Fasting recovers age-related hypertension in the rats: reset of renal renin-angiotensin system components and klotho

Abstract

Background

The renal renin-angiotensin system (RAS) plays a vital part in the control of blood pressure and is known to be affected by aging. This study aimed to investigate the effects of intermittent fasting on age-related hypertension and the expression of local renal RAS components.

Methods

The Wistar rats were categorized into three main age groups (young, middle aged, and elderly) and three dietary treatment models, including ad libitum feeding (AL), every other day fasting (EOD), and one day per week of fasting (FW). After three months, blood pressure (BP) was assessed. Some genes and proteins of the renal RAS system were measured by using Real time PCR and Western blot. α-klotho and Ang II proteins were assessed by ELISA method.

Results

Old rats exhibited significantly increase in BP and Ang II (P < 0.001 vs. young rats) and a significant reduction in circulating levels of α-klotho and kidney AT2R protein (P < 0.001, P < 0.01, vs. young rats, respectively). Additionally, they respond to aging by increasing the AT1aR/AT2R proteins ratio (P < 0.05). Two model of feeding reduced BP in old rats and circulating Ang II in middle-aged and older rats. Moreover, by fasting, ACE2 protein expression was elevated in old rats. EOD fasting also significantly elevated the AT2 receptor protein and reduced the AT1aR/AT2R proteins ratio in the older rats (P < 0.001, P < 0.01, respectively).

Conclusion

Our findings suggest that fasting, particularly EOD, can attenuate age-related hypertension, partly through reset of the local renal RAS and increase of klotho protein expression.

Introduction

Aging is associated with imbalances in hormonal and metabolic processes that contribute to homeostasis and enable the organism to adapt to changes in its environment. This, in turn, along with oxidative stress, activation of inflammation and disorders of lipid and glucose metabolism leads to structural changes and dysfunctional conditions in various body systems such as the cardiovascular system and kidneys [1, 2]. In this regard, one of the key control systems that changes during the aging process, is renin–angiotensin system (RAS), which is a critical control system that affects the regulation of blood pressure and sodium balance. During aging, RAS through over activation of AngII/Ang II type 1 receptor and overproduction of reactive oxygen species (ROS) and inflammatory responses acts as an accelerator in cell and organ senescence, and causes to hypertension [3], chronic kidney disease, atherosclerosis, and sarcopenia [4,5,6]. On the other hand, the other parts of RAS, AngII/Ang II type 2 receptor and ACE2 (angiotensin converting enzyme 2)/ Ang (1–7) /Mas receptor, modulate the harmful effect of ACE/Ang II/AT1 receptor and play a positive impact in RAS balance and delay senescence [4,5,6]. It seems that both local (tissue) and circulating RAS are involved in aging –related disease [6, 7].

Several studies suggest the anti-aging effects of ACE-inhibitors and angiotensin receptor blockers (ARBs) in rodent models [8,9,10,11]. Beneficial effects of RAS blockers on aging through increased klotho and sirtuin expression and activation of vitamin D signaling parallel the effects of calorie restriction (CR) in delaying aging [12]. Various models of fasting are common in several religious traditions such as Christianity, Judaism, Islam, as well as in ascetic practices in Hinduism, Buddhism, Jainism, etc [13,14,15,16]. Numerous evidences show that fasting has a beneficial role on human health by improving various metabolic markers. Our recent findings revealed that the restoration of RAS equilibrium in both the aorta and heart may be a part of involving mechanisms of fasting benefits on its cardiovascular rejuvenation [17].

As mentioned above, imbalance of RAS is assumed to play in the progression of aging and aging-related disorders such as diabetes, chronic kidney disease, dementia, osteoporosis, cancer [18] and hypertension. In addition, local RAS elements in different tissues such as brain, heart, and kidney contribute to regulatory process and the aging of these organs [19]. It seems that, effective interventions to delay local RAS age-related changes can be helpful in the prevention of age-related hypertension. Due to the lack of sufficient knowledge, in this study, age-related changes in kidney RAS components were evaluated. Then, the effect of a 3-month period of two fasting regimes, fasting one day per week (FW) or fasting every other day (EOD) on the components of renal RAS and arterial blood pressure in three age groups of rats was investigated.

Methods

Materials

Sodium thiopental was obtained from Sandoz (Austria). The QuantiTect Reverse Transcription Kit and Qiagen RNeasy Mini Kit were provided by Qiagen (GmbH). Primers were designed and provided by Sinaclon Co. (Tehran, Iran). Primary and secondary antibodies, were purchased from Abcam (Cambridge, United Kingdom). Beta-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and its secondary antibody was from Bio-Rad Laboratories. The α-Klotho and Ang II ELISA (enzyme-linked immunosorbent assay) kits were procured from Eastbiopharm (Hangzhou, China).

Ethics approval

The present study was carried out based on the guidelines set by the National Laboratory and received the approval of the Ethics Committee (permission No IR.KMU.REC.1395.313) at Kerman University of Medical Sciences, Kerman, Iran.

Animals and diet

54 male Wistar rats were accommodated in the Animal House with temperature control set between 22 and 24 °C, and a light-dark cycle exposed to 12 h of light alternated with 12 h of darkness. The rats were divided into three main age groups with 18 animals per group: 3 months (was considered young), 12 months (was considered middle-aged), and 22–24 months (was considered old). Each main group was divided to three subgroups as 6 animals per subgroup. Three dietary treatment models were randomly assigned to age subgroups which include unrestricted access to food (ad libitum, AL), every other day fasting (EOD), where they were allowed to eat freely on one day and underwent fasting on the following day and, fasting one day a week (FW), where animals unrestricted access to food for six days and then fasted for one day per week. Feeding or (fasting) time was at 8:30 a.m., and the duration of this protocol spanned three months prior to the sampling. During the three months of implementing the protocol, fortunately, we did not have any animal casualties. However, during carotid cannulation surgery and blood pressure measurement, 2 old animals died and were replaced with new animals.

Blood pressure measurement and sampling

24 h following the completion of the intervention, the rats were weighed and administered thiopental sodium (50 mg/kg) to induce anesthesia. A polyethylene catheter filled with saline contains anticoagulant substance was inserted into the right carotid artery, and then cannula connected to a pressure transducer to measure blood pressure once a stabilization period had passed. Blood samples were obtained via the tail vein using an appropriate technique. Plasma was obtained from the blood samples and kept at minus 80 °C to measure Ang II and α-klotho levels. Finally, the rats were euthanized under deep anesthesia using urethane (1000 mg/kg; Sigma), and their kidneys were extracted. The kidney tissue was snap-frozen and stored at minus 80 °C for measurement of renal RAS components.

Quantitative real-time PCR analysis

Total RNA was isolated utilizing the Qiagen RNeasy Mini Kit (Qiagen, GmbH). First-strand cDNA was synthesized from 1 µg of total RNA using the QuantiTect Reverse Transcription Kit (Qiagen, GmbH). For Quantitative real-time polymerase chain reaction (PCR)), a 10 µL total reaction volume was employed. This consisted of 1 µL of diluted cDNA, 5 µL of SYBR Green Supermix (BioFACT Co.), 3.2 µL of deionized H2O, and specific forward and reverse primers with a final volume of 0.8 µL. The design and selection of these primers were conducted by Sinaclon Co. The primer sequences utilized in this investigation can be found in Table 1.

Table 1 Sequences of the different primers

For PCR amplification, a StepOnePlus Real-Time PCR System (Applied Biosystems) was utilized, applying 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min, with an initial soak of 10 min at 95 °C. Each measurement was performed in duplicate and expressed relative to the detection of the standard GAPDH (glyceraldehyde-3-phosphate dehydrogenase). PCR was executed for the primer sets AT1aR, AT2R, and ACE2.

Western blot

A total of 30 milligrams of the upper part of the cortex tissue of the right kidney was removed and homogenized using lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 0.1% SDS), to extract total protein. The protein content in the resulting homogenate was determined using the Bradford method. The 30 µg protein samples were then subjected to boiling for 10 min and loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Electrophoresis was performed using a BioRad mini gel apparatus at 100 V for 90 min. 00The proteins on the gel were subsequently transferred onto a PVDF membrane using an electrophoretic apparatus at 60 V for 120 min.

After overnight blocking with 5% skimmed milk, the PVDF membrane was incubated with primary antibodies targeting AT1aR (1:1500, ab124505; Abcam), Ang II type 2 receptor (1:1000, ab92445; Abcam), ACE2 (1:1500, ab108252; Abcam), and beta-actin (1:2000, sc47778; Santa Cruz Biotechnology) for the kidney tissue. The incubation was carried out for 4 h at room temperature. Following this, the membrane was incubated with secondary antibodies (goat anti-rabbit IgG H&L [horseradish peroxidase (HRP)] (1:2000, ab6721; Abcam) and goat anti-mouse IgG (H + L)-HRP conjugate (1:2000, 170–6516; Bio-Rad Laboratories)). Protein bands were visualized using an ECL Western blot detection kit and quantified using Image Lab Software. The beta-actin protein level was utilized as the reference protein.

Enzyme-linked immunosorbent assay

The concentrations of a-klotho and angiotensin II in the blood were measured using commercially available Enzyme-Linked Immunosorbent Assay (ELISA) Kits (Eastbiopharm ELISA kit). The ELISA method was carried out as described in the manufacturer’s kit instructions, utilizing the biotin double-antibody sandwich technology [31].

Data analysis

The data was presented as mean ± standard error of the mean and p-value < 0.05 was considered as statistically significant. Statistical analysis was performed using SPSS software (version 22.0). A two-way ANOVA was initially used to examine the effects of age, diet, and their interactions. If significant effects were found, a one-way ANOVA was used to compare the means. If only the interaction between diet and age was significant, comparisons between groups were conducted using one-way ANOVA. The Tukey post hoc test was also employed in the analysis.

Results

Blood pressure

For systolic blood pressure (SBP) and diastolic blood pressure (DBP) the two-way ANOVA showed significant effect for age (F = 8.06, P < 0.01 for SBP; F = 58.85, P < 0.001 for DBP), diet (F = 45.62, P < 0.001 for SBP; F = 13.64, P < 0.001 for DBP), and age-diet treatment interaction (F = 12.65, P < 0.001 for SBP; F = 6.43, P < 0.001 for DBP). (Fig. 1A and B).

Fig. 1

(A) Systolic blood pressure (SBP), *P < 0.05 and ***P < 0.001 versus regimen-matched group. ###P < 0.001 vs. age-matched AL group. (B) diastolic blood pressure (DBP), *P < 0.05 and ***P < 0.001 versus regimen-matched group. #P < 0.05 vs. age-matched AL group and ###P < 0.001 vs. age-matched AL, FW group. (C) the mean arterial pressure (MAP) in different animal groups. * P < 0. 05 vs. regimen-matched young group, ***P < 0.001 vs. regimen-matched young and middle-aged group. ###P < 0.001 vs. age-matched AL group. (D) The plasma levels of Ang II in the rats. Values were measured using a rat Elisa kit. Data are represented as mean ± SEM, analysis was performed by one way ANOVA, 6 animals per group. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. regimen-matched young group. ##P < 0.01 and ###P < 0.001 vs. age-matched AL group. The measurements were from AL, ad libitum regimen; FW, fed ad libitum and fasted 1 day per week; EOD, fed ad libitum and fasted every other day

In three age groups with AL regimen, old rats were had higher systolic and diastolic blood pressure versus young and middle-aged rats (P < 0.001). Old animals were maintained on the FW or EOD, showed significantly reduce in systolic pressure versus regimen-matched young group (P < 0.001). With EOD feeding, DBP were lower in old rats versus age-matched AL group (P < 0.001). However, in three patterns of diet, DBP in old rats showed a significant increase versus regimen-matched young group.

For mean arterial pressure (MAP) analysis showed only the main effect of age and two factor interaction (age-diet) as significant with F = 3.78 and P < 0.01.

Old rats under AL diet had shown higher blood pressure compared to young and middle-age groups (P < 0.001). FW and EOD led to a decrease in mean arterial pressure in old rats (P < 0.01 and P < 0.001 vs. old rats with the AL regimen, respectively) so that, no significant difference was detected in MAP of EOD-fed animals (Fig. 1C).

Circulating level of angiotensin II

Analysis showed significant effect of diet (P < 0.001) and age-diet interaction (P < 0.001) on the plasma level of Angiotensin II. Among the three different age groups under the AL regimen, old animals showed greater levels of angiotensin II (P < 0.001 vs. young animals). FW and EOD fasting reduced this parameter in Middle-aged animals (P < 0.01) and older animals (P < 0.01 and P < 0.001, respectively) in comparison with age-matched animals on the AL diet.

FW fasting reduced the circulating level of angiotensin II in Middle-aged group (P < 0.05 vs. young group). With every other day fasting, the amount of Ang II was lower in the plasma of middle-age and old rats (P < 0.01 and P < 0.001 vs. young animals, respectively) (Fig. 1D).

AT1aR gene and protein expression

Analysis had not shown age, diet significantly effects and their interaction effect on AT1aR gene and protein expression in the kidney tissue. The values of AT1aR gene and protein expression are shown in Fig. 2A, B.

Fig. 2

The levels of (A) AT1aR mRNA and (B) AT1aR protein expression in the kidney of rats. The samples were from young, middle-aged and old animals. AT1aR mRNA levels in the kidney were measured by real-time PCR. AT1aR protein levels were measured by Western blot technique. Data are represented as mean ± SEM; analysis was performed by one-way ANOVA, 6 animals per group

AT2R gene and protein expression

Although there was no interaction between age and diets but, the results showed that age and diet had a significant effect (P < 0.05, P < 0.001, respectively) on AT2R gene expression in kidney.

As shown in Fig. 3A, the AT2R gene expression was enhanced in the FW group of middle and older rats (P < 0.01 and P < 0.001 vs. age-matched AL groups, respectively). Also, the EOD diet had an increasing effect on AT2R gene expression compared to AL group (P < 0.001). and also had an increasing effect versus young animals (P < 0.001 vs. regimen-matched young group) (Fig. 3A).

Fig. 3

The levels of (A) AT2R mRNA and (B) AT2R protein expression in the kidney of rats. The samples were from young, middle-aged and old animals. AT2R mRNA levels in the kidney were measured by real-time PCR. AT2R protein levels were measured by Western blot technique. Data are represented as mean ± SEM; analysis was performed by one-way ANOVA, 6 animals per group. * P < 0.05 vs. age-matched FW group, **P < 0.01 and ***P < 0.001 vs. age-matched AL group. #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. regimen-matched young group

Figure 3B showed a main effect of age (P = 0.033), a main effect of diet (P = 0.001) and interaction of age and diet (P = 0.018) on protein expression of AT2R in the kidney. The results showed that the expression of protein AT2R decreases with age, so that its expression was lower in AL old rats compared to AL young rats (P < 0.01).

The amount of AT2R protein was increased in the kidney of EOD old animals (P < 0.001 vs. age-matched AL group and P < 0.05 vs. age-matched FW group). But FW feeding couldn’t cause changes in comparison with the AL group. Also, the level of this protein was increased in the old group under the EOD diet compared to the young regimen matched animals (P < 0.05) (Fig. 3B).

ACE2 gene and protein expression

Analysis showed that ACE2 gene in the kidney is affected by age (P = 0.001) and diet (P = 0.012) regimen, but there was no marked effect for age-diet treatment interaction. The ACE2 gene expression was enhanced in EOD old group (P < 0.001 vs. EOD young and P < 0.01 vs. EOD middle-aged groups). Also, ACE2 mRNA of EOD old animals was higher than its corresponding AL group (P < 0.001) (Fig. 4A).

Fig. 4

The levels of (A) ACE2 mRNA and (B) ACE2 protein expression in the kidney of rats. The samples were from young, middle-aged, and old animals. ACE2 mRNA levels in the kidney were measured by real-time PCR. ACE2 protein levels were measured by Western blot technique. Data are represented as mean ± SEM; analysis was performed by one-way ANOVA, 6 animals per group. * P < 0.05 and ***P < 0.001 vs. age-matched AL group. **P < 0.01 vs. regimen-matched middle-aged group. #P < 0.05 and ###P < 0.001 vs. regimen-matched young group. ACE2, angiotensin-converting enzyme 2

Results indicated statistically significant age-diet interaction effects on ACE2 protein (P < 0.001). In Fig. 4B, in the middle- aged group, every other day fasting has been able to make a considerably increase in the ACE2 protein level (P < 0.05 vs. AL middle -aged animals). On the other hand, FW and every other day diet has shown a significant increase on expression of ACE2 protein compared to AL in the old group (P < 0.05). Comparison between the results of the young, middle- aged and old categories under the EOD fasting showed a higher level of ACE2 protein in middle-aged and old animals compared to young group (P < 0.05).

AT1aR/AT2R proteins ratio

Fasting significantly affected on AT1aR/AT2R proteins ratio in kidney (P < 0.001) and also there was age–diet interaction (P < 0.001). Pattern of AL feeding was associated with a significant increase in AT1aR/AT2R proteins ratio in old animals compared with the young animals (P < 0.05). Every other day fasting pattern showed a significant reduction on the AT1aR/AT2R proteins ratio in the AL and FW old animals (P < 0.01) (Fig. 5A).

Fig. 5

(A)The ratio of angiotensin II receptor 1a (AT1aR)/angiotensin II receptor 2 (AT2R) proteins in the kidney of rats. **P < 0.01 vs. age-matched AL, FW group. # P < 0.05 vs. regimen-matched young group. (B) The plasma levels of α- Klotho in different groups. *P < 0.05 and ***P < 0.001 vs. regimen-matched young and middle -aged group. ###P < 0.001 vs. age-matched AL, FW group. The samples were from young, middle-aged and old animals. AL, ad libitum regimen; FW, fed ad libitum and fasted 1 day per week; EOD, fed ad libitum and fasted every other day. Data are represented as mean ± SEM; analysis was performed by one-way ANOVA, 6 animals per group

Circulating level of α- klotho

Statistical analysis of data showed that circulating level of α- klotho is significantly affected by the fasting (P = 0.01), and also there was age-diet interaction regarding this factor (P < 0.001). The α-klotho showed age-related decrease, so that it was lower in older rats in comparison with middle-aged and young rats (P < 0.001). Every other day fasting enhanced the amount of this factor in older animals (P < 0.05) when compared with young and middle- aged animals. As shown in Fig. 5B, plasma α- klotho levels enhanced in EOD older animals (P < 0.001), and in FW older rats (P < 0.01) compared with the age-matched AL group (Fig. 5B).

Discussion

This study investigated the influences of age and two models of fasting, fasted 1 day per week (FW), or fasted every other day (EOD) on blood pressure and some RAS components of the kidney and the antiaging protein α-klotho in rat.

The results showed that changes in the blood pressure and kidney-RAS system were not significant until middle age. However, senescence was correlated with a significant increase in blood pressure, decrease in the amount of AT2R protein of kidney, a significant rise in the AT1R / AT2R proteins ratio of kidney and plasma Ang II level, and a significant decrease in klotho plasma level in older rats contrast to young rats.

On the other hand, especially EOD fasting reversed the aging effect on blood pressure and RAS, so that under EOD the mentioned parameters in old rats reduced to levels of young animals and also the ACE2 protein was significantly higher than young animals.

Consistent with our findings showing changes in RAS axis proteins in serum and kidney during the aging process, previous research has documented some of these age-induced changes in the renin-angiotensin system. An experimental study demonstrated that aging increases the AT1a and AT2 receptors expression in the kidney and elevated the AT1a receptor/AT2 receptor ratio in mesenteric artery [20]. Shulman et al. showed that the intrarenal AT1/AT2 receptor ratio is increased in aged rats and initiation of the intrarenal renin-angiotensin cascade promotes the progression of profound renal injury [21].

The other study confirmed that Wnt/β-catenin/RAS pathway mediates age-associated renal fibrosis and is linked to impaired mitochondrial function [22]. Hence, our findings along previous reports reveal that the balance of RAS elements is altered in the elderly kidneys. Also, association of arterial aging with enhance of PRR-ACE-Ang II-AT1R axis and reduction of ACE2-MasR axis is reported in mice. In the manner that ACE, ATR1 and ART1/ATR2 ratio increased and ACE2, ATR2 and MasR proteins expression decreased in aging mouse thoracic aorta [23].

RAS signaling has been reported in aging-related hypertension [3]. The involvement of renal AT1 receptors in increasing sodium retention and raising blood pressure occurs through the activation of renal tubular sodium transporters, including Na-K-ATPase, the Na/H exchanger, and the epithelial sodium channel [24,25,26]. In addition, the renal AT1 receptor appears to be overactive in the elderly and plays an important role in the development of blood pressure characteristics observed during the aging process [27]. Ang II exerts direct hemodynamic effects on the renal vasculature by induction of vasoconstriction in both the afferent and efferent arterioles. This in turn results in a reduction in renal blood flow, glomerular filtration rate, and sodium excretion [28] which overall increase blood pressure.

As mentioned above, the renin-angiotensin system assumes a central role among the various pathways involved in the aging of body organs, including the kidneys. Ang II is one of the key promoters in this system that essentially serves as the main product of the RAS and primary ligand of the Ang II receptors. A study shows that prolonged administration of Ang II reduces expression of klotho gene, and in vivo klotho gene delivery protects Ang II-induced renal impairment [29]. This research also found that losartan (an angiotensin II receptor blocker) inhibited Ang II–induced renal klotho mRNA downregulation [29]. In support of connection between RAS/ α- klotho, in vitro and in vivo studies showed that RAS-blockade increases the level of α- klotho [30]. These findings suggest that the involvement of Ang II is essential for the regulation of klotho gene expression.

Our prior investigation demonstrated that older rats exhibit significantly elevated blood pressure levels when compared to both middle-aged and young rats [31]. Conversely, reduced expression of renal klotho has been noted in diverse models of hypertension, including spontaneously hypertensive rats and also deoxy corticosterone acetate (DOCA)-salt hypertensive rats [32]. Present study indicated that middle-aged and old rats exhibited a decrease in plasma Ang II levels when subjected to both mild and severe fasting regimens. Another finding of this study was that fasting increased klotho expression after 3 months EOD diet in old rats, but had no effect on level of this protein in young animals. In addition, EOD increased AT2R, ACE2 and decreased AT1R/AT2R in aging rats. Our findings are in accordance with a study that shows that intermittent fasting increases AT2 receptor gene expression and ACE2 gene expression in the left ventricle of rats [33]. This increase in AT2R expression can be a resetting response caused by EOD to increase vasorelaxation and thus decrease blood pressure. Also others demonstrated that caloric restriction decreased the expression of ACE and AT1R genes in the heart of DS/obese rats [34]. Differential expression of Ang II receptors could be a possible interpretation for the augmented responsiveness of aging kidneys to EOD fasting. The relative expression of AT1R and AT2R are important in defining the impacts of EOD and may facilitate age-related susceptibility to renal aging. In present study, a reduction in renal AT1aR/AT2R ratio in response to EOD fasting regimes in old rats is mainly related to an increase in AT2R, and the anti-hypertensive effect of fasting at the renal level is mainly related to its effects on ACE2 and AT2R.

Previous evidence confirmed that the FGF-Klotho system has a significant contribution to the development of disorders associated with aging, such as metabolic disorders, malignant conditions, vascular diseases, and renal dysfunction [35]. Association of increase in levels of klotho and alterations in RAS components of plasma and kidney in aging rats following fasting raises the possibility that EOD treatment increase klotho protein via RAS balance regulation. In agreement with this assumption, Our recent findings showed a significant modulation of local RAS in the heart of aged rats following a three-month period of every other day fasting [17]. A previous experiment showed caloric restriction (CR) could induce klotho expression in the adult rat kidney [36]. Recently Dias et al. showed that longevity gene Klotho increased in the hippocampus by EOD feeding condition [37]. Klotho has been demonstrated to inhibit aging-related phenotype [38] and prolong the lifespan of mice [39] which both of them is two established consequences of intermittent fasting [40]. There is evidence suggesting that there are shared molecular targets that contribute to the anti-aging effects of both renin-angiotensin system blockade (RAS-bl) and calorie restriction, and they have a similar impact on mitochondria.

These mediators include peroxisome proliferator activated receptors (PPARs), klotho, Mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK) and sirtuins. It seems that anti-aging effects linked to calorie restriction and RAS blockade inserted through upregulation of klotho, sirtuins and PPARs and downregulation of mTOR gene expression and also activation of AMPK operation. It is believed that all of above processes can modulate the oxidant production and recover mitochondrial function and delay the deleterious effects of aging [12]. Previously we showed that, fasting increased the heart, kidney and circulatory sirtuins 1 and 3 of old rats [31]. Based on our findings and others existing evidence it can be assumed that a part of age-retarding effects of renin-angiotensin system blockade (RAS-bl), calorie restriction, and EOD is induced through a common pathway i.e., regulation of RAS and hence increasing the klotho and sirtuins as anti-aging proteins. Therefore, the finding of this study established that EOD through influence on RAS components expression and regulation of its balance and hence increase klotho as antiaging protein, may attenuates the aging process of the kidneys and age-related hypertension. It should be noted that weight loss may be one of the factors of blood pressure reduction following fasting, based on an idea that expresses during fasting, our bodies more use from our fat stores for energy. Because the availability of glucose decreases due to the decrease in the intake of carbohydrates, the fasting person may lose weight. However, some studies have shown that even without losing weight and alterations in the lipid and glucose profiles, fasting can reduce blood pressure [41]. In addition, we showed that EOD fasting induced significant weight loss in young rats [31], but it did not affect blood pressure, plasma levels of angiotensin II, and components of the renal RAS system in these animals. Therefore, the possibility of influence of eating style acclimation and malnutrition on the results of our study is small and role of other mechanisms especially RAS system is very important.

Conclusion

Overall, this study indicated that first; increase in the blood pressure and changes the kidney-RAS is not significant until middle age but it is obvious in older rats. Second; EOD fasting treatment can correct the age-related changes of RAS of kidney, plasma levels of Ang II and age-related hypertension. Third; effects of EOD partly mediated through rejuvenation of kidney-RAS, overexpression of the Klotho gene and their interaction.