Abstract Although men and male rats are at increased risk for age-related renal and cardiovascular disease compared with women and female rats, the mechanisms are unknown. However, castration of male normotensive rats protects against age-related glomerular sclerosis, and castration of spontaneously hypertensive rats (SHR) attenuates the age-related progression of hypertension to levels found in female SHR. The present study was performed to determine whether there are differences between male and female renal function in SHR with aging, and if so, the role that androgens play in the age-related changes in renal function in SHR. Renal hemodynamics, glomerular filtration rate (GFR), and proteinuria were measured in anesthetized male and female SHR aged 7 to 8, 14 to 15, and 30 to 32 weeks (n=5 to 7 per sex per age group). Studies were also performed in male SHR that had been castrated at 4 weeks of age and allowed to age to 7 to 8, 14 to 15, and 30 to 32 months of age (n=5 to 9 per age group). At 7 to 8 weeks of age when blood pressures are similar between male, female, and castrated male SHR, there were no differences in renal hemodynamics between male and female SHR. As the rats aged, mean arterial blood pressure increased in all groups and was higher in the intact male SHR than in females. Blood pressure in aging castrated males was similar to the pressure in aging female SHR. With advancing age, GFR in intact male SHR decreased by 20% to 30% at 14 to 15 and 30 to 32 weeks of age, respectively. Renal plasma flow (RPF) also tended to decrease in male SHR but not statistically so. There was no age-related reduction in GFR or RPF in female or castrated male SHR. With advancing age, renal vascular resistance (RVR) increased in all rats. RVR increased by 40% and 60% at 14 to 15 and 30 to 32 weeks in male SHR. In comparison, RVR increased by 20% and 40% in female SHR at similar ages. RVR in castrated male SHR was similar to RVR in females. The levels of urinary protein excretion increased with aging to 14 weeks in the male SHR, but in females and castrated males urinary protein excretion reached a plateau at 6 to 8 weeks that was maintained until 14 weeks of age and was approximately sixfold lower than in male SHR. These data suggest that androgens may play an important role in mediating the hypertension and the age-related alterations in renal function in the male SHR.
It is well known that men are at greater risk for cardiovascular and renal disease than are women of similar ages.1 For example, recent studies using the technique of 24-hour ambulatory monitoring have shown that blood pressure is higher in men than in women at similar ages.2 3 4 In addition, the incidence of end-stage renal failure caused by glomerulonephritis and hypertensive glomerular sclerosis is higher in men than women.1 Aging in men is also associated with greater decrements in renal function than in women.5 6
The mechanisms responsible for the promotion of cardiovascular and renal disease in men are unknown. Some studies have implicated that androgens such as testosterone may play an important role in the progression of cardiovascular and renal diseases. For example, we and others have found that castration of male SHR at a young age (3 to 5 weeks) attenuates the hypertension typically found in intact SHR males to the level found in SHR females.7 8 9 10 We have also found that castration of male normotensive rats results in protection against age-related renal injury as found in females.11 Since ovariectomy did not predispose the females to additional injury,11 it appears that the renal protection afforded aging females was not mediated by estrogen, but that the promotion of renal injury in males may be related to androgens such as testosterone.
The present study was performed to determine whether there are gender differences in renal hemodynamics in SHR with increasing age, and if so, to determine the role that androgens may play in mediating the gender differences in renal hemodynamics.
Male and female spontaneously hypertensive rats (SHR) were obtained at 5 weeks of age from Harlan SD (Indianapolis, IN). Male SHR that had been castrated at 4 weeks of age by the vendor were also obtained. Rats were maintained on standard rat diet (Teklad, Harlan SD) in a 12-hour/12-hour light/dark cycle and were allowed to age to 7 to 8 weeks, 14 to 15 weeks, or 30 to 32 weeks of age prior to study. All protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and were performed according to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and the guidelines of the Animal Welfare Act.
Renal Function Studies
Renal function was measured in SHR at 7 to 8 weeks, 14 to 15 weeks, and 30 to 32 weeks of age. Rats were anesthetized by intraperitoneal injection of the thiobarbiturate Inactin (100 to 110 mg/kg body wt; A. Lockwood, Sturtevant, Wisc) and placed on a temperature-regulated surgery table to maintain rectal temperature at 36°C 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 [Sigma Chemical Co] in Ringer’s solution) at 12.5 mL/kg/h for 45 minutes during the preparatory surgery and thereafter at 1.5 mL/kg/h throughout the experimental period to maintain a euvolemic preparation.13 14 A catheter was placed in the left jugular vein for infusion of 0.9% saline with or without 125I-iothalamate or 131I-hippuran at 1 mL/h. A tracheostomy was performed. A midline abdominal incision was then made, and a catheter was placed in the bladder for collections of urine samples into weighed microfuge tubes.
Following a 50-minute equilibration period, one 30-minute urine collection was made, and midpoint femoral arterial blood samples were taken. After the experiment the kidneys were removed and weighed.
Samples of urine (50 μL) and plasma (50 μL) from femoral arterial blood samples were counted by gamma counter.
These measurements allowed for the calculation of glomerular filtration rate (GFR), renal plasma flow, and renal vascular resistance, using standard equations.15 Data for GFR and renal plasma flow were factored for kidney weight.
Urinary Protein Excretion With Increasing Age
Male, female, and castrated male SHR (n=10 each gender) were placed in metabolism cages for 24 hours for measurement of urinary protein excretion at 4, 6, 8, 10, 12, and 14 weeks of age. Rats were fasted while in the metabolism cages but had unlimited access to water. Urinary protein was measured by the Bradford method using a commercially available dye reagent (Biorad) and bovine serum albumin as the standard as previously described.12 Data are expressed as milligrams of protein excreted per 24 hours.
The data were analyzed by ANOVA using Statview 512 software for the Macintosh. Significance was defined as P<.05. All data values are expressed as mean±SEM.
As shown in the Table⇓, body weights and kidney weights increased with age in all groups of SHR. Because kidney weights were different for all groups, renal hemodynamic measurements are factored for kidney weights.
The age-related changes in renal hemodynamics are shown in Fig 1⇓. GFR decreased with age in the intact male SHR by 20% at 14 to 15 weeks of age and by 26% at 30 to 32 weeks of age when compared with the value at 7 to 8 weeks. GFR in the female did not decrease with advancing age. GFR also did not change with aging in the castrated male SHR. Renal plasma flow tended to decrease with aging in the intact male SHR, but the decreases did not reach statistical significance. Renal plasma flow was not reduced with aging in female or castrated male SHR.
Renal vascular resistance (RVR) increased in all groups with aging (Fig 1⇑). RVR in male SHR increased by approximately 40% and 60% at 14 to 15 weeks and 30 to 32 weeks, respectively, compared with the value at 7 to 8 weeks. RVR in female SHR increased by approximately 20% and 40% at 14 to 15 weeks and 30 to 32 weeks of age, respectively. RVR tended to be lower in aging female SHR than in male SHR but was not statistically so. RVR was lower in castrated male SHR than in intact males at all age groups, and was also lower than RVR in females at 7 to 8 and 14 to 15 weeks of age.
As shown in Fig 2⇓, arterial pressure increased with aging in all groups of SHR. Mean arterial pressure was similar between groups at 7 to 8 weeks of age. However, at 14 to 15 weeks of age, mean arterial pressure was similar between female (157±2 mm Hg) and castrated male SHR (156±2 mm Hg) and both were significantly lower than the mean arterial pressure in intact males (174±3). There were similar differences between groups found at 30 to 32 weeks of age as well (female, 166±4; castrated male, 168±3; intact male, 181±3).
As shown in Fig 3⇓, urinary protein excretion began to increase at 6 weeks of age in the male SHRs, the time that serum testosterone levels begin to increase,10 and continued to increase as the rats aged to 14 weeks. We have previously found that serum testosterone reaches a peak at 12 weeks of age in the male SHR, and blood pressures are different between males and female and/or castrated male SHR beginning at 12 weeks of age.10 Protein excretion in females increased slightly from 4 to 8 weeks but declined at 10 weeks to values found in females at 6 weeks of age. Urinary protein excretion in castrated males was similar to the value in females until 8 weeks of age. After 8 weeks, the protein excretion rate in the castrated male SHR reached a plateau. At 14 weeks of age, urinary protein excretion was approximately fivefold to sixfold higher in male SHR than in female or castrated male SHR.
The present study was performed to determine whether there were gender differences in renal hemodynamics and protein excretion in SHR with age. At 7 to 8 weeks of age when mean arterial pressures are similar between males and females, there were no differences in GFR, renal plasma flow, or RVR between these two groups. At 14 to 15 and 30 to 32 weeks of age when arterial pressure is higher in males than females, GFR and renal plasma flow decreased in male SHR but was essentially preserved in female SHR. The male SHR also had higher urinary protein excretion rates than did females at all ages except 4 weeks. Thus, there are gender differences in renal hemodynamics and protein excretion with aging in SHR.
The second question addressed in the present studies was whether androgens played a role in mediating gender differences in renal hemodynamics, blood pressure regulation, and proteinuria in aging SHR. In castrated male SHR at 7 to 8 weeks of age, systemic and renal hemodynamics were similar to values in intact males and females, although RVR was lower. With aging, GFR and renal plasma flow were also preserved in castrated males as found in female SHR. Renal vascular resistance increased with aging in castrated males in parallel with the aging females. Urinary protein excretion in castrated male SHR was similar to the values found in females until 8 weeks of age, after which there was a slight increase. However, despite the slight increase, proteinuria was still fivefold lower in castrated males than in intact males. These data strongly suggest that androgens may promote age-related reductions in renal hemodynamics and increases in proteinuria and contribute to the renal dysfunction and injury associated with aging in SHR males.
Recent studies using the technique of 24-hour ambulatory blood pressure monitoring have shown that blood pressure is higher in men than in women at similar ages. Wiinberg and colleagues3 studied 352 Danish men and women aged 20 to 79 years who were divided into groups by gender 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 which blood pressures were numerically higher in the men but statistically similar for men and women.3 Staessen and colleagues2 performed meta-analysis using all ambulatory blood pressure data reported in English or French between 1980 and 1989 in men and women of mean age between 13 and 50 years.2 They found that 24-hour systolic and diastolic blood pressures were higher in men than in women. Anastos and colleagues4 have also shown that the incidence of uncontrolled hypertension is greater in men than women. Thus, epidemiological studies indicate that males experience higher blood pressures than do females. Although the severity of blood pressure is greater in males than females, the mechanism is unclear.
Studies in various strains of rats have also documented that blood pressure is higher in males than females.1 Crofton and colleagues16 and Rowland and Fregly17 found that arterial pressure increased more rapidly on high salt diet in male Dahl salt-sensitive rats than in females of this strain. Two other rat models of hypertension in which the hypertension progresses more rapidly in males than females are DOC-salt hypertension18 and the New Zealand genetically hypertensive rat.19 In previous studies we (and others) have shown that blood pressure is higher in male SHR than in female SHR beginning at 12 weeks of age,7 8 9 10 the time when serum testosterone levels reach a peak in the males.10 In the present studies we showed that the severity of hypertension is greater in male than female SHR. We also showed that castration reduces the severity of hypertension in the aging male SHR. These results indicate an important role for androgens in causing gender differences in blood pressure regulation.
Transplantation studies support the hypothesis that androgens may play an important role in promoting cardiovascular and renal disease. Harrap and colleagues20 found that when SHR male kidneys were transplanted into females, there was no significant rise in blood pressure. In contrast, when female SHR kidneys were transplanted into males, blood pressure increased as usual in the males; thus, the female kidney afforded no protection from the increases in blood pressure in the male SHR. These data strongly suggest that in addition to an intrinsic defect in the kidney of SHR to cause hypertension, there is also some factor, such as androgens, in male SHR that promotes blood pressure to higher levels than in the female SHR.
The rate of progression of chronic renal diseases is also more rapid in men than women. For example, the incidence of end-stage renal failure caused by glomerulonephritis and hypertensive glomerular sclerosis is higher in men than women.1 Men develop renal injury earlier than women in other chronic renal diseases, such as membranous nephropathy, IgA nephropathy, and polycystic kidney disease,1 and in general, aging in men is associated with greater decrements in renal function and increased glomerular sclerosis than in women.5 6 Thus, men are at greater risk for renal injury than are women.
With regard to chronic renal disease and aging in particular, male rats of many different strains, both normotensive and hypertensive, exhibit decreased GFR with aging5 and develop glomerular injury, including excessive proteinuria and sclerosis, at an earlier age than do female rats.5 In the present study we showed that the female was protected against the reductions in renal function with aging that is found in the male SHR. We also found that castration protected the male from aging-related reductions in renal function. Furthermore, we demonstrated that proteinuria was higher in male SHR than female SHR and that castration of the male reduced the protein excretion to levels that were somewhat higher than in female SHR but that were fivefold lower than found in male SHR. These data support the notion that androgens play an important role in mediating the age-related reduction in renal function and increase in proteinuria.
The mechanism(s) by which female rats are relatively protected against age-related renal injury and hypertension are unknown. It is possible that estrogen or other female sex steroids protect the female renal microvasculature from aging injury. We doubt that this is the mechanism, however, since we have previously shown that ovariectomy at 10 weeks of age was not responsible for an increment in glomerular sclerosis in normotensive females, aged 22 months.11 However, castration of male normotensive rats at 10 weeks of age prevented the age-related glomerular sclerosis found in the intact males.11 These data suggest then that estrogen and female hormones do not actively protect the female kidney from aging injury, but that testosterone or androgens may promote aging injury in the male kidney.
It is possible that androgens may mediate the hypertension and greater age-related decline in renal hemodynamics in male SHR by a mechanism involving the renin-angiotensin system. Chen and colleagues21 found that plasma renin activity was lower in female SHR than in males and could be increased with testosterone treatment in females. Furthermore, these investigators found that renin and angiotensinogen mRNA levels were lower in gonadectomized males or females when compared with intact animals and that testosterone treatment of gonadectomized male or female SHR resulted in increases in both renin and angiotensinogen mRNAs.21 Castration of male Wistar-Kyoto rats (WKY) also resulted in reductions in the levels of angiotensinogen mRNA to the low levels found in female WKY, and testosterone treatment of both intact female and castrated male WKY increased angiotensinogen mRNA to levels found in intact males.22 Future studies will be necessary to determine the interaction between androgens and the renin angiotensin system, and the role that androgens and the renin angiotensin system may play in the hypertension and the reduction in renal hemodynamics in aging male SHR.
This work was supported by National Institutes of Health grant HL51971 and by the American Heart Association, Mississippi Affiliate.
- Received March 15, 1997.
- Revision received April 16, 1997.
- Accepted April 17, 1997.
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Ichikawa I, Maddox DA, Cogan MG, Brenner BM. Dynamics of glomerular ultrafiltration in euvolemic Munich Wistar rats. Renal Physiol. 1978;1:121-131.
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Ouchi Y, Share L, Crofton JT, Iitake K, Brooks DP. Sex difference in the development of deoxycorticosterone-salt hypertension in the rat. Hypertension. 1987;9:172-177.
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Chen Y-F, Naftilan AJ, Oparil S. Androgen-dependent angiotensinogen and renin messenger RNA expression in hypertensive rats. Hypertension. 1992;19:456-463.