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

虽然小鼠肾脏缺血再灌注可导致肾小管上皮细胞死亡而导致严重的肾功能衰竭，但其确切的细胞机制尚不清楚。为探讨肾细胞死亡的时空演变及Fas/APO-1/CD95(Fas)在此过程中的作用，我们阻断小鼠左肾血管30、60、120min，然后再灌注24小时(每组4只)。琼脂糖凝胶电泳法分析提取的DNA，发现由180-200个碱基对的倍数组成的典型的梯形条带，这被认为是细胞凋亡的标志。条带的强度随缺血时间的延长而成比例增加。TUNEL组织化学分析显示，外髓远端肾小管可见DNA双链断裂的细胞核。我们还通过电子显微镜证实了细胞凋亡的存在。Northern印迹分析表明，在正常肾脏中有一条大小适中的Fas mRNA条带，在缺血再灌流的肾脏中该条带的表达增强。非放射性原位杂交显示远端肾小管上皮细胞外髓Fas基因表达阳性。免疫组织化学显示Fas抗原定位于外髓肾小管上皮细胞。LPR/LPR组小鼠肾脏缺血再灌注后的细胞凋亡率较低。我们的研究结果有力地表明，肾脏缺血再灌注通过Fas系统诱导外延髓特定区域的肾小管上皮细胞的凋亡。

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.