Identification and functional characterization of urinary protein biomarkers for RA

OBJECTIVES: To identify and validate novel urinary protein biomarkers associated with disease activity in RA, and to investigate their potential biological roles. METHODS: 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. RESULTS: 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. CONCLUSION: IGLV3-1 is a novel urinary protein biomarker that reflects RA disease activity and predicts clinical outcomes.