Mechanism of Urea, Water and NH3 Movement through Urea Transporter‐A2 Expressed in Lithobates catesbeianus Oocytes

Abstract

Tissues throughout the body generate ammonia as a result of protein degradation. This ammonia is detoxified by the liver into less toxic nitrogenous compounds, urea and glutamine, which travel to the kidney for excretion. While in the kidney, urea helps establish and maintain a high osmotic gradient in the medullary interstitium, important for the urinary concentrating mechanism, and glutamine is used by the proximal tubule cells to regenerate ammonia, which generates equimolar amounts of new HCO3−, important for the maintenance of acid‐base balance. Ammonia, as NH4+, is then secreted into the proximal fluid, reabsorbed in the thick ascending limb in Henle's loop, and secreted by the collecting duct (CD) through parallel NH3 and H+ transport, which react and re‐form NH4+ for excretion. Recent studies indicate that the basolateral and apical membranes of CD have low NH3 permeability, suggesting that NH3 transport in the CD is mediated by membrane proteins. Urea transporters (UTs) mediate the facilitated diffusion of urea across the plasma membrane. In the kidney, UTA1 and A3 are expressed in the apical and basolateral membranes of the inner medullary CD, respectively, UTA2 is expressed in the thin descending limb of the renal medulla, and UTB is expressed in the endothelial cells of the descending vasa recta. These transporters recycle and concentrate urea in the renal medulla, and also allow the excretion of urea with a minimal volume of water. However, there is some controversy over whether or not UTs also function as physiologically relevant H2O and NH3 channels in the kidney. This study explored the urea, H2O and NH3 permeabilities of mouse (m) UTA2 expressed in Lithobates catesbeianus oocytes, using Phloretin (Phl), an UT inhibitor, and site directed mutations of conserved threonine (T) residues (lining the selectivity filter of the three monomeric urea pores) to Valine (V). Lithobates oocytes were injected with cRNA encoding for c‐Myc tagged mUTA2WT (wild type), UTA2T176V, UTA2T338V, UTA2T176V/T338V or H2O. Heterologous expression was assessed by surface biotinylation and immunoblotting. Urea uptake (Jurea) was monitored using 14C‐urea and osmotic water permeability (Pf, cm/s) was calculated using video microscopy to monitor the rate of cell swelling in a hypotonic solution. A semequantitative index of NH3 permeability was evaluated by monitoring the maximum transient change in surface pH (ΔpHS(NH3)) caused by exposing the oocyte to 0.5 mM NH3/NH4+. The results show that mUTA2WT as well as all of the mutants were translated and translocated to the oocyte surface, mUTA2WT had Jurea, Pf and ΔpHS(NH3) values that were greater than all of the mutants and H2O‐injected control oocytes. Phl significantly reduced the Jurea and Pf of oocytes expressing mUTA2, but had no effect on the Jurea and Pf of the mutants or H2O oocytes. Thus, these data confirm that UTA2 conducts urea and show, for the first time, that UTA2 also conducts H2O and NH3. Furthermore, the Phr inhibitor and mutagenesis studies suggest that H2O and NH3 pass through the three monomeric urea pores in mUTA2. Thus, UTA2 (with their permeabilities to urea, NH3, and H2O) must be an important nexus for integrating the excretion of nitrogenous wastes, water and acid.Support or Funding InformationFAPESP, CNPqThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.