1 Department of Pharmaceutics, State University of New York at Buffalo, Amherst, New York 14260; and 2 Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland
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ABSTRACT |
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Decreased serum
sulfate concentrations are observed in hypothyroid patients. However,
the mechanism involved in thyroid hormone-induced alterations of renal
sulfate homeostasis is unknown. The objectives of this investigation
were to determine the effect of 6-propyl-2-thiouracil (PTU)-induced
hypothyroidism in rats on 1) the in
vivo serum concentrations, renal clearance, and renal reabsorption of
sulfate, 2) the in vitro renal
transport in brush-border membrane (BBM) and basolateral membrane (BLM)
vesicles, and 3) the cellular
mechanism of the hypothyroid-induced alteration in sulfate renal
transport. Serum sulfate concentrations, renal fractional reabsorption
of sulfate, and creatinine clearance were decreased significantly in
the hypothyroid group. The
Vmax values for
sodium-sulfate cotransport in BBM were significantly decreased in the
kidney cortex from the hypothyroid animals (0.90 ± 0.31 vs. 0.49 ± 0.08 nmol · mg1 · 10 s
1,
n = 5-6,
P < 0.05) without
changes in Km.
There were no significant differences in
Vmax and
Km for
sulfate/anion exchange transport in BLM. Sodium-dependent sulfate
transporter (NaSi-1) mRNA and protein levels were
significantly lower in the kidney cortex from hypothyroid rats.
Hypothyroidism did not alter the membrane motional order (fluidity) in
BBM and BLM, which indicates that the changes in the membrane fluidity
do not represent the mechanism for the altered renal transport. These
results demonstrate that PTU-induced hypothyroidism decreases
sodium-sulfate cotransport by downregulation of the NaSi-1 gene.
inorganic sulfate; sodium sulfate cotransport; NaSi-1; kidney
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INTRODUCTION |
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INORGANIC SULFATE is a physiological anion that is utilized in the sulfation of many endogenous and exogenous compounds. Xenobiotics, such as steroids, anti-inflammatory drugs, and adrenergic blockers and stimulants, undergo biotransformation by sulfate conjugation (30). Sulfate conjugation is essential for the biological activity of many endogenous compounds, such as heparin, heparan sulfate, dermatan sulfate, gastrin, and cholecystokinin (31, 33). Inorganic sulfate is also necessary for the biosynthesis of numerous structural components of membranes and tissues, such as sulfated glycosaminoglycans or cerebroside sulfate (17).
Inorganic sulfate can be absorbed from the diet or formed from the oxidation of the sulfur-containing amino acids, cysteine and methionine. It is eliminated from the body mainly in unchanged form by urinary excretion (48). Homeostasis of inorganic sulfate occurs predominantly by renal mechanisms. Inorganic sulfate enters into 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 (24, 47). The cDNA for the sodium-dependent sulfate transporter (NaSi-1), which contains 2,239 bp and encodes a protein of 595 amino acids, has been identified and cloned from rat kidney cortex (26). 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 counter ions (22, 36).
Thyroid hormone (triiodothyronine, T3) plays an important role in maturation of kidney growth and morphology (18) and can influence several transport processes in the kidney including that of sodium, phosphate, and adenosine (12, 27, 50). Serum sulfate concentrations are higher in hyperthyroid patients and lower in hypothyroid patients (43). However, the effect of T3 on sulfate renal transport is controversial. Tenenhouse et al. (44) demonstrated that sulfate uptake by sodium-dependent sulfate cotransport in renal BBM was significantly higher in T3-treated mice. However, Beers and Dousa (3) reported no effect of T3 treatment on sodium-sulfate cotransport in BBM isolated from both rats and mice. T3 also did not affect sodium-dependent sulfate uptake in opossum kidney cells (45). Thyroid hormone treatment may also alter sulfate/anion exchange in the kidney, possibly in a different manner. Chou et al. (8) reported that thyroid hormone treatment results in a decreased Vmax for lysosomal sulfate/anion exchange in the rat liver.
The objectives of this study were 1) to examine serum concentrations, renal clearance, and the fractional reabsorption of sulfate in a rat model of 6-propyl-2-thiouracil (PTU)-induced hypothyroidism; 2) to determine the renal transport of sulfate (both sodium-sulfate cotransport in BBM and sulfate/anion exchange in BLM) in membrane vesicle preparations isolated from the kidneys of hypothyroid rats; and 3) to determine the mechanism(s) involved in the hypothyroid-induced alterations in renal sulfate transport (NaSi-1).
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MATERIALS AND METHODS |
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Study design. Female Lewis rats weighing 200-220 g were used. Hypothyroid rats were produced by the administration of 0.05% PTU in the drinking water for 21 days (32). Control animals were given tap water during the treatment period. Food and water were provided to the rats ad libitum. Urine was collected for 12 h on the day before beginning the treatment (day 0) and on day 21. A blood sample was obtained at the midpoint of the urine collection period from the tail artery on days 0 and 21. Animals were weighed daily. Animals were killed by CO2 inhalation on day 21, and the kidneys were removed. Kidney cortex was trimmed and used for a membrane vesicle preparation or immediately frozen in liquid nitrogen for RNA and crude membrane preparations.
T3, sulfate, and creatinine analyses. T3 concentrations in serum were measured using a radioimmunoassay kit (Diagnostic Products, Los Angeles, CA). Serum and urinary sulfate concentrations were measured by single-column anion chromatography with a conductivity detector (Waters 431; Millipore, Milford, MA) and an anion-exchange precolumn and analytical column (Wescan Instruments, Santa Clara, CA) (29). 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 (11).
Renal vesicle preparation. BBM and BLM vesicles were prepared from kidney cortex by previously described procedures (4). The tissue from four to six animals in the same study group was combined for the membrane vesicle preparations. Briefly, the freshly isolated rat kidney cortex was homogenized in homogenizing buffer (250 mM sucrose and 10 mM triethanolamine-HCl, pH 7.6). BBM and BLM vesicles were separated using a Percoll (Sigma Chemical) density gradient centrifugation. The BBM fraction was further purified by MgCl2 precipitation. Both vesicle preparations were reconstituted in vesicle buffer (300 mM mannitol and 10 mM HEPES for the BBM; and 100 mM mannitol and 50 mM HEPES, pH 7.5 for the BLM). Alkaline phosphatase and Na+-K+-ATPase activities were measured as marker enzymes for BBM and BLM vesicles, respectively.
Sulfate transport studies. The sulfate transport was examined by measuring the uptake into membrane vesicles using a rapid filtration method (15). Freshly isolated membrane vesicles were used for the studies. 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 Na235SO4 (DuPont/NEN, Boston, MA) and various concentrations of K2SO4. Aliquots were filtered and rinsed with 5 ml ice-cold stop solution (300 mM mannitol and 10 mM HEPES for BBM; 200 mM mannitol and 50 mM HEPES for BLM, pH 7.5). All uptake studies were performed at room temperature.
The time course of sulfate uptake was evaluated at various time points (10, 30, 60, and 180 s, and 1 h). Preliminary studies demonstrated that uptake at 10 s represents the linear uptake process and that equilibrium conditions were obtained at 60 min in both BBM and BLM vesicle preparations. Therefore, 10-s uptake values were used for further concentration-dependent sulfate uptake studies to evaluate the Michaelis-Menten parameters. Sodium-dependent sulfate uptake was evaluated by measuring the various concentrations (0.1-8 mM) of sulfate uptake into BBM vesicles in the presence or absence of 100 mM NaCl. Sulfate uptake by anion-exchange transport was evaluated in the BLM vesicles using potassium bicarbonate as an anion exchanger (36). Diffusional uptake of sulfate was subtracted from total uptake by measuring uptake in BLM vesicles in the presence of potassium thiosulfate, a competitive inhibitor of the sulfate/anion exchanger, sat-1 (10). Vesicle protein concentrations were determined by the Coomassie blue binding method (6). Sulfate uptake rates from individual preparations were fitted to the Michaelis-Menten equation using the PCNONLIN nonlinear estimation program (Statistical Consultants, Lexington, KY) to obtain estimates of Km and Vmax. Uptake values were determined in triplicate uptake studies from one vesicle preparation, and studies were repeated five to six times.
Tissue RNA preparation. Total RNA was prepared from rat kidney cortex by the guanidium isothiocyanate method (7). The tissue from animals in the same group was combined and ground under liquid nitrogen before the initial homogenization step. Total RNA was prepared in duplicate from one tissue pool from each study group and used for the RT-PCR. Final RNA concentrations in the samples were determined by optical density at 260 nm.
RT-PCR. Primers, which were designed to produce a 700-bp DNA (native DNA), were prepared from the NaSi-1 cDNA (26). The 5' primer was constructed with a BamH I enzyme digestion site and a portion of the NaSi-1 cDNA corresponding to positions 492-513, CGTGGATCCACCAGTGCTGAAGCAGAGGCC. The 3' primer was constructed with a Pst I 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 a 100 bp of native cDNA located in the middle of the sequence. The cRNA transcribed in vitro from the deletion cDNA was added as an external standard to the RT-PCR mixture, and coamplified with sample RNA in the same reaction tube to correct for amplification efficiency. Fifty nanograms of tissue RNA and 10 fg deletion standard cRNA were reverse transcribed in the same tubes using SuperScript (Promega) at 42°C for 45 min. After the reverse transcriptase reaction, additional reactants for PCR including UlTma polymerase (Perkin-Elmer) 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 and transferred to hybridization matrices (Duralon-UV, Stratagene). The RT-PCR products were loaded on the gel in duplicate. The hybridization probe was a 301-bp NaSi-1 cDNA fragment (492-792 bp). The random primer labeling reaction was prepared using a random primer labeling kit (Prime-It; Stratagene, La Jolla, CA). Matrices were prehybridized for a minimum of 4 h and hybridized overnight in hybridizing solution (5× SSC, 1% SDS, 5× Denhardt's, 50% formamide, and 100 µg/ml sheared salmon sperm DNA) at 42°C. Matrices were washed five times in 2× SSC plus 0.1% SDS at room temperature, then twice in 0.1× SSC plus 0.1% SDS at room temperature followed by 0.1× SSC with 0.1% SDS at 65°C until the radioactivity was decreased to the background levels. 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 ELISA. Crude membrane fractions were prepared from kidney cortex to determine the protein expression levels in the tissue. 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 1,250 g for 10 min at 4°C. The supernatant was further centrifuged at 100,000 g for 30 min at 4°C (46). 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 method of Lowry et al. (23).
Sandwich-type ELISA procedure. NaSi-1 polyclonal and monoclonal antibodies were raised against rabbits and mice, respectively, as previously described (40). The antigen used for antibody production consisted of a recombinant protein containing 119 amino acids, which corresponded to amino acids 159-277 of the NaSi-1 protein. Western analysis showed that both the NaSi-1 polyclonal and monoclonal antibodies detected a 69-kDa protein in BBM. The size and the location of our immunoblot suggested that our antibodies recognized the NaSi-1 transporter. The NaSi-1 protein was detected by sandwich-type ELISA (39). The assay plates (polystyrene flat-bottom microtiter plates; Maxisorp, Nunc) 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 then incubated with samples (500 µg/well) at 4°C overnight. After incubation with NaSi-1 rabbit antiserum or preimmune serum (1:600 diluted in 0.3% BSA/PBS), wells were then incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG. Ten minutes after 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 in the tissue were calculated using a constructed standard curve using serial dilution of the NaSi-1 standard protein (6.58 to 164 fmol).
Evaluation of membrane motional order
(fluidity). The motional order of BBM and
BLM obtained from PTU-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 (1, 41). 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 a
spectrofluorometer (model 8000; SLM Aminco, Urbana, IL)
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]/ro,
where ro is the
maximal fluorescence anisotropy value in the absence of any rotational
motion (taken as 0.40) and r is the
steady-state anisotropy (35).
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 (GFR; estimated from
creatinine clearance), since 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 that was
reabsorbed was calculated by 1 (renal sulfate
clearance/GFR).
Statistics analysis. All results are expressed as means ± SD. A paired t-test was used to compare the values before and after the treatment in each group, and an unpaired t-test was used to compare the values between the groups.
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RESULTS |
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In vivo studies. Animals in both
groups gained 25-50 g after 21 days of the treatment, and there
were no differences in the weight gain in the two treatment groups.
There were no significant differences in all parameters measured on
day 0 between groups. Also there were
no significant differences in the control group in all parameters
(T3 values, creatinine clearance,
and sulfate disposition) evaluated before and after the treatment.
After 21 days of the treatment, animals that received PTU in their
drinking water exhibited significantly lower serum
T3 levels compared with the values
in control animals (Fig. 1). The creatinine
clearance was significantly lower in hypothyroid rats after 21 days of
treatment compared with the values on day
0 (2.9 ± 0.6 vs. 4.2 ± 1.5 ml · min1 · kg
1;
P < 0.01, n = 18). The mean values of serum
sulfate concentration, urinary excretion rate, renal sulfate clearance,
and renal reabsorption of sulfate on days
0 and 21 in each study
group are shown in Table 1. In the
hypothyroid rats, serum sulfate concentrations were significantly
decreased on day 21 compared with the
values for day 0 (P < 0.01, n = 26). There were no significant
differences in urinary excretion of sulfate and renal sulfate clearance
after PTU treatment. The fractional clearance of sulfate (sulfate
clearance/creatinine clearance) was significantly increased, and the
fraction of sulfate reabsorbed was significantly decreased in the
hypothyroid group on day 21 compared
with the values obtained on day 0 (P < 0.001, n = 15).
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Sulfate transport studies. The marker enzyme enrichment ratios (vesicles/homogenate) were similar to values reported previously (10). Enrichment ratios for alkaline phosphatase were 13.1 ± 6.5 (n = 6) and 14.1 ± 8.0 (n = 6) for control and hypothyroid, respectively. Enrichment ratios for Na+-K+-ATPase were 11.4 ± 5.2 (n = 6) and 12.4 ± 3.2 (n = 6) for control and hypothyroid, respectively. The time course for sulfate uptake was examined in BBM and BLM vesicles. Sulfate uptake exhibited a characteristic overshoot during the first minute of incubation in both BBM and BLM. Sulfate uptake in BBM by sodium-dependent sulfate transport was decreased at the 10-s time point in the hypothyroid group compared with that in the control group (0.43 ± 0.088 vs. 0.24 ± 0.087 nmol/mg protein; P < 0.01). There were no differences in the equilibrium uptake rates (evaluated at 60 min) in either membrane preparation, suggesting that there were no changes in sulfate binding or vesicle volume.
The BBM vesicles were incubated with or without sodium in the uptake
medium to determine kinetic parameters for sulfate transport in BBM.
Sulfate uptake into BBM vesicles increased linearly in the absence of
sodium. The difference between the two represents the sulfate uptake by
sodium-dependent sulfate cotransport.
Km and
Vmax 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.
2. The
Vmax value in the
hypothyroid group was significantly lower compared with the control
group (0.90 ± 0.31 and 0.49 ± 0.08 nmol · mg1 · 10 s
1 for control and
hypothyroid, respectively; P < 0.05, n = 5-6). The
Km for sulfate
transport in BBM was not significantly different between groups (0.44 ± 0.10 and 0.47 ± 0.19 mM for control and hypothyroid, respectively; n = 5-6). 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
Km (0.48 ± 0.21 vs. 0.30 ± 0.10 mM for control vs. hypothyroid, respectively,
n = 5-6) and
Vmax (0.52 ± 0.17 vs. 0.37 ± 0.09 nmol · mg
1 · 10 s
1 for control vs.
hypothyroid, respectively; n = 5-6) for the sulfate/anion exchange transport in BLM (Table
2).
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NaSi-1 mRNA abundance. Since the transport studies showed a difference in Vmax for sodium-dependent sulfate transport, the level of the mRNA for this transport gene (NaSi-1) was measured in kidney cortex and compared between the study groups. The southern hybridization blot is shown in Fig. 2A. The RT-PCR amplification efficiency was normalized by taking the hybridization signal ratio between native RNA (700 bp) and deletion standard cRNA (600 bp), and the ratio was normalized with the amount of total RNA loaded on the gel. The final values were expressed as the native vs. deletion standard DNA ratio per nanogram total RNA. The average of four lanes from the same study group was used for statistical analysis (Fig. 3). The steady-state mRNA levels in the hypothyroid group was significantly lower (decrease of 26.5%) compared with that in the control group (P < 0.01).
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NaSi-1 protein abundance. For the quantitative ELISA, the NaSi-1 protein standard was prepared from the purified NaSi-1 fusion protein. A linear relationship between the amounts of NaSi-1 standard protein vs. OD 490 was obtained (r2 = 0.99). A crude membrane fraction was isolated from the rat kidney cortex obtained from animals after 21 days of the treatment. ELISA was performed in triplicate from duplicate crude membrane preparations from each study group. The NaSi-1 protein abundance was significantly lower in the kidney cortex obtained from the hypothyroid animals than in the control group (decrease of 35.7%, P < 0.05, n = 6; Fig. 4).
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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 (1, 5). However, the PTU treatment did not produce any observable changes in membrane fluidity or the lipid order parameter for either BBM or BLM (Fig. 5).
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DISCUSSION |
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Thyroid hormone significantly influences some sodium-dependent transporters in the kidney, while having no effect on others. Sodium-phosphate cotransport in BBM isolated from rat and mouse kidneys is increased following T3 or T4 treatment due to an increased Vmax; no change in the Km for sodium-dependent phosphate transport occurs (13, 44, 49). This is consistent with the observation that hyperthyroid patients exhibit increased serum phosphate concentrations, whereas hypothyroid patients have lower serum phosphate levels (25). Sodium-dependent adenosine transport is decreased in BBM isolated from the kidney cortex of hypothyroid rats (27). However, unlike sodium-phosphate transport, this is due to a decreased affinity (Km) for the transport, with no reported change in the Vmax. T3 treatment can increase the activity of the Na+/H+ antiporter in rat kidney BBM (19), but it does not alter sodium-proline, sodium/D-glucose, or sodium-citrate cotransport processes (19, 49).
Early evidence of the hormonal regulation of sulfate by thyroid status was provided by Tallgren (43). He reported that clinical hyperthyroidism is associated with elevated serum sulfate concentrations and that hypothyroidism is associated with decreased serum sulfate concentrations. Tenenhouse et al. (44) demonstrated that sodium-dependent sulfate uptake in renal BBM is significantly increased in mice treated with a pharmacological dose of T3 compared with vehicle-treated controls due to an increased Vmax for sodium-sulfate cotransport. However, the effect of thyroid hormone on sodium-sulfate cotransport is controversial; Beers and Dousa (3) found no change in sodium-sulfate transport in renal BBM isolated from T3-treated rats or mice. Additionally, T3 does not stimulate sodium-dependent sulfate transport in opossum kidney cells (45).
The current investigation examined the effects of hypothyroidism on sulfate homeostasis and sulfate renal transport in the rat. Serum sulfate concentrations were significantly decreased in hypothyroid rats compared with those in control rats. This result is consistent with the lower serum sulfate concentrations reported for hypothyroid patients (43). GFR, estimated by creatinine clearance, was significantly decreased in hypothyroid rats compared with their pretreatment values, suggesting that amount of filtered sulfate was decreased in these animals. However, we observed no significant alterations in the urinary excretion or renal clearance of sulfate. Consequently, renal sulfate fractional reabsorption was significantly decreased in hypothyroid rats, supporting the findings of our in vitro study in membrane vesicles that showed a decreased Vmax for sodium-dependent sulfate uptake into BBM vesicles isolated from the hypothyroid rats. The lack of change in the renal clearance of sulfate in hypothyroid animals may be due to the changes in GFR observed in the present study, and which also occur in hypothyroid patients (18, 28). Decreased GFR results in elevated serum sulfate concentration, i.e., there is an inverse relationship between GFR and serum sulfate (2). One would expect decreased urinary excretion and renal clearance values in the presence of decreased GFR. However, it appears that the two opposing effects of decreased filtration and decreased reabsorption result in no apparent change in the renal clearance and urinary excretion of sulfate, although the renal clearance ratio (sulfate clearance/creatinine clearance) was significantly increased. It is also possible that the lack of change in the sulfate renal clearance in hypothyroid animals is partially due to reduced sulfate reabsorption in the papillary collecting duct. There is evidence indicating that sodium-sulfate cotransport-related mRNA and protein are expressed in collecting ducts (9).
We further investigated the mechanism of hypothyroidism-induced alterations on sodium-sulfate cotransport (NaSi-1). First, we investigated changes in Na+-K+-ATPase activity. Thyroid hormone regulates the activity of Na+-K+-ATPase in the kidneys possibly by direct upregulation of Na+-K+-ATPase in proximal tubule cells by a pretranslation mechanism (21). However, we found no significant difference in Na+-K+-ATPase activity between hypothyroid and control rats.
Second, we examined the membrane motional order (fluidity) in renal BBM and BLM, by examining the fluorescence polarization of DPH. Membrane fluidity affects the activity and kinetics of membrane-bound transport proteins and passive permeability properties (20). We have found that alterations in the membrane fluidity of Madin-Darby canine kidney (MDCK) cells due to preincubation with cholesterol or benzyl alcohol can alter sodium sulfate transport; the Vmax for sodium-sulfate cotransport increases with increased membrane fluidity (unpublished data). Thyroid hormones can influence the lipid composition of a number of cellular organelles in different tissues, resulting in changes in membrane fluidity. Thyroidectomy produces a decrease in the total cholesterol and phospholipid content of rat liver mitochondria (34). Hypothyroidism also alters the membrane fatty acid composition in rat brain mitochondria (42). However, the effect of hypothyroidism on membrane fluidity on kidney cortex BBM and BLM had not been previously examined. In this investigation, we found no significant differences in membrane fluidity in renal BBM and BLM isolated from the kidney of hypothyroid and control rats.
Third, we examined NaSi-1 mRNA and protein levels in kidney cortex of hypothyroid and control rats. NaSi-1 mRNA and protein levels were significantly lower in hypothyroid rats. It is possible that total tissue RNA and protein synthesis in the kidneys were lower in hypothyroid rats. Hayase et al. (16) reported that total tissue RNA and protein synthesis were lower in brain, kidney, and liver in hypothyroid rats. However, NaSi-1 mRNA and protein levels were significantly lower when normalized with total RNA and protein in tissue, suggesting that the NaSi-1 gene is regulated by thyroid hormone. These results indicate that the molecular mechanisms of the hypothyroidism-induced decrease of renal sulfate transport involves, at least in part, downregulation of NaSi-1 protein. NaSi-1 mRNA and protein levels are also significantly decreased in vitamin D deficiency (14) and following ibuprofen treatment (37) and increased following a low-sulfate diet (38).
In summary, significantly decreased serum sulfate concentrations, the renal fractional reabsorption of sulfate, and BBM sodium-sulfate cotransport were observed in experimentally induced hypothyroid rats. NaSi-1 mRNA and protein synthesis levels were significantly lower in these rats, suggesting that thyroid hormone is involved in renal cellular regulation of NaSi-1 by a mechanism not yet elucidated in detail.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. V. Balasubramanian [State University of New York (SUNY) at Buffalo] for performing the fluorescence polarization studies, Ms. Patricia Neubauer for technical assistance, and Dr. Richard R. Almon and Dr. Debra C. DuBois (SUNY at Buffalo) for advice and guidance with the RT-PCR and ELISA assays.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grant GM-40551, National Science Foundation Grant IBN 9629470, and grants from the Western New York Kidney Foundation/Upstate NY Transplant Services and the Kapoor Charitable Foundation at SUNY at Buffalo (for M. E. Morris), Swiss National Science Foundation (for H. Murer).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. E. Morris, 527 Hochstetter Hall, Dept. of Pharmaceutics, State Univ. of New York at Buffalo, Amherst, NY 14260.
Received 22 May 1998; accepted in final form 8 October 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Balasubramanian, S. V.,
R. M. Straubinger,
and
M. E. Morris.
Salicylic acid induces changes in the physical properties of model and native kidney membranes.
J. Pharm. Sci.
86:
199-204,
1997[Medline].
2.
Becker, E. L.,
H. O. Heinemann,
K. Igarashi,
J. E. Hodler,
and
H. Gershberg.
Renal mechanisms for the excretion of inorganic sulfate in man.
J. Clin. Invest.
39:
1909-1913,
1960.
3.
Beers, K. W.,
and
T. P. Dousa.
Thyroid hormone stimulates the Na+-PO4 symporter but not the Na+-SO4 symporter in renal brush border.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F323-F326,
1993
4.
Benincosa, L. J.,
and
M. E. Morris.
Inhibition of sulfate transport in rat kidney membrane vesicle preparations by nonsteroidal antiinflammatory drugs.
Drug Metab. Dispos.
21:
750-752,
1993[Medline].
5.
Benincosa, L. J.,
K. Sagawa,
and
M. E. Morris.
Renal adaptation to altered dietary sulfate in rats.
J. Pharmacol. Exp. Ther.
272:
248-255,
1995[Abstract].
6.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
7.
Chirgwin, J. M.,
A. E. Przybyla,
R. J. MacDonald,
and
W. J. Rutter.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[Medline].
8.
Chou, H.-F.,
M. Passage,
and
A. J. Jonas.
Regulation of lysosomal sulfate transport by thyroid hormone.
J. Biol. Chem.
269:
23524-23529,
1994
9.
Custer, M.,
H. Murer,
and
J. Biber.
Nephron localization of Na/SO24 cotransport-related mRNA and protein.
Pflügers Arch.
429:
165-168,
1994[Medline].
10.
Darling, I. M.,
M. L. Mammarella,
Q. Chen,
and
M. E. Morris.
Salicylate inhibits the renal transport of inorganic sulfate in rat membrane vesicle preparations.
Drug Metab. Dispos.
22:
318-323,
1994[Abstract].
11.
Darling, I. M.,
and
M. E. Morris.
Evaluation of "true" creatinine clearance in rats reveals extensive renal secretion.
Pharm. Res.
8:
1318-1322,
1991[Medline].
12.
Davis, P. J.,
and
F. B. Davis.
Nongenomic actions of thyroid hormone.
Thyroid
6:
497-504,
1996[Medline].
13.
Espinosa, R. E.,
M. J. Keller,
A. N. K. Yusufi,
and
T. P. Dousa.
Effect of thyroxine administration on phosphate transport across renal cortical brush border membrane.
Am. J. Physiol.
246 (Renal Fluid Electrolyte Physiol. 15):
F133-F139,
1984[Medline].
14.
Fernandes, I.,
G. Hampson,
X. Cahours,
P. Morin,
C. Coureau,
S. Couette,
D. Prie,
J. Biber,
H. Murer,
G. Friedlander,
and
C. Silve.
Abnormal sulfate metabolism in vitamin D-deficient rats.
J. Clin. Invest.
100:
2196-2203,
1997
15.
Goldinger, J. M.,
B. D. S. Khalsa,
and
S. K. Hong.
Photoaffinity of organic anion transport system in proximal tubule.
Am. J. Physiol.
247 (Cell Physiol. 16):
C217-C227,
1984[Abstract].
16.
Hayase, K.,
Y. Naganuma,
M. Moriyama,
A. Yoshida,
and
H. Yokogoshi.
Effect of a thyroid hormone treatment on brain protein synthesis in rats.
Biosci. Biotech. Biochem.
61:
1536-1540,
1997[Medline].
17.
Humphries, D. E.,
C. K. Silbert,
and
J. E. Silbert.
Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium.
J. Biol. Chem.
261:
9122-9127,
1986
18.
Katz, A. I.,
and
M. D. Lindheimer.
Actions of hormone on the kidney.
Annu. Rev. Physiol.
39:
97-133,
1977[Medline].
19.
Kinsella, J.,
and
B. Sacktor.
Thyroid hormones increase Na+-H+ exchange activity in renal brush border membranes.
Proc. Natl. Acad. Sci. USA
82:
3606-3610,
1985[Abstract].
20.
LeGrimellec, C.,
G. Friedlander,
E. H. E. Yandouzi,
P. Zlatkine,
and
M.-C. Giocondi.
Membrane fluidity and transport properties in epithelia.
Kidney Int.
42:
825-836,
1992[Medline].
21.
Lin, H. H.,
and
M. J. Tang.
Thyroid hormone upregulates Na-K-ATPase alpha and beta mRNA in primary cultures of proximal tubule cells.
Life Sci.
60:
375-382,
1997[Medline].
22.
Löw, I.,
T. Friedrich,
and
G. Buckhardt.
Properties on anion-exchanger in rat renal basolateral membrane vesicles.
Am. J. Physiol.
246 (Renal Fluid Electrolyte Physiol. 15):
F334-F342,
1984
23.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
24.
Lücke, H.,
G. Stange,
and
H. Murer.
Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex.
Biochem. J.
182:
223-229,
1979[Medline].
25.
Malamos, B.,
P. Sfikakis,
and
P. Pandos.
The renal handling of phosphate in thyroid disease.
J. Endocrinol.
45:
269-273,
1969[Medline].
26.
Markovich, D.,
J. Forgo,
G. Stange,
J. Biber,
and
H. Murer.
Expression cloning of rat renal Na+/SO24 cotransport.
Proc. Natl. Acad. Sci. USA
90:
8073-8077,
1993
27.
Martinez, F.,
M. Franco,
A. Quintana,
and
J. Herrera-Acosta.
Sodium-dependent adenosine transport is diminished in brush border membrane vesicles from hypothyroid rat kidney.
Pflügers Arch.
433:
269-275,
1997[Medline].
28.
Montenegro, J.,
O. Gonzalez,
R. Saracho,
R. Aguirre,
O. Gonzalez,
and
I. Martinez.
Changes in renal function in primary hypothyroidism.
Am. J. Kidney Dis.
27:
195-198,
1996[Medline].
29.
Morris, M. E.,
and
G. Levy.
Assay of inorganic sulfate in biologic fluids by nonsuppressed (single-column) ion chromatography.
Anal. Biochem.
172:
16-21,
1988[Medline].
30.
Mulder, G. J.
Sulfation availability in vivo.
In: Sulfation of Drugs and Related Compounds, edited by G. J. Mulder. Boca Raton, FL: CRC, 1981, p. 31-52.
31.
Mulder, G. J.
Sulfation in vivo and in isolated cell preparations.
In: Sulfation of Drugs and Related Compounds, edited by G. J. Mulder. Boca Raton, FL: CRC, 1981, p. 131-186.
32.
Murata, R.,
D. B. Haughey,
and
W. J. Jusko.
Effects of thyroid dysfunction on prednisolone and prednisone interconversion and disposition in the rat.
Drug Metab. Dispos.
18:
403-408,
1990[Abstract].
33.
Ofosu, F. A.,
G. J. Modi,
M. A. Blajchman,
M. R. Buchanan,
and
E. A. Johnson.
Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate.
Biochem. J.
248:
889-896,
1987[Medline].
34.
Parmar, D. V.,
M. A. Khandkar,
L. Pereira,
C. S. Bangur,
and
S. S. Katyare.
Thyroid hormones alter Arrhenius kinetics of succinate-2,6-dichloroindophenol reductase, and the lipid composition and membrane fluidity of rat liver mitochondria.
Eur. J. Biochem.
230:
576-581,
1995[Abstract].
35.
Pottel, H.,
W. van der Meer,
and
W. Herreman.
Correlation between the order parameter and the steady state fluorescence anisotropy of 1,6 diphenyl-1,3,5-hexatriene and an evaluation of membrane fluidity.
Biochim. Biophys. Acta
730:
181-186,
1983.
36.
Pritchard, J. B.,
and
J. L. Renfro.
Renal sulfate transport at the basolateral membrane is mediated by anion exchange.
Proc. Natl. Acad. Sci. USA
80:
2603-2607,
1983[Abstract].
37.
Sagawa, K., L. J. Benincosa, H. Murer, and M. E. Morris. Ibuprofen induced changes in sulfate renal
transport. J. Pharmacol. Exp. Ther. In
press.
38.
Sagawa, K., D. C. DuBois, R. R. Almon, H. Murer, and M. E. Morris. Cellular mechanisms of renal
adaptation of sodium dependent sulfate cotransport to altered dietary
sulfate in rats. J. Pharmacol. Exp.
Ther. In press.
39.
Sagawa, K.,
D. C. DuBois,
B. Han,
R. R. Almon,
J. Biber,
H. Murer,
and
M. E. Morris.
Detection and quantitation of a sodium- dependent sulfate cotransporter (NaSi-1) by sandwich-type enzyme linked immunosorbent assay.
Pflügers Arch.
437:
123-129,
1998[Medline].
40.
Sagawa, K.,
D. C. DuBois,
and
M. E. Morris.
Antibody production to sodium dependent sulfate transporter.
Pharm. Res.
14:
S334,
1997.
41.
Sarkar, S. N.,
S. V. Balasubramanian,
and
S. K. Sikdar.
Effect of fenvalerate, a pyrethroid insecticide on membrane fluidity.
Biochim. Biophys. Acta
1147:
137-142,
1993[Medline].
42.
Tacconi, M. T.,
G. Cizza,
G. Fumagalli,
P. S. Sartori,
and
M. Salmona.
Effect of hypothyroidism in adult rats on brain membrane fluidity and lipid content and composition.
Res. Commun. Chem. Pathol. Pharmacol.
71:
85-103,
1991[Medline].
43.
Tallgren, L. G.
Inorganic sulphate in relation to the serum thyroxine level and renal failure.
Acta Med. Scand. Suppl.
640:
1-100,
1980[Medline].
44.
Tenenhouse, H. S.,
J. Lee,
and
N. Harvey.
Renal brush-border membrane Na+-sulfate cotransport: stimulation by thyroid hormone.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F420-F426,
1991
45.
Tenenhouse, H. S.,
and
J. Martel.
Na+-dependent sulfate transport in opossum kidney cells is DIDS sensitive.
Am. J. Physiol.
265 (Cell Physiol. 34):
C54-C61,
1993
46.
Thomas, T. C.,
and
M. G. McNamee.
Purification of membrane proteins.
In: Guide to Protein Purification, edited by M. P. Deutscher. San Diego, CA: Academic, 1990, p. 499-520.
47.
Turner, R. J.
Sodium-dependent sulfate transport in renal outer cortical brush border membrane vesicles.
Am. J. Physiol.
247 (Renal Fluid Electrolyte Physiol. 16):
F793-F798,
1984
48.
Walser, M.,
D. W. Seldin,
and
A. Grollman.
An evaluation of radiosulfate for the determination of the volume of extracellular fluid in man and dogs.
J. Clin. Invest.
32:
299-311,
1953.
49.
Yusufi, A. N. K.,
N. Murayama,
M. J. Keller,
and
T. P. Dousa.
Modulatory effect of thyroid hormones on uptake of phosphate and other solutes across luminal brush border membrane of kidney cortex.
Endocrinology
116:
2438-2449,
1985[Abstract].
50.
Yusufi, A. N. K.,
M. Szczepanska-Konkel,
A. Hoppe,
and
T. P. Dousa.
Different mechanisms of adaptive increase in Na+-Pi cotransport across renal brush-border membrane.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F852-F861,
1989