| [Background] Urolithiasis is one of the most common diseases in urinary system.Epidemiological studies show that the incidence rate of urolithiasis is 5.8% in China’s adult population,of which 70%-80% calcium carbonate is the main component of calculus.At present,the main treatment for urolithiasis is surgery.However,the recurrence rate of urolithiasis is very high.The recurrence rate is about 50% in 5-10 years,and 75% in 20 years.At the same time,there is no effective means to inhibit the recurrence of stones.Most patients need to be treated many times because of the recurrence of kidney stones,which seriously affects the quality of life of patients and their families.Therefore,further revealing the exact pathogenesis of calcium oxalate stone,looking for new effective intervention targets,has important scientific research and clinical value.In the study of the pathogenesis of calcium oxalate stone,the theory of oxidative stress is generally accepted.When the concentration of oxalic acid in urine is too high,it will stimulate renal tubular epithelial cells to produce excessive ROS,leading to oxidative stress injury(OS).The injured renal tubular epithelial cells will provide calcium oxalate adhesion sites,and oxalate can combine with calcium ions and precipitate in the form of calcium oxalate crystals,then adhere,aggregate and grow on the damaged sites of cells,and finally form stones.Therefore,if there is a way to interfere with the formation of ROS in the kidney,it may be possible to interfere with the formation of calcium oxalate stones.ROS in human body is mainly produced by mitochondria and NADPH,and a large number of literatures have confirmed the role of ROS in the occurrence and development of calcium oxalate renal calculi.Our previous study found that chloroquine can inhibit oxidative stress injury by inhibiting ROS produced by mitochondria,and then inhibit calcium oxalate crystal deposition in rat kidney.However,whether chloroquine can regulate ROS production and oxidative stress injury through NADPH oxidase remains unclear.[Objective] The aim of this study was to confirm the inhibitory effect of chloroquine on hyperoxalic acid-induced oxidative stress injury in renal tubular epithelial cells and rat kidney tissue,and calcium oxalate crystal deposition in rat kidney.The role and mechanism of NAPDH oxidase in chloroquine inhibition of hyperoxalic acid-induced oxidative stress injury and calcium oxalate crystal deposition in renal tubular epithelial cells were also revealed.So as to further reveal the molecular mechanism of chloroquine inhibiting hyperoxalic acid-induced renal oxidative stress injury and calcium oxalate crystal deposition,and provide new experimental basis and intervention targets for clinical prevention and treatment of calcium oxalate stones.[Materials and Methods] Firstly,the model of oxidative stress injury and calcium oxalate crystal deposition in rat kidney was established by ethylene glycol method.The effects of chloroquine on renal oxidative damage,calcium oxalate crystal deposition and NADPH oxidase expression were detected by HE staining,silver nitrate staining,polarizing microscope and immunohistochemistry.Then,the effects of chloroquine pretreatment on oxidative stress injury related indicators of renal tubular epithelial cells induced by hyperoxalic acid were detected by kit,and the effects of chloroquine on NADPH expression and PKC signal pathway activation were detected by real-time fluorescent quantitative PCR and Western blot.Finally,the role of PKC signaling pathway in the regulation of NADPH expression,renal oxidative stress injury and calcium oxalate crystal deposition by chloroquine was further verified by rat model.[Results] Effect of chloroquine on calcium oxalate crystal deposition induced by ethylene glycol in rat kidney.There was no obvious crystal deposition in the control group and chloroquine treatment group,but a large number of calcium oxalate crystal deposition in the model group.Compared with the model group,the calcium oxalate crystal deposition of chloroquine intervention group was significantly reduced,while the crystal deposition of stone model + PMA group was significantly increased.Compared with the chloroquine intervention group,the kidney crystal deposition in the stone modeling + chloroquine + PMA group was significantly increased.Effect of chloroquine on oxidative stress injury of rat kidney induced by ethylene glycol.Compared with the control group,there was no significant change in the oxidative stress injury of the kidney tissue in the chloroquine group,and the oxidative stress injury of the kidney tissue in the stone model group was significantly aggravated.Compared with the model group,the oxidative stress injury in the chloroquine intervention group was significantly reduced,while the injury in the model + chloroquine + PMA group was significantly increased.Compared with the chloroquine intervention group,the oxidative stress injury of kidney tissue in the stone model + chloroquine + PMA group was significantly increased.Effect of chloroquine on expression of NADPH oxidase in rat kidney.Compared with the control group,the expression of NADPH oxidase NOX2,NOX4 and their subunits p22 phox,p47phox in the renal tissue of the model group were significantly increased,but the expression of Rac1 had no significant change.Compared with the model group,the expressions of NOX2,NOX4 and their subunits p22 phox,p47phox in chloroquine intervention group were significantly decreased,while the expressions of NOX2 and NOX4 in model + PMA group were significantly increased.Compared with the chloroquine intervention group,the expression of NOX2 and NOX4 in the kidney tissue of rats in the stone modeling + chloroquine + PMA group was significantly increased.Effect of chloroquine on hyperoxalic acid-induced oxidative stress injury in renal tubular epithelial cells(NRK-52E).High oxalic acid could significantly inhibit cell activity,promote the release of LDH,H2O2 and ROS,and reduce the content of GSH.Chloroquine pretreatment could significantly weaken the effect of high oxalic acid.Effect of chloroquine on the expression of NADPH oxidase in NRK-52 E cells.Chloroquine inhibited the expression of NOX2 and NOX4 in a time and dosedependent manner,and significantly attenuated the up regulation of NOX2 and NOX4 induced by hyperoxalic acid.Effect of chloroquine on PKC kinase activation and expression induced by hyperoxalic acid in NRK-52 E cells.Compared with the control group,hyperoxalic acid stimulation can significantly increase the phosphorylation level of PKCα and PKCδ,which significantly attenuated by Chloroquine pretreatment that were no significant difference in the expression of total protein.[Conclusion] 1.Chloroquine can significantly inhibit the oxidative stress injury of renal tubular epithelial cells and renal tissue induced by hyperoxalic acid.2.Chloroquine can significantly inhibit the deposition of calcium oxalate crystals induced by ethylene glycol in rat kidney.3.Chloroquine can decrease the expression of NADPH oxidase by inhibiting the activation of PKCα and PKCδ,so as to inhibit the oxidative stress injury of rat kidney induced by hyperoxalic acid. |
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