Proteomic profiling of engineered human immortalized podocyte cell model of Fabry disease

Renal involvment in Fabry disease(FD) is a frequent and early event with a significant morbidity and adverse prognosis, manifesting itself, initially, through microalbuminuria, evolving with a progressive reduction of the glomerular filtration rate and chronic kidney disease (CKD). Enzyme replacement therapyhas improved life quality of FD patients, although it does not stop the progression to CKD. The mechanisms by which the lysosomal accumulation of glycosphingolipids, chiefly Gb3, results in cell and organic dysfunction in FD are still unknown however, podocyteinjury plays an important role in the progression of this nephropathy. Podocytesare well-differentiated and specialized kidney cells with limited capacity for repair and replacement being the main glomerular cell involved in Fabry Nephropathy (FN). Thus, elucidating the molecular mechanisms of the podocytein the FD context is important. In this study, we have aimed to identify differentially abundant proteins in a model of immortalized human podocyteof FD, genetically edited with the aid of CRISPR/ Cas9technology using Two-dimensional Differential Gel Electrophoresis (2D-DIGE) and the proteins have been identified through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results have shown that proteins HSPD1, UCHL1 and Enolase1 are downregulated in FD podocytes. These proteins take part in signaling pathways involved in processes of tubulointerstitial fibrosis and autophagy. In order to confirm pro-fibrosis pathways, we have compared the expression of other proteins involved in this process by using Western Blot and our data have shown overexpression of proteins TGF-β (1.5x), Vimentin (2.3x) and VEGF-2 (1.5x) in Fabry podocytesas regards the controls. Additionally, proteins involved in proliferative pathways, e.g. PI3K 110a (2.5x) and PI3K 110b (1,45x), are also overexpressed. Therefore, the Fabry podocytesexpresses a pro-fibrosis proliferative pattern. These findings may improve our knowledge of Fabry podocitopathy aiming new strategy at intervention for preventing Fabry nephropathy.