ETM Experimental and Therapeutic Medicine 1792-0981 1792-1015 D.A. Spandidos 10.3892/etm.2018.6734 ETM-0-0-6734 Articles Calcimimetic R568 reduced the blood pressure and improved aortic remodeling in spontaneously hypertensive rats by inhibiting local renin-angiotensin system activity SunRuixia 1 * ZhangWenwen 1 2 * ZhongHua 1 WangLamei 3 TangNa 1 LiuYongmin 1 ZhaoYongli 1 ZhangTian 1 HeFang 1 Department of Pathophysiology/Key Laboratory of Education Ministry of Xinjiang Endemic and Ethnic Diseases, Medical College of Shihezi University, Shihezi, Xinjiang 832002, P.R. China Department of Emergency and Critical Care Medicine, The First Affiliated Hospital of Medical College of Shihezi University, Shihezi, Xinjiang 832002, P.R. China Centre of Medical Functional Experiments, Medical College of Shihezi University, Shihezi, Xinjiang 832002, P.R. China Correspondence to: Professor Fang He, Department of Pathophysiology/Key Laboratory of Education Ministry of Xinjiang Endemic and Ethnic Diseases, Medical College of Shihezi University, 59 North 2nd Road, Shihezi, Xinjiang 832002, P.R. China, E-mail: fangf2002shz@126.com

Contributed equally

 11 2018 13 09 2018 16 5 4089 4099 02032018 27072018 Copyright: © Sun et al. 2018 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Hypertension is a cardiovascular disease that seriously affects human health. Activation of the calcium-sensing receptor (CaSR) inhibits cyclic adenosine monophosphate (cAMP) formation by increasing [Ca2+]i and subsequently inhibiting renin release. The renin-angiotensin system (RAS) plays an important role in the development of essential hypertension (EH). The purpose of this study was to determine the effects of NPSR568 (R568)-activated CaSR on blood pressure (BP), proliferation, and remodeling of vascular smooth muscle cells, and the activity of the RAS in spontaneously hypertensive rats (SHRs). In this study, we treated SHR and Wistar-Kyoto rats with R568 for 8 weeks. The tail-cuff method was used to assess rat BP weekly. Morphological changes in the thoracic aorta were evaluated with hematoxylin-eosin and Masson staining. Western blotting and immunohistochemistry were used to detect the expression of RAS-related proteins and proliferative remodeling proteins in the thoracic aorta. An enzyme-linked immunosorbent assay was used to detect the content of cAMP, the RAS, and the CaSR in plasma and the thoracic aorta. Finally, we found that treatment with R568 for 8 weeks reduced the BP and inhibited arterial vascular proliferation remodeling in SHRs. R568 administration significantly suppressed the activity of local RAS in the thoracic aortas of SHRs. Moreover, R568 treatment reversed the low expression of CaSR in SHRs. R568 may serve as an effective strategy against EH.

 hypertension calcium-sensing receptor renin-angiotensin system proliferation and remodeling Introduction

Hypertension is one of the most important risk factors for cardiovascular disease. Epidemiologic studies have shown that patients with essential hypertension (EH) accounted for 90–95% of patients with hypertension (1). Although the etiology and pathogenesis of EH has not been determined, factors such as genetics and environment may have some impact on the occurrence and development of EH (2). Among the factors, the renin-angiotensin system (RAS) plays a crucial role in the pathogenesis of EH (3). Renin is the first rate-limiting enzyme in the RAS (4). Angiotensin II (Ang II) is a peptide hormone that mediates vasoconstriction and increases blood pressure (BP) by binding to the Ang II type 1 receptor (AT1R) and is involved in the development of hypertension (5).

The extracellular calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR), which is part of a superfamily of seven transmembrane domain receptors, the primary function of which is to regulate calcium homeostasis. The CaSR is mainly expressed in the thyroid, parathyroid, kidneys, and bone (6,7). There is also functional expression in the vascular wall, including vascular smooth muscle and endothelial cells (8–10). Increased extracellular Ca2+ (Ca2+]o) induces binding to the CaSR and activates the G-protein-phospholipase C (PLC)-inositol 1,4,5-trisphophate (IP3) receptor pathway, triggers an elevation in intracellular Ca2+ ([Ca2+]i), and is involved in the occurrence and development of hypertension (6). Studies on the relationship between calcium and BP have shown that proper intake of Ca2+ can effectively lower BP (11). This conclusion has been confirmed in animal experiments (12). Some researchers have also shown that elevated Ca2+ leads to vasodilation in vitro (13). Unfortunately, despite these findings, the molecular mechanisms underlying this association are not fully understood.

NPSR568 (R568), a calcimimetic, is an allosteric agonist of the CaSR. The mechanism of action of R568 is to increase the sensitivity of the CaSR to [Ca2+]o, and shift the calcium concentration response curve to the left. It is currently believed that the binding site of R568 to the CaSR is at the 7th transmembrane region of the receptor. The CaSR may be involved in BP regulation, and has become a current focus of research. Ogata et al (14) argued that R568 reduces BP in uremic rats and SHRs, but has no effect on normotensive rats. Rybczynska et al (15,16) reported that administration of NPS2143, an allosteric inhibitor of the CaSR, increased BP in normotensive rats; however, in rats with surgically removed parathyroid glands or an AT1R (e.g., losartan) in the presence of a calcium channel blocker or antagonist, no effect of elevated BP was observed. Atchison et al (11) and Ortiz-Capisano et al (17) suggest that the CaSR is expressed in juxtaglomerular cells, and that activation of the CaSR activates the ryanodine receptor (RyR) via the PLC/IP3 pathway to increase [Ca2+]i and inhibit cAMP formation, thereby inhibiting renin release; meanwhile, Maillard et al (18) also proved that the calcimimetic R568 can regulate renin release via CaSR. Our previous research also proved that reduced expression of the CaSR is associated with increased proliferation and remodeling of vascular smooth muscle cells (VSMCs) and promotes the development of EH via activation of the cAMP-RAS pathway (9).

The goal of this study was to determine the effect of CaSR activation on BP in spontaneously hypertensive rats (SHRs) and to partially elucidate the mechanism of action of the CaSR in regulating BP from the perspective of the RAS. Thus, we hypothesized that a R568-activated CaSR may lower BP and improve thoracic aortic proliferation and remodeling through the RAS pathway.

 Materials and methods <sec> <title>Animals

For this study, 42 male SHRs and age-matched male WKY (8-week old, 180–220 g, purchased from Vital River Laboratory Animal Science and Technology Co., Ltd, Beijing. License Number: SCXK2012-0001) were randomly divided into 4 groups: Group 1, WKY+NS (n=21); group 2, WKY+R568 (n=21); group 3, SHR+NS (n=21); and group 4, SHR+R568 group (n=21). R568 was dissolved in normal saline (NS) at a dose of 1.2 mg/kg/day in group 2/4, and was administered by intraperitoneal (i.p.) injection. At R568 treatment at 0, 4, and 8 weeks, rats were equivalent to 8, 12, and 16 weeks of age. Seven rats in each group were randomly selected and sacrificed to detect the corresponding indexes. In group 1/3, the rats received NS i.p., and treated as mentioned above. All animals were housed at a constant room temperature, humidity, and light cycle (alternating 12 h light/dark cycle), and had access to food and drinking water ad libitum. All experimental procedures in this study were approved by the Institutional Animal Research Committee of Shihezi Medical University, and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 86–23, revised 1986).

 Evaluation of EH

In the experiment, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure [MAP; (MAP=DBP+1/3 {SBP-DBP})] were measured once a week. At the same time of the week, the BP of rats was measured by tail-cuff method [BP-98A-L; Softron, Tokyo, Japan] (19), the room temperature was kept at a controlled temperature of 30°C, and repeated measurements were taken 3 times and averaged. At 0, 4, and 8 weeks with this treatment and after SBP, DBP, and MAP measurements, the rats were anesthetized, blood was extracted from the abdominal aorta and isolated plasma, then stored at −80°C for an enzyme-linked immunosorbent assay (ELISA). Rats were sacrificed and the thoracic aorta was isolated, 0.5 cm of the thoracic aorta was cut, placed in 10% neutral buffered formalin, and paraffin-embedded for histopathologic evaluation (hematoxylin and eosin and Masson staining) and immunohistochemistry analysis. The remaining part of the thoracic aorta tissue were stored at −80°C for subsequent protein extracted for western blotting and aorta homogenate for ELISA assay.

 Histologic evaluation

Thoracic aorta tissue was fixed in 10% formalin for more than 48 h, then dehydrated, embedded, sectioned, and stained with hematoxylin and eosin or Masson's trichrome. We then randomly selected three fields in each slice, calculated the parameters of the thoracic aorta vascular cross-sections by measuring the wall thickness, cross-sectional vessel area and wall area, total vessel wall thickness, and the percentages of medial wall thickness-to-the external diameter (WT%), cross-sectional vessel wall area-to-the total area (WA%), and collagen area-to-total vessel wall area (CA%). We captured the thoracic aorta images with an Olympus BX40 microscope and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics Inc., Buckinghamshire, UK).

 Immunohistochemistry analysis

Tissue sections were deparaffinized, washed, and antigen was retrieved with 0.01 M sodium citrate buffer (pH=6.0) in a pressure cooker. The sections were incubated with primary mouse polyclonal antibodies against CaSR (1:50; Abcam, MA, USA), calponin (1:600; Boster, Wuhan, China), smooth muscle actin α (α-SMA, 1:400; Boster), proliferating cell nuclear antigen (PCNA, 1:200; Boster), and primary rabbit monoclonal antibody against osteopontin (OPN, 1:100; Abcam) overnight at 4°C. Slides were washed three times in PBS (pH 7.4–7.6) and incubated with the corresponding secondary horseradish peroxidase-conjugated antibody (Invitrogen, Beijing, China) for 30 min at 37°C. A diaminobenzidine/peroxidase substrate was used to generate a brown signal. Negative controls were omitted with primary antibodies. To quantify protein levels, positive staining in tissue sections was further analyzed by Integral Optical Density (IOD)/vascular wall area using Image-Pro Plus 6.0 software. The same microscope and camera set was used for all images.

 Western blot analysis

Total protein was extracted from the thoracic aorta and quantified for protein concentration. The extracted protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred proteins to 0.45-mm SequiBlot PVDF membranes. After incubation with 5% non-fat milk, then incubated overnight at 4°C with primary antibodies against CaSR (1:1,000; Abcam), PCNA (1:250; Boster), α-SMA (1:400; Boster), calponin (1:300; Boster), OPN (1:1,000; Abcam), renin (1:250; Bioss, Beijing, China), AT1R (1:1,000; Abcam,), and β-actin (1:1,000; Boster). After being washed with TBST, the membranes were incubated with fluorescence conjugated goat anti-mouse or anti-rabbit IgGs (1:20,000; Boster) for approximately 2 h at room temperature. Protein was visualized using an enhanced chemiluminescence system (ECL, Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA USA). The intensity of protein bands was quantified using Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, USA) and the levels of protein detected were normalized to β-actin levels.

 Enzyme-linked immunosorbent assay

An ELISA kit (Wuhan Elabscience Biotechnology, Wuhan, China) was used to determine the levels of CaSR, cAMP, renin, and Ang II from plasma and thoracic aortic tissue homogenate, and the specific measurement method was according to the manufacturer's instructions. A microplate reader was used to read the absorbance at 450 nm (Bio-Rad Model 3550-UV).

 Drugs

R-568 has a chiral carbon atom and acts stereospecifically on CaSR. R568 was purchased from Tocris Bioscience Co., Ltd. (Minneapolis, MN, USA). The hydrophobic compounds were dissolved in normal saline at a dose of 1.2 mg/kg/day as a stock solution.

 Statistical analysis

Quantitative data were expressed as the mean ± SEM. For multiple experimental groups, we used one way analysis of variance (ANOVA) or Kruskal-Wallis test, then a Bonferroni post hoc test was used after ANOVA. For all analysis results, P<0.05 was considered statistically significant. Statistical analysis was performed using the SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

 Results <sec> <title>Effects of R568 on SBP, DBP, and mean arterial pressure (MAP) in SHRs

To investigate the effect of R568 on BP in SHRs, we measured the changes in SBP, DBP, and MAP using the tail-cuff method. Results showed that the BP of SHRs at different ages was higher than age-matched WKY rats (P<0.05), and the BP of the SHR16 group was significantly higher than the SHR8w group (P<0.05; Fig. 1). During 8 weeks of treatment, R568 reduced the SBP of SHRs at 16 weeks (P<0.05; Fig 1A), while R568 had little effect on the DBP and MAP (Fig. 1B and C).

 R568 attenuates aortic media remodeling in SHRs

The morphology of the thoracic aorta was determined with HE and Masson staining (Fig. 2). Compared with WKY rats, thoracic aorta endothelial cells were injured and the aortic media was thickened (increased WT% and WA%, P<0.05; Fig. 2A, C and D), and abnormal collagen accumulated (increase in CA%, P<0.05; Fig. 2B and E) as the BP increased. The above indicators in the SHR-16 group changes were more apparent than SHR-8 group (P<0.05; Fig. 2). After R568 treatment, 16w SHRs can reduce thoracic aortic proliferation and remodeling (P<0.05; Fig. 2).

 R568 inhibits proliferation and remodeling in thoracic aortas of SHRs

Immunohistochemistry and western blotting were used to analyze marker protein changes in hypertensive aortas. Compared with the age-matched WKY rats, two contractile/differentiated phenotype marker proteins (calponin and α-SMA) were decreased in the media of thoracic aortas in SHRs (P<0.05), and the expression of OPN, a synthetic/dedifferentiated phenotype marker protein, and PCNA was significantly increased (P<0.05; Figs. 3 and 4). The same trend was observed when comparing the SHR16w group with the SHR8w group (P<0.05); however, activation of the CaSR by R568 increased the expression of calponin and α-SMA, and reduced the expression of OPN and PCNA protein in the thoracic aortas of SHR16w group (P<0.05; Figs. 3 and 4). R568 had no significant effect on the expression of proliferative and remodeling proteins in WKY groups.

 R568 reverses the low expression of the CaSR in rat thoracic aortas

Western blotting and immunohistochemistry showed that expression of the CaSR in SHRs was lower than age-matched WKY rats (P<0.05). Compared with SHR16w group and SHR8w group, expression of the CaSR protein in the thoracic aortas of rats increased as the BP increased in SHRs (P<0.05; Fig. 5A-D); however, R568 reversed expression of the CaSR in SHRs at 16 weeks (P<0.05; Fig. 5A-D). R568 had no significant effect on CaSR protein expression in thoracic aortas of WKY rats.

 R568 reverses the low expression of the CaSR in plasma of SHRs and has no significant effect on aortic homogenates

The concentration of the CaSR in plasma and thoracic aortas of rats was determined with an enzyme-linked immunosorbent assay (ELISA). The CaSR concentration in SHRs plasma was significantly lower than age-matched WKY rats (P<0.05; Fig. 5E). The same trend was observed in comparing SHR16w group with SHR8w group (P<0.05; Fig. 5E). Interestingly, the content of the CaSR in SHR-16 group thoracic aortas was significantly higher than in WKY-16 group (P<0.05; Fig. 5F). After treatment with R568, the CaSR concentration was significantly elevated in the plasma of SHR16w group (P<0.05; Fig. 5E). However, R568 had no significant effect on the CaSR content in the thoracic aortas (Fig. 5F).

 R568 inhibits renin and AT1R protein increases while decreasing renin and Ang II levels in the SHR thoracic aorta

Western blotting and ELISA results showed that compared with age-matched WKY rats, renin, AT1R protein expression, and renin and Ang II concentration in thoracic aortas were increased in SHRs; the above indicators increased more significantly in SHR16w compared with those in SHR8w group (P<0.05; Fig. 6). R568 treatment reduced renin and AT1R protein expression, and renin and Ang II levels in the thoracic aortas of SHR16w group (P<0.05; Fig. 6). R568 had no significant effect on RAS-related protein expression in the thoracic aorta of WKY rats (Fig. 6).

 R568 has no significant effect on cAMP, renin, and Ang II levels in the plasma of rats

Our ELISA results showed that plasma levels of cAMP and Ang II increased and the renin levels decreased with increasing BP in SHRs compared with age-matched WKY rats (P<0.05; Fig. 7); the same trend can be observed compared with SHR16w and SHR8w groups (P<0.05; Fig. 7). There were no significant changes in plasma cAMP, renin, and Ang II levels in SHRs and WKY rats after treatment with R568 for 8 weeks (Fig. 7).

 Discussion

CaSR activation has a protective effect on EH and hypertension-induced aortic smooth muscle proliferation and remodeling. By analyzing the effects of a R568-activated CaSR on rat BP, aortic remodeling, and RAS, we demonstrated the following: i) decreased CaSR expression is involved in hypertension development; ii) low CaSR expression can activate circulating and vascular local RAS through the cAMP pathway and promote the development of hypertension; and iii) the CaSR may regulate BP by regulating local RAS activity. These data provide new insight into the regulatory mechanism of the CaSR on EH from the perspective of the RAS. To date, this is the first study to explore CaSR-regulated EH mechanisms through the RAS pathway.

The CaSR, a member of the GPCR superfamily, exerts biological effects by regulating the PLC-IP3 signaling pathway to induce an increase in [Ca2+]i (7). Recent studies have suggested that the CaSR agonist, R568, can reduce BP, but the underlying mechanism is not clear. Maillard et al (18) suggested that the calcimimetic R568 can regulate renin release via CaSR, and renin plays an important role in the occurrence of EH. Ogata et al (14) compared the effects of NPSR568 and parathyroidectomy on the progression of renal failure and showed that R568 decreased BP in uremic rats and SHRs, but had no effect on normotensive rats. Rybczyńska et al (20) also observed the effect of intravenous administration of R568 on the MAP of SHRs and WKY rats in the presence or absence of thyroparathyroidectomy and found that in the presence of parathyroid, calcimimetic R568 lowered the BP of SHRs, but has no effect on BP in WKY rats. In the current study, we first used SHRs and WKY rats of different ages to dynamically observe the anti-hypertensive effect of the CaSR agonist, R568. The results showed that R568 activation of CaSR reduced BP in SHRs but had no significant effect on BP in normotensive rats. Therefore, we believe that low CaSR expression is involved in the development of hypertension. Different views have also been proposed by other researchers. Nakagawa et al (21) observed the changes in MAP after acute injection of femoral vein R568 and the enantiomer, S568, in Sprague-Dawley (SD) rats. The results showed that R568 exerts an acute, CaSR-independent antihypertensive effect through vasodilation and negative degeneration at concentrations exceeding those required to modulate PTH secretion. Because S568 has little effect on the CaSR, Nakagawa et al (21) suggested that the effect of R568 on lowering BP may not be CaSR-mediated. From this point of view, the researchers believe that R568 is a phenylalkylamine compound, its effect on CaSR is basically stereotactic, and both isomers R568 and S568 are equivalent in blocking voltage or ligand-gated Ca2+ influx (21). The lack of stereoselectivity and the hypotensive effect of R568 suggest that exclusion of voltage-gated Ca2+ channels, except for CaSR-mediated activity, is still required; however, this remains to be demonstrated (21,22).

As an adaptive response, the progression of hypertension is often accompanied by changes in the structure and function of blood vessels [i.e., vascular remodeling (VR)], which is an independent risk factor for the increased incidence of cardiovascular events in hypertension (23). The central part of VR is the proliferation of VSMCs, migration to the sub-endocardium, and secretion of the extracellular matrix (24). VSMCs play an important role in maintaining BP, but progression of hypertension leads to phenotypic changes in VSMCs. The conversion of VSMCs from a contractile phenotype to a synthetic phenotype leads to thickening of the vessel wall. In this experiment, we determined the expression of SMAα and calponin (two contractile/differentiated phenotypic markers) (25), OPN (a synthetic/dedifferentiated phenotypic marker) (26), and PCNA (an important marker of cell proliferation) (27) in the thoracic aortas of rats. Protein expression in thoracic aortas in WKY rats and SHRs was detected with immunohistochemistry and western blotting, respectively. We observed that the expression of α-SMA and calponin decreased, while OPN and PCNA increased significantly with increasing age in SHRs; however, these changes were inhibited by the CaSR agonist, R568. HE and Masson staining showed that the WA%, WT%, and CA% of SHR thoracic aortas were significantly increased compared with WKY rats, which was reduced by R568. The above results indicate that R568 improved the proliferation and remodeling of thoracic aortas in SHRs.

Ziegelstein et al (28) suggested that the CaSR is expressed in aortic endothelial cells; whereas Smajilovic et al (29) demonstrated that the CaSR also exists in aortic VSMCs and that the CaSR can affect the proliferation of aortic VSMCs. Our previous study also put forward a similar view; expression of the CaSR protein in rat thoracic aorta VSMCs and the CaSR content were decreased with the increase in BP (9). In our experiments, similar results were obtained. We found that reduction in the CaSR could be reversed after treatment with R568 for 8 weeks in the plasma and thoracic aortas of SHRs rather than in thoracic aorta homogenates; however, the opposite result was observed in thoracic aorta homogenates when compared with SHR16w and WKY16w. Moreover, the same reverse trend was detected in CaSR protein expression in the thoracic aortas when SHR16w were compared with SHR8w. Unfortunately, no strong evidence has been found to explain this phenomenon. We presume this finding may be a compensatory manifestation for the increase of BP in SHRs, which provides a direction for further research in the future. Taken together, our results partially demonstrate that the CaSR agonist (R568) may play a protective role in EH, and EH evokes proliferation and remodeling of thoracic aortic cells in SHRs.

There is a complex network of contacts between the RAS, VR, and hypertension. Studies suggest that CaSR regulation of BP may involve six related pathways, but the molecular mechanism underlying this association has not been fully elucidated (30). To investigate the mechanism underlying the CaSR in hypertensive vascular remodeling, we selected the RAS pathway for further study, which is closely related to the development of clinical hypertension. Renin, which is closely linked to hypertension, is the first rate-limiting enzyme in the RAS, and cAMP plays a key role in this system (4). Alderman et al (31) divided hypertension into three types (high, low, and intermediate renin). At present, low renin hypertension (LRH), a subtype of EH, has been unanimously approved by researchers (32). The majority of EH patients in China are LRH (33). Angiotensinogen produces angiotensin I (Ang I) under the action of renin and angiotensin converting enzyme (ACE) acts on Ang I to produce angiotensin II (Ang II) (34). Ang II is the main active substance of the RAS, and binds to Ang II type 1 receptor (AT1R), thus causing vasoconstriction and cell proliferation (35). AT1R mediates the biological effects of Ang II and plays an important role in the regulation of BP (36). RAS is an important body fluid regulating system in the body. Circulating RAS mainly exists in plasma, and local RAS exists in the heart, aorta, brain, kidneys, and many other tissues (37). Recent studies have shown that intracellular RAS plays a more important role in the function and regulation of local tissues under certain cell types or pathologic conditions (38,39). Local RAS operates independently of circulating RAS and many of the adverse effects after AT1R activation are caused by locally-generated Ang II (40). Blaine et al (41) believes that the vascular local RAS, through the independent regulation of vascular function, promotes the occurrence of hypertension. Unger et al (42) also suggests that Angiotensin converting enzyme inhibitors (ACEIs), such as captopril, reduce BP in SHRs by inhibiting local RAS activity. In our experiments, it was observed that renin, Ang II, and AT1R in thoracic aortas increased with the increase in BP, whereas R568 inhibited the increase in these factors. In circulating blood, renin was lower in SHRs, whereas Ang II and cAMP exhibited the opposite trend, which is consistent with our previous study (9). Unfortunately, R568 treatment failed to significantly alter renin and Ang II levels in plasma. Li et al (43) believe that in the early stage of hypertension, circulatory RAS is activated and involved in the initial rise of BP, but after 8 weeks, circulating blood RAS is decreased, while BP continues to rise; at which time RAS in the heart, brain, kidneys, and other local tissues actively participate in EH and target organ damage maintenance, which is consistent with our point of view. Meanwhile, Ferrario et al (44) also noted that the effects of RAS blockers on Ang II concentrations in circulating plasma and local tissues were different, suggesting that local RAS intervention may be viable in the prevention and treatment of related diseases. As mentioned above, our results suggested that the CaSR agonist (R568) is viable to suppress local RAS activity to lower BP and improve VSMC proliferation and remodeling.

The limitations of our experiments were a lack of inhibition of the CaSR activity with NPS2314 or Calhex231, and further observations regarding the relationship between BP, thoracic aortic proliferation, and remodeling and RAS activity are needed. Despite these limitations, our study partially explains the basic mechanism by which the CaSR mediates BP through the RAS pathway.

In summary, our results indicate that low expression of the CaSR is involved in the development of hypertension by cAMP pathway activation of circulatory and vascular local RAS. Regulation of the CaSR on BP in SHRs, but not WKY rats, may be through regulation of local RAS activity. The clinically used ACEIs and Ang II receptor blockers lower BP by inhibiting RAS activity, but have significant side effects. We found that R568 can reduce BP by activating the CaSR to regulate RAS activity, thus preventing the side effects of these drugs. Continued studies are being conducted in our laboratory to further determine the mechanism by which the CaSR regulates BP at the molecular level. Our findings provide new insight into the pathogenesis of this complex disease and suggest that the CaSR may be a valuable pharmacologic target in patients with hypertension.

 Acknowledgements

Not applicable.

 Funding

The present study was supported by grants from the National Natural Science Foundation of China (grant no. 31560287) and the Xinjiang Graduate Student Research Innovation Project (grant no. XJGRI2017049).

 Availability of data and materials

All data generated or analyzed during this study are included in this published article.

 Authors' contributions

RS and FH contributed to the conception and design of the study. RS drafted the manuscript. WZ and NT performed the data analyses. HZ, LW and YL performed Masson's staining and immunohistochemical analysis. YZ and TZ performed and analyzed western blotting and ELISA. FH reviewed the manuscript and gave final approval to the submitted and final versions. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

The study was approved by the Ethics Committee of Shihezi Medical University (Shihezi, China).

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References Lloyd-JonesDAdamsRCarnethonMDe SimoneGFergusonTBFlegalKFordEFurieKGoAGreenlundKHeart disease and stroke statistics-2009 update: A report from the American Heart Association Statistics Committee and Stroke Statistics SubcommitteeCirculation119480486200910.1161/CIRCULATIONAHA.108.19125919171871 OsbornJWFinkGDSvedAFToneyGMRaizadaMKCirculating angiotensin II and dietary salt: Converging signals for neurogenic hypertensionCurr Hypertens Rep9228235200710.1007/s11906-007-0041-317519130 DasMPalSGhoshAAngiotensin converting enzyme gene polymorphism (insertion/deletion) and hypertension in adult asian indians: A population-based Study from Calcutta, IndiaHum Biol80303312200810.3378/1534-6617-80.3.30319130799 ChurchillPCSecond messengers in renin secretionAm J Physiol249F175F18419852411151 KangGLeeYRJooHKParkMSKimCSChoiSJeonBHTrichostatin a modulates angiotensin II-induced vasoconstriction and blood pressure via inhibition of p66shc activationKorean J Physiol Pharmacol19467472201510.4196/kjpp.2015.19.5.46726330760 BrownEMMacleodRJExtracellular calcium sensing and extracellular calcium signalingNat Rev Mol Cell Biol4530538200310.1038/nrm115412838336 WangRXuCZhaoWZhangJKYangBWuLCalcium and polyamine regulated calcium-sensing receptors in cardiac tissuesEur J Biochem27026802688200310.1046/j.1432-1033.2003.03645.x12787035 MolostvovGJamesSFletcherSBennettJLehnertHBlandRZehnderDExtracellular calcium-sensing receptor is functionally expressed in human arteryAm J Physiol Renal Physiol293F946F955200710.1152/ajprenal.00474.200617537980 QuYYJingHWangLMNaTHuaZLiuYMZhenLQianFFangHReduced expression of the extracellular calcium-sensing receptor (CaSR) is associated with activation of the renin-angiotensin system (RAS) to promote vascular remodeling in the pathogenesis of essential hypertensionPLoS One11e0157456201610.1371/journal.pone.015745627391973 WestonAHAbsiMWardDTOhanianJDoddRHDaubanPPetrelCRuatMEdwardsGEvidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231Cir Res97391398200510.1161/01.RES.0000178787.59594.a0 AtchisonDKOrtiz-CapisanoMCBeierwaltesWHAcute activation of the calcium-sensing receptor inhibits plasma renin activity in vivoAm J Physiol Regul Integr Comp Physiol299R1020R1026201010.1152/ajpregu.00238.201020660105 AyachiSIncreased dietary calcium lowers blood pressure in the spontaneously hypertensive ratMetabolism2812341238197910.1016/0026-0495(79)90136-7514084 CowDSome reactions of surviving arteriesJ Physiol42125143191110.1113/jphysiol.1911.sp00142816993057 OgataHRitzEOdoniGAmannKOrthSRBeneficial effects of calcimimetics on progression of renal failure and cardiovascular risk factorsJ Am Soc Nephrol14959967200310.1097/01.ASN.0000056188.23717.E512660330 RybczynskaALehmannAJurskajaskoABoblewskiKOrlewskaCFoksHDrewnowskaKHypertensive effect of calcilytic NPS 2143 administration in ratsJ Endocrinol191189195200610.1677/joe.1.0692417065402 RybczynskaAJurskajaskoABoblewskiKLehmannAOrlewskaCBlockade of calcium channels and AT1 receptor prevents the hypertensive effect of calcilytic NPS 2143 in ratsJ Physiol Pharmacol61163170201020436216 Ortiz-CapisanoMCReddyMMendezMGarvinJLBeierwaltesWHJuxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptorAm J Physiol Renal Physiol304F248F256201310.1152/ajprenal.00451.201223220722 MaillardMPTedjaniAPerregauxCBurnierMCalcium-sensing receptors modulate renin release in vivo and in vitro in the ratJ Hypertens2719801987200910.1097/HJH.0b013e32832f0d2219593209 WiddopRELiXCA simple versatile method for measuring tail cuff systolic blood pressure in conscious ratsClin Sci (Lond)93191194199710.1042/cs09301919337632 RybczyńskaABoblewskiKLehmannAOrlewskaCFoksHDrewnowskaKHoppeACalcimimetic NPS R-568 induces hypotensive effect in spontaneously hypertensive ratsAm J Hypertens18364371200510.1016/j.amjhyper.2004.10.01915797655 NakagawaKParekhNKoleganovaNRitzESchaeferFSchmittCPAcute cardiovascular effects of the calcimimetic R-568 and its enantiomer S-568 in ratsPediatr Nephrol2413851389200910.1007/s00467-009-1153-619280225 NemethEFCalcimimetic and calcilytic drugs: Just for parathyroid cells?Cell Calcium35283289200410.1016/j.ceca.2003.10.02015200152 RennaNFde Las HerasNMiatelloRMPathophysiology of vascular remodeling in hypertensionInt J Hypertens2013808353201310.1155/2013/80835323970958 LanTHHuangXQTanHMVascular fibrosis in atherosclerosisCardiovasc Pathol22401407201310.1016/j.carpath.2013.01.00323375582 BeamishJAHePMarchantREMolecular regulation of contractile smooth muscle cell phenotype: Implications for vascular tissue engineeringTissue Eng Part B Rev16467491201010.1089/ten.teb.2009.063020334504 WeberGFLettGSHaubeinNCOsteopontin is a marker for cancer aggressiveness and patient survivalBr J Cancer103861869201010.1038/sj.bjc.660583420823889 YiBCuiJNingJNWangGSQianGSLuKZOver-expression of PKGIα inhibits hypoxia-induced proliferation, Akt activation and phenotype modulation of human PASMCs: The role of phenotype modulation of PASMCs in pulmonary vascular remodelingGene492354360201210.1016/j.gene.2011.11.01022101188 ZiegelsteinRCXiongYHeCHuQExpression of a functional extracellular calcium-sensing receptor in human aortic endothelial cellsBiochem Biophys Res Commun342153163200610.1016/j.bbrc.2006.01.13516472767 SmajilovicSHansenJLChristoffersenTEHLewinESheikhSPTerwilligerEFBrownEMHaunsoSTfelt-HansenJExtracellular calcium sensing in rat aortic vascular smooth muscle cellsBiochem Biophys Res Commun34812151223200610.1016/j.bbrc.2006.07.19216919596 SmajilovicSYanoSJabbariRTfelt-HansenJThe calcium-sensing receptor and calcimimetics in blood pressure modulationBr J Pharmacol164884893201110.1111/j.1476-5381.2011.01317.x21410453 AldermanMHCohenHWSealeyJELaraghJHPlasma renin activity levels in hypertensive persons: Their wide range and lack of suppression in diabetic and in most elderly patientsAm J Hypertens1717200410.1016/j.amjhyper.2003.08.01514700504 EverettCMTurnerBLoboMPosterior reversible encephalopathy syndrome in (low renin) essential hypertensionJ R Soc Med100522523200710.1258/jrsm.100.11.52218048710 YuanWPanWKongJZhengWSzetoFLWongKECohenRKlopotAZhangZLiYC1,25-dihydroxyvitamin D3 suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin gene promoterJ Biol Chem2822982129830200710.1074/jbc.M70549520017690094 JoshiHBNewnsNStainthorpeAMacDonaghRPKeeleyFXJrTimoneyAGUreteral stent symptom questionnaire: Development and validation of a multidimensional quality of life measureJ Urol16910601064200310.1097/01.ju.0000049198.53424.1d12576846 de GasparoMCattKJInagamiTWrightJWUngerTInternational union of pharmacology. XXIII. The angiotensin II receptorsPharmacol Rev52415472200010977869 MogiMIwaiMHoriuchiMNew insights into the regulation of angiotensin receptorsCurr Opin Nephrol Hypertens18138143200910.1097/MNH.0b013e328324f5fa19430334 DanserAHLocal renin-angiotensin systemsMol Cell Biochem157211216199610.1007/BF002279008739248 SinghVPLeBBhatVBBakerKMKumarRHigh-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytesAm J Physiol Heart Circ Physiol293H939H948200710.1152/ajpheart.00391.200717483239 SinghVPLeBKhodeRBakerKMKumarRIntracellular angiotensin ii production in diabetic rats is correlated with cardiomyocyte apoptosis, oxidative stress and cardiac fibrosisDiabetes5732973306200810.2337/db08-080518829990 HunyadyLCattKJPleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin IIMol Endocrinol20953970200610.1210/me.2004-053616141358 BlaineEHSchornTWBogerJStatine-containing renin inhibitor. Dissociation of blood pressure lowering and renin inhibition in sodium-deficient dogsHypertension6I111I118198410.1161/01.HYP.6.2_Pt_2.I1116373590 UngerTGantenDLangRESchölkensBAIs tissue converting enzyme inhibition a determinant of the antihypertensive efficacy of converting enzyme inhibitors? Studies with the two different compounds, Hoe498 and MK421, in spontaneously hypertensive ratsJ Cardiovasc Pharmacol6872880198410.1097/00005344-198409000-000216209494 LiJGaoXZhangBKangLGuoZFanGAntagonistic effect of traditional Chinese medicine on RAS changes in experimental hypertensive ratsChin med New Drugs Clin Pharmacol1568702004(In Chinese) FerrarioCMJessupJChappellMCAverillDBBrosnihanKBTallantEADizDIGallagherPEEffect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin-converting enzyme 2Circulation11126052610200510.1161/CIRCULATIONAHA.104.51046115897343

Comparison of SBP, DBP, and MAP between different groups of rats. (A) Systolic blood pressure; (B) diastolic blood pressure; (C) mean arterial pressure. Data are means ± standard deviation (n=7). SHR+NS groups vs. the age-matched WKY+NS groups, *P<0.05; SHR16w+NS groups vs. SHR8w+NS groups, #P<0.05; SHR+R568 groups vs. the age-matched WKY+R568 groups, **P<0.05; SHR16w+R568 groups vs. SHR8w+R568 groups, ##P<0.05; SHR+R568 groups vs. SHR+NS groups, aP<0.05. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; NS, normal saline; R568, NPSR568. SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure.

Changes of proliferation and remodeling indices in thoracic aortic smooth muscle of rats. (A) HE staining. (B) Masson staining. (C) WT%, the wall thickness percentage of the external diameter. (D) WA%, total vessel wall area as a percentage of the total area (WA%). (E) CA%, total area occupied by collagen as a percentage of the total vessel wall area. Data are means ± standard deviation (n=7). SHR+NS groups vs. the age-matched WKY+NS groups, *P<0.05; SHR16w+NS groups vs. SHR8w+NS groups, #P<0.05; SHR+R568 groups vs. the age-matched WKY+R568 groups, **P<0.05; SHR16w+R568 groups vs. SHR8w+R568 groups, ##P<0.05; SHR+R568 vs. SHR+NS groups, aP<0.05. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; NS, normal saline; R568, NPSR568; WT%, the wall thickness percentage of the external diameter; WA%, total vessel wall area as a percentage of the total area (WA%); CA%, total area occupied by collagen as a percentage of the total vessel wall area. Scale bar, 50 µM.

Immunohistochemical analysis of proliferating remodeling protein expression in the thoracic aorta of rats. Representative images of (A) Calponin; (B) α-SMA, smooth muscle α-actin; (C) OPN, osteopontin; (D) PCNA, proliferating cell nuclear antigen. Quantification of (E) Calponin; (F) α-SMA, smooth muscle α-actin; (G) OPN, osteopontin; (H) PCNA, proliferating cell nuclear antigen. Data are means ± standard deviation (n=7). SHR+NS groups vs. the age-matched WKY+NS groups, *P<0.05; SHR16w+NS groups vs. SHR8w+NS groups, #P<0.05; SHR+R568 groups vs. the age-matched WKY+R568 groups, **P<0.05; SHR16w+R568 groups vs. SHR8w+R568 groups, ##P<0.05; SHR+R568 groups vs. SHR+NS groups, aP<0.05. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; NS, normal saline; R568, NPSR568; α-SMA, smooth muscle α-actin; OPN, osteopontin; PCNA, proliferating cell nuclear antigen. Scale bar, 50 µM.

Western blot analysis of proliferating remodeling protein expression in the thoracic aorta of rats. (A) Western blot analysis of the protein expression. (B) Calponin. (C) α-SMA, smooth muscle α-actin. (D) OPN, osteopontin. (E) PCNA, proliferating cell nuclear antigen. Data are means ± standard deviation (n = 7). SHR+NS groups vs. the age-matched WKY+NS groups, *P<0.05; SHR16w+NS groups vs. SHR8w+NS groups, #P<0.05; SHR+R568 groups vs. the age-matched WKY+R568 groups, **P<0.05; SHR16w+R568 groups vs. SHR8w+R568 groups, ##P<0.05; SHR+R568 groups vs. SHR+NS groups, aP<0.05. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; NS, normal saline; R568, NPSR568; α-SMA, smooth muscle α-actin; OPN, osteopontin; PCNA, proliferating cell nuclear antigen.

Detection of CaSR protein expression and content in plasma and thoracic aorta of rats. (A) Immunohistochemical analysis. (B) Western blotting analysis. (C) Densitometric analysis of A. (D) Densitometric analysis of B. (E) CaSR level in rat plasma (as detected by ELISA). (F) CaSR content in thoracic aorta (as detected by ELISA). Data are means ± standard deviation (n=7). SHR+NS groups vs. the age-matched WKY+NS groups, *P<0.05; SHR16w+NS groups vs. SHR8w+NS groups, #P<0.05; SHR+R568 groups vs. the age-matched WKY+R568 groups, **P<0.05; SHR16w+R568 groups vs. SHR8w+R568 groups, ##P<0.05; SHR+R568 groups vs. SHR+NS groups, aP<0.05. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; NS, normal saline; R568, NPSR568; CaSR; calcium-sensing receptor. Scale bar, 50 µM.

Determination of renin, AT1R protein expression and renin, Ang II levels in thoracic aorta of rats. (A) Western blotting analysis of Renin and AT1R expression. (B) Densitometric analysis of Renin expression. (C) Densitometric analysis of AT1R expression. (D) ELISA detection of renin concentration. (E) ELISA detection of Ang II concentration. Data are means ± standard deviation (n=7). SHR+NS groups vs. the age-matched WKY+NS groups, *P<0.05; SHR16w+NS groups vs. SHR8w+NS groups, #P<0.05; SHR+R568 groups vs. the age-matched WKY+R568 groups, **P<0.05; SHR16w+R568 groups vs. SHR8w+R568 groups, ##P<0.05; SHR+R568 groups vs. SHR+NS groups, aP<0.05. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; NS, normal saline; R568, NPSR568; Ang II, Angiotensin II; AT1R, Ang II type 1 receptor.

Determination of renin, Ang II and cAMP levels in plasma of rats. ELISA was used to detect the levels of (A) renin, (B) Ang II and (C) cAMP. Data are means ± standard deviation (n=7). SHR+NS groups vs. the age-matched WKY+NS groups, *P<0.05; SHR16w+NS groups vs. SHR8w+NS groups, #P<0.05; SHR+R568 groups vs. the age-matched WKY+R568 groups, **P<0.05; SHR16w+R568 groups vs. SHR8w+R568 groups, ##P<0.05. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; NS, normal saline; R568, NPSR568; Ang II, Angiotensin II; cAMP, cyclic adenosine monophosphate.