miR-21–Containing Microvesicles from Injured Tubular Epithelial Cells Promote Tubular Phenotype Transition by Targeting PTEN Protein

Renal fibrosis is inevitably progressive no matter what the initial insult is or whether the insult persists. In an experimental fibrosis model induced by unilateral ureteral obstruction, the accelerated pathological changes could hardly be explained by aggravated pressure caused by hydronephrosis after ligation. Moreover, at the initial stage, tubular phenotype transition and matrix deposition in obstructive kidneys are always local and scattered; however, these renal lesions expand and progress with time. In this study, cultured recipient tubular cells underwent phenotype transition after incubation with conditioned media derived from transforming growth factor-β1–treated donor tubular cells. Thus, it is reasonable to speculate that some secretable molecules from injured tubules contribute to the progression of renal fibrosis. Herein, we report that secreted miRNA-21 (miR-21) can serve as the molecule mediating intercellular communication. miR-21 was packaged into microvesicles, which enter and deliver miR-21 into recipient tubular cells, and exogenous miR-21 enhances Akt signaling by target depression of phosphatase and tensin homolog (PTEN) protein, and promotes tubular phenotype transition. These results demonstrate that tubular cells can secrete miR-21 and deliver it into recipient tubules by microvesicles, where the exogenous miR-21 can target PTEN protein and enhance Akt signaling in recipient cells. Microvesicle-mediated delivery of miR-21 among tubular epithelial cells might shed new light on the mechanism of progressive renal fibrosis. Renal fibrosis is inevitably progressive no matter what the initial insult is or whether the insult persists. In an experimental fibrosis model induced by unilateral ureteral obstruction, the accelerated pathological changes could hardly be explained by aggravated pressure caused by hydronephrosis after ligation. Moreover, at the initial stage, tubular phenotype transition and matrix deposition in obstructive kidneys are always local and scattered; however, these renal lesions expand and progress with time. In this study, cultured recipient tubular cells underwent phenotype transition after incubation with conditioned media derived from transforming growth factor-β1–treated donor tubular cells. Thus, it is reasonable to speculate that some secretable molecules from injured tubules contribute to the progression of renal fibrosis. Herein, we report that secreted miRNA-21 (miR-21) can serve as the molecule mediating intercellular communication. miR-21 was packaged into microvesicles, which enter and deliver miR-21 into recipient tubular cells, and exogenous miR-21 enhances Akt signaling by target depression of phosphatase and tensin homolog (PTEN) protein, and promotes tubular phenotype transition. These results demonstrate that tubular cells can secrete miR-21 and deliver it into recipient tubules by microvesicles, where the exogenous miR-21 can target PTEN protein and enhance Akt signaling in recipient cells. Microvesicle-mediated delivery of miR-21 among tubular epithelial cells might shed new light on the mechanism of progressive renal fibrosis. Renal fibrosis is an inevitable outcome of nearly all kinds of chronic kidney disease.1Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis.J Am Soc Nephrol. 2010; 21: 212-222Crossref PubMed Scopus (738) Google Scholar, 2Zeisberg M. 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Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition.J Clin Invest. 2007; 117: 3810-3820PubMed Google Scholar is one of the contributors to renal fibrogenesis; however, similarly as observed in clinical settings, injured tubules are always local and scattered at the initial stages and then expand and progress in murine obstructive kidneys. It indicates that injured tubules might influence the normal ones, which results in the progression of renal lesions. microRNA (miRNA), discovered as a family of noncoding RNAs, is responsible for regulating the expressions of up to 30% of mammalian protein-encoding genes. It has been proved in recent years that most proteins involved in renal fibrosis were regulated by miRNA,12Chandrasekaran K. Karolina D.S. Sepramaniam S. Armugam A. Wintour E.M. Bertram J.F. Jeyaseelan K. 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Microvesicles (MVs) are circular fragments of membrane that are released from the endosomal compartment as exosome or shed from the surface membranes of almost all kinds of cell types under both normal and pathological conditions.15Cocucci E. Racchetti G. Rupnik M. Meldolesi J. The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4.J Cell Sci. 2008; 121: 2983-2991Crossref PubMed Scopus (48) Google Scholar, 16Thery C. Zitvogel L. Amigorena S. Exosomes: composition, biogenesis and function.Nat Rev Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3968) Google Scholar, 17Peinado H. Alečković M. Lavotshkin S. Matei I. Costa-Silva B. Moreno-Bueno G. Hergueta-Redondo M. Williams C. García-Santos G. Ghajar C. Nitadori-Hoshino A. Hoffman C. Badal K. Garcia B.A. Callahan M.K. Yuan J. Martins V.R. Skog J. Kaplan R.N. Brady M.S. Wolchok J.D. Chapman P.B. Kang Y. Bromberg J. Lyden D. 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Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells.Kidney Int. 2012; 82: 412-427Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 24Gallo A. Tandon M. Alevizos I. Illei G.G. The majority of microRNAs detectable in serum and saliva is concentrated in exosomes.PLoS One. 2012; 7: e30679Crossref PubMed Scopus (848) Google Scholar We determine whether MV-mediated delivery of miR-21 regulates progression of renal fibrosis. In this study, we report that secreted miR-21 can serve as the molecule mediating intercellular communication. miR-21 was packaged into MVs, which enter and deliver miR-21 into recipient tubular cells, and exogenous miR-21 enhances Akt signaling by target depression of PTEN protein, and promotes tubular phenotype transition. These results demonstrate that tubular cells can secrete miR-21 and deliver it into recipient tubules by MVs, where the exogenous miR-21 can target PTEN protein and enhance Akt signaling in recipient cells. MV-mediated delivery of miR-21 among tubular epithelial cells might shed a new light on the mechanism of progressive renal fibrosis. A rat renal proximal tubular epithelial cell line (NRK-52E) was purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (Shanghai, China), which was originally obtained from ATCC (Manassas, VA; CRL-1571TM). Cells were cultured in Dulbecco's modified Eagle's medium–F12 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). For TGF-β1 treatment, NRK-52E cells were seeded at 80% confluence in complete medium containing 10% fetal bovine serum. Twenty-four hours later, the cells were changed to serum-free medium and incubated for 16 hours. Then, cells were treated with recombined human TGF-β1 (rhTGF-β1; R&D Systems, Minneapolis, MN) at a dose of 5 ng/mL. Conditioned medium was generated as follows: donor NRK-52E cells were incubated with rhTGF-β1 for 48 hours. The culture media were then changed into normal Dulbecco's modified Eagle's medium–F12 medium (without serum) to exclude the influence of rhTGF-β1 and incubated for another 48 hours. The later rhTGF-β1–free media were then collected and centrifuged at 300 × g, 1200 × g, and 10,000 × g for 5, 20, and 30 minutes separately, respectively, to harvest conditioned media. Meanwhile, the control medium was generated in the same procedure from donor cells that were not incubated with rhTGF-β1 in the first 48 hours. The recipient normal tubular cells were then incubated with control or conditioned media for various time periods or doses, as indicated, and then collected for further characterization. TGF-β neutralization antibody was obtained from R&D Systems. A matrix metalloprotease (MMP) inhibitor, C20H28N4O4, was obtained from Sigma-Aldrich (St. Louis, MO; M5939), which inhibits a variety of MMPs. Wortmannin and LY294002 were obtained from Sigma-Aldrich. NRK-52E cells were lysed with SDS sample buffer. The supernatants were collected after centrifugation at 13,000 × g at 4°C for 20 minutes. Protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich), and whole cell lysates were mixed with an equal amount of 2× SDS loading buffer. Samples were heated at 100°C for approximately 5 to 10 minutes before loading and were separated on precasted 10% or 5% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). Detection of protein expression by using Western blot analysis was performed according to the established protocols described previously.6Yang J. Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar The primary antibodies were as follows: anti–E-cadherin (BD Transduction Laboratories, Franklin Lakes, NJ); anti–α-smooth muscle actin (α-SMA), anti-actin, and anti-fibronectin (Sigma-Aldrich); anti–phospho-Smad3, anti-Smad3, anti-PTEN, anti-phospho Akt (Ser473), and anti-Akt (Cell Signaling Technology, Danvers, MA). Quantification was performed by measurement of the intensity of the signals with the use of ImageJ (NIH, Bethesda, MD). Indirect immunofluorescent staining was performed as previously described.6Yang J. Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar Briefly, cells cultured on coverslips were washed twice with cold PBS and fixed with cold methanol/acetone (1:1) for 10 minutes at −20°C. After three extensive washings with PBS, the cells were blocked with 0.1% Triton X-100 and 2% normal donkey serum in PBS buffer for 40 minutes at room temperature and then incubated with the specific primary antibodies previously described, followed by staining with fluorescein isothiocyanate–conjugated secondary antibody. Cells were double stained with DAPI to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i Epi-fluorescence microscope equipped with a digital camera (DS-Ri1; Nikon, Tokyo, Japan). In each experimental setting, immunofluorescent images were captured with identical exposure settings. MVs were isolated from conditioned media by differential centrifugation, according to previous publications.18Skog J. Wurdinger T. van Rijn S. Meijer D.H. Gainche L. Sena-Esteves M. Curry Jr., W.T. Carter B.S. Krichevsky A.M. Breakefield X.O. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3876) Google Scholar, 19Valadi H. Ekstrom K. Bossios A. Sjostrand M. Lee J.J. Lotvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (9455) Google Scholar, 20Zhang Y. Liu D. Chen X. Li J. Li L. Bian Z. Sun F. Lu J. Yin Y. Cai X. Sun Q. Wang K. Ba Y. Wang Q. Wang D. Yang J. Liu P. Xu T. Yan Q. Zhang J. Zen K. Zhang C.Y. Secreted monocytic miR-150 enhances targeted endothelial cell migration.Mol Cell. 2010; 39: 133-144Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar Briefly, after removing cells and other debris by centrifugation at 300 × g, 1200 × g, and 10,000 × g for 5, 20, and 30 minutes, respectively, the supernatant was centrifuged at 110,000 × g for 1 hour (all steps were performed at 4°C). MVs were collected from the pellet and resuspended in fetal bovine serum–free media. Total RNA of MVs derived from cells was then extracted using TRIzol LS reagent (Invitrogen). Cryoelectron microscopy and transmission electron microscopy were performed as previously described.20Zhang Y. Liu D. Chen X. Li J. Li L. Bian Z. Sun F. Lu J. Yin Y. Cai X. Sun Q. Wang K. Ba Y. Wang Q. Wang D. Yang J. Liu P. Xu T. Yan Q. Zhang J. Zen K. Zhang C.Y. Secreted monocytic miR-150 enhances targeted endothelial cell migration.Mol Cell. 2010; 39: 133-144Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar Briefly, for cryoelectron microscopy, a droplet of resuspended MV at the appropriate concentration was applied to carbon-coated holey film supported by a copper grid. After removing excess fluid by blotting, the grid was frozen by plunging it into liquid ethane. The vitrified specimens were stored in liquid nitrogen and transferred into a transmission electron microscope using a Gatan 626 cryoholder (Thomson Scientific Instruments Pty Ltd, Carlton, Australia). The specimens were imaged on an FEI (Burlington, VT) Tecnai 20 electron microscope with source LaB6, operated at 120 kV. The images were recorded by a Gatan UltraScan 894 charge-coupled device (Gatan, Warrandale, PA) in low-dose mode (30e/A2). For conventional transmission electron microscopy, the MV pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed. Samples were rinsed and postfixed in 1% osmium tetroxide. The samples were then embedded, fixed, and cut into several blocks (<1 mm3). The samples were dehydrated in increasing concentrations of alcohol and infiltrated with increasing concentrations of Quetol-812 epoxy resin (Nisshin EM, Tokyo, Japan) mixed with propylene oxide. Samples were embedded in pure, fresh, Quetol-812 epoxy resin and polymerized. Sections (100 nm thick) were cut using a Leica (Solms, Germany) UC6 ultramicrotome and poststained with uranyl acetate for 10 minutes and with lead citrate for 5 minutes at room temperature before observation in an FEI Tecnai T20 transmission electron microscope, operated at 120 kV. NRK-52E cells were labeled with Dil-C18 (a gift from Prof. Chen-yu Zhang, Nanjing University, Nanjing, Jiangsu, China) for 1 hour and then washed three times with PBS. The conditioned media, prepared as previously described, were collected and centrifuged to harvest MVs. MVs were resuspended in Dulbecco's modified Eagle's medium–F12 medium and incubated with cultured recipient NRK-52E cells. After incubation for various time periods, as indicated, cells were washed, fixed, and observed under a Nikon Eclipse 80i Epi-fluorescence microscope equipped with a digital camera. In each experimental setting, immunofluorescent images were captured with identical exposure settings. The procedure was conducted as previously described.25Chen X. Li Q. Wang J. Guo X. Jiang X. Ren Z. Weng C. Sun G. Wang X. Liu Y. Ma L. Chen J.Y. Wang J. Zen K. Zhang J. Zhang C.Y. Identification and characterization of novel amphioxus microRNAs by Solexa sequencing.Genome Biol. 2009; 10: R78Crossref PubMed Scopus (137) Google Scholar The complementary probes (in triplicate) against miRNAs were designed based on miRBase release 12.0 (http://www.mirbase.org). RNA labeling, microarray hybridization, and array scanning were performed as follows: briefly, 25 μg of total RNA was used to isolate low–molecular-weight RNA using polyethylene glycol solution precipitation. Subsequently, low–molecular-weight RNAs were labeled with Cy3 and hybridized with miRNA microarray (CapitalBio Corp, Beijing, China). Finally, hybridization signals were detected and quantified. Four independent adult amphioxus RNA samples were hybridized with miRNA microarray separately. Hybridization intensity values from individual amphioxus samples were filtered and global median normalized. We considered candidate miRNAs with a signal >3000 and P < 0.001 from a Student's test (compared with blank spotting solution) to be positive. The miRNAs were quantified as previously described, with minor modification.26Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. Lao K.Q. Livak K.J. Guegler K.J. Real-time quantification of microRNAs by stem-loop RT-PCR.Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (4152) Google Scholar Total RNA was prepared using a TRIzol isolation system, according to the instructions by the manufacturer (Invitrogen). To generate an miRNA cDNA library, the first strand of cDNA was synthesized using 1 μg of RNA in 20 μL of reaction buffer using miScript RT II buffer (Qiagen, Düsseldorf, Germany). The mix was incubated at 37°C for 60 minutes, followed by 95°C for 5 minutes. Subsequently, real-time quantification was performed using an Applied Biosystems 7300 Sequence Detection system (Applied Biosystems, Inc., Carlsbad, CA). The 20-μL PCR reaction system consisted of 1 μL RT product, 2 μL 10× miScript Universal Primer, 2 μL 10× miScript Primer Assay, 10 μL 2× QuantiTect SYBR Green PCR Master Mix, and RNase-free water (Qiagen). The mixtures were incubated at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All reactions were run in triplicate. The CT data were determined using default threshold settings, and the mean CT was calculated from the triplicate PCRs. The ratio of miRNAs in kidney with unilateral ureteral obstruction (UUO)/those in sham-control was calculated by using the equation 2-ΔCT, in which ΔCT=CTTreatment−CTSham-Control. All primers were purchased from Qiagen. U6 was used for normalization in miRNA quantitative PCR (qPCR) when total RNA was extracted from cell or tissue samples. The miRNA in situ hybridization (ISH) was performed using a mercury LNATM microRNA ISH optimization kit (Exiqon, Vedbaek, Denmark) for formalin-fixed, paraffin-embedded kidney samples, according to the protocol by the manufacturer. Briefly, sections (10 μm thick) were prepared, followed by deparaffinization in xylene and ethanol. The slides were incubated with 15 mg/mL of proteinase-K (Exiqon) for 20 minutes at 37°C. After washing and dehydrating, the slides were hybridized with double digoxigenin-labeled, LNATM miR-21 probe, LNATM-scrambled miRNA probe, LNATM U6 snRNA probe, and LNATM miR-126 probe (positive control) (Exiqon) for 1 hour at 55°C. The slides were washed with standard saline citrate buffer, and then incubated with blocking solution for 15 minutes, followed by incubation with anti-digoxigenin reagent for 60 minutes, alkaline phosphatase substrate for 2 hours at 30°C, and KTBT buffer twice for 5 minutes. The slides were mounted with mounting medium, and the results were analyzed by light microscopy (Nikon Eclipse 80i). Urine samples of obstructive kidney and sham-control were collected at different time points, as indicated. An equal volume of 200 μL of urine from each group was applied for total RNA isolation, and acidic phenol was used, followed by chloroform/isopropanol purification. The quantity of total RNA was then determined and normalized. A total of 1 μg of RNA was applied for reverse transcription to generate a urine miRNA cDNA library, using an miScript II RT kit (Qiagen) in 20 μL of reaction buffer. The mix was incubated at 37°C for 60 minutes, followed by 95°C for 5 minutes. Subsequently, real-time quantification was performed as previously described. The products were then size fractionated on agarose gel. miRNA-21 mimic, inhibitor, and their negative control (NC) RNA (purchased from Qiagen) were transfected into NRK-52E cells using Lipofectamine 2000 reagent (Invitrogen) following the protocols provided by the manufacturer. After transfection, cells were incubated at 37°C in a CO2 incubator for 24 hours until they were ready to assay for gene expression or further treatment. Male CD-1 mice, weighing 18 to 20 g, were purchased from the Shanghai Experimental Animal Center. They were housed in the animal facilities of the Experimental Animal Center of Nanjing Medical University with free access to food and water. Animals were treated humanely in accordance with National Medical Advisory Committee guidelines and by use of approved procedures of the Institutional Animal Use and Care Committee at the Nanjing Medical University. CD-1 mice were randomly assigned into five groups (with five mice per group): control group and UUO for 1, 3, 7, and 14 days groups. UUO was performed using an established procedure.6Yang J. Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar Briefly, under general anesthesia, complete left ureteral obstruction was performed by double ligation of the left ureter using 4-0 silk after a midline abdominal incision. Animals in the sham-control group had their ureter exposed and manipulated, but not ligated. Mice were euthanized at different time points, as indicated after surgery, and the obstructive kidneys were removed for further investigation. Kidney tissues were immersed in 4% neutral-buffered formaldehyde at 4°C for 48 hours. The tissues were paraffin embedded, processed for light microscopy, and divided into sections (3 μm thick). Sections were then stained with H&E for general histological analysis and Masson for extracellular matrix deposition. Pictures were taken with a Nikon Eclipse 80i microscope equipped with a digital camera. Kidney sections (3 μm thick) were deparaffinized and rehydrated by xylene, a graded alcohol series, and double-deionized water. Briefly, after blocking for 30 minutes at room temperature with blocking buffer, sections were incubated with anti-collagen I antibody (Santa Cruz Biotechnology, Santa Cruz, CA), then with horseradish peroxidase–conjugated secondary antibodies. Animals were randomly assigned to control and treatment groups. Statistical analysis was performed using SigmaStat software version 3.5 (Jandel Scientific Software, San Rafael, CA). Comparisons between groups were made using one-way analysis of variance, followed by the t-test. P < 0.05 was considered significant. In mice with obstructive kidney induced by UUO, the expansion rate of matrix deposition area was markedly accelerated in the later 4-day time compared with the initial 3-day time (Supplemental Figure S1), suggesting an unknown internal mechanism, rather than increased hydrostatic pressure promoting renal fibrosis progression. Tubular epithelial cells and tubulointerstitial spaces are the major locations of renal lesions. We, therefore, examined whether injured tubular cells were capable of promoting normal congener cells to undergo phenotype transition. The control and conditioned media were generated as described in Materials and Methods. Despite the removal of rhTGF-β1 for 48 hours, the donor tubular cells underwent phenotype transition when the conditioned media were harvested (data not shown). As shown in Figure 1A, after incubation with the conditioned media for various time periods, as indicated, normal NRK-52E cells were induced to undergo phenotype transition in a time-dependent manner, as demonstrated by the loss of E-cadherin, de novo expression of α-SMA, and fibronectin. The conditioned media were then used to incubate normal NRK-52E cells in different doses, as indicated, for 48 hours. Western blot analysis also shows that the recipient tubular cells underwent phenotype transition in a dose-dependent manner (Figure 1B). Immunofluorescent staining shows the phenotype transition of tubular epithelial cells after treatment with the conditioned media for 48 hours, which exhibited as total loss of the E-cadherin staining (Figure 1, C and F). Meanwhile, the α-SMA–positive microfilaments (Figure 1, D and G) appeared in the cytoplasm, and fibronectin was deposited in the intercellular area (Figure 1, E and H). All together, these data suggest that some secreted molecules from injured tubular cells probably mediated tubular epithelial cell-to-cell communication. The conditioned media of rhTGF-β1–treated NRK-52E cells should be a mixture of various ingredients, among which endogenously produced TGF-β1 and MMPs are probably two major profibrogenesis factors. We first investigated whether de novo–produced TGF-β1 by NRK-52E cells after rhTGF-β1 treatment accounted for the phenotype transition of epithelial cells. The rhTGF-β1–free conditioned media were collected as previously described. The concentration of TGF-β1 in the conditioned media was undetectable (data not shown). Neutralizin