Introduction

Increasingly, dramatic differences in physiology are being revealed between the sexes [1-3]. Indeed, sex differences are far more ubiquitous than originally thought, and these differences are important because they help not only explain why women or men are more or less prone to certain diseases, but also suggest that treatments should be tailored according to sex. The requirement that all phase III clinical trials must include women might, one would think, make questions regarding sex differences redundant. However, phase I trials, in which drug safety, tolerability, pharmacokinetics and dynamics are examined, and the larger phase II trials designed to test dosing and efficacy do not require the inclusion of women. Moreover, the underrepresentation of women in phase I and II clinical trials has been cited as the reason why eight out of ten drugs removed from the market exhibit greater adverse effects in women than men [4]. This sex imbalance in research is even more marked in the arena of basic science in animal models [5].

In this regard, it is well recognized that there are striking sex differences in the physiological mechanisms regulating arterial pressure, including modulation of renal and vascular function [6-9]. Thus, not all our knowledge of circulatory control, gained primarily in males, may be transferable to females where other factors may be in play.

Sex Differences in Arterial Pressure

In premenopausal women, arterial pressure is lower than in aged-matched men [10], a situation that is also observed in other adult mammalian species (Fig. 1). Women during pregnancy can double their blood volume and cardiac output, and yet arterial pressure declines, and this is associated with a reduced sensitivity to the actions of angiotensin II (AngII) [11]. If this normal pattern of events does not occur during pregnancy and hypertension develops, the consequences can be catastrophic for the mother and child [12]. Moreover, women are protected from cardiovascular and renal disease relative to men, prior to menopause [1, 3]. Thus, there are profound differences in the physiological and pathophysiological regulation of arterial pressure between the sexes.

Fig. 1
figure 1

Conscious basal mean arterial pressure (MAP, mean±SEM) measured in age-matched adult male and female mice (20 weeks of age; 72-h radiotelemetry recording, n = 8), rats (12 weeks of age; 72-h radiotelemetry recording; n = 10), rabbits (45 weeks of age; 1-h recording via ear artery catheter; n = 10) and sheep (1 year of age; 72-h recording via carotid loop catheter; n = 7). **P < 0.01 as compared to the respective male group (unpaired t-test with Bonferroni correction)

Androgens are known to be involved in the sex differences in the regulation of arterial pressure, with estrogen protecting against and testosterone exacerbating hypertension [6-8]. The reasons for these sex differences are incompletely understood, though it is suggested that sex hormones and sex chromosomes (genes) contribute by differentially modulating the renin-angiotensin system (RAS) [6-8, 13].

Renin-angiotensin System: New Pathways

The RAS is a master regulator of extracellular fluid homeostasis and thus renal function and arterial pressure. Major sex differences in the expression levels of components of the RAS have been identified, and there are also differences in the way males and females respond to stimulation and inhibition of the RAS under physiological and pathophysiological circumstances [14-17]. In the last 20 years there has been a resurgence of interest in the RAS with the discovery of angiotensin-converting enzyme 2 (ACE2) and additional receptors specific for angiotensin peptide fragments, suggesting the presence of a depressor arm of the RAS (ACE2/Ang(1-7)/MasR and AT2R), which counter-regulates the classical ACE/AngII/AT1R pathway [18].

Estrogen

Mounting evidence indicates that estrogen regulates all components of the RAS. Estrogen increases synthesis of angiotensinogen, while decreasing the synthesis of renin and ACE [19]. Estrogen decreases the expression of AT1Rs in target tissue but has the opposite effect on AT2R expression. In rat kidneys, ovariectomy decreased AT2R expression and estrogen replacement increased AT2R expression [20-22]. In males, testosterone amplifies the pressor pathways of the RAS [23]. This strongly suggests differential roles for these pathways between the sexes, with the balance tipped toward depressor pathways in females [24]. Our own work provides a powerful example of this. Remarkably, we have demonstrated that a chronic low-dose infusion of AngII decreased arterial pressure in female rats (~10 mmHg) at a dose that caused an increase in males [25••]. These startling findings illustrate the dual nature of AngII on arterial pressure and are consistent with the opposing effects of AT1R and AT2R activation [18]. We confirmed these findings in a second cohort of rats and went on to show that the action of AngII to decrease arterial pressure in females is mediated via the AT2R [26] and is estrogen-dependent [26]. Furthermore, we have extended these finding into AT2R knockout mice, demonstrating that female mice have an attenuated response to AngII infusion as compared to male wild-type and female AT2R knockout mice [27••]. Thus, we have demonstrated that the AT2R plays an enhanced role in regulating arterial pressure in females as compared to males. ACE2 has also been demonstrated to protect females, but not males, from the pressor effects of AngII infusion in studies utilizing ACE2-knockout mice [28••].

Clinically, congestive heart failure survival rates are better for women treated with angiotensin receptor blockers (which have the potential for an AT2R-mediated contribution) compared with ACE inhibitors, and the reverse is true for males [29, 30]. Also, there is evidence that the AT2R plays a pronounced role in arterial pressure regulation in premenopausal women. For example, during pregnancy when the RAS is upregulated arterial pressure actually falls, and this is associated with an increased AT2R/AT1R ratio, with evidence to suggest that this response is absent in pregnancy-induced hypertension [31••, 32]. Furthermore, on a background of AT1R blockade, the renal response to AngII infusion was attenuated more in women than in men and it was suggested that this was an AT2R-related effect [33-35]. Thus, evidence strongly supports a greater role for the depressor arm of the RAS in women than men.

Testosterone

The important role of testosterone in arterial pressure regulation has been well documented in testosterone removal and replacement studies. Arterial pressure is reduced following castration (testosterone removal) and restored with testosterone replacement in multiple animal models including spontaneously hypertensive rat (SHR), Dahl-salt-sensitive and Goldblatt hypertensive animal models [36-43]. In terms of renal function, the striking hormonal influence on renal control of arterial pressure is best demonstrated by renal transplantation studies. Renal transplantation from a hypertensive rat into a normotensive rat elevated arterial pressure in the normotensive rat post-transplantation [44]. However, following renal transplantation from a hypertensive female rat into a hypertensive male rat, the arterial pressure level remained unchanged despite the sex difference in arterial pressure [45]. These data indicate that it is not the kidney per se that determines the arterial pressure level but suggests that renal control of arterial pressure is influenced by the hormonal milieu. Testosterone directly interacts with the RAS, upregulating the classical constrictor pathway via upregulation of angiotensinogen gene expression, renin activity and AT1R expression [46-49]. The higher plasma renin levels seen in males is lowered following castration and increased following testosterone replacement, with a linear correlation between plasma renin activity and testosterone activity [7, 50]. In fact, the higher plasma renin levels may be a direct result of the shift in the renal pressure natriuresis/diuresis relationship. Specifically, the increased proximal tubular sodium reabsorption in the presence of testosterone and the reduction in delivery of sodium to the macula densa is hypothesized to lead to the increased renin release [51, 52]. Furthermore, ACE inhibition in male and female SHRs abolishes the sex difference in arterial pressure observed [53]. Taken together, these data demonstrate that testosterone interacts with the RAS, increasing activation of the classical pathway resulting in AT1R activation by AngII.

Sex Chromosomes

In addition to the well-documented influence of the sex hormones, it is becoming increasingly clear that the sex chromosome complement, XX for females and XY for males, can influence the RAS and arterial pressure regulation. The evidence for sex chromosome effects independent of hormonal effects has been illustrated by the Four Core Genotype (FCG) mouse model and the Y consomic rat model [54-56] (for review see Sampson et al. 2012 [57•]).

To date, studies involving these novel animal models have identified three key findings suggesting that the sex chromosomes influence arterial pressure regulation and renal function. Firstly, gonadectomized FCG mice with the XX chromosomal complement showed a greater pressor response to chronic AngII infusion than gonadectomized mice with the XY complement, independent of prior sex hormone and gonadal phenotype [58]. These data demonstrate that the sex chromosome complement significantly influences arterial pressure responses to AngII, independent of gonadal phenotype [58]. Secondly, the Y chromosome contributes around 10-15 mmHg arterial pressure, as demonstrated by a reduction in arterial pressure in hypertensive rats following introgression of the Y chromosome from a normotensive rat strain, and, vice versa, an increase in blood pressure in normotensive rats following introgression of the Y chromosome from a hypertensive rat strain [55, 56]. Thirdly, the Sry gene family, located on the Y chromosome, upregulates promoter activity of angiotensinogen, renin and ACE, and decreases promoter activity of ACE2 in vitro [59]. This results in a shift in the RAS with upregulation of the pro-hypertensive classical pathway and downregulation of the depressor arm of the RAS. While the physiological consequences of the interaction of the Y chromosome and the RAS remains unclear, the involvement of the kidney in this response is critical, with elegant studies demonstrating renal overexpression of Sry genes increases arterial pressure, renal sympathetic nerve activity, plasma renin activity and renal AngII content [59, 60]. In addition, both the AT2R and ACE2 genes are located on the X chromosome, suggesting a greater role of these depressor RAS arm components in females.

Sex Differences in Polymorphisms (SNPs) of the RAS

It is increasingly evident that sex-specific associations between hypertension and RAS gene polymorphisms exist. In males, ACE and AT1R gene polymorphisms are significantly associated with hypertension [61, 62], while in females angiotensinogen and ACE2 gene polymorphisms are associated with hypertension [63, 64]. This suggests that sex differences in responses to the RAS are present from birth. The most widely studied single nucleotide polymorphisms (SNPs) of the angiotensinogen gene are M235T and T174M [65]. Mohana et al. (2012) identified an increased frequency of these specific SNP genotypes in hypertensive females compared to normotensive females [64]. They suggest that in females, the risk of hypertension is increased when these genotypes are present. The T174M polymorphism encodes for a threonine substitution to methionine at codon 235. The heterozygous TM genotype (one copy encoding for threonine and one copy encoding for the substituted methionine) in females equated to a 2.48-fold increased risk of hypertension compared to both homozygous genotypes (TT+MM) [64]. Interestingly, there was no difference in frequency of any genotype between hypertensive and normotensive males [64].

The ACE insertion/deletion gene polymorphism (ACE I/D) at base pair 287 contributes around half the variance in plasma ACE levels in healthy individuals [66]. In contrast to the findings of the angiotensinogen polymorphisms, no associations between ACE I/D and hypertension have been reported in females [61]. Analysis of the Framingham Heart Study revealed an association between the ACE locus with diastolic blood pressure and hypertension in males but not in females, identifying that the presence of the homozygous DD (2 copies of the ACE gene with the deletion at base pair 287) is significantly associated with hypertension in males [61].

Given the location of ACE2 gene on the X chromosome and its highly polymorphic nature, it was hypothesized that sex differences in ACE2 gene polymorphisms would exist and show a possible sex-specific pattern of association to hypertension. A recent meta-analysis published by Lu et al. (2012) including over 11,000 subjects identified that the G8790A ACE2 variant was significantly associated with hypertension in females but not in males [63].

In males, a polymorphism of the AT1R, involving a substitution of adenosine to cytosine at base 1166 (A1166C), is associated with arterial pressure [62]. Specifically, males with the cytosine substitution (either homozygous or heterozygous) had higher arterial pressure than males with homozygous adenosine alleles [62]. While these data provide compelling evidence suggesting a genomic AT1R polymorphism increasing the risk of hypertension, these data were obtained from a small sample size, and, therefore, confirmation of this association in a larger sample size, across multiple ethnicities, is warranted. Regardless, these data provide evidence that the allelic association of AT1R gene polymorphisms and hypertension can be sexually dimorphic. Despite recent evidence linking AT2R polymorphisms with arterial pressure regulation [67], there are currently no data investigating sex-specific effects of AT2R polymorphisms. However, a recent analysis of RAS gene polymorphisms and association with renal function revealed a sex-specific association, with a significant association between the AT2R SNP (rs5950584) and urinary albumin excretion [68]. These data suggest that, as seen in other RAS gene components, there may be sex differences in the frequency or association of AT2R polymorphisms with hypertension.

Given the complex nature of arterial pressure regulation and the striking differences observed across ages and ethnicities, studies have reported contrasting findings with some reporting clear sex differences in the frequency of RAS gene polymorphisms and association with hypertension, while others report no difference between the sexes. What is clear is that sex differences in responses to RAS play an important role in arterial pressure regulation at a genomic as well as a non-genomic level and require further in depth investigation.

Recent Insights Into the Contribution of the RAS to Sex Differences in Kidney Function

The kidney, by maintaining an appropriate balance between fluid intake and renal excretion, plays a dominant role in the long-term regulation of arterial pressure [69]. Key components of the RAS are abundantly expressed throughout the kidney [70], and, as such, it is highly plausible that sex differences in the RAS contribute to the sexual dimorphism in hypertension. Certainly, distinct sex-related differences in renal function have been reported. Studies in normotensive and hypertensive rats have shown that females demonstrate a protective leftward shift in the pressure-natriuresis curve such that they excrete the same amount of sodium as males at a lower arterial pressure [42, 71••, 72]. With the significant advances that have recently been made with regards to our understanding of the functional role of the newly identified RAS depressor pathways in the regulation of arterial pressure, much emphasis in the past few years has been placed on elucidating the contribution of these RAS components to the sex differences in renal function.

The sex-specific role of the AT2R in the kidney has been attracting considerable attention given the mounting evidence of its contribution to sodium homeostasis and arterial pressure control. The AT2R has been detected in multiple sites throughout the renal tubules and vasculature, suggesting its involvement in the regulation of both renal tubular and hemodynamic function [73]. Moreover, the AT2R is differentially expressed in the male and female kidney, with a lower AT1R/AT2R ratio in females [20, 25••, 27••, 70, 74-76, 77••], suggesting that the receptor may play a sex-dependent role in the kidney. In support of this conjecture, it was recently shown that the male sex is associated with greater renal sensitivity to AngII. Schneider et al. [76] examined sex differences in acute renal hemodynamic responses in AngII-induced hypertensive mice during exposure to various salt-loading conditions to modulate renal AngII receptor expression. In addition to a greater pressor response to AngII, male mice demonstrated a greater renal vascular response to AngII when maintained on a normal or high-salt diet as compared to female mice, and this was associated, at least in part, with differences in renal vascular AT1R and AT2R expression. As mentioned earlier, in humans, sex differences in AngII-mediated renal hemodynamic responses have also been reported, and it was speculated that this may be attributable to differences in AngII receptor levels between males and females [33-35].

Several studies from our laboratory have provided strong evidence that the AT2R plays a significant functional role in the kidney and that its effects can be sexually dimorphic. In vivo studies in anesthetized normotensive rats showed that the AT2R enhances pressure-natriuresis in both sexes. During acute systemic AT2R blockade, the pressure-natriuresis-diuresis curves were shifted rightwards in both males and females [71]. This finding recapitulated previous findings in mice that the AT2R modulates pressure-natriuresis in males [78, 79], but was the first study to demonstrate that it does so to a similar extent in females. In addition, these studies revealed a sex-specific role for the AT2R in the female vasculature to provide protection against AT1R-mediated vasoconstriction by endogenous AngII. AT2R blockade blunted the autoregulation of the renal blood flow (RBF) and glomerular filtration rate (GFR) at low renal perfusion pressures in females but not in males [71]. This finding may be related to RAS activation, as indicated by increased plasma renin activity at these low pressures, and was further supported by examination of the RBF response to a graded AngII infusion where AT2R blockade enhanced the renal vasoconstrictor response to AngII in females alone [71]. Tubuloglomerular feedback (TGF) is a key regulator of renal vascular tone, and thus GFR, and therefore also contributes to setting pressure-natriuresis properties. Studies in AT2R-knockout mice showed that the sensitivity of TGF to AngII is also reduced by the presence of the AT2R in females but not males [27].

Collectively, these data provide strong evidence that the AT2R plays a significant role in the modulation of renal function, especially when the RAS is augmented in female rats and mice. As such, renal AT2R function could serve to limit hypertension particularly in females and may be a suitable therapeutic target to counterbalance the pressor actions of the RAS. Although, to date, the sex-dependent renal functional effects of AT2R stimulation in the setting of hypertension have not been explored, we recently showed in acute anesthetized studies in normotensive rats that pharmacological AT2R stimulation produces vasodilatory and natriuretic effects in both the male and female kidney [80•], providing further justification for prospective investigations into the sex-specific benefits of pharmacological AT2R stimulation in disease states.

The precise mechanism by which AT2R activation modulates natriuresis in males and females and protects against AngII-induced vascular alterations in females is still being investigated. Carey and colleagues have performed an extensive series of experiments providing substantial evidence to indicate that angiotensin III (AngIII) is the preferred AT2R ligand in the kidney, at least in the induction of natriuresis [81•, 82-85]. In addition to AngII, other biologically active peptides derived from AngI and AngII, including AngIII and Ang(1-7), can oppose classical AT1R-mediated pressor effects via their interaction with the AT2R [18]. Most recently, Kemp et al. reported that endogenous intrarenal AngIII, but not AngII or Ang(1-7), induces natriuresis via AT2R activation in the renal proximal tubule [81]. In female normotensive rats during systemic AT1R blockade, renal interstitial administration of AngIII induced natriuresis, and this response was abolished by concomitant AT2R blockade. This response was reportedly not reproducible with either AngII or Ang(1-7), in the presence or absence of ACE or aminopeptidase A inhibition to retard AngII and Ang(1-7) metabolism, respectively. Female rats were used in this study, as compared to male rats in their earlier work, on the basis that the sex differences in renal AT2R function that we reported were exclusive to renal hemodynamic function and did not affect sodium excretion [71, 80]. Further research, however, is required to clarify the role of the different angiotensin peptides in the sex-dependent regulation of renal hemodynamics.

As reviewed extensively by others [86-88], there is also growing evidence that the ACE2/Ang(1-7)/MasR axis elicits significant renal effects. This has triggered interest in determining whether this RAS pathway also plays a sex-specific role in the kidney. The MasR is expressed in both the tubular and vascular elements of the kidney, including cortical and proximal tubular cells, afferent arterioles and tubular epithelium [86]. Moreover, similar to the AT2R, the expression and activation of the ACE2/Ang(1-7)/MasR axis also differs between the sexes. We have identified greater renal ACE2 and MasR gene expression in female versus male normotensive rats [25, 70], further shifting the balance of RAS stimulation towards the depressor arm. Thus, in females, it is also likely that Ang(1-7) generation via ACE2 is enhanced. Indeed, Sullivan et al. [77] have reported that renal cortical levels of Ang(1-7) are significantly greater in female versus male SHR both basally and after exogenous AngII infusion. Pendergrass et al. [89] also reported greater plasma Ang(1-7) levels in female versus male mRen(2).Lewis hypertensive rats, despite lower renal cortical ACE2 activity in females. However, this may be attributed to greater neprilysin activity and protein in females leading to greater conversion of AngI or Ang(1-9) to Ang(1-7) [89]. Although Sullivan et al. [77] did not identify differential MasR expression in the renal cortex between male and female SHR, mRNA expression levels increased in response to AngII infusion in females only. In addition, MasR antagonism abolished the sex difference in the arterial pressure response to AngII infusion, providing substantial evidence that the differential MasR expression and Ang(1-7) levels in the kidney may contribute to the sex-dependent differences in physiological arterial pressure responses to AngII. In light of this new evidence, it is tempting to speculate that enhancement of the ACE2/Ang(1-7)/MasR axis in females may therefore also play a sex-dependent renoprotective role.

To our knowledge, studies reporting on the renal functional effects of this RAS pathway have so far been performed solely in males [90••, 91-93]. However, we have recently shown in acute anesthetized studies in normotensive rats that the MasR may also play a sex-specific role, at least in the renal vasculature [94]. RBF decreased significantly in female but not male Wistar rats when the MasR was blocked. However, MasR blockade did not alter the RBF response to AngII infusion in either sex. It is possible that the infused AngII, which has a low affinity for the MasR [18], was not converted within this acute setting (~20 min total) into Ang(1-7). Whether the MasR would differentially modulate renal hemodynamics under conditions of chronic elevations in AngII subsequently requires further interrogation. Moreover, we must also consider that previous studies reporting on the renal effects of Ang(1-7) have produced conflicting results, which may be explained by differences in experimental conditions [87, 88]. Therefore, studies are still needed to fully characterize the intrarenal ACE2/Ang(1-7)/MasR pathway in males and females in order obtain a complete picture of the physiological significance of this work and examine the possibility that targeting of the MasR may be therapeutically beneficial in males and/or females.

Conclusion

Sex differences exist in the regulation of arterial pressure and renal function by the RAS. Significantly, the counter-regulatory arm of the RAS, including ACE2/Ang(1-7)/MasR and AT2R, is upregulated in females. Evidence clearly demonstrates that premenopausal women as compared to aged-matched men are protected from hypertension, renal and cardiovascular diseases, and this differential balance of the RAS between the sexes likely contributes. Moreover, new components of the RAS continue to be identified, the angiotensin receptor interacting proteins and AngII receptor activating mechanisms, including receptor dimerization (see [95•]), while yet to be examined, these may also have sex-specific roles. With aging this cardiovascular protection in women is lost, and this may be related to loss of estrogen postmenopause, but the possible contribution of other sex hormones also needs to be further examined. Restoration of these RAS depressor pathways in older women, or upregulation of these pathways via gene therapy in males and females, represents therapeutic targets for the treatment of cardiovascular disease that are worth pursuing.