Exosomes are cell-derived vesicles present in urine. They contain protein and RNA and represent a reservoir for renal toxicity biomarkers. Exosomes can ‘shuttle’ microRNA between cells and represent a new signalling mechanism. To understand the mechanisms of exosome mediated signalling and to develop exosomes as biomarkers of organ-specific toxicity we explored exosome uptake by renal cells.
Exosomes were fluorescently labelled then co-cultured with murine cortical collecting duct (mCCD) cells. In a subset of studies mCCD cells were grown on transwell membranes to allow analysis of apical or basolateral compartments. The supernatant exosome concentration was measured by nanoparticle tracking analysis and the intracellular exosome concentration was measured by flow cytometry. Confocal microscopy was used for intracellular visualisation of labelled exosomes.
Labelled exosomes were internalised by mCCD cells and this was significantly increased by the vasopressin analogue desmopressin (dDAVP) (percentage of cells containing labelled exosomes: dDAVP 38% ± 8.4% vs control 16.7% ± 4.8%, P < 0.001). Tolvaptan, a V2 receptor antagonist, reduced exosome uptake following dDAVP (tolvaptan and dDAVP 15.6% ± 3.7% vs dDAVP 38% ± 8.4%, P < 0.001). Endothelin-1, a peptide that inhibits vasopressin, and dynamin inhibition, reduced exosome uptake following dDAVP stimulation. Using mCCD cell monolayers, the site of dDAVP action was confirmed to be basolateral, but exosome uptake was from apical and basolateral compartments.
In conclusion, this study is the first to demonstrate that vasopressin regulates exosome uptake in a V2 receptor mediated process occurring through clathrin-dependent endocytosis. The effect of drug toxicity on this process is now being explored.