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Role of Different Reactive Oxygen Species in Homocysteine‐induced NALP3 Inflammasome Activation in Mouse Podocytes
Author(s) -
Abais Justine,
Boini Krishna,
Xia Min,
Li Pin-Lan
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.691.9
Subject(s) - nalp3 , chemistry , inflammasome , reactive oxygen species , apocynin , biochemistry , catalase , superoxide , nadph oxidase , hydrogen peroxide , aim2 , dithiothreitol , xanthine oxidase , hydroxyl radical , uric acid , superoxide dismutase , caspase 1 , antioxidant , enzyme , receptor
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 (O 2 •− ) or other reactive oxygen species (ROS) are responsible for this activation. The present study tested the role of four common endogenous ROS – O 2 •− , hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO − ), and hydroxyl radical (OH) in NALP3 inflammasome activation by using their selective scavengers or enzyme inhibitors. Confocal microscopy showed that dismutation of O 2 •− 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, H 2 O 2 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 O 2 •− and ONOO − , but not H 2 O 2 or OH play an important role in mediating Hcys‐induced NALP3 inflammasome activation in podocytes (supported by NIH grants HL057244 and DK54927).