Protective Effects Of Exendin-4on Rat Mesangial Cells Cultured Under High Glucose Conditions And Its Potential Mechanism

BackgroundDiabetic nephropathy (DN) is one of the common microvascular complications in diabetes. The main pathological featuresof DN is glomerular and renal tubular basement membrane thickening, extracellular matrix (ECM) accumulation. DN is one of the main causes of death and disability in diabetic patients. In the western countries, the DN can often cause end-stage renal disease (ESRD).It brings a great financial burden for individuals and society, which has become a serious threat to human health worldwide public health problem. This situation forced people to go to further clarify the pathogenesis of DN and seek effective drug treatment of DN.The mesangial area is mainly composed of mesangial cells and extracellular matrix(ECM). The growing evidence suggests that mesangial cells play an important role in the pathogenesis of DN. Mesangial cell has many physiological effects as follows:1. It can support and protectthe glomerular capillary loops;2. It can secret ECM, inculdingcollagen type IV (Col IV), collagen type V (Col V) and fibronectin (FN), and so on.3.Mesangial cells can produce a variety of cytokines, in autocrine and paracrine pathways;4. Mesangial cells can control the blood flow of glomerular capillary, thus it can regulate glomerular filtration and the ion channel;5. It can transported macromolecular substance by phagocytosis;6. Mesangial cells with Fc receptor and C3b receptorcan participate in the immune response;7. Mesangial cells cansecretrenin. Under normal physiological conditions, mesangial cellskeep the balance of proliferation and apoptosis,and secret little ECM. But, many factors can promote mesangial cell proliferation, reduce cell apoptosisand matrix metalloproteinases secreted, which can increase the expression of FN and Col IV, eventually led to the excessive accumulation of ECM.Some clinical studies such as "diabetes control and complications trial (DCCT), epidemiology of diabetes interventions and complications (EDIC) and the British Prospective diabetes Study (UKPDS) showed thathyperglycemiais closely related to DN and the strict glycemic control can obviously reduce the incidence of DN.Some studies have found that high glucose can cause changes in mesangial cells as follows:1. Diglyceride (DAG) pathway:the glycolytic pathway can produce DAG, DAG further activate protein kinase C (PKC), which can cause a variety of biological effects.2. Advanced glycationend product (AGE):AGEcan produce reactive oxygen species (ROS), which eventually active TGFβ.3. Amino hexose pathways:High glucose make intracellular6-phosphoric acid fructose increase,which can cause more6-phosphoric acid fructose participate into the glycolytic pathway and produce more ECM.4. Aldose reductase pathway:high glucose participate into the aldose reductase pathway, leads to increased sorbitol, antioxidants and ROS,which further activate PKC,amp-activated protein kinase pathway (MAPK), JAK-STAT pathway and eventually induce a series of changes in the mesangial cells.Oxygencan produce some in the physiological station. These free radicals includingsuperoxide anion (O2-), hydroxyl radical (·OH), hydrogen peroxide (H2O2), and so on. Under normal circumstances, the generation of free radicalsmaintain dynamic balance. But in the high glucose and other factors can significantly increased the level of ROS and decreased antioxidant ability, which cause biological membrane lipid peroxidation, and denaturation of protein, DNA injured, finally resulting in tissue and cell damaged. ROS also as an important intracellular messenger, which can activate many signal transduction pathways, indirectly lead to injury of tissues and cells. Mesangial cell can produce ROSby mitochondrial respiratory chain, NADPH oxidase, xanthine oxidase and nitric oxide synthase, and so on. Under the high glucose ambient, mesangial cells generated too much ROS and then activated the polyol pathway, AGEs pathway, PKC pathway, which further promote the generation of free radicals in a vicious cycle.PKC is a family of serine-threonine kinases and it is in the center of multiple signaling pathways, which involved in somekinds of physiological and pathological activities in the body. Under normal circumstances, PKC mainly exist in an inactivated state in the celluar. when the external environment is changed, their specific substrate protein will attract PKC from cytoplasm to membrane, and the conformational of PKC will be changed by a series of phosphorylation of some kinds of protein. Finally the substrate is dissociated from the catalytic zone and activate PKC. The phenomenon which PKC transfer from cytosol to membrane is an important symbol of PKC activation.The mechanism responsible for the activation of PKC by high glucose appears to be related to an elevation of de novo DAG levels.The effect of high glucose on the expression of FN and type Ⅳ collagen can be prevented by general PKC inhibitors such as staurosporine or calphostin C. When mesangial cells treated with PKC agonists, the cells will secret type Ⅳ collagenand FNsuggesting that the effects of high glucose on increasing production of extracellular matrix proteins (ECM) are mediated through PKC activation. The activation of PKC increases the expression of TGF-β1, which is one of the most important growth factors in the regulation of extracellular matrix protein accumulation in diabetic nephropathy. The possible mechanism by which high glucose increases the expression and synthesis of TGF-β1and ECM is by PKC’s actions on inducing transcription factors c-fos and c-jun, which form complexes for activated protein-1(AP-1) binding site. Meanwhile the productin of ROS via NADPH oxidase depend on the activation of PKC.Transforming growth factor (TGF-β) as a kind of intercellular signaling proteins, adjust multiple target gene expression, cell proliferation, differentiation, apoptosis, immune reaction and extracellular matrix synthesis. Studies have shown that TGF-β can increase the ECM; Excessive expression of TGF-βcan arreste cell in G1phase, inhibite cell proliferation and result in cellhypertrophy; Many studies have shown that TGF-βcan result in kidney hypertrophy, glomerular sclerosis and renal tubular interstitial fibrosis. TGF-Pcan decrease the expression of matrix metalloproteinases, and increase fibrinolytic enzyme activators inhibitor PA1and matrix metalloproteinases inhibitor TIMPs which can reduce the degradation of the ECM. After transfection antisense TGF-β1, the expression of PAI-1mRNA of mesangial cells was reduced and the level of PAI-1was increased which can suppress the degradation of ECM. TGF-β1also has a strong effect on promoting fibrosis, the excessive ECM formation. High blood can activatie PKC, which increase the expression of the TGF-β. TGF-β can activate MAPK、P38MAPK which can further increase the expression ofthe TGF-β, and finally form a positive feedback path leading to continuingthe activation of TGF-β. Smads is a keysignal molecular of the downstream of TGF-β, which can bind to serine/threoninekinase receptors of mesangial cell membrane, and transduct signal from outside to the nucleus.The incretin hormones, including glucagon-like peptide-1(GLP-1), are secreted from the gastrointestinal tract. It was found in1980s. The physiological effects of GLP-1as follows.:Fistly,GLP-1can cause the glucose-dependent synthesis and secretion of insulin, inhibit islet β cell apoptosis, suppress glucagon secretion, reduce food intake, slow gastric emptying, enhance glucose metabolism in peripheral tissue and reduce hepatic glycogen output. Secondly, GLP-1regulatesglucose levelby increasing insulin secretion and inhibiting glucagon secretion via glucose-dependent way. In healthy individuals, fasting glucose is managed by tonic insulin/glucagon secretion, but excursions of postprandial glucoseare controlled by insulin and the incretin hormones. Some studies have demonstrated a strickly decline in postprandial GLP-1secretion, where individuals with normal glucose tolerance secrete more than those with impaired glucose tolerance, and individuals with type2diabetes demonstrate theimpairment in GLP-1secretion. But GLP-1can be rapidly degraded by the serine protease dipeptidyl peptidase-4(DPP-4).Exenatide is the first of the GLP-1receptor agonist that currently approved by the State Food and Drug Administration (SFDA), which mainly contains Exendin-4. Exendin-4is a naturally occurring peptide identified in the saliva of the Gila monster (Heloderma suspectum). The molecular structure of exendin-4makes it considerably more resistant than GLP-1to degradation by DPP-4. Exendin-4now has been used for the treatment of type2diabetes. Exenatide stimulates glucose-dependent insulin secretion, suppresses glucagon secretion, regulates fasten and postdainal glycemia and protects islet β cells.A recent study found that liraglutide, inhibits oxidative stress and albuminuria in streptozotocin (STZ)-induced type1diabetes mellitus rats, via a protein kinase A (PKA)-mediated inhibition of renal NAD(P)H oxidases.GLP-1receptor agonist ameliorates renal injury through its anti-inflammatory action without lowering blood glucose level in vivo. Long-term treatment of glucagon-like peptide-1agonist exendin-4ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice.The pathogenesis of DN is complex and is not very clear.Mesangial cells play a very important rolein glomerular sclerosis and fibrosis. Under the high glucose ambient, mesangial cell produce a series of changes, such as oxidative stress enhanced and PKC activation,which these signal molecules can activate TGFβ-smad signaling pathway, resulting in extracellular accumulation of mesangial area, glomerular sclerosis and fibrosis. Accumulating evidence has implicated that GLP-1may have a beneficial effect on renal diseases but the mechanism is not fully understood.The study aims to oberserving that effect of Exendin-4on mesangial cellsinjury induced by high glucoseand discussing the possible mechanism. Thus, we may provide some evidence for the protection effects of exendin-4beyond its glycemic control effect.Part1Effects of exendin-4on cell proliferation, cell cycle and ECM of rat mesangial cells under high glucose conditionsObjective:To study the protective effect of exendin-4on cell proliferation, cell cycle and ECM of mesangial cellscultured under high glucose ambient.Methods:l.The experiment object was rat mesangial cells (HBZY-1). Mesangial cells were cultured in DMEMcontaining10%FBS and5.6%glucose,100ug/ml penicillin and100ug/ml streptomycin at37℃, the cells were passaged every two days.2. The cells were divided into6groups according to different intervention.(1) Control group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM(2) HG group:Mesangial cells were cultured under30mmol/L(3) HG+E1group:Mesangial cells were culturedunder5.6mmol/L glucose-DMEM containing exendin-4(1nmol/L) for24h, then discarded the medium and cultured cells under30mmol/L glucose-DMEM(4) HG+E5group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM containing exendin-4(lnmol/L) for24h, then discarded the medium and cultured cells under30mmol/L glucose-DMEM(5) HG+E10group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM containing exendin-4(10nmol/L) for24h, then discarded the medium and cultured cells under30mmol/L glucose-DMEM(6) HG+E15group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM containing exendin-4(15nmol/L) for24h, then discarded the medium and cultured cells under30mmol/L glucose-DMEMMesangial cells of allgroups were culturedunder5.6mmol/L glucose-DMEM containing0.5%FBS for24hours before intervention to keep synchronized growth of cells.3. MTT method was used to detect the cell proliferation; Flow cytometry was used to detect the cell cycle. Elisa method was used to to detect the content of Col IV and FN in the supernatant of cells.4. Statistical analysis:All data are expressed as means±SD. Statistical analysis were performed using the SPSS13.0software Package. Statistical significance of differences among these groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. Multiple comparisons was analyzed by Dunnett’s T3method when P values less than0.05, P<0.05was considered as significantly.Results:1. Effects of exendin-4on cell proliferation of rat mesangial cellscultured under high glucose ambientAfter24and48h incubation, in comparison with control group, high glucose can significantly promote the proliferation of mesangial cells. After24hincubation, in comparison with HG group, Exendin-4(5nmol/L,10nmol/L,15nmol/L) can significantly inhibit the proliferation of mesangial cells; After48hincubation,in comparison with HG group, Exendin-4(1nmol/L,5nmol/L,10nmol/L,15nmol/L) can significantly inhibit the proliferation of mesangial cells2. Effects of exendin-4on cell cycle of rat mesangial cellscultured under high glucoseambient After48h incubation,the percentage of G1phase in HG group was significantlylower than control group and the percentage of S phase and G2phase was higher than Control group. Compared with HG group, Exendin-4(5nmol/L,10nmol/L,15nmol/L) significantly increased the percentage of G1and decreased the percentage of S phase. Meanwhile exendin-4(10nmol/L、15nmol/L) significantly decreased the percentage of SG2phase in a concentrationdependent manner.3. Effects of exendin-4on FN in the supernatant of rat mesangial cellscultured underhigh glucose ambient. After24h or48h incubation, the expression of FN in the supernatant in HG groupwas significantly increased compared with control group; After24h incubation, Exendin-4(10nmol/L,15nmol/L) can significance decrease the expression of FN in the supernatant in HG group.Exendin-4(5nmol/L,10nmol/L,15nmol/L) can significance decrease the expression of FN in the supernatant in HG group.4. Effects of exendin-4on COL IVin the supernatant of rat mesangial cellsculturedunder high glucose ambient. After24h or48h incubation, The expression of COL IV in the supernatant in HG group was significantly increased compared with control group; After24h incubation,Exendin-4(10nmol/L,15nmol/L) can striking decrease the expression of COL IV in the supernatant in HG group, After48h incubation,Exendin-4(5nmol/L,10nmol/L,15nmol/L) can significance decrease the expression of COL IV in the supernatant in HG group.Conclusions:Exendin-4can inhibit mesangial cell proliferation, arrest cell in G1phase. Meanwhile it can significantly reduce the expression of FN and COL IV in the supernatant in high glucose ambient. These results indicate that Exendin-4may be have some protective effects on DN. Part2Effects of exendin-4on TGFβ-Smad signaling pathway of rat mesangial cells under high glucose conditionsObjective:The purpose of this study was to determine whether exendin-4exert renoprotective effects via TGFβ-Smad signaling pathway.Methods:1. The experiment object was rat mesangial cells (HBZY-1). Mesangial cells were cultured in DMEM (5.6mmol/L glucose) containing10%FBS,100ug/ml penicillin and100ug/ml streptomycin at37℃,the cells were passaged every two days.2. The cells were divided into3groups according to different intervention:(1) Control group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM(2) HG group:Mesangial cells were cultured under30mmol/L(3) HG+E10group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM containing exendin-4(10nmol/L) for24h, then discarded the medium and cultured cells under30mmol/L glucose-DMEMMesangial cells of all these groups were cultured under5.6mmol/L glucose-DMEM containing0.5%FBS for24hours before intervention to keep synchronized growth of cells.3. Real time-PCR was used to detect the the expression of TGF-β1mRNA, Smad2mRNA, Smad3mRNA and Smad7mRNA in rat mesangial cellscultured under30mmol/L glucose ambient.Elisawas used to to detect the expression of TGF-β1in the supernatant of rat mesangial cellscultured under high glucose ambient and Western blottingwas used to to detect the expression of Smad2, Smad3and Smad7in rat mesangial cellscultured under high glucose ambient.4. Statistical analysis:All data are expressed as means±SD. Statistical analysis were performed using the SPSS13.0software Package. Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. Multiple comparisons was analyzed by Dunnett’s T3method when P values less than0.05, P<0.05was considered as significant.Results:1. Effects of exendin-4on TGF-β1mRNA of rat mesangial cellscultured under high glucose ambient.After24h incubation, the expression of TGF-β1mRNA in the supernatant in HG group was significantly increased compared with control group; Compared with HG group, exendin-4can decrease the expression of TGF-β1mRNA in HG group, but no statistical significance (P=0.064).2. Effects of exendin-4on Smad2mRNA of rat mesangial cellscultured under high glucose ambient.After24h incubation, the expression of Smad2mRNA in HG group was increased compared with control group, but no statistical significance; Compared with HG group, exendin-4can decrease the expression of Smad2mRNA in HG+E10group, but no statistical significance.3. Effects of exendin-4on Smad3mRNA of rat mesangial cellscultured under high glucose ambient.After24h incubation, the expression of Smad3mRNA in HG group was increased compared with control group, but no statistical significance; Compared with HG group, exendin-4can decrease the expression of Smad3mRNA in HG+E10group, but no statistical significance.4. Effects of exendin-4on Smad7mRNA of rat mesangial cellscultured under high glucose ambient.After24h incubation, the expression of Smad7mRNA in HG group was strikly decreased compared with control group. Compared with HG group, exendin-4wasstriklyincreased the expression of Smad7mRNA in HG+E10group.5. Effects of exendin-4on TGF-β1in the supernatant of rat mesangial cellscultured under high glucose ambient.After48h incubation, compared with control group the expression of TGF-β1in HG group was striking increased. Compared with HG group, exendin-4can significantly decrease the expression of TGF-β1in HG+E10group.6. Effects of exendin-4on the expression of Smad2and Smad3in rat mesangial cellscultured under high glucose ambient.After48h incubation, compared with control group, the expression of Smad2and Smad3in HG group was striking increased. Compared with HG group, exendin-4can significantly decrease the expression of Smad2and Smad3in HG+E10group.7. Effects of exendin-4on the expression of Smad7in rat mesangial cellscultured under high glucose ambient.After48h incubation, compared with control group, the expression of Smad7in HG group was striking decreased. Compared with HG group, exendin-4can significantly increase the expression of Smad7in HG+E10group.Conclusions:Exendin-4significantly inhibited the expression of TGFβ1,Smad2and Smad3in rat mesangial cellscultured under high glucose ambient.Meanwhile, Exendin-4significantly increased the expression of Smad7in rat mesangial cellscultured under high glucose ambient.which suggests that TGF-Smadpathway activation may involved in Exendin-4protection on mesangial cells. Part3Effects of exendin-4on oxidative stress of rat mesangial cells under high glucose conditionsObjective:The purpose of this study was to determine whether exendin-4exert renoprotective effects via ROS.Methods:1. The experiment object was rat mesangial cells (HBZY-1). Mesangial cells were cultured in DMEM (low glucose) containing10%fetal bovine serum (FBS),100ug/ml penicillin and100ug/ml streptomycin at37℃, the cells were passaged every two days.2. The cells were divided into3groups according to different intervention:(1) Control group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM(2) HG group:Mesangial cells were cultured under30mmol/L(3) HG+E10group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM containing exendin-4(10nmol/L) for24h, then discarded the medium and cultured cells under30mmol/L glucose-DMEMMesangial cells of all these groups were cultured under5.6mmol/L glucose-DMEM containing0.5%FBS for24hours before intervention to keep synchronized growth of cells.3. Spectrophotometric method was used to detect SOD activity in the supernatant of rat mesangial cellscultured under high glucose ambient.;Flow cytometry was used to detect ROS in rat mesangial cellscultured under high glucose ambient.; Elisa method was used to to detect the content of MDA and GSH in the supernatant of rat mesangial cellscultured under high glucose ambient.4. Statistical analysis:All data are expressed as means±SD. Statistical analysis were performed using the SPSS13.0software Package. Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. Multiple comparisons was analyzed by Dunnett’s T3method when P values less than0.05, P<0.05was considered as significant.Results:1. Effects of exendin-4on the content of MDA, GSH and the activity of SOD in the supernatant of in rat mesangial cellscultured under high glucose ambient.After48h incubation, compared with control group, the activity of SOD and the content of GSH in HG group was notably decreased,while the the content of MDA was significantly increased. Compared with HG group, exendin-4can significantly increased the activity of SOD and the content of GSHand decreased the content of MDAin the supernatant of in rat mesangial cellscultured under high glucose ambient.2. Effects of exendin-4on the fluorescence intensity of ROS in rat mesangial cellscultured under high glucose ambient.After48h incubation, compared with control group,the fluorescence intensity of ROS in rat mesangial cellsin HG group was notably increased. Compared with HG group, exendin-4can significantly decreased the fluorescence intensity of ROS inrat mesangial cells in HG+E10group.Conclusions:Exendin-4can increase the activity of SOD and the content of GSH in HG+E10group and can decrease the content of MDA in the supernatant of in rat mesangial cellscultured under high glucose ambient. Meanwhile, it can decrease fluorescence intensity of ROS in rat mesangial cellscultured under high glucose ambient. These results indicate that exendin-4may be exert renoprotective effects via ROS pathway. Part4Effects of exendin-4on the activation of PKCp II of rat mesangial cells under high glucose conditionsObjective: The purpose of this study was to determine whether exendin-4exert renoprotective effects via PKCβ Ⅱ signaling pathway.Methods:1. The experiment object was rat mesangial cells (HBZY-1). Mesangial cells were cultured in DMEM (low glucose) containing10%fetal bovine serum (FBS),100ug/ml penicillin and100ug/ml streptomycin at37℃, the cells were passaged every two days.2. The cells were divided into3groups according to different intervention:(1) Control group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM(2) HG group:Mesangial cells were cultured under30mmol/L(3) HG+E10group:Mesangial cells were cultured under5.6mmol/L glucose-DMEM containing exendin-4(10nmol/L) for24h, then discarded the medium and cultured cells under30mmol/L glucose-DMEMMesangial cells of all these groups were cultured under5.6mmol/L glucose-DMEM containing0.5%FBS for24hours before intervention to keep synchronized growth of cells.3. Western blotting method was used to detect the activation of PKCβⅡ of rat mesangial cellscultured under high glucose ambient4. Statistical analysis:All data are expressed as means±SD. Statistical analysis were performed using the SPSS13.0software Package. Statistical significance of differences among groups was evaluated by one-way ANOVA.Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. Multiple comparisons was analyzed by Dunnett’s T3method when P values less than0.05, P<0.05was considered as significant.Results:1. Effects of exendin-4on the activation of PKCβⅡ of rat mesangial cellscultured under high glucose ambient.After48h incubation, compared with control group, the activation of PKCβⅡ of rat mesangial cells culture in HG group was notably increased. Compared with HG group, exendin-4can significantly decreased the activation of PKCβⅡ of rat mesangial cells in HG+E10group.Conclusions:Exendin-4significantly inhibited the activation of PKCβⅡ of rat mesangial cells cultured under high glucose ambient., which suggests that inhibition of PKCβⅡ pathway activation may involved in Exendin-4protection on mesangial cells.