The present study investigated mechanisms contributing to sexual dimorphism in blood pressure in ageing rats. It is generally accepted that ageing affects kidney function as well as the autonomic nervous system and hormonal balance. Our original hypothesis was that the relative protection of renal and cardiovascular function in ageing females was due to the presence of ovarian steroids (primarily oestrogen) or detrimental effects of testosterone in males. Additionally, age-related changes in the autonomic balance of the sympathetic/parasympathetic nervous system might contribute to sex differences. The novel aspect of this work is that we gonadectomised animals in early adulthood and followed them as they aged. We looked at the renal function of intact and gonadectomised normotensive Wistar rats of both sexes in relation to blood pressure changes over a period of 18 months. The main findings of this study confirmed the view that females are protected against deteriorating kidney function and have lower arterial pressure than males. This appears to be largely explained by deleterious effects of testosterone and other androgens upon males, rather than protective effects of ovarian steroids in females. Androgens may partly exert their influence via the autonomic nervous system.
Sex steroids are responsible for the development of secondary characteristics in almost every species [29]. As expected, all rats in our study showed an increase in body mass across the life span, and there were clear differences between sexes and gonadectomised rats. Ablation of sex steroid synthesis by gonadectomy increased weight gain in females and reduced gain in males, consistent with other studies [30, 31]. Organ weights revealed that the absence of sex steroids had an impact on kidney and heart development. These organs were lighter in older animals, and they were smaller in gonadectomised animals (Table 1). Sex steroids appear to affect kidney development and function. Kidney size is postulated to be related to the number of glomeruli, and putatively, a decreased number of functioning nephrons in the male kidney may cause glomerular hypertension. The effect of testosterone on the number of the glomeruli was recently published [32]. Testosterone replacement therapy to castrated rats resulted in a higher kidney-to-body weight ratio but with reduced numbers of glomeruli. The number of glomeruli was highest in the castrated group indicating that testosterone promotes a progressive loss of glomeruli with ageing in males. In the present study, creatinine clearance, an estimation of glomerular filtration rate, indicated that kidney function declined with age but that females were relatively protected. Creatinine clearance was lower in males than in females which may suggest that ovarian steroids partly protect against age-related decline.
Under our conditions, we observed that females had lower levels of proteinuria compared to males, a difference which was abolished by castration but not by ovariectomy. Similarly, albuminuria was higher in males than in females, and interestingly, it was the highest in males in the oldest group of animals. Recent studies show that differences in proteinuria between male and female rat might be structural in origin. This further supports using Wistar model for the study of sex-dependent differences in glomerular filtration [33]. This is entirely consistent with earlier studies which show negative effects of testosterone on kidney function and glomerular filtration rate and renal blood flow [34–36]. The presence of proteinuria is a powerful indicator of probability of renal kidney disease in human [37, 38].
Regression analysis of ATGR1 expression and ED1 showed that they were associated (a positive moderate correlation r?=?0.4; P??0.001). Greater inflammation at 6 months of age was associated with a higher ATGR1/AGTR2 ratio (r?=?0.3; P??0.05). However, under our conditions, we did not see a clear relationship between the capacity to synthesise sex steroids and angiotensin II receptor expression across the 18-month period. ATG1R expression was highest at 6 months of age in all groups, again consistent with the phase of greatest inflammation. We are unsure why the inflammatory processes occurred in early adulthood but were dampened with ageing. It is possible that greater inflammation in earlier life left behind damage and functional deficit in the ageing animals, beyond the phase when inflammation had subsided. AGTR2 protein expression appeared to be higher in the gonadectomised females and then decrease with age in that group. Although AGTR1 protein expression was associated with greater inflammation, under our experimental conditions, the expression of AngII receptor proteins cannot explain the observed differences between the sexes or effects of gonadectomy upon renal function and blood pressure. Analysing AngII receptors expression by Western blot is challenging, and we must acknowledge some uncertainty about the validity of our measurements. Commercially available antibodies are not very well optimised and vary from batch to batch. Moreover, the type 1 receptor is heavily glycosylated in vivo and as a result has multiple molecular weights that exceed the single purified peptide. This influences findings regarding AGTR1 and AGTR2 expression and makes comparison between findings from different laboratories difficult. In this experiment, although we saw a single band with the size corresponding to the molecular size of the protein and we additionally used BLAST tool (from NCBI webpage) in order to check the specificity of the peptide used to produce the antibody, we were unable to assess specificity by more robust methods.
The telemetry blood pressure data showed that females have lower resting SBP (Fig. 5a) and also DBP (Fig. 5b). The difference between healthy normotensive males and females SBP was 4% and DBP 8%, and it was very consistent. DBP was lower in the sham animals compared to the gonadectomised, and this suggests that female and male hormones may play some role in the regulation of the blood pressure. The tail-cuff method did not replicate the differences in the SBP or DBP at 12 months of age observed by telemetry but did show a clear reduction in the SBP and DBP and increase in the HR of castrated males at 18 months suggesting that the observed change is very pronounced at this time point (Fig. 6a, b, c). However there was a tendency for SBP to be higher in males vs. in females (Fig. 6a, P?=?0.07). Additionally a comparison of SBP between 12 and 18 months in females (T test) showed a significant difference of 7 mmHg (P??0.01). This could be due to the impact of age-related decline in ovarian hormone production [39]. The discrepancy between blood pressures measured by telemetry and using an indirect tail-cuff method is of some interest. As described previously [22], tail-cuff pressures are prone to artefacts that are associated with stress but may also differ from central readings that are not obtained from resistance vessels due to factors such as the length of the arterial tree, length of the systole [40, 41], arterial stiffness [42] and activity of the autonomic nervous system. We might suspect a very distinct attenuation the vagal component of baroreflex sensitivity at 18 months of age in the castrated group as this was observed in a short-term study of castrated sexually matured male rats [43].
It is well established that circulation is affected by activity of the autonomic nervous system and that sympatho-vagal balance plays an important role in maintaining pressure [44]. Information on the autonomic regulation can be obtained by indirect (spectral analysis of the heart rate and systolic blood pressure) or direct (telemetry nerve recordings) measurements of the sympathetic nerve activity [24]. Circulation is affected by a cardiac cycle, respiration and vasomotor activity, and spectral analysis considers heart rate or arterial pressure as a sum of oscillatory components defined by their frequency and amplitude. In general, the low spectra are related to sympathetic and high spectra to the parasympathetic activities [45, 46].
In our study, gonadectomy did not affect cardiac sympatho-vagal balance in 12-month-old animals. Although we did not see any differences in the cardiac spectra, we did observe changes in the SBP spectra. Males and gonadectomised females show reduced HF% BP power which might suggests a sex difference in the effects of the respiration on the blood pressure. This would be in agreement with our data showing that the respiratory rates were higher in females than in males (Table 3). It seems that the VLF% were higher in males than in females suggesting that in males, sympathetic vasomotor tone might play an important role in the regulation of blood pressure. The increase in vasodilatory response in males compared to females has been shown in humans [47].
Our data show that spontaneous baroreflex gain was relatively higher in males but not affected by gonadectomy. Some of the human studies also show that males have higher baroreflex gain than females [48]. A more in-depth analysis of the blood pressure and heart rate variability at different ages, as well as contribution of such factors as the sympatho-adrenal nervous system to the regulation of the blood pressure, is needed to answer many remaining questions regarding sex differences in terms of regulating blood pressure.