OXIDATIVE METABOLISM AND TRANSPORT IN RENAL TUBULES

肾小管中的氧化代谢和转运

• 批准号: 2137931
• 负责人: LAZARO J MANDEL
• 金额: $ 33.77万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137931
• 关键词: adenosine triphosphate; aerobiosis; brush border membrane; cell membrane; cellular pathology; cytoskeletal proteins; enzyme activity; fluorescent dye /probe; immunocytochemistry; interference microscopy; kidney metabolism; laboratory rabbit; membrane structure; molecular cloning; protein isoforms; renal ischemia /hypoxia; renal tubular transport; tissue /cell culture

The long term objective of this laboratory is to obtain a detailed understanding of the cellular and molecular events underlying the cellular dysfunctions induced by renal ischemia. Past and continually expanding experience regarding the relationship between tubular metabolism and function as well as novel techniques will be used toward this goal. Two major specific aims are proposed far the next 5 years: In terms of the first aim, much experimental evidence suggests that a breakdown in the interactions between the plasma membrane and the scaffolding-like cytoskeletal structure may precede cell injury. The effects of ischemia on these interactions will be studied in various renal preparations, each one selected to address a different set of molecular mechanisms. The intact rat kidney will be used to study alterations in brush border structure caused by ischemia/reoxygenation, particularly investigating changes in actin, villin, and the 105kDa protein believed to link the actin filamentous bundle to the plasma membrane. At the same time, changes in the proximal tubule basolateral membrane structure will be studied, especially the role of ankyrin in this process. To this end, other experiments will seek to isolate the cDNAs encoding the isoform expressed in proximal renal tubules of the cytoskeletal linkage protein ankyrin, with the intention of cloning it and raising antibodies against this protein. Once these antibodies are available, they will be used to study the fate of this protein during ischemia and the role it plays in the structural changes that occur with oxygen deprivation. Freshly isolated tubular fragments will be used to focus on the plasma membrane- cytoskeletal breakdown that leads to membrane bleb formation during energy depletion. These studies will use a combination of video-enhanced differential interference contrast microscopy and fluorescence. Cultured renal cell lines will be used as models for specific aspects of ischemic injury, emphasizing the role of cytoskeletal and plasma membrane proteins and possible reversibility of function. The second aim deals with the adaptation of renal energy metabolism and transport to the culture environment. The search will continue for culture conditions that optimize oxidative metabolism and Na,K-ATPase activity, especially examining the expression of this enzyme.

该实验室的长期目标是获得详细的 了解细胞为基础的细胞和分子事件 肾脏缺血引起的功能障碍。过去，不断扩展 关于管状代谢与 功能和新技术将用于此目标。二 在接下来的5年中提出了主要的具体目标：就 第一个目的，许多实验证据表明 质膜与脚手架样之间的相互作用 细胞骨架结构可能在细胞损伤之前。缺血对 这些相互作用将在各种肾脏制剂中进行研究 被选中以解决不同的分子机制。完整 大鼠肾脏将用于研究刷子边框结构的变化 是由缺血/氧化引起的，尤其是研究了 肌动蛋白，Villin和105KDA蛋白被认为可以连接肌动蛋白 丝状束到质膜。同时，变化 将研究近端小管的基底外侧膜结构， 尤其是Ankyrin在此过程中的作用。为此，其他 实验将寻求隔离编码表达的同工型的cDNA 在细胞骨架连锁蛋白arnkyrin的近端肾小管中， 为了克隆它并提出针对此的抗体 蛋白质。一旦这些抗体可用，它们将用于研究 缺血期间该蛋白质的命运及其在 缺氧会发生的结构变化。刚隔离 管状碎片将用于关注质膜 - 细胞骨架分解，导致能量期间膜泡沫形成 消耗。这些研究将结合视频增强 差异干扰对比度显微镜和荧光。培养 肾细胞系将用作缺血特定方面的模型 损伤，强调细胞骨架和质膜蛋白的作用 以及功能的可逆性。 第二个目标涉及肾脏能量代谢的适应和 运输到文化环境。搜索将继续进行文化 优化氧化代谢和Na，K-ATPase活性的条件， 特别检查该酶的表达。