Role of Different Reactive Oxygen Species in Homocysteine‐induced NALP3 Inflammasome Activation in Mouse Podocytes

Abstract

Our recent studies have demonstrated the importance of NADPH oxidase‐mediated NALP3 inflammasome activation in both cultured podocytes and glomeruli of mice with hyperhomocysteinemia (hHcys). However, it remains unknown whether NADPH oxidase‐derived superoxide (O2•−) or other reactive oxygen species (ROS) are responsible for this activation. The present study tested the role of four common endogenous ROS – O2•−, hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical (OH) in NALP3 inflammasome activation by using their selective scavengers or enzyme inhibitors. Confocal microscopy showed that dismutation of O2•− by 4‐hydroxy‐2,2,6,6‐tetramethyl‐piperidinyloxy (TEMPOL) markedly reduced Hcysinduced colocalization of inflammasome protein NALP3 with ASC by 60.4% and NALP3 with caspase‐1 by 64.1% in podocytes. Size exclusion chromatography also demonstrated that TEMPOL inhibited Hcys‐induced NALP3 inflammasome formation by preventing ASC recruitment to higher molecular weight fractions. Scavenging of ONOO− by uric acid also prevented Hcys‐induced aggregation of NALP3 inflammasome proteins. Furthermore, TEMPOL and uric acid treatment were able to inhibit Hcys‐induced caspase‐1 activation, respectively by 40.5% and 39.2%, as well as IL‐1β production by 46.8% and 39.4%. By electromagnetic spin resonance spectrometry, Hcys was shown to increase superoxide production by 2.2 folds compared to control podocytes, which was inhibited by 71.5% by TEMPOL and 62.2% by uric acid. However, H2O2 decomposer, catalase and hydroxyl radical scavenger, tetramethylthiourea (TMTU) had no inhibitory effects on Hcys‐induced NALP3 inflammasome protein aggregation and increases in caspase‐1 activity and IL‐1β production. These results indicate that O2•− and ONOO−, but not H2O2 or OH play an important role in mediating Hcys‐induced NALP3 inflammasome activation in podocytes (supported by NIH grants HL057244 and DK54927).