Ultrastructural Localization of Carbonic Anhydrase II in ...

2 downloads 1 Views 3MB Size Report
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.

REFERENCES 1.Tisher

2.

CC, Madsen

KM:

Anatomy

of the

kidney.

Brenner BM, Rector FC Jr. eds. The Kidney. Philadelphia: The W.B. Saunders Co; 1990:3-75, L#{246}nnerholm G, Ridderstrale Y: Intracellular bution of carbonic anhydrase in the rat kidney. Int

In:

4th

ed.

distriKidney

1980:17:162-174,

255

Carbonic

Anhydrase

DC, Magill

3. Dobyan Bulger rabbit

in Intercalated

LS.

Cells

Friedman

PA,

RE: Carbonic anhydrase and mouse kidneys. Anat

Hebert

SC,

histochemistry Rec 1982:204:185-

Ridderstrale

Y, Kashgarian

Morphological duct. Kidney

5.

Int

M, Koeppen

heterogeneity 1988:34:655-670.

of

the

B, et at.:

rabbit

D, Roth J, Kumpulainen T, Orci L: Ultrastructural immunocytochemical localization of carbonic anhydrase. Presence in intercalated cells of the rat collecting tubule. Histochemistry 1982:75:209Brown

Kumpulainen

D,

munohistochemical in postnatal

and

1983;245:F1

7.

10-Fl

Brown localization

D,

sections 1985:83:153-158.

8.

Am

12.

J.

L: Imanhydrase J Physiol

with

Immunocytochemical on ultrathin

T: anhydrase

protein

A-gold.

anhydrase-rich

frozen

DC.

J Physiol

Verlander

G, Siegel rat

kidney 25.

1987:57:150-156.

Renal carbonic 1-F324.

1982:243:F31

anhydrase.

JW,

Madsen KM. Tisher CC: Effect of acute respiratory acidosis on two populations of intercalated cells in rat cortical collecting duct. Am J Physiol 1987;253:Fl 142-Fl 156. Brown D. Gluck S. Hartwig J: Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification as an H-ATPase. J Cell Biol 1987:105:1637-1648. Drenckhahn

D, Schl#{252}ter K, Allen

Localization

of

band

3 with

DP,

ankyrin

the basal membrane of intercalated kidney. Science 1985:230:1287-1289.

3 protein

in

the

rat

collecting

spectrin in the

cells

13. Verlander JW, Madsen KM, Low Tischer CC: Immunocytochemical band

Bennett

and

duct.

26.

Allen DP, localization of

15.

SL,

Brown opposite hal cell

D,

Natale

Hirsch

5,

Gluck

5:

An

W-ATPase

plasma membrane domains in kidney epithesubpopulations. Nature (Lond) 1988:331:622-

Schwartz of

17. 18.

256

functional

GJ,

Barasch epithelial

J, Al-Awqati polarity.

Q: Nature

Plasticity (Lond)

1985:318:368-371. Maren TH, Ellison AC: A study of renal carbonic anhydrase. Mol Pharmacol 1967:3:503-508. Wistrand PJ, Lmndahl 5, Walhstrand T: Human renal carbonic anhydrase. Purification and properties. EurJ Biochem 1975:57:189-195.

Renal

membrane-

Linser

PJ, Sorrentino

The

early peroxidase

Ultrastructural J Histochem

M, Moscona of

stages in

carbonic

AA:

of the

cy-

CytoCellular

anhydrase-C

and

glutamine synthetase in developing and mature mouse neural retina. Dev Brain Res 1984;13:65-71. Linser PJ, Perkins MS. Fitch FW, Moscona AA: Comparative characterization of monoclonal antibodies to carbonic anhydrase. Biochem Biophys Res Cornmun 1984:125:690-697. Laemmli UK: Cleavage of structural proteins during of the head 1970:227:680-685.

transfer

29.

30.

31.

in

32.

33.

34.

PF, Minier

of

bacteriophage

T4.

of proteins

from

LN, Lasher

RS:

Nature

28.

624.

16.

K-G:

electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: A method for their re-use in immunoatuoradiographic detection of antigens. J Immunol Methods 1982:51:241-249. Towbin H, Staehelmn T. Gordon J: Electrophoretic

J Physiol

15-F 125. J, Gluck 5, Lodish HF, Brown D: Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H-ATPase. Proc Natl Acad Sd USA 1989:86:5429-5433.

Alper

activity

Erickson

V: at rat

anhydrase

27.

1988:255:F1

14.

Knuuttila

Graham RC, Karnovsky MJ: absorption of injected horseradish proximal tubules of mouse kidney: tochemistry by a new technique. chem 1974:22:1077-1083.

assembly (Lond)

PS,

Am

PJ,

R: Carbonic

border and basal-lateral membranes cells. Pflugers Arch 1977:370:121-

compartmentalization

characteriza-

in the

Kinne

erythproper-

1083.

23.

Histochemistry

cells

Invest Bulger RE:

PJ,

and and

22.

24.

Lab

Wistrand

renal

bound carbonic anhydrase. Purification and properties. Kidney Int 1989:35:851-859. McLean 1W, Nakane PF: Periodate-lysine paraformaldehyde fixative: A new fixative for immunoelectron microscopy. J Histochem Cytochem 1974:22:1077-

Orci

of carbonic kidney. Am

Holth#{246}fer H, Schulte BA, Pasternack GJ, Spicer SS: Immunocytochemical

9. Dobyan

11.

Roth

T: Rat

Purification 1977:481:712-721.

Wistrand

18.

Kumpulamnen of carbonic

tion of carbonic collecting duct.

10.

T.

localization adult rat

Wahlstrand

21.

213.

6.

PJ,

of isolated brush of renal tubular 126.

collecting

Brown

Wistrand

rocyte carbonic anhydrases. ties. Biochim Biophys Acta

20.

197.

4.

19.

in

polyacrylamide

Quantitative

gels to nitro-

cellulose sheets: Procedure and some applications. Proc Natl Acad Sd USA 1979:76:4350-4354. L#{246}nnerholm G: Histochemical demonstration of carbonic anhydrase activity in the human kidney. Acta Physiol Scand 1973:88:455-468. Weiner ID, Hamm LL: Regulation of intracellular pH in the rabbit cortical collecting tubule. J Clin Invest 1990:85:274-281. Knepper MA, Good DW. Burg MB: Mechanism of ammonia secretion by cortical collecting duct of rabbits. Am J Physiol 1984;247:F729-F738. Schuster VL, Bonsib SM, Jennings ML: Two types of collecting duct mitochondria-rich (intercalated) cells: Lectin and band 3 cytochemistry. Am J Physiol 1986:251 :C347-C355. Schwartz GJ, Satlin LM. Bergmann JE: Fluorescent characterization of collecting duct cells: A second H-secreting type. Am J Physiol l988:255:F1003Fl 014. Madsen KM, Clapp WL, Verlander JW: Structure and function of the inner medullary collecting duct. Kidney Int 1988:34:441-454.

Volume

I

.

Number

3’

1990

Suggest Documents