CAJ | 학술논문

目的观察微炎性反应状态下低密度脂蛋白受体(low density lipoprotein receptor,LDLr)途径在糖尿病肾病(DN)足细胞损伤中的作用.方法选取8周龄雄性db/db小鼠及对照组db/m小鼠,分别按随机数字表法分为db/db、db/db+酪蛋白组和db/m、db/m+酪蛋白组,每组均为8只.参照本实验室已建立的方法制备糖尿病肾病微炎性反应模型,每周收集24 h尿液、检测24 h尿蛋白量,8周后处死,收集血清标本,留取肾组织,检测血清脂质谱的变化.透射电镜观察足细胞超微结构变化,激光共聚焦显微镜及Western印迹观察足细胞特异性标志蛋白WT-1、nephrin及间充质细胞表型标志物α平滑肌肌动蛋白(α-SMA)的表达情况.油红O染色和胞内游离胆固醇定量测定法观察肾小球中脂质积聚情况,免疫组化及Western印迹观察LDLr、固醇调节元件结合蛋白2(sterol regulatory element binding protein-2,SREBP-2)及其裂解激活蛋白(SREBP cleavage-activating protein,SCAP)在肾脏的表达情况,并且通过激光共聚焦显微镜观察WT-1与LDLr在肾脏的共表达情况.分析LDLr与nephrin、α-SMA表达的相关性.结果与db/db组相比,db/db+酪蛋白组小鼠的蛋白尿、足细胞结构损害及足细胞的丢失明显加重,足细胞α-SMA的蛋白表达量显著增加,肾小球内脂质积聚明显增多,肾脏LDLr、SREBP-2和SCAP的蛋白表达量也明显增加(均P＜0.05),并且LDLr的表达与nephrin的表达呈负相关(r=-0.855,P＜ 0.01),与α-SMA的表达呈正相关(r=0.768,P＜0.01).激光共聚焦荧光显微镜证实,小鼠肾脏的LDLr与足细胞特异性标志物WT-1存在共表达.结论炎性反应能够促进糖尿病小鼠足细胞损伤及表型改变,部分机制可能与炎性反应诱导肾脏足细胞LDLr表达上调,增加细胞内胆固醇摄入,导致足细胞内脂质堆积有关.
목적 관찰미염성반응상태하저밀도지단백수체(low density lipoprotein receptor,LDLr)도경재당뇨병신병(DN)족세포손상중적작용.방법 선취8주령웅성db/db소서급대조조db/m소서,분별안수궤수자표법분위db/db、db/db+락단백조화db/m、db/m+락단백조,매조균위8지.삼조본실험실이건립적방법제비당뇨병신병미염성반응모형,매주수집24 h뇨액、검측24 h뇨단백량,8주후처사,수집혈청표본,류취신조직,검측혈청지질보적변화.투사전경관찰족세포초미결구변화,격광공취초현미경급Western인적관찰족세포특이성표지단백WT-1、nephrin급간충질세포표형표지물α평활기기동단백(α-SMA)적표체정황.유홍O염색화포내유리담고순정량측정법관찰신소구중지질적취정황,면역조화급Western인적관찰LDLr、고순조절원건결합단백2(sterol regulatory element binding protein-2,SREBP-2)급기렬해격활단백(SREBP cleavage-activating protein,SCAP)재신장적표체정황,병차통과격광공취초현미경관찰WT-1여LDLr재신장적공표체정황.분석LDLr여nephrin、α-SMA표체적상관성.결과 여db/db조상비,db/db+락단백조소서적단백뇨、족세포결구손해급족세포적주실명현가중,족세포α-SMA적단백표체량현저증가,신소구내지질적취명현증다,신장LDLr、SREBP-2화SCAP적단백표체량야명현증가(균P＜0.05),병차LDLr적표체여nephrin적표체정부상관(r=-0.855,P＜ 0.01),여α-SMA적표체정정상관(r=0.768,P＜0.01).격광공취초형광현미경증실,소서신장적LDLr여족세포특이성표지물WT-1존재공표체.결론 염성반응능구촉진당뇨병소서족세포손상급표형개변,부분궤제가능여염성반응유도신장족세포LDLr표체상조,증가세포내담고순섭입,도치족세포내지질퇴적유관.
Objective To investigate the effects of low density lipoprotein receptor (LDLr) pathway on podocyte injury in diabetic nephropathy (DN) under inflammatory stress.Methods Male db/db mice and db/m mice were randomly divided into four groups (8 mice in each group):db/m group (control),casein injected db/m group (db/m + casein),db/db group (db/db),and casein injected db/db group (db/db + casein).An inflamed model of DN was established according to our previous study.24-hour urinary protein was measured every week.The plasma lipid profile was detected by clinical biochemistry assay.Podocyte changes were evaluated by electron microscope and immunofluorescent staining.Lipid accumulation in the kidney was evaluated by oil red O staining and intracellular cholesterol quantitative assay.The protein expression of Wilm's tumor-1 (WT-1),nephrin,α-smooth muscle actin (t-SMA),and molecules correlated with LDLr pathway were examined by immunohistochemical staining or Western blotting.The colocalized protein expression of LDLr with WT-1 was examined by immunofluorescent staining and laser confocal microscopy.Results There were no differences in plasma levels of LDL and HDL among four groups.Compared with db/db group,the db/db+ casein group showed markedly increased 24-hour urinary protein,more significant podocyte foot process effacement and podocyte damage,increased lipid droplet accumulation in kidneys,increased protein expressions of LDLr,SCAP and SREBP-2 in kidneys (all P ＜ 0.05).Interestingly,increased LDLr protein expression in kidneys of db/db mice was negatively correlated with decreased nephrin protein expression (r =-0.855,P ＜ 0.01) and positively correlated with increased α-SMA protein expression (r=0.768,P ＜ 0.01).Conclusions The disruption of LDLr pathway induced by inflammation contributes to podocyte injuries in diabetic nephropathy.