FL. P.J. Linser, The Whitney. Laboratory and the. Department of Anatomy and Cell ..... region. The principal cells have only faint immunostaining for CA II. Bar =.
Ultrastructural Subpopulations C. Craig
Jin Kim,1
Localization of Carbonic Anhydrase II in of Intercalated Cells of the Rat Kidney
Tisher,
Paul
J. Linser,
and
Kirsten
M. Madsen
was present in principal cells, tubule cells and inner medullary
J. Kim, CC. usher, KM. Madsen, Laboratory of Experimental Morphology, Division of Nephrology, Hypertension and Transplantation, University of Florida, Gainesville, FL
were negative
for carbonic
whereas
connecting
collecting
duct
anhydrase
II. These re-
P.J. Linser, The Whitney Laboratory and the Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL
sults demonstrate striking differences in and subcellular distribution of carbonic between type A and type B intercalated providing additional evidence for the at least two structurally and functionally
(J. Am. Soc. Nephrol.
ulations
1990; 1:245-256)
of
intercalated
cells
cells
in the
the intensity anhydrase II cells, thus existence of distinct
rat
pop-
collecting
duct. Key Words:
ABSTRACT At least two configurations of intercalated cells, type A and type B, ore present in the cortical collecting duct. Intercalated cells are rich in carbonic anhydrase.
However,
differences
it is not
in the
level
of this enzyme between lated cells. The purpose mine
the
relative
known
and
whether
subcellular
there
I
ore
content
and
intracellular
localize
hydrase tion
anhydrase,
II by
light
and electron microscopy by an indirect immunoperoxidase method. A Western immunoblot analysis of homogenates of rat kidney cortex and medulla with the carbonic anhydrose II antibody revealed a single polypeptide band at 29 kDa corresponding to the molecular size of carbonic onhydrase II. By both light and electron microscopy, carbonic anhydrase II immunoreactivity was present in all intercalated cells but the intensity of staining was much greater in type
A than in type B cells. In addition, type
A cells was especially
immunostaining
pronounced
in the apical
cytoplasm and apical microprojections type B cells, immunostaining was throughout
the
cytoplasm.
A third
in
configuration
of
intercalated cell with diffuse immunostaining for carbonic anhydrase II was occasionally observed in the connecting segment. Very weok immunostaining ‘correspondence Transplantation.
to Dr. .1. Kim, Box .1-224. JHMHc.
Division of Nophrology, Hypeilenslon and University of Florida. Gainesville, FL 32610.
1046-6673/0 103-0245$02.00/0 Journal at the American Society at Nephrology Copyright 0 1990 by the American Society of Nophralogy
Journal
of the American
Society
of Nephrology
Both
microscopy,
cells carbon
proton
studies
and
immuno-
high
enzyme dioxide
to
duct
and
immu-
demonstrated
levels
that
popu-
bicarbonate
(2-4) have
contain
(CA)-the of
in
histochemical
(5-8)
catalyzes
carbonic
that
of carbonic the
acid,
anhydra-
which
then
dissociates into protons and bicarbonate (reviewed in ref. 9). However, because most of these studies were performed at the light microscope level, little is known about the subcellular distribution of CA. Furthermore, it is not known whether specific types of intercalated cells exhibit different staining patterns for CA. Two distinct populations of intercalated cells, type A and
type
B.
have
been
described
in
the
cortical
collecting duct (CCD) of the rat (10). The type A intercalated cells are similar in ultrastructure to intercalated cells in the outer medullary collecting duct (OMCD). They contain an apical proton ATPase (11) and they
a
basolateral
are
intercalated
whereas in more diffuse
electron analysis
involved
(1).
intercalated
II in the various subpopcells in the rat collecting
carbonic
are
nocytochemical
duct. A rabbit polyclonal antibody directed against mouse erythrocyte carbonic anhydrase II was emto
they
transport
distribu-
duct,
immunob/ot
ntercalated cells constitute a heterogeneous lation of cells in the mammalian collecting
where
distribution
type A and type B intercaof this study was to deter-
tion of carbonic anhydrase ulations of intercalated
ployed
Collecting
cytochemistry.
anion
involved cells
exchanger
in
proton
are
present
(8.12-14),
secretion. only
and
The in
the
type CCD.
B Im-
munoreactivity for proton ATPase has been demonstrated in the basolateral membrane of type B cells in the rat (15). The function of the B cells is not known with certainty, but it has been suggested that they secrete bicarbonate (16). Recently. a third type of
intercalated
cell
with
apical
staining
for
proton
ATPase. but without a basolateral anion exchanger (14), has been described. The ultrastructural appearance of this cell has not been reported. It is known from biochemical studies that two different isozymes of CA are present in the kidney-a
245
Carbonic
Anhydrase
in Intercaiated
Cells
cytoplasmic enzyme, carbonic anhydrase II (CA II), previously designated carbonic anhydrase C. and a membrane-bound enzyme, carbonic anhydrase IV (CA IV) (9,17). Wistrand and co-workers have purified and characterized CA II from the supernatants of both
rat
and
human
renal
homogenates
more recently have proceeded terize CA IV from human kidney (20,21). The
relative in the rat
purpose
of
content various
kidney
by
this
study
(18,19)
to isolate membrane was
to
and
determine
and subcellular distribution populations of intercalated using
a
mouse erythrocyte CA and electron microscope
polyclonal
and
characfractions the
of CA II cells in the
antibody
II in combination immunocytochemistry.
against
with
light
METHODS
sections
Female Sprague-Dawley rats g were used in all experiments. tized ip with sodium pentobarbital wt).
Tissue
weighing 165 to 210 They were anesthe(50 mg/kg. body
Preservation
that
were
fixed
by
immersion
in
the
PLP
for
The rected
sections
were
washed
antibody against
terization
6
through of 50
three times (15 mm each) with 50 mM NH4CI in PBS. The sections were then permeabilized by a 4-h incubation in 0.05% saponin-1% ovalbumin-PBS (buffer A) and incubated overnight at 4#{176}C in the rabbit antiserum against mouse erythrocyte CA II diluted 1:400 in buffer A. Sections incubated without primary antibody or in normal rabbit serum served as controls. The tissue sections were washed six times (20 mm each) in 0.05% saponin-0. 1% ovalbumin-PBS (buffer B) and incubated for 3 h in peroxidase-conjugated goat anti-rabbit immunoglobulmn, Fab fragment (Pel-Freez), diluted 1:50 in buffer A. After being rinsed six times (3 mm each) in buffer B. the sections were fixed for 1 h in 1% glutaraldehyde in Tyrode buffer (pH 7.4) and washed sequentially with Tyrode buffer (pH 7.4) and 0.05 M Tris buffer (pH 7.6). In some experiments, 0.05% polyoxyethylene 9 lauryl
246
on
a
LKB
Nova
ultramicrotome.
of Antibody was mouse
of
this
a rabbit erythrocyte
(24).
blot against rat on a polyacrylamide perfused through
To
for
10 mm
to remove
removed, frozen on
separated
out
as
dry
described
standard
techniques was
all
the
blood.
into
ice. Western previously
mogenized in 5 volumes PAGE sample buffer and protein
been
insure
described
in
specificity
of the
reaction, a Western immunohomogenates was performed gel. The kidneys of two rats were the abdominal aorta with PBS (pH
7.4)
of
has
antibody diII. The charac-
kidney
then and
by
polyclonal CA
antibody
detail previously immunocytochemical
Immunocyfochemistry vibratome
cut
Characterization
h. Sections of tissue were cut transversely the entire kidney on a vibratome at a thickness zm and processed for immunocytochemistry.
The
were
One-micron sections were stained with toluidine blue and photographed on a Zeiss Photomicroscope II. Thin sections were stained with lead citrate and examined and photographed on a Zeiss 1OA transmission electron microscope.
The kidneys of six animals were perfused via the abdominal aorta, initially with phosphate-buffered saline (PBS) at pH 7.4 and then with the perlodatelysine-paraformaldehyde mixture (PLP) of McLean and Nakane (22) for 3 mm. After perfusion, the kidneys were removed and cut into 1- to 2-mm-thick slices
ether polidocanol was substituted for saponin as a detergent. For the detection of horseradish peroxidase, the sections were incubated in 0.1% diaminobenzidine in 0.05 M Tris buffer (pH 7.6) for 5 mm. after which H2O2 was added to a final concentration of 0.01% and incubation was continued for 10 mm (23). The sections were washed sequentially with 0.05 M Tris buffer (pH 7.6) and 0.1 M Na cacodylate buffer (pH 7.4), postfixed in 2% Os04 in 0.1 M Na cacodylate buffer (pH 7.4) for 1 h at 4#{176}C, dehydrated in graded ethanols. and embedded in Medcast resin. From all animals, 50-nm-thick vibratome sections through the entire kidney were mounted in Medcast resin on glass slides for light microscopy. Sections from the cortex, outer medulla, and inner medulla of four animals were embedded in Medcast resin for light and transmission electron microscopy. Plastic
per
kidneys
(25).
blotting Tissues
For lane.
were
and
(weight/volume) electrophoresed (26).
loaded
The
cortex
each
medulla, was carried were hoof SDSin 10% gels
sample,
Protein
g
32
was
deter-
mined by the Bio-Rad protein assay. Prestained molecular weight markers (Bethesda Research Laboratories. Inc.), which included bovine CA II, were coelectrophoresed with the tissue samples. Following electrophoresis, the proteins were electroblotted onto nitrocellulose (27). Blots were stained for 5 mm in 1% fast green10% acetic acid-50% methanol and then destained in this solution without the dye. After the protein patterns were photographed. the blots were transferred to 2.5% dry milk in Tris-buffered saline with 0.1% Tween 20 to block residual protein binding sites. Subsequent immunostamning of blots by the indirect immunoperoxidase method (28) was performed
diluted
with
1:200.
the
rabbit
Secondary
antiserum
antibodies
Voiume
to mouse
were
I ‘Number
CA
II,
horse-
3’
1990
Kim et ci
radish peroxidase ringer Mannheim).
conjugates
of goat
antisera
(Boeh-
RESULTS Immunoblot The enates
results of rat
of the renal
immunoblot cortex and
analysis medulla
of homogwith anti-
serum against mouse CA!! are shown In Figure 1. In both cortex and medulla, immunostaining was associated with a single polypeptide band that comigrated with bovine CA II, which was included with the commercial molecular weight marker proteins used in the analysis. Thus, the antiserum used in this study recognizes a protein of the molecular weight of CA in both cortex and medulla of the rat kidney.
II
Tissue Preservation The preservation of the ultrastructure of the kidney tubule cells was excellent in spite of the treatment with detergent, which was required to facilitate the penetration of antibodies into the tissue slices. There were no differences in ultrastructure or in immunoreactivity between tissue treated with saponm and tissue treated with polidocanol. In the tissue processed without detergent, there was poor penetration of the primary antibody and the intensity of immunostamning
from
with the
varied
distance
of the
cells
the tissue surface.
Light Microscopy Examination of 50-sm vibratome sections cut transversely through the kidney demonstrated a dark brown to black reaction product indicating the presence of CA II in a subpopulation of cells in the collecting duct (Figure 2). These cells had the appearance of intercalated cells, and they were present throughout the collecting duct except for the terminal segment of the inner medullary collecting duct (IMCD5). In the CCD and the connecting segment. two configurations of intercalated cells could be identified when viewed en face to reveal the circumference of the cells (Figure 2a and b). Some cells had a starshaped configuration with short, slender cytoplasmic processes
extending
laterally
from
the
main
cell
body
and exhibited strong immunoreactivity. Other cells had a more round, smooth configuration without lateral cell processes and exhibited less immunoreactivity. Only one configuration of intercalated cells was observed in the OMCD and the initial segment of the inner medullary collecting duct (IMCD1) (Figure 2c and d). These cells had a smooth or slightly angular circumference without lateral cell processes. and they exhibited intense CA II immunoreactivity.
Journal
of the American
Society
of Nephrology
Figure 1. Western blot analysis of homogenates of rat kidney cortex and medulla. Prestained molecular size markers and solubilized proteins of cortex and medulla were separated on 10% PAGE gels and then electroblotted to nitrocellulose. Panel A shows the blot after being stained with fast green. The molecular masses of the markers (in kilodoltons) are indicated adjacent to the lane designated with an asterisk. Proteins from cortex and medulla are shown in lanes c and m, respectively. In panel B, the nitrocellulose blot was washed to remove fast green staining and then immunostained for the localization of CA II via the indirect immunoperoxidose method. Note that the molecular size marker at 29 kDa is bovine CA II and shows strong immunoreactlvity. In both cortex and medulla, a single polypeptide is labeled with the antibody and this polypeptide comigrates with the marker CA II. Apparent staining of the 14.3-kDa molecular size marker Is residual dye from the prestolning and not immunoreactivlty. The molecular size markers (in kilodaitons) are: myosin H chain. 200; phosphorylase B, 97.4: BSA. 68; ovalbumin, 43; bovIne CA Il, 29; it-lactoglobulin, 18.4; lysozyme, 14.3 kDa.
Light microscope tic sections (Figure strong immunoreactivity connecting segment
examination of one-micron plas3) revealed that the cells with observed in the CCD and (Figure 3a) represented type A
247
Carbonic
Anhydrase
in intercalated
Cells
Figure 2. Light micrographs the initial cytoplasmic processes one
248
type
of 50-1km-thick vibratome sections demonstrating CA II immunoreactivity in intercalated cells in CCD (a), the CCD in the medullary ray (b). the OMCD (C), and the IMCD (d). In the CCD, cells with lateral processes and intense immunostaining represent type A intercalated cells (arrows) whereas cells without lateral and less intense immunostainirig represent type B intercalated cells (arrowheads). In the OMCD and IMCD, only
of intercalated
cell
is present.
Magniticotion,
x210.
Volume
I
.
Number
3’
1990
Kim et at
U
F
a
C
I 4’’
-
-
Figure 3. Light micrographs of 1-nm plastic sections from the CCD (a), OMCD (b), IMCD, (c), and IMCD, (d). Type intercalated cells in the CCD (arrows) and intercalated cells in the OMCD and IMCD exhibit strong immunoreactivity, comparison, type B intercalated cells in the CCD (arrowheads) exhibit much less immunoreactivity. There is no immunostaining of IMCD cells in the IMCD,. Magnification, x210.
Journal
of the
American
Society
of
Nephralogy
A In
249
Carbonic
Anhydrase
intercalated
in intercalated
cells,
and
tivity represented type of intercalated the CCD (14). Type on
one-micron
those
Cells
with
less
Electron
immunoreac-
type B cells and possibly a third cell which has been observed in A and type B cells were identified
plastic
sections
on
the
basis
of
their
overall cell configuration. Type A cells exhibited apical microprojections and lateral cytoplasmic processes, whereas type B cells lacked these features. In the connecting segment, large intercalated cells with variable immunoreactivity for CA II were observed in addition to the typical type A and type B intercalated cells. However, it was not possible to identify with certainty a specific third cell type on the basis of immunoreactivity for CA II alone. Intercalated cells in the OMCD and IMCD1 (Figure 3b and c) were similar in appearance
to type
A intercalated
cells
in the
CCD,
and they exhibited strong immunostaining for CA II. By light microscopy of both flat embedded vibratome sections and one-micron plastic sections, there was very faint immunostamning of principal cells in the CCD, OMCD, and IMCDI. There was no CA II immunostamning of IMCD cells which are present in the
IMCD4 (Figure 3b) or proximal tions above,
3d)
processed but
or of thick
ascending
without
primary
antibody,
no immunoreactivity.
Figure 4. Transmission electron micrograph intercalated cells. Note that the intensity (asterisks) and is especially pronounced Bar = 3 zm.
250
limbs
convoluted tubules (Figure in the same manner as
(Figure
3a). Secdescribed
demonstrated
Microscopy
By electron microscope immunocytochemistry, CA II immunoreactivity was observed in the cytoplasm of all intercalated cells. However, the intracellular distribution of reaction product and the intensity of immunostaining varied along the collecting duct and between type A and type B intercalated cells in the CCD (Figure 4). The two types of intercalated cells were identified by using previously established criteria (10). Although all intercalated cells exhibited diffuse cytoplasmic staining throughout the cell, the intensity of immunostamning was considerably less in type B than in type A intercalated cells (Figures 4 through 6). Furthermore, in the majority of type A cells (Figure 5), immunostamning was more pronounced in the apical region and over apical microprojections although some cells also exhibited heavy immunostaining in the basal cytoplasm. In addition, staining was especially dense in close proximity to the apical tubulovesicles (Figure 5). Sections cut through the basal portion of the type A intercalated cells parallel to the basement membrane revealed numerous cytoplasmic processes or micropedici (Figure 7) giving rise to a spiderlike appearance of the star-shaped configuration observed by light microscopy. In the type B intercalated cells (Figure 6), the
of the CCD demonstrating CA II immunoreactivity in both type A and type B of CA II immunoreactivity in type A cells is much stronger than in type B cells in the apical region. The principal cells have only faint immunostaining for CA II.
Volume
I
.
Number
3’
1990
Figure 5. Transmission electron micrograph of type A intercalated cell from the CCD. CA II immunoreactivity throughout the cytoplasm and is especially pronounced in the apical cytoplasm and microprojections cytoplasm and in close proximity to apical tubulovesicles. Note the lateral cytoplasmic process and immunoreactivity in mitochondria. Bar = I Mm.
is distributed and the basal the absence of
Figure 6. Transmission electron micrograph of type B intercalated cell from the CCD. The intensity of CA II immunostaining Is more uniform throughout the cell, although staining oflen appeared to be concentrated around cytoplasmic vesicles. There is no immunoreactivity in mitochondria. Bar 1 m. =
Carbonic
Anhydrase
in Intercalated
Figure 7. Transmission numerous cytoplasmic
intensity
of
throughout peared
electron micrograph of CCD illustrating processes or micropedici. Bar 2 Mm.
be
cells
cell
was
for of
in type
CA
was
Weak immunostamning clei in all intercalated no
immunoreactivity
in
any
staining Faint
252
components of
of the
plasma
immunostaining
in the con-
type
A
similar
and
a basal
to that
and
the
view
of a type
A cell
exhibiting
im-
in the
This
study
cellular tions
was
of
Light with
ity
between
In
addition,
observed
in
in the
vealed
that
dif-
always
and
IMCD
cells
the
to investigate in
the
in
the
the duct
antibody
against
striking
of immunostamning distribution
different
as
difwell
of immunoreactiv-
types
of
intercalated
immunoperoxidase configuration
of
immunocyto-
polyclonal
intensity
sub-
subpopula-
collecting
microscope II demonstrated
CA
the the
electron
a rabbit
erythrocyte
as
II was
was
immunoreactivity, of
cells
and
intracellular
mouse
and observed.
the
and
of CA in the various
intercalated
rat.
sysnot
CAll
undertaken
distribution
in the
was
no
immunostamning
ferences
examined,
between duct
DISCUSSION
cells CA
exhibited no
or
the
varied
collecting
IMCD.
vacuolar-lysosomal
membrane for
cells was
chemistry
in
staining
of the
over cell nuHowever, there
mitochondria
intercalated
but
markedly less intense than that of the intercalated cells. In the CCD and OMCD, the principal cells showed a weak cytoplasmic CA II staining (Figures 4 and 9). However, the nuclei of the principal cells usually appeared negative for CA II. The connecting
the
was observed examined.
cells, segments
there
However, interthe lateral mi-
cells
ferent
tubule
homogeneous.
cells of
the
lacked
of more
B intersimilar
exhibited weaker imdid type A cells. The in intercalated cells in
regions
characteristic
type was
stain-
of intercalated 8). It contained
9) and IMCD1 was A intercalated cells.
in these
munostamning
various
and II than
immunostamning
cells
cropedici
tem
A and segment
in the CCD. However,
the OMCD (Figure
was
of type connecting
vesi-
weak
a third configuration observed (Figure
intensity
calated
rather
segment, occasionally
munostamning
B cell
principal ap-
cytoplasmic
exhibited
mitochondria
uniform often
observed
numerous
observed
more
staining
around
B cells
ing. The immunostamning calated cells in the
to that necting
was
although
concentrated
Occasional
a type
=
immunostamning
the
to
des.
Cells
cells.
technique of
Volume
type
re-
A intercalated
I ‘Number
3
1990
Kim etal
cells
was
and
intercalated
tions
markedly
provide
functional various
different
cells further
and
from
in the support
structural
that
of type
OMCD. for
These
the
existence
heterogeneity
subpopulations
B cells
observaof both
among
of intercalated
cells
that
the have
been
described in the rat (1,10.13,14). The antibody used in this study recognized a polypeptide with the molecular weight of CA II on a Western blot analysis of solubilized proteins from both
cortex and medulla blot analysis does CA
of the
rat
kidney.
not distinguish particularly CA
isoenzymes,
have
nearly
identical
PAGE strated
analysis. that the
molecular
However, antiserum
recognized murine CAll ity with purified murine
The
immuno-
between
certain
CA II, which weights in SDS-
I and
the
kidney.
indi-
in in-
methods, both isozymes are stained. However, previous demonstration of diffuse cytoplasmic staining
in intercalated
could plasmic
Figure 8. Transmission electron micrograph of a third type cell size and numerous mitochondria. CA II immunoreactivity and type B intercalated cells. Bar 2 Mm.
observations
demonstrated
different isozymes of CA exist in the kidneyCA II, which is believed to be cytoplasmic, and CA IV, which is membrane bound (9,2 1). By histochemical
CA 1(24).
is evidence from both biochemical (18) and immunocytochemical (6) studies that CA I is not present in
these
Two
that at presence
there
together,
the immunoreactivity cells in this study
represents CA II. Previous histochemical studies using various modifications of the Hansson technique have demonstrated high activity of CA in intercalated cells of the rat (2), mouse (3), rabbit (3,4), and human (29) kidney.
previous work has demonused in the present study but showed no cross-reactiv-
Furthermore,
Taken
cate that tercalated
cells
by electron
least part of the of a cytoplasmic
membrane not
staining
microscopy
staining CA (2).
occurred
in
was
studies
with
of intercalated cell in the connecting is intermediate in intensity between
due
Whether intercalated
be determined because of staining (2). Immunofluorescence
munocytochemical
indicated
specific
to
the
or not cells
heavy
cyto-
and imantibodies
segment. Note that observed
the large in type A
=
Journal
of the
American
Society
of Nephrology
253
Carbonic
Anhydrase
in Intercalated
Cells
Figure 9. Transmission electron micrograph of an intercalated cell from the outer stripe of the outer medulla. The subcellular distribution of CA II and the intensity of immunostaining are very similar to those observed in the type A cells. Bar 1 Mm. =
against CA II have demonstrated immunoreactivity in intercalated cells in the CCD. OMCD, and IMCDI the rat kidney (6,8), thus confirming the presence CA II in intercalated cells. By using a horseradish peroxidase ers
(5)
detection
procedure.
demonstrated
Brown
and
immunoreactivity
proton
CA
of
plasmic (14,15).
immunostaining Physiologic
orescent
dyes
II in
intercalated cells of the rat CCD by electron microscope immunocytochemistry. However, none of these previous histochemical and immunocytochemical studies have described heterogeneity in the intensity and intracellular distribution of CA between different types of intercalated cells, probably because most of these studies were published at a time when subpopulations of intercalated cells had not yet been described in the collecting duct of the rat. Morphologic (10) and immunocytochemical (13-15) studies have now provided evidence for the presence of two and possibly three types of intercalated cells in
the
CCD
of
the
rat.
similar
in ultrastructure
OMCD.
They
cal
membrane
bicarbonate (13,14), and
254
both
A intercalated
cells
to intercalated possess
(11,15)
exchanger, they
Type
are
and
proton band
in the believed
cells
ATPase 3 protein,
basolateral to
be
are
in the
in the
api-
ence
cells
of
an
membrane
CCD
for studies
have
provided
apical
and
OMCD.
basolateral
or
Type
cyto-
the proton with pH-sensitive evidence
Ct7HCO3
B in-
diffuse
for
exchanger
ATPase flupres-
the in
peanut
(16.30). The demonstration in this study that type A intercalated cells exhibit greater immunoreactivity than do type B cells suggests the possibility that, under normal conditions, the acid-secreting type A intercalated cells may be more active with respect to the generation of protons and bicarbonate than are type B-intercalated cells. These observations are consistent with the results of physiologic studies (31) which have the and
provided isolated secrete
Although for
the
exhibit
lectmn-positive cells (type B intercalated cells) in the rabbit CCD. leading to the suggestion that these cells are involved in bicarbonate secretion in the CCD
bicarbonate
a chloride!
responsible
in
tercalated
co-work-
for
secretion
of
immunostamning
evidence perfused protons
even
secretion
type
that rabbit
acid-secreting CCD are always under
in the band
of
net
tubule.
B intercalated for
conditions
cells in active
cells
3 protein
Volume
I
do not exhibit (13,14,32), it has
.
Number
3
1990
Kim et al
bn
uMtM
cells
might
A nd tyi
that ty
represent
different
k !ntei1td
configurations
tiiodud within th ytiti1ffl
of the
cytoplasmic
CA II to the
it -1lfikIn membrane surface
same cell type that would change its polarity depending on the physiologic state of the animal (16). However, the absence of band 3 protein in type B intercalated cells together with the demonstration in this study of different cell configurations and different patterns of immunostamning for CA II in type A and type B cells support the existence of two distinct types of intercalated cells in the CCD. The observations in the connecting segment are more difficult to inter-
fixation. However, the dense immunostamning ical microprojections and apical cytoplasm served only in type A intercalated cells, that this staining pattern reflects specific tion of CA II immunoreactivity. A previous electron microscope study
pret. In intercalated
B to
heavy
of
tercalated
addition cells
to the typical with staining
type A and type patterns similar
those observed In the CCD, a third configuration intercalated observed. intercalated
cell,
a resting
intercalated
cell,
or
remains
to be
our
laboratory
tivity
for
of
an
established.
3 protein in ultrastructure this non-A, to
CA II was
a new either
type
the
of
B
form,
studies
absence
of
A or
intermediate
Preliminary an
from
immunoreac-
in this new cell. However, it from type B intercalated non-B intercalated cell most
band
corresponds
for
represents form
possibly
indicate
is different cells. Thus, likely
staining
cell with weak Whether this cell
a third
variety
of
intercalated
cell described by Alper and co-workers (14) in the CCD and connecting segment, where it displayed apical H-ATPase but no detectable basolateral band 3 protein. The
use
study
of the
immunoperoxidase
revealed
in the
basal
in the
CCD.
distinct
portion Such
technique
lateral
of the
type
processes
not
in type
B cells. More surprisingly, however, lateral cell processes were rarely observed in intercalated cells in the OMCD, suggesting the possibility that these cells may be different from type A cells. The functional significance of these differences is not known. However, it is of interest that functional differences have been reported between acid-secreting cells in the CCD and the
OMCD
of
the
rabbit
(33).
cells in the CCD which docytosis. a population OMCD bility
cells
was
The ing
of the rabbit (33). However,
not
contrast
did the
not possess structural
to
type
A
of lummnal encells in the endocytic identity
localization
apical
immunostamn-
of CAll
tubulovesicles
and
in apical
micro-
and
intercalated located close
apical
tubulovesicles
cells. CA to vesicles
which
contain the proton pump accentuated immunostamning to
the
may
Journal
membrane have
of the
resulted
American
of
(11).
II immunoreactivity
these from
Society
have
In type was
been
diffusion
reported
of Nephralogy
organelles of
the
cells
cells In the CCD were not examined.
present study. weak immunoreactivity over nuclei of all intercalated cells. was no staining cating that the
of mitochondria antibody against
study bonic
cross-react
does not anhydrase.
The
the
weak
of staining agreement with results and immunocytochemical ney.
Taken
CA
is not
together, present
with
in ref.
mitochondrial
in
principal
carcells
and
in the IMCD cells are in of previous histochemical (2) (6) studies in the rat kid-
these in
In the
was observed However, there
in any cells, indiCA II used in this
immunostamning
absence
the
observations IMCD,
suggest
which
that
is interesting
of the collecting duct in urine acidification
34).
this study reveals striking differences in the intensity and subcellular distribution of CA II immunoreactivity and in cell configuration between type A and type B intercalated cells. These findings
reaction
provide
further
support
for the
two structurally and functionally intercalated cells in the CCD and of the rat kidney.
existence
of at least
distinct connecting
types of segment
ACKNOWLEDGMENTS The authors acknowledge Cannon. Frederick Kopp,
TWO4
B
also
to
(15). It is possible that the we observed adjacent intracellular
the
of CAll in intercalated cells in the OMCD stripe of the outer medulla demonstrated localization of gold particles throughout the including over nuclei and mitochondria (7). In-
Health
projections of proton-secreting intercalated cells suggests that CA Ills located in close proximity to the proton pump which is associated with the apical
membrane
using
intracellular
the
technical
and Wendy Malis. The
assistance
of
James
K.
L. Wilber. and the secretarial authors also thank Dr. W.
support of Deborah S. Fischlschweiger. Director of the Electron Microscope Facility of the College of Dentistry at the University of Florida where the electron microscope studies were performed. This work was supported in part by National Institutes of Health Grant AM-28330. J. Kim is the recipient of a National Institutes of
capathose
of
established.
preferential
around
In
were capable of acid-secreting
the
In summary,
cells
present
to examine
of apwas obsuggesting localiza-
distribution in the inner
(reviewed
processes
A intercalated
were
technique
considering that this segment is also believed to be involved
in this
cytoplasmic
immunogold
f th during
Fogarty
International
Research
Fellowship
Award.
F05
198.
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Volume
I
.
Number
3’
1990
