ENaC Regulation in Mouse Cortical Collecting Duct Cells by EVs Released during the Inactive and Active Cycles of Salt-Loaded Hypertensive Diabetic db/db Mice

The downregulation of renal epithelial sodium channel (ENaC) activity in response to salt-loading in the diabetic kidney is important to prevent the exacerbation of diabetic kidney disease. An inflammatory response in diabetic db/db mice is associated with constitutively high ENaC activity even after salt-loading. During salt-loading, diabetic db/db mice release extracellular vesicles (EVs) into the urine during the active and inactive cycles that are enriched in bioactive molecules. A vast majority of these EVs are released across the luminal membrane of renal epithelial cells from each segment of the nephron and have the potential to module ENaC activity in recipient collecting duct cells. Our hypothesis was the distinct cargo that is enriched in EVs released during the inactive and active cycles of salt-loaded hypertensive diabetic db/db mice is responsible for increased ENaC membrane protein expression and activity in recipient collecting duct cells. In this study, we measured blood pressure by telemetry during the light (inactive) or dark (active) cycles. In addition, osmolality, electrolytes, and creatinine were measured and EV were isolated from urine samples collected during both cycles. Western blotting and densitometric analysis showed greater ENaC alpha protein expression in membrane fractions of mouse cortical collecting duct (mpkCCD) cells while single-channel patch clamp studies show greater ENaC activity in mpkCCD cells treated with uEVs isolated from the active cycle compared to the inactive cycle of hypertensive diabetic db/db mice. Various proteins and metabolites were found to be enriched in each of the two groups of EVs. Taken together, these data suggest the packaged cargo within EVs released after salt-loading diabetic db/db mice changes over a 24 hour cycle, and various molecules enriched in active cycle EVs contribute to the upregulation of renal ENaC protein expression and activity. Disclosure L.Yu: None. M.F.Gholam: None. A.A.Alli: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK123078-01A1 to A.A.A.)