JPWO2008068827A1 - Novel organic ion transporter with kidney specificity - Google Patents

Abstract

Human SLC22 useful as a research tool to develop drugs for the treatment of ketoacidosis that show kidney-specific expression and have the ability to transport nicotinic acid, salicylic acid, prostaglandin F2α or ketone bodies or the like A novel organic anion transporter of the family or mouse slc22 family.

Description

  The present invention relates to a novel organic ion transporter (membrane transporter) protein relating to the transport of ketone bodies and similar substances in the kidney, a DNA encoding the protein, and a screening method and kit for a compound that promotes or inhibits the activity of the protein. The present invention provides a therapeutic / ameliorating agent for ketoacidosis obtained by the screening method.

  Ketoacidosis is a condition caused by an absolute deficiency of insulin, a hormone that promotes glucose utilization in diabetic patients. It is likely to occur at the onset of type 1 DM patients or when IDDM patients interrupt insulin injection. If insulin falls into an absolute deficiency, tissues such as the liver and muscles will not be able to take in glucose in the blood, forcing it to metabolize fat in cells, etc., but in this metabolic process, β-hydroxybutyric acid A ketone body mainly composed of This acidic substance, a ketone body, causes acidosis (a state in which the blood is acidic). Here, human body fluid has a pH of 7.4, and if it becomes pH 7.0 or less, it can be life-threatening. However, when the ketone body increases and ketoacidosis progresses, the body fluid becomes pH 7.0 or less and dies. There is also a case. This ketoacidosis can occur based not only on diabetes but also on the side effects of drugs.

  Here, transfusion is the main treatment for ketoacidosis. Also, bicarbonate is administered in high-grade acidosis where arterial blood pH is below 7.0. However, treatment with infusion is cumbersome for patients, and when bicarbonate is used, in severe cases, salt water is excessive, and a sudden rise in carbon dioxide levels causes electrolyte imbalance and consciousness. May result in dullness, coma, or death. Based on this, as a new therapeutic means, Patent Document 1 proposes a therapeutic agent for diabetic ketoacidosis containing β-lactone alkylene carboxylic acid as an active ingredient.

The main object of the present invention is to provide a research tool for developing a drug for the treatment of ketoacidosis, which is superior to a β-lactone alkylene carboxylic acid, and a specific drug using the research tool is disclosed. It is a secondary purpose to provide.
JP-A-2-104523

  The present inventor first paid attention to the SLC22 organic ion transporter family (hereinafter referred to as “SLC22 family”) in solving the problems. Here, the SLC22 family includes many renal tubular drug transporters, and there are three subfamilies of organic anion transporter (OAT), organic cation transporter (OCT), and organic cation / carnitine transporter (OCTN). It is known to exist.

  Under such circumstances, the present inventor has been researching OAT, and as a result, a new organic anion transporter (new subfamily of OAT) having different properties from various organic anion transporters known so far. And the present invention has been completed.

Specifically, the human anion transporter of the human SLC22 family or mouse slc22 family according to the present invention is the same as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or one or more amino acids of the amino acid sequence Is a protein exhibiting kidney-specific expression having the ability to transport nicotinic acid, salicylic acid, prostaglandin F ₂ α, ketone bodies, or the like, having an amino acid sequence deleted, substituted or added. The DNA encoding the organic anion transporter of the human SLC22 family or mouse slc22 family according to the present invention is the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.

  Here, it was first confirmed from the search results of the genome database that the new OAT had low homology with other groups.

  Next, for the new OAT, a gene knockout mouse was prepared for the purpose of clarifying the function in vivo together with functional analysis in the measurement of RI-labeled compound uptake, and a diabetic mouse was prepared by administration of alloxan. When the physiological function was examined by examining the ketone body concentration, it was different from the known transport substrates of organic cation transporter (OCT), organic anion transporter (OAT), and zwitterion transporter (OCTN) belonging to SLC22. It was confirmed to show selectivity.

Moreover, for functional analysis of an in vitro expression system it was established stably expressing cell mouse cDNA of the new OAT introduced into mouse proximal renal tubule-derived strains S ₂ cells. Using this, uptake experiments of various organic anions and radiocationic compounds of organic cations were carried out, and showed remarkable transport activity for organic anions such as nicotinic acid, prostaglandin F ₂ α and salicylic acid. . On the other hand, organic anions (paraaminohippuric acid, estrone sulfate, cAMP, dicarboxylic acid) that are typical transport substrates of the OAT family, organic cations (tetraethylammonium, choline) that are typical transport substrates of the OCT family, It did not show significant transport of ambipolar organic ions (carnitine), a representative transport substrate. Above show different substrate selectivity to known SLC22 family transporters, since the structurally also belongs to a new subfamily, the gene product n ovel type o rganic a nion t ransporter 1 and (OATN1) Named.

  Furthermore, in Northern blotting, OATN1 showed kidney-specific expression, and in situ hybridization revealed expression in the proximal tubule. Furthermore, immunohistochemical studies using mouse kidneys showed that OATN1 was localized in the proximal tubular lumen membrane.

OATN1 is a transporter that exists in the luminal membrane of the renal proximal tubule and is responsible for reabsorption of ketone bodies including β-hydroxybutyric acid in the renal proximal tubule. It is assumed that the inhibitor of OATN1 suppresses reabsorption of ketone bodies from the renal proximal tubule and exerts a function as a ketone body excretion drug. Therefore, in stably expressing cell expressing mouse OATN1 the S ₂ cell, consider suppressing effects of various compounds on the nicotinic acid uptake is a substrate for OATN1, we explored the inhibitor OATN1. As a result, 2,4-dichlorophenyloxyacetic acid, 3-phenylpropionic acid, 4-phenylbutanoic acid or a compound containing a similar substance showed a great inhibitory effect.

  These transporter activity inhibitors act in vivo as inhibitors of OATN1, and can promote the excretion of ketones. That is, it can be used as a medicament for the treatment or improvement of ketoacidosis caused by excessive ketone bodies and a decrease in pH in the body. It can also be applied as a medicine for ketoacidosis caused by diabetes, starvation or side effects of drugs.

The present invention (1) includes nicotinic acid having the same amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids of the amino acid sequence are deleted, substituted or added, It is an organic anion transporter of the human SLC22 family or mouse slc22 family that has the ability to transport salicylic acid, prostaglandin F ₂ α or ketone bodies or the like. Here, the “deletion, substitution or addition of amino acids in the protein” may be such that the transport activity of the selected substance is not lost, and is usually 1 to about 110, preferably 1 to about 55. Such a protein usually has 1 to 80%, preferably 1 to 90% of amino acid sequence homology with the amino acid sequence shown in SEQ ID NO: 1 or 2. Further, the “ketone body” is a general term meaning β-hydroxybutyric acid, acetoacetic acid and acetone (mainly β-hydroxybutyric acid). Furthermore, “analogue” refers to a substance that has been significantly transported as a result of testing according to Example 7 of the present specification. “Organic anion transporter” and “OAT” do not mean conventional OAT in a narrow sense, but are broad OAT that means an organic anion transporter.

  The present invention (2) has the same amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or has an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence are deleted, substituted, or added. It is an organic anion transporter of the human SLC22 family or mouse slc22 family, which shows proper expression.

The present invention (3) is the same as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or nicotinic acid having an amino acid sequence in which one or more amino acids of the amino acid sequence are deleted, substituted or added, It is an organic anion transporter of the human SLC22 family or mouse slc22 family that shows kidney-specific expression and has the ability to transport salicylic acid, prostaglandin F ₂ α or ketone bodies or the like.

  The present invention (4) is the transporter according to any one of the inventions (1) to (3), wherein the transporter exhibits expression in a renal cortex and an outer layer of the renal medulla.

  The present invention (5) is the transporter according to any one of the inventions (1) to (4), wherein the transporter specifically expresses in a luminal membrane of a proximal tubule cell.

  The present invention (6) is the transporter according to any one of the inventions (1) to (5), which does not exhibit transport of paraaminohippuric acid, estrone sulfate, cAMP, dicarboxylic acid, tetraethylammonium, choline or carnitine. Here, “not showing transport” means that when transport activity is measured according to Example 7 of the present specification, the difference from Mock is less than twice.

  The present invention (7) is the human or mouse DNA encoding any one of the transporters of the inventions (1) to (6), or hybridizes with the DNA under stringent conditions and has the same ability as the transporter. And / or human or mouse DNA encoding a transporter indicating the expression site. Here, “hybridization under stringent conditions” usually means that hybridization is carried out in a hybridization solution of 5 × SSC or an equivalent salt concentration for about 12 hours at a temperature of 37 to 42 ° C. Alternatively, pre-washing may be performed as necessary with a solution having a salt concentration equivalent to this, and then washing is performed in a solution having a salt concentration equivalent to 1 × SSC.

  The present invention (8) is the DNA of the invention (7) comprising the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.

  In the present invention (9), a stably expressing cell in which the DNA of the invention (7) or (8) is incorporated, or the DNA of the invention (7) or (8) or a cRNA complementary to the DNA is introduced. Transiently expressing cells.

  The present invention (10) is a polyclonal or monoclonal antibody against the transporter according to any one of the inventions (1) to (6). Here, the antibody can be used as a medicine (antibody medicine), an assay (antibody for detection) or the like.

  The present invention (11) is a screening method for a substance that promotes or inhibits the activity of the transporter, comprising the step of using any one of the transporters of the inventions (1) to (6). Here, “use transporter” means not only direct use such as screening the activity promoting / inhibiting substance by expressing the transporter in cells, but also promoting / inhibiting activity based on the three-dimensional structure analysis of the transporter. Indirect use such as screening of substances (in silico analysis) is also included.

  The present invention (12) is a screening kit for a substance that promotes or inhibits the activity of the transporter comprising the transporter of any one of the inventions (1) to (6) or the cell of the invention (9). is there. The other components of the kit are not particularly limited, and examples include a culture medium, an uptake standard substance (RI-labeled substance), various salt buffers for uptake experiments, a solubilizer, a scintillator, a 24-well plate, and a vial. be able to.

  This invention (13) contains 4-phenyl butyric acid, 2, 4- dichlorophenyl oxyacetic acid, 3-phenyl propionic acid, or a substance similar to these, any one of said invention (1)-(6) It is a transporter activity inhibitor. Here, the “similar substance” means at least sulfinpyrazone, phenylbutazone, benzbromazone, valproic acid, butanoic acid, valeric acid, hexanoic acid, heptanoic acid, caprylic acid, nonanoic acid, decanoic acid, etc. It is intended to include compounds that exhibit the same action.

  The present invention (14) is an antibody drug or aptamer of any one of the transporters according to the inventions (1) to (6), wherein the activity of the transporter according to any one of the inventions (1) to (6) is inhibited. It is an agent.

  The present invention (15) is the activity inhibitor of the invention (13) or (14) for promoting ketone body excretion.

  The present invention (16) is the activity inhibitor according to any one of the inventions (13) to (15) for treating or improving ketoacidosis.

  The present invention (17) is the activity inhibitor of the invention (16), which is ketoacidosis due to diabetes or side effects of drugs.

  The present invention (18) comprises the step of analyzing the transporter of any one of the inventions (1) to (6) or the DNA of the invention (7) or (8) in urine. ) To (6) is a diagnostic method for determining the presence and / or severity of a disease associated with any one of the transporters.

  The present invention (19) relates to the transporter according to any one of the inventions (1) to (6), comprising a polyclonal antibody or a monoclonal antibody against any one of the transporters according to the invention (1) to (6). This is a diagnostic kit for judging the presence and / or severity of a disease.

  The present invention (20) includes the step of analyzing the transporter according to any one of the inventions (1) to (6) or the DNA of the invention (7) or (8) in a body fluid. ) To (6) a diagnostic method for judging a genetic abnormality of a transporter.

  The present invention (21) is a diagnostic kit for judging a genetic abnormality of any one of the transporters according to the inventions (1) to (6), comprising primers capable of knowing information on all exons.

  Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the present invention. In the following examples, unless otherwise specified, each operation is described in “Molecular Cloning: Published by Sambrook, J., Fritsh, EF and Maniatis, T., Cold Spring Harbor Press in 1989”. The method was used, or when a commercially available kit was used, it was used according to the instruction manual of the commercially available product.

Example 1 (Molecular cloning and analysis of OATN1)
A new isoform (OATN1) belonging to the organic ion transporter family (SLC22) was found (mouse OATN1 accession No .: NM — 133980.1, human OATN1) by NCBI genome database analysis using the base sequence of the translation region of human OAT1. accession No .: NM_004256.1 [GenBankTM / EBI / DDBJ]). Primers for PCR were designed with reference to the sequence of this database, and RT-PCR was performed on mRNA extracted from mouse and human kidneys. Here, the primer sequences are as follows.
Mouse Fw: CGG GAT CCC TCA GGA GTG AGA AGC AGA C
RV: GCT CTA GAG GGT ACT GTC CCT GAT ACT C
Human FW: CGG GAT CCG GAG GTA GTG ACT GGC ATA C
RV: GCT CTA GAA GCC ATG GGC CAG ATT GTG G
The target cDNA was obtained as a template by RT-PCR → cDNA amplification (molecular cloning of OATN1), and incorporated into a plasmid vector for later analysis. The OATN1 cDNA incorporated into a plasmid vector (pcDNA3.1) is used for preparation of stable expression cells, sequencing by DNA sequencing, probe preparation for in situ hybridization and Northern blot.
In the above, PCR products were introduced into the BamHI and XbaI sites, which are the multiple cloning sites of pcDNA3.1. Further, the full-length nucleotide sequence of the cDNA was determined by a dye terminator cycle sequencing method (Applied Biosystems) using a synthetic primer for determining the template base sequence (FIGS. 1 and 2). Further, the cDNA base sequence was analyzed by a conventional method to determine the translation region of the cDNA and the amino acid sequence of the protein encoded therein (FIGS. 3 and 4).

Example 2 {Expression of OATN1 gene in various tissues of mice (analysis by Northern blotting)}
Nylon obtained by labeling a cDNA fragment corresponding to the 1691-951st bases of the mouse OATN1 gene with ³² P-dCTP and using this as a probe for electrophoresis and blotting of total RNA extracted from various mouse tissues Northern hybridization was performed on the membrane as follows. Hybridization was performed overnight in a hybridization solution (Perfect Hybrid, Takara) containing an OATN1 cDNA fragment labeled with ³² P-dCTP at 58 degrees. The membrane was washed at 65 degrees with 0.1 × SSC containing 0.1% SDS. This was radioautographed on an imaging plate and detected with BAS2000 (Fuji Film) (FIG. 5). As a result, it was confirmed that the OATN1 gene showed kidney-specific expression.

Example 3 {OATN1 gene expression in kidney tissue (analysis by in situ hybridization)}
After fixing the mouse kidney by perfusing with 4% paraformaldehyde, it was minced and further fixed with 4% paraformaldehyde. The obtained mouse kidney was sliced to a thickness of 5 μm, and the obtained section was used for in situ hybridization. Sense cRNA and antisense cRNA were synthesized from full-length OATN1 cDNA using T7 or T3 RNA polymerase and used as probes. The sections were hybridized with the hybridization solution with the probe overnight, and washed with 0.1 × SSC for 30 minutes at 37 ° C. Color development was performed by an enzymatic method. As a result, in mouse kidney, OATN1 mRNA was expressed in the renal cortex and outer medullary outer zone (FIG. 6).

Example 4 (Production of N-terminal antibody of mouse OATN1 and human OATN1)
In the intracellular N-terminal region of mouse OATN1, mouse: 5-16th (AQVMAEVGDFGR), human: 2-16th (AQFVQVLAEIGDFGR) amino acid was peptide synthesized, conjugated to KLH (Keyhole limpet hemocyanin), and adjuvant After mixing with rabbits, rabbits were immunized. The first immunization was followed by a second immunization one month later, and a further two weeks later, and a serum was collected, which was used as an OATN1 antiserum. The antiserum was purified with an affinity column using an antigen to obtain an affinity antibody.

Example 5 (Immunohistochemical staining in kidney using anti-OATN1 antibody)
C57BL black mice were refluxed with phosphate buffered saline (PBS: 20 mM phosphoric acid, 140 mM sodium chloride, pH 7.4), followed by refluxing with a 4% paraformaldehyde solution, and then the kidneys were removed. The excised kidney was immersed in a 4% paraformaldehyde solution and fixed for 2 hours. Embedding using OCT compound to prepare frozen sections (5 μm). An antibody obtained by using the N-terminus of mouse OATN1 as an antigen (mouse OATN1 produced in Example 4) was diluted 500 times, reacted with a kidney section, and then a secondary antibody (Envison / HRP, conjugated with peroxidase). DAKO) was reacted to develop DAB (diaminobenzidine) color. As a result, it was confirmed that OATN1 was localized in the luminal membrane of the proximal tubule of the kidney in the mouse kidney (FIG. 7).

Example ₆ (Preparation of S 2 -OATN1 stably expressing cells)
The _{S 2} cell is a cell line derived from the proximal tubules were transfected using Lipofectamine 2000 with pcDNA3.1-OATN1 (Invitrogen), over a period of about a month selection at 400 mg / ml of geneticin Thus, cells stably expressing OATN1 were prepared. The culture conditions are as follows.
Culture tank: 33 degrees, 5% CO ₂
Culture medium: RITC 80-7 medium containing 5% fetal bovine serum, 10mg / ml transferring, 0.08 U / ml insulin, 10ng / ml recombinant epidermal growth factor, and 400mg / ml geneticin.

Example 7 (Measurement of transport activity of mouse OATN1)
S ₂ cells were plated on a 24-well cultured cell plate at 1 × 5 ¹⁰ cells / well, cultured for 2 days, and then Dulbecco's modified phosphate-buffered saline (D-PBS: 137 mM NaCl, 3 mM KCl, 8 mM NaHPO4 , 1 mM KH ₂ PO ₄ , 1 mM CaCl ₂ and 0.5 MgCl ₂ ; pH 7.4) and preincubated with D-PBS for 10 minutes. Incubation was carried out in an uptake solution containing each of the RI-labeled compounds, and after uptake, the uptake was immediately stopped by washing with D-PBS cooled 4 times. The cells were suspended in 0.1 M sodium hydroxide and measured with a liquid scintillation counter (LSC-3100; Aloka). As a result, as shown in FIGS. 8 and 9, S ₂ -OATN1 stably expressing cells in which OATN1 was expressed were β-hydroxybutyric acid ( ¹⁴ C-labeled) and nicotinic acid ( ³ H-labeled), which are the main components of the ketone body. ), Prostaglandin F ₂ α ( ³ H-labeled) and salicylic acid ( ¹⁴ C-labeled) were found to be incorporated into the cells. On the other hand, paraaminohippuric acid ( ^14C label), estrone sulfate ( ^14C label), α-ketoglutarate ( ^14C label), uric acid ( ^14C label), taurocholic acid ( ^14C ), which are organic anion transporter uptake substrates Label), ochratoxin A ( ³ H label), organic cation transporter uptake substrate tetraethylammonium ( ¹⁴ C label), organic zwitterion transporter uptake substrate carcinine ( ³ H label) (FIG. 10). FIG. 11 shows the results of examining the concentration-dependent uptake of β-hydroxybutyric acid by stably expressing cells (S ₂ ) of mouse OATN1.

Example 8 (Screening of inhibitors of OATN1)
According to the same method as in Example 7, a transport inhibition experiment was carried out by mixing 1 μM ³ H-nicotinic acid with 100 μM non-RI compound and the incorporation solution. As a result, the addition of anionic substances such as 2,4-dichlorophenyloxyacetic acid, 3-phenylpropionic acid, 4-phenylbutanoic acid, dichlorophenac, carboxylic acids having an alkyl substituent of 3 to 9 carbon atoms, etc. Inhibition of nicotinic acid uptake effect was confirmed (FIGS. 12 to 17).

Example 9 (Preparation of OATN1 knockout mouse)
By inserting the neomycin gene into the first exon of the mouse OATN1 gene and the intron portion before the first exon, which is predicted to be the promoter region thereof, by homologous recombination using a targeting vector, the expression regulatory site of the OATN1 gene and the first methionine The N-terminal translation region containing was destroyed.

Example 10 (Comparison of urinary β-hydroxybutyric acid concentration in diabetes model mice)
Diabetic mice were prepared by administering alloxan (100 mg / kg), which is known to induce diabetes, from the tail vein. Urine sugar was confirmed using test paper. Urine was collected on the 4th day after alloxan administration from diabetic wild-type mice and OATN1 knockout mice, and β-hydroxybutyric acid concentration in urine was determined using qualitative examination and biochemical techniques using test paper. Measured by quantitative examination. The result is shown in FIG. Here, FIG. 18 (1) shows the results of a urinary ketone body qualitative reaction test, in which the upper shows a wild type mouse, the lower shows an OATN1 knockout mouse, the left shows an alloxan-administered diabetes group, and the right shows a saline-administered group. . As can be seen from the figure, only the alloxan-treated diabetic group of OATN1 knockout mice developed color (purple). In the figure, the raw food administration group looks black because it is dark yellow but not purple. Next, FIG. 18 (2) shows the quantification result of urinary β-hydroxybutyric acid. As can be seen from the figure, β-hydroxybutyric acid was not detected in wild-type mice, whereas a large amount of β-hydroxybutyric acid was detected in OATN1 knockout mice. Moreover, FIG. 19 is a figure which showed the time-dependent change of the total amount of ketone excretion in urine after alloxan administration. As can be seen from the figure, the total ketone body excretion amount of the OATN1 knockout mouse was very large as compared with the wild type mouse. The “total ketone body” in this specification was measured by a well-known test method using a chemical reaction that reacts with a ketone group.

Example 11 (Urine ketone body excretion promotion confirmation test)
Among the inhibitors of OATN1, 4-phenylbutyric acid, 2,4-dichlorophenyloxyacetic acid, and 3-phenylpropionic acid were intraperitoneally administered, and the influence on urinary ketone body excretion was examined. Alloxan was administered to a male 8-week-old wild type mouse via the tail vein of 100 mg / kg bw (2% alloxan, 0.05 ml / 10 g bw), and 4 days after intravenous administration, an OATN1 inhibitor (above) was administered intraperitoneally. At 30 minutes, 60 minutes, 90 minutes and 180 minutes after administration, urine was collected and the ketone body concentration in urine was measured. The urinary ketone body concentration before administration of each inhibitor (inhibitor (−)) and after administration (inhibitor (+)) was compared. 4-Phenylbutyric acid, 2,4-dichlorophenyloxyacetic acid, and 3-phenylpropionic acid significantly increased urinary ketone body excretion (FIG. 20). Furthermore, when the time dependence of urinary ketone body concentration increase by 4-phenylbutyric acid was measured, an increase in urinary ketone body excretion was rapidly observed by administration of 4-phenylbutyric acid (FIG. 21). .

FIG. 1 shows the base sequence of mouse OATN1. FIG. 2 is a diagram showing the base sequence of human OATN1. FIG. 3 shows the amino acid sequence of mouse OATN1. FIG. 4 shows the amino acid sequence of human OATN1. FIG. 5 is a diagram showing the results of analyzing the expression of OATN1 gene mRNA in mouse organ tissues by Northern blotting. Of the organs examined, it was shown to be strongly expressed only in the kidney. FIG. 6 is a diagram showing the results of in situ hybridization of OATN1 using mouse kidney sections. Strong signals were confirmed in the cortical region and outer medullary outer zone. FIG. 7 shows the results of immunohistochemical staining using an anti-OATN1 antibody in mouse kidney sections. It was confirmed to be localized in the luminal membrane of the proximal tubule. FIG. 8 shows the results of an experiment of β-hydroxybutyric acid uptake by mouse OATN1 stably expressing cells (S ₂ -OATN1). Significant transport activity of β-hydroxybutyric acid by mouse OATN1 was confirmed. FIG. 9 is a diagram showing the results of an uptake experiment of compounds in which uptake other than β-hydroxybutyric acid was observed by mouse OATN1 stably expressing cells (S ₂ -OATN1). Significant transport activities of nicotinic acid, salicylic acid, and prostaglandin F ₂ α were confirmed. FIG. 10 is a diagram showing the results of an experiment for incorporating a representative substrate of the SLC22 family in which uptake of mouse OATN1 stably expressing cells (S ₂ -OATN1) was not observed. Representative substrates of organic ion transporters: paraaminohippuric acid, estrone sulfate, α-ketoglutaric acid, uric acid, ochratoxin A, taurocholic acid, representative substrates of organic cation transporters: tetraethylammonium, organic cation / ambipolar ion transporter No transport activity of carnitine, which is a representative substrate, was observed by OATN1. FIG. 11 is a graph showing the results of examining the concentration-dependent uptake of β-hydroxybutyric acid by mouse OATN1 stably expressing cells (S ₂ -OATN1). The result of the Eadie-Hofstee plot is shown on the side. FIG. 12 is a diagram showing the results of examining the effects of various organic anions added to the system in the nicotinic acid uptake experiment using mouse OATN1 stably expressing cells (S ₂ -OATN1). FIG. 13 is a graph showing the results of examining the effects of various diuretics added to the system in a nicotinic acid uptake experiment using stably expressing cells (S ₂ -OATN1) of mouse OATN1. FIG. 14 is a diagram showing the results of examining the effects of various organic anions added to the system in a nicotinic acid uptake experiment with stably expressing cells (S ₂ -OATN1) of mouse OATN1. FIG. 15 is a diagram showing the results of examining the effect of addition of a short-chain fatty acid to the system in an experiment of nicotinic acid uptake by a mouse OATN1 stably expressing cell (S ₂ -OATN1). FIG. 16 is a diagram showing the results of examining the effect of addition of a metabolite of a neurotransmitter to the system in a nicotinic acid uptake experiment using mouse OATN1 stably expressing cells (S ₂ -OATN1). FIG. 17 is a diagram showing the overall results of a nicotinic acid uptake experiment using cells stably expressing mouse OATN1 (S ₂ -OATN1) (data of some compounds not shown in FIGS. 12 to 16 are also shown). . FIG. 18 is a diagram showing the urinary β-hydroxybutyric acid concentration in diabetic wild-type mice and OATN1 knockout mice. FIG. 19 is a diagram showing a change with time of the total ketone excretion in urine after alloxan administration. FIG. 20 is a graph showing the total ketone body concentration in urine in diabetic WT mice after treatment with an OATN1 inhibitor. FIG. 21 is a diagram showing the time dependency of the total ketone body concentration in urine after treatment with 4-phenylbutyric acid (PBA).

Claims (21)

Nicotinic acid, salicylic acid, prostaglandin F ₂ having the same amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or having an amino acid sequence in which one or more amino acids are deleted, substituted, or added. An organic anion transporter of the human SLC22 family or mouse slc22 family having the ability to transport α or ketone bodies or the like.   Human SLC22, which has the same amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or has an amino acid sequence in which one or more amino acids of the amino acid sequence are deleted, substituted or added, and exhibits kidney-specific expression Family or mouse slc22 family organic anion transporters. Nicotinic acid, salicylic acid, prostaglandin F ₂ having the same amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or having an amino acid sequence in which one or more amino acids are deleted, substituted, or added. An organic anion transporter of the human SLC22 family or mouse slc22 family exhibiting kidney-specific expression with the ability to transport alpha or ketone bodies or the like.   The transporter according to any one of claims 1 to 3, wherein the transporter exhibits expression in a renal cortex and an outer layer of a renal medulla.   The transporter according to any one of claims 1 to 4, wherein the transporter specifically expresses in a luminal membrane of a proximal tubule cell.   The transporter according to any one of claims 1 to 5, which does not exhibit transport of paraaminohippuric acid, estrone sulfate, cAMP, dicarboxylic acid, tetraethylammonium, choline or carnitine.   Human or mouse DNA encoding the transporter according to any one of claims 1 to 6, or a transporter that hybridizes with the DNA under stringent conditions and exhibits the same ability and / or expression site as the transporter Human or mouse DNA encoding   The DNA according to claim 7, comprising the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.   A stable expression cell into which the DNA according to claim 7 or 8 is incorporated, or a transient expression cell into which the DNA according to claim 7 or 8 or a cRNA complementary to the DNA is introduced.   The polyclonal antibody or monoclonal antibody with respect to the transporter as described in any one of Claims 1-6.   The screening method of the substance which accelerates | stimulates or inhibits the activity of the said transporter including the process of using the transporter as described in any one of Claims 1-6.   A kit for screening for a substance that promotes or inhibits the activity of the transporter comprising the transporter according to any one of claims 1 to 6 or the cell according to claim 9.   The transporter activity inhibitor according to any one of claims 1 to 6, comprising 4-phenylbutyric acid, 2,4-dichlorophenyloxyacetic acid, 3-phenylpropionic acid or a similar substance thereto.   The transporter activity inhibitor according to any one of claims 1 to 6, which is the antibody drug or aptamer of the transporter according to any one of claims 1 to 6.   The activity inhibitor according to claim 13 or 14 for promoting ketone body excretion.   The activity inhibitor as described in any one of Claims 13-15 for a ketoacidosis treatment or improvement.   The activity inhibitor according to claim 16, which is ketoacidosis caused by diabetes or a side effect of a drug.   The transporter according to any one of claims 1 to 6, or the transporter according to any one of claims 1 to 6, comprising a step of analyzing the transporter according to any one of claims 1 to 6 or the DNA according to claim 7 or 8. Method for determining the presence and / or severity of a disease.   The presence or absence and / or severity of a disease associated with the transporter according to any one of claims 1 to 6, comprising a polyclonal antibody or a monoclonal antibody against the transporter according to any one of claims 1 to 6. Diagnostic kit to judge.   The transporter gene according to any one of claims 1 to 6, comprising a step of analyzing the transporter according to any one of claims 1 to 6 or the DNA according to claim 7 or 8 in a body fluid. A diagnostic method for judging abnormalities.   The diagnostic kit which judges the gene abnormality of the transporter as described in any one of Claims 1-6 containing the primer which can know the information of all the exons.

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