Proteomic analysis of Bradykinin signaling in podocytes

Abstract

Background and Aim: Diabetic end-stage nephropathy is the main cause of long-term death for diabetic patients. Podocyte damage is one of the hallmarks of diabetic nephropathy, where the loss of the podocyte filtration barrier leads to proteinuria and progressive renal damage. Research on Kallikrein Kinin System (KKS) has integrated its way into diabetic nephropathy research, where the fluctuations of the signaling molecules in this system have been shown to alter certain aspects of the disease. Bradykinin (BK) is a vasoactive peptide that was shown to play an important role in the progression of renal damage. In this study, we aim to evaluate the changes in protein expression profiles of rat podocytes upon the treatment with BK by Mass Spectrometry in conjunction with pathway enrichment analysis. This analysis will help in discovering core effectors whose upstream and downstream protein profile can elucidate the signaling mechanisms underlying BK’s modulation of the podocyte biology. Methods: Cultured immortalized rat podocytes were treated with BK at 10-7 M for 3 and 6 hours. Protein profile was evaluated by LC-MS-MS analysis, and pathway enrichment analysis was performed on the output data. The discovery of the upregulation of cyclo-oxygenase 2 (COX-2) prompted us to measure COX-2 protein and activity levels by western blot and enzyme immunoassay respectively, as well as identifying players in the BK-COX-2 signaling pathway by western blot. Results: BK treatment showed the up- and down-regulation of multiple proteins, where COX-2 was upregulated. Pathway enrichment analysis was performed on the proteomics data to broaden our understanding of the cellular processes occurring upon BK stimulation. We confirmed the upregulation of the COX-2 protein and activity levels using western blot and enzyme immuno-assay, and showed that the BK stimulation of COX-2 is downstream the MAPK pathway. We identified the downregulation of Nephrin upon to BK stimulation. Finally, using the enzyme immunoassay technique, we obse