NADPH oxidase-derived
reactive oxygen species (ROS) have been reported to activate NLRP3
inflammasomes resulting in podocyte and glomerular injury during
hyperhomocysteinemia (hHcys). However, the mechanism by which the
inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether
thioredoxin-interacting
protein (TXNIP), an endogenous inhibitor of the
antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3
inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by
siRNA or
verapamil prevented Hcys-induced TXNIP
protein recruitment to form NLRP3
inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1β production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers,
podocin and
desmin. In vivo, adult C57BL/6J male mice were fed a
folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by
verapamil (1 mg/ml in
drinking water) or by local
microbubble-ultrasound TXNIP
shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular
inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP
shRNA-transfected mice or those receiving
verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1β production in glomeruli of mice with hHcys. Correspondingly, TXNIP
shRNA transfection and
verapamil attenuated hHcys-induced
proteinuria,
albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3
inflammasome formation and activation and subsequent glomerular injury.