Nephrin-related phospho-protein network is associated with slit-diaphragm function

Abstract Background Minimal Change Disease (MCD) is characterized by nephrotic syndrome. In a subgroup of patients, pathogenic antibodies against the slit-diaphragm (SD) protein Nephrin were identified in blood and biopsy samples. Currently, mechanisms in podocytes induced by antibody binding to Nephrin are elusive. Methods Following antibody-mediated Nephrin clustering as a model of MCD, we analyzed the phospho-proteome in cell culture by mass spectrometry (MS). Corresponding genes to the differentially phosphorylated proteins identified by MS were tested for their necessity for an intact SD by a knockdown approach in Drosophila nephrocytes. The relevance of one candidate gene was characterized by knockout in the metanephric mesenchyme of mice which results in an early knockout in podocytes. Results We analyzed the phospho-proteome in cell culture employing an antibody-mediated Nephrin clustering model of MCD which revealed more than 430 differentially phosphorylated peptides. Gene set enrichment analysis showed that among others the biological process ‘regulation of focal adhesion (FA) assembly’ was enriched. We verified that many of the identified proteins were necessary for filtration in vivo employing a knockdown approach with consecutive analysis of filtration in Drosophila melanogaster nephrocytes. One of the phosphorylated proteins in the proteome analysis was Rapgef1 - an activating factor of the small GTPase Rap1. Rapgef1 knockout in the metanephric mesenchyme of mice resulted in nephrotic syndrome. RNA sequencing of Rapgef1 knockout mice glomeruli revealed significant regulation of genes involved in cell adhesion and extracellular matrix organization. Changes in glomerular basement membrane (GBM) and FA composition were confirmed by immunofluorescence analysis of Rapgef1 knockout mice glomeruli. Conclusions Following Nephrin clustering, we identified a Nephrin-associated phospho-protein network and showed that several corresponding genes were necessary for filtration in Drosophila nephrocytes. For Rapgef1 , we confirmed that it is not only essential for intact slit-diaphragms in Drosophila but also in mammals.