Paracrine interaction mediated by extracellular vesicles such as mesangial cells and podocytes in hypoxia and its contribution to fibrosis.

• Grant number: 17/03229-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: July 01, 2017
• End date: June 30, 2019
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Fernanda Teixeira Borges
• Grantee: Bianca Castino
• Host Institution: Pró-Reitoria de Pós-Graduação e Pesquisa. Universidade Cruzeiro do Sul (UNICSUL). São Paulo , SP, Brazil

Abstract

In hypoxia both tubulointerstitial and glomerular injury are observed. The podocyte is one of the first cells subjected to ischemia and, in the glomerulus, the mesangial cells are in opposition to the podocyte, separated only by the basement membrane. Thus, it is possible to suppose that a mechanism of intercellular communication occurs, especially in injurious conditions such as hypoxia. The aim of this work is to analyze the in vitro communication between podocytes and mesangial cells through extracellular vesicles under conditions of hypoxia and its effect on the TGF-²1 pathway and the production of extracellular matrix proteins.Podocytes will undergo hypoxia (1% O2, 5% CO2) and normoxia (21% O2 and 5% CO2) for 48h. Exosomes (50 ¼g/ml) will be extracted from the culture medium and used in the treatment of mesangial cells for 48h. Analysis of the gene expression for angiotensinogen and pre-pro-endothelin-1 will be evaluated by real-time PCR and protein synthesis for endothelin-1, angiotensin II, TGF-²1, collagen 1, fibronectin will be evaluated by western blot. The increase in intracellular calcium in mesangial cells will be analyzed by flow cytometry for the Fluo 4AM fluorescent marker and cell proliferation will be assessed by MTT. Podocytes will be transfected with GFP-labeled CD63 and used in co-culture with mesangial cells by means of a trans-well insert. The uptake of the exosomes by the mesangial cells will be evaluated by immunofluorescence for GFP. (AU)