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.

为了阐明受伤后足细胞存活的机制，我们分析了对损伤的反应，重点是细胞间连接。1。建立了足体损伤的体外评估：我们尝试了一种使用磁性珠和胶原酶的肾小球隔离方法，并成功地培养了Glomerereri的数量。通过这种方法，我们可以每只大鼠获得5×10^5的足细胞。大型矿物产生自由基在体外造成足细胞损伤。 50μm的梅拉丁损坏的足细胞在30分钟内暴露。 connexin43（CX43）基因是对足细胞损伤反应的最早反应基因之一，在培养的足细胞中被击倒。不幸的是，在Menadine暴露下，CX43-敲低的足细胞和对照足细胞之间的存活率没有显着差异。2。识别Podocytes的细胞间交界处的识别：当足细胞受伤时，紧密的连接和间隙结合在Podocytestes中形成了新形成。根据通过蛋白质印迹检测到的CX43的存在，将分离的肾小球的细胞质膜分馏。在一维凝胶电泳中，将包含Claudins的部分切除并通过LC-MSMS进行分析。因此，在分数中检测到Claudin-5，-6，-12和-15。免疫组织化学表明，Claudin-6位于足细胞和其他细胞中的其他。在这项研究中，我们可以建立对足细胞损伤的体外评估，并首次确定了足细胞紧密连接的跨膜成分。

项目成果

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