Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to crosslink actin microfilaments into higher order structures have been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and of CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the significant regenerative potential of injured glomeruli and that targeting the oligomerization cycle of dynamin represents an attractive potential therapeutic target to treat CKD.

BackgroundPodocytes 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 FindingsWe 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.ConclusionsBased 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.

Podocyte damage is the basis of many glomerular diseases with ultrastructural changes and decreased expression of components of the slit diaphragm such as nephrin and podocin. Under physiological conditions it is likely that the slit diaphragm underlies permanent renewal processes to indemnify its stability in response to changes in filtration pressure. This would require constant reorganization of the podocyte foot process and the renewal of slit diaphragm components. Thus far, the mechanisms underlying the turnover of slit diaphragm proteins are largely unknown. In this manuscript we examined a mechanism of nephrin endocytosis via CIN85/Ruk L -mediated ubiquitination. We can demonstrate that the loss of nephrin expression and onset of the proteinuria in CD2AP ؊/؊ mice correlates with an increased accumulation of ubiquitinated proteins and expression of CIN85/Ruk L in podocytes. In cultured murine podocytes CD2AP deficiency leads to an early ubiquitination of nephrin and podocin after stimulation with fibroblast growth factor-4. Binding assays with different CIN85/Ruk isoforms and mutants showed that nephrin and podocin are binding to the coiled-coil domain of CIN85/Ruk L . We found that in the presence of CIN85/Ruk L , which is involved in down-regulation of receptor-tyrosine kinases, nephrin is internalized after stimulation with fibroblast growth factor-4. Interestingly, coexpression of CIN85/Ruk L with CD2AP led to a decreased binding of CIN85/Ruk L to nephrin and podocin, which indicates a functional competition between CD2AP and CIN85/Ruk L . Our results support a novel role for CIN85/Ruk L in slit diaphragm turnover and proteinuria.Podocytes are highly specialized epithelial cells that cover the outer surface of the glomerular capillary tuft. They distend primary processes that further subdivide in numerous interdigitating foot processes. These foot processes form a specialized intercellular junction called "slit diaphragm." Many studies have emphasized the critical role of the slit diaphragm for maintaining the selective filtration barrier of the glomerulus (1, 2). Several proteins of the slit diaphragm have been identified that participate in common signaling pathways (3-5). One of the major components is nephrin. Nephrin is a transmembrane adhesion protein of the Ig superfamily, encoded by NPHS1. Humans and mice lacking nephrin are born without intact slit diaphragms and develop massive proteinuria already in utero (6, 7). There is cumulating evidence that nephrin is a signaling receptor molecule; nephrin forms with podocin and Neph1 a protein complex within the lipid raft that structurally functions as a transmembrane receptor (8). The intracellular human nephrin C terminus has several putative tyrosine phosphorylation sites that can be phosphorylated by the Src kinase Fyn. This receptor complex has been shown to interact with several protein kinases including Fyn, Yes, and phosphatidylinositol 3-kinase as well as with several adaptor proteins like Nck, Grb2, 2 and Crk (9, 10). A scaffolding protein ...

BackgroundMicroalbuminuria 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 FindingsIn this manuscript we analyzed the role of PKC-alpha (PKCα) on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCα-inhibitor (GÖ6976) leads to preserved nephrin expression and reduced proteinuria. In vitro, we found that high glucose stimulation would induce PKCα protein expression in murine and human podocytes. We can demonstrate that PKCα mediates nephrin endocytosis in podocytes and that overexpression of PKCα leads to an augmented endocytosis response. After PKC-activation, we demonstrate an inducible association of PKCα, PICK1 and nephrin in podocytes. Moreover, we can demonstrate a strong induction of PKCα in podocytes of patients with diabetic nephropathy.Conclusions/SignificanceWe therefore conclude that activation of PKCα is a pathomechanistic key event during the development of diabetic nephropathy. PKCα is involved in reduction of nephrin surface expression and therefore PKCα inhibition might be a novel target molecule for anti-proteinuric therapy.

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