Male spontaneously hypertensive rats (SHR) have a blunted pressure-natriuresis relationship and enhanced oxidative stress compared with female SHR. Furthermore, oxidative stress contributes to abnormal renal Na+ handling and renal damage in hypertension. The aim of this study was to determine whether a sex difference exists in renal inner medullary hydrogen peroxide (H2O2) levels and/or antioxidant systems in SHR and the influence of sex steroids on these systems. Thirteen-week-old intact and gonadectomized male and female SHR were placed in metabolic cages for 24-h urine collection. Renal inner medullas were isolated for antioxidant activity assays and Western blot analysis or for measurements of H2O2 using Amplex Red. Studies verified that male SHR had greater Na+ reabsorption compared with female SHR. Male SHR had enhanced urinary excretion of H2O2 compared with female SHR. Gonadectomy decreased H2O2 excretion in males and increased H2O2 excretion in females, suggesting that testosterone stimulates total body oxidative stress and estrogen suppresses levels of total body oxidative stress. There was not a sex difference in inner medullary H2O2 levels. Male SHR had a testosterone-dependent increase in inner medullary SOD activity, and both intact and gonadectomized males had high levels of inner medullary catalase activity compared with females. The results of this study showed that there was a sexual dimorphism in Na+ handling and oxidant status. We hypothesize that there is a testosterone-sensitive increase in whole body reactive oxygen species production that results in a compensatory increase in the inner medullary antioxidant capability possibly to normalize Na+ handling.
- oxidative stress
- free radicals
male spontaneously hypertensive rats (SHR) reportedly have a blunted pressure-natriuresis relationship compared with female SHR (26). Although the mechanism responsible for the sexual dimorphism in renal function is unknown, it may be related to a testosterone-dependent impairment in Na+ excretion in male SHR, as orchidectomy abolishes the observed sex difference. Oxidative stress contributes to the pathogenesis of hypertension by promoting abnormal renal Na+ handling and renal damage and is enhanced in male SHR (16, 20, 24). Therefore, a sex difference in oxidant status may contribute to the observed sex difference in renal function.
Recent studies have demonstrated a role for renal medullary hydrogen peroxide (H2O2) to influence the development of hypertension and renal function. The ability of renal H2O2 levels to affect Na+ handling has been underscored by Makino et al. (19), where direct infusions of H2O2 into the renal medulla results in decreased medullary blood flow and Na+ excretion. Furthermore, renal medullary interstitial H2O2 levels are greater in Dahl salt-sensitive rats compared with salt-resistant rats, supporting the hypothesis that medullary H2O2 levels may contribute to the development of hypertension (35). The renal inner medulla specifically is important in the fine-tuning of Na+ reabsorption; therefore, increased inner medullary H2O2 levels in male SHR could drive an increase in Na+ reabsorption.
An increase in oxidative stress may be the result of either an increase in the production of reactive oxygen species or a decrease in the ability to neutralize reactive oxygen species. Deficiencies in antioxidant systems have been shown in hypertension. In hypertensive patients and experimental animals, there is a reduction in total antioxidant status in whole blood and mononuclear cells (28, 29). This may be related to alterations in protein expression and/or activity of key antioxidant enzymes: superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). SOD catalyzes the dismutation of superoxide to molecular oxygen and H2O2. There are three isozymes of SOD, cytosolic and nuclear CuZn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), and extracellular EC-SOD (SOD3). Catalase catalyzes the dismutation of H2O2 to water and oxygen, and GPx catalyzes the reduction of hydroperoxides. SOD3 protein expression is lower in the kidney, and SOD1, SOD2, catalase, and GPx protein expression are lower in the myocardium of male SHR compared with male WKY (1, 6). Interestingly, treatment of SHR by intravenous injection of heparin-bound-SOD increases Na+ excretion, highlighting the importance of antioxidant systems in regulating renal function (10, 23). Additionally, coinfusion of an SOD mimetic and catalase has been shown to restore medullary blood flow and Na+ excretion, an effect not seen with infusion of an SOD mimetic alone, further supporting a role for H2O2, or the scavenging of H2O2, in Na+ handling (9, 19).
Sex influences oxidative stress. Epidemiological and experimental evidence suggests that oxidative stress is greater in the male sex (4, 11). Plasma levels of H2O2 are greater in hypertensive patients compared with normotensive patients, and the increase in H2O2 is more profound in men compared with women (18). A sex difference in oxidative stress may be related to a sexual dimorphism in antioxidant systems, as male rats have less SOD expression in the heart (3) and decreased SOD and catalase activities in macrophages compared with females (2). In contrast, male rats have been reported to have higher levels of GPx in the heart compared with females (3), although no difference has been observed in the activity of the enzyme in rat macrophages (2).
Although increased H2O2 levels have been linked to altered Na+ handling, there have been no previous studies examining the influence of sex hormones on antioxidant systems in hypertension. The aim of the study was to determine whether there is a sexual dimorphism in H2O2 levels and antioxidant systems in the renal inner medulla of hypertensive rats. We hypothesize that male SHR have a testosterone-dependent increase in Na+ retention and higher levels of inner medullary H2O2 compared with female SHR, in part because of a decrease in inner medullary antioxidant capability.
Male and female SHR were studied at 12–14 wk of age (Harlan Laboratories, Indianapolis, IN) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved and monitored by the Medical College of Georgia Institutional Animal Care and Use Committee. Rats were housed in temperature- and humidity-controlled, light-cycled quarters. Rats were placed in metabolic cages before death to facilitate 24-h urine collection and measurements of food and water intake. Subsets of animals were gonadectomized at 10 wk of age, as previously described (31) and allowed 3 wk to recover before use. Ovariectomy was confirmed as previously described (7). Blood pressure was measured by the tail-cuff method.
Assays and chemicals.
Urinary and plasma Na+ was measured using a Na+-selective electrode (ELISE, Beckman Instruments, Fullerton, CA). Urinary and inner medullary H2O2 levels were determined using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Briefly, the kidney was perfused with a Krebs/HEPES buffer, and the renal inner medulla was isolated. The intact medulla was incubated at 37°C for 1 h in the dark in 125-μl Amplex red solution plus 125-μl Krebs/HEPES. H2O2 levels were then measured in the buffer, in duplicate, using the Amplex Red kit. Plasma and tissue SOD, catalase, and GPx activities were determined according to manufacturer's instructions (Cayman Chemical, Ann Arbor, MI).
Western Blot analysis.
Inner medullas were homogenized, and Western blot analysis was performed, as previously described, with minor modifications (30). After transfer of protein onto PVDF, the membrane was blocked in 5% milk in TBS. Two-color immunoblots were performed using polyclonal primary antibodies to catalase (Abcam, Cambridge, MA), SOD1, SOD2, or SOD3 (Stressgen, Victoria, British Columbia Canada) in conjunction with a monoclonal antibody to actin (Sigma, St. Louis, MO). Specific bands were detected using the Odyssey Infrared Imager; IRDye800 (Rockland, Gilbertsville, PA) was used for the detection of anti-rabbit antibodies and AlexaFluor 680 was used for the detection of anti-mouse antibodies (Molecular Probes, Eugene, OR). Actin was used to verify equal protein loading, and all densitometric results are reported normalized to actin.
Urinary excretion and plasma data were compared using ANOVA followed by a Newman-Keuls post hoc comparison (Statistica, Tulsa, OK). Relative densitometric units for male/female and intact/gonadectomized comparisons were analyzed using an unpaired t-test with Welch's correction (Prism, San Diego, CA). For all comparisons, P < 0.05 was considered statistically significant.
Blood pressure was greater in male SHR compared with female SHR (P = 0.0006) (Table 1). Gonadectomy decreased blood pressure in male SHR to the level seen in females, whereas gonadectomy has no effect on blood pressure in females (male vs. ORX P = 0.0003). Similarly, male SHR had greater food and water intake compared with females (food, P < 0.0001, water P = 0.02), and this was decreased by orchidectomy (male vs. ORX: food, P < 0.0001; water, P = 0.02). Na+ intake was greater in male SHR compared with female SHR (P < 0.0001) and was decreased by gonadectomy in males (P < 0.0001). Na+ excretion was comparable between all groups. These data indicate a sexual dimorphism in Na+ handling that is sensitive to male sex hormones.
Total body H2O2 levels were determined by measuring H2O2 excretion. Male SHR had greater H2O2 excretion compared with females (P = 0.006, Fig. 1A). ORX decreased H2O2 excretion compared with intact males (P = 0.006) to the level in female SHR, abolishing the sex difference in total body H2O2 levels. OVX increased H2O2 excretion compared with gonad-intact females, but the effect was not statistically significant (P = 0.058). Despite differences in total body levels of H2O2, inner medullary H2O2 production was comparable in male and female SHR (Fig. 1B).
Alterations in antioxidant capability may also contribute to sex differences in oxidative stress. The activity and protein expression of SOD and catalase, as well as GPx activity, were examined in the renal inner medulla. Tissue SOD activity and expression are shown in Fig. 2. Males had greater total SOD activity in the renal inner medulla compared with females (P = 0.0006). Gonadectomy decreased SOD activity in males (P = 0.002), with no effect in females. Despite an increase in total SOD activity in males, SOD1, SOD2, and SOD3 protein expression were comparable between groups (relative densitometric values: SOD1-M, 0.21 ± 0.05; SOD1-R, 0.16 ± 0.05; SOD1-F, 0.18 ± 0.06; SOD1-V, 0.17 ± 0.05. SOD2-M, 0.36 ± 0.05; SOD2-R, 0.30 ± 0.04; SOD2-F, 0.34 ± 0.04; SOD2-V, 0.26 ± 0.02. SOD3-M, 0.04 ± 0.01; SOD3-R, 0.07 ± 0.01; SOD3-F, 0.1 ± 0.04; SOD3-V, 0.07 ± 0.03, where M is male, R is ORX, F is female, and V is OVX). Similarly, males had higher tissue catalase activity compared with females (P = 0.03); however, gonadectomy did not alter activity in either sex (Fig. 3). Inner medullary catalase protein expression was comparable between the four groups (relative densitometric values: M, 0.07 ± 0.03; R, 0.05 ± 0.02; F, 0.05 ± 0.02; V, 0.05 ± 0.02). Tissue GPx activity was comparable between the four groups (males: 8,570 ± 495; ORX: 10,148 ± 539; females: 9,623 ± 606; OVX: 9,561 ± 1,221 nmol·min−1·ml−1·μg−1). In contrast to the renal inner medulla, plasma SOD activity was greater in females compared with males (P = 0.003) and was unaffected by gonadectomy (Table 2). There were no differences in plasma catalase or GPx activities among the four groups.
The primary findings of this study are 1) male SHR have greater total body oxidative stress and a depressed plasma SOD capability compared with female SHR; 2) gonadectomy of male SHR decreased systolic blood pressure and total body oxidative stress, while gonadectomy of female SHR increased total body oxidative stress, although without any effect on blood pressure; and 3) renal inner medullary H2O2 levels are comparable between male and female SHR; however, male SHR have enhanced inner medullary antioxidant capability. We hypothesize that there is a tissue-specific increase in inner medullary antioxidant activity, as compensation for increases in oxidative stress, and as a result, inner medullary H2O2 is comparable in male and female SHR. The kidney is central to the maintenance of electrolyte balance and the control of fluid volume; in particular, the renal inner medulla is responsible for the fine-tuning of Na+ reabsorption. Because oxidative stress has been linked with altered Na+ handling, and direct infusion of H2O2 into the renal medulla decreases sodium excretion (18), we hypothesized that male SHR would have greater inner medullary H2O2 contributing to the observed sexual dimorphism in renal function. In fact, male SHR did not have a greater level of inner medullary H2O2, and this appears to be due to alterations in antioxidant capability.
It is well established that the male sex is associated with increased levels of oxidative stress (4, 11, 34). Our study supports these findings, as total body H2O2 levels were greater in male SHR, but with the caveat that tissue levels may be very different. Although H2O2 has previously been linked to changes in renal hemodynamics and Na+ excretion, this is the first study to examine the effects of sex hormones on H2O2 in hypertensive males and females. Although estradiol has been shown to decrease DNA breaks and damage induced by H2O2 (33), the ability of testosterone to influence levels of H2O2 in vivo had not previously been examined. Our data indicate that testosterone may promote, and estrogen may suppress, H2O2 production. The ability of estrogen to suppress indices of oxidative stress is not surprising, as sex differences in oxidative stress have often been linked to female sex hormones. Estrogen is thought to have direct antioxidant properties, and treatment with estrogen, and estrogen-like compounds, decrease oxidative stress in humans and experimental animals (12, 17, 22, 36). Although our data support a role for estrogen to protect against the development of oxidative stress, the mechanism is unclear. There were no differences in either plasma or inner medullary antioxidant activity or expression between gonad-intact and gonadectomized female SHR. In contrast to our findings, a positive correlation has been shown between estrogen levels and GPx activity in human erythrocytes, while whole blood SOD and catalase activities are higher in women (5, 21). Furthermore, total SOD and catalase activities are greater in macrophages from female compared with male Wistar rats (2). The difference in our findings and studies in the literature may be related to the fact that we are studying hypertensive animals, and most studies examining the role of estrogen as an antioxidant have been performed in normotensive animals. Male SHR have been shown to have a decreased antioxidant capability compared with normotensive male controls in both the whole kidney and heart and a decreased ability to upregulate antioxidant systems in response to an oxidative stress challenge in the whole kidney (1, 6, 13). Alterations in antioxidant potential may occur independent of sex in SHR and compromise the ability of female sex hormones to offer protection.
Consistent with previous reports, orchidectomy of male SHR decreased systolic blood pressure while ovariectomy of female SHR had no effect on systolic blood pressure (26, 31). Concomitant with a decrease in systolic blood pressure following orchidectomy was a decrease in total body oxidative stress in male SHR. What is uncertain from our study is the order in which these events occur. However, Fortepiani and Reckelhoff (13, 14) have shown that in male SHR levels of oxidative stress are positively correlated with mean arterial pressure. Therefore, we would hypothesize that the loss of male sex hormones following orchidectomy leads to a decrease in oxidative stress resulting in a lowering of blood pressure. Future studies will examine the mechanism responsible.
To our knowledge, this is the first study to examine whether tissue oxidative stress is related to a sexual dimorphism in antioxidant status. Our study indicates that male sex hormones influence oxidative stress and antioxidant capacity in the SHR. Little is known about the ability of male sex hormones to influence antioxidant status, especially outside the male reproductive tract. In the rat prostate, testosterone is associated with an increase in antioxidant potential; however, in the rat testis, testosterone decreases antioxidant potential (8, 32). Our data suggest that testosterone may be an important determinant of antioxidant capacity in the renal medulla, although the ability of either sex or sex hormones to regulate antioxidant capability may be tissue specific. In the presence of testosterone, inner medullary SOD activity was elevated independent of alterations in protein expression, although plasma SOD activity was greater in the female SHR and not sensitive to testosterone. Very little is known about the regulation of SOD in the kidney; however, there are known polymorphisms of SOD, as well as active and inactive forms of the enzyme determined by disulfide bridge patterns (15, 25). Whether testosterone may influence one of these parameters or act through a different mechanism to alter inner medullary SOD activity will have to be explored.
In conclusion, sexual dimorphisms in renal inner medullary function are not due to a sexual dimorphism in inner medullary H2O2. Despite male SHR having greater H2O2 excretion compared with females, there was an increase in inner medullary antioxidant capability that we hypothesize acts to diminish local oxidative stress levels. These data suggest that tissue-specific differences in antioxidant systems may be important in determining oxidant status (27).
This work was funded by grants HL60653 (to J. S. Pollock) AG024616 (to J. C. Sullivan) and a PhRMA predoctoral fellowship (to J. M. Sasser). J. S. Pollock is an Established Investigator of the American Heart Association.
The authors would like to acknowledge Heather Walker, Janet Hobbs, and Jacqueline Musall for their excellent technical assistance, and David M. Pollock for discussion and critically reading the manuscript.
Present address of J. M. Sasser: Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32611.
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