Analysis Of Lipid Metabolic Products Of Diabetic Nephropathy And Study On Pathogenic Mechanism

Because of ageing of population and changing of life-style,the incidence and prevalence of type 2 diabetes are rising globally.Diabetic nephropathy is the most consistent complication,from which 25%～-40%of diabetic patients are suffering. Latest data of China Renal Disease Association indicate that diabetic nephropathy is the second reason of end stage renal disease(ESRD).However,in most developed countries,such as Europe and America,the diabetic nephropathy has become the major reason of ESRD.Diabetic nephropathy is one of the dreaded microvascular complications of T2DM.Once diabetic patients suffer kidney disease and persistent albuminuria,whose pathogenetic condation will going on until RSED.Ever since a long time ago,nephrology scholars are always go in for the study of pathogenesy of diabetic nephrology.More and more studies demonstrate that,serum lipid disorders play an important role in the development of this dreaded disease.In 1936,Kimmelstiel and wilson found considerable lipids deposit in kidney arteriolae,glomerulus,and tubules.Animal experiments show that hypercholesteremia can result in focal glomeruloscerosis,and the damage of glomerulus can relieve after lowing down the serum lipids.Clinical research also confirm that,lipid metabolism disturbance is the independent risk factor of DN in type 2 diabetics.Many other evidences sign that,lipid metabolism disturbance closely related to the occurrence and development of DN.However,the definite mechanism of lipid metabolic products in renal damage is not clear.With regard to this,we chose KK-Ay mouse as DN animal model,which is a T2DM model and have severe dyslipidemia.The differences of lipid metabolic profiles in serum samples from KKAy mice and controls will be analysised by metabonomic technique,which is based on LC/MS.We except to find significant metabolic product,which contribute most to DN and have clinical research value.Besides,we also want to investigate their mechanism in DN.The whole study contains two parts.1.Analysis of lipid metabolic products of diabetic nephropathy by metabonomic techniqueKK-Ay mouse is a proper type 2 diabetic animal model to study dyslipidemia and diabetic nephropathy.We use metabonomic technique which bases on LC/MS and PCA to probe whether it can diagnose early,find significant biomaker and make clear the mechanism of dyslipidemia.We select male 6-week-age KK-Ay mice and nondiabetic control C57BL/6J mice which have the same genetic background.The animals will be fed for fourteen weeks,whose urine and blood samples will be collected in 8,12,14,16,18,20 weeks of age.Serum will be useed for testing blood glucose,cholesterol,triglyeride,low density lipoprotein,blood urea nitrogen and albumin by automatic biochemistry analyzer.Microalbumin will be tested in 24-hour-urine and spot urine by ELISA kit.3 mouse each group are sacrificed in 8,12,20 weeks of age,whose renal pathology will be investigated.Serum samples for metabolimic analysis must be deproteinizated with four times volume of acetonitrile.Then,analysis was performed on Agilent series for LC-MS.The sample were delivered into the ion source of mass spectrometry.The ESI-MS spectra were acquire in positive ion mode,which allowed much more metabolite information in profile.After gasification,ionization and some other detection and analysis,we get the data,which were preprocessed as follows:first divided the whole spectral into bins along the mass direction,calculated extracted ion chromatograms(XIC),then,matched filtration and detected peaks for each XIC;after that retention time alignment and peak matching algorithm were applied for all datum sets;finally,exported the results to a CSV file,which includes the information of the mass charge ratio,retention time and area of the peaks across all samples.The above procedure was carried out on R platform using XCMS toolbox that is freely under an open-source license.The preprocessing results were fed into MATLAB platform for further analysis.PCA was implemented herein to uncover the biochemical pattern of mice comparing transgenic and wild-type groups,and to suggest useful transgenic-related biomarkers.Prior to performing PCA,additional data preprocessing procedures were required.Normalization and Pareto scaling were used.After PCA, the Hotelling's T,was implemented to find out the most extreme variables in the Ioadings plot of PCA.All calculation programs implementing PCA were coded in MATLAB 7.0.The course of DN in KK-Ay mice were divided into three stage.8-week-age to 11-week-age is the stage without DN but only type 2 DM.12-week-age to 19-week-age is the early stage of DN.The mouse model represent typical symptom of DN after 20-week-age.This can be conformed to as the criterion of metabonomic diagnose.Score plot is one of the metabonomic results,which can distinguish the model and control group of each week precisely.Score plot of the first stage is not as good as expected.But score plots of stage 2 and 3 are perfect.Loading plot is also the result of metabonomics,which could find out the biomaker. Palmitoyllysophosphatidylcholine,oleoylglycerophosphocholine and stearoylglycerophosphocholine in the loading plots are contribute most to DN and raise dramatically in the KK-Ay mice,which can be recognized as the biomaker of DN for further study.According to the information of KEGG data base,we hypothesize that dyslipidemia ascribed to lysolecithin and phosphocholine is closely related to dysfunction of phosphocholine transferase and phospholipase.2.High density of oleic acid affect proliferation,fibration and inflammation of mesangial cells.Loading plot show that oleic acid contribute most to DN in 20-week-age.Besides, its peak area in KK-Ay mice is much bigger than in control group(P<0.05). Accordingly,this part will study the mechanism of oleic acid affecting proliferation, fibration and inflammation of mesangial cells.Disbalance of production and degradation of extracellular matrix is the basis of fibration.While,TIMP-1 can hole back the degradation of extracellular matrix. Results of RT-PCR indicate that,TIMP mRNA over-expressed after stimulated by oleic acid.TGF-βis acknowledged as a factor of fibration,and fibronectin(FN) is the major composition of extracellular matrix.Both of them are play an important part role in fibration,Immunocytochemistry can investigate the intracellular location and carry out semiquantitative analysis of TGF-βor FN.TGF-βcontained in supematant can also be tested by ELISA method.We find that,TGF-βexpress in endochylema mainly.The heigher density of oleic acid the yellow stained brighter.The result of FN shows the same trend.We can say that,TGF-βis able to stimulate MC to produce more FN, which may result in glomerular sclerosis.MTT results indicate that,oleic acid can make MC proliferation,but it is not remarkable.This may because of over expressed TGF-β,which can affect generation cycle of MC.Furthermore,inflammation is the key point in DN.We use EMSA methods to test the activity of NF-κB,and use RT-PCR method to investigate UXT and MCP-1mRNA.Stimulated by oleic acid,all the index are raise.That is to say,oleic acid can result in inflammatory and result in DN.On the whole,metabonomic technique can diagnose DN earlier in T2DM patients, find some significant biomakers for further study,and be applied to reveal the mechanism of dyslipidemia.Metabonomic analysis show that oleic acid is important to DN.Vitro experiments also confirm it can make MC proliferate,promote inflammation and fibration.