Acid-base Transport by the Renal Proximal Tubule

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AbstrAct

Each day, the kidneys filter 180 L of blood plasma, equating to some 4,300 mmol of the major blood buf-fer, bicarbonate (HCO3

). The glomerular filtrate enters the lumen of the proximal tubule (PT), and the majority of filtered HCO3

is reclaimed along the early (S1) and convoluted (S2) portions of the PT in a manner cou-pled to the secretion of H+ into the lumen. The PT also uses the secreted H+ to titrate non-HCO3

buffers in the lumen, in the process creating new HCO3

for trans-port into the blood. Thus, the PT along with more distal renal segments is largely responsible for regu-lating plasma [HCO3

]. In this review we first focus on the milestone discoveries over the past 50+ years that define the mechanism and regulation of acidbase transport by the proximal tubule. Further on in the re-view, we will summarize research still in progress from our laboratory, work that addresses the problem of how the PT is able to finely adapt to acidbase dis-turbances by rapidly sensing changes in basolateral levels of HCO3

and CO2 (but not pH), and thereby to exert tight control over the acidbase composition of the blood plasma.

Key words: Aquaporin, Na/HCO3 cotransporter, NBC, Out-of-equilibrium CO2/HCO3

solutions, Receptor pro-tein tyrosine phosphatase, RPTP

Department of Physiology and Biophysics, Case West-ern Reserve University, Cleveland, Ohio - USADepartment of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio - USADepartment of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio - USA

Lara A. Skelton, Walter F. Boron, Yuehan Zhou

Acid-base transport by the renal proximal tubule

ACId-BASE BALAnCE: BASIC ASPECTS

IntroductIon

The maintenance of a physiological ratio of the major blood-plasma buffer parameters, [HCO3

] and PCO2, is at the root of

stabilizing blood pH, as described by the Henderson-Has-selbalch equation:

pH = pK + log [HCO-3] [1]

s.PCO2

The lungs control plasma PCO2 by exhaling the CO2 pro-duced during aerobic respiration (1). The kidneys (2) regu-late plasma [HCO3

] by (i) reabsorbing the HCO3 filtered

in the glomeruli (~4,300 mmol/day) and (ii) transporting into the plasma new HCO3

that neutralizes the H+ aris-ing from sources such as the metabolic production non-volatile acids (~70 mmol/day).The renal proximal tubule (PT) is the major site of HCO3

reabsorption, reclaiming ~80% of the HCO3

filtered by the glomerulus. Nearly all of the remaining 20% is re-claimed along the distal nephron segments (3). As shown in Figure 1A, the PT cell secretes H+ from the cytosol across the microvillus apical membrane into the lumen via the Na-H exchanger (NHE), mainly NHE3 (4, 5), and the vacuolar-type H+-ATPase (6). Carbonic anhydrase IV is a membrane-associated carbonic anhydrase (CA) teth-ered by a glycosylphosphatidylinositol (GPI) anchor to the outer leaflet of the apical membrane along the tubule lumen, where it converts secreted H+ and luminal HCO3

to CO2 and H2O (7). The CO2 and H2O rapidly reenter the cell across the apical membrane, facilitated, as we will describe later, by the aquaporin 1 (AQP1) channel. In the cytosol, CO2 and H2O are converted back into HCO3

and H+ by CA II, the HCO3

exiting with Na+ across the baso-lateral membrane via the renal electrogenic Na+/HCO3

cotransporter (NBCe1-A) at a stoichiometry of 3:1 into the interstitial space and ultimately the blood (8). Other solutes (e.g., glucose, lactate, glutamine) move from the lumen into the PT cell via a variety of Na+-coupled trans-

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porters and other mechanisms, and subsequently either undergo metabolism in the PT cell or move into the blood (i.e., reabsorption). Cl and certain other solutes move from lumen to blood across tight junctions by diffusion and solvent drag. Accompanying the net movement of all of the above solutes is the osmotic movement of water, mainly through apical and basolateral AQP1 (9-11).The majority of the H+ secreted into the PT lumen titrates filtered HCO3

, the result of which is HCO3 reabsorption

(Fig. 1A). The remainder of the secreted H+ leads to the creation of new HCO3

, introduced above, because the efflux of an H+ across the apical membrane is coupled to the efflux of HCO3

across the basolateral membrane. Viewed from the perspective of the secreted H+ to which it is coupled, this new HCO3

has 3 components (Fig. 1B):(a) A tiny fraction remains free in the lumen and is there-by responsible for lowering luminal pH. The remainder titrates filtered buffers other than HCO3

, which renal physiologists have somewhat artificially divided into the following 2 classes.(b) When these buffers are anything other than NH3, we call the result formation of titratable acid. Examples of these filtered buffers are dibasic inorganic phosphate (HPO4

2), urate and creatinine. The PT (which can achieve a final luminal pH of ~6.8) is responsible for about half of the titration of HPO4

2 (pK 6.8) and a very small fraction of the titration of urate (pK 5.8) and creatinine (pK 5.0). Distal nephron segments make a far larger contribu-tion for these latter buffers.

(c) The final component of new HCO3 is coupled to am-

monium secretion. The PT is the principal site of renal ammonium synthesis (12), and the excretion of this NH4

+ is the major route for excreting H+ equivalents in the urine, in the following sequence of events. First, Na / amino acid cotransporters mediate the uptake of glutamine (Gln) across both the apical and basolateral membranes. The system B neutral amino acid transporters (SLC6A19 and SLC6A20) mediate the constitutive uptake of Gln across the apical membrane (13), whereas the system N amino acid transporter 3 (SNAT3 or SLC38A3) up-regulated by acidosis mediates Gln uptake across the basolateral membrane (13). Once inside the PT cell, the Gln enters the mitochondrion and undergoes hydrolysis via glutami-nase to form NH4

+ plus glutamate, which then undergoes oxidative deamination via glutamate dehydrogenase to produce NH4

+ plus -ketoglutarate (-KG2-). The newly formed NH4

+ dissociates to form intracellular H+ and NH3. The PT cell extrudes the H+ across the apical membrane via NHE3 and H+ pumps. The NH3 exits in parallel, prob-ably both through the membrane lipid and via AQP1 (14). Finally, the luminal H+ titrates the luminal NH3 to form NH4

+, much of which ultimately appears in the urine. The metabolism of -KG2- produces CO2 and glucose (glu-coneogenesis). In response to chronic metabolic acido-sis, the PT adaptively up-regulates ammonium synthesis to promote H+ excretion (12, 13, 15, 16).In addition to responding to chronic acidbase distur-bances, the PT also responds to acute disturbances. We

Fig. 1 - Model of acidbase metabolism by the proximal tubule (PT). A) Mechanism of HCO3 reabsorption in the early PT. The H+

pump (i.e., V-ATPase) and na-K pump are unlabeled. B) Formation of new HCO3. AQP1 = aquaporin 1; CA II and CA IV = carbon-

ic anhydrases 2 and 4; GdH = glutamate dehydrogenase; Gln = glutamine; Glu = glutamate; -KG2- = -ketoglutarate; nBCe1-A = electrogenic na/HCO3 cotransporter (1 variant A); nHE3 = na-H exchanger 3; OAA = oxaloacetate; PEP = phosphoenolpyruvate; PEPCK = phosphoenolpyruvate carboxykinase; SLC6A19 = system B neutral amino acid transporters; SnAT3 (aka SLC38A3) = system n amino acid transporter. *Chronic metabolic acidosis up-regulates nHE3, GA, GdH, PEPCK and SnAT3.

A B

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Skelton et al: Acidbase transport

will discuss these below in the section titled Regulation by acidbase parameters. To set the stage for this dis-cussion on regulation, we will first introduce the molecu-lar components of acidbase transport at the apical and basolateral sides of the PT cell.

ApIcAl H+ extrusIon

nHE3

The Na-H exchanger 3 (NHE3) is the most important H+-ex-truder along the PT apical membrane (17, 18). NHE3 utilizes the inward Na+ gradient created by the basolateral Na-K pump to exchange 1 H+ for 1 Na+, thereby extruding H+ against its electrochemical gradient (Fig. 1A). Adaptive re-sponses to chronic respiratory or metabolic acidosis involve increasing the abundance and activity of NHE3 protein in the apical membrane (19).The acute regulation of NHE3 involves the trafficking and recycling of NHE3 along the microvilli of the apical mem-brane via interaction between the C-terminal PDZ motif of NHE3 with the PDZ binding domain of the NHE regulatory factor-1 (NHERF-1). In turn NHERF-1 links NHE3 to the actin cytoskeleton through association with ezrin (20). Ezrin also serves as a low-affinity cAMP kinase (PKA) anchoring pro-tein (AKAP), enabling PKA to phosphorylate NHE3 (21). The actin-based motor myosin VI is involved in trafficking NHE3 from the tip to the base of the microvilli thereby acutely sup-pressing H+ extrusion (20). A complex combination of phos-pho-serine modifications at the NHE3 carboxy terminus be-tween amino acid residues 455 and 832 mediated both by PKA and protein kinase C (PKC) are involved in control-ling the majority of these acute regulatory stimuli. Generally, NHE3 activity is inhibited in response to hormonal or other stimuli that, via cAMP formation, enhance PKA. Conversely, NHE3 activity is stimulated in response to PKC activation, with serine modification being necessary (but not sufficient) for increased NHE3 activity (21).

Vacuolar type H+ pump or ATPase

Luminal acidification via the vacuolar-type H+-ATPase (V-ATPase) is required for a portion of the HCO3

reabsorption along the PT (Fig. 1A). This apical-membrane H+ pump is also the major H+-secreting protein in the thick ascend-ing limb (TAL) and the distal nephron, specifically in the -intercalated cells of the distal convoluted tubule (DCT), connecting tubule and the collecting duct (6). The multi-subunit V-ATPase consists of 2 major structures; an integral

membrane VO ring-like structure that mediates H+ move-

ment (subunits a-e) and a peripheral cytoplasmic V1 ATPase (subunits A-H). Mutations in the kidney-specific subunit B1 encoded by ATP6V1B1 are found in individuals with auto-somal-recessive distal renal tubular acidosis (dRTA) with deafness (type 1b). Mutations occurring in another kidney specific subunit a4, encoded by ATP6V0A4 cause dRTA with preserved hearing (22). The trafficking of the a4 subunit to the apical membrane along distal segments appears to be the major regulatory mechanism for V-ATPase activity in response to acid or NaHCO3 loading (23).

ApIcAl co2 uptAke

AQP1 discovered by Peter Agre and coworkers (24) is present in high abundance at the apical and basolateral membranes of the PT, where it plays a central role in the near-isotonic reabsorption of H2O across the PT epithe-lium (11, 25). Preliminary data suggest that AQP1 also is responsible for a major component of CO2 movement across the PT apical membrane, thereby playing a major role in HCO3

reabsorption.More than a century ago, Overton concluded that NH3 and other neutral amines compared with their positively charged conjugate weak acids (e.g., NH4

+) rapidly move through biological membranes (26). This observation and others with neutral weak acids supported Overtons hy-pothesis that the cell membrane is predominantly lipid. Others later concluded that all gases move through all membranes simply by dissolving in the membrane lipid (Overtons rule). However, in the mid-1990s, Waisbren et al demonstrated that the apical membranes of gastric pa-rietal and chief cells have no detectable permeability to either CO2 or NH3 (27) the first evidence that the century-old dogma is not entirely correct. In 1998, Nakhoul et al (28) as well as Cooper and Boron (29), showed that AQP1, overexpressed in Xenopus oocytes, serves a CO2 channel the first evidence for a gas channel. More evidence has accumulated that AQP1 behaves not only as a water channel but also as a CO2 channel (30-34).Some investigators have concluded that CO2 does not move through channels, based upon experimental or theoretical arguments (35-38). However, those conclu-sions have themselves been challenged (39, 40). It might be noted that most of the evidence regarding the gas-channel hypothesis is based on work with model systems. Thus, it would be timely to explore the gas-channel model in a physiologically relevant system. If AQP1 is really a gas channel, then as outlined in Figure 1A it ought to contribute to the diffusion of CO2 across the apical and

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basolateral membranes of PT cells. Moreover, the move-ment of CO2 through apical AQP1 ought to contribute to the reabsorption of HCO3

. Preliminary work on isolated, perfused mouse PTs (41) suggests that the knockout of AQP1 reduces maximal HCO3

reabsorption by ~50% CO2. Moreover, whereas the knockout of AQP1 has no ef-fect on the transepithelial flux of HCO3

from the basolat-eral solution (bath) to the lumen, it reduces the transepi-thelial flux of CO2 by ~60%. This work would be the first evidence that a channel plays a physiologically important role in a mammalian tissue.

bAsolAterAl Hco3 efflux

nBCe1-A (SLC4A4)

In 1983, using isolated perfused salamander PTs, Boron and Boulpaep were the first to demonstrate the existence of a Na/HCO3 cotransporter, and to show that an electrogenic Na/HCO3 cotransporter is the major route of HCO3

efflux across the PT basolateral membrane (42). This transporter is electrogenic because it mediates net exit of Na+ and more than 1 HCO3

. This cotransport is independent of Cl but blocked by stilbene derivatives such as 4-acetamido-4-isothiocyanato-2,2-stilbene disulfonate (SITS) (42).In mammalian cells, the Na+:HCO3

stoichiometry must be 1:3 to produce a net efflux of HCO3

from the PT cell. In-deed, Soleimani et al, working on basolateral membrane vesicles, measured a stoichiometry of 1:3 (8). In 1998, Romero and colleagues expression-cloned the cDNA that encodes the salamander renal electrogenic Na/HCO3 cotransporter (NBC) the first Na+-coupled HCO3

trans-porter to be cloned and found that NBC is in the same gene family as the anion exchangers AE1-AE3 (43). The original renal electrogenic NBC was eventually renamed NBCe1-A (44), following the discovery of 2 additional splice variants expressed in other tissues (45, 46) plus a second electrogenic NBC (47, 48) and 3 homologous elec-troneutral transporters (49-51). NBCe1-A is expressed at the basolateral membrane of the PT, at the highest lev-els in the S1 segment, consistent with its dominant role in mediating renal HCO3

reabsorption. Preliminary work (52) suggests that NBCe1-A, as expressed in Xenopus oocytes, actually transports carbonate (CO3

=). Mutations within the SLC4A4 gene that encodes human NBCe1-A 12 such mutations are known (53-61) produce a devas-tating phenotype that includes type 2 (proximal) RTA (44, 62) and, depending upon the mutation, defects in the eye, teeth and mental development.

AE2 (SLC4A2)

Using microelectrodes on isolated PTs, Kondo and Frmter demonstrated Cl-HCO3 exchange at the basolateral mem-brane of the S3 segment. Under physiological conditions, this transporter would couple the exit of 1 HCO3

into the interstitium to the uptake of 1 Cl (63). By semiquantitative polymerase chain reaction (PCR), Brosius et al detected AE2 in the convoluted and straight PT as well as more dis-tal segments of rat kidney (64). Thus, AE2 may account for the anion-exchange activity in the S3 segment.

cArbonIc AnHydrAses

The carbonic anhydrase enzymes effectively bypass the slow reaction in the sequence CO2 + H2O Slow H2CO3 Fast HCO3

+ H+, and thus are critical for HCO3 reabsorption

and the creation of new HCO3. Figure 1A summarizes the

disposition of CAs in the human PT.CA II is the archetypal mammalian-class CA. It is a 29-kDa cytosolic protein that is ubiquitously expressed, and is among the fastest of CAs, achieving a turnover rate for CO2 hydration of 1 106/s. Except for the thin ascending limb, CA II is present throughout the nephron, accounting for ~95% of the CA activity in the kidney (7). As illustrated in Figure 1A, cytosolic CA II plays a central role by con-verting the CO2 that enters across the apical membrane into H+ for secretion into the lumen plus HCO3

for export across the basolateral membrane. The importance of CA II is illustrated by the effect of inherited CA II deficiency, which causes mixed proximal and distal (type 3) RTA (22) accompanied by osteopetrosis (because of impaired os-teoclast function) and cerebral calcification (65).In humans, the 5% of renal CA activity not due to CA II is accounted for by 2 integral membrane proteins: CA IV and CA XII. The GPI-linked CA IV is localized both api-cally and basolaterally along the PT and TAL, and apically in -intercalated cells of the cortical collecting duct and cells of the medullary collecting duct (7). As shown in Fig-ure 1A, apical CA IV catalyses HCO3

dehydration, thereby consuming secreted H+ as well as generating membrane-permeant CO2 and H2O. Consistent with this role, CA IV has a higher HCO3

affinity and a greater HCO3 dehy-

dratase activity than CA II (66).The role of basolateral CA IV is unclear. Preliminary work suggests that CA IV minimizes the changes in surface pH caused by CO3

= transport mediated by NBCe1-A, but does not substantially change the activity of the cotrans-porter (52). Thus, CA IV may protect nearby proteins from extreme pH fluctuations caused by NBCe1-A.

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CA XII is a single-span transmembrane protein with its N terminus and catalytic domain in the extracellular space. This protein is exclusively basolateral, present in the hu-man PT but far more abundant in the TAL, DCT and prin-cipal cells (which reabsorb NaCl but not NaHCO3) of the collecting duct (67). In the TAL and DCT as suggested above for CA IV with CO3

= transport mediated by NBCe1-A CA XII might minimize pH fluctuations caused by H+ transporters.

regulAtIon of proxImAl-tubule AcId secretIon

Regulation by hormones

Angiotensin II

Angiotensin II (ANG II) is perhaps the most powerful hor-monal modulator of Na+, fluid, and HCO3

reabsorption by renal PTs. As we will see below (see the section Involve-ment of apical AT1 receptors, below), basolateral CO2 and HCO3

are equally powerful. Burg and Orloff were the first to examine the effect of ANG II on fluid reabsorption in isolated perfused PTs (68). Since then, many studies involving isolated tubules (69-74) and micropuncture (75-77) have reported that luminal or basolateral ANG II has biphasic concentration-dependent effects, increasing the fluid reabsorption rate (JV) and the HCO3

reabsorption rate (JHCO3) at low ANG II concentrations, but decreasing JV and JHCO3 at higher concentrations. ANG II acts via an-giotensin II receptors type 1 (AT1) (78, 79), which are G proteincoupled receptors (GPCRs), for both its stimula-tory and inhibitory effects (80, 81).

Dopamine

Acting via autocrine and paracrine effects, dopamine is a potent natriuretic hormone, acting in part by reducing api-cal NHE3 protein levels and thereby decreasing Na+ and volume reabsorption by the PT (82).

Endothelin

Endothelins are small peptides that induce vasoconstric-tion but also have other important physiological roles. In the renal PT, chronic metabolic acidosis increases en-dothelin-1 (ET-1) expression, enhancing its autocrine ac-tion on the apical endothelin-B receptor, which in turn stimulates NHE3 (83).

Nitric oxide

Nitric oxide (NO) is produced in renal tubular epithelium by the inducible isoform of nitric oxide synthase (iNOS). In situ microperfusion studies on mouse PTs show that the knock-out of iNOS reduces JV and JHCO3. Nevertheless, the mouse maintains normal acidbase status via unknown compensa-tory mechanisms (84).

Regulation by acidbase parameters

The 4 fundamental acidbase disturbances are metabolic alkalosis and acidosis, and respiratory alkalosis and aci-dosis defined below. Each of these disturbances can be acute or chronic. As discussed in conjunction with Equation 1, the renal or respiratory systems compen-sate for each disturbance by appropriately altering blood [HCO3

] or PCO2, thereby returning arterial pH toward nor-mal. In the process, a mixed-type acidbase disturbance develops (85). Here we will focus on the response of the PT to the 4 acute acidbase disturbances.Metabolic acidosis (i.e., a decrease in plasma [HCO3

] at a fixed PCO2, resulting in a fall in plasma pH) is compensated largely by an increase in alveolar ventilation, which lowers PCO2. If the source of the defect is extrarenal, the kidney including the PT (86, 87) will rapidly adapt by increasing H+ secretion. For example, Soleimani et al prepared brush-border or basolateral-membrane vesicles from rabbit PT suspensions preexposed for 2 hours to a pH 6.9 / 5% CO2 solution. They found that the acidosis increased both apical NHE3 and basolateral NBC activities (88).Metabolic alkalosis (i.e., an increase in plasma [HCO3

] at a fixed PCO2, causing a rise in pH) is compensated by changes opposite to those for metabolic acidosis. In isolated per-fused PTs, increases in basolateral [HCO3

] cause a fall in JHCO3 that is reversed by also raising PCO2 (89). Creating acute metabolic alkalosis in rats by infusing HCO3

causes a fall in JHCO3 as assessed by free-flow micropuncture (90).Respiratory alkalosis (i.e., a decrease in plasma PCO2, caus-ing a rise in pH) is compensated by a decrease in JHCO3. For example, Cogan found that hyperventilating a rat to reduce arterial PCO2 by ~20 mm Hg causes a marked fall in JHCO3, as determined by free-flow micropuncture (91).Respiratory acidosis (i.e., an increase in plasma PCO2, caus-ing a fall in pH) is compensated by an increase in JHCO3. More than half a century ago, a series of 3 papers demonstrated that acute respiratory acidosis in dogs rapidly stimulates re-nal acid secretion (92-94). Based on additional experiments, these authors concluded that it is most likely an increase in PCO2 rather than a decrease in pH that controls renal acid

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secretion in respiratory acidosis. In the more modern era, Chan and Giebisch, working specifically on microperfused PTs, showed that increasing basolateral (BL) pH either by lowering [CO2]BL or by increasing [HCO3

]BL causes JHCO3 to fall dramatically (95). Later work by Cogan, using free-flow micropuncture, showed that acute respiratory alkalosis in rats leads to a decrease in JHCO3, whereas acute respiratory acidosis has the opposite effect (91).In addition to the above work on acute acidbase distur-bances, others have investigated chronic effects. For exam-ple, working with cultured PT cells, Alpern and colleagues have found that chronic (1-7 days) metabolic or respiratory acidosis increases NHE3 activity in cultured cells in a pro-cess that involves activation of c-Src and autocrine signal-ing by endothelin (83, 96-99).As informative as the above studies have been, all of the maneuvers involved concomitant changes in at least 2 of the 3 key acidbase parameters: pH, [HCO3

] and PCO2. Thus, it was impossible to determine the extent to which any 1 of the 3 parameters produced the observed effects. Indeed, with classical approaches, it is impossible to change just 1 of the aforementioned 3 parameters as related in Equation 1.The conundrum of how to isolate the effects of the 3 acidbase parameters was finally addressed in 1995 by Zhao et al (100), who developed a technique for generating out-of-equilibrium (OOE) solutions to produce isolated changes in pH or [HCO3

] or [CO2], while holding the other 2 parameters constant. The technique was first applied to tubules in a se-

ries of experiments by Zhao et al (101), who introduced OOE solutions to the basolateral (BL) surface of rabbit S2 proxi-mal tubules. As described in the next 3 sections, Zhou et al (102) later monitored JHCO3 while systematically varying, one at a time, pHBL, [HCO3

]BL and [CO2]BL in isolated, perfused the rabbit proximal tubules (S2 segments).

Basolateral pH

In the first series of experiments (Fig. 2A, B), we increased pHBL from 6.8 to 8.0 while holding [HCO3

]BL at 22 mM and [CO2]BL at 5%. The middle symbol in Figure 2A (triangle) rep-resents the JHCO3 under standard, equilibrated conditions: a pHBL of 7.40, a [HCO3

]BL of 22 mM and a [CO2]BL of 5%. The triangle in Figure 2B represents the corresponding intracel-lular pH (pHi). Using OOE technology to lower pHBL to 6.8 or raise it to 8.0 always at a [HCO3

]BL of 22 mM and a [CO2]

BL of 5% produces a surprising result: no change in JHCO3 (Fig. 2A). Nevertheless, the isolated changes in pHBL pro-duce sizeable changes in pHi (Fig. 2B). Thus, the PT cell can not acutely regulate JHCO3 in response to changes in either pHBL or pHi per se.

Basolateral [HCO3]

In the second series of experiments (Fig. 2C, D), we in-creased basolateral [HCO3

] from 0 to 22 to 44 mM while holding pHBL at 7.40 and [CO2]BL at 5%. The middle symbol

Fig. 2 - Effect of isolated changes in basolateral (BL) pH and [HCO3

] on the rate of HCO3

reabsorption (JHCO3) and steady-state intracel-lular pH. A) Effect of isolated changes in pHBL, obtained us-ing out-of-equilibrium solutions, on JHCO3. B) Effect of isolated changes in pHBL on steady-state pHi. C) Effect of isolated changes in [HCO3

]BL on JHCO3. d) Effect of isolated changes in [HCO3

]BL on steady-state pHi. All experi-ments were performed at 37C. data from (102); reproduced with permission in accordance with terms of original publication, Copyright 2005 by the national Academy of Sciences.

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in Figure 2C represents the JHCO3 under the same standard, equilibrated conditions as in Figure 2A. As shown in Figure 2C, we found that graded increases in [HCO3

]BL cause a fall in JHCO3. As summarized in Figure 2D, the isolated increase of [HCO3

]BL from 0 to 22 mM also causes a substantial in-crease in pHi. In summary, isolated increases in [HCO3

]BL produce the appropriate compensatory response for whole-body acidbase balance: a fall in JHCO3.

Basolateral [CO2 ]

In the final series of these experiments (Fig. 3A, B), we in-creased [CO2]BL from 0 to 20% while holding pHBL at 7.40 and [HCO3

]BL at either 22 mM (solid symbols) or 0 mM (open symbols). The triangle in Figure 3A represents the same standard, equilibrated conditions as in Figure 2A and C. Fig-ure 3A shows that isolated increases in [CO2]BL from 0% to 20% cause graded increases in JHCO3. As shown in Figure

Fig. 3 - Effect of isolated changes in basolateral (BL) [CO2] on the rate of HCO3

reabsorption (JHCO3) and steady-state intra-cellular pH. A) Effect of isolated changes in [CO2]BL, obtained using out-of-equilibrium solutions on JHCO3. B) Effect of iso-lated changes in [CO2]BL on steady-state pHi. All experiments were performed at 37C. data represented by solid symbols from ref. (102); reproduced with permission in accordance with terms of original publication, Copyright 2005 by the national Academy of Sciences. data represented by open symbols are new.

3B, the graded increases in [CO2]BL also cause substantial decreases in pHi. The data obtained at a fixed [HCO3

]BL of 0 mM (open symbols) are similar, but translated along the y-axis. The 2 JHCO3 plots in Figure 3A show that isolated in-creases in [CO2]BL produce the appropriate compensatory response for whole-body acidbase balance: a rise in JHCO3. A comparison of the 2 JHCO3 plots in Figure 3A shows that a reduction in [HCO3

]BL causes an upward shift of the plot, consistent with the data in Figure 2C (compare data at 22 vs. 0 mM).Although not shown in Figure 2 or 3, none of the isolated changes in pHBL, [HCO3

]BL or [CO2]BL produced statistically significant effects on JV. Thus, the appropriate adjustments of JHCO3 to acidbase disturbances do not perturb fluid re-absorption in vitro, and thus presumably would not perturb blood pressure in vivo.In the 1980s, Al-Awqati and colleagues showed that in-creases in PCO2 lead to the insertion of H

+ pumps into apical membranes in turtle bladder (103) as well as PTs and corti-cal collecting tubules from rabbit (104). It is possible that the same fundamental processes are at work in both the Al-Awqati experiments and the OOE experiments in Figure 3. Al-Awqati and collaborators suggested that CO2 might act by lowering pHi and thereby modulating [Ca

2+]i. Indeed, changes in [Ca2+]i appear to be involved in the OOE ex-periments. Bouyer et al found that adding equilibrated 5% CO2 / 22 mM HCO3

(pH 7.40) has no effect on [Ca2+]i when added to the PT lumen but causes a slowly developing and sustained increase when added to the bath (105). More-over, switching the bath from a CO2/HCO3

-free solution to an OOE solution containing 22 mM HCO3

/pH 7.40, but virtually no CO2 (pure HCO3

) causes no change in [Ca2+]i, whereas switching the bath from a CO2/HCO3

-free solution to an OOE solution containing 5% CO2/pH 7.40 but virtu-ally no HCO3

(pure CO2) produces the same rise in [Ca2+]i

as does the equilibrated CO2/HCO3 solution. Thus, it is the

CO2 that causes [Ca2+]i to rise.

The first portion of Al-Awqatis hypothesis that a fall in pHi is the trigger for the insertion of H+ pumps appears not to be true in the OOE experiments. In Figure 4A, we replot the JHCO3 data from the previous 2 figures as a function of pHi. It is clear that pHi is not the unique determinant of JHCO3. In fact, the large changes in pHi produced by isolated changes in pHBL (diamonds) elicit no change in JHCO3. In retrospect, it is perhaps not surprising that pHi is not the unique deter-minant of JHCO3 because, from the perspective of pHi reg-ulation, one might have expected the H+ extruders at the apical membrane to have the opposite pHi dependency of the electrogenic Na/HCO3 cotransporter at the basolateral membrane. A further problem with using pHi as the unique

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signal for regulating pHBL is that different acidbase distur-bances can generate the same pHBL but different pHi values. In other words, the pHi signal is degenerate (i.e., knowl-edge of pHi cannot inform the cell about the status of pHBL or the identity of the acidbase disturbance). Furthermore, if pHi were the critical signal, the dynamics of pHi regula-tion (e.g., the recovery of pHi from an acute intracellular acid load; see (106-108)) would make JHCO3 quite unstable. The PT cell faced with the dilemma of selfishly regulating its own pHi while yet regulating blood pH as part of its raison dtre seems to have evolved the only way it could have: uncoupling pHi regulation from JHCO3.Figure 4B is similar to Figure 4A but a comparable plot with pHBL on the abscissa, shows that pHBL is also not the unique determinant of JHCO3 (see the vertical spread of JHCO3 data at pHBL = 7.4).Figure 4C is yet a third replot of the JHCO3 data in Figures

2 and 3, but this time as a function of the ratio [CO2]BL/[HCO3

]BL. Note the vertical spread of JHCO3 data at [CO2]BL/[HCO3

]BL = . Figure 4D is similar, but is a plot of JHCO3 versus the inverse ratio, [HCO3

]BL/[CO2]BL. Here, note the vertical spread of JHCO3 data at [HCO3

]BL/[CO2]BL = 0. Thus, neither of these ratio parameters is a unique determinant of JHCO3. In fact, the Henderson-Hasselbalch equation tells us that the ratio on the abscissa of Figure 4D is simply 10(pHBLpK). In other words, the abscissas in Figure 4B-D are merely transformations of one another. The PT cell clearly responds to isolated changes in [HCO3

]BL (Fig. 2C) and [CO2]BL (Fig. 3A). However, when faced with simultaneous changes in [HCO3

]BL and [CO2]BL, the cell evidently inte-grates this information in such a way that the [HCO3

]BL in-fluences the response to [CO2]BL as we saw in Figure 3A and vice versa. One possibility is that HCO3

competes with CO2 for binding to a common receptor.

Fig. 4 - Replots of data from Figures 2 and 3. A) dependence of JHCO3 on pHi for various out-of-equilibrium (OOE) protocols. Here we plot the JHCO3 data from Figure 2A versus the pHi data from Figure 2B, and do the same for Figure 2C versus Figure 2d, and for Figure 3A versus Figure 3B, respecting the symbols and colors in Figures 2 and 3. data represented by solid symbols from ref. (102); reproduced with permission in accordance with terms of original publication, Copyright 2005 by the national Academy of Sciences. B) dependence of JHCO3 on pHBL for various OOE protocols. As in panel A, we again replot the data from Figures 2 and 3, but here as a function of pHBL. C) dependence of JHCO3 on the [CO2]BL/[HCO3

]BL ratio for various OOE protocols. As in panels A and B, we replot the data from Figures 2 and 3, but here as a function of the ratio [CO2]BL/[HCO3

]BL. d) depen-dence of JHCO3 on the [HCO3

]BL/[CO2]BL ratio for various OOE protocols. As in panel C, we replot the data from Figures 2 and 3, but here as a function of the ratio [HCO3

]BL/[CO2]BL. none of the 4 parameters on the x-axes can uniquely predict JHCO3. BL = basolateral; JHCO3 = HCO3

reabsorption rate.

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Involvement of EGFR

In the wake of the above work with OOE solutions (102), a critical question is, how does the PT sense acute changes of [CO2]BL and/or [HCO3

]BL and transduce the signal(s) in the PT cell to regulate bicarbonate reabsorption? In studying the literature on gas-sensing by other organisms, Patrice Bouyer (then in our group) learned that Gilles-Gonzales et al (109) had demonstrated that the bacterium Sinorhizobium meliloti (formerly Rhizobium meliloti) sens-es low O2 levels using a 2-component system compris-ing the regulatory proteins, FixL and FixJ. Low O2 levels activate the His-kinase activity of the heme protein FixL, which activates FixJ, which in turn activates genes en-coding enzymes for nitrogen fixation (see (110)). In 1993, Chang et al found that the ability of the plant Arabidopsis to respond to ethylene, which acts as a hormone (111), depends on the protein ETR1 (112), the C-terminal portion of which is remarkably similar to both FixL and FixJ of the bacterial 2-component system. Because the mammalian cells do not have histidine kinases, Bouyer hypothesized that the CO2-sensing mechanism of renal PTs requires a receptor tyrosine kinase (RTK) or a receptor-associated (i.e., soluble) tyrosine kinase (sTK) that would interact with a membrane-bound CO2 sensor.Zhou and Bouyer began to test a variety of tyrosine-kinase

Fig. 5 - Model of CO2/HCO3 sensing and signal transduction

in the proximal tubule. The transporters are identified in the legend to Figure 1. AnG II = angiotensin II; AT1 = angiotensin receptor type 1; EGFR = epidermal growth factor receptor; PKC = protein kinase C; PLC = phospholipase C; Gq = G pro-tein q; RPTP = receptor protein tyrosine phosphatase .

inhibitors for their ability to inhibit the response of the rabbit PT to CO2. Luckily, the second drug on the list PD168393, a cell-permeant, highly specific and irreversible inhibitor of the ErbB family of receptor tyrosine kinases (113) blocked the JHCO3 response to changes in [CO2]BL (114). Another ErbB inhibitor, BPIQ-I, also inhibits the response to [CO2]BL. More-over, preliminary data suggest that PD168393 blocks the response to alterations in [HCO3

]BL (115). The PT expresses both ErbB1 (aka, EGFR or HER1) and ErbB2 (aka, HER2). Preliminary data suggest that exposing PT suspensions to CO2/HCO3

causes an increase in the tyrosine-phosphory-lation of ErbB1 and ErbB2 (116). Thus, the CO2/HCO3

sig-nal-transduction pathway may pass through ErbB1 and/or ErbB2. Our current signal-transduction model, upon which we will expand in the next several sections, is summarized in Figure 5.

Involvement of RPTP

We became interested in receptor protein tyrosine phos-phatase (RPTP) because it is a receptor protein tyrosine phosphatase (see (117)) that has an extracellular ligand-binding domain that is homologous to the canonical CAs. Joseph Schlessingers group cloned the cDNA encoding RPTP (118) and created a RPTP-knockout mouse (119). Barnea et al pointed out that the CA-like domain (CALD) of RPTP lacks 2 of the 3 His residues needed for coordi-nating Zn2+, and thus suggested that the CALD would be catalytically inactive (118). Preliminary work by Skelton et al (120) indeed suggests that RPTP lacks CA activity, but that a combination of 4 mutations (which render the CALD more like CA II) engenders CA activity. Moreover, prelimi-nary work by Zhou suggests that PTs from the RPTP-null mouse cannot respond to alterations in either [CO2]BL (121) or [HCO3

]BL (122). RPTP mRNA is present in kidney (123), and preliminary work that exploits a newly developed anti-body suggests that RPTP is expressed at the basal but not the lateral membrane of the PT (124). We hypothesize that the CALD of RPTP senses CO2 and/or HCO3 and that the phosphatase domain of RPTP then remodels ErbB1, ErbB2 and/or other proteins responsible for transmitting the CO2/HCO3

signal.

Involvement of apical AT1 receptors

We have already discussed the powerful role of ANG II which acts through apical and basolateral receptors in controlling JHCO3 in the PT (see the section Angiotensin II, above). An interesting aspect of ANG-II physiology is that the PT secretes an angiotensin-related substance into the

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lumen (125) (see also (126-128)). Working with rabbit PTs, we found that adding ANG II to the lumen (in addition to the amount secreted) or bath modulated the response to changes in [CO2]BL and vice versa (74). A follow-up study (129) showed that the luminal addition of saralasin, a pep-tide ANG II antagonist (130), or candesartan, a non-peptid-ic antagonist of specifically AT1 receptors (131), blocks the response of rabbit PTs to changes in [CO2]BL. Thus, the re-sponse to alterations in [CO2]BL requires an active apical AT1 receptor that is presumably stimulated by secreted ANG II. Because basolateral saralasin had no effect on the [CO2]BL dependence of JHCO3, we can conclude that the PT does not secrete ANG II basolaterally. Finally, PTs from the AT1A-null mouse (132, 133) exhibit no [CO2]BL-dependent changes in JHCO3. Thus, the JHCO3 response to altered [CO2]BL specifi-cally requires AT1A receptors at the apical membrane.To further explore the role of ANG II in the CO2 signal-trans-duction pathway, we added lisinopril, an antagonist of an-giotensin-converting enzyme (ACE), to the lumen. We were surprised to find that 240 nM luminal lisinopril has no ef-fect on the [CO2]BL dependence of JHCO3. However, this same dose of the ACE inhibitor, when added to the bath, totally eliminates the JHCO3 response, and even 60 nM basolateral lisinopril produces an inhibition. Thus, it is likely that the PT secretes preformed ANG II and that basolateral lisinopril acts by blocking the conversion of ANG I to ANG II within intra-cellular vesicles. We have begun using basolateral lisinopril to block the endogenous production of ANG II, allowing us to explore the isolated effect of adding ANG II to the lumen. Preliminary observations suggest that luminal 1011 M ANG II has little or no effect on JHCO3 when [CO2]BL is 0%, but that the effect of this dose of ANG II increases in a graded fash-ion as we raise [CO2]BL to 5% and then to 20% (134). Thus, at least part of the explanation for how basolateral CO2 con-trols JHCO3 is that CO2 may enhance the action of luminal ANG II. Note that basolateral CO2 has the opposite effect on the response to basolateral 1011 M ANG II: increasing levels of [CO2]BL reduce the stimulation by ANG II in a graded fash-ion (74). Thus, the signal-transduction pathways for [CO2]BL and ANG II interact in a complex way.

Involvement of protein kinase C (PKC)

It is generally accepted that the stimulatory effect of low-dose AT1A in the PT occurs through PKC. Indeed, PKC activation stimulates apical NHE3 (135), enhancing Na+ reabsorption (72), as well as HCO3

and fluid reabsorp-tion (136). Moreover, the stimulatory effect of low-dose ANG II appears to occur via PKC in the case of enhanced H+-pump activity (137), enhanced apical Na-H exchange

activity (138, 139) and enhanced HCO3 and water reab-

sorption (136). Evidence from rat PTs points to PKC- as being the critical isoform (139). In preliminary work on iso-lated perfused rabbit S2 PTs, a PKC inhibitor eliminates the CO2-evoked increase in JHCO3 (140). This result is con-sistent with the hypothesis that PKC plays an important role in the CO2 signal-transduction pathway.

Soluble adenylyl cyclase and G proteincoupled receptors

Although it appears to play no role in sensing acidbase disturbances in the PT, the cytoplasmic/soluble adenylyl cy-clase (sAC) is an evolutionarily conserved, cytosolic HCO3

chemosensor related to cyanobacterial adenylyl cyclases. Upon activation by HCO3

, sACs catalyze the conversion of ATP to cAMP (141). In the kidney, sAC has been local-ized to the TAL, distal tubule and collecting duct (142). In a collecting-duct cell model, sAC increases Na+ reabsorption in response to alkalosis (143). In the dogfish, sAC plays an important role in systemic acidbase homeostasis, specifi-cally in the gills. Here, alkalosis presumably by raising pHi and therefore [HCO3

]i stimulates V-ATPase insertion into the basolateral membrane, enhancing H+ absorption into the body (144). A potential conundrum, assuming that the acidbase sensitivity of sAC uniquely reflects changes in [HCO3

]i, is that both systemic metabolic alkalosis and sys-temic respiratory acidosis would raise [HCO3

]i.In 2003, Ludwig et al (145) were the first to report that ovarian cancer G proteincoupled receptor (OGR1) is a proton-sensing receptor that stimulates inositol phos-phate formation. Half-maximal activation of OGR1 oc-curred at pH 7.50, and was increasingly stimulated at more acidic pH, maximizing at pH 6.8. A related receptor, GPR4 is also proton sensitive in this case stimulating cAMP formation. Conserved extracellular histidine resi-dues in OGR1 and GPR4 are important for H+ sensing (145). The potential role for OGR1 or a related GPCR in acidbase sensing along the nephron is an attractive possibility. Finally, in Drosophila, a pair of GPCRs Gr21a and Gr63a act as a sensor for a CO2-related substance (146). OOE technology presumably could identify the true ligand of this insect sensor. It is not clear whether mammals have GPCRs with a similar function.

concludIng remArks

Every cellular and bodily function depends on pH, ev-erything from control of the cell cycle at one extreme to the muscle contraction that underlies exercise at the

S14


other. Thus, the regulation of intracellular pH and the whole-body acidbase homeostasis on which pHi regu-lation depends are of major importance. The past cen-tury has seen the defining of pH and buffering power, the realization that the lungs excrete CO2 and that the kidneys (including the proximal tubule) excrete acid into the urine, the discovery that acidbase status regulates these processes, the discovery of pHi regulation, the identification and cloning of the responsible acidbase transporters and advances in the understanding of regu-latory pathways. In the present review, we focus on the mechanism of HCO3

reabsorption by the PT cell (Fig. 1). Figure 5 summarizes our view of the acute regula-tion of this process. RPTP appears to be central in the response to alterations in [HCO3

]BL (Fig. 2C) and [CO2]BL (Fig. 3A), and the figure suggests that HCO3

and CO2 may compete for binding to RPTP. However, we really do not yet know the identities of the sensors for molecu-lar HCO3

and CO2. Although an RTK such as ErbB1/2 also appears to be essential for the response to altera-tions in [HCO3

]BL and [CO2]BL, we do not know how the signal crosses from the basolateral to the apical mem-brane. Following the action of luminal ANG II on the AT1A receptor, one can imagine how G protein q (Gq) might activate the apical H+-extrusion mechanisms. However,

it is not clear if or how the signal crosses back to the basolateral membrane to stimulate NBCe1-A. Although many unknowns remain, it is already clear that the acute regulation of acidbase transport by the PT depends not on blood pH per se but on the 2 parameters that define blood pH: [HCO3

]BL and [CO2]BL.

No human subjects were involved in this work.

Financial support: This work has been supported by a US

National Kidney Foundation Fellowship (FLB795) to L.A.S., US

National Institutes of Health grants NIH P01-DK17433 and R01-

DK081567 to W.F.B. and an American Heart Association Scientist

Development Grant 0735432N to Y.Z.

Conflict of interest statement: None declared.

Address for correspondence:Yuehan ZhouDepartment of Physiology and BiophysicsCase Western Reserve University School of Medicine10900 Euclid AvenueCleveland, OH 44106, [email protected]

references

1. Boron WF. Ventilation and perfusion of the lungs. In: Boron WF, Boulpaep EL, eds. Medical physiology: a cellular and molecular approach. 2nd ed. Philadelphia, PA: Saunders El-sevier; 2009:700-724.

2. Giebisch G, Windhager E. Transport of acids and bases. In: Boron WF, Boulpaep EL, eds. Medical physiology: a cellu-lar and molecular approach. Updated ed. Philadelphia, PA: Saunders Elsevier; 2009:851-865.

3. Boron WF. Acid-base transport by the renal proximal tubule. J Am Soc Nephrol. 2006;17:2368-2382.

4. Murer H, Hopfer U, Kinne R. Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney. Biochem J. 1976;154:597-604.

5. Wang T, Hropot M, Aronson PS, Giebisch G. Role of NHE iso-forms in mediating bicarbonate reabsorption along the nephron. Am J Physiol Renal Physiol. 2001;281:F1117-F1122.

6. Gluck SL, Underhill DM, Iyori M, Holliday LS, Kostrominova TY, Lee BS. Physiology and biochemistry of the kidney vacu-olar H+-ATPase. Annu Rev Physiol. 1996;58:427-445.

7. Purkerson JM, Schwartz GJ. The role of carbonic anhydras-es in renal physiology. Kidney Int. 2007;71:103-115.

8. Soleimani M, Grassl SM, Aronson PS. Stoichiometry of Na+-HCO3

- cotransport in basolateral membrane vesicles isolated from rabbit renal cortex. J Clin Invest. 1987;79:1276-1280.

9. Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science. 1992;256:385-387.

10. Nielsen S, Smith BL, Christensen EI, Knepper MA, Agre P. CHIP28 water channels are localized in constitutively water-permeable segments of the nephron. J Cell Biol. 1993;120:371-383.

11. Schnermann J, Chou CL, Ma T, Traynor T, Knepper MA, Verkman AS. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci U S A. 1998;95:9660-9664.

12. Good DW, Burg M. Ammonia production by individual seg-ments of the rat nephron. J Clin Invest. 1984;73:602-610.

13. Moret C, Dave MH, Schulz N, Jiang JX, Verrey F, Wagner CA. Regulation of renal amino acid transporters during metabolic acidosis. Am J Physiol Renal Physiol. 2007;292:F555-F566.

14. Musa-Aziz R, Jiang L, Chen LM, Behar KL, Boron WF. Con-

S15

EPHROLJN 2010; 23 (S16): S4-S18

centration-dependent effects on intracellular and surface pH of exposing Xenopus oocytes to solutions containing NH3/NH4+. J Membr Biol. 2009;228:15-31.

15. Wright PA, Knepper MA. Glutamate dehydrogenase activi-ties in microdissected rat nephron segments: effects of acid-base loading. Am J Physiol. 1990;259:F53-F59.

16. Curthoys NP, Lowry OH. The distribution of glutaminase isoenzymes in the various structures of the nephron in normal, acidotic, and alkalotic rat kidney. J Biol Chem. 1973;248:162-168.

17. Wang T, Yang CL, Abbiati T, et al. Mechanism of proximal tu-bule bicarbonate absorption in NHE3 null mice. Am J Physiol Renal Physiol. 1999;277:F298-F302.

18. Vallon V, Schwark JR, Richter K, Hropot M. Role of Na(+)/H(+) exchanger NHE3 in nephron function: micropuncture stud-ies with S3226, an inhibitor of NHE3. Am J Physiol Renal Physiol. 2000;278:F375-F379.

19. Ambuhl PM, Amemiya M, Danczkay M, et al. Chronic meta-bolic acidosis increases NHE3 protein abundance in rat kid-ney. Am J Physiol. 1996;271:F917-F925.

20. Leong PK, Devillez A, Sandberg MB, et al. Effects of ACE inhibition on proximal tubule sodium transport. Am J Physiol Renal Physiol. 2006;290:F854-F863.

21. Moe OW. Acute regulation of proximal tubule apical mem-brane Na/H exchanger NHE-3: role of phosphorylation, pro-tein trafficking, and regulatory factors. J Am Soc Nephrol. 1999;10:2412-2425.

22. Karet FE. Inherited distal renal tubular acidosis. J Am Soc Nephrol. 2002;13:2178-2184.

23. Stehberger PA, Schulz N, Finberg KE, et al. Localization and regulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of distal renal tubular acidosis. J Am Soc Nephrol. 2003;14:3027-3038.

24. Agre P. Nobel Lecture: aquaporin water channels. Biosci Rep. 2004;24:127-163.

25. Vallon V, Verkman AS, Schnermann J. Luminal hypotonic-ity in proximal tubules of aquaporin-1-knockout mice. Am J Physiol Renal Physiol. 2000;278:F1030-F1033.

26. Overton E. ber die osmotischen Eigenschaften der Zelle in ihrer Bedeutung fur die Toxicologie und Pharmacologie. Z Phys Chem. 1897;22:189-209.

27. Waisbren SJ, Geibel JP, Modlin IM, Boron WF. Unusu-al permeability properties of gastric gland cells. Nature. 1994;368:332-335.

28. Nakhoul NL, Davis BA, Romero MF, Boron WF. Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes. Am J Physiol. 1998;274:C543-C548.

29. Cooper GJ, Boron WF. Effect of PCMBS on CO2 permeabil-ity of Xenopus oocytes expressing aquaporin 1 or its C189S mutant. Am J Physiol. 1998;275:C1481-C1486.

30. Prasad GV, Coury LA, Fin F, Zeidel ML. Reconstituted aqua-porin 1 water channels transport CO2 across membranes. J Biol Chem. 1998;273:33123-33126.

31. Uehlein N, Lovisolo C, Siefritz F, Kaldenhoff R. The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiologi-cal functions. Nature. 2003;425:734-737.

32. Endeward V, Musa-Aziz R, Cooper GJ, et al. Evidence that Aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane. FASEB J. 2006;20:1974-1981.

33. Hub JS, de Groot BL. Does CO2 permeate through Aqua-porin-1? Biophys J. 2006;91:842-848.

34. Wang Y, Cohen J, Boron WF, Schulten K, Tajkhorshid E. Ex-ploring gas permeability of cellular membranes and mem-brane channels with molecular dynamics. J Struct Biol. 2007;157:534-544.

35. Fang X, Yang B, Matthay MA, Verkman AS. Evidence against aquaporin-1-dependent CO2 permeability in lung and kidney of mice. J Physiol (Lond). 2002;542:63-69.

36. Yang B, Fukuda N, Van Hoek A, Matthay MA, Ma T, Verkman AS. Carbon dioxide permeability of aquaporin-1 measured in erythrocytes and lung of aquaporin-1 null mice and in recon-stituted liposomes. J Biol Chem. 2000;275:2686-2692.

37. Missner A, Kugler P, Saparov SM, et al. Carbon di-oxide transport through membranes. J Biol Chem. 2008;283:25340-25347.

38. Missner A, Pohl P. 110 years of the Meyer-Overton rule: pre-dicting membrane permeability of gases and other small compounds. Chemphyschem. 2009;10:1405-1414.

39. Cooper GJ, Zhou Y, Bouyer P, Grichtchenko II, Boron WF. Transport of volatile solutes through AQP1. J Physiol. 2002;542:17-29.

40. Boron WF. The Sharpey-Schafer Lecture: gas channels. Exp Physiol. 2010 (in press).

41. Zhou Y, Bouyer P, Boron WF. Evidence that AQP1 is a func-tional CO2 channel in proximal tubules [abstract]. FASEB J. 2006;20:A1225.

42. Boron WF, Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander: basolateral HCO3

- transport. J Gen Physiol. 1983;81:53-94.

43. Romero MF, Hediger MA, Boulpaep EL, Boron WF. Expres-sion cloning and characterization of a renal electrogenic Na+/HCO3

- cotransporter. Nature. 1997;387:409-413.44. Romero MF, Fulton CM, Boron WF. The SLC4 family of HCO3

transporters. Pflgers Arch. 2004;447:495-509.

45. Abuladze N, Lee I, Newman D, et al. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J Biol Chem. 1998;273:17689-17695.

46. Bevensee MO, Schmitt BM, Choi I, Romero MF, Boron WF. An electrogenic Na/HCO3 cotransporter (NBC) with a novel C terminus, cloned from rat brain. Am J Physiol Cell Physiol. 2000;278:C1200-C1211.

47. Virkki LV, Wilson DA, Vaughan-Jones RD, Boron WF. Func-tional characterization of human NBC4 as an electrogenic Na+-HCO3

- cotransporter (NBCe2). Am J Physiol Cell Physiol. 2002;282:C1278-C1289.

S16


48. Sassani P, Pushkin A, Gross E, et al. Functional characteriza-tion of NBC4: a new electrogenic sodium- bicarbonate cotrans-porter. Am J Physiol Cell Physiol. 2002;282:C408-C416.

49. Choi I, Aalkjr C, Boulpaep EL, Boron WF. An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel. Nature. 2000;405:571-575.

50. Wang CZ, Yano H, Nagashima K, Seino S. The Na+-driven Cl-/HCO3

- exchanger: cloning, tissue distribution, and functional characterization. J Biol Chem. 2000;275:35486-35490.

51. Grichtchenko II, Choi I, Zhong X, Bray-Ward P, Russell JM, Boron WF. Cloning, characterization, and chromosomal mapping of a human electroneutral Na+-driven Cl-HCO3 ex-changer. J Biol Chem. 2001;276:8358-8363.

52. Grichtchenko II, Boron WF. Surface-pH measurements in voltage-clamped Xenopus oocytes co-expressing NBCe1 and CAIV: evidence for CO3

= transport [abstract]. FASEB J. 2002;16:A795.

53. Igarashi T, Inatomi J, Sekine T, et al. Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities. Nat Genet. 1999;23:264-266.

54. Horita S, Yamada H, Inatomi J, et al. Functional analy-sis of NBC1 mutants associated with proximal renal tubu-lar acidosis and ocular abnormalities. J Am Soc Nephrol. 2005;16:2270-2278.

55. Suzuki M, Vaisbich MH, Yamada H, et al. Functional analy-sis of a novel missense NBC1 mutation and of other muta-tions causing proximal renal tubular acidosis. Pflgers Arch. 2008;455:583-593.

56. Dinour D, Chang MH, Satoh J, et al. A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects. J Biol Chem. 2004;279:52238-52246.

57. Igarashi T, Inatomi J, Sekine T, et al. Novel nonsense mutation in the Na+/HCO3- cotransporter gene (SLC4A4) in a patient with permanent isolated proximal renal tubular acidosis and bilateral glaucoma. J Am Soc Nephrol. 2001;12:713-718.

58. Demirci FY, Chang MH, Mah TS, Romero MF, Gorin MB. Proxi-mal renal tubular acidosis and ocular pathology: a novel mis-sense mutation in the gene (SLC4A4) for sodium bicarbonate cotransporter protein (NBCe1). Mol Vis. 2006;12:324-330.

59. Lin SHP, Lo YF, Yang SS, Seki G. Severe metabolic acidosis causes early lethality in NBC1 W516X knock-in mice [ab-stract]. J Am Soc Nephrol. 2009;20:33A.

60. Inatomi J, Horita S, Braverman N, et al. Mutational and func-tional analysis of SLC4A4 in a patient with proximal renal tu-bular acidosis. Pflgers Arch. 2004;448:438-444.

61. Igarashi T, Inatomi J, Sekine T, et al. Mutational and functional analysis of the Na+/HCO3- cotransporter gene (SLC4AC) in patients with permanent isolated proximal renal tubular aci-dosis and ocular abnormalities [abstract]. J Am Soc Nephrol. 2003;14:302A.

62. Boron WF, Chen L, Parker MD. Modular structure of sodium-cou-pled bicarbonate transporters. J Exp Biol. 2009;212:1697-1706.

63. Kondo Y, Frmter E. Evidence of chloride/bicarbonate exchange mediating bicarbonate efflux from S3 segments of rabbit renal proximal tubule. Pflgers Arch. 1990;415:726-733.

64. Brosius FC III, Nguyen K, Stuart-Tilley AK, Haller C, Briggs JP, Alper SL. Regional and segmental localization of AE2 an-ion exchanger mRNA and protein in rat kidney. Am J Physiol. 1995;269:F461-F468.

65. Sly WS, Hewett-Emmett D, Whyte MP, Yu YS, Tashian RE. Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetro-sis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci U S A. 1983;80:2752-2756.

66. Baird TT Jr., Waheed A, Okuyama T, Sly WS, Fierke CA. Ca-talysis and inhibition of human carbonic anhydrase IV. Bio-chemistry. 1997;36:2669-2678.

67. Parkkila S, Parkkila AK, Saarnio J, et al. Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors. J Histochem Cytochem. 2000;48:1601-1608.

68. Burg MB, Orloff J. Control of fluid absorption in the renal proximal tubule. J Clin Invest. 1968;47:2016-2024.

69. Garvin JL. Angiotensin stimulates glucose and fluid absorp-tion by rat proximal straight tubules. J Am Soc Nephrol. 1990;1:272-277.

70. Harris PJ, Young JA. Dose-dependent stimulation and inhibi-tion of proximal tubule sodium reabsorption by angiotensin II in the rat kidney. Pflgers Arch. 1977;367:295-297.

71. Schuster VL, Kokko JP, Jacobson HR. Angiotensin II directly stimulates sodium transport in rabbit proximal convoluted tubules. J Clin Invest. 1984;73:507-515.

72. Du Z, Ferguson W, Wang T. Role of PKC and calcium in modulation of effects of angiotensin II on sodium trans-port in proximal tubule. Am J Physiol Renal Physiol. 2003;284:F688-F692.

73. Li L, Wang YP, Capparelli AW, Jo OD, Yanagawa N. Effect of luminal angiotensin II on proximal tubule fluid transport: role of apical phospholipase A2. Am J Physiol Renal Physiol. 1994;266:F202-F209.

74. Zhou Y, Bouyer P, Boron WF. Effects of angiotensin II on the CO2 dependence of HCO3

- reabsorption by the rab-bit S2 renal proximal tubule. Am J Physiol Renal Physiol. 2006;290:F666-F673.

75. Liu FY, Cogan MG. Angiotensin II stimulation of hydrogen ion secretion in the rat early proximal tubule. J Clin Invest. 1988;82:601-607.

76. Liu FY, Cogan MG. Angiotensin II stimulates early proximal bicarbonate absorption in the rat by decreasing cyclic ad-enosine monophosphate. J Clin Invest. 1989;84:83-91.

77. Wang T, Chan YL. Mechanism of angiotensin II action on proximal tubular transport. J Pharmacol Exp Ther. 1990;252:689-695.

78. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bern-stein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;351:233-236.

S17

EPHROLJN 2010; 23 (S16): S4-S18

79. Sasaki K, Yamano Y, Bardhan S, et al. Cloning and expres-sion of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 1991;351:230-233.

80. Baum M, Quigley R, Quan A. Effect of luminal angiotensin II on rabbit proximal convoluted tubule bicarbonate absorp-tion. Am J Physiol. 1997;273:F595-F600.

81. Zheng Y, Horita S, Hara C, et al. Biphasic regulation of renal proximal bicarbonate absorption by luminal AT(1A) receptor. J Am Soc Nephrol. 2003;14:1116-1122.

82. Bacic D, Kaissling B, McLeroy P, Zou L, Baum M, Moe OW. Dopamine acutely decreases apical membrane Na/H ex-changer NHE3 protein in mouse renal proximal tubule. Kid-ney Int. 2003;64:2133-2141.

83. Licht C, Laghmani K, Yanagisawa M, Preisig PA, Alpern RJ. An autocrine role for endothelin-1 in the regulation of proxi-mal tubule NHE3. Kidney Int. 2004;65:1320-1326.

84. Wang T. Role of iNOS and eNOS in modulating proximal tubule transport and acid-base balance. Am J Physiol Renal Physiol. 2002;283:F658-F662.

85. Boron WF. Acid-base physiology. In: Boron WF, Boulpaep EL, eds. Medical physiology: a cellular and molecular approach. 2nd ed. Philadelphia, PA: Saunders Elsevier; 2009:652-671.

86. Wu MS, Biemesderfer D, Giebisch G, Aronson PS. Role of NHE3 in mediating renal brush border Na+-H+ ex-change: adaptation to metabolic acidosis. J Biol Chem. 1996;271:32749-32752.

87. Nowik M, Lecca MR, Velic A, Rehrauer H, Brandli AW, Wag-ner CA. Genome-wide gene expression profiling reveals renal genes regulated during metabolic acidosis. Physiol Genom-ics. 2008;32:322-334.

88. Soleimani M, Bizal GL, McKinney TD, Hattabaugh YJ. Effect of in vitro metabolic acidosis on luminal Na+/H+ exchange and basolateral Na+:HCO3

- cotransport in rabbit kidney prox-imal tubules. J Clin Invest. 1992;90:211-218.

89. Sasaki S, Berry CA, Rector FC Jr. Effect of luminal and peri-tubular HCO3

- concentrations and PCO2 on HCO3- reabsorp-

tion in rabbit proximal convoluted tubules perfused in vitro. J Clin Invest. 1982;70:639-649.

90. Mello-Aires M, MacLaughlin MA, Malnic G. Proximal tubu-lar acidification in metabolic alkalosis. Braz J Med Biol Res. 1983;16:365-374.

91. Cogan MG. Effects of acute alterations in pCO2 on proxi-mal HCO3

-, Cl-, and H2O reabsorption. Am J Physiol. 1984;246:F21-F26.

92. Brazeau P, Gilman A. Effect of plasma CO2 tension on renal tubu-lar reabsorption of bicarbonate. Am J Physiol. 1953;175:33-38.

93. Dorman PJ, Sullivan WJ, Pitts RF. The renal response to acute respiratory acidosis. J Clin Invest. 1954;33:82-90.

94. Relman AS, Etsten B, Schwartz WB. The regulation of renal bicarbonate reabsorption by plasma carbon dioxide tension. J Clin Invest. 1953;32:972-978.

95. Chan YL, Giebisch G. Relationship between sodium and bi-carbonate transport in the rat proximal convoluted tubule. Am J Physiol. 1981;240:F222-F230.

96. Horie S, Moe O, Tejedor A, Alpern RJ. Preincubation in acid me-dium increases Na/H antiporter activity in cultured renal proximal tubule cells. Proc Natl Acad Sci U S A. 1990;87:4742-4745.

97. Laghmani K, Preisig PA, Moe OW, Yanagisawa M, Alpern RJ. Endothelin-1/endothelin-B receptor-mediated increases in NHE3 activity in chronic metabolic acidosis. J Clin Invest. 2001;107:1563-1569.

98. Yamaji Y, Amemiya M, Cano A, et al. Overexpression of csk inhibits acid-induced activation of NHE-3. Proc Natl Acad Sci USA. 1995;92:6274-6278.

99. Yamaji Y, Tsuganezawa H, Moe OW, Alpern RJ. Intracellular aci-dosis activates c-Src. Am J Physiol. 1997;272:C886-C893.

100. Zhao J, Hogan EM, Bevensee MO, Boron WF. Out-of-equi-librium CO2/HCO3

- solutions and their use in characterizing a new K/HCO3 cotransporter. Nature. 1995;374:636-639.

101. Zhao J, Zhou Y, Boron WF. Effect of isolated removal of either basolateral HCO3

- or basolateral CO2 on HCO3- reabsorption

by rabbit S2 proximal tubule. Am J Physiol Renal Physiol. 2003;285:F359-F369.

102. Zhou Y, Zhao J, Bouyer P, Boron WF. Evidence from renal proximal tubules that HCO3

- and solute reabsorption are acutely regulated not by pH but by basolateral HCO3

- and CO2. Proc Natl Acad Sci U S A. 2005;102:3875-3880.

103. Gluck S, Cannon C, Al-Awqati Q. Exocytosis regulates uri-nary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane. Proc Natl Acad Sci U S A. 1982;79:4327-4331.

104. Schwartz GJ, Al-Awqati Q. Carbon dioxide causes exo-cytosis of vesicles containing H+ pumps in isolated per-fused proximal and collecting tubules. J Clin Invest. 1985;75:1638-1644.

105. Bouyer P, Zhou Y, Boron WF. An increase in intracellular calcium concentration that is induced by basolateral CO2 in rabbit renal proximal tubule. Am J Physiol Renal Physiol. 2003;285:F674-F687.

106. Roos A, Boron WF. Intracellular pH. Physiol Rev. 1981;61:296-434.

107. Bevensee MO, Boron WF. Regulation of intracellular pH. In: Alpern RJ, Hebert SC, eds. Seldin and Giebischs the kidney: physiology and pathophysiology. 4th ed. Burlington, MA: Academic Press; 2008:1429-1480.

108. Boron WF. Regulation of intracellular pH. Adv Physiol Educ. 2004;28:160-179.

109. Gilles-Gonzalez MA, Ditta GS, Helinski DR. A hmoprotein with kinase activity encoded by the oxygen sensor of Rhizo-bium meliloti. Nature. 1991;350:170-172.

110. Rodgers KR. Heme-based sensors in biological systems. Curr Opin Chem Biol. 1999;3:158-167.

111. Ecker JR. The ethylene signal transduction pathway in plants. Science. 1995;268:667-675.

112. Chang C, Kwok SF, Bleecker AB, Meyerowitz EM. Arabidop-sis ethylene-response gene ETR1: similarity of product to two-component regulators. Science. 1993;262:539-544.

113. Fry DW, Bridges AJ, Denny WA, et al. Specific, irreversible

S18


inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc Natl Acad Sci U S A. 1998;95:12022-12027.

114. Zhou Y, Bouyer P, Boron WF. Role of a tyrosine kinase in the CO2-induced stimulation of HCO3 reabsorption by rabbit S2 proximal tubules. Am J Physiol Renal Physiol. 2006;291:F358-F367.

115. Zhou Y. Role of a receptor tyrosine kinase in the HCO3 in-duced inhibition of HCO3 reabsorption by rabbit S2 proximal tubule [abstract]. FASEB J. 2009;23:1012.5.

116. Skelton LA, Bouyer P, Boron WF. CO2/HCO3 induces EGFR phosphorylation in the proximal tubule [abstract]. J Am Soc Nephrol. 2006;17:578A.

117. Tiganis T, Bennett AM. Protein tyrosine phosphatase func-tion: the substrate perspective. Biochem J. 2007;402:1-15.

118. Barnea G, Silvennoinen O, Shaanan B, et al. Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP defines a new subfamily of receptor tyrosine phos-phatases. Mol Cell Biol. 1993;13:1497-1506.

119. Lamprianou S, Vacaresse N, Suzuki Y, et al. Receptor protein tyrosine phosphatase is a marker for pyramidal cells and sensory neurons in the nervous system and is not necessary for normal development. Mol Cell Biol. 2006;26:5106-5119.

120. Skelton LA, Musa-Aziz R, Qin X, Boron WF. Mutations that 'restore' enzymatic activity to the carbonic anhydrase-like domain (CALD) of receptor protein tyrosine phosphatase gamma (RPTP): importance of the shuttle His and CO2 bind-ing site. J Am Soc Nephrol. 2010 (in press).

121. Zhou Y, Boron WF. Effect of knocking out receptor protein tyrosine phosphatase (RPTP) in the CO2-induced stimula-tion of HCO3 reabsorption by mouse renal proximal tubule [abstract]. FASEB J. 2010;24:1024.7.

122. Zhou Y. Effect of knocking out receptor protein tyrosine phos-phatase (RPTP) in the HCO3-induced inhibition of HCO3 re-absorption by mouse renal proximal tubule [abstract]. FASEB J. 2010;24:1024.8.

123. Tsukamoto T, Takahashi T, Ueda R, Hibi K, Saito H, Takahashi T. Molecular analysis of the protein tyrosine phosphatase gamma gene in human lung cancer cell lines. Cancer Res. 1992;52:3506-3509.

124. Skelton LA, Boron WF. The receptor protein tyrosine phos-phatase gamma (RPTP) is located at the basal (but not lat-eral) membrane of proximal tubules. J Am Soc Nephrol. 2010 (in press).

125. Levens NR, Peach MJ, Carey RM. Role of the intrarenal renin-angiotensin system in the control of renal function. Circ Res. 1981;48:157-167.

126. Campbell DJ. Circulating and tissue angiotensin systems. J Clin Invest. 1987;79:1-6.

127. Carey RM, Siragy HM. Newly recognized components of the renin-angiotensin system: potential roles in cardiovascular and renal regulation. Endocr Rev. 2003;24:261-271.

128. Paul M, Poyan MA, Kreutz R. Physiology of local renin-angio-tensin systems. Physiol Rev. 2006;86:747-803.

129. Zhou Y, Bouyer P, Boron WF. Role of the AT1A receptor in the

CO2-induced stimulation of HCO3 reabsorption by renal proxi-mal tubules. Am J Physiol Renal Physiol. 2007;293:F110-F120.

130. Burnier M. Angiotensin II type 1 receptor blockers. Circula-tion. 2001;103:904-912.

131. Fabiani ME, Dinh DT, Nassis L, Casley DJ, Johnston CI. In vivo inhibition of angiotensin receptors in the rat kidney by candesartan cilexetil: a comparison with losartan. Am J Hy-pertens. 2000;13:1005-1013.

132. Ito M, Oliverio MI, Mannon PJ, et al. Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci U S A. 1995;92:3521-3525.

133. Sugaya T, Nishimatsu S, Tanimoto K, et al. Angiotensin II type 1a receptor-deficient mice with hypotension and hyper-reninemia. J Biol Chem. 1995;270:18719-18722.

134. Zhou Y, Boron WF. Effect of basolateral CO2 on the luminal ANG II sensitivity of HCO3 reabsorption by rabbit S2 proximal tubules [abstract]. FASEB J. 2008;22:760.2.

135. Weinman EJ, Shenolikar S. Protein kinase C activates the renal apical membrane Na+/H+ exchanger. J Membr Biol. 1986;93:133-139.

136. Liu FY, Cogan MG. Role of protein kinase C in proximal bicar-bonate absorption and angiotensin signaling. Am J Physiol. 1990;258:F927-F933.

137. Carraro-Lacroix LR, Malnic G. Signaling pathways involved with the stimulatory effect of angiotensin II on vacuolar H+-ATPase in proximal tubule cells. Pflgers Arch. 2006;452:728-736.

138. Houillier P, Chambrey R, Achard JM, Froissart M, Poggioli J, Paillard M. Signaling pathways in the biphasic effect of angiotensin II on apical Na/H antiport activity in proximal tu-bule. Kidney Int. 1996;50:1496-1505.

139. Karim ZG, Chambrey R, Chalumeau C, et al. Regulation by PKC isoforms of Na+/H+ exchanger in luminal membrane vesicles iso-lated from cortical tubules. Am J Physiol. 1999;277:F773-F778.

140. Zhou Y, Bouyer P, Boron WF. CO2-evoked HCO3 reabsorption in the rabbit S2 proximal tubule: Blockade by PKC inhibitor RO-31-8220 [abstract]. FASEB J. 2005;19:A156.

141. Chen Y, Cann MJ, Litvin TN, et al. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science. 2000;289:625-628.

142. Pastor-Soler N, Beaulieu V, Litvin TN, et al. Bicarbonate-regulat-ed adenylyl cyclase (sAC) is a sensor that regulates pH-depen-dent V-ATPase recycling. J Biol Chem. 2003;278:49523-49529.

143. Hallows KR, Wang H, Edinger RS, et al. Regulation of epi-thelial Na+ transport by soluble adenylyl cyclase in kidney collecting duct cells. J Biol Chem. 2009;284:5774-5783.

144. Tresguerres M, Parks SK, Salazar E, Levin LR, Goss GG, Buck J. Bicarbonate-sensing soluble adenylyl cyclase is an essential sensor for acid/base homeostasis. Proc Natl Acad Sci U S A. 2010;107:442-447.

145. Ludwig MG, Vanek M, Guerini D, et al. Proton-sensing G-protein-coupled receptors. Nature. 2003;425:93-98.

146. Jones WD, Cayirlioglu P, Kadow IG, Vosshall LB. Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature. 2007;445:86-90.

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Acid-base Transport by the Renal Proximal Tubule