Effects of Hematuria on the Proteomic Profile of Urinary Extracellular Vesicles: Technical Challenges

Hematuria is a common sign of many
renal and urologic pathologic
conditions and it may affect the proteomic analysis of urinary extracellular
vesicles (UEv), nanovesicles released from all cells in contact with
the urinary space. This condition hinders UEv based proteomic studies
aiming to discover biomarkers. Therefore, we studied the effects of
hematuria on the proteome of UEv and introduced a possible solution
to reduce its misleading impact. We mimicked hematuria by adding increasing
amount of blood to nonaffected urine and investigated its effects
on UEv isolation, purity, and proteomic composition. We proposed a
trypsin treatment able to reduce the impact of hematuria on UEv. The
effects of the treatment were investigated by evaluating the UEv proteomic
profile, the enrichment of known UEv markers, and by assessing differential
protein content by MS-based label-free quantification. Results showed
that as the blood contamination increased, it affected both the proteome
profile and the yield of UEv isolated from urine. Our treatment with
trypsin was able to counteract completely these effects for low/medium
levels of hematuria, which are most commonly encountered. This promising
finding could lead to the reliable use of hematuria samples for UEv
proteomic investigation.