Maturation of carbonic anhydrase IV expression in rabbit
kidney
Cornelia A.
Winkler,
Ann
M.
Kittelberger,
Richard H.
Watkins,
William M.
Maniscalco, and
George J.
Schwartz
Department of Pediatrics, University of Rochester School of
Medicine, Rochester, New York 14642
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ABSTRACT |
Carbonic anhydrase (CA) IV facilitates renal acidification by
catalyzing the dehydration of luminal H2CO3. CA
IV is expressed in proximal tubules, medullary collecting ducts, and
A-intercalated cells of the mature rabbit kidney (Schwartz GJ,
Kittelberger AM, Barnhart DA, and Vijayakumar S. Am J
Physiol 278: F894-F904, 2000). In view of the maturation of
HCO
transport in the proximal tubule and collecting
duct, the ontogeny of CA IV expression was examined. During the first 2 wk, CA IV mRNA was expressed in maturing cortex and medulla at ~20%
of adult levels. The maturational increase was gradual in cortex over
3-5 wk of age but surged in the medulla, so that mRNA levels
appeared higher than those in the adult medulla. In situ hybridization showed very little CA IV mRNA at 5 days, with increases in deep cortex
and medullary collecting ducts by 21 days. Expression of CA IV protein
in the cortex and medulla was minimal at 3 days of age but then
apparent in the juxtamedullary region, A-intercalated cells and
medullary collecting ducts by 18 days; there was little labeling of the
proximal straight tubules of the medullary rays. Thus CA IV
expression may be regulated to accommodate the maturational increase in
HCO
absorption in the proximal tubule. In the
medullary collecting duct, there is a more robust maturation of CA IV
mRNA and protein, commensurate with the high rate of
HCO
absorption in the neonatal segment.
kidney development; in situ hybridization; immunohistochemistry; Northern blot; medullary collecting duct; proximal tubule; intercalated
cell
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INTRODUCTION |
THE NEONATE IS
CHARACTERIZED by an immaturity in maintaining acid-base
homeostasis. During the first weeks of postnatal life, increasing
filtered loads stimulate the development of HCO
reabsorption capacity of the proximal tubule (9, 26, 29). Several processes involved in urine acidification, such as the expression of proton pumps (3, 15),
Na+/H+ exchangers (2, 4, 27), and
chloride-base exchange (28) undergo maturational changes.
There are also maturational changes in the distal nephron during
development (16, 21-22, 29), which contribute to the
overall maturation of urine acidification and electrolyte homeostasis.
In addition to maturational changes in structure and transporters,
there are increases in the nephron activities of alkaline phosphatase,
glucose-6-phosphatase, and 5'-nucleotidase (32). It is
likely that there are maturational increases in several of the carbonic
anhydrase (CA) enzymes, including CA II (5, 7) and CA IV
(25); however, the changes in specific nephron segments
have only been examined for CA II (12). Immaturity of CA
enzymes could impair the infant's ability to excrete an acid load and
contribute to some forms of metabolic acidosis in prematurity. Data
concerning the maturation of CA IV expression in specific nephron
segments, particularly in the medulla, are presently not available.
CA, a zinc metalloenzyme, catalyzes the hydration of CO2
and the dehydration of carbonic acid. Of the 14 isoenzymes isolated thus far, two of the major renal isozymes are cytosolic CA II, which
accounts for 95% of activity and membrane bound CA IV, which accounts
for much of the membrane-associated CA activity. CA IV facilitates
HCO
reabsorption in the proximal tubule by
catalyzing the dehydration of intraluminal carbonic acid, which is
formed by the combination of filtered HCO
with
secreted protons. By immunohistochemistry in the mature kidney, CA IV
expression is found primarily in the proximal tubule and inner
medullary collecting ducts, as well as in the apical membranes of type
A (H+-secreting) intercalated cells (24). In
addition, apical membrane CA activity has been identified in proximal
convoluted tubules (14), medullary collecting duct
(13, 20), as well as along the inner stripe of the rabbit
outer medullary collecting duct (31) and in the inner
medullary collecting duct (33). CA IV mRNA has also been
demonstrated in these segments by RT-PCR (30).
There have been few studies addressing the maturational changes of CA
isoenzymes in the developing kidney (5, 12, 25). A
previous maturational study (25) of CA IV protein
expression (by immunoblot) in rabbit kidney used an antibody that was
not satisfactory for immunohistochemistry. The purpose of the present study was to examine the development of CA IV mRNA and protein expression in specific nephron segments of maturing rabbit kidneys by
comparing results from Northern blots, in situ hybridization, and
immunohistochemistry. These studies were accomplished by using a newly
developed goat anti-rabbit CA IV antibody (24); we also used a full-length cDNA coding for rabbit CA IV (34),
which allowed us to perform Northern blots on maturing cortex and
medulla and to generate cRNA probes for in situ hybridization.
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METHODS |
Animals.
Pregnant New Zealand white rabbits, purchased from Hazelton-Dutchland
farms (Denver, PA) were allowed to deliver in our animal quarters to
provide newborns (1-7 days of age). Litters of 1- to 2-wk-old pups
were purchased with their mothers and allowed to grow to maturity in
our facility. The diet for the adult animals (weight: 1.8-3.1 kg)
consisted of standard laboratory chow (Purina Mills, Richmond, IN) and
freely accessible tap water. The pups were fed by and raised with their mothers.
For death, adult rabbits were first sedated by intramuscular injection
with xylazine (5 mg/kg) and ketamine (44 mg/kg) followed by an
intracardiac injection of pentobarbital (100 mg/kg), which resulted in
cardiac arrest. Baby rabbits were killed using an intraperitoneal
injection of pentobarbital (100 mg/kg). After harvest, the kidneys were
rinsed in ice-cold PBS and then cut into coronal slices of 1- to 2-mm
thickness. For in situ hybridization or immunohistochemistry, a kidney
was perfused via the renal artery with PBS until it blanched, followed
by fixation.
Preparation of total RNA from kidney and other tissues.
As previously described (34), coronal slices of 1- to 2-mm
thickness were dissected into cortical and inner medullary zones and
snap frozen in liquid nitrogen. In the first 3 wk of life, outer
medulla cannot be readily distinguished from inner medulla (25,
32), so that the medulla was cut 1 mm below the cortex. Approximately 1 mm of the papillary tip was removed from animals of all ages.
RNA was extracted from frozen tissue, which was homogenized in an acid
guanidinium thiocyanate-phenol chloroform solution (Tri Reagent,
Molecular Research Center, Cincinnati, OH). The integrity of the total
RNA was verified by fractionation on 1.5% agarose gels and
quantified spectrophotometrically by measuring the absorbance at 260 and 280 nm.
Northern blot hybridization.
We performed Northern analysis as previously described
(34). Briefly, 10 µg of total RNA from cortex and
medulla were denatured in 3% formaldehyde, size fractionated on 1.5%
agarose gels, transferred to nylon filters (Zeta-Probe, Bio-Rad), and
hybridized with DNA probes radiolabeled with 32P[dCTP] by
random hexanucleotide extension (Amersham, Arlington Heights, IL). The
DNA probes were rabbit CA IV (34) and rat
-actin
(housekeeping gene) (17), which were labeled to a specific activity of 1.5 × 109
counts · min
1 · µg
1.
Autoradiography was performed using Kodak XAR film at
80°C for
1-3 days.
In situ hybridization.
The perfused kidneys were fixed in 4% paraformaldehyde overnight at
4°C, dehydrated through a graded ethanol series, followed by
incubation in xylene. After impregnation in paraffin wax, the tissue
was cast into blocks. Serial sections of tissue of ~5- to 8-µm
thickness were cut on a microtome, placed on positively charged slides
(Superfrost Plus, VWR Scientific, Piscataway, NJ), and stored at
80°C.
Plasmid DNA containing full-length CA IV cDNA was linearized with
restriction enzymes BamHI or HindIII to make
antisense and sense templates for cRNA probes. As previously described,
sense and antisense probes were determined by sequence analysis
(34) and synthesized according to the protocol by Auten et
al. (1). Probes were radiolabeled with
[
-33P]UTP which was diluted with cold UTP to reduce
the specific activity of the probe to ~1.5 × 109
counts · min
1 · µg
1.
Alkaline hydrolysis reduced the probe length to ~200 bp.
Before hybridization, slides were deparaffinized by immersion in xylene
and then dehydrated through a graded ethanol series followed by
proteinase K (GIBCO) digestion for 30 min at 37°C. Slides were then
equilibrated with 100 mM triethanolamine-HCl (pH 8.0) and treated with
0.25% acetic anhydride. The slides were then washed in 2× saline
sodium citrate (SSC; 0.3 M NaCl, 0.03 M Na3 citrate),
dehydrated, and dried. Prehybridization was at 53°C for 3 h
followed by brief rinsing of the slides in 2× SSC. Hybridization was
performed for 16 h at 53°C using hybridization solution
containing 30 ng · kb
1 · ml
1 of probe.
The hybridization solution was 50% formamide, 300 mM NaCl, 10 mM
Tris · HCl (pH 8.0), 1 mM EDTA, 1× Denhardt's reagent, 10%
dextran sulfate, and 0.5 mg/ml yeast tRNA.
After hybridization, slides were rinsed, digested with RNase A, and
rinsed in RNase buffer. The slides were rinsed in 0.1× SSC at 67°C
and again in 0.1× SSC at room temperature. Then, the slides were
dehydrated by passing through graded ethanol washes, dipped in a 1:1
dilution of nitro blue tetrazolium-2 emulsion (Eastman Kodak, Rochester
NY) and exposed at 4°C for 2-3 wk, developed as described
(34), and counterstained with hematoxylin and eosin. Sections from at least three animals were examined at each age group (5 days, 18-21 days, and adult).
Immunohistochemistry.
Whole kidneys were perfused and harvested as described above. The
kidneys were cut into three sagittal sections and fixed overnight in
2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate (PLP) at pH
7.4 and then paraffin embedded. Sections (4-6 µm) were
deparaffinized with Propor (Anotech, Battle Creek, MI) and hydrated in
a decreasing ethanol series. Hydrogen peroxide 0.3% was used to remove
endogenous peroxidase. The membranes were permeabilized using 0.3%
Triton X-100 and tissue blocked with 10% horse serum. The primary
antibody was an affinity-purified goat anti-rabbit CA IV peptide
antibody (24) in a concentration of 1:125 in 5% horse
serum. The antibody had been generated against the
NH2-terminal amino acids numbered 73-88
(YDQREARLVENNGHSV) of the deduced 308-amino acid sequence of rabbit CA
IV. The secondary antibody was biotin horse anti-goat (Vector,
Burlingame, CA) in a concentration of 1:450 in 5% horse serum.
According to manufacturer's instructions, avidin-biotinylated
horseradish peroxidase (Vectastain Elite ABC kit) was applied followed
by reaction for 10 min with the substrate diaminobenzidine
tetrahydrochloride for brown color development. In some cases, no
counterstain was performed to better resolve the faint staining in the
immature kidneys. Competition studies were performed by preincubating
the antibody overnight at 4°C with the immunizing peptide at 10-fold
molar excess.
Images were obtained with a Polaroid Digital camera (model DMC 2.0),
imported into Adobe Photoshop (v 5.0), and further processed using
Powerpoint 97 (Microsoft, Seattle, WA) and Freehand 8 (Macromedia, San
Francisco, CA) software. Sections were examined from at least three
animals at each age group (3 days, 18 days, and adult).
Analysis and statistics.
The intensity of signals in 24- to 72-h autoradiograms was analyzed by
scanning densitometry (AlphaImager 2000, v.3.3, Alpha Innotech, San
Leandro, CA, or SigmaGel, Jandel, San Rafael, CA). To compensate for
differences in quantity of total RNA on each lane of the membrane, each
CA IV value was normalized to its respective value of
-actin. To
allow for differences in the intensity of signals among various
Northern analyses, one adult kidney cortex was run with each
maturational study. The adult cortex was set as 100%, and all other
samples were expressed as a percentage of this reference.
Densitometric data are presented as means ± SE. Data were grouped
by postnatal age in weeks (through 5 plus adult), and comparisons were
analyzed by one-way ANOVA plus Tukey-Kramer and Scheffé tests for
multiple comparisons (NCSS, Kaysville, UT). Significance was asserted
when P < 0.05.
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RESULTS |
Northern analysis of CA IV mRNA expression.
The maturational expression of CA IV in zones of the kidney was
examined by Northern analysis. As shown in the representative blot
depicted in Fig. 1, a single band of
~1.6 kb was detected at all ages in renal cortex and medulla.
Densitometric analysis revealed an increase of steady-state mRNA
expression after the first 2 wk of life in cortical tissue (Fig.
2). The expression in the first 2 wk
showed significant variability among individual pups and no tendency to
increase with age. After 2 wk of age there was a maturational increase;
the levels at 5 wk of age were not significantly different from adult
values.

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Fig. 1.
Northern analysis of 10 µg of total RNA from maturing
kidney cortex (Ctx) and inner medulla (IM). Blots were probed for
rabbit carbonic anhydrase (CA) IV mRNA (top) and then for
actin (bottom) to control for equal loading. The
left blots show that adult cortex expresses more CA IV than
does the medulla and more than either cortex or medulla of the
4-day-old rabbit, when normalized to actin. The right-hand
blots show that medullary CA IV expression in 18- to 27-day-old rabbit
kidneys appears more abundant than in adult medulla and is consistently
greater than the expression of the cortex at the same age.
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Fig. 2.
Densitometric analysis of total RNA from maturing rabbit
kidney cortex. Densitometry was performed on the CA IV and actin
signals, and ratios were determined for each tissue for each blot.
These ratios were normalized to the adult (set as 100%). The asterisks
indicate statistically different mean ratios from adult values by
Scheffé's and Tukey-Kramer tests, P < 0.05.
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The medullary expression (Fig. 3) showed
little change during the first 2 wk of life, followed by increases to 5 wk of age. Although the values at 4 and 5 wk numerically exceeded those
of the adults, these differences did not reach statistical significance by the Tukey-Kramer test; however, the 5-wk value significantly exceeded the adult value by the Scheffé test. In the third and fourth weeks of life the expression in the medulla exceeded that in the
cortex (Fig. 1), and this relationship appeared to reverse at maturity.

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Fig. 3.
Densitometric analysis of total RNA from maturing rabbit
kidney medulla. Densitometry was performed on the CA IV and actin
signals, and ratios determined for each tissue for each blot. These
ratios were normalized to the adult (set as 100%) that was run in the
same study. The asterisks indicate statistically different mean ratios
from the 5-wk medullary value by Scheffé's and Tukey-Kramer
tests. The adult value was significantly different from the 5-wk value
only by Scheffé's test, P < 0.05.
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In situ hybridization.
To localize CA IV mRNA expression patterns in the immature kidney, we
performed in situ hybridization. Figure 4
depicts the expression pattern in representative in situ
hybridization slides at three different maturational ages. At 5 days of
age, there was little CA IV mRNA expression in the outer cortex
(top left) and medulla (middle left), with the
glomeruli, inner medulla, and papilla (bottom left) being
completely negative for signal (left panels). Some
detectable signal was noted in the deep cortex at the cortico-medullary
junction (arrowheads). By 21 days (middle) the expression at
the cortico-medullary junction was heavier (top middle,
arrowhead), but the medullary rays did not express much CA IV mRNA
(arrow). Definite expression was detected in the inner medulla
(middle), most likely over medullary collecting ducts, with
negative expression in the terminal inner medulla and papilla (bottom middle). Compared with the positive findings with
the anti-sense CA IV probe in the inner medulla at 3 wk of age, the sense probe gave no signal (Fig. 5,
top panels). In adult kidneys (Fig. 4, right
panels), cortical expression was primarily along the medullary rays
(top right panel) and at the corticomedullary junction.
Medullary expression (Fig. 4, middle right panel) was most
abundant in the initial inner medulla, and primarily in the collecting
ducts, and less heavy in the outer medulla (top right panel
below medullary rays); the terminal inner medulla and papillary tip
remained negative (bottom right panel).

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Fig. 4.
Low-power views of in situ hybridization studies of CA IV
mRNA expression in maturing rabbit kidneys. Cortical, medullary, and
papillary regions of 5-day, 21-day, and adult rabbit kidneys are
visualized by using darkfield illumination. There is a faint signal for
CA IV in the 5-day kidney only at the corticomedullary junction
(arrowheads); expression is low in the medulla and not visible in the
papillary tip. In the cortex of the 21-day kidney, the heaviest signal
is in the juxtamedullary area (arrowhead) with some faint labeling
along the developing medullary rays (arrow). The outer medulla has no
signal (not shown), but there is strong signal over the inner medulla,
except for the papillary tip. The adult shows a prominent signal over
the medullary rays, minimal signal over the outer medulla, and heavy
signal in the inner medulla except for the papillary tip. Bar = 250 µm.
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Fig. 5.
Low-power comparisons of
positive and negative signals in maturing rabbit kidneys.
Top row shows in situ hybridization of 18-day kidney medulla
with anti-sense (left) and sense (right) CA IV
probes. The positive signal over medullary collecting ducts is not seen
using sense probe. Bar in right panel = 250 µm. The second
row of panels shows CA IV immunohistochemistry of 10-day kidney cortex,
and the third row shows the medulla using antibody (left)
and antibody plus 10-fold molar excess of competing peptide
(right). The peptide completely prevented the staining
pattern in the immature kidney. The fourth row of panels shows CA IV
immunohistochemistry of adult kidney cortex using antibody
(left) and antibody plus competing peptide (right). The
peptide completely inhibited the development of the brown positive
signal over the medullary rays. Bar in right panel of
row 4 = 250 µm and also applies to rows 2 and
3.
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Immunohistochemistry.
CA IV protein was demonstrated by immunohistochemistry (brown staining
in Fig. 6). The pattern of staining
appeared grossly similar to that seen by in situ hybridization (cf.
Fig. 4). The 3-day animal (Fig. 6, left panels) had minimal
expression in the medullary rays and in the nephrogenic zone (Fig.
6A). There was definite but faint expression at the
corticomedullary junction, which became more apparent by 10 days of age
(Fig. 5, second row, left panel). A higher power view of the
deep cortex of the 3-day-old kidney (Fig. 6C) showed faint
staining in some proximal convoluted tubules and no label in the
glomeruli. There was modest medullary expression (Fig. 6B,
left panel, and Fig. 5, third row, left panel) but no papillary expression. This staining could be eliminated by
preincubating the antibody with excess immunizing peptide (Fig. 5,
second and third rows, right panels). By 18 days (Fig. 6,
middle panels), there was stronger cortical expression
(A), especially at the corticomedullary junction but less
along the medullary rays. There was prominent expression in the medulla
(B), primarily along the medullary collecting ducts
(C), as in the mature kidney. In the adult animal (Fig. 6,
right panels), expression was prominent along medullary rays
in the cortex (A), primarily in proximal straight tubules
and less so in the proximal convoluted tubules of the cortical
labyrinth. Juxtamedullary proximal convoluted tubules also showed heavy
staining. This staining could be prevented by preincubating antibody
with peptide (Fig. 5, fourth row). There was strong expression in the
inner medulla (Fig. 6B, right panel) primarily
along the medullary collecting ducts (Fig. 6C). Glomeruli and papillary tip (Fig. 6D) were negative for CA IV
immunohistochemistry throughout all ages.

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Fig. 6.
Low-power views of CA IV
immunohistochemistry in maturing rabbit kidneys. Cortical, medullary,
and papillary regions of 3-day, 18-day, and adult rabbit kidneys are
visualized by using brightfield illumination of the brown DAB reaction
product. A: the nephrogenic outer cortical zone of the 3-day
rabbit kidney is negative for CA IV, but there is some staining at the
corticomedullary junction. The medullary rays are virtually negative at
this age. The 18-day kidney is more heavily labeled particularly at the
corticomedullary junction, and there is some staining in the medullary
rays. The adult shows prominent staining of medullary rays, and signal
is heaviest at the corticomedullary junction. B: the inner
medulla and papilla of the 3-day old show light staining. In the 18-day
old, the inner medulla is well stained particularly over the collecting
ducts (bottom part of figure); the outer medulla is negative
at this age (top). In the adult, the outer medulla
(top) is lightly stained as it makes a transition to the
heavier stained initial inner medulla. C: a higher power
view of the deep cortex of the 3-day-old kidney shows faint staining of
proximal convoluted tubules. Bar = 100 µm for this panel only. A
low-power view of the inner medulla shows the positive-staining
collecting ducts of the 18-day-old and the adult. D: the
papilla was negative in the adult, as well as in the younger animals
(not shown). Bar = 250 µm and applies to all other panels in this
figure other than C, left.
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Higher power views of the kidney are depicted in Fig.
7. In the 3-day-old kidney
(left panels), the nephrogenic zone (A) did not
express CA IV; there was no label in the vesicles, S-shaped bodies, or
in the ampullae of the nascent collecting ducts. Deeper in the cortex
(B) there was faint staining of proximal convoluted tubules,
predominately along the apical membrane (arrows). Glomeruli were
negative. In the medulla (C), there was faint staining of the apical membrane of medullary collecting ducts (arrows). Type A-intercalated cells with apical CA IV labeling (24) could
just barely be detected in cortical collecting ducts from 10-day-old animals (Fig. 8, top panel,
arrow). The 18-day-old kidney (Fig. 7, middle panels)
showed substantially greater CA IV expression. In the mid-deep cortex
(A), there was definite apical labeling of proximal
convoluted tubules (arrow); glomeruli were negative. The medullary rays
(B) showed faint apical labeling of proximal straight
tubules (arrows) and definite apical labeling of type A-intercalated
cells (Fig. 8, middle panel, arrows). The inner medulla
(Fig. 7C) revealed heavy apical and probably lateral or cytosolic staining of medullary collecting ducts.

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Fig. 7.
High power immunohistochemistry
of CA IV in 3-day, 18-day, and adult rabbit kidneys. A:
there is no staining of the nephrogenic zone of the 3-day rabbit
kidney. The 18-day old shows staining of the juxtamedullary proximal
convoluted tubules with more pronounced apical staining (arrow). In the
inner cortex, adult kidneys show heavier and more diffuse staining of
the proximal convoluted tubules with pronounced apical staining
(arrow), but also with cytosolic staining. Glomeruli are consistently
negative at all ages. B: deep cortical views show some faint
staining of proximal tubules in the 3-day-old (arrows) and
progressively heavier labeling in the medullary rays of the 18-day-old
(arrows) and adult; there is probably some basolateral staining of
adult proximal straight tubules. A collecting duct type
A-intercalated cell expressed apical CA IV (arrow). C:
labeling of inner medullary collecting ducts increases with age; the
staining in the younger animals is primarily apical (arrows), whereas
in the adult the staining is more diffuse, including apical and lateral
membranes and cytosol. Bar = 50 µm.
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Fig. 8.
High-power views of CA IV
immunocytochemistry in cortical collecting ducts from 10-day, 18-day,
and adult kidneys. Occasional cells show apical brown label of CA IV
(arrows) consistent with their being H+-secreting
A-intercalated cells (24). The staining pattern changes
from a fine apical line at 10 days (top, arrow) to a thicker
apical label at 18 days (middle, arrows), which is more
intense at maturity (bottom, arrows). Bar = 50 µm.
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Adult kidney (Fig. 7, right panels) showed heavier
expression of CA IV. In the inner cortex (Fig. 7A) there was
heavy labeling of proximal convoluted tubules (arrow) and negative
staining of the glomeruli; in the outer cortex, staining of proximal
convoluted tubules was less intense compared with the inner cortex (not
shown). Along the medullary rays (B), proximal straight
tubules abundantly expressed CA IV; although the staining was
predominately apical, brown reaction product was frequently observed
over most of the cell cytosol, as well as over the basolateral
membrane, as seen previously (24). In the medullary rays,
a few cells in collecting ducts expressed apical CA IV (Fig.
7B, right panel, arrow; Fig. 8, bottom
panel, arrows), indicating that they were type A-intercalated cells.
There was faint staining of collecting duct cells of the outer medulla
(not shown) but heavy staining of inner medullary collecting duct cells
(C); the pattern of brown reaction product was more apical
and cytosolic, as seen previously (24).
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DISCUSSION |
CA IV is an important enzyme in renal acid-base transport. Because
there are large maturational increases in proximal tubule (26) and medullary collecting duct (16)
HCO
reabsorption, it would be expected that there
would be concomitant increases in cortical and medullary CA IV. Indeed,
this has been recently demonstrated by immunoblotting of kidney
cortical and medullary membranes from maturing rabbits
(25); however, this antibody failed to show medullary
expression of CA IV protein by immunohistochemistry, despite the
previous demonstration of CA IV mRNA in the inner medullary collecting
duct (34). In addition, there have been no
nephron-specific studies of the maturation of CA IV gene expression.
For these reasons, we used a newly developed goat anti-rabbit CA IV
peptide antibody to perform immunohistochemistry and a CA IV cRNA probe
to perform in situ hybridization on kidney sections from maturing
rabbits. We also obtained densitometric data from Northern blots of
cortical and medullary kidney mRNA from maturing rabbits. In general,
there was excellent agreement between the in situ hybridization and
immunocytochemical patterns, and these paralleled the densitometric
data obtained from Northern blots of total RNA from cortex and medulla:
expression of CA IV was quite modest early in postnatal life and
increased after the second postnatal week, to reach mature levels early
in the second month of life.
CA IV mRNA and protein were expressed by the mature proximal tubule,
and the pattern showed heavy labeling of the medullary rays and
juxtamedullary tubules, with less staining of the early superficial
proximal tubules and even less of the S3 proximal straight tubules in
the medulla. The medullary ray pattern of CA IV expression early in
life was not observed, nor was there expression in the nephrogenic
zone. Expression of CA IV was most evident in convoluted tubules near
the juxtamedullary glomeruli, and more in the labyrinths than in the
medullary rays. This immature pattern was still evident at 18-21
days of age, and the medullary rays were just beginning to show CA IV
expression in the proximal straight tubules therein. This increase in
cortical CA IV expression was similar to that obtained from the
densitometric analysis of CA IV Northern blots of maturing kidney
cortex. This is not unexpected in view of the fact that proximal
tubules comprise the major cell type expressing CA IV in the kidney
cortex (6, 8).
We previously showed that there was a maturational surge in
juxtamedullary proximal tubular HCO
absorption
between 4 and 6 wk of age, such that mature levels of transport are
reached by the end of this period (26). We also concluded
from the morphological appearance of the maturing proximal tubules
(10) that the superficial proximal tubules lagged behind
the juxtamedullary tubules by at least 1 postnatal wk. Thus the
gradual increase in cortical CA IV mRNA expression is probably adequate
to mediate the maturational increases in transport that occur after 3 postnatal wk of age. The pattern of this maturational increase in CA IV
mRNA expression agrees well with the previously reported increase in CA
IV protein by Western blot (using a different antibody)
(25): expression was ~20% of the adult during the first
2 postnatal wk before steadily increasing to adult levels early in the
second month of life.
CA IV mRNA and protein were expressed by the mature medullary
collecting duct, especially the initial inner medullary collecting duct, as has been shown previously (24, 30, 34). The
neonatal medulla showed weak labeling of inner medullary collecting
ducts, but this appeared nearly mature by 18 days of age. Indeed, the densitometric expression of CA IV mRNA at 5 wk of age appeared numerically higher than that observed in the adult, and the intensity of the in situ hybridization signal over the medullary collecting ducts
at 18 days of age appeared to be comparable to that seen in mature
kidney inner medulla. Presumably, the high level of medullary CA IV
mRNA during the third to fifth week of life results in early maturation
of CA IV protein expression, as shown previously (25).
This pattern also reflects the early maturity of medullary collecting
duct HCO
absorption (16) and cell pH
(23). In addition, urinary concentrating ability, another major function of the medulla, matures after 21 days of age (11), parallel to the expression of CA IV.
The membrane distribution of CA IV expression in the proximal tubule
has previously been shown to be apical and basolateral (8, 24,
25). Because CA IV is a GPI-linked protein (18), it
is likely to be expressed only apically (19).
Perhaps, a non-GPI-linked form of CA IV or another isoenzyme of CA is
expressed basolaterally and was detected using our anti-peptide
antibody. Further studies are needed to determine the identity of the
basolateral expression.
In the present study, the staining pattern of CA IV in the immature
kidney differed from that of the adult. Whereas the pattern of CA IV
labeling in the immature kidney was typically apical in proximal
tubules, A-intercalated cells and in medullary collecting duct cells,
in mature kidneys the staining in the proximal tubule showed apical as
well as basolateral signals, and that in the medullary collecting duct
cells was more apical and cytosolic, as described previously
(24). Even though sections from all age groups were
batched to minimize intra-assay variability, it is not likely that the
change in staining pattern is due to overstaining of the mature kidney
or greater drift of the reaction product. The pattern in the mature
kidney did not change much after shorter color generation time (not
shown). At a minimum, we can conclude that early expression of CA IV in
renal epithelial cells was primarily apical, as expected for a
GPI-anchored protein. Whether the appearance of basolateral and
cytosolic CA IV signal in the mature kidney reflects a high rate of CA
IV synthesis, a different isoform of CA IV, another membrane-bound CA,
or an artifact of fixation will require further investigation.
In summary, these are the first studies to examine the maturational
expression of CA IV mRNA and protein in kidney sections, and in nephron
segments. There was good agreement between the results from in situ
hybridization and immunohistochemistry, and these correlated well with
the Northern blots. Expression of CA IV mRNA and protein was ~20% of
the adult during the first 2 wk of postnatal life, before increasing in
parallel with the previously observed increases in transport. The
maturation of the medulla appeared to precede that of the cortex, as
has been reported for a variety of functional studies.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health (NIH)
Grants DK-50603 (to G. J. Schwartz) and HL-54632 (to W. M. Maniscalco). C. A. Winkler was supported in part by NIH training grant T32 HD-07383.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: G. J. Schwartz, Div. of Pediatric Nephrology, Box 777, Univ. of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: George_Schwartz{at}urmc.rochester.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 11 August 2000; accepted in final form 17 January 2001.
 |
REFERENCES |
1.
Auten, RL,
Watkins RH,
Shapiro DL,
and
Horowitz S.
Surfactant apoprotein A (SP-A) is synthesized in airway cells.
Am J Respir Cell Mol Biol
3:
491-496,
1990[ISI][Medline].
2.
Baum, M.
Neonatal rabbit juxtamedullary proximal convoluted tubule acidification.
J Clin Invest
85:
499-506,
1990[ISI][Medline].
3.
Baum, M.
Developmental changes in rabbit juxtamedullary proximal convoluted tubule acidification.
Pediatr Res
31:
411-414,
1992[Abstract].
4.
Beck, JC,
Lipkowitz MS,
and
Abramson RG.
Ontogeny of Na/H antiporter activity in rabbit renal brush border membrane vesicles.
J Clin Invest
87:
2067-2076,
1991[ISI][Medline].
5.
Brion, LP,
Zavilowitz BJ,
Rosen O,
and
Schwartz GJ.
Changes in soluble carbonic anhydrase activity in response to maturation and NH4Cl loading in the rabbit.
Am J Physiol Regulatory Integrative Comp Physiol
261:
R1204-R1213,
1991[Abstract].
6.
Brion, LP,
Zavilowitz BJ,
Suarez C,
and
Schwartz GJ.
Metabolic acidosis stimulates carbonic anhydrase activity in rabbit proximal tubule and medullary collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F185-F195,
1994[Abstract/Free Full Text].
7.
Brown, D,
Kumpulainen T,
Roth J,
and
Orci L.
Immunohistochemical localization of carbonic anhydrase in postnatal and adult rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F110-F118,
1983[ISI][Medline].
8.
Brown, D,
Zhu XL,
and
Sly WS.
Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells.
Proc Natl Acad Sci USA
87:
7457-7461,
1990[Abstract].
9.
Edelmann, CM, Jr,
Rodriguez-Soriano J,
Boichis H,
Gruskin AB,
and
Acosta M.
Renal bicarbonate reabsorption and hydrogen ion excretion in infants.
J Clin Invest
46:
1309-1317,
1967[ISI].
10.
Evan, AP,
Gattone VHI,
and
Schwartz GJ.
Development of solute transort in rabbit proximal tubule. II. Morphological segmentation.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F391-F407,
1983[Abstract/Free Full Text].
11.
Forrest, JN, Jr,
and
Stanier MW.
Kidney composition and renal concentration ability in young rabbits.
J Physiol (Lond)
187:
1-4,
1966[ISI][Medline].
12.
Karashima, S,
Hattori S,
Ushijima T,
Furuse A,
Nakazato H,
and
Matsuda I.
Developmental changes in carbonic anhydrase II in the rat kidney.
Pediatr Nephrol
12:
263-268,
1998[ISI][Medline].
13.
Lonnerholm, G,
and
Wistrand PJ.
Membrane-bound carbonic anhydrase CA IV in the human kidney.
Acta Physiol Scand
141:
231-234,
1991[ISI][Medline].
14.
Lucci, MS,
Tinker JP,
Weiner IM,
and
DuBose TD, Jr.
Function of proximal tubule carbonic anhydrase defined by selective inhibition.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F443-F449,
1983[Abstract/Free Full Text].
15.
Matsumoto, T,
Fejes-Toth G,
and
Schwartz GJ.
Postnatal differentiation of rabbit collecting duct intercalated cells.
Pediatr Res
39:
1-12,
1996[Abstract].
16.
Mehrgut, FM,
Satlin LM,
and
Schwartz GJ.
Maturation of HCO
transport in rabbit collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
259:
F801-F808,
1990[Abstract/Free Full Text].
17.
Nudel, U,
Zakut R,
Shani M,
Neuman S,
Levy Z,
and
Yaffe D.
The nucleotide sequence of the rat cytoplasmic
-actin gene.
Nucleic Acids Res
11:
1759-1771,
1983[Abstract].
18.
Okuyama, T,
Waheed A,
Kusumoto W,
Zhu XL,
and
Sly WS.
Carbonic anhydrase IV: role of removal of C-terminal domain in glycosylphosphatidylinositol anchoring and realization of enzyme activity.
Arch Biochem Biophys
320:
315-322,
1995[ISI][Medline].
19.
Powell, SK,
Cunningham BA,
Edelman GM,
and
Rodriguez-Boulan E.
Targeting of transmembrane and GPI-anchored forms of N-CAM to opposite domains of a polarized epithelial cell.
Nature
353:
76-77,
1991[ISI][Medline].
20.
Ridderstrale, Y,
Kashgarian M,
Koeppen B,
Giebisch G,
Stetson D,
Ardito T,
and
Stanton B.
Morphological heterogeneity of the rabbit collecting duct.
Kidney Int
34:
655-670,
1988[ISI][Medline].
21.
Satlin, LM.
Postnatal maturation of potassium transport in rabbit cortical collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F57-F65,
1994[Abstract/Free Full Text].
22.
Satlin, LM,
Matsumoto T,
and
Schwartz GJ.
Postnatal maturation of rabbit renal collecting duct. III. Peanut lectin-binding intercalated cells.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F199-F208,
1992[Abstract/Free Full Text].
23.
Satlin, LM,
and
Schwartz GJ.
Postnatal maturation of the rabbit renal collecting duct: intercalated cell function.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F622-F635,
1987[Abstract/Free Full Text].
24.
Schwartz, GJ,
Kittelberger AM,
Barnhart DA,
and
Vijayakumar S.
Carbonic anhydrase IV is expressed in H+-secreting cells of the rabbit kidney.
Am J Physiol Renal Physiol
278:
F894-F904,
2000[Abstract/Free Full Text].
25.
Schwartz, GJ,
Olson J,
Kittelberger AM,
Matsumoto T,
Waheed A,
and
Sly WS.
Postnatal development of carbonic anhydrase IV expression in rabbit kidney.
Am J Physiol Renal Physiol
276:
F510-F520,
1999[Abstract/Free Full Text].
26.
Schwartz, GJ,
and
Evan AP.
Development of solute transport in rabbit proximal tubule. I. HCO
and glucose absorption.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F382-F390,
1983[ISI][Medline].
27.
Shah, M,
Gupta N,
Dwarakanath V,
Moe OW,
and
Baum M.
Ontogeny of Na+/H+ antiporter activity in rat proximal convoluted tubules.
Pediatr Res
48:
206-210,
2000[Abstract/Free Full Text].
28.
Shah, M,
Quigley R,
and
Baum M.
Maturation of rabbit proximal straight tubule chloride/base exchange.
Am J Physiol Renal Physiol
274:
F883-F888,
1998[Abstract/Free Full Text].
29.
Spitzer, A,
and
Schwartz GJ.
The kidney during development.
In: Handbook of Physiology. Renal Physiology Bethesda, MD: Am Physiol Soc, 1992, sect. 8, vol. I, chapt. 12, p. 475-544.
30.
Tsuruoka, S,
Kittelberger AM,
and
Schwartz GJ.
Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis.
Am J Physiol Renal Physiol
274:
F259-F267,
1998[Abstract/Free Full Text].
31.
Tsuruoka, S,
and
Schwartz GJ.
HCO
absorption in rabbit outer medullary collecting duct: role of luminal carbonic anhydrase.
Am J Physiol Renal Physiol
274:
F139-F147,
1998[Abstract/Free Full Text].
32.
Wachstein, M,
and
Bradshaw M.
Histochemical localization of enzyme activity in the kidneys of three mammalian species during their postnatal development.
J Histochem Cytochem
13:
44-56,
1965[ISI].
33.
Wall, SM,
Flessner MF,
and
Knepper MA.
Distribution of luminal carbonic anhydrase activity along the rat inner medullary collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F738-F748,
1991[Abstract/Free Full Text].
34.
Winkler, CA,
Kittelberger AM,
and
Schwartz GJ.
Expression of carbonic anhydrase IV mRNA in rabbit kidney: stimulation by metabolic acidosis.
Am J Physiol Renal Physiol
272:
F551-F560,
1997[Abstract/Free Full Text].
Am J Physiol Renal Fluid Electrolyte Physiol 280(5):F895-F903
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