Regulation of TRPML1 channel activity and inflammatory exosome release by endogenously produced reactive oxygen species 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 inflammatory cytokines produced by the NLRP3 inflammasome in podocytes after Hcy stimulation. We first demonstrated the remarkable elevation of endogenously produced ROS in podocytes treated with Hcy compared with control podocytes, which was abolished by pre-treatment with the NADPH oxidase inhibitors, gp91 ds-tat peptide and diphenyleneiodonium (DPI). In addition, Hcy induced activation in podocytes of NLRP3 inflammasomes and the formation of multivesicular bodies (MVBs) containing inflammatory cytokines, which were prevented by treatment with gp91 ds-tat or the ROS scavenger, catalase. Given the importance of the transient receptor potential mucolipin 1 (TRPML1) channel in Ca²⁺-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca²⁺ release through TRPML1 channels is inhibited by endogenously produced ROS in podocytes after Hcy stimulation. By GCaMP3 Ca²⁺ imaging, we confirmed the inhibition of TRPML1 channel activity by Hcy which was remarkably ameliorated by catalase and gp91 ds-tat peptide. By structured illumination microscopy (SIM) and nanoparticle tracking analysis (NTA), we found that ML-SA1, a TRPML1 channel agonist, significantly enhanced lysosome-MVB interaction and reduced exosome release in podocytes, which were attenuated by Hcy. Pre-treatment of podocytes with catalase or gp91 ds-tat peptide restored ML-SA1-induced changes in lysosome-MVB interaction and exosome secretion. Moreover, we found that hydrogen peroxide (H₂O₂) mimicked the effect of Hcy on TRPML1 channel activity, lysosome-MVB interaction, and exosome secretion in podocytes. Based on these results, we conclude that endogenously produced ROS importantly contributes to inflammatory exosome secretion from podocytes through inhibition of TRPML1 channel activity, which may contribute to the initiation of glomerular inflammation during hHcy.

Keywords: Exosome; Homocysteine; Lysosome; Podocyte; Reactive oxygen species; TRPML1 channel.

Fig. 1

Formation of reactive oxygen species induced by Hcy treatment of podocytes and blockade by NADPH oxidase inhibitors. A. Representative ESR spectra showing O2^•− production in different groups of podocytes. B. Summarized data showing O2^•− production in different groups of podocytes (n = 6). *p < 0.05 vs. Ctrl group, #p < 0.05 vs. Vehl-Hcy group. Ctrl, control; Vehl, vehicle; gp91 ds-tat, gp91 ds-tat peptide; DPI, diphenyleneiodonium; Hcy, homocysteine.

Fig. 2

NLRP3 inflammasome activation and MVB-inflammatory cytokine association after Hcy treatment of podocytes. A. Representative images and summarized data showing the colocalization of inflammasome markers in different groups of podocytes (n = 3–5). Scale bars = 20 μm. B. Representative images and summarized data showing the colocalization of MVB markers and IL-1β in different groups of podocytes (n = 4–5). Scale bars = 20 μm *p < 0.05 vs. Ctrl group, #p < 0.05 vs. Vehl-Hcy group. Ctrl, control; Vehl, vehicle; gp91 ds-tat, gp91 ds-tat peptide; Hcy, homocysteine.

Fig. 3

Enhanced exosome release from podocytes after Hcy and dependence on ROS and NADPH oxidase. A. Representative images showing exosome release from different groups of podocytes. The x axis is diameter (nm); the y axis is concentration; the z axis is intensity (a.u.). B. Summarized data showing exosome release from different groups of podocytes (n = 6). *p < 0.05 vs. Vehl group, #p < 0.05 vs. Ctrl group. Ctrl, control; Vehl, vehicle; gp91 ds-tat, gp91 ds-tat peptide; Hcy, homocysteine.

Fig. 4

Inhibition of lysosomal Ca²⁺ release through TRPML1 channel by Hcy. A. Representative images showing GCaMP3 fluorescence in different groups of podocytes. Scale bars = 40 μm. B. A representative curve showing that ML-SA1 induced remarkable elevation of GCaMP3 signal in podocytes under control condition. C. A representative curve showing that ML-SA1 induced small elevation of GCaMP3 signal in podocytes after Hcy stimulation. D. Summarized data showing GCaMP3 fluorescence in different groups of podocytes (n = 5–9). *p < 0.05 vs. Vehl group, #p < 0.05 vs. Ctrl group. Ctrl, control; Vehl, vehicle; gp91 ds-tat, gp91 ds-tat peptide; Hcy, homocysteine.

Fig. 5

Attenuation of lysosome-MVB interaction by Hcy treatment of podocytes. A. Representative images showing the colocalization of Rab7a-GFP and Lamp-1-RFP in different groups of podocytes. Scale bars = 5 μm. B. Summarized data showing the colocalization of Rab7a-GFP and Lamp-1-RFP in different groups of podocytes (n = 6–8). *p < 0.05 vs. Vehl group, #p < 0.05 vs. Ctrl group. Ctrl, control; Vehl, vehicle; gp91 ds-tat, gp91 ds-tat peptide; Hcy, homocysteine.

Fig. 6

Formation of NLRP3 inflammasomes and MVBs containing inflammatory cytokines in podocytes after H₂O₂ stimulation. A. Representative images and summarized data showing the colocalization of inflammasome markers in different groups of podocytes (n = 3–5). Scale bars = 20 μm. B. Representative images and summarized data showing the colocalization of MVB markers and IL-1β in different groups of podocytes (n = 4–5). Scale bars = 20 μm *p < 0.05 vs. Ctrl group. Ctrl, control; H₂O₂, hydrogen peroxide.

Fig. 7

Enhancement of exosome secretion from podocytes by H₂O₂. A. Representative images showing exosome release from different groups of podocytes. The x axis is diameter (nm); the y axis is concentration; the z axis is intensity (a.u.). B. Summarized data showing exosome release from different groups of podocytes (n = 6). *p < 0.05 vs. Vehl group, #p < 0.05 vs. Ctrl group. Ctrl, control; Vehl, vehicle; H₂O₂, hydrogen peroxide.

Fig. 8

Blockade of lysosomal TRPML1 channel-mediated Ca²⁺ release in podocytes by H₂O₂. A. A representative curve showing that ML-SA1 induced remarkable elevation of GCaMP3 signal in podocytes pre-treated with vehicle. B. A representative curve showing that ML-SA1 had no effects on GCaMP3 signal in podocytes pre-treated with H₂O₂. C. Summarized data showing GCaMP3 fluorescence in different groups of podocytes (n = 6–8). *p < 0.05 vs. Ctrl group, #p < 0.05 vs. Vehl group. Ctrl group. Ctrl, control; Vehl, vehicle; H₂O₂, hydrogen peroxide.

Fig. 9

Lysosome-MVB interaction in podocytes attenuated by H₂O₂. A. Representative images showing the colocalization of Rab7a-GFP and Lamp-1-RFP in different groups of podocytes. Scale bars = 5 μm. B. Summarized data showing the colocalization of Rab7a-GFP and Lamp-1-RFP in different groups of podocytes (n = 6). *p < 0.05 vs. Vehl group, #p < 0.05 vs. Ctrl group. Ctrl group. Ctrl, control; Vehl, vehicle; H₂O₂, hydrogen peroxide.

Fig. 10

Regulation of TRPML1 channel activity and inflammatory exosome release by endogenously produced ROS in mouse podocytes. In response to Hcy, NADPH oxidase is activated to produce ROS in podocytes. NLRP3 inflammasomes are activated by ROS to produce inflammatory cytokines such as IL-1β, which enter the late endosomes that form MVBs. At the same time, lysosomal Ca²⁺ release through TRPML1 channel is inhibited by ROS, leading to the impairment of lysosome trafficking and reduction of lysosome-MVB interaction, a process determining the MVB fate. Under such conditions, decreased lysosome degradation of MVBs leads to robust release of MVB contents as inflammatory exosomes from podocytes. Hcy, homocysteine; NOX, NADPH oxidase; ROS, reactive oxygen species; MVB; multivesicular body.