MicroRNA-195a-5p Regulates Blood Pressure by Inhibiting NKCC2A

Abstract

Background: Previous studies showed that miR-195a-5p was among the most abundant microRNAs (miRNAs) expressed in the kidney. Methods: Lentivirus silencing of tumor necrosis factor- (TNF) was performed in vivo and in vitro. Luciferase reporter assays confirmed that bumetanide-sensitive Na+-K+-2Cl-cotransporter isoform A (NKCC2A) mRNA is targeted and repressed by miR-195a-5p. Radiotelemetry was used to measure mean arterial pressure. Results: TNF upregulates mmu-miR-195a-5p, and -203 and downregulates mmu-miR-30c and -100 in the medullary thick ascending limb of male mice. miR-195a-5p was >3-fold higher in the renal outer medulla of mice given an intrarenal injection of murine recombinant TNF, whereas silencing TNF inhibited miR-195a-5p expression by ≈51%. Transient transfection of a miR-195a-5p mimic into medullary thick ascending limb cells suppressed NKCC2A mRNA by ≈83%, whereas transfection with Anti-miR-195a-5p increased NKCC2A mRNA. Silencing TNF in medullary thick ascending limb cells prevented increases in miR-195 induced by 400 mosmol/kg H2O medium, an effect reversed by transfection with a miR-195a-5p mimic. Expression of phosphorylated NKCC2 increased 1.5-fold in medullary thick ascending limb cells transfected with Anti-miR-195a-5p and a miR-195a-5p mimic prevented the increase, which was induced by silencing TNF in cells exposed to 400 mosmol/kg H2O medium after osmolality was increased by adding NaCl. Intrarenal injection of TNF suppressed NKCC2A mRNA, whereas injection of miR-195a-5p prevented the increase of NKCC2A mRNA abundance and phosphorylated NKCC2 expression when TNF was silenced. Intrarenal injection with miR-195a-5p markedly attenuated MAP after renal silencing of TNF in mice given 1% NaCl. Conclusions: The study identifies miR-195a-5p as a salt-sensitive and TNF-inducible miRNA that attenuates NaCl-mediated increases in blood pressure by inhibiting NKCC2A.