MicroRNA‐195 regulates blood pressure by inhibiting NKCC2A

Abstract

We previously showed that inhibition of renal tumor necrosis factor‐alpha (TNF) production induced by high salt intake increases Na+‐K+‐2Cl−cotransporter isoform A (NKCC2A) mRNA, protein, and phospho (p)NKCC2 expression. MicroRNA (miRNA) contributes to blood pressure regulation, however no studies have addressed the regulation of NKCC2 by miRNA. miR‐195 is one of the most abundant miRNAs expressed in the kidney and qRT‐PCR experiments showed that injection of TNF (5ng/g bw) directly into the kidney of male mice up‐regulates miR‐195 in the renal outer medulla (OM) by approximately 3.5‐fold (p<0.05). Subsequently, a potential miR‐195 binding site in the 3′‐UTR of NKCC2A was identified using the prediction tool TargetScan v5.1, and luciferase reporter gene assays confirmed that NKCC2A mRNA is directly targeted and repressed by miR‐195. Primary cultures of medullary thick ascending limb (mTAL) cells and freshly isolated mTAL tubules express NKCC2 isoforms A and F. However, only miR‐195 and NKCC2A, but not NKCC2F mRNA, were up‐regulated in mTAL cells exposed for 2 h to a change in osmolality from 300 to 500 mOsmol/kgH₂O, produced with NaCl. Transient transfection of mTAL cells with an shRNA vector targeting TNF prevented increases in miR‐195 induced by high NaCl concentration. Moreover, transfection of a miR‐195 mimic into mTAL cells suppressed NKCC2A mRNA, but not NKCC2F mRNA, by approximately 83% (p<0.05) in cells exposed to high NaCl. In contrast, transfection with anti‐miR‐195 increased NKCC2A mRNA without altering NKCC2F mRNA abundance. Laser‐scanning cytometry detected a 1.5 fold (p<0.05) increase in pNKCC2 protein expression in mTAL cells transfected with anti‐miR‐195, and a miR‐195 mimic prevented the increase of pNKCC2 induced by silencing TNF in cells exposed to high NaCl. Next, we determined whether miRNA‐195 regulates HS‐dependent changes in blood pressure by inhibiting NKCC2A. Both TNF and miR‐195 expression increased in OM from mice given 1% NaCl in the drinking water (HS) for 7 days. Intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6‐TNF‐ex4) inhibited the expression of miR‐195 (p<0.05), while increasing NKCC2A mRNA expression in mice ingesting HS. However, intrarenal injection of murine recombinant TNF significantly upregulated the expression of miR‐195 and suppressed the increases of NKCC2A mRNA in mice ingesting HS. Intrarenal administration of miR‐195 prevented the increase of NKCC2A mRNA abundance in the OM from mice injected with the TNF silencing lentivirus (U6‐TNF‐ex4) compared with the control lentivirus. Moreover, the HS‐induced increase in blood pressure detected by radiotelemetry after renal silencing of TNF was markedly attenuated in mice given an intrarenal injection of miR‐195. Collectively, the study identifies miR‐195 as a salt‐sensitive and TNF inducible miRNA that inhibits HS‐mediated increases in blood pressure by inhibiting NKCC2A.