Bladder filling and voiding affect umbrella cell tight junction organization and function

Abstract

Epithelial cells are continuously exposed to mechanical forces including shear stress and stretch, although the effect these forces have on tight junction (TJ) organization and function are poorly understood. Umbrella cells form the outermost layer of the stratified uroepithelium and undergo large cell shape and surface area changes during the bladder cycle. Here we investigated the effects of bladder filling and voiding on the umbrella cell TJ. We found that bladder filling promoted a significant increase in the length of the TJ ring, which was quickly reversed within 5 min of voiding. Interestingly, when isolated uroepithelial tissue was mounted in Ussing chambers and exposed to physiological stretch, we observed a 10-fold drop in both transepithelial electrical resistance (TER) and the umbrella cell junctional resistance. The effects of stretch on TER were reversible and dependent on the applied force. Furthermore, the integrity of the umbrella cell TJ was maintained in the stretched uroepithelium, as suggested by the limited permeability of biotin, fluorescein, and ruthenium red. Finally, we found that depletion of extracellular Ca(2+) by EGTA completely disrupted the TER of unstretched, but not of stretched uroepithelium. Taken together, our studies indicate that the umbrella cell TJ undergoes major structural and functional reorganization during the bladder cycle. The impact of these changes on bladder function is discussed.

Keywords: bladder; claudins; stretch; tight junction; uroepithelium.

Fig. 1.

Cell shape changes and claudin and ZO-1 expression in full, voided, and quiescent rat bladders. A: cross-sections of bladder tissue were stained with antibodies to ZO-1 (green), TRITC-phalloidin (red), and ToPro-3 (blue). B: tissues were stained with an antibody to claudin-8 (green) and TRITC-phalloidin (red). The position of the TJ is indicated by arrows. Umbrella cells are outlined with dashed lines. Bottom: higher magnification views of the boxed region. C: Western blots of claudin-8 and -4 in full (F), voided (V), and quiescent (Q) bladders. GAPDH and actin expression were used as loading controls.

Fig. 2.

Filling promotes expansion of the TJ ring, which is reversed upon voiding. A: en face view of the umbrella cell layer in full, voided, and quiescent bladders stained with an antibody to claudin-8. Image is a projection of a confocal Z-series. B: 3-D reconstruction of bladder epithelium stained with TRITC-phalloidin (red) and ToPro-3 (blue). C: 3-D reconstruction of umbrella cell layer from stretched tissues stained with antibodies to claudin-8 (green), TRITC-phalloidin (red), and ToPro-3 (blue). D: average length of TJ per umbrella cell (mean ± SE, n = 4). Values for voided and quiescent tissues are significantly different from those of full bladders (P < 0.05 using ANOVA and a Dunnett's post hoc test). The average length of TJ is not significantly different between voided and quiescent samples.

Fig. 3.

SEM analysis of the zippered apical membrane between adjacent umbrella cells. Tissues from full (A), voided (B), and quiescent (C) bladders were fixed and processed for SEM analysis. The boxed regions (left) are magnified on the right. The location and path of the zippered apical membrane is indicated by a dashed line.

Fig. 4.

Stretch augments uroepithelial ion transport. The rabbit bladder mucosa was mounted in Ussing chambers and equilibrated with a small, negative mucosal hydrostatic pressure, causing the tissue to bow inward. A: changes in the transepithelial voltage (V_T) in response to the addition (gray) or removal (black) of 0.2-ml aliquots of buffer from the mucosal compartment were recorded. A square current pulse of 10 μA was applied every 60 s to allow for determination of the TER. A representative tracing is shown. B: uroepithelial TER was plotted as a function of the volume in the mucosal compartment. Values are mean ± SE (n = 13 independent experiments). C: to mimic bladder filling and voiding, the uroepithelium was stretched by adding 15 aliquots of 0.2-ml of Krebs solution to the mucosal compartment over a period of 45 min and then the added fluid was removed at a rate of 1.5 ml/min. TER was measured 5 min after fluid removal. Values are mean ± SE (n = 7).

Fig. 5.

Stretch increases the junctional permeability of the uroepithelium. Isolated rabbit uroepithelium was mounted in Ussing chambers and the junctional resistance (R_J) was estimated as described in experimental procedures. A: representative recording showing the effect of gramicidin D (GrD) addition on the transepithelial voltage (V_T) of unstretched (control) and stretched uroepithelium. A square current pulse of 10 μA was applied every 60 s to allow for determination of TER. Gray arrows indicate the addition of 0.2-ml aliquots to the mucosal compartment; black arrows indicate the addition of GrD. B: estimation of the uroepithelial junctional resistance. V_T was plotted as function of TER following GrD addition for unstretched (open circles) and stretched uroepithelium (closed circles). The X- and Y-intercepts of the fit line equal the R_J and cellular electromotive force, respectively. C: R_J of unstretched (control) and stretched uroepithelium. Values are mean ± SE (n = 6–7 independent experiments; P < 0.001, using a Mann-Whitney test).

Fig. 6.

TJ integrity is maintained in stretched uroepithelium. Bladder mucosa mounted in Ussing chambers was maintained in a quiescent, unstretched condition (A) or subjected to stretch (B). Ruthenium red permeability was used to assess the integrity of the TJs by TEM. The location of the TJ is indicated by arrows, and the upper portion of the lateral membrane is indicated by arrowheads. C: bladder uroepithelium mounted in Ussing chambers remained in a quiescent state (control) or stretched in the absence or presence of EGTA. Following 30 min, sulfo-NHS-biotin was added to the mucosal hemichamber for 15 min, and the tissue was then fixed in situ. Cryosections of frozen tissue were labeled with Alexa-488-streptavidin (green), TRITC-phalloidin (red), and ToPro-3 (blue). Note the diffusion of biotin into the underlying tissues in control samples treated with EGTA. Images are representative of three independent experiments.

Fig. 7.

Remodeling of the paracellular pathway during experimental filling. A: representative recording showing the effect of EGTA on stretched uroepithelium. A square current pulse of 10 μA was applied every 60 s to allow for determination of TER. Gray arrows indicate the addition of 0.2-ml aliquots to the mucosal compartment; black arrow indicates the addition of EGTA. B: bladder uroepithelium mounted in Ussing chambers remained in an unstretched (control) state or was stretched (stretch). TER was measured at the beginning of the experiment (open bars; 1), after remaining unstretched or stretched (gray bars; 2), and 30 min after EGTA addition (closed bars; 3) (also see arrows in A). EGTA was added to the mucosal and serosal hemichambers to give a final concentration of 3 mM. Values are mean ± SE (n = 13–14 independent experiments; Mann-Whitney test, * P < 0.01 and **P < 0.001). C: tissue was mounted in Ussing chambers and remained in an unstretched (control) state or was stretched (stretch). Fluorescein was added to the mucosal hemichamber immediately after EGTA addition. Where indicated, EGTA was added to the mucosal and serosal hemichambers to give a final concentration of 3 mM. Fluorescein fluxes across the bladder uroepithelium were measured as described in experimental procedures. Values are mean ± SE (n = 4–6 independent experiments; Kruskal-Wallis followed by a Dunn's post hoc test, *P < 0.01). Differences between unstretched and stretched uroepithelium were not statistically significant (P > 0.05).