Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
Address correspondence to: Dr. Walter F. Boron, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8026. Phone: 203-785-4070; Fax: 203-785-4951; E-mail: walter.boron{at}yale.edu
One of the major tasks of the renal proximal tubule is to secreteacid into the tubule lumen, thereby reabsorbing approximately80% of the filtered HCO3– as well as generating new HCO3–for regulating blood pH. This review summarizes the cellularand molecular events that underlie four major processes in HCO3–reabsorption. The first is CO2 entry across the apical membrane,which in large part occurs via a gas channel (aquaporin 1) andacidifies the cell. The second process is apical H+ secretionvia Na-H exchange and H+ pumping, processes that can be studiedusing the NH4+ prepulse technique. The third process is thebasolateral exit of HCO3–via the electrogenic Na/HCO3co-transporter, which is the subject of at least 10 mutationsthat cause severe proximal renal tubule acidosis in humans.The final process is the regulation of overall HCO3– reabsorptionby CO2 and HCO3– sensors at the basolateral membrane.Together, these processes ensure that the proximal tubule respondsappropriately to acute acid-base disturbances and thereby contributesto the regulation of blood pH.
At the outset, allow me to acknowledge my mentors, without whomI would never have been in the position to summarize my HomerW. Smith Award Lecture. One limb of my scientific family treeis decidedly nonrenal: I did my Ph.D. research and 1 yr of postdoctoralwork with Albert Roos (a respiratory physiologist) in the Departmentof Physiology and Biophysics at Washington University in St.Louis. There, I learned to think critically and to begin tounderstand acid-base physiology. At the same time, Paul De Weer(a biophysicist/neurophysiologist) took me under his wing duringsummers in Woods Hole. There, I became acquainted with the squidgiant axon and the concept of transient changes in voltage andpH. Also at Woods Hole, I was befriended by John M. Russell.In our collaborations, I learned to think about links betweenHCO3– fluxes on the one hand and Na+ and Cl– fluxeson the other.
The other limb of my scientific family tree is of course quiterenal. I studied for two years as a fellow with Emile Boulpaepin the Department of Physiology (now, of course, Cellular andMolecular Physiology). There, I learned to think about kidneysand to perfuse renal tubules. Emile (1986 Homer W. Smith Award)was trained by Gerhard Giebisch (1971 Homer W. Smith Award),who in turn was trained by Robert Pitts (1964 Homer W. SmithAward), who in turn was trained by Homer W. Smith himself. Smithwas trained by Walter B. Cannon, who originated the conceptof homeostasis. In fact, while with Cannon, Smith studied—ofall things—intracellular pH (pHi). I suppose that withsuch an auspicious list forebears, I had no choice but to studyacid-base homeostasis. My group is interested in how the body,by controlling plasma [HCO3–] and PCO2, regulates bloodpH. One focus is the proximal tubule (PT), which, along withthe rest of the kidney, is largely responsible for controllingplasma [HCO3–]. A parallel focus is the regulation ofpHi by central nervous system neurons, which regulate ventilation,which in turn controls plasma PCO2. Along the way, we stumbledacross the concept of gas channels, which seem to be importantfor both respiration and acid-base homeostasis. In this review,I summarize some renal aspects of our work.
The PT is responsible for reabsorbing approximately 80% of thefiltered HCO3–. The PT also creates the "new HCO3–"that neutralizes the mineral acids that are generated by metabolism.The thick ascending limb reabsorbs another 10% of the filteredHCO3–, and the distal nephron another approximately 10%,so virtually no HCO3– is left in the final urine. Regardlessof whether the PT is reabsorbing HCO3– or creating newHCO3–, the fundamental mechanism (Figure 1) is the same(1–3): PT cells use the cytosolic enzyme carbonic anhydraseII (CA II) to convert CO2 and H2O to H+ and HCO3– (4).Mutations in CA II produce a proximal renal tubule acidosis(pRTA). The cells secrete the H+ into the tubule lumen, viathe apical Na-H exchanger NHE3 (5–8) and V-type H+ pumps(9,10). The cells move the HCO3– into the interstitialfluid and ultimately the blood, mainly via the renal splicevariant of the electrogenic Na/HCO3 co-transporter NBCe1-A (11,12).
Figure 1. Model of acid-base transport in the proximal tubule (PT). The PT reabsorbs HCO3– by using active-transport processes to secrete H+ into the tubule lumen and titrating HCO3– to CO2 and H2O. Thus, HCO3– reabsorption requires CO2 uptake across the apical membrane. Once inside the cell, CO2 and H2O recombine to regenerate HCO3–, which exits across the basolateral membrane. NHE3, Na-H exchanger 3; AQP1, aquaporin 1; CA II and CA IV, carbonic anhydrases II and IV; NBCe1-A, electrogenic Na/HCO3 co-transporter 1, splice variant A.
Most of the H+ that the PT secretes into the lumen titratesfiltered HCO3–, forming CO2 and H2O under the influenceof CA IV (4), which is tethered to the apical membrane. TheH2O crosses the apical membrane almost exclusively via the waterchannel aquaporin 1 (AQP1) (13), and this same protein—nowacting like a gas channel—seems to mediate the majorityof CO2 transport as well. The net effect of the processes thatwe have summarized is NaHCO3 reabsorption.
A small fraction of the H+ that the PT secretes into the lumentitrates a variety of luminal buffers (e.g., NH3, inorganicphosphate, creatinine), which we measure as NH4+ excretion andthe formation of titratable acid (14). The tiny amount of HCO3–that, in parallel, moves into the blood is the "new HCO3–."
In this review, for the sake of simplicity, I refer to PT transepithelialacid-base transport as "HCO3– reabsorption" (JHCO3). Infact, I mean the sum of JHCO3 and the creation of new HCO3–.We will see that these processes are under the powerful controlof a system that we are only beginning to understand, one thatacutely monitors plasma [CO2] and [HCO3–] but not plasmapH.
Effect of CO2 on pHi
Since the work of Jacobs on Symphytum flower petals (the pigmentof which is a pH indicator) in 1920 (15), it has been appreciatedthat CO2 can rapidly cross cell membranes and acidify the cytoplasm(reviewed in reference [16]). Thomas (17) and Boron and De Weer(18), both working with pH-sensitive microelectrodes, did thefirst work with physiologic levels of CO2 on animal cells. Bothstudies confirmed that CO2 does indeed cause a rapid fall inpHi (Figure 2A), as shown for the squid axon by the initialpart of the record in Figure 2B. If CO2 simply equilibratedacross the cell membrane, as shown in Figure 2A, then pHi wouldhave fallen—by an amount determined by the initial pHi,[CO2], and intracellular buffering power (16,19,20)—andstabilized. However, pHi recovers from this acute acid loadvia a mechanism that requires the input of energy. The experimentin Figure 2B was the first to demonstrate such a pHi recovery,that is, dynamic pHi regulation. We now know that a Na+-drivenCl-HCO3 exchanger (discussed below) is responsible for thispHi recovery—a metabolic compensation (i.e., HCO3–uptake) to a respiratory acidosis (i.e., CO2 influx). The withdrawalof CO2 causes pHi to rise substantially above its initial level,this overshoot being a direct reflection of the preceding metaboliccompensation. Subsequent work by scores of authors on countlesscell types, including the PT and other renal cells, shows thatthe response of virtually all cells to CO2/HCO3– is somevariation on the theme first demonstrated with the squid giantaxon (for review, see reference [21]).
Figure 2. Recovery of pHi from a CO2-induced acid load. (A) Model of CO2 equilibration across the cell membrane. Either the extrusion of H+ or the uptake of HCO3– would require energy. (B) Experimental record from a squid giant axon. The axon was cannulated at either end, and a glass, pH-sensitive microelectrode was inserted from one end, and a KCl-filled microelectrode was inserted from the other. pHi, intracellular pH; Vm, membrane potential (in mV). Data from reference (18).
Membranes with Negligible Gas Permeability
One of the core dogmas of physiology has been that all gasesrapidly cross all membranes by dissolving in the membrane lipid.In the case of CO2, the gas produces the effects that are shownin Figure 2B. For example, perfusing the lumen of a PT withCO2/HCO3– causes pHi to fall rapidly. Thus, when perfusingsingle gastric glands as in Figure 3A (22), using the techniquethat was introduced originally by Burg et al. (23), we weresurprised by the parietal cell data in Figure 3B (and chiefcell data; data not shown), which demonstrate that adding CO2/HCO3–to the luminal perfusate causes no detectable pHi change, eventhough far lower [CO2] levels—when presented to the basolateralsurface—evoke easily detectable pHi decreases (24). Theperfusate with 100% CO2 maintained its acidity along the entirelength of the lumen, ruling out the possibility that the CO2had totally escaped in the first few milliseconds of its journey.Furthermore, luminal CO2/HCO3– failed to alter pHi overa wide range of [CO2]/[HCO3–] ratios, ruling out the possibilitythat the separate influxes of CO2 and HCO3– might haveprecisely compensated for one another and yielded a null pHishift. These were the first membranes shown to have negligiblegas permeability. We also found that lowering luminal pH to1 failed to alter pHi (22).
Figure 3. Effect of luminal versus basolateral CO2/HCO3– on pHi in a parietal cell of an isolated, perfused gastric gland. (A) Perfusion of an isolated gastric gland. One pipette was used to bore a hole through the blind end of the gland. (B) Experimental record from a parietal cell. Unless otherwise indicated, the solutions were buffered with HEPES and contained no CO2 or HCO3–. pHi was measured with the fluorescent dye BCECF and a digital imaging system. Data from reference (24).
We similarly observed null pHi shifts, rather than the expectedpHi increase, when perfusing lumens of gastric gland (24) orcolonic crypts (25) with NH3/NH4+. Kikeri et al. (26), whenperfusing the lumen of a mouse medullary thick ascending limbwith NH3/NH4+, previously observed a substantial and paradoxicpHi decline, which proves that the pHi effects of the NH4+ influxoverwhelmed those (if any) of an NH3 influx. In fact, it wouldbe interesting to see whether the apical membranes of the medullarythick ascending limb, like those of the gastric gland, indeedare impermeable to CO2 and NH3.
AQP1: A Bifunctional Water/Gas Channel
In a seminar at the University of Pennsylvania, in which I presentedthe data in Figure 3B, I suggested that the unknown specializationof gastric-gland apical membranes that allows them to resistluminal pH values below 1 also renders these membranes impermeableto CO2 and NH3. Paul De Weer asked me whether I had consideredthe possibility that the difference between apical and basolateralgastric-gland membranes might be that the basolateral membranespossess "gas channels" that the apical membranes lack. At firstblush, I thought that the gas-channel suggestion was outlandish.De Weer soon forgot his comment, but I did not. After returninghome, I began to imagine where gas channels might exist, ifthey existed at all. As luck would have it, Peter Agre, aftera seminar at Yale, had generously sent us the cDNA encodingAQP1—discovered in red blood cells (RBC)—so thatwe might confirm the observation (27) that the water channelAQP1 is not permeable to H+ (see also reference [28]). The combinationof De Weer’s comment and Agre’s seminar provokedus to think about why RBCs—which transport gas, not water,for a living—should be such a rich reservoir of AQP1.Although there is no doubt that AQP1 is a water channel, mightits physiologic role in RBCs be as a gas channel?
To test this hypothesis, Nazih Nakhoul injected cRNA encodinghuman AQP1 (or water as a control) into Xenopus oocytes, whichhe later injected with CA II protein. The purpose of the CAII was to catalyze the intracellular reaction CO2 + H2O HCO3–+ H+ and thereby keep [CO2] low near the inner surface of thecell membrane, maximizing the CO2 influx. Nakhoul found thatwhen he exposed oocytes to CO2/HCO3–, those that expressedAQP1 exhibited a CO2-induced fall in pHi that was 40% fasterthan the control cells (29). This was the first evidence thatthe presence of a protein could enhance the movement of a dissolvedgas across a cell membrane.
Gordon Cooper extended Nakhoul’s observations in experimentssuch as those shown in Figure 4. An important difference inCooper’s experiments is that, rather than inject CA II,he enhanced CO2 influx by removing the oocyte’s vitellinemembrane (30). Focusing on the purple record in Figure 4, wesee that switching the extracellular solution from one bufferedwith HEPES to one buffered with 1.5% CO2/10 mM HCO3– causedpHi to fall slowly, at a rate of –9.6 x 10–4 pHunits/s. When Cooper subsequently transferred the oocyte todeionized water, osmosis caused it to lyse in 180 s. This oocytehad a relatively low level of AQP1 expression. The oocyte thatis represented by the orange record had a much higher rate ofacidification and a more rapid lysis. Finally, the oocyte thatis represented by the green record had an even higher acidificationrate and an even quicker lysis. These results showed that CO2entry paralleled AQP1 expression. Cooper additionally demonstratedthat p-chloromercuribenzene sulfonate (pCMBS), an organic mercurialthat blocks AQP1’s water permeability, also eliminatesthe statistical difference between AQP1 and control oocytes.Finally, Cooper found that the C189S mutant, which Preston etal. (31) showed to be mercury insensitive in terms of waterpermeability, also is pCMBS insensitive in terms of CO2 permeability.
Figure 4. Effect of AQP1 expression on the rate of CO2-induced acidification in Xenopus oocytes. The three records come from experiments on three different Xenopus oocytes, with vitelline membranes removed. pHi was measured with a liquid-membrane pH microelectrode in conjunction with a KCl-filled microelectrode. During the indicated period, the CO2/HCO3–-free HEPES solution was replaced with 1.5% CO2/10 mM HCO3– at a fixed pH of 7.50. The lysis time refers to the length of time required for the oocyte to begin to ooze when placed in deionized water. pHi/t is the slope of the CO2-induced acidification (pH units/s). Data from reference (30).
Prasad et al. (32) confirmed that AQP1 enhances CO2 permeability,studying purified AQP1 protein that was reconstituted into vesiclethat was made from Escherichia coli membranes. Yang et al. (33)published experiments that seem to disprove the hypothesis thatAQP1 acts as a conduit for CO2. However, as discussed by Cooperet al. (34), these experiments were designed in a way that precludeddetection of any enhancement of CO2 permeability by AQP1.
Using a mass-spectroscopy technique to measure the permeabilityof RBC to 12C18O16O, Forster et al. (35) in 1998 made the intriguingobservation that 4,4'-diisothiocyanatostilbene-2,2'-disulfonate(DIDS)—which, among other things, blocks the Cl-HCO3 exchangerAE1—greatly reduces CO2 permeability. In 2003, Blank andEhmke (36), using the fluorescent dye BCECF to monitor pH inRBC ghosts, showed that HgCl2 (presumably by blocking AQP1)and DIDS (presumably by blocking AE1) greatly reduced CO2 permeability.Also in 2003, Uehlein et al. (37) demonstrated that a homologueof AQP1 enhances CO2 permeability in plants, enhancing photosynthesis(the rate-limiting step for which is the availability of CO2)and the growth of leaves. More recently, Gros et al. (38), usingthe mass-spectroscopy approach with normal and AQP1-null humanRBCs, found that AQP1 is responsible for approximately 60% ofthe cells’ CO2 permeability. An unidentified RBC protein,possibly the Rh complex or AE1, is responsible for an additionalapproximately 30%. It is interesting that parallel experimentson oocytes showed that DIDS blocks approximately 50% of theCO2 flux through AQP1.
A final example of a physiologic role for AQP1 as a gas channelis provided by Zhou et al. (39), who worked with PT from wtversus AQP1-null mice (generously provided by Alan Verkman).Zhou et al. found that lack of AQP1 leads to a substantial deficitin HCO3– reabsorption, as would be predicted by the modelin Figure 1. He also performed another set of experiments thatrely on a novel rapid-mixing technique that was developed byZhao et al. (40) for creating out-of-equilibrium (OOE) CO2/HCO3–solutions. We describe this approach in more detail below. WhenZhou perfused the PT lumen with a CO2/HCO3–-free solutionand exposed the basolateral surface to a "pure" HCO3–solution (i.e., one with a physiologic [HCO3–] and pHbut virtually no CO2), it made no difference whether the PTwas from a wt or AQP1-null mouse: The backflux of "carbon" wasthe same. However, when he exposed the basolateral surface toa "pure" CO2 solution (i.e., one with a physiologic [CO2] andpH but virtually no HCO3–), the backflux, compared withwt tubules, was 60% lower in AQP1-null than in wild-type tubules.These data are consistent with the hypothesis that the CO2 permeabilityof AQP1 plays a major role in the reabsorption of HCO3–.
A lingering issue has been whether it is reasonable to expecta dissolved gas such as CO2 to pass through AQP1. Recent moleculardynamics simulations by Wang et al. (41) suggested that CO2could pass (1) through each of the four aquapores of an AQP1tetramer, single file with water and (2) through the centralpore formed by the four monomers. This central pore seems tobe a vacuum through which CO2 and O2 can move with great speed.
The NH4+ Prepulse
From his original work on the membrane-permeability propertiesof Spirogyra in the late 19th century, Overton understood thatNH3 in a solution that contains NH4+ (NH3 + H+ NH4+)—aswell as various neutral amines (R-NH2 + H+ R-NH3+) in solutionsthat contain their charged ammonium ions—crosses cellmembranes predominantly in their uncharged form. Working withRhododendron flower petals (the pigment of which shifts fromred to blue in response to alkalinity) and starfish eggs thatcontain neutral red, Jacobs (42) in 1922 recognized that theinflux of NH3—even in acidic solutions of NH4Cl—causesa rise in pH (Figure 5A). Many decades later, Thomas (17) useda pH-sensitive microelectrode to monitor the rise in pHi thatwas caused by exposing a snail neuron to a solution that containedNH4Cl. The left side of Figure 5B shows an experiment from 1976in which Boron and De Weer (18) exposed a squid giant axon fora relatively brief period to a solution that contained 10 mMNH4Cl. Although the NH3 in the solution was present at verylow levels compared with the NH4+, the influx of this NH3 neverthelessled to a consumption of cytoplasmic H+ and thus an increasein pHi. Removing the NH4Cl caused the pHi changes to reverse.However, pHi always fell to a value slightly below the initialone.
Figure 5. Effect of NH3/NH4+ on pHi, the NH4+ prepulse. (A) Model of NH3 equilibration across the cell membrane. (B) Experimental record from a squid giant axon. The axon was cannulated at either end, and a glass, pH-sensitive microelectrode was inserted from one end, and a KCl-filled microelectrode was inserted from the other. Data from reference (18).
The explanation for the pHi undershoot in the first part ofthe experiment becomes clear with a more protracted exposureto NH4Cl. The rapid initial NH3-induced pHi increase was followedby a much slower but sustained pHi decrease, which reflectsthe influx of the weak acid NH4+. Subsequently removing theNH4Cl, after this long exposure, caused pHi to undershoot thepre-NH4Cl value by an exaggerated amount. The magnitude of thisundershoot reflects the degree of NH4+ influx during the previousNH4Cl exposure. This was the first example of what has cometo be known as the "ammonium prepulse" technique for acid loadingcells. In fact, the axon will use its pHi-regulatory machineryto recover from the intracellular acid load in Figure 5B butonly in the presence of CO2/HCO3–, as Boron and De Weer(43) subsequently demonstrated. This later work was the firstpractical use of an NH4+ prepulse as a tool to acid load a celland the first demonstration that cells can use HCO3– uptaketo neutralize an acid load and thereby regulate pHi.
Apical Na-H Exchange
One of the first vertebrate cells to be the focus of a studythat exploited the NH4+ prepulse technique was the PT of thesalamander. Figure 6 shows the results of an experiment by Boronand Boulpaep (44), performed on an isolated, perfused tubule,cells of which were impaled with microelectrodes for monitoringpHi and basolateral membrane potential (basolateral Vm). Atfirst, both the lumen and the "bath" (i.e., the basolateralsolution) contained a Na+-free solution to block Na+-dependentprocesses for regulating pHi. In addition, the bath contained4-acetamido, 4'-isothiocyanato-2,2'-stilbene disulfonate (SITS;an analog of DIDS) to block the electrogenic Na/HCO3 co-transporteron the basolateral membrane. Applying NH4+ caused a rapid risein pHi, followed by a slower fall. Washing away the NH4+ causedpHi to fall substantially and then recover only very slightly.Subsequently returning 100 mM Na+ to the lumen caused pHi toreturn to its initial value. Other experiments showed that thispHi recovery is inhibited by amiloride, demonstrating that itwas an apical Na-H exchanger that was responsible for the pHirecovery. Murer et al. (45), in their studies of brush bordermembrane vesicles, had been the first to observe Na-H exchange.The groups of Aronson (46) and Saktor (47) also made seminalcontributions to our understanding of Na-H exchange by workingwith brush border membrane vesicles. The experiment shown Figure 6was the first demonstration of apical Na-H exchange by a livingepithelial cell.
Figure 6. Use of an NH4+ prepulse to acid-load an isolated, perfused salamander PT cell. pHi was measured with a recessed-tip glass pH microelectrode (17), in conjunction with a KCl-filled microelectrode. The tubule was exposed throughout the experiment, at both the luminal and the basolateral sides, to HEPES-buffered solution that lacked CO2/HCO3–. During the indicated time, the tubule was exposed from both the luminal and the basolateral (i.e., "bath") surfaces to a solution in which 20 mM NH4+ replaced 20 mM Na+, an NH4+ prepulse designed to acid-load the cell. Extracellular [Na+] was either 0 or 100 mM, as indicated. Vm, basolateral membrane potential; SITS, 4-acetamido, 4'-isothiocyanato-2,2'-stilbene disulfonate. Data from reference (44).
Initial Description
In the late 1970s and early 1980s, the mechanism of HCO3–exit across the PT basolateral membrane was a subject of considerableinterest (reviewed in references [2,3]). One suggestion wasthat HCO3– exited via Cl-HCO3 exchange, although the dominantview seems to have been that HCO3– exited via a conductivepathway, the simplest explanation for which would be a HCO3–channel. While exploring these options in an isolated, perfusedsalamander PT, Boulpaep and Boron (48) found that lowering basolateral[HCO3–] ([HCO3–]B) at a fixed [CO2]B causes pHito fall rapidly—an observation that was consistent withboth models. In addition, they found that this maneuver causesbasolateral Vm to shift rapidly in the positive direction, consistentwith the HCO3–-conductance model. However, when they removedbasolateral Na+, they made a startling observation (Figure 7):Although pHi fell as expected (e.g., reversing the basolateralNa-H exchanger would have caused an internal acidification),the basolateral Vm shifted rapidly in the positive direction.Lowering the concentration of an extracellular cation shouldhave caused basolateral Vm to shift in the negative direction.The observation that lowering [HCO3–]B and removing basolateralNa+ each caused basolateral Vm to shift in the positive direction—togetherwith other data—pointed to a new kind of transporter,an electrogenic Na/HCO3 co-transporter that moves more negativecharge as HCO3– (or CO3=) ions than positive charge asNa+. Like the previously discovered Cl-HCO3 exchanger of RBCsand the Na+-driven Cl-HCO3 exchanger of invertebrates, the Na/HCO3co-transporter is blocked by the disulfonic stilbenes SITS andDIDS. However, in contrast to the other two transporters, theelectrogenic Na/HCO3 co-transporter is independent of Cl–.Later work by Soleimani and Aronson on basolateral membranevesicles from rabbit renal cortex pointed to a Na+:HCO3–stoichiometry of 1:3 (49), which ensures that the transporternormally mediates the exit of Na+ and HCO3– across thebasolateral membrane—the basolateral step of HCO3–reabsorption. Later work by Frömter and his colleagues(50–52) showed that the electrogenic Na/HCO3 co-transporteris the dominant basolateral pathway for HCO3– in the S1and S2 segments of the PT, where the vast majority of the HCO3–reabsorption occurs, but that a basolateral Cl-HCO3 exchangeralso is important in the S3 segment.
Figure 7. The effect of basolateral Na+ removal in an isolated, perfused salamander PT, the electrogenic Na/HCO3 co-transporter. pHi was measured with a recessed-tip glass pH microelectrode (17), in conjunction with a KCl-filled microelectrode. The tubule was exposed throughout the experiment, at both the luminal and the basolateral sides, to a solution buffered with 1.5% CO2/10 mM HCO3–. During the indicated times, basolateral Na+ was replaced with an organic cation. Dotted red lines indicate expected Vm response in the absence of an electrogenic Na/HCO3 co-transporter. Data from reference (48).
Cloning
The absence of a naturally abundant and highly enriched sourceof the electrogenic Na/HCO3 co-transporter—unlike theCl-HCO3 exchanger AE1, which is highly expressed in RBCs—frustratedearly attempts to obtain the cDNA that encodes the electrogenicNa/HCO3 co-transporter. Michael Romero joined our laboratoryin 1992 with the goal of expression-cloning the transporterin Xenopus oocytes. Matthias Hediger in the laboratory of ErnestWright had perfected this approach in cloning the Na/glucosetransporter (53–55). Later, as an independent investigator,Hediger had already expression-cloned a wide range of membraneproteins, including a glutamate transporter (56), a subunitof heteromeric amino-acid transporters (57), a urea transporter(58), and an H-driven peptide transporter (59). We were mostfortunate that Hediger was interested in collaborating withRomero.
The first step in expression cloning is to inject size-selectedmRNA from a tissue source (rabbit kidneys in our case) intoXenopus oocytes and then assay for expression of the desiredfunction. Unfortunately, repeated attempts with rabbit mRNAwere fruitless. Before giving up, we decided to inject mRNAfrom salamander kidneys, reasoning that amphibian oocytes mightbetter express an amphibian mRNA. To our delight, this approachwas successful. Another key element in this study was our assay.We chose to remove extracellular Na+ in the presence of CO2/HCO3–and then look for a small, positive shift in Vm that is characteristicof the co-transporter (Figure 7), rather than the usual negativeshift. With Hediger, Romero generated a cDNA library from thesalamander mRNA and methodically searched through ever-smallergroups of clones to arrive at a single clone that can serveas the template for cRNA that encodes the protein that we namedNBC, for Na+ bicarbonate co-transporter (60).
We anticipated that NBC could be genetically related to anyof several known transporters, including the Na/glutamate transporters(subsequently grouped as part of the SLC1 family of solute-linkedcarriers [61]), the Cl-HCO3 exchangers AE1 through 3 (now partof the SLC4 family [11]), the Na/monocarboxylate co-transporters(which turned out to be part of the SLC5 family that includesthe Na/glucose co-transporters [62]), the Na/bile-salt transporters(SLC10 [63]), the cation-coupled Cl– co-transporters suchas the Na/Cl co-transporter (SLC12 family [64]), the Na/carboxylateco-transporters (SLC13 [65]), and the Na/phosphate co-transporters(SLC17 and SLC34 [66,67]). Upon sequencing the cDNA clone thatencodes NBC, we were surprised to learn that the deduced amino-acidsequence of salamander NBC is approximately 30% identical tothat for the three anion exchangers AE1 though AE3, which groupclosely in the dendrogram in Figure 8 (blue region) and alsoare known as SLC4A1 through SLC4A3. The cloning of the originalNBC (also now known as SLC4A4) led to the discovery of severalother Na+-coupled members of the SLC4 family, which group togetherin the gray region of Figure 8.
Figure 8. Dendrogram of the SLC4 family of HCO3– transporters. All of the transporters are members of the SLC4 family.
Burnham et al. (68) subsequently cloned human ortholog of thesalamander NBC, and Romero et al. (69) obtained the rat clone.It is interesting to note that even the pure rat clone failedto express robustly in Xenopus oocytes until subcloned intoa Xenopus expression vector that flanks both the 5' and 3' endsof the rat NBC with the corresponding untranslated regions ofthe Xenopus-globin mRNA. This result probably explains whywe and others failed in our attempts to expression clone NBCe1using mammalian mRNA. Bernhard Schmitt raised the first antibodiesto the electrogenic NBC, and he and his colleagues demonstratedthat the protein is expressed heavily at the basolateral membranesof the S1 and likely the early S2 segments of the renal PT (70).
Other Members of the SLC4 Family
With the discovery of at least one electroneutral NBC—theclone was identified by Pushkin et al. (71), and the encodedprotein was characterized as an electroneutral NBC by Choi etal. (72)—we appended the suffix "e" to designate electrogenicand "n" to designate electroneutral. Moreover, Virkki et al.(73) and Sassani et al. (74) demonstrated that one of four cDNAclones that previously were isolated by Pushkin et al. (75,76)actually encodes a second electrogenic NBC. Thus, the original,renal electrogenic NBC is known as NBCe1-A. A second splicevariant of the same gene—identified by Abuladze et al.(77) in pancreas and by Choi et al. (78) in heart—is knownas NBCe1-B. This splice variant seems to be the most widelyexpressed NBCe1 variant. A brain-specific splice variant thatwas identified by Bevensee et al. (79) is known as NBCe1-C.The second electrogenic NBC gene is NBCe2, and the electroneutralNBC is NBCn1.
In addition to NBCn1, at least two other electroneutral Na+-coupledHCO3– transporters exist. NDCBE, cloned and characterizedby Grichtchenko et al. (80), is a Na+-driven Cl-HCO3 exchangerthat is expressed heavily in brain but also in other tissues,including kidney. NCBE, cloned by Wang et al. (81), is electroneutral,but whether it transports Cl– still is controversial.Form follows function among mammalian transporters Na+-coupledHCO3– transporters, with the electrogenic NBC groupingtogether in the peach-colored subregion in the dendrogram inFigure 8 and the electroneutral Na+-coupled HCO3– transportersgrouping together in the green subregion.
Two members of the SLC4 family have deduced amino acid sequencesthat lie apart from the rest. AE4 originally was named as aCl-HCO3 exchanger (82). However, its function is unsettled,and key areas of its amino acid sequence are more reminiscentof a Na+-coupled HCO3– transporter. BTR1, originally clonedby Parker et al. (83), seems to be a Na+-coupled borate (i.e.,boron!) transporter (84), renamed NaBC1. Human mutations inNaBC1 cause a corneal condition that is known as congenitalhereditary endothelial dystrophy (85).
Structure Figure 9 is a model of the topology of NBCe1-A, based on studiesof the Cl-HCO3 exchanger AE1 (86). All members of the familyhave a large cytoplasmic N terminus (Nt) and a much smallercytoplasmic C terminus (Ct). The crystal structure of most ofthe Nt of AE1 has been solved (87) and shows that the Nt isa dimer. We now have crystallized the Nt of NBCe1-A, and a preliminaryx-ray diffraction analysis indicates that this Nt, too, is adimer (88).
Figure 9. Hypothetical topology of NBCe1-A, based on model of AE1 by Zhu et al. (86). The red circles indicate sites of reversible 4,4'-diisothyocyanatostilbene-2,2'-disulfonate (DIDS) binding; the brown circles with "C" indicate Cys residues on the third extracellular loop that are conserved among all Na+-coupled HCO3– transporters; the blue circles indicate actual N-glycosylation sites; the checkered circle indicates an unused consensus glycosylation site; and the green circles indicate the positions of known human mutations.
Various approaches (e.g., see references [89,90]) generallyhave yielded similar conclusions about the amino acid residuesthat contribute to transmembrane segments 1 through 5 (TM1 throughTM5). Therefore, investigators feel reasonably confident aboutthese assignments. However, investigators are far less comfortableabout the topology of the remainder of the molecule becausethe data sometimes are conflicting, presumably because the laterTMs are more flexible.
One characteristic of the Na+-coupled HCO3– transportersis that they all have a long extracellular loop between TM5and TM6 (11). Moreover, this loop is the site of consensus glycosylationsites—proved in the case of NBCe1-A (91). Another curiosityis the four heavily conserved cysteine residues in these loopsbut not those of other SLC4 family members. (It is interestingthat AE4 has these for Cys residues.) Work on AE1 shows thatlysine residues near the end of TM5 and TM12 can bind covalentlythe inhibitor DIDS. Preliminary work from our laboratory (92)indicates that these same regions are important for the reversible,noncovalent binding of DIDS.
Abuladze et al. (93) have mutated a large number of chargedresidues that are conserved among SLC4 members and have identifiedseveral mutations that interfere with function. Choi et al.(unpublished observations) have constructed a series of chimerasbetween NBCe1-A and one splice variant of NBCn1 (NBCn1-B) inwhich they exchanged the cytoplasmic Nt domain, third extracellularloop, and/or the cytoplasmic Ct domain. They found that noneof these three domains is important for determining the electrogenicityof the transporter but that both TM1 through 5 and TM6 through13 of NBCe1 must be present for the chimera to function as anelectrogenic co-transporter.
Naturally Human Mutations of NBCe1 (SLC4A4)
Investigators thus far have identified 10 mutations (94–100)in the human SLC4A4 gene that are associated with a varietyof defects that may include severe autosomal recessive, pRTA,ocular abnormalities, growth and mental retardation, and abnormaldentition. The sites of these mutations are indicated by greencircles in Figure 9. Three of these are nonsense mutations thatlead to premature truncation of the protein, and seven are missensemutations.
Of the three missense mutations, the first is Q29X (95), numberedfor the renal splice variant NBCe1-A, which results in a truncationvery early in the cytoplasmic Nt. The second is the deletionof nucleotide 2311 (97), which presumably causes a frameshiftin codon 721 and, after 27 anomalous amino acids, a prematurestop in the extracellular loop between TM7 and TM8. The thirdmissense mutation is a 67-nucleotide deletion at the boundaryof exon 23 and the following intron (100); it would lead toa truncated cytoplasmic Ct.
Of the seven missense mutations, R298S in the cytoplasmic Nt,S427L in TM1, and R510H near the extracellular end of TM4 allseem to reduce targeting of the co-transporter to the basolateralmembrane in polarized renal epithelial cells in culture (101).All three also exhibit decreased functional expression in Xenopusoocytes, although it is not clear to what extent this effectreflects decreased protein delivery to the plasma membrane (94,96,98,101,102).
Work from our laboratory suggests that R881C most likely causesa trafficking defect with normal intrinsic function (103), andA799V seems to cause defects in both trafficking and intrinsicfunction (104). The most recently discovered NBCe1 mutationis L522P in TM4, which does not traffic to the plasma membranein oocytes (99).
Initial Hints
More than a half century ago, Brazeau and Gilman (105) and Pittsand his colleagues (106) published classical experiments demonstratingthat respiratory acidosis elicits a rapid, compensatory increasein renal H+ secretion. However, the problem of determining howthe kidney senses acid-base disturbances had been refractoryto further dissection, mainly because the equilibrium CO2 +H2O HCO3– + H+ makes it difficult to attribute effectsto changes in [CO2] or to [HCO3–] or to [H+]. We becameinterested in this problem in a very indirect way.
Figure 10 shows the arrangement of an isolated, perfused PT.Figure 11 shows two experiments in which Nakhoul et al. (107)used the absorbance spectrum of a fluorescein dye to monitorpHi in the cells of an isolated, perfused S3 segment of a rabbitPT. In Figure 11A, switching only the luminal buffer from HEPESto CO2/HCO3– caused the expected decrease in pHi: Theinflux of CO2 produced the initial pHi decline, and the effluxof HCO3– across the basolateral membrane sustained theintracellular acidification. In Figure 11B, we see that makingthe same solution change on the basolateral side has a verydifferent effect. For a few seconds, pHi declined rapidly, asit did when we added CO2/HCO3– to the lumen. However,within 5 s, pHi began a rapid increase to a value that is farhigher than the initial pHi. One might argue that the sustainedpHi increase could have been due to HCO3– uptake. However,adding basolateral CO2/HCO3– triggers a similar pHi increaseeven when CO2/HCO3– is already present in the lumen, andunder these conditions, the tubule actively reabsorbs HCO3–.That is, the net traffic of HCO3– across the basolateralmembrane is outward. Therefore, as unlikely as it seems, CO2and/or HCO3– on the basolateral side of the cell somehowmust trigger the secretion of H+ across the membrane on theapical side.
Figure 11. Effect of apical versus basolateral CO2/HCO–3 on pHi in an isolated, perfused rabbit S3 PT. (A) Effect of adding 5% CO2/25 mM HCO–3 to the lumen. (B) Effect of adding 5% CO2/25 mM HCO–3 to the basolateral side (bath). pHi was computed from the absorbance spectrum of a fluorescein dye. Except as indicated, the luminal and basolateral solutions were buffered with HEPES and contained no CO2/HCO–3. The time bar applies to both panels. Data from reference (107).
Further experiments by Chen and Boron (108,109) showed thatadding CO2/HCO3– to the bath or to the bath and the lumenmarkedly stimulated both apical Na-H exchangers and proton pumps.However, adding CO2/HCO3– to the only lumen was withouteffect. These experiments directly demonstrated that a sensorfor CO2 and/or HCO3–—located at or near the basolateralmembrane—can stimulate H+ extrusion at the opposite sideof the cell. These experiments provided another surprise: Decreasesin pHi produce only very small increases in the rates of apicalNa-H exchange and H+ pumping in a living PT cell. The real regulationof H+ transport rates comes not from intracellular protons butfrom basolateral CO2 and/or HCO3–. But which?
OOE CO2/HCO3– Solutions
In the 1993 paper by Nakhoul et al. (107) is an appendix thatanalyzes the CO2/HCO3–-induced alkalinization of Figure 11Bin the context of the reactions that are responsible for theinterconversion of CO2 and HCO3–. The first of these reactionsis very slow, and the second is extremely fast:
Before submitting the paper, I wanted to review my calculationswith Robert Berliner, who at the time was Professor Emeritusin our department. After considering the above reaction ratesfor a couple of hours—and thinking about Figure 11B—Bobwistfully looked up at the ceiling and said that it was toobad that we had to add CO2 and HCO3– together, ratherthan one at a time. In an instant—primed by our discussionof the slow reaction governed by k1 and k–1—thethought came to me, "Of course we can!" All we need to do isto take advantage of that slow reaction ... and thus were bornout-of-equilibrium (OOE) solutions.
Figure 12A shows the approach for generating a solution withphysiologic levels of HCO3– and pH but virtually no CO2("pure" HCO3–), and Figure 12B shows the comparable approachfor generating a solution with physiologic levels of CO2 andpH but virtually no HCO3– ("pure" CO2). However, implementingOOE solutions proved to be more challenging than conceivingof them. Shortly after my conversation with Bob, Jinhua Zhaoarrived as a new postdoctoral fellow. For 2 yr, she: (1) laboredwith ever more powerful syringe pumps to deliver evenly twostreams of liquid, (2) employed various tricks for adequatelymixing these two streams, (3) dealt with issues of temperatureregulation, and (4) accomodated the chemistry of CO2 and HCO3–.We tested the system on squid giant axons (40), in which wewere able to create "pure" CO2 solutions that could acidifythe cell and increase intracellular buffering power as predictedas well as "pure" HCO3– solutions that could supply thesubstrate for a HCO3– transporter but not increase intracellularbuffering power.
Figure 12. Generating out-of-equilibrium (OOE) CO2/HCO3– solutions. (A) "Pure" HCO3–. (B) "Pure" CO2. In each case, a single syringe pump is used to deliver solutions evenly from two syringes. The two solutions meet at a "T" that is filled with a nylon mesh that promotes mixing. The total flow typically is 7 ml/min. The time from mixing to delivery of the OOE solution to the tubule to the removal of the solution is approximately 200 ms. Thus, the tubule is exposed continuously to a newly generated solution.
Zhao also applied her new technology to the isolated perfusedrabbit PT (110) and then introduced Yuehan Zhou to the project.He monitored JHCO3 as well as the rate of volume absorption(JV) as he systematically used OOE solutions to vary basolateralcomposition one parameter at a time (Figure 13A). The resultsshow that raising [HCO3–]B (at a fixed [CO2]B of 5% anda fixed pHB of 7.40) causes JHCO3 to fall (Figure 13B, left)—anappropriate response for the "metabolic" part of metabolic acid-basedisturbances. That is, the greater the plasma [HCO3–],the less HCO3– the kidney ought to reabsorb. Moreover,raising [CO2]B (at a fixed [HCO3–]B of 22 mM and a fixedpHB of 7.40) causes JHCO3 to rise (Figure 13B, middle)—anappropriate compensation for the "respiratory" part of respiratoryacid-base disturbances. That is, the greater the plasma [CO2],the more HCO3– the kidney ought to reabsorb. However,we were surprised to find that raising pHB (at a fixed [HCO3–]Bof 22 mM and a fixed [CO2]B of 5%) elicits no change in JHCO3(Figure 13B, right).
Figure 13. Effect on HCO3– reabsorption of varying—one at a time—basolateral [HCO3–], [CO2], and pH. (A) Model. (B) Experimental data obtained on isolated perfused rabbit S2 PT. The triangles indicate standard equilibrated conditions (5% CO2, 22 mM HCO3–, pH 7.40). All other symbols represent OOE solutions. JHCO3, rate of HCO3– reabsorption; JV, rate of volume reabsorption; B, basolateral. Data from reference (142).
In parallel experiments, we measured pHi under the conditionsof Figure 13B and found that, indeed, pHi changes substantiallyas one increases pHB from 6.8 to 8.0. However, these changesin pHB and pHi do not evoke changes in JHCO3. Instead, at leastin terms of its acute response to acid-base disturbances, thePT regulates plasma pH not by monitoring pH but by monitoringtwo surrogates: the main buffer components of the body, HCO3–and CO2 (Figure 14). This tactic of regulating a parameter thatthe body does not measure directly is not unique to acid-baseparameters and the PT. For example, the body uses stretch receptorsin vessels to gauge the adequacy of the effective circulatingblood volume.
Figure 14. Model of the regulation of JHCO3 and JV by basolateral CO2 and HCO3– in a renal PT. The green arrows represent stimulation, and the red arrows represent inhibition. We propose that basolateral CO2 stimulates the acid-base transporters but reciprocally inhibits the transporters that are responsible for the reabsorption of other solutes. HCO3– would have the opposite effect. Glc, glucose; Lac, lactate; Gln, glutamine; JOther, rate of reabsorption of solutes other than NaHCO3.
An extremely interesting observation in the above study is thatthe changes in JHCO3 occurred without the expected parallelchanges in JV. Because the fluid that the PT reabsorbs is approximatelyisosmotic, an increase in JHCO3, for example, should have beenaccompanied by a sizable increase in JV. It seems that the PTcell compensates for changes in NaHCO3 reabsorption by makingreciprocal changes in the reabsorption of other solutes, therebykeeping JV relatively constant (Figure 14). This response wouldhelp to maintain a stable BP as the tubule responds to acid-basedisturbances.
It is worth noting that in the above experiments, we appliedthe challenges only to the basolateral surface of the tubule.Therefore, it is possible that PT have a pH sensor at the apicalmembrane. Also, the above experiments were acute: The tubuleacclimated for approximately 5 min in the test solution, andthe entire period of data collection lasted approximately 30min. Alpern and colleagues showed that cultured PT cells thatwere subjected for 2 d to either metabolic or respiratory acidosisresponded by increasing their activity of Na-H exchange, a processthat requires new protein synthesis (111). Moreover, this effect,as well as increased expression of NHE3 mRNA, is blocked byoverexpressing Csk (112), a natural inhibitor of src kinases.
Role of Tyrosine Kinases
A critical question is how the tubule is able to sense changesin [CO2]B and transduce them into altered cell function. Thebacterium Rhizobium meliloti senses O2 (113) and the plant Arabidopsisthaliana senses ethylene (114–119) using membrane proteinsthat signal through a histidine kinase. Because animal cellslack histidine kinases, we hypothesized that PT cells mightuse a tyrosine kinase to signal an increase in CO2. Indeed,as shown in Figure 15, 35 nM PD168393, which alkylates a Cysresidue in the ATP binding pocket of tyrosine kinases in theerbB family (120), eliminates the ability of the PT to respondto changes in [CO2]B (121). Similarly, 10 nM BPIQ-I, which alsotargets members of the erbB family (122), blocks the abilityof the tubule to respond to increased [CO2]B. Indeed, preliminarywork suggests that exposing tubule suspensions to CO2/HCO3–leads to the phosphorylation of erbB1 (i.e., EGF receptor) atTyr residues (123). Although we do not yet know the moleculethat senses CO2, it seems that this dissolved gas signals througha receptor tyrosine kinase, perhaps in part through erbB1.
Figure 15. Effect of tyrosine-kinase inhibitors on the CO2-induced increase in JHCO3. Data from reference (121).
Role of Angiotensin II
The hormone that has the greatest stimulatory effect on JHCO3is angiotensin II (AngII), applied at a "low" dosage to eitherthe luminal or basolateral surface (124–127). When appliedat a "high" dosage to either surface, however, AngII actuallyreduces JHCO3 (126,128).
We examined the effect low-dosage (10–11 M) and of high-dosage(10–9 M) AngII on the response of JHCO3 to alterationsin [CO2]B (129). We found that low-dosage AngII, added to eitherthe lumen or the bath, tends to shift the JHCO3-versus-[CO2]Brelationship to the left, so that lower levels of [CO2]B tendto stimulate HCO3– reabsorption. Conversely, high-dosageAngII, added to either the lumen or the bath, tends to bluntthe JHCO3-versus-[CO2]B relationship.
The PT has all of the molecular machinery to generate its ownAngII (130–136), which actually appears in the tubulelumen. It is not yet established whether the tubule secretesangiotensinogen, AngI, or AngII. However, preliminary data fromour group on Prinivil, an inhibitor of the angiotensin-convertingenzyme, suggest that the tubule actually secretes preformedAngII (137). We decided to examine the effect of AngII receptorblockers on the JHCO3-versus-[CO2]B relationship. We were surprisedto find that in the absence of added AngII, 10–8 saralasin,a peptide that blocks both AT1 and AT2 receptors, reduces JHCO3to levels that normally are observed at 0% CO2 (see Figure 13B)and renders the tubule insensitive to changes in [CO2]B between0 and 20%. The AT1-specific blocker candesartan has a similareffect. Finally, in tubules from AT1A-null mice, we find a moderatedepression of JHCO3 at 5% CO2 and a total insensitivity to changesin [CO2]B.
Endogenous luminal AngII plays at least a critical permissiverole in the tubule’s response to alterations in [CO2]B.It would be interesting to know whether basolateral CO2 somehowaccentuates the endogenous AngII system, perhaps by increasingthe secretion of AngII, increasing the density or the sensitivityof apical AT1 receptors, or enhancing downstream signaling fromthe AT1 receptor to the acid-base transporters.
Looking back over the past 60 yr of progress in understandingacid secretion by the PT, it is sobering to realize that thepioneers in the field, working with technologies that are farmore primitive than those that are at our disposal today, wereable to assemble a good picture of the fundamental processes(138–141): The tubule exchanges luminal Na+ for cytosolicH+, carbonic anhydrase catalyzes the secreted H+ to titratefiltered HCO3– to CO2 and H2O, and these substances enterthe cell and regenerate HCO3– that then moves into theblood. Today, we understand many more of the details, includingthe dynamics of electrical and chemical gradients and the molecularidentities of the transporters. We know their amino acid sequences,and we even know many examples in which specific mutations ofthese proteins lead to human disease.
These advances notwithstanding, much remains to be learned.We still do not understand how the transporters function atan atomic level or the mechanisms of disease-causing mutations.Such understanding will come eventually through advances instructural biology. However, reading between the lines of thisreview, one can sense that many mysteries remain even at thecellular and molecular levels. Just how do CO2 and other gasesmove through aquaporins and other gas channels? Which moleculesdoes the PT cell use to sniff basolateral CO2 and HCO3–?How does the cell transduce these signals to the acid-base transporters?How does the cell reciprocally regulate the reabsorption ofNaHCO3 and of other solutes so as to stabilize JV? What roledoes locally generated AngII play in these processes? How doesthe PT cell integrate information from CO2 levels, locally versussystemically generated AngII, and other hormones such as parathyroidhormone and endothelin? With continued support from the NationalInstitutes of Health and other agencies and the enthusiasm ofbright young scientists, the renal community can look forwardto the day when other winners of the Homer W. Smith Award willanswer these questions.
Acknowledgments
The preparation of this review was supported by National Institutesof Health grants DK30344 and DK17433.
I thank the National Institutes of Health for 25 yr of support.I also thank agencies such as the American Heart Associationand the National Kidney Foundation for supporting my researchand the careers of fellows in my laboratory. I thank DuncanWong for computer support and Charleen Bertolini for administrativesupport.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
HCO3– + H+ and thereby keep [CO2] low near the inner surface of the cell membrane, maximizing the CO2 influx. Nakhoul found that when he exposed oocytes to CO2/HCO3–, those that expressed AQP1 exhibited a CO2-induced fall in pHi that was 40% faster than the control cells (29). This was the first evidence that the presence of a protein could enhance the movement of a dissolved gas across a cell membrane. 
NH4+)—as well as various neutral amines (R-NH2 + H+ 
-globin mRNA. This result probably explains why we and others failed in our attempts to expression clone NBCe1 using mammalian mRNA. Bernhard Schmitt raised the first antibodies to the electrogenic NBC, and he and his colleagues demonstrated that the protein is expressed heavily at the basolateral membranes of the S1 and likely the early S2 segments of the renal PT (70). 
pHi/
