Rhophilin-1 is a key regulator of the podocyte cytoskeleton and is essential for glomerular filtration.

Rhophilin-1 is a Rho GTPase-interacting protein, the biologic function of which is largely unknown. Here, we identify and describe the functional role of Rhophilin-1 as a novel podocyte-specific protein of the kidney glomerulus. Rhophilin-1 knockout mice were phenotypically normal at birth but developed albuminuria at about 2 weeks of age. Kidneys from severely albuminuric mice revealed widespread podocyte foot process effacement, thickening of the glomerular basement membrane, and FSGS-like lesions. The absence of any overt changes in the expression of podocyte proteins at the onset of proteinuria suggested that the primary cause of podocyte abnormalities in Rhpn1-null mice was the result of cell-autonomous, Rhophilin-1-dependent signaling events. In culture, Rhophilin-1 was detected at the plasma membrane leading edge of primary podocytes, where it elicited remodeling of the actin cytoskeleton network. This effect of Rhophilin-1 on actin cytoskeleton organization associated with inhibitory effects on Rho-dependent phosphorylation of the myosin regulatory light chain and stress fiber formation. Conversely, phosphorylation of myosin regulatory light chain increased in podocyte foot processes of Rhpn1(-/-) mice, implicating altered actinomyosin contractility in foot process effacement and compromised filtration capacity. Targeted deletion of RhoA in podocytes of Rhophilin-1 knockout mice exacerbated the renal injury. Taken together, our results indicate that Rhophilin-1 is essential for the integrity of the glomerular filtration barrier and that this protein is a key determinant of podocyte cytoskeleton architecture.

A remote cis-acting variant at 3q links glomerular NCK1 to diabetic nephropathy.

We have previously reported genetic association of a single nucleotide polymorphism (SNP), rs1866813, at 3q locus with increased risk of diabetic nephropathy (DN). The SNP is located approximately 70 kb downstream of a cluster of four genes. This raises a question how the remote noncoding polymorphism affects the risk of DN. In this study, we tested a long-range regulatory potential of this variant by a series of experiments. In a luciferase assay, two alleles of the SNP showed differential effects on the luciferase activity in transfected cells in vitro. Using transgenic zebrafish, we further demonstrated in vivo that two alleles of the SNP differentially regulated GFP expression in zebrafish podocytes. Immunofluorescence staining and Western blotting verified that only Nck1 of the four nearby genes was predominantly expressed in mouse glomeruli as well as in podocytes. Furthermore, genotypes of the SNP rs1866813 were correlated with NCK1 expression in immortalized lymphocytes from diabetic patients. The risk allele was associated with increased NCK1 expression compared to the non-risk allele, consistent with the results of the reporter-based studies. Interestingly, differential expression of glomerular Nck1 between mouse strains carrying the nephropathy-prone 129/Sv allele and nephropathy-resistant C57BL/6 allele was also observed. Our results show that the DN-associated SNP rs1866813 is a remote cis-acting variant differentially regulating glomerular NCK1 expression. This finding implicates an important role for glomerular NCK1 in DN pathogenesis under hyperglycemia.