We showed that intrarenal suppression of TNF (tumor necrosis factor) production under low salt (LS) conditions increases renal cortical AGT (angiotensinogen) mRNA and protein expression. Intrarenal injection of murine recombinant TNF attenuated increases of AGT in mice ingesting LS. Moreover, AGT mRNA and protein expression increased ≈6-fold and 2-fold, respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6-TNF-ex4). Silencing of TNF under normal salt and high salt (HS) conditions also resulted in increased AGT expression. Since renal TNF production decreases in response to LS and increases in response to HS, the data suggest that alterations in TNF production under these conditions modulate the degree of AGT expression. We also tested the hypothesis that TNF inhibits intrarenal AGT expression by a mechanism involving miR-133a. Expression of miR-133a decreased in mice given LS and increased in response to HS for 7 days. Intrarenal silencing of TNF reversed the effects of HS on miR-133a-dependent AGT expression. In contrast, intrarenal TNF administration increased miR-133a expression in the kidney. Collectively, the data suggest that miR-133a is a salt-sensitive microRNA that inhibits AGT in the kidney and is increased by TNF. The HS-induced increase in blood pressure observed following silencing of TNF was markedly reduced upon intrarenal administration of miR-133a suggesting that intrinsic effects of TNF in the kidney to limit the blood pressure response to HS include an increase in miR-133a, which suppresses AGT expression.

We have previously shown that TNF-α 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 high NaCl intake. As the role of TNF-α in the kidney is still being defined, the effects of low salt intake on TNF-α and NKCC2B expression were determined. Mice given a low-salt (0.02% NaCl) diet (LSD) for 7 days exhibited a 62 ± 7.4% decrease in TNF-α mRNA accumulation in the renal cortex. Mice that ingested the LSD also exhibited an ~63% increase in phosphorylated NKCC2 expression in the cortical thick ascending limb of Henle’s loop and a concomitant threefold increase in NKCC2B mRNA abundance without a concurrent change in NKCC2A mRNA accumulation. NKCC2B mRNA levels increased fivefold in mice that ingested the LSD and also received an intrarenal injection of a lentivirus construct that specifically silenced TNF-α in the kidney (U6-TNF-ex4) compared with mice injected with control lentivirus. Administration of a single intrarenal injection of murine recombinant TNF-α (5 ng/g body wt) attenuated the increases of NKCC2B mRNA by ~50% and inhibited the increase in phosphorylated NKCC2 by ~54% in the renal cortex of mice given the LSD for 7 days. Renal silencing of TNF-α decreased urine volume and NaCl excretion in mice given the LSD, effects that were reversed when NKCC2B was silenced in the kidney. Collectively, these findings demonstrate that downregulation of renal TNF-α production in response to low-salt conditions contributes to the regulation of NaCl reabsorption via an NKCC2B-dependent mechanism.

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 diverse stimuli activate the thick ascending limb of Henle’s loop (TAL) to synthesize tumor necrosis factor-alpha (TNF), which subsequently acts as an endogenous inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). Moreover, renal-specific silencing of TNF unmasks salt-dependent increases in blood pressure via a mechanism involving NKCC2A. The objective of the present study was to determine the effects of TNF on NKCC2 isoform and angiotensinogen (AGT) expression in the renal cortex under conditions of hypotonic stress. Mice given a low salt (0.02% NaCl) diet (LS) for 7 days exhibited a 62±7.4% decrease (p<0.5, n=4) in TNF mRNA expression in the renal cortex, compared with mice ingesting a normal salt diet (NSD), which was associated with a concomitant 4-fold increase in renal cortical AGT mRNA accumulation (p<0.05, n=4). Mice ingesting LS also exhibited about a 63% increase (p<0.05, n=4) in cortical TAL phospho-NKCC2 (pNKCC2) expression and a 3-fold increase in NKCC2B mRNA abundance without a concurrent change in NKCC2A mRNA accumulation. The increases in AGT and NKCC2B mRNA abundance were increased by 6-fold (p<0.05, n=4 ) and 5-fold (p<0.05, n=4), respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct designed to specifically silence TNF in the kidney (U6-TNF-ex4) compared with mice injected with control lentivirus (U6). The effects of a single intrarenal injection of murine recombinant TNF (5ng/g body weight) or saline control for 24 hr on renal AGT and NKCC2 mRNA levels were then determined in mice that ingested LS for 7 days. Administration of TNF inhibited the increase of AGT and NKCC2B mRNA abundance by approximately 42±5.9% and 49±6.5% respectively, p<0.05, n=4 ) in mice exposed to hypotonic stress. Similarly, intrarenal injection of TNF inhibited the increase in pNKCC2 by approximately 54% (p<0.05, n=4 ) in renal cortex from mice given LS for 7 days. Collectively, these findings suggest that downregulation of renal TNF production in response to hypotonic conditions contributes to the regulation of sodium chloride reabsorption via adaptive increases in AGT as well as a selective effect on NKCC2B, which is exclusively expressed by macula densa cells in the renal cortex.