MECHANISMS UNDERLYING SEGMENT-SPECIFIC NEPHROTOXICITY

特定部位肾毒性的潜在机制

• 批准号: 2155074
• 负责人: CHARLES E RUEGG
• 金额: $ 9.43万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2155074
• 关键词: acetaminophen; apical membrane; atomic absorption spectrometry; basolateral membrane; biotransformation; cadmium; high performance liquid chromatography; kidney cell; laboratory rabbit; liver metabolism; mercury; metal complex; mixed tissue /cell culture; renal toxin; renal tubular transport; renal tubule; toxicant interaction; toxin metabolism; transport proteins

Previous in vivo and in vitro research has demonstrated that most nephrotoxic chemicals induce selective injury within the kidney effecting either renal proximal straight (PST) or proximal convoluted (PCT) tubules. Selective injury observed in vitro suggests that innate cellular differences in metabolism and/or transport exist which may explain why each of these segments is susceptible to specific nephrotoxicants. Biochemical investigations to elucidate these innate cellular mechanisms in PST and PCT segments have been difficult to study due to the lack of tissue mass or a mixed population of tubular cell types in most preparations. Recently a new method was developed to isolate PST and PCT segments from one another in bulk making it possible to now compare and contrast basic mechanistic differences which render these segments innately susceptible to nephrotoxicant injury. The major goals of this proposal are to systematically investigate segment- specific metabolism- and transport-dependent mechanisms of nephrotoxicity by measuring the differential distribution or activity of several drug metabolizing enzymes and epithelial transport systems which may predispose specific nephron segments to nephrotoxic injury. this research should provide mechanistic information needed to develop rational approaches for preventing many forms of chemically-induced renal injuries. In these studies PST and PCT segments will be respectively isolated from the outer stripe region of the renal medulla or from the outer regions of the renal cortex using bulk dissection and standard Percoll gradient separation techniques. Once isolated and the purity characterized by marker enzyme analysis and histological examination, both fractions will be examined for various metabolic (e.g. glycolytic and gluconeogenic capacities, substrate utilization preferences), biochemical (e.g. rates of oxygen consumption, glutathione metabolism, phase I and II biotransformation enzyme activities), and transport (rates of organic acid, base, sugar and amino acid transport) differences. These measurements will be used to elucidate the differences in normal functions which might render the particular tubular segment susceptible to specific toxicant induced injury. In vitro exposure of both proximal tubular fractions to toxicants which effect either PST (acetaminophen, cis-platinum, hexachlorobutadiene, mercuric chloride) or PCT (hypoxia/anoxia, ethylene dibromide, potassium dichromate) will then be conducted to determine the mechanistic role of metabolism and transport as they relate to the pathophysiology of nephron specific injury. Additional studies will be conducted to: (1) manipulate the targeting of chemical gents to specific cell types (selective segmental delivery) through complexation of metals with specific carriers (cysteine or metallothionine); (2) evaluate the toxic consequences of delivering chemicals to the apical verses the basolateral cell surfaces; and (3) evaluate the interactive role between liver metabolism and nephrotoxicity in vitro by evaluating alterations in nephrotoxic responses in the presence and absence of liver tissue (co-incubations of liver and kidney tissues).

以前的体内和体外研究表明，大多数 肾毒性化学物质在肾脏内诱导选择性损伤 影响肾脏近端直（PST）或近端复杂的 （PCT）小管。 在体外观察到的选择性伤害表明先天 可能存在新陈代谢和/或运输的细胞差异 解释为什么这些细分都容易受到特定的影响 肾毒性。 生化研究以阐明这些先天的 PST和PCT段中的细胞机制很难研究 由于缺乏组织质量或管状细胞的混合种群 大多数准备工作中的类型。 最近开发了一种新方法 分离PST和PCT段，批量使其成为可能 现在比较和对比呈现的基本机械差异 这些段天生易受肾毒性损伤。 专业 该建议的目标是系统地研究细分市场 - 特定的代谢和运输依赖性机制 肾毒性通过测量的差分分布或活性 几种药物代谢酶和上皮运输系统 可能使特定的肾单位段诱发肾毒性损伤。 这 研究应提供开发所需的机械信息 预防多种形式的化学诱导的理性方法 肾脏受伤。 在这些研究中，PST和PCT段将是 分别从肾脏髓质的外条纹区域分离 或使用散装解剖和 标准Percoll梯度分离技术。 一旦孤立， 以标记酶分析和组织学为特征的纯度 检查，将检查两个分数的各种代谢（例如 糖酵解和糖原的能力，底物利用率 偏好），生化（例如，氧气消耗速率，谷胱甘肽 代谢，第一阶段和II生物转化酶的活动）和 运输（有机酸，碱，糖和氨基酸运输速率） 差异。 这些测量将用于阐明 正常功能的差异可能会导致特定 管状段易受特定毒物诱导的损伤。 在 两种近端管状级分的体外暴露于有毒物质 效应PST（对乙酰氨基酚，顺式铂，六氯丁烷， 氯化汞）或PCT（缺氧/缺氧，二溴化乙二醇，钾 然后将进行二分法）以确定 与之相关的代谢和运输与病理生理有关 肾脏特异性损伤。 其他研究将进行：（1） 操纵化学绅士对特定细胞类型的靶向 （选择性分段输送）通过金属络合 特异性载体（半胱氨酸或金属氨酸）； （2）评估有毒 将化学物质交付给顶端经文的后果 基底外侧细胞表面； （3）评估之间的互动作用 通过评估改变的肝脏代谢和肾毒性 在存在和不存在肝组织的肾毒性反应中 （肝脏和肾脏组织的共同结合）。