Advanced Oxidation Protein Products Induce Apoptosis In Podocytes Through Induction Of Endoplasmic Reticulum Stress

Backgroud and ObjectionDiabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) worldwide, and it is a serious public health concern. A prominent clinical manifestation of DN and an independent risk factor for DN progression is proteinuria, which is a marker of disease severity. Proteinuria results from the disruption of the glomerular filtration barrier, which include the innermost glomerular endothelium, the middle glomerular basement membrane(GBM), and the outermost podocyte. Podocytes, which are also referred to as glomerular epithelial cells, are considered to be the most important component of the glomerular filtration barrier. In the maintenance of the structure and function of the glomerular filtration barrier, a crucial role is played by podocytes. Podocytes are highly specialized, terminally differentiated cells that cannot proliferate, and thus podocyte loss through apoptosis might eventually lead to proteinuria in DN. Furthermore, accumulating evidence indicates that podocyte apoptosis and detachment from GBM play a key role in the development and progression of proteinuria and DN. However, the mechanisms responsible for podocyte apoptosis remain incompletely understood.Advanced oxidation protein products(AOPPs) are a class of dityrosine-containing protein cross-linking products that are formed during oxidative stress and are carried mainly by albumin in vivo. Witko-Sarsat et al first reported that the plasma level of AOPPs was elevated in uremia, which was the highest in patients on hemodialysis, followed by those on peritoneal dialysis and by undialyzed patients with advanced chronic renal failure. And, subsequently, accumulation of plasma AOPPs was found in patients with obesity, metabolic syndrome, primary renal diseases, IgA nephropathy and diabetes with or without microvascular complications. Notably, AOPPs levels increase with the deterioration of renal function. So AOPPs have recently emerged as novel renal pathogenic mediators. And elevated concentrations of plasma AOPPs have been widely reported to be closely associated with the pathogenesis of chronic kidney diseases, including DN. AOPPs-treated 5/6 Nx rats displayed greater proteinuria, higher serum creatinine, and lower creatinine clearance, and AOPPs challenge resulted in greater renal fibrosis in the remnant kidney. AOPPs-treated STZ-induced diabetic rats increases the urinary protein excretion. Furthermore, Zhou et al. reported that the accumulated AOPPs interacted with the receptor of advanced glycation end products (RAGE) to induce podocyte apoptosis through NADPH-dependent mechanisms in vivo and in vitro. However, the mechanisms underlying AOPP-induced podocyte apoptosis have not been completely elucidated.Endoplasmic reticulum stress (ERS) has recently reported to be a new pathway in the initiation of cellular apoptosis. The endoplasmic reticulum (ER) is responsible for several critical functions such as the synthesis, folding, assembly, modification, and transport of nascent proteins and the regulation of intracellular calcium levels. Numerous pathophysiological conditions that disturb ER function lead to accumulation of misfolded/unfolded proteins and alterations in calcium homeostasis in the ER, eventually resulting in ER stress. Subsequently, ER stress triggers the unfolded protein response (UPR), a series of integrative stress pathways. In response to ER stress, three UPR signal transduction pathways are initiated by three ER transmembrane proteins:protein kinase-like ER kinase (PERK), activating transcription factor 6(ATF6), and inositol requiring 1(IRE1). However, activation of the UPR either triggers an adaptive response to restore normal ER function and promote cell survival or induces apoptosis if the stress is prolonged or severe. So ERS is a physiological or pathological state.GRP78, which also known as BiP, is an ER specific molecular chaperone and a key regulator of the UPR; Normally,GRP78 binds with PERK^ ATF6 and IRE1,which keep them in an inactive state; When the ER stress occurs, GRP78 separates from the three ER-resident transmembrane transducers, and binds to unfolded or misfiled proteins within the ER lumen. So GRP78 is often regarded as an ER stress marker. After dissociating from GRP78, PERK and IRE1 are autophosphorylated to be activated, and ATF6 is translocated to Golgi, where it is cleaved for activation. Activated ATF6, IRE1, and PERK signals converge on the CHOP, the first molecule shown to regulate ER stress-mediated apoptosis. All three UPR pathways, and especially the PERK pathway, can trigger apoptosis by inducing CHOP. CHOP, which is also referred to as growth arrest-and DNA damage-inducible gene 153 (GADD153), is normally expressed at low levels in unchallenged conditions. ER stress also upregulates CHOP, and thus this molecule is used as another ER stress marker. CHOP-/- mice exhibit reduced levels of apoptosis in response to ER stress, and CHOP-/- cells are resistant to ERS induced apoptosis. So CHOP plays a crucial role in the commitment phase of ER stress induced apoptosis. CHOP-mediated apoptosis is widely accepted to involve the suppression of the anti-apoptotic protein Bcl-2. Caspase-12 is located in the ER membrane, and Caspase-12 activation is involved in the execution phase of ER stress-induced apoptosis. procaspase-12 is cleaved for activation after initiation of ER stress. Activated caspase-12 can activate caspase-9 and subsequently catalyze the cleavage of procaspase-3 in the ER stress-specific cascade.ER stress has been widely demonstrated to contribute to glomerular and tubular injury in kidney diseases. Furthermore, ER stress has been shown to be a mediator of podocyte apoptosis induced by various factors such as advanced glycation end products, albumin, saturated fatty acid palmitate, and high glucose. However, whether ER stress is involved in AOPP-induced podocyte apoptosis remains unclear. In our previous study, we found AOPPs could induce hypertrophy and epithelial-to-mesenchymal transition in human proximal tubular cells through induction of ER stress, and also might induce epithelial-to-mesenchymal transition in cultured podocytes through the induction of ER stress. So it suggested that ER stress is involved in AOPP-induced podocyte apoptosis. Furthermore, Zhou and colleagues have been demonstrated that NADPH oxidase-dependent O2-generation mediates AOPPs-induced podocyte apoptosis through interaction with RAGE rather than other scavenger receptors (SRs) such as CD36 and SR class A, which are expressed on the surface of podocytes. Recent studies have indicated that a close relationship exists between oxidative stress and ER stress. ROS production and oxidative stress are not only coincidental to ER stress, but are integral UPR components, contributing to support pro-apoptotic UPR signaling, which indicates that ROS generation is closely linked to ER stress. Collectively, it suggested that the ER stress involved in AOPPs-induced apoptosis in podocytes might be mediated by NADPH oxidase-dependent ROS generation through interaction with RAGE. This study aims at to demonstrate the our aboved hypothesis in vitro cultured mouse podocyte and further investigates the mechanisms of AOPPs-induced podocyte apoptosis.Methods and materials1.AOPPs-MSA preparation and content determinationAOPPs-MSA was prepared as previously described. Briefly, an MSA solution was exposed to 200mmol/L HOCl for 30 min at room temperature and then dialyzed against phosphate-buffered saline (PBS) at 4℃ for 24h to remove free HOCl. In the control incubation, native MSA was dissolved in PBS alone. All preparations were passed through a Detoxi-Gel columnin order to remove contaminating endotoxins. The endotoxin levels in the preparations were measured using the Amebocyte lysate assay kit and were determined to be<0.025EU/mL. The AOPP content was determined by measuring the absorbance at 340nm in an acidic condition, and it was calibrated using chloramine-T in the presence of potassium iodide. The AOPP contents in AOPPs-MSA and unmodified MSA were 70.2±3.41 and 0.23±0.05nmol/mg, respectively. The components of AGEs, including NE-(carboxylmethyl) lysine, pyridine, and pentosidine and glyoxal-, glycolaldehyde-, and glyceraldehydemodified proteins, were determined as previously described and were undetectable in the prepared samples.2. Cell cultureMurine podocytes were cultured as described. The cells were grown in RPMI 1640 containing 10%fetal bovine serum, streptomycin (100μg/mL), penicillin (100U/mL), sodium pyruvate (1mmol/L), sodium bicarbonate (0.075%), and HEPES buffer(10mmol/L). The cells were grown "undergrowth-permissive" conditions, which involved growing cells at 33℃ in the presence of interferon-y. To induce differentiation, cells were grown under "restrictive" conditions, at 37℃ for>12 days, in the absence of interferon-y. Experiments were performed using growth-restricted, conditionally immortalized podocytes of passages 10-18.In some experiments, the cells were treated with the ER stress inducer thapsigargin (0.25μM) for 24h. In the blocking experiments, the cells were pretreated with the ER stress inhibitor salubrinal(50μM), the ROS scavenger c-SOD (100U/mL), and the NADPH oxidase inhibitor DPI (10μM) for lh, respectively, before the cells incubated with 200μg/mL AOPPs for 24h.3. Real-time reverse transcription-polymerase chain reaction (RT-PCR)Total RNA was extracted from cells by using TRIzol reagent according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA according to PrimeScript RT reagent Kit Perfect Real Time. Quantitative real-time PCR was performed using a PCR SYBR Premix Ex TaqTMⅡ. The RT-PCR conditions were the following:95℃ for 2 min, followed by 40 cycles of 95℃ for 30s and 60℃ for 35s. The following sets of primers were used: β-actin:5’-GCCTTCCTTCTTGGGTATGG-3’(forward) and 5’-AGACAGCACTGTGTTGGCATAG-3’(reverse), GRP78:5’-CCTATTCCTGCGTCGGTGTG-3’(forward) and 5’-GCATCGAAGACCGTGTTCTC-3’(reverse), CHOP:5’-GGAAACGAAGAGGAAGAATC-3’(forward) and 5’-CTGGGCCATAGAACTCTGAC-3’(reverse), Bcl-2:5’-ATAACCGGGAGATCGTGATG-3’(forward) and 5’-GTTGCTCTCAGGCTGGAAGG-3’(reverse).All data were normalized using the internal control β-actin, and expression levels were analyzed using the 2-△△Ct method. 4. Western blot analysisCells were lysed in RIPA buffer, and the lysates were separated using SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk in TBST for 2h and incubated overnight at 4℃ with primary (rabbit) antibodies against the molecules PERK、IRE1、ATF6、p-PERK、p-IRE1、cleaved ATF6、CHOP、 Bcl-2、caspase-12、 cleaved caspase-3 and RAGE (dilution,1:1,000). The immunoblots were then incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1h. Proteins were detected using an enhanced chemiluminescence system, and band densities were quantified using Total Lab2.0 software. The internal control was β-actin.5. siRNA transfectionThe non-specific siRNA and siRNAs for RAGE, PERK, IRE1, and ATF6 were purchased from Shanghai Gene Pharma Co., Ltd (Shanghai, China). Podocytes were transfected with siRNA using Lipofectamine2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. Targeting sequences for each gene:RAGE:5’-GGUCAGAGCUGACAGUGAUTT-3’PERK:5’-GCAGGTCCTTGGTAATCAT-3’ATF6:5’-GCAAAGCAGCAGTCGATTA-3’IRE1:5’-CCAATGTATGTCACAGAAA-3’.The non-specific siRNA was used as a negative control in our experiments. The silencing effects of siRNAs for PERK, ATF6, or IRE1 were determined using Western blot analysis.6. TUNEL assayApoptosis in podocytes was assessed by using the DeadEnd TM Fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) System according to the manufacturer’s protocol. Briefly, cells were fixed in 4% paraformaldehyde at 4℃ for 30min, permeabilized with 0.1%Triton X-100 and 0.1% sodium citrate, and then incubated with terminal deoxynucleotidyl transferase and fluoresceinconjugated nucleotides at 37℃ for 60min. The localized green fluorescence of apoptotic cells, produced by fluorescein-12-dUTP, was detected using fluorescence microscopy. To control for false-positive cells, samples were counterstained with 4’,6’-diamino-2-phenylin-dole (DAPI, blue fluorescence; Molecular Probes) to identifythe nucleus.7. Measurement of intracellular ROSIntracellular ROS production was detected by Reactive Oxygen Species Assay Kit. After treatment with AOPPs(200μg/mL) for 24h, podocytes were washed with PBS for three times and incubated with 10μM of 2’,7’-dichlorofluorescin diacetate(DCFH-DA) for 20min at 37℃ in the dark. The cells were then washed three times with serum-free RMPI1640. The qualitative analysis of ROS generation was done using a plate reader at 488-nm excitation and 525-nm emission.8. Statistical analysisAll experiments were conducted in triplicate. Results are expressed as means±SD. Two independent samples were compared using Independent-Samples T Test. Multiple groups were compared using one-way ANOVA. For comparing two groups, the LSD method was used, or when the assumption of equal variance did not hold, Dunnett’s T3 method was used. Differences were considered statistically significant at P<0.05. Statistical analyses were performed using SPSS20.0 software.Results1. AOPPs induced ER stress in podocytesTo investigate whether AOPPs induced ER stress in podocytes, we examined the expression of GRP78 and CHOP by performing RT-PCR and Western blot assays. Exposure of podocytes to AOPPs increased the expression of GRP78 and CHOP at the mRNA and protein levels in a dose-and time-dependent manner. However, this effect was not observed in either untreated cells or in cells treated with unmodified MSA, indicating that the overexpression of GRP78 and CHOP was related to the advanced oxidation of MSA.2. AOPPs triggered podocyte apoptosis by inducing ER stresswe quantified the rates of podocyte apoptosis by using the TUNEL. The apoptotic rate of AOPPs-treated cells was significantly higher than that of untreated cells and cells treated with unmodified MSA, and this AOPPs effect was partly suppressed by salubrinal and reproduced by thapsigargin.3. AOPPs-induced ROS generation might be mediated by NADPH oxidaseThe cellular ROS levels of AOPPs-treated cells was significantly higher than that of untreated cells and cells treated with unmodified MSA, and AOPPs-triggered ROS generation was significantly suppressed by the NADPH oxidase inhibitor(DPI) and the scavenger of ROS(c-SOD).4. AOPPs-induced ER stress might be mediated by NADPH oxidase-dependent ROS generation.AOPPs-induced increase in expression of GRP78 and CHOP at the protein level was partly abolished by the NADPH oxidase inhibitor(DPI) and the scavenger of ROS(c-SOD) by Western blot assays.5. ER stress may contribute to AOPP-induced ROS generation.The cellular ROS levels of AOPPs-treated cells was significantly higher than that of untreated cells and cells treated with unmodified MSA, but AOPPs-induced ROS generation was partly reversed by salubrinal and reproduced by thapsigargin.6. AOPPs-induced podocyte apoptosis might be mediated by NADPH oxidase-dependent ROS generation.we determined the apoptotic rate of the cells treated with AOPPs in the presence or absence of DPI or c-SOD using the TUNEL method. The apoptotic rate of the cells treated with AOPPs in the presence of DPI or c-SOD was significantly lower than that of cells treated with AOPPs alone.7. The efficiency of siRNA targeting RAGE was evaluated.Cells were transfected with scramble siRNA or siRNA targeting RAGE. The efficiency of siRNA targeting RAGE was evaluated by Western blotting of RAGE. RAGE siRNA reduced the RAGE protein expression more than 70%.8. AOPPs-induced ROS generation and ER stress in podocytes were mediated by RAGE.Cells were transfected with scramble siRNA(siNC) or RAGE siRNA(siRNA) before AOPPs treatment, or the cells were transfected with scramble siRNA alone. The cellular ROS levels of siRNA was significantly lower than that of siNC. Western blotting results show that RAGE silencing significantly abrogated AOPPs-induced overexpression of GRP78 and CHOP.9. RAGE mediated AOPP-triggered apoptosis in podocytes.Cells were transfected with scramble siRNA or RAGE siRNA before AOPP treatment. Results of TUNEL assay showing that the apoptotic rate of the RAGE siRNA-transfected cells was significantly lower than that of the scramble siRNA transfected cells.10. AOPPs activated the PERK, ATF6, and IRE1 pathways of UPR in podocytes.We examined the expression of p-PERK,p-IREl and cleaved ATF6 by performing Western blot assays. AOPPs treatment induced phosphorylation of PERK and IRE1 and upregulated the protein level of cleaved ATF6 as compared with the corresponding levels in MSA-treated cells.11. The efficiency of siRNAs targeting PERK, ATF6, and IRE1 was evaluated.The cells were transfected with scramble siRNA or siRNAs targeting PERK, ATF6, or IRE1. Western blotting results show that siRNAs targeting PERK, ATF6, and IRE1, respectively, strongly down-regulated the protein expression of the three major ER stress sensors in podocytes compared with scramble siRNA.12. The all three UPR pathways, the PERK, ATF6, and IRE1 pathways, mediated AOPPs-induced podocyte apoptosis.Before AOPPs treatment, cells were transfected with scramble siRNA, PERK siRNA, ATF6 siRNA, or IRE1 siRNA, respectively. Knockdown of the three major ER stress sensors, respectively, significantly lowered the apoptotic rate of the cells compared with that of the scramble siRNA-transfected cells.13. CHOP-and caspase-12-dependent pathways were involved in ER stressmediated apoptosis, and CHOP-mediated apoptosis was regulated by Bcl-2.we examined the expression of CHOP, Bcl-2, caspase-12, and cleaved caspase-3 by performing Western blot and RT-PCR assays. Compared with the levels in untreated cells and native MSA-treated cells, the protein levels of CHOP, caspase-12, and cleaved caspase-3 were increased and those of Bcl-2 were decreased in AOPPs-treated podocytes. Moreover, the mRNA levels of CHOP were significantly upregulated and those of Bcl-2 were downregulated in AOPPs-treated podocytes. These effects of AOPPs were partly blocked when the cells were incubated with both AOPPs and salubrinal. By contrast, the aforementioned AOPPs effects were reproduced in cells treated with thapsigargin alone.Conclusionour results further suggest that AOPPs can induce ER stress in podocytes and, notably, they indicate that AOPPs trigger podocyte apoptosis through the induction of ER stress. Furthermore, our data indicates that the ER stress involved in AOPPs-induced podocyte apoptosis might be mediated by NADPH oxidase-dependent ROS generation through interaction with RAGE. Moreover, we show that all the three pathways of UPR, the PERK, ATF6, and IRE1 pathways, mediate ER stress-induced apoptosis in podocytes. Lastly, we have demonstrated that two ER stress-associated death pathways, the CHOP- and caspase-12-dependent pathways, mediate ER stress-induced podocyte apoptosis and that Bcl-2 suppression is involved in CHOP mediated apoptosis. Podocyte apoptosis plays a crucial role in the pathogenesis of DN, and the accumulation of AOPPs is prevalent in DN patients. Therefore, our findings could offer a novel strategy for treating DN by targeting ER stress.