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1 Department of Clinical Pharmacology, Jichi Medical School, Kawachi, Tochigi 329-0498, Japan; 2 Medical Service, Veterans Affairs Puget Sound Health Care System, University of Washington, Seattle, Washington 98108; and 3 Department of Pediatrics, 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 carbonic anhydrase (CA)
is critical to renal acidification. The role of CA activity on the
basolateral membrane of the proximal tubule has not been defined
clearly. To investigate this issue in microperfused rabbit proximal
straight tubules in vitro, we measured fluid and HCO3
absorption and cell pH before and after the extracellular CA inhibitor
p-fluorobenzyl-aminobenzolamide was applied in the bath to
inhibit only basolateral CA. This inhibitor was 1% as permeant as
acetazolamide. Neutral dextran (2 g/dl, molecular mass 70,000) was used
as a colloid to support fluid absorption because albumin could affect
CO2 diffusion and rheogenic HCO3 efflux.
Indeed, dextran in the bath stimulated fluid absorption by 55% over
albumin. Basolateral CA inhibition reduced fluid absorption (~30%) and markedly decreased HCO3 absorption
(~60%), both reversible when CA was added to the bathing solution.
In the presence of luminal CA inhibition, which reduced fluid (~16%)
and HCO3 (~66%) absorption, inhibition of
basolateral CA further decreased the absorption of fluid (to 74% of
baseline) and HCO3 (to 22% of baseline). CA
inhibition also alkalinized cell pH by ~0.2 units, suggesting the
presence of an alkaline disequilibrium pH in the interspace, which
would secondarily block HCO3 exit from the cell and
thereby decrease luminal proton secretion (HCO3
absorption). These data clearly indicate that basolateral CA has an
important role in mediating fluid and especially HCO3
absorption in the proximal straight tubule.
in vitro microperfusion; acidification; cell pH; inulin; dextran; para-fluorobenzyl-aminobenzolamide; hydratase assay
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CARBONIC ANHYDRASE (CA) is a renal enzyme that is critical to acid-base homeostasis. Up to 5% of renal CA activity is membrane bound, much of which corresponds to CA IV, whereas more than 95% is primarily cytosolic CA II (4, 16, 43, 44). However, in CA II-deficient patients and mice, inhibition of CA activity (presumably membrane-bound CA) diminishes renal acid excretion, indicating a major role in urinary acidification (3, 34). Functional studies in isolated nephron segments have clearly shown the presence of membrane-bound CA activity along the apical membranes of proximal tubules (14) and collecting ducts from the inner stripe of the outer medulla (18, 37) and inner medulla (42).
With respect to the proximal tubule, CA activity has been identified in brush-border and basolateral membranes (3, 16, 24, 43), and these findings have been confirmed by histochemistry in CA II-deficient mice (21). Using a variety of antibodies to membrane-bound CA IV, we (30, 31) and others (5) have detected CA IV on both apical and basolateral membranes of proximal tubules; labeling was heavier in straight (S2) than in convoluted (S1) segments.
The role of basolateral membrane CA in transepithelial fluid and
HCO3 transport by the proximal tubule has not been
functionally characterized. It was the purpose of this study to examine
whether inhibition of basolateral membrane-bound CA affected proximal
tubular handling of H+/HCO3. We
hypothesized that the following three properties would be sensitive to
inhibition of basolateral CA in isolated perfused proximal straight
tubules: 1) fluid absorption, 2) H+
secretion (HCO3 absorption), and 3) cell
pH. By increasing pH in the vicinity of the basolateral membrane and
intercellular space, HCO3 exit via the
Na+-HCO3 cotransporter would be inhibited
and HCO3 would accumulate in proximal tubule cells.
The results indicate that inhibition of basolateral CA results in an
increase in cell pH and a decrease in fluid and HCO3
absorption; the decrease in HCO3 absorption far
exceeded the decrease in fluid absorption. These effects were
completely reversed by adding CA (with the inhibitor) to the
basolateral medium.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolation of proximal straight tubules. Kidneys were obtained from female New Zealand White rabbits weighing 1.5-2.5 kg and maintained on standard laboratory chow plus free access to tap water. Death was accomplished using intracardiac injection of 130 mg pentobarbital sodium after premedication with ketamine (44 mg/kg) and xylazine (5 mg/kg).
Coronal slices (1-2 mm) of the kidneys were 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 (40). From the medullary rays in the mid- cortex, proximal straight tubule segments were isolated. Length was restricted to <1.5 mm to minimize axial differences along the proximal tubule.In vitro microperfusion. In vitro microperfusion was performed according to the method of Burg and Green (7), with modifications (27, 29, 40). An isolated proximal straight tubule segment was rapidly transferred to a 1.2-ml temperature- and environmentally controlled specimen chamber mounted on an inverted microscope and perfused 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 a pH of 7.4 at 37°C. The bathing solution was usually comprised of Burg's solution plus neutral dextran (molecular mass 70,000); initial control studies also used defatted bovine serum albumin at 6 g/dl (7). The specimen chamber was continuously suffused with 94% O2-6% CO2 to maintain the bath pH at 7.4. Bathing solution was continuously exchanged by a peristaltic pump at a rate of 14 ml/h to maintain constant solute concentration.
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 using a high-impedance electrometer (Duo 773, WPI).Fluid absorption.
The collecting end was sealed into a holding pipette using Sylgard 184 (Dow Corning, Midland, MI). The length of each segment was measured
using an eyepiece micrometer. Tubules were equilibrated for 20 min.
[14C]inulin was added to the perfusate at 10 µCi/ml,
yielding ~30 counts · min1 · nl1 and
equilibrated another 20 min. Samples (47 nl) were collected under
water-saturated mineral oil by timed filling of a calibrated volumetric
pipette. Collections were obtained in triplicate, placed in 1 ml of
water plus 6 ml of scintillation solution containing 4 mg Omnifluor
(Packard Bioscience, Groningen, The Netherlands) per milliliter in
toluene-Triton X-100 (2:1 vol/vol), and the beta emission of
14C was counted (Beckman LSC-3500; Aloka, Tokyo, Japan)
(39). Samples of perfusate were handled similarly with the
same pipette. Fluid absorption rate (Jv) was
calculated as
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HCO3 and fluid absorption.
The concentrations of inulin and total CO2 (assumed to be
equal to that of HCO3) in perfusate and collected
fluid were measured in a continuous flow microfluorimeter (Nanoflo;
WPI, Sarasota, FL) (45). Three 47-nl collections were made
per period and stored under water-saturated mineral oil. Aliquots (15 nl) of each collection were analyzed for total CO2 on the
day of the experiment and for inulin on the following day, using
procedures specified by the manufacturer. Samples of perfusate were
processed similarly. Perfusion rate was generally 5-8 nl/min.
HCO3 transport rate
(JHCO
3) was calculated as
![J<SUB>HCO<SUP>−</SUP><SUB>3</SUB></SUB><IT>=</IT>(<IT>V<SUB>i</SUB></IT>[HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>i</SUB>−<IT>V</IT><SUB>o</SUB>[HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>o</SUB>)/<IT>L</IT>](/content/vol280/issue1/fulltext/F146/img002.gif)
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]i and
[HCO3]o are HCO3
concentrations in the perfusate and collected fluid, respectively, and
L is tubular length in millimeters. Positive values of
JHCO3 indicate net
HCO3 absorption.
This experiment was designed to test the effect of inhibiting
basolateral CA on both fluid and HCO3 absorption.
After equilibration, baseline readings were obtained. Then 10 µM
p-fluorobenzyl-aminobenzolamide (38) was added
to the bathing solution, and collections was begun 10 min later for the
second period. This higher concentration of
p-fluorobenzyl-aminobenzolamide was used to inhibit most of
the basolateral enzyme [I50 at 25°C for CA IV is 2 µM
(E. R. Swenson, unpublished observations)]. In the third
period, 1 mg/ml CA (Sigma) was added with the
p-fluorobenzyl-aminobenzolamide to the bathing solution.
This concentration (33 µM) was chosen to exceed by threefold the
concentration of p-fluorobenzyl-aminobenzolamide and thereby
ensure complete binding of the drug and its removal from the bathing
solution. Reversal of the inhibition of the second period would prove
that it is specific to blocking extracellular CA activity. It would
also confirm that there is no residual cytosolic CA inhibition, which
could not be reversed by adding CA to the bathing solution.
An additional experiment was performed to show that
p-fluorobenzyl-aminobenzolamide did not permeate the
proximal tubule cells and thereby did not inhibit cytosolic CA. The
purpose of this experiment was to determine whether addition of the CA
inhibitor to the bath decreases fluid and HCO3
absorption in the presence of luminal
p-fluorobenzyl-aminobenzolamide. After equilibration,
baseline measurements of Jv and
JHCO3 were made. Then,
10 µM p-fluorobenzyl-aminobenzolamide were added to
the luminal fluid. In the third period, 10 µM
p-fluorobenzyl-aminobenzolamide were added to both the
luminal and bathing fluids. Finally, in the last period, in the
presence of 10 µM luminal and basolateral p-fluorobenzyl-aminobenzolamide, 5 mg/ml CA were added to
the bath. This concentration (165 µM) should have completely bound the drug and removed it from the bathing solution, thereby reversing the inhibition of basolateral
p-fluorobenzyl-aminobenzolamide.
Cell pH. After equilibration, a proximal straight tubule was perfused at 5 cm water pressure for 10-15 min with 5-10 µM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester or 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (Molecular Probes, Eugene, OR). Diffusion of the ester into the cytosol followed by de-esterification results in intracellular fluorescence. The distal end of the tubule was allowed to stick to the coverslip, which had been coated with poly-L-lysine (Sigma, St. Louis, MO); this maneuver reduced tubular movement and optimized fluorescent imaging. In general we focused on cells that were 100-200 µm from the tip of the perfusion pipette.
Cell pH was determined by excitation ratiofluorometry (490 nm/445 nm excitation; 520 nm emission), using an intracellular calibration with 10 µM nigericin-high potassium phosphate buffer (9, 27, 32) at the end of the experiment. Fluorescence was detected using a DAGE model 68 SIT camera, a Deltascan dual-monochrometer, and a quartz bifurcated fiberoptic illuminating system, using software provided by the manufacturer (Photon Technology, S. Brunswick, NJ). The system allowed us to examine multiple cells in duplicate 1-s readings by applying "regions of interest" to the captured images and to subtract background fluorescence from these images. By examining cells in focus close to the perfusion pipette and in the wall of the tubule, movement and contaminating fluorescent signals were minimized. Twenty to thirty minutes after washout of the dye, baseline readings were obtained. These were followed by readings 10-15 min after the addition of 10 µM p-fluorobenzyl-aminobenzolamide to the bathing solution (second period). In the third period the inhibitor was removed and baseline readings were obtained again to prove reversibility. In one experiment, CA (1 mg/ml) was added with the inhibitor to show reversibility.Viability. Evidence for viability was derived from the stability of Vte, a lack of inulin leak, and the absence of damaged cells assessed from the inclusion of 0.15 mg/ml Fast green dye. The experiment was discarded if there was any loss of voltage exceeding 1 mV, if leak of inulin exceeded 2%, or if there were green-staining cells in the wall of the tubule.
CA inhibitor assay. To determine whether basolaterally applied p-fluorobenzyl-aminobenzolamide enters the cell, we performed a CA inhibitor assay. Proximal straight tubules (1.5-2.8 mm) were microdissected in PBS containing calcium and transferred into a bath of PBS containing 100 µM of either the putatively impermeant inhibitor p-fluorobenzyl-aminobenzolamide or the very permeant inhibitor acetazolamide. The incubation was performed at room temperature to assure that there was no metabolic uptake of the drugs. After incubation for 15 min in the inhibitor, each tubule was briefly rinsed twice in PBS and transferred to a microcentrifuge tube. The tubules were freeze dried and then resuspended in a 1-2 µl of distilled water. The suspension was heated to 100°C to denature tubular CA activity and further lyse the cells.
The concentration of CA inhibitor was measured using a modification of the micro-method of Maren (15). In brief, one enzyme unit of purified bovine red cell CA was added to 60 µl of distilled water containing 25 mg/l bromthymol blue indicator at
10°C in a reaction
vessel that was continuously gassed with 100% CO2. The low
temperature was used to assure stronger binding of the inhibitor to the
cytosolic CA II in the reaction vessel to increase the assay's
detection of small amounts of inhibitor. The catalyzed CO2
hydration reaction was initiated by the addition of 40 µl of 50 mM
barbital buffer at pH 7.9, and the time was recorded to obtain a color
change of the indicator, which occurs at ~pH 6. Known amounts of the
two inhibitors or the unknowns were preincubated with CA in the
reaction vessel for 2 min with corresponding reductions in the amount
of distilled water to maintain a final volume of 100 µl after the
addition of 40 µl barbital buffer. The reaction times in the presence
of known concentrations of CA inhibitor were used to construct a
standard curve from which the unknown concentrations in the tubular
lysate were measured.
Statistics. Data are presented as means ± SE. Paired comparisons for each tubule were analyzed by paired t-test using statistical software (Microsoft Excel). Significance was asserted when P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Fluid absorption.
Nine proximal straight tubules averaging 1.0 ± 0.1 mm in length
were examined for the effect of dextran and basolateral CA inhibition
on fluid absorption (Fig. 1). Fluid
absorption in the presence of 6 g/dl bovine serum albumin in the bath
averaged 0.42 ± 0.02 nl · min1 · mm
tubule length1. The baseline voltage averaged 3.8 ± 0.1 mV. When albumin was replaced by 2 g/dl dextran, fluid
absorption was increased 55% to 0.66 ± 0.02 nl · min1 · mm tubule
length1 (P < 0.010). There was no
significant change in Vte (3.8 ± 0.1 mV). When 1 µM p-fluorobenzyl-aminobenzolamide was added
to the dextran bath, fluid absorption decreased 23% to 0.50 ± 0.02 nl · min1 · mm1
(P < 0.01) with no change in
Vte.
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absorption across the basolateral
membrane and thereby reduce overall transepithelial fluid and
HCO3 absorption.
Fluid and HCO3 absorption.
We examined nine proximal straight tubules averaging 1.2 ± 0.1 mm
in length for the effect of basolateral CA inhibition on the absorption
of fluid (Fig. 2A) and
HCO3 (Fig. 2B). The baseline data using a
2 g/dl dextran bath revealed a Jv of 0.65 ± 0.04 nl · min1 · tubule
length1 and a HCO3 absorption rate
(JHCO3) of
75 ± 1 pmol · min1 · mm1. In the
presence of 10 µM p-fluorobenzyl-aminobenzolamide in the
bath, there was a 31% decrease in fluid absorption (to 0.45 ± 0.04 nl · min1 · mm1,
P < 0.01) and a 62% decrease in
HCO3 absorption (to 29 ± 3 pmol · min1 · mm1,
P < 0.01). In addition, Vte
became slightly more negative with CA inhibition (from 3.5 ± 0.1 to 3.8 ± 0.1 mV, P < 0.01). When CA (1 mg/ml) was added to the bath with the inhibitor, transport was
restored to baseline (Jv, 0.75 ± 0.06 nl · min1 · mm1, and
JHCO3,
76 ± 2 pmol · min1 · mm1), as was
Vte (3.5 ± 0.1).
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Luminal and basolateral CA inhibition.
We determined in four tubules averaging 1.1 ± 0.1 mm in length
the additive effects of 10 µM
p-fluorobenzyl-aminobenzolamide added to the lumen and then
to the bath on the absorption of fluid (Fig.
3A) and HCO3
(Fig. 3B). The baseline data using a 2-g/dl dextran
bath revealed a Jv of 0.61 ± 0.01 nl · min1 · mm1 tubule
length and a JHCO3 of
72 ± 2 pmol · min1 · mm1. In the
presence of luminal p-fluorobenzyl-aminobenzolamide, there
was a 16% decrease in fluid absorption (to 0.51 ± 0.01 nl · min1 · mm1,
P < 0.01) and a 66% decrease in
HCO3 absorption (to 25 ± 3 pmol · min1 · mm1,
P < 0.01). In addition,
Vte became less negative with luminal CA
inhibition (from 3.6 ± 0.1 to 3.4 ± 0.1 mV,
P < 0.01). Then 10 µM
p-fluorobenzyl-aminobenzolamide were added to the bathing solution so that both luminal and basolateral CA were inhibited. There
was a further decrease in fluid absorption (to 0.45 ± 0.01 nl · min1 · mm1,
P < 0.05), HCO3 absorption (to
16 ± 2 pmol · min1 · mm1,
P < 0.01), and Vte (to
3.2 ± 0.1 mV, P < 0.01). With inhibition of
both luminal and basolateral CA, the residual rates of transport were
74% for fluid absorption and 22% for HCO3
absorption. When CA (5 mg/ml) was added to the bath with the inhibitor,
there was restoration of fluid (to 0.52 ± 0.01 nl · min1 · mm1,
P < 0.05) and HCO3 absorption (to
24 ± 3 pmol · min1 · mm1,
P < 0.01) to the level of solely luminal CA
inhibition. The increase in Vte did not quite
reach significance (to 3.3 ± 0.1 mV, 0.05 < P < 0.1).
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Cell pH.
Twenty-five cells in five tubules showed a baseline pH of 7.15 ± 0.03 units (Fig. 4A). The
addition of 10 µM p-fluorobenzyl-aminobenzolamide to the
bath caused a significant alkalinization to 7.34 ± 0.03 units
(P < 0.01). When the inhibitor was washed out, the
baseline pH of 7.15 ± 0.04 was restored. In six cells (Fig.
4B), the addition of 1 mg/ml CA to the bath in the presence
of p-fluorobenzyl-aminobenzolamide almost completely
restored cell pH back to the baseline (baseline, 7.15 ± 0.02; CA
inhibitor, 7.38 ± 0.02; inhibitor + CA, 7.21 ± 0.02, each change was significant).
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Comparison of p-fluorobenzyl-aminobenzolamide and acetazolamide
permeabilities.
Proximal straight tubules were exposed to 100 µM
p-fluorobenzyl-aminobenzolamide or acetazolamide for 15 min
and assayed for total cellular CA inhibitor (Table
1). Six tubules averaging 1.8 mm and
exposed to acetazolamide had a mean concentration of 96 ± 4 µM,
similar to the bathing concentration and suggesting complete
equilibration of the tubular cytoplasm. Seven tubules averaging 1.8 mm
and exposed to p-fluorobenzyl-aminobenzolamide had a mean
concentration of 1.4 ± 0.2 µM. Two of these tubules had
concentrations below 0.8 µM, the limit of the assay, but in the
calculations were considered to equal 0.8 µM. Therefore, the mean
concentration of p-fluorobenzyl-aminobenzolamide was at most 1.5% of that achieved with acetazolamide, indicating that the permeability of p-fluorobenzyl-aminobenzolamide was ~1/100
of that of acetazolamide.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Functional basolateral CA.
These studies have examined in four different ways the functional
presence of basolateral CA in the proximal straight tubule. We have
made use of a newly developed impermeant CA inhibitor, which has been
recently synthesized (38). In the first series we have
shown that basolateral p-fluorobenzyl-aminobenzolamide inhibited fluid absorption. In the second,
p-fluorobenzyl-aminobenzolamide inhibited the absorption of
fluid and HCO3 to a greater extent. The decrement in
Jv was 31%, whereas the decrease in
JHCO3 was 62%,
twice that for Jv. In the third series we have
shown an additive inhibition when
p-fluorobenzyl-aminobenzolamide was first applied to the perfusate and then to the bathing fluid, indicating the functional presence of CA on each membrane. The overall decrement in
Jv was only 26%, whereas the decrease
in JHCO3 was
78%, nearly three times that for Jv. The
sequential decreases with luminal and basolateral
p-fluorobenzyl-aminobenzolamide indicate separate roles for
both luminal and basolateral CA in mediating fluid and
HCO3 transport. In addition, this latter study shows
that the luminal and basolateral inhibitors are confined to the
membrane of application and do not permeate the cells.
3. The
nearly complete inhibition of
JHCO3 (compared
with 78% inhibition after both membrane CAs were inhibited) is
probably explained by the concomitant inhibition of cytosolic CA, as
well as membrane CAs. Presumably, these findings relate to the
dependence of HCO3 absorption on proton secretion via
the Na+/H+ exchanger, which requires cytosolic
CA to generate protons and HCO3 within the cell,
according to
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. Inhibition of the cytosolic enzyme would impair
the generation of protons for secretion into the lumen. In contrast,
fluid absorption is mediated by a variety of sodium-coupled ion
transporters, only one of which is the above-noted
Na+/H+ exchanger.
The reversal of CA inhibition, by adding CA to the inhibitor in the
bathing solution, indicates that only basolateral and not cytosolic CA
was inhibited. What would be the consequence of inhibiting basolateral
CA? To answer this question requires one to examine the
HCO3 exit step across the basolateral membrane. This
exit is primarily via the Na+-HCO3
cotransporter (22, 28, 36). It is likely that this
cotransporter involves the cotransport of Na+,
CO32, and HCO3 on
distinct sites (36).
Electrophysiological analyses (2, 6, 25) have indirectly
shown that membrane-bound CA is involved in passive rheogenic HCO3 transfer across the peritubular membrane. In
addition, HCO3 transport across basolateral membrane
vesicles can be inhibited by acetazolamide (11, 20, 35).
This finding suggests a role for membrane-bound CA in facilitating the
rheogenic transfer of HCO3 across the peritubular
membrane (6, 35). Inhibition of CA causes alkalinization
of proximal tubular cells (13), which would in turn
inhibit luminal H+ secretion. Indeed, we have observed
decreased HCO3 absorption (H+ secretion)
in the presence of basolateral CA inhibition (see Fig. 2B).
The mechanism for this decrease in proximal tubular HCO3 absorption in the setting of basolateral CA
inhibition has been reviewed by Seki et al. (33) and may
result from development of a disequilibrium
CO32 (alkaline pH disequilibrium) in the
basal labyrinth due to the accumulation of large amounts of
CO32 via the basolateral
Na+-HCO3 cotransporter (20).
Because the CO32 equilibrium
concentration in the physiological Ringer solution is only 20-80
µM, most of the CO32 generated must be
converted to HCO3, according to
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can only continue
when additional H+ are generated from H2O and
CO2. If the putative basolateral CA were inhibited, the
supply of H+ would be limited and local
CO32 gradients would build up in the
intercellular space to inhibit the
Na+-HCO3 cotransporter and secondarily
luminal H+ secretion. Indeed, there are recent data showing
that raising extracellular pH directly inhibits the activity of the
Na+-HCO3 cotransporter expressed in
oocytes (23), as suggested from our studies. By preventing
the rheogenic exit of HCO3, inhibition of basolateral
CA would also raise cell pH, as observed in our cell pH studies (see
Fig. 4A). Our results are compatible with the prediction
that CO32 is cotransported with
Na+ and HCO3 via the
Na+-HCO3 cotransporter (36).
Alkalinization of the cell by basolateral CA inhibition also may have a
direct effect on electroneutral NaCl and fluid absorption, mediated via
the basolateral Na+-dependent
Cl/HCO3 exchanger (1),
which is known to be functional in the proximal straight tubule
(26). As cell pH is increased, the driving force for
chloride exit is decreased, so that overall electroneutral NaCl
transport is inhibited. This could further decrease fluid and
HCO3 absorption.
Histological correlations. Correlation with histochemical or immunocytochemical evidence for basolateral CA certainly strengthens our findings. Ridderstrale et al. (21) used a modified cobalt phosphate method to detect CA activity in CA II-deficient mice. This histochemical method does not usually allow one to distinguish cytosolic from membrane-bound CA activity, but in the absence of the cytosolic enzyme, as in these mice, membrane staining is nearly unambiguous. They found clear evidence for both apical and basolateral staining of proximal tubules in the cortex. Careful attention to the figures suggests to us that the staining of the basolateral moiety was heavier than that of the brush border. Immunocytochemical studies using antibodies to CA IV have shown basolateral labeling in proximal tubules of rats (5) and rabbits (30, 31), but the staining appears to be heavier in the brush border than in the basolateral membranes.
At present it is not possible to reconcile these discrepancies between apical and basolateral membranes, but one possibility is that the CA activity on the latter membrane is not immunologically CA IV. The recent finding of other membrane-bound CAs [CA XII (41) and CA XIV (19)] suggests the possibility that one of these could be resident on the basolateral membrane. They could be present in addition to CA IV or could be cross-reacting with antibodies generated against CA IV. Further studies will be necessary to determine the molecular identity of the basolateral CA activity.Dextran and fluid absorption.
Proximal tubular fluid absorption is known to be enhanced by adding
albumin to the bathing solution (8, 10). However, our
studies utilizing CA inhibition in the interspace were likely to lead
to a buildup of carbonate and a possible disequilibrium alkaline pH. In
view of the interrelationship between HCO3 and
CO2 and the rheogenic nature of HCO3
exit, it seemed important to eliminate albumin from the bathing solution. Albumin is known to facilitate CO2 diffusion by
carrying protons in parallel with the diffusion of
HCO3 (12); this movement of albumin
would substantially boost the diffusion constant of CO2
diffusing across the interspace and might offset some of the inhibition
of CA. In addition, the negative charge of albumin might have
inhibitory effects on fluid and HCO3 absorption due
to the electrogenic nature of HCO3 exit via the
Na+-HCO3 cotransporter.
absorption; further studies are needed to
understand the mechanism for this increase in fluid transport.
In the presence of dextran, basolateral CA inhibition resulted in a
25-33% inhibition of fluid absorption, probably because of the
buildup of carbonate in the interspace. The alkalinization of the
interspace after CA inhibition also could have increased tight junction
permeability, resulting in a decrease in fluid absorption due to
enhanced backflux. Evidence against this possibility is derived from
the lack of change of the Vte. In the setting of
increased leakiness of the tight junction one would have expected to
observe a less negative Vte. However, we found
that Vte either became more negative or failed
to change after basolateral CA inhibition.
Permeability of p-fluorobenzyl-aminobenzolamide.
Our assay of CA-inhibitor permeability showed that
p-fluorobenzyl-aminobenzolamide is 1% as permeable as is
acetazolamide across the basolateral membrane of proximal straight
tubules. It was assumed that all of the drug had diffused into cellular cytoplasm; however, there could be some binding to the basolateral CA
as well. The contribution of membrane CA activity has been estimated to
be at most 10% of total CA hydratase activity (4), so
that the intracellular concentration is probably very close to the
measured concentration. Because the transport and cell pH experiments
were performed using 10 µM of
p-fluorobenzyl-aminobenzolamide, the estimated maximal
intracellular concentration would be 107 M, not high
enough to substantially inhibit cytosolic CA activity. Thus these
experiments are in agreement with other results obtained in this study,
which showed that CA applied to the bath medium reversed the inhibitory
effect of basolateral p-fluorobenzyl-aminobenzolamide. The
reversal could not have occurred if cytosolic CA were also inhibited by
the p-fluorobenzyl-aminobenzolamide. Thus
p-fluorobenzyl-aminobenzolamide is an impermeant CA inhibitor.
absorption
and secondarily affects cell pH. These results are consistent with
previous electrophysiological and membrane vesicle investigations, as
well as with more recent histochemical and immunocytochemical studies.
Alkalinization of the interspace is likely to inhibit basolateral
Na+-HCO3 cotransport, which would
decrease active transport. Alkalinization of the cell would decrease
the concentration of protons available for luminal
HCO3 reabsorption via the apical
Na+/H+ exchanger and decrease the cellular exit
of chloride, the latter reducing electroneutral NaCl transport. These
effects would lead to decreased fluid and HCO3
reabsorption. The magnitude of the effect of inhibiting basolateral CA
attests to the importance of membrane-bound CA in the region of the
proximal tubule cell interspace. Further studies are needed to
determine the molecular identity of the CA that is resident there.
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ACKNOWLEDGEMENTS |
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We are grateful to A. Kittelberger for technical assistance, Dr. M. Imai for providing microperfusion equipment, and Dr. A. Weinstein for critically reviewing the manuscript.
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FOOTNOTES |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50603 to G. J. Schwartz, National Heart, Lung, and Blood Institute Grant HL-45571 to E. R. Swenson, and a grant from the Ministry of Education, Science and Culture of Japan to S. Tsuruoka.
Address for reprint requests and other correspondence: G. J. Schwartz, Div. of Nephrology, Box 777 Univ. of Rochester School of Medicine, 601 Elmwood Ave, Rochester, NY 14642 (E-mail: george_schwartz{at}urmc.rochester.edu).
1
In one preliminary experiment in a medullary
collecting duct from the inner stripe, we titrated the effect of
p-fluorobenzyl-aminobenzolamide on net
HCO3 absorption. Previous studies (40)
have shown that benzolamide at 1 µM inhibited 96% of
HCO3 absorption, and this was reversed to 88% of
control levels by adding CA to the perfusate. The baseline
HCO3 absorptive rate in our medullary collecting duct
was 12.6 pmol · min1 · mm tubular
length1, and this was reduced to 5.6 pmol (45% of
baseline) with 1 µM p-fluorobenzyl-aminobenzolamide and to
0.7 pmol (5% of baseline) with 10 µM
p-fluorobenzyl-aminobenzolamide. Simultaneous perfusion of
10 µM p-fluorobenzyl-aminobenzolamide and 1 mg/ml CA
restored the flux to 11.2 pmol · min1 · mm1 (89% of
baseline). Voltage studies confirmed the decrease in electrogenic
H+ secretion by inhibiting CA: baseline, 3.5 mV; 1 µM
inhibitor, 2.7 mV; 10 µM inhibitor, 1.7 mV. The addition of CA (1 mg/ml) to the 10 µM inhibitor restored the voltage to 2.8 mV. These
data allowed us to choose a concentration of 10 µM
p-fluorobenzyl-aminobenzolamide to inhibit basolateral CA
and determine its role in mediating HCO3 absorption
by proximal straight tubules.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 May 2000; accepted in final form 19 September 2000.
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TOP
ABSTRACT
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METHODS
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DISCUSSION
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