Increased Production Of HMGB1,IL-6 Of HK-2 In The Exposure Of High Level Of Calcium In Vitro

Objective:To observe the injury of human renal tubular epithelial cells exposed in diverse calcium environment and the expression of inflammatory cytokines interleukin-6 and HMGB1 in vitro.Methods:Put calcium chloride dihydrate into DMEM-F12 medium getting different concentrations of calcium chloride solution(2.5 mM,5 mM,10 mM)as inducer.Cultured human renal tubular epithelial cells in vitro for 6 hours.The HK2 cell were divided into:a.normal control group(serum-free DMEM-F12medium);b.calcium I group(2.5 mM CaCl2 + DMEM-F12 medium);c.calcium II group(5 mM calcium CaCl2 + DMEM-F12 medium);d.calcium III group(10mM CaCl2 + DMEM-F12 medium).After cultured for 6 hours,using CCK-8assay to detect proliferation of HK2 cells.The content of LDH and interleukin-6in supernate were examined by microplate reader microfiltration and Elisa assay,respectively.The expression of HMGB1 in HK2 cells were detected by Western blot assay.Results:CCK-8 method in the microplate reader(450nm)measured OD value of group a,group b,group c,group d were 1.111 ± 0.063,1.020 ±0.048,0.981 ± 0.076,0.856 ± 0.075,respectively.There were significant difference between group a and group b,c and d.(p> 0.05).The OD value of group d was significantly higher than that of group b and group c(p <0.05).There was no significant difference between group b and group c(p> 0.05),suggesting that calcium ion solution could decrease cell proliferation.The concentrations of LDH in the supernatants of group a,b,c and d were(28.79±11.12)U/L、(50.93±11.72)U/L、(75.61±12.30)U/L、(211.32±10.40)U / L,respectively.There were significant difference between group a and group b,c and d(p <0.05).There was significant difference between group d and group b and c(p < 0.01).There was significant difference between group c and b(p <0.05).The concentrations of IL-6 in the supernatants of group a,b,c and d were(299.92 ± 14.13)pg / ml,(421.63 ± 20.56)pg / ml,(439.23 ± 14.95)pg /ml and(464.77 ± 24.95)pg/ml,respectively.There were significant difference between group a and group b,c and d(p <0.05).here was significant difference between group d and group b(p <0.05).There was no significant difference between group c and group d and b(p> 0.05).The relative expression of HMGB1 protein in b,c and d groups was significantly higher than that in group a(p <0.05).The expression of HMGB1 protein in group C and group d was significantly higher than that in group b(p <0.01).There was no significant difference between group d and group c(p> 0.05).Conclusion: Calcium environment will produce injury of human renal tubular epithelial cells and induce high expression of IL-6 and HMGB1.And this may be an important mechanism in the formation of calcium-containing kidney stones.