Effect Of Mcc950 In Different Concentrations Of Glucose And Uric Acid On NLRP3 Inflammatory Pathway In Human Renal Tubular Epithelial Cells

Background Diabetic kidney disease(DKD)is one of the common complications of type 2 diabetes with tubular injury playing an important role in its pathogenic process,but until now the underlying mechanism for renal tubular damage still remains unclear.NLRP3 inflammasome has been an established pathway for tubular injury,it may also be activated and induce secretion of IL-1βand IL-18 in cascade,finally lead to tubular damage by the chronic inflammatory attack.How the complicated inflammatory pathway exert its effectiveness on tubular epithelia has been not elucidated.We then tried to make the pathogenic mechanism more clear by cultivating tubular epithelia in different concentrations of glucose and uric acid by adding Mcc950,a specific blocker for NLRP3 inflammasome.Objective To investigate the selective inhibition of NLRP3 inflammasome in human renal tubular epithelial cell line(HK-2)by Mcc950 in the environment of high concentration of glucose and uric acid,and then to observe the expression and secretion levels of five inflammatory factors including NLRP3,ASC,Caspase-1,IL-1β and IL-18.Methods1)Human renal tubular epithelial cells(HK-2 cells)were cultured,including subculture, amplification,and partial cryopreservation.2)According to the concentration of glucose(5.5mmol/L and 25mmol/L),uric acid(0mmol/L,0.2 mmol/L,0.4 mmol/L)and Mcc950(0 mmol/L and 0.1 mmol/L),the 2×3×2factorial method was used to divide into 12 groups(as shown in table 1).3)Detection: 1)Real-time fluorescence quantitative PCR was used to detect the m RNA expression levels of the major components of NLRP3,ASC,Caspase-1,IL-1 and IL-18 in the inflammatory pathway of NLRP3 in each group;2)Expression levels of NLRP3,ASC,Caspase-1,IL-1 and IL-18 secreted protein in cells were detected by Elisa.Results(1)Gene expression levels of the five components of NLRP3 inflammasome in HK-2 cells and the release amounts of effector molecules IL-18 and IL-1 decreased with the increase of uric acid concentration in the n-glucose environment.(2)In the high-glucose environment,except for Caspase-1,gene expression levels of other components of NLRP3 inflammatory body in HK-2 cells and the release amounts of effector molecules IL-18 and IL-1 showed no obvious changes with uric acid concentration,but they were all reduced compared with those in the normal sugar environment.(3)In the normal glucose environment,the gene expression level of the five components of NLRP3 inflammatory body decreased when the uric acid was 0mmol/L in HK-2 cells added with Mcc950,but increased with the addition of uric acid.(4)In addition to Caspase-1 gene,Mcc950 had no significant effect on the gene expression level of NLRP3 inflammasome in the high-sugar environment.(5)The concentrations of IL-18 and IL-1 in HK-2 cell culture medium supplemented with Mcc950 were significantly decreased under the conditions of normal sugar and high sugar,but the concentrations of NLRP3 and ASC were not significantly changed.(6)The expression level of Caspase-1 gene in HK-2 cells in high glucose environment,the concentration of culture medium and whether Mcc950 was added were consistent with the trend in normal glucose environment.Conclusions(1)Renal tubular epithelial cells have a complete NLRP3 inflammatory system.(2)Both high uric acid and high glucose can activate the NLRP3 inflammatory pathway in HK-2 cells to produce IL-18 and IL-1;HK-2 cells have a compensatory mechanism that counter-regulates these activation.(3)Mcc950 can significantly inhibit the release of NLRP3 inflammatory system effector molecules mediated by uric acid in HK-2 cells in the environment of normal glucose and high glucose.