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Na+‐K+‐2Cl‐ cotransporter type 2 (NKCC2) isoform A regulates NFAT5 in medullary thick ascending limb (mTAL) cells
Author(s) -
Hao shoujin,
Zhao Hong,
Darzynkiewicz Zbigniew,
Battula Sailaja,
Ferreri Nicholas R.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1041.36
Subject(s) - cotransporter , medullary cavity , gene isoform , chemistry , microbiology and biotechnology , endocrinology , medicine , biology , anatomy , gene , biochemistry , sodium , organic chemistry
We postulate that NKCC2A is essential for the upregulation of nuclear factor of activated T cells isoform 5 (NFAT5) in mTAL cells exposed to high NaCl concentration. Primary cultures of mouse mTAL cells and freshly isolated mTAL tubules, both derived from outer medulla (outer stripe>inner stripe), express NKCC2 isoforms A and F. The abundance of NKCC2A, but not NKCC2F, mRNA increased approximately two‐fold when mTAL cells were exposed to an increase in osmolality from 300 to 500 mosmol/kg H 2 O, produced with NaCl. Subsequent to the increase in NKCC2A mRNA, the abundance of NFAT5 mRNA and endogenous NFAT5 protein expression increased approximately 2.5‐ and 2‐fold, respectively, after cells were exposed to 500 mosmol/kg H 2 O. Increases in NFAT5 mRNA accumulation and protein expression were abolished after mTAL cells transiently transfected with an shRNA vector targeting NKCC2A (U6‐N2A‐ex4), but not NKCC2F (U6‐N2F‐ex4), were exposed to hypertonic stress. NFAT5 transcriptional activity in response to high NaCl was dramatically reduced in mTAL cells transfected with U6‐N2A‐ex4, and partially reduced after transfection with U6‐N2F‐ex4 or NKCC2A‐to‐F mutant constructs, T216V, T226M, and A230C. Collectively, the data suggest that NKCC2A is a critical component of the signaling pathway required for NFAT5 mRNA accumulation, protein expression, and transcriptional activity in response to high NaCl concentration.