阿霉素诱导小鼠局灶性节段性肾小球硬化及其相关机制

Mice with focal segmental glomerular sclerosis are induced by adriamycin and its mechanism

目的 建立阿霉素诱导的局灶性节性段性肾小球硬化(FSGS)小鼠模型,观察氧化应激与凋亡相关的p38 MAPK信号通路在肾脏损伤中的表达以及二者之间的关系.方法 将8周雄性Balb/c小鼠随机分为FSGS组(n=20)和对照组(n=20),FSGS组以0.01 mg/g体重一次性尾静脉注射阿霉素,对照组给予等量的生理盐水.于3、7、14、22、32 d检测小鼠尿蛋白/尿肌酐;于22 d和32 d检测血肌酐、尿素氮;检测血、尿一氧化氮(NO)、活性氧(ROS)水平以及肾脏组织的ROS水平;HE染色观察肾组织病理形态变化;采用实时定量PCR方法检测NF-κB、CD36、IL-13、BAX、Bcl-2的mRNA水平;Western印迹检测NF-κB、p-p38、p-ERK 1/2蛋白表达.结果 与对照组比较,FSGS组小鼠尿蛋白/尿肌酐比值随时间逐渐升高并在第22天到达高峰(P＜0.05);FSGS组小鼠血肌酐、尿素氮、ROS、NO水平在22 d和32 d均高于对照组(均P＜ 0.05);32 d病理显示FSGS组系膜细胞、系膜基质增生,节段中度加重,足细胞增生明显,毛细血管袢受压狭窄;FSGS组肾脏组织中NF-κB、BAX、IL-13、CD36的mRNA水平随时间增强,第32天与对照组相比差异均有统计学意义(均P＜ 0.05);FSGS组NF-κB、p-p38MAPK蛋白表达均高于对照组(均P＜0.05),FSGS组p-ERK 1/2蛋白水平在22 d高于对照组(P＜0.05),但是在32 d却低于对照组(P＜0.05).结论 阿霉素成功诱导小鼠FSGS,其机制可能与氧化应激激活p38,上调NF-κB,促进炎性因子产生,及凋亡相关通路活化有关.

Objective To establish adriamycin-induced focal segmental glomerular sclerosis(FSGS) mice model,and observe the expressions of and relation between oxidative stress and p38 MAPK signal pathway in renal injury.Methods Eight-week-old male Balb/c mice were randomly divided into FSGS group (n=20) and control group (n=20).In FSGS group mice were intravenously injected with a single dose of adriamycin (0.01 rag/g),and mice in control group were received saline with the same dose.At day 3,7,14,22 and 32,urine protein-to-urine creatinine ratio (P/C) was detected.At day 22 and 32,serum creatinine,blood urea nitrogen,nitric oxide (NO) and reactive oxygen species (ROS) in blood and urine,and ROS in kidney tissues were detected;changes of pathological morphology in renal tissue were analyzed by HE stain;the expressions of NF-κB,CD36,IL-13,BAX and Bcl-2 mRNA were detected by real time quantitative PCR;the expressions of NF-κB,p-p38 and p-ERK1/2 protein were detected by Western blotting.Results Compared with that in control group,P/C was gradually increasing in FSGS group,and peaked at day 22 (P ＜ 0.05).At day 22 and 32,mice had higher creatinine,serum creatinine,urea nitrogen,ROS and NO in FSGS group than those in control group (all P ＜ 0.05).There were mild hyperplasia of mesangial cells and mesangial matrix,segment with moderate exacerbations,podocytes with significant proliferation,and the capillary loops of the stenosed in the glomerular in FSGS group at day 32.Compared with those in control group,the mRNA expression of NF-κB,BAX,IL-13 and CD36,and the protein expressions of NF-κB and p-p38 MAPK were gradually increased in FSGS group,all showed statistical differences at day 32 (all P＜ 0.05);the expression of p-ERK1/2 was increased at day 22 (P ＜ 0.05) but was reduced at day 32 (P ＜ 0.05).Conclusions Adriamycin has induced FSGS in mice successfully,which may through oxidative stress activating p38,up-regulating NF-κB,increasing the inflammatory cytokines and inducing apoptosis pathways.