MECHANISMS UNDERLYING SEGMENT-SPECIFIC NEPHROTOXICITY

先前的体内和体外研究表明，大多数 肾毒性化学物质在肾内诱导选择性损伤 影响肾近端直（PST）或近端回旋 (PCT)小管 在体外观察到的选择性损伤表明， 存在代谢和/或转运的细胞差异， 解释为什么这些部分中的每一个都容易受到特定的 肾毒物 生物化学研究，以阐明这些先天 PST和PCT节段的细胞机制一直难以研究 由于缺乏组织块或混合的管状细胞群 在大多数准备工作中。 最近开发了一种新方法， 批量隔离PST和PCT段， 现在来比较和对比基本的机械差异， 这些节段天生对肾毒性损伤敏感。 主要 该提案的目标是系统地调查细分市场- 特定的代谢和转运依赖机制 肾毒性通过测量的差异分布或活动 几种药物代谢酶和上皮转运系统， 可能使特定的肾单位节段易受肾毒性损伤。 这 研究应提供发展所需的机械信息， 预防多种形式的化学诱导的 肾损伤 在这些研究中，PST和PCT部分将 分别从肾髓质的外条纹区分离 或使用整体解剖从肾皮质的外部区域分离， 标准Percoll梯度分离技术。 一旦被隔离， 通过标记酶分析和组织学鉴定纯度 检查时，将检查两种组分的各种代谢（例如， 糖酵解和产酶能力，底物利用 偏好）、生化（例如耗氧率、谷胱甘肽 代谢、I期和II期生物转化酶活性），以及 运输（有机酸、碱、糖和氨基酸的运输速率） 差异 这些测量将用于阐明 正常功能的差异可能会使特定的 对特定毒物诱导的损伤敏感的管段。 在 将两个近端肾小管部分体外暴露于毒物， 对PST（对乙酰氨基酚、顺铂、六氯丁二烯 氯化汞）或PCT（缺氧/缺氧、二溴化乙烯、钾 重铬酸盐），然后将进行，以确定机械作用， 代谢和运输，因为它们涉及的病理生理学 肾单位特异性损伤。 将进行额外的研究，以：（1） 操纵化学药物靶向特定细胞类型 （选择性分段输送）通过金属与 特异性载体（半胱氨酸或金属硫代磷酸酯）;（2）评价毒性 将化学品输送到顶端的后果与 基底外侧细胞表面;和（3）评估之间的相互作用 通过评价改变进行体外肝代谢和肾毒性研究 在存在和不存在肝组织的情况下， （肝和肾组织的共孵育）。

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).