Urinary peptidomics reveals proteases involved in idiopathic membranous nephropathy

Abstract

Background: Idiopathic membranous nephropathy (IMN) is a cause of nephrotic syndrome that is increasing in incidence but has unclear pathogenesis. Urinary peptidomics is a promising technology for elucidating molecular mechanisms underlying diseases. Dysregulation of the proteolytic system is implicated in various diseases. Here, we aimed to conduct urinary peptidomics to identify IMN-related proteases.

Results: Peptide fingerprints indicated differences in naturally produced urinary peptide components among 20 healthy individuals, 22 patients with IMN, and 15 patients with other kidney diseases. In total, 1,080 peptide-matched proteins were identified, 279 proteins differentially expressed in the urine of IMN patients were screened, and 32 proteases were predicted; 55 of the matched proteins were also differentially expressed in the kidney tissues of IMN patients, and these were mainly involved in the regulation of proteasome-, lysosome-, and actin cytoskeleton-related signaling pathways. The 32 predicted proteases showed abnormal expression in the glomeruli of IMN patients based on Gene Expression Omnibus databases. Western blot revealed abnormal expression of calpain, matrix metalloproteinase 14, and cathepsin S in kidney tissues of patients with IMN.

Conclusions: This work shown the calpain/matrix metalloproteinase/cathepsin axis might be dysregulated in IMN. Our study is the first to systematically explore the role of proteases in IMN by urinary peptidomics, which are expected to facilitate discovery of better biomarkers for IMN.

Keywords: Idiopathic membranous nephropathy; Proteases; Urinary peptidomics.

Fig. 1

Workflow of the study. First, urine samples were collected from subjects; next, peptides were extracted from the samples and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Peptides differentially expressed in patients with idiopathic membranous nephropathy (IMN) and matched proteins were identified. Bioinformatics analyses were performed to predict kidney-originated proteins and reveal the expression pattern in kidneys based on the iProX database. Functional enrichment analysis was conducted using the Proteasix database to predict proteases. Finally, proteases were validated via Gene Expression Omnibus (GEO) database and western blot analyses. HC, healthy control; OD, other kidney disease

Fig. 2

Urinary peptide fingerprints. The X-axis indicates detected peptides smaller than 10,000 kDa, and the Y-axis denotes the intensity of the peptide signals. The peptide components differed among healthy control, idiopathic membranous nephropathy (IMN), IgA nephropathy (IgAN), focal segmental glomerulosclerosis (FSGS), and minimal change disease (MCN) groups

Fig. 3

Heat maps showing (A) the expression pattern of genes encoding the 158 kidney-originated proteins in single renal cells and (B) 55 differentially expressed proteins (DEP) in kidney tissue from patients with idiopathic membranous nephropathy (IMN) based on iProX database analysis

Fig. 4

Gene Ontology (GO) enrichment analysis of kidney-derived urine proteins. A Interaction network of biological processes. B Cellular components and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways

Fig. 5

Functional annotation of differentially expressed proteins validated in iProX. A Cellular components; (B) Biological processes; (C) Signaling pathways. Upregulated and downregulated pathways are indicated by red (right) and blue (left) bars

Fig. 6

Protease validation using Gene Expression Omnibus (GEO) database and western blot analyses. A Proteases differentially expressed in idiopathic membranous nephropathy (IMN). B and (C) Western blot showing relative expression of proteases. ^* P < 0.05; ^** P < 0.01; ns: P > 0.05