The bladder urothelium is generally considered to be poorly permeable blood-urine barrier. However, recent studies have shown that the urothelium expresses transmembrane water channels, aquaporins (AQPs). Currently, 13 AQPs (0‐12) subtypes have been identified in mammalian tissues, and of these subtypes, AQP1, AQP2, AQP3, AQP4, AQP7, AQP9 and AQP11 have been found in the urothelium of various species (1-3) indicating that AQPs could regulate urothelial cell volume and osmolarity, and thus determine the final composition of urine.  However, the exact functional role of AQPs and their cellular regulation and distribution under different osmotic conditions in bladder urothelium remains to be elucidated. Therefore, this study aimed to investigate the functional role and cellular distribution of AQP3 in adult pig bladder urothelium.

Bladders from ∼6-months old female pigs (Sus scrofa domestica) were obtained from a local abattoir (University of Bristol, Bristol, UK). Bladder urothelium/suburothelium (mucosa) membranes were used in an Ussing chamber system. The orientation of the urothelium as either basolateral or apical (bladder lumen) face was noted. Each half chamber (20 ml) was a circulating reservoir of Krebs solution, gassed with 95% O2/5% CO2 and maintained at 37°C by means of a thermostatted water jacket. After an equilibration period of 60-min, the basolateral side of the mounted mucosal strips were exposed to an isotonic Krebs solution whilst the apical side was exposed to a hypertonic or hypotonic Krebs solution containing 40% D2O in presence and absence of 300 µM mercuric chloride (HgCl2, a non-selective AQP inhibitor). The movement of D2O across the mucosa barrier was assessed by taking 1 ml samples from the basolateral side every 1 hour for a period of 8 hours. The samples were then analysed using a PerkinElmer FT-IR spectrometer. The integrity of the tissues was assessed at the end of the experiment using 2% Evans blue dye diluted in 1x phosphate buffer saline (PBS).
The change in concentration of D2O on the basolateral side of the mucosa over time,   in the presence and absence of HgCl2, was used to estimate the permeability (diffusion) coefficient (PD) using the equation:  PD = ᶲ/(A.∆C)   where ᶲ is the flux of the tracer across the membrane and is calculated from the net increase of the tracer in the basolateral surface, A is the area of the apical membrane, and ∆C is the concentration gradient for the D2O across the membrane. A paired Student’s t-test was used to compare the permeability coefficients in the presence and absence of HgCl2. Data are presented as mean±SEM. For immunocytochemistry, pig bladder mucosa, removed by blunt dissection, was incubated at 37°C with 1% HBSS, 5 mM EDTA and 10 mM HEPES for approximately 1 hour. Urothelial cells were released by gentle titration. Isolated cells were then were incubated in hypotonic and hypertonic Krebs solutions for 4 hours. Cells were then fixed with 100% methanol followed by incubation with primary antibodies for AQP3 and cytokeratin 20 (dilutions of 1:1000 and 1:500 respectively) for 1 hour followed by incubation with secondary antibodies (Alexa‐Fluor 488 and 647, dilution of 1:1000) for 45-min. Cells were then mounted on slides and viewed using Nikon eclipse TE300.

There was a gradual increase of D2O concentration on the basolateral side of the mucosa over time when the apical side was exposed to hypertonic or hypotonic solutions. The average permeability coefficient for D2O in the absence of HgCl2 (0.41±0.06 D2O%/hr.cm2, n=9) was significantly (p<0.01) higher than in the presence of HgCl2, 0.20±0.02 D2O%/hr.cm2).  AQP3 immunoreactivity was detected around the nucleus in urothelial cells that were not exposed to hypertonic or hypotonic solutions. However, after exposure to hypertonic and hypotonic solutions for 4 hours, AQP3 immunoreactivity was detected mainly in the cell membrane.

Water movement was detected across pig bladder urothelium which was significantly inhibited by HgCl2, demonstrating a potential role of AQPs in mediating transcellular movement of water across bladder urothelium. Immunofluorescent staining revealed cytoplasmic localisation of AQP3 in urothelial cells not exposed to osmotic gradients and its translocation to the cell membrane under osmotic stress.

AQPs in the urinary bladder urothelium may play a regulatory role in urothelial cell volume and modifying final urine composition depending on the requirements of fluid homeostasis.

Manso, M., Drake, M., Fry, C., Conway, M., Hancock, J. and Vahabi, B., 2019. Expression and localisation of aquaporin water channels in adult pig urinary bladder. Journal of Cellular and Molecular Medicine.
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