©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Kidney Cell-specific Activity of the Promoter of the Murine Renal Na-K-Cl Cotransporter Gene (*)

(Received for publication, January 18, 1996; and in revised form, February 7, 1996)

Peter Igarashi (1)(§) Dilys A. Whyte (2)(¶) Kui Li (1) Glenn T. Nagami (**)

From the  (1)Departments of Internal Medicine and (2)Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520 andMedical and Research Services, West Los Angeles Department of Veterans Affairs Medical Center, Los Angeles, California 90073

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The murine Nkcc2/Slc12a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5`-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5`-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A)bullet(C-T) microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5`-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na-K(NH(4))-Cl cotransport activity and endogenously expressed the 5.0-kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.


INTRODUCTION

Relatively little is known regarding molecular mechanisms of tissue-specific gene expression in the kidney, particularly when compared with other organs such as the liver. Recently, cDNAs encoding several proteins that are produced exclusively in the kidney have been cloned including Tamm-Horsfall protein (THP), (^1)aquaporin-2 water channel, V(2) vasopressin receptor, ClC-K1 chloride channel, vacuolar H-ATPase B1 subunit, cysteine conjugate beta-lyase, vitamin D(3) hydroxylase-associated protein, and kidney-specific cadherin. Also, promoters of the THP, aquaporin-2, and V(2) vasopressin receptor genes have been cloned and characterized(1, 2, 3) . However, to date, no enhancer elements or kidney-specific transcription factors that are responsible for tissue-specific expression of these genes have been identified.

We have examined the murine renal Na-K-Cl cotransporter gene (Nkcc2) as a model for kidney-specific gene expression. The Na-K-Cl cotransporters are a family of integral plasma membrane proteins that mediate coupled transport of Na, K, and Cl with a stoichiometry of 1:1:2 and are characteristically sensitive to inhibition by loop diuretics such as furosemide and bumetanide(4) . cDNAs that encode two distinct isoforms of bumetanide-sensitive Na-K-Cl cotransporters, termed NKCC1 and NKCC2, have been cloned. NKCC1 (also called BSC2) has been cloned from the shark rectal gland, mouse inner medullary collecting duct (mIMCD-3) cells, and human colonic carcinoma (T84) cells(5, 6, 7) . The Nkcc1 gene is expressed as a 6.5-7.4-kb transcript in many epithelial and nonepithelial tissues including colon, kidney, lung, stomach, brain, skeletal muscle, heart, and salivary gland(5, 6, 7) . In secretory epithelia, such as the shark rectal gland, the Nkcc1 gene product is a 195-kDa protein that is localized exclusively in the basolateral membrane and is activated by phosphorylation(4) . Thus, NKCC1 represents the secretory Na-K-Cl cotransporter that operates in series with apical Cl channels to achieve net Cl secretion. In nonepithelial cells, NKCC1 may be involved in cell volume regulation(4, 6) .

A second isoform of the Na-K-Cl cotransporter, designated NKCC2 (also called BSC1), is encoded by a distinct gene (Nkcc2, symbol Slc12a1) that is located on a different chromosome(8) . cDNAs encoding NKCC2 have been cloned from the kidney in rabbit, rat, and mouse(9, 10, 11) . The NKCC2 protein is predicted to contain 12 transmembrane segments, and the amino acid sequence of murine NKCC2 is 64% identical to murine NKCC1. Northern blot analysis of adult tissues revealed that Nkcc2, in contrast to Nkcc1, is expressed as a 5.0-kb transcript that is only detected in the kidney (9, 10, 11) . Moreover, within the kidney, expression of Nkcc2 is restricted to the thick ascending limb of the loop of Henle (TALH)(10, 11) . Recently, the rat NKCC2 (BSC1) protein was immunolocalized to the apical membrane of the cortical and medullary TALH(12) . Thus, NKCC2 represents the apical Na-K-Cl cotransporter that mediates active reabsorption of NaCl in the TALH and is the clinically important site of action of loop diuretics. Studies using in situ hybridization of mouse embryos revealed that Nkcc2 transcripts are expressed in the metanephros but are absent from all other nonrenal tissues, verifying that Nkcc2 is kidney-specific in both adult and developing animals(11) . In the developing metanephros, Nkcc2 transcripts are absent from the nephrogenic zone, which contains uninduced mesenchyme, ureteric buds, and early epithelial structures (renal vesicles, S-shaped bodies), but are highly expressed in more mature nephrons, specifically in distal limbs of developing loops of Henle(11) . Northern blot analysis of embryonic mouse kidneys confirmed that Nkcc2 is induced at 14.5 days postcoitus, which corresponds to stages of nephrogenesis after development of the first S-shaped bodies. Taken together, these results indicate that Nkcc2 is a kidney-specific gene that is a marker for differentiation of the loop of Henle during kidney development.

The present study was undertaken to begin examining the molecular basis for kidney-specific expression of the murine Nkcc2 gene. The specific aims were to determine whether kidney-specific expression of Nkcc2 had a transcriptional basis, to clone and sequence the promoter of the Nkcc2 gene, and to evaluate whether the activity of the Nkcc2 promoter was kidney cell-specific.


EXPERIMENTAL PROCEDURES

Materials

Mice (males, age 35 days or older, strain CD-1 or C57BL/6J) were obtained from Charles River or the Jackson Laboratory. RNase H-free murine Moloney leukemia virus reverse transcriptase (SuperScript II) was obtained from Life Technologies, Inc. Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs or Boehringer Mannheim. pGL3 plasmids, luciferin, and reporter lysis buffer were from Promega. Human growth hormone assay kits were from Nichols Institute Diagnostics. Nylon filters (Hybond-N) were from Amersham Corp., and nitrocellulose filters were from Schleicher & Schuell. Radionucleotides were from Amersham or DuPont NEN. Other reagents were of molecular biological grade from Sigma, Promega, Boehringer Mannheim, or U.S. Biochemical Corp.

Nuclear Run-off Assays

Nuclear run-off assays were performed using an adaptation of the method of Celano et al.(14) . Nuclei were harvested from tissues (1 g) by homogenization with a Dounce homogenizer (10 strokes, pestle B) in medium containing 0.32 M sucrose, 3 mM CaCl(2), 2 mM magnesium acetate, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM DTT, 10 mM Tris-Cl (pH 8.0). All experiments were conducted at 0-4 °C in the presence of 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was filtered through cheesecloth and centrifuged briefly, and the supernatant was rehomogenized in the same medium without Triton X-100 (10 strokes). The sucrose concentration was adjusted to 1.37 M, and the presence of free nuclei was verified by phase contrast microscopy. Nuclei in a final volume of 8 ml were layered over a 3-ml cushion containing 2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, and 10 mM Tris-Cl (pH 8.0). Samples were centrifuged at 107,000 times g for 45 min, and the nuclear pellet was resuspended in 100 µl of storage buffer containing 25% glycerol, 5 mM magnesium acetate, 0.1 mM EDTA, 5 mM DTT, and 50 mM Tris-Cl (pH 8.0). Nuclei were used immediately or stored frozen overnight at -70 °C, which gave equivalent results. Run-off transcription was performed by incubating nuclei (5 times 10^7) for 1 h at 26 °C in medium containing 35% glycerol; 10 mM Tris-Cl (pH 7.5); 5 mM MgCl(2); 80 mM KCl; 0.1 mM EDTA; 0.5 mM DTT; 1 µl of RNAsin (Promega); 4 mM each ATP, GTP, CTP; and [alpha-P]UTP (200 µCi, 3000 µCi/mmol). The reaction was stopped by the addition of 200-500 units of RNase-free DNase I and CaCl(2) (1 mM final) followed by incubation at 26 °C for 30 min. Proteinase K (20 µg), yeast tRNA (50 µg), and SDS (0.5% final) were added, and samples were incubated for an additional 30 min at 37 °C. RNA was isolated by extraction with RNAzol B (Biotecx Labs) and chloroform and precipitation of the aqueous phase with isopropyl alcohol. Following centrifugation and washing with 70% ethanol, the pellet was resuspended in 10 mM Tris-Cl (pH 7.2), 1 mM EDTA, and 0.1% SDS. Radiolabeled transcripts (1 times 10^7 cpm/ml) were hybridized in glass scintillation vials with nitrocellulose filters spotted with 5 µg of denatured plasmid encoding murine NKCC2 (clone 3`-R27, nucleotides 1277-4655 in GenBank data base accession number U20973), pRGAPD-13 encoding rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or empty pBluescript. Hybridization was performed for 36 h at 65 °C in medium containing 10 mM TES (pH 7.4), 10 mM EDTA, 0.2% SDS, 0.6 M NaCl, and 5 times Denhardt's reagent. Filters were washed with 1 times SSC containing 0.1% SDS at room temperature for 1 h, 0.1 times SSC containing 0.5% SDS at 65 °C for 1 h, then exposed in a PhosphorImager (Molecular Dynamics).

Library Screening

A mouse genomic library in FIXII (strain 129/Sv, catalog number 946305) was purchased from Stratagene and screened by filter hybridization with a P-labeled murine Nkcc2 cDNA (clone 8) as described previously (11) . Clones that were positive on duplicate filters were plaque-purified, and the genomic inserts were restriction-mapped using single or double digests with SacI, XbaI, or SalI. Restriction fragments were transferred to nylon membranes, and end fragments and exon-containing fragments were identified as described previously(11) . A 3.5-kb XbaI/SnaBI restriction fragment that contained sequences at the 5` end of the transcript was subcloned into XbaI/EcoRV-digested pBluescript II KS(+) (Stratagene) and sequenced completely. Overlapping sequence of both strands was obtained using a combination of manual and automated methods(11) . Sequence analysis was performed using MacVector version 4.1 software (Eastman Kodak Co.). Unless indicated otherwise, consensus sequences of eukaryotic promoter elements were from compilations by Locker (15) or Faisst and Meyer(16) .

Primer Extension Analysis

The 5` ends of the murine Nkcc2 transcripts were identified using ligation-anchored PCR and primer extension analysis. Ligation-anchored PCR was performed as described previously(11) . PCR products were isolated on low melting agarose gels, cloned into the plasmid pCRII (Invitrogen), and sequenced. Primer extension analysis was performed using an antisense oligonucleotide (GGCTTCGGTTTTAGATGACCCGATA) that was complementary to the 5`-untranslated region of mouse Nkcc2 (nucleotides -107 to -131 with respect to the translation start codon, Fig. 5B). The oligonucleotide was end-labeled with [-P]ATP using polynucleotide kinase and then incubated overnight at 30 °C with 10 µg of poly(A) RNA in hybridization medium containing 40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA, and 60% formamide. Hybridization products were precipitated with ethanol and resuspended in medium containing 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl(2); 1 mM DTT; 10 mM each dATP, dCTP, dGTP, and TTP; and 1.2 µl of RNAsin (Promega). RNase H-free murine Moloney leukemia virus reverse transcriptase (200 units) was added, and samples were incubated for 2 h at 42 °C. The reaction was quenched with EDTA, extracted with phenol-chloroform, and precipitated with ethanol. Reaction products were analyzed on 6% polyacrylamide sequencing gels.


Figure 5: Sequence of the 5` end of the murine Nkcc2 gene. Panel A, partial restriction map of genomic clone LMH8. Positions of restriction sites for SacI, XbaI, and SalI are indicated by the vertical bars. The bent arrow indicates the transcription initiation site. The shaded bar indicates the cloned 5`-flanking region. Panel B, proximal 5`-flanking sequence (uppercase) and transcribed sequences (lowercase) of the mouse Nkcc2 gene obtained from genomic clone LMH8. Nucleotide positions are numbered to the right with respect to the transcription initiation site (bent arrow) at +1. Boxes enclose consensus sequences for known regulatory elements. Single underlined nucleotides indicate restriction sites used for deletion analysis. Double underlined nucleotides indicate the GA microsatellite. Dotted underlined sequence was identical to cDNA sequence. Nucleotides indicated in boldface are invariant in splice donor and acceptor sites, and slashes indicate the splice sites. Only a portion of the sequence of the first intron is shown. The straight arrow indicates oligonucleotide used for primer extension analysis. CREB, cAMP-response element binding protein; GAF, interferon- activation factor; ISGF-3, interferon-alpha-stimulated gene factor 3; ER, estrogen receptor.



Northern Blot Analysis

Poly(A) RNA was purified from tissues and confluent monolayers of cells as described previously(11, 17) . RNA was electrophoresed on denaturing formaldehyde gels and transferred to nylon filters (Hybond-N). Northern blots were hybridized with an antisense P-labeled riboprobe that was transcribed from the murine Nkcc2 cDNA clone 10 as described previously(11) . Hybridization was performed overnight at 60 °C in medium containing 50% formamide, 5 times SSC, 5 times Denhardt's reagent, 0.2% SDS, 200 µg/ml denatured salmon sperm DNA, 200 µg/ml yeast tRNA, and 2 times 10^6 cpm/ml P-labeled riboprobe. Northern blot was washed twice for 30 min at 68 °C in 0.1 times SSC containing 0.1% SDS and then exposed to Kodak X-Omat AR film at -70 °C with a single intensifying screen.

Mammalian Cell Culture and Transport Assays

TAL cells were obtained by microdissection of TALH segments from kidneys of a transgenic mouse (Tg(SV40E)Bri7) carrying the SV40 large T antigen, as described previously(18) . The presence of the large T antigen immortalizes the expressing cells. Initially, microdissected cells were grown on fibronectin-coated plates in Dulbecco's modified Eagle's medium/Ham's F-12 medium (50:50) supplemented with 10% fetal bovine serum. After passage 8, TAL cells were grown in medium supplemented with 7% fetal bovine serum, 5 nM sodium selenite, and 0.03 nM insulin under an atmosphere containing 35% O(2) and 5% CO(2). Growth in the presence of elevated oxygen tension was essential for the expression of differentiated properties. NIH 3T3 fibroblasts and HeLa cells were obtained from the American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. S1 cells, an established line derived from the proximal tubule of the Tg(SV40E)Bri7 mouse, were grown as described previously(18) .

Na-K-Cl cotransport activity in TAL cells was measured as furosemide-sensitive NH(4) influx. TAL cells were grown to confluence on plastic coverslips and then incubated at 37 °C in 140 mM NaCl, 25 mM HEPES (pH 7.4), 5 mM KCl, 1 mM MgCl(2), 1 mM NaH(2)PO(4), and 1 mM CaCl(2). All solutions were gassed with 100% O(2), and experiments were performed in the nominal absence of HCO(3). Intracellular pH (pH(i)) was measured using the permeant, pH-sensitive fluorescent dye 2-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) as described previously(19) . The cells on coverslips were preloaded with BCECF-AM for 15 min, washed with the HEPES-buffered solution, and then mounted in a microfluorometer. After a steady base-line fluorescence ratio was obtained, 10 mM NH(4)Cl was added to the medium (isosmotically replacing NaCl) in the presence or absence of 1 mM furosemide. pH(i) was calculated from the ratio of emission fluorescence at 535 nm obtained at alternating excitation wavelengths of 495 and 440 nm, and calibration was performed at the completion of each experiment using the nigericin-high potassium technique.

Construction of Reporter Plasmids

Reporter plasmids used in this study were derived from pGL3-Basic (Promega). The 3.5-kb XbaI/SnaBI restriction fragment of genomic clone LMH8 (see ``Results'') was excised from pBluescript II KS(+) by digestion with XbaI and SalI and then subcloned into unique NheI and XhoI sites in pGL3-Basic. The resulting plasmid (designated pGL3B-NKCC2) contained 2255 bp of proximal 5`-flanking region cloned in the sense orientation upstream to a promoterless luciferase gene. pGL3-Control, which contains a luciferase gene driven by the SV40 early region promoter/enhancer, and empty pGL3-Basic were used as positive and negative controls, respectively. To generate plasmids containing nested deletions of the proximal 5`-flanking region, most of the 5`-untranslated region including the first intron was first removed from pGL3B-NKCC2 using the unique site elimination method of Deng and Nickoloff(20) . The mutagenic oligonucleotide was GCTTACTTAGATCGCAGATCTGCAGCCTTCTTCTGAGC, which deleted the sequence 3` to nucleotide +23 (numbered with respect to the transcription start site). The selection oligonucleotide was GTAAGAGCTCGATATCTATCGATAG, which converted a unique, nonessential KpnI site in pGL3 into an EcoRV site. The primers were annealed to denatured pGL3B-NKCC2, and the gaps were filled with native T7 DNA polymerase and T4 DNA ligase. DNA was digested with KpnI and transformed into a mismatch repair-defective (mutS) strain of Escherichia coli. Plasmids were recovered by alkaline lysis minipreps, redigested with KpnI, and retransformed into E. coli strain XL1-Blue. The resulting plasmid was sequenced to confirm removal of 5`-untranslated region and transfected into TAL cells to verify that luciferase activity was unaffected (data not shown). The plasmid was then digested with EcoRV and PvuII, SmaI, XhoI, SacI, or SpeI; end-filled with T4 DNA polymerase if necessary; and recircularized to generate plasmids containing segments of the Nkcc2 gene extending from +23 to -1529, -1087, -835, -469, or -190, respectively. The 5` termini of the fragments were verified by dideoxynucleotide sequencing. Plasmid DNA for transfections was purified by alkaline lysis and passage over silica columns (Qiagen).

Transient Transfection and Reporter Gene Assays

Luciferase reporter plasmids were introduced into cultured mammalian cells (TAL cells, NIH 3T3 fibroblasts) using cationic liposomes (LipofectAMINE, Life Technologies). Cells were plated on 100-mm plastic dishes at a density of 0.5-1 times 10^6 cells/dish. After 24-48 h, the cells achieved 50-60% confluency and were washed once with Opti-MEM I reduced serum medium (Life Technologies) immediately prior to use. Each dish of cells was transfected with 4 µg of reporter plasmid and 4 µg of pXGH5 encoding human growth hormone as a control for transfection efficiency(21) . Plasmid DNA (8 µg total) and cationic liposomes (48 µl) were first added to Opti-MEM I in separate tubes to a total volume of 300 µl each. The solutions were combined and incubated at room temperature for 20 min. An additional 2.4 ml of Opti-MEM I was added, and the mixture was applied to one dish of cells, which was then returned to the tissue culture incubator. After 6 h of incubation at 37 °C, 3 ml of standard culture medium containing twice the usual concentration of fetal bovine serum were added. 24 h after transfection, the medium was replaced with standard growth medium, and cells were incubated for an additional 48 h prior to assay.

Luciferase activity was measured in cell lysates using methods similar to those described previously(22) . Cells were washed twice with Ca-, Mg-free phosphate-buffered saline and then lysed by incubation for 20 min at room temperature in 900 µl of reporter lysis buffer (Promega). Lysed cells were scraped into microcentrifuge tubes and freeze-thawed once using a dry ice-ethanol bath. After brief vortexing and centrifugation at 14,000 times g to remove cell debris, 20 µl of supernatant was aliquoted into 75 times 12-mm plastic tubes (Sarstedt). The reaction was initiated by rapid addition of 100 µl of luciferase assay reagent (Promega) containing 20 mM of Tricine (pH 7.8), 1.07 mM (MgCO(3))(4)bulletMg(OH)(2)bullet5H(2)0, 2.67 mM MgSO(4), 0.1 mM EDTA, 33.3 mM DTT, 270 µM coenzyme A, 530 µM ATP, and 470 µM luciferin. Light output was integrated over 10 s at room temperature using an Optocomp I photon-counting luminometer (MGM Instruments). Data were normalized for growth hormone concentration, which was measured in the conditioned medium using a solid phase radioimmunoassay according to the manufacturer's directions (Nichols Institute Diagnostics). Luciferase measurements were performed in triplicate, and measurements of growth hormone were performed in duplicate. Standard curves were generated in each experiment to insure that results were within the linear range of the assay.

Statistical Analysis

The mean data from independent experiments using different cell preparations are reported. Error bars are ± S.E. Data were analyzed using Student's t test for paired data. Statistical significance was defined as p < 0.05.


RESULTS

Kidney-specific Transcription of the Nkcc2 Gene

Expression of Nkcc2 transcripts is kidney-specific in the adult and embryonic mouse(11) . In most cases, tissue-specific expression is regulated at the level of initiation of gene transcription. However, there also exist examples in which tissue specificity is primarily due to post-transcriptional regulation(23, 24) . This was potentially the case for Nkcc2 in which tissue-specific alternative splicing has been observed(9, 11) . Accordingly, to determine whether transcription of the Nkcc2 gene was kidney-specific, nuclear run-off assays were performed. Nuclei were harvested from mouse renal medulla (which expresses Nkcc2 transcripts) and mouse liver (which does not express Nkcc2), and nascent transcripts were elongated in vitro in the presence of [alpha-P]UTP. Since no new initiation of transcription occurs under these conditions, the incorporation of radiolabel into specific transcripts accurately reflects the rate of gene transcription at the time of nuclear isolation. Radiolabeled transcripts were detected by hybridization to an excess of filter-immobilized cDNAs encoding rat GAPDH (positive control), empty pBluescript (negative control), or murine NKCC2. As shown by the dot blot in Fig. 1, run-off transcripts encoding GAPDH were produced in nuclei from both liver and kidney. The relative rates of Gapdh transcription in liver and kidney were comparable with those observed previously in the rat(24) . In contrast, run-off transcripts encoding NKCC2 were produced in nuclei from renal medulla but not in nuclei from liver. No run-off transcripts hybridizing to empty pBluescript were detected in either tissue. These experiments showed that the Nkcc2 gene was transcribed in nuclei from renal medulla but not in nuclei from liver and, therefore, that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific transcription of the Nkcc2 gene. The possibility that post-transcriptional regulation may also have contributed to kidney-specific expression of Nkcc2 was not explored.


Figure 1: Measurement of the rate of transcription of the Nkcc2 gene using nuclear run-off assays. Nuclei were isolated from liver (left) and renal medulla (right), and nuclear run-off assays were performed as described under ``Experimental Procedures.'' Radiolabeled transcripts (5 times 10^6 cpm) were hybridized to 5 µg of filter-immobilized cDNAs encoding NKCC2 (top), GAPDH (middle), or empty pBluescript (bottom). Three independent experiments gave identical results, and a representative autoradiogram is shown. Exposure in a PhosphorImager was for 7 days.



Expression of Nkcc2 in TAL Cells

To generate a murine cell line that endogenously expressed Nkcc2, TALH were microdissected from a transgenic mouse carrying the SV40 large T antigen (Tg(SV40E)Bri7), and the microdissected cells were established in culture using methods similar to those described previously(18) . The stable cell line that was produced (called TAL cells) has been grown and maintained in culture for more than 1 year. TAL cells exhibited an epithelial phenotype, formed monolayers when grown on solid substratum, and produced ``domes'' indicative of transepithelial solute transport. When grown in medium containing 7% fetal bovine serum under an atmosphere of 35% O(2) and 5% CO(2), TAL cells exhibited properties of the differentiated TALH including synthesis of preproepidermal growth factor and Tamm-Horsfall protein (data not shown). In a preliminary study, TAL cells have been successfully used to functionally express the promoter of the preproepidermal growth factor gene(25) . To determine whether TAL cells produced an apical Na-K-Cl cotransporter, transport assays were performed. In the isolated perfused thick ascending limb, exposure to luminal NH(4)/NH(3) causes intracellular acidification, rather than the more typical alkalinization, due to the higher membrane permeability to NH(4) compared with NH(3) in this nephron segment(26) . The relatively increased apical permeability to NH(4) reflects, in part, rapid transport of NH(4) via the Na-K-Cl cotransporter, in which NH(4) can substitute for K(27) . Fig. 2shows that confluent monolayers of TAL cells exhibited intracellular acidification following exposure to apical NH(4)/NH(3). Moreover, intracellular acidification was inhibited by furosemide, a competitive inhibitor of coupled Na-K-Cl cotransport, so that intracellular pH remained above base-line levels. These results were consistent with the presence of a furosemide-sensitive Na-K(NH(4))-Cl cotransporter on the apical membrane in these cells.


Figure 2: Measurement of apical Na-K(NH(4))-Cl cotransport activity in murine TAL cells. Intracellular pH was measured in confluent monolayers of TAL cells as described under ``Experimental Procedures.'' The arrow indicates replacement with medium containing 10 mM NH(4)Cl (isosmotically substituted for NaCl). The left trace (A) is in the absence of 1 mM furosemide, while the right trace (B) is in the presence of 1 mM furosemide (solid line). Results of a representative experiment are shown.



To determine whether TAL cells expressed the Nkcc2 gene, Northern blot analysis was performed. As shown in Fig. 3, TAL cells expressed an Nkcc2 transcript that was identical in size (5.0 kb) to the transcript expressed in native kidney. In contrast, no Nkcc2 transcripts were detected in NIH 3T3 fibroblasts (Fig. 3), whereas transcripts encoding GAPDH were present in both cell types (not shown). It is important to note, however, that the amount of RNA subjected to analysis was 10-fold greater in TAL cells compared with native kidney. Moreover, since TALH comprises a minority population of tubules in the kidney, the level of expression of Nkcc2 in TAL cells was considerably less than in native thick ascending limb cells. Nevertheless, experiments shown in Fig. 6and Fig. 7indicate that the level of expression was adequate for studies of the Nkcc2 promoter when a sufficiently sensitive reporter gene assay was used.


Figure 3: Northern blot analysis of expression of Nkcc2 in TAL cells. Poly(A) RNA from TAL cells (15 µg), NIH 3T3 fibroblasts (15 µg), and mouse kidney (1.5 µg) was hybridized with a P-labeled antisense Nkcc2 riboprobe. The autoradiogram was exposed overnight. Positions of molecular weight standards (in kb) are shown on the left. The arrow indicates the 5-kb Nkcc2 transcript.




Figure 6: Functional expression of the Nkcc2 promoter in TAL cells and NIH 3T3 fibroblasts. NIH 3T3 fibroblasts (columns 1-3) or TAL cells (columns 4-6) were co-transfected with 4 µg of pXGH5 and 4 µg of pGL3-Basic (columns 1 and 4), 4 µg pGL3-Control (columns 2 and 5), or 4 µg pGL3B-NKCC2 (columns 3 and 6). 72 h after transfection, cells were lysed and assayed for luciferase activity and growth hormone. Normalized light output is shown. Data are mean (±S.E.) of four separate experiments. n.s., not significantly different from column 2 (p = 0.26, t test). *, significantly greater than column 3 (p < 0.001, t test).




Figure 7: Deletion analysis of the proximal 5`-flanking region of the Nkcc2 gene. Panel A, recombinant plasmids containing nested deletions of the proximal 5`-flanking region of Nkcc2 (in pGL3-Basic) were generated by site-directed mutagenesis and restriction digestion. The bent arrows indicate transcription initiation sites, and the closed bars indicate the putative HNF-1 site at nucleotide position -211. Plasmids were transfected into TAL cells, and luciferase activity was measured in cell lysates after 72 h. To control for transfection efficiency, cells were co-transfected with pXGH5, and growth hormone was measured in the conditioned medium. Panel B, shaded bars indicate normalized light output relative to the plasmid containing 2.3 kb of proximal 5`-flanking region. Data are mean (±S.E.) of six independent experiments.



Cloning of the Murine Nkcc2 Gene and Identification of the Transcription Initiation Site

Because nuclear run-off assays indicated that kidney-specific expression of Nkcc2 had a transcriptional basis, our next objective was to isolate a genomic clone containing the proximal 5`-flanking region of the Nkcc2 gene including the gene promoter. A murine genomic library in bacteriophage FIXII was screened with an Nkcc2 cDNA, and three overlapping clones of 20.1, 16.7, and 13.6 kb were obtained that together contained 34 kb of the sequence of the gene. Southern blot analysis (not shown) indicated that the 20.1-kb genomic clone (called LMH8) contained sequences at the 5`-end of the Nkcc2 cDNA and was therefore likely to contain the desired 5`-flanking region. The transcription initiation site was mapped using primer extension analysis and ligation-anchored PCR. Fig. 4shows results of primer extension analysis in which an end-labeled antisense oligonucleotide complementary to the 5`-untranslated region of Nkcc2 was annealed to poly(A) RNA from mouse kidney and elongated with reverse transcriptase. The major product in kidney was 124 bp, which indicated that the 5` end of the Nkcc2 mRNA was located 230 nucleotides from the start codon. A product of similar size was observed in TAL cells, which indicated that the transcription initiation sites were similar in TAL cells and native kidney. Importantly, the relative autoradiographic densities of the bands correlated with the abundance of Nkcc2 transcripts in TAL cells and kidney observed by Northern blot analysis (Fig. 3). Primer extension products were not observed in NIH 3T3 fibroblasts (Fig. 4) or yeast tRNA (not shown), further verifying the specificity of the assay.


Figure 4: Mapping of Nkcc2 transcription initiation sites by primer extension analysis. An antisense oligonucleotide complementary to the 5`-untranslated region of the mouse Nkcc2 cDNA (nucleotides -107 to -131 numbered with respect to the translation start codon, Fig. 5B) was end-labeled, annealed to 10 µg of poly(A) RNA from mouse kidney (left lane), NIH 3T3 fibroblasts (middle lane), or TAL cells (right lane) and then elongated with reverse transcriptase. Products were analyzed on 6% polyacrylamide sequencing gels, and a representative autoradiogram is shown. Lanes between samples were intentionally left blank to preclude the possibility of sample spillover. The arrow indicates the major primer extension products. Positions of molecular size standards (in bp) are indicated on the left.



To confirm the location of the transcription initiation site using an independent method and to obtain the sequence of the 5` end of the transcript, ligation-anchored PCR was performed. Amplification using a gene-specific primer that annealed 241 bp 3` to the translation start codon produced cDNAs that were 471 bp in length, which confirmed that the authentic 5` end of the Nkcc2 transcript was located 230 bp 5` to the start codon (not shown). The ligation-anchored PCR product was cloned and sequenced and compared with the sequence of LMH8. The nucleotide sequences were identical in the region of overlap (indicated by dotted underlined nucleotides in Fig. 5B), verifying the identity of the genomic clone. However, beginning at nucleotide +35, the sequence of LMH8 contained an additional 1101 bp of sequence that was not present in the cDNA. This sequence was flanked by consensus splice donor (MAGGTRAGT) and acceptor (Y(n)NYAGG) sites(15) . These results indicated that the first exon of Nkcc2 was 34 bp in length and noncoding. The first intron was 1101 bp, and the second exon contained the translation start codon. The 5` end of the transcript that was identified by ligation-anchored PCR and primer extension analysis corresponded to a single transcription initiation site in the gene that was located 1330 bp upstream to the start codon. The genomic clone LMH8 contained an additional 13.5 kb of 5`-flanking sequence that was upstream to this transcription initiation site (Fig. 5A).

Sequence of the Proximal 5`-Flanking Region of the Murine Nkcc2 Gene

A 3.5-kb XbaI/SnaBI restriction fragment of LMH8 that contained 2.3 kb of the proximal 5`-flanking region of the murine Nkcc2 gene was subcloned and sequenced completely. Fig. 5B shows a portion of the nucleotide sequence that was obtained. The bent arrow depicts the transcription initiation site as mapped by primer extension analysis and ligation-anchored PCR. The proximal 5`-flanking region contained typical eukaryotic promoter elements including the sequence TTACTGT from nucleotides -2 to +5 (numbered with respect to the transcription initiation site), which matched 7/8 positions with a consensus initiator (Inr) element (YYANWYY)(28) . A TATA box was located at position -29, which would be in the appropriate location to direct initiation of transcription from the observed site at +1. Also present were two upstream CCAAT boxes at positions -75 and -115, one of which (at -75) contained extended homology to a consensus CP1 binding site. Although no canonical Sp1 sites were found upstream to the transcription initiation site, a variant GC box was present at position -36. A simple sequence repeat consisting of (G-A)bullet(C-T) was identified at position -411.

The proximal 5`-flanking region contained consensus recognition sequences for several transcription factors that are known to be involved in tissue-specific gene expression. At positions -261 and -290 there were the sequences TTGTGCAAT and TGAAGCAAT that exactly matched the consensus binding site (TKNNGYAAK) (15) for CCAAT/enhancer binding proteins (C/EBP), which mediate tissue-specific expression in liver and adipocytes. Two other sequences at positions -704 and -1184 (TTAGGAAAT and TGGGGAAAT) matched the consensus C/EBP site at 8/9 nucleotides. At positions -169, -294, -1070, and -1081 there were the sequences CAGTTG, CAACTG, CACTTG, and CAACTG, which contained the E-box motif (CANNTG) that was first identified in the enhancers of B-cell-specific genes(29) . At position -211, there was the sequence GATTAATGATTTACT that matched 13/15 nucleotides with a consensus binding site for hepatocyte nuclear factor 1 (HNF-1), a transcription factor that is involved in tissue-specific expression in the liver and other organs(30) .

Several consensus binding sites were identified for transcription factors that function as effector molecules in signal transduction pathways. These included the sequence TGACGTAG at nucleotide -1111, which exactly matched the consensus recognition site (TGACGYMR) (31) for cAMP-response element binding protein. Also present were consensus binding sites for NF-kappaB (GGGRHTYYCC) (15) at -138; interferon- activation factor (TTNCNNNAA) (31) at -93, -245, and -714; interferon-alpha-stimulated gene factor 3 (YAGTTTCWYTTTYCC) (15) at -359; activator protein-1 (TGASTMA) (16) at -111, -838, -971, -1039, and -1118; and activator protein-2 (CCCMNSSS) (16) at -432, -477, and -1180. Although three half-sites for estrogen receptor (AGGTCA) (16) were identified at -561, -640, and -726, no consensus binding sites for glucocorticoid or mineralocorticoid receptors were found.

The sequence of the Nkcc2 promoter was aligned with the sequences of cloned promoters from other kidney-specific genes including human aquaporin-2 (2) and rat V2 vasopressin receptor(3) , but no significant regions of homology were identified (data not shown). Of particular interest was the alignment with the gene encoding THP, which is expressed in the same nephron segment (TALH) as Nkcc2. The human, bovine, and rat THP promoters have been cloned, sequenced, and functionally expressed in vitro(1) . The rat THP gene also contains a consensus HNF-1 binding site that is located 280 bp 5` to the transcription initiation site within a phylogenetically conserved region. However, no other extended regions of sequence similarity between the THP and Nkcc2 genes were found (not shown).

Cell-specific Activity of the Nkcc2 Promoter

To determine whether the proximal 5`-flanking region contained a functional gene promoter, transient expression in TAL cells was performed. The 3.5-kb XbaI/SnaBI fragment of the genomic clone LMH8 containing the transcription initiation site and 2255 bp of proximal 5`-flanking region was cloned upstream to a promoterless luciferase reporter gene in the plasmid pGL3-Basic. The resulting plasmid (designated pGL3B-NKCC2) was transfected into TAL cells or NIH 3T3 fibroblasts using cationic liposomes. 72 h after transfection, the cells were lysed and assayed for luciferase activity. As a control for transfection efficiency, cells were co-transfected with pXGH5, and human growth hormone was assayed in the cell supernatants using a radioimmunoassay. Promoter activity was inferred from light output normalized for differences in transfection efficiency (which were minimal). Fig. 6shows the normalized light output following transfection of NIH 3T3 fibroblasts or TAL cells with pGL3-Basic (as a negative control), pGL3-Control containing luciferase driven by the SV40 early region promoter/enhancer (as a positive control), and pGL3B-NKCC2. Transfection with pGL3-Basic produced low levels of luciferase in both cell types (columns 1 and 4). Transfection with pGL3-Control resulted in higher luciferase levels that were comparable in the two cell types (columns 2 and 5). In contrast, transfection with pGL3B-NKCC2 produced minimal luciferase in NIH 3T3 fibroblasts but considerably greater luciferase (130-fold higher) in TAL cells (column 6 versus column 3). Similarly, transfection of pGL3B-NKCC2 into murine proximal tubule cells (S1 cells) and HeLa cells, which do not endogenously express Nkcc2, produced only 20.0 ± 4.4% and 6.6 ± 2.2%, respectively, of the luciferase activity of pGL3-Control (not shown). Taken together, these results demonstrated that the cloned 3.5-kb fragment contained a promoter that was highly active in TAL cells and that the Nkcc2 promoter exhibited cell-specific activity. These results also corroborated the results of nuclear run-off assays that showed that transcriptional regulation was at least partly responsible for the relative abundance of Nkcc2 transcripts in expressing and nonexpressing cells.

Deletion Analysis of the Proximal 5`-Flanking Region of the Nkcc2 Gene

To begin identifying the locations of regulatory elements that were required for Nkcc2 promoter activity in TAL cells, deletion analysis was performed. Luciferase reporter plasmids were constructed that contained a nested set of deletions extending from nucleotide +23 within the first exon to convenient restriction sites within the proximal 5`-flanking region. Plasmids were transfected into TAL cells, and normalized luciferase activity was measured. As shown in Fig. 7, deletion from -2255 to -1529 resulted in a 2.3-fold stimulation of luciferase activity. Luciferase activity was high and quite similar in cells transfected with the promoter having deletions from -1529 to -469. However, further deletion of the promoter to -190 resulted in a 76% reduction in luciferase activity. These results indicated that positive regulatory elements that controlled Nkcc2 promoter activity in TAL cells were likely to be located from -469 to -190. This region contained the putative HNF-1 site at -211, the GA microsatellite (at -411), two consensus C/EBP binding sites (at -261 and -290), and an E-box (at -294). In addition, the stimulation of luciferase activity that was observed following deletion from -2255 to -1529 suggested that a negative regulatory element may be present between these two nucleotide positions.


DISCUSSION

Previous studies using Northern blot analysis and in situ hybridization indicated that expression of transcripts encoding the apical Na-K-Cl cotransporter (NKCC2) was restricted to the kidney in both adult and developing mammals(9, 10, 11) . In the present study, nuclear run-off assays demonstrated that kidney-specific expression was due, at least in part, to kidney-specific transcription of the Nkcc2 gene. The cis-acting regulatory elements that govern initiation of transcription may be interspersed throughout the gene. However, in most cases the proximal promoter region contains sufficient information for efficient and tissue-specific gene transcription, although distal enhancers may be required to achieve maximal levels of expression. For example, regulatory elements located within the first 300 bp 5` to the transcription initiation site are sufficient to mediate tissue-specific expression of the albumin, beta-globin, and growth hormone genes in hepatocytes, erythroid cells, and somatotrophs, respectively(32) . These proximal regulatory elements represent binding sites for tissue-specific or tissue-enriched proteins that activate gene transcription. Accordingly, we first focused on elements within the proximal 5`-flanking region that may be responsible for kidney-specific transcription of Nkcc2.

We isolated a 20.1-kb genomic clone that contained 13.5 kb of the proximal 5`-flanking region of the Nkcc2 gene. The sequence of the proximal 5`-flanking region contained a TATA box and two CCAAT boxes, which are typical eukaryotic promoter elements in tissue-specific genes but are frequently absent from genes that are expressed ubiquitously. Although the recognition sequences of novel transcription factors that mediate kidney-specific gene expression remain unknown, consensus binding sites were identified for several transcription factors that regulate tissue-specific expression in other organs and are also present in the kidney. Four potential binding sites for members of the C/EBP family were identified. C/EBPalpha, C/EBPbeta (NF-IL6, LAP), and albumin D site-binding protein are basic leucine zipper proteins that activate a variety of tissue-specific genes, particularly during terminal differentiation in liver and adipocytes (33) . Recent studies suggest that tissue-specific expression of genes encoding aldolase B and cytosolic phosphoenolpyruvate carboxykinase in the renal proximal tubule may also involve C/EBP(34, 35) . Although C/EBP family members have been detected in the renal cortex and medulla (34, 36) , the levels of expression are very low compared with liver, and the resolution of existing studies is insufficient to ascertain whether these transcription factors are present in the loop of Henle. Four E-boxes were identified in the Nkcc2 promoter that could represent binding sites for basic helix-loop-helix (bHLH) proteins. Tissue-specific bHLH proteins (e.g. MyoD, MASH1) are important for gene expression in muscle, neurons, lymphocytes, and pancreas(29) . In general, tissue-specific (class B) bHLH proteins bind DNA preferentially as heterodimers with ubiquitous (class A) bHLH proteins (e.g. E12, E47). Although the E2A gene products, E12 and E47, are expressed in the kidney(37) , no class B proteins that are potential binding partners and kidney-specific have been identified. Inhibitory HLH proteins (e.g. Id-1) that bind to class B proteins but lack a basic domain and cannot bind DNA are also produced in the kidney. The down-regulation of Id-1 expression that occurs during nephron development has been taken as indirect evidence for the existence of class B proteins that function during terminal differentiation in the kidney(38) .

Of potential importance was the presence of a consensus binding site for HNF-1 at -211. The HNF-1 family consists of two distinct proteins, HNF-1alpha (also called HNF-1 or LF-B1) and HNF-1beta (also called vHNF-1 or LF-B3), which are transcription factors that regulate liver-specific expression of genes such as alpha(1)-antitrypsin, albumin, and fibrinogen(30) . HNF-1alpha and HNF-1beta are diverged homeodomain proteins that bind to DNA as dimers and recognize an inverted palindrome that contains the consensus sequence RGTTAATNATTAACM. In addition to liver, HNF-1alpha and HNF-1beta are also highly expressed in certain extrahepatic tissues, including the kidney. Recent studies using transgenic mice indicate that tissue-specific expression of the cytosolic phosphoenolpyruvate carboxykinase gene in the renal proximal tubule requires a promoter element (P2) that contains a consensus HNF-1 binding site(39) . In the developing mouse kidney, expression of HNF-1alpha is stage-dependent and immediately precedes expression of Nkcc2; HNF-1alpha is absent from uninduced mesenchyme and early nephrogenic structures (ureteric buds, renal vesicles, comma-shaped bodies) but is induced at 14.5 days postcoitus in late S-shaped bodies and remains highly expressed in differentiated renal tubules, including the loops of Henle(40) . The presence of a consensus HNF-1 binding site in the Nkcc2 promoter and the overlapping patterns of expression of Nkcc2 and HNF-1alpha in the developing kidney raise the possibility that Nkcc2 may be a target for transcriptional activation by HNF-1alpha. Consistent with this hypothesis, deletion of a 280-bp DNA segment containing the putative HNF-1 site caused a 76% reduction in promoter activity (see below). Recent preliminary studies suggest that HNF-1alpha can bind to the site at -211 and transactivate promoter activity. (^2)

Several consensus binding sites were identified for transcription factors that function as effector molecules in signal transduction pathways. These included the cAMP-response element binding protein. Although apical Na-K-Cl cotransport activity in the murine TALH is stimulated by arginine vasopressin via a pathway that presumably involves cAMP(41) , it is currently not known whether this effect entails changes in Nkcc2 gene expression. Thus, the importance of the binding site for cAMP-response element binding protein remains uncertain. As well, the Nkcc2 promoter contained consensus recognition sites for NF-kappaB, which mediates the transcriptional response to cytokines; interferon- activation factor and interferon-alpha stimulated gene factor 3, which consist of oligomers of STAT proteins that mediate the response to interferons; AP-1, which consists of heterodimers of Fos and Jun proteins; and AP-2, which is inducible by protein kinase C. However, no information is currently available regarding regulation of Nkcc2 gene expression by any of these signaling pathways.

Interestingly, the 5`-flanking region of murine Nkcc2 contained a simple sequence repeat consisting of (G-A)bullet(C-T). Microsatellites have occasionally been found in the 5`-flanking regions of vertebrate genes, where their function, if any, remains unknown. Because they are also found in introns, 3`-flanking region, and intergenic regions, the presence of a microsatellite in the proximal 5`-flanking sequence of Nkcc2 may be coincidental. However, in Drosophila, GA repeats are present in the promoters of the heat shock protein 26 and heat shock protein 70 genes, where they comprise a DNase I hypersensitive site that is essential for inducible promoter activity. Moreover, a Drosophila protein, called GAGA factor, has been identified that binds to the GA sequence and appears to alter chromatin structure during gene activation(42) . In mammals, a corresponding transcription factor that binds to double-stranded sequences containing GA repeats has not yet been identified. However, polypyrimidine tracts can also form triple helical H-DNA via Hogness base pairing, and a protein that binds to the resulting single-stranded DNA of the CT repeat has been identified in several mammalian species(43) . Taken together, these results raise the possibility that the GA-rich sequence may be important for altering chromatin structure during transcription of the Nkcc2 gene. Alternatively, since GA repeats are also genetically unstable, the sequence may contribute to hypermutation of the nonrepetitive flanking region.

To verify that the cloned 5`-flanking region contained a functional gene promoter, transient transfection experiments were performed. These studies utilized a stable cell line (TAL cells) that was derived from the murine TALH. TAL cells exhibited furosemide-sensitive intracellular acidification following exposure to apical NH(4)/NH(3), expressed a 5-kb Nkcc2 transcript by Northern blot analysis, and produced a primer extension product that was similar in size to the product from native kidney. Taken together, these results demonstrated that TAL cells expressed authentic NKCC2 and were, therefore, appropriate recipient cells for studies of the promoter. Transfection experiments showed that the cloned Nkcc2 promoter was highly active in cells that expressed endogenous Nkcc2 (TAL cells) but was relatively inactive in cells that did not express Nkcc2 (NIH 3T3 fibroblasts, HeLa cells, and S1 proximal tubule cells). These results suggested that cell type-specific promoter activity may contribute to kidney-specific and nephron segment-specific expression of Nkcc2. Moreover, regulatory elements that were required for cell-specific promoter activity were likely to be contained within the cloned 3.5-kb fragment. However, these experiments do not exclude the possibility that additional distal regulatory elements may be required for high level, tissue-specific expression of Nkcc2 in vivo. Further experiments utilizing transgenic mice will be required to verify whether the cloned Nkcc2 promoter contains regulatory elements that are sufficient to confer kidney-specific expression in an intact organism. To begin identifying the locations of regulatory elements that were required for promoter activity in TAL cells in vitro, deletion analysis was performed. These studies indicated that critical cis-acting regulatory elements were located between 190 and 469 bp 5` to the transcription start site. This region was interesting because it contained the putative HNF-1 site, GA microsatellite, two consensus C/EBP binding sites, and an E-box, further underscoring the potential importance of one or more of these elements for expression of Nkcc2.

In conclusion, we have demonstrated that kidney-specific expression of Nkcc2 is due, at least in part, to kidney-specific gene transcription. We have identified a stable murine cell line (TAL cells) that exhibits apical Na-K(NH(4))-Cl cotransport activity and endogenously expresses the 5-kb Nkcc2 transcript. We have cloned and sequenced the murine Nkcc2 promoter and observed that the promoter exhibits kidney cell-specific activity. Deletion analysis has identified a 280-bp DNA segment that is required for maximal promoter activity and contains consensus recognition sequences for several transcription factors expressed in the kidney.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1 DK-42921 (to P. I.) and RO1 DK-44122 (to G. T. N.) and by a grant-in-aid from the American Heart Association (to P. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A preliminary account of this work has been published in abstract form (13) .

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U45313[GenBank].

§
An Established Investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed: Section of Nephrology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4185; Fax: 203-785-7068; peter.igarashi{at}yale.edu.

Supported by National Institutes of Health Postdoctoral Training Grant T32 DK-07276.

**
Recipient of a Veterans Administration Clinical Investigator Award.

(^1)
The abbreviations used are: THP, Tamm-Horsfall protein; NKCC, Na-K-Cl cotransporter; BSC, bumetanide-sensitive cotransporter; Slc12a1, solute carrier family 12 (sodium/potassium/chloride transporters), member 1; TAL, thick ascending limb; TALH, thick ascending limb of the loop of Henle; kb, kilobase pair(s); bp, base pair(s); DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; SSC, standard sodium citrate (0.15 M NaCl, 0.015 M sodium citrate (pH 7.0)); BCECF-AM, 2-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester; C/EBP, CCAAT/enhancer-binding protein; HNF-1, hepatocyte nuclear factor 1; NF-kappaB, nuclear factor kappa B; AP-1 and AP-2, activator protein-1 and -2, respectively; STAT, signal transduction and transcription; bHLH, basic helix-loop-helix; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid (systematic); PIPES, 1,4-piperazinediethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

(^2)
D. A. Whyte, S. E. Quaggin, and P. Igarashi, unpublished observations.


ACKNOWLEDGEMENTS

We thank Jacqueline Kusnezov, Lisa Harrison, Ilyssa Okrent, and Delta Mishler for expert technical assistance. We thank Michele Pucci for expert secretarial assistance. We thank Drs. Biff Forbush, Greg Vanden Heuvel, Sue Quaggin, and Hugh Taylor for critically reviewing the manuscript and for many helpful discussions. The Tg(SV40E)Bri7 mouse was a generous gift from Dr. Darrell Fanestil (University of California, San Diego).


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