Hypertonic NaCl intake induces renal TNF production by a pathway involving NKCC2A and NFAT5

Abstract

TNF is produced by the medullary thick ascending limb (mTAL), however, the effect of hypertonicity on production of this cytokine has not been determined. Mice given 1% NaCl in the drinking water for 3 days exhibited a two‐fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. A concomitant 3‐fold increase in NKCC2A and 2‐fold increase in NFAT5 mRNA accumulation was observed in mTAL tubules from mice given 1% NaCl. Increases in urinary TNF levels were abolished after treatment with furosemide or intrarenal injection of a lentivirus shRNA construct that knocked down NKCC2A (EGFP‐N2A‐ex4); plasma levels of TNF did not change. NFAT5 mRNA abundance also decreased after injection of EGFP‐N2A‐ex4. TNF production by primary cultures of mTAL cells increased approximately 6‐fold in response to high NaCl concentration (400 mosmol/kg H2O) and was inhibited in cells treated with bumetanide or transiently transfected with a dnNFAT5 construct (3.0 ± 0.9 to 0.9±0.2 pg/μg protein; p<0.05). Transduction of mTAL cells with EGFP‐N2A‐ex4 prevented increases in TNF production in response to high NaCl concentration and reduced transcriptional activity of an NFAT5 promoter construct by more than 60%. Since NKCC2A expression is restricted to the TAL segment of the nephron, NKCC2A‐dependent activation of NFAT5 is part of a pathway contributing to TNF production by the TAL in response to hypertonic NaCl intake.