Proteomic Analysis Of Urine Extracellular Vesicles In IgA Nephropathy

Aim:Immunoglobulin A nephropathy(IgAN)is one of the most prevalent forms of primary glomerulonephritis,which usually progresses to end-stage renal disease(ESRD).Renal biopsy is the gold standard for IgAN diagnosis,but the bleeding rate after renal biopsy is higher.In this study,proteomics analysis of urine extracellular vesicles in IgAN patients was conducted to explore a safe,simple and effective non-invasive method for the diagnosis of IgAN.Methods:In this study,subjects were divided into normal control and IgAN group.The differentially expressed proteins in urinary extracellular vesicles of the two groups were identified by liquid chromatography-mass spectrometry(LC-MS/MS),and the functions of the differentially expressed proteins were analyzed from different perspectives by bioinformatics analysis.The differentially expressed proteins were selected as candidate protein markers,which were up-regulated in the IgAN group,and might have a crucial role in the pathophysiology of IgAN according to current studies.Then,we enlarged the sample size for semi-quantitatively determining the expression of candidate protein markers in the normal control group and IgAN group by western blot.Results:A total of 2934 proteins were identified by LC-MS/MS in the study,of which,2428 proteins contained quantitative information.1.5 times was defined as the threshold of differential expression change,and t-test p<0.05 was used as the significance threshold.Among the quantified proteins,when p<0.05,the change of differential expression levels over 1.5 in IgAN/normal control ratio was defined as the threshold for significant up-regulation,and that less than 1/1.5 was defined as the threshold for significant down-regulation.As a result,we found that 180 proteins were up-regulated and 157 were down-regulated in IgAN.Then,the functions and characteristics of these differential proteins were annotated from the following aspects:1.Gene Ontology(GO),which comprised biological process,molecular function and cell composition,was applied to elucidate the biological function of proteins.2.Protein metabolism pathway was annotated by the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.3.Protein domain.4.Clusters of Orthologous Groups of proteins(COG)functional classification.5.Subcellular structure localization.The enrichment analysis of differentially expressed proteins in two groups was conducted at diverse levels,including GO classification,KEGG pathway and protein domain,and it was found that the differentially expressed proteins had a significant enrichment in some functional types.For example,GO enrichment analysis revealed that differentially expressed proteins were highly enriched in the inflammatory response and immune complex formation,and KEGG pathway enrichment analysis suggested that differentially expressed proteins were significantly enriched in the NF-κB signaling pathway and complement and coagulation cascades.All of these were important in the development of IgAN.Ceruloplasmin,Haptoglobin and Serotransferrin were screened as candidate protein markers through bioinformatics analysis and PubMed database.Western blotting was used to determine the expression levels of the three candidate proteins in the two groups.It was found that the expressions of Ceruloplasmin,Haptoglobin and Serotransferrin in the IgAN group were significantly higher than those in the normal control group.Conclusions:In summary,Ceruloplasmin,Haptoglobin and Serotransferrin in urinary extracellular vesicles of IgA nephropathy may be novel biomarkers in the diagnosis of IgA nephropathy.