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Title: Endothelial derived vesicles mediate podocyte dysfunction
Author: Hill, Nicola Rachel
ISNI:       0000 0004 7969 8762
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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The transfer of intracellular components, such as mRNA and miRNA via extracellular vesicles (EVs) is thought to facilitate cell-to-cell communication and may play a role in modulation and progression of kidney disease. EVs can bind to or be incorporated into a recipient cell, where they deliver their functional intracellular content which can result in molecular changes to the recipient cell. Several miRNAs have important roles in the pathogenesis of different types of chronic kidney disease (CKD) including various types of glomerulonephritis (GN). The kidneys glomerular filtration barrier (GFB) is composed of podocytes, glomerular basement membrane (GBM) and endothelium with its glycocalyx. Podocytes are crucial in maintaining the integrity of the GBM, and a decline in renal function occurs when podocytes become damaged or are lost. Podocytes and glomerular endothelial cells can communicate during renal disease; however, very little is known about how extracellular signals from the microenvironment regulate podocyte activity. This study proposes that GEnCs secrete EVs; these small vesicles, approximately 100nm in diameter were visualised by LM10 NanoSight technology microscopy and purified by ultracentrifugation. As detected by western blotting, EVs expressed protein markers, CD63 and TSG101 and whether unstimulated or stimulated with glucose, LPS or puromycin aminonucleoside (PAN), glomerular endothelial cells secreted equivalent numbers of EVs. Upon incubation with activated glomerular endothelial cell EVs, podocyte miRNA and mRNA expression changed significantly. Podocyte function was modulated, including dysregulation of the actin cytoskeleton, increased OXPHOS, mitochondrial activation, ROS production, alongside a decrease in VEGF secretion. miR-200C-3p was upregulated in podocytes upon incubation with EVs isolated from glucose and PAN treated glomerular endothelial cells. Overexpression of miR-200C-3p caused a downregulation of podocyte VEGF production. From the data, I conclude that activated glomerular endothelial cell EVs cause podocyte functional changes including downregulation of VEGF and may deliver a variety of miRNA's including miR-200C-3p.
Supervisor: Woollard, Kevin ; Pusey, Charles Sponsor: Nadhmi Auchi Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral