活性维生素D通过调节糖尿病肾病大鼠巨噬细胞M1及M2表型活化防治足细胞损伤

Active vitamin D prevents podocyte injury via regulation of macrophage M1 and M2 phenotype in diabetic nephropathy rats

目的探讨活性维生素D（VD）能否通过调节肾组织巨噬细胞M1及M2表型活化从而防治糖尿病。肾病（DN）大鼠足细胞损伤。方法利用腹腔注射链脲菌素（STZ）建立糖尿病大鼠模型。将SD雄性大鼠按随机数字表法分为4组：对照组1（NC-1组，n=8）、对照组2（NC-2组，n=8，对照＋骨化三醇0．1μg·kg^-1·d^-1灌胃）、DN组（n=24）、DN＋VD干预组（VD组，n=24，DN＋骨化三醇0．1μg·kg^-1·d^-1灌胃），定期检测血糖、体质量，收集尿标本，分别于干预后8周、14周、18周末处死动物，检测Scr、BUN和尿蛋白变化；PAS染色观察肾脏病理改变；免疫组化法检测肾组织CD68^＋巨噬细胞浸润数量；Western印迹法检测nephrin、podocin、CD68以及M1巨噬细胞特异性标志物诱导型氮氧化物合酶（iNOS）、肿瘤坏死因子α（TNF—α）和M2巨噬细胞特异性标志物CD163、精氨酸酶1（Arg-1）、甘露糖受体（MR）表达。结果（1）与两对照组相比，DN组Scr、BUN、24h尿蛋白量及肾小球系膜基质增生程度显著增加（P〈0．05），podocin、nephrin蛋白表达下降（P〈0．05）。VD干预后能明显改善上述病理现象（均P〈0．05）。（2）与两对照组相比，DN组肾组织CD68^＋巨噬细胞浸润数量明显增加，呈时间依赖性。VD干预后能显著减少CD68^＋巨噬细胞浸润数量（P〈0．05）。（3）进一步确定肾组织巨噬细胞M1、M2活化表型发现，8周、14周、18周末DN组iNOS、TNF-α蛋白表达较对照组显著升高（均P〈0．05），VD干预后能明显抑制同期DN肾组织iNOS、TNF-α表达（均P〈0．05）；8周、14周末VD组CD163、Arg-1、MR蛋白表达与DN组相比差异无统计学意义（均P〉0．05），而18周末VD组CD163、Arg-1、MR蛋白表达较DN组显著升高（均P〈0．05），CD163／CD68蛋白表达比例亦显著增加（P〈0．05）。（4）相关分析显示，M1标志物iNOS与nephrin、podocin蛋白表达均呈负相关（r=-0．707，P〈0．01；r=-0．712，P〈0．01）；M2标志物CD163与nephrin、podocin蛋白表达均呈正相关（r=0．627，P〈0．01；r=0．613，P〈0．01）。结论活性维生素D具有调节巨噬细胞表型活化的能力，通过抑制巨噬细胞M1型活化并增强M2型活化，进而发挥足细胞保护作用。

Objective To investigate the effect of active vitamin D （VD） on macrophage M1 and M2 phenotype and its role in protecting podocyte impairment in diabetic nephropathy （DN）. Methods Diabetes mellitus rats were established by intraperitoneal injection with streptozocin. Rats were randomly divided into four groups： normal- 1 （NC- 1, n=8）, normal- 2 （NC- 2, n=8, normal rats treated with calcitriol 0.1μg·kg^-1·d^-1 by gavages）, DN （n=24） and VD （n=24, DN＋calcitriol 0.1 μg·kg^-1·d^-1 by gavages）. Blood glucose and body weight were assessed, and 24-hour urine was collected regularly. Blood and urine samples were taken for biochemical study, and kidney tissues were used for PAS staining to assess histological changes. Immunohistochemical staining was used to detect number of CD68 ^＋ macrophage. Western blotting was used to detect protein expressions of nephrin, podocin, CD68, M1 specific marker of inducible nitric oxide synthase （iNOS）, TNF-α and M2 specific marker of CD163, arginase 1 （Arg- 1）, mannose receptor （MR）. Results （1） In DN group, levels of BUN, Scr, urinary protein and glomerular mesangial matrix proliferation were significantly higher （P 〈 0.05）, and the expressions of nephrin, podocin were significantly decreased compared with NC groups （P 〈 0.05）. These above changes were significantly improved in VD group （P 〈 0.05）. （2）Number of CD68 ^＋ macrophage infiltration in DN group was increased in a time dependent manner compared with NC groups, which was significantly reduced in VD group （P 〈 0.05）. （3）To further definite M1 and M2 macrophage activation phenotype, the protein expressions of iNOS and TNF-α was increased in DN group at 8^th, 14^th, 18^th weeks compared with NC groups （P 〈 0.05）, which were significantly decreased in VD group （P 〈 0.05）. Although, there were no significant difference of protein expressions of CD163, Arg- 1 and MR between VD and DN group at both 8^th and 14^th week （P 〉 0.05）, the protein expressions of CD163, Arg- 1 and MR were higher in VD group at 18^th week than that in DN group （P 〈 0.05）, and the ratio of CD163/CD68 was also enhanced in VD group （P〈0.05）. （4）Moreover, the protein expression of iNOS was negatively correlated with expression of either nephrin or podocin （r = -0.707, P 〈 0.01; r = -0.712, P 〈 0.01）, whereas the protein expression of CD163 was positively correlated with expression of either nephrin or podocin （r=0.627, P〈 0.01; r=0.613, P〈 0.01）. Conclusion Vitamin D can regulate macrophage phenotype, via inhibiting M1 macrophage activation and enhancing M2 macrophage activation to protect podocyte impairment.