PKCα Mediates β-Arrestin2-dependent Nephrin Endocytosis in Hyperglycemia
2011; Elsevier BV; Volume: 286; Issue: 15 Linguagem: Inglês
10.1074/jbc.m110.204024
ISSN1083-351X
AutoresIvo Quack, Magdalena Woznowski, Sebastian A. Potthoff, Romy Palmer, Eva Königshausen, Sema Sivritas, Mario Schiffer, Johannes Stegbauer, Oliver Vonend, Lars Christian Rump, Lorenz Sellin,
Tópico(s)Pancreatic function and diabetes
ResumoNephrin, the key molecule of the glomerular slit diaphragm, is expressed on the surface of podocytes and is critical in preventing albuminuria. In diabetes, hyperglycemia leads to the loss of surface expression of nephrin and causes albuminuria. Here, we report a mechanism that can explain this phenomenon: hyperglycemia directly enhances the rate of nephrin endocytosis via regulation of the β-arrestin2-nephrin interaction by PKCα. We identified PKCα and protein interacting with c kinase-1 (PICK1) as nephrin-binding proteins. Hyperglycemia induced up-regulation of PKCα and led to the formation of a complex of nephrin, PKCα, PICK1, and β-arrestin2 in vitro and in vivo. Binding of β-arrestin2 to the nephrin intracellular domain depended on phosphorylation of nephrin threonine residues 1120 and 1125 by PKCα. Further, cellular knockdown of PKCα and/or PICK1 attenuated the nephrin–β-arrestin2 interaction and abrogated the amplifying effect of high blood glucose on nephrin endocytosis. In C57BL/6 mice, hyperglycemia over 24 h caused a significant increase in urinary albumin excretion, supporting the concept of the rapid impact of hyperglycemia on glomerular permselectivity. In summary, we have provided a molecular model of hyperglycemia-induced nephrin endocytosis and subsequent proteinuria and highlighted PKCα and PICK1 as promising therapeutic targets for diabetic nephropathy. Nephrin, the key molecule of the glomerular slit diaphragm, is expressed on the surface of podocytes and is critical in preventing albuminuria. In diabetes, hyperglycemia leads to the loss of surface expression of nephrin and causes albuminuria. Here, we report a mechanism that can explain this phenomenon: hyperglycemia directly enhances the rate of nephrin endocytosis via regulation of the β-arrestin2-nephrin interaction by PKCα. We identified PKCα and protein interacting with c kinase-1 (PICK1) as nephrin-binding proteins. Hyperglycemia induced up-regulation of PKCα and led to the formation of a complex of nephrin, PKCα, PICK1, and β-arrestin2 in vitro and in vivo. Binding of β-arrestin2 to the nephrin intracellular domain depended on phosphorylation of nephrin threonine residues 1120 and 1125 by PKCα. Further, cellular knockdown of PKCα and/or PICK1 attenuated the nephrin–β-arrestin2 interaction and abrogated the amplifying effect of high blood glucose on nephrin endocytosis. In C57BL/6 mice, hyperglycemia over 24 h caused a significant increase in urinary albumin excretion, supporting the concept of the rapid impact of hyperglycemia on glomerular permselectivity. In summary, we have provided a molecular model of hyperglycemia-induced nephrin endocytosis and subsequent proteinuria and highlighted PKCα and PICK1 as promising therapeutic targets for diabetic nephropathy. IntroductionDiabetes mellitus is a major health problem in Western countries. Despite therapeutic advances, diabetic nephropathy remains the leading cause of end stage renal disease (1Rossing P. Curr. Diab. Rep. 2006; 6: 479-483Crossref PubMed Scopus (128) Google Scholar). Chronic hyperglycemia causes glomerular damages such as endothelial dysfunction, loss of negative charges in the basement membrane, and podocyte damage (2Ziyadeh F.N. Wolf G. Curr. Diabetes Rev. 2008; 4: 39-45Crossref PubMed Scopus (312) Google Scholar). Microalbuminuria is an early symptom of a "leaky" glomerular barrier and is associated with a markedly increased cardiovascular risk (3Kalaitzidis R. Bakris G. J. Clin. Hypertens. 2009; 11: 636-643Crossref Scopus (22) Google Scholar).Podocytes are visceral epithelial cells wrapped around glomerular capillaries and are connected by the slit diaphragm. This specialized cell junction is a dynamic multiprotein complex that functions as a size-selective sieve to prevent the loss of plasma proteins through urine (4Huber T.B. Benzing T. Curr. Opin. Nephrol. Hypertens. 2005; 14: 211-216Crossref PubMed Scopus (195) Google Scholar, 5Tryggvason K. Patrakka J. Wartiovaara J. N. Engl. J. Med. 2006; 354: 1387-1401Crossref PubMed Scopus (440) Google Scholar). Nephrin serves as the backbone of this diaphragm by functioning as a regulator of podocyte signaling and mediator of actin dynamics (6Jones N. Blasutig I.M. Eremina V. Ruston J.M. Bladt F. Li H. Huang H. Larose L. Li S.S. Takano T. Quaggin S.E. Pawson T. Nature. 2006; 440: 818-823Crossref PubMed Scopus (385) Google Scholar, 7Welsh G.I. Saleem M.A. Pathology. 2010; 220: 328-337Crossref Scopus (98) Google Scholar, 8Gerke P. Huber T.B. Sellin L. Benzing T. Walz G. J. Am. Soc. Nephrol. 2003; 14: 918-926Crossref PubMed Scopus (145) Google Scholar). Several podocyte signaling abnormalities have been noted in diabetes. For example, mice and humans with diabetes show altered distribution and expression of nephrin (9Kim J.J. Li J.J. Jung D.S. Kwak S.J. Ryu D.R. Yoo T.H. Han S.H. Choi H.Y. Kim H.J. Han D.S. Kang S.W. J. Am. Soc. Nephrol. 2007; 18: 2303-2310Crossref PubMed Scopus (38) Google Scholar, 10Koop K. Eikmans M. Baelde H.J. Kawachi H. De Heer E. Paul L.C. Bruijn J.A. J. Am. Soc. Nephrol. 2003; 14: 2063-2071Crossref PubMed Scopus (265) Google Scholar, 11Doublier S. Salvidio G. Lupia E. Ruotsalainen V. Verzola D. Deferrari G. Camussi G. Diabetes. 2003; 52: 1023-1030Crossref PubMed Scopus (314) Google Scholar), and hyperglycemia increases diacylglycerol generation, causing PKC activation (2Ziyadeh F.N. Wolf G. Curr. Diabetes Rev. 2008; 4: 39-45Crossref PubMed Scopus (312) Google Scholar). However, it is not fully understood how "hyperglycemic" signaling in diabetes affects the expression and distribution of nephrin.Menne et al. (12Menne J. Meier M. Park J.K. Boehne M. Kirsch T. Lindschau C. Ociepka R. Leitges M. Rinta-Valkama J. Holthofer H. Haller H. Kidney Int. 2006; 70: 1456-1462Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) reported a major step elucidating podocyte signaling in diabetes: they showed that albuminuria does not develop in diabetic PKCα-deficient mice and concluded that decreased nephrin expression in diabetes is a result of altered transcriptional regulation. All of the studies until now were conducted in chronic models of diabetes (13Meier M. Menne J. Haller H. Diabetologia. 2009; 52: 765-775Crossref PubMed Scopus (38) Google Scholar). Therefore, their conclusions were based on the changes occurring in long term hyperglycemia. However, considering the highly dynamic structure of the slit diaphragm, we hypothesized that elevated blood glucose levels have an immediate impact on the molecular composition of the slit diaphragm. Our hypothesis is supported by the recent study of Axelsson et al. (14Axelsson J. Rippe A. Rippe B. Am. J. Physiol. Renal Physiol. 2010; 298: F1306-F1312Crossref PubMed Scopus (36) Google Scholar), who demonstrated that acute hyperglycemia impairs glomerular permselectivity in previously normoglycemic rats. Therefore, nephrin might be involved in the changes in glomerular permeability induced by high blood glucose levels.We have recently demonstrated that nephrin endocytosis occurs in a β-arrestin2-dependent manner (15Quack I. Rump L.C. Gerke P. Walther I. Vinke T. Vonend O. Grunwald T. Sellin L. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 14110-14115Crossref PubMed Scopus (86) Google Scholar). Regulation of highly dynamic protein complexes such as the slit diaphragm typically involves changes in their phosphorylation pattern (16Garg P. Verma R. Nihalani D. Johnstone D.B. Holzman L.B. Mol. Cell Biol. 2007; 27: 8698-8712Crossref PubMed Scopus (118) Google Scholar). However, the kinase mediating the binding of β-arrestin2 to nephrin has not yet been identified. Studies have evidenced that β-arrestin2 binding is regulated by serine/threonine phosphorylation of its target proteins (17Xiang B. Yu G.H. Guo J. Chen L. Hu W. Pei G. Ma L. J. Biol. Chem. 2001; 276: 4709-4716Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Lorenz S. Frenzel R. Paschke R. Breitwieser G.E. Miedlich S.U. Endocrinology. 2007; 148: 2398-2404Crossref PubMed Scopus (55) Google Scholar). In our efforts to identify candidate kinases, sequence analysis of the nephrin C terminus revealed a PKC consensus site within the β-arrestin2 interaction motif. The available evidence on PKC signaling in diabetes and the presence of the PKC consensus site in close proximity to the β-arrestin2 interaction motif support our hypothesis that hyperglycemia influences nephrin endocytosis via PKC signaling. In the present study, we examined the role of PKC in β-arrestin2-mediated nephrin endocytosis and its effect on the slit diaphragm integrity in vitro and in vivo to elucidate the mechanism underlying the loss of surface nephrin and subsequent albuminuria upon hyperglycemia in diabetes mellitus.DISCUSSIONThis study shows that a short duration of hyperglycemia is sufficient to induce proteinuria, a finding supporting our hypothesis that a high blood glucose level induces leakiness of the glomerular filter by increased nephrin endocytosis. This result is also in line with earlier findings that diabetic podocytes show a change in their nephrin staining pattern from surface expression to cytoplasmic distribution, suggesting increased internalization of nephrin (29Aaltonen P. Luimula P. Aström E. Palmen T. Grönholm T. Palojoki E. Jaakkola I. Ahola H. Tikkanen I. Holthöfer H. Lab. Invest. 2001; 81: 1185-1190Crossref PubMed Scopus (128) Google Scholar). As we have previously demonstrated, nephrin endocytosis is mediated by the interaction of β-arrestin2 and nephrin (15Quack I. Rump L.C. Gerke P. Walther I. Vinke T. Vonend O. Grunwald T. Sellin L. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 14110-14115Crossref PubMed Scopus (86) Google Scholar). By our present study, we have shown for the first time that a high glucose milieu rapidly enhances the interaction of β-arrestin2 and nephrin in a dose-dependent manner.The interaction of β-arrestin2 is usually regulated by the phosphorylation of serine or threonine residues of its targets (30DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1143) Google Scholar). A sequence scan of the nephrin intracellular domain has revealed a PKC consensus site within the predicted β-arrestin2 binding domain (31Pearson R.B. Kemp B.E. Methods Enzymol. 1991; 200: 62-81Crossref PubMed Scopus (866) Google Scholar). However, the PKC family comprises 12 isozymes, and at least three contribute to diabetic kidney damage (13Meier M. Menne J. Haller H. Diabetologia. 2009; 52: 765-775Crossref PubMed Scopus (38) Google Scholar). Several lines of evidence support PKCα as the candidate kinase involved in nephrin endocytosis in hyperglycemia. First, PKCα mediates β-arrestin2 binding to other single transmembrane receptors such as the TGFβ receptor (23Tossidou I. Starker G. Kruger J. Meier M. Leitges M. Haller H. Schiffer M. Cell Physiol. Biochem. 2009; 24: 627-634Crossref PubMed Scopus (39) Google Scholar). Second, PKCα deficiency prevents albuminuria in diabetic mice (12Menne J. Meier M. Park J.K. Boehne M. Kirsch T. Lindschau C. Ociepka R. Leitges M. Rinta-Valkama J. Holthofer H. Haller H. Kidney Int. 2006; 70: 1456-1462Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Third, hyperglycemia increases the formation of diacylglycerol, which activates PKC (32Noh H. King G.L. Kidney Int. Suppl. 2007; 106: S49-S53Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Indeed, suppression of PKCα activity or knockdown with siRNA abrogated the effect of a high glucose concentration, because no further increase in the β-arrestin2-nephrin interaction could be measured in our experimental setup. In line with these results, no β-arrestin2 binding was observed in PKCα-deficient podocytes.Sequence analysis of the nephrin intracellular domain predicted a PKC recognition motif ((S/T)XX(K/R)) and a putative β-arrestin2 binding motif ((S/T)X4–5(S/T); nephrin amino acids 1120–1125; TGERDT) in close proximity. This indicated that PKCα regulates the binding of β-arrestin2 by the phosphorylation of threonine residues within the β-arrestin2 binding motif in the nephrin intracellular domain. Indeed, the in vitro and in vivo experiments showed that PKCα was recruited to the nephrin intracellular domain and phosphorylated threonine residues 1120 and 1125. Simultaneous mutation of threonine residues 1120 and 1125 abrogated the binding of β-arrestin2 to nephrin completely, confirming the relevance of the nephrin TGERDT motif as the binding motif of β-arrestin2. In other transmembrane receptor systems phosphorylated by PKCα, PICK1 serves as an adaptor molecule for PKCα (26Hanley J.G. Pharmacol. Ther. 2008; 118: 152-160Crossref PubMed Scopus (100) Google Scholar). Indeed, we observed recruitment of PICK1 to the complex of nephrin, PKCα, and β-arrestin2. Because our results suggested that high glucose concentrations markedly enhance the interaction of nephrin and β-arrestin2, we examined whether stronger binding of β-arrestin2 leads to increased endocytosis of nephrin. An increase in the glucose concentration enhanced nephrin endocytosis significantly. Depletion of PICK1 or PKCα attenuated the endocytosis and prevented the loss of nephrin surface expression nearly completely. In accordance with our results, the role of PKCα in diabetic nephrin endocytosis was confirmed very recently by Tossidou et al. (33Tossidou I. Teng B. Menne J. Shushakova N. Park J.K. Becker J.U. Modde F. Leitges M. Haller H. Schiffer M. PLoS One. 2010; 5: e10185Crossref PubMed Scopus (63) Google Scholar). It has been recently reported that nephrin traffics via raft- or clathrin-mediated endocytosis (34Qin X.S. Tsukaguchi H. Shono A. Yamamoto A. Kurihara H. Doi T. J. Am. Soc. Nephrol. 2009; 20: 2534-2545Crossref PubMed Scopus (69) Google Scholar). From other receptors, it is known that β-arrestin2-dependent endocytosis mainly utilizes the clathrin pathway. As presumed, we noted markedly decreased endocytosis of nephrin after inhibition of the clathrin-dependent pathway.According to our results, acute hyperglycemia causes albuminuria. De facto, we demonstrated that mice with an episode of severe hyperglycemia over 24 h develop a significant increase in urinary albumin excretion. Our observations are backed by the very recent finding that hyperglycemia induced by glucose infusion increases glomerular permeability in nondiabetic rats; the change in glomerular size selectivity was not related to hyperosmolarity and was reversed after the glucose infusion was ceased (14Axelsson J. Rippe A. Rippe B. Am. J. Physiol. Renal Physiol. 2010; 298: F1306-F1312Crossref PubMed Scopus (36) Google Scholar).In summary, the present study shows that acute hyperglycemia in diabetes increases nephrin endocytosis in a PKCα- and PICK-dependent manner and thus causes albuminuria (Fig. 8). Conceivably, acute events such as increased nephrin endocytosis serve as an ignition spark for chronically altered slit diaphragm signaling in long standing hyperglycemia. We are convinced that PKCα and PICK1 are promising therapeutic targets for diabetic nephropathy. IntroductionDiabetes mellitus is a major health problem in Western countries. Despite therapeutic advances, diabetic nephropathy remains the leading cause of end stage renal disease (1Rossing P. Curr. Diab. Rep. 2006; 6: 479-483Crossref PubMed Scopus (128) Google Scholar). Chronic hyperglycemia causes glomerular damages such as endothelial dysfunction, loss of negative charges in the basement membrane, and podocyte damage (2Ziyadeh F.N. Wolf G. Curr. Diabetes Rev. 2008; 4: 39-45Crossref PubMed Scopus (312) Google Scholar). Microalbuminuria is an early symptom of a "leaky" glomerular barrier and is associated with a markedly increased cardiovascular risk (3Kalaitzidis R. Bakris G. J. Clin. Hypertens. 2009; 11: 636-643Crossref Scopus (22) Google Scholar).Podocytes are visceral epithelial cells wrapped around glomerular capillaries and are connected by the slit diaphragm. This specialized cell junction is a dynamic multiprotein complex that functions as a size-selective sieve to prevent the loss of plasma proteins through urine (4Huber T.B. Benzing T. Curr. Opin. Nephrol. Hypertens. 2005; 14: 211-216Crossref PubMed Scopus (195) Google Scholar, 5Tryggvason K. Patrakka J. Wartiovaara J. N. Engl. J. Med. 2006; 354: 1387-1401Crossref PubMed Scopus (440) Google Scholar). Nephrin serves as the backbone of this diaphragm by functioning as a regulator of podocyte signaling and mediator of actin dynamics (6Jones N. Blasutig I.M. Eremina V. Ruston J.M. Bladt F. Li H. Huang H. Larose L. Li S.S. Takano T. Quaggin S.E. Pawson T. Nature. 2006; 440: 818-823Crossref PubMed Scopus (385) Google Scholar, 7Welsh G.I. Saleem M.A. Pathology. 2010; 220: 328-337Crossref Scopus (98) Google Scholar, 8Gerke P. Huber T.B. Sellin L. Benzing T. Walz G. J. Am. Soc. Nephrol. 2003; 14: 918-926Crossref PubMed Scopus (145) Google Scholar). Several podocyte signaling abnormalities have been noted in diabetes. For example, mice and humans with diabetes show altered distribution and expression of nephrin (9Kim J.J. Li J.J. Jung D.S. Kwak S.J. Ryu D.R. Yoo T.H. Han S.H. Choi H.Y. Kim H.J. Han D.S. Kang S.W. J. Am. Soc. Nephrol. 2007; 18: 2303-2310Crossref PubMed Scopus (38) Google Scholar, 10Koop K. Eikmans M. Baelde H.J. Kawachi H. De Heer E. Paul L.C. Bruijn J.A. J. Am. Soc. Nephrol. 2003; 14: 2063-2071Crossref PubMed Scopus (265) Google Scholar, 11Doublier S. Salvidio G. Lupia E. Ruotsalainen V. Verzola D. Deferrari G. Camussi G. Diabetes. 2003; 52: 1023-1030Crossref PubMed Scopus (314) Google Scholar), and hyperglycemia increases diacylglycerol generation, causing PKC activation (2Ziyadeh F.N. Wolf G. Curr. Diabetes Rev. 2008; 4: 39-45Crossref PubMed Scopus (312) Google Scholar). However, it is not fully understood how "hyperglycemic" signaling in diabetes affects the expression and distribution of nephrin.Menne et al. (12Menne J. Meier M. Park J.K. Boehne M. Kirsch T. Lindschau C. Ociepka R. Leitges M. Rinta-Valkama J. Holthofer H. Haller H. Kidney Int. 2006; 70: 1456-1462Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) reported a major step elucidating podocyte signaling in diabetes: they showed that albuminuria does not develop in diabetic PKCα-deficient mice and concluded that decreased nephrin expression in diabetes is a result of altered transcriptional regulation. All of the studies until now were conducted in chronic models of diabetes (13Meier M. Menne J. Haller H. Diabetologia. 2009; 52: 765-775Crossref PubMed Scopus (38) Google Scholar). Therefore, their conclusions were based on the changes occurring in long term hyperglycemia. However, considering the highly dynamic structure of the slit diaphragm, we hypothesized that elevated blood glucose levels have an immediate impact on the molecular composition of the slit diaphragm. Our hypothesis is supported by the recent study of Axelsson et al. (14Axelsson J. Rippe A. Rippe B. Am. J. Physiol. Renal Physiol. 2010; 298: F1306-F1312Crossref PubMed Scopus (36) Google Scholar), who demonstrated that acute hyperglycemia impairs glomerular permselectivity in previously normoglycemic rats. Therefore, nephrin might be involved in the changes in glomerular permeability induced by high blood glucose levels.We have recently demonstrated that nephrin endocytosis occurs in a β-arrestin2-dependent manner (15Quack I. Rump L.C. Gerke P. Walther I. Vinke T. Vonend O. Grunwald T. Sellin L. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 14110-14115Crossref PubMed Scopus (86) Google Scholar). Regulation of highly dynamic protein complexes such as the slit diaphragm typically involves changes in their phosphorylation pattern (16Garg P. Verma R. Nihalani D. Johnstone D.B. Holzman L.B. Mol. Cell Biol. 2007; 27: 8698-8712Crossref PubMed Scopus (118) Google Scholar). However, the kinase mediating the binding of β-arrestin2 to nephrin has not yet been identified. Studies have evidenced that β-arrestin2 binding is regulated by serine/threonine phosphorylation of its target proteins (17Xiang B. Yu G.H. Guo J. Chen L. Hu W. Pei G. Ma L. J. Biol. Chem. 2001; 276: 4709-4716Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Lorenz S. Frenzel R. Paschke R. Breitwieser G.E. Miedlich S.U. Endocrinology. 2007; 148: 2398-2404Crossref PubMed Scopus (55) Google Scholar). In our efforts to identify candidate kinases, sequence analysis of the nephrin C terminus revealed a PKC consensus site within the β-arrestin2 interaction motif. The available evidence on PKC signaling in diabetes and the presence of the PKC consensus site in close proximity to the β-arrestin2 interaction motif support our hypothesis that hyperglycemia influences nephrin endocytosis via PKC signaling. In the present study, we examined the role of PKC in β-arrestin2-mediated nephrin endocytosis and its effect on the slit diaphragm integrity in vitro and in vivo to elucidate the mechanism underlying the loss of surface nephrin and subsequent albuminuria upon hyperglycemia in diabetes mellitus.
Referência(s)