摘要目的 探讨进展性肾病大鼠模型肾皮质能量代谢的变化及其相关分子机制.方法 以5/6肾切除模型大鼠为进展性肾病模型,分为手术组和假手术组,于造模完成后1周和12周处死大鼠,留取血液、尿液及肾脏标本.检测血尿素氮、血肌酐和24 h尿蛋白量以评估肾功能.PAS染色和天狼星红染色检测肾组织病理改变.液相色谱-质谱串联靶向代谢组学定量分析肾皮质能量代谢物的表达.实时荧光定量PCR检测肾皮质炎性因子(白细胞介素1β、白细胞介素6)、纤维化因子(纤连蛋白、Ⅰ型胶原)、糖酵解及三羧酸循环相关酶的mRNA表达.Western印迹检测肾皮质纤维化因子(纤连蛋白、Ⅰ型胶原)、沉默信息调节因子(SIRT-1)和肝脏激酶B1(LKB1)的蛋白表达.结果 与假手术组比较,手术组大鼠造模后1周和12周肾功能指标升高,肾组织病理损伤明显,肾皮质炎性因子及纤维化因子的mRNA表达上调,同时纤维化蛋白表达也上调(均P＜ 0.05),肾损伤随时间延长而加重.与假手术组比较,手术组大鼠1周时肾皮质糖酵解代谢物乳酸,三羧酸循环代谢物柠檬酸、异柠檬酸、草酰乙酸表达均上调,氧化磷酸化代谢物还原型辅酶Ⅰ表达上调(均P＜ 0.05),三磷酸腺苷(ATP)无明显变化,而12周时异常代谢物表达进一步增加,如糖酵解代谢物丙酮酸、氧化型辅酶Ⅰ及ATP表达下调(均P＜ 0.05).两组间磷酸戊糖途径代谢物还原型及氧化型辅酶Ⅱ差异均无统计学意义(均P＞ 0.05).与假手术组比较,手术组大鼠1周时肾皮质的糖酵解酶己糖激酶2、乳酸脱氢酶a的mRNA表达上调,三羧酸循环相关酶丙酮酸脱氢酶α、丙酮酸脱氢酶β、琥珀酸脱氢酶的mRNA表达下调(均P＜0.05),12周时异常代谢酶mRNA表达进一步增加.1周时两组大鼠肾皮质LKB1和SIRT-1的蛋白表达差异无统计学意义,而12周时手术组LKB1、SIRT-1蛋白表达低于假手术组(均P＜ 0.05).结论 在大鼠肾病进展过程中伴随肾纤维化和炎性反应,其肾皮质发生能量代谢改变.代谢改变表现为糖酵解增强,氧化磷酸化功能下降,且随病程进展而加重,其机制与能量调节蛋白LKB1及SIRT-1受抑制有关.

abstractsObjective To explore the changes of renal cortical energy metabolism and its related molecular mechanisms in rats with progressive kidney disease.Methods A rat model of 5/6 nephrectomy was established as the model of progressive nephropathy.Rats were divided into surgical group (5/6Nx group) and sham-operated group (Sham group).Respectively,the rats were sacrificed at 1 week and 12 weeks after completing the model,and their blood,urine sample and kidney specimens were collected.Blood urea nitrogen,serum creatinine and 24 h urine protein were used to evaluate the renal function.Pathological changes in renal tissue were detected by PAS staining and Sirius red staining.The renal cortical energy metabolites were made quantitative analysis by liquid chromatography-mass spectrometry-based targeted metabolomics.The mRNA expressions of inflammatory cytokines (IL-6,IL-1β),fibrosis factors (fibronectin,collagen-1),glycolytic and tricarboxylic acid (TCA) cycle related enzymes were confirmed by real-time PCR.The protein expressions of fibrotic proteins (fibronectin,collagen-1),silent information regulator 1 (SIRT-1) and liver kinase B1 (LKB1) were tested by Western blotting.Results Compared with those in Sham group,the renal function indexes increased,the renal tissue pathological damage was obvious,the mRNA expressions of renal cortical inflammatory and fibrosis factors increased,and fibrotic proteins also increased in 5/6Nx group rats at 1 week and 12 weeks (all P ＜ 0.05),meanwhile,kidney damage worsened over time.Compared with those in Sham group,in the renal cortex of 5/6Nx group glycolytic metabolite lactate,the TCA cycle metabolites (citrate,isocitrate,oxaloacetate) and the oxidized phosphorylation metabolite reduced coenzyme Ⅰ were up-regulated (all P ＜ 0.05),but adenosine triphosphate (ATP) was no change at 1 week,then the abnormal metabolites increased further at 12 weeks,such as the down-regulation of pyruvate,oxidized coenzyme Ⅰ and ATP (all P ＜ 0.05).The pentose phosphate pathway metabolites (reduction and oxidized coenzyme Ⅱ) shows no statistical significant difference in the two group (all P ＞ 0.05).Compared with those in Sham group,in the 5/6Nx group the mRNA expressions of glycolytic enzyme hexokinase 2 and lactate dehydrogenase a were upregulated in the renal cortex at 1 week,whereas the mRNA expressions of pyruvate dehydrogenase α,pyruvate dehydrogenase β and succinate dehydrogenase of the TCA cycle related enzymes were downregulated (all P ＜ 0.05).Meanwhile,renal abnormal metabolic enzyme mRNA expressions were further increased in the 5/6Nx group at 12 weeks.The protein levels of SIRT-1 and LKB1 were not significantly different in the renal cortex of two group rats at 1 week,while SIRT-1 and LKB1 levels decreased in 5/6Nx group than those in Sham group at 12 weeks (all P＜ 0.05).Conclusions During the progression of nephropathy,rats accompanied with renal fibrosis and inflammatory have energy metabolism changes in the renal cortex which accompanies.The features of metabolic changes are manifested as enhanced glycolysis and decreased oxidative phosphorylation,which is aggravated gradually.Its mechanism is related to the inhibition of energy-regulating proteins LKB1 and SIRT-1.