In patients with chronic kidney disease (CKD), loss of cellular proteins increases the risks of morbidity and mortality. Persistence of muscle protein catabolism in CKD results in striking losses of muscle proteins as whole-body protein turnover is great; even small but persistent imbalances between protein synthesis and degradation cause substantial protein loss. No reliable methods to prevent CKD-induced muscle wasting currently exist, but mechanisms that control cellular protein turnover have been identified, suggesting that therapeutic strategies will be developed to suppress or block protein loss. Catabolic pathways that cause protein wasting include activation of the ubiquitin–proteasome system (UPS), caspase-3, lysosomes and myostatin (a negative regulator of skeletal muscle growth). These pathways can be initiated by complications associated with CKD, such as metabolic acidosis, defective insulin signalling, inflammation, increased angiotensin II levels, abnormal appetite regulation and impaired microRNA responses. Inflammation stimulates cellular signalling pathways that activate myostatin, which accelerates UPS-mediated catabolism. Blocking this pathway can prevent loss of muscle proteins. Myostatin inhibition could yield new therapeutic directions for blocking muscle protein wasting in CKD or disorders associated with its complications.

Muscle wasting in chronic kidney disease (CKD) begins with impaired insulin/IGF-1 signaling, causing abnormal protein metabolism. In certain models of muscle atrophy, reduced satellite cell function contributes to atrophy, but how CKD affects satellite cell function is unknown. Here, we found that isolated satellite cells from mice with CKD had less MyoD, the master switch of satellite cell activation, and suppressed myotube formation compared with control mice. In vivo, CKD delayed the regeneration of injured muscle and decreased MyoD and myogenin expression, suggesting that CKD impairs proliferation and differentiation of satellite cells. In isolated satellite cells from control mice, IGF-1 increased the expression of myogenic genes through an Akt-dependent pathway. CKD impaired Akt phosphorylation in satellite cells after muscle injury. To test whether impaired IGF-1 signaling could be responsible for decreased satellite cell function in CKD, we created an inducible IGF-1 receptor knockout mouse and found impaired satellite cell function and muscle regeneration. In addition, both CKD and IGF-1 receptor knockout mice developed fibrosis in regenerating muscles. Taken together, impaired IGF-1 signaling in CKD not only leads to abnormal protein metabolism in muscle but also impairs satellite cell function and promotes fibrosis in regenerating muscle. These signaling pathways may hold potential therapeutic targets to reduce CKD-related muscle wasting.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle crosssectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-b, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-b3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-b pathway proteins.

The mechanisms underlying the muscle wasting that accompanies CKD are not well understood. Animal models suggest that impaired differentiation of muscle progenitor cells may contribute. Expression of the myogenesis-suppressing transcription factor Ying Yang-1 increases in muscle of animals with CKD, but the mechanism underlying this increased expression is unknown. Here, we examined a profile of microRNAs in muscles from mice with CKD and observed downregulation of both microRNA-29a (miR-29a) and miR-29b. Because miR-29 has a complementary sequence to the 3Ј-untranslated region of Ying Yang-1 mRNA, a decrease in miR-29 could increase Ying Yang-1. We used adenovirus-mediated gene transfer to express miR-29 in C2C12 myoblasts and measured its effect on both Ying Yang-1 and myoblast differentiation. An increase in miR-29 decreased the abundance of Ying Yang-1 and improved the differentiation of myoblasts into myotubes. Similarly, using myoblasts isolated from muscles of mice with CKD, an increase in miR-29 improved differentiation of muscle progenitor cells into myotubes. In conclusion, CKD suppresses miR-29 in muscle, which leads to higher expression of the transcription factor Ying Yang-1, thereby suppressing myogenesis. These data suggest a potential mechanism for the impaired muscle cell differentiation associated with CKD.

Uremic cardiomyopathy and muscle atrophy are associated with insulin resistance and contribute to chronic kidney disease (CKD)-induced morbidity and mortality. We hypothesized that restoration of miR-26a levels would enhance exosome-mediated microRNA transfer to improve muscle wasting and cardiomyopathy that occur in CKD. Methods: Using next generation sequencing and qPCR, we found that CKD mice had a decreased level of miR-26a in heart and skeletal muscle. We engineered an exosome vector that contained Lamp2b, an exosomal membrane protein gene fused with a muscle-specific surface peptide that targets muscle delivery. We transfected this vector into muscle satellite cells and then transduced these cells with adenovirus that expresses miR-26a to produce exosomes encapsulated miR-26a (Exo/ miR-26a ). Exo/ miR-26a was injected once per week for 8 weeks into the tibialis anterior (TA) muscle of 5/6 nephrectomized CKD mice. Results: Treatment with Exo/ miR-26a resulted in increased expression of miR-26a in skeletal muscle and heart. Overexpression of miR-26a increased the skeletal muscle cross-sectional area, decreased the upregulation of FBXO32/atrogin-1 and TRIM63/MuRF1 and depressed cardiac fibrosis lesions. In the hearts of CKD mice, FoxO1 was activated, and connective tissue growth factor, fibronectin and collagen type I alpha 1 were increased. These responses were blunted by injection of Exo/ miR-26a . Echocardiograms showed that cardiac function was improved in CKD mice treated with Exo/ miR-26a . Conclusion: Overexpression of miR-26a in muscle prevented CKD-induced muscle wasting and attenuated cardiomyopathy via exosome-mediated miR-26a transfer. These results suggest possible therapeutic strategies for using exosome delivery of miR-26a to treat complications of CKD.