Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1^fl/fl/Podo^Cre) 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^fl/fl/Podo^Cre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1^fl/fl/Podo^Cre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1^fl/fl/Podo^Cre 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^fl/fl/Podo^Cre mice compared to WT/WT mice. In the podocytes from Asah1^fl/fl/Podo^Cre 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 Ca²⁺-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca²⁺ release through TRPML1 channels is inhibited in the podocytes of Asah1^fl/fl/Podo^Cre mice. By GCaMP3 Ca²⁺ imaging, it was found that lysosomal Ca²⁺ 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^fl/fl/Podo^Cre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1^fl/fl/Podo^Cre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1^fl/fl/Podo^Cre 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.

足细胞病和相关肾病综合征 (NS) 已在具有酸性神经酰胺酶 (Ac) 的 α 亚基（主要催化亚基）的足细胞特异性缺失的敲除小鼠品系 (Asah1 ^fl/fl / Podo ^{Cre ) 中报告。}然而，这些小鼠足细胞病的发病机制仍然未知。本研究测试了 Asah1 基因敲除是否会增强足细胞外泌体的释放，这可能是 Asah1 ^fl/fl / Podo ^Cre小鼠足细胞病和相关 NS 的致病机制。我们首先证明了 Asah1 ^fl/fl / Podo ^{Cre中尿外泌体排泄的显着升高}小鼠与WT / WT小鼠相比，通过免疫组织化学检测， Asah1 ^{fl / fl} / Podo ^Cre小鼠的肾小球中伴有显着的膜联蛋白-II（外泌体标记物）积累。在细胞研究中，我们还证实，与 WT/WT 小鼠相比，Asah1 基因敲除增强了从 Asah1 ^fl/fl / Podo ^{Cre小鼠分离的足细胞原代培养物中外泌体的释放。}在来自 Asah1 ^fl/fl / Podo ^{Cre的足细胞中}与对照组相比，溶酶体和多泡体（MVB）的相互作用被证明是减少的，这表明MVB降解减少可能导致外泌体释放增加。鉴于瞬时受体电位粘蛋白 1 (TRPML1) 通道在 Ca ²⁺依赖性溶酶体运输和随后的溶酶体-MVB 相互作用中的关键作用，我们测试了通过 TRPML1 通道释放的溶酶体 Ca ²⁺在 Asah1 ^fl/fl的足细胞中是否受到抑制/Podo ^Cre老鼠。通过 GCaMP3 Ca ²⁺成像，发现溶酶体 Ca ²⁺在 Asah1 基因缺失的足细胞中，通过 TRPML1 通道的释放受到显着抑制。作为 Ac 产品，发现鞘氨醇可挽救 TRPML1 通道活性，从而恢复溶酶体-MVB 相互作用并减少 Asah1 ^fl/fl / Podo ^Cre小鼠足细胞的外泌体释放。N , N-二甲基鞘氨醇 (DMS)（一种有效的鞘氨醇激酶抑制剂）和鞘氨醇的组合显着抑制 Asah1 ^fl/fl / Podo ^Cre小鼠的尿外泌体排泄。^{此外，拯救 Asah1 fl/fl} / Podo ^Cre足细胞中的 Aash1 基因表达小鼠表现出正常的神经酰胺代谢和外泌体分泌。基于这些结果，我们得出结论，Ac 的正常表达对控制 TRPML1 通道活性、溶酶体-MVB 相互作用以及随后的足细胞外泌体释放具有重要意义。Asah1 基因缺陷抑制 TRPML1 通道活性，从而增强外泌体释放，这可能有助于足细胞病和相关 NS 的发展。