Uremic toxin indoxyl sulfate induces the imbalance of cellular iron metabolism and oxidative stress to interfere with myogenesis in myoblasts

Chronic kidney disease (CKD) leads to the accumulation of uremic toxins such as indoxyl sulfate (IS), which has been linked to myopathy. Iron is essential for muscle growth and differentiation, with ferrous iron (Fe²⁺) contributing to intracellular oxidative stress. Although IS known to affect muscle differentiation and regeneration, the underlying mechanisms remain poorly understood. Both iron overload and deficiency can negatively impact muscle growth. We hypothesized that IS impairs myoblast differentiation by disrupting the balance between intracellular oxidative stress and iron metabolism. To test this, we exposed C2C12 myoblasts and primary human skeletal muscle myoblasts to IS during the proliferation phase and maintained IS exposure throughout the differentiation process. IS treatment reduced both intracellular reactive oxygen species (ROS) and free Fe²⁺ levels during differentiation. It also altered intracellular iron metabolism and upregulated the gene expression and activity of antioxidant-related enzymes, maintaining the cells in a high-antioxidant state and establishing a new oxidative balance. Unexpectedly, Fe²⁺ (FeSO₄) supplementation, with or without IS, significantly increased ROS levels and further exacerbated the inhibition of myoblast differentiation induced by IS, suggesting that cellular redox homeostasis was disrupted. These findings reveal that IS induces an imbalance in cellular iron metabolism and oxidative stress, providing new insights into an alternative mechanism by which IS inhibits muscle differentiation and regeneration.