New perspectives in transplantation therapy

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Introduction and Aims: Exosomes are microvescicles secreted from various types of activated cells.Urinary exosomes are mainly derived from cells of the nephron and carry the surface protein markers of the cells of origin.In this perspective urinary exosomes can be an easly available source of information about the renal condition and the damage degree.The aim of this study was the evaluation of the expression of different markers of inflammation, tissue damage and regeneration by exosomes present in the urine of kidney transplanted patients.Methods: Urinary exosomes were isolated from 11 control transplanted patients, 9 patients with slow graft function (SGF, defined ascreatinine level> 3 mg/dL on day 5 post transplant, but no need for dialysis) and 10 healthy subjects matched for sex and age.Urine samples were collected 1 (day 1) and 7 (day7) days after the transplant.Exosomes were obtained from the second fresh morning urine treated with protetase inhibitor, centrifuged to eliminate cell debris and Tamm-Horsfall protein and ultracentrifugated at 100000g for 1h.As exosomes are smaller than the lower sensitivity limit of the cytofluorimeter, cytofluorimeter analysis was performed after adsorption of isolated vesicles on 4 μm aldehyde-sulphate latex beads.Expression of CD45, CD56 and VEGFR2 was indicated as geometric mean and values normalized on the expression of CD24 (urine exosome marker).microRNA were isolated using mirVana kit and analysed by quantitative RT-PCR.Results: A panel of several markers was tested on urinary exosomes.CD45 and CD56 were used as markers of inflammation, and VEGFR2 as marker of endothelial damage.At day 1 after transplant CD45, CD56 and VEGFR2 were higher in transplanted patients than in normal subjects.Moreover, CD45 and CD56 were higher in SGF patients in respect to controls.At day 7, CD45, CD56 and VEGFR2 decreased in control patients and returned to basal levels.In contrast, no decrease was observed in SGF-patients, who maintained higher values.Finally, at day 7, an increase in microRNA involved in renal regeneration were observed in both control and SGF patients.Conclusions: These findings suggest that markers of inflammatory cells, of endothelial damage and of cell regeneration can be detected in the urinary exosomes of transplanted patients.Their levels are correlated with the pathophysiological condition of the graft function and its cellular/molecular causes.These data suggest an interesting diagnostic role for the urinary exosomes.

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