ARTICLE |
Correspondence to: Dale R. Abrahamson, Dept. of Cell Biology, U. of Alabama at Birmingham, 6th Floor Volker Hall, 1670 University Blvd, Birmingham, AL 35294-0019.
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Summary |
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During glomerular development, subendothelial and -epithelial basement membrane layers fuse to produce the glomerular basement membrane (GBM) shared by endothelial cells and epithelial podocytes. As glomeruli mature, additional basement membrane derived from podocytes is spliced into the fused GBM and loose mesangial matrices condense. The mechanisms for GBM fusion, splicing, and mesangial matrix condensation are not known but might involve intermolecular bond formation between matrix molecules. To test for laminin binding sites, we intravenously injected mouse laminin containing 1-, ß1-, and
1-chains into 2-day-old rats. Kidneys were immunolabeled for fluorescence and electron microscopy with domain-specific rat anti-mouse laminin monoclonal antibodies (MAbs), which recognized only mouse and not endogenous rat laminin. Intense labeling for injected laminin was found in mesangial matrices and weaker labeling was seen in GBMs of maturing glomeruli. These patterns persisted for at least 2 weeks after injection. In control newborns receiving sheep IgG, no binding of injected protein was observed and laminin did not bind adult rat glomeruli. To assess which molecular domains might mediate binding to immature glomeruli, three proteolytic laminin fragments were affinity-isolated by MAbs and injected into newborns. These failed to bind glomeruli, presumably owing to enzymatic digestion of binding domains. Alternatively, stable incorporation may require multivalent laminin binding. We conclude that laminin binding sites are transiently present in developing glomeruli and may be functionally important for GBM assembly and mesangial matrix condensation. (J Histochem Cytochem 46:291300, 1998)
Key Words: glomerular basement, membranes, matrix, laminin, monoclonal antibodies, bamacan, collagen Type IV, entactin, nidogen, perlecan
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Introduction |
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On the basis of extensive reconstitution experiments with purified components, current biochemical evidence indicates that basement membranes consist of two distinct but interwoven networks of polymerized collagen Type IV and laminin, (reviewed by
The kidney glomerular basement membrane (GBM) is unlike most basement membranes found elsewhere in the body. During early glomerular development, the basement membrane layer beneath ingrowing endothelial cells fuses with a layer beneath immature podo-cytes to yield a common, double-thickness basement membrane located between two adherent cell sheets. Subsequently, during glomerular capillary loop expansion, additional basement membrane material, derived mainly if not exclusively from podocytes, is inserted or spliced into the fused GBM (reviewed in 1-, ß1-, and
1-chains and is known to be expressed in the immature kidney (
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Materials and Methods |
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Laminin Purification and Digestion
Laminin was purified from the mouse EnglebrethHolmSwarm tumor matrix by sequential salt extraction, DEAE chromatography, and gel filtration, using a modification of the method originally reported by
Affinity Purification of Laminin Fragments
Rat anti-mouse laminin MAb IgGs 5A2, 5C1, and 5D3 were purified from ascites fluid obtained from nude mice and were characterized by Western blotting and rotary shadow electron microscopy of affinity-purified laminin digests as previously described (
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Detection of Laminin Binding Sites In Vivo
All experiments involving animals met institutional and NIH guidelines. Undigested mouse laminin was injected IV into the left saphenous vein of ether-anesthetized infant SpragueDawley rats. The amounts of laminin injected varied with age of the animals, and for most experiments averaged ~510 µg laminin/g body weight. After recovering from anesthesia, rats were returned to their litters. One day later the same rats received a second injection of laminin via the right saphenous vein, were allowed to recover, and returned to their litters again. Separate groups of infant rats received IV injections of crude elatase or pepsin digests of laminin, or the MAb 5C1 affinity-purified pepsin fragment of laminin, the MAb 5D3 affinity-purified elastase fragment, or the MAb 5A2 elastase fragment, following an identical injection protocol as that described for intact laminin. In addition, 60 and 72 µg of sheep IgGs were injected sequentially into infant rats as controls. Likewise, mature ~225 g rats received two daily IV injections of 1.13 mg of intact mouse laminin.
Two to 14 days after the second IV injection the rats were re-anesthetized and kidneys were removed and promptly frozen in 2-methylbutane chilled in a dry-ice/acetone bath. Unfixed cryostat sections were then incubated sequentially with MAb 5A2, 5C1, or 5D3, and then with goat anti-rat IgGrhodamine (OrganonTeknika Cappel; Durham, NC). Sections from newborn rats that received IV injections of sheep IgG as controls were incubated with rabbit anti-sheep IgGfluorescein (OrganonTeknika Cappel).
In addition to IV administration of mouse laminin into newborn rats, unfixed cryostat sections of infant rat kidneys were incubated in a humid chamber for 1 hr at room temperature with purified laminin ranging in concentration from 10 to 240 µg/ml. Sections were washed with PBS, treated with rat anti-mouse laminin MAbs, and processed for indirect immunofluorescence microscopy.
Immunoelectron Microscopy
Each anti-laminin MAb was also conjugated directly to activated horseradish peroxidase (HRP), exactly as described previously (
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Results |
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Preliminary studies showed that, after a single IV injection of intact mouse laminin into anesthetized newborn rats, there was no detectable binding of the injected protein to immature rat glomeruli in vivo. However, after two daily injections of exogenous laminin into 1-, 2-, or 3-day-old rats, immunofluorescence examination showed intense localization of injected laminin specifically to maturing glomeruli (Figure 2). As shown in Figure 2b, Figure 2c, and Figure 2d, anti-laminin MAbs 5A2, 5C1, and 5D3 all labeled glomeruli in similar patterns. The injected laminin bound abundantly to developing mesangial matrices and lengths of peripheral capillary loop GBMs were positive as well (Figure 2). However, less mature glomeruli located beneath the capsule were only weakly labeled or unlabeled (Figure 2a). Because considerable nephrogenesis continues in rats and mice during the first week after birth, the immature glomeruli in superficial layers of the cortex that contained only small amounts of injected laminin were just beginning to form when the first laminin injection was given 4 days earlier. There was no binding of injected laminin to developing tubule basement membranes, perivascular matrices, or other structures in the infant rat kidney (Figure 2).
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The ability to distinguish between the extrinsic mouse laminin that we injected and the intrinsic native laminin synthesized within the rat kidney was crucial for our experimental approach. As shown in Figure 3a, when cryostat sections from rat kidneys were incubated with rat anti-mouse laminin MAbs there was no binding whatsoever of the MAbs to basement membranes or other structures in the sections. This result therefore shows that the rat anti-mouse laminin MAbs did not recognize autologous rat laminin. Importantly, when glomeruli from newborn animals that had received two IV injections of sheep IgG as controls were then labeled with anti-sheep IgGfluorescein, no binding of the injected IgG was observed (Figure 3b). This shows that the binding of injected laminin seen in Figure 2 did not merely reflect nonspecific assimilation of injected protein into developing glomeruli. Likewise, when adult rats that had received the same amount of injected laminin/g body weight as the newborns were assessed, no binding of injected laminin was seen in kidneys taken from mature animals (data reviewed but not shown). In addition, to assess whether the laminin binding sites identified in vivo (Figure 2) might be accessible ex vivo, we incubated unfixed cryosections of newborn rat kidneys at room temperature with mouse laminin ranging in concentration from 10 to 240 µg/ml. In all cases there was no binding of the extrinsic laminin to newborn kidney sections in vitro (reviewed but not shown).
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To examine the in vivo binding of laminin in greater detail, newborn and adult rats that had received the same amount of injected laminin/g body weight over a 2-day period were injected with MAb 5D3HRP conjugates. As shown in Figure 4a, an intense peroxidase reaction product was seen in glomeruli of newborn rats, especially within mesangial matrices. A weak reaction product was also seen in peripheral GBMs of many capillary loops, and the same binding patterns were seen after in vivo labeling with 5A2 and 5C1HRP. Those GBMs that were weakly labeled were found in loops that were still undergoing basement membrane splicing and podocyte foot process development, whereas capillary loops within the same glomeruli that appeared ultrastructurally mature did not contain injected laminin (Figure 4a). In adult rats there was no binding of injected laminin to mesangial matrices, GBMs, or elsewhere in kidneys (Figure 4b), and this was consistent with the immunofluorescence results.
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To investigate the relative stability of the bound laminin, kidneys from rats that had received IV injections of laminin 2 days after birth were removed 3, 5, 7, 9, or 14 days after the second injection. Immunofluorescence labeling showed that over this time period considerable amounts of injected laminin remained bound within glomeruli (Figure 5). Mesangial areas continued to be intensely labeled and linear GBM labeling was also found in glomerular capillary loops (Figure 5). The labeling patterns seen 2 weeks after the last laminin injection were the same for MAbs 5A2, 5C1, and 5D3 (Figure 5).
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Having shown that IV injected laminin bound specifically to developing GBMs and mesangial matrices and remained bound for at least 2 weeks after injection, we sought to identify the molecular domains on laminin that mediate this binding. Laminin was digested with pepsin or elastase and newborn rats received IV injections of either crude enzyme digests or separate proteolytic laminin fragments purified by MAb affinity chromatography. As shown in Figure 6, however, none of the injected laminin fragments could be detected within developing GBMs or mesangial matrices.
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The ages, numbers of animals injected with various preparations of intact laminin and proteolytic fragments of the molecule, and whether or not these injections resulted in glomerular binding are summarized in Table 1. The dominant variable for detection of injected laminin in glomeruli was the age of the animal at the time of injection. Animals that were 1, 2, or 3 days old when they received the first injection always contained injected laminin in their glomeruli. Beginning ~5 days after birth, however, injected laminin was variably seen in glomeruli of young rats and there was no incorporation of extrinsic laminin in adults (Table 1).
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Discussion |
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Several conclusions can be drawn from our results. First, injections of mouse laminin-1 into newborn rats resulted in incorporation of this protein into mesangial matrices and GBMs of immature glomeruli. In contrast, injected laminin did not bind to other basement membranes of developing kidneys and, when injected into adult rats, there was no association with glomerular matrices or other tissues in the mature kidney. These findings therefore demonstrate that laminin binding sites are transiently present within basement membranes of developing glomeruli. Moreover, these sites were specific for intact laminin, and other proteins injected as controls, as well as proteolytic fragments of laminin, failed to bind to developing glomerular matrices. Binding of injected laminin to the developing GBM was stable and there were no perceptible changes in glomerular patterns of bound laminin for at least 2 weeks after injection.
Second, binding sites for laminin in immature glomeruli were identified only after IV administration of the protein in vivo. When unfixed kidney sections from newborn rats were incubated with mouse laminin in vitro, there was no detectable adherence to basement membranes or to other structures. In addition, abundant binding of injected laminin to immature glomeruli of newborn rats in vivo was first seen after two daily IV injections, whereas little binding was detected after a single injection. These results therefore differ markedly from what is seen after the injection of cationized ferritin (
Third, injected laminin bound abundantly to glomeruli of 13-day-old rats, in which extensive GBM and mesangial matrix formation is still taking place. In contrast, only variable glomerular binding was seen when laminin was injected into 510 day-old rats, in which glomerular development is concluding. No binding was detected when laminin was injected into adults. The absence of binding of injected laminin in older animals might have been due to the progressive acquisition of permselective sieving properties of the mature glomerular capillary wall, which would therefore minimize binding of circulating laminin to potential GBM binding sites. However, mesangial areas of adult glomeruli are considered to be much more permeable to circulating macromolecules than the GBM, but injected laminin localized only to mesangial matrices and GBMs in developing rats, and this was never seen in adults. Laminin binding in newborns was also not immediate, and only those developing glomeruli that had been exposed to circulating laminin over a period of several days were positive. Considering these findings, we believe that the in vivo glomerular binding of injected laminin in infant rats reflects accumulation of this protein in GBMs and mesangial matrices undergoing extracellular assembly.
Northern analysis, ribonuclease protection assays, and/or in situ hybridization studies have shown that mRNAs encoding the 1- and
2-chains of collagen Type IV, the core protein of perlecan, and the
1-, ß1-, and
1-chains of laminin are all present at relatively high levels during kidney development (
1- and
-2-chains of collagen Type IV along with a "fetal" isoform of laminin containing
1, ß1 and
1 laminin chains (
3-,
4-, and
5-chains of collagen Type IV and the
5, ß2, and
1 laminin chains (
What regulates the appearance and disappearance of different GBM protein isoforms during development is not known, but these changes are important for the morphological and physiological maturation of the glomerulus. For example, homozygote mutant mice lacking the 3-chain of Type IV collagen die of renal failure 34 months after birth (
3(IV)-chain in these mice, there is also an absence of collagen
4- and
5(IV)-chains and abnormally increased amounts of collagen
1- and
2(IV)-chains, collagen VI, and perlecan (
3(IV) collagen deficiencies, however, GBMs in these animals are ultrastructurally normal and contain the appropriate complement of GBM collagens and perlecan. In contrast, the laminin ß1-chain, which ordinarily is replaced by the ß2-chain, persists in mature glomeruli of these ß2 chain mutants, and unusually broad podocyte foot processes are also present (
3(IV) collagen deficiency, mechanisms accounting for dysregulation of other basement membrane gene products in laminin ß2 mutants are not known but may reflect sensory feedback at the level either of gene expression or of post-translational GBM protein assembly (
When we examined young rats that had received the last injection of mouse laminin 2 weeks earlier, we found that injected laminin remained associated with GBMs of mature glomeruli. Furthermore, labeling with MAb 5A2, which recognizes an epitope on the short arm (
The domains on laminin known to bind basement membrane proteins with high affinity in vitro include the highly homologous N-termini (Domain VI) of the -, ß-, and
-chains, which interact with other laminin molecules to form laminin polymers (
chain that binds to the G3 globular domain at the C-terminus of entactin (
-chain (
In conclusion, what we have shown in this study is that, when injected into newborn rats, intact mouse laminin bound specifically to GBMs and mesangial matrices of developing glomeruli. This injected laminin remained bound for at least 2 weeks after injection, and the ß1-chain of this laminin, as recognized by the binding of MAb 5A2, also persisted. We suspect that the binding sites we have identified in developing glomeruli are probably crucial for the formation of intermolecular crosslinks during extracellular basement membrane assembly and may be important specifically for GBM fusion and mesangial matrix condensation in vivo. Because three separate proteolytic fragments of laminin failed to bind, however, our results suggest further that multivalent linkages between laminin and its ligands are required for stable incorporation into matrices. Future studies aimed at determining precisely which laminin domains mediate its binding, and to which molecules these laminin domains bind, should provide a much better understanding of basement membrane assembly and organization in vivo.
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Acknowledgments |
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Supported by NIH grants DK34972 and DK52483. The hybridoma core facility at UAB is supported by AR20614, and the electron microscopy core is a shared facility of the Comprehensive Cancer Center (CA13148).
We thank Barry Robert for critically reviewing an earlier version of the manuscript.
Received for publication June 20, 1997; accepted October 13, 1997.
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