Redox Regulation of TRPML1 Channel Activity and Lysosome Trafficking in Podocytes

Abstract

Recent studies have indicated that redox signaling is importantly involved in the control of exosome secretion from a variety of cells. However, the mechanism by which reactive oxygen species (ROS) regulates exosome release remains unknown. In the present study, we hypothesized that ROS attenuate TRPML1 channel‐mediated Ca2+ release in podocytes through inhibition of acid ceramidase (AC) and thereby reduce lysosome trafficking, leading to prolongation of MVB fate to promote exosome secretion. Using GCaMP3 Ca2+ imaging technique, it was found that ML‐SA1‐induced Ca2+ release from lysosomes in podocytes was almost completely blocked by H2O2. However, sphingosine as an AC product of ceramide hydrolysis increased TRPML1 channel‐mediated Ca2+ release from lysosome of podocytes, which was not prevented by pre‐treatment with H2O2. Given the fact that Hcy induces NADPH oxidase‐derived ROS production in podocytes, we tested whether Hcy‐induced endogenous ROS production inhibits TRPML1 channel activity in these cells. It was found that pre‐treatment of podocytes with Hcy for 24 hours completely blocked TRPML1 channel‐mediated Ca2+ release. In the presence of catalase, Hcy‐induced blockade of lysosomal TRPML1 channel activation was prevented. Moreover, inhibition of NADPH oxidase by gp91 ds‐tat peptide or diphenyleneiodonium also abolished the inhibitory effects of Hcy on ML‐SA1‐induced TRPML1 channel‐mediated Ca2+ release. By confocal microscopy, we observed that ML‐SA1 enhanced the lysosome‐MVB interaction in podocytes, which was diminished by pre‐treatment with H2O2. However, when podocytes were pretreated with sphingosine, ML‐SA1 remained to enhance lysosome‐MVB interaction in the presence or absence of H2O2. Functionally, ML‐SA1 was found to enhance lysosome trafficking in podocytes towards the perinuclear region, which was prevented by pre‐treatment of these cells with H2O2. From these results, we conclude that both exogenous and endogenous ROS inhibit TRPML1 channel activity in lysosome of podocytes through inhibition of AC, which may cause derangement of lysosome trafficking and lysosome‐MVB interaction, leading to increased exosome secretion.Support or Funding InformationThis study was supported by NIH grants DK54927, DK120491, and HL057244.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.