BACKGROUND: Previous studies showed that miR-195a-5p was among the most abundant miRNAs expressed in the kidney. METHODS: Lentivirus silencing of tumor necrosis factor-α (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 H 2 O 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 H 2 O 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.

We previously showed that TNF produced by renal epithelial cells inhibits NKCC2 activity as part of a mechanism that attenuates increases in blood pressure in response to increases in sodium intake. We also showed that ambient urine osmolality was higher in TNF deficient mice compared with age‐ and sex‐matched wild type (WT) mice. As the role of TNF in the kidney is still being defined, the effect of TNF produced by renal epithelial cells on aquaporin‐2 (AQP2) expression was determined in this study. TNF levels were higher in urine, but not plasma, of mice that ingested 1% NaCl (HS) in the drinking water for three days compared with tap water (40±4 vs 18±5 pg/24 h). The increase in urinary TNF levels was associated with an approximately 5‐fold increase in TNF mRNA abundance in the inner medulla excised from mice given HS. Interestingly, we observed that miR‐137 levels also increased approximately 2‐fold in the inner medulla isolated from mice given HS. Six days after intrarenal injection of a lentivirus construct designed to silence TNF, EGFP‐shTNF‐ex4, renal but not splenic TNF mRNA levels were significantly reduced. Injection of control lentivirus (U6) did not alter TNF mRNA in either kidney or spleen. RT‐PCR analysis showed that AQP2 mRNA levels were significantly decreased in the inner medullary tissue from mice given HS. However, renal silencing of TNF in mice given HS exhibited a 3‐fold increase in AQP2 mRNA and protein expression in the inner medulla compared with WT. In contrast, EGFP‐shTNF‐ex4 but not U6 decreased miR‐137 levels in the inner medulla in mice given HS. Moreover, target seed regions of miR‐137 were conserved in the 3′‐untranslated region of mouse AQP2 mRNA. Accordingly, intrarenal injection of miR‐137 inhibited the increase of AQP2 mRNA abundance in the inner medulla induced by silencing TNF in the kidneys of mice given HS. As TNF production in the kidney was induced by administration of HS for 3 days, the effects of TNF silencing on urine volume were determined. Mice were acclimated in metabolic cages for 3 days after receiving intrarenal injections of either EGFP‐shTNF‐ex4 or U6. Food, body weight, water intake, and urine output was then measured in mice given HS or tap water for 3 days. Both water intake and urine output were reduced in mice injected with EGFP‐shTNF‐ex4 compared with mice given U6 lentivirus. The decrease in urine volume was associated with an increase in AQP2 mRNA accumulation. Collectively, these findings suggest that the intrinsic effects of TNF produced within the kidney in response to HS intake include an inhibitory effect on AQP2 expression that is mediated via a mechanism involving miR‐137. Support or Funding Information NIH(HL133077) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.