MOLECULAR MECHANISMS OF RENAL CELL INJURY

急性肾缺血是一种常见的临床事件，与相当大的发病率和死亡率相关。 被称为急性肾小管坏死（ATN）的临床诊断与尿量减少和血清肌酐进行性增加有关。 在从ATN恢复期间，观察到三至五天的多尿，多尿定义为每天尿量大于2升。 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酶的正常膜分布重新建立，ATN的多尿期就消退。然而，ATN的多尿期延长不能与估计肾上皮细胞膜更新率的最佳可用数据相一致。 这表明，新合成的Na，K-ATP酶可能是错误的排序或在缺血性损伤恢复的肾上皮细胞中靶向错误的质膜结构域。 本提案中概述的实验旨在测试缺血性损伤或ATP耗竭破坏上皮细胞的分选和靶向机制的假设。 具体而言，缺血性损伤导致转运囊泡形成的动力学延迟，从而允许分选的蛋白质从其特异性转运复合物中逃逸。 这将导致在从损伤恢复期间蛋白质的显著错误分选。 将通过检查从ATP耗竭恢复期间各种顶侧和基底侧膜蛋白和糖脂的蛋白分选特征来检验这一假设。 将使用低水平光学显微镜、荧光标记蛋白、图像处理和生化分离技术在体内检查蛋白质的分选和转运。

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 re-established 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 a kinetic delay in transport vesicle formation that permits sorted proteins to escape from their specific transport complex. 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 examined in vivo using low level light microscopy, fluorescent tagged proteins, image processing and biochemical isolation techniques.