Introduction and Aim: Glomerular epithelial cells (podocytes) are essential for kidney filter function. Podocyte defects are central to many chronic glomerular diseases and are characterized by aberrant intercellular junctions (slit diaphragms) and actin cytoskeletal dynamics. Patients expressing mutated variants of the slit diaphragm protein Nephrin fail to develop functional slit diaphragms, due to signaling defects to the podocyte actin cytoskeleton. Drosophila nephrocytes form a filtration barrier, express the Nephrin orthologue Sticks and stones (Sns), and serve as a genetically tractable model for mammalian podocytes. The aim of our study is to analyze Nephrin signal transduction.
Methods: An assay was established to analyze the role of genes of interest in Drosophila nephrocytes. In the experimental strain, the secreted protein Atrial Natriuretic Protein (ANP)-GFP-GFP was ectopically expressed by the ubiquitin promoter (ubi::ANP-GFP-GFP). This line also contained a sns-Gal4 transgene. Nephrocytes filter and then take up proteins of defined size, here ANP-GFP-GFP. This allows testing the impact of genes of interest on nephrocyte filter function by crossing in respective UAS-dsRNA strains. Standard immunofluorescence and co-immunoprecipitation assays were employed to establish a novel protein interaction.
Results: Immunohistochemistry indicates that Nephrin and the Rap GEF C3G co-localized in human podocytes. The interaction of the two proteins was confirmed by co-immunoprecipitating Nephrin and C3G in HEK cell lysates. To test the functional relevance of these proteins we used the Drosophila system where cell type specific gene silencing experiments can be performed. Nephrocyte specific knockdown of the Nephrin orthologue sns as well as C3G compromised filtration and protein uptake as shown by defective ANP-GFP-GFP uptake. We demonstrated that the C3G effector Rap1 was necessary for Drosophila nephrocyte function.
Discussion and conclusion: Drosophila melanogaster is a powerful model system to study nephrocyte function and Nephrin signaling. C3G interacts with Nephrin and is required for nephrocyte function.