Urinary Biomarkers Identify Children with Vesicoureteral Reflux

Background: Vesicoureteral reflux (VUR) in childhood may cause severe renal injury leading to kidney failure. The methods for diagnosis and monitoring this process comprise expensive invasive imaging and management is not straight forward. Sensitive and specific biomarkers of VUR severity, progression, or scarring would aid in decision making regarding therapy and intervention. Recent studies indicate that a panel of biomarkers will be more sensitive to distinguish between various renal diseases and aid in disease staging.

Hypothesis: We hypothesize that the urinary biomarker profile will be able to distinguish between VUR and healthy age/gender-matched controls.

Design/Methods: Children (birth to 4 years) with VUR Grades 3-5 and healthy age-matched controls were enrolled in a prospective cohort study to identify urinary cytokines. Urine is collected at study entrance, yearly thereafter and pre and post surgical procedure. Urine cytokines are identified using an antibody array kit that detects 174 cytokines and injury markers (RayBiotech, Inc.,G-2000 Array Series). Cy3 fluorescence intensity is detected by laser scanner and normalized to sample’s urine creatinine. Fluorescence intensity data is used to compare cytokine concentration and relative quantities between groups. The cytokine’s relative fluorescence intensity was a semi-
quantitative means of comparison. Custom Elisa with 10 chosen biomarkers from screening array (Trail- R3, IL-8, VE-Cadherin, DR6, MMP1, MMP13, IL-2Rα, Groα, bFGF, MIF) was utilized for quantitation of marker concentration.

Results: 28 markers of 174 from screening array were found to be significantly higher in VUR patients compared to controls. 10 of 28 markers were chosen for validation. Of the 10 markers chosen for validation 3 (IL-8, MIF, Trail-R3) were found to be significantly different between VUR and controls. When utilizing logistic regression, the ROC curve of the combination of markers (IL-8, MIF, Trail-R3) is highly able to distinguish reflux from control with AUC of 0.896.

Conclusions: Three markers used in conjunction (IL-8, MIF, Trail-R3) identify children with reflux. These markers provide more evidence in the process of reflux leading to scarring. This may allow us in the future to decide which patients should undergo testing with VCUG.