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，但除了极端病例外，草酸盐在尿石症中的确切作用尚不清楚。在确定与特发性高草酸尿症或尿石症有关的遗传缺陷方面进展甚微，部分原因是草酸对肾单位的影响尚不清楚。体内模型允许在细胞间关联和调节级联完整的情况下研究草酸对肾上皮的影响。我们将使用分级系列高血氧大鼠模型：1)载药控制；(2)高草酸尿，（3)高草酸尿+结晶尿，(4)高草酸尿+结晶尿+晶体沉积在肾脏）和微阵列分析，以建立坚实的数据基础，作为未来研究的跳板。在具体目标1中，我们将建立一种新的模型，用于研究高血氧大鼠通过渗透微型泵皮下输送草酸盐。特异性目标2将通过确定每个组织池所需的大鼠数量、池/处理组的数量以及池必须运行的芯片数量来验证微阵列分析，以控制技术和生物变异性。它还将通过比较微阵列分析和RT-PCR获得的结果，确定检测对照和治疗大鼠之间变化的灵敏度极限。Specific Aim 3将使用微阵列分析和上述开发的模型来确定当高草酸尿症进展为尿石症时基因表达模式如何变化，特别强调在没有晶体的情况下草酸盐调节的那些基因。数据将通过SAM（统计分析）和数据挖掘程序SpotFire和genesspring进行分析。肾功能将通过肌酐清除率和钠排泄分数来监测。肾脏形态将通过光镜和电子显微镜进行评估。肾小管功能的标志物将在尿液和肾脏中进行评估，以确定对草酸损害最敏感的肾元部分。根据不同的标记，将在信息（RT-PCR, Northern blot）和蛋白质（Western blot，免疫组织化学或酶分析）的水平上测量表达。这项工作的长期目标是建立草酸盐引起肾脏损伤的机制。

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.