Postnatal Ontogeny of Angiotensin Receptors and ACE2 in Male and Female Rats

GENDER MEDICINE/VOL. 9, NO. 1, 2012

Postnatal Ontogeny of Angiotensin Receptors and ACE2 in Male and Female Rats Amanda K. Sampson, PhD1; Karen M. Moritz, PhD2; and Kate M. Denton, PhD1 1

Department of Physiology, Monash University, Melbourne, Victoria, Australia; and Biological Sciences, University of Queensland, Brisbane, Queensland, Australia

2

Department of

ABSTRACT Background: Sex differences in the expression of the angiotensin (Ang) II receptors and angiotensinconverting enzyme 2 (ACE2) have been hypothesized to be a potential mechanism contributing to sex-specific differences in arterial pressure. Currently, sex differences in the expression of the angiotensin receptors and ACE2 remain undefined. Objectives: The aim of this study was to define the postnatal ontogeny of mRNA expression, from birth to adulthood, of the Ang II and Ang-(1-7) receptors and ACE2 in male and female rats. Methods: Kidney and heart tissue was collected from male and female Sprague Dawley rats and snapfrozen at postnatal days (PNDs) 1, 30, 42, 70, and 110 (adult), and real-time polymerase chain reaction was performed to determine relative expression of the Ang II and Ang-(1-7) receptors (AT1aR, AT1bR, AT2R, and MasR) and ACE2. Results: All these components of the renin-angiotensin system (RAS) were detected in the kidney and left ventricle, although expression levels differed significantly between the sexes and across organs. Gene expression of most components of the RAS was high at birth and decreased with age in both sexes, except for ACE2 expression, which increased in the left ventricle with age (PAge ⬍ 0.001). Low levels of AT2R were observed in the ventricles in both sexes as adults. Most notably, AT2R expression was greatest in female kidneys and lowest in male kidneys compared with the left ventricle (PAge*Sex ⬍ 0.05). Interestingly, MasR expression in the female kidney was similar to the level of AT2R expression. Left ventricular MasR expression was greater than AT2R expression in both sexes but was not different between the sexes. The highest level of ACE2 expression was observed in adult female kidneys (PAS ⬍ 0.05). Conclusions: The enhanced mRNA expression of the vasodilatory arm of the renal RAS (ACE2, AT2R) in females observed in the present study may contribute to sex differences in the regulation of arterial pressure and the incidence of cardiovascular disease in women. (Gend Med. 2012;9:21–32) © 2012 Elsevier HS Journals, Inc. All rights reserved. Key words: angiotensin-converting enzyme 2, angiotensin receptors, sex differences, gene expression/ regulation, heart, kidney. Accepted for publication December 27, 2011. © 2012 Elsevier HS Journals, Inc. All rights reserved.

doi:10.1016/j.genm.2011.12.003 1550-8579/$ - see front matter

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INTRODUCTION Sex-specific differences in the regulation of arterial pressure are well recognized and particularly evident during the reproductive years.1 Differential expression of the angiotensin (Ang) II receptors in males and females has been hypothesized to be a potential mechanism contributing to sex-specific differences in arterial pressure.2 The 2 main Ang II receptors, AT1R and AT2R, exert opposing effects on the cardiovascular system.3,4 All the classic excitatory effects evoked by Ang II (vasoconstriction, sodium reabsorption, vascular growth) result from AT1R stimulation, whereas AT2R stimulation causes vasodilation, natriuresis, and antiproliferation, although these effects may be masked by the overriding effects of AT1R stimulation.4 Thus, a role for AT2R in the control of arterial pressure is disputed because the expression level of AT2R is reported to be low compared with AT1R, and AT2R effects have been difficult to expose. However, it is now appreciated that there is a low level of AT2R expression in cardiovascular tissue.4 Ang II acts at both AT1R and AT2R subtypes with equal affinity.5 However, in males, at least, AT1R expression greatly exceeds AT2R expression, and thus vasoconstriction predominates. The major physiologic role of the recently discovered angiotensin-converting enzyme 2 (ACE2) is the cleavage of Ang II to form Ang-(1-7),6 and it has been demonstrated in vitro that ACE2 converts Ang II to Ang-(1-7) with high catalytic efficiency.7 It is evident that Ang(1-7) can act via the AT2R receptor or its own receptor, the Mas receptor (MasR),8 to mediate vasodilation.6 What is clear is that the ratio of AT1R to AT2R and the balance of ACE and ACE2, dictating the rate of formation of the various Ang peptides, will determine whether vasoconstriction or vasodilation predominates. Although there is a growing body of evidence of sex differences in the renin-angiotensin system (RAS),9 it is not possible from the literature to obtain a clear picture of the relative balance of the vasoconstrictor and vasodilator arms of the RAS in males and females. Just as importantly, it is difficult to assess the importance of these counterregulatory mechanisms in different tissues with the local RAS. Primarily, this is because few studies

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have directly examined differences in the RAS between males and females, and even fewer have looked at ⬎1 organ. Thus, a comparison of studies that report relative expression levels is impossible because they are performed at different ages, with different diets, in different tissue, and in different species and often in only 1 sex.10 –20 The present study aimed to characterize the postnatal ontogeny from birth to adulthood of AT1aR, AT1bR, AT2R, MasR, and ACE2 gene expression in the kidney and heart, organs particularly affected by cardiovascular disease with differing incidences between the sexes, in male and female rats and to elucidate any potential sex-specific differences in the expression of components of the RAS. It was our hypothesis that mRNA expression of the vasodilator components (AT2R, MasR, and ACE2) of the RAS would be enhanced in females.

METHODS Animals and Tissue Collection All experiments were approved by the Monash University Animal Ethics Committee in accordance with the guidelines of the National Health and Medical Research Council of Australia. Timemated pregnant Sprague Dawley rats (Animal Resource Centre, Western Australia) gave birth normally, and offspring were used for tissue collection at postnatal day (PND) 1. Sprague Dawley male and female rats were obtained (Animal Resource Centre, Western Australia) and fed standard chow (0.25% NaCl) and water ad libitum until tissue collection at PNDs 30, 42, 70, and 110 as outlined in the following.

Measurement of Arterial Pressure, Heart Rate, and Activity Male and female Sprague Dawley rats 14 weeks old (12 male and 12 female) were obtained and maintained on a sodium-controlled diet (0.25% wt/wt sodium chloride; Glen Forrest Stockfeeders, Western Australia) with water ad libitum. The rats were individually housed with a 12-hour light/ dark cycle at a temperature of 22–25°C. Rats were acclimatized to these conditions for 1 week before implantation of the telemetry transmitter. Briefly, animals were anesthetized (isoflurane, 2%– 4% O2),

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and a telemetry transmitter (TAI-PAC40; Data Sciences International, St. Paul, Minnesota) was implanted in the abdominal aorta, as previously described.21 After a 7-day recovery period, recordings of diastolic and systolic pressures were made (Dataquest Labpro Version 3.0; Data Sciences International) and were recorded as 10-second averages every 10 minutes for a period of 1 week. Mean arterial pressure (MAP) was calculated from the 10-second averages of diastolic and systolic pressures. The data were then analyzed as 24-hour averages.

CAT TCC REV: GCC GAA GCG ATC TTA CAT AGG TG), MasR (predesigned assay, Taqman Gene Expression Assays, Applied Biosystems) and ACE2 (predesigned assay, Taqman Gene Expression Assays, Applied Biosystems, Mulgrave, Vic, Australia) were multiplexed with 18S; however, AT2R (predesigned assay, Taqman Gene Expression Assays, Applied Biosystems) was run in separate wells to 18S after optimization experiments revealed CT value interference when these genes are multiplexed. 18S expression was analyzed using a 2-way ANOVA, with the factors age and sex, and did not differ between any age or sex.

Tissue Collection Calculations of Relative Gene Expression

Kidneys and left ventricles were collected from male and female offspring at ages PND 1, 30, 42, 70, and 110. These ages were chosen to represent birth, post-weaning, early puberty, late puberty, and post-puberty/sexual maturity, as shown previously.22–24 For tissue collection at age PND 1, pups were decapitated, and kidneys and hearts were excised and snap-frozen for RNA extractions. For tissue collection at all other ages (PNDs 30, 42, 70, and 110), the offspring were euthanized with an overdose of sodium pentobarbitone (325 mg mL; Lethabarb, Virbac Pty Ltd., Regents Park, NSW, Australia), and the kidneys and hearts were immediately excised and snap-frozen for RNA extractions. For each sex and at each time point, between 6 and 20 tissue samples were collected.

The CT value for 18S was subtracted from the CT value for the gene of interest to give a ⌬CT for each sample. All samples were run in duplicate with the average ⌬CT used for each sample. The ⌬CT of the calibrator group (the mean ⌬CT of the female PND 1 group for AT1aR, AT1bR, MasR, and ACE2 for the AT2R expression the corresponding PND 1 group for each sex) was then subtracted from each sample to give a ⌬⌬CT value. This was then substituted into the equation 2–⌬⌬CT to generate expression relative to the calibrator group. Fold differences in expression between the sexes were determined using comparisons of the ⌬CT values given that 1 cycle greater ⌬CT value is equivalent to a 2-fold lower expression level.

Gene Expression Studies

Statistical Analysis

Total RNA was extracted using RNeasy extraction kits (Qiagen, Germantown, Maryland). One microgram of RNA was then reverse-transcribed into cDNA, as previously described.21,25 Gene expression of AT1aR, AT1bR, AT2R, MasR, and ACE2 was determined using an Eppendorf Mastercycler Realplex (North Ryde, NSW, Australia) real-time machine.21,25 18S was used as the internal housekeeping gene, and a comparative cycle of threshold fluorescence (CT) method was used. AT1aR (probe: ACC GCT GGC CCT TCG GCA A, primers: FWD: GGG CAG TCT ATA CCG CTA TGG AG; REV: GCC GAA GCG ATC TTA CAT AGG TG), AT1bR (probe: CGG CCA AGT CAC ACG CAG GCT, primers: FWD: CCT CCA GCT TCT GAA ATA

All data are presented as mean (SEM). MAPs were analyzed using a 2-way ANOVA using the factors sex (PS) and diurnal time period (PD) and the interaction between sex and diurnal time period (PSD) with a post hoc Tukey test for pairwise comparisons. All gene expression data were analyzed using a 2-way ANOVA using the factors age (PA) and sex (PS) and the interaction between age and sex (PAS). A post hoc analysis using the pairwise multiple comparison Tukey test was performed to test for the differences between sexes at different ages. Body and organ weight data were analyzed using an unpaired t test between males and females for each age group. Statistical significance was accepted at P ⱕ 0.05.

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Table I. Body and organ weights. BW, g

Total KID, g

KID:BW, %

LV, g

LV:BW, g

Male PND PND PND PND PND

1 (n ⫽ 20) 30 (n ⫽ 8) 42 (n ⫽ 11) 70 (n ⫽ 9) 110 (n ⫽ 10)

6.4 (0.2) 85 (4) 185 (5) 354 (7) 380 (8)

0.08 (0.01) 0.9 (0.05) 1.7 (0.05) 2.6 (0.05) 2.6 (0.20)

1.3 (0.03) 1.1 (0.01) 0.9 (0.01) 0.7 (0.01) 0.7 (0.04)

0.04 (0.01) 0.3 (0.02) 0.6 (0.03) 0.97 (0.02) 0.86 (0.05)

0.67 (0.02) 0.4 (0.02) 0.3 (0.02) 0.3 (0.01) 0.2 (0.01)

Female PND PND PND PND PND

1 (n ⫽ 14) 30 (n ⫽ 8) 42 (n ⫽ 10) 70 (n ⫽ 12) 110 (n ⫽ 10)

5.9 (0.2) 74 (4)* 150 (2)† 223 (5)† 243 (9)†

0.08 (0.01) 0.84 (0.01) 1.36 (0.04)† 1.6 (0.04)† 1.5 (0.04)†

1.4 (0.04)* 1.1 (0.02) 0.91 (0.03) 0.7 (0.01) 0.6 (0.01)*

0.04 (0.01) 0.31 (0.01) 0.5 (0.02)† 0.7 (0.04)† 0.7 (0.04)*

0.7 (0.02) 0.42 (0.02) 0.3 (0.01) 0.3 (0.01) 0.3 (0.01)‡

BW ⫽ body weight; KID ⫽ kidney; LV ⫽ left ventricle; PND ⫽ postnatal day. All data are presented as the mean (SEM). P values as compared with age-matched males. *P ⱕ 0.05. † P ⱕ 0.005. ‡ P ⱕ 0.01.

RESULTS Body Weights and Organ Weights At birth (PND 1), there was no difference in body weight, total kidney weight, or left ventricular weight between males and females; however, when corrected for body weight, females had a significantly greater kidney:body weight ratio (P ⬍ 0.05, Table I). Females weighed significantly less than males at all other ages (P ⬍ 0.04). Females also had lower total kidney weights (P ⬍ 0.005) than males from PND 42 onward, and when corrected for body weight, adult females (PND 110) had a marginally lower kidney:body weight ratio (P ⬍ 0.05). Left ventricular weights were also significantly lower in females compared with their male counterparts from PND 42 onward (P ⬍ 0.04), but no differences were apparent when corrected for body weight for ages PND 1, 30, 42, or 70 (Table I). At PND 110, females had a significantly greater left ventricular weight when corrected for body weight (Table I).

AT1aR Gene Expression Relative AT1aR gene expression decreased significantly with age in both kidney and heart tissue (Figure 1). In the adult (PND 110), AT1aR expression was ⱖ4-fold greater in the kidney than in the left ventricle. In the kidney, there were no differ-

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ences between expression levels in males and females throughout postnatal life. The same profile was observed in the left ventricle, with no effect of sex on the expression of the AT1aR (Figure 1).

Figure 1. Relative AT1aR mRNA gene expression. Data for kidney and left ventricular expression in females (open columns) and males (filled columns) are expressed relative to postnatal day 1 females for each organ and are presented as the mean (SEM). Data were analyzed using a 2-way ANOVA with the factors age (PA), sex (PS), and the interaction between age and sex (PAS).

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sion of AT2R decreased with age, reaching very low levels by PND 42, whereas female levels remained at ⬃40% of PND 1 levels (PAS ⬍ 0.04, Figure 3). Left ventricular AT2R gene expression profiles differed between the sexes with age (PAS ⬍ 0.001), but decreased to a similar low level in adult males and females (Figure 3).

MasR Gene Expression

Figure 2. Relative AT1bR mRNA gene expression. Data for kidney and left ventricular expression in females (open columns) and males (filled columns) are expressed relative to postnatal day 1 females for each organ and are presented as the mean (SEM). Data were analyzed using a 2-way ANOVA with the factors age (PA), sex (PS), and the interaction between age and sex (PAS). *P ⱕ 0.005, †P ⱕ 0.05 compared with age-matched males via post hoc analysis.

Renal MasR expression was not different between males and females from birth to PND 70. At PND 110, however, females had significantly greater renal MasR expression than age-matched males (3-fold greater in females, P ⬍ 0.001; Figure 4). In PND 110 females, expression of MasR was equal to AT2R expression. Left ventricular MasR expression did not change significantly with age in either sex and was not different between males and females at any age (Figure 4). Left ventricular MasR expression at PND 110 was significantly greater than AT2R expression in both sexes, with AT2R expression in the heart ⬍1% of left ventricular MasR expression.

AT1bR Gene Expression The expression level of AT1bR in the adult was ⱖ8-fold greater in the kidney than in the left ventricle. In the kidney, AT1bR gene expression was similar at PND 1 in males and females, and there was a significant effect of age on the expression levels (PA ⬍ 0.02, Figure 2). The expression profiles over the postnatal period were also different between the sexes, with females showing little change in expression of AT1bR from birth to adulthood, but males showing a significant decrease (PAS ⬍ 0.003). Thus, in the kidney, female rats expressed higher levels of the AT1bR in adulthood than males (PS ⫽ 0.02, Figure 2). In the left ventricle, expression of AT1bR significantly decreased with age (PA ⬍ 0.001, Figure 2); again females maintained a higher level of expression than males in adulthood (PS ⬍ 0.05).

AT2R Gene Expression Distinct differences in AT2R expression were noted between the sexes and organs. Renal expression levels of AT2R were similar between the sexes at PND 1 (Figure 3). In males, the renal expres-

Figure 3. Relative AT2R mRNA gene expression. Data for kidney and left ventricular expression in females (open columns) and males (filled columns) are expressed relative to postnatal day 1 females for each organ and are presented as the mean (SEM). Data were analyzed using a 2-way ANOVA with the factors age (PA), sex (PS), and the interaction between age and sex (PAS). *P ⱕ 0.05, †P ⱕ 0.005 compared with age-matched males via post hoc analysis.

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[1] mm Hg vs 91 [1] mm Hg; day ⫽ 89 [1] mm respectively; PS ⫽ 0.0027, Figure 6).

DISCUSSION

Figure 4. Relative MasR mRNA gene expression. Data for kidney and left ventricular expression in females (open columns) and males (filled columns) are expressed relative to postnatal day 1 females for each organ and are presented as the mean (SEM). Data were analyzed using a 2-way ANOVA with the factors age (PA), sex (PS), and the interaction between age and sex (PAS). *P ⱕ 0.005 compared with age-matched males via post hoc analysis.

This study characterized the postnatal ontogeny of Ang II and Ang-(1-7) receptors AT1aR, AT1bR, AT2R, MasR, and ACE2 gene expression. Temporal expression of all these components of the RAS were detected in the kidney and left ventricle, and expression levels differed significantly between the sexes and across organs. mRNA gene expression of all components of the RAS was high at birth and decreased with age in males and females, except for ACE2 expression, which increased in the left ventricle with age. There were 2 major findings. First, expression of AT2R, MasR, and ACE2 was significantly greater in adult female kidneys compared with male kidneys, demonstrating an enhanced vasodilator arm of the RAS in female kidneys.26,27 Second, this is the first study to characterize the postnatal ontogeny of ACE2 in males and females. Although ACE2 and MasR were highly expressed in the adult left ventricle in both sexes, AT2R ex-

ACE2 Gene Expression ACE2 was expressed at high levels from birth to adulthood in the kidney and left ventricle (Figure 5). The expression level of ACE2 across the organs in the adult was ⱖ2-fold greater in the kidney than in the left ventricle. In the kidney, ACE2 expression decreased significantly with age (PA ⫽ 0.008, Figure 5). In females, the renal levels of expression were greater at PND 110 than in age-matched males (P ⫽ 0.006, Figure 5). Interestingly, ACE2 expression in the left ventricle increased with age, similarly in males and females (PA ⬍ 0.001, Figure 5).

MAP in PND 110 Male and Female Rats MAP was significantly higher in the nighttime period compared with the daytime period in both PND 110 males and females, with a decrease of 6 mm Hg in both sexes (PD ⬍ 0.001, Figure 6). PND 110 males had a significantly higher MAP during the nighttime period compared with diurnally matched PND 110 females, a difference that was maintained during the daytime period (night ⫽ 95

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Figure 5. Relative angiotensin-converting enzyme 2 mRNA gene expression. Data for kidney and left ventricular expression in females (open columns) and males (filled columns) are expressed relative to postnatal day 1 females for each organ and are presented as the mean (SEM). Data were analyzed using a 2-way ANOVA with the factors age (PA), sex (PS), and the interaction between age and sex (PAS). *P ⱕ 0.01, † P ⱕ 0.05 compared with age-matched males via post hoc analysis.

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Figure 6. Mean arterial pressure in postnatal day 110 female (open columns) and male (filled columns) rats. Data are presented as the mean (SEM). Data were analyzed using a 2-way ANOVA with the factors sex (PS), diurnal time period (PD), and the interaction between sex and diurnal time period (PSD). *P ⱕ 0.05 compared with diurnal time period–matched female, †P ⱕ 0.05 compared with same-sex nighttime mean arterial pressure via post hoc analysis.

pression was low. The primary function of ACE2 has been suggested to be the generation of Ang-(1-7). Our findings suggest that Ang-(1-7) formed by ACE2 does not act via an AT2R-mediated mechanism in the left ventricle but likely through its other wellrecognized receptor, MasR.8 Consistent with previous work, the present study showed that the expression levels of AT2R in the kidney were high in males and females at birth, with expression levels decreasing dramatically with age.17 However, here we provide evidence of markedly greater renal AT2R expression in female compared with male rats at 16 weeks of age (PND 110) maintained on a 0.25% NaCl diet, a difference that was less apparent at 10 weeks of age (PND 70). This is essentially in agreement with previous studies that showed that males have very low renal AT2R expression and a higher level of expression in females.28,29 However, the degree to which AT2R is differentially expressed in males and females was not previously appreciated because few studies directly compared the sexes. To put this in perspective, in the present study, AT2R expression in the adult male kidney was ⬍1% of that in the females. The present study examined whole-kidney homogenates, and thus it is not possible to specify in what

renal structures AT2R is greater in females than in males. However, AT2R has been localized to glomerular podocytes, proximal tubules, collecting ducts, and the renal vasculature,30 with higher receptor densities noted, particularly in the vasculature and tubular epithelium in females.31,32 MasR is expressed in multiple tissues including tissues involved in blood pressure regulation such as the brain, heart, and kidney.33 We observed no difference in expression from birth to PND 70 in males or females; however, at PND 110, females had significantly greater renal MasR expression than age-matched males. This is in contrast to the findings of Sullivan et al,34 with no difference in MasR expression between the sexes reported. This is likely due to the fact that we characterized the MasR ontogeny in normotensive Sprague Dawley rats as opposed to spontaneously hypertensive rats as used by Sullivan et al,34 with spontaneously hypertensive rats shown to have increased renal MasR expression compared with Sprague Dawley rats.33 We observed no difference in left ventricular MasR expression with age or between the sexes. There was, however, greater MasR expression in the left ventricle than AT2R expression in both sexes, supporting the growing evidence in the literature that MasR plays a greater role in the vasodilatory arm of the RAS in the heart than AT2R.35 The discovery of ACE2 has provided support for the emerging vasodilatory arm of the RAS that counterregulates the action of Ang II at the AT1R.6 Previous studies showed that ACE2, like AT2R, is particularly abundant in tubular epithelium.36,37 To our knowledge, this is the first study to characterize the postnatal ontogeny of ACE2 in males and females. In the kidney, we showed that expression levels are high at birth and decrease to a greater extent in males with age than in females such that higher levels of ACE2 are present in the adult female kidney (⬃2.5-fold). Thus, in the female kidney, the concurrent high levels of ACE2 and AT2R suggest a role in sodium and water balance. We recently demonstrated that direct activation of AT2R, via the agonist compound 21, resulted in a greater renal vasodilation in female compared with male rats.38

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Renal AT1aR and AT1bR expression decreased with postnatal age in males, which is consistent with previous reports.18 In females, renal expression of AT1aR also decreased with age to a similar level in adulthood as in males. Interestingly, although renal AT1bR gene expression decreased with age, the residual level of AT1bR expression was greater in the female kidney in adulthood compared with the male kidney. The physiologicl significance of this is unclear because AT1aR has been reported to be the predominant receptor subtype in all renal vascular and tubular elements,39,40 except for the renal glomerulus where AT1bR is more abundantly expressed,41 albeit in males. Therefore, taken together, with no difference in renal AT1aR expression and greater renal expression of both AT2R and ACE2 in females, the balance of renal vasoconstrictor to vasodilator components of the RAS is markedly enhanced in females in adulthood and may contribute to the sex-specific differences in the regulation of kidney function and arterial pressure under physiologic and pathophysiologic conditions. In juxtaposition to the findings in the kidney, the left ventricle had relatively high adult (male and female) expression levels of ACE2, but low expression levels of AT2R, in agreement with previous studies in males.42 It is well recognized that Ang-(1-7) acts via AT2R and MasR, and there is a significant body of evidence to suggest that MasR may be the receptor for Ang-(1-7) in the heart.43 This distribution of ACE2, MasR, and AT2R in the heart in the present study suggests that Ang-(1-7) formed by ACE2 in the heart is not primarily acting via AT2R, which supports the evidence suggesting the predominant role of MasR in the activity of Ang-(1-7) in the heart. On the other hand, AT2R has consistently been shown to play a protective role in cardiovascular hypertrophy and fibrosis, particularly in aged animals.44 AT2R knockouts generally exhibit vascular hypertrophy and increased cardiac fibrosis compared with wild-type littermates, and greater AT2R-mediated vasoprotective remodeling is seen in wild-type females.45 A protective role for ACE2 in heart disease has also been proposed, and the present study provides evidence supporting the possibility that targeting the

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ACE2–Ang-(1-7) axis in the heart may be beneficial in males and females. Sex differences in MAP during the reproductive years are well reported in humans.1 Animal models of disease, including spontaneously hypertensive rats, Dahl salt-sensitive rats, and Ang II–induced hypertensive rats, have also consistently demonstrated sex differences.46 – 48 Evidence of the presence of sex differences in MAP in normotensive animal models has been conflicting, with some studies reporting significant differences and others showing no differences.46 –50 We observed a small but significant sex difference in MAP in both the daytime and nighttime periods at PND 110. This is consistent with evidence from Sartori-Valinotti et al,48 who observed a similar magnitude of difference in arterial pressure in male compared with female Sprague Dawley rats, as well as numerous other studies that found sex differences in animal models in diseased states.46,47,49,51 We suggest that, given the small magnitude of difference observed between the sexes, previous studies may have been underpowered and therefore unable to detect such a small difference. A limitation of our study is that although we performed mRNA expression analysis, we did not confirm the translation to protein. This was attempted, but, in our hands, the available commercial antibodies were nonspecific, resulting in multiple bands on each Western blot. In addition to multiple bands, we were unable to consistently replicate Western blots for AT2R and AT1R. Furthermore, AT2R antibodies produced immunostaining in tissue from AT2R knockout mice, providing further evidence of the nonspecificity. Our protein expression results, although disappointing, are not entirely surprising, given the difficulties reported in the measurement of G protein– coupled receptors (such as AT1R and AT2R) in the literature.52,53 In summary, we have shown markedly higher renal AT2R, MasR, and ACE2 expression in adult females compared with males. This finding is in line with findings of our previous studies demonstrating an enhanced AT2R action in female rats.21,27,29,38 Taken together, this provides evidence of a shift in the renal vasoconstrictor:vaso-

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dilator ratio of the RAS in favor of vasodilation in females. Given the fact that the local renal RAS plays a key role in arterial pressure regulation, the sex-specific differences in this expression may well contribute to the sex-specific differences observed in arterial pressure in adulthood. One obvious explanation for the sex-specific differences in gene expression is the influence of the sex hormones.31 It has been shown that sex hormones can interact directly with the RAS, with testosterone able to upregulate the expression of AT1R and estrogen involved in the downregulation of AT1R, renin and ACE and upregulation of AT2R.28,31,54 There is limited evidence detailing the effect of estrogen on ACE2 expression with conflicting results. It has been shown that ACE2 gene expression is significantly increased in pregnancy in rats, which is consistent with our current hypothesis.55 In contrast, Liu et al56 reported that ovariectomy increased renal ACE2 expression with estrogen replacement in ovariectomized mice, resulting in reduced renal ACE2 expression. To date, there is no reported evidence of the effect of testosterone on kidney or left ventricular expression of ACE2; however, the level of testosterone has been shown to have no effect on ACE2 levels in the testis, which may suggest that testosterone plays little role in ACE2 regulation.57 Our data suggest a potential sex-specific regulation of the RAS, which requires further investigation to determine the functional consequences of these alterations.

vasodilation in the adult female kidney, with enhanced ACE2 and AT2R expression. These distinctions in the RAS between males and females may underlie some of the puzzling differences that have emerged between the sexes, not only in the rates of cardiovascular disease, but also in terms of the symptoms, risk factors, and, importantly, the response to treatment in women.

ACKNOWLEDGMENTS This work was supported by grant G06M2640 from the National Heart Foundation of Australia and grant 334031 from the National Health and Medical Research Council of Australia Project. Dr. Sampson was supported by an Australian Postgraduate Award, and Dr. Denton was supported by National Health and Medical Research Council of Australia Fellowship 490918. Drs. Denton, Moritz, and Sampson contributed to study design, literature search, data interpretation, figure creation, and drafting of the manuscript. Dr. Sampson conducted the data collection for this study.

CONFLICTS OF INTEREST The authors have indicated that they have no conflicts of interest regarding the content of this article.

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CONCLUSIONS

24-h ambulatory blood pressure in 352 normal

These studies highlight 2 important issues. The first is that differences in age may explain disparities in outcomes among studies examining the RAS because basal expression of components of the RAS vary considerably with age. Perhaps most importantly, we have shown marked differences between 10 (PND 70) and 16 (PND 110) weeks of age in the RAS, a window within which most studies are performed. The second feature of these studies is the distinct differences in expression of the components of the RAS between males and females and also across different organs. Most notably, the balance of the vasoconstrictor to vasodilator components of the RAS is tipped in favor of

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Address correspondence to: Amanda K. Sampson, PhD, Department of Vascular Pharmacology, Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria, Australia 3004. E-mail: Amanda.sampson@ bakeridi.edu.au

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