Paracrine interaction mediated by extracellular vesicles in glomerular cells under hypoxia and its contribution to fibrosis.

Abstract

In hypoxia is observed the tubulointerstitial and glomerular injury characterized by accumulation of extracellular matrix, however the glomerular lesion, more moderate, is less studied. The podocyte is one of the first cells subject to hypoxia and, in the glomerulus, mesangial and glomerular endothelial cells are in opposition to the podocyte, separated by the glomerular basement membrane. It is possible to suppose that intercellular communication, especially in conditions such as hypoxia, also mediates glomerular injury. The objective of this work is to analyze a new mechanism of interglomerular communication, mediated by extracellular vesicles (VEC), mainly exosomes, under conditions of hypoxia in vitro, focusing its contribution to glomerular dysfunction.Immortalized human podocytes will be submitted to conditions of hypoxia (1% O2, 5% CO2) and normoxia (21% O2 and 5% CO2) for 48h. VEC (50 ¼g/ml) will be extracted from the culture medium and used in the treatment of human mesangial and endothelial cells in normoxia for 48h. In the exosomes we will evaluate the presence of mRNA for angiotensinogen, TGF-²1 and pre-pro-endothelin. In the mesangial cells treated or not with exosomes the dysfunction will be evaluated the protein synthesis of angiotensin II, TGF-²1, collagen 1 and fibronectin by western blot, the capacity of contraction will be evaluated by the increase in intracellular calcium, analyzed by flow cytometry and viability by MTT assay. In endothelial cells, treated or untreated, cell dysfunction will be assessed by the synthesis of endothelin-1, transwell migration capacity, matrigel tube formation capacity and cell proliferation. VEC-mediated communication will be evaluated by the co-culture of green fluorescent protein (GFP) labeled podocytes and their uptake by endothelial cells and mesangial cells in culture. (AU)