MicroRNAs (miRNAs) are non-coding RNAs that bind target mRNA transcripts and modulate gene expression. In the cortical collecting duct (CCD), aldosterone stimulates the expression of genes that increase activity of the epithelial sodium channel (ENaC); in the early phase of aldosterone induction, one such gene is serum and glucocorticoid regulated kinase 1 (SGK1). We hypothesized that aldosterone regulates the expression of miRNAs in the early phase of induction to control the expression of target genes that stimulate ENaC activity. We treated mpkCCDc14 cells with aldosterone or vehicle for 1 hour and used a miRNA microarray to analyze differential miRNA expression. We identified miR-466g as a miRNA that was decreased by 57% after 1 hour of aldosterone treatment. Moreover, we identified a putative miR-466g binding site in the 3'-UTR of SGK1. We constructed an SGK1 3'-UTR luciferase reporter and found that co-transfection of miR-466g suppressed luciferase activity in HEK293 cells in a dose dependent manner. Deletion or introduction of point mutations that disrupt the miR-466g target site attenuated miR-466g-directed suppression of luciferase activity. Finally, we generated stably transduced mpkCCDc14 cell lines over-expressing miR-466g. Cells over-expressing miR-466g demonstrated 12.9 fold lower level of SGK1 mRNA compared to control cells after 6 hours of aldosterone induction; moreover, cells over-expressing miR-466g exhibited 25% decrease in amiloride-sensitive current after 6 hours of aldosterone induction and complete loss of amiloride-sensitive current after 24 hours of aldosterone induction. Our findings implicate miR-466g as a novel aldosterone responsive miRNA that regulates SGK1 and ENaC in CCD cells.
- cortical collecting duct
- Copyright © 2016, American Journal of Physiology - Renal Physiology