Role of Rac1 in renal tubular epithelial cell oxidative injury induced by oxalate

Abstract

Objective To explore the potential role of ras-related C3 botulinum toxin substrate (Rac1) in renal tubular epithelial cell oxidative injury induced by oxalate.Methods The Madin-Darby canine kidney (MDCK) cells were cultured in vitro and divided randomly into group A (blank control group),B (Rae1 inhibitor group),C (oxalate group) and D (Rae1 inhibitor and oxalate group).Group B and D were exposed in medium with Rac1 inhibitor NSC23766 (100 μmol/L) thirty minutes,and then 5 mmol/L oxalate were added to group C and D.In other control procedures,cells were treated with PBS.Four hours later,hydrogen peroxide (H2O2) and lactate dehydrogenase (LDH) were measured in each medium and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity were measured in cells,Rac1 expression were determined using immunocytochemical staining,and calcium oxalate crystallization adhered to cells were observed microscopically after adding 0.75 mmol/L calcium oxalate to the medium.Results H2 O2,LDH and NADPH oxidase activity in cells of group A were (24.23 ± 2.44) mmol/L,(0.62 ± 0.06) U/ml,(1 042.83 ±61.00) RLU/ (min·mg),group B were (24.46 ±3.39) mmol/L,(0.56 ± 0.06) U/ml,(1 096.67 ± 66.12) RLU/ (min· mg),group C were (67.84 ± 4.25) mmol/L,(1.71 ± 0.08) U/ml,(2 852.50 ± 105.71) RLU/ (min· mg),group D were (34.79 ± 3.07) mmol/L,(0.96 ± 0.07) U/ml,(1118.83 ±57.56) RLU/ (min·mg).Compared with group A,the level of Rac1 expression,NADPH oxidase activity,H2 O2 generation and LDH release in cells of group C increased significantly (all P ＜ 0.05),and the number of calcium oxalate adhered to cells in group C also increased,while compared with group C,the above indicators observed in group D decreased significantly (all P ＜ 0.05).Conclusion Oxalate cause renal tubular epithelial cell oxidative injury via Rac1 regulated-NADPH oxidase.Inhibition of Rac1 results in decreased reactive oxygen species (ROS) production and a reduction in cell injury. Key words: Oxalate; Renal tubular epithelial cell; Ras-related C3 botulinum toxin substrate; Nicotinamide adenine dinucleotide phosphate oxidase; Oxidative injury