Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome

Abstract

Recurrent urinary tract infections (UTIs) pose a significant burden on the health care system. Underlying mechanisms predisposing children to UTIs and associated changes in the urinary proteome are not well understood. We aimed to investigate the urinary proteome of a subset of children who have vesicoureteral reflux (VUR) and recurrent UTIs because of their risk of developing infection-related renal damage. Improving diagnostic modalities to identify UTI risk factors would significantly alter the clinical management of children with VUR. We profiled the urinary proteomes of 22 VUR patients with low grade VUR (1-3 out of 5), a history of recurrent UTIs, and renal scarring, comparing them to those obtained from 22 age-matched controls. Urinary proteins were analyzed by mass spectrometry followed by protein quantitation based on spectral counting. Of the 2,551 proteins identified across both cohorts, 964 were robustly quantified, as defined by meeting criteria with spectral count (SC) ≥2 in at least 7 patients in either VUR or control cohort. Eighty proteins had differential expression between the two cohorts, with 44 proteins significantly up-regulated and 36 downregulated (q <0.075, FC ≥1.2). Urinary proteins involved in inflammation, acute phase response (APR), modulation of extracellular matrix (ECM), and carbohydrate metabolism were altered among the study cohort.

Keywords: Mass spectrometry; acute phase response; clinical proteomics; infectious disease; renal function or biology; urinary tract infection; urine analysis; vesicoureteral reflux.

Graphical abstract

Fig. 1.

Volcano Plot of VUR to Control Cohort Ratios for All Quantified Proteins. Of the 964 robustly identified and quantified proteins in the VUR and control cohorts, 80 proteins were deemed differentially regulated candidate markers by meeting criteria of q-value <7.5% and FC ≥+1.2 (up-regulated in VUR; green) or ≤−1.2 (down-regulated in VUR; red). Specific candidate proteins of interest as determined by biological network analysis are indicated. FC, fold change.

Fig. 2.

Hierarchical Clustering of Differentially Regulated Proteins Demonstrates Segregation of the VUR and Control Cohorts. Eighty proteins met criteria of corrected q-value FDR <7.5% and FC ≥1.2. Hierarchical clustering utilized the normalized SC for these proteins in each sample, with color intensity based on SC value. The resulting dendrograms demonstrated segregation and clustering for the majority of the VUR and control samples (horizontal axis). The candidate markers also clustered and segregated based on whether they were up- or downregulated in the VUR cohort (vertical axis).

Fig. 3.

Functional Characterization of VUR-regulated Proteins. A, Overrepresented biological pathways from all regulated proteins in the urine of the VUR patients. Percentage of proteins indicates the VUR-altered proteins over the entire proteins in the pathway (bars), while significance of the enrichment is represented by the p value (enrichment and reference 0.05 p value). B, Representative molecular function was separately assigned to the urinary proteins that were up- (green) or downregulated (red). Asterix represents p value <0.05. Functional enrichment was performed via FunRich version 3.0. ECM, extracellular matrix; n.s., not significant; VUR, vesicoureteral reflux.

Fig. 4.

ELISA Quantification of ORM1 in ValidationCohort. A validation cohort consisting of VUR (n = 20) and control (n = 20) samples separate from the discovery cohort were tested. Mean levels with standard error are shown, demonstrating a statistically significant elevation of ORM1 levels in the VUR cohort by Mann-Whitney-Wilcoxon test. ORM, orosomucoid; VUR, vesicoureteral reflux.