Urinary Proteins Of IgA Nephropathy And Henoch-Schonlein Purpura Nephritis In Children With High-resolution Mass Spectrometry And Clinical Study

Background and objective:Primary IgA nephropathy(IgAN)is one of the most common glomerulonephritis in children and a common cause of chronic kidney disease in children and adolescents.Its pathology was characterized by IgA glomerular deposition.Glomerular deposition of IgA also characterizes other renal disorders,including Henoch-Schonlein purpura nephritis(HSPN)and other immune-complex glomerulonephritis afflicting patients due to chronic liver disease.Although the prognosis is not the same exactly,recent studies have suggested that IgAN and HSPN have similar pathogenesis.Renal biopsy is the gold standard for diagnosis of IgAN and HSPN,but due to its invasion and bleeding risk,it is not suitable for primary clinical first-line medical personnel to master and repeat operations.Clinical approaches to noninvasive biomarkers are urgently needed.At present,urine proteomics has been widely used in the study of kidney diseases and biomarkers.Here,we detected the differentially expressed proteome of patients with IgAN and HSPN in urine by liquid chromatography-tandem mass spectrometry(LC-MS/MS)in the data-independent acquisition(DIA)mode for the first time.The results may provide important clues for the pathogenesis,progression and prognosis of the two diseases,as well as provide references for urinary biomarkers.Methods:Children diagnosed with IgAN(group A,n=54)and HSPN(group B,n=54)by renal biopsy were studied from the Department of Pediatrics,Jinling hospital,the First School of Clinical Medicine,Southern Medical University between June 2018 and December 2019.Clinical data were recorded for these patients(including gender,age of onset,onset time,pathology,treatment outcome and laboratory parameters,such as urine protein quantification,urine erythrocyte count,serum IgA,serum IgE,serum IgM,serum creatinine and so on).In addition,healthy children without underlying diseases(group C,n=34))were selected as the control group.Midstream urine in the morning was collected from all participants.Among them,the urine specimens of 4 cases in group A,4 cases in group B and 4 cases in group C were analyzed by high-resolution mass spectrometry.The obtained protein profiles of group A and group B were compared with those of group C respectively to define the differentially expressed proteins(the disease-specific proteins)of the two groups.Then,the differentially expressed proteins of the two groups were analysed by Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Besides that,preliminary validations were performed for screening potential biomarkers in the urine specimens of 50 cases in group A,50 cases in group B and 50 cases in group C using enzyme-linked immunosorbent assay(ELISA).Then,the validated results were statistically correlated with the above clinicopathologic indicators.Results:(1)A total of 276 urinary proteins were found to be differentially expressed in children with IgAN compared with the healthy controls(p<0.05).By GO analysis and KEGG pathway annotation,the differentially expressed proteins were significantly enriched in cell adhesion molecules,ECM-receptor interactions and cholesterol metabolism(P<0.05).In addition,the PI3K-Akt signalling pathway,complement and coagulation cascade,platelet activation and regulation of actin cytoskeleton were involved(2)A total of 125 urinary proteins were found to be differentially expressed in children with HSPN compared with the healthy controls(P<0.05).By GO analysis and KEGG pathway annotation,the differentially expressed proteins were significantly enriched in aldosterone synthesis and secretion(P<0.05).In addition,cell adhesion molecules,ECM-receptor interactions,the PI3K-Akt signalling pathway,complement and coagulation cascade,platelet activation,cholesterol metabolism and regulation of actin cytoskeleton were involved.(3)From the integrated information of mass spectrometry,there were 90 commonly signalling pathways and 74 differentially expressed urinary proteins in these pathways,which all existed in the IgAN group and HSPN group.The core proteins ITGB1,TNC,A1BG and AFM involved in the pathogenesis of IgAN and HSPN were selected for preliminary verification by ELISA.The results showed that the levels of urinary ITGB1 and urinary TNC were down-regulated significantly in the IgAN group and HSPN group compared with that of the healthy group,while the levels of urinary A1BG and urinary AFM were significantly up-regulated in the IgAN group and HSPN group compared with that of the healthy group.The above results were consistent with the results of mass spectrometry(P<0.05).In addition,the levels of urinary A1BG and urinary AFM in the two groups were positively correlated with 24-hour urinary protein quantification(P<0.05).In IgAN group,the level of urinary A1BG was correlated with glomerular segural sclerosis,and the level of urinary AFM in IgAN group was correlated with crescent formation(P<0.05)Conclusions:In paper,we elucidated the specifically expressed urine proteins and their biological signalling pathways in children with IgAN and HSPN,and we found the common involved pathways and their differentially expressed proteins in the two groups.Our study provides new insights into the assessment of pathogenesis,progression,and prognosis in children with IgAN and HSPN,as well as new potential biomarkers.