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IL-1 Up-Regulates Osteopontin Expression in Experimental Crescentic Glomerulonephritis in the Rat

1999; Elsevier BV; Volume: 154; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)65330-8

ISSN

1525-2191

Autores

Xue Yu, Jun-Ming Fan, David J. Nikolic‐Paterson, Nianshen Yang, Wei Mu, Raimund Pichler, Richard J. Johnson, Robert C. Atkins, Hui Y. Lan,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Osteopontin (OPN) is a macrophage chemotactic and adhesion molecule that acts to promote macrophage infiltration in rat anti-glomerular basement membrane (GBM) glomerulonephritis. The present study investigated the role of interleukin-1 (IL-1) in the up-regulation of renal OPN expression in this disease model. Accelerated anti-GBM glomerulonephritis was induced in groups of six rats. Animals were treated by a constant infusion of the IL-1 receptor antagonist or saline (control) over days −1 to 14 (induction phase) or days 7 to 21 (established disease). In normal rat kidney, OPN was expressed in a few tubules ( 5 cells per glomerular cross-section) and tubular epithelial cells (50–75% OPN-positive). Up-regulation of OPN expression was associated with macrophage accumulation within the kidney, severe proteinuria, loss of renal function, and severe histological damage including glomerular crescentic formation and tubulointerstitial fibrosis. In contrast, IL-1 receptor antagonist treatment of either the induction phase of disease or established disease significantly reduced OPN mRNA and protein expression in glomeruli (↓75–85%, P < 0.001) and tubules (↓45–60%, P < 0.001). The reduction in OPN expression was associated with significant inhibition of macrophage accumulation and progressive renal injury. In vitro, the addition of IL-1 to the normal rat tubular epithelial cell line NRK52E up-regulated OPN mRNA and protein levels, an effect that was dose-dependent and inhibited by the addition of IL-1 receptor antagonist, thus demonstrating that IL-1 can act directly to up-regulate renal OPN expression. In conclusion, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and support for the argument that inhibition of OPN expression is one mechanism by which IL-1 receptor antagonist treatment suppresses macrophage-mediated renal injury. Osteopontin (OPN) is a macrophage chemotactic and adhesion molecule that acts to promote macrophage infiltration in rat anti-glomerular basement membrane (GBM) glomerulonephritis. The present study investigated the role of interleukin-1 (IL-1) in the up-regulation of renal OPN expression in this disease model. Accelerated anti-GBM glomerulonephritis was induced in groups of six rats. Animals were treated by a constant infusion of the IL-1 receptor antagonist or saline (control) over days −1 to 14 (induction phase) or days 7 to 21 (established disease). In normal rat kidney, OPN was expressed in a few tubules ( 5 cells per glomerular cross-section) and tubular epithelial cells (50–75% OPN-positive). Up-regulation of OPN expression was associated with macrophage accumulation within the kidney, severe proteinuria, loss of renal function, and severe histological damage including glomerular crescentic formation and tubulointerstitial fibrosis. In contrast, IL-1 receptor antagonist treatment of either the induction phase of disease or established disease significantly reduced OPN mRNA and protein expression in glomeruli (↓75–85%, P < 0.001) and tubules (↓45–60%, P < 0.001). The reduction in OPN expression was associated with significant inhibition of macrophage accumulation and progressive renal injury. In vitro, the addition of IL-1 to the normal rat tubular epithelial cell line NRK52E up-regulated OPN mRNA and protein levels, an effect that was dose-dependent and inhibited by the addition of IL-1 receptor antagonist, thus demonstrating that IL-1 can act directly to up-regulate renal OPN expression. In conclusion, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and support for the argument that inhibition of OPN expression is one mechanism by which IL-1 receptor antagonist treatment suppresses macrophage-mediated renal injury. Osteopontin (OPN) is a highly acidic glycoprotein that contains an adhesive arginine-glycine-aspartic acid sequence.1Butler WT Structural and functional domains of osteopontin.Ann N Y Acad Sci. 1995; 760: 6-11Crossref PubMed Scopus (74) Google Scholar OPN functions as a cell adhesion and migration molecule which can bind to a number of ligands including the αvβ3 integrin (vitronectin receptor), CD44, collagen type I, and fibronectin.1Butler WT Structural and functional domains of osteopontin.Ann N Y Acad Sci. 1995; 760: 6-11Crossref PubMed Scopus (74) Google Scholar, 2Reinholt FP Hultenby K Oldberg A Heinegard D Osteopontin: a possible anchor of osteoclasts to bone.Proc Natl Acad Sci USA. 1990; 87: 4473-4475Crossref PubMed Scopus (693) Google Scholar, 3Weber GF Ashkar S Glimcher MJ Cantor H Receptor-ligand interaction between CD44 and osteopontin (Eta-1).Science. 1996; 271: 509-512Crossref PubMed Scopus (800) Google Scholar A wide range of cell types including osteoclasts, some epithelia, macrophages, T cells, smooth muscle cells, and some tumors has been shown to express OPN in a constitutive or inducible fashion.2Reinholt FP Hultenby K Oldberg A Heinegard D Osteopontin: a possible anchor of osteoclasts to bone.Proc Natl Acad Sci USA. 1990; 87: 4473-4475Crossref PubMed Scopus (693) Google Scholar, 3Weber GF Ashkar S Glimcher MJ Cantor H Receptor-ligand interaction between CD44 and osteopontin (Eta-1).Science. 1996; 271: 509-512Crossref PubMed Scopus (800) Google Scholar, 4Brown L F Berse B Van de Water L Papadopoulos-Sergiou A Perruzzi CA Manseau EJ Dvorak HF Senger DR Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces.Mol Biol Cell. 1992; 3: 1169-1180Crossref PubMed Scopus (392) Google Scholar, 5Lopez CA Hoyer JR Wilson PD Waterhouse P Denhardt DT Heterogeneity of osteopontin expression among nephrons in mouse kidneys and enhanced expression in sclerotic glomeruli.Lab Invest. 1993; 69: 355-363PubMed Google Scholar, 6Miyazaki Y Setoguchi M Yoshida S Higuchi Y Akizuki S Yamamoto S The mouse osteopontin gene: expression in monocytic lineages and complete nucleotide sequence.J Biol Chem. 1990; 265: 14432-14438Abstract Full Text PDF PubMed Google Scholar, 7Patarca R Freeman GJ Singh RP Wei FY Durfee T Blattner F Regnier DC Kozak CA Mock BA Morse III, HC Jerrells TR Cantor H Structural and functional studies of the early T lymphocyte activation 1 (Eta-1) gene: definition of a novel T cell-dependent response associated with genetic resistance to bacterial infection.J Exp Med. 1989; 170: 145-161Crossref PubMed Scopus (293) Google Scholar, 8Giachelli C Bae N Lombardi D Majesky M Schwartz S Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR).Biochem Biophys Res Commun. 1991; 177: 867-873Crossref PubMed Scopus (176) Google Scholar, 9Senger DR Perruzzi CA Gracey CF Papadopoulos A Tenen DG Secreted phosphoproteins associated with neoplastic transformation: close homology with plasma proteins cleaved during blood coagulation.Cancer Res. 1988; 48: 5770-5774PubMed Google Scholar The adhesive functions of OPN are thought to be involved in diverse biological activities such as bone absorption, tumor metastasis, and inhibition of renal stone formation.2Reinholt FP Hultenby K Oldberg A Heinegard D Osteopontin: a possible anchor of osteoclasts to bone.Proc Natl Acad Sci USA. 1990; 87: 4473-4475Crossref PubMed Scopus (693) Google Scholar, 9Senger DR Perruzzi CA Gracey CF Papadopoulos A Tenen DG Secreted phosphoproteins associated with neoplastic transformation: close homology with plasma proteins cleaved during blood coagulation.Cancer Res. 1988; 48: 5770-5774PubMed Google Scholar, 10Ross FP Chappel J Alvarez JI Sander D Butler WT Farach-Carson MC Mintz KA Robey PG Teitelbaum SL Cheresh DA Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin αvβ3 potentiate bone resorption.J Biol Chem. 1993; 268: 9901-9907Abstract Full Text PDF PubMed Google Scholar, 11Shiraga H Min W VanDusen WJ Clayman MD Miner D Terrell CH Sherbotie JR Foreman JW Przysiecki C Neilson EG Hoyer JR Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily.Proc Natl Acad Sci USA. 1992; 89: 426-430Crossref PubMed Scopus (383) Google Scholar A functional role for OPN in monocyte infiltration at sites of inflammation has recently been established. OPN, which binds avidly to macrophages, induces prominent monocyte infiltration when injected subcutaneously in mice.12Singh RP Patarca R Schwartz J Singh P Cantor H Definition of a specific interaction between the early T lymphocyte activation 1 (Eta-1) protein and murine macrophages in vitro and its effect upon macrophages in vivo.J Exp Med. 1990; 171: 1931-1942Crossref PubMed Scopus (211) Google Scholar In addition, macrophage accumulation induced by intradermal injection of the chemoattractant N-formyl-met-leu-phe in rats is inhibited by administration of a neutralizing anti-OPN antibody.13Giachelli CM Lombardi D Johnson RJ Murry CE Almeida M Evidence for a role of osteopontin in macrophage infiltration in response to pathological stimuli in vivo.Am J Pathol. 1998; 152: 353-358PubMed Google Scholar Macrophage infiltration is thought to play an important role in mediating renal injury in both immune and nonimmune forms of kidney disease.14Nikolic-Paterson DJ Lan HY Atkins RC Macrophages in immune renal injury.in: Neilson EG Couser WG Immunologic Renal Diseases. Lippincott-Raven, Philadelphia1977: 575-592Google Scholar A clear association between up-regulation of OPN expression and macrophage infiltration has been described in a wide range of experimental models of glomerular and interstitial nephritis.15Pichler R Giachelli CM Lombardi D Pippin J Gordon K Alpers CE Schwartz SM Johnson RJ Tubulointerstitial disease in glomerulonephritis: potential role of osteopontin (uropontin).Am J Pathol. 1994; 144: 915-926PubMed Google Scholar, 16Giachelli CM Pichler R Lombardi D Denhardt DT Alpers CE Schwartz SM Johnson RJ Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis.Kidney Int. 1994; 45: 515-524Crossref PubMed Scopus (216) Google Scholar, 17Pichler RH Franceschini N Young BA Hugo C Andoh TF Burdmann EA Shankland SJ Alpers CE Bennett WM Couser WG Johnson RJ Pathogenesis of cyclosporine nephropathy: roles of angiotensin II and osteopontin.J Am Soc Nephrol. 1995; 6: 1186-1196PubMed Google Scholar, 18Diamond JR Kees-Folts D Ricardo SD Pruznak A Eufemio M Early and persistent up-regulated expression of renal cortical osteopontin in experimental hydronephrosis.Am J Pathol. 1995; 146: 1455-1466PubMed Google Scholar, 19Eddy AA Giachelli CM Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria.Kidney Int. 1995; 47: 1546-1557Crossref PubMed Scopus (360) Google Scholar, 20Kleinman JG Worcester EM Beshensky AM Sheridan AM Bonventre JV Brown D Upregulation of osteopontin expression by ischemia in rat kidney.Ann N Y Acad Sci USA. 1995; 760: 321-323Crossref PubMed Scopus (47) Google Scholar, 21Magil AB Pichler R Johnson RJ Osteopontin in chronic puromycin aminonucleoside nephrosis.J Am Soc Nephrol. 1997; 8: 1383-1390PubMed Google Scholar, 22Lan HY Yu XQ Yang N Nikolic-Paterson DJ Mu W Pichler R Johnson RJ Atkins RC De novo glomerular osteopontin expression in rat crescentic glomerulonephritis.Kidney Int. 1998; 53: 136-145Crossref PubMed Scopus (74) Google Scholar A functional role for OPN in promoting macrophage-mediated renal injury has recently been demonstrated in rat crescentic anti-glomerular basement membrane (GBM) glomerulonephritis.23Yu XQ Nikolic-Paterson DJ Mu W Giachelli CM Atkins RC Johnson RJ Lan HY A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat.Proc Assoc Am Physicians. 1998; 110: 50-64PubMed Google Scholar Administration of a neutralizing anti-OPN antibody during the induction phase of the disease (days 0 to 7) significantly inhibited glomerular and interstitial macrophage and T cell infiltration and the associated glomerular injury (proteinuria), loss of renal function, and histological damage including glomerular crescent formation and tubulointerstitial lesions. Furthermore, delaying administration of the anti-OPN antibody until disease was established caused a partial reversal of renal injury and damage.23Yu XQ Nikolic-Paterson DJ Mu W Giachelli CM Atkins RC Johnson RJ Lan HY A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat.Proc Assoc Am Physicians. 1998; 110: 50-64PubMed Google Scholar Having established the functional importance of OPN in promoting macrophage-mediated renal injury, the next issue is to identify the factors that up-regulate OPN expression within the kidney. We postulate that the cytokine interleukin-1 (IL-1) may be an important inducer of renal OPN expression, based on two observations. First, IL-1 has been shown to up-regulate OPN gene expression in chrondocytes and osteoblasts.24Margerie D Flechtenmacher J Buttner FH Karbowski A Puhl W Schleyerbach R Bartnik E Complexity of IL-1 β induced gene expression pattern in human articular chondrocytes.Osteoarthritis Cartilage. 1997; 5: 129-138Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 25Jin CH Miyaura C Ishimi Y Hong MH Sato T Abe E Suda T Interleukin 1 regulates the expression of osteopontin mRNA by osteoblasts.Mol Cell Endocrinol. 1990; 74: 221-228Crossref PubMed Scopus (53) Google Scholar Second, blocking IL-1 activity inhibits macrophage infiltration and renal injury in rat anti-GBM disease.26Lan HY Nikolic-Paterson DJ Zarama M Vannice JL Atkins RC Suppression of experimental glomerulonephritis by the IL-1 receptor antagonist.Kidney Int. 1993; 43: 479-485Crossref PubMed Scopus (159) Google Scholar, 27Lan HY Nikolic-Paterson DJ Mu W Vannice JL Atkins RC Interleukin-1 receptor antagonist halts the progression of established rat crescentic glomerulonephritis in the rat.Kidney Int. 1995; 47: 1303-1309Crossref PubMed Scopus (87) Google Scholar Therefore, the current study examined whether IL-1 is an important inducer of OPN expression in experimental crescentic glomerulonephritis. This study used archival material from two previous studies.26Lan HY Nikolic-Paterson DJ Zarama M Vannice JL Atkins RC Suppression of experimental glomerulonephritis by the IL-1 receptor antagonist.Kidney Int. 1993; 43: 479-485Crossref PubMed Scopus (159) Google Scholar, 27Lan HY Nikolic-Paterson DJ Mu W Vannice JL Atkins RC Interleukin-1 receptor antagonist halts the progression of established rat crescentic glomerulonephritis in the rat.Kidney Int. 1995; 47: 1303-1309Crossref PubMed Scopus (87) Google Scholar Accelerated autologous anti-GBM glomerulonephritis was induced in inbred male Sprague-Dawley rats (150–200 g) by subcutaneous immunization with 5 mg normal rabbit IgG in Freund's complete adjuvant, followed 5 days later (termed day 0) by an intravenous injection of 10 ml/kg rabbit anti-rat GBM serum (12.5 mg IgG/ml). In the first experiment, disease was induced in two groups of six rats which were treated from day −1 until being killed at day 14 (induction phase of disease) by a constant infusion of either human recombinant IL-1 receptor antagonist (IL-1ra) (Amgen, Boulder, CO) or saline by means of an Alzet 2002 miniosmotic pump implanted under the skin of the back. In the second experiment, disease was induced in three groups of six rats each. One group was killed on day 7 with no treatment. The other two groups were treated with IL-1ra or saline starting on day 7 and maintained until the animals were killed on day 21 (established disease). One group of six normal rats was also studied. Immunohistochemical staining for OPN protein and macrophage accumulation was performed on formalin-fixed, paraffin-embedded sections using a microwave antigen retrieval method.22Lan HY Yu XQ Yang N Nikolic-Paterson DJ Mu W Pichler R Johnson RJ Atkins RC De novo glomerular osteopontin expression in rat crescentic glomerulonephritis.Kidney Int. 1998; 53: 136-145Crossref PubMed Scopus (74) Google Scholar, 23Yu XQ Nikolic-Paterson DJ Mu W Giachelli CM Atkins RC Johnson RJ Lan HY A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat.Proc Assoc Am Physicians. 1998; 110: 50-64PubMed Google Scholar, 28Lan HY Mu W Nikolic-Paterson DJ Atkins RC A novel, simple, reliable, and sensitive method of multiple immunoenzymic staining: use of microwave oven heating to block antibody cross-reactivity and retrieve antigens.J Histochem Cytochem. 1995; 43: 97-102Crossref PubMed Scopus (342) Google Scholar Sections were dewaxed and treated with 10 minutes of microwave oven heating in 400 ml of 0.01 mol/L sodium citrate, pH 6.0, at 2450 MHz and 800W. After preincubation with 10% fetal calf serum (FCS) and 10% normal goat serum in PBS for 20 minutes, sections were drained and then labeled with mouse mAbs to rat OPN (MPIIIB10, obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA)29Liaw L Almeida M Downey W Hart CE Schwartz SM Giachelli CM Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro.Circ Res. 1994; 74: 214-224Crossref PubMed Scopus (372) Google Scholar, 30Liaw L Lombardi DM Almeida MM Schwartz SM deBlois D Giachelli CM Neutralizing antibodies directed against osteopontin inhibit rat carotid neointimal thickening after endothelial denudation.Arterioscler Thromb Vasc Biol. 1997; 17: 188-193Crossref PubMed Scopus (95) Google Scholar or rat macrophages (ED1)31Dijkstra CD Dopp EA Joling P Kraal G The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3.Immunology. 1985; 54: 589-599PubMed Google Scholar, 32Damoiseaux JG Dopp EA Calame W Chao D MacPherson GG Dijkstra CD Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1.Immunology. 1994; 83: 140-147PubMed Google Scholar for 60 minutes, washed 3 times in phosphate-buffered saline (PBS) and endogenous peroxidase inactivated by incubation in 0.3% H2O2 in methanol. Sections then were washed in PBS, incubated with peroxidase-conjugated goat anti-mouse IgG, washed in PBS, incubated with mouse peroxidase antiperoxidase complexes, and developed with 3,3-diaminobenzidine to produce a brown color. Double immunostaining was used to detect OPN and macrophages within the same section. After staining with the ED1 mAb was completed, as described above, sections were given a second round of microwave oven heating to block antibody cross-reactivity and enhance detection of OPN. Following precincubation as above, sections were incubated with the MPIIIB10 mAb, then incubated sequentially with alkaline phosphatase-conjugated goat anti-mouse IgG and mouse alkaline phosphatase anti-alkaline phosphatase complexes and developed with Fast Blue BB Salt (Ajax Chemicals, Melbourne, Australia). No staining was seen in negative control sections using the 73.5 IgG1 (anti-human CD45R) irrelevant isotype control mAb. All peroxidase- and alkaline phosphatase-conjugated antibodies and complexes were purchased from Dakopatts (Glostrup, Denmark). A 1-kb cRNA probe was generated from the rat smooth muscle osteopontin cDNA clone 2B7.8Giachelli C Bae N Lombardi D Majesky M Schwartz S Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR).Biochem Biophys Res Commun. 1991; 177: 867-873Crossref PubMed Scopus (176) Google Scholar Sense and antisense cRNA probes were labeled with either [35S] or digoxigenin (DIG)-UTP using a RNA polymerase kit (Boehringer Mannheim, Mannheim, Germany). A 358-bp cRNA antisense riboprobe for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also DIG-labeled. Probe labeling was determined by liquid scintillation counting (35S-labeled probes) or by dot blotting (DIG-labeled probes). In situ hybridization was performed on 4-μm paraffin sections of formalin-fixed tissues using a radioactive method as described previously.33Wilcox JN Smith KM Schwartz SM Gordon D Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque.Proc Natl Acad Sci USA. 1989; 86: 2839-2843Crossref PubMed Scopus (1069) Google Scholar Tissue sections were hybridized with 300,000 cpm/section of sense or anti-sense OPN cRNA probe at 55°C. After washing, the hybridized probe was detected by emulsion photography. Only low levels of background hybridization were seen using the sense probe (0 to 2 grains/cell). Positively stained cells were quantitated in tissue sections as previously described.22Lan HY Yu XQ Yang N Nikolic-Paterson DJ Mu W Pichler R Johnson RJ Atkins RC De novo glomerular osteopontin expression in rat crescentic glomerulonephritis.Kidney Int. 1998; 53: 136-145Crossref PubMed Scopus (74) Google Scholar Briefly, the number of cells labeled with the antisense OPN cRNA probe (defined as >5 grains per cell) or the different mAbs were counted under high power in at least 50 glomerular cross-sections (gcs) per animal. The number of tubules labeled with the antisense OPN cRNA probe or the different mAbs were scored under high power in at least 1000 cortical tubules. All scoring was performed on coded slides. Data are expressed as the mean ± SE for groups of six animals. A normal rat kidney epithelial-derived cell line, NRK52E, was obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's minimal essential medium (DMEM) containing 2% fetal calf serum (FCS). Cells were grown to confluence in 125-cm2 plastic tissue culture flasks, the medium was changed to serum-free, and the cells were then cultured with or without recombinant mouse IL-1α (1, 10, or 20 ng/ml) for up to 5 days. Preincubation of cells with IL-1ra (20μg/ml) for 30 minutes was used specifically to block IL-1 stimulation of cultured cells. Northern blotting was performed as previously described.22Lan HY Yu XQ Yang N Nikolic-Paterson DJ Mu W Pichler R Johnson RJ Atkins RC De novo glomerular osteopontin expression in rat crescentic glomerulonephritis.Kidney Int. 1998; 53: 136-145Crossref PubMed Scopus (74) Google Scholar, 34Hattori M Nikolic-Paterson DJ Lan HY Kawaguchi H Ito K Atkins RC Up-regulation of ICAM-1 and VCAM-1 expression during macrophage recruitment in lipid induced glomerular injury in ExHC rats.Nephrology. 1995; 1: 221-232Crossref Scopus (20) Google Scholar Briefly, total cellular RNA from cultured NRK52E cells was extracted using the RNAzol reagent (Gibco BRL, Gaithersburg, MD) and 20-μg samples denatured with glyoxal and dimethylsulfoxide, size-fractionated on 1.2% agarose gels, and capillary-blotted onto positively charged nylon membranes (Boehringer Mannheim). Membranes were hybridized overnight at 68°C with DIG-labeled cRNA probes in DIG Easy Hyb solution (Boehringer Mannheim). Following hybridization, membranes were washed finally in 0.1 × SSC/0.1% sodium dodecyl sulfate (SDS) at 68°C. Bound probes were detected using sheep anti-DIG antibody (Fab) conjugated with alkaline phosphatase and development with CPD-Star enhanced chemiluminescence (Boehringer Mannheim). Chemiluminescence emissions were captured on Kodak XAR film and densitometry analysis performed using the Gel-Pro Analyzer program (Media Cybernetics, Silver Spring, MD). NRK52E cells were grown in 125-cm2 flasks with or without IL-1 and analyzed by Western blotting as previously described.35Nikolic-Paterson DJ Jun Z Tesch GH Lan HY Foti R Atkins RC De novo CD44 expression by proliferating mesangial cells in anti-Thy-1 nephritis in the rat.J Am Soc Nephrol. 1996; 7: 1006-1014PubMed Google Scholar Cells were washed in PBS and then lysed in 1 ml of 1% Nonidet P-40, 25 mmol/L Tris-HCl, 150 mmol/L NaCl, 10 mmol/L EDTA, pH 8.0, containing a 1:50 dilution of a protease inhibitor cocktail (P2714, Sigma-Aldrich Co., Castle Hill, Australia) for 30 minutes on ice. Samples were centrifuged at 14,000 g for 5 minutes to pellet cell debris. Samples (20 μg) were mixed with SDS polyacrylamide gel electrophoresis sample buffer, boiled for 5 minutes, electrophoresed on a 10% SDS polyacrylamide gel, and electroblotted onto Hybond-ECL nitrocellulose membrane (Amersham International, Buckinghamshire, UK). The membrane was blocked in PBS containing 5% skimmed milk powder, 1% FCS, and 0.02% Tween 20 and then incubated for 1 hour with 5μg/ml of MPIIIB10 mAb diluted in the above buffer. After washing, the membrane was incubated with a 1:20,000 dilution of peroxidase-conjugated goat anti-mouse IgG in PBS containing 1% normal goat serum and 1% FCS. The blot was then developed using the ECL detection kit (Amersham) to produce a chemiluminescence signal which was captured on X-ray film. In situ hybridization and immunostaining identified OPN mRNA and protein expression by some glomerular parietal epithelial cells in normal rat kidney (Figure 1A). In addition, constitutive OPN expression was seen in the thick ascending limbs of the loop of Henle, accounting for less than 5% (3.3 ± 1.8%) of cortical tubules (Figure 1, Figure 2).Figure 2Quantitation of OPN mRNA and protein expression in rat anti-GBM disease. The number of glomerular cells and tubules expressing OPN mRNA or protein was scored in stained tissue sections from saline-treated (closed bars) or IL-1ra-treated (hatched bars) anti-GBM disease or normal rats (open bars). A-B: Experiment 1 with IL-1ra or saline treatment over days 0 to 14 of anti-GBM disease. C-D: Experiment 2 with IL-1ra or saline treatment over days 7 to 21 of anti-GBM disease. One group of animals received no treatment before being killed on day 7 (dotted bars). The number of glomerular cells expressing OPN mRNA or protein (A and C) and the percent of cortical tubules expressing OPN mRNA or protein (B and D) were scored. Data are mean ± SE for six animals. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by ANOVA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There was marked up-regulation of OPN mRNA and protein expression on day 14 of rat anti-GBM glomerulonephritis in saline-treated rats. In glomeruli, both podocytes and infiltrating macrophages (shown by double immunostaining) expressed OPN, whereas crescents showed strong OPN mRNA and protein staining (Figure 1, Figure 2, Figure 3). There was also a striking increase in tubular OPN expression (50.6 ± 3.2% OPN-positive cortical tubules), most particularly in proximal tubular epithelial cells, which was associated with focal infiltration of ED1-positive macrophages and tubular damage (Figure 1, Figure 2, Figure 3). IL-1ra treatment significantly inhibited glomerular and tubular up-regulation of OPN expression on day 14 of anti-GBM disease (Figure 1, Figure 2, Figure 3). This was associated with reduced macrophage accumulation within the glomeruli (10 ± 2.1 versus4.8 ± 1.9 cells/gcs in saline and IL-1ra treated rats, respectively; P < 0.01) and in the cortical tubulointerstitium (170 ± 41 versus 29 ± 2.5 cells/mm2; P < 0.01). As previously described,26Lan HY Nikolic-Paterson DJ Zarama M Vannice JL Atkins RC Suppression of experimental glomerulonephritis by the IL-1 receptor antagonist.Kidney Int. 1993; 43: 479-485Crossref PubMed Scopus (159) Google Scholar IL-1ra treatment suppressed proteinuria (332 ± 80 versus 260 ± 62 mg/24hrs; P < 0.05), prevented a decline in creatinine clearance (0.66 ± 0.1 versus 0.96 ± 0.25 ml/min; P < 0.01), and reduced histological injury, including glomerular crescent formation (13 ± 11.2% versus 1.2 ± 1.6%, P < 0.01). The ability of IL-1 to up-regulate OPN expression during the progressive phase of established renal injury was examined in Experiment 2. Crescentic disease was established by day 7 after anti-GBM serum administration and was associated with significant up-regulation of glomerular and tubular OPN expression (Figure 1, Figure 2). Saline treatment from days 7 to 21 saw a further increase in glomerular and tubular OPN mRNA and protein expression (Figure 1, Figure 2, Figure 3) in association with a further decline in creatinine clearance and a progressive increase in proteinuria as previously described.27Lan HY Nikolic-Paterson DJ Mu W Vannice JL Atkins RC Interleukin-1 receptor antagonist halts the progression of established rat crescentic glomerulonephritis in the rat.Kidney Int. 1995; 47: 1303-1309Crossref PubMed Scopus (87) Google Scholar Double immunohistochemistry showed a tight association between OPN expression and macrophage accumulation in areas of severe tissue damage, such as glomerular crescent formation and tubulointerstitial lesions (Figure 3C). IL-1ra treatment of established anti-GBM disease from days 7 to 21 caused a significant reduction in glomerular and tubular OPN mRNA and protein expression (Figure 1, Figure 2, Figure 3). Indeed, the number of glomerular OPN-positive cells was reduced to levels below that seen on day 7 of disease, before the induction of IL-1ra treatment (Figure 2C). This was associated with recovery of normal renal function, a minor reduction in proteinuria, and prevention of further histological damage.27Lan HY Nikolic-Paterson DJ Mu W Vannice JL Atkins RC Interleukin-1 receptor antagonist halts the progression of established rat crescentic glomerulonephritis in the rat.Kidney Int. 1995; 47: 1303-1309Crossref PubMed Scopus (87) Google Scholar To investigate whether IL-1 can act directly to up-regulate OPN expression in the development of rat anti-GBM glomerulonephritis, we examined the effect of adding IL-1 to the level of OPN mRNA expression in the normal rat epithelial cell line NRK52E. This cell line was chosen because tubular epithelial cells are the major site of OPN production within the injured kidney. Northern blot analysis showed that the NRK52E cell line constitutively expresses low levels of OPN mRNA (Figure 4). Within 6 hours of IL-1α stimulation, there was an increase in OPN mRNA levels that peaked after 24 hours (threefold induction). The IL-1-induced up-regulation of OPN mRNA levels was dose-dependent and was maintained for a 5-day culture period (Figure 4, Figure 5). Preincubation of cells with an excess of the IL-1ra completely blocked IL-1 up-regulation of OPN mRNA expression, demonstrating specificity of the effect. Western blotting showed that NRK52E cells constitutively express OPN protein of approximately 68 kd, consistent with previous studies of NRK52E cells and rat mesangial cells.36Malyankar UM Almeida M Johnson RJ Pichler RH Giachelli CM Osteopontin regulation in cultured rat renal epithelial cells.Kidney Int. 1997; 51: 1766-1773Crossref PubMed Scopus (64) Google Scholar, 37Nagasaki T Ishimura E Shioi A Jono S Inaba M Nishizawa Y Morii H Otani S Osteopontin gene expression and protein synthesis in cultured rat mesangial cells.Biochem Biophys Res Commun. 1997; 233: 81-85Crossref PubMed Scopus (18) Google Scholar IL-1 increased OPN protein levels within NRK52E cells in a dose-dependent fashion and this was completely inhibited by preincubation of cells with the IL-1ra (Figure 6).Figure 5Interleukin-1 causes sustained up-regulation of OPN mRNA by cultured rat kidney epithelial cells. A: NRK52E cells were stimulated with various concentrations of murine IL-1α for 3 or 5 days in the presence or absence of 50μg/ml human IL-1ra. Total cellular RNA was analyzed by Northern blotting using OPN and GAPDH probes. B: The normalized OPN to GAPDH mRNA ratio is shown. Data are mean ± SE. *, P < 0.05; **, P < 0.01 versus the unstimulated control by t-test with Welch's correction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6IL-1 up-regulates OPN protein expression by cultured rat kidney epithelial cells. NRK52E cells were cultured with various concentrations of murine IL-1α for 3 days in the presence or absence of 50μg/ml IL-1ra. Twenty micrograms of cell lysates were analyzed by Western blotting using the MPIIIB10 anti-OPN mAb. A predominant band of approximately 68 kd was seen.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This study has shown that IL-1 is an important factor in up-regulating OPN gene expression in experimental crescentic glomerulonephritis. The ability of IL-1ra treatment to inhibit the induction of OPN expression and to down-regulate OPN expression in established anti-GBM disease clearly demonstrates a functional role for IL-1 in the up-regulation of OPN in experimental crescentic glomerulonephritis. Indeed, the inhibition of renal macrophage infiltration seen with IL-1ra treatment is entirely consistent with the ability of anti-OPN antibody treatment to suppress macrophage infiltration and subsequent renal injury in this model.23Yu XQ Nikolic-Paterson DJ Mu W Giachelli CM Atkins RC Johnson RJ Lan HY A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat.Proc Assoc Am Physicians. 1998; 110: 50-64PubMed Google Scholar, 26Lan HY Nikolic-Paterson DJ Zarama M Vannice JL Atkins RC Suppression of experimental glomerulonephritis by the IL-1 receptor antagonist.Kidney Int. 1993; 43: 479-485Crossref PubMed Scopus (159) Google Scholar, 27Lan HY Nikolic-Paterson DJ Mu W Vannice JL Atkins RC Interleukin-1 receptor antagonist halts the progression of established rat crescentic glomerulonephritis in the rat.Kidney Int. 1995; 47: 1303-1309Crossref PubMed Scopus (87) Google Scholar Furthermore, the ability of IL-1 to increase OPN mRNA levels rapidly in cultured tubular epithelial cells demonstrates that IL-1 acts directly to up-regulate OPN expression in renal cells. This is the first demonstration that IL-1 can up-regulate OPN expression in kidney cells. The ability of IL-1 to up-regulate OPN expression in renal epithelial cells is consistent with previous studies reporting that IL-1 increases OPN mRNA expression by cultured chrondocytes and osteoblasts.24Margerie D Flechtenmacher J Buttner FH Karbowski A Puhl W Schleyerbach R Bartnik E Complexity of IL-1 β induced gene expression pattern in human articular chondrocytes.Osteoarthritis Cartilage. 1997; 5: 129-138Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 25Jin CH Miyaura C Ishimi Y Hong MH Sato T Abe E Suda T Interleukin 1 regulates the expression of osteopontin mRNA by osteoblasts.Mol Cell Endocrinol. 1990; 74: 221-228Crossref PubMed Scopus (53) Google Scholar However, it should be noted that regulation of OPN gene expression varies greatly between different cell types. For example, IL-1 failed to increase OPN gene expression in cultured rat mesangial cells.37Nagasaki T Ishimura E Shioi A Jono S Inaba M Nishizawa Y Morii H Otani S Osteopontin gene expression and protein synthesis in cultured rat mesangial cells.Biochem Biophys Res Commun. 1997; 233: 81-85Crossref PubMed Scopus (18) Google Scholar In addition, vitamin D can induce or suppress OPN mRNA levels in osteoblasts depending on their state of differentiation.38Gerstenfeld LC Zurakowski D Schaffer JL Nichols DP Toma CD Broess M Bruder SP Caplan AI Variable hormone responsiveness of osteoblast populations isolated at different stages of embryogenesis and its relationship to the osteogenic lineage.Endocrinology. 1996; 137: 3957-3968Crossref PubMed Scopus (33) Google Scholar Furthermore, platelet-derived growth factor induces OPN expression in vascular smooth muscle cells and marrow stromal cells, but not in NRK52E tubular epithelial cells.36Malyankar UM Almeida M Johnson RJ Pichler RH Giachelli CM Osteopontin regulation in cultured rat renal epithelial cells.Kidney Int. 1997; 51: 1766-1773Crossref PubMed Scopus (64) Google Scholar, 39Green RS Lieb ME Weintraub AS Gacheru SN Rosenfield CL Shah S Kagan HM Taubman MB Identification of lysyl oxidase and other platelet-derived growth factor-inducible genes in vascular smooth muscle cells by differential screening.Lab Invest. 1995; 73: 476-482PubMed Google Scholar, 40Tanaka H Liang CT Effect of platelet-derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats.J Cell Physiol. 1995; 164: 367-375Crossref PubMed Scopus (49) Google Scholar It was interesting that the increase in renal OPN mRNA and protein production seen in rat crescentic glomerulonephritis was very similar to the pattern of up-regulation of renal IL-1β expression described previously in this disease model,41Tesch GH Yang N Yu H Lan HY Foti R Chadban SJ Atkins RC Nikolic-Paterson DJ Intrinsic renal cells are the major source of interleukin-1β synthesis in normal and diseased rat kidney.Nephrol Dial Transplant. 1997; 12: 1109-1115Crossref PubMed Scopus (77) Google Scholar suggesting that up-regulation of OPN expression within the injured kidney may be due to local IL-1 production. In particular, strong IL-1β and OPN mRNA and protein expression were seen in glomerular and tubular epithelial cells, including glomerular crescents and areas of tubulointerstitial injury. Whereas double staining showed that some glomerular macrophages expressed OPN, consistent with in vitro studies,6Miyazaki Y Setoguchi M Yoshida S Higuchi Y Akizuki S Yamamoto S The mouse osteopontin gene: expression in monocytic lineages and complete nucleotide sequence.J Biol Chem. 1990; 265: 14432-14438Abstract Full Text PDF PubMed Google Scholar podocytes were the major cell type expressing OPN in the glomerulus.22Lan HY Yu XQ Yang N Nikolic-Paterson DJ Mu W Pichler R Johnson RJ Atkins RC De novo glomerular osteopontin expression in rat crescentic glomerulonephritis.Kidney Int. 1998; 53: 136-145Crossref PubMed Scopus (74) Google Scholar Therefore, the suppression of glomerular OPN expression by IL-1ra treatment is largely attributed to inhibition of podocyte OPN production, with a lesser effect due to the reduction in macrophage infiltration and macrophage OPN expression. It was intriguing that the rapid induction of OPN mRNA expression seen following the addition of IL-1 to cultured tubular epithelial cells was sustained for 5 days, in contrast with the 2 days required to detect TGF-β-induced up-regulation of OPN mRNA in this cell line.36Malyankar UM Almeida M Johnson RJ Pichler RH Giachelli CM Osteopontin regulation in cultured rat renal epithelial cells.Kidney Int. 1997; 51: 1766-1773Crossref PubMed Scopus (64) Google Scholar Since IL-1 can induce TGF-β synthesis by tubular epithelial cells, it may be the case that the sustained IL-1 up-regulation of OPN expression by the cultured tubular epithelial cells reflects a secondary effect of IL-1-induced production of TGF-β. This possibility warrants further investigation. IL-1 plays a crucial role in inducing renal macrophage infiltration.42Nikolic-Paterson DJ Lan HY Atkins RC IL-1 receptor antaogonism.Semin Nephrol. 1996; 16: 583-590PubMed Google Scholar Indeed, IL-1 induces a broad range of chemokines and leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1, which, acting in concert, facilitate the extravasation of blood leukocytes and their subsequent focal accumulation within the kidney. Blocking the action of just one IL-1-inducible adhesion or chemotactic molecule can inhibit leukocyte infiltration in experimental kidney disease,43Nishikawa K Guo YJ Miyasaka M Tamatani T Collins AB Sy MS McCluskey RT Andres G Antibodies to intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 prevent crescent formation in rat autoimmune glomerulonephritis.J Exp Med. 1993; 177: 667-677Crossref PubMed Scopus (193) Google Scholar, 44Lloyd CM Minto AW Dorf ME Proudfoot A Wells TN Salant DJ Gutierrez-Ramos JC RANTES, and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation, and interstitial fibrosis.J Exp Med. 1997; 185: 1371-1380Crossref PubMed Scopus (436) Google Scholar thereby demonstrating the interdependence of these molecules. This is illustrated by the ability of anti-OPN antibody treatment to suppress leukocyte infiltration and subsequent renal injury in rat anti-GBM glomerulonephritis without affecting up-regulation of ICAM-1 expression.23Yu XQ Nikolic-Paterson DJ Mu W Giachelli CM Atkins RC Johnson RJ Lan HY A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat.Proc Assoc Am Physicians. 1998; 110: 50-64PubMed Google Scholar This implies that ICAM-1 is necessary for leukocyte-endothelial interactions but that renal OPN expression is required for migration and localization of macrophages and T cells within the kidney, resulting in local tissue damage. In summary, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and argues that inhibition of OPN expression is one of the mechanisms by which IL-1ra treatment suppresses macrophage-mediated renal injury in experimental crescentic glomerulonephritis.

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