Age- and Gender-Related Differences in Renal Vascular Responses to Angiotensin II in Rats: The Role of the Mas Receptor

Background Renal hemodynamic is influenced by both gender difference and age. Also, the Mas receptor (MasR) as one of the depressor components of the renin-angiotensin system which has more expression in females could postpone some dysfunctions associated with age, although the association between MasR and age in renal vascular responses to angiotensin II (Ang II) in male and female rats was well undefined. Therefore, the current study examined the effects of age and sex on systemic and renal vascular responses to graded doses of Ang II in Wistar rats with or without MasR antagonists (A779). Materials and Methods Anesthetized Wistar male and female rats with two age ranges of 8–12 and 24–28 weeks were exposed to cannulate venous and arterial vessels. After stability, mean arterial pressure (MAP), renal perfusion pressure (RPP), renal vascular resistance (RVR), and renal blood flow (RBF) were measured in response to the infusion of Ang II with or without A779. Results There were no significant differences in the base values of MAP, RPP, RBF, and RVR between the two genders in both the age ranges of 8–12 and 24–28 weeks. In addition, no significant gender difference was observed in the age ranges of the above mentioned parameters among the groups receiving vehicle or A779. Also, the infusion of vehicle or A779 could not significantly change the base values. On the other hand, the responses of RBF and RVR to Ang II revealed gender differences among 8–12-week groups (P < 0.05) but not in 24–28-week groups, while the blockade of MasR could not influence the responses in the age ranges. Conclusion It was concluded that age could impress sex difference in RBF and RVR responses to Ang II infusion and that MasR alone could not participate in these responses. In other words, MasR is not active under normal and acutely elevated Ang II levels.


Introduction
Age is an important factor that afects kidney hemodynamics as well as the renin-angiotensin system (RAS) activity. Both kidneys and RAS participate in the regulation of blood pressure, and advancing age could predispose the prevalence of cardiovascular and kidney diseases in elderly individuals [1]. RAS has two vasoconstrictor and vasodilator arms including angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor 1 (AT1R) and ACE2/angiotensin 1-7 (Ang 1-7)/Mas receptor (MasR) axes, respectively [2,3]. Advancing age increases and reduces the expression of vasoconstrictor and vasodilator arms, respectively [4]. Besides kidney hemodynamics and RAS, gender diferences could be overshadowed by age. It was documented that premenopause women have a lower blood pressure than age-matched men, while the gender diference is eliminated by aging [5]. In addition, the serum activity of RAS enzymes such as ACE and ACE2 is variable with respect to age and gender [6].
Several studies have exhibited the role of MasR in systemic and renal vascular responses to Ang II infusion in hypertensive and partial renal ischemia-reperfusion models [7][8][9]. Considering that MasR may participate to postpone endothelial dysfunction induced by aging [10], the role of MasR in gender-and age-related vascular responses to Ang II in normotensive rats still was not clearly defned. Terefore, the present study investigated the efects of age and gender in systemic and renal vascular responses to the administration of graded doses of Ang II in Wistar rats with or without MasR antagonist (A779).

Materials and Methods
2.1. Animals. Wistar male and female rats used in this study were born and grown in the animal room of the Water and Electrolytes Research Center with a temperature of 23-25°C and a 12 h light/12 h dark cycle. Te birth date of each animal was recorded. After weaning, the ofspring were classifed randomly into two age categories of 8-12 and 24-28 weeks. Te animals were housed with free access to water and food until experiment day. Te experimental procedures were approved by the Ethics Committee of Isfahan Medical Sciences University (Code # IR. MUI.RESEARCH. REC. 1399.564).

Experimental Groups
Te animals with two age ranges of 8-12 weeks (n � 22: 10 males and 12 females) and 24-28 weeks (n � 24: 12 males and 12 females) were divided randomly into the following eight experimental groups: Groups 1 and 2: 8-12-week male (group 1; n � 5) and female (group 2; n � 6) rats received vehicle before (30 min before Ang II injection) and along with Ang II administration.
3.1. Surgical Procedure. Te animals were anesthetized by urethane (1.7 g/kg; intraperitoneally). After tracheostomy, the carotid and femoral arteries were cannulated by using polyethylene tubes (PE9658 for the carotid artery and PE8040 for the femoral artery, Microtube Extrusions; Australia) flled with saline-heparin solution to measure the mean arterial pressure (MAP) and renal perfusion pressure (RPP), respectively. In addition, to infuse Ang II and A779 (or vehicle), a polyethylene tube flled with saline solution was implanted into the jugular vein. After placing the animal on the right side, an incision was made on the left fank by using an electrosurgical instrument (Erbe Elektromedizin GmbH; Germany), and the left kidney was exposed and warily separated from the surrounding adipose tissues with special care. Ten, the left renal artery was carefully isolated, and a fow probe (Transonic Systems Inc.; USA) connected to a perivascular fowmeter system (TS420, Transonic System Inc; USA) was placed around it for measuring renal blood fow (RBF). In order to control RPP during Ang II infusion, an adjustable occluder was fxed around the abdominal aorta just before the branching of the renal artery. Finally, carotid and femoral cannulas connected to the pressure transducers were attached to the power lab system (ADInstruments; Australia). To provide oxygen and ventilation during the experiment, an oxygen tank was connected to the tube implanted into the trachea to use oxygen as needed.

Experimental Design.
At frst, MAP, RPP, and RBF were measured continuously for at least 30 minutes to stabilize the conditions, and the recording of the last fve minutes was considered basal data. Ten, groups 3, 4, 7, and 8 received a blouse dose of A779 (50 μg/kg) followed by a continuous dose infusion of A779 (50 μg/kg/h) via a microsyringe pump (New Era Pump Systems, Inc; USA). Also, groups 1, 2, 5, and 6 received vehicle instead of A779. Te data of last 3-5 minutes were assigned for evaluating antagonist (or vehicle) efect. After that, all animals received Ang II at doses of 30, 100, 300, and 1000 ng/kg/min using a microsyringe pump without discontinuing A779 (or vehicle) infusion. Each dose was infused for 15 minutes. Te recording of the last 3-5 minutes for each dose was considered to evaluate MAP, RPP, and RBF. After ending the experiment, the animals were sacrifced humanely, and the kidneys were immediately removed and weighed. Renal vascular resistance (RVR) was calculated by the RPP/RBF ratio.

Statistical
Analysis. Data were analyzed by using SPSS software, version 25 and expressed as the mean ± standard error of the mean (SEM). Te analysis of one-way ANOVA followed by the LSD posttest was used to analyze the values of body weight (BW) as well as the weight of the right and left kidneys among groups in both the two age categories. Te kidney weight (KW) was represented as both values of raw and normalized based on 100 g BW (KW/100 g BW).
Te baseline data of MAP, RPP, RBF, and RVR were analyzed by the independent t-test. In addition, RBF and RVR responses to A779 or vehicle were reported as the percentage of change from the basal data. Also, RBF and RVR responses to Ang II were reported as the percentage of change from the values after A779 or vehicle infusion, before Ang II infusion. Moreover, repeated measure analysis followed by the LSD posttest was used to analyze all hemodynamic parameters in both stages of A779 (or vehicle) and response to Ang II. Te values of P < 0.05 were considered signifcant. Data were expressed as the mean ± standard error of the mean. * P < 0.05 and * * * P < 0.001 represent signifcant diferences in comparison to the 8-12-week male receiving vehicle; # P < 0.05 and ## P < 0.01 show signifcant diferences in comparison to the 8-12-week male receiving A779; ∆∆∆ P < 0.001 shows a signifcant change compared with the 24-28-week male receiving vehicle; $$ P < 0.01 and $$$ P < 0.001 reveal signifcant diferences in comparison to the 24-28-week male receiving A779.

BW, KW, and KW/100 g BW Measurements.
Te values of BW, KW, and KW/100 g BW are shown in Table 1. Tere were signifcant diferences in BW among groups in both the 8-12-and 24-28-week categories (P < 0.05). In addition, the values of left and right KWs demonstrated signifcant changes among the 8-12-week groups (P < 0.05). Te comparison of the values of left and right KWs found signifcant diferences among the 24-28-week groups (P < 0.05), although, no signifcant diference was observed in the values of the right and left KW/100 g BW (Table 1).

Baseline
Measurements. MAP, RPP, RBF, and RVR were measured in 10 males and 12 females of 8-12-week rats (Figures 1 and 2). Also, similar parameters were measured in 12 females and 12 males of 24-28-week rats (Figures 1 and  2). Tere were no signifcant changes in MAP and RPP between female and male groups in both the age ranges ( Figure 1). Te values of RBF and RVR either absolute or normalized by KW showed no signifcant changes between the 8-12-week male and female rats (Figure 2), but there was a signifcant diference in RBF/KW in the 24-28-week groups (P < 0.05) (Figure 2(e)).

Antagonist (A779) Efect Measurements.
Te fndings showed that the infusion of A779 or vehicle had no signifcant efect on MAP, RPP, RBF, and RVR in all groups, regardless of age and sex (Figures 3 and 4). Also, no signifcant diferences in MAP and RPP were observed between 8-12-week males and females receiving either vehicle or A779 ( Figure 3). Similar observations were seen among the 24-28-week groups ( Figure 3). Moreover, the percentage change of RBF and RVR did not exhibit any signifcant changes between the two genders, regardless of age and treatment ( Figure 4).

Vascular Response to Ang II Administration.
Te administration of Ang II graded doses increased MAP in 8-12week groups receiving either vehicle or A779 (P dose < 0.05) ( Figure 5(a)). In addition, a similar fnding was observed in all 24-28-week groups (P dose < 0.05) ( Figure 5(c)). Te administration of vehicle or A779 induced no signifcant changes in MAP among the groups in both the age ranges (Figures 5(a) and 5(c)). Also, there were no signifcant changes in RPP values during the infusion of Ang II graded doses in all 8-12-week groups ( Figure 5(b)  changes in all 24-28-week groups (P dose < 0.05) ( Figure 5(d)). Tere were no signifcant diferences in RPP among all groups in both the age ranges ( Figures 5(b) and 5(d)). On the other hand, all experimental groups revealed signifcant changes in RBF and RVR responses to Ang II administration (P dose < 0.05, Figure 6). In addition, a signifcant diference was observed in the percentage change of RBF in response to Ang II between 8-12-week male and female groups receiving either vehicle or A779 (P group < 0.05) (Figure 6(a)). In other words, 8-12-week females exhibited more decrement in RBF percentage change than 8-12-week males (Figure 6(a)), and the gender diference was eliminated by increasing age (Figure 6(c)). Moreover, 8-12-week females exhibited more increment in RVR percentage change in response to Ang II than 8-12week males with or without receiving A779 (P group < 0.05) (Figure 6(b)), and the increasing age removed the sex difference among the groups (Figure 6(d)).

Discussion
Te current study's aim was to determine whether the renal hemodynamic responses to the infusion of Ang II with or without receiving A779 were infuenced by age and gender. Te fndings were organized and discussed in some issues.
First, the present study exhibited the increment of MAP and RVR as well as the decrement of RBF in response to the infusion of Ang II in all groups receiving vehicle or A779. Te literature has documented the dose-dependent pressor responses to Ang II [12][13][14]. Ang II, as one of the most important peptides of RAS, elicits vasoconstrictor efects mediated by AT1R [2]. Second, the present study revealed that the infusion of Ang II with or without A779 signifcantly induced a gender diference in the responses of RVR and RBF in the age range of 8-12 weeks with more intensity in female gender. In this regard, some studies examined the AT1aR participation of vascular smooth muscle cells (VSMCs) in renal vascular responses to Ang II in female [15] and male [16] mice. Wolf et al. found that the removal of VSMC-AT1aR in female mice was accompanied with a decrement of 50-75% in vasoconstrictive responses to Ang II [15], and another study reported that the elimination of VSMC-AT1aR in male mice induced a decrease of 20% in vasoconstrictive responses to Ang II [16]. Terefore, the current study hypothesizes that female gender has prominent VSMC-AT1aR in acute peripheral vasoconstrictive responses and suggests further investigations in this regard.
In addition, a clinical study reported that normotensive healthy young women had a greater decrement and increment in GFR and RVR in response to Ang II than age-    Figure 4: Hemodynamic data of the percentage (%) changes of renal blood fow (RBF) and renal vascular resistance (RVR) before (base) and after (treat) vehicle or A779 infusion in 8-12-week (a, b) and 24-28-week (c, d) animals in both genders. Data were expressed as the mean ± standard error of the mean. Tere were no signifcant diferences between age-matched males and females receiving vehicle or A779.

8-12 weeks 24-28 weeks
matched men, respectively. Te responses exhibited that both gender and AT1R gene polymorphism could determine the changes involved in glomerular fltration, whereas MAP values in response to Ang II were equal in both genders [17].
On the other hand, there were some pieces of evidence against the fndings of the current study. One study performed on 8-12-week mice receiving a normal sodium diet indicated the greater responses of MAP and RVR to Ang II in males than in females but without gender diference in RBF response to Ang II, as well as in the expression of AT1R and AT2R and eNOS in the kidney vessels [18]. Tis contrast is probably due to the lower doses of Ang II in the current study. Tird, the current study found no gender diference in vascular responses to Ang II in the age range of 24-28 weeks. It seems that renal vascular responses to Ang II increased with age in the male sex. Evidence showed that aging induced higher contractile responses to Ang II in males than in females which were paralleled with decreasing the expression of AT2R, while there were not any changes in ACE and AT1R [19]. Aging has destructive efects on the endothelial function. Costa et al. exhibited that the inhibition of NOS decreased contractile responses to Ang II in aged male mice than in aged female mice, whereas the inhibition of NOS increased contractile responses to Ang II in young male and female mice [19]. Also, a decrease in the eNOS expression was observed in the aorta of old male mice [19]. Endothelin also, as a vasoconstrictor agent investigated in gender diference-related subjects [20], could be proposed in renal vascular responses to Ang II with age, especially in the male gender. One study reported that endothelin vasoconstrictor tone is infuenced by increasing age in men [21], and therefore, the subject could be known as a risk factor for the prevalence of cardiovascular disease in men with age. Fourth, concerning the role of MasR, the present study achieved an interesting fnding. Te presence or absence of A779 as a MasR antagonist had no signifcant efect on vascular responses to Ang II, regardless of age and sex. It was expected that the blockade of MasR intensifed the vasoconstrictive efects of Ang II. However, one study reported that the blockade of MasR alone could not infuence the vascular response to Ang II in 16-week male and female rats, but the simultaneous blockade of MasR and AT2R attenuated the reduction in vascular response in females only  as the mean ± standard error of the mean. Tere was no signifcant diference between age-matched males and females receiving vehicle or A779. [13]. Terefore, the present study hypothesized that MasR alone was not able to impress vascular responses to Ang II in male and female rats in both 8-12-and 24-28-week age ranges. However, the interaction of MasR and other receptors, especially AT2R, must be examined in future studies. Moreover, the current study suggests that MasR is not active under normal and acutely elevated Ang II levels. It seems that MasR regulates renal hemodynamics only under more chronic conditions such as pregnancy, hypertension, or diabetes. In this regard, one study evidenced that the blockade of MasR alters the RBF and RVR responses to Ang II after moderate renal ischemia reperfusion in female rats, but not in male rats [8]. Another study also reported that the blockade of MasR intensifed renal vascular responses to Ang II after partial renal ischemia reperfusion in hypertensive rats [11]. In addition, in spite of decreasing ACE2/ Ang 1-7/MasR axis expression with age [4], evidence reported that double deletion of MasR and AT2R, but not single deletion of MasR and AT2R, could participate in the appearance of consequences induced by aging [22]. Terefore, it seems that both MasR and AT2R could take part in attenuating consequences induced by aging. Overall, the present study suggests that the interaction between receptors may involve in the vascular responses to vasoactive agents in various age ranges, and it is recommended that more investigations should be conducted.

Limitation.
Tere is a limitation in this study that could be addressed in future research studies. Tis study used 8-12-and 24-28-week animals. It is recommended that future studies should use animals with greater age intervals.

Conclusion
Te current study concluded that age could impress sex diference in RBF and RVR responses to Ang II infusion and that MasR alone could not participate in these responses. In   other words, MasR is not active under normal and acutely elevated Ang II levels. It is suggested to evaluate the efects of dual blockade of MasR and another receptor, especially AT2R.

Data Availability
Te data used to support the fndings of the study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.