Abnormal podocyte TRPML1 channel activity and exosome release in mice with podocyte-specific Asah1 gene deletion.

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1/Podo) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1/Podo mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1/Podo mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1/Podo mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1/Podo mice compared to WT/WT mice. In the podocytes from Asah1/Podo mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca release through TRPML1 channels is inhibited in the podocytes of Asah1/Podo mice. By GCaMP3 Ca imaging, it was found that lysosomal Ca release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1/Podo mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1/Podo mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1/Podo mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.