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目的探讨骨化三醇对高糖状态下肾小管上皮细胞肾素表达的影响及其机制。方法按随机数字表法将24只6~8周龄Wistar大鼠随机分为对照组、糖尿病组、骨化三醇组(0.03μg·kg~(-1)·d~(-1)),各组8只。16周后处死各组大鼠,检测尿蛋白、尿肌酐、内生肌酐清除率(endogenous creatinine clearance rate,CCr),并应用实时荧光定量法(real time-polymerase chain reaction,RT-PCR)和蛋白印迹法(Western blot)检测肾脏组织肾素及磷酸化细胞外信号调节激酶(phosphorylated extracellularsignal-regulated kinase,p-ERK)/细胞外信号调节激酶(extracellularsignal-regulated kinase,ERK)表达情况。NRK-52E细胞分组:①正常对照组;②高糖组(糖浓度为25 mmol/L,不同时间点);③高糖+骨化三醇(10~8 mol/L、10~9 mol/L、10~10 mol/L)组;④高糖+ERK抑制剂(FR18020410~5 mol/L)组。RT-PCR法检测肾素mRNA表达,Western印记法检测肾素蛋白、p-ERK/ERK蛋白表达。结果与对照组比较,糖尿病组大鼠尿β2微球蛋白(β2-microglobulin,β2-MG)、尿白蛋白/尿肌酐升高(P<0.05),CCr降低(P<0.05);与糖尿病组比较,骨化三醇组大鼠尿β2-MG、尿白蛋白/肌酐降低(P<0.05),CCr升高(P<0.05)。与对照组比较,糖尿病组大鼠肾组织肾素、p-ERK/总ERK表达均明显升高,骨化三醇能显著减轻糖尿病大鼠上述蛋白的高表达(P<0.05)。高糖可诱导NRK-52E细胞ERK蛋白磷酸化(P<0.05),诱导肾素mRNA及蛋白表达上调,并呈时间依赖性(P<0.05)。骨化三醇可浓度依赖性地降低高糖诱导的NRK-52E细胞ERK蛋白磷酸化及肾素mRNA、蛋白的高表达(P<0.05)。ERK抑制剂能部分抑制高糖诱导的肾素蛋白高表达(P<0.05)。结论骨化三醇降低糖尿病大鼠蛋白尿,具有肾脏保护作用,其机制可能与抑制ERK通路降低高糖诱导的肾素高表达有关。
Objective To investigate the effect of calcitriol on renin expression in renal tubular epithelial cells under high glucose condition and its mechanism. Methods Twenty-four Wistar rats aged 6-8 weeks were randomly divided into control group, diabetic group and calcitriol group (0.03μg · kg -1 · d -1) according to random number table method. 8 in each group. After 16 weeks, the rats in each group were sacrificed and the urinary protein, urinary creatinine and endogenous creatinine clearance rate (CCr) were measured. Real time-polymerase chain reaction (RT-PCR) and protein Western blot was used to detect the expression of renin and phosphorylated extracellular signal-regulated kinase (p-ERK) / extracellular signal-regulated kinase (ERK) in renal tissues. NRK-52E cells were divided into the following groups: ① normal control group; ② high glucose group (glucose concentration 25 mmol / L at different time points); ③ high glucose + calcitriol (10 -8 mol / L, 10-9 mol / L, 10 ~ 10 mol / L) group; ④ high glucose + ERK inhibitor (FR18020410 ~ 5 mol / L) group. The expression of renin mRNA was detected by RT-PCR and the protein expression of renin and p-ERK / ERK was detected by Western blot. Results Compared with the control group, the urinary β2-microglobulin (β2-MG), urinary albumin / creatinine increased (P <0.05) and the CCr decreased Compared with the calcitriol group, urinary β2-MG, urinary albumin / creatinine were decreased (P <0.05) and CCr was increased (P <0.05). Compared with the control group, the expression of renin, p-ERK / total ERK in diabetic rats increased significantly, calcitriol significantly reduced the above protein expression in diabetic rats (P <0.05). High glucose could induce phosphorylation of ERK protein in NRK-52E cells (P <0.05), and induce upregulation of mRNA and protein expression of renin in a time-dependent manner (P <0.05). Calcitriol decreased phosphorylation of ERK protein and mRNA and protein expression of NRK-52E cells (P <0.05) in a concentration-dependent manner. ERK inhibitors partially inhibited high glucose-induced high expression of renin (P <0.05). Conclusion Calcitriol can reduce the proteinuria in diabetic rats, and its mechanism may be related to the inhibition of ERK pathway and the decrease of high glucose-induced high renin expression.