Reduced Serum Klotho Levels in Disuse-Induced Skeletal Muscle Atrophy: An Experimental Study Related to Orthopedic Immobilization

Abstract

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 alterations remains unclear. In this experimental study, sixteen male Wistar rats (3 months old) were randomly assigned to a control group (n = 8) or an intermittent hindlimb unloading group (3 h/day for 15 consecutive days) (n = 8). Skeletal muscle morphology was evaluated in the extensor digitorum longus (EDL) and soleus muscles using hematoxylin-eosin staining, and muscle fiber cross-sectional area (CSA) was quantified with ImageJ software. Interstitial connective tissue changes were qualitatively assessed using Masson’s trichrome staining, and serum Klotho concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Intermittent unloading induced histological features consistent with early disuse-associated muscle remodeling in both EDL and soleus muscles, accompanied by a modest reduction in muscle fiber CSA compared with controls. Serum Klotho levels were significantly lower in the unloading group (7.44 ± 0.54 vs 8.21 ± 0.81, p = 0.045), and a moderate positive correlation was observed between circulating Klotho concentrations and mean muscle fiber CSA (r = 0.58, p = 0.02). Mild interstitial connective tissue expansion was qualitatively noted in the soleus muscle following unloading. These findings suggest that short-term intermittent hindlimb unloading in young adult rats is associated with reduced circulating Klotho levels and mild skeletal muscle remodeling. Serum Klotho may represent a candidate biomarker of early disuse-associated muscle alterations; however, further studies are required to confirm its clinical applicability. These results may have potential implications for orthopedic conditions characterized by immobilization, including fracture treatment, casting, and postoperative rehabilitation.