Urine Peptidomics Research Of Idiopathic Membranous Nephropathy Based On Liquid Chromatography-tandem Mass Spectrometry

Objective: Proteinuria and proteinuria-related symptoms in patients with idiopathic membranous nephropathy(IMN)are considered as manifestations of renal dysfunction,but the pathogenesis and progression of IMN are not fully elucidated.Deregulation of proteolytic system is a known pathway leading to diseases manifestation,we hypothesized that abnormal expression of protease in IMN may be related to the production of urine peptides.The development of high-throughput mass spectrometry has promoted the application of proteomics in disease research,urinary proteomics provide insights for exploring the pathogenesis of IMN.This work aimed at analyzing urinary peptidomics and exploring the significance of related proteasess of IMN.Methods: In this study,the naturally produced peptides in the urine of 20 heathy individuals,22 IMN and 15 other kidney disease were analyzed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),proteins matched with peptides specifically were identified and differentially expressed proteins in IMN specificity were screened out.Proteasix bioinformatics database was used to predict the proteases that cleavge these proteins to produce endogenous peptides.Then,the public proteomics databases i Pro X and genomics databases GEO were used to validate the expression levels of the screened IMN differentially expressed proteins and proteases.Finally,Western Blot was used to validate the expression level of several key proteases calpain,cathepsin and matrix metalloproteinase in the kidney tissue of IMN patients.Results: 1.A total of 1080 peptide-specific matching proteins were identified,279 urine differentially expressed proteins of IMN were screened,32 proteases involved with endogenous peptides were predicted;2.55 urine differentially expressed proteins of IMN were also differentially expressed in kidney tissues,which mainly participated in the regulation of proteasome,lysosome and actin cytoskeleton,antigen processing and presentation and other signal pathways;3.Abnormal expression of some 32 predicted proteases in glomerulus of IMN was revealed;4.Western Blot showed that the expression levels of calpain CAPN1,matrix metalloproteinase MMP14 and tissue metalloproteinase CTSS were increased in the kidney tissue of IMN patients.Conclusion: 1.There were differences in urine peptides components among IMN patients,healthy individuals and other patients with kidney diseases.Urine peptidomics can reflect the changes of urinary peptide components of IMN;2.Urine peptidomics may reflect the change of molecular of IMN at the tissue level.3.The changes of renal calpain,matrix metalloproteinase and tissue metalloproteinase expression in IMN patients may be related to the production of urine peptides and kidney tissue injury.