Disuse-Induced Skeletal Muscle Atrophy Is Associated with Reduced Serum Klotho Levels in a Rat Model

Disuse-Induced Skeletal Muscle Atrophy Is Associated with Reduced Serum Klotho Levels in a Rat Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Disuse-Induced Skeletal Muscle Atrophy Is Associated with Reduced Serum Klotho Levels in a Rat Model Sefa Key, Omer Esmez, Anıl Agar, Osman sedat Tanyeri, Gözde Arkalı, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8535331/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 Background: Klotho is an anti-aging protein involved in phosphate homeostasis, oxidative stress regulation, and tissue regeneration. Although circulating Klotho levels decline with chronological aging, its response to acute disuse-induced skeletal muscle atrophy remains poorly understood. Methods: Sixteen male Wistar rats (3 months old) were randomly assigned to a control group (n = 8) or a disuse group subjected to a tail suspension–based unloading protocol (3 h/day for 15 days) (n = 8). Skeletal muscle atrophy was evaluated histologically in the extensor digitorum longus (EDL) and soleus muscles using hematoxylin–eosin staining, and muscle fiber cross-sectional area (CSA) was quantified with ImageJ. Interstitial connective tissue changes were assessed qualitatively using Masson’s trichrome staining. Serum Klotho concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Results: Rats exposed to disuse demonstrated histological findings consistent with early disuse-related skeletal muscle alterations in both EDL and soleus muscles, accompanied by a significant reduction in muscle fiber CSA compared with controls. Serum Klotho levels were significantly lower in the disuse group (p < 0.05). Correlation analysis revealed a moderate positive association between circulating Klotho concentrations and mean muscle fiber CSA (r = 0.58, p = 0.02). Mild interstitial connective tissue expansion was qualitatively observed in the soleus muscle following disuse. Conclusions: Short-term disuse-induced skeletal muscle atrophy is associated with a reduction in circulating Klotho levels, independent of chronological ageing. These findings suggest that serum Klotho may serve as a candidate biomarker reflecting early disuse-related muscle degeneration. Further mechanistic studies are warranted to clarify the role of Klotho in skeletal muscle atrophy and regeneration. Biological sciences/Biochemistry Health sciences/Medical research Biological sciences/Physiology Klotho disuse-induced muscle atrophy skeletal muscle immobilization tail suspension rat model Figures Figure 1 Figure 2 Figure 3 INTRODUCTION The anti-ageing gene klotho was first identified in 1997 and was named after the mythological Greek goddess who spins the thread of life [ 1 ]. Experimental studies have demonstrated that klotho-deficient mice develop a constellation of phenotypes resembling accelerated ageing, including reduced lifespan, skin atrophy, osteoporosis, sarcopenia, atherosclerosis, infertility, and emphysema [ 1 ]. In contrast, overexpression of klotho has been shown to extend lifespan by approximately 20–30% in mice, highlighting its central role in longevity and tissue homeostasis [ 2 ]. Klotho is a type I transmembrane protein predominantly expressed in the distal renal tubules and, to a lesser extent, in other tissues such as the brain and liver, where it functions as an important metabolic and anti-ageing regulator [ 1 ]. Although the kidney represents the principal source of circulating klotho, expression has also been reported in the parathyroid gland and choroid plexus [ 3 ]. The extracellular domain of membrane-bound klotho is cleaved by a disintegrin and metalloproteinase (ADAM), releasing soluble klotho into the renal interstitium and subsequently into the systemic circulation [ 4 , 5 ]. Circulating klotho interacts with several membrane-associated receptors, including those involved in transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF) signalling pathways [ 2 , 6 ]. Through inhibition of IGF signalling, klotho induces the expression of antioxidant enzymes such as superoxide dismutase, while simultaneously suppressing Wnt and TGF-β signalling pathways to limit fibrotic processes [ 2 , 7 ]. Soluble klotho is detectable in blood, urine, and cerebrospinal fluid [ 8 ] and plays an essential role in calcium and phosphate homeostasis. In the kidney, calcium reabsorption in the distal tubule is mediated primarily by transient receptor potential vanilloid type 5 and type 6 (TRPV5 and TRPV6) channels [ 9 – 11 ]. The secreted form of klotho enhances the activity of these channels, and klotho-deficient mice consequently exhibit increased urinary calcium excretion [ 12 , 13 ]. This renal calcium loss may secondarily promote increased calcium mobilization from bone, thereby linking klotho deficiency to skeletal fragility. Klotho exists in two major forms: circulating α-klotho (c-α-klotho) and membrane-bound α-klotho (m-α-klotho). Membrane-bound klotho acts as a co-receptor for fibroblast growth factor-23 (FGF-23), a hormone produced by osteocytes and osteoblasts, and together they exert a phosphaturic effect in the renal tubular epithelium [ 8 , 14 , 15 ]. Animals lacking either FGF-23 or α-klotho display a phenotype resembling accelerated ageing and shortened lifespan [ 1 , 15 ]. Dietary phosphate restriction has been shown to attenuate this ageing-like phenotype and prolong survival in α-klotho–deficient mice, suggesting that phosphate dysregulation plays a key role in klotho-mediated ageing processes [ 16 ]. However, the molecular mechanisms through which circulating α-klotho exerts its systemic anti-ageing effects remain incompletely understood. Emerging evidence indicates that circulating α-klotho may also influence skeletal muscle homeostasis. The c-α-klotho protein has been shown to inhibit TGF-β1 signalling by blocking the type II serine/threonine kinase receptor (TbRII), thereby attenuating fibrosis [ 6 ]. In addition to TGF-β1, c-α-klotho appears to suppress other muscle-wasting TGF-β family members, including myostatin, which is a well-established negative regulator of muscle mass [ 17 ]. Through these shared signalling pathways, klotho may exert protective effects against muscle wasting and degeneration. Sarcopenia is traditionally defined as an age-related condition characterized by progressive loss of skeletal muscle mass and strength [ 18 ]. Skeletal muscle constitutes more than 40% of total body mass in young adults; however, muscle mass declines by approximately 0.1–0.5% per year after the age of 30 [ 19 ]. Epidemiological studies estimate that sarcopenia affects nearly 40% of individuals over 80 years of age and approximately 25% of those under 70 [ 20 ]. The clinical consequences of sarcopenia include reduced mobility, increased risk of falls and fractures, loss of independence, and increased mortality in both humans and experimental animals [ 21 , 22 ]. Importantly, experimental studies in mice have demonstrated that age-related conditions such as osteopenia and sarcopenia are closely associated with disrupted klotho expression [ 23 ]. In humans, low circulating klotho levels have been linked to reduced bone mineral density and diminished grip strength, further supporting a role for klotho in musculoskeletal health [ 24 , 25 ]. While the relationship between klotho and age-related muscle loss has been increasingly explored, the effects of acute muscle disuse on circulating klotho levels remain poorly defined. Disuse-induced muscle atrophy, resulting from immobilization or unloading, is a common and clinically relevant musculoskeletal problem encountered in prolonged bed rest, hospitalization, and critical illness. Although disuse atrophy has been proposed to contribute to sarcopenia-like phenotypes, little is known about whether short-term disuse alters circulating klotho levels independently of chronological ageing. Therefore, the aim of the present study was to investigate serum klotho levels in a rat model of disuse-induced skeletal muscle atrophy and to examine the association between circulating klotho and muscle morphological changes. By addressing this gap, the present study seeks to provide experimental evidence linking klotho to early disuse-related muscle degeneration. METHODS Animals and experimental design The study protocol was approved by the Laboratory Animal Care and Use Committee of Fırat University Faculty of Medicine. Sixteen male Wistar albino rats ( Rattus norvegicus ), aged 3 months and weighing 220 ± 20 g, were included. Animals were randomly assigned to either a control group (n = 8) or a disuse group subjected to tail suspension–based unloading (n = 8) (Fig. 1). Rats were housed four per cage under standard laboratory conditions (12:12 h light–dark cycle, temperature 22 ± 2°C, humidity 50 ± 5%) with ad libitum access to food and water. Disuse was induced using a partial tail suspension protocol adapted from previously described unloading models. Anesthesia was used only briefly for tail harness application, and animals were fully awake during the 180-min unloading period [ 26 ]. Briefly, rats in the disuse group were anesthetized with an intraperitoneal injection of ketamine (75–90 mg/kg) combined with xylazine (5–10 mg/kg). Adequate depth of anesthesia was verified by the absence of the pedal withdrawal reflex. Animals were then suspended by the tail for 180 min once daily for 15 consecutive days, with the hindlimbs elevated and without ground contact, while allowing forelimb locomotion for access to food and water. Control animals underwent identical daily handling and tail-harness procedures; however, they were not suspended. Anesthesia was used only briefly for tail harness application, and both groups were fully awake during the unloading/handling period. Food and water intake were monitored daily throughout the experimental period, and no significant differences were observed between groups, indicating that muscle changes were unlikely to be attributable to reduced nutritional intake. Ethics approval and reporting guidelines All animal procedures were carried out in accordance with relevant guidelines and regulations for the care and use of laboratory animals. The study protocol was approved by the Laboratory Animal Care and Use Committee of Fırat University Faculty of Medicine (approval date: 17 July 2023; approval number: 17217). All methods are reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments; https://arriveguidelines.org ). No ether was used for anesthesia or euthanasia at any stage of the study. Anesthesia was induced with ketamine (75–90 mg/kg) and xylazine (5–10 mg/kg) administered intraperitoneally. Euthanasia was performed under deep anesthesia by exsanguination via cardiac puncture, and death was confirmed by cessation of heartbeat and respiration. All efforts were made to minimize animal suffering and to reduce the number of animals used. Blood sampling and serum Klotho measurement At the end of the 15-day intervention, blood samples were collected via cardiac puncture under deep anesthesia induced with ketamine (75–90 mg/kg) and xylazine (5–10 mg/kg) administered intraperitoneally, between 09:00 and 11:00 a.m. to minimize circadian variation. Adequate depth of anesthesia was confirmed by the absence of the pedal withdrawal reflex. Euthanasia was performed under deep anesthesia by exsanguination via cardiac puncture, and death was confirmed by cessation of heartbeat and respiration. Samples were allowed to clot at room temperature for 30 min and were then centrifuged at 3000 rpm for 15 min at 4°C. Serum was aliquoted and stored at − 80°C until analysis. Serum Klotho concentrations were measured using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit specific for rat Klotho (Immuno-Biological Laboratories, Japan), according to the manufacturer’s instructions. All samples were analyzed in duplicate, and investigators performing ELISA measurements were blinded to group allocation. Histopathological analysis Histological analyses were performed by investigators blinded to the experimental groups. Extensor digitorum longus (EDL) and soleus muscle samples were fixed in 10% buffered formalin for 48 h, dehydrated through graded ethanol series, cleared in xylol, and embedded in paraffin. Transverse sections (5–6 µm) were obtained and routinely stained with hematoxylin–eosin (H&E) for evaluation of muscle morphology [ 27 ]. For morphometric analysis, six non-overlapping microscopic fields per muscle (EDL and soleus) per animal were randomly selected and imaged at 40× magnification. Muscle fiber cross-sectional area (CSA) was quantified on H&E-stained sections using ImageJ software (ImageJ 1.54p, National Institutes of Health, USA) [ 26 ]. Individual fiber borders were manually traced after calibration with a stage micrometer, and CSA values were calculated automatically (µm²). Prespecified exclusion criteria included obliquely sectioned fibers, longitudinally cut fibers, necrotic or split fibers, and fields containing artifacts. Across all animals, a total of 1,922 EDL fibers and 1,834 soleus fibers were analyzed. Masson’s trichrome staining was performed on adjacent sections to qualitatively assess interstitial collagen deposition as an indicator of fibrosis. Trichrome-stained sections were evaluated descriptively and were not used for CSA measurements. Statistical analysis Statistical analyses were performed using IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, USA). Data distribution was assessed using the Shapiro–Wilk normality test. Comparisons between control and disuse groups were conducted using an independent samples t -test for normally distributed variables. Data are presented as mean ± standard deviation (SD). All statistical tests were two-tailed, and a p value < 0.05 was considered statistically significant. Given the exploratory nature of this experimental study and the absence of prior data on disuse-related changes in circulating Klotho levels, no a priori power calculation was performed. The study was designed to detect early morphological and biochemical alterations associated with short-term mechanical unloading. RESULTS Quantitative morphometric analysis demonstrated a reduction in muscle fiber cross-sectional area (CSA) in both the extensor digitorum longus (EDL) and soleus muscles following partial tail suspension. Compared with control animals, the tail-suspended group exhibited a 2.74% decrease in mean CSA in EDL fibers and a 4.57% decrease in soleus fibers (Fig. 2). The relatively greater reduction observed in the soleus muscle is consistent with its postural function and higher sensitivity to unloading-related changes. Histological examination of hematoxylin–eosin (H&E)–stained sections from control animals revealed preserved muscle architecture, characterized by relatively uniform polygonal fibers with peripheral nuclei and the absence of prominent inflammatory infiltration or degenerative features (Fig. 4A, C). In contrast, muscle sections from tail-suspended rats demonstrated histopathological findings consistent with early disuse-related muscle alteration, including mildly reduced fiber caliber, focal architectural irregularity, and occasional mononuclear cell presence within the endomysial and perimysial compartments in both EDL and soleus muscles (Fig. 4B, D). Masson’s trichrome staining showed no apparent interstitial fibrosis in either EDL or soleus muscles of control rats (Fig. 5A, C). In the tail-suspended group, a mild increase in interstitial connective tissue was qualitatively observed, particularly in the soleus muscle (Fig. 5B, D), suggesting early extracellular matrix remodeling in response to mechanical unloading. Because collagen deposition was evaluated qualitatively, these observations should be interpreted descriptively rather than quantitatively. Visualization of individual CSA values using scatter and distribution plots demonstrated partially overlapping distributions between groups; however, a statistically significant between-group difference was observed for both muscles (Fig. 2), indicating a consistent trend toward smaller fiber size in the disuse group. Serum Klotho concentrations were significantly lower in the tail-suspended group compared with control animals (Fig. 3, p < 0.05). Individual data points showed a consistent reduction across animals exposed to disuse. Correlation analysis revealed a moderate positive association between circulating Klotho levels and mean muscle fiber CSA (r = 0.58, p = 0.02), indicating that lower serum Klotho concentrations were associated with smaller muscle fiber size across experimental groups. Collectively, these results demonstrate that short-term partial unloading induces early morphological changes in both fast- and slow-twitch skeletal muscles, accompanied by a significant reduction in circulating Klotho levels. The observed association between serum Klotho concentration and muscle fiber size supports a potential link between systemic Klotho availability and disuse-related skeletal muscle alterations. Mean muscle fiber CSA values (µm²) with corresponding standard deviations are provided in Fig. 2; despite modest percentage reductions, between-group differences were statistically significant. DISCUSSION Population ageing represents a major global public health challenge. It is estimated that by 2035 nearly one quarter of the European population will be aged 65 years or older, accompanied by a substantial increase in age-related morbidity and mortality [ 28 ]. This demographic shift has intensified the search for reliable biological markers that reflect ageing processes and predict age-associated functional decline. Among these biomarkers, klotho has emerged as a key longevity-associated protein, with circulating levels shown to decline progressively from mid-adulthood onward [ 29 ]. Klotho is predominantly synthesized in the distal convoluted tubules of the kidney but is also expressed at lower levels in several extrarenal tissues, including the brain, parathyroid gland, adipose tissue, liver, pancreas, and skeletal muscle [ 30 ]. Although the precise cellular localization and functional role of klotho within skeletal muscle remain incompletely understood, growing evidence suggests that it plays an important role in regulating muscle mass, strength, and regenerative capacity. In clinical conditions such as chronic obstructive pulmonary disease, klotho has been associated with muscle weakness and has been shown to localize to both the nucleus and plasma membrane of muscle fibers [ 31 ]. Experimental models further demonstrate that impaired klotho signaling reduces myogenic capacity and disrupts normal muscle development [ 31 ]. Several experimental studies have shown that klotho deficiency—whether due to genetic deletion, ageing, or disease—is associated with reduced muscle mass and impaired myogenesis [ 2 ]. Conversely, klotho overexpression has been reported to increase satellite cell numbers, attenuate muscle atrophy, and enhance muscle regeneration following injury in dystrophic and aged muscle models [ 32 ]. These findings collectively support the concept that klotho exerts direct or indirect protective effects on skeletal muscle integrity. Sarcopenia, characterized by age-related loss of muscle mass and function, is a major contributor to frailty, disability, and mortality in older adults. Progressive reductions in muscle strength result in impaired mobility, increased fall risk, and loss of independence. Importantly, circulating klotho levels decline with age and have been associated with reduced grip strength, poor physical performance, and increased all-cause mortality [ 33 ]. A large population-based ageing study proposed a mechanistic link between sarcopenia and reduced circulating klotho, suggesting that klotho deficiency may reflect diminished physiological reserve [ 34 ]. In this context, the moderate positive correlation observed in the present study between serum klotho levels and muscle fiber cross-sectional area supports the hypothesis that systemic klotho deficiency may also be relevant in early disuse-related muscle atrophy. Exercise is a well-recognized stimulus for increasing circulating klotho levels, although the magnitude of this response varies with age and fitness status. Beyond sarcopenia, ageing is characterized by impaired skeletal muscle regeneration following injury, resulting in prolonged recovery and increased susceptibility to recurrent damage. Experimental evidence indicates that klotho enhances regenerative capacity by modulating muscle stem cell function and suppressing excessive Wnt and TGF-β signaling pathways [ 34 ]. In klotho hypomorphic mice, depletion of muscle stem cells and severe impairment of regeneration have been demonstrated, whereas recombinant klotho supplementation restores stem cell function by inhibiting aberrant Wnt signaling in aged muscle [ 35 ]. In the present study, partial tail suspension induced clear histological features of disuse-related atrophy in both EDL and soleus muscles, accompanied by a significant reduction in serum klotho levels. This finding suggests that klotho decline may occur independently of chronological ageing and may reflect acute unloading-induced muscle degeneration. Consistent with this observation, klotho deficiency has previously been associated with reduced body weight, impaired muscle strength, and decreased physical performance in adult mice [ 36 ]. Although molecular markers of muscle atrophy such as atrogin-1 or MuRF-1 were not assessed in this study, previous investigations using similar suspension models have demonstrated activation of ubiquitin–proteasome pathways under comparable conditions. The partial suspension protocol used in this study represents a milder unloading model compared with continuous hindlimb unloading, which is considered the gold standard for inducing disuse atrophy and simulating microgravity conditions. Nevertheless, this approach effectively produced early histological signs of muscle atrophy while minimizing animal distress and systemic stress. The mild increase in interstitial connective tissue observed, particularly in the soleus muscle, likely reflects early extracellular matrix remodeling; however, fibrosis assessment was qualitative, and quantitative collagen analysis was not performed. The magnitude of CSA reduction observed in the present study was relatively small; however, this finding is consistent with the use of a short-term, intermittent unloading protocol designed to capture early disuse-related changes rather than advanced atrophy. Previous studies employing continuous hindlimb unloading have reported more pronounced muscle loss, whereas partial or intermittent suspension models preferentially induce subtle but biologically relevant early alterations. In this context, the concurrent reduction in circulating Klotho levels despite modest structural changes suggests that Klotho may be a sensitive systemic indicator of early muscle disuse. Several limitations of the present study should be acknowledged. The sample size was modest, and causal relationships between klotho reduction and muscle atrophy cannot be inferred. Histological evaluation relied on H&E and Masson’s trichrome staining without immunohistochemical confirmation, limiting precise differentiation between degenerative and regenerative processes. In addition, serum klotho levels were measured without parallel assessment of intramuscular klotho expression at the mRNA or protein level. Despite these limitations, the present findings suggest that circulating klotho may be sensitive to acute muscle disuse and could serve as an early biomarker of unloading-related muscle degeneration. It has been shown that klotho is essential for an adequate regenerative response to acute muscle injury and that systemic α-klotho supplementation promotes myofiber regeneration in aged mice when administered at appropriate doses and intervals [38]. Consistent with these findings, age-related declines in circulating α-klotho have been shown to impair muscle regeneration through mitochondrial dysfunction in progenitor cells, whereas restoration of α-klotho levels improves regenerative capacity in aged skeletal muscle [ 37 ]. Further mechanistic studies incorporating tissue-specific klotho analyses and interventional models are warranted to clarify the role of klotho in muscle degeneration and regeneration. CONCLUSION In conclusion, partial tail suspension induced early disuse-related muscle atrophy in both fast- and slow-twitch muscles and was associated with a significant reduction in circulating klotho levels. These findings suggest that serum klotho may reflect acute changes in muscle integrity independent of chronological ageing. Although causality cannot be established, the observed association between reduced klotho levels and muscle fiber atrophy supports the potential role of klotho as a biomarker of muscle disuse and early degeneration. Further studies integrating molecular analyses and klotho-targeted interventions are needed to determine whether modulation of klotho signaling may represent a therapeutic strategy for preventing or attenuating disuse-related muscle loss. Declarations Ethics approval and consent to participate All animal procedures were carried out in accordance with relevant guidelines and regulations for the care and use of laboratory animals. The study protocol was approved by the Laboratory Animal Care and Use Committee of Fırat University Faculty of Medicine (approval date: 17 July 2023; approval number: 17217). All methods are reported in accordance with the ARRIVE guidelines ( https://arriveguidelines.org ). Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Fırat University Scientific Research Projects Unit. Author Contribution • **SK:** Conceptualization, study design, supervision of experimental protocol, critical revision of the manuscript, final approval.• **ÖE:** Conceptualization, study design, data acquisition, histopathological interpretation, statistical analysis, manuscript drafting, final approval.• **AA:** Conceptualization, study design, supervision of experimental protocol, critical revision of the manuscript, final approval.• **OST:** Experimental procedures, physiological data interpretation, laboratory supervision, manuscript editing, final approval.• **GA:** Data collection, laboratory support, data verification, literature review, manuscript editing, final approval.• **SÇ:** Histopathological evaluation, interpretation of tissue findings, methodological support, critical manuscript revision, final approval.• **AY:** Experimental design support, physiological interpretation, data analysis assistance, manuscript revision, final approval.All authors read and approved the final manuscript. Acknowledgements The authors gratefully acknowledge the support of the Fırat University Scientific Research Projects Unit. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Kuro-o, M. et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390 , 45–51 (1997). Kurosu, H. et al. Suppression of aging in mice by the hormone Klotho. Science 309 , 1829–1833 (2005). Hu, M. et al. Renal production, uptake, and handling of circulating α-Klotho. J. Am. Soc. Nephrol. 27 , 79–90 (2016). Takenaka, T., Watanabe, Y., Inoue, T., Miyazaki, T. & Suzuki, H. Fibroblast growth factor 23 enhances renal klotho abundance. Pflugers Arch. 465 , 935–943 (2013). Takenaka, T. et al. Antialbuminuric actions of calcilytics in the remnant kidney. Am. J. Physiol. Ren. Physiol. 309 , F216–F226 (2015). Doi, S. et al. 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Klotho regulates myogenic response. Exp. Physiol. 108 , 1531–1547 (2023). Semba, R. D. et al. Plasma klotho and mortality. J. Gerontol. Biol. Sci. Med. Sci. 66 , 794–800 (2011). Carlson, M. E. et al. TGF-β and Wnt in muscle aging. Aging Cell. 8 , 676–689 (2009). Ahrens, H. E., Huettemeister, J., Schmidt, M., Kaether, C. & Vom Maltzahn, J. Klotho and muscle stem cell function. Skelet. Muscle . 8 , 11 (2018). Phelps, M., Pettan-Brewer, C., Ladiges, W. & Yablonka-Reuveni, Z. Muscle decline in klotho-deficient mice. Biogerontology 14 , 729–739 (2013). Sahu, A. et al. Age-related declines in α-Klotho. Nat. Commun. 9 , 4859 (2018). Additional Declarations No competing interests reported. Supplementary Files 20231309Dr.r.yesiSefaKEY11.pdf figure4.jpeg Supplementary Figure 4. Representative hematoxylin–eosin (H&E)–stained sections of EDL and soleus muscles from control and tail-suspended rats (200×), demonstrating muscle fiber size reduction and structural alterations following disuse. figure5.jpeg Supplementary Figure 5. Representative Masson’s trichrome–stained sections of EDL and soleus muscles from control and tail-suspended rats (200×), illustrating mild interstitial connective tissue changes after unloading. 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8535331","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":576982869,"identity":"9f32e4ce-109d-495a-b71b-419af9b23cc4","order_by":0,"name":"Sefa Key","email":"","orcid":"","institution":"Fırat University","correspondingAuthor":false,"prefix":"","firstName":"Sefa","middleName":"","lastName":"Key","suffix":""},{"id":576982870,"identity":"f137577f-5c9c-476e-840e-fa1b4248f6a3","order_by":1,"name":"Omer 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15:22:30","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":234528,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/a56eaada5e55b040c71ccce1.png"},{"id":100697915,"identity":"905ae588-331a-4f6d-b44e-ad4b4243dcc7","added_by":"auto","created_at":"2026-01-20 15:18:49","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":233075,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/6943b009e85b8418acc833a6.png"},{"id":100697912,"identity":"0f98434e-4b7a-4bc1-ad1a-be2be8defcb6","added_by":"auto","created_at":"2026-01-20 15:18:47","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77521,"visible":true,"origin":"","legend":"","description":"","filename":"9d668c03031b49e585c6b338f42a30161structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/24e424e9652fa00152c724e7.xml"},{"id":100698001,"identity":"823affa5-d272-44e5-b670-c18d022c6927","added_by":"auto","created_at":"2026-01-20 15:19:47","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91586,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/f240db2423d2d6f43bc7c649.html"},{"id":100698002,"identity":"51509a21-449a-40e9-931e-2a6adbeb1bfe","added_by":"auto","created_at":"2026-01-20 15:19:50","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124504,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental workflow of the study. Rats were allocated to control or partial tail suspension groups (3 h/day for 15 days). Serum samples were collected for Klotho measurement by ELISA, followed by histological and morphometric evaluation of extensor digitorum longus (EDL) and soleus muscles.\u003c/p\u003e","description":"","filename":"figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/99d1efcec5382207d5c20a93.jpeg"},{"id":100698309,"identity":"f31c86c0-c8fa-4e13-8665-852312fe72d9","added_by":"auto","created_at":"2026-01-20 15:23:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219664,"visible":true,"origin":"","legend":"\u003cp\u003eMuscle fiber cross-sectional area (CSA) in the extensor digitorum longus (EDL) and soleus muscles of control and tail-suspended rats. Each dot represents an individual animal (n = 8 per group).\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/b03f93a09825fcc7ac5c828b.jpg"},{"id":100698266,"identity":"77a8fe33-c1f9-400b-b4b8-51bb225ca454","added_by":"auto","created_at":"2026-01-20 15:22:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121013,"visible":true,"origin":"","legend":"\u003cp\u003eSerum Klotho concentrations measured by ELISA in control and tail-suspended rats. Data are presented as mean ± SD with individual animal values (n = 8 per group). p \u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/2db1d3ea1b67764cc7eebf2c.jpg"},{"id":103507953,"identity":"03fef969-6f60-40ca-bd57-697f8a00daa3","added_by":"auto","created_at":"2026-02-26 13:46:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1060298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/615a005d-b8a0-404e-8850-89a404c84a20.pdf"},{"id":100698000,"identity":"da88ae7a-386b-4c85-8d15-ffd625d4dfd7","added_by":"auto","created_at":"2026-01-20 15:19:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":128865,"visible":true,"origin":"","legend":"","description":"","filename":"20231309Dr.r.yesiSefaKEY11.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/696702b713778943742a5370.pdf"},{"id":100697980,"identity":"3a52d287-3af1-436e-833c-a6c8ed567714","added_by":"auto","created_at":"2026-01-20 15:19:20","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":360143,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 4. Representative hematoxylin–eosin (H\u0026amp;E)–stained sections of EDL and soleus muscles from control and tail-suspended rats (200×), demonstrating muscle fiber size reduction and structural alterations following disuse.\u003c/p\u003e","description":"","filename":"figure4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/43793099eceaebd5291ffc3b.jpeg"},{"id":100697983,"identity":"fe0f5ad8-d7b3-421a-86a9-dc06903623ae","added_by":"auto","created_at":"2026-01-20 15:19:21","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":265742,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 5. Representative Masson’s trichrome–stained sections of EDL and soleus muscles from control and tail-suspended rats (200×), illustrating mild interstitial connective tissue changes after unloading.\u003c/p\u003e","description":"","filename":"figure5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8535331/v1/aaf09a57d61c5da53d0aa0d1.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Disuse-Induced Skeletal Muscle Atrophy Is Associated with Reduced Serum Klotho Levels in a Rat Model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe anti-ageing gene klotho was first identified in 1997 and was named after the mythological Greek goddess who spins the thread of life [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Experimental studies have demonstrated that klotho-deficient mice develop a constellation of phenotypes resembling accelerated ageing, including reduced lifespan, skin atrophy, osteoporosis, sarcopenia, atherosclerosis, infertility, and emphysema [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In contrast, overexpression of klotho has been shown to extend lifespan by approximately 20\u0026ndash;30% in mice, highlighting its central role in longevity and tissue homeostasis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKlotho is a type I transmembrane protein predominantly expressed in the distal renal tubules and, to a lesser extent, in other tissues such as the brain and liver, where it functions as an important metabolic and anti-ageing regulator [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although the kidney represents the principal source of circulating klotho, expression has also been reported in the parathyroid gland and choroid plexus [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The extracellular domain of membrane-bound klotho is cleaved by a disintegrin and metalloproteinase (ADAM), releasing soluble klotho into the renal interstitium and subsequently into the systemic circulation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Circulating klotho interacts with several membrane-associated receptors, including those involved in transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF) signalling pathways [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Through inhibition of IGF signalling, klotho induces the expression of antioxidant enzymes such as superoxide dismutase, while simultaneously suppressing Wnt and TGF-β signalling pathways to limit fibrotic processes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSoluble klotho is detectable in blood, urine, and cerebrospinal fluid [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and plays an essential role in calcium and phosphate homeostasis. In the kidney, calcium reabsorption in the distal tubule is mediated primarily by transient receptor potential vanilloid type 5 and type 6 (TRPV5 and TRPV6) channels [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The secreted form of klotho enhances the activity of these channels, and klotho-deficient mice consequently exhibit increased urinary calcium excretion [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This renal calcium loss may secondarily promote increased calcium mobilization from bone, thereby linking klotho deficiency to skeletal fragility.\u003c/p\u003e \u003cp\u003eKlotho exists in two major forms: circulating α-klotho (c-α-klotho) and membrane-bound α-klotho (m-α-klotho). Membrane-bound klotho acts as a co-receptor for fibroblast growth factor-23 (FGF-23), a hormone produced by osteocytes and osteoblasts, and together they exert a phosphaturic effect in the renal tubular epithelium [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Animals lacking either FGF-23 or α-klotho display a phenotype resembling accelerated ageing and shortened lifespan [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Dietary phosphate restriction has been shown to attenuate this ageing-like phenotype and prolong survival in α-klotho\u0026ndash;deficient mice, suggesting that phosphate dysregulation plays a key role in klotho-mediated ageing processes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the molecular mechanisms through which circulating α-klotho exerts its systemic anti-ageing effects remain incompletely understood.\u003c/p\u003e \u003cp\u003eEmerging evidence indicates that circulating α-klotho may also influence skeletal muscle homeostasis. The c-α-klotho protein has been shown to inhibit TGF-β1 signalling by blocking the type II serine/threonine kinase receptor (TbRII), thereby attenuating fibrosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition to TGF-β1, c-α-klotho appears to suppress other muscle-wasting TGF-β family members, including myostatin, which is a well-established negative regulator of muscle mass [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Through these shared signalling pathways, klotho may exert protective effects against muscle wasting and degeneration.\u003c/p\u003e \u003cp\u003eSarcopenia is traditionally defined as an age-related condition characterized by progressive loss of skeletal muscle mass and strength [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Skeletal muscle constitutes more than 40% of total body mass in young adults; however, muscle mass declines by approximately 0.1\u0026ndash;0.5% per year after the age of 30 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Epidemiological studies estimate that sarcopenia affects nearly 40% of individuals over 80 years of age and approximately 25% of those under 70 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The clinical consequences of sarcopenia include reduced mobility, increased risk of falls and fractures, loss of independence, and increased mortality in both humans and experimental animals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, experimental studies in mice have demonstrated that age-related conditions such as osteopenia and sarcopenia are closely associated with disrupted klotho expression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In humans, low circulating klotho levels have been linked to reduced bone mineral density and diminished grip strength, further supporting a role for klotho in musculoskeletal health [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While the relationship between klotho and age-related muscle loss has been increasingly explored, the effects of acute muscle disuse on circulating klotho levels remain poorly defined.\u003c/p\u003e \u003cp\u003eDisuse-induced muscle atrophy, resulting from immobilization or unloading, is a common and clinically relevant musculoskeletal problem encountered in prolonged bed rest, hospitalization, and critical illness. Although disuse atrophy has been proposed to contribute to sarcopenia-like phenotypes, little is known about whether short-term disuse alters circulating klotho levels independently of chronological ageing. Therefore, the aim of the present study was to investigate serum klotho levels in a rat model of disuse-induced skeletal muscle atrophy and to examine the association between circulating klotho and muscle morphological changes. By addressing this gap, the present study seeks to provide experimental evidence linking klotho to early disuse-related muscle degeneration.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and experimental design\u003c/h2\u003e \u003cp\u003e The study protocol was approved by the Laboratory Animal Care and Use Committee of Fırat University Faculty of Medicine. Sixteen male Wistar albino rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e), aged 3 months and weighing 220\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g, were included. Animals were randomly assigned to either a control group (n\u0026thinsp;=\u0026thinsp;8) or a disuse group subjected to tail suspension\u0026ndash;based unloading (n\u0026thinsp;=\u0026thinsp;8) (Fig.\u0026nbsp;1). Rats were housed four per cage under standard laboratory conditions (12:12 h light\u0026ndash;dark cycle, temperature 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, humidity 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%) with ad libitum access to food and water.\u003c/p\u003e \u003cp\u003eDisuse was induced using a partial tail suspension protocol adapted from previously described unloading models. Anesthesia was used only briefly for tail harness application, and animals were fully awake during the 180-min unloading period [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, rats in the disuse group were anesthetized with an intraperitoneal injection of ketamine (75\u0026ndash;90 mg/kg) combined with xylazine (5\u0026ndash;10 mg/kg). Adequate depth of anesthesia was verified by the absence of the pedal withdrawal reflex. Animals were then suspended by the tail for 180 min once daily for 15 consecutive days, with the hindlimbs elevated and without ground contact, while allowing forelimb locomotion for access to food and water. Control animals underwent identical daily handling and tail-harness procedures; however, they were not suspended. Anesthesia was used only briefly for tail harness application, and both groups were fully awake during the unloading/handling period. Food and water intake were monitored daily throughout the experimental period, and no significant differences were observed between groups, indicating that muscle changes were unlikely to be attributable to reduced nutritional intake.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthics approval and reporting guidelines\u003c/h3\u003e\n\u003cp\u003e All animal procedures were carried out in accordance with relevant guidelines and regulations for the care and use of laboratory animals. The study protocol was approved by the Laboratory Animal Care and Use Committee of Fırat University Faculty of Medicine (approval date: 17 July 2023; approval number: 17217). All methods are reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNo ether was used for anesthesia or euthanasia at any stage of the study. Anesthesia was induced with ketamine (75\u0026ndash;90 mg/kg) and xylazine (5\u0026ndash;10 mg/kg) administered intraperitoneally. Euthanasia was performed under deep anesthesia by exsanguination via cardiac puncture, and death was confirmed by cessation of heartbeat and respiration. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\n\u003ch3\u003eBlood sampling and serum Klotho measurement\u003c/h3\u003e\n\u003cp\u003eAt the end of the 15-day intervention, blood samples were collected via cardiac puncture under deep anesthesia induced with ketamine (75\u0026ndash;90 mg/kg) and xylazine (5\u0026ndash;10 mg/kg) administered intraperitoneally, between 09:00 and 11:00 a.m. to minimize circadian variation. Adequate depth of anesthesia was confirmed by the absence of the pedal withdrawal reflex. Euthanasia was performed under deep anesthesia by exsanguination via cardiac puncture, and death was confirmed by cessation of heartbeat and respiration. Samples were allowed to clot at room temperature for 30 min and were then centrifuged at 3000 rpm for 15 min at 4\u0026deg;C. Serum was aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003e Serum Klotho concentrations were measured using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit specific for rat Klotho (Immuno-Biological Laboratories, Japan), according to the manufacturer\u0026rsquo;s instructions. All samples were analyzed in duplicate, and investigators performing ELISA measurements were blinded to group allocation.\u003c/p\u003e\n\u003ch3\u003eHistopathological analysis\u003c/h3\u003e\n\u003cp\u003eHistological analyses were performed by investigators blinded to the experimental groups. Extensor digitorum longus (EDL) and soleus muscle samples were fixed in 10% buffered formalin for 48 h, dehydrated through graded ethanol series, cleared in xylol, and embedded in paraffin. Transverse sections (5\u0026ndash;6 \u0026micro;m) were obtained and routinely stained with hematoxylin\u0026ndash;eosin (H\u0026amp;E) for evaluation of muscle morphology [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor morphometric analysis, six non-overlapping microscopic fields per muscle (EDL and soleus) per animal were randomly selected and imaged at 40\u0026times; magnification. Muscle fiber cross-sectional area (CSA) was quantified on H\u0026amp;E-stained sections using ImageJ software (ImageJ 1.54p, National Institutes of Health, USA) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Individual fiber borders were manually traced after calibration with a stage micrometer, and CSA values were calculated automatically (\u0026micro;m\u0026sup2;). Prespecified exclusion criteria included obliquely sectioned fibers, longitudinally cut fibers, necrotic or split fibers, and fields containing artifacts. Across all animals, a total of 1,922 EDL fibers and 1,834 soleus fibers were analyzed.\u003c/p\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining was performed on adjacent sections to qualitatively assess interstitial collagen deposition as an indicator of fibrosis. Trichrome-stained sections were evaluated descriptively and were not used for CSA measurements.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, USA). Data distribution was assessed using the Shapiro\u0026ndash;Wilk normality test. Comparisons between control and disuse groups were conducted using an independent samples \u003cem\u003et\u003c/em\u003e-test for normally distributed variables. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). All statistical tests were two-tailed, and a \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Given the exploratory nature of this experimental study and the absence of prior data on disuse-related changes in circulating Klotho levels, no a priori power calculation was performed. The study was designed to detect early morphological and biochemical alterations associated with short-term mechanical unloading.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eQuantitative morphometric analysis demonstrated a reduction in muscle fiber cross-sectional area (CSA) in both the extensor digitorum longus (EDL) and soleus muscles following partial tail suspension. Compared with control animals, the tail-suspended group exhibited a 2.74% decrease in mean CSA in EDL fibers and a 4.57% decrease in soleus fibers (Fig.\u0026nbsp;2). The relatively greater reduction observed in the soleus muscle is consistent with its postural function and higher sensitivity to unloading-related changes.\u003c/p\u003e \u003cp\u003eHistological examination of hematoxylin\u0026ndash;eosin (H\u0026amp;E)\u0026ndash;stained sections from control animals revealed preserved muscle architecture, characterized by relatively uniform polygonal fibers with peripheral nuclei and the absence of prominent inflammatory infiltration or degenerative features (Fig.\u0026nbsp;4A, C). In contrast, muscle sections from tail-suspended rats demonstrated histopathological findings consistent with early disuse-related muscle alteration, including mildly reduced fiber caliber, focal architectural irregularity, and occasional mononuclear cell presence within the endomysial and perimysial compartments in both EDL and soleus muscles (Fig.\u0026nbsp;4B, D).\u003c/p\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining showed no apparent interstitial fibrosis in either EDL or soleus muscles of control rats (Fig.\u0026nbsp;5A, C). In the tail-suspended group, a mild increase in interstitial connective tissue was qualitatively observed, particularly in the soleus muscle (Fig.\u0026nbsp;5B, D), suggesting early extracellular matrix remodeling in response to mechanical unloading. Because collagen deposition was evaluated qualitatively, these observations should be interpreted descriptively rather than quantitatively.\u003c/p\u003e \u003cp\u003eVisualization of individual CSA values using scatter and distribution plots demonstrated partially overlapping distributions between groups; however, a statistically significant between-group difference was observed for both muscles (Fig.\u0026nbsp;2), indicating a consistent trend toward smaller fiber size in the disuse group.\u003c/p\u003e \u003cp\u003eSerum Klotho concentrations were significantly lower in the tail-suspended group compared with control animals (Fig.\u0026nbsp;3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Individual data points showed a consistent reduction across animals exposed to disuse.\u003c/p\u003e \u003cp\u003eCorrelation analysis revealed a moderate positive association between circulating Klotho levels and mean muscle fiber CSA (r\u0026thinsp;=\u0026thinsp;0.58, p\u0026thinsp;=\u0026thinsp;0.02), indicating that lower serum Klotho concentrations were associated with smaller muscle fiber size across experimental groups.\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that short-term partial unloading induces early morphological changes in both fast- and slow-twitch skeletal muscles, accompanied by a significant reduction in circulating Klotho levels. The observed association between serum Klotho concentration and muscle fiber size supports a potential link between systemic Klotho availability and disuse-related skeletal muscle alterations. Mean muscle fiber CSA values (\u0026micro;m\u0026sup2;) with corresponding standard deviations are provided in Fig.\u0026nbsp;2; despite modest percentage reductions, between-group differences were statistically significant.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePopulation ageing represents a major global public health challenge. It is estimated that by 2035 nearly one quarter of the European population will be aged 65 years or older, accompanied by a substantial increase in age-related morbidity and mortality [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This demographic shift has intensified the search for reliable biological markers that reflect ageing processes and predict age-associated functional decline. Among these biomarkers, klotho has emerged as a key longevity-associated protein, with circulating levels shown to decline progressively from mid-adulthood onward [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKlotho is predominantly synthesized in the distal convoluted tubules of the kidney but is also expressed at lower levels in several extrarenal tissues, including the brain, parathyroid gland, adipose tissue, liver, pancreas, and skeletal muscle [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although the precise cellular localization and functional role of klotho within skeletal muscle remain incompletely understood, growing evidence suggests that it plays an important role in regulating muscle mass, strength, and regenerative capacity. In clinical conditions such as chronic obstructive pulmonary disease, klotho has been associated with muscle weakness and has been shown to localize to both the nucleus and plasma membrane of muscle fibers [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Experimental models further demonstrate that impaired klotho signaling reduces myogenic capacity and disrupts normal muscle development [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral experimental studies have shown that klotho deficiency\u0026mdash;whether due to genetic deletion, ageing, or disease\u0026mdash;is associated with reduced muscle mass and impaired myogenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Conversely, klotho overexpression has been reported to increase satellite cell numbers, attenuate muscle atrophy, and enhance muscle regeneration following injury in dystrophic and aged muscle models [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These findings collectively support the concept that klotho exerts direct or indirect protective effects on skeletal muscle integrity.\u003c/p\u003e \u003cp\u003eSarcopenia, characterized by age-related loss of muscle mass and function, is a major contributor to frailty, disability, and mortality in older adults. Progressive reductions in muscle strength result in impaired mobility, increased fall risk, and loss of independence. Importantly, circulating klotho levels decline with age and have been associated with reduced grip strength, poor physical performance, and increased all-cause mortality [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A large population-based ageing study proposed a mechanistic link between sarcopenia and reduced circulating klotho, suggesting that klotho deficiency may reflect diminished physiological reserve [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this context, the moderate positive correlation observed in the present study between serum klotho levels and muscle fiber cross-sectional area supports the hypothesis that systemic klotho deficiency may also be relevant in early disuse-related muscle atrophy.\u003c/p\u003e \u003cp\u003eExercise is a well-recognized stimulus for increasing circulating klotho levels, although the magnitude of this response varies with age and fitness status. Beyond sarcopenia, ageing is characterized by impaired skeletal muscle regeneration following injury, resulting in prolonged recovery and increased susceptibility to recurrent damage. Experimental evidence indicates that klotho enhances regenerative capacity by modulating muscle stem cell function and suppressing excessive Wnt and TGF-β signaling pathways [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In klotho hypomorphic mice, depletion of muscle stem cells and severe impairment of regeneration have been demonstrated, whereas recombinant klotho supplementation restores stem cell function by inhibiting aberrant Wnt signaling in aged muscle [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, partial tail suspension induced clear histological features of disuse-related atrophy in both EDL and soleus muscles, accompanied by a significant reduction in serum klotho levels. This finding suggests that klotho decline may occur independently of chronological ageing and may reflect acute unloading-induced muscle degeneration. Consistent with this observation, klotho deficiency has previously been associated with reduced body weight, impaired muscle strength, and decreased physical performance in adult mice [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Although molecular markers of muscle atrophy such as atrogin-1 or MuRF-1 were not assessed in this study, previous investigations using similar suspension models have demonstrated activation of ubiquitin\u0026ndash;proteasome pathways under comparable conditions.\u003c/p\u003e \u003cp\u003eThe partial suspension protocol used in this study represents a milder unloading model compared with continuous hindlimb unloading, which is considered the gold standard for inducing disuse atrophy and simulating microgravity conditions. Nevertheless, this approach effectively produced early histological signs of muscle atrophy while minimizing animal distress and systemic stress. The mild increase in interstitial connective tissue observed, particularly in the soleus muscle, likely reflects early extracellular matrix remodeling; however, fibrosis assessment was qualitative, and quantitative collagen analysis was not performed.\u003c/p\u003e \u003cp\u003eThe magnitude of CSA reduction observed in the present study was relatively small; however, this finding is consistent with the use of a short-term, intermittent unloading protocol designed to capture early disuse-related changes rather than advanced atrophy. Previous studies employing continuous hindlimb unloading have reported more pronounced muscle loss, whereas partial or intermittent suspension models preferentially induce subtle but biologically relevant early alterations. In this context, the concurrent reduction in circulating Klotho levels despite modest structural changes suggests that Klotho may be a sensitive systemic indicator of early muscle disuse.\u003c/p\u003e \u003cp\u003eSeveral limitations of the present study should be acknowledged. The sample size was modest, and causal relationships between klotho reduction and muscle atrophy cannot be inferred. Histological evaluation relied on H\u0026amp;E and Masson\u0026rsquo;s trichrome staining without immunohistochemical confirmation, limiting precise differentiation between degenerative and regenerative processes. In addition, serum klotho levels were measured without parallel assessment of intramuscular klotho expression at the mRNA or protein level.\u003c/p\u003e \u003cp\u003eDespite these limitations, the present findings suggest that circulating klotho may be sensitive to acute muscle disuse and could serve as an early biomarker of unloading-related muscle degeneration. It has been shown that klotho is essential for an adequate regenerative response to acute muscle injury and that systemic α-klotho supplementation promotes myofiber regeneration in aged mice when administered at appropriate doses and intervals [38]. Consistent with these findings, age-related declines in circulating α-klotho have been shown to impair muscle regeneration through mitochondrial dysfunction in progenitor cells, whereas restoration of α-klotho levels improves regenerative capacity in aged skeletal muscle [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Further mechanistic studies incorporating tissue-specific klotho analyses and interventional models are warranted to clarify the role of klotho in muscle degeneration and regeneration.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, partial tail suspension induced early disuse-related muscle atrophy in both fast- and slow-twitch muscles and was associated with a significant reduction in circulating klotho levels. These findings suggest that serum klotho may reflect acute changes in muscle integrity independent of chronological ageing. Although causality cannot be established, the observed association between reduced klotho levels and muscle fiber atrophy supports the potential role of klotho as a biomarker of muscle disuse and early degeneration. Further studies integrating molecular analyses and klotho-targeted interventions are needed to determine whether modulation of klotho signaling may represent a therapeutic strategy for preventing or attenuating disuse-related muscle loss.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eAll animal procedures were carried out in accordance with relevant guidelines and regulations for the care and use of laboratory animals. The study protocol was approved by the Laboratory Animal Care and Use Committee of Fırat University Faculty of Medicine (approval date: 17 July 2023; approval number: 17217). All methods are reported in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the Fırat University Scientific Research Projects Unit.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e\u0026bull; **SK:** Conceptualization, study design, supervision of experimental protocol, critical revision of the manuscript, final approval.\u0026bull; **\u0026Ouml;E:** Conceptualization, study design, data acquisition, histopathological interpretation, statistical analysis, manuscript drafting, final approval.\u0026bull; **AA:** Conceptualization, study design, supervision of experimental protocol, critical revision of the manuscript, final approval.\u0026bull; **OST:** Experimental procedures, physiological data interpretation, laboratory supervision, manuscript editing, final approval.\u0026bull; **GA:** Data collection, laboratory support, data verification, literature review, manuscript editing, final approval.\u0026bull; **S\u0026Ccedil;:** Histopathological evaluation, interpretation of tissue findings, methodological support, critical manuscript revision, final approval.\u0026bull; **AY:** Experimental design support, physiological interpretation, data analysis assistance, manuscript revision, final approval.All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors gratefully acknowledge the support of the Fırat University Scientific Research Projects Unit.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKuro-o, M. et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e390\u003c/b\u003e, 45\u0026ndash;51 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurosu, H. et al. Suppression of aging in mice by the hormone Klotho. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e309\u003c/b\u003e, 1829\u0026ndash;1833 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, M. et al. Renal production, uptake, and handling of circulating α-Klotho. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 79\u0026ndash;90 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakenaka, T., Watanabe, Y., Inoue, T., Miyazaki, T. \u0026amp; Suzuki, H. 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Commun.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 4859 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"Klotho, disuse-induced muscle atrophy, skeletal muscle, immobilization, tail suspension, rat model","lastPublishedDoi":"10.21203/rs.3.rs-8535331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8535331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eKlotho is an anti-aging protein involved in phosphate homeostasis, oxidative stress regulation, and tissue regeneration. Although circulating Klotho levels decline with chronological aging, its response to acute disuse-induced skeletal muscle atrophy remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eSixteen male Wistar rats (3 months old) were randomly assigned to a control group (n\u0026thinsp;=\u0026thinsp;8) or a disuse group subjected to a tail suspension\u0026ndash;based unloading protocol (3 h/day for 15 days) (n\u0026thinsp;=\u0026thinsp;8). Skeletal muscle atrophy was evaluated histologically in the extensor digitorum longus (EDL) and soleus muscles using hematoxylin\u0026ndash;eosin staining, and muscle fiber cross-sectional area (CSA) was quantified with ImageJ. Interstitial connective tissue changes were assessed qualitatively using Masson\u0026rsquo;s trichrome staining. Serum Klotho concentrations were measured by enzyme-linked immunosorbent assay (ELISA).\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eRats exposed to disuse demonstrated histological findings consistent with early disuse-related skeletal muscle alterations in both EDL and soleus muscles, accompanied by a significant reduction in muscle fiber CSA compared with controls. Serum Klotho levels were significantly lower in the disuse group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Correlation analysis revealed a moderate positive association between circulating Klotho concentrations and mean muscle fiber CSA (r\u0026thinsp;=\u0026thinsp;0.58, p\u0026thinsp;=\u0026thinsp;0.02). Mild interstitial connective tissue expansion was qualitatively observed in the soleus muscle following disuse.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eShort-term disuse-induced skeletal muscle atrophy is associated with a reduction in circulating Klotho levels, independent of chronological ageing. These findings suggest that serum Klotho may serve as a candidate biomarker reflecting early disuse-related muscle degeneration. Further mechanistic studies are warranted to clarify the role of Klotho in skeletal muscle atrophy and regeneration.\u003c/p\u003e","manuscriptTitle":"Disuse-Induced Skeletal Muscle Atrophy Is Associated with Reduced Serum Klotho Levels in a Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 12:41:46","doi":"10.21203/rs.3.rs-8535331/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":"09a232ec-75e6-446e-b2b1-bdea91ac84ec","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61386476,"name":"Biological sciences/Biochemistry"},{"id":61386477,"name":"Health sciences/Medical research"},{"id":61386478,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-02-26T03:40:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 12:41:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8535331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8535331","identity":"rs-8535331","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}