PODOCYTES & STRESS RESPONSE IN NEPHROTIC SYNDROME

肾病综合征是儿童肾脏疾病最常见的形式之一。它的特点是蛋白质大量泄漏穿过肾脏的过滤屏障，足细胞发生戏剧性的结构变化，这是构成屏障的一部分。这些变化包括富含肌动蛋白的足细胞足突的收缩(消失)及其肌动蛋白细丝的破坏，并可通过使用活性氧分子清除剂治疗而减弱，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一种肌动蛋白聚合的调节因子，热休克蛋白27(Hsp27)，并报道了在NS期间肾小球中Hsp27的诱导。我们推测，HSP27通过调节肌动蛋白细丝动力学，在介导NS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起重要作用，通常用于治疗NS的治疗方法通过改变Hsp27的表达和/或磷酸化来保护足细胞免受氧化剂诱导的损伤。为了验证这些假说，我们将：1)确定诱导的足细胞Hsp27表达和/或磷酸化是否保护NS，2)鉴定肾小球Hsp27结合蛋白并测量在NS期间Hsp27和识别的蛋白之间相互作用的变化，3)测量诱导的足细胞Hsp27改变对足细胞应激反应的保护作用，并将这些影响与足细胞用皮质类固醇、环孢素A和环磷酰胺(NS的常用治疗方法)所产生的效果进行比较。我们将使用体内(大鼠PAN肾病)和体外(培养的“分化”足细胞的PAN和鱼精蛋白处理)NS模型来确定体内(全动物热疗，Hsp27转基因动物)或体外(Hsp27正义/反义/磷酸化突变稳定转染)诱导的Hsp27是否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双杂交文库将用于鉴定和定义Hsp27结合蛋白，并将通过生化分析来确定它们在NS期间与Hsp27相互作用的变化。与用于NS的药物处理后相比，将Hsp27正/反义/磷酸化突变体导入培养的“分化”足细胞，用与NS相关的特定生物应激源(氧化应激、肌动蛋白细丝断裂、热休克)和细胞应激反应(存活、肌动蛋白细丝结构、热休克蛋白和抗氧化剂)进行处理。确定Hsp27在调节NS的足细胞结构中具有重要的生物学作用，将有助于开发针对这种非常常见的肾脏疾病的更高靶向性和毒性更低的治疗方法。

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.