Increased Podocyte Exosome Release in Glomerular Injury induced by NLRP3 Inflammasome Activation during Hyperhomocysteinemia

Abstract

The NOD‐like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis to elevated homocysteine (Hcys) levels in vitro and in vivo. However, it remains unknown how the products of NRLP3 inflammasome activated in cytoplasm are secreted out of podocytes and whether exosome release serves as a critical mechanism to mediate the action of NLRP3 inflammasome activation in Hcys‐induced kidney injury. In the present study, we tried to answer these questions using Smpd1trg/Podocre (a podocye‐specific sphingomyelin phosphodiesterase 1 (Smpd1) transgenic mouse strain) because Smpd1 encodes acid sphingomyelinase that produces ceramide in lysosomes and regulates lysosome function and potentially controls exosome release. It was found that Smpd1trg/Podocre mice had obvious overexpression of Smpd1 gene and remarkable increase in ceramide concentrations in podocytes. These Smpd1trg/Podocre mice were fed a folate‐free (FF) diet to induce hyperhomocysteinemia (hHcys), and ~ 3 folds increase in colocalization of inflammasome proteins, NLRP3 with ASC or with caspase‐1 and ~ 4 folds of IL‐1β production were found in the glomeruli of these FF‐treated Smpd1trg/Podocre mice, suggesting NLRP3 inflammasome formation and activation. Increased IL‐1β production in the glomeruli was accompanied by decreased podocin levels, but enhanced desmin expression in podocytes with glomerular injury as shown by morphological examinations. We also observed significantly increased colocalization of Annexin‐2 and alkaline phosphatase (exosome markers) in glomeruli of FF‐treated Smpd1trg/Podocre mice than their control littermates, indicating the increased exosome release in mice with Smpd1 gene overexpression in podocytes. By Nanoparticle Tracking Analysis, we found that urinary exosomes (60–140 nm in size) were significantly increased especially from 90nm to 140nm in FF‐treated Smpd1trg/Podocre mice, but not in their control littermates. These increased exosomes in urine from FF‐treated Smpd1trg/Podocre mice contained more detectable podocin protein than control littermates, meaning enrichment of podocyte‐derived exosomes in urine from the podocyte‐specific Smpd1 transgenic mice. Taken together, our results suggest that increased ceramide in podocytes due to Smpd1 gene overexpression may increase exosome release and thereby enhance excretion of inflammasome products to induce podocyte dysfunction and glomerular injury.Support or Funding Informationsupported by NIH grants DK54927 to P.L. and, DK107991 to N.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.