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Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

2013; Elsevier BV; Volume: 183; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2013.07.030

ISSN

1525-2191

Autores

Kirankumar Katta, Miriam Boersema, Saritha Adepu, Heleen Rienstra, Johanna W.A.M. Celie, Rik Mencke, Grietje Molema, Harry van Goor, Jo H. M. Berden, Gerjan Navis, Jan‐Luuk Hillebrands, Jacob van den Born,

Tópico(s)

Liver physiology and pathology

Resumo

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non–FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction. Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non–FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction. Renal chronic transplant dysfunction (CTD) is the leading cause of long-term loss of transplanted kidneys.1Kouwenhoven E.A. IJzermans J.N. de Bruin R.W. Etiology and pathophysiology of chronic transplant dysfunction.Transpl Int. 2000; 13: 385-401Crossref PubMed Google Scholar, 2Chapman J.R. O'Connell P.J. Nankivell B.J. Chronic renal allograft dysfunction.J Am Soc Nephrol. 2005; 16: 3015-3026Crossref PubMed Scopus (364) Google Scholar CTD is the result of tissue remodeling in the intrarenal arteries, glomeruli, and tubulointerstitium that leads to transplant vasculopathy, focal glomerulosclerosis (FGS), interstitial fibrosis, and tubular atrophy.2Chapman J.R. O'Connell P.J. Nankivell B.J. 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Sheaff M. Weening J.J. Banff '05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN').Am J Transplant. 2007; 7: 518-526Crossref PubMed Scopus (923) Google Scholar These lesions are characterized by accumulation of extracellular matrix, activation of mesangial cells, interstitial myofibroblasts, tubular epithelial cells, and chronic inflammation. To date, in the absence of knowledge of the pathogenetic mechanisms leading to development of these lesions, no effective therapies are available to prevent or treat renal CTD. Progressive loss can only be retarded by antihypertensive and antiproteinuric treatment in combination with lipid-lowering drugs. Recently, our research group reported accumulation of the heparan sulfate proteoglycans (HSPGs) collagen type XVIII and perlecan during glomerular and vascular tissue remodeling in CTD in rats.5Rienstra H. Katta K. Celie J.W. van Goor H. Navis G. van den Born J. Hillebrands J.L. Differential expression of proteoglycans in tissue remodeling and lymphangiogenesis after experimental renal transplantation in rats.PLoS One. 2010; 5: e9095Crossref PubMed Scopus (34) Google Scholar However, whether and how these proteoglycans mediate tissue remodeling in CTD is as yet unknown. In native kidney diseases and ischemia–reperfusion injury, we have already demonstrated that HSPGs are involved in leukocyte influx and proteinuria-mediated renal injury.6Celie J.W. Reijmers R.M. Slot E.M. Beelen R.H. Spaargaren M. Ter Wee P.M. Florquin S. van den Born J. Tubulointerstitial heparan sulfate proteoglycan changes in human renal diseases correlate with leukocyte influx and proteinuria.Am J Physiol Renal Physiol. 2008; 294: F253-F263Crossref PubMed Scopus (36) Google Scholar, 7Celie J.W. Rutjes N.W. Keuning E.D. Soininen R. Heljasvaara R. Pihlajaniemi T. Dräger A.M. Zweegman S. Kessler F.L. Beelen R.H. Florquin S. Aten J. van den Born J. Subendothelial heparan sulfate proteoglycans become major L-selectin and monocyte chemoattractant protein-1 ligands upon renal ischemia/reperfusion.Am J Pathol. 2007; 170: 1865-1878Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Zaferani A. Vives R.R. van der Pol P. Hakvoort J.J. Navis G.J. van Goor H. Daha M.R. Lortat-Jacob H. Seelen M.A. van den Born J. Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease.J Biol Chem. 2011; 286: 5359-5367Crossref PubMed Scopus (49) Google Scholar Perlecan is a large, modular, pericellular HSPG with a spatial distribution that enables control over major cell-signaling events such as migration, proliferation, and differentiation in response to bound growth factors. Perlecan is composed of five different domains, of which the C-terminal domain, named endorepellin, is best known for its antiangiogenic properties.9Whitelock J.M. Melrose J. Iozzo R.V. Diverse cell signaling events modulated by perlecan.Biochemistry. 2008; 47: 11174-11183Crossref PubMed Scopus (207) Google Scholar, 10Iozzo R.V. Sanderson R.D. Proteoglycans in cancer biology, tumour environment and angiogenesis.J Cell Mol Med. 2011; 15: 1013-1031Crossref PubMed Scopus (409) Google Scholar The carbohydrate side chains (ie, the glycosaminoglycans) are attached to proteoglycan core proteins and can bind a variety of ligands, depending on their highly variable composition (mainly variations in patterns of N- and O-sulfation).11Lortat-Jacob H. The molecular basis and functional implications of chemokine interactions with heparan sulphate.Curr Opin Struct Biol. 2009; 19: 543-548Crossref PubMed Scopus (90) Google Scholar Along with chondroitin sulfate and dermatan sulfate proteoglycans, HSPGs form the large majority of the proteoglycan family. Potential ligands include chemokines11Lortat-Jacob H. The molecular basis and functional implications of chemokine interactions with heparan sulphate.Curr Opin Struct Biol. 2009; 19: 543-548Crossref PubMed Scopus (90) Google Scholar and growth factors such as basic fibroblast growth factor (bFGF/FGF2).12Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4.J Biol Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 13Gallagher J.T. Turnbull J.E. Heparan sulphate in the binding and activation of basic fibroblast growth factor.Glycobiology. 1992; 2: 523-528Crossref PubMed Scopus (93) Google Scholar Proteoglycans are highly involved in morphogenesis and in tissue remodeling processes.14Schwabiuk M. Coudiere L. Merz D.C. SDN-1/syndecan regulates growth factor signaling in distal tip cell migrations in C. elegans.Dev Biol. 2009; 334: 235-242Crossref PubMed Scopus (22) Google Scholar, 15Olivares G.H. 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Basement membrane proteoglycans: modulators par excellence of cancer growth and angiogenesis.Mol Cells. 2009; 27: 503-513Crossref PubMed Scopus (174) Google Scholar, 19Aviezer D. Hecht D. Safran M. Eisinger M. David G. Yayon A. Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis.Cell. 1994; 79: 1005-1013Abstract Full Text PDF PubMed Scopus (489) Google Scholar Studies in nontransplantation models of renal and vascular disease have identified potential roles of FGF2 in FGS and neointima formation.20Nakamura T. Ebihara I. Fukui M. Osada S. Nagaoka I. Horikoshi S. Tomino Y. Koide H. Messenger RNA expression for growth factors in glomeruli from focal glomerular sclerosis.Clin Immunol Immunopathol. 1993; 66: 33-42Crossref PubMed Scopus (44) Google Scholar, 21Floege J. Kriz W. Schulze M. Susani M. Kerjaschki D. Mooney A. Couser W.G. Koch K.M. Basic fibroblast growth factor augments podocyte injury and induces glomerulosclerosis in rats with experimental membranous nephropathy.J Clin Invest. 1995; 96: 2809-2819Crossref PubMed Scopus (143) Google Scholar, 22Kriz W. Hähnel B. Rösener S. Elger M. Long-term treatment of rats with FGF-2 results in focal segmental glomerulosclerosis.Kidney Int. 1995; 48: 1435-1450Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 23Chan J. Prado-Lourenco L. Khachigian L.M. Bennett M.R. Di Bartolo B.A. Kavurma M.M. TRAIL promotes VSMC proliferation and neointima formation in a FGF-2-, Sp1 phosphorylation-, and NFkappaB-dependent manner.Circ Res. 2010; 106: 1061-1071Crossref PubMed Scopus (67) Google Scholar, 24Camozzi M. Zacchigna S. Rusnati M. Coltrini D. Ramirez-Correa G. Bottazzi B. Mantovani A. Giacca M. Presta M. Pentraxin 3 inhibits fibroblast growth factor 2-dependent activation of smooth muscle cells in vitro and neointima formation in vivo.Arterioscler Thromb Vasc Biol. 2005; 25: 1837-1842Crossref PubMed Scopus (86) Google Scholar These findings led us to hypothesize that interaction of FGF2 with proteoglycans also affects tissue remodeling processes in CTD. To test this hypothesis, we used an experimental rat CTD model. We microdissected glomeruli, the arterial media, and the neointima and performed low-density quantitative RT-PCR (RT-qPCR) analysis for matrix and cell-surface proteoglycans and FGF2. In addition, we profiled the heparan sulfate (HS) polysaccharide side chains by anti-HS monoclonal antibodies (mAbs) and their binding capacity for FGF2 and L-selectin. Functionally, we investigated the HSPG involvement of FGF2-driven mesangial proliferation. Our data indicate spatial proteoglycan involvement in CTD and thus suggest a potential target for intervention therapy in the future. Inbred female Dark Agouti (DA) rats (175 to 210 g) were obtained from Harlan Laboratories (Horst, the Netherlands; Indianapolis, IN) and inbred male Wistar Furth (WF) rats (240 to 295 g) from Charles River Laboratories International (l'Arbresle, France; Wilmington, MA). All animals received care in compliance with the NIH Guide for the Care and Use of Laboratory Animals (revised 1985), the University of Groningen guidelines for animal husbandry, and the Dutch Law on Experimental Animal Care. Female DA kidney allografts were orthotopically transplanted into male Wistar–Furth recipients, as described previously.5Rienstra H. Katta K. Celie J.W. van Goor H. Navis G. van den Born J. Hillebrands J.L. Differential expression of proteoglycans in tissue remodeling and lymphangiogenesis after experimental renal transplantation in rats.PLoS One. 2010; 5: e9095Crossref PubMed Scopus (34) Google Scholar, 25Rienstra H. Boersema M. Onuta G. Boer M.W. Zandvoort A. van Riezen M. Rozing J. van Goor H. Navis G.J. Popa E.R. Hillebrands J.L. Donor and recipient origin of mesenchymal and endothelial cells in chronic renal allograft remodeling.Am J Transplant. 2009; 9: 463-472Crossref PubMed Scopus (26) Google Scholar, 26Boersema M. Katta K. Rienstra H. Molema G. Nguyen T.Q. Goldschmeding R. Navis G. van den Born J. Popa E.R. Hillebrands J.L. Local medial microenvironment directs phenotypic modulation of smooth muscle cells after experimental renal transplantation.Am J Transplant. 2012; 12: 1429-1440Crossref PubMed Scopus (14) Google Scholar Cold ischemic time ranged from 16 to 38 minutes; warm ischemic time ranged from 19 to 32 minutes. Recipients received 5 mg/kg s.c. cyclosporin A (Sandimmune; Novartis, Basel, Switzerland) on the first 10 days after transplantation. The contralateral kidney was removed 8 to 14 days after transplantation. Total follow-up time was 12 weeks, unless animals had to be sacrificed sooner because of renal failure. Allografts that developed severe CTD were used in this study (n = 5). Further characteristics of the model are described elsewhere.5Rienstra H. Katta K. Celie J.W. van Goor H. Navis G. van den Born J. Hillebrands J.L. Differential expression of proteoglycans in tissue remodeling and lymphangiogenesis after experimental renal transplantation in rats.PLoS One. 2010; 5: e9095Crossref PubMed Scopus (34) Google Scholar, 25Rienstra H. Boersema M. Onuta G. Boer M.W. Zandvoort A. van Riezen M. Rozing J. van Goor H. Navis G.J. Popa E.R. Hillebrands J.L. Donor and recipient origin of mesenchymal and endothelial cells in chronic renal allograft remodeling.Am J Transplant. 2009; 9: 463-472Crossref PubMed Scopus (26) Google Scholar, 26Boersema M. Katta K. Rienstra H. Molema G. Nguyen T.Q. Goldschmeding R. Navis G. van den Born J. Popa E.R. Hillebrands J.L. Local medial microenvironment directs phenotypic modulation of smooth muscle cells after experimental renal transplantation.Am J Transplant. 2012; 12: 1429-1440Crossref PubMed Scopus (14) Google Scholar Nontransplanted DA kidneys (n = 5) and DA-to-DA isografted kidneys (n = 5) served as controls. Laser microdissection, RNA isolation, and RT-qPCR were performed essentially according to Asgeirsdottir et al.27Asgeirsdottir S.A. Werner N. Harms G. Van Den Berg A. Molema G. Analysis of in vivo endothelial cell activation applying RT-PCR following endothelial cell isolation by laser dissection microscopy.Ann N Y Acad Sci. 2002; 973: 586-589Crossref PubMed Scopus (9) Google Scholar Glomeruli and various layers (including the tunica media and the neointima) of larger arteries were separately dissected from nine serial sections per kidney. An average of 194 (range, 71 to 308) glomeruli and 63 (range, 25 to 117) arteries were dissected from each kidney. Glomeruli and the various arterial layers were isolated from allografted, isografted, and nontransplanted kidneys (n = 5 per group). Total RNA was also isolated from whole-kidney sections from allograft, isograft, and nontransplanted animals (n = 3 per group). Total RNA was isolated from microdissected structures and whole kidney using an RNeasy micro kit (Qiagen, Hilden, Germany; Valencia, CA). Reverse transcription was performed using Invitrogen SuperScript III Reverse Transcriptase (Life Technologies, Breda, the Netherlands; Carlsbad, CA) and random hexamer primers (Promega, Leiden, the Netherlands; Madison, WI). Gene expression was analyzed with a custom-made microfluidic card–based low-density array (Life Technologies–Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands) using an ABI Prism 7900HT sequence detection system (Life Technologies–Applied Biosystems). Relative mRNA levels were calculated as 2−ΔCT, where ΔCT = CT(gene of interest) − CT(β-actin). CT values that were beyond detection level were set manually to 50. Composition of the low-density array is presented in Table 1.Table 1Composition of Low-Density ArrayGene nameProtein short nameGene symbolAssay IDMatrix molecules Collagen, type I, alpha 1Collagen ICol1a1Rn01463848_m1 Collagen, type IV, alpha 1Collagen IVCol4a1Rn01482925_m1Proteoglycans PerlecanPerlecanLOC313641Rn01515780_g1 AgrinAgrinAgrnRn00598349_m1 VersicanVersicanVcanRn01493755_m1 BiglycanBiglycanBgnRn00567229_m1 Syndecan 1Syndecan-1Sdc1Rn00564662_m1 Syndecan 4Syndecan-4Sdc4Rn00561900_m1Growth factors Fibroblast growth factor 2FGF2Fgf2Rn00570809_m1 Transforming growth factor, beta 1TGF-β1Tgfb1Rn01475963_m1Reference genes Beta actinβ-ActinActbRn00667869_m1 Beta 2 microglobulinB2MB2mRn00560865_m1 Eukaryotic 18S rRNA18S18SHs99999901_s1 Open table in a new tab Frozen sections (4 μm thick) were fixed in acetone or 4% formaldehyde and were blocked for endogenous peroxidase activity with 0.03% H2O2 if appropriate. Sections were blocked with normal goat or rabbit serum. Sections were incubated for 1 hour with the following primary antibodies: mouse anti-human FGF2 (PeproTech, London, UK; Rocky Hill, NJ), mouse anti-HS mAb JM-403,28van den Born J. Gunnarsson K. Bakker M.A. Kjellén L. Kusche-Gullberg M. Maccarana M. Berden J.H. Lindahl U. Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody.J Biol Chem. 1995; 270: 31303-31309Crossref PubMed Scopus (133) Google Scholar mouse anti-HS stub mAb 3G10 (Seikagaku, Tokyo, Japan),29David G. Bai X.M. Van der Schueren B. Cassiman J.J. Van den Berghe H. Developmental changes in heparan sulfate expression: in situ detection with mAbs.J Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (407) Google Scholar and mouse anti-rat perlecan (clone 10B2; kindly provided by J.R. Couchman, University of Copenhagen). Binding of primary antibodies was detected by incubating the sections for 30 minutes with secondary antibodies diluted in PBS with 5% normal rat serum: goat anti-mouse IgG1 horseradish peroxidase (HRP) (SouthernBiotech, Birmingham, AL), rabbit anti-mouse IgM HRP (Dako, Heverlee, Belgium; Carpinteria, CA), or goat anti-mouse IgG1 Alexa Fluor 488 (Life Technologies). HRP activity was visualized using a tyramide signal amplification tetramethylrhodamine system (TSA; PerkinElmer, Waltham, MA). All fluorescence microscopy was performed using a Leica DMLB microscope (Leica Microsystems, Rijswijk, the Netherlands; Wetzlar, Germany) equipped with a Leica DC300F camera and Leica QWin software version 2.8. Frozen sections (4 μm thick) were fixed in 2% paraformaldehyde for 10 minutes and then were permeabilized in 0.5% Triton X-100 for 5 minutes. Sections were incubated for 1 hour with mouse anti-rat perlecan (mIgG1, clone 10B2) or a cocktail of mouse anti-rat CD90 (alias Thy-1) (mIgG1, clone OX7, tissue culture supernatant) and polyclonal rabbit anti–Ki-67 (Novocastra NCL-Ki67p; Leica Biosystems, Wetzlar, Germany) antibodies. After primary antibody incubation, sections were blocked for endogenous peroxidase activity with 0.03% H2O2 if appropriate. Binding of primary antibodies was detected by incubating the sections for 30 minutes with appropriate secondary antibodies diluted in PBS–1% bovine serum albumin with 3% normal rat serum. The secondary antibodies used were goat anti-mouse IgG1 Alexa Fluor 488 (Life Technologies) and goat anti-rabbit IgG HRP (Dako). HRP activity was visualized using the TSA tetramethylrhodamine system (PerkinElmer). Nuclei were counterstained with DAPI. Slides were mounted with Aqua PolyMount medium (Polysciences, Warrington, PA). Images were acquired with an Axio Observer Z1 inverted microscope (Carl Zeiss Microscopy, Jena, Germany) equipped with TissueFAXS acquisition software version 2.0.4 (TissueGnostics, Vienna, Austria; Tarzana, CA). To detect capacity of renal proteoglycans to bind FGF2, formalin-fixed rat renal sections were incubated with 1 μg/mL recombinant human FGF2 (recFGF2; PeproTech) for 60 minutes. After a washing step, staining was continued according to the FGF2 staining protocol as described above. Use of formalin fixation essentially avoids recognition of endogenous renal FGF2 by anti-FGF2 antibodies. Similarly, paraformaldehyde-fixed rat renal sections were incubated with 1 μg/mL L-selectin–Fc recombinant chimeric protein and visualized as described previously.30Celie J.W. Beelen R.H. van den Born J. Effect of fixation protocols on in situ detection of L-selectin ligands.J Immunol Methods. 2005; 298: 155-159Crossref PubMed Scopus (17) Google Scholar To confirm that the observed binding pattern was mediated by HS proteoglycans, the sections were pretreated with 0.05 U/mL heparitinase I (EC 4.2.2.8, Flavobacterium heparinum; Seikagaku) for 1 hour at 37°C in a humidified chamber. In an enzyme-linked immunosorbent assay (ELISA) approach, we evaluated the capability of fluid phase HS-like polysaccharides to compete for the interaction of recFGF2 with immobilized perlecan. To this end, MaxiSorp 96-well plates (Nalge Nunc International, Rochester, NY) were coated overnight in PBS with 5 μg/mL perlecan (Sigma-Aldrich, St. Louis, MO). After a washing in PBS with 0.05% Tween 20, wells were blocked with 5% nonfat milk powder in Tris-buffered saline for 1 hour. In a separate microtiter plate, 0.5 μg/mL recFGF2 was incubated for 30 minutes with a dilution range of different HS-like polysaccharides and then was transferred to the ELISA plate after the wells had been washed again. Incubation in the wells took 1 hour. The wells were washed again, and monoclonal mouse anti-FGF2 antibodies were added to the wells (0.5 μg/mL Tris-buffered saline–Tween 20). Secondary antibody was added after a washing step (HRP-labeled rabbit anti-mouse IgG, 1:5000; Dako). Secondary antibody was detected with 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich) for 15 minutes in the dark, and the reaction was stopped by adding 1.5 N H2SO4. Absorbance was measured at 450 nm in a microplate reader. All incubations were performed in a volume of 100 μL per well at room temperature. Polysaccharides used in the FGF2 competition ELISA were heparin from porcine intestinal mucosa (Sigma-Aldrich) and HS from bovine kidney (Seikagaku); N-sulfation of HS from bovine kidney and N- and O-desulfation of heparin from porcine intestine were performed as described before.8Zaferani A. Vives R.R. van der Pol P. Hakvoort J.J. Navis G.J. van Goor H. Daha M.R. Lortat-Jacob H. Seelen M.A. van den Born J. Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease.J Biol Chem. 2011; 286: 5359-5367Crossref PubMed Scopus (49) Google Scholar HS from human aorta was isolated essentially according to the method of Iverius.31Iverius P.H. Coupling of glycosaminoglycans to agarose beads (Sepharose 4B).Biochem J. 1971; 124: 677-683Crossref PubMed Scopus (234) Google Scholar HS from Engelbreth–Holm–Swarm murine sarcoma was obtained from Seikagaku. To evaluate FGF2 binding by perlecan from renal lysates, a slightly modified ELISA approach was followed. Perlecan from renal lysates (10 μg protein/mL carbonate buffer) was immunocaptured on monoclonal mouse anti-rat perlecan antibody (1:1000 in PBS; mAb 10B2; provided by J.R. Couchman) immobilized in MaxiSorp 96-well plates. After appropriate blocking, captured renal perlecan was incubated with 0.5 μg/mL recFGF2, followed by biotinylated anti-FGF2 mAb (clone JKFb-2, 1 μg/mL Tris-buffered saline–Tween 20; Novus Biologicals, Cambridge, UK) and then by HRP-conjugated streptavidin (1:5000 in Tris-buffered saline–Tween 20). Substrate reaction and plate reading was as described above. Rat mesangial cells (positive for Thy 1.1, perlecan, and α-smooth muscle actin; passage 11 to 15) were cultured in 24-well plates in Dulbecco's modified Eagle's medium supplemented with 25 mmol/L HEPES, 4.5 mg/mL glucose, pyridoxine, 1 mmol/L pyruvate, 50 ng/mL insulin, and 10% fetal bovine serum. Before stimulation with FGF2, cells were grown until confluency, serum-deprived (0.5% serum) for 24 hours, and then incubated with 0.031 to 8 ng/mL of FGF2 for 24 hours. For measurement of proliferation, 0.5 μCi/mL [3H]thymidine (GE Healthcare) was added to the cultures. After 24 hours, 5% trichloroacetic acid precipitable material was dissolved in 0.1% SDS, OptiPhase HiSafe 3 liquid scintillation cocktail (PerkinElmer) was added, and radioactivity was counted in a Wallac 1214 Rackbeta liquid scintillation counter (PerkinElmer). To study whether FGF2-induced proliferation of mesangial cells was reduced in the presence of exogenous HS, mesangial cells were stimulated with 0.5 ng/mL FGF2 for 24 hours in the presence of various concentrations (0, 8, 32, and 128 μg/mL) of exogenous HS from bovine kidney (HSBK; Seikagaku). Proliferation was measured as described above. To study whether proteoglycan sulfation was reduced by chlorate, mesangial cells were cultured for 24 hours in the presence of 5 to 25 mmol/L sodium chlorate (Sigma-Aldrich) and 2 μCi/mL [35S]sulfate (GE Healthcare, Little Chalfont, UK). Incorporation of [35S]sulfate into proteoglycans was quantified as described above for [3H]thymidine incorporation. Finally, to analyze whether chlorate impairs FGF2-induced proliferation of mesangial cells, cells were stimulated with 0.5 ng/mL FGF2 for fixed times (0, 0.5, 1, 2, 4, and 24 hours) in the presence or absence of 25 mmol/L sodium chlorate and in the presence of 0.5 μCi/mL [3H]thymidine. Total culture time was 24 hours. Proliferation was determined as described above. Before stimulation with FGF2, cells were grown until confluency, serum-deprived (0.5% serum) for 24 hours, and incubated with FGF2 for 24 hours. Proliferation was measured by adding 0.5 μCi/mL [3H]thymidine (GE Healthcare) for 24 hours to the cultures. After 24 hours, 5% trichloroacetic acid precipitable material was dissolved in 0.1% SDS, OptiPhase HiSafe 3 cocktail (PerkinElmer) was added, and radioactivity was counted in a Wallac 1214 Rackbeta liquid scintillation counter (PerkinElmer). Incorporation of [35S]sulfate into proteoglycans was quantified similarly as described above for [3H]thymidine incorporation. Rat mesangial cells were cultured as described above. After expansion, cells were seeded on coverslips and serum-starved for 24 hours in medium containing 0.5% fetal calf serum (FCS) after attachment. Subsequently, cells were stimulated for 48 hours in medium containing either 2% or 10% FCS. After stimulation, cells were fixed in 2% paraformaldehyde and double-stained for α-SMA (mIgG2a, clone 1A4; Dako) and perlecan (mIgG1, clone 10B2) as described above. Binding of primary antibodies was detected by incubating the sections for 30 minutes with goat anti-mouse IgG1 Alexa Fluor 488 and goat anti-mouse IgG2a Alexa Fluor 555 (Life Technologies). Nuclei were counterstained with DAPI. Slides were mounted with Aqua PolyMount medium (Polysciences). Confocal microscopy was performed using an inverted microscope (Zeiss LSM 780 NLO; Axio Observer Z1). To quantify total cell numbers and numbers of perlecan expressing mesangial cells, coverslips were scanned using TissueFAXS acquisition software (TissueGostics) on a Zeiss Axio Observer Z1 inverted microscope. Quantitative analyses were performed using TissueQuest fluorescence analysis software (TissueGnostics). mRNA expression levels were analyzed using a one-way analysis of variance with Tukey's post hoc test. P values of <0.05 were considered statistically significant (IBM SPSS software version 18; IBM, Armonk, NY). Statistical outliers, as detected by Grubbs' test for outliers, were excluded from analyses. Mesangial cell culture data were expressed as means ± SEM and analyzed by one-way analysis of variance. If overall P < 0.05, Bonferroni's multiple compa

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