Kurihara H, Anderson J M, Kerjaschki D, Farquhar M G
Division of Cellular and Molecular Medicine, University of California, San Deigo, La Jolla 92093-0651.
Am J Pathol. 1992 Oct;141(4):805-16.
Nephrosis induced in rats by puromycin aminonucleoside treatment (PAN) results in the apical displacement of the glomerular filtration slit membrane by newly formed, intercellular occluding-type junctions. Similar changes can also be induced by acute kidney perfusion with protamine sulfate (PS). We have investigated the molecular nature of these altered junctions using an antibody to ZO-1, a protein found exclusively in tight junctions. Immunoblotting demonstrates ZO-1, a 225-kd band, in glomerular extracts of normal, PAN-, and PS-treated rats. By immunofluorescence, ZO-1 was localized at the base of podocytes outlining the capillary loops of glomeruli from all three experimental groups. At the electron microscope level, using immunoperoxidase or immunogold labeling, ZO-1 was concentrated along the cytoplasmic surfaces of the slit diaphragms of normal rats. In PAN or PS rats, it was concentrated along both the newly formed occluding-type junctions and the remaining slit diaphragms. When podocalyxin (the major membrane sialoprotein of the podocyte) was similarly localized, it was found exclusively apical to the displaced slit membrane. Based on morphology and the presence of ZO-1, the altered junctions seen in PAN and PS rats appear to represent bona fide tight junctions. Their rapid (15-minute) induction in PS-treated rats suggests that on neutralization of the cell surface charge by polycation perfusion, discontinuous tight junctions form from a preexisting pool of junctional proteins. These findings raise the possibility that glomerular hydraulic conductivity may be regulated in part by regulating the relative patency and width of the filtration slits through focal tight junction assembly.
用嘌呤霉素氨基核苷处理（PAN）诱导大鼠产生的肾病会导致肾小球滤过裂隙膜被新形成的细胞间封闭型连接向顶端移位。用硫酸鱼精蛋白（PS）对大鼠进行急性肾脏灌注也能诱导出类似变化。我们使用针对紧密连接中特有的一种蛋白质ZO-1的抗体，研究了这些改变的连接的分子性质。免疫印迹显示，正常、PAN处理和PS处理的大鼠肾小球提取物中存在225-kd条带的ZO-1。通过免疫荧光，ZO-1定位于足细胞基部，勾勒出所有三个实验组肾小球的毛细血管袢。在电子显微镜水平，使用免疫过氧化物酶或免疫金标记，ZO-1集中在正常大鼠裂隙隔膜的细胞质表面。在PAN或PS处理的大鼠中，它集中在新形成的封闭型连接和剩余的裂隙隔膜上。当足细胞表面蛋白（足细胞的主要膜唾液蛋白）进行类似定位时，发现它仅位于移位裂隙膜的顶端。基于形态学和ZO-1的存在，在PAN和PS处理的大鼠中看到的改变的连接似乎代表真正的紧密连接。它们在PS处理的大鼠中迅速（15分钟）诱导形成，这表明通过聚阳离子灌注中和细胞表面电荷后，不连续的紧密连接从预先存在的连接蛋白池中形成。这些发现增加了一种可能性，即肾小球水力传导率可能部分通过局部紧密连接组装来调节滤过裂隙的相对通畅性和宽度来进行调节。
