YAP 在特发性膜性肾病中的表达及其对 C5b-9 诱导足细胞损伤的作用
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1.海口市人民医院肾内科,海口 570208; 2.中山市博爱医院肾病风湿科,广东 中山 510603

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R-33

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Expression of YAP in idiopathic membranous nephropathy and its role in C5b-9-induced podocyte injury
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1.Department of Nephrology, Haikou People’s Hospital, Haikou 570208, China. 2. Department of Nephrology and Rheumatology, Bo’ai Hospital, Zhongshan 510603

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    摘要:

    目的 在特发性膜性肾病(IMN)中探究转录共激活因子 Yes 相关蛋白(YAP)的表达及其在膜攻击复合物 C5b-9 诱导足细胞损伤的作用。 方法 免疫组织化学染色检测 YAP 蛋白在 IMN 患者肾组织及正常对照肾组织中的表达;选用亚溶解剂量的膜攻击复合物 C5b-9(以下简称 C5b-9)在体外建立人足细胞系 MPC5 的损伤模型,Western blot 检测随 C5b-9 刺激时间的延长,足细胞中 YAP 蛋白的表达变化;使用 siRNA 瞬时转染技术,靶向沉默足细胞中 YAP 基因表达,qRT-PCR 及 Western blot 检测转染效率;按足细胞处理的不同将其分为 4 组,正常培养的 Control 组,经 C5b-9 处理的 C5b-9 组,C5b-9 处理转染 si-NC 或 si-YAP 的的 C5b-9+si-NC 组与 C5b-9+si-YAP 组; 应用 CCK-8 实验检测上述各组细胞的增殖活力,Anenxin V-FITC 双染色联合流式细胞数检测各组细胞的凋亡发生 率,DCFH-DA 法检测细胞中活性氧(ROS)含量,Western blot 检测各组细胞中足细胞标志性蛋白 Nephrin、WT1 与紧密连接蛋白 ZO-1、表型转换蛋白 Snail 及纤连蛋白 Fibronectin 的蛋白表达水平。 结果 与正常对照肾组织相比, YAP 在 IMN 中的表达显著增加(P<0. 05);随 C5b-9 刺激时间的延长,足细胞中 YAP 蛋白的表达水平逐渐升高;转染 si-YAP 后,足细胞中 YAP mRNA 及蛋白水平均显著降低(P<0. 05);与 Control 组相比,C5b-9 组、C5b-9+si-NC 组 及 C5b-9+si-YAP 组细胞的增殖活力均显著降低(P<0. 05),而凋亡率及 ROS 含量均显著增加(P<0. 05);但与C5b-9 组或 C5b-9+si-NC 组相比,C5b-9+si-YAP 组细胞增殖活力显著增加(P<0. 05),凋亡率及 ROS 含量显著减少(P< 0. 05);Western blot 检测各组细胞中相关蛋白的表达结果显示,与 Control 组相比,C5b-9 组、C5b-9+si-NC 组及 C5b- 9+si-YAP 组细胞中 Nephrin、WT1 及 ZO-1 蛋白表达均显著降低(P<0. 05),Snail 与 Fibronectin 蛋白表达均显著增加 (P<0. 05);而与 C5b-9 组或 C5b-9+si-NC 组相比,C5b-9+si-YAP 组细胞中 Nephrin、WT1 及 ZO-1 蛋白表达显著增加 (P<0. 05),而 Snail 与 Fibronectin 蛋白表达显著减少(P<0. 05)。 结论 本研究结果表明 YAP 蛋白在 IMN 患者肾小球中的表达显著增加,而沉默足细胞中 YAP 基因表达能够明显改善 C5b-9 诱导的细胞功能损伤,并抑制其凋亡的发生。

    Abstract:

    Objective To investigate expression of YAP in idiopathic membranous nephropathy (IMN). Methods Expression of YAP protein in IMN patients and normal renal tissues was detected by immunohistochemistry. An injury model of the human podocyte line MPC5 was established in vitro with C5b-9. Expression of YAP protein in podocytes was detected by Western blot. siRNA transient transfection was used to silence YAP gene expression in podocytes. The transfection efficiency was assessed by qRT-PCR and Western blot. Podocytes were divided into four groups: control, C5b- 9, C5b-9+si-NC and C5b-9+si-YAP groups. A CCK-8 assay was used to assess the proliferation activity, annexin V-FITC double staining combined with flow cytometry was used to assess apoptosis, DCFH-DA staining was used to detect the content of reactive oxygen species (ROS). Western blot was used to measure the apoptosis rate. The expression of podocyte marker protein nephrin, WT1, tight junction protein ZO-1, phenotype transition protein snail and fibronectin was detected by Western blot. Results Compared with normal renal tissue, the expression of YAP in IMN was increased significantly (P< 0.05). With prolongation of the C5b-9 stimulation time, the expression level of YAP protein was increased in podocytes (P< 0.05). After si-YAP transfection, the mRNA and protein levels of YAP in podocytes were decreased significantly (P<0.05). Compared with the control group, cell proliferation in C5b-9, C5b-9+si-NC and C5b-9+si-YAP groups was decreased significantly, while the apoptosis rate and ROS content were increased significantly ( P< 0.05). However, compared with C5b-9 and C5b-9+si-NC groups, cell proliferation in the C5b-9+si-YAP group was increased significantly and the ROS content in cells was decreased significantly (P<0.05). Expression of nephrin, WT1 and ZO-1 in C5b-9, C5b-9+ si-NC and C5b-9 + si-YAP groups was significantly lower than that in the control group ( P< 0.05). Compared with C5b-9 and C5b-9+si-NC groups, the protein expression of nephrin, WT1 and ZO-1 in the C5b-9+si-YAP group was increased significantly, while the protein expression of Snail and fibronectin was decreased significantly ( P< 0.05). Conclusions This study showed that YAP protein expression in glomeruli of IMN patients is significantly increased and silencing YAP gene expression in podocytes significantly improves C5b-9-damaged induced functions and inhibits apoptosis.

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史添立,罗 贞,焦 石. YAP 在特发性膜性肾病中的表达及其对 C5b-9 诱导足细胞损伤的作用[J].中国比较医学杂志,2022,32(3):1~8.

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  • 收稿日期:2021-08-19
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  • 在线发布日期: 2022-04-18
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