Urinary extracellular vesicles as a source of protein-based biomarkers in feline chronic kidney disease and hypertension

Abstract

Objectives To validate a methodology for isolating feline urinary extracellular vesicles (uEVs) and characterise the uEV population and proteome in cats with normal renal function and cats with normotensive or hypertensive chronic kidney disease Methods Feline uEVs were isolated using three different methods (precipitation alone, precipitation followed by size exclusion chromatography, and ultrafiltration followed by size exclusion chromatography, which were compared via transmission electron microscopy and nanoparticle tracking analysis . Cats with normal renal function (n = 9), normotensive CKD (n = 10) and hypertensive CKD (n = 9) were identified and uEVs isolated from patient urine samples via ultrafiltration followed by size exclusion chromatography. EV size and concentration were determined using nanoparticle tracking analysis, and subsequently underwent proteomic analysis using LC-MS/MS to identify differences in protein expression between categories.

Results uEV preparations contained particles of the expected size and morphology, and those obtained by UF+SEC had a significantly higher purity (highest particle: protein ratio). The uEV proteomes contained EV markers and proteins originating from all nephron segments. UEV concentration and size were unaffected by renal disease or hypertension. There were no differentially expressed proteins detected when comparing uEVs derived from cats in the healthy category with the combined CKD category, but five differentially expressed proteins were identified between the normotensive CKD and hypertensive CKD categories.

Clinical Significance Feline uEVs can be successfully isolated from stored urine samples. Differentially expressed uEV proteins were discovered in cats with hypertensive CKD, and warrant further investigation into their utility as biomarkers or therapeutic targets.