Pathogenesis of ischemia induced golgi dysfunction

急性肾缺血是一种常见的临床事件，具有相当高的发病率和死亡率。临床诊断为急性肾小管坏死(ATN)，与尿量减少和血肌酐进行性升高有关。在ATN的恢复过程中，多尿症被定义为每天的尿量超过2升，持续三到五天。ATN的病理特征与肌动蛋白细胞骨架的改变和膜极性的丧失有关。ATN的多尿相被认为与功能性Na，K-ATPase的心尖位置有关。一旦Na，K-ATPase的正常膜分布重建，ATN的多尿相就会分解。然而，ATN延长的多尿期并不能与估计肾上皮细胞膜周转率的最佳数据相一致。提示新合成的Na，K-ATPase在肾上皮细胞缺血损伤后的恢复过程中可能被错误定位或定位于错误的质膜结构域。这项提案中概述的实验旨在检验这样一种假设，即缺血损伤或ATP耗竭扰乱了上皮细胞的分选和靶向机制。具体来说，缺血损伤导致货物(整体膜)蛋白从其特定的运输复合体中逃逸。一种互补的可能性是，t-陷阱和/或sec6/sec8基侧对接复合体的随机分布是ATP缺乏的后遗症。这将导致在受伤恢复过程中蛋白质的严重错误分类。这一假说将通过检测在从ATP耗竭中恢复过程中各种顶端和基底侧膜蛋白和糖脂的蛋白质分类特征来检验。蛋白质的分类和运输将使用低水平光学显微镜、荧光标记蛋白质、图像处理和生化分离技术在体内进行检查。将用免疫组织化学方法和先进的成像技术研究sec6/sec8对接复合体和Synaxin亚型的亚细胞分布。

Acute renal ischemia is a common clinical occurrence associated with considerable morbidity and mortality. The clinical diagnosis known as acute tubular necrosis (ATN) is associated with decreased urine output and a progressive increase in the serum creatinine. During the recovery from ATN, polyuria defined as a urine output greater than 2 liters per day is observed for three to five days. Pathological features of ATN have been associated with alterations in the actin cytoskeleton and a loss of membrane polarity. The polyuric phase of ATN has been suggested to be causally related to the apical location of functional Na, K-ATPase. Once the normal membrane distribution of Na, K-ATPase is reestablished the polyuric phase of ATN resolves. However the prolonged polyuric phase of ATN cannot be reconciled with the best available data estimating rates of membrane turnover in renal epithelial cells. This suggests that newly synthesized Na, K-ATPase may be mis-sorted or targeted to the wrong plasma membrane domain in renal epithelial cells recovering from ischemic injury. The experiments outlined in this proposal, are designed to test the hypothesis that ischemic injury or ATP depletion, disrupts the sorting and targeting machinery of epithelial cells. Specifically, ischemic injury causes cargo (integral membrane) proteins to escape from their specific transport complex. A complementary possibility is that a randomized distribution of t-snares and/or the sec6/sec8 basolateral docking complex is a sequelae of ATP deprivation. This would lead to significant mis-sorting of proteins during recovery from injury. This hypothesis will be tested by examining the protein sorting characteristics of a variety of apical and basolateral membrane proteins and glycolipids during the recovery from ATP depletion. The sorting and transport of proteins will be examine din vivo using low level light microscopy, fluorescent tagged proteins, image processing and biochemical isolation techniques. The subcellular distribution of the sec6/sec8 docking complex and syntaxin isoforms will be studied by immunohistochemical methods and advanced imaging techniques.