Oxalate & Crystal Renal Phenotypes: A Microarray Study

草酸盐

• 批准号: 6508619
• 负责人: SUSAN RUTH MARENGO
• 金额: $ 15.3万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6508619
• 关键词: disease /disorder model; drug delivery systems; electron microscopy; gene expression; genetic regulation; kidney; kidney function; laboratory rat; light microscopy; microarray technology; model design /development; northern blottings; oxalates; polymerase chain reaction; primary hyperoxalurias; renal cortex; renal medulla; renal tubule; urinary calculi; western blottings

DESCRIPTION (provided by applicant): Despite the fact that hyperoxaluria is an established risk factor for calcium oxalate (CaOx) urolithiasis, except for extreme cases, oxalate's exact role in urolithiasis is not well understood. There has been little progress in determining the genetic defects involved in idiopathic hyperoxaluria or urolithiasis, in part because oxalate's effects on the nephron are not well understood. In vivo models allow study of oxalate's effects on the renal epithelium with the intercellular associations and regulatory cascades intact. We will use a graded series of hyperoxaluric rat models 1) vehicle control; 2) hyperoxaluria, 3) hyperoxaluria+crystalluria and 4) hyperoxaluria+crystalluria+crystals deposited in the kidneys) and microarray analysis to develop a solid data base to be used as a springboard for future studies. In Specific Aim I we will develop a new model for the study of hyperoxaluric rats by using osmotic minipumps to deliver the oxalate subcutaneously. Specific Aim 2 will validate the microarray analysis by determining the number of rats needed for each pool of tissue, the number of pools/treatment group and the number of chips on which a pool must be run to control for technical and biological variability. It will also determine the limits of sensitivity for detecting changes between control and treated rats, by comparing the results obtained by microarray analysis vs RT-PCR. Specific Aim 3 will use microarray analysis and the models developed above to determine how the pattern of gene expression changes as hyperoxaluria progresses into urolithiasis, with particular emphasis on those genes regulated by oxalate in the absence of crystals. Data will be analyzed by SAM(statistical analysis) and the data mining programs SpotFire & GeneSpring. Kidney function will be monitored by creatinine clearance and fraction sodium excretion. Renal morphology will be evaluated by light and electron microscopy. Markers of tubular function will be evaluated in the urine and kidney to determine those segments of the nephron that are the most sensitive to damage by oxalate. Depending on the marker, expression will be measured at the level of the message (RT-PCR, Northern blot) and protein (Western blot, immunohistochemistry or enzymatic assays). The long term objective of this work is to establish the mechanism of oxalate induced damage in the kidney.

描述（由申请人提供）：尽管高草酸尿症是草酸钙（CaOx）尿石症的既定危险因素，但除极端情况外，草酸盐在尿石症中的确切作用尚不清楚。在确定特发性高草酸尿症或尿石症所涉及的遗传缺陷方面进展甚微，部分原因是草酸盐对肾单位的影响尚不清楚。体内模型可以研究草酸盐对肾上皮的影响，同时细胞间关联和调节级联完好。我们将使用一系列分级的高草酸尿大鼠模型1）载体控制； 2）高草酸尿，3）高草酸尿+晶体尿和4）高草酸尿+晶体尿+沉积在肾脏的晶体）和微阵列分析，以开发一个坚实的数据库，作为未来研究的跳板。在具体目标 I 中，我们将开发一种新模型，通过使用渗透微型泵皮下输送草酸盐来研究高草酸尿大鼠。具体目标 2 将通过确定每个组织池所需的大鼠数量、池/治疗组的数量以及池必须运行以控制技术和生物变异性的芯片数量来验证微阵列分析。通过比较微阵列分析与 RT-PCR 获得的结果，它还将确定检测对照大鼠和治疗大鼠之间变化的灵敏度极限。具体目标 3 将使用微阵列分析和上面开发的模型来确定随着高草酸尿症进展为尿石症，基因表达模式如何变化，特别强调在没有晶体的情况下受草酸盐调节的基因。数据将通过 SAM（统计分析）和数据挖掘程序 SpotFire 和 GeneSpring 进行分析。通过肌酐清除率和钠排泄分数来监测肾功能。将通过光学和电子显微镜评估肾脏形态。将在尿液和肾脏中评估肾小管功能标志物，以确定肾单位对草酸盐损伤最敏感的部分。根据标记的不同，将在信息（RT-PCR、Northern blot）和蛋白质（Western blot、免疫组织化学或酶测定）水平上测量表达。这项工作的长期目标是建立草酸诱导肾脏损伤的机制。