NaDC3过表达对人肾小管细胞能量代谢和活性氧产生的影响

Effects of NaDC3 over-expression on energy metabolism and ROS generation in human renal tubular cells

目的:研究三羧酸循环中间代谢产物转运蛋白-高亲和力钠依赖性二羧酸转运蛋白(NaDC3)基因过表达对人肾小管细胞能量代谢与活性氧(ROS)产生的影响。方法:用重组NaDC3逆转录病毒载体感染人肾小管上皮细胞HKC。用Western blotting检测细胞中外源性NaDC3蛋白的表达水平。通过液体闪烁法测定细胞内同位素[3H]标记琥珀酸(NaDC3的转运底物)的浓度。分别用Clark电极法和反相高压液相法测定细胞内氧耗量和ATP含量。分别用荧光探针JC-1和CM-H2DCFDA处理细胞后通过激光共聚焦显微镜检测线粒体膜电位和细胞内ROS含量。结果:Western blotting结果显示导入外源NaDC3基因后可使细胞内NaDC3蛋白的表达水平明显增加。转运实验显示NaDC3病毒感染细胞内的[3H]-琥珀酸水平明显高于未感染和对照载体感染细胞。NaDC3病毒感染细胞中的氧耗量和ATP含量明显高于未感染和对照载体感染细胞。激光共聚焦检测显示,与未感染和对照载体感染细胞相比,NaDC3感染细胞的线粒体膜电位和ROS水平明显增加。结论:NaDC3过表达可通过转运过多的三羧酸循环中间代谢产物进入细胞内而加快肾小管上皮细胞能量代谢的速率、增加细胞内ROS的产生水平。

AIM ： To study the effects of over - expression of tricarbexylic acid cycle intermediates transporter NaDC3 （high affinity sodiumdependent dicarbexylate transporter） on energy metabolism and ROS generation in human renal tubular cells. METHODS ： Recombinant retrovirus vector containing NaDC3 gene was constructed and used for infecting human renal tubular epithelial cell HKC. Control vector containing Neo gene was also constructed and infected cells. Liquid scintillation method was used to determine the level of [ 3H ] -succinate （as a transport substrate of NaDC3 ） in the cells. Clark electrode method and reverse phase HPLC were used to detect oxygen consumption and ATP content intracellulaxly, respectively. Mitochondrial membrane potential and reactive oxygen species （ROS） content in HKC were determined with laser confocal microscope after treatment with fluorescent probe JC - 1 and CM - H2DCFDA, respectively. RESULTS ： Western blotting analysis showed that the expression of NaDC3 protein in uninfected - and control vector - infected cells was at lower level. After infection with recombinant NaDC3 vector, expression level of NaDC3 protein in HKC cells was increased markedly. Transport assay revealed that the level of [ 3H ] -succinate in NaDC3 -infected cells was noticeably increased. Oxygen consumption and ATP content in NaDC3 -infected HKC were significantly higher than those in uninfected - and control vector - infected cells. Laser eonfocal analysis revealed that mitochondfial membrane potential and ROS level in NaDC3 - infected HKC were increased, compared with uninfected - and control vector - infected cells. CONCLUSION ： Over - expression of NaDC3 accelerates the speed rate of energy metabolism and increases intracellular ROS generation by transporting an overdose of tricarboxylic acid cycle intermediates in human renal tubular epithelial cells.