Regulation of NLRP3 Inflammasome Activation and Inflammatory Exosome Release in Podocytes by Acid Sphingomyelinase During Obesity

Abstract

The activation of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been reported to importantly contribute to glomerular inflammation and injury under different pathological conditions such as obesity. However, the mechanism mediating NLRP3 inflammasome activation in podocytes and subsequent glomerular injury remains poorly understood. Given that the ceramide signaling pathway has been reported to be implicated in obesity-related glomerulopathy (ORG), the present study was designed to test whether the ceramide-producing enzyme, acid sphingomyelinase (ASM), determines NLRP3 inflammasome activation and inflammatory exosome release in podocytes leading to glomerular inflammation and injury during ORG. In Smpd1^trg/Podo^cre mice, podocyte-specific overexpression of Smpd1 gene which encodes ASM significantly exaggerated high-fat diet (HFD)-induced NLRP3 inflammasome activation in podocytes and immune cell infiltration in glomeruli compared to WT/WT mice. Smpd1 gene deletion, however, blocked these pathological changes induced by HFD in Smpd1^-/- mice. Accompanied with NLRP3 inflammasome activation and glomerular inflammation, urinary excretion of exosomes containing podocyte marker and NLRP3 inflammasome products (IL-1β and IL-18) in Smpd1^trg/Podo^cre mice on the HFD was much higher than that in WT/WT mice. In contrast, Smpd1^-/- mice on the HDF had significantly lower urinary exosome excretion than WT/WT mice. Correspondingly, HFD-induced podocyte injury, glomerular sclerosis, and proteinuria were more severe in Smpd1^trg/Podo^cre mice, but milder in Smpd1^-/- mice compared to WT/WT mice. Using podocytes isolated from these mice, we demonstrated that visfatin, a prototype pro-inflammatory adipokine, induced NLRP3 inflammasome activation and enrichment of multivesicular bodies (MVBs) containing IL-1β in podocytes, which was much stronger in podocytes from Smpd1^trg/Podo^cre mice, but weaker in those from Smpd1^-/- mice than WT/WT podocytes. By quantitative analysis of exosomes, it was found that upon visfatin stimulation, podocytes from Smpd1^trg/Podo^cre mice released much more exosomes containing NLRP3 inflammasome products, but podocytes from Smpd1^-/- mice released much less exosomes compared to WT/WT podocytes. Super-resolution microscopy demonstrated that visfatin inhibited lysosome-MVB interaction in podocytes, indicating impaired MVB degradation by lysosome. The inhibition of lysosome-MVB interaction by visfatin was amplified by Smpd1 gene overexpression but attenuated by Smpd1 gene deletion. Taken together, our results suggest that ASM in podocytes is a crucial regulator of NLRP3 inflammasome activation and inflammatory exosome release that instigate glomerular inflammation and injury during obesity.

Keywords: NLRP3 inflammasome; exosome.; obesity-related glomerulopathy; podocyte.