Liraglutide Inhibits The Proliferation Of Glomerular Extracellular Matrix In Diabetic Nephropathy Through Regulation Of Wnt/β-catenin Signaling Pathway

Background:Diabetic nephropathy(DN),one of the most common microvascular complications of diabetes,is also the main cause of end stage renal disease worldwide.The most important pathological change in the progression of DN is the progressive accumulation of extracellular matrix(ECM)in the mesangial region of the glomerulus,leading to glomerular sclerosis.Recent studies have shown that the Wnt/β-catenin signaling pathway of glomerular mesangial cells is impaired in a high-glucose environment,which is involved in the production of glomerular extracellular matrix and the progression of glomerular fibrosis.The glucagon-like peptide-1(GLP-1)receptor agonist liraglutide has a protective effect on DN,but the specific mechanism is not yet clear.Objective:To explore whether the GLP-1 receptor agonist liraglutide can inhibit the proliferation of glomerular ECM and improve DN by regulating the Wnt/β-catenin signaling pathway of glomerular mesangial cells.To provide a new theoretical basis for the clinical application of liraglutide and the search for therapeutic targets for DN.Methods:Cell experiments: 1.The concentration gradient of GLP-1 receptor agonist liraglutide was used to interfere with human mesangial cells(HMCs)under high glucose state for 24 hours.Western blot was used to detect the expression of ECM-related proteins,as well as detecting the expression of Wnt/β-catenin signaling pathway proteins.Immunofluorescence was used to detect the distribution of β-catenin protein in cells.2.Liraglutide was used to interfere with HMCs under high glucose state,then β-catenin-specific inhibitor XAV-939 was used to inhibit β-catenin signaling.Western blot was used to detect the expression of ECM-related proteins.3.Liraglutide was used to interfere with HMCs under high glucose state,then knock down β-catenin expression by transiently transfecting β-catenin si RNA.Western blot was used to detect the expression ofβ-catenin protein and ECM-related proteins.Animal experiment: Diabetes was induced by a single intraperitoneal injection of 65 mg/kg streptozotocin(STZ)in SD rats.Three days after STZ injection,rats with a fasting blood glucose(FBG)level of > 16.7mmol/L were identified as diabetic model.Diabetic rats then continue received a normal diet for another 7 weeks with 24 h urinary microalbumin level >400μg were judged as DN model.Animal groups: normal control group(NC),diabetic nephropathy group(DN),liraglutide treatment group(DN+Lir).Each experimental group includes 6mice.Eight weeks after STZ injection,Rats in the liraglutide treated group received subcutaneous injection with liraglutide at 200μg/kg/12 h,and rats in the DN group were given the same volume of physiological saline solution through the same route.After 8 weeks of continuous administration,the FBG,body weight,kidney weight,serum creatinine,urea nitrogen,24 h urine albumin and urine creatinine of the rats were measured.HE,PAS,and Masson staining were performed to observe the pathological changes of kidney tissues in each group.Immunohistochemical staining was used to detect the expression of glomerular FN,Col-IV,α-SMA,Wnt4,Wnt5 a,p-GSK-3β and β-catenin protein.Results:Cell experiments:1.High glucose intervention for 24 hours induced the expression of extracellular matrix related proteins Fibronectin(FN),Collagen IV(Col-IV)andα-SMA protein in HMCs.Treatments with liraglutide significantly reduced the8expression of FN,Col-IV and α-SMA protein,and it had a certain concentration dependence.2.High glucose intervention in HMCs for 24 hours significantly inhibited the expression of Wnt/β-catenin signaling pathway proteins including Wnt4,Wnt5 a,p-GSK-3β,β-catenin total protein and β-catenin nuclear protein,and reduced the distribution of β-catenin in the nucleus.Treatments with liraglutide for 24 hours significantly upregulated the expression of Wnt4,Wnt5 a,p-GSK-3β,β-catenin total protein and β-catenin nuclear protein,and increased the distribution ofβ-catenin in the nucleus.3.After the application of β-catenin inhibitor XAV-939 combined with liraglutide for 24 hours under high glucose state,the inhibitory effect of liraglutide on the expression of FN,Col-IV and α-SMA protein induced by high glucose in HMCs was significantly attenuated.4.Application of β-catenin si RNA transfection for 48 h under high glucose state significantly knocked down β-catenin expression.At the same time,the inhibitory effect of liraglutide on high glucose-induced FN,Col-IV and α-SMA protein expression was significantly attenuated in HMCs.Animal experiment:1.The FBG level was significantly increased and body weight significantly decreased in DN rats induced by STZ.There was no significant difference in FBG and body weight after 8 weeks of liraglutide treatment.2.Serum creatinine,urea nitrogen,24 h urine albumin,urine albumin/urine creatinine ratio and kidney weight/body weight ratio were significantly increased in the DN rats.Liraglutide treatment of rats with DN significantly reduced serum creatinine,urea nitrogen,24 h urine albumin,urine albumin/urine creatinine ratio,and kidney weight/body weight ratio.3.The renal cortex of DN rats showed obvious glomerular hypertrophy,increased renal tubular epithelial cell vacuolization,glomerular basement membrane thickening,mesangial expansion,and a large amount of collagen deposition in the glomerulus and tubular interstitium.All those pathological changes were significantly reduced after liraglutide treatment.The glomerular area was significantly reduced,the mesangial area proliferation was reduced,and the collagen deposition was significantly reduced.4.The immunohistochemical staining results of glomerular showed that the expression levels of glomerular FN,Col-IV and α-SMA protein in the DN rats were significantly increased,and the expression levels of glomerular Wnt4,Wnt5 a,p-GSK-3β and β-catenin protein were significantly reduced.Liraglutide treatment significantly reduced FN,Col-IV and α-SMA expression and restored expression of Wnt4,Wnt5 a,p-GSK-3β and β-catenin.Conclusion:1.GLP-1 receptor agonist liraglutide inhibit high glucose-induced extracellular matrix proteins production in HMCs by up-regulating the Wnt/β-catenin signaling pathway.2.GLP-1 receptor agonist liraglutide treatment can improve renal function and glomerular pathological damage in rats with diabetic nephropathy,reduce glomerular fibrosis,and exert renal protection besides hypoglycemic effect.Its mechanism of action may be related to the activation of Wnt/β-catenin signaling pathway.