WO2000023100A2

WO2000023100A2 - Genes and proteins predictive and therapeutic for renal disease and associated disorders

Info

Publication number: WO2000023100A2
Application number: PCT/US1999/024723
Authority: WO
Inventors: Richard A. Shimkets
Original assignee: Curagen Corporation
Priority date: 1998-10-22
Filing date: 1999-10-22
Publication date: 2000-04-27
Also published as: WO2000023100A8; DE69911226D1; CA2347625A1; AU1449500A; EP1124572B1; WO2000023100A3; JP2002527489A; EP1124572A2; ATE249236T1; HK1039279A1; WO2000023100A9

Abstract

The present invention discloses 16 genes that are differentially expressed in animal models of hypertension relative to non-hypertensive controls. In particular, 16 genes were identified which are differentially-expressed in the fawn-hooded rat (FHR) and the closely-related IRL rat strain, in comparison to the control ACI strain of rat. Also disclosed are therapeutic and prophylactic methods for treating or preventing renal failure, platelet storage-pool disease, hypertension, insulin-dependent diabetes mellitus (IDDM) and/or other associated diseases or disorders using these genes and nucleic acids encoding these genes. Methods for diagnosis, prognosis, and screening, by the detection of the differentially-expressed proteins and nucleic acids, as well as derivatives, fragments and analogs thereof, are also disclosed herein.

Description

GENES AND PROTEINS PREDICTIVE AND THERAPEUTIC FOR RENAL DISEASE AND ASSOCIATED DISORDERS

RELATED APPLICATIONS

This application claims priority to USSN 09/177,264, filed October 22, 1998, which is incorporated by reference in its entirety.

FIELD OF THE INVENTION The invention relates to nucleic acids and, more particularly, to genes expressed in renal disease.

BACKGROUND OF THE INVENTION

Hypertension, hyperlipidemia and diabetes mellitus affect hundreds of millions of individuals world-wide and account for a significant fraction of morbidity and mortality, particularly among older individuals. These conditions can increase the risk for the development of conditions such as end-stage renal disease (ESRD), coronary heart disease (CHD) and stroke. For example, more than 70% of ESRD has been shown to be associated with hypertension. Not all individuals suffering from hypertension, hyperlipidemia and diabetes mellitus develop ESRD, CHD or stroke. The underlying mechanism for this variability is currently unknown.

It has been suggested that the progression for ESRD, CHD or stroke is determined at least in part by one or more genetic factors.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of genes differentially expressed in an animal model of hypertension. Accordingly, disclosed herein is a set of 16 differentially- expressed genes [hereinafter "GENE SET"], as well as derivatives, fragments, analogs and homologs thereof, which were demonstrated to be differentially-expressed within awn-hooded rat (FHR) and IRL rodent models of renal disease, in comparison to control rat strain. These differentially-expresseα _foenes are illustrated in Table 1 and include: Zi. peptidase (Aminopeptidase N); RTl .B-1 (alpha) chain of the integral membrane protein; δ subunit of FIFO ATPase; keratin 19; brain calbindin-d28k (CaBP28K); the inhibitor protein of metalloproteinase 3 (TIMP-3); integral membrane protein 1 (Itml); isovaleryl-CoA dehydrogenase (IVD); rab GDI-β; IRPR (IFN-β); organic cation transporter (OCT2); bile mayaliculus domain-specific glycoprotein; L-arginine:glycine amidinotransferase; protein phosphatase 1-β (PPl-β); renal kallikrein and the p8 protein.

The present invention also discloses therapeutic and/or prophylactic methods for treating or preventing renal failure, platelet storage-pool disease, hypertension and/or other associated diseases or disorders.

Also disclosed by the present invention are methods for diagnosis, prognosis, and screening, by the detection of the differentially-expressed GENE SET proteins and nucleic acids, as well as derivatives, fragments and analogs thereof. Diagnostic, prognostic and screening kits are also disclosed herein. In an additional embodiment of the present invention, assays which screen for the therapeutic value of GENE SET proteins, nucleic acids and derivatives (and fragments and analogs thereof), as well as anti-GENE SET antibodies are also provided. Additionally, the present invention also discloses methods for the screening of modulators (i.e., activators or inhibitors) of the GENE SET protein or nucleic acid activity which affect renal disease, platelet storage-pool disease, hypertension and/or associated disease or disorders.

DESCRIPTION OF THE FIGURES

In order that the present invention disclosed herein is better understood and appreciated, the following detailed description is set forth.

Figure 1 : illustrates the differential-expression of IRPR (IFN-β) mRNA in the FHR, IRL and ACI (control) rodent strains at 8 and 32 weeks-of-age.

Figure 2: illustrates the confirmation of differential-expression of IRPR (IFN-β) mRNA in the FHR and ACI (control) rodent strains at 32 weeks-of-age by the Oligo Poisoning^™ methodology. Table 1 : A list of _feenes which were demonstrated be differential!., -expressed with the FHR and IRL strains of rat, in comparison to the control ACI strain. GenBank Accession Number, common name, and fold modulation relative to the controls are provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses the novel finding of a group of genes (hereinafter designated "GENE SET" genes) which are demonstrated to be differentially-expressed within experimental animal models (i.e., the FHR and IRL strains of rat) for renal failure. A total of 16 differentially-expressed GENE SET genes are characterized. In specific embodiments of the present invention, GENE SET proteins or nucleic acids (and derivatives, fragments or analogs thereof), as well as anti-GENE SET antibodies are utilized as therapeutics for the treatment or prevention of human platelet storage-pool disease, hypertension and, preferably, renal disease. These differentially-expressed genes are illustrated in Table 1 and include: Zn-peptidase (Aminopeptidase N): RTl .B-1 (alpha) chain of the integral membrane protein; δ subunit of FIFO ATPase; keratin 19: brain calbindin-d28k (CaBP28K); the inhibitor protein of metalloproteinase 3 (TIMP-3); integral membrane protein 1 (Itml) isovaleryl-CoA dehydrogenase (IVD); rab GDI-β; IRPR (IFN-β); organic cation transporter (OCT2); bile mayaliculus domain-specific glycoprotein; L-arginine:glycine amidinotransferase; protein phosphatase 1-β (PPl-β); renal kallikrein and the p8 protein. Additionally, pharmaceutical compositions are also disclosed herein.

Other embodiments of the present invention relate to methods of diagnosis, prognosis and screening for existing, or future impairment of renal function by the detection of differential expression of human nucleic acid or amino acid sequences which are homologous to the GENE SET for diagnostic purposes. In one specific embodiment, subjects are screened for dysregulation of the GENE SET genes.

The present invention also discloses methods of assaying the GENE SET for the ability to affect the predisposition to, or onset of. renal impairment and to methods of the screening of GENE SET modulators (i.e., agonists, antagonists and inhibitors of the GENE SET).

(1) Nucleic Acids and Encoded Proteins of the GENE SET

The GENE SET proteins (and derivative, fragments, analogs and homologs thereof) and the nucleic acids encoding the GENE SET proteins (and derivatives, fragments and analogs thereof) are provided b> _Lne present invention. GENE SET proteins an nucleic acids may be obtained by any methodology known within the art. The GENE SET amino acid and nucleotide sequences for, inter alia, human, rat. hamster, dog. mouse, bovine, porcine. Drosophila melanogaster, Xenopus. horse, and dogfish are available in various public-access databases (e.g., GenBank, EMBL, and the like).

In the practice of the present invention, any eukaryotic cell may potentially serve as the nucleic acid source for the isolation of GENE SET nucleic acids. These GENE SET nucleic acids may be obtained by standard procedures known within the art including, but not limited to: (/) chemical synthesis; (ii) by cDNA cloning or (Hi) by the cloning of genomic DNA, or fragments thereof, purified from the desired cell. See e.g., Sambrook. et al., 1989. Molecular Cloning, A Laboratory Manual, 2d Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Glover, 1985. DNA Cloning: A Practical Approach (MRL Press, Ltd., Oxford. U.K.). It should be noted however, that clones which are derived from genomic DNA may contain regulatory and non-coding (intronic) DNA regions in addition to coding (exonic) regions; whereas clones which are derived from complementary DNA (cDNA) will contain only coding, exonic sequences.

In the molecular cloning of a gene from cDNA, cDNA is generated from total cellular RNA, or mRNA, by methods that are well-known within the art. The gene may also be obtained from genomic DNA, where DNA fragments are generated (e.g., using restriction enzymes or by mechanical shearing), some of which will possess the desired genomic sequence. The linear DNA fragments may then be separated according to size by standard techniques including, but not limited to, agarose and polyacrylamide gel electrophoresis and size-exclusion chromatography.

Once the DNA fragments are generated, identification of the specific DNA fragment containing all or a portion of the GENE SET gene may be accomplished in a number of ways. A preferred methodology for isolating a GENE SET gene is by amplification by the polymerase chain reaction (PCR), which may be used to amplify the desired GENE SET sequence in a genomic or cDNA library or, alternately, from genomic DNA or cDNA which has not been incorporated into a library. Oligonucleotide primers that hybridize to GENE SET sequences may be utilized as primers in PCR-mediated amplification reactions. The nucleic acid sequences of the oligonucleotide primers that are utilized are dependent upon the sequence of the specific fragment to be amplified, and may be readily ascertained by one who is skilled within the art. Such synthetic ongonucleotides may be utilized as pπmers in PM-mediated amplification of sequences of interest (RNA or DNA) which are derived, preferably, from a cDNA library. PCR may be preformed, for example, by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp^®). In the practice of the present invention, one may elect to synthesize several different degenerate primers for use in the PCR reactions. It is also possible to vary the stringency of hybridization conditions utilized in priming the PCR reactions, thus allowing for greater or lesser degrees of nucleotide sequence homology between the known GENE SET nucleotide sequence, and the nucleic acid sequence of a GENE SET homolog being isolated. In specific embodiments, low stringency conditions are preferred for cross species hybridization; whereas for same species hybridization, moderately stringent conditions are preferable.

Following the successful amplification of a segment of a GENE SET homolog, that segment of interest may be molecularly-cloned and sequenced. and subsequently utilized as a probe in the isolation of a complete cDNA or genomic clone. This, in turn, will permit the determination and isolation of the gene's complete nucleotide sequence. Alternately, PCR amplification may also be utilized to detect and quantitate GENE SET mRNA levels (e.g., for use in the diagnostic, prognostic and screening methods described Section 4, infra).

In one embodiment of the present invention, a portion of the GENE SET gene (derived from any species), or its specific mRNA, or a fragment thereof, may be isolated and labeled. The generated DNA fragments may then be screened by nucleic acid hybridization to a labeled probe (see e.g., Benton & Davis, 1977. Science 196:180; Grunstein & Hogness, 1975. Proc. Natl. Acad. Sci. U.S.A. 72:3961) and those DNA fragments possessing substantial homology to the probe will hybridize. Alternately, in another embodiment, an oligonucleotide probe may be synthesized and labeled, and the generated DNA fragments may be screened by nucleic acid hybridization to the labeled oligonucleotide probe. In yet another embodiment, GENE SET nucleic acids may be also identified and isolated by expression-cloning using, for example, anti- GENE SET antibodies for the initial selection.

In alternative embodiments of the present invention, methods known within the art may be utilized to obtain GENE SET DNA by cloning or amplification. These methods include, but are not limited to: (/^') chemically synthesizing the gene sequence itself from the known GENE SET sequence; (//) generating a cDNA to the mRNA species which encodes the GENE SET protein and other methods within the scope of the invention. Once a clone has been obtained, its identity may be confirmed by nucleic acid sequencing, by any method well-known in the art, and compared to known GEnE SET sequences. DNA sequence analysis ..nods include, but are not limited to: (i) the chemical method (see e.g., Maxam & Gilbert. 1980. Meth. Enzymol. 65:499-560); (//^') the dideoxynucleotide chain-termination method (see e.g., Sanger, et al, 1911. Proc. Natl. Acad. Sci. U.S.A. 74:5463); (Hi) the use of T₇ DNA polymerase (see e.g., Tabor & Richardson, U.S. Patent No. 4J95.699); (iv) use of an automated DNA sequenator (e.g., Applied Biosystems; Foster City, CA) or (v) the method described in PCT Publication WO 97/15690. dated May 1, 1997 to Nandabalan. et al.

Nucleic acids, which are hybridizable to a GENE SET nucleic acid or to a nucleic acid encoding a GENE SET derivative, may be isolated by nucleic acid hybridization under conditions of low, high, or moderate stringency. By way of example, and not of limitation, hybridization procedures using such conditions of low stringency are as follows (see also e.g., Shilo & Weinberg, 1981. Proc. Natl. Acad. Sci. USA 78:6789-6792): filters containing immobilized DNA are pre-hybridized for 6 hours at 40°C in a solution containing: 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA. 0.1% PVP, 0.1% Ficoll, 1% BSA and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll. 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt vol) dextran sulfate and 5-20 x 10⁶ cpm ³²P-labeled probe. The filters are then incubated in hybridization mixture for 18-20 hours at 40°C, and washed for 1.5 hours at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS. The wash solution is then replaced with fresh solution and the filters are incubated an additional 1.5 hours at 60°C. The filters are blotted dry and autoradiographed. If necessary, the filters are washed for a third time at 65-68°C and re-exposed to X-ray film. Other conditions of low stringency hybridization, which are well-known in the art (e.g., as employed for cross- species hybridizations), may also be employed in the practice of the present invention. By way of example, and not of limitation, procedures using such conditions of moderate stringency hybridization are as follows: filters containing immobilized DNA are pre-hybridized for 6 hours at 55°C in a solution containing: 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Hybridizations are performed in the same solution and 5- 20 x 10⁶ cpm ³²P-labeled probe is used. The filters are incubated in hybridization mixture for 18- 20 hours at 55°C, and then washed twice for at 37°C for 1 hour in a solution containing 2X SSC, 0.1% SDS. The filters are then blotted dry and autoradiographed. Other conditions of moderate stringency, which are well-known within the art, may be employed in the practice of the present invention. Again, by way o_* example, and not of limitation, procedures us_^ such conditions of high stringency hybridization are as follows: pre-hybridization of filters containing immobilized DNA is performed for 8 hours to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris- HCl (pH 7.5), 1 mM EDTA. 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C in pre- hybridization mixture containing: 100 μg/ml denatured salmon sperm DNA and 5-20 x 10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is then followed by a third wash in 0.1X SSC at 50°C for 45 minuets prior to autoradiography. Other conditions of high stringency hybridization, which are well-known within the art, may also be utilized in the practice of the present invention.

Nucleic acids which encode derivatives and analogs of GENE SET proteins, GENE SET anti-sense nucleic acids and primers that may be utilized to detect GENE SET alleles and GENE SET gene expression are additionally disclosed by the present invention. GENE SET proteins (and derivatives, analogs and fragments of GENE SET proteins) may be obtained by any method known within the art including, but not limited to, recombinant expression methods, purification from natural sources and chemical synthesis. In one embodiment of the present invention. GENE SET proteins may be obtained by recombinant protein expression techniques; wherein the GENE SET gene of interest (or portion thereof) is ligated into an appropriate cloning vector for subsequent expression within a particular host cell. A large number of vector-host systems are known with in the art. and include, but are not limited to: bacteriophages (e.g., λ bacteriophage and derivatives) or bacterial plasmids (e.g., pBR322 or pUC plasmid derivatives or the Bluescript^® vector (Stratagene: La Jolla, CA)). The insertion into a cloning vector may, for example, be accomplished by ligating the DNA fragment into a cloning vector that possesses complementary, cohesive termini. However, if the complementary restriction site for the restriction endonuclease (RE) utilized to digest the insert DNA are not present within the cloning vector, the ends of the DNA molecules may be enzymatically modified (e.g., Klenow fragment of DNA polymerase I). Alternatively, any site desired may be produced by ligating oligonucleotide sequences (i.e., linkers) onto the DNA termini. Moreover, these ligated linkers may also comprise specific, chemically-synthesized oligonucleotides encoding RE recognition sequences. DNA sequence-specific DNA-binding protein binding sites, and the like. In an alternative embodiment of the present invention, the RE-digested vector and GENE SET gene of interest may be modified by homopolymeric tailing with terminal deoxynucleotidyl transK.ase (TdT). The recombinant molecules^* may . . n be introduced into host cells via transformation, transfection, infection, electroporation, etc.. so that a plurality of copies of the gene sequence are generated.

In an alternative embodiment, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot-gun^"" approach. Enrichment for the desired gene (e.g., by size fractionation) may be performed prior to the insertion of the sequence of interest into the cloning vector.

It should be noted that the molecular-cloning and expression of both cDNA and genomic sequences are within the scope of the present invention. In specific embodiments thereof, transformation of host cells with recombinant DNA molecules which incorporate the isolated

GENE SET gene, cDNA or synthesized DNA sequence enables generation of multiple copies of the gene. Accordingly, the GENE SET gene sequence may be obtained in large quantities by in vitro culture of the transformants. isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

The nucleotide sequence encoding a GENE SET protein (or a functionally-active analog, fragment or derivative thereof) may then be inserted into an appropriate expression vector (i.e., a vector which contains the necessary (exogenous) regulatory elements for the transcription and translation of the inserted protein-coding sequence). In an alternate specific embodiment, the required transcriptional and translational regulatory signals may be supplied by the native

(endogenous) GENE SET gene and or its flanking regions . A variety of host- vector systems may be utilized to express the protein-coding sequence including, but are not limited to: (/^') mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); (/ ) insect cell systems infected with virus (e.g., baculovirus); (///) microorganisms such as yeast containing yeast vectors or (iv) bacteria transformed with bacteriophage. DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities, and depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements may be utilized.

Similarly, any of the methods utilized for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods include, but are not limited to, in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequence encoding a GENE SET protein or . tide fragment may be regulated by a second . _cleϊc acid sequence such that the GENE SET protein (or derivative, fragment or analog) is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a GENE SET protein may be controlled by any promoter/enhancer element known within the an. In a specific embodiment of the present invention, the promoter is not native to the genes for the GENE SET proteins.

Promoters which may be utilized include, but are not limited to: (0 the SV40 early promoter (see e.g., Bernoist & Chambon, 1981. Nature 290:304-310); (ii) the promoter contained in the 3'- terminus long terminal repeat of Rous sarcoma virus (see e.g.. Yamamoto. et al., 1980. Cell 22:787-797); (///) the Herpesvirus thymidine kinase promoter (see e.g., Wagner, et al., 1981. Proc. Natl. Acad. Sci. USA 78:1441-1445); (iv) the regulatory sequences of the metallothionein gene (see e.g., Brinster, et al., 1982. Nature 296:39-42); (v) prokaryotic expression vectors such as the β-lactamase promoter (see e.g., Villa-Kamaroff, et al.. 1978. Proc. Natl. Acad. Sci. USA 75:3727-3731) or (vi) the tac promoter (see e.g., DeBoer, et al. 1983. Proc. Natl. Acad. Sci. USA 80:21-25. In addition, animal transcriptional control regions which exhibit tissue specificity and have been utilized in transgenic animals may also be utilized. These transcriptional control regions include, but are not limited to: (0 the elastase I gene control region which is active in pancreatic acinar cells (see e.g., Swift, et al., 1984. Cell 38:639-646: (//) the insulin gene control region which is active in pancreatic β-cells (see e.g., Hanahan. et al., 1985. Nature 315:115-122); (Hi) the immunoglobulin gene control region which is active in lymphoid cells (see e.g., Alexander, et al., 1987. Mol. Cell Biol. 7: 1436-1444); (iv) the mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (see e.g., Leder, et al., 1986. Cell 45:485-495); (v) the α-fetoprotein gene control region which is active in liver (see e.g., Krumlauf. et al., 1985. Mol. Cell. Biol. 5:1639-1648); (vi) the β-globin gene control region which is active in myeloid cells (see e.g., Kollias. et al., 1986. Cell 46:89-94) and (vii) the myosin light chain-2 gene control region which is active in skeletal muscle (see e.g., Sani, 1985. Nature 314:283-286).

In a specific embodiment of the present invention, a vector is utilized which comprises: (0 a promoter operably-linked to nucleic acid sequences encoding the GENE SET protein, or a fragment, derivative or homolog thereof; (//) one or more origins of replication and optionally, ( ) one or more selectable markers (e.g., an antibiotic resistance gene).

In a preferred embodiment of the present invention, a vector is utilized which comprises a promoter operably-linked to nucleic acid sequences encoding a GENE SET protein, one or more origins of replication, and one or more selectable markers. For example, in a specific embodiment, an express. n construct is made by subcloning a GENE - _^ T coding sequence into- the EcoRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith & Johnson. 1988. Gene 7:31-40), thus allowing the expression of the GENE SET protein product from the subclone in the correct reading frame. In specific embodiments of the present invention, expression vectors containing GENE

SET gene inserts may be identified by three general approaches: (/) nucleic acid hybridization. (ii) presence or absence of "marker^" gene functions, and (Hi) expression of inserted sequences. In the first approach, the presence of a GENE SET gene inserted in an expression vector may be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted GENE SET gene. In the second approach, the recombinant vector/host system may be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, and the like) caused by the insertion of a GENE SET gene in the vector. For example, if the GENE SET gene is inserted within the marker gene sequence of the vector, recombinants containing the GENE SET insert may be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors may be identified by assaying the GENE SET product expressed by the recombinant. Such assays may be based, for example, on the physical or functional properties of the GENE SET protein in in vitro assay systems (e.g., binding with anti-GENE SET antibody or the GENE SET receptor). Once a particular recombinant DNA molecule is identified and isolated, several methods known within the art may be utilized for propagation. Once a suitable host system and growth conditions are established, recombinant expression vectors may be propagated and prepared in quantity. As previously explained, the expression vectors which may be used include, but are not limited to, the following vectors or their derivatives: (/) human or animal viruses (e.g., vaccinia virus or adenovirus); (//^') insect viruses (e.g., baculovirus): (/ /) yeast vectors; (iv) bacteriophage vectors (e.g., lambda); (v) plasmid and cosmid DNA vectors and the like.

In a further embodiment, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific manner desired. Expression from certain promoters may be elevated in the presence of certain inducers; thus, expression of the genetically engineered GENE SET protein may be controlled.

Furthermore, different types of host cells possess characteristic and specific mechanisms for the translational and post-translational processing and modification (e g. , glycosylation, phosphorylation of proteins and the like). Appropriate cell lines or host systems may be chosen to ensure the desired modification and processing of the foreign proteii. .xpressed. For example-, expression in a bacterial system may be used to produce a non-glycosylated core protein product; whereas expression in yeast will produce a glycosylated product. Expression in mammalian cells may be used to ensure "native" glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect these processing mechanisms to varying extents. In other specific embodiments of the present invention, the GENE SET protein (or derivative, fragment or analog) may be expressed as a fusion, or chimeric protein product (i.e., comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence of a different protein). Such a chimeric product may be produced by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to one another by methods (in the proper coding frame), and expressing the chimeric product by methods which are well-known within the art. In an alternate embodiment, a chimeric product is generated by protein synthetic techniques (e.g., by use of a peptide synthesizer).

The GENE SET protein may also be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and partition chromatography), centrifugation, differential solubility or by any other standard technique for the purification of proteins known within the art. The functional properties of the isolated proteins may then be ascertained and evaluated by use of any suitable assay. Alternatively, the protein of interest may be synthesized by the numerous chemical methods known within the art (see e.g., Hunkapiller, et al., 1984. Nature 310:105-11 1).

In another alternate embodiment of the present invention, native GENE SET proteins may be purified from natural sources utilizing standard methods such as those described supra (e.g., immunoaffinity purification).

(2) Methods of Treatment of Renal Disease The present invention discloses methods of treating and preventing renal diseases

(primarily renal failure and, preferably, acute renal failure) and platelet storage-pool disease by the administration of one or more therapeutic compounds (hereinafter designated a "Therapeutic") of the present invention. In specific embodiments, the "Therapeutics" of the present invention may include and of the GENE SET proteins including: Zn-peptidase (Aminopeptidase N); RTl .B-1 (alpha) chain of the integral membrane protein; δ subunit of FIFO ATPase: keratin 19; brain calbindin-d28k (CaBP28K); the inhibitor protein of metalloproteinase 3 (TIMP-3); integral membrane protein 1 (Itml); isovaleryl-CoA dehydrogenase (IVD); rab GDI-β; IRPR (IFN-β); Oiganic cation transporter (OCT2); bile mayalic .us domain-specific!:' glycoprotein; L-arginine:glycine amidinotransferase: protein phosphatase 1-β (PPl-β); renal kallikrein and the p8 protein (and derivatives, fragments analogs and homologs thereof), as well as the nucleic acid sequences (i.e.. the GENE SET genes) encoding the proteins (and derivatives, fragments and analogs thereof).

In the practice of the present invention, the subject to which the Therapeutic is administered is preferably an animal, such as including, but not limited to, cows, pigs, horses, chickens, cats, dogs, etc. and is more preferably a mammal. In the most preferred embodiment of the present invention, the subject is a human. Generally, the administration of the products of a species origin or species reactivity (in the case of antibodies) which is derived from the same species as that of the subject, is a preferred embodiment. Thus, in a preferred embodiment, a human GENE SET protein (or derivative, fragment or analog thereof) or a nucleic acid (or derivative, fragment or analog thereof, including anti-sense nucleic acid sequences thereof) are therapeutically or prophylactically administered to a human patient. In a preferred embodiment. Therapeutics of the present invention are administered therapeutically, and preferably, prophylactically, to patients who are suffering from, or who are in danger of suffering from, renal failure or a renal disease, preferably, acute renal failure.

Therapeutics of the present invention may be administered either alone or in combination with other therapies (e.g., pharmaceutical compositions which are effective in the treatment or prevention of renal impairment). Therapeutics may also be concomitantly administered with drugs which treat or ameliorate the effect of certain risk factors, including, but not limited to, therapeutics which reduce cholesterol levels, treat obesity, treatment insulin-dependent and non- insulin-dependent diabetes mellitus (IDDM, NIDDM)) and the like. In a preferred embodiment of the present invention, a Therapeutic is administered with one or more anti-hypertensive drugs, including, but not limited to: (/^') sympatholytics (e.g., propranolol, atenolol, nadolol, labetalol, prazosin, terazosin, doxazosin, clonidine, gugeneacine, methyldopa, reserpine, etc.); (ii) angiotensin inhibitors (e.g., benazepril, captopril, enalapril. losartan); (Hi) calcium channel blockers (e.g., diltiazem, felodipine. isradipine, nifedipine. verapamil); (iv) diuretics (e.g., thiazides - such as bendioflumethiazide, benzthiazide and hydrocholorothiazide; loop diuretics - such as bumetanide, ethacrynic acid, furosemide. and torsemide; potassium-sparing diuretics - such as amiloride, spironolactone and triametrene and various other types of diuretics and vasodilators - such as hydralazine and minoxidil). It should also be noted that it is within the skill of those within ... art to monitor and adjust the treatment or prophylactic regimen for the treatment or prevention of renal disease, while concomitantly treating or preventing other, potentially associated diseases or disorders (e.g., hypertension).

(A) Gene Therapy

In a specific embodiment of the present invention, nucleic acids comprising a sequence encoding a GENE SET (or derivative, fragment or analog thereof) or a GENE SET anti-sense nucleic acid, are administered utilizing gene therapy methods. Gene therapy refers to a therapy which is performed by the administration of a specific nucleic acid (or derivative, fragment or analog) or an anti-sense nucleic acid, to a subject in need of such treatment. In the embodiment of the present invention, the nucleic acid produces its encoded protein or an anti-sense nucleic acid that mediates a therapeutic effect.

Any of the methods for gene therapy known within the art may be utilized in the practice of the present invention. For general reviews of the methods of gene therapy see e.g., Goldspiel, et al, 1993. Clin. Pharmacy .12:488-505; Wu & Wu, 1991. Biotherapy 3: 87-95; Mulligan, 1993. Science 260:926-932; .Wu & Wu, 1991. Biotherapy 3:87-95: Tolstoshev, 1993. Ann. Rev. Pharmacol. Toxicol. 32:573-596; Morgan & Anderson, 1993. Ann. Rev. Biochem. 62:191-217 and Morgan & Anderson, 1993. TIBTECH U :l 55-215.

In a preferred aspect, the Therapeutic comprises a GENE SET nucleic acid which is part of an expression vector that expresses a GENE SET protein (or derivative, fragment, analog or homolog thereof), a chimeric protein, preferably comprising a GENE SET protein (or derivative, fragment, analog or homolog thereof) or a GENE SET anti-sense nucleic acid thereof, within a suitable host. In a specific embodiment, such a nucleic acid possesses a promoter which is operably-linked to the GENE SET coding region, or to a sequence encoding a GENE SET anti- sense nucleic acid; wherein said promoter is inducible or constitutive and, optionally, tissue- specific. In another particular embodiment, a nucleic acid molecule is utilized in which the GENE SET coding sequences (and any other desired sequences) are flanked by regions which promote homologous recombination at a desired site within the genome, thus providing for intra- chromosomal expression of the GENE SET nucleic acid. See e.g.. Koller & Smithies, 1989. Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra. et al, 1989. Nature 342:435-438.

In a preferred embodiment of the present invention, the Therapeutic comprises a GENE SET nucleic acid which is part of an expression vector that expresses the GENE SET proteins (or derivatives, fragment, analogs or chimeric proteins thereof) Within „ suitable host. In particular, such a nucleic acid possesses a promoter operably-linked to the GENE SET coding region(s) or, less preferably, a separate promoter operably-linked to the GENE SET protein- coding region, wherein said promoter is inducible or constitutive and, optionally, tissue-specific. Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy. In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This may be accomplished by any of numerous methods known in the art including, but not limited to, constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular by: (/^') infection using a defective or attenuated retroviral or other viral vector (see e.g., U.S. Patent No. 4.980J86); (H) direct injection of naked DNA: (/ ) use of microparticle bombardment (e.g., a gene gun - Biolistic, DuPont); (iv) coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules: (v) by administering it in linkage to a peptide which is known to enter the nucleus; (vi) administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu & Wu, 1987. J. Biol. Chem. 262:4429- 4432) which can be used to target cell types specifically-expressing the receptors and the like. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid may be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor. See e.g., PCT Publications WO 93/14188; WO 93/20221. Alternatively, the nucleic acid may be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination. See e.g., Koller & Smithies, 1989. Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra, et al., 1989. Nature 342:435-438.

In a one embodiment of the present invention, a viral vector that contains the GENE SET nucleic acid or, alternately, codes for GENE SET anti-sense nucleic acid, may be utilized. For example, a retroviral vector may be used (see e.g., Miller, et al. 1993. Meth. Enzymol. 217:581- 599 (1993) which have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. Hence, the GENE SET nucleic acid to be utilized in gene therapy may be cloned into the vector, which facilitates delivery of the gene into α patient. A specific application of this techno^gymay be'found'in Boesen, et al.. 1994. Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdr gene to hematopoietic stem cells in order to increase their resistivity to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include: Clowes, et al, 1994. J. Clin. Invest. 93:644-651; Kiem, et al, 1994. Blood 83:1467-1473 and Salmons & Gunzberg, 1993. Human Gene Therapy 4: 129-141.

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia where the virus naturally infects to cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. In addition, adenoviruses have the advantage of being capable of infecting non-dividing cells. See e.g., Kozarsky & Wilson, 1993. Curr. Opin. Genet. Develop. 3:499-503. Adeno-associated virus (AAV) has also been proposed for use in gene therapy. See e.g., Walsh, et al. 1993. Proc. Soc. Exp. Biol. Med. 204:289-300. Another approach to gene therapy involves transferring a gene into cells in tissue culture by such methods as electroporation. lipofection, calcium phosphate-mediated transfection, or viral infection. Generally, the method of transfer includes the transfer of a selectable marker to the cells which are then placed under selection to isolate those cells which have taken-up and are expressing the transferred gene and only those selected cells are then delivered to a patient. In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art including, but not limited to, transfection, electroporation. microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome- mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like (see e.g., Loeffler & Behr, 1993. Meth. Enzymol 217:599-618; Cohen, et al, 1993. Meth. Enzymol 217:618-644) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique chosen should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny. The resulting recombinant cells may be delivered to a patient by any of the methods well-known within the art.

With respect to the administration of the specific gene therapy agent, in a preferred embodiment of the present invention, epithelial cells are injected (e.g., subcutaneously). In another embodiment, recombinant skin cells may be applied as a skin . t onto the patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and may be determined by one skilled within the art. Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type and include, but are not limited to: epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes. blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils. eosinophils. megakaryocytes. granulocytes, various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc). In a preferred embodiment of the present invention, the cell utilized for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, a GENE SET nucleic acid or nucleic acid encoding a GENE SET anti-sense nucleic acid is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which may be isolated and maintained in vitro may potentially be used in accordance with this embodiment of the present invention. Such stem cells include but are not limited to hematopoietic stem cells (HSC). stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells (see e.g., PCT Publication WO 94/08598) and neural stem cells (see e.g., Stemple & Anderson, 1992. Cell 71:973-985).

Embryonic stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures. See e.g.. Rheinwald, 1980. Meth. Cell Bio. 2L229-237. In stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells within the lining of the gut provide for a rapid renewal rate of this tissue. ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture. See e.g., Pittelkow & Scott, 1986. Mayo Clinic Proc. 61J71-782. If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody administration to promote moderate immunosuppression) may also be used. W th respect to hematopoietic stem cells (HSC), any technique which provides for the isolation, propagation, and maintenance in vitro of HSCs may be used in this embodiment of the present invention. Techniques by which this may be accomplished include, but are not limited to: ( ) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor or (if) the Use of previously established long-term HSC cultures, which may be allergenic or xenogeneic. Non- autologous HSC are used preferably in conjunction with a method of suppressing transplantation immune reactions of the future host patient. In a specific embodiment of the present invention. human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration. See e.g., Kodo, et al, 1984. J. Clin. Invest. 73:1377-1384.

In a preferred embodiment of the present invention, the HSCs may be made highly enriched or in substantially pure form. This enrichment may be accomplished before, during or after long-term culturing, and may be performed by any techniques known in the art. Long-term cultures of bone marrow cells may be established and maintained by using, for example, modified Dexter cell culture techniques (see Dexter, et al, 1977. J. Cell Physiol 9J J35) or Witlock-Witte culture techniques (see Witlock & Witte, 1982. Proc. Natl. Acad. Sci. USA 79:3608-3612). In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

Additional methods which may be utilized, or adapted for utilization, for the delivery of a nucleic acid encoding a GENE SET protein (or functional derivative, fragment or analogs thereof) are described in Section 4, infra.

(B) Antibodies

In one embodiment of the present invention, as discussed hereinabove, antibodies which possess the ability to bind GENE SET proteins (and derivatives, fragments or analogs thereof) may be utilized to treat or prevent renal failure or acute disease or, preferably, acute renal failure. Anti-GENE SET antibodies may also be utilized in the diagnostic, prognostic and screening methods disclosed by the present invention (e.g., as described in Section 3, infra). Such anti- GENE SET antibodies include, but are not limited to, polyclonal. monoclonal, chimeric, single- chain, F_ab fragments and an F_ab expression library. In a specific embodiment, antibodies specific for human GENE SET proteins are disclosed. In another specific embodiment, antibodies which possess the ability to reduce or inhibit GENE SET activity in vitro and/or in vivo, are also disclosed.

Various methods well-known within the art may be utilized for the production of polyclonal antibodies to a GENE SET protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies specific for an epitope of a GENE SET protein or a nucleic acid encoding a GENE SET protein (and derivatives, fragments or analogs thereof) may be obtained. For the production of antibody, various host animals may be immunized by injection with the native GENE SET protein (or a synthetic version, derivative, fragment or analog thereof) including, but not limited to, rabbits, mice, rats, primates, etc. In addition, various adjuvants may be utilized to increase the immunological response, depending on the host species, and include, but are not limited to: Freund's (complete and incomplete); mineral gels (e.g., aluminum hydroxide); surface active substances (e.g., lysolecithin); pluronic polyols; polyanions; peptides; oil emulsions; keyhole limpet hemocyanins: dinitrophenol and potentially useful human adjuvants (e.g., bacilli Calmette-Guerin (BCG) and corynebacterium parvum). For preparation of monoclonal antibodies which are specific for a GENE SET protein sequence (or derivative, fragment or analog thereof), any methodology which provides for the production of antibody molecules by continuous in vitro cell lines may be used. These methods include, but are not limited to: (/) the hybridoma technique (see e.g., Kohler & Milstein, 1975. Nature 256:495-497); (ii) the trioma technique (Cole, et al, 1985. In: Monoclonal Antibodies and Cancer Tlierapy (Alan R. Liss, Inc.); (Hi) the human B-cell hybridoma technique (see e.g., Kozbor, et al, 1983. Immunology Today 4:72) and the EBV hybridoma technique to produce human monoclonal antibodies (see e.g., Cole, et al, 1985. In: Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc.). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing a recently developed technology (see e.g., PCT Patent Publication U590/02545). Also within the scope of the present invention are the utilization of human antibodies which may be obtained through the use of human hybridomas (see e.g., Cote, et al, 1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or, as previously discussed, by transforming human B cells with EBV virus in vitro (see e.g., Cole, et al, 1985. In: Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc.).

In yet another embodiment, the techniques developed for the production of "chimeric antibodies" (see e.g., Morrison, et al, 1984. Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger, et al, 1984. Nature 3JJ:604-608; Takeda, et al. 1985. Nature 314:452-454) by splicing of the genes from a mouse antibody molecule specific for GENE SET together with genes from a human antibody molecule of appropriate biological activity may be utilized, and within the scope of the present invention. Alternately, non-human antibodies may be "humanized" by known methods (see e.g., U.S. Patent No. 5.225,539). Also disclosed by the present invention are techniques for the production of single-chain antibodies (see e.g., U.S. Patent No. 4.946,778) may be adapted to produce GENE SET-specific single-chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of F_ab expression libraries (see e.g.. Huse, et al, 1989. Science 246:1275- 1281) to allow rapid and efficacious identification of monoclonal F_ab fragments with the desired specificity for GENE SET proteins (or derivatives or analogs thereof). In an alternative embodiment, antibody fragments which contain the idiotype of the molecule may be generated by known techniques. For example, such fragments include but are not limited to: (0 the F(_ab)₂ fragment which may be produced by pepsin digestion of the antibody molecule; (ii) the F_ab. fragments which may be generated by reducing the disulfide bridges of the F(_ab.)₂ fragment; (Hi) the F_ab fragments which may be generated by treating the antibody molecule with papain and a reducing agent) and (iv) the F_v fragments.

Screening for the desired antibody may be accomplished by any of the techniques which are well-known within the art including, but not limited to, enzyme-linked immunosorbent assay (ELISA). For example, to select antibodies which recognize a specific region (e.g., active site, transmembranal region and the like) of a GENE SET protein, the generated hybridomas may be may be examined for a product which binds to a GENE SET fragment containing such a specific region. Similarly, for the selection of an antibody that may reduce or inhibit GENE SET activity, one may assay the antibody using any of the assays for GENE SET activity described in Section 6, infra.

(C) Anti-Sense Nucleic Acids

In one embodiment of the present invention, GENE SET function may be reduced or inhibited through the use of GENE SET anti-sense nucleic acids, to treat or prevent renal failure or acute disease, preferably, acute renal failure. In a specific embodiment, nucleic acids of at least six nucleotides which are anti-sense to a gene or cDNA encoding a GENE SET protein (or a portion thereof) are used in a therapeutic or prophylactic manner. A GENE SET "anti-sense" nucleic acid, as used herein, refers to a nucleic acid which is capable of hybridizing to a portion of a GENE SET RN A (preferably mRNA) by virtue of some sequence complementarily. The anti-sense nucleic acid may be complementary to a coding and/or noncoding region of a GENE SET mRNA.

The GENE SET anti-sense nucleic acids of the present invention are comprised of at least six nucleotides and are, preferably, oligonucleotides (ranging from 6-150 nucleotides, or more preferably, 6 to 50 nucleotides). In specific embodiments, the oligonucleotide utilized in the practice of the present invention is at least 10 nucleotides. at least 15 nucleotides. at least 100 nucleotides, or at least 125 nucleotides. The ohgonucleotides may be DNA, RNA or chimeric mixtures (or derivatives or modified versions thereof), and may be either single-stranded or double-stranded. In addition, the oligonucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may also include other appending groups such as: (/) peptides facilitating transport across the cell membrane (see e.g., PCT Publication No. WO 88/09810) or blood-brain barrier (see e.g., PCT Publication No. WO 89/10134); (ii) hybridization-triggered cleavage agents (see e.g., Krol, et al. 1988. BioTechniques 6:958- 976) or (Hi) intercalating agents (see e.g.. Zon, 1988. Pharm. Res. 5:539-549). The GENE SET anti-sense nucleic acid is preferably an oligonucleotide, more preferably of single-stranded DNA. In another preferred embodiment, the oligonucleotide comprises a sequence anti-sense to a portion of human GENE SET. The oligonucleotide may be modified at any position on its structure with substituents generally known within the art. In yet another embodiment, the oligonucleotide is an β-anomeric oligonucleotide which forms specific double- stranded hybrids with complementary RNA in which (contrary to the usual β- units) the strands run parallel to each other. See e.g.. Gautier, et al, 1987. Nucl. Acids Res. 15:6625-6641. The oligonucleotide may also be conjugated to another molecule (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.).

The anti-sense ohgonucleotides of the invention may be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer (such as are commercially available from Biosearch. Applied Biosystems. and the like). As examples, but not of limitations, phosphorothioate ohgonucleotides may be synthesized by the method of Stein, et al. (1988. Nucl. Acids Res. j_6:3209) and methylphosphonate ohgonucleotides may be prepared by use of controlled pore glass polymer supports (see e.g., Sarin, et al, 1988. Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451).

In a specific embodiment of the present invention, the GENE SET anti-sense oligonucleotide comprises catalytic RNA, or a ribozyme (see e.g., PCT Publication WO 90/11364; Sarver, et al, 1990. Science 247:1222-1225). In another embodiment, the oligonucleotide is a 2'-0-methylribonucleotide (see e.g., Inoue, et al, 1987. Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (see e.g.. Inoue. et al, 1987. FEBS Lett. 215:327-330).

In an alternative embodiment of the present invention, the GENE SET anti-sense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector may be introduced in vivo such that it is taken up by a cell, within which cell the vector (or a portion thereof) is transcribed, resulting in the production of an anti-sense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the GENE SET anti-sense nucleic acid and may either remain episomal or become chromosomally integrated, so long as it is capable of being transcribed to produce the desired anti-sense RNA. Such vectors may be constructed by recombinant DNA technology methods standard within the art and may include, but not be limited to, plasmid. viral, or like vectors, which are used for replication and expression in mammalian cells. Expression of the sequence encoding the GENE SET anti-sense RNA may be by any promoter known within the art to function within mammalian or, preferably, human, cells. Such promoters may be inducible or constitutive and include, but are not limited to: (/^') the SV40 early promoter region (see e.g., Bernoist & Chambon, 1981. Nature 290:304-310); (ii) the promoter contained in the 3'-terminus long terminal repeat of Rous sarcoma virus (see e.g., Yamamoto. et al, 1980. Cell 22:181 -191); (Hi) the Herpesvirus thymidine kinase promoter (see e.g., Wagner, et al, 1981. Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445) and (iv) the regulatory sequences of the metallothionein gene (see e.g., Brinster, et al, 1982. Nature 296:39-42).

The anti-sense nucleic acids of the present invention comprise a sequence complementary to at least a portion of an RNA transcript of a GENE SET gene, preferably a human GENE SET gene. However, absolute complementarily, although preferred, is not required. The term "a sequence complementary to at least a portion of an RNA,^"" as utilized herein, is defined as a sequence possessing sufficient complementarily to enable it to hybridize with the RNA. resulting in the formation of a stable duplex: in the case of double-stranded GENE SET anti-sense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability of the anti-sense nucleic acid to hybridize will depend upon both the degree of complementarily and the length of the anti-sense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a GENE SET RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled within the art may ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. The present invention further provides pharmaceutical compositions comprising an effective amount of the GENE SET anti-sense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra. In a specific embodiment, pharmaceutical compositions comprising GENE SET anti-sense nucleic acids may be administered via liposomes. microparticles. or microcapsules. In alternate embodiments, it may be useful to use such compositions to achieve sustained release of the GENE SET anti-sense nucleic acids. Additional methods that may be adapted for use in the delivery of a GENE SET anti-sense nucleic acid of the present invention will be disclosed in Section 5, infra.

The amount of GENE SET anti-sense nucleic acid which will be effective in the treatment or prevention of acute disease will be dependant upon the nature of the disease, and may be determined by standard clinical techniques. Where possible, it is desirable to determine the anti-sense cytotoxicity in cells in vitro, and then in useful animal model systems, prior to testing and in vivo use in humans.

(3) Methods of Diagnosis, Prognosis and Screening

The present invention also discloses methods of diagnosis, prognosis and screening for renal failure or acute disease or, preferably, acute renal failure in individuals who include, but are not limited to, those subjects having renal disease or renal failure, having previously suffered an cerebrovascular event or exhibit one or more "risk factors" for renal failure or one or more conditions associated with renal failure.

In one embodiment of the present invention, anti-GENE SET-antibodies are used to detect and quantitate GENE SET levels in one or more tissues (e.g., blood) of a subject in immunoassays. In particular, such an immunoassay is performed by use of a method comprising contacting a sample derived from a patient with an anti-GENE SET antibody under conditions such that immunospecific-binding may occur, and subsequently detecting or measuring the amount of any immunospecific binding by the antibody. The particular amino acid deletion, insertion or substitution in the GENE SET may change the epitope recognized by a specific anti- (wild-type) GENE SET antibody such that antibody binds the GENE SET to a lesser extent or not at all. Additionally, antibodies may be produced (e.g., as described in Section 2(B), supra) against the GENE SET protein (or portion thereof) which possess the ability to immunospecifically-bind to the particular GENE SET protein, but not the wild type GENE SET protein (as determined by the in vitro immunoassay methods described below). These specific anti- GENE SET antibodies may be used to detect the presence of. for example, GENE SET proteins by measuring the immunospecific-binding by the anti-GENE SET protein antibodies and, optionally, lack of immunospecific binding by the anti-(wild-type) GENE SET protein antibodies. Additionally, GENE SET proteins possessing deletion or insertion mutations may be detected by either an increase or decrease in protein size by, for example, but not limited to, Western blot analysis using an anti-GENE SET protein antibody that recognizes a constituent protein of the GENE SET.

Immunoassay methods of the present invention which may be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RJA). enzyme linked immunosorbent assay(ELISA), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and the like. Kits for diagnostic or screening use are also provided by the present invention which comprise, in one or more containers, an anti-GENE SET antibody and (optionally) a labeled binding partner to the antibody. Alternatively, the anti-GENE SET antibody may be labeled with a detectable marker (e.g.. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety). A kit is also provided which comprises, in one or more containers, a nucleic acid probe capable of hybridizing to GENE SET RNA or, preferably, capable of specifically hybridizing to a GENE SET gene. In a specific embodiment, a kit may comprise, in one or more containers, a pair of oligonucleotide primers (e.g.. each in the size range of 6-30 nucleotides) which are capable of priming amplification of at least a portion of a GENE SET nucleic acid by: (/^') polymerase chain reaction (PCR; see e.g., Innis, et al, 1990. PCR Protocols (Academic Press, Inc., San Diego, CA); (ii) ligase chain reaction; (Hi) use of Qβ replicase; (iv) cyclic probe reaction, or other methods known in the art, under the appropriate reaction conditions. A kit may, optionally, further comprise, in one or more containers, a predetermined amount of a purified GENE SET protein or nucleic acid (e.g., for use as a standard or control).

(4) Assays for the GENE SET Proteins or Modulators of the GENE SET Proteins and Nucleic Acids

A variety of methods are available within the art for use in assaying the activity of the GENE SET proteins (and derivatives, analogs, fragments and homologs thereof) and nucleic acids encoding the GENE SET proteins (and derivatives, analogs and fragments thereof). Methods are also available for the screening of putative GENE SET modulators (e.g., GENE SET protein agonists, antagonists and inhibitors). Such modulators of GENE SET protein activity include, but are not limited to. GENE SET anti-sense nucleic acids, anti-GENE SET antibodies, and competitive inhibitors of GENE SET for binding to the GENE SET protein receptor.

In vitro methods for assaying GENE SET proteins (and derivatives, fragments, homologs and analogs thereof), the nucleic acids encoding these GENE SET proteins, and putative modulators of GENE SET proteins (e.g., agonists, antagonists or inhibitors of GENE SET protein activity) include, but are not limited to: ( ) GENE SET receptor binding assays (see e.g., Vesely, et al, 1992. Renal Phys. Biochem. 15:23-32; Iwashina. et al. 1994. J Biochem. 115:563- 567; Chang, et al, 1996. Curr. Eye Res. 15:137-143; (/^{' '}) measurement of changes in cGMP concentrations in cells possessing GENE SET receptors (see e.g., Schulz, et al, 1989. Cell 58:1155-1162; Wedel, et al, 1997. Proc Natl. Acad. Sci. U.S.A. 21:459-462 and (Hi) changes in intracellular Ca²⁺ resulting from GENE SET receptor signaling in response to GENE SET binding (see e.g., Nascimento-Gomes. et al, 1995. Brazil J. of Med. Biol. Res. 28:609-613. However, it should be noted that any measurement of GENE SET receptor activity elicited by GENE SET binding may be used to assay GENE SET activity in vitro. The activity of the GENE SET proteins (and derivatives, fragments, analogs and homologs thereof), the nucleic acids encoding these GENE SET proteins (and derivatives, fragments, analogs and homologs thereof) and putative modulators of GENE SET activity may also be ascertained in vivo. By way of example, and not of limitation, the infusion of GENE SET proteins in humans causes significant increases in cGMP levels in both plasma and urine (see e.g., Vesely, et al, 1995. Am. J. Med. Sci 310:143-149; Vesely, et al, 1996. Metabolism: Clin. & Exp. 45:315-319). In addition, the administration of GENE SET proteins to humans also elicits significant diuresis and reduction in blood pressure (see e.g., Vesely. et al, 1996. Life Sciences 59:243-254); similar effects have also been previously demonstrated in rodents (see e.g., Garcia, et al, 1989. Hypertension JJ:567-574). Accordingly, the GENE SET proteins and nucleic acids (and derivatives, analogs, fragments and homologs thereof), as well as putative

GENE SET modulators, may be assayed by administration of the test compound to a test animal (preferably a non-human test animal such as a rat or mouse), followed by the subsequent measurement of the one or more of the physiological parameters described above (e.g., cGMP levels in urine and/or plasma, diuretic effect, decrease in blood pressure, and the like). In a preferred embodiment of the present invention, rats derived from crosses with the fawn-hooded rat (FHR) may be used to assay for GENE SET protein, nucleic acid or modulator activity. For example, rats which possess the renal failure-predisposing locus on chromosome 1 with the concomitant lack of the renal failure-protective locus on chromosome 5 (which maps to the GENE SET gene) and, optionally, the other renal failure-protective locus on chromosome 4, may be used to screen for putative GENE SET modulators and antagonists. In a specific embodiment of the present invention, nucleic acids containing the nucleotide sequence encoding a GENE SET protein may be introduced into the rats possessing the chromosome 1 renal failure predisposing locus but not the renal failure protective loci. In accord, the GENE SET protein useful for treatment and prevention of renal failure would increase renal failure latency when either administered or transgenically-expressed in the renal failure prone rats lacking both the protective loci and fed a high salt diet to induce hypertension.

In another specific embodiment of the present invention, a putative modulator of GENE SET activity, or of latency or predisposition to renal failure, may be screened by:

(/^') administering a putative modulator of GENE SET activity to an animal prone to renal failure and (ii) measuring one or more physiological parameters associated with GENE SET activity; wherein a change in one or more of the parameters, relative to an animal not administered the putative modulator, would be indicative of the putative modulator possessing the ability to modulate GENE SET activity or latency or predisposition to renal failure. In yet another specific embodiment, the animal prone to renal failure is fed a high salt diet. In a preferred embodiment of the present invention, the physiological parameter which may be measured is renal failure latency. Additionally, GENE SET modulators may be screened using a recombinant test animal which expresses a GENE SET transgene or expresses a GENE SET protein under the control of a promoter which is not the native (endogenous) GENE SET gene promoter, at an increased level relative to a wild-type test animal.

Another embodiment of the present invention discloses a methodology for screening a GENE SET protein, nucleic acid or modulator for the ability to alter GENE SET activity comprising: (/) administering the GENE SET protein, nucleic acid or modulator to a test animal prone to renal failure and (ii) measuring renal failure latency in the test animal in which renal failure latency is indicative of GENE SET activity. In a specific embodiment, a recombinant test animal which expresses a GENE SET transgene or expresses GENE SET protein under the control of a promoter that is not the native GENE SET gene promoter at an increased level relative to a wild-type test animal is used to screen the GENE SET for a change in GENE SET activity.

In yet another specific embodiment, a method for screening for a modulator of GENE SET activity or of latency or predisposition to renal failure is provided which comprises measuring renal failure latency in a renal failure-prone animal which recombinantly expresses a putative modulator of GENE SET activity, wherein a change in renal failure latency relative to an analogous renal failure-prone animal which does not recombinantly express the putative modulator is indicative of the putative modulator possessing the ability to modulate GENE SET activity or latency or predisposition to renal failure.

(5) Pharmaceutical Compositions

The present invention discloses methods of treatment and prophylaxis by the administration, to a subject in need of such treatment, an effective amount of a Therapeutic of the present invention. In a preferred embodiment, the Therapeutic is substantially-purified. The subject is preferably an animal, including, but not limited to, animals such as cows, pigs, horses, chickens, cats, dogs, etc.. and is preferably a mammal, and most preferably human.

Formulations and methods of administration which may be employed in the practice of the present invention when the Therapeutic comprises a nucleic acid are described in Section 2(A) and Section 2(C) supra. Additional appropriate formulations and routes of administration may be selected from among those described hereinbelow.

Various delivery systems are known and can be used to administer a Therapeutic of the invention including, but not limited to: encapsulation in liposomes. microparticles, microcapsules, recombinant cells capable of expressing the Therapeutic, receptor-mediated endocytosis (see e.g., Wu & Wu, 1987. J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, and the like. Methods of introduction include, but are not limited to: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal. epidural. and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa. rectal and intestinal mucosa. etc.) and may be administered together with other biologically active agents. Administration may be either systemic or local. Furthermore, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection, intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir (e.g.. an Ommaya reservoir). Pulmonary administration may also be employed (e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent).

In another specific embodiment of the present invention, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, this may be achieved by, for example, and not by way of limitation, local infusion during surgery. topical application (e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers). In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue. In another embodiment, the Therapeutic may be delivered in a vesicle, in particular a liposome (see e.g., Treat, et al, In: Liposomes in the Therapy of Infectious Disease and Cancer (Liss, New York, NY). In yet another embodiment, the Therapeutic may be delivered in a controlled release system. In one specific embodiment, a pump may be utilized. See e.g., Sefton, 1987. CRC Crit. Ref. Biomed. Eng. 14:201. In another embodiment, polymeric materials can be used (see e.g., Medical Applications of Controlled Release 1984. (CRC Pres., Boca Raton, FL). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target (e.g., the brain), thus requiring only a fraction of the systemic dose. In a specific embodiment where the Therapeutic is a nucleic acid encoding a protein Therapeutic, the nucleic acid may be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, (e.g., by use of a retroviral vector; see U.S. Patent No. 4,980,286), or by direct injection; or by use of microparticle bombardment (e.g., a gene gun, Biolistic, DuPont), or coating with lipids or cell-surface receptors or transfecting agents; or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot. et al. 1991. Proc. Natl. Acad. Sci USA 88:1864-1868), and the like. Alternatively, a nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable ' as used herein, means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term "carrier." as used herein, refers to a diluent, adjuvant, excipient. or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil. soybean oil, mineral oil. sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt. rice, flour, chalk, silica gel. sodium stearate. glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol. propylene. glycol. water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington 's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic

(preferably in a substantially purified form) together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The Therapeutics of the invention can be formulated as neutral or salt forms.

The amount of the Therapeutic of the present invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined in a quantitative manner by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.

The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 μg of active compound/kg of patient body weight. Suitable dosage ranges for intranasal administration are generally about

0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; whereas oral formulations preferably contain 10% to 95% active ingredient.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

(6) Animal Models

The present invention also provides animal models. In a specific embodiment, animal models for renal failure or ischemic disease, specifically acute renal failure are provided. In one embodiment, FHRs may be bred with normal or non-renal failure-prone rats not possessing a GENE SET allele. Rats may then be selected which possess the chromosome 1 locus for renal failure predisposition but do not have the chromosome 5 locus (i.e.. possess a wild-type GENE SET locus) or, optionally, the chromosome 4 locus, demonstrated to be protective for renal failure in the FHR strain. Such animals may be used to test for GENE SET proteins with reduced activity or for GENE SET antagonists as described in Section 6, supra.

In another embodiment of the present invention, transgenic animals may be bred or produced through molecular-biological means, which over-express or under-express one or more of the GENE SET genes (e.g., by introducing a member or members of the GENE SET gene under the control of a heterologous promoter or a promoter which facilitates the expression of GENE SET proteins and/or nucleic acids in tissues which do not normally express GENE SET components. Additionally, "knockout" mice may be initially produced by promoting homologous recombination between a GENE SET gene in its chromosome and an exogenous GENE SET gene that has been rendered biologically inactive, preferably by insertion of a heterologous sequence (e.g., an antibiotic resistance gene) or by non-homologous recombination. In a preferred embodiment of the present invention, introduction of heterologous DNA is carried out by transforming embryo-derived stem (ES) cells with a vector containing the insertionally-inactivated GENE SET gene or a GENE SET gene which was under the control of a heterologous promoter, followed by injecting the ES cells into a blastocyst and implanting the blastocyst into a "foster mother" animal. Accordingly, the resulting mice are chimeric animals ("knockout animal" or "transgenic animal") in which an GENE SET gene has been inactivated or overexpressed or misexpressed (see e.g., Capecchi, 1989. Science 244:1288-1292). The chimeric animal can then be bred to produce additional knockout or transgenic animals. Such chimeric/transgenic animals include, but are not limited to, mice, hamsters, sheep, pigs, cattle, etc., and are, preferably, non-human mammals. Transgenic and knockout animals can also be made in D. melanogaster, C. elegans. and the like, by methods which are commonly-known within the art.

(7) Specific Examples

The quantitative expression analysis (QEA^®) methodology (see PCT Publication WO 97/15690, dated May 1, 1997 to Nandabalan, et al), which has now been registered as the trademark GeneCalling^®, was utilized to identify and characterize genes which were, putatively, differentially-expressed within the FHR and IRL rodent strains (i.e., renal disease animal models) as compared to the ACI (control) rodent strain. It should be noted, however, that RNA reverse transcriptase polymerase chain reaction (RT-PCR) may also be, preferably, utilized to identify the differentially-expressed GENE SET genes/gene products in the practice of the present invention. Both the GeneCalling^® and RT-PCR methods will be discussed in detail in the following sections.

Prior to utilization in the assays disclosed herein, the degree of proteinuria exhibited by both the FHR and IRL rodent strains, in comparison to the control ACI strain, is quantitatively determined. Both the FHR and IRL rodent strains exhibited marked proteinuria. (A) Experimental Methods and Materials of the Present Invention

(I) Identification of GENE SET Genes by GeneCalling⁸

(/^') Tissue Dissection

Kidney tissue derived from the FHR, IRL and ACI (control) rodent strains were analyzed by the proprietary GeneCalling-⁸ methodology.

In brief, FHR, IRL and ACI rats were maintained on normal rat chow and water ad libitum. FHR and the control ACI rats were sacrificed at 1.5 and 7.5 months-of-age, whereas the IRL is sacrificed only at 1.5 months-of-age. While the 1.5 month rats appeared "normal" as a result of laboratory blood-work which is performed, the FHR by 7.5 months-of-age is demonstrated to have developed pronounced proteinuria. This finding is in agreement with previous experimental results which found that the median age of onset of proteinuria (and other renal-related disorders) in the FHR was approximately 4 months-of-age. Following the abnormal laboratory results indicating the onset of proteinuria, the animals were sacrificed and their kidneys removed and quick-frozen in liquid nitrogen immediately after dissection. The kidneys were stored at -70°C until utilized in the subsequent GeneCalling^® protocols.

( /) Isolation of Total Cellular and Poly(AV RNA Total cellular RNA was extracted from 5 mg of heart, liver, fat, kidney, or brain tissue by initially grinding the tissue into a fine powder in liquid nitrogen. The powdered tissue was then transferred to a tube containing 500 μl Triazol Reagent^® (Life Technologies; Gaithersburg, MD) and was dispersed using a Polytron homogenizer (Brinkman Instruments; Westbury, NY). See e.g., Chomszynski, et al. 1987. Annal Biochem. 162 156-159; Chomszynski, et al, 1993. Biotechniques 15:532-533, 536-537. The total cellular RNA fraction was then extracted with 50 μl BCP (l-bromo-3-chloropropane: Molecular Research; Cincinnati. OH) to facilitate phase separation. The extraction mixture was centrifuged for 15 minutes at 4°C at 12,000 x G, and the aqueous phase was removed and transferred to a fresh tube. The RNA was then precipitated with 0.5 volume of isopropanol per original volume of Triazol Reagent^® used, and the sample was re- centrifuged at room temperature for 10 minutes at 12,000 x G. The supernatant was then discarded, the pellet washed with 70% ethanol and re-centrifuged at room temperature for 5 minutes at 12,000 x G. Finally the 70% ethanol was removed and the centrifuge tube was inverted and let stand to dry in this position. The resulting RNA pellet was re-suspended in 100 μl water (i.e., 1 μl/mg of original tissue weight) and heated to 55°C until completely dissolved. The final concentration of total cellular RNA was quantitated by fluorometry with OliGreen^® (Molecular Probes: Eugene. OR). In addition, the quality of the total cellular RNA was determined by both spectrophotometry and formaldehyde agarose gel electrophoresis. Poly(A)^' mRNA was prepared from 100 μg of total cellular RNA by use of affinity chromatography with oligo(dT) magnetic beads (PerSeptive: Cambridge, MA) or with the Dynabeads mRNA Direct Kit^® (Dynal; Oslo, Norway) as directed by the manufacturer. The Poly(A)⁺ mRNA was harvested in a small volume of sterile water, and the final yield quantified by OD₂₆₀ measurement and fluorometry with OliGreen^® (Molecular Probes; Eugene, OR). The Poly(A)^* mRNA was stored at -20°C for subsequent utilization in GeneCalling^® protocols.

(ii) cDNA Synthesis

The RNA samples were then treated with DNase to remove endogenous, contaminating DNA. 28 μl of 5X reverse transcriptase buffer (Life Technologies; Gaithersburg, MD), 10 μl 0J M DTT, 5 units RNAguard^® (Pharmacia Biotech, Upsala. Sweden) per 100 mg tissue and 1 unit RNase-free DNase I^® (Pharmacia Biotech) per 100 mg tissue, were added to the re-suspended RNA samples. The reaction mixture was then incubated at 37°C for 20 minutes. The total RNA concentration was quantified by measuring OD₂₆₀ of a 100-fold dilution and the samples stored at -20°C. cDNA was synthesized from the Poly(A)^T RNA as follows: the Poly(A)⁺ RNA was mixed with 50 ng random hexanucleotides (50 ng/μl) in 10 μl of water. The mixture was heated to 70°C for 10 minutes, quick-chilled in ice-water slurry, and kept on ice for 1-2 minuets. The condensate was collected by centrifugation in a microfuge for 10 seconds. In an alternate embodiment of the present invention. 200 pmols oligo(dT)„V (where V = A, C or G) was utilized in place of the random hexanucleotide primers.

The first strand synthesis was carried out by adding a reaction mixture consisting of the following reagents: 4 μl 5X first strand buffer (BRL; Grand Island NY), 2 μl 100 mM DTT, 1 μl 10 mM dNTP mix and 2 μl water to the primer-annealed RNA. The reaction mixtures were then incubated at 37°C for 2 minuets and 1 μl of Superscript IP reverse transcriptase (BRL) was added following the manufacturer's recommendation and the reactions were then incubated at 37°C for 1 hour.

To synthesize the second cDNA strand, the samples were placed on ice, 30 μl of 5X Second strand buffer, 90 μl of cold water, 3 μl of 10 mM dNTP, 1 μL (10 units) of E. coli DNA ligase (BRL), 4 μl (40 units) of E. coli DNA polymerase (BRL). and 1 μl (3.5 units) of E. coh

RNase H (BRL) were added to the tubes, and the reactions were incubated for 2 hours at 16°C.

The resulting cDNA was then incubated with 2 μl of T4 DNA polymerase (5 units) at 16°C for 5 minuets. The resulting cDNA was dephosphorylated with Arctic Shrimp Alkaline Phosphatase

("SAP"; US Biochemicals; Cleveland. OH) by the addition of 20 μl 10X SAP buffer. 25 μl of water, and 5 μl (5 units) of SAP to the reaction mixtures which were then incubated at 37°C for

30 minuets.

The cDNA was extracted with phenol-chloroform (50:50 v/v), chloroform-isoamyl alcohol (99:1 v/v), and precipitated from the aqueous phase by the addition of NaOAc (pH 5.0) to a final concentration of 0J M, 20 μg glycogen and 2 volumes of ethanol. The reactions were incubated at -20°C for 10 minuets and the cDNA was collected by centrifugation at 14,000 x g for 10 minuets The supernatant was aspirated and the cDNA pellet washed with 75% ethanol. resuspended in TΕ, and the yield of cDNA was estimated using fluorometry with Picogreen¹ (Molecular Probes, Eugene OR)..

(Hi) GeneCalling^® Expression Analysis For subsequent GeneCalling^® expression analysis, 75 ng cDNA was transferred to a separate tube, resuspended in TE to a concentration 600 ng/ml and stored at -20°C. GeneCalling^® analysis was performed as disclosed in PCT Publication WO 97/15690, dated May 1, 1997 to Nandabalan, et al By way of example, and not of limitation, an exemplar GeneCalling^® analysis performed on the PC4 gene encoding IRPR INF-β) will be disclosed herein. Restriction endonuclease (RE) digestion of the INF-β-encoding nucleic acid was performed with BsrFI and Bglll restriction endonucleases. Adapter molecules for the subsequent GeneCalling^® analysis were prepared from linker and primer ohgonucleotides. For the "sticky" termini generated by the BsrFI restriction endonuclease, the linker oligonucleotide: 5'-GGCCCGAAGTACA-3' [SEQ ID NO:l] and the primer oligonucleotide: 5'-GGCCCGAAGTAC-3' [SEQ ID NOJ], were used. For the "sticky" termini generated by the Bglll restriction endonuclease, the linker oligonucleotide: 5 -GGCCCAGCCACT-3' [SEQ ID NOJ] and the primer oligonucleotide: 5'-GGCCCAGCCAC-3- [SEQ ID NO:4] were used. One set of the primers was labeled with a FAM fluorescent label and the other set was labeled with a biotin moiety. The adapters were prepared by mixing the linker and primer ohgonucleotides together in water at a concentration ratio of 1 JO (linker to primer) with the primer held at a total concentration of 50 pm μl. The reaction mixture was incubated at 50°C for 10 minutes and then allowed to cool slowly to room temperature to anneal the linkers and primers. The adapters were stored at -20°C. It should be noted that other oligonucleotide linkers and primers would be required for use with the nucleic acid sequences encoding one of the other GENE SET proteins. By necessity, the sequences of these oligonucleotide molecules would be dependent upon the nucleic acid sequences of the specific GENE SET sequences, and would be readily ascertainable by individuals skilled within the art.

The GeneCalling^® reactions were performed using an automated GeneCalling^® procedure. Reactions were preformed in a standard 96-well thermal cycler format using a Beckman Biomek 2000^® robot (Beckman; Sunnyvale, CA). The cDNA samples were analyzed in triplicate with BsrFI and Bglll restriction enzymes. All steps were performed by the robot, including solution mixing (from user provided stock reagents) and temperature profile control.

The RE/ligase reaction contained the following components per reaction: 1 U each of BsrFI and Bglll (New England Biolabs; Beverly, MA), 1 μl of each annealed adapter prepared as above (10 pm), 0J μl T₄ DNA ligase [1 Unit μl] (Life Technologies, Gaithersburg, MD), 1 μl 10 mM ATP (Life Technologies), 5 ng of the prepared cDNA, 1.5 μl 10X NEB 2 buffer (New England Biolabs), 0.5 μl of 50 mM MgCl₂ and water to bring the total volume to 10 μl. The reactions were then transferred to thermal cycler.

The robot performed the RE/ligation reaction in a PTC- 100^® Thermal Cycler equipped with a mechanized lid (MJ Research: Watertown, MA) with the following temperature profile: 15 minutes at 37^°C, ramp down 21^°C in 5 minutes, 16°C for 30 minutes, 37^°C for 10 minutes and 65^°C for 10 minutes.

The PCR reaction mix contained the following components per reaction: 10 μl 5X E-Mg (300 mM Tris-HCl pH 9.0, 75 mM (NH₄)₂SO₄), 100 pm of BsrFI- and Bglll-primers [SEQ ID NO: 16 and NO: 18, respectively], 1 μl 10 mM dNTP mix (Life Technologies), 2.5 Units of 50: 1 dilution of KlenTaq polymerase (Life Technologies):PFU polymerase (Stratagene, La Jolla, CA), and water to being the total reaction volume to 35 μl per PCR reaction.

The PCR reaction was then heated to 72^°C and 35 μl was transferred to each separate digestion/ligation reaction. The PTC- 100^® Thermal Cycler then performed the PCR reaction with a thermal profile of 72^°C for 10 minutes, 15 cycles of 95°C for 30 seconds and 68°C for 1 minute, and then 72°C for 10 minutes, and finally holding the reactions at 4°C.

Prior to further analysis, the GeneCalling^® products were subjected to a post-PCR clean up protocol as follows: MPG^® streptavidin magnetic beads (CPG: Lincoln Park. NJ) were prepared (3 μl of beads for every 5 μl of GeneCalling^® reaction product) by pre-washing the beads in 10 μl binding buffer (5 M NaCl, 10 mM TRIS, pH 8.0, 1 mM EDTA) per 5 μl original volume of GeneCalling^® reaction product. 10 μl of washed beads was dispensed in a 96 well FALCON^® TC plate for even' GeneCalling^® sample processed. GeneCalling^® products were added to the beads, mixed well and incubated for 30 minutes at 50^°C. The sample volume was made 100 μl with binding buffer, the plate placed on a 96 well magnetic particle concentrator, and the beads allowed to migrate for 5 minutes. The liquid was then removed, and 200 μl washing buffer (10 mM Tris. pH 7.4. 10 mM EDTA) added per well. The washing step was then repeated. For analysis, the beads were resuspended in 5 μl loading buffer (80% deionized formamide, 20% 25 mM EDTA. pH 8.0, 50 mg/ml Blue Dextran) per 5 μl of beads, and the supernatant was then analyzed by electrophoresis on an ABI 377^® (Applied Biosystems, Inc.) automated sequencer under denaturing conditions using the GeneSmay^® computer software (ABI) for analysis. A GeneSmay 500^® ROX ladder (diluted 1 JO in gel sample loading buffer) was utilized to a size control during the subsequent GeneCalling^® analysis. The results obtained by the GeneCalling^® methodology for the differential -expression of the PC4 gene encoding IRPR (INF-β) is illustrated in Figure 1.

"Oligonucleotide poisoning^"" was then performed to confirm that the putative differentially-expressed fragment were the genes which were originally predicted.

(iv) Confirmation of the GeneCalling⁰ Results bv the Oligo-Poisoning^™ Methodology The present invention uses a positive confirmation methodology, known as Oligo-

Poisoning^™, that identifies nucleic acids containing putatively identified sequences predicted to generate observed GeneCalling^® signals, that are actually present in the sample. This method confirms the presence of a specific, defined flanking nucleic acid subsequence which is adjacent to the "target" subsequence recognized by the probing means within a nucleic acid-containing sample. Importantly, Oligo-Poisoning^™ is also equally applicable to confirming putative sequence identifications in any sample of nucleic acid fragments which possess a certain generic sequence structure or motif. This generic structure only limits fragments to have known terminal subsequences capable of acting as PCR primers. Oligo-Poisoning proceeds by initially performing PCR amplification of, for example

GeneCalling^® reaction products, so as to produce detectable results for all nucleic acid fragments contained within the GeneCalling^® reaction which do not possess the putatively identified subsequence. In the preferred embodiment of the present invention, this is achieved by adding a molar excess of a "poisoning^" primer designed to amplify only those nucleic acid fragments having the putatively identified subsequence. The poisoning primer may, preferably, be unlabeled or it may be labeled so as to allow it to be differentiated from any other type of label utilized in the PCR amplification reaction. Following PCR amplification, the resulting reaction products are then separated by electrophoresis and the electrophoretic mobilities of the various fragments are examined. As those nucleic acid fragments containing the putatively identified subsequence which have undergone amplification will be, preferably, unlabeled, they will not generate a detectable signal.

Generally, the parameters of the PCR amplification reactions utilized in Oligo- Poisoning^™ confirmation methodology are, preferably, similar or identical to those used in the generation of the initial GeneCalling^® signals. This is especially advantageous in the case of application of Oligo-Poisoning^™ to GeneCalling,^® due to the fact that the "poisoned" signals may be readily compared to the initial GeneCalling^® signals. Details of exemplary, preferred PCR protocols are described in following sections. In particular, for example, it is preferable that a hot start PCR method be used, and the preferable hot start method utilizing the wax layering technique will be subsequently described in further detail, infra.

In the practice of the wax laying technique of the present invention. PCR reaction vessels are set up by placing dNTPs and water in the lower portion of a reaction vessel; layering wax on top of this dNTP solution; and placing the remainder of the PCR reaction mix on top on the wax layer. As previously described, the wax used preferably melts rapidly at near but less than 72°C, the temperature preferred for the extension phase of the PCR amplification. During PCR amplification, the first thermal cycle begins with a denaturing temperature of approximately 96°C, which is adequate to melt the wax, cause mixing of the reagent compartments, and initiate amplification. The PCR thermal profile is performed, as described in the following section with a preferred stringent annealing temperature of at least approximately 57°C. Also, one primer of the pair of regular primers used in the PCR amplification may be biotinylated, thus allowing the utilization of the magnetic bead separation technique to facilitate the removal remove fragments from the input sample. The last GeneCalling^® step is separation according to length of the amplified fragments followed by detection the fragment lengths and end labels (if any). Lengths of the fragments excised from a cDNA sample typically span a range from a few tens of base pairs to perhaps 1000 bp. Any separation method with adequate length resolution, preferably at least to three bp in a 1000 base pair sequence, can be used. It is preferred to use gel electrophoresis in any adequate configuration known in the art. Gel electrophoresis is capable of resolving separate fragments which differ by three or more base pairs and, with knowledge of average fragment composition and with correction of composition induced mobility differences, of achieving a length precision down to 1 bp. A preferable electrophoresis apparatus is an ABI 377^™ (Applied Biosystems, Inc.) automated sequencer using the Gene Scan^™ software (ABI) for analysis. The electrophoresis can be done by suspending the reaction products in a loading buffer, which can be non-denaturing, in which the dsDNA remains hybridized and carries the labels (if any) of both primers. The buffer can also be denaturing, in which the dsDNA separates into single strands that typically are expected to migrate together (in the absence of large average differences in strand composition or significant strand secondary structure).

The length distribution is detected with various detection means. If no labels are used, means such as antigen (Ag) and antibody (Ab) staining and intercalating dyes can be used. Here, it can be advantageous to separate reaction products into classes, according to the previously described protocols, in order that each band can be unambiguously identified as to its target end subsequences. In the case of fluorochrome labels, since multiple fluorochrome labels can be typically be resolved from a single band in a gel, the products of one recognition reaction with several REs or other recognition means or of several separate recognition reactions can be analyzed in a single lane. However, where one band reveals signals from multiple fluorochrome labels, interpretation can be ambiguous: is such a band due to one fragment cut with multiple REs or to multiple fragments each cut by one RE. In this case, it can also be advantageous to separate reaction products into classes.

Oligo-Poisoning^™ confirmation methodology is comprised of an unlabeled oligonucleotide possessing a nucleotide sequence which is capable of hybridizing to the GENE SET sequence(s) of interest was included in a PCR reaction using the GeneCalling^® reaction products as substrate, thus preventing amplification with the labeled primers. Specifically, each of the oligonucleotide poisoning reaction mixtures contained: 1 μl of a 1 : 100 dilution of the GeneCalling^® reaction products, 5 μl TB 2.0 (500 mM TRIS-HC1 (pH 9.15), 160 mM (NH₄)₂S0₄, 20 mM MgCl₂, 2 μl 10 mM equimolar mixture of dNTPs. 0J μl each BsrFI and Bglll primers (100 pm/ml), 2 μl GENE SET "oligonucleotide poisoning" primer (1000 pm/ml), 1 μl 5 M betaine, 1 μl NEB 2 buffer (10 mM TRIS-HC1, 10 mM MgCl₂, 50 mM NaCl, 1 mM DTT (pH 7.9 at 25°C), 0.25 μl (25 U/μl) of a 16:1 dilution of KlenTaq:PFU and 38 μl water.

The following PCR amplification protocol was performed for a total of 13 cycles in a thermal cycler: 96°C for 30 seconds; 57^°C for 1 minute; 72^°C for 2 minutes. The amplified products were then held at 4^;C for subsequent analysis utilizing automated sequencing apparatus as previously described, supra.

The confirmatory, Oligo-Poisoning^™ results for the PC4 gene encoding IRPR (INF-β) is illustrated in Figure 2. .Analogous reactions were then performed for the remaining, differentially-expressed GENE SET genes/gene products.

(II) Identification of GENE SET Genes by RNA Reverse Transcriptase Polvmerase Chain Reaction (RT-PCR)

(i) Tissue Dissection Kidney tissue derived from the FHR, IRL and ACI (control) rodent strains may also be analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR) amplification methodology. FHR, IRL and ACI rats were maintained on normal rat chow and water ad libitum. FHR and the control ACI rats were sacrificed at 1.5 and 7.5 months-of-age, whereas the IRL was sacrificed only at 1.5 months-of-age. While the 1.5 month rats appeared "normal" as a result of laboratory blood-work which was performed, the FHR by 7.5 months-of-age was demonstrated to have developed pronounced proteinuria. This finding is in agreement with previous experimental results which found that the median age of onset of proteinuria (and other renal- related disorders) in the FHR was approximately 4 months-of-age. Following the abnormal laboratory results indicating the onset of proteinuria, the animals were sacrificed and their kidneys removed and quick-frozen in liquid nitrogen immediately after dissection. The kidneys were stored at -70°C until utilized in the subsequent GeneCalling^® protocols.

(ii) Isolation of Total Cellular RNA Total cellular RNA was extracted from 5 mg of heart, liver, fat. kidney, or brain tissue by initially grinding the tissue into a fine powder in liquid nitrogen. The powdered tissue was then transferred to a tube containing 500 μl Triazol Reagent^® (Life Technologies; Gaithersburg, MD) and was dispersed using a Polytron homogenizer (Brinkman Instruments; Westbury, NY). See e.g., Chomszynski, et al. 1987. Annal Biochem. 162 156-159; Chomszynski, et al, 1993. Biotechniques J_5_:532-533, 536-537. The total cellular RNA fraction was then extracted with 50 μl BCP (l-bromo-3-chloropropane: Molecular Research; Cincinnati, OH) to facilitate phase separation. The extraction mixture was centrifuged for 15 minutes at 4°C at 12,000 x G, and the aqueous phase was removed and transferred to a fresh tube. The RNA was then precipitated with 0.5 volume of isopropanol per original volume of Triazol Reagent^® used, and the sample was re- centrifuged at room temperature for 10 minutes at 12,000 x G. The supernatant was then discarded, the pellet washed with 70% ethanol and re-centrifuged at room temperature for 5 minutes at 12,000 x G. Finally the 70% ethanol was removed and the centrifuge tube was inverted and let stand to dry in this position. The resulting RNA pellet was re-suspended in 100 μl water (i.e., 1 μl/mg of original tissue weight) and heated to 55°C until completely dissolved. The final concentration of total cellular RNA was quantitated by fluorometry with OliGreen^® (Molecular Probes; Eugene, OR). In addition, the quality of the total cellular RNA was determined by both spectrophotometry and formaldehyde agarose gel electrophoresis. The total cellular RNA was stored at -20°C for subsequent utilization in the RT-PCR protocols.

(Hi) RNA Reverse Transcription (RT) Reactions

The initial reverse transcription reactions were performed as follows: 1 μg of total cellular RNA was mixed in 11 μl of RNase-free water (Ambion) with 1 μl (20:1 dilution; 100 pmoles/μl) oligo(dTX₅V primer (where V = A, C or G; Amitoff). Any RNA secondary structure was denatured by heating at 70°C for 10 minuets, followed by quick-chilling on ice. The denatured RNA was then collected by centrifugation for 15 seconds in a microfuge and to each tube was added: 4 μl 5X first-strand reaction buffer (BRL); 2 μl 0J mM DTT and 1 μl 10 mM dNTP mixture (Pharmacia). The reaction mixture was heated to 37°C for 2 minutes and 1 μl Superscript II reverse transcriptase (BRL) was added, followed by continued incubation at 37C for 1 hour.

(iv) PCR-Mediated Amplification of the RT Products Following reverse transcription, each of the samples were then subjected to PCR- mediated amplification. For a total of 10 samples, the following PCR reaction mixture was prepared: 50 μl 10X PCR buffer; lOμl dNTP mixture; 10 μl "sense" Primer (100 pmoles/μl; Pharmacia); 10 μl "anti-sense" Primer (100 pmoles/μl; Pharmacia); 2 μl KlenTaq (Life Technologies) and 418 μl RNase-free water. It should be noted that individuals skilled within the art may easily design both "sense" and "anti-sense" primers which would possess homology for the sequence(s) of interest. For the RT-PCR reactions. 49 μl of the PCR reaction mixture was added to 1 μl of the initial RT reaction and PCR amplification was performed for a total of 30 cycles in a thermal cycler under the following conditions: 96 C for 30 seconds; 57 C for 1 minute; 72°C for 2 minutes. The amplified products were then held at 4'C for subsequent analysis and agarose gel electrophoresis was performed to confirm the quality of the RT-PCR products.

Prior to further analysis, the RT-PCR products were subjected to a post-PCR clean up protocol as follows: MPG^® streptavidin magnetic beads (CPG: Lincoln Park, NJ) were prepared (3 μl of beads for every 5 μl of RT-PCR reaction product) by pre-washing the beads in 10 μl binding buffer (5 M NaCl, 10 mM TRIS, pH 8.0, 1 mM EDTA) per 5 μl original volume of RT- PCR reaction product. 10 μl of washed beads were dispensed in a 96 well FALCON^® TC plate for every RT-PCR sample processed. RT-PCR products were added to the beads, mixed well and incubated for 30 minutes at 50^°C. The sample volume was made 100 μl with binding buffer, the plate placed on a 96 well magnetic particle concentrator, and the beads allowed to migrate for 5 minutes. The liquid was then removed, and 200 μl washing buffer (10 mM Tris, pH 7.4, 10 mM EDTA) added per well. The washing step was then repeated.

For analysis, the beads were resuspended in 5 μl loading buffer (80% deionized formamide, 20% 25 mM EDTA, pH 8.0, 50 mg/ml Blue Dextran) per 5 μl of beads, and the supernatant was then analyzed by electrophoresis on an ABI 377^® (Applied Biosystems, Inc.) automated sequencer under denaturing conditions using the GeneSmay^® computer software (ABI) for analysis. A GeneSmay 500^® ROX ladder (diluted 1 : 10 in gel sample loading buffer) was utilized to a size control during the subsequent RT-PCR analysis.

(B) Physiological and Biochemical Significance of the Experimental Results

A total of 20,000 gene fragments, generated from approximately 10,000 genes, were compared, in triplicate, between renal tissue samples derived from each of the three rat strains.

A comparison of previously-characterized, "common" differentially-expressed genes between the diseased animals (i.e., the FHR and IRL strains) and the control (i.e., ACI strain) animals across time points was then performed. A total of 36 gene fragments were identified using this technique.

Subsequently, further analysis was provided through the utilization of the GeneCalling¹ methodology. By use of the GeneCalling^® methodology, the present invention discloses a total of 16 genes (GENE SET) which were found to be differentially-expressed among these rat strains. The differentially-expressed genes comprising the GENE SET are illustrated in Table 1. (i) The Zn-Peptidase (Aminopeptidase N

As illustrated in Table 1 a 45-fold decrease in mRNA expression of the Zn-peptidase (Aminopeptidase N; GenBank Ace. No. Z25073) were found in the FHR and ACI animals, in comparison to the mRNA levels in the ACI control rodents.

The Zn-peptidase (Aminopeptidase N) is a cell surface peptidase composed of a single type of subunit with characteristics typical of ectoenzymes. Ectoenzymes are integral plasma membrane proteins with the majority of the molecule containing catalytic sites exposed to the external, non-cytoplasmic surface. See e.g., Kenny & Turner. 1987. In: Mammalian Ectoenzymes, pp. 1-13 (Elsevier Scientific Publishing Co., Amsterdam). These enzymes, acting upon extracellular substrates, participate in the metabolism of secreted regulatory molecules and intestinal dietary substrates, as well as in the modulation of cell-cell interactions. See e.g., Luzio, et al, 1987. In: Mammalian Ectoenzymes, pp. 11 1-137 (Elsevier Scientific Publishing Co., Amsterdam). The Zn-peptidase (Aminopeptidase N) is anchored in the cell membrane via a hydrophobic domain which is adjacent to a small cytoplasmic region at the amino-terminus of the protein (see e.g., Feracci, et al. 1982. Biochim. Biophys. Ada 684:133-136) a short "stalklike" projection connects the transmembranal domain to the hydrophilic, extracellular region which comprises the majority of the molecule (see e.g., Hussain. et al, 1981. Biochem. J. 199:179-186). The catalytic activity of Zn-peptidase (Aminopeptidase N), which preferentially removes neutral amino-terminus amino acid residues from oligopeptides, is present in the extracellular region. See e.g., Louvard, et al, 1975. Biochim. Biophys. Ada 389:389-400. Zn- peptidase (Aminopeptidase N) has been demonstrated to be widely distributed in numerous tissue (including the central nervous system) and is particularly abundant in the kidney and intestinal microvilli. In a comparison of numerous tissues in the rat. Zn-peptidase

(Aminopeptidase N) transcripts were found to be approximately 5-fold higher in the kidney than in the next most abundant tissue, the lung. See e.g., Watt & Yip, 1989. J. Biol. Chem. 264:5480- 5487. These findings have led to the proposal that Aminopeptidase N functions to cleave dietary substrates prior to absorption in the intestine and to regulate the action of hormones and neurotransmitters by inactivating such peptides at the cell surface (see e.g., Turner, et al, 1987. In: Mammalian Ectoenzymes. pp. 21 1-248 (Elsevier Scientific Publishing Co., Amsterdam).

Human Zn-peptidase (Aminopeptidase N) has been shown to be encoded by a single gene (designated KZP), localized on chromosome 15. See e.g., Watt & Yip, 1989. J. Biol Chem. 264:5480-5487. In several mammalian species, including humans, two mRNAs of approximately 3.4 and 3.9 Kb. whose relative concentrations varied among different tissues, have been demonstrated. These multiple transcripts do not appear to be due to the presence of a pseudogene. The difference in mRNA size may be due either to: (i) varying lengths of 3'- or 5'-intronic region(s) or (ii) alternative splicing of a primary transcript from the same gene.

The 45-fold decrease in Zn-peptidase (Aminopeptidase N) mRNA expression in the FHR and IRL animals is interesting due to the normally high levels of this transcript in the kidney. As disclosed by the present invention, the FHR and IRL rat strains possess a dramatic decrease in the expression of the Zn-peptidase (Aminopeptidase N) transcripts. Accordingly, exogenous administration of this enzyme may function to ameliorate some of the deleterious physiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of Zn- peptidase (Aminopeptidase N) mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(ii) δ subunit of FlFo ATPase

As illustrated in Table 1 a 10-fold increase in the expression of δ subunit of FlFo ATPase (GenBank Ace. No. U00926) mRNA was demonstrated in the FHR and IRL animal strains, in comparison to the levels of this transcript in the ACI control animals.

The FlFo ATPase enzyme has been localized to the inner surface of the cytoplasmic membrane, where it catalyzes the interconversion of cellular ATP with the energy in the transmembranal electrochemical gradient of protons. The intrinsic membrane-bound proton channel (Fo) is comprised of 3 subunits: a, b and c; whereas the extrinsic catalytic sector (Fl) is comprised of 5 subunits: α, β. δ, ε and γ. See e.g., Senior, 1990. Ann. Rev. Biophys. Biophys. Chem. 19:7-41. The γ subunit plays an extremely important role in the catalytic function of the enzyme and has been implicated in both the assembly of the Fl sector (see e.g., Klionsky &

Simoni, 1985. J. Biol. Chem. 260:1 1200-11206) and in the gating of protons between the Fo and Fl sectors (see e.g., Yoshida, et al. 1977. Proc. Natl. Acad. Sci. U.S.A. 74:936-940). (Hi) Keratin 19

As illustrated in Table 1. a 7-fold increase in the expression of the keratin 19 gene (GenBank Ace. No. x81449) was demonstrated in the FHR and IRL strains, in comparison to the levels of this transcript in the ACI control animals. Keratin 19 is an intermediate filament polypeptide found in diverse types of epithelial cells, particularly in simple epithelia. See e.g., Moll, et al. 1982. Cell 3L11-24. Keratin 19 possesses a molecular weight of 40 kDal and an isoelectric point of 5J (classified as an acidic polypeptide), thus making it the smallest of the known, major cytokeratins.

The human urogenital tract epithelium (e.g., prostate and kidney) has been shown to contain fairly high levels of keratin 19 by immunohistochemical staining methods. See e.g.,

Nagle, et al, 1991. Am. J. Path. 138:119-128. In the prostate, keratin 19 was demonstrated to be expressed in a heterogeneous manner and occurred in both the basal and luminal cells of normal, dysplastic and benign hyperplastic tissues. See e.g., Peehl. et al, 1996. Cell Tissue Res. 285:171-176. Interestingly, cells derived directly from prostatic carcinoma and from several cell lines derived from neoplastic epithelium (e.g., PC-3 prostatic carcinoma cell line) have also been demonstrated to contain keratin 19. although in a minority of cells. See e.g., Sherwood, et al, 1990. J. Urol 143:167-171.

(iv) Brain Calbindin-d28k (CaBP28K) As illustrated in Table 1 , the expression of the brain calbindin-d28k (CaBP28K) gene

(GenBank Ace. No. m27839) was demonstrated to be decreased 6-fold in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain.

Brain calbindin-d28k (CaBP28K) is a member of the EF-hand family of Ca -binding proteins (CaBPs) which participate in many physiological processes involved in the sequestering and modulation of free, intracellular Ca²⁺. See e.g., Leathers, et al, 1990. J. Biol. Chem. 265:9838-9841; Ren & Ruda, 1994. Brain Res. Rev. 19:163-179. While the majority of localization studies have been limited to the central nervous system, recent experimental findings have demonstrated brain calbindin-like protein in several tissues including the kidney. See e.g., Frantz, et al, 1994. J. Neurosc Res. 37:287-302. Brain calbindin is frequently co-localized with other CaBPs including: calretinin and parvalbumin. See e.g., Arai, et al, 1994. Histochemistry 101 :9- 12.

Although little is currently known of the function of CaBPs (e.g., calbindin) in the central nervous system, and especially in other tissue types, they are primarily thought to function as Ca^2τ buffering and sequestering proteins. Microinjection of high concentrations of calbindin and/or parvalbumin have been shown to reduce the transient increase of free Ca^2* concentration produced by brief depolarizations in dorsal root ganglion neurons. See e.g., Chard, et al, 1993. . J. Physiol 472:341-357. In addition, calbindin in oxytocin magnocellular neurons in the hypothalamus was demonstrated to be expressed in extremely high levels, thus allowing for a high degree of Ca²⁺ buffering action. See e.g., Cobbett, et al, 1986. Brain Res. 362:7-16. These high levels of expression are though to protect the neurons against the neurotoxic effects of excess Ca²⁺ during prolonged periods of intense neuronal activity where intracellular levels of Ca²⁺ are markedly elevated. Similarly, level of various ions and electrolytes (e.g., Ca²J are altered in hypertension, renal disease, protenuria and the like.

Hence, the decreased levels of calbindin transcripts (and possibly the calbindin protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. Due to the ability of calbindin to modulate intracellular Ca^2÷ concentration, administration of exogenous calbindin to individuals suffering from renal disease and/or associated disorders may help ameliorate some of the deleterious physiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of calbindin mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(v) Tissue Inhibitor of Metalloproteinase 3 (TIMP-3)

As illustrated in Table 1, the expression of the tissue inhibitor of metalloproteinase 3 (TIMP-3) gene (GenBank Ace. No. u27839) was demonstrated to be decreased 5-fold in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain. Tissue inhibitors of the matrix metalloproteinases (MMPs) include TIMP-1, TIMP-2 and

TIMP-3. See e.g., Anand, et al, 1996. Biochem. Cell Bio. 74:854-8861. Although all the TIMPs are similar to one another to the extent of only 35-40% amino acid identity, some key similarities suggest significant tertiary structural conservation with only subtle differences in surface and internal topography. For example, all TIMPs possess 12 cysteine amino acid residues at conserved locations and. in the case of TIMP-3, it has been shown that these residues participate in the formation of 6 intrachain disulfide bonds (see e.g., Apte. et al, 1994. Genomics 19:86-90). It is also assumed that this complex folding is responsible, in-part. for the thermodynamic stability of TIMP-3. Additionally, all TIMPs share the property of MMP inhibition (e.g., inhibition of MMP-1, MMP-2, MMP-3 and MMP-9), although there appears to be subtle differences in their interactions with the zymogen form of the MMP enzyme. For example, TIMP-3 possesses poor aqueous solubility and is localized in the extracellular matrix (ECM), but in cultured cells TIMP-3 is found only in the substratum and not within the conditioned medium. See e.g., Blenis & Hawkes, 1984. J. Biol. Chem. 259:1 1563-11570. While the ECM ligand(s) of TIMP-3 have not yet been identified, it has been suggested that one possible ligand may be hyaluronic acid. See e.g., Alexander, et al, Development (Cambridge) 122:1723-1736. Therefore, the affinity of TIMP-3 for the ECM, as well as its expression within a number of epithelia, has led to the proposal that TIMP-3 may be a component of the basement membrane. See e.g., Apte, et al,

1994. Genomics 19:86-90. Accordingly, it has been hypothesized that the more variant carboxyl- terminal region of the TIMPs may subserve the distinctive properties of each TIMP species and that the carboxyl-terminal region of TIMP-3 possesses the ECM-binding domain.

The tissue inhibitor of metalloproteinase 3 (TIMP-3) is a transiently-expressed, secreted 24-25 kDal protein 188 amino acid residues in length. See e.g., Apte, et al, 1994. ev. Dyn. 200:177-197. TIMP-3 is a highly basic protein, with a pi of approximately 9.0. An N-linked glycosylation site, highly-conserved in all species examined, suggests that TIMP-3 may exist in a glycosylated native form (see e.g., Apte, et al, 1994. Genomics 19:86-90), although the biological significance of this glycosylation is unclear at the present time. The human TIMP-3 gene has been localized to chromosomal position 22ql3J and the complete structure of the human TIMP-3 gene has been determined and is comprised of a total of 5 exonic regions and is at least 30 kb in size. See e.g., Apte, et al. 1994. Genomics 19:86-90. Multiple TIMP-3 transcripts (i.e., 2.4, 2.8 and 5.0 kb in human placenta) have been identified (see e.g., Apte, et al, 1995. J. Biol. Chem. 270:14313-14318) and are thought to result from the differential utilization of poly-adenylation signals within the TIMP-3 gene (see e.g., Byrne, et al,

1995. Mol. Med. 1:418-427). Interestingly, the human TIMP-3 gene has been found to be a TATA-less gene which, nonetheless, commences transcription at a single site (see e.g., Apte, et al, 1995. J. Biol. Chem. 270:14313-14318). although multiple transcriptional start sites have been found in the murine TIMP-3 gene. Developmental analysis studies have demonstrated that the human TIMP-3 gene is stringently regulated in vivo and are expressed in such a manner as to minimize overlap in gene expression. See e.g., Anand-Apte, et al. 1996. Biochem. Cell Bio. 74:854-861. Unlike the other TIMPs, TIMP-3 is predominantly expressed in high levels in many epithelial tissues and, in adult tissues, the highest levels of TIMP-3 expression are found in the kidney. See e.g., Apte. et al. 1994. Genomics 19:86-90.

Hence, the decreased levels of TIMP-3 transcripts (and possibly the TIMP-3 protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. As the levels of TIMP-3 are normally high in human kidney, administration of exogenous TIMP-3 may help ameliorate some of the deleterious physiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of TIMP-3 mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(vi) Integral Membrane Protein 1 (Itml) and the RTl.B-lα chain of Itml As illustrated in Table 1. the expression of the tissue inhibitor of integral membrane protein 1 (Itml) gene (GenBank Ace. No. L34260) was demonstrated to be decreased 5-fold in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain. In contrast, as illustrated in Table 1, the expression of the RTl.B-lα mRNA (GenBank Ace. No. X14879) was increased 20-fold in the FHR and IRL animals.

The integral membrane family of proteins are primarily involved in the synthesis of numerous bioactive polypeptides. See e.g., Chen & Shields, 1996. J. Biol. Chem. 271 :5297- 5300; Miller, et al, 1992. J. Cell Biol. 118:267-283. The human Itml gene has been localized to 1 Iq23-q24 (see e.g., Hong, et al. 1996. Genomics 31J95-300), a chromosomal region associated with translocations. several oncogenes and the human congenital pre-neoplastic syndrome, ataxia telangiectasia (see e.g., Gatti. et al, 1988. Nature 336:577-580). For example, the rare translocation (1 l,14)(q23, q32) translocation associated with malignant non-Hodgkin lymphoma has been shown to be associated with this chromosomal region. See e.g., Bloomfield, et al, 1983. Cancer Res. 43:2975-2884. In addition, the cloning of a novel 1 lq23 breakpoint from a non-Hodgkin lymphoma and the mapping of this breakpoint to this region on chromosome 11 was recently reported. See e.g., Meerabux. et al, 1994. Oncogene 9:893-898. Both the human and murine integral membrane protein 1 (Itml) genes have been shown to encode a 2.1 kb mRNA transcript which appears to be differentially polyadenylated, as revealed by the isolation of cDNA clones with poly(A)-tails at various locations within the molecule (see e.g., Hong, et al, 1996. Genomics 3J J95-300). There appears, however, to be one primary site of polyadenylation in both the human and murine Itml transcripts. The biological significance of this differential polyadenylation is not clear at this time. In addition, the Itml promoter has been partially characterized and is comprised of a degenerated TATA box and several potential binding sites for transcriptional factors SP1, GCF, E2A. Myb and PPAR. See e.g., Ness. et al, 1989. Cell 59:1 1 15-1125. Both the human and murine Itml proteins are comprised of 705 amino acid residue protein with average molecular weights of 80.597 and 80.572 daltons, respectively. See e.g., Hong, et al, 1996. Genomics 3L295-300. One of the most striking structural feature of the Itml protein is the presence of a total of 10-14 computer-predicted transmembranal domains which is highly suggestive of Itml being a transmembranal protein. There is a remarkable degree of amino acid sequence conservation between the human and murine proteins, with over a 98.5% homology. This finding suggests that Itml is under severe structural constraints to exert is biological function(s), Homology searches performed within public DNA and protein databases have identified Itml homology with the T12A2J gene from C. elegans and the STT3 gene from S. cerevisiae. See e.g., Hong, et al. 1996. Genomics 31J95-300. In addition, a short human cDNA sequence deposited as an EST Sequence (Z13858) is believed to be the human counterpart of the STT3 gene from S. cerevisiae. See e.g., Hong, et al. 1996. Genomics 31 :295-300.

The putative biological function(s) of Itml gene product are unclear at the present time. A recent study has shown that no homology exists between the human Itml gene product and various members of a large, family of cell surface receptors possessing up to 7 transmembranal domains. See e.g., Fei, et al, 1994. Nature 368:563-566. In addition, Itml does not appear to contain domains with enzymatic activity. Thus, while Itml is most probably not directly involved in transmembranal signaling, its plurality of transmembranal domains is a characteristic feature of transporter proteins such as the ABC (ATP binding cassette) superfamily of active transporters (see e.g., Howard, 1993. In: Molecular Biology of Membranes: Structure and Function pp. 27-32 (Plenum Press, New York, NY). Specific examples of non-ABC transporter proteins which possess greater than 7 transmembranal domains (TMDs) include: (i) the 11 TMD NaVglucose co-transport protein (see e.g., Hediger, et al. 1987. Nature 330:379-381); (ii) the 12 TMD proton-coupled oligopeptide transport protein (see e.g., Fei. et al, 1994. Nature 368:563- 566) and (Hi) the 10 TMD vasopressin-regulated urea transport protein (see e.g., You, et /., 1993. Nature 365:844-847. Thus, it is believed to be highly likely that the Itml protein functions as a novel type of permease/transporter transmembranal protein.

Hence, the decreased levels of Itml transcripts (and possibly the Itml gene product) and/or the increased levels of the RTl .B-lα chain of Itml which are disclosed by the present invention, within the FHR and IRL renal disease animal models, may play a role in the etiology of renal disease and associated disorders. For example, the concomitant increase in the levels of the RTl.B-lα chain of Itml and the decreased levels of the full-length transcript of Itml may be illustrative of a defects in transcriptional promotion, mRNA splicing or the like. It is possible that the administration of exogenous Itml or, conversely, the administration of an antagonist of

RTl.B-lα chain of Itml (should such markedly-elevated levels prove physiologically harmful) may help ameliorate some of the deleterious physiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of Itml mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(vii) rab GDI-β

As illustrated in Table 1. there was a 4-fold decrease in the expression of the rab GDI-β gene (GenBank Ace. No. x74401) gene in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain.

The rab i_^nily of small, GTP-binding proteins (G-proteins) regulate various formi-tJf intracellular vesicular transport such as exocytosis, endocytosis and transcytosis. See e.g., Novick & Brennwald, 1993. Cell 75:597-601. A synopsis of one proposed mode of action (see e.g., Zerial & Stenmark, 1993. Cwrr. Opin. Cell Biol. 5:613-620) of rab small G-proteins is as follow: when the GDP-bound form of rab small G-protein in the cytosol is converted to the

GTP-bound form, it interacts and transports its specific vesicle to its specific adaptor membrane. Following the fusion of the vesicle with the adapter membrane, the GTP-bound form of the rab small G-protein is converted to the GDP-bound form, which is then translocated from the membrane to the cytosol. In this model, the conversion of the rab small G-protein between the GTP-bound and GDP-bound forms and its cyclical translocation between the vesicle/membrane and cytosolic fractions are essential for biological function.

The rab GDP-disassociation inhibitor (rab GDI) is a cytosolic protein which has been shown to inhibit the disassociation of GDP from, and the subsequent binding of GTP to, the rab3 A protein. See e.g., Sasaki, et al, 1990. J. Biol Chem. 265:2333-2337. It has been further shown that rab GDI forms a stable ternary complex with the GDP-bound form of the lipid- modified rab3A protein, but neither with the GTP -bond form of the lipid-modified rab3A, nor with the GDP-bound or GTP-bound form of the lipid now-modified rab3A protein. See e.g., Araki. et al, 1991. Mol. Cell. Biol. U : 1438-1447. In addition, rab GDI possesses activities to: (i) inhibit the binding of the GDP-bound form of the lipid-modified rab3A, but not the GTP- bound forms, to membranes and (ii) induce the disassociation of the GDP-bound for of the lipid- modified rab3A protein from membranes. See e.g., Araki, et al, 1990. J. Biol Chem. 265:13007-13015. Moreover, rab GDI is active not only on rab3A. but also on all other members of the rab protein family, thus far characterized, as well. See e.g., Beranger, et al, 1994. J. Biol. Chem. 269:13637-13643; Soldati, et al, 1993. Mol Biol. Cell. 4:425-434. Accordingly, on the basis of these biochemical findings, the function of rab GDI as a regulatory protein for the cyclical translocation of the rab small G-proteins. has become a well-established tenet. Two forms of the rab GDI protein have now been characterized from the rat ~ rab GDI-α and GDI-β. See e.g., Nishimura, et al, 1994. J. Biol. Chem. 269:14191-14198. While the rat rab GDI-α proteins appears to be the counterpart of bovine rab GDI (see e.g., Matsui. et al, 1990. Mol. Cell. Biol. 10:4116-4122), the rat rab GDI-β protein appears to belong to a different isoform. A recent study (see e.g., Araki, et al, 1995. Biochem. Biophys. Res. Comm. 211 :296- 305) has examined the biochemical characteristics and functions of rat rab GDI-β. In general, the biochemical characteristics of rab GDI-β are indistinguishable from those of rab GDI-α, including such characteristics as: (i) the inhibitory effect on the disassociation of GDP from rab3A; (ii) the substrate specificity,; (Hi) the requirement of the post-translational lipid modifications of rab3A; (iv) the stoichiometric interaction with the GDP-bound form ofrab3A; (v) the inhibitory effect on the binding of rab3A to the membrane and (vi) the stimulatory effect on the dislocation of rab3A from the membrane. In addition, the concentration of rab GDI-β required for the inhibition of the GTP/GDP exchange reactions is similar to that of rab GDI-α.

It is believed that the two different isoforms (i.e., αand β) of rab GDI primarily exist as a function of differences in organ/tissue distribution and intracellular localization. See e.g., Araki, et al, 1995. Biochem. Biophys. Res. Comm. 21 1 :296-305. For example, rab GDI-α is expressed in high levels in the brain and in much lower levels in other tissues; whereas rab GDI-β are ubiquitously expressed. See e.g., Shisheva, et al. 1994. Mol. Cell. Biol. 14:3459-3468. Similarly, rab GDI-α is a totally cytosolic protein; whereas a high concentration rab GDI-β is found to be associated with membranes. See e.g., Shisheva, et al. 1994. J. Biol. Chem. 269:23865-23868. On the basis of these differences, it is though that the rab GDI-α and rab GDI-β isoforms may have different biological function(s). See e.g., Shisheva, et al, 1994. J. Biol Chem. 269:23865-23868. Specifically, rab GDI-β has recently been demonstrated to deliver the rab small G-protein to their specific acceptor membranes, accompanied with their GTP/GDP exchange reactions. See e.g.. Soldati & Shapiro. 1994. Nature 369:76-78. Thus, it is possible that the interactions of each isoform with other proteins such as a GDI-displacement factor (see e.g., Soldati & Shapiro. 1994. Nature 369:76-78) or a stimulatory GEP which functions to stimulate the GDP/GTP exchange reaction, may be different. Hence, the decreased levels of rab GDI-β transcripts (and possibly the rab GDI-β protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. As rab GDI-β is ubiquitously expressed in numerous tissues, the administration of exogenous rab GDI-β may serve to ameliorate some of the deleterious physiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of rab GDI-β mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(viii) PC4 Gene Encoding IRPR (IFN-β) As illustrated in Table 1, there was a 3.4-fold decrease in the expression of the PC4 gene which encodes IRPR (IFN-β; GenBank Ace. No. J04511) in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain.

The interferons (IFNs) are a heterogeneous family of secreted polypeptides which possess multiple biological functions. While IFNs are essential components of the host defense mechanism against viral infections, they also play a critical role in cell growth and differentiation, as well as in other immunoregulatory functions. See e.g., Seen & Lengyel, 1992. J. Biol. Chem. 267:5017-5020.

The IFN family has been divided into two major groups based upon differences in their structure, function and/or modes of synthesis. The first group includes the IFN-α/β family (also known as type I IFN) which consists of 20 highly-similar genes encoding IFN-α localized on chromosome 9 in humans and a total of 3 genes encoding IFN-β localized on chromosomes 2, 5 and 9 in humans. See e.g., Thanos. 1996. Hypertension 27:1025-1029. The second ground (also known as type II IFN) consists of a single gene encoding IFN-γ. The group I IFNs (IFN-α and IFN-β) are rapidly induced in almost every cell type following viral infection; whereas the group II IFN (IFN-γ) is produced by activated T-cells and natural killer cells. See e.g., Seen & Lengyel, 1992. J. Biol. Chem. 267:5017-5020.

Interferon-β (IFN-β) is (primarily) synthesized by fibroblastic and epithelial cells and possesses a 30% homology to INF-α. As previously discussed, the genes encoding human IFN-β have been localized to chromosomes 2, 5 and 9: whereas the gene encoding the glycoprotein receptor for IFN-β have been localized to chromosome 21. Following synthesis. IFN-β is sequestered into vesicles and secreted. See e.g., Miyamoto, et al. 1988. Cell 54;903- 913. The majority of study of the biological effects of IFN-β have been as anti-viral (see e.g., Mims & White, 1984. In: Viral Pathogenesis and Immunology (Oxford Press; Blackwell, Great Britain)) and as anti-neoplastic (see e.g.. Vilcek, 1990. In: Handbook of Experimental Pharmacology (Springer- Verlag; Berlin. FRG)) agents.

The inducible enhancer of the IFN-β gene is comprised of overlapping regulatory elements which are recognized by a distinct set of transcriptional factors that may be activated not only by viral infection, but by various other extracellular signals. See e.g., Goodbourn & Maniatis, 1988. Proc. Natl. Acad. Sci U.S.A. 85:1447-1451. Additional studies have also demonstrated that direct protein-protein interactions are involved in the transcriptional synergism between the various, distinct elements of the IFN-β promoter. See e.g., Du. et al, 1993. Cell 74:887-898. Interestingly, the related IFN. IFN-γ has been found to cause upregulation of Class 1 major histocompatibility complex (MHC) expression on the β-cells of the pancreatic islets of Langerhans in insulin-dependent diabetes mellitus (IDDM). See e.g., Thomas, et al, 1998. J. Clin. Investig. 102:1249-1257. The in vitro effects of various cytokines (including IFN-β) have putatively been linked to the pathogenesis of IDDM (see e.g.. Rabinovitch, 1998. Roles of Cell- Mediated Immunity and Cytokines in the Pathogenesis of Insulin-Dependent Diabetes Mellitus, In: Diabetes Mellitus: A Fundamental and Clinical Text (Lippincott-Raven Publishers, Philadelphia, PA), although, in many cases, their exact in vivo biological function(s) is unclear at the present time.

The decreased levels of IFN-β transcripts (and possibly the IFN-β protein) disclosed in the present invention, within the FHR and IRL renal disease animal models, may play a role in the etiology of renal disease and associated disorders. Although the expression of IFN-β is primarily upregulated in response to viral infection, this protein is ubiquitously expressed in numerous tissues and serves as a cell growth/differentiation molecule. Hence, the administration of exogenous IFN-β may serve to ameliorate some of the deleterious physiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of IFN-β mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders. (ix) Organic Cation Transporter Protein-2 (OCT2)

As illustrated in Table 1. there was a 2.5-fold decrease in the expression of the organic cation transporter protein-2 (OCT2) gene (GenBank Ace. No. d83044) in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain. In a recent study (see e.g., Grundermann, et al, 1994. Nature 372:549-552) using functional expression cloning, a cDNA encoding an organic cation transporter protein 1 (OTCl) has been isolated and characterized from the rat kidney. OTCl was demonstrated to be a 556 amino acid residue protein which mediates the active, unidirectional transcellular transport of cationic drugs (e.g., tetraethylammonium, procainmide and cimetidine) and various endogenous metabolites (e.g., N'-methylnicotinamide) through the plasma membrane of the proximal tubular cells of the kidney. In related study (see e.g., Okuda, et al, 1996. Biochem. Biophys. Res. Comm. 224:500-507), a related organic cation transporter protein (OTC2) in the rat kidney was isolated and examined for functional expression and tissue distribution. The rat kidney OTC2 was demonstrated to be a 593 amino acid residue protein with a molecular weight of approximately 66 kDal (including a poly(A) tail). Overall amino acid identity with OTCl was found to be 67%; whereas homology within the highly-conserved α-helical regions was on the order of 85%. Hydrophobicity analysis indicated that OCT2 possesses a total of 12 putative transmembranal α-helices and 2 putative N-linked glycosylation sites. In addition, 4 putative protein kinase A phosphorylation sites and 2 putative protein kinase C phosphorylation sites were identified with the predicted intracellular domains of the OTC2 protein.

Tissue distribution studies of the OCT2 protein demonstrated high levels of expression within the kidney, especially in the medulla. In contrast, OCTl mRNA levels were found to be markedly higher in the cortical region of the kidney, rather than in the medulla (see e.g., Grundermann, et al, 1994. Nature 372:549-552). Interestingly, while OCT2 mRNA was not detected in the brain, heart, lung, liver, small intestine or spleen; OCTl was found in the kidney, liver, small intestine and liver (see e.g., Grundermann, et al, 1994. Nature 372:549-552). Therefore, OCT2 demonstrated a markedly different tissue distribution pattern in comparison to that found for OCTl.

Studies examining the biological function of the OCT2 protein established a dramatic inhibition of tetraethylammonium uptake in the presence of cimetidine, procainmide and quinidine. This inhibition was attenuated somewhat by an increase in pH from 5.4 to 8.0. In accord, this data suggests that OCT2 is independent of the proton gradient, medium pH or HVorganic ion concentration and is likely to be distinct from the H /organic ion antiporter protein

(see e.g., Okuda, et al, 1996. Biochem. Biophys. Res. Comm. 224:500-507).

The decreased levels of OCT2 transcripts (and possibly the OCT2 protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. A standard methodology to ascertain the role of physiological role of transporter proteins (e.g., OCT2) involves the blocking of the active transport mechanism of the transporter protein. For example, "libraries" of small molecules may be screened for their ability to inhibit or augment transporter protein function. The active transport of cationic drugs and various endogenous metabolites may be decreased as a result of the low levels of OCT2 expression in renal disease, thus concomitantly reducing the effectiveness of pharmacological treatments and/or causing the intracellular build-up of potentially deleterious metabolites. Therefore, as OCT2 is expressed in high levels within the non-pathologic kidney, the administration of exogenous OCT2 may serve to ameliorate some of the pathophysiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of OCT2 mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(x) L-Arginine:Glvcine Amidinotransferase (AT)

As illustrated in Table 1, there was a 16-fold increase in the expression of the L-arginine:glycine amidinotransferase gene (GenBank Ace. No. U07971) in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain.

L-arginine:glycine amidinotransferase (Human AT; GenBank Ace. No. X86401) catalyzes the transfer of the amidino group from L-arginine to glycine. The resultant guanidinoacetic acid is the intermediate precursor of creatine. See e.g., Walker, 1973. Enzymes 9:497-509. Creatine and its phosphorylated from play an important role in energy metabolism of numerous tissues, acting as a dynamic reservoir of high-energy phosphate which serves to buffer the rapid fluctuations of the ATP/ADP ratio during periods of high cellular activity (e.g., action potentials in neurons). See e.g., Walker, 1979. Adv. Enzymol. Relat. Areas Mol. Biol. 50:177- 242. While the highest tissue concentrations of creatine and creatine phosphate are found in the skeletal muscle, heart, spermatozoa and the photoreceptor cells of the retina, most creatine is not synthesized in these tissues but is taken-up from the blood. In contrast, the major site of creatine synthesis are the kidney, liver and pancreas, where the AT is located in the cytoplasm and in the intermembrane space of the mitochondria. See e.g.. Margi, et al. 1975. FEBS Lett.

55:91-93.

The human AT gene has been mapped to the locus D15S109E on the distal part of the chromosome band 15ql5J (see e.g.. Fougerousse. et al, 1994. Hum. Mol. Genet. 3:285-293). Two isoforms of the AT enzyme have been isolated, and are believed to represent the cytosolic and mitochondria versions of this enzyme which are derived from the same gene by alternative splicing. See e.g., Humm, et al, 1997. Biochem. J. 322:771-776. Additionally, the AT gene lies within a chromosomal region which demonstrates a significant linkage disequilibrium for limb- girdle muscular dystrophy type 2A (LGMD2A; see e.g., Chiannilkulchai, et al, 1995. Hum. Mol. Genet. 4:717-725). AT is inhibited in gyrate atrophy of the choroid and retina with concomitant hyperornithinaemia, due to the absence of L-ornithine-2-oxoacid aminotransferase, resulting in a 10- to 20-fold increase in plasma and urinary concentrations of ornithine. See e.g., Sipila, 1980. Biochim. Biophys. Ada 613:79-84. In contrast. AT expression is markedly down-regulated in Wilms tumor of the kidney. See e.g.. Austruy, et al, 1993. Cancer Res. 53:2888-2894. The markedly increased levels of L-arginine:glycine amidinotransferase (AT) transcripts

(and possibly the AT protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. As the kidney is normally involved in a high rate of creatine and creatine phosphate biosynthesis in a non-pathologic state, the elevated level of AT may serve to dramatically upregulate the in situ biosynthesis of creatine and creatine phosphate within the renal disease kidney. It should also be noted that the levels of both plasma creatine and creatine phosphate are typically elevated in both renal disease and hypertension. Accordingly, therefore, the administration of an AT antagonist or inhibitor may serve to ameliorate some of the pathophysiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of AT mRNAs may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(xi) Protein Phosphatase 1-β

As illustrated in Table 1. there was a 4-fold increase in the expression of the protein phosphatase 1-β (GenBank Ace. No. s78218) gene in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain.

Protein phosphatase 1 (PP1) has pleiotropic actions within eukaryotic cells. Although it was initially identified as a key enzyme in the hormonal regulation of glycogen metabolism, it is known to play important roles in the control of muscle contraction and protein synthesis, as well as being essential for the completion of cell division (see e.g . Cohen, 1989. Ann. Rev. Biochem. 54:453-508; Cohen & Cohen, 1989. J. Biol. Chem. 264:21435-21438). PP1 functions in the dephosphorylation of serine and threonine residues and is inhibited by thermostable proteins, inhibitor 1 and inhibitor 2, the tumor-promoter okadaic acid and the hepatotoxin microcystin. The PP1 catalytic subunit has been demonstrated to be complexed with a variety of proteins in vivo, which are involved in the folding of the active site amino acids (inhibitor 1; see e.g., Alessi, et al, 1993. Eur. J. Biochem. 213:1055-1066) and in targeting it to particular subcellular locations, such as glycogen particles (the G subunit) and the muscle contractile apparatus (the M subunit). See e.g., Hubbard & Cohen. 1993. Trends Biochem. Sci. 18:112-111.

A total of isoforms of the catalytic subunit have been identified in rodents (PPl-α, PPl-β and PPl-γ; see e.g., Ohkura, et al. 1989. Cell 57:997-1007); whereas currently only PPl-α and PPl-γ have been isolated and sequenced in humans (see e.g.. Barker, et al, 1993. Biochim. Biophys. Acta 1178:228-233). A recent study (see e.g., Barker, et al, 1994. Biochim. Biophys. Acta 1220:212-218) has localized the human PPl-β gene to chromosomal position 2p23, and this PPl-β gene is unlinked to those gene encoding human PPl-α (1 lql3) and PPl-γ (12q24). The human PPl-β gene possesses an open reading frame (ORE) of 981 nucleotides encoding a protein with an approximate molecular weight of 37 kDal. These findings are identical to that reported for rabbit PPl-β (see e.g., Dombradi, et al, 1990. Eur. J. Biochem. 194:739-745) and rat PPl-γ (see e.g., Sasaki, et al, 1990. Jpn. J. Cancer Res. 81:1272-1280). It should be noted that human PPl-β possesses a 90% homology to human PPl-α and PPl-γ; whereas human PPl-α and PPl-γ are even more closely related to one another possessing a 93-94% homology. See e.g., Barker, et al, 1994. Biochim. Biophys. Acta 1220:212-218. Several mRNA species (i.e.. 5.4 kb. 3.0 kb and 2.0 kb) have been demonstrated for human PPl-β, and may represent alternative splicing of the human PPl-β mRNA. Additionally, these mRNA species are differentially-expressed within tissues. For example, in the kidney the ration of the 5.4 kb to 3.0 kb mRNA species is 0.4: 1. See e.g., Barker, et al, 1994. Biochim. Biophys. Acta 1220:212-218. The increased levels of PPl-β transcripts (and possibly the PPl-β protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. Therefore, the administration of an PPl-β antagonist or inhibitor may serve to ameliorate some of the pathophysiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of PPl-β mRNA may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(xii) Kallikrein

As illustrated in Table 1, there was a 3.5-fold decrease in the expression of the kallikrein protein (GenBank Ace. No. ml 9647) gene in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain.

Kallikreins are a family of proteolytic enzymes (i.e., serine proteases) with a substrate preference for cleaving arginine amino acid residues. See e.g., Schachter, 1979. Pharmacol. Rev. 3 _.:1-17. There are two primary groups of kallikreins - glandular/tissue kallikrein and plasma kallikrein. Plasma kallikrein circulates as an inactive precursor, which is synthesized in the liver and activated via the Hageman factor. See e.g., Scicli & Carretero. 1986. Kidney Int. 29:120- 130. Tissue kallikreins differ in both size and specificity from the plasma-based enzyme, and may be detected in pancreas, submandibular glands, brain, reproductive organs, heart, blood vessels and the kidney. See e.g., Nolly, et al, 1990. Hypertension 16:436-440. Tissue kallikreins possess molecular weights of 24-45 kDal, where the majority of the variation in molecular weight is due to differences in post-translational glycosylation (e.g., the human renal kallikrein is ~ 20% carbohydrate). Although these enzymes are highly homogeneous, they nonetheless exhibit distinct differences in substrate recognition, which is reflected in identifiable differences in the amino acid sequences of the enzymes. See e.g., MacDonald, et al, 1988. Biochem. J. 253:313-321. The active site amino acid residue triad which is considered to be essential for kallikrein enzymatic activity is comprised of His-41, Asp-96 and Ser-189, and is the primary determinant of the enzyme's cleavage specificity. See e.g., Bothwell, et al, 1979. J.

Biol. Chem. 254:7287-7294. The purified renal kallikrein is initially synthesized as a zymogen (designated pro-kallikrein) with an attached 17 amino acid residue signal peptide preceding a 7 amino acid residue activation sequence, which must be enzymatically-cleaved prior to activation of the enzyme. Nonetheless, in all species thus far examined, a single gene encodes the enzyme which has been designated "true tissue kallikrein" (EC 3.4.21.35). which is the predominant kinnogen-cleaving enzyme in most tissues, including the kidney. See e.g., Scicli & Carretero, 1986. Kidney Int. 29:120-130. This kallikrein has been shown to be identical to the enzyme species found in the urine. See e.g., Baker & Shine, 1985. DNA 4:445-450. The tissue kallikreins are encoded by a highly, conserved clusters of genes which vary in number between the different mammalian species. For example, the kallikrein multigene family is comprised of a total of 20 and 24 genes in the rat and mouse, respectively. See e.g., Wines, et al, 1989. J. Biol. Chem. 264:7653-7662; Gerald, et al, 1986. Biochim. Biophys. Acta 866:1-14. Clements, et al, 1990. J. Biol. Chem. 265: 1077- 1081). All tissue kallikrein genes, regardless of species, consist of 5 exonic and 4 intronic regions. See e.g., Wines, et al, 1989. J. Biol. Chem. 264:7653-7662. In humans, the kallikrein gene has been localized to chromosomal position 3q26- qter, in close proximity to the two related genes encoding the α-HS glycoprotein and the histidine-rich glycoprotein See e.g.. Mϋller-Esterl & Nakaniski, 1986. Trends Biochem. Sci. JJ J36-339.

At present, only a limited number of kallikrein family gene products have been identified and characterized. Other characterized enzymes of the rodent kallikrein enzyme family include: (i) rat tonin, which can cleave angiotensin and other polypeptide hormone precursors in vitro (see e.g., Lazure, et al, 1987. Biochem. Cell Biol. 65:321-337); (ii) the γ subunit of murine nerve growth factor (NGF) which processes both the NGF precursor and the murine epidermal growth factor (EGF)-binding protein, which subsequently processes the precursor of EGF (see e.g., Drinkwater, et al, 1987. Biochemistry 26:6750-6756).

In humans, prior to the present invention, a total of only 3 tissue kallikrein-like genes or gene products have been identified, comprising the hRKALL, hGK-1 and PSA genes tandemly- arranged on the long-arm of chromosome 19 at ql3J-13.4 (see e.g., Evans, et al, 1988. Biochemistry 27:3124-3129), in a position analogous to the murine kallikrein locus on chromosome 7 (see e.g., Murray, et al, 1990. J. Cardiovascular Pharmacol 15(Supple. 6):S7- S15). Although the kallikrein gene has been cloned and sequenced and its expression detected in several other glandular tissues and organs (see e.g., Baker & Shine. 1985. DNA 4:445-450), prior to the present invention, intra-renal expression of the "true" kallikrein gene had previously not been demonstrated in humans. Tissue kallikreins which are produced by such organs as the submandibular glands, have been shown to reach the systemic circulation, where it was able to be filtered from the plasma and reabsorbed from the lumen by renal cells. See e.g., Rabito, et al, 1983. Circ Res. 52:635-641. This finding may help explain the absence of intra-renal gene expression. An alternative hypothesis for the lack of intra-renal expression is that, under basal conditions, the renal kallikrein gene is not constitutively expressed, but when renal release of kallikrein is stimulated (by physiological or pathological stimuli), de novo expression occurs to restore tissue stores. See e.g., Cumming, et al. 1994. Clin. Sci. 87:5-11. The different members of the kallikrein family are believed to play diverse enzymatic roles by selectively converting peptide pro-hormones and growth factors into biologically-active molecules. For example, kallikrein functions to release the potent vasodilatory peptides, the kinins (e.g., bradykinin and lysl-bradykinin), from a plasma globulin kinnogen (see e.g., Fuller & Funder, 1986. Kidney Int. 29:953-964). The kallikrein-kinin system, through its marked effects on cardiovascular and renal function, may be directly involved in the pathogenesis of hypertension and renal disease/failure. See e.g., Regoli & Barabe. 1980. Pharmacol. Rev. 32:1- 46; Margolius, 1980. Prog. Biochem. Pharmacol. 17:116-122.

Although the biological function of the tissue kallikreins is not completely understood, it is thought that they may function to increase local organ blood flow, promote flow of glandular secretions and/or process various pro-enzymes (e.g., pro-renin). See e.g., Sealey, et al, 1978. Nature (London) 275:144-145. For example, in the kidney (tissue) kallikrein has been localized to the tubular cells of the distal nephron of various mammals, including humans (see e.g., Figueroa, et al, 1988. Histochem. 89:437-442). This finding suggests an intrinsic renal kallikrein-kinin system, which may inhibit water and sodium readsoφtion (see e.g., Mills, 1982. Q. J. Exp. Physiol 67:393-399) and promote renal vasodilation (see e.g., Proud, et al, 1985. Kidney Int. 25:880-885). A more recent study has also demonstrated rat renal kallikrein mRNA to be localized to the vascular pole of the glomerulus, thus suggesting that renal kallikrein may have a role in the function of the proximal nephron. See e.g., Xiong, et al, 1989. Kidney Int. 35:1324-1329. Although renal kallikrein has been found to be decreased in many forms of hypertension in both human and laboratory animals (see e.g., Gilboa, et al, 1984. Lab. Invest. 50:72-78) and is believed to be involved in the regulation of renal blood flow, sodium and water excretion and blood pressure, as well as in the pathogenesis of experimental and clinical hypertension (see e.g., Margolius, 1996. Diabetes 45(Supple. 1):S14-S19). the administration of exogenous kallikrein, prior to the present invention, has not been suggested as a potential therapeutic modality for the prevention or treatment of renal disease, platelet storage pool disease, hypertension, or other renal disease-associated diseases or disorders. Hypotensive hemorrhage has been shown to be a is a potent stimulus to renal kallikrein release, with a fall in blood pressure from 100 to 80 mm Hg resulting in a 4-fold increase in renal kallikrein release (see e.g., Maier, et al, 1981. Circ. Res. 48:386-392). Additionally, a possible functional association with the renin-angiotensin system has also been proposed, whereby renal kallikrein activates prorenin, but generates kinins, which concomitantly antagonize the actions of angiotensin II. See e.g.. Mills, 1982. 0. J. Exp. Physiol 67:393-399); Proud, et al. 1985. Kidney

Int. 25:880-885.

The decreased levels of kallikrein transcripts (and possibly the kallikrein protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. Therefore, the administration of exogenous a kallikrein may serve to ameliorate some of the pathophysiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of kallikrein mRNA may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

(xii) p8 Protein

As illustrated in Table 1, there was a 3 -fold increase in the expression of the p8 protein (GenBank Ace. No. afO 14503) gene in the FHR and IRL animals, in comparison to the levels of this transcript in the control ACI strain. The p8 protein and gene have recently been isolated and characterized in the rat pancreatic acinar cells during both the acute phase of pancreatitis and pancreatic development/regeneration. See e.g.. Mallo, et al, 1991. J. Biol. Chem. 272:32360-32369. Additional in vitro studies have demonstrated that the p8 protein is induced in both pancreatic and non-pancreatic cells (e.g., the kidney) in response to some apoptotic stimuli during periods of cellular stress or pathological aggression, and it is this overexpression which functions to promote cellular growth. See e.g.. Mclntosh, et al, 1995. Neuropathol Appl. Neurobiol. 21 :477- 479; Brown, 1995. Neuropathol Appl. Neurobiol. 21:473-475: Das. et al, 1995. J. Mol. Cell. Car diol. 27:181-193. The p8 protein is especially interesting as it undergoes an extremely rapid induction in response to the apoptotic stimuli. See e.g., Mclntosh. et al, 1995. Neuropathol. Appl. Neurobiol. 21 :411-419; Mallo. et al, 1997. J. Biol. Chem. 272:32360-32369. It should be noted that this finding is not unexpected, as mammalian cells generally respond to such stressors by altering their normal pattern of protein synthesis and gene expression. See e.g., Kogure & Kato, 1993. Stroke 24:2121-2127. Such a changeover in gene expression is characterized by dramatic increases in the production of stress proteins with a concomitant decrease in the production of the normal array of cellular proteins. Stress proteins are not novel components of the physiologically-stressed cells, as the majority of these proteins are expressed within cells under normal conditions. See e.g.. Schlesinger, 1990. J. biol. Chem. 265:12111-12114. In contrast, however, the levels of both the p8 mRNA and protein are markedly reduced within normal adult tissues and organs. See e.g., Mallo. et al, 1997. J. Biol Chem. 272:32360-32369.

Primary structural analysis (see e.g., Brennan & Matthws, 1989. Trends Biochem. Sci. 14:286-290) of the p8 protein has demonstrated that the protein possesses neither a signal peptide nor a transmembranal region(s). However, the presence of: (i) a highly-conserved bipartite motif of nuclear targeting in its carboxyl-terminal region; (ii) a basic helix-turn-helix motif and (Hi) a moderate degree of homology with various homeodomains suggest that the p8 protein may function within the nucleus as a DNA-binding protein, most probably as a transcriptional factor. See e.g., Brennan & Matthws. 1989. Trends Biochem. Sci. 14:286-290. Moreover, the p8 protein possesses the ability to be phosphorylated by various kinases (i.e., 3 potential phosphorylation sites for protein kinase C and one site for casein kinase II). See e.g., Woodget, et al, 1986. Eur. J. Biochem. 61 : 177-184; Pinna, 1990. Biochim. Biophys. Acta 1045:267-284. These observations have led to the hypothesis that the p8 protein may be involved in a phosphorylation/ dephosphorylation signaling pathway involving an initial translocation of p8 to the nucleus followed by a sequence-specific binding to DNA. See e.g., Mallo. et al, 1997. J. Biol. Chem. 272:32360-32369.

The p8 protein has also been shown to function as a promoter of cellular growth factor(s) in vitro, when its cDNA is transfected into COS-7 and AR4-2J cells. This finding lends additional credence to the hypothesis that the p8 protein putatively functions as a transcriptional factor which can regulate the growth of organs such as the pancreas, liver, kidney, small intestine, lungs and heart. See e.g., Mallo, et al, 1997. J. Biol. Chem. 272:32360-32369.

The increased levels of p8 transcripts (and possibly the p8 protein) disclosed in the present invention within the FHR and IRL renal disease animal models may play a role in the etiology of renal disease and associated disorders. As previous studies have demonstrated that p8 expression is upregulated by various physiological stressors (i.e., within the pancreas during the acute phase of pancreatitis), the increased levels of this protein within the renal disease animals may be due to an analogous pathological/stress-induction mechanism. In addition, p8 has been shown to function as a growth factor within these same pancreatic cells. Hence, the administration of exogenous p8 protein, an inducer to upregulate p8 expression or the like, or, in contrast, should the increased levels of the p8 protein be subsequently found to be physiologically harmful, an antagonist or inhibitor may be administered to help ameliorate some of the pathophysiological effects of renal disease and/or associated disorders. Additionally, quantitation of the level of p8 mRNA within various cells/tissues may be useful in prognostic tests for a predisposition to kidney disease or in the diagnosis of early/sub-clinical renal disease or associated disorders.

It should be noted that the present invention is not to be limited in scope by the specific embodiments disclosed herein. Indeed, various modifications of the invention, in addition to those disclosed herein, will become readily apparent to those individuals skilled within the art from the foregoing disclosure and accompanying figures. Such modifications are fully intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing renal disease, said method comprising administering, to a subject in which such treatment or prevention is desired one or more proteins selected from the group consisting of: Zn-peptidase (Aminopeptidase N): RTl .B-l(alpha) chain of the integral membrane protein; δ subunit of FIFO ATPase; keratin 19: brain calbindin-d28k (CaBP28K); the inhibitor protein of metalloproteinase 3 (TIMP-3); integral membrane protein 1 (Itml); isovaleryl-CoA dehydrogenase (IVD): rab GDI-β; IRPR (IFN-β); organic cation transporter (OCT2); bile mayaliculus domain-specific glycoprotein; L-arginine:glycine amidinotransferase; protein phosphatase 1-β (PPl-β); renal kallikrein and the p8 protein, in an amount sufficient to treat or prevent renal disease.

2. The method of claim 1. wherein one or more of the proteins is a human protein.

3. A method of treating or preventing renal disease, said method comprising administering to a subject in which such treatment or prevention is desired one or more of the following:

(a) antibodies specific for one or more of the proteins recited in the method of claim 1;

(b) nucleic acids which encode one or more of the proteins recited in the method of claim l;

(c) anti-sense nucleic acid derivatives of the nucleic acids encoding one or more of the proteins recited in the method of claim 1 ;

(d) antibodies specific for one or more of the proteins recited in the method of claim 1 ; and

(e) antibodies specific for the anti-sense nucleic acid derivatives of the nucleic acids encoding one or more of the proteins recited in the method of claim 1, in an amount sufficient to treat or prevent renal disease.

4. A method of treating or preventing platelet storage-pool disease, said method comprising administering to a subject in which such treatment or prevention is desired one or more of the following:

(b) nucleic acids which encode one or more of the proteins recited in the method of claim l; (c) anti-sense nucleic acid derivatives of the nucleic acids encoding one or more of the proteins recited in the method of claim 1 ;

(d) antibodies specific for one or more of the proteins recited in the method of claim 1; and

(e) antibodies specific for the anti-sense nucleic acid derivatives of the nucleic acids encoding one or more of the proteins recited in the method of claim 1, in an amount sufficient to treat or prevent platelet storage-pool disease.

5. A method of treating or preventing hypertension, said method comprising administering to a subject in which such treatment or prevention is desired one or more of the following:

(c) anti-sense nucleic acid derivatives of the nucleic acids encoding one or more of the proteins recited in the method of claim 1;

(e) antibodies specific for the anti-sense nucleic acid derivatives of the nucleic acids encoding one or more of the proteins recited in the method of claim 1, in an amount sufficient to treat or prevent hypertension.

6. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of one or more of the following:

(a) an isolated, protein as recited in the method of claim 1 ;

(b) antibodies specific for one or more of the proteins recited in the method of claim 1;

(c) nucleic acids encoding for one or more of the proteins recited in the method of claim l;

(d) anti-sense nucleic acid derivatives of the nucleic acids encoding for one or more of the proteins recited in the method of claim 1;

(e) antibodies specific for one or more of said nucleic acids which encode the proteins recited in the method of claim 1; and

(f) antibodies specific for one or more of said anti-sense nucleic acid derivatives of the nucleic acids encoding the proteins recited in the method of claim 1 , and a pharmaceutically acceptable carrier.

7. A kit, comprising in one or more containers, a therapeutically or prophylactically effective amount of the pharmaceutical composition of claim 6.

8. The pharmaceutical composition of claim 6, wherein said nucleic acid is a nucleic acid vector.

9. The pharmaceutical composition of claim 6, wherein said anti-sense nucleic acid derivative is a nucleic acid vector.

10. A method of screening for a modulator of renal disease, platelet storage-pool disease, hypertension or related diseases or disorders of one or more of the proteins recited in the method of claim 1, said method comprising:

(a) contacting a cell with a test compound;

(b) measuring the levels of a protein recited in the method of claim 1 in said cell;

(c) measuring the level of the protein in a control cell not contacted with said test compound; and

(d) comparing the levels of the protein in the cells of step (b) and (c), wherein an alteration in the level of proteins in the cells indicates that the test compound is a modulator of renal disease, platelet storage-pool disease, hypertension and/or related diseases or disorders.

11. A method of screening for a modulator of renal disease, platelet storage-pool disease, hypertension or related diseases or disorders, said method comprising:

(a) administering a test compound to a test animal which is predisposed to developing or has already developed renal disease, platelet storage-pool disease, hypertension or related diseases or disorders;

(b) administering the test compound to a matched control animal which is predisposed to developing or has already developed renal disease, platelet storage-pool disease, hypertension or related diseases or disorders;

(c) measuring the level of the protein or nucleic acid of claim 1 or a nucleic acid encoding the nucleic acid of claim 1 in the animals of step (a) and step (b); and (d) comparing the levels of the protein or nucleic acid in the test and matched control animals, wherein a change the relative levels indicates that the test compound is a modulator of renal disease, platelet storage-pool disease, hypertension and/or related diseases or disorders.

12. The method of claim 11 , wherein said test animal is a recombinant test animal which expresses a transgene selected from the group consisting of:

(a) a Zn-peptidase (Aminopeptidase N) transgene or expresses Zn-peptidase (Aminopeptidase N) under the control of a promoter which is not the native Zn-peptidase (Aminopeptidase N) gene promoter;

(b) an RTl.B-1 (alpha) chain of the integral membrane protein transgene or expresses the RTl.B-1 (alpha) chain of the integral membrane protein under the control of a promoter which is not the native RTl .B-l (alpha) chain of the integral membrane protein gene promoter;

(c) an δ subunit of FIFO ATPase transgene or expresses the δ subunit of FIFO ATPase under the control of a promoter which is not the native δ subunit of FIFO ATPase gene promoter;

(d) a keratin 19 transgene or expresses keratin 19 under the control of a promoter which is not the native keratin 19 gene promoter;

(e) an inhibitor protein of metalloproteinase 3 (TIMP-3) transgene or expresses an inhibitor protein of metalloproteinase 3 (TIMP-3) under the control of a promoter which is not the native inhibitor protein of metalloproteinase 3 (TIMP-3) gene promoter;

(f) an integral membrane protein 1 (Itml) transgene or expresses integral membrane protein 1 (Itml) under the control of a promoter which is not the native integral membrane protein 1 (Itml) gene promoter;

(g) an isovaleryl-CoA dehydrogenase (IVD) transgene or expresses isovaleryl-CoA dehydrogenase (IVD) under the control of a promoter which is not the native isovaleryl- CoA dehydrogenase (IVD) gene promoter;

(h) expresses a rab GDI-β transgene or expresses rab GDI-β under the control of a promoter which is not the native rab GDI-β gene promoter:

(i) an IRPR (IFN-β) transgene or expresses IRPR (IFN-β) under the control of a promoter which is not the native IRPR (IFN-β) gene promoter; (j) an organic cation transporter (OCT2) transgene or expresses organic cation transporter

(OCT2) under the control of a promoter which is not the native organic cation transporter (OCT2) gene promoter:

(k) a bile mayaliculus domain-specific protein transgene or expresses bile mayaliculus domain-specific protein under the control of a promoter which is not the native bile mayaliculus domain-specific protein gene promoter:

(1) a L-arginine:glycine amidinotransferase transgene or expresses L-arginine:glycine amidinotransferase under the control of a promoter which is not the native L- arginine:glycine amidinotransferase gene promoter;

(m) a protein phosphatase 1-β (PPl-β) transgene or expresses protein phosphatase 1-β (PPl-β) under the control of a promoter which is not the native protein phosphatase 1-β (PPl-β) gene promoter;

(n) a renal kallikrein transgene or expresses renal kallikrein under the control of a promoter which is not the native renal kallikrein gene promoter; and (o) a p8 protein transgene or expresses p8 protein under the control of a promoter which is not the native p8 protein gene promoter; at an increased level relative to a wild-type test animal.

13. A recombinant, non-human ammal comprising a non-native gene sequence selected from the group consisting of:

(a) a Zn-peptidase (Aminopeptidase N) gene sequence;

(b) an RTl.B-1 (alpha) chain of the integral membrane protein gene sequence;

(c) a δ subunit of FIFO ATPase gene sequence;

(d) a keratin 19 gene sequence;

(e) a brain calbindin-d28k (CaBP28K) gene sequence;

(f) an inhibitor protein of metalloproteinase 3 (TIMP-3) gene sequence;

(g) an integral membrane protein 1 (Itml) gene sequence; (h) an isovaleryl-CoA dehydrogenase (IVD) gene sequence; (i) a rab GDI-β gene sequence:

(j) an IRPR (IFN-β) gene sequence;

(k) an organic cation transporter (OCT2) protein gene sequence; (1) a bile mayaliculus domain-specific glycoprotein gene sequence; (m) an L-arginine:glycine amidinotransferase gene sequence; (n) a protein phosphatase 1-β (PPl-β) gene sequence: (o) a renal kallikrein gene sequence; and (p) a p8 protein gene sequence.

14. The method according to any one of claims 1-5, 10 and 11, further comprising administering a drug selected from the group consisting of:

(a) a sympatholytic;

(b) an angiotensin inhibiting drug;

(c) a calcium channel blocking drug;

(d) a diuretic; and

(e) a vasodilator.

15. A kit comprising in one or more containers a therapeutically or prophylactically effective amount of the pharmaceutical composition of claim 6.

16. A method for diagnosing the susceptibility or presence of renal disease, platelet storage- pool disease, hypertension or related diseases or disorders comprising:

(a) providing a tissue sample from a subject;

(b) measuring the level of one or more of the proteins recited in claim 1 or nucleic acid recited in claim 3 in said subject or measuring the level of a nucleic acid encoding the protein of claim 1 in said subject; and

(c) comparing the amount of protein or nucleic acid in said test sample with the level of protein or nucleic acid in a control sample, wherein the control sample is taken from an individual not suffering or susceptible to platelet storage-pool disease, hypertension or related diseases or disorders, wherein an alteration in the level of the protein or nucleic acid in the subject relative tot he control indicates the subject is susceptible to or suffers from renal disease, platelet storage-pool disease, hypertension or related diseases or disorders.

17. The method of claim 15, wherein said protein is measured using an antibody for said protein.