan sulfate proteoglycan in the glomerular basement membrane in type 1 diabetes mellitus. Kidney Int 41: 1070eC1080, 1992
Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 83: 253eC307, 2003
Tsilibary EC: Microvascular basement membranes in diabetes mellitus. J Pathol 200: 537eC546, 2003
Kretzler M: Regulation of adhesive interaction between podocytes and glomerular basement membrane. Microsc Res Tech 57: 247eC253, 2002
Kojima K, Kerjaschki D: Is podocyte shape controlled by the dystroglycan complex Nephrol Dial Transplant 17 (Suppl. 9): 23eC24, 2002
Tryggvason K: Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm. J Am Soc Nephrol 10: 2440eC2445, 1999
Wartiovaara J, Ofverstedt LG, Khoshnoodi J, Zhang J, Makela E, Sandin S, Ruotsalainen V, Cheng RH, Jalanko H, Skoglund U, Tryggvason K: Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography. J Clin Invest 114: 1475eC1483, 2004
Tryggvason K, Ruotsalainen V, Wartiovaara J: Discovery of the congenital nephrotic syndrome gene discloses the structure of the mysterious molecular sieve of the kidney. Int J Dev Biol 43: 445eC451, 1999
Khoshnoodi J, Sigmundsson K, Ofverstedt LG, Skoglund U, Obrink B, Wartiovaara J, Tryggvason K: Nephrin promotes cell-cell adhesion through homophilic interactions. Am J Pathol 163: 2337eC2346, 2003
Saleem MA, Ni L, Witherden I, Tryggvason K, Ruotsalainen V, Mundel P, Mathieson PW: Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation. Am J Pathol 161: 1459eC1466, 2002
Huber TB, Hartleben B, Kim J, Schmidts M, Schermer B, Keil A, Egger L, Lecha RL, Borner C, Pavenstadt H, Shaw AS, Walz G, Benzing T: Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol 23: 4917eC4928, 2003
Foster RR, Saleem MA, Mathieson PW, Bates DO, Harper SJ: Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol 288: F48eCF57, 2004
Griffin SV, Petermann AT, Durvasula RV, Shankland SJ: Podocyte proliferation and differentiation in glomerular disease: role of cell-cycle regulatory proteins (Review). Nephrol Dial Transplant 18 (Suppl. 6): vi8eCv13, 2003
Weinstein T, Cameron R, Katz A, Silverman M: Rat glomerular epithelial cells in culture express characteristics of parietal, not visceral, epithelium. J Am Soc Nephrol 3: 1279eC1287, 1992
Mundel P, Reiser J, Ze瞚ga-Meje-Borja A, Pavenstdt H, Davidson G, Kriz W, Zeller R: Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248eC258, 1997
Jat PS, Sharp PA: Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature. Mol Cell Biol 9: 1672eC1681, 1989
Steffes MW, Leffert JD, Basgen JM, Brown DM, Mauer SM: Epithelia cell foot process width in intact and uninephrectomized diabetic and nondiabetic rats. Lab Invest 43: 225eC230, 1980
Ellis EN, Steffes MW, Chavers B, Mauer SM: Observations of glomerular epithelial cell structure in patients wit type I diabetes mellitus. Kidney Int 32: 736eC741, 1987
Berg UB, Torbjornsdotter TB, Jaremko G, Thalme B: Kidney morphological changes in relation to long-term renal function and metabolic control in adolescents with IDDM. Diabetologia 41: 1047eC1056, 1998
Steffes MW, Schmidt D, McCrery R, Basgen JM: Glomerular cell number in normal subjects and in type 1 diabetic patients. Kidney Int 59: 2104eC2113, 2001
Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, Meyer TW: Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 99: 342eC348, 1997
White KE, Bilous RW, Marshall SM, El Nahas M, Remuzzi G, Piras G, De Cosmo S, Viberti G: Podocyte number in normotensive type 1 diabetic patients with albuminuria. Diabetes 51: 3083eC3089, 2002
Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, Fioretto P: Is podocyte injury relevant in diabetic nephropathy Studies in patients with type 2 diabetes. Diabetes 52: 1031eC1035, 2003
Nelson RG, Meyer TW, Myers BD, Bennett PH: Clinical and pathological course of renal disease in non-insulin-dependent diabetes mellitus: the Pima Indian experience. Semin Nephrol 17: 124eC131, 1997
Meyer TW, Bennett GL, Nelson RG: Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria. Diabetologia 42: 1341eC1344, 1999
White KE, Bilous RW: Structural alterations to the podocyte are related to proteinuria in type 2 diabetic patients. Nephrol Dial Transplant 19: 1437eC1440, 2004
Nakamura T, Ushiyama C, Suzuki S, Hara M, Shimada N, Ebihara I, Koide H: Urinary excretion of podocytes in patients with diabetic nephropathy. Nephrol Dial Transplant 15: 1379eC1383, 2000
Smithies O: Why the kidney glomerulus does not clog: a gel permeation/diffusion hypothesis of renal function. Proc Natl Acad Sci U S A 100: 4108eC4113, 2003
Patari A, Forsblom C, Havana M, Taipale H, Groop PH, Holthofer H: Nephrinuria in diabetic nephropathy of type 1 diabetes. Diabetes 52: 2969eC2974, 2003
Doublier S, Salvidio G, Lupia E, Ruotsalainen V, Verzola D, Deferrari G, Camussi G: Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II. Diabetes 52: 1023eC1030, 2003
Koop K, Eikmans M, Baelde HJ, Kawachi H, De Heer E, Paul LC, Bruijn JA: Expression of podocyte-associated molecules in acquired human kidney diseases. J Am Soc Nephrol 14: 2063eC2071, 2003
Benigni A, Gagliardini E, Tomasoni S, Abbate M, Ruggenenti P, Kalluri R, Remuzzi G: Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy. Kidney Int 65: 2193eC2200, 2004
Langham RG, Kelly DJ, Cox AJ, Thomson NM, Holthofer H, Zaoui P, Pinel N, Cordonnier DJ, Gilbert RE: Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition. Diabetologia 45: 1572eC1576, 2002
Gassler N, Elger M, Kranzlin B, Kriz W, Gretz N, Hahnel B, Hosser H, Hartmann I: Podocyte injury underlies the progression of focal segmental glomerulosclerosis in the fa/fa Zucker rat. Kidney Int 60: 106eC116, 2001
Coimbra TM, Janssen U, Grone HJ, Ostendorf T, Kunter U, Schmidt H, Brabant G, Floege J: Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int 57: 167eC182, 2000
Mifsud SA, Allen TJ, Bertram JF, Hulthen UL, Kelly DJ, Cooper ME, Wilkinson-Berka JL, Gilbert RE: Podocyte foot process broadening in experimental diabetic nephropathy: amelioration with renin-angiotensin blockade. Diabetologia 44: 878eC882, 2001
Kelly DJ, Aaltonen P, Cox AJ, Rumble JR, Langham R, Panagiotopoulos S, Jerums G, Holthofer H, Gilbert RE: Expression of the slit-diaphragm protein, nephrin, in experimental diabetic nephropathy: differing effects of anti-proteinuric therapies. Nephrol Dial Transplant 17: 1327eC1332, 2002
Ortiz A, Ziyadeh FN, Neilson EG: Expression of apoptosis-regulatory genes in renal proximal tubular epithelial cells exposed to high ambient glucose and in diabetic kidneys. J Investig Med 45: 50eC56, 1997
Murata I, Takemura G, Asano K, Sano H, Fujisawa K, Kagawa T, Baba K, Maruyama R, Minatoguchi S, Fujiwara T, Fujiwara H: Apoptotic cell loss following cell proliferation in renal glomeruli of Otsuka Long-Evans Tokushima Fatty rats, a model of human type 2 diabetes. Am J Nephrol 22: 587eC595, 2002
Kumar D, Zimpelmann J, Robertson S, Burns KD: Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney. Nephron Exp Nephrol 96: e77eCe88, 2004
Kumar D, Robertson S, Burns KD: Evidence of apoptosis in human diabetic kidney. Mol Cell Biochem 259: 67eC70, 2004
Ding G, Reddy K, Kapasi AA, Franki N, Gibbons N, Kasinath BS, Singhal PC: Angiotensin II induces apoptosis in rat glomerular epithelial cells. Am J Physiol Renal Physiol 283: F173eCF180, 2002
Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke P, Mundel P, Bottinger EP: Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 108: 807eC816, 2001
Yoo J, Ghiassi M, Jirmanova L, Balliet AG, Hoffman B, Fornace AJ Jr, Liebermann DA, Bottinger EP, Roberts AB: Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation. J Biol Chem 278: 43001eC43007, 2003
Schiffer M, Mundel P, Shaw AS, Bottinger EP: A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta-induced apoptosis. J Biol Chem 279: 37004eC37012, 2004
Vogelmann SU, Nelson WJ, Myers BD, Lemley KV: Urinary excretion of viable podocytes in health and renal disease. Am J Physiol Renal Physiol 285: F40eCF48, 2003
Korhonen M, Ylanne J, Laitinen L, Virtanen I: Distribution of beta 1 and beta 3 integrins in human fetal and adult kidney. Lab Invest 62: 616eC625, 1990
Adler S: Characterization of glomerular epithelial cell matrix receptors. Am J Pathol 141: 571eC578, 1992
Chen HC, Chen CA, Guh JY, Chang JM, Shin SJ, Lai YH: Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes. Life Sci 67: 2345eC2353, 2000
Regoli M, Bendayan M: Alterations in the expression of the alpha 3 beta 1 integrin in certain membrane domains of the glomerular epithelial cells (podocytes) in diabetes mellitus. Diabetologia 40: 15eC22, 1997
Kitsiou PV, Tzinia AK, Stetler-Stevenson WG, Michael AF, Fan WW, Zhou B, Tsilibary EC: Glucose-induced changes in integrins and matrix-related functions in cultured human glomerular epithelial cells. Am J Physiol Renal Physiol 284: F671eCF679, 2003
Kagami S, Border WA, Ruoslahti E, Noble NA: Coordinated expression of beta 1 integrins and transforming growth factor-beta-induced matrix proteins in glomerulonephritis. Lab Invest 69: 68eC76, 1993
Kumar NM, Sigurdson SL, Sheppard D, Lwebuga-Mukasa JS: Differential modulation of integrin receptors and extracellular matrix laminin by transforming growth factor-beta 1 in rat alveolar epithelial cells. Exp Cell Res 221: 385eC394, 1995
Gross ML, El-Shakmak A, Szabo A, Koch A, Kuhlmann A, Munter K, Ritz E, Amann K: ACE-inhibitors but not endothelin receptor blockers prevent podocyte loss in early diabetic nephropathy. Diabetologia 46: 856eC868, 2003
Bonnet F, Cooper ME, Kawachi H, Allen TJ, Boner G, Cao Z: Irbesartan normalises the deficiency in glomerular nephrin expression in a model of diabetes and hypertension. Diabetologia 44: 874eC877, 2001
Davis BJ, Cao Z, de Gasparo M, Kawachi H, Cooper ME, Allen TJ: Disparate effects of angiotensin II antagonists and calcium channel blockers on albuminuria in experimental diabetes and hypertension: potential role of nephrin. J Hypertens 21: 209eC216, 2003
Toblli JE, DeRosa G, Cao G, Piorno P, Pagano P: ACE inhibitor and angiotensin type I receptor antagonist in combination reduce renal damage in obese Zucker rats. Kidney Int 65: 2343eC2359, 2004
Gloy J, Henger A, Fischer KG, Nitschke R, Mundel P, Bleich M, Schollmeyer P, Greger R, Pavenstdt H: Angiotensin II depolarizes podocytes in the intact glomerulus of the rat. J Clin Invest 99: 2772eC2781, 1997
Hoffmann S, Podlich D, Hahnel B, Kriz W, Gretz N: Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats. J Am Soc Nephrol 15: 1475eC1487, 2004
Abbate M, Zoja C, Morigi M, Rottoli D, Angioletti S, Tomasoni S, Zanchi C, Longaretti L, Donadelli R, Remuzzi G: Transforming growth factor-beta1 is up-regulated by podocytes in response to excess intraglomerular passage of proteins: a central pathway in progressive glomerulosclerosis. Am J Pathol 161: 2179eC2193, 2002
Durvasula RV, Petermann AT, Hiromura K, Blonski M, Pippin J, Mundel P, Pichler R, Griffin S, Couser WG, Shankland SJ: Activation of a local tissue angiotensin system in podocytes by mechanical strain. Kidney Int 65: 30eC39, 2004
Huang XR, Chen WY, Truong LD, Lan HY: Chymase is upregulated in diabetic nephropathy: implications for an alternative pathway of angiotensin II-mediated diabetic renal and vascular disease. J Am Soc Nephrol 14: 1738eC1747, 2003
Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813eC820, 2001
Evans JL, Goldfine ID, Maddux BA, Grodsky GM: Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 23: 599eC622, 2002
Vega-Warner V, Ransom RF, Vincent AM, Brosius FC, Smoyer WE: Induction of antioxidant enzymes in murine podocytes precedes injury by puromycin aminonucleoside. Kidney Int 66: 1881eC1889, 2004
Kojima K, Matsui K, Nagase M: Protection of alpha(3) integrin-mediated podocyte shape by superoxide dismutase in the puromycin aminonucleoside nephrosis rat. Am J Kidney Dis 35: 1175eC1185, 2000
Adler S: Structure-function relationships associated with extracellular matrix alterations in diabetic glomerulopathy. J Am Soc Nephrol 5: 1165eC1172, 1994
Kanwar YS, Liu ZZ, Kashihara N, Wallner EI: Current status of the structural and functional basis of glomerular filtration and proteinuria. Semin Nephrol 11: 390eC413, 1991
Kashihara N, Watanabe Y, Makino H, Wallner EI, Kanwar YS: Selective decreased de novo synthesis of glomerular proteoglycans under the influence of reactive oxygen species. Proc Natl Acad Sci U S A 89: 6309eC6313, 1992
Brinkkoetter PT, Holtgrefe S, van der Woude FJ, Yard BA: Angiotensin II type 1-receptor mediated changes in heparan sulfate proteoglycans in human SV40 transformed podocytes. J Am Soc Nephrol 15: 33eC40, 2004
Yard BA, Kahlert S, Engelleiter R, Resch S, Waldherr R, Groffen AJ, van den Heuvel LP, van der Born J, Berden JH, Kroger S, Hafner M, van der Woude FJ: Decreased glomerular expression of agrin in diabetic nephropathy and podocytes, cultured in high glucose medium. Exp Nephrol 9: 214eC222, 2001
Neufeld G, Cohen T, Gengrinovitch S, Potorak Z: Vascular endothelial growth factor and its receptors. FASEB J 13: 9eC22, 1999
De Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH: Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. J Am Soc Nephrol 12: 993eC1000, 2001
Flyvbjerg A, Dagnaes-Hansen F, De Vriese AS, Schrijvers BF, Tilton RG, Rasch R: Amelioration of long-term renal changes in obese type 2 diabetic mice by a neutralizing vascular endothelial growth factor antibody. Diabetes 51: 3090eC3094, 2002
Schrijvers BF, Flyvbjerg A, De Vriese AS: The role of vascular endothelial growth factor (VEGF) in renal pathophysiology. Kidney Int 65: 2003eC2017, 2004
Cooper ME, Vranes D, Youssef S, Stacker SA, Cox AJ, Rizkalla B, Casley DJ, Bach LA, Kelly DJ, Gilbert RE: Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes 48: 2229eC2239, 1999
Wendt TM, Tanji N, Guo J, Kislinger TR, Qu W, Lu Y, Bucciarelli LG, Rong LL, Moser B, Markowitz GS, Stein G, Bierhaus A, Liliensiek B, Arnold B, Nawroth PP, Stern DM, D’Agati VD, Schmidt AM: RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy. Am J Pathol 162: 1123eC1137, 2003
Iglesias-de la Cruz MC, Ziyadeh FN, Isono M, Kouahou M, Han DC, Kalluri R, Mundel P, Chen S: Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes. Kidney Int 62: 901eC913, 2002
Chen S, Kasama Y, Lee JS, Jim B, Marin M, Ziyadeh FN: Podocyte-derived vascular endothelial growth factor mediates the stimulation of 3(IV) collagen production by transforming growth factor-1 in mouse podocytes. Diabetes 53: 2939eC2949, 2004
Rizkalla B, Forbes JM, Cooper ME, Cao Z: Increased renal vascular endothelial growth factor and angiopoietins by angiotensin II infusion is mediated by both AT1 and AT2 receptors. J Am Soc Nephrol 14: 3061eC3071, 2003
Kakizawa H, Itoh Y, Imamura S, Matsumoto T, Ishiwata Y, Ono Y, Yamamoto K, Kato T, Hayakawa N, Oda N, Goto Y, Nagasaka A, Senda T, Itoh M: Possible role of VEGF in the progression of kidney disease in streptozotocin (STZ)-induced diabetic rats: effects of an ACE inhibitor and an angiotensin II receptor antagonist. Horm Metab Res 36: 458eC464, 2004
Freeburg PB, Robert B, St. John PL, Abrahamson DR: Podocyte expression of hypoxia-inducible factor (HIF)-1 and HIF-2 during glomerular development. J Am Soc Nephrol 14: 927eC938, 2003
Wolf G, Schroeder R, Stahl RA: Angiotensin II induces hypoxia-inducible factor-1 alpha in PC 12 cells through a posttranscriptional mechanism: role of AT2 receptors. Am J Nephrol 24: 415eC421, 2004
Cohen MP, Sharma K, Jin Y, Hud E, Wu VY, Tomaszewski J, Ziyadeh FN: Prevention of diabetic nephropathy in db/db mice with glycated albumin antagonists: a novel treatment strategy. J Clin Invest 95: 2338eC2345, 1995
Daniels BS, Brandt C, Aggarwal M: Increased glomerular H202 production in diabetes: role of glycated proteins (Abstract). J Am Soc Nephrol 4: 791A, 1993
Foster RR, Hole R, Anderson K, Satchell SC, Coward RJ, Mathieson PW, Gillatt DA, Saleem MA, Bates DO, Harper SJ: Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes. Am J Physiol Renal Physiol 284: F1263eCF1273, 2003
Sung SH, Chen S, Laping NJ, Ziyadeh FN: Albuminuria is ameliorated by an inhibitor of VEGF receptors, SU5416, in diabetic db/db mice but is unaffected in Smad3-knockout mice rendered diabetic with streptozotocin (Abstract). J Am Soc Nephrol 15: 720AeC721A, 2004
Chen S, Jim B, Ziyadeh FN: Diabetic nephropathy and transforming growth factor-beta: transforming our view of glomerulosclerosis and fibrosis build-up. Semin Nephrol 23: 532eC543, 2003
Ziyadeh FN: Mediators of diabetic renal disease: the case for TGF- as the major mediator. J Am Soc Nephrol 15 (Suppl. 1): S55eCS57, 2004
Wang S-N, Hirschberg R: Growth factor ultrafiltration in experimental diabetic nephropathy contributes to interstitial fibrosis. Am J Physiol Renal Physiol 278: F554eCF560, 2000
Wang S-N, LaPage J, Hirschberg R: Role of glomerular ultrafiltration of growth factors in progressive interstitial fibrosis in diabetic nephropathy. Kidney Int 57: 1002eC1014, 2000
Hong SW, Isono M, Chen S, Iglesias-de la Cruz MC, Han DC, Ziyadeh FN: Increased glomerular and tubular expression of TGF-beta1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 158: 1653eC1663, 2001
Ziyadeh FN, Hoffman BB, Han DC, Iglesias-de la Cruz MC, Hong SW, Isono M, Chen S, McGowan TA, Sharma K: Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor- antibody in db/db diabetic mice. Proc Natl Acad Sci U S A 97: 8015eC8020, 2000
Kopp JB, Factor VM, Mozes M, Nagy P, Sanderson N, Bottinger EP, Klotman PE, Thorgeirsson SS: Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease. Lab Invest 74: 991eC1003, 1996
Sharma R, Khanna A, Sharma M, Savin VJ: Transforming growth factor-beta1 increases albumin permeability of isolated rat glomeruli via hydroxyl radicals. Kidney Int 58: 131eC136, 2000
Benigni A, Zoja C, Corna D, Zatelli C, Conti S, Campana M, Gagliardini E, Rottoli D, Zanchi C, Abbate M, Ledbetter S, Remuzzi G: Add-on anti-TGF-beta antibody to ACE inhibitor arrests progressive diabetic nephropathy in the rat. J Am Soc Nephrol 14: 1816eC1824, 2003
Fujimoto M, Maezawa Y, Yokote K, Joh K, Kobayashi K, Kawamura H, Nishimura M, Roberts AB, Saito Y, Mori S: Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy. Biochem Biophys Res Commun 305: 1002eC1007, 2003
Massaguee J: TGF- signal transduction. Annu Rev Biochem 67: 753eC791, 1998(Gunter Wolf, Sheldon Chen)