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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 Ca 2+ -dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca 2+ release through TRPML1 channels is inhibited in the podocytes of Asah1 fl/fl /Podo Cre mice. By GCaMP3 Ca 2+ imaging, it was found that lysosomal Ca 2+ 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., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Lysosomal acid ceramidase (Ac) has been shown to be critical for ceramide hydrolysis and regulation of lysosome function and cellular homeostasis. In the present study, we generated a knockout mouse strain (Asah1 fl/fl /Podo Cre ) with a podocyte-specific deletion of the α subunit (main catalytic subunit) of Ac. Although no significant morphologic changes in glomeruli were observed in these mice under light microscope, severe proteinuria and albuminuria were found in these podocyte-specific knockout mice compared with control genotype littermates. Transmission electron microscopic analysis showed that podocytes of the knockout mice had distinctive foot process effacement and microvillus formation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice bearing the Asah1 podocyte-specific gene deletion. Ceramide accumulation determined by liquid chromatography-tandem mass spectrometry was demonstrated in isolated glomeruli of Asah1 fl/fl /Podo Cre mice compared with their littermates. By crossbreeding Asah1 fl/fl /Podo Cre mice with Smpd1 -/- mice, we also produced a double knockout strain, Smpd1 -/- /Asah1 fl/fl /Podo Cre , that also lacks Smpd1, the acid sphingomyelinase that hydrolyzes sphingomyelin to ceramide. These mice exhibited significantly lower levels of glomerular ceramide with decreased podocyte injury compared with Asah1 fl/fl /Podo Cre mice. These results strongly suggest that lysosomal Ac in podocytes is essential for the maintenance of the structural and functional integrity of podocytes., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1 fl/fl /SM Cre ) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1 fl/fl /SM Cre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1 fl/fl /SM wt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1 fl/fl /SM Cre mice, high dose of P i led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1 fl/fl /SM Cre CASMCs as shown by GCaMP3 Ca 2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.

The transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca 2+ release that is involved in Ca 2+ -dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested whether the TRPML1 channel in podocytes mediates lysosome trafficking, which is essential for multivesicular body (MVB) degradation by lysosomes. We first demonstrated the abundant expression of TRPML1 channel in podocytes. By GCaMP3 Ca 2+ imaging, we characterized the lysosomal specificity of TRPML1 channel-mediated Ca 2+ release in podocytes. Given the important role of acid ceramidase (AC) in lysosome function and podocyte injury, we tested whether AC regulates this TRPML1 channel-mediated Ca 2+ release and consequent lysosome-dependent MVB degradation in podocytes. Pharmacologically, it was found that TRPML1 channel activity was remarkably attenuated by the AC inhibitor carmofur. Sphingosine, as an AC product, was demonstrated to induce TRPML1-mediated Ca 2+ release, which was inhibited by a TRPML1 blocker, verapamil. Using a Port-a-Patch planar patch-clamp system, we found that AC-associated sphingolipids, sphingomyelin, ceramide, and sphingosine had different effects on TRPML1 channel activity in podocytes. Functionally, the inhibition of AC or blockade of TRPML1 channels was found to suppress the interaction of lysosomes and MVBs, leading to increased exosome release from podocytes. These results suggest that AC is critical for TRPML1 channel-mediated Ca 2+ release, which controls lysosome-MVB interaction and exosome release in podocytes.

The pannexin-1 (Panx1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether Panx1 is present in podocytes and whether this channel in podocytes mediates ATP release leading to glomerular inflammation or fibrosis. To answer these questions, we first characterized the expression of Panx channels in podocytes. Among the three known pannexins, Panx1 was the most enriched in podocytes, either cultured or native in mouse glomeruli. Using a Port-a-Patch planar patch-clamp system, we recorded a large voltage-gated outward current through podocyte membrane under the Cs + in/Na + out gradient. Substitution of gluconate or aspartate for chloride in the bath solution blocked voltage-gated outward currents and shifted the reversal potential of Panx1 currents to the right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage-gated outward currents were substantially attenuated by specific Panx1 channel inhibitors. Given the anti-inflammatory and intracellular ATP restorative effects of adiponectin, we tested whether this adipokine inhibits Panx1 channel activity to block ATP release. Adiponectin blocked Panx1 channel activity in podocytes. Mechanistically, inhibition of acid ceramidase (AC) remarkably enhanced Panx1 channel activity under control conditions and prevented the inhibition of Panx1 channel by adiponectin. Correspondingly, intracellular addition of AC products, sphingosine or sphingosine-1-phosphate (S1P), blocked Panx1 channel activity, while elevation of intracellular ceramide had no effect on Panx1 channel activity. These results suggest that adiponectin inhibits Panx1 channel activity in podocytes through activation of AC and associated elevation of intracellular S1P., (Published by Elsevier B.V.)