Fluid reabsorption across pig urinary bladder

Introduction and aims: 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 (AQP0, AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, AQP9, AQP10, AQP11, AQP12) subtypes have been identified in mammalian tissues, and from these subtypes, AQPs 1-4, AQP7, AQP9 and AQP11 have been found in the urothelium of various species indicating that AQPs could regulate urothelial cell volume and osmolarity, determine the final composition of urine. However, the exact function of AQPs, their cellular regulation and distribution under different osmotic conditions in bladder urothelium remain to be elucidated. Therefore, this study aimed to i) investigate the expression of AQPs in pig urinary bladder, ii) the potential role of AQPs in mediating water transport across pig bladder urothelium in response to osmotic stress iii) investigate the effect of hypotonic and hypertonic solution on AQP3 intracellular localisationMethods: Bladders obtained from (~6 months old) female pigs, as well as tissues used as positive control were obtained from the local abattoir and used to investigate AQPs. PCR was carried out on mRNA isolated from bladder mucosa and on urothelium cell suspensions, as well as tissues used as positive control to identify AQPs 1-11. Localisation of AQPs was determined using immunohistochemistochemical method on paraffin embedded mucosa sections. Movement of deuterium oxide (D2O) fluxes across the bladder urothelium/suburothelium (mucosa) membranes were assessed using an Ussing chamber system. AQP3 intracellular localisation in urothelial cells after exposure to changes in tonicity was asses using immunoflourescence. Results: AQPs 1, 3, 9, 11 mRNA expression was found in the mucosa of pig bladder, AQP 3, 9, 11 expression was also found specifically in urothelium. Immunohistochemistry showed AQP1 labelling in the endothelium of lamina propria blood vessels. AQPs 3 and 11 were expressed throughout the urothelium and AQP9 immunoreactivity was detected in upper region of the urothelium. Ussing chamber experiment shows gradual increase in D2O concentration on the basolateral side of the mucosa over time when osmotic gradient was created between the apical and basolateral side of the mucosa tissue section with a known D2O concentration on the apical side. D2O diffusion rate was halted when tissue was treated with HgCl2. The average diffusion rate for D2O in absence of HgCl2 and in the presence of HgCl2 was calculated using Newman-keuls test. AQP3 immunoreactivity was detected around the nucleus in untreated urothelial cells, however, after exposure to hypertonic and hypotonic solutions for 4 and 5 hours, AQP3 immunoreactivity was detected mainly in the cell membrane.Conclusions. Four different AQPs subtypes were identified in adult pig bladder, suggesting that AQPs may play a regulatory role in urothelial cell volume and water transport across the urothelium. Water movement was detected across pig bladder urothelium which was significantly inhibited by HgCl2, demonstrating a possible role of AQPs in mediating transcellular movement of water across bladder urothelium. Immunofluorescent staining revealed cytoplasmic localisation of AQP3 in untreated urothelial cells 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 the final urine composition depending on the overall fluid homeostasis