Angiotensin Type 2 Receptor Stimulation Increases Renal Function in Female, but Not Male, Spontaneously Hypertensive RatsNovelty and Significance
Accumulating evidence suggests that the protective pathways of the renin–angiotensin system are enhanced in women, including the angiotensin type 2 receptor (AT2R), which mediates vasodilatory and natriuretic effects. To provide insight into the sex-specific ability of pharmacological AT2R stimulation to modulate renal function in hypertension, we examined the influence of the AT2R agonist, compound 21 (100–300 ng/kg per minute), on renal function in 18- to 19-week-old anesthetized male and female spontaneously hypertensive rats. AT2R stimulation significantly increased renal blood flow in female hypertensive rats (PTreatment<0.001), without influencing arterial pressure. For example, at 300 ng/kg per minute of compound 21, renal blood flow increased by 14.3±1.8% from baseline. Furthermore, at 300 ng/kg per minute of compound 21, a significant increase in urinary sodium excretion was observed in female hypertensive rats (+180±59% from baseline; P<0.05 versus vehicle-treated rats). This was seen in the absence of any major change in glomerular filtration rate, indicating that the natriuretic effects of AT2R stimulation were likely the result of altered renal tubular function. Conversely, we did not observe any significant effect of AT2R stimulation on renal hemodynamic or excretory function in male hypertensive rats. Finally, gene expression studies confirmed greater renal AT2R expression in female than in male hypertensive rats. Taken together, acute AT2R stimulation enhanced renal vasodilatation and sodium excretion without concomitant alterations in glomerular filtration rate in female hypertensive rats. Chronic studies of AT2R agonist therapy on renal function and arterial pressure in hypertensive states are now required to establish the suitability of AT2R as a therapeutic target for cardiovascular disease, particularly in women.
- receptor, angiotensin, type 2
- renal circulation
- renin–angiotensin system
- sex characteristics
See Editorial Commentary, pp 227–228
The development and progression of hypertension differs between men and women. Before menopause, arterial pressure is lower in women than in men of similar age.1 However, after menopause, this cardioprotection in women is lost, and a higher proportion of women than men have hypertension after the age of 65 years.1 Similar differences in arterial pressure control have been observed in other mammalian species, including spontaneously hypertensive rats (SHRs).2,3 These sex-related differences in the development of hypertension have been strongly linked to sexual dimorphism in the renin–angiotensin system (RAS), which plays a pivotal role in the long-term regulation of arterial pressure.3,4
Classically, angiotensin II (AngII) acts via the angiotensin type 1 receptor (AT1R) to cause vasoconstriction and sodium retention. Activation of the pressor RAS is a key mediator in the development of hypertension, and drugs that target this system are mainstays of current antihypertensive therapy. More recently, a depressor arm of the RAS has been recognized, which counterbalances the actions of AngII at AT1R. AngII together with other biologically active angiotensin peptides, including angiotensin (1–7) and angiotensin III, interact with angiotensin type 2 receptor (AT2R) to evoke vasodilatation and natriuresis. Overwhelming evidence suggests that the depressor RAS is upregulated in women by estrogen, and this contributes to sexual dimorphism in arterial pressure control.3,4 It is, therefore, plausible that enhancement of the depressor RAS, and in particular the AT2R, may represent a novel therapeutic target in the treatment of hypertension, particularly in women.
Our recent data from normotensive rats support the notion that AT2R plays an integral, yet sexually dimorphic, functional role in the kidney. Acute systemic blockade of AT2R shifted the pressure–natriuresis relationship rightwards in both female and male rats.5 Moreover, AT2R blockade blunted the autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR) at low renal perfusion pressures and enhanced the renal vasoconstrictor response to AngII in female rats only.5 This led to our conclusion that AT2R modulates sodium excretion in both sexes and may contribute to the mechanisms by which female sex is protected against AngII-induced vascular alterations and hypertension. In addition, we recently demonstrated that acute administration of the selective AT2R agonist, compound 21 (C21), produced significant vasodilatory and natriuretic effects in the kidney of both female and male normotensive rats.6 The ability of C21 to induce natriuresis was similar between the sexes; however, C21 induced a greater increase in RBF in female rats. Remarkably, these effects were observed in the absence of any AT1R blockade, demonstrating the ability of C21 to directly modulate renal function in male and female normotensive rats.6
Despite this evidence for a significant functional role of AT2R in the kidney in male and female normotensives, the sex-specific ability of pharmacological AT2R stimulation to modulate renal function in hypertension remains unknown. Therefore, we aimed to provide further insight into the ability of pharmacological AT2R stimulation using C21 to modulate renal hemodynamic and excretory function in hypertension using the SHR model.
Eighteen- to 19-week-old SHRs were anesthetized and instrumented for measurement of mean arterial pressure (MAP; carotid catheter) and RBF (transonic flow probe). Renal hemodynamic and excretory function was examined in response to vehicle, graded infusion of C21 (100–300 ng/kg per minute), or C21 combined with the AT2R antagonist PD123319 (1 mg/kg bolus plus 1 mg/kg per hour). GFR was measured via [3H]-inulin clearance. In a separate cohort of rats, renal angiotensin receptor mRNA expression was measured by real-time reverse transcription polymerase chain reaction. Detailed methods are in the online-only Data Supplement.
Body weight and left kidney wet weight did not differ significantly between any of the male or female treatment groups (Table). Furthermore, uterine weight was similar for each in the female treatment groups. The female treatment groups also consisted of similar numbers of rats in estrus and anestrus, as identified by vaginal smear.
Basal levels of MAP, RBF, and GFR were not significantly different between the male and female treatment groups. However, although baseline urine flow (UF) and urinary sodium excretion (UNa+V) levels did not differ significantly for any in the male treatment group, a difference in baseline UF and UNa+V was detected between the female C21-treated and C21 plus PD123319-treated groups (PGroup<0.01).
Influence of C21 on Renal Hemodynamic Function
In female and male vehicle-treated, C21-treated, and C21 plus PD123319-treated SHRs, MAP did not change significantly from baseline (Figure 1A and 1D). However, in the male SHR treatment group, we observed a small but significant decrease (≈7–9 mm Hg) in MAP over time (Figure 1D; PTime<0.001).
In female SHRs, no significant change in RBF was observed in response to vehicle treatment. Whereas, in response to C21 infusion, RBF increased significantly from baseline at each dose of C21 administered, as compared with the female vehicle-treated SHRs (Figure 1B; PGroup<0.001). There was a trend for dose-dependent increase in RBF in response to C21 in female SHRs (PDose=0.066). Furthermore, this renal vasodilatory response to C21 in the absence of any change in MAP was reflected by a significant reduction in renal vascular resistance, as compared with female vehicle-treated SHRs (Figure 1C; PGroup<0.01). Each of these responses was completely abolished by coinfusion of C21 with the AT2R antagonist, PD123319 (Figure 1C; PGroup<0.001 for RBF; PGroup<0.01 for renal vascular resistance).
Unlike the female SHRs, no significant change in RBF was observed in response to C21 in male SHRs. Instead, RBF remained close to baseline levels, similar to that observed in the male vehicle-treated group (Figure 1E; PGroup>0.05). Accordingly, no significant effect of C21 on renal vascular resistance in male SHRs was observed (Figure 1F).
Influence of C21 on Renal Excretory Function
Renal excretory function was examined during baseline and experimental periods with 300 ng/kg per minute of C21. In female SHRs, a time-dependent reduction in GFR was observed in response to vehicle treatment. However, a similar reduction in GFR was observed in the female C21-treated group (Figure 2). Overall, this indicates that there was no significant effect of C21 on GFR in female SHRs. Subsequently, there was a greater reduction in filtration fraction in female C21-treated versus vehicle-treated SHRs (P<0.05; Figure 2). In contrast, because no significant change in RBF or GFR was observed in response to C21 in male SHRs, no significant difference in filtration fraction was observed between male vehicle-treated and C21-treated SHRs (Figure 2).
Finally, no significant change in UF, UNa+V, or fractional sodium excretion (FENa+) was observed in either female or male SHRs in response to vehicle treatment (Figure 3). However, each of these variables increased significantly in response to C21 in female SHRs (each PGroup<0.05; Figure 3). Importantly, these responses in female SHRs to C21 were completely abolished by coinfusion of C21 with PD123319. On the contrary, no significant effect of C21 was observed on UF, UNa+V, or FENa+ in male SHRs.
Renal angiotensin type 1a receptor, angiotensin type 1b receptor, and AT2R mRNA Expression
Renal angiotensin type 1a receptor expression was significantly greater (≈1.4-fold) in female SHRs as compared with male SHRs (P<0.05; Figure 4A). There was no significant difference in renal angiotensin type 1b receptor expression between male and female SHRs (Figure 4B). Renal AT2R expression was also significantly greater (≈4.3-fold) in female SHRs as compared with male SHRs (P<0.05; Figure 4C).
The major findings of this study were that pharmacological stimulation of AT2R induced renal vasodilatory and natriuretic effects in female SHRs. In contrast, no significant renal vasodilatory or natriuretic response to C21 was observed in male SHRs. In addition, we confirmed that renal AT2R mRNA expression was significantly greater in female than in male SHRs. Together, this knowledge provides a strong rationale for subsequent studies to investigate the long-term and sex-specific renal functional effects of pharmacological AT2R stimulation to establish whether AT2R agonist therapy represents a novel therapeutic approach for hypertension, particularly in women.
Given the dominant role of kidneys in the long-term regulation of arterial pressure, the preservation of renal function is a major goal in the identification of novel therapeutic targets for hypertension. In the current study, C21 induced significant renal vasodilatory effects in female SHRs, in the absence of any significant change in arterial pressure. These responses were completely abolished by AT2R blockade, thus confirming the role of AT2R in mediating this response while providing further support for the selectivity of this dose range of C21 for AT2R. The fact that C21 had no significant effect on arterial pressure yet induced renal vasodilatation in female SHRs might reflect greater AT2R expression (or a greater AT2R-to-AT1R ratio) in the renal vasculature as compared with the systemic circulation in female SHRs. Certainly, studies in both humans and experimental models have demonstrated a functional role for AT2R in numerous vascular territories. This includes resistance vessels from the renal, mesenteric, uterine, adrenal, coronary, and peripheral circulations, as well as large capacitance vessels such as the aorta.7
This observation of a sex-specific renal vascular response to C21 in the present study is consistent with our previous observations in rodents that AT2R plays a greater functional role in female than in male renal vasculature, providing protection against the vasoconstrictor effects of AngII.5,6,8 Furthermore, our data support the notion that differences in the renal response to C21 between male and female SHRs is attributable to sex differences in renal AT2R expression. We observed significantly greater basal AT2R mRNA expression in the kidneys of female SHRs as compared with male SHRs, in agreement with the previous findings of Silva-Antonialli et al.2 In addition, although we observed significantly greater renal angiotensin type 1a receptor expression in female SHRs as compared with their male counterparts, the relative AT2R-to-AT1R ratio was much greater in female than in male SHRs, although such sex differences in basal renal AT1R or AT2R mRNA expression are not always reported.9 Overall, it is highly plausible that the lack of response to C21 we observed in male SHRs is attributable to significantly lower renal expression of AT2R, as compared with their female counterparts. Subsequently, the dominant AT1R-mediated effects of endogenous AngII may mask any AT2R-mediated vasodilatory responses induced by C21 in male sex. Certainly, we have previously shown in conscious male SHRs that C21 alone administered >4 hours did not elicit any significant effect, at least on arterial pressure. However, when given in combination with the AT1R antagonist, candesartan, C21 lowered arterial pressure in SHRs,10 which fits with this hypothesis.
The possibility also exists that high levels of endogenous AngII may result in significant AT2R stimulation and thus limit an additional vasodilator effect of C21 administration, as suggested recently by Brouwers et al.11 Similar to the present study, this group did not identify any significant effect of intravenous bolus doses of C21 on RBF in male SHRs,11 which may reflect the presence of anesthesia, unlike other studies,10 although AT2R-mediated vasodilatation occurred in anesthetized female SHRs in the current study. Interestingly, during combined angiotensin-converting-enzyme inhibition with captopril to inhibit the endogenous production of AngII, Brouwers et al11 reported that C21 induced dose-dependent renal vasodilatation in male SHRs in the absence of any change in arterial pressure, thus supporting their proposition that the inhibition of endogenous AngII is required in male SHRs for AT2R-mediated vasodilatory responses to be manifest.
In the current study, we also observed significant increases in both UF and UNa+V in female SHRs in response to acute C21 administration. This was observed in the absence of a concomitant increase in GFR, suggesting that AT2R-mediated natriuresis induced by C21 in female SHRs was more than likely because of changes in the renal tubular reabsorption of sodium, rather than intrarenal hemodynamic changes. This ability of C21 to increase RBF in the absence of any major change in GFR is similar to what we observed previously in male and female normotensive rats.6 As discussed in this previous study, there are several possible mechanisms that could explain no change in GFR while RBF increased. First, similar vasodilatory effects of C21 at the pre- and postglomerular vessels occurred such that RBF can increase in the absence of any change in GFR. Second, given the reports that AT2R expression on podocytes contributes to the maintenance and function of the glomerular filtration barrier,12 it is also possible that C21 is able to induce the relaxation of podocytes to reduce the surface area available for filtration, thus leading to a reduction in GFR. Subsequently, a reduction in glomerular capillary ultrafiltration coefficient could counteract any AT2R-mediated increases in glomerular capillary pressure, thus preventing any significant change in GFR.
On the contrary, we did not observe any significant effect of C21 administration on UNa+V in male SHRs. This finding is somewhat surprising given that we and others have previously reported significant natriuretic effects of AT2R stimulation alone in numerous male rodent models, including normotensive Sprague–Dawley rats, uninephrectomized rats, and obese Zucker rats.6,13–15 This finding suggests that the lesser renal AT2R expression we and others have identified in male versus female SHRs may be responsible for the lack of natriuretic response of male SHRs to C21 in the present study.2 Moreover, renal AT2R expression is likely lesser in male SHRs as compared with these other male rodent models in which C21-mediated natriuretic effects have been observed. Overall, concomitant blockade of AT1R or angiotensin-converting enzyme inhibition may, therefore, also be required for AT2R-mediated natriuresis in male SHRs to manifest and should be addressed in future studies.
Although our studies demonstrate the ability of acute AT2R stimulation to modulate renal function, at least, in female SHRs, caution should be applied when interpreting these findings given that they were performed in anesthetized and renal denervated animals. Prospective studies should investigate the chronic effects of pharmacological AT2R stimulation on renal function and arterial pressure control in the hypertensive setting to confirm the physiological significance of this work. In addition, in light of the knowledge that the enhancement of the depressor RAS in women is modulated by estrogen, it will also be important to take into account how aging and age-related changes in sex hormone balance influence the potential of AT2R as a therapeutic target in female and male sexes. This is of course essential to the clinical translation of this work given that the population most frequently in need of clinical treatment for hypertension and associated disease is the elderly subjects.4 Certainly, it is highly plausible that the protective role of AT2R in the female kidneys is lost with age in association with a reduction in estrogen levels, such that menopausal status may be a critical factor when it comes to establishing the suitability of AT2R as a therapeutic target in women.
It will also be of interest to determine the ability of pharmacological AT2R stimulation to improve the efficacy and utility of existing RAS-targeted therapies. Certainly, there is significant evidence in literature of a biological cross-talk between the angiotensin receptors. Human and animal studies have shown that AT2R expression is increased during AT1R blockade16–18 and that AT2R contributes, at least in part, to the antihypertensive effects of angiotensin receptor blockers. For example, Savoia et al18 reported that peripheral resistance arteries from diabetic hypertensive patients treated with an AT1R antagonist exhibit enhanced AT2R expression and associated AT2R-mediated vasodilatation in response to AngII. In addition, studies have shown that AT2R activation reduces AT1R expression and function.16,19 Taken together, these findings support the postulate that AT2R stimulation in the presence of AT1R blockade would likely provide complementary therapeutic benefit and potentiate the long-term antihypertensive effects of existing angiotensin receptor blockers. Moreover, in the context of the present study, it is likely that greater renal vasodilatory and natriuretic effects may be observed in response to C21 treatment against a background of AT1R blockade. As previously mentioned, we have already demonstrated the ability of acute C21 administration to reduce arterial pressure in male SHRs during simultaneous AT1R blockade, and this was observed to a significantly greater extent compared with AT1R blockade alone.10 However, we are yet to establish the chronic renal functional and antihypertensive effects of this combination therapy and whether or not these effects are enhanced in females and/or males.
In summary, the present study demonstrates that AT2R stimulation enhances renal vasodilatation and natriuresis in female SHRs, without concomitant alterations in GFR. Chronic studies of AT2R agonist therapy on renal function and arterial pressure in hypertensive states are now required to establish whether pharmacological stimulation of AT2R with highly selective AT2R agonists such as C21 could, therefore, represent a suitable therapeutic target for the treatment of hypertension and associated renal disease, at least in women. This includes investigations into whether AT2R agonist therapy can improve the efficacy and utility of existing antihypertensive therapies.
Sources of funding
This work was supported by National Health and Medical Research Council grants 606652, 490918, and 490919.
U.M. Steckelings received modest research support from Vicore Pharma. The other authors report no conflicts.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.113.02809/-/DC1.
- Received November 11, 2013.
- Revision received December 3, 2013.
- Accepted April 10, 2014.
- © 2014 American Heart Association, Inc.
- Go AS,
- Mozaffarian D,
- Roger VL,
- et al
- Silva-Antonialli MM,
- Tostes RC,
- Fernandes L,
- Fior-Chadi DR,
- Akamine EH,
- Carvalho MH,
- Fortes ZB,
- Nigro D
- Hilliard LM,
- Nematbakhsh M,
- Kett MM,
- Teichman E,
- Sampson AK,
- Widdop RE,
- Evans RG,
- Denton KM
- Hilliard LM,
- Jones ES,
- Steckelings UM,
- Unger T,
- Widdop RE,
- Denton KM
- Brown RD,
- Hilliard LM,
- Head GA,
- Jones ES,
- Widdop RE,
- Denton KM
- Sullivan JC,
- Bhatia K,
- Yamamoto T,
- Elmarakby AA
- Brouwers S,
- Smolders I,
- Massie A,
- Dupont AG
- Hakam AC,
- Hussain T
- Kemp BA,
- Bell JF,
- Rottkamp DM,
- Howell NL,
- Shao W,
- Navar LG,
- Padia SH,
- Carey RM
- Savoia C,
- Tabet F,
- Yao G,
- Schiffrin EL,
- Touyz RM
- Cosentino F,
- Savoia C,
- De Paolis P,
- Francia P,
- Russo A,
- Maffei A,
- Venturelli V,
- Schiavoni M,
- Lembo G,
- Volpe M
- Savoia C,
- Touyz RM,
- Volpe M,
- Schiffrin EL
Novelty and Significance
What Is New?
This is the first study to demonstrate the sex-specific ability of pharmacological stimulation of angiotensin type 2 receptor (AT2R) to modulate renal function in hypertension.
What Is Relevant?
Acute AT2R stimulation induced renal vasodilatation and increased natriuresis in the female spontaneously hypertensive rat kidney. This was observed in the absence of any significant change in arterial pressure or glomerular filtration rate.
No significant effect of acute AT2R stimulation on renal hemodynamic or excretory function was observed in male spontaneously hypertensive rats.
The sex difference in renal response to C21 may be attributable to significantly greater renal AT2R mRNA expression in the kidneys of female versus male spontaneously hypertensive rats.
AT2R stimulation enhances renal vasodilatation and sodium excretion without concomitant alterations in glomerular filtration rate in female spontaneously hypertensive rats. Chronic studies of the sex-specific effects of AT2R agonist therapy in hypertensive states are now required to establish the potential of AT2R stimulation as a novel therapeutic target for the treatment of cardiovascular disease, at least in women.