PHARMACOLOGICAL RESCUE OF MUTANT LAMININ IN NEPHROTIC SYNDROME

描述（由申请人提供）：肾病综合征（NS）的特征是大量蛋白尿、低白蛋白血症和进行性肾功能丧失的风险增加。它导致严重的发病率和高死亡率，占美国流行终末期肾脏疾病的15%，每年花费超过30亿美元。大多数与基因突变相关的NS病例没有有效的治疗方法。我们的研究重点是由NS中最常见的突变基因之一LAMB2的错义突变引起的单基因NS。LAMB2编码的层粘连蛋白ß2是层粘连蛋白521 （α5ß2γ1）的一个组成部分，是肾小球基底膜（GBM）的主要层粘连蛋白三聚体。通过我们建立的细胞和敲除/转基因小鼠模型，我们发现足细胞向GBM分泌错误折叠的C321R-LAMB2受到抑制，从而导致足细胞内质网（ER）应激导致蛋白尿。此外，突变体在GBM中的积累增加可减轻蛋白尿。因此，恢复突变层粘连蛋白的缺陷运输和挽救错误折叠的2蛋白以达到其功能目标是有希望的治疗策略。本研究的总体目标是研究一种新的策略，以治疗干预遗传形式的NS。为了实现我们的研究目标，我们建立了一个NS小鼠模型（Lamb2-/-； NEPH- C321R-LAMB2小鼠），在该模型中，通过小鼠肾素启动子（NEPH），足细胞靶向表达突变型大鼠Lamb2 (C321R-LAMB2)，取代野生型小鼠Lamb2。我们的初步数据表明，错误折叠的突变蛋白更容易被蛋白质降解，这是由泛素-蛋白酶体途径介导的。更令人兴奋的是，与单独的化学伴侣相比，化学伴侣纠正蛋白质错误折叠和蛋白酶体抑制剂阻断蛋白质降解的组合在体外产生了更明显的促分泌作用。因此，我们将在NS小鼠模型中进一步研究联合治疗对蛋白质错误折叠和降解的调节作用。拟议的研究将在NS动物模型中分析化学伴侣与蛋白酶体抑制剂联合治疗的可行性。该结果可能对治疗由异常内质网蛋白酶和足细胞内质网功能障碍引起的家族性或散发性NS患者具有重要的治疗意义。

DESCRIPTION (provided by applicant): Nephrotic syndrome (NS) is characterized by heavy proteinuria, hypoalbuminemia, and increased risk of progressive loss of kidney function. It causes serious morbidity and high mortality, accounting for 15% of prevalent end-stage renal disease at an annual cost of more than $3 billion in the US. There is no effective treatment for most cases of NS associated with genetic mutations. Our research has been focused on a monogenic form of NS caused by missense mutations of LAMB2, one of the most commonly mutated genes in NS. Laminin ß2 encoded by LAMB2 is a component of laminin-521 (α5ß2γ1), the major laminin trimer of the glomerular basement membrane (GBM). Using our established cell and knockout/transgenic mouse models, we have found that inhibited secretion of misfolded C321R-LAMB2 from podocytes to the GBM and the resulting podocyte endoplasmic reticulum (ER) stress lead to proteinuria. In addition, increased accumulation of the mutant in the GBM alleviates proteinuria. Therefore, restoring defective trafficking of the mutant laminin and rescuing misfolded 2 proteins to reach their functional destination are hopeful therapeutic strategies. The overall goal of this research is to investigate a novel strategy for therapeutic intervention in genetic forms of NS. To accomplish our research goal, we have generated a NS mouse model (Lamb2-/-; NEPH- C321R-LAMB2 mice) in which podocyte-targeted expression of the mutant form of rat LAMB2 (C321R-LAMB2) via the mouse nephrin promoter (NEPH) replaces the wild-type mouse LAMB2. Our preliminary data show that the misfolded mutant protein is much more susceptible to protein degradation, which is mediated by the ubiquitin-proteasome pathway. More excitingly, a combination of a chemical chaperone correcting protein misfolding and a proteasome inhibitor blocking protein degradation elicit a much more pronounced pro- secretion effect as compared to the chemical chaperone alone in vitro. Thus, the effect of combined treatment regulating both protein misfolding and degradation will be further investigated in our NS mouse model. The proposed study will assay the feasibility for therapeutic use of chemical chaperones in combination with proteasome inhibitors in a NS animal model. The results may have significant therapeutic implications for the treatment of familial or sporadic NS patients caused by aberrant ER proteostasis and podocyte ER dysfunction.