Am J Physiol Regul Integr Comp Physiol 302: R274 –R282, 2012.
First published November 2, 2011; doi:10.1152/ajpregu.00546.2011.
Oxidative stress contributes to sex differences in angiotensin II-mediated
hypertension in spontaneously hypertensive rats
Kanchan Bhatia,1 Ahmed A. Elmarakby,2,3 Azza El-Remessey,2,4 and Jennifer C. Sullivan1,2
1
Department of Medicine, 2Department of Pharmacology and Toxicology, 3Department of Oral Biology, Georgia Health
Sciences University and Program in Clinical and Experimental Therapeutics, University of Georgia 4College of Pharmacy,
Augusta, Georgia
Submitted 26 September 2011; accepted in final form 31 October 2011
superoxide; kidney; apocynin; NADPH oxidase
system (RAAS) is important in the regulation of body fluid and blood pressure homeostasis, and overactivation of the RAAS contributes to the
development of numerous pathophysiological processes (20,
21, 40). There is an expanding literature regarding sex differences in the expression of RAAS components, as well as in
functional responses to RAAS activation to both acute and
chronic ANG II infusion (24, 25, 36, 38, 46). We recently
published that greater levels of the vasodilatory peptide ANG
(1–7) contribute to sex differences in the hypertensive response
to chronic ANG II infusion in spontaneously hypertensive rats
(SHR), although it is unknown whether there are additional
molecular mechanism(s) contributing to sex differences in the
blood pressure response to ANG II. Activation of angiotensin
type 1 (AT1) receptors mediate most well-known biological
THE RENIN-ANGIOTENSIN-ALDOSTERONE
Address for reprint requests and other correspondence: J. C. Sullivan, 1459
Laney Walker Blvd., Georgia Health Sciences Univ., Augusta, GA 30912
(e-mail: [email protected]).
R274
functions of ANG II, including the stimulation of oxidative
stress, and female SHR have fewer AT1 receptors than male
SHR (43). However, little is known regarding the relative
contribution of ANG II-mediated oxidative stress on blood
pressure regulation in male vs. female rats.
Reactive oxygen species (ROS) are generated by all cell
types and act as intercellular and intracellular signaling molecules. However, the highly reactive nature of ROS also poses
potential deleterious effects on cellular structure and integrity,
and increases in oxidative stress contribute to the development
and progression of numerous cardiovascular diseases, including hypertension (11, 15, 35). In addition to known sex differences in the RAAS, there are also sex differences in oxidative
stress levels. Both clinical and experimental studies have
shown that oxidative stress levels are higher in males compared
with females (10, 21, 23, 26), and sex differences in oxidative
stress have been implicated in the sexual dimorphism in hypertension (10, 21, 26). Oxidative stress is determined by the
balance of the production of free radicals and the ability of
antioxidants to neutralize them. Deficiencies in antioxidant
systems have been reported in hypertensive patients and in
experimental models of hypertension (8, 30, 41, 42), and
females tend to have greater antioxidant capacity compared
with males (5, 58). Thus, the differential balance in the prooxidant and antioxidant status may play an important role in
blunting oxidative stress-mediated cardiovascular pathologies
in females.
Evidence in the literature suggests that there are sex differences in ANG II-induced increases in oxidative stress. An
acute dose of ANG II (0.1 ␮mol/l) mediated greater superoxide
(O⫺
2 ) and hydrogen peroxide (H2O2) production in isolated
cerebral arteries from male C57BL6/J mice than females (12),
and chronic ANG II infusion increased plasma thiobarbituric
acid reactive substances (TBARS), a marker of oxidative
stress, in male, but not female, C57BL/6 mice (13). It is well
established in male experimental animals that ANG II-mediated increases in oxidative stress contribute to the development
of hypertension (28, 59), and increased NADPH oxidase activity has been reported as a source of the excess O⫺
2 levels
following ANG II infusion (27, 29, 21, 13). Although studies
have suggested that males are more susceptible to ANG IImediated increases in oxidative stress, the contribution of
increases in oxidative stress to the development of ANG II
hypertension in both sexes has not been clearly examined.
We hypothesize that there is a sex difference in ANG
II-mediated increases in ROS generation and in the compensatory activation of antioxidant capacity in SHR, which contributes to sex differences in ANG II-induced hypertension.
Studying male and female SHR allows for the assessment of
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Bhatia K, Elmarakby AA, El-Remessey A, Sullivan JC. Oxidative stress contributes to sex differences in angiotensin II-mediated
hypertension in spontaneously hypertensive rats. Am J Physiol Regul
Integr Comp Physiol 302: R274 –R282, 2012. First published November 2, 2011; doi:10.1152/ajpregu.00546.2011.—NADPH oxidase has
been implicated in ANG II-induced oxidative stress and hypertension
in males; however, the contribution of oxidative stress to ANG II
hypertension in females is unknown. In the present study, we tested
the hypothesis that greater antioxidant capacity in female spontaneously hypertensive rats (SHR) blunts ANG II-induced oxidative stress
and hypertension relative to males. Whole body and renal cortical
oxidative stress levels were assessed in female and male SHR left
untreated or following 2 wk of chronic ANG II infusion. Chronic
ANG II infusion increased NADPH oxidase enzymatic activity in the
renal cortex of both sexes; however, this increase only reached
significance in female SHR. In contrast, male SHR demonstrated a
greater increase in all measurements of reactive oxygen species
production in response to chronic ANG II infusion. ANG II infusion
increased plasma superoxide dismutase activity only in female SHR
(76 ⫾ 9 vs. 190 ⫾ 7 Units·ml⫺1·mg⫺1, P ⬍ 0.05); however, cortical
antioxidant capacity was unchanged by ANG II in either sex. To
assess the functional implication of alterations in NADPH enzymatic
activity and oxidative stress levels following ANG II infusion, additional experiments assessed the ability of the in vivo antioxidant
apocynin to modulate ANG II hypertension. Apocynin significantly
blunted ANG II hypertension in male SHR (174 ⫾ 2 vs. 151 ⫾ 1
mmHg, P ⬍ 0.05), with no effect in females (160 ⫾ 11 vs. 163 ⫾ 10
mmHg). These data suggest that ANG II hypertension in male SHR is
more dependent on increases in oxidative stress than in female SHR.
SEX DIFFERENCES IN ANG II-INDUCED HYPERTENSION IN SHR
MATERIALS AND METHODS
Animals. Male and female SHR were used in this study (Harlan
Laboratories, Indianapolis, IN). All experiments were conducted in
accordance with the National Institutes of Health’s Guide for the Care
and Use of Laboratory Animals and were approved and monitored by
the Georgia Health Sciences University Institutional Animal Care and
Use Committee. A subset of male and female SHR (n ⫽ 9) were
implanted with telemetry transmitters (Data Sciences International, St.
Paul, MN) at 11 wk of age, as previously described (46). Rats were
allowed 1 wk to recover before they were placed on telemetry
receivers for the measurement of baseline blood pressure. After 5
days, rats were randomized to receive one of the following treatments:
chronic ANG II infusion (200 ng·kg⫺1·min⫺1 for 14 days; Phoenix
Pharmaceuticals, Burlingame, CA) via subcutaneous osmotic minipump (Alzet, Cupertino, CA; n ⫽ 5) or the in vivo antioxidant
apocynin (1.5 mmol/l) in the drinking water for 7 days prior to ANG
II infusion, and throughout the infusion period (n ⫽ 4).
Additional male and female SHR (11–12 wk, respectively) were
left untreated (n ⫽ 5– 8) or received chronic ANG II infusion as
described above (n ⫽ 5). Rats were placed in metabolic cages weekly
for 24-h urine collection. Following 2 wk of ANG II infusion, rats
(13–14 wk) were anesthetized with ketamine/xylazine (48 mg/kg and
6.4 mg/kg ip, respectively; Phoenix Pharmaceuticals, St. Joseph,
MO), a terminal blood sample was taken, and plasma was separated
by centrifugation. Kidneys were removed, and the renal cortexes were
isolated and snap frozen in liquid nitrogen.
Lucigenin chemiluminescence assay. Renal cortexes were homogenized for determination of NADPH oxidase enzymatic activity and
basal O⫺
2 levels via lucigenin chemiluminescence, as previously
described (47). NADPH oxidase activity was measured in the presence of 100 ␮mol/l NADPH (Sigma, St. Louis, MO) and incubated
with lucigenin (Sigma; 5 ␮mol/l). All counts were normalized to total
protein: 35 ␮g/well for the determination of NADPH oxidase activity;
350 ␮g/well for basal determination of O⫺
2 . The data were expressed
as counts per minute per milligram protein. Specificity of the assay
was confirmed by the inclusion of the superoxide scavenger, tempol
(Sigma; 10 mmol/l).
Dihydroethidine staining. Glomeruli were isolated from the renal
cortex (n ⫽ 5) and stained with dihydroethidine (DHE; Invitrogen,
Carlsbad, CA) for the detection of O⫺
2 generation, as previously
described with slight modifications (14). Briefly, the kidney cortexes
were minced and filtered through successive mesh filters (200 –50
␮m) in ice-cold 0.9% NaCl solution. Glomeruli retained on the mesh
were washed (4°C, 2,000 g) and suspended in 50 mM Tris-HCl (pH
7.4). The purity of the preparation was assessed by light microscopy.
One-hundred microliters of the isolated glomeruli suspension was
fixed on slides, and slides were incubated in 2 ␮M DHE for 30 min
at 37°C. Images were obtained with a LSM 510 META confocal
microscope, with an excitation of 488 nm and emission of 574 to 595 nm
or a 560-nm long pass filter. The slides were blind scored by three
examiners, and the intensity of fluorescence was quantified in each group
using MetaMorph software (Molecular Devices, Sunnyvale, CA).
Nitrotyrosine slot blot. Slot-blot analysis was used as described
previously (1). Briefly, 100 ␮g of renal cortical homogenate (n ⫽ 5 or
6) was immobilized onto a nitrocellulose membrane. After blocking
with PBST containing 5% nonfat milk, membranes were reacted with
antibody against nitrotyrosine (Calbiochem, San Diego, CA) using
1:500 dilutions in PBST containing 5% nonfat milk and applied
overnight at 4°C. Primary antibody was detected by 1:5,000 dilutions
of horseradish peroxidase-conjugated sheep anti-mouse antibody in
PBST and enhanced-chemiluminescence (GE Healthcare, Piscataway,
NJ). The optical density of the samples was quantified using densitometry software (Alpha Innotech, San Leandro, CA) and compared
with that of controls and expressed as relative optical density.
Western blot analysis. Renal cortical samples (n ⫽ 6/group) were
homogenized, and Western blotting was performed as previously
described (49). Following homogenization, protein concentrations
were determined by standard Bradford assay (Bio-Rad, Hercules, CA)
using BSA as the standard. Briefly, after proteins were transferred
onto polyvinylidene difluoride membranes and blocked in 5% milk in
Tris-buffered saline, two-color immunoblots were performed using
polyclonal primary antibodies to CuZnSOD, MnSOD, EcSOD (polyclonal; Stressgen Bioreagents, Victoria, BC, Canada), and catalase
(polyclonal; Abcam, Cambridge, MA). Specific bands were detected
using the Odyssey infrared imager in conjunction with the appropriate
IRDye secondary antibodies (LI-COR Biosciences, Lincoln, NE).
␤-actin (monoclonal; Sigma) was used to verify equal protein loading,
and all densitometric results are reported normalized to ␤-actin.
Kits and assays. All assays were performed according the manufacturer’s instructions. Plasma 8-isoprostane levels were measured at
a wavelength of 410 nm, and data were expressed as pg/ml (Cayman
Chemicals, Ann Arbor, MI). For the detection of H2O2 levels and
TBARS, urine samples were spun at 3,000 rpm for 10 min to remove
particulate matter prior to assay, and levels were expressed as nanomoles excreted/day (H2O2; Invitrogen Amplex Red, Eugene, OR; and
TBARS: Cayman Chemicals, Ann Arbor, MI). Total antioxidant
potential was measured in the kidney cortex and urine samples, with
the data expressed as millimoles copper-reducing equivalents per
milligram protein (BIOXYTECH AOP-450, Oxis Research, Foster
City, CA). Catalase and superoxide dismutase (SOD) activity were
measured in plasma and renal cortical samples by ELISA kits, and the
data were expressed as units per milliliter per milligram protein
(Cayman Chemicals, Ann Arbor, MI).
Statistical analysis. All data are presented as means ⫾ SE. Mean
arterial pressure (MAP) and urinary protein excretion data were
analyzed using ANOVA for repeated measurements. Urinary TBARS
excretion rates were compared using a one-way ANOVA. The rest of
the data was analyzed using two-way ANOVA, factor 1 was sex of the
animal, and factor 2 was ANG II treatment. Differences were considered statistically significant with P ⬍ 0.05. Analyses were performed
using GraphPad Prism version 4.0 software (GraphPad Software, La
Jolla, CA).
RESULTS
Total body oxidative stress measurements. Plasma 8-isoprostane levels and urinary H2O2 excretion rates were measured in untreated and ANG II-infused male and female SHR,
as measures of total body oxidative stress (Table 1). Basal
8-isoprostane levels were comparable in untreated male and
female SHR. Chronic ANG II infusion increased plasma 8-iso-
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the effect of sex on oxidative stress and antioxidant systems in
essential hypertension, as well as in response to chronic ANG
II infusion. There are sex differences in renal cortical oxidative
stress levels and in inner medullary antioxidant capacity in
SHR (49, 50), and Fortepiani and Reckelhoff (15) showed that
male SHR have a decrease in basal blood pressure in response
to the antioxidant tempol, while female SHR do not. Thus, the
first goal of this study was to assess whole body and renal
cortical measures of oxidative stress and antioxidant capacity
in male and female SHR. Animals were studied under basal
conditions and following ANG II infusion to determine how
direct stimulation of the RAAS alters oxidative stress. We
found that following ANG II infusion, male SHR had a more
robust increase in oxidative stress levels than female SHR. On
the basis of these observations, additional studies were designed to test the hypothesis that ANG II-induced hypertension
in male SHR is more dependent on increases in oxidative stress
than in female SHR.
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SEX DIFFERENCES IN ANG II-INDUCED HYPERTENSION IN SHR
Table 1. Total Body oxidative stress measurements in untreated and ANG II infused male and female SHR
Oxidative Stress Parameters
Male
Male⫹ANG II
Female
Female⫹ANG II
Plasma 8-isoprostane, pg/ml
Urinary H2O2 excretion, nmol/day
80 ⫾ 18
20 ⫾ 2
164 ⫾ 26
78 ⫾ 20
81 ⫾ 12
9⫾2
68 ⫾ 3
26 ⫾ 5
Values are presented as means ⫾ SE using two-way ANOVA; n ⫽ 5– 8. Plasma 8-isoprostane-sex, P ⫽ 0.013; ANG II, P ⫽ 0.0014; interaction, P ⫽ 0.05.
Urinary H2O2 excretion-sex, P ⫽ 0.0068; ANG II, P ⫽ 0.0016; interaction, P ⫽ 0.07.
even after ANG II infusion levels remained greater in male
SHR (two-way ANOVA: effect of sex, P ⬍ 0.0001; effect of
ANG II, P ⫽ 0.01; interaction, P ⫽ 0.6). Cortical nitrotyrosine
content also tended to be greater in untreated male SHR
compared with females. Chronic ANG II infusion increased
nitrotyrosine content in the renal cortex of male SHR (36%
increase) with no change in females (two-way ANOVA:
effect of sex, P ⫽ 0.13; effect of ANG II, P ⫽ 0.026;
interaction, P ⫽ 0.19; Fig. 1C). Sex differences in oxidative
stress levels were also indicated in isolated glomeruli. DHE
fluorescence was greater in glomeruli from untreated male
SHR than females, and ANG II infusion increased DHE
intensity only in males (24% increase; two-way ANOVA:
effect of sex, P ⬍ 0.001; effect of ANG II, P ⫽ 0.09;
interaction, P ⫽ 0.006; Fig. 2, A–E).
Total body antioxidant potential (AOP), SOD, and catalase
activity. Antioxidant capacity is an important determinant of
oxidative stress levels. Urinary AOP tended to be less in
untreated male SHR compared with female SHR. Infusion of
ANG II decreased urinary AOP in both male (41% decrease)
and female SHR (39% decrease); however, AOP levels tended
to remain higher in females (two-way ANOVA: effect of sex,
P ⫽ 0.18; effect of ANG II, P ⫽ 0.009; interaction, P ⫽ 0.54;
Table 2). Plasma SOD activity was also less in male SHR
compared with females, and chronic ANG II infusion increased
SOD activity only in the female SHR (150% increase; two-way
ANOVA: effect of sex, P ⬍ 0.0001; effect of ANG II, P ⬍
0.0001; interaction, P ⬍ 0.0001). Plasma catalase activity was
comparable in all groups (two-way ANOVA: effect of sex,
P ⫽ 0.53; effect of ANG II, P ⫽ 0.39; interaction, P ⫽ 0.28).
Renal cortical antioxidant potential, SOD, and catalase
activity. Renal cortical AOP was comparable in untreated male
and female SHR and unaffected by ANG II infusion (two-way
Fig. 1. Renal cortical NADPH oxidase enzymatic activity (A) and basal cortical O⫺
2 production (B) in untreated and ANG II-treated male (M) and female (F)
spontaneously hypertensive rats, as measured by lucigenin chemiluminescence. C: nitrotyrosine content was measured in the renal cortex as an oxidative stress
marker by slot blot analysis; n ⫽ 5– 6.
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prostane levels in male (105% increase), but not female, SHR
(two-way ANOVA: effect of sex, P ⫽ 0.013; effect of ANG II,
P ⫽ 0.0014; interaction, P ⫽ 0.05). Basal H2O2 excretion rates
tended to be greater in males compared with females. H2O2
excretion increased with chronic ANG II infusion in both male
(290% increase) and female SHR (189% increase); however,
even after ANG II infusion, excretion rates remained greater in
male SHR (two-way ANOVA: effect of sex, P ⫽ 0.0068;
effect of ANG II, P ⫽ 0.0016; interaction, P ⫽ 0.07).
NADPH oxidase enzymatic activity. NADPH oxidase enzymatic activity was assessed by adding exogenous NADPH to
renal cortical homogenates; therefore, this assay measures the
potential of the enzyme complex to produce O⫺
2 and is not a
measure of NADPH oxidase-derived O⫺
production
in vivo.
2
Consistent with our previous data (50), NADPH oxidase enzymatic activity was comparable in untreated male and female
SHR. Chronic ANG II infusion increased NADPH oxidase
enzymatic activity in both male (43% increase) and female
(154% increase) SHR; however, the increase was greater in
female SHR (two-way ANOVA: effect of sex, P ⫽ 0.015;
effect of ANG II, P ⫽ 0.014; interaction, P ⬍ 0.0001, Fig. 1A).
Renal cortical oxidative and nitrative stress measurements.
Additional experiments were performed to assess whether
increases in renal cortical NADPH oxidase enzymatic activity
translated into comparable increases in tissue oxidative stress
levels. Renal cortical O⫺
2 levels were measured using lucigenin
chemiluminescence, cortical nitrotyrosine levels were assessed
using slot blot analysis, and glomerular O⫺
2 levels were measured using DHE staining. Consistent with our previous report
(50), basal O⫺
2 levels were greater in the renal cortex of male
SHR compared with females (Fig. 1B). Renal cortical O⫺
2
levels increased with chronic ANG II infusion in both male
(30% increase) and female SHR (48% increase), however,
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SEX DIFFERENCES IN ANG II-INDUCED HYPERTENSION IN SHR
ANOVA: effect of sex, P ⫽ 0.54; effect of ANG II, P ⫽ 0.83;
interaction, P ⫽ 0.22; Table 2). Total SOD and catalase
activity were also measured in renal cortical homogenates.
Neither sex of the animal or ANG II infusion altered SOD
activity (two-way ANOVA: effect of sex, P ⫽ 0.18; effect of
ANG II, P ⫽ 0.56; interaction, P ⫽ 0.93; Table 2). Catalase
activity was higher in the renal cortex of male SHR than
females in untreated rats, and ANG II infusion tended to
increase catalase activity in female SHR (75% increase; twoway ANOVA: effect of sex, P ⬍ 0.001; effect of ANG II, P ⫽
0.068; interaction, P ⫽ 0.67).
CuZnSOD, MnSOD, and EcSOD protein expression levels
were also determined and consistent with the activity data;
protein expression levels were similar between groups (twoway ANOVA: CuZnSOD-effect of sex, P ⫽ 0.55; effect of
ANG II, P ⫽ 0.49; interaction, P ⫽ 0.53; MnSOD-effect of
sex, P ⫽ 0.89; effect of ANG II, P ⫽ 0.59; interaction, P ⫽
0.56; EcSOD-effect of sex, P ⫽ 0.96; effect of ANG II, P ⫽
0.85; interaction, P ⫽ 0.80; Fig. 3, A–C). Catalase protein
expression was also comparable between all groups (two-way
ANOVA: effect of sex P ⫽ 0.62; effect of ANG II P ⫽ 0.25;
interaction P ⫽ 0.28; Fig. 3D).
Mean arterial pressure measurements. Consistent with our
previous report, male SHR had higher blood pressures than
females at baseline and a greater increase in blood pressure in
response to ANG II infusion than female SHR at all time points
(47). Compared with baseline, ANG II infusion significantly
increased blood pressure in males after 5 days of ANG II
infusion and after 8 days in females (percent increase in MAP:
males, 19 ⫾ 3%; females, 12 ⫾ 2%; P ⬍ 0.05). To determine
Table 2. Total body and renal cortical measurements for antioxidant potential, SOD, and catalase activity in untreated and
ANG II-infused male and female spontaneously hypertensive rats
Antioxidant Parameters
AOP, mM copper-reducing equivalents/mg protein
Urine
Cortex
SOD activity, units/ml
Plasma
Cortex
Catalase activity, ␮M 䡠 min⫺1 䡠 ml⫺1 䡠 mg protein⫺1
Plasma
Cortex
Male
Male⫹ANG II
Female
Female⫹ANG II
2.4 ⫾ 0.1
0.16 ⫾ 0.01
1.4 ⫾ 0.2
0.17 ⫾ 0.01
2.8 ⫾ 0.7
0.16 ⫾ 0.01
1.7 ⫾ 0.5
0.14 ⫾ 0.01
54 ⫾ 1
3.8 ⫾ 0.5
56 ⫾ 1
3.6 ⫾ 0.4
76 ⫾ 9
3.2 ⫾ 0.4
190 ⫾ 7
3.0 ⫾ 0.2
5⫾2
11 ⫾ 2
6⫾1
13 ⫾ 1
8⫾1
4⫾1
7⫾1
7⫾1
Values are presented as means ⫾ SE, using two-way ANOVA; n ⫽ 6 – 8. Urinary antioxidant potential (AOP)-sex, P ⫽ 0.18; ANG II, P ⫽ 0.009; interaction,
P ⫽ 0.54; cortex AOP-sex, P ⫽ 0.54; ANG II, P ⫽ 0.83; interaction, P ⫽ 0.22. Plasma SOD activity-sex, P ⬍ 0.0001; ANG II, P ⬍ 0.0001; interaction, P ⬍
0.0001; cortex SOD activity-sex, P ⫽ 0.18; ANG II, P ⫽ 0.56; interaction, P ⫽ 0.93. Plasma catalase activity-sex, P ⫽ 0.53; ANG II, P ⫽ 0.39; interaction,
P ⫽ 0.28; cortex catalase activity-sex, P ⬍ 0.001; ANG II, P ⫽ 0.068; interaction, P ⫽ 0.67
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Fig. 2. Dihydroethidine (DHE) staining for O⫺
2 generation in isolated glomeruli. Average intensity of fluorescence quantified by MetaMorph software (A) with
representative images from an untreated male (M; B), a male treated with ANG II (M⫹ANG II; C), an untreated female (F; D) and a female treated with ANG
II (F⫹ANG II; E) below. n ⫽ 5.
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SEX DIFFERENCES IN ANG II-INDUCED HYPERTENSION IN SHR
Fig. 3. Antioxidant protein expression in the renal cortex of untreated and ANG II-treated male (M) and female (F) spontaneously hypertensive rats (SHR).
Protein expression of CuZn SOD (A), MnSOD (B), EcSOD (C), and catalase (D) measured by Western blot analysis in the renal cortex normalized to
actin; n ⫽ 6.
DISCUSSION
Although there is an ever expanding literature base supporting the existence of sex differences in chronic ANG II-mediated hypertension, the molecular mechanisms responsible have
yet to be fully elucidated. Ample evidence suggests that ANG
II-mediated increases in oxidative stress contribute to ANG
II-driven pathologies in males (Fig. 5); the role of oxidative
stress in ANG II hypertension in females is unknown. This
becomes especially intriguing in light of recent reports of sex
differences in ANG II-induced oxidative stress. As such, the
primary novel finding of this study is that despite a robust
increase in NADPH oxidase, enzymatic activity in the renal
cortex and modest increases in indices of oxidative stress in
female SHR, treatment with the antioxidant apocynin did not
alter ANG II-induced hypertension. In contrast, apocynin significantly attenuated ANG II hypertension in male SHR,
thereby abolishing the sex difference in blood pressure and
verifying that differences in ANG II-induced oxidative stress in
males vs. females contribute to sex differences in ANG IImediated increases in blood pressure.
Males are known to have higher levels of ROS compared
with females under basal conditions, and there is sufficient
evidence in the literature showing that ANG II infusion increases oxidative stress in males (10, 15, 44, 49). Consistent
with our results, chronic ANG II infusion has been shown to
induce increases in urinary H2O2 excretion and plasma 8-isoprostanes levels in male Sprague-Dawley rats (17, 33, 35, 38,
39, 45, 56). Although less is known regarding the effect of
chronic ANG II infusion in female experimental animals,
available data support our results that females are less susceptible to ANG II-induced increases in oxidative stress. ANG II
infusion in C57BL/6J mice for 14 or 28 days resulted in
Fig. 4. Effect of apocynin on ANG II hypertension. Twenty-four-hour mean arterial pressure (MAP) was measured by telemetry and in male and female SHR.
Apocynin treatment was begun on day 8 with ANG II infusion initiated on day 15 in male (A) and female SHR (B). #Significant difference from untreated,
P ⬍ 0.05. *Significant difference from same-sex baseline blood pressure, P ⬍ 0.05; n ⫽ 4 or 5.
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the contribution of oxidative stress to the increase in blood
pressure in response to chronic ANG II infusion, additional
male and female SHR were treated with the in vivo antioxidant
apocynin. Treatment with apocynin alone for 1 wk did not alter
basal blood pressure in either sex (Fig. 4, A and B). Infusion of
ANG II in the presence of apocynin increased blood pressure
in both sexes (percent increase in MAP: males, 9 ⫾ 2%;
females, 13 ⫾ 5%). Apocynin attenuated an ANG II-induced
increase in blood pressure in male SHR (Fig. 4A), with no
effect on ANG II-mediated increases in blood pressure in
female SHR (Fig. 4B), thereby abolishing the sex difference in
the ANG II blood pressure response.
To assess the effectiveness of apocynin to decrease markers
of oxidative stress, urinary TBAR excretion rates were measured. In male SHR, ANG II infusion increased urinary TBAR
excretion from 76 ⫾ 5 to 112 ⫾ 12 nmol/day (P ⬍ 0.05), and
apocynin attenuated this increase (87 ⫾ 9 nmol/day; P ⬍ 0.05;
n ⫽ 4). TBAR excretion rates were comparable among all
three groups of female SHR (untreated: 43 ⫾ 3 nmol/day;
ANG II treated: 47 ⫾ 7 nmol/day; apocynin⫹ANG II: 48 ⫾ 5
nmol/day; not significant, n ⫽ 4).
SEX DIFFERENCES IN ANG II-INDUCED HYPERTENSION IN SHR
increases in plasma TBARS and cardiac NADPH-driven generation of O⫺
2 in male, but not female, mice (13). Similarly,
chronic ANG II infusion causes cerebral endothelial dysfunction in male MnSOD-deficient mice, but not in female mice,
suggesting greater ANG II-mediated increases in oxidative
stress in male mice (9). In the current study, we found that
ANG II infusion resulted in modest increases in oxidative
stress in females, although the absolute levels of oxidative
stress attained remained less in females relative to their male
counterparts. The increase in oxidative stress seen in females in
the current study is likely related to the use of SHR as opposed
to normotensive mice as the background for the ANG II
infusion. Regardless, the modest increases in systemic and
tissue levels of oxidative stress did not translate into an
exaggerated increase in blood pressure following ANG II
infusion in females.
NADPH oxidase has been implicated as the primary source
of increases in O2·⫺ production in the vasculature and kidneys
of male experimental animals infused with ANG II (10, 12, 31,
55), and increased NADPH oxidase activity has been reported
in isolated vascular smooth muscle cells from male SHR
treated with ANG II (6). Sex differences in NADPH oxidase
have also been reported, with male C57BL/6 mice having
greater cardiac NADPH oxidase enzymatic activity following
ANG II infusion than females (13). Surprisingly, in the present
study, we found that renal cortical NADPH oxidase enzymatic
activity was increased to a greater degree in female SHR
following 2 wk of ANG II infusion than in males. Despite this
finding, female SHR did not have correspondingly greater
increases in tissue levels of oxidative stress following ANG II
infusion compared with male SHR. We speculate that the
disconnect between NADPH enzymatic activity and oxidative
stress in female SHR may be related to lower levels of AT1
protein expression or lowered efficiency of AT1 receptor coupling to the NADPH oxidase activation compared with male
SHR. Future studies will directly address this question. ANG II
stimulation of NADPH oxidase has been shown to reduce nitric
oxide (NO) bioavailability in kidneys of male SHR (2), and we
have previously published that female SHR have greater NO
bioavailability in the renal cortex than male SHR, independent
of alterations in NO synthase activity or expression (48). We
proposed that greater NO bioavailability in females was the
result of less scavenging of NO by O2·⫺ (50); therefore, we
hypothesize that enhanced NO bioavailability may mitigate
ANG II-induced increases in blood pressure (BP) to a greater
extent in female SHR.
To begin to assess the molecular mechanism responsible for
the observed sex differences in the degree of ANG II-induced
oxidative stress, we assessed the primary antioxidant systems.
It is anticipated that the ability of antioxidants to neutralize free
radicals is of equal importance to the production of free
radicals in the determination of oxidative stress levels (18, 22).
We postulated that sex differences in oxidative stress following
chronic ANG II infusion were due to sex differences in
antioxidant capacity. There are clinical and experimental reports of greater antioxidant potential in females compared with
males. Under basal conditions, female Wistar rats have greater
liver mitochondrial MnSOD protein expression than males (5),
and female imprinting control region mice have greater aortic
Cu/Zn-SOD and EC-SOD protein expression than males (51).
Female Wistar rats also have greater catalase activities in
macrophages compared with males (3). Of particular relevance
to the current study, increasing antioxidant capacity has been
shown to be critical in mediating physiological responses to
ANG II in experimental animals. Overexpression of human
superoxide dismutase 1 or an infusion of an SOD mimetic both
inhibit ANG II-mediated increases in oxidative stress in male
mice (55, 58) and rats (33). In the present study, we found that
plasma SOD activity was significantly increased following
ANG II infusion in female SHR. This increase in plasma SOD
activity was accompanied by a decrease in plasma 8-isoprostane levels; however, there was not a corresponding increase in
catalase activity, which may account for the increase in H2O2
excretion in females following ANG II infusion. Although not
directly tested in this study, EC-SOD has been reported to be
the primary SOD isoform in plasma, interstitial fluid, lymph,
and synovial fluid and has a long circulatory half-life (16).
Therefore, we hypothesize that EC-SOD mediates the increase
in plasma total SOD activity measured in female SHR. Consistent with other studies in female C57BL/6J mice and albino
Wistar rats, there was no evidence of increased antioxidant
capacity at the tissue level in either males or females following
ANG II infusion (15, 53). Our findings suggest that enhanced
antioxidant potential in females is not solely responsible for
sex differences in ANG II-mediated oxidative stress levels in
the renal cortex; however, we cannot rule out alternative
tissue-specific alterations in antioxidant capacity. Although not
examined in this study, it should be noted that sex differences
in ANG II-induced oxidative stress may be related to female
sex hormones. Chronic central administration of 17␤-estradiol
in conjunction with ANG II infusion blocks ROS generation in
male C57BL/6J mice in the subfornical organ (58), and ovariectomy of female C57BL/6J mice results in greater ANG
II-mediated increases in cardiac NADPH oxidase-derived O2·⫺
production compared with intact females (13). Future studies
will determine the role of sex steroids in regulating ANG
II-mediated oxidative stress.
Similar to previous reports from our laboratory and others,
males had a greater increase in blood pressure with chronic
ANG II infusion than females (46, 53, 57). We have previously
published that greater levels of ANG (1–7) contribute to the
lower blood pressure maintained by female SHR during ANG
II infusion (46). However, is the lower level of ANG (1–7) the
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00546.2011 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.2 on June 17, 2017
Fig. 5. Schematic flow chart of the impact of ANG II-mediated increases in
oxidative stress on blood pressure in male and female SHR.
R279
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SEX DIFFERENCES IN ANG II-INDUCED HYPERTENSION IN SHR
referred to apocynin as an in vivo antioxidant throughout this
article. In addition, we cannot attest to any other nonspecific
effects of apocynin, as additional parameters were not examined in our study; however, indices of oxidative stress were
reduced by apocynin in male SHR. Finally, all biochemical
analyses were made in both plasma and urine as measures of
whole body oxidative stress or directly in the kidney. Since
apocynin was delivered systemically, we cannot differentiate
the relative role of renal oxidative stress in mediating the blood
pressure response to ANG II compared with increases in
oxidative stress that may also occur in other tissues and organs
that also modulate blood pressure.
Perspectives and Significance
We recently published that greater levels of the vasodilatory
peptide ANG (1–7) in female SHR contributes to the lower BP
response to chronic ANG II infusion in females; however,
whether there were additional prohypertensive factors that
contributed to the greater increase in blood pressure in males
was unknown. As a result, the current study focused on
oxidative stress, and indeed, we found that male SHR have
greater increases in ANG II-mediated oxidative stress contributing to their hypertension. Although female SHR had lower
levels of ANG II-mediated increases in renal cortical oxidative
stress, there were only modest changes in antioxidant capacity,
suggesting additional mechanisms may be mediating oxidative
stress levels in female SHR. Of interest, ANG-(1–7) has been
reported to attenuate ANG II-mediated NADPH oxidase activation and ROS production in glomeruli and mesangial cells
from type 2 diabetic mice (32) and decrease renal NADPH
oxidase activity in the diabetic SHR kidney (4), thus implicating a role for ANG (1–7) in suppressing oxidative stress.
Therefore, we speculate that greater levels of ANG (1–7) in
ANG II-infused female SHR antagonizes ANG II-mediated
increases in oxidative stress, thereby limiting increases in
blood pressure.
ACKNOWLEDGMENTS
We are grateful to Dr. David Pollock for assistance with telemetry studies.
The authors would like to acknowledge the assistance of Krystal Brinson and
Chet Joyner for glomeruli isolation and Western blot analysis.
GRANTS
This study was funded by National Institutes of Health Grant 1R01
HL093271– 01A1 to J. C. Sullivan.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: K.B., A.A.E., and A.E.-R. performed experiments;
K.B. and J.C.S. analyzed data; K.B. and J.C.S. interpreted results of experiments; K.B. prepared figures; K.B. drafted manuscript; K.B., A.A.E., A.E.-R.,
and J.C.S. edited and revised manuscript; J.C.S. conception and design of
research; J.C.S. approved final version of manuscript.
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