The effect of high calcium-induced renal epithelial autophagy on the expressions of MCP-1 and HMGB1 in vitro
YI Kai
ZHAO Jia-wen
LI Cheng-yang
CHENG Ji-wen
ZHAO Yu-tong
MA Chen-jun
LIAO Nai-kai
DENG Yao-liang
Abstract：Objective To investigate the effect of high calcium induced autophagy on the cellular productions of inflammatory cytokines MCP-1 and HMGB1 in HK-2 cells. Methods HK-2 cells were treated with different concentrations of calcium medium (0 mmol/L, 5 mmol/L, and 10 mmol/L) for 12 h, and subsequently incubated with indicated high calcium medium (10 mmol/L) for 0 h, 6 h, and 12 h, respectively. After the specified time, the culture cells were collected for further analysis. Western blot was used to assess the levels of intracellular LC3 B-II/LC3 B-I (protein marker of autophagy) and Beclin1 protein. Also, the expression of HMGB1 and MCP-1 mRNA were tested by real-time PCR (RT-PCR). The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) were used to pre-treated HK-2 cells for 6 h, and then the cells were exposed to different concentration of calcium medium (0 mmol/L, 5 mmol/L, and 10 mmol/L) for 12 h. The MCP-1 and HMGB1 mRNA expression were tested by RT-PCR. In addition, after pre-treatment with different concentration of CQ (10 μmol/L, 20 μmol/L and 30 μmol/L), HK-2 cells were cultured with medium supplemented with calcium of 10 mmol for 12 h. MTT assay was used to detect the effect of CQ on cell proliferation.Results Compared with the control, high calcium treatment significantly increased the protein levels of LC3 B-Ⅱ/LC3 B-Ⅰ and Beclin 1 in concentration-and time-dependent manners (P＜0.05). Meanwhile, the productions of MCP-1 mRNA and HMGB1 mRNA in HK-2 cells exposed to high level of calcium were significantly increased (P＜0.05). Pretreatment with 3-MA or CQ significantly down-regulated the expression of MCP-1 mRNA but not HMGB1 mRNA in HK-2 cells. In addition, pretreatment with CQ resulted in the improvement of proliferation of HK-2 cells exposed to high calcium. Conclusion The production of MCP-1 and HMGB1 in HK-2 cells exposed to high level of calcium are increased, in accordance with autophagy. Pretreatment with autophagy inhibitors 3-MA or CQ suppresses the production of MCP-1 but not HMGB1 in the HK-2 cells, and improves the cells proliferation.
Keywords：hypercalciuriahuman renal epithelial cellsautophagyMCP-1HMGB1
Publication Date：2019-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 483-487 )