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JNEPHROL 2010; 23 (S16): S4-S18

Acid-Base balance: Basic aspects

Acid-base transport by the renal proximal tubule

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

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


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

Each day, the kidneys filter 180 L of blood plasma,

equating to some 4,300 mmol of the major blood buffer, 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 coupled 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 transport into the blood. Thus, the PT along with more
distal renal segments is largely responsible for regulating 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 review, 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 disturbances 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.

pH = pK + log


The lungs control plasma PCO by exhaling the CO2 pro2
duced during aerobic respiration (1). The kidneys (2) regulate 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+ arising from sources such as the metabolic production nonvolatile 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 reclaimed 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) tethered 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 basolateral 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-

Key words: Aquaporin, Na/HCO3 cotransporter, NBC,

Out-of-equilibrium CO2/HCO3 solutions, Receptor protein tyrosine phosphatase, RPTP

The maintenance of a physiological ratio of the major bloodplasma buffer parameters, [HCO3] and PCO , is at the root of


2010 Societ Italiana di Nefrologia - ISSN 1121-8428

JNEPHROL 2010; 23 (S16): S4-S18

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 = carbonic 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.

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 thereby 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
(HPO42), urate and creatinine. The PT (which can achieve
a final luminal pH of ~6.8) is responsible for about half of
the titration of HPO42 (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 contribution for these latter buffers.

(c) The final component of new HCO3 is coupled to ammonium 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 glutaminase 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, probably 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 (gluconeogenesis). In response to chronic metabolic acidosis, the PT adaptively up-regulates ammonium synthesis
to promote H+ excretion (12, 13, 15, 16).
In addition to responding to chronic acidbase disturbances, the PT also responds to acute disturbances. We

Skelton et al: Acidbase transport

will discuss these below in the section titled Regulation

by acidbase parameters. To set the stage for this discussion on regulation, we will first introduce the molecular components of acidbase transport at the apical and
basolateral sides of the PT cell.

Apical H+ extrusion
The Na-H exchanger 3 (NHE3) is the most important H+-extruder 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 responses 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 membrane 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 protein (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 suppressing H+ extrusion (20). A complex combination of phospho-serine modifications at the NHE3 carboxy terminus between amino acid residues 455 and 832 mediated both by
PKA and protein kinase C (PKC) are involved in controlling 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 (VATPase) 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 ascending 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 multisubunit V-ATPase consists of 2 major structures; an integral

membrane VO ring-like structure that mediates H+ movement (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 autosomal-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 epithelium (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 hypothesis 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 parietal and chief cells have no detectable permeability to
either CO2 or NH3 (27) the first evidence that the centuryold 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 conclusions have themselves been challenged (39, 40). It might
be noted that most of the evidence regarding the gaschannel 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

JNEPHROL 2010; 23 (S16): S4-S18

basolateral membranes of PT cells. Moreover, the movement 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 effect on the transepithelial flux of HCO3 from the basolateral solution (bath) to the lumen, it reduces the transepithelial 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-4isothiocyanato-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. Indeed, 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 transporter 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 electroneutral transporters (49-51). NBCe1-A is expressed at
the basolateral membrane of the PT, at the highest levels 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 devastating 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 membrane 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 distal 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 29kDa 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 converting 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 osteoclast 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 apically 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 Figure 1A, apical CA IV catalyses HCO3 dehydration, thereby
consuming secreted H+ as well as generating membranepermeant CO2 and H2O. Consistent with this role, CA IV
has a higher HCO3 affinity and a greater HCO3 dehydratase 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 cotransporter (52). Thus, CA IV may protect nearby proteins from
extreme pH fluctuations caused by NBCe1-A.


Skelton et al: Acidbase transport

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 human PT but far more abundant in the TAL, DCT and principal 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 NBCe1A CA XII might minimize pH fluctuations caused by H+

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 knockout of iNOS reduces JV and JHCO . Nevertheless, the mouse
maintains normal acidbase status via unknown compensatory mechanisms (84).

Regulation by acidbase parameters

Regulation of proximal-tubule acid


Regulation by hormones

Angiotensin II
Angiotensin II (ANG II) is perhaps the most powerful hormonal modulator of Na+, fluid, and HCO3 reabsorption by
renal PTs. As we will see below (see the section Involvement 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 (7577) have reported that luminal or basolateral ANG II has
biphasic concentration-dependent effects, increasing the
fluid reabsorption rate (JV) and the HCO3 reabsorption
rate (JHCO ) at low ANG II concentrations, but decreasing
JV and JHCO at higher concentrations. ANG II acts via an3
giotensin II receptors type 1 (AT1) (78, 79), which are G
proteincoupled receptors (GPCRs), for both its stimulatory and inhibitory effects (80, 81).

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

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

The 4 fundamental acidbase disturbances are metabolic

alkalosis and acidosis, and respiratory alkalosis and acidosis defined below. Each of these disturbances can
be acute or chronic. As discussed in conjunction with
Equation 1, the renal or respiratory systems compensate for each disturbance by appropriately altering blood
[HCO3] or PCO , thereby returning arterial pH toward nor2
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 PCO , resulting in a fall in plasma pH) is compensated
largely by an increase in alveolar ventilation, which lowers
PCO . 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 brushborder 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 PCO , causing a rise in pH) is compensated by changes
opposite to those for metabolic acidosis. In isolated perfused PTs, increases in basolateral [HCO3] cause a fall in
JHCO that is reversed by also raising PCO (89). Creating acute
metabolic alkalosis in rats by infusing HCO3 causes a fall in
JHCO as assessed by free-flow micropuncture (90).
Respiratory alkalosis (i.e., a decrease in plasma PCO , caus2
ing a rise in pH) is compensated by a decrease in JHCO . For
example, Cogan found that hyperventilating a rat to reduce
arterial PCO by ~20 mm Hg causes a marked fall in JHCO , as
determined by free-flow micropuncture (91).
Respiratory acidosis (i.e., an increase in plasma PCO , caus2
ing a fall in pH) is compensated by an increase in JHCO . More
than half a century ago, a series of 3 papers demonstrated
that acute respiratory acidosis in dogs rapidly stimulates renal acid secretion (92-94). Based on additional experiments,
these authors concluded that it is most likely an increase in
PCO rather than a decrease in pH that controls renal acid

JNEPHROL 2010; 23 (S16): S4-S18

Fig. 2 - Effect of isolated changes

in basolateral (BL) pH and [HCO3]
on the rate of HCO3 reabsorption
(JHCO3) and steady-state intracellular pH. A) Effect of isolated
changes in pHBL, obtained using 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 experiments 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.

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 JHCO to
fall dramatically (95). Later work by Cogan, using free-flow
micropuncture, showed that acute respiratory alkalosis in
rats leads to a decrease in JHCO , whereas acute respiratory
acidosis has the opposite effect (91).
In addition to the above work on acute acidbase disturbances, others have investigated chronic effects. For example, 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 process that involves activation of c-Src and autocrine signaling 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 PCO . 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 acid
base parameters was finally addressed in 1995 by Zhao et
al (100), who developed a technique for generating out-ofequilibrium (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 proximal tubules. As described in the next 3 sections, Zhou et al
(102) later monitored JHCO 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) represents the JHCO 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 intracellular 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]
of 5% produces a surprising result: no change in JHCO
(Fig. 2A). Nevertheless, the isolated changes in pHBL produce sizeable changes in pHi (Fig. 2B). Thus, the PT cell can
not acutely regulate JHCO in response to changes in either
pHBL or pHi per se.

Basolateral [HCO3]
In the second series of experiments (Fig. 2C, D), we increased basolateral [HCO3] from 0 to 22 to 44 mM while
holding pHBL at 7.40 and [CO2]BL at 5%. The middle symbol

Skelton et al: Acidbase transport

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

the rate of HCO3 reabsorption (JHCO3) and steady-state intracellular pH. A) Effect of isolated changes in [CO2]BL, obtained
using out-of-equilibrium solutions on JHCO3. B) Effect of isolated 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.

in Figure 2C represents the JHCO 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 JHCO . As summarized in Figure 2D, the isolated increase
of [HCO3]BL from 0 to 22 mM also causes a substantial increase in pHi. In summary, isolated increases in [HCO3]BL
produce the appropriate compensatory response for wholebody acidbase balance: a fall in JHCO .

Basolateral [CO2 ]
In the final series of these experiments (Fig. 3A, B), we increased [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. Figure 3A shows that isolated increases in [CO2]BL from 0% to
20% cause graded increases in JHCO . As shown in Figure


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 JHCO plots in Figure 3A show that isolated in3
creases in [CO2]BL produce the appropriate compensatory
response for whole-body acidbase balance: a rise in JHCO .
A comparison of the 2 JHCO 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 JHCO to acidbase disturbances do not perturb fluid re3
absorption in vitro, and thus presumably would not perturb
blood pressure in vivo.
In the 1980s, Al-Awqati and colleagues showed that increases in PCO lead to the insertion of H+ pumps into apical
membranes in turtle bladder (103) as well as PTs and cortical 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 [Ca2+]i. Indeed,
changes in [Ca2+]i appear to be involved in the OOE experiments. 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). Moreover, 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 virtually 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
JHCO data from the previous 2 figures as a function of pHi.
It is clear that pHi is not the unique determinant of JHCO . In
fact, the large changes in pHi produced by isolated changes
in pHBL (diamonds) elicit no change in JHCO . In retrospect, it
is perhaps not surprising that pHi is not the unique determinant of JHCO because, from the perspective of pHi reg3
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

JNEPHROL 2010; 23 (S16): S4-S18

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) Dependence 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.

signal for regulating pHBL is that different acidbase disturbances can generate the same pHBL but different pHi values.
In other words, the pHi signal is degenerate (i.e., knowledge 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 regulation (e.g., the recovery of pHi from an acute intracellular acid
load; see (106-108)) would make JHCO 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 JHCO .
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 JHCO (see the vertical spread of JHCO data at
pHBL = 7.4).
Figure 4C is yet a third replot of the JHCO data in Figures

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

[HCO3]BL. Note the vertical spread of JHCO data at [CO2]BL/
[HCO3]BL = . Figure 4D is similar, but is a plot of JHCO
versus the inverse ratio, [HCO3]BL/[CO2]BL. Here, note the
vertical spread of JHCO data at [HCO3]BL/[CO2]BL = 0. Thus,
neither of these ratio parameters is a unique determinant
of JHCO . 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 integrates this information in such a way that the [HCO3]BL influences 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.

Skelton et al: Acidbase transport

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 JHCO response to changes in [CO2]BL (114). Another ErbB
inhibitor, BPIQ-I, also inhibits the response to [CO2]BL. Moreover, 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-phosphorylation of ErbB1 and ErbB2 (116). Thus, the CO2/HCO3 signal-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.
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 protein q; RPTP = receptor protein tyrosine phosphatase .

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 GillesGonzales et al (109) had demonstrated that the bacterium
Sinorhizobium meliloti (formerly Rhizobium meliloti) senses low O2 levels using a 2-component system comprising 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 encoding 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

Involvement of RPTP
We became interested in receptor protein tyrosine phosphatase (RPTP) because it is a receptor protein tyrosine
phosphatase (see (117)) that has an extracellular ligandbinding 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 coordinating 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, preliminary 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 antibody 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 JHCO 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

JNEPHROL 2010; 23 (S16): S4-S18

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 peptide ANG II antagonist (130), or candesartan, a non-peptidic antagonist of specifically AT1 receptors (131), blocks the
response of rabbit PTs to changes in [CO2]BL. Thus, the response 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 JHCO , 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
JHCO . Thus, the JHCO response to altered [CO2]BL specifi3
cally requires AT1A receptors at the apical membrane.
To further explore the role of ANG II in the CO2 signal-transduction pathway, we added lisinopril, an antagonist of angiotensin-converting enzyme (ACE), to the lumen. We were
surprised to find that 240 nM luminal lisinopril has no effect on the [CO2]BL dependence of JHCO . However, this same
dose of the ACE inhibitor, when added to the bath, totally
eliminates the JHCO 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 intracellular 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 JHCO when [CO2]BL is 0%, but that
the effect of this dose of ANG II increases in a graded fashion as we raise [CO2]BL to 5% and then to 20% (134). Thus,
at least part of the explanation for how basolateral CO2 controls JHCO 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 fashion (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 lowdose AT1A in the PT occurs through PKC. Indeed, PKC
activation stimulates apical NHE3 (135), enhancing Na+
reabsorption (72), as well as HCO3 and fluid reabsorption (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 reabsorption (136). Evidence from rat PTs points to PKC- as
being the critical isoform (139). In preliminary work on isolated perfused rabbit S2 PTs, a PKC inhibitor eliminates
the CO2-evoked increase in JHCO (140). This result is con3
sistent with the hypothesis that PKC plays an important
role in the CO2 signal-transduction pathway.

Soluble adenylyl cyclase and G proteincoupled

Although it appears to play no role in sensing acidbase
disturbances in the PT, the cytoplasmic/soluble adenylyl cyclase (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 localized 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, specifically 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 systemic 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 phosphate formation. Half-maximal activation of OGR1 occurred 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 residues 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, everything from control of the cell cycle at one extreme
to the muscle contraction that underlies exercise at the

Skelton et al: Acidbase transport

other. Thus, the regulation of intracellular pH and the

whole-body acidbase homeostasis on which pHi regulation depends are of major importance. The past century 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 regulatory 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 regulation 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 molecular HCO3 and CO2. Although an RTK such as ErbB1/2
also appears to be essential for the response to alterations in [HCO3]BL and [CO2]BL, we do not know how the
signal crosses from the basolateral to the apical membrane. 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,

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basolateral membrane to stimulate NBCe1-A. Although
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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 R01DK081567 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 Zhou
Department of Physiology and Biophysics
Case Western Reserve University School of Medicine
10900 Euclid Avenue
Cleveland, OH 44106, USA

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