Extracellular Vesicles as Local Messengers in Glomerular Crosstalk
Hasmik Soloyan, M.D., Paola Aguiari, Ph.D., Roger De Filippo, M.D., Laura Perin, Ph.D., Sargis Sedrakyan, Ph.D..
Children's Hospital Los Angeles, Los Angeles, CA, USA.
BACKGROUND: The crosstalk between the podocyte and glomerular endothelial cells (GEC) is vital for the maintenance of glomerular function. Any slight alteration of their communication propagates injury and disruption of glomerular function, commonly observed during CKD. Extracellular vesicles (EV) have key paracrine functions in physiological and pathological processes. Thus, we hypothesize that EVs play important role in glomerular crosstalk through horizontal transfer of their cargo to the target cells, especially during CKD when the glomerular filtration barrier is damaged. In particular, this study focuses on the transfer and paracrine signaling of GEC-EV to podocytes.
METHODS: We isolated EV from GEC and characterized them by Nanosight analyzer, PCR, WB and flow cytometry for size distribution, RNA and protein content, and for surface marker expression. We used a no-cell-contact co-culture model to establish the transfer and intake of GEC-EV by podocytes, which was further confirmed in isolated glomeruli by the Cre-lox method. Podocytes exposed to GEC-EV were analyzed by PCR. To study EV transfer in vivo we used a transgenic model of CKD, Alport syndrome - characterized by a deficiency in the type IVα5 collagen and expression of tdTomato in endothelial cells, including GEC. TEM studies using immunogold co-labeling for tdTomato and CD63 were used to identify EV origin and uptake.
RESULTS: GEC-EVs expressed exosomal CD9, C63, cell-adhesion and angiogenic markers, especially the VEGF receptors type I and II. In vitro, transfer of normal GEC-EV to podocytes was confirmed by the Cre-lox activation of the tdTomato in podocytes by the Cre protein delivered via the GEC-EV. Transfer of GEC-EVs maintained podocyte morphology, WT1, podocin, nephrin and VEGF expression, while podocytes exposed to EVs isolated from Alport GEC showed alteration of these markers, thus possibly indicating that Alport GEC-EVs present a different cargo than normal GEC-EVs. In vivo, TEM data showed a presence of GEC-EV in podocytes, confirming EV transfer across the glomerular filtration barrier.
CONCLUSIONS: Our data suggest that EV transfer across the glomerular filtration barrier is an essential physiological event that regulates cellular function in podocytes. Identification of novel mediators of endothelial-podocyte crosstalk may lead to the development of more effective treatments for glomerular kidney disease.
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