Study On Biomarkers Of Renal Injury In Henoch Purpura By TMT Technique

Background:Henoch-Schonlein purpura(HSP),also known as immunoglobulin A vasculitis(IgAV),is one of the common autoimmune diseases in children,characterized by systemic vasculitis.About 90%of cases occur in children between the ages of 2 and 10,with the onset peaking between the ages of 4 and 7.The lesions mainly involve mucous membrane,skin,gastrointestinal tract,joints and other parts.If it involves kidney,it is called henoch-schonlein’s purpura nephritis,HSPN),also known as IgA vasculitis with nephritis(IgAVN).HSPN occurs in 30%to 50%of patients with HSP within 4 to 6 weeks of their first onset.It is usually mild,with a small percentage of patients developing nephrotic syndrome or renal failure.The long-term prognosis depends on the degree of renal injury.At present,the gold standard for the diagnosis of HSPN is still renal biopsy,but it is difficult to be widely applied due to its invasive examination,relatively high cost,poor patient compliance and other problems.In recent years,in addition to the discovery of new biomarkers through traditional laboratory detection methods,proteomics technology has also provided the possibility for HSPN to discover novel biomarkers.This technology has the advantages of high throughput,high sensitivity and less need for biological fluid samples,and is expected to make greater contribution to the screening of promising biomarkers of purpura nephritis.Objective:In this study,the tandem mass tags for relative and absolute quantitation(TMT)protein quantitative technology based on the tandem mass spectrometry method was used to detect the differential expression of proteome in serum and urine of children with purpura and purpura nephritis.In order to discover biomarkers of early kidney damage in children with HSP,more clearly understand the pathogenesis of this disease,and provide important clues for the diagnosis,treatment and prognosis of this disease.Methods:Children in HSPN group(N group,n=15)and HSP group(P group,n=25)who were admitted to the Department of Pediatrics of the First Affiliated Hospital of China Medical University from June 2021 to February 2023 and confirmed by renal biopsy were selected as the research objects.Clinical data(including gender,age of onset,urinary red blood cell count,24h urinary protein quantification and other laboratory indicators)were collected.Serum and 24-hour urinary supernatant of all children were collected before treatment,and serum and urine samples of 2 patients in each group were mixed in equal amounts respectively for quantitative analysis of TMT proteins.To identify proteins that are differentially expressed in serum and urine between the two groups of disease.Then,the differentially expressed proteins in serum and urine were analyzed by gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),pathway enrichment,pathway classification and protein network interaction.In addition,13 cases in the HSPN group and 23 cases in the HSP group were selected for western blot verification analysis of the differential proteins,in order to further clarify their significance as biomarkers for early diagnosis.Results:(1)Compared with the HSP group,106 proteins were differentially expressed in serum of the HSPN group,among which 59 proteins were up-regulated and 47 proteins were down-regulated(p<0.05).The GO and KEGG analysis showed that the major pathways involved in these differential proteins include protein binding,protein kinase binding,copper ion binding,transmembrane signaling receptor activity,cancer pathway,estrogen signaling pathway,toll-like receptor signaling pathway,Th17 cell differentiation,leukocyte transendothelial migration,IL-17 signaling pathway,NF-κB signaling pathway,and PI3K-A kt signaling pathway,TGF-β signaling pathway,complement and coagulation cascade.(2)Compared with the HSP group,290 proteins were found to be differently expressed in the urine of the HSPN group,among which 107 proteins were up-regulated and 183 proteins were down-regulated(p<0.05).The GO and KEGG analysis showed that The major pathways involved in these differential proteins include receptor regulatory activity,cationic binding,signal receptor binding,metal ion binding,protein binding,tight junctions,leukocyte migration across endothelial cells,protein digestion and absorption,ECM-receptor interactions,adhesion plaques,PI3K-Akt signaling pathway,estrogen signaling pathway,cell adhesion molecules,complement,and coagulation levels Synthetical,metabolic,etc.(3)Based on the above mass spectrum information,11 proteins were found to be differentially expressed in serum and urine,including KRT2,KRT9,KRT1,CP,ALB,OAF,MMP2,IGHM and IGFBP4.There were 24 signaling pathways that were jointly involved,including leukocyte migration across endothelial cells,cytokine-cytokine receptor interaction,GnRH signaling pathway,relaxin signaling pathway,AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,PPAR signaling pathway,complement and coagulation cascade,etc.(4)MMP2 was selected for preliminary western blot verification from the proteins of common differential expression in serum and urine.The results showed that compared with the HSP group,the serum MMP2 expression level was significantly upregulated in the HSPN group,while the urine MMP2 expression level was significantly down-regulated in the HSPN group,with statistical significance(p<0.05).The results are in agreement with those of mass spectrometry.Conclusion:In this paper,we elucidated the proteins differentially expressed in serum and urine of children with HSPN and their related signaling pathways,found common proteins differentially expressed in serum and urine and common biological pathways,and verified the protein MMP2,which proved the accuracy of this mass spectrometry analysis.Our study provides insights into the pathogenesis,diagnosis,prognosis assessment,screening of new biomarkers and discovery of new therapeutic targets for HSPN in children.