| BackgroundDiabetic nephropathy (DN) is a most common cause of end-stage renal disease worldwide. A prominent clinical characteristic of DN and an independent risk factor for the progression of DN is proteinuria, which results from the disruption of the glomerular filtration barrier. Glomerular endothelial cells are an essential component of glomerular filtration barrier and play a crucial role in regulating glomerular permeability. Injury of glomerular endothelial cells will cause dysfunction of glomerular filtration, and then lead to proteinuria. Furthermore, experimental and clinical studies have indicated that endothelial dysfunction plays a critical role in the pathogenesis of DN. Moreover, endothelial-to-mesenchymal transition (EndMT/EndoMT), a specific form of epithelial-mesenchymal transition (EMT), has been suggested to play an essential role in the development of endothelial dysfunction and contributes to the development and progression of glomerulosclerosis and tubulointerstitial fibrosis, which is a critical morphological feature of DN. During EndMT, endothelial cells lose their endothelial cell markers, including vascular endothelial cadherin (VE-cadherin), CD31, and claudin 5, and express increased levels of mesenchymal markers such asa-smooth muscle actin (a-SMA), vimentin, and fibroblast-specific protein 1 (FSP-1). EndMT was first reported in the embryonic development of the heart, however, emerging evidence demonstrates that EndMT contributes to the pathogenesis of renal fibrosis. Zeisberg et al. reported for the first time that EndMT contributes to the accumulation of myofibroblasts, which is considered to be the key mediators of renal fibrosis, using 3 distinct mouse models of renal disease, including the streptozocin-induced diabetic nephrology. Subsequent studies conducted by Li et al. demonstrated that EndMT occurs and contributes to the early development and progression of diabetic kidney fibrosis. Collectively, EndMT plays critical roles in the pathogenesis of diabetic kidney fibrosis. Thus, understanding the mechanisms of the EndMT program and targeting them might yield novel therapeutic strategies for DN. However, the mechanisms underlying EndMT program remain poorly understood.Numerous pathologic processes can cause glomerular endothelial alterations. Advanced oxidation protein products (AOPPs) have been shown to activate vascular endothelial cells through a receptor of advanced glycation end products (RAGE)-mediated signaling pathway. AOPPs, which were firstly reported by Witko-Sarsat et al., are dityrosine-containing, crosslinking products that are formed as a result of the reaction between plasma albumin and chlorinated oxidants during oxidative stress. AOPPs have been shown to play a pathogenic role in the progression of chronic kidney disease, including DN. However, the biological effects of AOPPs on renal glomerular endothelial cells and the mechanisms underlying the aforementioned processes remain unclear.Endoplasmic reticulum (ER) stress has been widely demonstrated to play a crucial role in renal pathophysiology. ER stress is a physiological or pathological state that results from a variety of disturbances such as glucose deprivation, hypoxia, and viral infection. The accumulation of unfolded/misfolded proteins in the ER leads to ER stress and, subsequently, to the unfolded protein response (UPR), which is recognized as an adaptive response. However, when ER stress is prolonged or severe, the UPR might also trigger apoptotic pathways. ER stress has been shown to induce EMT in lung epithelial cells, PC C13 thyroid cells, and proximal renal tubular epithelial cells. More importantly, ER stress has also been shown to contribute to the damage of renal glomerular endothelial cells. However, the exact role of ER stress in AOPP-induced damage of renal glomerular endothelial cells has not been previously reported.ObjectivesIn this study, we investigated whether AOPPs induce EndMT in human renal glomerular endothelial cells (HRGECs), and we also examine whether AOPPs trigger ER stress in HRGECs. Furthermore, we investigated whether AOPP-induced EndMT in HRGECs is mediated by ER stress.Methods1. AOPPs preparation and content determinationAOPPs-BSA was prepared as previously described. Briefly, a 100 mg/mL BSA solution was exposed to 200 mmol/L of HOC1 for 30 min at room temperature and then dialyzed overnight against phosphate-buffered saline (PBS) to remove free HOC1. The AOPPs preparation was passed through a Detoxi-Gel column in order to remove contaminating endotoxins. The levels of endotoxin in the preparations were detected using the Amebocyte lysate assay kit and were examined to be<0.025 EU/mL. The content of AOPPs was measured as described previously. The AOPPs content in AOPPs-BSA and unmodified BSA were 65.2 ± 2.12 and 0.2 ± 0.04 nmol/mg, respectively.2. Cell culture of HRGECsThe HRGECs were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and cultured (at 37 ℃, in a 5% CO2 atmosphere) in an endothelial cell medium (ScienCell) containing 5% fetal bovine serum,1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution. In the experiments, we used approximately 80%-confluent cells from passages 2-5; these cells were were transferred to serum-free medium for 24 h before use in all experiments.3. AOPPs induced EndMT in HRGECsHRGECs were incubated with control medium, native BSA (200 μg/mL), or the indicated concentrations of AOPPs (50,100,200, and 400 μg/mL) for 24 h, or with 200 μg/mL AOPPs for the indicated times (12,24,48 h). The mRNA expression of the endothelial markers, VE-cadherin, CD31, and claudin 5, and mesenchymal markers, a-SMA, vimentin and FSP-1, were determined using quantitative Real-Time PCR (qPCR) analysis.4. AOPPs triggered ER stress in HRGECsHRGECs were cultured in control medium, medium containing unmodified BSA (200 μg/mL), or medium containing the indicated concentrations of AOPPs (50,100, 200, and 400 μg/mL) for 24 h, or with medium containing 200 μg/mL AOPPs for the indicated times (12,24,48 h). The mRNA expression of ER stress markers were determined using qPCR analysis.5. AOPPs induced EndMT in HRGECs through the induction of ER stressHRGECs were treated with AOPPs or native BSA (both at 200 μg/mL) for 24 h in the presence or absence of salubrinal (50 μmol/L), or the cells were treated with thapsigargin (0.25 μmol/L) alone. The mRNA and protein expressions of the GRP78, CHOP, VE-cadherin, CD31, claudin 5, a-SMA, vimentin, and FSP-1 were determined using qPCR analysis and western blotting, respectively.6. Statistical analysisAll experiments were conducted in triplicate. Results are expressed as means ± SD. Multiple groups were compared using one-way ANOVA. For comparing 2 groups, the LSD method was used, or when the assumption of equal variance did not hold, the Dunnett’s T3 method was used. Differences were considered statistically significant at P< 0.05. Statistical analyses were conducted using SPSS 13.0.Results1. AOPPs induced EndMT in HRGECsTo determine whether AOPPs induce EndMT in HRGECs, we cultured HRGECs in the presence of AOPPs or BSA and then used qPCR to measure the expression of the endothelial markers, CD31, VE-cadherin, and claudin5 and the mesenchymal markers, FSP-1, a-SMA, and vimentin. AOPP treatment downregulated the mRNA levels of VE-cadherin, CD31, and claudin 5 but upregulated those of a-SMA, vimentin, and FSP-1 in a concentration-and time-dependent manner. The expression of these endothelial and mesenchymal markers was not changed in control cells and in cells treated with unmodified BSA, which suggested that the loss of endothelial markers and the overexpression of mesenchymal markers were associated with the advanced oxidation of BSA. Collectively, these results indicated that AOPPs induced EndMT in HRGECs.2. AOPPs triggered ER stress in HRGECsTo examine whether AOPPs triggered ER stress in HRGECs, we evaluated the expression of ER stress markers, GRP78 and CHOP, by performing qPCR. Compared with the levels in control cells, the mRNA levels of GRP78 and CHOP were significantly increased in a concentration-and time-dependent manner in AOPP-treated HRGECs. The expression of GRP78 and CHOP was not changed in control cells and in cells treated with unmodified BSA, which indicated that the overexpression of GRP78 and CHOP was associated with the advanced oxidation of BSA. These results suggested that AOPPs triggered ER stress in HRGECs.3. AOPPs induced EndMT in HRGECs through the induction of ER stressHRGECs were treated with AOPPs or native BSA (both at 200 μg/mL) for 24 h in the presence or absence of ER stress inhibitor salubrinal, or the cells were treated with ER stress inducer thapsigargin alone. Whereas salubrinal treatment partly suppressed the AOPP-induced increase in the expression of GRP78 and CHOP at the mRNA and protein levels, the effects of AOPP treatment were reproduced in cells exposed to thapsigargin alone. Furthermore, salubrinal exposure partly reversed AOPP-dependent downregulation of VE-cadherin, CD31, and claudin 5 expressions and upregulation of a-SMA, vimentin, and FSP-1 expression at the mRNAand protein levels, whereas treatment of cells with thapsigargin alone induced the AOPP effects.Collectively, our data indicated that AOPP-induced EndMT in HRGECs was mediated by ER stress.ConclusionIn this study, we have presented results showing that AOPPs induced EndMT in HRGECs and also triggered ER stress in these cells. Unlike AOPPs, unmodified BSA had not affect on HRGECs, which suggests that the effects were induced as a result of protein modification and were not triggered by BSA or other contaminations. More importantly, our findings indicate for the first time that AOPP-induced EndMT in HRGECs was mediated by ER stress. Our findings further elucidate the mechanisms underlying AOPPs contribute to the progression of DN and provide the theory evidences for the prevention and treatment of DN. |
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