Abstract P218: Downregulation of Renal Tumor Necrosis Factor-Alpha Production in Response to Hypotonic Stress Facilitates Adaptive Increases in Renal Cortical Angiotensinogen and Nkcc2b

Abstract

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.