Remodeling the cellular profile of collecting ducts by chronic
carbonic anhydrase inhibition
Corinne
Bagnis,
Vladimir
Marshansky,
Sylvie
Breton, and
Dennis
Brown
Program in Membrane Biology, Massachusetts General Hospital and
Department of Medicine, Harvard Medical School, Boston, Massachusetts
02120
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ABSTRACT |
Factors regulating the differentiated
phenotype of principal cells (PC) and A- and B-intercalated cells (IC)
in kidney collecting ducts are poorly understood. However, we have
shown previously that carbonic anhydrase II (CAII)-deficient mice have
no IC in their medullary collecting ducts, suggesting a potential role for this enzyme in determining the cellular composition of this tubule
segment. We now report that the cellular profile of the collecting
ducts of adult rats can be remodeled by inhibiting CA activity in rats
by using osmotic pumps containing acetazolamide. The 31-kDa subunit of
the vacuolar H+-ATPase, the sodium/hydrogen exchanger
regulatory factor NHE-RF, and the anion exchanger AE1 were used to
identify IC subtypes by immunofluorescence staining, while aquaporin 2 and aquaporin 4 were used to identify PC. In the cortical collecting
ducts of animals treated with acetazolamide for 2 wk, the percentage of B-IC decreased significantly (18 ± 2 vs. 36 ± 4%,
P < 0.01) whereas the percentage of A-IC increased
(82 ± 2 vs. 64 ± 4%, P < 0.01) with no
change in the percentage of total IC in the epithelium. In some treated
rats, B-IC were virtually undetectable. In the inner stripe of the
outer medulla, the percentage of IC increased in treated animals
(48 ± 2 vs. 37 ± 3%, P < 0.05) and the
percentage of PC decreased (52 ± 2 vs. 63 ± 3%,
P < 0.05). Moreover, IC appeared bulkier, protruded
into the lumen, and showed a significant increase in the length of
their apical (20.8 ± 0.5 vs. 14.6 ± 0.4 µm,
P < 0.05) and basolateral membranes (25.8 ± 0.4 vs. 23.8 ± 0.5 µm, P < 0.05) compared with
control rats. In the inner medullary collecting ducts of treated
animals, the number of IC in the proximal third of the papilla was
reduced compared with controls (11 ± 4 vs. 40 ± 11 IC/mm2, P < 0.05). These data suggest that
CA activity plays an important role in determining the differentiated
phenotype of medullary collecting duct epithelial cells and that the
cellular profile of collecting ducts can be remodeled even in adult
rats. The relative depletion of cortical B-IC and the relative increase
in number and hyperplasia of A-IC in the medulla may be adaptive
processes that would tend to correct or stabilize the metabolic
acidosis that would otherwise ensue following systemic carbonic
anhydrase inhibition.
acetazolamide; carbonic anhydrase II; rat kidney; hydrogen-adenosine 5'-triphosphatase; sodium/hydrogen exchanger
regulatory factor; AE1 anion exchanger; aquaporin 2; immunocytochemistry; principal cell; intercalated cell
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INTRODUCTION |
THE COLLECTING DUCT EPITHELIUM of
the mammalian kidney is a remarkably heterogeneous structure that shows
structural and functional adaptive changes during development as well
as in the mature animal. The role of the mature collecting duct in the
control of acid/base status (20), sodium/potassium
balance, and fluid homeostasis (53) is reflected by its
cellular composition. Intercalated cells are involved in acid/base
homeostasis, and principal cells are responsible for sodium and water
balance. Both cell types are involved in potassium transport processes.
At least three clearly distinct types of collecting duct cells have
been identified: A-intercalated cells (A-IC), B-intercalated cells
(B-IC), and principal cells (PC). A-IC are involved in acid secretion
whereas B-IC secrete bicarbonate (2, 52). All IC have high
cytosolic levels of carbonic anhydrase type II (CAII) (14,
27). A-IC cells can be identified by the presence of the
H+-adenosine triphosphatase (H+-ATPase) in
their apical plasma membrane and the kidney variant of the band 3 Cl
/HCO
exchanger (AE1) in their
basolateral plasma membrane. B-IC have either basolateral, apical, or
bipolar/diffuse H+-ATPase distribution but do not contain
detectable AE1 (5, 39). We have recently shown that the
56-kDa subunit of the H+-ATPase is a
PSD-95/Disks-large/ZO-1 (PDZ)-binding protein and that the PDZ-domain
protein NHE-RF is colocalized with the H+-ATPase in B- but
not in A-IC in the collecting duct (10). Thus NHE-RF is a
marker for AE1-negative B-IC, and its selective expression may be
related to the ability of B-cells to modulate the polarized expression
of H+-ATPase by allowing transport vesicles to interact
with components of the cytoskeleton. PC can be identified by the
presence of the vasopressin-regulated water channel aquaporin 2 (AQP2)
on their apical plasma membrane and aquaporin-4 (AQP4) on their
basolateral plasma membrane (1).
Approximately 40% of the cells in the connecting tubule (CNT),
cortical collecting duct, and the outer medullary collecting duct
(OMCD) are IC in adult rats (13). A-IC and B-IC constitute 60 and 40%, respectively, of the IC in the renal cortex of adult kidney. B-IC are rare in the outer stripe of the outer medulla, and
absent from the inner stripe (IS) and the inner medullary collecting
duct (IMCD). A-IC gradually disappear from the proximal third and are
virtually absent in the terminal two-thirds of the IMCD (13,
28). However, both A-IC and B-IC are found in the IMCD of
newborn rats. They progressively disappear from the deep IMCD during
the first 3 wk after birth (28), but IC gradually increase
in number in other collecting duct regions, including the cortex during
postnatal development.
Little information is available concerning the mechanism by which
different cell types arise in the renal collecting duct system. In the
developing kidney, growth factors and hormones as well as changes in
the pH, osmolarity, and variations in the extracellular electrolyte
composition (45) play important roles in the
differentiation of IC and PC. Because both A-IC and B-IC are present in
the fetal rat kidney, these subtypes of IC seem to proliferate as a
result of programmed differentiation during development
(28). Furthermore, the cause of the selective depletion of
IC from the tip of the papilla during postnatal development remains
poorly understood (31), although Kim et al.
(25) showed that IC appear to be deleted from the
medullary collecting duct by two distinct mechanisms: type B-IC undergo
apoptosis and subsequent phagocytosis by neighboring PC,
whereas type A-IC are eliminated by extrusion into the tubule lumen.
We have previously shown that IC are greatly depleted and replaced by
PC in medullary collecting ducts of CAII-deficient mice (8). This suggests a potential role for CA in regulating
cell-type diversity in collecting ducts. In this study, we examined the effect of pharmacological inhibition of CA activity on rat kidney collecting ducts. By using antibodies against cell-type-specific proteins, we show that a significant remodeling of the cellular profile
of this tubule segment occurs in adult rats after 2 wk of acetazolamide
treatment via osmotic pumps.
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METHODS |
Animals.
Adult Sprague-Dawley rats were maintained on a standard diet and had
free access to water. They were implanted with Alzet osmotic pumps and
treated with acetazolamide (15 mg · kg
1 · day
1), a CA
inhibitor, for up to 2 wk. The animals were anesthetized with Nembutal
(Abbott Laboratories, North Chicago, IL, 40 mg/kg body weight ip), and
the osmotic pumps were implanted beneath the skin at the nape of the
neck. The concentration of acetazolamide used to fill the pumps was
calculated on the base of the average pump rate provided by the
manufacturer, the body weight of the animals, and the dose required.
The osmotic pumps were checked at the time the pumps were removed, and
all of them had delivered the drug that was initially loaded.
Systemic and urinary biological parameters.
In an initial set of experiments, six animals were implanted with
osmotic pumps and treated for up to 2 wk (acetazolamide-treated rats,
n = 6; control rats, n = 4). After 7 and 14 days, they were put into metabolic cages for 48 h to
monitor weight, plasma creatinine, bicarbonate and chloride levels, and
urinary creatinine, sodium, potassium, and pH.
Tissue fixation and preparation.
Animals were anesthetized with Nembutal (65 mg/kg body wt, ip) and
perfused through the left ventricle first with PBS (0.09% NaCl in 10 mM phosphate buffer, pH 7.4) followed by PLP fixative (4%
paraformaldehyde, 10 mM sodium periodate, 10 mM lysine, and 5% sucrose
in 0.1 mM sodium phosphate) as described previously (9,
15). Before PLP perfusion, the left renal artery and vein were
clamped and the left kidney was removed and frozen in liquid nitrogen
for Western blot analysis. The right kidney was then perfusion-fixed
for 5 min in situ, and slices were further fixed by immersion overnight
in PLP at 4°C, washed three times in PBS, and stored until use in the
same buffer containing 0.02% sodium azide. Tissues were cryoprotected
by immersion in 0.9 M (30%) sucrose in PBS for at least 1 h,
mounted for cryosectioning in Tissue-Tek (Miles, Elkhart, IN) before
freezing in liquid nitrogen and sectioning at 4 µm with a
Reichert-Jung 2800 Frigocut cryostat (Spencer Scientific, Derry, NH).
Sections were picked up on Fisher Superfrost Plus charged glass slides
(Fisher Scientific, Pittsburg, PA).
Immunofluorescence microscopy.
Fixed sections were hydrated in PBS for 10 min and treated for 5 min
with 1% SDS in PBS, an antigen retrieval technique that we described
previously (16). Sections were washed 3 × 5 min in
PBS and blocked in a solution of 1% BSA/PBS/sodium azide for 15 min.
Primary antibodies, diluted as detailed below, were applied for 1 h at room temperature. Sections were then washed 2 × 5 min in
high-salt PBS (PBS containing 0.27% NaCl to reduce background staining) and 1 × 5 min in normal PBS. Secondary antibodies were applied for 1 h at room temperature, followed by washes as above.
Primary antibodies were used as follows: 1) a chicken
polyclonal affinity-purified antibody against the 31-kDa subunit of the
vacuolar H+-ATPase, at a 1:20 dilution; 2) an
affinity-purified rabbit polyclonal antibody against the COOH-terminal
dodecapeptide sequence of the AE2 anion exchanger at a dilution of
1:800 (kindly provided by Dr. Seth Alper, Beth Israel Hospital, Boston,
MA); this antibody also recognizes the COOH terminal of the AE1 anion
exchanger (5) and will be referred to as the anti-AE1
antibody; 3) an affinity-purified rabbit polyclonal antibody
IC270 raised against glutathione S-transferase-NHE-RF fusion
protein amino-acids 270-358; this antibody has been characterized previously (21) and was kindly provided by Dr. Vijaya
Ramesh, Massachusetts General Hospital, Boston, MA; 4) a rabbit
polyclonal antibody raised against CAII from red blood cells (kindly
provided by Dr. W. Sly, Wash. Univ., St. Louis, MO) used at a 1:100
dilution; 5) a rabbit polyclonal antibody raised against the
second external loop of AQP2 at a 1:100 dilution (22); and
6) a rabbit polyclonal affinity-purified antibody raised
against the COOH-terminal decapeptide of AQP4 at a dilution of 1:25
(49).
Secondary antibodies used were either goat anti-rabbit immunoglobulin
(IgG) coupled to FITC (Kirkegaard & Perry, Gaithersburg, MD), goat
anti-rabbit IgG coupled to indocarbocyanine (CY3, Jackson ImmunoResearch, West-Grove, PA), and donkey anti-chicken IgG coupled to
FITC or CY3 (Jackson ImmunoResearch, West-Grove, PA). Some 4-µm
sections were double stained to confirm the identity of positive and
negative cell types that were detected with single staining and for the
purpose of morphological measurements on the cells of interest. Both
primary antibodies were applied at the same time by using the
appropriate final dilutions. In a second step, both secondary
antibodies were also applied simultaneously.
Some sections were double stained with anti-NHE-RF and anti-AE1
antibodies. Because both antibodies were raised in rabbit, an
amplification procedure was used to allow staining of sections with two
primary antibodies raised in the same species. Briefly, the first
affinity-purified antibody, anti-NHE-RF, was applied at a dilution of
1:10, a concentration that was too low to be detectable by conventional
application of a secondary fluorescent antibody in these sections, as
determined in preliminary experiments. The dilute anti-NHE-RF antibody
was detected by using a tyramide amplification kit (NEN Life Science
Products) with tyramide-FITC as a fluorescent reagent, according to the
manufacturer's instructions. The sections were then incubated
conventionally with anti-AE1 and secondary goat-anti-rabbit CY3 as
described above. No cross reactivity between the two sets of reagents
was detectable under these conditions.
Slides were mounted in a 2:1 mixture of Vectashield (Vector
Laboratories, Burlingame, CA) mounting medium and 1.5 M Tris solution (pH 8.9). Some sections were examined with a Nikon Eclipse 800 epifluorescence photomicroscope (Nikon Instruments, Garden City, NY)
and photographed using Kodak TMAX 400 black and white film push-processed to 1600 ASA. Other images were acquired digitally from
the Nikon 800 by using a Hamamatsu Orca charge-coupled device (CCD)
camera or from a Nikon FXA photomicroscope with an Optronics 3-bit CCD
color camera. They were stored on an Apple Macintosh Power PC 8500 and
analyzed by using IPLab Spectrum version 3.1a image capture and
analysis software (Signal Analytics, Vienna, VA). Image montages were
arranged by using Adobe Photoshop 4.0, and hardcopies were produced by
using an Epson Stylus 600 ink jet printer.
Quantification in the cortex by using H+-ATPase and
AE1 antibodies.
The quantitative studies were performed on one randomly chosen section
from each animal for each set of incubations. Tubules that were
sectioned perpendicularly to the basement membrane were used for the
quantification. The number of IC was counted in the cortex of kidney
sections double stained for H+-ATPase and AE1
(acetazolamide treated n = 6, control n = 6). A total of 50 pictures were digitally acquired (28 from the
control group and 22 from the acetazolamide-treated group).
Double-stained cells (apical H+-ATPase and basolateral AE1)
were counted as A-IC. Cells with staining for H+-ATPase but
no AE1 were counted as B-IC. Nonstained cells were identified as PC.
The percentages of A-IC and B-IC were calculated from the number of
A-IC and B-IC relative to the total number of IC in each tubule.
Quantification in the inner stripe of the outer medulla by using
H+-ATPase, AQP2, and AQP4 antibodies.
The number of stained IC and PC in the inner stripe was counted by
using the H+-ATPase and AQP2 antibodies, respectively, as
cell-specific markers. Digital images, showing IS sections, were
counted for each group of animals (1-wk acetazolamide treated
n = 2, 6 pictures; control n = 3, 9 pictures; 2-wk acetazolamide treated n = 6; 12 pictures; control n = 6, 11 pictures). A total of 38 digital images were examined. Collecting ducts in the IS were
distributed regularly throughout each image, and the total number of
cells were counted and expressed per total area of each photograph. The
percentages of IC and PC were then estimated from the number of
positive cells relative to the total number of cells in each tubule
stained with H+-ATPase and AQP2 antibodies, respectively.
The number of IC was also counted on 23 black and white prints from the
IS at a final magnification of ×98. Apical and basolateral membrane
length of IC and PC was measured by using IPLab Spectrum software on
digitized images. The apical length was measured in IC and PC stained
for H+-ATPase and AQP2, respectively, and the basolateral
length was measured in IC and PC stained with AE1 and AQP4,
respectively, to highlight the membrane domain of interest. A total of
341 cells were counted (1-wk acetazolamide-treated rats,
n = 3; controls for 1-wk rats, n = 2;
2-wk acetazolamide-treated rats, n = 6; controls for
2-wk rats, n = 6).
Quantification in the IMCD by using antibodies against the 31-kDa
subunit of the H+-ATPase.
The number of IC in the IMCD was counted on sections of the inner
medulla stained for the 31-kDa subunit of the H+-ATPase.
Two black and white prints taken with a Nikon 800 microscope (final
magnification ×98) were counted for each animal (2-wk acetazolamide treated n = 9, controls n = 9). The
first set of pictures was taken from the proximal region of the inner
medulla and included the initial 1 mm that extended from the beginning
of the inner medulla toward the tip of the papilla. The second set of
pictures was taken more distally and included an additional 1 mm.
Together, both pictures accounted for about one-third of the total
length of the inner medulla (i.e., the region where IC are located in the IMCD). Thirty-six prints were examined overall. Data were expressed
as the total number of IC per unit area for the first and second set of
pictures separately, and for both sets of images combined.
Electron microscopy.
For electron microscopy, some kidneys were fixed in 2.5%
glutaraldehyde containing 0.1-M cacodylate buffer, pH 7.4. Tissues were
chopped into smaller pieces (about 1 mm3) and immersed in
the same fixative, as described previously (8). They were
then postfixed for 1 h in 2% osmium tetroxide, stained en bloc
with uranyl acetate, dehydrated in graded ethanol, and embedded in Epon
(Electron Microscopy Sciences, Ft. Washington, PA). Thin sections were
stained with uranyl acetate and lead citrate prior to examination with
a Philips model CM10 electron microscope (Philips, Mahwah, NJ).
Western blotting.
Rats were perfused through the left ventricle with PBS (pH 7.4) as
described above. A kidney was removed, cut into smaller pieces with a
razor blade, transferred into ice-cold homogenization buffer, and
weighed. Kidney samples were homogenized in 10 ml of homogenization
buffer (250 mM sucrose, 1 mM EDTA, 18 mM HEPES-Tris, pH 7.4) per gram
of tissue in the presence of Complete, a cocktail of protease
inhibitors (Boehringer Mannheim, Germany). Tissue was homogenized by
using 20 strokes with a glass potter (model C-925, Thomas) equipped
with a tight-fitting Teflon pestle. Homogenates were centrifuged for 10 min (1,000 g, 4°C). The supernatant (S1) was either
aspirated or further centrifuged (30 min, 10,000 g, 4°C).
The resulting pellet (P2) was kept on ice for SDS-PAGE, and the
supernatant S2 was centrifuged for 1 h (100,000 g,
4°C). Protein concentration was measured after solubilization of the membranes in 0.1% SDS with the Pierce-bicinchoninic acid protein assay
reagent (Pierce, Rockford, IL) by using albumin as standard (44). All pellets and supernatants were diluted 3:1 in 4×
Laemmli (reducing) sample buffer (Boston BioProducts, Ashland, MA) and boiled for 5 min. Samples were loaded at 10 µg protein/lane onto SDS-polyacrylamide (12%) minigels (Bio-Rad, Richmond, CA) and separated by using the Laemmli method (29). Proteins were
transferred onto Immobilon-P transfer membrane (Millipore Bedford, MA)
in a Bio-Rad (Richmond, CA) semidry transfer cell. Membranes were blocked in buffer (5% nonfat dry milk in 15 mM NaCl, 5 mM
Tris · HCl, 0.3% Tween 20, pH 7.0) for 1 h at 20°C.
Membranes were then incubated with the primary antibodies
(H+-ATPase, CAII, and AQP2) diluted 1:500, 1:1000, and
1:1,000 respectively. Goat anti-chicken or anti-rabbit HRP-conjugated
secondary antibodies were diluted 1:12,000 and 1:10,000 respectively.
Washes between and after incubations were repeated four times.
Detection of antibody binding was performed with the enhanced
chemiluminescence method (Amersham Life Sciences, Buckinghamshire, UK)
by using Kodak X-Omat blue XB-1 film. Films were scanned and
quantitatively analyzed by using NIH Image 1.62 software.
Statistics.
Data were analyzed by using IPLab Spectrum 3.1a running on a Power
Macintosh 8500. Values are means ± SE, and significance levels
were calculated by using the two-tailed Student's t-test for unpaired samples using Statview software version 4.5 1.1 (Abacus Concepts, Berkeley, CA).
 |
RESULTS |
Systemic and urinary biological parameters.
The blood and urine parameters of control and acetazolamide-treated
animals after 7 and 14 days are summarized in Tables
1 and 2,
respectively. No difference in plasma bicarbonate was observed after 7 and 14 days between the acetazolamide-treated and the control animals.
No change in renal function was observed. Acetazolamide induced a
significant but transient increase in diuresis and in natriuresis
(day 7, P < 0.05). Urinary pH did not
change significantly compared with controls.
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Table 1.
Plasma parameters and weight of control and acetazolamide-treated
animals after 7 and 14 days, respectively
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A-IC and B-IC in the cortical collecting duct.
The 31-kDa subunit of the vacuolar H+-ATPase was
distributed in IC, either on the apical membrane, on the basolateral
membrane, or on cytoplasmic vesicles. IC exhibiting apical staining for H+-ATPase associated with a basolateral staining for AE1
were identified as A-IC. IC exhibiting positive staining for
H+-ATPase and no staining for AE1 were considered to be
B-IC (Fig. 1). The percentage of IC and
PC relative to the total number of cells was similar in all groups
(Fig. 2A). In control rats,
the percentages of A-IC and B-IC were 64 ± 4 and 36 ± 4%,
respectively, which agrees with previously published data (Fig.
2B) (12). However, in acetazolamide-treated
animals, the percentage of A-IC increased significantly and that of
B-IC decreased significantly (82 ± 2 and 18 ± 2%,
P < 0.01 vs. control) (Fig. 2B).

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Fig. 1.
Double staining of the 31-kDa subunit of vacuolar
H+-ATPase (green) and anion exchanger AE1 (red) in a 4-µm
section of control (A) and acetazolamide-treated kidney
cortex (B). Arrows indicate B-intercalated cells (IC)
stained with anti-H+-ATPase antibodies, diffusely or on the
basolateral membrane (some B-IC also have bipolar H+-ATPase
staining). A-IC are stained on their apical membrane with
anti-H+-ATPase and on their basolateral membrane with
anti-AE1 antibodies, respectively. The number of B-IC decreased
significantly in cortical collecting ducts of acetazolamide-treated
animals. Bar = 10 µm.
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Fig. 2.
Effect of acetazolamide administration on the percentage
of IC and principal cells (PC; A) and on the percentage of
A-IC and B-IC (B) in the cortical collecting duct (CCD).
Cell types were identified by using a double staining for AE1 and
H+-ATPase (means ± SE). *P < 0.01 vs. control.
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To confirm that the H+-ATPase-positive but AE1-negative IC
identified in our quantitative study were indeed B cells, we examined the qualitative pattern of expression of NHE-RF in control and acetazolamide-treated rat kidneys. We have previously reported that
NHE-RF, a PDZ-domain protein originally identified as a regulatory factor for the sodium/hydrogen exchanger NHE3 (54) is
specifically expressed in B-IC but not A-IC in cortical collecting
ducts (10). As shown in Fig.
3A, NHE-RF positive IC were
always AE1 negative as previously described and were readily detectable
in control collecting ducts. However, in collecting ducts from
acetazolamide-treated rats, NHE-RF-positive cells were much less
abundant. In the example illustrated in Fig. 3B (from an
animal that had a dramatic loss of B-IC based on data from the
quantitative study described above), they were virtually undetectable.
In contrast, AE1-positive IC were numerous in cortical collecting ducts
from this rat.

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Fig. 3.
Identification of B-type intercalated cells was confirmed
by performing a double staining to detect sodium/hydrogen exchanger
regulatory factor (NHE-RF; green) and AE1 (red). NHE-RF-positive
B-cells were frequently seen in control collecting ducts (A,
arrows). Acetazolamide-treated collecting ducts (B) showed a
striking decrease in the number of NHE-RF-positive cells, whereas
AE1-positive A-IC were abundant. The arrowhead indicates an
AE1-positive red blood cell in A. The proximal tubule brush
border shows a variable amount of NHE-RF staining in both controls and
acetazolamide-treated rats. Bar = 15 µm.
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IC in the inner stripe of the outer medulla.
In the inner stripe of control rats, the percentages of IC and PC in
the collecting duct were 37 ± 3 and 63 ± 3%, respectively (Fig. 4A). In 2-wk
acetazolamide-treated animals (Fig. 4B), the percentage of
IC increased significantly (48 ± 2%, P < 0.05 vs. control group) and the percentage of PC decreased significantly (52 ± 2%, P < 0.05 vs. control group), whereas
the total number of cells remained unchanged (Fig.
5).

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Fig. 4.
Double staining of the 31-kDa subunit of vacuolar
H+- ATPase (green) and aquaporin-2 (red) in 4-µm
sections of control (A) and acetazolamide-treated kidney
inner stripe (IS) (B). Quantitative studies showed that the
number and the size of IC increased significantly in
acetazolamide-treated animals. Bars = 10 µm.
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Fig. 5.
Effect of acetazolamide on the percentage of IC and PC in
collecting ducts from the IS of the outer medulla (means ± SE),
after 7 days (D7) or 14 days (D14). * P < 0.05 vs.
control group.
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As shown in Figs. 6 and 7A, IC
from 2-wk acetazolamide-treated animals appeared bulkier, protruded
markedly into the lumen, and showed a significant increase in their
apical membrane length compared with control rats (20.9 ± 0.5 vs.
14.6 ± 0.4 µm, P < 0.05). In addition, a
significant increase in the length of the IC basolateral membrane was
observed in acetazolamide-treated animals after 1 wk (23.6 ± 0.9 vs. 21.4 ± 0.5 µm, P < 0.05) and 2 wk
(25.8 ± 0.4 vs. 23.7 ± 0.5, P < 0.05) of
treatment (Fig. 7B).

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Fig. 6.
Double staining of the 31-kDa subunit of the vacuolar
H+-ATPase (red) and AE1 (green) in a 4-µm section of
control (A) and acetazolamide-treated kidney IS of the outer
medulla (B). These antibodies stained IC in the collecting
ducts. IC were more numerous in the acetazolamide-treated animals than
in controls. They appeared also bulkier, increased in size, and
protruded into the tubule lumen. Bars = 10 µm.
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Fig. 7.
Effect of acetazolamide on the apical (A) and
basolateral (B) membrane lengths of IC in the outer
medullary collecting duct (OMCD) in the IS (means ± SE) after D7
and D14. *P < 0.05 vs. control group.
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IC in the IMCD.
In the inner medulla, the number of IC was lower in rats treated with
acetazolamide for 2 wk compared with control animals (Fig.
8). This reduction was marginally
significant in the proximal region that included the first 1 mm of the
inner medulla (113 ± 22 vs. 155 ± 22 IC/mm2,
P = 0.09). However, a clearly significant reduction was
observed in the second 1 mm located more distally (11 ± 4 vs.
40 ± 11 IC/mm2, P < 0.05) (Fig.
9).

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Fig. 8.
Double staining using H+-ATPase (green) and
AE1 antibodies (red) on control (A) and acetazolamide
(B) sections of the inner medulla (IM). The number of IC in
the distal part of the inner medulla/papilla decreased markedly in the
acetazolamide-treated rats. Bar = 250 µm.
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Fig. 9.
Effect of acetazolamide on the number of IC in the inner
medullary collecting ducts (IMCD) (means ± SE). Proximal region
(Prox) includes the first 1 mm of the inner medulla. Distal region
(Dist) includes the second 1 mm located more distally. Total, both
regions pooled together. *P < 0.05 vs. control
group.
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Electron microscopy.
By conventional thin-section electron microscopy, IC and PC could be
clearly distinguished in all regions of the collecting duct. In control
rats, IC had morphological features similar to those that have been
previously described, with numerous mitochondria, a large number of
intracellular vesicles, and apical microvilli (Fig.
10A). In 2-wk
acetazolamide-treated animals, IC were increased in size, bulkier, and
protruded markedly in the lumen (Fig. 10B). The number of
intracellular vesicles seemed to be decreased in these cells although
this was not evaluated quantitatively. Apical microvilli were more
developed in acetazolamide-treated rats.

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Fig. 10.
Electron micrographs showing morphological features of
intercalated cells from control (A) and acetazolamide
(B) collecting ducts in the IS. Control IC are smaller, with
fewer apical microvilli and numerous apical vesicles. Acetazolamide
induces a large amplification of the apical surface, which has many
microvilli. There are few vesicles in the cytoplasm. The cell protrudes
into the tubule lumen. Bar = 1 µm.
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Western blotting.
S3, P3, and P2 fractions were obtained from kidney inner stripe
(corresponding to the cytosolic, microsomal, and plasma membrane fractions of the cells, respectively). An S1 fraction (total
homogenate) was obtained from whole papilla. Western blotting with the
anti H+-ATPase antibody showed a single band in all
samples. As shown in Figs. 11 and
12, this band was more intense in
the membrane and cytosolic fractions of acetazolamide-treated rats
compared with control rats. Quantitative studies showed a significant
increase in the amount of protein detected in the lanes corresponding
to the cytosolic fraction of 2-wk acetazolamide-treated rats
(P < 0.05) and a difference which did not reach
statistical significance in the membrane fraction. No significant
difference was observed between control and acetazolamide-treated rats
when samples were probed with antibodies against CAII and AQP2 (data
not shown).

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|
Fig. 11.
Western blot showing the effect of acetazolamide on the
amount of protein detected with an anti-31-kDa subunit of the
H+-ATPase in the membrane fraction of kidney homogenates
from the IS (means ± SE). Results are shown from 3 different rats
for each condition. While an increase is observed after treatment it
did not reach significance (P = 0.0618).
|
|

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|
Fig. 12.
Western blot showing the effect of acetazolamide on the
amount of protein detected with an anti-31-kDa subunit of the
H+-ATPase in the cytosolic fraction of kidney homogenates
of the IS (means ± SE). Results from 3 different rats are shown.
A significant increase in the amount of 31-kDa subunit occurs after
acetazolamide treatment. *P < 0.05 vs. control.
|
|
 |
DISCUSSION |
We have previously shown that IC are absent from medullary
collecting ducts of CAII-deficient mice (8), which
suggests a potential role for CA in determining the cellular
composition of this segment of the urinary tubule. Our present study
shows that chronic (2-wk) CA inhibition by acetazolamide induces a
marked remodeling of collecting ducts in all regions of the kidney. In the cortex of acetazolamide-treated animals there was a significant decrease in the number of B-IC and a corresponding increase in the
percentage of A-IC compared with control rats. In some extreme cases,
B-IC were rarely found in the cortical collecting duct (see Fig.
1B). However, the ratio of total IC to PC was no different in acetazolamide-treated rats and control rats, suggesting that CA
inhibition favors the acid-secreting IC phenotype to the detriment of
bicarbonate-secreting B cells. In the inner stripe of the outer medulla, acetazolamide induced a significant increase in the number of
IC and a decrease in the number of PC. The size of IC was also increased. These changes were associated with a higher level of expression of the H+-ATPase in cytosolic fractions measured
by Western blotting, although the amount associated with membrane
fractions was not increased significantly. A previous study on the
effect of acetazolamide on collecting duct morphology was performed by
Lonnerholm et al. (30). By using CA as a marker of IC,
their major conclusion was that IC from acetazolamide-treated rats
appeared bulkier than in control rats. Our study supports this finding
in the inner stripe collecting duct.
The present data suggest that acetazolamide treatment triggers a series
of events that would tend to increase net proton secretion in both
cortical and OMCDs. Morphological alterations in IC similar to
those described here occur after induction of acute and chronic metabolic and respiratory acidosis in rats (32). In the
inner medulla, however, acetazolamide treatment reduced the number of IC. We have previously reported a similar, but more pronounced, depletion of inner medullary IC in CAII-null mice (8).
However, CA inhibition for 2 wk did not result in a loss of IC from
other collecting duct segments. On the contrary, IC were more numerous and more "developed" in the inner stripe of acetazolamide-treated rats. Although it is possible that more prolonged treatment with acetazolamide might induce a more widespread loss of IC in medullary collecting ducts, it is also possible that a lack of (or inhibition of)
CA activity has different effects on collecting duct cells during
postnatal renal development and in adult animals.
It has been known for many decades that the collecting duct undergoes
morphological adaptation to acid-base disturbances (32, 53). Metabolic acidosis induces changes consistent with
increased activity of A-IC and decreased activity of B-IC (6, 7,
39, 50), whereas the opposite changes are seen in systemic
alkalosis (6, 7, 39, 51). However, in most previous
studies, the population of A-IC vs. B-IC has appeared to remain
constant, implying that the observed changes occurred in populations of
cells with a fixed A or B phenotype. Chronic metabolic acidosis or
alkalosis in late pregnancy or during initial lactation in rats led to
a decrease and an increase, respectively, in the percentage of B-IC in
the pups as defined by the location of plasma membrane "studs" (37), an ultrastructural marker of the vacuolar
H+-ATPase (11). After ammonium chloride
loading, a significant increase in apical H+- ATPase
staining and apical membrane area in AE1-positive IC was observed in
addition to a reduction in the number of IC with basolateral
H+-ATPase staining (50). Morphological
modifications of A-IC and B-IC have also been observed in the kidneys
of K+-depleted rats (38, 43, 46). Although in
our study plasma potassium data were not available, urinary potassium
was similar in both groups, suggesting that potassium balance was
similar in acetazolamide-treated rats and in control animals.
Interpretation of some previous studies is confounded, however, by the
existence of AE1-negative cells that express apical H+-ATPase, as well as some cells expressing intermediate
patterns of H+-ATPase staining (5, 7, 26). In
the absence of double-staining for both AE1 and H+-ATPase,
these cells would be identified as A cells based simply on
morphological criteria or H+-ATPase staining alone.
Furthermore, A-IC can have very low levels of AE1 staining under some
conditions (39). Our conclusion obtained from AE1- and
H+-ATPase-stained sections, i.e., that B cells are reduced
in number in acetazoamide-treated rats, was, therefore, reevaluated in
sections stained for NHE-RF, a recently identified marker of B-IC
(10). Qualitative examination of the sections revealed a
marked reduction in the number of cells stained for NHE-RF in the
cortical collecting ducts of acetazolamide-treated animals, consistent
with a loss of B-IC and supporting our data using AE1 and
H+-ATPase staining patterns to distinguish A-IC and B-IC.
It is not surprising that different parts of the collecting duct
exhibit different morphological responses to acetazolamide treatment.
Rabbit outer and inner medullary collecting ducts vary in
susceptibility to CA inhibitors and in their response to stimulation with CO2 (35, 36). It is likely that the
concentration of acetazolamide in the inner medulla is higher than in
the outer medullary or cortical collecting ducts, and more complete CA
inhibition in the inner medulla could lead to the observed decrease in
the number of IC in this region. How might CA inhibition affect the differentiated phenotype of an epithelial cell? Whereas acetazolamide treatment results in a transient systemic acidosis of variable duration
(23, 33, 34) and could, therefore, mimic the effects of
metabolic acidosis on IC, it also induces an acute increase in
intracellular pH (pHi) in IC (40), whereas the
pHi of PCs does not change. The effect of chronic
acetazolamide treatment on pHi in IC is not known, but
pHi changes can affect many cellular processes, including
regulation of acid-base transporter trafficking and insertion (4,
19), as well as gene expression (17, 42, 48).
In mammals, the acute response to CA inhibition is an increase in the
excretion of bicarbonate, sodium and potassium, an increase in urinary
flow, and titratable acid. However, the loss of bicarbonate and sodium
is self-limited on continued administration of the drug, probably
because the initial acidosis resulting from bicarbonate loss activates
bicarbonate reabsorption by CA-independent mechanisms. In our study,
major morphological modifications of A-IC were observed after 2 weeks
of acetazolamide treatment despite the fact that no significant
systemic acidosis was observed compared with controls. Therefore, the
persistence of an "activated" form of the A-IC phenotype in the
cortex and outer medulla does not seem to require the continued stimuli
of systemic CO2, plasma bicarbonate, or pH imbalance.
Although our present study did not screen for an initial transient
acidosis at early time points after CA inhibition, as shown in previous
reports (23, 33, 34), our data suggest that 1)
a possible transient initial systemic acidosis induced by acetazolamide
triggered a series of events that leads to the development of
"activated" A-IC, and "inhibition" of the B-IC phenotype, which
persisted after 2 wk; 2) the continued inhibition of CA at
the cellular level was itself responsible for the establishment of the
"activated" A-IC phenotype, perhaps by altering pHi
regulation and modifying gene expression (see above); and 3)
the systemic acid/base parameters providing feedback to IC are either
too small to measure accurately after renal compensation has occurred,
or are oscillating and were missed by our sampling procedure. It has
been shown that acute acid/base disturbances can alter IC characteristics after just a few hours (39, 41), but for
how long these changes can be maintained after the initial stimulus was
not examined. Indeed, acidosis, when occurring either in animals or in
humans after acetazolamide treatment, peaks usually after 1.5 to 5 days
(23). It seems paradoxical that A-IC developed morphological characteristics compatible with a high rate of proton secretion (31, 32) under conditions in which one of the
key components of their acidification mechanism, CAII, was inhibited. However, this could reflect a compensatory upregulation of one component of the proton secretory machinery in response to the complete
or partial inactivation of another component, i.e., CAII. This would at
least partially explain the apparently normal acid/base status of the
acetazolamide-treated animals, as well as in most acetazolamide-treated
patients (24).
Whether the reduction in the number of B-IC observed in the cortex of
acetazolamide-treated rats could be attributed to an interconversion of
B-IC into A-IC or was associated with a selective depletion of B-IC
together with an increased proliferation of A-IC remains uncertain. In
vitro, IC have been reported to switch polarity, with
bicarbonate-secreting cells transforming into proton-secreting cells
under some culture conditions (3, 47). Furthermore, there
are several older studies in the literature addressing the issue of
phenotype switching (i.e., conversion between principal and IC) in
collecting duct epithelial cells, dating back to the previous century
(see Ref. 53 for a review of this literature). So far,
however, no clear evidence in favor of this process has been obtained
by using the new generation of cell-specific antibodies and probes. The
fact that IC and PC could have a common cellular origin has also been
proposed by Fejes-Toth et al. (18). Our present data
clearly show that the cellular profile of collecting ducts can be
modulated in adult animals by CA inhibition. This result sets the stage
for future studies aimed at identifying the mechanism(s) by which this
process takes place.
 |
ACKNOWLEDGEMENTS |
We thank Mary McKee for technical assistance with electron
microscopy. We are grateful to Yvette Adabra, Claude Jacquiaud, and
Marie Chantal Jaudon (Pitié Salpétrière Hospital,
Paris, France) who performed the biological studies in vivo.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-42956 (D. Brown and V. Marshansky), a
research fellowship from the Institut National de la Santé et de
la Recherche Médicale, the Fondation Arthur Sachs (as part of the
Fullbright Program), L'Assistance Publique, and the Association des
Femmes Diplômées des Universités (C. Bagnis). S. Breton was partially supported by a Claflin Distinguished Scholarship
from the Massachusetts General Hospital and by National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-38452.
Address for reprint requests and other correspondence:
Corinne Bagnis, Renal Unit, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail:
bagnis{at}receptor.mgh.harvard.edu).
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 30 May 2000; accepted in final form 10 November 2000.
 |
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