Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro

Zhou et al. Stem Cell Research & Therapy 2013, 4:34 http://stemcellres.com/content/4/2/34 RESEARCH Open Access Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro Ying Zhou1,2†, Huitao Xu1,2†, Wenrong Xu1,3*, Bingying Wang1, Huiyi Wu2, Yang Tao4, Bin Zhang1, Mei Wang1, Fei Mao1, Yongmin Yan1, Shuo Gao1, Hongbing Gu4, Wei Zhu1 and Hui Qian1* Abstract Introduction: Administration of bone marrow mesenchymal stem cells (MSCs) or secreted microvesicles improves recovery from acute kidney injury (AKI). However, the potential roles and mechanisms are not well understood. In the current study, we focused on the protective effect of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-ex) on cisplatin-induced nephrotoxicity in vivo and in vitro. Methods: We constructed cisplatin-induced AKI rat models. At 24 h after treatment with cisplatin, hucMSC-ex were injected into the kidneys via the renal capsule; human lung fibroblast (HFL-1)-secreted exosomes (HFL-1-ex) were used as controls. All animals were killed at day 5 after administration of cisplatin. Renal function, histological changes, tubular apoptosis and proliferation, and degree of oxidative stress were evaluated. In vitro, rat renal tubular epithelial (NRK-52E) cells were treated with or without cisplatin and after 6 h treated with or without exosomes. Cells continued to be cultured for 24 h, and were then harvested for western blotting, apoptosis and detection of degree of oxidative stress. Results: After administration of cisplatin, there was an increase in blood urea nitrogen (BUN) and creatinine (Cr) levels, apoptosis, necrosis of proximal kidney tubules and formation of abundant tubular protein casts and oxidative stress in rats. Cisplatin-induced AKI rats treated with hucMSC-ex, however, showed a significant reduction in all the above indexes. In vitro, treatment with cisplatin alone in NRK-52E cells resulted in an increase in the number of apoptotic cells, oxidative stress and activation of the p38 mitogen-activated protein kinase (p38MAPK) pathway followed by a rise in the expression of caspase 3, and a decrease in cell multiplication, while those results were reversed in the hucMSCs-ex-treated group. Furthermore, it was observed that hucMSC-ex promoted cell proliferation by activation of the extracellular-signal-regulated kinase (ERK)1/2 pathway. Conclusions: The results in the present study indicate that hucMSC-ex can repair cisplatin-induced AKI in rats and NRK-52E cell injury by ameliorating oxidative stress and cell apoptosis, promoting cell proliferation in vivo and in vitro. This suggests that hucMSC-ex could be exploited as a potential therapeutic tool in cisplatin-induced nephrotoxicity. Keywords: Acute kidney injury, Apoptosis, Cisplatin, Exosome, Human umbilical cord mesenchymal stem cells, Oxidative stress, Proliferation * Correspondence: ; † Equal contributors 1 School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu, China 3 The Affiliated Hospital, Jiangsu University, 228 Jiefang Road, Zhenjiang, Jiangsu, China Full list of author information is available at the end of the article © 2013 Zhou et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhou et al. Stem Cell Research & Therapy 2013, 4:34 http://stemcellres.com/content/4/2/34 Introduction Previous studies have shown that mesenchymal stem cells (MSCs) from different sources, including human bone marrow, cord blood, embryo and fetal membranes can be applied in tissue repair, such as promoting recovery from acute kidney injury (AKI) induced by various causes [1-4]. Similarly, our laboratory has demonstrated that MSCs derived from human umbilical cord and rat bone marrow mesenchymal stem cells can ameliorate ischemia/reperfusion-induced AKI and mouse hepatic injury [5-8]. Moreover, MSCs have been used in clinical trials [9,10]. Recently, microvesicles (MVs) derived from MSCs have been widely applied to experimental research, including AKI. Some studies have demonstrated that microvesicles released by human bone marrow MSCs contribute to repairing AKI induced by glycerol and ischemia-reperfusion [11,12] and improved survival in a lethal model of AKI induced by cisplatin in severe combined immunodeficiency (SCID) mice [13]. Exosomes are microvesicles of multivesicular bodies (MVBs) released from various cells into the extracellular space when intracellular MVBs fuse with the plasma membrane of the cell. Consequently, exosomes are present in cell culture supernatants. Exosomes are composed of a lipid bilayer, and contain proteins, mRNA and miRNA [14,15]. Previous research has indicated that exosomes play a biological role mainly in transmission of proteins, mRNA and miRNA. CD9, CD63 and CD81 have been used as characteristic markers of exosomes [15-17]. In view of these findings, more and more research has been conducted with the aim of investigating the functions of exosomes. The effect of exosomes on AKI, hepatic injury and myocardial ischemia/reperfusion injury has been demonstrated previously [11-13,18,19]. Cisplatin, as an anticancer drug, is often used to induce an animal model of AKI [1,13]. The mechanism of cisplatin renal toxicity is not fully understood; the main reason for AKI occurrence may be renal tubular apoptosis, and the increase of lipid peroxidation and reactive oxygen species (ROS) in the kidney, leading to kidney oxidative damage and renal cell death. At the same time, oxidative damage is one of the triggers for renal cell apoptosis [20-22]. It has also been reported that antioxidant stress pathways are stimulated by mesenchymal stromal cells in renal repair after ischemia-reperfusion injury [23]. Thus, we suggest exosomes derived from MSCs may ameliorate AKI caused by antioxidant stress pathways. Although exosomes derived from bone marrow mesenchymal stem cells have been successfully applied in improving survival in a lethal model of AKI induced by cisplatin in SCID mice [13], the mechanism and the protective effect of exosomes in an immunocompetent model are unclear. Moreover, the therapeutic potential Page 2 of 13 of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-ex) on kidney injury has not been reported so far. Based on previous studies, the aim of the present study was to investigate the effect of hucMSC-ex on cisplatininduced AKI. Our findings suggest that exosomes released from hucMSCs could suppress cisplatin-induced AKI and NRK-52E cell injury by improving oxidative stress and cell apoptosis, promoting cell proliferation in vivo and in vitro. Methods Isolation and charac (...truncated)