Objective: To explore the effect of high glucose-based peritoneal dialysis fluids on NLRP3-IL-1β in human peritoneal mesothelial cells.
Methods: HMrSV5 cells (SV40 immortalized human peritoneal mesothelial cell line) were grown in type I collagen-coated dishes in DMEM/F12 containing 10% fetal calf serum (FCS). All experiments on HMrSV5 cells were performed between passages 5 and 10. The cells were divided into 7 groups: control, 1.5% dextrose, 2.5% dextrose, 4.25% dextrose, rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A. Immunoblotting was used to evaluate the expression of IL-1β. Small interfering RNA (siRNA) targeting NLRP3 was used to downregulate the expression of NLRP3 and Western blot was used to evaluate the expression of IL-1β in human peritoneal mesothelial cells exposed to 4.25% dextrose. In the meanwhile, resveratrol (RSV) was used to induce autophagy, 3-methyladenine (3-MA) and siRNA against Beclin 1 or ATG5 were used to block auto-phagy, flow cytometric was used to analyze the respiring (mitotracker deep red), total (mitotracker green) and reactive oxygen species (ROS)-generating mitochondria (mitoSOX); Western blot was used to evaluate the expression of IL-1β.
Results: The IL-1β relative expressions were 0, 0.175±0.082, 0.418±0.163, 2.357±0.288, 2.642±0.358, 3.271±0.462, and 0.123±0.091, indicating that the cells exposed to high glucose-based peritoneal dialysis fluids and cells treated with mitochondria respiratory chain key enzyme complex I, and complex III inhibitors increased the IL-1β expression. And we found that NLRP3 knock-down significantly blocked the upregulation of IL-1β. In addition, the fluorescence intensity of total mitochondria and ROS-generating mitochondria in the following groups: control, negative control, RSV, 3-MA, ATG5 siRNA, Beclin1 siRNA were 1.76±0.42, 1.83±0.55, 1.85±0.62, 7.36±0.92, 5.35±0.77, 5.06±0.62 and 821.68±95.12, 868.15±102.82, 723.39±92.56, 1 660.08±113.65, 1 433.01±107.24, 1 562.36±112.88 respectively. The increased concentrations of mitochondrial ROS and IL-1β upregulation were confirmed in the inhibition but not the induction of auto-phagy. We also found that downregulation of ATG5 and Beclin1 sensitized cells for the release of IL-1β induced by MSU (monosodium urate) or nigericin which was the NLRP3 inflammasome activator. RSV treatment attentuated this effect.
Conclusion: Long-term application of high glucose-based peritoneal dialysis fluids can trigger the consistent activation of NLRP3-IL-1β in peritoneal mesothelial cells. Timely initiation of autophagy may block the NLRP3-IL-1β activation and provide a basis for the further development of a potential therapeutic strategy for delay of chronic inflammation and peritoneal fibrosis associated with peritoneal dialysis.