Urinary protein analysis in mice lacking major urinary proteins

Abstract

Mouse urine contains major urinary proteins (MUPs) that are not found in human urine. Therefore, even healthy mice exhibit proteinuria, unlike healthy humans, making it challenging to use mice as models for human diseases. It was also unknown whether dipsticks for urinalysis could measure protein concentrations precisely in urine containing MUPs. To resolve these problems, we produced MUP-knockout (Mup-KO) mice by removing the Mup gene cluster using Cas9 proteins and two guide RNAs and characterized the urinary proteins in these mice. We measured the urinary protein concentrations in Mup-KO and wild-type mice using a protein quantitation kit and dipsticks. We also examined the urinary protein composition using SDS-PAGE and two-dimensional electrophoresis (2DE). The urinary protein concentration was significantly lower ( P <0.001) in Mup-KO mice (17.9 ± 1.8 mg/dl, mean ± SD, n=3) than in wild-type mice (73.7 ± 8.2 mg/dl, n=3). This difference was not reflected in the dipstick values, perhaps due to the low sensitivity to MUPs. This suggests that dipsticks have limited ability to measure changes in MUPs with precision. SDS-PAGE and 2DE confirmed that Mup-KO mice, like humans, had no MUPs in their urine, whereas wild-type mice had abundant MUPs in their urine. The absence of the masking effect of MUPs in 2DE would enable clear comparisons of urinary proteins, especially low-molecular-weight proteins. Thus, Mup-KO mice may provide a useful model for human urinalysis.

The major urinary protein gene cluster knockout mouse as a novel model for translational metabolism research

“…With regard to previous reports that have shown inhibition of Mup expression by aging 53 , fasting 8 , 9 , dietary restriction 10 , overfeeding, and obesity 8 , 9 , studying these conditions in KO mice is expected to further improve the understanding of the metabolic implications of Mup in mice. Furthermore, the KO mouse model has recently been suggested as a useful model for human urinalysis 54 . In the KO mice, the urinary protein status was comparable to that of humans and eliminated the Mup-related masking of other urinary proteins.…”

The Role of Collagen VIII in the Aging Mouse Kidney

“…In contrast to humans, mouse urine contains major urinary proteins (MUPs) resulting in a physiological proteinuria. The high level of MUPs in urine may mask the presence or absence of urinary proteins of similar sizes in the analyses of urinary proteins, making the determination of proteinuria in mice difficult [31]. The measurement of cystatin C in urine (UcysC) has been shown to be a suitable and non-invasive method to detect renal pathologies; increased UcysC concentrations allow for the accurate detection of tubular dysfunction among pure and mixed nephropathies [22].…”

Characterization of the Entner-Douderoff Pathway inPseudomonas aeruginosaCatheter-associated Urinary Tract Infections

“…Third, the study involving UPEC also studied the fitness of other central carbon metabolism pathways in vivo and identified colonization defects in the gluconeogenesis and TCA mutants. This observation could be attributed to the fact that mouse urine contains a higher concentration of proteins compared to glucose (36, 37). Therefore, the significance of the E-D pathway appears to vary across different strains and infection sites.…”