Analysis of pathomechanisms of defective podocyte adhesion in FSGS.

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The podocyte is central in the pathogenesis of proteinuric glomerular diseases such as focal segmental glomerulosclerosis (FSGS). FSGS is one of the leading causes of chronic kidney disease. Podocytes form the outer part of the filtration barrier with their intercellular contacts called slit diaphragms. Slit diaphragms are formed by the essential adhesion proteins Nephrin and Neph1. We dissected that Nephrin transmits signals to Integrin β1 at focal adhesions (FA) via the small GTPase Rap1 and its activating factor RAPGEF1 (C3G). This signaling pathway may influence podocyte adhesion and thus podocyte loss in glomerular disease. To analyze molecular mechanisms of Nephrin signaling, we employ the versatile in vivo Drosophila nephrocyte model of podocytes. Nephrocytes exhibit slit diaphragm-like structures consisting of orthologs of mammalian adhesion proteins such as Nephrin. We showed in several mouse models that knockout (KO) of key genes of this pathway in podocytes leads to FSGS. In mass spectrometry analysis of the phospho-proteome of podocytes after activation of Nephrin signaling, we identified over 180 differentially phosphorylated proteins. We will now pursue the overarching hypothesis that Nephrin transmits signals from the slit diaphragm to focal adhesions thereby influencing the composition of the extracellular matrix. In the first work package, we will test whether Nephrin and Integrin α3β1 interact genetically by crossing heterozygous Nephrin KO with podocyte-specific Integrin α3 or β1 mice and analyzing the modulation of the slit diaphragm phenotype. We will dissect the mechanisms by single-nucleus sequencing. In the second part, we will investigate the hypothesis in the Drosophila model that Nephrin affects the composition of the extracellular matrix. In the phospho-proteome analysis after activation of Nephrin signal transduction, we found among others proteins of the Epidermal Growth Factor Receptor (EGFR) signaling pathway enriched phosphorylated. In the third part, we will pursue the hypothesis that Nephrin and EGFR signaling are integrated to affect focal adhesions.