Small Extracellular Vesicles with a High Sphingomyelin Content Isolated from Hypertensive Diabetic db/db Mice Inhibits Calcium Mobilization and Augments Amiloride-Sensitive Epithelial Sodium Channel Activity.

Extracellular vesicles (EVs) contain bioactive lipids that play a key role in pathophysiology. We hypothesized that EVs released from salt-loaded hypertensive diabetic db/db mice have increased bioactive lipid content that inhibits intracellular calcium mobilization and increases the activity of renal epithelial sodium channels (ENaC). An enrichment of sphingomyelins (SMs) was found in small urinary EVs (uEVs) isolated from salt-loaded hypertensive diabetic db/db mice (n = 4) compared to non-salt loaded db/db mice with diabetes alone (n = 4). Both groups of mice were included in the same cohort to control for variability. Cultured mouse cortical collecting duct (mpkCCD) cells loaded with a calcium reporter dye and challenged with small uEVs from hypertensive diabetic db/db mice showed a decrease in calcium mobilization when compared to cells treated with small uEVs from diabetic db/db mice. The amiloride-sensitive transepithelial current was increased in mpkCCD cells treated with small uEVs with abundant sphingomyelin content from hypertensive diabetic db/db mice in a dose- and time-dependent manner. Similar results were observed in mpkCCD cells and Xenopus 2F3 cells treated with exogenous sphingomyelin in a time-dependent manner. Single-channel patch clamp studies showed a decrease in ENaC activity in cells transiently transfected with sphingomyelin synthase 1/2 specific siRNA compared to non-targeting siRNA. These data suggest EVs with high sphingomyelin content positively regulate renal ENaC activity in a mechanism involving an inhibition of calcium mobilization.