Autophagic Deficiency and Dedifferentiation in Podocytes of Mice Lacking Acid Ceramidase (Asah1) Gene

Abstract

Listen

Translate article

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain with a podocyte‐specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Asah1fl/fl/Podocre, Asah1 is a gene code of mouse acid ceramidase). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether autophagic deficiency and associated dedifferentiation of podocytes contribute to the pathogenesis of podocytopathy in Asah1fl/fl/Podocre mice. By Western blot analysis and immunofluorescent staining, we demonstrated the significant reduction of epithelial markers (ZO‐1 and P‐cadherin) and remarkable elevation of mesenchymal markers (α‐SMA and FSP‐1) in podocytes lack of Asah1 gene in vivo and in vitro. Also, we found that Asah1 gene knockout induced autophagic deficiency in podocytes, which were evidenced by autophagosome accumulation and decreased lysosome‐autophagosome interaction in these cells. Given the vital role of transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and interaction with other vehicles, we tested whether this lysosomal channel is implicated in autophagic deficiency in podocytes lacking Asah1 gene. As a selective TRPML1 channel agonist, ML‐SA5 enhanced lysosome‐autophagosome interaction in WT/WT podocytes, but only had partial effects on this interaction in Asah1fl/fl/Podocre podocytes. Autophagosome accumulation was also attenuated by ML‐SA5 in podocytes lacking Asah1 gene. On the contrary, ML‐SI1 as a TRPML1 channel inhibitor impaired autophagic flux in WT/WT podocytes and amplified autophagic deficiency in Asah1fl/fl/Podocre podocytes. The enhancements of lysosome trafficking and lysosome‐autophagosome interaction by ML‐SA5 were blocked by dynein inhibitors, EHNA and ciliobrevin D, indicating the contribution of dynein to lysosome movement in response to TRPML1 channel‐mediated Ca2+ release. Pathologically, Asah1 gene deletion‐induced podocyte dedifferentiation, reduction of slit diaphragm proteins, and proteinuria were inhibited by ML‐SA5 but enhanced by ML‐SI1. Based on these results, we conclude that Asah1 gene deletion induces autophagic deficiency and dedifferentiation in podocytes through blockade of lysosomal TRPML1 channel, thereby leading to the development of podocytopathy and nephrotic syndrome in Asah1fl/fl/Podocre mice.