JP2001505995A - Hepatocyte growth factor antagonist - Google Patents

Abstract

  (57) [Summary] Provided is a method of treating a human having chronic kidney disease, comprising administering to the human a hepatocyte growth factor antagonist.

Description

DETAILED DESCRIPTION OF THE INVENTION Hepatocyte growth factor antagonist Field of the invention The field of the invention is the treatment of chronic kidney disease. Background of the Invention Hepatocyte growth factor (HGF) is a heterodimeric molecule derived from a 728 amino acid prepro-precursor protein that is proteolytically processed by a specific protease to form mature HGF (Miyazawa et al., 1993, J. Am. Biol. Chem. 268: 100 24-1028). HGF binds to cells via the tyrosine kinase receptor c-met. Binding of HGF to c-met has the effect of stimulating renal epithelial cell growth; increasing cell motility; inducing renal tubule formation (Santos et al., 1993, Dev. Biol. 159: 535-548; Cantley et al., 1994, Am. J. Physiol. 267: F271-F280). Thus, HGF is characterized as a mitogen, motogen and morphogen, respectively, and is thought to play a role in kidney development. In addition, HGF has been implicated in renal remodeling following injury, such as that caused by increased levels of HGF and its receptor c-met in the kidney after nephrectomy or ischemia (Joannidis et al., 1994, Am. J. Physiol. .267: F231-F236). In addition, HGF has been observed to result in an improvement in renal function following mercuric chloride challenge or ischemia (Kawaida et al., 1994, Proc. Natl. Acad. Sci. USA 91: 4357-4361; Miller et al., 1994). , Am. J. Physiol. 266: Fl29-F134). Glomerular hypertrophy occurs during renal remodeling in many chronic kidney diseases. The events are associated with glomerular basement membrane swelling, glomerular stromal and epithelial cell proliferation, and collagen and fibronectin accumulation. The role of HGF in glomerular cell matrix expansion is unknown. Chronic renal dysfunction is a progressive loss of functional renal mass with compensatory growth and remodeling. Molecular and cellular events that occur during chronic renal dysfunction include growth factor release, glomerular stromal cell proliferation and extracellular matrix swelling (Klahr et al., 1988, New Engl. J. Med. 318). Striker et al., 1989, In: Klahr, (Ed.) Seminars in Nephrology, pp. 318, Philadelphia, WB. Saunders); E bihara et al., 1993, J. Am. Am. Soc. Nephrol. 3: 1387-1397). Components of the extracellular matrix that change during chronic renal insufficiency include collagen, fibronectin and laminin (Ebihara et al., Supra; Tarsio et al., 1988, Diabetes 37: 532-539; Yoshioka et al., 1989, Kidney Int. 35: 1203-1211). If chronic renal insufficiency is established, in a nephrectomy model, fibronectin and collagen a1 immunocytochemical staining is improved in the glomerular cellularity-increased area 2 and 6 weeks after nephrectomy, 5/6 of the kidney volume (Foelge et al., 1992, Lab. Invest. 66: 485-497). Several protein factors have been implicated in stimulating mitogenesis or extracellular matrix production of glomerular stromal cells during chronic renal insufficiency. These include thrombin (Xu et al., 1995, Am. J. Pathol. 146: 101-110; Sraer et al., 1993, Ren. Fail. 15: 343-348; Albrightson et al., 1992, J. Pharmacol. Exp. Ther. 2563: 404-412), epidermal growth factor (Byyny et al., 1972, Endocrinology 90: 1261-1266; Ki1lion et al., 1993, J. Urol. 150: 1551-1556), insulin-like growth factor 1 (Stiles et al. 1985, Endocrinology 117: 2397-2401; Fagin et al., 1987, Endocrinology 120: 718-724), and transforming growth factor b1 (Coimbra et al., 1991, Am. J. Pathol. 138: 223-234; Okuda et al.). , 1990, J. Clin. Invest. 86: 453-462). There remains a need for compositions for the treatment of chronic kidney disease that will help reduce or eliminate renal dysfunction and diseases leading to death or dependence on dialysis. Summary of the Invention The present invention relates to a method of treating a human having chronic renal disease comprising administering a hepatocyte growth factor antagonist to the human. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the extracellular acidity of transformed mouse glomerular stromal cells (MMC-SV40 cells) (panels AC) or normal human glomerular stromal cells (panel D) measured by the microphysiological assay. 5 is a series of graphs showing conversion rates. Panel A: MMC-SV40 cells were treated with HGF (100 ng / ml) for 10 minutes in the presence or absence of 0.1% bovine serum albumin (BSA) or 0.5% fetal bovine serum (FBS). Panel B: MMC-SV40 cells were treated with HGF (100 ng / ml) for 2, 5, 10 and 15 minutes. Panel C: MMC-SV40 cells were treated with four different concentrations of HGF. Panel D: Normal human glomerular stromal cells were treated with 100 ng / ml HGF; two traces are shown. FIG. 2 is a graph showing the rate of extracellular acidification of MMC-SV40 cells treated with HGF (50 ng / ml). Cells were cultured in control medium or different concentrations of Birchall et al. Pharmacol. Exp. Ther. 268: 922-929 perfused with media containing the protein kinase inhibitor RO-32-0432. FIG. 3 shows mouse threads transfected with normal human glomerular stromal cells (HMC), MMC-SV40 cells or luciferase reporter plasmid controlled by collagen a1 (IV) promoter, treated with different concentrations of HGF for 24 hours. It is a graph which shows the incorporation of tritiated thymidine into spherical stromal cells (MMC-COL cells). FIG. 4 is a graph showing collagen a1 (IV) promoter activity in MMC-COL cells in response to different concentrations of HGF. Data are means of four replicates ± SD. FIG. 5 is a graph showing creatinine clearance in lean and obese (diabetic) mice measured 21 days after injection of either vehicle or HGF. HGF significantly reduced creatinine levels in obese (diabetic) mice ( ^* <0.05; n = 5-6). LV-fat-free vehicle; LH-fat-free HGF; OV-obese vehicle; OH-obese HGF-treated mice. Detailed description of the invention According to the present invention, it has been found that chronic administration of HGF results in chronic kidney disease. Thus, while HGF may be useful in the treatment of acute kidney disease, renal function is impaired when the compound is administered to milk animals for extended periods of time. It was also found that treating cells with HGF resulted in increased fibronectin mRNA and transcription in glomerular stromal and epithelial cells. Thus, the present invention is based on the discovery that HGF contributes partially to renal disease by activating fibronectin and collagen synthesis, thereby causing glomerulosclerosis. The present invention relates to a method for treating chronic kidney disease in a mammal, comprising administering an antagonist of HGF to a mammal. By the term "chronic kidney disease" as used herein is meant a progressive loss of renal function as measured by glomerular filtration rate. Antagonists of HGF include, but are not limited to, small non-peptide molecules, peptides comprising specific portions of HGF, peptide portions having anti-HGF activity (peptides), antibodies directed against HGF, Includes nucleic acids having sequences complementary to all or part of the nucleic acids encoding HGF and small chemical compounds that inhibit HGF-specific proteases. As used herein, the term "HGF antagonist activity" refers to a compound that inhibits the normal activity of HGF as measured, for example, in any one or more of the assays described herein. means. As an example, treatment of mouse glomerular stromal cells with HGF increases the rate of acidification of these cells. Thus, a compound having HGF antagonist activity is defined as a compound that inhibits HGF-induced increase in mouse glomerular stromal cells. To identify antagonists of HGF, test compounds were evaluated for HGF antagonist activity in one or more of the assays described in the Examples section herein or in other assays for measuring HGF function. Preferably, compounds with HGF antagonist activity are first identified using in vitro tests. Such in vitro tests include, but are not limited to, test compounds for HGF binding to the cell membrane of cells known to respond to HGF, the rate of HGF-induced acidification of glomerular stromal cells. And a test to evaluate the effect of a test compound on HGF-induced extracellular matrix gene expression. To evaluate the effect of test compounds on HGF binding, [ ¹²⁵ I] -labeled HGF is incubated with or without test compound and cell membranes from A498 cells attached to scintillation beads. If the compound inhibits the binding of HGF to the membrane, ¹²⁵ I] -labeled HGF is not near the scintillation beads and a reduction in signal will be obtained. The second assay used below determines whether the compound is an agonist or antagonist. To assess the effect of test compounds on the rate of cell acidification, mesangial cells are incubated with or without test compounds in the presence or absence of HGF. The rate of cell acidification is measured by microphysiometry as described herein to determine the effect of the test compound on HGF-induced cell acidification. A decrease in the rate of acidification of cells treated with HGF and the test compound as compared to the rate of acidification of cells treated with HGF alone indicates that the test compound has HGF antagonist activity. To assess the effect of a test compound on extracellular matrix gene expression, incubate the cells with or without the test compound under the conditions described in the Examples herein, in the presence or absence of HGF . The expression of mRNA associated with extracellular matrix genes (eg, collagen (IV) and fibronectin) is measured and compared between various cells. A decrease in extracellular matrix mRNA expression in cells treated with HGF and the test compound as compared to extracellular matrix mRNA expression in cells treated with HGF alone further implies that the test compound has HGF antagonist activity. I do. Test compounds may also be tested for HGF antagonist activity in an in vivo assay. As is evident from the data shown in the experimental examples, long-term treatment of mice with HGF induces renal failure. Thus, another specific method of assessing the HGF antagonist activity of a test compound consists of administering HGF alone and HGF to which the test compound has been added to mice over a period of at least 21 days. Additional controls include mice that have received the appropriate playavo compound. Creatine clearance is evaluated in each set of mice as a measure of renal function. An increase in creatine clearance in mice administered HGF together with the test compound compared to creatine clearance in mice administered HGF alone indicates that the test compound has additional HGF antagonist activity. HGF antagonists comprising a peptide having a specific portion of HGF include, but are not limited to, those that include a first krinkle region near the N-terminus of HGF. One example of such a peptide is the truncated HGF peptide NK2 (Chan et al., 1991, Science 254: 1382-1 385). To obtain a preparation of a peptide comprising a substantially pure HGF moiety, the peptide can be produced by cloning and expressing HGF DNA encoding the desired portion of HGF. HGF DNA and amino acid sequences are described in Miyazawa et al., 1991, Eur. J. Biochem. 197: 15-22 (SEQ ID NOS: 1 and 2). The isolated DNA encoding the desired portion of HGF is cloned into an expression vector and the protein is then expressed. The procedures for cloning and expressing peptides are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York). The peptide thus expressed can be obtained using general peptide purification means well known in the art. As used herein, the term "substantially pure" refers to one that is naturally isolated from a component that accompanies the compound, eg, a protein or polypeptide. Typically, at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, of the total material in the sample, Most preferably, if at least 98% is the compound of interest, the compound is substantially pure. For example, in the case of a polypeptide, the purity can be measured by any appropriate method by column chromatography, gel electrophoresis, or HPLC analysis. A compound, eg, a protein, is also substantially purified when it is essentially free of naturally bound components, or when it separates its natural contaminants, including its natural counterparts. The present invention also includes analogs of the peptides obtained according to the method of the present invention. Analogs can be distinguished from naturally occurring peptides by conservative amino acid sequence differences, or by modifications that do not affect sequence, or both. For example, conservative amino acid modifications may be made that change the primary sequence of the peptide, but usually do not change its function. Conserved amino acid sequences typically include the following groups of substitutions: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. Modifications (which usually do not alter the primary sequence) include in vivo or in vitro chemical derivatization of the peptide, for example, acetylation or carboxylation. In addition, modifications of glycosylation, such as enzymes that modify the pattern of glycosylation during the synthesis of the peptide and process or in a further processing step, e.g., affecting the glycosylation of the peptide, e.g., Also included are modifications made by exposure to mammalian glycosylation or deglycosylation enzymes. Also included are sequences having phosphorylated amino acid residues, eg, phosphotyrosine, phosphoserine or phosphothreonine. Also included are peptides that have been modified using conventional biological techniques to improve their resistance to proteolytic properties or to optimize solubility properties. Analogs of such peptides include those containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the present invention are not limited to the exemplary products listed herein. Thus, the present invention also relates to an active fragment of HGF having HGF antagonist activity. As described herein, if a particular polypeptide inhibits the action of HGF, the polypeptide is considered to have HGF antagonist activity. As used herein, the term "fragment" as applied to an HGF peptide is generally at least about 10 contiguous amino acids in length, typically at least about 20 contiguous amino acids, and more typically Refers to at least about 50 contiguous amino acids, usually at least about 78 contiguous amino acids. A nucleic acid sequence complementary to HGF can be obtained using the HGF sequence described in Nakamura et al. (Supra). Currently, administration of antisense oligonucleotides to mammals is common in the art and can be performed using any of the administration methods described herein. Nucleic acid complementary to nucleotides 78-950 of HGF (SEQ ID NO: 1) or a portion thereof is such that HGF DNA or one thereof is such that the RNA is expressed in HGF mRNA in an antisense orientation (ie, complementarily). Can be obtained by cloning the part into an expression vector. As used herein, "isolated DNA" refers to a DNA sequence, segment or fragment that has been purified from its flanking sequence in its naturally occurring state, for example, the sequence that is usually adjacent to the fragment. For example, a DNA fragment separated from sequences adjacent to the fragment in the naturally occurring genome. The term also applies to DNA that has been substantially purified from other components that naturally accompany the DNA, such as RNA, DNA or protein, that accompany it in cells. As used herein, the term "complementary" refers to subunit sequence complementarity between two nucleic acids, for example, two DNA or RNA molecules. If the nucleotide position of both molecules is occupied by nucleotides capable of base-pairing with each other, then the nucleic acids are considered to be mutually complementary at that position. That is, two nucleic acids are interconnected if a substantial number (at least 50%) of the corresponding positions in each molecule form mutually normal base pairs (eg, A: T and G: C nucleotide pairs). Is complementary to The present invention should be understood to include DNA that is substantially homologous to HGF DNA or a portion thereof, which DNA is useful for producing HGF complementary nucleic acids or HGF peptides. Preferably, DNA that is substantially homologous is about 50% homologous, preferably about 70% homologous, more preferably about 80% homologous to DNA obtained using the methods of the present invention. And most preferably about 90% homologous. As used herein, "homologous" refers to subunit sequence similarity between two macromolecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules. If a subunit position in both two molecules is occupied by the same monomeric subunit, for example, if two DNA molecules are occupied by adenine, they are homologous at that position. Homology between two sequences is a linear function of the number of matching or homologous positions, for example, half the positions in two compound sequences (eg, five positions in a 10-unit long polymer). Are homologous, two sequences are 50% homologous, and two sequences share 90% homology if 90% of the positions are matched or homologous, eg, nine out of ten. . For example, the DNA sequences 3'ATTGCC5 'and 3'TATGCG5' share 50% homology. Anti-HGF antibodies are readily obtained by immunizing a mammal with the HGF peptides identified herein. Protocols for obtaining antibodies (either monoclonal or polyclonal) to known peptides are described in Harlow et al. (1988, In: Antibodies, A Labpratory Manual, Cold Spring Harbor, NY), and the protocol can be easily performed by those skilled in the art. Polyclonal antibodies to HGF are raised in any suitable mammal, such as a mouse or rabbit. Monoclonal anti-HGF antibodies are obtained by immunization of mice with HGF peptide, followed by production of hybridoma cells capable of secreting anti-HGF antibodies. Other means of producing monoclonal antibodies, such as those expressed by bacteriophages, are now well known in the art and are described, for example, in Marks et al. (1991, J. Mol. Biol. 222: 581-597), Barbas (1995, Nature Medicine 1: 837-839), de Kruif et al. (1995, J. Mol. Biol. 248: 97-105) and Sternberg et al. (1995, Proc. Natl. Acad. Sci. USA 92: 1609-1613). Peptidomics having HGF-antagonist-like activity are also designed and used according to the present invention. Further information describing the administration of the peptide methic is provided in PCT application PCT / US93 / 01201 and US Pat. No. 5,334,702, which is incorporated herein by reference. Any of the techniques described in either of these two documents may be used in the present invention for the administration of the peptidomic. HGF antagonists also include small molecules that are not peptide or nucleic acid molecules and that have HGF antagonist activity as defined herein. Examples of HGF antagonists useful in the methods of the present invention include, but are not limited to, Faletto et al. (PCT application WO 94/06909), Roosetal. (PCT application WO94 / 06456), Japanese Patent Application Publication No. 5-208998, and Aaronson et al. (PCT application WO92 / 05184). Kidney diseases that can be treated using the methods of the present invention include, but are not limited to, polycystic disease, diabetic nephropathy, focal segmental glomerulosclerosis, hypertension-induced nephropathy, adrenal gland, and the like. Protocols for treating mammals with chronic nephropathy, including the administration of antagonists of HGF, will be apparent to those skilled in the art and will vary with the type of disease and the type and age of the mammal. Contemplated treatment regimes include those administered once or once an hour, once a day, once a week, once a month, or once a year. Dosage may vary from 1-1000 mg of antagonist per kg of body weight, and is in a form suitable for delivery of the compound. The route of administration will also vary with the disorder to be treated. The present invention contemplates administration of an HGF antagonist to a human to reduce or eliminate chronic nephropathy. The protocol described below for the administration of HGF to humans is given as an example of a method for administering HGF to humans. This protocol should not be construed as the only protocol that can be used, but rather merely as an example. Other protocols will be apparent to those of skill in the art in possession of the present invention. In effect, for administration to humans, the HGF antagonist is dissolved in about 1 ml of saline and a dose of 1-1000 mg / kg body weight is administered orally or intravenously once to several times daily. You. Renal function is monitored throughout the treatment. Antagonists of HGF are prepared for administration by suspending or dissolving in a pharmaceutically acceptable carrier, such as saline, saline, or other formulation apparent to those of skill in such administration. The compositions of the present invention can be administered to mammals in one of the conventional ways, either in sustained release formulations using biodegradable biocompatible polymers or by on-site delivery using micelles, gels and liposomes ( For example, it is administered orally, parenterally, transdermally or transmucosally) or rectally (eg, by suppository or enema) or nasal (eg, by nasal spray). Thus, an HGF antagonist is administered to a mammal by any route in order to reach the target area in the mammal, ie, the kidney, when it exerts its effects. Suitable pharmaceutically acceptable carriers will be apparent to the skilled artisan and will depend on the route of administration. Compounds having HGF antagonist activity also include those compounds that are formulated to target a specific type of cell. For example, it is known in the art to encapsulate or formulate compounds such that they are directed to specific receptors on cells. Such preparations include antibody labeling preparations, receptor ligand binding preparations, and the like. The present invention is further described by the description of the following examples. These examples are for illustrative purposes only and are not limiting unless otherwise specified. Thus, the present invention should not be construed in any way as being limited to the following examples, but rather should include all modifications that become apparent as a result of the teachings described herein. It is. The examples described herein indicate that antagonists of HGF may be used, as treatment of cells in vitro with HGF results in synthesis in the production of compounds related to chronic nephropathy according to the data described herein. Methods and results are provided that are useful for treating chronic nephropathy. Similarly, chronic treatment of animals in vivo with HGF results in reduced renal function. Example Cell culture 8-10 weeks old SJL / J (H-2 ^B ) A mouse mesangial cell culture was established from glomeruli obtained from mouse kidney (Wolf et al., 1992, Am. J. Pathol. 140: 95-107). 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) containing 2 mM L-glutamine and 180 mg / dl glucose, supplemented with 10% FBS and 100 U / ml penicillin / streptomycin. _Two The cells were grown at 37 ° C. in the medium. Cells were subcultured by washing with phosphate buffered saline (PBS) and incubating with 0.05% trypsin supplemented with 20 mM EDTA. Mouse mesangial cells (Wolf et al., Supra) transformed with non-capsidated SV40 virus to establish a permanent cell line are referred to as MMC-SV40 cells. These cells exhibit many characteristics of differentiated mesangial cells (Wolfetal., Supra). Stable transfection was performed on MMC-SV40 cells using a reporter construct HB35 that expresses a luciferase driven by the "minigene" consisting of the 5 'flanking of the murine COL4A1 gene and the first intron region ( Fumo et al., 1994, Am. J. Physiol. 267: F632-F638). The stable transformants are called MMC-COL cells. These cells exhibited a similar pattern of growth and protein synthesis in response to elevated glucose concentrations as MMC-SV40 cells (Fumoetal., Supra). Cryopreserved human mesangial cells (passage 3) were purchased from Clonetics Corp. (San Diego, Calif.), 5% FBS, 50 mg / ml gentamicin (Gentamicin) and 50 ng / ml amphotericin (Amphotericin). )-Growing in Clonetics Mesangial Cell Growth Medium (MsGM) supplemented with -B. Human mesangial cells were grown according to the methods provided by Clonetics Corporation and used in these experiments during passages 6-8. Microphysiology Measurement Cell Sensor (cytosensor) microphysiology instruments are based on a pH-sensitive silicon sensor that is part of a microvolume flow chamber in which cells are immobilized (McConnel et al., 1992, Science). 257: 1906-1912). Mouse mesangial cell 150cm ^Two The flasks were passaged using trypsin into capsule caps with a 3 mm pore size polycarbonate membrane (Molecular Devices, Inc., Sunnyvale, CA) at a density of 300,000 cells / cap. Attached for 24 hours in media specific for each cell type. After fitting the spacer ring and insert cap into the capsule cap, the assembled unit is transferred to the sensor chamber and 100 ml with bicarbonate-free RPMI 1640 medium (Molecuarar Devices, Inc.) in a microphysiological instrument. Per minute. The acidification rate was measured by the change in pH overtime, which was measured when the pump was stopped at 2 minute intervals for 30 seconds. Mitogenesis assay The mitogenic effects of HGF in mouse mesangial cells were incorporated into newly synthesized DNA [ ^Three [H] thymidine. Cells were passaged into 24 well dishes (2.5 x 10 ^Three Cells / well) and incubated for 72 hours in growth media. The combined cultures were quiesced for 48 hours in DMEM medium containing 2 mM L-glutamine and 100 mg / dl glucose and supplemented with 3% FBS, 100 / ml penicillin and 100 mg / ml streptomycin. HGF was diluted in unsupplemented DMEM medium and added to the wells in triplicate for 24 hours. During the last 4 hours of this 24-hour culture, ^Three [H] thymidine was applied. Cells were washed with PBS and 5% trichloroacetic acid was added to precipitate proteins and nucleic acids, which were not incorporated [ ^Three [H] thymidine was removed. The precipitate was then dissolved by the addition of 0.5N NaOH, 400 ml of which was added to the scintillation fluid and counted in a Taurus liquid scintillation counter (ICN Biomedicals, Inc., Huntsville, AL). Luciferase assay MMC-COL cells were cultured in growth media at 24, 000 plates at 25,000 cells / well for 48 hours. The cells were then incubated in the same medium used to quiescent the cells in the proliferation assay and incubated for an additional 48 hours before adding HGF. At the times indicated, pH 7.0 containing 0.1 mM potassium phosphate and 1 mM dithiothreitol, 1% Triton X-100. Cells were lysed (luciferase activity) using buffer 2 or trypsinized (cell number). Lysed cells were centrifuged and 100 ml of the supernatant was placed in duplicate in wells of a 96-well microtiter plate. Luminescence was measured directly at room temperature using a Microlumat LB96P luminometer (Wallac Inc., Gaithersburg, MD), and 100 ml of the luciferin reaction mixture was automatically injected and integrated over 20 seconds. This mixture contains 0.1 mM potassium phosphate, 10 mM ATP, 20 mM MgCl _Two And 1 mM dithiothreitol stock buffer (pH 7.2) and newly added 0.8 mg / ml D-luciferin (Boehringer Mannheim, Indianapolis, IN). Luciferin activity was expressed in relative light units per cell number (RLU) measured from cell cultures similarly treated in a well, trypsinized and counted (Coulter Electronic LTD, Luton, Beds, England). Northern blot hybridization MMC-SV40 cells 150 cm ^Two Cultured in flasks and incubated in the above growth medium for 3-4 days. 48 hours before HGF addition, the medium was changed to DMEM medium containing 2 mM L-glutamine and 100 mg / dl glucose and supplemented with 3% FBS, 100 U / ml penicillin and 100 mg / ml streptomycin. Cells were incubated with HGF for 2, 6 and 16 hours. Total RNA was extracted from mouse cells by guanidium thiocyanate denaturation and phenol-chloroform extraction (Chomczynski et al., 1987, Anal. Biochem. 162: 156-159). Total RNA (10 mg / lane) was fractionated on a 0.2 M formaldehyde-1% agarose gel and transferred to a nylon membrane (Nylon-1; Bethesda Research Laboratories, Bethesda, MD) in 4 × SSC. Equivalent loading and transfer was confirmed by methylene blue staining. Randomly primed that recognizes a 7.6 kb mRNA [ ³² [P] DNA probes were generated for fibronectin. Fibronectin was purchased from ATCC. Hybridization was performed using 50% formamide, 225 mM NaCl, 20 mM NaH. _Two PO _Four , 1.5 mM EDTA, 1% sodium dodecyl sulfate, 0.5% dry milk, 100 mg / ml yeast, total RNA and salmon DNA. ⁶ Performed at 42 ° C. for 16 hours using cpm / ml. The blot was washed at 65 ° C. with 0.2 × SSC final stringency, 0.2% sodium dodecyl sulfate. The membrane was exposed to a phosphor imaging plate and bands were quantified with ImageQuant software (Molecular Dynamics, Inc.). Animal experiments Alzet miniosmotic pumps (Alza, Palo Alto, CA) were filled with 14.6 ng / ml HGF or vehicle (350 mM NaCl, 10 mM phosphate pH 7.3, 625 mg / ml human serum albumin). C57BL / Ks lean and obese mice with recessive db mutations were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were implanted intraperitoneally under Ketalar (60 mg / kg) and killed 21 days later. On the day of sacrifice, urine samples were collected in metabolic cages for 24 hours. When killed, blood was collected. Urine and serum creatinine levels were measured on a Synchron Clinical System AS8 (Beckman; Columbia, MD). Creatinine clearance was calculated to measure renal function. HGF-mediated acidification rate To assess the effect of FBS or BSA on HGF-mediated acidification rate, MMC-SV40 cells were pretreated with 0.1% BSA, 0.5% FBS or both for 30 minutes. This experiment was performed because recombinant HGF from R & D Systems, Minneapolis, MN occurs in a single-chain precursor form that requires enzyme activation. Proteases in serum or secreted by cells can activate HGF, or proteases contained in FBS may be required. In addition, like other growth factors, HGF tends to adhere to plastic tubes and may require a carrier molecule to deliver HGF to cells. After the preincubation, cells were exposed to 100 mg / ml HGF for 10 minutes in the presence or absence of 0.1% BSA or 0.5% FBS. In the presence of FBS, the baseline response was set 30% higher than before HGF administration, but a weaker HGF-induced acidification was observed. BSA dramatically attenuated the peak response of HGF in the presence or absence of FBS (FIG. 1, panel A). Therefore, in the remaining experiments, HGF was administered in running medium in the absence of FBS or BSA. The effect of the length of time the cells were exposed to HGF on the acidification rate was assessed by treating MMC-SV40 cells with 100 ng / ml HGF for 2, 5, 10 and 15 minutes. Incubation of the cells in the presence of HGF for 5, 10 and 15 minutes each resulted in similar peaks of acidification rate, but further experiments required 5 minutes for rapid recovery of cells after this treatment. (FIG. 1, Panel B). HGF dose response was evaluated by treating MMC-SV40 cells with 3, 10, 30, or 100 ng / ml HGF. A concentration of 30 ng / ml HGF showed the greatest increase in acidification rate, and no further increase was seen at 100 ng / ml HGF (FIG. 1, panel C). Primary human mesangial cells were treated at passage 7 to determine if untransformed mesangial cells also respond to HGF. 100 ng / ml HGF induced an increase in the rate of acidification of human Nemesangial cells (FIG. 1, panel D). The involvement of the PKC-mediated second messenger system in the response of mesangium to HGF was evaluated using the PKC inhibitor RO-32-0432. Transformed mouse mesangial cells were pretreated with 1, 3, or 5 mM RO-32-0432 for 30 minutes, and HGF at a concentration of 50 ng / ml was added for 5 minutes in the presence of the pretreatment medium. RO-32-0432 partially blocked the rate of HGF-induced acidification in a concentration-dependent manner (FIG. 2). Effect of HGF on mitogenesis Proliferation of normal human cells and immobilized mouse mesangial cells was assessed by incorporation of tritiated thymidine. Human renal glomerular mesangial cells were serum-starved for 48 hours. Mouse mesangial cells were grown in 3% serum. Each set of cells was treated for 24 hours with increasing doses of HGF up to 100 ng / ml. HGF did not affect tritiated thymidine incorporation in both normal human cells and immobilized mouse mesangial cells at any of the doses tested (FIG. 3). Effect of HGF on extracellular matrix gene expression The effect of HGF on extracellular gene expression was assessed by assessing collagen al (IV) promoter activation of luciferase reporter gene in MMC-COL cells and collagen al (IV) in MMC-SV40 cells. And fibronectin mRNA were assessed by Northern blot hybridization analysis. HGF-induced activation of the collagen promoter in mouse mesangial cells was dose-dependent. In the presence of 30 ng / ml HGF, the activity of the collagen promoter was twice as high as that of the untreated cells (FIG. 4). MMC-SV40 cells were cultured at 50 ng / min in the presence or absence of 1 mM 5-amino-2- (4-aminoanilino) benzenesulfonic acid (ICN Biomedicals, Inc., Costa Mesa, Calif.), A protein kinase C inhibitor. Treated with ml of HGF. Northern blots showed that HGF increased collagen al (IV) mRNA levels in mouse mesangial cells and that the inhibitor blocked HGF-induced increases (data not shown). MMC-SV40 cells were also treated with 50 ng / ml HGF for 2, 6 and 16 hours in the presence of 3% serum. Northern blots showed that HGF induced an increase in fibronectin mRNA levels at 6 and 16 hours after HGF administration (data not shown). Effect of HGF on renal function The effect of HGF on renal function was determined in normal and diabetic mice by assessing creatinine clearance after 21 days of prolonged administration of HGF. Creatinine clearance in normal and diabetic vehicle-transplanted mice was 400 and 454 ml / min / 100 g, respectively. HGF increased creatinine clearance in both groups, 283 and 287, respectively. ^* ml / min / 100g (based on vehicle group) ^* p <0.05; n = 5-6 mice; FIG. 5). The data presented herein confirm that glomerular stromal cells respond to changes exhibited by HGF in acidification rates and extracellular matrix gene expression. The effect of HGF on glomerular stromal cells is mediated in part by the protein kinase C second messenger system. In contrast to the mitogenic activity of HGF on endothelial cells, HGF has no effect on the growth of mouse or human glomerular stromal cells. HGF has different effects on glomerular stromal cells during chronic renal dysfunction. Furthermore, in addition to other proteins known to act on renal dysfunction, the data of the present invention indicate that HGF also contributes to matrix production in glomerular stromal cells and thus to chronic kidney damage Indicates that In summary, HGF promotes extracellular acidification of glomerular stromal cells and increases fibronectin and collagen al (IV) gene expression in glomerular stromal cells. Therefore, HGF contributes to glomerulosclerosis by activating glomerular interstitial cells and increasing extracellular matrix deposition. In addition, treatment of animals with HGF for 21 days (long term treatment) reduced creatinine clearance, suggesting reduced renal function. Therefore, the chronically high HGF observed in many chronic kidney diseases appears to contribute to decreased renal function by causing extracellular matrix expansion in the glomeruli. The disclosure of each and every patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety. While the invention will be disclosed with reference to particular embodiments, it will be apparent that other embodiments and variations of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. There will be. It is intended that the appended claims be constructed to include all such embodiments and equivalent variations.

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Claims (1)

[Claims] 1. A method for identifying an antagonist of HGF activity, comprising:   Treating the cells with HGF in the presence or absence of the test compound;   Comparing levels of fibronectin mRNA in cells, where testing Test compound compared to the level of fibronectin mRNA in the absence of the compound Lower levels of fibronectin mRNA in the presence of An F antagonist.   A method that includes: 2. A method for identifying an antagonist of HGF activity, comprising:   Treating the cells with HGF in the presence or absence of the test compound;   Compare acidification rates in cells, where acidification in the absence of test compound The lower acidification rate in the presence of the test compound as compared to the Indicating an F antagonist. 3. A method for identifying an antagonist of HGF activity, comprising:   Administering HGF to the mouse in the presence or absence of the test compound;   Compare creatine clearance rates in mice, where HGF is HGF compared to the rate of creatine clearance in mice administered alone Creatine clearance rate in mice treated with Adding indicates that the test compound is an HGF antagonist.   A method that includes: 4. An HGF antigen identified by the method according to claim 1, 2 or 3. Antagonist. 5. A method of treating a human with chronic renal disease, comprising the steps of: Administering to the subject. 6. Anti-HGF antibodies, peptidemetics and polypeptide fragments of HGF HGF antibody selected from the group consisting of