人脐带间充质干细胞对糖尿病肾病大鼠肾脏HIF-1α表达的影响

Effect of HUMSC on HIF-1α expression in diabetic nephropathy rats

摘要:

目的 观察人脐带间充质干细胞（HUMSC）对糖尿病肾病大鼠肾脏缺氧诱导因子（HIF-1α）表达的影响。方法 50只健康清洁级8周龄雄性SD大鼠，随机选取20只为健康对照组，其余30只采用链脲菌素复制糖尿病肾病大鼠模型（模型组），两组大鼠饲养12周。第12周，随机选取两组大鼠各3只分别尾静脉注射DiR-HUMSCs，代谢12～16 h后在小动物活体光学3D成像系统下观察DiR-HUMSCs在大鼠体内的分布。随机选取9只模型组大鼠进行HUMSCs移植（HUMSCs移植组）。HUMSCs移植采用尾静脉注射方式移植浓度为1×10⁶个/mL HUMSCs 500 μL至大鼠体内，每周1次，连续4周，共28 d。检测3组大鼠的24 h尿蛋白定量（24 h UPro）、血清肌酐（Scr）、血尿素氮（BUN）、尿肌酐（Ucr）、尿白蛋白与肌酐比值（UACR）水平；酶联免疫吸附试验（ELISA）检测3组大鼠血清HIF-1α水平；采用PAS和Masson染色进行肾脏组织病理检测；免疫荧光法检测3组大鼠肾组织HIF-1α、Slc12A3和Aquaporin1蛋白的表达。结果 移植HUMSCs治疗4周后，与健康对照组比较，模型组和HUMSCs移植组大鼠Ucr水平降低（P <0.05），Scr、24 h UPro、BUN、UCAR均升高（P <0.05）；与模型组比较，HUMSCs移植组大鼠Ucr水平差异无统计学意义（P >0.05），Scr、24 h UPro、BUN、UCAR均降低（P <0.05）。糖尿病肾病大鼠病理损伤缓解，系膜增生和基底膜增厚改善，小管空泡变性减少，间质纤维化减轻。模型组大鼠血清HIF-1α水平较健康对照组升高（P <0.05），HUMSCs移植组血清HIF-1α水平较模型组下降（P <0.05）。与健康对照组比较，模型组大鼠肾脏远端小管中HIF-1α蛋白水平增加（P <0.05）；HUMSCs移植组的HIF-1α蛋白水平较模型组降低（P <0.05）。模型组远端小管标记蛋白Slc12A3水平低于健康对照组（P <0.05），HUMSCs移植组Slc12A3水平较模型组升高（P <0.05）。HUMSCs移植组的Aquaporin1蛋白水平较模型组和健康对照组均降低（P <0.05）。糖尿病肾病大鼠近端小管中无HIF-1α表达。结论 HIF-1α主要在糖尿病肾病大鼠肾脏远端小管表达，且HUMSCs可通过抑制HIF-1α的表达修复肾小管的损伤。

Abstract:

Objective To observe the effect of human umbilical cord mesenchymal stem cells (HUMSCs) on the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in the kidneys of rats with diabetic nephropathy.Methods Fifty healthy clean-grade male SD rats aged 8 weeks were selected, with 20 serving as the healthy control group and the remaining 30 used to replicate a diabetic nephropathy rat model using streptozotocin. After 12 weeks of rearing, three rats from each group were randomly selected and injected intravenously with DiR-labeled HUMSCs. The distribution of DiR-HUMSCs within the rats was observed 12-16 hours later using a small animal live optical 3D imaging system. Nine model group rats underwent HUMSC transplantation, receiving a 500 μL intravenous injection of 1×10⁶ cells/mL HUMSCs weekly for four weeks. The levels of 24-hour urinary protein (24 h UPro), serum creatinine (Scr), blood urea nitrogen (BUN), urinary creatinine (Ucr), and urinary albumin to creatinine ratio (UACR) were measured. Serum HIF-1α levels were detected by enzyme-linked immunosorbent assay (ELISA). Renal tissue pathology was examined using PAS and Masson staining, and the expression of HIF-1α, Slc12A3, and Aquaporin1 proteins in renal tissues was detected using immunofluorescence.Results After four weeks of HUMSC treatment, Ucr levels decreased in the model and HUMSC transplantation groups compared to the healthy control group (P <0.05). Scr, 24 h UPro, BUN, and UCAR increased (P <0.05). Compared to the model group, there was no significant difference in Ucr levels in the HUMSC transplantation group (P >0.05), while Scr, 24 h UPro, BUN, and UCAR decreased (P <0.05). Pathological damage in diabetic nephropathy rats was alleviated, with reduced mesangial proliferation, basal membrane thickening, tubular vacuolization, and interstitial fibrosis. Serum HIF-1α levels were higher in the model group than in the healthy control (P <0.05), and significantly lower in the HUMSC transplantation group compared to the model group (P <0.05). HIF-1α protein levels were higher in the distal tubules of the model group (P <0.05) and reduced in the HUMSC transplantation group (P <0.05). Slc12A3 levels were lower in the model group compared to the healthy control (P <0.05) and higher in the HUMSC transplantation group compared to the model group (P <0.05). Aquaporin1 levels in the HUMSC transplantation group were lower than in both the model and healthy control groups (P <0.05). There was no expression of HIF-1α in the proximal tubules of diabetic nephropathy rats.Conclusion HIF-1α is primarily expressed in the distal tubules of diabetic nephropathy rats, and HUMSCs can repair tubular damage by inhibiting the expression of HIF-1α.