摘要
目的探讨核因子-κB受体活化因子（RANK）在足细胞损伤中的作用及其对蛋白尿的影响。方法用Cre-LoxP重组酶系统建立足细胞RANK特异性敲除小鼠（RANK-/-，KO组），琼脂糖电泳法鉴定其基因型。设野生型小鼠（wT）和普通C57小鼠（各6只）为对照组。用脂多糖（LPS）腹腔注射方法建立短暂蛋白尿小鼠模型。检测各组小鼠尿白蛋白，尿肌酐等指标；模型建立后48h处死小鼠取肾脏行病理检查。免疫组化法检测肾脏足细胞数量；电镜下观察足细胞足突融合情况；免疫荧光法检测整合素（integrin）β1及integrinβ3等相关蛋白表达变化；Western印迹法检测integrinβ1、integrinβ3及尿激酶型纤溶酶原激活物受体（uPAR）等相关蛋白的表达变化。结果3组小鼠均于造模后第2天出现明显蛋白尿，KO组小鼠尿白蛋白／肌酐比值明显低于对照组wT组及C57组[（23．70±9．90）比（107．56±22．32）；（23．70±9．90）比（132．13±14．26），P〈0．0011，差异有统计学意义。免疫组化结果显示，造模后3组小鼠足细胞数量均有减少，KO组小鼠足细胞减少程度比其他两组轻。电镜结果亦显示，造模后KO组小鼠足细胞足突融合程度较对照组轻。免疫荧光结果显示，与造模前组相比，造模后各组小鼠肾组织integrinpl表达均有下降，KO组较对照组下降幅度更大；integrinB3表达升高，KO组升高幅度较对照组较少（均P〈0．05）。Western印迹结果显示，与造模前组相比，造模后各组小鼠。肾组织integrinB1表达均下降，KO组小鼠下降幅度更大；各组小鼠integrinB3及uPAR表达均升高，KO组升高幅度较其余两组较小（均P〈0．05）。结论敲除小鼠足细胞特异性RANK基因后可以减少LPS诱导的蛋白尿，提示RANK参与了由足细胞损伤导致蛋白尿的发生，其机制与调节integfinpl，integrin β3及uPAR等分子的表达有关。
Objective To investigate the role of RANK in injured podocyte and its effects on proteinuria. Methods PCR analysis was carried out to identify the genotypes of podocyte-specific RANK knockout mice. Six （6-8 weeks old） female RANK-/- mice were chosen as KO group. Six （6-8 weeks old） littermates and six （6-8 weeks old） C57/B6j mice were chosen as WT group and C57 group respectively. Each group was injected with LPS to create podocyte-injured proteinuria model. 24h urine was collected before and after injection. Urinary albumin, urine creatinine and UACR were detected by automatic biochemical analyzer. The mice were sacrificed after 48 h. Cortical kidney tissue samples were stained by PAS and collected for transmission electron microscopy to detect the foot process effacement. The number of podocytes was examined at different time points by immunohistochemistry . The expression of intergrin-β1, integrin- β3 and uPAR was detected by immunofluorescence and westernblot. Results （1） Comparing KO group, WT group and C57 group, it was found that there was no difference in weight, morphology, function of kidney and ACR. （2） After LPS injection, proteinuria and ACR were increased in three groups, but less significantly in KO group （23.70±9.90）, compared to those in WT group （107.56±22.32） and C57 group （132.13±14.26） （P 〈 0.001）. （3） In three groups, the number of podocytes was decreased after LPS injection and the decrease in the KO group was the slightest. （4） Compared to WT and C57 group, podocyte foot process effacement was less obvious in KO group after LPS injection in transmission electron microscopy. （5） Immunofluorescence results showed that after LPS injection, integrin-β1 was decreased in three groups, but most significantly in KO group. Integrin-β3 was increased in three groups, but less obvious in KO group. （6） Westernblot results showed consistency with immunofluorescence. The expression of uPAR protein was increased in three groups, with the increase in KO group being the slightest. Conclusions Podocytespecific knockout of RANK reduces LPS-induced proteinuria, suggesting that RANK might be involved in the development of proteinuria in podocyt injury. Its mechanism is probably related with integrin-β1, integrin-β3 and uPAR.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2016年第3期187-194,共8页
Chinese Journal of Nephrology
基金
“十二五”国家科技支撑计划项目（2011BAI10B06）
国家自然科学基金项目（81470930,81370808）
广东省自然科学基金（2014A030313544）
广州市科技计划攻关项目（2012J4300084）
