A multiplexed analysis approach identifies new association of inflammatory proteins in patients with overactive bladder

Abstract

Overactive bladder (OAB) is a common debilitating bladder condition with unknown etiology and limited diagnostic modalities. Here, we explored a novel high-throughput and unbiased multiplex approach with cellular and molecular components in a well-characterized patient cohort to identify biomarkers that could be reliably used to distinguish OAB from controls or provide insights into underlying etiology. As a secondary analysis, we determined whether this method could discriminate between OAB and other chronic bladder conditions. We analyzed plasma samples from healthy volunteers (n = 19) and patients diagnosed with OAB, interstitial cystitis/bladder pain syndrome (IC/BPS), or urinary tract infections (UTI; n = 51) for proinflammatory, chemokine, cytokine, angiogenesis, and vascular injury factors using Meso Scale Discovery (MSD) analysis and urinary cytological analysis. Wilcoxon rank-sum tests were used to perform univariate and multivariate comparisons between patient groups (controls, OAB, IC/BPS, and UTI). Multivariate logistic regression models were fit for each MSD analyte on 1) OAB patients and controls, 2) OAB and IC/BPS patients, and 3) OAB and UTI patients. Age, race, and sex were included as independent variables in all multivariate analysis. Receiver operating characteristic (ROC) curves were generated to determine the diagnostic potential of a given analyte. Our findings demonstrate that five analytes, i.e., interleukin 4, TNF-α, macrophage inflammatory protein-1β, serum amyloid A, and Tie2 can reliably differentiate OAB relative to controls and can be used to distinguish OAB from the other conditions. Together, our pilot study suggests a molecular imbalance in inflammatory proteins may contribute to OAB pathogenesis.

Keywords: OAB; UTI; biomarkers; bladder pain syndrome; interstitial cystitis.

Fig. 1.

Urine Inflammatory cell scores do not distinguish overactive bladder (OAB) from other bladder diseases. Urine sediments from OAB, interstitial cystitis/bladder pain syndrome (IC/BPS), acute urinary tract infcetion (UTI), and controls were spun onto slides and analyzed under a microscope. A semiquantitative polymorphonuclear cell (PMN) score ranging from 0 to 4 was assigned to each sample, represented by the bar graph.

Fig. 2.

Selective pro- and anti-inflammatory markers can differentiate OAB from healthy controls. Box plots are shown of the normalized expressions of the 5 analytes [IL-4, TNF-α, macrophage inflammatory protein (MIP-1β), Tie2, and serum amyloid A (SAA)] measured in pg/ml using Meso Scale Discovery technology comparing overactive bladder patients (n = 17) with healthy controls (n = 19). Using Wilcoxon rank-sum analysis, levels of IL4, TNF-α, Tie2, and SAA in OAB patients were statistically different from healthy controls, with P < 0.05. Using multivariate linear models controlling age, race, and sex, IL4, MIP-1β, and Tie2 in OAB patients were statistically different from healthy controls, with P < 0.05.

Fig. 3.

Receiver operating characteristic (ROC) curves can distinguish OAB patients from healthy controls. ROC curves for TNF-α, MIP-1β, Tie2, and SAA can differentiate OAB patients (n = 17) from healthy controls (n = 19).