Therapeutic agent for nephropathy and renal function improving agent

Abstract

The purpose of the present invention is to provide a novel therapeutic agent for nephropathy. The present invention is a therapeutic agent for nephropathy comprising a mesenchymal stem cell culture supernatant. The mesenchymal stem cell culture supernatant is preferably a culture supernatant obtained by culturing adipose tissue-derived, umbilical cord tissue-derived or bone marrow tissue-derived mesenchymal stem cells. In addition, the mesenchymal stem cell culture supernatant is preferably obtained by culturing mesenchymal stem cells with a culture medium containing at least one substance selected from the group consisting of EGF, bFGF, albumin, transferrin, and insulin.

Description

Technical Field

[0001] The present invention relates to a kidney disease therapeutic agent and a renal function improving agent. Background Art

[0002] Renal failure includes acute renal damage and acute renal failure with a sharp decline in renal function, and chronic kidney disease and chronic renal failure with a slow decline in renal function. When renal failure progresses, waste products in the body cannot be fully excreted, uremic substances accumulate in the body, and uremic symptoms such as edema, hypertension, hyperkalemia, and anemia appear. The methods for treating uremia include hemodialysis, continuous portable peritoneal dialysis (CAPD), and kidney transplantation. Although hemodialysis and CAPD can replace the kidneys, they cannot replace all the functions of the kidneys, and cannot secrete hematopoietic stimulating hormones, activate vitamin D, regulate blood pressure, inactivate unnecessary hormones, etc. In addition, hemodialysis needs 2-3 times a week, 4-5 hours / time, and must be maintained for life, which becomes a great burden on patients. CAPD is to inject dialysate into the peritoneal cavity through a catheter implanted in the peritoneal cavity, and then drain it after 5 to 6 hours of storage. Therefore, except for the time of changing the bag, the patient can move freely, but there is a risk of peritonitis and infection at the outlet of the catheter subcutaneous channel, causing hernia, peritoneal thickening and other problems. Kidney transplantation is an effective treatment method, but there are fewer kidney donors than people who want to undergo kidney transplantation, and donors for living kidney transplantation are limited to relatives, which also places a heavy burden on the donor side.

[0003] On the other hand, renal failure is also a problem in animals other than humans. For example, it is reported that 81% of cats over 15 years old suffer from chronic kidney disease, and it is said that most elderly cats suffer from this disease. However, it is said that symptoms of renal disease only appear when the kidneys lose about two-thirds of their function, and it is a disease that is difficult to detect in the early stage. Similarly, in dogs, renal failure is also the third leading cause of death, and it often occurs in old age. It is known that dog breeds such as bull terriers, English cocker spaniels, cavaliers, West Highland white terriers, boxers, and Shar-Pei are more likely to suffer from renal disease. In addition, there is no effective treatment for chronic kidney disease in dogs and cats, and the current focus is on prolonging the remaining renal function and delaying the progression of the disease. Therefore, there is a desire to develop new and effective renal failure treatment drugs that are applicable not only to humans but also to non-human animals.

[0004] Mesenchymal stem cells are progenitor cells with multi-differentiation ability that were first isolated from bone marrow by Friedenstein (1982) (see non-patent document 1). Mesenchymal stem cells have been shown to exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected to be a new method for treating various intractable diseases (see patent documents 1-2). Recently, it has been known that there are cells with equivalent functions to mesenchymal cells in adipose tissue, placenta, umbilical cord, and egg membranes. Therefore, mesenchymal stem cells are sometimes referred to as interstitial cells.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2012-157263

[0008] Patent Document 2: Japanese Patent Application No. 2012-508733

[0009] Non-patent literature

[0010] Non-patent document 1: Pittenger FM et al., Science 284, pp.143-147, 1999 Summary of the invention

[0011] Problem that the invention aims to solve

[0012] In view of the above circumstances, an object of the present invention is to provide a new therapeutic agent for kidney disease.

[0013] Solutions for solving problems

[0014] As a result of intensive studies to solve the above problems, the present inventors found that culture supernatant of mesenchymal stem (stromal) cells (MSCs) can treat kidney disease, thereby completing the present invention. That is, the gist of the present invention is as follows.

[0015] [1] A kidney disease therapeutic agent comprising a mesenchymal stem cell culture supernatant.

[0016] [2] The therapeutic agent for renal disease according to [1], wherein the mesenchymal stem cell culture supernatant is a culture supernatant obtained by culturing adipose tissue-derived, umbilical cord tissue-derived, or bone marrow tissue-derived mesenchymal stem cells.

[0017] [3] The therapeutic agent for kidney disease according to [1] or [2], which is a mesenchymal stem cell culture supernatant obtained by culturing mesenchymal stem cells in a culture medium containing at least one selected from the group consisting of EGF, bFGF, albumin, transferrin, and insulin.

[0018] [4] The kidney disease therapeutic agent according to [3], characterized in that the content of exosomes in the mesenchymal stem cell culture supernatant is high.

[0019] [5] The kidney disease therapeutic agent according to [3], characterized in that the nitric oxide content in the mesenchymal stem cell culture supernatant is low.

[0020] [6] A renal function improving agent comprising a mesenchymal stem cell culture supernatant.

[0021] Effects of the Invention

[0022] According to the present invention, a new therapeutic agent for kidney disease can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a graph showing the number of particles in each culture supernatant.

[0024] Figure 2 It is a graph showing the particle size in each culture supernatant.

[0025] Figure 3 It is a graph showing the protein concentration in each culture supernatant.

[0026] Figure 4 It is a graph showing the content of CD9 / CD63-positive extracellular vesicles (exosomes) in each culture supernatant.

[0027] Figure 5 It is a graph showing the nitric oxide (NOx) content in each culture supernatant.

[0028] Figure 6 It is a graph showing the pH of each culture supernatant.

[0029] Figure 7 It is a graph showing the blood creatinine level, urea nitrogen level, and potassium level after administration of a culture medium or one type of culture supernatant to acute renal injury model rats.

[0030] Figure 8 Representative photographs showing the pathological tissues of the kidneys of rats with acute renal injury model after administration of a medium or a culture supernatant.

[0031] Fig. 9 The graphs show the pathological scores of the kidneys of rats with acute renal injury model after administration of a medium or a culture supernatant.

[0032] Fig.10 It is a graph showing the blood creatinine level, urea nitrogen level, and potassium level after administration of each culture supernatant to acute renal injury model rats. DETAILED DESCRIPTION

[0033] The novel therapeutic agent for kidney disease of the present invention is described in detail below.

[0034] [Renal disease treatment agent]

[0035] The therapeutic agent for kidney disease of the present invention is characterized by comprising a specific mesenchymal stem cell culture supernatant. The therapeutic agent for kidney disease of the present invention exhibits an excellent effect of treating kidney disease by comprising a specific mesenchymal stem cell culture supernatant.

[0036] (Mesenchymal stem cell culture supernatant)

[0037] In the present invention, the mesenchymal stem cell culture supernatant refers to the liquid obtained by separating the culture medium that is in direct contact with the cells or in indirect contact with the cells through a semipermeable membrane when the mesenchymal stem cells are cultured for a certain period of time. The culture supernatant is sometimes also called the culture medium acclimation medium or the conditioned medium.

[0038] In the present invention, the mesenchymal stem cell culture supernatant is the culture supernatant obtained when culturing mesenchymal stem cells, and has the characteristic of high exosome content.

[0039] Exosomes refer to cell-derived vesicles with a diameter of 50nm to 150nm that exist outside cells, and are present in large quantities in all body fluids such as blood, saliva, and urine. Exosomes contain lipids and proteins derived from cell membranes on their surface and nucleic acids and proteins such as mRNA and miRNA inside, and contain information from released cells. Therefore, the information contained in exosomes can also be used as biomarkers for diagnostic markers, etc. In addition, it is known that bioinformatics molecules containing these nucleic acids and proteins act in cells that have ingested exosomes, and it is known that intercellular communication is caused by the exchange of nucleic acids and proteins with the help of exosomes. Therefore, it is believed that exosomes can be used not only for diagnosis, but also for the prevention and treatment of diseases.

[0040] In the present invention, when specifying the diameter or particle size of an extracellular vesicle or exosome, since these are not necessarily spherical, it refers to the maximum size of the extracellular vesicle. The diameter or particle size can be measured using existing technologies for measuring nanoparticle size, such as microscopy techniques (e.g., transmission electron microscopy) or light scattering techniques. Furthermore, it can also be measured using nanoparticle tracking analysis (NTA) based on the analysis of both light scattering and Brownian motion. In addition, regarding the diameter or particle size of extracellular vesicles and exosomes, for extracellular vesicles with a particle size of 50 nm or more, NanoSight (LM10, Quantum Design) can be used for measurement, and for extracellular vesicles with a particle size of less than 50 nm, a transmission electron microscope can be used for measurement. In addition, the number of exosomes can be counted using NanoSight (LM10, Quantum Design), Exosome Counting System (ExoCounter, JVC Kenwood), etc. The Exosome Counting System is based on the following method: using antibodies of disks and nanostrains to specifically perform sandwich detection on surface antigens that are exosome markers.

[0041] The method for measuring the exosome content in the culture supernatant is not particularly limited, and an example of a method for measuring the content of CD9 / CD63-positive extracellular vesicles can be exemplified. CD9 / CD63 is a family of four transmembrane proteins present on the surface of exosomes and is a surface marker of exosomes. It should be noted that, herein, "CD9 / CD63" refers to CD9 and CD63.

[0042] The mesenchymal stem cell culture supernatant of the present invention may have a higher exosome content than other cell culture supernatants. Specifically, the mesenchymal stem cell culture supernatant of the present invention may have a higher CD9 / CD63-positive extracellular vesicle content than the culture supernatant obtained by culturing mesenchymal stem cells using a conventional mesenchymal stem cell culture method (e.g., a culture method using a DMEM medium containing 10% FBS). Preferably, the exosome content is 5 times higher than that of the conventional mesenchymal stem cell culture supernatant, more preferably 10 times higher, further preferably 50 times higher, and particularly preferably 100 times higher.

[0043] The mesenchymal stem cell culture supernatant of the present invention may contain more exosomes than the culture supernatant obtained by culturing mesenchymal stem cells using a conventional mesenchymal stem cell culture method (e.g., a culture method using a DMEM medium containing 10% FBS). The method for measuring the number of exosomes in the culture supernatant is as described above.

[0044] The mesenchymal stem cell culture supernatant of the present invention has a characteristic of low nitric oxide content compared with other cell culture supernatants. Specifically, the mesenchymal stem cell culture supernatant of the present invention has a lower nitric oxide content than the culture supernatant obtained by culturing mesenchymal stem cells using a previous mesenchymal stem cell culture method (for example, a culture method using a DMEM culture medium containing 10% FBS). It should be noted that the determination of nitric oxide content in the culture supernatant can be carried out using a commercially available kit or the like. As such a commercially available kit, for example, Nitric Oxide Assay kit (Cat. No. D2NO-100, Bio Assay Systems) and the like can be cited.

[0045] As the mesenchymal stem cell culture supernatant of the present invention, the liquid obtained by separation from the cells can be used directly, or some treatment can be performed. The treatment of the mesenchymal stem cell culture supernatant is not particularly limited, and purification treatment, concentration treatment, drying and powdering treatment, etc. can be listed. In the present invention, as the mesenchymal stem cell culture supernatant, a liquid can be used, and as needed, a drying treatment such as reduced pressure drying, freeze drying, spray drying (spray-dry), evaporation drying, high pressure method or ultra-high pressure method can be used to reduce or remove the liquid component, thereby concentrating and liquefying, semi-solidifying, solidifying or powdering.

[0046] In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into more than one cell (osteocyte, cardiomyocyte, chondrocyte, tendon cell, adipocyte, etc.) belonging to the mesenchymal system and can proliferate while maintaining the ability. The term mesenchymal stem cells used in the present invention refers to the same cells as interstitial cells, without specifically distinguishing between the two. In addition, sometimes referred to as mesenchymal cells. As tissues containing mesenchymal stem cells, adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. can be listed. Mesenchymal stem cells in the present invention include mesenchymal stem cells derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. Among them, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred, and adipose tissue-derived mesenchymal stem cells are further preferred.

[0047] The mesenchymal stem cells in the present invention may be of the same species or a different species than the subject to be treated with the renal disease therapeutic agent of the present invention. The species of the mesenchymal stem cells in the present invention include humans, horses, cattle, sheep, pigs, dogs, cats, rabbits, mice, and rats.

[0048] Mesenchymal stem cells, for example, can be cells provided by PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, etc., or cells prepared by methods well known to those skilled in the art. In addition, mesenchymal stem cells can be primary cells isolated from donor tissues, or cells of cell lines.

[0049] The mesenchymal stem cells in the present invention can be cells that are appropriately repeatedly cryopreserved and thawed. In the present invention, cryopreservation can be performed by suspending the mesenchymal stem cells in a cryopreservation solution known to those skilled in the art and cooling them. Suspension can be performed by stripping the cells with a stripping agent such as trypsin, transferring them to a cryopreservation container, and adding the cryopreservation solution after appropriate treatment.

[0050] In the present invention, the medium used for culturing mesenchymal stem cells is not particularly limited as long as it can culture the mesenchymal stem cells in a good state. For example, a medium that can proliferate human mesenchymal stem cells while maintaining the ability to differentiate into osteocytes, chondrocytes, and adipocytes is preferred.

[0051] The culture medium used in the present invention can be prepared by adding serum and / or components other than serum to a basal medium.

[0052] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential MEM (EMEM) medium, αMEM medium, Dulbecco's Modified Eagle's MEM (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, MCDB201 medium, and mixed medium thereof.

[0053] As the above-mentioned serum, for example, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum, etc. can be listed, but it is not limited to these. When using serum, 5v / v% to 15v / v%, preferably 10v / v% can be added to the basal medium.

[0054] As components other than the serum, one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, and thioglycerol can be added to prepare the serum. In addition, lipids, amino acids, proteins, sugars such as glucose, polysaccharides, vitamins such as pantothenic acid, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts such as sodium chloride and magnesium sulfate can be further added as needed.

[0055] Examples of the fatty acids include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of the lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of the amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of the proteins include, but are not limited to, colicin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharides include, but are not limited to, glycosaminoglycans, and hyaluronic acid and heparan sulfate are particularly examples of glycosaminoglycans, but are not limited to them. Examples of the above growth factors include, but are not limited to, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF).

[0056] From the viewpoint of use in treatment of humans or animals, the medium for culturing mesenchymal stem cells used in the present invention is preferably a medium that does not contain xenogeneic components such as serum (xeno-free). Examples of such culture media include Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use), manufactured by PromoCell, Mesenchymal Stem Cell Growth Medium XF (Ready-to-use), manufactured by PromoCell, MSCGM BulletKit™, MSCGM™ Mesenchymal Stem Cell Growth Medium BulletKit™ (manufactured by Lonza), xeno-free medium for human mesenchymal stem cells (MSC NutriStem (registered trademark) XF, manufactured by Biological Industries, Inc.), (MesenCult-ACF Plus (manufactured by Veritas), StemX Vivo™ Serum-Free Human MSC Expansion Media (manufactured by R&D Systems, Corning), serum-free medium for adipose-derived stem cells (KBM ADSC-4, manufactured by KOHJIN BIO), serum-free medium for mesenchymal stem cells (R: STEM Medium for hMSC A medium prepared in advance and provided as a culture medium for mesenchymal stem cells (stromal cells) such as those manufactured by HighGrowth and Rohto Corporation.

[0057] In particular, from the perspective of the therapeutic effect of the kidney disease therapeutic agent of the present invention, the culture medium for culturing mesenchymal stem cells preferably contains at least one, preferably two, more preferably three, further preferably four, and particularly preferably five selected from the group consisting of EGF, bFGF, albumin, transferrin and insulin.

[0058] (Preparation of mesenchymal stem cell culture supernatant)

[0059] The supernatant of mesenchymal stem cells obtained by the following method can be used as the mesenchymal stem cell culture supernatant in the present invention. In addition, the supernatant obtained by removing unnecessary components by means of dialysis, ultrafiltration, etc., the fraction obtained by fractionating the supernatant using a column, etc., the fraction selected using antibodies against specific molecules, etc., the fraction obtained by centrifugation, etc. can also be used as the mesenchymal stem cell culture supernatant in the present invention.

[0060] As a culture medium used when obtaining the culture supernatant, a culture medium similar to that used to culture mesenchymal stem cells can be used. The method for obtaining the culture supernatant is not particularly limited as long as it is a method suitable for culturing each mesenchymal stem cell, for example, a method for obtaining the culture supernatant by culturing mesenchymal stem cells at a temperature of 20°C to 37°C, a 2% to 7% CO2 environment, and a 5% to 21% O2 environment, preferably at room temperature to 37°C, and a 5% CO2 environment.

[0061] The mesenchymal stem cell culture supernatant of the present invention can be any one in which the mesenchymal stem cells are in contact with the culture medium. The washing liquid obtained by washing the mesenchymal stem cells with the culture medium can also be used as the culture supernatant in the present invention. The contact time between the mesenchymal stem cells and the culture medium is, for example, within 14 days, preferably within 10 days, more preferably within 7 days, and more preferably within 5 days. The culture used to obtain the culture supernatant can be a flat culture attached to a flask, or a suspension and stirring culture attached to microbeads, etc.

[0062] The kidney disease therapeutic agent of the present invention may contain other ingredients in addition to the mesenchymal stem cell culture supernatant within the range that does not hinder the effect of the present invention. Other ingredients include dimethyl sulfoxide (DMSO), protective agents such as serum albumin, antibiotics, vitamins, carriers, excipients, disintegrants, buffers, emulsifiers, stabilizers, preservatives, preservatives, physiological saline, etc.

[0063] Regarding the dosage of the nephropathy therapeutic agent of the present invention, it can be adjusted according to the state (weight, age, symptoms, physical condition, etc.) of the patient and the animal suffering from the nephropathy therapeutic agent of the present invention, the dosage form, the route of administration, etc., from the viewpoint of exerting a sufficient disease treatment effect, there is a tendency to prefer a large amount, on the other hand, from the viewpoint of suppressing the occurrence of side effects, there is a tendency to prefer a small amount. Usually, the dosage when administering to human adults is 0.01mL~3L / time, preferably 0.1mL~2L / time, more preferably 0.5mL~1L / time, and further preferably 1mL~500mL / time. In addition, the dosage per unit weight of the patient is 0.0001~100mL / kg, preferably 0.002~40mL / kg, more preferably 0.01~20mL / kg, and further preferably 0.02~10mL / kg. The dosage when administered to a cat or dog varies according to the dog species and age, and is usually 0.001 to 2L / time, preferably 0.01mL to 1L / time, more preferably 0.1mL to 500mL / time, and further preferably 1mL to 250mL / time. In addition, the dosage per unit body weight of the affected animal is 0.0001 to 100mL / kg, preferably 0.002 to 40mL / kg, more preferably 0.01 to 20mL / kg, and further preferably 0.02 to 10mL / kg. It should be noted that this dosage can be set as one amount and administered multiple times, or this dosage can be divided into multiple times and administered.

[0064] The renal therapeutic agent of the present invention can be administered together with one or more other agents. As other agents, any agent that can be used as a renal therapeutic agent can be listed, for example, immunosuppressants such as adrenocortical hormone, mizoribine, cyclosporine, tacrolimus, cyclophosphamide, etc.; antiplatelet drugs such as dipyridamole and dilazoline dihydrochloride hydrate; anticoagulants such as heparin and warfarin potassium; antihypertensive drugs such as angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists (ARBs), and calcium antagonists; loop diuretics such as furosemide and azosemide; albumin concentrate preparations, dyslipidemia improving drugs, phosphate adsorbents, potassium adsorbents, erythropoietin preparations, beraprost sodium, etc.

[0065] The kidney disease therapeutic agent of the present invention can be mixed in an infusion solution for use. Here, the "infusion solution" in the present invention refers to an artificial solution used in a therapeutic method of continuously infusing an artificial solution through a non-oral route (mainly intravenous) to replenish water, electrolytes (salts), calories, nutrients, etc. Examples of the infusion solution in the present invention include physiological saline, Japanese Pharmacopoeia physiological saline, 5% glucose solution, Japanese Pharmacopoeia glucose injection, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactated Ringer's solution, acetic Ringer's solution, solution No. 1 (initial solution), solution No. 2 (dehydration supplement), solution No. 3 (maintenance solution), solution No. 4 (postoperative recovery solution), etc.

[0066] When the renal disease therapeutic agent of the present invention is a liquid, the pH of the renal disease therapeutic agent is not particularly limited as long as it is within the pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable range, and as an example, it can be 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0.

[0067] When the kidney disease therapeutic agent of the present invention is a liquid, there is no particular limitation on the osmotic pressure of the kidney disease therapeutic agent as long as it is within the range acceptable to the organism. As an example of the osmotic pressure ratio of the composition of the present invention, a range of preferably 0.7 to 5.0, more preferably 0.8 to 3.0, and further preferably 0.9 to 1.4 can be cited. The osmotic pressure can be adjusted by methods known in the art using inorganic salts, polyols, sugar alcohols, sugars, etc. Regarding the osmotic pressure ratio, it can be set as the ratio of the osmotic pressure of the sample to the osmotic pressure of 286mOsm (0.9w / v% sodium chloride aqueous solution) based on the fifteenth revision of the Japanese Pharmacopoeia. The osmotic pressure can be measured with reference to the osmotic pressure determination method (freezing point depression method) recorded in the Japanese Pharmacopoeia. It should be noted that the standard solution for osmotic pressure ratio determination (0.9 w / v% sodium chloride aqueous solution) is prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500-650°C for 40-50 minutes, naturally cooling it in a desiccator (silica gel) and accurately weighing 0.900 g and dissolving it in purified water to make a fixed volume of 100 mL, or using a commercially available standard solution for osmotic pressure ratio determination (0.9 w / v% sodium chloride aqueous solution).

[0068] The administration routes of the therapeutic agent for kidney disease of the present invention to a subject include oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intramedullary administration, intraperitoneal administration, sublingual administration, rectal administration, vaginal administration, intraocular administration, nasal administration, inhalation, transdermal administration, implantation, spraying onto the kidney surface, and direct administration via a patch, etc. From the viewpoint of the effectiveness of the therapeutic agent for kidney disease of the present invention, implantation, administration into the renal artery, intravenous administration, spraying onto the kidney surface, and direct administration via a patch, etc. are preferred, and from the viewpoint of reducing the burden on the subject, intravenous administration is more preferred.

[0069] The target species of the therapeutic agent for kidney disease of the present invention may be any mammal, and examples thereof include humans, horses, cattle, sheep, pigs, dogs, cats, rabbits, mice, rats, and rare animals.

[0070] The kidney disease therapeutic agent of the present invention can be preferably used for the treatment of kidney diseases, and in particular, it is more preferably used for the treatment of acute renal failure, chronic renal failure, acute glomerulonephritis, glomerulonephritis such as chronic glomerulonephritis, interstitial nephritis, acute nephritic syndrome, recurrent persistent hematuria, chronic nephritic syndrome, nephrotic syndrome, IgA nephropathy, lupus nephritis, diabetic nephropathy, or acute progressive nephritic syndrome.

[0071] Example

[0072] The present invention will be described in detail below with reference to Examples and Test Examples, but the present invention is not limited to these Examples and the like.

[0073] [Preparation of mesenchymal stem cell culture supernatant]

[0074] Adipose tissue-derived mesenchymal stem cells (Lonza, hereinafter referred to as "ADSC") were cultured for 4 days with RIM medium (serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin and insulin, Rohto) or Rstem medium (serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin and insulin, Rohto) to obtain culture supernatant (hereinafter referred to as "RIM culture supernatant" and "RS culture supernatant"). In addition, after ADSC was cultured with RIM medium for 3 days, the medium was exchanged and replaced with RMFS1 medium (Rohto), and cultured for 2 days to obtain culture supernatant (hereinafter referred to as "RMFS1 culture supernatant"). Furthermore, ADSC was cultured with DMEM (Sigma) containing 10% FBS (ThemoFisher) for 3 days, and the medium was exchanged and replaced with DMEM, and cultured for 2 days to obtain culture supernatant (hereinafter referred to as "DMEM culture supernatant"). It should be noted that the culture conditions at this time were 5% CO 2 and 37°C.

[0075] [Analysis of mesenchymal stem cell culture supernatant-1]

[0076] The number and size of particles in the RIM culture supernatant, RS culture supernatant, RMFS1 culture supernatant, and DMEM culture supernatant were measured using the nanoparticle analysis system NanoSight (LM10, Quantum Design). It was confirmed that the number of particles in the RIM culture supernatant, RS culture supernatant, and RMFS1 culture supernatant was larger than that in the DMEM culture supernatant ( Figure 1 and Figure 2 ).

[0077] [Analysis of mesenchymal stem cell culture supernatant-2]

[0078] The protein concentrations of RIM culture supernatants, RS culture supernatants, RMFS1 culture supernatants, and DMEM culture supernatants were measured using MocroBCAPProtein Assay kit (Cat. No. 23235, Bio Assay Systems). It was confirmed that the protein content of RIM culture supernatants, RS culture supernatants, and RMFS1 culture supernatants was lower than that of DMEM culture supernatants ( Figure 3 ).

[0079] [Analysis of mesenchymal stem cell culture supernatant-3]

[0080] The content of CD9 / CD63 positive extracellular vesicles (exosomes) in RIM culture supernatant, RS culture supernatant, RMFS1 culture supernatant and DMEM culture supernatant was measured using ELISA kit (Cat. No. C#: EXH0102EL, Cosmobio). It was confirmed that the content of CD9 / CD63 positive extracellular vesicles (exosomes) (ng / mL) in RIM culture supernatant, RS culture supernatant and RMFS1 culture supernatant was higher than that in DMEM culture supernatant ( Figure 4 ). In addition, it was confirmed that the RIM culture supernatant and RS culture supernatant contained EGF, IGF1, M-CSF, TGFb, and VEGF.

[0081] [Analysis of mesenchymal stem cell culture supernatant-4]

[0082] The nitric oxide (NOx) content in the supernatant of RIM culture, RS culture, RMFS1 culture, and DMEM culture was measured using a nitric oxide analysis kit (Cat. No. D2NO-100, Bio Assay Systems). It was confirmed that the nitric oxide content in the supernatant of RIM culture, RS culture, and RMFS1 culture was lower than that in the supernatant of DMEM culture ( Figure 5 ).

[0083] [Physical properties of mesenchymal stem cell culture supernatant-1]

[0084] The pH of the RIM culture supernatant, RS culture supernatant, RMFS1 culture supernatant, and DMEM culture supernatant was measured using a small pH meter (LAQUAtwin, pH-22B, Horiba Advanced Techno Co., Ltd.). It was confirmed that the pH of the RIM culture supernatant, RS culture supernatant, and RMFS1 culture supernatant was closer to neutral than that of the DMEM culture supernatant ( Figure 6 ).

[0085] [Physical properties of mesenchymal stem cell culture supernatant-2]

[0086] The viscosity of the RIM culture supernatant, RS culture supernatant, RMFS1 culture supernatant, and DMEM culture supernatant was measured using a viscometer (TV-100EL, Toki Sangyo). It was confirmed that the RIM culture supernatant, RS culture supernatant, and RMFS1 culture supernatant had higher viscosity than the DMEM culture supernatant (Table 1).

[0087] [Table 1]

[0088] sample Viscosity(mPa) RIM culture supernatant 1.126 RS culture supernatant 1.13 RMFS1 culture supernatant 1.063 DMEM culture supernatant 1.059

[0089] [Effects of mesenchymal stem cell culture supernatant on kidney disease]

[0090] For rat (SD, female, 5 week old, Japanese SLC company), under isoflurane inhalation anesthesia, cut the right ventral part, after the right renal artery and vein are blocked with suture ligation and blood flow, remove the right kidney, close the abdomen. After the right kidney is removed for 14 days, under isoflurane inhalation anesthesia, cut the left ventral part, the blood flow of the left kidney is blocked with silk thread for 30 minutes. After blocking for 30 minutes, blood flow is restored, thus causing acute renal injury. For this rat, before 60 minutes (before 30 minutes of blood flow blocking) of blood flow recovery, with 1mL / intravenous administration RIM culture medium or RIM culture supernatant. After 24 hours of ischemia-reperfusion of left kidney, blood sampling 0.2-0.3mL from subclavian vein, after 48 hours of ischemia-reperfusion of left kidney, remove kidney, carry out formalin fixation. From the obtained blood samples, blood urea nitrogen (BUN) was measured by the urease-GLDH method, creatinine (CRE) was measured by the enzymatic method, and potassium (K) was measured by the ion selective electrode method. It should be noted that in each test, L-type Wako CRE·M (Fujifilm Wako Pure Chemical Industries, Ltd.) and Hitachi ion electrode reagent (Fujifilm Wako Pure Chemical Industries, Ltd.) were used as reagents. It was found that administration of RIM culture supernatant reduced BUN, CRE and K and improved pathological images compared with RIM culture medium ( Figure 7 , Figure 8 and Fig. 9 ).

[0091] [Effects of mesenchymal stem cell culture supernatant on kidney disease 2]

[0092] For rats (SD, female, 5 weeks old, Japanese SLC company), under isoflurane inhalation anesthesia, the right ventral part was cut open, and after the right renal artery and vein were ligated with sutures and blood flow was blocked, the right kidney was removed and the abdomen was closed. After the right kidney was removed for 14 days, the left ventral part was cut open under isoflurane inhalation anesthesia, and the blood flow of the left kidney was blocked with silk thread for 30 minutes. After blocking for 30 minutes, blood flow was restored, thus causing acute kidney injury. For this rat, 60 minutes before blood flow recovery (30 minutes before blood flow blocking), RIM culture supernatant, RS culture supernatant, RMFS1 culture supernatant or DMEM culture supernatant were administered intravenously with 1mL / . After 24 hours of ischemia-reperfusion of the left kidney, 0.2-0.3mL of blood was collected from the subclavian vein, and about 2mL of blood was collected from the abdominal vein under isoflurane inhalation anesthesia after 48 hours of ischemia-reperfusion of the left kidney. About 2 mL of blood was collected from the abdominal vein under isoflurane inhalation anesthesia 48 hours after ischemia-reperfusion of the left kidney, and blood urea nitrogen (BUN) was measured by the urease-GLDH method, creatinine (CRE) was measured by the enzymatic method, and potassium (K) was measured by the ion-selective electrode method. In addition, blood glucose level (GLU) was measured by the HK-G6PDH method. It should be noted that in each test, L-type Wako CRE·M (Fujifilm Wako Pure Chemical Industries, Ltd.), Hitachi ion electrode reagent (Fujifilm Wako Pure Chemical Industries, Ltd.), and Quick Auto Neo GLU-HK (Shino test) were used as reagents. It can be seen that compared with the DMEM culture supernatant administration group, the RIM culture supernatant administration group, the RS culture supernatant administration group, and the RMFS1 culture supernatant administration group reduced BUN and CRE of the left kidney 24 hours after ischemia-reperfusion, and K of the left kidney 48 hours after ischemia-reperfusion ( Fig.10 In addition, body weight, food intake, and water intake were measured every day from the day before to 48 hours after ischemia-reperfusion of the left kidney. The results also showed that the RIM culture supernatant, RS culture supernatant, and RMFS1 culture supernatant administration groups had the effect of alleviating weight loss, food intake reduction, and water intake increase compared with the DMEM culture supernatant.

[0093] Industrial Applicability

[0094] According to the present invention, a novel therapeutic agent for kidney disease can be provided. In addition, the therapeutic agent for kidney disease of the present invention can be preferably used not only for humans but also for kidney disease in animals other than humans for which there is currently no effective treatment.

Claims

1. A kidney disease therapeutic agent comprising a mesenchymal stem cell culture supernatant.

2. The therapeutic agent for kidney disease according to claim 1, wherein The mesenchymal stem cell culture supernatant is a culture supernatant obtained by culturing adipose tissue-derived, umbilical cord tissue-derived, or bone marrow tissue-derived mesenchymal stem cells.

3. The therapeutic agent for kidney disease according to claim 1 or 2, wherein The mesenchymal stem cell culture supernatant is obtained by culturing mesenchymal stem cells in a culture medium containing at least one selected from the group consisting of EGF, bFGF, albumin, transferrin, and insulin.

4. The therapeutic agent for kidney disease according to claim 3, characterized in that The content of exosomes in the culture supernatant of mesenchymal stem cells is high.

5. The therapeutic agent for kidney disease according to claim 3, characterized in that The nitric oxide content in the culture supernatant of mesenchymal stem cells is low.

6. A renal function improving agent comprising a mesenchymal stem cell culture supernatant.

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