OXIDATIVE METABOLISM AND TRANSPORT IN RENAL TUBULES

肾小管中的氧化代谢和转运

• 批准号: 3228041
• 负责人: LAZARO J MANDEL
• 金额: $ 29.09万
• 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 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3228041
• 关键词: adenosine triphosphate; brush border membrane; calcium metabolism; cell membrane; chelating agents; cytoplasm; cytoskeleton; enzyme induction /repression; free radicals; glycolysis; histochemistry /cytochemistry; hypoxanthines; hypoxia; immunochemistry; immunocytochemistry; ionophores; kidney metabolism; laboratory rabbit; laboratory rat; membrane lipids; membrane permeability; mitochondria; oxidation reduction reaction; oxygen tension; oxygen transport; phospholipids; renal ischemia /hypoxia; renal tubular transport; renal tubule; singlet oxygen; tissue /cell culture; xanthine oxidase; adenosine triphosphate; brush border membrane; calcium metabolism; cell membrane; chelating agents; cytoplasm; cytoskeleton; enzyme induction /repression; free radicals; glycolysis; histochemistry /cytochemistry; hypoxanthines; hypoxia; immunochemistry; immunocytochemistry; ionophores; kidney metabolism; laboratory rabbit; laboratory rat; membrane lipids; membrane permeability; mitochondria; oxidation reduction reaction; oxygen tension; oxygen transport; phospholipids; renal ischemia /hypoxia; renal tubular transport; renal tubule; singlet oxygen; tissue /cell culture; xanthine oxidase

We propose to apply the concepts learned from previous studies on the interrelationship between metebolism and transport in renal tubules to two broad areas: the effects of anoxia on proximal renal function and the metabolism of cultured renal proximal tubules. We will utilize suspensions of proximal tubules obtained from the rabbit and rat kidneys to study the tubular effects of anoxia independently from hemodynamic factors present during and after clamp-induced ischemia. Previous experiments in our laboratory subjecting the rabbit tubule suspension to anoxia and reoxygenation suggest that most of the cellular damage occurs during anoxia. The mechanism and sites of this damage will be investigated with a variety of interrelated approaches. The involvement of individual variables, such as calcium, ATP, and oxygen-derived free radicals will be evaluated. Cytosolic free calcium (Caf) will be measured using calcium-sensitive dyes and Caf will be modulated by use of calcium-selective ionophores and chelators. The effects of oxygen-free radicals will be tested during anoxia and reoxygenation by comparing rabbit and rat tubules, by addition of hypoxanthine and xanthine oxidase to the medium, and by the use of protective agents. Mitochondrial function will continue to be assessed independently from plasma membrane integrity. New techniques will be developed to measure membrane damage. Immunocytochemistry will be used to visualize the organization of cytoskeletal proteins within the microvilli and terminal web in association with the plasma membrane. Brush border membrane enzyme activity and plasma membrane phospholipid breakdown will also be measured during anoxia. This information is expected to have clinical relevance, since the added basic knowledge concerning the dysfunctions and protective mechanisms should suggest clinical interventions to ameliorate the dysfunctions. We have recently developed an improved rabbit proximal tubule suspension that has a normal viability of 1-3 days. This suspension will be used to study the conditions in the culture environment that cause a widely observed adaptation of cellular energy metabolism, such that oxidative metabolism decreases and glycotytic capacity is considerably enhanced. We will study the metabolic changes that occur as a function of partial oxygen pressure, metabolic substrates and mitogens, focusing on the conditions necessary for glycolytic enzyme induction. This knowledge will be used to attain our long term goal of developing the methodology to grow renal cells in monolayer culture that express normal oxidative metabolism.

我们建议运用从以前的研究中学到的概念 元重复性与肾脏运输之间的相互关系 小管到两个宽区域：缺氧对近端的影响 肾功能和培养的肾脏近端的代谢 小管。 我们将利用获得的近端小管的悬浮液 从兔子和大鼠肾脏研究 缺氧独立于存在的血液动力学因素 在夹紧诱发的缺血之后。 我们以前的实验 实验室对兔小管悬浮液的悬浮症进行缺氧和 重氧表明大多数细胞损伤发生 在缺氧期间。 这种伤害的机制和部位将是 通过各种相互关联的方法进行了调查。 这 涉及单个变量，例如钙，ATP和 将评估氧气的自由基。 无胞质 钙（CAF）将使用钙敏感染料和 CAF将通过使用钙选择性离子载体和 螯合剂。 将测试无氧自由基的影响 通过比较兔子和大鼠，在缺氧和重氧过程中 小管，通过将低黄嘌呤和黄嘌呤氧化酶添加到 中等，并通过使用保护剂。 线粒体 功能将继续与血浆独立评估 膜完整性。 新技术将开发到 测量膜损伤。 免疫细胞化学将使用 可视化细胞骨架蛋白的组织 与等离子体的微绒毛和终端网 膜。 刷边界膜活性和等离子体 膜磷脂分解也将在 缺氧。 该信息有望具有临床相关性， 由于有关功能障碍的附加基础知识和 保护机制应提示临床干预措施 改善功能障碍。 我们最近开发了一个改善的兔子近端小管 正常生存能力为1-3天。 这个悬架 将用于研究文化环境中的条件 这会引起广泛观察到的细胞能的适应 代谢，氧化代谢降低，并且 糖基质能力大大提高。 我们将研究 随着部分氧的函数发生的代谢变化 压力，代谢底物和有丝分裂剂，专注于 糖酵解酶诱导所需的条件。 这 知识将用于实现我们发展的长期目标 在单层培养中种植肾细胞的方法论 表达正常的氧化代谢。