JP7580739B2 - Prevention and treatment of urinary stones by controlling oncostatin M receptor signaling - Google Patents

Description

The present invention relates to a novel agent for preventing and treating urinary tract stones that can inhibit stone formation. More specifically, the present invention relates to an agent for inhibiting urinary tract stone formation, which comprises a substance that inhibits the function or expression of oncostatin M or its receptor.

Urolithiasis is a disease in which stones form in the urinary tract from the kidneys to the urethra. When stones become stuck in the ureter, they can cause colic attacks and hematuria, as well as postrenal renal failure due to hydronephrosis. The annual incidence of urinary stones in Japan is on the rise due to the Westernization of diet and lifestyle, and the aging of the population. The lifetime incidence is also high, with one in seven men and one in 15 women suffering from urinary stones at least once in their lifetime (Non-Patent Document 1).

The five-year recurrence rate for urinary stones is high, at 40-50%, placing a strain on the medical economy. The reason for this is that current treatments for urinary stones mainly involve promoting stone passage with oral medication or breaking up stones through surgical treatment, and there is no established treatment to prevent stone formation itself. Therefore, there is a need for the development of new drugs that can prevent stone formation.

Oncostatin M (OSM) is a cytokine belonging to the IL-6 family that shares gp130 in its receptor complex. OSM was discovered as a factor that inhibits the proliferation of human tumor cells, but it has become clear that it is a molecule that has a variety of functions in addition to antitumor effects. The inventors previously reported that OSM is involved in pain via its receptor, and that pain can be suppressed by administering an OSM antagonist or by selectively removing nociceptive neurons that express the OSM receptor (OSMR) (Patent Document 1). They also reported that OSM improves adipose tissue inflammation and insulin resistance in obesity via its receptor (Patent Document 2). However, nothing is known about the relationship between OSM or OSMR and urinary tract stones.

JP 2005-247836 A JP 2013-67593 A

Yasui, T. et al., Urology, 71(2): 209-213 (2008)

The object of the present invention is to provide a novel drug for the treatment of urolithiasis that can inhibit the formation of stones.

Urinary stones are formed by calcium oxalate crystals binding to crystal-binding proteins (osteopontin, annexin 2, etc.). In order to identify molecules involved in the formation of urinary stones, the present inventors investigated the changes in gene expression in the kidneys of glyoxylate-treated mice, a urolithiasis model, and found that when calcium oxalate crystals appeared in the renal tubules, the production of OSM increased in the renal tubules and OSMR was expressed in the renal tubule epithelial cells. When glyoxylate was administered to oncostatin M receptor beta (OSMRβ)-deficient mice, calcium oxalate crystal formation was hardly observed. In OSMRβ-deficient mice, the increase in the expression of inflammation-related genes in the kidney after glyoxylate administration was suppressed, and the number of macrophages (especially inflammation-inducing M1 macrophages) was significantly reduced. Furthermore, in OSMRβ-deficient mice, the expression of crystal-binding proteins in the kidney after glyoxylate administration was suppressed. On the other hand, they found that stimulation of renal tubular epithelial cells derived from wild-type mice with OSM increased the expression of crystal-binding proteins, and stimulation of fibroblasts derived from wild-type mice with OSM increased the expression of the inflammatory cytokine TNF-α.
Based on these findings, the inventors concluded that the appearance of calcium oxalate crystals increases the expression of OSM in renal tubular epithelial cells, activating signaling via OSMR in the cells, which results in the induction of expression of various crystal-binding proteins that bind to calcium oxalate crystals and form urinary stones (see upper panel of Figure 8). They demonstrated that blocking signals from OSMR can suppress the expression of crystal-binding proteins, thereby preventing the formation of urinary stones, i.e., the onset and recurrence of urolithiasis (see lower panel of Figure 8), and thus completed the present invention.

That is, the present invention is as follows.
[1] An agent for suppressing urinary stone formation, comprising a substance that inhibits the function or expression of oncostatin M or its receptor.
[2] A substance that inhibits the function of oncostatin M is
(a) an antibody against oncostatin M, or (b) an antagonist against oncostatin M,
A substance that inhibits the expression of oncostatin M
(c) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the oncostatin M gene;
The agent according to [1], which is (d) an antisense nucleic acid against a transcription product of the oncostatin M gene, or (e) a ribozyme nucleic acid against a transcription product of the oncostatin M gene.
[3] A substance that inhibits the function of the oncostatin M receptor,
(a) an antibody against oncostatin M receptor β, or (b) an antagonist against oncostatin M receptor β,
A substance that inhibits the expression of oncostatin M receptors
(c) a nucleic acid or a precursor thereof having RNAi activity against a transcription product of the oncostatin M receptor β gene;
The agent according to [1], which is (d) an antisense nucleic acid against a transcription product of the oncostatin M receptor β gene, or (e) a ribozyme nucleic acid against a transcription product of the oncostatin M receptor β gene.
[4] The agent according to [3], wherein the substance that inhibits the function of the oncostatin M receptor is an antibody against oncostatin M receptor β, and the antibody is in the form of a conjugate with a cytotoxin.
[5] The agent according to any one of [1] to [4], which is for the prevention or treatment of urinary tract stones.
[6] A diagnostic agent for determining the risk of urinary tract stones, comprising a substance that specifically binds to oncostatin M.
[7] A method for screening for a drug for inhibiting urinary tract stone formation, comprising:
(1) contacting oncostatin M with its receptor in the presence and absence of a test substance;
(2) comparing the binding between oncostatin M and its receptor under both of the above conditions; and (3) selecting a test substance that reduces the binding between oncostatin M and its receptor as a candidate drug for inhibiting urinary stone formation.
[8] A method for screening for a drug for inhibiting urinary tract stone formation, comprising:
(1) contacting a cell expressing the Oncostatin M receptor with Oncostatin M in the presence and absence of a test substance;
(2) comparing oncostatin M receptor signaling under both of the above conditions; and (3) selecting a test substance that reduces oncostatin M receptor signaling as a candidate drug for inhibiting urinary stone formation.

According to the present invention, by blocking OSMR signaling, the expression of crystal-binding proteins can be suppressed and the formation of urinary stones can be prevented, making it possible to fundamentally prevent and treat urinary stones (inhibiting progression and preventing recurrence). Furthermore, the OSM-OSMR signaling system can be used to discover new drugs for preventing and treating urinary stones.

FIG. 1 shows the expression of OSM (A) and OSMRβ (B) in the kidneys of a urolithiasis model mouse. Expression of OSM and OSMRβ in kidneys 6 days after glyoxylate administration. Arrowheads indicate double positive cells. Scale bar = 100 μm. Crystal formation after glyoxylate administration in OSMRβ-deficient mice. *P<0.05 Scale bar = 500 μm The lower left corner of Fig. 3A is an enlargement of the boxed area. Changes in expression of inflammation-related genes (TNF-α (A), IL-1β (B), F4/80 (C), and MCP-1 (D)) after glyoxylate administration in OSMRβ-deficient mice. *P<0.05 This figure shows the change in the number of renal total macrophages (A) and M1 macrophages (B) after glyoxylate administration in OSMRβ-deficient mice. *P<0.05 Changes in expression of crystal-binding proteins (osteopontin (OPN; A), annexin a1 (ANXa1; B), annexin a2 (ANXa2; C), and KIM1 (D)) after glyoxylate administration in OSMRβ-deficient mice. *P<0.05 Changes in gene expression of crystal-binding proteins in renal tubular epithelial cells. (A) Expression of OSMRβ and osteopontin (OPN) in the kidney 6 days after the start of glyoxylate treatment. (B) Expression of OPN, annexin a1 (ANXa1), and annexin a2 (ANXa2) in renal tubular epithelial cells from wild-type mice stimulated with OSM. (C) Expression of TNF-α in fibroblasts, macrophages, and renal tubular epithelial cells (PRECs) from wild-type mice stimulated with OSM. *P<0.05 Scale bar = 100 μm This is a schematic diagram of the normal process of urinary stone formation (upper panel) and the mechanism by which urinary stone formation is inhibited by blocking OSMRβ signaling. Figure showing the inhibitory effect of anti-OSMRβ antibody (clone 7D2) on crystal formation caused by glyoxylate administration. (A) Medgel containing 7D2 (80 μg) or its isotype control antibody (Isotype) (80 μg) was implanted subcutaneously on the back of 8-week-old wild-type mice (C57BL/6J), and glyoxylate (80 mg/kg body weight) was administered intraperitoneally every day for 6 days starting 2 days after implantation. (B) The area of crystals positive for pizolato staining was corrected for the area of the entire kidney and quantified. Scale bar = 500 μm This is a graph showing the inhibitory effect of anti-OSMRβ antibody (7D2) on the expression of crystal-binding proteins (osteopontin (OPN), annexin a1 (ANXa1), annexin a2 (ANXa2)) and inflammation-related molecules (TNF-α, IL-1β, F4/80, MCP-1) after glyoxylate administration. *P<0.05

The present invention provides an agent for inhibiting the formation of urinary stones (hereinafter also referred to as the "stone-inhibiting agent of the present invention"), which comprises a substance that inhibits the function or expression of oncostatin M or its receptor.

In this specification, the term "urinary stones" is not particularly limited as long as it is a stone that occurs in the urinary tract from the kidneys to the urethra, and examples thereof include calcium oxalate stones, calcium phosphate stones, uric acid stones, magnesium ammonium phosphate stones, cystine stones, and stones that are a mixture of these stones, but calcium stones are preferred, and calcium oxalate stones are more preferred.

(Oncostatin M (OSM))
Oncostatin M (OSM), one of the target molecules of the stone-inhibiting agent of the present invention, is a cytokine belonging to the IL-6 family. In humans, OSM is translated into a precursor having an amino acid sequence of 252 amino acids as shown in SEQ ID NO: 2, and then the signal peptide at positions 1-25 and 31 amino acids at the C-terminus are removed to form a mature form consisting of 196 amino acids from positions 26-221.

As used herein, "oncostatin M (OSM)" refers to a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 2. As used herein, proteins and peptides are written in accordance with the convention of peptide designation, with the N-terminus (amino terminus) at the left and the C-terminus (carboxyl terminus) at the right.
"An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2" means
(a) the amino acid sequence of an ortholog in another warm-blooded animal (e.g., guinea pig, rat, mouse, chicken, rabbit, dog, pig, sheep, cow, monkey, etc.) of human OSM consisting of the amino acid sequence set forth in SEQ ID NO: 2; or (b) the amino acid sequence of a natural allelic variant or genetic polymorphism of human OSM consisting of the amino acid sequence set forth in SEQ ID NO: 2 or the ortholog of (a) above.
Preferably, the OSM is human OSM consisting of the amino acid sequence set forth in SEQ ID NO:2 or a naturally occurring allelic variant or polymorphism thereof, such as, but not limited to, the SNP registered in dbSNP as rs5763919, which substitutes Thr (ACG) at position 9 for Met (ATG).

(Oncostatin M Receptor (OSMR))
Oncostatin M receptor (OSMR), the other target molecule of the stone inhibitor of the present invention, is a cell surface receptor that binds to OSM and transmits signals to downstream factors, and is a heterodimer of OSM-specific oncostatin M receptor β (OSMRβ) and gp130, which is common to IL-6 family receptors. OSM can bind to both OSMRβ and gp130 with low affinity, but cannot transmit signals into cells when bound alone. OSMRβ is a single-pass transmembrane protein, and in humans, it has an amino acid sequence of 979 amino acids represented by SEQ ID NO: 4, of which positions 1-27 are a signal peptide, positions 28-740 are an extracellular domain, positions 741-761 are a transmembrane domain, and positions 762-979 are an intracellular domain.

As used herein, "OSMRβ" refers to a protein that contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4. "An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4" means
(a) the amino acid sequence of an ortholog in another warm-blooded animal (e.g., guinea pig, rat, mouse, chicken, rabbit, dog, pig, sheep, cow, monkey, etc.) of human OSMRβ consisting of the amino acid sequence represented by SEQ ID NO: 4; or (b) the amino acid sequence of a natural allelic variant or genetic polymorphism of human OSMRβ consisting of the amino acid sequence represented by SEQ ID NO: 2 or the ortholog of (a) above.
Preferably, OSMRβ is human OSMRβ consisting of the amino acid sequence set forth in SEQ ID NO: 4, or a natural allelic variant or genetic polymorphism thereof. Examples of such genetic polymorphisms include, but are not limited to, the SNP registered in dbSNP as rs34675408 in which His (CAT) at position 187 is replaced with Gln (CAG).

(OSM function inhibitor)
In the present invention, a "substance that inhibits the function of OSM" may be any substance that inhibits the stone formation-promoting function of OSM once it has been functionally produced (e.g., induction of OSMR expression in renal tubular epithelial cells, induction of crystal-binding protein expression via OSMR in renal tubular epithelial cells, promotion of crystal formation in the kidney, induction of inflammation-related gene expression in the kidney, induction of M1 macrophages in the kidney, induction of TNF-α expression in renal fibroblasts, etc.), and examples of this include substances that block OSMR signaling by binding to OSM and inhibiting its binding to OSMR.

Specifically, examples of substances that inhibit the function of OSM include antibodies against OSM. The antibodies may be either polyclonal or monoclonal. These antibodies may be produced according to known methods for producing antibodies or antisera. The isotype of the antibody is not particularly limited, but is preferably IgG, IgM or IgA, and is particularly preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to OSM. In addition to complete antibody molecules, the antibody may be, for example, fragments such as Fab, Fab', F(ab') ₂ , genetically engineered conjugate molecules such as scFv, scFv-Fc, minibodies, diabodies, or derivatives thereof modified with molecules having a protein stabilizing effect such as polyethylene glycol (PEG).

In a preferred embodiment, an antibody against OSM is used as a pharmaceutical intended for administration to humans, and therefore the antibody (preferably a monoclonal antibody) is an antibody with reduced risk of exhibiting antigenicity when administered to humans, specifically a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc., and is particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced by genetic engineering in accordance with standard methods. Although fully human antibodies can also be produced from human-human (or mouse) hybridomas, in order to provide large amounts of antibodies stably and at low cost, it is desirable to produce them using human antibody-producing mice or phage display methods.

A substance that inhibits the function of OSM may also be, for example, an antagonist that binds to OSM competitively with the receptor OSMR. Examples of such antagonists include the extracellular domain of OSMRβ or gp130, or a complex thereof, or a fragment thereof that contains the binding domain with OSM. Antagonists against OSM can also be obtained by constructing a competitive assay system using OSM and OSMR and screening a compound library.

In another preferred embodiment, substances that inhibit the function of OSM include, for example, the low molecular weight compounds described in Tables 1 and 2 of U.S. Patent Application Publication No. 2015/0093391.

(OSM expression inhibitor)
In the present invention, the term "substance that inhibits the expression of OSM" refers to any substance that acts at any stage of the OSM gene, such as the transcription level, the level of post-transcriptional regulation, the level of translation into protein, or the level of post-translational modification. Thus, substances that inhibit the expression of OSM include, for example, substances that inhibit the transcription of the OSM gene (e.g., antigenes), substances that inhibit the processing of an initial transcription product into mRNA, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of mRNA into protein (e.g., antisense nucleic acids, miRNA) or that degrade mRNA (e.g., siRNA, ribozymes, miRNA), substances that inhibit the post-translational modification of an initial translation product, and the like. Substances that act at any stage can be used, but substances that bind complementarily to mRNA to inhibit translation into protein or degrade mRNA are preferred.

A preferred example of a substance that specifically inhibits translation of OSM gene mRNA into protein (or degrades mRNA) is a nucleic acid that contains a nucleotide sequence complementary to the nucleotide sequence of the mRNA, or a part of it.
A nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene means a nucleotide sequence having a degree of complementarity that can bind to a target sequence of the mRNA and inhibit its translation (or cleave the target sequence) under physiological conditions, specifically, for example, a nucleotide sequence having a homology of 90% or more, preferably 95% or more, more preferably 97% or more, and particularly preferably 98% or more with a nucleotide sequence that is completely complementary to the nucleotide sequence of the mRNA (i.e., the nucleotide sequence of the complementary strand of the mRNA) in the overlapping region. The "nucleotide sequence homology" in the present invention can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3).

More specifically, a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene is a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO:1. Here, "stringent conditions" refers to, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, such as hybridization in 6xSSC (sodium chloride/sodium citrate)/45°C, followed by one or more washes in 0.2xSSC/0.1% SDS/50-65°C, but a person skilled in the art can appropriately select hybridization conditions that provide equivalent stringency.

Preferred examples of mRNA for the OSM gene include the mRNA for human OSM comprising the nucleotide sequence set forth in SEQ ID NO:1, or its orthologues in other warm-blooded animals, as well as naturally occurring allelic variants or genetic polymorphisms thereof.

"A portion of a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene" is not particularly limited in length or position, so long as it can specifically bind to the mRNA of the OSM gene and inhibit protein translation from the mRNA (or degrade the mRNA), but in terms of sequence specificity, it should contain a portion complementary to the target sequence that is at least 10 bases long, preferably 15 bases long, and more preferably 19 bases long.

Specifically, preferred examples of nucleic acids containing a portion of a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene include any of the following (a) to (c):
(a) a nucleic acid or a precursor thereof having RNAi activity against the mRNA of the OSM gene
(b) an antisense nucleic acid to the mRNA of the OSM gene;
(c) a ribozyme nucleic acid for the mRNA of the OSM gene;

(a) Nucleic acid or precursor thereof having RNAi activity against OSM gene mRNA In this specification, double-stranded RNA consisting of an oligoRNA complementary to the OSM gene mRNA and its complementary strand, so-called siRNA, is defined as being included in nucleic acids containing a nucleotide sequence or a part thereof complementary to the nucleotide sequence of the OSM gene mRNA.

siRNA can be designed based on the cDNA sequence information of the target gene, for example, according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)). In addition, short hairpin RNA (shRNA), which is a precursor of siRNA, can be obtained by appropriately selecting any linker sequence (e.g., about 5-25 bases) capable of forming a loop structure and linking the designed sense strand and antisense strand via the linker sequence.

The sequences of siRNA and/or shRNA can be searched for using search software provided free of charge on various websites. Examples of such websites include, but are not limited to, the siDESIGN Center provided by Dharmacon (http://dharmacon.horizondiscovery.com/jp/design-center/?rdr=true&LangType=1041&pageid=17179928204) and the siRNA Target Finder provided by GenScript (https://www.genscript.com/tools/sirna-target-finder).

In the present specification, microRNAs (miRNAs) that target the mRNA of the OSM gene are also defined as nucleic acids that contain a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene or a part thereof. miRNAs can be searched for using target prediction software provided free of charge on various websites. Examples of such websites include, but are not limited to, TargetScan (http://www.targetscan.org/vert_72/) published by the Whitehead Institute in the United States and DIANA-micro-T-CDS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index) published by the Alexander Fleming Center for Biomedical Sciences in Greece. Alternatively, miRNAs that target OSM mRNA can be searched for using TarBase (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=tarbasev8/index), a database of miRNAs that have been experimentally proven to act on target mRNAs, published by Cesaret University, Institut Pasteur, and other institutions.

The nucleotide molecules constituting the siRNA and/or shRNA, or the miRNA and/or pre-miRNA may be natural RNA or DNA, but may contain various chemical modifications to improve stability (chemical and/or enzymatic) and specific activity (affinity with RNA). For example, in order to prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide constituting the antisense nucleic acid may be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. In addition, the hydroxyl group at the 2'-position of the sugar (ribose) of each nucleotide may be replaced with -OR (R=CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ CH ₂ NHC(NH)NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position.

The sugar conformation of RNA is predominantly C2'-endo (S type) and C3'-endo (N type), and in single-stranded RNA, these two conformations exist in equilibrium, but when double-stranded, they are fixed to the N type. Therefore, in order to confer strong binding ability to target RNA, BNA (LNA) (Imanishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, J.S. et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003), which are RNA derivatives in which the conformation of the sugar moiety is fixed to the N type by bridging the 2' oxygen and 4' carbon, can also be preferably used.

siRNA can be prepared by synthesizing the sense and antisense strands of the target sequence on mRNA using an automatic DNA/RNA synthesizer, denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then annealing them at about 30 to about 70°C for about 1 to about 8 hours. siRNA can also be prepared by synthesizing shRNA, which is the precursor of siRNA, and cleaving it using a dicer. miRNA and pre-miRNA can be synthesized using an automatic DNA/RNA synthesizer based on their sequence information.

In the present specification, a nucleic acid designed to generate siRNA or miRNA against the mRNA of the OSM gene in vivo is also defined as being included in the nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene or a part thereof. Such a nucleic acid includes an expression vector constructed to express the above-mentioned shRNA or siRNA, or miRNA or pre-miRNA. As a promoter, a polIII promoter is generally used to accurately transcribe short RNA. Examples of the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, etc. In addition, a sequence of four or more consecutive T's is used as a transcription termination signal. Expression cassettes for miRNA and pre-miRNA can also be prepared in the same manner as for shRNA.
The siRNA or shRNA, or miRNA or pre-miRNA expression cassette thus constructed is then inserted into a plasmid vector or a viral vector, such as a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, or Sendai virus, or an animal cell expression plasmid.

(b) Antisense nucleic acid against the mRNA of the OSM gene In the present invention, an "antisense nucleic acid against the mRNA of the OSM gene" is a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a part thereof, and has the function of inhibiting protein synthesis by binding to the target mRNA to form a specific and stable double strand.
The antisense nucleic acid may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or a DNA:RNA hybrid, and may further include known modifications. Modified nucleotides may have modified sugar moieties. As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA/RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H to cause selective degradation of the target RNA. Thus, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only a sequence in the mRNA, but also a sequence in an intron region in the initial translation product of the OSM gene.

The target region of the antisense nucleic acid of the present invention is not particularly limited in length, so long as the hybridization of the antisense nucleic acid results in inhibition of translation into protein, and may be the entire sequence or a partial sequence of the mRNA encoding the protein, with a short length of about 10 bases and a long length of the entire sequence of the mRNA or initial transcription product. Considering the ease of synthesis, antigenicity, intracellular transport issues, etc., oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred, but are not limited thereto.

The nucleotide molecules constituting the antisense nucleic acid may also be modified in the same manner as in the case of siRNA, etc., described above, to improve stability, specific activity, etc.

The antisense oligonucleotides of the present invention can be prepared by determining the target sequence of the mRNA or initial transcription product based on the cDNA sequence or genomic DNA sequence of the OSM gene, and synthesizing a complementary sequence to this using a commercially available automated DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.). In addition, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by known methods.

(c) a ribozyme nucleic acid for the mRNA of the OSM gene;
Other examples of nucleic acids containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene or a part thereof include ribozyme nucleic acids capable of specifically cleaving the mRNA within the coding region. In the narrow sense, the term "ribozyme" refers to RNA having an enzymatic activity for cleaving nucleic acids, but in this specification, the term is used to include DNA as long as it has sequence-specific nucleic acid cleavage activity.

Nucleic acids containing nucleotide sequences or parts thereof complementary to the nucleotide sequence of the mRNA of the OSM gene can be provided in specialized forms such as liposomes, microspheres, for gene therapy applications, or in adduct form. Such adducts include hydrophobic groups such as polycationic bodies such as polylysine, which act to neutralize the charge of the phosphate backbone, and lipids (e.g., phospholipids, cholesterol, etc.) that enhance the interaction with cell membranes and increase the uptake of the nucleic acid. Preferred lipids for adducts include cholesterol and its derivatives (e.g., cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3' or 5' end of the nucleic acid and can be attached via base, sugar, or intramolecular nucleoside bonds. Other groups include capping groups specifically located at the 3' or 5' end of the nucleic acid to prevent degradation by nucleases such as exonucleases and RNases. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

The substance that inhibits the expression of OSM in the present invention is not limited to a nucleic acid that contains a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene as described above, or a part thereof, but may be other substances such as low molecular weight compounds, so long as they directly or indirectly inhibit the production of OSM protein.

(OSMR function inhibitor)
In the present invention, a "substance that inhibits the function of OSMR" may be any substance that inhibits the stone formation-promoting function of OSMR once it has been functionally produced (e.g., induction of expression of crystal-binding protein in tubular epithelial cells, promotion of crystal formation in the kidney, induction of expression of inflammation-related genes in the kidney, induction of M1 macrophages in the kidney, induction of TNF-α expression in renal fibroblasts, etc.), and examples of such substances include those that bind to OSMR and inhibit its binding to OSM, thereby blocking the transmission of signals from OSM.

Specifically, examples of substances that suppress the function of OSMR include antibodies against OSMRβ and/or gp130. The antibodies may be either polyclonal or monoclonal. These antibodies can be produced according to known methods for producing antibodies or antisera. The isotype of the antibody is not particularly limited, but is preferably IgG, IgM or IgA, and particularly preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to the extracellular region of OSMR (OSMRβ, gp130 or a complex thereof), and may be a complete antibody molecule, or a derivative thereof modified with a _molecule having a protein stabilizing effect, such as polyethylene glycol (PEG).

Since gp130 is a receptor subunit common to receptors of cytokines belonging to the IL-6 family, antibodies that recognize gp130 alone may also act on other cytokine receptors of the IL-6 family and have undesirable effects. Therefore, the antibody against OSMR is preferably an antibody against OSMRβ or an antibody against a complex of OSMRβ and gp130.

In a preferred embodiment, the antibody against OSMR is used as a pharmaceutical intended for administration to humans, and therefore the antibody (preferably a monoclonal antibody) is an antibody with reduced risk of exhibiting antigenicity when administered to humans, specifically a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc., and is particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced by genetic engineering in accordance with standard methods. Although fully human antibodies can also be produced from human-human (or mouse) hybridomas, in order to provide large amounts of antibodies stably and at low cost, it is desirable to produce them using human antibody-producing mice or phage display methods.

In another preferred embodiment, the antibody against OSMR may be provided in the form of a conjugate with a cytotoxin. By selectively removing OSMR-expressing renal tubular epithelial cells or renal interstitial fibroblasts, the same effect as blocking OSMR-mediated signaling can be achieved. Therefore, by using an antibody against OSMR, a cytotoxin can be selectively delivered to OSMR-expressing renal tubular epithelial cells or renal interstitial fibroblasts, thereby selectively damaging the cells. Examples of cytotoxins include, but are not limited to, saporin, diphtheria toxin, ricin, and herpes thymidine kinase.

A substance that inhibits the function of OSMR may also be, for example, an antagonist that binds to OSMR competitively with the ligand OSM. Examples of such antagonists include dominant-negative mutants of OSM that bind to OSMRβ and/or gp130 but do not transmit signals to OSMR. Antagonists to OSMR can also be obtained by constructing a competitive assay system using OSMR and OSM and screening a compound library.

(OSMR expression inhibitor)
In the present invention, the term "substance that suppresses the expression of OSMR" may be one that acts at any stage of the OSMRβ gene and/or gp130 gene, preferably the OSMRβ gene, such as the transcription level, the post-transcriptional regulation level, the translation level into protein, or the post-translational modification level. Thus, examples of substances that suppress the expression of OSMR include substances that inhibit the transcription of the OSMRβ gene and/or gp130 gene, preferably the OSMRβ gene (e.g., antigene), substances that inhibit the processing of an initial transcription product into mRNA, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of mRNA into protein (e.g., antisense nucleic acid, miRNA) or that degrade mRNA (e.g., siRNA, ribozyme, miRNA), and substances that inhibit the post-translational modification of an initial translation product. Any substance that acts at any stage can be used, but a substance that binds complementarily to mRNA to inhibit translation into protein or degrades mRNA is preferred.

A substance that specifically inhibits translation of the OSMRβ gene and/or gp130 gene, preferably the OSMRβ gene, from mRNA to protein (or degrades the mRNA) preferably includes a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a part thereof.
A nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSMRβ gene and/or gp130 gene, preferably the OSMRβ gene, means a nucleotide sequence having such a degree of complementarity that it can bind to a target sequence of the mRNA and inhibit its translation (or cleave the target sequence) under physiological conditions, specifically, for example, a nucleotide sequence having a homology of 90% or more, preferably 95% or more, more preferably 97% or more, particularly preferably 98% or more with a nucleotide sequence that is completely complementary to the nucleotide sequence of the mRNA (i.e., the nucleotide sequence of the complementary strand of the mRNA) in the overlapping region. The "nucleotide sequence homology" in the present invention can be calculated in the same manner as described above.

More specifically, a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSMRβ gene is a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 3. Here, "stringent conditions" refers to, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, such as hybridization in 6xSSC (sodium chloride/sodium citrate)/45°C, followed by one or more washes in 0.2xSSC/0.1% SDS/50-65°C, but a person skilled in the art can appropriately select hybridization conditions that provide equivalent stringency.

Preferred examples of mRNA for the OSMRβ gene include the mRNA for human OSM comprising the nucleotide sequence set forth in SEQ ID NO:3, or its orthologs in other warm-blooded animals, as well as naturally occurring allelic variants or genetic polymorphisms thereof.

"A portion of a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSMRβ gene" is not particularly limited in length or position, so long as it can specifically bind to the mRNA of the OSMRβ gene and inhibit protein translation from the mRNA (or degrade the mRNA), but from the standpoint of sequence specificity, it contains a portion complementary to the target sequence that is at least 10 bases long, preferably 15 bases long, and more preferably 19 bases long.

Specifically, preferred examples of nucleic acids containing a portion of a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSMRβ gene include any of the following (a) to (c):
(a) a nucleic acid or a precursor thereof having RNAi activity against the mRNA of the OSMRβ gene
(b) an antisense nucleic acid to the mRNA of the OSMRβ gene;
(c) Ribozyme nucleic acids against the mRNA of the OSMRβ gene The specific aspects of these nucleic acids and their production methods are the same as those of the above-mentioned "nucleic acids containing a portion of a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the OSM gene," except that the target mRNA is replaced with the mRNA of the OSMRβ gene.

(Drugs containing antibodies against OSM or OSMR, or small molecule compounds that suppress the expression or function of OSM or OSMR)
Antibodies against OSM or OSMR, or small molecule compounds that inhibit the expression or function of OSM or OSMR, can block OSMR signaling, inhibit the expression of crystal-binding proteins, and inhibit the formation of urinary stones. Therefore, these substances can be used as drugs to inhibit the formation of urinary stones, and thus as drugs to prevent and/or treat urolithiasis. Here, "prevention" includes not only preventing the onset of urinary stones, but also delaying their onset. Furthermore, "treatment" includes inhibiting the progression of formed urinary stones and preventing their recurrence.

The pharmaceutical containing the above-mentioned antibody or low molecular weight compound can be administered orally or parenterally (e.g., intravascular administration, subcutaneous administration, etc.) as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form to humans or other warm-blooded animals (e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, chickens, etc.), preferably humans, particularly to high-risk groups for urolithiasis (e.g., patients with hypercalciuria and/or hyperuricosuria, those with a family history of urolithiasis) or those with a history of urolithiasis.

The above-mentioned antibodies and low molecular weight compounds may be administered as they are, or may be administered as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the above-mentioned antibodies or low molecular weight compounds or salts thereof and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.

For compositions for parenteral administration, for example, injections, suppositories, intranasal preparations, etc. are used, and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. Such injections can be prepared according to known methods. For example, the injections can be prepared by dissolving, suspending, or emulsifying the antibody or low molecular weight compound of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid that is usually used for injections. As aqueous liquids for injection, for example, physiological saline, isotonic liquids containing glucose and other adjuvants, etc. are used, and may be used in combination with appropriate solubilizing agents such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As oily liquids, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with benzyl benzoate, benzyl alcohol, etc. as solubilizing agents. The prepared injection solution is preferably filled into a suitable ampoule. Suppositories for rectal administration may be prepared by mixing the antibody or its salt with a conventional suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

The above-mentioned parenteral or oral pharmaceutical compositions are conveniently prepared in a dosage unit form that matches the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories. The antibody or low molecular weight compound is preferably contained in an amount of 0.1 to 500 mg per dosage unit form, particularly 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.

The dosage of the above-mentioned medicine containing the above-mentioned antibody or low molecular weight compound or a salt thereof varies depending on the subject, symptoms, route of administration, etc., but for example, a single dose of an antibody or low molecular weight compound is usually about 0.0001 to 20 mg/kg body weight, and low molecular weight compounds are administered orally or parenterally about 1 to 5 times a day, while antibodies are administered by intravenous injection once a day to once every few months. Similar amounts can also be administered in the case of other parenteral and oral administrations. If symptoms are particularly severe, the dosage may be increased according to the symptoms.

The above-mentioned compositions may contain other active ingredients as long as they do not cause undesirable interactions when combined with the above-mentioned antibodies or low molecular weight compounds. For example, other active ingredients include, but are not limited to, magnesium, citric acid, uric acid production inhibitors such as alloprilol, thiazides, etc.

(Drugs containing antisense nucleic acids, ribozyme nucleic acids, siRNAs and their precursors)
The antisense nucleic acids (or miRNAs) of the present invention that can bind complementarily to the transcripts of the OSM or OSMR genes and inhibit protein translation from the transcripts, siRNAs (or ribozymes, miRNAs) that can target homologous (or complementary) base sequences in the transcripts of the OSM or OSMR genes and cleave the transcripts, and furthermore, shRNAs and pre-miRNAs that are precursors of the siRNAs and miRNAs (hereinafter collectively referred to as "nucleic acids of the present invention") can be used as inhibitors of urinary tract formation and, ultimately, as drugs for the prevention and/or treatment of urolithiasis.

The pharmaceutical containing the nucleic acid of the present invention can be administered orally or parenterally (e.g., intravascular administration, subcutaneous administration, etc.) as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form to humans or non-human warm-blooded animals (e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, chickens, etc.), preferably humans, particularly to high-risk groups for urolithiasis (e.g., patients with hypercalciuria and/or hyperuricosuria, those with a family history of urolithiasis) or those with a history of urolithiasis.

When the nucleic acid of the present invention is used as a drug for inhibiting the formation of urinary stones, it can be formulated and administered according to a method known per se. That is, the nucleic acid of the present invention can be used alone, or can be functionally inserted into an expression vector for suitable mammalian cells, such as a retrovirus vector, an adenovirus vector, or an adenovirus-associated virus vector. The nucleic acid can be administered as it is, or together with an adjuvant for promoting uptake, by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be made into an aerosol and administered locally into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of intracellular uptake, the nucleic acid may be formulated (injectable) alone or together with a carrier such as liposome, and administered intravenously, subcutaneously, etc.

The nucleic acid of the present invention may be administered per se or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration.

For compositions for parenteral administration, for example, injections, suppositories, intranasal preparations, etc. are used, and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Such injections can be prepared according to known methods. For example, the injections can be prepared by dissolving, suspending, or emulsifying the nucleic acid of the present invention in a sterile aqueous or oily liquid that is usually used for injections. As aqueous liquids for injection, for example, physiological saline, isotonic liquids containing glucose and other adjuvants, etc. are used, and may be used in combination with appropriate solubilizing agents, for example, alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As oily liquids, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with benzyl benzoate, benzyl alcohol, etc. as solubilizing agents. The prepared injection solution is preferably filled into a suitable ampoule. Suppositories for rectal administration may be prepared by mixing the nucleic acid with a conventional suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

The above-mentioned parenteral or oral pharmaceutical compositions are conveniently prepared in a dosage unit form that matches the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid of the present invention is preferably contained in an amount of, for example, about 0.01 to 500 mg per dosage unit form.

The dosage of the above-mentioned pharmaceutical containing the nucleic acid of the present invention varies depending on the subject, symptoms, route of administration, etc., but for example, it is convenient to administer a single dose of the nucleic acid of the present invention by intravenous injection, usually at about 0.0001 to 20 mg/kg body weight, once every day to once every six months. Similar amounts can also be administered in other parenteral and oral administrations. When symptoms are particularly severe, the dosage may be increased according to the symptoms.

The above-mentioned compositions may contain other active ingredients as long as they do not cause undesirable interactions when combined with the nucleic acid of the present invention. Examples of other active ingredients include various compounds that have a therapeutic effect against urinary tract stones. Examples of other active ingredients include, but are not limited to, magnesium, citric acid, uric acid production inhibitors such as alloprilol, and thiazides.

(Diagnosis agent for urinary stone risk)
The present invention also provides a diagnostic agent for the risk of urolithiasis, comprising a substance that specifically binds to OSM. Since the amount of OSM secreted by renal tubular epithelial cells increases significantly during the formation of urolithiasis, it is believed that the amount of OSM in urine increases in patients with urolithiasis. Thus, for example, urine is collected from a test animal, preferably a human, more preferably a human suspected of having urolithiasis, and the amount of OSM in the urine sample is measured and compared with that of a healthy individual, thereby diagnosing the risk of developing or recurring urolithiasis.

The "substance that specifically binds to OSM" contained in the diagnostic agent of the present invention may be an antibody against OSM or an antagonist against OSM as described above. Expression of OSM can be detected by various immunochemical techniques, preferably using an antibody against OSM.

Labeling agents used in the measurement method using a labeling substance include, for example, radioisotopes, enzymes, fluorescent substances, and luminescent substances. Radioisotopes include, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], and [ ¹⁴ C]. As the enzyme, it is preferable to use one that is stable and has a large specific activity, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase. As fluorescent substances, for example, fluorescamine, fluorescein isothiocyanate (FITC), and phycoerythrin (PE) are used. As luminescent substances, for example, luminol, luminol derivatives, luciferin, and lucigenin are used.

Antibodies to OSM may be directly or indirectly labelled with a labelling substance. In a preferred embodiment, the anti-OSM antibody is unlabelled and OSM can be detected with a labelled secondary antibody, such as an antiserum or anti-Ig antibody raised against the animal in which the anti-OSM antibody was produced. Alternatively, a biotinylated secondary antibody can be used to form an OSM-anti-OSM antibody-secondary antibody complex which can then be visualised using labelled streptavidin.

The anti-OSM antibody can be dissolved in a suitable buffer solution known per se and frozen for storage. The diagnostic agent of the present invention can also be made into a kit by combining the anti-OSM antibody with a labeled secondary antibody, a reaction buffer, a blocking solution, a washing solution, etc.

The present invention also provides a screening method for drugs that inhibit urinary stone formation using inhibition of the binding between OSM and OSMR as an index (screening method (I) of the present invention). The method comprises the steps of:
(1) contacting OSM with OSMR in the presence and absence of a test substance;
(2) comparing the binding between OSM and OSMR under both of the above conditions; and (3) selecting a test substance that reduces the binding between OSM and OSMR as a candidate drug for inhibiting urinary stone formation.
Here, as the OSMR, either OSMRβ or gp130 can be used alone, but considering the affinity with OSM, it is preferable to use a complex of OSMβ and gp130. Furthermore, the OSMR may be used in its entirety, or a fragment containing the domain required for binding to OSM, such as the extracellular domain, may be used.

For example, OSMR is immobilized on a solid phase, and OSM is brought into contact with the solid phase and incubated in the presence and absence of a test substance. The liquid phase is then separated and removed, and the solid phase is reacted with an anti-OSM antibody labeled with an enzyme or fluorescent substance. The amount of OSM bound to the solid phase is measured using the sandwich method. If the amount of OSM is significantly reduced in the presence of the test substance, the test substance can be selected as a candidate drug for inhibiting the formation of urinary stones.

The present invention also provides a screening method for a drug for suppressing urinary stone formation using inhibition of OSMR signaling as an index (screening method (II) of the present invention). The method comprises the steps of:
(1) contacting a cell expressing OSMR with OSM in the presence and absence of a test substance;
(2) comparing the OSMR signaling under both of the above conditions; and (3) selecting a test substance that reduces OSMR signaling as a candidate drug for inhibiting urinary stone formation.
Here, since the use of OSMRβ or gp130 alone does not result in signal transduction from OSM, a complex of OSMβ and gp130 is used as OSMR. Examples of cells that express OSMR include renal tubular epithelial cells and fibroblasts.

For example, when using ureteral epithelial cells expressing OSMR, OSM is added to the culture medium of the cells in the presence and absence of the test substance, and the cells are incubated. Then, for example, the expression level of the crystal-binding protein (e.g., osteopontin, annexin a1, annexin a2, KIM1, etc.) gene in the cells is measured by RT-PCR or immunological assays using various antibodies. If the expression level of the gene is significantly reduced in the presence of the test substance, the test substance can be selected as a candidate for a drug to suppress the formation of urinary stones. Alternatively, a construct in which a reporter gene such as GFP or luciferase is linked downstream of the promoter of the crystal-binding protein gene is introduced into the cells, and the cells are stimulated with OSM in the same manner. The expression level of the reporter is compared in the presence and absence of the test substance, and if the expression level of the reporter gene is significantly reduced in the presence of the test substance, the test substance can be selected as a candidate for a drug to suppress the formation of urinary stones.

The present invention will be explained in more detail below with reference to the following examples, but these are merely illustrative and do not limit the scope of the present invention in any way.

Materials and Methods
animal
Eight-week-old male C57BL/6J mice were purchased from Japan SLC (Hamamatsu, Japan). The protocol for generating OSMRβ ^−/− was as previously described (Tanaka, M., et al. Blood 102: 3154-3162, 2003). Heterozygous breeding pairs were used to generate OSMRβ ^+/+ (WT; wild-type) and OSMRβ ^−/− littermates from our breeding colony. All mice were housed in an SPF facility under controlled conditions of 12-h light/dark cycle, temperature 22-25°C, and humidity 50-60% relative humidity. Mice were allowed free access to food (MF; Oriental Yeast, Tokyo, Japan) and water. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised 1978) or the Ministry of the Environment's standards for the care and management of laboratory animals and the experience of pain, and were approved by the Animal Care and Use Committee of Wakayama Medical University in accordance with the guidelines for animal experiments.

Glyoxylate (GOx) injection into mice As previously reported (Okada, A., et al. Urological research 35: 89-99, 2007), mice were intraperitoneally injected with 80 mg/kg glyoxylate (GOx; dissolved in PBS, Sigma-Aldrich, St. Louis, MO) once a day for 3, 6, 9, 12, or 15 days to create a mouse model of kidney stone disease.

Administration of anti-OSMRβ antibody (clone 7D2) In this study, we used anti-OSMRβ antibody (clone 7D2; Esashi E., et al. J Immunol 173: 4360-4367, 2004) and its isotype control antibody (R&D Systems, Minnesota, USA). Medgel Sheet II (Nitta Gelatin, Osaka, Japan), a hydrogel for sustained release, was used for administration of the antibodies. Each antibody (80 μl of antibody at a concentration of 1 mg/ml) was dropped onto 8 mg of Medgel and allowed to stand overnight at 4°C to allow the antibody to swell into the Medgel Sheet II. The Medgel Sheet II with the swollen antibody was embedded in the dorsal skin of mice, and two days later, glyoxylate (80 mg/kg) was administered to the mice every day for 6 days.

Pizzolato staining and quantification of crystal deposits. Mice were deeply anesthetized with isoflurane and perfused transcardially with ice-cold 0.9% NaCl followed by 4% paraformaldehyde. Kidney specimens were then rapidly removed and fixed in the same fixative for 3 or 4 h at 4°C, and cryoprotected in 30% sucrose in 0.1 M PBS for 16 h. The specimens were embedded in optimal cutting temperature (OCT) embedding medium (Sakura Finetek, Torrance, CA), flash frozen in cold hexane on dry ice, and stored at -80°C. Frozen sections were cut at 6 μm thickness using a cryostat. Pizzolato staining was performed to detect oxalate-containing crystals as previously described (Pizzolato P. The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 12: 333-336, 1964). Crystal deposition was quantified by measuring the area of positive Pizolato staining using Image J (US National Institutes of Health, Bethesda, MD) and expressed as a percentage of the total tissue area of renal cross sections. Multiple sections spaced 120 μm apart were selected for each kidney.

Immunohistochemical staining Mice were deeply anesthetized with isoflurane and transcardially perfused with ice-cold 0.9% NaCl followed by ice-cold modified Zamboni fixative (2% paraformaldehyde and 0.2% picric acid in 0.1 M PBS). Kidney specimens were quickly removed and fixed in the same fixative at 4°C for 3 h. All specimens were then immersed in 20% sucrose in 0.1 M PBS for 16 h. The specimens were embedded in OCT embedding medium, flash frozen in cold hexane on dry ice, and stored at -80°C. Frozen sections were prepared at 6 μm thickness using a cryostat. Immunofluorescent histological staining was then performed as previously reported (Komori, T., et al. J Biol Chem 287: 19985-19996, 2012). Briefly, sections were preincubated with 5% normal donkey serum for 1 h at room temperature, and then incubated with primary antibodies for 16 h at 4°C. Primary antibodies were used at the following dilutions: goat anti-OSMRβ (1:200; R&D Systems, Minneapolis, MN), goat anti-OSM (1:100; Abcam, Cambridge, UK), rabbit anti-EpCAM (1:100; Abcam), rabbit anti-PDGFRβ (1:100; Abcam), rat anti-F4/80 (1:50; Serotec, Oxford, UK), and rabbit anti-OPN (1:100; Abcam). Sections were then incubated with Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h at room temperature. Sections were counterstained with 4',6-diamino-2-phenylindole (DAPI). Immunofluorescence images were obtained using a fluorescence microscope (Olympus) equipped with a digital CCD camera (Olympus, Tokyo).

Preparation of single cell suspensions from mouse kidneys. Mice were deeply anesthetized with isoflurane and kidneys were rapidly removed. Kidneys were minced and digested with type II collagenase (Sigma-Aldrich) in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 2% fetal calf serum (FCS) using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were then passed through a nylon mesh (100 μm pore size; BD Biosciences) and centrifuged at 1200 rpm for 5 min. Cells in the pellet were resuspended in DMEM supplemented with 2% FCS and used as samples for flow cytometry and for isolation of primary renal epithelial cells (PRECs), macrophages, and fibroblasts.

Flow cytometry. Cells isolated from kidneys were incubated with anti-CD16/CD32 antibody (1:100, BD Biosciences, San Jose, CA) for 20 min at 4°C to block Fc binding, and then incubated with fluorescently labeled primary antibodies or control IgG for 30 min at 4°C. FITC-conjugated anti-CD45 antibody, PE-conjugated anti-CD11b antibody, APC-conjugated anti-F4/80 antibody, and PE-Cy7-conjugated anti-Ly6C antibody were purchased from eBiosciences (San Diego, CA). Stained cells were analyzed using a BD FACSVerse flow cytometer (BD Biosciences). Dead cells were removed using 7-aminoactinomycin-D (7-AAD) staining (eBioscience, San Diego, CA). Flow cytometry results were analyzed using FlowJo software suites (Tree Star, Ashland, OR). A forward scatter vs. side scatter plot was used as the first gate to exclude aggregates and debris. A second gate based on side scatter vs. 7-AAD was then used to identify individual live cells. CD45, F4/80, and CD11b triple positive cells were then sorted as macrophages in the kidney. Ly6C positive cells in the macrophage fraction were identified as M1 macrophages as previously reported (Taguchi K., et al. J Am Soc Nephrol 25: 1680-1697, 2014). Single color controls were used for compensation and gate setting.

Isolation of PRECs, macrophages, and fibroblasts from kidneys. PRECs and macrophages were isolated using magnetic activated cell sorting (MACS) anti-APC Multisort kit (Miltenyi Biotech) and autoMACS Pro Separator (Miltenyi Biotech). Cells isolated from kidneys were reacted with anti-CD16/CD32 antibody, followed by APC-conjugated anti-EpCAM antibody (eBioscience) and APC-conjugated anti-F4/80 antibody (eBioscience), to obtain both EpCAM-stained and F4/80-stained cells. Sorted PRECs and macrophages were cultured for 3 days in DMEM supplemented with 10% FCS, 100 U/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen). The cells were then treated with vehicle or 50 ng/ml OSM and cultured for 1 and 2 hours. All cells were cultured at 37°C in a humidified atmosphere of 5% _CO2 .
For the isolation of renal fibroblasts, cells isolated from kidneys were resuspended in DMEM supplemented with 10% FCS, 100 U/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen) and seeded onto collagen-coated 6-well plates (Corning, Corning, NY). When cells reached confluence, they were passaged, and the fourth passage cells were used as renal fibroblasts.

Quantitative PCR
Quantitative real-time PCR was performed as previously reported (Komori T., et al. J Biol Chem288: 21861-21875, 2013) with modifications. Briefly, total RNA was extracted and prepared from kidneys and cultured renal tubular epithelial cells, macrophages, and renal fibroblasts using TRI reagent (Molecular Research Center, Cincinnati, OH). cDNA was synthesized from total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).
The following TaqMan Gene Expression Assays (Applied Biosystems) were used: OSM (Mm01193966_m1), OSMRβ (Mm00495424_m1), F4/80 (Mm00802529_m1), TNF-α (Mm00443258_m1), IL-1β (Mm00434228_m1), monocyte chemoattractant protein-1 (MCP-1) (Mm00441242_m1), OPN (Mm00436767_m1), ANXa1 (Mm00440225_m1), ANXa2 (Mm01150673_m1), KIM1 (Mm00506686_m1), and 18S (Hs99999901_s1).
Quantitative real-time PCR was performed for each gene using Rotor Gene Probe PCR Kits (QIAGEN) on a Rotor Gene Q (QIAGEN, Hilden, Germany). The PCR amplification protocol was 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 60°C for 45 s. Analysis was performed using the ΔΔCT method, and the amount of mRNA for each transcript was normalized by the amount of 18S ribosomal RNA.

Statistical analysis Results are expressed as mean ± SEM. Comparisons between two groups were analyzed using Student's t-test. For multiple comparisons between groups, ANOVA was followed by post-hoc Bonferroni test. For all statistical tests, the threshold for significance was set at p < 0.05.

(result)
Example 1 Expression of OSM and OSMRβ in the kidney of a mouse model of urolithiasis As a mouse model of urolithiasis, 8-week-old wild-type mice (C57BL/6J) were used, in which glyoxylate (80 mg/kg body weight), an oxalate precursor, was administered intraperitoneally every day. Gene expression of OSM and OSMRβ in the kidney of this mouse model was examined by real-time PCR. The results are shown in Figure 1. Both OSM (Figure 1A) and OSMRβ (Figure 1B) increased from the third day after glyoxylate administration, and OSM reached its maximum on the sixth day, and OSMRβ reached its maximum on the third day. In addition, the expression of OSM and OSMRβ in the kidney on the sixth day after glyoxylate administration is shown in Figure 2. Both OSM and OSMRβ were expressed in the renal tubule epithelium, and OSMRβ was expressed in EpCAM-positive renal tubules. OSMRβ was also expressed in PDGFRβ-positive fibroblasts.

Example 2 Crystal formation after glyoxylate administration in OSMRβ-deficient mice
Glyoxylate (80 mg/kg body weight) was administered intraperitoneally daily to OSMRβ-deficient mice (OSMRβ ^-/- ) and control wild-type mice (WT), and crystal formation in the kidney was examined. The results are shown in Figure 3. In wild-type mice, numerous crystals positive for pizolato staining were observed at the corticomedullary junction 6 days after glyoxylate administration (Figure 3A). In contrast, almost no crystal formation was observed in OSMRβ ^-/- mice (Figure 3A). Figure 3B shows the quantification results of the area of pizolato staining-positive crystals corrected for the total kidney area.

Example 3: Changes in expression of inflammation-related genes after glyoxylate administration in OSMRβ-deficient mice
Glyoxylate (80 mg/kg body weight) was administered intraperitoneally daily to OSMRβ-deficient mice (OSMRβ ^-/- ) and control wild-type mice (WT), and the expression of TNF-α, IL-1β, F4/80, and MCP-1 in the kidneys was examined. The results are shown in Figure 4. Decreased expression of TNF-α (Figure 4A) and IL-1β (Figure 4B) was observed in the kidneys of OSMRβ ^-/- mice 3 and 6 days after glyoxylate administration. In addition, decreased expression of F4/80 (Figure 4C) and MCP-1 (Figure 4D) was observed in the kidneys of OSMRβ ^-/- mice 6 days after glyoxylate administration.

Example 4 Changes in renal macrophage cell number after glyoxylate administration in OSMRβ-deficient mice
Glyoxylate (80 mg/kg body weight) was administered intraperitoneally every day to OSMRβ-deficient mice (OSMRβ ^-/- ) and control wild-type mice (WT), and the numbers of total macrophages and M1 macrophages in the kidneys were analyzed by flow cytometry. The results are shown in Figure 5. Six days after glyoxylate administration, the numbers of total macrophages (Figure 5A) and M1 macrophages (Figure 5B) were decreased in the kidneys of OSMRβ ^-/- mice.

Example 5: Changes in expression of crystal-binding proteins after glyoxylate administration in OSMRβ-deficient mice
Glyoxylate (80 mg/kg body weight) was administered intraperitoneally daily to OSMRβ-deficient mice (OSMRβ ^-/- ) and their control wild-type mice (WT), and the expression of the crystal-binding proteins osteopontin (OPN), annexin a1 (ANXa1), annexin a2 (ANXa2), and KIM1 in the kidneys was examined. The results are shown in Figure 6. Decreased expression of OPN (Fig. 6A), ANXa1 (Fig. 6B), ANXa2 (Fig. 6C), and KIM1 (Fig. 6D) was observed in OSMRβ ^-/- mice 3 and 6 days after glyoxylate administration.

Example 6: Changes in gene expression of crystal-binding proteins in renal tubular epithelial cells The expression of OSMRβ and osteopontin (OPN) in the kidney was examined 6 days after the start of glyoxylate administration. The results are shown in Figure 7A. Osteopontin was expressed in OSMRβ-positive renal tubular epithelium. In addition, EpCAM-positive renal tubular epithelial cells were collected from wild-type mice, and the expression of OPN, annexin a1 (ANXa1), and annexin a2 (ANXa2) was examined 1 hour and 2 hours after stimulation with OSM (50 ng/ml). The results are shown in Figure 7B. Increased expression of OPN, ANXa1, and ANXa2 was observed due to OSM. Furthermore, fibroblasts, F4/80-positive macrophages, and EpCAM-positive renal tubular epithelial cells (PRECs) were collected from wild-type mice, and TNF-α expression was examined 1 and 2 hours after stimulation with OSM (50 ng/ml). The results are shown in Figure 7C. OSM increased TNF-α expression in fibroblasts.

Example 7 Inhibitory effect of anti-OSMRβ antibody (clone 7D2) on glyoxylate-induced crystal formation
Medgel containing 7D2 (80 μg) or its isotype control antibody (80 μg) was implanted subcutaneously on the back of 8-week-old wild-type mice (C57BL/6J), and glyoxylate (80 mg/kg body weight) was administered intraperitoneally for 6 consecutive days starting 2 days after the implantation. The amount of crystal formation in the kidney was examined. The results are shown in Figure 9. In the isotype control group (Isotype), many crystals positive for pizolato staining were observed at the corticomedullary junction 6 days after glyoxylate administration, whereas the formation of crystals was reduced in the 7D2 group (7D2) (Figure 9A). Figure 9B shows the quantification results of the area of pizolato staining-positive crystals corrected for the total kidney area.

Example 8 Inhibitory effect of anti-OSMRβ antibody (7D2) on the expression of crystal-binding proteins and inflammation-related molecules after glyoxylate administration
Medgel containing 7D2 (80 μg) or its isotype control antibody (80 μg) was implanted subcutaneously in the back of 8-week-old wild-type mice (C57BL/6J). Starting two days after implantation, glyoxylate (80 mg/kg body weight) was administered intraperitoneally for six consecutive days. Gene expression of crystal-binding proteins (OPN, ANXa1, ANXa2) and inflammation-related molecules (TNF-α, IL-1β, F4/80, MCP-1) in the kidney was examined. The results are shown in Figure 10. Compared to the isotype control group, the expression of crystal-binding proteins (OPN, ANXa1, ANXa2) and F4/80 was significantly decreased in the 7D2 group (7D2), and other inflammation-related molecules (TNF-α, IL-1β, MCP-1) also showed a tendency to decrease.

These results strongly suggest that the appearance of calcium oxalate crystals increases the expression of OSM in renal tubular epithelial cells, activating signaling via OSMR in the cells, which in turn induces the expression of various crystal-binding proteins that bind to calcium oxalate crystals and lead to urinary stone formation (Fig. 8, upper panel). Furthermore, it was suggested that OSM acts on renal fibroblasts to induce the expression of TNF-α, further eliciting an inflammatory response in OSMR-expressing cells (Fig. 8, upper panel). On the other hand, blocking signals from OSMR suppresses the expression of crystal-binding proteins and inhibits urinary stone formation (Fig. 8, lower panel).

The inhibitor of the present invention is extremely useful in that it can suppress the expression of crystal-binding proteins by blocking OSMR signaling, thereby preventing the formation of urinary stones, and therefore enables the fundamental prevention and treatment of urolithiasis (inhibition of progression and prevention of recurrence). In addition, the screening method of the present invention can use the OSM-OSMR signaling system to search for and identify novel preventive and therapeutic drugs for urolithiasis, and is therefore useful as a drug discovery tool for the development of a cure for urolithiasis.

Claims (7)

An agent for suppressing the formation of urinary stones, comprising a substance that inhibits the function or expression of oncostatin M , wherein the substance that inhibits the function of oncostatin M is
(a) an antibody against oncostatin M, or
(b) the extracellular domain of oncostatin M receptor or gp130, or a complex thereof
and
A substance that inhibits the expression of oncostatin M
(c) a nucleic acid having RNAi activity against a transcript of the oncostatin M gene;
(d) an antisense nucleic acid against a transcription product of the oncostatin M gene, or
(e) a ribozyme nucleic acid for a transcription product of the oncostatin M gene;
That is, a drug . An agent for suppressing the formation of urinary stones, comprising a substance that inhibits the function or expression of an oncostatin M receptor,
Substances that inhibit the function of the oncostatin M receptor
(a) Antibody against oncostatin M receptor β
and
A substance that inhibits the expression of oncostatin M receptors
( b ) a nucleic acid having RNAi activity against a transcript of the oncostatin M receptor β gene;
( c ) an antisense nucleic acid against a transcription product of the oncostatin M receptor β gene, or ( d ) a ribozyme nucleic acid against a transcription product of the oncostatin M receptor β gene. The agent according to claim 2 , wherein the substance that inhibits the function of the oncostatin M receptor is an antibody against the oncostatin M receptor β, and the antibody is in the form of a conjugate with a cytotoxin. The agent according to any one of claims 1 to 3 , which is for preventing or treating urolithiasis. A diagnostic agent for the risk of urinary tract stones, comprising an antibody against oncostatin M. A method for screening a drug for inhibiting the formation of urinary stones, comprising:
(1) contacting oncostatin M with its receptor in the presence and absence of a test substance;
(2) comparing the binding of oncostatin M to its receptor under both of the above conditions; and (3) selecting a test substance that reduces the binding of oncostatin M to its receptor as a candidate drug for inhibiting urinary stone formation. A method for screening a drug for inhibiting the formation of urinary stones, comprising:
(1) contacting a cell expressing the Oncostatin M receptor with Oncostatin M in the presence and absence of a test substance;
(2) comparing oncostatin M receptor signaling under both of the above conditions; and (3) selecting a test substance that reduces oncostatin M receptor signaling as a candidate drug for inhibiting urinary stone formation.