Lysosome‐MVB Encountering and Inflammatory Exosome Release Controlled by Acid Sphingomyelinase in Podocytes during Hyperhomocysteinemia

Abstract

Previous studies have demonstrated that overexpression of acid sphingomyelinase (ASM) enhances NLRP3 inflammasome activation and podocyte‐derived inflammatory exosome release during hyperhomocysteinemia (hHcy). However, the mechanism underlying the enhancement of inflammatory exosome release remains unclear. The present study tested a hypothesis that overexpression of ASM blocks lysosome‐MVB encountering via inhibition of lysosome trafficking, leading to enhanced inflammatory exosome release from podocytes during hHcy. Nanoparticle tracking analysis showed a largely increased urinary exosomes in WT/WT mice with hHcy, but not in Smpd1−/− (ASM gene knockout) mice. In Smpd1trg/Podocre (podocyte‐specific ASM gene overexpression) mice, this increase in urinary exosomes was not only during hHcy, but also on the ND. Increased exosomes were podocytes‐derived by detection of podocin as podocyte marker and CD63 as exosome marker. Biochemical analyses showed that NLRP3 products, IL‐1β and IL‐18 significantly increased in hHcy in WT/WT and Smpd1trg/Podocre mice, but not in Smpd1−/− mice. In vitro, we found that Hcy stimulation enhanced release of exosomes out of podocytes from WT/WT and Smpd1trg/Podocre mice, but it had no effect on exosome release from podocytes of Smpd1−/− mice. Exosomes were isolated with ultracentrifugation and purified with exosome purification kit from podocyte culture medium. It was found that Hcy increased CD63 in purified exosomes from WT/WT and Smpd1trg/Podocre podocytes, but it had no effect in Smpd1−/− podocytes. ELISA analysis showed that these increased exosomes contain a lot of IL‐1β and IL‐18 when podocytes were stimulated by Hcy. Super‐resolution microscopy showed the interaction of lysosomes with MVBs. Lysosomes encountered and fused to MVBs much less in podocytes from WT/WT and Smpd1trg/Podocre mice when they were treated with Hcy. In Smpd1−/− podocytes, Hcy failed to change the lysosome‐MVB encounters and interactions. ML‐SA1 as a cell‐permeable lysosome trafficking stimulator via activation of lysosomal TRPML1 channel markedly increased lysosomes movement toward nucleus. Hcy significantly reduced lysosomes movements to nucleus and overexpression of Smpd1 gene enhanced the effect of Hcy. In Smpd1−/− podocytes, ML‐SA1‐induced lysosome movement toward nucleus remained unchanged during Hcy treatment. These results suggest that ASM‐mediated sphingolipid signaling controls lysosome function determining inflammatory exosome release from podocytes during hHcy.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.