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 disuseinduced 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.