Department of Pediatrics, University of Rochester School of Medicine, Rochester, New York 14642
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ABSTRACT |
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The connecting
tubule (CNT) contains
-(H+-secreting) and
-(HCO
3-secreting) intercalated
cells and is therefore likely to contribute to acid-base homeostasis.
To characterize the mechanisms of HCO
3
transport in the rabbit CNT, in which there is little definitive data
presently available, we microdissected the segments from the
superficial cortical labyrinth, perfused them in vitro, measured net
HCO
3 transport (JHCO
3)
by microcalorimetry, and examined the effects of several experimental
maneuvers. Mean ± SE basal
JHCO
3 was
3.4 ± 0.1 pmol · min
1 · mm
1
(net HCO
3 secretion), and
transepithelial voltage was
13 ± 1 mV
(n = 47). Net
HCO
3 secretion was markedly inhibited
by removal of luminal Cl
or
application of basolateral
H+-ATPase inhibitors (bafilomycin
or concanamycin), maneuvers that inhibit
-intercalated cell
function. Net HCO
3 secretion was not
affected by inhibitors of
-intercalated cell function (basolateral
Cl
removal, basolateral
DIDS, or luminal H+-ATPase
inhibitors). Net HCO
3 secretion was stimulated by isoproterenol and inhibited by acetazolamide. These data
indicate that 1) CNTs secrete
HCO
3 via an apical DIDS-insensitive
Cl
/HCO
3
exchanger, mediated by a basolateral bafilomycin- and
concanamycin-sensitive H+-ATPase;
2) inhibition of cytosolic carbonic
anhydrase decreases HCO
3 secretion;
and 3) stimulation of
-adrenergic receptors increases HCO
3 secretion.
The failure to influence net HCO
3
transport by inhibiting
-intercalated cell apical
H+-ATPases or basolateral
Cl
/HCO
3
exchange suggests that the CNT has fewer functioning
-intercalated
cells than the cortical collecting duct. These are the
first studies to examine the rate and mechanisms of
HCO
3 secretion by the rabbit CNT; this
is clearly an important segment in mediating acid-base homeostasis.
intercalated cell; distal nephron; collecting duct; hydrogen-adenosine 5'-triphosphatase; carbonic anhydrase; chloride/bicarbonate exchange; isoproterenol; microperfusion in vitro; bafilomycin; concanamycin; acetazolamide; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; Sch-28080
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INTRODUCTION |
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THE CONNECTING TUBULE (CNT) joins distal tubules to the cortical collecting duct (CCD) (16). The CNT contains two types of cells: 1) the CNT cell and 2) the intercalated cell. The ratio of CNT cells to intercalated cells is ~5:4 (16). With this relatively large number of intercalated cells, it is likely that the CNT plays an important role in acid-base homeostasis.
There are two major types of intercalated cells:
1) -intercalated cells secrete
H+, and
2)
-intercalated cells secrete
HCO
3. Therefore, the relative
distribution of intercalated cell subtypes in the rabbit CNT may
determine the overall direction of acid-base transport. A morphological
characterization of the CNT by Verlander et al. (43) using
colocalization of carbonic anhydrase II and band 3 (anion exchanger
AE1) to identify
-intercalated cells and carbonic anhydrase II and
peanut lectin to identify
-intercalated cells showed that ~50% of
the intercalated cells in the CNT were
-intercalated cells. On the
other hand, Muto et al. (27) published a microelectrode study using
luminal reduction of chloride to hyperpolarize
-intercalated cells,
but not
-cells, and showed that 97% of intercalated cells were
-type. Using a monoclonal antibody (B63) directed against the apical
membrane of peanut lectin agglutinin-selected cells of the rabbit
kidney cortex, Fejes-Toth et al. (9) found substantial labeling of
-intercalated cells in both the CNT and CCD.
Several studies have compared other functions of the CNT with those of
the CCD. Imai (15) showed that the transepithelial voltage of the CNT
was 27.0 ± 2.7 mV compared with
3.5 ± 2.1 mV in
the CCD. Also, the voltage response to isoproterenol is at least an
order of magnitude more sensitive in the CNT. Compared with the CCD
(32), the rate of Na+ absorption
is approximately three to four times larger in the CNT (1), and that of
K+ secretion is at least as large
in the CNT (17). Thus the CNT appears to play a major role in the renal
handling of several electrolytes.
In view of our interest in the types of functional intercalated cell
types present in this segment, we chose to measure net HCO3 transport in isolated perfused
CNTs. We also attempted to characterize the mechanisms of
HCO
3 transport and the relative
contributions of
- and
-intercalated cells to the overall
HCO
3 flux. Lastly, we tested whether
-adrenergic agents stimulate HCO
3
secretion in this segment.
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MATERIALS AND METHODS |
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Animals. Female New Zealand White rabbits weighing 1.8-2.8 kg and maintained on normal laboratory chow (Purina lab diet 5326; Purina Mills, Richmond, IN) plus free access to tap water were used. The animals were killed by intracardiac injection of 130 mg pentobarbital sodium after premedication with ketamine (44 mg/kg) and xylazine (5 mg/kg).
Tubule isolation and microperfusion. 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 (41, 42). CNT segments were microdissected from the superficial cortical labyrinth, because of the infrequent branching of the superficial compared with the deep cortical CNT (15).
In vitro microperfusion was performed according to the method of Burg and Green (5) with modifications (40-42). An isolated CNT 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/kg H2O, and gassed with 94% O2-6% CO2, yielding a pH 7.4 at 37°C (40-42). The specimen chamber was continually suffused with 94% O2-6% CO2 to maintain pH at 7.4 (34). The collecting end of the segment was sealed into a holding pipette using Sylgard 184 (Dow Corning, Midland, MI). The perfused 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 were generally made in triplicate for each experimental period. The bathing solution was exchanged by a peristaltic pump at a rate of 14 ml/h to maintain constant solute concentrations.HCO3 transport.
The concentration of total CO2
(assumed to be equal to that of HCO
3)
in the 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 CNT (15, 17, 31, 37) the rate of
HCO
3 transport
(JHCO
3) was calculated as
JHCO
3 = (C0
CL) × (VL/L),
where VL is the rate of collection of
tubular fluid (~1.5 nl/min), L is
tubular length (in mm), and J is in
pmol · min
1 · mm
1
tubular length. When
JHCO
3
is more than 0 there is net HCO
3
absorption, when
JHCO
3 is less than 0 there is net HCO
3
secretion. The sensitivity of the Picapnotherm was 10-20
counts/pmol HCO
3, so that for samples
of 14.5 nl, there were 145-290 counts/mM. The coefficient of
variation for a 20 mM standard measured in quadruplicate was <1%
(<45 counts/sample of 4,500 counts). This level of sensitivity
allowed us to reliably detect differences of 1 mM
HCO
3 between perfused and collected fluids. In practice, we perfused tubules at ~1.5 nl/min, which corresponded to a flow rate of 3.5-4
nl · min
1 · mm
1
and generally resulted in a difference of ~1 mM between perfused and
collected fluids under basal conditions. Each sample was determined in
the Picapnotherm immediately after collection. The perfusate HCO
3 concentration, measured at the
beginning and end of each experiment, averaged 24.4 mM.
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, Sarasota, FL). Collections of tubular fluid were initiated once the Vte had stabilized (45-60 min), and readings were recorded at the conclusion of each collection.
Viability. Evidence for damaged cells and gross leak of perfusate was continually assessed by the inclusion of 0.15 mg/ml Fast green dye (Sigma) to each perfusate during the study (40, 41). The experiment was discarded if tubular damage was detected.
Experimental protocols.
Net HCO3 flux was first measured under
basal conditions (Burg's solution in lumen and bath) and then after removal of Cl
or another
experimental maneuver. Calcium concentration was raised to 6 mM in
chloride-free solutions to allow for additional complexing by the
substitute gluconate (37, 40, 41).
Chemicals. Bafilomycin A1, DIDS, isoproterenol, and acetazolamide were purchased from Sigma Chemical (St. Louis, MO); concanamycin was purchased from Fluka Biochemical (Ronkonkoma, NY). All other reagents and chemicals were of analytic grade and purchased from Sigma. Each agent was dissolved in DMSO at 0.1% final concentration.
Analysis and statistics. Data are presented as means ± SE; n is the number of animals studied. Paired comparisons for each tubule were analyzed by paired t-test, and comparisons between tubules from control vs. other segments were analyzed by unpaired t-tests and one-way ANOVA plus the Tukey-Kramer or Bonferroni tests for multiple comparisons. Statistical software was used (Number Cruncher Statistical Software, Kaysville, UT). Significance was asserted when P < 0.05.
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RESULTS |
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Baseline data.
Net HCO3 secretion was observed in all
47 CNT segments (Fig. 1, Table
1). The mean net
HCO
3 flux was
3.38 ± 0.09 pmol · min
1 · mm
1,
not significantly different from
3.87 ± 0.23 pmol · min
1 · mm
1
observed recently in 29 control CCDs (40). Both CNT and CCD were
significantly different from our previously published results in outer
medullary collecting ducts from the inner stripe, which absorbed net
HCO
3 at a rate of 12.8 ± 0.3 pmol · min
1 · mm
1
(n = 25) (41).
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Effect of luminal Cl removal.
To test the Cl
dependence
of HCO
3 secretion in perfused CNT
segments, we replaced all luminal
Cl
by gluconate (Fig.
2, Table 1). This maneuver would presumably inhibit the secretion of HCO
3 in
exchange for Cl
in
-intercalated cells. The secretion of
HCO
3 was markedly inhibited in the
absence of luminal Cl
(
3.52 ± 0.15 pmol · min
1 · mm
1
to
0.17 ± 0.04, n = 5, P < 0.001), and there was no
evidence for underlying net H+
secretion (positive HCO
3 flux) by
-intercalated cells. Associated with the loss of
HCO
3 secretion was a decrease in
collected fluid HCO
3 concentration from 25.5 ± 0.1 mM to virtually the perfusate concentration of 24.3 ± 0.1 mM. Allowing for the generation of a liquid junction potential of 6.8 mV (38), there was no significant change in Vte with
Cl
removal (
12.7 ± 0.7 mV to
12.3 ± 0.6). This result was consistent with
electroneutral HCO
3 secretion mediated by
-intercalated cells in the CNT.
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Effect of bath Cl removal.
We next tested whether inhibition of basolateral anion exchange,
which is normally active in
-intercalated cells, would have any
effect on net HCO
3 flux (Fig.
3, Table 1). There was no significant
change in the rate of HCO
3 secretion
(
3.41 ± 0.22 pmol · min
1 · mm
1
to
3.43 ± 0.23, n = 5) or
collected fluid HCO
3 concentration
(25.4 ± 0.1 mM to 25.4 ± 0.1) with bath
Cl
removal; however there
was a small significant decrease in
Vte (
11.7 ± 0.6 mV to
11.4 ± 0.6). The relative magnitude of the increase in HCO
3 secretion, as
-intercalated cell H+ secretion
was presumably inhibited by bath
Cl
removal, was <1%. The
results of this protocol suggested that countervailing
H+ secretion by
-intercalated
cells was not detectable.
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Effect of luminal DIDS.
We used 50 µM of the stilbene DIDS to try and inhibit the apical
anion exchanger of -intercalated cells (Table 1), realizing that
this agent does not inhibit HCO
3
secretion by CCD
-intercalated cells (30). Indeed, DIDS in the lumen did not significantly change net HCO
3
flux (
3.82 ± 0.22 pmol · min
1 · mm
1
to
3.82 ± 0.23, n = 4) or
Vte
(
12.8 ± 0.6 mV to
12.8 ± 0.6). There
was a small decrease in collected fluid
HCO
3 concentration (25.5 ± 0.1 mM
to 25.3 ± 0.1, P < 0.01) that
was probably associated with a minimal increase in flow rate. The results of this protocol suggested that the apical anion exchanger in
-intercalated cells was DIDS-insensitive in the CNT, as in the CCD.
Effect of bath DIDS.
To further examine the role of -intercalated cells in contributing
to net HCO
3 transport, we inhibited the basolateral anion exchanger AE1 with 50 µM DIDS (Table 1). Indeed, bath DIDS had no significant effect on net
HCO
3 flux (
2.61 ± 0.23 pmol · min
1 · mm
1
to
2.66 ± 0.31, n = 6),
collected fluid HCO
3 concentration
(25.1 ± 0.1 mM to 25.1 ± 0.2), or
Vte (
13.7 ± 0.6 mV to
13.0 ± 0.7). The relative magnitude of the
increase in HCO
3 secretion, as
-intercalated cell H+ secretion
was inhibited by DIDS, was only 2% and not significant. These results
suggested that
-intercalated cells did not contribute substantially
to the overall net HCO
3 flux.
Effect of luminal bafilomycin.
By inhibiting the luminal
H+-ATPase with bafilomycin, we
were able to further assess the contribution of -intercalated cells to net HCO
3 transport (Table 1). In
keeping with the previous studies showing little effect of inhibiting
-intercalated cell function, luminal bafilomycin caused a small (1%) but significant increase in net
HCO
3 secretion (
3.91 ± 0.27 pmol · min
1 · mm
1
to
3.95 ± 0.27, n = 4, P < 0.05), and no change in
Vte (
14.4 ± 0.3 mV to
14.3 ± 0.3). There was a minimal decrease in
collected fluid HCO
3 concentration
(25.5 ± 0.1 mM to 25.4 ± 0.2, P < 0.01), which was also associated
with a comparably lower perfusate HCO
3
concentration (24.4 ± 0.1 mM vs. 24.2 ± 0.2, P < 0.05). These data did not
suggest a major contribution of
-intercalated cells to mediating
HCO
3 transport in the CNT.
Effect of bath bafilomycin and concanamycin.
It is believed that -intercalated cells have a basolateral
H+-ATPase to generate
HCO
3 for secretion into the luminal
fluid. If this pump were inhibited by 5 nM bafilomycin, then net
HCO
3 secretion by
-intercalated
cells would also be inhibited (Fig. 4,
Table 1). Indeed, bath bafilomycin completely inhibited net
HCO
3 secretion (
3.63 ± 0.34 pmol · min
1 · mm
1
to
0.14 ± 0.05, n = 5, P < 0.001), reduced collected fluid
HCO
3 concentration to the level of the
perfusate concentration (25.5 ± 0.1 mM to 24.3 ± 0.1, P < 0.01) and diminished the
Vte (
13.3 ± 1.1 mV to
5.0 ± 1.4, P < 0.001). The inhibition of the
electrogenic basolateral H+-ATPase
would be expected to reduce luminal electronegativity in addition to
obliterating HCO
3 secretion.
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Effect of luminal Sch-28080.
It is believed that -intercalated cells have an apical gastric-like
H+-K+-ATPase
that may also mediate H+ transport
and would be sensitive to the inhibitor Sch-28080 at 10 µM (36, 46).
If this pump were inhibited, then net
HCO
3 secretion by
-intercalated
cells would be stimulated (Table 1). We found, however, that luminal
Sch-28080 had no significant effect on net
HCO
3 transport (
3.62 ± 0.32 pmol · min
1 · mm
1
to
3.64 ± 0.31, n = 5) or
collected fluid HCO
3 concentration
(25.3 ± 0.1 mM to 25.3 ± 0.1), and a minimal effect on
Vte (
13.4 ± 0.7 mV to
13.2 ± 0.7, P < 0.05). These results showed that
the apical
H+-K+-ATPase
did not play a major role in HCO
3
transport under baseline conditions in the CNT.
Effect of bath Sch-28080.
The possibility that the
H+-K+-ATPase
might be located basolaterally in these intercalated cells was
considered by adding 10 µM Sch-28080 to the bathing solution. In this
case the inhibition of a basolateral
H+-K+-ATPase
would result in diminished HCO3
secretion in perfused CNT segments (Table 1). We found that basolateral Sch-28080 had no effect on net HCO
3
transport (
3.25 ± 0.12 pmol · min
1 · mm
1
to
3.23 ± 0.15, n = 4),
collected fluid HCO
3 concentration
(25.4 ± 0.1 mM to 25.5 ± 0.1), or
Vte (
12.5 ± 0.5 mV to
12.4 ± 0.5). These results showed that a
putative basolateral H+-K+-ATPase
did not play a major role in mediating net
HCO
3 secretion in the CNT under
baseline conditions.
Effect of bath isoproterenol.
Data from the perfused CCD indicate that isoproterenol stimulates
HCO3 secretion and apical
Cl
/HCO
3
exchange (12, 30). Also, isoproterenol has been found to decrease the
lumen- negative voltage of the perfused CCD, perhaps by stimulating
electrogenic Cl
transport
out of the lumen (13). In addition, the
Vte of the perfused CNT is more sensitive than the CCD to isoproterenol in the
micromolar range (15). We therefore examined the effect of 1 µM
isoproterenol added to the bathing solution on net
HCO
3 transport in CNT segments (Fig.
6, Table 1). Isoproterenol caused a large
decrease in lumen-negative
Vte (
14.0 ± 0.6 mV to
7.8 ± 0.2, n = 6, P < 0.001), while also stimulating
net HCO
3 secretion by 72%
(
3.48 ± 0.21 pmol · min
1 · mm
1
to
5.98 ± 0.34, P < 0.001) and increasing collected fluid
HCO
3 concentration by 0.7 mM (25.4 ± 0.1 mM to 26.1 ± 0.2, P < 0.01). These results indicated that isoproterenol significantly
stimulated net HCO
3 secretion by
-intercalated cells of the CNT, as in the CCD. Both segments appear
to be comprised primarily of
- rather than
-intercalated cells,
at least by function; but the CNT, unlike the CCD (42), did not appear
to show measurable
-cell function after inhibition of the
-cells.
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Effect of bath acetazolamide.
The secretion of HCO3 is known to be
dependent on cytosolic carbonic anhydrase II, since inhibition of
carbonic anhydrase with the permeant agent acetazolamide eliminates net HCO
3 secretion in perfused CCDs
without affecting lumen-negative
Vte (24). We
examined the effect of adding 10 µM acetazolamide to the bath on net
HCO
3 transport in perfused CNT
segments (Fig. 7, Table 1); this lower
concentration (24, 34, 35) was used to minimize nonspecific effects and yet inhibit most of the carbonic anhydrase. We found that acetazolamide inhibited 84% of net HCO
3 secretion
(
3.42 ± 0.25 pmol · min
1 · mm
1
to
0.56 ± 0.10, n = 5, P < 0.001), reduced collected
HCO
3 concentration (25.4 ± 0.1 mM
to 24.6 ± 0.1, P < 0.01), and
inhibited Vte
(
13.1 ± 0.9 mV to
6.1 ± 1.0, P < 0.01). These results showed that
HCO
3 secretion by the perfused CNT was dependent on cytosolic carbonic anhydrase, and inhibition of this enzyme also blocked the basolateral electrogenic
H+ pump and thereby reduced
luminal Vte.
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DISCUSSION |
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Our studies demonstrate that the CNT plays a major role in acid-base
homeostasis. In addition to exhibiting higher rates of Na+ absorption (1) and
K+ secretion (17) than the CCD,
the CNT secretes HCO3 at rates
comparable to that of the contiguous CCD. This finding indicates that
the CNT contributes HCO
3 to the alkaline urine of rabbits. Indeed, in the rabbit with 200,000 nephrons
per kidney (16) and at least 1 mm of CNT segments per nephron in the
cortical labyrinth, there would be ~1.4 µmol
HCO
3 secreted per min or 2 mmol/day.
The CNT may secrete more HCO
3 than the
CCD, which averages a similar transport rate over 3-4 mm of length
but drains an average of six nephrons (16). Thus the CNT may be the
most important HCO
3-secreting segment
in the rabbit kidney.
We did not expect such a high rate of
HCO3 secretion in the CNT because of
the relatively large numbers of band 3-positive intercalated cells
found in the CNT by immunocytochemistry (43). Indeed, if 50% of the
intercalated cells were
-type (band 3 positive) and 50% were
-type (peanut lectin positive) (43), then the CNT segment would be
expected to secrete HCO
3 or
H+, depending on which
intercalated cell function could be inhibited, much like the CCD (40).
The mechanisms of HCO3 transport in
the CNT have not been previously examined. Our data show (Table
2) that luminal
Cl
removal or bath
bafilomycin or bath concanamycin each markedly inhibited net
HCO
3 secretion; acetazolamide inhibited nearly all HCO
3 secretion.
These findings indicate that in the presence of cytosolic carbonic
anhydrase II in
-intercalated cells (3, 4, 18, 22, 28), cell water
and CO2 generate carbonic acid,
which dissociates to H+ and
HCO
3. Protons are secreted across the
basolateral membrane via a bafilomycin-sensitive vacuolar
H+-ATPase, and
HCO
3 is secreted by a
Cl
-dependent process,
presumably
Cl
/HCO
3
exchange, across the apical membrane. Evidence for the electrogenic
secretion of H+ across the
basolateral membrane in
-intercalated cells was noted in the
reduction in electronegativity of
Vte with
inhibitors of the H+-ATPase or of
H+ pumping. There was no
demonstrable effect on HCO
3 transport
or Vte with
luminal DIDS, suggesting that this apical Cl
/HCO
3
exchanger was not DIDS sensitive, as has been established for the
apical anion exchanger of CCD
-intercalated cells (9, 30).
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These findings are compatible with the microelectrode studies of Muto
et al. (27), which demonstrated functionally that 97% of the
intercalated cells were of the -type. We would assume that a failure
to observe net H+ secretion
(HCO
3 absorption) during inhibition of
HCO
3 secretion (Tables 1 and 2) is
consistent with a lack of many functioning
-intercalated cells.
Clearly, the histological finding of band 3-labeled cells in the CNT
would suggest otherwise; immunocytochemical identification of subunits of the vacuolar H+-ATPase in the
CNT is certainly required. Perhaps, these "
-intercalated cells" do not express the H+
pumps required for net HCO
3
absorption. Furthermore, the lack of effect of luminal bafilomycin, a
specific H+-ATPase inhibitor at
nanomolar concentrations (6, 23), tends to rule out a significant
contribution to the net HCO
3 flux from
apical H+ pumps. The lack of
effect of basolateral DIDS, an inhibitor of band 3, also suggests
minimal H+ secretion from the
-intercalated cells. It is possible that these cells secrete protons
not via a vacuolar H+-ATPase but
rather via a gastric-type
H+-K+-ATPase
(36, 44, 46). The failure of Sch-28080 to affect net
HCO
3 flux suggested that this type of
H+-K+-ATPase
does not contribute significantly to the net transport of
HCO
3.
The removal of Cl from the
bath would be expected to inhibit basolateral
Cl
/HCO
3
exchange, presumably of
-intercalated cells. Inasmuch as no effect
of bath Cl
removal, bath
DIDS, or luminal bafilomycin was noted (see Table 2), the contribution
of
-intercalated cells was probably small. Although both types of
intercalated cells are believed to have basolateral
Cl
conductances (10, 27),
the removal of bath Cl
did
not affect net HCO
3 transport. If
-intercalated cells were not contributing much to net
HCO
3 secretion, then this maneuver to
decrease Cl
-dependent
HCO
3 efflux across the basolateral membrane of these cells would not be expected to have much effect. Regarding
-intercalated cells in the CCD, the removal of bath Cl
would cause a decrease
in cell Cl
, which enhances
apical
Cl
/HCO
3
exchange and therefore net HCO
3 secretion (25, 47); the increase in
HCO
3 secretion then causes a loss of
cellular base and a decrease in cell pH (29), which would then
stimulate the basolateral H+ pump.
Such a stimulation in net HCO
3
secretion was not observed in the CNT, despite a detectable basolateral Cl
conductance (27),
suggesting that the apical
Cl
/HCO
3
exchanger might have been operating at close to maximal capacity, or
the Cl
conductances were
small, or cell Cl
was
already very low and not greatly affected by this maneuver. In
addition, the use of DIDS, which blocks
Cl
channels (21) in
addition to its inhibition of band 3-like Cl
/HCO
3
exchangers, failed to affect net HCO
3 flux. Measurements of the DIDS- and
Cl
-induced changes in
intercalated cell pH in perfused CNT segments might help us to better
understand the role of Cl
in mediating HCO
3 secretion.
An examination of the CCD's response to isoproterenol provides further
information about the mechanism of
HCO3 secretion in the CNT. It is well
known that isoproterenol and other
-agonists increase cAMP formation
in
-intercalated cells (8). Schuster (30) found that 1 µM
isoproterenol in the bath increased CCD
HCO
3 secretion by 44% and depolarized the Vte by 85%.
There was also a significant correlation between baseline
HCO
3 secretory rates and the
increments in HCO
3 secretion induced
by isoproterenol (30). Iino et al. (13) showed that isoproterenol
decreased Vte by 51% but did not affect isotopic lumen-to-bath
Na+ flux; however, isoproterenol
stimulated Cl
absorption by
145%. Interestingly, acetazolamide abolished the effect of
isoproterenol on
Vte, suggesting
that the
-agonist was stimulating some form of
Cl
absorption that depended
on
H+/HCO
3
transport. Kimmel and Goldfarb (19) found that 1 µM isoproterenol
caused K+ secretion to decrease by
41% and Vte by
53% in the CCD and that removing
Cl
from the bath and
perfusate attenuated the ability of isoproterenol to reduce
K+ secretion and
Vte. Fluorescent
cell pH studies indicate that isoproterenol causes a decrease in pH of
-intercalated cells (7, 12), and the pH change was prevented if
Cl
was removed from the
luminal fluid. Finally, isoproterenol stimulates Cl
/Cl
self-exchange (39). Each of these results would indicate that the
apical
Cl
/HCO
3
exchanger is required for the action of isoproterenol. Thus
isoproterenol stimulates HCO
3 secretion and Cl
absorption
via
Cl
/HCO
3
exchange in
-intercalated cells of the CCD. The mechanism for the
depolarization of
Vte may become
apparent in the analysis of induced
Cl
conductances.
In the turtle bladder, cAMP stimulates
HCO3 secretion along with a reversal
of the Vte (from
serosa negative to positive) (39). These authors believed that cAMP may
induce electrogenic HCO
3 secretion by
opening an apical Cl
(also
HCO
3) conductance. If the induced
apical anion channel allowed the passage of
HCO
3, in addition to the
electroneutral transport via the apical
Cl
/HCO
3
exchanger, then there would be electrogenic HCO
3 secretion.
If such an apical anion channel were induced in the nephron segment by isoproterenol, via elevating cell cAMP (2, 14), then the Vte would tend to become more negative with anion entry into the lumen. This has not been observed in the CCD (13, 15, 19, 30) or in the CNT (15); rather, the Vte becomes depolarized (less negative) after isoproterenol is added to the bath. Our studies in the CNT confirm this observation.
Perhaps the change in
Vte in response
to isoproterenol may be better understood in the context of increasing
Cl absorption. If the
putative apical Cl
conductance were induced by isoproterenol, then there would be, in
addition to increased electroneutral
Cl
absorption, increased
electrogenic Cl
absorption,
as shown by Iino et al. (13): the flow of negative charges out of the
lumen would depolarize
Vte as observed
by us and others (13, 15, 19, 30). On the other hand, if a basolateral Cl
conductance were
activated by cAMP-isoproterenol in
-intercalated cells, then there
would be increased apical
Cl
entry via
Cl
/HCO
3
exchange due to the increased driving force for intracellular
Cl
. This would result in
increased electroneutral HCO
3 secretion and Cl
absorption, probably with little change in
Vte, which was
not observed in the CNT. Thus, in response to isoproterenol, the
induction of an apical Cl
conductance allowing for electrogenic
Cl
absorption, in addition
to increased electroneutral HCO
3 secretion and Cl
absorption, appears to be required to explain the findings observed in
the CCD and CNT. The additional opening of the basolateral Cl
conductance would
further help to stimulate the transcellular fluxes of
Cl
and possibly
HCO
3 and could therefore contribute to
the luminal depolarization, as well as to the increase in
HCO
3 secretion and
Cl
absorption.
In summary, we have shown that the CNT segment plays a major role in
acid-base homeostasis. This segment secretes
HCO3 at rates comparable to that of
the contiguous CCD. The secretion of
HCO
3 requires apical
Cl
, indicating the
probability that it is mediated by apical
Cl
/HCO
3
exchange. It also requires cytosolic carbonic anhydrase. The pump
driving HCO
3 secretion appears to be a
basolaterally located H+-ATPase.
From our functional studies we conclude that these are
-intercalated
cells. The contribution of
-intercalated cells to baseline
HCO
3 transport appears to be small, because net transport was not substantially influenced by luminal H+-ATPase inhibitors or inhibition
of basolateral
Cl
/HCO
3
exchange. These studies indicate that the CNT should be included with
the collecting duct segments as important contributors to renal
acid-base homeostasis and could be quantitatively the most important
HCO
3-secreting segment in the kidney.
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ACKNOWLEDGEMENTS |
---|
We are grateful for the technical assistance of A. Kittelberger and D. Barnhart.
![]() |
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 the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50603.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. J. Schwartz, Div. of Pediatric Nephrology, Dept. of Pediatrics, PO Box 777, Univ. of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: George_Schwartz{at}URMC.Rochester.edu).
Received 12 February 1999; accepted in final form 26 May 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Almeida, A. J.,
and
M. B. Burg.
Sodium transport in the rabbit connecting tubule.
Am. J. Physiol.
243 (Renal Fluid Electrolyte Physiol. 12):
F330-F334,
1982
2.
Blot-Chabaud, M.,
M. Laplace,
F. Cluzeaud,
C. Capurro,
R. Cassingena,
A. Vandewalle,
N. Farman,
and
J. P. Bonvalet.
Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance.
Kidney Int.
50:
367-376,
1996[Medline].
3.
Breton, S.,
S. L. Alper,
S. L. Gluck,
W. S. Sly,
J. E. Barker,
and
D. Brown.
Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F761-F774,
1995
4.
Brown, D.,
T. Kumpulainen,
J. Roth,
and
L. Orci.
Immunohistochemical localization of carbonic anhydrase in postnatal and adult rat kidney.
Am. J. Physiol.
245 (Renal Fluid Electrolyte Physiol. 14):
F110-F118,
1983[Medline].
5.
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
6.
Drose, S.,
K. U. Bindseil,
E. J. Bowman,
A. Siebers,
A. Zeeck,
and
K. Altendorf.
Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases.
Biochemistry
32:
3902-3906,
1993[Medline].
7.
Emmons, C.,
and
J. B. Stokes.
Cellular actions of cAMP on HCO3-secreting cells of rabbit CCD: dependence on in vivo acid-base status.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F528-F535,
1994
8.
Fejes-Toth, G.,
and
A. Naray-Fejes-Toth.
Isolated principal and intercalated cells: hormone responsiveness and Na+-K+-ATPase activity.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F742-F750,
1989
9.
Fejes-Toth, G.,
A. Naray-Fejes-Toth,
L. M. Satlin,
F. M. Mehrgut,
and
G. J. Schwartz.
Inhibition of bicarbonate transport in peanut lectin-positive intercalated cells by a monoclonal antibody.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F901-F910,
1994
10.
Furuya, H.,
M. D. Breyer,
and
H. R. Jacobson.
Functional characterization of - and
-intercalated cell types in rabbit cortical collecting duct.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F377-F385,
1991
11.
Gifford, J. D.,
L. Rome,
and
J. H. Galla.
H+-K+-ATPase activity in rat collecting duct segments.
Am. J. Physiol.
262 (Renal Fluid Electrolyte Physiol. 31):
F692-F695,
1992
12.
Hayashi, M.,
Y. Yamaji,
M. Iyori,
W. Kitajima,
and
T. Saruta.
Effect of isoproterenol on intracellular pH of the intercalated cells in the rabbit cortical collecting ducts.
J. Clin. Invest.
87:
1153-1157,
1991[Medline].
13.
Iino, Y.,
J. L. Troy,
and
B. M. Brenner.
Effects of catecholamines on electrolyte transport in cortical collecting tubule.
J. Membr. Biol.
61:
67-73,
1981[Medline].
14.
Ikeda, M.,
M. Iyori,
K. Yoshitomi,
M. Hayashi,
M. Imai,
T. Saruta,
and
K. Kurokawa.
Isoproterenol stimulates Cl current by a Gs protein-mediated process in
-intercalated cells isolated from rabbit kidney.
J. Membr. Biol.
136:
231-241,
1993[Medline].
15.
Imai, M.
The connecting tubule: a functional subdivision of the rabbit distal nephron segments.
Kidney Int.
15:
346-356,
1979[Medline].
16.
Kaissling, B.,
and
W. Kriz.
Structural analysis of the rabbit kidney.
Adv. Anat. Embryol. Cell Biol.
56:
1-121,
1979[Medline].
17.
Kaufman, J. S.,
and
R. J. Hamburger.
Potassium transport in the connecting tubule.
Miner. Electrolyte Metab.
22:
242-247,
1996[Medline].
18.
Kim, J.,
C. C. Tisher,
P. J. Linser,
and
K. M. Madsen.
Ultrastructural localization of carbonic anhydrase II in subpopulations of intercalated cells of the rat kidney.
J. Am. Soc. Nephrol.
1:
245-256,
1990[Abstract].
19.
Kimmel, P. L.,
and
S. Goldfarb.
Effects of isoproterenol on potassium secretion by the cortical collecting tubule.
Am. J. Physiol.
246 (Renal Fluid Electrolyte Physiol. 15):
F804-F810,
1984[Medline].
20.
Koeppen, B. M.
Electrophysiology of collecting duct H+ secretion: effect of inhibitors.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F79-F84,
1989
21.
Light, D. B.,
E. M. Schwiebert,
G. Fejes-Toth,
A. Naray-Fejes-Toth,
K. H. Karlson,
F. V. McCann,
and
B. A. Stanton.
Chloride channels in the apical membrane of cortical collecting duct cells.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F273-F280,
1990
22.
Matsumoto, T.,
and
G. J. Schwartz.
Novel method for performing carbonic anhydrase histochemistry and immunocytochemistry on cryosections.
J. Histochem. Cytochem.
40:
1223-1227,
1992
23.
Mattsson, J. P.,
K. Vaananen,
B. Wallmark,
and
P. Lorentzon.
Omeprazole and bafilomycin, two proton pump inhibitors: differentiation of their effects on gastric, kidney and bone H+-translocating ATPases.
Biochim. Biophys. Acta
1065:
261-268,
1991[Medline].
24.
McKinney, T. D.,
and
M. B. Burg.
Bicarbonate secretion by rabbit cortical collecting tubules in vitro.
J. Clin. Invest.
61:
1421-1427,
1978[Medline].
25.
Mehrgut, F. M.,
L. M. Satlin,
and
G. J. Schwartz.
Maturation of HCO3 transport in rabbit collecting duct.
Am. J. Physiol.
259 (Renal Fluid Electrolyte Physiol. 28):
F801-F808,
1990
26.
Muto, S.,
M. Imai,
and
Y. Asano.
Further electrophysiological characterization of the - and
-intercalated cells along the rabbit distal nephron segments: effects of inhibitors.
Exp. Nephrol.
1:
301-308,
1993[Medline].
27.
Muto, S.,
K. Yasoshima,
K. Yoshitomi,
M. Imai,
and
Y. Asano.
Electrophysiological identification of - and
-intercalated cells and their distribution along the rabbit distal nephron segments.
J. Clin. Invest.
86:
1829-1839,
1990[Medline].
28.
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].
29.
Satlin, L. M.,
T. Matsumoto,
and
G. J. Schwartz.
Postnatal maturation of rabbit renal collecting duct. III. Peanut lectin-binding intercalated cells.
Am. J. Physiol.
262 (Renal Fluid Electrolyte Physiol. 31):
F199-F208,
1992
30.
Schuster, V. L.
Cyclic adenosine monophosphate-stimulated bicarbonate secretion in rabbit cortical collecting tubules.
J. Clin. Invest.
75:
2056-2064,
1985[Medline].
31.
Schwartz, G. J.,
J. Barasch,
and
Q. Al-Awqati.
Plasticity of functional epithelial polarity.
Nature
318:
368-371,
1985[Medline].
32.
Schwartz, G. J.,
and
M. B. Burg.
Mineralocorticoid effects on cation transport by cortical collecting tubules in vitro.
Am. J. Physiol.
235 (Renal Fluid Electrolyte Physiol. 4):
F576-F585,
1978
33.
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
34.
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
35.
Shimizu, T.,
K. Yoshitomi,
M. Nakamura,
and
M. Imai.
Site and mechanism of action of trichlormethiazide in rabbit distal nephron segments perfused in vitro.
J. Clin. Invest.
82:
721-730,
1988[Medline].
36.
Silver, R. B.,
P. A. Mennitt,
and
L. M. Satlin.
Stimulation of apical H-K-ATPase in intercalated cells of cortical collecting duct with chronic metabolic acidosis.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F539-F547,
1996
37.
Star, R. A.,
M. B. Burg,
and
M. A. Knepper.
Bicarbonate secretion and chloride absorption by rabbit cortical collecting ducts. Role of chloride/bicarbonate exchange.
J. Clin. Invest.
76:
1123-1130,
1985[Medline].
38.
Stone, D. K.,
D. W. Seldin,
J. P. Kokko,
and
H. R. Jacobson.
Anion dependence of rabbit medullary collecting duct acidification.
J. Clin. Invest.
71:
1505-1508,
1983[Medline].
39.
Tago, K.,
V. L. Schuster,
and
J. B. Stokes.
Regulation of chloride self exchange by cAMP in cortical collecting tubule.
Am. J. Physiol.
251 (Renal Fluid Electrolyte Physiol. 20):
F40-F48,
1986[Medline].
40.
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
41.
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
42.
Tsuruoka, S.,
and
G. J. Schwartz.
HCO3 absorption in rabbit outer medullary collecting duct: role of luminal carbonic anhydrase.
Am. J. Physiol.
274 (Renal Physiol. 43):
F139-F147,
1998
43.
Verlander, J. W.,
K. M. Madsen,
and
C. C. Tisher.
Axial distribution of band 3-positive intercalated cells in the collecting duct of control and ammonium chloride-loaded rabbits.
Kidney Int.
50:
S-137-S-147,
1996.
44.
Weiner, I. D.,
and
A. E. Milton.
H+-K+-ATPase in rabbit cortical collecting duct B-type intercalated cell.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F518-F530,
1996
45.
Weiner, I. D.,
A. E. Weill,
and
A. R. New.
Distribution of Cl/HCO
3 exchange and intercalated cells in rabbit cortical collecting duct.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F952-F964,
1994
46.
Wingo, C. S.,
K. M. Madsen,
A. Smolka,
and
C. C. Tisher.
H-K-ATPase immunoreactivity in cortical and outer medullary collecting duct.
Kidney Int.
38:
985-990,
1990[Medline].
47.
Yasoshima, K.,
L. M. Satlin,
and
G. J. Schwartz.
Adaptation of rabbit cortical collecting duct to in vitro acid incubation.
Am. J. Physiol.
263 (Renal Fluid Electrolyte Physiol. 32):
F749-F756,
1992
48.
Zhou, X.,
and
C. S. Wingo.
Stimulation of total CO2 flux by 10% CO2 in rabbit CCD: role of an apical Sch-28080- and Ba-sensitive mechanism.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F114-F120,
1994