Proteomic and mRNA Profiling of Urine and Urine Sediment

描述（由申请人提供）：本项目的目的是验证尿蛋白和基因表达谱作为工具，从肾小球肾炎患者的尿液中获得新的机制和诊断信息。虽然以前的研究已经检查了尿液，以了解肾脏疾病的发病机制，并确定疾病活动，严重程度或预后的临床相关标志物，这些研究仅限于分析相对较少的预选候选蛋白。据推测，通过检查整个尿蛋白质组在肾小球肾炎，以及蛋白质和基因表达的白细胞存在于肾炎尿，以前未被识别的蛋白质表达模式将出现。这些蛋白质网络的调查将导致新的见解肾小球疾病的发病机制。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。这些主题将在两个具体目标中发展。在目的1中，双向聚丙烯酰胺凝胶电泳（2D-PAGE）将被用来映射一个良好的特征性队列的SLE肾炎患者的肾脏疾病复发之前，期间和之后的尿蛋白。这些研究将确定尿蛋白质组如何从非活动性疾病变化到活动性SLE。来自这些疾病活动的不同阶段的差异表达的蛋白质将通过质谱法（MS）进一步鉴定。据预测，在复发前参与的蛋白质网络将反映疾病激活的机制，而在活动性疾病中表达的蛋白质将代表肾损伤的介质，并且在治疗期间表达的蛋白质将决定治疗的成功或失败。目的2将蛋白质组学扩展到活动性SLE肾炎患者尿液中的白细胞。此外，cDNA微阵列分析将用于表征这些白色血细胞表达的基因。将尿液基因和蛋白表达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。该项目的结果预计将证明尿蛋白质组学和cDNA微阵列分析的相关性，通过识别以前未被识别的蛋白质网络活跃在SLE肾炎，挑战现有的肾脏疾病发病机制的范例。

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.