Elsevier

Free Radical Biology and Medicine

Volume 67, February 2014, Pages 211-220
Free Radical Biology and Medicine

Original Contributions
Contribution of endogenously produced reactive oxygen species to the activation of podocyte NLRP3 inflammasomes in hyperhomocysteinemia

https://doi.org/10.1016/j.freeradbiomed.2013.10.009Get rights and content

Highlights

  • Scavenging of O2radical dot and H2O2 prevents Hcys-induced NLRP3 inflammasome formation and activation in podocytes.

  • Endogenous O2radical dot and H2O2 are needed for hHcys-induced NLRP3 inflammasome activation.

  • O2radical dot and H2O2 contribute to NLRP3 inflammasome activation and promote glomerular injury during hHcys.

Abstract

Hyperhomocysteinemia (hHcys) is an important pathogenic factor contributing to the progression of end-stage renal disease. Recent studies have demonstrated the implication of nicotinamide adenine dinucleotide phosphate oxidase-mediated NLRP3 inflammasome activation in the development of podocyte injury and glomerular sclerosis during hHcys. However, it remains unknown which reactive oxygen species (ROS) are responsible for this activation of NLRP3 inflammasomes and how such action of ROS is controlled. This study tested the contribution of common endogenous ROS including superoxide (O2radical dot), hydrogen peroxide (H2O2), peroxynitrite (ONOO), and hydroxyl radical (radical dotOH) to the activation of NLRP3 inflammasomes in mouse podocytes and glomeruli. In vitro, confocal microscopy and size-exclusion chromatography demonstrated that dismutation of O2radical dot by 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol) and decomposition of H2O2 by catalase prevented Hcys-induced aggregation of NLRP3 inflammasome proteins and inhibited Hcys-induced caspase-1 activation and IL-1β production in mouse podocytes. However, scavenging of ONOO or radical dotOH had no significant effect on either Hcys-induced NLRP3 inflammasome formation or activation. In vivo, scavenging of O2radical dot by Tempol and removal of H2O2 by catalase substantially inhibited NLRP3 inflammasome formation and activation in glomeruli of hHcys mice as shown by reduced colocalization of NLRP3 with ASC or caspase-1 and inhibition of caspase-1 activation and IL-1β production. Furthermore, Tempol and catalase significantly attenuated hHcys-induced glomerular injury. In conclusion, endogenously produced O2radical dot and H2O2 primarily contribute to NLRP3 inflammasome formation and activation in mouse glomeruli resulting in glomerular injury or consequent sclerosis during hHcys.

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 O2radical dot and H2O2 levels prevented Hcys-induced NLRP3 inflammasome formation in podocytes

Using the O2radical dot dismutase mimetic Tempol, H2O2 decomposer catalase, or radical dotOH 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 O2radical dot and H2O2 levels attenuated Hcys-induced NLRP3 inflammasome formation and suppressed downstream caspase-1 activation and IL-1β production. In vivo, dismutation of O2radical dot 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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