Testosterone Exacerbates Hypertension and Reduces Pressure-Natriuresis in Male Spontaneously Hypertensive Rats
Studies were performed in intact male and female, gonadectomized male and female, and gonadectomized female rats given testosterone for 5 weeks to investigate the role played by testosterone in altered blood pressure control and pressure-natriuresis in male SHR. Serum testosterone levels reached a peak at 12 weeks of age in intact male SHR. Systolic blood pressure, measured weekly from 5 to 20 weeks of age, was similar between groups until 12 weeks of age when blood pressure became higher in males (195±3 mm Hg) than in females (168±3 mm Hg) or males castrated at 4 weeks (173±4 mm Hg). At 17 to 19 weeks direct measurement of arterial pressure in anesthetized rats confirmed that mean arterial pressure was higher in male (182±1 mm Hg) than in female (159±2 mm Hg) and castrated male SHR (159±2 mm Hg). In addition, testosterone (5 mg in Silastic pellets, SC for 5 weeks) administered to ovariectomized (ovx+T) females caused arterial pressure to increase by mm11% (175±2 mm Hg), which was significantly higher than in intact female, castrated male, or untreated ovariectomized (ovx) female SHR (158±2 mm Hg). Acute pressure-natriuresis was blunted in male SHR compared with females, castrated males, or ovx females, in which this relationship was similar. Pressure-natriuresis was also blunted in ovx+T females as found in intact male SHR. These data support the hypothesis that male sex hormones contribute to the exacerbation of hypertension in SHR by reducing pressure-natriuresis.
- GFR = glomerular filtration rate
- ovx = ovariectomized
- ovx+T = ovariectomized females that received testosterone for 5 weeks
- RPF = renal plasma flow
- SHR = spontaneously hypertensive rat(s)
Men are at greater risk for cardiovascular and renal disease at an earlier age than are women. Recent studies using the technique of 24-hour ambulatory blood pressure monitoring in normotensive populations have shown that blood pressure is higher in men than in women at similar ages.1–3⇓⇓ Wiinberg et al1 studied 352 Danish men and women, aged 20 to 79 years, who were divided into groups by sex and age. Blood pressure increased with aging in both men and women, but the men had higher 24-hour mean blood pressures than did the women for all age groups except the 70- to 79-year-old individuals in whom blood pressures were numerically higher in the men but statistically similar for men and women.1 Staessen et al2 performed meta-analysis using all ambulatory blood pressure data reported in English or French between 1980 and 1989 in men and women aged 13 to 50 years. They also found that 24-hour systolic and diastolic blood pressures were higher in men than in women. Anastos et al3 have also shown that the incidence of uncontrolled hypertension is greater in men than in women. Thus, epidemiological studies indicate that men have higher blood pressures than do women. The mechanisms for the sex difference in blood pressure regulation in humans are unknown.
The sex-associated differences in blood pressure regulation observed in humans have also been documented in various animal models. For example, male SHR have higher blood pressures than do females of similar ages.4–7⇓⇓⇓ Crofton et al8 and Rowland and Fregly9 found that arterial pressure in male Dahl salt-sensitive rats increased more rapidly on a high-sodium chloride diet than in females. Two other models in the rat in which hypertension progresses more rapidly in males are the deoxycorticosterone-salt model of hypertension10 and the New Zealand genetically hypertensive rat.11
Although higher blood pressure has been documented in men, the mechanisms responsible for the sex difference have yet to be determined. Some studies have implied that androgens may play an important role in the progression of cardiovascular diseases. For example, several studies have shown that elevations in blood pressure are attenuated by gonadectomy of the males in animal models of hypertension, such as SHR and Dahl salt-sensitive rats.8,9⇓ Although studies point to androgens as a possible mediator in the progression of hypertension in male animals, the mechanisms whereby male hormones may increase blood pressure remain uncertain.
To date all forms of hypertension have been found to be associated with a reduction in the pressure-natriuresis relationship.12 Previous studies by other investigators have documented a shift in the pressure-natriuresis relationship in SHR.13,14⇓ If androgens in fact contribute to the exaggerated rise in blood pressure in male SHR, then they also should have an effect on the pressure-natriuresis relationship. The present study, therefore, was designed to test this hypothesis by measuring the testosterone-mediated component of pressure-natriuresis in the male SHR.
All studies were performed using male and female SHR, purchased from Harlan Sprague-Dawley. Male and female SHR at 3 weeks of age were castrated or ovx by the vendor and received at 4 weeks of age. Rats were maintained on standard rat diet (Teklad; Harlan Sprague-Dawley) in a 12-hour:12-hour light/dark cycle. All studies were approved by the Animal Care and Use Committee, University of Mississippi Medical Center, using National Institutes of Health Guidelines.
Serial Conscious Blood Pressure Measurements
Starting at 5 weeks of age blood pressure was measured weekly for 7 weeks in intact male, female, and castrated male SHR (n=16 each sex) by tail plethysmography (IITC, Inc).
Testosterone Treatment of ovx Female SHR and Serum Testosterone Assays
After arrival at 4 weeks of age, ovx female SHR were allowed to mature to 12 to 13 weeks of age and were then implanted subcutaneously in the shoulder area with 0.19-mm Silastic pellets (0.062 in ID, 0.125 in OD; Dow Corning) containing 5 mg of testosterone.15 After 3 weeks the pellets were replaced.
Serum testosterone was measured at the time of renal function studies in ovx females treated with testosterone and in anesthetized intact male SHR at 2-week intervals as they matured from 6 to 14 weeks of age (n=5 per age group), using a commercially available radioimmunoassay kit (Coat-A-Count Total Testosterone Assay kit, Diagnostic Products Corporation).
Renal Function and Acute Pressure-Natriuresis Studies
At 17 to 19 weeks of age, male (n=9), female (n=7), castrated male (n=8), ovx female (n=9), and ovx+T females (n=7) were anesthetized by intraperitoneal injection of the thiobarbiturate, Inactin (100 to 110 mg/kg body weight; A. Lockwood) and placed on a temperature-regulated surgery table to maintain rectal temperature at 36 to 38°C. Catheters were placed in the femoral artery (for continuous monitoring of blood pressure and for blood sampling) and in the femoral vein for infusion of isoncotic artificial rat plasma (2.5 g/dL bovine immunoglobulin, 2.5 g/dL bovine serum albumin in Ringer’s solution) at 12.5 mL/kg per hour for 45 minutes during the preparatory surgery and thereafter at 1.5 mL/kg per hour throughout the experimental period to maintain an euvolemic preparation.16,17⇓ A catheter was placed in the left jugular vein for infusion of 0.9% saline with [125I]iodoiothalamate (Glofil 0.05 μCi/kg/min; Cypros) and [131I]iodohippuran (0.1 μCi/kg/min; Syncor International) at 1 to 2 mL/h (depending on body weight). A tracheostomy was performed. A midline abdominal incision was then made, and catheters were placed in both ureters for collection of urine samples into weighed microfuge tubes.
After a 50-minute equilibration for isotope infusion and at the ambient blood pressure of the rat, a timed (15 to 30 minutes) urine sample and a midpoint arterial blood sample were collected. After this period, a snare was placed around the aorta above the renal arteries, and the snare was tightened in a stepwise fashion to produce graded decreases in renal perfusion pressure of approximately 20 mm Hg down to 110±5 mm Hg. Each decrease in blood pressure was followed by a 10-minute equilibration period and a 15- to 30-minute period during which urine and midpoint blood samples were again taken. After the experiment the kidneys were removed and weighed.
Samples of urine (25 to 50 μL) and femoral arterial plasma (50 μL) were counted by gamma counter (Searle, Tm Analytic). Urinary and plasma sodium were measured by atomic absorption (Instrumentation Laboratory).
These measurements allowed for the calculation of GFR, RPF, renal vascular resistance,18 and urinary sodium excretion.
The data were analyzed by ANOVA for repeated measures within groups, using Statview 512 software for the Macintosh. Significance was defined as P<.05. All data values are expressed as mean±SEM.
As shown in Fig 1A, blood pressure as measured by tail plethysmography, increased with maturity in all SHR. However, at 12 weeks of age and thereafter, blood pressures in male SHR were significantly higher than in females. Castration prevented the blood pressure from increasing in males so that the blood pressure was the same as in females.
Testosterone levels (Fig 1B) in intact male SHR were below detectable limits at 6 weeks of age but started to increase by 8 weeks and reached a peak at 12 weeks of age. This coincides with the time when blood pressures in male SHR became significantly higher than in females or castrated males.
As shown in the Table and Fig 2, in anesthetized rats, as in conscious animals, mean arterial pressure at 17 to 19 weeks of age was higher in intact males than in females. Castrated males had blood pressures similar to females, whereas testosterone treatment of ovariectomized female SHR for 5 weeks resulted in a significant increase in arterial pressure. Serum testosterone levels in ovx+T females at the time of the experiment were similar to values in intact male SHR at 9 to 10 weeks of age (16±6 ng/mL). Ovariectomy alone had no effect on mean arterial pressure in female SHR.
GFR and RPF, when factored for kidney weight, were similar in all groups of rats at ambient blood pressure (shown in the Table). In addition, GFR and RPF were similar in all groups at the reduced renal perfusion pressures.
As shown in Fig 3, there was a hypertensive shift in the acute pressure-natriuresis relationship in male SHR when compared with females. Castration of the male shifted the pressure-natriuresis relationship back to values found in intact females. Moreover, ovariectomized female SHR treated with testosterone had a blunted pressure-natriuresis that was not dissimilar from intact male SHR. Ovariectomy alone had no effect on pressure-natriuresis.
The major findings of the present study are that androgens mediate the exaggerated blood pressure and hypertensive shift in the pressure-natriuresis relationship in male SHR.
During maturation, we found that blood pressure increases in all SHR but becomes significantly higher in males than females at 12 weeks of age. In addition we report that castration of males reduces blood pressure to levels that were not different from blood pressures in females. Our data are consistent with previous studies by Masubuchi et al5 who also documented that female SHR had lower blood pressures than males and that castration attenuated the hypertension in males. The present study extends these findings by demonstrating that the exacerbation of the hypertension in the male SHR occurs at a time when serum testosterone levels have reached a peak at 12 weeks of age. We also found that androgens play a role in mediating the exacerbation of hypertension in male SHR, since 5 weeks of treatment with testosterone increased blood pressure in ovariectomized female SHR by 10% when compared with intact or ovariectomized females or castrated males. In fact, these blood pressure levels were only 4% lower than in intact males.
Several lines of evidence indicate that there are sex differences in blood pressure regulation in humans. In addition to the studies showing sex differences in blood pressure with aging in adults,1,2⇓ Bachmann et al19 documented that blood pressure in a German population of children, aged 4 to 18 years, increased with age in both boys and girls, but the increase was significantly steeper during adolescence in males than in the preadolescent period. Harschfield et al20 made similar findings in a population of American children, aged 10 to 18 years. These investigators also found that blood pressures were higher in boys than girls in all age groups while awake and was higher in male adolescents, aged 16 to 18, while asleep, a time when testosterone levels are highest. These studies suggest that androgens may play a role in mediating the increase in blood pressure with puberty in male adolescents.
Although studies have implicated androgens such as testosterone in the progression of hypertension, the mechanism(s) by which androgens could affect blood pressure are unknown. The kidney has long been known to play an important role in mediating the hypertension of SHR. Cross-transplantation studies have shown that transplantation of the SHR kidney into normotensive F1 hybrids of SHR and Wistar rats resulted in hypertension in the F1 rats.21,22⇓ Conversely, the hypertension was attenuated in SHR when they were transplanted with a kidney from the normotensive Wistar or the F1 hybrid.21,22⇓ Furthermore, independent studies by Khraibi and Knox13 and Roman14 have shown that there are alterations in the renal pressure-natriuresis relationship in SHR as found in all other models of hypertension.12 Whether sex differences in blood pressure regulation are also associated with alterations in pressure-natriuresis, however, has not been shown previously. Indeed we found that intact male SHR excrete a lower level of sodium at equivalent blood pressures than do females or castrated male SHR. Moreover, testosterone treatment of ovx female SHR also resulted in a hypertensive shift in the pressure-natriuresis relationship, and this suggests that it is specifically testosterone, or a metabolite, that plays a role in the blunted pressure-natriuresis relationship in male SHR compared with female SHR.
Although we showed in the present study that androgens cause a blunting in the pressure-natriuresis relationship in SHR, our studies do not address the exact mechanism(s) by which androgens may mediate this change. Testosterone or its metabolites, mediated via androgen receptors, could have a direct effect on tubular reabsorption of sodium and thereby cause a shift in the pressure-natriuresis relationship. We have preliminary data that androgen receptors are located in nuclei of renal proximal tubule cells.23 Alternatively, testosterone could affect pressure-natriuresis by indirect mechanisms such as activation of the renin-angiotensin system. Plasma renin activity has been shown to be higher in men than in women.24 Furthermore, male rats have higher angiotensinogen levels than females, and testosterone treatment increases and castration decreases angiotensinogen mRNA and plasma renin.25,26⇓ Future studies will be necessary to determine the mechanism(s) by which androgens shift the pressure-natriuresis relationship and increase blood pressure in SHR.
Sex differences in blood pressure have been suggested to be due to protection of the female by estrogens. To determine the role that estrogens may play in the progression of hypertension in female SHR, we compared blood pressure in intact and ovariectomized female SHR. There was no difference in the mean arterial pressures between the two groups. Furthermore, ovariectomy did not affect renal pressure-natriuresis. These data indicate that the presence of female hormones such as estrogen are not responsible for attenuating the hypertension and causing protection in the female SHR.
In summary, blood pressure in male SHR becomes significantly higher than in females at 12 weeks of age, a time when serum testosterone levels in the male reach their peak. Castration of the male SHR attenuated the increase in blood pressure. Moreover, testosterone treatment of ovariectomized female SHR results in increases in blood pressure as found in intact male SHR. These data implicate androgens in mediating the progression of hypertension in the male. The pressure-natriuresis relationship is also blunted in the male SHR compared with female or castrated male SHR. Furthermore, testosterone treatment of ovariectomized female SHR results in blunting of the pressure-natriuresis relationship as found in intact male SHR. These data support the hypothesis that male sex hormones contribute to the exacerbation of hypertension in the male SHR by reducing pressure-natriuresis.
This work was supported by funds from National Institutes of Health (Grants HL 51971 and HL 38499) and American Heart Association, Mississippi Affiliate (Grant 9307720S).
- Received September 7, 1997.
- Revision received October 15, 1997.
- Accepted October 24, 1997.
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