1INSERM U652, IFR58, Institut des Cordeliers, Université René Descartes, 2INSERM U665, Institut National de la Transfusion Sanguine, 3UMR 7134 CNRS-Université Pierre et Marie Curie, 4Departement de Physiologie and 6Service de Biochimie, Hôpital Européen Georges Pompidou, AP-HP, 5INSERM U439, Hôpital Lariboisière, and 7Département de Physiologie, Hôpital Necker-Enfant Malades, AP-HP, Paris, France
Submitted 21 April 2005 ; accepted in final form 27 July 2005
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ABSTRACT |
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rhesus protein; acid-base; tubular acidosis; ammonia
In aqueous solution, total ammonia exists in two forms: the cation NH4+ and the gas NH3. NH3 can reversibly react with a proton to form NH4+. The pKa of this buffer reaction is 9.03 at physiological pH and temperature of plasma (3). Consequently, NH4+ represents the predominant form of total ammonia at physiological pH. Before identification of Rh glycoproteins as putative NH3/NH4+ transporters, it was assumed that NH3 gas freely crosses cell membranes by nonionic diffusion, whereas the NH4+ ion is impermeant and must share membrane transport proteins with other cations, in particular K+, to cross cell membranes (11). Several groups have attempted to assess the mechanism of total ammonia (i.e., NH3 + NH4+) transport through Rh glycoproteins by heterologous expression in Xenopus laevis oocytes to determine whether these proteins mediate NH3 or NH4+ transport (2, 24, 28, 42). Although all these studies concluded that Rh glycoproteins increase total ammonia transport, they differed in their conclusions with respect to the nature of the transported species, as well as to the mechanism of transport. Some reported the electrogenic transport of NH4+ down its electrochemical gradient in Rhbg- or RhCG-expressing oocytes (2, 28), others the exchange of NH4+ against a proton in an NH4+/H+ countertransport in RhAG- or RhBG-expressing oocytes (24, 42), and Bakouh et al. (2), in addition to an electrogenic NH4+ transport pathway, also suggested that NH3 could be directly transported by RhCG. More recently, two groups reported the crystal structure of the AmtB protein, an Rh glycoprotein paralog from Escherichia coli, and proposed a model in which proteins from the Mep/Amt/Rh superfamily are NH3 channels (16, 47). Stopped-flow analyses to study the total ammonia transport in red blood cells from human and mouse genetic variants with various defects of proteins, that comprise or interact with the Rh complex, demonstrated at the same time that RhAG/Rhag facilitates NH3 transfer across the membranes (30), in agreement with the AmtB model. More recently, using the same approach with Madin-Darby canine kidney and human embryonic kidney (HEK293) cells transfected with RhBG and RhCG, Ripoche and co-workers (48) also confirmed that these proteins accelerate NH3 flux across cell membranes.
Despite compelling evidence that Rh glycoproteins can transport NH3, the physiological functions of these proteins in mammals remain largely unknown. We and others have previously reported that RhBG/Rhbg and RhCG/Rhcg are expressed in the kidney in the collecting duct (7, 29, 37), a nephron segment where total ammonia transport occurs in the NH3 form in parallel to H+ secretion (9, 10, 18). Rhbg was also found to be expressed in the liver in the plasmic membrane of the perivenous hepatocytes, which take up NH3 to be incorporated into glutamine (41). Therefore, it has been anticipated that Rhbg in the kidney may serve to accelerate NH3 transport because nonionic diffusion of NH3 through the lipid bilayer may possibly be too slow to support the high rate of NH3 transport required by this nephron segment (17). RhBG/Rhbg could also provide a molecular target for regulating NH3 transport through collecting duct epithelium and might therefore be critical for renal acid-base regulation (17, 29). In the liver, because perivenous hepatocytes represent a high-affinity, low-capacity NH4+ clearance pathway, in contrast to midzonal and periportal hepatocytes, which convert NH4+ into urea by a high-capacity, low-affinity pathway, it has been proposed that Rhbg-mediated NH3 transport may be critical to maintaining plasma NH4+ concentration in the micromolar range to avoid hepatic encephalopathy (17, 41).
Therefore, to gain insight into Rhbg physiological function, we generated mice with targeted gene disruption of RHbg and examined whether the mice displayed hallmarks of distal tubular acidosis or disturbed hepatic glutamine and nitrogen metabolism.
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METHODS |
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The EcoRI linearized vector RHbg 2.3EcoRI was electroporated into 129/Ola embryonic stem (ES) cells. ES cell growth, electroporation (800 V, 0.3 µF, 80 x 106 cells for 100 µg of linearized targeting vector), and puromycin selection were performed at genOway (Lyon, France) according to standard procedures. PCR was used to screen for targeted ES cell clones. For the 3'-end 2.9-kb product, the forward primer 5'-ATG GCT TCT GAG GCG GAA AGA ACC AG-3' (from the targeting vector) and reverse primer 5'-GAG GCT GTT ATG CCT GGG GAA TTC TA-3' (from the deleted 1.7-kb fragment) were used. For the 5'-end 3.7-kb fragment, primers were forward: 5'-GCC TTC TTA CTC TCC AGA CTC CCT CCT T-3' (from the deleted 1.7-kb fragment) and reverse: 5'-GAT CGG TGC GGG CCT CTT CGC TAT TAC-3' (from the targeting vector; see blue arrows in Fig. 1B). Insertional targeting was confirmed by Southern blot analysis with BglII using a 513-bp PCR fragment from the gapped region as a probe (primers were forward: 5'-ACT GAG GAA CAC CAC CCC AC-3' and reverse: 5'-GCC AAG GCT TTA CCC ACA AA-3'). In targeted clones, an 18.3-kb BglII fragment was present in addition to the wild-type 7.7-kb BglII fragment.
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Immunohistochemistry.
Kidneys or livers from RHbg/ mice and from their respective WT control littermates were fixed in vivo by perfusion with 4% paraformaldehyde, embedded in paraffin, and used for different staining procedures as described previously (7, 29). A rabbit anti-Rhbg (29), a rabbit anti-Rhcg (29), a rabbit anti-56-kDa 1-subunit of the H+-ATPase (a kind gift from Sylvie Breton and Dennis Brown, Harvard Medical School, Boston, MA) (5), or an antibody recognizing AE1 (a kind gift from Seth L. Alper, Harvard Medical School) (4) was used for these studies. All these antibodies have been used and characterized previously in mouse.
Physiological parameter measurements. All experiments were performed using age- and sex-matched littermates. All animals were treated in compliance with French and European Union animal care guidelines. For the determination of basal-state physiological parameters, adult RHbg+/+ (WT) and RHbg/ mice were housed in metabolic cages. Mice were allowed to adapt to these cages for 7 days, and then 24-h urine samples were collected with light mineral oil in the urine collector to determine daily urinary electrolyte excretion. The mice were given deionized water ad libitum and fed with standard laboratory chow (M20, Dietex). The same procedure was used for the acid-loading experiments, except that mice were given 0.28 M NH4Cl in place of water. Arterial pH, PCO2, and PO2 were measured with a pH/blood-gas analyzer (AVL Compact 1, AVL Instruments Médicaux, Eragny-sur-Oise, France). Serum and urine electrolytes and creatinine were measured by standard methods with a Beckman LX20 autoanalyzer (Coulter-Beckman, Villepinte, France) and Olympus AU 400 (Olympus, Rungis, France). Plasma NH4+ was measured by the standard enzymatic method. Plasma amino acid levels were determined by ion-exchange chromatography with ninhydrin detection. Urinary pH and bicarbonates were measured with a pH/blood-gas analyzer (ABL 555, Radiometer, Copenhagen, Denmark). Urinary NH4+ and titratable acid were measured by titration with a DL 55 titrator (Mettler Toledo, Viroflay, France).
Measurement of bone mineral density by dual-energy X-ray absorptiometry. Analysis of all animals by dual-energy X-ray absorptiometry (DXA) was carried out under anesthesia. Total body, whole femur, and caudal vertebral bone mineral content (BMC; mg) and bone mineral density (BMD; mg/cm2) of age-matched female mice were measured using a PIXImus instrument (version 1.44; Lunar). An ultra-high-resolution mode (resolution, x0.18 mm) was used (20). The precision and reproducibility of the instrument were previously evaluated by calculating the coefficient of variation of repeated DXA measurements. The coefficient of variation was <2% for all evaluated parameters. A phantom was scanned daily to monitor the stability of the measurements.
Isolated, perfused tubule studies with WT and RHbg/ mice.
Cortical collecting ducts (CCD) were dissected from female WT and RHbg/ mice at 2332 wk of age and microperfused in vitro (10). The basic approach used to determine NH3 permeability involved construction of a transepithelial gradient of NH3 and measurement of the resulting NH3 flux as previously described by Flessner et al. (10). Mice were allowed free access to autoclaved standard chow (M20, Dietex) and distilled water until the time of the experiments. Mice were anesthetized with 50 mg/kg pentobarbital sodium. Both kidneys were cooled in situ with control bath solution for 1 min and then removed and cut into thin coronal slices for tubule dissection. To obtain CCD segments, medullary rays were dissected from the slices at 10°C under a Wild M-8 dissecting microscope in the control bath solution of the experiment (see below). The isolated tubule was transferred to the bath chamber on the stage of an inverted microscope (Axiovert 100, Carl Zeiss) and mounted on concentric glass pipettes for microperfusion at 37°C. Bath solution was delivered at a rate of 20 ml/min and warmed to 37 ± 0.5°C by a water jacket immediately upstream of the chamber. The perfusion rate was adjusted by hydrostatic pressure to 10 nl/min. For most tubules, three to four collections were made. Two reliable collections were considered a minimum. The tubules were equilibrated for 2030 min at 37°C before the beginning of collections. To construct a transepithelial NH3 gradient, the perfusion (lumen) solution contained (in mM) 140 Na+, 1 NH4+, 5 K+, 2 HCO3, and 143 Cl; the bath solution contained (in mM) 140 Na+, 1 NH4+, 5 K+, 23 HCO3, and 122 Cl; in addition, both solutions contained (in mM) 5.5 glucose, 2 Ca2+, 1.2 Mg2+, 1.2 SO42, 2.5 HPO42, and 10 HEPES. The osmolarity of the solution was 295 ± 5 mosmol/kgH2O. All solutions were equilibrated with 95% O2-5% CO2. Once the solutions were gassed and the pH checked, they were placed in a reservoir and continuously bubbled with 95% O2-5% CO2. The actual pH of the solutions was monitored several times during experiments, and the pH of solutions was checked at the end of the experiment to ensure that changes did not occur. Carbonic anhydrase (no. C2522, Sigma, Saint Quentin Falavier, France) was added to the perfusate solution (1 mg/10 ml of solution). The purpose of carbonic anhydrase was to prevent any pH disequilibrium that might arise from proton secretion or NH3 transport. Transepithelial voltage was measured with a FD-223 differential electrometer (World Precision Instruments) by the use of a Ag/AgCl electrode connected to the perfusion pipette via a 0.15 M NaCl-agar bridge; a 0.15 M NaCl-agar bridge also connected the peritubular bath to an Ag/AgCl electrode. Transepithelial voltage was measured at the tip of the perfusion pipette during each period. Total ammonia concentration was measured in 10- to 12-nl samples of peritubular, perfused, and collected fluids using an NH3 diagnostic kit (Sigma) and the flow-through microfluorometer Nanoflo apparatus (World Precision Instruments) (45). To determine the initial and end-luminal pH, measurement of luminal pH with fluorescence microspectrometry utilizing BCECF (Molecular Probes, Eugene, OR) diluted in the luminal fluid at a 10 µM final concentration. The dye was excited alternatively at 490 and 440 nm with a 100-W halogen lamp and a computer-controlled chopper assembly. Emitted light was collected through a dichroic mirror, passed through a 530-nm filter, and focused onto a CCD camera (ICCD 2525F, Videoscope International) connected to a computer. The measured light intensities were digitalized with 8-bit precision (256 grey level scale) for further analysis. For each tubule, two regions of interest were drawn (one for initial luminal pH, the other for end-luminal pH), and the mean grey level for each excitation wavelength was calculated with Starwise Fluo software (Imstar, Paris, France). Background fluorescence was subtracted from fluorescence intensity at each excitation wavelength to obtain intensities of intraluminal fluorescence. The 490- to 440-nm ratio was used as an indicator of intraluminal pH. The following procedure was performed to calibrate the dye. The dye was diluted at the same concentration as in lumen in HCO3/CO2-free, HEPES-buffered solutions titrated to 6.3, 6.5, 6.7, or 6.9. A sample of each solution was introduced in a glass tube with the same inner diameter as the tubules, and the dye was excited as explained above. A linear calibration curve was derived from these measurements, and used to determine initial and end-luminal pH values.
Calculations of transepithelial NH3 permeability.
Assuming an absence of osmotic or hydrostatic pressure gradients across the epithelium and therefore an absence of net fluid transport, the passive transepithelial transport of total ammonia (Am) may be described by
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To calculate the permeability to NH3, the equation is rearranged as follows
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The net rate of transport JAm is calculated as follows
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As may be calculated as Ld, where d (mm) is the inner tubule diameter.
The total ammonia concentration ([Am]) is equal to the sum of the concentrations of the two species NH3 and NH4+ and is the quantity actually measured by the microfluorimetric assay. The equilibrium between the two species is defined by the Henderson-Hasselbalch equation
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The pKa equals 9.03 at physiological pH and temperature. Knowing the values for pH and [Am], the values for [NH3] and [NH4+] may be determined simultaneously.
Intracellular pH measurement.
CCD cells were loaded with the fluorescent probe BCECF, prepared as a 20 mM stock in DMSO, by exposing the cells for 20 min at room temperature to the control bath solution containing 10 µM BCECF. Loading was continued until the fluorescence intensity at 440-nm excitation wavelength was at least one order of magnitude higher than background fluorescence. The loading solution was then washed out by initiation of bath flow and the tubule was equilibrated with dye-free control bath solution for 510 min. Bath solution was delivered at a rate of 20 ml/min and warmed to 37 ± 0.5°C by water jacket immediately upstream to the chamber. Perfusion rate was adjusted by hydrostatic pressure to
20 nl/min to prevent axial changes in the composition of luminal fluid.
Intracellular dye was excited alternatively at 490 and 440 nm with a 100-W halogen lamp and a computer-controlled chopper assembly. Emitted light was collected through a dichroic mirror, passed through a 530-nm filter, and focused onto a CCD camera (ICCD 2525F, Videoscope International) connected to a computer. The measured light intensities were digitized with 8-bit precision (256 grey level scale) for further analysis. For each tubule, a region of interest was drawn and the mean grey level for each excitation wavelength was calculated with the Starwise Fluo software (Imstar). Background fluorescence was subtracted from fluorescence intensity at each excitation wavelength to obtain intensities of intracellular fluorescence. The 490- to 440-nm ratio was used as an indicator of intracellular pH (pHi).
The control solution composition was (in mM) 142 Na+, 4 K+, 1.5 Ca2+, 1.2 Mg2+, 145 Cl, 1.2 SO42, 2 HPO42, 5.5 glucose, 5 alanine, and 10 HEPES. After a 120-s recording, the peritubular solution was changed for a solution containing 4 NH4+, 138 Na+, 4 K+, 1.5 Ca2+, 1.2 Mg2+, 145 Cl, 1.2 SO42, 2 HPO42, 5.5 glucose, 5 alanine, and 10 HEPES, whereas the luminal solution was left unchanged. All solutions were adjusted to pH 7.40 with Tris and continuously bubbled with 100% O2 passed through a 3 M KOH CO2 trap.
Intracellular dye was calibrated at the end of each experiment using the high [K+]-nigericin technique. Tubules were perfused and bathed with a HEPES-buffered, 95 mM K+ solution containing 10 µM of the K+/H+ exchanger nigericin. Four different calibration solutions, titrated to 6.9, 7.2, 7.5, or 7.7, were used.
Statistics. All data are represented as means ± SE. Comparisons were assessed by unpaired or paired Student's t-test, as appropriate. Differences were considered significant at P < 0.05.
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RESULTS |
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The other physiological parameters measured in RHbg/ and WT mice are summarized in Tables 1 and 2. There were no detectable differences between RHbg/ and WT mice. In particular, liver nitrogen metabolism was normal with the absence of hyperammonemia, and there was normal plasma [glutamine] (see Table 1). There was also no significant difference in plasma urea, ornithine, citrulline, and arginine concentrations, and in urea urinary excretion, indicating that it is unlikely that an increase in urea synthesis compensated for a defect in glutamine synthesis (Table 1).
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Measurement of BMD of WT and RHbg/ mice. Incomplete distal tubular acidosis in human patients (i.e., impairment of distal renal acidification with no overt acidosis) is frequently discovered as isolated osteoporosis and hypercalciuria (39, 40). Therefore, we assessed BMD of 16 WT and 22 RHbg/ mice, at 4 mo of age, by DEXA. There was no evidence for bone demineralization of RHbg/ vs. WT mice, with a BMD value of 0.082 ± 0.002 mg/cm2 in RHbg/ mice vs. 0.078 ± 0.002 mg/cm2 in WT when measured at the femur (P = 0.13), and 0.064 ± 0.001 mg/cm2 in RHbg/ mice vs. 0.062 ± 0.006 mg/cm2 in WT, when measured at the caudal vertebrae (P = 0.35). Total BMD was also not different (0.056 ± 0.001 mg/cm2 in RHbg/ mice vs. 0.054 ± 0.001 mg/cm2 in WT, P = 0.16). Values of BMC were also not different (not shown). RHbg/ mice did also not exhibit hypercalciuria with respect to WT (0.78 ± 0.13 mmol/mmol creatinine in KO mice vs. 1.28 ± 0.3 mmol/mmol creatinine in controls, n = 10 for both groups, NS).
Immunolocalization of Rhcg, AE1, and the 56-kDa 1-subunit of the H+ pump in kidney sections from RHbg/ and WT mice.
We next examined whether RHbg inactivation disrupted the expression of the other proteins involved in NH4+ transport in the collecting duct, i.e., the basolateral Cl/HCO3 exchanger AE1 and the apical H+ pump, or of Rhcg, the other renal Rh glycoprotein isoform, or whether regulation of these proteins could have accounted for a compensatory process masking the impairment in Rhbg-mediated NH3 transport. As shown in Fig. 3, we could find no alteration in the expression level and subcellular localization of either Rhcg (Fig. 3A compared with 3B), H+-ATPase (Fig. 3C compared with 3D), or AE1 (Fig. 3E compared with 3F) by immunohistochemistry, despite RHbg disruption.
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DISCUSSION |
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We used an insertional gene-targeting strategy to knock out the RHbg gene (46). Southern blot analysis of the DNA from RHbg/ mice indicated that the targeting event was effective. The insertion of the vector resulted in duplication of a part of the RHbg gene, leaving the initial five exons (of a total of 10) intact (see Fig. 1). Immunohistochemical experiments using an antibody recognizing the COOH-terminal end of the Rhbg protein (29) demonstrated that the sequence downstream of the vector was not expressed. Accordingly, only a truncated RNA species encompassing exons 15, and not the full-length Rhbg RNA, could be detected by RT-PCR analysis of the KO mice transcripts (not shown). It is very unlikely that a cell surface-expressed truncated protein could be translated from the transcript from exons 15 because this protein would lack the COOH-terminal cytoplasmic domain recently shown to be critical for membrane expression of RhBG (22). Furthermore, even if this truncated protein could be expressed, it is anticipated that the lack of transmembrane-spanning domains M8, M9, M10, and M11, including numerous highly conserved residues, such as H318, an amino acid that is involved in the stabilization of NH3 within the pore-forming channel (see Fig. 1 in Ref. 16), will preclude any transport activity.
We did not detect any impairment in renal NH4+ excretion in RHbg/ mice. However, in humans, impairment of distal tubule acidification (i.e., distal NH4+ transport function) does not always lead to overt metabolic acidosis, and patients with incomplete distal tubular acidosis present with normal urinary pH and NH4+ excretion (31). In these patients, the administration of an acid load unmasks the renal acidification disorder by demonstrating their inability to increase their net acid excretion normally, mainly as urinary NH4+ (8, 27, 32, 43). However, in the present study, RHbg/ mice exhibited a normal response to the chronic acid loading. There was also no evidence of bone demineralization, another classic feature of incomplete renal tubular acidosis.
In the collecting duct, Rhcg, the apical ortholog of Rhbg, is normally expressed at the apical pole of the same cells that express Rhbg at the basolateral membrane (29), and NH3 transport is driven by the pH gradient generated by H+ secretion (19) that occurs through the combined action of the basolateral Cl/HCO3 exchanger AE1 and the apical H+ pump (36). Rhcg was not altered by RHbg disruption and remained restricted to the apical membrane of the cells, and thus it is unlikely that Rhcg has compensated for the absence of Rhbg transport function. It is also unlikely that the other mechanisms involved in net NH4+ transport by the collecting duct could have participated in a compensatory process masking a defect in Rhbg-mediated NH3 transport. Indeed, we found no evidence for the stimulation of the main proteins involved in H+ secretion, urinary pH was identical in RHbg+/+ and RHbg/ mice under basal conditions and decreased to the same extent after acid loading, and there was no difference in the amount of titratable acid excreted by RHbg/ and WT mice despite RHbg disruption. Finally, to rule out the possibility than a compensatory mechanism has masked a defect in Rhbg-dependent NH3/NH4+ transport, we also assessed directly NH3 or NH4+ transport across the basolateral membrane of CCD cells and NH3 diffusive permeability across CCD epithelium by in vitro microperfusion. We chose to study the CCD rather than the other segments of the collecting duct because we and others have described that Rhbg is expressed in a larger amount in the cortex than in the medulla (29, 37). It is noteworthy that this higher amount of Rhbg protein parallels a higher permeability to NH3 of the CCD than that of medullary segments, as demonstrated by Flessner et al. (10). Thus we hypothesized that a defect in Rhbg-mediated NH3 transport would have a more dramatic effect that would be more easily detectable in the CCD than in medullary portions of the collecting duct. However, once again we could not find any evidence for impaired NH3 or NH4+ transport consecutive to Rhbg disruption in these studies. There were also no evidences of alteration in liver NH4+ extraction and detoxification and of glutamine metabolism. KO mice did not exhibit obvious differences in their behavior and survived normally, indicating that it was unlikely that they were suffering from hepatic encephalopathy. The alternate pathway of NH3 extraction and detoxification from the plasma by the liver is the urea cycle that occurs in midzonal and periportal hepatocytes (12). Urea synthesis and glutamine synthesis from NH3 are two independent pathways, differentially regulated (12). Thus we hypothesized that a shift from glutamine biosynthesis by perivenous hepatocytes to urea synthesis by the other hepatocytes might have limited the accumulation of NH3 in the plasma. However, there was no significant difference in plasma urea and in the concentrations of the main amino acids involved in the urea cycle, namely, ornithine, citrulline, and arginine, indicating that it is unlikely that an increase in urea synthesis compensated for a defect in glutamine synthesis.
The conclusion that Rhbg is not critical for NH4+ transport in the kidney and the liver goes counter to expectations from heterologous expression studies (2, 24, 28, 42), crystallography data (16, 47), and stopped-flow analyses of red blood cells (30). However, all these studies were performed in vitro or ex vivo and could not therefore determine the physiological impact of Rh glycoprotein-mediated transport in vivo. Moreover, they were aimed at determining the precise molecular transport mechanism of NH4+, NH3, or of its derivative, methylammonium. Because they did not test transport specificity of the different Rh glycoproteins studied, they do not rule out the possibility that, in vivo, transport of another substrate occurs preferentially to NH3 through Rh glycoproteins. CO2 is another gas that has also a high diffusion permeability through cell membranes, and it has been suggested that membrane transport proteins may account for the high CO2 permeability of some cells (6). It has also been proposed that the Mep/Amt/Rh protein family represents a family of gas channels that may also accept CO2 as a substrate (3335). However, this hypothesis remains speculative and certainly requires a more direct demonstration. Nonetheless, if Rhbg transports CO2 into the collecting duct cells, mice would probably display an acidification defect similar to that observed when the Cl/HCO3 exchanger is inactivated (1). Indeed, CO2 must enter the intercalated cells to be hydrated to yield an H+ and an HCO3, a reaction catalyzed by the enzyme carbonic anhydrase. This reaction is believed to provide the principle source of H+ required for H+-ATPase function and to provide HCO3 that is basolaterally extruded through AE1 to replenish the body's HCO3 stores (19). Accordingly, it has been demonstrated that acetazolamide (21, 38), an inhibitor of carbonic anhydrase, or the nominal absence of CO2/HCO3 (38) is able to abolish H+ secretion of isolated collecting ducts microperfused in vitro. Thus, although we did not directly measure CO2 transport across the basolateral membrane of collecting duct cells, our experiments demonstrating that RHbg/ mice do not display any urinary acidification defect render unlikely the possibility that Rhbg is a CO2 transporter.
Apart from their NH3 transport properties, several observations about the Mep/Amt/ Rh proteins are striking. It has been demonstrated that MEP2, a Rh glycoprotein paralog of yeast, and AmtB, in Escherichia coli, are NH4+-sensing receptors (14, 23). A possible role of Rh glycoproteins in cell signaling or proliferation has also been suggested. The first report of an RhCG sequence in GenBank was as human PDRC2 (accession no. AF081497), identified as a tumor-related protein. More convincing is the recent identification of RHbg and RHcg as candidate cancer-causing genes in a murine model of glioblastoma (15). The authors identified a total of 66 candidate cancer-causing loci from 108 independent tumors. Rhbg and Rhcg were both tagged, each in three independent tumors (15). Thus it is tempting to speculate that the Rh glycoproteins might not function as epithelial transporters but are rather involved in cell signaling.
In summary, we have demonstrated that RHbg inactivation does not lead to distal tubular acidosis or to hyperammonemic hepatic encephalopathy, arguing against a critical role of this protein in NH3 epithelial transport.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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