ABSTRACT
Background
The vitamin D level in the blood is associated with the incidence of hypertension. The present study investigated whether or not calcitriol, an active form of vitamin D, reverses age-related hypertension.
Methods
Young (3-month-old) and aged (12-month-old) C57BL/6 male mice were administered with or without calcitriol at 150 ng/kg per day by oral gavage for 8 weeks. Blood pressure was measured by tail-cuff plethysmography and telemetry, and superoxide production in renal tissue was assessed by fluorescence imaging, and the protein expression of AP1/AT1R signaling pathway was examined by Western blot.
Results
We showed that 24-hour renal sodium excretion was impaired and blood pressure was increased in aged mice, which was related to the enhancement of renal AT1R expression and function. In addition, the expression of transcription factor AP1 (a dimer of c-Fos and c-Jun) and the binding of AP1 to the AT1R promoter region was significantly enhanced, accompanied by decreased nuclear translocation of Nrf2, abnormal mitochondrial function including decreased ATP production, NAD+/NADH ratio and mtDNA copy numbers, and increased reactive oxygen species. Calcitriol increased 24-hour urinary sodium excretion and reduced blood pressure in aged mice. Mechanically, calcitriol increased the nuclear translocation of Nrf2, improved mitochondrial function, reduced AP1 binding ability to AT1R promoter, which reversed enhanced AT1R expression and function, and lowered blood pressure in aged mice.
Conclusions
Our findings indicated that calcitriol reversed age-related hypertension via downregulating renal AP1/AT1R pathway through regulating mitochondrial function. Thus, calcitriol may be a valuable therapeutic strategy for age-related hypertension.
Hypertension is an important risk factor for cardiovascular and cerebrovascular diseases (Citation1). It is widely prevalent in the world, and the control rate of hypertension in China is not optimistic (Citation2). A large number of epidemiological data show that the prevalence rate of hypertension in China is as high as 23.2%–44.7%, but the control rate is only 5.6%–15% (Citation3). Due to the large population base in China, people over 60 years old account for 17.3% of the total population, and the prevalence of hypertension is as high as 50% (Citation4). Therefore, with the increasing aging of the population, the burden of hypertension and cardiovascular disease is an urgent problem that needs to be addressed.
The renin–angiotensin system (RAS) plays a pivotal role in regulation of blood pressure (Citation5). Most of the physiological and pathophysiological effects of RAS are mediated by the angiotensin II type 1 receptor (AT1R). The expression and activity of AT1R were significantly enhanced in most hypertensive population and animal models including age-related hypertension (Citation6,Citation7). Patients with age-related hypertension are often complicated with multiple organ dysfunctions, especially renal insufficiency (Citation8). Related clinical evidence also demonstrates that AT1R blocker is one of the best options for the treatment of age-related hypertension, which are associated with a reduction in morbidity and mortality as well as an improvement in quality of life (Citation9). Therefore, a comprehensive understanding of the regulation of the AT1R is important for developing new drug for the treatment of age-related hypertension.
Calcitriol is the major active form of vitamin D, which can protect the cardiovascular system and prevent the occurrence of cardiovascular diseases by reducing oxidative stress (Citation10). Previous studies have shown that calcitriol improves mitochondrial function and reduces inflammation in placentae of obese women (Citation11), and protects against myocardial reperfusion injury through attenuating mitochondrial impairment (Citation12). However, it is uncertain whether or not calcitriol can reverse age-related hypertension through regulating mitochondrial function. The present experiments determined whether or not calcitriol lowers age-related hypertension via downregulating renal AP1/AT1R pathway through regulating mitochondrial function.
Materials and methods
Animal protocols
C57BL/6 mice were obtained from Charles River laboratory. This study was approved by the Committee on Animal Care of the Ganzhou Municipal Hospital and was carried out in accordance with the NIH guidelines for the care and use of laboratory animals. Animals were fed with a standard diet provided by the animal facilities and had access to food and water ad libitum. Young (3-month-old) and aged (12-month-old) C57BL/6 male mice were randomly assigned to control group and calcitriol group. Control group were treated with vehicle (dimethyl sulfoxide, DMSO, Sigma, MO, USA) for 8 weeks, and calcitriol group were treated with calcitriol (Sigma, MO, USA) at 150 ng/kg per day by oral gavage for 8 weeks.
Blood pressure measurement
Blood pressure was measured weekly by the tail-cuff method (BP-2010A, Softron, Japan) for 8 weeks. Briefly, a cuff with a constant temperature of 37°C was placed around the tail and blood pressure was recorded when showing a stable sinusoidal waveform. The mean systolic blood pressure was calculated from at least three values with a difference <10 mmHg. Blood pressure was also measured by telemetry (DSI, Harvard Bioscience). Briefly, the catheter, connected to the transmitter, was implanted into the abdominal aorta via the iliac artery. The blood pressure was recorded every 1 min and data were analyzed using Dataquest software.
Renal function measurement
The renal function was assessed by measuring 24 h-natriuresis, serum creatinine (Cr) and blood urea nitrogen (BUN). For measuring 24 h-natriuresis, the mice were placed in metabolic cages with free access to food and drink for 24 h urine collection. Urine electrolytes and chemistries were measured by a flame photometer 480 (Ciba Corning Diagnostics, MA, USA). Serum creatinine (Cr) and blood urea nitrogen (BUN) were measured using an automated Beckman Analyzer (Beckman Instruments GmbH, Munich, Germany).
Surgical and experimental procedures for renal function studies
The function of sodium excretion was observed by infusion of Candesartan (Sigma) in vivo. Briefly, the mice were anesthetized with pentobarbital (50 mg/kg, intraperitoneally; Sigma) and the left jugular vein was catheterized with PE-10 tubing to infuse vehicle or reagent. A midline abdominal incision was made and the bladder was exposed. The bladder was catheterized (PE10) to collect urine. To determine the effect of Candesartan-induced diuresis and natriuresis, Candesartan (10 μg/kg/min) was infused through the left jugular vein for 40 min. Urine flow and urinary sodium excretion (UNaV) were recorded during Candesartan infusion. Sodium and potassium concentrations in urine samples were measured by a flame photometer 480 (Ciba Corning Diagnostics). The rats from these separate groups were euthanized using CO2 inhalation for further studies.
Inflammatory and oxidative stress markers measurement
Inflammatory markers including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and oxidative stress markers including malondialdehyde (MDA) and superoxide dismutase (SOD) in renal tissues were measured by enzyme linked immunosorbent assay (ELISA) kit (Beyotime, Shanghai, China).
ROS and Ang II measurement
The kidney was frozen and sliced into sections (10 μm) using a Leica CM 100 cryostat. The sections were incubated with the fluorescent dye dihydroethidium (DHE, 5 μmol/L, Beyotime) for 15 min at 37°C. The renal tissues were briefly washed and imaged by an Olympus Fluoview FV1000 laser scanning confocal system. Ang II concentrations in renal tissues were measured by enzyme immunoassay kit (Cloud Clone, Wuhan, China).
Cell culture and treatment
In vitro experiments utilized primary cultures of renal proximal tubular (RPT) cells from young and aged mice. RPT cells were isolated as described previously (Citation13). At ≈ 85% confluency, the RPT cells were treated with or without calcitriol (10−7M, Sigma), ML385 (Nrf2 antagonist, 10 μmol/L, Sigma) for 24 hours. DMSO was used as a vehicle control. After treatment with the reagent or vehicle, the cells were used for further studies.
ATP content and NAD+/NADH measurements
Renal tissues (20 mg) or RPT cells were taken to detect the ATP content, according to the ATP kit instructions (Beyotime). Briefly, renal tissues were homogenized with 200 μl lysate for 30 min and centrifuged at 12 000 g for 5 min. The relative light unit (RLU) value was measured by luminometer after adding 100 µl ATP detection solution and 20 µl samples into the test tube at an interval of 2 seconds. The ATP content of each sample was standardized through the protein concentration to obtain the relative content of ATP. NAD+ and NADH levels in renal tissues were measured via NAD+/NADH assay kit (Beyotime).
Mitochondrial DNA (mtDNA) copy number measurement
Total DNA was extracted from renal tissue or RPT cells using the QIAamp DNA Mini Kit (Qiagen, Germany), and quantitative real-time PCR was performed with mitochondrial D-loop primers. The specific primers used in the study as follows: D-loop, forward, 5’-CCTCCGTGAAATCAACAACC-3,’ reverse, 5’-TAAGGGGAACGTATGGACGA-3;’ GAPDH, forward, 5’-AGACAGCCGCATCTTCTTGT-3,’ reverse, 5’-CTTGCCGTGGGTAGAGTCAT-3.’ Data were normalized to the mRNA of GAPDH and were analyzed using the 2−△△Ct method.
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed to measure the interaction of AP1 with AT1R promoter, using ChIP Kit (Millipore, MA, USA). Briefly, renal tissues or RPT cells were minced with small scissors and homogenized with nucleus/chromatin preparation buffer I to collect nuclei. Chromatin was sheared by 10 seconds of sonication with repeated 10 times to generate chromatin fragment with an average size of 0.3 kb. Chromatin lysate subjected to immunoprecipitation with ChIP-grade antibody against c-Fos (Abcam, Cambridge, UK), c-Jun (Abcam), IgG (Abcam). The immunoprecipitates were extracted after incubation with RNase A and proteinase K and then used for real-time quantitative PCR to evaluate the enrichment of AP1 on promoter region of AT1R. The following primers were used for ChIP-PCR, AT1R: forward, 5’-CGGTTCTCTCCTTCTACCTTTG-3;’ reverse, 5’-GTTCTTATCGGTCCAACCATCT-3.’
Real-time quantitative RT-PCR (qRT-PCR) analysis
qRT-PCR was used to determine mRNA levels of ACE and AT1R in the renal cortex by using specific primers. The following primers were used for qRT-PCR, ACE: forward, 5’-TTGACGTGAGCAACTTCCAG-3;’ reverse, 5’-CAGATCAGGCTCCAGTGACA-3.’ AT1R: forward, 5’-CAAAAGGAGATGGGAGGTCA-3;’ reverse, 5’-TGACAAGCAGTTTGGCTTTG-3.’ GAPDH: forward, 5’-AGACAGCCGCATCTTCTTGT-3;’ reverse, 5’-CTTGCCGTGGGTAGAGTCAT-3.’ Total RNA was extracted using TRIzol reagent and cDNA was synthesized using reverse transcript reagents from Bio-Rad Laboratories. The relative mRNA expression was measured by the 2−ΔΔCT method and was normalized to GAPDH mRNA levels.
Western blotting
The cytoplasmic and nuclear fractions from renal tissues were isolated using a Nuclear and Cytoplasmic Extraction Reagents kit (Beyotime) to determine expression of AP1 (c-Fos and c-Jun) and Nrf2. Equal amounts of protein were loaded onto an 8%–12% SDS-PAGE gel for separation and transferred onto nitrocellulose membranes. Membranes were blocked with 8% fat-free milk in TBS (Tris-buffered saline) with 0.5% Tween-20 and probed with primary antibodies against Nrf2 (1:300, Abcam), c-Fos (1:300, Abcam), c-Jun (1:300, Abcam), AT1R (1:300, Abcam), GAPDH (1:1000, Abcam) and H3 (1:1000, Abcam) at 4°C overnight. After washing blots, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10000, Li-Cor Bioscience, Bad Homburg, Germany). The intensity of protein bands was analyzed using Quantity One image analysis software.
Statistical analysis
Data were presented as mean ± SEM. All experiments were repeated at least three times. In the experiments comparing multiple time points, separate t-tests were used for each time point. Differences were analyzed using one-way ANOVA followed by Student-Newman-Keuls test for three or more samples. P < .05 was defined as statistically significant.
Results
Calcitriol lowered blood pressure and improved impaired natriuresis in aged mice
In agreement with previous studies (Citation13), age is an important risk factor for elevated blood pressure, and the blood pressure, especially SBP, of aged mice which was measured by noninvasive tail-cuff method and telemetry was significantly higher than that of young mice (). Urinary sodium excretion, a key process in the long-term regulation of blood pressure, was markedly reduced in aged mice, relative to young mice (). Calcitriol treatment for 8 weeks reduced the levels of blood pressure and increased 24 h-urinary volume output and urinary sodium excretion in aged mice (). Calcitriol treatment had no significant effect on blood pressure and renal sodium excretion in young mice. In addition, serum Cr and BUN were measured and no significant differences were found among the groups ().
Figure 1. Calcitriol lowered blood pressure and improved impaired natriuresis in aged mice. The young and aged mice were treated with vehicle or calcitriol (150 ng/kg/day) for 8 weeks. Tail-cuff systolic blood pressure (SBP) (a) was measured once a week for 8 weeks and 24-hour ambulatory SBP (b) was measured after 8 weeks of vehicle or calcitriol treatment. 24-hour urinary volume output (c), sodium excretion (d), serum creatinine (Cr) (e) and blood urea nitrogen (BUN) (f) were measured after 8 weeks of vehicle or calcitriol treatment. Data are expressed as the means ± S.E.M (n=6/group). *P <.05 vs. others.
Calcitriol down-regulated renal AT1R expression and improved AT1R-mediated sodium excretion function
Due to the important role of RAS component on renal function, we determined the levels of RAS component in kidney. The Ang II and ACE mRNA levels in renal tissues from aged mice were higher than young mice (). Moreover, compared with young mice, the mRNA and protein expressions of AT1R in kidney were higher in aged mice (). Calcitriol treatment for 8 weeks significantly decreased Ang II, ACE and AT1R levels in kidney from aged mice, not from young mice (). The increased AT1R expression was pathophysiological significance. Compared with young mice, AT1R antagonist Candesartan-mediated natriuretic and diuretic effects were significantly higher in aged mice (). Calcitriol treatment for 8 weeks normalized Candesartan-mediated natriuresis and diuresis in aged mice ().
Figure 2. Effect of calcitriol on renal RAS component expression and function in aged mice. The young and aged mice were treated with vehicle or calcitriol (150 ng/kg/day) for 8 weeks. (a) Ang II levels in renal tissues were measured by enzyme immunoassay kit. Renal ACE (b) and AT1R mRNA (c) were determined by qRT-PCR and AT1R protein expression (d) was determined by immunoblotting. Urine flow e) and urinary sodium excretion (UNaV) (f) were recorded during the vehicle or Candesartan (10 μg/kg/minute) infusion via the left jugular vein of mice. Data are expressed as the means ± S.E.M (n=6/group). *P <.05 vs. others.
Calcitriol inhibited the activation of AP1 and reduced the binding level of AP1 on the AT1R promoter
Previous studies have reported that AP1, as a transcription factor, binds to the AT1R promoter to promote its transcriptional activity (Citation14). The AP-1 transcription factor is a dimer of c-Fos and c-Jun. We checked the expression levels of AP1 in nuclear fraction and found that the expression levels of c-Fos and c-Jun were increased in aged mice, relative to young mice (). Moreover, the results of CHIP assay showed that c-Fos and c-Jun binding to the AT1R promoter was higher in aged mice, relative to young mice (). Calcitriol treatment for 8 weeks reduced the expression levels of c-Fos and c-Jun in nuclear fraction and decreased the binding of c-Fos and c-Jun with AT1R promoter in aged mice ().
Figure 3. Effect of calcitriol on the binding level of AP1 on the AT1R promoter in aged mice. The young and aged mice were treated with vehicle or calcitriol (150 ng/kg/day) for 8 weeks. The protein expressions of c-Fos (a) and c-Jun (b) were determined by immunoblotting. (c and d) the binding levels of c-Fos and c-Jun on the AT1R promoter were determined by chromatin immunoprecipitation-quantitative real-time polymerase chain reaction. Data are expressed as the means ± S.E.M (n=6/group). *P <.05 vs. others.
Calcitriol reduced inflammation and oxidative stress in kidney of aged mice
Given role of inflammation and oxidative stress in AP1 activation (Citation14), we checked the levels of inflammation and oxidative stress in those mice. The results found that the levels of inflammation makers including IL-1β and TNF-α were increased in aged mice, relative to young mice (). Calcitriol treatment for 8 weeks reduced IL-1β and TNF-α level in kidney from aged mice (). Consistent with inflammatory change, oxidative stress was also increased in aged mice, relative to young mice. The results showed that MDA level was increased and SOD level was decreased in aged mice (). Calcitriol treatment for 8 weeks reduced the levels of oxidative stress in kidney from aged mice, but not in young mice ().
Figure 4. Effect of calcitriol on inflammation and oxidative stress in kidney of aged mice. The young and aged mice were treated with vehicle or calcitriol (150 ng/kg/day) for 8 weeks. (a and b) the inflammatory indexes including IL-1β and TNF-α were determined by ELISA. (c and d) the marker of oxidative stress including MDA and SOD were determined by ELISA. Data are expressed as the means ± S.E.M (n=6/group). *P <.05 vs. others.
Calcitriol improved mitochondrial function in kidney of aged mice
Previous studies have shown that the activation of inflammation and oxidative stress is closely related to abnormal mitochondrial function (Citation15). The level of reactive oxygen species (ROS) was detected by DHE staining, which is an important indicator of mitochondrial function. We found that the kidney of aged mice, relative to young mice, had increased ROS production ( a1 and a2). Moreover, we also found that ATP production, mtDNA copy number and NAD+/NADH ratio were decreased in aged mice, relative to young mice (). Calcitriol treatment for 8 weeks reduced ROS production, increased ATP production, mtDNA copy number and NAD+/NADH ratio in aged mice, but not in young mice ().
Figure 5. Effect of calcitriol on mitochondrial function in kidney of aged mice. The young and aged mice were treated with vehicle or calcitriol (150 ng/kg/day) for 8 weeks. (a1 and a2) representative images and quantified DHE fluorescence in kidney of mice. ATP production (b), mtDNA copy numbers (c) and NAD+/NADH ratio (d) in renal tissue from young and aged mice with or without calcitriol treatment. Data are expressed as the means ± S.E.M (n=6/group). *P <.05 vs. others.
Calcitriol improved mitochondrial function and reduced AT1R expression in kidney from aged mice dependent on Nrf2 signaling
Previous studies have shown that Nrf2 activation improves mitochondrial function, which maintains the cellular redox poise (Citation16). We found that the expression of Nrf2 in the nucleus was reduced in aged mice, however, treatment with calcitriol promoted the nucleus translocation of Nrf2 (). ML385 (Nrf2 antagonist) blocked the ability of calcitriol to increase ATP production and mtDNA copy number, decrease the binding of c-Fos and c-Jun with AT1R promoter and AT1R protein expression in RPT cells from aged mice (). These results suggested that calcitriol improved mitochondrial function and reduced AT1R expression in kidney from aged mice dependent on Nrf2 signaling.
Figure 6. The role of Nrf2 in the improvement of mitochondrial function and reduction of AT1R expression by Celastrol. The RPT cells from young and aged mice were treated with or without calcitriol (10−7M), ML385 (Nrf2 antagonist, 10 μmol/L) for 24 hours. (a) protein abundance of Nrf2 in nuclear fraction of RPT cells was determined by immunoblotting. Data are expressed as the means ± S.E.M (n=6/group). *P <.05 vs. others. ATP production (b) and mtDNA copy numbers (c) were detected in RPT cells from young and aged mice. (d and e) the binding levels of c-Fos and c-Jun on the AT1R promoter were determined by chromatin immunoprecipitation-quantitative real-time polymerase chain reaction. (f) AT1R protein expression was determined by immunoblotting. Data are expressed as the means ± S.E.M (n=6/group). *P <.05 vs. young+Vehicle; #P <.05 vs. aged+Calcitriol.
Discussion
With the aging of the global population, the incidence of age-related hypertension is rising sharply, and the related complications caused by it are a problem that cannot be ignored (Citation17). However, the pathological mechanisms are still elusive and treatment strategies for age-related hypertension are still limited. In the present study, we showed for the first time that calcitriol reversed age-related hypertension via downregulating renal AP1/AT1R pathway through regulating mitochondrial function.
The pathogenesis of age-related hypertension is complex, and multiple organs and signaling mechanisms may be involved. The mechanisms reported so far include overactivation of sympathetic nerve (Citation18), peripheral vascular systolic and diastolic dysfunction (Citation19), abnormal remodeling of vascular smooth muscle cells (Citation20), and activation of renal RAS system (Citation21), podocyte senescence and glomerular sclerosis (Citation22). It has been reported that the increase of reactive oxygen species is closely related to the development of age-related hypertension. Oxidative stress can cause vasoconstriction and diastolic dysfunction (Citation23), lower renal blood flow and decreased renal vascular density (Citation24), and impaired angiogenesis (Citation25) in age-related hypertensive animal models. In addition, oxidative stress causes abnormal urinary sodium excretion function mediated by renal dopamine D1 receptor or AT1 receptors, which is also one of the important pathogenesis of age-related hypertension (Citation26). Mitochondria produce reactive oxygen species in the process of biological oxidation and energy conversion (Citation27). When there is an imbalance between the generation of reactive oxygen species and the body’s antioxidant defense system, mitochondria will produce oxidative stress (Citation28). The increased oxidative stress caused by mitochondrial dysfunction may be one of the important factors for the enhanced expression of AT1R in hypertensive animal models. In our present study, we found that blood pressure in aged mice was significantly higher than in younger mice, accompanied by significantly enhanced renal AT1R expression and function. However, the mechanism regulating AT1R in aged mice is not clear.
Previous studies have shown a significant negative correlation between vitamin D levels and the incidence of cardiovascular diseases, including heart failure (Citation29), hypertension (Citation30), and atherosclerosis (Citation31). For patients with chronic renal insufficiency, supplementation of vitamin D can significantly reduce the incidence of cardiovascular adverse events and improve the long-term prognosis (Citation32). Some studies have reported that vitamin D is a potent endogenous suppressor of the RAS, due to its ability to inhibit renin transcription in vitro and in vivo (Citation33). Calcitriol is the major active form of vitamin D (Citation34). In this study, we found that calcitriol can reduce blood pressure in aged mice, accompanied by inhibiting renal AT1R expression and function. Due to the important role of oxidative stress in RAS activation, oxidative stress may act as a bridge between calcitriol and AT1R expression.
Previous studies have shown that calcitriol can play a role in the prevention and treatment of cardiovascular diseases by reducing oxidative stress levels (Citation10). Vitamin D deficiency attenuates endothelial function by reducing antioxidant activity and vascular eNOS expression in the rat microcirculation (Citation35). Calcitriol Supplementation ameliorates microvascular endothelial dysfunction in vitamin D-deficient diabetic rats, and autonomic dysfunction and hypertension in spontaneously hypertensive rats by reducing oxidative stress (Citation35,Citation36). Mitochondria are important sources of reactive oxygen species, and mitochondrial dysfunction is one of the important pathogenesis of cardiovascular diseases (Citation37). Mitochondrial dysfunction causes an increased production of ROS, which leads to loss of nitric oxide signaling, loss of endothelial barrier function and infiltration of leukocytes to the vascular wall, explaining the low-grade inflammation characteristic for the vasculature in mice with age-related hypertension (Citation38). The crucial pathway Nrf2 plays a key role in cellular defense against mitochondrial dysfunction (Citation16). Vitamin D receptor activation could attenuate age-related osteoporosis by promoting the accumulation and nuclear translocation of Nrf2 (Citation39). The present study showed that calcitriol could increase the nuclear translocation of Nrf2, which improved mitochondrial function and reduced oxidative stress in aged mice.
The present study found that chronic oral administration of calcitriol reversed age-related hypertension. Whether or not there is a relationship between low plasma levels of vitamin D and elevated arterial blood pressure in aged patients remains unclear. More clinical data are needed to further confirm the correlation between vitamin D levels and age-related hypertension.
In conclusion, chronic treatment with calcitriol reversed age-related hypertension. The calcitriol-induced protective effect is likely to be mediated by downregulating renal AP1/AT1R pathway through regulating mitochondrial function.
Author contribution
HR conceived and designed the experiments; LB wrote the manuscript; HW, ZH and LY performed the experiments; XQ and ZL analyzed the data; PF provided the project funding and approved the final version of the manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Additional information
Funding
References
- Pedelty L, Gorelick PB. Management of hypertension and cerebrovascular disease in the elderly. Am J Med. 2008;121:S23–10. doi:10.1016/j.amjmed.2008.05.019.
- Yin R, Yin L, Li L, Silva-Nash J, Tan J, Pan Z, Zeng J, Yan LL. Hypertension in China: burdens, guidelines and policy responses: a state-of-the-art review. J Hum Hypertens. 2022;36(2):126–34. doi:10.1038/s41371-021-00570-z.
- Bundy JD, He J. Hypertension and related cardiovascular disease burden in China. Ann Global Health. 2016;82(2):227–33. doi:10.1016/j.aogh.2016.02.002.
- Liu P, Li Y, Zhang Y, Mesbah SE, Ji T, Ma L. Frailty and hypertension in older adults: current understanding and future perspectives. Hypertens Res. 2020;43(12):1352–60. doi:10.1038/s41440-020-0510-5.
- Arnold AC, Gallagher PE, Diz DI. Brain renin–angiotensin system in the nexus of hypertension and aging. Hypertens Res. 2013;36(1):5–13. doi:10.1038/hr.2012.161.
- Chugh G, Pokkunuri I, Asghar M. Renal dopamine and angiotensin II receptor signaling in age-related hypertension. Am J Physiol Renal Physiol. 2013;304:F1–7. doi:10.1152/ajprenal.00441.2012.
- Mowry FE, Peaden SC, Stern JE, Biancardi VC. TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats. Pharmacol Res. 2021;174:105877. doi:10.1016/j.phrs.2021.105877.
- Gil FZ, Lucas SR, Gomes GN, Cavanal Mde F, Coimbra TM. Effects of intrauterine food restriction and long-term dietary supplementation with L-arginine on age-related changes in renal function and structure of rats. Pediatr Res. 2005;57(5 Part 1):724–31. doi:10.1203/01.PDR.0000159514.06939.7E.
- Trofimiuk E, Wielgat P, Braszko JJ. Candesartan, angiotensin II type 1 receptor blocker is able to relieve age-related cognitive impairment. Pharmacol Rep. 2018;70:87–92. doi:10.1016/j.pharep.2017.07.016.
- Renke G, Starling-Soares B, Baesso T, Petronio R, Aguiar D, Paes R. Effects of vitamin D on cardiovascular risk and oxidative stress. Nutrients. 2023;15(3):769. doi:10.3390/nu15030769.
- Phillips EA, Hendricks N, Bucher M, Maloyan A. Vitamin D supplementation improves mitochondrial function and reduces inflammation in placentae of obese women. Front Endocrinol (Lausanne). 2022;13:893848. doi:10.3389/fendo.2022.893848.
- Yao T, Ying X, Zhao Y, Yuan A, He Q, Tong H, Ding S, Liu J, Peng X, Gao E, et al. Vitamin D receptor activation protects against myocardial reperfusion injury through inhibition of apoptosis and modulation of autophagy. Antioxid Redox Signal. 2015;22:633–50.
- Frame AA, Wainford RD. Mechanisms of altered renal sodium handling in age-related hypertension. Am J Physiol Renal Physiol. 2018;315:F1–F6. doi:10.1152/ajprenal.00594.2017.
- Liu D, Gao L, Roy SK, Cornish KG, Zucker IH. Role of oxidant stress on AT1 receptor expression in neurons of rabbits with heart failure and in cultured neurons. Circ Res. 2008;103:186–93. doi:10.1161/CIRCRESAHA.108.179408.
- Zhao M, Wang Y, Li L, Liu S, Wang C, Yuan Y, Yang G, Chen Y, Cheng J, Lu Y, et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021;11(4):1845–63. doi:10.7150/thno.50905.
- Esteras N, Abramov AY. Nrf2 as a regulator of mitochondrial function: energy metabolism and beyond. Free Radic Biol Med. 2022;189:136–53. doi:10.1016/j.freeradbiomed.2022.07.013.
- Fuchs FD, Whelton PK. High blood pressure and cardiovascular disease. Hypertension. 2020;75(2):285–92. doi:10.1161/HYPERTENSIONAHA.119.14240.
- Balasubramanian P, Branen L, Sivasubramanian MK, Monteiro R, Subramanian M. Aging is associated with glial senescence in the brainstem - implications for age-related sympathetic overactivity. Aging (Albany NY). 2021;13(10):13460–73. doi:10.18632/aging.203111.
- Berenyiova A, Balis P, Kluknavsky M, Bernatova I, Cacanyiova S, Puzserova A, Murdoch C. Age- and hypertension-related changes in NOS/NO/sGC-derived vasoactive control of rat thoracic aortae. Oxid Med Cell Longev. 2022;2022:1–13. doi:10.1155/2022/7742509.
- Chi C, Li DJ, Jiang YJ, Tong J, Fu H, Wu YH, Shen FM. Vascular smooth muscle cell senescence and age-related diseases: state of the art. Biochim Biophys Acta Mol Basis Dis. 2019;1865(7):1810–21. doi:10.1016/j.bbadis.2018.08.015.
- Pasanen L, Launonen H, Siltari A, Korpela R, Vapaatalo H, Salmenkari H, Forsgard RA. Age-related changes in the local intestinal renin-angiotensin system in normotensive and spontaneously hypertensive rats. J Physiol Pharmacol. 2019;70(2). doi:10.26402/jpp.2019.2.03.
- Fang Y, Chen B, Liu Z, Gong AY, Gunning WT, Ge Y, Malhotra D, Gohara AF, Dworkin LD, Gong R. Age-related GSK3β overexpression drives podocyte senescence and glomerular aging. J Clin Invest. 2022;132(4). doi:10.1172/JCI141848.
- Brunt VE, Gioscia-Ryan RA, Casso AG, VanDongen NS, Ziemba BP, Sapinsley ZJ, Richey JJ, Zigler MC, Neilson AP, Davy KP, et al. Trimethylamine-N-Oxide promotes age-related vascular oxidative stress and endothelial dysfunction in mice and healthy humans. Hypertension. 2020;76(1):101–12. doi:10.1161/HYPERTENSIONAHA.120.14759.
- Pushpakumar S, Ren L, Juin SK, Majumder S, Kulkarni R, Sen U. Methylation-dependent antioxidant-redox imbalance regulates hypertensive kidney injury in aging. Redox Biol. 2020;37:101754. doi:10.1016/j.redox.2020.101754.
- Wei T, Huang G, Gao J, Huang C, Sun M, Wu J, Bu J, Shen W. Sirtuin 3 deficiency accelerates hypertensive cardiac remodeling by impairing angiogenesis. J Am Heart Assoc. 2017;6. doi:10.1161/JAHA.117.006114.
- Chugh G, Lokhandwala MF, Asghar M. Oxidative stress alters renal D1 and AT1 receptor functions and increases blood pressure in old rats. Am J Physiol Renal Physiol. 2011;300(1):F133–138. doi:10.1152/ajprenal.00465.2010.
- Dan Dunn J, Alvarez LA, Zhang X, Soldati T. Reactive oxygen species and mitochondria: a nexus of cellular homeostasis. Redox Biol. 2015;6:472–85. doi:10.1016/j.redox.2015.09.005.
- Zhang B, Pan C, Feng C, Yan C, Yu Y, Chen Z, Guo C, Wang X. Role of mitochondrial reactive oxygen species in homeostasis regulation. Redox Rep. 2022;27:45–52. doi:10.1080/13510002.2022.2046423.
- Djousse L, Cook NR, Kim E, Bodar V, Walter J, Bubes V, Luttmann-Gibson H, Mora S, Joseph J, Lee IM, et al. Supplementation with vitamin D and omega-3 fatty acids and incidence of heart failure hospitalization: VITAL-Heart failure. Circulation. 2020;141(9):784–86. doi:10.1161/CIRCULATIONAHA.119.044645.
- Chen S, Sun Y, Agrawal DK. Vitamin D deficiency and essential hypertension. J Am Soc Hypertens. 2015;9:885–901. doi:10.1016/j.jash.2015.08.009.
- Carbone F, Liberale L, Libby P, Montecucco F. Vitamin D in atherosclerosis and cardiovascular events. Eur Heart J. 2023;44:2078–94. doi:10.1093/eurheartj/ehad165.
- Jean G, Souberbielle JC, Chazot C. Vitamin D in chronic kidney disease and dialysis patients. Nutrients. 2017;9(4):328. doi:10.3390/nu9040328.
- Deng X, Cheng J, Shen M. Vitamin D improves diabetic nephropathy in rats by inhibiting renin and relieving oxidative stress. J Endocrinol Invest. 2016;39(6):657–66. doi:10.1007/s40618-015-0414-4.
- Vieth R. Vitamin D supplementation: cholecalciferol, calcifediol, and calcitriol. Eur J Clin Nutr. 2020;74(11):1493–97. doi:10.1038/s41430-020-0697-1.
- Wee CL, Mokhtar SS, Singh KKB, Yahaya S, Leung SWS, Rasool AHG, Ghosh R. Calcitriol supplementation ameliorates microvascular endothelial dysfunction in Vitamin D-deficient diabetic rats by upregulating the vascular eNOS protein expression and reducing oxidative stress. Oxid Med Cell Longev. 2021;2021:1–11. doi:10.1155/2021/3109294.
- Xu ML, Yu XJ, Zhao JQ, Du Y, Xia WJ, Su Q, Du MM, Yang Q, Qi J, Li Y, et al. Calcitriol ameliorated autonomic dysfunction and hypertension by down-regulating inflammation and oxidative stress in the paraventricular nucleus of SHR. Toxicol Appl Pharmacol. 2020;394:114950.
- Peoples JN, Saraf A, Ghazal N, Pham TT, Kwong JQ. Mitochondrial dysfunction and oxidative stress in heart disease. Experiment Molecul Med. 2019;51(12):1–13. doi:10.1038/s12276-019-0355-7.
- Mikhed Y, Daiber A, Steven S. Mitochondrial oxidative stress, mitochondrial DNA damage and their role in age-related vascular dysfunction. Int J Mol Sci. 2015;16:15918–53. doi:10.3390/ijms160715918.
- Xu P, Lin B, Deng X, Huang K, Zhang Y, Wang N. VDR activation attenuates osteoblastic ferroptosis and senescence by stimulating the Nrf2/GPX4 pathway in age-related osteoporosis. Free Radic Biol Med. 2022;193(Pt 2):720–35. doi:10.1016/j.freeradbiomed.2022.11.013.