Lysosome dysfunction in podocytopathy and associated hypertension

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Project Summary Podocytopathy is an important pathogenic basis for different glomerular diseases such as minimal change disease (MCD), diffuse mesangial sclerosis, focal segmental glomerulosclerosis, collapsing glomeru- lonephropathy and global glomerulosclerosis associated with hyperhomocysteinemia (hHcy), obesity and diabetes mellitus. These glomerular diseases have been reported to account for the vast majority of end-stage renal disease and kidney-associated hypertension and cardiovascular diseases. Recent studies have indicated that normal autophagy is a critical cellular process to control podocyte function and even its life span and that deficient autophagy and associated autophagosome (AP) accumulation or increases in exosome release produce podocyte injury and podocytopathy. We have shown that a sphingolipid-mediated signaling pathway is importantly implicated in lysosome dysfunction, autophagic flux deficiency and ultimate podocytopathy and glomerular sclerosis. The present grant proposal will test a central hypothesis that lysosomal acid ceramidase (AC)-mediated sphingolipid metabolism critically controls lysosome trafficking or fusion to APs or multivesicular body (MVB) and subsequent degradation process determining the normal phenotype and function of podocytes. AC gene defect or functional deficiency may disturb lysosome degradation of APs and MVBs, which induces AP accumulation and exosome release from MVBs leading to podocyte phenotypic transition, effacement and ultimate MCD. To test this hypothesis, three Specific Aims are proposed. Specific Aim 1 will determine whether autophagic flux and exosome excretion in podocytes are fine controlled by lysosomal AC activity and whether the deficiency of this AC regulation causes podocytopathy and MCD in Asah1fl/fl/Podocre mice, but not in their littermates. Specific Aim 2 attempts to elucidate the central role of lysosomal AC- mediated sphingolipid signaling in the regulation of lysosome trafficking to and fusion with APs and MVBs for their degradation using podocyte-specific Asah1 gene deletion, rescuing and silencing. In Specific Aim 3, we will explore the mechanisms by which lysosome trafficking or fusion in podocytes is regulated by AC- associated sphingolipids via gating lysosomal TRPML1 channels and associated Ca2+ release using patch clamping of isolated lysosomes and lysosome-specific Ca2+ imaging with GCaMP3-ML as an indicator. These proposed studies will present a novel mouse model for podocytopathy and MCD and use this model to explore associated molecular mechanisms triggering podocytopathy. The grant proposal represents the first effort in the research field to investigate the AC-mediated lysosome regulation of autophagic flux and exosome excretion in podocytes and associated pathogenic role in potocytopathy. The findings may make paradigm shift in understanding pathogenesis of podocytopathy and MCD and help identify lysosomal AC as a therapeutic target for prevention or treatment of MCD and other glomerular diseases.