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).

此前的体内和体外研究表明，大多数 肾毒性化学物质导致肾脏内选择性损伤 影响肾近端直(PST)或近端曲张 (%)小管。体外观察到的选择性损伤表明，先天的 新陈代谢和/或运输方面存在细胞差异，这可能 解释为什么这些细分市场中的每一个都容易受到特定 肾毒药。生物化学研究以阐明这些先天的 PST和PCT节段的细胞机制一直很难研究 由于缺乏组织块或混合的肾小管细胞群 大多数制剂中的类型。最近，一种新的方法被开发出来 批量隔离PST和PCT数据段，使之成为可能 现在比较和对比基本的机制差异，这些差异导致 这些节段天生就容易受到肾毒性损伤。少校 这项提案的目标是系统地调查细分市场- 依赖于代谢和运输的特定机制 通过测量肾毒性的不同分布或活性 几种药物代谢酶和上皮运输系统 可能使特定的肾单位节段发生肾毒性损伤。这 研究应提供开发所需的机械性信息 预防多种形式化学诱发疾病的合理途径 肾脏损伤。在这些研究中，PST和PCT段将 分别从肾髓质外纹区分离 或从肾皮质的外部区域进行大量解剖和 标准Percoll梯度分离技术。一旦与世隔绝， 标记酶分析和组织学鉴定纯度 检查时，将检查这两个组分的各种代谢(如： 糖酵解和糖异生能力、底物利用率 偏好)、生化(例如耗氧率、谷胱甘肽 代谢，第一阶段和第二阶段生物转化酶活性)，以及 转运(有机酸、碱、糖和氨基酸的转运速率) 不同之处。这些测量将被用来阐明 正常功能的差异可能会使特定的 对特定毒物引起的损伤敏感的管段。在……里面 两种近端肾小管部分体外暴露于 PST(扑热息痛、顺铂、六氯丁二烯、 氯化汞)或PCT(缺氧/缺氧、二溴乙烷、钾 重铬酸盐)，以确定其机械作用 代谢和转运与心脏的病理生理学 肾单位特异性损伤。我们会进行更多研究，以：(1) 操纵化学物质对特定细胞类型的靶向 (选择性分段递送)通过与金属络合 特定载体(半胱氨酸或金属硫氨酸)；(2)毒性评估 将化学物质输送到顶端诗句的后果 基侧细胞表面；以及(3)评估细胞表面之间的相互作用 评价肝肾毒性的体外肝代谢和肾毒性 在有无肝组织的情况下的肾毒性反应 (肝和肾组织的共同孵化)。