Investigation of transplantation cell therapy for acute renal endothelial cell injury.

在日本，政府每年为医疗保险提供的资金约为3000万日元。ESRD的人口是20万人，每个患者每年的费用是500万日元。因此，目前每年用于ESRD的费用为1亿日元。鉴于进入终末期肾病的主要是2型糖尿病肾病，有必要开发控制进行性肾脏疾病的治疗方法。在本项目中，首先研究了内皮移植治疗肾脏疾病的疗效。在裸鼠肾动脉超选择性注射抗肾小球内皮抗体，建立血栓性微血管病(TMA)模型。血管内皮细胞损伤在注射后即刻开始，3-4天后随着内皮细胞的增殖而部分恢复。因此，在缺损区补充内皮细胞可能会增加再生肾小球的数量，加速…的恢复更多的过程，并保存肾功能。TMA开始3天后，经左颈动脉导管将人脐静脉内皮细胞(HUVE)注入动物体内。注射HJVEC一周后采血，取出完整肾脏。24小时后检测BUN的升高，发现TMA肾引起的BUN升高有统计学意义。当HUVEC移植到TMAA肾脏时，这种增加被抑制。当注射大鼠肾小叶间动脉内皮细胞而不是人脐静脉内皮细胞时，这种增加进一步降低。移植后1周仍可在肾小球束内观察到注入细胞。电子显微镜也证实了这一点。应用活体视频摄像机观察内皮移植的血流动力学效应，有效地揭示了缺血再灌注损伤中肾小管周围无血流现象。血管内皮细胞移植后几分钟，肾小球红细胞流量增加，最初为低流量。因此，内皮移植在TMA模型中的疗效部分来源于改善肾小球血流。在这些实验的同时，我们建立了绿色荧光大鼠，观察了骨髓移植对TMA、缺血性急性肾功能衰竭、慢性间质性肾病、SHC-肾功能衰竭和新月体肾炎等几种肾脏疾病模型的疗效。这些项目目前正在进行中。在这个项目中，至少有一个小组报告了骨髓移植对缺血性急性肾功能衰竭的疗效，尽管我们可以在这短时间内发现病情恶化。因此，骨髓细胞的排泄因子被认为比移植的骨髓细胞更为关键。在所研究的因子中，G-CSF是与干细胞移植相关的有希望的因子之一，对缺血再灌注损伤的恢复过程有效，其机制目前正在研究中。除了这些细胞移植实验外，我们还阐明了羟基自由基清除剂对缺血再灌注损伤的疗效，并于最近获得了发表。在研究期间，成功地开展了这一项目，并提出了有价值的意见。较少

The governmental resource for medicare is about 3000 million yen per year in Japan. The population of ESRD is 200 thousand and the expense for each patient is 5 million yen per year. Thus 100 million yen is currently spent for ESRD in each year. Given the fact that the entry to ESRD is predominantly increasing from type 2 DM nephropathy, it is necessary to develop the therapeutic approach controlling the progressive renal disease.In this project, the efficacy of endothelial transplantation to renal disease was firstly investigated. The thrombotic micro-angiopathy (TMA) was induced to athymic nude rats injecting anti glomerular endothelial antibody super selectively to renal artery. The endothelial injury is initiated immediately after the injection, while its recovery process partly starts 3-4 days later as the endothelial proliferation. Therefore, the supplementation of endothelial cells to the defective sites may increase the number of regenerating glomeruli, accelerate the recovery … More process, and preserve renal function. Three days after the initiation of TMA, human umbilical venous endothelial cells (HUVE) were arterially injected to animals via catheter thru left jugular artery. One week after the injection of HJVEC, blood sampling was performed and the intact kidney was removed. After 24 h, the increase of BUN was examined and found the statistically significant increase because of the TMA kidney. This increase was reduced when HUVEC was transplanted to TMAA kidney. When rat renal interlobular arterial endothelial cells were injected instead of HUVEC, this increase was further decreased. The injected cells were observed at glomerular tuft even one week after the transplantation. It was also confirmed by electron microscopy. The hemodynamic effect of endothelial transplantation was examined using intravital video CCD camera which efficiently elucidated the peritubular no flow phenomena in ischemia reperfusion injury (ref 12). The glomerular red blood cell flow, initially low flow, was increased several minutes after the endothelial transplantation. Therefore the efficacy of endothelial transplantation in TMA model is partly derived from the improvement of glomerular blood flow.In parallel to these experiments, we developed green fluorescent rats to investigate the efficacy of bone marrow transplantation to several renal disease model, such as TMA, ischemic acute renal failure, chronic interstitial renal disease, SHC-renal failure, and crescentic glomerular nephritis. These projects are currently ongoing. During this project, at least one group reported the efficacy of bone marrow transplantation to ischemic acute renal failure though we could find deterioration during this short period. Therefore, the excretion factors from bone marrow cells were considered rather pivotal instead of transplanted bone marrow cells. Among the factors investigated, G-CSF is one of the promising factors related to stem cell transplantation, that was effective for the recovery process of ischemia-reperfusion injury, and that mechanism is currently investigating. In addition to these cell transplantation experiments, we elucidated the efficacy of hydroxyl radical scavenger to ischemia-reperfusion injury and got the acceptance of publication recently. This project was successfully conducted and generated valuable observations during the research periods. Less