Robustness of urinary extracellular vesicle-derived miRNA profiles over multiple days and the impact of urine concentration

Urinary extracellular vesicle (EV)-derived microRNAs (miRNAs) have emerged as promising noninvasive biomarkers for disease prediction. However, fluctuations in urine concentration throughout the day may influence miRNA abundance and profiles, potentially limiting their clinical utility. To ensure the broader applicability of urinary miRNAs in disease prediction, it is essential that their profiles remain stable regardless of collection time. In this study, we systematically examined factors influencing urinary miRNA abundance and assessed the stability of miRNA profiles. We collected serial urine samples from healthy individuals over three days, and performed small RNA sequencing to analyze urinary miRNA profiles in relation to various urine concentration parameters. Total miRNA counts were negatively correlated with urine volume and positively correlated with urine concentration indicators, including specific gravity and creatinine levels. miRNA profiles remained stable across different times and days when samples with low miRNA counts were excluded. These findings indicate that urine dilution—primarily due to fluid intake—is a major cause of variation in urinary miRNA abundance. Our results highlight the importance of collecting adequately concentrated samples and provide foundational insights for developing standardized urine collection protocols to enhance the reliability of urinary miRNAs as biomarkers. miRNA-seq of urinary miRNA over 3 days from 6 healthy individuals.