Advanced Oxidation Protein Products Induce Hypertrophy And Epithelial-To-Mesenchymal Transition In Human Proximal Tubular Cells Through Induction Of Endoplasmic Reticulum Stress

Chronic kidney disease (CKD) often leads to end-stage renal disease and it is a severe global public health problem. CKD is known to progress to end-stage renal disease through the main pathological basis and the final common pathway of renal interstitial fibrosis. Renal fibrosis includes glomerular sclerosis and renal interstitial fibrosis, which are closely linked and are directly related to the degree of decline in renal function. Hypertrophy of tubular cells is the main cause of renal hypertrophy, because the tubulointerstitium makes up almost 90% of the kidney volume. One of the important mechanism is that the compensatory hypertrophy of the remanent nephron is a mechanism of progressive development of chronic renal failure. The renal tubular epithelial cells which initiated compensatory hypertrophy demonstrate hypermetabolism, increased oxygen consumption, increased formation of oxygen radicals, leading to further aggravating renal tubular interstitial injury; at the same time, the hypertrophic renal tubular epithelial cells regulate the proliferation, differentiation and chemotaxis of renal cell and monocyte/macrophage and transform into myofibroblast by secreted cytokines, chemical chemokines, vasoactive substances and expressed adhesion molecules,promoting the occurrence of renal fibrosis and progressive deterioration of chronic renal failure. Moreover, previous studies demonstrated that initial adaptive tubular hypertrophy will become maladaptive and lead to tubular atrophy and interstitial fibrosis.A growing number of studies revealed that renal inherent cells, such as mesangial cells, renal tubular epithelial cells, would lose their inherent cell phenotypes and turn into myofibroblast under the condition of inflammation, damage, hypoxia, which played a role in the renal interstitial fibrosis. A large number of reserches showed that the epithelial-to-mesenchymal transition(EMT) would lead to renal intersititial fibrosis. Although the cellular mechanisms that facilitate tubulointerstitial fibrosis and the contribution of tubular EMT to renal interstitial fibrosis are still controversial, tubular EMT is through to play a role in the pathogenesis of CKD. Epithelial-mesenchymal transition (EMT) occurs in the early stage of renal fibrosis and it is a reversible process. Therefore, exploring the mechanism of tubular EMT and preventing the EMT of renal tubular epithelial cells is critical for preventing and interrupting the accelerated progression of CKD.Advanced oxidation protein products (AOPPs), which were first reported by Witko-Sarsat et al. in 1996, are dityrosine-containing and cross-linking protein products that are mainly formed as a result of the reaction of plasma albumin with chlorinated oxidants during oxidative stress. Previous studies have suggested that AOPPs can be observed in the plasma of patients with early-stage CKD and that elevated levels of AOPPs are correlated positively with lesions of renal function. Furthermore, growing evidence suggests that AOPPs play a crucial role in the progression of CKD. AOPPs have been reported to induce podocyte apoptosis, renal tubular epithelial injury, and mesangial cell proliferation and differentiation. However, although numerous factors might induce hypertrophy in renal tubular epithelial cells and initiate EMT in the kidney, whether AOPPs induce the hypertrophy and EMT of renal tubular epithelial cells remains poorly understood.The endoplasmic reticulum (ER) functions as the major processing center where newly synthesized, secreted and membrane-associated proteins are correctly folded and assembled. Adverse environmental conditions such as hypoxia affect the function of the ER and cause an imbalance between the protein-folding capacity of the ER and its protein content, which results in the accumulation of unfolded or misfolded proteins in the ER lumen and leads to ER stress. ER stress is a self-protection mechanism of cells that can restore homoeostasis within the ER. Unfolded protein response, which triggers both adaptive and apoptotic pathways, is initiated after ER stress. Moderate ER stress can enhance the tolerance of stress, but severe or prolonged ER stress can lead to cell hypertrophy,hyperplasia,differentiation and apoptpsis. ER stress has been increasingly recognized to participate in the development or pathology of several kidney diseases such as diabetic nephropathy, renal tubular interstitial lesions induced by toxins or drugs, renal ischemia-reperfusion injury, and CKD. Moreover, AOPPs have been shown to induce inflammation in adipocytes by inducing ER stress in vitro. However, whether AOPPs induce ER stress and how ER stress affects renal tubular epithelial cells are questions that remain poorly studied.At present, literature showed that reactive oxygen species (ROS) plays a very important role in the process of the development of chronic kidney disease (CKD), and increased oxidative stress state is considered to be one of the main pathogenic factors in chronic kidney disease patients.Reactive oxygen species (ROS) can induce cell proliferation, hypertrophy and apoptosis. In normal physiological conditions, the kidney itself will generate some reactive oxygen species, including superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl free radical (OH") and so on, which can be effectively eliminated by superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX) and vitamin E. When the generation of reactive oxygen species in the body is more than antioxidant system load, oxidative stress would cause tissue damage, thus, the balance of active oxygen species plays a vital role to maintain the structure and function of the kidney. As is known to all, oxidative stress can activate the endoplasmic reticulum stress, but whether oxidative stress take an part in the advanced oxidation protein product-induced hypertrophy and epithelial-to-mesenchymal transition in human proximal tubular cells still needs further research.Based on the above backgrounds, the HK-2 cells were cultured with AOPPs in vitro. The cell cycle distribution was detected by flow cytometry. The mRNA and protein content of CHOP, GRP78, a-SMA,E-cadherin and p27 were evaluated by quantitative real-time PCR and western blot, respectively. After pretreating with DPI, SOD, salubrinal and thapsigargin, We observed the change of the above indicators. In this study, we investigated whether AOPPs induce hypertrophy and EMT in HK-2 cells (a human proximal tubular epithelial cell line), and whether the process is mediated by ER stress. Moreover, we examined whether oxidative stress is involved in this process and the relationship between ER stress and oxidative stress. Our data will provide new experimental basis for early interruptting of CKD. PurposeThis research is mainly to explore whether AOPPs induce the hypertrophy and EMT of renal tubular epithelial cells and the mechanism in the processes. Moreover,it explore whether oxidative stress take an part in the Advanced Oxidation Protein Product-induced Hypertrophy and Epithelial-To-Mesenchymal Transition in Human Proximal Tubular Cells and the relationship betweem ER stress and oxidative stress. Our data will provide new theraputic target for early interruptting of CKD. Method1. AOPP preparationThe bull serum albumin (BSA) and hypochlorite solution were mixtured at the molar ratio of 1:140,25 to 30℃,30 minute, and then dialyzed by PBS for 24 hours to remove any free HOC1 in the solution. In the control incubation, native BSA was dissolved in PBS alone. All preparations were passed through a Detoxi-Gel column (Pierce, Rockford, IL, USA) in order to remove contaminating endotoxins. The endotoxin levels in the preparations were measured using the Amebocyte lysate assay kit (Sigma, St. Louis, MO, USA) and were determined to be <0.025 EU/mL. The AOPPs’content was determined in an acidic condition by testing absorbance at 340nm and was calibrated with Chloramines-T.2.cell cultureA human proximal tubular epithelial cell line was abtained from ATCC(Manassas,VA,USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM/F12) which contains 10% Fetal Bovine Serum,50 U/ml penicillin and 50μg/ml streptomycin(37℃,5% CO2). The cells were trypsinized using 0.25% trypsin-EDTA when growing to 80%-90% confluence, and then incubated in 6-well plates until they reached approximately 70%-80% confluence for all experiments.3.dose-dependent effectIn brief,cells were treated with AOPP in concentrations of 50,100,200 and 400μg/ml or 50μg/ml BSA for 24h,then the protein levels of CHOP,GRP78,p27,a-SMA,E-cadherin and Total Protein were analyzed by western blot;the mRNA levels of CHOP,GRP78,p27,a-SMA,E-cadherin were analyzed by Real Time Quantitative PCR(RT-PCR)。Cell cycle was detected by flow cytometry.4.time-dependent effectIn brief,cells were treated with 200μg/ml AOPP or 50μg/ml BSA for different time(0.5h,1h,3hrs,6hrs,12hrs and 24hrs),then the protein levels ofCHOP,GRP78,p27,a-SMA,E-cadherin and Total Protein were analyzed by western blot;the mRNA levels of CHOP,GRP78,p27,a-SMA,E-cadherin were analyzed by Real Time Quantitative PCR(RT-PCR)。Cell cycle was detected by flow cytometry. 5.the role of ER stress in advanced oxidation protein product-induced hypertrophy and epithelial-to-mesenchymal transition in human proximal tubular cellsCells were treated with 200μg/ml AOPP or 50μg/ml BSA or pretreated with 50μmol/L salubrinal or 0.25μmol/L thapsigargin before AOPP. After incubating for 24hrs, the expression of CHOP,GRP78,p27,a-SMA,E-cadherin were assessed by Western blot and RT-PCR respectively. Cell cycle was detected by flow cytometry. 6.the role of oxidative stress in Advanced Oxidation Protein Product-induced Hypertrophy and Epithelial-To-Mesenchymal Transition in Human Proximal Tubular CellsCells were treated with 200μg/ml AOPP or 50μg/ml BSA or pretreated with 10μmol/L an NADPH oxidase inhibitor (diphenyleneiodonium,DPI) or 200μmol/L superoxide dismutase(SOD) before AOPP. After incubating for 24hrs,the expression of CHOP,GRP78,p27,α-SMA,E-cadherin were assessed by Western blot and RT-PCR respectively. Cell cycle was detected by flow cytometry.Results1. AOPPs induced hypertrophy in HK-2 cells in dose and time dependent mannerTo investigate whether AOPPs induce HK-2-cell hypertrophy, we measured the total protein content in HK-2 cells. AOPP stimulation significantly increased the total protein content in HK-2 cells (Fig.1). Additionally, we also measured the protein and mRNA expression of p27 and cell-cycle distribution of HK-2 cells. Treatment of the cells with AOPPs induced the overexpression of p27 at the protein (Fig.2, A & B) and gene (Fig.2, C & D) levels and increased the percentage of HK-2 cells arrested in G1 phase (Fig.2 E & F) in a dose- and time-dependent manner. Although the maximal expression of p27, the total protein content, and the maximal number of cell arrested in Gl phase were not detected in the 24-h AOPP-treatment group, their levels in this group were significantly higher than that in the control group. Moreover, this effect was not observed in either the control group or the unmodified-BSA group. These results indicated that the overexpression of p27, the accumulation of total proteins, and the increased percentage of HK-2 cells arrested in G1 phase were all associated with the advanced oxidation of BSA. Taken together, these data suggested that AOPPs induced HK-2-cell hypertrophy through induction of the overexpression of p27 and G1 phase arrest.2. AOPPs induced EMT in HK-2 cellsTo determine whether AOPPs induce tubular EMT, we measured the expression of E-cadherin and a-SMA. Treatment with AOPPs downregulated the mRNA and protein levels of E-cadherin but upregulated those of a-SMA in a concentration-and time-dependent manner (Fig.3). The AOPP-induced increase in the mRNA levels of a-SMA peaked at 12 h and then decreased by 24 h, but the level was still significantly more than that in the control (P< 0.05). The expression of E-cadherin and a-SMA was not changed in control cells and in cells treated with unmodified BSA, which indicated that the loss of E-cadherin and the overexpression of a-SMA were associated with the advanced oxidation of BSA. Collectively, these results indicated that AOPPs induced EMT in HK-2 cells.3. AOPPs induced ER stress in HK-2 cellsIn this study, to examine whether AOPPs triggered ER stress, we examined the expression of GRP78 and CHOP by performing RT-PCR and western-blot assays. Compared with the levels in control cells and in cells treated with unmodified BSA, the protein and mRNA levels of GRP78 and CHOP were significantly increased in AOPP-treated HK-2 cells (Fig.4). Although the maximal expression of CHOP and GRP78 at both gene and protein levels was not in the 24-h AOPP-treatment group, the expression levels were still at significantly higher than those in the control group. Collectively, these data indicated that AOPPs induced ER stress in HK-2 cells.4. AOPPs induced hypertrophy and EMT in HK-2 cells through induction of ER stressTo investigate the role of ER stress in AOPP-induced hypertrophy and EMT in HK-2 cells, we measured the expression of CHOP, GRP78, p27, E-cadherin, and a-SMA in cells treated without or with AOPPs and an ER stress inhibitor (salubrinal), or in cells treated with an ER stress inducer (thapsigargin) alone. AOPP again induced the overexpression of CHOP, GRP78, p27, and a-SMA and suppressed the expression of E-cadherin at the transcriptional and translational levels, and these effects of AOPPs were partly reversed when the cells were treated with both AOPPs and the ER stress inhibitor. By contrast, the aforementioned effects of AOPPs were reproduced when cells were treated with only the ER stress inducer (Fig.5). Furthermore, the AOPP-induced increases in the total protein content in HK-2 cells and the percentage of HK-2 cells arrested in Gl phase were also partly reversed following treatment with the ER stress inhibitor, whereas both of these increases were produced in cells after treatment with the ER stress inducer alone (Fig.6). Collectively, these results indicated that AOPP-induced hypertrophy and EMT in HK-2 cells was mediated by the activation of ER stress.5. ER stress is associated with oxidative stressTo determine the role of oxidative stress in ER stress in HK-2 cells, we measured the expression of CHOP, GRP78, p27, a-SMA, and E-cadherin in the presence or absence of AOPPs and either an NADPH oxidase inhibitor (diphenyleneiodonium, DPI) or superoxide dismutase (SOD), a cell-permeable scavenger of reactive oxygen species (ROS). Our results showed that AOPP-induced loss of E-cadherin and overexpression of a-SMA, CHOP, and GRP78 were partly suppressed after treatment with the NADPH oxidase inhibitor and the ROS scavenger (Fig.5). Moreover, the increases in the total protein content in HK-2 cells and the percentage of HK-2 cells arrested in G1 phase after AOPP treatment were also partly reversed by DPI and SOD (Fig.6). Taken together, these data suggested that oxidative stress played a role upstream or downstream of ER stress and participated in the process of AOPP-induced HK-2-cell hypertrophy and EMT.ConclusionOur results suggest that AOPPs induced HK-2-cell hypertrophy and EMT by inducing ER stress, which was likely mediated by ROS. These findings could facilitate the development of novel therapeutic strategies for suppressing the progression of early CKD.