Differential regulation of NFAT 5 by NKCC 2 isoforms in medullary thick ascending limb 3 ( mTAL ) cells 4 5

26 27 The effects of Na-K-2Cl cotransporter type 2 (NKCC2) isoforms on the regulation of nuclear 28 factor of activated T cells isoform 5 (NFAT5) was determined in mouse medullary thick ascending 29 limb (mTAL) cells exposed to high NaCl concentration. Primary cultures of mTAL cells and freshly 30 isolated mTAL tubules, both derived from outer medulla (outer stripe>inner stripe), express NKCC2 31 isoforms A and F. The relative expression of NKCC2A mRNA was approximately two-fold greater 32 than NKCC2F in these preparations. The abundance of NKCC2A mRNA, but not NKCC2F mRNA, 33 increased approximately two-fold when mTAL cells were exposed for 2 hr to a change in osmolality 34 from 300 to 500 mosmol/kg H2O, produced with NaCl. Total NKCC2 protein expression also 35 increased. Moreover, a 2.5-fold increase in NFAT5 mRNA accumulation was observed after cells 36 were exposed to 500 mosmol/kg H2O for 4 hr. Laser scanning cytometry (LSC) detected a two-fold 37 increase in endogenous NFAT5 protein expression in response to high NaCl concentration. 38 Pretreatment with the loop diuretic bumetanide dramatically reduced transcriptional activity of the 39 NFAT5-specific reporter construct, TonE-Luc, in mTAL cells exposed to high NaCl. Transient 40 transfection of mTAL cells with shRNA vectors targeting NKCC2A prevented increases in NFAT5 41 mRNA abundance and protein expression, and inhibited NFAT5 transcriptional activity in response to 42 hypertonic stress. Silencing of NKCC2F mRNA did not affect NFAT5 mRNA accumulation but 43 partially inhibited NFAT5 transcriptional activity. These findings suggest that NKCC2A and 44 NKCC2F exhibit differential effects on NFAT5 expression and transcriptional activity in response to 45 hypertonicity produced by high NaCl concentration. 46 47 48