The role of anionic charge of the glomerular basement membrane.

尽管评估肾小球基底膜(GBM)阴离子位置的尝试越来越多，但目前还没有一种确定的方法来准确地定量GBM阴离子电荷的变化。本工作旨在建立一种定量测定大鼠肾小球基底膜阴离子电荷的方法，并对其电荷屏障的变化进行评价。以含铜铜蓝为阳离子探针。左肾用含铜蓝的醋酸钠缓冲液灌流。灌流后，用过滤法分离基底膜，用原子吸收光谱仪测定铜含量。分离的基底膜的电子显微镜观察显示致密层两侧均有染色阳性的纤维。超声波等分离步骤不影响GBM中铜的含量。PAN肾炎大鼠肾小球基底膜与铜的结合度明显降低。注射PAN后第9天，随着蛋白尿的恢复，铜与GBM的结合度显著降低，第42天恢复到对照水平。在分离的GBM的体外研究中，研究了pH对CuProlinic Blue结合的影响。PH在8.0~4.0时，异丙基蓝与GBM的结合率无显著差异，但当pH在3.0~4.0时，结合率开始下降。Cuplrolinic Blue的pH依赖解离曲线表明，该染料优先与GBM中的硫酸盐基团反应。这些结果表明，我们发展的方法可以用来量化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.