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Original Article

Hormonal Regulation of Sodium/Sulfate Co-Transport in Renal Epithelial Cells

Hwa Jeong Lee^*, Kazuko Sagawa^*, Wei Shi^*, Heini Murer and Marilyn E. Morris^*,1

^* Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo, Amherst, New York 14260; and
Institute of Physiology, University of Zürich, CH-8057 Zürich, Switzerland

	Abstract

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Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17ß-estradiol (EST) on renal sodium/sulfate co-transport. ³⁵S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inorganic sulfate is an important physiological anion that is used in metabolic processes producing biological sulfate conjugates, which are crucial for human growth and development (1-3). The increased serum sulfate concentrations in infants, young children, and pregnant women (4-7) are due, at least in part, to an enhanced sulfate renal reabsorption (8) that represents the predominant mechanism involved in sulfate homeostasis (9). Sulfate renal reabsorption occurs predominantly in the proximal tubule (10). Inorganic sulfate is actively transported through the brush border membrane (BBM) by a sodium-dependent process (11, 12). The cDNA involved in renal sodium/sulfate co-transport at BBM (NaSi-1) has been identified by Markovich et al. (13, 14). Sulfate exits from the proximal tubular cells across the basolateral membrane (BLM) via an anion exchange mechanism that is electroneutral and saturable (15, 16).

Little is known regarding the hormonal regulation of sulfate homeostasis. Sulfate serum concentrations are elevated in infants and young children (mean serum concentration of 0.47 mM in newborns compared with 0.33 mM in children over 3 years and adults) (7), and studies in animals have demonstrated that there is increased sodium/sulfate co-transport in the proximal tubule of the kidneys (8). The role of hormones involved in growth and development, such as growth hormone (GH) and insulin-like growth factor–1 (IGF-1) is not known. Renal sulfate reabsorption has been reported to be increased in a patient with acromegaly and gigantism (17) and in dogs following GH administration (18), suggesting a role in sulfate regulation. The GH-stimulated increase in sulfate renal reabsorption may be mediated by IGF-1 because the changes in renal plasma flow and glomerular filtration rate (GFR) after GH administration parallel the increase in plasma concentrations of IGF-1 (19). Additionally, sulfate uptake by cartilage of Xenopus laevis tadpoles is significantly stimulated by IGF-1 and IGF-2 but not by GH, suggesting that the cartilage growth-promoting activity of GH is mediated by insulin-like growth factors (20).

Serum sulfate concentrations are also elevated in pregnant women (mean serum concentration of 0.43 mM during the third trimester vs 0.32 mM in adult controls) (4-6), and the renal reabsorption of sulfate is increased in pregnant women (21) and animals (9). 17ß-Estradiol (EST) and progesterone (PG) serum concentrations increase with increasing gestational period during pregnancy (22), but their influence on sulfate renal reabsorption has not been examined. Serum sulfate concentrations and renal fractional reabsorption of sulfate are decreased in postmenopausal women, although altered sulfate homeostasis in menopause does not seem to be reversed by estrogen supplementation (23).

The objective of the present study was to investigate the effects of GH, IGF-1, PG, and/or EST on sodium/sulfate co-transport in Madin-Darby canine kidney (MDCK) cells that have been stably transfected with NaSi-1 cDNA (24) and in opossum kidney (OK) cells that exhibit endogenous sodium/sulfate co-transport.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials.
Dexamethasone, 17ß-estradiol, and progesterone were obtained from Sigma Chemical Co. (St. Louis, MO). Mouse anti-rabbit IgG conjugated with horseradish peroxidase was purchased from Sigma Immunochemicals (St. Louis, MO). Recombinant human insulin-like growth factor–1 (IGF-1) and human growth hormone were kindly donated from Genentech, Inc. (San Francisco, CA). ³⁵SO₄^2– (as Na₂SO₄, 1050–1600 Ci/mmol) was obtained from New England Nuclear Research Products (DuPont Company, Boston, MA). Biodegradable counting scintillant was supplied from Amersham Co. (Arlington Heights, IL). Commassie blue dye reagent concentrate and bovine plasma -globulin protein standard were purchased from Bio-Rad (Richmond, CA). Dulbecco's modified Eagle's medium, fetal bovine serum, TRIzol reagent, and trypsin were obtained from Gibco BRL (Buffalo, NY). RNase inhibitor (RNasin) and SuperScript were supplied from Promega (Madison, WI). UlTma polymerase was obtained from Perkin Elmer (Branchburg, NJ). 1,6-Diphenyl-1,3,5-hexatriene (DPH) was supplied from Molecular Probes (Eugene, OR).

Cell Culture Conditions.
MDCK/NaSi-1 cells (MDCK cells transfected with rat NaSi-1 cDNA under the control of a dexamethasone-inducible promoter (24)) were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 22 mM NaHCO₃, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO₂/95% air at 37°C. MDCK/NaSi-1 cells were induced by incubating with 1 µM dexamethasone for 16 hr (24) before hormone treatment and were used up to 15 passages for the studies. OK cells (American Type Cell Culture, Rockville MD) were maintained in DMEM F-12 medium containing 2 mM L-glutamine, 10% fetal bovine serum, and 50 IU/ml penicillin and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO₂/95% air at 37°C (25).

Hormone Treatment of MDCK/NaSi-1 and OK Cells.
MDCK/NaSi-1 cells were preincubated with GH (dissolved in water) at concentrations of 10^–8–10^–12 M and IGF-1 (diluted in 0.9% NaCl solution) at concentrations of 10^–6–10^–9 M that represents the range of physiological to pharmacological concentrations of these hormones (17, 18, 20). PG and/or EST (dissolved in ethanol) at concentrations of 10^–6–10^–10 M, the range of physiological concentrations during the menstrual cycle (estrous cycle) and late pregnancy in humans and guinea pigs (22, 26, 27), were added to cells in culture. MDCK/NaSi-1 cells were preincubated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST at various times to examine the time course of the hormonal effect on sulfate uptake. Similar concentrations and incubation times were used for the OK cell studies. All hormones were preincubated with the cells in serum-free medium. The effect of the hormones on sodium-dependent and sodium-independent sulfate uptake was determined in MDCK/NaSi-1 cells in the presence and absence of dexamethasone (which is used to induce NaSi-1 expression).

Sulfate Uptake Studies.
Inorganic sulfate uptake was examined in MDCK/NaSi-1 and OK cells grown to confluency on culture dishes (35 mm), as previously described (24, 25). Uptake studies were done at room temperature in a buffered solution (137 mM NaCl, 5.3 mM KCl, 2.8 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES/Tris, pH 7.4) containing 0.5 mM K₂SO₄ and tracer amounts of radiolabeled sulfate (2 µCi/ml). For studies done in the absence of sodium, NaCl was replaced by an equimolar amount of N-methyl-D-glucamine/HCl. At the end of the incubation period, the uptake buffer was removed by suction, and the cells were washed rapidly three times with ice-cold stop solution consisting of 137 mM NaCl and 10 mM Tris/HCl (pH 7.4). Cells were then lyzed with 1% Triton X-100 (for MDCK/NaSi-1 cells) or 0.5% Triton X-100 (for OK cells) for 1 hr. Radioactivity and protein concentrations were determined by liquid scintillation counting and the Coomassie blue binding method (28), respectively.

Cell RNA Preparation.
Total RNA was extracted from MDCK/NaSi-1 cells using TRIzol reagent according to the manufacturer's protocol. Final RNA concentrations in samples were determined by measuring the optical density at 260 nm.

The primers were derived from the NaSi-1 cDNA identified by Markovich et al. (14) and were designed to produce a 700 base-pair DNA (native DNA), as described by Sagawa et al. (29). A deletion standard cDNA (600 base pairs) was prepared by depleting 100 base pairs of native DNA located in the middle of the sequence. The cRNA in vitro transcribed from the depletion cDNA was added as an external standard to the RT-PCR mixture, and coamplified with sample RNA to correct for amplification efficiency.

For the reverse transcriptase reaction, the reactant containing 10 ng total RNA extracted from MDCK/NaSi-1 cells, 600 fg deletion standard cRNA, 5 mM DTT, and 0.1 mM dNTP were mixed in buffer containing 50 mM Tris/HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl₂, and were denatured at 75°C for 5 min before two units of RNase inhibitor, 10 units of SuperScript, and 0.5 µM primers 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 PCR (10 mM Tris (pH 9.3), 0.4 µM primers, 40 nM dNTP and 3 U/100 µl UlTma polymerase) were 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. The final extension was at 72°C for 7 min, and samples were kept at 4°C.

Crude Cell Membrane Preparation for ELISA.
Crude membrane fractions were prepared from MDCK/NaSi-1 cells to determine the protein expression levels in the cell using a modification of the procedure of Biber et al. (30). The cells were scraped in brush border membrane (BBM) buffer (300 mM mannitol, 20 mM HEPES/Tris, pH 7.4) and centrifuged at 31,000g for 15 min at 4°C. The pellet was resuspended in BBM buffer and homogenized with a polytron homogenizer (Type PTA-10S, setting 5, Kinematica, Switzerland) for 2 min at 4°C. The resulting homogenate was centrifuged at 130,000g for 30 min at 4°C. The final pellet was resuspended in 2.5% Triton X-100 in phosphate buffered saline solution (PBS) to gently extract proteins. Protein concentrations were determined by the method of Bradford (28). All samples were stored at –80°C until assayed.

Sandwich Type ELISA Procedure.
The NaSi-1 polyclonal and monoclonal antibodies were raised against rabbits and mice, respectively (31). An ELISA was performed, as previously described by Sagawa et al. (31). Briefly, assay plates (polystyrene flatbottom microtiter plates, 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 adsorption. 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 horseradish peroxidase conjugated mouse anti-rabbit IgG. 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 optical density at 490 nm was measured using a Microkinetics Reader (Bio-Tek Instruments, Winooski, VT). The amounts of NaSi-1 protein in MDCK/NaSi-1 cells were calculated using a standard curve obtained by a serial dilution of the NaSi-1 standard protein (6.58–164 fmoles).

Measurement of Membrane Motional Order.
The membrane fluidity of intact MDCK/NaSi-1 cells preincubated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST was determined by measuring the fluorescence polarization of DPH. MDCK/NaSi-1 cells were diluted with 2 ml of PBS (pH 7.4), and 5 µl of 1 mg/ml DPH in tetrahydrofuran were added to the cells. Fluorescence polarization measurements were conducted using an SLM Aminco (SLM Aminco, Urbana, IL) 8000 spectrofluorometer with film polarizers (FP110) at temperatures of 25°C and 37°C with excitation wavelength of 355 nm and the emission wavelength of 430 nm (32). The correction for light scattering was performed by control experiments performed without the added probe (33).

Statistical Analysis.
All results are presented as the mean ± SD, unless otherwise indicated. The data were compared by one-way ANOVA followed by a Tukey's test among more than two groups and by an unpaired Student's t test between two groups. The differences were considered to be statistically significant when the P-value was less than 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of Sulfate Uptake into MDCK/NaSi-1 Cells.
Sulfate (0.5 mM) uptake into MDCK/NaSi-1 cells was determined at various time points. Sodium-dependent sulfate uptake into MDCK/NaSi-1 cells increased linearly with time, up to 20 min of incubation, and reached a plateau after 60 min, indicating equilibrium. Sodium/sulfate co-transport in the cells was saturable over a wide range of sulfate concentrations (0.1 mM–6 mM) (Fig. 1). The V_max and K_m for sodium-dependent sulfate uptake in MDCK/NaSi cells were 12.0 ± 0.9 nmol/mg protein/5 min and 464 ± 55.9 µM, respectively (n = 3). In all studies, sulfate uptake in MDCK/NaSi-1 cells was significantly increased in the presence of sodium compared with that in the absence of sodium, suggesting the sodium dependence of the transport process. Preincubation of cells with 1 µM dexamethasone produced a 27.5-fold increase in the sodium-dependent transport of sulfate (0.5 mM), from 0.19 ± 0.10 nmol/mg protein/5 min to 5.22 ± 0.54 nmol/mg protein/5 min, mean ± SD (n = 11–12).

View larger version (12K): [in this window] [in a new window]   Figure 1.  Concentration-dependent uptake of sulfate into MDCK/NaSi-1 cells. Sodium-dependent sulfate uptake was calculated as the difference between sulfate uptake rates at 5 min determined with and without sodium. The data were fitted to the Michaelis-Menten equation using nonlinear regression analysis. Each data point is the mean ± SD from three separate experiments, with triplicate determinations of uptake in each preparation.

View larger version (24K): [in this window] [in a new window]   Figure 2.  Sulfate uptake into OK cells. Sulfate uptake was determined in OK cells in the presence and absence of sodium (an equimolar amount of N-methyl-D-glucamine/HCl was substituted for NaCl) at a sulfate concentration of 0.5 mM. The data represent the mean ± SD, n = 9. Uptake in the presence of sodium was significantly greater (P < 0.001) than in the absence of sodium.

View larger version (58K): [in this window] [in a new window]   Figure 3.  Concentration-dependent effect of GH on sodium-dependent sulfate uptake in MDCK/NaSi-1 cells. The data represent the difference between sulfate uptake rates at 5 min determined in the presence and absence of sodium. Cells were preincubated with varying concentrations of GH for 24 hr. The data are the mean ± SD from four separate preparations, with triplicate determinations of uptake in each preparation. *P < 0.05 compared with control.

View larger version (54K): [in this window] [in a new window]   Figure 4.  Concentration-dependent effect of IGF-1 on sodium-dependent sulfate uptake in MDCK/NaSi-1 cells. Sodium/sulfate co-transport was determined from the difference between uptake rates at 5 min measured in the presence and absence of sodium. Cells were preincubated with varying concentrations of IGF-1 for 5 hr. The data are the mean ± SD from three separate experiments in which triplicate determinations were obtained. *P < 0.05, **P < 0.01 compared with control.

View larger version (47K): [in this window] [in a new window]   Figure 5.  Effect of PG, EST, GH, and IGF-1 on sodium-dependent sulfate uptake in MDCK/NaSi cells in the absence of dexamethasone-induced expression of NaSi-1. MDCK/NaSi-1 cells were preincubated with PG (10 nM), EST (10 nM), or GH (0.1 nM) for 24 hr, with IGF-1 (1000 nM) for 5 hr or with dexamethasone (1 µM) for 16 hr. These concentrations of hormones and times of preincubation have been shown to produce the maximal effects on sodium/sulfate co-transport. The data represent the mean ± SE of n = 4–12 determinations. *P < 0.05, **P < 0.001 compared with control.

View larger version (61K): [in this window] [in a new window]   Figure 6.  Concentration-dependent effect of PG/EST on sodium-dependent sulfate uptake in MDCK/NaSi-1 cells. Sodium-dependent sulfate uptake was calculated as the difference between sulfate uptake rates at 5 min determined with and without sodium. The data are the mean ± SD from four separate experiments, with triplicate determinations of sulfate uptake in each experiment. *P < 0.05 compared with control.

View larger version (74K): [in this window] [in a new window]   Figure 7.  Effect of GH, IGF-1, PG, and EST on sodium-dependent sulfate uptake in OK cells. OK cells were preincubated with GH (10–3 nM), PG (10–1 and 103 nM), EST (10–1 and 103 nM) and PG/EST (10–1 nM of each) for 24 hr or IGF-1 (102 nM) for 5 hr. The data represents the mean ± SE for the sodium-dependent sulfate uptake determined at 5 min in two to four separate experiments, with triplicate determinations of sulfate uptake in each experiment. *P < 0.05, **P < 0.001 compared with control.

View larger version (61K): [in this window] [in a new window]   Figure 8.  NaSi-1 mRNA levels in MDCK/NaSi-1 cells preincubated with GH, IGF-1, PG, and/or EST. The data are the mean ± SD of duplicate determinations of two RNA preparations of the cells. MDCK/NaSi-1 cells were preincubated with 0.1 nM GH, 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST for 24 hr or with 100 nM IGF-1 for 5 hr. The mRNA values were compared as RT-PCR products that were expressed as the volume ratio of co-amplified NaSi-1 DNA and deletion DNA and normalized by the amount of total RNA. *P < 0.001, **P < 0.0005 compared with control.

View larger version (66K): [in this window] [in a new window]   Figure 9.  NaSi-1 protein levels in MDCK/NaSi-1 cells preincubated with GH, IGF-1, PG, and/or EST. The data are the mean ± SD of four measurements from each of two crude membrane preparations of the cells. MDCK/NaSi-1 cells were preincubated with 0.1 nM GH, 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST for 24 hr or with 100 nM IGF-1 for 5 hr. *P < 0.05, **P < 0.01 compared with control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The use of cultured renal epithelial cells is widely accepted as a powerful tool to investigate the regulatory mechanisms for the functional alterations of transport systems such as sodium/phosphate co-transport and sodium/glucose co-transport (30). Regulation of sulfate homeostasis is mediated by altered sodium/sulfate co-transport at the BBM. The present study investigated the effects of hormones, those involved in growth, development and pregnancy, on sodium-dependent sulfate transport in OK cells and on sodium-dependent transport and NaSi-1 mRNA and protein expression in MDCK/NaSi-1 cells. MDCK cells show negligible or very little sodium-dependent sulfate or phosphate transport (24). GH, IGF-1, EST, or PG may be responsible for the increased sulfate reabsorption in young and pregnant mammals.

In MDCK/NaSi-1 cells, sulfate uptake was significantly increased in the presence of sodium compared with that in the absence of sodium, indicating the sodium dependence of the transport process, and that the dexamethasone-inducible sodium/sulfate co-transport activity was predominantly expressed at the apical membrane (BBM) of the cells (24). Sodium-dependent sulfate uptake was saturable with a V_max of 12.0 ± 0.9 nmol/mg protein/5 min and K_m of 464 ± 55.9 µM. The K_m value for sodium/sulfate co-transport in MDCK/NaSi-1 cells was similar to the values for this transport process in BBM vesicles isolated from the kidney cortex of rats (39) and guinea pigs (8).

GH and IGF-1 increased sodium/sulfate co-transport activity in OK cells, which exhibited endogenous sodium/sulfate co-transport activity, and in the transfected renal cell line, MDCK/NaSi-1. The maximal effect of GH in MDCK/NaSi-1 cells occurred at a concentration of 0.1 nM whereas IGF-1 stimulated sodium-dependent sulfate uptake into the cells in a concentration-dependent manner. The mRNA level of NaSi-1 was significantly enhanced by 69% and 87% in the MDCK/NaSi-1 cells preincubated with GH and IGF-1, respectively. The NaSi-1 protein level was also increased by 49% and 36% in GH-treated and IGF-1–treated cells, respectively, which corresponds to the increase in sodium/sulfate co-transport activity observed in the cells treated with these hormones. In the absence of dexamethasone pretreatment of MDCK/NaSi-I cells, IGF-1 had no effect on sodium/sulfate uptake, indicating that it does not affect the expression of NaSi-1 from the dexamethasone-inducible promoter. GH had a modest but significant effect on NaSi-1 expression (3.0% of that produced by dexamethasone); however, GH increased sodium-dependent sulfate transport by a mean value of 0.15 nmol/mg protein/5 min in the absence of dexamethasone compared with an increase of 1.78 nmol/mg protein/5 min in the presence of dexamethasone. Therefore, the effect of GH on sodium/sulfate uptake cannot be explained by considering only its effect on the dexamethasone-inducible promoter. The effect of IGF-1 on sodium/sulfate co-transport is similar to that of IGF-1 on sodium/phosphate co-transport. IGF-1 stimulates sodium-dependent phosphate transport in a dose-dependent manner in opossum kidney (OK) cells via a mechanism involving de novo protein synthesis (40).

PG and EST increased sodium-dependent sulfate uptake in MDCK/NaSi-1 cells, but only PG or the combination of PG and EST increased sulfate uptake in OK cells. These hormones stimulated sulfate uptake in a concentration-dependent fashion up to 10 nM in MDCK/NaSi-1 cells; further increases in the hormone concentrations reduced the sodium/sulfate co-transport activity. These observations are consistent with the findings of Beck et al. (41) that PG increased the sulfate uptake in a dose-dependent manner up to 10 nM in EST-primed endometrial epithelial cells, and at higher PG concentrations there was a decrease in the uptake of sulfate. An additive or synergistic effect of PG and EST on sodium/sulfate co-transport was not observed in MDCK/NaSi-I cells, which was consistent with the findings of Beck et al. (41) in guinea pig endometrial epithelial cells. The cells preincubated with PG, EST, or PG/EST exhibited NaSi-1 mRNA levels that were increased by 23%, 51%, and 45%, respectively, and NaSi-1 protein levels that were enhanced by 36%, 50%, and 49%, respectively. Preincubation of cells with PG or EST, in the absence of dexamethasone, had no effect on sodium/sulfate uptake, suggesting that these hormones do not affect the dexamethasone-inducible promoter. The present investigation provides the first evidence of gonadal hormonal regulation of sodium/sulfate co-transport in the kidney.

It has been reported that estrogen decreases the fluidity of rabbit ileal basolateral membrane (42) and of rat sinusoidal liver plasma membrane (43). In addition, progesterone reduces the motional order of the Rana oocyte plasma membrane (44) and of hamster spermatozoal plasma membrane (45). In this study, a significant decrease in membrane fluidity was observed in the MDCK/NaSi-1 cells preincubated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control (untreated) cells at both 25°C and 37°C. A decreased membrane fluidity of MDCK/NaSi-1 cells preincubated with cholesterol reduced sodium/sulfate co-transport activity (46). Therefore, the decrease in membrane fluidity of the cells treated with these hormones would be expected to produce a reduction of sodium-dependent sulfate transport. However, it appears that the increased NaSi-1 protein levels in the cells produced by these hormones has a greater influence on sodium/sulfate co-transport and negates the opposing effect of decreased cell membrane fluidity.

In conclusion, GH, IGF-1, and PG/EST increased the activity of sodium/sulfate co-transport in MDCK/NaSi-1 and OK cells cultured in vitro. These observations suggest that GH and IGF-1 may play important roles in the regulation of renal sulfate reabsorption during growth and development. In addition, the stimulation of renal sulfate reabsorption during pregnancy could be mediated by PG and possibly EST. The increased steady-state levels of NaSi-1 mRNA and NaSi-1 protein produced by hormone treatment may represent the mechanism responsible for the enhanced sodium-dependent sulfate transport in MDCK/NaSi-1 cells preincubated with GH, IGF-1, PG, and/or EST.

	Footnotes

 
This work was supported by NSF grant IBN 9629470 and grants from the Western New York Kidney Foundation/Upstate Transplant Services and the Kapoor Charitable Foundation at SUNY at Buffalo (M.E.M.). Additional support was provided by the Swiss National Science Foundation (H.M.). The material in this manuscript was presented as a poster session at the annual meeting of the American Society of Pharmaceutical Scientists in 1997, and an abstract of this presentation was published (Pharm Res 14:S334, 1997).

¹ To whom requests for reprints should be addressed at the Department of Pharmaceutics, 527 Hochstetter Hall, State University of New York at Buffalo, Amherst, NY 14260. E-mail: memorris{at}acsu.buffalo.edu

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