Mutant Laminin Chains and Nephrotic Syndrome

描述（由申请人提供）：层粘连蛋白（LAMB2）是肾小球基底膜（GBM）的主要层粘连蛋白三聚体Laminin-521的一个组成部分。LAMB2的零突变导致Pierson综合征，以先天性肾病综合征为特征，伴有严重的眼部和神经缺陷。相反，大多数错义LAMB2突变，如R246Q，导致孤立性先天性肾病综合征。为了研究错义突变导致蛋白尿的机制，我们培育了转基因小鼠，其中R246Q突变体(2)在足细胞中以不同水平表达，并取代GBM中的正常(2)。我们的初步数据显示，这些小鼠表现出蛋白尿，可以通过增加突变体R246Q-LAMB2沉积到GBM中来减轻。此外，在体外，与野生型相比，突变体LAMB2蛋白的分泌受到严重抑制。因此，我们假设致病性LAMB2点突变导致蛋白错误折叠、内质网应激和体内外分泌缺陷。化学伴侣，如牛磺酸去氧胆酸（TUDCA），是一种低分子量化合物，可以稳定蛋白质构象并挽救有运输缺陷的错误折叠蛋白质。我们已经证明，对表达突变体的细胞进行TUDCA处理可显著增加突变体LAMB2蛋白在培养基中的分泌。因此，我们提出，化学伴侣可以改善突变体LAMB2蛋白的折叠，减少内质网应激，并改善突变体层粘胶蛋白三聚体向GBM的分泌，从而减轻LAMB2 -/-中的蛋白尿；mutLAMB2转基因小鼠。为了验证这些假设，我们将：

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.