Impact of Renal Denervation on Circadian Variations of Blood Pressure and Clock Gene Expression in Spontaneously Hypertensive Rats

Impact of Renal Denervation on Circadian Variations of Blood Pressure and Clock Gene Expression in Spontaneously Hypertensive Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Impact of Renal Denervation on Circadian Variations of Blood Pressure and Clock Gene Expression in Spontaneously Hypertensive Rats Guan-Ying Yang, Shi-Kun Chen, Hong-Yin Chen, Ruo-Nan Liu, Jie-Ying Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6164221/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hypertension is often associated with elevated nighttime blood pressure (BP), a significant risk factor for cardiovascular and cerebrovascular diseases. This study explores the effects of renal denervation (RDN) on circadian BP rhythms and clock gene expression in spontaneously hypertensive rats (SHR). Ten-week-old SHR were randomized into RDN and sham surgery (Sham) groups, with Wistar-Kyoto rats (WKY) as controls. BP was measured at rest (14:00) and during activity (02:00) biweekly, and BP variability was analyzed. RDN significantly reduced BP, particularly during the resting phase, thereby enhancing circadian BP variation. The Sham group displayed minimal circadian variation in plasma and renal norepinephrine levels, whereas the RDN group exhibited an overall reduction in norepinephrine, with lower levels during rest than during activity. Furthermore, the Sham group showed no significant circadian variation in the renin-angiotensin-aldosterone system (RAS), RDN restored circadian rhythms in ACE1, Ang II, ACE2, and Ang1-7. Additionally, the Sham group demonstrated consistently high renal BMAL1 protein expression, whereas RDN exhibited reduced BMAL1 expression during the resting phase, indicating restored circadian variation. These findings suggest that RDN not only lowers BP but also improves its circadian rhythm, likely through modulation of sympathetic nervous activity, the RAS system, and the circadian clock gene BMAL1. Health sciences/Medical research/Experimental models of disease Health sciences/Medical research/Translational research Renal denervation Hypertension Circadian variation BMAL1 RAS system Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Hypertension represents a predominant risk factor contributing to the global incidence and mortality of cardiovascular diseases( 1 ). Epidemiological data indicate that approximately 33% of the world's population, equivalent to 2.64 billion individuals among 8 billion, are affected by hypertension, thereby establishing it as a substantial global public health concern( 2 , 3 ). The circadian rhythm, an endogenous biological oscillator operating on an approximately 24-hour cycle, governs various physiological parameters including blood pressure, heart rate, and body temperature, all of which demonstrate distinct circadian variations( 4 , 5 ). The physiological blood pressure rhythm typically manifests as an elevation upon morning awakening, maintenance during diurnal activity, and subsequent nocturnal reduction (with nighttime blood pressure averaging 10%-20% lower than daytime measurements)( 6 ). Based on nocturnal blood pressure variations, circadian blood pressure patterns can be classified into four distinct categories: dipper (nocturnal blood pressure reduction > 10%), extreme dipper (nocturnal reduction > 20%), non-dipper (nocturnal reduction ≤ 10%), and reverse dipper (nocturnal blood pressure exceeding daytime levels)( 7 , 8 ). Clinical observations have demonstrated a higher prevalence of abnormal blood pressure rhythms among hypertensive patients( 9 , 10 ), particularly in cases of resistant hypertension where non-dipper patterns are more pronounced( 11 ). Notably, non-dipper hypertensive patients exhibit more severe target organ damage compared to their dipper counterparts( 12 , 13 ). The molecular regulation of circadian rhythms involves central clock genes located in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral clock genes distributed throughout various tissues( 14 , 15 ). These biological clocks are primarily regulated through transcription-translation feedback loops comprising both positive and negative regulatory clock genes( 4 , 16 ). The aryl hydrocarbon receptor nuclear translocator-like protein (BMAL1), alternatively designated as Arntl3 in murine models and MOP3 in humans, serves as a principal regulator of the mammalian molecular clock( 17 , 18 ). Genetic ablation of BMAL1 has been shown to disrupt 24-hour activity patterns, a fundamental measure of circadian output( 19 ). Peripheral circadian clock genes demonstrate tissue-specific expression patterns, with aortic BMAL1 playing a crucial role in the circadian regulation of blood pressure and heart rate( 20 ). Furthermore, BMAL1 in perivascular adipose tissue modulates resting-phase blood pressure through transcriptional regulation of angiotensinogen( 21 ). However, the specific role of renal BMAL1 in circadian blood pressure regulation remains to be fully elucidated, despite the kidney's established importance in blood pressure homeostasis. Beyond genetic regulation, the sympathetic nervous system contributes significantly to the circadian modulation of blood pressure( 22 ). Scientific evidence confirms the association between elevated morning blood pressure and sympathetic nervous system activity( 23 ). In patients with primary hypertension, nocturnal norepinephrine levels exhibit characteristic elevation during sleep( 24 , 25 ). Experimental studies utilizing 6-hydroxydopamine-induced sympathectomy in Wistar rats have demonstrated the abolition of circadian blood pressure rhythms and significant reduction in heart rate variability( 26 ). Renal denervation (RDN) has emerged as a promising therapeutic intervention for hypertension in recent years, primarily through modulation of sympathetic nervous system activity( 27 ). In metabolic syndrome rat models, RDN has been shown to restore normal circadian blood pressure patterns( 28 ). Nevertheless, the efficacy of RDN in regulating circadian blood pressure in spontaneously hypertensive rats remains to be determined. The present study aims to investigate the effects of RDN on circadian blood pressure regulation and the expression patterns of the core clock gene BMAL1 in spontaneously hypertensive rats. Materials and Methods 1. Animals And Ethics Approval Eight-week-old male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats purchased by Vital River Laboratory Animal Technology Company (Beijing, China), with the animal experiment quality certificate number: SCXK (Jing) 2021-0006. The laboratory utilized artificial lighting, with a 12-hour light/dark cycle. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. This study was conducted in accordance with the ARRIVE guidelines. All animal handling procedures comply with the regulations have been approved by the Experimental Animal Ethics Committee of Fujian Medical University, with the ethical review number: IACUCFJMU2022-0569. 2. Surgical Procedures Following a two-week period of adaptive feeding, the SHR rats were randomly assigned to either the renal denervation (RDN) group or the sham surgery group. The rats were anesthetized via intraperitoneal injection of carbamate (800 mg/kg) and α-chloralose (40 mg/kg). A dorsal-lateral incision was made to expose the renal artery and vein sheath. Under a dissecting microscope, the renal artery and vein were carefully isolated from surrounding connective tissue, and visible nerve fibers were severed. Sterile gauze soaked in a 10% phenol- ethanol solution was applied to the renal vessels for 10 minutes to perform renal sympathetic nerve ablation, while the sham group received saline application to the renal artery, and WKY rats underwent no treatment. The contralateral side was treated similarly. 3. Blood Pressure Measurement Non-invasive tail-cuff plethysmography (BP-300, Chengdu Taimeng Science and Technology Company, China) was used to measure the resting (14:00) and active (02:00) systolic blood pressure (SBP) of each rat both preoperatively and biweekly postoperatively until the conclusion of the experiment (16 weeks of age). The blood pressure variability was calculated using the formula: variability = (active period blood pressure - resting period blood pressure) / active period blood pressure. To register the pulsations of the tail artery, the rats were kept in a thermostatic chamber at 30℃ for 10 minutes before each measurement, averaged from three readings. 4. Euthanasia Process, Sample Collection and Preservation At 16 weeks of age, after measuring the blood pressure, each group of rats were anesthetized at 14:00 and 02:00 after intraperitoneal injection of carbamate (800 mg/kg) and α-chloralose (40 mg/kg) for anesthesia. Euthanasia is by cervical dislocation under anesthesia. Before the rats were euthanized, blood was collected from the abdominal aorta, after which the kidneys were harvested. One kidney was fixed in formaldehyde, while the other was rapidly frozen in liquid nitrogen and subsequently stored at -80°C for future analysis. Plasma samples were isolated from whole blood and stored at -80°C. 5. Detection of norepinephrine (NE)、Ang II and Angiotensin-(–) in Kidney and Plasma 50mg samples were taken from renal cortex and added to 0.5ml Phosphate Buffered Saline (PBS solution). The mixture was homogenized using an electric homogenizer at a rate of 7000 r/min for 15 seconds, and this procedure was repeated thrice. The obtained homogenate was then centrifuged at a rate of 3000 r/min for 20 minutes at 4°C, and the supernatant was collected. NE ELISA kits (YJ1002827), Ang II ELISA kits (YJ002823), Ang1-7 ELISA kits (YJ920741) all from Enzyme-Link Biotechnology Company in China were used to measure the NE, Ang II, Ang1-7 content in the plasma and kidney, following the instructions outlined in the kit manual. 6. Western Blot Analysis Kidney cortex tissues were ground using RIPA lysate (P0013B, beyotime, China) containing protease inhibitors (P1050, beyotime, China), and protein concentrations were determined using the BCA Protein Concentration Measurement Kit (AR1189, BOSTER, China) to determine protein concentration. Protein expression was analyzed by Western blot analysis as described previously( 29 ). Antibodies and dilutions are as follows: ACE1, 1;1000 dilution (A11357, ABclonal, China), ACE2, 1:1000 dilution (A12737, ABclonal, China), AT1R, 1:1000 dilution (25343-1-AP, Proteintech, China), MasR, 1:800 dilution (sc-390453, Santa Cruz, America). 7. Immunohistochemical (IHC) staining Kidneys were fixed using paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4µm with a slicer. Then, the slides were deparaffinized and incubated with citrate buffer, after being treated with H 2 O 2 to inhibit endogenous peroxidase, the sections were blocked with diluted BSA, the Tyrosine hydroxylase antibodies were used for IHC staining. 8. Statistical Analysis Data analysis was conducted using the software SPSS 25, Student’s t-test or one-way ANOVA followed by Fisher's least significant difference (LSD) post hoc test was used to assess the differences between groups. All data are presented as mean ± SEM. P < 0.05 was considered statistically significant. Active phase (Night) of WKY, Sham and RDN groups compared to their respective resting phase (Day), a P < 0.05; the overall level of Sham group compared to WKY group, b P < 0.05; the overall level of RDN group compared to Sham group, c P < 0.05. Results Impact of RDN on circadian variation of blood pressure At baseline assessment conducted at 10 weeks of age, no statistically significant differences in systolic blood pressure (SBP) were observed between the Sham group and the RDN group during either the resting or active phases. However, both experimental groups demonstrated significantly elevated SBP values compared to the WKY control group. RDN intervention resulted in a significant reduction of SBP in SHR during both circadian phases, with maximal therapeutic efficacy observed at 12 weeks post-intervention. Comprehensive quantitative data are presented in Tables 1 and 2 . Table 1 Changes in resting systolic blood pressure (SBP/mm Hg) (n = 8) Age(weeks) WKY Sham RDN 10 129.9 ± 7.6 176.8 ± 6.9 b 178.1 ± 6.7 b 12 128.5 ± 4.8 181.9 ± 9.5 b 145.0 ± 7.1 bc 14 132.6 ± 4.4 196.4 ± 9.7 b 151.2 ± 6.9 bc 16 131.0 ± 5.2 197.8 ± 7.6 b 152.0 ± 6.8 bc 1mm Hg = 0.133kPa. Table 2 Changes in active systolic blood pressure (SBP/mm Hg) (n = 8) Age(weeks) WKY Sham RDN 10 145.1 ± 6.8 189.4 ± 8.3 b 190.0 ± 9.3 c 12 141.6 ± 5.6 193.3 ± 11.0 b 160.2 ± 6.1 bc 14 146.8 ± 4.2 207.8 ± 11.9 b 164.7 ± 5.9 bc 16 146.0 ± 5.5 206.5 ± 7.6 b 166.0 ± 3.6 bc The WKY control group maintained stable blood pressure variability at approximately 10% throughout the experimental period. Initial measurements at 10 weeks of age revealed no significant differences in blood pressure variability between the Sham and RDN groups. However, by 12 weeks of age, the RDN group demonstrated a significant enhancement in circadian blood pressure rhythm, exhibiting a variability of 9.05 ± 2.44%, which was markedly higher than that of the Sham group (5.84 ± 1.68%; p < 0.05). This improvement in circadian regulation persisted at 16 weeks of age, with the RDN group maintaining a blood pressure variability of 8.49 ± 2.66%, significantly exceeding the Sham group's variability of 4.18 ± 2.83% (p < 0.05). Detailed statistical data are provided in Table 3 . Table 3 Changes in blood pressure variability (%) (n = 8) Age(weeks) WKY Sham RDN 10 10.50 ± 2.96 6.43 ± 3.09 b 5.91 ± 2.12 b 12 9.26 ± 1.19 5.84 ± 1.68 b 9.05 ± 2.44 c 14 9.62 ± 2.72 5.42 + 2.68 b 8.25 ± 1.40 16 10.26 ± 2.05 4.18 ± 2.83 b 8.49 ± 2.66 c All data are presented as mean ± SEM. P < 0.05 was considered statistically significant. Active phase (Night) of WKY, Sham and RDN groups compared to their respective resting phase (Day), a P < 0.05; the overall level of Sham group compared to WKY group, b P < 0.05; the overall level of RDN group compared to Sham group, c P < 0.05. Impact of RDN on the circadian variations in sympathetic nerve activity Tyrosine hydroxylase (TH), a marker for sympathetic nerve fibers, serves as a reliable indicator of sympathetic nervous system excitability. Immunohistochemical staining was employed to evaluate TH protein expression in the kidneys of experimental rats during both resting and active phases. The Sham group exhibited elevated TH expression levels during both phases, whereas the RDN group demonstrated an overall reduction in TH expression, with daytime levels significantly lower than nighttime levels (Fig. 1 A). To further investigate circadian differences in sympathetic nerve activity, norepinephrine (NE) levels were assessed in both renal tissue and plasma. The Sham group exhibited significantly higher overall levels of renal and plasma NE compared to WKY rats, with no notable circadian variation. In contrast, RDN treatment reduced overall NE content in hypertensive rats, particularly during the resting phase, thereby enhancing the circadian variation of NE levels (Fig. 1 B, C). Impact of RDN on the circadian variations of Ang II and Ang1-7 in the kidneys and plasma. Ang II and Ang1-7 are the primary bioactive components of the RAS. In WKY rats, both renal and plasma Ang II exhibited a distinct circadian rhythm, with significantly elevated levels during the active phase compared to the resting phase. The Sham group displayed overall higher levels of renal and plasma Ang II compared to the WKY group, with no discernible circadian variation. RDN effectively reduced Ang II levels, particularly during the resting phase, and restored circadian rhythmicity in Ang II expression (Fig. 2 A, B). Similarly, WKY rats demonstrated a circadian rhythm in renal and plasma Ang1-7 levels. In the Sham group, no significant circadian variation in Ang1-7 was observed, and overall expression levels were relatively low. Following RDN treatment, SHR exhibited a pronounced circadian variation in renal and plasma Ang1-7, with levels during the active phase being lower than those during the resting phase, and overall expression surpassing that of the Sham group (Fig. 2 C, D). Impact of RDN on the circadian variations of RAS-related proteins in rat kidneys. In the Sham group of SHR, renal angiotensin-converting enzyme (ACE1) levels were significantly higher than those in the WKY group, with no detectable circadian variation. In contrast, the WKY group exhibited a pronounced circadian pattern, characterized by lower levels during the day and higher levels at night. RDN significantly reduced ACE1 levels in SHR, particularly during the resting phase (Fig. 3 A, B). The Sham group also exhibited markedly lower levels of angiotensin-converting enzyme 2 (ACE2), which catalyzes the conversion of Ang II to the vasodilatory peptide Ang1-7, compared to the WKY group, with consistently low levels during both phases. RDN significantly enhanced overall ACE2 expression, particularly restoring its levels during the resting phase (Fig. 3 A, C). Collectively, these findings indicate that RDN restored circadian rhythmicity in renal ACE1 and ACE2 expression in hypertensive rats. Regarding the angiotensin II type 1 receptor (AT1R), overall expression in the Sham group was significantly higher than in the WKY group. RDN resulted in an overall reduction in AT1R expression in SHR, with no significant circadian variation observed in any group (Fig. 3 A, D). Similarly, the expression of the angiotensin 1–7 receptor (MasR) showed no significant circadian variation across groups. However, the Sham group exhibited significantly lower overall MasR expression compared to the WKY group, while RDN increased MasR protein levels in SHR (Fig. 3 A, E). Impact of RDN on the circadian variation of BMAL1 protein In WKY rats, BMAL1 protein expression exhibited a significant circadian rhythm, with lower levels during the day and higher levels at night. In contrast, the hypertensive Sham group displayed consistently high BMAL1 expression throughout both phases, with no discernible circadian variation. RDN treatment reduced daytime BMAL1 expression in hypertensive rats, thereby restoring circadian rhythmicity (Fig. 4 ). Discussion Our findings demonstrate that hypertensive rats exhibit significantly attenuated circadian variations in blood pressure compared to normotensive controls, with this reduction becoming more pronounced with advancing age. RDN not only effectively reduced overall blood pressure in hypertensive rats but also restored circadian blood pressure rhythmicity. This improvement was associated with the modulation and stabilization of circadian sympathetic activity, as well as the restoration of circadian variations in the renal RAS and the core clock gene BMAL1. Notably, this study provides the first evidence of a mechanistic link between sympathetic nervous system activity and BMAL1 regulation in the context of hypertension. Blood pressure is inherently characterized by a well-defined circadian rhythm( 7 , 30 ), and disruptions in this rhythm are strongly associated with the pathogenesis of cardiovascular( 31 , 32 ), cerebral( 33 , 34 ), and renal diseases( 35 , 36 ). Clinical studies have demonstrated that hypertensive patients with reverse dipper or non-dipper patterns experience more severe structural and functional cardiac damage compared to those with dipper patterns( 37 , 38 ). Specifically, non-dipper hypertension has been linked to impaired left ventricular systolic function, as evidenced by reduced myocardial strain( 39 ). Furthermore, among hypertensive patients with a history of cerebral infarction, the prevalence of non-dipper hypertension is approximately 62.16%, significantly higher than the 35.85% observed in patients without such sequelae( 40 ). Aging further exacerbates these patterns, with hypertensive patients increasingly exhibiting dipper and non-dipper profiles as they age( 41 ). Consistent with these clinical observations, our experimental data revealed that hypertensive rats exhibit diminished circadian blood pressure variations, which progressively decline with age. Importantly, our study highlights that RDN not only achieves blood pressure reduction but also restores physiological circadian blood pressure rhythms, offering a potential therapeutic strategy for managing hypertension-related circadian dysregulation. The critical role of the sympathetic nervous system in regulating the circadian rhythm of blood pressure is well-documented( 42 , 43 ). Clinical studies have demonstrated that RDN effectively modulates circadian blood pressure profiles, improving circadian rhythmicity and reducing cardiovascular risk in patients with resistant hypertension( 44 ). For instance, in unilateral nephrectomized patients, the prevalence of dipper patterns decreased, while non-dipper and riser patterns increased post-nephrectomy, highlighting the influence of renal innervation on circadian blood pressure regulation( 45 ). Preclinical studies further support these findings; for example, long-term RDN has been shown to normalize circadian blood pressure rhythms in metabolic syndrome rats, a phenomenon associated with enhanced urinary sodium excretion and suppression of renal Na+-Cl cotransporter upregulation( 28 ). While our findings align with these observations by demonstrating that RDN restores circadian blood pressure rhythms in SHRs, our study uniquely identifies that this effect is primarily mediated through the modulation of sympathetic nervous system activity and its interaction with the core clock gene BMAL1. This distinction underscores the pivotal role of sympathetic overactivation, a well-established contributor to hypertension, in disrupting circadian blood pressure regulation. Clock genes are fundamental to circadian regulation, and BMAL1, as a core component of the molecular clock, plays a critical role in maintaining blood pressure circadian rhythms( 17 , 18 ). BMAL1 exhibits tissue- and sex-specific effects on blood pressure regulation. For instance, mice lacking smooth muscle BMAL1, but not cardiomyocyte BMAL1, exhibit moderately reduced blood pressure and diminished circadian amplitude( 46 ). Similarly, collecting duct-specific deletion of BMAL1 lowers blood pressure in male mice but not in females, highlighting the gene's sex-dependent regulatory role( 47 ). While these studies elucidate the importance of BMAL1 in blood pressure regulation, they primarily focus on normotensive models and do not address its circadian expression patterns in hypertensive conditions. Our study addresses this gap by demonstrating that SHR exhibit elevated renal BMAL1 expression with blunted day-night variations compared to normotensive WKY rats. RDN not only reduced overall renal BMAL1 expression but also restored its circadian rhythmicity in hypertensive rats. The interplay between BMAL1 and the sympathetic nervous system further underscores its regulatory role. Previous research has shown that BMAL1 knockout mice exhibit reduced expression of phenylethanolamine N-methyltransferase, a key enzyme in catecholamine synthesis, leading to decreased epinephrine and norepinephrine levels( 48 ). This suggests that BMAL1 positively regulates catecholamine production, linking circadian gene activity to sympathetic tone. In our study, the observed changes in BMAL1 expression following RDN may reflect a negative feedback mechanism by which the sympathetic nervous system modulates BMAL1 activity, thereby influencing circadian blood pressure rhythms. These findings provide novel insights into the complex interplay between sympathetic activity, circadian gene regulation, and blood pressure control in hypertension. The renin-angiotensin system is a pivotal regulatory mechanism in blood pressure homeostasis, exhibiting distinct circadian variations( 49 ). In transgenic hypertensive (TGR) rats, plasma ACE1 activity is significantly elevated and demonstrates an inverted rhythm compared to normotensive controls( 50 ). In rats subjected to restraint stress, Ang II infusion has been shown to reverse the 24-hour blood pressure rhythm( 51 ). Clinical studies have also highlighted the role of RAS in circadian dysregulation, patients with non-dipper primary hypertension exhibit significantly elevated nighttime urinary angiotensinogen (U-AGT) levels, indicative of nocturnal RAS activation( 10 ). In the present study, we investigated the circadian variations of renal and plasma RAS components. Our findings demonstrate that RDN significantly reduced Ang II levels in both renal tissue and plasma, as well as downregulated renal ACE1 expression, particularly during the daytime, thereby enhancing circadian rhythmicity. Concurrently, RDN increased Ang1-7 levels in the kidneys and plasma, along with upregulating renal ACE2 expression, especially during the nighttime, restoring physiological circadian differences. These results suggest that the rebalancing of the ACE1/Ang II/AT1R and ACE2/Ang1-7/MASR axes represents a key mechanism through which RDN modulates circadian blood pressure variations. Conclusions In summary, RDN effectively ameliorated circadian blood pressure dysregulation in hypertensive rats, likely through the modulation of sympathetic nervous system activity, the restoration of clock gene BMAL1 expression, and the enhancement of circadian dynamics in the ACE1/Ang II/AT1R and ACE2/Ang1-7/MASR pathways. These findings suggest that achieving both blood pressure reduction and the restoration of circadian blood pressure rhythms could represent a novel therapeutic goal in hypertension management. Additionally, timing RDN interventions to coincide with periods of peak sympathetic activity may further optimize clinical outcomes. However, this study is not without limitations. The reliance on animal models, while informative, cannot fully replicate the complexities of human hypertension. Moreover, blood pressure regulation is a multifactorial process involving numerous interconnected systems, with the local renal RAS representing only one of many contributing factors. Similarly, while BMAL1 plays a critical role in circadian blood pressure regulation, it is likely that other rhythmic genes and pathways also contribute to this intricate physiological process. Future studies should explore these additional mechanisms and validate these findings in human populations to advance our understanding of hypertension and its treatment. Declarations CONFLICT OF INTEREST The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. FUNDING This work was supported by the Science and Technology Project of Fujian Province, China (No.2020Y0023) and Fujian Provincial Finance Project (BPB-2023SJZ). Author Contribution JZ-S and WQ-C designed and directed the study. GY-Y and SK-C wrote and revised the manuscript and carried out main experiments, HY-C and RN-L carried out this animal experiment, including animal feeding, model building and blood pressure measuring. JY-L, JS-D and SQ-L calculated and analyzed the experimental data. All authors reviewed the manuscript Acknowledgement We gratefully acknowledge all the participants in this study and staff members of the follow-up team. Data Availability The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors. References Fuchs FD, and Whelton PK . High Blood Pressure and Cardiovascular Disease. Hypertension (Dallas, Tex : 1979) 75: 285-292, 2020. Mills KT, Stefanescu A, and He J . The global epidemiology of hypertension. Nature reviews Nephrology 16: 223-237, 2020. 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Altered Circadian Timing System-Mediated Non-Dipping Pattern of Blood Pressure and Associated Cardiovascular Disorders in Metabolic and Kidney Diseases. Int J Mol Sci 19: 2018. C S, L G, P F, Z W, and L J . The correlation between the circadian rhythms of blood pressure and heart rate and the cardiac structure and function in patients with hypertension. Chinese Hypertension 26: 535-540, 2018. L L, SL L, L A, Y Z, and S C . The correlation between the circadian rhythm of blood pressure in patients with hypertension and cardiac structure and function. Journal of Integrated Traditional and Western Medicine for Cardiovascular Diseases 22: 1081-1085, 2024. Cakal S, and Cakal B . Evaluation of global left ventricular systolic function in dipper and newly diagnosed nondipper hypertensive patients. European heart journal 42: 2021. Z J, Z X, W L, and W X . The relationship between circadian variations in blood pressure, heart rate variability, and the occurrence of sequelae following cerebral infarction in elderly patients with hypertension. . Hainan Medicine 34: 3064-3068, 2023. D L, W Y, X J, G Y, and W C . The relationship between circadian rhythms of blood pressure and time of day in elderly patients with hypertension. Chinese Hypertension 31: 764-768, 2023. Head GA . The sympathetic nervous system in hypertension: assessment by blood pressure variability and ganglionic blockade. J Hypertens 21: 1619-1621, 2003. Grassi G, Bombelli M, Seravalle G, Dell'Oro R, and Quarti-Trevano F . Diurnal blood pressure variation and sympathetic activity. Hypertension research : official journal of the Japanese Society of Hypertension 33: 381-385, 2010. Moiseeva A, Caraus A, Abras M, Ciobanu N, Moscalu V, Surev A, Cociu M, Caraus M, and Falkovskaya A . THE EFFECT OF RENAL DENERVATION ON THE CIRCADIAN BLOOD PRESSURE PATTERN IN PATIENTS WITH RESISTANT HYPERTENSION %J Journal of Hypertension. 40: e245-e245, 2022. Ohashi N, Isobe S, Ishigaki S, Suzuki T, Motoyama D, Sugiyama T, Nagata M, Kato A, Ozono S, and Yasuda H . The Effects of Unilateral Nephrectomy on Blood Pressure and Its Circadian Rhythm. Internal medicine (Tokyo, Japan) 55: 3427-3433, 2016. Zhongwen X, Wen S, Shu L, Guogang Z, Karyn E, A. SE, Mellani L, Zhenheng G, and C. GM . Role of Smooth Muscle BMAL1 in Time-of-day Vasoconstriction Variation and Blood Pressure Circadian Rhythm %J HYPERTENSION. 64: 2014. Zhang D, Jin C, Obi IE, Rhoads MK, Soliman RH, Sedaka RS, Allan JM, Tao B, Speed JS, Pollock JS, and Pollock DM . Loss of circadian gene Bmal1 in the collecting duct lowers blood pressure in male, but not female, mice. American journal of physiology Renal physiology 318: F710-f719, 2020. Curtis AM, Cheng Y, Kapoor S, Reilly D, Price TS, and Fitzgerald GA . Circadian variation of blood pressure and the vascular response to asynchronous stress. Proc Natl Acad Sci U S A 104: 3450-3455, 2007. Takimoto-Ohnishi E, and Murakami K . Renin-angiotensin system research: from molecules to the whole body. The journal of physiological sciences : JPS 69: 581-587, 2019. Lemmer B, Witte K, Enzminger H, Schiffer S, and Hauptfleisch S . Transgenic TGR(mREN2)27 rats as a model for disturbed circadian organization at the level of the brain, the heart, and the kidneys. Chronobiology international 20: 711-738, 2003. Schiffer S, Pummer S, Witte K, and Lemmer B . Cardiovascular regulation in TGR(mREN2)27 rats: 24h variation in plasma catecholamines, angiotensin peptides, and telemetric heart rate variability. Chronobiology international 18: 461-474, 2001. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6164221","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432126389,"identity":"4ddda8d3-6fc1-4d9e-85f5-7c876364ea1e","order_by":0,"name":"Guan-Ying Yang","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guan-Ying","middleName":"","lastName":"Yang","suffix":""},{"id":432126390,"identity":"3dfda58c-7dd8-4ff1-9eb4-d09f2ad3073b","order_by":1,"name":"Shi-Kun Chen","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shi-Kun","middleName":"","lastName":"Chen","suffix":""},{"id":432126391,"identity":"104f78e4-fef9-4060-875e-4b382ad74855","order_by":2,"name":"Hong-Yin Chen","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong-Yin","middleName":"","lastName":"Chen","suffix":""},{"id":432126392,"identity":"0e7896c2-ac21-4e20-be2a-31fabd7b4239","order_by":3,"name":"Ruo-Nan Liu","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruo-Nan","middleName":"","lastName":"Liu","suffix":""},{"id":432126393,"identity":"bd80ba81-ea05-40da-b724-0927c700e976","order_by":4,"name":"Jie-Ying Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie-Ying","middleName":"","lastName":"Li","suffix":""},{"id":432126394,"identity":"1c2a3826-bdfa-42e9-ab64-a723e44a74b8","order_by":5,"name":"Jia-Shi Ding","email":"","orcid":"","institution":"The First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Shi","middleName":"","lastName":"Ding","suffix":""},{"id":432126395,"identity":"da5ddc95-9470-41aa-89fa-247b1b1a3839","order_by":6,"name":"Si-Qi Luo","email":"","orcid":"","institution":"The First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Si-Qi","middleName":"","lastName":"Luo","suffix":""},{"id":432126396,"identity":"9dcd8f4e-96d2-4023-80d5-0bf44cb9d35b","order_by":7,"name":"Wenqin Cai","email":"","orcid":"","institution":"The First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenqin","middleName":"","lastName":"Cai","suffix":""},{"id":432126397,"identity":"4e7150a6-14fb-46c3-a11d-564cfcaa3d31","order_by":8,"name":"Jinzi Su","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYFACHgaGDzZsYKYE0VoYZ6SRqoWZJ42BBC3yM3IPPrZJ4Is2OMB88DYPg10eQS0GN/KSjXMS2HI3HGBLtuZhSC4mrEUix0w69wdIC4+ZNA/DgcQGwg4DarEA28L/jTgtDDeAWhjAWnjYiNNicOZdsmEPUMvMw2zGlnMMkolwWHvuwQc/Eo7l9h1vfnjjTYUdEQ6DgGMMDMxgS4lUDwQ1xCsdBaNgFIyCkQcA06w3tlsLT8AAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Fujian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jinzi","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2025-03-05 16:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6164221/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6164221/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79102478,"identity":"7d8f8b87-221d-4808-9d20-61e36b0bca7d","added_by":"auto","created_at":"2025-03-24 12:30:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":171013,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of renal denervation (RDN) on circadian differences in sympathetic nerve activity. (A) Immunohistochemical staining of TH, (B) the concentration of NE in plasma, (C) the concentration of NE in kidney. A(n=3); B、C( n=6) Day: resting phase; Night: active phase. Compared to the resting phase (Day), \u003csup\u003ea \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; in comparison to the overall WKY group, \u003csup\u003eb \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; and compare to the overall Sham group, \u003csup\u003ec \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6164221/v1/97287cafe0a9cec1f27545d5.png"},{"id":79102476,"identity":"e8934a0f-d286-404b-8e92-905c32ab3269","added_by":"auto","created_at":"2025-03-24 12:30:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":300501,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of RDN on the circadian expression differences of renal and plasma Ang II and Ang1-7 (n=6). (A) Plasma Angiotensin II, (B) Renal Angiotensin II, (C) Plasma Angiotensin 1-7, (D) Renal Angiotensin 1-7. Day: resting phase, Night: active phase. Compared to the resting phase (Day), \u003csup\u003ea \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; in comparison to the overall WKY group, \u003csup\u003eb \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; and compare to the overall Sham group, \u003csup\u003ec \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6164221/v1/e22d54fb6cbcfc07851f1cde.png"},{"id":79101499,"identity":"5a873526-68a5-4cbe-b5c0-266ba80aa238","added_by":"auto","created_at":"2025-03-24 12:22:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155325,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of RDN on the circadian expression of renal RAS components in various groups of rats (n=8). (A) Representative images of ACE1, ACE2, AT1R, and MasR proteins, along with their quantitative analysis. (B) Angiotensin-converting enzyme 1 (ACE1), (C) Angiotensin-converting enzyme 2 (ACE2), (D) Angiotensin II type 1 receptor (AT1R), and (E) Angiotensin 1-7 receptor (MasR). Day: resting phase, Night: active phase. Compared to the resting phase (Day), \u003csup\u003ea \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; in comparison to the overall WKY group, \u003csup\u003eb \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; and compare to the overall Sham group, \u003csup\u003ec \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Original blots are presented in Supplementary Material.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6164221/v1/c049d4d1dc6d069c9bc90cd5.png"},{"id":79101502,"identity":"06dd3570-46dc-475c-bfea-19cbda5b839a","added_by":"auto","created_at":"2025-03-24 12:22:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148543,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of RDN on the circadian expression differences of BMAL1(n=8). Representative Western blot images along with quantitative analysis are presented. Day: Resting phase; Night: Active phase. Compared to the resting phase (Day), \u003csup\u003ea \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; in comparison to the overall WKY group, \u003csup\u003eb \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; and compare to the overall Sham group, \u003csup\u003ec \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Original blots are presented in Supplementary Material.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6164221/v1/261bb84989b890c438c9400c.png"},{"id":84661420,"identity":"3994afd9-0751-42ca-b50f-953387513753","added_by":"auto","created_at":"2025-06-16 04:31:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2665658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6164221/v1/7b189037-dcce-4d14-b2f0-f0d2ec547670.pdf"},{"id":79101498,"identity":"398a7ddf-48ac-4098-bd87-093f17a2415f","added_by":"auto","created_at":"2025-03-24 12:22:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":466429,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6164221/v1/9d3e18c68a095484cceb4dee.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of Renal Denervation on Circadian Variations of Blood Pressure and Clock Gene Expression in Spontaneously Hypertensive Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertension represents a predominant risk factor contributing to the global incidence and mortality of cardiovascular diseases(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Epidemiological data indicate that approximately 33% of the world's population, equivalent to 2.64\u0026nbsp;billion individuals among 8\u0026nbsp;billion, are affected by hypertension, thereby establishing it as a substantial global public health concern(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The circadian rhythm, an endogenous biological oscillator operating on an approximately 24-hour cycle, governs various physiological parameters including blood pressure, heart rate, and body temperature, all of which demonstrate distinct circadian variations(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The physiological blood pressure rhythm typically manifests as an elevation upon morning awakening, maintenance during diurnal activity, and subsequent nocturnal reduction (with nighttime blood pressure averaging 10%-20% lower than daytime measurements)(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Based on nocturnal blood pressure variations, circadian blood pressure patterns can be classified into four distinct categories: dipper (nocturnal blood pressure reduction\u0026thinsp;\u0026gt;\u0026thinsp;10%), extreme dipper (nocturnal reduction\u0026thinsp;\u0026gt;\u0026thinsp;20%), non-dipper (nocturnal reduction\u0026thinsp;\u0026le;\u0026thinsp;10%), and reverse dipper (nocturnal blood pressure exceeding daytime levels)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Clinical observations have demonstrated a higher prevalence of abnormal blood pressure rhythms among hypertensive patients(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), particularly in cases of resistant hypertension where non-dipper patterns are more pronounced(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Notably, non-dipper hypertensive patients exhibit more severe target organ damage compared to their dipper counterparts(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe molecular regulation of circadian rhythms involves central clock genes located in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral clock genes distributed throughout various tissues(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). These biological clocks are primarily regulated through transcription-translation feedback loops comprising both positive and negative regulatory clock genes(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The aryl hydrocarbon receptor nuclear translocator-like protein (BMAL1), alternatively designated as Arntl3 in murine models and MOP3 in humans, serves as a principal regulator of the mammalian molecular clock(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Genetic ablation of BMAL1 has been shown to disrupt 24-hour activity patterns, a fundamental measure of circadian output(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Peripheral circadian clock genes demonstrate tissue-specific expression patterns, with aortic BMAL1 playing a crucial role in the circadian regulation of blood pressure and heart rate(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Furthermore, BMAL1 in perivascular adipose tissue modulates resting-phase blood pressure through transcriptional regulation of angiotensinogen(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, the specific role of renal BMAL1 in circadian blood pressure regulation remains to be fully elucidated, despite the kidney's established importance in blood pressure homeostasis.\u003c/p\u003e \u003cp\u003eBeyond genetic regulation, the sympathetic nervous system contributes significantly to the circadian modulation of blood pressure(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Scientific evidence confirms the association between elevated morning blood pressure and sympathetic nervous system activity(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In patients with primary hypertension, nocturnal norepinephrine levels exhibit characteristic elevation during sleep(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Experimental studies utilizing 6-hydroxydopamine-induced sympathectomy in Wistar rats have demonstrated the abolition of circadian blood pressure rhythms and significant reduction in heart rate variability(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Renal denervation (RDN) has emerged as a promising therapeutic intervention for hypertension in recent years, primarily through modulation of sympathetic nervous system activity(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In metabolic syndrome rat models, RDN has been shown to restore normal circadian blood pressure patterns(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Nevertheless, the efficacy of RDN in regulating circadian blood pressure in spontaneously hypertensive rats remains to be determined. The present study aims to investigate the effects of RDN on circadian blood pressure regulation and the expression patterns of the core clock gene BMAL1 in spontaneously hypertensive rats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Animals And Ethics Approval\u003c/h2\u003e \u003cp\u003eEight-week-old male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats purchased by Vital River Laboratory Animal Technology Company (Beijing, China), with the animal experiment quality certificate number: SCXK (Jing) 2021-0006. The laboratory utilized artificial lighting, with a 12-hour light/dark cycle. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. This study was conducted in accordance with the ARRIVE guidelines. All animal handling procedures comply with the regulations have been approved by the Experimental Animal Ethics Committee of Fujian Medical University, with the ethical review number: IACUCFJMU2022-0569.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. Surgical Procedures\u003c/h3\u003e\n\u003cp\u003eFollowing a two-week period of adaptive feeding, the SHR rats were randomly assigned to either the renal denervation (RDN) group or the sham surgery group. The rats were anesthetized via intraperitoneal injection of carbamate (800 mg/kg) and α-chloralose (40 mg/kg). A dorsal-lateral incision was made to expose the renal artery and vein sheath. Under a dissecting microscope, the renal artery and vein were carefully isolated from surrounding connective tissue, and visible nerve fibers were severed. Sterile gauze soaked in a 10% phenol- ethanol solution was applied to the renal vessels for 10 minutes to perform renal sympathetic nerve ablation, while the sham group received saline application to the renal artery, and WKY rats underwent no treatment. The contralateral side was treated similarly.\u003c/p\u003e\n\u003ch3\u003e3. Blood Pressure Measurement\u003c/h3\u003e\n\u003cp\u003eNon-invasive tail-cuff plethysmography (BP-300, Chengdu Taimeng Science and Technology Company, China) was used to measure the resting (14:00) and active (02:00) systolic blood pressure (SBP) of each rat both preoperatively and biweekly postoperatively until the conclusion of the experiment (16 weeks of age). The blood pressure variability was calculated using the formula: variability = (active period blood pressure - resting period blood pressure) / active period blood pressure. To register the pulsations of the tail artery, the rats were kept in a thermostatic chamber at 30℃ for 10 minutes before each measurement, averaged from three readings.\u003c/p\u003e\n\u003ch3\u003e4. Euthanasia Process, Sample Collection and Preservation\u003c/h3\u003e\n\u003cp\u003eAt 16 weeks of age, after measuring the blood pressure, each group of rats were anesthetized at 14:00 and 02:00 after intraperitoneal injection of carbamate (800 mg/kg) and α-chloralose (40 mg/kg) for anesthesia. Euthanasia is by cervical dislocation under anesthesia. Before the rats were euthanized, blood was collected from the abdominal aorta, after which the kidneys were harvested. One kidney was fixed in formaldehyde, while the other was rapidly frozen in liquid nitrogen and subsequently stored at -80\u0026deg;C for future analysis. Plasma samples were isolated from whole blood and stored at -80\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003e5. Detection of norepinephrine (NE)、Ang II and Angiotensin-(–) in Kidney and Plasma\u003c/h3\u003e\n\u003cp\u003e50mg samples were taken from renal cortex and added to 0.5ml Phosphate Buffered Saline (PBS solution). The mixture was homogenized using an electric homogenizer at a rate of 7000 r/min for 15 seconds, and this procedure was repeated thrice. The obtained homogenate was then centrifuged at a rate of 3000 r/min for 20 minutes at 4\u0026deg;C, and the supernatant was collected. NE ELISA kits (YJ1002827), Ang II ELISA kits (YJ002823), Ang1-7 ELISA kits (YJ920741) all from Enzyme-Link Biotechnology Company in China were used to measure the NE, Ang II, Ang1-7 content in the plasma and kidney, following the instructions outlined in the kit manual.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e6. Western Blot Analysis\u003c/h2\u003e \u003cp\u003eKidney cortex tissues were ground using RIPA lysate (P0013B, beyotime, China) containing protease inhibitors (P1050, beyotime, China), and protein concentrations were determined using the BCA Protein Concentration Measurement Kit (AR1189, BOSTER, China) to determine protein concentration. Protein expression was analyzed by Western blot analysis as described previously(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Antibodies and dilutions are as follows: ACE1, 1;1000 dilution (A11357, ABclonal, China), ACE2, 1:1000 dilution (A12737, ABclonal, China), AT1R, 1:1000 dilution (25343-1-AP, Proteintech, China), MasR, 1:800 dilution (sc-390453, Santa Cruz, America).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e7. Immunohistochemical (IHC) staining\u003c/h3\u003e\n\u003cp\u003eKidneys were fixed using paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4\u0026micro;m with a slicer. Then, the slides were deparaffinized and incubated with citrate buffer, after being treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to inhibit endogenous peroxidase, the sections were blocked with diluted BSA, the Tyrosine hydroxylase antibodies were used for IHC staining.\u003c/p\u003e\n\u003ch3\u003e8. Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eData analysis was conducted using the software SPSS 25, Student\u0026rsquo;s t-test or one-way ANOVA followed by Fisher's least significant difference (LSD) post hoc test was used to assess the differences between groups. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Active phase (Night) of WKY, Sham and RDN groups compared to their respective resting phase (Day), \u003csup\u003ea\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; the overall level of Sham group compared to WKY group, \u003csup\u003eb\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; the overall level of RDN group compared to Sham group, \u003csup\u003ec\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImpact of RDN on circadian variation of blood pressure\u003c/h2\u003e \u003cp\u003eAt baseline assessment conducted at 10 weeks of age, no statistically significant differences in systolic blood pressure (SBP) were observed between the Sham group and the RDN group during either the resting or active phases. However, both experimental groups demonstrated significantly elevated SBP values compared to the WKY control group. RDN intervention resulted in a significant reduction of SBP in SHR during both circadian phases, with maximal therapeutic efficacy observed at 12 weeks post-intervention. Comprehensive quantitative data are presented in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in resting systolic blood pressure (SBP/mm Hg) (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(weeks)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWKY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRDN\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e129.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e176.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e178.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e128.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e181.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e145.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e132.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e196.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e131.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e197.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e152.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e1mm Hg\u0026thinsp;=\u0026thinsp;0.133kPa.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in active systolic blood pressure (SBP/mm Hg) (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(weeks)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWKY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRDN\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e145.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e189.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e190.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e141.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e193.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e160.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e146.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e207.8\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e164.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e146.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e206.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e166.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe WKY control group maintained stable blood pressure variability at approximately 10% throughout the experimental period. Initial measurements at 10 weeks of age revealed no significant differences in blood pressure variability between the Sham and RDN groups. However, by 12 weeks of age, the RDN group demonstrated a significant enhancement in circadian blood pressure rhythm, exhibiting a variability of 9.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44%, which was markedly higher than that of the Sham group (5.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This improvement in circadian regulation persisted at 16 weeks of age, with the RDN group maintaining a blood pressure variability of 8.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66%, significantly exceeding the Sham group's variability of 4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Detailed statistical data are provided in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in blood pressure variability (%) (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(weeks)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWKY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRDN\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.42\u0026thinsp;+\u0026thinsp;2.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Active phase (Night) of WKY, Sham and RDN groups compared to their respective resting phase (Day), \u003csup\u003ea\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; the overall level of Sham group compared to WKY group, \u003csup\u003eb\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; the overall level of RDN group compared to Sham group, \u003csup\u003ec\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eImpact of RDN on the circadian variations in sympathetic nerve activity\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTyrosine hydroxylase (TH), a marker for sympathetic nerve fibers, serves as a reliable indicator of sympathetic nervous system excitability. Immunohistochemical staining was employed to evaluate TH protein expression in the kidneys of experimental rats during both resting and active phases. The Sham group exhibited elevated TH expression levels during both phases, whereas the RDN group demonstrated an overall reduction in TH expression, with daytime levels significantly lower than nighttime levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo further investigate circadian differences in sympathetic nerve activity, norepinephrine (NE) levels were assessed in both renal tissue and plasma. The Sham group exhibited significantly higher overall levels of renal and plasma NE compared to WKY rats, with no notable circadian variation. In contrast, RDN treatment reduced overall NE content in hypertensive rats, particularly during the resting phase, thereby enhancing the circadian variation of NE levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of RDN on the circadian variations of Ang II and Ang1-7 in the kidneys and plasma.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAng II and Ang1-7 are the primary bioactive components of the RAS. In WKY rats, both renal and plasma Ang II exhibited a distinct circadian rhythm, with significantly elevated levels during the active phase compared to the resting phase. The Sham group displayed overall higher levels of renal and plasma Ang II compared to the WKY group, with no discernible circadian variation. RDN effectively reduced Ang II levels, particularly during the resting phase, and restored circadian rhythmicity in Ang II expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003eSimilarly, WKY rats demonstrated a circadian rhythm in renal and plasma Ang1-7 levels. In the Sham group, no significant circadian variation in Ang1-7 was observed, and overall expression levels were relatively low. Following RDN treatment, SHR exhibited a pronounced circadian variation in renal and plasma Ang1-7, with levels during the active phase being lower than those during the resting phase, and overall expression surpassing that of the Sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of RDN on the circadian variations of RAS-related proteins in rat kidneys.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the Sham group of SHR, renal angiotensin-converting enzyme (ACE1) levels were significantly higher than those in the WKY group, with no detectable circadian variation. In contrast, the WKY group exhibited a pronounced circadian pattern, characterized by lower levels during the day and higher levels at night. RDN significantly reduced ACE1 levels in SHR, particularly during the resting phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003eThe Sham group also exhibited markedly lower levels of angiotensin-converting enzyme 2 (ACE2), which catalyzes the conversion of Ang II to the vasodilatory peptide Ang1-7, compared to the WKY group, with consistently low levels during both phases. RDN significantly enhanced overall ACE2 expression, particularly restoring its levels during the resting phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C). Collectively, these findings indicate that RDN restored circadian rhythmicity in renal ACE1 and ACE2 expression in hypertensive rats.\u003c/p\u003e \u003cp\u003eRegarding the angiotensin II type 1 receptor (AT1R), overall expression in the Sham group was significantly higher than in the WKY group. RDN resulted in an overall reduction in AT1R expression in SHR, with no significant circadian variation observed in any group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, D). Similarly, the expression of the angiotensin 1\u0026ndash;7 receptor (MasR) showed no significant circadian variation across groups. However, the Sham group exhibited significantly lower overall MasR expression compared to the WKY group, while RDN increased MasR protein levels in SHR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImpact of RDN on the circadian variation of BMAL1 protein\u003c/h2\u003e \u003cp\u003eIn WKY rats, BMAL1 protein expression exhibited a significant circadian rhythm, with lower levels during the day and higher levels at night. In contrast, the hypertensive Sham group displayed consistently high BMAL1 expression throughout both phases, with no discernible circadian variation. RDN treatment reduced daytime BMAL1 expression in hypertensive rats, thereby restoring circadian rhythmicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings demonstrate that hypertensive rats exhibit significantly attenuated circadian variations in blood pressure compared to normotensive controls, with this reduction becoming more pronounced with advancing age. RDN not only effectively reduced overall blood pressure in hypertensive rats but also restored circadian blood pressure rhythmicity. This improvement was associated with the modulation and stabilization of circadian sympathetic activity, as well as the restoration of circadian variations in the renal RAS and the core clock gene BMAL1. Notably, this study provides the first evidence of a mechanistic link between sympathetic nervous system activity and BMAL1 regulation in the context of hypertension.\u003c/p\u003e \u003cp\u003eBlood pressure is inherently characterized by a well-defined circadian rhythm(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and disruptions in this rhythm are strongly associated with the pathogenesis of cardiovascular(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), cerebral(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and renal diseases(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Clinical studies have demonstrated that hypertensive patients with reverse dipper or non-dipper patterns experience more severe structural and functional cardiac damage compared to those with dipper patterns(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Specifically, non-dipper hypertension has been linked to impaired left ventricular systolic function, as evidenced by reduced myocardial strain(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Furthermore, among hypertensive patients with a history of cerebral infarction, the prevalence of non-dipper hypertension is approximately 62.16%, significantly higher than the 35.85% observed in patients without such sequelae(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Aging further exacerbates these patterns, with hypertensive patients increasingly exhibiting dipper and non-dipper profiles as they age(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Consistent with these clinical observations, our experimental data revealed that hypertensive rats exhibit diminished circadian blood pressure variations, which progressively decline with age. Importantly, our study highlights that RDN not only achieves blood pressure reduction but also restores physiological circadian blood pressure rhythms, offering a potential therapeutic strategy for managing hypertension-related circadian dysregulation.\u003c/p\u003e \u003cp\u003eThe critical role of the sympathetic nervous system in regulating the circadian rhythm of blood pressure is well-documented(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Clinical studies have demonstrated that RDN effectively modulates circadian blood pressure profiles, improving circadian rhythmicity and reducing cardiovascular risk in patients with resistant hypertension(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). For instance, in unilateral nephrectomized patients, the prevalence of dipper patterns decreased, while non-dipper and riser patterns increased post-nephrectomy, highlighting the influence of renal innervation on circadian blood pressure regulation(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Preclinical studies further support these findings; for example, long-term RDN has been shown to normalize circadian blood pressure rhythms in metabolic syndrome rats, a phenomenon associated with enhanced urinary sodium excretion and suppression of renal Na+-Cl cotransporter upregulation(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). While our findings align with these observations by demonstrating that RDN restores circadian blood pressure rhythms in SHRs, our study uniquely identifies that this effect is primarily mediated through the modulation of sympathetic nervous system activity and its interaction with the core clock gene BMAL1. This distinction underscores the pivotal role of sympathetic overactivation, a well-established contributor to hypertension, in disrupting circadian blood pressure regulation.\u003c/p\u003e \u003cp\u003eClock genes are fundamental to circadian regulation, and BMAL1, as a core component of the molecular clock, plays a critical role in maintaining blood pressure circadian rhythms(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). BMAL1 exhibits tissue- and sex-specific effects on blood pressure regulation. For instance, mice lacking smooth muscle BMAL1, but not cardiomyocyte BMAL1, exhibit moderately reduced blood pressure and diminished circadian amplitude(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Similarly, collecting duct-specific deletion of BMAL1 lowers blood pressure in male mice but not in females, highlighting the gene's sex-dependent regulatory role(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). While these studies elucidate the importance of BMAL1 in blood pressure regulation, they primarily focus on normotensive models and do not address its circadian expression patterns in hypertensive conditions. Our study addresses this gap by demonstrating that SHR exhibit elevated renal BMAL1 expression with blunted day-night variations compared to normotensive WKY rats. RDN not only reduced overall renal BMAL1 expression but also restored its circadian rhythmicity in hypertensive rats. The interplay between BMAL1 and the sympathetic nervous system further underscores its regulatory role. Previous research has shown that BMAL1 knockout mice exhibit reduced expression of phenylethanolamine N-methyltransferase, a key enzyme in catecholamine synthesis, leading to decreased epinephrine and norepinephrine levels(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). This suggests that BMAL1 positively regulates catecholamine production, linking circadian gene activity to sympathetic tone. In our study, the observed changes in BMAL1 expression following RDN may reflect a negative feedback mechanism by which the sympathetic nervous system modulates BMAL1 activity, thereby influencing circadian blood pressure rhythms. These findings provide novel insights into the complex interplay between sympathetic activity, circadian gene regulation, and blood pressure control in hypertension.\u003c/p\u003e \u003cp\u003eThe renin-angiotensin system is a pivotal regulatory mechanism in blood pressure homeostasis, exhibiting distinct circadian variations(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). In transgenic hypertensive (TGR) rats, plasma ACE1 activity is significantly elevated and demonstrates an inverted rhythm compared to normotensive controls(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In rats subjected to restraint stress, Ang II infusion has been shown to reverse the 24-hour blood pressure rhythm(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Clinical studies have also highlighted the role of RAS in circadian dysregulation, patients with non-dipper primary hypertension exhibit significantly elevated nighttime urinary angiotensinogen (U-AGT) levels, indicative of nocturnal RAS activation(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In the present study, we investigated the circadian variations of renal and plasma RAS components. Our findings demonstrate that RDN significantly reduced Ang II levels in both renal tissue and plasma, as well as downregulated renal ACE1 expression, particularly during the daytime, thereby enhancing circadian rhythmicity. Concurrently, RDN increased Ang1-7 levels in the kidneys and plasma, along with upregulating renal ACE2 expression, especially during the nighttime, restoring physiological circadian differences. These results suggest that the rebalancing of the ACE1/Ang II/AT1R and ACE2/Ang1-7/MASR axes represents a key mechanism through which RDN modulates circadian blood pressure variations.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, RDN effectively ameliorated circadian blood pressure dysregulation in hypertensive rats, likely through the modulation of sympathetic nervous system activity, the restoration of clock gene BMAL1 expression, and the enhancement of circadian dynamics in the ACE1/Ang II/AT1R and ACE2/Ang1-7/MASR pathways. These findings suggest that achieving both blood pressure reduction and the restoration of circadian blood pressure rhythms could represent a novel therapeutic goal in hypertension management. Additionally, timing RDN interventions to coincide with periods of peak sympathetic activity may further optimize clinical outcomes.\u003c/p\u003e \u003cp\u003eHowever, this study is not without limitations. The reliance on animal models, while informative, cannot fully replicate the complexities of human hypertension. Moreover, blood pressure regulation is a multifactorial process involving numerous interconnected systems, with the local renal RAS representing only one of many contributing factors. Similarly, while BMAL1 plays a critical role in circadian blood pressure regulation, it is likely that other rhythmic genes and pathways also contribute to this intricate physiological process. Future studies should explore these additional mechanisms and validate these findings in human populations to advance our understanding of hypertension and its treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis work was supported by the Science and Technology Project of Fujian Province, China (No.2020Y0023) and Fujian Provincial Finance Project (BPB-2023SJZ).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJZ-S and WQ-C designed and directed the study. GY-Y and SK-C wrote and revised the manuscript and carried out main experiments, HY-C and RN-L carried out this animal experiment, including animal feeding, model building and blood pressure measuring. JY-L, JS-D and SQ-L calculated and analyzed the experimental data. All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe gratefully acknowledge all the participants in this study and staff members of the follow-up team.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eFuchs FD, and Whelton PK\u003c/strong\u003e. 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This study explores the effects of renal denervation (RDN) on circadian BP rhythms and clock gene expression in spontaneously hypertensive rats (SHR). Ten-week-old SHR were randomized into RDN and sham surgery (Sham) groups, with Wistar-Kyoto rats (WKY) as controls. BP was measured at rest (14:00) and during activity (02:00) biweekly, and BP variability was analyzed. RDN significantly reduced BP, particularly during the resting phase, thereby enhancing circadian BP variation. The Sham group displayed minimal circadian variation in plasma and renal norepinephrine levels, whereas the RDN group exhibited an overall reduction in norepinephrine, with lower levels during rest than during activity. Furthermore, the Sham group showed no significant circadian variation in the renin-angiotensin-aldosterone system (RAS), RDN restored circadian rhythms in ACE1, Ang II, ACE2, and Ang1-7. Additionally, the Sham group demonstrated consistently high renal BMAL1 protein expression, whereas RDN exhibited reduced BMAL1 expression during the resting phase, indicating restored circadian variation. These findings suggest that RDN not only lowers BP but also improves its circadian rhythm, likely through modulation of sympathetic nervous activity, the RAS system, and the circadian clock gene BMAL1.\u003c/p\u003e","manuscriptTitle":"Impact of Renal Denervation on Circadian Variations of Blood Pressure and Clock Gene Expression in Spontaneously Hypertensive Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 12:22:45","doi":"10.21203/rs.3.rs-6164221/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57d4d740-b6e5-4a19-a1ce-868b93d461a6","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46028667,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":46028668,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2025-06-16T04:23:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-24 12:22:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6164221","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6164221","identity":"rs-6164221","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}