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Histochemical and molecular-biological study on apoptosis in the model of renal failure

肾衰竭模型细胞凋亡的组织化学和分子生物学研究

基本信息

批准号：
07671231
负责人：
SAITO Takao
金额：
$ 1.47万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Although ischemia-reperfusion of mouse kidney is known to cause severe renal failure due to tubular cell death, the exact cellular mechanism responsible for this phenomenon is not clear. In order to investigate the spatial and temporal development of renal cell death and the role of Fas/APO-1/CD95 (Fas) in this process, we occluded the left renal vessels in a group of mice for 30,60 or 120 min followed by reperfusion for 24h (N=4 for each group). Analysis of the isolated DNA in agarosegel electrophoresis revealed a typical ladder pattern of bands consisting of multiples of 180-200 base pairs, considered as the hallmark of apoptosis. The intensity of the bands increased proportionately with the duration of ischemia. Histochemical analysis using TUNEL showed the presence of nuclei with DNA double-strand breaks specifically in distal renal tubules of the outer medulla. We also confirmed the presence of apoptosis by electron microscopy. Analysis of total RNA by Northern blotting revealed one appropriate-sized band for Fas mRNA in the normal kidney which intensified in the ischemia-reperfused kidney. Moreover, nonradioactive in situ hybridization revealed that distal renal tubular epithelial cells were positive for Fas mRNA in the outer medulla. Fas antigen was also localized to the renal tubular epithelial cells of the outer medulla by immunohistochemistry. The number of apoptotic cells in the ischemia-reperfusion kidney of the lpr/lpr mouse was low. Our findings strongly indicate that ischemia-reperfusion of the kidney induces apoptosis of a specific area of tubular epithelial cells in the outer medulla through the Fas system.

虽然已知小鼠肾脏的缺血-再灌注会由于肾小管细胞死亡而导致严重的肾衰竭，但导致这种现象的确切细胞机制尚不清楚。为探讨肾细胞死亡的时间和空间发展过程以及Fas/APO-1/CD 95（Fas）在这一过程中的作用，我们将一组小鼠左肾血管阻断30、60和120 min，然后再灌注24 h（每组N=4）。在琼脂糖凝胶电泳中分析分离的DNA揭示了由180-200个碱基对的倍数组成的典型的梯状条带模式，被认为是细胞凋亡的标志。随着缺血时间的延长，条带的强度逐渐增强。TUNEL法组织化学分析表明，存在的细胞核DNA双链断裂，特别是在远端肾小管的外髓质。我们还证实了细胞凋亡的存在，通过电子显微镜。北方印迹法分析总RNA，发现正常肾组织中Fas mRNA有一条大小合适的条带，缺血再灌注肾组织中Fas mRNA的条带增强。此外，非放射性原位杂交显示，远端肾小管上皮细胞外髓部Fas mRNA呈阳性。Fas抗原也定位于外髓质的肾小管上皮细胞的免疫组化。lpr/lpr小鼠缺血再灌注肾脏中凋亡细胞数量较少。我们的研究结果有力地表明，缺血再灌注的肾脏诱导凋亡的特定区域的肾小管上皮细胞外髓质通过Fas系统。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Shoji Nogae, Masanobu Miyazaki, Nobuyuki Kobayashi Takao Saito, Keishi Abe, Hiroshi Saito, Paul K Nakane Yoshinobu Nakanishi and Takehiko Koji: "Induction of apoptosis in ischemia-reperfusion model of mouse kidney : Possible involvement of Fas." J Am Soc

Shoji Nogae、Masanobu Miyazaki、Nobuyuki Kobayashi Takao Saito、Keishi Abe、Hiroshi Saito、Paul K Nakane Yoshinobu Nakanishi 和 Takehiko Koji：“小鼠肾脏缺血再灌注模型中细胞凋亡的诱导：Fas 可能参与”。