Effects of NaDC3 over-expression on energy metabolism and ROS generation in human renal tubular cells / 中国病理生理杂志

ABSTRACT

AIM: To study the effects of over-expression of tricarboxylic acid cycle intermediates transporter NaDC3 (high affinity sodium-dependent dicarboxylate 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 intracellularly, 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-H_2DCFDA, 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 confocal analysis revealed that mitochondrial 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.