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

Urinary extracellular vesicle (uEV)-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 uEV-derived miRNAs in disease prediction, it is essential that their profiles remain stable regardless of collection time. In this study, we systematically examined factors influencing miRNA abundance in uEVs 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 uEV-derived 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 is a major cause of variation in miRNA abundance. Our results highlight the importance of collecting adequately concentrated samples and provide foundational insights for developing standardized urine collection protocols, thereby facilitating the use of urinary uEV-derived miRNAs as robust and reliable biomarkers for disease prediction.