Pre-incubation with hucMSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy
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The administration of cisplatin is limited due to its nephrotoxic side effects, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury. In this study, we demonstrated that the pretreatment of human umbilical cord MSC-derived exosomes (hucMSC-Ex) can prevent the development of cisplatin-induced renal toxicity by activation of autophagy in vitro and in vivo.
In vitro, rat renal tubular epithelial (NRK-52E) cells were pre-incubated with exosomes from hucMSC or HFL1 (human lung fibroblast cells; as control) for 30 min, and 3-methyladenine (an autophagic inhibitor) and rapamycin (an autophagic inducer) for 1 h before cisplatin treatment for 8 h, respectively. Cells were harvested for apoptosis assay, enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, we constructed cisplatin-induced acute kidney injury rat models. Prior to treatment with cisplatin for 0.5 h, hucMSC-Ex or HFL1-Ex were injected into the kidneys via the renal capsule. 3-methyladenine and rapamycin were injected under the kidney capsule before hucMSC-Ex. All animals were sacrificed at 3 days after cisplatin injection. Renal function, Luminex assay, tubular apoptosis and proliferation, and autophagy response were evaluated.
hucMSC-Ex inhibited cisplatin-induced mitochondrial apoptosis and secretion of inflammatory cytokines in renal tubular epithelial cells in vitro. hucMSC-Ex increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in NRK-52E cells. Rapamycin mimicked the effects of hucMSC-Ex in protecting against cisplatin-induced renal injury, while the effects were abrogated by the autophagy inhibitor 3-methyladenine in the animals.
Our findings indicate that the activation of autophagy induced by hucMSC-Ex can effectively relieve the nephrotoxicity of cisplatin. Therefore, pre-treatment of hucMSC-Ex may be a new method to improve the therapeutic effect of cisplatin.
KeywordsHuman umbilical cord mesenchymal stem cell Exosome Cisplatin Nephrotoxicity Autophagy
Acute kidney injury
Blood urea nitrogen
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorting
Human fetal lung fibroblast-1
Human umbilical cord-derived mesenchymal stem cell
Human umbilical cord-derived mesenchymal stem cell exosomes
Low-glucose Dulbecco’s modified Eagle’s medium
Minimum essential medium
Mesenchymal stem cell
Mammalian target of rapamycin
Nanoparticle tracking analysis
Quantitative real-time polymerase chain reactiuon
Tumor necrosis factor
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling
Cisplatin is a platinum inorganic complex, extensively used in clinical chemotherapy for many solid tumors. Administration of a high dose of cisplatin is limited due to its nephrotoxic side effects [1, 2]. Therefore, preventive measures are needed to counteract the renal damage induced by cisplatin. It is now recognized that exosomes/microvesicles as nanoparticles are an integral part of the intercellular microenvironment , and may play a pivotal role in cell-to-cell communication . Microvesicles (MVs) are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes of most cell types. Exosomes/microvesicles may interact with target cells by surface-expressed ligands and transfer surface receptors, and deliver proteins, mRNA, and bioactive lipids between cells [5, 6]. Recently, many studies have revealed that mesenchymal stem cell (MSC)-derived exosomes or microvesicles can promote tissue regeneration. MVs from human bone marrow-derived MSCs contribute to favor functional and morphological recovery in rodent models of acute kidney injury (AKI) induced by glycerol , ischemia/reperfusion , and cisplatin . The research implies that exosomes from MSCs may be a novel stem cell-based therapy for kidney diseases. Our previous works indicate that human umbilical cord-derived MSCs (hucMSCs) improve the recovery of ischemia/reperfusion-induced rat renal injury via anti-apoptotic and anti-inflammatory mechanisms [10, 11, 12]. We also demonstrated that hucMSC-derived exosomes (hucMSC-Ex) protect against cisplatin-induced renal oxidative stress and apoptosis, alleviate CCl4-induced liver fibrosis, and enhance cutaneous wound healing [13, 14, 15, 16, 17]. All the above studies show that hucMSC-Ex can improve the tissue injury. However, it is unknown whether hucMSC-Ex administration before injury can prevent kidney damage in the early stages.
Studies have demonstrated that autophagy is critical for normal proximal tubule function and protection against acute tubular injury [18, 19]. There is a dynamic feedback between autophagy and cellular energy balance . Autophagy not only plays a principal role in the supply of nutrients for cell survival, but also plays an active role in cellular homeostasis, where it acts as a cytoplasmic quality-control regulator to eliminate long-lived or unfolded proteins and damaged organelles [21, 22]. These reports revealed that the activation of autophagy plays an important role in relieving tissue damage. Whether hucMSC-Ex can promote autophagy activation is unclear.
Herein, we show that hucMSC-Ex had a markedly preventative effect against cisplatin-induced renal toxicity, and we explored the mechanism of action. Our findings indicate that hucMSC-Ex promoted autophagy in renal tubule epithelial cells and kidney tissues by cisplatin induced through the inhibition of mTOR, thus alleviating cell apoptosis and inflammatory response in the early stages.
The study was approved by the ethical committee of Jiangsu University (2012258).
Isolation and culture of hucMSCs
After obtaining parental and ethics committee consent, the fresh umbilical cords were collected and processed within 6 h as described previously . Umbilical cords were cut into pieces and floated in low glucose Dulbecco’s modified Eagle’s medium (LG-DMEM) containing 10% fetal bovine serum (FBS; ExCell Biology, China) and 1% penicillin and streptomycin. Cord pieces were subsequently incubated at 37 °C in humid air with 5% CO2. When well-developed colonies of fibroblast-like cells reached 80% confluency, cultures were trypsinized into new flasks for further expansion. hucMSCs were identified by fluorescence-activated cell sorting (FACS) and differentiation experiments.
FACS analysis and differentiation studies of hucMSCs
hucMSCs were resuspended in phosphate-buffered saline (PBS). Cell aliquots (300 μl) were incubated with fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated monoclonal antibodies specific for CD13, CD29, CD44, CD90, CD105, HLA-1, CD45, HLA-DR, and CD34 on ice for 30 min. FITC or PE mouse nonimmune isotypic IgG were used as controls. All antibodies were purchased from BD Company. The pluripotency of hucMSCs was confirmed by their ability to differentiate into osteocytes and adipocytes, as described previously .
Isolation and characterization of exosomes from hucMSCs and HFL1 cells
As control cells, human fetal lung fibroblast-1 (HFL1) cells were obtained from Cell Bank, Type Culture Collection Committee, the Chinese Academy of Sciences (Shanghai, China). In order to allow exosome separation, HFL1 cells were cultured with serum-free α-modified minimum essential medium (α-MEM); hucMSCs were cultured with serum-free LG-DMEM. The conditioned medium was collected at 48 h. Density gradient centrifugations were executed and the procedure for the extraction and purification of exosomes was performed as described previously [13, 14]. The purified exosomes were stored at –86 °C until use. The expression of the exosomal makers CD81, CD9, and CD63 was detected using Western blot. The purified exosomes were further identified by nanoparticle tracking analysis (NTA) system (Version 2.3 Build 0006 BETA2) and transmission electron microscopy.
NTA and transmission electron microscopy
In order to analyze the characteristics of the exosomes, particle size, substantial shape, and the relative-intensity three-dimensional plot of hucMSC-Ex were tested using the NTA system. The purified hucMSC-Ex were applied to glow-discharged carbon-coated copper grids to detect the precise shape. PTA (phosphor-tungsten acid) staining was conducted for 30 min. The grids were then rinsed with droplets of deionized water and dried. Ultra-thin sections of NRK-52E cells were prepared to observe the formation of autophagic bodies. Transmission electron micrographs were recorded using a Tecnai 12 (Philips, Holland).
In vitro experiment
Normal rat kidney epithelial (NRK-52E) cells were obtained from Cell Bank (Shanghai, China). NRK-52E cells were pre-incubated with exosomes from hucMSC (200 μg/ml) or exosomes from HFL1 (200 μg/ml) for 30 min, followed by treatment with cisplatin (8 μM) for 8 h. 3-methyladenine (3MA; 1 mg/ml; Sigma, USA) or rapamycin (200 μg/ml; Sigma) was given for 0.5 h before exosomes treatment and 1 h before cisplatin treatment in NRK-52E cells, respectively.
In vivo rat model
Female Sprague-Dawley rats at 6–8 weeks old, weighing 210–250 g, were used. The animals were kept under standard laboratory conditions (12 h light/12 h dark cycle, and 21 ± 2 °C). Animals were divided into seven groups of six rats each and treated as follows: 1) control group (no cisplatin treatment); 2) PBS group (intraperitoneal injection of a single dose of 5 mg/kg cisplatin); 3) hucMSC-Ex group (0.5 h before cisplatin administration both kidneys in one rat received a renal capsule injection of 200 μg hucMSC-Ex); 4) HFL1-Ex group (0.5 h before cisplatin administration both kidneys in one rat received a renal capsule injection of 200 μg HFL1-Ex); 5) hucMSC-Ex + 3MA group (500 μg/kidney 3MA was injected under the kidney capsule before hucMSC-Ex and cisplatin administration); 6) 3MA group (500 μg/kidney 3MA was injected under the kidney capsule before cisplatin administration); and 7) Rapa group (20 μg/kidney 3MA was injected under the kidney capsule before cisplatin administration).
All animals were sacrificed at 3 days after cisplatin injection. Kidney tissue specimens were divided into two. One was quickly excised, rinsed in ice-cold saline, and fixed in 4% paraformaldehyde, and the paraffin-embedded tissues were sliced and stained with hematoxylin and eosin (H&E). Sections were analyzed and at least 20 random fields were scored by a nephropathologist in a blinded manner. Tubular cell necrosis, tubular dilation, and tubular protein casts (200× magnification) in sections were observed and analyzed. Abnormalities were graded by a semiquantitative score from 0 to 4: 0, no abnormalities; 1, changes affecting less than 25% of the sample; 2, changes affecting 25 to 50%; 3, changes affecting 50 to 75%; 4, changes affecting more than 75%. The other specimen was used immediately or frozen at –86 °C until further biochemical analysis. Blood samples were collected before and after cisplatin injection (0, 1, 2, and 3 days). Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured by an automatic biochemical analyzer (AU2700; Olympus). Serum levels of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL6 were determined by Luminex assays (Luminex 200; Millipore).
Mitochondrial membrane potential assay
NRK-52E cells seeded in 24-well plates were treated as described for the in vitro experiment. After treatment with cisplatin for 8 h, the cells were fixed with 4% paraformaldehyde. Mitochondrial membrane potential was measured by a JC-1 kit according to the manufacturer’s protocol (Beyotime, China).
RNA extraction and quantitative real-time PCR
Total RNA was extracted with Trizol reagent (Invitrogen, USA) from NRK-52E cells and kidney tissues; cDNA was synthesized using a reverse transcription kit according to the manufacturer’s instructions (Vazyme, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of ATG5, ATG7, and β-actin genes. All samples were examined in triplicate, and all reactions were repeated three times independently using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was used to detect the apoptotic cells. hucMSCs, HFL1 cells, NRK-52E cells, and the kidney tissues were examined using the TdT-FragEL DNA fragmentation detection kit according to the manufacturer's protocol (Boster, China). Positive cells were identified as dark brown nuclei under a light microscope (Nikon eclipse TE 3000-U, Japan). The number of apoptotic cells was counted in 20 randomly selected visual fields of blinded samples, at 200× magnification.
Cells, exosomes, and tissues were lysed in RIPA buffer. Protein concentration was determined using the BCA assay kit (Pierce, USA). Sources and dilution factors of primary antibodies were: rabbit polyclonal CD63 (1:1000; Bioworld, USA), CD9 (1:1000; Bioworld), CD81 (1:1000; Epitomics, USA), PCNA (1:1000; Bioworld), BCL-XL (1:100; SAB, USA), BCL-2 (1:1000; Bioworld), Bax (1:1000; Bioworld), Cytochrome C (1:500; Abcam, USA), caspase-3 (1:500; Bioworld), IL-1β (1:500; Bioworld,), LC3B (1:500; Abcam), Beclin-1 (1:600; Proteintech, USA), mTOR (1:500; SAB), p-mTOR (1:500; SAB), 4EBP1 (1:200; SAB), p70S6K (1:200; SAB), and mouse monoclonal GAPDH (1:3000; Kang Chen, China). The nucleoprotein and plasma protein was separated by the nuclear and plasma protein isolation kit (Vazyme, China), with the primary antibody NF-kB-P65 in the nucleus (1:500; SAB), primary antibody nucleoprotein Histone (1:1000; SAB). After incubation with the primary antibodies overnight at 4 °C, membranes were washed three times with Tris-buffered saline with 0.05% Tween-20 and challenged with HRP-conjugated goat anti-rabbit or goat anti-mouse antibody (1:2000; Bioworld). Western blot was performed by Luminata™ crescendo western HRP substrate (Millipore, USA) and analyzed using MD Image Quant Software.
Immunocytochemistry and immunohistochemistry
NRK-52E cells, treated as described for the in vitro experiment, were fixed in 4% paraformaldehyde and permeabilized with HEPES-Triton X100 buffer. The paraffin-embedded kidney tissue sections were dewaxed. Endogenous peroxidase activity was then inhibited by exposure to 3% hydrogen peroxide for 10 min. Subsequently, the sections were boiled for 10 min in citrate buffer (pH 6.0, 10 mM) for antigen retrieval. The sections were then blocked with 5% BSA (Boster Bioengineering, Co. Ltd., Wuhan, China) and incubated with TNF-α (1:100; Bioworld) and PCNA (1:100; Bioworld) primary antibody at 37 °C for 1 h. After the sections were washed with PBS, they were then incubated with diluted secondary antibody for 20 min. Finally, cells were visualized using diaminobenzidine (DAB) substrate and counterstained with hematoxylin for microscopic examination. The number of PCNA-positive cells were counted in 20 randomly selected visual fields of blinded samples, at 200× magnification.
Enzyme-linked immunosorbent assay
NRK-52E cells were treated as mentioned above. Culture supernatants were collected and cell debris was removed by centrifugation. Supernatants were examined using the rat TNF-α enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's protocol (ExCell Biology, China).
Female Sprague-Dawley rats were treated as mentioned above. The serum inflammatory cytokines TNF-α, IL-1β, and IL6 were measured using Luminex kits (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.
Cell transfection and structured illumination microscopy assay
NRK-52E cells were seeded in 24-well plates and cultured for 24 h, then transfected with mRFP-GFP-LC3 adenovirus according to the manufacturer's protocol (Han Heng Biology, China). Then, cells were cultured on coverslips and divided into four groups: control; PBS; pre-treated hucMSC-Ex; and pre-treated HFL1-Ex. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with HEPES-Triton X100 buffer. Finally, the cells were stained with Hoechst33342 for nuclear staining, and the images were acquired with a structured illumination microscopy (Nikon, SIM).
All data from different experiments are expressed as mean ± SD. Statistical analysis was performed by Student’s t test or by analysis of variance (ANOVA) with Newmann-Keuls multicomparison or Dunnett’s post hoc tests as appropriate. A two-tailed P value <0.05 was considered statistically significant.
Characterization of hucMSC and hucMSC-Ex
MSCs isolated from the umbilical cord were characterized by FACS analysis (see Additional file 1: Figure S1A) and induced differentiation (Additional file 1: Figure S1B and C). Exosomes were extracted and purified from hucMSC and were identified by morphology and exosomal markers. The particle size and concentration of exosomes were measured by an NTA system, peaking at 102 nm diameter (see Additional file 2: Figure S2Aa), a relative-intensity three-dimensional plot (shown in Additional file 2: Figure S2Ab), and electron micrograph of phosphotungstanic acid-stained exosomes (Additional file 2: Figure S2Ac). The results of transmission electron microscopy showed that the hucMSC-Ex had a fingerprint-like membrane structure (Additional file 2: Figure S2Ba) and a spheroid shape (Additional file 2: Figure S2Bb). The purified hucMSC-Ex expressed exosomal marker proteins such as CD81, CD9, and CD63 (Additional file 2: Figure S2C). TUNEL stain showed that the number of apoptotic cells was not significantly different between normal cultured conditions and serum-free cultured conditions at 48 h (Additional file 2: Figure S2D). Electron microscope examination showed that hucMSC-Ex expressed CD9 colloidal gold (Additional file 2: Figure S2E).
hucMSC-Ex can prevent cisplatin-induced AKI in vivo
hucMSC-Ex inhibits cisplatin-induced apoptosis and inflammatory response in vitro
hucMSC-Ex activated autophagy in vitro
hucMSC-Ex prevents cisplatin-induced apoptosis and secretion of inflammatory cytokines by activating autophagy in vitro
hucMSC-Ex-mediated activation of autophagy is required for the prevention of cisplatin-induced AKI in vivo
Increasing evidence suggests that the administration of exogenous mesenchymal stem cells (MSCs) improves the recovery of injured tissue, including acute kidney injury (AKI) [10, 11, 12, 27, 28, 29, 30, 31]. Several studies have demonstrated that the administration of MSCs could reverse kidney injury through paracrine mechanisms rather than by MSC transdifferentiation [32, 33]. MSC exosomes might be such a paracrine mechanism for cell-to-cell communication. Exosomes are small vesicles released by cells bearing the surface antigens characteristic of the cell of origin [34, 35]. These vesicles may play critical roles in cell communication by transferring RNA, proteins, and bioactive lipids . Exosomes may be more advantageous than stem cells in regenerative medicine due to the avoidance of possible long-term pathologic differentiation of engrafted cells or tumor generation [37, 38]. The release of exosomes may be constitutive, or a consequence of cell activation by soluble agonists, or physical and chemical stresses such as oxidative stress and hypoxia, or by shear stress . We demonstrated in this study that pre-treatment with hucMSC-Ex could inhibit cisplatin-induced apoptosis and the secretion of inflammatory cytokines in renal proximal tubule epithelial cells. hucMSC-Ex pretreatment suppressed the increase of BUN and serum Cr levels, as well as the deterioration of proximal tubule epithelial cells induced by cisplatin.
The role of autophagy as a degradative pathway is critical in regenerative medicine. Many reports show that basal or physiological autophagy contributes to the maintenance of cellular homeostasis and quality control of proteins and subcellular organelles. Under pathological conditions or cell stress, autophagy is induced which may serve as an adaptive and protective mechanism for cell survival. Autophagy is essential to the homeostasis and physiological function of podocytes in the kidney . Notably, autophagy induction as a self-protection mechanism has been demonstrated in renal tubular cells in experimental models of AKI caused by ischemia-reperfusion and nephrotoxicants such as cisplatin and cyclosporine [41, 42, 43, 44]. Recent studies also report that autophagy play an important role in MSC-promoted tissue regeneration [45, 46]. Baixauli et al.  note that the emerging function of exosomes as a means of alleviating intracellular stress conditions in coordination with the autophagy-lysosomal pathway is essential for preserving intracellular protein and RNA homeostasis. However, whether hucMSC-Ex can activate target cell autophagy in advance to prevent tissue injury is not reported. Here, we found that hucMSC-derived exosomes can induce target cell autophagy to prevent nephrotoxicity in the early injury stage. In our study, we found that hucMSC-Ex upregulated the expression of the autophagic marker protein LC3B through the inhibition of mTOR phosphorylation. mTOR is an evolutionarily conserved nutrient-sensing serine/threonine protein kinase, with a critical role in regulating protein synthesis and autophagy [48, 49]. mTOR signaling negatively regulates autophagy, and mTOR suppression by rapamycin contributes to the induction of autophagy . We found that hucMSC-Ex downregulated the expression of phosphorylated mTOR and changed the levels of expression of p70S6K and 4EBP1, suggesting that hucMSC-Ex may activate autophagy by inhibiting the mTOR signaling pathway.
In order to prove whether hucMSC-Ex-mediated autophagy is essential for the inhibition of apoptosis and secretion of inflammatory cytokines, we pre-treated NRK-52E cells with the specific inhibitor of autophagy, 3-methyladenine (3MA). Inhibition of autophagy reduces the effects of hucMSC-Ex on anti-apoptosis and the inhibition of inflammatory cytokines. On the other hand, the autophagic inducer rapamycin has a similar effect on renal protection to that of hucMSC-Ex both in vitro and in vivo, suggesting that induction of autophagy is essential for the protective role of hucMSC-Ex in cisplatin-induced renal injury.
This study reveals that hucMSC-derived exosomes prevent against cisplatin-induced AKI through an autophagy-related mechanism. These findings provide a basis for the future use of exosomes as a new biological therapeutic approach for renal diseases and injuries.
This work was supported by the National Natural Science Foundation of China (grant nos. 81272481, 31140063, and 30840053), the Major Research Plan of Jiangsu Higher Education (grant 15KJA320001), Jiangsu Province “333 Project” fund (grant BRA2015399), Jiangsu Province for Outstanding Sci-Tech Innovation Team in Colleges and Universities (grant SJK2013-10), the opening project of the Key Laboratory of Embryo Molecular Biology, Ministry of Health of China, and Shanghai Key Laboratory of Embryo and Reproduction Engineering (grant KF201601), and Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
The National Natural Science Foundation of China (81272481); the National Natural Science Foundation of China (31140063); and the National Natural Science Foundation of China (30840053).
Availability of data and materials
BW: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing. HJ: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing. BZ: collection and/or assembly of data and data analysis, and manuscript writing. JW: collection and/or assembly of data and data analysis. CJ: data analysis and interpretation. XZ: provision of study material and interpretation. YY: data analysis and interpretation. LY: collection and/or assembly of data. JY: data analysis and interpretation. HQ: study design, data analysis and interpretation, manuscript writing, and final approval of manuscript. WX: study design, data analysis and interpretation. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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Ethical approval and consent to participate
The study was approved by the ethical committee of Jiangsu University (2012258).
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