Renal miR‐195 mediates TNF‐dependent inhibition of NKCC2

Abstract

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.