Advanced Oxidation Protein Products Induce Epithelial-to-mesenchymal Transition In Human Proximal Tubular Epithelial Cells Through Oxidative Stress

Renal tubular interstitial fibrosis(TIF) is a common feature of chronic kidney disease which caused by many diseases.The degree of renal tubular interstitial fibrosis keeps pace with renal function decreased during the prognosis of diseases. Renal interstitial fibrosis which includes the imbalances of inhibition and promoting fibrosis cytokine, the enhanced oxidative stress, inflammatory reaction, renal inherent cells and immune cells apoptosis, is charactered with renal tubular atrophy, extracellular matrix(ECM) accumulation, chronic inflammation of tubulointerstitium.In the process of renal tubular interstitial fibrosis,accompany with the renal tisuue irreversible damaged, renal function gradually decline and develop to end stage renal failure.Epithelial-to-mesenchymal transition (EMT) has been widely accepted as a crucial factor by which injured renal tubular epithelial cells transform into mesenchymal cells that contribute to the development of fibrosis in chronic renal failure.In2002,the technology of conditional gene targeting knockout was applied to mice in order to express the specific LacZ gene on renal tubular epithelial cells, by tracing the LacZ labeled tubular cells in the unilateral ureteric obstruction (UUO) model in mice, about36percent of fibroblasts were found form from renal tubular epithelial cells. Renal tubular epithelial cells are polar cells. E-cadherin is linked to the generation of the polarized epithelial phenotype.Cell-cell junctions depenting on E-cadherin are crucial to maintain cell and tissue polarity and integrity,and also are the primary cellular determinant of epithelial barrier function. The process of EMT is chatacterized by differentiated epithelial cells undergo a phenotypic conversion that gives rise to the matrix-producing fibroblasts and myofibroblasts.Epithelial cells lose their marker proteins such as E-cadherin and cytokeratin (CK), indicating the intercellular junctional contacts and their contacts with the basement membrane are lost,and gain of a mesenchymal phenotype with expression of mesenchymal proteins including α-smooth muscle actin(α-SMA) and vimentin (Vim). Without the tight junctions,tubular epithelial cells detach from the basement membrane,and rearrange cytoplasmic cytoskelet.Fibroblasts that associated with renal function and prognostic can synthesis and secrete ECM,which obtained by transdifferentiated renal tubular epithelial cells and lead to exaggetate ECM are secreted but degradated less,and eventually leading to renal interstitial fibrosis.Renal epithelial cells can be activated by modified proteins such as the products of lipid peroxidation and advanced glycation end products(AGEs),the expression of fibrogenic growth factors such as TGF-β increase,and exgressive ECM are secreted,resulting in renal interstitial fibrosis.Advanced oxidation protein products (AOPP), containing two tyrosine and carbonyl groups, is formed by the crosslinking products oxidation system of protein oxidative damage, and has similar structure with AGEs. AOPP is not only found in plasma of hemodialysis patients because of chronic renal failure (CRF), but also in non-diabetes patients, metabolic syndrome, aging, IgA nephropathy, acute renal injury induced by CKD,etc. The level of AOPP in plasma is along with the deterioration of renal function and can be considered as an important index to evaluate the therapeutic effect and prognosis of IgA nephropathy. AOPP is though to be a new kind of uremic toxins,leading to chronic renal failure (CRF) immune function disorder and promoting the progress of oxidative stress. AOPP is an inflammatory reaction medium as monocyte respiratory burst, mediated large inflammatory cytokines such as TNF-α, IL-1synthesising and releasing, cascading and worsening the inflammatory response. Studies have found that AOPP induced mouse podocyte EMT,downregulating the expression of E-cadherin,nephrin and pococy,and upregulating the expression of a-SMA,which resulting in proteinuria.AOPP also induced inflammatory response and insulin resistance in cultured adipocytes,involved in adipocyte dysfunction in metabolic syndrome and type2diabetes.Oxidative stress is the imbalance of intracellular oxidation and antioxidant,which is responsible to many diseases. The activation of Nicotinamide adenine nucleoside phosphate(NADPH) oxidase and the accumulation of reactive oxygen species(ROS) may have an important pathophysiological role in the process of tissue oxidative injuried.The ROS,including superoxide, nitric oxide radical, hydroxyl radical and hydrogen peroxide, accumulate in the body or within cells and increase the level of non-enzymatic glycation antioxidant enzymes, whereas antioxidant enzyme activity decreased in cells.Cellular membranes are damaged,causing perturbation in the fluidity and lipid membrane liquid bilayer structure that lead to cell oxidative damage, lipid membrane peroxidation, intracellular protein and enzyme denaturation and DNA damagement.ROS-mediated activation of Src kinase increses tyrosine phosphorylation of p120-catenin,that is bant with E-cadherin, and then leads to translocation of p120-catenin,which results in the activation of Rho/Rho kinase pathway that leading to the dissociation of cell-cell contact.ROS also act as second messengers for several signal transduction cascade pathways and transcription factors, such as nuclear factor-κB (NF-κB),and aggravate the oxidative stress. ROS come from a wide range of enzyme system,including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, xanthine oxidase system, mitochondrial pathway, nitric oxide synthase and cyclooxygenase system.NADPH oxidase not only exists in the phagocytic cells, but also exist in non-phagocytic cells such as renal tubular epithelial cells.Resent studies have shown that the predominant source of ROS generation inside renal tubular epithelial cells is NADPH-dependent O2-generation,supported by NADH,serveing as substrate,and NADHP,offering electronics.Oxidative stress can be activated by many factors.Proteinuria activation increase the ROS generation in activated renal tubular epithelial cells.High glucose induce ROS generation via activation of the PKC, hexosamine, polyol and AGEs pathway.ROS accumulation increase the expression of PKC, mitogen activated protein kinase,proto cytokines and transcription factors, upregulating expression of ECM gene.Given transforming growing factor-β1(TGF-β1) to rat renal tubular epithelial cells increase production of ROS and upregulate the expression of NADPH oxidase subunit,whereas could be blockde by NADPH oxidase inhibitors.Since ROS have short half life period and are difficult to be detected in vitro,Malondialdehyde(MDA),which is a stable lipid metabolites product of oxidative stress,can be used as a good index of oxidative stress. So are antioxidant enzymes superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GSH-px) activity.To sum up, we hypothesized that AOPP induce EMT via oxidative stress in human proximal renal tubular epithelial cells in vitro.To invesgate the effects and mechanism of AOPP in EMT,We used AOPP,made by bovine serum albumin (BSA)oxidized by sodium hypochlorite,to stimulate human proximal tubular epithelial cells(HK-2), the blank control and physiological dose of serum albumin group as negative control.Expressions of α-SMA and E-cadherin were observed both by Western blot and real-time Polymerase Chain Reaction,and index of oxidative stress such as level of MDA,activity of SOD,CAT and GSH-px were measured with or without NADPH oxidase inhibitor or oxygen free radical scavenger.(一) PurposeTo investigate the effects of AOPP on expression of α-SMA and E-cadherin and index of oxidative stress such as level of MDA,activity of SOD,CAT and GSH-px in cultured human proximal tubular epithelial cells(HK-2) with or without DPI or C-SOD.This finding may thrown light on further treatment for EMT and renal tubulointerstital fibrosis.(二) Methods1.Preparatin of AOPP Bovine serum albumin (BSA) was incubated with hypochlorous acid (HOCl) at the molar ratio of1:140at room temperature for30min, and then dialyzed against PBS for24h to remove any free HOCl in the solution. AOPP were removed endotoxin by Detoxi-Gel gel column. The content of AOPP were determined by measuring absorbance at340nm.2.Cells culturedHK-2cells were pelleted,resuspended and plated in DMEM supplementde with10%fetal bovine serum,50U/ml penicillin,and50μg/ml streptomycin,culture flasks were kept in a95%air-5%CO2enviroment at37℃.3.Dose-dependent effectConfluent HK-2Cells were treated with increased levels of AOPP(50,100,200and400μg/ml) or50μg/ml blood uman serum albumen for24h,then the expression of a-SMA and E-cadherin were measured by Western blot and RT-qPCR respectively.4. Time-dependent effectHK-2Cells were treated by200μg/ml AOPP or50μg/ml BSA for different time(30min,lh,3h,6h,12h and24h),a-SMA and E-cadherin expression were assessed by Western blot and RT-qPCR respectively.5.Oxidative stressConfluent HK-2Cells were treated by200μ g/ml AOPP,50μ g/ml BSA,or pretreated with100μmol/L diphenyleneiodonium(DPI) or200U/ml cytoplasmic superoxide dismutase(C-SOD) before stimulating AOPP,expression of a-SMA and E-Cadherin were analysised at24h,and the malondialdehyde(MDA) level,superoxide dismutase (SOD) activity,catalase(CAT) activity and glutathione peroxidase(GSH-px) activity were measured and calculated for different time(30min,lh and24h) respectively.(三) Results1.Compared with the blank control group and BSA group,different concentrations of AOPP,showing dose dependent effect,upregulated the expression of a-SMA and downregulated that of E-cadherin in HK-2cells. 2.200μg/ml AOPP downregulated the expression of E-cadherin in30min,and upregulated that of α-SMA in6h, showing time dependent effect.3.The MDA level was increased,while the SOD,CAT and GSH-px activity were decreased in HK-2cells after treated by AOPP.DPI or C-SOD can partly prevent the expression changes of a-SMA and E-cadherin,decreased MDA level,and increased the activities of SOD,CAT,GSH-px.(四) ConclusionsAOPP induced EMT in renal tubular epithelial cells via oxidative stress.This effects could be prevented by inhibiting the activation of NADPH Oxidase or antioxidant,and thus the process of EMT and RIF could be postponed.