中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2014年
6期
429-436
,共8页
郭银凤%宋志霞%周敏%张晓良
郭銀鳳%宋誌霞%週敏%張曉良
곽은봉%송지하%주민%장효량
维生素D%糖尿病肾病%巨噬细胞%足细胞%表型
維生素D%糖尿病腎病%巨噬細胞%足細胞%錶型
유생소D%당뇨병신병%거서세포%족세포%표형
Vitamin D%Diabetic nephropathies%Macrophages%Podocytes%Phenotype
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0.1 μg· 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、24 h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),CD 163/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.
目的 探討活性維生素D(VD)能否通過調節腎組織巨噬細胞M1及M2錶型活化從而防治糖尿病腎病(DN)大鼠足細胞損傷.方法 利用腹腔註射鏈脲菌素(STZ)建立糖尿病大鼠模型.將SD雄性大鼠按隨機數字錶法分為4組:對照組1(NC-1組,n=8)、對照組2(NC-2組,n=8,對照+骨化三醇0.1μg·kg-1·d-1灌胃)、DN組(n=24)、DN+VD榦預組(VD組,n=24,DN+骨化三醇0.1 μg· kg-1·d-1灌胃),定期檢測血糖、體質量,收集尿標本,分彆于榦預後8週、14週、18週末處死動物,檢測Scr、BUN和尿蛋白變化;PAS染色觀察腎髒病理改變;免疫組化法檢測腎組織CD68+巨噬細胞浸潤數量;Western印跡法檢測nephrin、podocin、CD68以及M1巨噬細胞特異性標誌物誘導型氮氧化物閤酶(iNOS)、腫瘤壞死因子α(TNF-α)和M2巨噬細胞特異性標誌物CD163、精氨痠酶1(Arg-1)、甘露糖受體(MR)錶達.結果 (1)與兩對照組相比,DN組Scr、BUN、24 h尿蛋白量及腎小毬繫膜基質增生程度顯著增加(P＜0.05),podocin、nephrin蛋白錶達下降(P＜0.05).VD榦預後能明顯改善上述病理現象(均P＜0.05).(2)與兩對照組相比,DN組腎組織CD68+巨噬細胞浸潤數量明顯增加,呈時間依賴性.VD榦預後能顯著減少CD68+巨噬細胞浸潤數量(P＜0.05).(3)進一步確定腎組織巨噬細胞M1、M2活化錶型髮現,8週、14週、18週末DN組iNOS、TNF-α蛋白錶達較對照組顯著升高(均P＜0.05),VD榦預後能明顯抑製同期DN腎組織iNOS、TNF-α錶達(均P＜0.05);8週、14週末VD組CD163、Arg-1、MR蛋白錶達與DN組相比差異無統計學意義(均P＞0.05),而18週末VD組CD163、Arg-1、MR蛋白錶達較DN組顯著升高(均P＜0.05),CD 163/CD68蛋白錶達比例亦顯著增加(P＜0.05).(4)相關分析顯示,M1標誌物iNOS與nephrin、podocin蛋白錶達均呈負相關(r=-0.707,P＜0.01;r=-0.712,P＜0.01);M2標誌物CD163與nephrin、podocin蛋白錶達均呈正相關(r=0.627,P＜ 0.01;r=0.613,P＜ 0.01).結論 活性維生素D具有調節巨噬細胞錶型活化的能力,通過抑製巨噬細胞M1型活化併增彊M2型活化,進而髮揮足細胞保護作用.
목적 탐토활성유생소D(VD)능부통과조절신조직거서세포M1급M2표형활화종이방치당뇨병신병(DN)대서족세포손상.방법 이용복강주사련뇨균소(STZ)건립당뇨병대서모형.장SD웅성대서안수궤수자표법분위4조:대조조1(NC-1조,n=8)、대조조2(NC-2조,n=8,대조+골화삼순0.1μg·kg-1·d-1관위)、DN조(n=24)、DN+VD간예조(VD조,n=24,DN+골화삼순0.1 μg· kg-1·d-1관위),정기검측혈당、체질량,수집뇨표본,분별우간예후8주、14주、18주말처사동물,검측Scr、BUN화뇨단백변화;PAS염색관찰신장병리개변;면역조화법검측신조직CD68+거서세포침윤수량;Western인적법검측nephrin、podocin、CD68이급M1거서세포특이성표지물유도형담양화물합매(iNOS)、종류배사인자α(TNF-α)화M2거서세포특이성표지물CD163、정안산매1(Arg-1)、감로당수체(MR)표체.결과 (1)여량대조조상비,DN조Scr、BUN、24 h뇨단백량급신소구계막기질증생정도현저증가(P＜0.05),podocin、nephrin단백표체하강(P＜0.05).VD간예후능명현개선상술병리현상(균P＜0.05).(2)여량대조조상비,DN조신조직CD68+거서세포침윤수량명현증가,정시간의뢰성.VD간예후능현저감소CD68+거서세포침윤수량(P＜0.05).(3)진일보학정신조직거서세포M1、M2활화표형발현,8주、14주、18주말DN조iNOS、TNF-α단백표체교대조조현저승고(균P＜0.05),VD간예후능명현억제동기DN신조직iNOS、TNF-α표체(균P＜0.05);8주、14주말VD조CD163、Arg-1、MR단백표체여DN조상비차이무통계학의의(균P＞0.05),이18주말VD조CD163、Arg-1、MR단백표체교DN조현저승고(균P＜0.05),CD 163/CD68단백표체비례역현저증가(P＜0.05).(4)상관분석현시,M1표지물iNOS여nephrin、podocin단백표체균정부상관(r=-0.707,P＜0.01;r=-0.712,P＜0.01);M2표지물CD163여nephrin、podocin단백표체균정정상관(r=0.627,P＜ 0.01;r=0.613,P＜ 0.01).결론 활성유생소D구유조절거서세포표형활화적능력,통과억제거서세포M1형활화병증강M2형활화,진이발휘족세포보호작용.
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 0.1 μg· 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 8th,14th,18th 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 8th and 14th week (P ＞ 0.05),the protein expressions of CD163,Arg-1 and MR were higher in VD group at 18th 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 M 1 macrophage activation and enhancing M2 macrophage activation to protect podocyte impairment.
