PODOCYTES & STRESS RESPONSE IN NEPHROTIC SYNDROME

足细胞

基本信息

批准号：
6921134
负责人：
WILLIAM E SMOYER
金额：
$ 7.55万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
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

项目摘要

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。其特点是蛋白质大量渗漏穿过肾脏的过滤屏障，以及部分构成屏障的足细胞发生巨大的结构变化。这些变化包括富含肌动蛋白的足细胞足突的收缩（消失）及其肌动蛋白丝的破坏，并且可以通过活性氧分子清除剂的治疗来减弱，这表明 NS 与足细胞氧化损伤之间存在联系。我们最近在正常足细胞中检测到了肌动蛋白聚合的调节剂热休克蛋白 27 (hsp27)，并报告了 NS 期间肾小球中 hsp27 的诱导。我们假设 hsp27 通过调节肌动蛋白丝动力学，在介导 NS 中发生的足细胞结构变化中发挥重要作用。我们还假设 hsp27 在足细胞对氧化应激的反应中具有重要作用，并且常用于治疗 NS 的疗法通过改变 hsp27 表达和/或磷酸化来保护足细胞免受氧化剂诱导的损伤。为了检验这些假设，我们将：1) 确定足细胞 hsp27 表达和/或磷酸化的诱导变化是否可以预防 NS，2) 鉴定肾小球 hsp27 结合蛋白并测量 NS 期间 hsp27 与已鉴定蛋白质之间相互作用的变化，以及 3 ) 测量足细胞 hsp27 诱导改变对足细胞应激反应的保护作用，并将这些作用与用皮质类固醇治疗足细胞产生的作用进行比较，环孢素 A 和环磷酰胺（NS 的常见治疗方法）。我们将使用 NS 的体内（大鼠 PAN 肾病）和体外（PAN 和鱼精蛋白处理培养的“分化”足细胞）模型来确定 hsp27 的诱导是在体内（整个动物热疗、hsp27 转基因动物）还是在体外（hsp27 有义/反义/磷酸化突变体稳定转染）保护足细胞免受足突消失和 NS 的影响。来自大鼠肾小球的酵母二杂合文库将用于鉴定和定义 hsp27 结合蛋白，并通过生化分析确定它们在 NS 期间与 hsp27 相互作用的变化。转染 hsp27 有义/反义/磷酸化突变体的培养的“分化”足细胞将接受与 NS（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、诱导热休克蛋白和抗氧化剂）与使用 NS 药物治疗后的结果进行比较。鉴定 hsp27 在调节 NS 足细胞结构中的生物学重要作用将有助于为这种非常常见的肾脏疾病开发更具针对性和毒性较小的疗法。