Identification and functional characterization of urinary protein biomarkers for RA.

Abstract

To identify and validate novel urinary protein biomarkers associated with disease activity in RA, and to investigate their potential biological roles. We employed a multi-phase strategy integrating proteomic discovery, biomarker validation, population-based analysis and mechanistic studies. Differentially expressed proteins (DEPs) were first identified using data-independent acquisition-based proteomic profiling in a discovery set comprising RA patients and healthy controls (HCs). Candidate proteins were subsequently validated by ELISA in two independent validation cohorts. Population-based associations were assessed in two RA cohorts (n = 301 cross-sectional; n = 214 longitudinal), with disease activity evaluated using VAS and DAS28 scores. Functional relevance was further examined in synovial tissues, fibroblast-like synoviocytes (FLS) and cytokine assays. A total of 209 DEPs were identified between RA patients and HCs, with enrichment analyses highlighting immune-related pathways. Among these, IGLV3-1 demonstrated high diagnostic potential (AUC = 0.99) and was consistently validated in two independent cohorts. In population-based analyses, IGLV3-1 levels were positively associated with both VAS (β = 1.070, 95% CI: 0.264-1.875) and DAS28 scores (β = 0.672, 95% CI: 0.029-1.315) and predicted a reduced likelihood of VAS pain improvement (OR = 0.188, 95% CI: 0.047-0.746). Mechanistically, IGLV3-1 was upregulated in RA synovial tissues and FLS, and positively correlated with IL-6, IL-8 and IL-12p70 levels. Its knockdown via siRNA in FLS led to reduced expression of these pro-inflammatory cytokines at both mRNA and protein levels. IGLV3-1 is a novel urinary protein biomarker that reflects RA disease activity and predicts clinical outcomes.