Our ultimate goal is to develop a non-invasive procedure for the diagnosis of IgA nephropathy (IgAN). The specific goal of the studies proposed in this application is to identify urinary polypeptides that can be used as biomarkers of IgAN. In preliminary studies, we have demonstrated elevated levels of IgA and IgG immunoglobulins and IgA-IgG complexes in the urine of IgAN patients using immune complex-specific ELISA. Furthermore, we have identified several potential urinary polypeptide biomarkers for IgAN by capillary electrophoresis-mass spectrometry (CE-MS) and sequenced some of these polypeptides using tandem mass spectrometry. Based on these data, we hypothesize that the urine proteome of IgAN patients contains disease-specific biomarkers. We propose to test this hypothesis by first confirming our preliminary data through analysis of archival urine samples collected during studies of IgAN from well-characterized patients and controls. We will determine the levels of immunoglobulins and immune complexes using classical and immune complex-specific ELISA protocols with quality assurance and verification by western blot analysis. These data will then be extended by analysis using CE-MS to identify positive and negative biomarkers of disease in which the software tool, MosaCluster, with support vector machines, will be used for classification of samples. This approach will define a membership, with positive values (disease-associated marker) and negative values (marker for normal). The results of ELISA and CE-MS will be compared and correlated with the clinical findings. Candidate biomarkers identified by CE-MS will be further characterized by Fourier transform-ion cyclotron resonance MS to define the amino acid sequence and post- translational modifications. Finally, candidate biomarkers will be verified using an independent cohort of patients and controls. This study is designed to identify markers that can be used to distinguish IgAN from other renal diseases as well as biomarkers of disease progression and severity. Relevance: IgAN is the most common primary glomerulonephritis worldwide. Currently, diagnosis requires an invasive renal biopsy and IgAN is frequently diagnosed at a very late stage. The identification of biomarkers that can be used as the basis of a noninvasive diagnostic test and/or used to monitor the course of the disease would be extremely beneficial.
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