ARTICLE |
Correspondence to: Lambert P. van den Heuvel, Dept. of Pediatrics, Univ. of Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
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Summary |
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Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)
Key Words: agrin, perlecan, heparan sulfate proteoglycan, glomerular basement, membrane
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Introduction |
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The glomerular basement membrane (GBM) is a highly organized network of extracellular proteins such as collagen IV (3,
4, and
5), laminins (
5,
1, ß2,
1), nidogen/entactin, and heparan sulfate proteoglycans (HSPGs) (
Since the characterization of perlecan as an HSPG component of basement membranes (
In rat and mouse, the agrin core protein has an approximate molecular mass of 210 kD (
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Materials and Methods |
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Antibodies
Characteristics of the antibodies used are as follows. Anti-agrin antiserum PAb 707 was raised in rabbit against full-length chick agrin from a recombinant source (
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Indirect Immunofluorescence
Adult human kidney cortex tissue obtained from post-trauma donors was snap-frozen in polybrene, cooled with liquid nitrogen. Two-µm sections were stored at -80C until use. Skeletal muscle tissue was obtained from adult rat hamstrings and frozen in polybrene immediately after dissection. Six-µm sections were prepared as above. Immunohistochemistry was performed as described elsewhere (-bungarotoxin was added to the secondary antibody mixture at a final dilution of 1:400. In double immunofluorescence studies, MAbs were detected by goat anti-mouse immunoglobulins coupled to Texas Red (Sanbio; Uden, The Netherlands).
Immunoelectron Microscopy
Incubations were performed at 22C, unless otherwise indicated. Kidney cortex slices were immersed for 3 hr with PLP fixative (a mixture of periodate-lysine and 2% paraformaldehyde), rinsed with PBS (pH 7.4), cryoprotected in 2.3 M sucrose for 45 min, and snap-frozen in liquid nitrogen. Twenty-five-µm cryostat sections were incubated with PAb 707 diluted 1:100 in PBS containing 1% BSA for 18 hr at 4C and then washed three times for 30 min with PBS. This was also done with MAb 95J10 (diluted 1:5) and MAb M215 (diluted 1:10). After rinsing, the sections were incubated for 90 min with the appropriate peroxidase-conjugated secondary antibody (goat anti-rabbit immunoglobulins, Dakopatts; rabbit anti-mouse immunoglobulins, BioSys) diluted 1:70 in PBS containing 1% BSA. After three 30-min washes in PBS, the sections were preincubated for 10 min in PBS containing 0.05% diaminobenzidine. Subsequently, the sections were stained for 10 min with the same medium containing 0.03% hydrogen peroxide. For immunogold labeling, goat anti-rabbit and goat anti-mouse immunoglobulins coupled to colloidal gold of 1 nm (Nanoprobes; Stony Brook, NY) were diluted 50-fold in PBS containing 1% BSA. The sections were incubated for 90 min at 22C, then washed three times for 30 min in distilled water, and the gold signal was enhanced using HQ silver (Nanoprobes) for 5 min. After washing in distilled water for 18 hr at 4C, the sections were postfixed in 1% osmium for 20 min at 22C, dehydrated, and embedded in Epon 812. Thin sections were prepared on a Reichert ultratome and examined in a Jeol 1200 EX electron microscope.
Preparation of Glomerular Extract
Glomeruli were isolated from human kidney cortex by the sieving method (
Enzyme-linked Immunosorbent Assay
For quantitative determination of HSPGs by ELISA, crude glomerular extract was diluted 40-fold in coating buffer (
For semiquantitative ELISA, wells were uniformly coated with 0.5 µg/well purified GBM HSPG (
Nitrous Acid Treatment and Immunoblotting
Five µg GBM HSPG (
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Results |
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Immunofluorescence Microscopy
The presence of agrin in the glomerulus was investigated by indirect immunofluorescence. Cryosections of human renal cortex were stained with antibodies against agrin, perlecan, and "GBM-HSPG," a previously described HSPG isolated from the human GBM (
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Because agrin is concentrated at the NMJ, we next compared staining patterns of anti-agrin and anti-GBM-HSPG antibodies in rat skeletal muscle sections. Junctions were identified by staining with rhodamine--bungarotoxin, recognizing the acetylcholine receptor clusters on the postsynaptic membrane (Figure 2A). Staining of the same section with MAb Agr-33 (directed against rat agrin) resulted in a strong signal localized at the synaptic cleft of the NMJ (Figure 2B). All junctions identified by rhodamine-
-bungarotoxin staining showed strong immunofluorescence. In addition, extrajunctional patches were observed that were strongly stained by Agr-33 but not by rhodamine-
-bungarotoxin. Furthermore, a weak staining of basement membranes surrounding the muscle fibers was present. Comparably, sections stained by rhodamine-
-bungarotoxin (Figure 2C) were immunostained with anti-GBM-HSPG antiserum K42 (Figure 2D). In agreement with Agr-33, the K42 antiserum stained all neuromuscular junctions together with some focal accumulations outside the junction and weak staining of the basement membrane that surrounds the muscle fibers.
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Immunoelectron Microscopy
To study the distribution of basement membrane HSPGs at the ultrastructural level, immunoelectron microscopy was performed on human kidney cortex. The distributions of agrin (top panel), perlecan (middle panel), and the GBM-HSPG recognized by M215 (bottom panel) are shown in Figure 3. With anti-agrin antiserum a strong linear staining was seen along the full length of the GBM (Figure 3A-C). The entire width of the GBM was stained, but the signal was markedly stronger at both edges of the GBM. This localization at both the endothelial and the epithelial side of the GBM was confirmed by immunogold labeling (Figure 3C). We could also observe faint staining of the mesangial matrix. The anti-perlecan MAb 95J10 produced strong staining at the interface of the mesangium and the capillary endothelium (Figure 3D-F). The overall GBM staining was mild and was restricted to the endothelial side in a nonlinear pattern. In addition, by immunogold labeling perlecan staining was found exclusively on the endothelial side of the GBM. With MAb M215, strong linear staining was observed throughout the entire length and width of the GBM (Figure 3G-I). Immunogold labeling also demonstrated that the M215 epitope was present throughout the GBM. Occasionally, mild staining of the mesangial matrix was observed.
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The linear distribution of the agrin-like HSPG along the GBM corresponds with the homogenous linear staining by immunofluorescence in this and previous studies (Figure 1B) (
Immunoreactivity of Anti-Agrin Antiserum with Purified GBM-HSPG
The described results show a co-localization of agrin-like and GBM-HSPG-like immunoreactivity in both the GBM and the NMJ. Given the unknown identity of the purified GBM-HSPG and the identification of agrin as a HSPG, we asked whether both proteoglycans could be identical. To address this, isolated GBM-HSPG was coated into microtiter wells. Antisera raised against agrin showed a strong reaction with this proteoglycan, whereas the corresponding preimmune sera were negative (Figure 4, open squares and circles). In addition, we tested the capability of anti-agrin antiserum to compete in ELISA with four different MAbs against human GBM-HSPG (including M215 and M138, all recognizing the same core protein). The binding of three MAbs was not blocked. Because the anti-agrin antiserum was raised against chick agrin, this suggests that the corresponding epitopes of human agrin are not immunogenic or absent in chick agrin. However, the binding of M138 to native GBM-HSPG was inhibited by anti-agrin (Figure 4, solid squares). These results show that the isolated GBM-HSPG is recognized by both anti-GBM-HSPG and anti-agrin antibodies.
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Western blot analysis of isolated GBM-HSPG was performed with antibodies against agrin and GBM-HSPG (Figure 5). All antibodies recognized a large proteoglycan with a characteristically smeared appearance and similar molecular mass. The antigen was sensitive to nitrous acid treatment, indicating the presence of heparan sulfate residues. The nitrous acid-treated core protein was recognized by all antibodies and displayed a molecular mass of approximately 200-210 kD. In addition to this prominent band, two additional bands were weakly stained by M215 and M138. These bands are presumably degradation products and could also be visualized with PAb 707 after prolonged exposure (not shown).
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Quantitative Detection of HSPGs in Isolated Glomeruli
The strong staining of agrin in immunofluorescence and immunoelectron microscopy suggests that it is highly concentrated in the GBM. To determine the relative contribution of each molecule to the total HSPG content of the glomerulus, quantitative ELISA was performed with 95J10 and M215. A standard curve for 95J10 was constructed using an affinity-purified recombinant fragment comprising Domains I and II of human perlecan (
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Discussion |
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In this study we have used an antiserum against chick agrin (
The quantitation of the agrin-like GBM-HSPG and perlecan in extract from isolated glomeruli could be misleading in two ways. First, the standard used for quantitation by M215 cannot be considered absolutely contaminant-free, even though it was extensively purified (
What functions might agrin serve in the human GBM? Although many aspects of the structure and function of agrin isoforms have been studied in detail, the presence of agrin in the mammalian kidney and GBM has not been previously described. One study reported the occurrence of agrin-like proteins in the tubular structures of chick kidney and showed that these molecules have only little AChR clustering activity (
Second, agrin may provide a cytoskeletal link for the GBM through its interaction with -dystroglycan (
-dystroglycan does not appear to be the receptor involved in AChR clustering (
-dystroglycan binding region of agrin is mapped to the C-terminal half of the molecule, an N-terminal fragment of 130 amino acids was shown to bind to laminin-1, -2, and -4 (
1, ß2 and
1 (
A third possible function of agrin in the GBM is suggested by the presence of nine Kazal-type protease inhibitor domains (
In contrast to the early expression of agrin in synaptogenesis, the onset of expression in the GBM may be delayed to later stages of development. Agrin-deficient mutant mice show aberrations in the development of neuromuscular junctions and die shortly before birth. At this stage, no prominent kidney malformations were mentioned (
From this study, we conclude that agrin is a major HSPG component of the human GBM. This finding has drastic implications for the investigation of structural and functional properties of the GBM. Changes in agrin structure and content may be important for glomerular function and may play a role in various types of glomerulopathy. The structure of the agrin isoform that is present in the GBM and its relevance for cell-matrix interactions are subjects for further investigation.
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Acknowledgments |
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Supported by grant C93.1309 from the Dutch Kidney Foundation (AJG,CAB,LAM,JHV,LPH) and by grant #31-33697.92 from the Swiss National Science Foundation (MAR).
Received for publication February 10, 1997; accepted July 22, 1997.
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