Gender difference in the Oatp1-mediated tubular reabsorption
of estradiol 17
-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
 |
ABSTRACT |
The gender
difference in the urinary excretion of estradiol-17
-glucuronide
(E2-17
G) was examined in rats. The urinary clearance of
E2-17
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 E2-17
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
E2-17
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 E2-17
G is a typical substrate, was found in the
kidney membrane fractions after these treatments. These results suggest
that urinary E2-17
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
 |
INTRODUCTION |
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
17
-D-glucuronide (E2-17
G) is a substrate
of rat Oatp families that include Oatp1 (15), Oatp2
(Slc21a5) (26), and Oatp4 (Slc21a10)
(8). E2-17
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 E2-17
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 E2-17
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.
 |
MATERIALS AND METHODS |
Materials.
[3H]E2-17
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
-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]E2-17
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]E2-17
G injection solution. The
infusion of 1,4SL (50 mg · h
1 · kg
1) was started
4 h before the start of the
[3H]E2-17
G injection, followed by the
coadministration of [3H]E2-17
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]E2-17
G). For analysis of the tissue
uptake of [3H]E2-17
G, blood was collected
from the jugular vein after bolus administration of
[3H]E2-17
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]E2-17
G in tissue to its blood
concentration vs. the area under the blood concentration-time curve
(AUC)/blood concentration (CB) of
[3H]E2-17
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]E2-17
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%
-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
|
(1)
|
where the hematocrit was assumed to be 0.45, and the GFR was
taken as the CLtotal of inulin.
Metabolism of [3H]E2-17
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]E2-17
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
-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 E2-17
G. From the
mass-balance equation between x and
x+
x
|
(2)
|
where x, C(x), Q(x), AR,
P1, P2, Pw, Cp,
fp, and
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)
|
(3)
|
Differentiation of Eq. 3 gives
|
(4)
|
The urine flow rate (U) can be regarded as Q(x =
l), where l is the length of the nephron
|
(5)
|
From Eqs. 2-5, the drug concentration in the
urine [C(x = l)] can be obtained by
|
(6)
|
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
|
(7)
|
In the simulation study, PS1, PS2, and
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.
 |
RESULTS |
Gender differences in systemic elimination, excretion, and plasma
protein binding of E2-17
G.
After intravenous administration of
[3H]E2-17
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 E2-17
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 E2-17
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 E2-17
G. In female rats, most of the radioactivity
appeared at the position of E2-17
G with other minor
bands, including estradiol, whereas the E2-17
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-17 -glucuronide
([3H]E2-17 G) in male and female rats after
iv bolus injection. Rats received iv bolus injections of
[3H]E2-17 G (180 pmol/kg), and the amount
of E2-17 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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Fig. 2.
Time profiles of the plasma concentration (A)
and urinary (B) and biliary (C) excretion of
[3H]E2-17 G in male and female rats during
iv infusion. Rats received an iv priming dose of
[3H]E2-17 G (0.4 µCi · ml 1 · kg 1) and a
subsequent infusion at 10 µCi · h 1 · kg 1. The
amount of E2-17 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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Fig. 3.
HPTLC analysis of the radioactivity excreted into the
urine during iv infusion of E2-17 G.
[3H]E2-17 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]E2-17 G (STD) was also applied, and the
positions corresponding to [3H]E2-17 G and
estradiol (E2) are indicated. Typical results obtained from
3 rats are shown.
|
|
Tissue uptake profile of E2-17
G.
To check the gender difference in tissue uptake, the
CLuptake of E2-17
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]E2-17 G.
The time profiles for the plasma and tissue concentrations of
[3H]E2-17 G were measured after iv bolus
injection, and data were expressed as integration plots of
the ratio (Kp) of [3H]E2-17 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
E2-17
G excretion.
To assess the involvement of certain transport systems in the renal
disposition of E2-17
G, we next examined the effect of coadministration of DBSP, which is an inhibitor of organic anion transporters, on the CLurine,p of E2-17
G.
Coinfusion of DBSP increased the CLurine,p of
E2-17
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
E2-17
G in both sexes (Table 2). The radioactivity
corresponding to the position of E2-17
G appeared to
increase in the HPTLC analysis of urine from male rats after
coadministration of DBSP (Fig. 3). Mannitol was also coinfused with
E2-17
G to increase the urine flow rate and,
subsequently, to affect the renal handling of E2-17
G. Although mannitol infusion increased the CLurine,p of
E2-17
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
-glucuronidase, was coinfused with E2-17
G (Table 2).
The CLurine,p of E2-17
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).
Gender difference in the urinary excretion of E2-17
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 E2-17
G was
reduced in both male and female EHBR compared with the control rats
(Table 2).
Metabolism of E2-17
G in kidney homogenate.
The metabolism of E2-17
G was examined in kidney
homogenate. E2-17
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).
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
E2-17
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
. 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
for E2-17
G has
not yet been experimentally determined, although it has been reported
that the
is almost unity when the molecular weight is higher than
170 (19). Therefore,
was set at 0.009, 0.09, 0.9, or
0.99 (Fig. 6). When the
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 E2-17
G was too great. At an
of 0.99 and a PS1 of 10 ml · min
1 · kg
1, the
CLurine,p of E2-17
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
E2-17
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 E2-17 G. The
PS1, PS2, and reflection coefficient ( ) 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 |
In the present study, we found a marked gender difference in the
urinary excretion profile of E2-17
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
E2-17
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 E2-17
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 E2-17
G from the blood side.
It should be noted that the CLurine,p of
E2-17
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 E2-17
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
E2-17
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 E2-17
G, mannitol treatment cannot
completely eliminate the gender difference in the CLurine,p of E2-17
G (Table 2), and further studies were performed
to analyze the renal handling of E2-17
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 E2-17
G is
a substrate of Oatp1 (15) is considered, the gender
difference in E2-17
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
E2-17
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 E2-17
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 E2-17
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
E2-17
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
E2-17
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 E2-17
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
E2-17
G transport properties.
We have previously reported that Mrp2 is mainly involved in the biliary
excretion of E2-17
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
E2-17
G excretion in female rats in the present study
(Table 2). The CLbile,p of E2-17
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 E2-17
G in the kidney. When we
consider that the gender difference in the CLurine,p of
E2-17
G can still be observed in EHBR (Table 2), Mrp2
does not seem to be involved in the gender difference of urinary
E2-17
G excretion.
In both the bolus and infusion studies, the CLtotal of
E2-17
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 E2-17
G
by processes other than excretion exhibits only a small gender
difference. Renal
-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 E2-17
G. However, we did not find any clear gender difference in the metabolism of either phenolphthalein glucuronide or
E2-17
G in whole kidney homogenate (Table 4). In
addition, we could not detect any metabolism of E2-17
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 E2-17
G in the
renal proximal tubules of male rats.
In conclusion, this study demonstrated a considerable gender difference
in urinary excretion of E2-17
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.
 |
REFERENCES |
1.
Ballantyne, B,
and
Bright JE.
Variations in mammalian renal cortical beta-glucuronidase activity according to species, strain and sex.
Cell Mol Biol
23:
369-377,
1978[ISI][Medline].
2.
Bardelli, A,
Ponzetto C,
and
Comoglio PM.
Identification of functional domains in the hepatocyte growth factor and its receptor by molecular engineering.
J Biotechnol
37:
109-122,
1994[ISI][Medline].
3.
Bergwerk, AJ,
Shi X,
Ford AC,
Kanai N,
Jacquemin E,
Burk RD,
Bai S,
Novikoff PM,
Stieger B,
Meier PJ,
Schuster VL,
and
Wolkoff AW.
Immunologic distribution of an organic anion transport protein in rat liver and kidney.
Am J Physiol Gastrointest Liver Physiol
271:
G231-G238,
1996[Abstract/Free Full Text].
4.
Bottaro, DP,
Rubin JS,
Faletto DL,
Chan A,
Kmiecik TE,
Vande Woude GF,
and
Aaronson SA.
Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.
Science
251:
802-804,
1991[ISI][Medline].
5.
Braunlich, H,
Meinig T,
and
Grosch U.
Postnatal development of sex differences in renal tubular transport of p-aminohippurate (PAH) in rats.
Exp Toxicol Pathol
45:
309-313,
1993[ISI][Medline].
6.
Brunelle, FM,
and
Verbeeck RK.
Conjugation-deconjugation cycling of diflunisal via beta-glucuronidase catalyzed hydrolysis of its acyl glucuronide in the rat.
Life Sci
60:
2013-2021,
1997[ISI][Medline].
7.
Buchler, M,
Konig J,
Brom M,
Kartenbeck J,
Spring H,
Horie T,
and
Keppler D.
cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats.
J Biol Chem
271:
15091-15098,
1996[Abstract/Free Full Text].
8.
Cattori, V,
Hagenbuch B,
Hagenbuch N,
Stieger B,
Ha R,
Winterhalter KE,
and
Meier PJ.
Identification of organic anion transporting polypeptide 4 (Oatp4) as a major full-length isoform of the liver-specific transporter-1 (rlst-1) in rat liver.
FEBS Lett
474:
242-245,
2000[ISI][Medline].
9.
Crofton, JT,
Ratliff DL,
Brooks DP,
and
Share L.
The metabolic clearance rate of and pressor responses to vasopressin in male and female rats.
Endocrinology
118:
1777-1781,
1986[Abstract].
10.
Dawborn, JK.
Application of Heyrovsky's inulin method to automatic analysis.
Clin Chim Acta
12:
63-66,
1965[ISI][Medline].
11.
Eckhardt, U,
Schroeder A,
Stieger B,
Hochli M,
Landmann L,
Tynes R,
Meier PJ,
and
Hagenbuch B.
Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells.
Am J Physiol Gastrointest Liver Physiol
276:
G1037-G1042,
1999[Abstract/Free Full Text].
12.
Griffin, RJ,
Godfrey VB,
Kim YC,
and
Burka LT.
Sex-dependent differences in the disposition of 2,4-dichlorophenoxyacetic acid in Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters.
Drug Metab Dispos
25:
1065-1071,
1997[Abstract/Free Full Text].
13.
Harris, RZ,
Benet LZ,
and
Schwartz JB.
Gender effects in pharmacokinetics and pharmacodynamics.
Drugs
50:
222-239,
1995[ISI][Medline].
14.
Hart, CD,
Flozak AS,
and
Simmons RA.
Modulation of glucose transport in fetal rat lung: a sexual dimorphism.
Am J Respir Cell Mol Biol
19:
63-70,
1998[Abstract/Free Full Text].
15.
Kanai, N,
Lu R,
Bao Y,
Wolkoff AW,
Vore M,
and
Schuster VL.
Estradiol 17 beta-D-glucuronide is a high-affinity substrate for oatp organic anion transporter.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F326-F331,
1996[Abstract/Free Full Text].
16.
Kato, Y,
Akhteruzzaman S,
Hisaka A,
and
Sugiyama Y.
Hepatobiliary transport governs overall elimination of peptidic endothelin antagonists in rats.
J Pharmacol Exp Ther
288:
568-574,
1999[Abstract/Free Full Text].
17.
Kino, I,
Kato Y,
Lin JH,
and
Sugiyama Y.
Renal handling of biphosphonate alendronate in rats.
Biopharm Drug Dispos
20:
193-198,
1999[ISI][Medline].
18.
Kling, L,
Schaumann W,
Kaufmann B,
Sponer G,
and
Bartsch W.
Sex difference in saluretic and diuretic activity of torasemide in Sprague-Dawley rats (Abstract).
Naunyn Sch Arch Pharmacol
344, Suppl 2:
R114,
1991.
19.
Komiya, I.
Urine flow dependence of renal clearance and interrelation of renal reabsorption and physicochemical properties of drugs.
Drug Metab Dispos
14:
239-245,
1986[Abstract].
20.
Lin, JH,
Chiba M,
Chen IW,
Nishime JA,
and
Vastag KJ.
Sex-dependent pharmacokinetics of indinavir: in vivo and in vitro evidence.
Drug Metab Dispos
24:
1298-1306,
1996[Abstract].
21.
Lin, JH,
Chiba M,
Chen IW,
Nishime JA,
and
Vastag KJ.
Sex-dependent pharmacokinetics of indinavir in vivo and in vitro evidence.
Drug Metab Dispos
24:
1298-1306,
1996[Abstract].
22.
Lu, R,
Kanai N,
Bao Y,
Wolkoff AW,
and
Schuster VL.
Regulation of renal oatp mRNA expression by testosterone.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F332-F337,
1996[Abstract/Free Full Text].
23.
Meffin, PJ,
Brooks PM,
and
Sallustio BC.
Alternations in prednisolone disposition as a result of time of administration, gender and dose.
Br J Clin Pharmacol
17:
395-404,
1984[ISI][Medline].
24.
Morikawa, A,
Goto Y,
Suzuki H,
Hirohashi T,
and
Sugiyama Y.
Biliary excretion of 17beta-estradiol 17beta-D-glucuronide is predominantly mediated by cMOAT/MRP2.
Pharm Res
17:
546-552,
2000[ISI][Medline].
25.
Mugford, CA,
and
Kedderis GL.
Sex-dependent metabolism of xenobiotics.
Drug Metab Rev
30:
441-498,
1998[ISI][Medline].
26.
Noe, B,
Hagenbuch B,
Stieger B,
and
Meier PJ.
Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain.
Proc Natl Acad Sci USA
94:
10346-10350,
1997[Abstract/Free Full Text].
27.
Ogawa, K,
Suzuki H,
Hirohashi T,
Ishikawa T,
Meier PJ,
Hirose K,
Akizawa T,
Yoshioka M,
and
Sugiyama Y.
Characterization of inducible nature of MRP3 in rat liver.
Am J Physiol Gastrointest Liver Physiol
278:
G438-G446,
2000[Abstract/Free Full Text].
28.
Paigen, K.
Mammalian beta-glucuronidase: genetics, molecular biology, and cell biology.
Prog Nucleic Acid Res Mol Biol
37:
155-205,
1989[ISI][Medline].
29.
Schaub, TP,
Kartenbeck J,
Konig J,
Vogel O,
Witzgall R,
Kriz W,
and
Keppler D.
Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules.
J Am Soc Nephrol
8:
1213-1221,
1997[Abstract].
30.
Simon, FR,
Fortune J,
Iwahashi M,
Bowman S,
Wolkoff A,
and
Sutherland E.
Characterization of the mechanisms involved in the gender differences in hepatic taurocholate uptake.
Am J Physiol Gastrointest Liver Physiol
276:
G556-G565,
1999[Abstract/Free Full Text].
31.
Sorrentino, D,
Licko V,
and
Weisiger RA.
Sex differences in sulfobromophtalein-glutathione transport by perfused rat liver.
Biochem Pharmacol
37:
3119-3126,
1988[ISI][Medline].
32.
Tanaka, E.
Gender-related differences in pharmacokinetics and their clinical significance.
J Clin Pharm Ther
24:
339-346,
1999[ISI][Medline].
33.
Tanaka, Y,
Deguchi Y,
Ishii I,
and
Terai T.
Sex differences in excretion of zenarestat in rat.
Xenobiotica
21:
1119-1125,
1991[ISI][Medline].
34.
Tang-Liu, DD,
Tozer NT,
and
Riegelman S.
Urine flow-dependence of theophylline renal clearance in man.
J Pharmacokinet Biopharm
10:
351-364,
1982[ISI][Medline].
35.
Terashita, S,
Sawamoto T,
Deguchi S,
Tokuma Y,
and
Hata T.
Sex-dependent and independent renal excretion of nivadipine metabolites in rat: evidence for a sex-dependent active secretion in kidney.
Xenobiotica
25:
37-47,
1995[ISI][Medline].
36.
Torres, AM.
Gender-differential liver plasma membrane affinities in hepatic tetrabromosulfonephthalein (TBS) uptake.
Biochem Pharmacol
51:
1117-1122,
1996[ISI][Medline].
37.
Urakami, Y,
Nakamura N,
Takahashi K,
Okuda M,
Saito H,
Hashimoto Y,
and
Inui K.
Gender differences in expression of organic cation transporter OCT2 in rat kidney.
FEBS Lett
461:
339-342,
1999[ISI][Medline].
38.
Walters, HC,
Craddock AL,
Fusegawa H,
Willingham MC,
and
Dawson PA.
Expression, transport properties, and chromosomal location of organic anion transporter subtype 3.
Am J Physiol Gastrointest Liver Physiol
279:
G1188-G1200,
2000[Abstract/Free Full Text].
39.
Yamada, T,
Niinuma K,
Lemaire M,
Terasaki T,
and
Sugiyama Y.
Carrier-mediated hepatic uptake of the cationic cyclopeptide, octreotide, in rats. Comparison between in vivo and in vitro.
Drug Metab Dispos
25:
536-543,
1997[Abstract/Free Full Text].
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