Activation Of Renal Tubule Epithelia Cell Autophagy Induced By High Glucose And The Regulating Effect Of Liraglutide On It

Diabetic nephropathy is one of the serious complications for diabetes mellitus(DM) patients. It was reported that almost half of patients who had type 1 and 15% who had type 2 DM patients could become endstage renal disease. Tubular dysfunction has been reported in DM, which suggests a pivotal role of tubulo-interstitium in diabetic nephropathy development. Nevertheless, the exact mechanism underlying tubular dysfunction in DM remains unknown.Autophagy is a self-degradative process that is important for balancing sources of energy at critical times. Autophagy also plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria, endoplasmic reticulum and peroxisomes, as well as eliminating intracellular pathogens. Thus, autophagy is generally thought of as a survival mechanism. Misregulation of autophagy can result in a variety of pathological conditions in human beings. Although our understanding of regulatory pathways that control autophagy is still limited, an increasing number of studies have shed light on the importance of autophagy in a wide range of physiological processes and human diseases. Autophagy is necessary to maintain architecture and function of pancreatic beta cells. Altered autophagy is also involved in pancreatic beta cell death.As autophagy plays a protective or harmful role in diabetes is still not clear, its role on the pathogenesis of diabetic nephropathy is unknow too.Liraglutide is an analogy of human GLP-1. GLP-1 binds with GLP-1 receptor(GLP-1R) to stimulate glucose dependent insulin secretion; potentiates β cell response to glucose; stimulates β cell neogenesis and proliferation;inhibits β cell apoptosis and inhibits glucagon secretion. The regulating effect of GLP-1 on autophagy is not clear yet.Part 1 High glucose induced HK-2 cell defectObjective: To explore the influence of high glucose on HK-2 cell. Method:HK-2 cells were incubated in different concentrion of glucose medium for 72h; incubated in 40 m M glucose for up to 72 h.Then HK-2 cells were divided into six groups,cultured in DMED with normal glucose(N); 40mmol/l glucose(HG); 40mmol/l glucose with different concentetion of Liraglutide(HG+1nm/l LA; HG+10nm/l LA; HG+100nm/l LA)and40mmol/l glucose with 100nm/l Liraglutide and 1000nm/l exendin-39(HG+100 nm/l LA+1000nm/l EX) for 72 h. Cell viability was assessed by MTS assay.The dead cells were stained with Trypan Blue. Changes of ultra-structure in HK-2 cells induced by high glocose and examined under TEM. The expression of GLP1-R were detected by western blot. Results: Cell viability deceased in a glucose concentration-dependent manner and a time-dependent manner. There is exact expression of GLP-1R in HK-2 cells.Liraglutide reduced the effect of high glucose on cell viability in a concentration-dependent manner. Exposure to the high glucose for 72 h, dead-cell significantly increased. The ultra-structure of HK-2 cells is injured. Conclusion: High-glucose induced toxicity in HK-2 cells. Liraglutide can protect cells from this harm.Part 2 High glucose induced activation of autopahgy in HK-2cells and regulating effect of Liraglutide on itObjective: To explore if high glucose can induced activation of autopahgy in HK-2 cells and the regulating effect of Liraglutide on it. Methods: After HK-2 cells were incubated with different mediem(N, HG, HG+100nmol/l LA, HG+100nmol, l LA+1000nmol/l EX) for 72 h. Autophagosome were examined under TEM.Real time PCR, western blot and Immunofluorescence staining were used to detect the expression Of GLP-1R. The accumulation of acidic vesicular organelles were visualized by acridine orange staining.LC3 and beclin-1 were detected by western blot. Results: Autophagosome were observed in cells treated with high glucose.Hk-2 cells stimulated with 40 mmol/l high glucose for 72 h showed significantly decreased GLP1-R expression. Liraglutide(LA) significantly enhanced GLP1-R expression. The accumulation of acidic vesicular organelles enhanced in group of HG, Liraglutide. Depressed its accumulation. Hk-2 cells stimulated with 40 mmol/l high glucose for 72 h showed significantly enhanced LC3 and beclin1 expression. Liraglutide can suppressed their protein expression.GLP-1R antagonist(exendin-39) can inhibited the effects of Liraglutide in above all. Conclutions: High glucose reduced the ablitily of GLP1-R. Autophagy were activated by high glucose and Liraglutide can suppressed this affect by up regulating GLP1-R expression.Part 3 High glucose induced activation of autopahgy in type 1diabetic rats and regulating effect of Liraglutide on itObjective: To explore if state of high glucose can induced activation of autopahgy in type 1 diabetic rats and the regulating effect of Liraglutide on it. Methods: SD rats were divided randomly into 4 groups:(1)non-diabetes(N)(2)diabetes(DM)(3)diabetes treated with Liraglutide(DM+LA)(4)diabetes treated with Liraglutide and GLP1-R agonist exendin-39(DM+LA+EX). Type 1 diabetic model was induced by intravenous injection of STZ. Make a collection of specimes and detected the metabolic variables. Hematoxylin and eosin stain were used to observe the form of kindey. Western blot and immunoperoxidase staining were used to detect the expression of GLP1-R and LC3. The expression of beclin1 was measured by western blot.Results: Compared with normal group, the serum glucose, relative kidney weight and food intake increased significantly in DM group; the body weight were decreased significantly in diabetic rats and there is no significant effect of Liraglutide on these change. The rise of serum creatinine and urea nitrogen is significantly. Hematoxylin and eosin stain of renal shows glomerulus magnified, vacuolar degeneration appeard in kidney tubules. Liraglutide can improved this situation.The diabetic rats showed significantly depressed GLP1-R expression. Liraglutide significantly enhanced GLP1-R protein expression. The diabetic rats showed significantly enhanced LC3 and beclin1 expression. Liraglutide can decreased their expression. GLP-1R antagonist(exendin-39) can inhibited the effects of Liraglutide on above all. Conclusion: Glucose toxicity do the harm to the kindey of diabetic rats and reduced the ablitily of GLP1-R. Autophagy were activated in the kindey of diabetic rats and Liraglutide can suppressed this affect by up regulating GLP1-R expression.