P0707UREMIC TOXINS IMPAIR SKELETAL MUSCLE REGENERATION PROCESS INDUCING CELL CYCLE ARREST AND APOPTOSIS IN CULTURED MYOBLASTS

Abstract Background and Aims The loss of muscle mass and function has been related to chronic kidney disease (CKD). About 37% of dialysis patients show symptoms of sarcopenia and this has been related to an increased risk of mortality. Changes in sarcopenic muscle include the loss of its regenerative capacity due to a reduction in the number and function of satellite cells, the muscle stem cells. The concentration of serum uremic toxins (UT) increases in parallel to a decline in the glomerular filtration rate in patients with CKD and this uremia may be involved in the development of sarcopenia. Previous studies showed as serum concentration of UT found in the early stages of CKD inhibits myogenic differentiation of cultured myoblasts. Nevertheless, the effect of those concentrations found in the advanced stages of CKD has not been described. The study aimed to analyse whether UT affect the muscular regeneration process by modifying the proliferation capacity of myoblasts (activated satellite cells). Method Cultured mouse myoblasts C2C12 cells were used for all experiments. Cells were grown with 0% or 10% FBS culture media in the presence or absence of indoxyl sulphate and para-cresol at doses of 100µg/ml each one, which are similar to ones found in the advanced stages of CKD. Proliferation was evaluated by scratch wound healing and cell cycle by flow cytometry with propidium iodide and the fluorescent probe CFSE, an intracellular protein binding dye that is divided equally between daughter cells, allowing the discrimination of successive rounds of cell division. Chromosome condensation was assessed by immunofluorescence staining by confocal microscopy. Apoptosis was analysed by annexin V staining. Results C2C12 cells treated with UT shown a significant decrease in the proliferation rate. A significant delay in wound closure was observed in cells treated with UT compared to control cells. Myoblasts treated with UT suffered a significant decrease in the proliferation rate since the probe remained higher than in the vehicle-treated cells. Proliferating cells treated with UT suffered a dramatic cell cycle arrest between the phases S and G2/M. Chromosome condensation was also analysed, finding that in the presence of colcemid, vehicle-treated cells condensed their chromosomes, as expected, whereas UT-treated cells did not, suggesting that UT stop the cell cycle at any point before the entry of cells in the mitosis phase. Besides, there was strong phosphorylatio