Contribution of Gasdermin D Pore Formation to NLRP3 Inflammasome Product Secretion and Pyroptosis in Podocytes during Obesity-Induced Glomerulopathy

NLRP3 inflammasome activation in podocytes has been reported to play an important role in the development of glomerular inflammation and sclerosis during obesity. Although, the molecular mechanism mediating the secretion of NLRP3 inflammasome products to induce local inflammatory response remains poorly understood. Given that gasdermin D (GSDMD) has been found to form oligomeric pores on plasma membrane for release of inflammasome product and execution of pyroptosis, the present study tested whether GSDMD pore contributes to NLRP3 inflammasome product secretion and pyroptosis in podocytes and thereby initiates glomerular inflammation and sclerosis in obesity-induced glomerulopathy (ORG). By Western blot analysis, we demonstrated that palmitic acid (PA) as an obesity-related danger factor dose-dependently stimulated the reduction of total GSDMD and elevation of cleaved GSDMD (GSDMD-NT) in podocytes. In vivo, glomerular decrease in total GSDMD and increase in GSDMD-NT were remarkable in wild type (WT/WT) mice on high-fat diet (HFD) compared to control mice. Urinary excretion of NLRP3 inflammasome products was also significantly increased by HFD treatment. Moreover, we found that pre-treatment with amitriptyline or MCC950 blocked NLRP3 inflammasome activation as well as GSDMD pore formation on plasma membrane of podocytes, while disulfiram prevented GSDMD pore formation without affecting NLRP3 inflammasome activation. Since acid sphingomyelinase (ASM)-ceramide signaling pathway plays a vital role in the development of ORG, we examined whether ASM activity determines GSDMD pore formation in podocytes. By atom force microscopy, a 3D topography map of a single podocyte was generated, and pore-like structures were observed in the membrane of PA-treated WT/WT podocytes. Podocytes lacking Smpd1 (gene code of ASM) gene did not show pore formation, but Smpd1 gene overexpression amplified PA-induced pore formation in podocytes. Moreover, it was found that HFD-induced GSDMD cleavage and pyroptosis in glomeruli were blocked by Smpd1 gene deletion in Smpd1−/− mice. On the contrary, podocyte-specific Smpd1 gene overexpression amplified GSDMD-NT production and pyroptosis in glomeruli. Correspondingly, HFD-induced podocyte foot process effacement and glomerular sclerosis in mice were inhibited by Smpd1 gene knockout but exaggerated by podocyte-specific Smpd1 gene overexpression. Taken together, our findings suggest that ASM-ceramide signaling pathway may control GSDMD pore formation in podocytes to determine glomerular inflammation and sclerosis in ORG. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.