Expression of Agrin, Dystroglycan, and Utrophin in Normal Renal Tissue and in Experimental Glomerulopathies
2000; Elsevier BV; Volume: 156; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65046-8
ISSN1525-2191
AutoresC.J.I. Raats, Jacob van den Born, Marinka A.H. Bakker, Birgitte Oppers‐Walgreen, Brenda J.M. Pisa, Henry Dijkman, Karel J.M. Assmann, Jo H. M. Berden,
Tópico(s)Diabetic Foot Ulcer Assessment and Management
ResumoThe dystrophin-glycoprotein complex, which comprises α. and β-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against α- and β-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane. The dystrophin-glycoprotein complex, which comprises α. and β-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against α- and β-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane. Dystroglycan (DG) is an important member of the dystrophin-glycoprotein complex (DGC) which links the subsarcolemmal cytoskeleton to the basal lamina in skeletal muscle.1Henry MD Campbell KP Dystroglycan: an extracellular matrix receptor linked to the cytoskeleton.Curr Opin Cell Biol. 1996; 8: 625-631Crossref PubMed Scopus (237) Google Scholar The importance of this link becomes clear from the severe muscular dystrophies resulting from mutations in genes that encode different members of the DGC.2Campbell KP Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage.Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (755) Google Scholar, 3Worton R Muscular dystrophies: diseases of the dystrophin-glycoprotein complex.Science. 1995; 270: 755-756Crossref PubMed Scopus (178) Google Scholar, 4Ervasti JM Ohlendieck K Kahl SD Gaver MG Campbell KP Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle.Nature. 1990; 345: 315-319Crossref PubMed Scopus (812) Google Scholar, 5Cote PD Moukhles H Lindenbaum M Carbonetto S Chimaeric mice deficient in dystroglycans develop muscular dystrophy and have disrupted myoneural synapses.Nat Genet. 1999; 23: 338-342Crossref PubMed Scopus (201) Google Scholar DG is synthesized as a large precursor protein and is posttranslationally cleaved into α-DG, a heavily glycosylated peripheral membrane protein and the transmembrane protein β-DG. In skeletal muscle, α-DG is a major binding protein for agrin as well as for laminins.6Bowe MA Deyst KA Leszyk JD Fallon JR Identification and purification of an agrin receptor from torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans.Neuron. 1994; 12: 1173-1180Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 7Gee SH Montanaro F Lindenbaum MH Carbonetto S Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor.Cell. 1994; 77: 675-686Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 8Ibraghimov-Beskrovnaya O Ervasti JM Leveille CJ Slaughter CA Sernett SW Campbell KP Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1184) Google Scholar, 9Yamada H Denzer AJ Hori H Tanaka T Anderson LVB Fujita S Fukuta-Ohi H Shimizu T Ruegg MA Matsumura K Dystroglycan is a dual receptor for agrin and laminin-2 in Schwann cell membrane.J Biol Chem. 1996; 271: 23418-23423Crossref PubMed Scopus (100) Google Scholar α-DG remains noncovalently linked to β-DG, which through its cytoplasmic tail, binds directly to the C-terminal portion of dystrophin10Rosa G Ceccarini M Cavaldesi M Zini M Petrucci TC Localization of the dystrophin binding site at the carboxyl terminus of beta-dystroglycan.Biochem Biophys Res Commun. 1996; 223: 272-277Crossref PubMed Scopus (67) Google Scholar, 11Suzuki A Yoshida M Hayashi K Mizuno Y Hagiwara Y Ozawa E Molecular organization at the glycoprotein-complex-binding site of dystrophin. Three dystrophin-associated proteins bind directly to the carboxy-terminal portion of dystrophin.Eur J Biochem. 1994; 220: 283-292Crossref PubMed Scopus (219) Google Scholar whereas the N-terminal domain of dystrophin binds to the subsarcolemmal actin cytoskeleton.12Yoshida M Ozawa E Glycoprotein complex anchoring dystrophin to sarcolemma.J Biochem. 1990; 108: 748-752Crossref PubMed Scopus (449) Google Scholar, 13Way M Pope B Cross RA Kendrick Jones J Weeds AG Expression of the N-terminal domain of dystrophin in E. coli and demonstration of binding to F-actin.FEBS Lett. 1992; 301: 243-245Abstract Full Text PDF PubMed Scopus (130) Google Scholar Utrophin is an autosomal homologue of dystrophin and can also bind β-DG.14Matsumura K Ervasti JM Ohlendieck K Kahl SD Campbell KP Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle.Nature. 1992; 360: 588-591Crossref PubMed Scopus (442) Google Scholar Recent in vitro studies support the role of the DGC in adhesion. Adhesion of a rat schwannoma cell line to laminin could be inhibited by antibodies against α-DG in vitro.15Matsumura K Chiba A Yamada H Fukuta-Ohi H Fujita S Endo T Kobata A Anderson LVB Kanazawa I Campbell KP Shimizu T A role for dystroglycan in schwannoma cell adhesion to laminin.J Biol Chem. 1997; 272: 13904-13910Crossref PubMed Scopus (85) Google Scholar Furthermore, myotubules of patients with Duchenne muscular dystrophy were unable to adhere to laminin α2 in vitro.16Angoli D Corona P Baresi R Mora M Wanke E Laminin-alpha-2 but not -alpha-1-mediated adhesion of human (Duchenne) and murine (mdx) dystrophic myotubes is seriously defective.FEBS Lett. 1997; 408: 341-344Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (GBM).17Raats CJI Bakker MAH Hoch W Tamboer WPM Groffen AJA Van den Heuvel LPWJ Berden JHM Van den Born J Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.J Biol Chem. 1998; 273: 17832-17838Crossref PubMed Scopus (72) Google Scholar Various other components of the DGC were also identified in the kidney.18Durbeej M Larsson E Ibraghimov-Beskrovnaya O Roberds SL Campbell KP Ekblom P Non-muscle alpha-dystroglycan is involved in epithelial development.J Cell Biol. 1995; 130: 79-91Crossref PubMed Scopus (187) Google Scholar, 19Durbeej M Henry MD Ferletta M Campbell KP Ekblom P Distribution of dystroglycan in normal adult mouse tissues.J Histochem Cytochem. 1998; 46: 449-457Crossref PubMed Scopus (154) Google Scholar, 20Love DR Morris GE Ellis JM Fairbrother U Marsden RF Bloomfield JF Edwards YH Slater CP Parry DJ Davies KE Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse.Proc Natl Acad Sci USA. 1991; 88: 3243-3247Crossref PubMed Scopus (114) Google Scholar Therefore, we hypothesized that glomerular visceral epithelial cells or podocytes are linked to the GBM in a similar way as the myocyte to the basal lamina. The podocytes and GBM, together with the fenestrated endothelium form the glomerular capillary wall (GCW), the barrier preventing passage of plasma proteins into the urinary space during glomerular ultrafiltration. During heavy proteinuria in various human and experimental glomerulopathies, the podocytes show dramatic morphological changes like fusion of foot processes and/or detachment from the GBM.21Grishman E Churg J Focal glomerular sclerosis in nephrotic patients: an electron microscopic study of glomerular podocytes.Kidney Int. 1975; 7: 111-122Crossref PubMed Scopus (138) Google Scholar, 22Powell HR Relationship between proteinuria and epithelial cell changes in minimal lesion glomerulopathy.Nephron. 1976; 16: 310-317Crossref PubMed Scopus (34) Google Scholar, 23Murphy WM Moretta FL Jukkola AF Epithelial foot-process effacement in patients with proteinuria.Am J Clin Pathol. 1979; 72: 529-532Crossref PubMed Scopus (7) Google Scholar, 24Bertani T Poggi A Pozzoni R Delaini F Sacchi G Thoua Y Mecca G Remuzzi G Donati MB Adriamycin-induced nephrotic syndrome in rats: sequence of pathologic events.Lab Invest. 1982; 46: 16-23PubMed Google Scholar, 25Bohman SO Jaremko G Bohlin AB Berg U Foot process fusion and glomerular filtration rate in minimal change nephrotic syndrome.Kidney Int. 1984; 25: 696-700Crossref PubMed Scopus (48) Google Scholar, 26Gabbai FB Gushwa LC Wilson CB Blantz RC An evaluation of the development of experimental membranous nephropathy.Kidney Int. 1987; 31: 1267-1278Crossref PubMed Scopus (24) Google Scholar Several observations suggest that podocyte dysfunction and subsequent detachment contributes to the development of proteinuria. In adriamycin nephropathy (ADN), puromycin aminonucleoside nephrosis, and serum sickness nephritis, proteinuria correlates with podocyte detachment,27Messina A Davies DJ Dillane PC Ryan GB Glomerular epithelial abnormalities associated with the onset of proteinuria in aminonucleoside nephrosis.Am J Pathol. 1987; 126: 220-229PubMed Google Scholar, 28Inokuchi S Shirato I Kobayashi N Koide H Tomino Y Sakai T Re-evaluation of foot process effacement in acute puromycin aminonucleoside nephrosis.Kidney Int. 1996; 50: 1278-1287Crossref PubMed Scopus (56) Google Scholar but not with changes in GBM charge density.29Whiteside C Prutis K Cameron R Thompson J Glomerular epithelial detachment, not reduced charge density, correlates with proteinuria in adriamycin and puromycin nephrosis.Lab Invest. 1989; 61: 650-659PubMed Google Scholar Passage of albumin through the GCW was localized to regions of saponin-induced detachment of podocytes in the single nephron model.30Laurens W Battaglia C Foglieni C De Vos R Malanchini B Van Damme BJC Vanrenterghem YF Remuzzi G Remuzzi A Direct podocyte damage in the single nephron leads to albuminuria in vivo.Kidney Int. 1995; 47: 1078-1086Crossref PubMed Scopus (44) Google Scholar In another study it was found that injection of a monoclonal antibody (mAb) directed against a component of the slit diaphragm resulted in an acute massive proteinuria.31Orikasa M Matsui K Oite T Shimizu F Massive proteinuria in rats by a single intravenous injection of a monoclonal antibody.J Immunol. 1988; 141: 807-814PubMed Google Scholar, 32Kawachi H Matsui K Orikasa M Morioka T Oite T Shimizu F Quantitative studies of monoclonal antibody 5–1-6-induced protein-uric state in rats.Clin Exp Immunol. 1992; 87: 215-219Crossref PubMed Scopus (27) Google Scholar Therefore, it is generally assumed that the extent of podocyte detachment is related to the severity of proteinuria. However, no conclusive data are available on the mechanism of podocyte detachment during proteinuria. The present study focuses on the distribution of agrin, α- and β-DG, and utrophin in the kidney. Expression and localization was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR. on RNA isolated from renal cortex, isolated glomeruli, and cultured podocytes and mesangial cells, and by immunofluorescence (IF) and immunoelectron microscopy (IEM) with monoclonal antibodies against the N- and C-terminus of agrin, α-DG, β-DG, and utrophin. These studies show that apart from agrin, α- and β-DG and utrophin are also present in the GCW and suggest a link between the podocyte and the GBM. Furthermore, the expression of the DGC was studied in two experimental models of proteinuric glomerulopathy: ADN in rats, which serves as a model for the nephrotic syndrome, characterized by extensive fusion of podocyte foot processes; and passive Heymann nephritis (PHN), a model for human membranous nephropathy, characterized by IgG depositions subepithelially in the GCW. Only in ADN were changes seen in the distribution of DG. The experimental protocol for the animal studies was approved by the local ethical committee. For induction of ADN and PHN and for the isolation of mesangial cells, we used male Wistar-Unilever rats that were bred at our animal laboratory and weighed ∼150 g at the start of the experiments. The animals were given standard food and tap water ad libitum. For primary podocyte culture, female Sprague-Dawley rats (Charles-River, Sulzfeld, Germany) ∼120 g were used. ADN was induced in four rats as described.33Desassis JF Raats CJI Bakker MAH Van den Born J Berden JHM Anti-proteinuric effect of cyclosporin A in adriamycin nephropathy in rats.Nephron. 1997; 75: 336-341Crossref PubMed Scopus (28) Google Scholar As controls, four rats were injected with the same volume of saline. Antibodies against renal tubular epithelium raised in a sheep were used for the induction of PHN. Serum of four normal sheep was pooled and used as control. The immunoglobulin (Ig) from the antiserum and the normal sheep serum was purified by ammonium sulfate precipitation followed by ion-exchange chromatography using DEAE-Sepharose. The Ig was dialyzed extensively against phosphate-buffered saline (PBS), concentrated to one-fifth of the original volume, passed through a 0.2-μm filter and used for injection. PHN was induced in four rats by three intravenous injections on subsequent days of 40 mg IgG of this purified sheep anti-renal tubular epithelium antiserum. As a control, four rats were injected with the same amount of normal sheep IgG. Urinary albumin excretion was measured as described previously.34Raats CJI Bakker MAH Van den Born J Berden JHM Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome.J Biol Chem. 1997; 272: 26734-26741Crossref PubMed Scopus (99) Google Scholar The primary culture of rat podocytes was performed as described by Mundel and co-workers.35Mundel P Reiser J Kriz W Induction of differentiation in cultured rat and human podocytes.J Am Soc Nephrol. 1997; 8: 697-705PubMed Google Scholar Briefly, glomeruli were isolated from female Sprague-Dawley rats by the differential sieving procedure and cultured for 4 days in RPMI 1640 medium supplemented with 10. fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mmol/L glutamine, 1 mmol/L pyruvate (all from Gibco, Paisley, Scotland). The outgrowing epithelial cells were trypsinized and passed through a sieve with a pore size of 32 μm to remove the remaining glomerular cores. The epithelial cells were cultured in plastic tissue culture flasks (Greiner, Frickenhausen, Germany) or plastic slide flasks (Nunc, Roskilde, Denmark) coated with collagen I (Seromed, Berlin, Germany) for 3 to 4 weeks without splitting. Medium was changed twice a week. Isolation and culture of rat mesangial cells was performed as described by Wolthuis and co-workers.36Wolthuis A Boes A Rodemann HP Grond J Vasoactive agents affect growth and protein synthesis of cultured rat mesangial cells.Kidney Int. 1992; 41: 124-131Crossref PubMed Scopus (77) Google Scholar For IF and RT-PCR mesangial cells between passage 12 and 15 were used. Total RNA was extracted from kidney cortex and glomeruli by RNAzol (Campro Scientific, Veenendaal, The Netherlands) according to the instructions of the manufacturer. RNA extractions from podocytes and mesangial cells were performed using the guanidinium isothiocyanate acid phenol chloroform procedure37Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63082) Google Scholar with small modifications. In all samples, RNA was dissolved in DEPC-treated water for 10 minutes at 65°C and stored at −80°C. RNA concentration was determined by measuring the absorbance of a diluted aliquot at 260 nm. Quality of the RNA samples was checked by electrophoresis on a 0.8% agarose gel and staining with ethidium bromide. No degradation was observed. Primers and probes listed in Table 1 were designed using the Primer Express 1.0 program (Perkin-Elmer Cetus, Norwalk, CT) based on the rat agrin and dystroglycan cDNA sequence38Rupp F Payan DG Magill-Solc C Cowan DM Scheller RH Structure and expression of a rat agrin.Neuron. 1991; 6: 811-823Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 39Rupp F Ozcelik T Linial M Peterson K Francke U Scheller RH Structure and chromosomal localization of the mammalian agrin gene.J Neurosci. 1992; 12: 3535-3544Crossref PubMed Google Scholar, 40Lee NH Weinstock KG Kirkness EF Earle-Hughes JA Fuldner RA Marmaras S Glodek A Gocayne JD Adams MD Kerlavage AR Fraser CM Venter JC Comparative expressed sequence tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment.Proc Natl Acad Sci U S A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar and the homological stretches of mouse and human utrophin cDNA sequence.41Guo WX Nichol M Merlie JP Cloning and expression of full length mouse utrophin: the differential association of utrophin and dystrophin with AChR clusters.FEBS Lett. 1996; 398: 259-264Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 42Tinsley JM Blake DJ Roche A Fairbrother U Riss J Byth BC Knight AE Kendrick-Jones J Suthers GK Love DR Edwards YH Davies KE Primary structure of dystrophin-related protein.Nature. 1992; 360: 591-593Crossref PubMed Scopus (349) Google Scholar The primer sets for the agrin N-terminus and C-terminus are exon-spanning (based on the mouse gene), with introns of 165 and 270 bp, respectively. Primers and probes were synthesized and purified by the Eurogentec Corporation (Seraing, Belgium). Synthesis of single-stranded cDNA was performed in a solution containing 1 μg of total RNA, 50 mmol/L Tris-HCl (pH 8.3), 7.5 mmol/L KCl, 6 mmol/L MgCl2, 10 mmol/L DTT, 0.2 mmol/L of dNTPs (Boehringer Mannheim, Mannheim, Germany), 2.5 μmol/L of random hexamers (Perkin-Elmer Cetus), 1000 U/ml RNase inhibitor (RNasin. Promega, Madison, WI), 20,000 U/ml Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) in a total volume of 20 μl. Reactions were overlaid with mineral oil and were subsequently incubated in a DNA thermal cycler (Eppendorf Master Cycler 5330, Eppendorf, Hamburg, Germany) at 20°C for 10 minutes, 42°C for 50 minutes, and 95°C for 5 minutes. Amplification of cDNA was performed by addition of 80 μl of a mixture containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.9), various concentrations of MgCl2 ranging between 0.1 and 2.5 mmol/L (see Table 1), 0.2 mmol/L of dNTPs, 150 nmol/L of upstream primer, 150 nmol/L of downstream primer, and 2.5 U/ml Thermoperfect DNA polymerase (Integro, Zaandam, The Netherlands). Amplification started with an initial denaturation step at 94°C for 2 minutes, followed by 34 cycles of denaturation at 94°C for 1.5 minutes, annealing at various temperatures ranging between 54 and 62°C (see Table 1) for 1.5 minutes, and extension at 72°C for 1.5 minutes. After the last cycle, the extension phase was prolonged for 10 minutes at 72°C and the samples were cooled to 15°C. To optimize the PCR reaction, for each product the MgCl2 concentration and the annealing temperature were varied and the conditions yielding maximal signal together with minimal background were chosen for the experiments.Table 1Primers and Probes Used in RT-PCR and Southern BlotReference no.GenBank no.Oligo sequence 5′→3′Base pair numberFragment lengthMgCl2 concentrationAnnealing temp. (°C)Agrin N-terminus38Rupp F Payan DG Magill-Solc C Cowan DM Scheller RH Structure and expression of a rat agrin.Neuron. 1991; 6: 811-823Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 39Rupp F Ozcelik T Linial M Peterson K Francke U Scheller RH Structure and chromosomal localization of the mammalian agrin gene.J Neurosci. 1992; 12: 3535-3544Crossref PubMed Google ScholarM64780Forward primerGCC GTA TAG GTG CAA CCC G998–10161012.562Reverse primerTAC GGA GTT AAA CTG GCA GGT CT1098–1076ProbeAAA GTA CGC TCT GGT CAA TGC CAA1033–1056Agrin C-terminus38Rupp F Payan DG Magill-Solc C Cowan DM Scheller RH Structure and expression of a rat agrin.Neuron. 1991; 6: 811-823Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 39Rupp F Ozcelik T Linial M Peterson K Francke U Scheller RH Structure and chromosomal localization of the mammalian agrin gene.J Neurosci. 1992; 12: 3535-3544Crossref PubMed Google ScholarM64780Forward primerTTC GAA TCA GGG CTC ACA GG5744–57631010.562Reverse primerGTC CAG TTG CGT GGC ACC5844–5827ProbeAGC CCC TGT GAC TGG ATC TTC C5799–5820Dystroglycan40Lee NH Weinstock KG Kirkness EF Earle-Hughes JA Fuldner RA Marmaras S Glodek A Gocayne JD Adams MD Kerlavage AR Fraser CM Venter JC Comparative expressed sequence tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment.Proc Natl Acad Sci U S A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google ScholarH35660Forward primerTTT GGA AGA AAC CAT TTT GAG CAT95–1181011.054Reverse primerCAG GGA AGG GAT ACA TTA TTG CA195-173ProbeGTA CCT TTT AGG GAG GAA TGC CTT TT148–173Utrophin41Guo WX Nichol M Merlie JP Cloning and expression of full length mouse utrophin: the differential association of utrophin and dystrophin with AChR clusters.FEBS Lett. 1996; 398: 259-264Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 42Tinsley JM Blake DJ Roche A Fairbrother U Riss J Byth BC Knight AE Kendrick-Jones J Suthers GK Love DR Edwards YH Davies KE Primary structure of dystrophin-related protein.Nature. 1992; 360: 591-593Crossref PubMed Scopus (349) Google ScholarY12229/ForwardGCC CAC AAT GAC ATA TTT AAA AGC AT7492–7517 (Y12229)1541.054X69086primer7501–7526 (X69086)ReverseCCC TGA TGC TAG CAG ATT TTG C7645–7624 (Y12229)primer7654–7633 (X69086)ProbeCGG CAG AAG ATG GTG AAA GCT CT7528–7550 (Y12229) Open table in a new tab After amplification, 25 μl of PCR products were analyzed on a 1.5% agarose gel and stained with ethidium bromide. To check the specificity of the PCR products, they were blotted to a Hybond N+ nylon membrane (Amersham, Buckinghamshire, UK), using the alkali blotting procedure according to the manufacturer. Probes were 5′ end-labeled with γ-32P-dATP using T4 kinase (Boehringer Mannheim). Unincorporated radioactivity was removed using a Sephadex G-25 spin column. The Southern blots were prehybridized with 1 mg of herring sperm DNA in 0.5 mol/L NaH2PO4/Na2HPO4, 7% SDS, 1 mmol/L EDTA buffer (pH 7.2; hybridization solution) for 1 hour at 58°C, and then 50 pmol/L radiolabeled oligomer probe was added and incubation was continued overnight at 58°C. To remove unbound probes, blots were washed with 0.5 mol/L NaH2PO4/Na2HPO4, 1% SDS, 1 mmol/L EDTA (pH 7.2), for 10 minutes at 58°C and with 0.25 mol/L NaH2PO4/Na2HPO4, 1% SDS, 1 mmol/L EDTA (pH 7.2), for 10 minutes at 58°C and exposed to X-ray film (Eastman-Kodak, Rochester, NY) for 1 hour at −80°C. Indirect IF was performed as described previously34Raats CJI Bakker MAH Van den Born J Berden JHM Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome.J Biol Chem. 1997; 272: 26734-26741Crossref PubMed Scopus (99) Google Scholar on 2-μm cryostat sections of rat or human kidney and rat soleus muscle or human quadriceps muscle. Sections were fixed in acetone at 4°C during 10 minutes, except for incubations with the anti-dystrophin and anti-sarcoglycan antibodies, which was performed on nonfixed sections. Details on the used antibodies are given inTable 2.17Raats CJI Bakker MAH Hoch W Tamboer WPM Groffen AJA Van den Heuvel LPWJ Berden JHM Van den Born J Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.J Biol Chem. 1998; 273: 17832-17838Crossref PubMed Scopus (72) Google Scholar, 43Hoch W Campanelli JT Harrison S Scheller RH Structural domains of agrin required for clustering of nicotinic acetylcholine receptors.EMBO J. 1994; 13: 2814-2821Crossref PubMed Scopus (100) Google Scholar, 44Tsen G Halfter W Kroger S Cole GJ Agrin is a heparan sulfate proteoglycan.J Biol Chem. 1995; 270: 3392-3399Crossref PubMed Scopus (236) Google Scholar, 45Ervasti JM Campbell KP A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin.J Cell Biol. 1993; 122: 809-823Crossref PubMed Scopus (1165) Google Scholar, 46Bewick GS Nicholson LVB Young C Slater CR Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle.Neuromuscul Disord. 1993; 3: 503-506Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 47Bewick GS Nicholson LVB Young C O'Donnell E Slater CR Different distributions of dystrophin and related proteins at nerve-muscle junctions.Neuroreport. 1992; 3: 857-860Crossref PubMed Scopus (124) Google Scholar, 48Nicholson LV Johnson MA Davison K O'Donnell E Falkous G Barron M Harris JB Dystrophin or a "related protein" in Duchenne muscular dystrophy?.Acta Neurol Scand. 1992; 86: 8-14Crossref PubMed Scopus (32) Google Scholar Primary antibodies were diluted in PBS containing 1% bovine serum albumin (BSA) and 0.05% sodium azide (IF-buffer), fluorescein isothiocyanate-labeled secondary antibodies were diluted in IF-buffer containing 10% normal rat serum (for IF on rat tissue), all antibodies were incubated during 45 minutes at room temperature. To evaluate the deposition of sheep and rat IgG and complement factor C3c in the GCW of rats with PHN, sections were directly incubated with fluorescein isothiocyanate-labeled rabbit anti-sheep IgG (Southern, Birmingham, AL. diluted 1:100 in IF-buffer, goat anti-rat IgG and goat anti-rat C3c (both from Nordic, Tilburg, The Netherlands) both diluted 1:50 in IF-buffer. After incubation with antibodies, the sections were washed with PBS, fixed with 1% paraformaldehyde in PBS for 15 minutes, washed, and embedded in Vectashield mounting medium H-1000 (Vector Laboratories Inc., Burlingame, CA) and examined with a Zeiss Axioskop microscope equipped with an epi-illuminator. The staining of the antibodies in the GCW of the rats (four ADR rats and four controls. four PHN rats and four controls) were evaluated in 25 glomeruli on a scale between 0 and 4+ by two independent observers on coded sections and the mean of the two scores was used for further analysis.Table 2Antibodies Used to Localize the Various Members of the DGC by IF and IEMAntibodyAntigenReference no.Dilution IFDilution IEMConjugate*Conjugated to FITC for IF, to peroxidase for IEM.DilutionMI-90Agrin N-terminus17Raats CJI Bakker MAH Hoch W Tamboer WPM Groffen AJA Van den Heuvel LPWJ Berden JHM Van den Born J Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.J Biol Chem. 1998; 273: 17832-17838Crossref PubMed Scopus (72) Google Scholar400100Goat anti-hamster‡Jackson, West Grove, PA.50Agr-131Agrin C-terminus43Hoch W Campanelli JT Harrison S Scheller RH Structural domains of agrin required for clustering of nicotinic acetylcholine receptors.EMBO J. 1994; 13: 2814-2821Crossref PubMed Scopus (100) Google Scholar1000250Goat anti-mouse IgG2a§Southern, Birmingham, AL.8045Agrin C-terminus44Tsen G Halfter W Kroger S Cole GJ Agrin is a heparan sulfate proteoglycan.J Biol Chem. 1995; 270: 3392-3399Crossref PubMed Scopus (
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