Expression of the Ammonia Transporter, Rh C Glycoprotein, in Normal and Neoplastic Human Kidney
Ki-Hwan Han*,
Byron P. Croker,,
William L. Clapp,,
Dietrich Werner,
Manisha Sahni,
Jin Kim||,
Hye-Young Kim,
Mary E. Handlogten and
I. David Weiner,¶
* Department of Anatomy, Ewha Womans University, Seoul, Korea; Department of Pathology and Division of Nephrology, Hypertension and Transplantation, University of Florida, and Department of Pathology and ¶ Nephrology Section, North Florida/South Georgia Veterans Health System, Gainesville, Florida; and || Department of Anatomy and Medical Research Center for Cell Death Disease Research Center, The Catholic University of Korea, Seoul, Korea
Address correspondence to: Dr. I. David Weiner, Division of Nephrology, Hypertension and Transplantation, University of Florida College of Medicine, P.O. Box 100224, Gainesville, FL 32610-0224. Phone: 352-273-5358; Fax: 352-271-4518; E-mail: weineid{at}ufl.edu
Received for publication February 21, 2006.
Accepted for publication July 10, 2006.
Recent studies have identified the presence of a novel Mep/Amt/Rhglycoprotein family of proteins that may play an important rolein transmembrane ammonia transport. One of the mammalian membersof this family, Rh C glycoprotein (RhCG), transports ammonia,is expressed in distal nephron sites that are critically importantfor ammonia secretion, exhibits increased expression in responseto chronic metabolic acidosis, and originally was cloned asa tumor-related protein. The purpose of our studies was to determinethe localization of RhCG in the normal and neoplastic humankidney. Immunoblot analysis of human renal cortical proteinlysates demonstrated RhCG protein expression with a molecularweight of approximately 52 kD. Immunohistochemistry revealedboth apical and basolateral Rhcg expression in the distal convolutedtubule, connecting segment, and initial collecting tubule andthroughout the collecting duct. Co-localization with calbindin-D28k,H+-ATPase, aquaporin-2, and pendrin showed that distal convolutedtubule and connecting segment cells, A-type intercalated cells,and non-A, non-B cells express RhCG and that B-type intercalatedcells, principal cells, and inner medullary collecting ductcells do not. In renal neoplasms, RhCG was expressed by chromophoberenal cell carcinoma and renal oncocytoma but not by clear cellrenal cell carcinoma or by papillary renal cell carcinomas.These studies suggest that RhCG contributes to both apical andbasolateral membrane ammonia transport in the human kidney.Furthermore, renal chromophobe renal cell carcinoma and renaloncocytoma seem to originate from the A-type intercalated cell.
Renal ammonia metabolism is the primary component of net acidexcretion and thereby is critical for acid-base homeostasis(1–4). Ammonia is produced by the proximal tubule andis secreted preferentially into the luminal fluid. The thickascending limb of the loop of Henle reabsorbs the majority ofluminal ammonia into the renal interstitium, where the collectingduct secretes it into the luminal fluid. Under normal conditions,70 to 80% of total urinary ammonia is secreted by the collectingduct, and almost the entire increase in net acid excretion inresponse to chronic metabolic acidosis is due to increased ammoniaexcretion. Importantly, this increased renal ammonia excretionis associated with substantial increases in collecting ductammonia secretion. Accordingly, understanding the regulationof collecting duct ammonia transport is important.
Recent studies have identified a Mep/Amt/Rh glycoprotein familyof proteins, which we have termed the ammonia transporter family,that may mediate an important role in ammonia transport (5–7).The mammalian members of this family include the erythroid-specificprotein Rh A glycoprotein (RhAG/Rhag) and the nonerythroid proteinsRh B glycoprotein (RhBG/Rhbg) and Rh C glycoprotein (RhCG/Rhcg).Early studies identified that RhAG and RhCG were homologousto Mep/Amt proteins and that they restored ammonia-dependentgrowth in yeast that was deficient in endogenous ammonia transporters,suggesting that RhAG and RhCG could function as ammonia transporters(8). Further studies demonstrated that RhAG can transport ammoniaand have characterized the transport as being functionally equivalentto facilitated NH3 transport and/or NH4+/H+ exchange (9–11).
The nonerythroid Rh glycoproteins, RhBG and RhCG, also transportammonia (10–16) and are expressed in a wide variety oftissues in which ammonia metabolism is important, includingkidneys, liver, central nervous system, gastrointestinal tract,and skeletal muscles (17–23). In the kidney, RhCG is expressedin the distal convoluted tubule (DCT), connecting segment (CNT),initial collecting tubule (ICT), and collecting duct in bothrat and mouse kidneys (19,21,24), and Rhcg expression in theouter medullary collecting duct (OMCD) and inner medullary collectingduct (IMCD) increases in response to chronic metabolic acidosis(24,25). Therefore, RhCG may be important in renal ammonia transport.
To understand the specific role of RhCG in renal ammonia transport,it is important to know both its cellular and its membrane expressionin the human kidney. Studies in the mouse and rat have suggestedthat there may be species-specific differences in the locationof RhCG. In the mouse kidney, only apical immunoreactivity forRhCG has been reported (19). One study in the rat kidney alsoidentified only apical RhCG immunoreactivity (21), whereas otherstudies, using both light microscopic immunohistochemistry andimmunogold electron microscopy, have demonstrated RhCG expressionin both the apical and basolateral plasma membranes (24,25).Therefore, it is possible that RhCG/Rhcg may play a centralrole in both apical and basolateral ammonia transport in themammalian kidney. However, no previous studies have examinedthe expression of these proteins in the human kidney. Giventhe important differences in the expression of Rhcg, apicalversus apical and basolateral, in the rat and mouse kidneys,respectively, understanding RhCG’s expression in the humankidney is important for understanding its specific role in humanammonia transport. Therefore, the purpose of our studies wasto examine RhCG expression in the normal human kidney. We usednephrectomy specimens that were obtained as part of treatmentfor renal carcinomas and examined RhCG expression in uninvolvedregions of the kidney and in neoplastic regions. To determinethe specific cell types that express RhCG, we performed double-labelingimmunohistochemistry using antibodies against calbindin D28K,H+-ATPase, pendrin, and aquaporin-2 (AQP-2).
Antibodies
Affinity-purified antibodies to RhCG that were generated inthis laboratory have been characterized previously (17,19,20,24,25).Antibodies to H+-ATPase were obtained from Santa Cruz Biotechnology(SC-20943; Santa Cruz, CA), and antibodies to AQP-2 and calbindin-D28kwere obtained from Chemicon (Temecula, CA). Antibodies to pendrinwere provided by Ines E. Royaux, PhD (National Human GenomeResearch Institute, National Institutes of Health, Bethesda,MD).
Human Kidney Tissue
Normal human kidney tissues were obtained from the pathologylaboratory of the Gainesville Veterans Affairs Medical Center(GVAMC; Gainesville, FL). Samples were obtained from unusedportions of nephrectomy specimens that resulted from treatmentof renal cell carcinoma. Tissue was fixed in 10% buffered formalin.Tissue from a total of 15 kidneys was used. These studies wereapproved by Subcommittee for Clinical Investigation, GVAMC,and the University of Florida.
Human Renal Cortical Protein Lysate
Normal human renal cortical protein lysates were obtained fromBecton-Dickinson Biosciences (San Jose, CA).
Immunoblot Analysis
Immunoblot analysis was performed as described previously indetail (17,19,20,26). Briefly, renal tissue lysate protein (25µg/lane) was electrophoresed on 10% PAGE ReadyGels (Bio-Rad,Hercules, CA). Proteins were transferred electrophoreticallyto nitrocellulose membranes; membranes then were blocked andincubated for 5 h with 7 µg/ml affinity-purified primaryantibody. After washing, membranes were exposed to secondaryantibody (goat anti-rabbit IgG conjugated to horseradish peroxidase;Promega, Madison, WI) diluted 1:5000. Sites of antibody-antigenreaction were visualized using enhanced chemiluminescence anda Kodak Image Station 440CF digital imaging system (Perkin-ElmerLife Sciences, Boston, MA).
Immunohistochemistry
Immunolocalization of RhCG was accomplished using immunoperoxidaseprocedures and a commercially available kit (Dako, DakoCytomation,Glostrup, Denmark) as we have reported in detail previously(17,19,20,24). RhCG antibody was used at either 1:1000 or 1:2000dilution. Similar results were obtained with each dilution.In some experiments, sections were counterstained with hematoxylin.Sections were photographed using a Nikon E600 microscope equippedwith DIC optics, a DXM1200F digital camera, and ACT-1 software(Nikon USA, Melville, NY).
Double-Labeling Procedure
Co-localization of RhCG with other proteins was performed usingsequential immunoperoxidase procedures and a commercially availablekit (Dako, DakoCytomation). The tissue sections were dewaxed;had endogenous peroxidase quenched using DAKO Peroxidase solutionfor 30 min; then were washed, incubated with DAKO serum-freeblocking solution, washed, and incubated with RhCG antibodyovernight at 4°C in a humidified chamber. The sections thenwere washed, incubated with biotinylated anti-mouse and anti-rabbitsecondary antibody (DAKO LSAB2 kit) for 30 min, washed, incubatedwith streptavidin for 30 min, washed, exposed to diaminobenzidinefor 5 min, and then washed. The above procedure then was repeatedwith the substitution of a second primary antibody (H+-ATPase,1:200; AQP-2, 1:200; or, calbindin D28K, 1:5000) and the substitutionof Vector SG for diaminobenzidine.
Nomenclature
The term ammonia is used to refer to the combination of thetwo molecular species, NH3 and NH4+. When referring specificallyto the molecular species NH3, we specifically state "NH3," andwhen referring to NH4+ we specifically state "NH4+". Accordingto standard nomenclature, RhAG refers to the human Rh A glycoprotein,and Rhag refers to nonhuman Rh A glycoproteins, and we use asimilar pattern for Rh B and C glycoproteins. In the initialreport, the term RhGK (Rh glycoprotein kidney) was used (8).RhCG and RhGK are alternative names for the same protein andmRNA. RhCG is the term that is used most frequently currentlyand is used in this report.
Immunoblot Analysis
Immunoblot analysis of normal human renal cortical protein lysateidentified expression of a 52-kD protein that was identicalin apparent molecular weight to that observed in the normalmouse kidney (Figure 1A). A minor band with a molecular weightof approximately 48 kD also was present. This latter band isconsistent with alternative glycosylation of RhCG, which wasreported previously in the rat kidney (21). Specificity of theimmunoblot immunoreactivity was confirmed both by using affinity-purifiedantibodies, which prevented the nonspecific immunoreactivityin mouse kidney protein that was reported previously when unpurifiedantisera were used (19), and by showing that preincubating theantibody with the immunizing peptide blocked protein recognition(Figure 1B).
Figure 1. Immunoblot of normal human kidney protein. (A) Immunoblot of normal human and mouse renal cortical protein revealed expression of a 52-kD protein that was identical in size to mouse kidney Rh C glycoprotein (Rhcg). Lesser abundance of an approximately 48-kD protein also is observed, consistent with alternative glycosylation of RhCG. (B) Immunoblot specificity by showing the effect of preincubating the antibody with the immunizing peptide.
RhCG Immunolocalization
Immunohistochemical localization of RhCG in human kidney revealedexpression in the DCT, CNT, and ICT and throughout the collectingduct (Figure 2). Preincubating the affinity-purified antibodywith the immunizing peptide blocked detectable immunoreactivity(Figure 2B). Tissues that were treated with secondary antibodyalone showed no detectable immunoreactivity (data not shown).
Figure 2. Low-power micrograph of RhCG expression in the human kidney. (A) Low-power magnification of RhCG immunoreactivity in human kidney cortex. Immunoreactivity is present in a subset of convoluted tubule segment and in collecting duct epithelia. No expression is observed in proximal tubule epithelia (PT) or glomeruli (G). (B) Preincubation of the antibody with the immunizing peptide blocked immunoreactivity. (C) Rhcg expression in convoluted tubule segments in the human cortex. Both apical and basolateral Rhcg immunoreactivity is clearly present. Essentially all cells express Rhcg, and a subset of cells demonstrates increased immunoreactivity. (D) Expression in the cortical collecting duct (CCD). In contrast to convoluted tubule and initial collecting duct segments, only a subset of CCD cells expresses detectable RhCG immunoreactivity. These cells tend to be larger, protrude into the tubule lumen, and have morphologic characteristics suggestive of intercalated cells. (E) Expression in the outer medullary collecting duct in the inner stripe. Both apical and basolateral immunoreactivity is present in only a subset of cells (arrows). Basolateral immunoreactivity in these cells appears to be more intense than the apical immunoreactivity. (F) Expression in the inner medullary collecting duct. Only a minority of cells expresses Rhcg immunoreactivity (arrows); both apical and basolateral Rhcg immunoreactivity is present in these cells.
High-power observation of RhCG expression showed both apicaland basolateral immunoreactivity. In the CNT and DCT, almostall cells expressed RhCG immunoreactivity (Figure 2C). In thecortical collecting duct (CCD), OMCD, and IMCD, only a minorityof cells expressed detectable RhCG immunoreactivity (Figure 2,D through F). In general, basolateral RhCG immunoreactivityseemed more intense than apical immunoreactivity. No expressionwas observed in the glomerulus, proximal tubule, loop of Henle,or erythrocytes. The segmental distribution of RhCG in humankidney seems to be similar to the mouse and rat kidney (19,21,24).
Co-Localization with Calbindin-D28k
To confirm that the convoluted tubule segments that expressedRhCG were the DCT and CNT and not proximal tubule segments,we performed co-localization with calbindin-D28k, which labelsDCT, CNT, and CCD but not the proximal tubule (27). Figure 3Ashows representative findings in which calbindin-D28k–positivecells expressed RhCG, thereby confirming that RhCG is expressedby the DCT and CNT.
Figure 3. Co-localization of RhCG with calbindin-D28k, H+-ATPase, pendrin, and aquaporin-2 (AQP-2). (A) Co-localization of RhCG (brown) with the distal convoluted tubule (DCT)- and connecting segment (CNT)-specific marker calbindin-D28k (blue). Convoluted tubule segments that expressed calbindin-D28k (arrows) also expressed RhCG, whereas calbindin-D28k–negative cortical segments (*) did not. (B) Co-localization of H+-ATPase (blue) with RhCG (brown) in the CNT. Both cells with apical H+-ATPase immunoreactivity (arrows; the A-type intercalated cell and non-A, non-B cell) and cells without detectable H+-ATPase immunoreactivity (arrowheads; DCT and CNT cells) express apical and basolateral RhCG immunoreactivity. Cells with diffuse H+-ATPase immunoreactivity (B-type intercalated cells) do not (white arrows). (C) In the cortical collecting duct (CCD), cells with apical H+-ATPase immunoreactivity (blue) express RhCG (arrows), whereas cells with diffuse H+-ATPase immunoreactivity do not (white arrow). In contrast to the CNT, H+-ATPase–negative CCD principal cells do not express detectable RhCG immunoreactivity. (D) In the outer medullary collecting duct, H+-ATPase–positive cells (intercalated cells) express RhCG immunoreactivity (arrows), whereas H+-ATPase–negative cells (principal cells) do not. (E) In the CNT, both cells with apical (arrows) and without detectable apical pendrin (brown) express RhCG (blue). (F) CCD pendrin-positive cells (arrows) do not express detectable RhCG, and RhCG-positive cells do no express detectable pendrin (arrowheads). (G) Co-localization of AQP-2 (blue) with RhCG (brown) in the DCT (left tubule) and initial collecting tubule (ICT; right tubule). In the DCT, both apical AQP-2–positive (arrowhead) and AQP-2–negative (arrows) cells express RhCG, although AQP-2–negative cells, in general, express more intense RhCG immunoreactivity. Rare AQP-2–and RhCG-negative cells (yellow arrow) most likely are B-type intercalated cells. In the ICT (right tubule), AQP-2–positive cells (white arrows) do not exhibit significant RhCG immunoreactivity, whereas AQP-2–negative cells (white arrowheads) do. (H) A CCD in which AQP-2–positive principal cells (arrows) do not exhibit detectable RhCG immunoreactivity. AQP-2–negative cells (intercalated cells), in general, express intense RhCG immunoreactivity (arrowhead). Cells without either detectable AQP-2 or RhCG (white arrow) most likely are B-type intercalated cells. In the outer medulla (I), only AQP-2–negative cells (arrowheads) express detectable RhCG immunoreactivity; AQP-2–positive cells do not (arrows).
Co-Localization of RhCG with H+-ATPase
At least two fundamentally different cell types are presentin the CNT and the collecting duct: The principal cell and theintercalated cell. Intercalated cells are involved in H+ secretionand are regulated by acid-base balance, are characterized byintense H+-ATPase expression, and in the mouse and rat kidneysare the sites of increased RhCG expression (19,21,24,25). Co-localizationof RhCG with H+-ATPase in the human kidney showed increasedRhCG expression in intercalated cells. CNT cells with apicalH+-ATPase (A-type intercalated cells and non-A, non-B cells)expressed intense RhCG immunoreactivity (Figure 3B). Cells withdiffuse H+-ATPase (B-type intercalated cells) did not expressdetectable RhCG immunoreactivity. H+-ATPase–negative cellsexpressed intermediate RhCG immunoreactivity. In the CCD, onlyintercalated cells with apical H+-ATPase immunoreactivity (A-typeintercalated cells) expressed RhCG (Figure 3C). Cells with diffuseH+-ATPase immunoreactivity (B-type intercalated cells) did notexpress detectable RhCG immunoreactivity; neither was RhCG immunoreactivityidentified in H+-ATPase–negative principal cells. In theOMCD, only H+-ATPase–positive A-type intercalated cellsexpressed RhCG; H+-ATPase–negative principal cells didnot (Figure 3D).
Co-Localization of RhCG with Pendrin
These observations suggest that A-type and/or non-A, non-B intercalatedcells but not B-type intercalated cells express RhCG. To confirmthis, we co-localized RhCG with pendrin, an apical Cl–/HCO3–exchanger that is present in the B-type intercalated cell andthe non-A, non-B cell but not in the A-type intercalated cell(28–30). In the CNT, where the majority of pendrin-positivecells are non-A, non-B cells, the majority of pendrin-positivecells expressed RhCG immunoreactivity (Figure 3E). This, combinedwith the observation that cells in the CNT with apical H+-ATPaseimmunoreactivity express RhCG, identifies that CNT non-A, non-Bintercalated cells express RhCG.
In the CCD, where the majority of pendrin-positive intercalatedcells are B-type intercalated cells, pendrin-positive cellsdid not express RhCG immunoreactivity (Figure 3F). This, combinedwith the observation that CCD intercalated cells with diffuseH+-ATPase immunoreactivity did not express RhCG, identifiesthat B-type intercalated cells do not express RhCG. We alsoobserved that some CCD had many pendrin-positive cells, whereasother CCD had few, even in the same kidney. The explanationfor this observation is not clear.
Co-Localization of RhCG with AQP-2
The observation that RhCG is not expressed by H+-ATPase–negativecells in the CCD, OMCD, and IMCD suggests that the principalcell and the IMCD cell do not express RhCG. To confirm this,we performed co-localization of RhCG with AQP-2, a principalcell and IMCD cell marker. AQP-2–positive cells in theCNT expressed RhCG (Figure 3G), whereas AQP-2–positivecells in the CCD, OMCD, and IMCD did not (Figure 3, H and I).In the CNT, both principal cells and nonprincipal cells expressedRhCG. In contrast, CCD, OMCD principal cells, and IMCD cellsdid not express RhCG.
RhCG Expression in Renal Carcinomas
Because of the high level of RhCG expression in the kidney,we examined whether RhCG might be expressed in renal tumors.Both chromophobe renal cell carcinoma specimens (n = 2) andrenal oncocytoma (n = 2) specimens expressed plasma membraneRhCG immunoreactivity (Figure 4, A and B). Apparent tubule-likeprofiles occasionally were evident in renal oncocytomas in whichall cells expressed both apical and basolateral RhCG immunoreactivity(Figure 4C). Tubular profiles were not observed in chromophoberenal cell carcinomas. RhCG immunoreactivity intensity in bothchromophobe renal cell carcinoma and renal oncocytoma was lessintense than in RhCG-positive cells in adjacent portions ofuninvolved renal parenchyma.
Figure 4. RhCG expression in renal carcinomas. (A) Rhcg expression in chromophobe renal cell carcinoma and plasma membrane RhCG immunoreactivity in this pleomorphic tumor. No immunoreactivity was seen in tumor sections that were stained with an unrelated antibody. (B and C) Rhcg immunoreactivity in human oncocytoma. Occasional tubular profiles (*) demonstrated RhCG immunoreactivity in all cells, consistent with origin from collecting duct A-type intercalated cells (C), and expressed both apical and basolateral Rhcg immunoreactivity. RhCG immunoreactivity in both chromophobe renal cell carcinoma and in oncocytoma was less intense than in adjacent normal renal parenchyma (data not shown). (D and E) Immunohistochemical examination of RhCG immunoreactivity in papillary renal cell carcinoma (D) and clear cell renal cell carcinoma (E). No detectable RhCG immunoreactivity was observed in either tumor type. RhCG immunoreactivity in adjacent uninvolved renal parenchyma (arrow, D) was normal.
No detectable RhCG immunoreactivity was observed in either papillarycell carcinoma (n = 4) or clear cell renal cell carcinoma (n= 5). RhCG immunoreactivity was easily visible in adjacent normalrenal structures, thereby providing internal validation thatconfirms efficacy of the labeling procedure in these studies(Figure 4, D and E). Thus, RhCG is expressed by chromophoberenal cell carcinoma and renal oncocytoma but not by eitherrenal cell or clear cell renal cell carcinomas.
These studies provide the first examination of the expressionof the nonerythroid ammonia transporter family member, RhCG,in the normal and neoplastic human kidney. RhCG is present inthe DCT, CNT, and ICT and throughout the collecting duct, andit exhibits both apical and basolateral expression. In the cortex,CNT cells, A-type intercalated cells, and non-A, non-B cellsbut not B-type intercalated cells express RhCG. In the CCD,OMCD, and IMCD, only A-type intercalated cells express RhCG;there was no detectable expression by either principal cells,B-type intercalated cells, or IMCD cells. This expression patternhas important differences from both the mouse and the rat kidneyand has important implications for the mechanisms of ammoniasecretion by the human kidney. Finally, chromophobe renal cellcarcinoma and renal oncocytomas, but not papillary renal cellcarcinoma or clear cell renal cell carcinomas express RhCG.
The first major finding in this study is that RhCG is expressedin the human kidney in distal nephron sites involved in ammoniasecretion. Ammonia is produced in the proximal tubule throughglutamine metabolism, reabsorbed in the thick ascending limbof the loop of Henle, and then secreted by the collecting duct(1–3). Under normal conditions, the collecting duct secretesapproximately 70 to 80% of total urinary ammonia, and collectingduct ammonia secretion is substantially stimulated in responseto chronic metabolic acidosis (1,4). Moreover, RhCG expressionis stimulated by chronic metabolic acidosis, at least in theOMCD and the IMCD, consistent with RhCG’s mediating acritical role in ammonia secretion (24). The finding that thehuman kidney expresses RhCG in distal nephron sites that areinvolved in ammonia secretion is consistent with RhCG’scontributing to renal ammonia metabolism.
The observation that RhCG is expressed in collecting duct intercalatedcells supports existing hypotheses regarding ammonia secretion.In animal models, luminal acidification, including the generationof a luminal disequilibrium pH, results from apical H+ secretionby H+-ATPase and H+-K+-ATPase and is both necessary for andregulates transepithelial ammonia secretion (31–36). Collectingduct ammonia transport seems to involve both diffusive and transporter-mediatedcomponents (26,37), and the transporter-mediated component ofapical transport is stimulated by luminal H+ (37). This maybe relevant to maximizing the efficiency of ammonia secretion.H+ secretion in the absence of luminal carbonic anhydrase activitygenerates a luminal disequilibrium pH. This accelerates ammoniasecretion to a greater degree than is explainable by the meanluminal pH (33,35,38). Because intercalated cells are the primarymechanism of collecting duct H+ secretion, the luminal disequilibriumpH is greatest in the region adjacent to the intercalated cellapical membrane. The close proximity of the greatest degreeof luminal acidification with apical RhCG may increase apicalmembrane ammonia transport. Thus, the concomitant expressionof both apical H+ and ammonia transport mechanisms in the samecells may enable synergistic H+ and ammonia secretion.
An important new observation is that the cellular expressionof RhCG in the human kidney differs from that observed in ratand the mouse kidney. In the rat and mouse kidneys, principalcells in the CCD and OMCD express Rhcg (19,21,24), and principalcell Rhcg expression, at least in the OMCD, is stimulated bychronic metabolic acidosis (25). This contrasts with observationsin the human kidney (our study) in which no detectable RhCGimmunoreactivity was observed in these cells. This could reflecteither fundamental differences in functional role of the CCDand OMCD principal cell in the rat and mouse as compared withthe human kidney or RhCG expression in the human principal cellsbelow the level of detectability. It is important to note thatthe H+-K+-ATPase isoforms HK2a and HK2c have been identifiedin rat and rabbit principal cells (39,40), that rabbit outermedullary collecting duct in the inner stripe (OMCDi) principalcells possess multiple physiologically regulated apical H+ transportmechanisms (41,42), and that chronic metabolic acidosis increasesrat OMCDi principal cell RhCG expression (25). Thus, in therat, mouse, and rabbit kidneys, principal cells seem to contributeto transporter-mediated transepithelial H+ and ammonia secretion.It is possible that differences in principal cell RhCG immunoreactivitybetween human and rodent kidneys could reflect principal cellscontributing to acid-base and ammonia metabolism in the rodentkidney but not in the human kidney.
A second important observation is that human renal RhCG expressionis predominantly basolateral, with a lesser degree of apicalimmunoreactivity. In the mouse kidney, only apical immunoreactivityhas been observed (18,19). In the rat, one study found onlyapical RhCG immunoreactivity (18), whereas we observed, usingboth light and immunogold electron microscopy, that rat collectingduct cells express both apical and basolateral plasma membraneRhCG (24,25) and that basolateral plasma membrane Rhcg expressionin the OMCDi exceeds apical plasma membrane expression underbasal conditions (25). Chronic metabolic acidosis increasesrat basolateral plasma membrane Rhcg expression, suggestingthat basolateral Rhcg mediates a physiologically relevant rolein increased ammonia secretion in physiologically relevant conditions(25). Therefore, it is possible that basolateral RhCG in thehuman kidney contributes to basolateral ammonia uptake and,thereby, to transepithelial ammonia secretion.
Differences in RhCG expression in the B-type intercalated celland the non-A, non-B cell may provide important insights intothe physiologic role of these cell types. The B-type intercalatedcell generally is believed to mediate important roles in bothbicarbonate secretion in response to metabolic alkalosis andchloride reabsorption in response to chloride depletion and/ormineralocorticoid stimulation (30,43–45). The absenceof RhCG in this cell type is consistent with the lack of necessityfor transcellular ammonia secretion in any of these conditions.In contrast, the non-A, non-B cell expresses apical H+-ATPasein conjunction with apical pendrin and apical RhCG, suggestingthat this cell type may contribute to both proton secretionand ammonia secretion and to regulation of Cl– homeostasis.
Another observation from these studies is that RhCG is expressedby chromophobe renal cell carcinoma and renal oncocytomas butnot by either papillary renal cell carcinoma or clear cell renalcell carcinomas. Renal oncocytomas are benign neoplasms witha granular eosinophilic cytoplasm that contains numerous mitochondria(46). Chromophobe renal cell carcinomas are low-grade malignantneoplasms with a mixture of eosinophilic cells and cells withclear cytoplasm and numerous microvesicles and variable numbersof mitochondria (46,47). Each tumor accounts for up to 5% ofrenal neoplasms (48,49). Despite their different neoplasticpotential, both express carbonic anhydrase II (CA II) and AE1and lack immunoreactivity for high molecular weight cytokeratin,vimentin, CD10, and peanut lectin (50–53). RhCG expressionby both of these tumors, as demonstrated in our study, in combinationwith previous evidence of CA II and AE1 expression, stronglysuggests that they derive from collecting duct A-type intercalatedcells. Because DCT and CNT cells, which express RhCG, do notexpress CA II or AE1, chromophobe renal cell carcinomas andrenal oncocytomas are unlikely to originate from either DCTor CNT cells. Because differentiating chromophobe renal cellcarcinoma from renal cell carcinoma using standard histologiccriteria sometimes is difficult, examining RhCG immunoreactivitymay be helpful in selected cases. The lack of RhCG immunoreactivityin clear cell renal cell carcinoma and papillary renal cellcarcinoma is consistent with previous studies, suggesting thatthese tumors likely derive from proximal tubule cells (54,55).
Although RhCG originally was cloned as a tumor-related cDNA(56), RhCG immunoreactivity in chromophobe renal cell carcinomaand renal oncocytoma was less intense than in adjacent uninvolvedrenal parenchyma. Decreased RhCG expression has been observedin other epithelial cancers (57). Therefore, decreased, notincreased, RhCG overexpression may be characteristic of humantumors. It is interesting that the level of RhCG downregulationmay have prognostic significance in the response of some tumorsto treatment (58). Whether similar correlations between RhCGexpression and treatment response will be possible for chromophoberenal cell carcinoma and renal oncocytoma is unknown at present.
It is important to recognize that RhCG has not been shown definitivelyto contribute in vivo to ammonia transport. Extensive in vitroevidence shows that all mammalian Rh glycoproteins, RhAG/Rhag,RhBG/Rhbg, and RhCG/Rhcg, can transport ammonia (8–16,59)and that collecting duct ammonia transport exhibits characteristicsthat are consistent with transport by Rh glycoproteins (26,37).However, disruption of the Rhbg gene does not detectably alterbasal or acidosis-stimulated renal ammonia metabolism (60).Therefore, either Rhbg is not involved in renal ammonia transport,or compensatory mechanisms exist in its absence. An alternativerole for Rh glycoproteins is CO2 transport, particularly inview of evidence in the green algae, Chlamydomonas reinhardtii,that the Rh glycoprotein Rh1 may be involved in CO2 transport(61,62).
Another potential limitation of these studies is that they usetissue from kidneys with coexisting carcinoma. Because patientswith renal carcinomas generally have normal acid-base balanceand normal renal function, at least before surgical nephrectomy,systemic pH alterations or impaired GFR are unlikely to havealtered RhCG expression in the tissues studied. It is possiblebut unlikely that there might be alterations at the molecularor cellular level of uninvolved kidney that predisposed to thedevelopment of renal carcinoma that could alter RhCG expression.Against this possibility, however, is that different abnormalitiesprobably are present in differing renal tumors and the similarityof RhCG expression that we observed in kidneys with multipledifferent types of renal carcinomas.
Our study identifies several important features of RhCG expressionin the normal and neoplastic human kidney. Similar to the mouseand rat kidney, human renal RhCG is found in the DCT, CNT, ICT,and the collecting duct, sites that are responsible for secretingthe majority of renal ammonia secretion. In contrast to themouse and rat, however, in the collecting duct, RhCG is foundonly in intercalated cells that express apical H+-ATPase, theA-type intercalated cell, and the non-A, non-B cell, suggestingthat coordinated proton and ammonia transport and generationof a luminal disequilibrium pH are critically important fortransepithelial ammonia secretion. RhCG is not found in theB-type intercalated cell, consistent with this cell type havinga primary role in the human kidney in bicarbonate excretionand chloride reabsorption and not in urine acidification orammonia secretion. Finally, both chromophobe renal cell carcinomaand renal oncocytoma but not clear cell renal cell carcinomaor papillary renal cell carcinomas express RhCG, consistentwith the former two but not the latter two originating fromcollecting duct intercalated cells.
Acknowledgments
These studies were supported by funds from the National Institutesof Health (DK45788 and NS47624 to I.D.W.), the Department ofVeterans Affairs Merit Review Program (to I.D.W.), and the KoreaResearch Foundation (grant KRF-2005-003-E00006 to K.-H.H.) andby an International Society of Nephrology Fellowship Award (toH.Y.K.).
Portions of this work were published in abstract form previously(J Am Soc Nephrol 16: 344A, 2005).
We thank Gina Cowsert for secretarial assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Hamm LL, Simon EE: Roles and mechanisms of urinary buffer excretion.
Am J Physiol 253
: F595
–F605, 1987[Medline]
DuBose TD Jr, Good DW, Hamm LL, Wall SM: Ammonium transport in the kidney: New physiological concepts and their clinical implications.
J Am Soc Nephrol 1
: 1193
–1203, 1991[Abstract]
Good DW, Knepper MA: Ammonia transport in the mammalian kidney.
Am J Physiol 248
: F459
–F471, 1985[Medline]
Sajo IM, Goldstein MB, Sonnenberg H, Stinebaugh BJ, Wilson DR, Halperin ML: Sites of ammonia addition to tubular fluid in rats with chronic metabolic acidosis.
Kidney Int 20
: 353
–358, 1982[CrossRef]
Nakhoul NL, Hamm LL: Non-erythroid Rh glycoproteins: A putative new family of mammalian ammonium transporters.
Pflugers Arch 447
: 807
–812, 2004[CrossRef][Medline]
Weiner ID: The Rh gene family and renal ammonium transport.
Curr Opin Nephrol Hypertens 13
: 533
–540, 2004[Medline]
Weiner ID, Verlander JW: Renal and hepatic expression of the ammonium transporter proteins, Rh B glycoprotein and Rh C glycoprotein.
Acta Physiol Scand 179
: 331
–338, 2003[CrossRef][Medline]
Marini AM, Matassi G, Raynal V, Andre B, Cartron JP, Cherif-Zahar B: The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast.
Nat Genet 26
: 341
–344, 2000[CrossRef][Medline]
Ripoche P, Bertrand O, Gane P, Birkenmeier C, Colin Y, Cartron JP: Human Rhesus-associated glycoprotein mediates facilitated transport of NH3 into red blood cells.
Proc Natl Acad Sci U S A 101
: 17222
–17227, 2004[Abstract/Free Full Text]
Westhoff CM, Siegel DL, Burd CG, Foskett JK: Mechanism of genetic complementation of ammonium transport in yeast by human erythrocyte Rh-associated glycoprotein (RhAG).
J Biol Chem 279
: 17443
–17448, 2004[Abstract/Free Full Text]
Westhoff CM, Ferreri-Jacobia M, Mak DD, Foskett JK: Identification of the erythrocyte Rh-blood group glycoprotein as a mammalian ammonium transporter.
J Biol Chem 277
: 12499
–12502, 2002[Abstract/Free Full Text]
Mak DD, Dang B, Weiner ID, Foskett JK, Westhoff CM: Characterization of transport by the kidney Rh glycoproteins, RhBG and RhCG.
Am J Physiol Renal Physiol 290
: F297
–F305, 2006[Abstract/Free Full Text]
Zidi-Yahiaoui N, Mouro-Chanteloup I, D’Ambrosio AM, Lopez C, Gane P, Le van Kim C, Cartron JP, Colin Y, Ripoche P: Human Rhesus B and Rhesus C glycoproteins: Properties of facilitated ammonium transport in recombinant kidney cells.
Biochem J 391
: 33
–40, 2005[CrossRef][Medline]
Bakouh N, Benjelloun F, Hulin P, Brouillard F, Edelman A, Cherif-Zahar B, Planelles G: NH3 is involved in the NH4+ transport induced by the functional expression of the human Rh C glycoprotein.
J Biol Chem 279
: 15975
–15983, 2004[Abstract/Free Full Text]
Nakhoul NL, DeJong H, Abdulnour-Nakhoul SM, Boulpaep EL, Hering-Smith K, Hamm LL: Characteristics of renal Rhbg as an NH4+ transporter.
Am J Physiol Renal Physiol 288
: F170
–F181, 2004[Medline]
Ludewig U: Electroneutral ammonium transport by basolateral Rhesus B glycoprotein.
J Physiol 559
: 751
–759, 2004[Abstract/Free Full Text]
Handlogten ME, Hong SP, Zhang L, Vander AW, Steinbaum ML, Campbell-Thompson M, Weiner ID: Expression of the ammonia transporter proteins, Rh B glycoprotein and Rh C glycoprotein, in the intestinal tract.
Am J Physiol Gastrointest Liver Physiol 288
: G1036
–G1047, 2005[Abstract/Free Full Text]
Quentin F, Eladari D, Cheval L, Lopez C, Goossens D, Colin Y, Cartron JP, Paillard M, Chambrey R: RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.
J Am Soc Nephrol 14
: 545
–554, 2003[Abstract/Free Full Text]
Verlander JW, Miller RT, Frank AE, Royaux IE, Kim YH, Weiner ID: Localization of the ammonium transporter proteins, Rh B Glycoprotein and Rh C glycoprotein, in the mouse kidney.
Am J Physiol Renal Physiol 284
: F323
–F337, 2003[Abstract/Free Full Text]
Weiner ID, Miller RT, Verlander JW: Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein in the mouse liver.
Gastroenterology 124
: 1432
–1440, 2003[CrossRef][Medline]
Eladari D, Cheval L, Quentin F, Bertrand O, Mouro I, Cherif-Zahar B, Cartron JP, Paillard M, Doucet A, Chambrey R: Expression of RhCG, a new putative NH3/NH4+ transporter, along the rat nephron.
J Am Soc Nephrol 13
: 1999
–2008, 2002[Abstract/Free Full Text]
Liu Z, Peng J, Mo R, Hui CC, Huang CH: Rh type B glycoprotein is a new member of the Rh superfamily and a putative ammonia transporter in mammals.
J Biol Chem 276
: 1424
–1433, 2001[Abstract/Free Full Text]
Liu Z, Chen Y, Mo R, Hui Cc, Cheng JF, Mohandas N, Huang CH: Characterization of human RhCG and mouse Rhcg as novel nonerythroid Rh glycoprotein homologues predominantly expressed in kidney and testis.
J Biol Chem 275
: 25641
–25651, 2000[Abstract/Free Full Text]
Seshadri RM, Klein JD, Kozlowski S, Sands JM, Kim YH, Handlogten ME, Verlander JW, Weiner ID: Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis.
Am J Physiol Renal Physiol 290
: F397
–F408, 2006[Abstract/Free Full Text]
Seshadri RM, Klein JD, Smith T, Sands JM, Handlogten ME, Verlander JW, Weiner ID: Changes in the subcellular distribution of the ammonia transporter Rhcg, in response to chronic metabolic acidosis.
Am J Physiol Renal Physiol 290
: F1443
–F1452, 2006[Abstract/Free Full Text]
Handlogten ME, Hong SP, Westhoff CM, Weiner ID: Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3).
Am J Physiol Renal Physiol 287
: F628
–F638, 2004[Abstract/Free Full Text]
Biner HL, Arpin-Bott MP, Loffing J, Wang X, Knepper M, Hebert SC, Kaissling B: Human cortical distal nephron: Distribution of electrolyte and water transport pathways.
J Am Soc Nephrol 13
: 836
–847, 2002[Abstract/Free Full Text]
Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, Verlander JW: Localization of pendrin in mouse kidney.
Am J Physiol Renal Physiol 284
: F229
–F241, 2003[Abstract/Free Full Text]
Kim YH, Kwon TH, Frische S, Kim J, Tisher CC, Madsen KM, Nielsen S: Immunohistochemical and immunoelectron microscopic localization of pendrin in intercalated cell subtypes in rat and mouse kidney.
Am J Physiol Renal Physiol 283
: F744
–F754, 2002[Abstract/Free Full Text]
Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, Green ED: Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion.
Proc Natl Acad Sci U S A 98
: 4221
–4226, 2001[Abstract/Free Full Text]
Flessner MF, Knepper MA: Ammonium transport in collecting ducts.
Miner Electrolyte Metab 16
: 299
–307, 1990[Medline]
Star RA, Burg MB, Knepper MA: Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct.
Am J Physiol 252
: F1148
–F1157, 1987[Medline]
Star RA, Kurtz I, Mejia R, Burg MB, Knepper MA: Disequilibrium pH and ammonia transport in isolated perfused cortical collecting ducts.
Am J Physiol 253
: F1232
–F1242, 1987[Medline]
Hamm LL, Trigg D, Martin D, Gillespie C, Buerkert J: Transport of ammonia in the rabbit cortical collecting tubule.
J Clin Invest 75
: 478
–485, 1985[Medline]
Knepper MA, Good DW, Burg MB: Ammonia and bicarbonate transport by rat cortical collecting ducts perfused in vitro.
Am J Physiol 249
: F870
–F877, 1985[Medline]
Knepper MA, Good DW, Burg MB: Mechanism of ammonia secretion by cortical collecting ducts of rabbits.
Am J Physiol 247
: F729
–F738, 1984[Medline]
Handlogten ME, Hong SP, Westhoff CM, Weiner ID: Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3).
Am J Physiol Renal Physiol 289
: F347
–F358, 2005[Abstract/Free Full Text]
Flessner MF, Wall SM, Knepper MA: Ammonium and bicarbonate transport in rat outer medullary collecting ducts.
Am J Physiol 262
: F1
–F7, 1992[Medline]
Verlander JW, Moudy RM, Campbell WG, Cain BD, Wingo CS: Immunohistochemical localization of H-K-ATPase alpha(2c)-subunit in rabbit kidney.
Am J Physiol Renal Physiol 281
: F357
–F365, 2001[Abstract/Free Full Text]
Ahn KY, Kone BC: Expression and cellular localization of mRNA encoding the "gastric" isoform of the H+-K+ subunit alpha subunit in rat kidney.
Am J Physiol 268
: F99
–F109, 1995[Medline]
Weiner ID, Frank AE, Wingo CS: Apical proton secretion by the inner stripe of the outer medullary collecting duct.
Am J Physiol Renal Physiol 276
: F606
–F613, 1999[Abstract/Free Full Text]
Weiner ID, Wingo CS, Hamm LL: Regulation of intracellular pH in two cell populations of the inner stripe of the rabbit outer medullary collecting duct.
Am J Physiol 265
: F406
–F415, 1993[Medline]
Quentin F, Chambrey R, Trinh-Trang-Tan MM, Fysekidis M, Cambillau M, Paillard M, Aronson PS, Eladari D: The Cl-/HCO3- exchanger pendrin in the rat kidney is regulated in response to chronic alterations in chloride balance.
Am J Physiol Renal Physiol 287
: F1179
–F1188, 2004[Abstract/Free Full Text]
Wall SM: Recent advances in our understanding of intercalated cells.
Curr Opin Nephrol Hypertens 14
: 480
–484, 2005[Medline]
Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, Verlander JW: NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: Role in Cl- conservation.
Hypertension 44
: 982
–987, 2004[Abstract/Free Full Text]
Krishnan B, Truong LD: Renal epithelial neoplasms: The diagnostic implications of electron microscopic study in 55 cases.
Hum Pathol 33
: 68
–79, 2002[CrossRef][Medline]
Cheville JC, Lohse CM, Zincke H, Weaver AL, Blute ML: Comparisons of outcome and prognostic features among histologic subtypes of renal cell carcinoma.
Am J Surg Pathol 27
: 612
–624, 2003[CrossRef][Medline]
Alanen KA, Ekfors TO, Lipasti JA, Nurmi MJ: Renal oncocytoma: The incidence of 18 surgical and 12 autopsy cases.
Histopathology 8
: 731
–737, 1984[Medline]
Bonsib SM, Bromley C: Immunocytochemical analysis of band 3 protein in renal cell carcinoma, nephroblastoma, and oncocytoma.
Arch Pathol Lab Med 118
: 702
–704, 1994[Medline]
Kollert-Jons A, Wagner S, Hubner S, Appelhans H, Drenckhahn D: Anion exchanger 1 in human kidney and oncocytoma differs from erythroid AE1 in its NH2 terminus.
Am J Physiol 265
: F813
–F821, 1993[Medline]
Storkel S, Steart PV, Drenckhahn D, Thoenes W: The human chromophobe cell renal carcinoma: Its probable relation to intercalated cells of the collecting duct.
Virchows Arch B Cell Pathol Incl Mol Pathol 56
: 237
–245, 1989[Medline]
Storkel S, Pannen B, Thoenes W, Steart PV, Wagner S, Drenckhahn D: Intercalated cells as a probable source for the development of renal oncocytoma.
Virchows Arch B Cell Pathol Incl Mol Pathol 56
: 185
–189, 1988[Medline]
Chen B, Xu Z, Xu X, Cai Y, Han Y, Wang J, Xia S, Hu H, Wei F, Wu M, Wang M: RhCG is downregulated in oesophageal squamous cell carcinomas, but expressed in multiple squamous epithelia.
Eur J Cancer 38
: 1927
, 2002[CrossRef][Medline]
Luthra R, Wu TT, Luthra MG, Izzo J, Lopez-Alvarez E, Zhang L, Bailey J, Lee JH, Bresalier R, Rashid A, Swisher SG, Ajani JA: Gene expression profiling of localized esophageal carcinomas: Association with pathologic response to preoperative chemoradiation.
J Clin Oncol 24
: 259
–267, 2006[Abstract/Free Full Text]
Benjelloun F, Bakouh N, Fritsch J, Hulin P, Lipecka J, Edelman A, Planelles G, Thomas SR, Cherif-Zahar B: Expression of the human erythroid Rh glycoprotein (RhAG) enhances both NH3 and NH4+ transport in HeLa cells.
Pflugers Arch 450
: 155
–167, 2005[CrossRef][Medline]
Chambrey R, Goossens D, Bourgeois S, Picard N, Bloch-Faure M, Leviel F, Geoffroy V, Cambillau M, Colin Y, Paillard M, Houillier P, Cartron JP, Eladari D: Genetic ablation of Rhbg in mouse does not impair renal ammonium excretion.
Am J Physiol Renal Physiol 289
: F1281
–F1290, 2005[Abstract/Free Full Text]
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 U S A 101
: 7787
–7792, 2004[Abstract/Free Full Text]
Soupene E, King N, Feild E, Liu P, Niyogi KK, Huang CH, Kustu S: Rhesus expression in a green alga is regulated by CO(2).
Proc Natl Acad Sci U S A 99
: 7769
–7773, 2002[Abstract/Free Full Text]
Related Article
This Month’s Highlights
J. Am. Soc. Nephrol. 2006 17: 2635-2636.
[Full Text][PDF]
This article has been cited by other articles:
H.-Y. Kim, C. Baylis, J. W. Verlander, K.-H. Han, S. Reungjui, M. E. Handlogten, and I. D. Weiner Effect of reduced renal mass on renal ammonia transporter family, Rh C glycoprotein and Rh B glycoprotein, expression
Am J Physiol Renal Physiol,
October 1, 2007;
293(4):
F1238 - F1247.
[Abstract][Full Text][PDF]
K.-H. Han, H.-Y. Kim, B. P. Croker, S. Reungjui, S.-Y. Lee, J. Kim, M. E. Handlogten, C. A. Adin, and I. D. Weiner Effects of ischemia-reperfusion injury on renal ammonia metabolism and the collecting duct
Am J Physiol Renal Physiol,
October 1, 2007;
293(4):
F1342 - F1354.
[Abstract][Full Text][PDF]
,
Top
Abstract
Introduction
2a and HK
