Role Of Racl In Renal Tubular Epithelial Cell Oxidative Injury Induced By Oxalate

Objective:To observe the variation of the nicotinamide adenine dinucleotide phosphate oxidase activity, the content of active oxygen species in the Madin-Darby canine kidney (MDCK) cells, and the adhesion conditions of calcium oxalate crystal to the surface of MDCK cells after inhibiting the expression of ras-related C3botulinum toxin substrate (Rac1) in MDCK cells exposed in oxalic acid, so as to explore the possible role of Racl in MDCK cells oxidative injury induced by oxalic acid. To offer new angles for the research on first-time occurrence and recurrence mechanism of calcium oxalate kidney stones by verifying our hypothesis that Racl has a vital influence in the first-time episodes and recurrence of calcium oxalate stones.Methods:The MDCK cells were cultured in vitro and divided randomly into group A(blank control group), B(Racl inhibitor group), C(oxalate group) and D(Racl inhibitor and oxalate group). Group B and D were exposed in medium with Racl inhibitor NSC23766(100umol/L) group A and C were exposed in DMEM medium thirty minutes, and then5mmol/L oxalate were added to group C and D. In other control procedures, cells were treated with phosphate buffer solution (PBS). Four hours later, hydrogen peroxide (H2O2) content and lactate dehydrogenase (LDH) activity were measured in each medium and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity were measured in cells by chemoluminescence, Racl expression were determined using immunocytochemical staining, and calcium oxalate crystallization adhered to cells were observed microscopically after adding0.75mmol/L calcium oxalate to the medium.Results:H2O2, LDH and NADPH oxidase activity in cells of group A were (24.23±2.44) mmol/L,(0.62±0.06) U/mL,(1042.83±61.00) RLU/min/mg, group B were (67.84±4.25) mmol/L,(1.71±0.08) U/mL,(2852.50±105.71) RLU/min/mg, and 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 Rac1expression, NADPH oxidase activity, H2O2generation and LDH release in cells of group C increased significantly (all p<0.05), and the quantity of calcium oxalate attached to the renal tubular epithelial cell in group C also increased (p<0.05), while compared with group C, the above indicators observed in group D decreased significantly (all p<0.05). Conclusion:Oxalate causes renal tubular epithelial cells injury via Racl regulated-NADPH oxidase and enhances the adhering capacity of calcium oxalate crystals to renal tubular epithelial cells. Inhibition of Rac1expression can distinctly reduce the generation of reactive oxygen species (ROS) and the degree of cell oxidative injury, and weaken the crystallization adhesion to the cell surface. Therefore, Racl has significant influence on the injury of renal tubular epithelial cells exposed in oxalic acid and promotes the formation and recurrence of calcium oxalate kidney stone.