Ceramide‐Mediated Enhancement of Inflammatory Exosome Secretion from Podocytes during Hyperhomocysteinemia

Abstract

Exosome secretion has been shown increased during podocyte injury and associated glomerular diseases, and this increased exosome secretion may be important in mediating release of inflammasome products to activate glomerular inflammatory response leading to chronic glomerular disease. Given the crucial role of ceramide in the control of exosome biogenesis and release, the present study used a podocyte‐specific sphingomyelin phosphodiesterase 1 (Smpd1) transgenic mouse strain (Smpd1trg/Podocre mice) to test whether ceramide production enhanced by Smpd1 gene overexpression promotes inflammatory exosome release from podocyte leading to glomerular inflammation during Hcys. We demonstrated that in Smpd1trg/Podocre mice, hHcy (induced by the FF diet) produced significant podocyte inflammasome activation accompanied by large increases in urinary podocyte‐derived exosomes. By immunohistochemical detection, we found that the glomerular inflammatory responses was more remarkably activated by hHcy in Smpd1trg/Podocre mice than their littermates, as shown by increased staining of lymphocyte and T‐cell markers such as CD45 and CD43. Under electronic microscopy, we observed that hHcy induced more severe podocyte injury and exosome release around these podocytes in Smpd1trg/Podocre mice than their littermates. Using confocal microscopy, about 2.8 folds increase colocalization of multivesicle body marker (MVB) Vps16 and NLRP3 inflammasome product IL‐1β, but about 0.4 folds decrease interaction of Vps16 with lysosome (Lamp‐1) (a process determining MVB fate and exosome release) were found in the glomeruli of Smpd1trg/Podocre mice with hHcy compared to their littermates. In cultured podocytes, the nanoparticle tracking analysis confirmed that L‐Hcy treatment significantly increased microparticle release into supernatants especially from 50nm to 150nm and they were characterized as podocytes‐derived IL‐1β‐containing exosomes as shown by detection of synaptopodin, CD63 and IL‐1β via Western blot analysis. This L‐Hcy‐induced podocyte release of IL‐1β exosomes was mimicked by AC inhibitor carmofur, but attenuated by AC inducer, genistein. It was also found that L‐Hcy‐induced decreases in Vps16 and Lamp‐1 interaction could be simulated by carmofur and inhibited by genistein. All these results together suggest that increased ceramide in podocytes enhances release of inflammatory exosomes during hHcy by reduction of lysosome‐MVBs interaction and prolongation of MVBs to facilitate exosome secretion.Support or Funding Informationsupported by NIH grant DK54927, DK120491 and HL057244This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.