Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosis: Rhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.
ABSTRACT: Ammonia secretion by the collecting duct (CD) is critical for acid-base homeostasis and, when defective, causes distal renal tubular acidosis (dRTA). The Rhesus protein RhCG mediates NH(3) transport as evident from cell-free and cellular models as well as from Rhcg-null mice. Here, we investigated in a Rhcg mouse model the metabolic effects of Rhcg haploinsufficiency, the role of Rhcg in basolateral NH(3) transport, and the mechanisms of adaptation to the lack of Rhcg. Both Rhcg(+/+) and Rhcg(+/-) mice were able to handle an acute acid load, whereas Rhcg(-/-) mice developed severe metabolic acidosis with reduced ammonuria and high mortality. However, chronic acid loading revealed that Rhcg(+/-) mice did not fully recover, showing lower blood HCO(3)(-) concentration and more alkaline urine. Microperfusion studies demonstrated that transepithelial NH(3) permeability was reduced by 80 and 40%, respectively, in CDs from Rhcg(-/-) and Rhcg(+/-) mice compared with controls. Basolateral membrane permeability to NH(3) was reduced in CDs from Rhcg(-/-) mice consistent with basolateral Rhcg localization. Rhcg(-/-) responded to acid loading with normal expression of enzymes and transporters involved in proximal tubular ammoniagenesis but reduced abundance of the NKCC2 transporter responsible for medullary accumulation of ammonium. Consequently, tissue ammonium content was decreased. These data demonstrate a role for apical and basolateral Rhcg in transepithelial NH(3) transport and uncover an incomplete dRTA phenotype in Rhcg(+/-) mice. Haploinsufficiency or reduced expression of RhCG may underlie human forms of (in)complete dRTA.
Project description:BACKGROUND: Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties. METHODOLOGY/PRINCIPAL FINDINGS: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy. CONCLUSIONS/SIGNIFICANCE: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.
Project description:High dietary protein imposes a metabolic acid load requiring excretion and buffering by the kidney. Impaired acid excretion in CKD, with potential metabolic acidosis, may contribute to the progression of CKD. Here, we investigated the renal adaptive response of acid excretory pathways in mice to high-protein diets containing normal or low amounts of acid-producing sulfur amino acids (SAA) and examined how this adaption requires the RhCG ammonia transporter. Diets rich in SAA stimulated expression of enzymes and transporters involved in mediating NH4 (+) reabsorption in the thick ascending limb of the loop of Henle. The SAA-rich diet increased diuresis paralleled by downregulation of aquaporin-2 (AQP2) water channels. The absence of Rhcg transiently reduced NH4 (+) excretion, stimulated the ammoniagenic pathway more strongly, and further enhanced diuresis by exacerbating the downregulation of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC2) and AQP2, with less phosphorylation of AQP2 at serine 256. The high protein acid load affected bone turnover, as indicated by higher Ca(2+) and deoxypyridinoline excretion, phenomena exaggerated in the absence of Rhcg. In animals receiving a high-protein diet with low SAA content, the kidney excreted alkaline urine, with low levels of NH4 (+) and no change in bone metabolism. Thus, the acid load associated with high-protein diets causes a concerted response of various nephron segments to excrete acid, mostly in the form of NH4 (+), that requires Rhcg. Furthermore, bone metabolism is altered by a high-protein acidogenic diet, presumably to buffer the acid load.
Project description: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.
Project description:Mammalian glycosylated rhesus (Rh) proteins include the erythroid RhAG and the nonerythroid RhBG and RhCG. RhBG and RhCG are expressed in multiple tissues, including hepatocytes and the collecting duct (CD) of the kidney. Here, we expressed human RhAG, RhBG and RhCG in Xenopus oocytes (vs. H2O-injected control oocytes) and used microelectrodes to monitor the maximum transient change in surface pH (DpHS) caused by exposing the same oocyte to 5 % CO?/33 mM HCO?? (an increase) or 0.5 mM NH?/NH?? (a decrease). Subtracting the respective values for day-matched, H?O-injected control oocytes yielded channel-specific values (*). (?pH*(S))(CO?) and (-?pH*(S))(NH?) were each significantly >0 for all channels, indicating that RhBG and RhCG--like RhAG--can carry CO? and NH?. We also investigated the role of a conserved aspartate residue, which was reported to inhibit NH? transport. However, surface biotinylation experiments indicate the mutants RhBG(D178N) and RhCG(D177N) have at most a very low abundance in the oocyte plasma membrane. We demonstrate for the first time that RhBG and RhCG--like RhAG--have significant CO? permeability, and we confirm that RhAG, RhBG and RhCG all have significant NH? permeability. However, as evidenced by (?pH*(S))(CO?)/ (-?pH*(S))(NH?) values, we could not distinguish among the CO?/ NH? permeability ratios for RhAG, RhBG and RhCG. Finally, we propose a mechanism whereby RhBG and RhCG contribute to acid secretion in the CD by enhancing the transport of not only NH? but also CO? across the membranes of CD cells.
Project description:In humans, NH(3) transport across cell membranes is facilitated by the Rh (rhesus) family of proteins. Human Rh C glycoprotein (RhCG) forms a trimeric complex that plays an essential role in ammonia excretion and renal pH regulation. The X-ray crystallographic structure of human RhCG, determined at 2.1 A resolution, reveals the mechanism of ammonia transport. Each monomer contains 12 transmembrane helices, one more than in the bacterial homologs. Reconstituted into proteoliposomes, RhCG conducts NH(3) to raise internal pH. Models of the erythrocyte Rh complex based on our RhCG structure suggest that the erythrocytic Rh complex is composed of stochastically assembled heterotrimers of RhAG, RhD, and RhCE.
Project description:Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.
Project description:Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the ?-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1.
Project description:Background and Aims: Esophageal squamous cell carcinoma (ESCC), a major histologic subtype of esophageal cancer, is increasing in incidence, but the genetic underpinnings of this disease remain unexplored. The aim of this study is to identify the recurrent genetic changes, elucidate their roles and discover new biomarkers for improving clinical management of ESCC. Methods: Western blotting and immunohistochemistry were performed to detect the expression level of RHCG. Bisulfite genomic sequencing (BGS) and methylation-specific PCR (MSP) were used to study the methylation status in the promoter region of RHCG. The tumor-suppressive effect of RHCG was determined by both in-vitro and in-vivo assays. Affymetrix cDNA microarray was used to identify the underlying molecular mechanism. Results:RHCG was frequently downregulated in ESCCs, which was significantly correlated with poor differentiation (P = 0.001), invasion (P = 0.003), lymph node metastasis (P = 0.038) and poorer prognosis (P < 0.001). Demethylation treatment and bisulfite genomic sequencing analyses revealed that the downregulation of RHCG in both ESCC cell lines and clinical samples was associated with its promoter hypermethylation. Functional assays demonstrated that RHCG could inhibit clonogenicity, cell motility, tumor formation and metastasis in mice. Further study revealed that RHCG could stabilize I?B by decreasing its phosphorylation, and subsequently inhibit NF-?B/p65 activation by blocking the nuclear translocation of p65, where it acted as a transcription regulator for the upregulation of MMP1 expression. Conclusions: Our results support the notion that RHCG is a novel tumor suppressor gene that plays an important role in the development and progression of ESCC.
Project description:Mutations of the human ATP6V1B1 gene cause distal renal tubular acidosis (dRTA; OMIM #267300) often associated with sensorineural hearing impairment; however, mice with a knockout mutation of Atp6v1b1 were reported to exhibit a compensated acidosis and normal hearing. We discovered a new spontaneous mutation (vortex, symbol vtx) of Atp6v1b1 in an MRL/MpJ (MRL) colony of mice. In contrast to the reported phenotype of the knockout mouse, which was developed on a primarily C57BL/6 (B6) strain background, MRL-Atp6v1b1vtx/vtx mutant mice exhibit profound hearing impairment, which is associated with enlarged endolymphatic compartments of the inner ear. Mutant mice have alkaline urine but do not exhibit overt metabolic acidosis, a renal phenotype similar to that of the Atpbv1b1 knockout mouse. The abnormal inner ear phenotype of MRL- Atp6v1b1vtx/vtx mice was lost when the mutation was transferred onto the C57BL/6J (B6) background, indicating the influence of strain-specific genetic modifiers. To genetically map modifier loci in Atp6v1b1vtx/vtx mice, we analysed ABR thresholds of progeny from a backcross segregating MRL and B6 alleles. We found statistically significant linkage with a locus on Chr 13 that accounts for about 20% of the hearing threshold variation in the backcross mice. The important effect that genetic background has on the inner ear phenotype of Atp6v1b1 mutant mice provides insight into the hearing loss variability associated with dRTA caused by ATP6V1B1 mutations. Because MRL-Atp6v1b1vxt/vtx mice do not recapitulate the metabolic acidosis of dRTA patients, they provide a new genetic model for nonsyndromic deafness with enlarged vestibular aqueduct (EVA; OMIM #600791).
Project description:The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.