Transfer of microRNA-486-5p from human endothelial colony forming cell–derived exosomes reduces ischemic kidney injury
Authors: Jose L. Viñas, Dylan Burger, Joseph Zimpelmann, Randa Haneef, William Knoll, Pearl Campbell, Alex Gutsol, Anthony Carter, David S. Allan, Kevin D. Burns
Journal: Kidney International (2016)

Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Akt phosphorylation. Exposure of hypoxic endothelial cells to conditioned medium from ECFCs pretreated with anti–miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti–miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury. Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Akt phosphorylation. Exposure of hypoxic endothelial cells to conditioned medium from ECFCs pretreated with anti–miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti–miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury. Ischemia/reperfusion (IR) is a major cause of acute kidney injury (AKI) in humans, and is associated with tubular cell necrosis and endothelial cell dysfunction and loss.1Basile D.P. The endothelial cell in ischemic acute kidney injury: implications for acute and chronic function.Kidney Int. 2007; 72: 151-156Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 2Basile D.P. Anderson M.D. Sutton T.A. Pathophysiology of acute kidney injury.Compr Physiol. 2012; 2: 1303-1353Crossref PubMed Scopus (665) Google Scholar, 3Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1326) Google Scholar No treatments have been shown to accelerate kidney repair in humans. In experimental AKI administration of certain stem or progenitor cells improves kidney histologic and functional recovery with no requirement for cell engraftment, indicating that repair pathways may be activated by paracrine factors released by the cells.4Morigi M. Benigni A. Mesenchymal stem cells and kidney repair.Nephrol Dial Transplant. 2013; 28: 788-793Crossref PubMed Scopus (83) Google Scholar, 5Patschan D. Patschan S. Muller G.A. Endothelial progenitor cells in acute ischemic kidney injury: strategies for increasing the cells' renoprotective competence.Int J Nephrol. 2011; 2011 ([e-pub ahead of 2011 Apr 27]) (Accessed May 6, 2015): 828369http://dx.doi.org/10.4061/2011/828369Crossref PubMed Google Scholar, 6Togel F.E. Westenfelder C. 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Tada T. et al.Endothelial dysfunction in ischemic acute renal failure: rescue by transplanted endothelial cells.Am J Physiol Renal Physiol. 2002; 282: F1140-F1149Crossref PubMed Scopus (258) Google Scholar In this regard, endothelial colony-forming cells (ECFCs) are early lineage endothelial progenitors that can be isolated from human cord blood or peripheral blood.8Yoder M.C. Mead L.E. Prater D. et al.Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.Blood. 2007; 109: 1801-1809Crossref PubMed Scopus (1241) Google Scholar We recently demonstrated that administration of human cord blood ECFCs to nonobese diabetic severe combined immunodeficient (NOD-SCID) mice with IR AKI exerted protective effects, with diminished levels of tubular necrosis, apoptosis, oxidative stress, and inflammatory cell infiltrate.9Burger D. Viñas J.L. Akbari S. et al.Human endothelial colony forming cells protect against acute kidney injury: role of exosomes.Am J Pathol. 2015; 185: 2309-2323Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Notably, delivery of small extracellular vesicles (exosomes) derived from ECFCs to mice also protected against kidney injury and inhibited apoptosis in cultured endothelial cells exposed to hypoxia/reoxygenation (HR).9Burger D. Viñas J.L. Akbari S. et al.Human endothelial colony forming cells protect against acute kidney injury: role of exosomes.Am J Pathol. 2015; 185: 2309-2323Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar These findings are consistent with the beneficial effects of stem cell– or progenitor cell–derived vesicles on kidney recovery in other rodent models of AKI.10Bruno S. Grange C. Deregibus M.C. et al.Mesenchymal stem cell-derived microvesicles protect against acute tubular injury.J Am Soc Nephrol. 2009; 20: 1053-1067Crossref PubMed Scopus (999) Google Scholar, 11Bruno S. Grange C. Collino F. et al.Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury.PLoS One. 2012; 7: e33115Crossref PubMed Scopus (478) Google Scholar, 12Cantaluppi V. Gatti S. Medica D. et al.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 (416) Google Scholar, 13Sanchez M.B. Bruno S. Grange C. et al.Human liver stem cells and derived extracellular vesicles improve recovery in a murine model of acute kidney injury.Stem Cell Res Ther. 2014; 5: 124Crossref PubMed Scopus (78) Google Scholar MicroRNAs (miRNAs) are 18- to 25-nucleotide RNA species that have profound effects on protein translation via binding to complementary regions of mRNA, usually in the 3′ untranslated region (UTR).14Mendell J.T. Olson E.N. MicroRNAs in stress signaling and human disease.Cell. 2012; 148: 1172-1187Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar Extracellular vesicles selectively package miRNA species, and the transfer of miRNAs from cell-derived vesicles to recipient cells can regulate protein expression and contribute to reparative signaling responses.15Das S. Halushka M.K. Extracellular vesicle microRNA transfer in cardiovascular disease.Cardiovasc Pathol. 2015; 24: 199-206Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar In experimental AKI, depletion of proangiogenic miR-126 and miR-296 from extracellular vesicles derived from human endothelial progenitor cells inhibited their kidney reparative effects in vivo and reduced their antiapoptotic effects in cultured cells.12Cantaluppi V. Gatti S. Medica D. et al.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 (416) Google Scholar In mice with glycerol-induced AKI, global depletion of miRNAs from mesenchymal stromal cell–derived extracellular vesicles by Drosha knockdown blocked their beneficial effects on kidney functional and morphologic recovery.16Collino F. Pomatto M. Camussi G. et al.AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs.J Am Soc Nephrol. 2015; 26: 2349-2360Crossref PubMed Scopus (183) Google Scholar Thus, miRNA transfer from extracellular vesicles may be involved in accelerating repair after AKI. In the current studies, we determined the role of exosomal miRNA transfer in the protective effects on IR injury. Using Next Generation Sequencing, we defined the differential miRNA expression pattern in 2 classes of ECFC-derived extracellular vesicles: exosomes (40- to 100-nm diameter) and microparticles (MPs, 100- to 1000-nm diameter). Our results indicate an abundance of miR-486-5p within ECFC exosomes and suggest a role in recovery from IR injury in vivo and protection against endothelial cell apoptosis in vitro via miRNA transfer and targeting of phosphatase and tensin homolog (PTEN), with activation of Akt. We first characterized small RNA libraries from ECFCs and their exosomes and MPs. Exosomes had a mean diameter of 91 nm and expressed the surface marker TSG101, whereas MPs had a mean diameter of 224 nm and lacked TSG101 (Figure 1). After eliminating transfer RNA, 158 miRNAs were isolated by Next Generation Sequencing. Although the majority of miRNAs was common to vesicles and ECFCs, a smaller fraction was unique to exosomes, MPs, or ECFCs (Supplementary Figure S1). Exosome and MP miRNAs were ranked according to enrichment based on data from 2 independent sets of miRNA libraries. In ECFC exosomes, miR-486-5p was found at levels 289-fold higher than in MPs (and 30-fold higher than ECFC levels) (Figure 2). The next highest differentially expressed exosome miRNAs were found at levels ∼4-fold higher than in MPs. The differential levels of miR-486-5p between exosomes and MPs were also evident by real-time polymerase chain reaction (PCR) (P < 0.001) (Figure 2). Further studies therefore focused on the role of exosomal miR-486-5p in ischemic injury.Figure 2Endothelial colony-forming cell (ECFC) exosomes are highly enriched in miR-486-5p. (a) Table shows the 10 most abundant microRNAs (miRNAs) in ECFC exosomes compared with microparticles (MP). (b) miR-486-5p levels in ECFC exosomes (EX) and MP, by real-time polymerase chain reaction. *P <0 .001; n = 3.View Large Image Figure ViewerDownload (PPT) In cultured human umbilical vein endothelial cells (HUVECs) exposed to HR, administration of ECFC-derived conditioned medium or exosomes significantly increased miR-486-5p levels, whereas conditioned medium from ECFCs transfected with antagomiR to miR-486-5p blocked this increase (Figure 3a). PTEN, a potential target of miR-486-5p, is involved in apoptotic signaling through the prosurvival Akt pathway.17Small E.M. O'Rourke J.R. Moresi V. et al.Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486.Proc Natl Acad Sci U S A. 2010; 107: 4218-4223Crossref PubMed Scopus (318) Google Scholar, 18Alexander M.S. Casar J.C. Motohashi N. et al.MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy-associated symptoms.J Clin Invest. 2014; 124: 2651-2667Crossref PubMed Scopus (105) Google Scholar HR significantly increased PTEN expression, an effect blocked by ECFC-derived exosomes (Figure 3b). Phosphorylation of Akt was not significantly altered in HUVECs exposed to HR. However, administration of ECFC-derived exosomes stimulated Akt phosphorylation (Figure 3c). We reported that ECFC-derived exosomes (but not MPs) inhibit caspase-3 activation induced by HR in HUVECs.9Burger D. Viñas J.L. Akbari S. et al.Human endothelial colony forming cells protect against acute kidney injury: role of exosomes.Am J Pathol. 2015; 185: 2309-2323Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar To determine the potential role of miR-486-5p transfer from ECFC exosomes in endothelial protection, HUVECs were subjected to HR and exposed to conditioned medium from ECFCs that had been transfected with or without antagomiR to miR-486-5p. As shown in Figure 4a, HR-stimulated PTEN expression was blocked by ECFC-derived conditioned medium. By contrast, conditioned medium from ECFCs transfected with antagomiR to miR-486-5p failed to inhibit HR-mediated increases in PTEN. ECFC-derived conditioned medium increased Akt phosphorylation in HUVECs subjected to HR, whereas conditioned medium from ECFCs transfected with antagomiR to miR-486-5p blocked this effect (Figure 4b). HR induced a significant increase in caspase-3 activity in HUVECs, and ECFC-derived conditioned medium inhibited this effect (Figure 4c). On the other hand, conditioned medium from ECFCs transfected with antagomiR to miR-486-5p did not affect HR-stimulated caspase-3 activity. The transfer of miR-486-5p was further studied in co-cultures of ECFCs and HUVECs, exposed to either the inhibitor of exosome secretion GW4869,19Kosaka N. Iguchi H. Yoshioka Y. et al.Secretory mechanisms and intercellular transfer of microRNAs in living cells.J Biol Chem. 2010; 285: 17442-17452Crossref PubMed Scopus (1504) Google Scholar, 20Alexander M. Hu R. Runtsch M.C. et al.Exosome-delivered microRNAs modulate the inflammatory response to endotoxin.Nat Commun. 2015; 6: 17321Crossref Scopus (518) Google Scholar or the inhibitor of exosome uptake 5-(N-ethyl-N-isopropyl)amiloride (EIPA).21Khalil I.A. Kogure K. Akita H. Harashima H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery.Pharmacol Rev. 2006; 58: 32-45Crossref PubMed Scopus (1064) Google Scholar, 22Taverna S. Amodeo V. Saieva L. et al.Exosomal shuttling of miR-126 in endothelial cells modulates adhesive and migratory abilities of chronic myelogenous leukemia cells.Mol Cancer. 2014; 13: 169Crossref PubMed Scopus (112) Google Scholar, 23Tian T. Zhu Y.L. Zhou Y.Y. et al.Exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating miR-21 delivery.J Biol Chem. 2014; 289: 22258-22267Crossref PubMed Scopus (424) Google Scholar Significant increases in HUVEC levels of miR-486-5p were observed in co-culture compared with HUVECs cultured alone (Figure 5). Incubation of co-cultured cells with GW4869 or EIPA blocked this increase. Transfection of ECFCs with pre–miR-486-5p (which requires processing to generate mature miR-486-5p) before co-culture with HUVECs caused marked increases in HUVEC miR-486-5p levels (∼700-fold), which were significantly inhibited by GW4869 or EIPA. Tracking of ECFC-derived exosomes into HUVECs was studied using the red fluorescent dye PKH26. In phalloidin-stained HUVECs incubated with exosomes prelabeled with PKH26, red fluorescent particles were detected within cytoplasm by confocal microscopy (Figure 6). Incubation of HUVECs with PKH-labeled exosomes in the presence of EIPA significantly inhibited cytoplasmic red fluorescent staining, whereas GW4869 had no effect on uptake of exosomes. To test whether exosomal miR-486-5p directly targets PTEN in HUVECs, a dual-reporter luciferase assay was performed, using a reporter vector containing a complementary miR-486-5p sequence in its 3′-UTR (Figure 7a). When HUVECs transfected with the reporter vector were incubated with ECFC exosomes or with miR-486-5p mimic, luciferase activity was significantly reduced (Figure 7b). The inhibitory effect of exosomes on HUVEC luciferase activity was blocked when HUVECs were first transfected with antagomiR to miR-486-5p. In contrast, neither exosomes nor transfection of HUVECs with a miR-486-5p mimic reduced HUVEC luciferase activity when cells were transfected with a mutated vector of the PTEN 3′-UTR (Figure 7c). To determine the role of PTEN in endothelial cell apoptosis, experiments were conducted in HUVECs in which PTEN expression was decreased by small interfering RNA (siRNA) before exposure to HR and treatment with ECFC exosomes. Transfection of HUVECS with siRNA to PTEN reduced PTEN protein expression (Supplementary Figure S2) and eliminated caspase-3 activation in response to HR. Furthermore, in HUVECs with knockdown of PTEN, exosomes had no effect on caspase-3 activation in response to HR (Figure 8). In separate experiments, transfection of HUVECs with scrambled siRNA or incubation with the transfection vehicle Lipofectamine (Life Technologies Inc., Carlsbad, CA) had no effect on caspase-3 activation in response to HR (data not shown, n = 3). The potential impact of exosomal miR-486-5p transfer in protection from kidney IR injury in vivo was studied in immunocompetent FVB mice. Mice were subjected to kidney IR (or sham surgery) and treated with or without ECFC exosomes by jugular venous infusion at reperfusion. One group of mice received exosomes derived from ECFCs that were first transfected with antagomiR to miR-486-5p, which resulted in significant depletion of exosomal miR-486-5p levels (Supplementary Figure S3). Kidney levels of miR-486-5p in FVB mice tended to increase 24 hours after IR, although this did not reach statistical significance. Exosome administration caused a further significant increase (Figure 9). By contrast, treatment with exosomes derived from ECFCs transfected with antagomiR did not induce a change in kidney miR-486-5p levels. Kidney PTEN expression was significantly increased after IR, and exosome administration inhibited expression. This inhibition was not observed in mice treated with exosomes from ECFCs transfected with antagomiR. Kidney Akt phosphorylation was increased in mice treated with exosomes, but not in mice that received exosomes from antagomiR-transfected ECFCs. We previously showed that administration of ECFCs or their exosomes to immunoincompetent NOD-SCID mice with kidney IR injury has renal functional and histologic protective effects.9Burger D. Viñas J.L. Akbari S. et al.Human endothelial colony forming cells protect against acute kidney injury: role of exosomes.Am J Pathol. 2015; 185: 2309-2323Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Accordingly, we measured levels of miR-486-5p, PTEN, and phosphorylated Akt in kidneys from these mice. Kidney miR-486-5p levels increased significantly 24 hours after IR in NOD-SCID mice, and treatment with ECFCs or exosomes after IR induced further significant increases (Supplementary Figure S4). As with FVB mice, exosomes (and ECFCs) inhibited kidney PTEN expression and stimulated Akt phosphorylation in NOD-SCID mice compared with IR alone. As shown in Figure 10, administration of exosomes to FVB mice with IR caused potent protection against kidney injury after 24 hours, as evidenced by normalization of plasma creatinine and blood urea nitrogen, decreased infiltration of neutrophils, and diminished histologic injury. Histologic evidence of apoptosis and caspase-3 activation were also reduced by exosome treatment (Figure 11). These protective effects were significantly attenuated in mice that received exosomes derived from ECFCs that were transfected with antagomiR to miR-486-5p.Figure 11Protective effects of endothelial colony-forming cell (ECFC) exosomes on apoptosis in FVB mice with kidney ischemia/reperfusion (IR) injury: role of miR-486-5p. (a) Graph depicts semiquantitative assessment of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining in kidneys from sham-treated FVB mice (Sham), FVB mice with kidney IR, FVB mice with kidney IR treated with ECFC exosomes at the time of reperfusion (IR+EX), and FVB mice with kidney IR treated with exosomes derived from ECFCs transfected with antagomiR (AmiR) to miR-486-5p (IR+EX+AmiR) 24 hours after IR. Results are depicted as TUNEL-positive nuclei per high-powered field (HPF). *P < 0.001 versus Sham, IR+EX; n = 5–7. (b) Representative photomicrographs of kidney TUNEL imaging from FVB mice. Arrows indicate TUNEL-positive nuclei, indicative of apoptosis (sham kidneys had only few TUNEL-positive nuclei). Original magnification ×600. Bar = 10 μm. (c) Graph depicts kidney caspase-3 activity in the 4 groups of FVB mice 24 hours after IR. *P < 0.05 versus Sham, IR+EX; n = 5–7. AFU, arbitrary fluorescent units.View Large Image Figure ViewerDownload (PPT) Delivery of extracellular vesicles derived from endothelial progenitor cells protects against experimental kidney IR injury, and transfer of miRNA to recipient cells has been implicated in the reparative process.9Burger D. Viñas J.L. Akbari S. et al.Human endothelial colony forming cells protect against acute kidney injury: role of exosomes.Am J Pathol. 2015; 185: 2309-2323Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 12Cantaluppi V. Gatti S. Medica D. et al.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 (416) Google Scholar, 16Collino F. Pomatto M. Camussi G. et al.AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs.J Am Soc Nephrol. 2015; 26: 2349-2360Crossref PubMed Scopus (183) Google Scholar The current studies indicate that transfer of miR-486-5p from ECFC-derived exosomes to endothelial cells plays an important role in preventing apoptosis after kidney IR. This conclusion is supported by several observations: (i) ECFC-derived exosomes are highly enriched in miR-486-5p, and delivery of exosomes to 2 separate mouse strains with kidney IR injury or to cultured endothelial cells exposed to hypoxic injury increases miR-486-5p levels, inhibits PTEN expression, and enhances Akt phosphorylation; (ii) ECFC-derived conditioned medium inhibits hypoxia-induced apoptotic responses in cultured endothelial cells associated with inhibition of PTEN expression and Akt activation, effects that are blocked by miR-486-5p antagomiR; (iii) transfer of miR-486-5p from ECFCs to endothelial cells occurs in co-culture and is blocked by inhibitors of exosomal release and uptake; (iv) miR-486-5p directly targets PTEN in cultured endothelial cells, and knockdown of PTEN expression prevents HR-induced apoptosis; and (v) ECFC exosomes potently protect against kidney IR injury in mice, an effect that is lost when ECFCs are first transfected with antagomiR to miR-486-5p. Several miRNA candidates have been implicated in kidney IR injury including proangiogenic miR-126 and miR-296,12Cantaluppi V. Gatti S. Medica D. et al.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 (416) Google Scholar proapoptotic miR-24,24Lorenzen J.M. Kaucsar T. Schauerte C. et al.MicroRNA-24 antagonism prevents renal ischemia reperfusion injury.J Am Soc Nephrol. 2015; 25: 2717-2729Crossref Scopus (115) Google Scholar and miR-21 (antiapoptotic).25Godwin J.G. Ge X. Stephan K. et al.Identification of a microRNA signature of renal ischemia reperfusion injury.Proc Natl Acad Sci U S A. 2010; 107: 14339-14344Crossref PubMed Scopus (316) Google Scholar We did not detect miR-296 or miR-24 in our study, and although miR-126 and miR-21 were found in ECFC-derived exosomes, levels were not increased compared with ECFCs or MPs. However, using an unbiased approach, we demonstrated that ECFC exosomes are highly enriched in miR-486-5p compared with ECFCs or MPs. Although other differentially expressed miRs, mRNAs, or proteins in exosome cargo could play a role in the protective effects on IR injury, miR-486-5p is a compelling candidate for mediating antiapoptotic effects in vivo and in vitro. Thus, miR-486-5p has been shown to target PTEN in skeletal muscle, which, in turn, enhances the antiapoptotic phosphoinositide 3-kinase/Akt pathway.18Alexander M.S. Casar J.C. Motohashi N. et al.MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy-associated symptoms.J Clin Invest. 2014; 124: 2651-2667Crossref PubMed Scopus (105) Google Scholar This pathway is particularly relevant to AKI because enhanced Akt phosphorylation is associated with improved kidney function in mice with IR injury.26Xu M.J. Feng D. Wang H. et al.IL-22 ameliorates renal ischemia-reperfusion injury by targeting proximal tubule epithelium.J Am Soc Nephrol. 2014; 25: 967-977Crossref PubMed Scopus (67) Google Scholar Our data indicate direct targeting of endothelial cell PTEN by exosomal miR-486-5p, suggesting that this leads to enhanced Akt phosphorylation and inhibition of apoptosis. Thus, delivery of exosomes to mice with kidney IR injury was associated with reduced kidney expression of PTEN and enhanced Akt phosphorylation, accompanied by increased levels of miR-486-5p. In cultured HUVECs exposed to HR, ECFC exosomes or conditioned media increased miR-486-5p levels, and the effects of ECFC conditioned media on PTEN, Akt, and caspase-3 activity were reversed by pretransfection of ECFCs with miR-486-5p antagomiR. Knockdown of PTEN in HUVECs was associated with decreased apoptotic response. Despite these data, the earliest time point of Akt activation by exosomes in relation to PTEN suppression was not defined in our study. Thus, although Akt phosphorylation appears to be a downstream event related to PTEN inhibition,17Small E.M. O'Rourke J.R. Moresi V. et al.Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486.Proc Natl Acad Sci U S A. 2010; 107: 4218-4223Crossref PubMed Scopus (318) Google Scholar, 18Alexander M.S. Casar J.C. Motohashi N. et al.MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy-associated symptoms.J Clin Invest. 2014; 124: 2651-2667Crossref PubMed Scopus (105) Google Scholar the link between these 2 signaling targets in apoptosis after kidney IR requires further study. Although our data suggest that reduced PTEN expression may be protective immediately after IR, the long-term consequences of PTEN suppression remain unclear. In a rat model of kidney IR, Lan et al.27Lan R. Geng H. Polichnowski A.J. et al.PTEN loss defines a TGF-β-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis.Am J Physiol Renal Physiol. 2012; 302: F1210-F1223Crossref PubMed Scopus (94) Google Scholar showed that persistent loss of PTEN in proximal tubules leads to growth arrest and a profibrotic phenotype, which may contribute to chronic kidney disease after AKI. Optimal dosing and timing of delivery of exosomes enriched in miR-486-5p after AKI should therefore be further investigated to mitigate such potential late adverse effects. In our in vivo studies, FVB mice had increased kidney PTEN expression after IR, despite a tendency for increased miR-486-5p levels, compared with sham-treated mice. In NOD-SCID mice with kidney IR, we also observed increased PTEN expression, with significantly enhanced kidney miR-486-5p levels. Thus, miR-486-5p levels may have to achieve a threshold before significantly affecting PTEN expression in the kidney after IR. Moreover, in addition to PTEN, other potential targets of miR-486-5p should be considered in the overall protective response. Alternate targets for miR-486-5p include the transcription factor FoxO1, which has been implicated in miR-486/PTEN/Akt signaling and muscle wasting in chronic kidney disease,28Xu J. Rongshan L. Biruh W. et al.Transcription factor FoxO1, the dominant mediator of muscle wasting in chronic kidney disease, is inhibited by microRNA-486.Kidney Int. 2012; 82: 401-411Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar and SMAD2, which is associated with attenuation of transforming growth factor-β1–induced fibroblast proliferation and experimental lung fibrosis.29Ji X. Wu B. Fan J. et al.The anti-fibrotic effects and mechanisms of microRNA-486-5p in pulmonary fibrosis.Sci Rep. 2015; 5: 14131Crossref PubMed Scopus (75) Google Scholar In hepatocellular carcinoma tissue, miR-486-5p directly targets phosphoinositide K3R1, leading to suppression of phosphoinositide 3-kinase/Akt activation and inhibition of cell growth.30Huang X.P. Hou J. Shen X.Y. et al.MicroRNA-486-5p, which is downregulated in hepatocellular carcinoma, suppresses tumor growth by targeting PIK3R1.FEBS J. 2015; 282: 579-594Crossref PubMed Scopus (86) Google Scholar Our study focused on endothelial cells as major targets