title: Differential regulation of NFAT5 by NKCC2 isoforms in medullary thick ascending limb (mTAL) cells.
abstract: The effects of Na(+)-K(+)-2Cl(-) cotransporter type 2 (NKCC2) isoforms on the regulation of nuclear factor of activated T cells isoform 5 (NFAT5) were determined in mouse medullary thick ascending limb (mTAL) cells exposed to high NaCl concentration. Primary cultures of mTAL cells and freshly isolated mTAL tubules, both derived from the outer medulla (outer stripe>inner stripe), express NKCC2 isoforms A and F. The relative expression of NKCC2A mRNA was approximately twofold greater than NKCC2F in these preparations. The abundance of NKCC2A mRNA, but not NKCC2F mRNA, increased approximately twofold when mTAL cells were exposed for 2 h to a change in osmolality from 300 to 500 mosmol/kgH₂O, produced with NaCl. Total NKCC2 protein expression also increased. Moreover, a 2.5-fold increase in NFAT5 mRNA accumulation was observed after cells were exposed to 500 mosmol/kgH₂O for 4 h. Laser-scanning cytometry detected a twofold increase in endogenous NFAT5 protein expression in response to high NaCl concentration. Pretreatment with the loop diuretic bumetanide dramatically reduced transcriptional activity of the NFAT5-specific reporter construct TonE-Luc in mTAL cells exposed to high NaCl. Transient transfection of mTAL cells with shRNA vectors targeting NKCC2A prevented increases in NFAT5 mRNA abundance and protein expression and inhibited NFAT5 transcriptional activity in response to hypertonic stress. Silencing of NKCC2F mRNA did not affect NFAT5 mRNA accumulation but partially inhibited NFAT5 transcriptional activity. These findings suggest that NKCC2A and NKCC2F exhibit differential effects on NFAT5 expression and transcriptional activity in response to hypertonicity produced by high NaCl concentration.
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, New York, USA.
authors.forename: Shoujin
authors.initials: S
authors.lastname: Hao
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
authors.forename: Sailaja
authors.initials: S
authors.lastname: Battula
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Ferreri
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D007679:Kidney Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008138:Loop of Henle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1
chemical_list: D050778:NFATC Transcription Factors; D020033:Protein Isoforms; D012333:RNA, Messenger; C578715:Slc12a1 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D012965:Sodium Chloride