Renal miR‐195 mediates TNF‐dependent inhibition of NKCC2

Abstract

We previously showed that tumor necrosis factor‐alpha (TNF) produced by renal epithelial cells inhibits Na+‐K+‐2Cl− cotransporter (NKCC2) activity as part of a mechanism that attenuates increases in blood pressure in response to elevated in sodium intake. However, the mechanisms by which TNF inhibits NKCC2 isoform expression and activity have not been studied. Previous miRNA array studies have found that miR‐195 is one of the most abundant microRNAs expressed in the kidney. In the present study, we demonstrate by qRT‐PCR that TNF up‐regulates mmu‐miR‐146a, ‐195, and ‐203 and down‐regulates mmu‐miR‐30c and ‐100 in the medullary thick ascending limb (mTAL). Most notably, qRT‐PCR analysis indicated that the level of miR‐195 was 4‐fold higher in mTAL tubules isolated from the renal medulla of male mice given a single intrarenal injection of murine recombinant TNF (5ng/g body weight) compared with the control group (saline injection). Data obtained using a TAL‐specific TNF knockout (KO) mouse model showed that miR‐195 abundance in the mTAL tubules from these mice was significantly decreased by 58% (P=0.04, n=4) compared with control mice (floxed TNF littermate controls) in response to ingestion of high salt (HS; 1% NaCl in drinking water) for 3 days. Concomitantly, NKCC2A isoform mRNA abundance was increased 3‐fold while phospho (p)NKCC2 expression increased approximately 64% in the renal medulla of the TAL‐specific TNF KO mice compared with littermate control mice. Collectively, the data suggest that TNF‐derived from the TAL in response to HS intake is an important inducer of miR‐195 levels. Using the prediction tool TargetScan v5.1, we identified a potential miR‐195 binding site in the 3′‐UTR of NKCC2A. Accordingly, intrarenal injection of miR‐195 inhibited the HS‐mediated increases in NKCC2A mRNA and concurrent pNKCC2 expression by 81% and 59%, respectively, in the renal medulla from TAL‐specific TNF KO mice. Consistent with the in vivo effects of TNF on miR‐195, treatment of primary cultures of mouse mTAL cells with 1 nM TNF for 4 hr increased miR‐195 expression more than 3‐fold (P=0.01; n=4). Moreover, NKCC2A mRNA and pNKCC2 expression was inhibited by 76% (P=0.02; n=4) and 61% (P=0.01; n=4), respectively, in mTAL cells exposed to hypertonic conditions (400 mOsmol/kg H2O) following cotreatment with miR‐195 and a short hairpin (sh)TNF silencing lentivirus construct. Silencing of TNFR1 in mTAL cells with a siRNA oligonucleotide inhibited the TNF‐mediated increase of miR‐195 expression and prevented the inhibitory effects of TNF by 73% (P=0.03; n=4), as well as suppressing the increase in pNKCC2 expression. These data identify miR‐195 as an important downstream target of TNFR1‐dependent signaling, which suppresses NKCC2A and pNKCC2 expression in mTAL cells. In summary, the present findings uncover a novel physiological mechanism in which miR‐195 induction via activation of TNFR1‐dependent signaling suppresses NKCC2 expression and activity, suggesting that miR‐195 expression may contribute to the regulation of salt and water excretion and blood pressure via modulation of NKCC2A isoform expression and activity.Support or Funding InformationThis work was supported by NIH grant R01HL133077