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形式之一。 它的特征是蛋白质在肾脏的过滤屏障中大规模泄漏以及足细胞中巨大的结构变化，部分构成了屏障。 这些变化包括富含肌动蛋白的足球脚部过程的缩回（递增），并破坏其肌动蛋白丝，并可以通过用活性氧分子清除剂进行治疗来减弱，这表明NS与氧化剂损伤与足细胞之间的联系之间存在联系。 最近，我们检测到正常足细胞中据报道的肌动蛋白聚合热休克蛋白27（HSP27）的调节剂，并在NS期间报告了Hsp27诱导Hsp27。 我们假设HSP27通过调节肌动蛋白丝动力学来介导NS中发生的足细胞结构变化具有重要作用。 我们还假设Hsp27在对氧化剂应激的足细胞反应中具有重要作用，并且通常通过改变HSP27表达和/或磷酸化来保护足细胞来保护足细胞来治疗NS ACT的疗法。 为了检验这些假设，我们将：1）确定诱导的足细胞HSP27表达和/或磷酸化对NS的预防变化，2）确定识别肾小球HSP27结合蛋白，并测量Hsp27和Hsp27与NS中鉴定的蛋白质之间的相互作用变化用皮质类固醇，环孢菌素A和环磷酰胺（NS的常见治疗方法）对足细胞治疗的人的影响。 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 ERDERMENT和NS。 来自大鼠肾肾小球的酵母两个混合文库将用于识别并定义HSP27结合蛋白，并且在NS期间与HSP27相互作用的变化将通过生化分析确定。 用HSP27感官/反义/磷酸化突变体转染的培养的“分化”足细胞将用与NS（氧化剂应激，肌动蛋白细丝破坏，热休克）和细胞应激反应（肌动蛋白的静态，肌动蛋白结构，肌动蛋白细胞结构，诱导HSPS和抗毒剂相比）与药物相比的胁迫进行处理。 鉴定HSP27在调节NS中调节足细胞结构中的生物学上重要作用将允许为这种非常常见的肾脏疾病形式开发更靶向较高的毒性疗法。