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3 absorption in rabbit outer
medullary collecting duct: role of luminal carbonic anhydrase
Departments of Pediatrics and Medicine, University of Rochester School of Medicine, Rochester, New York 14642
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing
the dehydration of carbonic acid into
CO2 plus water, facilitates H+ secretion in the renal outer
medullary collecting duct from the inner stripe
(OMCDi). To examine the role of
CA IV on H+ secretion, we measured
net HCO
3 transport in perfused
OMCDi segments and
examined the effect on transport of two extracellular CA inhibitors,
benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer,
polyoxyethylene bis(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg.
Chem. 24: 262-272, 1996)]. These agents
would inhibit only the luminal CA enzyme. Dose titration curves for net
HCO3 flux were performed for each
drug. Basal HCO3 absorptive flux was
12 pmol · min1 · mm1
in control segments and significantly increased to 16 pmol · min1 · mm1
in segments from 3-day acid-treated animals. The concentrations of
benzolamide and F-3500 that inhibited
HCO3 absorption by 50% were ~0.1
and ~5 µM, similar to the
Ki for CA IV
inhibition by these agents (0.2 and 4.0 µM, respectively; T. Maren,
C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98-104, 1997).
Adding exogenous CA to the inhibitor in the perfusate nearly restored
basal HCO3 transport, suggesting that
cytosolic CA II was not inhibited by these impermeant inhibitors. In
OMCDi segments from acidotic
rabbits, the concentrations of benzolamide and F-3500 that inhibited
HCO3 absorption by 50% were 50 and
500 µM, respectively, >100 times the
Ki for CA IV
inhibition and for inhibition of HCO3 transport in control tubules. Thus, in the
OMCDi, doses of extracellular CA
inhibitors that inhibited ~50% of CA IV activity also comparably inhibited HCO3 transport, indicating
that H+ secretion depends in part
on the availability of luminal CA IV activity. Acidosis substantially
decreased the sensitivity of HCO3
transport to CA inhibition.
inner stripe of outer medulla; kidney; benzolamide; aminobenzolamide; acid-base homeostasis; carbonic anhydrase IV
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE KIDNEY PLAYS a major role in regulating acid-base
homeostasis, with the final control occurring in the collecting duct. Inhibition of luminal carbonic anhydrase (CA) results in an acid disequilibrium pH in the outer medullary collecting duct (OMCD) (40),
indicating that H+ secretion,
rather than direct HCO3 absorption, is
the major mechanism of acidification in this segment. Acid excretion
there is mediated by H+ secretion
via a vacuolar
H+-adenosinetriphosphatase
(H+-ATPase) and a P-type
gastric-like
H+-K+-ATPase
(1, 2, 43). Under the pathophysiological condition of metabolic
acidosis, the cortical collecting duct reverses the polarity of its flux from net secretion of
HCO3 to net proton secretion (3, 27,
35, 41), whereas the OMCD (43) and inner medullary collecting duct
(IMCD) (5, 12, 46) increase their
H+ secretion rates. Adaptation to
acidosis is likely to occur at the level of the
H+-secreting cell along the
collecting duct (4, 18-20, 44).
Urinary acidification is believed to be facilitated by the enzyme CA, which exists in two isoforms. More than 95% of the activity is located in the cytosol as CA II, whereas up to 5% is membrane bound and corresponds to CA IV (7, 26, 48). CA IV catalyzes the dehydration of intraluminal carbonic acid that results from the secretion of protons into the lumen (11) in the reaction
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(1) |
The purpose of the present study was to use extracellular CA inhibitors
to investigate the role of CA IV in the maintenance of
H+ secretion
(HCO3 absorption) at the level of the
isolated perfused OMCDi of rabbit
kidney. Similarly, a second aim was to examine the role of
CA IV in the OMCDi taken from
acid-treated animals, since increases in
H+ secretion (43) and in CA IV
mRNA have been demonstrated in OMCDi during chronic metabolic
acidosis (42). We made use of a newly synthesized membrane-impermeant,
high-molecular-weight CA inhibitor to inhibit just the luminal enzyme
in many of the transport experiments.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animals
Female New Zealand White rabbits weighing 1.5-3 kg and maintained on normal laboratory chow (Purina lab diet no. 5326; Purina Mills, Richmond, IN) plus free access to tap water were used as normal control rabbits in this study. An additional group of rabbits of comparable weight was acid treated for 3 days by providing 75 mM NH4Cl added to a 7.5% sucrose drinking solution, which yielded an acid-equivalent load of 10-15 meq · kg1 · day1,
and by limiting food intake to 2% of body weight (7, 33, 35). The
animals were killed by intracardial injection of 130 mg pentobarbital
sodium after premedication with ketamine (44 mg/kg) and xylazine (5 mg/kg).
Tubule Isolation
Kidneys were removed, and 1- to 2-mm coronal slices were made and transferred to chilled dissection medium containing (in mM) 145 NaCl, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 L-alanine, pH 7.4, 290 ± 2 mosmol/kgH2O (33). From the corticomedullary rays, OMCDi segments were isolated under a dissecting microscope using sharpened forceps (43). Attention was made to obtain the ducts from deep within the OMCDi (below the termination of straight proximal tubule segments and adjacent to the medullary thick ascending limbs of Henle's loop). To maximize the reproducibility of this isolation in such a heterogeneous epithelium (19, 30, 34, 36), relatively short segments (0.5-0.7 mm) were obtained.In Vitro Microperfusion
In vitro microperfusion was performed by the method of Burg and Green (9), as previously described (33, 35, 41). An isolated OMCDi was rapidly transferred to a 1.2-ml temperature- and environmentally controlled chamber mounted on an inverted microscope and perfused and bathed at 37°C with Burg's solution containing (in mM) 120 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 L-alanine, 290 ± 2 mosmol/kgH2O, and gassed with 94% O2-6% CO2, yielding pH 7.4 at 37°C (33, 35). The specimen chamber was continuously suffused with 94% O2-6% CO2 to maintain pH at 7.4 (37). The collecting end of the segment was sealed into a holding pipette using Sylgard 184 (Dow Corning, Midland, MI). The length of each segment was measured using an eyepiece micrometer. Fourteen-nanoliter samples of tubular fluid were collected under water saturated mineral oil by timed filling of a calibrated volumetric pipette. Collections during each period were made in triplicate.Bathing solution was exchanged by a peristaltic pump at a rate of 14 ml/h to maintain constant solute concentrations.
Bicarbonate Transport
The concentration of total CO2 (assumed to be equal to that of HCO3) in perfusate (C0) and collected fluid (CL) was measured by microcalorimetry (Picapnotherm; Microanalytical Instrumentation, Mountain View, CA). Because there is no net water absorption in the OMCD (1, 13, 15), the rate of HCO3 transport
(JHCO3)
was calculated as
JHCO3 = (C0 
CL) × (VL/L),
where VL was the
rate of collection of tubular fluid (~2 nl/min),
L was the tubular length (in mm), and
J was in
pmol · min1 · mm
tubular length1. When
JHCO3 > 0, there was net HCO3 absorption
equivalent to net H+ secretion.
The sensitivity of the Picapnotherm was 10-20 counts/pmol total
CO2
(tCO2), so that for samples of
14.5 nl, there were 145-290 counts/mM
tCO2. The coefficient of
variation for a 20 mM standard measured in quadruplicate
was <0.5% (<23 counts/sample of 4,500 counts). This level of
sensitivity allowed us to reliably detect HCO3 differences of 1 mM between
perfused and collected fluids. In practice, we perfused tubules at
3-4
nl · min1 · mm1,
which generally resulted in a difference of ~5 mM between perfused and collected fluids.
Transepithelial Voltage
Transepithelial voltage (Vte) was measured using the perfusion pipette as an electrode. The voltage difference between calomel cells connected via 3 M KCl agar bridges to perfusing and bathing solutions was measured with a high-impedance electrometer (World Precision Instruments, New Haven, CT). Collections of tubular fluid were initiated once the Vte had stabilized (30-45 min), and readings were recorded at the conclusion of each collection.Viability
Evidence of damaged cells and gross leak of perfusate was continually assessed by the inclusion of 0.15 mg/ml FD & C green dye to each perfusate during the study (41). The experiment was discarded if tubular damage was detected.Experimental Microperfusion Protocols
Net HCO3 flux was first measured under
basal conditions (Burg's solution in lumen and bath), then again after
the application of luminal CA inhibitors, and sometimes after the
addition of exogenous CA (CA from bovine erythrocytes; Sigma Chemical,
St. Louis, MO) to the luminal CA inhibitor. Collections were made 5 min
after each maneuver and in triplicate. Two different CA inhibitors were
used: the relatively impermeant benzolamide (kindly provided by Dr. T. DuBose) and a high-molecular-weight agent, F-3500 (10), prepared by
covalently linking the closely related CA inhibitor, aminobenzolamide,
to the nontoxic polymer, polyoxyethylene
bis(acetic acid) (3,350 Da,
synthesized and kindly provided by Drs. C. Conroy and T. Maren). Each
inhibitor was used sequentially in the same tubule at 10-fold higher
concentrations until a substantial inhibition of
HCO3 absorption was detected. The
Ki at 37°C
for F-3500 is 4 µM against CA IV; that of benzolamide is 0.2 µM
(10, 24). These sulfonamide inhibitors are ~20-fold more active
(lower Ki)
against CA II (24). Control experiments were also performed using the
3,350-Da polymer that was not coupled to aminobenzolamide. Once
HCO3 transport was inhibited
substantially, 1 or 5 mg/ml (33 or 167 µM, respectively) CA was added
to the CA inhibitor, and collections were resumed in the same tubule.
Analysis and Statistics
Data are presented as means ± SE. Paired comparisons for each tubule were analyzed by paired t-test, and comparisons between tubules from control vs. acid-treated animals were analyzed by unpaired t-tests using statistical software (NCSS, Kaysville, UT). Significance was asserted when P < 0.05.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Whole Animal Data
Blood taken from the heart at the time of death confirmed a metabolic acidosis (control, 7.36 ± 0.01 pH units; plasma HCO3, 23.8 ± 0.3 mM,
n = 23; acid, 7.18 ± 0.02 pH units; plasma HCO3, 15.6 ± 0.8 mM, n = 13;
P < 0.01 for both comparisons), and
urine taken by postmortem bladder aspiration showed appropriate
acidification during acidosis (control, 7.80 ± 0.12 pH units; acid,
4.57 ± 0.08 pH units; P < 0.01).
Benzolamide Studies
OMCD HCO3 transport in
control vs. acid-treated animals. As we
have recently shown (43), acid treatment induced
H+ secretion
(HCO3 absorption) in isolated perfused OMCDi segments. Control segments
absorbed HCO3 at 12.5 ± 0.2 pmol · min1 · mm1
(n = 23) compared with 15.5 ± 0.2 pmol · min1 · mm1
(n = 15, P < 0.01) from acid-treated animals.
Consistent with the induction of electrogenic
H+ secretion by in vivo acidosis
(43), there was a more positive Vte in OMCD
segments from acid-treated vs. control animals (5.2 ± 0.4 mV vs.
3.8 ± 0.1 mV, P < 0.05).
Benzolamide titration in control
tubules. A benzolamide titration of increasing
concentration by 10-fold was performed in control tubules (Table
1). In four paired studies, luminal
108 M benzolamide reduced
HCO3 transport from 12.9 ± 0.1 to
12.2 ± 0.1 pmol · min1 · mm1
(94% of basal rate, Fig. 1), whereas
107 M reduced it to 6.0 ± 0.1 pmol · min1 · mm1
(46% of basal rate), and
106 M reduced it to 0.5 ± 0.1 pmol · min1 · mm1
(4% of basal rate, with each change significant at
P < 0.05). Associated with the
inhibition was the progressively increasing concentration of
HCO3 in collected fluid, despite comparable flow rates, such that, at
106 M, the perfused and
collected fluids had equal concentrations of
HCO3. With the reduction of
electrogenic H+ secretion (43),
luminal CA inhibition reduced
Vte from a
baseline of 3.6 ± 0.1 mV to 3.5 ± 0.1 with
108 M, 3.1 ± 0.4 mV
with 107 M, and 2.5 ± 0.1 mV with 106 M (each
change significant at P < 0.05).
Addition of CA (5 mg/ml or 167 µM) to the luminal fluid containing
106 M benzolamide reversed
the inhibition of transport and restored it to 11.3 ± 0.2 pmol · min1 · mm1
(88% of basal rate but significantly lower,
P < 0.01) and restored collected
fluid HCO3 concentration to 20.5 ± 0.2 mM (P < 0.01). Voltage was also
increased by luminal CA to 3.3 ± 0.1 mV
(P < 0.01 but significantly less
than baseline; P < 0.05). The
approximate Ki
for inhibition of HCO3 transport by
benzolamide was 107 M.
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Benzolamide titration in tubules from acidotic
animals. A similar benzolamide titration was performed
in OMCD segments from acid-treated animals; basal transport rate was
higher than controls by 25% (P < 0.05) (Table 1, Fig. 1). In contrast to what was observed in tubules
from control animals, 108 M
benzolamide had no effect on HCO3
transport in tubules from acid-treated animals (data not shown).
Luminal 107 M benzolamide
only slightly decreased HCO3 transport
from 16.1 ± 0.1 to 16.0 ± 0.1 pmol · min1 · mm1
(99.4% of basal rate, P < 0.05),
whereas 106 M inhibited
transport to 14.4 ± 0.1 pmol · min1 · mm1
(89% of basal rate), and
105 M reduced it to 4.1 ± 0.2 pmol · min1 · mm1
(25% of the basal rate, P < 0.01 for the latter two changes). In keeping with the inhibition of
electrogenic H+ secretion, luminal
CA inhibition reduced
Vte from a
baseline of 5.2 ± 0.4 to 5.1 ± 0.3 mV with
107 M benzolamide, 4.9 ± 0.3 mV with 106 M,
and 3.9 ± 0.2 mV with
105 M (each change
significant at P < 0.05). Collected
fluid concentrations under basal conditions were 1 mM below those of
control tubules and increased with high concentrations of benzolamide
but not >23 mM. Addition of CA (5 mg/ml or 167 µM) to the luminal
fluid containing 105 M
benzolamide reversed the inhibition of transport and restored it to
14.3 ± 0.4 pmol · min1 · mm1
(89% of basal but significantly lower,
P < 0.01) and restored collected fluid HCO3 concentration to
18.9 ± 0.2 mM (P < 0.01).
Voltage was also increased by luminal CA to 4.9 ± 0.4 mV
(P < 0.01 but did not quite reach
baseline levels, P < 0.05). The approximate
Ki for inhibition
of HCO3 transport in acid-treated
OMCDi segments by benzolamide was
5 × 105 M.
F-3500 Studies
F-3500 titration in control tubules. An F-3500 titration of increasing concentration by 10-fold was performed in control tubules (Table 2, Fig. 2). In three paired studies, 1 µM F-3500 in the luminal fluid decreased HCO3 absorption from 12.6 ± 0.2 to
10.7 ± 0.2 pmol · min1 · mm1
(85% of basal rate, P < 0.01),
whereas 10 µM decreased it to 3.6 ± 0.4 pmol · min1 · mm1
(29% of basal rate, P < 0.01), 100 µM decreased it to 2.4 ± 0.1 pmol · min1 · mm1
(19% of basal rate, P < 0.01), and
1 mM decreased it to 2.1 ± 0.2 pmol · min1 · mm1
(17% of basal rate, P < 0.01). With
comparable flow rates, the titration of inhibitor resulted in
increasing collected fluid HCO3
concentrations, which peaked at 1 mM below that of the perfused
HCO3 concentration. The estimated
Ki for inhibition
of HCO3 absorption by F-3500 was 5 µM.
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Consistent with electrogenic H+
secretion mediating HCO3 absorption in
this segment (43), basal
Vte was likewise
inhibited by F-3500. The basal voltage was 3.8 ± 0.1 mV,
which decreased to 3.5 ± 0.1 mV with 1 µM F-3500, to 2.6 ± 0.2 mV with 10 µM, to 2.3 ± 0.2 mV with 100 µM, and to 2.1 ± 0.2 mV with 1 mM F-3500 (all decreases significant,
P < 0.05).
F-3500 titration in tubules from acidotic
animals. In tubules taken from acid-treated animals,
the basal rate of HCO3 absorption was
higher, and the degree of inhibition of
HCO3 absorption by F-3500 was less
than that observed for control tubules (Table 2, Fig. 2), as was seen
for benzolamide. In one segment, 1 µM F-3500 decreased
HCO3 absorption to 99% of basal rate
(data not shown), whereas 10 µM decreased
HCO3 absorption in two segments from
15.9 ± 0.3 to 15.5 pmol · min1 · mm1
(97% of basal rate). In three segments, 100 µM F-3500 decreased HCO3 absorption from 15.9 ± 0.3 to
10.8 ± 0.1 pmol · min1 · mm1
(68% of basal, P < 0.01), and 1 mM
decreased it to 2.7 ± 0.1 pmol · min1 · mm1
(17% of basal rate, P < 0.01). At
comparable flow rates, collected fluid
HCO3 concentrations increased with
increasing dose of inhibitor. The estimated
Ki for inhibiting
HCO3 absorption by F-3500 was 500 µM.
Vte decreased from a baseline value of 4.9 ± 0.2 to 3.9 ± 0.1 mV with 100 µM F-3500 and to 2.8 ± 0.4 mV with 1 mM inhibitor (P < 0.05 for both comparisons).
In tubules from both control and acid-treated animals, the inhibition
at 1 mM (presumed to inhibit 99.2% of CA IV activity) (24) reduced the
HCO3 flux to ~17% of the basal
rate. Interestingly, the residual (uninhibited) flux from acid-treated
animals was still 29% greater than that from control animals,
proportional to the increase in net
HCO3 absorptive flux observed in
tubules from acid-treated animals noted above and previously (43).
Effect of F-3500 and exogenous CA on
HCO3
transport. In these experiments, we attempted to
determine whether exogenous CA could reverse the effects of luminal CA
IV inhibition and thus determine better a role for this membrane-bound
enzyme in mediating transepithelial H+ secretion
(HCO3 absorption). In 10 OMCDi segments, the basal rate of
net HCO3 transport was 12.1 ± 0.3 pmol · min1 · mm1
(Table 3), and this was decreased to 3.8 ± 0.1 pmol · min1 · mm1
(31% of basal rate, P < 0.01),
whereas collected fluid HCO3 concentration was increased 2.5 mM to 23.1 ± 0.1 mM by 10 µM
F-3500. Concomitant with the inhibition of electrogenic
H+ secretion,
Vte decreased
from a baseline of 3.4 ± 0.2 to 1.9 ± 0.2 mV
(P < 0.01). The addition of 1 mg/ml
(33 µM) of exogenous CA (to the luminal fluid containing the CA
inhibitor) restored HCO3 flux to
9.4 ± 0.3 pmol · min1 · mm1,
decreased collected fluid HCO3
concentration to 21.0 ± 0.2 mM, and increased voltage to 2.6 ± 0.2 mV. When 5 mg/ml (167 µM) CA was added in the presence of the
inhibitor, reversal of CA inhibition was nearly complete: flux was 11.2 ± 0.3 pmol · min1 · mm1
(93% of basal), collected fluid HCO3
concentration reached basal levels, and voltage increased to 2.8 ± 0.2 mV. However, both the flux and voltage in the presence of CA + F-3500 were still significantly less than basal values
(P < 0.05).
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Similar experiments addressed the role of CA in mediating
HCO3 transport in eight
OMCDi tubules from acid-treated rabbits (Table 3). The basal rate of
HCO3 transport was 15.0 ± 0.3 pmol · min1 · mm1,
and Vte averaged
4.8 ± 0.3 mV. Compared with control tubules, the inhibition of
HCO3 transport and voltage by 10 µM
F-3500 was quite modest in OMCDi
segments from acid-treated animals [flux, 14.6 ± 0.3 pmol · min1 · mm1
(P < 0.01) but 97% of basal rate;
collected fluid HCO3 concentration,
19.1 ± 0.3 mM; voltage, 4.1 ± 0.3 mV
(P = NS)]. Increasing F-3500 to
100 µM reduced HCO3 flux to 10.5 ± 0.2 pmol · min1 · mm1
(70% of basal rate, P < 0.01),
increased collected fluid HCO3 concentration by 1.5 mM (P < 0.05),
and reduced voltage to 2.7 ± 0.3 mV
(P < 0.01). Adding 5 mg/ml (167 µM) of exogenous CA (to the luminal fluid containing the CA
inhibitor) restored the HCO3 flux to
14.5 ± 0.3 pmol · min1 · mm1
(97% of basal rate), reduced collected fluid
HCO3 to basal levels, and restored the
voltage to 3.2 ± 0.3 mV. However, both the flux and voltage in the
presence of CA + F-3500 were still significantly less than basal values
(P < 0.05).
Polyoxyethylene bis(Acetic Acid) Polymer: Effect of Polymer and
Exogenous CA on HCO3 Transport
in Control Tubules
3 transport, we examined six OMCDi
segments using 10 µM polymer, followed by the addition of exogenous
CA at 1 mg/ml (33 µM; Table 4, Fig.
3). The basal flux was 12.9 ± 0.4 pmol · min1 · mm1,
with a collected fluid HCO3
concentration of 20.1 ± 0.3 mM and
Vte of 3.3 ± 0.2 mV. Addition of polymer had no effect on flux (12.9 ± 0.5 pmol · min1 · mm1),
HCO3 concentration, or voltage (3.1 ± 0.2 mV). Addition of exogenous CA to the perfused polymer also
had no effect on flux (12.8 ± 0.4 pmol · min1 · mm1),
collected HCO3 concentration, or
voltage (3.2 ± 0.2 mV). These studies ruled out a toxic effect of
the polymer to which had been coupled aminobenzolamide. These data also
showed that exogenous CA failed to stimulate
HCO3 absorption, indicating that
endogenous luminal CA was more than adequate to support the observed
rate of transport.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In the lumen of the proximal tubule (8, 29, 31), membrane-bound CA IV
catalyzes the dehydration of carbonic acid (formed from the reaction
between filtered HCO3 and secreted
H+) into
CO2 + water, thereby favoring
further H+ secretion
(HCO3 absorption). Inhibition of this
enzyme with a dextran-bound CA inhibitor reduced
HCO3 reabsorption by 80% (17). These
data suggested that inhibition of the membrane-bound CA would cause an
acid disequilibrium pH to be generated, the opposing gradient of which
would secondarily slow down the rate of
H+ secretion that mediates
HCO3 reabsorption. In the proximal
tubule, inhibition of luminal CA reduced net
HCO3 reabsorption to the same extent
as did a permeant CA inhibitor, but only the former caused an acid
disequilibrium pH because H+
secretion was not substantially inhibited (17). Thus a functional luminal CA is necessary for optimal
HCO3 reabsorption in the proximal
tubule.
In the OMCDi, there is functional
evidence that membrane-bound carbonic anhydrase (CA IV) is present on
the luminal membrane of the OMCDi
(30, 40) to facilitate H+
secretion. There are no studies reported to test the role of luminal CA
on mediating H+ secretion
(HCO3 absorption) in this segment. Previous studies (15, 40) using isolated perfused OMCD segments have
shown that permeant CA inhibitors such as acetazolamide can markedly
inhibit net HCO3 absorption. However, in one study in the outer stripe of the outer medullary collecting duct
(OMCDo), CA inhibition had
little effect on HCO3 transport (28),
despite use of high concentrations of the more potent and lipid-soluble
ethoxzolamide. Presumably, these inhibitors affected both
membrane-bound and cytosolic CA. It was not possible to reconcile these
findings with others showing sensitivity of HCO3 transport to CA inhibitors and
with studies identifying CA in the cytosol and lumen of the OMCD.
Furthermore, none of the studies reported the use of an impermeant CA
inhibitor to separate the roles of luminal and cytosolic CA. Our
studies represent the first to formally establish a role for luminal CA in mediating H+ secretion
(HCO3 absorption) in the rabbit
OMCDi.
In mice genetically deficient in cytosolic CA II, administration of a
CA inhibitor increased urinary HCO3 excretion (6), indicating a major role for CA IV in mediating HCO3 reabsorption. However, a
titration of the luminal enzyme to elicit a
Ki for
HCO3 transport has not been performed.
Benzolamide is a relatively impermeant CA inhibitor (17, 22, 23) that
enters the cell to a lesser extent than does acetazolamide; yet,
benzolamide can still not be rigorously excluded from inhibiting
cytosolic CA as well (14).
The high-molecular-weight CA inhibitor F-3500 was especially prepared
to inhibit solely the luminal enzyme CA IV (10, 24). Rigorous testing
has shown that the agent is not actively taken up by the kidney, does
not cross cell membranes, and is excluded from the intracellular fluid
(24). Because F-3500 has residual contamination by unreacted free
aminobenzolamide up to a maximum of 0.2%, one must calculate whether
cytosolic CA could be inhibited by the free drug. At concentrations of
F-3500 that inhibited most of HCO3
absorption in control tubules (10 µM), the maximal free drug
concentration would be 0.02 µM, not nearly sufficient to fully
inhibit cytosolic CA II.
The residual HCO3 absorptive flux in
OMCD segments from control animals averaged 0.5 ± 0.1 pmol · min1 · mm1
at a benzolamide concentration of
106 M and comprised <5%
of the basal H+ secretion rate.
The titration of benzolamide concentration vs. HCO3 absorptive rate indicated that
the Ki for reducing transport by 50% was ~0.1 µM. This concentration of
benzolamide would be expected to inhibit ~50% of luminal CA of the
tubule (22-24), and, therefore, the substantial inhibition might
reflect inhibition of cytosolic CA II as well. If the concentration of 1 µM benzolamide were achieved in the cytosol, it would be expected to inhibit 95% of cytosolic CA II. On the other hand, the rapid reversal of inhibition with CA perfusion (see
RESULTS and below) tends to rule out
substantial cytosolic CA inhibition. The residual HCO3 absorptive flux in OMCD segments
from control animals that were treated with 1 mM F-3500 was 2.1 ± 0.2 pmol · min1 · mm1,
and this was considered the membrane-bound CA IV uncatalyzed H+ secretory rate. This rate was
16% of the basal rate, similar to the 22% observed in proximal
tubules perfused with a macromolecular CA inhibitor (17). The
Ki for reducing
by 50% the transport with F-3500 was ~5 µM, similar to the
Ki for inhibiting
CA IV (24), indicating that approximately one-half of the luminal membrane activity was inhibited. Thus
H+ secretion
(HCO3 absorption) was extremely
sensitive to luminal CA inhibition within the concentration range of
the Ki.
The rationale for the perfusion of CA simultaneously with CA inhibitor
was to confirm that the depression of transport could be reversed by
competing for binding to the luminal enzyme. In addition, this
experiment allowed us to examine the extent of cytosolic CA inhibition,
which would not be reversible by perfusing with exogenous CA. Indeed,
perfusion with 1 µM benzolamide and 1 mg/ml (33 µM) CA restored
HCO3 absorption to 11.3 ± 0.2 pmol · min1 · mm1
(88% of basal rate), indicating nearly complete reversibility. It also
meant that little cytosolic CA could have been inhibited by
benzolamide. Similarly, with the large CA inhibitor, F-3500, at 10 µM, perfusion with 5 mg/ml (167 µM) CA restored
HCO3 absorption to 11.2 ± 0.3 pmol · min1 · mm1 (93% of basal rate),
indicating near complete reversibility of this inhibitor. It is likely
that little cytosolic CA II was inhibited by free aminobenzolamide in
view of the high percentage of basal rate achieved. On the other hand,
the failure to completely restore HCO3
absorption might indicate residual cytosolic CA II inhibition by
permeant (benzolamide) or unreacted (aminobenzolamide) CA inhibitor.
The secretion of protons by OMCD segments taken from acid-treated
animals was relatively resistant to CA inhibition. As noted previously
(43), OMCDi segments from
acid-treated rabbits absorbed HCO3 at
a 30% higher basal rate than did segments from control animals. With
benzolamide, the
Ki for inhibiting HCO3 transport was increased from 0.1 to 50 µM in acid-treated tubules, whereas, with F-3500, the
Ki was increased from 5 to 500 µM. From the F-3500 titration, the residual flux was
2.7 ± 0.1 pmol · min1 · mm1
or 17% of the basal flux, and this was considered the uncatalyzed H+ secretion
(HCO3 absorption) rate. The residual flux after 105 M
benzolamide was 4.1 ± 0.2 pmol · min1 · mm1
or 25% of the basal flux in a separate set of tubules; the near identity of these percentages suggests that ~20% of the basal flux
is uncatalyzed by luminal CA IV. When CA was perfused simultaneously with benzolamide, HCO3 absorptive flux
was restored to 14.3 ± 0.4 pmol · min1 · mm1
(89% of basal rate); with F-3500, the flux was restored to 14.5 ± 0.3 pmol · min1 · mm1
(97% of basal rate). These studies suggest that there was little inhibition of cytosolic CA II, despite the high concentrations of
inibitors used. The nearly complete reversibility also ruled out a
toxic effect of the agents on tubule function. Whether the CA
inhibitors could have directly inhibited the luminal
H+-ATPase was not formally tested;
however, a previous study of proton pumping in rat renal cortical
endocytotic vesicles (32) showed that ethoxzolamide did not inhibit
proton pumping into endocytotic vesicles. This finding would suggest
that the CA inhibitors did not directly inhibit the luminal
H+-ATPase.
Chronic metabolic acidosis has been shown to result in a threefold
increase in CA IV mRNA in the kidney cortex and outer medulla (47) and
a more than twofold increase in SDS-resistant hydratase activity
(presumably CA IV) in the cortex, particularly in the proximal
convoluted tubule (7); the 138% increase in the OMCD activity failed
to reach statistical significance (7). It is unlikely that such
increases in luminal CA activity could mediate a >50-fold decrease in
sensitivity of HCO3 transport to CA
inhibition in OMCDi segments taken
from acid-treated rabbits.
Studies using the polymer without the sulfonamide also showed no toxic
effect, confirming that the depression of
HCO3 absorption was not due to tubular
damage by the polymer. Because of the stability of
HCO3 transport in the presence of the
polymer (see Fig. 3), plus the previous findings of stable HCO3 absorption after a 3-h incubation
(43), time-control studies without inhibitors were not performed. In addition, we failed to show that exogenous CA further stimulated HCO3 absorption (see Table 4),
indicating adequacy of the endogenous luminal membrane-bound CA IV
activity. Similar findings for HCO3
transport were previously observed in the
OMCDo, which does not express
luminal CA IV (40); however, exogenous CA eliminated the observed
disequilibrium pH. These findings would suggest that the disequilibrium
pH was not large enough to inhibit
HCO3 absorption in the OMCDo. But in the
OMCDi, where luminal CA IV has
been detected functionally, the amount of enzyme is adequate to limit
any fall in luminal pH due to H+
secretion (40), thereby reducing the opposing pH gradient. These and
other studies suggest that luminal CA plays an important role in
mediating HCO3 absorption in segments with high intrinsic rates of H+
secretion, including the S1 and S2 proximal tubules and
OMCDi.
Carbonic anhydrase inhibitors cause metabolic acidosis at doses that
maximally inhibit renal carbonic anhydrase (21, 22). It is known (21,
25) that renal bicarbonaturia induced by acetazolamide is markedly
reduced during acidosis. Indeed, the greater the acidosis, the less the
diuretic effect of CA inhibition. The mechanism for this renal
resistance has not been determined, but it has not been attributed to
decreased filtered load of HCO3. Maren
(21) originally suggested that the participation of CA in renal
acidification is diminished during metabolic acidosis, but the observed
increases in CA activity (7) would suggest a bigger, not smaller, role
for CA during acidosis. Further studies are needed to characterize
luminal CA IV and its sensitivity to inhibition before and during
chronic metabolic acidosis. Alternatively, the pumps secreting protons
in tubules from acidotic animals may be more resistant than controls to
the disequilibrium pH induced by the inhibitors. A formal examination
of proton pumping as a function of luminal pH in the perfused
OMCDi is required to investigate this alternative explanation.
In summary, we have used nonpermeant CA inhibitors to show, for the
first time, that inhibition of luminal CA at a concentration similar to
that of the Ki
resulted in a major reduction in HCO3 absorption in isolated perfused
OMCDi segments. These findings would suggest that luminal CA IV, which comprises <5% of total kidney CA activity (7, 26, 48), might indeed be rate limiting when
renal acidification is increased. Perhaps the modest increase observed
in CA IV activity (7) reflects an appropriate adaptation to chronic
metabolic acidosis. This would be in contrast to the situation for CA
II, which is present in major excess in the kidney and requires a
>99% inhibition for an effect on renal acid-base transport to be
observed (22). In this case, small increases in CA II mRNA and activity
(7, 38) might not be physiologically important in the adaptation to
chronic metabolic acidosis. We have also shown for the first time a
resistance of HCO3 transport to CA
inhibition in tubules taken from rabbits with chronic metabolic
acidosis: the Ki
for reducing HCO3 absorption by 50%
increased by a factor of 100 for F-3500 and by a factor of 500 for
benzolamide. The mechanism for this apparent resistance to CA
inhibition is not entirely clear but may involve changes in
conformation, secondary structure, or glycosylation, which could affect
substrate binding, alter membrane characteristics, or change the
interactions with specific cofactors. Any of these possibilities could
protect the enzyme from inhibition.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful for the technical assistance of A. Kittelberger and J. Toner. We thank Drs. M. Flessner and A. Weinstein for helpful discussions and for reading the manuscript.
| |
FOOTNOTES |
|---|
S. Tsuruoka was supported by a postdoctoral fellowship award from the American Heart Association New York State Affiliate. G. J. Schwartz was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50603.
Address for reprint requests: G. J. Schwartz, Div. of Pediatric Nephrology, Depts. of Pediatrics and Medicine, PO Box 777, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642.
Received 11 June 1997; accepted in final form 22 September 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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|---|
1.
Armitage, F. E.,
and
C. S. Wingo.
Luminal acidification in K-replete OMCDi: contributions of H-K-ATPase and bafilomycin-A1-sensitive H-ATPase.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F450-F458,
1994
2.
Armitage, F. E.,
and
C. S. Wingo.
Luminal acidification in K-replete OMCDi: inhibition of bicarbonate absorption by K removal and luminal Ba.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F116-F124,
1995
3.
Atkins, J. L.,
and
M. B. Burg.
Bicarbonate transport by isolated perfused rat collecting ducts.
Am. J. Physiol.
249 (Renal Fluid Electrolyte Physiol. 18):
F485-F489,
1985
4.
Bastani, B.,
H. Purcell,
P. Hemken,
D. Trigg,
and
S. Gluck.
Expression and distribution of renal vacuolar proton-translocating adenosine triphosphatase in response to chronic acid and alkali loads in the rat.
J. Clin. Invest.
88:
126-136,
1991.
5.
Bengele, H. H.,
J. H. Schwartz,
E. R. McNamara,
and
E. A. Alexander.
Chronic metabolic acidosis augments acidification along the inner medullary collecting duct.
Am. J. Physiol.
250 (Renal Fluid Electrolyte Physiol. 19):
F690-F694,
1986.
6.
Brechue, W. F.,
E. Kinne-Saffran,
R. K. H. Kinne,
and
T. H. Maren.
Localization and activity of renal carbonic anhydrase (CA) in CA-II deficient mice.
Biochim. Biophys. Acta
1066:
201-207,
1991[Medline].
7.
Brion, L. P.,
B. J. Zavilowitz,
C. Suarez,
and
G. J. Schwartz.
Metabolic acidosis stimulates carbonic anhydrase activity in rabbit proximal tubule and medullary collecting duct.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F185-F195,
1994
8.
Brown, D.,
X. L. Zhu,
and
W. S. Sly.
Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells.
Proc. Natl. Acad. Sci. USA
87:
7457-7461,
1990
9.
Burg, M.,
and
N. Green.
Bicarbonate transport by isolated perfused rabbit proximal tubules.
Am. J. Physiol.
233 (Renal Fluid Electrolyte Physiol. 2):
F307-F314,
1977
10.
Conroy, C. W.,
G. C. Wynns,
and
T. H. Maren.
Synthesis and properties of two new membrane-impermeant high-molecular-weight carbonic anhydrase inhibitors.
Bioorg. Chem.
24:
262-272,
1996.
11.
Dobyan, D. C.,
and
R. E. Bulger.
Renal carbonic anhydrase.
Am. J. Physiol.
243 (Renal Fluid Electrolyte Physiol. 12):
F311-F324,
1982
12.
Graber, M. L.,
H. H. Bengele,
E. Mroz,
C. Lechene,
and
E. A. Alexander.
Acute metabolic acidosis augments collecting duct acidification rate in the rat.
Am. J. Physiol.
241 (Renal Fluid Electrolyte Physiol. 10):
F669-F676,
1981
13.
Hamm, L. L.,
K. S. Hering-Smith,
and
V. M. Vehaskari.
Control of bicarbonate transport in collecting tubules from normal and remnant kidneys.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F680-F687,
1989
14.
Kunau, R. T., Jr.
The influence of the carbonic anhydrase inhibitor, benzolamide (CL-11,366), on the reabsorption of chloride, sodium, and bicarbonate in the proximal tubule of the rat.
J. Clin. Invest.
51:
294-306,
1972.
15.
Lombard, W. E.,
J. P. Kokko,
and
H. R. Jacobson.
Bicarbonate transport in cortical and outer medullary collecting tubules.
Am. J. Physiol.
244 (Renal Fluid Electrolyte Physiol. 13):
F289-F296,
1983.
16.
Lonnerholm, G.,
and
P. J. Wistrand.
Membrane-bound carbonic anhydrase CA IV in the human kidney.
Acta Physiol. Scand.
141:
231-234,
1991[Medline].
17.
Lucci, M. S.,
J. P. Tinker,
I. M. Weiner,
and
T. D. DuBose, Jr.
Function of proximal tubule carbonic anhydrase defined by selective inhibition.
Am. J. Physiol.
245 (Renal Fluid Electrolyte Physiol. 14):
F443-F449,
1983
18.
Madsen, K. M.,
and
C. C. Tisher.
Response of intercalated cells of rat outer medullary collecting duct to chronic metabolic acidosis.
Lab. Invest.
51:
268-276,
1984[Medline].
19.
Madsen, K. M.,
and
C. C. Tisher.
Structural-functional relationships along the distal nephron.
Am. J. Physiol.
250 (Renal Fluid Electrolyte Physiol. 19):
F1-F15,
1986
20.
Madsen, K. M., J. W. Verlander, J. Kim, and
C. C. Tisher. Morphological adaptation of the collecting
duct to acid-base disturbances. Kidney
Int. 40, Suppl. 33:
S-57-S-63, 1991.
21.
Maren, T. H.
Carbonic anhydrase inhibition. IV. The effects of metabolic acidosis on the response to Diamox.
Bull. Johns Hopkins Hosp.
98:
159-183,
1956.
22.
Maren, T. H.
Carbonic anhydrase: chemistry, physiology and inhibition.
Physiol. Rev.
47:
595-781,
1967
23.
Maren, T. H.
Benzolamide: a renal carbonic anhydrase inhibitor.
In: Orphan Drugs, edited by F. E. Karch. New York: Dekker, 1982, p. 90-115.
24.
Maren, T. H.,
C. W. Conroy,
G. C. Wynns,
and
D. R. Godman.
Renal and cerebrospinal fluid formation pharmacology of a high-molecular weight carbonic anhydrase inhibitor.
J. Pharmacol. Exp. Ther.
280:
98-104,
1997
25.
Maren, T. H.,
B. C. Wadsworth,
E. K. Yale,
and
L. G. Alonso.
Carbonic anhydrase inhibition. III. Effects of Diamox on electrolyte metabolism.
Bull. Johns Hopkins Hosp.
95:
277-321,
1954.
26.
McKinley, D. N.,
and
P. L. Whitney.
Particulate carbonic anhydrase in homogenates of human kidney.
Biochim. Biophys. Acta
445:
780-790,
1976[Medline].
27.
McKinney, T. D.,
and
M. B. Burg.
Bicarbonate transport by rabbit cortical collecting tubules.
J. Clin. Invest.
60:
766-768,
1977.
28.
McKinney, T. D.,
and
K. K. Davidson.
Bicarbonate transport in collecting tubules from outer stripe of outer medulla of rabbit kidneys.
Am. J. Physiol.
253 (Renal Fluid Electrolyte Physiol. 22):
F816-F822,
1987
29.
Rector, F. C., Jr.,
N. W. Carter,
D. W. Seldin,
and
A. C. Nunn.
The mechanism of bicarbonate reabsorption in the proximal and distal tubules of the kidney.
J. Clin. Invest.
44:
278-290,
1965.
30.
Ridderstrale, Y.,
M. Kashgarian,
B. Koeppen,
G. Giebisch,
D. Stetson,
T. Ardito,
and
B. Stanton.
Morphological heterogeneity of the rabbit collecting duct.
Kidney Int.
34:
655-670,
1988[Medline].
31.
Ridderstrale, Y.,
P. J. Wistrand,
and
R. E. Tashian.
Membrane-associated carbonic anhydrase activity in the kidney of CA II-deficient mice.
J. Histochem. Cytochem.
40:
1665-1673,
1992[Abstract].
32.
Sabolic, I.,
and
G. Burckhardt.
Characteristics of the proton pump in rat renal cortical endocytotic vesicles.
Am. J. Physiol.
250 (Renal Fluid Electrolyte Physiol. 19):
F817-F826,
1986
33.
Satlin, L. M.,
and
G. J. Schwartz.
Cellular remodeling of HCO3-secreting cells in rabbit renal collecting duct in response to an acidic environment.
J. Cell Biol.
109:
1279-1288,
1989
34.
Schuster, V. L.,
G. Fejes-Toth,
A. Naray-Fejes-Toth,
and
S. Gluck.
Colocalization of H+ ATPase and band 3 anion exchanger in rabbit collecting duct intercalated cells.
Am. J. Physiol.
260 (Renal Fluid Electrolyte Physiol. 29):
F506-F517,
1991
35.
Schwartz, G. J.,
J. Barasch,
and
Q. Al-Awqati.
Plasticity of functional epithelial polarity.
Nature
318:
368-371,
1985[Medline].
36.
Schwartz, G. J.,
L. M. Satlin,
and
J. E. Bergmann.
Fluorescent characterization of collecting duct cells: a second H+-secreting type.
Am. J. Physiol.
255 (Renal Fluid Electrolyte Physiol. 24):
F1003-F1014,
1988
37.
Schwartz, G. J.,
A. M. Weinstein,
R. E. Steele,
J. L. Stephenson,
and
M. B. Burg.
Carbon dioxide permeability of rabbit proximal convoluted tubules.
Am. J. Physiol.
240 (Renal Fluid Electrolyte Physiol. 9):
F231-F244,
1981
38.
Schwartz, G. J.,
C. A. Winkler,
B. J. Zavilowitz,
and
T. Bargiello.
Carbonic anhydrase II mRNA is induced in the rabbit kidney cortex during chronic metabolic acidosis.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F764-F772,
1993
39.
Sly, W. S.,
M. P. Whyte,
T. Krupin,
and
V. Sundaram.
Positive renal response to intravenous acetazolamide in patients with carbonic anhydrase II deficiency.
Pediatr. Res.
19:
1033-1036,
1985[Medline].
40.
Star, R. A.,
M. B. Burg,
and
M. A. Knepper.
Luminal pH disequilibrium ammonia transport in outer medullary collecting duct.
Am. J. Physiol.
252 (Renal Fluid Electrolyte Physiol. 21):
F1148-F1157,
1987
41.
Tsuruoka, S.,
and
G. J. Schwartz.
Metabolic acidosis stimulates carbonic anhydrase (CA) IV in rabbit outer medullary collecting duct of the inner stripe (OMCDi) (Abstract).
J. Am. Soc. Nephrol.
7:
1262,
1996.
42.
Tsuruoka, S.,
and
G. J. Schwartz.
Adaptation of rabbit cortical collecting duct HCO3 transport to metabolic acidosis in vitro.
J. Clin. Invest.
97:
1076-1084,
1996[Medline].
43.
Tsuruoka, S.,
and
G. J. Schwartz.
Metabolic acidosis stimulates H+ secretion in the rabbit outer medullary collecting duct (inner stripe) of the kidney.
J. Clin. Invest.
99:
1420-1431,
1997[Medline].
44.
Verlander, J. W.,
K. M. Madsen,
J. K. Cannon,
and
C. C. Tisher.
Activation of acid-secreting intercalated cells in rabbit collecting duct with ammonium chloride loading.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F633-F645,
1994
45.
Wall, S. M.,
M. F. Flessner,
and
M. A. Knepper.
Distribution of luminal carbonic anhydrase activity along the rat inner medullary collecting duct.
Am. J. Physiol.
260 (Renal Fluid Electrolyte Physiol. 29):
F738-F748,
1991
46.
Wall, S. M.,
J. M. Sands,
M. F. Flessner,
H. Nonoguchi,
K. R. Spring,
and
M. A. Knepper.
Net acid transport by isolated perfused inner medullary collecting ducts.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F75-F84,
1990
47.
Winkler, C. A.,
A. M. Kittelberger,
and
G. J. Schwartz.
Expression of carbonic anhydrase IV mRNA in rabbit kidney: stimulation by metabolic acidosis.
Am. J. Physiol.
272 (Renal Physiol. 41):
F551-F560,
1997
48.
Wistrand, P. J.,
and
K.-G. Knuuttila.
Renal membrane-bound carbonic anhydrase. Purification and properties.
Kidney Int.
35:
851-859,
1989[Medline].
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