Vincent Annette S, Lim Beng Gek, Tan Jasmine, Whiteman Matthew, Cheung Nam Sang, Halliwell Barry, Wong Kim Ping
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore.
Kidney Int. 2004 Feb;65(2):393-402. doi: 10.1111/j.1523-1755.2004.00391.x.
Chronic renal failure has been associated with oxidative stress. Serum sulfite, sulfate, cysteine, homocysteine, cysteine sulfinic acid, and gamma-glutamylcysteine are elevated in patients on hemodialysis, suggesting an accelerated catabolism of sulfur-containing amino acids or a reduced elimination of sulfite/sulfate, or both. Administration of metabisulfite has also been shown to damage kidney cells.
Measurement of reactive oxygen species (ROS) was performed with the fluorescence of dichlorofluorescein (DCF), and that of intracellular ATP was by the luciferin-luciferase reaction. Oxidation of sulfite and succinate by isolated mitochondria from rat kidney was monitored polarographically. The fluorescent probe, 5, 5', 6, 6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was employed to assess any loss in membrane potential in energized respiring mitochondria. Activities of glutamate and malate dehydrogenases (GDH, MDH, respectively) were assayed by the spectrophotometric measurement of NADH. Sulfite was determined by HPLC-fluorimetric measurement of monochlorobimane-sulfite and cell viability was by the MTT procedure.
An immediate increase in ROS followed exposure of Madin-Darby canine kidney (MDCK), type II, and opossum kidney (OK) cells to 5-500 micromol/L sulfite. Depletion of intracellular ATP was also observed. A low rate of oxidation of 100 micromol/L sulfite was observed polarographically in isolated kidney mitochondria, but ADP-stimulated State 3 respiration was not apparent. ATP biosynthesis from the oxidation of glutamate in rat kidney mitochondria was significantly inhibited by coincubation with 100 micromol/L sulfite; this was not the case with malate, succinate, and TMPD/ascorbate. However, activities of both GDH and MDH in kidney mitochondrial extract were inhibited. The mitochondrial membrane potential and cell viability were not compromised.
Micromolar sulfite elicited an immediate increase in ROS in MDCK, type II, and OK cells. This was accompanied by a depletion of intracellular ATP, which could be explained by its inhibitory effect on mitochondrial GDH. Although MDH was similarly inhibited, the impact was buffered by the high level of this enzyme in kidney mitochondria.
慢性肾衰竭与氧化应激有关。血液透析患者血清中的亚硫酸盐、硫酸盐、半胱氨酸、同型半胱氨酸、半胱氨酸亚磺酸和γ-谷氨酰半胱氨酸水平升高，提示含硫氨基酸分解代谢加速或亚硫酸盐/硫酸盐清除减少，或两者皆有。亚硫酸氢盐的给药也已被证明会损害肾细胞。
用二氯荧光素（DCF）荧光法测定活性氧（ROS），用荧光素-荧光素酶反应测定细胞内ATP。用极谱法监测大鼠肾分离线粒体中亚硫酸盐和琥珀酸盐的氧化。使用荧光探针5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物（JC-1）评估活跃呼吸线粒体膜电位的任何损失。通过分光光度法测定NADH来检测谷氨酸脱氢酶和苹果酸脱氢酶（分别为GDH、MDH）的活性。通过高效液相色谱-荧光法测定单氯双硫醚-亚硫酸盐来测定亚硫酸盐，通过MTT法测定细胞活力。
将II型马-达二氏犬肾（MDCK）细胞和负鼠肾（OK）细胞暴露于5-500 μmol/L亚硫酸盐后，ROS立即增加。还观察到细胞内ATP耗竭。在分离的肾线粒体中，用极谱法观察到100 μmol/L亚硫酸盐的氧化速率较低，但ADP刺激的状态3呼吸不明显。与100 μmol/L亚硫酸盐共同孵育显著抑制了大鼠肾线粒体中谷氨酸氧化产生的ATP生物合成；苹果酸、琥珀酸以及四甲基对苯二胺/抗坏血酸则不然。然而，肾线粒体提取物中GDH和MDH的活性均受到抑制。线粒体膜电位和细胞活力未受影响。
微摩尔浓度的亚硫酸盐可使MDCK II型细胞和OK细胞中的ROS立即增加。这伴随着细胞内ATP的耗竭，这可以用其对线粒体GDH的抑制作用来解释。尽管MDH同样受到抑制，但肾线粒体中该酶的高水平缓冲了这种影响。
