Effects of testosterone on the rat renal medullary vasopressin receptor concentration and the antidiuretic response

Life Sciences, Vol. 56, No. 14, pp. 1215-1222, 1995 Copyright @ 1995 Elswicr Science Ltd Printed in the USA. All rights resewed oQ24-32oS/9s $950 + .oo

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EFFECTS OF TESTOSTERONE ON THE RAT RENAL MEDULLARY VASOPRESSIN RECEPTOR CONCENTRATION AND THE ANTIDIURETIC RESPONSE I. Pave, Cs. Varga*, M. Sztks**, F. L&z10‘***, M. Sztcsi, J. Gardi and F. A. Laszlo* Endocrine Unit and ***lst Department of Medicine, Albert Szent-Gyorgyi Medical University, 6720 Szeged, Koranyi fasor 8, Hungary *Institute of Comparative Physiology, Szeged University, and **Institute of Biochemistry, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary

H-

(Received in final form January 13, 1995)

Summary The renal concentrating ability declines with age in humans and animals. Studies suggest that the concentrating defect is due to a decrease in renal vasopressin sensitivity. With ageing, expression of the renal vasopressin V2 receptor in rat is impaired; the normal receptor expression is restored by testosterone treatment. The effect of testosterone on the renal sensitivity to vasopressin was investigated in young rats. Male rats after orchidectomy and chronic antiandrogen cyproterone acetate treatment, and female rats after chronic testosterone phenylpropionate treatment, were used. The plasma arginine-vasopressin (AVP) and testosterone concentrations, and the antidiuretic responses to AVP and the V2 agonist deamino[8-D-argininel-vasopressin (dDAVP) after volume loading were measured, and the renal [3H]AVP binding density was determined. The plasma AVP level decreased slightly, but not significantly, in male rats after orchidectomy and cyproterone acetate treatment, but did not alter in female rats after testosterone treatment. The AVP and dDAVP sensitivities decreased in male rats after orchidectomy and cyproterone acetate administration, and increased in female rats treated with testosterone, as compared with the animals with a normal gonadal function. [3H]AVP binding to the renal inner medullary membranes was decreased following orchidectomy or antiandrogen treatment in male rats, and increased in testosteronetreated female rats. The results suggest that testosterone may play a physiological role in maintenance of the V2 vasopressin receptor expression and hence in the normal urinary concentrating ability in rat. Key Words: testosterone,

vasopressin, dDAVP, cyproterone acetate, V, vasopressin receptor

The urinary concentrating ability decreases with ageing in humans (1, 2). The argininevasopressin (AVP) response to dehydration is maintained, and relative resistance to AVP in elderly people has therefore been postulated (3). In the aged rat, a membrane binding assay revealed a decrease in renal binding sites for AVP (4), and there was a decrease in AVP-dependent CAMP

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generation in renal papillary slices (5). The plasma testosterone level decreases testosterone treatment restores the AVP binding sites in the aged male rat (6).

14, 199.5

with age, and

The roles of testosterone in the urinary concentrating ability and the renal medullary vasopressin receptor expression were examined in young animals following orchidectomy or antiandrogen cyproterone acetate treatment in male Wistar rats, and following testosterone phenylpropionate treatment in female Wistar rats. The [3H]AVP binding to the renal inner medullary membranes reflects mainly the concentration of the antidiuretic V2 receptors. Materials and Methods The experiments were carried out on 3-month-old male and female Wistar rats (ZOO-250 g). Bilateral orchidectomy was carried out on males under ether anesthesia 4 weeks before the experimentation. Another group of male rats was treated with the antiandrogen cyproterone acetate (Androcur, Schering) through a gastric tube, in an oral dose of 2.5 mg/day in 2 ml water once daily for 4 weeks. Testosterone phenylpropionate (Retandrol, Richter) was injected subcutaneously into a group of female rats, in a dose of 2.5 mg/day once daily for 3 weeks. After decapitation of the animals at the end of the treatment period, plasma AVP and testosterone levels were determined by RIA (7). Measurement of the Antidiuretic Effects of AVP and dDAVP on Volume Loaded Animals. Following deprivation of food for 12 h, 5 ml/100 g body weight 2% ethanol was administered into male rats. Simultaneously, 1 pg AVPflOO g body weight or 25 pg deamino-(&D-arginine]vasopressin (dDAVP)/lOO g body weight was injected intraperitoneally. 30-min urine fractions were collected, of which the osmolalities (using the technique of freezing point depression) and the volumes were measured. The antidiuretic potencies of AVP and dDAVP in female rats were determined by the method of De Wied (8). Following deprivation of food for 12 h, the animals were hydrated with a solution of 2% ethanol in water (5 ml/l00 g body weight) via a gastric tube. After 60 min, the animals were anesthetized with the same volume of 10% ethanol, and 2 h after the beginning of the experiment the animals were again hydrated with 2% ethanol. After this pretreatment, the urine bladder was cannulated, and the volumes and osmolalities of the urine excreted in intervals of 10 min were measured. 2% ethanol solution was administered orally to replace excreted urine. Different amounts of AVP or dDAVP in 0.1 ml isotonic solution were administered into the tail vein, and the resulting diuresis decrease was expressed as a percentage. One animal was used 2 or 3 times after the diuresis had returned to the starting level. Prenaration of Renal Medullarv Membranes. The method used was a modification of that of Herzberg et al. (6) and Marching0 et al. (9). Rats were killed by decapitation. The medullopapillary region of the kidneys was dissected and homogenized with a motor-driven homogenizer (Teflon pestle, 10 strokes) in 50 vol. ice-cold 100 rnM Tris-HCl (pH 7.4), 5 mM MgC12 solution. After gauze filtration, the suspension was centrifuged at 1000 g for 10 min, and the supernatant was recentrifuged at 20,000 g for 30 min. The pellet was resuspended in the same buffer to achieve a final protein concentration of approximately l-2 mg/ml. 13HlAVP Bindine to Renal Plasma Membranes. The incubation of 10 nM [3H]AVP (Amersham, Buckinghamshire, specific activity 17 Ci/mmol) and renal medullary membranes (0.2-0.3 mg protein) in binding buffer (100 mM Tris-HCl (pH 7.4), 5 mM MgC12, end volume 0.5 ml) was performed at 25 oC for 2 h in the presence or absence of several concentrations of AVP (homologous displacement) (10). To terminate the binding tests, the mixtures were diluted with 5 ml ice-cold washing buffer (10 mM Tris-HCl (pH 7.4), 0.5 mM MgC12 and 0.1% BSA),

Testosterone on VasopressinSensitivity

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immediately filtered under vacuum through Whatman GF/C filters, and rinsed twice with 5 ml icecold washing buffer, The filters were placed into counting vials with scintillation liquid and analyzed by liquid scintillation spectrometry. Bmax and KD values were determined by the iterative nonlinear model-fitting program LIGAND (11). Statistical Analvses. All results are expressed as means differences was calculated by the two-tailed Student t-test.

+ S.E.M. The significance

of

Results Plasma A lower pretreatment plasma AVP level was detected in female rats than in male rats. Orchidectomy or cyproterone acetate treatment slightly, but not significantly, decreased the AVP level in males. In females, testosterone treatment did not change the plasma AVP concentration (Fig. 1). The plasma testosterone level was decreased significantly orchidectomy (co. 1 ng/ml), cyproterone acetate treatment decreased only slightly 12

male control

CA orchidectomy treatment

female control

testosterone treatment

FIG. 1. Plasma AVP levels following orchidectomy or cyproterone acetate (CA) treatment in male rats, and following testosterone phenylpropionate treatment in female rats (n = 15-30, means it S.E.M., *p < 0.05) TABLE 1. Volume and total content of osmotically active compounds of the urine fractions (0- 150 min) following volume loading and AVP administration in orchidectomized or cyproterone acetate-pretreated male rats

Animals

male control CA treatment orchidectomy

Urine volume (ml/100 g)

1.2 f 0.2 3.7 f 0.3 2.2 f 0.5

Values are means of 12-15 independent measurements procedures, see Materials and Methods.’

Osmotically active compounds (mosmol)

0.67 f 0.19 0.71 f 0.28 0.68 f 0.24 +- S.E.M. For experimental

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(2.1 + 0.3 nghnl) as compared with the male controls (2.8 + 0.4 ng/ml). Testosterone phenylpropionate treatment resulted in a higher plasma testosterone level as compared with that of the female controls (83 k 4- and 0.2 f 0.1 ng/ml, respectively). Antidiuretic Activities of AVP and dDAVP in Testosterone-Deficient Male and in Testosterone-Treated Female Rats. The effective doses of AVP and the renal V2 selective agonist dDAVP caused significantly milder antidiuresis in orchidectomized or cyproterone acetatepretreated male rats (Fig. 2A and B). The time-dependent diuresis curves show that dDAVP was long-acting in this experimentai model as compared with AVP. In testosterone-pretreated female rats, AVP and dDAVP induced a more pronounced antidiuresis as compared with non-pretreated controls (Fig. 3A and B). There was no significant difference in the amount of excreted

‘““1 B

0

30

60

120

150

0

30

*

60

90

120

150

180

210

240

mill

270 Ill,"

FIG. 2. Diuretic curves for male rats following orchidectomy (0) or cyproterone acetate pretreatment (*) and for control male rats (x). The animals were hydrated and simultaneously treated with 1 ug/lOO g body weight AVP (A) or 25 pg/lOO g body weight dDAVP (3, n = 12-15, means +-S.E.M., *p < 0.05) 100

0

female control

25 pg AVP testosterone treatment

75 pg AVP female testosterone control treatment

3.0 fg DDAVP female testosterone control treatment

9.0 fg DDAVP female testosterone control treatment

FIG. 3. Percentage changes in diuresis following administration of different doses of AVP (A) or dDAVP (B), as indicated, in controls and testosterone-pretreated female rats (n = lo- 12, means f S.E.M., *p < 0.05)

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osmotically active compounds (Table 1). In both males and females, in the absence of AVP or dDAVP, there was no significant difference in diuresis between the pretreated and control animal groups. Renal Medullarv Vasooressin ReceDtor Concentration in Testosterone-Deficient Male and Testosterone-Treated Female Rats. The concentration of the renal medullary vasopressin (mainly V2) receptor was decreased in orchidectomized or cyproterone acetate-pretreated male rats (Fig. 4) as compared with non-treated male controls. The decrease was more pronounced in the cyproterone acetate-pretreated animals, but the difference between the two pretreated groups was

male control

CA orchidectomy treatment

female control

testosterone treatment

FIG. 4. Specific [3H]AVP binding to renal medullary plasma membranes following orchidectomy or cyproterone acetate (CA) treatment in male rats, and following testosterone phenylpropionate treatment in female rats. The results are means of 5 independent measurements f S.E.M. (*p < 0.05)

TABLE 2. Apparent dissociation constants (KD values) of the renal medullary vasopressin receptors following orchidectomy or cyproterone acetate (CA) treatment in male rats, and testosterone phenylproprionate treatment in female rats

Animals

Apparent dissociation

constant (KD, nmol)

male control CA treatment orchidectomy

4.5 + 1.9 6.7 + 2.4 6.4 f 2.2

female control testosterone treatment

2.6 + 1.7 4.1 * 2.0

Values are means of 5 independent determinations procedures, see Materials and Methods.

f S.E.M. For experimental

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not significant. The female rats treated with testosterone phenylpropionate had an elevated receptor concentration as compared with the female controls. These animals had the highest receptor concentration among the investigated animals. The apparent dissociation constant (KD) did not differ significantly in any group (Table 2). Discussion Orchidectomy or cyproterone acetate treatment slightly, but not significantly, reduced the plasma AVP level in male rats. Female animals had a lower plasma AVP level than males, and testosterone phenylpropionate treatment did not alter it. These observations correspond to previous results (12- 14), which reported that the basal AVP secretion was higher in male than in female rats, and that the difference was abolished after orchidectomy and restored by treatment with testosterone. However, Thornton et al. (15) found the plasma AVP levels to be unaffected by testosterone propionate treatment in castrated goats. A reduced renal AVP sensitivity in aged subjects has been described by several authors (3-6, 16). In rats, this phenomenon may be caused by a reduced number of renal V2 binding sites, as determined by membrane receptor binding assay (6) or by immunocytochemical staining (17), probably through the testosterone deficiency, because testosterone restores V2 receptor expression (6). Another possibility may be that an enhanced plasma vasopressin level on ageing could downregulate the renal vasopressin binding sites (18, 19). However, the data relating to vasopressin secretion with ageing are contradictory. Ageing is accompanied only by a tendency to a reduction in plasma AVP level, whereas testosterone treatment increases the plasma AVP level (6). Other investigators did not find a significant difference between vasopressin levels in young and old rats (17). Testosterone seems to increase the AVP content in the senescent rat brain (20). Terwel et al. (21) and Miller (19), however, detected an increase in plasma AVP level and depleted AVP concentrations in several brain structures of the aged rat (21). The urinary AVP excretion is also elevated in aged animals (22). These observations of different results can be explained by the different experimental methods employed, but also suggest that it is not possible to explain impaired urinary concentration ability in terms of the changes in plasma AVP level alone. Our data indicate that a positive effect of testosterone on the renal medullary vasopressin receptor, mostly V2, expression is independent of age: young male rats after orchidectomy or chronic treatment with the antiandrogen cyproterone acetate exhibited a reduced [3H]AVP binding site concentration, while young female rats following chronic testosterone phenylpropionate treatment exhibited an enhanced [3H]AVP binding site concentration in a renal inner medullary plasma membrane preparation. Correspondingly, the antidiuretic effects of AVP and the V2 selective agonist dDAVP were significantly decreased in orchidectomized or cyproterone acetatetreated male rats, and enhanced in female rats treated with testosterone. The altered concentrating ability could not be explained in terms of the altered plasma AVP levels measured before the experiments, for in the experimental models used in this study the initial volume loading suppresses endogenous AVP secretion and hence its effects. The dose of testosterone phenylpropionate for the female animals was pharmacological and resulted in a high plasma testosterone level (see Results). We used this treatment because of its effectivity in causing vasopressin-induced renal cortical necrosis (23). The results suggest a correlation between the change in the number of antidiuretic vasopressin receptors and the antidiuretic responses to AVP and dDAVP. Our results do not tend to support the view that the down- or upregulation of the receptors may be caused by the alteration in plasma AVP level. A significant pressor effect through the Vl vascular receptors can also be

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excluded, because similar results were obtained in the experiments performed with AVP, which unselectively activates both Vl and V2 receptors, and the V2 selective agonist dDAVP (24). Testosterone can modify vasopressin (V2) receptor expression in the kidney, probably through the binding to its own renal receptors (25). The androgen receptors may also be responsible for the trophic effect of testosterone described in the kidney tissues (26). Testosterone was recently shown to have a positive effect on vasopressin receptor expression in other tissues, investigated by binding assay or autoradiography (27, 28). It increases the number and the affinity of vasopressin receptors in the uterus of the chicken (29) and the number of vasopressin binding sites in the canary brain (30). In another autoradiographical study (31) however, testosterone caused an elevation only in the number of oxytocin, but not vasopressin (Vl) binding sites in the rat brain, which are presumed to play an important role in behavioral processes (32). The differences can be explained by the different receptor subtypes. Other steroid hormones also interact with the vasopressin receptor expression. Through the glucocorticoid receptors, dexamethasone treatment increases the number of Via vasopressin binding sites in a rat mammary tumor cell line, affecting its synthesis or the translation of its mRNA (33). In contrast, the mineralocorticoid deoxycorticosterone acetate (DOCA) reduces the hepatic Vl and renal V2 receptors, whereas the renal Vl receptor concentration is elevated by DOCA administration (34). Our present results suggest that testosterone may play a physiological role in maintenance of the renal medullary vasopressin (V2) receptor expression and in the normal urinary concentrating ability in young rat. Acknowledeements The authors would like to thank Dr. Gabor Toth for the AVP and Dr. Istvan Toth for the fruitful discussions. This work was supported by grants of the Hungarian Foundation for Scientific Research, OTKA T 1406 l/3, T 5491 l/4, 1350 and 895.

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