Redox Triggering of Podocyte NLRP3 Inflammasomes and Glomerular Injury in Hyperhomocysteinemia

Hyperhomocysteinemia (hHcys), an important pathogenic factor contributing to the progression of end-stage renal disease (ESRD), has been shown to activate NOD-like receptor protein 3 (NLRP3) inflammasomes and cause podocyte dysfunction and glomerular sclerosis. hHcys induces aggregation of the three inflammasome components – NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1 – and its activation is indicated by increased caspase-1 activity and secretion of interleukin-1β (IL-1β). The aims of the present study sought to elucidate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling in hHcys-induced NLRP3 inflammasome activation, to dissect the contribution of common endogenous reactive oxygen species (ROS) including superoxide (O2•−), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical (•OH), and to explore the molecular mechanisms by which the NLRP3 inflammasome senses changes in oxidative stress through thioredoxin-interacting protein (TXNIP). Specific inhibition of the gp91phox subunit of NADPH oxidase markedly reduced Hcys-induced caspase-1 activity and IL-1β production in cultured podocytes. Concurrently, gp91phox−/− or administration of a gp91ds-tat peptide also exhibited diminished glomerular inflammasome formation and activation in mice fed a folate-free (FF) diet to induce hyperhomocysteinemia and displayed glomerular protection as shown by prevention of hHcys-induced proteinuria, albuminuria and glomerular sclerosis. Interestingly, dismutation of O2•− by 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl and administration of H2O2 decomposer catalase either in cultured podocytes or hyperhomocysteinemic mice inhibited hHcys-induced NLRP3 inflammasome aggregation and activation. Hyperhomocysteinemic mice also demonstrated a significant increase in glomerular TXNIP binding to NLRP3, confirmed by confocal microscopy, size-exclusion chromatography, and co-immunoprecipitation studies. Blockade of TXNIP by genetic interference or by the calcium channel blocker verapamil prevented this hHcys-induced TXNIP-NLRP3 binding, NLRP3 inflammasome formation and activation, as well as protected hyperhomocysteinemic mice from glomerular dysfunction and damaged morphology. In conclusion, hHcys-induced NADPH oxidase activation is importantly involved in the switching on of NLRP3 inflammasomes in podocytes, where NADPH oxidase-derived O2•− and H2O2 primarily contribute to NLRP3 inflammasome activation. TXNIP binding to NLRP3 is a key signaling mechanism allowing NLRP3 inflammasome to sense these changes in oxidative stress. These findings greatly enhance our understanding of the early pathogenesis of hHcys-induced glomerular sclerosis, which may identify new therapeutic targets for prevention or treatment of ESRD