NA+/K+ ATPASE & POLARITY IN ADPKD CYST FORMATION

一种用于分析人类常染色体显性遗传的明确培养系统 多囊肾病(ADPKD)上皮细胞已被设计并 由这位调查员在上一次拨款期间所表现出来的。 从单个来源的单层培养进行比较 显微解剖ADPKD囊肿与年龄匹配的正常近端和远端 (CCT)肾小管生长异常，基底膜 ADPKD细胞的合成、激素反应和酶活性。一个 囊性增大的主要后果的改变增加 ADPKD中NAK-ATPase活性与顶端细胞表面错位的关系 细胞。这可能是反向向量运输的原因。 钠进入囊性小管腔，导致渗透液蓄积。 这一可能性在ADPKD单层生长中得到了实验证实 可渗透膜支持分离心尖室和基底室，因为 有80%的~(22)Na从基底膜输送到根尖中层。现在 提案将侧重于描述这些内容的精确和详细的性质 NAK-ATPase与膜极性缺陷。假设ADPKD ADPKD上皮细胞表达NAK-ATPase异常将被检测 测定催化活性的动力学；分子亚基 组成；亚基合成、细胞加工和 降解；信使RNA水平和体外翻译活性。至 检查膜极性缺陷的全部范围：分布和 顶端和基底侧部酶的活性水平与NHS-生物素标记 模式将被确定。改变NAK位置的假说- ATPase到顶膜的原因是分选机制存在缺陷 ADPKD将通过检测推定的多肽信号进行测试 并通过检查脂肪酸(~3H-肉豆蔻酸、~3H-肉豆蔻酸)的模式 棕榈酸酯和14C-乙醇胺)标记。以确定在多大程度上 顶膜改变是由于脂肪酸酰化缺陷所致， 硫代酯-棕榈酸酯、硫代酯酰胺的特异性裂解敏感性 和糖基磷脂酰肌醇的键将被测试。整个纸巾， 培养的正常和ADPKD上皮细胞、根尖膜和基底膜 囊泡将被使用并接受脉冲追逐和NHS-生物素标记 蛋白质提取、SDS-PAGE、Western blotting和 免疫沉淀、酶分析、电子免疫和细胞化学 定位；RNA提取；Northern分析；体外翻译。

A defined culture system for the analysis of human autosomal dominant polycystic kidney disease (ADPKD) epithelia has been devised and characterized by this investigator during the last grant period. Comparisons made of monolayer cultures derived from individually microdissected ADPKD cysts and age-matched normal proximal (PST) and distal (CCT) tubules showed several abnormalities of growth, basement membrane synthesis, hormone response, and enzyme activities in ADPKD cells. An alteration of major consequence for cystic enlargement was increased activity of NaK-ATPase and mislocation to the apical cell surfaces in ADPKD cells. This could be responsible for reversed vectorial transport of sodium into cystic tubule lumens and lead to osmotic fluid accumulation. This possibility was confirmed experimentally in ADPKD monolayers grown on permeable membrane supports separating apical and basal compartments since 80% of 22Na was transported from the basal to apical media. The present proposal will focus on delineating the precise and detailed nature of these NaK-ATPase and membrane polarity defects. The hypothesis that ADPKD the NaK-ATPase expressed by ADPKD epithelia is abnormal will be tested by examining the kinetics of catalytic activity; molecular subunit composition; rates of subunit synthesis, cellular processing and degradation; messenger RNA levels and translational activity in vitro. To examine the full extent of membrane polarity defects: the distribution and activity levels of apical and basolateral enzymes and NHS-biotin labelling patterns will be determined. The hypothesis that altered location of NaK- ATPase to apical membranes is caused by defective sorting mechanisms in ADPKD will be tested by examination of the putative peptide signal sequence; and by examination of patterns of fatty acid (3H-myristate, 3H- palmitate and 14C-ethanolamine) labelling. To determine to what extent apical membrane alterations are due to defects in fatty acid acylation, susceptibility to specific cleavage of thioester-palmitate, thioester-amide and glycosyl phosphatidylinositol bonds will be tested. Whole tissues, cultured normal and ADPKD epithelial cells, apical and basolateral membrane vesicles will be used and subjected to pulse chase and NHS-biotin labelling protein extraction, characterization by SDS-PAGE, Western blotting and immunoprecipitation, enzyme assays, electron immuno-and cytochemical localization; RNA extraction; Northern analysis; and in vitro translation.