WO2001016354A1 - Modulation de he4 dans des maladies inflammatoires et renales - Google Patents

Modulation de he4 dans des maladies inflammatoires et renales Download PDF

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WO2001016354A1
WO2001016354A1 PCT/US2000/024282 US0024282W WO0116354A1 WO 2001016354 A1 WO2001016354 A1 WO 2001016354A1 US 0024282 W US0024282 W US 0024282W WO 0116354 A1 WO0116354 A1 WO 0116354A1
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nucleic acid
agent
acid molecule
igan
seq
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PCT/US2000/024282
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William E. Munger
Hong-Wei Sun
Ronald J. Falk
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Gene Logic, Inc.
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Priority to AU73473/00A priority Critical patent/AU7347300A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors

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  • This invention relates to methods of correlating gene expression with inflammatory diseases and renal disorders in healthy and disease samples and methods for drug screening using identified gene product candidates as targets. Specifically, this invention relates to methods which monitor the expression of HE4, including an HE4 which has been identified in kidney tissue from a patient diagnosed with immunoglobulin A nephropathy (IgAN).
  • IgAN immunoglobulin A nephropathy
  • Such monitoring can serve 1) as a predictor of glomerulonephritis, including mesangial proliferation; inflammation; necrotizing crescentic glomerulonephritis (NCGN); minimal change disease and sclerosis, 2) as a means of identifying agents that modulate HE4 expression in mesangial and other tissues and 3) broadly as a general marker for renal diseases and inflammatory diseases including noninfectious systemic inflammatory responses (SIRS).
  • SIRS noninfectious systemic inflammatory responses
  • Kidney disease affects many millions of people in the United States and worldwide. In 1990 the US had greater than 200,000 patients in end stage renal disease (ESRD) programs, largely as a result of renal involvement by glomerular diseases.
  • ESRD end stage renal disease
  • kidney disease The mechanisms of glomerular diseases are quite varied. Although certain common mechanisms may underlie blood or protein in the urine (e.g., loss of the glomerular charge barrier), the nature of the processes initiating renal injury differ. For example, immune- mediated renal injury is a major pathogenic mechanism of glomerular damage. In diabetic nephropathy, other mechanism are clearly at work. At this time kidney disease usually cannot be cured. Once the glomeruli (filtering units) are damaged, they cannot be repaired. Therefore, treatment focuses on slowing the progression of the disease and preventing complications.
  • kidney biopsy While diagnosis of the renal patient may involve urinanalysis, microscopic examination and radiologic studies, when these procedures fail to give a diagnosis or are not indicated for clinical reasons (e.g., patient cannot cooperate, has bleeding abnormalities; only a solitary kidney exists or blood pressure is too high), the use of kidney biopsy may be required (Cecil Textbook of Medicine 20th edition, 1996, (Bennett et al. eds.) p. 517, W.B. Saunders Company, Philadelphia, PA).
  • Renal biopsy is performed to: (1) help establish a histologic diagnosis; (2) help estimate prognosis and potential reversibility or progression of the renal lesion; (3) estimate the value of therapeutic modalities; and (4) determine the natural history of renal diseases (The Merck Manual 16th edition, 1992).
  • IgA Nephropathy IgA nephropathy is the most common form of glomerulonephritis world wide
  • IgAN causes little to no pain, about 30% of patients will develop ESRD after 20 years, particularly those who present with hypertension, heavy proteinuria or renal insufficiency (Galla JH., 1995). IgAN can also cause a vasculitis which can lead to necrotizing crescentic glomerulonephritis (NCGN), the latter ultimately resulting in renal failure and end-stage renal disease.
  • NCGN necrotizing crescentic glomerulonephritis
  • Glomerular disorders such as IgAN that progress to renal failure are characterized by mesangial cell proliferation and accumulation of mesangial matrix.
  • Mesangial cell are cells found within the glomerular lobules of kidneys, where they serve as structural supports, regulate blood flow, are phagocytic and may act as accessory cells presenting antigen in immune responses.
  • Factors that control mesangial cell function include cytokines and growth mediators, matrix components including integrins, and interactions with other cells such as the endothelial and epithelial cells.
  • IgAN is thought to involve abnormalities in the production and/or catabolism of IgA.
  • Some polymorphism evidence suggests that genetic factors determine the susceptibility to and rate of progression of IgAN.
  • genes involving blood pressure regulation especially a polymorphism for angiotensin converting enzyme, have been found to be associated with disease progression (Schmidt et al, Ann Med Interne (Paris) (1999) 150(2):86-89 and Chen et al, Chin MedJ(Engl) (1997) 110(7):526-529).
  • IgAN pathogenesis While some studies have focused on genetic factors, others have attempted to characterize IgAN pathogenesis by examining gene expression using RT-PCR and/or Northern blotting. For example, as cytokines have been found to play a relevant role in the pathogenesis of IgAN, gene expression of proinflammatory cytokines, immuno-regulatory cytokines and growth factors have been analyzed (Yano et al, J Clin Immunol (1997) 17(5):396-403). In renal tissue, mRNA for IL-l ⁇ and PDGF-B have both been shown to be increased in biopsies from IgA patients. Further, expression of TGF- ⁇ l (Yang et al, Nephron (1997) 77(3):290-297), ⁇ , ⁇ -T cell receptor V regions (Olive et al, Kidney Int
  • SIRS Systemic inflammatory response syndrome
  • ARDS respiratory distress syndrome
  • Hensel et al Anesthesiology (1998) 89(1):93-104)
  • preterm labor syndrome Dudley., Am J Obstet Gynecol (1999) 180:(1 Pt 3):S251-256
  • intraperitoneal hyperthermic perfusion Sumida et al, Anest Analg (1999) 88(4):771-776
  • Human epididymal proteins represent a near ubiquitous mammalian family of proteins which have been localized in epididymal epithelium, within the lumen of the epididymal duct and vas deferens, and also on the surface of spermatozoa (Ellerbrock et al, Int JAndrol (1994) 17(6):314-323; Uhlenbruck et al, Int JAndrol (1993) 16(1):53-61; Pera et al, IntJ Androl (1994) 17(6):324-330; Xu et al, Arch Androl (1996) 37(2):135-141). As is the case in other species, a link to a specific function for human epididymal proteins such a maturation or storage has not be conclusively demonstrated.
  • HE4 sequence has been identified in nucleic acid samples from diseased kidney biopsies.
  • the diagnosis of many renal diseases, such as IgAN continue to rely on highly invasive techniques (i.e., renal biopsy) to establish histo logical involvement as there is no sufficiently reliable blood or urine test for diagnosis.
  • highly invasive techniques i.e., renal biopsy
  • demonstration of such expression in nucleic acid samples obtained from living patients can serve as a diagnostic for detection of IgAN, necrotizing crescentic glomerulonephritis (NCGN) and minimal change disease, potentially replacing present invasive modalities.
  • cells in which HE4 can be induced can be used to screen compounds which modulate the expression of this gene.
  • the novel HE4 sequence may be a protective response and therefore its regulation therapeutically could ameliorate damage to the kidneys. More broadly, because many progressive disease responses reflect an unhealthy physiologic and biochemical imbalance as opposed to a necessary state of homeostasis for health, either lowered or elevated HE4 production in general or lowered or elevated HE4 production outside a range that is homeostatically healthy could be deleterious. Therefore, measuring the level of HE4 could provide important diagnostic and prognostic information. Relatedly, modulating HE4 therapeutically up or down could provide a significant means of treating diseases in which HE4 plays a role.
  • the novel HE4 sequence is also expressed in salivary gland, tests, lung, utems, trachea, prostate, thyroid, heart, pancreas, colon, and other tissues (see Figure 4). Therefor, it might play a significant role in disease processes associated with these tissues, particularly disease processes that involve inflammation and scarring.
  • the invention includes an isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO:2; (b) an isolated nucleic acid molecule comprising SEQ ID NO:l; and (c) an isolated nucleic acid molecule which encodes an HE4 protein containing a substitution or deletion of at least one amino acid corresponding to residue 71, 73 or 102 of sequence of SEQ ID NOs: 3 and 4.
  • the invention also includes vectors containing the nucleic acid molecules, host cells transformed with these nucleic acid molecules and recombinant methods of expressing the encoded proteins.
  • the invention also includes an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2 and an HE4 protein containing a substitution or deletion of at least one amino acid corresponding to residue 71, 73 or 102 of sequence of SEQ ID NOs: 3 and 4.
  • the invention includes methods of detecting HE4 in patient fluid or tissue samples as well as methods for screening for agents which modulate the expression or function of HE4.
  • FIG. 1 Autoradiographic image of the distribution of HE4 (JT22962) expression in various human tissues determined by Northern blot analysis.
  • FIG. 1 Graphic representation of HE4 expression in renal biopsy samples. RT-PCR is performed on nucleic acids from the biopsied renal tissues listed. Relative expression is determined by comparing the amount of HE4 PCR product in any given sample to that of GAP-DH expression.
  • FIG. 3 Expression profile of HE4 sequence in renal disease using Q-RT-PCR.
  • RT- PCR is performed on nucleic acids from minimum change disease; mild, moderate and severe forms of IgAN; and NCGN.
  • Relative expression is determined by comparing the amount of HE4 PCR product in any given sample to that of GAP-DH expression.
  • FIG. 4 Graphic representation of HE4 expression in various tissues. RT-PCR is performed on nucleic acids from the tissues listed. Relative expression is determined by comparing the amount of the HE4 PCR product in any given sample to that of GAP-DH expression.
  • the present invention is based in part on identifying genes that are differentially regulated or expressed in human diseased kidney tissue compared to normal kidney tissue.
  • a novel HE4 sequence has been identified that is differentially regulated in inflammatory and renal diseases and whose expression can be correlated, for example, with minimal change disease, IgAN (including correlation with degrees of severity of the disease), and NCGN.
  • monitoring of expression is considered to be indicative of treatment efficacy as well as predictive of end stage renal disease development.
  • the gene, as well as the peptides they encode, can serve as targets for agents that can be used to modulate the activity of HE4.
  • agents may be identified which modulate biological processes associated with injury to the kidney such as mesangial cell injury by cytokine/macrophage infiltration. Agents may also be identified which modulate the biological processes associated with recovery from IgA injury to the kidney.
  • Sclerosis refers to a thickening or hardening of a body part. Sclerosis also refers to a disease characterized by this thickening or hardening.
  • IgA Nephropathy or Berger's Disease refers to a group of disorders featuring recurrent episodes of macroscopic hematuria (blood in urine), mild proteinuria (protein in urine), glomerular changes, with or without progression to renal failure.
  • Minimal Change Disease refers to a disorder of the kidneys which largely affects the glomerulus, the blood filtering structure. This disorder is one common cause of nephrotic syndrome in children affecting 2 to 3 children per 100,000 population under age 16 in the U.S. Minimal change disease is also seen rarely in adults. The cause is unknown but may be related to an autoimmune illness. Presently, minimal change disease can only be diagnosed by renal biopsy.
  • Non-alcoholic Crescentic Glomerulonephropathy refers to a relatively uncommon form of acute glomerulonephritis that results in damage within the glomerulus of the kidney. There is rapid loss of kidney function with the formation of crescents on microscopic analysis (kidney biopsy). This disorder may result in acute glomerulonephritis or nephrotic syndrome and ultimately results in renal failure and end-stage renal disease.
  • Mesangial cells refer to cells found within the glomerular lobules of mammalian kidney, where they serve as structural supports, may regulate blood flow, are phagocytic and may act as accessory cells, presenting antigen in immune responses.
  • Ischemia herein refers to a decrease in blood supply to a body organ, tissue or part caused by constriction or obstmction of blood vessels.
  • Modulate refers to the inhibition, induction, agonism and/or antagonism of the expression or function of an HE4 gene or gene product.
  • Nucleic acid includes DNA and RNA molecules and is used synonymously with the terms “nucleic acid sequence” and “polynucleotide.”
  • Transcriptional factors refer to a class of proteins that bind to a promoter or to a nearby sequence of DNA to facilitate or prevent transcription initiation.
  • Polypeptide refers to an amino acid sequence including, but not limited to, proteins and protein fragments, naturally derived or synthetically produced.
  • Transcriptional profiling refers to any assay method or technique which is capable of analyzing, quantitatively and/or qualitatively, one or more mRNA species found in a cell or a nucleic acid sample.
  • assays include but are not limited to RT-PCR, quantitative PCR (Q-PCR), RNase protection assays, subtractive hybridization, READS and Northern blots.
  • WAP Whey Acidic Protein
  • HE4 identified in IgAN renal tissue has two WAP domains:Gly-Phe-Thr-Leu-Val-Ser-Gly-Thr-Gly-Ala-Glu-Lys-Thr-Gly-Val-Cys-Pro-Glu- Leu-Gln-Ala-Asp-Gln-Asn-Cys-Thr-Gln-Glu-Cys-Val-Ser-Asp-Ser-Glu-Cys-Ala-Asp-Asn- Leu-Lys-Cys-Cys-Ser-Ala-Gly-Cys-Ala-Thr-Phe-Cys (WAP I) (amino acids 21 to 70 of SEQ ID NOs:
  • Epididymal proteins are known proteins whose function has not been firmly established. However they are thought to perform a maturation function in the epididymis of mammals and may be extracellular proteinase inhibitors based on sequence similarity of proteins having such a function in genital tract mucous secretions (Kirchoff et al, 1991).
  • Attributes characteristic of HE4 and stmctural homologues of epididymal proteins in other species are: (i) they possess two whey acid protein domains; (ii) they belong to the "four disufide core" family of proteins; (iii) they can be found and isolated from epididymal epithelium, within the lumen of the epididymal duct and vas deferens, and also on the surface of spermatozoa; and (iv) they are glycosylated (Kirchoff et al, 1991).
  • HE4 and its stmctural homologues share extensive homology (See, e.g., Ellerbrock et al.1991 ; Uhlenbruck et al, 1993; Pera et al, 1994; XU et al, 1996).
  • probes from HE4 DNA can be used to screen Northern blots containing boar, bull and stallion mRNA to identify structurally homologous epididymal gene products.
  • antibodies recognizing HE4 proteins have been shown to react specifically in the epididymis of the boar and the bull (Uhlenbruck et al, 1993).
  • homology or identity can be determined by BLAST (Basic Local
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • HE4 DNA comprises the nucleic acid sequence as set forth in SEQ ID NO:l, wherein said DNA encodes the amino acid sequence as set forth in SEQ ID NO:2.
  • HE4 DNA is not limited to the sequences defined by SEQ ID NO: 1 , and includes those nucleic acid sequences which encode the present novel HE4 sequence which differs from the HE4 of Kirchoff et al. (SEQ ID NO: 3).
  • HE4 DNA encodes a polypeptide comprising 124 amino acids as set forth in SEQ ID NO:2 versus 125 amino acids as shown in Kirchoff et. al (1991) (SEQ ID NOs: 3 and 4).
  • HE4 DNA encodes a polypeptide comprising a Ser-71 (as set forth in SEQ ID NO:2) where Leu is present at the equivalent position of the encoded HE4 of Kirchoff et al (1991) (SEQ ID NOs: 3 and 4). Still further, between the equivalent Leu-72 and Pro-73 of SEQ ID NO:2, the HE4 of Kirchoff et al. has a cystinyl residue. Still further, the Ser-101 of the SEQ ID NO:2 is occupied by a threonyl residue in the HE4 of Kirchoff et al (1991) (SEQ ID NOs: 3 and 4). Any HE4 sequence, however, may be used in the methods of the invention, including natural variants, amino acid sequence variants, etc.
  • One means of diagnosing renal disease using the nucleic acids of the present invention involves obtaining kidney tissue from living subjects. Obtaining tissue samples from living sources is not problematic for tissues such as kidney as biopsy has become routine for certain procedures. For example, such procedures are used for dialysis and during transplant typing. (Yang et al, 1997).
  • diagnosing renal disease can be performed using body fluids.
  • HE4 protein or nucleic acid may be monitored in non-renal, accessible tissue (e.g., blood) and other body fluids which comprise renal filtrates (e.g., urine).
  • blood can be isolated and analyzed by transcriptional profiling to determine if HE4 expression has been modulated relative to a control.
  • IgAN can be accompanied by pyuria (i.e., the production of urine which contains white blood, see Ibels LS, et al, Medicine (Baltimore) (1994) 63(l):269-276), these cells may also be used to assay for HE4 levels.
  • Such a determination may then be used to differentiate between urinary tract infection, tuberculosis, cancer and acute glomerulonephritis or severity of IgAN observed (e.g., mild, moderate, severe).
  • rDNA molecules Containing a Nucleic Acid Molecule The present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence.
  • a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989).
  • a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.
  • vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the stmctural gene included in the rDNA molecule.
  • Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
  • the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
  • the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • vector plasmids Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N. J.
  • Expression vectors compatible with eukaryotic cells can also be used to form a rDNA molecules that contains a coding sequence.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided cont- ⁇ -ing convenient restriction sites for insertion of the desired DNA segment.
  • Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al, J. Mol. Anal. Genet. (1982) 1:327-341).
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.
  • the present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described.
  • the production of a recombinant form of a protein typically involves the following steps:
  • nucleic acid molecule that encodes a protein of the invention, such as the nucleic acid molecule having SEQ ID NO: 1 or nucleotides 23-397 of SEQ ID NO: 1. If the encoding sequence is unintermpted by introns, it is directly suitable for expression in any host.
  • the nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
  • the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • Each of the foregoing steps can be done in a variety of ways.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein.
  • Another embodiment of the present invention provides methods for identifying agents that modulate the expression of HE4.
  • Such assays may utilize any available means of monitoring for changes in the expression level of HE4 mRNA.
  • an agent is said to modulate the expression of HE4, if it is capable of up- or down-regulating expression of an appropriate nucleic acid in a cell.
  • cell lines that contain reporter gene in-frame fusions between the HE4 promoter and any assayable fusion partner may be prepared.
  • cell which express HE4 physiologically may be used.
  • Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a protein of the invention, for example, a protein having SEQ ID NO:2. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention.
  • Cell lines are exposed to the agent to be tested under appropriate conditions and time periods, after which total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed.
  • Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from HE4 nucleic acids. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and the potential probe:non-target hybrids.
  • Probes may be designed from HE4 nucleic acids through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly known, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, 1989) or Ausubel et al. (Current Protocols in Molecular Biology, Greene Publishing Co., NY, 1995).
  • “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium citrate/0.1% SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine semm albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 rnM sodium citrate at 42°C.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine semm albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 rnM sodium citrate at 42°C.
  • Another example is use of 50% formamide, 5 x SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2 x SSC and 0.1% SDS.
  • a skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. Hybridization conditions are modified using known methods, such as those described by Sambrook et al.
  • Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the nucleic acids encoding HE4 under conditions in which the probe will specifically hybridize.
  • nucleic acid fragments comprising at least one, or part of one of the nucleic acids encoding HE4 can be affixed to a solid support, such as a silicon wafer or a porous glass wafer.
  • the wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize.
  • a solid support such as a silicon wafer or a porous glass wafer.
  • Such glass wafers and hybridization methods are widely available, for example, those disclosed by Beattie (WO 95/11755).
  • Silicon wafers and hybridization methods are also widely available, for example, those disclosed by Rava et al (U.S. Patent No: 5,874,219).
  • Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using a RNase Protection Assay (i.e., RPA, see Ma et al, Methods (1996) 10:273-
  • RPA RNase Protection Assay
  • an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription.
  • a phage specific DNA dependent RNA polymerase promoter e.g., T7, T3 or SP6 RNA polymerase
  • the labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45 °C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA.
  • the resulting hybrids are then digested in a buffer comprising 40 ⁇ g/ml ribonuclease A and 2 ⁇ g/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.
  • cells or cell lines In another assay format for identification of agents which effect the expression of the instant gene products, cells or cell lines would first be identified which express said gene products physiologically. Cell and or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agents with appropriate surface transduction mechanisms and or the cytosolic cascades.
  • such cells or cell lines may be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) comprising an operable non-translated 5'-promoter containing end of the stmctural gene encoding the instant gene product fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag.
  • an expression vehicle e.g., a plasmid or viral vector
  • an operable non-translated 5'-promoter containing end of the stmctural gene encoding the instant gene product fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptide
  • the agent comprises a pharmaceutically acceptable excipient and is contacted with cells comprised in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising semm or conditioned media comprising PBS or BSS and/or semm incubated at 37 °C .
  • PBS phosphate buffered saline
  • BSS Eagles balanced salt solution
  • PBS or BSS comprising semm or conditioned media comprising PBS or BSS and/or semm incubated at 37 °C .
  • Said conditions may be modulated as deemed necessary by one of skill in the art.
  • the cells will be dismpted and the polypeptides of the dismptate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot).
  • immunological assay e.g., ELISA, immunoprecipitation or Western blot.
  • the pool of proteins isolated from the "agent contacted” sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent contacted” sample compared to the control will be used to distinguish the effectiveness of the agent.
  • the agents identified by the above methods can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents.
  • tissues may be assayed under conditions which model IgAN, NCGN, minimal change disease, SIRS or another inflammatory disease, particularly inflamamatory diseases of the lungs.
  • IgAN model systems include incubation of cultured cells with IL-1 and 11-6 (Lin et al, JLab Clin Med (1999) 133(l):55-63). Further, exposure of cells to ethanol (Smith et al, Alcohol (1993) 10(6):477-480) or gliadin (Amore et al, Am J Kidney Dis (1994) 23(2):290-301) have been shown to be adequate models for IgAN.
  • assays which incubate cells under conditions that simulate IgAN in vitro include, but are not limited to, for example, incubation of mesangial cells with cytokines, growth factors, antigens or primary alcohols.
  • Assays which simulate IgAN in vivo include, but are not limited to oral immunization in animal models (Amore et al, 1994 and Trachtman et al, 1996), including oral administration of vomitoxin (Yan et al, Food Chem Toxicol (1998) 36(12):1095-1106) or gram-negative bacteria (or LPS, See Endo et al, Nephron (1993) 65(2): 196-205).
  • NCGN can be simulated in vivo and in vitro by stimulation of rats or isolated mesengial cells with specific cytokines (e.g., INF- ⁇ ; see Coers et al.). Further, NCGN can be simulated by renal ischemia (see Brouwer et al, Kidney Int (1995) 47(4): 1121- 1129) or by immunization with myeloperoxidase (Brouwer et al, JExp Med (1993) 177(4): 905 -914).
  • specific cytokines e.g., INF- ⁇ ; see Coers et al.
  • cytokines e.g., INF- ⁇
  • NCGN can be simulated by renal ischemia (see Brouwer et al, Kidney Int (1995) 47(4): 1121- 1129) or by immunization with myeloperoxidase (Brouwer et al, JExp Med (1993) 177(4): 905 -914).
  • SIRS is simulated by exercise (Iannoli et al, Adv Exp Med Biol (1997) 428:333-341) or by modulation of inflammatory cytokines similar to that seen for IgAN (e.g., IL-6, IL-8 and TGF ⁇ modulation).
  • minimal change disease can be simulated in vivo and in vitro by treatment of rats or rat podocytes with puromycin aminonucleoside (Bertram et al, Cell Tissue Res (1990) 260(3):555-563) or by removing ⁇ 2-6 linked sialic acids from glomerular filtration barriers (Gelberg et al, Lab Invest (1996) 74(5):907-920).
  • Candidate peptide agents can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instmmentation and produced recombinantly using standard recombinant production systems (e.g., PCR and cloning). Peptide production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included in the candidate peptide product.
  • Another embodiment of the present invention includes methods for identifying agents that modulate at least one activity of the HE4 protein. Such methods or assays may utilize any means of monitoring or detecting the desired protein(s).
  • the relative amounts of protein between a cell population exposed to an agent to be tested compared to an un-exposed control cell population may be assayed.
  • probes such as specific antibodies are used to monitor the differential expression of the protein(s) in different cell populations.
  • Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
  • Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.
  • Antibodies directed against some HE4 polypeptides are known in the art (see Uhlenbruck et. al, 1993) and such antibodies may be used as probes. Further, antibodies against HE4 protein, for instance the protein of SEQ ID NO:2, may be prepared by immunizing suitable mammalian hosts using the peptides, polypeptides or proteins alone or conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation.
  • Preferred peptides for preparing antibodies include sequences spanning amino acids 71, 73 or 101 of SEQ ID NO.:2 or 4.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the gene products can also be produced in the context of chimeras with multiple species origin.
  • the HE4 specific antibody can be humanized antibodies or human antibodies, as described in U. S. Patent No. 5,585,089 by Queen et al. See also Riechmann et al, Nature (1988) 332: 323-27.
  • Agents which modulate the binding activity of the protein are identified by assaying the binding activity of the protein from the exposed cell line or population and a control, unexposed cell line or population, thereby identifying agents which modulate the binding activity of the protein. Binding assays to measure the ability of the agent to modulate the binding activity of proteins are widely available such as the assays disclosed by Morris et al, Oncogene (1991) 6(12):2339-48 and Cibelli et al, EurJBiochem (1996) 237(1):311-7.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the an HE4 protein alone or with its associated substrates, binding partners, etc.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of HE4 proteins. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies a sdescribed above.
  • the proteins and nucleic acids of the invention are expressed in various tissues including differential expression in renal disease.
  • Agents that modulate, up-or-down-regulate the expression of the protein or agents such as agonists or antagonists of at least one activity of the protein may be used to modulate biological and pathologic processes associated with the protein's function and activity.
  • modulating HE4 therapeutically up or down could provide a significant means of treating diseases in which HE4 plays a role.
  • the novel HE4 sequence is also expressed in salivary gland, tests, lung, uterus, trachea, prostate, thyroid, heart, pancreas, colon, and other tissues (see Figure 4). Therefor, it might play a significant role in disease processes associated with these tissues, particularly disease processes that involve inflammation and scarring.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of the invention.
  • the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect.
  • expression of a protein of the invention may be associated with kidney cell growth regeneration and/or recovery from kidney disease.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • kidney damage or ESRD may be prevented or disease progression modulated by the administration of agents which up-regulate or modulate in some way the expression or at least one activity of a protein of the invention.
  • agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process.
  • an agent of the present invention can be administered in combination with other known drugs or may be combined with dialysis or anti-rejection drugs used during transplantation.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt. The preferred dosages comprise 0.1 to 10 ⁇ g/kg body wt. The most preferred dosages comprise 0.1 to 1 ⁇ g/kg body wt.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, symps or inhalations and controlled release forms thereof.
  • the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
  • the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
  • the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
  • Another embodiment of the present invention provides methods for isolating and identifying binding partners of proteins of the invention.
  • a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention.
  • peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture.
  • the binding partner that bound to the protein of the invention can then be removed and further analyzed.
  • the entire protein for instance a protein comprising the entire amino acid sequence of SEQ ID NO: 2 can be used.
  • a fragment of the protein can be used.
  • Another embodiment includes protease inhibitor assays.
  • HE4 can be tested against a panel of proteases; e.g., ficin, papain, cathepsin H, cathepsin B (see Minakata et al, Biol Chem Hoppe Seyler (1985) 366(1):15-18 or elastase (Moriyama et al, Mol Hum Reprod (1998) 4(10):946-950). Briefly, the protease is incubated with its protease substrate in the presence and absence of HE4 protein or polypeptide.
  • N ⁇ -benzoyl-DL arginine-2-napthylamide (substrate) is incubated under appropriate conditions in the presence and absence of HE4.
  • relative inhibition is determined by absorbance at 530 nM (Minakata et al, 1985).
  • a cellular extract refers to a preparation or fraction which is made from a lysed or dismpted cell.
  • the preferred source of cellular extracts will be cells derived from human kidney tissue, for instance, renal biopsy tissue or tissue culture cells.
  • cellular extracts may be prepared from normal human kidney tissue or available cell lines, particularly kidney derived cell lines.
  • a variety of methods can be used to obtain an extract of a cell.
  • Cells can be disrupted using either physical or chemical dismption methods.
  • physical dismption methods include, but are not limited to, sonication and mechanical shearing.
  • chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis.
  • a skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.
  • the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur.
  • conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell.
  • Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.
  • the bound complex is separated from the mixture.
  • techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used.
  • the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
  • the protein of the invention can be immobilized on a solid support.
  • the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract.
  • the identified binding partners can be either a single protein or a complex made up of two or more proteins.
  • binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al, Methods Mol Biol (1997) 69:171-84 or Sauder et al, J Gen Virol (1996) 77(5):991-6 or identified through the use of epitope tagged proteins or GST fusion proteins.
  • the nucleic acid molecules of the invention can be used in a yeast two- hybrid system.
  • the yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.
  • Transgenic animals containing mutant, knock-out or modified genes corresponding to HE4 or the cDNA sequence of SEQ ID NO:l are also included in the invention.
  • Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene".
  • the nucleic acid sequence of the transgene in this case a form of SEQ ID NO: 1 , may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No.
  • mice A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S.
  • Patent No. 5,602,307 possess a bovine growth hormone gene (Clutter et al, Genetics (1996) 143(4):1753-1760); or, are capable of generating a fully human antibody response (McCarthy The Lancet (1997) 349(9049):405).
  • mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
  • Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al, Mol ReprodDev (1997) 46(4):515-526; Houdebine Reprod Nutr Dev (1995) 35(6):609-617; Petters Reprod Fertil Dev (1994) 6(5):643-645; Schnieke et al, Science (1997) 278(5346):2130-2133; and Amoah J.4n.ma/ Science (1997) 75(2):578-585).
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules.
  • Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
  • Total cellular RNA was prepared from the kidney tissue described above as well as from control, non-IgAN kidney tissue using the procedure of Yang et al, 1997
  • cDNA was synthesized according to the protocol described in the GIBCO/BRL kit for cDNA synthesis.
  • the reaction mixture for first-strand synthesis included 6 ⁇ g of total RNA, and 200 ng of a mixture of anchored oligo(dT) primers degenerate at nl
  • reaction mixture may include lO ⁇ g of total RNA, and 2 pmol of 1 of the 2 base-anchored oligo(dT) primers with a heel such as RP5.0 (CTCTCAAGGATCTTACCGCT(T) 18 AT) (SEQ ID NO: 6), or
  • RP6.0 (TAATACCGCGCCACATAGCA(T) 18 CG) (SEQ ID NO: 7), or RP9.2 (CAGGGTAGACGACGCTACGC(T) 18 GA) (SEQ ID NO: 8) along with other components for first-strand synthesis reaction except reverse transcriptase.
  • This mixture was then layered with mineral oil and incubated at 65 °C for 7 min followed by 50 °C for another 7 min.
  • 2 l of SUPERSCRIPT REVERSE TRANSCRIPTASE® 200 units/ ⁇ l; GIBCO/BRL was added quickly and mixed, and the reaction continued for 1 hr at 45-50°C.
  • Second-strand synthesis was performed at 16°C for 2 hr.
  • oligonucleotide A2 was first phosphorylated at the 5 ' end using T4 polynucleotide kinase (PNK). After phosphorylation, PNK was heat denatured, and l ⁇ g of the oligonucleotide Al was added along with 10 ⁇ annealing buffer (1 M NaCl/100 mM
  • the following sets of primers were used for PCR amplification of the adapter ligated
  • oligonucleotide Al or Al.l was 5' -end-labeled using 15 ⁇ l of [ ⁇ - 32 P]ATP (Amersham; 3000 Ci/mmol) and PNK in a final volume of 20 ⁇ l for 30 min at 37°C. After heat denaturing PNK at 65 °C for 20 min, the labeled oligonucleotide was diluted to a final concentration of 2 ⁇ M in 80 ⁇ l with unlabeled oligonucleotide Al.l.
  • PCR This "hot start” PCR was done to avoid artefactual amplification arising out of arbitrary annealing of PCR primers at lower temperatures during transition from room temperature to 94°C in the first PCR cycle.
  • PCR consisted of 5 cycles of 94°C for 30 sec, 55°C for 2 min, and 72°C for 60 sec followed by 25 cycles of 94°C for 30 sec, 60°C for 2 min, and 72° C for 60 sec. A higher number of cycles resulted in smeary gel patterns.
  • PCR products (2.5 ⁇ l) were analyzed on 6% polyacrylamide sequencing gel.
  • SEQ. ID NO:l is the full-length cDNA sequence which corresponds to the differentially expressed band of Figure 5.
  • SEQ ID NO:2 is the amino acid sequence of the encoded HE4 protein.
  • Northern blot and PCR Expression Analysis are prepared using a probe derived from SEQ ID NO:l with hybridization conditions as described by Sambrook et al. (1989), see Figure 1.
  • Biopsy tissue is obtained from normal and diseased kidneys, wherein extracted tissues are lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al.
  • Real time PCR detection is accomplished by the use of the ABI PRISM 7700 Sequence Detection System. The 7700 measures the fluorescence intensity of the sample each cycle and is able to detect the presence of specific amplicons within the PCR reaction.
  • Each sample is assayed for the level of GAPDH and clone JT22962 (HE4).
  • GAPDH detection is performed using Perkin Elmer part#402869 according to the manufacturer's directions.
  • Primers are designed for clone JT22962 (HE4) using Primer Express, a program developed by PE to efficiently find primers and probes for specific sequences. These primers are used in conjunction with SYBR green (Molecular Probes), a nonspecific double stranded DNA dye, to measure the expression level of clone JT22962 (HE4), which is normalized to the GAPDH level in each sample (see Figure 2 and Figure 3).
  • SYBR green Molecular Probes
  • Tissue Distribution Tissues are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. PCR is carried out as described above. Quantative expression data was not normalized to GAPDH in each sample (see Figure 3).
  • Example 3 Method of Screening for Modulators of Kidney HE4 Expression Using IL-1 and IL-6 Using human cultured mesangial cells, IL-1 and 11-6 are administered to cells in culture according to the method of Lin et al. (1999). At specific time points pre- or at post- administration, candidate agents and diluent (i.e., carrier minus agent; control) are contacted with the human mesangial cells. Cells are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling means to determine whether the candidate agent modulates the expression of HE4. Agents which up- or down-regulate the expression of HE4 are then designated as modulators of the gene.
  • candidate agents and diluent i.e., carrier minus agent; control
  • Isolated nucleic acids are then assayed by a transcriptional
  • Ethanol is administered to cultured contractile mesangial cells in culture according to the method of Smith et al (1993).
  • candidate agents and diluent i.e., carrier minus agent; control
  • control i.e., carrier minus agent
  • Control and test cells are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. (1989).
  • Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4.
  • Agents which up- or down- regulate the expression of HE4 will then be designated as modulators of the gene.
  • Gliadin is administered to cultured contractile mesangial cells in culture according to the method of Amore et al (1994).
  • candidate agents and diluent i.e., carrier minus agent; control
  • control i.e., carrier minus agent
  • Control and test cells are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. (1989).
  • Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4.
  • Agents which up- or down- regulate the expression of HE4 will then be designated as modulators of the gene.
  • IgAN Oral Immunization animal models for IgAN are well documented (Amore et al, 1994; Endo et al, 1993; Yan et al, 1998; and Tractman et al, 1996).
  • IgAN is induced in animals by oral administration of LPS (Endo et al, 1993), bovine gamma globulin (Trachtman et al, 1996), or vomitoxin (Yan et al, 1998).
  • gliadin maybe used (Amore et al, 1994).
  • agents are administered to the animals (including carrier-only for controls).
  • kidneys of the animals are removed for isolation of nucleic acids by standard methods as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4. Agents which up- or down-regulate HE4 will then be designated as modulators of the gene.
  • ddY mice A spontaneous model for IgAN exists in mice (e.g., ddY mice, see, Nakamura et al, 1992). Agents are administered to ddY mice at various time points or at various concentrations at a single time point. The kidneys of the animals are then removed for isolation of nucleic acids by standard methods as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4. Agents which up- or down- regulate HE4 will then be designated as modulators of the gene.

Abstract

L'invention concerne de manière générale des changements dans l'expression génique dans des maladies inflammatoires et rénales, telles que la néphropathie à dépôts mésangiaux d'IgA et la glomérulonéphrite à croissants épithéliaux nécrosante. L'invention concerne tout particulièrement une nouvelle séquence de HE4, identifiée dans un tissu rénal atteint de néphropathie à dépôts mésangiaux d'IgA. L'invention concerne encore des procédés de diagnostic de maladies inflammatoires et rénales, telle que la néphropathie à dépôts mésangiaux d'IgA, ces procédés consistant à mesurer les taux d'HE4 dans des échantillons d'acide nucléique ou cellulaires prélevés sur des patients. L'invention concerne enfin un procédé de criblage de modulateurs de HE4.
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