Gender difference in the Oatp1-mediated tubular reabsorption of estradiol 17beta -D-glucuronide in rats

Yasumasa Gotoh1, Yukio Kato1,2, Bruno Stieger3, Peter J. Meier3, and Yuichi Sugiyama1,2

1 Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033; 2 Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan; and 3 Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8901 Zurich, Switzerland


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The gender difference in the urinary excretion of estradiol-17beta -glucuronide (E2-17beta G) was examined in rats. The urinary clearance of E2-17beta G was >250 times lower in male than in female rats. No such major gender difference was observed in its biliary excretion or metabolism in kidney homogenate. Both plasma protein binding and inulin clearance were comparable in male and female rats, suggesting that this gender difference cannot be explained by glomerular filtration. The urinary clearance with respect to the plasma unbound E2-17beta G in male rats was <1% of the glomerular filtration rate, indicating its potential reabsorption by the kidney, and this increased to a level comparable with that found in female rats when dibromosulfophthalein was coinfused. A marked increase in E2-17beta G urinary excretion was also observed in male rats that had undergone orchidectomy. Testosterone injections given to female rats reduced the urinary excretion to a level comparable with that of control male rats. The concomitant change in the expression of the gene product for organic anion-transporting polypeptide Oatp1, of which E2-17beta G is a typical substrate, was found in the kidney membrane fractions after these treatments. These results suggest that urinary E2-17beta G excretion is subject to hormonal regulation and that the large gender difference can be explained by regulation in Oatp1-mediated reabsorption.

gender difference; urinary excretion; reabsorption; organic anion-transporting polypeptide 1; multispecific resistance-associated protein 2


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THE DISPOSITION PROFILES of a number of xenobiotics exhibit gender differences, especially as far as animals are concerned (9, 12-14, 21, 23, 31-33, 35, 36). Hormonal regulation of the expression of certain types of metabolizing enzymes has been demonstrated. For example, CYP2C11 and 2C12 isoforms in liver microsomes are controlled by the plasma concentration profile of growth hormone, which is negatively and positively regulated by somatostatin and growth hormone-releasing factor, respectively, after stimulation by testosterone and estrogen (25). In another case, the metabolism of indinavir, probably due to CYP3A isoforms, exhibits a larger gender difference in rats, whereas the difference is relatively small in both monkeys and humans (20).

Gender differences have also been reported in hepatic and renal uptake and/or excretion. The Na+-dependent hepatic uptake of taurocholate is higher in male than in female rats (30). The uptake of tetraethylammonium, but not para-aminohippuric acid, by isolated kidney slices is higher in male than in female rats (5). Urinary excretion of xenobiotics, especially organic anions such as torasemide and zenarestat, shows gender differences: these compounds are excreted into the urine in much higher concentrations by female than by male rats, whereas this gender difference is minimal in humans (18, 33, 35).

Although the molecular mechanism for such gender differences in xenobiotic excretion is not yet fully characterized, several transporters have been shown to exhibit gender-dependent expression. The expression of the gene product for the Na+-taurocholate cotransporting polypeptide is higher in the liver of male than of female rats (30). The gene expression of the organic cation transporter OCT2 (gene symbol: Slc22a2) in the kidney is also higher in male than in female rats, whereas OCT1 (Slc22a1), OCT3 (Slc22a3), and organic anion transporter OAT1 (Slc22a6) exhibit only minimal gender differences (37). The gene expression of the organic anion-transporting polypeptide Oatp1 (Slc21a1) in rat kidney is regulated by testosterone and is about one-fifth less in female than in male rats (22).

Immunomorphological examination has revealed that the gene product detected by Oatp1 antibody is localized at the sinusoidal plasma membrane of the liver and at the apical plasma membrane of the kidney in the S3 segment of the proximal tubule of the outer medulla (3), although the physiological function of renal Oatp1 is still unclear. Oatp1 is believed to exhibit multispecific substrate recognition, since transfection of the gene encoding Oatp1 into mammalian cell lines results in the functional expression of the uptake system for a variety of organic anions (11). Therefore, these findings prompted us to examine the possibility of gender differences in the urinary excretion of typical substrates of Oatps if Oatp1 is involved in the secretion and/or reabsorption of its substrates at renal apical membranes. Estradiol 17beta -D-glucuronide (E2-17beta G) is a substrate of rat Oatp families that include Oatp1 (15), Oatp2 (Slc21a5) (26), and Oatp4 (Slc21a10) (8). E2-17beta G is mainly excreted into the bile after systemic injection in rats, and it is possible that Oatps are involved in its transport at the sinusoidal membrane, whereas at the canalicular membrane, multispecific resistance-associated proteins Mrp2 (Abcc2) are mainly involved in its transport (7, 15, 24). Mrp2 is also expressed at the apical membrane in the kidney (29). The contribution of Mrp2 to the urinary excretion of E2-17beta G seems to be small in rats (24), whereas the involvement of Oatps is still unknown as far as the kidney is concerned. In the present study, we examined the gender difference in the urinary excretion of E2-17beta G and found a >250-fold higher urinary clearance in female compared with male rats. We also attempted to clarify the possible relevance of such a gender difference in urinary excretion as well as in the expression of Oatp1.


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Materials. [3H]E2-17beta G (55 or 44 µCi/nmol) was purchased from New England Nuclear (Boston, MA). Inulin, mannitol, D-saccharic acid 1,4-lactone (1,4SL), and testosterone were purchased from Wako Pure Chemical Industries (Osaka, Japan). Dibromosulfophthalein (DBSP) was obtained from the Société d'Etudes et de Recherches Biologiques (Paris, France). The beta -glucuronidase activity assay kit was purchased from Sigma Chemical (St. Louis, MO). HPLC-grade methanol, chloroform, and acetic acid were purchased from Wako Pure Chemical Industries. All other chemicals and reagents were commercial products of analytical grade. Seven-week-old male and female Sprague-Dawley (SD) rats were purchased from Charles River Japan (Tokyo, Japan). Three-week-old male rats and 4-wk-old female rats were purchased for orchidectomy and testosterone treatment, respectively. Seven-week-old male Eisai hyperbilirubinemic rats (EHBR), which have a hereditary defect in Mrp2, were purchased from Japan SLC (Shizuoka, Japan). All animals had free access to water and food. The studies reported in this article have been carried out in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the US National Institutes of Health.

In vivo study. Rats underwent bile duct cannulation by use of polyethylene tubing (PE-10, 0.61 mm ID; Becton-Dickinson, Bedford, MA), and the bladder was catheterized by use of a different type of polyethylene tubing (no. 8, 2.33 mm OD; Hibiki, Tokyo, Japan). The body temperature of the rats was maintained by suitable lighting. The intravenous bolus injection study was performed as described previously (24). In the constant infusion study, [3H]E2-17beta G, dissolved in saline, was administered through the femoral vein at a priming dose of 0.4 µCi · ml-1 · kg-1, followed by a sustained infusion of 10 µCi · h-1 · kg-1 at a rate of 6 ml/h with a Terufusin syringe pump (model STC-531, Terumo, Tokyo, Japan), which was fitted with an extension tube (Safeed, Terumo). Inulin was administered as a priming dose of 16 mg · ml-1 · kg-1, followed by a sustained infusion of 20 mg/h. Blood was collected at 20, 40, 60, 80, 100, and 120 min from the jugular vein by use of a syringe (SS-01T, Terumo) without cannulation. Bile and urine specimens were taken at 0-20, 20-40, 40-60, 60-80, 80-100, and 100-120 min. The urinary bladder was catheterized with polyethylene tubing (no. 8, 2.33 mm OD; Hibiki), and the urine was collected by use of 0.2-0.5 ml of saline. Total radioactivity in plasma, bile, and urine was measured in a liquid scintillation counter (Tricarb 1900CA, Packard; Tokyo, Japan). In the inhibition study, DBSP (2.5 µmol · min-1 · kg-1) was injected after mixing it with the [3H]E2-17beta G injection solution. The infusion of 1,4SL (50 mg · h-1 · kg-1) was started 4 h before the start of the [3H]E2-17beta G injection, followed by the coadministration of [3H]E2-17beta G. This infusion rate of 1,4SL was set so that the amount injected per hour was almost the same as that in the report of Brunelle and Verbeeck (6), in which 1,4SL was injected as 11 boluses (50 mg/kg) at -1, -0.5, 0, 1, 2, 3, 4, 5, 6, 7, and 8 h. Mannitol was infused after mixing it at a concentration of 20% (wt/vol) with the infused solution. Plasma, urine, and bile samples were subjected to the following analysis to determine the parent compound ([3H]E2-17beta G). For analysis of the tissue uptake of [3H]E2-17beta G, blood was collected from the jugular vein after bolus administration of [3H]E2-17beta G (10 µCi/kg) via the femoral vein. At 0.5, 1, 3, and 5 min after administration, rats were killed, and liver, kidney, small intestine, and muscle were excised immediately. A portion of each tissue was weighed, and the radioactivity was counted. The tissue uptake clearance (CLuptake) was estimated as the initial slope of the plot (integration plot) of the ratio (Kp) of [3H]E2-17beta G in tissue to its blood concentration vs. the area under the blood concentration-time curve (AUC)/blood concentration (CB) of [3H]E2-17beta G (39). The plasma unbound fraction (fp) and the blood-to-plasma concentration ratio (Rb) were determined in vitro at a blood concentration of 150 pM, as described previously (16).

Determination of [3H]E2-17beta G and inulin. Plasma and urine were deproteinized with 2 volumes of methanol; then aliquots were transferred to Eppendorf tubes and lyophilized. Kidney and liver were mixed with 4 volumes (wt/vol) of saline and homogenized at 4°C. The homogenate was deproteinized with 2 volumes of methanol, and then aliquots were lyophilized. The dried residues were dissolved in methanol and spotted onto high-performance thin-layer chromatography (HPTLC) plates (10 × 10 cm, Whatman; Clifton, NJ). Bile specimens were spotted directly onto HPTLC plates, and then chromatography was carried out with a chloroform-methanol-acetic acid (10:4:1, vol/vol/vol) mixture. The radioactivity associated with each spot on the HPTLC plates was quantified using an image analyzer (BAS-2500, Fuji Photo Film, Tokyo, Japan). The detection limit was defined as 200 dpm. Inulin concentration in plasma was analyzed by spectrophotometric assay by a modification of the method of Heyrovsky (10). Briefly, 10 µl of sample were added to 2 µl of 0.5% beta -indolylacetic acid in ethanol and 80 µl of concentrated hydrochloride. After 24 h at ambient temperature, the samples were measured at 530 nm in a Spectramax 190 spectrometer (Molecular Devices).

Calculation of in vivo kinetic parameters. The plasma concentration-time profile after bolus injection was fitted to a two-exponential equation, and the AUC was estimated by integration. The total clearance (CLtotal) was calculated as the dose/AUC. The urinary and biliary clearance values (CLbile,p and CLurine,p, respectively) were calculated as the ratio of the cumulative excreted amount (up to 120 min) to the AUC. In the infusion study, the CLtotal was calculated as the infusion rate divided by the steady-state plasma concentration (Cpss), which was estimated as the mean value of the plasma concentrations at 80, 100, and 120 min. The CLurine,p and CLbile,p were obtained as the urinary or biliary excretion rate, which was assessed as the excretion rate between 80 and 120 min after the start of infusion divided by the Cpss. The intrinsic clearance (PSuptake) for tubular uptake from the extracellular space in the kidney was determined from CLuptake, fp, glomerular filtration rate (GFR), and Rb by use of the following equation
CL<SUB>uptake</SUB> − f<SUB>p</SUB>GFR = Q<SUB>p</SUB>f<SUB>p</SUB>PS<SUB>uptake</SUB>/ (1)

(Q<SUB>p</SUB> + f<SUB>p</SUB> PS<SUB>uptake</SUB>(1 − hematocrit)/R<SUB>b</SUB>)
where the hematocrit was assumed to be 0.45, and the GFR was taken as the CLtotal of inulin.

Metabolism of [3H]E2-17beta G in kidney homogenate. Kidney was added to 2 volumes (wt/vol) of 0.1 M acetic acid buffer (pH 5.0) and homogenized at 4°C. The rat kidney homogenate was incubated with [3H]E2-17beta G (0.5 µCi/ml) in the presence or absence of DBSP (300 µM) at 37°C for 30 min. The kidney homogenate obtained from male and female rats was also incubated with phenolphthalein monoglucuronide (6 mM). To assess the efficacy of the 1,4SL treatment in vivo, the kidney was resected after constant intravenous infusion of 1,4SL (50 mg · h-1 · kg-1) for 6 h, and then kidney homogenate was prepared. After incubation, samples were deproteinized with 2 volumes of methanol and then centrifuged. For the determination of [3H]estradiol, the supernatant was applied to an HPTLC plate and developed with CHCl3-MeOH-AcOH (10:4:1, vol/vol/vol). The metabolite of phenolphthalein monoglucuronide was determined using a beta -glucuronidase activity measurement kit (Sigma).

Orchidectomy in male rats and testosterone treatment of female rats. The experimental procedure followed that described in previous reports (22). Orchidectomy was performed in 3-wk-old male rats, which were then used for the experiments at 2 wk postsurgery. Testosterone was dissolved in polyethylene glycol 400 at a concentration of 25 µg/ml and then given to female rats at 4 wk of age for 1 wk by use of two subcutaneous osmotic minipumps (ALZET model 2001, ALZA, CA). The sham groups received polyethylene glycol 400 alone.

Western blot analysis of Oatp1 in liver and kidney membranes. The membrane fraction was obtained from the liver and kidney, and Western blot analysis was performed as described previously (27). The Oatp1 antibody used was raised against a fusion protein with the 40 COOH-terminal amino acids, and maltose-binding protein was described in the study by Eckhardt et al. (11). Although this antibody cross-reacts with rat Oatp3 (D. Sugiyama, unpublished observation), such a reaction could not affect the present finding on the renal expression of Oatp1, because the expression of Oatp3 was reported not to be detected in the kidney of male rats (38).

Simulation study for the urine flow-dependent increase in CLurine,p based on a physiological model. The physiological model was constructed by using a modification of the tube model for drug and water reabsorption proposed by Komiya (19). Although the previous model assumed only unidirectional transtubular transport (reabsorption) of the drug (19), here we considered bidirectional transport (i.e., secretion and reabsorption) of E2-17beta G. From the mass-balance equation between x and x+Delta x
Q(<IT>x</IT>)C(<IT>x</IT>) − Q(<IT>x+&Dgr;x</IT>)C(<IT>x+&Dgr;x</IT>) = &Dgr;AR(<IT>x</IT>)·P<SUB>1</SUB>·C<SUB>p</SUB>·f<SUB>p</SUB> (2)

− &Dgr;AR(<IT>x</IT>)·P<SUB>2</SUB>·C(<IT>x</IT>) − &Dgr;AR(<IT>x</IT>)·(1 − &rgr;)·P<SUB>w</SUB>·C(<IT>x</IT>)
where x, C(x), Q(x), AR, P1, P2, Pw, Cp, fp, and rho  are the length from the glomerular filtration site along the nephron, drug concentration in tubular fluid at x, tubular fluid flow rate at x, surface area of tubule at x, permeability of drug secretion, permeability of drug reabsorption, permeability of water, plasma concentration, plasma unbound fraction, and the reflection coefficient, respectively. Because the tubular fluid flow rate at x = 0 was assumed to be the same as the GFR (19)
Q(<IT>x</IT>) = GFR − AR(<IT>x</IT>)·P<SUB>w</SUB> (3)
Differentiation of Eq. 3 gives
dQ(<IT>x</IT>)/dAR(<IT>x</IT>) = −P<SUB>w</SUB> (4)
The urine flow rate (U) can be regarded as Q(x = l), where l is the length of the nephron
U = Q(<IT>x = l</IT>)<IT> =</IT> GFR <IT>− </IT>AR(<IT>l</IT>)<IT>·</IT>P<SUB>w</SUB> (5)
From Eqs. 2-5, the drug concentration in the urine [C(x = l)] can be obtained by
C(<IT>x = l</IT>)<IT> = </IT>[(GFR − U)f<SUB>p</SUB>C<SUB>p</SUB>&rgr; + f<SUB>p</SUB>C<SUB>p</SUB>PS<SUB>1</SUB> − f<SUB>p</SUB>C<SUB>p</SUB>PS<SUB>2</SUB>]/ (6)

[(GFR − U)&rgr; − PS<SUB>2</SUB>] · (U/GFR)<SUP>(PS<SUB>2</SUB>/(GFR−U) − &rgr;)</SUP>

− (f<SUB>p</SUB>C<SUB>p</SUB>PS<SUB>1</SUB>)/[(GFR−U)&rgr; − PS<SUB>2</SUB>]
where PS1 and PS2 are equal to P1 · AR(l) and P2 · AR(l), respectively. Because the CLurine,p can be regarded as C(x = l) · U/Cp
CL<SUB>urine,p</SUB> = f<SUB>p</SUB> · U · [(GFR−U)&rgr; + PS<SUB>1</SUB> − PS<SUB>2</SUB>]/ (7)

[(GFR−U)&rgr; − PS<SUB>2</SUB>) · (U/GFR)<SUP>(PS<SUB>2</SUB>/(GFR−U)−&rgr;)</SUP>

− (f<SUB>p</SUB> · U · PS<SUB>1</SUB>)/[(GFR−U)&rgr; − PS<SUB>2</SUB>]
In the simulation study, PS1, PS2, and rho  were set at various constant values, whereas GFR and fp were set at the mean values for the male and female rats observed in the present study.


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Gender differences in systemic elimination, excretion, and plasma protein binding of E2-17beta G. After intravenous administration of [3H]E2-17beta G, its elimination profile in plasma and excretion profile into urine and bile were followed (Fig. 1). Both the plasma concentration and biliary excretion profiles of E2-17beta G were slightly higher in male than in female rats (Fig. 1, A and C), whereas the urinary excretion was much less in male than in female rats (Fig. 1B). To normalize the gender difference in systemic elimination, both CLbile,p and CLurine,p were determined (Table 1). The CLurine,p in females was >250 times higher than that in males, whereas the gender difference in CLbile,p was not so obvious (Table 1). A similar phenomenon was also observed when E2-17beta G was infused intravenously (Fig. 2). The CLurine,p was >330 times higher in female than in male rats (Table 2). Figure 3 shows the HPTLC pattern of the radioactivity excreted into the urine during the intravenous infusion of E2-17beta G. In female rats, most of the radioactivity appeared at the position of E2-17beta G with other minor bands, including estradiol, whereas the E2-17beta G radioactivity was very low in male rats, with other minor bands (Fig. 3). Values for fp , Rb, and GFR did not exhibit such a large gender difference (Table 1). Compared with fpGFR, the CLurine,p in male rats was <1% of the fpGFR, whereas that in female rats was almost comparable with the fpGFR (Tables 1, 2).


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Fig. 1.   Time profiles of the plasma concentration (A) and cumulative urinary (B) and biliary (C) excretion of [3H]estradiol-17beta -glucuronide ([3H]E2-17beta G) in male and female rats after iv bolus injection. Rats received iv bolus injections of [3H]E2-17beta G (180 pmol/kg), and the amount of E2-17beta G in plasma, urine, and bile was determined by high-performance thin-layer chromatography (HPTLC). Urinary excretion in male rats was below the detection limit (B). Each point and vertical bar represent the mean ± SE of 3 rats.


                              
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Table 1.   Kinetic parameters of [3H]E2-17beta G after iv administration of [3H]E2-17beta G to male and female SD rats



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Fig. 2.   Time profiles of the plasma concentration (A) and urinary (B) and biliary (C) excretion of [3H]E2-17beta G in male and female rats during iv infusion. Rats received an iv priming dose of [3H]E2-17beta G (0.4 µCi · ml-1 · kg-1) and a subsequent infusion at 10 µCi · h-1 · kg-1. The amount of E2-17beta G in plasma, urine, and bile was determined by HPTLC. Each point with vertical bar represents the mean ± SE of 5 rats.


                              
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Table 2.   Kinetic parameters of [3H]E2-17beta G during constant iv infusion of [3H]E2-17beta G to male and female SD rats



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Fig. 3.   HPTLC analysis of the radioactivity excreted into the urine during iv infusion of E2-17beta G. [3H]E2-17beta G was infused at 10 µCi · h-1 · kg-1 in the presence or absence of dibromosulfophthalein (DBSP; 2.5 µmol · min-1 · kg-1). The urine was collected from 100 to 120 min after the start of the infusion and subjected to HPTLC. Authentic [3H]E2-17beta G (STD) was also applied, and the positions corresponding to [3H]E2-17beta G and estradiol (E2) are indicated. Typical results obtained from 3 rats are shown.

Tissue uptake profile of E2-17beta G. To check the gender difference in tissue uptake, the CLuptake of E2-17beta G by each tissue was examined. The CLuptake, assessed as the initial slope of the integration plot, exhibited a minimal gender difference in the kidney, liver, small intestine, and muscle (Fig. 4). The CLuptake in male and female rats was 536 and 590, 810 and 884, 27.5 and 25.7, and 17.9 and 12.5 µl · min-1 · g-1 in the kidney, liver, small intestine, and muscle, respectively (mean of three rats). The CLuptake in the kidney was approximately four times higher than the fpGFR in either male or female rats (Table 2). On the basis of Eq. 1, the PSuptake, which represents the intrinsic clearance for tubular uptake from the renal extracellular space (17), was calculated. The PSuptake was 37.4 and 49.6 ml · min-1 · kg body wt-1 in male and female rats, respectively.


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Fig. 4.   Tissue uptake profile of [3H]E2-17beta G. The time profiles for the plasma and tissue concentrations of [3H]E2-17beta G were measured after iv bolus injection, and data were expressed as integration plots of the ratio (Kp) of [3H]E2-17beta G in tissue to its blood concentration vs. the area under the blood concentration-time curve (AUC)/blood concentration (CB). The initial slope represents the uptake clearance (CLuptake) by each tissue. Data points and vertical and horizontal bars represent means ± SE of 3 independent experiments.

Effect of several treatments on the gender difference in E2-17beta G excretion. To assess the involvement of certain transport systems in the renal disposition of E2-17beta G, we next examined the effect of coadministration of DBSP, which is an inhibitor of organic anion transporters, on the CLurine,p of E2-17beta G. Coinfusion of DBSP increased the CLurine,p of E2-17beta G in male rats up to a level comparable with that of the control females, whereas the effect of DBSP was minimal in females (Table 2). DBSP reduced the CLbile,p of E2-17beta G in both sexes (Table 2). The radioactivity corresponding to the position of E2-17beta G appeared to increase in the HPTLC analysis of urine from male rats after coadministration of DBSP (Fig. 3). Mannitol was also coinfused with E2-17beta G to increase the urine flow rate and, subsequently, to affect the renal handling of E2-17beta G. Although mannitol infusion increased the CLurine,p of E2-17beta G in female rats, the CLurine,p in male rats was still below the detection limit in the presence of mannitol (Table 2). As a control experiment, the CLurine,p of theophylline was also examined, and this was found to be 1.36 and 0.598 ml · min-1 · kg-1, respectively, in the presence or absence of mannitol in male rats. Such a twofold increase in the CLurine,p of theophylline is compatible with a previous report (19). Under the same conditions, the GFR was 6.39 and 7.38 ml · min-1 · kg-1, respectively. The CLurine,p in male rats was also below the detection limit when 1,4SL, an inhibitor of beta -glucuronidase, was coinfused with E2-17beta G (Table 2).

The CLurine,p of E2-17beta G in male rats after orchidectomy was almost the same as the control level in female rats, whereas the effect of orchidectomy on the CLbile,p was relatively smaller (Table 3). In female rats, testosterone treatment reduced the CLurine,p to a level below the detection limit, with only a minimal effect on CLbile,p (Table 3).

                              
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Table 3.   Effects of androgen hormone

Gender difference in the urinary excretion of E2-17beta G in both normal and EHBR. To assess the involvement of Mrp2 in the gender difference, a similar analysis was performed in EHBR. A gender difference in CLurine,p was observed in EHBR as in the case of normal rats (Table 2). The CLbile,p of E2-17beta G was reduced in both male and female EHBR compared with the control rats (Table 2).

Metabolism of E2-17beta G in kidney homogenate. The metabolism of E2-17beta G was examined in kidney homogenate. E2-17beta G metabolism exhibited a minimal gender difference and was not affected by DBSP at 300 µM (Table 4). The metabolism of phenolphthalein glucuronide also exhibited a minimal gender difference (Table 4). After 1,4SL treatment in vivo, the kidney was resected, and the metabolism of phenolphthalein glucuronide was also examined. Such metabolic activity was reduced to approximately one-fourth of that of the controls (Table 4).

                              
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Table 4.   beta -Glucuronidase activity in rat kidney homogenate

Expression of the gene product for Oatp1 in liver and kidney. The membrane fraction was obtained from the liver and kidney of rats after orchidectomy and control male rats (5 wk of age) and testosterone-treated females and control females (5 wk of age). Expression of Oatp1 in female rat kidney was not detectable, although it was much higher in male rat kidney (Fig. 5). Oatp1 expression in the kidney of male rats after orchidectomy was much lower than in control rats, whereas Oatp1 expression in the kidney of testosterone-treated female rats increased compared with control rats (Fig. 5). The Oatp1 expression in the liver did not show any clear gender difference (Fig. 5). These results were compatible with previous findings on the change in Oatp1 mRNA level reported by Lu et al. (22). The renal Oatp1 migrated as peptides of 38 and 40 kDa protein in the presence of 1,4-dithiothreitol (DTT), but liver Oatp1 migrated as 85 kDa protein, as reported by Bergwerk et al. (3). A gender difference in renal Oatp1 expression was also observed in EHBR (at 7 wk of age), whereas only a minimal gender difference was observed in the liver of EHBR (Fig. 5).


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Fig. 5.   Expression of the gene product for organic anion transporting polypeptide 1 (Oatp1) in the kidney and liver. Expression of Oatp1 was examined in Sprague-Dawley (SD) rat controls (male; 12, 6, 2.4 µg/lane, female; 12 µg/lane), in SD male rats following orchidectomy (12 µg/lane), in testosterone-treated female rats (12 µg/lane), and in Eisai hyperbilirubinemic rats (EHBR; 12 µg/lane). DTT, 1,4-dithiothreitol. Membrane fractions were prepared from 3 rats, and a typical result is shown.

Simulation of urine flow dependence in the CLurine,p of E2-17beta G. To explain the gender difference in the CLurine,p by membrane transport processes involved in renal secretion and/or reabsorption, a simulation study was performed by varying PS1, PS2, and rho . Because the PS1 represents the permeability clearance for the net secretion from the blood side to the urine side, this PS1 should be equal to or less than the PSuptake that represents only the clearance for the tubular uptake from the blood to the tubules. In the present simulation study, PS1 was set at 0.01, 0.1, 1, or 10 ml · min-1 · kg-1 (Fig. 6). The rho  for E2-17beta G has not yet been experimentally determined, although it has been reported that the rho  is almost unity when the molecular weight is higher than 170 (19). Therefore, rho  was set at 0.009, 0.09, 0.9, or 0.99 (Fig. 6). When the rho  was 0.009, 0.09, and 0.9, all the simulated lines were under the observed CLurine,p in female rats at any of the PS1 and PS2 values examined (data not shown), probably because the effect of convection flow on the reabsorption of E2-17beta G was too great. At an rho  of 0.99 and a PS1 of 10 ml · min-1 · kg-1, the CLurine,p of E2-17beta G at the control level of U (~ 20 µl · min-1 · kg-1) in female rats can be simulated when PS2 is 1 ml · min-1 · kg-1 (Fig. 6D). The observed CLurine,p of E2-17beta G in the presence of DBSP was also simulated in both sexes when PS1 and PS2 were 10 and 1 ml · min-1 · kg-1, respectively (Fig. 6D). In this case, both the simulated and observed CLurine,p in female rats did not increase as much as when the U increased (Fig. 6D). On the other hand, when the PS2 was increased and set at 9-216 ml · min-1 · kg-1, the simulated CLurine,p clearly depended on the U (Fig. 6D). At a PS2 of 216 ml · min-1 · kg-1, the simulated CLurine,p was still below the limit of detection when the U was set at the observed value in the presence of mannitol (Fig. 6D). Thus the simulated CLurine,p at a PS1 and a PS2 of 10 and 216 ml · min-1 · kg-1, respectively, was compatible with the present finding that the CLurine,p in male rats was below the detection limit in the presence and absence of mannitol (Table 2).


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Fig. 6.   Simulation study of the effect of intrinsic clearances for secretion (PS1) and reabsorption (PS2) on the urinary clearance (CLurine,p) of E2-17beta G. The PS1, PS2, and reflection coefficient (rho ) were set at constant values, as shown in A-D, whereas urine flow rate (U) was changed. The observed value for CLurine,p in male and female rats in the presence of mannitol or DBSP, or in their absence, is also shown. The detection limit for CLurine,p is also shown as a broken line. Note that the observed CLurine,p in control and mannitol-treated male rats was below the detection limit; therefore, the observed U alone is indicated by an arrow.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we found a marked gender difference in the urinary excretion profile of E2-17beta G in rats (Fig. 1). When we consider that the plasma concentration profile was not very different between the two sexes (Fig. 1A), such a gender difference in urinary excretion cannot be explained by its systemic elimination and may possibly be due to renal handling of E2-17beta G. In addition, because the fp, Rb, and GFR are also comparable in males and females (Table 1), the gender difference should be minimal as far as the glomerular filtration of E2-17beta G is concerned. Because the CLuptake was almost comparable for both sexes (Fig. 2), there should be only a minimal gender difference in the renal tubular uptake of E2-17beta G from the blood side.

It should be noted that the CLurine,p of E2-17beta G in male rats was <1% (Tables 1, 2) of the fpGFR, whereas the CLurine,p was almost comparable with the fpGFR in female rats. This result indicates extensive reabsorption of E2-17beta G in the renal tubules of male rats. To obtain further evidence for such reabsorption, the effect of mannitol on the CLurine,p was examined, since the efficiency of reabsorption should be reduced when the urine flow rate is increased due to the osmotic effect produced by mannitol (19, 34). Actually, the CLurine,p of E2-17beta G was increased 1.5-fold by mannitol in female rats, whereas that in male rats was still below the detection limit even in the presence of mannitol (Table 2). Thus, although the increase in CLurine,p by mannitol suggests that there is reabsorption of E2-17beta G, mannitol treatment cannot completely eliminate the gender difference in the CLurine,p of E2-17beta G (Table 2), and further studies were performed to analyze the renal handling of E2-17beta G.

Lu et al. (22) reported that the expression of mRNA for Oatp1 in kidney is ~6 times higher in male than in female rats. The Oatp1 gene product detected by the same antibody as that used in the present study was localized at the apical membrane in the kidneys of male rats (3). When the fact that E2-17beta G is a substrate of Oatp1 (15) is considered, the gender difference in E2-17beta G excretion could at least partially be explained by different expression levels of Oatp1 in male and female rats. To test this hypothesis, both the urinary excretion of E2-17beta G (Table 3) and the expression of Oatp1 protein in the kidney (Fig. 5) were examined both in male rats subjected to orchidectomy and in female rats treated with testosterone. Testosterone-treated female rats were included because Lu et al. demonstrated hormonal regulation of Oatp1 mRNA expression (22). After orchidectomy, male rats had a CLurine,p value that was almost comparable with that of female control rats, and the testosterone-treated female rats had a CLurine,p comparable with that of control male rats (Table 3). A similar change produced by these treatments was also observed in the expression of Oatp1 gene product in the kidney (Fig. 5). These results support testosterone-dependent regulation of both urinary excretion of E2-17beta G and apical Oatp1 expression in the proximal tubules. In addition, the Oatp1 inhibitor DBSP increased the CLurine,p in male rats showing higher Oatp1 expression but had a weaker effect in female rats with lower Oatp1 expression (Table 2). Therefore, it is most likely that the gender difference in the urinary excretion of E2-17beta G is caused by the gender difference in renal Oatp1 expression, although the present data cannot exclude the possibility of a contribution from other unknown E2-17beta G transporter(s), the expression of which would also be under testosterone control. Lu et al. have reported that mRNA for Oatp1 is slightly increased by oophorectomy, and administration of estradiol to oophorectomized female rats moderately suppresses renal Oatp1 mRNA expression, suggesting presumably weaker estrogen control. To characterize in greater detail the hormonal regulation of urinary E2-17beta G excretion, further studies, including oophorectomy in females, are needed.

The expression of Oatp1 in the liver did not show any clear gender difference (Ref. 22 and Fig. 5 of this study), which supports previous findings by Simon et al. (30). These results are compatible with a minimal gender difference in the CLuptake of E2-17beta G in the liver (Tables 1, 2). The gene product detected by the Oatp1 antibody used in the present study has, as indicated by Bergwerk et al. (3), at least two forms as far as the disulfide bond is concerned: reduction during SDS-PAGE affected the migration of the reactive band in the kidney, whereas it did not affect the migration of reactive protein in the liver. It remains to be investigated whether the kidney and liver Oatp1 isoforms have distinct substrate specificities and different E2-17beta G transport properties.

We have previously reported that Mrp2 is mainly involved in the biliary excretion of E2-17beta G, whereas its contribution to renal excretion seems to be minor as evidenced by similar excretion profiles in normal rats and EHBR (24). Because the latter analysis was performed only in male rats, we also analyzed the E2-17beta G excretion in female rats in the present study (Table 2). The CLbile,p of E2-17beta G was also much higher in normal female rats than in female EHBR, whereas the difference in the CLurine,p was minimal only between the two rat strains (Table 2). These results suggest that Mrp2 is not the major transporter of E2-17beta G in the kidney. When we consider that the gender difference in the CLurine,p of E2-17beta G can still be observed in EHBR (Table 2), Mrp2 does not seem to be involved in the gender difference of urinary E2-17beta G excretion.

In both the bolus and infusion studies, the CLtotal of E2-17beta G was slightly higher in female than in male rats (Tables 1, 2). Because the CLbile,p did not show a clear gender difference and the CLurine,p accounts, at most, for a low percentage of the CLtotal (Tables 1, 2), these findings suggest that the systemic elimination of E2-17beta G by processes other than excretion exhibits only a small gender difference. Renal beta -glucuronidase is reported to be under androgenic control (28). Ballantyne and Bright (1) reported a gender difference in the activity of rat kidney, although this difference was only approximately twofold. Hence, it might be possible that a gender difference also exists in the renal metabolism of E2-17beta G. However, we did not find any clear gender difference in the metabolism of either phenolphthalein glucuronide or E2-17beta G in whole kidney homogenate (Table 4). In addition, we could not detect any metabolism of E2-17beta G in male and female urine during a 2-h sampling period.

Finally, a simulation study based on a physiological model also suggested that the observed gender difference in CLurine,p can simply be explained by alterations in the tubular reabsorption process (Fig. 6). When the PS1 was fixed at 10 ml · min-1 · kg-1, the observed CLurine,p in control female rats could be simulated at a PS2 of 1 ml · min-1 · kg-1 (Fig. 6D). On the other hand, the CLurine,p in control male rats was below the detection limit (Tables 1, 2), and this can also be simulated when the PS1 was fixed at the same value, whereas the PS2 increased to 216 ml · min-1 · kg-1 (Fig. 6D). In addition, the urine flow dependence in the CLurine,p of male and female rats was also compatible with the simulated values under the same PS1 and PS2 values (Fig. 6D). Furthermore, the observed CLurine,p in male rats was increased by DBSP compared with that of female rats (Table 2), and this can also be simulated when the PS2 was reduced, because of inhibition of the reabsorption process by DBSP, from 216 to 1 ml · min-1 · kg-1 (Fig. 6D). These observations are compatible with the tubular reabsorption process being responsible for gender difference in CLurine,p, and thus they support a significant causative role of Oatp1-mediated reabsorption of E2-17beta G in the renal proximal tubules of male rats.

In conclusion, this study demonstrated a considerable gender difference in urinary excretion of E2-17beta G in male and female rats. This gender difference is most probably caused by testosterone-dependent upregulation of Oatp1 at the apical membrane of renal proximal tubular epithelial cells and cannot be explained by glomerular filtration, renal tubular uptake from the basolateral side, and/or Mrp2-mediated secretion into the urine. It remains to be investigated whether Oatp1 and/or other apical renal organic anion transporters contribute also to gender differences in the urinary excretion of other drugs and drug metabolites.


    ACKNOWLEDGEMENTS

We thank Hiroyuki Kusuhara for help with the Western blot analysis.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. Sugiyama, Graduate School of Pharmaceutical Sciences, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp).

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. Section 1734 solely to indicate this fact.

First published February 19, 2002;10.1152/ajpendo.00363.2001

Received 9 August 2001; accepted in final form 6 February 2002.


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