The role of anionic charge of the glomerular basement membrane.

尽管越来越多的尝试，以评估阴离子网站在肾小球基底膜（GBM），还没有一个既定的方法，准确定量的改变的阴离子电荷的GBM。本工作旨在建立一种定量测定嘌呤霉素肾炎大鼠肾小球基底膜（GBM）阴离子电荷的方法，并估计电荷屏障的变化。以含铜的铜离子蓝作为阳离子探针。左肾用含铜灵蓝的醋酸钠缓冲液灌注。灌流后，通过筛分技术分离GBM，用原子吸收分光光度计测定铜含量。电子显微镜观察显示，GBM致密层两侧均可见染色阳性纤维。超声等分离方法对GBM中铜含量无影响。PAN肾炎大鼠GBM铜结合程度明显降低。PAN注射后铜与GBM结合的程度在第9天显著降低，并在第42天恢复到对照水平，同时伴有白蛋白尿的恢复。在使用分离的GBM的体外研究中，研究了pH对铜灵蓝结合的影响。在pH 8.0 ~ 4.0范围内，铜灵蓝与GBM的结合率无显著差异，但在pH 3.0 ~ 4.0范围内，铜灵蓝与GBM的结合率开始下降。该pH依赖性的解离曲线表明该染料优先与GBM中的硫酸根基团反应。这些结果表明，我们开发的方法可能是有用的定量GBM的阴离子电荷的变化。

Despite the growing number of attempts to evaluate anionic site in glomerular basement membrane (GBM), there has not been an established method which accurately quantifies an alteration of the anionic charge of the GBM. The present work was performed to develop a method to quantify the anionic charge of the GBM and to estimate the change of the charge barrier in puromycin aminonucleoside (PAN) nephritis in rats. Cuplorinic blue which contains copper was used as a cationic probe. Left kidney was perfused with sodium acetate buffer containing cuprolinic blue. After perfusion, GBM was isolated by sieving technique and the copper content was measured with an atomic absorption spectrophotometer. Electron microscopic study of isolated GBM revealed the dye-positive fibrils on both side of the lamina densa. Isolation procedure, such as sonication did not affect the copper content of GBM. The degree of copper binding to GBM was markedly reduced in PAN nephritis rats. The degree of copper binding to GBM after PAN injection was markedly reduced at day 9 and returned to the control level at day 42, concomitantly with the recovery of albuminuria. In vito study using isolated GBM, the effects of pH on the cuprolinic blue binding was studied. At pH between 8.0 and 4.0, no significant difference was observed in the cuprolinic blue binding to GBM, but at pH between 3.0 and 4.0, the binding rate began to decrease. This pH-dependent dissociation curve of cuplrolinic blue suggested that the dye preferentially reacted with the sulfate groups in GBM. These results demonstrated that the method which we developed may be useful to quantifies an alteration of the anionic charge of GBM.