AmericatnJournal ofPathology, Vol. 137, No. 4, October 1990 Copyright C) American Association ofPathologists

Monoclonal Antibodies Against Membrane Proteins of the Rat Glomerulus Immunochemical Specificity and Immunofluorescence Distribution of the Antigens

Aaro Miettinen,* Gerhard Dekan,t and Marilyn Gist Farquhart From the Department ofBacteriology and Immunology, University ofHelsinki, Helsinki, Finland*; and the Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticutt

Monoclonal antibodies (MAbs) were generated to detergent-solubilized glomerular extracts to identify new epithelial and endothelial membrane proteins and to study the possible role of the corresponding antigens in theformation of immune deposits. Triton X- 114 extracts of isolated glomeruli were subjected to phase separation, and the resultant detergent and aqueous phases were used to immunize mice. Monoclonal antibodies were prepared by standard techniques, and hybridomas secreting antibodies (IgGs) that recognize glomerular cell surface antigens were selected by enzyme immunoassay (EIA) and indirect immunofluorescence. The IgGs of 13 MAbs selectedfor study recognized antigens of different molecular weights (45 to 350 kd) by immunoprecipitation and immunoblotting and had different distributions in the glomerulus and in other renal structures by immunofluorescence. Several proved to recognize known antigens-ie, podocalyxin (MAbs IA, 5A, I IA, and 20A), gp330 (20B), and dipeptidylpeptidase IV (26C). Others recognized antigens not previously characterized that fell into four groups: 1) those that were detected mainly in glomeruli; 2) those present in both glomeruli and peritubular capillaries; 3) those present in both glomeruli and tubule epithelia; and 4) those detected in all these sites. The pattern ofglomerular staining also varied, but most of the antigens appeared to be expressed on either the endothelium or the epithelium, or on both. 27A IgG was specificforpodocytes and weakly precipitated a 103-kd protein. 7A and 13A IgG precipitated a 120-kd protein and stained

glomeruli as well as the basal aspects of distal tubules. 23A IgG recognized a more-than 350-kd antigen that appeared to be specific for endothelial cells in rat kidney and in all other organs studied. 14A IgG precipitated a 150-kdprotein and stained glomeruli, proximal tubule brush borders, and endothelial and epithelial cells in rat kidney and in several other organs. 4B and 9B IgG gave a granular cytoplasmic staining in all cells. When injected intravenously into rats, all of the MAbs except 4B and 9B rapidly bound to glomeruli, demonstrating that the respective antigens are exposed at the cell surface and represent potential targets for antibody-mediated immune injury. It is concluded that selective detergent extraction ofglomeruli is a useful approach for generation of antibodies that recognize native, nondenatured membrane components of glomerular endothelial and epithelial cells. (Am JPathol 1990, 13 7:929-944)

To understand the normal functions and pathologic reactions of glomerular cells requires characterization of their cell-surface proteins that mediate such interactions as adherence to the glomerular basement membrane (GBM) and mesangial matrix, cell-to-cell contacts, and binding of circulating antibodies. To date relatively few surface proteins of rat glomerular cells have been isolated and characterized. So far the list includes gp330, the Heymann nephritis antigen,14 podocalyxin,5 podoendin,6 di peptidylpeptidase IV (DPPIV),7 8 and 51 -kd9 and 1 15/107Supported by NIH grant DK1 7724 and a gift from RJR Nabisco, Inc., 19861989 (to M. G. Farquhar), and by grants from the Paulo Foundation and the Finnish Kidney Foundation (to A. Miettinen). Dr. Farquhar and Dr. Dekan's present address is: Division of Cellular and Molecular Medicine, M-051, University of California San Diego, La Jolla, CA 92093. Accepted for publication May 25, 1990. Address reprint requests to Marilyn Gist Farquhar, PhD, Division of Cellular and Molecular Medicine, M-051, University of California at San Diego, La Jolla, CA 92093.

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kdl° proteins of unknown function. In this study we set out to generate monoclonal antibodies (MAbs) to membrane proteins of glomerular endothelial and epithelial cells. Our approach was to sequentially extract isolated rat glomeruli to obtain a fraction enriched in membrane proteins to use as an immunogen. In this report we describe and characterize by immunochemical and immunofluorescence methods 13 monoclonal antibodies (IgGs) against both novel and previously known glomerular proteins. We also demonstrate that, after intravenous injection, 11 of the MAbs bind to glomeruli, indicating that the antibodies recognize exposed epitopes on the corresponding antigens. In a companion article,1" we demonstrate the immunoelectron microscopic localization of the antigens in situ and the distribution of bound IgG after intravenous injection.

immunofluorescence was from Dakopatts (Glostrup, Denmark) or Zymed (South San Francisco, CA). [1251]iodine (carrier-free, 12.9 mCi/4g), [35S]cysteine (1200 Ci/mmol), and ['251]protein A (1260 Ci/mmol) were from Amersham Corp. (Arlington Heights, IL). lodobeads and lodogen were from Pierce Chemical Co. (Rockford, IL), and Limbro multi-well tissue culture plates were from Flow Laboratories (MacLean, VA). RPMI 1640, Dulbecco's Modified Eagles Medium (DMEM), fetal calf serum, Iglutamine, thymidine, hypoxanthine, penicillin, and streptomycin were from GIBCO (Long Island, NY). NS-Nutridoma was from Boehringer Mannheim Biochemicals (Indi-

anapolis, IN).

Animals

Materials and Methods Materials Antipain, pepstatin A, leupeptin, benzamidine, bovine serum albumin (BSA), fluorescamine, Ponceau S, diaminobenzidine, p-phenylenediamine, Triton X-1 14, poly-l-lysine (molecular weight [MW] = 40,500), 2, 5-diphenyloxazole (PPO), dimethyl sulfoxide (DMSO), and the subclass-specific goat anti-mouse IgG kit were from Sigma Chemical Co. (St. Louis, MO). Triton X-100 was from Eastman Kodak (Rochester, NY), and diisopropylfluorophosphate from Aldrich (Milwaukee, WI). Sodium dodecyl sulfate (SDS), acrylamide, bis-acrylamide, TEMED, ammonium persulfate, high- and low-molecular-weight standards, and Coomassie Brilliant Blue R-250 were from Bio-Rad Laboratories (Richmond, CA). Nitrocellulose (0.45-,q pore size) was from Schleicher & Schuell (Keene, NH). Goat anti-mouse IgG (gamma chain-specific) conjugated to rhodamine isothiocyanate (TRITC), goat antimouse IgG (gamma chain specific), horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG, HRP-conjugated swine anti-rabbit IgG, and rabbit anti-rat C3 coupled to fluorescein isothiocyanate (FITC) were from Cappel Laboratories (Cochraneville, PA). F(ab')2 fragments of goat anti-mouse IgG conjugated to TRITC, FITC-conjugated sheep anti-mouse IgG, and F(ab')2 fragments of goat anti-rabbit IgG conjugated to TRITC were obtained from TAGO (San Mateo, CA). Rabbit anti-mouse IgG (H + L) and sheep anti-rabbit IgG (Fc) conjugated to alkaline phosphatase were from Promega Biotec (Madison, WI); the BCIP/NBT substrate for alkaline phosphatase was from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). Protein A Sepharose CL-4B and PD-10 columns were from Pharmacia Fine Chemicals (Piscataway, NJ, or Uppsala, Sweden). Rabbit anti-mouse IgG (H + L) used as a bridge for immunoprecipitation, immunoblotting, and

Male and female Sprague-Dawley rats (weighing 120 to 170 g) used for the preparation of glomerular extracts and 2-day-old baby rats used for metabolic labeling of renal cortices were from Camm Research Laboratory Animals (Wayne, NJ). Female Sprague-Dawley rats (150 to 200 g) for in vivo binding studies were from Camm or from the Department of Bacteriology and Immunology, University of Helsinki. Female BALB/c and C/B6 Fl mice (4 weeks old) used for immunizations and for production of ascites were from Jackson Laboratories (Bar Harbor, ME) or from the Department of Bacteriology and Immunology, University of Helsinki.

Detergent Extraction of Rat Glomeruli Rats were anesthetized with ether, kidneys were perfused with ice-cold phosphate-buffered saline (PBS), pH 7.3, containing protease inhibitors (1 mmol/l each antipain, pepstatin A, leupeptin, benzamidine, and diisopropyfluorophosphate) after which isolated glomeruli were prepared by sieving.3 All isolation steps, unless otherwise stated, were performed at 4°C in the presence of protease inhibitors. Glomeruli were incubated in 0.2% Triton X100 in PBS at 20°C for 10 minutes on a Nutator to extract cytoplasmic proteins. After centrifugation (600g for 3 minutes) and washing, the pellet was suspended in cold 2% Triton X-1 14 in TRIS-buffered saline (TBS) and incubated at 4°C for 15 minutes on a Nutator to extract membrane proteins. After centrifugation (13,000g for 15 minutes), the supernatant was subjected to Triton X-1 14 phase separation.12 Protein concentrations were determined by the fluorescamine assay.13 The extraction steps were monitored by electron microscopy and SDS polyacrylamide gel electrophoresis (SDS-PAGE).

Monoclonal Antibodies Against Rat Glomerulus Proteins 931 AJP October 1990, Vol. 13 7, No. 4

Preparation of Monoclonal Antibodies to Glomerular Cell Membranes

turer's instructions. Free iodine was removed and the detergent exchanged on a PD-10 column equilibrated with PBS containing 0.5% Triton X-100 and protease inhibi-

BALB/c and C/B6 Fl mice were immunized intraperitoneally with 20 to 100 ,ug protein (absorbed to potassiumalum) from either the detergent or aqueous phase of the Triton X-1 14 extract and boosted by intraperitoneal injection at regular intervals. The mice received 22 X 109 killed Haemophilus pertussis bacteria as adjuvant 2 hours before antigen injection and 20 ,ug of antigen in 0.2 ml of PBS intravenously 3 days before fusion. Spleen cells from mice showing the highest serum titer for antiglomerular antibodies by indirect immunofluorescence and enzyme immunoassay (EIA) were fused with X63/Ag8.653 myeloma cells. The resultant hybridomas were subsequently cloned by the Hybridoma Facility of the Howard Hughes Institute of Immunology, Yale University School of Medicine. Initial screening of IgG-producing hybridomas and clones was performed by EIA using 96-well flat bottom tissue culture plates coated overnight with the Triton X1 14 extract (20 ,ug protein/ml, diluted in PBS), followed by incubation with culture supernatant (2 hours), goat antimouse IgG, gamma chain-specific (2 hours), and HRPconjugated rabbit anti-goat IgG (2 hours), and o-phenylenediamine substrate. Culture supernatants that were positive by EIA were screened for glomerular staining by indirect immunofluorescence on acetone-fixed cryostat sections of rat kidney. Positive supernatants were tested on aldehyde-fixed semithin (0.5 ,t) frozen sections to identify those IgGs that bound to fixed antigen and were suitable for immunocytochemistry. Selected clones were expanded in RPMI 1640 supplemented with 2 mmol/l 1-glutamine, hypoxanthine, thymidine, and either 10% fetal calf serum or Nutridoma-NS and characterized in more detail. IgG was prepared from ascites by affinity purification on Protein A-Sepharose CL-4B14 and from culture supernatants by ammonium sulfate precipitation followed by purification on Protein A-Sepharose. The IgG subclass was determined by double immunodiffusion in 1% agarose against mouse IgG subclass-specific antisera. To determine their isoelectric spectrotypes, isolated IgGs were subjected to agarose isoelectric focusing, and transferred to nitrocellulose.15,16 Strips of the latter were incubated with rabbit anti-mouse IgG in 2% BSA-PBS (12 to 16 hours), followed by HRP-labeled swine anti-rabbit IgG (12 to 16 hours), and HRP substrate as described above.

tors.

Radioiodination of Detergent Extracts Triton X-1 14 extracts (detergent and aqueous phases) of glomeruli were iodinated in an ice bath using lodobeads or lodogen-coated glass tubes according to the manufac-

Metabolic Labeling of Kidney Slices and Preparation of Cortical Extracts Kidneys from 2-day-old baby rats were flushed via the left ventricle with RPMI medium; 125-y thick slices were cut with a Smith-Farquhar, Sorvall TC-11 tissue chopper, and incubated in a cysteine-free RPMI medium in a shaking waterbath at 37°C under continuous gassing with 95% 02-5% CO2. After an equilibration period (15 minutes), [35S]cysteine (200 to 400 uCi/ml) was added followed by incubation (3 hours) in the same medium plus 0.3% BSA and cysteine (5 mg/liter). At the end of the labeling period the slices were washed several times (ice-cold TBS with protease inhibitors), homogenized at 4°C (3 X 20 seconds) with a Tissumizer (Tekmar, Cincinnati, OH) and centrifuged (500g for 10 minutes). The resulting postnuclear supernatant was centrifuged (100,000g for 1 hour), and the pellet extracted in 0.2% Triton X-1 00 in TBS with protease inhibitors (3 hours). The extract was cleared by centrifugation and used for immunoprecipitation.

Immunoprecipitation Varying amounts of culture supernatants, ascitic fluids, or isolated IgGs (5 to 50 /Ag) were added to iodinated glomerular detergent extracts (5 X 105-106 cpm) or metabolically labeled cortical extracts and incubated on a Nutator for 4 hours at room temperature or overnight at 40C. Rabbit anti-mouse IgG (20 ,ug/ml) was added (2 hours), followed by 5 mg preswollen Protein A-Sepharose (2 hours). After pelleting, the beads were washed overnight (five changes of 1% Triton X-1 00 in TBS plus protease inhibitors) and rinsed once with 20 mmol/l TRIS-phosphate buffer, pH 6.8. Alternatively several MAbs (1 3A, 23A, 27A) were incubated overnight with postnuclear supernatants from homogenized kidney slices, 1% Triton X-100 was added (1 hour), the postnuclear supernatant was cleared by centrifugation in a Beckman Microfuge (Beckman, Palo Alto, CA), and processed as above.

SDS-PAGE Freshly isolated glomeruli, Triton X-1 14 extracts from isolated glomeruli, and immunoprecipitates were heated for 5 minutes in SDS sample buffer (10% SDS, 36 mmol/l dithiothreitol, 9 mmol/l ethylenediaminetetra-acetic acid

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(EDTA), 80 mmol/l TRIS-phosphate buffer, pH 6.8) and spun 5 minutes in a Microfuge. The proteins present in the supernatants were separated on 5% to 12% gradient gels,17 and either stained with Coomassie blue or silver5 or transferred to nitrocellulose for immunoblotting (see below). For autoradiography the gels were stained, dried, and exposed at -70°C with preflashed x-ray film (Kodak XAR-5). [35S]cysteine-labeled immunoprecipitates were treated with PPO/DMSO before exposure.

Immunoblotting Glomerular proteins separated by SDS-PAGE were transferred to nitrocellulose (200 mA for 16 hours at 40C) and stained with Ponceau S. Strips of nitrocellulose were incubated for 1 hour in Blotto (5% nonfat dry milk, 1% Tween 20, 0.5 mol/l NaCI, 20 mmol/l PBS, pH 7.5), followed by incubation overnight with monoclonal or polyclonal antibodies. The bound IgG was detected by incubation for 1 hour in alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG (diluted 1:7500 in Blotto), followed by nitroblue tetrazolium (100 ,ug/ml) and 5-bromo-4-chloro-3indolyl phosphate (50 ,ug/ml) in TRIS buffer (0.1 mol/l TRIS, 0.1 mol/l NaCI and 5 mmol/l MgCI2, pH 9.5). When [1251]protein A was used, the nitrocellulose strips were incubated (1 hour) in rabbit anti-mouse IgG (1:1000 in Blotto), followed by [1251]protein A (1 hour) and autoradiography.

Depletion and Affinity Purification of 1A and 20B IgG To test the specificity of 1 A and 20B IgG by depletion on their specific respective antigens, 1 A and 20B IgG were mixed (0.5 ,g/ml) in 3 ml PBS containing 2% BSA and incubated overnight with either podocalyxin transferred to nitrocellulose18 or gp330 coupled to Sepharose 4B.3 For affinity purification nitrocellulose-bound podocalyxin and gp330 bound to Sepharose 4B were incubated overnight with 50 ,ug/ml 1 A or 20B IgG, and the bound antibodies were eluted with 0.2 mol/l glycine-HCI."8 After neutralization aliquots of the affinity-depleted and affinity-purified IgGs were tested by immunofluorescence on rat kidney cryostat sections. Controls consisted of sections incubated with undepleted 1 A and 20B IgG (0.5 ,ug/ml) alone or mixed together and PBS containing 2% BSA.

Preparation of Tissues for Immunofluorescence and Electron Microscopy For immunofluorescence on cryostat sections, rat kidneys were flushed with DMEM by retrograde perfusion via

the abdominal aorta. Blocks of kidney and other tissues (brain, tongue, salivary gland, lung, heart, liver, spleen, small intestine, pancreas, epididymis, and testis) were snap frozen in isopentane cooled with liquid nitrogen. Kidney samples from mice, rabbits, dogs, and chicken and from normal areas of human kidneys (removed because of renal carcinoma) were similarly prepared. For immunofluorescence on semithin (0.5-a) frozen sections, rat kidneys were flushed with DMEM and fixed by perfusion for 5 to 10 minutes with 3% paraformaldehyde/0.05% glutaraldehyde in 0.1 mol/l sodium phosphate buffer. They were then removed, cut into pieces, and the latter further fixed for a total of 1 hour, after which they were cryoprotected by infiltration (1 hour) with 2.3 mol/l sucrose in phosphate buffer containing 50% polyvinylpyrrolidone,19 mounted on aluminum nails, and frozen in liquid nitrogen. For electron microscopy, isolated and extracted glomeruli were fixed in 1% glutaraldehyde/4% formaldehyde in 0.1 mol/l sodium cacodylate (1 hour) at 200C, postfixed with OS04, stained in block with uranyl acetate, dehydrated, and embedded in Epox 812 (Fullam, Latham, NY).

Immunofluorescence Cryostat sections (5 Iu) prepared from unfixed tissues were mounted on glass slides and fixed in acetone for 10 minutes at -20°C. The sections were incubated for 30 minutes at room temperature with the culture supernatants or IgGs (5 to 10 Ag/ml in PBS, 1% ovalbumin, pH 7.4) followed by TRITC- or FITC-labeled anti-mouse IgG (30 minutes) and mounted in 90% glycerol containing phenylenediamine.20 To test for complement binding, cryostat sections of normal rat kidney were incubated (30 minutes) with the MAbs, followed by fresh rat serum (diluted 1 :10 in PBS) and FITC-conjugated anti-rat C3. Semithin (0.5 g) frozen sections were cut from aldehyde-fixed tissues at -80,C21 on a Reichert FC-4 Ultramicrotome E equipped with a cryoattachment. They then were transferred to glass slides coated with poly-l-lysine (1 mg/ml), incubated with culture supernatants or isolated IgGs (2 hours at room temperature or overnight at 40C), followed by a TRITC-conjugated goat anti-mouse (Fab')2. In some cases, incubation with the primary antibody was followed by a rabbit anti-mouse bridging antibody and TRITC-conjugated goat anti-rabbit IgG. The anti-mouse IgG and IgG conjugates were affinity depleted of crossreactivity with rat IgG on a rat IgG-Sepharose CL-4B column. Slides were examined by epifluorescence and photographed on a Zeiss Photomicroscope IlIl using Kodak Tri-X Pan (ASA 400) film.

Monoclonal Antibodies Against Rat Glomerulus Proteins 933 AJP October 1990, Vol. 13 7, No. 4

Intravenous Injection of Monoclonal IgGs Five milligrams of isolated IgG in 1 ml of PBS was injected into rats via the external jugular vein. Kidney samples were taken at 5 minutes to 1 hour after injection, cryostat sections were prepared, and the bound monoclonal IgG was detected directly by incubating the sections with TRITC- or FITC-labeled anti-mouse IgG (30 minutes).

Results

Preparation of Immunogen: Monitoring of Glomerular Extraction A sequential extraction procedure was devised to obtain a fraction enriched in glomerular membrane proteins. Isolated glomeruli (Figure 1 A) were first treated with 0.2% Triton X-100, which serves to partially permeabilize the cells, releasing soluble cytoplasmic proteins and retaining many membrane proteins. As shown in Figure 1 B, the partially extracted endothelium and foot process layer, including the slit membranes, remained attached to the GBM after this treatment. When the pellet was further extracted with 2% Triton X-1 14, the endothelium and foot process layer were not seen-presumably the proteins were solubilized and released into the supernatant. Only the GBM with attached membrane fragments remained behind in the pellet (Figure 1 C). Thus the supernatant obtained after Triton X-1 14 extraction would be expected to be enriched in membrane proteins and partially depleted of cytoplasmic proteins and insoluble GBM components. The Triton X-1 14 supernatant then was subjected to phase separation, which serves to partially separate hydrophilic proteins (enriched in the aqueous phase) from hydrophobic integral membrane proteins (enriched in the detergent phase).12 The yield of glomerular proteins extracted in 2% Triton X-1 14 was - 118 ,ug protein/rat, of which 60% partitioned into the detergent phase and 40% into the aqueous phase (Figure 2). Of the mice selected for fusion, one was immunized with proteins from the detergent phase and the other with those from the aqueous phase.

General Properties of Monoclonal Antibodies Of the 1 146 growing hybridomas obtained, 201 produced IgGs that bound to a glomerular extract in the EIA assay, and of these 159 stained glomeruli by indirect immunofluorescence on acetone-fixed cryostat sections of adult rat kidney. Thirty of the latter were selected for further subcloning, and 13 clones were characterized in more detail. Eleven clones (1 A to 20A) originated from mice immu-

nized with glomerular antigens that partitioned into the aqueous phase, and three (clones 23A to 27A) originated from those immunized with the detergent phase. The characteristics of the 13 monoclonal antibodies selected for further study are shown in Table 1. It is evident that the IgGs had different isoelectric points (4.0 to 7.6), were of different IgG subclasses, gave different staining patterns, and recognized proteins of different molecular weights (65 to 350 kd). Only 27A IgG activated rat complement, as assessed by the indirect immunofluorescence assay. By isoelectric focusing, many monoclonal IgGs showed a family of bands of different isoelectric points. The pH range within a given family seemed to vary with the genetic background of the hybridoma cells, being greatest for those derived from C/B6F1 mice (clones 23A, 26C and 27A) and less for those derived from BALB/c mice (clones 1 A to 20B). 4B, 9B, and 1 3A IgG precipitated proteins in the low ionic strength region of the focusing gel, and their isoelectric points could not be determined.

Immunoprecipitation and Immunoblotting When the antigen specificity of the monoclonal antibodies was determined by immunoprecipitation and/or immu-

noblotting techniques (Figure 3), several recognized known glomerular proteins. For example, the IgGs of clones 1 A, 5A, 1 1 A, and 20A all recognized podocalyxin by immunoblotting as they bound to a 1 40-kd polypeptide in glomerular extracts, and several that were tested (clone 1 A and 5A) precipitated a 125-kd protein from iodinated extracts of isolated glomeruli or from extracts of metabolically labeled renal cortical slices (Figure 3). The latter comigrated with the protein precipitated by polyclonal antipodocalyxin.5 Another monoclonal IgG, 20B, proved to recognize the Heymann nephritis antigen gp330, as it precipitated a protein from labeled cortical extracts comigrating with that precipitated by our reference anti-gp330 antibodies2 3 and gave the same staining pattern (see below). The remaining antibodies proved to recognize other, unknown proteins. Clone 27A IgG precipitated a faint 103kd band and usually also another -50-kd band but did not recognize any proteins by immunoblotting. The IgGs of clones 7A and 1 3A recognized faint bands of 120 and 45 kd by immunoblotting, and 13A precipitated a similar 120-kd protein. Antibodies of clone 23A precipitated a large (more than 350 kd) protein that migrated more slowly on SDS-PAGE than gp330 (compare 23A with 20B in Figure 3). Clone 26C and 14A IgGs recognized 105and -150-kd peptides, respectively, by immunoprecipitation or immunoblotting of glomerular extracts, and clones 4B and 9B recognized a 65-kd protein by immunoblotting.

c Figure 1. Results of sequenitial detergenit extraction of isolated glomeruli. A: Isolated glomerulus before extraction demonstrating that glomerular compotnentts, intcluding the foot processes (fp), slit membranes (arrow), and peripheralfentestrated endothelial layer (En), are intact. B: Pellet obtained after extraction with 0.2% Triton X-100. The footprocess layer anld the endothelium are partially detached from the glomerular basemetnt membrane (B3M). 7he cell membranes ofthefootprocess are no longer intact and their cjytoplasm appears partlj extracted, buit the overall architecture ofthefootprocesse-s and slit membratne-s (arrows) ispreserved. C: Pellet obtained after Triton X- 114 extraction. The endothelial andfoot process layers have been detached and tbe proteins presumably solubilized and released into the supernzatant. Only the basement membrane with residualparticulate and membratnousfragments remains after this step. Cap, former capillary lumen (X35, 000).

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ijia

i;il

200 kd -

116 kd 97 kd

4-

-

66 kd -

4-

45 kd -

A B C Figure 2. SDS-PAGE profiles of a total rat glomerular extract (lane A) and the Triton X-114 aqueous phase (lane B) or detergent phase (lane C) extracts used as immunogens. A: Isolated rat glomeruli were directly, boiled in SDS-sample buffer, electrophoresed otn a 5% to 12% gradient gel, and stained with silverfollowed by Coomassie blue. B and C: Isolated glomeruli were sequentially extracted in Triton X- 100 and Triton X- 114; the 7riton X- 114 extract was subjected to phase separation, as described in Methods, and the aqueous and detergent phases separated as in lane A. The extracts are relatively depleted of cytoskeletal proteins, such as myosin (broad 200-kd band in lane A) and are selectively enriched in other proteins. For example, bands of 330 kd and 50 kd are enriched in the aqueous phase, and a 100-kd protein is enriched in the detergent phase (indicated by, arrows).

Staining of Rat Kidney Sections When the monoclonal antibodies were used to localize their antigens by indirect immunofluorescence on cryostat sections of adult rat kidney, several different staining patterns were seen (Figure 4): One antibody, 27A (anti103 kd), stained only glomeruli (Figure 4A). Two others, 7A and 13A (anti-120 kd), stained glomeruli (Figure 4B, C), and the basolateral aspects of some distal tubules

(Figure 6A). A third group, 23A (anti-350 kd) (Figure 4E), 1 A and 5A (Figure 4D), 1 1 A and 20A (anti-podocalyxin) stained glomeruli, and blood vessels but did not stain tubules. A fourth group, 14A (anti-1 50 kd) (Figure 4F), 20B (anti-gp330) (Figure 4G), and 26C (anti-1 05 kd) (Figure 4H) stained glomeruli and the brush borders of proximal tubules. The last group, 4B (Figure 41) and 9B, stained all renal cells and blood vessels: there was strong cytoplasmic staining of distal tubules and faint staining of proximal tubules, glomeruli, and vascular smooth muscle cells. When the distribution of the antigens was determined in semithin (0.5 A) frozen sections, where the ability to localize antigens among glomerular structures is greatly improved, clone 27A IgG gave an intense, granular pattern (Figure 5A), suggesting staining of cell membranes and intracellular structures of podocytes. IgGs from clones 7A and 13A gave a linear fluorescence pattern suggesting localization of the antigen(s) they recognize to the glomerular basement membrane and/or podocytes (Figure 5B). The staining pattern obtained with 23A IgG suggested that its antigen is largely or exclusively restricted to endothelial cells (Figure 5C). With 1 A, 5A, 1 1 A, and 20A IgGs (anti-podocalyxin), both endothelial cells and podocytes appeared to be stained (Figure 5D). The latter pattern is identical to that obtained with polyclonal anti-podocalyxin. 20B IgG gave a fine, punctate staining of glomeruli and a strong reaction of the brush borders of proximal tubules, similar to that obtained with anti-gp330 antibodies.23 The pattern obtained with IgGs of clones 1 4A (Figure 5E) and 26C (Figure 5F) was primarily epithelial in origin, but with 26C the endothelium of glomerular capillaries was also weakly reactive. When 1 A IgG was affinity depleted by incubation with podocalyxin and 20B IgG by incubation with gp330, there was a drastic reduction in their titer compared with the titer of the simultaneously present second IgG. Affinitypurified 1 A and 20B IgGs yielded the typical podocalyxin and gp330 immunofluorescence patterns (not shown). Most of the MABs did not stain glomeruli of species other than the rat. However 20B (anti-gp330) IgG reacted with the brush borders of mouse proximal tubule (Figure 6B). Background staining due to endogenous mouse IgG made it difficult to determine whether these antibodies also bind to mouse glomeruli. Only 4B and 9B IgGs bound to and gave an identical staining of kidneys of all species tested.

Staining of Extrarenal Tissues When the ability of the monoclonal antibodies to recognize antigens present in various rat tissues was tested by indirect immunofluorescence, most proved to recognize antigens that are widely distributed in other tissues. Inter-

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Table 1. Characteristics ofMonoclonalAntibodies to Glomerular Proteins Immunofluorescence of Rat Kidney MAb Glomerulus Proximal tubule Distal tubule Blood vessels 27A ++ 7A ++ + 13A ++ + 23A + + 1A ++ + ++ 5A + 11A ++ + 20A ++ + ++ 14A ++ + + 20B ++ 26C ++ ++ + 4B + + + +/++ + 9B + + +/++ * ND, not determined.

estingly, however, 27A IgG did not stain any of the extrarenal tissues tested, suggesting that this antibody recognizes an antigen specific for glomerular podocytes. Clones 7A and 1 3A IgG stained the basal aspects of epithelia from several tissues tested, including those of the stratum germinativum of the epidermis (Figure 6C), bronchi, mucous membranes, and chorioid plexus. Clone 23A IgG (Figure 7A through C) and 1 A, 5A, 1 1 A, and 20A (antipodocalyxin) IgG recognized endothelial cells of all blood vessels in all tissues tested, as reported earlier for polyclonal anti-podocalyxin.522 These IgGs also stained the sinusoidal endothelium of the liver and the spleen. No reaction was detected with other cell types. Clone 26C IgG reacted with the luminal or apical aspects of several epithelia such as the bile canalicular domain of hepatocytes (Figure 7D), brush borders of enterocytes (Figure 7E), and the epithelium of pancreatic and salivary ducts. This IgG also stained the cells of the perineural sheaths of peripheral nerves and the luminal aspects of the endothelium of some blood vessels, including that of the liver (Figure 7D) and spleen. Clone 1 4A IgG stained epithelial tissues and blood vessels in an almost identical manner to that of 26C IgG (Figure 7F). However the antibodies also bound to erythrocytes. Clone 20B (anti-gp330) reacted with the apical aspects of some (but not all) epithelial tissues; the reaction was most intense at the luminal surface of the epididymal epithelium (not shown). Clones 4B and 9B gave a similar granular intracellular staining in all tissues and, after permeabilization, all cultured cells tested (Figure 6D). The pattern of distribution suggested staining of mitochondria.

Antigen (kd) 103 120 120 350 140 140 140 140 150 330 105 65 65

Antibody Isotype

IgG3, k IgG,, k IgG,, k IgG2b, k IgG2b, k IgG,, k IgG,, k IgG,, k IgG,, k

IgGi, k

IgG,, k

IgG2b, k

IgG2b, k

pI 6.9; 7.2; 7.4; 7.6 n.d.* n.d. 6.8; 7.0 4.0 6.6; 7.0 6.8; 7.0 6.0 7.0; 7.5 5.8; 6.0 6.5; 6.7; 6.8 n.d.

n.d.

into rats. When kidney samples were taken at 5 minutes to 1 hour after injection and the bound mouse IgGs were detected by immunofluorescence, all except 4B (Figure

81) and 9B IgGs bound to their antigens in vivo. After injection of 5 mg 27A, 7A, and 1 3A IgGs, the antibodies bound to glomeruli, giving a strong, linear fluorescence pattern suggesting binding to the GBM (Figure 8A through C). Interestingly 13A IgG also bound to the basolateral aspects of some distal but not proximal tubules and to the smooth muscle layer of some arteries (results not shown). After injection of 5A (anti-podocalyxin) (Figure 8D) and 23A IgG (Figure 8E), staining of glomeruli and blood vessels of all sizes was seen, suggesting binding to endothelial cells. 14A and 26C IgGs bound to glomeruli (Figure 8F, H) and to a lesser extent to peritubular capillaries. 20B IgG (anti-gp330) also bound to glomeruli (Figure 8G), in a pattern identical to that seen after intravenous injection of polyclonal anti-gp330 IgG.3

Discussion

Binding of Monoclonal Antibodies to Kidney In Vivo

In this study we have raised a group of MAbs against a detergent lysate of glomeruli. Our purpose was to attempt to identify new antigens located at the surface of glomerular endothelial and epithelial cells to use the antibodies 1) to characterize the surface proteins of these cells, and 2) to study the fate of the corresponding IgGs after in vivo injection. Toward this end we developed a sequential extraction procedure designed to provide an immunogen at least partially depleted of cytoplasmic proteins and GBM components and enriched in glomerular membrane proteins maintained in their quasi-native conformational state. The approach was successful in that we suc-

To test whether the MAbs could bind to their antigenic epitopes in vivo, we injected purified IgGs intravenously

ceeded in generating MAbs that recognize glomerular endothelial and epithelial cell surface proteins, and all of these IgGs bound to glomeruli after in vivo injection. Sev-

. :u ,

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IMMUNOPRECIPITATION [35S]Cys-labelled Kidney Slices

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Figure 6. Inidirect immunolluorescence of crostat sectionis of various rat tissues reacted with AAbs. A: Distal tubules of rat kidney stainzed with 13.4 IgG, demonstrating the anitigen at the base of the cells. B: Mouse kidney reacted u'ith 20B (anti-gpt3.30) IgG. The brush border of the proximal tubule is stainied Glomeruilar (G) fluorescence is due to endogeniouis deposits of nmouse IgG. C: Epitheliumni (Ep) of rat tonigue stainied with 7A IgG. A linearfluorescence is seenI at the base of the stratum germinativum (arrow) where itfaces the dermis (D). D: Cuilturied NRK cells permeabilized anid reacted with 413 IgG. The immunlofluorescenice patterni suggests mitochondrial staining A and C, X480; B, X200; D, X 750.

Figure 7. Inidirect imnmqojfluorescencce of semithini sections of several rat organis. 23A stains the endothelium (arrows) of liver (A), panicreas (B), anid intestine (C). 266 (anti-DI5TlV) strong/v stainis the endothelium and bile canialiculi of the liver (D). 26C (E) also reacts o'ith the bruish border oJlthe initestinie as cloes 14A (F). v, vein (X480).

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Figure 8. Cryostat sections of kidneys from rats killed 1 hour after inijection of monoclonal IgGs and reacted with FITC anti-mouse IgG. A: 27A. B: 7A. C: 13A. D: 5A. E: 23A. F: 14A. G: 20B. H: 26C. I: 4B. All except 4B strongly bind to glomeruli. 14A, which was localized 5 minutes after inijectiotn, is weaker than the rest. 5A and 23A also bind to peritubular capillaries, and 14A and 20B IgG are filtered antd bind to the proximal tubule brutsh border (X300).

One of the IgGs we generated, 26C, appears to recognize the enzyme dipeptidylpeptidase IV (DPPIV) as it has similar properties,8 and it precipitates DPPIV activity from a brush border fraction (Biemesderfer et al: manuscript in preparation). This 90- to 108-kd hydrolase is present on proximal tubule cells, podocytes, the apical aspects of several epithelia and endothelia, and- also on most Iymphocytes.7,25 29Neutral endopeptidase or enkephalinase,30 another brush border hydrolase of similar molecular weight (-90 kd) that is homologous to the common acute lymphoblastic leukemia antigen CALLA,3' is also expressed on podocytes.32 Several other monoclonal IgGs proved to recognize novel proteins that have not been previously described:

23A IgG recognizes a large more-than 350-kd protein that appears to be restricted to vascular endothelia. Like podocalyxin, it stains the endothelium of glomerular and peritubular capillaries and that of all other organs studied (liver, pancreas, heart, intestine, and skin). It may prove to be useful as a general endothelial cell marker. Another MAb, 14A, stains glomeruli and proximal tubule brush borders and precipitates a -150-kd protein. Its distribution in other organs is somewhat variable: it stains the brush border of the intestine and both the sinusoids and bile canaliculi of the liver. Thus it has properties resembling those of aminopeptidase N as described in the proximal tubule of the rabbit32 and pig33 kidney, but its identity remains to be established by appropriate assays.

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Two other MAbs, 7A and 13A, proved to recognize novel -120-kd and 45-kd proteins by immunoblotting and immunoprecipitation. These give a linear staining pattern in the glomerulus at the base of the cells of the distal tubule and of epithelia of several other tissues. This pattern resembles that obtained with antibodies to s-laminin34 or the M283+++ fragment of type IV collagen,3 but the respective molecular weights of the latter (190 and 28 kd) are different from that of the antigen recognized by 7A and 13A IgG. Of particular interest is MAb 27A, which faintly precipitates novel -50 and 103 kd proteins that appear to be restricted to the glomerular epithelium and are not found in any other structure in the adult rat kidney or other tissues. It recognizes an antigen that is present in high concentrations inside the podocyte. Its granular intracellular staining pattern resembles that of podoendin6; however the size of the 27A antigen is different and, unlike podoendin, it is not detectable on endothelia. The fact that treatment of cryostat sections with ethanol or methanol inhibits glomerular staining with 27A IgG, together with other recent data, suggest that the main antigen recognized by this MAb may be a carbohydrate epitope of a glycolipid,36 and this epitope may be shared with one or more glycoproteins. 4B and 9B IgGs precipitated a 65-kd protein that appears to be an ubiquitous component present in all cells of all tissues and species tested. Its fluorescence staining pattern suggests that it is present in mitochondria. -

Variability in Kidney Cell Types Recognized by the MAb All of the MAbs we characterized have in common that they are expressed in the glomerulus; however their distribution among glomerular components and among other cell types in the kidney differs greatly. One, 23A, appears to be exclusively an endothelial marker. Another, 27A, appears to be exclusively a glomerular epithelial cell marker, as it is not found on any other kidney or extrarenal cell type examined. Podocalyxin and podoendin6 are proteins that are shared by both the glomerular epithelium and the glomerular and peritubular endothelia, but are not expressed on other renal cells. Several proteins, including gp330 (20B), DPPIV (26C), and the 14A antigen, are found on both glomerular and proximal tubular epithelia as well as on epithelia of several other organs. Finally the 7A and 13A proteins are expressed on glomerular, distal tubule, and several extrarenal epithelia. Based on the information available, the podocyte appears to be unique among epithelia in terms of the cytoskeletal proteins and the surface molecules it expresses: First, it expresses vimentin instead of cytokera-

tin, which is the intermediate filament protein expressed by most other epithelial cells.37 Second, it expresses several proteins (podocalyxin, 27A antigen) not found on any other epithelium. Third, it shares several proteins in common with the endothelium (podocalyxin, podoendin) and lymphocytes (CALLA or neutral endopeptidase).

MAbs Generated by Others Several investigators have been successful in generating MAbs to glomerular and proximal tubule proteins of the rat,1'4'9'25'38-42 human4348 rabbit,-2 49 or mouse-' kidney. In the case of the rat, solubilized brush border proteins,1'425 whole kidney homogenates,42 cortical homogenates,10'41 whole glomeruli,39 a crude membrane fraction,3 or collagenase-digested glomerular proteins9 have been used as the immunogen. Podocalyxin, gp330, and DPPIV appear to be highly immunogenic, as antibodies to these proteins occur with high frequency in MAbs generated against brush border fractions1' 4 glomeruli,38 or glomerular lysates (this report).

References 1. Bhan AK, Schneeberger EE, Baird LG, Collins AB, Kamata K, Bradford D, Erikson ME, McCluskey RT: Studies with monoclonal antibodies against brush border antigens in Heymann nephritis. Lab Invest 1985, 53:421-432 2. Kerjaschki D, Farquhar MG: The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border. Proc Natl Acad Sci USA 1982, 79: 5557-5561 3. Kerjaschki D, Farquhar MG: Immunocytochemical localization of the Heymann nephritis antigen (gp330) in glomerular epithelial cells of normal Lewis rats. J Exp Med 1983, 157: 667-686 4. Ronco P, Melcion C, Geniteau M, Ronco E, Reininger L, Galceran M, Verroust P: Production and characterization of monoclonal antibodies against rat brush border antigens of the proximal convoluted tubule. Immunology 1984, 53:8795 5. Kerjaschki D, Sharkey DJ, Farquhar MG: Identification and characterization of podocalyxin-The major sialoprotein of the renal glomerular epithelial cell. J Cell Biol 1984, 98:1591 1596 6. Huang TW, Langlois JC: Podoendin: A new cell surface protein of the podocyte and endothelium. J Exp Med 1985, 162: 245-267 7. Natori Y, Hayakawa I, Shibata S: Identification of gp108, a pathogenic antigen of passive Heymann nephritis, as dipeptidyl peptidase IV. Clin Exp Immunol 1987, 70:434-439 8. Verroust P, Ronco P, Chatelet F: Antigenic targets in membranous glomerulonephritis. Springer Semin Immunopathol 1987, 9:341-358

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9. Orikasa M, Matsui K, Oite T, Shimizu F: Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. J Immunol 1988,141:807-814 10. Mendrick DL, Rennke HG: I. Induction of proteinuria in the rat by a monoclonal antibody against SGP-1 15/107. Kidney Int 1988, 33:818-830 11. Dekan G, Miettinen A, Schnabel E, Farquhar MG: Binding of monoclonal antibodies to glomerular endothelium, slit membranes and epithelium after in vivo injection. Am J Pathol

1990,137:913-927 12. Bordier C: Phase separation of integral membrane proteins in Triton X-1 14 solution. J Biol Chem 1981, 256:1604-1607 13. Udenfriend S, Stein S, Bohlen P, Dairman W, Leimgruber W, Weigele M: Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 1972,178:871-872 14. Campbell AM: Monoclonal antibody technology. Laboratory Techniques in Biochemistry and Molecular Biology. New York, Elsevier, 1984,13:176-178 15. Olsson T, Kostulas V, Link H: Improved detection of oligoclonal IgG in cerebrospinal fluid by isoelectric focusing in agarose, double-antibody peroxidase labeling and avidin-biotin amplification. Clin Chem 1984, 30:1246-1249 16. Elovaara I, Seppala I, Palo J, Sulkava R, Erkinjuntti T: Oligoclonal immunoglobulin bands in cerebrospinal fluid of patients with Alzheimer's disease and vascular dementia. Acta Neurol Scand 1988, 77:397-401 17. Maizel JV: Polyacrylamide gel electrophoresis of viral proteins. Methods Virol 1971, 5:179-246 18. Schnabel E, Dekan G, Miettinen A, Farquhar MG: Biogenesis of podocalyxin-the major glomerular sialoglycoprotein-in the newborn rat kidney. Eur J Cell Biol 1989, 48:313-326 19. Tokuyasu KT: Use of polyvinylpyrrolidone and polyvinyl alcohol for cryoultramicrotomy. Histochem J 1989, 21:163-171 20. Platt JL, Michael AF: Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine. J Histochem Cytochem 1983, 31:840-842 21. Tokuyasu KT: Application of cryomicrotomy to immunocytochemistry. J Microsc 1986,143:139-149 22. Horvat R, Hovorka A, Dekan G, Poczewski H, Kerjaschki D: Endothelial cell membranes contain podocalyxin-the major sialoprotein of visceral glomerular epithelial cells. J Cell Biol

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30:649-655 26. Doxsey S, Kerjaschki D, Farquhar MG: A large membrane glycoprotein (gp330) is a resident of coated pits of several absorptive epithelia. J Cell Biol 1983, 97:1 78a

27. Chatelet F, Brianti E, Ronco P, Roland J, Verroust P: Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli: II. Extrarenal distribution. Am J Pathol 1986,122:512-519 28. Sawada H, Stukenbrok H, Kerjaschki D, Farquhar MG: Epithelial polyanion (podocalyxin) is found on the sides but not the soles of the foot processes of the glomerular epithelium. Am J Pathol 1986,125:309-318 29. Chatelet F, Brianti E, Ronco P, Roland J, Verroust P: Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli: I. Renal distribution. Am J Pathol 1986,122:500-511 30. Malfroy B, Schofield PR, Kuang W, Seeburg PH, Mason AJ, Henzel WJ: Molecular cloning and amino acid sequence of rat enkephalinase. Biochem Biophys Res Comm 1987,144: 59-66 31. Shipp MA, Vijayaraghavan J, Schmidt EV, Masteller EL, D'Adamio L, Hersh LB, Reinherz EL: Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 ("enkephalinase"): Direct evidence by cDNA transfection analysis. Proc Natl Acad Sci USA 1989,

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Acknowledgments The authors thank Sue Ann Mentone for technical assistance in the immunofluorescence. They thank llkka Seppala, MD, University of Helsinki, Finland, for valuable help in the isoelectric focusing.