维生素D受体在糖尿病肾病足细胞损伤及蛋白尿缓解中的作用

Role of vitamin D receptor in podocyte injury and proteinuria of diabetic kidney disease

目的探讨维生素D受体(VDR)在糖尿病肾病(DKD)足细胞中的表达水平及在足细胞损伤及蛋白尿缓解中的作用。方法(1)本研究纳入了65例诊断患有2型糖尿病(伴或不伴蛋白尿)的患者,并纳入了25例年龄和性别相匹配的健康体检者为对照组。根据白蛋白/肌酐(ACR)的尿排泄比例对2型糖尿病患者进行分组,分别为无蛋白尿(ACR<30 mg/g,n=24)、微量白蛋白尿(ACR 30~300 mg/g,n=18)和临床蛋白尿(ACR>300 mg/g,n=23)。另选择25例经肾活检确诊的DKD患者作为DKD组。正常肾脏组织标本均取自泌尿外科同一时期肾脏肿瘤切除患者10例。将各组检测指标进行对比,同时采用实时定量PCR、ELISA法和免疫组化法检测VDR在各组患者的血液、尿液样本和肾脏组织中的表达情况,以及使用Pearson相关分析分析VDR与尿蛋白的相关性。(2)在2型糖尿病肾病小鼠模型中对上述结果进行验证,将遗传背景均为C57BLKs/J的雄性db/db小鼠及同窝出生的db/m小鼠,随机分为正常对照组(A组)、DKD对照组(B组)、DKD二甲基亚砜处理组(C组)、DKD帕立骨化醇(VDR激动剂)处理组(D组),C、D组连续腹腔注射处理8周,对照组不做任何处理。小鼠10周龄时开始连续干预8周,在小鼠22周龄(开始干预后12周)检测各组小鼠体重、血、尿生化指标对比;Western印迹法检测β⁃catenin、VDR的变化;免疫荧光观察足细胞标志蛋白podocin及足细胞损伤蛋白α⁃SMA的表达变化。结果(1)与正常健康对照组相比,无蛋白尿组、微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿组的糖尿病患者相比,微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05)。(2)与正常健康对照组相比,无蛋白尿糖尿病组和DKD组患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿糖尿病组患者相比,DKD组患者血浆中VDR的mRNA和蛋白水平亦较低(均P<0.05)。(3)免疫组化结果显示,DKD组肾组织中VDR的表达明显少于正常对照组。(4)DKD患者血浆中VDR mRNA相对水平与ACR呈负相关(r=-0.342,P<0.05)。(5)各组尿液上清液中VDR的水平与血浆中的水平呈相反趋势。(6)Western印迹结果显示,B组、C组肾小球足细胞β⁃catenin蛋白表达高于D组(均P<0.05),VDR蛋白的表达低于D组(均P<0.05);免疫荧光结果显示,B组、C组肾小球足细胞podocin的表达低于D组(均P<0.05),α⁃SMA的表达高于D组(均P<0.05)。结论VDR高表达缓解DKD足细胞损伤及蛋白尿。

Objective To investigate the expression level of vitamin D receptor(VDR)in podocytes of diabetic kidney disease(DKD)and its role in podocyte injury and proteinuria.Methods(1)Sixty⁃five patients who had been diagnosed with type 2 diabetes mellitus(with or without albuminuria)were enrolled in this study and 25 age⁃and sex⁃matched healthy control subjects were enrolled.According to the ratio of urinary excretion of albumin/creatinine(ACR),the type 2 diabetes mellitus patients were classified into without proteinuria group(ACR<30 mg/g,n=24),microalbuminuria group(ACR 30-300 mg/g,n=18)and clinical proteinuria group(ACR>300 mg/g,n=23).Another 25 patients with DKD confirmed by renal biopsy were selected as the DKD group.Normal kidney tissue samples were taken from the same period of urinary surgical department for 10 cases of renal tumors in patients with renal resection.The test indicators in each group were compared.The VDR expression in blood,urine samples and kidney tissues of patients was detected by real⁃time quantitative PCR,ELISA and immunohistochemistry,and then were compared among different groups.The correlation between VDR and ACR was analyzed by Pearson correlation analysis.(2)Male db/db mice with genetic background of C57BLKs/J and db/m mice born in littermates were randomly divided into normal control group(group A),DKD control group(group B),DKD treated with dimethyl sulfoxide group(group C),DKD treated with paricalcitol(VDR agonist)group(group D).The C and D groups were treated by continuous intraperitoneal injection for 8 weeks,and the group A and B were not treated.The mice were started to intervene continuously for 8 weeks at the age of 10 weeks.At 22 weeks of age(12 weeks after starting intervention),the biochemical indexes of the mice's body weight,blood and urine were compared.The changes ofβ⁃catenin and VDR were detected by Western blotting.The expressions of podocyte marker protein podocin and podocyte injury proteinα⁃SMA were observed by immunofluorescence.Results(1)Compared with the normal healthy control group,the plasma levels of VDR mRNA and protein in diabetic patients without proteinuria,microalbuminuria and clinical proteinuria were lower(all P<0.05).Compared with the diabetic patients without proteinuria,the plasma levels of VDR mRNA and protein in diabetic patients with microalbuminuria and clinical proteinuria were lower(all P<0.05).(2)Compared with the normal healthy control group,the plasma levels of VDR mRNA and protein in diabetic patients without proteinuria and DKD patients were lower(all P<0.05).Compared with diabetic patients without proteinuria,the plasma levels of VDR mRNA and protein in the DKD group were also lower(both P<0.05).(3)Immunohistochemical results showed that the expression of VDR in kidney tissue of DKD group was significantly lower than that of normal control group.(4)The relative level of VDR mRNA in plasma of patients with DKD was negatively correlated with ACR(r=-0.342,P<0.05).(5)The levels of VDR in urine supernatant of each group showed opposite trends with the plasma levels.(6)Western blotting results showed that the expression ofβ⁃catenin protein in groups B and C was higher than that in group D(both P<0.05),and the expression of VDR protein was lower than that in group D(both P<0.05).Immunofluorescence results showed that the expression of podocin in groups B and C was lower than that in group D(both P<0.05),and the expression ofα⁃SMA was higher than that in group D(both P<0.05).Conclusion VDR overexpression relieves podocyte injury and proteinuria in DKD.