NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake

Abstract

Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving the Na ϩ -K ϩ -2Cl Ϫ cotransporter (NKCC2). A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in nuclear factor of activated T cells 5 (NFAT5) mRNA and protein expression was observed in the outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in the outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately sixfold in response to an increase in osmolality to 400 mosmol/kgH2O produced with NaCl and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake. TNF; NFAT5; Ton/EBP; NKCC2; thick ascending limb; hypertonic stress TUMOR NECROSIS FACTOR-␣ (TNF) is produced by several cell types along the nephron including the medullary thick ascending limb of Henle's loop (mTAL), which reabsorbs ϳ25% of filtered NaCl and is the site of action for loop diuretics (12, 41). TNF acts in an autocrine manner to inhibit in vitro correlates of Na ϩ reabsorption by a mechanism involving induction of cyclooxygenase-2 (COX-2), and the contribution of this cytokine to renal function is still being defined (17). For instance, recent studies have shown that TNF exhibits natriuretic effects, exerts a tonic inhibitory effect on the Na ϩ -K ϩ -2Cl Ϫ cotransporter (NKCC2), and inhibits endothelial nitric oxide synthase expression in the TAL (2,50,53,54). The mTAL produces TNF in response to several stimuli; however, knowledge regarding the pathways that contribute to TNF production by the kidney is limited (1,12,18,22,41,59).Renal medullary tonicity modulates regulatory systems that control tubular and microcirculatory function in the kidney; the molecules involved in these processes are still being explored (51). The rate of NaCl transport in the TAL is an important determinant of medullary hypertonicity, and the majority of this transport occurs via NKCC2 a 12-transmembrane-helix transport protein expressed exclusively...

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NFAT5 Is Protective Against Ischemic Acute Kidney Injury

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MAD2B-mediated SnoN downregulation is implicated in fibroblast activation and tubulointerstitial fibrosis

“…Two days before UUO model establishment, shRNA-MAD2B or their negative controls (scrambled) were delivered into the mice kidney by means of intraparenchymal injections. The procedure for intraparenchymal lentivirus delivery was performed as described before (9,11). Briefly, after anesthesia the renal pedicle of the mice was temporarily occluded, and then a 31-G needle was inserted from the lower pole of the kidney parallel to the long axis and was carefully pushed toward the upper pole.…”

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