Association between NDUFS1 from urinary extracellular vesicles and decreased differential renal function in children with ureteropelvic junction obstruction

Abstract

Background: Ureteropelvic 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.

Methods: Preoperative 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.

Results: After 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).

Conclusions: Reduced expression of NDUFS1 in uEVs might indicate the decline of DRF in children with UPJO.

Keywords: Differential renal function; NDUFS1; Ureteropelvic junction obstruction; Urinary extracellular vesicles.

Fig. 1

Identification of urinary extracellular vesicles. A, B Transmission electron microscopy showed that the uEVs had a discoid vesicle-like structure with an intact lipid bilayer. C Nanoparticle tracking analysis: the size range of the particles. D Western blot experiments showed that calnexin was not expressed in uEVs but that TSG101 and CD63 were expressed in uEVs. Full-length blots/gels are presented in Additional file 2

Fig. 2

Proteomic analysis of urinary extracellular vesicles. A Volcano plot of proteins with FDR values < 0.05 and absolute fold change values > 1.5. B Clustering diagram of the major differentially expressed proteins between samples

Fig. 3

Bioinformatics analysis of the differentially expressed proteins. A Cluster map of the GO enrichment analysis results for the DEPs. The color from green to red indicates higher expression. The color bars on the left are used to distinguish biological processes, cellular components, and molecular functions. B DEPs-based KEGG concentration bubble map. The left side of this figure is the Sankey plot, representing the proteins contained in each pathway, and the right side is the conventional bubble plot, where the bubble size indicates the number of proteins to which the pathway belongs, and the bubble color indicates the P-value

Fig. 4

Screening of hub protein. A PPI network diagram of the differentially expressed proteins according to the degree algorithm. Darker colors and larger bubbles represent greater connectivity. B GO enrichment STRING diagram of major differentially expressed proteins. The enrichment results are presented using chordal plots, with genes on the left, GO terms on the right, and the middle line indicating affiliation. The proteins ranked by − log₁₀(P) value are shown by the Circro diagrams

Fig. 5

Relationship between NDUFS1 expression and DRF in children with UPJO. A, B Decreased expression of uEVs-derived NDUFS1 in children with unilateral UPJO and an SFU level of 4 with DRF < 40%. Full-length blots/gels are presented in Additional file 2. C Correlation between the relative uEVs expression levels of NDUFS1 and DRF. D Correlation between urinary NDUFS1 levels in the bladder and changes in DRF. *P < 0.05, **P < 0.01