Abstract
Abstract: :
Purpose: This study was designed to explore the role for aldose reductase (AR) in high glucose-induced oxidative stress in human retinal pigment epithelial (RPE) cells. Methods: The experiments were performed in human RPE-47 cells and RPE-47 cells stably transformed to overexpress the human AR gene (RPE-75) with resulting 4-fold increase in the AR gene expression and activity. The cells were cultured in 5 mM glucose or 30 mM glucose with/without one of two AR inhibitors (ARIs), sorbinil (100 µM) or fidarestat (1 µM) added for 3 h at the end of a 48 h-experiment. In a separate set, non-transfected RPE-47 cells were cultured in 5 mM glucose with/without one of four pro-oxidants, buthionine sulfoximine (BSO,100 µM), artemisinin (50 µM), primaquine (50 µM), or diethyl maleate (DEM, 60 µM), that cause oxidative stress through four different mechanisms not including sorbitol pathway activation. Intracellular abundance of reactive oxygen species (ROS) was assessed with the molecular fluorescent probe 5-(and-6-)-chloromethyl-2',7'-dichlorodi-hydrofluorescein diacetate (CM-H2DCFDA), the dichlorofluorescein derivative with the best retention properties among all the studied analogs, added for 30 min before the end of experiments. Then the cells have been washed, trypsinized and the CM-DCF fluorescence, the index of ROS generation, was measured by flow cytometry. Sorbitol concentration was measured spectrofluorometrically by enzymatic procedure. Results: High glucose induced sorbitol pathway hyperactivity in both RPE-47 and RPE-75 cells. Intracellular sorbitol concentration was about 10-fold higher in RPE-75 cells cultured for 48 h in 30 mM glucose than in RPE-47 cells processed similarly. Correspondingly, intracellular ROS abundance was 3-fold higher in RPE-47 cells in 30 mM glucose vs 5 mM glucose (p<0.01), and 13-fold higher in RPE-75 cells in 30 mM glucose vs 5 mM glucose (p<0.01). Sorbitol accumulation and ROS production in RPE-47 and RPE-75 cells cultured in 30 mM glucose were decreased by both ARIs to the levels observed in 5 mM glucose. Treatment of RPE-47 cells cultured in 5 mM glucose with one of four pro-oxidants resulted in 3- (artemisinin), 6-(BSO and DEM) and 14-fold(primaquine) increase in ROS production. These were not affected by either sorbinil or fidarestat thus indicating that two ARIs do not have direct antioxidant properties. Conclusion: Increased AR activity has a key role in high glucose-induced ROS generation in RPE cells. Further studies are needed to sort out the complex relations between AR, oxidative stress and the function of outer blood-retinal barrier in diabetes.
Keywords: 567 retinal pigment epithelium • 504 oxidation/oxidative or free radical damage • 387 diabetes