The Role And Mechanism Of CD36 In Palmitate Induced Diabetic Nephropathy Glomerular Fibrosis And The Protective Effects Of Astragaloside ?

Diabetic nephropathy?DN?is one of the most common microvascular complications of Diabetes mellitus?DM?.Patients with DN will eventually develop end-stage renal disease?ESRD?and DN has become the primary cause of ESRD worldwide.Glomerular fibrosis is one of the important pathological features of DN progression and caused by increased extracellular matrix secretion of mesangial cells?MCs?.The activation of TGF-?1/Smad signaling pathway induces collagen IV?Col 4?and fibronectin?FN?secretion of MCs,which is considered to be the core mechanism of DN fibrosis.In addition to hyperglycemia,elevated levels of free fatty acids?FFAs?are considered to be one of the risk factors for DN:FFAs induce podocyte apoptosis and MCs fibrosis.Cluster of differentiation 36?CD36?induces increased FFAs absorption,which is one of the main mechanisms of cell damage caused by FFAs.In addition,it has been reported that the activation of transient receptor potential channel 6?TRPC6?plays an important role in renal fibrosis.Therefore,FFAs activate TRPC6 and its downstream transcription factor activated nuclear factor 2?NFAT2?through CD36,which may be the potential mechanism of glomerular fibrosis in DN.Astragaloside IV?AS-IV?is the main active ingredient of Chinese traditional medicine Astragalus membranaceus,and has various pharmacological activities such as anti-inflammatory and anti-oxidation.Our group has confirmed the protective effect of AS-IV on type 1 DN rats and high glucose-stimulated MCs.Whether AS-IV has protective effects on FFAs-induced MCs fibrosis and type 2 DN rats remains unclear.The aim of this study is to investigate the mechanisms of FFAs-induced MCs fibrosis and the protective effects of AS-IV.Part I The role and mechanism of CD36/TRPC6/NFAT2 pathway in palmitate induced diabetic nephropathy glomerular fibrosisObjective:The role of CD36/TRPC6/NFAT2 signaling pathway on fibrosis was explored in a human glomerular mesangial cells?HMCs?stimulated by palmitate?PA?.A preliminary validation was performed in a high-fat diet?HFD?combined with low-dose streptozotocin?STZ?-induced type 2 DN rats.Methods:1.HMCs stimulated by PA?200?M?was model group.The control group contained equal amounts of bovine serum albumin?BSA?.The experimental design was as follows:1.1 The cultured HMCs were divided into?1?control group,?2?PA 2h group,?3?PA6h group,?4?PA 12h group,and?5?PA 24h group.Western blotting was used to detect the expression of TGF-?1,p-Smad2/3,FN,Type IV Alpha 1 Chain?Col4A1?,CD36 and TRPC6.Western blotting and immunofluorescence?IF?were used to detect NFAT2expression.Oil red staining was used to observe the intracellular lipid deposition.1.2 The cultured HMCs were divided into:?1?control group,?2?PA 0.5h group,?3?PA 2h group,and?4?PA 6h group.Fluorescence microscopy was used to detect intracellular Ca²⁺levels at various time points.Flow cytometry was used to detect intracellular Ca²⁺levels after 6 h of PA stimulation.1.3 The cultured HMCs were divided into:?1?control group,?2?PA 6h group,?3?PA6h+SKF96365 group,?4?Flufenamic acid?FA?group,?4?PA 6h+FA group,?5?Sulfo-N-succinimidyl oleate?SSO?group,?6?PA 6h+SSO group,?7?PA 6h+CD36siRNA group.Fluorescence microscopy was used to detect intracellular Ca²⁺levels.1.4 The cultured HMCs were divided into:?1?control group,?2?PA 24h group,?3?PA 24h+SKF96365 group,?4?FA group,?5?PA 24h+FA group.Western blotting was used to detect the expression of TGF-?1,p-Smad2/3,FN,Col4A1,TRPC6 and NFAT2.1.5 The cultured HMCs were divided into:?1?control group,?2?PA 24h group.IF detected FN,NFAT2;CD36,NFAT2 co-expression.1.6 The cultured HMCs were divided into:?1?control group,?2?PA 24h group,?3?PA 24h+NFAT2 siRNA group.The expression of TGF-?1,p-Smad2/3,FN and Col4A1was detected by Western blotting.1.7 The cultured HMCs were divided into:?1?control group,?2?PA 24h group,?3?PA 24h+SSO group.Western blotting was used to detect the expression of TGF-?1,p-Smad2/3,FN,Col4A1 and NFAT2.The BODIPY^TM FL C16 probe detected the FFAs absorption.1.8 The cultured HMCs were divided into:?1?control group,?2?PA 24h group,?3?SSO group,?4?PA 24h+SSO group.Oil red staining was used to detect lipid deposition in HMCs.1.9 The cultured HMCs were divided into:?1?control group,?2?PA 24h group,?3?PA 24h+CD36 siRNA group.Western blotting was used to detect the expression of TGF-?1,p-Smad2/3,FN,Col4A1 and NFAT2.Oil red staining and BODIPY^TM FL C16probe were used to detect lipid deposition and FFAs absorption in HMCs,respectively.2.SD rats were fed with HFD and DN rat model was induced by intraperitoneal injection?ip?low-dose STZ?35 mg/kg?six weeks later.After confirming the success of rat model,rats were fed with HFD for another 8 weeks.The control group received a standard die.During the experiment,the general condition?mental state,diet and urine?of each group was observed.The body weight and blood glucose?BG?levels were measured regularly.After 8 weeks,the 24 hours urine were collected by metabolic cage and blood sampling were collected form abdominal aorta.24 h urine protein?Upro?,microalbuminuria?ALB?,urine creatinine?UCr?content,serum creatinine?SCr?,blood urea nitrogen?BUN?,triglyceride?TG?,total cholesterol?TC?content and FFAs were measured.Morphological changes of the kidney were observed by HE staining;renal fibrosis was observed by PASM,Masson staining and electron microscopy.The expression of CD36 and NFAT2 in the kidneys was detected by immunohistochemistry?IHC?.Western blotting was used to detect the expression of TGF-?1,p-Smad2/3,FN,Col4A1,CD36 and nuclear NFAT2 in renal tissues of each group.Results:1.After PA stimulation,the expression of TGF-?1,p-Smad2/3,Col4A1,FN,CD36and nuclear NFAT2 were significantly increased in a time-dependent manner.2.After PA stimulation,intracellular lipid deposition increased in a time-dependent manner.The intracellular Ca²⁺began to increase after PA stimulation for 2 h,and the intracellular Ca²⁺level increased significantly after PA stimulation for 6h.3.TRPC6 channel inhibitor SKF96365 and siRNA significantly inhibited PA-induced expression of TGF-?1,p-Smad2/3,Col4A1,FN and nuclear NFAT2.4.The TRPC6 activator FA up-regulated the expression of TGF-?1,p-Smad2/3,Col4A1,FN and nuclear NFAT2,enhanced the effect of PA on fibrosis.5.SKF96365 inhibited PA-induced intracellular Ca²⁺elevation;FA enhanced PA-induced elevation of intracellular Ca²⁺levels.6.The expression of TRPC6 protein showed a characteristic of negative feedback regulation in the PA stimulation HMCs:the expression of TRPC6 decreased in a time-dependent manner after PA stimulation,while SKF96365 inhibited this process.However,FA down regulated TRPC6 expression in HMCs.7.NFAT2 siRNA inhibited PA-induced TGF-?1,p-Smad2/3,Col4A1 and FN expression.8.CD36 inhibitor SSO reduced the absorption of FFAs and inhibited PA-induced TGF-?1,p-Smad2/3,Col4A1,FN and NFAT2 expression.9.SSO induced intracellular lipid deposition of HMCs and up-regulated Ca²⁺levels.10.CD36 siRNA inhibited the FFAs uptake,reduced intracellular lipid deposition and Ca²⁺elevation,suspressed PA-induced TGF-?1,p-Smad2/3,Col4A1,FN,CD36 and NFAT2 expression.11.After STZ injection,rats in the DN group showed signs of hairiness,dullness,weight loss,increased water intake and urine output.BG,TG,TC,FFAs and renal function related indicators:24h Upro,ALB,SCr,and BUN were significantly increased than control group,creatinine clearance?CCr?decreased.Renal index increased in DN group,and HE staining showed significant proliferation of mesangial cells.PASM,Masson staining and electron microscopy showed that glomerular basement membrane thickened and mesangial matrix increased in DN rats.Western blotting and immunohistochemistry showed that the expression of TGF-?1,p-Smad2/3,FN,Col4A1,CD36 and NFAT2 significantly increased in DN rats.Conclusions:1.PA induced fibrosis of HMCs through activating TGF-?1/Smad.2.Inhibition of TRPC6/NFAT2 signaling pathway suspressed PA-induced HMCs fibrosis.3.PA up-regulated CD36 expression,increased intracellular lipid deposition and FFAs uptake.4.Inhibition of CD36 suspressed PA-induced fibrosis of HMCs,and its mechanism might be related to inhibition of the activity of TRPC6/NFAT2 pathway.Part?The role of CD36 in the effect of astragaloside IV on type 2diabetic nephropathy glomerular fibrosis and oxidative stress.Objective:The effects of AS-IV on fibrosis,oxidative stress and CD36 were investigated in type2 DN rats and PA-stimulated HMCs.Methods:1.The cultured HMCs were divided into?1?control group,?2?PA 24h group,?3?PA24h+AS-IV different dose groups?20,40,80???.The expression of TGF-?1,p-Smad2/3,FN,Col4A1,NADPH oxidase 4?NOX4?,p22 ^phox and CD36 were detected by Western blotting.Oil red staining was used to detect lipid deposition in HMCs.The levels of intracellular ROS were measured by DCFH-DA and DHE probe.2.The cultured HMCs were divided into?1?control group,?2?PA 24h group,?3?PA24h+SSO group,?4?PA 24h+AS-IV group?80???,?5?PA 24h+SSO+AS-IV group?80???.The expression levels of TGF-?1,p-Smad2/3,FN,Col4A1,NOX4 and p22^phox were detected by Western blotting.BODIPY^TM FL C16 probe was used to detect FFAs uptake in HMCs.The levels of intracellular ROS were measured by DCFH-DA and DHE probe.3.Copying the Type 2 DN model was in the same way as the first part.After confirming the success of STZ intraperitoneal injection,rats were randomly divided into DN model group?DN group?,AS-IV?20,40,80 mg/kg?treatment group,and normal control group?Control group?,n=8.General observation,biochemical indicators and renal pathology were the same as the first part.Western blotting and IHC were used to detect the expression of TGF-?1,p-Smad2/3,FN,Col4A1,NOX4,p22^phox and CD36 in renal tissues of each group.Results:1.AS-IV inhibited PA-induced intracellular lipid deposition,FFAs uptake in HMCs,down-regulated TGF-?1,p-Smad2/3,FN,Col4A1,NOX4,p22^phox and CD36expression,and reduced ROS production.SSO also inhibited PA-induced oxidative stress.Nevertheless,the combined use of SSO and AS-IV did not enhance the efficacy.2.BG,TG,TC,FFAs and renal function related indexes:24h Upro,ALB,SCr and BUN in AS-IV treatment group were significantly dereased than DN group,CCr increased.Renal HE,PASM and Masson staining showed that mesangial hyperplasia and basement membrane thickening in AS-IV treatment groups were significantly decreased than DN group.The Western blotting and IHC results indicated that the expression of TGF-?1,p-Smad2/3,FN,Col4A1,NOX4,p22^phoxhox and CD36 in AS-IV group were significantly decreased than that in the DN group.Conclusions:1.AS-IV had protective effects on renal fibrosis and oxidative stress in type 2 DN rats.2.AS-IV had protective effects on PA-induced intracellular lipid deposition,FFAs uptake,fibrosis and oxidative stress in HMCs,and its mechanism was related to the down-regulation of CD36 expression.