Median values of CD24⁺ and CD133⁺ EV levels in the urine of transplanted patients at day 1 and 7 after transplant (D1 and D7), divided according to the graft vascular lesions (VS), as described in Material and methods.

<p>No increase in urinary CD133⁺ EVs was observed in VS2 patients. Statistical analysis was performed as described in the Materials and methods section. * = p<0.05.</p

Urinary CD133⁺ Extracellular Vesicles Are Decreased in Kidney Transplanted Patients with Slow Graft Function and Vascular Damage

<div><p>Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133⁺ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133⁺ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133⁺ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133⁺ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133⁺ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.</p></div

Characteristics of the transplanted patients, ESRD patients and normal subjects in the study.

<p><b>ECD:</b> expanded criteria donors according to Crystal City Meeting criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104490#pone.0104490-Rosengard1" target="_blank">[35]</a>. <b>GFR:</b> glomerular filtration rate. <b>Histological score:</b> performed on pretransplant biopsies according to a semiquantitative method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104490#pone.0104490-Remuzzi1" target="_blank">[25]</a>. <b>APKD:</b> adult polycystic kidney disease; <b>Gn:</b> glomerulonephritis; <b>LD:</b> living donor; <b>DD:</b> deceased donor. <b>ID:</b> immunosuppressive therapy. <b>ESRD:</b> end-stage kidney disease. Continuous variables are express as mean ± SD.</p

Characterization of urinary EVs in transplanted patients.

<p>Urinary EVs were obtained from patients (n = 25) undergoing renal transplant. <b>Panel A</b>. Mean number of EVs/day in the urine of transplanted patients were similar at day 1 and 7 after transplant, counted as described in Material and Methods. Data are mean ± SD of all samples. <b>Panel B and C</b>. Characterization of EVs showing expression of CD9 and CD81 exosome markers and NanoSight analysis of size distribution profile. Similar results were obtained for samples from all transplanted patients at day 1 and 7. <b>Panel D</b>. Representative western blot analysis showing the donor origin of the extracellular vesicle using typing sera containing antibodies directed against HLA class I antigens of the donor not shared by the recipient, at 1 and 7 days after transplant. In the table are reported the HLA typing of the recipient and the donor and the HLA-antigen recognized by the typing serum (TORP1262). HLA-B5 (bold) is present in the donor and not in the recipient. Three patients were tested with similar results.</p

Detection of CD133⁺ EVs in transplanted patients.

<p>Panel A and B. Representative dot plots showing the gating strategy of latex bead-conjugated EVs (A) and the fluorescence detection of isotype or CD133 on EVs from a transplanted patients at day 1 or 7.</p

CD24⁺ and CD133⁺ EVs in the urine of normal subjects, ESRD and transplanted patients.

<p>Panel A. Levels of urinary CD24⁺ and CD133⁺ EVs in normal subjects (H), ESRD patients with residual diuresis and transplanted patients at day 1, 7 or 30 after transplant (D1, D7 and D30, respectively). A significant reduction of CD133⁺ EVs but not of CD24⁺ EVs was observed in ESRD and transplanted patients at day 1. Data are expressed as % positive beads and are mean ± SD of all samples. <b>Panel B and C</b>. Representative cytofluorimetric histograms (B) and median values (C) of CD24⁺ and CD133⁺ EV levels in the urine of patients with EGF (n = 13) and SGF (n = 12) at day 1 and 7 after transplant. Urinary CD133⁺ EVs but not CD24⁺ EVs were lower in EGF patients at day 1, and significantly increased at day 7. Statistical analysis was performed as described in the Materials and methods section. * = p<0.05.</p

MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury.

<p>Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.</p

<i>In vitro</i> anti-apoptotic effects of MVs on TECs.

<p>A) The percentage of apoptotic TECs after incubation with 5 µg/ml of cisplatin was evaluated by the Tunel assay. TECs were incubated in the presence of cisplatin with or without different doses of MVs derived from BM-MSCs or fibroblasts (10 or 30 µg/ml) and 3% FCS (Ctrl = TECs incubated 48 hours in the presence of 3% FCS only). Results are expressed as mean±SD of 4 different experiments. Analyses of variance with Newmann-Keuls multicomparison test was performed: *p<0.05 MVs (30 µg) <i>vs</i> vehicle alone. B) Histograms showing the relative expression (Rq) of different anti-apoptotic genes in cisplatin (TEC CIS) and cisplatin-MV treated tubular cells (TEC CIS+MV) in respect to control cells treated with vehicle alone (TEC). Experiments are performed in triplicate. Data was analysed via a Student’s <i>t</i> test (unpaired, 2-tailed); * <i>p<</i>0.05 TEC CIS vs TEC; ** <i>p</i><0.05 TEC CIS+MV vs TEC CIS; *** <i>p</i><0.05 TEC CIS+MV vs TEC. C) Histograms showing the relative expression (Rq) of pro-apoptotic genes in cisplatin (TEC CIS) and cisplatin-MV treated tubular cells (TEC CIS+MV) in respect to control cells (TEC). Experiments are performed in triplicate. Data was analysed via a Student’s <i>t</i> test (unpaired, 2-tailed); * <i>p<</i>0.05 TEC CIS vs TEC; ** <i>p</i><0.05 TEC CIS+MV vs TEC CIS.</p

Body weight, survival, renal function and morphology in SCID mice injected with cisplatin and different regimens of MVs.

<p>Results are expressed as mean±SD; ANOVA with Dunnet’s multicomparison test: </p><p>* <i>p<</i>0.05 siMV and miMV treatments <i>vs</i> cisplatin (CIS); </p><p>† <i>p<</i>0.05 miMV <i>vs</i> siMV.</p><p>CIS = cisplatin injection; CIS+siMV = cisplatin treated with single injection of MVs; CIS+RNase-MV = cisplatin treated with injection of MV pre-treated with RNase; CIS+miMV = cisplatin treated with multiple injection of MVs.</p

List and function of genes that significantly differ between cisplatin-treated human tubular cells stimulated or not with MVs.

<p>List and function of genes that significantly differ between cisplatin-treated human tubular cells stimulated or not with MVs.</p