JP2012082181A - Endogenous erythropoietin expression-enhancer and organic ion transporter expression-enhancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To clarify how uremia substances accumulated along with the lowering of estimated glomerular filtration rate (eGFR) which is evaluation of a kidney function is involved with renal anemia which is one of main complications of chronic kidney disease (CKD).SOLUTION: It was found out that indoxyl sulfate being one of the uremia substances enhancing the expression of GAT3, suppresses the expression of organic anion transporter SLCO4CI, and suppresses the expression of erythropoietin. Further, it was clarified that spherical absorbing coal (KREMEZIN (R)) being one of activated charcoal has a function for enhancing endogenous erythropoietin expression and can be used as a therapeutic agent of the renal anemia, further has a function for enhancing the expression of the organic anion transporter (SLCO4CI), and can be used as the uremia substance discharge enhancer.

Description

  The present invention relates to an endogenous erythropoietin expression enhancer, an organic ion transporter expression enhancer, and the like, which contain a uremic substance adsorbent such as adsorbed charcoal as an active ingredient.

  The number of patients with renal diseases such as patients with renal failure, especially patients undergoing renal dialysis, has been increasing year by year. One of the functions of the kidney is the excretion of taking drugs and uremic substances that are flowing into the blood, sending them out into the urine and excreting them outside the body. Therefore, when kidney function is lowered, drugs and uremic substances are likely to accumulate in the body, and kidney cells are damaged, causing further decline in kidney function. In addition, it is known that a person with impaired kidney function is more likely to have a stroke or myocardial infarction than a normal person. Therefore, maintaining the function of excreting uremic substances is very important in preventing the deterioration of kidney damage, and is also important in terms of preventing stroke and heart disease. Currently, 200,000 patients are undergoing dialysis treatment to artificially remove uremic substances, and more than 30,000 people are newly introduced to dialysis every year. Since dialysis therapy costs about 5 million yen per person per year, medical expenses of about 1 trillion yen are constantly required for 200,000 people, which is also a social problem.

  In recent years, the concept of chronic kidney disease (CKD) has been proposed, and recognition of the importance of its prevention and treatment is increasing. CKD is a condition in which renal impairment and chronic decline in renal function continue chronically, and end-stage renal failure occurs if appropriate treatment is not performed, and it is impossible to survive without artificial dialysis or kidney transplantation. . End-stage renal failure continues to increase worldwide, and it is said that there are currently about 13.3 million CKD patients in Japan, which is about 1 in 8 adults. In addition, the number of patients undergoing artificial dialysis continues to increase as described above, and early detection and early treatment of CKD is an urgent issue. Under these circumstances, if a method for preventing symptoms of renal diseases such as renal failure and CKD, or a treatment that alleviates such symptoms and delays the introduction of dialysis, the patient's QOL (Quality of Life) will be developed. In addition to contributing to society, it is expected that medical costs can be significantly reduced, contributing greatly to society.

  Renal anemia is one of the major complications in CKD, and it is known that the pathology of renal anemia is based on a decrease in endogenous erythropoietin (Epo) production. It is not clear how a decrease in renal function affects erythropoietin production, but uremic substances may be one of the causes because of reports that hemodialysis has improved renal anemia Sex has been suggested. Erythropoietin is one of the hematopoietic promoting factors that promote red blood cell production, and erythropoietin deficiency contributes to anemia. Since erythropoietin is mainly produced in the kidney, the expression level of erythropoietin decreases due to chronic renal failure and anemia occurs. Therefore, the main treatment for renal anemia is administration of erythropoietin, but erythropoietin preparations are expensive and require frequent injections.

  So far, the inventors have been involved in the excretion of the uremic substance, an organic anion transporter SLCO4C1 that is expressed only on the side of the human renal proximal tubule, and controls the excretion of the uremic substance by regulating the expression of SLCO4C1. The possibility of the new CKD treatment was reported (nonpatent literature 1).

  It is also known that the expression of erythropoietin is enhanced by a GATA3 inhibitor because a GATA transcription factor binds to the GATA sequence upstream of the erythropoietin gene and negatively regulates erythropoietin gene expression. And it is reported that L-NMMA (NG-monomethyl L-arginine) accumulated due to renal failure increases GATA binding activity and mRNA expression level, thereby suppressing erythropoietin gene expression and causing renal anemia. (Non-Patent Document 2).

  One of the preparations for renal failure currently used clinically is a form of activated carbon, spherical adsorbed charcoal cremedin (registered trademark: Kureha Chemical Co., Ltd.), which is an orally administrable uremic substance adsorbing function. Cremedin is a black spherical particle obtained by oxidizing and reducing spherical microporous carbon derived from petroleum hydrocarbons at a high temperature, adsorbing harmful substances present in the digestive tract without being absorbed into the body, It is an oral adsorbent that is used for the purpose of eliminating uremic substances and suppressing the decrease in renal function by being excreted together with feces. Cremedin is also known to enhance the action of a recombinant human erythropoietin preparation (rHuEPO) administered for the treatment of renal anemia, but the detailed mechanism is not clear (Patent Document 1). .

  The inventors of the present application analyzed the serum of 41 CKD patients using a capillary electrophoresis mass spectrometer (CE-MS), and estimated glomerular filtration rate (eGFR), which is an evaluation of renal function. 52 substances (including 36 new substances) including indoxyl sulfate were identified as uremic substances accumulating with a decrease (Non-patent Document 3).

JP-A-6-199689

Toyohara T. et al., J Am Soc Nephrol., 20: 2546-2555 (2009) Imagawa S. et al., Kidney International. 61. 396-404 (2002) Toyohara T. et al., Hypertens Res. 2010 Sep; 33 (9): 944-52. (2010)

  The present inventors have identified uremic substances that accumulate with a decrease in eGFR, which is an evaluation of renal function (Japanese Patent Application No. 2009-205033, Non-Patent Document 3), but how these substances are involved in the pathology of CKD. I didn't know if I was doing it. Therefore, an object of the present invention is to clarify how these uremic substances are involved in renal anemia, which is one of the main complications of CKD.

  The present inventors investigated the effect of indoxyl sulfate (IS; Indoxyl Sulfate), one of the uremic substances, on the expression of GATA3 and the expression of SLCO4C1 transporter, and verified the effect on erythropoietin production and its mechanism. did. Furthermore, the inventors have found that activated carbon (cremedin) enhances the expression of SLCO4C1 transporter and enhances the expression of endogenous erythropoietin, thereby completing the present invention.

  That is, the present invention relates to (1) an endogenous erythropoietin expression enhancer comprising a uremic substance adsorbent as an active ingredient, and (2) an endogenous erythropoietin expression enhancer according to (1) above, wherein the uremic substance adsorbent is activated carbon. And (3) the endogenous erythropoietin expression enhancer according to (2) above, wherein the activated carbon is a spherical adsorbed charcoal, and (4) the endogenous erythropoietin expression enhancer according to any one of (1) to (3) above The present invention relates to a therapeutic agent for renal anemia.

  The present invention also relates to (5) an organic ion transporter expression enhancer comprising an uremic substance adsorbent as an active ingredient, and (6) an organic ion transporter expression described in (5) above, wherein the uremic substance adsorbent is activated carbon. (7) The organic ion transporter expression enhancer described in (6) above, wherein the activated carbon is a spherical adsorption charcoal, and (8) the organic ion transporter is an organic anion transporter SLCO4C1 The organic ion transporter expression enhancer according to any one of (7) and (9) a uremic substance excretion enhancer comprising the organic ion transporter expression enhancer according to any one of (5) to (8) above.

  According to the endogenous erythropoietin expression enhancer comprising the activated carbon of the present invention as an active ingredient, it is possible to enhance the amount of erythropoietin expressed in vivo and treat renal anemia. Moreover, according to the organic anion transporter SLCO4C1 expression enhancer which uses the activated carbon of this invention as an active ingredient, the amount of SLCO4C1 can be increased and the uremic substance excretion action can be enhanced.

It is a figure which shows the result of having added the uremic substance (anion) to erythropoietin production human hepatocyte cell line Hep3B, and having investigated the influence on erythropoietin production ability. It is a figure which shows the result of having added the uremic substance (cation) to erythropoietin production human hepatocyte cell line Hep3B, and having investigated the influence on erythropoietin production ability. It is a figure which shows that indoxyl sulfate (IS) suppresses SLCO4C1 mRNA expression in HK-2 cells. FIG. 4 shows that indoxyl sulfate (IS) enhances GATA3 mRNA expression in HK-2 cells. It is a figure which shows that the mRNA expression of SLCO4C1 is enhanced by the administration of cremedin in the renal failure rat. It is a figure which shows that indoxyl sulfate (IS) and 1-methyl adenosine (1-Methyladenosine) suppress the expression of erythropoietin at a transcription | transfer level in ACHN cell. It is a figure which shows that indoxyl sulfate (IS) suppresses the expression of SLCO4C1 at a transcription level (left graph) and enhances the expression of GATA3 at a transcription level (right graph) in ACHN cells. It is a figure which shows that the expression of erythropoietin is enhanced by the administration of cremedin to a renal anemia patient. It is a figure showing the mechanism in which the expression of SLCO4C1 transporter and erythropoietin is enhanced by adsorbing and eliminating uremic substances and suppressing suppression of GATA3 expression and suppression of SLCO4C1 transporter expression by uremic substances.

  As the uremic substance adsorbent in the present invention, the uremic substance is adsorbed and discharged out of the body, thereby reducing the uremic substance in the body, thereby reducing the expression of GATA3 which has been enhanced by the uremic substance. The substance that adsorbs a uremic substance that can be used for the purpose of enhancing the expression of endogenous erythropoietin after the suppression of the expression of erythropoietin by is released, and the suppression of the expression of the organic ion transporter by GATA3 is released. Any substance that adsorbs a uremic substance that can be used for the purpose of enhancing expression is not particularly limited. Examples thereof include activated carbon, organic polymers, inorganic adsorbents such as zeolite, clay, clay, and oral adsorbents such as starches and celluloses, chitin and chitosan, among which activated carbon is preferable. Further, among activated carbons, black particles obtained by oxidation / reduction treatment of petroleum hydrocarbon-derived microporous carbon at high temperature, 0.05 to 2 mm in diameter, preferably 0.1 to 1 mm in diameter, more preferably Is preferably a spherical adsorbed carbon which is a high-purity carbon fine particle having an infinite number of pores having a diameter of 0.2 to 0.4 mm. Such spherical adsorbed charcoal can be obtained as a commercially available product, Kremezin (registered trademark), for example, “Kremezin (registered trademark) fine grain” or “Kremezin (registered trademark) capsule 200” manufactured by Kureha Chemical Industries, Ltd. “Spherical adsorption charcoal fine granule Mylan” manufactured by Mylan Pharmaceutical Co., Ltd. can be used. In this specification, these spherical adsorbed charcoal may be collectively referred to as cremedin.

  A preferred embodiment of the endogenous erythropoietin expression enhancer of the present invention comprises a spherical adsorbent as an active ingredient as a uremic substance adsorbent, and the expression of erythropoietin by administering an effective amount thereof to mammals including humans. The amount can be increased, and when used in combination with a carrier such as an excipient, a drug or a supplement can be obtained. By administering this drug or supplement to mammals including humans with renal insufficiency, the expression level of erythropoietin in vivo increases and can be used for the prevention or treatment of renal anemia. Examples of such erythropoietin include mouse erythropoietin gene (NM_007942; GenBank accession number), rat erythropoietin gene (NM_017001), human erythropoietin gene (NM_000799), and proteins expressed from these.

  A preferred embodiment of the organic ion transporter expression enhancer of the present invention comprises a spherical adsorbent charcoal as an active ingredient as a uremic substance adsorbent, and an organic ion is administered by administering an effective amount thereof to mammals including humans. It is a drug that can enhance the expression level of the transporter. This drug can increase the excretion of uremic substances by increasing the expression level of the organic ion transporter by administering it to mammals including humans with renal failure. Examples of the organic ion transporter include SLCO4C1, OCT1 (organic cation transporter1), OCT2 and OCT3, OCTN1 (novelorganic cation transporter1), OCTN2, OAT1 (organic anion transporter1), OAT2 and OAT3. SLCO4C1 can be preferably shown. Examples of such SLCO4C1 include rat SLCO4C1 gene (NM_001002024), mouse SLCO4C1 gene (NM_172658), human SLCO4C1 gene (AY273896), and expressed proteins expressed from these.

  The therapeutic agent for renal anemia of the present invention containing the above-described endogenous erythropoietin expression enhancer of the present invention and the uremic substance excretion enhancer of the present invention including the above-described organic ion transporter expression enhancer of the present invention are orally administered. However, the dose depends on the subject (animal or human), age, individual difference, medical condition and the like. For example, the dose when orally administering spherical adsorbed charcoal (clemedin) to humans is usually 0.1 to 100 g, preferably 1 to 50 g, more preferably 3 to 20 g per day. It may be taken in 4 divided doses, and the dose may be appropriately increased or decreased depending on the symptoms. For example, activated carbon such as cremedin can be given in any dosage form such as granules, tablets, dragees, capsules, sticks, sachets or suspensions. In addition, the administration target of the therapeutic agent for renal anemia and the uremia substance excretion enhancer in this specification includes not only humans but also other non-human animals, but humans and non-human mammals are preferable, and humans are more preferable.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

  The inventors of the present application analyzed the serum of 41 CKD patients using a capillary electrophoresis mass spectrometer (CE-MS), and estimated glomerular filtration rate (eGFR), which is an evaluation of renal function. 52 (including 36 new substances) including indoxyl sulfate were identified as uremic substances accumulating with a decrease (Non-patent Document 3, Japanese Patent Application No. 2009-205033). Of these, 31 types of uremic substance compounds were obtained, cells in which erythropoietin production was induced under a low oxygen condition were cultured in a medium containing these compounds, and the effect on erythropoietin production ability was evaluated by the following method. did.

Erythropoietin-producing human hepatocyte cell line Hep3B (obtained from ATCC (American Type Culture Collection)) is 100 U / mL penicillin (GIBCO), 100 μg / mL streptomycin (GIBCO), 10% fetal calf serum (GIBCO) And 5% CO ₂ /95% air at 37 ° C. with Dulbecco's modified Eagle's medium (DMEM) (manufactured by GIBCO). In order to examine the effect of various drugs on erythropoietin production ability, 1 × 10 ⁵ Hep3B cells were seeded per well in a 12-well cell culture plate, and 24 hours later, the culture solution was used as a test substance (each 1 mM, 1-methyl Adenosine was changed to a culture solution containing 0.1 mM) or 1% DMSO or water. The control group is a normal cultured cell apparatus under the conditions of 5% CO ₂ /95% air and 37 ° C., and the erythropoietin production induction group is 5% CO ₂ /1% O ₂ and 37 ° C. hypoxia using a low oxygen chamber. Cultured for 24 hours under conditions. After culturing for 24 hours, the concentration of erythropoietin in the supernatant was measured using a human erythropoietin ELISA kit manufactured by Bender Med Systems, and the protein concentration of the cells was measured using a BCA protein measurement kit manufactured by Bio-Rad. did. The value of the amount of erythropoietin in the measured supernatant was divided by the value of the amount of protein in the cell, and the amount of erythropoietin produced per certain amount of cell was calculated. Furthermore, each test substance was added with a culture solution containing 1% DMSO or water, 5% CO ₂ /1% O _2, and erythropoietin production per fixed cell amount when cultured at 37 ° C. as 100%. The amount of erythropoietin produced in each case was determined. FIG. 1 shows a graph of results obtained when an anionic test substance is used, and FIG. 2 shows a graph of results obtained when a cationic test substance is used.

  As a result, indoxyl sulfate (IS), isocitrate (Isocitrate), citraconic acid (Citraconate), N-acetyl-L-glutamic acid (N-Ace-L-Glu), homovanillic acid (Homovanilate), N-acetyl-aspartic acid-glutamic acid (N-Ace-Asp-Glu), mandelate, L-cysteine-S-sulfate, cation as hypotaurine, 1- Thirteen substances (1-asterisk and sharp in FIGS. 1 and 2): 1-Methyladenosine, Indole-3-acetate, Kynurenine, N-monomethyl-arginine (NMMA) ), A significant decrease in erythropoietin-producing ability was observed with the addition.

[Inhibition of SLCO4C1 expression by indoxyl sulfate]
The inhibitory action of SLCO4C1 expression by indoxyl sulfate (IS) in HK-2 cells was verified. 5 × 10 ⁵ HK-2 cells were seeded in a 6-well cell culture plate and then incubated for 24 hours. Thereafter, the medium was replaced with a medium containing indoxyl sulfate (0.3, 1, 3 mM, 3 concentrations) or water, and RNA was extracted after incubation for 24 hours. After preparing cDNA using SuperScript III (manufactured by Invitrogen), the expression of SLCO4C1 mRNA was measured by quantitative PCR using GAPDH as an internal standard. Renal epithelial cell culture kit (manufactured by Lonza) is used as the cell culture medium, and TaqMan (registered trademark) Gene Expression Assays is used for quantitative PCR (GAPDH; Assay ID: Rn99999916_s1, SLCO4C1; Assay ID: Rn01427754_m1 TaqMan (registered) (Trademark) probe (Applied Biosystems) was used) and Step One Plus Real-Time PCR System (Applied Biosystems). As a result, it was shown that the expression of SLCO4C1 mRNA was suppressed depending on the concentration of indoxyl sulfate (FIG. 3). That is, it was suggested that indoxyl sulfate, which is one of uremic substances, suppresses the expression of SLCO4C1, thereby suppressing the discharge of uremic substances and enhancing renal failure.

[Induction action of GATA3 expression by indoxyl sulfate]
The effect of inducing GATA3 expression by indoxyl sulfate (IS) in HK-2 cells was verified. 5 × 10 ⁵ HK-2 cells were seeded in a 6-well cell culture plate and then incubated for 24 hours. Thereafter, the medium was replaced with a medium containing indoxyl sulfate (0.3, 1, 3 mM, 3 concentrations) or water, and RNA was extracted after incubation for 24 hours. After preparing cDNA using SuperScript III (manufactured by Invitrogen), the expression of GATA3 mRNA was measured by quantitative PCR using GAPDH as an internal standard. Renal epithelial cell culture kit (manufactured by Lonza) was used as the cell culture medium, and TaqMan (registered trademark) Gene Expression Assays was used for quantitative PCR (GAPDH; Assay ID: Rn99999916_s1, GATA3; Assay ID: Hs00231122_m1 TaqMan (registered) (Trademark) probe (Applied Biosystems) was used) and Step One Plus Real-Time PCR System (Applied Biosystems). As a result, it was shown that the expression of GATA3 mRNA was induced depending on the concentration of indoxyl sulfate (FIG. 4). That is, it was suggested that indoxyl sulfate, which is one of uremic substances, may suppress the expression of erythropoietin by inducing the expression of GATA3, which is a factor that suppresses the expression of erythropoietin.

[SLCO4C1 expression inducing effect by administration of cremedin in rats with renal failure]
Next, the effect of klemedin, a spherical adsorbent charcoal that has a function of discharging uremic substances out of the body, on SLCO4C1 mRNA expression in renal failure rats was examined. Chronic renal failure rats were prepared by performing 5/6 nephrectomy on 10-week-old male Wistar rats. Blood pressure, blood sampling, and 24-hour urine collection were performed 10 weeks after nephrectomy, and were distributed to control group 13 and cremedin group 11 and started to be administered cremedin (8% mixed with powdered food). After administration for 4 weeks, blood pressure measurement, blood collection, and 24-hour urine collection were performed again, followed by euthanasia. After RNA was extracted from the isolated kidney and cDNA was prepared using SuperScript III (Invitrogen), SLCO4C1 mRNA expression was measured using GAPDH as an internal standard (FIG. 5). The expression level of SLCO4C1 mRNA was assayed by t-test in rats (control 10, Cremedin 8) having a Ccr (creatinine clearance) of 1.0 ml / min to 2.0 ml / min after 4 weeks of administration of cremedin. However, a significant difference was confirmed. In other words, it was confirmed that SLCO4C1 expression is enhanced by administration of cremedin to renal failure rats.

[Inhibition of erythropoietin expression by indoxyl sulfate and 1-methyladenosine]
In order to examine the influence of indoxyl sulfate and 1-methyladenosine on erythropoietin transcriptional activity, a luciferase assay using the 5 ′ upstream region of the mouse erythropoietin gene was performed by the following method.

[Plasmid DNA]
The plasmid vector HE-mPro-luc for the promoter activity luciferase assay of the 5 'upstream transcriptional regulatory region of the mouse erythropoietin gene restricts the region from -571 to +53 bp in length starting from the translation start point of exon 1 of the mouse erythropoietin gene It was excised with enzymes XbaI and SacI and inserted into a luciferase reporter vector pXP2 (Nordeeen SK. BioTechniques. 6: 454-453.1988).

[Cultivated cells]
Human kidney cancer-derived cultured cell line ACHN is 10% fetal bovine serum (FBS) (GIBCO), 100 IU / ml penicillin and 10 μg / ml streptomycin (GIBCO) at a final concentration in RPMI 1640 medium (GIBCO). And cultured under conditions of 37 ° C. and 5% CO ₂ concentration.

[Transfection and luciferase reporter assay]
Hep3B cells were subcultured in a 24-well cell culture plate at a density of 10 × 10 ⁴ cells per well, and luciferase reporter assay was performed on Hep3B cells that had a cell density of 70-80% under a microscope. The plasmid DNA for transfection was performed. The solution composition for transfection was 2 μg HE-mPro-luc per well, 50 ng pRh-TK (Promega) and 2 μl Lipofectamine 2000 (Invitrogen) mixed with 100 μl OptiMEM-I (GIBCO) at room temperature. And then added to 24-well cell culture plate ACHN cells pre-incubated with 0.5 ml of serum-free OptiMEM-I medium, followed by conditions of 37 ° C., 5% CO ₂ /95% air Incubated for 4 hours without serum and without antibiotics. Thereafter, the medium was replaced with normal medium (RPMI1640 / 10% FBS / 100 IU / ml penicillin, 10 μg / ml streptomycin) supplemented with indoxyl sulfate (10 μM), 1-methyladenosine (1 μM), or DMSO (0.1%). Furthermore, it was incubated at 37 ° C. under low oxygen conditions of 5% CO ₂ /1% O ₂ for 36 hours. Thereafter, the medium was replaced with 0.5 ml of serum-free PBS, the cells were gently washed, and the work of removing PBS by suction was performed twice. Then, 100 μl of passive lysis buffer (Promega) was added for 20 minutes at room temperature. And dissolved. The Luciferase Reporter Assay used Dual Luciferase Reporter Assay System (Promega), and the measurement was carried out using a Luminometer Lumat LB9507 (NERTHOLD TECHNOLOGIES) to measure the amounts of Fire fly and Renilla luciferases. The transcriptional activity of the erythropoietin gene was calculated by dividing the amount of Fire fly luciferase by the amount of Renilla luciferase.

  As a result, the transcriptional activity of the erythropoietin gene was reduced to 53% by the addition of indoxyl sulfate and 63% of the transcriptional activity of the erythropoietin gene by the addition of 1-methyladenosine, compared to the control to which DMSO was added (FIG. 6). That is, it has been clarified that indoxyl sulfate and 1-methyladenosine, which are uremic substances, significantly reduce the transcriptional activity of the erythropoietin gene.

[Inhibition of SLCO4C1 expression and GATA3 expression induction by indoxyl sulfate]
Next, in order to examine the influence of indoxyl sulfate and 1-methyladenosine on the transcriptional activity of SLCO4C1 gene, luciferase for detecting the promoter activity of the 5 ′ upstream transcriptional regulatory region of human SLCO4C1 gene and the 5 ′ upstream transcriptional regulatory region of GATA3 gene The assay was performed as follows.

[Plasmid DNA]
The plasmid vector SLCO4C1-3886 / + 110-pGL3c for the promoter activity luciferase assay of the 5 ′ upstream transcriptional regulatory region of the SLCO4C1 gene was amplified by PCR using a PCR method from −3886 to +110 bp in length. It was prepared by inserting into pGL3 basic luciferase expression vector (Promega). The plasmid vector pGATA3-luc for the promoter activity luciferase assay of the 5 ′ upstream transcriptional regulatory region of the GATA3 gene is excised from the 3.5 kb of the GATA3 promoter region with the restriction enzymes BamHI and NcoI and continuous with the 2 kb gene cassette encoding the luciferase protein. And inserted into a pBluscriptSK + (Novagen) vector.

[Transfection and luciferase reporter assay]
ACHN cells were maintained by the above-mentioned method, subcultured at a density of 20 × 10 ⁴ cells per well in a 24-well cell culture plate, and ACHN having a cell density of 70-80% under a microscope. Cells were transfected with plasmid DNA for luciferase reporter assay. The solution composition for transfection was Fire fly luciferase vector (SLCO4C1-3886 / + 110-pGL 1.2 μg or pGATA3-luc 2 μg) per well, pRh-TK (Promega) 50 ng as Renilla Luciferase Reporter Vector, Lipofectamine 2000 (manufactured by Invitrogen) as a reaction reagent was mixed with 100 μl of serum-free medium OptiMEM-I (manufactured by GIBCO) and then incubated at room temperature for 20 minutes. This transfection solution was added to ACHN cells in a 24-well cell culture plate pre-incubated with 0.5 ml of serum-free OptiMEM-I medium, serum-free at 37 ° C., 5% CO ₂ and without addition of antibiotics. Incubated for 4 hours. Thereafter, the medium was replaced with a normal medium (10% FBS, 100 IU / ml penicillin, RPMI 1640 medium containing 10 μg / ml streptomycin) supplemented with indoxyl sulfate (100 μM) or DMSO, and further replaced with 44 ° C. at 37 ° C., 5% CO ₂ . Incubated for hours. Thereafter, the medium was replaced with 0.5 ml of serum-free PBS, and the cells were gently washed and the PBS was removed by suction. Then, 100 μl of passive lysis buffer (Promega) was added, and the mixture was warmed to room temperature for 20 minutes. And mixed to dissolve. Luciferase reporter assay was carried out using Dual Luciferase Reporter Assay System (manufactured by Promega), and luciferase levels of Fire fly and Renilla were measured using a luminometer Lumat LB9507 (manufactured by NERTHOLD TECHNOLOGIES). Then, the transcriptional activity of SLCO4C1 gene and GATA3 gene was calculated by dividing the amount of Fire fly luciferase by the amount of Renilla luciferase.

  FIG. 7 shows the transcriptional activity of the SLCO4C1 gene or GATA3 gene when indoxyl sulfate is added when the transcriptional activity of the control sample added with DMSO is 1. As a result, it was shown that the addition of indoxyl sulfate decreased the transcriptional activity of SLCO4C1 gene and increased the transcriptional activity of GATA3. That is, it has been shown that indoxyl sulfate reduces SLCO4C1 gene expression at the transcription level and controls to increase GATA3 gene expression.

[Erythropoietin expression-enhancing effect by administration of cremedin to patients with renal anemia]
A total of 4 patients, 3 adult males and 1 female with renal insufficiency disease showing renal anemia, were administered cremidine in 6 g, 3 divided doses over 22 months. Blood was collected before and after administration of cremedin, and the concentration of erythropoietin in the blood was measured using a human erythropoietin ELISA kit manufactured by Bender Med Systems. As a result, the amount of erythropoietin in the blood increased in three people after the administration of cremedin compared with that before the administration of cremedin, and there was no change in one person (FIG. 8). It is known that the amount of erythropoietin decreases due to renal failure, that is, the increase in the expression level of endogenous erythropoietin by the administration of cremedin prevented the decrease in the amount of erythropoietin, suggesting an increase.

  From the above, it has been clarified that indoxyl sulfate, which is one of uremic substances, enhances the expression of GATA3, suppresses the expression of SLCO4C1 transporter, and further suppresses the expression of erythropoietin. And it was shown that the administration of cremedin enhanced the expression of SLCO4C1 transporter and also enhanced the expression of erythropoietin. This is because, by adsorbing and eliminating the uremic substance on cremedin, the expression enhancement of GATA3 by the uremic substance and the suppression of the expression of SLCO4C1 transporter are suppressed, whereby the inhibition of the expression of erythropoietin by GATA3 is released, It is considered that endogenous erythropoietin expression is also enhanced by promoting the excretion of the uremic substance by the SLCO4C1 transporter by enhancing the expression of the SLCO4C1 transporter (FIG. 9).

[CE-MS analysis of substances fluctuating by administration of cremedin in rats with renal failure]
Chronic renal failure rats were prepared by performing 5/6 nephrectomy on 10-week-old male Wistar rats. Chronic renal failure rats (n = 12) fed the basic diet, control group, chronic renal failure rats (n = 10) fed the diet supplemented with kremezin (mixed with 8% powdered diet), kremedin group, and kremeji added Normal Wistar rats fed the prepared diet (mixed with 8% powdered diet) were designated as the Sham group (n = 3). After rearing for 4 weeks, plasma samples were collected and subjected to CE-MS analysis by the following method. A methanol solution prepared with an internal standard substance is added to each plasma sample and stirred. After further adding chloroform and stirring, the mixture is centrifuged at 4600 g for 5 minutes, and then the supernatant is collected in an ultrafiltration filter (fractionated molecular weight 5000). After centrifuging at 4100C and 9100 g for 2 hours, the filtrate was concentrated by centrifugation and measured by CE-TOFMS.

  As a result, 6 anionic substances having a significant difference between the control group and the cremedin group were 4-oxopentanoate, o-hydroxybenzoate, hippurate, and 3-indoxyl. They were sulfuric acid (3-Indoxyl sulfate), N-acetylneuraminic acid (N-Acetylneuraminate), and cholic acid (Cholate), all of which were lower in the cremedin group than in the control group. In addition, 15 cations having a significant difference between the control group and the cremedin group include glycine (Glycine) trimethylamine N-oxide, hypotaurine, cytosine, nicotinamide, and imidazole. -4-acetic acid (Imidazole-4-acetate), creatine (Creatine), asparagine (Asparagine), γ-guanidinobutyrate (γ-Guanidinobutyrate), γ-butyrobetaine (γ-Butyrobetaine), glutamine (Glutamine), allantoin (Allantoin) ), N-γ-Ethylglutamine, Tryptophan, Glycerophosphorylcholine, of which trimethylamine N-oxide and tryptophan are significantly higher in the cremedin group than in the control group. there were. In addition, cations 5-methylcytosine and anthranilate (also known as o-aminobenzoic acid) were detected only in the control group, but were hardly detected in the Sham and cremedin groups.

  The present invention can be suitably used in the fields of therapeutic drugs for renal anemia and excretion of uremic substances.

Claims (9)

  An endogenous erythropoietin expression enhancer comprising a uremic substance adsorbent as an active ingredient.   2. The endogenous erythropoietin expression enhancer according to claim 1, wherein the uremic substance adsorbent is activated carbon.   The endogenous erythropoietin expression enhancer according to claim 2, wherein the activated carbon is a spherical adsorbed charcoal.   A therapeutic agent for renal anemia comprising the endogenous erythropoietin expression enhancer according to any one of claims 1 to 3.   An organic ion transporter expression enhancer comprising a uremic substance adsorbent as an active ingredient.   6. The organic ion transporter expression enhancer according to claim 5, wherein the uremic substance adsorbent is activated carbon.   The organic ion transporter expression enhancer according to claim 6, wherein the activated carbon is a spherical adsorption charcoal.   The organic ion transporter expression enhancer according to any one of claims 5 to 7, wherein the organic ion transporter is an organic anion transporter SLCO4C1.   A uremic substance excretion enhancer comprising the organic ion transporter expression enhancer according to claim 5.