| ObjectiveDiabetic kidney disease(DKD),a common microvascular complication of diabetes,has becomes a major cause of end-stage renal disease(ESRD)worldwide.Hypoglycemic strategy is considered as the main way to protect kidney,however,the renal function in some patients still deteriorates after effective hypoglycemic treatment.Thus,it is urgent to find a drug that can not only effectively reduce blood glucose,but also prevent the progression of DKD.As a kind of dipeptidyl peptidase-4(DPP-4)inhibitor,saxagliptin has become a new drug for type 2 diabetes(T2DM).Studies have shown that saxagliptin has a protective effect on kidney,but the exact mechanism is not clear.DPP-4 has enzyme activity.SDF-1α is the natural substrate of DPP-4,and DPP-4 can cleave active SDF-1α into inactive SDF-1α,which can be prevented by saxaglitptin as a kind of DPP-4 inhibitor.SDF-1α,which has a protective effect on kidney,can promote PKA phosphorylation.Oxidative stress is closely related to the physiology and pathology of DKD and can be suppressed by PKA phosphorylation.Podocyte epithelial-to-mesenchymal transition(EMT)is a potential pathway leading to proteinuria,and oxidative stress induced podocyte EMT.We studied high fat diet(HFD)and streptozotocin(STZ)induced T2 DM rats and conditionally immortalized mouse podocyte clone,and through experiments in vivo and in vitro,we investigated whether saxagliptin prevented DKD by inhibiting podocyte EMT through inhibition of DPP-4,cleavage of SDF-1α,and oxidative stress.Method1.Animal experiment in vivo: in this study,after 45 male Sprague Dawley(SD)rats were given 1-week adaptive feeding,15 rats were randomly fed with chow diet(CD group).The other 30 rats were fed with high fat diet(HFD group).After 10-week feeding,body weight,blood glucose,serum lipid levels,renal function,liver fuction were monitored and insulin resistance was assessed by intraperitoneal glucose tolerance test(IPGTT).Rats in HFD group were then injected with a small dose of STZ 25mg/kg through tail vein.Establishment of the T2 DM model was considered successful when the blood glucose level was more than 16.7 mmol/l.After T2 DM model was successfully induced,the T2 DM rats were divided into diabetic group(DM)and diabetic rats orally administered saxagliptin group(DM+Sax).Moreover,the 15 rats which were fed with chow diet continued to be allowed free access to the chow diet as normal control group(NC).Body weight and blood glucose were monitored weekly,and after 12-week treatment,24 h urine samples were collected for measurement of urine microalbumin(UMA).All rats were sacrificed after 12 weeks of saxagliptin treatment,and blood samples were collected to examine serum lipid levels,renal function and liver function.Left kidneys were immediately removed and weighed.Levels of oxidative stress marker,8-OHdG(8-hydroxy-2-deoxyguanosine)and podocyte injury marker,Mindin in urine from rats were detected by ELISA(enzyme linked immunosorbent assay).The basic structure of kidneys and extracellular matrix accumulation in rats from each group were observed by hematoxylin-eosin(HE)staining and periodic acid-Schiff staining(PAS).The basic structure of podocyte from rats was observed through electron microscope.The enzyme activity of DPP-4 in serum and kidneys was determined through DPPIV/CD26 assay kit for biological samples.The expression of Desmin,Podocin,Nephrin and DPP-4 from different groups was observed by immunofluorescence staining.The expression of Fsp1,Fibronectin,SDF-1α and NOX2 in renal sections was detected via immunohistochemical staining.The ROS levels in rats from each group were detected by DHE staining.The protein levels of DPP-4,SDF-1α,p-PKA,PKA,NOX2,p-ERK1/2,ERK1/2,TGF-β1,α-SMA,Desmin,Nephrin,Podocin were tested by western blot.2.Cell experiment in vitro: conditionally immortalized mouse podocytes were cultured in medium medium containing IFN-γ at 33℃ to induce proliferate,and were cultured in medium medium without IFN-γ at 37 ℃ for 10 to 14 days to induce differentiation.The cells were then divided into 5 groups:(1)normal glucose group(NG)as controls incubated in RPMI 1640 containing 5.6mmol/l D-glucose;(2)mannitol group(MA)as an isosmotic controls incubated in RPMI 1640 containing 5.6mmol/l D-glucose and 24.4mmol/l mannitol;(3)high glucose group(HG)incubated in RPMI 1640 containing 30mmol/l D-glucose;(4)high glucose and saxaglitpin group(HG+Sax)incubated in RPMI 1640 containing 30mmol/l D-glucose and 500nmol/l saxagliptin;(5)high glucose,saxaglitpin and AMD3100 group(HG+AMD3100+Sax)incubated in RPMI 1640 containing 30mmol/l D-glucose and 500nmol/l saxagliptin after SDF-1α receptor antagonist AMD3100 20 μg/ml treatment.The activity of DPP-4 enzyme in podocytes was measured by DPPIV/CD26 assay kit for biological samples.ROS levels were detected by fluorescent probe DCFH-DA.Protein levels of DPP-4,SDF-1α,p-PKA,PKA,NOX2,p-ERK1/2,ERK1/2,TGF-β1,Desmin,Nephrin and Podocin were detected by western blot.ResultI.Animal experiment results1.The model of type 2 dibetic rats was successfully establishedAfter 10 weeks of administration with chow diet and high fat diet respectively,the body weight and serum lipid of SD rats in HFD group was significantly increased(P < 0.05).Results from IPGTT showed insulin resistance in HFD group,and after they were injected with a small dose of STZ in the tail vein,rats had obvious symptoms of polydipsia,polyuria,polyphagia and weight loss.Random blood glucose ≥ 16.7mmol/l suggested that the model of T2 DM rats was successfully established.2.Saxagliptin reduced UMA in type 2 dibetic ratsAfter 12-week treatment of saxaglitpin,the blood glucose in DM group and DM +Sax group was significantly higher than that of NC group(P<0.05),and the blood glucose after saxaglitpin treatment was lower than DM group,but it was not significant(P>0.05).Compared with rats from NC group,24 h UMA was notably upregulated in rats from both DM group and DM+Sax group(P<0.05),and it was significantly improved after saxagliptin treatment(P<0.05).3.Saxagliptin improved extracellular matrix accumulation in glomeruli and podocyte structure in type 2 dibetic ratsThe results from HE and PAS staining showed that,there were no obvious abnormalities in kidneys from rats in NC group,and they presented clear structure of glomeruli and tubules,no obvious abnormalities in glomerular size.Moreover,we observed diffuse proliferation of mesangial matrix in rats from DM rats,while it was improved after saxaglitpin treatment.Electron microscopy revealed no obvious abnormal structure of podocytes in NC group,while we found effacement of foot processes in DM group which were recovered after saxaglitpin administration to a certain extent.4.Saxagliptin inhibited podocyte EMT in type 2 dibetic ratsResults from immunofluorescence,immunohistochemistry and western blot showed that,compared with rats from NC group,the levels of Fibronectin,Fsp1,TGF-β1,α-SMA and Desmin were significantly increased(P<0.05),while the levels of Nephrin and Podocin were significantly decreased(P<0.05),suggesting podocyte EMT existed in T2 DM rats.However,compared with rats from DM group,the levels of Fibronectin,Fsp1,TGF-β1,α-SMA and Desmin were significantly decreased(P<0.05),while the levels of Nephrin and Podocin were significantly increased after saxagliptin treatment(P<0.05),suggesting saxagliptin inhibited podocyte EMT in type 2 dibetic rats.5.Saxaglitpin inhibited DPP-4 enzyme activity and SDF-1α cleavage in type 2 diabetic ratsCompared with NC group,DPP-4 enzyme activity in serem and kidneys of rats from DM group was significantly increased(P<0.05),while DPP-4 enzyme activity in DM+Sax group was significantly decreased compared with DM group(P<0.05).Through immunofluorescence,immunohistochemistry and western blot,we observed the expression of DPP-4 in DM group was significantly higher than that in NC group(P<0.05),while the expression of SDF-1α was significantly lower than that in NC group(P<0.05).After saxaglitpin treatment,the expression of DPP-4 was decreased obviously,while the expression of SDF-1α was increased significantly(P<0.05),indicating that saxagliptin inhibited DPP-4 enzyme activity and SDF-1α cleavage.6.Saxaglitpin inhibited oxidative stress in diabetic rats.Immunohistochemical staining,DHE staining and western blot showed that,compared with rats in NC group,the phosphorylated levels of PKA in rats from DM group were significantly decreased(P<0.05),while the levels of NOX2,ROS and p-ERK1/2 were significantly increased(P<0.05).Compared with rats in DM group,the phosphorylated levels of PKA were increased significantly after treatment with saxaglitpin(P<0.05),while the levels of NOX2,ROS and p-ERK1/2 were significantly decreased(P<0.05).Ⅱ.Results of cell experiments1.Saxagliptin inhibited high glucose induced podocyte EMT.Results from DPPIV/CD26 assay kit for biological samples,fluorescent probe DCFH-DA and western blot showed that,saxagliptin significantly inhibited the enzyme activity and protein levels of DPP-4(P<0.05),inhibited the cleavage of SDF-1α(P<0.05),promoted the phosphorylation of PKA(P<0.05),down-regulated the expression of NOX2,ROS,p-ERK1/2(P<0.05),and supressed the expression of TGF-β1 and Desmin(P<0.05),improved the expression of Nephrin and Podocin(P<0.05).2.Saxagliptin inhibited high glucose induced podocyte EMT through SDF-1αResults from fluorescent probe DCFH-DA and western blot showed that,compared with HG+Sax group,the phosphorylated levels of PKA were obviously decreased(P<0.05),the levels of NOX2,ROS,p-ERK1/2,TGF-β1 and Desmin were significantly increased(P<0.05),the levels of Nephrin and Podocin were clearly decreased(P<0.05)after treatment with saxagliptin combined with AMD3100(P<0.05).Conclusion1.Saxagliptin improved UMA in T2 DM rats independent of hypoglycemic effects.2.Saxagliptin prevented the development of DKD by inhibiting the enzyme activity of DPP-4,inhibiting SDF-1α cleavage,improving oxidative stress and inhibiting EMT in podocytes. |
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