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Published online before print May 31, 2007
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From the Renal Division,* and the Department of Cell Biology and Physiology,|| Washington University School of Medicine, St. Louis, Missouri; the Division of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; the Division of Nephrology and Immunology, Rheinisch-Westfälische Technische Hochschule, Aachen, Germany; the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan; and The Jackson Laboratory,¶ Bar Harbor, Maine
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Anionic sites can be detected based on their affinity for cationic probes and have been found in association with each layer of the capillary wall. The anionic glycocalyx of podocytes and endothelial cells that is formed largely by podocalyxin5 may contribute to the barrier. Podocalyxin serves a critical role in dictating podocyte foot process architecture, presumably through charge-related repulsive effects.6 However, the glomerular basement membrane (GBM) is generally considered to be of primary importance. GBM charge is imparted by sulfated glycosaminoglycan (GAG) side chains of proteoglycans and to a lesser extent by carboxyl and sialyl groups of glyco-proteins. GBM anionic sites are distributed in a quasi-regular pattern along both laminae rarae but are most prominent along the subepithelial aspect.7,8 These were identified as heparan sulfate proteoglycans (HSPGs) based on their susceptibility to enzymatic digestion.9 Other sulfated GAGs are not present in the GBM in significant amounts.
Charge barrier dysfunction is considered an important factor in the pathogenesis of glomerular disease.10-13 This may be brought about by decreased expression or undersulfation of GBM-HSPGs.14-16 In animal models, enzymatic removal of glomerular heparan sulfate (HS) or charge neutralization results in proteinuria and promotes the permeability of anionic tracers.4 However, a recent study has challenged the notion that GBM-HS is important for glomerular permselectivity.17
Three genetically distinct basement membrane (BM)-HSPGs are recognized: perlecan, collagen XVIII, and agrin. Perlecan and collagen XVIII are both found in the glomerulus but are localized primarily to the mesangial matrix and Bowman’s capsule and are only prominent in the GBM during development.15,18 Mice lacking the attachment sites for HS on perlecan have normal glomerular ultrastructure and no renal disease but show increased susceptibility to protein-overload proteinuria.19,20 Collagen XVIII mutants have mild mesangial expansion and only slightly elevated serum creatinine levels compared with controls.21
Agrin has been identified as the predominant GBM-HSPG in all species studied, prompting speculation that it may be a critical determinant of the charge barrier.22,23
It is characterized by an 
2000-residue core protein of 220 kd that carries at least two GAG chains, bringing its mass to 400 kd. Agrin is generally classified as an HSPG, but it can carry both heparan and chondroitin sulfate (CS) GAGs.24,25
Sites for GAG attachment have been mapped experimentally in chick agrin to one site located between the seventh and eighth follistatin-like domains that carries exclusively HS and to a second in the serine/threonine-rich region that carries predominantly CS.25
Alternative promoters give rise to two isoforms of agrin that are either BM or cell associated, with the latter being specific to neurons.26
BM-associated agrin binds to the laminin 
1 chain via a globular domain (NtA) at its N terminus and to dystroglycan and integrin receptors through its C terminus.27-29
By virtue of these interactions, agrin is thought to be an integral part of the molecular complex linking podocytes to the GBM.30
Agrin-deficient mice are grossly normal but die at birth with severe neuromuscular defects.31 The objective of this study was to assess the role of agrin in glomerular development and function through the study of agrin mutant mice. To overcome the perinatal lethal phenotype, we generated conditional knockouts in which Agrn was ablated in podocytes. Here, we demonstrate that agrin contributes significantly to GBM anionic charge but is not needed for glomerular function.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Kidneys were studied from agrin-deficient mice homozygous for a knockout allele (Agrntm4Jrs; denoted Agrndel) in which genomic sequence from within exon 6 to intron 33 (numbering according Rupp et al32 ) is replaced by a loxp-flanked PGK-neo cassette31 or a gene trap allele (AgrnGt(p1.8TM)192Wcs; denoted Agrnß-geo) that disrupts Agrn 3' of the last NtA-encoding exon.26 A conditional allele harboring loxP sites within introns 6 and 33 (Agrntm1Rwb; denoted Agrnfl) was generated through "recombineering" in EL350 cells,33 followed by electroporation of the construct into R1 embryonic stem cells. Embryonic stem cell clones were injected into C57BL/6J blastocysts to generate germline chimeras that were bred to obtain heterozygous and homozygous mice. In some cases, these were crossed to FLPe transgenic mice34 to eliminate the frt-flanked neo selectable marker.
Conditional knockouts were generated by crossing to mice that express Cre recombinase from the human NPHS2 promoter (2.5P-Cre35
) or the murine Pax3 promoter (P3pro-Cre36
). Genotyping was performed by polymerase chain reaction (PCR) using primers for the following alleles: Agrnfl, 5'-AGCCCGGAAACTCTGGATTCC-3' (exon 33) and 5'-CAAAGTGGTTGCTCTGCAGCG-3' (exon 34); Agrnfl
neo, 5'-CGGACACACATATGCTAGTGA-3' (exon 6) and 5'-ACTGTCCAGCTGAGCACACAGC-3' (exon 7); Agrndel, 5'-TGCCAAGTTCTAATTCCATCAGAAGCTGAC-3' (neo) and 5'-GGGCTAACACCAACAACAATGCAACAAAGG-3' (intron 33), or 5'-CAGTGAAGAATGGGAAAGCTG-3' (exon 5) and the exon 34 primer for Agrndelneo; Agrnß-geo, 5'-GGATTGGTGGCGACGACTCC-3' and 5'-AATGGGCAGGTAGCCGGATCAAGCG-3'; Cre, 5'-CGGTCGATGCAACGAGTGATGAG-3' and 5'-ACGAACCT GGTCGAAATCAGTGCG-3'; and FLP, 5'-GTGGATCGATCCTACCCCTTGCG-3' and 5'-GGTCCAACTGCAGCCCAAGCTTCC-3'. Mice were studied on a mixed 129 x C57BL/6J genetic background. Animal experiments were approved by the Washington University Animal Studies Committee.
Analysis of Mutant mRNA Transcripts
Total RNA extracted with Tri-Reagent (Molecular Research Center, Cincinnati, OH) was used to synthesize cDNA with the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, CA). PCR reactions contained 2 µl of cDNA template, 125 µmol/L dNTPs, 2.5 U of Taq polymerase (Bioline, Randolph, MA) in the supplied buffer, 0.2 µmol/L each of a forward primer in exon 5 or 6 (as above), and a reverse primer in exon 34 (as above), exon 35 (5'-GCCCACCTGAAGGGAACC-3'), or exon 36 (5'-CACAAAACCCGTGCCATAG-3'). Amplicons were cloned in pCR2.1-TOPO (Invitrogen) and sequenced.
Expression of Recombinant Agrin
A cDNA (GenBank accession no. BP758133) encoding mouse agrin in pBC SK+ (Stratagene, La Jolla, CA) was provided by Dr. Susumu Seino (Kobe University, Kobe, Japan). A 3632-bp KpnI-EcoRI fragment of this clone that begins with 56 bp of the 5'-untranslated region and terminates within exon 18 was subcloned into pcDNA3.1/myc-His (Invitrogen). The resulting construct, agrin1–1171, encodes an 1171-amino acid (128 kd) epitope-tagged fragment that includes both GAG attachment sites identified in the chick sequence. A truncated construct was generated that recapitulates the form of agrin expressed from the Agrnfl allele after Cre recombination. A cDNA of 1825 bp was generated by PCR using agrin1–1171 as template, 0.2 µmol/L of the primers 5'-CCAAGCTTCGCCATGGTCCGCCCGCGGC-3' and 5'-CAGAATTCTGGCAGTGTCCGGCTGAGGCC-3' mismatched (underlined) to introduce HindIII and EcoRI adapters, 200 µmol/L dNTPs, and 5 U of Herculase Taq polymerase (Stratagene) in the supplied buffer. The PCR product was cloned into the HindIII and EcoRI sites of pcDNA3.1/myc-His. The resulting construct, agrin1–602, comprises all coding sequence up to the 3' end of exon 6 and encodes a 602-amino acid (70 kd) epitope-tagged fragment of agrin.
Cell Culture, Transfection, and Western Blotting
COS-7 and 293 cells maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum were transiently transfected with agrin1–1171, agrin1–602, or the empty vector using Lipofectamine (Invitrogen). After culturing in serum-free Opti-MEM (Invitrogen), conditioned medium was recovered and cleared by centrifugation (300 x g for 15 minutes at 4°C). Cells lysates were prepared in 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) containing Complete protease inhibitor (Roche, Indianapolis, IN) and cleared by centrifugation (16,000 x g for 15 minutes at 4°C). Samples were subjected to reducing SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Imobillon-P membranes (Millipore, Bedford, MA). Blots were blocked with 3% bovine serum albumin (BSA) in 10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.1% Tween 20 and incubated with mouse anti-human Myc antibody 9E10 (Calbiochem, San Diego, CA) diluted 1:2000 or rat anti-mouse perlecan antibody (Chemicon, Temecula, CA) diluted 1:1000. After washing in Tris-buffered saline/Tween 20, blots were probed with biotinylated anti-mouse or anti-rat antibodies diluted 1:1000, then incubated in ABC-horseradish peroxidase reagent (Vector Laboratories, Burlingame, CA), and detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL). For deglycosylation, cell lysates or media (buffered with the addition of NaOAc, pH 7.0, to 50 mmol/L and CaCl2 to 2 mmol/L) were incubated with PNGase F (0.1 U/ml; Prozyme, San Leandro, CA), heparinase III (2 U/ml; Sigma, St. Louis, MO), or chondroitinase ABC (0.4 U/ml; Seikagaku, Tokyo, Japan) overnight at 37°C.
Histology and Immunostaining
Formalin-fixed, paraffin-embedded kidney sections were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff reagent. Kidneys were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA) and frozen in 2-methylbutane cooled in a dry-ice/ethanol bath. Unfixed cryosections were blocked with 1.5% goat serum in phosphate-buffered saline and stained with the antibodies indicated in Table 1
. Phosphate-buffered saline was substituted with 20 mmol/L Tris-HCl, pH 7.4, and 100 mmol/L NaCl for detection of 
-dystroglycan. For HS staining, sections were fixed in acetone for 10 minutes at 4°C and blocked with 10% goat serum and 1% BSA in phosphate-buffered saline using the Mouse-on-Mouse kit (Vector Laboratories). Neuromuscular junctions were labeled with rhodamine-conjugated -bungarotoxin (Molecular Probes, Eugene, OR) diluted 1:200.
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Minced cortex was immersion-fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.2, postfixed in 1% OsO4, and then dehydrated and embedded in Polybed (Polysciences, Warrington, PA). Sections were counterstained with uranyl acetate/lead citrate and examined with a CX-100 electron microscope (JEOL, Tokyo, Japan). For polyethyleneimine (PEI) labeling, samples were incubated 30 minutes in 0.5% PEI (1.8 kd; Sigma) in 0.9% NaCl, pH 7.3. After washing in 0.1 mol/L cacodylate, specimens were fixed in the same buffer containing 2.5% glutaraldehyde and 2% phosphotungstic acid, pH 7.3, and then postfixed and embedded as above. Glomerular capillary loops were photographed at magnification x14,000 using a blinded experimental design, and the number of PEI aggregates per micrometer along each aspect of the GBM was counted. Analysis of E17.5-P0 Agrndel/del (n = 4) and control littermates (n = 4) was based on 40 micrographs representing 
22 glomeruli for each group. For podocyte-specific knockouts (n = 7) and controls (n = 8) 7 weeks to 11 months of age, 97 micrographs representing 38 glomeruli for each group were used.
Clinical Chemistry, Ficoll Clearance Studies, and Protein-Overload Proteinuria
Urine samples were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. Urinary protein and creatinine concentrations were measured using Biuret and Jaffé reactions, respectively, on a Cobas Mira Plus analyzer (Roche). Enzyme-linked immunosorbent assays were used to quantify albumin (Exocell, Philadelphia, PA) and total IgG (Alpha Diagnostics, San Antonio, TX). To assess glomerular charge selectivity, mice were given a bolus of fluorescein isothiocyanate-labeled carboxymethyl Ficoll-70 (250 µg/g body weight; TdB Consultancy, Uppsala, Sweden) in 0.9% NaCl by tail vein injection and then housed 24 hours in metabolic cages (Hatteras Instruments, Cary, NC). The concentration of the tracer in urine was determined by diluting samples and standards in 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, and measuring the fluorescence with a QuantaMaster fluorimeter (
ex 488 nm, em 529 nm; Photon Technology International, Lawrenceville, NJ). The fraction of the dose excreted over 24 hours was calculated for comparison. To induce overload proteinuria, mice were given daily intraperitoneal injections of endotoxin-free BSA (15 mg/g body weight; Sigma) in 0.9% NaCl for 5 days. Urine was collected at 24-hour intervals before injection.
Statistical Analyses
Minitab v13.1 statistical software (State College, PA) was used to analyze PEI and urinalysis data using two-sample t-tests, and a general linear model for analysis of variance was applied to analyze the protein-overload data. Differences were considered significant at P < 0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Agrin is essential for neuromuscular synaptogenesis and has been extensively studied in this context. However, the early lethality of agrin-deficient mice poses a barrier to studies that might define important roles for agrin during postnatal life. We therefore generated a conditional allele (Agrnfl; Figure 1A
) by introducing loxP sites within introns 6 and 33 of the mouse Agrn gene. Agrnfl/fl mice were phenotypically normal, indicating agrin was properly expressed from the "floxed" allele. Excision of the frt-flanked neo cassette (Agrnflneo) had no effect; alleles containing or lacking this insert were used interchangeably and are hereafter referred to as Agrnfl.
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) in which genomic sequence from within exon 6 to intron 33 was replaced by a loxP-flanked PGK-neo cassette.31
Deletion of the neo insert (Agrndelneo) by crossing to ß-actin-Cre mice37
had no phenotypic effect, and both forms of this allele are hereafter referred to as Agrndel, unless otherwise noted. Finally, we used a gene trap allele (Agrnß-geo; Figure 1C
) that disrupts the Agrn gene 3' of the NtA-encoding exons.26 Agrn Knockout Mice Synthesize N-Terminal Truncated Forms of Agrin
Each mutant allele retains the capacity to encode variants of agrin with biological activity. Agrnß-geo/ß-geo mutants, for example, lack BM-associated agrin but express normal levels of the neuron-specific isoform.26
The Agrndel and recombined Agrnfl alleles represent extensive and nearly identical genomic deletions, but both could theoretically encode truncated forms of agrin, because they retain multiple intact exons. Importantly, the splicing of mutant transcripts from either exon 5 or 6 to exon 36 would result in an in-frame mRNA encoding a form of "miniagrin" that could be biologically active.38
Reverse transcriptase-PCR using primers in exons 6 and 36 yielded a single product from podocyte-specific knockout kidney (Agrnfl/fl;2.5P-Cre; see below) that was not amplified from liver of the same mice or from wild-type kidney. Sequencing revealed splicing occurred from exons 6 to 34, thereby introducing a frameshift that disrupts the C terminus of any protein derived from a recombined Agrnfl allele (Figure 1A)
. No products were amplified from fetal Agrndel/del kidney using a forward primer in exon 5 and reverse primers in exons 34, 35, or 36 due to the intervening neo cassette, but the same reactions using RNA from Agrndelneo/+ kidney yielded amplicons spliced from exons 5 to 34, which also introduces a frameshift (Figure 1B)
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Agrin is present in the GBM of early capillary loop stage glomeruli in fetal human kidney.39
Here, normal fetal mouse kidney was stained with a panel of three different antisera raised against the C terminus of agrin. Each gave the same pattern, labeling the BM and vascular cleft of S-shaped nephrons and pre-capillary loop-stage glomeruli (Figure 2A)
. At later stages of glomerular development, there was prominent staining of the GBM (Figure 2B)
. The BMs of collecting ducts, peritubular capillaries, and vascular smooth muscle cells were positive with all antisera. Agrndel/del and Agrnß-geo/ß-geo kidneys failed to stain with C-terminal antibodies (Figure 2, C and D
, respectively), confirming that they are agrin specific.
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. Unexpectedly, MI-91 also stained Agrndel/del mutant kidney in a manner indistinguishable from controls (Figure 2G)
, but its failure to label Agrnß-geo/ß-geo kidney (Figure 2H)
proves its specificity for agrin. These findings were confirmed using a polyclonal N-terminal antibody, GR-14 (results not shown). Thus, although the Agrndel allele is functionally null, it encodes an N-terminal fragment of agrin that is properly localized to BMs. Based on the structure of the mutant alleles, the epitopes recognized by MI-91 and GR-14 must be located within the first five follistatin-like domains. Podocytes Are Responsible for Deposition of Agrin in Mature GBM
Transgenic mice expressing Cre from the Pax3 promoter (P3pro-Cre) were used to mutate Agrn broadly in developing kidney. P3pro-Cre is expressed by cells of the metanephric mesenchyme and targets all of their descendants, including glomerular and tubular epithelial cells.41 A potentially complicating issue is that the transgene is also expressed at other sites throughout the embryo.36 Agrnfl/fl;P3pro-Cre mutants phenocopied agrin-deficient mice; mutants were stillborn, and their diaphragms lacked neuromuscular junctions (results not shown). This suggests that Cre was active in motoneurons, and it establishes that the Agrnfl allele is functionally null upon recombination. Nevertheless, these mutants proved useful for investigating the cellular origin of agrin in the GBM. In late fetal Agrnfl/fl;P3pro-Cre kidneys labeled with C-terminal agrin antibodies, the BM of S-shaped nephrons was negative, and the GBM of mature glomeruli was very weakly positive. Ureteric bud and collecting duct BMs were positive, because P3pro-Cre is not expressed in ureteric bud derivatives. The small amount of agrin detected in mutant GBM is probably endothelial-derived, because the BM of earlier avascular S-shaped nephrons was already negative. These findings suggest that glomerular endothelial cells, at least in late fetal kidney, are not a significant source of GBM agrin.
Transgenic mice that express Cre under the human NPHS2 promoter (2.5P-Cre) were used to specifically target podocytes. In this system, recombination occurs when Cre is expressed by immature podocytes in early capillary loop stage glomeruli.35
Mice lacking podocyte-derived agrin displayed no overt phenotype. Mutants were fertile, lived until at least 1 year of age, and had body weights not significantly different from sex-matched control littermates. The GBM of normal adults was intensely labeled with C-terminal agrin antibodies (Figure 2, I and J)
. As in fetal mice, all BMs in normal adult kidney were labeled with the N-terminal mAb MI-91 (Figure 2, M and N)
. The GBM of Agrndel/fl;2.5P-Cre mutants analyzed between 1 and 3 weeks of age showed a marked reduction in staining with C-terminal antibodies. By 3 weeks, labeling of mutant GBM was segmental, with only short stretches of some capillary loops weakly positive. The GBM of conditional mutants beyond 3 weeks of age failed to stain with C-terminal antibodies (Figure 2, K and L)
, implicating podocytes as the source of agrin in mature GBM. However, GBM agrin levels were clearly influenced by the mutant genotype. In Agrndel/fl;2.5P-Cre and Agrnß-geo/fl;2.5P-Cre mice 2 to 9.5 months of age, the GBM was either negative (n = 23) or showed weak segmental staining (n = 4). GBM labeling was more prevalent in Agrnfl/fl;2.5P-Cre mutants, as noted in 8 of 11 adult mice examined. We hypothesize that this reflects inefficient recombination of both Agrnfl alleles in some podocytes, resulting in early deposition of agrin in the GBM that persists because of slow turnover. To avoid this confounding issue, the studies herein used only conditional mutants that were confirmed to lack GBM agrin by immunostaining. All BMs in conditionally mutant kidneys were labeled with the N-terminal mAb MI-91 (Figure 2, O and P)
, irrespective of genotype, because the recombined Agrnfl allele, like the Agrndel allele, encodes an N-terminal fragment of agrin.
Truncated Forms of Agrin Expressed by Mutants Are Not Glycanated
Mouse agrin contains many potential sites for GAG attachment. Although the truncated mutant proteins lack the sites identified in other species, they retain others that could carry GAG chains in vivo (Figure 3A)
. To investigate this, in vitro studies were performed using an epitope-tagged construct, agrin1–602, that recapitulates the form synthesized from the recombined Agrnfl allele. This cDNA includes all of the coding sequence of the Agrndel allele. A longer construct, agrin1–1171, that includes both GAG attachment sites identified in chick served as a control.
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. This size is slightly greater than that predicted from the deduced amino acid sequence (128 kd), a discrepancy explained in part by N-linked glycosylation, because PNGase F digestion shifted this band to 134 kd (results not shown). In the medium, agrin1–1171 was detected as a smear typical of proteoglycans that ranged from 190 to 250 kd. Chondroitinase digestion shifted this species to a more discrete band of 172 kd, indicating that agrin1–1171 carries predominantly CS-GAGs (Figure 3C)
. A diffuse smear remained that may be explained by incomplete digestion or by the presence of chondroitinase-insensitive GAGs. This protein was also N-glycosylated, because PNGase F digestion further reduced its size to 163 kd. Heparinase digestion had little effect on secreted agrin1–1171, indicating that it does not carry HS-GAG (Figure 3C)
. As a control, blotting for perlecan revealed a high-molecular weight species in undigested samples of media that shifted to a discrete band of 400 kd after heparinase digestion, consistent with the size of the perlecan core protein (Figure 3D)
. This proves that COS-7 cells possess the necessary pathways for biosynthesis of HSPGs and confirms that heparinase digestion was efficient.
Recombinant agrin1–602 was detected as a band of 79 kd in cell lysates (Figure 3B)
. PNGase F digestion shifted the band to 74 kd, closer to its predicted mass of 70 kd (not shown). The protein was secreted to the medium, where it was detected as a band of 86 kd (Figure 3B)
. The fact that it was detected as a discrete species suggests that agrin1–602 does not carry GAGs; consistent with this, it was insensitive to digestion with chondroitinase (Figure 3C)
and heparinase (results not shown). The same findings were made using transfected 293 cells.
Loss of GBM-HS in Podocyte-Specific Knockouts
Perlecan was detected in all BMs of normal fetal kidney and colocalized with agrin in the vascular cleft of S-shaped nephrons and the nascent GBM of early capillary loop stage glomeruli. GBM labeling (Figure 4A)
diminished as glomeruli matured, and perlecan became prominent in the mesangium. The same staining pattern was noted in Agrndel/del (Figure 4B)
and Agrnß-geo/ß-geo mutants (results not shown). In adult glomeruli, only the mesangial matrix and Bowman’s capsule BM were significantly labeled (Figure 4C)
. In podocyte-specific knockouts up to 7 months of age, glomerular staining for perlecan was properly localized to the mesangium (Figure 4D)
.
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. In adult conditional mutants, staining of the GBM was either reduced (n = 1) or absent (n = 7), consistent with a loss of GBM-HS (Figure 4F)
. This finding was supported by staining with mAb NAH46 directed against N-acetylated domains in precursor forms of HS (results not shown). Dual-labeling with mAb MI-91 revealed glomeruli present in both control and mutant tissue sections (Figure 4, G and H
, respectively). CS-GAGs were detected using mAb CS-56.44
In normal adults, it diffusely stained the interstitium, Bowman’s capsule, and tubular BMs but did not label the GBM. The staining pattern in adult conditional knockouts was identical, indicating that no CSPG carrying the CS-56 epitope is aberrantly deposited in mutant GBM (results not shown). Distribution of Other Renal BM Components and Their Receptors in Agrin Mutants
Laminin in mature GBM is a heterotrimer consisting of the 5, ß2, and 1 chains. An antibody to the ß2 chain labeled the GBM and vascular smooth muscle in normal fetal mice (Figure 5A)
. In Agrnß-geo/ß-geo mutants, laminin ß2 was detected in these sites in a linear BM-like distribution but also in a punctate pattern consistent with an intracellular localization (Figure 5B)
. Punctate labeling was observed for all other laminin isoforms evaluated in Agrnß-geo/ß-geo mutants (1, 2, 5, ß1, and 1) but not in Agrndel/del mice, where the staining pattern for each was indistinguishable from controls (results not shown). The Agrnß-geo allele encodes a chimeric protein consisting of the agrin NtA domain fused to ß-geo. This is expected to be membrane-bound in the ER in a configuration that exposes the NtA to the lumen where it would be free to interact with its only known binding partner, laminin 1; we hypothesize that this leads to "co-trapping" of laminin trimers containing 1, as previously shown.45
In contrast, nidogen-1 and the type IV collagen chains of mature GBM (3, 4, and 5) were detected in the normal pattern and at normal levels in agrin-deficient mice (Figure 5, C and D
; results not shown). All of these BM components were expressed normally in adult conditional knockout kidney.
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3 was detected in normal fetal glomeruli in a podocyte distribution, and there was comparable labeling of Agrndel/del mutants (Figure 5, E and F
, respectively). Staining was also normal in adult conditional mutants (results not shown). Dystroglycan is expressed by all epithelial cells in normal fetal kidney, including podocytes (Figure 5G)
. There was comparable labeling in Agrndel/del mutants (Figure 5H)
and in 3-week-old conditional knockouts (results not shown), so agrin is not necessary for proper dystroglycan localization. Agrin Mutants Show No Significant Renal Histological or Ultrastructural Abnormalities
Compared with normal controls (Figure 6, A and B)
, no pathological changes were noted in Agrndel/del (Figure 6, D and E)
or Agrnß-geo/ß-geo (not shown) kidneys. Glomerular ultrastructure in Agrndel/del (Figure 6F)
and Agrnß-geo/ß-geo mutants (not shown) was also indistin-guishable from controls (Figure 6C)
; thus, agrin is not required for nephrogenesis. Conditional mutants (Figure 6J)
showed no histopathology compared with controls (Figure 6G)
until at least 8 months of age. Likewise, glomerular ultrastructure in conditional mutants was indistinguishable from controls up to 4 months of age (Figure 6, K and H
, respectively). GBM irregularities, characterized by focal thickening and epithelial protrusions of GBM material, were noted in mutants beyond 4 months (Figure 6L)
. Although these changes were occasionally observed in control mice, they were more prominent in mutants, especially when comparisons were made between littermates (Figure 6I)
. Morphometric analyses were not performed and may be confounded by the mixed genetic background of these mice. In any event, agrin is dispensable for the structural organization and integrity of the glomerular capillary wall.
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Anionic sites were visualized by EM after labeling with the cationic probe PEI. In mature glomeruli of normal fetal mice, there was punctate labeling of the GBM concentrated in a regular pattern along both the subepithelial and subendothelial laminae rarae (Figure 7, A and C)
. This staining pattern presumably reflects concentration of the probe at sites with the greatest negative charge density. In littermate Agrndel/del mutants, subendothelial labeling was comparable with controls, but there was a marked reduction in the number of anionic sites underlying podocytes (Figure 7, B and D)
. Quantitation of PEI aggregates per micrometer of GBM length (Figure 7E)
revealed a significant reduction in subepithelial anionic sites compared with controls (5.41 ± 0.24 versus 13.95 ± 0.33; P < 0.001). There was also a small reduction in subendothelial anionic sites in mutants (11.25 ± 0.33 versus 12.67 ± 0.36; P = 0.005). Labeling of mesangial matrix, Bowman’s capsule, and tubular BMs was comparable in all mice and provided an internal control for probe penetration.
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. PEI labeling was reduced throughout the GBM in adult conditional mutants (Figure 7, H and I)
. Quantitative analysis revealed subepithelial anionic sites per micrometer of normal adult GBM numbered 15.39 ± 0.27 (Figure 7J)
. Subepithelial labeling was significantly reduced in adult conditional mutants (8.00 ± 0.26; P < 0.001), whereas subendothelial anionic sites were equivalent in number to controls (9.29 ± 0.21 versus 8.99 ± 0.19; P = 0.29). In addition, many subepithelial anionic sites were less intensely labeled in the mutant than in the control, a qualitative difference that probably reflects reduced charge density in the GBM. The charge defects in mutants show that podocyte-derived agrin contributes significantly to the fixed negative charge of adult GBM. The Glomerular Filtration Barrier Is Not Compromised in Agrin Mutants
Agrin-deficient mice die shortly after birth and fail to fill their bladders, precluding urinalysis as a means of assessing renal function. To test this indirectly, amniotic fluid from E16.5 to E18.5 embryos was analyzed by SDS-PAGE. Albumin was detected as a prominent 70-kd band present at equivalent levels in samples from mutant and control mice (results not shown).
By SDS-PAGE and determination of protein-, albumin-, and IgG-to-creatinine ratios, conditional mutants displayed no evidence of significantly elevated urinary protein levels up to 10 months of age (Figure 8, A–C
; results not shown). Mutant and control urines were also indistinguishable when analyzed by nondenaturing PAGE to assess differences relating to protein charge (results not shown). In mutants, tubular resorption of filtered proteins may be sufficient to compensate for a loss of glomerular permselectivity. To test the glomerular barrier in the setting of an experimental challenge, mice were injected with BSA for 5 consecutive days to induce protein-overload proteinuria. Mutant and control mice responded with a marked increase in urinary protein/creatinine ratios, but at no time point were these values significantly different between groups (Figure 8D)
. Finally, glomerular charge selectivity was assessed by measuring the clearance of carboxymethyl Ficoll-70. The amount of this inert and highly anionic tracer recovered in urine directly reflects its glomerular permeability and would be expected to differ between mutant and controls only if the intrinsic negative charge of the GBM serves as a physiologically relevant barrier. The fraction of the administered dose excreted over 24 hours by conditional mutants was not significantly different from controls (Figure 8E)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Much of our studies involved defining the nature of the truncated agrin proteins produced by the Agrndel and recombined Agrnfl alleles. Both mutant proteins are expected to be composed of the NtA domain and roughly one-half the complement of follistatin-like domains; the interaction between the NtA and the laminin 1 chain27
is apparently sufficient for incorporation of the truncated proteins into BMs. It is a formal possibility that the mutant proteins retain unknown functions despite their lack of GAGs and the C terminus that binds cellular receptors. To resolve this may ultimately require the generation of a conditional allele that deletes the entire coding region.
Agrin is present in most renal BMs as revealed by N-terminal antibodies, but it is not required in these sites for nephrogenesis. Kidney development is also normal in mice lacking collagen XVIII21 and perlecan-HS.19 In contrast, HS itself is critical for this process, because deficiency of certain enzymes involved in HS biosynthesis causes renal agenesis.46,47 Presumably, this dramatic phenotype reflects the fact that all forms of HS linked to both BM- and cell-associated proteoglycans (eg, syndecans and glypicans) are disrupted.
The GBM is assembled from the same basic repertoire of components found in other BMs, but it contains specific isoforms essential for its role as a filtration barrier. Mature GBM isoforms of laminin (5ß21) and type IV collagen (345) were detected in mutants, indicating that the well-defined developmental transitions involving these proteins48,49
proceed normally in the absence of agrin. Perlecan was not deposited ectopically to compensate for the loss of agrin, so we expected the amount of HS in mutant GBM to be disrupted. This was assessed by immunostaining using antibodies JM-403 and NAH46 that recognize specific HS structures in the GBM. The former has been used to demonstrate the loss of GBM-HS in human and experimental kidney disease, including human membranous nephritis, lupus nephritis, minimal change disease, and diabetic nephropathy14,39,50
and rat adriamycin nephropathy and Heymann nephritis.51,52
Although these studies support the theory that reductions in GBM-HS contribute directly to loss of barrier function, labeling with mAb JM-403 was recently reported to be normal in diabetic humans and rats with microalbuminuria, and it was concluded that loss of GBM-HS may be a secondary event that occurs in advanced disease.53
Here, immunostaining with both antibodies revealed that adult conditional mutants lack GBM-HS. This confirms that mouse agrin exists as a HSPG in vivo but questions the importance of GBM-HS for kidney function.
Anionic sites in the laminae rarae of the GBM were described decades ago and attributed to HSPGs based on their susceptibility to GAG-degrading enzymes, but the specific forms that impart charge to the GBM have never been formally identified. Here, we show that the majority of anionic sites in the lamina rara externa of fetal and adult GBM represent agrin. GBM charge was not disrupted in adult perlecan-HS mutants,20
which in light of the present study can be explained by the presence of agrin. Agrin mutants showed a 50% reduction in the number of subepithelial anionic sites, and on a qualitative basis, those that remained appeared less intensely stained by PEI. This charge defect is, in both respects, as profound as that typically attributed to be a causative factor in human and experimental kidney disease; reported alterations range from a 63% reduction in human congenital nephrotic syndrome,54
to 57% in membranous nephritis, to 52 to 20% in focal segmental glomerulosclerosis, to 21% in minimal change disease,55,56
and to 28 to 18% in rat models of diabetic nephropathy and puromycin nephrosis.57,58
The key finding of this work is that agrin mutant mice have normal renal function despite a severe disruption of GBM charge. To test the glomerular barrier in the setting of an experimental challenge, we used a model of protein-overload proteinuria. This choice was based on an earlier study of perlecan-HS-deficient mice that used this technique to reveal a role for perlecan in glomerular filtration.20 No such defect was found in agrin mutants under similar experimental conditions. These findings are difficult to reconcile, especially considering the restriction of perlecan to the mesangium in adults. We also performed a clearance study using the anionic tracer carboxymethyl Ficoll to test glomerular charge selectivity. Clearance rates were nearly identical in mutant and control mice, suggesting that if a charge barrier exists, it is probably associated with cellular constituents of the capillary wall.
Although agrin is not needed for glomerular filtration, it may play important roles in other aspects of renal pathophysiology. By virtue of their charge, HSPGs are thought to influence the deposition and localization of circulating immune complexes and promote planting of cationic antigens that can elicit in situ formation of immune deposits.16,59-61 Conditional agrin knockout mice may provide a useful model to investigate the impact of GBM charge on these processes.
Beyond excluding a role for agrin in glomerular filtration, our findings challenge the notion that the GBM serves as a charge barrier and should force a reevaluation of this concept. In doing so, they direct attention to other sites, particularly the anionic glycocalyx of podocytes and glomerular endothelial cells. Although a role for the former in maintaining the elaborate architecture of podocytes has been defined, attention is only now becoming focused on the endothelial glycocalyx proper as a component of the filtration barrier.62 Dissecting these factors will hopefully advance our understanding of the pathogenesis of human kidney disease and lead to new approaches for its treatment.
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Supported by an American Heart Association Established Investigator Award and National Institutes of Health grant R01DK064687 (to J.H.M.), an Alzheimer’s Association New Investigator Research Award (to R.W.B.), a National Kidney Foundation Fellowship (to S.J.H.), and Dutch Kidney Foundation grant C05.5152. Mice were housed in a facility supported by National Institutes of Health grant C06RR015502.
Accepted for publication April 12, 2007.
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