TNF inhibits AQP2 expression via a miR137-dependent pathway

We previously showed that production of tumor necrosis factor-alpha (TNF) by the kidney, under normotensive, non-inflammatory conditions, inhibits expression of angiotensinogen in the proximal tubule and NKCC2 isoform A in the thick ascending limb of Henle’s loop via micro-RNA (miRNA)-dependent mechanisms. As miR-137 is a regulator of aquaporin-2 (AQP2) expression and TNF inhibits the expression of several extrarenal aquaporins, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. Target seed regions of mmu-miR-137 are conserved in the 3'-untranslated region (3’-UTR) of mouse AQP2 mRNA and luciferase assays showed that mmu-miR-137 directly targeted the 3’-UTR of mouse AQP2. Moreover, AQP2 expression was decreased by approximately 70% (P<0.05) in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, indicating that miR-137 directly targets AQP2 mRNA in these cells. The decrease in AQP2 mRNA accumulation was accompanied by a 53% decrease (P<0.05) in AQP2 protein expression when mmu-miR-137 was overexpressed in IMCD cells. Exposure of IMCD cells for 2 hr to 400 mosmol/kg H2O medium, made by increasing the osmolality by adding NaCl, increased mmu-miR-137 mRNA expression about 2-fold (P<0.05) as determined by quantitative RT-PCR analysis. These conditions also increased TNF production approximately 2-fold (P<0.05). To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, cells were transfected with either a lentivirus construct to silence TNF or a control construct. The silencing construct, EGFP-shTNF-ex4, but not U6 control, decreased mmu-miR-137 mRNA expression in the IMCD cells exposed to 400 mosmol/kg H2O medium by approximately 63% (P<0.05) indicating that TNF upregulates mmu-miR-137 mRNA expression in IMCD cells. The levels of miR-137 also increased approximately 2-fold (P<0.05) in inner medulla isolated from age- and sex-matched male or female mice given 1% NaCl in the drinking water (HS) for 3 days. Six days after intrarenal injection of lentivirus EGFP-shTNF-ex4 to silence TNF, renal AQP2 mRNA levels increased approximately 3-fold (P<0.05) in inner medullary tissue isolated from male or female mice given HS compared with their respective control groups. In addition, renal silencing of TNF in male or female mice given HS exhibited an increase in AQP2 protein expression the inner medulla compared with control group as determined by Western blot analysis. Similarly, EGFP-shTNF-ex4, but not U6, decreased miR-137 levels in the inner medulla in male or female mice given HS. Taken together, these data reveal a novel mechanism by which TNF mediates the inhibition of AQP2 via miR-137, indicating that miR-137 may be important for studying TNF-regulated effects involving the AQP2 pathway on renal water excretion and blood pressure regulation. NIH grants R01 HL133077 and HL153525 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.