American Journal ofPathology, Vol. 137, No. 4, October 1990 Copyright © American Association ofPathologists

Binding of Monoclonal Antibodies to Glomerular Endothelium, Slit Membranes, and Epithelium After In Vivo Injection Localization of Antigens and Bound IgGs by Immunoelectron Microscopy Gerhard Dekan,* Aaro Miettinen,t Eva Schnabel,* and Marilyn Gist Farquhar* From the Department of Cell Biology, Yale Unitversity School ofMedicine, New Haven, Connecticut*; and the Department of Bacteriology and Immunology, UJniversity of Helsinki, Hevlsinki, Finlandt

The antigens recognized by seven monoclonal antibodies (MAbs) raised against rat glomerular proteins were localized, and the sites of binding of the MAbs after in vivo injection were determined by immunoelectron microscopy. The antigens were localized in situ by immunoperoxidase and immunogold labeling to different domains and microdomains of the glomerular endothelium and epithelium. 23A recognized an antigen expressed exclusively on the luminal (apical) domain of the endothelium. 5A (anti-podocalyxin) and 26C (anti-DPPIV) recognized antigens expressed on the apical domains of both the endothelium andpodocytes. 13A, 14A, 20B (anti-gp330), and 27A recognized antigens restricted to podocytes in the glomerulus. The 13A antigen was present on their basal surface and the 27A and 14A antigens were expressed on both their apical and basal domains. The 14A antigen also was associated with the filtration slit membranes. All these MAbS bound to their antigens after injection in vivo. Those that recognize endothelial antigens were rapidly cleared from the circulation and rapidly disappearedfrom glomeruli, whereas those that recognize epithelial antigens persisted in the circulation and were detectable in glomerulifor hours or days. The sites of binding of the MA bs differed: 23A and 5A IgG (antipodocalyxin) bound exclusively to the luminal domain of the endothelium, whereas 26C IgG (antiDPPIV) bound to both the luminal endothelial membrane and the apical and basal domains ofpodocytes. The MAbs that recognize podocyte antigens bound to different domains of the podocyte plasmalemma: 13A and 27A IgGs to the basal do-

main, 14A to the slit membranes, and 20B to coated pits on the entire plasma membrane. 27A IgG led to the formation of small subepithelial immune deposits that remained up to 10 days. It is concluded that 1) glomerular membrane proteins vary considerably in their distribution among plasmalemmal domains and microdomains of endothelial and epithelial cells; 2) virtually all structures in the glomerulus and all domains and microdomains of the endothelium and podocyte are accessible to circulating antibodies; and 3) thefate of immune complexes formed by binding to glomerular components varies with the location of the antigen within the glomerulus, with those that bind to the basal domain and slit membranes of the podocyte persisting longer than the others. (Am J Pathol 1990, 13 7:913-92 7)

Using selective extraction and Triton X-1 14 phase separation of membrane proteins, we have recently generated a family of monoclonal antibodies (MAbs) to glomerular proteins that proved to recognize primarily endothelial and epithelial cell-surface components. In a previous paper1 we characterized these IgGs and determined their immunochemical specificities and distribution in the kidney and in a number of other organs by immunofluorescence. We have further demonstrated that all these MAbs, except two that recognize an intracellular protein, bind to glomeruli after injection, indicating that the corresponding antigens are accessible in vivo. This indicates that our antiSupported by a Max Kade Fellowship, 1987-88, (to G. Dekan), NIH Grant DK17724, and a gift, 1986-1989, from RJR Nabisco, Inc. (to M. G. Farquhar), and by grants from the Paulo Foundation and the Finnish Kidney Foundation (to A. Miettinen). Dr. Farquhar's 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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bodies represent useful tools for studies on the mechanisms of formation of immune deposits in glomeruli. In this paper, we have studied the specific sites of binding of seven of these antibodies after in vivo injection and have compared the fate of several of the IgGs that recognize antigens present exclusively on the glomerular epithelium (1 3A, 20B, 27A) with that of those that recognize antigens present on the glomerular endothelium (23A) or are shared by both endothelial and epithelial cells (5A, 14A, 260).

Materials and Methods Materials Polyvinyl alcohol (MW = 10,000), polyvinylpyrrolidone (MW = 10,000), and mouse IgG were obtained from Sigma Chemical Co. (St. Louis, MO). Protein A was purchased from Pharmacia Fine Chemicals (Piscataway, NJ), and gold chloride from Fisher (Fair Lawn, NJ). Glutaraldehyde was from EM Sciences (Fort Washington, PA) and Epox 812 from Fullam (Latham, NY). The rabbit antimouse IgG used as a bridging antibody for immunoperoxidase and immunogold labeling was affinity depleted of cross-reactivity with rat IgG on a rat IgG Sepharose CL4B column. Fab fragments of sheep anti-mouse and antirabbit IgG conjugated to horseradish peroxidase (HRP) were from Biosys (Compiegne, France). Goat anti-rabbit IgG coupled to 5-nm colloidal gold (GAR 5) was from Janssen Life Science Products (Piscataway, NJ). Protein A gold conjugates (5 and 10 nm) were prepared as described by Slot and Geuze.2 The sources of animals and other reagents are the same as given in the accompanying report.1

fused with DMEM followed by perfusion with PLP fixative (2% paraformaldehyde, 0.75 mol/l [molar] lysine, 0.01 mol/l sodium periodate in phosphate buffer, pH 7.4)4 for 5 to 10 minutes. Pieces of kidney cortex were prepared and further fixed for a total of 6 hours in PLP. They then were cryoprotected (10% DMSO, 1 hour) and snap frozen in isopentane cooled with liquid nitrogen as described in detail elsewhere.5 For immunogold labeling on ultrathin frozen sections, perfusion was with 3% paraformaldehyde-0.05% glutaraldehyde in 0.1 mol/l sodium phosphate buffer, pH 7.4, for 5 to 10 minutes, after which tissue blocks were prepared and fixed for a total of 1 hour in the same fixative. They then were cryoprotected by infiltration with 2.3 mol/l sucrose in phosphate buffer containing 50% polyvinylpyrrolidone (1 hour), mounted on aluminum nails, and frozen in liquid nitrogen.6

Immunofluorescence Semithin (0.5 ,u) frozen sections were prepared as described in the accompanying report' and incubated with rabbit anti-mouse IgG (1:50 in phosphate-buffered saline [PBS], 0.1% ovalbumin, pH 7.4) for 2 hours at room temperature or overnight at 4°C, followed by rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit F(ab')2 (1:50, 1 hour). The sections were mounted (90% glycerol in PBS containing 0.1% p-phenylenediamine), examined by epifluorescence, and photographed in a Zeiss Photomicroscope IlIl using Kodak Tri-X Pan film (ASA 400; Eastman Kodak Co., Rochester, NY).

Immunoperoxidase Procedure Antibody Preparation IgG was prepared either from delipidated ascites by protein A affinity chromatography with stepwise pH gradients3 or from culture supernatants by ammonium sulfate precipitation followed by protein A purification. The protein concentration was determined by measuring the optical density (OD) at 280 nm.

Preparation of Tissues for Immunocytochemistry and Electron Microscopy The methods for the collection of kidney tissue and for preparation of cryostat sections and semithin (0.5-,u) frozen sections were described in the companion paper.1 For immunoperoxidase labeling on cryostat sections, the kidneys of normal or antibody-injected rats were per-

For localization in situ of the antigens recognized by the MAb's, cryostat sections (20 to 30 ,g) were cut from PLPfixed kidney tissue, and the sections were incubated overnight with monoclonal IgGs (5 to 50 ,ug/ml), followed by Fab fragments of sheep anti-mouse IgG conjugated to HRP (1: 100, 2 hours). Alternatively, rabbit anti-mouse IgG (1:25, 2 hours) was used as a bridging antibody before incubation with Fab fragments of sheep anti-rabbit IgG conjugated to HRP. After the latter incubation, the sections were fixed in 3% glutaraldehyde in 0.1 mol/l sodium cacodylate buffer, reacted with diaminobenzidine (DAB), and processed for electron microscopy as described previously.5 For the detection of mouse IgG bound in vivo, cryostat sections were prepared from the kidneys of rats injected with IgGs (see below) and incubated directly in either the anti-mouse peroxidase conjugate or in the rabbit antimouse bridging antibody after the anti-rabbit peroxidase conjugate, and processed as above.

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Table 1. Localization ofAnitigens Recogniized by MAbs olPlasmalemmalDomains and Microdomains of Glomerular anid Proximal Tubiule Cells Glomerular Proximal tubule brush endothelium Podocyte border SlSIt MAb Basal Basal* Coated pits Apicalt membranes Microvilli Coated pits Apicalt 23A + 5A + + _ + +11 26C + ++ 13A + 14A + + + ++ 20B + + 27A + -+ *

Surface facing the GBM.

t Luminal surface including the membrane of the fenestra. t Surface facing the urinary spaces above the level of the slit diaphragms. 11 Detectable on peritubular but not glomerular capillaries. T Detectable by binding in vivo.

Immunogold Labeling of Ultrathin Frozen Sections Ultrathin cryosections were cut at -1 1 00C on a Reichert Ultracut E equipped with an FC-4E cryoattachment following the techniques of Tokuyasu,7 as recently described.8 Ultrathin sections were transferred to hexagonal nickel grids (200 mesh), which had been coated with formvar and carbon. Subsequent incubations and washing steps were carried out by floating the grids on droplets of the filtered solutions. After quenching with 10% fetal calf serum (FCS) containing 0.01 mol/l glycine (to block free aldehyde groups), the sections were incubated for 1 hour with the mouse monoclonal IgGs (diluted in PBS with 10% FCS) followed by rabbit anti-mouse IgG (diluted 1:50 in PBS + 10% FCS, 1 hour) and goat anti-rabbit IgG conjugated to 5 nm colloidal gold (30 minutes). To detect in vivo bound mouse IgG, the first antibody incubation was omitted. The sections then were postfixed (10 minutes) with 2% glutaraldehyde in PBS, stained with either neutral uranyl acetate (2% uranyl acetate, 0.15 mol/l oxalic acid, pH 7.4) or 1% OS04 for 15 minutes, followed by acidic uranyl acetate (2%) for 15 minutes. Finally they were absorption stained for 5 minutes with 0.002% lead citrate in 2.2% polyvinyl alcohol, as recently described by Tokuyasu.6

In Vivo Binding of MAb Female Sprague-Dawley rats (1 00 to 150 g) were injected intravenously via the external jugular vein with IgG (1 to 5 mg) from clones 5A, 13A, 14A, 20B, 23A, 26C, and 27A in 1 ml PBS, pH 7.3. Normal mouse IgG and nonspecific IgG from 27A ascites depleted of 27A IgG served as controls. A total of 30 animals were injected. Blood samples were taken for the detection of mouse IgG. Kidneys were collected at 5 to 60 minutes, and (except for 14A, 23A, and control IgG), 1 day or 10 days after injection, and

processed for immunofluorescence and immunoelectron microscopy. Urine samples were collected from experimental rats and albumin concentration age-matched controls at 1 to 10 days after injection and albumin concentration measured by radial immunodiffusion as described.9

Detection of Mouse IgG in Rat Sera To monitor the disappearance of injected mouse IgG from the circulation, blood samples were taken at 10 minutes to 10 days after injection and assayed by indirect immunofluorescence using unfixed cryostat sections of normal rat kidney as described.9 The serum titer at which specific staining of renal structures occurred was used as the measure of the amount of specific antibody remaining in the circulation.

Results Localization of Glomerular Antigens in Normal Rat Kidney by Immunoelectron Microscopy To determine their precise distribution within the glomerulus, it was first necessary to localize the endogenous antigens recognized by each MAb in the normal rat kidney by immunoelectron microscopy. Particular attention was paid to the differential distribution of the antigens on specific domains and microdomains of each of the cell types (Table 1). Toward this end, two immunolabeling methods were used-immunoperoxidase on cryostat sections and immunogold on ultrathin frozen sections. The advantages and disadvantages of these two different but complementary methods have been discussed in detail elsewhere.'01'

Localization of a Presumptive Endothelial Marker, MAb 23A Results obtained by indirect immunofluorescence on semithin sections suggested that the 350-kd antigen rec-

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Figure 1. Indirect immunoperoxidase localization of the antigens recognized bjy MAbs 26C or anti-DPPIV(A) and 23A (B) in the rat A: Both the eyndotheliuim (En) and the visceral epithelium (Ep) express DPPIV Staininzg is restricted mainly to the luminal or apical surface of the endothelium anid to the apical surface of the podocytesfaicing the urinary spaces above the slit diaphragms (arrows). The antigen is also detected within two multivesicular bodies (ml') within the epithelial cytoplasm (X 19, 000). B: The 23A antigen is located exclusively on7 the luminal surface of the endothelium (X3 1,000). Cryostat sections were incubated with primary antibody followed by a sheep anti-mouse Fab-peroxidase coniugate before embedding. Cap, capillary lumen; US, urinart space.

glomerulus.

ognized by this MAb might be restricted in its distribution to endothelia. When the antigen was localized at the ultrastructural level by immunoperoxidase, it was found exclusively on the luminal (apical) surfaces of the glomerular

(Figure 1 B) and peritubular capillary endothelium. It was not detected on the basal domain of the endothelial cell membrane facing the basement membrane (Table 1). The absence of labeling of the basal domain was con-

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cipitate a - 105-kd protein from cortical extracts and to deplete DPPIV activity from brush border fractions.1 By immunoperoxidase the antigen has a very similar distribution to that of podocalyxin in the glomerulus (Figure 1 A). It also represents an apical domain marker for the proximal tubule (Table 1) and several other epithelia.1 Within the brush border it is restricted mainly to the microvillar microdomain, and it is absent from the coated pits (Table 1).

Localization of Antigens Present on the Glomerular Epithelium, MAbs 13A, 14A, 20B, and 27A

.... .:

B. Figure 2. Distribution of the 1 3A antigen (A) and bounld 1,3A IgG(B) by immunoelectron microscopy. A: Imninioperoxidase of the 1,3A antigen in the glomeruluiis oJ a normal rat. Reactioni produict is concentrated at the base of the epithelial (Ep)foot processes (arrous) and extends uip to the level of the slit diaphragms (X 16, 500). B: Immiunogold labeling of a glomertultus from a rat sacrificed 5 minuites after in ectioni of 1.3A IgG. The boiund antibodjy has the same distribuition as the antigen (A). Gold particles are seen oier the lamina rara externia of the GBAM (BM) and alonig the plasma membrane at the base of the epithelial (Ep) foot processes. The gold particles ofteni seem to be concentrated in or near filtration slits (arrous). An ultrathin frozeni section was incuibated with rabbit aniti-mouise IgG folloued bJy a 5-n m gold-goat anti-rabbit IgG conjugater

By immunofluorescence the 120-kd antigen recognized by 13A was found in the glomerulus and at the base of distal tubule and several other epithelia.1 By immunoelectron microscopy, this antigen was detected along the basal domain of the podocytes (Figure 2A). Peroxidase reaction product was found in the lamina rara externa extending up to the level of the slit diaphragms, and adhering to the basal surfaces of the foot processes. It could not be detected on the apical (urinary) surfaces of the epithelium. Attempts to localize the antigen by immunogold labeling on ultrathin frozen sections were unsuccessful.

(X529, 000).

firmed by immunogold localization on ultrathin frozen sections (not shown).

Localization of Glomerular Antigens Present on Both the Endothelium and Epithelium, MAbs 5A (Anti-podocalyxin) and 26C (Anti-DPPIV) Monoclonal antibody 5A IgG, as well as 1 A, 1 1 A, and 20A IgGs, were shown to recognize podocalyxin as they immunoprecipitated a -125-kd protein that comigrated with podocalyxin and had an identical distribution to podocalyxin in the adult1 and newborn8 rat kidney: As shown elsewhere,8'10 podocalyxin is an apical domain marker for both the glomerular endothelium and epithelium-ie, it is found on the luminal surface of the glomerular endothelium and the urinary surfaces of the podocytes down to the level of the slit membrane (Table 1), with the podocyte plasma membrane being 5 to 10 times more heavily labeled than that of the endothelial cell. Monoclonal antibody 26C recognizes dipeptidyl peptidase IV based on its localization and on its ability to pre-

Figure 3. Inclirect immunyioperoxidase localization of the 14A anitigeni in the rat glomerulus. Labeling is founid on both the apical domain of the podocyte cell membrane facing the urinary spaces (US) anid on the basal domain of thefootprocesses facing the GBM. Stainiitng is accentuated at the level of the slit diaphragms (arrowheads) between neighboring foot processes. A multivesicular body (m') in the epithelial cjytoplasm (Ep) is also labeled. The endothelium (Eui) is niot stained. Cap,

capillary lumen (X25,000).

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14A IgG precipitated a -150-kd band from cortical extracts and stained podocytes, proximal tubule brush borders, and the endothelium of peritubular, liver, intes-

Effects of Antibodies on Glomerular Permselectivity

tine, and pancreatic capillaries.1 By immunoperoxidase the antigen was found in the glomerulus on all surfacesboth apical and basal-of podocytes, but not on the glomerular endothelium. Interestingly, staining often appeared to be accentuated at slit diaphragms (Figure 3). 20B IgG was shown to recognize gp330, the main antigen of Heymann nephritis.1 By immunoelectron microscopy, the 20B antigen, like gp330,12 was restricted to clathrin-coated pits-the latter are found on both basal and apical domains of the podocyte cell membrane (ie, cell body and the basal and lateral surfaces of the foot processes), and on the intermicrovillar microdomain of the proximal tubule brush border. 27A IgG precipitated weakly a 103-kd glomerular protein. By immunofluorescence it stained only the podocytes.1 By immunoperoxidase labeling at the ultrastructural level, it was evident that the antigen was present within all organelles of the exocytic pathway-ie, cisternae of the endoplasmic reticulum (ER) and Golgi complex (Figure 4A). It was also detected on the apical domain of the podocyte facing the urinary spaces, where it had a patchy distribution, being present in some locations and not others. Vesicular profiles present in the urinary spaces also were heavily labeled on their outer surfaces with peroxidase reaction product. Results of immunogold localization (Figure 4B) confirmed the immunoperoxidase results. These results suggest that 27A recognizes an abundant membrane-bound antigen that is synthesized by glomerular epithelial cells at a high rate, delivered to the cell surface, and eventually shed into the urinary spaces.

The binding of those MAbs tested (5A, 20B, 26C, and 27A) to their glomerular antigens did not significantly increase urinary albumin excretion. The only exception was in the case of one of six rats that received 5 mg 27A IgG where albumin (4 mg/24 hours) was detected, suggesting that binding of 27A IgG can cause albuminuria under some circumstances.

Disappearance of Injected Mouse Antibodies from the Circulation All the MAbs tested that recognize cell-surface components on vascular endothelium (ie, 5A, 23A, and 26C) disappeared rapidly from rat blood, as none of these IgGs were detected in rat sera by the indirect immunofluorescence assay 1 day after injection of up to 5 mg IgG. Disappearance of 5A (podocalyxin) appeared to be the most rapid, as by 1 hour undiluted sera from rats injected with this MAb were negative. By contrast, three MAbs that recognize epithelial antigens, 27A, 1 3A, and 20B (anti-gp330), disappeared more slowly from the circulation. At 1 hour, 27A IgG was detected at a 1 :1000 dilution, after 1 day at 1: 10, and none was found after 10 days. The 1 3A titer remained high, ie, more than 1:1000 at all time intervals from 1 hour to 10 days and 20B IgG decreased slowly from 1:10,000 at 1 hour to 1 :100 at 10 days, as reported earlier for polyclonal

anti-gp330 IgG.13

The Endothelial Marker (23A) Binds Predominantly to the Endothelium By immunofluorescence on semithin (0.5-Ju) frozen sections, binding of 23A was very strong 1 hour after injection (Figure 5A). Bound IgG outlined the peripheral loops of both glomerular and peritubular capillaries. In addition, weak binding was seen to the proximal tubule brush border, suggesting that 23A IgG is filtered. The fate of the bound antibody was not studied after 1 hour. By immunoperoxidase localization, it was evident that within the glomerulus 23A IgG bound exclusively to the luminal (apical) surface of the endothelial plasma membrane. The pattern was very similar to that seen after localization of the antigen in in situ (Figure 1 B), except that instead of being distributed in a uniform layer, the reaction product was present in clumps, suggesting that patching of immune complexes14.15 might have occurred.

The Sites of Binding of Two MAbs (5A and 26C) That Recognize Both Endothelial and Epithelial Membrane Proteins Differ By immunofluorescence, the pattern of binding of antipodocalyxin (5A) was very similar to that described for the endothelial marker, 23A: At 5 to 60 minutes after injection, the bound antibody strongly outlined the peripheral loops of glomerular and peritubular capillaries (Figure 5B). Sometimes mononuclear leukocytes and platelets were found adhering to the endothelium at 1 hour and 1 day. After 1 day, bound 5A IgG was barely detectable by immunofluorescence and was absent after 10 days. This is in keeping with the rapid decline in its serum titer. By immunoperoxidase, the bound mouse IgG was seen to be restricted to the luminal plasma membrane and endothelial fenestrae of glomerular capillaries, where it occurred in clumps or patches. The endothelium often appeared to be swollen at early (5 to 60 minutes) time points (Figure 6A). Often DAB reaction product appeared to have diffused into the inner layers of the glomerular basement membrane (GBM) near heavily reacted endothelial fenestrae. The fact that binding in vivo was largely re-

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AW

B Figure 4. Distribuition of the antigeu recogniized b) MAb 97A in the rat glomerulus b immunoperoxidase localization on a cryostat section (A) and immuniiogold labelinig oni an ultrathin frozent sectioni (B). A: DAB reaction prodtct is associated exclusively with i'isceral glomeruilar epithelial cells (Lp). Ihe re ispatchy surface staining (arrows) of the plasma membranze along the podocyte cell body andidbot processes as well as oni vesicles located ini the uriniary spaces (arroubheads). Cisterntae of the entdoplasmic reticulum (er) and Golgi coniplex (Gc) are also labeled. B: Gold particles are seeni oi'er the entdoplasmic reticulum (er), numerous ivesicles (i'e) in the Golgi regioni (Gc), the Golgi stacks themselves(arrow), a multii'esicular body (mi), and ol theplasma membrale (pm). In the case of the latter, clusters of gold particles are separated by bare regionis. 7he iniset depicts iesicular profiles decorated unith golcdparticles (arrowheads) in the urinaryspaces. Cap, capillary lumeni. A, X 19,000; B, X66, OOO; (inset, X25, 000).

stricted to the endothelium is in sharp contrast to the distribution of podocalyxin in situ, where it is most prominent on the urinary surfaces of podocytes. By immunofluorescence (Figure 5C), staining at 5 to 60 minutes after injection for bound 26C (anti-DPPIV) was weaker, more spotty, and was confined largely to the glo-

merulus. Staining of peritubular capillaries was much weaker and barely detectable. Bound 26C IgG disappeared quickly from glomeruli and was not detected at 1 or 10 days. By immunoelectron microscopy, this MAb was seen to bind very strongly to the luminal (apical) surfaces of the glomerular endothelium (Figures 7A, 8A).

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eQs=^Evf~ isz-tEW:;fm

Immunlofluorescetnce localization on semithin (0. 5 Mm)frozen sectiots of six MAbs in rat glomeruli 5 to 60 minutes after inljectionI. Binldinlg to glomerrular capillaries is seen with all MAbs. 23A (A) and 5A (B) bind strongly to both glomerular (arrowheads) aid peritubular (arrous) capillaries otutliniing their lumina, suggesting binding to the endothelim. 26C(C), 14A (D), and 27A (F) IgGs bind to glomeruilar capillaries in a more interrupted pattern. 14A, and sometimes also 27A bind to the proximal tubuile brush border (PT). 1.3A IgG (E) binds exclusively to the glomerulus, producing a bright, linear stain ing patterni. X500. Figure 5.

in vivo

However, unlike the situation with anti-podocalyxin, 26C IgG also crossed the GBM and bound at lower concentrations to both the basal and urinary (apical) surfaces of the podocyte, where it could be detected by both immunoperoxidase (Figure 7A) and immunogold (Figure 8A) procedures. Dimethylaminoazobenzene reaction product was present in clumps or patches in all these locations (Figure 7A). The binding of 26C IgG to the basal domain of

the epithelium was unexpected, as the antigen could not be reliably localized to these cell surfaces in situ (Figure 1 A). It can be concluded that the behavior of MAbs 5A and 26C, which recognize antigens expressed on the apical surface of the glomerular and extraglomerular endothelium as well as on the apical or urinary surfaces of podocytes, is quite different after injection in vivo. At doses of up to 5 mg, 5A binds predominantly to the apical domain

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Figure 6. Immunszoperoxidase localization of MAbs 5A or anti-podocalyxin (A) and 14A (B) in the rat glomerulus 1 hour after in vivo injection. A: Binidinig ofjanti-podocalyxin is restricted to the luminzal surfce of the glomerular endothelium (En). No binding to the surfaces ofpodocytes is detected, uwhich is in striking conitrast to the stronig staining ofthe podocyteplasma membrane after localizationi oJ podocalyxin in situ.8 B: Binding of 14A IgG is restricted to the jltration slits ofpodocytes, u'hich are cut in a partly grazing section to the lceft (arrous) anid in normial sectionI to the right. It is nIot detected on the enidothelium (En) or bound to the basal sturfaces of the podocytes. A and B, X 19,000.

of the endothelium, whereas 26C binds to the apical domains of both the endothelium and epithelium. Whether these differences in behavior reflect the relative amount of the respective antigen that is present, its accessibility, or some other properties of the antigen or immune complexes remains to be determined.

MAb 14A Binds to the Epithelial Slit Diaphragms At 60 minutes after injection, staining for 14A IgG was found both in the glomerulus and on proximal tubule brush borders, indicating that this IgG is filtered and able

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Figure 7. Immunoperoxidase localization of in4jected MAb 26C (A) and 20B (B) in the rat glomerulus. A: Binding of anti-DPPIV (26C) is detected on the apical surface of the endothelium (En) facing the capillary lumen (Cap) and in the endothelialfenestrae (arrows) 1 hour after injectiont. It is also detected at lower concentration on the uri-

nary surfaces (arrowheads) of podocytes

(Ep) (X 17,500). B: Anti-gp330 IgG (20B) is found in dotlike clumps in coated pits (arrows) at the base ofafootprocess (fi) facing the urinary spaces (US) and in a multivesicular body (mv') in the podocyte cytoplasm 1

day after injection (X34,000). to bind to its antigen in the proximal tubule. The fate of 14A IgG was not followed after 60 minutes. By immunoperoxidase, the pattern of binding of this MAb in the glo-

merulus was unique in that it was found predominantly under the slit diaphragms bridging the filtration slits, which appeared narrowed (Figure 6B). It rarely bound to the

Figure 8. Distribution ofbound 26C(A) and 27A IgG (B) in the glomerulus 1 hour after injection as seen hj' immunogold labeling on ultrathin frozen sections. A: Gold particles are found on the luminal plasma-

lemma of the endothelium (arrows), at the base of the foot processes (arrowheads), andsometimes on the lateral membrane of the foot processes (arrowhead, right) facing the urinary space (US). B: One hour after injection of 27A IgG the filtration slits

(between arrowheads)

are

narrowed,

deepened, and densely labeled with gold particles, demonstrating the presence of bound 27A IgG (A and B, X 73,000).

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Figure 9. Dev,elopmenlt of glomerular subepithelial immutne deposits as seen by immuntoperoxidase labeling after itjection of27A IgG. A: At I hour after inijectiont the slits between the podocyte foot processes are niarrowed or obliterated, and the foot processes appear swolleni ancd distorted. Reactioni product isfounid mainly in the obliterated slits (arrows). B: At 10 daj's after injectionl, small immune deposits have]frmed iutnder the slit diaphragms and under thejbot processes (arrows). The slits are open, and the epithelial foot processes are more normal in shape (A and B, X 15,000).

basal domain of the foot processes facing the GBM. This is somewhat surprising, as the antigen was localized in situ to this domain. In addition, there was strong binding

to the luminal surfaces of the peritubular capillary endothelium, and to basolateral membranes of proximal tubule

cells (not shown). These results confirm the in situ local-

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ization, indicating that 1) the 1 4A antigen is most concentrated near or on the slit diaphragms of the glomerular epithelium, and 2) it is present on the peritubular but not the glomerular capillary endothelium. Up to now, only one other antigen has been localized to the epithelial slit membranes.16

MAB 13A Binds to the Base of the Foot Processes and Persists Up to 10 Days 13A IgG bound very rapidly and selectively to glomeruli, yielding a strong, linear, basement membranelike signal (Figure 5E) by 5 minutes after injection. By immunofluorescence, there was no major qualitative or quantitative change in the signal up to 10 days. By immunogold labeling of cryostat sections, the bound IgG was detected over the lamina rara externa of the GBM (Figure 2B) in the same location as the antigen was detected (compare with Figure 2A). It was impossible to discern whether the antigen was associated with the basal surface of the epithelial cell membrane or with components of the basement membrane itself.

Monoclonal Anti-gp330 IgG Binds to Epithelial Coated Pits But Does Not Lead to Formation of Immune Deposits After injection of MAb 20B, bound anti-gp330 IgG was detected by immunofluorescence in a granular pattern in glomeruli and in proximal tubule brush borders at all time points.1 Its detection on the proximal tubule brush border indicates that this MAb also crosses the glomerular capillary wall and binds to its antigen in the brush border. When the bound antibody was localized by immunoperoxidase (Figure 7B), it was found in small dot-like aggregates in coated pits located at the base or on the sides of the foot processes-ie, in exactly the same locations as the antigen. After longer periods (1 or 10 days), the IgG was detected within multivesicular bodies in the podocyte cytoplasm. These observations suggest that monoclonal anti-gp330 IgG penetrates the GBM, passes into the urinary spaces, and binds to its antigen in coated pits on both the basal and urinary surfaces of the epithelium while in transit. Thereafter, at least part of the bound IgG is taken up into podocytes by endocytosis and disposed of via the lysosomal system.

27A IgG Forms Subepithelial Immune Deposits At early intervals after injection (5 to 60 minutes), 27A IgG (Figure 5F) bound to glomeruli in a linear pattern. This pat-

tern contrasts sharply with the mostly intracellular location of the antigen as seen by indirect immunofluorescence' and immunoelectron microscopy (Figures 3A, B) in normal glomeruli. At 1 day, the distribution of the IgG had changed to a more granular pattern, which was still demonstrable, but weaker, after 10 days. When the distribution of the bound 27A IgG was studied by immunoelectron microscopy in rats killed at 5 to 60 minutes after injection, it was localized at the base of the foot processes and in filtration slits under the slit membranes (Figures 8B, 9A). Although the antigen could not be reliably detected on the basal domain of the podocyte by immunoperoxidase or immunogold labeling in situ, the rapidity (less than 5 minutes) with which the antibody bound to the basal surface of the podocyte membrane suggests that the antigen is normally present in this location (Table 1). Antibody binding led to a striking swelling of the foot processes and obliteration of many of the filtration slits (Figures 8B, 9A). After 1 day these changes were less prominent, and the deposited IgG was located mainly under the slit diaphragms, which explains the change from a linear to a granular immunofluorescence staining pattern. At 10 days, glomerular morphology had returned to normal, but the subepithelial immune deposits persisted (Figure 9B). The kidneys of rats injected with 27A ascites depleted of 27A IgG and those injected with control mouse IgG were negative by immunofluorescence and immunoperoxidase (not shown).

Discussion In this study we have injected MAbs raised against detergent solubilized, non-denatured glomerular proteins into normal rats to gain insights into the factors that affect the deposition of immune complexes in glomeruli. In particular, we were interested in determining the accessibility of antigens located at different sites in the glomerular capillary wall to circulating antibodies and the effects of the location of the antigen on the fate of the immune complexes thus formed. Toward this end, we injected antibodies that recognize exclusively an endothelial cell-surface antigen, those that recognize both endothelial and epithelial glomerular antigens, and those that recognize only epithelial antigens. The main results obtained were the following: 1) Each of the antibodies bound to its glomerular antigen in vivo regardless of the location of the latter; 2) the behavior of the antibodies over time differed according to the antigen's location: IgGs that recognize endothelial cell-surface antigens were rapidly cleared from the blood and rapidly disappeared from the glomerulus, whereas those that recognize exclusively epithelial antigens remained in the circulation for longer periods and also persisted in glomeruli; 3) the behavior of those that

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recognized antigens expressed on both endothelial cells and podocytes was variable: one (anti-podocalyxin) bound predominantly to the apical or luminal domain of the endothelium, whereas another (anti-DPPIV) bound to the apical domains of both the endothelium and epithelium; 4) one MAb (14A) that recognized an antigen present on both the apical and basal domains of the podocyte bound exclusively to the epithelial slit membranes after in vivo injection; and 5) antibody charge and isotype were not major factors in influencing the ability of the MAbs to cross the glomerular capillary wall and bind to the glomerular and tubular epithelial antigens.

The Luminal (Apical) Membrane of the Glomerular Endothelium Is a Primary Antibody-binding Site Monoclonal antibodies that were known or suspected to recognize endothelial cell-surface components bound in vivo to the luminal endothelial cell surface. This was the case for MAb 23A, which exclusively recognizes a novel 350-kd antigen of unknown function that is widely distributed on endothelia of all blood vessels in all organs studied.1 It was also true for those that recognize antigens present on both the endothelium and the podocytes-ie, 5A, which recognizes podocalyxin, and 26C, which recognizes DPPIV. However other than the fact that they all bound to the endothelium, the behavior of these MAbs was quite different: anti-podocalyxin rapidly disappeared from the blood and its binding was restricted to the endothelium. Apparently, the number of antigenic sites available on the luminal surface of the glomerular and peritubular capillary endothelium and the endothelia throughout the rest of the vasculature were sufficient to rapidly (within 1 hour) deplete this MAb from the circulation, so that none reached sites on the podocytes when amounts of antibody up to 5 mg were injected. Preliminary experiments suggest that with doses of IgG >5 mg, the endothelial binding capacity is exceeded, as there is some epithelial

staining. The situation was quite different with 26C (anti-DPPIV) whose antigen, like podocalyxin, is widely distributed on endothelial cells. Curiously 26C IgG crossed the glomerular capillary and bound to the entire podocyte cell membrane, including the domain facing the glomerular basement membrane. A special case is represented by 14A, which is widely distributed on the luminal domain of capillary endothelia, including that of peritubular capillaries, and is present on the basal and apical domain of the podocyte, but is not detectable on the glomerular endothelium. Surprisingly, after intravenous injection, the MAb bound predominantly to filtration slits at the level of the slit

diaphragms.

The difference in the behavior of anti-podocalyxin and 26C IgG (anti-DPPIV) is surprising and unexpected. It suggests that the amount of antibody that bound to the endothelium in vivo in the case of 26C was much less, perhaps because of its lower avidity or to the presence of fewer available epitopes. Differences in charge cannot be invoked to explain the difference in behavior of the MAbs, as their net charges are similar (pl = -6.5 to 6.8). The behavior of our anti-DPPIV was similar to that of an antiDPPIV MAb raised by Ronco et al.17 The rapid disappearance of the MAbs' antigen-antibody complexes bound to the endothelial cell surface is most likely due to the fact that they are shed from the cell surface into the circulation and are eventually cleared by phagocytic cells in the spleen and other sites, as described for antibodies against angiotensin-converting en-

zyme.15

Persistence of Anti-epithelial Antibodies in the Glomerulus In contrast to antibodies that recognize endothelial antigens, those that recognize exclusively epithelial antigens persisted in the circulation and remained bound as long as there was detectable IgG in the circulation. Perhaps the most interesting findings were those obtained with MAbs 1 3A and 27A, both of which, based on the sites of antibody binding, appear to recognize antigens located on the basal domain of the foot processes. 13A IgG persisted in the circulation at high concentration up to 10 days and remained bound to the glomerulus up to 10 days, the longest interval studied. By immunoelectron microscopy, it could be seen that this IgG bound uniformly to the lamina rara externa of the GBM. It is not clear whether the antigen recognized by 1 3A is a cell-surface component of the podocyte membrane at the base of the foot processes or a basement membrane component, as it is impossible to tell where one leaves off and the other begins. However, the fact that the linear pattern persists and the complexes do not rearrange and take on a granular pattern is in keeping with the behavior of complexes formed by antibodies to structural components that are fixed to the GBM (eg, heparan sulfate proteoglycans).9 The presence of the antigen at the basal aspects of several other epithelia (eg, those of the distal tubule, bronchus, and choroid plexus) and its absence from the lamina rara interna suggest that the antigen is epithelial in origin. Our ability to immunoprecipitate the antigen after metabolic labeling of cortical slices in vitro1 demonstrates that it is synthesized by renal cells.

Monoclonal Anti-gp330 IgG Does Not Form

Subepithelial Immune Deposits We have previously established12,13 that after injection of polyclonal anti-gp330 IgG, subepithelial immune deposits

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are formed by the following sequence of events. The circulating IgG binds to its antigen located in coated pits at the base of the epithelial foot processes; the immune complexes thus formed are shed into the lamina rara externa and become rapidly cross-linked to the GBM. As a result they become firmly attached to the GBM,13 and thereafter grow in size by a repetition of this sequence of events. Monoclonal anti-gp330 IgG also binds to its antigen in coated pits after injection but, as reported earlier,18'19 no immune deposits are formed. Small aggregates that represent immune complexes are seen in coated pits; however they do not appear to become cross-linked to the GBM, as no subepithelial deposits were found by electron microscopy and the fine granular deposits seen by immunofluorescence disappeared rapidly after the antibodies disappeared from the circulation. At least some of the IgG was taken up by endocytosis and presumably disposed of via lysosomes by the podocytes. As already suggested,18 the simplest explanation for this difference in behavior between monoclonal and polyclonal anti-gp330 IgGs is that recognition of multiple epitopes is required for lattice formation and the development of immune deposits.

MAb 27A, a Specific Marker for Podocytes, Forms Subepithelial Immune Deposits One of the most intriguing antibodies is MAb 27A, which recognizes an antigen that is restricted exclusively to the podocyte, because it was not found in any other organ tested.' This IgG weakly precipitated a 103-kd protein from biosynthetically labeled glomeruli, but recent results2o suggest that the main antigen recognized by these antibodies is a carbohydrate epitope of a glycolipid and this epitope may be shared with one or more proteins. By immunoelectron microscopy, we detected the antigen in biosynthetic compartments (rough ER and Golgi) and on the plasma membrane of podocytes, and in vesicular profiles in the urinary spaces. Its localization and abundance demonstrate that it is synthesized by podocytes-probably at a high rate-and delivered to the plasma membrane, where it is somehow shed from the cell surface into the urinary spaces. When 27A IgG was injected in vivo, it bound rapidly (within 5 minutes) to rat glomeruli and caused swelling of the foot processes and narrowing of the filtration slits, suggesting that the 27A antigen is located at the base and along the sides of the foot processes. The findings were reminiscent of those obtained after neutralization of the surface charge of the podocytes by infusion of polycations.2' It is of interest that only this antibody resulted in activation of complement in vitro,' and one rat (of six injected) developed proteinuria. Interestingly, by 1 day af-

ter the injection, immune deposits similar to those seen in passive Heymann nephritis after binding polyclonal antigp330 IgG13 appeared under the slit diaphragms and foot processes. Although the immune deposits did not grow in size, they remained for at least 10 days, ie, until the injected IgG disappeared from the circulation. Apparently these immune complexes did not become cross-linked to the GBM. However their persistence even for 10 days is unique, as generally binding of monoclonal antibodies, including those to gp330, does not lead to formation of recognizable immune deposits. In summary we have used MAbs to glomerular components to determine the distribution of the antigens they recognize and to compare the pattern of antigen localization in situ with that of the antibody bound in vivo. The seven antibodies studied proved to have different specificities for different glomerular cell types and for different podocyte plasmalemmal domains. Several of the antigens, ie, those detected by MAb 23A and 27A appear to be new and exclusive markers for the endothelium and podocyte, respectively, and may prove to be useful for the identification of these cell types. Of interest was the fact that after in vivo injection, the MAbs selectively bound to different cell types; in the case of the podocyte, they bound to specific microdomains of the cell membrane, and the sites of binding did not always coincide with the distribution of the antigen in situ. These results stress the important connection between the location of the antigens and the accessibility of their antigenic determinants in the deposition of immune complexes in the glomerular capillary wall.

References 1. Miettinen A, Dekan G, Farquhar MG: Production of monoclonal antibodies against membrane proteins of rat glomerular podocytes and endothelial cells. Am J Pathol 1990, 137:

929-944 2. Slot JW, Geuze HJ: A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 1985, 38: 87-93 3. Campbell AM: Monoclonal antibody technology. Laboratory Techniques in Biochemistry and Molecular Biology. Vol 13. New York, Elsevier, 1984, pp 176-178 4. McLean W, Nakane PF: Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J Histochem Cytochem 1974, 22:1077-1083 5. Brown WJ, Farquhar MG: The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae. Cell 1984, 36:295-307 6. Tokuyasu KT: Use of polyvinylpyrrolidone and polyvinyl alcohol for cyroultramicrotomy. Histochem J 1989, 21:163-171 7. Tokuyasu KT: Application of cryomicrotomy to immunocytochemistry. J Microsc 1986,143:139-149

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8. 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 9. Miettinen A, Stow JL, Mentone S, Farquhar MG: Antibodies to basement membrane heparan sulfate proteoglycans bind to the laminae rarae of the glomerular basement membrane (GBM) and induce subepithelial GBM thickening. J Exp Med 1986,163:1064-1084 10. 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 11. Kerjaschki D, Sawada H, Farquhar MG: Immunoelectron microscopy in kidney research: Some contributions and limitations. Kidney Int 1986, 30:229-245 12. 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 13. Kerjaschki D, Miettinen A, Farquhar MG: Initial events in the formation of immune deposits in passive Heymann nephritis: Gp330-antigp330 immune complexes form in epithelial coated pits and rapidly become attached to the GBM. J Exp Med 1987,166:109-128 14. Andres G, Brentjens JR, Caldwell PRB, Camussi G, Matsuo S: Formation of immune deposits and disease. Lab Invest 1986, 55:510-520 15. Matsuo S, Fukatsu A, Taub ML, Caldwell PRB, Brentjens JR, Andres G: Glomerulonephritis induced in the rabbit by antiendothelial antibodies. J Clin Invest 1987, 79:1798-1811

16. 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 17. Ronco P, Allegri L, Melcion C, Pirotsky E, Appay M-D, Bariety J, Pontillon F, Verroust P: A monoclonal antibody to brush border and passive Heymann nephritis. Clin Exp Immunol 1984, 55:319-332 18. Allegri L, Brianti E, Chatelet F, Manara GC, Ronco P, Verroust P: Polyvalent antigen-antibody interactions are required for the formation of electron-dense immune deposits in passive Heymann nephritis. Am J Pathol 1986,126:1-6 19. Ronco P, Neale TJ, Wilson CB, Galceran M, Verroust P: An immunopathologic study of a 330-kd protein defined by monoclonal antibodies and reactive with anti-RTEa5 antibodies and kidney eluates from active Heymann nephritis. J Immunol 1986,136:125-130 20. Miettinen A, Reivinen J, Holthofer H, Laitinen J, Rauvala H, Dekan G, Farquhar MG: Monoclonal antibodies (MAB) to nephritogenic sialoglycolipids of rat glomerular podocytes. Kidney Int (In press) 21. Seiler MW, Rennke HG, Venkatachalam MA, Cotran RS: Pathogenesis of polycation-induced alterations ("fusion") of glomerular epithelium. Lab Invest 1977, 36:48-61

Acknowledgment The authors thank Sue Ann Mentone for the technical assistance in the immunoperoxidase procedures.

Binding of monoclonal antibodies to glomerular endothelium, slit membranes, and epithelium after in vivo injection. Localization of antigens and bound IgGs by immunoelectron microscopy.

The antigens recognized by seven monoclonal antibodies (MAbs) raised against rat glomerular proteins were localized, and the sites of binding of the M...
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