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Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1−/− mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1−/− mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect. NEW & NOTEWORTHY: In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects. [ABSTRACT FROM AUTHOR]

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome. • Asah1 gene deletion impairs autophagic flux and thereby induces autophagosome accumulation in podocytes. • Asah1 gene knockout inhibits lysosome trafficking through blockade of TRPML1 channel. • Asah1 gene deletion induces podocyte dedifferentiation. • Enhancement of TRPML1 channel activity attenuates autophagosome accumulation and dedifferentiation in podocytes lacking Asah1 gene. [ABSTRACT FROM AUTHOR]

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis in response to hyperhomocysteinemia (hHcy). However, it remains unknown how the products of NLRP3 inflammasome in cytoplasm are secreted out of podocytes. In the present study, we tested whether exosome release serves as a critical mechanism to mediate the action of NLRP3 inflammasome activation in hHcy-induced glomerular injury. By various approaches, we found that hHcy induced NLRP3 inflammasome activation and neutrophil infiltration in glomeruli of WT/WT mice. Lysosome-MVB interaction in glomeruli remarkably decreased in WT/WT mice fed with FF diet, leading to elevation of urinary exosome excretion of these mice. Podocyte-derived exosomes containing pro-inflammatory cytokines increased in urine of WT/WT mice in response to hHcy. The release of inflammatory exosomes from podocytes was prevented by Smpd1 gene deletion but enhanced by podocyte-specific Smpd1 gene overexpression (Smpd1 encodes Asm in mice). Pathologically, hHcy-induced podocyte injury and glomerular sclerosis were blocked by Smpd1 gene knockout but amplified by podocyte-specific Smpd1 gene overexpression. Taken together, our results suggest that Asm-ceramide signaling pathway contributes to NLRP3 inflammasome activation and robust release of inflammatory exosomes in podocytes during hHcy, which together trigger local glomerular inflammation and sclerosis. • Exosomes mediate the secretion of NLRP3 inflammasome products from podocyte. • Smpd1 gene overexpression enhances inflammatory exosome release from podocytes. • Smpd1 gene knockout inhibits inflammatory exosome release from podocytes. • Asm contributes to exosome release via inhibition of lysosome-MVB interaction. [ABSTRACT FROM AUTHOR]

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1fl/fl/PodoCre) 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 Asah1fl/fl/PodoCre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1fl/fl/PodoCre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1fl/fl/PodoCre 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 Asah1fl/fl/PodoCre mice compared to WT/WT mice. In the podocytes from Asah1fl/fl/PodoCre 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 Asah1fl/fl/PodoCre 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 Asah1fl/fl/PodoCre mice. Combination of N , N -dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1fl/fl/PodoCre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1fl/fl/PodoCre 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. • Podocyte-specific Asah1 gene knockout elevates urinary exosome excretion. • Asah1 gene knockout inhibits lysosome-MVB interaction and thereby enhances exosome release from podocytes. • TRPML1 channel activity is inhibited by Asah1 gene knockout. • Enhancement of TRPML1 channel activity attenuates exosome secretion from podocytes with Asah1 gene knockout. [ABSTRACT FROM AUTHOR]

Podocytes are visceral epithelial cells covering the outer surface of glomerular capillaries in the kidney. Blood is filtered through the slit diaphragm of podocytes to form urine. The functional and structural integrity of podocytes is essential for the normal function of the kidney. As a membrane-bound organelle, lysosomes are responsible for the degradation of molecules via hydrolytic enzymes. In addition to its degradative properties, recent studies have revealed that lysosomes may serve as a platform mediating cellular signaling in different types of cells. In the last decade, increasing evidence has revealed that the normal function of the lysosome is important for the maintenance of podocyte homeostasis. Podocytes have no ability to proliferate under most pathological conditions; therefore, lysosome-dependent autophagic flux is critical for podocyte survival. In addition, new insights into the pathogenic role of lysosome and associated signaling in podocyte injury and chronic kidney disease have recently emerged. Targeting lysosomal functions or signaling pathways are considered potential therapeutic strategies for some chronic glomerular diseases. This review briefly summarizes current evidence demonstrating the regulation of lysosomal function and signaling mechanisms as well as the canonical and noncanonical roles of podocyte lysosome dysfunction in the development of chronic glomerular diseases and associated therapeutic strategies. [ABSTRACT FROM AUTHOR]