Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the field of the invention is treatment of chronic renal disease.
• Hepatocyte growth factor is a heterodimeric molecule derived from a preproprecursor protein of 728 amino acids, which is proteolytically processed by a specific protease to form mature HGF (Miyazawa et al . , 1993, J. Biol. Chem. 268:10024-10028).
• HGF binds to cells through the tyrosine kinase receptor c-met. Binding of HGF to c-met has the following effects: Growth of renal epithelial cells is stimulated; the motility of cells is enhanced; and, renal tubule formation is induced (Santos et al . , 1993, Dev. Biol. 159:535-548; Cantley et al . , 1994, Am. J. Physiol. 267 :F271-F280) .
• HGF may be characterized as a mitogen, a motogen and a morphogen, respectively, and is believed to play a role in renal development.
• HGF is involved in renal remodeling following injury as reflected by increased levels of HGF and its receptor c-met in the kidney following nephrectomy or ischemia (Joannidis et al . , 1994, Am. J. Physiol. 267 : F231-F236) . Further, HGF has been observed to effect an improvement in renal function following mercuric chloride assault or ischemia (Kawaida et al . , 1994, Proc. Natl. Acad. Sci. USA 91:4357-4361; Miller et al., 1994, Am. J. Physiol. 266 : F129-F134) . Glomerular hypertrophy occurs during renal remodeling in many chronic renal diseases. This event is associated with glomerular basement membrane expansion, proliferation of mesangial and epithelial cells, and the accumulation of collagen and fibronectin. The role of HGF in glomerular extracellular matrix expansion is not known.
• Chronic renal failure is the progressive loss of functional renal mass, accompanied by compensatory growth and remodeling.
• the molecular and cellular events that take place during chronic renal failure include release of growth factors, proliferation of glomerular mesangial cells and expansion of extracellular matrix (Klahr et al . , 1988, New Engl . J. Med. 318:1657-1666; Striker et al . , 1989, In: Klahr, (Ed.) Seminars in Nephrology, pp. 318, Philadelphia, W.B. Saunders) ; Ebihara et al . , 1993, J. Am. Soc . Nephrol. 3:1387-1397).
• the components of the extracellular matrix which change during chronic renal failure include collagen, fibronectin, and laminin
• thrombin Xu et al . , 1995, Am.
• compositions for treatment of chronic renal disease which serve to diminish or ablate disease leading to renal failure and either death or dependence upon dialysis.
• the invention relates to a method of treating a human having chronic renal disease comprising administering a hepatocyte growth factor antagonist to the human .
• Figure 1 is a series of graphs depicting extracellular acidification rates of transformed mouse mesangial cells (MMC-SV40 cells) (Panels A-C), or normal human mesangial cells (Panel D) determined by microphysiometry .
• Panel A MMC-SV40 cells 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 treated with HGF (100 ng/ml) for 2, 5, 10, and 15 minutes.
• Panel C MMC-SV40 cells treated with 4 concentrations of HGF.
• Panel D Normal human mesangial cells treated with 100 ng/ml HGF; duplicate traces are shown.
• Figure 2 is a graph depicting extracellular acidification rates of MMC-SV40 cells treated with HGF (50 ng/ml) . Cells were perfused with control medium or medium containing different concentrations of RO-32- 0432, a protein kinase inhibitor described by Birchall et al., 1994, J. Pharmacol. Exp . Ther . 268:922-929.
• FIG 3 is a graph depicting tritiated thymidine incorporation of normal human mesangial cells (HMC) , MMC-SV40 cells, or mouse mesangial cells transfected with a luciferase reporter plasmid driven by the collagen al (IV) promoter (MMC-COL cells) treated with different concentrations of HGF for 24 hours.
• Figure 4 is a graph depicting collagen al (IV) promoter activity in MMC-COL cells in response to different concentrations of HGF. Data are the means ⁇ SD of quadruplicates .
• HGF histoneum growth factor
• the invention relates to a method of treating chronic renal disease in a mammal comprising administering an antagonist of HGF to the mammal.
• chronic renal disease as used herein, is meant 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, peptidometics having anti-HGF activity, antibodies directed against HGF, nucleic acids having a sequence which is complementary to all or a portion of the nucleic acid encoding HGF and small chemical compounds which inhibit HGF-specific protease.
• HGF antagonist activity is meant a compound which inhibits the normal activity of HGF, such as determined, for example, in any one or more of the assays described herein.
• treatment of mouse mesangial cells with HGF results in an increase in the acidification rate of these cells.
• a compound having HGF antagonist activity is defined as one which inhibits an HGF-induced increase in acidification rate in mouse mesangial cells.
• a test compound is assessed for HGF antagonist activity in one or more of the assays described herein in the experimental examples section, or in any other assay for measurement of HGF function.
• an in vi tro test is used to identify a compound having HGF antagonist activity.
• Such in vi tro tests include, but are not limited to, tests which assess the affect of the test compound on HGF binding to cell membranes of cells which are known to respond to HGF, on HGF-induced acidification rate of mesangial cells and tests which assess the affect of the test compound on HGF-induced extracellular matrix gene expression.
• [ 125 I] -labeled HGF is incubated with cell membranes from A498 cells attached to scintillation beads plus or minus the -test compound. If the compound inhibits HGF binding to the membrane, then the [125j_]_ labeled HGF will not be in the proximity of the scintillation bead resulting in decreased signal. A secondary assay used below would determine if this compound is an agonist or antagonist.
• mesangial cells are incubated in the presence or absence of HGF plus or minus the test compound.
• the acidification rates of the cells are measured by microphysiometry as described herein and the effect of the test compound on HGF-induced cell acidification is determined.
• a reduction in the acidification rates of cells treated with HGF and the test compound, compared with the acidification rates in cells treated with HGF alone, is an indication that the test compound has HGF antagonist activity.
• test compound On extracellular matrix gene expression, cells are incubated in the presence or absence of HGF plus or minus the test compound under the conditions described herein in the experimental examples section.
• the expression of mRNA associated with extracellular matrix genes e.g., collagen al(IV) and fibronectin; is measured and compared among the different sets of cells.
• the test compound may also be tested for HGF antagonist activity in an in vivo assay.
• long term treatment of mice with HGF induces renal failure.
• Another exemplary way to assess the HGF antagonist activity of a test compound is to administer HGF alone HGF plus test compound to mice over at least a 21 day period. Additional controls include mice which are administered a suitable placebo compound. Creatine clearance is assessed in each set of mice as a measure of renal function. An increase in creatine clearance in mice administered HGF plus the test compound, compared with creatine clearance in mice administered HGF alone, is another indication that the test compound has HGF antagonist activity.
• HGF antagonists which comprise peptides comprising specific portions of HGF include, but are not limited to, those which encompass the first krinkle domain near the N-terminal of HGF.
• An example of such a peptide is the truncated HGF peptide NK2 (Chan et al . , 1991, Science 254:1382-1385).
• the peptide may be produced by cloning and expressing HGF DNA encoding the desired portion of HGF.
• HGF DNA and amino acid sequences are provided in Miyazawa et al . , 1991, Eur . J. Biochem. 197:15-22 (SEQ ID NOs : 1 and 2).
• An isolated DNA encoding the desired portion of HGF is cloned into an expression vector and the protein is expressed therefrom. Procedures for cloning and expression of 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
• Peptide so expressed may be obtained using ordinary peptide purification procedures well known in the art .
• substantially pure describes a compound, e.g., a protein or polypeptide which has been separated from components which naturally accompany it.
• a compound is substantially pure when 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%, and most preferably at least 98% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e . g. , in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis.
• a compound, e.g., a protein is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state .
• the present invention also includes analogs of peptides obtained according to the methods of the invention.
• Analogs can differ from naturally occurring peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both.
• conservative amino acid changes may be made, which although they alter the primary sequence of the peptide, do not normally alter its function.
• Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.
• Modifications include in vivo, or in vi tro chemical derivatization of peptides, e . g. , acetylation, or carboxylation. Also included are modifications of glycosylation, e . g.
• glycosylation patterns of a peptide those made by modifying the glycosylation patterns of a peptide during its synthesis and processing or in further processing steps; e.g., by exposing the peptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
• enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes.
• sequences which have phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine .
• peptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties .
• Analogs of such peptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.
• the peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.
• the present invention also relates to active fragments of HGF having HGF antagonistic activity.
• a specific polypeptide is considered to have HGF antagonistic activity if it inhibits the action of HGF as described herein.
• fragment as applied to a HGF peptide, will ordinarily be at least about 10 contiguous amino acids, typically at least about 20 contiguous amino acids, more typically at least about 50 continuous amino acids and usually at least about 78 contiguous amino acids in length.
• Nucleic acid sequence complementary to HGF may be generated using the sequence of HGF provided in Nakamura et al . ( supra) .
• Administration of antisense oligonucleotides to mammals is now common in the art and may be accomplished by using any of the administration techniques described herein.
• Nucleic acid complementary to nucleotides 78-950 of HGF (SEQ ID NO: 1) or portions thereof may be obtained by cloning HGF DNA or portions thereof into an expression vector such that RNA is expressed therefrom in the antisense orientation (i.e., complementary) to HGF mRNA.
• isolated DNA refers to a DNA sequence, segment, or fragment which has been purified from the sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
• the term also applies to DNA which has been substantially purified from other components which naturally accompany the DNA, e.g., RNA or DNA or proteins which naturally accompany it in the cell.
• the invention should also be construed to include DNAs which are substantially homologous to HGF DNA or portions thereof, which DNAs are useful for the production of HGF complementary nucleic acid, or for the production of HGF peptides.
• DNA which is substantially homologous is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to DNA obtained using the method of the invention.
• Homologous refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
• the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
• the DNA sequences 3 ⁇ TTGCC5' and 3 ' TATGCG5 ' share 50% homology.
• Anti-HGF antibodies are easily generated by immunization of a mammal with the HGF peptide identified herein. Protocols for the generation of antibodies (either monoclonal or polyclonal antibodies) to a known peptide are described in Harlow et al . (1988, In:
• Polyclonal antibodies to HGF may be raised in any suitable mammal, such as a mouse or a rabbit.
• Monoclonal anti-HGF antibodies are generated by immunization of a mouse with HGF peptide followed by production of hybridoma cells capable of secreting anti- HGF antibody.
• Other means of producing monoclonal antibodies, such as antibodies which are expressed by bacteriophage, are now also 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) .
• Peptidometics having HGF-antagonist-like activity may also be designed and used according to the present invention. Additional information describing administration of peptidometics is provided in PCT/US93/01201 and U.S. Patent No. 5,334,702, which are hereby incorporated herein by reference. Any of the techniques described in either of these two references may be employed in the present invention for the administration of peptidometics.
• An HGF antagonist may also include small molecules having HGF antagonist activity as defined herein which are not peptide or nucleic acid molecules.
• HGF antagonists useful in the method of the present invention include, but are not limited to, those described in Faletto et al . (WO 94/06909) Roos et al. (WO94/06456) , JP05208998 and Aaronson et al . , (WO 92/05184) .
• Kidney diseases which are treatable using the method of the invention include, but are not limited to, polycystic disease, diabetic nephropathy, focal segmental glomerulosclerosis , hypertension-induced nephropathy, hypernephroma, and the like.
• Protocols for treatment of mammals with a chronic renal disease involving administration of an antagonist of HGF will be apparent to those skilled in the art and will vary depending upon- the type of disease and the type and age of the mammal .
• Treatment regimes which are contemplated include a single dose or dosage which is administered hourly, daily, weekly or monthly, or yearly. Dosages may vary from 1-1000 mg/kg of body weight of the antagonist, and will be in a form suitable for delivery of the compound. The route of administration may also vary depending upon the disorder to be treated.
• the invention contemplates administration of an HGF antagonist to humans for the purpose of alleviating or ablating chronic renal disease.
• the protocol which is described below for administration of HGF to a human is provided as an example of how to administer HGF to a human. This protocol should not be construed as being the only protocol which can be used, but rather, should be construed merely as an example of the same. Other protocols will become apparent to those skilled in the art when in possession of the instant invention.
• the HGF antagonist is dissolved in about 1 ml of saline and doses of 1-1000 mg per kg of body weight are administered orally or intra-venously once per day to several times per day. Renal function is monitored throughout the administration period.
• the antagonist of HGF is prepared for administration by being suspended or dissolved in a pharmaceutically acceptable carrier such as saline, salts solution or other formulations apparent to those skilled in such administration.
• a pharmaceutically acceptable carrier such as saline, salts solution or other formulations apparent to those skilled in such administration.
• the compositions of the invention may be administered to a mammal in one of the traditional modes (e.g., orally, parenterally, transdermally or transmucosally) , in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema) or nasally (e.g., by nasal spray) .
• an HGF antagonist may be administered to the mammal by any route in order that it eventually reaches the target area in the mammal, i.e., the kidney, wherein it exerts its effects.
• the appropriate pharmaceutically acceptable carrier will be evident to those skilled in the art and will depend upon the route of administration .
• Compounds having HGF antagonist activity also include compounds which are formulated so as to target specific types of cells. For example, it is now known in the art to encapsulate or otherwise formulate compounds such that they are directed to specific receptors on cells. Such formulations include antibody- tagging formulations, receptor-ligand binding formulations and the like.
• MMC-SV40 cells Mouse mesangial cells transformed with noncapsid forming SV40 virus to establish a permanent cell line (Wolf et al . , supra) are designated as MMC-SV40 cells. These cells exhibit many features of differentiated mesangial cells (Wolf et al . , supra) .
• a stable transfection was performed on MMC-SV40 cells with a reporter construct HB35, which expresses luciferase driven by a "minigene" comprised of the 5' flanking and first intron regions of the murine COL4A1 gene (Fumo et al., 1994, Am. J. Physiol. 267 : F632-F638) .
• the stable transformants are designated as MMC-COL cells. These cells exhibited patterns of growth and protein synthesis in response to elevated glucose concentration similar to MMC-SV40 cells (Fumo et al . , supra) .
• Cryopreserved human mesangial cells (passage 3) were purchased from Clonetics Corp. (San Diego, CA) and were grown in Clonetics Mesangial Cell Growth Medium (MsGM) supplemented with 5% FBS, 50 mg/ml Gentamicin and 50 ng/ml Amphotericin-B. Human mesangial cells were grown according to the methods provided by Clonetics Corp. and were used in these experiments between passage 6-8. Microphysiometry
• the cytosensor microphysiometer is based on a pH sensitive silicon sensor which is part of a microvolume flow chamber in which cells are immobilized (McConnel et al., 1992, Science 257:1906-1912).
• Mouse mesangial cells were subcultured with trypsin from 150 cm 2 flasks into capsule cups (Molecular Devices, Inc., Sunnyvale, CA) having a polycarbonate membrane of 3 mm pore size at a density of 300,000 cells per cup. Cells were allowed to attach for 24 hours in the medium specified for each cell type.
• the mitogenic effect of HGF on mouse mesangial cells was measured as the amount of [ ⁇ H] thymidine incorporated into newly synthesized DNA.
• Cells were subcultured into 24 well dishes (2.5 x 10 3 cells per well and Incubated in growth media for 72 hours.
• Subconfluent cultures were made quiescent by placing them for 48 hours in DMEM medium containing 2 mM L- glutamine and 100 mg/dl glucose, the medium being further supplemented with 3% FBS and 100/ml penicillin and 100 mg/ml streptomycin.
• HGF was diluted in unsupplemented DMEM medium and then added to wells in triplicate for 24 hours.
• Cells were pulsed with [ 3 H] thymidine during the last 4 hours of the 24 hour incubation period. Cells were washed with PBS, and then 5% trichloroacetic acid was added to precipitate proteins and nucleic acids and to remove unincorporated
• MMC-COL cells were cultured in 24 well plates at 25,000 cells per well in growth medium for 48 hours. Cells were then incubated in the same medium used to make the cells quiescent in the proliferation assay and were incubated for an additional 48 hours before HGF was added. At the times indicated, cells were lysed using 500 ml of a buffer containing 0.1 mM potassium phosphate and 1 mM dithiothreitol, pH 7.2 with 1 % Triton X-100 (luciferase activity) or were trypsinized (cell number). The lysed cells were centrifuged and 100 ml of the supernatant was added in duplicate to wells of a 96 well microliter plate.
• Light emission was measured directly at room temperature using a Microlumat LB96P luminometer (Wallac Inc., Gaithersburg, MD) and integrated over a 20-s period following the automated injection of 100 ml of a luciferin reaction mixture.
• This mixture contained a stock buffer of 0.1 mM potassium phosphate, 10 mM ATP, 20 mM MgCl2 and 1 mM dithiothreitol, pH 7.2, and freshly added 0.8 mg/ml of D-luciferin (Boehringer Mannheim, Indianapolis, IN) .
• Luciferase activity was expressed as relative light units (RLU) per number of cells as determined from similarly treated cultures of cells in wells which were trypsinized and counted (Coulter Electronic LTD, Luton, Beds, England). Northern blot hybridization
• MMC-SV40 cells were" cultured in 150 cm 2 flasks and incubated for 3 to 4 days in the growth medium described above .
• the medium was changed to DMEM medium containing 2 mM L-glutamine and 100 mg/dl glucose and supplemented with 3% FBS and lOOU/ml penicillin and 100 mg/ml streptomycin for 48 hours before the addition of HGF.
• Cells were incubated for 2, 6, and 16 hours with HGF.
• Total RNA was extracted from mouse cells by guanidinium thiocyanate denaturation and acidified phenol-chloroform extraction (Chomczynski et al . , 1987, Anal. Biochem. 162:156-159).
• RNA (10 mg/lane) was fractionated on 0.2 M formaldehyde-1% agarose gels and transferred to nylon membranes (Nylon-1; Bethesda Research Laboratories, Bethesda, MD) in 4X SSC . Equivalent loading and transfer were verified by methylene blue staining. Random primed [32p]DNA. probes were made for fibronectin that recognize an mRNA of 7.6 kb. The fibronectin clone was purchased from ATCC .
• Hybridizations were performed with 10 ⁇ cpm/ml of labeled DNA in 50% formamide, 225 mM NaCl, 20 mM NaH 2 P0 4 , 1.5 mM EDTA, 1% sodium dodecyl sulfate, 0.5% dry milk, 100 mg/ml yeast total RNA and 300 mg/ml salmon DNA, at 42 °C for 16 hours. Blots were washed with a final stringency of 0.2 X SSC, 0.2% sodium dodecyl sulfate at 65°C.
• Membranes were exposed to phosphor imaging plate and bands were quantified with ImageQuant software (Molecular Dynamics, Inc.).
• Alzet mini-osmotic pumps (Alza, Palo Alto, CA) were filled with either HGF at 14.6 ng/ml or vehicle (350 mM NaCl, 10 mM phosphate pH 7.3, 625 mg/ml human serum albumin.
• Lean and obese mice of the C57BL/Ks strain with the recessive db mutation were purchased from the Jackson laboratory (Bar Harbor, ME) .
• Mice received the implants in the peritoneal cavity under Ketalar (60 mg/kg) and were killed 21 days later. Twenty-four hour urine samples were collected in metabolic cages on the day of sacrifice. At the time of sacrifice, blood was also collected.
• Urine and serum creatinine levels were determined by a Synchron Clinical System AS8 (Beckman; Columbia, MD) . Renal function was determined by calculating creatinine clearance.
• MMC-SV40 cells were pretreated with 0.1% BSA, 0.5% FBS or both for 30 minutes .
• This experiment was performed because recombinant HGF obtained from R&D Systems, Minneapolis, MN occurs in the single chain precursor form which requires enzymatic activation. Proteases in the serum or proteases secreted by cells may activate HGF, or proteases contained in FBS may be required. In addition, HGF, like other growth factors, tends to adhere to plastic tubing and thus, a carrier molecule for delivery of HGF to the cells may be required. Following the pre-incubation period, cells were exposed to 100 mg/ml HGF for 10 minutes in the presence or absence of 0.1 % BSA or 0.5% FBS.
• SV40 cells for 5 minutes with 3, 10, 30, or 100 ng/ml of HGF.
• a concentration of 30 ng/ml HGF elicited the maximum increase in acidification rate with no further increase at 100 ng/ml HGF.
• Figure 1, Panel C To determine if non-transformed mesangial cells also respond to HGF, primary human mesangial cells were treated at passage 7. HGF at a concentration of 100 ng/ml induced an increase in the acidification rate of human mesangial cells ( Figure 1, Panel D) .
• the involvement of PKC mediated second messenger systems in mesangial responses to HGF was evaluated using the PKC inhibitor RO-32-0432.
• Transformed mouse mesangial cells were pre-treated 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 HGF-induced acidification rate in a concentration dependent fashion ( Figure 2).
• HGF extracellular matrix gene expression was evaluated by assessing collagen al(IV) promoter activation of a luciferase reporter gene in MMC-COL cells and by northern blot hybridization analysis of collagen al(IV) and fibronectin mRNA in MMC- SV40 cells.
• MMC-SV40 cells were treated with 50 ng/ml HGF in the presence or absence of 1 mM 5-amino-2- (4- aminoanilino)benzenesulfonic acid (ICN Biomedicals, Inc., Costa Mesa, CA) , a protein kinase C inhibitor.
• Northern blots indicated that collagen al (IV) mRNA levels were increased in the mouse mesangial cells by HGF and that the inhibitor blocked the HGF-induced increase (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 indicated that HGF induced an increase in fibronectin mRNA levels at 6 and 16 hours following HGF administration (data not shown) .
• the data presented herein establish that glomerular mesangial cells respond to HGF exhibiting changes in acidification rate and extracellular matrix gene expression. The effect of HGF on mesangial cells is mediated in part by protein kinase C second messenger systems.
• HGF has no effect on proliferation of mouse or human mesangial cells. HGF has distinct effects on glomerular mesangial cells during chronic renal failure. Moreover, in addition to other proteins known to affect renal malfunction, the present data demonstrate that HGF also contributes to matrix production in mesangial cells and therefore chronic renal disease.
• HGF stimulates the extracellular acidification rate of mesangial cells and increases gene expression of fibronectin and collagen al(IV) in glomerular mesangial cells. Therefore, HGF contributes to glomerulosclerosis by activating mesangial cells to increase extracellular matrix deposition.
• HGF long term treatment
• TELECOMMUNICATION INFORMATION (A TELEPHONE: 610-270-5096 (B TELEFAX: 610-270-5090 (C TELEX:
• MOLECULE TYPE cDNA
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• FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:
• Tyr lie Arg Asn Cys lie lie Gly Lys Gly Arg Ser Tyr Lys Gly Thr 125 130 135
• Arg Gly Leu Met Asp His Thr Glu Ser Gly Lys lie Cys Gin Arg Trp 220 225 230 235 GAT CAT CAG ACA CCA CAC CGG CAC AAA TTC TTG CCT GAA AGA TAT CCC 830 Asp His Gin Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro 240 245 250
• MOLECULE TYPE protein
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE internal
• ORIGINAL SOURCE

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• 1997
  • 1997-11-03 EP EP97948148A patent/EP0941111A4/de not_active Withdrawn
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  • 1997-11-03 JP JP52162598A patent/JP2001505995A/ja active Pending
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