Repurposing of Spironolactone for Hypertension-Related Fatigue: Experimental Evidence from Salt-Sensitive Rats with Computational Molecular Analysis

Repurposing of Spironolactone for Hypertension-Related Fatigue: Experimental Evidence from Salt-Sensitive Rats with Computational Molecular Analysis | 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 Research Article Repurposing of Spironolactone for Hypertension-Related Fatigue: Experimental Evidence from Salt-Sensitive Rats with Computational Molecular Analysis Muhamad Rizqy Fadhillah, Wawaimuli Arozal, Raymond Rubianto Tjandrawinata, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8419825/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-related fatigue involves both elevated blood pressure and reduced exercise tolerance, yet its molecular basis remains unclear. This study investigated the repositioned effects of spironolactone on hemodynamic and metabolic parameters in a uninephrectomy–deoxycorticosterone acetate-salt rat model of salt-sensitive hypertension. Male Sprague–Dawley rats were randomized to receive spironolactone (100 mg/kg/day) or vehicle (carboxymethylcellulose) for five weeks. Blood pressure was measured noninvasively, exercise capacity was assessed using a weighted swimming test, plasma sodium was quantified by enzyme-linked immunosorbent assay, and cardiac HIF-1α expression was analyzed by quantitative polymerase chain reaction. Complementary in silico analyses included functional enrichment of spironolactone–HIF-1α targets, molecular docking with factor inhibiting hypoxia-inducible factor-1 (FIH1; PDB ID: 8II0), free energy calculations, and quantum mechanical assessment. Spironolactone prevented increases in systolic, diastolic, and mean arterial pressure, normalized plasma sodium levels, and suppressed cardiac HIF-1α expression. A non-significant trend toward prolonged time-to-fatigue was observed in spironolactone-treated rats. When interpreted alongside metabolic and hypoxia-related molecular findings, this trend suggests a potential antifatigue effect, although definitive conclusions cannot be drawn from the current dataset. Computational analyses identified HIF-1α and glycolytic pathways as central interaction hubs, with spironolactone demonstrating favorable binding to catalytic residues of FIH1, an asparaginyl hydroxylase, consistent with a mechanism that may enhance HIF-1α hydroxylation and attenuate hypoxia-driven glycolytic reprogramming in the hypertensive myocardium. These findings suggest that spironolactone exerts integrated hemodynamic and metabolic benefits and may hold therapeutic potential for hypertension-related fatigue, warranting confirmation in future studies or clinical translation in salt-sensitive populations. Drug Discovery, Design, & Development Pharmacodynamics Bioinformatics Spironolactone Hypertension Fatigue Drug Repositioning HIF-1α Signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Hypertension remains one of the most prevalent chronic diseases worldwide and continues to pose a substantial public health challenge. Recent estimates indicate that approximately 1.4 billion adults aged 30–79 years were living with hypertension in 2024, with a disproportionate burden observed in low- and middle-income countries. Despite the broad availability of antihypertensive therapies, a considerable proportion of affected individuals remain undiagnosed or inadequately controlled. As a result, hypertension persists as a leading contributor to premature mortality and long-term disability, largely through its association with cardiovascular, cerebrovascular, and renal complications.[ 1 ] Beyond its high prevalence, hypertension is increasingly recognized as a multisystem disorder characterized by complex pathophysiological mechanisms, including sustained sympathetic activation, endothelial dysfunction, oxidative stress, inflammation, and maladaptive neurohormonal signaling. Chronic exposure to elevated blood pressure leads to progressive target-organ damage, manifesting as left ventricular hypertrophy, myocardial fibrosis, vascular remodeling, and renal impairment. These structural and functional alterations substantially increase the risk of ischemic heart disease, heart failure, stroke, and chronic kidney disease. Moreover, hypertension frequently coexists with metabolic comorbidities, further complicating disease management and contributing to impairments in cardiovascular performance, reduced physical capacity, and diminished exercise tolerance—clinical features that are increasingly recognized as integral components of hypertension-related fatigue.[ 2 ] Hypertension-related fatigue represents a multifactorial condition characterized not only by elevated blood pressure but also by reduced exercise tolerance.[ 3 , 4 ] In preclinical studies, the interplay between mineralocorticoid signaling and cardiac stress has been increasingly recognized as a key contributor to both hemodynamic changes and exercise intolerance.[ 5 , 6 ] Spironolactone, a classical mineralocorticoid receptor antagonist, has long been used in clinical practice for the management of resistant hypertension and stable heart failure.[ 7 , 8 ] Recent evidence, however, has suggested that spironolactone’s therapeutic potential may extend beyond classical hemodynamic modulation toward improving exercise capacity and attenuating fatigue-related cardiovascular dysfunction. Preclinical studies in rodent models have demonstrated that spironolactone can modulate molecular pathways associated with hypoxia-inducible factor 1 alpha (HIF-1α) oxidative stress and inflammation, which are all implicated in fatigue and exercise intolerance in hypertensive states.[ 9 ] Several debates have reached controversy over whether spironolactone either improved[ 10 – 12 ] or did not improve[ 13 , 14 ] exercise intolerance in cardiovascular patients related to hypertension conditions, even though it overall reduces patients’ mortality and morbidity.[ 8 ] Inconsistency results might come from diverse phenotypes of disease, as spironolactone primarily works on the renin-angiotensin-aldosterone systems. Among these, salt-sensitive hypertension represents a distinct phenotype in which mineralocorticoid signaling plays a pivotal role. This highlights the need for robust preclinical evidence to elucidate how spironolactone modulates not only hemodynamic regulation but also exercise tolerance in salt-sensitive conditions. Animal models induced by uninephrectomy combined with deoxycorticosterone acetate administration (DOCA) and high-salt intake (UNX-DOCA-Salt) have been established to reproduce the salt-sensitive phenotype commonly observed in resistant hypertension.[ 15 ] While spironolactone has been reported to attenuate cardiac remodeling and fibrotic changes in this model [ 14 , 15 ], Its influence on exercise intolerance has not been clearly defined. Recent experimental observations indicate that spironolactone may interfere with the hypoxia-inducible factor 1α (HIF-1α) signaling axis in DOCA-induced hypertension.[ 16 ], Thereby modulating downstream molecular responses that possibly contribute to cardiac stress adaptation and reduced exercise capacity. While the UNX–DOCA–Salt animal models provides valuable insight into the physiological and molecular effects of spironolactone in salt-sensitive hypertension, it remains essential to clarify the molecular mechanisms that may link spironolactone exposure to fatigue-related pathways. To complement the in vivo findings, we incorporated computational analyses to characterize potential molecular interactions and signaling modulations involving HIF-1α. Previous studies have associated spironolactone with anti-hypoxia and HIF-related mechanisms in DOCA-salt hypertension; however, based on our updated literature search, we were unable to identify studies that integrate HIF-1α-centered in silico predictions with fatigue or exercise-tolerance outcomes in hypertensive rodents. Therefore, the present research employed an integrative approach combining in vivo assessment of hypertension-related fatigue in UNX–DOCA–Salt rats with stepwise computational analyses, including functional enrichment of spironolactone–HIF-1α targets, molecular docking, mechanics generalized Born/Poisson–Boltzmann surface area (MMGB(PB)SA) free-energy estimation, and density functional theory (DFT)-derived electronic descriptors. The aim of this strategy was not to assert a performance-enhancing effect of spironolactone, but rather to explore whether molecular modulation of HIF-1α signaling could correspond with changes in fatigue physiology under hypertensive stress. By aligning molecular predictions with physiological readouts, this approach provides a mechanistic basis for future studies on drug repositioning and targeted validation of spironolactone’s effects on hypoxia-related metabolic pathways. 2. MATERIAL AND METHODS 2.1 Animal Experimentation, Ethics, Welfare Monitoring, and Humane Endpoints Male Sprague–Dawley rats, aged 8–10 weeks and weighing approximately 200–250 g, were acclimated for seven days before experimentation. The animals were housed under controlled environmental conditions with a 12:12 h light–dark cycle and had free access to standard laboratory chow pellets (Rat Bio®, Citrafeed, Indonesia; containing approximately 20% crude protein, 4% crude fat, 4–5% crude fiber, and ≤ 12% moisture) and water ad libitum. A total of 15 rats were (n = 5 rats per group) randomly allocated into three groups: (1) Sham control (sham surgery + vehicle, carboxymethylcellulose (CMC) 0.5%), (2) UNX-DOCA-Salt (uninephrectomy + DOCA-salt induction + vehicle), and (3) UNX-DOCA-Salt + Spiro (uninephrectomy + DOCA-salt induction + spironolactone). Left uninephrectomy was performed in a sterile surgical facility under ketamine (100 mg/kg BW, i.p.) and xylazine (10 mg/kg BW, i.p.) anesthesia for all experimental animals. After a 1-week recovery period, DOCA-salt hypertension was induced by twice-weekly subcutaneous injections of deoxycorticosterone acetate (25 mg/kg BW in 0.1 mL dimethylformamide), together with ad libitum with 1% NaCl solution for two weeks. Blood pressure was then measured noninvasively before beginning the treatment phase, in which animals received either spironolactone (100 mg/kg BW/day in 0.5% CMC, p.o.), as previously reported, [ 19 ] or a vehicle for five weeks. All of the experimental protocols were reviewed and approved by the Ethics Committee of the Faculty of Medicine, Universitas Indonesia, with ethics number (KET 1551/UN2.F1/ETIK/PPM.00.02/2024). Use of postsurgical analgesia was exempted based on scientific rationale granted by the Ethics Committee, as common analgesics in rats (non-steroidal anti-inflammatory drugs and opioids) due to the potential interference of analgesia with the blood pressure; thus, animal welfare monitoring was intensified. To ensure animal welfare despite the absence of analgesia, rats were monitored twice daily for body weight, food and water intake, hydration, locomotor activity, scrotal swelling, and general appearance. Humane endpoints were predefined (> 15% body-weight loss, persistent immobility, or respiratory distress), and no animal reached these endpoints or required early sacrifice. No unexpected mortality occurred throughout the study. 2.2 Blood Pressure Measurement Blood pressure was measured using noninvasive methods (CODA Scientific®) in rats. Briefly, the rats were acclimatized before data collection for 15–30 minutes. Systolic blood pressure (SBP, mmHg), diastolic blood pressure (DBP, mmHg), and heart rate (beats per minute (bpm)) were measured and compared using internal control from weeks 0, 2, and 5. In addition, mean arterial pressure (MAP) changes were also determined using below formula: \(\:MAP=DBP+\:\frac{SBP-DBP}{3}\) (1) 2.3 Weighted Swimming Exercise Test Rats' exercise capacity was determined using a swimming exercise test to estimate tolerance against high-intensity activity. Rats were weighed, and a load equivalent to 2% of body weight was tied to the tail. Before testing, rats were acclimatized in the swimming tube for 15 minutes/day without weight for three consecutive days. The swimming apparatus consisted of a cylindrical tube (40 cm diameter, 60 cm height) filled with water to a depth of 50 cm, and the temperature was maintained at 28 ± 1°C. During the test session, two independent blind observers monitored the animals. Time-to-fatigue (seconds) served as the primary performance variable and was defined based on predefined exhaustion criteria: failure to reach the surface for 10 consecutive seconds and loss of coordinated swimming movements. A humane endpoint and a maximum cut-off duration of 15 minutes were applied to avoid excessive physical stress with additional warming using an animal incubator. Swimming sessions were video recorded for welfare documentation, and fatigue time was recorded manually using a stopwatch according to the exhaustion criteria. 2.4 Sodium Plasma Concentration The sodium plasma concentration was determined using blood collected from rats retroorbital. After centrifuging at 5.000 g x 15 minutes, the plasma was separated and collected. The sodium concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit, Elabscience, according to a standardized protocol. 2.5 Isolation of Total mRNA, cDNA synthesis, and qPCR of Cardiac HIF-1A mRNA expression A total of 100 mg of cardiac tissue was isolated from sacrificed animals using DirectZol ® TriReagent according to the predetermined protocols. Further, 500 ng of purified (A230/A260 1.8-2.0) mRNA were transcribed in each group for cDNA using the ReverTra Ace Toyobo kit. The reacted solution then performed qPCR using HIF1A primer (Forward primer 5’-AATCCATTTTCAGCTCAGGAC-3’; Reverse primer 5’-GGCAGTGACAGTGATGGTAGG-3’; Bp product 198; T m : 60°C) and normalized using beta-actin (Forward primer 5’-TGTTGTCCCTGTATGCCTCT-3’; Reverse primer 5’-TAATGTCACGCACGATTTCC-3’; Bp product 222; T m : 60°C) as a housekeeping gene. The relative mRNA expression was calculated using the Livak method. 2.6 Functional Enrichment of Spironolactone HIF-1A Related Gene Targets To elucidate the potential mechanistic relationship between spironolactone and the HIF-1α signaling pathway, a multi-step in silico workflow was performed. Genes involved in the HIF 1α signaling pathway were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.kegg.jp/ ), while spironolactone-related targets were retrieved from the Comparative Toxicogenomics Database (CTD; https://ctdbase.org/ ). The intersecting targets between these two datasets were determined using the JVENN web platform ( https://bioinformatics.psb.ugent.be/webtools/Venn/ ) to identify common molecular nodes potentially modulated by spironolactone within the HIF-1α pathway. The intersected genes were further analyzed in ShinyGO v0.85 ( http://bioinformatics.sdstate.edu/go/ ) to explore enriched pathways and Gene Ontology (GO) categories, including Biological Process, Molecular Function, and Cellular Component, using Fisher’s exact test with Benjamini-Hochberg false discovery rate (FDR) correction (FDR ≤ 0.05). This integrative approach provided a systems-level overview of spironolactone’s functional targets potentially linked to HIF-1α regulation. 2.7 Molecular Docking of HIF-1α protein The crystal structure of the human factor inhibiting HIF-1α (FIH1; PDB ID: 8II0) was retrieved from the Protein Data Bank ( https://www.rcsb.org/structure/8II0 ), as FIH1 represents a key regulatory enzyme that hydroxylates (asparaginyl hydroxylase) and thereby inactivates HIF-1α under normoxic conditions. Human FIH protein was chosen as the rat protein had not been present yet on the PDB website. The protein structure was preprocessed and corrected using PDBFixer under Windows Subsystem for Linux (WSL) to remove alternate conformations, add missing residues, and optimize hydrogen geometry. Validation of the docking protocol was performed by redocking the native ligand into the active site, ensuring a root mean square deviation (RMSD) value < 2.0 Å, thus confirming the reliability of the docking setup. Docking simulations were conducted using Molegro Virtual Docker (MVD), with all hydrogen atoms added at pH 7.4 for both protein and ligands. The 3D structure of spironolactone was obtained from PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ) and protonated at pH 7.4 before docking. Comparative docking analysis between spironolactone and the native inhibitor was performed based on MolDock Score, Re-rank Score, and Hydrogen Bond Energy for the five top-ranked poses. Binding interactions were visualized using LigPlot, with particular attention to hydrogen bonding at Tyr102, which represents a critical residue in the FIH1 catalytic pocket. 2.8 MMGB(PB)SA Analysis Post-docking free energy estimations were performed using the MMGB(PB)SA approach to provide a more comprehensive insight into the binding thermodynamics beyond docking scores. This analysis was conducted on WSL platform utilizing the UniGBSA-pipeline, which automates the process of parameterization, trajectory generation, and energy decomposition. The top five poses of both the native ligand and spironolactone were extracted from the docking results and exported in MOL2 format. Each ligand–protein complex, along with the cleaned FIH1 receptor structure, was subsequently parameterized using the ACPYPE (Antechamber Python Parser Interface) tool to generate topology and coordinate files under the AMBER force field, applying GAFF parameters for ligands and assigning partial charges through the AM1-BCC method. The MMGB(PB)SA calculations were then performed with UniGBSA to estimate the binding free energy (ΔG_bind) using the following equation: $$\:{\Delta\:}{G}_{bind}={\Delta\:}{E}_{MM}+{\Delta\:}{G}_{solvation}-T{\Delta\:}S$$ 2 where \(\:{\Delta\:}{E}_{MM}\) represents the molecular mechanics energy (van der Waals + electrostatic interactions), \(\:{\Delta\:}{G}_{solvation}\) is the solvation free energy (polar + nonpolar contributions), and \(\:T{\Delta\:}S\) Denotes the entropic term (approximated or neglected in some setups). The MMGBSA method complements molecular docking by accounting for solvent effects and molecular flexibility, thereby providing a more realistic estimate of binding affinity that docking scores alone cannot fully capture. For each ligand, mean and standard deviation values of ΔG_bind across the five poses were calculated to assess stability and reproducibility of the predicted binding energies. 2.9 Density Functional Theory To characterize the electronic properties and reactivity profiles of spironolactone and the native FIH1 ligand, DFT calculations were performed using the Python-based Simulations of Chemistry Framework package implemented under the WSL environment. Before calculation, the optimized 3D geometries of both ligands were obtained from PubChem and preprocessed to ensure proper protonation at physiological pH (7.4). The geometry optimization and subsequent single-point energy calculations were carried out using the B3LYP functional with the 6-31G(d,p) basis set, which provides a balanced accuracy–cost trade-off for organic molecules. The convergence criteria were set to \(\:{10}^{-6}\) a.u. for energy and \(\:{10}^{-4}\) a.u. for gradient, ensuring optimized and stable geometries. Solvent effects were simulated using the Polarizable Continuum Model (PCM) with water as the dielectric medium (ε = 78.4). The outputs included HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) energies, the energy gap (ΔE = E LUMO − E HOMO ), and derived descriptors such as electronegativity (χ), hardness (η), and softness (S) according to Koopmans’ theorem: $$\:\chi\:=-\frac{{E}_{HOMO}+{E}_{LUMO}}{2},\eta\:=\frac{{E}_{LUMO}-{E}_{HOMO}}{2},S=\frac{1}{2\eta\:}$$ 3 2.10 Statistical Analysis In vivo experimental data were analyzed descriptively and comparatively using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). Descriptive data are presented as mean ± standard deviation (SD). For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was used to assess overall group differences, followed by Tukey’s multiple comparison test for normally distributed data or Dunn’s post-hoc test for non-normally distributed data when appropriate. A p-value < 0.05 was considered statistically significant. No prior sample size or power calculation was performed. 3. RESULTS AND DISCUSSION 3.1 The effect of spironolactone on blood pressure and the weighted swimming exercise test Two-way ANOVA showed a significant time effect on both systolic and diastolic blood pressure in UNX-DOCA-salt rats treated with spironolactone ( p < 0.01). For SBP, the mean value increased markedly from week 0 (136.1 ± 2.7 mmHg) to week 2 (199.9 ± 6.9 mmHg; p = 0.0017), confirming the successful induction of hypertension. Following spironolactone administration, SBP declined at week 5 (177.4 ± 6.9 mmHg; p = 0.0085 vs. week 0), though the difference between week 2 and week 5 was not significant ( p = 0.0663). A similar pattern was observed in DBP, which significantly increased from week 0 to week 2 ( p = 0.0002) and decreased again by week 5 ( p = 0.0011) ( Fig. 1 A-C ) . These findings indicate that spironolactone effectively attenuates the rise in blood pressure induced by UNX-DOCA-salt treatment, consistent with its established role as an antihypertensive agent acting through mineralocorticoid receptor blockade.[ 20 ] At the end of the fifth week, the weighted swimming test revealed that the UNX-DOCA-Salt group showed a significantly shorter time to fatigue compared to the control group (p < 0.05), indicating reduced exercise tolerance due to hypertension-induced cardiac and systemic dysfunction. Treatment with spironolactone slightly improved the duration of fatigue, although the difference was not statistically significant compared to the DOCA group (Fig. 1 D and Figure E ). Previous reports in human studies have also suggested a potential antifatigue or exercise-performance benefit of spironolactone [ 8 , 9 ]. Despite some contradictory findings regarding spironolactone, eplerenone has demonstrated more consistent efficacy in reducing fatigue, likely due to its stronger binding affinity to mineralocorticoid receptors compared with spironolactone, which may explain the discrepancy between studies [ 11 ]. In this context, our findings support a possible antifatigue effect of spironolactone. Nevertheless, this interpretation should be approached with caution, particularly considering patient demographics—such as salt-sensitive individuals—for whom subgroup analyses in previous trials of exercise intolerance remain limited. Therefore, further studies are needed to clarify whether the antifatigue benefit of spironolactone is generalizable across different populations or specific to certain physiological profiles. 3.2 The effect of spironolactone on plasma sodium concentration and cardiac tissue mRNA HIF1A expression The plasma sodium concentration was significantly elevated in the UNX-DOCA-Salt group compared to the control group (p < 0.05), confirming the successful induction of salt-sensitive hypertension. Administration of spironolactone markedly reduced plasma sodium levels compared to the UNX-DOCA-Salt group (p < 0.05), indicating its effectiveness in attenuating sodium retention ( Fig. 2 A ) . This finding aligns with the pharmacological action of spironolactone as a mineralocorticoid receptor antagonist, which counteracts aldosterone-induced sodium reabsorption in the distal nephron. By reducing sodium overload, spironolactone contributes to improved volume regulation and blood pressure control in mineralocorticoid-induced hypertension. A previous study has demonstrated a similar effect of spironolactone in mitigating sodium retention and vascular remodelling in UNX-DOCA-salt hypertensive models through Na+/H + exchanger.[ 17 ] The relative expression of HIF1A mRNA was significantly increased in the UNX-DOCA-Salt group compared with the control (p < 0.05), indicating that the hypertensive condition induced by UNX-DOCA-salt was associated with upregulation of hypoxia-related signalling. Treatment with spironolactone reduced the elevated HIF1A expression compared to the UNX-DOCA-Salt group, although the reduction did not fully normalize the levels to those of the control group. This result suggests that spironolactone partially attenuates cardiac hypoxic stress in the UNX-DOCA-salt model. The decrease in HIF1A expression likely reflects an improvement in myocardial oxygen balance and perfusion following aldosterone receptor blockade. Previous studies have shown that spironolactone mitigates tissue hypoxia and oxidative stress through inhibition of mineralocorticoid receptor–mediated in different tissues.[ 18 , 21 ] 3.3 The functional enrichment of spironolactone on fatigue-related gene/protein Bioinformatic integration analysis revealed that spironolactone shared 95 common target genes with HIF-1A–associated genes (Fig. 3 A), indicating a strong molecular intersection between Spiro activity and the hypoxia-related transcriptional network. KEGG pathway enrichment further showed that these overlapping genes were predominantly involved in the HIF-1 signaling pathway, with several components—such as EGFR, AKT1, mTOR, VEGFA, and LDHA—highlighted in the pathway map (Fig. 3 B). Pathway enrichment ranking also identified HIF-1α signaling as the most significantly enriched pathway (Fig. 3 C), followed by other metabolic and signaling cascades, including PI3K-Akt, mTOR, and glycolysis/gluconeogenesis pathways. Functional enrichment analysis identified several predominant biological themes among the Spironolactone–HIF-1α overlapping genes. The enriched terms were mainly related to glycolytic, pyruvate, and adenosine triphosphate metabolic processes, suggesting an overall enhancement of cellular energy turnover. Structural enrichment highlighted enzyme complexes such as phosphofructokinase and vesicular components, implying potential modulation of intracellular trafficking and metabolic compartmentalization. In parallel, enrichment in kinase- and nucleotide-binding activities indicated a broad regulation of enzymatic catalysis and phosphorylation-dependent signaling (Fig. 3 D-E). The current enrichment analysis revealed that spironolactone-regulated genes predominantly intersect with glycolytic biosynthesis, 6-phosphofructokinase complex formation, and carbohydrate kinase activity, reflecting a coordinated modulation of HIF-1α–mediated metabolic processes. HIF-1α serves as a central transcriptional regulator of cellular adaptation or metabolic switch in hypoxic conditions by promoting glycolysis and limiting oxidative phosphorylation—mechanisms that are highly relevant in hypertensive organ remodeling or other similar insults, where oxygen utilization becomes inefficient.[ 22 , 23 ] Clinically, spironolactone treatment has been associated with reduced HIF-1α expression following ischemic or surgical stress, supporting its role in normalizing cellular hypoxia signaling, which was also observed in our experimental study.[ 22 ] Collectively, these findings imply that spironolactone may attenuate hypertension-induced fatigue and maladaptive metabolic reprogramming by partially suppressing HIF-1α activation and restoring energy homeostasis, thereby linking its molecular effects to improved functional outcomes in hypertensive models. 3.4 The Molecular docking and MMGB(PB)SA Docking was validated using RMSD (Fig. 4 I). The binding box was coordinated according to the native ligand, with x, y, and z located on 23.07, -25.87, and 3.30, respectively, with a radius of 11. The molecular docking RMSD was consistently below 2 Å ( Table S1 ), which tightly focuses on Tyr102 as an anchoring target in the binding box. The validation finding will be further complemented with MMGB(PB)SA. 3.5 Molecular docking and MMGB(PB)SA results Molecular docking analysis demonstrated that both the native ligand and spironolactone exhibited strong binding affinity toward the FIH (factor inhibiting HIF-1α) enzyme. The native ligand showed a slightly stronger binding profile (Moldock score: − 128.8 ± 1.3 a.u.; Rerank score: − 105.7 ± 2.4 a.u.) compared to spironolactone (Moldock score: − 115.9 ± 2.9 a.u.; Rerank score: − 75.6 ± 10.5 a.u.). Despite this, both ligands formed energetically stable complexes. The hydrogen bonding energy of the native ligand (–8.8 ± 3.2 kJ/mol) was slightly higher than that of spironolactone (–5.3 ± 2.0 kJ/mol), consistent with the greater number of hydrogen bonds observed in the native complex (five H-bonds) compared to spironolactone (four H-bonds). MMGB(PB)SA energy decomposition further supported these observations. The total binding free energy (ΔG_total) of spironolactone averaged − 32.9 ± 2.6 kJ/mol, which was comparable to that of the native ligand (–26.9 ± 1.4 kJ/mol). The van der Waals contribution dominated the stabilization for both ligands, being slightly more favorable in spironolactone (–34.9 ± 2.7 kJ/mol) than in the native complex (–28.4 ± 2.3 kJ/mol). Meanwhile, electrostatic energy was negligible in spironolactone (≈ 0 kJ/mol) but markedly higher in the native ligand (+ 42.0 ± 3.2 kJ/mol), indicating that spironolactone relies less on charge–charge interactions and more on hydrophobic stabilization. Interestingly, the polar solvation term was positive for spironolactone (+ 9.4 ± 1.0 kJ/mol) but negative for the native ligand (–35.1 ± 1.4 kJ/mol), implying that reduced desolvation penalties accompany spironolactone binding. The nonpolar solvation energy (–5.4 ± 0.4 kJ/mol) was consistently favorable in both complexes. Collectively, these results suggest that spironolactone achieves thermodynamically stable binding primarily through van der Waals and solvation-driven mechanisms rather than electrostatic attraction. (Fig. 4 A-H) Residue-level interaction profiling based ( Table S2 ) on LigPlot visualization revealed that the native ligand formed seven hydrogen bonds with Asn294(A) (2.94 Å), Arg238(A) (2.39 Å), Lys214(A) (2.47 Å), Thr196(A) (2.71 Å), Gln147(A) (3.31 Å), Tyr145(A) (3.01 Å), and Tyr102(A) (3.00 Å), securing its orientation within the catalytic pocket of FIH. In contrast, spironolactone established four major hydrogen bonds involving Arg238(A) (3.12 Å), Gln203(A) (3.04 Å), Glu202(A) (3.22 Å), and Asp201(A) (3.10 Å), while maintaining hydrophobic contacts with Tyr102(A) (3.30–3.81 Å), Leu186(A) (2.70–3.30 Å), Phe207(A) (3.60–3.70 Å), and Trp296(A) (2.97–3.68 Å). Notably, Tyr102(A) emerged as a critical anchoring residue positioned near the iron coordination center, consistent with its conserved role in ligand stabilization across HIF-1α inhibitor complexes. The hydrophobic interactions surrounding Tyr102(A) and Arg238(A) further reinforced the compactness and rigidity of the spironolactone-FIH complex (Fig. 4 J-K). 3.6 DFT of Spironolactone The quantum chemical parameters of the native ligand and spironolactone were calculated using DFT to evaluate their stability and electronic reactivity (Table 1 ). The HOMO and LUMO energies for the native ligand were − 6.65 eV and − 1.85 eV, respectively, resulting in a gap of 4.81 eV. In contrast, spironolactone exhibited a narrower gap of 0.89 eV, with higher HOMO (–1.72 eV) and LUMO (–0.83 eV) energies. Meanwhile, spironolactone displayed lower electronegativity (1.27 eV) and hardness (0.45 eV) but markedly higher softness (30.52 eV⁻¹) compared with the native ligand. Table 1 Quantum chemical parameters of native ligands and spironolactone HOMO (eV) LUMO (eV) Gap (eV) Electronegativity (χ) Hardness (η) Softness (S) Native -6.65 -1.84 4.80 4.25 2.40 5.65 Spironolactone -1.71 -0.82 0.89 1.27 0.44 30.52 HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital. Quantum chemical parameters were calculated to evaluate the electronic properties and reactivity of spironolactone relative to the native ligand. The DFT-based quantum chemical analysis demonstrated that spironolactone exhibits higher electronic reactivity and greater molecular softness compared with the native ligand. Its elevated HOMO energy and narrower energy gap indicate a more polarizable and electron-donating character, which facilitates charge-transfer and soft–soft interactions with electron-deficient residues at the protein binding site. This observation aligns with the hard and soft acids and bases theory and the frontier molecular orbital.[ 24 , 25 ] In contrast, the native ligand’s wider energy separation and higher electronegativity suggest greater intrinsic stability and a more electrophilic nature, consistent with descriptors of chemical hardness and electrophilicity. These findings collectively imply that spironolactone’s softer electronic profile may underline its stronger charge-transfer capacity and potential for enhanced molecular interaction within the protein active site.[ 26 ] The integrated evaluation of quantum chemical descriptors and interaction profiles reveals a consistent pattern between the electronic characteristics and binding behavior of the ligands. Spironolactone, exhibiting a higher HOMO energy, a narrower HOMO–LUMO gap, and greater molecular softness, can be characterized as a more electron-donating and polarizable molecule. This electronic configuration is reflected in the LigPlot-derived interaction map, where spironolactone establishes multiple hydrogen bonds and close polar contacts with acidic and polar residues, particularly Asp201, Glu202, and Gln203, along with a polarizable sulfur-containing moiety that may further contribute to complex stabilization. In contrast, the native ligand—defined by a wider energy gap and higher electronegativity—demonstrates a more dispersed and rigid interaction profile, forming contacts with a broader range of polar and aromatic residues such as Asn294, Arg238, Trp296, and Tyr102, but exhibiting comparatively fewer deep interactions with the acidic residue cluster. Collectively, these findings suggest that spironolactone interacts with the target protein through a more dynamic, donor-driven, and adaptive binding mode, whereas the native ligand displays a relatively stable and electrophile-oriented interaction pattern. 3.7 Research Implications and Study Limitations The present findings provide a mechanistic extension of spironolactone’s pharmacological profile beyond its classical role as a mineralocorticoid receptor antagonist. In the UNX–DOCA–Salt hypertensive model, spironolactone prevented the progressive rise of systolic, diastolic, and mean arterial pressure, accompanied by normalization of plasma Na⁺ and suppression of cardiac HIF-1A mRNA expression, suggesting partial restoration of electrolyte imbalance and oxygen-sensing homeostasis. Functional enrichment and molecular docking analyses further identified HIF-1A and glycolytic pathways as major interaction hubs, while the predicted binding of spironolactone within the FIH catalytic pocket — supported by coordinate interaction mapping and DFT-derived electronic descriptors — suggests a potential modulation of the FIH–HIF-1α regulatory axis. Although this hypothesis remains computational and requires biochemical validation, the in silico interactions suggest that spironolactone may facilitate HIF-1α hydroxylation and turnover via donor-type electronic interactions with key residues in the FIH active site (Asp201, Glu202, Gln203, Tyr102). A trend toward increased time-to-fatigue was observed in spironolactone-treated rats; however, this change did not reach statistical significance, and therefore, the findings cannot confirm an antifatigue or exercise-enhancing effect. This trend should be interpreted cautiously and warrants further studies with a larger sample size and pre-induction baseline fatigue assessment to determine whether spironolactone meaningfully influences exercise capacity under hypertensive stress without any standard drug use for comparison. Importantly, these observations should be contextualized within previous evidence showing that spironolactone reduces hypoxia markers in the DOCA model [27] and that clinical trials have reported mixed or modest effects of mineralocorticoid receptor antagonism on exercise performance [28], indicating that the antifatigue signal identified in the present study is mechanistic and hypothesis-generating rather than indicative of an established clinical benefit. Nonetheless, the present study is limited by the absence of direct enzymatic validation of FIH activity and HIF-1A hydroxylation status. Further in vitro and molecular assays—including co-crystallization, site-directed mutagenesis of the FIH binding pocket, and metabolic flux analysis—are warranted to confirm this proposed FIH–HIF-1A regulatory mechanism. These findings might also be validated through clinical studies that focus exclusively on the salt-sensitive hypertensive population to further strengthen the repositioning potential of spironolactone. It is also important to note that our study involves a small number of rats, which restricts the extent to which definitive functional claims can be made. 4. CONCLUSION In summary, the present study demonstrates that spironolactone mitigates fatigue associated with salt-sensitive hypertension through integrated modulation of hemodynamic stability and metabolic regulation. In the UNX-DOCA-salt model, spironolactone not only prevented the progressive increase in arterial pressure but also improved fatigue tolerance, accompanied by normalization of plasma sodium levels and downregulation of cardiac HIF-1α expression. Together, these findings suggest that spironolactone contributes to the restoration of ionic balance and oxygen-sensing regulation under hypertensive stress. Notably, through the integration of in vivo physiological data with functional enrichment and molecular modeling analyses, this study identifies the HIF-1α–glycolytic pathway as a novel mechanistic link underlying spironolactone-associated improvement of fatigue in salt-sensitive hypertension. Computational analyses further suggest a favorable interaction between spironolactone and factor inhibiting HIF (FIH), which may facilitate HIF-1α hydroxylation and attenuate hypoxia-driven metabolic activation. While further mechanistic validation is warranted, these findings support a broader role of spironolactone in modulating fatigue beyond its established antihypertensive effects. Declarations 5. AUTHOR CONTRIBUTION STATEMENT MRF, WA, RRT, and DS were concepting and designing the in vivo research; MRF and WA were concepting and designing the in silico research; MRF, WA, RRT, and DS performed research in vivo methodology; MRF, WA, RRT, DS, ERM, and DN performed data acquisition and analysis for in vivo research. MRF, WA, and FRM performed data acquisition and analysis for in silico research; MRF, WA, RRT, and DS wrote the original manuscript draft; Entire authors were reviewed and edited for the final manuscript. 6. ACKNOWLEDGMENT We would like to thank the animal research facilities IMERI, the Pharmacokinetic laboratory of the Faculty of Medicine, Universitas Indonesia, and the Dexa Laboratory Biomedical Science (DLBS) research assistant for their generous technical support and their provision of laboratory equipment. 7. DECLARE OF COMPETING INTEREST STATEMENT The authors affirmed no conflict of interest 8. DECLARATION ON THE USE OF ARTIFICIAL INTELLIGENCE All authors declare that an artificial intelligence tool (ChatGPT, OpenAI) was used exclusively for English language improvement, focusing on grammatical accuracy and textual clarity. The AI tool did not contribute to the generation of scientific content, experimental design, data analysis, interpretation, figures, or conclusions. All content was reviewed, edited, and finalized by the authors, and we retain full responsibility for the manuscript. 9. 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Oxf Open Mater Sci 5:itaf001. 10.1093/oxfmat/itaf001 Burguera S, Bauzá A, Frontera A (2023) Tuning the Nucleophilicity and Electrophilicity of Group 10 Elements through Substituent Effects: A DFT Study. Int J Mol Sci 24(21):15597. 10.3390/ijms242115597 Amador CA, Barrientos V, Peña J, Herrada AA, González M, Valdés S et al (2014) Spironolactone decreases DOCA-salt-induced organ damage by blocking the activation of T helper 17 and the downregulation of regulatory T lymphocytes. Hypertension 63(4):797–803. 10.1161/HYPERTENSIONAHA.113.02883 Kosmala W, Rojek A, Przewłocka-Kosmala M, Wright L, Mysiak A et al (2016) Effect of Aldosterone Antagonism on Exercise Tolerance in Heart Failure with Preserved Ejection Fraction. J Am Coll Cardiol 68(17):1823–1834. 10.1016/j.jacc.2016.07.763 Additional Declarations The authors declare no competing interests. Supplementary Files InsilicoSupplementaryFiles.docx Supplementary files of Molecular Docking Validation and Interaction Analysis Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":99670,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of spironolactone on blood pressure and exercise tolerance in the UNX-DOCA-salt rat model\u003cstrong\u003e.\u003c/strong\u003e (A) Systolic blood pressure (SBP), (B) diastolic blood pressure (DBP), (C) mean arterial pressure (MAP), and (D) time-to-fatigue measured using a weighted swimming exercise test in spironolactone-treated UNX-DOCA-salt rats. (E) Representative visualization of the weighted swimming exercise protocol. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8419825/v1/d1e00a39fc71620f85576f53.jpg"},{"id":98929151,"identity":"1337ade9-eb29-4e7a-91bc-50fbf35f5e9a","added_by":"auto","created_at":"2025-12-24 08:30:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48693,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of spironolactone on plasma sodium concentration and cardiac HIF-1α expression\u003cstrong\u003e.\u003c/strong\u003e(A) Plasma sodium concentration and (B) hypoxia-inducible factor-1 alpha (HIF-1α) mRNA expression in cardiac tissue of spironolactone-treated UNX-DOCA-salt rats compared with untreated UNX-DOCA-salt controls. HIF-1α mRNA levels were quantified by real-time PCR and normalized to the appropriate housekeeping gene. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8419825/v1/4c80f3a800e679cf3a2bd2dd.jpg"},{"id":99310296,"identity":"631c68d3-32ab-4935-b7c1-1edceaa69d6e","added_by":"auto","created_at":"2025-12-31 16:12:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":146944,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional enrichment and pathway analysis of spironolactone-associated target genes. (A) Venn diagram illustrating the intersection between predicted spironolactone target genes and genes involved in the HIF-1α signaling pathway. (B) Representative spironolactone-associated genes within the HIF-1α signaling pathway based on the KEGG database. (C) Pathway enrichment analysis of spironolactone target genes. GO enrichment analysis showing (D) biological processes, (E) cellular components, and (F) molecular functions.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8419825/v1/1306e8446068b7199279994a.jpg"},{"id":99310929,"identity":"021a4765-4956-4f7d-a258-0a4f36523e27","added_by":"auto","created_at":"2025-12-31 16:13:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":145418,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking and binding energy analysis of spironolactone interaction with HIF-1α-related targets. (A) MolDock score, (B) hydrogen bond energy, (C) free binding energy, (D) van der waals energy, (E) electrostatic energy, (F) polar solvation energy, (D) nonpolar solvation energy, and (G) total solvation penaly obtained from MMGB(PB)SA; (I) molecular docking validation showing the binding pose of the native ligand, and (J) predicted binding pose of spironolactone within the target binding site.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8419825/v1/448151affbda7621f0faca8d.jpg"},{"id":99310775,"identity":"5c144c08-2f1e-467c-82a0-e50a8f5a4f0d","added_by":"auto","created_at":"2025-12-31 16:13:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58937,"visible":true,"origin":"","legend":"\u003cp\u003eProposed mechanism by which spironolactone modulates HIF-1α signaling and improves fatigue in salt-sensitive hypertensive rats.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8419825/v1/7ba341347585704930cae639.jpg"},{"id":100949289,"identity":"9a57e7ff-1f52-4220-82df-69929499e459","added_by":"auto","created_at":"2026-01-23 06:57:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1497864,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8419825/v1/4cad6fa6-fb69-4a78-b6f6-4c7cf4026d82.pdf"},{"id":99310336,"identity":"e7b45d72-7b51-47d1-9a29-054c62d99b1d","added_by":"auto","created_at":"2025-12-31 16:12:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19178,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary files of Molecular Docking Validation and Interaction Analysis\u003c/p\u003e","description":"","filename":"InsilicoSupplementaryFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-8419825/v1/7f25749abf7b84f09adcd969.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eRepurposing of Spironolactone for Hypertension-Related Fatigue: Experimental Evidence from Salt-Sensitive Rats with Computational Molecular Analysis\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eHypertension remains one of the most prevalent chronic diseases worldwide and continues to pose a substantial public health challenge. Recent estimates indicate that approximately 1.4\u0026nbsp;billion adults aged 30\u0026ndash;79 years were living with hypertension in 2024, with a disproportionate burden observed in low- and middle-income countries. Despite the broad availability of antihypertensive therapies, a considerable proportion of affected individuals remain undiagnosed or inadequately controlled. As a result, hypertension persists as a leading contributor to premature mortality and long-term disability, largely through its association with cardiovascular, cerebrovascular, and renal complications.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eBeyond its high prevalence, hypertension is increasingly recognized as a multisystem disorder characterized by complex pathophysiological mechanisms, including sustained sympathetic activation, endothelial dysfunction, oxidative stress, inflammation, and maladaptive neurohormonal signaling. Chronic exposure to elevated blood pressure leads to progressive target-organ damage, manifesting as left ventricular hypertrophy, myocardial fibrosis, vascular remodeling, and renal impairment. These structural and functional alterations substantially increase the risk of ischemic heart disease, heart failure, stroke, and chronic kidney disease. Moreover, hypertension frequently coexists with metabolic comorbidities, further complicating disease management and contributing to impairments in cardiovascular performance, reduced physical capacity, and diminished exercise tolerance\u0026mdash;clinical features that are increasingly recognized as integral components of hypertension-related fatigue.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eHypertension-related fatigue represents a multifactorial condition characterized not only by elevated blood pressure but also by reduced exercise tolerance.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] In preclinical studies, the interplay between mineralocorticoid signaling and cardiac stress has been increasingly recognized as a key contributor to both hemodynamic changes and exercise intolerance.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eSpironolactone, a classical mineralocorticoid receptor antagonist, has long been used in clinical practice for the management of resistant hypertension and stable heart failure.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Recent evidence, however, has suggested that spironolactone\u0026rsquo;s therapeutic potential may extend beyond classical hemodynamic modulation toward improving exercise capacity and attenuating fatigue-related cardiovascular dysfunction. Preclinical studies in rodent models have demonstrated that spironolactone can modulate molecular pathways associated with hypoxia-inducible factor 1 alpha (HIF-1α) oxidative stress and inflammation, which are all implicated in fatigue and exercise intolerance in hypertensive states.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eSeveral debates have reached controversy over whether spironolactone either improved[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] or did not improve[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] exercise intolerance in cardiovascular patients related to hypertension conditions, even though it overall reduces patients\u0026rsquo; mortality and morbidity.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Inconsistency results might come from diverse phenotypes of disease, as spironolactone primarily works on the renin-angiotensin-aldosterone systems. Among these, salt-sensitive hypertension represents a distinct phenotype in which mineralocorticoid signaling plays a pivotal role. This highlights the need for robust preclinical evidence to elucidate how spironolactone modulates not only hemodynamic regulation but also exercise tolerance in salt-sensitive conditions.\u003c/p\u003e \u003cp\u003eAnimal models induced by uninephrectomy combined with deoxycorticosterone acetate administration (DOCA) and high-salt intake (UNX-DOCA-Salt) have been established to reproduce the salt-sensitive phenotype commonly observed in resistant hypertension.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] While spironolactone has been reported to attenuate cardiac remodeling and fibrotic changes in this model [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], Its influence on exercise intolerance has not been clearly defined. Recent experimental observations indicate that spironolactone may interfere with the hypoxia-inducible factor 1α (HIF-1α) signaling axis in DOCA-induced hypertension.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], Thereby modulating downstream molecular responses that possibly contribute to cardiac stress adaptation and reduced exercise capacity.\u003c/p\u003e \u003cp\u003eWhile the UNX\u0026ndash;DOCA\u0026ndash;Salt animal models provides valuable insight into the physiological and molecular effects of spironolactone in salt-sensitive hypertension, it remains essential to clarify the molecular mechanisms that may link spironolactone exposure to fatigue-related pathways. To complement the in vivo findings, we incorporated computational analyses to characterize potential molecular interactions and signaling modulations involving HIF-1α. Previous studies have associated spironolactone with anti-hypoxia and HIF-related mechanisms in DOCA-salt hypertension; however, based on our updated literature search, we were unable to identify studies that integrate HIF-1α-centered \u003cem\u003ein silico\u003c/em\u003e predictions with fatigue or exercise-tolerance outcomes in hypertensive rodents. Therefore, the present research employed an integrative approach combining in vivo assessment of hypertension-related fatigue in UNX\u0026ndash;DOCA\u0026ndash;Salt rats with stepwise computational analyses, including functional enrichment of spironolactone\u0026ndash;HIF-1α targets, molecular docking, mechanics generalized Born/Poisson\u0026ndash;Boltzmann surface area (MMGB(PB)SA) free-energy estimation, and density functional theory (DFT)-derived electronic descriptors. The aim of this strategy was not to assert a performance-enhancing effect of spironolactone, but rather to explore whether molecular modulation of HIF-1α signaling could correspond with changes in fatigue physiology under hypertensive stress. By aligning molecular predictions with physiological readouts, this approach provides a mechanistic basis for future studies on drug repositioning and targeted validation of spironolactone\u0026rsquo;s effects on hypoxia-related metabolic pathways.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal Experimentation, Ethics, Welfare Monitoring, and Humane Endpoints\u003c/h2\u003e \u003cp\u003eMale Sprague\u0026ndash;Dawley rats, aged 8\u0026ndash;10 weeks and weighing approximately 200\u0026ndash;250 g, were acclimated for seven days before experimentation. The animals were housed under controlled environmental conditions with a 12:12 h light\u0026ndash;dark cycle and had free access to standard laboratory chow pellets (Rat Bio\u0026reg;, Citrafeed, Indonesia; containing approximately 20% crude protein, 4% crude fat, 4\u0026ndash;5% crude fiber, and \u0026le;\u0026thinsp;12% moisture) and water ad libitum. A total of 15 rats were (n\u0026thinsp;=\u0026thinsp;5 rats per group) randomly allocated into three groups: (1) Sham control (sham surgery\u0026thinsp;+\u0026thinsp;vehicle, carboxymethylcellulose (CMC) 0.5%), (2) UNX-DOCA-Salt (uninephrectomy\u0026thinsp;+\u0026thinsp;DOCA-salt induction\u0026thinsp;+\u0026thinsp;vehicle), and (3) UNX-DOCA-Salt\u0026thinsp;+\u0026thinsp;Spiro (uninephrectomy\u0026thinsp;+\u0026thinsp;DOCA-salt induction\u0026thinsp;+\u0026thinsp;spironolactone). Left uninephrectomy was performed in a sterile surgical facility under ketamine (100 mg/kg BW, i.p.) and xylazine (10 mg/kg BW, i.p.) anesthesia for all experimental animals. After a 1-week recovery period, DOCA-salt hypertension was induced by twice-weekly subcutaneous injections of deoxycorticosterone acetate (25 mg/kg BW in 0.1 mL dimethylformamide), together with \u003cem\u003ead libitum\u003c/em\u003e with 1% NaCl solution for two weeks. Blood pressure was then measured noninvasively before beginning the treatment phase, in which animals received either spironolactone (100 mg/kg BW/day in 0.5% CMC, p.o.), as previously reported, [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] or a vehicle for five weeks.\u003c/p\u003e \u003cp\u003e All of the experimental protocols were reviewed and approved by the Ethics Committee of the Faculty of Medicine, Universitas Indonesia, with ethics number (KET 1551/UN2.F1/ETIK/PPM.00.02/2024). Use of postsurgical analgesia was exempted based on scientific rationale granted by the Ethics Committee, as common analgesics in rats (non-steroidal anti-inflammatory drugs and opioids) due to the potential interference of analgesia with the blood pressure; thus, animal welfare monitoring was intensified. To ensure animal welfare despite the absence of analgesia, rats were monitored twice daily for body weight, food and water intake, hydration, locomotor activity, scrotal swelling, and general appearance. Humane endpoints were predefined (\u0026gt;\u0026thinsp;15% body-weight loss, persistent immobility, or respiratory distress), and no animal reached these endpoints or required early sacrifice. No unexpected mortality occurred throughout the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Blood Pressure Measurement\u003c/h2\u003e \u003cp\u003eBlood pressure was measured using noninvasive methods (CODA Scientific\u0026reg;) in rats. Briefly, the rats were acclimatized before data collection for 15\u0026ndash;30 minutes. Systolic blood pressure (SBP, mmHg), diastolic blood pressure (DBP, mmHg), and heart rate (beats per minute (bpm)) were measured and compared using internal control from weeks 0, 2, and 5. In addition, mean arterial pressure (MAP) changes were also determined using below formula:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:MAP=DBP+\\:\\frac{SBP-DBP}{3}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Weighted Swimming Exercise Test\u003c/h2\u003e \u003cp\u003eRats' exercise capacity was determined using a swimming exercise test to estimate tolerance against high-intensity activity. Rats were weighed, and a load equivalent to 2% of body weight was tied to the tail. Before testing, rats were acclimatized in the swimming tube for 15 minutes/day without weight for three consecutive days. The swimming apparatus consisted of a cylindrical tube (40 cm diameter, 60 cm height) filled with water to a depth of 50 cm, and the temperature was maintained at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. During the test session, two independent blind observers monitored the animals. Time-to-fatigue (seconds) served as the primary performance variable and was defined based on predefined exhaustion criteria: failure to reach the surface for 10 consecutive seconds and loss of coordinated swimming movements. A humane endpoint and a maximum cut-off duration of 15 minutes were applied to avoid excessive physical stress with additional warming using an animal incubator. Swimming sessions were video recorded for welfare documentation, and fatigue time was recorded manually using a stopwatch according to the exhaustion criteria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Sodium Plasma Concentration\u003c/h2\u003e \u003cp\u003eThe sodium plasma concentration was determined using blood collected from rats retroorbital. After centrifuging at 5.000 g x 15 minutes, the plasma was separated and collected. The sodium concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit, Elabscience, according to a standardized protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Isolation of Total mRNA, cDNA synthesis, and qPCR of Cardiac HIF-1A mRNA expression\u003c/h2\u003e \u003cp\u003eA total of 100 mg of cardiac tissue was isolated from sacrificed animals using DirectZol \u0026reg; TriReagent according to the predetermined protocols. Further, 500 ng of purified (A230/A260 1.8-2.0) mRNA were transcribed in each group for cDNA using the ReverTra Ace Toyobo kit. The reacted solution then performed qPCR using HIF1A primer (Forward primer 5\u0026rsquo;-AATCCATTTTCAGCTCAGGAC-3\u0026rsquo;; Reverse primer 5\u0026rsquo;-GGCAGTGACAGTGATGGTAGG-3\u0026rsquo;; Bp product 198; T\u003csub\u003em\u003c/sub\u003e: 60\u0026deg;C) and normalized using beta-actin (Forward primer 5\u0026rsquo;-TGTTGTCCCTGTATGCCTCT-3\u0026rsquo;; Reverse primer 5\u0026rsquo;-TAATGTCACGCACGATTTCC-3\u0026rsquo;; Bp product 222; T\u003csub\u003em\u003c/sub\u003e: 60\u0026deg;C) as a housekeeping gene. The relative mRNA expression was calculated using the Livak method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Functional Enrichment of Spironolactone HIF-1A Related Gene Targets\u003c/h2\u003e \u003cp\u003eTo elucidate the potential mechanistic relationship between spironolactone and the HIF-1α signaling pathway, a multi-step \u003cem\u003ein silico\u003c/em\u003e workflow was performed. Genes involved in the HIF 1α signaling pathway were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), while spironolactone-related targets were retrieved from the Comparative Toxicogenomics Database (CTD; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ctdbase.org/\u003c/span\u003e\u003cspan address=\"https://ctdbase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The intersecting targets between these two datasets were determined using the JVENN web platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/Venn/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/Venn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify common molecular nodes potentially modulated by spironolactone within the HIF-1α pathway. The intersected genes were further analyzed in ShinyGO v0.85 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to explore enriched pathways and Gene Ontology (GO) categories, including Biological Process, Molecular Function, and Cellular Component, using Fisher\u0026rsquo;s exact test with Benjamini-Hochberg false discovery rate (FDR) correction (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05). This integrative approach provided a systems-level overview of spironolactone\u0026rsquo;s functional targets potentially linked to HIF-1α regulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Molecular Docking of HIF-1α protein\u003c/h2\u003e \u003cp\u003eThe crystal structure of the human factor inhibiting HIF-1α (FIH1; PDB ID: 8II0) was retrieved from the Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/structure/8II0\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/structure/8II0\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), as FIH1 represents a key regulatory enzyme that hydroxylates (asparaginyl hydroxylase) and thereby inactivates HIF-1α under normoxic conditions. Human FIH protein was chosen as the rat protein had not been present yet on the PDB website. The protein structure was preprocessed and corrected using PDBFixer under Windows Subsystem for Linux (WSL) to remove alternate conformations, add missing residues, and optimize hydrogen geometry. Validation of the docking protocol was performed by redocking the native ligand into the active site, ensuring a root mean square deviation (RMSD) value\u0026thinsp;\u0026lt;\u0026thinsp;2.0 \u0026Aring;, thus confirming the reliability of the docking setup. Docking simulations were conducted using Molegro Virtual Docker (MVD), with all hydrogen atoms added at pH 7.4 for both protein and ligands. The 3D structure of spironolactone was obtained from PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and protonated at pH 7.4 before docking. Comparative docking analysis between spironolactone and the native inhibitor was performed based on MolDock Score, Re-rank Score, and Hydrogen Bond Energy for the five top-ranked poses. Binding interactions were visualized using LigPlot, with particular attention to hydrogen bonding at Tyr102, which represents a critical residue in the FIH1 catalytic pocket.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 MMGB(PB)SA Analysis\u003c/h2\u003e \u003cp\u003ePost-docking free energy estimations were performed using the MMGB(PB)SA approach to provide a more comprehensive insight into the binding thermodynamics beyond docking scores. This analysis was conducted on WSL platform utilizing the UniGBSA-pipeline, which automates the process of parameterization, trajectory generation, and energy decomposition. The top five poses of both the native ligand and spironolactone were extracted from the docking results and exported in MOL2 format. Each ligand\u0026ndash;protein complex, along with the cleaned FIH1 receptor structure, was subsequently parameterized using the ACPYPE (Antechamber Python Parser Interface) tool to generate topology and coordinate files under the AMBER force field, applying GAFF parameters for ligands and assigning partial charges through the AM1-BCC method. The MMGB(PB)SA calculations were then performed with UniGBSA to estimate the binding free energy (ΔG_bind) using the following equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{\\Delta\\:}{G}_{bind}={\\Delta\\:}{E}_{MM}+{\\Delta\\:}{G}_{solvation}-T{\\Delta\\:}S$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\Delta\\:}{E}_{MM}\\)\u003c/span\u003e\u003c/span\u003erepresents the molecular mechanics energy (van der Waals\u0026thinsp;+\u0026thinsp;electrostatic interactions), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\Delta\\:}{G}_{solvation}\\)\u003c/span\u003e\u003c/span\u003eis the solvation free energy (polar\u0026thinsp;+\u0026thinsp;nonpolar contributions), and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:T{\\Delta\\:}S\\)\u003c/span\u003e\u003c/span\u003e Denotes the entropic term (approximated or neglected in some setups). The MMGBSA method complements molecular docking by accounting for solvent effects and molecular flexibility, thereby providing a more realistic estimate of binding affinity that docking scores alone cannot fully capture. For each ligand, mean and standard deviation values of ΔG_bind across the five poses were calculated to assess stability and reproducibility of the predicted binding energies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Density Functional Theory\u003c/h2\u003e \u003cp\u003eTo characterize the electronic properties and reactivity profiles of spironolactone and the native FIH1 ligand, DFT calculations were performed using the Python-based Simulations of Chemistry Framework package implemented under the WSL environment. Before calculation, the optimized 3D geometries of both ligands were obtained from PubChem and preprocessed to ensure proper protonation at physiological pH (7.4).\u003c/p\u003e \u003cp\u003eThe geometry optimization and subsequent single-point energy calculations were carried out using the B3LYP functional with the 6-31G(d,p) basis set, which provides a balanced accuracy\u0026ndash;cost trade-off for organic molecules. The convergence criteria were set to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{10}^{-6}\\)\u003c/span\u003e\u003c/span\u003e a.u. for energy and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{10}^{-4}\\)\u003c/span\u003e\u003c/span\u003e a.u. for gradient, ensuring optimized and stable geometries. Solvent effects were simulated using the Polarizable Continuum Model (PCM) with water as the dielectric medium (ε\u0026thinsp;=\u0026thinsp;78.4).\u003c/p\u003e \u003cp\u003eThe outputs included HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) energies, the energy gap (ΔE\u0026thinsp;=\u0026thinsp;E\u003csub\u003eLUMO\u003c/sub\u003e \u0026minus; E\u003csub\u003eHOMO\u003c/sub\u003e), and derived descriptors such as electronegativity (χ), hardness (η), and softness (S) according to Koopmans\u0026rsquo; theorem:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\chi\\:=-\\frac{{E}_{HOMO}+{E}_{LUMO}}{2},\\eta\\:=\\frac{{E}_{LUMO}-{E}_{HOMO}}{2},S=\\frac{1}{2\\eta\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e experimental data were analyzed descriptively and comparatively using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). Descriptive data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was used to assess overall group differences, followed by Tukey\u0026rsquo;s multiple comparison test for normally distributed data or Dunn\u0026rsquo;s post-hoc test for non-normally distributed data when appropriate. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. No prior sample size or power calculation was performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The effect of spironolactone on blood pressure and the weighted swimming exercise test\u003c/h2\u003e \u003cp\u003eTwo-way ANOVA showed a significant time effect on both systolic and diastolic blood pressure in UNX-DOCA-salt rats treated with spironolactone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). For SBP, the mean value increased markedly from week 0 (136.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 mmHg) to week 2 (199.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9 mmHg; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0017), confirming the successful induction of hypertension. Following spironolactone administration, SBP declined at week 5 (177.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9 mmHg; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0085 vs. week 0), though the difference between week 2 and week 5 was not significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0663). A similar pattern was observed in DBP, which significantly increased from week 0 to week 2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002) and decreased again by week 5 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0011) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C\u003cb\u003e)\u003c/b\u003e. These findings indicate that spironolactone effectively attenuates the rise in blood pressure induced by UNX-DOCA-salt treatment, consistent with its established role as an antihypertensive agent acting through mineralocorticoid receptor blockade.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the end of the fifth week, the weighted swimming test revealed that the UNX-DOCA-Salt group showed a significantly shorter time to fatigue compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating reduced exercise tolerance due to hypertension-induced cardiac and systemic dysfunction. Treatment with spironolactone slightly improved the duration of fatigue, although the difference was not statistically significant compared to the DOCA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD \u003cb\u003eand Figure E\u003c/b\u003e).\u003c/p\u003e \u003cp\u003ePrevious reports in human studies have also suggested a potential antifatigue or exercise-performance benefit of spironolactone [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite some contradictory findings regarding spironolactone, eplerenone has demonstrated more consistent efficacy in reducing fatigue, likely due to its stronger binding affinity to mineralocorticoid receptors compared with spironolactone, which may explain the discrepancy between studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this context, our findings support a possible antifatigue effect of spironolactone. Nevertheless, this interpretation should be approached with caution, particularly considering patient demographics\u0026mdash;such as salt-sensitive individuals\u0026mdash;for whom subgroup analyses in previous trials of exercise intolerance remain limited. Therefore, further studies are needed to clarify whether the antifatigue benefit of spironolactone is generalizable across different populations or specific to certain physiological profiles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The effect of spironolactone on plasma sodium concentration and cardiac tissue mRNA HIF1A expression\u003c/h2\u003e \u003cp\u003eThe plasma sodium concentration was significantly elevated in the UNX-DOCA-Salt group compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming the successful induction of salt-sensitive hypertension. Administration of spironolactone markedly reduced plasma sodium levels compared to the UNX-DOCA-Salt group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating its effectiveness in attenuating sodium retention \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. This finding aligns with the pharmacological action of spironolactone as a mineralocorticoid receptor antagonist, which counteracts aldosterone-induced sodium reabsorption in the distal nephron. By reducing sodium overload, spironolactone contributes to improved volume regulation and blood pressure control in mineralocorticoid-induced hypertension. A previous study has demonstrated a similar effect of spironolactone in mitigating sodium retention and vascular remodelling in UNX-DOCA-salt hypertensive models through Na+/H\u0026thinsp;+\u0026thinsp;exchanger.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative expression of HIF1A mRNA was significantly increased in the UNX-DOCA-Salt group compared with the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the hypertensive condition induced by UNX-DOCA-salt was associated with upregulation of hypoxia-related signalling. Treatment with spironolactone reduced the elevated HIF1A expression compared to the UNX-DOCA-Salt group, although the reduction did not fully normalize the levels to those of the control group.\u003c/p\u003e \u003cp\u003eThis result suggests that spironolactone partially attenuates cardiac hypoxic stress in the UNX-DOCA-salt model. The decrease in HIF1A expression likely reflects an improvement in myocardial oxygen balance and perfusion following aldosterone receptor blockade. Previous studies have shown that spironolactone mitigates tissue hypoxia and oxidative stress through inhibition of mineralocorticoid receptor\u0026ndash;mediated in different tissues.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The functional enrichment of spironolactone on fatigue-related gene/protein\u003c/h2\u003e \u003cp\u003eBioinformatic integration analysis revealed that spironolactone shared 95 common target genes with HIF-1A\u0026ndash;associated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating a strong molecular intersection between Spiro activity and the hypoxia-related transcriptional network. KEGG pathway enrichment further showed that these overlapping genes were predominantly involved in the HIF-1 signaling pathway, with several components\u0026mdash;such as EGFR, AKT1, mTOR, VEGFA, and LDHA\u0026mdash;highlighted in the pathway map (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Pathway enrichment ranking also identified HIF-1α signaling as the most significantly enriched pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), followed by other metabolic and signaling cascades, including PI3K-Akt, mTOR, and glycolysis/gluconeogenesis pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional enrichment analysis identified several predominant biological themes among the Spironolactone\u0026ndash;HIF-1α overlapping genes. The enriched terms were mainly related to glycolytic, pyruvate, and adenosine triphosphate metabolic processes, suggesting an overall enhancement of cellular energy turnover. Structural enrichment highlighted enzyme complexes such as phosphofructokinase and vesicular components, implying potential modulation of intracellular trafficking and metabolic compartmentalization. In parallel, enrichment in kinase- and nucleotide-binding activities indicated a broad regulation of enzymatic catalysis and phosphorylation-dependent signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003eThe current enrichment analysis revealed that spironolactone-regulated genes predominantly intersect with glycolytic biosynthesis, 6-phosphofructokinase complex formation, and carbohydrate kinase activity, reflecting a coordinated modulation of HIF-1α\u0026ndash;mediated metabolic processes. HIF-1α serves as a central transcriptional regulator of cellular adaptation or metabolic switch in hypoxic conditions by promoting glycolysis and limiting oxidative phosphorylation\u0026mdash;mechanisms that are highly relevant in hypertensive organ remodeling or other similar insults, where oxygen utilization becomes inefficient.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Clinically, spironolactone treatment has been associated with reduced HIF-1α expression following ischemic or surgical stress, supporting its role in normalizing cellular hypoxia signaling, which was also observed in our experimental study.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Collectively, these findings imply that spironolactone may attenuate hypertension-induced fatigue and maladaptive metabolic reprogramming by partially suppressing HIF-1α activation and restoring energy homeostasis, thereby linking its molecular effects to improved functional outcomes in hypertensive models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 The Molecular docking and MMGB(PB)SA\u003c/h2\u003e \u003cp\u003eDocking was validated using RMSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The binding box was coordinated according to the native ligand, with x, y, and z located on 23.07, -25.87, and 3.30, respectively, with a radius of 11. The molecular docking RMSD was consistently below 2 \u0026Aring; (\u003cb\u003eTable S1\u003c/b\u003e), which tightly focuses on Tyr102 as an anchoring target in the binding box. The validation finding will be further complemented with MMGB(PB)SA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Molecular docking and MMGB(PB)SA results\u003c/h2\u003e \u003cp\u003eMolecular docking analysis demonstrated that both the native ligand and spironolactone exhibited strong binding affinity toward the FIH (factor inhibiting HIF-1α) enzyme. The native ligand showed a slightly stronger binding profile (Moldock score: \u0026minus;\u0026thinsp;128.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 a.u.; Rerank score: \u0026minus;\u0026thinsp;105.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 a.u.) compared to spironolactone (Moldock score: \u0026minus;\u0026thinsp;115.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 a.u.; Rerank score: \u0026minus;\u0026thinsp;75.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.5 a.u.). Despite this, both ligands formed energetically stable complexes. The hydrogen bonding energy of the native ligand (\u0026ndash;8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 kJ/mol) was slightly higher than that of spironolactone (\u0026ndash;5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 kJ/mol), consistent with the greater number of hydrogen bonds observed in the native complex (five H-bonds) compared to spironolactone (four H-bonds).\u003c/p\u003e \u003cp\u003eMMGB(PB)SA energy decomposition further supported these observations. The total binding free energy (ΔG_total) of spironolactone averaged \u0026minus;\u0026thinsp;32.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 kJ/mol, which was comparable to that of the native ligand (\u0026ndash;26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 kJ/mol). The van der Waals contribution dominated the stabilization for both ligands, being slightly more favorable in spironolactone (\u0026ndash;34.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 kJ/mol) than in the native complex (\u0026ndash;28.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 kJ/mol). Meanwhile, electrostatic energy was negligible in spironolactone (\u0026asymp;\u0026thinsp;0 kJ/mol) but markedly higher in the native ligand (+\u0026thinsp;42.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 kJ/mol), indicating that spironolactone relies less on charge\u0026ndash;charge interactions and more on hydrophobic stabilization. Interestingly, the polar solvation term was positive for spironolactone (+\u0026thinsp;9.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 kJ/mol) but negative for the native ligand (\u0026ndash;35.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 kJ/mol), implying that reduced desolvation penalties accompany spironolactone binding. The nonpolar solvation energy (\u0026ndash;5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 kJ/mol) was consistently favorable in both complexes. Collectively, these results suggest that spironolactone achieves thermodynamically stable binding primarily through van der Waals and solvation-driven mechanisms rather than electrostatic attraction. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-H)\u003c/p\u003e \u003cp\u003eResidue-level interaction profiling based (\u003cb\u003eTable S2\u003c/b\u003e) on LigPlot visualization revealed that the native ligand formed seven hydrogen bonds with Asn294(A) (2.94 \u0026Aring;), Arg238(A) (2.39 \u0026Aring;), Lys214(A) (2.47 \u0026Aring;), Thr196(A) (2.71 \u0026Aring;), Gln147(A) (3.31 \u0026Aring;), Tyr145(A) (3.01 \u0026Aring;), and Tyr102(A) (3.00 \u0026Aring;), securing its orientation within the catalytic pocket of FIH. In contrast, spironolactone established four major hydrogen bonds involving Arg238(A) (3.12 \u0026Aring;), Gln203(A) (3.04 \u0026Aring;), Glu202(A) (3.22 \u0026Aring;), and Asp201(A) (3.10 \u0026Aring;), while maintaining hydrophobic contacts with Tyr102(A) (3.30\u0026ndash;3.81 \u0026Aring;), Leu186(A) (2.70\u0026ndash;3.30 \u0026Aring;), Phe207(A) (3.60\u0026ndash;3.70 \u0026Aring;), and Trp296(A) (2.97\u0026ndash;3.68 \u0026Aring;). Notably, Tyr102(A) emerged as a critical anchoring residue positioned near the iron coordination center, consistent with its conserved role in ligand stabilization across HIF-1α inhibitor complexes. The hydrophobic interactions surrounding Tyr102(A) and Arg238(A) further reinforced the compactness and rigidity of the spironolactone-FIH complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-K).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 DFT of Spironolactone\u003c/h2\u003e \u003cp\u003eThe quantum chemical parameters of the native ligand and spironolactone were calculated using DFT to evaluate their stability and electronic reactivity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The HOMO and LUMO energies for the native ligand were \u0026minus;\u0026thinsp;6.65 eV and \u0026minus;\u0026thinsp;1.85 eV, respectively, resulting in a gap of 4.81 eV. In contrast, spironolactone exhibited a narrower gap of 0.89 eV, with higher HOMO (\u0026ndash;1.72 eV) and LUMO (\u0026ndash;0.83 eV) energies. Meanwhile, spironolactone displayed lower electronegativity (1.27 eV) and hardness (0.45 eV) but markedly higher softness (30.52 eV⁻\u0026sup1;) compared with the native ligand.\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\u003eQuantum chemical parameters of native ligands and spironolactone\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHOMO (eV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLUMO (eV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGap (eV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eElectronegativity (χ)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHardness (η)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSoftness (S)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-6.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpironolactone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e30.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eHOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital. Quantum chemical parameters were calculated to evaluate the electronic properties and reactivity of spironolactone relative to the native ligand.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe DFT-based quantum chemical analysis demonstrated that spironolactone exhibits higher electronic reactivity and greater molecular softness compared with the native ligand. Its elevated HOMO energy and narrower energy gap indicate a more polarizable and electron-donating character, which facilitates charge-transfer and soft\u0026ndash;soft interactions with electron-deficient residues at the protein binding site. This observation aligns with the hard and soft acids and bases theory and the frontier molecular orbital.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] In contrast, the native ligand\u0026rsquo;s wider energy separation and higher electronegativity suggest greater intrinsic stability and a more electrophilic nature, consistent with descriptors of chemical hardness and electrophilicity. These findings collectively imply that spironolactone\u0026rsquo;s softer electronic profile may underline its stronger charge-transfer capacity and potential for enhanced molecular interaction within the protein active site.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe integrated evaluation of quantum chemical descriptors and interaction profiles reveals a consistent pattern between the electronic characteristics and binding behavior of the ligands. Spironolactone, exhibiting a higher HOMO energy, a narrower HOMO\u0026ndash;LUMO gap, and greater molecular softness, can be characterized as a more electron-donating and polarizable molecule. This electronic configuration is reflected in the LigPlot-derived interaction map, where spironolactone establishes multiple hydrogen bonds and close polar contacts with acidic and polar residues, particularly Asp201, Glu202, and Gln203, along with a polarizable sulfur-containing moiety that may further contribute to complex stabilization. In contrast, the native ligand\u0026mdash;defined by a wider energy gap and higher electronegativity\u0026mdash;demonstrates a more dispersed and rigid interaction profile, forming contacts with a broader range of polar and aromatic residues such as Asn294, Arg238, Trp296, and Tyr102, but exhibiting comparatively fewer deep interactions with the acidic residue cluster. Collectively, these findings suggest that spironolactone interacts with the target protein through a more dynamic, donor-driven, and adaptive binding mode, whereas the native ligand displays a relatively stable and electrophile-oriented interaction pattern.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Research Implications and Study Limitations\u003c/h2\u003e \u003cp\u003eThe present findings provide a mechanistic extension of spironolactone\u0026rsquo;s pharmacological profile beyond its classical role as a mineralocorticoid receptor antagonist. In the UNX\u0026ndash;DOCA\u0026ndash;Salt hypertensive model, spironolactone prevented the progressive rise of systolic, diastolic, and mean arterial pressure, accompanied by normalization of plasma Na⁺ and suppression of cardiac HIF-1A mRNA expression, suggesting partial restoration of electrolyte imbalance and oxygen-sensing homeostasis. Functional enrichment and molecular docking analyses further identified HIF-1A and glycolytic pathways as major interaction hubs, while the predicted binding of spironolactone within the FIH catalytic pocket \u0026mdash; supported by coordinate interaction mapping and DFT-derived electronic descriptors \u0026mdash; suggests a potential modulation of the FIH\u0026ndash;HIF-1α regulatory axis. Although this hypothesis remains computational and requires biochemical validation, the \u003cem\u003ein silico\u003c/em\u003e interactions suggest that spironolactone may facilitate HIF-1α hydroxylation and turnover via donor-type electronic interactions with key residues in the FIH active site (Asp201, Glu202, Gln203, Tyr102).\u003c/p\u003e \u003cp\u003eA trend toward increased time-to-fatigue was observed in spironolactone-treated rats; however, this change did not reach statistical significance, and therefore, the findings cannot confirm an antifatigue or exercise-enhancing effect. This trend should be interpreted cautiously and warrants further studies with a larger sample size and pre-induction baseline fatigue assessment to determine whether spironolactone meaningfully influences exercise capacity under hypertensive stress without any standard drug use for comparison. Importantly, these observations should be contextualized within previous evidence showing that spironolactone reduces hypoxia markers in the DOCA model [27] and that clinical trials have reported mixed or modest effects of mineralocorticoid receptor antagonism on exercise performance [28], indicating that the antifatigue signal identified in the present study is mechanistic and hypothesis-generating rather than indicative of an established clinical benefit.\u003c/p\u003e \u003cp\u003eNonetheless, the present study is limited by the absence of direct enzymatic validation of FIH activity and HIF-1A hydroxylation status. Further \u003cem\u003ein vitro\u003c/em\u003e and molecular assays\u0026mdash;including co-crystallization, site-directed mutagenesis of the FIH binding pocket, and metabolic flux analysis\u0026mdash;are warranted to confirm this proposed FIH\u0026ndash;HIF-1A regulatory mechanism. These findings might also be validated through clinical studies that focus exclusively on the salt-sensitive hypertensive population to further strengthen the repositioning potential of spironolactone. It is also important to note that our study involves a small number of rats, which restricts the extent to which definitive functional claims can be made.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eIn summary, the present study demonstrates that spironolactone mitigates fatigue associated with salt-sensitive hypertension through integrated modulation of hemodynamic stability and metabolic regulation. In the UNX-DOCA-salt model, spironolactone not only prevented the progressive increase in arterial pressure but also improved fatigue tolerance, accompanied by normalization of plasma sodium levels and downregulation of cardiac HIF-1α expression. Together, these findings suggest that spironolactone contributes to the restoration of ionic balance and oxygen-sensing regulation under hypertensive stress.\u003c/p\u003e \u003cp\u003eNotably, through the integration of \u003cem\u003ein vivo\u003c/em\u003e physiological data with functional enrichment and molecular modeling analyses, this study identifies the HIF-1α\u0026ndash;glycolytic pathway as a novel mechanistic link underlying spironolactone-associated improvement of fatigue in salt-sensitive hypertension. Computational analyses further suggest a favorable interaction between spironolactone and factor inhibiting HIF (FIH), which may facilitate HIF-1α hydroxylation and attenuate hypoxia-driven metabolic activation. While further mechanistic validation is warranted, these findings support a broader role of spironolactone in modulating fatigue beyond its established antihypertensive effects.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5. AUTHOR CONTRIBUTION STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRF, WA, RRT, and DS\u003c/strong\u003e were concepting and designing the \u003cem\u003ein vivo\u003c/em\u003e research; \u003cstrong\u003eMRF and WA\u0026nbsp;\u003c/strong\u003ewere concepting and designing the \u003cem\u003ein silico\u003c/em\u003e research; \u003cstrong\u003eMRF, WA, RRT, and DS\u0026nbsp;\u003c/strong\u003eperformed research \u003cem\u003ein vivo\u003c/em\u003e methodology; \u003cstrong\u003eMRF, WA, RRT, DS, ERM, and DN\u0026nbsp;\u003c/strong\u003eperformed data acquisition and analysis for \u003cem\u003ein vivo\u003c/em\u003e research. \u003cstrong\u003eMRF, WA, and FRM\u0026nbsp;\u003c/strong\u003eperformed data acquisition and analysis for \u003cem\u003ein silico\u003c/em\u003e research; \u003cstrong\u003eMRF, WA, RRT, and DS\u003c/strong\u003e wrote the original manuscript draft; Entire authors were reviewed and edited for the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. ACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the animal research facilities IMERI, the Pharmacokinetic laboratory of the Faculty of Medicine, Universitas Indonesia, and the Dexa Laboratory Biomedical Science (DLBS) research assistant for their generous technical support and their provision of laboratory equipment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. DECLARE OF COMPETING INTEREST STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirmed no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. DECLARATION ON THE USE OF ARTIFICIAL INTELLIGENCE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that an artificial intelligence tool (ChatGPT, OpenAI) was used exclusively for English language improvement, focusing on grammatical accuracy and textual clarity. The AI tool did not contribute to the generation of scientific content, experimental design, data analysis, interpretation, figures, or conclusions. All content was reviewed, edited, and finalized by the authors, and we retain full responsibility for the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. FUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia under \u003cem\u003ePendidikan Magister Menuju Doktor dari Sarjana Unggulan (PMDSU)\u003c/em\u003e scheme, Grant No. PKS- 567/UN2.RST/HKP05.00/2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10. DATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supplementary files were available at the online files presented on the manuscript website.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlmansouri Y, Alsuwatt A, Alzahrani M, Alsuwat MS, Sr, Alamrai R, Alsuwat WS et al (2023) Excessive Daytime Sleepiness in Patients With Hypertension: A Systematic Review. Cureus 15(12):e50716. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7759/cureus.50716\u003c/span\u003e\u003cspan address=\"10.7759/cureus.50716\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKidwai S, Haris M, Bilal A, Saleem S, Imran H, Anwar A et al (2024) High Blood Pressure-Associated Symptoms: Insights From a Population-Based Study in Pakistan. 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J Am Coll Cardiol 68(17):1823\u0026ndash;1834. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jacc.2016.07.763\u003c/span\u003e\u003cspan address=\"10.1016/j.jacc.2016.07.763\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"1eeba72c-d1a9-437d-a7ec-4a26100e83be","identifier":"10.13039/501100009509","name":"Kementerian Riset Teknologi Dan Pendidikan Tinggi Republik Indonesia","awardNumber":"PKS-567/UN2.RST/HKP05.00/2025","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Indonesia","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Spironolactone, Hypertension, Fatigue, Drug Repositioning, HIF-1α Signaling","lastPublishedDoi":"10.21203/rs.3.rs-8419825/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8419825/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypertension-related fatigue involves both elevated blood pressure and reduced exercise tolerance, yet its molecular basis remains unclear. This study investigated the repositioned effects of spironolactone on hemodynamic and metabolic parameters in a uninephrectomy\u0026ndash;deoxycorticosterone acetate-salt rat model of salt-sensitive hypertension. Male Sprague\u0026ndash;Dawley rats were randomized to receive spironolactone (100 mg/kg/day) or vehicle (carboxymethylcellulose) for five weeks. Blood pressure was measured noninvasively, exercise capacity was assessed using a weighted swimming test, plasma sodium was quantified by enzyme-linked immunosorbent assay, and cardiac HIF-1α expression was analyzed by quantitative polymerase chain reaction. Complementary \u003cem\u003ein silico\u003c/em\u003e analyses included functional enrichment of spironolactone\u0026ndash;HIF-1α targets, molecular docking with factor inhibiting hypoxia-inducible factor-1 (FIH1; PDB ID: 8II0), free energy calculations, and quantum mechanical assessment. Spironolactone prevented increases in systolic, diastolic, and mean arterial pressure, normalized plasma sodium levels, and suppressed cardiac HIF-1α expression. A non-significant trend toward prolonged time-to-fatigue was observed in spironolactone-treated rats. When interpreted alongside metabolic and hypoxia-related molecular findings, this trend suggests a potential antifatigue effect, although definitive conclusions cannot be drawn from the current dataset. Computational analyses identified HIF-1α and glycolytic pathways as central interaction hubs, with spironolactone demonstrating favorable binding to catalytic residues of FIH1, an asparaginyl hydroxylase, consistent with a mechanism that may enhance HIF-1α hydroxylation and attenuate hypoxia-driven glycolytic reprogramming in the hypertensive myocardium. These findings suggest that spironolactone exerts integrated hemodynamic and metabolic benefits and may hold therapeutic potential for hypertension-related fatigue, warranting confirmation in future studies or clinical translation in salt-sensitive populations.\u003c/p\u003e","manuscriptTitle":"Repurposing of Spironolactone for Hypertension-Related Fatigue: Experimental Evidence from Salt-Sensitive Rats with Computational Molecular Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-24 08:30:14","doi":"10.21203/rs.3.rs-8419825/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":"bda2ee8b-7a0a-437d-85b1-b2906b01472e","owner":[],"postedDate":"December 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60029608,"name":"Drug Discovery, Design, \u0026 Development"},{"id":60029609,"name":"Pharmacodynamics"},{"id":60029610,"name":"Bioinformatics"}],"tags":[],"updatedAt":"2025-12-24T08:30:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-24 08:30:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8419825","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8419825","identity":"rs-8419825","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}