Phenotype analysis of perlecan mutated mice in which heparan sulfates are removed by the use of ES cell.

在肾小球滤过过程中，肾小球基底膜（glomerular basal membrane， GBM）作为电荷屏障，将带负电荷的大分子保留在循环中。在分子基础上，GBM硫酸肝素蛋白聚糖（HSPG）被认为是构成屏障的关键成分。这种负电荷理论长期以来被普遍接受。然而，在分子水平上没有直接的证据表明屏障的崩溃与蛋白尿的发生有关。Perlecan是一种主要的GBM HSPG。Perlecan包含五个分子结构域。第一个结构域的外显子3编码硫酸肝素的附着位点。本研究旨在从不同的角度来评价负电荷理论。利用胚胎干细胞技术，在不移帧的情况下去除外显子3。结果，产生了具有突变蛋白分子的纯合子小鼠。我们在转录后水平的生化分析表明，从纯合子获得的成纤维细胞产生的无硫酸肝素的perlecan具有完整的核心分子大小。虽然纯合子没有自发表现出大量蛋白尿，但腹膜内白蛋白超载导致2周后尿蛋白排泄显著增加。我们的免疫电镜检查显示，纯合子GBM中的PEI染色位点与野生型对照没有区别。结果表明，其他GBM HSPG可以弥补perlecan硫酸肝素的损失。另一个GBM HSPS， Agrin，不是候选。

In the process of glomerular filtration, glomerular basement membranes (GBM) serve as a charge barrier to retain negatively-charged macromolecules in the circulation. On the molecular basis, GBM heparan sulfate prteoglycans (HSPG) have been thought of as key components that constitute the barrier. This negative charge theory has long been generally accepted. However, there is no direct evidence at the molecular level which links the collapse of the barrier to the development of proteinuria. Perlecan is a major GBM HSPG. Perlecan encompasses five molecular domains. Exon 3 of the first domain encodes heparan sulfate attachment sites. The present study was designed to evaluate negative charge theory from a different perspective. By the use of embryonic stem (ES) cell technology, the exon 3 was removed without frame shift. As a result, homozygous mouse was produced having mutated perlecan molecule. Our biochemical analysis at the post-transcriptional level demonstrated fibroblast obtained from the homozygote produed perlecan without heparan sulfates having an intact size of the core molecule. Although homozygotes did not spontaneously show massive proteinuria, intra-peritoneal albumin overload resulted in significant increase in urinary protein excretion after 2 weeks. Our immunoelectron microscopic investigation disclosed PEI stained sites in the GBM of homozygotes was not different from those of wild-type controls. The results strongly indicated other GBM HSPG compensate the loss of perlecan heparan sulfate. Agrin, another GBM HSPS, was not a candidate.