The Chronobiome–RAAS Axis: A Proposed Framework Linking Circadian Rhythms, Gut Microbiota, and Blood Pressure Regulation — A Scoping Review Protocol

The Chronobiome–RAAS Axis: A Proposed Framework Linking Circadian Rhythms, Gut Microbiota, and Blood Pressure Regulation — A Scoping Review Protocol Registration Type: Prospective (registered before literature search commencement) License: Creative Commons Attribution 4.0 International (CC BY 4.0) | Category: Medicine and Health Sciences Keywords: Circadian rhythm; Chronobiome; Gut microbiota; Renin-angiotensin-aldosterone system; Hypertension; Blood pressure; Scoping review Authors and Affiliations # Author Affiliation ORCID Email 1 Dr Asjed Khan Jadoon Dept. of Pharmacology, Faculty of Pharmacy & Pharmaceutical Sciences, University of Karachi; Sheikh Mohammad Bin Zayed Al-Nahyan Institute of Cardiology, Quetta, Pakistan 0009-0009-8598-3176 [email protected] 2 Dr Syeda Afroz Dept. of Pharmacology, Faculty of Pharmacy & Pharmaceutical Sciences, University of Karachi, Pakistan 0009-0009-6377-5413 [email protected] 3 Dr Noman-Ul-Haq Dept. of Pharmacy Practice, Faculty of Pharmacy & Health Sciences, University of Balochistan, Quetta, Pakistan 0000-0002-9785-3661 [email protected] 4 Dr Nighat Dept. of Oral and Maxillofacial Surgery, Bolan Medical Complex Hospital Unit 2, Quetta, Pakistan 0009-0000-5211-4905 [email protected] 5 Dr Misbah Dept. of Operative and Conservative Dentistry, Bolan Medical Complex Hospital Unit 2, Quetta, Pakistan 0009-0006-2579-2317 [email protected] 6 Dr Asfand Yar Khaliq Sheikh Mohammad Bin Zayed Al-Nahyan Institute of Cardiology, Quetta, Pakistan 0009-0007-7570-8205 [email protected] 7 Dr Uswah Dawood Sheikh Mohammad Bin Zayed Al-Nahyan Institute of Cardiology, Quetta, Pakistan 0009-0009-5024-762X [email protected] Corresponding Author: Dr Asjed Khan Jadoon — Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi-75270, Pakistan. Phone: +92-336-3332710. Email: [email protected]. ORCID: https://orcid.org/0009-0009-8598-3176 Author Contributions Author Role Dr Asjed Khan Jadoon Principal Investigator: conceived and designed the study, drafted the protocol, will lead acquisition of data Dr Syeda Afroz Academic supervision; critical revision of the protocol Dr Noman-Ul-Haq Academic supervision; critical revision of the protocol Dr Nighat Drafting and critical revision of the protocol Dr Misbah Drafting and critical revision of the protocol Dr Asfand Yar Khaliq Will contribute to acquisition of data (second screener) Dr Uswah Dawood Drafting and critical revision of the protocol All authors have read and approved this protocol. Registration Status This protocol is being registered prospectively, prior to execution of the literature search described in Section 3.4. As of the registration date, no records have been identified, screened, or included, and no data have been extracted or synthesized. Activity Status Protocol developed Complete Literature search executed Not started Screening Not started Data extraction Not started Data synthesis Not started Manuscript writing Not started Any deviation from the methods described in this protocol during the conduct of the review will be disclosed and justified in the final manuscript, in line with PRISMA-ScR reporting standards. 1. Background and Rationale 1.1 Background Hypertension remains a leading preventable cause of cardiovascular morbidity and mortality worldwide, affecting more than one billion individuals. Despite advances in antihypertensive pharmacotherapy, a substantial proportion of patients fail to achieve optimal blood pressure control, suggesting that additional biological mechanisms may contribute to disease pathogenesis and therapeutic responsiveness [1]. The renin-angiotensin-aldosterone system (RAAS) is a principal endocrine mechanism regulating blood pressure, vascular tone, and fluid-electrolyte balance. The classical RAAS pathway involves renin-mediated conversion of angiotensinogen to angiotensin I, followed by angiotensin-converting enzyme (ACE)-dependent generation of angiotensin II (Ang II), which acts on AT1 receptors to promote vasoconstriction, sodium retention, and aldosterone release. The counter-regulatory ACE2/Ang-(1-7)/Mas axis opposes these effects, promoting vasodilation. RAAS activity follows circadian rhythms, with fluctuations in plasma renin activity, angiotensin II, aldosterone, and renal sodium handling contributing to physiological blood pressure variability [2]. Circadian rhythms are endogenous biological oscillations with approximately 24-hour periodicity that synchronize physiological processes with environmental cycles, coordinated by the suprachiasmatic nucleus and by peripheral molecular clocks operating in the gut, liver, kidney, and cardiovascular system [3]. The gut microbiota has emerged as a regulator of host circadian physiology and cardiovascular health. Intestinal microbial communities undergo diurnal oscillations influenced by feeding behavior, sleep-wake cycles, light exposure, dietary composition, and host circadian clocks, altering nutrient metabolism, intestinal barrier integrity, immune activation, and endocrine signaling [4]. Microbial metabolites — including short-chain fatty acids (SCFAs), bile acids, indole derivatives, and trimethylamine-N-oxide (TMAO) — may function as signaling molecules influencing peripheral clock gene expression, inflammatory pathways, vascular biology, and pathways relevant to RAAS activity [5]. 1.2 Rationale Circadian biology, gut microbiota, and RAAS physiology have largely been reviewed separately. Preclinical evidence indicates these systems may be interconnected: microbial metabolites may influence RAAS-related pathways through immune modulation, endothelial signaling, and renal physiology, and RAAS-targeting medications have been reported to alter intestinal microbial composition and intestinal circadian-associated gene expression in animal models [6]. This remains an emerging, largely preclinical body of evidence rather than an established causal pathway, which is precisely why a scoping review — designed to map heterogeneous, developing literature rather than pool effect sizes — is the appropriate review type here. ● Map the current evidence landscape across circadian, microbiome, and RAAS domains ● Identify what is established, what is emerging, and what remains hypothetical ● Identify knowledge gaps and propose a conceptual framework to guide future primary research ● Highlight potential (not yet validated) opportunities for chronotherapy and microbiota-directed interventions 1.3 Research Questions Primary Question: What is the nature and extent of the evidence on bidirectional interactions among circadian rhythms, gut microbiota, and the renin-angiotensin-aldosterone system in relation to blood pressure regulation? ● What evidence exists for circadian regulation of RAAS components (classical and counter-regulatory)? ● What evidence exists for diurnal rhythms in gut microbial composition and function? ● What evidence exists for microbial metabolites influencing cardiovascular physiology and RAAS-related pathways? ● What evidence exists for interactions between circadian rhythms, gut microbiota, and RAAS in hypertension, including nondipping and salt-sensitive phenotypes? ● What evidence exists for chronopharmacology and chrononutrition relevant to RAAS-targeting drugs? ● What evidence exists for microbiota-targeted interventions in hypertension? 1.4 Population–Concept–Context (PCC) Framework Component Description Population Humans and animal models (rats, mice) Concept Circadian rhythms, gut microbiota, microbial metabolites, RAAS (classical and ACE2/Ang-(1-7)/Mas), blood pressure, hypertension, chronotherapy Context Cardiovascular physiology, hypertension, chronobiology, microbiome research, pharmacology 2. Objectives Primary Objective: To systematically map and synthesize available evidence on bidirectional interactions among circadian rhythms, gut microbiota, and the RAAS in blood pressure regulation, and to grade this evidence by strength rather than assume a unified pathway is already established. ● Describe the evidence for circadian regulation of RAAS components, including the ACE2/Ang-(1-7)/Mas axis ● Describe the evidence for diurnal rhythms in gut microbial composition and function ● Describe the evidence for microbial metabolites influencing cardiovascular physiology and RAAS-related pathways, distinguishing direct RAAS evidence from downstream vascular/renal effects ● Identify evidence for interactions between circadian rhythms, gut microbiota, and RAAS in hypertension, explicitly separating association from demonstrated mechanism ● Describe the evidence for chronopharmacology and chrononutrition relevant to RAAS-targeting drugs ● Identify knowledge gaps and propose a conceptual framework (explicitly labeled as proposed/hypothesis-generating) for future research 3. Methods 3.1 Review Design This review will be conducted as a scoping review following the methodological framework of Arksey and O'Malley, as refined by the Joanna Briggs Institute (JBI) [7,8], and will be reported according to PRISMA-ScR guidelines [9]. A scoping review, rather than a systematic review or meta-analysis, was selected because the literature spans heterogeneous study types (animal experiments, human observational studies, fecal microbiota transplantation studies, pharmacological studies) that are not suited to effect-size pooling, and because a primary aim is to map the evidence landscape and identify gaps rather than answer a single pooled-effect question. 3.2 Eligibility Criteria Inclusion criterion Description Study types Original experimental investigations, animal studies, human observational studies, clinical trials, mechanistic investigations Participants Humans and animal models (rats, mice) Concept Studies investigating circadian rhythms, gut microbiota, microbial metabolites, RAAS physiology, hypertension, or blood pressure regulation Context Cardiov