The Effect Of Nox4 Subunit NADPH Oxidase On Renal Interstitial Fibrosis In Lupus Nephritis And Its Intervention Therapy

BackgroundLupus nephritis(LN)is one of the most common complications of systemic lupus erythematosus(SLE),and eventually progresses to end-stage renal disease(ESRD),as the renal fibrosis has been the common final pathway of chronic kidney disease(CKD).In the process of renal fibrosis,epithelial-mesenchymal Transmition(EMT)is the important way for the production of a great quantity of fiber cells and myofibroblasts and extracellular matrix.In the LN procession,a lot of autoantibodies and immune complex formation can cause oxidative stress in kidney,and the production of large amounts of reactive oxygen species(ROS),can induce the renal tubulointerstitial EMT and the final renal fibrosis.Nicotinamide adeninedinucleotide phosphate oxidase system(NADPH Oxidase)is a major source of ROS,Nox4 subunit,is the main type in renal tubular epithelial cells,however,whether the ROS resources of Nox4 involve the EMT in renal tubular epithelial cells remains to be seen.Study found the E3 ubiquitin ligase Cbl-c can start the Ubiquitin-proteasome system(UPS)and promote the degradation of Nox4 in alveolar epithelial cell so as to delay the pulmonaary fibrosis,but whether Cbl-c can play a similar role in kidney is still unknown.Our study aims to investigate what role the Nox4 subunit NADPH oxidase plays in renal fibrosis of lupus nephritis in and the degradation effect of Nox4 ubiquitination.ObjectiveTo investigate the role and the degradation effect of Nox4 subunit NADPH oxidase in the progression of EMT of human renal tubular epithelial cells in lupus nephritis(LN),in order to provide new ideas and clinical treatment to lupus nephritis.Methods1.From January 2014-August 2015,31 renal tissues with LN were collected in nephrology department of Henan province people hospitalin from 31 LN patients who received renal biopsy,taking its kidney puncture organization as experiment group.While,3 renal tissues were collected from tumor next organization of kidney tumor patients as normal control group.The expression levels of Nox4 subunit NADPH oxidase(Nox4),alpha smooth muscle actin(?-SMA),and fibronectin(FN)protein were measured using immune fluorescence method and the semi-quantitative analysis.2.Stimulating human renal tubular epithelial cells(HK-2)with anti ds-DNA antibody to simulate the process of renal interstitial fibrosis in lupus nephritis(LN),the normal cultured human renal tubular epithelial cells were divided into four groups:(1).normal control group(DMEM-F12 medium with 10% FBS);(2).antibody group(DMEM-F12 medium with 10% FBS + 100ug/ml anti-ds-DNA antibody);(3).transfection group(DMEM-F12 medium with 10% FBS +Cbl-c cDNA transfection);(4).transfection+antibody group(DMEM-F12 medium with 10% FBS + Cbl-c cDNA transfection + 100ug/ml anti-ds-DNA antibody).Following test will be detected after dealing with 72 hours to these group cells.(1)Cell proliferation were detected by MTT.(2)The levels of TGF-1 in cell supernatant of different group were measured by enzyme linked immunosorbent assay(ELISA).(3)The levels of ROS were deteced using DCFH-DA fluorescent probe method.(4)The levels of epithelial mesenchymal transdifferentiation pathway related protein Nox4,E-cadherin,?-SMA,and FN were measured by Western blotting.Results1.Renal tissue immunofluorescence results showed that LN patients had a weak expression of Nox4 in tubules epithelium,while large amounts of ?-SMA and FN were tested in LN patients of glomerular basement membrane zone and renal tubule interstitial,and their expression levels were related to the pathological types,patients with pathological type ?and ? LN tended to have high expression of above proteins where as patients with pathological type II LN tended to have low expression of above proteins.However,the normal control group showed none expression.2.1.HK-2 cells proliferation activity decrease after the incubation with 100ug/ml anti-dsDNA antibody,and we observed cell morphology changing from polygonal to spindle shaped,and the intercellular space increasing under the microscope.The proliferation activity of HK-2 cells was increased in the transfected + antibody group than that in the antibody group.Besides,the anti-dsDNA antibody can increase the TGF-?1 concentration heavily,while the TGF-beta1 in the transfected + antibody group was significantly less than that in the antibody group,which suggests that there are a great deal of TGF-?1 in LN as the profibrotic cytokines,and the overexpression of Cbl-c had no special effect on the secretion of TGF-beta 1.2.2.The expression of protein Nox4 of HK-2 cells was significcantly raised,and coming with the increasing ROS after the incubation with anti-dsDNA antibody,suggesting that the overexpression of Nox4 subunit NADPH oxidase can increase the genaration of ROS in patients with lupus nephritis.The transfection of Cbl-c could significantly reduce the expression of Nox4.Compared with the antibody group,the expression of Nox4 and ROS generation decreased evidently in transfection+antibody group,which suggested that promoting the degradation of Nox4 subunit NADPH oxidases can effectively reduce the production of ROS.2.3.Compared with the control group,the expression level of mesenchymal cell marker protein ?-SMA and FN increased highly,while the epithelial cell marker E-cadherin decreasing significantly in the antibody group.The expression of protein ?-SMA and FN decreased obviously,while the E-Cadherin raised in the transfection+antibody group,comparing to the antibody group.Our results indicate that the epithelial cell in LN can occur epithelial-mesenchymal transmition(EMT),which should be closely related to the activation of the Nox4 subunit NADPH oxidase,and the promotion of the degradation of Nox4 can effectively reduce the generation of ROS and inhibit the occurrence of EMT.Conclusions1.The activation of Nox4 subunit NADPH Oxidase / ROS oxidation stress pathway can induce the epithelial mesenchymal transmition of renal tubular epithelial cells in Lupus Nephritis.2.The ubiquitinated degradation of Nox4 subunit NADPH oxidase can suppress ROS production and EMT progression of renal tubular epithelial cells in Lupus nephritis,so as to delay the renal fibrosis progression.