Plasma Metabonomics Study On Differential Diagnosis Between IgA Nephropathy And Membranous Nephropathy

Objectives:IgA nephropathy(IgAN)and membranous nephropathy(MN)remain the most common glomerulonephritis..Traditional clinical biomarkers of kidney function,such as urea and creatinine,are difficult to discriminate between IgAN and MN.Only when the kidney is seriously damaged,these biomarkers will be abnormal significantly.Currently,it is a gold standard by renal biopsy in diagnosis of IgAN and MN.But renal biopsy is an invasive examination,which can not only bring physical suffering to patients,but also may cause complications like bleeding,arterio-venous fistula and so on.Besides,many patients cannot receive renal biopsy due to complex disease conditions.For examples,patients are suffering from severe hypertension,evident hemorrhagic tendency and solitary kidney.Metabonomics is to reveal the complex biological regulation mechanism in biological system by qualitative and quantitative analysis of small molecule metabolites.Metabolites are the final products of various physiological and pathological process and they can directly reflect the physical condition.The progress of kidney diseases is closely related to the metabolism in the organism.Therefore,the objective of this study is to find different metabolites between IgAN patients and MN patients by gas chromatography-mass spectrometry(GC-MS).Further more,we reveal the relevance between these metabolites and diseases.It will reduce the economical and psychological burden of patients.This study will provide a new insight for discriminating between IgAN and MN based on metabonomics method.Methods:1.Research objects and experimental groups: we selected 86 patients of the nephrology department in our hospital as study objects.According to the pathological results,they were divided into two groups.42 IgAN patients are in the first group.44 MN patients are in the second group.Plasma samples of all subjects were collected in the morning.42 IgAN patients and44 MN patients were all proved by pathological diagnosis.All patients did not receive any hormone or immune inhibitor treatment and none of them had other systematic complications.2.Plasma analyzed by GC-MS: Firstly,protein was removed by precooling methanol.Secondly,tridecanoic acid(20ug/ml)was added as internal label.And then,metabolites were extracted and freeze-dried.100 ?L of methoxyamine pyridine were added into the freeze-dried samples.Vortex to dissolve thoroughly.Put the samples in37? water bath for 120 min.Then,MSTFA was added into the samples.37? water bath of silylation reaction lasted for 60 min.Centrifuged for 15 min at the speed of 1,5000 rpm,4?.The supernatant will be analyzed by GC-MS.After separation by GC,mass spectrum scanning was used to collect peak compounds at each time point.3.Screening and identification of biomarkers: after noise filtering,retention time alignment,chromatographic peak detection and matching processing,we searched for the information of metabolites through NIST05,Wiley,Replib and Fiehn library.Then they were analyzed by principal component analysis(PCA)and partial least square discriminant analysis(PLS-DA)to find out biomarkers between IgAN and MN.Value of p(bilateral significance testing)<0.05 was concerned as the obvious difference.After logistic regression analysis,we make a ROC curve.Besides,the different metabolites were further confirmed by HDMB.Results:1.In this study,plasma metabolic profiles of IgAN and MN patients were analyzedby GC-MS.We find out biomakkers between IgAN and MN by multi-dimensional statistical method.2.In the primary screening,110 compounds were firstly detected in this study.After statistical analysis,we found that 15 different metabolites can be used to make diagnosis for IgAN and MN.There are 9 amino acids,including leucine,alanine,serine,glutamic acid,aspartic acid,cysteine,pyroglutamic acid,homoserine and glycine.The others are ethanolamine,carbamic acid,1,2-propanediol,1,5 – pentanediol,oxalic acid,2-ketoisocaproic acid,butanedioic acid,3-phenylacrylonitrile and mono-methy phosphate.3.Mono-methy phosphate and 3-phenylacrylonitrile are the protective factors of IgAN.2-ketoisocaproic acid and butanedioic acid are risk factors of IgAN.But it is all opposite to MN.Conclusions:Metabonomics study provide new potential biomarkers for the diagnosis of IgA nephropathy and membranous nephropathy.