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Molecular and functional study of the movement of water, urea or ammonia (NH3/NH4+) through the urea transporters (UTs) expressed in oocytes of Lithobates catesbeianus (American bullfrog)

Grant number: 18/22855-1
Support Opportunities:Regular Research Grants
Duration: May 01, 2019 - October 31, 2021
Field of knowledge:Biological Sciences - Physiology - Physiology of Organs and Systems
Principal Investigator:Raif Musa Aziz
Grantee:Raif Musa Aziz
Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil


Tissues throughout the body generate ammonia as a result of protein degradation. Upon entering circulation, this ammonia travels to the liver, where it is converted into less toxic nitrogenous compounds, urea and glutamine, which can then enter the circulation and travel to the kidneys to be excreted in the urine. In the kidney, urea contributes to maintain a high osmolarity gradient in the medullary interstitium-which is important for the urinary concentrating mechanism-and glutamine is used by the proximal tubule cells to regenerate ammonia in a metabolic process that generates equimolar concentration of new HCO3--which is important to maintain acid-base homeostasis. Ammonia, as NH4+, is then secreted into the proximal fluid, reabsorbed by the thick ascending limb of the Henles loop, and secreted by the collecting duct (CD) through parallel NH3 and H+ transport, which combine and re-form NH4+ that will then be excreted. Recent observations, from our laboratory and others, indicated that the basolateral and apical membranes of CD have low NH3 permeability, suggesting that membrane proteins are likely involved in NH3 (dissolved gas) transport in the CD cell. Mammalian urea transporters (UTs) belong to the SLC14 family of solute carriers and are responsible for the facilitated diffusion of urea across plasma membrane. There are two UT genes: SLC14A1, which encodes UT-B, and SLC14A2, which generates splice variants UT-A1, A2 and A3. UT-B is highly expressed in the red blood cell membrane, and is also localized in endothelial cells of the descending vasa recta and liver. UT-A1 and A3 are expressed, respectively, on the apical and basolateral membranes of the inner medullary CD. UT-A2 is expressed in the thin descending limb of the Henle´s loop. These transporters recycle and thereby concentrate urea in the renal medulla to maintain hyperosmolar interstitium, which is necessary for maximal urine concentration, and also allow the excretion of urea with a minimal volume of water. However, there is some controversy over whether or not UTs are also physiologically relevant for H2O (during dehydration) and NH3 (during acidosis) transport in the kidney. Previous work has shown that mammalian UT-B, heterologously expressed in Xenopus laevis oocytes, not only transport urea but are also capable of transporting H2O and NH3. The crystal structure of the bovine UT-B show that UTs are homotrimeric, proteins similar to the homotetrameric structure of AQP1. Each monomer consists of two protomers (6 helices each) that fold together to form a hydrophilic urea channel. The N- and C-termini of each monomer are located in the cytosol. At the center of the three monomers is a hydrophobic pore, blocked by lipid molecules. We hypothesize that UT-As can, in addition to urea, transport H2O and NH3, using the monomeric urea channel. The present study will measure the urea, H2O, and NH3 permeabilities of mouse (m) UT-As expressed in Lithobates catesbeianus oocytes, a heterologous expression system developed and standardized by our laboratory. Briefly, urea uptake will be monitored using 14C-urea, osmotic water permeability (Pf) will be computed using video microscopy after placing the oocytes in a hypotonic solution to monitor the rate of cell swelling, and NH3 permeability will be measured using a microelectrode with a blunt tip to record the maximum transient change in pH at the surface of the oocyte (DpHS) caused by NH3 influx. We believe that the results from this study will provide valuable insights into the role(s) of UTs-with their permeabilities to urea, water and NH3 are an important nexus for integrating the excretion of nitrogenous wastes, water, and acid. Furthermore, deepening the knowledge of the mechanisms involved in the excretion of water and acid (ammonia) will improve our understanding of basic kidney function, and could potentially aid in the treatment of hydroelectrolytic and acid-base disorders. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
KABUTOMORI, J.; PINA-LOPES, N.; MUSA-AZIZ, R.. Water transport mediated by murine urea transporters: implications for urine concentration mechanisms. BIOLOGY OPEN, v. 9, n. 8, p. 9-pg., . (18/22855-1)
WANG, JIN-LIN; WANG, XIAO-YU; WANG, DENG-KE; PARKER, MARK D.; MUSA-AZIZ, RAIF; POPPLE, JACOB; GUO, YI-MIN; MIN, TIAN-XIN; XIA, TIAN; TAN, MIN; et al. Multiple acid-base and electrolyte disturbances upregulate NBCn1, NBCn2, IRBIT and L-IRBIT in the mTAL. JOURNAL OF PHYSIOLOGY-LONDON, v. 598, n. 16, . (18/22855-1)

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