The Regulatory Effects Of Macrophage On The Renal Tubular Epithelial Cell-Calcium Phophate Coculture System

Objectives:To investigate the regulatory effects of macrophage in the human renal tubular epithelial cell (HK-2)-calcium phosphate crystal coculture system and the relationships between Fetuin-A and HMGB1 in the system.Methods:In this experimental research, the human renal tubular epithelial cells were exposed to calcium phosphate crystals with the presence or absence of macrophage and/or HMGB1, subsequently, the expression levels of Fetuin-A in human renal tubular epithelial cells and the MCP-lin the supernatants were determined. In the primary part, it was divided into 3 groups which included group A、group B and group C. Group A:HK-2; Group B:HK-2+CaP (100ug/ml); Group C:M(?)+HK-2+CaP(100ug/ml); Group C, which included M0-HK-2-CaP that consist a coculture system, was established by the Transwell cocultrue system. After incubating at 37℃ in 5% CO2 air atmosphere for 6h、 9h、12h, before the harvest, there was a morphological observation of the human renal tubular epithelial cells in different groups by inverted phase contrast microscope, and then the renal tubular epithelial cells in all groups were harvested for the subsequent Real-Time Polymerase Chain Reaction (RT-PCR). And the supernatants of the culture media were collected and then centrifuged with 2000rpm for 10min for the subsequent ELISA assay, and the contents of MCP-1 in the supernatants were evaluated. Additionally, in the other part, the renal tubular epithelial cells were exposed to the Human recombinant HMGB1 protein(rHMGB1) with different concentrations which ranged from Ong/ml to 2000ng/ml(0ng/ml,500ng/ml, 1000ng/ml,2000ng/ml), after incubating for 24h, the renal tubular epithelial cells were harvested and the expression levels of the Fetuin-A protein in the tubular epithelial cells were determined by RT-PCR.Results:the human renal tubular epithelial cells in Group B、group C showed no significant difference in contrast to group A in morphology. And the differences of Fetuin-A expression level among Group A, Group B and Group C showed statistic significance(P<0.05), of which that of Group C showed a lowest level when compared to the others, and that of Group B appeared a lower level than that of Group A, the decrease expressions of Fetuin-A in tubular epithelial cells in the three groups were in a time-dependent manner (p<0.05). And the content of MCP-1 in Group C is apparently higher than that of the other groups (p<0.05). The expression levels of Fetuin-A in tubular epithelial cells that exposed to different concentrations of HMGB1 increased in a concentration-dependent manner according to RT-PCR, of which that of Group 1000ng/ml and Group 2000ng/ml were higher than that of Group 500ng/ml and the control(P<0.05), and that of Group 2000ng/ml showed a highest level than that of others(P<0.05).Conclusions:1.In the coculture system that established by Transwell which simulates the homeostasis of the human renal papillae, the interaction between renal tubular epithelial cells and calcium phosphate crystals did induce the occurrence of inflammation and the release of inflammatory cytokines, and the down-regulation of Fetuin-A in renal tubular epithelial cells possibly associated with inflammation.2. In the coculture system that established by Transwell which simulates the homeostasis of the human renal papillae, macrophage showed significant regulatory effects on the cytokines in the calcium phosphate-renal tubular epithelial cell coculture system, which up-regulated the expression levels of inflammation cytokines such as MCP-1, while the Fetuin-A in the tubular epithelial cell was suppressed.3. The expression of Fetuin-A in the tubular epithelial cell was regulated by HMGB1, and, possibly, there may exist different mechanisms in regulating the expression of Fetuin-A in different phases of inflammation that different kinds of inflammatory cytokines were released.