Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype

Abstract

Bladder urothelium senses and communicates information about bladder fullness. However, the mechanoreceptors that respond to tissue stretch are poorly defined. Integrins are mechanotransducers in other tissues. Therefore, we eliminated β1-integrin selectively in urothelium of mice using Cre-LoxP targeted gene deletion. β1-Integrin localized to basal/intermediate urothelial cells by confocal microscopy. β1-Integrin conditional-knockout (β1-cKO) mice lacking urothelial β1-integrin exhibited down-regulation and mislocalization of α3- and α5-integrins by immunohistochemistry but, surprisingly, had normal morphology, permeability, and transepithelial resistance when compared with Cre-negative littermate controls. β1-cKO mice were incontinent, as judged by random urine leakage on filter paper (4-fold higher spotting, P<0.01; 2.5-fold higher urine area percentage, P<0.05). Urodynamic function assessed by cystometry revealed bladder overfilling with 80% longer intercontractile intervals (P<0.05) and detrusor hyperactivity (3-fold more prevoid contractions, P<0.05), but smooth muscle contractility remained intact. ATP secretion into the lumen was elevated (49 vs. 22 nM, P<0.05), indicating abnormal filling-induced purinergic signaling, and short-circuit currents (measured in Ussing chambers) revealed 2-fold higher stretch-activated ion channel conductances in response to hydrostatic pressure of 1 cmH2O (P<0.05). We conclude that loss of integrin signaling from urothelium results in incontinence and overactive bladder due to abnormal mechanotransduction; more broadly, our findings indicate that urothelium itself directly modulates voiding.

Figure 1.

UPII-Cre mice specifically excise flanking loxP sites in urothelium, and UPII-Cre/β1^fl/fl mice lose expression in the basal cells of the urothelium. A) Schematic showing construct used to create UPII-Cre mice. B, C) Rosa-R26R-YFP floxed reporter mouse crossed with UPII-Cre mouse reveals specific expression of YFP in Cre-expressing urothelium (C) but not in Cre⁻ littermate control (B). D) Immunofluorescent localization of β1-integrin in bladder shows modest expression in urothelium (delineated by AQP3 staining in red) located primarily along the basal surface (yellow in merged image). U, urothelium; LP, lamina propria; SM, smooth muscle; BV, blood vessel. E) Integrin expression in control mice (green) seen as yellow signal in urothelium (integrin colocalizing with red AQP3; Cre⁻ littermates of β1-cKOs). F) Loss of integrin expression in urothelium of β1-cKOs. Scale bars = 50 μm (C, D); 10 μm (E, F).

Figure 2.

Immunoblotting confirms a reduction in β1-integrin expression. A) Urothelial lysates (50 μg) from control and β1-cKO bladders were immunoblotted with β1-integrin rabbit monoclonal antibody. WB, whole bladder. B) Quantitation of β1-integrin expression by densitometry was normalized to β-actin signal (n=4 bladders/group, means ± sem). *P < 0.05.

Figure 3.

Immunoperoxidase staining of α3-integrin reveals mislocalization and down-regulation of α3-integrin in β1-cKO bladders. A, B) Control bladders. C, D) β1-cKO bladders. U, urothelium; LP, lamina propria.

Figure 4.

Bladder morphology looks normal for β1-cKO mice. Control (A, B, C) and β1-cKO mouse bladders (D, E, F) were examined by H&E staining (A, D), TEM (B, E), and SEM (C, F). Urothelial morphology and ultrastructure look normal in all groups. B, E) UC, umbrella cell; n, nuclei of intermediate and basal cells. C, F) Adjacent UCs shown by asterisks. No evidence indicates loss of surface cells, injury, or dedifferentiation. Tight junctions appear normal. Scale bars = 50 μm (A, D); = 10 μm (C–F).

Figure 5.

Barrier function is unimpaired in β1-cKO mice. Bladders from control mice and β1-cKO mice were mounted in modified Ussing chambers for measurements of permeability to isotopic water (A) and urea (B) and for transepithelial resistance (C). Permeability coefficients were calculated from the flux rates across the urothelium. Data are shown as means ± sem, with n = 5–8 mice/group. There were no significant differences in permeability or TER.

Figure 6.

β1-cKO mice are incontinent. Voiding behavior was analyzed by quantitation of urine spot patterns on filter paper. A–D) Representative urine spot patterns for control (A, B) and β1-cKO mice (C, D). Control mice void in a controlled manner in one or two areas, while β1-cKOs have uncontrolled random urine leakage. E–G) Summary data for 4 mice/group that were tested on 3 separate days. Urine spot number (E) and spot area (F) as a percentage of the filter paper area were significantly greater in the β1-cKO mice; frequency distribution of urine spot volumes (G) showed β1-cKO mice had a greater proportion of urine deposits that were moderately large (0.8–4 μl). *P < 0.05, **P < 0.01.

Figure 7.

β1-cKO mice have overactive bladder and overfill. Cystometry was performed on anesthetized mice, and intrabladder pressure was monitored with a pressure transducer throughout multiple fill and void cycles. A, B) Representative cystometrograms of a control mouse (A) and a β1-cKO mouse (B). Asterisks indicate the point at which voiding occurs; time between voids is the intercontractile interval (ICI). C) ICI is greater for β1-cKO mice than for controls. *P < 0.05. D) Prevoiding contractions were much more frequent in β1-cKO mice than controls. *P < 0.01. E) Voiding pressure changes were not different. F) ATP concentrations in voided cystometry fluid were higher in β1-cKO mice. *P < 0.05. Data in panels C–F are shown as means ± sem. n = 6–7 mice/group (C–E); 4 mice/group (F).

Figure 8.

β1-cKO bladders exhibit higher stretch-activated ion conductances than controls: A) Bladders were mounted in Ussing chambers with continuous monitoring of I_sc. On exposure to 1 cmH₂O hydrostatic pressure on the luminal side (arrows), I_sc increased dramatically. The magnitude of the increase was significantly greater for β1-cKO mouse bladders. B) Summary of I_sc changes with pressure (n=4 mice/group). *P < 0.05.