Inhibitory Effect of Podocyte‐Specific Silencing of Acid Sphingomyelinase Gene on NLRP3 Inflammasome Activation and Glomerular Injury During Hyperhomocysteinemia

Abstract

Acid Sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)‐induced NLRP3 inflammasome activation in podocytes and glomerular sclerosis. The present study was designed to test whether somatic podocyte‐specific silencing of ASM gene (its mouse code is Smpd1) attenuates hHcy‐induced NLRP3 inflammasome activation and associated exosome release in podocytes and thereby suppresses glomerular inflammatory response and injury. We first constructed a floxed Smpd1 shRNA construct (Vigene Bioscience, Cat# IS100001) that has both RFP and GFP as reporter genes, where RFP is expressed if there is no cre excision. If cre excision occurs, GFP is expressed. This floxed Smpd1 shRNA was transfected into podocytes isolated from WT/Podocre mice, which decreased ASM mRNA and protein levels by 90% and 60%, respectively. This suggests efficient gene silencing in podocytes with cre gene. When this floxed Smpd1 shRNA was transfected into mouse kidney by intravenous infusion and ultrasound‐microbubble directed introduction, ASM expression decreased by 65% in WT/Podocre mice and 51% in Smpd1trg/Podocre mice after 8 weeks of shRNA transfection. It was also found that transfection of floxed Smpd1 shRNA substantially attenuated hHcy‐induced increase in NLRP3 inflammasome product, IL‐1β. By nanoparticle tracking analysis, floxed Smpd1 shRNA transfection was shown to significantly block hHcy‐induced elevation of urinary exosome excretion, an indicative of NLRP3 inflammasome product release from the cells. In Smpd1trg/Podocre mice, hHcy was found to induce exaggerated NLRP3 inflammasome activation, exosome excretion and glomerular inflammatory response such as immune cell infiltration compared to WT/WT mice. This exaggerated glomerular pathological response to hHcy was also significantly suppressed by podocyte‐specific Smpd1 gene silencing. Besides, podocyte‐specific Smpd1 gene deletion substantially attenuated proteinuria and albuminuria. Taken together, our results suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and associated exosome release and that podocyte‐specific silencing of ASM gene may be an important therapeutic strategy for treatment of glomerular injury and ultimate sclerosis during hHcy.