The rhesus protein RhCG: a new perspective in ammonium transport and distal urinary acidification
2010; Elsevier BV; Volume: 79; Issue: 2 Linguagem: Inglês
10.1038/ki.2010.386
ISSN1523-1755
AutoresCarsten A. Wagner, Olivier Devuyst, Hendrica Belge, Soline Bourgeois, Pascal Houillier,
Tópico(s)Metabolism and Genetic Disorders
ResumoUrinary acidification is a complex process requiring the coordinated action of enzymes and transport proteins and resulting in the removal of acid and the regeneration of bicarbonate. Proton secretion is mediated by luminal H+-ATPases and requires the parallel movement of NH3, and its protonation to NH4+, to provide sufficient buffering. It has been long assumed that ammonia secretion is a passive process occurring by means of simple diffusion driven by the urinary trapping of ammonium. However, new data indicate that mammalian cells possess specific membrane proteins from the family of rhesus proteins involved in ammonia/μm permeability. Rhesus proteins were first identified in yeast and later also in plants, algae, and mammals. In rodents, RhBG and RhCG are expressed in the collecting duct, whereas in humans only RhCG was detected. Their expression increases with maturation of the kidney and accelerates after birth in parallel with other acid–base transport proteins. Deletion of RhBG in mice had no effect on renal ammonium excretion, whereas RhCG deficiency reduces renal ammonium secretion strongly, causes metabolic acidosis in acid-challenged mice, and impairs restoration of normal acid–base status. Microperfusion experiments or functional reconstitution in liposomes demonstrates that ammonia is the most likely substrate of RhCG. Similarly, crystal structures of human RhCG and the homologous bacterial AmtB protein suggest that these proteins may form gas channels. Urinary acidification is a complex process requiring the coordinated action of enzymes and transport proteins and resulting in the removal of acid and the regeneration of bicarbonate. Proton secretion is mediated by luminal H+-ATPases and requires the parallel movement of NH3, and its protonation to NH4+, to provide sufficient buffering. It has been long assumed that ammonia secretion is a passive process occurring by means of simple diffusion driven by the urinary trapping of ammonium. However, new data indicate that mammalian cells possess specific membrane proteins from the family of rhesus proteins involved in ammonia/μm permeability. Rhesus proteins were first identified in yeast and later also in plants, algae, and mammals. In rodents, RhBG and RhCG are expressed in the collecting duct, whereas in humans only RhCG was detected. Their expression increases with maturation of the kidney and accelerates after birth in parallel with other acid–base transport proteins. Deletion of RhBG in mice had no effect on renal ammonium excretion, whereas RhCG deficiency reduces renal ammonium secretion strongly, causes metabolic acidosis in acid-challenged mice, and impairs restoration of normal acid–base status. Microperfusion experiments or functional reconstitution in liposomes demonstrates that ammonia is the most likely substrate of RhCG. Similarly, crystal structures of human RhCG and the homologous bacterial AmtB protein suggest that these proteins may form gas channels. The kidneys excrete ∼70 mmol of acids per day from the body. Only a minute fraction is excreted as free protons, but most acids are in the form of ammonium (about 2/3) and titratable acids (about 1/3), such as phosphate. The importance of renal acid elimination is underlined by a variety of syndromes of acquired or inherited forms of renal tubular acidosis.1.Fry A.C. Karet F.E. Inherited renal acidoses.Physiology (Bethesda). 2007; 22: 202-211Crossref PubMed Scopus (65) Google Scholar, 2.Wagner C.A. Devuyst O. Bourgeois S. et al.Regulated acid-base transport in the collecting duct.Pflugers Arch. 2009; 458: 137-156Crossref PubMed Scopus (131) Google Scholar Chronic metabolic acidosis represents a major morbidity and mortality risk factor and may even accelerate deterioration of renal function in patients with early stages of renal disease.3.de Brito-Ashurst I. Varagunam M. Raftery M.J. et al.Bicarbonate supplementation slows progression of CKD and improves nutritional status.J Am Soc Nephrol. 2009; 20: 2075-2084Crossref PubMed Scopus (552) Google Scholar, 4.Bailey J.L. Metabolic acidosis: an unrecognized cause of morbidity in the patient with chronic kidney disease.Kidney Int Suppl. 2005; 96: S15-S23Abstract Full Text Full Text PDF PubMed Google Scholar Type A intercalated cells (A-ICs) in the collecting duct system (late distal convoluted tubule to the initial third of the inner medullary collecting duct) mediate the removal of acids (protons and ammonium), as well as the de novo generation of bicarbonate.2.Wagner C.A. Devuyst O. Bourgeois S. et al.Regulated acid-base transport in the collecting duct.Pflugers Arch. 2009; 458: 137-156Crossref PubMed Scopus (131) Google Scholar Cytosolic carbonic anhydrase II hydrates CO2 to form H+ and HCO3-, which in turn is released into the interstitium involving the basolateral and A-IC-specific chloride/bicarbonate exchanger AE1.5.Alper S.L. Genetic diseases of acid-base transporters.Annu Rev Physiol. 2002; 64: 899-923Crossref PubMed Scopus (155) Google Scholar, 6.Stehberger P.A. Shmukler B.E. Stuart-Tilley A.K. et al.Distal renal tubular acidosis in mice lacking the AE1 (band3) Cl-/HCO3- exchanger (slc4a1).J Am Soc Nephrol. 2007; 18: 1408-1418Crossref PubMed Scopus (94) Google Scholar H+-ATPases localized at the luminal pole of A-IC excrete protons,7.Wagner C.A. Finberg K.E. Breton S. et al.Renal vacuolar H+-ATPase.Physiol Rev. 2004; 84: 1263-1314Crossref PubMed Scopus (336) Google Scholar thereby acidifying urine. However, H+-ATPases can establish a maximal pH gradient of about 2–2.5 units pH between the intracellular compartment (∼pH 7.2) and urine, thus limiting removal of hydrogen ions. The daily amount of acids removed is about 1 mEq per kg body weight (about 70 mEq in a healthy adult person). The excretion of this amount of acid in an unbuffered solution would thus require several hundred liters of urine (1 l of unbuffered urine, pH 4.5, containing maximally 30 μM protons). Titratable acids (mainly phosphate, to a lesser extent citrate, and creatinine) can help buffer protons (about 1/3 of the daily acid load). A major fraction of protons, however, is buffered by ammonia after parallel secretion into urine (approximately 2/3 of the daily acid load). Ammonia secretion occurs along the entire length of the collecting duct system, but increases substantially in the later parts.8.Knepper M.A. Packer R. Good D.W. Ammonium transport in the kidney.Physiol Rev. 1989; 69: 179-249Crossref PubMed Scopus (222) Google Scholar, 9.Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (112) Google Scholar In 1945, Robert Pitts10.Pitts R.F. The renal regulation of acid base balance with special reference to the mechanism for acidifying the urine. Ii.Science. 1945; 102: 81-85Crossref PubMed Scopus (6) Google Scholar, 11.Pitts R.F. The renal regulation of acid base balance with special reference to the mechanism for acidifying the urine.Science. 1945; 102: 49-54Crossref PubMed Scopus (13) Google Scholar had described in two seminal papers the role of ammonium in renal acid secretion and postulated that ammonium secretion is a passive process driven by the ammonia concentration gradient, the diffusion of ammonia across the luminal membrane, and the subsequent trapping of ammonium in urine after protonation. This hypothesis remained textbook knowledge until recently. Further work demonstrated that ammoniagenesis occurs from metabolism of glutamine in the proximal tubule regenerating the bicarbonate lost while buffering protons stemming from metabolism.12.Curthoys N.P. Renal ammonium ion production and excretion.in: Alpern R.J. Hebert S.C. Seldin and Giebisch's The Kidney Physiology and Pathophysiology. 4th edn. Academic Press, New York2007: 1601-1619Google Scholar, 13.Busque S.M. Wagner C.A. Potassium restriction, high protein intake, and metabolic acidosis increase expression of the glutamine transporter SNAT3 (Slc38a3) in mouse kidney.Am J Physiol Renal Physiol. 2009; 297: F440-F450Crossref PubMed Scopus (41) Google Scholar, 14.Ibrahim H. Lee Y.J. Curthoys N.P. Renal response to metabolic acidosis: role of mRNA stabilization.Kidney Int. 2008; 73: 11-18Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Ammonium is then secreted into urine at the level of the proximal tubule, is mostly actively reabsorbed in the thick ascending limb, and finally accumulates in the interstitium with a high cortico-medullary gradient (Figure 1).8.Knepper M.A. Packer R. Good D.W. Ammonium transport in the kidney.Physiol Rev. 1989; 69: 179-249Crossref PubMed Scopus (222) Google Scholar This interstitial high concentration, together with a pH gradient (from inside the cells of the collecting duct into urine), provides the driving force for ammonia transport by intercalated cells. Ammonia secretion in the collecting duct is mediated by intercalated cells, but principal cells may also contribute although their permeability is lower.15.Yip K.P. Kurtz I. NH3 permeability of principal cells and intercalated cells measured by confocal fluorescence imaging.Am J Physiol. 1995; 269: F545-F550PubMed Google Scholar NH3/NH4+ uptake from interstitium by intercalated cells may be mediated by several pathways, including the Na+/K+/2Cl-cotransporter NKCC1, the Na+/K+-ATPase, and might involve also the RhCG protein (for a review, see Weiner and Hamm9.Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (112) Google Scholar). Preliminary data from our group show reduced basolateral NH3 permeability in RhCG KO mice. The final step of ammonia secretion into urine had been studied in great detail by Knepper and colleagues8.Knepper M.A. Packer R. Good D.W. Ammonium transport in the kidney.Physiol Rev. 1989; 69: 179-249Crossref PubMed Scopus (222) Google Scholar using microperfusion experiments and demonstrating that it involved high apical ammonia permeability. Whether this is an active process or involves transport proteins has remained elusive. The discovery of Marini et al.16.Marini A.M. Matassi G. Raynal V. et al.The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast.Nat Genet. 2000; 26: 341-344Crossref PubMed Scopus (290) Google Scholar that the mammalian homologs (RhAG and RhGK/RhCG) of the yeast methyl-ammonia permeases ammonium transporters could also mediate transport of ammonia/um opened the possibility that these molecules might participate in renal ammonium elimination. Similar molecules were also found in plants, algae, and fish. Expression of rhesus proteins RhAG, RhBG, and RhCG in various heterologous cell models induced ammonia/ammonium transport. However, the mode of transport and exact substrate (NH3 or NH4+), as well as the coupling to other ions (counter- or cotransport of protons) and stoichiometry, have remained controversial (see below and for a review, Weiner and Hamm9.Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (112) Google Scholar). Moreover, deletion of the algae Rh1 protein suggested even the possibility that rhesus proteins might be involved in CO2 permeability of biological membranes.17.Soupene E. Inwood W. Kustu S. Lack of the Rhesus protein Rh1 impairs growth of the green alga Chlamydomonas reinhardtii at high CO2.Proc Natl Acad Sci USA. 2004; 101: 7787-7792Crossref PubMed Scopus (138) Google Scholar In fish, four homologous proteins, fRhag, fRhbg, Rhcg1, and Rhcg2, are expressed in gills, transport ammonia in heterologous expression system, and are thought to mediate active ammonia excretion.18.Nakada T. Westhoff C.M. Kato A. et al.Ammonia secretion from fish gill depends on a set of Rh glycoproteins.FASEB J. 2007; 21: 1067-1074Crossref PubMed Scopus (149) Google Scholar, 19.Wright P.A. Wood C.M. A new paradigm for ammonia excretion in aquatic animals: role of Rhesus (Rh) glycoproteins.J Exp Biol. 2009; 212: 2303-2312Crossref PubMed Scopus (252) Google Scholar In mammals, members of the rhesus protein family are expressed in various organs and distinct cells. RhAG is mainly detected in erythrocytes, RhBG in the liver, kidney, and ovary, and RhCG in the kidney, liver, brain, skeletal muscle, prostate, and pancreas.9.Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (112) Google Scholar However, recent data suggest that species differences may exist. RhBG protein was detected in mouse and rat kidney, but not in human kidney.20.Brown A.C. Hallouane D. Mawby W.J. et al.RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.Am J Physiol Renal Physiol. 2009; 296: F1279-F1290Crossref PubMed Scopus (26) Google Scholar In the kidney, RhBG and RhCG have been localized exclusively to the distal tubule, connecting tubule, and cortical and medullary collecting duct.9.Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (112) Google Scholar, 20.Brown A.C. Hallouane D. Mawby W.J. et al.RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.Am J Physiol Renal Physiol. 2009; 296: F1279-F1290Crossref PubMed Scopus (26) Google Scholar, 21.Quentin F. Eladari D. Cheval L. et al.RhBG and RhCG, the Putative Ammonia Transporters, Are Expressed in the Same Cells in the Distal Nephron.J Am Soc Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (127) Google Scholar Small differences in the localization have been reported from different laboratories using different antibodies and different species. RhBG was detected in mouse and rat kidney on the basolateral side of various cell types. RhBG is found in the distal convoluted tubule in intercalated cells (and possibly also in distal convoluted tubule cells), in the connecting tubule in all cell types, in the cortical collecting duct in A-ICs and principal cells in mouse, whereas in rat expression might be restricted to A-ICs. In the outer medullary collecting duct and inner medullary collecting duct, only A-ICs express RhBG.21.Quentin F. Eladari D. Cheval L. et al.RhBG and RhCG, the Putative Ammonia Transporters, Are Expressed in the Same Cells in the Distal Nephron.J Am Soc Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (127) Google Scholar, 22.Verlander J.W. Miller R.T. Frank A.E. et al.Localization of the ammonium transporter proteins RhBG and RhCG in mouse kidney.Am J Physiol Renal Physiol. 2003; 284: F323-F337Crossref PubMed Scopus (149) Google Scholar The localization of RhCG has been reported in mouse, rat, and human kidney. However, the exact distribution is controversial. Quentin et al.21.Quentin F. Eladari D. Cheval L. et al.RhBG and RhCG, the Putative Ammonia Transporters, Are Expressed in the Same Cells in the Distal Nephron.J Am Soc Nephrol. 2003; 14: 545-554Crossref PubMed Scopus (127) Google Scholar described only apical staining for RhCG in rat kidney, whereas the laboratory of D. Weiner has reported both apical and basolateral staining for RhCG in human, rat, and mouse kidney (for references, see detailed review9.Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (112) Google Scholar). In human kidney, RhCG localization has been reported for basolateral and apical membranes, but RhBG could not be detected.9.Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (112) Google Scholar, 20.Brown A.C. Hallouane D. Mawby W.J. et al.RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.Am J Physiol Renal Physiol. 2009; 296: F1279-F1290Crossref PubMed Scopus (26) Google Scholar Our own results support the notion of RhCG localization at both poles of cells in mouse and human kidney. RhCG is found in the late distal convoluted tubule, in the connecting tubule and cortical collecting duct, and in the outer stripe of the outer medulla in all cell types (possibly excluding non-A-ICs), whereas in the late outer medullary in collecting duct and inner medullary collecting duct only A-ICs are stained.20.Brown A.C. Hallouane D. Mawby W.J. et al.RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.Am J Physiol Renal Physiol. 2009; 296: F1279-F1290Crossref PubMed Scopus (26) Google Scholar, 22.Verlander J.W. Miller R.T. Frank A.E. et al.Localization of the ammonium transporter proteins RhBG and RhCG in mouse kidney.Am J Physiol Renal Physiol. 2003; 284: F323-F337Crossref PubMed Scopus (149) Google Scholar, 23.Eladari D. Cheval L. Quentin F. et al.Expression of RhCG, a New Putative NH3/NH4+ Transporter, along the Rat Nephron.J Am Soc Nephrol. 2002; 13: 1999-2008Crossref PubMed Scopus (111) Google Scholar, 24.Kim H.Y. Baylis C. Verlander J.W. et al.Effect of reduced renal mass on renal ammonia transporter family, Rh C glycoprotein and Rh B glycoprotein, expression.Am J Physiol Renal Physiol. 2007; 293: F1238-F1247Crossref PubMed Scopus (55) Google Scholar Ammonia excretion along the collecting duct varies in the different subsegments.8.Knepper M.A. Packer R. Good D.W. Ammonium transport in the kidney.Physiol Rev. 1989; 69: 179-249Crossref PubMed Scopus (222) Google Scholar During acidosis, the cortical collecting duct becomes a major site of ammonia secretion,25.Sajo I.M. Goldstein M.B. Sonnenberg H. et al.Sites of ammonia addition to tubular fluid in rats with chronic metabolic acidosis.Kidney Int. 1981; 20: 353-358Abstract Full Text PDF PubMed Scopus (65) Google Scholar coinciding with the strongest staining for RhCG. Staining by RhCG is only weak in the inner stripe of the outer medulla and inner medulla, segments that secrete considerable amounts of ammonia.8.Knepper M.A. Packer R. Good D.W. Ammonium transport in the kidney.Physiol Rev. 1989; 69: 179-249Crossref PubMed Scopus (222) Google Scholar One explanation might be that RhCG is required in the portions of the collecting duct (cortex) in which the ammonium gradient from interstitium to lumen is less steep and RhCG facilitates NH3 transport, whereas in the medullary regions the gradient is steeper and ammonium excretion is less dependent on transport pathways. During pre- and postnatal nephrogenesis, the expression and maturation of several transport proteins implicated in the final urinary acidification is tightly regulated in order to compensate the acid-generating process of growth.26.Bonnici B. Wagner C.A. Postnatal expression of transport proteins involved in acid-base transport in mouse kidney.Pflugers Arch. 2004; 448: 16-28Crossref PubMed Scopus (33) Google Scholar, 27.Jouret F. Auzanneau C. Debaix H. et al.Ubiquitous and kidney-specific subunits of vacuolar H+-ATPase are differentially expressed during nephrogenesis.J Am Soc Nephrol. 2005; 16: 3235-3246Crossref PubMed Scopus (35) Google Scholar, 28.Smith A.N. Jouret F. Bord S. et al.Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.J Am Soc Nephrol. 2005; 16: 1245-1256Crossref PubMed Scopus (51) Google Scholar These changes are paralleled by the maturation of intercalated cells, which includes the acquisition of the subcellular localization of H+-ATPases similar to the adult kidney and removal of non-A-ICs from the inner medulla and the inner stripe of the outer medulla.7.Wagner C.A. Finberg K.E. Breton S. et al.Renal vacuolar H+-ATPase.Physiol Rev. 2004; 84: 1263-1314Crossref PubMed Scopus (336) Google Scholar, 26.Bonnici B. Wagner C.A. Postnatal expression of transport proteins involved in acid-base transport in mouse kidney.Pflugers Arch. 2004; 448: 16-28Crossref PubMed Scopus (33) Google Scholar, 29.Song H.K. Kim W.Y. Lee H.W. et al.Origin and fate of pendrin-positive intercalated cells in developing mouse kidney.J Am Soc Nephrol. 2007; 18: 2672-2682Crossref PubMed Scopus (21) Google Scholar These features correlate with the fact that mutations in ATP6V0A4 and ATP6V1B1 genes, encoding the intercalated cell-specific V0 a4 and V1 B1 subunits, respectively,7.Wagner C.A. Finberg K.E. Breton S. et al.Renal vacuolar H+-ATPase.Physiol Rev. 2004; 84: 1263-1314Crossref PubMed Scopus (336) Google Scholar have been associated with early-onset cases of distal renal tubular acidosis (dRTA), suggesting that the segmental distribution of intercalated cell-specific isoforms of H+-ATPases is acquired at birth or during early infancy. Jouret et al.27.Jouret F. Auzanneau C. Debaix H. et al.Ubiquitous and kidney-specific subunits of vacuolar H+-ATPase are differentially expressed during nephrogenesis.J Am Soc Nephrol. 2005; 16: 3235-3246Crossref PubMed Scopus (35) Google Scholar showed that the intercalated cell-specific a4 and B1 subunits were induced from E15.5 in the mouse developing kidney, following the onset of expression of the forkhead transcription factor, Foxi1. From E15.5, Foxi1 mRNA was detected in intercalated cells, where it co-distributed with B1 in late nephrogenesis. Our preliminary investigations in mouse kidney30.Aydin A. Geffers L. Parreira K. et al.Ontogeny of the Rhesus proteins, Rhcg and Rhbg, during mouse nephrogenesis and kidney maturation.J Am Soc Nephrol. 2009; 20: 378AGoogle Scholar reveal that both RhBG and RhCG show an early (E13.5) and progressive increase in the renal expression, followed by a strong induction after birth (Figure 2). A similar expression pattern was detected for the intercalated cell markers (a4 and B1 subunits of H+-ATPase, AE1, pendrin) and the intercalated cell-specific transcription factor Foxi1. By in situ hybridization, RhCG was detected at E14.5 in kidney tubules. From E17.5 on, RhBG and RhCG are expressed in the distal convoluted and connecting tubules and collecting ducts. The adult kidney displayed a strong signal in the connecting tubule and cortical collecting ducts and in some cells lining the outer-medullary and inner-medullary collecting ducts. Immunostaining failed to identify RhBG at the embryonic stages analyzed, whereas RhCG expression was observed in developing collecting ducts at E17.5. After birth, RhBG and RhCG are expressed in cortex and medulla, in which they show distinct basolateral (RhBG) and basolateral and apical (RhCG) reactivity in intercalated cells, like in adult kidneys. Similar observations were made in rat kidney, with the difference that RhBG was detectable before birth and that RhCG was first detected in basolateral membranes and only later also in apical membranes.31.Han K.H. Mekala K. Babida V. et al.Expression of the gas-transporting proteins, Rh B glycoprotein and Rh C glycoprotein, in the murine lung.Am J Physiol Lung Cell Mol Physiol. 2009; 297: L153-L163Crossref PubMed Scopus (31) Google Scholar To date, no detailed information is available on the mechanisms of transcriptional regulation of RhCG in the mammalian kidney. RhBG-deficient mice were reported in 2005 by Chambrey et al.32.Chambrey R. Goossens D. Bourgeois S. et al.Genetic ablation of Rhbg in the mouse does not impair renal ammonium excretion.Am J Physiol Renal Physiol. 2005; 289: F1281-F1290Crossref PubMed Scopus (75) Google Scholar These mice did not show altered urinary ammonium excretion under basal conditions or after an acid load. Basolateral NH3/NH4+ permeabilities, as well as transepithelial ammonia fluxes were similar in wildtype and RhBG KO mice. Thus, presently the physiological significance of RhBG remains unknown. The fact that RhBG does not have a major role in renal ammonium handling could be explained by the basolateral expression of RhCG or the existence of alternate basolateral entry pathways for ammonium. In contrast, genetic ablation of RhCG in at least three different mouse models demonstrates a critical role for this protein in urinary ammonium excretion.33.Biver S. Belge H. Bourgeois S. et al.A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility.Nature. 2008; 456: 339-343Crossref PubMed Scopus (136) Google Scholar, 34.Lee H.W. Verlander J.W. Bishop J.M. et al.Collecting duct-specific Rh C glycoprotein deletion alters basal and acidosis-stimulated renal ammonia excretion.Am J Physiol Renal Physiol. 2009; 296: F1364-F1375Crossref PubMed Scopus (70) Google Scholar, 35.Bourgeois S. Aydin A. Mihailova M. et al.Rhesus protein Rhcg is essential for proton excretion by the kidneys.J Am Soc Nephrol. 2009; 20: 34AGoogle Scholar Two mouse models of complete RhCG deficiency33.Biver S. Belge H. Bourgeois S. et al.A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility.Nature. 2008; 456: 339-343Crossref PubMed Scopus (136) Google Scholar, 35.Bourgeois S. Aydin A. Mihailova M. et al.Rhesus protein Rhcg is essential for proton excretion by the kidneys.J Am Soc Nephrol. 2009; 20: 34AGoogle Scholar show only mildly reduced ammonium excretion under basal conditions and normal blood acid–base parameters. However, acid-loading mice with HCl or NH4Cl in food induced more severe metabolic acidosis and KO mice had a very significant defect in their maximal capacity to increase urinary ammonium excretion as compared with wild-type mice. The excretion of titratable acidity is not affected by the loss of RhCG. Lower urinary ammonium excretion is most likely not due to the effect of reduced ammoniagenesis on the level of the proximal tubule, as the expression of ammoniagenic enzymes, and the concentration of blood glutamine were similar in all mice. At the cellular level, microperfusion experiments in the cortical and outer medullary collecting duct from acid-loaded mice demonstrated that the NH3, but not the NH4+ permeability of the apical membrane was reduced by about 60%. Similarly, when we measured total transepithelial NH3 permeability, we found a 60% reduction.33.Biver S. Belge H. Bourgeois S. et al.A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility.Nature. 2008; 456: 339-343Crossref PubMed Scopus (136) Google Scholar To further test the possible significance of basolaterally localized RhCG, we also performed experiments assessing the basolateral NH3 and NH4+ permeabilities and found a reduction of the basolateral NH3 permeability by about 40%, suggesting that RhCG contributes to basolateral NH3 fluxes, but that additional pathways exist. Thus, RhCG is critical for urinary ammonium excretion and is required for collecting duct NH3 secretion. A mouse model with partial deletion of RhCG only in the cortical and medullary collecting duct, but not in the connecting tubule (due to the choice of Cre-deleted mice under the control of the Ksp-cadherin promoter that is not expressed in the connecting tubule) shows similar features to the total KO mouse models with reduced urinary ammonium excretion under basal conditions and during an HCl acid load.34.Lee H.W. Verlander J.W. Bishop J.M. et al.Collecting duct-specific Rh C glycoprotein deletion alters basal and acidosis-stimulated renal ammonia excretion.Am J Physiol Renal Physiol. 2009; 296: F1364-F1375Crossref PubMed Scopus (70) Google Scholar However, the reduction is less severe than in total KO mice. Partial KO mice had normal urinary pH, whereas total KO mice displayed more alkaline urine under all conditions.33.Biver S. Belge H. Bourgeois S. et al.A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility.Nature. 2008; 456: 339-343Crossref PubMed Scopus (136) Google Scholar These discrepancies may be explained by the important contribution of the late distal tubule and particularly the connecting tubule to overall renal acid excretion.36.Kovacikova J. Winter C. Loffing-Cueni D. et al.The connecting tubule is the main site of the furosemide-induced urinary acidification by the vacuolar H+-ATPase.Kidney Int. 2006; 70: 1706-1716Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Taken together, these data indicate that RhCG is required for normal ammonium excretion, that on the levels of the apical and basolateral membranes RhCG is involved in mediating NH3 fluxes, and that free diffusion, as postulated by Pitts, does not account for the majority of NH3 excretion. What mediates the remaining NH3 fluxes—free diffusion or membrane proteins—remains to be established. Also, RhAG KO mice have been generated and show greatly reduced ammonia and methyl-ammonia fluxes in red blood cells,37.Goossens D. Trinh-Trang-Tan M.M. Debbia M. et al.Generation and characterisation of Rhd and Rhag null mice.Br J Haematol. 2009; 148: 161-172Crossref PubMed Google Scholar resembling patients with inherited disorders of the red blood cells rhesus complex.38.Ripoche P. Bertrand O. Gane P. et al.Human Rhesus-associated glycoprotein mediates facilitated transport of NH(3) into red blood cells.Proc Natl Acad Sci USA. 2004; 101: 17222-17227Crossref PubMed Scopus (140) Google Scholar In zebrafish, knock-down experiments with morpholinos reduced expression of fRhag, fRhbg, or fRhcg and ammonia secretion across gills, further supporting a role of rhesus proteins in ammonia transport in other species.39Braun M.H. Steele S.L. Ekker M. et al.Nitrogen excretion in developing zebrafish (Danio rerio): a role for Rh proteins and urea transporters.Am J Physiol Renal Physiol. 2009; 296: F994-F1005Crossref PubMed Scopus (102) Google Scholar The functional data from the isolated collecting duct indicate that RhCG is required for NH3 fluxes, but the question remains whether RhCG is a channel or transporter for NH3 or NH4+. Several lines of evidence from functional experiments and structural data suggest that RhCG and related rhesus proteins function as gas channels. First, functional data from heterologous expression systems have yielded controversial results whether RhCG mediates NH3 uniport or NH4+/H+ antiport. Heterologously expressed RhCG in mammalian cell lines, as well as in Xenopus oocytes, provided evidence that both NH3 and NH4+ interact with the protein.40.Zidi-Yahiaoui N. Mouro-Chanteloup I. D'Ambrosio A.M. et al.Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells.Biochem J. 2005; 391: 33-40Crossref PubMed Scopus (73) Google Scholar, 41.Bakouh N. Benjelloun F. Hulin P. et al.NH3 is involved in the NH4+ transport induced by the functional expression of the human Rh C glycoprotein.J Biol Chem. 2004; 279: 15975-15983Crossref PubMed Scopus (106) Google Scholar Experiments in Xenopus oocytes expressing several aquaporine water channels and rhesus family members demonstrated NH3 permeability in the rhesus, but not in aquaporine family members.42.Musa-Aziz R. Chen L.M. Pelletier M.F. et al.Relative CO2/NH3 selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG.Proc Natl Acad Sci USA. 2009; 106: 5406-5411Crossref PubMed Scopus (189) Google Scholar The in vitro microperfusion experiments in the cortical collecting duct and outer medullary collecting duct from wild-type and RhCG-deficient mice are consistent with NH3 fluxes, but do not rule out other transport modes. Second, the related RhAG protein mediates NH3 fluxes in human and in mouse red blood cells.38.Ripoche P. Bertrand O. Gane P. et al.Human Rhesus-associated glycoprotein mediates facilitated transport of NH(3) into red blood cells.Proc Natl Acad Sci USA. 2004; 101: 17222-17227Crossref PubMed Scopus (140) Google Scholar, 43.Goossens D. Trinh-Trang-Tan M.M. 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Smith T. et al.Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis.Am J Physiol Renal Physiol. 2006; 290: F1443-F1452Crossref PubMed Scopus (62) Google Scholar, 53.Seshadri R.M. Klein J.D. Kozlowski S. et al.Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis.Am J Physiol Renal Physiol. 2006; 290: F397-F408Crossref PubMed Scopus (73) Google Scholar demonstrated that RhCG protein abundance increases and that more RhCG protein is shifted to the apical membrane. In contrast to a membrane freely permeable to NH3, the presence of specific transport proteins, such as RhCG, may provide the kidney with the ability to regulate and rapidly adapt urinary ammonium excretion by controlling its transport rates. Clearly, understanding the acute and chronic regulation of ammonium transport and the role of RhCG will require further experiments. Recent experiments from our group using microperfusion of isolated collecting duct segments indicate that the genetic ablation of RhCG is associated with a strong reduction in H+-ATPase activity in A-ICs.35.Bourgeois S. Aydin A. Mihailova M. et al.Rhesus protein Rhcg is essential for proton excretion by the kidneys.J Am Soc Nephrol. 2009; 20: 34AGoogle Scholar However, at light microscopy level the localization of several H+-ATPase subunits appeared normal. It remains speculative at this time whether RhCG may have an additional role as regulator of H+-ATPase activity or whether deprotonation of NH4+ by RhCG may provide a major source of protons for the H+-ATPase. Nevertheless, it may explain why RhCG-deficient mice have more alkaline urine. Various genes have been identified that cause dRTA in humans or rodent models, including the AE1 exchanger, the a4 and B1 subunits of the H+-ATPase, carbonic anhydrase II, or proteins involved in collecting duct sodium reabsorption and its regulation by aldosterone.1.Fry A.C. Karet F.E. Inherited renal acidoses.Physiology (Bethesda). 2007; 22: 202-211Crossref PubMed Scopus (65) Google Scholar, 5.Alper S.L. Genetic diseases of acid-base transporters.Annu Rev Physiol. 2002; 64: 899-923Crossref PubMed Scopus (155) Google Scholar It is tempting to speculate that RhCG may be another candidate gene for dRTA in humans, in those patients in whom no mutations in any of these known genes have been identified to date. Direct sequencing of patients with recessive forms of dRTA yielded no evidence for RhCG mutations so far. Arguably, such patients may be difficult to detect, as testing for an incomplete distal RTA remains challenging and is not a standard procedure in most centers. Of interest, our recent data suggest that also heterozygous mice lacking only one allele of RhCG develop a form of incomplete dRTA.35.Bourgeois S. Aydin A. Mihailova M. et al.Rhesus protein Rhcg is essential for proton excretion by the kidneys.J Am Soc Nephrol. 2009; 20: 34AGoogle Scholar Similarly, in a rat model of cyclosporine-induced renal tubular acidosis, reduced expression of RhCG was reported.54.Lim S.W. Ahn K.O. Kim W.Y. et al.Expression of ammonia transporters, Rhbg and Rhcg, in chronic cyclosporine nephropathy in rats.Nephron Exp Nephrol. 2008; 110: e49-e58Crossref PubMed Scopus (17) Google Scholar Thus, reduced levels of RhCG expression (such as in haploinsufficiency of RhCG) and/or activity due to either genetic inactivation or dysregulation may be involved in specific syndromes of complete or incomplete dRTA. The discovery that the rhesus proteins RhAG, RhBG, and RhCG are involved in mediating cellular NH3/NH4+ transport has opened a new field of investigations into the role of these interesting proteins. In the kidney, RhCG appears to be an important molecule in ammonium excretion and urinary acidification along the collecting duct. Our picture of how the collecting duct excretes ammonium has to be revised with these new findings (Figure 3). However, many open questions remain, such as the exact transport mechanism, the acute and long-term regulation of expression and activity, its functional and physical interaction with other proteins, or its role in other extrarenal organs. Obviously, NH3 can be excreted to some extent even in the complete absence of RhCG. What mediates NH3 permeability—free diffusion or a transporter/channel—will be interesting to test. The role of RhCG in renal diseases of inborn or acquired forms of impaired urinary acid excretion is to be unraveled. The fact that RhCG appears to be highly regulated makes it likely that its regulation and dysregulation may contribute to specific forms of renal tubular acidosis. The role of RhCG in other tissues such as epididymis, lung, or liver has not been examined in much detail and may link this protein to important functions, such as ammonium detoxification or male fertility. We thank Dominique Eladari and Regine Chambrey for many valuable and critical discussions. The work in the laboratories of the authors has been supported by the 7th EU Framework Project EUNEFRON (GA#201590), the Swiss National Science Foundation, the Zurich Center for Integrative Human Physiology, the Belgian agencies FNRS and FRSM, the 'Fondation Alphonse & Jean Forton', an Inter-university Attraction Pole (IUAP P6/05), the DIANE project (Communauté Française de Belgique), the Institut National de la Santé et de la Recherche Médicale, and the Facility for Renal Phenotyping at the Centre de Recherche des Cordeliers.
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