URINARY PROTEOMIC ANALYSIS IN HEALTHY SUBJECTS AND HYPERTENSIVE PATIENTS:APPLICATIONS FOR BIOMARKER DISCOVERY

Several recent studies have implemented urinary proteomic approach for biomarker discovery. Human urine is one of the most interesting fluids, where proteomic analysis helps to identify differential expression of proteins in patients for early disease detection for better therapeutic outcome. Interestingly, urinary proteomics has focused more attention for the biomarker discovery than serum proteomics, because of the limited protein content, simpler composition, and low abundance of protease concentration. Recent studies demonstrated that proteins in the urine are highly stable after repeated freeze and thaw cycles, and the length of sample handling at room temperature does not affect the urine proteome. Proteomics analysis in the renal field has been mainly focused on the detection and identification of urinary proteins that change in pathologic or inflammatory conditions. Exosome isolation helps to reduce the complexity of the proteome to discover less abundant proteins but clinically relevant. In this thesis urinary proteomic analysis was successfully applied for the identification of novel protein biomarkers from urine and urinary exosomes. A “traditional” proteomic approach was applied to the study of the changes in urinary proteins related to hypertension (and in particular primary aldosteronism, PA) and hormonal cycle in women. Several urine samples from healthy controls and patients with hypertensive diseases were collected for the comparative proteomics analysis. After these analyses a “putative” biomarker was identified, the serine protease inhibitor serpin B3 (SB3), and further studied by means of different methodologies (ELISA, western immunoblotting, immunohistochemistry, Real Time PCR etc.), in a bigger set of samples. Additionally urine and tissue samples were obtained from primary aldosteronism subtype (aldosterone-producing adenoma, APA, and bilateral adrenal hyperplasia, BAH) patients for the detection of serpin B3. Interestingly the data obtained from these samples suggest a role of aldosterone in the modulation of serpin B3, giving new insights for the understanding of the mechanisms involved in primary aldosteronism. We successfully applied a method for urinary exosomes isolation (by nanomembrane concentrator application) with the aim of isolating exosomal protein and RNA for the detection of potential biomarkers such as prostasin and serpin B3. Exosomes isolation protocol was confirmed by electron microscopy as well as the presence of urinary exosome specific marker protein aquaporin 2. Additionally, the behavior of such protein was investigated, together with other components related to Na-water balance, in a “circadian rhythm” study on 6 healthy subjects. Taken all together, these data point toward the possible application of such approach to the use of proteomics approach as valuable tool for biomarker discovery and analysis, with a potential clinical usefulness.