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.
