Aquaporin-2 (AQP2) is the main vasopressin-regulated water channel in the kidney connecting tubule and collecting ducts and is responsible for the regulation of final urine output. Previous studies on transgenic mice have demonstrated a crucial role of AQP2 in water handling in the collecting duct, but so far the precise role of AQP2 in the connecting tubule is unknown. Until recently, it was believed that regulation of water reabsorption occurred exclusively in the collecting duct, based on studies in rabbits, showing a very low water permeability and an absence of AQP2 expression in the CNT. However, rat, mouse and humans were shown to express AQP2 in the CNT, which is regulated by vasopressin. Besides this, micropuncture studies showed a substantial water reabsorption in the CNT. To study the role of AQP2 in the CNT, AQP2-CNT-KO mice were generated by mating mice harboring loxP sites around exon 3 of the AQP2 gene with mice expressing Cre recombinase driven by the promoter region of the B1 subunit of V-ATPase. It was shown previously that this leads to Cre activity in 50% of the principal cells in the CNT. Knockout and wildtype mice were kept in metabolic cages for a total of 5 days. After 2 days of acclimatization, body weight, food and water intake and 24 hr urine were measured for 2 days. Animals where then challenged by a 24 hr water restriction, providing around 55% of baseline water intake in gelled food. Confocal laser scanning immunofluorescence microsopy demonstrated an absence of AQP2 immunolabeling in the CNT of the knockout animals. Under basal conditions, the 24 hour urine output was significantly higher in the knockout animals compared to the wildtype (3.0±0.3 vs. 1.9±0.3 ml/20 g BW), while urine osmolality was significantly decreased (1179±107 vs. 1790±146 mOsm/l), suggesting a mild urinary concentrating defect. There was no difference in bodyweight, food intake or osmolar excretion. The mean drinking volume was higher in the knockout group. However, this difference was not statistically significant. A 24-hr water restriction decreased urine volume in both the wildtype and knockout animals, while urine osmolality was increased. There was no significant difference in urine volume between the groups (0.69±0.11 ml/20 g BW in the knockout animals vs. 0.56±0.11 in the wildtype), while urine osmolality was still lower in the knockout animals (2087±169 mOsm/l vs. 2678±144). Water reabsorption was maximally stimulated by intraperitoneal injection of dDAVP. A significant difference in urine osmolality was observed before injection (873±129 mOsm/l in the knockout animals vs. 1387±163 in the wildtype). Four hours after injection, osmolality was increased to around 2700 mOsm/l in both groups (2616±188 mOsm/l in knockout animals vs. 2758±177 in the wildtype). Altogether, these data show that the AQP2-CNT-KO mice demonstrate a mild urinary concentrating defect. However, when challenged with an injection of dDAVP, the knockout mice were able to concentrate their urine to the same extent as the wildtype mice, showing that the CNT only plays a minor role in urine concentrating ability or that this can be compensated for by an increased water reabsorption in the collecting duct.
Acta Dermato-venereologica, Supplementum, 2011
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43rd Sandbjerg Meeting on Membrane Transport, 2011