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Department of Physiology (J.L.R., A.A.), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, D.F. 07000; Department of Pharmacy (E.M.), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México, D.F. 11340; and Department of Pharmacology and Physiology (F.J.-J.), Centro Básico, Universidad Autónoma de Aguascalientes, Mexico, D.F. 20100
Address all correspondence and requests for reprints to: Jose L. Reyes, M.D., Ph.D., Department of Physiology and Biophysics, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, P.O. Box 14–740, México, D.F. 07000, Mexico. E-mail: jreyes{at}fisio.cinvestav.mx
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pharmacokinetic studies
Rats were anesthetized with pentobarbital (30 mg/kg BW, ip). The
trachea was isolated, and a catheter was inserted to allow adequate
ventilation. The carotid artery and the jugular vein were both
catheterized to obtain samples and to administrate test compounds,
respectively. For the kinetic studies, 300-µl blood samples were
obtained at 2, 5, 10, 15, 30, 60, 90, 120, and 150 min after
administration of the test compound. A blood sample obtained at the
beginning of the experiment was used as a blank and to determine the
initial hematocrit count. A final sample for hematocrit was obtained,
once the sampling for kinetics was completed. An equivalent volume of
isotonic saline solution was infused to restore the amount extracted in
the samples.
PAH has been extensively used as an indicator of the renal secretory pathway of organic anions, because it is not biotransformed and is secreted by the proximal tubule. This compound was chosen to estimate its disappearance rate from plasma in male and female rats and to look for differences possibly attributable to sexual dimorphism. A single bolus of PAH (10 mg/kg BW, aqueous solution, iv) was administered. This dose does not saturate the transport system (11); therefore, the kinetics can be adequately estimated.
The size of the experimental groups was determined with basis on the method for sample size estimation in infinite populations (12), and each group was formed by 20 rats. All experimental procedures were performed in accordance with international recommendations for the use and care of laboratory animals.
Measurement of PAH in plasma samples
Concentrations of PAH in plasma were measured using the method
described by Bratton and Marshall (13), modified for small samples.
Briefly, 300 µl trichloroacetic acid (10%) were added to 300 µl
blood. The mixture was agitated and centrifuged for 3 min at 3500 rpm.
A 400-µl aliquot of the protein-free filtrate was obtained, added
with 50 µl trichloroacetic acid and 150 µl of HCl (1.2 N),
agitated, and centrifuged at 3500 rpm for 3 min. The supernatant was
separated, and 150 µl NaNO2 (0.1%) were added. Ammonium
sulfamate (0.5%, 150 µl) was added, followed by the addition of 150
µl N-1-naphthylethylenediamine dihydrochloride (0.1%).
Samples were kept in the dark for 20 min. Sample absorbance was
measured at 540 nm in a spectrophotometer (Beckman DU50, Mexico City,
Mexico). This modification to the method of Bratton and Marshall
allowed the use of small-volume (300 µl) blood samples; the percent
recovery was 94, with a detection limit of 0.1 mg/ml. We used plasma of
the same animal as a blank to correct for the absorbance caused by
plasma components other than PAH.
Effect of testosterone on the PAH kinetics in female rats
Renal cells are sensitive to the effect of testosterone. Among
other effects, testosterone promotes protein synthesis in mice (14).
The effect of exogenous testosterone was assessed in intact adult
female rats. Testosterone (10 mg/kg BW, im, in sesame oil) was given to
20 rats (169.7 ± 11.6 g BW) for 7 days. This treatment was
similar to that described by Köening et al. (15) to
induce synthesis of renal enzymes in female mice. At the end of
treatment, PAH kinetic studies were performed in these animals. PAH was
administered as a single bolus (10 mg/kg BW, iv). Sampling was
performed as previously described.
Acute effect of testosterone in male rats
Janne et al. (16) reported that testosterone induced
protein synthesis in the mouse kidney, which was detectable 2 h
after administration. Similarly, creatinine clearance increased
150–330 min after intramuscular administration of testosterone to rats
(17). To study the acute effect of testosterone on PAH kinetics in the
male rat, the hormone was administered to 20 rats in a single dose (20
mg/kg BW, im) 170 min before the PAH kinetic studies described above.
This dose is 20-fold higher than the dose required to induce an
androgen effect in rats (18).
Effect of orchiectomy on PAH kinetics in rats
To induce an acute reduction of the androgen effect, 20
45-day-old rats were subjected to orchiectomy 15 days before the PAH
kinetic studies. This period was chosen because 10 days after
castration, the serum concentration of testosterone declines by 90%
(19). The experimental procedure followed in the kinetic analysis was
the same as described above.
Effect of acute administration of testosterone on the kinetics of
PAH in orchiectomized rats
To study the effect of a substitutive androgen therapy in
orchiectomized rats, testosterone (20 mg/kg BW, im) was administered to
20 rats on day 15 after surgery. PAH kinetic studies were performed 170
min after administration. The testosterone dose used in this
experimental series is higher than that employed by Fedor et
al. (20) for substitutive therapy in orchiectomized rats.
Effect of castration on the renal tubular secretion of PAH in
female rats
Some metabolic effects of testosterone may be mediated by the
interaction of the hormone with estrogen receptors in the target cells.
We considered it of interest to study whether ovariectomy might affect
PAH kinetics. Female adult Wistar rats were ovariectomized, and kinetic
studies were performed in these animals.
Kinetic model for the elimination of PAH
Kinetic analysis was done considering a model of two open
compartments, because the data (fitted by an automated least-squares
procedure) were in agreement with this model (21), which is similar to
those for indomethacin (22) and arginine vasopressin in rats (23).
Concentration decreases in the central compartment after a
biexponential function, represented by the equation:
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and ß represent the disappearance rates for the
distribution and elimination components, respectively, and their values
are given by the slopes of each of the adjusted curves. A and B
represent the initial values of the distribution and elimination
components, respectively, extrapolated from the y-axis intercept.
The biological half-time was calculated from:
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Time-course of PAH uptake by renal cortical slices from
orchiectomized or intact male rats
To prevent hemodynamic systemic factors from participating in
the presumable differences between orchiectomized and intact male rats,
we measured PAH uptake in cortical renal slices. Orchiectomized rats
were used 15 days after surgery. Cortical slices were obtained from
intact male or orchiectomized rats. The slices were incubated for 5,
10, 20, 40, 60, 90, and 120 min in Ringer solution containing
14C-PAH (10-6 M; SA, 41.3
mCi/mmol; New England Nuclear, Mexico City, Mexico), under gentle
stirring, at 25 C, and gassed with 95% O2-5%
CO2. At the end of each period, one group of slices was
retired from the incubation media and blotted. Slices were dried
overnight at 80 C. Dry weight of tissue pieces was recorded in a
Mettler (Mexico City, Mexico) M-5 balance, and results are expressed as
dpm per mg dry weight. Samples were digested with a commercial
solubilizer (NCS, Amersham Co., Mexico City, Mexico) at 50 C for 60 min
in separate vials, and 10 ml scintillation liquid (Instagel, Packard,
Mexico City, Mexico) were added after cooling the digested material.
Radioactivity was measured in a liquid scintillation counter (Packard
Tri Carb, mod 3255, Instrumental Technion, Mexico City, Mexico), and
corrections for quenching and background were made. Nonspecific uptake
was determined in another group of slices incubated in the presence of
radioactive PAH (10-6 M) and probenecid
(10-4 M, Sigma Chemical Co., St. Louis, MO),
as previously described (25).
Binding of PAH to plasma proteins
Pharmacokinetics is highly dependent on the degree of drug
binding to plasma proteins. Therefore, we decided to study whether the
binding of PAH to these proteins might explain the differences observed
between male and female rats in renal excretion of PAH.
Sixty-day-old male and female rats were anesthetized with ether. Blood was obtained by cardiac puncture, and 9 ml were mixed with 1 ml sodium citrate (3.8%). This mixture was centrifuged at 2000 rpm for 8 min. Aliquots of 1 ml of plasma were incubated in the presence of 14C-PAH (SA, 41.3 mCi/mmol; New England Nuclear) at 5, 10, and 20 mM, at 37 C, under constant stirring for 30 min. Then, duplicates of 100 µl were obtained, and the remaining 800 µl were put into dialysis bags. Plasma samples in the bags were introduced into vials containing 10 ml of phosphate buffer solution (pH 7.4), which were then incubated for 24 h, at 37 C, under gentle stirring. After incubation, duplicated aliquots (100 µl) were obtained from the bags contents and from the incubation media. These samples were mixed with 500 µl of a commercial solubilizer and 10 ml of scintillation solution (New England Nuclear). Radioactivity was measured in a liquid scintillation spectrophotometer (Tri-Carb, Packard). Corrections for background and quenching were made.
Renal clearance studies
Female and male Wistar rats, weighing 256 ± 8 and 306
± 17 g BW, respectively, were anesthetized with pentobarbital (30
mg/kg, ip). The jugular vein, the femoral artery, and the trachea were
catheterized with polyethylene tubing. The bladder was catheterized
with a double polyethylene tube, allowing for air injection to expedite
emptying. After the surgical preparation, plasma separated from blood
collected from other rats was injected (1.4 ml/100 g BW) at a rate of
0.1 ml/min, to compensate for blood loss during surgery. Inulin and PAH
prime and sustaining infusions were given via the jugular vein, and 3%
mannitol in saline solution was infused at a rate of 0.1 ml/min during
the experiments.
Inulin and PAH were measured as previously described (26).
Statistical analysis
The data were analyzed with one-way ANOVA by means of a
commercial software. A value of P < 0.05 was
considered as statistically significant. In addition, to study
similarities among groups, we performed the Duncan’s multiple-ranks
test. Values for elimination half-time (t1/2el) are
expressed as mean ± SD. All other values are
mean ± SEM
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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), suggesting that the tubular handling
of PAH is susceptible to being modulated by this hormone in the intact
female rat.
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), reaching values similar to those of
female rats (castrated male: 28.1 ± 7.1, and female rat:
25.7 ± 4.5 min, respectively). These findings suggest that PAH
secretion was dependent on testosterone activity.
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). Administration of testosterone to
intact male rats did not change the PAH t1/2el (intact
male: 16.2 ± 2.1 vs. testosterone-treated male:
18.6 ± 2.6 min). Our results show that substitutive treatment
with testosterone offsets the increase in the PAH t1/2el
produced by orchiectomy, in line with the suggestion that testosterone
activity is critical for the maintenance of the functional sexual
dimorphism in the kidney.
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Duncan’s multiple ranks test showed that the experimental series can be fitted into two groups: the first group formed by intact males, testosterone-treated females, orchiectomized males treated with testosterone, and intact males also treated with testosterone. All these experimental conditions depicted an accelerated t1/2el for PAH, and among these groups there were no statistical differences. The second group would be formed by intact females, orchiectomized rats, and ovariectomized rats. The animals under these hormonal conditions depict slow t1/2els for PAH, which were statistically different from those of the first group (P < 0.01).
Effect of orchiectomy on the PAH uptake by cortical slices
To examine the tubular component of PAH excretion, in the absence
of confounding factors such as renal hemodynamics and hepatic
metabolism or excretion of this compound, we studied the in
vitro uptake of PAH by cortical slices obtained from
orchiectomized or intact male rats. Uptake was significantly higher in
slices from intact male rats than in those from orchiectomized rats
(Fig. 5). Kinetic analysis of the initial
uptake rate showed that this parameter was similar in slices from
intact males and in those from orchiectomized rats, k = 0.066
min-1 and k = 0.059 min-1, respectively
(Fig. 5, insert). These results suggest that the differences between
intact males and orchiectomized rats are caused by a greater number of
transporting sites for PAH in the intact animals than in the
orchiectomized ones. However, the analyses of the initial uptake rates
indicate that these transporting sites have the same affinity in both
groups of animals. Therefore, the main effect of testosterone is to
modulate the number of functional transporting molecules in the cell
membrane, without producing any evident changes in the transport speed
of each of these carriers.
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). These results
indicate that renal hemodynamics does not contribute to the differences
observed in PAH kinetics between intact male and female rats.
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), which indicates that protein binding
does not contribute to the more rapid elimination of PAH by male
rats.
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). The final
hematocrit counts were lower than initial values, and this change was
similar in all the series. There was an average decrease of 6.9 ±
0.2% for all the groups. There was no apparent relationship between
the observed differences in the kinetics of PAH and the degree of
change in the hematocrit count (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed a shorter t1/2el in intact male rats than in orchiectomized or female rats. This effect of orchiectomy was offset by testosterone administration at doses intended to compensate for the decrement of the hormone induced by castration (20). These results provide evidence for a stimulatory effect of this hormone on the renal excretory function. Administration of testosterone to intact male rats did not significantly increase the elimination rate of PAH. Because the administration of testosterone followed a schedule similar to that used with female and castrated male rats, in which a clear reduction in the PAH t1/2el was observed, we can be confident that this lack of significant stimulation was not caused by a short time of observation for the hormone’s action or to a low dose; and this suggests that, under physiological conditions, testosterone concentrations in males’ plasma are adequate to exert its modulatory action on this renal secretory pathway.
We studied the effects of testosterone on other kidney functions, such as the glomerular filtration rate, measured in terms of inulin clearance. This parameter was similar in male and female rats, suggesting that the effects of testosterone on the renal capabilities show some selectivity and are not influenced to the same extent. We did not observe differences in PAH clearance between male or female rats. It must be emphasized that PAH and inulin renal clearance are indicators of renal hemodynamics (PAH clearance for renal plasma flow and inulin clearance for glomerular filtration rate). Therefore, our results suggest that testosterone does not play a major role in renal hemodynamics in the intact male or female rat.
Kleinman et al. (3) reported that PAH transport was higher in unanesthetized male rats than in female rats. We did not observe this sort of difference in PAH clearances, possibly because of a disparity in experimental approach; Kleinman et al. (3) studied PAH transport under maximum capacity conditions using PAH doses higher than those used by us. These observations stress the convenience of performing complete kinetic studies to disclose sex differences in the kidney function. Furthermore, in addition to the selectivity of the stimulatory effect of testosterone on renal secretion, but not on glomerular filtration rate or plasma renal flow, there also are differences that depend on the target organ. For instance, liver, submaxillary glands, and kidney are all androgen-responsive nonsexual organs. However, in the study reported by Paigen and Peterson (27), ß-glucoronidase activity in mice was induced in the kidney but not in the liver or in the submaxillary glands. This induction was age-dependent. In 10-day-old mice, there was no effect of testosterone, but there was an 8-fold increase in the activity of this enzyme in 30-day-old mice. Similar results were obtained with dihydrotestosterone.
An additional explanation might be put forward for the lack of gender differences in PAH clearance. The liver has an active transporting system for PAH, which is similar in its cell mechanisms to that of the kidney. Therefore, because we used a relatively low amount of PAH in the clearance studies, part of this material might have been captured by the liver, and this could have contributed to the lack of difference in renal clearances (28).
It should be emphasized that the distribution component of PAH kinetics did not differ in male, female, or castrated male rats. Therefore, the differences observed between male and female rats in PAH kinetics must be caused by the testosterone effects on the elimination component of the process, which is mostly dependent on the excretory capacity of the kidney. The degree of binding of some drugs to plasma proteins is an important factor that has to be taken into consideration when kinetic studies are performed (29, 30). Nevertheless, the differences in PAH kinetics observed between male and female rats in our study cannot be explained by the binding to plasma proteins, because this was similar in both groups.
In the in vivo studies, the higher capacity of male rats to eliminate PAH might be attributable to several factors, such as systemic or hepatic excretion of PAH through bile. Because we were interested in disclosing the effects of testosterone on epithelial cells, particularly those of the proximal tubule, we studied the effect of testosterone on renal cortical slices. Renal slices possess the secretory mechanism to transport PAH, and under these conditions, this mechanism can be studied in the absence of hemodynamic or other systemic factors. The measurements of in vitro PAH uptake by cortical slices showed that slices from orchiectomized rats had a lower ability to concentrate PAH than those from intact male rats. This finding suggests that testosterone has a positive effect on the modulation of the functional state of PAH-carrying molecules. Because the initial rates of PAH uptake were similar in slices from intact males and orchiectomized rats, we suggest that the hormone does not modify the affinity of the transporting molecules and that the main effect of testosterone is to increase the number of functional transporting molecules in the cell membrane. Further studies are required to fully clarify this issue.
Other renal effects of testosterone involve mechanisms that are important in the development of toxicity in this organ. Administration of hexachlorobutadiene, a nephrotoxic drug, was shown to induce more damage to proximal tubules in male than in female rats (31). N-acetyltransferase activity in male mice showed a 2-fold increase in kidney by 30 days post natal, whereas the kidney activity of this enzyme remained unchanged in female mice. Castration reduced the activity of this enzyme in the male kidney to female levels, whereas testosterone replacement restored original activity. Because N-acetylation participates in the transformation of hydrazine drugs and arylamine carcinogens into cytotoxic and carcinogenic products, differences in the activity of N-acetyltransferase associated with testosterone might explain, at least in part, the higher susceptibility of male mice to 2-acetylaminofluorene mutagenicity and carcinogenicity (32). It should be emphasized that testosterone does not always produce a stimulatory effect on the kidney. Suzuki et al. (33) reported that this hormone decreased the activity of carbonic anhydrase in a dose-dependent manner. In the same study, the activity of the brush border Mg2+/HCO3--adenosine triphosphatase (another protein involved in cell transport mechanisms) did not change after testosterone administration.
Sciarra et al. (19) reported that castration reduced circulating levels of testosterone by approximately 90%. The decrease in renal secretory ability showed by castrated animals in our study is in agreement with the expected effect from a reduction in testosterone circulating levels. The recovery of a high excretory capacity in castrated animals that received restoration treatment with testosterone only 170 min before being tested provides evidence of the plasticity of the renal secretory mechanisms.
In female rats, treatment with testosterone increased their secretory capacity to levels similar to those of intact male rats, thus suggesting that this hormone is responsible for the sexual dimorphism observed in the renal function of the rat. It is remarkable that the increased secretory capacity was not related to a higher body mass, because rats treated with testosterone had a lower average weight than the control female rats.
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Footnotes |
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Received July 24, 1997.
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) or female (
) rats.
Calculations were made from the equation: t1/2el =
0.693/ß. Values are mean ± SD. Means ±
SEM are shown in the plotting, n = 20 for each
group.
) male
rats. Experiments in the castrated animals were performed 15 days after
surgery. Means ± SEM are shown, n = 20 for each
group. Values for t1/2el are mean ± SD.
