The Effects And Mechanism Of Glucagon-like Peptide-1 (GLP-1) On Apoptosis Of Podocytes Induced By Advanced Oxidation Protein Products (AOPPs)

[Background]With the development of economy, the prevalence of diabetes mellitus is increasing year by year, resulting from living standards improved, diet structure changed and body activities decreased. It was estimated by 2009 there has been 9,2400 thousand people diagnosed with diabetes mellitus. Diabetic nephropathy (DN) was considered as severe microvascular complication of diabetes mellitus, the development and progression of diabetic nephropathy leads to end-stage renal disease, contributing greatly for the mortality and disability of diabetes. DN has been the first disease to cause end-stage renal disease in developing countries. In China, DN ranks the second after glomerular nephritis. The cost of treatment for end-stage renal disease is too high to bring a heavy burden no matter on fiscal or family. Thus, it’s significantly important to make emphasis on early intervention for DN.Research on the mechanism of DN was initially focused on the accumulation of mesangial matrix and thickening of glomerular basement membrane, while it can’t explain the changes of urine protein excretion and high filtration completely. In recent years, damage in the structure and function of glomerular filtration barriers, especially podocytes as major component, has become new hotspots. Both clinical and experimental data suggest that podocytes play an important role in the development of DN.Podocyte, or glomerular visceral cell, is one of the major cell types in the glomerulars, which forms a critical part of the glomerular filtration barrier and functions to prevent urinary protein leakage and to maintain glomerular capillary loop integrity. Podocytes depletion leads to area of denuded glomerular basement membrane, culminating in proteinuria and development of glomerulo-sclerosis.Numerous factors have been implicated in the pathogenesis of podocyte injury and depletion:hyperglycemia, hypertension, hyperlipidemia, angiotensin Ⅱ, reactive oxygen species (ROS), transforming growth factor-P,and receptor for advanced glycation end products (RAGE) have been extensively characterized. The mechanism of upregulation of RAGE, as well as its upstream and downstream pathways has become hotpot.Recently, a family of oxidized protein compounds, termed "advanced oxidative protein products (AOPPs)", has emerged as a novel class of renal pathogenic mediators. AOPPs are a class of dityrosine-containing protein products formed during oxidative stress and carried mainly by albumin in vivo. The accumulation of plasma AOPPs was first identified in patients who underwent dialysis and was subsequently found in subjects with diabetes, metabolic syndrome, and nondiabetic chronic kidney disease. An increase in the concentration of plasma AOPPs to the level that has been found in diabetic patients increases urinary excretion of albumin in both normal and streptozotoin-induced diabetic rats. Consistent with these observations, data from a clinical study have shown that plasma AOPP level is a strong predictor for the prognosis of IgA nephropathy.Zhou LL and her team members have studied on the underlying cellular and molecular mechanisms of AOPPs accumulation in the progression of proteinuria and glomerulosclerosis. They found that accumulation of AOPPs promotes podocyte deletion by the induction of podocyte apoptosis, which is mainly mediated by NADPH oxidase-dependent O-2 generation.Advanced oxidative protein products (AOPPs) play a central role in the pathogenesis of diabetic nephropathy by mediating the injury and apoptosis of podocytes via a receptor for advanced glycation endproducts (RAGE) enhancing oxidative stress. Recently studies have identified that glucagon-like peptide-1 (GLP-1) can protect cells against AOPPs-induced oxidative damage via down-regulating the expression of RAGE.GLP-1 is a polypeptide secreted by the intestinal L cell. GLP-1 is best known as a safe and effective insulinotropic hormone and has been proposed as prospective approach to clinical treatment of type 2 diabetes mellitus (T2DM). Besides the well-known actions that in pancreatic cells, GLP-1 stimulate survival and proliferation, and increased insulin secretion, GLP-1 also plays an important role in diabetic cardiovascular complications. GLP-1 inhibits adhesion of monocytes to aortic endothelial cells, in order to alleviate inflammatory injury and inhibit AS progression. GLP-1 can reduce TNF-alpha-induced inflammatory, in order to protect endothelial cells against hypercoagulation and inflammatory. Therefore, the long-acting GLP-1analogue, liraglutide, has been suggested to be used for improve the endothelial cell dysfunction associated with premature atherosclerosis identified in type 2 diabetic patients. Moreover, GLP-1 receptor agonist exendin-4 promotes endothelial repair quickly due to stimulating proliferation of vascular endothelial cells.Our preliminary study in vitro confirmed that GLP-1 can suppress the expression of RAGE protein and mRNA leval in renal mesangial cells and vascular endothelium cells, lower intracellular ROS production and inhibit apoptosis. Thus we hypothesize that GLP-1 could ameliorate early injuries and apoptosis of podocytes by blocking AOPPs-RAGE pathway, consequently delay or suspend the advance and onset of DN.[Objectives]In this project we aim on the base of apoptotic model induced by AOPPs, to observe the effects of GLP-1 on apoptosis of podocytes induced by AOPPs, and investigate the effects of GLP-1 on apoptotic related molecules pathways.[Content]The whole project includes three parts:Part Ⅰ The effects of GLP-1 on apoptosis of podocytes induced byAOPPsObjectivesTo establish the apoptotic model by treating podocytes with AOPP-RSA, and analyze the effects of GLP-1 on apoptosis of podocytes induced by AOPPs.Methods1. Preparation of AOPP-RSA in vitroRSA was dissolved in phosphate-buffered saline (PBS; pH=7.4) with D-glucose, then incubated for 12 weeks in the dark. AOPP-RSA was identified using a fluorescence spectrophotometer.2. Culture of podocytesPodocytes were donated from Nanfang Hospital, extended into RPMI 1640 which do not contain γ-interferon at 37℃ in a humidified atmosphere of 5% CO2 after cell culture incubator to differentiation.3. Cell viability was assessed by CCK8.Groups:AOPP-RSA treated the cells at concentrations of 200 μg/mL for 48h;. The protective effect of GLP-1 on AOPPs-induced apoptosis was tested by co-incubation with different concentrations GLP-1 (0,10,50,100,200nmol/L) for 48h.The cells are planted in 96-well plates. After each experimental treatment, add CCK8 solution to each hole, and add 200μL DMSO to each hole. Absorption was measured at 570 nm with a microplate reader. Meanwhile, Then, set up blank holes which only contain culture medium and without cells, regulate to zero based on blank cells while comparing the colors. The absorbance measured by CCK8 reflects cells injury.4. Morphology of cells apoptosis was observed by Hoechst 33258 staining.5. Flow cytometry assay apoptosisCells were divided into four groups:control group,200μg/mLAOPP-RS A group, 200μg/mLAOPPs+100nmol/LGLP-1 group,100 nmol/LGLP-1 group. After incubation for 48h, cells were collected, centrifuged and drop the supernatant, washed twice with PBS. The cells were resuspended in PBS and stained with Annexin V and PI for 15 min at room temperature. Flow cytometric analyses were performed on a FACSCalibur flow cytometer, and the early apoptotic rate were analyzed with the Cell Quest analysis program.Statistical AnalysisAll analyses were carried out with SPSS 20.0 software. Data are expressed as mean ± standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. We used Spearman correlation analysis for correlation analysis. Statistical significance was defined as two-sided p<0.05.Results1. The results of GLP-1 inhibiting cytotoxicity induced by AOPPs showed that the OD value of AOPPs is significantly different with control ones (P=0;000). The OD value of AOPPs+GLP-1 (10,50,100,200nmol/L) groups were 0.1±0.01、 0.16±0.02、0.21±0.03、0.23±0.02 respectively, and compared with AOPPs group, results showed that the OD value of in AOPPs+GLP-1 (50,100,200nmol/L) groups were significantly higher than AOPPs group (AOPPs vs.50 nmol/LGLP-1+AOPPs, P=0.000; AOPPs vs.100,200 nmol/LGLP-1+AOPPs, P=0.000). However, there was no statistical difference between AOPPs+10nmol/L GLP-1 and AOPPs group(P=0.932). The OD value of control was higher than the other five groups (P=0.000). Spearman correlation analysis showed that there was a significant positive correlation exists between the OD value and GLP-1 concentrations in which 10, 50,100,200nmol/L GLP-1+AOPPs groups (r=0.901, P=0.000).2. The apoptotic cells in AOPPs groups which showed typical morphological changes were observed by using Hoechst 33258 staining. Apoptotic cells were observed as unintact round nuclei and fragmented or condensed nuclei.3.The results obtained from FCM showed that the apoptotic rate in AOPPs group is (26.78±2.58)%, which significantly higher than that of control (7.27±0.55)% (P=0.000), and higher than AOPPs+GLP-1 group (14.43±0.93)%(P=0.001).ConclusionCertain concentration range (50-200nmol/L) GLP-1 can restore the cell viability inhibited by AOPPs, and partly attenuate the apoptosis induced by AOPPs in a dose dependent fashion.Part Ⅱ The molecular mechanisms of the anti-apoptotic action of GLP-1Section I The effects of GLP-1 on oxidative stress and important caspases induced by AOPPs in podocytesObjectivesWe analyzed the level of oxidative stress by confocal laser, and tested the activities of caspase-9,-3 using enzyme-linked immunosorbent assay (ELISA), in order to analyze the mechanisms of the anti-apoptotic action of GLP-1.Methods1. The level of reactive oxygen species (ROS) was quantified by 2’, 7’-dichlorofluorescein diacetate assay.2. The activities of caspase-9 and -3 were detected by ELISA.Statistical AnalysisAll analyses were carried out with SPSS 20.0 software. Data are expressed as mean ± standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. GLP-1 inhibits the ROS generation induced by AOPPsDCF fluorescence in cells exposed to AOPPs was 64.60±2.91, strikingly increased compared with control group (30.58±2.06, P=0.000). DCF fluorescence of AOPPs+GLP-1 group was 48.93±2.99, significantly lower than that of AOPPs (P=0.000), but still significantly higher than control group (P=0.000).2. Caspase-9,-3 activities of AOPPs group were significantly higher than that of control group (P=0.000,0.000) and AOPPs+GLP-1 group (P=0.000,0.000), respectively.ConclusionExposure to AOPPs for 48h caused a significant increase in ROS generation and activities of caspase-9 and -3, but co-incubation with GLP-1 attenuate these AOPPs-induced effects in podocyte. Therefore, we speculated that oxidative stress and caspase-9,-3 may contribute to the anti-apoptotic actions of GLP-1 against AOPPs.Section II The effects of GLP-1 on RAGE pathway induced by AOPPs in podocytesObjectivesTo analyze the effects of GLP-1 on RAGE pathway and the downstream Bcl-2, Bax level in AOPPs-treated podocytes apoptosis.Methods1. RT-PCR assay RAGE mRNA.2. Flow cytometry assay apoptosis. Use the same method with that of Chapter I.3. Western-blot assay RAGE, Bcl-2, Bax.Cells with various treatments were collected and lysed in protein lysis buffer. The supernatant contained the cell extracts and the protein concentration was measured using the BCA protein assay. Equal amounts of protein (20 μg) from each sample were separated on 12% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% non-fat milk for 2h and incubated with either anti-p-RAGE (1:2,000), anti-Bcl-2 (1:1,000), anti-Bax (1:2,000). Secondary specific horseradish-peroxidase-linked antibodies (1:5,000) were added for 1 h, and immune complexes were detected by ECL chemiluminescence, the band intensity was measured and quantified with ImAOPP J software.Statistical AnalysisAll analyses were carried out with SPSS 20.0 software. Data are expressed as mean ± standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. We used Spearman correlation analysis for correlation analysis. Statistical significance was defined as two-sided p<0.05.Results1. The effects of cotreatment with exendin9-39 on the anti-apoptotic action of GLP-1 against AOPPsApoptotic rate in AOPPs group was (27.57±1.11)%, which was significantly higher than that of control group (6.55±0.31)%, (P=0.000), and also higher than that of AOPPs+GLP-1 group (9.53±0.64)(P=0.002). Apoptotic rate in exendin9-39 treatment group was (18.41±1.16)%, which was significantly higher than that of AOPPs+GLP-1 group (P=0.000).3. The effects of pretreatment with exendin9-39 on the expression of Bcl-2, Bax and RAGE in podocytes.AOPPs significantly increased the expression of Bax and RAGE (P=0.006, 0.000 vs. control), and markedly decreased the expression of Bcl-2 (P=0.000, vs. control). The expression of Bax, RAGE was higher than AOPPs+GLP-1 group (P=0.014,0.002), while the expression of Bcl-2 was lower (P=0.001, vs. AOPPs+GLP-1 group). Cotreatment with exendin9-39 partially attenuated the inhibition effects of GLP-1 on upregulation of Bax and RAGE and reduced the stimulation effects of GLP-1 on upregulation of Bcl-2 (P=0.004 vs. AOPPs+GLP-1).ConclusionThis study revealed that GLP-1 could prevent cell apoptosis of podocytes expose to AOPPs. We also have provided that GLP-1 may play an antiapoptotic role through regulating the expression of RAGE、Bcl-2、Bax and caspase ensyme.