Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Chronic Kidney Disease

Abstract

Alterations in mineral metabolism involve changes in serum levels of phosphate as well as its key regulators, fibroblast growth factor 23 (FGF23), vitamin D, parathyroid hormone and klotho. Chronic Kidney Disease (CKD) is a public health epidemic that affects an estimated 37 million Americans, and is associated with elevated serum levels of phosphate (hyperphosphatemia) and FGF23 as well as various pathologies, such vascular calcification, cardiac hypertrophy, and skeletal muscle atrophy. We previously found that elevated FGF23 directly targets cardiac myocytes and contributes to cardiac hypertrophy. Furthermore, elevated phosphate has direct effects on smooth muscle cells and induces vascular calcification. In order to determine whether FGF23 and/or phosphate might also contribute to CKD-associated skeletal muscle atrophy, we tested if skeletal muscle cells can respond to elevations of FGF23 or phosphate. Since the skeletal muscle phenotype in CKD is not well characterized, we analyzed skeletal muscle on a functional, histological, and molecular level in two established mouse models of CKD. We studied the C2C12 skeletal muscle cell line during the differentiation process from myoblasts to myotubes as well as fully differentiated myotubes. Myoblast differentiation was induced with horse serum; cells were co-treated with 25 ng/ml FGF23 or 5 mM phosphate for 5 days, followed by the expression analysis of differentiation markers by qPCR. In a separate study, differentiated C2C12 myotubes were treated with FGF23 or phosphate for 24 hours, followed by the qPCR analysis of atrophy genes (atrogenes), including MT1, Murf1, and Fbox32. Furthermore, we studied mice with global deletion of collagen 4a3 (Col4a3-/-) and wildtype littermates at 10 weeks of age, as well as C57Bl/6 mice receiving an adenine-rich (0.2%) diet or regular chow for 12 weeks. In all mice we studied grip strength, hindlimb circumference by MRI, cross-sectional area of individual muscle fibers that were immuno-labeled with anti-laminin by fluorescence microscopy, and expression levels of atrogenes by qPCR. Phosphate but not FGF23 affected the differentiation process of C2C12 myoblasts and differentiated C2C12 myotubes. Phosphate treatments significantly reduced expression levels of MyoD and MyoG and increased expression of MT1. In both mouse models, grip strength and areas of hindlimb and myotubes were significantly reduced, and expression levels of MT1 and Murf1 were significantly elevated when compared to their controls. FGF23 does not target skeletal muscle cells suggesting that it cannot directly contribute to skeletal muscle atrophy. Elevated phosphate impairs myoblast differentiation and induces atrophy in vitro. We have characterized two CKD mouse models with associated skeletal muscle atrophy, where we will now determine the contribution of hyperphosphatemia to the injury process. Pharmacological approaches targeting phosphate uptake or excretion, or phosphate's direct actions on tissues might alleviate various CKD-associated pathologies.