Mutant Laminin Chains and Nephrotic Syndrome

描述(申请人提供)：层粘连蛋白2(LAMB2)是层粘连蛋白-521的一种成分，层粘连蛋白-521是肾小球基底膜(GBM)的主要层粘连蛋白三聚体。LAMB2零突变导致Pierson综合征，以先天性肾病综合征为特征，并伴有严重的眼和神经缺陷。相反，大多数错义LAMB2突变，如R246Q，会导致孤立的先天性肾病综合征。为了研究错义突变导致蛋白尿的机制，我们建立了转基因小鼠，其中R246Q突变(2)在不同水平的足细胞中表达，并取代了正常(2)在GBM中的表达。我们的初步数据显示，这些小鼠的蛋白尿可以通过增加突变的R246Q-LAMB2在GBM中的沉积来减轻。此外，在体外，与野生型相比，突变型LAMB2蛋白的分泌受到严重抑制。因此，我们假设致病的LAMB2点突变在体外和体内都会导致蛋白质错误折叠、内质网应激和分泌缺陷。化学伴侣，如牛磺酸脱氧胆酸(TUDCA)，是一种低分子化合物，可以稳定蛋白质构象，挽救运输缺陷的错误折叠蛋白质。我们已经证明，TUDCA处理表达突变体的细胞显著增加了突变LAMB2蛋白分泌到培养液中。因此，我们认为化学伴侣可以改善突变的LAMB2蛋白的折叠，减少内质网应激，并促进突变的层粘连蛋白三聚体分泌到基底膜，从而减轻Lamb2-/-；muLAMB2转基因小鼠的蛋白尿。为了检验这些假设，我们将： 1.研究LAMB2点突变是否导致蛋白质错误折叠和内质网应激 在体外和体内都有。 2.确定化学伴侣是否能促进LAMB2点的分泌 并在体外减轻内质网应激。 3.研究化学伴侣是否促进突变层粘连蛋白(2和 减轻Lamb2-/-；muLAMB2小鼠的蛋白尿。 这些研究将有助于我们了解先天性肾病综合征发病的分子机制，并在相关的动物模型中分析化学伴侣治疗的可行性。我们的结果可能对携带LAMB2错义突变患者的先天性肾病综合征的治疗具有重要的治疗意义。 公共卫生相关性：先天性肾病综合征是一种重要的儿童肾脏疾病，可导致蛋白尿和进行性肾功能衰竭。我们正在研究给予具有药理活性的伴侣是否可以改善动物模型中的蛋白尿。这一结果可能为肾脏蛋白尿疾病提供一种新的治疗方案。

DESCRIPTION (provided by applicant): Laminin (2 (LAMB2) is a component of Laminin-521, the major laminin trimer of the kidney glomerular basement membrane (GBM). Null mutations in LAMB2 cause Pierson syndrome, characterized by congenital nephrotic syndrome with severe ocular and neurological defects. In contrast, most missense LAMB2 mutations, such as R246Q, cause isolated congenital nephrotic syndrome. To investigate the mechanisms whereby missense mutations cause proteinuria, we have generated transgenic mice in which R246Q mutant (2 is expressed in podocytes at different levels and replaces normal (2 in the GBM. Our preliminary data show that these mice exhibit proteinuria that can be attenuated by increased deposition of mutant R246Q-LAMB2 into the GBM. Moreover, in vitro, secretion of mutant LAMB2 proteins is severely inhibited compared to wild-type. Therefore, we hypothesize that pathogenic LAMB2 point mutations cause protein misfolding, ER stress, and secretion defects both in vitro and in vivo. Chemical chaperones, such as taurodeoxycholic acid (TUDCA), are low molecular weight compounds that can stabilize protein conformation and rescue trafficking-defective misfolded proteins. We have shown that TUDCA treatment of cells expressing the mutants significantly increases the secretion of mutant LAMB2 proteins into the medium. We propose, therefore, that chemical chaperones can improve the folding of mutant LAMB2 proteins, reduce ER stress, and improve the secretion of mutant laminin trimers into the GBM, thereby attenuating proteinuria in Lamb2-/-; mutLAMB2 transgenic mice. To test these hypotheses, we will: 1. Examine whether LAMB2 point mutations cause protein misfolding and ER stress both in vitro and in vivo. 2. Determine whether chemical chaperones can improve the secretion of LAMB2 point mutants and reduce ER stress in vitro. 3. Investigate whether chemical chaperones improve secretion of mutant laminin (2 and attenuate proteinuria in Lamb2-/-; mutLAMB2 mice. These studies will help us to understand the molecular mechanisms underlying the pathogenesis of congenital nephrotic syndrome and assay the feasibility for therapeutic use of chemical chaperones in a relevant animal model. Our results may have significant therapeutic implications for the treatment of congenital nephrotic syndrome in patients carrying missense LAMB2 mutations. PUBLIC HEALTH RELEVANCE: Congenital nephrotic syndrome is an important childhood kidney disease which causes proteinuria and progressive renal failure. We are studying whether the administration of pharmacologically active chaperones can ameliorate proteinuria in an animal model. The results may indicate a new treatment scheme for renal proteinuric diseases.