Involvement Of Macrophages In The Regulation Of Calcium Oxalate Calculi In Renal Papillary Calcification

The First Part:the Expression of Macrophages and HMGB1 in Renal Papilla Calcified Tissue and Interaction AnalysisObjective:To investigate the role of macrophages and HMGB1 in the formation of renal papilla calcification, nidus for future development of CaOx kidney stone. Methods:collect 13 renal papilla calcified tissue samples of patients with calcium oxalate kidney stones as experimental group,12 normal renal papillary tissue samples from patients underwent radical nephrectomy of tumors as control group. The expression and distribution of macrophages (CD68) and HMGB1 in renal tissue papillae were detected by real-time fluorescence quantitative PCR and immunohistochemical method. Macrophages, renal tubular epithelial cells and calcium oxalate crystals in the renal papilla calcified tissue were observed by transmission electron microscope technique. Results:The expressions of CD68 and HMGB1 in renal papilla calcified tissues of patients with calcium oxalate kidney stones were higher than renal tumor patients. Macrophages were expressed in the small vessel cavity and interstitial tissue, HMGB1 was mainly expressed in the renal tubular epithelial cells and interstitial tissue. Electron microscopy showed that the macrophages infiltrated and phagocytized calcium oxalate crystal in the interstitial tissue of renal calcified papilla. Conclusion:macrophages and HMGB1 may be involved in the formation of renal papilla calcification in CaOx kidney stone.The Study of Interaction Between Macrophages and calcium oxalate monohydrate in vitroObjective:To observe the interaction of the calcium oxalate monohydrate crystals (COM) with macrophages in vitro and investigate the expression of HMGB1 in macrophages stimulated by COM. Methods:the process of macrophages phagocytized COM was observed by live cell imaging technology, the morphology changes of macrophages were observed by phase contrast microscope. Macrophages were stimulated by 100ug/ml COM stimulation, for 0, 6,12,24h and 36h, the HMGB1 m RNA expression in the cells were detected by RT-PCR. HMGB1 protein content in total cell proteins and the cytoplasm of cellswere detected by Western blot. After the cells were stimulated by COM for 0,1,2h and 4h, TNF-alpha and IL-6 content in cell culture supernatant detected by ELISA. Results:1.we have observed the adhesion and phagocytosis of COM crystals by the macrophages under live cell imaging.2. RT-PCR results showed that, after the cells stimulated by COM at 0-12h, HMGB1 mRNA expression had no obvious change. At 18-24h, HMGB1 mRNA expression in cells were significantly increased. At 0-6h, content of HMGB1 protein in the cytoplasm of macrophages was low. But, at 12-36h, HMGB1 protein in cytoplasm increased gradually. After COM stimulated by 0-6h, there were no obvious changes of HMGB1 protein in total protein content, but it began to increase when the cells stimulated by 12h. At 24-36h, it maintained at a higher level.3.The results of ELISA showed that TNF-α and IL-6 in cell culture supernatant of macrophages increased significantly for 2h, reached the peak at 4H. Conclusion:1.the phenomenon that macrophage phagocytosised and cleared COM, which may be a protective immune mechanism strigered by the crystal deposition in kidney.2. COM can induce the expression of HMGB1 in macrophage, TNF- alpha and IL-6, inflammatory factors; the secretion time of HMGB1 was later than that of TNF- alpha and IL-6, this may be one of the important mechanisms of renal chronic inflammatory injury induced by crystals.The Third Part:experimental research on the hmgbi expression of renal tubular epithelial cells stimulated by calcium phosphate crystalsObjective:To study the expression of HMGB 1 mRNA and protein in human renal tubular epithelial cell (HK-2) stimulated by calcium phosphate crystals.Methods:Morphological changes of renal tubular epithelial cells stimulated by calcium phosphate phase were observed by the contrast microscope.when the cells were stimulated by 100 ug/ml calcium phosphate for 18 h,24 h,30h, HMGB1mRNA was detected by RT-PCR.After the cells were stimulated by 100ug/ml calcium phosphate for 0,3,6,9,12,18,24 and 48h, the content of HMGB1 protein in cells total protein were detected by Western blot.Results: LAt18-30h,HMGB1mRNA showed a time dependent increase and reached the peak at 30h,there was statistical significance (p<0.05). At 3,6,9h,there was no significant difference of HMGB 1 proteincontents compared with Oh, but 18-48h, the contents of HMGB 1 protein showed a time dependent increase and reached the peak at 48h.There was statistical significance, compared with Oh (p<0.05).Conclusion:calcium phosphate stimulated the HMGB1 expression of renal tubular epithelial cells.this process last for quite a long time, which may be one of the important causes of renal papilla calcification.The Fourth Part:the synergistic effects of hmgbi in inflammatory factors release of macrophages induced by calcium phosphate crystalsObjective:To investigate the synergistic effect of HMGB1 on the release of IL-1β, IL-6, TNF-a, MCP-1 early inflammatory cytokines of macrophages induced by calcium phosphate crystals and its possible mechanism. Method: 1. Macrophages were divided into blank group, 100ug/mlCaP group, 100ng/mlHMGB1 group, 100ug/mlCaP+100ng/mlHMGB1 group, intervented for 1,2,4h, the cell supernatant was collected, The IL-1β, IL-6, TNF-a, MCP-1 content were detected by ELISA 2. Macrophages were divided into 100ug/mlCaP group, 100ug/mlCaP+10ng/mlHMGB1 group, 100ug/mlCaP+50ng/mlHMGB1 group, 100ug/mlCaP+100ng/ml group, intervented for 4h, the cell supernatant was collected. The IL-1β, IL-6, TNF-a, MCP-1 content were detected by ELISA 3. The macrophages were stimulated by 100ug/mlCaP for 1,2,4h. NF-KB mRNA of macrophages was detected by RT-PCR.4, Macrophages were stimulated by 100ug/mlCaP+different concentration of HMGB1(100ng/ml,500ng/ml, 1000ng/ml) 1h, NF-KB mRNA of macrophages was detected by RT-PCR. Results:1.ELISA results showed that the group 100ug/mlCaP,100ng/mlHMGB1, IL-1β, IL-6, TNF-a, MCP-1 content in cell culture supernatant were higher than the blank group, 100ug/mlCaP+100ng/mlHMGB1 group, IL-1β, IL-6, TNF-a, MCP-1 in cell culture supernatant were higher than those in the blank group, 100ug/mlCaP group, 100ng/mlHMGB1 group, P<0.05, and was time dependent.2.The IL-1β, IL-6, TNF-a, MCP-1 content in cell culture supernatant of different concentrations of HMGB1+100ug/mlCaP group were higher than 100ug/mlCaP group, P<0.05, in a dose-dependent manner.3. the macrophages were stimulated by 100ug/mlCap for 1,2,4h, compared with the control group,3 groups of NF-KB mRNA relative expression lever in macrophage were increased, and was time dependent,reached the peak at 4h, p<0.05. The NF-KB mRNA relative expression lever in macrophages of 100 ug/mlCap+different concentration of HMGB1 groups were higher than 100 ug/mlCap group, p<0.05. Conclusion:1. HMGB1 acts in synergy with CaP in activating macrophages to secrete proinflammatory cytokines IL-1β、IL-6, and TNF-a, MCP-1.2 The synergistic effect of HMGB1 on the release of proinflammatory cytokines in macrophages induced by calcium phosphate crystals may be related to the activation of NF-KB signal pathway.The Fifth Part:the regulation effects of macrophages in the calcium phosphate-renal tubular epithelial cells reaction systemObjective:to study the migration of macrophages in calcium phosphate-macrophage, renal tubular epithelial cell co-culture system. to investigate the regulation of macrophage on inflammatory factor MCP-1、TGF-β in calcium phosphate-renal tubular epithelial cells reaction system. Methods:by using Transwell cell co-culture system, A group:macrophageswere inoculated in upper chamber, renal tubular epithelial cells in lower chamber.B group:macrophages were inoculated in upper chamber, renal tubular epithelial cells and calcium phosphate in lower chamber, macrophages migration was observed in the two groups at different time(6h,9h,12h). MCP-1, TGF-β in the cell culture supernatant of C group (calcium phosphate+renal tubular epithelial cells)and B group were detected by ELISA.Results:For 6h and 9h, the number of macrophage migration in group B was gradually increased, compared with A (p<0.05). But for 12h, there was no significant difference between the two groups. The concentration of MCP-1, TGF-β in the culture supernatant of group B was higher than group B and D, When the two index were detected by ELISAat 1,3,6h and 24,48,72h respectively.RT-PCR showed that same results in the TGF-β, MCP-1mRNA expression of renal tubular epithelial cells (P<0.05).Conclusion:1. Calcium phosphate crystal has a promoting effect on macrophage migration,it may be related to the MCP-1 induced by Renal tubular epithelial cells.2. In the regulation of macrophages,TGF-β、MCP-1 inflammatory cytokines in calcium phosphate-renal tubular epithelial cells reaction system increased obviously. Which may be associated with the release of HMGB1 in renal tubular epithelial cells stimulated by calcium phosphate, and HMGB1 would activate the NF-KB signal pathway of macrophages.