Cross-Species Identification of Urinary Small Extracellular Vesicle-Associated miR-223-3p as a Candidate Biomarker for Nephrolithiasis in a Discovery Cohort.

Abstract

Background: Nephrolithiasis remains a major global health burden with limited non-invasive tools for early diagnosis and monitoring. Urinary small extracellular vesicle (sEV)-associated microRNAs (miRNAs) are stable molecular signatures that reflect renal pathophysiological shifts. This study aims to identify candidate urinary sEV-associated miRNAs for nephrolithiasis in a clinical discovery cohort and prioritize candidate biomarkers by integrating human urinary sEV miRNA profiles with publicly available murine nephrolithiasis datasets.

Methods: Small RNA sequencing was performed on RNA isolated from urinary sEVs obtained from a clinical discovery cohort (25 patients with nephrolithiasis and 8 healthy controls) to identify candidate biomarkers associated with nephrolithiasis. Differentially expressed miRNAs (DEmiRNAs) were identified using a |log₂ fold change| > 1 and adjusted P < 0.05 threshold. Functional enrichment (GO/KEGG) was conducted on predicted target genes. To prioritize biologically relevant candidates, human DEmiRNAs were intersected with an available murine kidney miRNA dataset (GSE186793) and the ExoCarta database. The lead candidate, miR-223-3p, was further validated via RT-qPCR in human urinary sEV samples and urinary sEVs isolated from an independent CaOx-induced mouse model.

Results: We identified 111 significantly dysregulated urinary sEV-associated miRNAs (105 upregulated, 6 downregulated) in patients with nephrolithiasis. Functional analysis linked these miRNAs to endocytosis, mitochondrial organization, and the MAPK/Wnt signaling pathways. Integrative analysis of the human urinary sEV, the murine kidney miRNA datasets (GSE186793), and the ExoCarta database prioritized a conserved five-miRNA signature (miR-223-5p, miR-223-3p, miR-342-3p, let-7i-5p, and miR-29c-3p). Among these candidates, miR-223-3p demonstrated the strongest translational potential and was therefore selected for further validation. RT-qPCR confirmed that miR-223-3p was significantly upregulated in both human urinary sEVs and urinary sEVs from the CaOx-induced mouse model.

Conclusion: Our findings characterize a distinct urinary sEVs-associated miRNA landscape within this discovery cohort and identify miR-223-3p as a promising candidate biomarker for nephrolithiasis. Given the relatively small, clinically heterogeneous discovery cohort and the absence of an independent validation cohort, these findings should be considered hypothesis-generating and require validation in larger, independent, clinically stratified prospective studies before clinical application.