Oxidative Stress Induces Fibroblast Activation And Renal Fibrosis

BackgroundKidney fibrosis,characterized by excessive extracellular matrix（ECM）deposition leading to scarformation in renal parenchyma,which is produced by activated fibroblasts,is generally considered as the final common consequence of a wide variety of chronic kidney diseases（CKD）.Oxidative stress plays an important role in renal pathology,but the specific effects on fibroblasts remains unclear.In this experiment,we mainly want to study how oxidative stress acts on fibroblasts to regulate renal fibrosis.ObjectiveTo investigate the role of oxidative stress produced by NADPH oxidase in renal fibrosis and to explore its underlying mechanisms.MethodsThe expression of kidney Nox4 was detected by immunohistochemistry（IHC）in a variety of animal models of chronic kidney injury.We established an oxidative stress model by unilateral ischemic/reperfusion injury（UIRI）plus AOPP,and down-regulated the expression of Nox4 gene in the kidney through hydrodynamic-based gene delivery of interfering sequence.Furthermore,WB,IHC and other methods were used to detect the changes in renal fibrosis,kidney injury and renal function related indicators caused by oxidative stress induced by Nox4.In vitro,we stimulated the normal rat kidney interstitial fibroblast（NRK-49F）with AOPP to detect the express of NADPH oxidase,fibrosis and proliferation-related proteins,and downstream signaling molecules.Changes in NRK-49F were then observed after using Nox4 small interfering RNA（siRNA）and inhibitors of signaling molecules.At the same time,the effects of oxidative stress induced by Nox4 on the proliferation and activation of fibroblasts were further verified by extracting primary fibroblasts.ResultsThe expression of Nox4 was significantly up-regulated in the kidneys of various CKD animal models.In the UIRI model,exogenous knockdown of Nox4 attenuated AOPP-induced aggravation of cell damage,development of renal fibrosis,and the deterioration of renal function.In vitro experiments,AOPP-stimulated NRK-49F caused an increase in intracellular ROS,activated pkca and MAPK signaling pathways,and promoted the proliferation and activation of NRK-49F.On the other hand,the use of Nox4 small interfering RNA（siRNA）and inhibitors of p-p38 and p-p42/44 inhibited the proliferation activation of NRK-49F.At the same time,the expression of α-SMA-positive primary fibroblast abstracted after UIRI Nox4 was also increased,which further confirmed that oxidative stress promoted fibroblast activation.ConclusionsThese results indicate that Nox4 promotes the development and progression of renal fibrosis through oxidative stress,and targeting Nox4 expression may be a new strategy for the treatment of renal fibrosis.