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.

nfat5 or tonicity-responsive enchancer/osmotic-response element-binding protein (TonEBP/OREBP), a transcription factor crucial for cellular responses to hypertonic stress (33, 38), is expressed in the kidney and contributes to induction of genes that increase the accumulation of organic osmolytes, which protect cells against damage in a hypertonic environment (2). For instance, high concentrations of NaCl stimulate nuclear factor of activated T cells isoform 5 (NFAT5), which increases transcription of several genes important for the function of the renal medulla (2, 14, 29, 32) including those encoding transporters and biosynthetic enzymes for cellular accumulation of organic osmolytes: sodium-dependent myo-inositol transporter (SMIT), Na-Cl-betaine cotransporter, Na-Cl-taurine cotransporter, aldose reductase, which converts glucose into sorbitol, and an esterase that produces glycerophosphocholine from phosphatidylcholine. The importance of this mechanism is underscored by the severe atrophy in the renal medulla of NFAT5-deficient mice, which are unable adapt to hypertonicity (32).

Limited direct information is available regarding the regulation and function of NFAT5 in epithelial cells from the medullary thick ascending limb (mTAL) (22), although the importance of NFAT5 in the outer medulla of the kidney is evident from several studies (2, 14, 29, 37). The majority of NaCl uptake (∼75%) across the apical membrane of the mTAL occurs by a cotransport process in which the influx of Na⁺ drives the uptake of Cl⁻ and K⁺ (7, 23). This process is mediated by NKCC2 (also termed mBSC1 for bumetanide-sensitive cotransporter) (18, 41). NKCC2 loss-of-function mutations lead to a severe form of salt wasting in neonates, termed Bartter's syndrome (49), and disruption of the NKCC2 gene by homologous recombination in mice causes a similar syndrome (50). Immunolocalization experiments revealed that NKCC2 and NFAT5 are sequentially expressed in the kidney during development (30). In addition, inhibition of NKCC2 activity by furosemide dramatically reduced expression of NFAT5 and its target genes (48).

Slc12a1, the gene encoding for NKCC2 (18, 24), gives rise to several NKCC2 transcripts derived from differential splicing (45, 52). Three major NKCC2 isoforms, NKCC2A, NKCC2B, and NKCC2F, exhibit subtle exon sequence differences, are spatially distributed along the TAL and display distinct transport characteristics (7, 21, 40, 47). NKCC2A has effects on the concentrating and diluting ability of the kidney, and, in its absence reduced Cl⁻ absorption was observed in the context of high Cl⁻ concentrations (7, 43). Thus an important role for NKCC2A is postulated in the mTAL during conditions where NaCl concentrations are elevated. In this study, we determined the contributions of NKCC2A and NKCC2F to the expression and activity of NFAT5 in mTAL cells exposed to hypertonicity induced by an elevated concentration of NaCl. NFAT5, in addition to its function as a regulator of genes for organic osmolytes in the outer medulla of the kidney, is required for TNF gene transcription in the mTAL (1, 22). Consideration of the interaction between these molecules will be crucial to understanding novel mechanisms that participate in physiological regulatory pathways and protective mechanisms in the TAL.

MATERIALS AND METHODS

Animals.

Male C57BL/6J mice (8–12 wk) purchased from Jackson Laboratory were maintained on a standard diet and given tap water ad libitum. Experimental procedures were conducted in accordance with institutional and international guidelines for the welfare of animals (animal welfare assurance no. A3362-01, Office of Laboratory Animal Welfare, Public Health Service, National Institutes of Health).

Chemicals and reagents.

Unless otherwise noted, all chemicals were of the highest grade commercially available. An anti-NFAT5 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a 1:1,000 dilution for immunoblot analysis. The antibody for NKCC2 (Chemicon) was used at a 1:3,000 dilution; the antibody for Tamm-Horsfall glycoprotein (THP; AbD Serotec) was used at a 1:1,000 dilution. Tissue culture media was obtained from Life Technologies (Grand Island, NY). Collagenase (type 1A) was from Sigma (St. Louis, MO), and polyvinylidene difluoride (PVDF) membranes were obtained from Amersham (Arlington Heights, IL). The luciferase assay kit was from Promega (Madison, WI).

Isolation of mTAL tubules and cells.

mTAL tubules and cells (>98% purity, see Fig. 1A) were isolated in a manner similar to that previously described (5, 12, 36). Briefly, male C57BL/6J mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (0.065 mg/10 g body wt). The kidneys were perfused with sterile 0.9% saline via retrograde perfusion of the aorta, removed, and cut along the corticopapillary axis. The outer medulla was excised, minced with a sterile blade, and incubated for 10 min at 37°C in a 0.01% collagenase solution gassed with 95% oxygen. The suspension was sedimented on ice, mixed with Hanks' balanced salt solution (HBSS) containing 2% BSA, and the supernatant containing the crude suspension of tubules was collected. The collagenase digestion step was repeated three times; the remaining undigested tissue and the combined supernatants were centrifuged for 10 min, resuspended in HBSS, and filtered through a 52-μm nylon mesh membrane (Fisher Scientific, Springfield, NJ). The filtered solution was discarded, and tubules retained on the mesh were resuspended in HBSS and centrifuged at 500 rpm for 10 min; pelleted tubules were used in experiments, or to establish primary cultures of mouse mTAL cells. Cells were grown in six-well plates using renal epithelial cell basal medium (REBM; Cambrex), containing renal epithelial cell growth medium (REGM; Cambrex) consisting of rhEGF, insulin, hydrocortisone, GA-1000 (gentamycin sulfate and amphotericin B), FBS, epinephrine, T3 (triiodothronine), and transferrin. After 6–7 days, monolayers of cells were 70–80% confluent. The cells were quiesced for 24 h in RPMI containing 0.42 mM CaCl₂ and 0.5% FBS, l-glutamine (2 mM), 100 U/ml streptomycin/penicillin (GIBCO), MEM nonessential amino acids (GIBCO), MEM sodium pyruvate, and β-mercaptoethanol before their use (53).

Fig. 1.

Identification of Na⁺-K⁺-2Cl⁻ cotransporter type 2 (NKCC2) isoforms. A: medullary thick ascending limb (mTAL) cells (>98%) stained with anti-Tamm-Horsfall glycoprotein (THP) antibody (Alexa 488; green) and 4,6-diamidino-2-phenylindole (DAPI; blue) and excited sequentially at different wavelengths. Slides were examined using a Nikon Microphot FXA microscope equipped for epifluorescence illumination. B: cDNA fragments for NKCC2 isoforms were generated by RT-PCR and evaluated by separation on a 1% agarose gel stained with ethidium bromide. Panels represent total RNA from murine primary mTAL cells, whole kidney, cortex, and outer medulla. Lanes 1–3: cDNA fragments for NKCC2 isoforms A, B, and F, respectively; M, markers.

DNA preparation and plasmid constructs.

All constructs were generated using standard cloning procedures and verified by restriction enzyme analysis and DNA sequencing. U6-shRNA scrambled (U6) and targeting constructs for NKCC2A were designed in a manner similar to that used to engineer NFAT5 (U6-N5 ex8), as described previously (13, 22). PCRs for NKCC2A silencing and nonsilencing shRNA control employed a common primer for the 5′-end, forward: gcagaattcGATCCGACGCCGCCATCTCT; the 3′-end for scrambled nonsilencing shRNA was gcagctagcCTCGAGAAAAAAGAACGTTCGATAATGGATCCTACACAAAGATCCATTATCGAACGTTCAAACAAGGCTTTTCTCCAAGGGATA. The inhibitory construct for NKCC2A (U6-N2A ex4), which was cloned into pcDNA3.1, was designed by targeting exon 4 of the Slc12a1 gene; the NKCC2A reverse primer for the 3′-end was gcagctagcCTCGAGAAAAAACCCAGTGATAGAGGTTACCCTACACAAAGGTAACCTCTATCACTGGGAAACAAGGCTTTTCTCCAAGGGATA (43). Silencing of NKCC2A or NKCC2F mRNA also was accomplished using the lentiviral vector psiLv-U6 (GeneCopoeia). The target sequence of the inhibitory construct for NKCC2A (U6-N2A ex4) was GGTAACCTCTATCACTGGG; the target sequence of the inhibitory construct for NKCC2F (U6-N2F ex4) was GTGACAACACTCACAGGTA; both constructs were designed by targeting exon 4 of the Slc12a1 gene. The pTonE_Luc reporter (originally from Dr. Steffan N. Ho) (51) was kindly provided by Dr. Feng Cheng (Washington University, St. Louis, MO).

Gene transfection and transduction.

After murine mTAL cells were cultured to 70–80% confluence in six-well plates with membrane inserts (cell culture inserts, BD Biosciences) as indicated (11), the medium was removed and cells were placed in 1 ml of serum-free OPTI-MEM medium containing different plasmid DNA constructs and 10 μl lipofectamine reagent (Life Technologies) or Lipofectamine 2000 (Invitrogen) for 4 h at 37°C/5% CO₂. Flow cytometric analysis of mTAL cells revealed ∼60% transfection efficiency with pcDNA3.1 constructs (22). mTAL cells were transduced in 0.5 ml of serum-free OPTI-MEM medium for 4 h at 37°C/5% CO₂ with 20 μl of 1 × 10⁸ TU/ml containing lentivirus constructs to knock down NKCC2A (psiLV-U6-N2A ex4) or NKCC2F (psiLV-U6-N2F ex4) mRNA (GeneCopoeia). Following the transduction period, 1.5 ml of REGM containing 20% FBS in the presence of 8 μg of Polybrene (Sigma)/ml was added, and cells were incubated overnight at 37°C/5% CO₂. The medium was then removed, and cells were cultured for an additional 12–48 h in REGM containing 10% FBS. Lentivirus transduction efficiency was >95% as determined by flow cytometry analysis (not shown).

Isolation of total RNA and amplification of cDNA fragments.

Total RNA was isolated from mouse mTAL tubules and primary cultures of mTAL cells by adding 1 ml TRIzol Reagent and incubating at room temperature for 10 min. Chloroform (0.2 ml) was then added at room temperature for 2–3 min followed by centrifugation for 15 min at 12,000 rpm and 4°C. Isopropanol (3 vol) was added to the recovered supernatant, and the mixture was incubated at room temperature for 10 min, then centrifuged at 4°C at 12,000 rpm for 15 min. The supernatant was discarded, the pellet was washed in 1 ml of 75% EtOH, mixed gently, and centrifuged for 5 min at 7,500 rpm at 4°C; the supernatant was removed, and the pellet was dried for 5–10 min. Finally, the RNA pellet was resuspended in 50 μl of RNase-free dH₂O and stored at −70°C. After total RNA was treated with DNAse I for 30 min, a 3-μg aliquot was used for cDNA synthesis using the Superscript Preamplification system (Life Technologies) in a 20-μl reaction mixture containing Superscript II reverse transcriptase (200 U/μl) and random hexamers (50 ng/μl). The reaction was incubated at room temperature for 10 min to allow extension of the primers by reverse transcriptase, then at 42°C for 50 min, 70°C for 15 min, and 4°C for 5 min. cDNA fragments were size fractionated on a 1% agarose gel and stained with ethidium bromide.

Quantitative real-time RT-PCR analysis.

A 0.5-μg aliquot of total RNA was converted to cDNA using random primers and PowerScript RT (Clontech) according to the manufacturer's protocol. The cDNA from each RNA sample was put in a 20 μl RT-PCR mixture using a FastStart DNA Master SYBR Green I kit (Roche) supplemented with 3 mM MgCl₂ and Platinum Taq polymerase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was used to determine the accumulation of mRNA. The specific primer pairs for murine NKCC2 isoforms and NFAT5 are provided in Table 1. Input cDNAs were normalized using the housekeeping gene β-actin, and the efficiency of primer pair amplification was determined using a standard curve generated using protocols described previously (44, 46). The 2(−ΔΔ C_T) method was used to evaluate changes in mRNA accumulation for NKCC2 isoforms and NFAT5 (31).

Table 1.

Oligonucleotide-specific primers for PCR of NKCC2 isoforms and NFAT5

Primer	Sequence	PCR Size, bp	Position
F-NKCC2A (m)	5-GGTAACCTCTATCACTGGGT-3		883–902
R-NKCC2A (m)	5-GTCATTGGTTGGATCCACCA-3	237	1120–1101
F-NKCC2B (m)	5-GCCGTGACAGTGACAGCCAT-3		873–892
R-NKCC2B (m)	5-GGATCCACCATCATTGAATCG-3	235	1108–1088
F-NKCC2F (m)	5-GTCGGCTATTTGCACAAACG-3		910–929
R-NKCC2F (m)	5-ATGGAACCGATGATGCGGAT-3	230	1140–1121
F-NFAT5 (m)	5-AACATTGGACAGCCAAAAGG-3		804–823
R-NFAT5 (m)	5-GCAACACCACTGGTTCATTA-3	223	1027–1008

NKCC2, Na⁺-K⁺-2Cl⁻ cotransporter type 2; NFAT, nuclear factor of activated T cells isoform 5; F, forward; R, reverse.

Protein and reporter assay.

Nuclear extracts were prepared by a modification of the method of Dignam et al. (1, 9). One day after transfection, cells were quiesced overnight in RPMI medium containing 0.5% FBS and treated with appropriate reagents for the indicated times. After treatment, cells were harvested with RIPA buffer into 1.5-ml Eppendorf tubes and spun for 5 min at 4,000 rpm and 4°C. The cell pellets were lysed in CE buffer [in mM: 10 Tris, pH 8.0, 60 KCl, 2 MgCl₂, 1 DTT, 0.1 EDTA, and 0.5 phenylmethylsulfonyl fluoride (PMSF), as well as 10 μg/ml aprotinin, 25 μM leupeptin, 2 μM pepstatin A, and 0.3% Nonidet P-40 at 4°C] and centrifuged for 5 min at 4,000 rpm and 4°C. The nuclei were kept on ice and washed in 0.5 ml CE buffer without Nonidet P-40 for 5 min at 4,000 rpm. Nuclear proteins were extracted under high-salt conditions in a solution containing 20 mM Tris, pH 7.8, 0.42 M NaCl, 1.5 mM MgCl₂, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 10 μg aprotinin per ml, 25 μM leupeptin, 2 μM pepstatin A, and 25% (vol/vol) glycerol for 30 min at 4°C. After centrifugation at 12,000 rpm for 30 min, the protein concentration in the supernatant was determined with a Bio-Rad protein assay kit. In the NFAT5 reporter assay experiments, cells were transfected using DEAE-dextran with luciferase reporter plasmids (1 μg) and TK-Renilla (0.6 μg), together with different plasmid constructs (7 μg) or corresponding empty plasmid vector (pcDNA3.1). For experiments using bumetanide, mTAL cells were quiesced overnight in RPMI medium containing 0.5% FBS, treated with different concentrations of bumetanide (2.5, 25, 250 μM) for 15 min, then exposed to 500 mosmol/kgH₂O by adding NaCl for 6 h. The luciferase activities of cell extracts were determined using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega). Luciferase activity was calculated as relative light units from reporter luciferase normalized to Renilla reniformis luciferase values.

Western blot analysis.

Cells were solubilized with sucrose isolation buffer for total NKCC2 (10) or lysis buffer [0.4 M NaCl, 0.5 mM EGTA, 1.5 mM MgCl₂, 10 mM HEPES, pH 7.9; 5% (vol/vol) glycerol, and 0.5% (vol/vol) Nonidet P-40] for NFAT5 (22), respectively, after protease inhibitors (Roche Diagnostics) were added. The protein samples were heated at 60°C in loading buffer, and protein concentration was determined with a Bio-Rad protein assay kit. Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Following blocking at room temperature for 1 h with 5% skim milk, membranes were probed at 4°C overnight with appropriate primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Pharmacia Biotech). Membranes were washed, and proteins were detected by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL).

Immunofluorescence.

The cells were directly seeded onto two-chamber tissue culture-treated glass slides (BD Bioscience), washed several times with PBS, fixed with freshly prepared 4% paraformaldehyde in PBS for 1 h, rinsed several times with fresh PBS, and stored in culture-treated glass slides at 4°C. For staining, cells were permeabilized with 0.1% Triton X-100 in PBS for 1 h at room temperature. After each sequence with either NFAT5 primary (goat), THP primary (sheep), or secondary antibody (donkey anti-sheep-conjugated with Alexa Fluor 488; Invitrogen), slides were washed five times with a high-salt solution containing 1% BSA and 2.3% sodium chloride in PBS, followed by a single wash with PBS. Cells were then washed three times and stained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 min followed by a single wash with PBS. The slides were examined using a Nikon Microphot FXA microscope equipped for epifluorescence illumination. Laser-scanning cytometry (LSC; iCys; CompuCyte, Cambridge, MA) was used to measure expression of nuclear NFAT5. In brief, nuclear and cytoplasmic fluorescence was measured by LSC using UV and 488-nm wavelength argon ion lasers to excite the fluorescence of DAPI and Alexa Fluor 488, respectively. Nuclear contouring was based on the blue fluorescence of DAPI. The intensity of blue (DAPI) and green (Alexa Fluor 488) fluorescence emission was measured by separate photomultipliers. The integrated value of green fluorescence representing NFAT5 immunofluorescence was measured in the nucleus and cytoplasm, which was defined by integration and peripheral contour settings, respectively, as described previously (8).

Statistics.

All data are presented as means ± SD. Statistical analyses were performed using one-way ANOVA followed by Tukey's multiple comparisons test or unpaired t-test as appropriate.

RESULTS

Identification of NKCC2 isoforms in the mTAL.

The mouse mTAL cell preparation used in the present study was >98% positive for the TAL-specific marker Tamm-Horsfall glycoprotein; no staining was observed when cells were incubated in the absence of the primary antibody (Fig. 1A). Specific primer pairs for murine NKCC2A, NKCC2B, and NKCC2F were designed using Epicentre software according to the cDNA sequences of mutually exclusive cassette exons A, B, and F for mouse NKCC2 (Table 1). The positions and sequences of these primer pairs were confirmed using NCBI BLAST, and cDNA amplification yielded fragment sizes consistent with those predicted by the primers sets used for each isoform (Table 1 and Fig. 1B). Analysis of PCR products reveals that mTAL cells and the outer medulla contain mRNA for NKCC2 A and F isoforms while NKCC2 A and B isoforms are present in the cortex (Fig. 1B). These data indicate that NKCC2 isoform expression in primary mTAL cells mirrors that of the outer medulla. In control experiments, total RNA was amplified before cDNA synthesis to exclude the possibility of contamination with genomic DNA (not shown). The DNA sequences for each of the PCR fragments derived from mTAL cells were identical to those previously published for the corresponding NKCC2 isoforms (not shown).

High NaCl concentration increases the NKCC2A isoform in mTAL cells.

The relative abundance of mRNA levels for NKCC2 A and F isoforms in mTAL tubules and cells was determined using qRT-PCR. Evaluation of the relative expression indicated that NKCC2A mRNA was ∼2.5-fold greater than NKCC2F mRNA in mTAL tubules and primary cells (Fig. 2A). The relative abundance of NKCC2A mRNA increased approximately twofold when mTAL cells were exposed to an increase in osmolality from 300 to 500 mosmol/kgH₂O, produced with NaCl for 2 h (Fig. 2B). In contrast, NKCC2F mRNA accumulation did not change in response to an increase in osmolality (Fig. 2C). Although isoform-specific antibodies for NKCC2 are not available, an increase in total NKCC2 protein expression was detected after incubation of mTAL cells for 3 h with 500 mosmol/kgH₂O produced with NaCl (Fig. 2D). These data indicate that NKCC2A mRNA accumulation is selectively and transiently increased in mTAL cells, after exposure to high NaCl concentration and suggest that an increase in total NKCC2 protein expression reflects an increase in NKCC2A expression as part of an adaptive response to high osmolality.

Fig. 2.

Relative abundance of NKCC2 isoform mRNA and expression of total NKCC2 protein in the mTAL/response to high NaCl concentration. A: accumulation of NKCC2A and F isoform mRNA in mTAL cells and freshly isolated tubules evaluated by qRT-PCR. Accumulation of mRNA for NKCC2A (B) and NKCC2F (C) in mTAL cells after osmolality was increased from 300 to 500 mosmol/kgH₂O by adding NaCl; values are means ± SD from 3 independent experiments (*P < 0.05; n = 3). D: Western blot analysis of total NKCC2 expression in mTAL cells after osmolality was increased from 300 to 500 mosmol/kgH₂O by adding NaCl; values are means ± SD from 3 independent experiments (*P < 0.05; n = 3).

High NaCl concentration increases NFAT5 in mTAL cells.

Raising the osmolality to 500 mosmol/kgH₂O increased NFAT5 mRNA in mTAL cells more than twofold (Fig. 3A). Moreover, the increase was maximal 4 h after exposure to NaCl and occurred subsequent to the peak accumulation of NKCC2A mRNA (Fig. 2B). An increase in endogenous NFAT5 protein expression also was observed after cells were incubated with 500 mosmol/kgH₂O (Fig. 3B). No staining was detected in cells when the primary antibody was omitted (Fig. 3B, top). Quantitative assessment by LSC showed that NFAT5 protein expression increased more than twofold when mTAL cells were exposed to an increase in osmolality from 300 to 500 mosmol/kgH₂O (Fig. 3C). The bivariate DNA content vs. NFAT5 distributions shows the increase was observed in cells in all phases of the cell cycle. The contribution of NKCC2 transport function to the transcriptional activity of NFAT5 in response to exposure to high NaCl was evaluated in mTAL cells transfected with an NFAT5-specific reporter construct (TonE-Luc) (22, 51). Transcriptional activity of NFAT5 increased ∼12-fold in mTAL cells exposed to hypertonic stress induced by NaCl (Fig. 3D). The effects of high NaCl on NFAT5 transcriptional activity was inhibited by pretreating mTAL cells with the loop diuretic bumetanide (Fig. 3D). These data are consistent with the notion that an increase in NFAT5 mRNA accumulation and, subsequently, protein synthesis and transcriptional activity in response to hypertonic stress occur following an increase in NKCC2A and are dependent on the ion transport function of NKCC2.

Fig. 3.

High NaCl increases nuclear factor of activated T cells isoform 5 (NFAT5) mRNA and protein in mTAL cells. A: abundance of NFAT5 mRNA as osmolality was increased from 300 to 500 mosmol/kgH₂O by adding NaCl (*P < 0.05; n = 3). B: immunofluorescence analysis of cells exposed to a change in osmolality from 300 to 500 mosmol/kgH₂O for 24 h. Cells were sequentially excited at different wavelengths after they were stained with anti-NFAT5-polyclonal antibody (Alexa 488; green), and DNA was counterstained with DAPI (blue). Slides were examined using a Nikon Microphot FXA microscope equipped for epifluorescence illumination. Images were obtained at a magnification of ×40; shown is a representative figure from 4 similar experiments. C: change in NFAT5 expression in cells exposed to osmolality altered from 300 to 500 mosmol/kgH₂O for 24 h. Values in left and middle (X, shown in bold) are the mean NFAT5 immunofluorescence intensity of measured cells in a representative figure from 3 similar experiments; summation of data from these experiments is shown on the right. The dashed vertical lines outline cells in the G₁, S, and G₂M phases of the cell cycle. D: inhibition of NFAT5 transcriptional activity after mTAL cells were preincubated with bumetanide, then exposed to an increase in osmolality from 300 to 500 mosmol/kgH₂O for 6 h. Relative luciferase activity was measured and normalized using Renilla activity.

Regulation of NFAT5 by NKCC2 isoforms.

Plasmid DNA and lentivirus-based shRNA vectors targeting a region in exon 4 of the NKCC2 gene (Fig. 4A) and subjected to a BLAST search to ensure specificity were used to assess the contribution of NKCC2A and F isoforms to the regulation of NFAT5 in mTAL cells exposed to high NaCl concentration. Each of the pcDNA3.1 and psiLV-U6 vectors specifically targeted the appropriate NKCC2 isoform. For instance, the NKCC2A shRNA construct (U6-N2A-ex4) specifically knocks down NKCC2A but not NKCC2F mRNA in mTAL cells (Fig. 4B). Additionally, lentivirus construct psiLV-U6-N2F-ex4 specifically targets the NKCC2F isoform (Fig. 4C), while psiLV-U6-N2A-ex4 specifically targets NKCC2A (Fig. 4D). Accordingly, mTAL cells were transfected with either NKCC2A shRNA (U6-N2A-ex4) or negative control shRNA (U6), then exposed to hypertonic conditions. The increase in NFAT5 mRNA accumulation induced by NaCl was abolished in mTAL cells transfected with NKCC2A shRNA; transfection with control shRNA had no effect (Fig. 5A). Similarly, the increase in NFAT5 protein in response to high NaCl concentration was negated in cells transfected with NKCC2A shRNA but not control shRNA (Fig. 5B). In contrast, knockdown of NKCC2F did not affect the increase in NFAT5 mRNA accumulation induced by exposure to high NaCl (Fig. 5C). Collectively, these data suggest that NKCC2A, but not NKCC2F, is required for the increase in NFAT5 mRNA accumulation and protein expression in mTAL cells exposed to high NaCl concentration.

Fig. 4.

Construction and validation of NKCC2 isoform targeting vectors. A: DNA sequences and construction of NKCC2A and NKCC2F shRNA. B: specific knockdown of NKCC2A isoform mRNA in mTAL cells transfected with plasmid U6-N2A-ex4. U6, control shRNA. C: specific knockdown of NKCC2F with psiLV-U6-N2F-ex4. D: specific knockdown of NKCC2A with psiLV-U6-N2A-ex4.

Fig. 5.

Effects of NKCC2 isoform knockdown on NFAT5 mRNA abundance and protein expression. NFAT5 mRNA abundance (A) and protein expression (B) were determined in mTAL cells exposed to 300 or 500 mosmol/kgH₂O for 4 h following transfection with either NKCC2A shRNA (U6-N2A-ex4) or control construct (U6). C: knockdown of NKCC2F with psiLV-U6-N2F-ex4 does not affect NFAT5 mRNA abundance. Values are means ± SD (n = 3).

NKCC2A and NKCC2F contribute to NFAT5 transcriptional activity in response to high NaCl concentration.

The contribution of NKCC2A and NKCC2F to the transcriptional activity of NFAT5 was evaluated in mTAL cells cotransfected with NKCC2 isoform-specific lentivirus constructs and an NFAT5-specific reporter construct (TonE-Luc) (22, 51). Transcriptional activity of NFAT5 increased ∼12-fold in mTAL cells grown on membrane inserts and exposed to hypertonic stress induced by NaCl (Fig. 6). Specific knockdown of NKCC2A mRNA with psi-LV-U6-N2A-ex4 dramatically reduced the increase in NFAT5 transcriptional activity induced by exposure to high NaCl while the control lentivirus construct was without effect (Fig. 6). Interestingly, specific knockdown of NKCC2F mRNA with psi-LV-U6-N2F-ex4 also reduced NFAT5 transcriptional activity. Collectively, these findings suggest that while NKCC2 A and F isoforms contribute to the NFAT5 transcriptional response to high NaCl, increases in the amount of NFAT5 mRNA and protein in mTAL cells is NKCC2A dependent.

Fig. 6.

NKCC2A and NKCC2F contribute to NFAT5 transcriptional activity in mTAL cells. mTAL cells were grown on membrane inserts in 6-well plates. Cells were transfected with pTonE-Luc, pRL-TK, and lentivirus constructs targeting NKCC2A or NKCC2F. Cells were then challenged with NaCl (500 mosmol/kgH₂O) for 24 h. TonE-luciferase activity was determined and normalized using corresponding Renilla reniformis luciferase activity. Values are means ± SD expressed as fold-change relative to untreated cells; n = 4.

DISCUSSION

We demonstrated that the NKCC2A isoform contributes to the regulation of NFAT5 expression and transcriptional activity in mTAL cells subjected to hypertonic stress. In contrast, NKCC2F is part of a mechanism that regulates NFAT5 transcriptional activity without affecting mRNA accumulation. The response of mTAL cells to high NaCl concentration included a selective increase in NKCC2A mRNA accumulation and total NKCC2 protein expression that subsequently was linked to an increase in NFAT5 mRNA accumulation, protein expression and transcriptional activity. NFAT5 transcriptional activity was contingent upon NKCC2 transport activity, as pretreatment of mTAL cells with bumetanide inhibited the increase in the NFAT5-specific reporter construct TonE-Luc in response to high NaCl concentration. The present study demonstrates that both NKCC2A and NKCC2F contribute to NFAT5 transcriptional activity in mTAL cells and suggests that the additional function of NKCC2A to increase the abundance of NFAT5 may be part of a mechanism critical for adaptation to medullary hypertonicity as well as other NFAT5-dependent functions in the mTAL.

The TAL is responsible for reabsorption of ∼25% of filtered Na⁺ and contributes to the generation of the osmotic gradient that drives vasopressin-dependent water reabsorption by the collecting duct (23). NKCC2 is selectively expressed along the TAL and macula densa, where it subserves critical functions in these nephron cell types (24, 34, 35, 45). Several studies have addressed the expression pattern and function of three alternatively spliced isoforms of NKCC2 (24, 40). For instance, in rabbits the B isoform was principally expressed in the cortex and the F isoform was predominantly expressed in the medulla, whereas the A isoform was equally distributed in the cortex and medulla (45). Studies in mice revealed that NKCC2 isoforms exhibited different patterns of expression in the mature TAL as follows: isoform F was most abundant in the inner stripe of the outer medulla, isoform A was most highly expressed in the outer stripe of the outer medulla, and isoform B was most highly expressed in the renal cortex (24, 43). Collectively, the data suggest the pattern of expression is similar in rabbit and mouse TAL. The preparation of mTAL tubules and primary cultured cells used in the present study contained the NKCC2 A and F isoforms. Similarly, the medulla from which the tubules and cultured cells were derived also expressed only the A and F isoforms. In contrast, the B isoform was present in the cortex, while whole kidney contained all three isoforms. These data are consistent with others (6, 24) showing that isoforms A and F are present in mTAL cells, while isoform B is localized primarily to the cortical TAL (cTAL) and macula densa. Importantly, within the outer medulla of the mouse kidney, NKCC2A mRNA abundance in the outer stripe of the outer medulla is about fourfold greater than that in the cortex or inner stripe of the outer medulla (43). Meanwhile, NKCC2A mRNA in the outer stripe of the outer medulla also is abundant in the human kidney (4). Conversely, the inner stripe of the outer medulla contains more than fourfold greater NKCC2F mRNA than in the cortex or outer stripe of the outer medulla in the mouse kidney (43). In the present study, the A isoform was twice as abundant as the F isoform, suggesting that many of the mTAL tubules, and primary cells derived from these tubules, were from the outer stripe of the outer medulla. The absence of the B isoform suggests that cTAL and macula densa cells were not present in appreciable numbers. Accordingly, the findings in the present study regarding the relative abundance of NKCC2 isoforms in mTAL cells are consistent with results obtained by in situ hybridization analysis in mice (17, 43, 45).

The cDNA encoding the cotransporter Slc12a1 gene in the mammalian kidney was identified in 1994 (18, 45). Although notable successes have been achieved in studies using heterologous expression systems such as HEK293 and Xenopus laevis oocytes, the present study is the first to address the relative expression and regulation of NKCC2 isoforms in mTAL cells exposed to hypertonicity (28, 39, 45). The abundance of NKCC2 isoform A mRNA increased approximately twofold when mTAL cells were exposed to a change in osmolality from 300 to 500 mosmol/kgH₂O, produced with NaCl. This selective response may be part of an adaptive mechanism to high NaCl concentrations and reflects an important task of alternatively spliced transcripts as a means of regulating function in the mTAL. The response of primary mTAL cells to high NaCl concentration suggests that upregulation of NKCC2A may be part of a protective mechanism involving NFAT5 (TonEBP), based on the established protective role of this transcription factor in the kidney. It also is interesting to note that 40 mg/day furosemide infused by an osmotic minipump, which resulted in a decrease in medullary tonicity due to low sodium concentration, was associated with blunted expression of TonEBP (44). These data are consistent with the present finding that bumetanide inhibited NFAT5 transcriptional activity in mTAL cells. Moreover, NFAT5 transactivation decreases in response to hypotonicity but increases in response to hypertonicity (15, 29), and NFAT5 was increased by stabilizing its mRNA in response to hypertonic conditions in mouse inner medullary collecting duct cells (mIMCD3) (3). Together, these data support the notion that NKCC2A may be linked to the regulation of a transcription factor critical to TAL function.

NKCC2 is a 12-transmembrane-helix transport protein with extensive cytoplasmic N- and C-terminal regions. Although NKCC2 is encoded by a single gene (Slc12a1), differential splicing of NKCC2 pre-mRNA results in formation of three isoforms arising from variable exon 4, including NKCC2A, NKCC2B, and NKCC2F (21, 40). A prominent difference between the splice isoforms, contributed by sequence differences in the second transmembrane domain and adjacent intracellular loop segment, is their relative affinities for Na⁺, K⁺, and Cl⁻. For instance, mouse NKCC2B and NKCCA exhibit higher affinities for Na⁺ and K⁺ compared with NKCC2F (47). Although a role for NKCC2B in the regulation of renin has been demonstrated, the profile of physiological responses attributed to other NKCC2 isoforms is likely to expand (7, 42). Sequence-specific silencing of NKCC2 isoforms was used to assess the contribution of NKCC2A and F to the regulation of NFAT5 in mTAL cells exposed to hypertonic stress. The selective increase in NKCC2A in response to hypertonicity induced by high NaCl concentration is consistent with previous reports that this isoform exhibits a high-capacity transport function (16, 20, 47). The ability of this isoform to regulate NFAT5 expression as well as transcriptional activity is complementary to high-capacity transport as it may provide a sensitive mechanism that protects TAL cells from increases in hypertonicity. Important functional, pharmacological, and kinetic differences between NKCC2 isoforms may contribute to isoform-specific physiological responses. For instance, perhaps the high-capacity, high-affinity transport function of NKCC2A, relative to NKCC2F, contributes to the mechanism by which NKCC2A elicits an effect on NFAT5 expression, as this effect is not shared by the low-affinity, low-capacity NKCC2F isoform. How these features of the NKCC2A molecule translate into increased NFAT5 protein expression remains to be determined. Exposure of mTAL cells to high NaCl concentration triggered a sequential increase in NKCC2A and NFAT5 mRNA abundance, suggesting that the mTAL may respond to local hypertonicity, via an NKCC2A-dependent mechanism that increases the amount of NFAT5 present, in addition to contributing to an increase in its activity via an increase in ion transport activity. In the macula densa, NKCC2A activity may be required for NaCl sensing in response to high concentrations of chloride; a role for NFAT5 has not been addressed in this regard (43).

In summary, NKCC2A and NKCC2F contribute to the activation of NFAT5 in mTAL cells. However, while both isoforms increase NFAT5 transcriptional activity in response to high NaCl, NKCC2A was selectively increased in response to high NaCl and subsequently contributes to an increase in NFAT5 abundance in mTAL cells. The advantage conferred upon these cells by this additional function of NKCC2A may relate to mechanisms that minimize damage initiated by the stress of hyperosmolality in the medullary interstitium and also may be important for physiological functions dependent on NFAT5 activity in mTAL cells.

GRANTS

This work was supported by National Institutes of Health Grants HL085439 and HL34300.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.