The osmotic H2O‐permeability of human urea transporters UT‐A1, A2, A3 and B in Rana catesbeiana oocytes
Authors: Kabutomori Jessica , Geyer R.Ryan , Musa‐Aziz Raif 
Published: November 2023

The ability of the kidney to excrete urine more concentrated than the extracellular fluid depends on the creation of a hyperosmotic renal medullary interstitium that provides the osmotic driving force for water reabsorption in the collecting duct (CD) under the influence of antidiuretic hormone (ADH). In the presence of ADH, the CD is highly permeable to water due to insertion of apical membrane aquaporin 2 water channels, allowing water to be reabsorbed into interstitium driven by osmotic gradient. Most of the osmotic gradient is due to the accumulation of NaCl and urea in the inner medulla. Urea is mainly synthesized in the liver via the urea cycle and transported to the kidney and ultimately excreted in the urine. Mammalian urea transporters (UTs) are members of the SLC14 family and facilitate the diffusion of urea across the kidney plasma membranes: UT‐A1 is expressed on the apical membrane and UT‐A3 on the basolateral membrane of the inner medullary CD, UT‐A2 is expressed in the thin descending limb of the renal outer and inner medulla, and UT‐B is expressed in the endothelial cells of the descending vasa recta and red blood cells. Although it is clear that human UTs (hUTs) play a critical role in the urinary concentrating mechanisms, there is some debate as to whether hUTs also facilitate water transport in the kidney. Previously, it was shown that hUT‐B can function as a water channel. This study explores the heterologous expression and osmotic water permeability (Pf) of hUT‐A1 and hUT‐A2, hUT‐A3, hUT‐B c‐Myc‐tagged using Rana catesbeiana oocytes. Using surface biotinylation and western blot analysis, we showed that Rana oocytes injected with H2O (control) have no detectable signal, whereas oocytes injected with cRNA encoding for hUTs express the hUT protein at the oocyte surface. Using video microscopy, we monitored the rate of oocyte swelling, as we reduced the extracellular osmolality from 195 to 90 mOSm, and thereby compute Pf (cm/s). The Pf values for hUT‐A1 (0.0009 ± 0.0002, n=30) and H2O (0.0006 ± 0.0001, n=46) oocytes were not significantly different from one another. The mean Pf value for hUT‐A2 (0.0021 ± 0.0003, n=20), hUT‐A3 (0.0013 ± 0.0002, n=6) and hUT‐B (0.0019 ± 0.0002, n=39) expressing oocytes were all significantly greater than that of H2O‐injected control (0.0006 ± 0.0001, n=46) oocytes. In addition, preliminary data show that phloretin treatment (0.5 mM for 20 min) significantly reduces Pf of oocytes injected with hUT‐A2 (to 0.0014 ± 0.0003, n=6), hUT‐A3 (to 0.0009 ± 0.0002, n=5) and hUT‐B cRNA (to 0.0012 ± 0.0003, n=6), but does not affect the Pf of oocytes injected with H2O (to 0.0007 ± 0.0001, n=24) or hUT‐A1 cRNA (to 0.0006 ± 0.00004, n=5). Thus, our data confirm that hUT‐B conducts water, similar to the observation seen by others using Xenopus oocytes, and indicate for the first time that hUT‐A2 and hUT‐A3‐expressing Rana oocytes have significant water permeability. Therefore, if the observed water transport activity of hUT‐A2, hUT‐A3 and hUT‐B in Rana oocytes is predictive of their function in renal epithelia, then the water permeability of these hUTs may contribute to the formation of concentrated urine.Support or Funding InformationFAPESP & CNPq, Brazil.