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

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

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

<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

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

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

Over-expression of miR-100 in HG-treated MCs impairs mTOR and TGFβ expression and collagen production.

<p><b>(A)</b> miR-100 expression was evaluated by qRT-PCR on 48h LG- and HG-cultured MCs, either alone or in combination with MSC-EVs or HLSC-EVs for 18h. Data normalized to RNU6B are representative of four different experiments performed in triplicate (n = 4). (<i>p = 0</i>.<i>04</i>, LG vs HG treated MCs). <b>(B)</b> Gain-of-function experiments were performed on MCs cultured in LG or HG medium for 48h, using pre-miR-100 oligonucleotides. Pre-miR negative control (neg c) oligonucleotides were used as control. miR-100 expression was evaluated by qRT-PCR and normalized to RNU6B (five experiment performed in triplicate, n = 5) (<i>p = 0</i>.<i>003</i>, pre-miR-100 vs pre miR neg in LG; <i>p = 0</i>.<i>002</i> pre-miR-100 vs pre miR neg in HG. <b>(C)</b> Cell extracts from MCs cultured in either a LG or HG medium and transfected with either pre-miR neg c or pre-miR-100 were subjected to SDS-PAGE and analysed for mTOR, TGFβ and collagen type IV content, normalized to β-actin. <b>(D)</b> Densitometric analysis was performed on western blots of MCs treated as above (n = 6) (LG pre-miR neg c vs HG pre-miR neg c, <i>p = 0</i>.<i>02</i> for mTOR, <i>p = 0</i>.<i>03</i> for TGFβ and <i>p = 0</i>.<i>01</i> for collagen type IV content; HG pre-miR neg c vs HG pre-miR-100, <i>p<0</i>.<i>001</i> for mTOR, TGFβ and collagen type IV content).</p

Stem Cell-Derived, microRNA-Carrying Extracellular Vesicles: A Novel Approach to Interfering with Mesangial Cell Collagen Production in a Hyperglycaemic Setting

<div><p>Extracellular vesicles (EVs) that are derived from stem cells are proving to be promising therapeutic options. We herein investigate the therapeutic potential of EVs that have been derived from different stem cell sources, bone-marrow (MSC) and human liver (HLSC), on mesangial cells (MCs) exposed to hyperglycaemia. By expressing a dominant negative STAT5 construct (ΔNSTAT5) in HG-cultured MCs, we have demonstrated that miR-21 expression is under the control of STAT5, which translates into Transforming Growth Factor beta (TGFβ) expression and collagen production. A number of approaches have been used to show that both MSC- and HLSC-derived EVs protect MCs from HG-induced damage via the transfer of miR-222. This resulted in STAT5 down-regulation and a decrease in miR-21 content, TGFβ expression and matrix protein synthesis within MCs. Moreover, we demonstrate that changes in the balance between miR-21 and miR-100 in the recipient cell, which are caused by the transfer of EV cargo, further contribute to providing beneficial effects. Interestingly, these effects were only detected in HG-cultured cells. Finally, it was found that HG reduced the expression of the nuclear encoded mitochondrial electron transport chain (ETC) components, CoxIV. It is worth noting that EV administration can rescue CoxIV expression in HG-cultured MCs. These results thus demonstrate that both MSC- and HLSC-derived EVs transfer the machinery needed to preserve MCs from HG-mediated damage. This occurs via the horizontal transfer of functional miR-222 which directly interferes with damaging cues. Moreover, our data indicate that the release of EV cargo into recipient cells provides additional therapeutic advantages against harmful mitochondrial signals.</p></div