| Acute kidney injury (AKI) is a common clinical disease, whose mortality rate is 50-80%. Despite advancements in the research of pathogenesis of AKI, no effective treatment is available at present. Supportive therapy remains to be the major option for the treatment of AKI patients who are waiting for recovery of renal function. This calls for the development of new and more effective treatments for AKI.Stem cell is defined as a cell that has the potential of self-renewal and multilineage differentiation, and has broad prospects in the treatment of acute renal failure. Many studies have confirmed that stem cell is effective for the treatment of acute renal failure.Mesenchymal stem cell (MSC) is now commonly used in research. Adult bone marrow-derived MSC is most prevalent, and with the in-depth study, more sources of MSC are available from adult or embryonic and fetal tissue and cord blood. But as differentiation and proliferation ability of stem cells from different sources are different, little is known about what kind of stem cell is best for disease treatment or new drug research. Although adult bone marrow MSC has a wide variety of sources, its use is limited, partly because the amount of MSC in adult bone marrow is very low, accounting for 0.001-0.0001% of the total number of nucleated cells. With the growth of donor age, the number of bone marrow MSC reduce and the differentiation capacity of MSC is iminished. Compared with adult-derived MSC, embryo-derived MSC have greater expansion and differentiation potentials. Immunologically, histocompatibility antigens are less intensively expressed in embryo-derived cells of early gestation terms than in adult cells.There are still controversies over the mechanism of how stem cells mediate the therapeutic actions. Although some early studies believed that this protective effect is attributed to the replacement of damaged cells by differentiated stem cells, recent studies suggest that only a small percentage of repaired tubular cells are stem cell-derived [1]. A more recent opinion is that stem cells exert their therapeutic effect in AKI by some paracrine/endocrine mechanism. Regulating the paracrine function would be a new method to enhance the therapeutic efficacy of MSC.Vascular endothelial growth factor (VEGF) can induce proliferation and anti-apoptotic response in renal tubular epithelial cells, and exert proangiogenic effects in AKI as well. Knockdown of VEGF by siRNA reduced the effectiveness of MSC in the treatment of ischemic AKI in a rat model, indicating that VEGF secreted by MSC plays an important renoprotective role in AKI when stem cell treatment is used.We therefore hypothesized that up-regulation of VEGF could strengthen the renal protective effect of MSC. The aim of the present study was to see whether the protective effect of stem cells could be enhanced by increasing VEGF secretion in a cisplatin-induced tubular cell damage model in vitro and a nude-mouse model of cisplatin-induced AKI in vivo.This study consists of 3 sections:Section 1:Adenoviral vector tansfection of hMSCAd.CMV-VEGF165 (provided by Dr. YuDong Zhang from the department of cardiothoracic surgery of the affiliated hospital of Nantong University, Nantong, JS, China) was used to infect hMSC. Ad.CMV-GFP was used to define the optimal transfection MOI. The result showed that the cell grown rate of hMSC infected with Ad-EGFP at MOI 50 was similar to that of regular hMSC without infection, and that hMSC remained to be a homogenous population of fibroblast-shaped cells. Therefore, the adenoviral vector was used to infect hMSC at MOI 50 in the subsequent experiments due to high efficiency and low toxicity. It was found that the peak level appeared at day 4-6 after transduction. At day 4 of transduction, VEGF level in the medium of VEGF-hMSC increased significantly, about 4 times that of uninfected hMSC. Adenovirus-mediated VEGF gene transfer was safe and effective, and transfected hMSC exhibited a high level of VEGF expression of.Section 2:Protection of VEGF-hMSC on cisplatin-impaired TCMK-1 in a co-culture systemTCMK-1 were pretreated with cisplatin for 24 h and cocultured with hMSC, VEGF-hMSC and AD-hMSC. Three days after cisplatin pretreatment, TCMK-1 slowed decreased regeneration, increased apoptosis and PCNA expression as compared with normal TCMK-1 (p< 0.01).Compared with TCMK-1 pretreatment with cisplatin alone, co-culturing with every kind of hMSC ceased the inhibitory effect of cisplatin on TCMK-1 growth, reduced apoptosis, and increased PCNA expression. These effects were most pronounced in VEGF-transfected hMSC. VEGF transfecton improved the ability of hMSC to impair cisplatin induce TCMK-1 injury. The repair mechanism may be related to promoting the proliferation and anti-apoptosis of hMSC. Section 3:Protection of VEGF-hMSC on cisplatin-induced renal injuryAKI was induced by subcutaneous injection of cisplatin (18 mg/kg).24 h after cisplatin administration, nude mice were divided into five groups:group A, normal group; group B, saline group; group C, Ad.CMV-VEGF165 transfected hMSC (VEGF-hMSC) group; group D, Ad.CMV-con transfected hMSC (AD-hMSC) group; and group E, hMSC group.Renal function and the tubular structure were impaired in group B 4 days after cisplatin administration.. The HE staining tubular necrosis count, tube count and the level of serum BUN were lower in VEGF-hMSC, AD-hMSC and hMSC groups than those in saline group.The PANC and TUNEL staining was increased in the kidney of all groups after AKI compared with that in normal kidney. PCNA count was higher in VEGF-hMSC, AD-hMSC and hMSC groups than that in saline group, while TUNEL count was lower. PCNA count in VEGF-hMSC group was the highest and TUNEL count was the lowest of the three groups. All hMSC treatment groups had a higher area percentage of peritubular capillary than the saline group (p<0.05). VEGF-hMSC therapy significantly restored the area percentage of peritubular capillary (p<0.01, versus AD-hMSC and hMSC).This study demonstrated that hMSC transfection with VEGF gene improved their therapeutic benefits in healing cisplatin-induced renal epithelial injury. VEGF-modified hMSC enhanced the renoprotective activity in the AKI nude-mice model compared to regular hMSC and vacant advirus transfected hMSC.In this part of the experiment, we successfully established a cisplatin-induced acute renal injury model in nude mice. VEGF gene transfer increased the ability of hMSC in repairing AKI. The specific mechanism may be related to the fact that VEGF up-regulation can improve stem cells to promote mitosis, anti-apoptosis and protection of the vascular micro-environment effects.This study indicates that implantation of VEGF-modified hMSC could provide advanced benefits in protection against renal tubular injury by anti apoptosis, improving microcirculation and proliferation in vitro and in vivo, thus providing a new source of stem cells for clinical use and a new method of improving the therapeutic effect of stem cells in AKI.
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