Pathogenesis of ischemia induced golgi dysfunction

急性肾缺血是一种常见的临床疾病，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）与尿量减少和血清肌酐逐渐升高有关。在ATN恢复期间，多尿定义为每天尿量大于2升，持续3至5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的多尿期已被认为与功能性Na， k - atp酶的顶端位置有因果关系。一旦Na， k - atp酶的正常膜分布被重建，ATN的多尿期就会结束。然而，ATN延长的多尿期与肾上皮细胞膜更新率的最佳可用数据不一致。这表明，在缺血损伤后的肾上皮细胞中，新合成的Na， k - atp酶可能被错误地分类或靶向到错误的质膜结构域。本提案中概述的实验旨在验证缺血性损伤或ATP耗竭会破坏上皮细胞的分选和靶向机制的假设。具体来说，缺血性损伤导致货物（整体膜）蛋白从其特定的运输复合物中逃逸。一种补充的可能性是，t-snare和/或sec6/sec8基底外侧对接复合体的随机分布是ATP剥夺的后遗症。这将导致损伤恢复过程中蛋白质的严重错误分类。这一假设将通过检查各种顶端和基底外侧膜蛋白和糖脂在ATP耗竭恢复过程中的蛋白质分选特性来验证。蛋白质的分类和运输将使用低水平光显微镜，荧光标记蛋白质，图像处理和生化分离技术进行体内检查。将通过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syntaxin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.