BackgroundUreteropelvic junction obstruction (UPJO) is the most common cause of pediatric congenital hydronephrosis, and continuous kidney function monitoring plays a role in guiding the treatment of UPJO. In this study, we aimed to explore the differentially expressed proteins (DEPs) in the urinary extracellular vesicles(uEVs) of children with UPJO and determine potential biomarkers of uEVs proteins that reflect kidney function changes.MethodsPreoperative urine samples from 6 unilateral UPJO patients were collected and divided into two groups: differential renal function (DRF) ≥ 40% and DRF < 40%.We subsequently used data-independent acquisition (DIA) to identify and quantify uEVs proteins in urine, screened for DEPs between the two groups, and analyzed biofunctional enrichment information. The proteomic data were evaluated by Western blotting and enzyme-linked immunosorbent assay (ELISA) in a new UPJO testing cohort.ResultsAfter one-way ANOVA, a P adj value < 0.05 (P-value corrected by Benjamin–Hochberg) was taken, and the absolute value of the difference multiple was more than 1.5 as the screening basis for obtaining 334 DEPs. After analyzing the enrichment of the DEPs according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment combined with the protein–protein interaction (PPI) network results, we selected nicotinamide adenine dinucleotide-ubiquinone oxidoreductase core subunit S1 (NDUFS1) for further detection. The expression of NDUFS1 in uEVs was significantly lower in patients with DRF < 40% (1.182 ± 0.437 vs. 1.818 ± 0.489, P < 0.05), and the expression level of NDUFS1 was correlated with the DRF in the affected kidney (r = 0.78, P < 0.05). However, the NDUFS1 concentration in intravesical urine was not necessarily related to the change in DRF (r = 0.28, P = 0.24).ConclusionsReduced expression of NDUFS1 in uEVs might indicate the decline of DRF in children with UPJO.