The Protective Effect Of Exendin-4 And Its Mechanism In Damages Of Renal Tubular Epithelial Cells Induced By Advanced Glycation End Products

BackgroundAccording to the 2019 International Diabetes Federation Diabetes Atlas,there are 463 million people suffering from diabetes in the world and 116.4 million in China.Diabetes and its complications has brought heavy pressure to human health and society.Diabetic kidney disease(DKD),one of the microvascular complication of diabetes mellitus,is the main causes of end-stage renal disease,which seriously lower the quality of patien’s life.Therefore,it is important to further study the pathogenesis of DKD and find effective treatment and preventive measures.Autophagy is a kind of adaptive response to stress,which is a universal regulatory way in cells.In this process,the cytotoxic proteins and organelles can be degraded,and thus the energy resources was reused in the cells to cope with nutrient depletion.Research increasingly suggests that the activation and recovery of autophagy may be related to the improvement of DKD,which is helpful to delay the progression of this disease.AMPK/mTOR signaling pathway may be the regulatory target of autophagy to improve renal function.Glucagon like peptide-1(GLP-1)is a gastrointestinal hormone secreted by L cells in the intestine,which can promote the glucose-induced insulin secretion and increase satiety.GLP-1 can also act on a variety of tissues and organs besides pancreas,so it has a wide and important biological effect on brain,lung,kidney,stomach,heart and other tissues.In recent years,many clinical trials have shown that GLP-1 may play a renal protective role in DKD.In animal experiments,it was observed that GLP-1 can improve high glucose mediated renal tissue damage by reducing oxidative stress level.However,whether GLP-1 can protect renal function in DKD by regulating autophagy and its specific mechanism has not been determined.ObjectiveIn this study,we established an AGEs-induced renal tubular epithelial cells injury model with HKC-8.The aim of this study was to observe the effect of GLP-1 analogue exendin-4 on AGEs-induced changes in cell proliferation and oxidative stress,as well as its effect on autophagy related proteins and AMPK/mTOR pathway,so that to have an preliminary research on the protective effect and mechanism of exendin-4 on AGEsinduced renal tubular epithelial cells injury.MethodHKC-8 was selected as the renal tubular epithelial cell model,and the GLP-1 receptor agonist exendin-4 was used in this study.The cells were pretreated with AGEs of 200 μg/ml to establish renal tubular epithelial cell injury model,and then were intervened with different concentrations of exendin-4.Cell proliferation was detected by CCK8 kit,intracellular oxidative stress level and the distribution of Nrf2 were detected by immunofluorescence technique,and the expression levels of LC3,p62 and AMPK/mTOR pathway related proteins were detected by western blot.Result1.GLP-1 receptor was expressed in HKC-8 cells according to the results of Western blot and immunofluorescence.2.AGEs decreased the proliferation level of HKC-8 cells.Exendin-4 significantly enhanced the proliferation ability of HKC-8 cells after AGEs mediated injury in a concentration dependent manner,while this effect could be blocked by GLP-1 receptor antagonists.3.Intracellular ROS level was increased significantly after AGEs treatment.Exendin4 can reduce the elevated intracellular ROS level induced by AGEs,while this effect could be blocked by GLP-1 receptor antagonists.4.Compared with the control group,p-AMPK/AMPK was decrease in the AGEs group,the AMPK signal pathway was inhibited.While exendin-4 treatment increased p-AMPK/AMPK,the AMPK signaling pathway was activated.5.Compared with the control group,p-mTOR/mTOR was increased in the AGEs group,the mTOR signaling pathway was activated.While exendin-4 treatment could significantly decrease p-mTOR/mTOR,the mTOR signaling pathway was inhibited,and its effect could be blocked by GLP-1 receptor antagonists.6.Compared with the control group,the expression of autophagy related index LC3B II was decreased,and expression of p62 tend to increase in the AGEs group.Compared with the AGEs group,LC3BII/LC3BI was significantly increased,and expression of P62 was significantly reduced in the exendin-4(100nM)group,and its effect could be blocked by GLP-1 receptor antagonists.7.Nrf2 was mainly distributed in the cytoplasm after AGEs treatment,while exendin4 promoted Nrf2 transfer into nucleus.8.The effect of Exendin-4 decreasing intracellular ROS level was enhanced by rapamycin,an inhibitor of mTOR;while blocked by 3BDO,an activator of mTOR.ConclusionIn conclusion,this experiment demonstrated that the GLP-1 analogues exendin-4 can alleviate damage of renal tubular epithelial cells induced by AGEs.Exendin-4 play a renal protective effect through activation of autophagy by regulating AMPK/mTOR signaling pathway to reduce intracellular oxidative stress.