Expression and function of NFAT5 in medullary thick ascending limb (mTAL) cells

Abstract

The contribution of nuclear factor of activated T cells 5 (NFAT5) to the regulation of tumor necrosis factor-alpha (TNF) production in medullary thick ascending limb (mTAL) cells is unclear. RT-PCR analysis was performed on primary cultures of mouse mTAL cells and freshly isolated mTAL tubules to determine which NFAT isoforms are present in this nephron segment. Primer pairs were designed, based on published sequences for mouse NFAT1-5, to produce fragments of approximately 200 bp. Analysis of PCR products by gel electrophoresis and subsequent DNA sequencing indicated that cells and tubules contained mRNA for all five NFAT isoforms. The relative expression of NFAT isoforms was then determined using quantitative real-time RT-PCR. The data indicate that NFAT isoforms 5 >/= 1 are the predominant isoforms present in mTAL cells and tubules. Western blot analysis demonstrated constitutive expression of NFAT5 in nuclear extracts from mTAL tubules and primary culture cells; expression in mTAL cells also was detected by immunofluorescence. Expression of NFAT5 was increased in mTAL cells transiently transfected with an NFAT5 overexpression vector (pcDNA3.1-NFAT5), resulting in increased basal and calcium-sensing receptor (CaR)-mediated TNF production. Transient transfection of mTAL cells with a small hairpin RNA vector that targeted exon 8 of NFAT5 (U6-N5 ex8) significantly inhibited TNF promoter activity. Transient transfection with U6-N5 ex8 also reduced nuclear expression of NFAT5, TNF mRNA accumulation, and attenuated CaR-mediated activation of Cl(-) entry into polarized mTAL cells. Collectively, these data suggest that activation of NFAT5 is part of a TNF-dependent pathway that inhibits apical Cl(-) influx in the mTAL after activation of CaR.

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

“…We previously showed that NFAT5 is involved in calcium sensing receptor-dependent TNF production in mTAL cells (22). As NKCC2A is part of a pathway that regulates NFAT5 in cultured mTAL cells (23) and in vivo (Fig.…”

“…All constructs were generated using standard cloning procedures and verified by restriction enzyme analysis and DNA sequencing. A NFAT5-dominant negative (NFAT5-DN) expression plasmid was generated by cloning a NFAT5 cDNA fragment containing the NFAT5 DNA binding domain (DBD) into pcDNA3.1 vector (Invitrogen); a cDNA (nucleotides 1074 -1694) encoding amino acids 175-471 (NM_018823.1) was used in this study (22). The cDNA was amplified from the pBluescript SK ϩ vector containing full-length NFAT5 using restriction enzymes BamHI and XhoI and ligated into pcDNA3.1 vector.…”

“…The medium was then removed, and the cells were cultured for an additional 12-48 h in REGM containing 10% FBS. Transfection efficiency was evaluated by flow cytometry as described previously (22).…”

“…We recently identified NFAT5 as a predominant NFAT isoform in the mTAL and showed it is integral to TNF gene transcription induced by activation of the calcium sensing receptor (22). We also showed that NKCC2 isoform A (NKCC2A) contributes to the regulation of NFAT5 in primary cultures of mTAL cells exposed to elevated NaCl concentration (23).…”

“…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). …”