Irini Tossidou

Mutations in the Gene That Encodes the F-Actin Binding Protein Anillin Cause FSGS

FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1-associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm-associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.

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.

Parietal epithelia cells in the urine as a marker of disease activity in glomerular diseases

BACKGROUND The detection of viable podocytes in the urine of patients with proteinuric diseases has been described as a non-invasive method to monitor disease activity. Most of the published studies use podocalyxin (PDX) as a podocyte specific marker. METHODS We examined the excretion of viable PDX-positive cells in a random set of spot urine from patients with biopsy-proven focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MGN) or membranoproliferative glomerulonephritis (MPGN) and characterized the excreted cells for podocyte and parietal epithelia markers as well as for proliferation activity. RESULTS We found that untreated patients with active disease excrete high numbers of PDX-positive cells in their urine. In contrast to that we were not able to detect significant amounts of PDX-positive cells in the urine of patients with active minimal change disease (MCD) and patients with FSGS or MGN in full remission. When we further characterized the cells we rarely detected expression of podocyte specific markers in the PDX-positive cells, but at least 50% of the PDX-positive cells were double positive for cytokeratin (CK8-18). Immunohistochemistry of the corresponding renal biopsies showed that 100% of podocytes and parietal cells stained positive for PDX. Semiquantitative analysis revealed that 45% of parietal cells were positive for CK8-18 and 100% of proximal tubular cells. No cells of the glomerular epithelial layer stained positive for CK8-18. CONCLUSIONS PDX-positive cells are lost in the urine in disease states that require podocyte regeneration and are a useful non-invasive marker for glomerular disease activity. These cells are possibly derived from the parietal epithelial layer.

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 (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/Significance We 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.

The balance of autocrine VEGF-A and VEGF-C determines podocyte survival

Podocytes are an important component of the glomerular filtration barrier and are the major source of vascular endothelial growth factor (VEGF) in the glomerulus. The role of VEGF for the phenotype of the glomerular endothelium has been intensely studied; however, the direct effects of autocrine VEGF on the podocyte are largely unknown. In this study we characterized the expression of VEGF isoforms and VEGF receptors in cultured human podocytes and examined direct effects on cell signaling and apoptosis after stimulation with exogenous VEGF or ablation of autocrine VEGF. We identified VEGF-A and VEGF-C as the dominant isoforms in human podocytes and showed that autocrine levels of both are important for the intracellular activation of antiapoptotic phosphoinositol 3-kinase/AKT and suppression of the proapoptotic p38MAPK via VEGFR-2. We demonstrated that ablation of VEGF-A or VEGF-C as well as treatment with bevacizumab or a VEGFR-2/-3 tyrosine kinase inhibitor led to reduced podocyte survival. In contrast, ablation of VEGF-B had no effect on podocyte survival. Treatment with exogenous VEGF-C reversed the effect of VEGF-A neutralization, and exogenous VEGF-A abrogated the effect of VEGF-C ablation in human podocytes. Our results underline the importance of autocrine VEGF for podocyte survival and indicate the delicate balance of VEGF-A and VEGF-C to influence progression of glomerular diseases.

CIN85/RukL Is a Novel Binding Partner of Nephrin and Podocin and Mediates Slit Diaphragm Turnover in Podocytes

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/RukL-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/RukL 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/RukL. We found that in the presence of CIN85/RukL, which is involved in down-regulation of receptor-tyrosine kinases, nephrin is internalized after stimulation with fibroblast growth factor-4. Interestingly, coexpression of CIN85/RukL with CD2AP led to a decreased binding of CIN85/RukL to nephrin and podocin, which indicates a functional competition between CD2AP and CIN85/RukL. Our results support a novel role for CIN85/RukL in slit diaphragm turnover and proteinuria.

Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models.

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 cross-link actin microfilaments into higher-order structures has 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 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 substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.

IGF-Binding Protein-3 Modulates TGF-β/BMP-Signaling in Glomerular Podocytes

Podocyte apoptosis initiates progressive glomerulosclerosis in TGF-beta1 transgenic and CD2AP-knockout (CD2AP-/-) mice. It was previously shown that in both mouse models, activation of the TGF-beta pathway is the key event during development of podocyte apoptosis. Furthermore, CD2AP is an important modifier of TGF-beta-induced survival signaling via activation of the phosphoinositol 3-kinase/AKT signaling pathway. This article presents IGF-binding protein-3 (IGFBP-3) as a new modulator of apoptosis and survival signaling in glomerular podocytes. High expression of IGFBP-3 protein in the urine of diseased CD2AP-/- mice was discovered, and IGFBP-3 expression in glomerular podocytes and parietal cells was detected. IGFBP-3 can induce changes in podocyte actin cytoskeleton, leads to apoptosis in cultured murine podocytes, and can enhance TGF-beta1-induced apoptosis in vitro. For studying this process on a molecular level, proapoptotic p38 mitogen-activated protein kinase pathways and antiapoptotic phosphoinositol 3-kinase/AKT pathways were examined in cultured murine podocytes. It was found that IGFBP-3 increments the level of TGF-beta1-induced phosphorylated p38 mitogen-activated protein kinase and decreases the phosphorylation of antiapoptotic AKT. This effect is specific for the co-stimulation of IGFBP-3 with TGF-beta1 because a combination of IGFBP-3 with bone morphogenic protein-7 (BMP-7), another member of the TGF-beta superfamily, results in apoptosis opposing signaling effects with a strong increase of phosphorylated AKT and subsequent functional effects. These results demonstrate that the IGF/IGFBP axis plays an important role in the development of podocyte apoptosis by modulation of TGF-beta and BMP-7-induced pro- and antiapoptotic signals.

PKC-alpha Modulates TGF-β Signaling and Impairs Podocyte Survival

Background: Progressive loss of podocytes has been documented as an early lesion in the development of glomerular disease. In a variety of glomerular diseases, including diabetic nephropathy the activation of transforming growth factor-beta (TGF-β) has been demonstrated to promote podocyte death and the development of glomerulosclerosis. In this manuscript we analyzed the role of PKC-alpha (PKCα) on TGF-β1 induced apoptosis in podocytes. Methods: To accomplish this we generated stable murine PKCα deficient podocyte cell lines and examined survival- and pro-apoptotic signaling signatures as well as caspase activation after stimulation with TGF-β. Results: After stimulation with TGF-β we can demonstrate an enhanced and prolonged activation of PI3K/AKT and ERK1/2 in PKCα-knockout (PKCα-/-) podocytes compared to PKCα-wildtype (PKCα+/ +) podocytes, whereas proapoptotic signaling via p38MAPK is significantly reduced. Interestingly, activation of the Smad-pathway is also prolonged in the PKCα-/-podocytes. When we analyzed the underlying mechanisms we found a TGF-β inducible interaction of PKCα with the TGF-β-type-I-receptor (TGFβRI). Moreover, endocytosis assays showed that the TGFβRI is less internalized in PKCα-/- podocytes. Conclusion: Since we can demonstrate a key role for PKCα in the signaling response after stimulation with TGF-β we conclude that PKCα might be an interesting target molecule as a “podocyte protective” therapy.

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.