WNK kinases regulate thiazide-sensitive Na-Cl cotransport (original) (raw)

DNA constructs. Human WNK4 sequence was used to perform a TBLASTN search of the mouse GenBank expressed sequence tag (EST) database. Three unique EST clones were identified and obtained from Research Genetics (Huntsville, Alabama, USA). One clone contained an open reading frame encoding 1,222 amino acids that was greater than 88% identical to human WNK4. WNK4 was amplified from mouse (BALB/c) kidney cDNA by PCR. The resulting product was sequenced, confirming identity with the sequence above, and then subcloned into a Xenopus expression vector, pgh19. This mouse WNK4 sequence was reported to GenBank (accession no. AY 184228). PHAII-causing mutations were introduced into WNK4 using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California, USA). The fidelity of the mutations was confirmed by sequencing at The Vollum Institute at Oregon Health and Science University.

We previously cloned mouse NCC (6). An N-terminal flag–tagged NCC was generated in pgh19 using PCR and sequenced to confirm the results. Rat WNK1, a generous gift of Melanie Cobb (University of Texas Southwestern Medical School, Dallas, Texas, USA), was subcloned into pgh19. Serum- and glucocorticoid-induced kinase (SGK) was a generous gift of David Pearce, University of California, San Francisco (San Francisco, California, USA).

Generation of anti-WNK4 antibody. An anti–mouse WNK4 synthetic peptide antibody was generated in rabbits by immunizing with a peptide representing amino acids 644–662 of mouse WNK4 (ASGLSDMGEGGQMRKNPVK). The antibody was produced by Alpha Diagnostic International (San Antonio, Texas, USA).

Sodium uptake. DNA template (NCC, WNK1, and WNK4) was linearized downstream from the 3′ UTR using _Not_I or AclI. The DNA was then treated with proteinase K (mCAP mRNA Capping Kit; Stratagene) for 1 hour at 37°C, extracted twice with equal volumes of phenol and chloroform, and then with chloroform alone. The DNA was precipitated in 0.1 volume of 3 M sodium acetate and 100% ethanol (1:25) in dry ice. The DNA was resuspended in RNase-free buffer, and the size and amount were checked by agarose gel electrophoresis. T7 RNA polymerase (mMES-SAGE mMACHINE; Ambion Inc., Austin, Texas, USA) was used to transcribe cRNA’s. Transcript yield, size, and quantity were checked by spectroscopy and RNA agarose gel electrophoresis.

For injection, RNA was heated at 65°C for 2 minutes, quick spun, and aspirated into a pipette. Sorted Xenopus oocytes were injected with 50 nl of water each or 12.5 ng each of NCC, WNK1, WNK4, or SGK cRNA individually or in combination. When multiple cRNA’s were injected into oocytes, 12.5 ng of each was injected, but the total injected volume remained 50 nl. After injection, oocytes were stored in Barth’s storage solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) for 3–5 days at 18°C, with fresh solution changes daily.

Injected oocytes were transferred to ND96 Cl– free solution (96 mM Na+ isethionate, 2 mM K+ gluconate, 1.8 mM Ca2+ gluconate, 1 mM Mg2+ gluconate, 5 mM HEPES, 2.5 mM Na+ pyruvate, and 5 mg/dl gentamicin, pH 7.4) 24 hours prior to the uptake assay. Thirty minutes prior to the addition of uptake medium, the oocytes were added to ND96 (Cl– and K+ free) with inhibitors (1 mM ouabain, 100 μM amiloride, and 100 μM bumetanide), according to the protocol of Gamba (7). The oocytes were then transferred to isotonic uptake medium (58 mM NaCl, 38 mM _N_-methyl-D-glucamine, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, with inhibitors, pH 7.4) and 10 μCi/ml final concentration of 22Na and incubated at 30°C for 1 hour. The oocytes were washed five times with 3 ml ice-cold isotonic uptake medium, lysed in 0.1% SDS, added to 5 ml of liquid scintillant (Hionic-Fluor; Perkin-Elmer Life Sciences, Wellesley, Massachusetts, USA), and counted for 2 minutes in a liquid scintillation counter. Fifteen to thirty oocytes were analyzed for each uptake condition during each experiment. Mean values reflect five to ten repetitions of each experiment (each using 15–30 oocytes per experimental condition).

Immunoblot. Groups of five oocytes injected 4 days prior with cRNA were transferred to Eppendorf tubes and chilled on ice for 5 minutes. Incubation buffer was then replaced with 500 μl of homogenization buffer (20 mM Tris-HCl, pH 7.6, 100 mM NaCl, and 2% NP-40) and the oocytes were lysed by repeated vortexing and pipeting. The yolk and cellular debris were pelleted at 3,600 g for 10 minutes and the supernatant was centrifuged at 3,600 g three times to remove additional yolk. All centrifugation steps were performed at 4°C. Oocyte homogenate samples were stored at –70°C prior to use.

Immunoblotting was essentially as described previously (6). Oocyte homogenates were solubilized in sample buffer (100 mM Tris-HCl, pH 7.6, 3% glycerol, 2% SDS, and 0.01% β-mercaptoethanol) and heated at 60°C for 10 minutes. The proteins were then separated on a 3–8% NuPAGE Tris-Acetate Gel (Novex no. EA0378; Invitrogen Corporation, Carlsbad, California, USA) and transferred to PVDF paper. The blot was blocked with Tween (Blotto-T) (5% nonfat dried milk in PBS-T) and then incubated with anti-NCC (8) (diluted 1:1,000 in Blotto-T) or anti-WNK4 antibody (diluted 1:1,000). The blot was then washed, incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG, diluted 1:2,000; Zymed Laboratories Inc., South San Francisco, California, USA). Protein was detected using the ECL Plus kit (Amersham Life Sciences Inc., Arlington Heights, Illinois, USA) according to the manufacturer’s instructions. Detected bands were analyzed densitometrically using NIH’s ImageJ image processing program (available at http://rsb.info.nih.gov/ij/docs/index.html) (8).

Immunofluorescence. Four days after injection, oocytes were fixed with 3% paraformaldehyde in PBS for 4 hours at 4°C. Cryosections 6 μm thick were placed on chrom alum gelatin-coated glass slides, blocked with PBS buffer containing 0.1% BSA, and incubated with anti–mouse NCC (8) diluted in PBS buffer containing 0.1% BSA and 0.05% Tween 20 (1:100) at 4°C overnight. Sections were rinsed with PBS four times, incubated with FITC-conjugated goat anti-rabbit antibody IgG, H+L (1:200, Zymed Laboratories Inc.) for 1 hour at 22°C, and rinsed with PBS buffer four times. The slides were then viewed on a Zeiss Axiophot phase-contrast microscope (Carl Zeiss Inc., Thornwood, New York, USA). Membrane NCC staining was evaluated by two observers blinded to the injected material.

Immunoprecipitation. Oocytes injected with flag-tagged NCC were prepared as described above for Western blot. Rabbit anti–mouse NCC (2 μl) was added to oocyte homogenate (100 μl) and incubated at 22°C for 1 hour. Thirty microliters of protein G–Sepharose (Amersham Life Sciences Inc.) was added and incubated at 4°C overnight. The reaction mixture was then centrifuged and the beads were washed five times in homogenizing buffer. Immunoprecipitated NCC protein was eluted from the protein G–Sepharose by heating the mixture to 60°C for 10 minutes with sample buffer containing 2.5% SDS and 2.5% β-mercaptoethanol. Immunoprecipitates were analyzed by SDS-PAGE.

Cell-surface biotinylation. Cell-surface biotinylation was performed as described (9, 10), with modifications. Oocytes, prepared as above, were washed five times with ND96 and then incubated with 1 mg/ml Sulfo-NHS-LC-Biotin (Pierce Biotechnology Inc., Rockford, Illinois, USA) for 1 hour at 4°C. The mixture was then washed five times, quenched with 100 mM glycine for 1 hour at 4°C, and prepared as described above for immunoprecipitation. ImmunoPure Streptavidin Beads (Pierce Biotechnology Inc.) were added and the mixture was incubated at 4°C for 2 hours. The beads were washed five times and eluted with SDS sample buffer, as above. The precipitated proteins were analyzed by Western blot using the anti–mouse NCC antibody.

Statistics. Group comparisons were made by unpaired Student t test. When multiple comparisons were made, the Bonferroni correction was used. A value of P < 0.05 was considered significant.