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

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

<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

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

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

Values of the LF differences between baseline and last 30 minutes of hemodialysis.

<p>Values of the LF differences between baseline and last 30 minutes of hemodialysis in the three groups by excluding diabetes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120167#pone.0120167.t003" target="_blank">Table 3</a>). The circles represent the singular patients. The errorbar represents the median and the 25^th and 75^th percentile (patients are grouped by the tertiles of FO_pre/ECW% values). Note the increase in LF in the first tertile. The outliers are inserted in the picture at a different scale in order to focus the attention to the errorbars.</p

Data_Sheet_1_Human Liver Stem Cell-Derived Extracellular Vesicles Prevent Aristolochic Acid-Induced Kidney Fibrosis.docx

<p>With limited therapeutic intervention in preventing the progression to end-stage renal disease, chronic kidney disease (CKD) remains a global health-care burden. Aristolochic acid (AA) induced nephropathy is a model of CKD characterised by inflammation, tubular injury, and interstitial fibrosis. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) have been reported to exhibit therapeutic properties in various disease models including acute kidney injury. In the present study, we aimed to investigate the effects of HLSC-EVs on tubular regeneration and interstitial fibrosis in an AA-induced mouse model of CKD. NSG mice were injected with HLSC-EVs 3 days after administering AA on a weekly basis for 4 weeks. Mice injected with AA significantly lost weight over the 4-week period. Deterioration in kidney function was also observed. Histology was performed to evaluate tubular necrosis, interstitial fibrosis, as well as infiltration of inflammatory cells/fibroblasts. Kidneys were also subjected to gene array analyses to evaluate regulation of microRNAs (miRNAs) and pro-fibrotic genes. The effect of HLSC-EVs was also tested in vitro to assess pro-fibrotic gene regulation in fibroblasts cocultured with AA pretreated tubular epithelial cells. Histological analyses showed that treatment with HLSC-EVs significantly reduced tubular necrosis, interstitial fibrosis, infiltration of CD45 cells and fibroblasts, which were all elevated during AA induced injury. At a molecular level, HLSC-EVs significantly inhibited the upregulation of the pro-fibrotic genes α-Sma, Tgfb1, and Col1a1 in vivo and in vitro. Fibrosis gene array analyses revealed an upregulation of 35 pro-fibrotic genes in AA injured mice. Treatment with HLSC-EVs downregulated 14 pro-fibrotic genes in total, out of which, 5 were upregulated in mice injured with AA. Analyses of the total mouse miRnome identified several miRNAs involved in the regulation of fibrotic pathways, which were found to be modulated post-treatment with HLSC-EVs. These results indicate that HLSC-EVs play a regenerative role in CKD possibly through the regulation of genes and miRNAs that are activated during the progression of the disease.</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