Clopidogrel Improves Diabetes-induced Renal Fibrosis And The Underlying Mechanism

Background:Diabetes mellitus is a chronic progressive disease and diabetic nephropathy(Diabetic nephropathy,DN)is one of the main complications of it.DN is one of the most common microvascular complications in diabetes mellitus,and is the main cause of death in diabetic patients with complications.The ultimate result of DN progression is renal fibrosis,scar tissue formation,and finally end-stage renal disease.The typical pathological changes are progressive thickening of the basement membrane of glomerular capillaries and progressive dilatation of the mesangial membrane caused by gradual deposition of the extracellular matrix(Extracellular matrixc,ECM)in the mesangial region.The ECM is mainly composed of Fibronectin(Fibronectin,FN),Collagen(including Collagen?and Collagen?),Laminin(Laminin)and proteoglycan.Elevated blood glucose levels lead to an imbalance between the synthesis and breakdown of ECM,which in turn speeds up glomerular sclerosis and leads to increased renal fibrosis.Therefore,how to effectively prevent renal complications in diabetic patients and improve diabetic nephropathy induced renal fibrosis is the key to solve the problem.Clopidogrel,a structural thienopyridine antiplatelet agent,specifically inhibits adenosine diphosphate(Adenosine diphosphate,ADP)-dependent platelet aggregation and adhesion by inhibiting the P2Y₁₂ receptor.Studies have shown that the occurrence of diabetic nephropathy is related to the increase of activated platelet and platelet hyperresponsiveness.After platelet aggregation and activation,a large amount of platelet derived growth factor(Platelet derived growth factor,PDGF)is released and transforming growth factor-?(Tissue growth factor beta,TGF-?)is activated.The up-regulation of PDGF and TGF-?can promote the overexpression of FN.The stimulation of TGF-?on rat mesangial cells elicits the increased expressions of type I collagen and FN.However,it is still not clear whether clopidogrel treatment can inhibit the expression of FN and improve diabetes induced kidney fibrosis in type I diabetes mice model.Objiective:To test the potential therapeutic effects of clopidogrel on diabetic nephropathy(DN)in streptozotocin induced type 1 diabetic mice,and to further explore that the protective mechanism of clopidogrel against DN is associated with its inbibition of fibronection in diabetes-induced renal fibrosis.Method:(1)Type 1 diabetes were induced in mice by continuously seven intraperitoneal injections of STZ.Fasting blood glucose was measured at 7th day after the last intraperitoneal injection of STZ.According to the blood glucose value,mice were divided into 2 groups,control group(Vehicle),and diabetes group(STZ),then set this time point as diabetic initial time.(2)After three months(diabetic condition for 3months),five of diabetic mice(DM-3M)and five of control mice(CTRL-3M)were randomly selected and were sacrificed.All blood and kidneys were collected.(3)The other mice were randomly divided into four groups according to blood glucose:normal control group(C TRL),clopidogrel normal control group(CTRL+CLO),diabetes control group(DM)and clopidogrel-treated diabetes group(DM+CLO).Mice in C TRL+CLO group and DM+CLO group were fed with clopidogrel at a dose of 20mg/kg/d in drinking water for three months.The other two groups were given normal drinking water.(4)After 3 months of clopidogrel treatment,all mice at six diabetic condition were scrificed,and blood and kidney were collected in groups.(5)The body weight,blood glucose,renal tissue weight,tibia length,and other values of mice in each group were monitored.(6)The expression levels of target proteins such as TGF-?,CTGF,FN,laminin,type I collagen and type IV collagen in mouse kidneys at 3 and 6 months were detected by Western blotting,quantitative real-time PCR and tissue staining.Result:(1)At 3 months and 6 months,compared with control mice,diabetic mice showed significant changes in renal pathology due to the progression of diabetes,including decreased urinary creatinine,enlarged glomerular size,and increased renal tibia ratio.At 6 months,there were no significant changes in glomerular diameter between clopidogrel-treated diabetic mice(DM+CLO)and diabetic control mice(DM),but there were significant reductions in renal tibial ratio in clopidogrel-treated diabetic mice(DM+C LO).(2)The expressiona of TGF-?,CTGF,FN and laminin in the kidneys of diabetic mice was increased by immunoimprinting method compared with the control mice at 3 months.While the expression of type IV collagen was not significantly changed.At 6 months,the protein expressions of TGF-?,CTGF,type I collagen and type IV collagen in the kidney of diabetic control mice were not significantly changed compared with those of normal control mice.The expression of laminin showed a tendency to increase,and the protein expression of FN was significantly increased.At 6 months,laminin expression was decreased in clopidogrel-treated diabetic mice compared with diabetic control mice,and FN protein expression was significantly decreased.(3)Real-time quantitative PCR showed no significant changes in the expression levels of TGF-?mRNA,FN mRNA,type I collagen mRNA and type IV collagen mRNA in the kidneys of diabetic mice compared with the normal control mice at 3 months.Compared with the normal control group at 6 months,the mRNA expressions of TGF-?and type IV collagen in the kidneys of diabetic control group were not significantly changed,while the mRNA expressions of FN and type I collagen were significantly increased in diabetic control group at 6 months,and their expressions were significantly decreased in clopidogrel-treated diabetic group.(4)In immunohistochemical staining results,the expressions of TGF-?and laminin in the kidneys of diabetic mice was significantly increased compared with that of normal control mice at 3 months.The expressions of laminin,type I collagen,type IV collagen,and fibronectin were significantly increased in diabetic control mice compared with normal control mice at 6 months.Significantly,these proteins expressions were decreased in clopidogrel-treated diabetic mice.(5)In Western blotting results,the expressions of ERK1/2,Akt and P38 in the kidneys of diabetic mice were increased compared with those of normal control mice at 3 months,while the expressions of JNK,Smad2/3 and Smad4 were not significantly changed.There was no significant change in the expression of these proteins at 6 months.Conclusion:(1)The degree of renal fibrosis in diabetic mice increased gradually with the progression of the disease.(2)C lopidogrel shows the protective and ameliorative effects in the process of diabetes-induced kidney fibrosis.(3)C lopidogrel may ameliorates renal fibrosis by reducing FN expression through an independent mechanism.(4)Induction of TGF-?and CTGF and their upstream signaling are most obvious in the kidneys of diabetic mice at 3 months.As early markers of fibrosis,their expressions arenot significantly changed at 6 months.However,renal fibrosis still worsens with the progression of the disease.