Urinary proteomics of Henoch-Schönlein purpura nephritis in children using liquid chromatography-tandem mass spectrometry

Abstract

Background: Henoch-Schönlein purpura nephritis (HSPN) is the principal cause of morbidity and mortality in children with Henoch-Schönlein purpura (HSP). However, the criteria for risk assessment currently used is not satisfactory. The urine proteome may provide important clues to indicate the development of HSPN.

Methods: Here, we detected and compared the urine proteome of patients with HSPN and healthy controls by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the data-independent acquisition (DIA) mode. The differentially expressed proteins were analysed by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. For validation, enzyme-linked immunosorbent assay (ELISA) was used to analyse the selected proteins.

Results: A total of 125 proteins (29 upregulated and 96 downregulated) were found to be differentially expressed in children with HSPN compared with the controls. Forty-one proteins were predicted to have direct interactions. The enriched pathways mainly included focal adhesion, cell adhesion molecules, the PI3K-Akt signalling pathway, ECM-receptor interactions and so on. Cell adhesion related to the pathogenesis of HSPN was the main biological process identified in this study. The decrease in two proteins (integrin beta-1 and tenascin) was validated by ELISA.

Conclusions: Our study provides new insights into the assessment of HSPN progression in children, as well as new potential biomarkers. The data confirm the value of the urinary proteome in capturing the emergence of HSPN.

Keywords: Children; Henoch-Schönlein purpura nephritis; Mass spectrometry; Proteomics; Urine.

Fig. 1

Cluster analysis of 125 differentially expressed proteins. The hierarchical clustering results are represented as a tree heat map, with the ordinate representing significantly differentially expressed proteins and the abscissa representing sample information. Significant differences in protein expression in the different numerical expression quantities (log2 expression) of the samples with different colours are shown in the heat map, where red represents significantly upregulated proteins, green represents significantly downregulated proteins, and grey represents no quantitative information for proteins. HS-1, 2, 3, and 4 represent patients with Henoch-Schönlein purpura nephritis, and Con-1, 2, 3, and 4 represent the healthy control group

Fig. 2

GO enrichment analysis of 125 differentially expressed proteins. The abscissa represents enriched GO function classifications, which are divided into three major categories: biological process (BP), molecular function (MF) and cellular component (CC). The ordinate represents the number of different proteins under each functional classification. The colour bars represent the significance of the enriched GO functional classification. The colour gradient represents the size of the p value (p < 0.05). The label at the top of the bar graph shows the enrichment factor (richFactor ≤ 1), which represents the ratio of the number of differentially expressed proteins annotated to a certain GO functional category to the number of identified proteins annotated to that GO functional category

Fig. 3

Top 20 enriched pathways according to KEGG functional classification analysis of 125 differentially expressed proteins. The ordinate represents the number of differentially expressed proteins contained in each KEGG pathway. The x-coordinate represents the significantly enriched KEGG pathway

Fig. 4

KEGG pathway enrichment analysis of 125 differentially expressed proteins. The ordinate represents the significantly enriched KEGG pathway. The x-coordinate represents the number of differentially expressed proteins contained in each KEGG pathway. The colour bars represent the significance of the enriched KEGG pathways. The colour gradient represents the size of p values (p < 0.05). The label at the top of the bar graph shows the enrichment factor (richFactor ≤ 1), which represents the proportion of the number of differentially expressed proteins involved in a KEGG pathway among all identified proteins

Fig. 5

Differentially expressed protein interaction networks. In the protein interaction network, nodes represent proteins and lines represent protein–protein interactions. Yellow nodes are differentially expressed proteins. The number of proteins directly interacting with protein A is called the linkage degree of protein A. P24821 tenascin, P05556 integrin beta-1, A0A024RAQ9 versican, P02452 collagen alpha-1(I), P02671 Fibrinogen alpha, A0A024R462 fibronectin-1

Fig. 6

Urinary levels of integrin beta-1 and tenascin by ELISA in the validation cohort. a The urine concentration level of integrin beta-1 using ELISA kits. b The urine concentration level of tenascin using ELISA kits. The ordinate represents the concentration of proteins. The x-coordinate represents the group of validation. HSPN represents the group of Henoch-Schönlein purpura nephritis, Normal represents the healthy control group, and HSP represents the group of Henoch-Schönlein purpura