高血糖在高草酸尿环境下促进肾草酸钙结石形成的机制研究

The mechanism of hyperglycemia promoting the formation of renal calcium oxalate stone under hyperoxaluria

目的通过细胞及动物实验,探究高血糖促进草酸钙结石形成的机制。方法八周龄Wistar雄性大鼠(共40只)随机分为4组:阴性对照组、高草酸尿模型组,高血糖模型组,高血糖合并高草酸尿模型组;每组10只。采用q RT-PCR检测在含有不同浓度葡萄糖培养基下HK2细胞中OPN、MCP-1、Cbfa1、BMP-2 m RNA的表达;酶联免疫吸附测定(ELISA)检测OPN与MCP-1在培养基中的浓度;钙盐染色检测各组大鼠肾组织中草酸钙结石晶体;细胞凋亡试验检测各组大鼠肾组织中肾小管上皮细胞凋亡。免疫组织化学染色法(IHC)检测OPN在各组大鼠肾组织中的表达;ELISA检测MCP-1在各组大鼠24小时尿液中的含量。结果高浓度葡萄糖环境中,HK-2细胞的OPN与MCP-1表达上调。高血糖合并高草酸尿模型组肾组织中有大量草酸钙沉积,大量肾小管上皮细胞凋亡,OPN表达显著增加,其尿液中MCP-1总量明显增加,与其他组相比有统计学差异(P<0.05)。结论高血糖能促使肾小管上皮细胞炎症趋化因子OPN、MCP-1表达增加。在高草酸尿环境中,高血糖能促进肾小管上皮细胞凋亡及草酸钙结石形成。高血糖可能是通过炎症趋化因子加重局部炎症反应,促进肾小管上皮细胞凋亡及草酸钙结石形成。

Objective To explorethe mechanism of hyperglycemia promoting the formation of calcium oxalate stones through cell and animal experiments.Methods Eight-week-old W istar male rats(40 rats in total)were randomized into 4 groups:negative control group,hyperoxaluria model group,hyperglycemic model group,hyperglycemia group with high oxalic acid urine model group;10 rats in each group.The mRNA levels of the OPN,MCP-1,Cbfa1 and BMP-2 were analyzed with real-time reverse transcription polymerase chain reaction.The protein levels of OPN and MCP-1 in culture medium of renal tubular epithelial cells were detected by enzyme-linked immunosorbent adsorption assay.Detecting calcium oxalate stones in renal tissue of rats from each group by calcium salt staining.Detecting the apoptosis of renal tubular epithelial cells in each group by apoptosis test.Detecting the expression of OPNin rat kidney tissues from each groupby immunohistochemistry.Detecting the expression ofkidney tissues from each groupby immunohistochemistry.Detecting the expression of MCP-1 in rats 24 hours urine from each group by enzyme linked immunosorbent adsorption assay.Results PCR and enzyme linked immunosorbent assay showed that OPN and MCP-1 were up-regulated in both mRNA and protein levels in high concentrations of glucose medium.Therewere large amounts of calcium salt deposition in renal tubules,large number of renal tubular epithelial cell apoptosis and OPN increased significantly in kidney tissues in hyperglycemia combined with hyperoxaluria model group,MCP-1 was increased in 24 hours urine from hyperglycemia combined with hyperoxaluria model group compare to other groups.Conclusion Hyperglycemia can induce the expression of chemokine,OPN and MCP-1,in renal tubular epithelial cells.Underhyperoxaluria,hyperglycemia can lead to the increase of apoptosis of renal tubular epithelial cells,and the formation of calcium oxalate stone increases.Hyperglycemia may contribute to the formation of calcium oxalate stones by increasing the local inflammatory response.