Spontaneous voiding by mice reveals strain-specific lower urinary tract function to be a quantitative genetic trait

Abstract

Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18-24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations.

Keywords: animal model; bladder; heritability; lower urinary tract symptoms; micturition.

Fig. 1.

Image processing of void spot filters. A: photographic image of a urine-stained filter paper on an ultraviolet illuminator. B: thresholded and outlined image. C: corner voids are indicated by dotted regions and represent a total of 20% of the filter paper area. D: center voids are indicated by the dotted region and represent 40% of the total area. E: controlled delivery of urine in different volumes (1, 2 5, 10, 25, 50, and 100 μl) onto filter paper repeated in triplicate. F: image processing and quantitation of the spot areas from E allowing construction of an area-to-volume standard curve.

Fig. 2.

Individual mice exhibit reproducible patterns of urination that are strain unique. Top: photographs showing filters collected over 4 days [days 1–4 (D1–D4)] from two individual mice (mouse 804 and mouse 792) of two different inbred strains. Bottom: quantitative data extracted from four mice of each strain assayed on 4 consecutive days in the afternoon. All three parameters (urine spot number, percent area in the primary void, and total urine spot area) were highly statistically different by Student's t-test (P values are indicated).

Fig. 3.

Void spot data from all eight Collaborative Cross (CC) founder strains assayed in the morning, afternoon, and night. A: urine spot number. B: percent area in the primary void. C: total urine volume. D: percent volume in the corners. E: percent volume in the center. Each bar represents 3–5 consecutive assays/mouse averaged and 3–4 mice/strain averaged. Error bars are SEs.

Fig. 4.

Cystometry on all eight CC founder strains. A: representative cystometrograms (CMGs) from each strain. All tracings are displayed on the same x- and y-axes. B: summary data for intercontractile intervals (ICIs). C: summary data for pressure amplitude, defined as peak void pressure minus baseline pressure immediately after micturition. D: voiding duration, defined as the width of the voiding peak at 50% maximal pressure amplitude. E: mouse weights at 12–16 wk of age. Data were obtained from 3 or 4 mice of each strain; error bars are SEs.

Fig. 5.

Individual filters from four NZO/H1LtJ mice.

Fig. 6.

Effect of age on void spot data and CMGs. A and B: void spot filters from two 6-wk-old C57BL/6J mice. C and D: void spot filters from two 24-mo-old C57BL/6J mice. E: urine spot number summary data from young and old mice (n = 5). *P < 0.05. F: CMG from an old mouse.

Fig. 7.

Effect of urothelial injury on urine spotting. Urine spotting was assayed in 10 female C57BL/6J mice for 2 days before they received intravesical PBS (n = 5) or protamine sulfate (PS; 10 mM, n = 5). Mice were then reassayed on filter paper at 1, 24, and 48 h after instillation. Top: filter paper images showing the effect of PS administered at time 0 in one mouse. Bottom: graph showing the data summary for urine spot number at different time points. **P < 0.01.

Fig. 8.

Effect of enforced diuresis on void spots and CMGs. C57BL/6J mice were given water (control) or 5% sucrose to drink for 8 wk. A: void spots from a control mouse. B: void spots from a 5% sucrose-fed mouse. C: CMG from a control mouse. D: CMG from a 5% sucrose-fed mouse.