Integrin α1β1 and Transforming Growth Factor-β1 Play Distinct Roles in Alport Glomerular Pathogenesis and Serve as Dual Targets for Metabolic Therapy
2000; Elsevier BV; Volume: 157; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64802-x
ISSN1525-2191
AutoresDominic Cosgrove, Kathryn D. Rodgers, Daniel T. Meehan, Caroline Miller, Karen Bovard, Amy Gilroy, Humphrey Gardner, Victor Kotelianski, Phillip J. Gotwals, Aldo Amatucci, Raghu Kalluri,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoAlport syndrome is a genetic disorder resulting from mutations in type IV collagen genes. The defect results in pathological changes in kidney glomerular and inner-ear basement membranes. In the kidney, progressive glomerulonephritis culminates in tubulointerstitial fibrosis and death. Using gene knockout-mouse models, we demonstrate that two different pathways, one mediated by transforming growth factor (TGF)-β1 and the other by integrin α1β1, affect Alport glomerular pathogenesis in distinct ways. In Alport mice that are also null for integrin α1 expression, expansion of the mesangial matrix and podocyte foot process effacement are attenuated. The novel observation of nonnative laminin isoforms (laminin-2 and/or laminin-4) accumulating in the glomerular basement membrane of Alport mice is markedly reduced in the double knockouts. The second pathway, mediated by TGF-β1, was blocked using a soluble fusion protein comprising the extracellular domain of the TGF-β1 type II receptor. This inhibitor prevents focal thickening of the glomerular basement membrane, but does not prevent effacement of the podocyte foot processes. If both integrin α1β1 and TGF-β1 pathways are functionally inhibited, glomerular foot process and glomerular basement membrane morphology are primarily restored and renal function is markedly improved. These data suggest that integrin α1β1 and TGF-β1 may provide useful targets for a dual therapy aimed at slowing disease progression in Alport glomerulonephritis. Alport syndrome is a genetic disorder resulting from mutations in type IV collagen genes. The defect results in pathological changes in kidney glomerular and inner-ear basement membranes. In the kidney, progressive glomerulonephritis culminates in tubulointerstitial fibrosis and death. Using gene knockout-mouse models, we demonstrate that two different pathways, one mediated by transforming growth factor (TGF)-β1 and the other by integrin α1β1, affect Alport glomerular pathogenesis in distinct ways. In Alport mice that are also null for integrin α1 expression, expansion of the mesangial matrix and podocyte foot process effacement are attenuated. The novel observation of nonnative laminin isoforms (laminin-2 and/or laminin-4) accumulating in the glomerular basement membrane of Alport mice is markedly reduced in the double knockouts. The second pathway, mediated by TGF-β1, was blocked using a soluble fusion protein comprising the extracellular domain of the TGF-β1 type II receptor. This inhibitor prevents focal thickening of the glomerular basement membrane, but does not prevent effacement of the podocyte foot processes. If both integrin α1β1 and TGF-β1 pathways are functionally inhibited, glomerular foot process and glomerular basement membrane morphology are primarily restored and renal function is markedly improved. These data suggest that integrin α1β1 and TGF-β1 may provide useful targets for a dual therapy aimed at slowing disease progression in Alport glomerulonephritis. Alport syndrome is a hereditary basement membrane disease affecting approximately one in 5,000 people.1Flinter F Alport syndrome.J Med Genet. 1992; 34: 326-330Crossref Scopus (48) Google Scholar The disease is manifest by juvenile to adult onset progressive glomerulonephritis usually associated with a high-frequency-specific sensorineural hearing loss, dot and fleck retinopathy, and lens abnormalities. No effective drug therapy exists for this disease, which is currently treated by dialysis and renal transplant.1Flinter F Alport syndrome.J Med Genet. 1992; 34: 326-330Crossref Scopus (48) Google Scholar, 2Gregory MC Terreros DA Barker DF Fain PN Denison JC Atkin CL Alport syndrome—clinical phenotypes, incidence, and pathology.Contrib Nephrol. 1996; 117: 1-28Crossref PubMed Google Scholar The most common form of the disease is X-linked, and caused primarily by mutations in the collagen α5(IV) gene,3Barker DE Hostikka SL Zhou J Show 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; 248: 1224-1227Crossref PubMed Scopus (672) Google Scholar accounting for ∼80% of the cases. Mutations in the collagen α3(IV) or α4(IV) genes lead to the recessive forms of the disease.4Lemmink HH Mochizuki T van den Heuvel LPWJ Schroder CH Barrientos A Monnens LAH van Oost BA Brunner HG Reeders ST Smeets HJM Mutations in the type IV collagen α3 (COLCOL4A3) gene in autosomal recessive Alport syndrome.Hum Mol Genet. 1994; 3: 1269-1273Crossref PubMed Scopus (197) Google Scholar, 5Mochizuki T Lemmink HH Mariyama M Antignac C Gubler MC Pirson Y Verellen-Dumoulin C Chan B Schroder CH Smeets HJ Reeders ST Identification of mutations in the α3(IV) and α4(IV) collagen genes in autosomal recessive Alport syndrome.Nat Genet. 1994; 8: 77-82Crossref PubMed Scopus (445) Google Scholar The absence of any one of these type IV collagen chains can result in the absence of all three chains in the glomerular basement membrane (GBM), presumably due to an obligatory association of the three chains in forming the type IV collagen superstructure.6Kashtan CE Kim Y Distribution of the α1 and α2 chains of collagen IV and of collagens V and VI in Alport syndrome.Kidney Int. 1992; 42: 115-126Crossref PubMed Scopus (103) Google Scholar, 7Gubler M-C Knebelmann B Beziau A Broyer M Pirson Y Haddoum F Kleppel MM Antignac C Autosomal recessive Alport syndrome: immunohistochemical study of type IV collagen chain distribution.Kidney Int. 1995; 47: 1142-1147Crossref PubMed Scopus (168) Google Scholar Normal distribution of the three α chains is observed in approximately one third of patients.8Mazzucco G Barsotti P Muda AO Fortunato M Mihatsch M Torri-Tarelli L Renieri A Faraggiana T De Marchi M Monga G Ultrastructural and immunohistochemical findings in Alport's syndrome: a study of 108 patients from 97 Italian families with particular emphasis on COL4A5 gene mutation correlations.J Am Soc Nephrol. 1998; 9: 1023-1031PubMed Google Scholar The adult GBM contains a thin subendothelial network of collagen α1(IV) and α2(IV) chains, and a thick subepithelial network of collagen α3(IV), α4(IV), and α5(IV) chains.9Desjardins M Bendayan M Ontogenesis of glomerular basement membrane: structural and functional properties.J Cell Biol. 1991; 113: 689-700Crossref PubMed Scopus (47) Google Scholar These networks are thought to be physically separate from one another.10Kleppel MM Fan WW Cheong HI Michael AF Evidence for separate networks of classical and novel basement membrane collagen.J Biol Chem. 1992; 267: 4137-4142Abstract Full Text PDF PubMed Google Scholar, 11Gunwar S Ballester F Noelken ME Ninomiya Y Hudson B Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of α, 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 (178) Google Scholar In Alport syndrome the entire width of the GBM is comprised of collagen α1(IV. and α2(IV) chains, which is the normal collagen composition of the embryonic GBM.12Miner JH Sanes JR Collagen IV α3, α4, and α5 chains in rodent basal lamina: sequence, distribution, association with laminins, and developmental switches.J Cell Biol. 1994; 127: 879-891Crossref PubMed Scopus (357) Google Scholar, 13Kalluri R Shield III, CF Todd P Hudson BG Nielson 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 (265) Google Scholar These changes result in progressive loss of glomerular function because of alterations in the GBM, podocyte effacement, and mesangial matrix expansion. Type IV collagen networks comprised of only α1(IV) and α2(IV. chains are more susceptible to endoproteolysis than GBM containing all five type IV collagen chains,13Kalluri R Shield III, CF Todd P Hudson BG Nielson 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 (265) Google Scholar which is likely because of the greater number of crosslinks formed in a network of collagen α3(IV), α4(IV), and α5(IV) chains.11Gunwar S Ballester F Noelken ME Ninomiya Y Hudson B Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of α, 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 (178) Google Scholar Based on these observations, it has been proposed that the irregular ultrastructure of Alport GBM might be attributed to focal endoproteolysis of the GBM. Two independently produced gene knockout murine models for Alport syndrome have been described,14Cosgrove 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. 1996; 10: 2981-2992Crossref PubMed Scopus (299) Google Scholar, 15Miner JH Sanes JR Molecular and functional defects in kidneys of mice lacking collagen α3(IV): implications for Alport syndrome.J Cell Biol. 1996; 135: 1403-1413Crossref PubMed Scopus (256) Google Scholar as well as one resulting from a random transgene insertion event.16Lu W Phillips CL Killen PD Hlaing WR Elder FF Miner JH Overbeek PA Meisler MH Insertional mutation of the collagen genes Col4a3 and Col4a4 in a mouse model for Alport syndrome.Genomics. 1999; 15: 113-124Crossref Scopus (65) Google Scholar These models have proven to have progressive renal disease that is remarkably similar to that in humans. Expansion of the mesangial matrix occurs early in Alport renal pathogenesis. The most abundant integrin on mesangial cells is the α1β1 heterodimer.17Patey N Halbwachs-Mecarelli D Droz D LeSavre P Noel LH Distribution of integrin subunits in normal human kidney.Cell Adhes Commun. 1994; 2: 159-167Crossref PubMed Scopus (36) Google Scholar, 18Sterk LM deMelker AA Kramer D Kuikman I Chand A Claessen N Weening JJ Sonnenberg A Glomerular extracellular matrix components and integrins.Cell Adhes Commun. 1998; 5: 177-192Crossref PubMed Google Scholar An α1 integrin knockout has been produced that shows no renal abnormalities and no phenotype detrimental to the survival of the animal.19Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin α1 by homologous recombination permits normal murine development, but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (230) Google Scholar Considering the recently described roles for α1β1 integrin in collagen-dependent cell proliferation, cell adhesion, mesangial matrix remodeling, and mesangial cell migration,19Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin α1 by homologous recombination permits normal murine development, but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (230) Google Scholar, 20Pozzi A Wary KK Giancotti FG Gardner HA Integrin α1β1 mediates a unique collagen-dependent proliferation pathway in vivo.J Cell Biol. 1999; 142: 587-594Crossref Scopus (252) Google Scholar, 21Kagami S Kondo S Loster K Reutter W Kuhara T Yatsumoto K Kuroda Y α1β1 integrin-mediated collagen matrix remodeling by rat mesangial cells is differentially regulated by transforming growth factor-β and platelet-derived growth factor-BB.J Am Soc Nephrol. 1999; 10: 779-789PubMed Google Scholar we suspected that integrin α1β1 might play a specific role in Alport renal disease progression. To test this notion, we produced a mouse null at both the collagen α3(IV) gene (Alport mouse) and the α1 integrin gene. These double-knockout mice have delayed onset and slowed progression of glomerular disease, attenuated expansion of the mesangial matrix, and markedly improved foot process architecture, illustrating a major role for α1β1 integrin in Alport glomerular disease progression. Transforming growth factor (TGF)-β has been shown to promote accumulation of extracellular matrix in both wound repair and fibrotic diseases, including glomerulonephritis.22Border WA Ruoslahti E Transforming growth factor-β in disease: the dark side of tissue repair.J Clin Invest. 1992; 90: 1-7Crossref PubMed Scopus (1049) Google Scholar In recent studies, we demonstrated a likely role for TGF-β1 in Alport glomerular and tubulointerstitial disease.23Sayers R Kalluri R Rodgers KD Shield III, CF Meehan DT Cosgrove D Role for TGF-β in Alport renal disease progression.Kidney Int. 1999; 56: 1662-1673Crossref PubMed Scopus (80) Google Scholar Herein, we extend these earlier studies by illustrating that inhibition of TGF-β1, by injecting a type II TGF-β soluble receptor as a competitive inhibitor, prevents the irregular thickening of the GBM. Treating the double knockouts with the TGF-β1 soluble receptor provides synergistic benefits, restoring podocyte foot process architecture, inhibiting matrix deposition in the GBM, and slowing mesangial matrix expansion. Based on this new evidence, we conclude that renal pathogenesis in Alport syndrome involves biochemical pathways modulated by TGF-β1 and integrin α1β1, and that the two pathways affect distinct aspects of glomerular pathology. The collagen α3(IV) knockout mice were described previously.14Cosgrove 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. 1996; 10: 2981-2992Crossref PubMed Scopus (299) Google Scholar These mice, which were originally in the 129 Sv/J background, were successively back-crossed 10 times with 129 Sv mice. Heterozygotes were then crossed with homozygote integrin α1 knockouts, also described previously,19Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin α1 by homologous recombination permits normal murine development, but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (230) Google Scholar which were on a pure 129 Sv background. A breeding stock of mice heterozygous for the collagen α3(IV) mutation and homozygous for the integrin α1 mutation was established. No difference in the kinetics of renal pathogenesis was observed for Alport mice on the 129 Sv/J versus 129 Sv backgrounds. Semiquantitative measurements of urinary protein were performed using Albustix reagent strips (Bayer Corporation, Elkhart, IN), and reading the relative amounts from the color chart provided with the kit. Successive readings were performed weekly on fresh urine from the same experimental animals. Microhematuria was assessed using Hemastix reagent strips (Bayer Corporation). Fractions of the above urine samples were subjected to gel analysis. Samples (the equivalent of 0.5 μl of undiluted urine) were fractionated by electrophoresis through 10% denaturing acrylamide gels. Urine creatinine levels were measured when sufficient urine was collected for meaningful readings (>100 μl). We observed no significant effect of the TGF-β1 inhibitor on urine creatinine levels, and these levels fluctuated by no more than 20% throughout the time course. The protein in the gels was stained with Coomassie blue and photographed. Bovine serum albumin was used as a molecular weight standard. End-stage renal disease was defined as the point in the disease progression where a weight loss of >15% of the body mass was observed. Animals were euthanized at this point and end stage was confirmed by visual examination. Blood urea nitrogen levels at this stage were consistently >200 mg/dl. The kidneys are visibly smaller with a granular surface and are pale in color. When such visual characteristics were present, histological examination invariably confirmed advanced fibrosis. Fresh external renal cortex was minced in 4% paraformaldehyde, allowed to fix for 2 hours, and stored at 5°C in phosphate-buffered saline (PBS). The tissue was washed extensively (5 times for 10 minutes each at 4°C) with 0.1 mol/L Sorenson's buffer (Sorenson's buffer was made by combining 100 ml of a 200 mmol/L monobasic sodium phosphate with 400 ml of 200 mmol/L dibasic sodium phosphate, with 500 ml of water, and adjusted to pH 7.4), and postfixed in 1% osmium tetroxide in Sorenson's buffer for 1 hour. The tissue was then dehydrated in graded ethanol (70%, then 80%, then 90%, then 100% for 10 minutes each), and finally in propylene oxide and embedded in Poly/Bed 812 epoxy resin (Polysciences, Inc., Warrington, PA) following the procedures described by the manufacturer. Glomeruli were identified in 1-μm sections stained with toluidine blue, and thin sections were cut at 70-nm thickness using a Reichert Jung Ultracut E ultramicrotome (Cambridge Instrument Co., Vienna, Austria). Sections were mounted onto grids and stained with uranyl acetate and lead citrate. Grid-mounted sections were examined and photographed using a Phillips CM10 electron microscope. Small pieces (approximately 2-mm cubes) of kidney cortex were fixed in 3% phosphate-buffered glutaraldehyde, then postfixed in 1. phosphate-buffered osmium tetroxide. Samples were then dehydrated in graded ethanols, and critical point-dried in carbon dioxide. The cubes were then cracked in pieces by stressing them with the edge of a razor blade, and mounted with glue onto stubs with the cracked surface facing upward. The surface was sputter-coated using gold/palladium and visualized with a scanning electron microscope. Fresh kidneys were removed and embedded in Tissue Tek OCT aqueous compound. They were frozen at −150°C and sectioned at 4 μm on a Microm cryostat. Slides were fixed in cold 100% acetone for 10 minutes then air-dried overnight. Slides were washed three times in cold 1× PBS. Primary antibodies were diluted together in 7% nonfat dry milk (1:200 for entactin, 1:10 for laminin α2), applied to the sections, and incubated overnight at 4°C in a humidified dish. Slides were then washed successively for 5, 7, and 10 minutes with cold 1× PBS. Secondary antibodies were diluted together at 1:00 (Texas red α-rabbit for lam-α2 and fluorescein isothiocyanate α-rat for entactin) in the blocking agent and applied for 4 hours at 4°C. After PBS washes, Vectashield (Vector Laboratories, App Imaging, Santa Clara, CA) anti-fade mounting media was applied and sections were coverslipped, sealed, and imaged. Images were collected using a Cytovision Ultra image analysis system (Applied Imaging, Inc. interfaced with an Olympus BH-2 fluorescence microscope. For the remaining laminin chain-specific antibodies, slides were brought to room temperature, then postfixed in cold (−20°C) acetone for 15 minutes, and air-dried overnight at 5°C. The specimens were rehydrated by three successive washes in PBS for 10 minutes each at room temperature, then denatured by immersing them in 0.1% sodium dodecyl sulfate at 37°C for 45 minutes. This step greatly enhanced immunoreactivity, presumably by exposing the masked laminin epitopes (B. Patton, personal communication). The specificity of the laminin chain-specific antibodies have been established in previous publications.24Miner JH Patton BL Lentz SI Gilbert DJ Snider WD Jenkens NA Copeland NG Sanes JR The laminin α chains: expression, developmental transitions, and chromosomal locations of α1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel α3 isoform.J Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (584) Google Scholar, 25Patton BL Miner JH Chiu AY Sanes JH Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice.J Cell Biol. 1997; 139: 1507-1521Crossref PubMed Scopus (379) Google Scholar The anti-laminin α1 (8B3) and β1 (5A2) antibodies were mouse monoclonals, and a gift from D. Abrahamson (Department of Anatomy and Cell Biology, University of Kansas Medicine Center, Kansas City, KS).26Abrahamson DR Irwin MH St John PL Perry EW Accavitti MA Heck LW Couchman JR Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains.Cell Biol. 1989; 109: 2477-2491Crossref Scopus (82) Google Scholar Anti-laminin α2 chain-specific rabbit antibodies were a gift from Dr. Peter Yurchenco (Robert Wood Johnson Medical School, Piscataway, New Jersey).27Vachon PH Loechel F Xu H Wewer UM Engvall E Merosin and laminin in myogenesis: specific requirement for merosin in myotube stability and survival.J Cell Biol. 1996; 134: 1483-1497Crossref PubMed Scopus (196) Google Scholar The anti-laminin α3 chain-specific antibodies were a gift from Dr. Bob Burgeson (anti laminin-5 rabbit antiserum No. 4101) and described previously.28Rousselle P Lundstrum GP Keene DR Burgeson RE Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.J Cell Biol. 1991; 114: 567-576Crossref PubMed Scopus (659) Google Scholar, 29Marinkovich MP Lunstrum GP Keene DR Burgeson RE The dermal-epidermal junction of human skin contains a novel laminin variant.J Cell Biol. 1992; 119: 695-703Crossref PubMed Scopus (235) Google Scholar, 30Sanes JR Engvall E Butkowski R Hunter DD Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere.J Cell Biol. 1990; 111: 1685-1699Crossref PubMed Scopus (505) Google Scholar The anti-laminin α4 (C0877) and α5 (8948) rabbit antisera were provided by Jeff Miner (Washington University School of Medicine Department of Nephrology, St Louis, MO).24Miner JH Patton BL Lentz SI Gilbert DJ Snider WD Jenkens NA Copeland NG Sanes JR The laminin α chains: expression, developmental transitions, and chromosomal locations of α1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel α3 isoform.J Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (584) Google Scholar The anti-laminin β2 (Gpb1. chain-specific guinea pig antisera were a gift from Joshua Sanes (Washington University School of Medicine Department of Anatomy and Neurobiology, St. Louis, MO).31Utani A Kopp JB Kozak CA Matsuki Y Amizuka N Sugiyama S Yamada Y Mouse kalinin B1 (laminin β3 chain): cloning and tissue distribution.Lab Invest. 1995; 72: 300-310PubMed Google Scholar Anti-laminin β3 antibodies were provided by Yoshi Yamada (National Institute of Dental and Craniofacial Research, Bethesda, MD).31Utani A Kopp JB Kozak CA Matsuki Y Amizuka N Sugiyama S Yamada Y Mouse kalinin B1 (laminin β3 chain): cloning and tissue distribution.Lab Invest. 1995; 72: 300-310PubMed Google Scholar A rat monoclonal antibody for the laminin γ1 chain was purchased from Chemicon (Temecula, CA). All secondary reagents were purchased from Vector Laboratories. For fibronectin immunostaining, the antibody was purchased from Sigma (St. Louis, MO; catalogue no. F3648). The same procedure was used as used for the laminin α2 chain. The extracellular domain of the murine TGF-β type II receptor was amplified from a murine lung cDNA library (Clontech, Palo Alto, CA. by PCR, and engineered to contain a 5′ Not I, and a 3′ Sal I restriction site. The Fc region of murine IgG2a was amplified by PCR from a murine hybridoma, and engineered to contain a 5′ Sal I restriction site and a 3′ Not I restriction site. The receptor and Fc fragments were purified, digested with the appropriate restriction enzymes, and ligated into the expression vector pEAG347, which contains tandem SV40, and adenovirus major late promoters; the SV40 late polyA termination signal; an ampicillin resistance gene; and a pSV2 dihydrofolate reductase-derived selection marker. The resulting construct (pAA002) was transformed into competent JM109, and plasmids were selected for the correct orientation of the receptor-Fc fusion gene. Proper sequence and alignment was confirmed by DNA sequencing. pAA002 was transfected in Chinese hamster ovary cells (CHO DUKX-B1) by electroporation. After selection in 200 nmol/L methotrexate, single clones were selected and screened for the expression of mTGFβR: Fc. The clone with the highest titer was picked for expansion to 20 L fermentors, and the expressed protein was purified over protein A-Sepharose (Pharmacia, Piscataway, NJ), under sterile, endotoxin-free conditions. The protein is >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and contains <1 U endotoxin per mg protein. Activity of the mTGFβR: Fc was assessed in the mink lung epithelial cell assay as described.32Tsang ML-S Zhou L Zheng BL Wenker J Fransen G Humphrey J Smith JM O'Connor-McCourt M Lucas R Weatherbee JA Characterization of recombinant soluble human transforming growth factor-β Type II (rhTGF-bsRII).Cytokine. 1995; 7: 389-397Crossref PubMed Scopus (75) Google Scholar, 33O'Connor-McCourt M Segarini P Grothe S Tsang ML-S Weatherbee JA Analysis of the interaction between two TGF-β binding proteins and three TGF-β isoforms using surface plasmon resonance.Ann NY Acad Sci. 1995; 766: 300-302Crossref PubMed Scopus (15) Google Scholar, 34Smith JD Bryant SR Couper LL Vary CPH Gotwals PJ Koteliansky VE Lindner V Soluble transforming growth factor-β type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial regrowth.Circ Res. 1999; 84: 1212-1222Crossref PubMed Scopus (192) Google Scholar Briefly, Mv1Lu cells (ATCC CCL-64) were maintained in minimal essential medium supplemented with 100 U/ml penicillin, 100 Tg/ml streptomycin, 10% fetal bovine serum, and 4 mmol/L l-glutamine. To test, serial dilutions of TGFβR: Fc were incubated with 0.1 ng/ml TGF-β1, 0.5 ng/ml TGF-β2, and 0.05 ng/ml TGF-β3 (R&D Systems, Minneapolis, MN) for 1 hour in assay medium (minimal essential medium supplemented with 100 U/ml of penicillin, 100 Tg/ml of streptomycin, and 10% fetal bovine serum) in a 96-well microtiter tissue culture plate. Mv1Lu cells were resuspended in assay medium and added to the assay plate at a concentration of 6,000 cells per well. The cells were incubated at 37°C for 48 hours and pulsed with [3H]thymidine (70 to 86 Ci/mmol; Amersham) for an additional 6 hours. DNA synthesis, which reflects cell proliferation, was determined by measuring incorporation of [3H]thymidine. As reported for similar TGF-β receptor antagonists,32Tsang ML-S Zhou L Zheng BL Wenker J Fransen G Humphrey J Smith JM O'Connor-McCourt M Lucas R Weatherbee JA Characterization of recombinant soluble human transforming growth factor-β Type II (rhTGF-bsRII).Cytokine. 1995; 7: 389-397Crossref PubMed Scopus (75) Google Scholar, 33O'Connor-McCourt M Segarini P Grothe S Tsang ML-S Weatherbee JA Analysis of the interaction between two TGF-β binding proteins and three TGF-β isoforms using surface plasmon resonance.Ann NY Acad Sci. 1995; 766: 300-302Crossref PubMed Scopus (15) Google Scholar mTGFβR: Fc blocks TGF-β1 (IC50 = 1 nmol/L), and TGF-β3 (IC50 = 1 nmol/L), but not TGF-β2-mediated inhibition of Mv1Lu cell proliferation. The collagen α3(IV)-deficient mouse developed in this laboratory14Cosgrove 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. 1996; 10: 2981-2992Crossref PubMed Scopus (299) Google Scholar was crossed with the integrin α1-deficient mouse19Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin α1 by homologous recombination permits normal murine development, but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (230) Google Scholar to produce an animal null at both alleles. Comparative analysis of renal disease pathogenesis of Alport mice versus these double-knockout mice revealed a markedly slower rate of renal disease in the double knockouts. Both the time of onset and the rate of progression of proteinuria was delayed relative to the Alport mice (Figure 1A and Table 1), suggesting improved function of the glomerular filter. The data in Table 1 represent successive analysis of urinary albumin in three sets of experimental animals, and illustrate that differences in onset and progression of proteinuria in the Alport mouse versus the double knockout are highly reproducible. Microhematuria was also assessed, and did not vary significantly among the experimental groups. The mean age of end-stage renal failure (as defined in the Methods section), based on analysis of 10 experimental sets (10 controls and 10 Alport, 10 integrin α1 null mice, and 10 double-knockout animals) was extended from 8.5 ± 0.5 weeks in the Alport mice to 14.5 ± 0.9 weeks in the double knockouts.Table 1Inactivating α1β1 Integrin and TGF-β1 Results in Additive Improvement in Slowing Both the Rate of Onset and Progression of Albuminuria in the Alport Mouse SystemAge (Wks)12345678910Control−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Control Sol Rec−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Alport−−±++++++++++Renal failure−−±++++++++++−−±++++++++++DKO−−−−±++++++++−−−−±+++++++−−−−−+++++++DKO Sol Rec−−−−−−±±±+−−−−−−±±±+−−−−−−−±±±Semiquantitative assessment of proteinuria was performed using Albustrix (Bayer Corporation, Elkhart, IN) at the indicated times. Each symbol represents an individual urine analysis. The data represents successive analysis of three individual animals for each of the indicated experimental groups. Approximate albumin concentrations: (−) = negative; (±) = trace; (+) = about 0.3 mg/ml; (++) = about 1 mg/ml; (+++) = greater than 3 mg/ml. DKO = double knockout; SolRec = animals treated with mTGFβR:Fc. Open table in a new tab Semiquantitative assessment of proteinuria was per
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