Increases In MCP-1 And HMGB1 Production In HK-2 Cells Exposed To High Level Of Calcium In Vitro

ObjectiveTo investigate the effect of high level of calcium on the expression of inflammatory cytokines MCP-1 and HMGB1 in human kidney epithelial cells (HK-2).MethodsCultured HK-2 cells were divided into ① control group (+normal media); ② Ca I group (+5 mmol/L Ca2+ media); ③CaⅡ group (+10 mmol/L Ca2+ media); ④ Ca Ⅲ group (+15 mmol/L Ca2+ media). The cells were incubated with or without additives for 3h、6h and 9h. The cell activity of each groups was measured by MTT assay at 3h、6h、9 h, respectively. At 6 h, DAPI staining was used to observe the cell apoptosis. LDH activities, H2O2 contents and 8-IP concentrations in the media were determined by microplate reader microfiltration and ELISA, respectively. The mRNA expression of MCP-1 and HMGB1 in the cells were determined by RT-qPCR, and the proteins of MCP-1、HMGB1 in the media were measured by ELISA.ResultsThe results of MTT assay showed that the inhibition rate of cell activity increased significantly with the increase in calcium concentration in media. The results of DAPI staining showed that the apoptotic cells increased with the increase in calcium concentration in the media. The LDH activities, H2O2 and 8-IP concentrations in the medias all increased in accordance with the increased concentrations of calcium supplemented. Compared with control group, the mRNA expression of MCP-1 and HMGB1 in the HK-2 cells increased significantly in the 3 calcium supplemented groups (P<0.05). The protein concentrations of MCP-1 in the media of control group and Ca I, Ca Ⅱ, Ca III groups were 13.48±1.60 pg/mL,22.93±2.86 pg/mL,40.57±1.63 pg/mL,30.39±1.61 pg/mL, respectively with significantly statistical differences among the 4 groups (P<0.05). The protein concentrations of HMGB1 in the media of control group and Ca I, Ca II, Ca III groups were 138.17±5.99 pg/mL, 181.82±4.54 pg/mL,346.81±20.26 pg/mL,223.15±16.0 pg/mL, respectively with significantly statistical differences among the 4 groups (P<0.05).ConclusionsThe productions of MCP-1 and HMGB1 in the HK-2 cells exposed to high level of calcium significantly increase, in accordance with the oxidative cell injury.