Oxalate & Crystal Renal Phenotypes: A Microarray Study

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，但除了极端情况外，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还不清楚。体内模型可以研究草酸对肾上皮细胞的影响，而细胞间的联系和调节级联保持不变。我们将使用一系列分级的高草酸大鼠模型1)车辆对照组；2)高草酸尿，3)高草酸+晶体尿和4)高草酸+晶体+晶体沉积在肾脏中)和微阵列分析来建立坚实的数据库，作为未来研究的跳板。在特定的目的一，我们将建立一个新的模型研究高草酸尿大鼠使用渗透微泵给药草酸皮下。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在其上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。它还将通过比较微阵列分析和RT-PCR获得的结果，确定检测对照组和治疗组大鼠之间变化的敏感度界限。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，基因表达模式是如何变化的，特别是在没有晶体的情况下受草酸调控的基因。数据将通过SAM(统计分析)和数据挖掘程序Spotfire&GeneSpring进行分析。肾功能将通过内生肌酐清除量和钠排泄分数进行监测。肾脏形态将通过光镜和电子显微镜进行评估。将在尿液和肾脏中评估肾小管功能的标志物，以确定肾单位中对草酸损伤最敏感的部分。根据标记的不同，将在消息(RT-PCR、Northern印迹)和蛋白质(Western印迹、免疫组织化学或酶分析)水平上测量表达。这项工作的长期目标是建立草酸引起肾脏损害的机制。

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.