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Program in Membrane Biology, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02120
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 · kg1 · day1), 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).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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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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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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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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