Primers used in qRT-PCR experiments.

<p>Primers used in qRT-PCR experiments.</p

<i>De novo in vitro</i> and <i>in vivo</i> expression of human proteins after cisplatin and MVs treatment.

<p>A) Representative confocal micrographs showing the nuclear and cytoplasmatic expression of human POLR2E and SUMO-1 proteins <i>in vivo,</i> in kidney sections of cisplatin-AKI (AKI-CIS) mice treated or not with MVs and sacrificed 48 hours later, and <i>in vitro</i> by TECs treated with cisplatin and cultured in the absence (vehicle) or in the presence of 50 µg of MVs (MV) for 24 hours. Nuclei were counterstained with Hoechst dye. Original magnification: ×400 for kidney sections and ×630 for TECs. B) 1×10⁵ TECs treated with cisplatin and cultured in the absence (CIS) or in the presence of two different preparations of MVs (+MV) for 1 hour were analysed by RT-PCR for specific human mRNA <i>POLR2E</i>. Bands of PCR products specific for human <i>POLR2E</i> of the expected size (90 pb) were detected in a 4% agarose gel electrophoresis. As positive control the extract of human bone marrow-derived MSCs (BM-MSC) was used. The * indicates the control without cDNA.</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

Renal cell apoptosis and proliferation in cisplatin-AKI mice untreated or treated with different regiments of MVs.

<p>A) Quantification of Tunel-positive cells/high power field (hpf) at different time points. Data are expressed as mean ±SD of 8 different mice for each experimental condition. ANOVA with Dunnet’s multicomparison test was performed: * <i>p</i><0.05 siMVs or miMVs <i>vs</i> CIS; ** <i>p</i><0.05 miMVs <i>vs</i> siMVs. Representative micrographs of Tunel staining of renal sections of cisplatin mice given vehicle alone (day 4) and of cisplatin mice treated with different regiments of MVs at different time points (4, 14 and 21 days). Original magnification: ×400. B) Quantification of PCNA positive cells/hpf and of BrdU positive cells/hpf at different time points. BrdU was injected intraperitoneally for 2 successive days before mice being killed. Data are expressed as mean ±SD of 8 different mice for each experimental condition. ANOVA with Dunnet’s multicomparison test was performed: * <i>p</i><0.05 siMVs versus CIS. Abbreviations: Ctrl = healthy mice; CIS = cisplatin treated mice injected with vehicle alone; MV = cisplatin treated mice with single injection of MVs.</p

RNase treatment does not modify MV size, but reduces RNA content of MVs.

<p>A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of <i>SUMO-1</i>, <i>POLR2</i> and <i>Act B</i> transcripts in MVs treated or not with RNase, express as 2^-δCt, as described in material and methods.</p

Schematic representation of the protocol of cisplatin induced AKI and MV administration regimens and survival curves.

<p>A) Graph showing time-points of cisplatin administration, siMVs or miMVs and the time-points of sacrifice. B) Survival curves of SCID mice with cisplatin induced AKI treated with different regiments of MVs administration. All mice receiving vehicle alone died within 5 days. Mice that received siMVs or miMVs injections survived significantly longer than control mice treated with vehicle alone or with a si(RNase-inactivated)MVs. Data was analysed via a log-rank test: * <i>p</i><0.05 siMV <i>vs</i> CIS; ** <i>p</i><0.05 miMV <i>vs</i> siMV. Abbreviations: vehicle = cisplatin treated mice injected with vehicle alone; siMV = cisplatin treated mice with single injection of MVs; miMV = cisplatin treated mice with multiple injection of MVs; RNase MV = cisplatin treated mice injected with a single dose of MVs pre-treated with RNase.</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

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

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

Values of time and frequency domain indices estimated during the first 15 min (baseline) and the last 30 min of the HD session.

<p>Patients are grouped in tertiles of pre-dialysis FO/ECW%. Diabetic patients are excluded.</p><p>Comparisons between baseline (BL) and last 30 min of hemodialysis (HD)</p><p># Wilcoxon signed rank test p-value< 0.05, or</p><p>§ p-value<0.01 (italics)</p><p>Values of time and frequency domain indices estimated during the first 15 min (baseline) and the last 30 min of the HD session.</p