Introduction

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The homeostasis of inorganic sulfate, a physiological anion that is utilized in conjugation reactions of exogenous and endogenous compounds, is maintained predominantly by active reabsorption in the renal proximal tubule. Inorganic sulfate enters the proximal tubule cell across the brush-border membrane (BBM) by sodium-dependent sulfate cotransport. This transport system is distinct from sodium-dependent amino acid, phosphate, or glucose cotransport (Lücke et al., 1979; Turner, 1984). The cDNA for the sodium-dependent sulfate transporter (NaSi-1), which contains 2239 base pairs (bp) and encodes a protein of 595 amino acids, has been identified and cloned from rat kidney cortex (Markovich et al., 1993). Sulfate exits from the cell across the basolateral membrane (BLM) through sulfate anion exchange transport for which hydroxyl ions, bicarbonate, and oxalate can serve as counterions (Löw et al., 1984; Pritchard, 1987). Recently, Karniski et al. (1998) identified a rat renal sulfate anion transporter (sat-1) from a renal cortex cDNA library using rat liver sat-1 as a screening probe. Western blot and immunohistochemistry analysis using sat-1 monoclonal antibodies showed the sat-1 protein is localized in the basolateral membrane but not the apical membrane of the proximal tubule (Karniski et al., 1998).

Renal sodium-dependent sulfate transport in the BBM is altered under various physiological and pharmacological conditions. Sulfate-deficient rats demonstrate a decreased renal clearance of sulfate in vivo, an increased V_max for sodium sulfate cotransport without changes in the K_m, and increased levels of NaSi-1 mRNA and protein in kidney cortex (Benincosa et al., 1995; Sagawa et al., 1998b). Opposite changes are observed in rats with experimentally induced hypothyroidism (Tenenhouse et al., 1991; Sagawa et al., 1999) and with vitamin D deficiency (Fernandes et al., 1997). Infants and young humans and animals, as well as pregnant women, exhibit an increased reabsorption of sulfate compared with adult men and women (Cole et al., 1985; Pena and Neiberger, 1992; Lee et al., 1999a). Treatment with the nonsteroidal anti-inflammatory drug ibuprofen or chronic potassium depletion results in an increased renal clearance of sulfate, a decreased V_max value for sodium sulfate cotransport in BBM, and decreased levels of NaSi-1 mRNA and protein in the kidney cortex compared with that in control animals (Sagawa et al., 1998a; Markovich et al., 1999).

Little is known regarding the hormonal regulation of sulfate homeostasis. Various hormones, including glucocorticoids, regulate sodium-dependent transport systems. Glucocorticoids enhance Na⁺-H⁺ exchange transport (Kinsella et al., 1985) and sodium-dependent bile acid transport in ileal BBM (Nowicki et al., 1997) and inhibit sodium-dependent phosphate uptake, but not the sodium-dependent uptake of glucose or proline, in renal BBM vesicles (Levi et al., 1995; Loffing et al., 1998). Renfro et al. (1989) reported that dexamethasone pretreatment significantly decreases sodium-dependent sulfate uptake in chick renal BBM. However, the in vivo clearance of sulfate after glucocorticoid treatment and the mechanism involved in changes in the renal reabsorption of sulfate have not been examined. Therefore, the objectives of this study were to determine the effect of treatment with the glucocorticoid methylprednisolone (MPL) on 1) the serum concentrations, urinary excretion, and renal clearance of sulfate, 2) sulfate transport in kidney cortex BBM and BLM vesicles, and 3) the cellular mechanism of the MPL-induced alterations in sulfate renal transport. MPL was used in these studies because it is a glucocorticoid that is widely used clinically for its anti-inflammatory and immunosuppressant properties and it exhibits only minimal mineralocorticoid activity.

	Materials and Methods

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Animal Treatment. Male adrenalectomized Wistar rats weighing 225 to 250 g were used and maintained with regular rat chow and 0.9% NaCl drinking water. One day before the study, right jugular vein cannulas were implanted under ether anesthesia. Animals received 50 mg/kg MPL sodium succinate (Solu-Medrol; Upjohn Co., Kalamazoo, MI) over 30 s through the cannula. Control animals had jugular vein cannulas implanted but received only the vehicle. For the determination of inorganic sulfate and creatinine renal clearance values, urine samples were collected for 12 h after drug administration with a blood sample (0.8 ml) obtained at 6 h. To examine sulfate transport in kidney membrane vesicles, animals were administered either 50 mg/kg MPL or control vehicle and the kidneys were removed after exsanguination under ether anesthesia at 6 h. Additionally, animals were sacrificed by exsanguination under ether anesthesia at 2, 4, 6, and 12 h (three animals per time point), kidneys were removed from the animals, and kidney cortex was trimmed immediately. Tissue samples were frozen immediately in liquid nitrogen and stored at 80°C for RNA or membrane preparations.

Sulfate and Creatinine Assay. Serum and urinary sulfate concentrations were measured by single-column anion chromatography with a conductivity detector (Water 431; Millipore Co., Milford, MA) and an anion exchange precolumn and analytical column (Wescan Instruments, Santa Clara, CA) (Morris and Levy, 1988). 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).

Renal Vesicle Preparation. BBM and BLM vesicles were prepared from whole kidney cortex as previously described (Benincosa and Morris, 1993; Darling et al., 1994). The tissues from four animals in the same study group were combined for the membrane vesicle preparations. Briefly, the freshly isolated rat kidney cortex was homogenized in homogenizing buffer (250 mM sucrose, 10 mM triethanolamine-HCl, pH 7.6). BBM and BLM vesicles were separated using a Percoll (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation. The BBM fraction was further purified by MgCl₂ precipitation.

Sulfate Transport Studies. The sulfate transport was examined by measuring the uptake into membrane vesicles using a rapid filtration method (Goldinger et al., 1984; Darling et al., 1994). Experiments were begun by diluting vesicles 1:10 (to yield a final protein concentration of 0.6 mg/ml) with the uptake medium (100 mM mannitol, 10 mM HEPES, 100 mM NaCl or KCl for BBM; 200 mM mannitol, 50 mM HEPES, with or without 20 mM potassium thiosulfate for BLM; pH 7.5) containing [³⁵S]Na₂SO₄ (DuPont-New England Nuclear, Boston, MA) and various concentrations of K₂SO₄. All uptake studies were performed at room temperature.

Evaluation of Membrane Motional Order (Fluidity). The motional order of BBM and BLM obtained from MPL-treated or control rat kidney cortex was determined by examining the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) as previously described (Balasubramanian et al., 1997; Lee et al., 1999b). To incorporate the probe, 2 µl of 2 mM DPH in tetrahydrofuran was added to the membrane vesicles, and incubated at 37°C for 1 h. Fluorescence polarization measurements were done on an SLM Aminco (Urbana, IL) 8000 spectrofluorometer with film polarizers (FPl 110). Samples were excited at 355 nm, and the emission was monitored at 430 nm with 4-nm excitation and emission slits. The lipid order parameter (S) was calculated from the steady-state polarization value by the equation: S² = [(4r/3)  0.1]/r_o, where r_o is the maximal fluorescence anisotropy value in the absence of any rotational motion (taken as 0.40) and r is the steady-state anisotropy.

Tissue RNA Preparation. Total RNA was prepared from rat kidney cortex using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instruction. Tissue obtained from the animals at the same time point was pooled. Final RNA concentrations in sample were determined by absorbance at 260 nm.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR). NaSi-1 mRNA in total RNA isolated from kidney cortex was measured as described previously (Sagawa et al., 1998b). The size of the amplified native NaSi-1 mRNA was 700 bp. Amplification efficiency, as well as amount loaded and transferred, was normalized with an externally added deletion standard (600 bp). The deletion standard cDNA was prepared by deleting a 100 bp of native cDNA located in the middle of the sequence. For the reverse transcriptase reaction, 10 ng tissue RNA and 700 fg deletion standard cRNA were coamplified using SuperScript (Promega, Madison, WI) at 42°C for 45 min. After the reverse transcriptase reaction, additional reactants for PCR, including UlTma polymerase, were directly added to the same tubes. After the 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 and transferred to hybridization matrices (Duralon-UV; Stratagene, La Jolla, CA), as previously described (Sagawa et al., 1998b). The RT-PCR products were loaded on the gel in duplicate. Hybridization probe was 300 bp NaSi-1 cDNA at positions 492 to 792. The random primer labeling reaction was prepared using a random primer labeling kit (Prime-It; Stratagene). Hybridization signals were visualized and analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The RT-PCR 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 Enzyme-Linked Immunosorbent Assay. Crude membrane fractions were prepared from kidney cortex to determine the protein expression levels in the tissue. Approximately 0.50 g of ground tissue powder was homogenized in the homogenizing buffer (250 mM sucrose, 10 mM triethanolamine-HCl, pH 7.6) and centrifuged at 1250g for 10 min at 4°C. The supernatant was further centrifuged at 100,000g for 30 min at 4°C (Thomas and McNamee, 1990). The pellet containing crude membrane fractions was resuspended in 2.5% Triton X-100 in 1× PBS (sample buffer) to gently extract proteins. Protein concentrations were measured by the Lowry method (Lowry et al., 1951).

Sandwich-Type Enzyme-Linked Immunosorbent Assay Procedure. NaSi-1 polyclonal and monoclonal antibodies were raised against rabbits and mice (Sagawa et al., 1998c). NaSi-1 transport protein level was measured in crude membrane fraction isolated from kidney cortex as previously described (Sagawa et al., 1998c). A 500-µg sample was added to each well for the assay. Serial dilutions of the NaSi-1 standard protein (6.58-164 fmol) were used to construct a standard curve. The amounts of NaSi-1 present in tissue were determined using this standard curve.

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 glomerular filtration rate (creatinine clearance) because the serum protein binding of sulfate is negligible (Berglund, 1960). 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 that was reabsorbed was calculated as 1  (renal sulfate clearance/glomerular filtration rate).

Statistical Analysis. All results are expressed as the mean ± S.D. unless indicated otherwise. Serum concentrations, urinary excretion rates, and clearance values for both sulfate and creatinine and the V_max and K_m values determined in membrane preparations were compared between study groups using unpaired t tests. The differences in NaSi-1 mRNA and protein levels between study groups were compared using one-way ANOVA. Post hoc tests were calculated with the Dunnett multiple comparisons test.

	Results

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Serum Concentrations and Urinary Excretion of Sulfate. Serum sulfate concentrations were analyzed and compared between control animals and animals that received MPL. There was no significant difference in serum sulfate concentrations after MPL administration (Table 1). Creatinine serum concentrations, urinary excretion rates, and clearance values were not significantly different between control and MPL-treated animals. However, both sulfate urinary excretion rates (determined over 12 h) and renal clearance values increased significantly, and the fraction reabsorbed in the kidney decreased significantly with MPL treatment (Table 1).

Sulfate Transport Studies. The BBM vesicles were incubated with or without sodium in the uptake medium to determine kinetic parameters for sodium-dependent sulfate transport in BBM. Sulfate uptake into BBM vesicles increased linearly in the absence of sodium. Preliminary time course studies indicated that the 10-s uptake point represented linear uptake. The difference in uptake in the presence and absence of sodium represents the sulfate uptake by sodium-dependent sulfate cotransport. The K_m and V_max values were estimated by fitting the data using the Michaelis-Menten equation. A representative fit of the sodium-dependent sulfate uptake process using nonlinear regression analysis is shown in Fig. 1. The V_max value in the MPL group was significantly lower compared with the control group (P < .02, n = 3; Table 2). The K_m value for sulfate transport in BBM was not significantly different between groups. The BLM vesicles were incubated in the absence and presence of the competitive inhibitor thiosulfate. The difference between the two represents sulfate uptake by the bicarbonate-driven sulfate anion exchange transporter in BLM. There were no significant differences in K_m and V_max values for the sulfate anion exchange transport in BLM (Fig. 2, Table 2).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Sodium-dependent sulfate uptake into kidney cortex BBM isolated from control (adrenalectomized) and MPL-treated rats. The data represent the difference between sulfate uptake rates determined in the presence and absence of sodium. The mean data obtained from one preparation were fitted to the Michaelis-Menten equation using nonlinear regression analysis.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Sulfate anion exchange in kidney cortex BLM isolated from control (adrenalectomized) and MPL-treated rats. The data represent the difference between uptake rates determined in the absence and presence of thiosulfate. All BLM vesicle preparations contained 50 mM KHCO3 in the internal buffer as the driving force for the anion exchanger. The mean data obtained from one preparation were fitted to the Michaelis-Menten equation using nonlinear regression analysis.

Membrane Motional Order (Fluidity). The fluorescence polarization studies with DPH demonstrated that BLM and BBM differ from one another, in that the motional order of BBM is less than that of BLM. This is consistent with previous reports that examined the fluidity of these membranes (Balasubramanian et al., 1997). However, treatment of the animals with MPL did not produce any changes in membrane fluidity or the lipid order parameter for either BBM or BLM (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Fluorescence polarization of DPH in BBM and BLM isolated from control (adrenalectomized) and MPL-treated rats. Four measurements were taken per point for each preparation. Each data point represents the mean value obtained. The S.D. for each point ranged from 0.003 to 0.008. There was no difference in membrane fluidity between groups.

NaSi-1 mRNA and Protein Abundance. NaSi-1 mRNA levels in kidney cortex were measured before and 2, 4, 6, and 12 h after a single dose (50 mg/kg) of MPL (Fig. 4). Significantly decreased NaSi-1 mRNA levels were observed at the 4- and 6-h time points (P < .01). There was no significant difference in NaSi-1 mRNA levels between control and MPL-treated animals at 12 h after MPL administration. NaSi-1 protein expression level in tissue was significantly decreased 4, 6, and 12 h after the administration of a single dose (50 mg/kg) of MPL (P < .05, .01, and .05, respectively; Fig. 5).

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Comparison of NaSi-1 mRNA levels in kidney cortex of MPL-treated and control (adrenalectomized) rats. The mRNA levels were compared as RT-PCR products that were expressed as the volume ratio of coamplified NaSi-1 DNA and deletion DNA and normalized by the amount of total RNA. Values are presented as mean ± S.D. (n = 4). NaSi-1 mRNA values were significantly decreased at 4 and 6 h compared with control (**P < .01).

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Comparison of NaSi-1 protein levels in crude membrane fractions isolated from the kidney cortex of MPL-treated and control (adrenalectomized) rats. The data represent mean ± S.D. determined in triplicate from one sample. Values are presented as mean ± S.D. (n = 4). NaSi-1 protein values were significantly decreased at 4, 6, and 12 h compared with control (*P < .05, **P < .01).

	Discussion

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The current investigation demonstrated that MPL-pretreated animals have decreased NaSi-1 mRNA and protein synthesis in the kidney cortex. This gene-mediated inhibition of NaSi-1 transport significantly increased in vivo urinary excretion and renal clearance of sulfate, resulting in a decreased renal reabsorption of sulfate. The lack of change in serum sulfate concentrations was not unexpected; even after the administration of a sulfate-deficient diet, changes in sulfate renal reabsorption are able to maintain serum sulfate concentrations within the normal range (Benincosa et al., 1995; Sagawa et al., 1998b). The renal clearance of sulfate in the adrenalectomized rats was about 10-fold lower than literature values for female Sprague-Dawley (Frick et al., 1986), male Fischer 344 (Bakhtian et al., 1993), or female Lewis (Morris et al., 1988; Sagawa et al., 1998b, 1999) rats, all of which average about 0.7 ml/min/kg. Additionally, the fraction reabsorbed in the adrenalectomized Wistar rats was 0.983, which is greater than the previously reported values of 0.75 to 0.90 in untreated Sprague-Dawley, Fischer 344, or Lewis rats. This suggests either there are significant strain differences in inorganic sulfate disposition in rats or that a deficiency of adrenal hormones results in an increased reabsorption of inorganic sulfate. The latter hypothesis is supported by reported increases in the renal reabsorption of phosphate in adrenalectomized rats (Frick and Durasin, 1980).

Renfro et al. (1989) examined sulfate transporters in chick BBM and BLM isolated from chick kidney pretreated with another synthetic glucocorticoid, dexamethasone, and demonstrated that dexamethasone caused a significant decrease in sodium sulfate cotransport in BBM; the anion exchange process in BLM was not altered. Our results in adrenalectomized Wistar rats receiving a single dose of MPL confirm the results of Renfro et al. (1989). The V_max value for sodium/sulfate cotransport in the BBM was significantly decreased with no change in the K_m value; nor was sulfate anion exchange in the BLM altered. The decreased V_max value for sodium/sulfate cotransport is consistent with the changes in sulfate reabsorption observed in vivo: regulation of sulfate reabsorption occurs via changes in sodium/sulfate cotransport (Markovich and Knight, 1998; Sagawa et al., 1998b, 1999; Lee et al., 1999a; Markovich et al., 1999).

Glucocorticoids are known to be important regulators of renal sodium/phosphate cotransport (Levi et al., 1995; Loffing et al., 1998). Glucocorticoid excess due to disease or the administration of pharmacological doses of glucocorticoids results in the decreased tubular reabsorption of phosphate due to a decreased V_max value for sodium/phosphate cotransport. Cell studies have demonstrated that glucocorticoids have a direct and selective effect on sodium/phosphate cotransport: glucocorticoids do not affect sodium/glucose or sodium/proline transport in the proximal tubule, and the effect on sodium/phosphate cotransport is independent of glucocorticoid-induced changes in renal hemodynamics and/or other hormones. Dexamethasone treatment results in decreased NaPi-2 mRNA and protein expression, as well as significant changes in BBM lipid composition, including increased glucosylceramide and sphingomyelin concentrations, and a subsequent decrease in BBM fluidity. These alterations in lipid composition have been demonstrated to modulate sodium/phosphate cotransport activity and NaPi-2 protein abundance. In the present investigation, we found that the mechanism underlying the glucocorticoid-induced decrease in sodium/sulfate cotransport involved the decreased expression of NaSi-1 mRNA and protein, similar to that reported with sodium/phosphate cotransport. We did not evaluate membrane lipid composition but did determine the fluorescence polarization of DPH as a measure of the membrane motional order in the hydrophobic core of the membrane lipid bilayer, a value that is inversely related to membrane fluidity (Balasubramanian et al., 1997). In contrast to that observed after dexamethasone administration, we did not observe any alteration in membrane fluidity after a single dose of MPL. Changes in membrane fluidity have been shown to alter sodium/sulfate cotransport in kidney cells and membrane vesicles (Lee et al., 1999b). The lack of effect of MPL on membrane fluidity in the present study may reflect differences in glucocorticoid administration between our study and that of Levi et al. (1995). We administered a single dose of MPL to rats and examined membrane fluidity 6 h later, whereas Levi et al. administered pharmacological doses of dexamethasone over a 4-day period. However, we cannot rule out glucocorticoid-specific changes in membrane lipid composition and fluidity.

MPL administration (50 mg/kg i.v.) to adrenalectomized Wistar rats results in peak MPL concentrations of about 40 µg/ml; plasma concentrations decline biexponentially with a terminal half-life of 0.57 h (Xu et al., 1995). MPL plasma concentrations fall to about 10 ng/ml (the assay detection limit) by 6 h (Xu et al., 1995). However, our results indicated that the NaSi-1 transport protein level is still inhibited at 12 h after MPL administration. This may be due to the delayed responses by MPL. Generally, gene-mediated effects of glucocorticoids exhibit delayed responses due to the induction or inhibition of protein synthesis after steroid receptor binding (Xu et al., 1995). The mouse NaSi-1 gene and promoter region have recently been characterized (Beck and Markovich, 2000). The promoter region contains a number of cis-acting elements, including five putative glucocorticoid responsive elements. This supports our present findings that indicate that glucocorticoids may be involved in the regulation of sulfate homeostasis. The relatively small changes in NaSi-1 protein expression may be due to the fact that we examined changes after a single dose of a glucocorticoid and not after chronic treatment.

In conclusion, the current investigation demonstrated the first evidence that the mechanism underlying glucocorticoid regulation of renal sulfate transport involves down-regulation of the NaSi-1 gene, resulting in decreased NaSi-1 mRNA and protein expression in the rat kidney cortex.