MOLECULAR MECHANISMS IN NEPHROTIC SYNDROME

肾病综合征（NS）是儿童常见的肾脏疾病， 特征为远端“足”突的回缩（消失）， 肾小球毛细血管周围的肾小球上皮细胞（GEC）。 足突是肾脏滤过屏障的重要组成部分， 它们的结构主要由肌动蛋白微丝调节， 在足突中大量存在 肌动蛋白和GEC本身被认为是 锚定肾小球基底膜的复合肌动蛋白相关 蛋白质和β 1整联蛋白粘附分子。 GEC足消失 过程与GEC肌动蛋白分布的改变有关 微丝和增加肾小球表达和磷酸化 热休克蛋白27是一种调节肌动蛋白聚合的热休克蛋白。 我们 假设：1）表达和/或 肌动蛋白微丝的相互作用，肌动蛋白相关蛋白 黏着斑蛋白、talin、α-肌动蛋白和β 1整联蛋白粘附分子， 对于GEC足突消失的发展至关重要， 肾病综合征，和2）这些变化是由改变介导 GEC hsp 27的表达。 为了验证这些假设，我们计划确定; 1)如果肌动蛋白的表达或相互作用发生特定变化， 肌动蛋白相关蛋白黏着斑蛋白、talin和α-肌动蛋白以及β 1 GECs中的整合素是足突发育所必需的 消除和NS，并确定这些之间的关系 GEC足突消失的改变和不同程度，以及2）如果 肾小球hsp 27表达的诱导变化导致发育 或保护免受GEC足突消失和NS。 使用两者在 体内（大鼠PAN肾病）和体外（分离的 肾小球）模型的GEC足突消失，我们将分析变化 在F-和G-肌动蛋白中，肌动蛋白相关蛋白（黏着斑蛋白、talin、α-肌动蛋白） 辅肌动蛋白）和β 1整合素在肾小球中使用半定量共聚焦显微镜 显微镜、北方和西方印迹和免疫沉淀研究。 表达的变化与它们之间的相互作用之间的关系 分子和GEC足突在多个阶段消失的程度 将使用电子显微镜来确定疾病。 的影响 将在体外研究hsp 27对足突消失的诱导， (heat肾小球休克和hsp 27转染）和体内（全身 热疗和诱导型正义和反义hsp 27 cDNA转基因小鼠）。 GEC调节中特异性分子改变的鉴定 结构，这是关键的发展GEC脚的过程 擦除和NS将允许开发更具体和更少的 NS的毒性治疗。

Nephrotic syndrome (NS), a common kidney disease in children, is characterized by retraction (effacement) of the distal "foot" processes of glomerular epithelial cells (GEC) that surround glomerular capillaries. Foot processes are an essential part of the kidney's filtration barrier, and their structure is regulated primarily by actin microfilaments which are abundant in foot processes. Actin and the GEC itself are thought to be anchored the glomerular basement membrane by a complex of actin-associated proteins and beta1 integrin adhesion molecules. Effacement of GEC foot processes has been linked to altered distributions of GEC actin microfilaments and increased glomerular expression and phosphorylation of hsp27, a heat shock protein reported to regulate actin polymerization. We hypothesize that: 1) Specific changes in the expression and/or interaction of actin microfilaments, the actin-associated proteins vinculin, talin, and alpha-actin, and beta1 integrin adhesion molecules are critical for the development of GEC foot process effacement during nephrotic syndrome, and 2) These changes are mediated by altered expression of GEC hsp27. To test these hypotheses we plan to determine; 1) If specific changes in the expression or interaction of actin, the actin-associated proteins vinculin, talin and alpha-actinin, and beta1 integrins in GECs are necessary for the development of foot process effacement and NS, and to determine the relationship between these alterations and variable degrees of GEC foot process effacement, and 2) If induced changes in the expression of glomerular hsp27 result in development of, or protection from, GEC foot process effacement and NS. Using both in vivo (PAN nephrosis in rats) and in vitro (protamine treatment of isolated glomeruli) models of GEC foot process effacement, we will analyze changes in F- and G-actin, actin-associated proteins (vinculin, talin, alpha- actinin), and beta1 integrins in glomeruli using semiquantitative confocal microscopy, Northern, and Western blots, and immunoprecipitation studies. The relationship between changes in the expression and interaction of these molecules and the extent of GEC foot process effacement at multiple stages of disease will be determined using electron microscopy. The effect of hsp27 induction on foot process effacement will be studied both in vitro (heat shock and hsp27 transfections of glomeruli) and in vivo (whole body hyperthermia and inducible sense and antisense hsp27 cDNA transgenic mice). Identification of specific molecular alterations in the regulation of GEC structure which are critical for the development of GEC foot process effacement and NS would permit the development of more specific and less toxic therapies for NS.