The role of membrane protein proteolysis in membranous nephropathy

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Membranous nephropathy (MN) is an autoimmune disease of the glomerulus, mediated by autoantibodies that target antigens expressed by glomerular podocytes. 2009, PLA2R1 was identified as the first podocyte antigen, to which 70% of patients developed autoantibodies in MN. 2014 we identified THSD7A as the second target to autoimmunity in 10% of patients with MN. In MN autoantibodies bind to the transmembrane antigen situated at podocyte foot processes. As a consequence, foot processes efface and subepithelial immune deposits develop, both morphological hallmarks of MN. Patients develop a nephrotic syndrome, characterized by proteinuria over 3.5 g/day, edema, and hyperlipidemia. The mechanisms of foot process effacement and sub epithelial immune deposition are not clear in MN, but most likely involve the proteolysis of podocyte membrane proteins such as cell adhesion, cell-cell contact proteins, and of podocyte membrane proteins serving as antigen(s). It has been described from other systems that both PLA2R1 and THSD7A undergo proteolysis, resulting (at least in the case of THSD7A) in the generation of a soluble extracellular protein fragment and a small intracellular fragment, which we can detect in podocytes. We identified the membrane-anchored protease A Disintegrin And Metalloproteinase (ADAM) 10 to be expressed at podocyte foot processes. ADAM10 proteolytic activity results in the shedding of numerous membrane-bound substrates such as Notch, L-Selectin, E-cadherin, VE-cadherin, CXCL16, and L1 adhesion molecule and thereby regulates many biological processes such as cell migration and proliferation, neurite and axon guidance, and immunity. Some known ADAM10 targets are relevant for podocyte survival, adhesion and signaling, therefore favoring an important role for ADAM10 in podocytes. In order to investigate, whether ADAM10 is involved in the shedding of podocyte membrane proteins in MN, we generated mice with podocyte-specific ADAM10 deficiency. These mice exhibited no obvious developmental phenotype and are healthy until adulthood. In this proposal we hypothesize that ADAM10 is involved in the development of foot process effacement and subepithelial antibody/antigen deposition and will address this hypothesis in four aims. We will investigate the factors resulting in ADAM10 up regulation and activation in podocytes in MN (aim 1), the pathophysiologic role of ADAM10 for subepithelial antibody/antigen deposition in the THSD7A-specific MN model developed by us (aim 2), the pathophysiologic role of ADAM10 for foot process effacement in the well established model of adriamycin nephritis and protein overload model (aim 3), and we will aim at identifying new podocyte-specific ADAM10 targets by cell surface proteomics of glomerular membrane preparations and of in vivo biotinylated glomerular proteins (aim 4). These investigations will clarify the role of ADAM10 for podocyte membrane proteolysis in membranous nephropathy.