The Role Of ROS From Mitochodria And Nox Isoforms In Oxalate-mediated Oxidative Stress And Cell Injury In Renal Tubular Cells

Part ?The role of ROS from mitochondria and Nox isoforms in oxalate-mediated oxidative stress and cell injury in NRK-52E cellsObjctive To indentify the potential sources of reactive oxygen species(ROS)production and investigate the role of ROS from various sources in renal tubular cells exposure to oxalate.Methods Oxalate-induced cytotoxicity by viability with CCK-8 assay and lactate dehydrogenase(LDH)release.DCF-DA and mitoSOX Red were used to determine intracellular and mitochondrial ROS(mtROS)production,respectively.Mitochondrialmembrane potential(??m)was measured to evaluate mitochondrial function.The protein expression of Nox4,Nox2 and p22 were also detected to explore the effect of oxalate on NADPH oxidase.And the effect of mitochondria targeted antioxidant and specific NADPH oxidase subtype inhibitors on oxalate-induced renal tubular cytotoxicity were determined.Results Our results revealed that oxalate significantly decreased the viability of NRK-52E cells.Intracellular ROS generation was associated with the changes of protein expression of p22 and Nox4.The mitochondria were also a source of ROS production.Inhibitor of Nox2 or scavenging of mtROS rather than inhibition of Nox4/1 prevented oxalate induced cell injury.Conclusions ROS from different sources may play unique role in oxalate-mediated renal tubular cell injury.And we identify some potential targets for prevent oxalate-induced renal tubular cells injury.Part ?MitoTEMPO prevents oxalate induced injury in NRK-52E cells via inhibiting mitochondrial dysfunction and modulating oxidative stressObjective As one of major risks for urolithiasis,hyperoxaluria can be caused by genetic defect or diatery intake.And high oxalate induced renal epithelial cells injury is related to oxidative stress and mitochondrial dysfunction.Here,we investigated whether mitoTEMPO,a mitochondria-targeted antioxidant could protect against oxalate mediated injury in NRK-52E cells via inhibiting mitochondrial dysfunction and modulating oxidative stress.Methods MitoSOX Red was used to determine mitochondrial ROS(mtROS)production.Mitochondrial membrane potential(??m)and quantification of ATP synthesis were measured to evaluate mitochondrial function.The protein expression of Nox4,Nox2 and p22 were also detected to explore the effect of oxalate and mitoTEMPO on NADPH oxidase.Results Our results revealed that pretreatment with mitoTEMPO significantly inhibited oxalate-induced lactate dehydrogenase(LDH)and malondialdehyde(MDA)release,and decreased oxalate-induced mtROS generation.Further,mitoTEMPO pre-treatment restored disruption of ??m and decreased ATP synthesis mediated by oxalate.In addition,mitoTEMPO altered the protein expression of Nox4 and p22,and decreased the protein expression of IL-6 and osteopontin(OPN)induced by oxalate.Conclusions We concluded that mitoTEMPO may be a new candidate to protect against oxalate induced kidney injury as well as urolithiasis.Part ?The effect of mitoTEMPO on hyperoxaluria-induced oxidative stress and calcium oxalate crystal deposition in kidneyObjective Oxidative stress and mitochondrial dysfunction play important role in hyperoxaluria-induced renal injury and calcium oxalate crystal deposition.This study was designed to investigate the effect of mitoTEMPO,a mitochondria-targeted antioxidant,on oxidative stress injury and calcium oxalate crystal deposition in experimental rat model of hyperoxaluria.Methods We used a modified method to establish animal models of hyperoxaluria and nephrolithiasis by intraperitoneal injection of glyoxylic acid.TUNEL stain was used to detect the apoptosis of renal tubular cells.The oxidative fluorescent dye dihydroethidium was used to evaluate the superoxide production in renal tissue.Western blot was used to detect the expression of major NADPH oxidase subunits in renal tissue.Immunohistochemistry and western blot were used to detect the expression of OPN in the kidney.Von Kossa stain was used to detect the calcium oxalate crystal deposition in the kidney.Results MitoTEMPO treatment could reduce the apoptosis of renal tubular cells,and mitoTEMPO significantly reduced the increase of ROS and lipid peroxidation in renal tissue of hyperoxaluric rat.The expression of Nox2,Nox4,p22 and OPN significantly increased in renal tissue of hyperoxaluric rat,while mitoTEMPO significantly reversed the above effect.Also,mitoTEMPO treatment inhibited the formation of calcium oxalate crystals in the kidney mildly.Conclusions We concluded that mitoTEMPO can mitigate renal oxidative injury and may regulate the expression of modulators of urinary stone formation.MitoTEMPO can be used as a candidate to prevent renal injury and calcium oxalate crystal deposition mediated by hyperoxaluria.