Original ContributionsContribution of endogenously produced reactive oxygen species to the activation of podocyte NLRP3 inflammasomes in hyperhomocysteinemia
Section snippets
Cell culture
Kindly provided by Dr. Paul E. Klotman (Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA), a conditionally immortalized mouse podocyte cell line was cultured undifferentiated with 10 U/ml recombinant mouse interferon-γ at 33 °C on collagen I-coated flasks in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Passaged podocytes were allowed to differentiate at 37 °C for 10–14 days in the absence of
Reduction of intracellular O2− and H2O2 levels prevented Hcys-induced NLRP3 inflammasome formation in podocytes
Using the O2− dismutase mimetic Tempol, H2O2 decomposer catalase, or OH scavenger TMTU, we tested whether Hcys-induced inflammasome formation and activation can be altered. By confocal microscopy analysis, we demonstrated that Hcys induced colocalization (yellow spots) of inflammasome molecules (NLRP3 (green) vs ASC or caspase-1 (red)) in podocytes, compared to control cells, suggesting increased formation of NLRP3 inflammasomes (Fig. 1A). However, prior treatment of podocytes with Tempol or
Discussion
The goal of this study was to dissect which endogenously produced ROS in response to increased Hcys in podocytes in vitro and in vivo contribute to hHcys-induced NLRP3 inflammasome formation and activation. Studies using cultured podocytes revealed that reduction of intracellular O2− and H2O2 levels attenuated Hcys-induced NLRP3 inflammasome formation and suppressed downstream caspase-1 activation and IL-1β production. In vivo, dismutation of O2− and decomposition of H2O2 in
Acknowledgment
This work was supported by Grants DK54927, HL075316, and HL57244 (to P.L.) and 1F31AG043289-01 (to J.M.A.) from the National Institutes of Health.
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