输尿管梗阻肾脏差异蛋白质的筛选和鉴定

Screening and identifying of differential proteins in kidney after unilateral ureteral obstruction

目的 筛选输尿管完全梗阻（complete unilateral ureteral obstruction,CUUO）后不同时间点大鼠肾脏的差异蛋白质,并检测部分差异蛋白在输尿管梗阻大鼠肾脏及儿童先天性肾积水肾脏中的表达.方法 应用双向电泳及质谱分析技术,检测2个月龄SD大鼠CUUO后12h、24 h、72 h以及对照组四组肾脏组织差异蛋白质的表达.应用蛋白质印迹（Western blot）方法检测筛查出的差异蛋白质ANXA4和ETFB在CUUO组和正常对照组大鼠肾脏组织以及15例肾积水患儿肾脏组织及15例肾胚瘤患儿瘤旁正常肾脏组织中的表达.结果 69个表达量相差2倍以上的差异蛋白质点,经质谱分析和蛋白质数据库检索成功鉴定出39个蛋白质,涉及细胞凋亡、线粒体能量代谢及线粒体损伤、氧化应激等方面.在动物模型中ANXA4的表达随梗阻时间延长呈持续上调：CUUO12 h为0.7809±0.036,CUUO24 h为0.9385±0.013,CUUO72 h为1.0218±0.032,Sham为0.7058±0.009,组间差异有统计学意义（P＜0.05）;随梗阻时间的延长ETFB的表达逐渐下调：CUUO12h为0.9301±0.017,CUUO24h为0.6488±0.021,CUUO72 h为0.5452±0.018,组间差异有有统计学意义（P＜0.05）,而在CUUO72h组和正常对照组表达无明显差异（CUUO72 h 0.5452±0.018比Sham0.5419±0.035,P＞0.05）.在肾积水患儿肾脏组织中这两种蛋白的表达均显著上调（ETFB：积水组0.9322±0.014比对照组0.8325±0.012,ANXA4：积水组1.100±0.018比对照组0.8815±0.007P＜0.05）.结论 运用双向电泳技术结合质谱分析及生物功能信息分析筛选并鉴定出39个在CUUO大鼠肾脏中差异表达的蛋白质,这些蛋白质可能与梗阻性肾病的发生、发展有关.其中ANXA4和ETFB可能在梗阻性肾病的发生、发展过程中发挥作用.

Objective To screen the differential proteins at different timepoints in rat kidney with complete unilateral ureteral obstruction （CUUO） and verify the partial differential expressions in animal model and clinical hydronephrosis.Methods Two dimensional electrophoresis （2-DE） and MALDI-TOF/TOF MS were employed to separate and identify the differential proteins in the groups of unilateral ureteral obstruction （CUUO） and corresponding sham rat kidney tissues.Western blot was used to verify the differential protein expressions of ANXA4 and ETFB in rat kidney and clinical renal tissues,including hydronephrosis （n =15） and Wilm＇s tumor （n =15）.Results A total of 39 differential proteins whose expression levels significantly increased or decreased by over 2 folds were successfully identified by peptide mass fingerprinting.And they were involved in cell apoptosis,energy metabolism,mitochondrial injuries and oxidative stress,etc.In rat kidney,the expression of ANXA4 was continuously up-regulated with CUUO persisting （CUUO12 h：0.7809 ± 0.036,CUUO24 h：0.9385 ± 0.013,CUUO72 h：1.0218 ± 0.032,sham：0.7058 ± 0.009,P＜0.05） and ETFB was continuously down-regulated with CUUO persisting （CUUO12 h：0.9301 ± 0.017,CUUO24 h：0.6488 ± 0.021,CUUO72 h：0.5452 ± 0.018,P＜0.05） and the expression in CUUO72 h had no significant differences with that in control group （CUUO72 h：0.5452 ± 0.018 vs sham 0.5419 ± 0.035,P＞ 0.05）.In clinical tissues,two proteins were both up-regulated in hydronephrosis versus control group （ETFB in hydronephrosis：0.9322 ± 0.014 vs 0.8325 ± 0.012 in control,ANXA4 in hydronephrosis：1.100 ± 0.018 vs 0.8815 ± 0.007 in control,P＜0.05）.Conclusions A total of 39 differentially expressed proteins probably associated with the genesis and development of obstructive nephropathy have been successfully screened from CUUO and corresponding sham kidney tissues.And ANXA4 and ETFB may be involved in the development and progression of obstructive nephropathy.