Fujigaki Y, Nagase M, Hidaka S, Matsui K, Shirai M, Nosaka H, Kawachi H, Shimizu F, Hishida A
First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu; Department of Medicine, Teikyo University School of Medicine, Tokyo.
Kidney Int. 1998 Nov;54(5):1491-500. doi: 10.1046/j.1523-1755.1998.00131.x.
We previously reported that monoclonal antibody (mAb) 5-1-6 bound to renal filtration slits induces massive proteinuria without causing ultrastructural changes in the glomerulus. This study evaluated the underlying mechanisms of the increase in glomerular permeability.
The distribution of endogenous albumin and IgG in the glomerular basement membrane (GBM) was studied in in situ drip-fixed glomeruli of Munich-Wistar rats by use of immunogold immunocytochemistry in the presence and absence of mAb 5-1-6. The density of foot process glycocalyx components was estimated by labeling with Limax fluvus lectin- or Helix pomatia lectin-gold complexes. Anionic sites in the GBM were examined by labeling with cationic gold at pH 2.0 or 7.4. Carboxyl groups, which also furnish an anionic charge to the GBM, were examined by specific biotinylation and colloidal gold probe methods. In addition, the infusion-staining of anionic sites was performed by use of ruthenium red in both Munich-Wistar and Wistar rats.
The urinary excretion of albumin and IgG was increased markedly in the treated rats, indicating a non-selective barrier defect. In the control rats, albumin and IgG molecules were mainly located along the inner half of the GBM, and to a lesser degree in the lamina rara externa. In the treated rats, the albumin and IgG moieties were more equally distributed throughout the width of the GBM. Newly appearing, small dense peaks at the outer side of the GBM were evident, indicating a barrier function of outer zone of the GBM and/or epithelial cell layer. No intergroup differences in the density of lectin binding sites on foot processes were seen. The reduction in the number of ruthenium red-positive anionic sites and cationic gold (pH 2. 0)-labeled anionic sites in the lamina rara externa was significant in the treated rats at day 3, indicating a possible alteration of charged proteoglycan in the lamina rara externa. No such changes were seen with cationic gold (pH 7.4)-labeled anionic sites in the GBM. The density of labeled carboxyl groups was significantly reduced in the treated rats relative to the controls.
These results show that the injection of mAb 5-1-6 induced a perturbation of the charge- and probably the size-selective glomerular filtration barrier. The observed reduction in the levels of various negatively charged substances resulted in massive proteinuria, implying that alteration of target antigens can affect the integrity of the GBM constituents maintaining the normal barrier function.
我们之前报道过，与肾滤过裂隙结合的单克隆抗体（mAb）5-1-6可诱导大量蛋白尿，而不会引起肾小球超微结构改变。本研究评估了肾小球通透性增加的潜在机制。
在有或无mAb 5-1-6存在的情况下，通过免疫金免疫细胞化学方法，在慕尼黑-维斯塔大鼠原位滴注固定的肾小球中研究内源性白蛋白和IgG在肾小球基底膜（GBM）中的分布。通过用黄蛞蝓凝集素或苹果螺凝集素-金复合物标记来估计足突糖萼成分的密度。通过在pH 2.0或7.4下用阳离子金标记来检查GBM中的阴离子位点。通过特异性生物素化和胶体金探针方法检查也为GBM提供阴离子电荷的羧基。此外，在慕尼黑-维斯塔大鼠和Wistar大鼠中均使用钌红进行阴离子位点的灌注染色。
治疗组大鼠白蛋白和IgG的尿排泄量显著增加，表明存在非选择性屏障缺陷。在对照大鼠中，白蛋白和IgG分子主要位于GBM的内半部分，在外疏松层中的分布较少。在治疗组大鼠中，白蛋白和IgG部分在GBM的整个宽度上分布更为均匀。在GBM外侧出现新的小致密峰，表明GBM外层和/或上皮细胞层具有屏障功能。足突上凝集素结合位点的密度在组间未见差异。在第3天，治疗组大鼠外疏松层中钌红阳性阴离子位点和阳离子金（pH 2.0）标记的阴离子位点数量减少显著，表明外疏松层中带电荷的蛋白聚糖可能发生了改变。GBM中阳离子金（pH 7.4）标记的阴离子位点未见此类变化。与对照组相比，治疗组大鼠标记羧基的密度显著降低。
这些结果表明，注射mAb 5-1-6会引起电荷选择性和可能的大小选择性肾小球滤过屏障的扰动。观察到的各种带负电荷物质水平的降低导致大量蛋白尿，这意味着靶抗原的改变会影响维持正常屏障功能的GBM成分的完整性。
