431 Defective uromodulin polymerization and peptide excretion in a natural canine model of kidney stones

Abstract

OBJECTIVES/GOALS: Using a natural canine model of kidney stone disease, we previously identified a pathogenic variant in the uromodulin gene (UMOD) that imparts a dramatic risk for calcium oxalate (CaOx) stones. This study was designed to characterize the effects of the pathogenic variant on uromodulin processing, specifically polymerization and peptide excretion. METHODS/STUDY POPULATION: Uromodulin polymerization status and peptides were measured in random urine samples from CaOx stone-forming dogs with the pathogenic UMOD variant and breed-, sex-, and age-matched healthy control dogs. Polymerization status was determined using an ultracentrifugation protocol and Western blotting in 6 CaOx cases and 3 controls; relative abundance of the polymerizing and nonpolymerizing forms was evaluated. Uromodulin peptide abundances were measured by LC-MS/MS with 4 dogs per group; results were summed to determine total uromodulin peptide excretion for each dog, and individual peptide abundances were calculated as a percentage of the total. Polymerization status and peptides were compared between groups. RESULTS/ANTICIPATED RESULTS: Dogs with the pathogenic UMOD variant had abnormalities in both uromodulin polymerization and peptide processing. The polymerization data showed that the polymerizing form of uromodulin was abundant in all healthy controls but absent or severely reduced in most dogs with the variant. In contrast, nonpolymerizing uromodulin was detected in all dogs with no observed difference between those with and without the variant. The peptidomics data showed that stone-forming dogs with the pathogenic UMOD variant lacked a peptide cleavage site, resulting in the loss of two common peptides that terminate at that site and the presence of longer peptides that span the site. DISCUSSION/SIGNIFICANCE: These findings implicate uromodulin polymerization and peptide processing defects in kidney stone risk. Future studies will define the mechanisms through which these defects affect stone formation, ultimately informing development of novel preventative therapies.