The regeneration of renal microcirculation system with mononuclear-derived cells in chronic renal injury

在慢性肾损伤中，肾小管间质损害的进展预示着肾功能不全。本研究的初步目的是通过单核细胞植入重建慢性肾损伤动物模型的微循环系统，并评价其对肾功能的影响。我们采用habuv -venome静脉给药后低剂量血管紧张素II渗透泵皮下给药作为慢性大鼠肾损伤模型，在该模型中，肾小球硬化和小管间质纤维化等有害变化持续4 ~ 6周。血尿素氮和血清肌酐显著升高（约为对照组的3倍）。最初，细胞移植使用来自骨髓的绿色荧光（PKH2-G1）标记的单核细胞进行。然而，由于肾组织，特别是小管和间质中fitc波长的非特异性信号非常高，我们无法清楚地确定pkh2 - g1阳性细胞。因此，我们修改了移植人外周血单核细胞的策略。细胞培养7 d后，采用微泡介导的超声法将Lac Z基因转染到细胞中，转染效率达到50%。转染后的细胞经肾静脉穿刺移植。移植后第4 ~ 7天，冷冻肾组织进行X-gal染色，追踪移植细胞。β-半乳糖苷酶阳性细胞局部见于部分小管，但未见于小管周围区域。此外，用β-半乳糖苷酶、水通道蛋白-1（AQP-1）抗体和von Willebrand （vW）因子抗体的免疫荧光研究显示，单层小管上皮中部分细胞β-半乳糖苷酶和AQP-1均阳性，但vW因子未阳性。接下来，我们将这些细胞移植到Habu-venome和AngII慢性肾损伤模型中，并随访移植效果至6周。然而，我们没有观察到移植后明显的组织学改善和功能改变。总之，来源于外周血的人单核细胞至少部分可以分化为小管上皮细胞。为了获得组织学和功能上的效果，移植方法的进一步改进是必要的。少

In chronic renal injury, the progression of tubulo-interstitial damages predicts the renal dysfunction. The initial goal of this study was to regenerate the microcirculation system in animal model of chronic renal injury with mononuclear cells implantation and to evaluate the significance in renal function by the implantation. We used intravenous Habu-venome administration followed by the low-dose of Angiotensin II administration subcutaneously with osmotic pump as a chronic rat renal injury model In this model, the deleterious changes such as glomerular sclerosis and tubulo-interstitial fibrosis for 4 to 6 weeks. In addition, blood urea nitrogen and serum creatinine significantly increased (approximately 3-folds increase above the control rats). Initially, the cell transplantation was performed with green fluorescence (PKH2-G1)-labeled mononuclear cells, which were derived from bone marrow. However, we could not clearly determine PKH2-G1-positive cells, because non-specific signals in … More FITC-wave length in renal tissues, especially in tubules and interstitium were quite high. Therefore, we modified the strategy in transplantation with human mononuclear cells from peripheral blood. After 7 days of cell culture, Lac Z gene was transferred to the cells using microbubble-mediated ultrasonopolation method, which achieved 50% transfection efficiency. The transfected cells were transplanted via renal vein puncture. To trace the transplanted cells, X-gal staining was carried out with frozen tissue of kidney on forth to seventh day after transplantation. β-galactosidase-positive cells were focally detected in some tubules, but not in peritubular regions. In addition, the imuunofluorescence studies with antibodies for β-galactosidase, Aquaporine-1(AQP-1), and von Willebrand (vW) factor showed that some cells in the single layer of tubular epithelium were positive for both β-galactosidase and AQP-1, but not with vW factor. Next, we transplanted these cells into chronic renal injury model with Habu-venome and AngII and followed the effects of transplantation up to 6 weeks. However, we could not observe the significant histological improvements as well as functional changes by transplantation. In conclusion, human mononuclear cells-derived from peripheral blood may, at least in part, differentiate to tubular epithelial cells. To gain histological and functional effects, further improvement in transplantation method will be necessary. Less