PODOCYTES & STRESS RESPONSE IN NEPHROTIC SYNDROME

足细胞

• 批准号: 6786993
• 负责人: WILLIAM E SMOYER
• 金额: $ 6.53万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6786993
• 关键词: actins; binding proteins; corticosteroids; cyclophosphamide; cyclosporines; gene expression; genetically modified animals; heat shock proteins; kidney disorder chemotherapy; laboratory rat; membrane permeability; nephrotic syndrome; nonhuman therapy evaluation; oxidative stress; phosphorylation; protein protein interaction; renal glomerulus; tissue /cell culture; yeast two hybrid system

Nephrotic syndrome is one of the most common forms of kidney disease in children. It is characterized by massive leakage of protein across the kidney's filtration barrier and dramatic structural changes in podocytes, which in part comprise the barrier. These changes include retraction (effacement) of the actin-rich podocyte foot processes with disruption of their actin filaments, and can be attenuated by treatment with reactive oxygen molecule scavengers, suggesting a link between NS and oxidant injury to podocytes. We recently detected a reported regulator of actin polymerization, heat shock protein 27 (hsp27), in normal podocytes, and reported induction of hsp27 in glomeruli during NS. We hypothesize that hsp27 has an important role in mediating the podocyte structural changes which occur in NS, via regulation of actin filament dynamics. We also hypothesize that hsp27 has an important role in the podocyte response to oxidant stress, and that the therapies commonly used to treat NS act by protecting podocytes from oxidant-induced injury via alterations in hsp27 expression and/or phosphorylation. To test these hypotheses we will: 1) Determine if induced changes in podocyte hsp27 expression and/or phosphorylation protect against NS, 2) Identify glomerular hsp27-binding proteins and measure changes in the interaction between hsp27 and the identified proteins during NS, and 3) Measure the protective effects of induced alterations in podocyte hsp27 on the podocyte stress response, and compare these effects to those resulting from podocyte treatment with corticosteroids, cyclosporine A, and cyclophosphamid (common treatments for NS). We will use both in vivo (PAN nephrosis in rats) and in vitro (PAN and protamine treatment of cultured "differentiated" podocytes) models of NS to determine if induction of hsp27 in vivo (whole animal hyperthermia, hsp27 transgenic animals) or in vitro (hsp27 sense/antisense/phosphorylation mutant stable transfections) protects podocytes against foot process effacement and NS. A yeast two hybrid library from rat kidney glomeruli will be used to identify, and define hsp27-binding proteins, and alterations in their interactions with hsp27 during NS will be determined by biochemical analyses. Cultured "differentiated" podocytes transfected with hsp27 sense/antisense/phosphorylation mutants will be treated with stressors with specific biological relevance to NS (oxidant stress, actin filament disruption, heat shock) and the cellular stress response (survival, actin filament structure, induction of hsps and antioxidants) compared to that after treatment with drugs used for NS. Identification of a biologically important role for hsp27 in regulating podocyte structure in NS would permit the development of more highly targeted and less toxic therapies for this very common form of kidney disease.

肾病综合征是儿童最常见的肾脏疾病之一。 其特征在于蛋白质大量渗漏穿过肾脏的滤过屏障，足细胞的结构发生显著变化，足细胞部分构成了滤过屏障。 这些变化包括收缩（消失）的肌动蛋白丰富的足细胞足的过程与破坏他们的肌动蛋白丝，并可以通过与活性氧分子清除剂治疗衰减，表明NS和氧化剂对足细胞损伤之间的联系。 最近，我们发现了一个报道的调节肌动蛋白聚合，热休克蛋白27（HSP 27），在正常足细胞，并报告诱导HSP 27在肾小球NS。 我们推测，热休克蛋白27有一个重要的作用，在介导足细胞的结构变化发生在NS，通过调节肌动蛋白丝的动力学。 我们还假设热休克蛋白27在足细胞对氧化应激的反应中具有重要作用，并且通常用于治疗NS的疗法通过改变热休克蛋白27表达和/或磷酸化来保护足细胞免受氧化剂诱导的损伤。 为了验证这些假设，我们将：1）确定足细胞hsp 27表达和/或磷酸化的诱导变化是否保护免受NS，2）鉴定肾小球hsp 27结合蛋白并测量NS期间hsp 27与鉴定的蛋白之间的相互作用的变化，和3）测量足细胞hsp 27的诱导改变对足细胞应激反应的保护作用，并将这些效应与用皮质类固醇、环孢霉素A和环磷酰胺（NS的常见治疗）治疗足细胞所产生的效应进行比较。 我们将使用NS的体内（大鼠PAN肾病）和体外（培养的“分化的”足细胞的PAN和鱼精蛋白处理）模型来确定hsp 27的体内诱导（整个动物高热，hsp 27转基因动物）或体外诱导（hsp 27正义/反义/磷酸化突变稳定转染）是否保护足细胞免受足突消失和NS。 将使用来自大鼠肾小球的酵母双杂交文库来鉴定和定义热休克蛋白27结合蛋白，并通过生化分析确定NS期间它们与热休克蛋白27相互作用的改变。 与用用于NS的药物处理后相比，用与NS具有特定生物学相关性的应激物（氧化应激、肌动蛋白丝破坏、热休克）和细胞应激反应（存活、肌动蛋白丝结构、hsps和抗氧化剂的诱导）处理用hsp 27正义/反义/磷酸化突变体转染的培养的“分化的”足细胞。 Hsp 27在NS中调节足细胞结构的生物学重要作用的鉴定将允许开发针对这种非常常见的肾脏疾病形式的更高靶向和毒性更小的疗法。