Fujigaki Y, Batsford S, Yamashita F, Yonemura K, Hishida A, Kawachi H, Shimizu F, Vogt A
First Department of Medicine, Hamamatsu University School of Medicine, Japan.
Virchows Arch. 2001 Feb;438(2):136-45. doi: 10.1007/s004280000295.
Proteinuria in passive Heymann nephritis (PHN) results from complement-mediated glomerular injury, since complement depletion with cobra venom factor (CVF) prevents proteinuria. However, there are no comprehensive morphological studies identifying the sites of injury leading to onset of proteinuria. To address this issue, we attempted to locate sites of injury involved in the onset of proteinuria in PHN. PHN was induced in intact Munich-Wistar rats (PHN-rats, examined at days 3, 5, and 7) and in complement-depleted rats (CVF treated, PHN-CVF-rats, examined at days 3 and 5). The distribution of endogenous albumin in the glomerular basement membrane (GBM) was studied in in situ drip-fixed glomeruli using immunogold immunocytochemistry, and glomerular anionic sites were visualized by polyethyleneimine staining. In addition, the ultrastructural localization of an epitope recognized by a proteinuria-inducing monoclonal antibody (called 5-1-6) directed against the slit diaphragm was examined. Significant proteinuria was seen in intact PHN-rats, starting at day 5. The intensity of gold labeling for endogenous albumin was significantly increased at the outermost site of the GBM (GBM interfacing foot process and the filtration slit, designated area O) at day 3 in both PHN-rats and PHN-CVF-rats in comparison to untreated controls. At day 5, labeling for albumin in area O was decreased in PHN-rats, but not in PHN-CVF-rats, where it was then higher; in PHN-rats, some areas between epithelial cells and subepithelial deposits were almost free of albumin labeling at day 7. There was no evidence of epithelial cell detachment in any group at day 5, but on day 7 limited focal detachment was seen exclusively in PHN-rats. In proteinuric rats, amorphous material that stained for albumin could be seen in the urinary space, without any exocytosis of labeling by glomerular epithelial cells. A significant reduction of intensity of staining for anionic sites was seen in parallel in both groups, but only in the regions of the lamina rara externa adjacent to subepithelial deposits. This local loss of charge might contribute to enhanced permeability to albumin in both PHN- and PHN-CVF-rats. Changes in the appearance of the filtration slits and in the density and distribution of antigen recognised by monoclonal antibody 5-1-6 were similar in PHN- and PHN-CVF-rats at day 5. Complement depletion prevented neither the reduction in anionic sites of the GBM nor the changes in the slit diaphragm observed. These data suggest that albumin leakage between the epithelial cell and the GBM (area O) could occur in PHN-rats, perhaps as a result of epithelial foot-process changes. This may be the final link in the chain of events responsible for the onset of proteinuria in PHN.
被动型海曼肾炎（PHN）中的蛋白尿是由补体介导的肾小球损伤所致，因为用眼镜蛇毒因子（CVF）消耗补体可预防蛋白尿。然而，尚无全面的形态学研究来确定导致蛋白尿发生的损伤部位。为解决这一问题，我们试图定位PHN中与蛋白尿发生相关的损伤部位。在完整的慕尼黑-维斯塔大鼠（PHN大鼠，于第3、5和7天进行检查）和补体消耗的大鼠（经CVF处理，PHN-CVF大鼠，于第3和5天进行检查）中诱导出PHN。使用免疫金免疫细胞化学方法，在原位滴注固定的肾小球中研究内源性白蛋白在肾小球基底膜（GBM）中的分布，并通过聚乙烯亚胺染色观察肾小球阴离子位点。此外，还检查了一种针对裂孔隔膜的诱导蛋白尿的单克隆抗体（称为5-1-6）所识别的表位的超微结构定位。完整的PHN大鼠在第5天开始出现明显的蛋白尿。与未处理的对照组相比，在第3天，PHN大鼠和PHN-CVF大鼠GBM最外层部位（GBM与足突和滤过裂隙交界处，称为区域O）内源性白蛋白的金标记强度均显著增加。在第5天，PHN大鼠中区域O的白蛋白标记减少，但PHN-CVF大鼠中未减少，且此时更高；在PHN大鼠中，第7天时上皮细胞与上皮下沉积物之间的一些区域几乎没有白蛋白标记。在第5天，任何组均未发现上皮细胞脱离的证据，但在第7天，仅在PHN大鼠中观察到有限的局灶性脱离。在蛋白尿大鼠的尿腔中可见到染白蛋白的无定形物质，而肾小球上皮细胞无标记物的胞吐现象。两组中阴离子位点染色强度均平行显著降低，但仅在与上皮下沉积物相邻的外疏松层区域。这种局部电荷丧失可能导致PHN大鼠和PHN-CVF大鼠中白蛋白通透性增强。在第5天，PHN大鼠和PHN-CVF大鼠中滤过裂隙的外观以及单克隆抗体5-1-6所识别抗原的密度和分布变化相似。补体消耗既不能阻止GBM阴离子位点的减少，也不能阻止观察到的裂孔隔膜变化。这些数据表明，PHN大鼠中上皮细胞与GBM之间（区域O）可能发生白蛋白渗漏，这可能是上皮足突变化的结果。这可能是PHN中导致蛋白尿发生的一系列事件中的最后一环。
