Materials and Methods

Dietary treatment.

The sulfur-free diet (a diet without cysteine, methionine and inorganic sulfate) (Table1) was purchased from Teklad (Madison, WI). Four different diets were prepared by adding various amounts ofl-methionine, l-cysteine or inorganic sulfate (Sigma Chemical Co., St. Louis, MO). These include; 1) a diet without cysteine, methionine and inorganic sulfate (0% methionine diet), 2) a diet without cysteine and inorganic sulfate but with 0.3% methionine (0.3% methionine diet), 3) a diet containing 0.82% methionine 0.34% cysteine and 0.12% inorganic sulfate (0.82% methionine; control diet), 4) a diet without cysteine and inorganic sulfate but with 2.46% methionine (2.46% methionine diet). Inorganic sulfate was added to the control diet as potassium sulfate and sodium sulfate in a ratio of 50:50. The concentrations of potassium and sodium were increased 13.5 and 27.5%, respectively when 0.12% sulfate was added to the control diet.

Study design.

Female Lewis rats (Charles River, Wilmington, MA) weighing 180 to 200 g were randomly divided into four groups and kept in individual cages. The rats received regular laboratory chow (Ralston Purina Co., St. Louis, MO) for 3 days, and then were fed the control synthetic test diet for an additional 3 days. Thereafter the rats received the test diets and deionized drinking water for 8 days. Food and water were provided to the rats ad libitum. Urine was collected for 12 hr on the day before beginning the diets (day 0) and on days 4 and 7. During the urine collection, animals were kept in individual metabolic cages from 7:00 a.m. to 7:00p.m., and received their drinking water. The diets were restricted during this period to avoid contamination of urine samples with sulfate ion from the diets. A blood sample was obtained at the midpoint of the urine collection period from the tail artery on days 0, 4 and 7. Animals were weighed daily. Animals were sacrificed by CO₂ inhalation on day 8 of the study and the kidneys were removed. Kidney cortex was trimmed and immediately frozen in liquid nitrogen. Gastrocnemius muscle was also removed from both right and left legs of the animals, and weighed.

Sulfate and creatinine analysis.

Serum and urinary sulfate concentrations were measured by single column anion chromatography (Morris and Levy, 1988) with a conductivity detector (Waters 431, Millipore Co., Milford, MA) and an anion-exchange precolumn and analytical column (Wescan Instruments, Santa Clara, CA). A mobile phase of 4 mM potassium hydrogen phthalate, pH 4.2 at a flow rate of 1.6 ml/min was used. The internal standard was potassium iodide. Serum and urinary creatinine concentrations were measured by an alkaline picrate assay described by Darling and Morris (1991).

Tissue RNA preparation.

Total RNA was prepared from rat kidney cortex by the guanidium isothiocyanate method (Chirgwin et al., 1979). The tissues isolated from the animals in the same group were combined and ground under liquid nitrogen before the initial homogenization step. Final RNA concentrations in samples were determined by measuring the optical density at 260 nm.

RT-PCR.

The primers were derived from the published NaSi-1 cDNA (Markovich et al., 1993) and were designed to produce a 700-bp DNA (native DNA). The 5′ primer was constructed with aBamH-1 enzyme digestion site and a portion of the NaSi-1 cDNA corresponding to positions 492–512; CGTGGATCCACCAGTGCTGAAGCAGAGGCC. The 3′ primer was constructed with aPst-1 enzyme digestion site and a portion of the cDNA corresponding to positions 1172–1192; TGCCTGCAGGCAACTAAGGCAACAGTTGAA. A deletion standard cDNA (600 bp) was prepared by deleting 100 bp of native cDNA located in the middle of the sequence. As a result, the primers used to amplify the native DNA and deletion standard were identical. The cRNA in vitro transcribed from the deletion cDNA was added as an external standard to RT-PCR mixture, and coamplified with sample RNA in the same reaction tube to correct for amplification efficiency.

For the reverse transcriptase reaction, the reactants containing 10 ng tissue RNA, 200 fg deletion standard RNA, 5 mM dithiothreitol, 0.5 μM primers, and 0.1 mM deoxynucleotide triphosphate (dNTP) were mixed in the first strand buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂) and first denatured at 75°C for 5 min before 2 U of RNase inhibitor (RNasin, Promega, Madison, WI) and 10 U of SuperScript (Promega) were added. The total reaction volume was 20 μl and the reaction was carried out at 42°C for 45 min. After the reverse transcriptase reaction, additional reactants for polymerase chain reaction (PCR) (10 mM Tris (pH 9.3), 0.4 μM primers, 40 nM dNTP and 3 U/100 μl UlTma polymerase) were directly added to the same tubes. After first heating at 95°C for 1 min, 25 cycles were run as follows; 95°C for 1 min, 65°C for 1 min and 72°C for 1 min. Final extension was at 72°C for 7 min and samples were kept at 4°C.

Southern hybridization.

The RT-PCR products were size separated on 1.5% agarose gel in TAE (Tris-acetate/ethylenediamine tetraacetic acid [EDTA]) buffer and transferred to hybridization matrices (Duralon-UV, Stratagene, La Jolla, CA) using a positive pressure transfer apparatus (Posiblot Transfer Apparatus, Stratagene). The hybridization probe was chosen to contain the sequence present in both native and external standard. The probe (300-bp NaSi-1 cDNA fragment; 492–792 bp) was prepared and amplified by PCR. The random primer labeling reaction was performed using a random primer labeling kit (Prime-It, Stratagene). Matrices were prehybridized for a minimum of 4 hr and hybridized overnight in hybridizing solution (5× SSC, 1% sodium dodecylsulfate (SDS), 5× Denhardt’s 50% formamide, 100 μg/ml sheared salmon sperm DNA) at 42°C. Matrices were washed five times in 2× SSC, 0.1% SDS at room temperature, then two times in 0.1 × SSC, 0.1% SDS at room temperature followed by 0.1 × SSC, 0.1% SDS at 65°C until the radioactivity was decreased to background levels. Hybridization signals were visualized and quantitated using a PhosphoImager (Molecular Dynamics, Sunnyvale, CA). The RT-PCT results were expressed as a ratio between amplified NaSi-1 mRNA and amplified deletion standard cRNA, added as an external standard, normalized by the amount of total RNA.

Crude membrane preparation for ELISA.

Crude membrane fractions were prepared from kidney cortex to determine the protein expression levels in the tissue. Animals were sacrificed and tissue was harvested and stored in the same manner as described for the total RNA preparations. Approximately 0.25 g of ground tissue powder was homogenized in the homogenizing buffer (250 mM sucrose, 10 mM triethanolamine-HCl, pH 7.6) and centrifuged at 1250 ×g for 10 min at 4°C. The supernatant was further centrifuged at 100,000 × g for 30 min at 4°C (Thomas and McNamee, 1990). The pellet containing crude membrane fractions was resuspended in 2.5% Triton-X in 1 × PBS (sample buffer) to gently extract proteins. Protein concentrations were measured by the method of Lowry et al. (1951). All samples were frozen in liquid nitrogen and stored at −80°C until assayed.

Sandwich-type ELISA procedure.

The NaSi-1 polyclonal and monoclonal antibodies were raised against rabbits and mice, respectively (Sagawa et al., 1997). The antigen used for antibody production consisted of a recombinant protein containing 119 amino acids that corresponded to amino acids 159 to 277 of the NaSi-1 protein. The specificity of the raised antibodies was examined by Western analysis using BBM and BLM purified from rat kidney cortex. Both NaSi-1 polyclonal and monoclonal antibodies detected a 69-kDa protein in BBM. The size of the band agreed with a previous report (Lötscher et al., 1996a). The size and the location of our immunoblot suggested that our antibodies recognized NaSi-1 transporter. The NaSi-1 protein was detected by sandwich-type ELISA (Sagawa et al, 1998). The assay plates (polystyrene flat-bottom microtiter plates, Maxisorp, Nunc, Denmark) were coated with the NaSi-1 monoclonal antibody (10 μg/ml), then incubated with 5% Blotto/PBS overnight at 4°C to block nonspecific absorption. Wells were washed and incubated with samples or sample buffer only (negative control) at 4°C overnight. The wells were incubated with NaSi-1 antiserum or preimmune serum (1:600 diluted in 0.3% BSA/PBS), then incubated with horse radish peroxidase conjugated mouse anti-rabbit IgG (Sigma Immunochemicals, St. Louis, MO). After washing, freshly prepared substrate solution (0.5 mg/ml o-phenylenediamine dihydrochloride, 0.045% H₂O₂) was added. The reaction was stopped with 2 M sulfuric acid and the OD at 490 nm was measured using a Microkinetics Reader (Bio-Tek instruments, Winooski, VT). The amounts of NaSi-1 protein in the tissue were calculated using a standard curve obtained by a serial dilution of the NaSi-1 standard protein (6.58–164 fmol).

Data analysis.

Renal sulfate and creatinine clearances were calculated as the urinary excretion rate divided by the midpoint serum concentration. The sulfate filtration rate was determined from the product of the serum sulfate concentrations and GFR (creatinine clearance) because the serum protein binding of sulfate is negligible. The amount of sulfate reabsorbed was calculated as the amount of sulfate excreted in urine subtracted from the total amount filtered. The fraction of the filtered sulfate which was reabsorbed was calculated by 1-CL_R/GFR.

Statistical analysis.

All results are expressed as the mean ± S.D. The data obtained after each diet were compared to day 0 values with a paired t test. The values between the groups were compared using one-way analysis of variance. Posttests were performed with the Dunnett multiple comparisons test.