水蛭素对高糖环境下足细胞顶膜区蛋白的影响

Effects of hirudin on the apical membrane domain protein in high glucose-cultured podocytes

目的:观察水蛭素对体外高糖环境下足细胞顶膜区蛋白的影响,探索其对足细胞的保护作用及可能的作用机制。方法:条件性永生化小鼠足细胞在33℃/γ-IFN存在条件下,细胞增殖;37℃/无γ-IFN条件下,细胞分化。免疫细胞化学法鉴定分化成熟的细胞核是否表达足细胞特异性蛋白WT-1。MTT法筛选最佳药物浓度。将分化后足细胞分为正常糖组(含D-葡萄糖5.5mmol/L)、高糖组(含D-葡萄糖30mmol/L)、高渗组(含D-葡萄糖5.5mmol/L+D-甘露醇24.5mmol/L)和高糖加最佳浓度水蛭素组,分别干预24、48、72h,免疫细胞化学法、Realtime PCR、Western Blot检测足细胞顶膜区podocalyxin、GLEPP1表达。结果:鉴定结果显示分化成熟的细胞核表达足细胞特异性蛋白WT-1。MTT法筛选最佳水蛭素浓度为10-5mol/L。免疫细胞化学结果显示podocalyxin、GLEPP1均表达于足细胞膜,与高糖组比较,高糖加水蛭素组podocalyxin、GLEPP1表达有所增加。与同时间点高糖组比较,高糖加水蛭素组各时间点podocalyxin mRNA和GLEPP1蛋白表达均显著增加(P<0.01),高糖加水蛭素组72h后GLEPP1 mRNA表达显著增加(P<0.05)。高糖加水蛭素组48、72h后podocalyxin蛋白表达显著增加(P<0.05)。结论:水蛭素能增加高糖环境下足细胞顶膜区podocalyxin、GLEPP1表达,从而保护足细胞,维持肾小球滤过屏障结构和功能的完整性,这可能对减少糖尿病肾病尿蛋白、延缓肾脏病进展具有一定的作用。

Objective:To observe the effect of hirudin on the apical membrane domain protein in high glucosecultured podocytes and to explore the potential mechanism of podocyte-protecting effect.Methods:Conditional immortalized mouse podocytes proliferated under 33℃/γ-IFN condition,and podocytes differentiated under 37℃/noγ-IFN condition.Identified whether adult podocyte nucleus express specific protein WT-1 by immunocytochemistry.Screened the optimum drug concentration by MTT assay.Differentiated podocytes were divided into normal glucose group(NG,D-glucose concentration 5.5 mmol/L),high glucose group(HG,D-glucose concentration 30 mmol/L),mannitol group(MG,D-glucose concentration 5.5 mmol/L+D-mannitol concentration 24.5 mmol/L)and hirudin group.After intervention for 24 h,48 h and 72 h,the expression of podocyte apical membrane domain protein podocalyxin and GLEPP1 was detected by immunocytochemistry,Real-time PCR and Western Blot.Results:Immunocytochemistry identification showed that the cell nucleus expressed podocyte specific protein WT1.MTT assay showed that the optimum concentration of hirudin was 10-5 mol/L.Immunocytochemistry showed that podocalyxin,GLEPP1 expressed in the podocyte membrane,and compared with HG,hirudin group podocalyxin,GLEPP1 expression increased.Hirudin group podocalyxin mRNA and GLEPP1 protein expression increased at the three time points(P<0.01);after 72 h intervention GLEPP1 mRNA expression increased(P<0.05);after 48 h,72 h hirudin intervention podocalyxin protein expression increased(P<0.05).Conclusion:Hirudin can increase the apical membrane domain podocalyxin,GLEPP1 expression in high glucose-cultured podocytes,thus protect podocyte,and maintain the integrity of glomerular filtration barrier;it may have some effect on reducing urinary protein and delaying diabetic nephropathy progress.