Intrarenal silencing of TNF facilitates salt‐dependent increases in blood pressure

Abstract

We previously showed that the thick ascending limb (TAL) produces tumor necrosis factor‐alpha (TNF) in response to diverse stimuli and acts as an endogenous inhibitor of NKCC2. The effect of renal TNF production on blood pressure and adaptation to high NaCl (HS) intake was determined in this study. We designed lentivirus constructs suitable for intrarenal administration and silencing of TNF and the NKCC2A isoform. Silencing of TNF was validated in primary cultures of mTAL cells challenged with high NaCl concentrations. For in vivo silencing of TNF purified U6‐TNF‐ex4, or control lentivirus (U6), was injected directly into each kidney. Three days later qRT‐PCR analysis showed that TNF mRNA was markedly reduced in kidney but not spleen. Accordingly, we administered 1% NaCl in the drinking water to study the intrinsic effects of TNF produced within the kidney on blood pressure regulation under normal and high salt conditions. Baseline SBP was similar in mice ingesting tap water and a normal salt diet (NSD) that were given control lentivirus (U6) and U6‐TNF‐ex4. However, silencing TNF increased SBP (127.9±4.3 mmHg) when mice were switched to 1% NaCl for 3 days compared with mice injected with U6 control (117.6±3.2 mmHg). The increases in blood pressure, which were continuously observed for 7 days and were detected concomitantly with an increase in renal but not systemic TNF production, promptly returned to baseline levels when mice were switched from 1% NaCl to tap water. Renal silencing of TNF also induced a specific 2‐fold increase in NKCC2A mRNA levels while NKCC2B and NKCC2F levels were unchanged. Analysis of daytime and nighttime periods by radiotelemetry showed that silencing of TNF not only increased MAP (108.2±4.1mmHg) compared with control (96.4±2.3 mmHg) during daytime but also increased MAP (118.7±5.0 mmHg) compared with control (107.0±2.9 mmHg) during nighttime in mice ingesting 1% NaCl. Knockdown of TNF in the kidney did not disturb the diurnal regulation of blood pressure but caused a further sensitivity to NaCl‐induced increases in blood pressure in mice with 1 kidney. Importantly, the NaCl‐mediated increases in blood pressure were completely absent when NKCC2A and TNF were concomitantly silenced in the kidney. Additionally, studies were performed using mice placed in metabolic cages that received intrarenal injections of control (U6), U6‐TNF‐ex4, or a mixture of U6‐TNF‐ex4 and U6‐N2A‐ex4. Renal silencing of TNF elicited a decrease in urine volume, sodium, and chloride excretion, while potassium excretion increased compared with mice that received injections of control lentivirus. The diuresis and natriuresis observed in response to renal TNF silencing was abolished when NKCC2A was concurrently silenced suggesting this isoform contributes to the transition from a salt‐resistant to salt‐sensitive phenotype. The data indicate that the effects of renal TNF silencing and the subsequent increase in NKCC2A set in motion changes in transporters that increase diuresis and natriuresis. Collectively, the increase in blood pressure in response to HS when TNF is silenced in the kidney suggests that deficiency of locally derived TNF production is sufficient to render normotensive healthy mice salt‐sensitive as well as alter electrolyte excretion.Support or Funding InformationThis work was supported by grants from NIH R01 HL133077, NYMC/Touro Bridge grant, and the George M. O'Brien Kidney Center at Yale, NIH P30 DK079310.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.