Role for Macrophage Metalloelastase in Glomerular Basement Membrane Damage Associated with Alport Syndrome
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.050896
ISSN1525-2191
AutoresVelidi H. Rao, Daniel T. Meehan, Duane Delimont, Motowo Nakajima, Takashi Wada, Michael Ann Gratton, Dominic Cosgrove,
Tópico(s)Platelet Disorders and Treatments
ResumoAlport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease. Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease. Alport syndrome has become a leading model for genetic disorders affecting basement membranes. The gene frequency is about 1 in 5000 people, making it among the more prevalent of known genetic disorders.1Atkin CL Gregory MC Border WA Schrier RW Gottschalk CW Alport Syndrome. Diseases of the Kidney. 4. Little, Brown, Boston1988: 617-641Google Scholar, 2Pescucci C Longo I Bruttini M Mari F Renieri A Type-IV collagen related diseases.J Nephrol. 2003; 16: 314-316PubMed Google Scholar It has been determined that X-linked Alport syndrome is caused by any of a series of mutations in the COL4A5gene.3Barker DF Hostikka SL Zhou J Chow LT Oliphant AR Gerken SC Gregory MC Skolnick MH Atkin CL Tryggvason K Identification of mutations in the COL4A5 collagen gene in Alport syndrome.Science. 1990; 348: 1224-1227Crossref Scopus (653) Google Scholar Hundreds of different mutations in the gene have been identified in families carrying the disease thus far.4Kashtan CE Alport syndrome. An inherited disorder of renal, ocular, and cochlear basement membranes.Medicine (Baltimore). 1999; 78: 338-360Crossref PubMed Scopus (164) Google Scholar, 5Kashtan CE Familial hematuria due to type IV collagen mutations: Alport syndrome and thin basement membrane nephropathy.Curr Opin Pediatr. 2004; 16: 177-181Crossref PubMed Scopus (51) Google Scholar The autosomal form of Alport syndrome, which displays the same range of phenotypes as the X-linked form of the disease, is due to mutations in either basement membrane collagen genes COL4A3 or COL4A4.6Lemmink HH Mochizuki T van den Heuvel LP Schroder CH Barrientos A Monnens LA van Oost BA Brunner HG Reeders ST Smeets HJ Mutations in the type IV collagen α3 (COL4A3) gene in autosomal recessive Alport syndrome.Hum Mol Gen. 1994; 3: 1269-1273Crossref PubMed Scopus (196) Google Scholar, 7Mochizuki T Lemmink HH Mariyama M Antignac C Gubler MC Pirson Y Verellen-Dumoulin C Chan B Schroder CH Smeets HJ Identification of mutations in the α3(IV) and α4(IV) collagen genes in autosomal recessive Alport syndrome.Nat Genet. 1994; 8: 77-81Crossref PubMed Scopus (435) Google Scholar Alport syndrome is characterized by a juvenile onset of proteinuria. The protein in the urinary space of the glomerulus precedes changes in glomerular cell types, including effaced podocyte foot processes and expansion of the mesangium. These changes culminate in accumulation of extracellular matrix associated with progressive glomerulonephritis. The glomeruli eventually become fibrotic, resulting in a decreased capacity of the kidney to filter the blood. This ultimately results in a fatal uremia. Current therapy is limited to dialysis and transplantation, with a higher risk than normal patients of anti-glomerular basement membrane disease in the transplanted organ due to immune reaction against the type IV collagen chains. A hallmark and unique characteristic of Alport glomerular disease is an irregular thickening, thinning, and splitting of the glomerular basement membrane (GBM).8Rumpelt HJ Alport's syndrome: specificity and pathogenesis of glomerular basement membrane alterations.Pediatr Nephrol. 1987; 3: 422-427Crossref Scopus (43) Google Scholar The progressive GBM damage is associated with podocyte foot process effacement. The mechanism underlying this phenotype is unknown; however, it has been suggested that thickened regions might represent areas of matrix deposition.9Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Sayers R Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev V. 1996; 10: 2981-2992Crossref PubMed Scopus (295) Google Scholar, 10Cosgrove D Rodgers K Meehan D Miller C Bovard K Gilroy A Gardner H Kotelianski V Gotwals P Amatucci A Integrin alpha1-beta1 and transforming growth factor-beta1 play distinct roles in Alport glomerular pathogenesis and serve as dual targets for metabolic therapy.Am J Pathol. 2000; 157: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 11Abrahamson DR Prettyman AC Robert B St John PL Laminin-1 reexpression in Alport mouse glomerular basement membranes.Kidney Int. 2003; 63: 826-834Crossref PubMed Scopus (69) Google Scholar Alternatively, it has been shown that type IV collagen matrix from Alport kidneys is more susceptible to endoproteolytic cleavage than that from normal kidneys.12Kalluri R Shield CF Todd P Hudson B Neilson EG Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis.J Clin Invest. 1997; 99: 2470-2478Crossref PubMed Scopus (262) Google Scholar This is presumably due to a significant reduction of interchain disulfide cross-links resulting from differences in collagen chain composition.13Gunwar S Ballester F Noelken ME Sado Y Ninomiya Y Hudson BG Identification of a novel disulfide-cross-linked network of alpha3, alpha4 and alpha5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome.J Biol Chem. 1998; 273: 8767-8775Crossref PubMed Scopus (174) Google Scholar Homeostatic extracellular matrix turnover is a delicately balanced system of coupled biosynthetic and degradative processes. The matrix metalloproteinase (MMP) family consists of over 25 members that collectively can degrade all components of the extracellular matrix. MMP activity is associated with several normal processes of tissue remodeling. Dysregulation of the MMPs may contribute to disease processes. The control and regulation of the extracellular matrix degradation has been shown to be complex, and knowledge of the system in Alport syndrome is rudimentary. Preliminary evidence implicates a role for MMPs in renal pathogenesis associated with Alport syndrome.14Rao VH Lees GE Kashtan CE Nemori R Singh RK Meehan DT Rodgers K Berridge BR Bhattacharya G Cosgrove D Increased expression of MMP-2, MMP-9 (type IV collagenases/gelatinases) and MT1-MMP in canine X-linked Alport syndrome (XLAS).Kidney Int. 2003; 63: 1736-1748Crossref PubMed Scopus (52) Google Scholar, 15Rao VH Lees GE Kashtan CE Delimont DC Singh R Meehan DT Bhattacharya G Berridge BR Cosgrove D Dysregulation of renal MMP-3 and MMP-7 in canine X-linked alport syndrome.Pediatric Nephrology. 2005; 20: 732-739Crossref PubMed Scopus (12) Google Scholar, 16Rodgers KD Rao V Meehan DT Fager N Gotwals P Ryan ST Koteliansky V Nemori R Cosgrove D Tissue monocytes may promote myofibroblast accumulation and tubular epithelial cell death in renal fibrosis.Kidney Int. 2003; 63: 1338-1355Crossref PubMed Scopus (46) Google Scholar MMP-12 (metalloelastase) is a potent protease with broad matrix substrate specificity that has long been associated with macrophages and lung disease.17Chandler S Cossins J Lury J Wells G Macrophage metalloelastase degrades matrix and myelin proteins and processes a tumour necrosis factor-α fusion protein.Biochem Biophys Res Commun. 1996; 228: 421-429Crossref PubMed Scopus (135) Google Scholar To date, expression of MMP-12 has only been demonstrated in macrophages,18Kaneko Y Sakatsume M Xie Y Kuroda T Igashima M Narita I Gejyo F Macrophage metalloelastase as a major factor for glomerular injury in anti-glomerular basement membrane nephritis.J Immunol. 2003; 170: 3377-3385PubMed Google Scholar, 19Vos CM van Haastert ES de Groot CJ van der Valk P de Vries HE Matrix metalloproteinase-12 is expressed in phagocytotic macrophages in active multiple sclerosis lesions.J Neuroimmunol. 2003; 138: 106-114Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar hypertrophic osteoclasts,20Hou P Troen T Ovejero MC Kirkegaard T Andersen TL Byrjalsen I Ferreras M Sato T Shapiro SD Foged NT Matrix metalloproteinase-12 (MMP-12) in osteoclasts: new lesson on the involvement of MMPs in bone resorption.Bone. 2004; 34: 37-47Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar vascular smooth muscle cells,21Wu L Fan J Matsumoto S Watanabe T Induction and regulation of matrix metallopro6teinase 12 by cytokines and CD40 CD40 signaling in monocytes/macrophages.Biochem Biophys Res Commun. 2000; 269: 808-815Crossref PubMed Scopus (50) Google Scholar and some cancer cells.22Zucker S Vacirca J Role of matrix metalloproteinases (MMPs) in colorectal cancer.Cancer Metastasis Rev. 2004; 23: 101-117Crossref PubMed Scopus (426) Google Scholar, 23Ding Y Shimada Y Gorrin-Rivas MJ Itami LZ Hong T Maeda M Komoto I Kawabe A Kaganoi J Clinicopathological significance of human macrophage metalloelastase expression in esophageal squamous cell carcinoma.Oncology. 2002; 63: 378-384Crossref PubMed Scopus (32) Google Scholar Here we show MMP-12 is markedly induced in the glomeruli of Alport mice. The degree of induction in glomeruli from Alport mice correlates well with the progression of glomerular disease. We provide evidence to suggest that the cellular mechanism of MMP-12 induction might be linked to monocyte chemoattractive protein-1 (MCP-1)-mediated activation of the CCR2 receptor on glomerular podocytes. Inhibition of MMP-12 with either the small molecule inhibitor MMI270, or the CCR2 receptor antagonist propagermanium, attenuates GBM thickening and preserves the integrity of the glomerular filter. These data suggest that irregular thickening of the GBM in Alport syndrome may be caused, in part, by proteolytic degradation of the GBM due to elevated expression of MMP-12 in glomerular podocytes. The cellular mechanism of MMP-12 induction (MCP-1 activation of CCR2 on glomerular podocytes) is quite unexpected and only previously described in macrophages. The Alport mouse model is in the 129 Sv background and has been described previously.9Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Sayers R Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev V. 1996; 10: 2981-2992Crossref PubMed Scopus (295) Google Scholar Wild-type mice are normal for both collagen α3(IV) alleles and are a product of double heterozygote crosses for the Alport mutation. The use of animals in this study was performed in accordance with an approved Institutional Animal Care and Use Committees protocol. Extreme care was taken to minimize pain and discomfort. MMP inhibitors were administered between 4 and 7 weeks of age. All drugs were freshly prepared before administration. BAY-129566 was emulsified in suspension with 0.5% carboxymethylcellulose, and 4 mg was given once a day by oral gavage. MMI270 was solubilized in 0.9% saline and administered by intraperitoneal injection (10 mg/kg body weight) twice a day. Mouse Engelbreth-Holm-Swarm tumor type IV collagen was obtained from BD Biosciences Discovery Labware (Two Oak Park, Bedford, MA). Protein substrates were incubated with recombinant mouse MMP-12 (a kind gift from Dr. Y. Kaneko, Nigata, Japan) in 40 μl of assay buffer (0.1 mol/L Tris-HCl, pH 8.0, 0.1 mol/L NaCl, 10 mmol/L CaCl2, 1.0 mmol/L ZnCl2, 0.05% Brij 35, and 0.02% NaN3) at 37°C for 24 hours. The inhibitors used were MMI270 and BAY-129566 at 50 nmol/L. Reactions were stopped with 4× loading buffer containing dithiothreitol and heated in boiling water for 5 minutes. Sodium dodecyl sulfate-PAGE using 5% gels was followed by staining with Coomassie R-250 and destaining. Isolation of mouse glomeruli was performed as described previously.24Takemoto M Asker N Gerhardt H Lundkvist A Johansson BR Saito Y Betsholtz C A new method for large scale isolation of kidney glomeruli from mice.Am J Pathol. 2002; 161: 799-805Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar The procedure involves cardiac perfusion with deactivated 4.5 μmol/L Dynabeads (Dynal Biotech, Oslo, Sweden) followed by collagenase digestion and glomerular isolation using a magnet. We found these preparations to be consistently >97% pure, allowing reliable assessment of glomerular-specific gene expression in mice. Total RNA samples were treated with RNase-free DNase I (Gibco-BRL, Gaithersburg, MD) at 37°C for 1 hour to remove any contaminating genomic DNA before reverse transcription (RT). Total RNA was reverse-transcribed by using Superscript II (Gibco-BRL) with oligo(dT) primers. To ensure that the quantitation of MMP transcripts in serial samples was not affected by differences in the amount of RNA added, integrity of RNA, or sample to sample differences in levels of RT-polymerase chain reaction (PCR) inhibition, an internal control reaction targeting the GAPDH gene was run in multiplex with each reaction and used to normalize results for MMP transcripts. Primers and TaqMan probes for murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems (catalogue no. 4308313, Foster City, CA) and used as per the manufacturer's instructions. The data were analyzed using comparative threshold cycle (CT) method. The mRNA quantity for the control is expressed as 1× sample, and all other quantities from Alport samples are expressed as -fold differences relative to the controls. No measurable fluorescence signal was detected in repeated RT-PCR runs in which the reverse transcriptase was omitted from the reaction mixture. Primers were tested by standard endpoint RT-PCR, and the single band obtained was sequence-verified. Real-time RT-PCR was performed on a TaqMan ABI 7000 Sequence Detection System (Applied Biosystems). PCR was performed with TaqMan Universal PCR Master Mix (Applied Biosystems), which contained AmpliTaq Gold DNA polymerase, AmpErase urasil-N-glycosylate, dNTPs with dUTP, and optimized buffer components. AmpErase urasil-N-glycosylate treatment prevented the possible reamplification of carryover PCR products. Each target molecule was co-amplified with primers and TaqMan probe for GAPDH in the same PCR tube. The total volume of the PCR reaction was 50 μl. The final concentration of each oligonucleotide in the PCR reaction was as follows: GAPDH primers, 100 nmol/L; primers for target molecules, 900 nmol/L; TaqMan probe for GAPDH, 200 nmol/L; and TaqMan probe for the target molecules, 250 nmol/L. The primers and probes are as follows. MMP-2: Sense, 5′-GTT TAT TTG GCG GAC AGT GAC A-3′; Antisense, 5′-AGA ATG TGG CCA CCA GCA A-3′; Probe, 5′-6FAM-CCA CGT GAC AAG CC-MGBNFQ-3′; MMP-9: Sense, 5′-CCA AGG GTA CAG CCT GTT CCT-3′; Antisense, 5′-GCA CGC TGG AAT GAT CTA AGC-3′; Probe, 5′-6FAM-ACT CGT GCG CTG CC-MGBNFQ-3′; MMP-12: Sense, 5′-GCC ACA CTA TCC CAG GAG CAT ATA-3′; Antisense, 5′-AGC TGC ATC AAC CTT CTT CAC A-3′; Probe, 5′-6FAM-ATG CAG AGA AGC CC-MGBNFQ-3′; MMP-14: Sense, 5′-GAG GAG AGA TGT TTG TCT TCA AGG A-3′; Antisense, 5′-GGG TAT CCA TCC ATC ACT TGG TTA-3′; Probe, 5′-6FAM-TCC TCA CCC GCC AGA G-MGBNFQ-3′. TaqMan Rodent GAPDH Control Reagents (catalogue no. 4308313) containing the primers and VIC-probe were purchased from Applied Biosystems. Thermal cycling was initiated with incubation at 50°C for 2 minutes and 95°C for 10 minutes for optimal EmpErase UNG activity and activation of AmpliTaq Gold DNA polymerase, respectively. After this initial step, 40 cycles were performed. Each PCR cycle consisted of heating at 95°C for 15 seconds for melting and 60°C for 60 seconds for annealing and extension. All controls consisting of ddH2O were negative for target and housekeeping genes. Results of PCR are as follows: CCR2: annealed at 58°C for 35cycles, 199 bp (Forward: 5′-CAC GAA GTA TCC AAG AGC TT-3′; Reverse: 5′-CAT GCT CTT CAG CTT TTT AC-3′); MCP-1: annealed at 60°C for 30 cycles, 519 bp (Forward: 5′-AGA GAG CCA GAC GGA GGA AG-3′; Reverse: 5′-GTC ACA CTG GTC ACT CCT AC-3′). Data are expressed as mean ± SD. Differences between means were tested for significance using Student's t-test. Differences were considered significant at the level of P < 0.05. Cryosections (4 μmol/L) of kidneys from 7-week-old wild-type and Alport mice were air-dried, fixed by immersion in ice-cold acetone, and subjected to immunohistochemical staining analysis. Antibodies used were specific for MMP-12 (goat polyclonal antibody against mouse MMP-12; Santa Cruz Biotechnology, Santa Cruz, CA, used at 1:100 dilution), type IV collagen α1/2 chains (rabbit polyclonal against mouse type IV collagen; Biodesign, Inc., Saco, ME, used at 1:200 dilution), fibronectin (rabbit polyclonal against human plasma fibronectin; Sigma-Aldrich, St. Louis, MO, used at 1:200). Anti-CD11b antibodies were directly conjugated to Alexa 568 (Molecular Probes, Eugene, OR) and purchased from Cedarlane Laboratories (Hornby, ON, Canada). For dual immunofluorescence immunostaining, this antibody was added to the mixture containing the secondary antibody. All antibodies were diluted into 7% nonfat dry milk in phosphate-buffered saline (PBS) to reduce nonspecific binding. Primary antibodies were allowed to react for 2 hours at room temperature in a humidified chamber. After three 5-minute washes in PBS, slides were incubated with fluorescein isothiocyanate-conjugated secondary antibodies for 1 hour at room temperature (goat anti-rabbit; Vector Laboratories, Burlingame, MA, used at 1:200). The sections were covered with coverslips, sealed, and imaged. Images were collected using a Cytovision Ultra Image analysis system interfaced with an Olympus BH-2 fluorescence microscope. Northern blots analysis was performed as described previously.9Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Sayers R Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev V. 1996; 10: 2981-2992Crossref PubMed Scopus (295) Google Scholar Ten micrograms of total glomerular RNA was fractionated on 1% agarose formaldehyde gels and transferred to nylon membranes. Probes were either a gel-purified PCR fragment of the MMP-12 transcript using a specific primer set (Sense: 5′-AAG CAA CTG GGC AAC TGG ACA ACT C-3′ and antisense: 5′-TGG TGA CAG AAA GTT GAT GGT GGA C-3′, annealing at 60°C for 30 cycles) or the DECA template for mouse β-actin (Ambion, Inc., Austin TX). Probes were labeled with 32P-dCTP using either random primers or the DECA method provided by the manufacturer. Hybridizations were performed overnight at 50°C using ULTRAhyb hybridization buffer (Ambion), and the membranes were washed according to the manufacturer's instructions. Membranes were exposed to X-ray film overnight. For riboprobe preparation, a 631-bp fragment of the mouse MMP-12 cDNA and a 199-bp fragment of the mouse CCR2 cDNA were amplified from reverse-transcribed RNA using the primers listed above for Northern blot analysis. The resulting fragments were cloned into the pCRII TOPO cloning vector (Invitrogen) and sequence-verified. Fifteen micrograms of the plasmids was linearized using HindIII to provide a 5′ overhang. DNA was isolated using phenol/chloroform extractions. One μg of linear DNA was transcribed as recommended in the Boehringer Mannheim DIG Labeling Kit using T7 polymerase. The yield of labeled probe was estimated by spotting the probe and labeled control onto nylon membrane and developing as recommended in the nonradioactive in situhybridization application manual (Roche Applied Science, Mannheim, Germany). For hybridization, 6-μm paraffin sections were digested in 3 μg/ml proteinase K in 0.1 mol/L Tris, pH 7.5, for 10 minutes at 37°C. They were prehybridized for 1 hour at 45°C in 50% deionized formamide, 2× standard saline citrate, 10 Tween 20, and 1 mg of Escherichia coli tRNA. The hybridization solution consisted of 50 ng heat-denatured riboprobe, 50% deionized formamide, 8% dextran sulfate, 10% Tween 20, 2× standard saline citrate, 20% tRNA, and 10 mg/ml boiled salmon sperm. Slides were hybridized at 45°C overnight. The DIG Wash and Block Buffer Set was used to develop the slides in conjunction with the color substrate solution to which we added 25 mmol/L Levamisole. Transmission electron microscopy was performed as previously described.9Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Sayers R Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev V. 1996; 10: 2981-2992Crossref PubMed Scopus (295) Google Scholar Ultrastructural localization studies of type IV collagen were performed essentially as previously described.25Bhattacharya G Miller C Kimberling WJ Jablonski MM Cosgrove D Localization and expression of usherin: a novel basement membrane protein defective in people with Usher syndrome type IIa.Hear Res. 2002; 3476: 1-11Crossref Scopus (64) Google Scholar Kidneys from 7-week-old wild-type and Alport mice were fixed by heart perfusion with 2% paraformaldehyde in PBS and postfixed in this same solution overnight. Ultrathin (70-nm) sections were reacted for 4 hours at room temperature with goat anti-collagen IV antibodies (Southern Biotechnology, Birmingham, AL). After six 10-minute washes in PBS, specimen were reacted for 2 hours at room temperature with 10-nm gold-conjugated anti-rabbit antibodies (Vector Laboratories). Grids were washed as before, air-dried, counterstained with uranyl acetate, and examined using transmission electron microscopy. Isolated glomeruli were lysed in radioimmune precipitation assay lysis buffer (consisting of 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, 1% Nonidet P-40, 100 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.4) containing protease inhibitors (P8340; Sigma-Aldrich). An aliquot was assayed for total protein (Pierce Biochemicals, Rockford, IL). From 20 μg of total protein, CCR2 protein was immunoprecipitated using goat anti-human CCR2 antibody (Santa Cruz Biotechnology) as described previously.14Rao VH Lees GE Kashtan CE Nemori R Singh RK Meehan DT Rodgers K Berridge BR Bhattacharya G Cosgrove D Increased expression of MMP-2, MMP-9 (type IV collagenases/gelatinases) and MT1-MMP in canine X-linked Alport syndrome (XLAS).Kidney Int. 2003; 63: 1736-1748Crossref PubMed Scopus (52) Google Scholar, 15Rao VH Lees GE Kashtan CE Delimont DC Singh R Meehan DT Bhattacharya G Berridge BR Cosgrove D Dysregulation of renal MMP-3 and MMP-7 in canine X-linked alport syndrome.Pediatric Nephrology. 2005; 20: 732-739Crossref PubMed Scopus (12) Google Scholar The immunoprecipitated protein was electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane that was first incubated with the antibody against CCR2 (1:1000) followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich). The protein bands were visualized using the ECL plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ). Propagermanium (3-oxygemylpropinic acid polymer; Sanwa Kagaku Kenkyusho Co., Nagoya, Japan) was administered orally (10 mg/kg in 1% gelatin) by gavage once daily starting at 4 weeks of age, and kidneys were harvested at 7 weeks of age. Three animals per group, Alport mice and wild-type littermates, were gavaged with drug or vehicle only and analyzed. 129sv wild-type and 4α3 KO mice were back-crossed into transgenic mice containing a γ-interferon-inducible, temperature-sensitive mutant of the SV-40 large T-antigen.26Jat PS Noble MD Ataliotis P Tanaka Y Yannoutsos N Larsen L Kioussis D Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse.Proc Natl Acad Sci USA. 1991; 88: 5096-5100Crossref PubMed Scopus (627) Google Scholar Glomeruli were isolated as described above. The decapsulated glomeruli were resuspended in 0.1% trypsin, 0.05% collagenase I in PBS and digested for up to 2 hours at 37°C with moderate shaking.27Kreisberg JI Hoover RL Karnovsky MJ Isolation and characterization of rat glomerular epithelial cells in vitro.Kidney Int. 1978; 14: 21-30Crossref PubMed Scopus (162) Google Scholar The glomeruli were monitored visually every 30 minutes, at which time the suspension was pipetted to alleviate clumping. After the glomeruli were disassociated, media containing FCS was added, and the suspension was spun for 10 minutes at 1500 rpm at 4°C. The pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 containing 10% fetal calf serum (FCS), penicillin, streptomycin, gentamicin, and glutamine and plated on a 10-cm tissue culture dish at 37°C. The culture was left undisturbed for several days. The trypsinized culture was then passed through a sieve with 25-μm pore size to remove mesangial and endothelial cells.28Mundel P Reiser J Kriz W Induction of differentiation on cultured rat and human podocytes.J Am Soc Nephrol. 1997; 8: 697-705PubMed Google Scholar The one or more “sieved” cultures were then resuspended in Renal Basal Media with supplements (BioWhittaker, Inc., San Diego, CA) in the presence of 100 U/ml of mouse recombinant γ-interferon (Calbiochem, La Jolla, CA) and placed at 33°C. The one ore more proliferative cultures were passed and gradually weaned to DMEM/Ham's F-12 containing 10% FCS and 10 U/ml γ-interferon at 33°C. Cells were placed at 37°C in DMEM/Ham's F-12 containing 5% FCS without γ-interferon and allowed to differentiate for at least 2 weeks. Observations on proliferative nature and morphology were noted. The culture(s) were then validated after 2 weeks of the “nonpermissive” conditions by markers of differentiated podocytes in vivo.29Pavenstadt H Kriz W Kretzler M Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307PubMed Google Scholar Differentiated podocytes grown on slides were characterized using immunofluorescence. Cells were fixed with −20°C acetone for 5 minutes (air-dry for 2 hours) or 25°C 2% paraformaldehyde and 4% sucrose for 10 minutes. Cells were washed once with PBS for 5 minutes, permeabilized with 0.3% Triton X-100 for 10 minutes, washed again, and then blocked with 2% FCS, 2% bovine serum albumin, 0.2% fish gelatin for at least 30 minutes. The slides were rinsed once with PBS and incubated with primary antibodies (1:50) of interest in blocking solution at 4°C overnight. The slides were washed three times with PBS at 25°C and incubated with the appropriate Alexa-labeled secondary antibody (1:150) in blocking solution at 25°C for 2 hours. Slides were washed, mounted, and visualized under an Olympus BH2 fluorescence microscope and captured with a Spot-RT digital camera model 2.3.1. Primary antibodies included CD2AP (H-290), WT-1 (C-19), and VWF (H-300), all from Santa Cruz Biotechnology. Nephrin (extracellular domain) was a gift from R. Kalluri. Glomeruli were isolated and dissociated as above for podocyte cell cultures using wild-type mice (non-immortomouse). After the glomeruli were disassociated, the suspension was transferred to a 15-ml conical tube containing 10 ml of media plus FCS and spun for 10 minutes at 4°C at 1500 rpm. The pellet was resuspended in 12.5 ml of DMEM/Ham's F-12 containing 20% FCS, penicillin, streptomycin, gentamicin, glutamine, insulin, and selenium followed by transfer and plating to a 10-cm tissue culture dish. The culture was left undisturbed for several days. Cells were grown to near confluency, trypsinized, and passed 1:3 in growth media. Within 24 hours, the culture media was replaced with d-valine containing DMEM (eliminating fibroblast contamination). Under these conditions the mesangial cells quickly outgrow all other cells in the mixture. The cells were qualified as me
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