Proteomic and mRNA Profiling of Urine and Urine Sediment

描述(由申请人提供)：该项目的目标是验证尿蛋白和基因表达谱作为从肾炎患者的尿液中获得新的机制和诊断信息的工具。尽管以前的研究通过检测尿液来深入了解肾脏疾病的发病机制，并确定疾病活动性、严重性或预后的临床相关标记物，但此类研究仅限于分析相对较少的预先选择的候选蛋白。据推测，通过检查肾小球肾炎期间的整个尿蛋白组，以及肾炎尿液中白细胞表达的蛋白质和基因，将出现以前未知的蛋白质表达模式。对这些蛋白质网络的研究将为肾小球疾病的发病机制带来新的见解。此外，通过表征与疾病活动的不同阶段相关的蛋白质表达模式，将确定新的预后指标。这些主题将以两个具体目标为基础。在目标1中，二维聚丙烯酰胺凝胶电泳法(2D-PAGE)将用于绘制一组具有良好特征的SLE肾炎患者在肾脏疾病复发前、期间和之后的尿蛋白图谱。这些研究将确定尿液蛋白质组如何从非活动期疾病转变为活动期SLE。来自这些疾病活动不同阶段的差异表达的蛋白质将通过质谱学(MS)进一步鉴定。据预测，复发前参与的蛋白质网络将反映疾病的激活机制，而在活动期疾病中表达的蛋白质将代表肾脏损伤的介质，而在治疗过程中表达的蛋白质将决定治疗的成败。目的2将蛋白质组学扩展到活动期SLE肾炎患者尿液中的白细胞。此外，还将使用基因芯片分析来表征这些白细胞表达的基因。尿液基因和蛋白的表达将与同时获得的外周血单核细胞进行比较。预计差异表达的蛋白质和基因将反映肾内炎症事件。该项目的结果有望通过鉴定以前未被识别的活跃在SLE肾炎中的挑战肾脏疾病发病机制的现有范式的蛋白质网络，来证明尿蛋白组学和cDNA微阵列分析的相关性。

DESCRIPTION (provided by applicant): The objective of this project is to validate urine protein and gene expression profiling as tools to derive novel mechanistic and diagnostic information from the urine of patients with glomerulonephritis. Although previous investigations have examined urine to gain insight into the pathogenesis of renal disease, and to identify clinically relevant markers of disease activity, severity, or prognosis, such studies were limited to analysis of a relatively small number of pre-chosen candidate proteins. It is postulated that by examining the entire urinary proteome during glomerulonephritis, as well as the proteins and genes expressed by leukocytes present in nephritic urine, previously unrecognized patterns of protein expression will emerge. Investigation of these protein networks will lead to new insights into the pathogenesis of glomerular disease. Furthermore, by characterizing patterns of protein expression associated with distinct phases of disease activity, novel prognostic indicators will be identified. These themes will be developed in two Specific Aims. In Aim 1, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) will be used to map the urine proteins of a well-characterized cohort of patients with SLE nephritis before, during, and after relapse of renal disease. These studies will define how the urinary proteome changes from inactive disease through active SLE. Differentially expressed proteins from these different phases of disease activity will be further identified by mass spectrometry (MS). It is predicted that the protein networks engaged before relapse will reflect mechanisms of disease activation, while proteins expressed in active disease will represent mediators of renal injury, and proteins expressed during treatment will determine therapeutic success or failure. Aim 2 will extend proteomics to the leukocytes present in the urine of patients with active SLE nephritis. Additionally, cDNA microarray analysis will be used to characterize genes expressed by these white blood cells. Urinary gene and protein expression will be compared to that of simultaneously obtained peripheral blood mononuclear cells. It is anticipated that differentially expressed proteins and genes will reflect intra-renal inflammatory events. The results of this project are expected to demonstrate the relevance of urinary proteomic and cDNA microarray analyses by identifying previously unrecognized protein networks active in SLE nephritis that challenge existing paradigms of renal disease pathogenesis.