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(GBM)起到电荷屏障的作用，将带负电荷的大分子保留在循环中。在分子基础上，GBM硫酸乙酰肝素原聚糖(HSPG)被认为是构成屏障的关键成分。这种负电荷理论早已被普遍接受。然而，在分子水平上没有直接的证据将屏障的崩溃与蛋白尿的发展联系起来。Perlecan是一位主要的GBM HSPG。Perlecan包括五个分子结构域。第一个结构域的外显子3编码硫酸乙酰肝素结合位点。本研究旨在从不同的角度评价负电荷理论。利用胚胎干细胞技术，去除了外显子3而不移码。结果，产生了纯合子的小鼠，其Perlecan分子发生了突变。我们在转录后水平的生化分析表明，从纯合子生产的不含硫酸乙酰肝素的Perlecan获得的成纤维细胞具有完整的核心分子大小。虽然纯合子不会自发出现大量蛋白尿，但在2周后，腹膜白蛋白超负荷导致尿蛋白排泄显著增加。我们的免疫电子显微镜观察发现，纯合子和野生型对照的基底膜中的PEI染色部位没有差别。结果有力地表明，其他GBM HSPG弥补了硫酸肝素的损失。另一位GBM HSP阿格林不是候选人。

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.