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From the Periphery of the Glomerular Capillary Wall Toward the Center of Disease: Podocyte Injury Comes of Age in Diabetic Nephropathy

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From the Periphery of the Glomerular Capillary Wall Toward the Center of Disease: Podocyte Injury Comes of Age in Diabetic Nephropathy
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   Perspectives in Diabetes From the Periphery of the Glomerular Capillary WallToward the Center of Disease Podocyte Injury Comes of Age in Diabetic Nephropathy  Gunter Wolf, 1 Sheldon Chen, 2 and Fuad N. Ziyadeh 2 Nephropathy is a major complication of diabetes. Alter-ations of mesangial cells have traditionally been thefocus of research in deciphering molecular mechanismsof diabetic nephropathy. Injury of podocytes, if recog-nized at all, has been considered a late consequencecaused by increasing proteinuria rather than an eventinciting diabetic nephropathy. However, recent biopsy studies in humans have provided evidence that podo-cytes are functionally and structurally injured very earlyinthenaturalhistoryofdiabeticnephropathy.Thediabetic milieu, represented by hyperglycemia, nonen-zymatically glycated proteins, and mechanical stressassociated with hypertension, causes downregulation of nephrin,animportantproteinoftheslitdiaphragmwithantiapoptotic signaling properties. The loss of nephrinleads to foot process effacement of podocytes and in-creasedproteinuria.Akeymediatorofnephrinsuppres-sionisangiotensinII(ANGII),whichcanactivateothercytokine pathways such as transforming growth fac-tor-   (TGF-  ) and vascular endothelial growth factor(VEGF) systems. TGF-  1 causes an increase in mesan-gial matrix deposition and glomerular basement mem-brane (GBM) thickening and may promote podocyteapoptosis or detachment. As a result, the denuded GBMadheres to Bowman’s capsule, initiating the develop-ment of glomerulosclerosis. VEGF is both produced by and acts upon the podocyte in an autocrine manner tomodulate podocyte function, including the synthesis of GBM components. Through its effects on podocyte biol-ogy, glomerular hemodynamics, and capillary endothe-lial permeability, VEGF likely plays an important role indiabetic albuminuria. The mainstays of therapy, glyce-mic control and inhibition of ANG II, are key measuresto prevent early podocyte injury and the subsequentdevelopment of diabetic nephropathy.  Diabetes  54:1626–1634, 2005 D iabetic nephropathy is the leading cause of end-stage renal disease and is clinically charac-terized by proteinuria and progressive renalinsufficiency. Dating back to the first descrip-tion by Kimmelstiel and Wilson (1), histological analyseshave focused on the increase in mesangial matrix as themain lesion of diabetic glomerulopathy. In addition, glo-merular basement membrane (GBM) thickening has beenconsidered an important pathophysiological event in thedisease. These pathological alterations have been linked tofunctional consequences, first described in landmarkstructure-function studies done in patients with type 1 aswell as type 2 diabetes (2–4). Specifically, mesangialmatrix expansion correlates closely with both proteinuria and deterioration of renal function. It has been proposedthat accumulation of matrix in the mesangial area reducesthe capillary surface area available for filtration, therebycontributing to the progressive loss of renal function (2).Renal failure may also arise from nephron dropout due totubulointerstitial fibrosis (5), and this process is aggra- vated by the harmful downstream effects of proteinuria onthe tubules (6). However, the genesis of proteinuria indiabetes is not readily explained by the associated mesan-gial matrix expansion. Rather, consideration should begiven to alterations of the glomerular filtration barrier,which is composed of the glomerular endothelium, theGBM, and the podocyte (glomerular visceral epithelialcell). Widespread endothelial dysfunction is believed toresult in proteinuria (7), which is exacerbated by intraglo-merular hemodynamic stress (8). Although it is highlyfenestrated, the glomerular endothelium might pose somehindrance to protein permeability (9,10). As for the GBM,its conspicuous thickening in diabetes, perhaps due toaccumulation of collagen IV and alterations in its architec-ture and composition (11), would seem to constitute a more effective barrier to the filtration of proteins but is infact more porous to proteins (12). While loss of chargeselectivity in the GBM has been proposed to partly explainthe proteinuria (13), a decrease in negatively charged From the  1 Department of Internal Medicine (Klinik fu¨r Innere Medizin III),University Hospital, Jena, Germany; and the  2 Department of Medicine, Renal-Electrolyte and Hypertension Division, Penn Center for the Molecular Studiesof Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania. Address correspondence and reprint requests to Fuad N. Ziyadeh, MD,Professor of Medicine, Renal-Electrolyte and Hypertension Division, Univer-sity of Pennsylvania, 700 Clinical Research Building, 415 Curie Blvd., Phila-delphia, PA 19104-4218. E-mail: ziyadeh@mail.med.upenn.edu.Received for publication 7 December 2004 and accepted in revised form19 January 2005. AGE, advanced glycation end product; ANG II, angiotensin II; CD2AP,CD2-associated protein; GBM, glomerular basement membrane; HSPG, hepa-ran sulfate proteoglycans; RAGE, receptor for AGE; ROS, reactive oxygenspecies; STZ, streptozotocin; TGF-  , transforming growth factor-  ; VEGF, vascular endothelial growth factor.© 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 1626 DIABETES, VOL. 54, JUNE 2005   proteoglycans occurs late in the course of diabetic ne- phropathy, sometimes long after the appearance of mi-croalbuminuria (14). This leads to the conclusion that thefinal barrier restricting plasma proteins to the vasculatureis the slit diaphragm of the podocyte; little research,however, has been conducted on podocyte biology indiabetes until recently. The advent of improved cell cul-ture techniques, the discovery of key podocyte-specificmolecules, and advances in transgenic technology haverevolutionized the study of podocytes in health and dis-ease (15). The purpose of this review is to summarize theemerging evidence that podocytopathy plays a pivotal rolein the manifestations of diabetic glomerulopathy, expand-ing upon the “mesangiocentric” dogma of diabetic glomer-ular disease. PODOCYTE BIOLOGY   A comprehensive description of the biology of this highlydifferentiated cell is beyond the scope of this article, but a basic understanding is necessary to appreciate how podo-cyte malfunction contributes to diabetic nephropathy (15).Podocytes extend long processes toward the GBM towhich they affix by cell surface adhesion proteins such asthe   3  1 integrin and dystroglycan (16–18). The foot processes of adjacent podocytes interdigitate and areseparated by narrow spaces (30–40 nm) that are bridgedby a porous membrane called the slit diaphragm. Thesemembranes contain pores that are freely permeable towater 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 bar-rier, and knowledge of its molecular architecture will helpelucidate the role that the slit diaphragm plays in protein-uria (15). A major advance was the discovery of nephrin,whose gene is mutated in the congenital nephrotic syn-drome of the Finnish type, a rare form of hereditarynephrosis characterized by diffuse foot process effacementof the podocytes (21). Nephrin, a transmembrane proteinwith 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 pro-tein (CD2AP) interact with nephrin in cholesterol-richregions of the cytoplasm called lipid rafts and anchor nephrin to actin filaments of the podocyte cytoskeleton(15,23). CD2AP links nephrin and podocin to phosphoino-sitide 3-OH kinase, and this complex has cell-signaling properties that stimulate Akt, a serine-threonine kinase(24). One target of nephrin/CD2AP-induced phosphoryla-tion is Bad, a proapoptotic protein of the Bcl-2 family (24).Upon phosphorylation, Bad is inactivated and apoptosisdoes not occur. Thus, afferent signaling coming from intactslit diaphragms prevents podocyte apoptosis (24,25), a beneficial effect given that terminally differentiated podo-cytes fail to proliferate and are not easily replaced (26).The inability of podocytes to proliferate may be secondaryto upregulation of cell cycle inhibitory proteins, p57 and p27 (22). Expression of p27 in cultured podocytes can bestimulated by high glucose, and this further prevents cellreplication (22).Because it does not normally divide, the podocyterepresents one of the last unexplored frontiers in renal cellculture research. Podocytes in primary culture can becoaxed into reentering the cell cycle, but they invariablylose their differentiated characteristics. Besides, the cellhomogeneity in primary culture is questionable becauseglomerular parietal epithelial cells often contaminate andovergrow the podocytes (27). To circumvent these diffi-culties, “conditional immortalization” has been developedto obtain a pure population of podocytes that can either  proliferate or differentiate (28). The technique involves theintroduction of an oncogene whose gene product is tem- perature sensitive, such as the tsA58 variant of the SV40large tumor antigen (TAg) (29). The TAg protein com- plexes with and disables p53 and retinoblastoma proteins,removing two major barriers to cell cycle progression andallowing the podocyte to be propagated in culture. Whendifferentiated podocytes are needed for experimentation,the cells can be “thermoshifted” to a higher temperaturethat 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 describedfoot process widening of podocytes (30), a finding later confirmed in patients with relatively advanced nephropa-thy due to type 1 diabetes (31). More recent investigationsalso described an increase in foot process width in mi-croalbuminuric type 1 diabetic subjects, and foot processwidth correlated directly with the urinary albumin excre-tion rate (32). In addition to foot process widening, thenumber and density of podocytes have been reported to bemarkedly reduced (podocytopenia) in diabetic patients,whether afflicted with type 1 or type 2 diabetes (33–36).While an ideal protocol for counting podocytes in biopsyspecimens has not been developed yet, it has been arguedthat modern morphometric techniques can yield reason-able approximations. For instance, it has been estimatedthat the podocyte number is significantly reduced, even indiabetes of short duration (33). In Pima Indians with type2 diabetes, podocyte density is drastically reduced, and theremaining foot processes are widened to maintain GBMcoverage (34,37). Among all glomerular morphologicalcharacteristics, the decreased number of podocytes per glomerulus was the strongest predictor of progressive re-nal disease, with fewer cells predicting more rapid pro-gression (38). Additionally, a recent morphometric study(36) in European type 2 diabetic patients has shown that a diminution of podocyte density rather than absolute re-duction in podocyte number is more predictive of the presence and magnitude of albuminuria. The numericaldensity of podocytes per glomerulus was reduced in patients with microalbuminuria and was decreased evenfurther in patients with overt proteinuria (36). A cross-sectional study also reported a significant inverse correla-tion between proteinuria and both podocyte number anddensity per glomerulus (39). Consistent with the observedloss of podocytes in diabetes, podocytes were present inthe urine in 53% of type 2 diabetic patients with microalbu-minuria and in 80% with macroalbuminuria, while nor-moalbuminuric patients and healthy control subjects hadundetectable levels of urinary podocytes (40). Interest- G. WOLF, S. CHEN, AND F.N. ZIYAHDEH DIABETES, VOL. 54, JUNE 2005 1627  ingly, treatment with an ACE inhibitor reduced the num-ber of urinary podocytes (40).Podocytopenia may exacerbate the development of pro-teinuria because a denuded GBM can come into contactwith Bowman’s capsule and promote synechiae formation,an initial step in the development of glomerulosclerosis(15). Morphologic changes in the podocyte are also be-lieved to engender proteinuria. With foot process widen-ing, the decrease in slit diaphragm length might impedethe filtration of water and lower the glomerular filtrationrate. 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 thetubule’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 func-tion, a characteristic of diabetic nephropathy, but thetheory remains unproven and controversial (41). Even so,the worsening proteinuria could induce tubular atrophyand interstitial fibrosis, and these irreversible tubulointer-stitial changes, together with glomerulosclerosis, then leadto 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 in-stance 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 withmacroalbuminuria, whereas none of the nondiabetic sub- jects had nephrinuria (42). These findings were not corre-lated with biopsies, but they suggest that increased urinarynephrin equates with early podocyte injury, even beforethe onset of microalbuminuria. Another study (43) foundthat nephrin staining was extensively reduced in renalbiopsy specimens from nephropathic patients with type 1diabetes. Several studies have evaluated nephrin mRNA and protein expression in type 2 diabetes. In general,nephrin protein production was downregulated in thediabetic subjects compared with the nondiabetic controlsubjects (44–46), and the decrease in nephrin correlatedwith the broadening of the foot process widths. In con-trast, CD2AP expression was not reduced in podocytesfrom diabetic patients, suggesting that the reduction innephrin was not due to widespread podocyte loss or injury(45); this underscores the importance of nephrin in main-taining podocyte integrity. Finally, type 2 diabetic patientswho were treated with the ACE inhibitor perindopril for 2 years exhibited a nephrin mRNA expression that was closeto normal when compared with nondiabetic control sub- jects; patients not treated with the ACE inhibitor hadsignificantly reduced nephrin transcripts (46). PODOCYTE DYSFUNCTION IN EXPERIMENTALDIABETES  Animal experiments allow for the repeated sampling of kidney tissue over time, furnishing a longitudinal record of the development of diabetic nephropathy and the efficacyof pharmacological interventions. Serial investigations(47) on kidneys from obese Zucker   fa/fa  rats, a model of type 2 diabetes that develops segmental glomerulosclero-sis, revealed that nephropathy started with damage to podocytes, manifesting as foot process effacement andcytoplasmic accumulation of lipid droplets. Early podo-cyte damage antedated the development of glomeruloscle-rosis and tubulointerstitial damage in this model (48). Inthe 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 INHUMANS AND ANIMAL MODELS The exact etiology for podocyte loss in diabetes remainsspeculative, but two mechanisms can be suggested: apo- ptosis and cell detachment. Evidence for a primary role of apoptosis in either human or experimental diabetic ne- phropathy is scant (51–54). However, podocyte apoptosiscan be demonstrated in cell culture. For instance, angio-tensin II (ANG II) induces apoptosis in cultured rat glo-merular epithelial cells, and this effect is mediated bythe transforming growth factor-   (TGF-  ) system sinceit is inhibited by an anti–TGF-   antibody (55). In TGF-  1–overexpressing transgenic mice, albeit nondiabetic,the podocyte undergoes apoptosis in situ shortly after thesclerotic lesion appears in the glomerulus (56). Thus, theheightened intraglomerular activity of TGF-  1 that ischaracteristic of diabetes may theoretically be responsiblefor apoptosis (57), a process that can be mediated throughSmad7, which inhibits the nuclear translocation of the cellsurvival factor nuclear factor-  B (56,58).The other mechanism of podocyte loss in diabetes mayrelate to the detachment of podocytes from the GBM. Thisscenario does not exclude a role for apoptosis, since cellsmay first detach and then undergo apoptosis because of interruption of viability cues deriving from cell-matrixinteractions (17). Alternatively, detached cells can be shedin the urine as live podocytes (59). In fact, podocyturia worsens with the progression from normoalbuminuria tomicroalbuminuria to overt proteinuria (40). Loss of cellanchorage to the GBM may result from downregulation of the   3  1 integrin receptor, the principal adhesion com- plex that attaches the podocyte to the GBM (16,17,60,61).Several studies (62–64) have shown that the   3  1 integrinis decreased in the podocytes of humans and rats withdiabetes. Furthermore, high glucose media in cultured rator human podocytes decreases the expression of    3  1integrin (62,64); this downregulation is perhaps mediatedby increased levels of TGF-  1 (65,66). ROLE OF ANG II IN NEPHRIN EXPRESSION ANDPODOCYTE INJURY  Pharmacological interventions in animal models have sup- ported the view that an increased ANG II activity isinvolved in podocyte injury in diabetes. ACE inhibition or  AT 1  receptor antagonism attenuated podocyte foot pro-cess broadening in rats with STZ-induced diabetes (49). Inanother study (67) involving the STZ-induced diabetic rat,therapy with an ACE inhibitor, but not an endothelinreceptor antagonist, prevented loss of podocytes and podo-cyte injury. Similarly, an ACE inhibitor, but not aminogua-nidine, an inhibitor of advanced glycation end product(AGE) formation, attenuated the diabetes-associated re- PODOCYTOPATHY IN DIABETIC NEPHROPATHY  1628 DIABETES, VOL. 54, JUNE 2005  duction in nephrin expression (50). Likewise, an AT 1 receptor antagonist, but not the calcium channel blocker amlodipine, normalized the reduced nephrin expression in podocytes from spontaneously hypertensive rats with su- perimposed STZ-induced diabetes (68,69). Finally, therenal damage that occurs in obese Zucker rats was moreeffectively controlled by combined treatment with an ACEinhibitor and an AT 1  receptor antagonist than either mono-therapy alone (70). Thus, it may be concluded that sup- pression of nephrin expression in podocytes can becaused by increased renal ANG II activity in diabetes, butthe molecular mechanisms are incompletely understood.Podocytes express the AT 1  and probably the AT 2  recep-tors after injury and therefore could respond to stimula-tion with ANG II (15,71). Transgenic rats with targetedoverexpression of the podocyte’s AT 1  receptor showed pseudocysts in podocytes, followed by foot process ef-facement and local detachment, with subsequent progres-sion to focal segmental glomerulosclerosis (72). Highglucose concentrations induce ANG II formation in podo-cytes through upregulation of angiotensinogen expression(15). Furthermore, ANG II production in podocytes can beactivated by proteinuria, likely by the transit of proteinsthrough the filtration barrier (73) and by mechanicalstretch, which mimics the hemodynamic effects of intra-glomerular hypertension (74). Interestingly, ANG II forma-tion as a consequence of mechanical stretch appears to beindependent of ACE (74). As an alternative to ACE for theconversion of ANG I to ANG II, chymase has been shownto be upregulated in the glomeruli of patients with ne- phropathy due to type 2 diabetes (75). Because chymase isnot blocked by ACE inhibitors, glomeruli in the diabeticstate may still generate ANG II despite ACE inhibition.Some evidence indicates that the increase in angiotensino-gen expression is signaled by intracellular reactive oxygenspecies (ROS) (74). Oxidative stress is a leading initiator of cellular dysfunction in diabetes complications (76,77), andincreased ROS generation can induce podocyte dysfunc-tion (78,79). ROLE OF HEPARAN SULFATE PROTEOGLYCANS  A decrease in the GBM content of negatively chargedHSPG contributes to the loss of charge selectivity in theglomerular filtration barrier and to the progression of pro-teinuria (80,81). One potential mechanism for decreasedde novo synthesis of proteoglycans is the increased gen-eration of ROS (81,82). Although the predominant proteo-glycan in the GBM was thought to be perlecan, it hasbecome more clear that agrin is the more abundant HSPG(83). Synthesis of proteoglycans occurs in all three glo-merular cell types, but podocytes are an especially im- portant source of these negatively charged molecules.Proteoglycan synthesis in podocytes is differentially influ-enced by high ambient glucose and ANG II (83,84); highglucose suppresses the production of agrin’s core protein,whereas ANG II decreases synthesis of the core proteinand 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 antiproteinuriceffect of ACE inhibitors and angiotensin receptor blockersin diabetic nephropathy. ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR Losses of nephrin and proteoglycan are not the only pathological changes fostering proteinuria in diabetic ne- phropathy. Vascular endothelial growth factor (VEGF), a survival and angiogenic factor with strong microvascular  permeabilizing properties (85), may increase the perme-ability of the glomerular filtration barrier to circulating proteins. The most convincing evidence that VEGF over-expression is involved in the proteinuria of diabetes comesfrom studies in type 1 diabetic (STZ-induced) rats and type2 diabetic  db/db  mice. Neutralization of VEGF with a systemically administered anti-VEGF antibody reducedthe urinary albumin excretion by at least 50% comparedwith the untreated diabetic controls (86,87). In addition, VEGF induces endothelial nitric oxide synthase, thereby promoting the vasodilation and hyperfiltration that aretypical of early diabetic nephropathy (86,88).The expression of VEGF in the glomerulus is most pronounced in the podocytes, and experimental diabetesincreases VEGF mRNA and protein expression (89,90). Incultured podocytes, VEGF expression can be stimulatedby high glucose concentrations (91), by TGF-  1 (91,92),and by ANG II acting via AT 1  and AT 2  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-induc-ible factor-1   expression is stimulated by ANG II in a  posttranscriptional mechanism involving AT 2  receptors(96). This increase is caused by downregulation of a prolylhydroxylase (SM-20/PHD3), the enzyme responsible for initiating hypoxia-inducible factor-1   degradation (96).Upregulation of podocyte-derived VEGF in diabetes maydepend 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 sig-nificantly depressed VEGF expression in the kidney andameliorated albuminuria and glomerulosclerosis (90). Pre-cursors of AGE such as Amadori-glycated serum albuminmay mediate diabetic proteinuria (97) and promote glo-merular production of hydrogen peroxide (98). Whether increased ROS production stimulates podocyte VEGF ex- pression remains to be investigated. An intriguing concept that has come to attention is that podocytes not only produce VEGF but are also acted uponby VEGF (92,99). This VEGF autocrine loop may playimportant roles in podocyte biology since VEGF decreasesintracellular calcium concentrations and protects againstcytotoxicity (99), possibly via the antiapoptotic actions of nephrin (25). These cytoprotective effects were reversedwhen endogenous VEGF was blocked by either a classIII tyrosine kinase inhibitor PTK787/ZK222584 (99) or a monoclonal anti-VEGF antibody (25). VEGF also stimulates the podocyte to produce the   3chain 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 wasstimulated by TGF-  1 treatment, the production of    3(IV)collagen increased (92). Blockade of endogenous VEGFaction by a specific inhibitor of VEGF receptor kinases,SU5416, reduced the TGF-  1–induced expression of   3(IV) collagen by  50%, establishing a pivotal role for the G. WOLF, S. CHEN, AND F.N. ZIYAHDEH DIABETES, VOL. 54, JUNE 2005 1629   VEGF autocrine system in at least one aspect of theregulation of GBM composition by podocytes (92).It may be speculated that VEGF-stimulated podocyte production of    3(IV) collagen contributes to diabeticthickening of the GBM and its altered permselectivity.Extrapolating from our in vitro work in cultured podo-cytes, we treated diabetic  db/db  mice with intraperitonealinjections of SU5416 at 2 mg/kg body wt, given twice a week for 8 weeks (100). SU5416 treatment, which had noadverse effects, significantly prevented GBM thickeningand albuminuria without affecting blood glucose levels(100 and S.H. Sung, S.C., F.N.Z., unpublished data). Thesefindings with SU5416 compare favorably with those of theanti-VEGF antibody in  db/db  mice (87); however, thatstudy did not report the effect of anti-VEGF therapy onGBM thickening. That SU5416 particularly benefited the podocyte was also suggested by the preservation of neph-rin protein, assayed by immunostaining (S.H. Sung, S.C.,F.N.Z., unpublished data). Glomerular nephrin levels, whichare typically reduced in the  db/db  mouse, were restoredalmost to normal by SU5416 treatment (S.H. Sung, S.C.,F.N.Z., unpublished data), suggesting that nephrin preser- vation represents another mechanism whereby VEGFblockade might ameliorate diabetic albuminuria. ROLE OF TGF-  1 TGF-   plays an integral role in the pathogenesis of dia-betic nephropathy (101). This fibrogenic cytokine is stim-ulated by the diabetic state, is increased in the kidneyduring diabetes, and is able to recapitulate the hypertro- phic and sclerotic features of diabetic renal disease (102).Elevated levels of TGF-   have been measured in theglomeruli of STZ-induced diabetic rats by micropuncturetechniques (103,104), and an intracellular transducer of TGF-   signaling, Smad3, has been shown to translocateinto the glomerular nuclei of diabetic  db/db  mice (105),attesting to the overactivity of the TGF-   system in dia-betic nephropathy. Inhibition of TGF-   by a panselectiveneutralizing antibody in diabetic  db/db  mice preventeddiabetic renal hypertrophy, mesangial matrix expansion,and the development of renal insufficiency (106). How-ever, there was no significant effect on albuminuria in thediabetic mice (106). A clear role for TGF-   in the pathogenesis of diabeticalbuminuria cannot be established at present because of conflicting studies. In nondiabetic models, overexpressionof active TGF-  1 in transgenic mice caused mesangialexpansion, interstitial fibrosis, renal insufficiency, and progressive proteinuria (107). In addition, an increase inalbumin permeability was observed when isolated glomer-uli 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 albumin-uria after 8 weeks of treatment with a neutralizing anti–TGF-   antibody, perhaps because VEGF expression in thekidney cortex was kept elevated by other diabetic factors(106). Interestingly, the work of Benigni et al. (109) inuninephrectomized STZ-induced diabetic rats revealed FIG. 1. Overview of the mechanisms of podocyte injury leading to diabetic nephropathy. Metabolic factors in the diabetic milieu (TGF-  , highglucose, glycated proteins, ROS, and ANG II) and hemodynamic factors (via mechanical stretch) converge on the podocyte to increase VEGF and ANG II production. Other effects include upregulation of the TGF-   type II receptor and downregulation of the cell surface   3  1 integrins.Podocyte-derived VEGF operating in an autocrine loop, perhaps via VEGFR-1 signaling, stimulates the production of    3(IV) collagen, leading toGBM thickening, and suppresses the expression of nephrin, favoring apoptosis and foot process widening/effacement. Paracrine actions of VEGFontheendotheliummayincreaseglomerularcapillarypermeabilityandrelaxafferentarteriolartone(throughendothelialnitricoxidesynthase),generating hemodynamic forces that can injure podocytes. ANG II also suppresses nephrin expression, and it decreases production of thenegatively charged HSPG. ANG II and high glucose upregulate the TGF-  type II receptor and may augment the podocyte’s response to paracrineTGF-  , such as that coming from the mesangial cell. The TGF-   /type II receptor interaction stimulates extracellular matrix production by thepodocyte (contributing to GBM thickening) and by the mesangium (leading to mesangial matrix expansion). The TGF-   system in the podocytealso promotes apoptosis and decreases integrin expression, which can lead to podocyte detachment and podocyturia. As the results of the aboveprocesses, the podocytopenia, foot process widening with loss of nephrin, GBM dysfunction, decreased HSPG, and hemodynamic stress allprovoke or exacerbate diabetic proteinuria. Worsening proteinuria coupled with profibrotic stimuli (exemplified by the TGF-   system) induceglomerulosclerosis and tubulointerstitial fibrosis, leading relentlessly to progressive renal insufficiency. PODOCYTOPATHY IN DIABETIC NEPHROPATHY  1630 DIABETES, VOL. 54, JUNE 2005
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