Pathomechanismen der fokal segmentalen Glomerulosklerose (FSGS) : Charakterisierung von "Survival"- und Apoptose-Signalwegen

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Role of Protein Kinase C in Podocytes and Development of Glomerular Damage in Diabetic Nephropathy

The early glomerular changes in diabetes include a podocyte phenotype with loss of slit diaphragm proteins, changes in the actin cytoskeleton and foot process architecture. This review focusses on the role of the Protein Kinase C family in podocytes and points out the differential roles of classical, novel and atypical PKCs in podocytes. Some PKC-isoforms are indispensable for proper glomerular development and slit diaphragm maintenance whereas others might be harmful when activated in the diabetic milieu. Therefore some might be interesting treatment targets in the early phase of diabetes

CD2AP Regulates SUMOylation of CIN85 in Podocytes

Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85

Podocytic PKC-Alpha Is Regulated in Murine and Human Diabetes and Mediates Nephrin Endocytosis

Background: Microalbuminuria is an early lesion during the development of diabetic nephropathy. The loss of high molecular weight proteins in the urine is usually associated with decreased expression of slit diaphragm proteins. Nephrin, is the major component of the glomerular slit diaphragm and loss of nephrin has been well described in rodent models of experimental diabetes as well as in human diabetic nephropathy. Methodology/Principal Findings: In this manuscript we analyzed the role of PKC-alpha (PKCa) on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCa-inhibitor (GÖ6976) leads to preserved nephrin expression and reduced proteinuria. In vitro, we found that high glucose stimulation would induce PKCa protein expression in murine and human podocytes. We can demonstrate that PKCa mediates nephrin endocytosis in podocytes and that overexpression of PKCa leads to an augmented endocytosis response. After PKC-activation, we demonstrate an inducible association of PKCa, PICK1 and nephrin in podocytes. Moreover, we can demonstrate a strong induction of PKCa in podocytes of patients with diabetic nephropathy. Conclusions/Significance: We therefore conclude that activation of PKCa is a pathomechanistic key event during the development of diabetic nephropathy. PKCa is involved in reduction of nephrin surface expression and therefore PKC

Def-6, a Novel Regulator of Small GTPases in Podocytes, Acts Downstream of Atypical Protein Kinase C (aPKC) λ/ι

Supplemental Data Supplemental Figure S1 Characterization of WT and aPKC-deficient podocytes. A–C: Genomic DNA isolated from deficient and control cell lines was tested for the presence of Cre recombinase (A), floxed and WT alleles of PKCλ/ι (B), or WT and knockout alleles of PKCζ (C). As controls, genomic DNA samples of tail biopsies were used. D: Differentiated deficient or control cells were stained with antibodies against synaptopodin or WT-1. All used cell lines were positive for the tested podocyte markers. Scale bars = 50 μm. Download Supplemental Figure S2 Relative mRNA and protein expression of PKCλ/ι, PKCζ, and Def-6 in deficient and control podocytes. A–C: Real-time PCR measurements and Western blot analysis of PKCλ/ι- and PKCζ-deficient cells in comparison with control cells. A: PKCλ/ι mRNA and protein are reduced in the PKCλ/ι −/− cells. B: PKCζ mRNA and protein are reduced in the PKCζ −/− cells. C: Def-6 mRNA is up-regulated in the PKCλ/ι −/− cells but not in PKCζ −/− cells. mRNA level is normalized for HPRT-1. Def-6 protein expression is not changed. ∗∗P < 0.01. Download Supplemental Table S1 Download Supplemental Table S2 Download Supplemental Table S3 Download Supplemental Table S4 Download Supplemental Table S5 Download Supplemental Table S6 Download Supplemental Table S7 Download Supplemental Table S8 Download Supplemental Data Supplemental material for this article can be found at . The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes

CD2AP/CIN85 balance determines receptor tyrosine kinase signaling response in podocytes

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes

Cofilin-1 inactivation leads to proteinuria--studies in zebrafish, mice and humans.

BACKGROUND: Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration. METHODS AND FINDINGS: We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus. CONCLUSIONS: Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development