Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in the Absence and Presence of Chronic Kidney Disease

Abstract

BackgroundChronic Kidney Disease (CKD) is a public health epidemic that is associated with elevated serum levels of phosphate (hyperphosphatemia) as well as skeletal muscle atrophy, but this interconnection is poorly understood. Elevated phosphate (Pi) has direct effects on smooth muscle cells and induces vascular calcification. Pi also induces the production of the hormone fibroblast growth factor (FGF) 23 in bone. We wanted to test if Pi induces atrophy and/or FGF23 expression in skeletal muscle cells. Furthermore, we analyzed skeletal muscle on a functional, histological and molecular level in three models of hyperphosphatemia – two CKD models, i.e. mice with global deletion of collagen 4a3 (Col4a3‐/‐) and wildtype mice receiving an adenine‐rich diet, as well as wildtype mice on a high Pi diet with normal kidney function. Finally, we determined the effect of a low Pi diet on skeletal muscle in Col4a3‐/‐ mice.MethodsC2C12 myotubes were treated with 1‐5 mM Pi for 24 hours, followed by qPCR expression analysis of atrophy genes (atrogenes), including MT1, Trim63, and Fbox32, as well as FGF23. Furthermore, we studied Col4a3‐/‐ mice receiving normal chow or a 0.2% Pi diet at 10 weeks of age. We also analyzed C57Bl/6 mice receiving an adenine‐rich (0.2%) diet for 14 weeks or a 3% Pi diet for 3 months. We analyzed grip strength, hindlimb area by MRI, muscle wet weight, cross‐sectional area of individual muscle fibers immuno‐labeled with anti‐laminin by fluorescence microscopy, and expression levels of atrogenes by qPCR and of FGF23 by qPCR and ELISA.ResultsPi treatments increased the expression levels of atrogenes as well as FGF23 in C2C12 myotubes. In the three mouse models, grip strength and cross‐sectional area of myofibers were significantly reduced, and the expression levels of atrogenes were significantly elevated when compared to respective controls. Furthermore, we detected elevations in the mRNA and protein levels of FGF23 in the hindlimb muscles of all models. Additionally, the two CKD models showed significant reductions in muscle weight and hindlimb area. Administration of a 0.2% Pi diet protected Col4a3‐/‐ mice from developing skeletal muscle atrophy.ConclusionElevated Pi induces myotube atrophy and FGF23 expression in vitro. Mouse models with hyperphosphatemia not only develop skeletal muscle atrophy, but also produce FGF23 in skeletal muscle tissue in the presence and absence of CKD, and a low Pi diet protects the skeletal muscle in CKD mice. Pharmacological approaches targeting Pi uptake or excretion, or inhibition of Pi’s direct actions on tissues might alleviate various CKD‐associated pathologies. Future studies need to determine whether skeletal muscle‐derived FGF23 contributes to tissue injury or is protective against Pi‐induced damage.