Redox Regulation of Lysosome‐MVB Interaction and Inflammatory Exosome Release during NLRP3 Inflammasome Activation by High Homocysteine in Mouse Podocytes

Abstract

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome in podocytes has been implicated in the initiation of glomerular inflammation during hyperhomocysteinemia (hHcy). However, the mechanism by which NLRP3 inflammasome products are released from podocytes remains unknown. The present study tested whether exosome secretion from podocytes is enhanced by NADPH oxidase-produced reactive oxygen species (ROS), which may serve as a pathogenic mechanism mediating the release of products by NLRP3 inflammasome activation in podocytes. We first demonstrated the remarkable elevation of endogenously produced ROS in podocytes treated with homocysteine (Hcy) compared with control podocytes, which was abolished by pre-treatment with catalase, a hydrogen peroxide (H2O2) scavenger or gp91 ds-tat peptide, a NADPH oxidase inhibitor. Hcy also induced activation of NLRP3 inflammasome and formation of MVBs containing the products of NLRP3 inflammasome activation such as IL-1β in podocytes, which were prevented by catalase and gp91 ds-tat peptide. By super-resolution microscopy and nanoparticle tracking analysis (NTA), we found that ML-SA1, a TRPML1 channel agonist that increases lysosome trafficking by calcium release, significantly enhanced lysosome-MVB interaction and reduced exosome release in podocytes, which attenuated Hcy effects. In the presence of catalase or gp91 ds-tat peptide, moreover, Hcy-induced reduction of lysosome-MVB interaction and exosome secretion was restored. We also tested whether exogenous ROS can mimic the effects of Hcy on exosome release in podocytes. It was found that H2O2 activated NLRP3 inflammasome and induced formation of MVBs containing inflammatory cytokines in a dose-dependent manner. Furthermore, we confirmed that H2O2 decreased lysosome-MVB interaction and increased exosome release in podocytes. Based on these results, we conclude that endogenously produced ROS importantly contributes to inflammatory exosome secretion from podocytes during activation of NLRP3 inflammasome in response to high Hcy due to inhibition of lysosome-MVB interaction. This redox regulation of Hcy-induced inflammatory exosome release may be a critical molecular mechanism triggering glomerular inflammatory response during hHcy (supported by NIH grant DK120491).