Disruption of Glomerular Basement Membrane Charge through Podocyte-Specific Mutation of Agrin Does Not Alter Glomerular Permselectivity
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.061116
ISSN1525-2191
AutoresScott J. Harvey, George Jarad, Jeanette M. Cunningham, Angelique L. Rops, Johan van der Vlag, Jo H. M. Berden, Marcus J. Moeller, Lawrence B. Holzman, Robert W. Burgess, Jeffrey H. Miner,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoGlomerular charge selectivity has been attributed to anionic heparan sulfate proteoglycans (HSPGs) in the glomerular basement membrane (GBM). Agrin is the predominant GBM-HSPG, but evidence that it contributes to the charge barrier is lacking, because newborn agrin-deficient mice die from neuromuscular defects. To study agrin in adult kidney, a new conditional allele was used to generate podocyte-specific knockouts. Mutants were viable and displayed no renal histopathology up to 9 months of age. Perlecan, a HSPG normally confined to the mesangium in mature glomeruli, did not appear in the mutant GBM, which lacked heparan sulfate. Moreover, GBM agrin was found to be derived primarily from podocytes. Polyethyleneimine labeling of fetal kidneys revealed anionic sites along both laminae rarae of the GBM that became most prominent along the subepithelial aspect at maturity; labeling was greatly reduced along the subepithelial aspect in agrin-deficient and conditional knockout mice. Despite this severe charge disruption, the glomerular filtration barrier was not compromised, even when challenged with bovine serum albumin overload. We conclude that agrin is not required for establishment or maintenance of GBM architecture. Although agrin contributes significantly to the anionic charge to the GBM, both it and its charge are not needed for glomerular permselectivity. This calls into question whether charge selectivity is a feature of the GBM. Glomerular charge selectivity has been attributed to anionic heparan sulfate proteoglycans (HSPGs) in the glomerular basement membrane (GBM). Agrin is the predominant GBM-HSPG, but evidence that it contributes to the charge barrier is lacking, because newborn agrin-deficient mice die from neuromuscular defects. To study agrin in adult kidney, a new conditional allele was used to generate podocyte-specific knockouts. Mutants were viable and displayed no renal histopathology up to 9 months of age. Perlecan, a HSPG normally confined to the mesangium in mature glomeruli, did not appear in the mutant GBM, which lacked heparan sulfate. Moreover, GBM agrin was found to be derived primarily from podocytes. Polyethyleneimine labeling of fetal kidneys revealed anionic sites along both laminae rarae of the GBM that became most prominent along the subepithelial aspect at maturity; labeling was greatly reduced along the subepithelial aspect in agrin-deficient and conditional knockout mice. Despite this severe charge disruption, the glomerular filtration barrier was not compromised, even when challenged with bovine serum albumin overload. We conclude that agrin is not required for establishment or maintenance of GBM architecture. Although agrin contributes significantly to the anionic charge to the GBM, both it and its charge are not needed for glomerular permselectivity. This calls into question whether charge selectivity is a feature of the GBM. The glomerular capillary wall is thought to function as both a size- and charge-selective barrier. The concept of charge selectivity emerged from a series of now classic studies examining the clearance of tracers differing in charge.1Chang RL Deen WM Robertson CR Brenner BM Permselectivity of the glomerular capillary wall: III. 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These types of studies have been challenged on several fronts, particularly on the basis of deformation, degradation, or selective uptake of the differentially charged tracers. Whether charge selectivity exists and is important for glomerular function is a subject of intense debate.4Comper WD Glascow EF Charge selectivity in kidney ultrafiltration.Kidney Int. 1995; 47: 1242-1251Crossref PubMed Scopus (101) Google Scholar Nevertheless, the concept remains a cornerstone of renal physiology. 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 podocalyxin5Kerjaschki D Sharkey DJ Farquhar MG Identification and characterization of podocalyxin: the major sialoprotein of the renal glomerular epithelial cell.J Cell Biol. 1984; 98: 1591-1596Crossref PubMed Scopus (389) Google Scholar may contribute to the barrier. 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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.15Groffen AJ Veerkamp JH Monnens LA van den Heuvel LP Recent insights into the structure and functions of heparan sulfate proteoglycans in the human glomerular basement membrane.Nephrol Dial Transplant. 1999; 14: 2119-2129Crossref PubMed Scopus (72) Google Scholar, 18Saarela J Rhen M Oikarinen A Autio-Harmainen H Pihlajaniemi T The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans.Am J Pathol. 1998; 153: 611-626Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar 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.19Rossi M Morita H Sormunen R Airenne S Kreivi M Wang L Fukai N Olsen BR Tryggvason K Soininen R Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney.EMBO J. 2003; 22: 236-245Crossref PubMed Scopus (194) Google Scholar, 20Morita H Yoshimura A Inui K Ideura T Watanabe H Wang L Soininen R Tryggvason K Heparan sulfate of perlecan is involved in glomerular filtration.J Am Soc Nephrol. 2005; 16: 1703-1710Crossref PubMed Scopus (93) Google Scholar Collagen XVIII mutants have mild mesangial expansion and only slightly elevated serum creatinine levels compared with controls.21Utriainen A Sormunen R Kettunen M Carvalhaes LS Sajanti E Eklund L Kaappinen R Kitten GT Pihlajaniemi T Structurally altered basement membranes and hydrocephalus in a type XVIII collagen deficient mouse line.Hum Mol Genet. 2004; 13: 2089-2099Crossref PubMed Scopus (114) Google Scholar 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.22Groffen AJ Ruegg MA Dijkman H van de Velden TJ Buskens CA van den Born J Assmann KJ Monnens LA Veerkamp JH van den Heuvel LP Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.J Histochem Cytochem. 1998; 46: 19-27Crossref PubMed Scopus (148) Google Scholar, 23Groffen AJ Buskens CA van Kuppevelt TH Veerkamp JH Monnens LA van den Heuvel LP Primary structure and high expression of human agrin in basement membranes of adult lung and kidney.Eur J Biochem. 1998; 254: 123-128Crossref PubMed Scopus (70) Google Scholar 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.24Tsen G Halfter W Kroger S Cole GJ Agrin is a heparan sulfate proteoglycan.J Biol Chem. 1995; 270: 3392-3399Crossref PubMed Scopus (237) Google Scholar, 25Winzen U Cole GJ Halfter W Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters.J Biol Chem. 2003; 278: 30106-30114Crossref PubMed Scopus (49) Google Scholar 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.25Winzen U Cole GJ Halfter W Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters.J Biol Chem. 2003; 278: 30106-30114Crossref PubMed Scopus (49) Google Scholar Alternative promoters give rise to two isoforms of agrin that are either BM or cell associated, with the latter being specific to neurons.26Burgess RW Skarnes WC Sanes JR Agrin isoforms with distinct amino termini: differential expression, localization, and function.J Cell Biol. 2000; 151: 41-52Crossref PubMed Scopus (141) Google Scholar BM-associated agrin binds to the laminin γ1 chain via a globular domain (NtA) at its N terminus and to dystroglycan and integrin receptors through its C terminus.27Kammerer RA Schulthess T Landwehr R Schumacher B Lustig A Yurchenco PD Ruegg MA Engel J Denzer AJ Interaction of agrin with laminin requires a coiled-coil conformation of the agrin binding site within the laminin γ1 chain.EMBO J. 1999; 18: 6762-6770Crossref PubMed Google Scholar, 28Gesemann M Brancaccio A Schumacher B Ruegg MA Agrin is a high-affinity binding protein of dystroglycan in non-muscle tissue.J Biol Chem. 1998; 273: 600-605Crossref PubMed Scopus (118) Google Scholar, 29Martin PT Sanes JJ Integrins mediate adhesion to agrin and modulate agrin signaling.Development. 1997; 124: 3909-3917PubMed Google Scholar By virtue of these interactions, agrin is thought to be an integral part of the molecular complex linking podocytes to the GBM.30Raats CJ van Den Born J Bakker MA Oppers-Walgreen B Pisa BJ Dijkman HB Assmann KJ Berden JH Expression of agrin, dystroglycan and utrophin in normal renal tissue and in experimental glomerulopathies.Am J Pathol. 2000; 156: 1749-1765Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Agrin-deficient mice are grossly normal but die at birth with severe neuromuscular defects.31Lin W Burgess RW Dominguez B Pfaff SL Sanes JR Lee KF Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse.Nature. 2001; 410: 1057-1064Crossref PubMed Scopus (450) Google Scholar 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. 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 al32Rupp F Ozcelik T Linial M Peterson K Francke U Scheller R Structure and chromosomal localization of the mammalian agrin gene.J Neurosci. 1992; 12: 3535-3544Crossref PubMed Google Scholar) is replaced by a loxp-flanked PGK-neo cassette31Lin W Burgess RW Dominguez B Pfaff SL Sanes JR Lee KF Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse.Nature. 2001; 410: 1057-1064Crossref PubMed Scopus (450) Google Scholar or a gene trap allele (AgrnGt(p1.8TM)192Wcs; denoted Agrnβ-geo) that disrupts Agrn 3′ of the last NtA-encoding exon.26Burgess RW Skarnes WC Sanes JR Agrin isoforms with distinct amino termini: differential expression, localization, and function.J Cell Biol. 2000; 151: 41-52Crossref PubMed Scopus (141) Google Scholar A conditional allele harboring loxP sites within introns 6 and 33 (Agrntm1Rwb; denoted Agrnfl) was generated through "recombineering" in EL350 cells,33Lee EC Yu D Martinez de Velasco J Tessarollo L Swing DA Court DL Jenkins NA Copeland NG A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.Genomics. 2001; 73: 56-65Crossref PubMed Scopus (1000) Google Scholar 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 mice34Rodríguez CI Buchholz F Galloway J Sequerra R Kasper J Ayala R Stewart AF Dymecki SM High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP.Nat Genet. 2000; 25: 139-140Crossref PubMed Scopus (897) Google Scholar 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-Cre35Moeller MJ Sanden SK Soofi A Wiggins RC Holzman LB Podocyte-specific expression of Cre recombinase in transgenic mice.Genesis. 2003; 35: 39-42Crossref PubMed Scopus (249) Google Scholar) or the murine Pax3 promoter (P3pro-Cre36Li J Chen F Epstein JA Neural crest expression of Cre recombinase directed by the proximal Pax3 promoter in transgenic mice.Genesis. 2000; 26: 162-164Crossref PubMed Scopus (137) Google Scholar). 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); AgrnflΔneo, 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 AgrndelΔneo; Agrnβ-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 × C57BL/6J genetic background. Animal experiments were approved by the Washington University Animal Studies Committee. 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. 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. 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 × 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 × 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. 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 α-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 α-bungarotoxin (Molecular Probes, Eugene, OR) diluted 1:200.Table 1Antibodies Used for ImmunohistochemistryAntibodyDilutionSpeciesSupplierAgrin 4,8 (C terminus)300Rabbit anti-humanRegeneron Pharmaceuticals (Tarrytown, NY)Agrin LG (C terminus)25,000Rabbit anti-mouseT. Sasaki (Max Planck Institute, Martinsried, Germany)Agrin (C terminus)300Hamster anti-ratP. Noakes (University of Queensland, Brisbane, Australia)*Hamster antiserum raised against rat agrin fusion proteins.63Agrin (N terminus; mAb MI-91)300Hamster anti-ratJ. Berden (Radboud University, Nijmegen, The Netherlands)Agrin (N terminus; GR-14)800Sheep anti-ratJ. Berden (Radboud University)Perlecan (mAb 1948)300Rat anti-mouseChemicon, Temecula, CAHS (mAb JM-403)100Mouse anti-ratJ.H. Berden (Radboud University)HS (mAb NAH46)100Mouse anti-E. coliSeikagaku, Tokyo, JapanCS (mAb CS-56)200Mouse anti-chickenSigma, St. Louis, MOIntegrin α3200Rabbit anti-chickenM. DiPersio (Albany Medical College, Albany, NY)α-Dystroglycan (mAb IIH6)100Mouse anti-rabbitK. Campbell (University of Iowa, Iowa City, IA)Laminin β22000Rabbit anti-mouseT. Sasaki (Max Planck Institute)Nidogen (mAb 1946)1000Rat anti-mouseChemiconThe following secondary antibodies were used for detection: Cy3-conjugated goat anti-rabbit IgG (1:1500), Cy3- or Cy2-conjugated goat anti-hamster IgG (1:1500 or 1:200, respectively), Cy3-conjugated goat anti-rat IgG (1:1500), fluorescein isothiocyanate-conjugated goat anti-mouse IgM (1:100; Jackson Immunoresearch, West Grove, PA); Alexa Fluor 488-conjugated donkey anti-sheep IgG or goat anti-mouse IgM (1:200; Invitrogen); and Alexa 488 Fluor-conjugated goat anti-mouse IgG1 (1:200; Molecular Probes).* Hamster antiserum raised against rat agrin fusion proteins.63Campanelli JT Hoch W Rupp F Kreiner T Scheller RH Agrin mediates cell contact-induced acetylcholine receptor clustering.Cell. 1991; 67: 909-916Abstract Full Text PDF PubMed Scopus (106) Google Scholar Open table in a new tab The following secondary antibodies were used for detection: Cy3-conjugated goat anti-rabbit IgG (1:1500), Cy3- or Cy2-conjugated goat anti-hamster IgG (1:1500 or 1:200, respectively), Cy3-conjugated goat anti-rat IgG (1:1500), fluorescein isothiocyanate-conjugated goat anti-mouse IgM (1:100; Jackson Immunoresearch, West Grove, PA); Alexa Fluor 488-conjugated donkey anti-sheep IgG or goat anti-mouse IgM (1:200; Invitrogen); and Alexa 488 Fluor-conjugated goat anti-mouse IgG1 (1:200; Molecular Probes). 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 ×14,000 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. 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 (λex 488 nm, λem 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. 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. 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 (AgrnflΔneo) had no effect; alleles containing or lacking this insert were used interchangeably and are hereafter referred to as Agrnfl. The experiments herein also made use of previously described "null" alleles. The first was a targeted knockout (Agrndel; Figure 1B) in which genomic sequence from within exon 6 to intron 33 was replaced by a loxP-flanked PGK-neo cassette.31Lin W Burgess RW Dominguez B Pfaff SL Sanes JR Lee KF Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse.Nature. 2001; 410: 1057-1064Crossref PubMed Scopus (450) Google Scholar Deletion of the neo insert (AgrndelΔneo) by crossing to β-actin-Cre mice37Lewandoski M Meyers EN Martin GR Analysis of fgf8 gene function in vertebrate development.Cold Spring Harb Symp Quant Biol. 1997; 62: 159-168Crossref PubMed Google Scholar 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 (Agrnβ-geo; Figure 1C) that disrupts the Agrn gene 3′ of the NtA-encoding exons.26Burgess RW Skarnes WC Sanes JR Agrin isoforms with distinct amino termini: differential expression, localization, and function.J Cell Biol. 2000; 151: 41-52Crossref PubMed Scopus (14
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