Roles of Intercellular junction of podocytes in surviving injuries

足细胞细胞间连接在损伤幸存中的作用

• 批准号: 17590822
• 负责人: YAOITA Eishin
• 金额: $ 2.37万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590822/
• 关键词: kidney; glomerulus; podocyte; culture; connexin43; claudin; gap junction; tight junction; ZO-1; 足細胞; 細胞間接着装置; 細胞傷害

To clarify the mechanism of survival of podocytes after injuries, we analyzed the podocyte responses to injuries focusing on the intercellular junctions.1.Establishment of in vitro assay for podocyte injury : We tried a glomerular isolation method with magnetic beads and collagenase, and succeeded in getting a number of glomeruli suitable for primary podocyte culture. By this method, we could get 5×10^5 podocytes per rat. Podocyte injury was caused in vitro by menadione producing free radicals. 50μM of menadione damaged podocytes within 30min exposure. Connexin43 (Cx43) gene, one of earliest response genes responding to podocyte injury, was knocked down in cultured podocytes by siRNA. Unfortunately there was no significant difference of survival rate under the menadione exposure between the Cx43-knockdown podocytes and control podocytes.2.Identification of components of intercellular junctions of podocytes : When podocytes are injured, tight junctions and gap junctions are newly formed in podocytes. Cytoplasmic membranes of isolated glomeruli were fractionated according to the presence of Cx43 detected by Western blotting. In one-dimensional gel electrophoresis, the portion which contained claudins was cut out and analyzed by LC-MSMS. Consequently, claudin-5,-6,-12 and -15 were detected in the fraction. Immunohistochemistry demonstrated claudin-6 localized in podocytes and the others in endothelial cells.In this study, we could establish the in vitro assay for podocyte injury, and identified the transmembrane component of the tight junction of podocytes for the first time.

为了阐明足细胞损伤后的存活机制，我们重点分析了足细胞对细胞间连接处损伤的反应。1.足细胞损伤体外检测方法的建立：我们尝试了磁珠和胶原酶的肾小球分离方法，成功获得了一批适合原代足细胞培养的肾小球。通过这种方法，每只大鼠可以获得5×10^5的足细胞。足细胞损伤是由产生自由基的甲萘醌在体外引起的。 50μM甲萘醌在暴露30分钟内损伤足细胞。 Connexin43 (Cx43) 基因是响应足细胞损伤的最早反应基因之一，在培养的足细胞中被 siRNA 敲除。不幸的是，Cx43敲低足细胞和对照足细胞在甲萘醌暴露下的存活率没有显着差异。2.足细胞细胞间连接成分的鉴定：当足细胞受伤时，足细胞中新形成紧密连接和间隙连接。根据蛋白质印迹检测到的 Cx43 的存在，对分离的肾小球的细胞质膜进行分级。在一维凝胶电泳中，切出含有密蛋白的部分并通过LC-MSMS进行分析。结果，在级分中检测到紧密蛋白-5、-6、-12和-15。免疫组织化学证实claudin-6定位于足细胞，其他蛋白定位于内皮细胞。本研究建立了足细胞损伤的体外检测方法，并首次鉴定了足细胞紧密连接的跨膜成分。

项目成果

Role of Fat1 in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney

Fat1在嘌呤霉素氨基核苷肾病和新生儿肾足细胞接触形成中的作用

• 发表时间: 2005
• 期刊: Kidney Int 68
• 作者: Eishin;Yaoita
• 通讯作者: Yaoita

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

• DOI: 10.1002/pmic.200401075
• 发表时间: 2005-03-01
• 期刊: PROTEOMICS
• 影响因子: 3.4
• 作者: Yoshida, Y;Miyazaki, K;Yamamoto, T
• 通讯作者: Yamamoto, T

Role of Fatl in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney.

Fatl 在嘌呤霉素氨基核苷肾病和新生儿肾足细胞接触形成中的作用。

• 发表时间: 2005
• 期刊: Kidney Int. 68
• 作者: Yao J;Kitamura M;Zhu Y;Meng Y;Kasai A;Hiramatsu N;Morioka T;Takeda M;Oite T;Yaoita E
• 通讯作者: Yaoita E

An improved method for primary culture of rat podocytes

• DOI: 10.1038/sj.ki.5000398
• 发表时间: 2006-06-01
• 期刊: KIDNEY INTERNATIONAL
• 影响因子: 19.6
• 作者: Katsuya, K.;Yaoita, E.;Yamamoto, T.
• 通讯作者: Yamamoto, T.

Glomerular podocytes express protocadherin 17

肾小球足细胞表达原钙粘蛋白 17

• 发表时间: 2007
• 期刊: Acta Med. Biol 55
• 作者: Kosugi R;Shioi T;Watanabe-Maeda K;Yoshida Y;Takahashi K;Machida Y;Izumi T.;Mizuno J;Takada Takuma
• 通讯作者: Takada Takuma