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• W2765781134 abstract "Ammonium, stemming from renal ammoniagenesis, is a major urinary proton buffer and is excreted along the collecting duct. This process depends on the concomitant secretion of ammonia by the ammonia channel RhCG and of protons by the vacuolar-type proton-ATPase pump. Thus, urinary ammonium content and urinary acidification are tightly linked. However, mice lacking Rhcg excrete more alkaline urine despite lower urinary ammonium, suggesting an unexpected role of Rhcg in urinary acidification. RhCG and the B1 and B2 proton-ATPase subunits could be co-immunoprecipitated from kidney. In ex vivo microperfused cortical collecting ducts (CCD) proton-ATPase activity was drastically reduced in the absence of Rhcg. Conversely, overexpression of RhCG in HEK293 cells resulted in higher proton secretion rates and increased B1 proton-ATPase mRNA expression. However, in kidneys from Rhcg-/- mice the expression of only B1 and B2 subunits was altered. Immunolocalization of proton-ATPase subunits together with immuno-gold detection of the A proton-ATPase subunit showed similar localization and density of staining in kidneys from Rhcg+/+ and Rhcg-/-mice. In order to test for a reciprocal effect of intercalated cell proton-ATPases on Rhcg activity, we assessed Rhcg and proton-ATPase activities in microperfused CCD from Atp6v1b1-/- mice and showed reduced proton-ATPase activity without altering Rhcg activity. Thus, RhCG and proton-ATPase are located within the same cellular protein complex. RhCG may modulate proton-ATPase function and urinary acidification, whereas proton-ATPase activity does not affect RhCG function. This mechanism may help to coordinate ammonia and proton secretion beyond physicochemical driving forces. Ammonium, stemming from renal ammoniagenesis, is a major urinary proton buffer and is excreted along the collecting duct. This process depends on the concomitant secretion of ammonia by the ammonia channel RhCG and of protons by the vacuolar-type proton-ATPase pump. Thus, urinary ammonium content and urinary acidification are tightly linked. However, mice lacking Rhcg excrete more alkaline urine despite lower urinary ammonium, suggesting an unexpected role of Rhcg in urinary acidification. RhCG and the B1 and B2 proton-ATPase subunits could be co-immunoprecipitated from kidney. In ex vivo microperfused cortical collecting ducts (CCD) proton-ATPase activity was drastically reduced in the absence of Rhcg. Conversely, overexpression of RhCG in HEK293 cells resulted in higher proton secretion rates and increased B1 proton-ATPase mRNA expression. However, in kidneys from Rhcg-/- mice the expression of only B1 and B2 subunits was altered. Immunolocalization of proton-ATPase subunits together with immuno-gold detection of the A proton-ATPase subunit showed similar localization and density of staining in kidneys from Rhcg+/+ and Rhcg-/-mice. In order to test for a reciprocal effect of intercalated cell proton-ATPases on Rhcg activity, we assessed Rhcg and proton-ATPase activities in microperfused CCD from Atp6v1b1-/- mice and showed reduced proton-ATPase activity without altering Rhcg activity. Thus, RhCG and proton-ATPase are located within the same cellular protein complex. RhCG may modulate proton-ATPase function and urinary acidification, whereas proton-ATPase activity does not affect RhCG function. This mechanism may help to coordinate ammonia and proton secretion beyond physicochemical driving forces. The renal collecting duct excretes acid into urine by parallel secretion of protons (H+) and ammonia (NH3) by vacuolar H+-ATPases (H+-ATPase) and the ammonia transporter RhCG.1Weiner I.D. Hamm L.L. Molecular mechanisms of renal ammonia transport.Annu Rev Physiol. 2007; 69: 317-340Crossref PubMed Scopus (116) Google Scholar, 2Wagner C.A. Devuyst O. Bourgeois S. et al.Regulated acid-base transport in the collecting duct.Pflugers Arch. 2009; 458: 137-156Crossref PubMed Scopus (142) Google Scholar, 3Wagner C.A. Devuyst O. Belge H. et al.The rhesus protein RhCGa new perspective in ammonium transport and distal urinary acidification.Kidney Int. 2011; 79: 154-161Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Renal acid excretion is critical for maintaining normal acid-base homeostasis and is increased during acidosis or following a systemic acid-load.2Wagner C.A. Devuyst O. Bourgeois S. et al.Regulated acid-base transport in the collecting duct.Pflugers Arch. 2009; 458: 137-156Crossref PubMed Scopus (142) Google Scholar Highly conserved through evolution from bacteria to humans, RhCG belongs to the Rh (Rhesus) glycoprotein family and has been characterized by in vitro and ex vivo studies as a channel selectively permeable to NH3 but not to ammonium (NH4+).4Gruswitz F. Chaudhary S. Ho J.D. et al.Function of human Rh based on structure of RhCG at 2.1 A.Proc Natl Acad Sci U S A. 2010; 107: 9638-9643Crossref PubMed Scopus (158) Google Scholar, 5Mouro-Chanteloup I. Cochet S. Chami M. et al.Functional reconstitution into liposomes of purified human RhCG ammonia channel.PLoS One. 2010; 5: e8921Crossref PubMed Scopus (38) Google Scholar, 6Zidi-Yahiaoui N. Callebaut I. Genetet S. et al.Functional analysis of human RhCGcomparison with E. coli ammonium transporter reveals similarities in the pore and differences in the vestibule.Am J Physiol Cell Physiol. 2009; 297: 537-547Crossref PubMed Scopus (24) Google Scholar In the kidney, RhCG expression specifically spreads from the late distal convoluted tubule to the inner medullary collecting duct, on both basolateral and apical poles of most epithelial cells (with the exception of selective apical expression in non–type-A intercalated cells).7Han K.H. Croker B.P. Clapp W.L. et al.Expression of the ammonia transporter, rh C glycoprotein, in normal and neoplastic human kidney.J Am Soc Nephrol. 2006; 172: 670-2679Google Scholar, 8Kim H.Y. Verlander J.W. Bishop J.M. et al.Basolateral expression of the ammonia transporter family member, Rh C Glycoprotein, in the mouse kidney.Am J Physiol Renal Physiol. 2009; 296: F543-F555Crossref PubMed Scopus (51) Google Scholar, 9Verlander 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 (156) Google Scholar, 10Eladari 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 (110) Google Scholar, 11Bourgeois S. Bounoure L. Christensen E.I. et al.Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosisRhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.J Biol Chem. 2013; 288: 5518-5529Crossref PubMed Scopus (29) Google Scholar We recently showed that RhCG is responsible for a major part of NH3 transported through apical and basolateral membranes of the collecting duct resulting in a drastic reduction of transepithelial NH3 transport in collecting ducts from Rhcg−/− mice.11Bourgeois S. Bounoure L. Christensen E.I. et al.Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosisRhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.J Biol Chem. 2013; 288: 5518-5529Crossref PubMed Scopus (29) Google Scholar, 12Biver 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 (147) Google Scholar As a consequence of this defect, chronically acid-loaded Rhcg−/− mice cannot eliminate NH4+ into urine and develop a severe incomplete distal renal tubular acidosis characterized by low blood pH and HCO3− concentration.11Bourgeois S. Bounoure L. Christensen E.I. et al.Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosisRhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.J Biol Chem. 2013; 288: 5518-5529Crossref PubMed Scopus (29) Google Scholar, 12Biver 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 (147) Google Scholar Similarly, mice with selective deletion of Rhcg from only the collecting duct or intercalated cells show reduced urinary ammonium excretion.13Lee H.W. Verlander J.W. Bishop J.M. et al.Effect of intercalated cell-specific Rh C glycoprotein deletion on basal and metabolic acidosis-stimulated renal ammonia excretion.Am J Physiol Renal Physiol. 2010; 299: F369-F379Crossref PubMed Scopus (47) Google Scholar, 14Lee H.W. Verlander J.W. Handlogten M.E. et al.Effect of collecting duct-specific deletion of both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on renal response to metabolic acidosis.Am J Physiol Renal Physiol. 2014; 306: F389-F400Crossref PubMed Scopus (30) Google Scholar While RhCG is responsible for luminal NH3 secretion, the renal H+-ATPase is critical to actively secrete H+ into urine.15Wagner C.A. Finberg K.E. Breton S. et al.Renal vacuolar H+-ATPase.Physiol Rev. 2004; 84: 1263-1314Crossref PubMed Scopus (367) Google Scholar, 16Breton S. Brown D. Regulation of luminal acidification by the V-ATPase.Physiology (Bethesda). 2013; 28: 318-329Crossref PubMed Scopus (142) Google Scholar H+-ATPases are ubiquitous multi-subunit proteins composed of a cytosolic V1 catalytic domain, responsible for ATP hydrolysis and a membrane-associated V0 domain mediating H+ translocation.16Breton S. Brown D. Regulation of luminal acidification by the V-ATPase.Physiology (Bethesda). 2013; 28: 318-329Crossref PubMed Scopus (142) Google Scholar, 17Forgac M. Vacuolar ATPasesrotary proton pumps in physiology and pathophysiology.Nat Rev Mol Cell Biol. 2007; 8: 917-929Crossref PubMed Scopus (1081) Google Scholar In the collecting duct, H+-ATPases are localized at the luminal side of type-A intercalated cells and basolateral side of non–type-A intercalated cells. The V1 domain contains 8 subunits A–H, including the tissue-specific subunit isoforms B1 and B2. The B1 isoform is highly enriched in intercalated cells of kidney and its genetic mutation or deletion cause a form of distal renal tubular acidosis.18Karet F.E. Finberg K.E. Nelson R.D. et al.Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness.Nat Genet. 1999; 21: 84-90Crossref PubMed Scopus (579) Google Scholar, 19Finberg K.E. Wagner C.A. Bailey M.A. et al.The B1 subunit of the H+ATPase is required for maximal urinary acidification.Proc Natl Acad Sci U S A. 2005; 102: 13616-13621Crossref PubMed Scopus (115) Google Scholar, 20Dhayat N.A. Schaller A. Albano G. et al.The vacuolar H+-ATPase B1 subunit polymorphism p.E161K associates with impaired urinary acidification in recurrent stone formers.J Am Soc Nephrol. 2016; 27: 1544-1554Crossref PubMed Scopus (36) Google Scholar The V0 domain contains 6 different subunits, a, d, c, c', c'', and e, including a1, a2, a3, and a4. Also, the a4 isoform is highly expressed in kidney21Schulz N. Dave M.H. Stehberger P.A. et al.Differential localization of vacuolar H+-ATPases containing a1, a2, a3, or a4 (ATP6V0A1–4) subunit isoforms along the nephron.Cell Physiol Biochem. 2007; 20: 109-120Crossref PubMed Scopus (27) Google Scholar, 22Stehberger P. Schulz N. Finberg K.E. et al.Localization and regulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of distal renal tubular acidosis.J Am Soc Nephrol. 2003; 14: 3027-3038Crossref PubMed Scopus (73) Google Scholar and mutations in humans and its deletion in mice are associated with renal tubular acidosis.23Smith A.N. Skaug J. Choate K.A. et al.Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing.Nat Genet. 2000; 26: 71-75Crossref PubMed Scopus (5) Google Scholar, 24Hennings J.C. Picard N. Huebner A.K. et al.A mouse model for distal renal tubular acidosis reveals a previously unrecognized role of the V-ATPase a4 subunit in the proximal tubule.EMBO Mol Med. 2012; 4: 1057-1071Crossref PubMed Scopus (45) Google Scholar, 25Norgett E.E. Golder Z.J. Lorente-Canovas B. et al.Atp6v0a4 knockout mouse is a model of distal renal tubular acidosis with hearing loss, with additional extrarenal phenotype.Proc Natl Acad Sci U S A. 2012; 109: 13775-13780Crossref PubMed Scopus (50) Google Scholar Urinary acidification and ammonium content are closely linked.26Wrong O. Davies H.E. The excretion of acid in renal disease.Q J Med. 1959; 28: 259-313PubMed Google Scholar Secretion of NH3 and H+ by RhCG and H+-ATPases may be coordinated, as protonation of NH3 and subsequent trapping of NH4+ are critical for the efficient excretion of NH4+.26Wrong O. Davies H.E. The excretion of acid in renal disease.Q J Med. 1959; 28: 259-313PubMed Google Scholar, 27Weiner I.D. Verlander J.W. Ammonia transporters and their role in acid-base balance.Physiol Rev. 2017; 97: 465-494Crossref PubMed Scopus (67) Google Scholar, 28Pitts 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 (7) Google Scholar, 29Pitts 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 (16) Google Scholar The partial overlap of both proteins in type-A intercalated cells may allow even for a direct functional coupling or interaction.30Quentin 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 (133) Google Scholar We have recently shown in 2 differently constructed mouse models lacking Rhcg that urinary pH is more alkaline during stimulation of urinary acidification and NH4+ excretion.11Bourgeois S. Bounoure L. Christensen E.I. et al.Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosisRhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.J Biol Chem. 2013; 288: 5518-5529Crossref PubMed Scopus (29) Google Scholar, 12Biver 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 (147) Google Scholar Because Rhcg−/− mice have highly reduced NH3 secretion, the lack of NH3 buffering capacity would be expected to result in a rather more acidic urinary pH, as shown in another mouse model with intact Rhcg-mediated NH3 secretion but reduced medullary NH4+ accumulation.31Stettner P. Bourgeois S. Marsching C. et al.Sulfatides are required for renal adaptation to chronic metabolic acidosis.Proc Natl Acad Sci U S A. 2013; 110: 9998-10003Crossref PubMed Scopus (46) Google Scholar Thus, we hypothesized that the absence of Rhcg may reduce proton secretion by H+-ATPases. To assess whether RhCG and H+-ATPase were located within the same cellular protein complex in intercalated cells, we performed co-immunoprecipitation of RhCG and B1 and B2 H+-ATPase subunits from native rat kidney medulla (Figure 1). Immunoprecipitation with antibodies against RhCG or B1 (Figure 1a and b) or RhCG or B2 (Figure 1c and d) after chemical crosslinking yielded protein complexes that contained both proteins, whereas immunoprecipitation with control IgG did not detect RhCG or the B subunits. Thus, RhCG, B1, and B2 containing H+-ATPases are part of the same protein complex in kidney. We further confirmed partial colocalization of Rhcg and B1 subunits at the apical pole of intercalated cells in mouse kidney by immunohistochemistry (Figure 1e). We recently showed in ex vivo microperfused collecting ducts from Rhcg−/− mice a reduced NH3 transepithelial flux due to decrease in apical and basolateral NH3 permeabilities.11Bourgeois S. Bounoure L. Christensen E.I. et al.Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosisRhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.J Biol Chem. 2013; 288: 5518-5529Crossref PubMed Scopus (29) Google Scholar, 12Biver 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 (147) Google Scholar Here, using the same approach, we assessed H+ transport activity in microperfused cortical collecting ducts (CCDs) from Rhcg+/+ and Rhcg−/− mice challenged for 2 days with an HCl load in food to maximize transport activities. Luminal NH4Cl prepulses were performed and H+ transport activity was assessed from the steep intracellular pH (pHi) recovery rates after maximal intracellular acidification when NH4Cl was removed from the lumen.32Roos A. Boron W.F. Intracellular pH.Physiol Rev. 1981; 61: 296-434Crossref PubMed Scopus (2288) Google Scholar, 33Wagner C.A. Lukewille U. Valles P. et al.A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.Pflugers Arch. 2003; 446: 623-632Crossref PubMed Scopus (40) Google Scholar The initial steep rate of alkalinization reflects H+ extrusion, mostly by H+-ATPases33Wagner C.A. Lukewille U. Valles P. et al.A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.Pflugers Arch. 2003; 446: 623-632Crossref PubMed Scopus (40) Google Scholar (Figure 2a and b). We also observed in wild-type (WT) mice an early, almost immediate but slow pHi recovery that was not observed in knockout mice, suggesting that it is related to the function of Rhcg. As the initial pHi values were slightly different in CCDs from Rhcg−/− and Rhcg+/+ mice, we also measured intracellular buffering power and calculated H+ fluxes (JH+) across the membrane to directly compare transport rates. Similar to pHi recovery rates, JH+ was greatly reduced in CCDs from Rhcg−/− by 89% (Rhcg+/+: 32.6 ± 12.8 pmol/mm/min vs. Rhcg−/−: 3.6 ± 0.63 pmol/mm/min, P ≤ 0.0001) (Figure 2c). Thus, the more alkaline urine observed in Rhcg−/− during acid-loading results from impaired proton secretion along the collecting duct. Next, we investigated whether the reduced H+ flux observed in microperfused CCDs from Rhcg−/− mice could be linked to altered expression of H+-ATPase subunits. At the mRNA level, we could not detect any variation of the intercalated cell-enriched B1 H+-ATPase isoform (Supplementary Figure S1A), and the more ubiquitous B2 (Supplementary Figure S1B) and a4 (Supplementary Figure S1C) H+-ATPase isoforms between Rhcg+/+ and Rhcg−/− mice. In membrane fractions from medullary kidney tissue to enrich the proportion of H+-ATPases originating from intercalated cells, the amount of the intercalated cell-specific B1 isoform (Figure 3a) was increased, whereas the B2 isoform (Figure 3b) was decreased in kidney tissue from Rhcg−/− mice (P ≤ 0.01). However, the protein abundance of the ubiquitous E2 (Figure 3c) and a1 isoforms (Figure 3c), as well as the kidney-enriched a4 (Figure 3d) subunits of the H+-ATPase, was not different between the 2 genotypes. Reduced H+-ATPase activity may result from altered subcellular localization of H+-ATPases. Immunostaining for the B1, B2, E, and a4 H+-ATPase subunits showed no apparent difference in subcellular localization of the 3 proteins in type-A intercalated cells in Rhcg+/+ and Rhcg−/− kidneys (Figure 4). Electron microscopy with immunogold staining for the ubiquitous A subunit similarly detected no difference in the subcellular localization of this H+-ATPase subunit (Figure 5). The absence of Rhcg did not affect the immunoreactivity for the A subunit in type-A intercalated cells of Rhcg−/− mice. H+-ATPase staining was observed mostly in intercalated cells and was associated with the luminal membrane as well as with intracellular vesicles, as described previously.16Breton S. Brown D. Regulation of luminal acidification by the V-ATPase.Physiology (Bethesda). 2013; 28: 318-329Crossref PubMed Scopus (142) Google Scholar Thus, the absence of Rhcg is not associated with a redistribution of H+-ATPases in type-A intercalated cells.Figure 5Subcellular localization of H+-ATPases in type-A intercalated cells is similar in Rhcg+/+ and Rhcg−/− mice. Immunogold staining for the A subunit of the H+-ATPase revealed a similar density of H+-ATPases at the apical membrane and in apically localized vesicles of type-A intercalated cells in Rhcg+/+ (upper panels) and Rhcg−/− (lower panels) mice. Magnification of the boxed areas of the overview electron micrograph are shown on the right. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test if the expression of RhCG was associated with stimulation of H+-ATPase activity, we used HEK293 cells, which express H+-ATPases and exhibit H+-ATPase–mediated H+ secretion across the plasma membrane.34Lang K. Wagner C.A. Haddad G. et al.Intracellular pH activates membrane-bound Na+/H+ exchanger and vacuolar H+-ATPase in human embryonic kidney (HEK) cells.Cell Physiol Biochem. 2003; 13: 257-262Crossref PubMed Scopus (41) Google Scholar Nontransfected HEK293 cells and HEK293 cells stably transfected with human RhCG were used to examine the effect of RhCG expression on H+-ATPase expression and activity. Quantitative reverse-transcriptase polymerase chain reaction (PCR) confirmed high expression levels of RhCG in RhCG-transfected HEK293 cells as reported previously (Figure 6a).5Mouro-Chanteloup I. Cochet S. Chami M. et al.Functional reconstitution into liposomes of purified human RhCG ammonia channel.PLoS One. 2010; 5: e8921Crossref PubMed Scopus (38) Google Scholar Next, we compared the expression levels of transcripts encoding different subunits of H+-ATPase, ATP6V1B1, ATP6V1B2, and ATP6V0A1, and found an exclusive increase in mRNA expression of ATP6V1B1 in HEK-RhCG compared to HEK-WT cells (Figure 6b–d). Following NH4Cl prepulse acidification in a HCO3− and Na+ free solution, we measured intracellular pH recovery rates, similar to experiments in microperfused CCDs (Figure 7a). We have previously demonstrated that this pHi recovery is mediated solely by H+-ATPases and sensitive to the H+-ATPase blocker bafilomycin.34Lang K. Wagner C.A. Haddad G. et al.Intracellular pH activates membrane-bound Na+/H+ exchanger and vacuolar H+-ATPase in human embryonic kidney (HEK) cells.Cell Physiol Biochem. 2003; 13: 257-262Crossref PubMed Scopus (41) Google Scholar The initial pHi of both groups of cells measured was in the same range (pH 7.16 ± 0.001 for nontransfected HEK293 cells and pH 7.18 ± 0.002 for RhCG-expressing HEK293 cells, Figure 7a) which allowed a direct comparison of ΔpHi/Δt. In HEK-RhCG cells, the rate of pHi recovery after NH4Cl removal was 52% higher than the one observed in nontransfected HEK (HEK-RhCG: [0.666 ± 0.001]*10–3 pH units/min vs. nontransfected HEK: [0.322 ± 0.006]*10–3 pH units/min, P ≤ 0.01) (Figure 7b). This result suggests that the presence of RhCG can stimulate H+ secretion and is in agreement with our data showing that H+ secretion is impaired in collecting ducts from Rhcg−/− mice. To assess whether H+-ATPase activity could affect Rhcg activity, we used a genetic animal model with reduced H+-ATPase activity, mice lacking the Atp6v1b1 (B1) subunit.19Finberg K.E. Wagner C.A. Bailey M.A. et al.The B1 subunit of the H+ATPase is required for maximal urinary acidification.Proc Natl Acad Sci U S A. 2005; 102: 13616-13621Crossref PubMed Scopus (115) Google Scholar, 35Rothenberger F. Velic A. Stehberger P.A. et al.Angiotensin II stimulates vacuolar H+-ATPase activity in renal acid-secretory intercalated cells from the outer medullary collecting duct.J Am Soc Nephrol. 2007; 18: 2085-2093Crossref PubMed Scopus (66) Google Scholar, 36Paunescu T.G. Russo L.M. Da Silva N. et al.Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice.Am J Physiol Renal Physiol. 2007; 293: F1915-F1926Crossref PubMed Scopus (59) Google Scholar Atp6v1b11−/− mice challenged with an HCl-containing diet showed a more alkaline urinary pH compared with Atp6v1b1+/+ mice (Figure 8a; Supplementary Tables S1 and S2), as demonstrated previously.19Finberg K.E. Wagner C.A. Bailey M.A. et al.The B1 subunit of the H+ATPase is required for maximal urinary acidification.Proc Natl Acad Sci U S A. 2005; 102: 13616-13621Crossref PubMed Scopus (115) Google Scholar However, urinary NH4+ excretion was similar between both genotypes before and during acute HCl treatment. But, after 4 days of acid-inducing diet, Atp6v1b1−/− presented with a significant 23% reduction of NH4+ excretion (Figure 8b). To assess whether collecting ducts from Atp6v1b11−/− mice had altered NH3 permeability, we investigated NH3 membrane permeabilities and H+ secretion in ex vivo microperfused isolated CCDs from Atp6v1b1+/+ and Atp6v1b1−/− mice receiving HCl diet for 2 days. We confirmed the expected reduction in H+ secretion in Atp6v1b1−/− mice (Figure 8c), but did not detect any differences in NH3 membrane permeability across the apical (Figure 8d) or basolateral (Figure 8e) membranes. Thus, in this genetic model, reduced H+-ATPase activity is not paralleled by reduced NH3 permeability. The present study provides a new and additional explanation for the well-known association between urinary acidification and ammonium excretion.26Wrong O. Davies H.E. The excretion of acid in renal disease.Q J Med. 1959; 28: 259-313PubMed Google Scholar We demonstrate that the ammonia channel RhCG is required for maximal urinary acidification and that RhCG stimulates H+-ATPase activity. Secretion of protons acidifies urine, which provides a major driving force for NH3 secretion and trapping of NH4+ after protonation. Thus, in this physico-chemical model, H+-secretion is the primary process that then affects the chemical gradient for NH3 secretion. Urinary acidification depends on H+ excretion along the collecting duct mediated mostly by H+-ATPases and to a lesser extent by H+-K+-ATPases. In intercalated cells, H+-ATPases are the primary pump at the apical membrane mediating type-A intercalated cell H+-excretion after an intracellular acid-load.33Wagner C.A. Lukewille U. Valles P. et al.A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.Pflugers Arch. 2003; 446: 623-632Crossref PubMed Scopus (40) Google Scholar, 37Milton A.E. Weiner I.D. Intracellular pH regulation in the rabbit cortical collecting duct A-type intercalated cell.Am J Physiol. 1997; 273: F340-F347PubMed Google Scholar The B1 H+-ATPase subunit is specifically represented and colocalizes with RhCG at the luminal side of renal type-A intercalated cells.10Eladari 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 (110) Google Scholar Based on previous observations that Rhcg null mice had more alkaline urine despite reduced NH3 secretion rates,11Bourgeois S. Bounoure L. Christensen E.I. et al.Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosisRhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.J Biol Chem. 2013; 288: 5518-5529Crossref PubMed Scopus (29) Google Scholar, 12Biver 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 (147) Google Scholar we hypothesized that Rhcg was required for maximal urinary acidification. Here, we observed a reduced apical H+ secretion rate in acid-loaded Rhcg−/− mice suggesting a form of regulation of H+-ATPases by RhCG or its activity. In Rhcg−/− mice, reduced H+-ATPase activity was not caused by downregulation of B1 or other H+-ATPase subunits at mRNA or protein level. On the contrary, B1 was upregulated, which may represent a compensatory process. The B2 H+-ATPase subunit was downregulated and as previously been shown to exhibit counterregulation in B1-deficient mice.38Paunescu T.G. Da Silva N. Marshansky V. et al.Expression of the 56-kDa B2 subunit isoform of the vacuolar H+-ATPase in proton-secreting cells of the kidney and epididymis.Am J Physiol Cell Physiol. 2004; 287: C149-C162Crossref PubMed Scopus (82) Google Scholar These data suggest that the reduction in apical H+ flux observed in Rhcg−/− mice collecting ducts is not the consequence of a lower amount of H+-ATPases formed but rather explained by H+-ATPases that are not fully functional due to either an inadequate assembling of the subunits or improper insertion of the pumps at the plasma membrane. Immunohistochemistry and immunogold staining for several subunits of the V1 and V0 domains of the H+-ATPase at the apical pole of intercalated cells did not find any evidence for altered subcellular localization and the presence of both V0 and V1 domain subunits suggests also intact assembly of pumps. In vitro studies in HEK293 cells revealed that overexpression of RhCG stimulates H+ extrusion paralleled by higher mRNA expression of the B1 H+-ATPase subunit. HEK293 cells express H+-ATPases and exhibit H+ extrusion only after intracellular acidification in a slow, time dependent manner that is independent from trafficking of H+-ATPases to the plasma membrane.34Lang K. Wagner C.A. Haddad G. et al.Intracellular pH activates membrane-bound Na+/H+ exchanger and vacuolar H+-ATPase in human embryonic kidney (HEK) cells.Cell Physiol Bioch" @default.
• W2765781134 created "2017-11-10" @default.
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• W2765781134 date "2018-02-01" @default.
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• W2765781134 title "The ammonia transporter RhCG modulates urinary acidification by interacting with the vacuolar proton-ATPases in renal intercalated cells" @default.
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