AmericanJournal of Pathology, Vol. 137, No. 1,July 1990 Copyright© American Association ofPathologists
Loss and Rearrangement of Glomerular Basement Membrane Laminin During Acute Nephrotoxic Nephritis in the Rat
Vijittra Leardkamolkarn,* David J. Salant,t and Dale R. Abrahamson*t From the Department of Cell Biology andAnatomy,* and the Nephrology Research and Training Center,* University ofAlabama at Birmingham, Birmingham, Alabama; and Evans Memorial Department of Clinical Research, and the Department ofMedicine, t Boston University Medical Center, Boston, Massachusetts
Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils andproteinuria (nephrotoxic nephritis orNTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experimentspresented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined bypostfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces ofdetaching endothelial cells, demonstrating the removal of at least some laminin from the
GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN. (Am JPathol 1990, 13 7:187-198)
Nephrotoxic nephritis (NTN) is a classic experimental model of human glomerulonephritis that is induced in rats by intravenous injections of serum from sheep or rabbits immunized with rat glomerular basement membrane (GBM) preparations (reviewed in Wilson and Dixon'). The experimental disease can be divided into two stages. First, an acute, heterologous phase is caused by the binding of injected sheep or rabbit anti-GBM antibody to the GBM. Injury during this phase may be mediated by the activation of host complement, infiltration of neutrophils into glomeruli, and/or by the direct action of antibody.1-3 Second, a chronic, autologous phase begins approximately 5 days later when an immune response against the injected antibody develops and host IgG binds to the GBM-fixed heterologous antibody. This leads to an infiltration of monocytes that contribute to glomerular damage.' 2 In both stages, GBM barrier function is lost and proteinuria occurs. Despite considerable research on NTN in the past 60 years, the specific GBM components to which nephrotoxic antibodies (NTAbs) bind are still unclarified. In addition, although an injurious role for compleAccepted for publication February 16, 1990. Funded by the National Institutes of Health (DK 30932, DK 34972, and DK 39258) and the American Heart Association. Drs. Salant and Abrahamson are Established Investigators of the American Heart Association. Address reprint requests to Dale R. Abrahamson, PhD, Department of Cell Biology and Anatomy, University of Alabama at Birmingham, UAB Station, Box 302, Birmingham, AL 35294.
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ment'13 and neutrophil-derived reactive oxygen species (reviewed in Shah4) and proteases1' 5-8 has been demonstrated, precisely how the GBM is affected during this pathologic condition is not known. In the studies presented here, we examined the ultrastructural distribution of immunoreactive laminin in the GBM as an indicator of possible alterations in GBM composition and architecture in acute NTN. For one approach, we processed normal and nephritic GBM for posffixation, postembedding immunogold electron microscopy. We also labeled the GBM in vivo, by intravenously injecting anti-laminin, both before and after injections of NTAbs, and performed immunoperoxidase and immunogold electron microscopy. Our evidence from all approaches indicates a loss and redistribution of GBM laminin during NTN.
Materials and Methods Proteins and Reagents Laminin was purified from the murine Englebreth-HolmSwarm (EHS) tumor by a modification of the method originally developed by Timpl et al9 and characterized as described previously.'1 Sheep and rabbit anti-laminin IgGs (anti-Lam) were purified by affinity chromatography as before.9'1112 Nephrotoxic antibodies were collected from sheep that had been immunized with whole rat glomeruli.13 Horseradish peroxidase (HRP, type VI) was purchased from Sigma Chemical Co. (St. Louis, MO) and conjugated directly to affinity-purified sheep and rabbit anti-Lam IgGs using methods described by Nakane and Kawaoi.14 Horseradish peroxidase-conjugated sheep anti-rabbit and rabbit anti-sheep IgGs and fluorochromeconjugated antibodies against sheep IgG, rabbit IgG and rat C3 were obtained from Organon Teknika-Cappel, West Chester, PA. Anti-rabbit IgG conjugated to 10-nm diameter colloidal gold was purchased from Janssen Life Science Products (Piscataway, NJ).
Inhibition ELISAs Enzyme-linked immunosorbent assay plates were coated with purified EHS tumor laminin or bovine serum albumin (BSA) (20 ug/ml), overnight at 4°C, washed with phosphate-buffered saline (PBS), and blocked with 5% horse serum in PBS for 1 hour at room temperature. Serial dilutions of sheep NTAbs were plated first and incubated at room temperature for 1 hour, and the plates were washed extensively before reincubation with rabbit anti-Lam antibodies for another 1 hour. The plates were washed and
rabbit anti-sheep IgG-HRP or sheep anti-rabbit IgG-HRP, diluted 1:1000, was added for 1 hour. In other cases, sheep anti-Lam-HRP was added directly to plates that had been preincubated with sheep NTAbs. Plates were washed extensively with PBS and then developed for peroxidase activity.15 Control experiments included incubating laminin- or BSA-coated plates with sheep NTAbs or sheep or rabbit anti-Lam alone and reincubation with appropriate secondary antibody-HRP conjugates.
Animal Experiments The goal of the overall experimental design was to examine the distribution of laminin within the GBM during acute stages of experimental anti-GBM disease using immunofluorescence, immunoperoxidase, and immunogold microscopy. Male Sprague-Dawley rats weighing 120 to 150 g were used throughout. For fluorescence microscopy, rats received dual injections of (1) sheep NTAbs (0.2 mg/ rat) followed 3 hours later by (2) affinity-purified rabbit antiLam (2 mg/rat) via saphenous veins. Kidney tissues were examined 1 hour after the second injection by immunofluorescence, as described below. For immunoperoxidase, five rats received injections of anti-Lam-HRP 1 hour before the administration of NTAbs. Kidneys were then fixed 3 hours later and processed for peroxidase histochemistry. A reverse labeling procedure was also carried out in which NTAbs were injected into five rats 3 hours before the administration of anti-Lam-HRP. In these cases, tissues were fixed 1 hour after the second injection. Anti-Lam-HRP was injected into normal rats as controls. A similar injection protocol was followed for colloidal gold studies. Three rats that had received NTAbs 3 hours earlier received injections of unconjugated rabbit antiLam. Kidneys were lightly fixed, embedded in Lowicryl resin, and sections were labeled with anti-rabbit IgG-colloidal gold, as described below. In addition, three rats re-
ceived anti-Lam 1 hour before receiving NTAbs. Control rats received anti-Lam alone 1 hour and 4 hours before they were killed, respectively (one rat each). For indirect labeling experiments, kidneys from one normal rat and two rats that had received NTAbs were lightly fixed and embedded in Lowicryl. Finally, kidneys from 3 normal rats, 3 nephritic rats, and 3 anti-Lam-injected rats were fixed in Karnovsky's fixative and processed through osmium and epoxy resin for routine morphology.
Immunofluorescence Microscopy Unfixed kidneys from experimental animals were rapidly frozen in 2-methyl butane cooled in a dry ice-acetone bath
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and sectioned at -20°C in a cryostat. Sections, 4 gm thick, were air dried, treated with fluorochrome conjugated-anti-sheep IgG, anti-rabbit IgG, or anti-rat C3 for 5 minutes, washed with PBS, pH 7.4, and mounted in glycerol:PBS (1:1). Slides were examined in a Leitz Ortholux 11 or Orthoplan photomicroscope (E. Leitz Inc., Rockleigh, NJ) equipped for epifluorescence using appropriate filter cubes.
Immunoperoxidase Electron Microscopy Kidney tissues from anti-Lam-HRP-injected rats were fixed in 1.6% paraformaldehyde and 3% glutaraldehyde in 0.1 mol/l (molar) phosphate buffer, pH 7.416 for 2 hours, and washed in 0.1 mol/l buffer containing 35 mg/ml sucrose. Sections, 40 gm thick, were obtained with a Vibratome (Ted Pella, Inc; Redding, CA) and processed for peroxidase histochemistry in 0.01% hydrogen peroxide and 0.05% diaminobenzidine,17 as previously described.18 Sections were postfixed in 2% osmium before embedding in epoxy resin. Ultrathin sections were stained with lead citrate for 2 minutes and examined by electron microscopy in a JEOL 100 CX (JEOL USA Inc., Peabody, MA) or Hitachi H 7000 instrument (Hitachi Scientific Instruments, Mountain View, CA).
Postembedding Immunogold Electron Microscopy Kidneys from nephritic, anti-Lam-injected, and normal uninjected rats were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 mol/l phosphate buffer for 2 hours at 40C and washed in 0.1 mol/l phosphate buffer containing 35 mg/ml sucrose. Tissues were processed and embedded in Lowicryl K4M resin (Polysciences, Warrington, PA) at 40C using methods previously published.18 19 Thin sections, '70 nm thick, were picked up on uncoated 400 mesh nickel grids and blocked with ammonium chloride and bovine serum albumin as before.20 Sections were labeled as follows: a) Sequential labeling. Ultrathin Lowicryl sections from normal or nephritic rats that had not received antiLam injections were labeled first with affinity-purified rabbit anti-Lam (15 ,ug/ml) for 18 hours at 40C. The grids were washed thoroughly with PBS and then reincubated with anti-rabbit IgG-colloidal gold for 3 hours at room temperature. Sections were then washed, briefly stained with 1% uranyl acetate and lead citrate, and examined by electron microscopy. b) Direct labeling. Lowicryl sections from rats that had received injections of anti-Lam were labeled directly with goat anti-rabbit IgG-colloidal gold (1:3 dilution) for 72 hours at 40C. Grids were washed, stained, and examined.
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Figure 1. Graph showing the relative amount of bound antibodies on laminin-coated ELISA plates. Sheep nephrotoxic antibodies (S NTAbs) have little binding activity compared to sheep or rabbit anti-laminin antibodies (S or Rb anti-Lam). Preincubation ofplates with S NTAbs also does not inhibit the binding of S or Rb anti-Lam. Each bar represents the mean of three measurements, +/- the standard error.
Morphometry Randomly photographed fields of Lowicryl sections from normal and nephritic glomeruli were printed and areas of cross-sectioned GBM were measured using a SigmaScan digitizing tablet (Jandel Scientific, Sausalito, CA). Gold particles were counted manually. In addition, perpendicular distances across the GBM, as measured between the basal endothelial cell membranes and the bases of the podocyte foot processes, were determined. Average widths of GBM, lengths of basal podocyte cell surface, and widths of slit pores in normal and acutely nephritic kidneys were also measured in sections from routinely processed and epoxy-embedded tissues. Results are expressed as mean ± the standard error. Statistical analyses were carried out using Student's t-test.
Results Inhibition ELISA To determine whether the sheep NTAbs contained significant amounts of anti-Lam, an inhibition ELISA was car-
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Figure 2. Immunofluorescence micrographs of glomeruli from a rat that received a dual injection of sheep NTAbs and rabbit anti-Lam. Cryostat sections were stained with anti-sheep lgGfluorescein (a), anti-rabbit IgG rhodamine (b), at. 4 anti-rat C3 (c). NTAbs and anti-Lam bind in linearpatterns to the GBM (a and b) and there is abundant deposition ofcomplement (c). Separate immunofluorescence control experiments showed that the anti-rabbit IgG did not cross-react with the sheep NTAbs. a, b, c X550.
ried out. Figure 1 shows that there was little cross-reactivity between NTAbs and anti-Lam. When laminin-coated plates were incubated with sheep NTAbs, only very low
anti-laminin activities were detected as compared to those seen with sheep or rabbit anti-Lam (Figure 1). Furthermore, preincubation of laminin-coated plates with NTAbs did not reduce the binding of sheep or rabbit antiLam (Figure 1).
Immunofluorescence Light Microscopy Both nephrotoxic and anti-laminin antibodies bound to the GBM in the same linear pattern (Figures 2a and b). However, the presence of NTAbs on the GBM did not block the binding of anti-Lam in vivo (Figure 2b). Unlike what has been seen in previous studies on the failure of antiLam to activate complement in vivo,10,'1 here the binding of NTAbs initiated host complement deposition in glomeruli (Figure 2c).
Immunoperoxidase Electron Microscopy As seen previously,10'1121 the injection of anti-Lam-HRP into otherwise normal rats resulted in a specific and even deposition of peroxidase reaction product across the full width of the GBM in all capillary loops. The binding of injected anti-Lam to the GBM did not lead to an acute inflammatory response within glomeruli, however, and normal glomerular architecture was observed for up to 2 weeks after injection.10 1121 In contrast, when NTAbs were injected into rats that had received anti-Lam-HRP 1 hour earlier, there was a rapid and massive influx of neutrophils into glomeruli (Figure 3). The neutrophil infiltrates were accompanied by widespread endothelial cell detachment from the GBM and podocyte foot process effacement (Figure 3). Also unlike what was seen in normal rats, there was uneven laminin immunoreactivity within the GBM in many capillary loops of acutely nephritic rats (Figures 3 and 4). Although reaction product was present across the full thickness of the nephritic GBM, HRP was often more pronounced in the lamina rara interna (Figures 3 and 4), especially in areas of endothelial cell detachment (Figure 5). In regions in which neutrophils were closely apposed or adherent to denuded GBM, however, there were no apparent breaks or discontinuities in binding of anti-LamHRP (Figures 3 and 4). Where endothelial cells were detaching from the GBM, clumps of some peroxidase-positive, basement membranelike material remained associated with the basal endothelial cell surface (Figure 5), as if basement membrane elements were being removed from the GBM during cell detachment. An identical set of results was obtained in rats that received NTAbs 3 hours before in vivo labeling with anti-Lam-HRP.
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Figure 3. Electron micrograph ofglomerulusfrom a rat that received an intravenous injection of rabbit anti-Lam-HRP 1 hour before receiving NTAbs. Tissue was.fixed 3 hours later. Several neutrophils (PMN) are present within capillary loops and widespread loss of the endothelium has occurred. Damage to epithelialpodocytes (Ep) is also indicated by foot process widening (arrowheads) and surface blebbing (*). Although HRP reaction product extends across the GBM, the most intense labeling frequently occurs on the inner layer (arrows). No abrupt discontinuities in labeling are observed along lengths of the GBM, however. RBC, red blood cell. X 11,500.
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_5, Figure 4. Higher magnification electron micrograph ofglomerular capillary loop from a nephritic rat labeled with anti-Lam-HRP, as in Figure 3. HRP reaction productpersists beneath a neutrophil (PMN) lying close to the GBM (arrows) and is more concentrated in the lamina rara interna. Note foot process effacement in the epithelial layer (Ep). X22,500. Figure 5. Electron micrograph of glomerular capillary wallfrom a rat given NTAbs 3 hours before anti-Lam-HRP. Some laminin immunoreactivity is present on the basal surfaces (arrows) ofendothelial cells (En) detachingfrom the GBM. Intense HRP reaction product is also present in the lamina rara interna of the GBM (arrowheads). CL, capillary lumen; Ep, epithelialpodocytefootprocesses. X27,500.
Immunogold Electron Microscopy and Morphometry To evaluate further any changes in laminin distribution, Lowicryl sections from normal and nephritic rats were se-
quentially labeled with rabbit anti-Lam and anti-rabbit IgGcolloidal gold (Figure 6). In addition, sections from normal and nephritic rats that had received intravenous injections of rabbit anti-Lam were labeled directly with immunogold (Figure 7). With both labeling protocols there appeared to
Laminin Redistribution During Nephritis 193 AJPJuly 1990, Vol. 13 7, No. 1
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Figure 6. Electron micrographs offormaldebyde-fixed, Lowicryl-embedded kidney sections from a normal rat (a) or a rat given NTAbs (b). Sections were labeled sequentially with rabbit anti-Lam and anti-rabbit IgG-colloidal gold, as described in Materials and Methods. A marked reduction in labeling is observed in the nephritic GBM. Note also the increased distance between the endothelial cells and podocytes in the nephritic glomerulus (bars measure 0. 18g1m in (a) and 0.3 ,um in (b). CL, capillary lumen; En, endothelium; Ep, foot processes of epithelium. a, b X90,000.
be a reduction in colloidal gold binding to the GBM of acutely nephritic rats when compared with normal (Figure 6) or anti-Lam-injected controls (Figure 7). To quantitate these observations, GBM-bound gold particles were counted, expressed as the number of particles bound/ JAm,2 and compared between groups. Significantly less gold bound to nephritic GBMs than to those of normal rats or those injected with anti-Lam (Figure 8). In addition, as shown in Figure 8, the same amount of rabbit IgG was detected in rats that received anti-Lam 1 hour before NTAbs, as in those labeled after NTAb injection. These results were consistent with our inhibition ELISA and immunofluorescence findings presented earlier, indicating that the bound NTAbs did not inhibit the binding of rabbit anti-Lam to the GBM in vivo.
Several additional morphometric measurements were taken from the same Lowicryl sections used for particle counting as well as from sections from routinely fixed and epoxy-embedded kidneys. In rats that received NTAbs, the width of the GBM averaged 0.28 ,m in Lowicryl sections and 0.18 gm in epoxy sections (Figure 9). The marked difference between the Lowicryl- and epoxy-embedded tissue was likely due to the comparative instability of Lowicryl in the electron beam as well as to the weaker fixation conditions used for postembedding immunolabeling. Nevertheless, the values from rats that received NTAbs were significantly greater (P < 0.001) than GBM widths from either uninjected or anti-Lam-injected rats, regardless of the embedding media (Figure 9). Interestingly, the GBMs of anti-Lam-injected rats, although much
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Figure 7. Electron micrographs of Lowicryl sections ofkidneysfrom rats injected with (a) rabbit anti-Lam alone, (b) rabbit antiLam followed by NTAbs, and (c) NTAbs followed by rabbit anti-Lam. Sections were labeled directly with anti-rabbit IgG-colloidal gold. Less gold binds in (b) and (c) than in (a). CL, capillary lumen; En, endothelium; Ep, footprocesses ofpodocytes. a, b, c X90,000.
Laminin Redistribution During Nephritis
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Our posffixation labeling as well as in vivo prefixation immunolabeling results clearly indicated changes in GBM laminin immunoreactivities during acute stages of antiGBM disease in the rat. Whereas the reduction in laminin immunoreactivity might have been due to proteolytic degradation of laminin by infiltrating neutrophils, we believe that another reason for the observed decrease was the
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Figure 8. Histograms comparing relative laminin immunoreactivities within the GBM. Graph on left reflects measurements taken from rats that receivedNTAbs andfrom normal controls. Lowicryl sections were labeled sequentially with rabbit antiLam and anti-rabbit IgG-colloidal gold. Graph on right reflects measurements taken from rats that had received NTAbs followed by rabbit anti-Lam intravenously, anti-Lam followed by
significantly greater distance across the GBM between endothelial cells and podocytes. This widening could have been caused by the detachment of glomerular endothelial cells, loosening of the GBM meshwork, and also possibly to the effacement of podocyte foot processes. lThe end result was a redistribution of laminin over a greatly expanded matrix. We did not detect significant levels of anti-laminin activity in the NTAbs used in this study. Correspondingly, previous efforts to simulate the heterologous phase of
NTN using antibodies directed specifically against individual GBM components, such as laminin,'" 1,21-23 collagen type IV,22-3 or heparan sulfate proteoglycans24-5 generally
NTAbs, or anti-Lam alone. In these instances, sections were labeled directly with anti-rabbit IgG-colloidal gold. In all cases,
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narrower than those that received NTAbs, were wider than in normal animals (Figure 9). In contrast, the ratio of total basal foot process surface/unit length GBM did not differ between rats that received anti-Lam and normal rats and averaged 0.83 in epoxy sections (Figure 10). However, this ratio was much greater (P < 0.001) in nephritic rats, reflecting the effacement of foot processes (Figure 10). There was also a corresponding decrease (P
Loss and Rearrangement of Glomerular Basement Membrane Laminin During Acute Nephrotoxic Nephritis in the Rat
Vijittra Leardkamolkarn,* David J. Salant,t and Dale R. Abrahamson*t From the Department of Cell Biology andAnatomy,* and the Nephrology Research and Training Center,* University ofAlabama at Birmingham, Birmingham, Alabama; and Evans Memorial Department of Clinical Research, and the Department ofMedicine, t Boston University Medical Center, Boston, Massachusetts
Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils andproteinuria (nephrotoxic nephritis orNTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experimentspresented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined bypostfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces ofdetaching endothelial cells, demonstrating the removal of at least some laminin from the
GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN. (Am JPathol 1990, 13 7:187-198)
Nephrotoxic nephritis (NTN) is a classic experimental model of human glomerulonephritis that is induced in rats by intravenous injections of serum from sheep or rabbits immunized with rat glomerular basement membrane (GBM) preparations (reviewed in Wilson and Dixon'). The experimental disease can be divided into two stages. First, an acute, heterologous phase is caused by the binding of injected sheep or rabbit anti-GBM antibody to the GBM. Injury during this phase may be mediated by the activation of host complement, infiltration of neutrophils into glomeruli, and/or by the direct action of antibody.1-3 Second, a chronic, autologous phase begins approximately 5 days later when an immune response against the injected antibody develops and host IgG binds to the GBM-fixed heterologous antibody. This leads to an infiltration of monocytes that contribute to glomerular damage.' 2 In both stages, GBM barrier function is lost and proteinuria occurs. Despite considerable research on NTN in the past 60 years, the specific GBM components to which nephrotoxic antibodies (NTAbs) bind are still unclarified. In addition, although an injurious role for compleAccepted for publication February 16, 1990. Funded by the National Institutes of Health (DK 30932, DK 34972, and DK 39258) and the American Heart Association. Drs. Salant and Abrahamson are Established Investigators of the American Heart Association. Address reprint requests to Dale R. Abrahamson, PhD, Department of Cell Biology and Anatomy, University of Alabama at Birmingham, UAB Station, Box 302, Birmingham, AL 35294.
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ment'13 and neutrophil-derived reactive oxygen species (reviewed in Shah4) and proteases1' 5-8 has been demonstrated, precisely how the GBM is affected during this pathologic condition is not known. In the studies presented here, we examined the ultrastructural distribution of immunoreactive laminin in the GBM as an indicator of possible alterations in GBM composition and architecture in acute NTN. For one approach, we processed normal and nephritic GBM for posffixation, postembedding immunogold electron microscopy. We also labeled the GBM in vivo, by intravenously injecting anti-laminin, both before and after injections of NTAbs, and performed immunoperoxidase and immunogold electron microscopy. Our evidence from all approaches indicates a loss and redistribution of GBM laminin during NTN.
Materials and Methods Proteins and Reagents Laminin was purified from the murine Englebreth-HolmSwarm (EHS) tumor by a modification of the method originally developed by Timpl et al9 and characterized as described previously.'1 Sheep and rabbit anti-laminin IgGs (anti-Lam) were purified by affinity chromatography as before.9'1112 Nephrotoxic antibodies were collected from sheep that had been immunized with whole rat glomeruli.13 Horseradish peroxidase (HRP, type VI) was purchased from Sigma Chemical Co. (St. Louis, MO) and conjugated directly to affinity-purified sheep and rabbit anti-Lam IgGs using methods described by Nakane and Kawaoi.14 Horseradish peroxidase-conjugated sheep anti-rabbit and rabbit anti-sheep IgGs and fluorochromeconjugated antibodies against sheep IgG, rabbit IgG and rat C3 were obtained from Organon Teknika-Cappel, West Chester, PA. Anti-rabbit IgG conjugated to 10-nm diameter colloidal gold was purchased from Janssen Life Science Products (Piscataway, NJ).
Inhibition ELISAs Enzyme-linked immunosorbent assay plates were coated with purified EHS tumor laminin or bovine serum albumin (BSA) (20 ug/ml), overnight at 4°C, washed with phosphate-buffered saline (PBS), and blocked with 5% horse serum in PBS for 1 hour at room temperature. Serial dilutions of sheep NTAbs were plated first and incubated at room temperature for 1 hour, and the plates were washed extensively before reincubation with rabbit anti-Lam antibodies for another 1 hour. The plates were washed and
rabbit anti-sheep IgG-HRP or sheep anti-rabbit IgG-HRP, diluted 1:1000, was added for 1 hour. In other cases, sheep anti-Lam-HRP was added directly to plates that had been preincubated with sheep NTAbs. Plates were washed extensively with PBS and then developed for peroxidase activity.15 Control experiments included incubating laminin- or BSA-coated plates with sheep NTAbs or sheep or rabbit anti-Lam alone and reincubation with appropriate secondary antibody-HRP conjugates.
Animal Experiments The goal of the overall experimental design was to examine the distribution of laminin within the GBM during acute stages of experimental anti-GBM disease using immunofluorescence, immunoperoxidase, and immunogold microscopy. Male Sprague-Dawley rats weighing 120 to 150 g were used throughout. For fluorescence microscopy, rats received dual injections of (1) sheep NTAbs (0.2 mg/ rat) followed 3 hours later by (2) affinity-purified rabbit antiLam (2 mg/rat) via saphenous veins. Kidney tissues were examined 1 hour after the second injection by immunofluorescence, as described below. For immunoperoxidase, five rats received injections of anti-Lam-HRP 1 hour before the administration of NTAbs. Kidneys were then fixed 3 hours later and processed for peroxidase histochemistry. A reverse labeling procedure was also carried out in which NTAbs were injected into five rats 3 hours before the administration of anti-Lam-HRP. In these cases, tissues were fixed 1 hour after the second injection. Anti-Lam-HRP was injected into normal rats as controls. A similar injection protocol was followed for colloidal gold studies. Three rats that had received NTAbs 3 hours earlier received injections of unconjugated rabbit antiLam. Kidneys were lightly fixed, embedded in Lowicryl resin, and sections were labeled with anti-rabbit IgG-colloidal gold, as described below. In addition, three rats re-
ceived anti-Lam 1 hour before receiving NTAbs. Control rats received anti-Lam alone 1 hour and 4 hours before they were killed, respectively (one rat each). For indirect labeling experiments, kidneys from one normal rat and two rats that had received NTAbs were lightly fixed and embedded in Lowicryl. Finally, kidneys from 3 normal rats, 3 nephritic rats, and 3 anti-Lam-injected rats were fixed in Karnovsky's fixative and processed through osmium and epoxy resin for routine morphology.
Immunofluorescence Microscopy Unfixed kidneys from experimental animals were rapidly frozen in 2-methyl butane cooled in a dry ice-acetone bath
Laminin Redistribution During Nephritis 189 AJPJuly 1990, Vol. 137, No. 1
and sectioned at -20°C in a cryostat. Sections, 4 gm thick, were air dried, treated with fluorochrome conjugated-anti-sheep IgG, anti-rabbit IgG, or anti-rat C3 for 5 minutes, washed with PBS, pH 7.4, and mounted in glycerol:PBS (1:1). Slides were examined in a Leitz Ortholux 11 or Orthoplan photomicroscope (E. Leitz Inc., Rockleigh, NJ) equipped for epifluorescence using appropriate filter cubes.
Immunoperoxidase Electron Microscopy Kidney tissues from anti-Lam-HRP-injected rats were fixed in 1.6% paraformaldehyde and 3% glutaraldehyde in 0.1 mol/l (molar) phosphate buffer, pH 7.416 for 2 hours, and washed in 0.1 mol/l buffer containing 35 mg/ml sucrose. Sections, 40 gm thick, were obtained with a Vibratome (Ted Pella, Inc; Redding, CA) and processed for peroxidase histochemistry in 0.01% hydrogen peroxide and 0.05% diaminobenzidine,17 as previously described.18 Sections were postfixed in 2% osmium before embedding in epoxy resin. Ultrathin sections were stained with lead citrate for 2 minutes and examined by electron microscopy in a JEOL 100 CX (JEOL USA Inc., Peabody, MA) or Hitachi H 7000 instrument (Hitachi Scientific Instruments, Mountain View, CA).
Postembedding Immunogold Electron Microscopy Kidneys from nephritic, anti-Lam-injected, and normal uninjected rats were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 mol/l phosphate buffer for 2 hours at 40C and washed in 0.1 mol/l phosphate buffer containing 35 mg/ml sucrose. Tissues were processed and embedded in Lowicryl K4M resin (Polysciences, Warrington, PA) at 40C using methods previously published.18 19 Thin sections, '70 nm thick, were picked up on uncoated 400 mesh nickel grids and blocked with ammonium chloride and bovine serum albumin as before.20 Sections were labeled as follows: a) Sequential labeling. Ultrathin Lowicryl sections from normal or nephritic rats that had not received antiLam injections were labeled first with affinity-purified rabbit anti-Lam (15 ,ug/ml) for 18 hours at 40C. The grids were washed thoroughly with PBS and then reincubated with anti-rabbit IgG-colloidal gold for 3 hours at room temperature. Sections were then washed, briefly stained with 1% uranyl acetate and lead citrate, and examined by electron microscopy. b) Direct labeling. Lowicryl sections from rats that had received injections of anti-Lam were labeled directly with goat anti-rabbit IgG-colloidal gold (1:3 dilution) for 72 hours at 40C. Grids were washed, stained, and examined.
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Figure 1. Graph showing the relative amount of bound antibodies on laminin-coated ELISA plates. Sheep nephrotoxic antibodies (S NTAbs) have little binding activity compared to sheep or rabbit anti-laminin antibodies (S or Rb anti-Lam). Preincubation ofplates with S NTAbs also does not inhibit the binding of S or Rb anti-Lam. Each bar represents the mean of three measurements, +/- the standard error.
Morphometry Randomly photographed fields of Lowicryl sections from normal and nephritic glomeruli were printed and areas of cross-sectioned GBM were measured using a SigmaScan digitizing tablet (Jandel Scientific, Sausalito, CA). Gold particles were counted manually. In addition, perpendicular distances across the GBM, as measured between the basal endothelial cell membranes and the bases of the podocyte foot processes, were determined. Average widths of GBM, lengths of basal podocyte cell surface, and widths of slit pores in normal and acutely nephritic kidneys were also measured in sections from routinely processed and epoxy-embedded tissues. Results are expressed as mean ± the standard error. Statistical analyses were carried out using Student's t-test.
Results Inhibition ELISA To determine whether the sheep NTAbs contained significant amounts of anti-Lam, an inhibition ELISA was car-
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Figure 2. Immunofluorescence micrographs of glomeruli from a rat that received a dual injection of sheep NTAbs and rabbit anti-Lam. Cryostat sections were stained with anti-sheep lgGfluorescein (a), anti-rabbit IgG rhodamine (b), at. 4 anti-rat C3 (c). NTAbs and anti-Lam bind in linearpatterns to the GBM (a and b) and there is abundant deposition ofcomplement (c). Separate immunofluorescence control experiments showed that the anti-rabbit IgG did not cross-react with the sheep NTAbs. a, b, c X550.
ried out. Figure 1 shows that there was little cross-reactivity between NTAbs and anti-Lam. When laminin-coated plates were incubated with sheep NTAbs, only very low
anti-laminin activities were detected as compared to those seen with sheep or rabbit anti-Lam (Figure 1). Furthermore, preincubation of laminin-coated plates with NTAbs did not reduce the binding of sheep or rabbit antiLam (Figure 1).
Immunofluorescence Light Microscopy Both nephrotoxic and anti-laminin antibodies bound to the GBM in the same linear pattern (Figures 2a and b). However, the presence of NTAbs on the GBM did not block the binding of anti-Lam in vivo (Figure 2b). Unlike what has been seen in previous studies on the failure of antiLam to activate complement in vivo,10,'1 here the binding of NTAbs initiated host complement deposition in glomeruli (Figure 2c).
Immunoperoxidase Electron Microscopy As seen previously,10'1121 the injection of anti-Lam-HRP into otherwise normal rats resulted in a specific and even deposition of peroxidase reaction product across the full width of the GBM in all capillary loops. The binding of injected anti-Lam to the GBM did not lead to an acute inflammatory response within glomeruli, however, and normal glomerular architecture was observed for up to 2 weeks after injection.10 1121 In contrast, when NTAbs were injected into rats that had received anti-Lam-HRP 1 hour earlier, there was a rapid and massive influx of neutrophils into glomeruli (Figure 3). The neutrophil infiltrates were accompanied by widespread endothelial cell detachment from the GBM and podocyte foot process effacement (Figure 3). Also unlike what was seen in normal rats, there was uneven laminin immunoreactivity within the GBM in many capillary loops of acutely nephritic rats (Figures 3 and 4). Although reaction product was present across the full thickness of the nephritic GBM, HRP was often more pronounced in the lamina rara interna (Figures 3 and 4), especially in areas of endothelial cell detachment (Figure 5). In regions in which neutrophils were closely apposed or adherent to denuded GBM, however, there were no apparent breaks or discontinuities in binding of anti-LamHRP (Figures 3 and 4). Where endothelial cells were detaching from the GBM, clumps of some peroxidase-positive, basement membranelike material remained associated with the basal endothelial cell surface (Figure 5), as if basement membrane elements were being removed from the GBM during cell detachment. An identical set of results was obtained in rats that received NTAbs 3 hours before in vivo labeling with anti-Lam-HRP.
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Figure 3. Electron micrograph ofglomerulusfrom a rat that received an intravenous injection of rabbit anti-Lam-HRP 1 hour before receiving NTAbs. Tissue was.fixed 3 hours later. Several neutrophils (PMN) are present within capillary loops and widespread loss of the endothelium has occurred. Damage to epithelialpodocytes (Ep) is also indicated by foot process widening (arrowheads) and surface blebbing (*). Although HRP reaction product extends across the GBM, the most intense labeling frequently occurs on the inner layer (arrows). No abrupt discontinuities in labeling are observed along lengths of the GBM, however. RBC, red blood cell. X 11,500.
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_5, Figure 4. Higher magnification electron micrograph ofglomerular capillary loop from a nephritic rat labeled with anti-Lam-HRP, as in Figure 3. HRP reaction productpersists beneath a neutrophil (PMN) lying close to the GBM (arrows) and is more concentrated in the lamina rara interna. Note foot process effacement in the epithelial layer (Ep). X22,500. Figure 5. Electron micrograph of glomerular capillary wallfrom a rat given NTAbs 3 hours before anti-Lam-HRP. Some laminin immunoreactivity is present on the basal surfaces (arrows) ofendothelial cells (En) detachingfrom the GBM. Intense HRP reaction product is also present in the lamina rara interna of the GBM (arrowheads). CL, capillary lumen; Ep, epithelialpodocytefootprocesses. X27,500.
Immunogold Electron Microscopy and Morphometry To evaluate further any changes in laminin distribution, Lowicryl sections from normal and nephritic rats were se-
quentially labeled with rabbit anti-Lam and anti-rabbit IgGcolloidal gold (Figure 6). In addition, sections from normal and nephritic rats that had received intravenous injections of rabbit anti-Lam were labeled directly with immunogold (Figure 7). With both labeling protocols there appeared to
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Figure 6. Electron micrographs offormaldebyde-fixed, Lowicryl-embedded kidney sections from a normal rat (a) or a rat given NTAbs (b). Sections were labeled sequentially with rabbit anti-Lam and anti-rabbit IgG-colloidal gold, as described in Materials and Methods. A marked reduction in labeling is observed in the nephritic GBM. Note also the increased distance between the endothelial cells and podocytes in the nephritic glomerulus (bars measure 0. 18g1m in (a) and 0.3 ,um in (b). CL, capillary lumen; En, endothelium; Ep, foot processes of epithelium. a, b X90,000.
be a reduction in colloidal gold binding to the GBM of acutely nephritic rats when compared with normal (Figure 6) or anti-Lam-injected controls (Figure 7). To quantitate these observations, GBM-bound gold particles were counted, expressed as the number of particles bound/ JAm,2 and compared between groups. Significantly less gold bound to nephritic GBMs than to those of normal rats or those injected with anti-Lam (Figure 8). In addition, as shown in Figure 8, the same amount of rabbit IgG was detected in rats that received anti-Lam 1 hour before NTAbs, as in those labeled after NTAb injection. These results were consistent with our inhibition ELISA and immunofluorescence findings presented earlier, indicating that the bound NTAbs did not inhibit the binding of rabbit anti-Lam to the GBM in vivo.
Several additional morphometric measurements were taken from the same Lowicryl sections used for particle counting as well as from sections from routinely fixed and epoxy-embedded kidneys. In rats that received NTAbs, the width of the GBM averaged 0.28 ,m in Lowicryl sections and 0.18 gm in epoxy sections (Figure 9). The marked difference between the Lowicryl- and epoxy-embedded tissue was likely due to the comparative instability of Lowicryl in the electron beam as well as to the weaker fixation conditions used for postembedding immunolabeling. Nevertheless, the values from rats that received NTAbs were significantly greater (P < 0.001) than GBM widths from either uninjected or anti-Lam-injected rats, regardless of the embedding media (Figure 9). Interestingly, the GBMs of anti-Lam-injected rats, although much
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Discussion
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Our posffixation labeling as well as in vivo prefixation immunolabeling results clearly indicated changes in GBM laminin immunoreactivities during acute stages of antiGBM disease in the rat. Whereas the reduction in laminin immunoreactivity might have been due to proteolytic degradation of laminin by infiltrating neutrophils, we believe that another reason for the observed decrease was the
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significantly greater distance across the GBM between endothelial cells and podocytes. This widening could have been caused by the detachment of glomerular endothelial cells, loosening of the GBM meshwork, and also possibly to the effacement of podocyte foot processes. lThe end result was a redistribution of laminin over a greatly expanded matrix. We did not detect significant levels of anti-laminin activity in the NTAbs used in this study. Correspondingly, previous efforts to simulate the heterologous phase of
NTN using antibodies directed specifically against individual GBM components, such as laminin,'" 1,21-23 collagen type IV,22-3 or heparan sulfate proteoglycans24-5 generally
NTAbs, or anti-Lam alone. In these instances, sections were labeled directly with anti-rabbit IgG-colloidal gold. In all cases,
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narrower than those that received NTAbs, were wider than in normal animals (Figure 9). In contrast, the ratio of total basal foot process surface/unit length GBM did not differ between rats that received anti-Lam and normal rats and averaged 0.83 in epoxy sections (Figure 10). However, this ratio was much greater (P < 0.001) in nephritic rats, reflecting the effacement of foot processes (Figure 10). There was also a corresponding decrease (P
