Original Paper Acta Anat 1992;144:213-224
P. Okot-Opiro11 W. Baier’ M.R. Celio ' - c
Monoclonal Antibodies against Rat Kidney Antigens
Institute of Anatomy. University of Kiel, FRG: Institute of Anatomy, University of Zürich. Switzerland: Institute of Histology. University of Fribourg. Pcrolles, Fribourg. Switzerland
Key Words
Abstract
Immunohistochemistry Hybridomas Brush border Proximal tubule Basolateral membrane
The proximal tubule of the nephron is subdivided into three structurally and functionally distinct segments, which can be differentiated with the help of spe cial methods. With the aim of producing selective markers for these three por tions of the proximal tubule, we raised monoclonal antibodies against the brush border membranes of the rat kidney. Immunohistochemistry was carried out with eleven different monoclonal antibodies to sections of rat kidney and other tissues at the light- and electron-microscopical level. These monoclonal antibod ies mainly detect antigens located on the brush border of the proximal tubule, and they allow a distinction between its three segments. However, some anti bodies also recognize other portions of the nephron, or even the glomerulus or stromal elements. Sites recognized by the antibodies are not limited to the kid ney, but staining is observed on the intestinal brush border, the intralobular ducts of the pancreas, the bile canaliculi of the liver and on the macrophage clustersof the spleen. These antibodies are interesting reagents which can be applied to study biochemical differences between brush border membranes. In addition, they recognize antigenically related sites in other organs with reabsorptive or secretory tasks.
glucose transport, albumin uptake and phosphate transport
Introduction
12]-
Received: February 20. 1992 Accepted: March 2. 1992
All these transport processes are driven by specialized membrane proteins of, as yet, largely unknown structures, located in the brush border of the apical membrane of the epithelial cells. To understand the role of these proteins, it would be necessary either to purify them or to block their activity in vitro. Both experiments can be performed using highly spe cific monoclonal antibodies.
Marco R. Celio. MD Institute of Histology University of Fribourg. Pirolles CH-1700 Fribourg (Switzerland)
© 1992 Karger AG. Basel 0001-5180/92/1443-0213 $ 2.75/0
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The proximal tubule has been very intensively investi gated in various species [1], and its ultrastructural appear ance is found to differ over its length [2. 3). Three different segments of the proximal tubule have been distinguished according to their ultrastructural characteristics [2]. This structural heterogeneity is correlated with the gradual func tional changes along the proximal tubule, which are not only associated with a proximodistal decrease in the rcabsorptivc capacity for sodium, but also with alterations in
Table 1. Monoclonal antibodies against rat kidney antigens
Antibodies
IgG class
Renal corpuscle and Bowman’s capsule
Proximal convoluted tubule
Proximal straight tubule
Distal straight tubule
Macula densa
142
IgGl
+ parietal layer
S2: + BB juxtamcdullary
S3: + + + BB
-
144
IgG 1
S: + BB vacuolar apparatus
146
IgGl
—
148
IgGl
-
150
IgGl
—
S2: + + (+ ) BB BLM: + S3: + + + BB. BLM OS: + + S2: + + + BB S3: + + + BB S2: + + + BB S3: + + + BB S2: + BB
MTAL: LuM: + CTAL: LuM: + P. mac. S: LuM: + CTAL: LuM: + P. mac. S: LuM: + BLM: +
151
IgGl
pedicels: + BM: +
153
lgG2b
—
155
IgGl
157
IgGl
159
IgGl
161
IgGl
—
-
SI: S2: SI: S2: SI: S2: SI: S2:
+ + + BB + + + BB + + + BB + + + BB + BB + BB + + + BB + + + BB
SI: + + + BB S2: + + + BB vacuolar apparatus: + (seldom) SI: + + + BB S2: + + BB SI: + + + BB S2: + + BB
S2: + + BB S3: + + BB OS: + + BB S2: + + + BB S3: + + + BB S2 (vacuolar apparatus) S2: + + + BB S3: + + + BB S2: + + + BB S3: + + BB
S2: + + BB S3: + + + BB
S2: + + BB S3: + + BB
CTAL: LuM: + P. mac. S: LuM: + MTAL: LuM: + BLM: + —
~
single LuM: +
—
—
“ P. mac. S: LuM: + CTAL: + BLM: + LuM: + P. mac. S: + LuM: +
“
.
LuM: + BLM: + (few cells)
BB = Brush border; BLM = basolateral membrane; BM = basement membrane; CNTC=cortical connecting tubule cells; CTAL = cortical thick ascending limb: Co = cortex: IC = interstitial cells; IL=intralobular ducts; IS = inner stripe; LuM = luminal membrane; MC = macrophage clusters; Med. ray = medullary ray: MTAL= medullary thick ascending limb; OS = outer stripe; P. mac. S = postmacula S segment; SI. S2, S3 = segments of the proximal tubule: -F=weak staining; + + =strong staining; + + + = very strong staining.
Materials and Methods Brush borders were isolated and purified according to standard techniques. Dissolved in complete Freund's adjuvant, they were injected subcutaneously into Balb/c mice. After 8 months of monthly immunization to boost production, the animals were sacrificed, and the spleens used for cell fusion with AG 8-653 myeloma cells. The pro
214
Okot-Opiro/Baier/Cclio
tocol for the production of monoclonal antibodies follows that already published by others [4J. Culture supernatants were screened on cryo stat sections of the rat kidney and fixed with 4% paraformaldehyde in 0 .1 M phosphate buffer (pH 7.3). To obtain a consistent supply of monoclonal antibodies, the desired and stable hybrids were subcloned twice by limiting dilution. A solid-phase antibody assay was employed to determine the lgG subclasses. Immunohistochcmistry and immunoelectron microscopy to ascer tain the distribution of these eleven antigens in the kidney and other tissues of the rat were performed using ascitic fluid. For this purpose, healthy, adult Wistar rats of both sexes, weighing 160-250 g, were anesthetized with 4% chloralhydrate in 0.1 M phosphate buffer. pH 7.3 (I ml/100 g). After having opened the thoracic cavity, an incision was made in the left ventricle, and perfusion started via a catheter, which was in serted in the left part of the heart. Blood was washed out with 0.1 M phosphate buffer (pH 7.3). and the tissues were then fixed with 4% paraformaldehyde in 0 .1 M phosphate buffer (pH 7.3). This was done by maintaining the liquid flow (25 ml/min) and the pressure low and
New Kidney Antigens
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In this paper, we describe the exact distribution of the staining pattern of eleven monoclonal antibodies directed against brush border antigens. These occur mainly on the glycocalyx of the brush border of the proximal tubule and can be used to differentiate its three portions. These anti bodies are, however, never absolutely specific for the brush border; they recognize additional sites in the nephron and other organs.
Table 2. Monoclonal antibodies against rat kidney antigens
Antibodies
Distal convoluted tubule
Connecting tubule
Cortical collecting duct
Outer medullary collecting duct
Inner medullary collecting duct
142
BLM: + (seldom) BLM: + (seldom)
IC: LuM: +
IC: LuM: +
IC: LuM: + +
-
—
—
IC: LuM: +
—
IC: LuM: + CNTC: LuM: + + IC: LuM: + + BLM: +
IC: LuM: +
IC: LuM: +
—
IC: LuM: +
IC: LuM: +
—
—
IC: LuM: + (seldom) IC: LuM: + IC: LuM: + +
—
~
IC: LuM: + IC: LuM: + +
-
-
-
IC: LuM: + (seldom) IC: LuM: + IC: LuM: + CD: LuM: + + BLM: + IC: LuM: +
—
IC: LuM: + + CNTC: LuM: + BLM: +
IC: LuM: + (seldom) IC: LuM: + CD: LuM : + + BLM: +
144 146 148
—
150
—
151 153
-
155
~
157 159
-
161
-
IC: LuM: + + IC: LuM: + + CNTC: LuM: + BLM: + IC: LuM: + (seldom) -
IC: LuM: + +
IC: LuM: + IC: LuM: + CD; LuM; +-f BLM; + IC: LuM: +
For abbreviations, see table 1.
horse anti-mouse IgG (H + L) for I h at room temperature. Finally, they were incubated with ABC reagent for 1.5 h [6]. The peroxidase reaction was developed with 1.3.3.-diaminobenzidine HCI in 0.05 M Tris-buffered saline (pH 7.2) and 30% hydrogen peroxide solution (10 ml:200pl:l.S pl/ml) under visual control. Control slides were similarly treated, except that they did not receive the primary antibodies. In one experiment, the sections were incubated with sialidasc (a neuraminidase enzyme of A rihrobacter ttreafaciens |7|) dissolved in 0.2 M acetate buffer. pH 5. for 30 min. before the primary antibodies were applied to them. Sections were dehydrated in a graded series of ethanol and finally coverslipped with DcPcX (Merck). For electron-microscopic studies, 50-pm-thick sections of fixed kidney were cut on a Lancer vibratome and treated following the immunoperoxidase technique. Subsequently, they were postfixed with 2.5% glutaraldehyde for I h. washed with 0.1 M phosphate buffer (pH 7.3) for 30 min and further postfixed with 1% OsOj for 2 h. After dehydration in a graded series of ethanol. for 15 min in each dilution, they were immersed in 1,2-propylene oxide (for 30 min) and embed ded in Aralditc overnight. Thin sections were cut with a diamond knife. Unstained and stained sections were viewed and photographed in a Zeiss electron microscope. Lastly, a passive immunization test was performed to establish a pathogenetic role of some of these antibodies |8. 9], Three pairs of male Wistar rats (180-200 g) were each injected intraperitoneally with antibodies 151. 157 and 144. respectively. Two and 14 days after the application of these antibodies, a rat from each pair was sacrificed.
215
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constant for 20 min. The tissues of interest (kidney, intestine, pan creas. liver and spleen) were removed, cut into slices and postfixed with the same fixative for 1 h. The tissues were then transferred and kept in 18% sucrose until use. All the steps described above were car ried out at room temperature and applied to all other animals (rabbits, hamsters, guinea pigs. Balb/c mice and monkeys). Human and pig kid ney were treated following exactly the same procedure except that they were perfused and fixed through the renal artery'. Kidney tissues, derived from 18-day-old rat fetuses, were removed by sacrificing them in utero by an overdose of 4% chloralhydrate to the mother. Others were obtained from 1-, 7-, II- and 16-day-old rats. Since the kidneys at these stages are small, they were not perfused, but were immersed in a fixative of 4% paraformaldehyde in 0.1 M phos phate buffer (pH 7.3). After a couple of hours, they were then trans ferred and kept in 18% sucrose until sectioning. Fixed tissues, frozen in liquid nitrogen, were then sectioned at -18 °C with a Reichert-Jungcryostate and mounted on glass slides, coated with chrome-alum gelatine. Serial sections (10 pm thick) of kidney, pancreas, intestine, liver and spleen of normal rats, as well as kidneys of the other animals men tioned earlier, were used for indirect immunoperoxidase staining (5). In a series of experiments, l-um-thin sections were cut with a Reichert cryoultramicrotome. After 15 min of washing, they were incubated with the eleven different clones for 3 days at 4 °C. The ascites from each antibody were initially diluted with 10% fetal calf serum in Tris-buffered saline. After having tested various dilutions of the primary antibodies, a working dilution of 1:500 was chosen. Subsequently, the sections were incubated with biotinylated
Table 3. Monoclonal antibodies against rat kidney antigens
Anti bodies
Interstitium fibroblast
142 144 146 148 150 151 153 155 157 159 161
_
Results
Our investigations describe the distribution of antigens in the proximal tubule of the rat kidney, demonstrated by eleven monoclonal antibodies. The immunohistochemical reaction shows that the expression of these antigenic deter minants is not only restricted to the brush border mem brane of the rat kidney, but also occurs in other tissues and organs of the rat (tables 1-3). The eleven monoclonal anti bodies which recognize these determinants are species spe cific and do not cross-react with rabbit, pig, guinea pig, hamster, monkey and human kidney. They all belong to the IgG subclass with the exception of antibody 153 which is of the IgG2b subclass (tables 1-3). Pretreatment of kid ney sections by sialidase did not produce observable changes in the staining patterns of the eleven different clones. Immunostaining was not possible on sections embedded in Paraplast® with the exception of antibody 153 (IgG2b subclass). The topographical distribution of the antigenic determinants recognized by the sum of the anti bodies extends to all three segments (SI. S2. S3) of the proximal tubule. Since some antibodies show similarities in staining, they were arranged in groups. Antibody 150 stains the SI and S2 segments in the cortical labyrinth and the upper parts of the medullary rays (fig. 1), antibody 155 stains the S2 segment in the labyrinth and the upper parts of the medullary rays (fig.2). and antibodies 144 (fig.3) and 142 (fig.4) as well as 161 stain the S2 and S3 segments in the labyrinth, in the medullary rays and in the outer stripe. Six antibodies (146, 148,151,153, 157and 159) stain all three segments (SI, S2, S3) in the cortical labyrinth, in the upper parts of the med ullary rays and in the outer stripe (e.g. 153; fig.5). The boundaries of these segments are overlapping. Subcellularly. a number of different staining patterns (tables 1-3) could be observed with the eleven antibodies, all of which stain, preferentially and predominantly, the brush border of the proximal tubules (fig.6-10. 12, 15. 16). Nevertheless, moderate-to-strong staining was observed in
216
-
Med. ray: + Co. OS: +
Intestine
Liver
+ + BB
_
-
-
+ + BB + + BB
+ + BC + + BC
-
-
+ + BB + + BB
-
-
+ + BC + + BC + + BC
-
+ + BB
-
-
IS: +
-
-
+ + BB
Spleen
_
_
-
+ 1L
-
-
Pancreas
-
-
MC -
-
-
-
-
+ IL -
-
1L + IL
-
Med. ray = Medullary ray; BB = brush border; BC = biIe canaliculi; IL=intralobular ducts: MC=macrophage clusters; Co, OS = cortex, outer stripe; IS = inner stripe.
Okot-Opiro/Baier/Celio
the distal tubules (fig.8, 10, 11, 13) and in the basolatcral membranes (fig. 15, 16) as well as in intracytoplasmic gran ules (fig. 14). The glomerulus (fig.6). the parietal layer of Bowman’s capsule (fig. 12), the thin Henlc’s loops (fig. 18) and the collecting ducts (fig. 17) show a moderate reaction. These antigens were detectable as early as the 18th ges tational day. Immunoreaction covers the apical border and the basolateral membranes of the metanephrogenic blas tema (fig. 19). Kidney sections from 1-, 7-. 11- and 16-dayold rats show similarly distributed immunoreactive sites. Examination of the distribution of the antigenic determi nants in different rat organs revealed the following patterns (table 1): (1) positive immunohistochemical reaction along the intralobular duct of the exocrine pancreas with anti-
Fig. 1-5. General distribution of antigens on longitudinal cryostat sections (10 gnr thick) of the whole rat kidney fixed with 4% paraformaldehyde. These images were made with a photographic enlarger by projecting sections directly on to photographic paper (‘histography'). C = Cortex; M = medulla; P = Pelvis: SI. S2. S3 = seg ments of the proximal tubule. x 8 . 1 A faint lebelingof the SI and the S2 segments with no distinctive demarcations in the cortex (C). Anti body 150. 2 This kidney section shows a faint staining which is restricted to the S2 segment only. Antibody 155. 3 The intensive label ing of the S2 and the S3 portions is apparent. These portions begin in the cortex and end sharply at the boundary between the cortex and the medulla. Antibody 144. 4 Kidney section showing the distribution of antigens in the S2 and the S3 segments similar to that in figure 3. but with less intense labeling. Antibody 142. 5 The distribution of the anti gen in this kidney section extends to all the three portions (S I. S2. S3) of the proximal tubule. Antibody 148.
New Kidney Antigens
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perfused and fixed. The tissue sections were treated immunohistochemically. omitting the primary antibody. To ascertain whether there was an increase in the thickness of the glomerular basal lamina or a modification of the expression of the renal brush border, kidney sections from all the three pairs of rats were processed for standard electron microscopy, omitting the peroxidase reaction. To detect similar antigens in the urine, a ‘spot test’ was performed [ 10] on nitrocellulose paper with the urine of an adult rat deprived of water for 2 days.
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217
218
Okot-Opiro/Baier/Cclio
New Kidney Antigens
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(For legends see p. 221.)
219
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(For legends see p. 221.)
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'
BLM
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v ♦
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i
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y
V
V
20
BG
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22 ; v
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: vv IVlG
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'M:-
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■
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Sft
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New Kidney Antigens
'V,.. - \% . *V>.‘ '
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\
Fig. 6. Light micrograph of a renal corpuscle, showing the affer ent arteriole (AA) at the vascular pole and the immunolabelcd glo merulus (G). Bowman's space is confluent with the proximal tubule (PT) at the urinary pole. The luminal membrane of the S2 and the brush border (BB) of the SI segments of the proximal tubule are stained. Antibody 151. X375. Fig. 7. The same antibody as in figure 6 stains the brush border (PTBB) of the longitudinally cut proximal tubule (PT). 1-pm-thick ultracryostat sections, x 600. Fig. 8. Positive reactions are seen on the brush border of the proximal tubule (PTBB) and on the luminal membrane of the outer colecting tubules (OCT). The lumen of the collecting tubules are so narrow that their luminal membranes collapse together and form sin gle lines. Kidney section (I pm thick). Antibody 151. x600. Fig. 9. Micrograph from a 1-pm-thick kidney section of the rat shows immunoreaclivc sites in the brush border of the proximal tubule (SI and S2). UP = Urinary pole. Antibody 153. x60
P. Okot-Opiro11 W. Baier’ M.R. Celio ' - c
Monoclonal Antibodies against Rat Kidney Antigens
Institute of Anatomy. University of Kiel, FRG: Institute of Anatomy, University of Zürich. Switzerland: Institute of Histology. University of Fribourg. Pcrolles, Fribourg. Switzerland
Key Words
Abstract
Immunohistochemistry Hybridomas Brush border Proximal tubule Basolateral membrane
The proximal tubule of the nephron is subdivided into three structurally and functionally distinct segments, which can be differentiated with the help of spe cial methods. With the aim of producing selective markers for these three por tions of the proximal tubule, we raised monoclonal antibodies against the brush border membranes of the rat kidney. Immunohistochemistry was carried out with eleven different monoclonal antibodies to sections of rat kidney and other tissues at the light- and electron-microscopical level. These monoclonal antibod ies mainly detect antigens located on the brush border of the proximal tubule, and they allow a distinction between its three segments. However, some anti bodies also recognize other portions of the nephron, or even the glomerulus or stromal elements. Sites recognized by the antibodies are not limited to the kid ney, but staining is observed on the intestinal brush border, the intralobular ducts of the pancreas, the bile canaliculi of the liver and on the macrophage clustersof the spleen. These antibodies are interesting reagents which can be applied to study biochemical differences between brush border membranes. In addition, they recognize antigenically related sites in other organs with reabsorptive or secretory tasks.
glucose transport, albumin uptake and phosphate transport
Introduction
12]-
Received: February 20. 1992 Accepted: March 2. 1992
All these transport processes are driven by specialized membrane proteins of, as yet, largely unknown structures, located in the brush border of the apical membrane of the epithelial cells. To understand the role of these proteins, it would be necessary either to purify them or to block their activity in vitro. Both experiments can be performed using highly spe cific monoclonal antibodies.
Marco R. Celio. MD Institute of Histology University of Fribourg. Pirolles CH-1700 Fribourg (Switzerland)
© 1992 Karger AG. Basel 0001-5180/92/1443-0213 $ 2.75/0
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/12/2020 7:43:00 AM
The proximal tubule has been very intensively investi gated in various species [1], and its ultrastructural appear ance is found to differ over its length [2. 3). Three different segments of the proximal tubule have been distinguished according to their ultrastructural characteristics [2]. This structural heterogeneity is correlated with the gradual func tional changes along the proximal tubule, which are not only associated with a proximodistal decrease in the rcabsorptivc capacity for sodium, but also with alterations in
Table 1. Monoclonal antibodies against rat kidney antigens
Antibodies
IgG class
Renal corpuscle and Bowman’s capsule
Proximal convoluted tubule
Proximal straight tubule
Distal straight tubule
Macula densa
142
IgGl
+ parietal layer
S2: + BB juxtamcdullary
S3: + + + BB
-
144
IgG 1
S: + BB vacuolar apparatus
146
IgGl
—
148
IgGl
-
150
IgGl
—
S2: + + (+ ) BB BLM: + S3: + + + BB. BLM OS: + + S2: + + + BB S3: + + + BB S2: + + + BB S3: + + + BB S2: + BB
MTAL: LuM: + CTAL: LuM: + P. mac. S: LuM: + CTAL: LuM: + P. mac. S: LuM: + BLM: +
151
IgGl
pedicels: + BM: +
153
lgG2b
—
155
IgGl
157
IgGl
159
IgGl
161
IgGl
—
-
SI: S2: SI: S2: SI: S2: SI: S2:
+ + + BB + + + BB + + + BB + + + BB + BB + BB + + + BB + + + BB
SI: + + + BB S2: + + + BB vacuolar apparatus: + (seldom) SI: + + + BB S2: + + BB SI: + + + BB S2: + + BB
S2: + + BB S3: + + BB OS: + + BB S2: + + + BB S3: + + + BB S2 (vacuolar apparatus) S2: + + + BB S3: + + + BB S2: + + + BB S3: + + BB
S2: + + BB S3: + + + BB
S2: + + BB S3: + + BB
CTAL: LuM: + P. mac. S: LuM: + MTAL: LuM: + BLM: + —
~
single LuM: +
—
—
“ P. mac. S: LuM: + CTAL: + BLM: + LuM: + P. mac. S: + LuM: +
“
.
LuM: + BLM: + (few cells)
BB = Brush border; BLM = basolateral membrane; BM = basement membrane; CNTC=cortical connecting tubule cells; CTAL = cortical thick ascending limb: Co = cortex: IC = interstitial cells; IL=intralobular ducts; IS = inner stripe; LuM = luminal membrane; MC = macrophage clusters; Med. ray = medullary ray: MTAL= medullary thick ascending limb; OS = outer stripe; P. mac. S = postmacula S segment; SI. S2, S3 = segments of the proximal tubule: -F=weak staining; + + =strong staining; + + + = very strong staining.
Materials and Methods Brush borders were isolated and purified according to standard techniques. Dissolved in complete Freund's adjuvant, they were injected subcutaneously into Balb/c mice. After 8 months of monthly immunization to boost production, the animals were sacrificed, and the spleens used for cell fusion with AG 8-653 myeloma cells. The pro
214
Okot-Opiro/Baier/Cclio
tocol for the production of monoclonal antibodies follows that already published by others [4J. Culture supernatants were screened on cryo stat sections of the rat kidney and fixed with 4% paraformaldehyde in 0 .1 M phosphate buffer (pH 7.3). To obtain a consistent supply of monoclonal antibodies, the desired and stable hybrids were subcloned twice by limiting dilution. A solid-phase antibody assay was employed to determine the lgG subclasses. Immunohistochcmistry and immunoelectron microscopy to ascer tain the distribution of these eleven antigens in the kidney and other tissues of the rat were performed using ascitic fluid. For this purpose, healthy, adult Wistar rats of both sexes, weighing 160-250 g, were anesthetized with 4% chloralhydrate in 0.1 M phosphate buffer. pH 7.3 (I ml/100 g). After having opened the thoracic cavity, an incision was made in the left ventricle, and perfusion started via a catheter, which was in serted in the left part of the heart. Blood was washed out with 0.1 M phosphate buffer (pH 7.3). and the tissues were then fixed with 4% paraformaldehyde in 0 .1 M phosphate buffer (pH 7.3). This was done by maintaining the liquid flow (25 ml/min) and the pressure low and
New Kidney Antigens
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In this paper, we describe the exact distribution of the staining pattern of eleven monoclonal antibodies directed against brush border antigens. These occur mainly on the glycocalyx of the brush border of the proximal tubule and can be used to differentiate its three portions. These anti bodies are, however, never absolutely specific for the brush border; they recognize additional sites in the nephron and other organs.
Table 2. Monoclonal antibodies against rat kidney antigens
Antibodies
Distal convoluted tubule
Connecting tubule
Cortical collecting duct
Outer medullary collecting duct
Inner medullary collecting duct
142
BLM: + (seldom) BLM: + (seldom)
IC: LuM: +
IC: LuM: +
IC: LuM: + +
-
—
—
IC: LuM: +
—
IC: LuM: + CNTC: LuM: + + IC: LuM: + + BLM: +
IC: LuM: +
IC: LuM: +
—
IC: LuM: +
IC: LuM: +
—
—
IC: LuM: + (seldom) IC: LuM: + IC: LuM: + +
—
~
IC: LuM: + IC: LuM: + +
-
-
-
IC: LuM: + (seldom) IC: LuM: + IC: LuM: + CD: LuM: + + BLM: + IC: LuM: +
—
IC: LuM: + + CNTC: LuM: + BLM: +
IC: LuM: + (seldom) IC: LuM: + CD: LuM : + + BLM: +
144 146 148
—
150
—
151 153
-
155
~
157 159
-
161
-
IC: LuM: + + IC: LuM: + + CNTC: LuM: + BLM: + IC: LuM: + (seldom) -
IC: LuM: + +
IC: LuM: + IC: LuM: + CD; LuM; +-f BLM; + IC: LuM: +
For abbreviations, see table 1.
horse anti-mouse IgG (H + L) for I h at room temperature. Finally, they were incubated with ABC reagent for 1.5 h [6]. The peroxidase reaction was developed with 1.3.3.-diaminobenzidine HCI in 0.05 M Tris-buffered saline (pH 7.2) and 30% hydrogen peroxide solution (10 ml:200pl:l.S pl/ml) under visual control. Control slides were similarly treated, except that they did not receive the primary antibodies. In one experiment, the sections were incubated with sialidasc (a neuraminidase enzyme of A rihrobacter ttreafaciens |7|) dissolved in 0.2 M acetate buffer. pH 5. for 30 min. before the primary antibodies were applied to them. Sections were dehydrated in a graded series of ethanol and finally coverslipped with DcPcX (Merck). For electron-microscopic studies, 50-pm-thick sections of fixed kidney were cut on a Lancer vibratome and treated following the immunoperoxidase technique. Subsequently, they were postfixed with 2.5% glutaraldehyde for I h. washed with 0.1 M phosphate buffer (pH 7.3) for 30 min and further postfixed with 1% OsOj for 2 h. After dehydration in a graded series of ethanol. for 15 min in each dilution, they were immersed in 1,2-propylene oxide (for 30 min) and embed ded in Aralditc overnight. Thin sections were cut with a diamond knife. Unstained and stained sections were viewed and photographed in a Zeiss electron microscope. Lastly, a passive immunization test was performed to establish a pathogenetic role of some of these antibodies |8. 9], Three pairs of male Wistar rats (180-200 g) were each injected intraperitoneally with antibodies 151. 157 and 144. respectively. Two and 14 days after the application of these antibodies, a rat from each pair was sacrificed.
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constant for 20 min. The tissues of interest (kidney, intestine, pan creas. liver and spleen) were removed, cut into slices and postfixed with the same fixative for 1 h. The tissues were then transferred and kept in 18% sucrose until use. All the steps described above were car ried out at room temperature and applied to all other animals (rabbits, hamsters, guinea pigs. Balb/c mice and monkeys). Human and pig kid ney were treated following exactly the same procedure except that they were perfused and fixed through the renal artery'. Kidney tissues, derived from 18-day-old rat fetuses, were removed by sacrificing them in utero by an overdose of 4% chloralhydrate to the mother. Others were obtained from 1-, 7-, II- and 16-day-old rats. Since the kidneys at these stages are small, they were not perfused, but were immersed in a fixative of 4% paraformaldehyde in 0.1 M phos phate buffer (pH 7.3). After a couple of hours, they were then trans ferred and kept in 18% sucrose until sectioning. Fixed tissues, frozen in liquid nitrogen, were then sectioned at -18 °C with a Reichert-Jungcryostate and mounted on glass slides, coated with chrome-alum gelatine. Serial sections (10 pm thick) of kidney, pancreas, intestine, liver and spleen of normal rats, as well as kidneys of the other animals men tioned earlier, were used for indirect immunoperoxidase staining (5). In a series of experiments, l-um-thin sections were cut with a Reichert cryoultramicrotome. After 15 min of washing, they were incubated with the eleven different clones for 3 days at 4 °C. The ascites from each antibody were initially diluted with 10% fetal calf serum in Tris-buffered saline. After having tested various dilutions of the primary antibodies, a working dilution of 1:500 was chosen. Subsequently, the sections were incubated with biotinylated
Table 3. Monoclonal antibodies against rat kidney antigens
Anti bodies
Interstitium fibroblast
142 144 146 148 150 151 153 155 157 159 161
_
Results
Our investigations describe the distribution of antigens in the proximal tubule of the rat kidney, demonstrated by eleven monoclonal antibodies. The immunohistochemical reaction shows that the expression of these antigenic deter minants is not only restricted to the brush border mem brane of the rat kidney, but also occurs in other tissues and organs of the rat (tables 1-3). The eleven monoclonal anti bodies which recognize these determinants are species spe cific and do not cross-react with rabbit, pig, guinea pig, hamster, monkey and human kidney. They all belong to the IgG subclass with the exception of antibody 153 which is of the IgG2b subclass (tables 1-3). Pretreatment of kid ney sections by sialidase did not produce observable changes in the staining patterns of the eleven different clones. Immunostaining was not possible on sections embedded in Paraplast® with the exception of antibody 153 (IgG2b subclass). The topographical distribution of the antigenic determinants recognized by the sum of the anti bodies extends to all three segments (SI. S2. S3) of the proximal tubule. Since some antibodies show similarities in staining, they were arranged in groups. Antibody 150 stains the SI and S2 segments in the cortical labyrinth and the upper parts of the medullary rays (fig. 1), antibody 155 stains the S2 segment in the labyrinth and the upper parts of the medullary rays (fig.2). and antibodies 144 (fig.3) and 142 (fig.4) as well as 161 stain the S2 and S3 segments in the labyrinth, in the medullary rays and in the outer stripe. Six antibodies (146, 148,151,153, 157and 159) stain all three segments (SI, S2, S3) in the cortical labyrinth, in the upper parts of the med ullary rays and in the outer stripe (e.g. 153; fig.5). The boundaries of these segments are overlapping. Subcellularly. a number of different staining patterns (tables 1-3) could be observed with the eleven antibodies, all of which stain, preferentially and predominantly, the brush border of the proximal tubules (fig.6-10. 12, 15. 16). Nevertheless, moderate-to-strong staining was observed in
216
-
Med. ray: + Co. OS: +
Intestine
Liver
+ + BB
_
-
-
+ + BB + + BB
+ + BC + + BC
-
-
+ + BB + + BB
-
-
+ + BC + + BC + + BC
-
+ + BB
-
-
IS: +
-
-
+ + BB
Spleen
_
_
-
+ 1L
-
-
Pancreas
-
-
MC -
-
-
-
-
+ IL -
-
1L + IL
-
Med. ray = Medullary ray; BB = brush border; BC = biIe canaliculi; IL=intralobular ducts: MC=macrophage clusters; Co, OS = cortex, outer stripe; IS = inner stripe.
Okot-Opiro/Baier/Celio
the distal tubules (fig.8, 10, 11, 13) and in the basolatcral membranes (fig. 15, 16) as well as in intracytoplasmic gran ules (fig. 14). The glomerulus (fig.6). the parietal layer of Bowman’s capsule (fig. 12), the thin Henlc’s loops (fig. 18) and the collecting ducts (fig. 17) show a moderate reaction. These antigens were detectable as early as the 18th ges tational day. Immunoreaction covers the apical border and the basolateral membranes of the metanephrogenic blas tema (fig. 19). Kidney sections from 1-, 7-. 11- and 16-dayold rats show similarly distributed immunoreactive sites. Examination of the distribution of the antigenic determi nants in different rat organs revealed the following patterns (table 1): (1) positive immunohistochemical reaction along the intralobular duct of the exocrine pancreas with anti-
Fig. 1-5. General distribution of antigens on longitudinal cryostat sections (10 gnr thick) of the whole rat kidney fixed with 4% paraformaldehyde. These images were made with a photographic enlarger by projecting sections directly on to photographic paper (‘histography'). C = Cortex; M = medulla; P = Pelvis: SI. S2. S3 = seg ments of the proximal tubule. x 8 . 1 A faint lebelingof the SI and the S2 segments with no distinctive demarcations in the cortex (C). Anti body 150. 2 This kidney section shows a faint staining which is restricted to the S2 segment only. Antibody 155. 3 The intensive label ing of the S2 and the S3 portions is apparent. These portions begin in the cortex and end sharply at the boundary between the cortex and the medulla. Antibody 144. 4 Kidney section showing the distribution of antigens in the S2 and the S3 segments similar to that in figure 3. but with less intense labeling. Antibody 142. 5 The distribution of the anti gen in this kidney section extends to all the three portions (S I. S2. S3) of the proximal tubule. Antibody 148.
New Kidney Antigens
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perfused and fixed. The tissue sections were treated immunohistochemically. omitting the primary antibody. To ascertain whether there was an increase in the thickness of the glomerular basal lamina or a modification of the expression of the renal brush border, kidney sections from all the three pairs of rats were processed for standard electron microscopy, omitting the peroxidase reaction. To detect similar antigens in the urine, a ‘spot test’ was performed [ 10] on nitrocellulose paper with the urine of an adult rat deprived of water for 2 days.
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217
218
Okot-Opiro/Baier/Cclio
New Kidney Antigens
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(For legends see p. 221.)
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(For legends see p. 221.)
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Fig. 6. Light micrograph of a renal corpuscle, showing the affer ent arteriole (AA) at the vascular pole and the immunolabelcd glo merulus (G). Bowman's space is confluent with the proximal tubule (PT) at the urinary pole. The luminal membrane of the S2 and the brush border (BB) of the SI segments of the proximal tubule are stained. Antibody 151. X375. Fig. 7. The same antibody as in figure 6 stains the brush border (PTBB) of the longitudinally cut proximal tubule (PT). 1-pm-thick ultracryostat sections, x 600. Fig. 8. Positive reactions are seen on the brush border of the proximal tubule (PTBB) and on the luminal membrane of the outer colecting tubules (OCT). The lumen of the collecting tubules are so narrow that their luminal membranes collapse together and form sin gle lines. Kidney section (I pm thick). Antibody 151. x600. Fig. 9. Micrograph from a 1-pm-thick kidney section of the rat shows immunoreaclivc sites in the brush border of the proximal tubule (SI and S2). UP = Urinary pole. Antibody 153. x60
