Medicine for preventing or treating kidney damage

Abstract

To provide means that can universally inhibit the progress of nephropathy such as chronic kidney disease.SOLUTION: One embodiment of the present invention relates to a pharmaceutical for preventing or treating nephropathy, containing a compound having the activity of inhibiting the bonding of a Toll-like receptor expressed in the proximal renal tubule and calciprotein particles in the urine as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical for preventing or treating nephropathy. In particular, one aspect of the present invention relates to a pharmaceutical for preventing or treating nephropathy, comprising, as an active ingredient, a compound having activity of inhibiting binding between a Toll-like receptor expressed in the proximal tubule and calciprotein particles in urine. [Background technology]

[0002] Chronic kidney disease, a type of kidney disorder, often occurs as a renal complication of lifestyle-related diseases such as diabetes and hypertension, as well as kidney diseases such as chronic glomerulonephritis. The only way to prevent the progression of chronic kidney disease is to identify the underlying disease in each patient and thoroughly treat that disease. Therefore, there is a need to identify universal therapeutic targets common to all patients with chronic kidney disease and develop treatments.

[0003] A common pathological condition observed during the progression of chronic kidney disease is a decrease in the number of functional nephrons. Nephrons are functional units of the kidney, consisting of tubules and glomeruli. When the number of nephrons decreases, a compensatory mechanism is activated that increases the amount of substances excreted in urine per nephron. Phosphorus becomes a problem in this situation. When the amount of phosphorus excreted per nephron increases, the phosphorus concentration in the primary urine increases, leading to the formation of microparticles containing calcium phosphate crystals (hereinafter also referred to as "calciprotein particles") within the lumen of the renal tubules. Calciprotein particles are nanoparticles composed of complexes of calcium phosphate crystals and the serum protein fetuin A. Calciprotein particles are known to have the ability to damage renal tubule cells. Therefore, the formation of calciprotein particles can cause kidney damage.

[0004] For example, Patent Document 1 describes a method for measuring calciprotein particles and a method for assisting in the examination of chronic kidney disease. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6566697 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, although knowledge about the pathology of chronic kidney disease has been gained, no means capable of universally suppressing the progression of chronic kidney disease has been developed at present.

[0007] Therefore, an object of the present invention is to provide a means capable of universally suppressing the progression of renal disorders such as chronic kidney disease. [Means for solving the problem]

[0008] The present inventors have investigated various means for solving the above-mentioned problems. They have found that calciprotein particles in primary urine can be a universal therapeutic target for renal disorders such as chronic kidney disease. They have also found that calcium phosphate particles, which are components of calciprotein particles, can specifically bind to Toll-like receptors expressed in proximal tubules. They have also found that the addition of fetuin A to a human proximal tubule cell line in a high-phosphate medium significantly improves the cell viability of the cell line. Based on these findings, the present inventors have completed the present invention.

[0009] That is, the present invention includes the following aspects and embodiments. (1) A pharmaceutical for preventing or treating kidney disorders, comprising as an active ingredient a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine. (2) The pharmaceutical composition according to the above embodiment (1), wherein the renal disorder is one or more disorders selected from the group consisting of chronic kidney disease, renal tubular disorder, renal fibrosis, inflammatory cell infiltration into the interstitium, and nephrocarcinosis. (3) The pharmaceutical according to the above embodiment (1) or (2), wherein the compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine is fetuin A or an analog thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. [Effects of the Invention]

[0010] The present invention makes it possible to provide a means capable of universally suppressing the progression of renal disorders such as chronic kidney disease. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the results of surface plasmon resonance analysis performed by scanning the extracellular domain of TLR4 protein over synthetic calcium phosphate particles immobilized on a sensor chip in Test I. In the figure, the horizontal axis represents time (seconds) and the vertical axis represents response (RU). [Figure 2] Figure 2 shows the relative mRNA levels of various markers in the kidneys of mice homozygous for the Tlr4 floxed allele carrying the Cre transgene (Tlr4-Cre) and mice carrying only the Cre transgene (Cre) fed a high-phosphate diet in Study II. Data are shown as mean ± standard deviation. ** indicates P < 0.01 vs. Cre by Mann-Whitney U test. [Figure 3] Figure 3 shows the relative mRNA levels of various markers in wild-type mice (WT) and TLR4-deficient mice (Tlr4lps-del) fed a normal diet (NP) or a high-phosphate diet (HP) in Study II. Data are shown as mean ± standard deviation (N = 10, 8, 8, and 13 for WT;NP, WT;HP, Tlr4lps-del;NP, and Tlr4lps-del;HP, respectively). * and ** indicate P < 0.05 and 0.01, respectively, for WT;NP by Mann-Whitney U test. # and ## indicate P < 0.05 and 0.01, respectively, for WT;HP by Mann-Whitney U test. [Figure 4] 4 is a graph showing the cell viability of a human proximal tubule cell line cultured in a control medium or a high-phosphate medium when fetuin A or alendronate was added to the cell line in Test III. In the figure, the horizontal axis represents the concentration (mg / mL) of fetuin A or alendronate added, and the vertical axis represents the relative cell viability. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present inventors have discovered that calciprotein particles in primary urine can be a universal therapeutic target for renal disorders such as chronic kidney disease. They have also found that calcium phosphate particles, a component of calciprotein particles, can specifically bind to Toll-like receptors expressed in proximal tubules. Furthermore, they have found that the addition of fetuin A to a human proximal tubule cell line in a high-phosphate medium significantly improves the cell viability of the cell line. Therefore, one aspect of the present invention relates to a pharmaceutical for preventing or treating renal disorders, which contains as an active ingredient a compound that inhibits the binding of Toll-like receptors expressed in proximal tubules to calciprotein particles in urine. This pharmaceutical can universally suppress the progression of renal disorders.

[0013] In each embodiment of the present invention, calciprotein particles refer to nanoparticles of a complex of calcium phosphate crystals and the serum protein fetuin A. As the number of functional nephrons decreases, a pathological condition commonly observed during the progression of chronic kidney disease, the amount of phosphorus excreted per nephron increases, leading to an increase in the phosphorus concentration in the primary urine and the formation of calciprotein particles in the lumen of the renal tubules.

[0014] In each embodiment of the present invention, the Toll-like receptor expressed in the proximal tubule refers to a Toll-like receptor (hereinafter also referred to as "TLR"), a membrane-bound receptor involved in innate immunity, that is specifically expressed in the proximal tubule. Examples of Toll-like receptors expressed in the proximal tubule include TLR4. TLR4 is known as a pattern recognition receptor that mediates the inflammatory response induced by calciprotein particles in macrophages (Koppert, S., et al. Front Immunol 9, 1991 (2018)). In addition, TLR4 is expressed on the apical side of the proximal tubule, which is distributed at the corticomedullary junction (El-Achkar, T.M., et al. Am J Physiol Renal Physiol 295, F534-544 (2008)).

[0015] The active ingredient of the pharmaceutical of this embodiment, a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine, is preferably fetuin A or an analog thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. Fetuin A is a serum protein that associates with calcium phosphate crystals to form calciprotein particles. By administering the pharmaceutical of this embodiment, which contains as an active ingredient a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine, to a subject, for example, a human patient, the progression of kidney damage can be universally suppressed.

[0016] The reasons why each embodiment of the present invention exhibits the above-described effects can be explained as follows. However, each embodiment of the present invention is not limited to the following effects and principles. Calciprotein particles induce renal tubular cell injury by binding to Toll-like receptors expressed in the proximal tubule, such as TLR4 expressed on the luminal side of the proximal tubule, and undergoing endocytosis. As shown in the following examples, adding fetuin A to a proximal tubule cell line can suppress cytotoxicity induced by calciprotein particle formation in a high-phosphate medium. Fetuin A is also known to bind to Toll-like receptors (Pal, D., et al. Nat Med 18, 1279-1285, 2012). Therefore, it is speculated that fetuin A competitively inhibits the binding of Toll-like receptors to calciprotein particles, thereby suppressing cytotoxicity in proximal tubule cell lines. Furthermore, mice lacking TLR4 specifically in renal tubule cells do not suffer from tubular injury even when the amount of phosphorus excreted per nephron increases and calciprotein particles are formed in the primary urine. Therefore, the progression of kidney damage can be universally suppressed by administering to a subject, for example, a human patient, a pharmaceutical of this embodiment containing, as an active ingredient, a compound that inhibits the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine.

[0017] In each aspect of the present invention, the activity of an active ingredient compound to inhibit the binding of Toll-like receptors expressed in proximal tubules to calciprotein particles in urine can be determined, for example, by measuring the binding inhibitory activity upon addition of the active ingredient compound in a surface plasmon resonance analysis of the binding between Toll-like receptors and calciprotein particles in urine, or by adding a Toll-like receptor extracellular domain protein and the active ingredient compound to a multi-well plate on which calciprotein particles have been immobilized, and then directly or indirectly quantifying the amount of Toll-like receptor protein that binds to the immobilized calciprotein particles.

[0018] In each aspect of the present invention, the renal disorder to be prevented or treated is preferably one or more disorders selected from the group consisting of chronic kidney disease, tubular damage, renal fibrosis, inflammatory cell infiltration into the interstitium, and nephrocarcinosis. By administering a pharmaceutical agent of this aspect, which contains as an active ingredient a compound that inhibits the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles, to subjects with renal disorders such as those exemplified above, for example, human patients with such renal disorders, the progression of renal disorders can be universally suppressed.

[0019] In each aspect of the present invention, the preventive or therapeutic effect on renal damage in a subject with renal damage, for example, a human patient with renal damage, can be determined, for example, by using the amount of blood fibroblast growth factor 23 (FGF23), blood creatinine, blood urea nitrogen, urinary L-fatty acid binding protein (L-FABP), urinary N-acetylglucosaminidase (NAG), urinary beta2-microglobulin, urinary creatinine, or the like as an indicator.

[0020] As used herein, "prevention" means substantially preventing the occurrence (development or manifestation) of a symptom or disorder in a subject with renal impairment, e.g., a human patient with renal impairment. Also, as used herein, "treatment" means suppressing (e.g., suppressing progression of), alleviating, repairing, and / or curing a symptom or disorder that has developed (developed or manifested) in a subject with renal impairment, e.g., a human patient with renal impairment.

[0021] In each aspect of the present invention, the compound used as an active ingredient includes not only the compound itself but also its salt. When the compound used as an active ingredient is in the form of a salt, it is preferably a pharmaceutically acceptable salt. Counter ions of the salt of the compound used as an active ingredient include, but are not limited to, cations such as sodium ion, potassium ion, calcium ion, magnesium ion, or substituted or unsubstituted ammonium ion, or chloride ion, bromide ion, iodide ion, phosphate ion, nitrate ion, sulfate ion, carbonate ion, bicarbonate ion, perchlorate ion, formate ion, acetate ion, trifluoroacetate ion, propionate ion, lactate ion, maleate ion, hydroxymaleate ion, methylmaleate ion, fumarate ion, adipate ion, benzoate ion, 2-acetoxybenzoate ion, p-aminobenzoate ion, and the like. Preferred anions include benzoate, nicotinate, cinnamate, ascorbate, pamoate, succinate, salicylate, bismethylenesalicylate, oxalate, tartrate, malate, citrate, gluconate, aspartate, stearate, palmitate, itaconate, glycolate, glutamate, benzenesulfonate, cyclohexylsulfamate, methanesulfonate, ethanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate, and naphthalenesulfonate. When the compound used as an active ingredient is in the form of a salt with the above counter ions, the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubule to calciprotein particles in urine can be substantially equivalent to that of the compound itself.

[0022] In each aspect of the present invention, the compound used as an active ingredient includes not only the compound itself but also solvates of the compound or its salt. When the compound or its salt used as an active ingredient is in the form of a solvate, it is preferably a pharmaceutically acceptable solvate. Solvents that can form solvates with the compound or its salt are, but are not limited to, water or organic solvents such as methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine, acetonitrile, or ethyl acetate. When the compound or its salt used as an active ingredient is in the form of a solvate with the above solvent, the activity of inhibiting the binding of Toll-like receptors expressed in proximal tubules to calciprotein particles in urine can be substantially equivalent to that of the compound itself.

[0023] In each embodiment of the present invention, the compounds used as active ingredients also include mixtures of stereoisomers of the compounds, such as individual enantiomers and diastereomers of the compounds, as well as racemates.

[0024] In the pharmaceutical composition of this embodiment, the compound used as the active ingredient may be used alone or in combination with one or more pharmaceutically acceptable ingredients. The pharmaceutical composition of this embodiment can be formulated into various dosage forms commonly used in the art depending on the desired administration method. Therefore, the pharmaceutical composition of this embodiment can also be provided in the form of a pharmaceutical composition containing a compound having activity inhibiting the binding of Toll-like receptors expressed in the proximal tubule to urinary calciprotein particles and one or more pharmaceutically acceptable carriers. In this embodiment, the pharmaceutical composition may contain, in addition to the above ingredients, one or more pharmaceutically acceptable additives such as one or more pharmaceutically acceptable vehicles (e.g., solvents such as sterile water or solutions such as physiological saline), excipients, binders, vehicles, solubilizers, preservatives, stabilizers, disintegrants, disintegration inhibitors, bulking agents, lubricants, surfactants, emulsifiers, oily liquids (e.g., vegetable oils), suspending agents, buffers, soothing agents, antioxidants, sweeteners, and flavoring agents.

[0025] The dosage form of the pharmaceutical of this embodiment is not particularly limited, and may be a formulation for use in parenteral administration, a formulation for use in transmucosal (e.g., nasal, sublingual, or oral mucosal), transdermal, transanal (enema), or transvaginal administration, or a formulation for use in oral administration. Furthermore, the dosage form of the pharmaceutical of this embodiment may be a formulation in a unit dose form or a formulation in a multiple dose form. Examples of formulations for use in parenteral administration include injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. Additives that can be mixed into injections include, but are not limited to, vehicles such as isotonic solutions containing physiological saline, glucose, or other auxiliary agents (e.g., D-sorbitol, D-mannitol, or sodium chloride), solubilizers such as alcohols (e.g., ethanol or benzyl alcohol), esters (e.g., benzyl benzoate), and polyalcohols (e.g., propylene glycol or polyethylene glycol), nonionic surfactants such as polysorbate 80 or polyoxyethylene hydrogenated castor oil, oily solutions such as sesame oil or soybean oil, buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as benzalkonium chloride or procaine hydrochloride, stabilizers such as human serum albumin or polyethylene glycol, preservatives, and antioxidants. Prepared injections are usually filled into appropriate containers (e.g., vials or ampoules) and stored under appropriate conditions until use.

[0026] The medicament of this embodiment can also be used in combination with one or more other medicaments useful as a medicament. In this case, the medicament of this embodiment may be provided in the form of a single medicament containing a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine and one or more other medicaments, or may be provided in the form of a pharmaceutical combination or kit containing multiple formulations in which a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine and one or more other medicaments are separately formulated. In the case of the pharmaceutical combination or kit form, the respective formulations can be administered simultaneously or separately (for example, consecutively).

[0027] In the pharmaceutical of this embodiment, the compound used as the active ingredient includes not only the compound itself but also pharmaceutically acceptable salts of the compound and pharmaceutically acceptable solvates thereof. Pharmaceutically acceptable salts of the compound used as the active ingredient and pharmaceutically acceptable solvates thereof are not limited, but are preferably, for example, the salts or solvates exemplified above. When the compound used as the active ingredient is in the form of the salt or solvate, the compound can be used for the desired pharmaceutical application.

[0028] When a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubule to urinary calciprotein particles is applied to pharmaceutical purposes, the compound encompasses not only the compound itself but also prodrug forms of the compound. As used herein, "prodrug" refers to a compound that is converted into a parent drug in vivo. In this embodiment of the pharmaceutical, prodrug forms of the compound used as an active ingredient include, but are not limited to, esters of the hydroxyl group of the compound with any carboxylic acid, amides of the hydroxyl group with any amine, and amides of the amino group of the compound with any carboxylic acid. When the compound used as an active ingredient is in the prodrug form, the pharmacokinetics of the prodrug form upon administration to a subject can be improved without substantially reducing the activity of the parent drug.

[0029] The pharmaceutical of this embodiment can be applied to various subjects requiring prevention or treatment of kidney damage. The subject is preferably a human or non-human mammalian subject or patient (e.g., a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, hamadryas baboon, or chimpanzee), and more preferably a human patient. By administering the pharmaceutical of this embodiment to the subject, kidney damage can be prevented or treated in the subject.

[0030] When administering the pharmaceutical of this embodiment to a subject, particularly a human patient, the exact dosage and administration method should ultimately be determined by the attending physician, taking into account many factors, such as the subject's age and sex, the symptoms to be prevented or treated, the exact state (e.g., severity) of the disease and / or disorder, and the route of administration. Therefore, in the pharmaceutical of this embodiment, the active ingredient, a compound having activity inhibiting the binding of Toll-like receptors expressed in the proximal tubules to calciprotein particles in urine, is administered to the subject at a therapeutically effective dosage and administration method (e.g., dosage amount and administration route). For example, when administering the pharmaceutical of this embodiment to a subject, particularly a human patient, the dosage of the compound used as the active ingredient is typically in the range of 0.01 to 1000 μg / kg body weight / day, for example, 0.5 to 200 μg / kg body weight / day.

[0031] The medicament of this embodiment may be administered by any administration route, for example, by intravenous administration, enema administration, subcutaneous administration, intramuscular administration, oral administration, or intraperitoneal administration.

[0032] A compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles, which is used as an active ingredient of the medicament of this embodiment, can be used to prevent or treat nephropathy in subjects with nephropathy, for example, human patients with nephropathy. Therefore, another aspect of the present invention relates to a prophylactic or therapeutic agent for nephropathy, which contains as an active ingredient a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles. The prophylactic or therapeutic agent of this embodiment has the same characteristics as the medicament of this embodiment described above. Furthermore, the prophylactic or therapeutic agent of this embodiment can be used in the same dosage and administration as the medicament of this embodiment described above. The prophylactic or therapeutic agent of this embodiment can be used to prevent or treat nephropathy in subjects with nephropathy, for example, human patients with nephropathy. In the prophylactic or therapeutic agent of this embodiment, the nephropathy is preferably one or more disorders selected from the group consisting of chronic kidney disease, tubular damage, renal fibrosis, inflammatory cell infiltration into the interstitium, and nephrocarcinosis. The above-mentioned nephropathy can be prevented or treated by administering an effective amount of a compound having activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to a subject with nephropathy, for example, a human patient with nephropathy.

[0033] Another aspect of the present invention is a method for preventing or treating renal damage, comprising administering to a subject with renal damage, e.g., a human patient with renal damage, an effective amount of a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles. The compound administered in the method of this aspect has the same characteristics as the compound used as the active ingredient of the medicament of this aspect described above. Furthermore, the method of this aspect can be carried out using the same dosage and administration method as the medicament of this aspect described above. In the method of this aspect, the renal damage is preferably one or more disorders selected from the group consisting of chronic kidney disease, tubular damage, renal fibrosis, inflammatory cell infiltration into the interstitium, and nephrocarcinosis. The above-mentioned renal damage can be prevented or treated by administering to a subject with renal damage, e.g., a human patient with renal damage, an effective amount of a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles.

[0034] Another aspect of the present invention is a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles, for use in the prevention or treatment of nephropathy in a subject with nephropathy, for example, a human patient with nephropathy. Yet another aspect of the present invention is the use of a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles in the manufacture of a medicament for the prevention or treatment of nephropathy in a subject with nephropathy, for example, a human patient with nephropathy. Yet another aspect of the present invention is the use of a compound having the activity of inhibiting the binding of Toll-like receptors expressed in the proximal tubules to urinary calciprotein particles, for the prevention or treatment of nephropathy in a subject with nephropathy, for example, a human patient with nephropathy. The compound of this aspect has the same characteristics as the compound used as the active ingredient in the medicament of this aspect described above. Furthermore, the compound of this aspect can be used in the same dosage and administration as the medicament of this aspect described above. In the compound or use of this embodiment, the renal disorder is preferably one or more disorders selected from the group consisting of chronic kidney disease, renal tubular damage, renal fibrosis, inflammatory cell infiltration into the interstitium, and nephrocarcinosis. The above-mentioned renal disorders can be prevented or treated by administering an effective amount of a compound having activity that inhibits the binding of Toll-like receptors expressed in the proximal tubules to a subject with renal disorder, for example, a human patient with renal disorder. [Example]

[0035] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0036] <Test I: Binding of calciprotein particles to Toll-like receptors> We hypothesized that the cytotoxic signal of calcium phosphate particles might be mediated by a cell surface receptor to explain the mechanism by which calcium phosphate particles damage renal tubular cells. A putative receptor for calcium phosphate particles must meet at least two criteria: first, it must bind to calcium phosphate crystals and therefore likely belong to the pattern recognition receptor family. Second, it must be expressed at the corticomedullary junction of the kidney. We identified Toll-like receptor 4 (TLR4) as a candidate that fulfills these two criteria. TLR4 is known to mediate the inflammatory response induced by calcium phosphate particles in macrophages (Koppert, S., et al. Front Immunol 9, 1991 (2018)). Additionally, TLR4 is expressed on the apical side of proximal tubules, which are distributed at the corticomedullary junction (El-Achkar, TM, et al. Am J Physiol Renal Physiol 295, F534-544 (2008)). To determine whether calcium phosphate particles can physiologically interact with TLR4 in vitro, surface plasmon resonance analysis was performed by scanning the extracellular domain of TLR4 protein on synthetic calcium phosphate particles immobilized on a sensor chip. Surface plasmon resonance analysis was performed using a ProteOn XPR36 (Bio-Rad). Alendronate was immobilized on a sensor chip (ProteOn GLC Sensor Chip, Bio-Rad) by amine coupling. The synthetic calcium phosphate particles were added to the chip in running buffer (20 mM HEPES, pH 7.4, 135 mM NaCl, 1 mM NaHPO, 2 mM CaCl, 0.005% Tween 20) and immobilized on the chip. After washing with running buffer, the extracellular domain of human TLR4 (R&D Systems) was added at a predetermined concentration (8.8, 17.6, 35.2, 70.5, or 141 nM). Kinetic analysis was performed using the Langmuir model. The results of surface plasmon resonance analysis are shown in Figure 1. In the figure, the horizontal axis represents time (seconds) and the vertical axis represents response (RU).

[0037] As a result of kinetic analysis, the association rate constant (ka) was 6.24 × 10 4 M -1 s -1 , dissociation rate constant (kd)=6.01×10 4 M -1 s -1 , and the equilibrium dissociation constant (K D )=9.63×10 -9 Furthermore, as shown in Figure 1, direct interaction between calcium phosphate particles and the TLR4 ectodomain was observed with a dissociation constant of 9.63 nM in the absence of myeloid differentiation factor-2 (MD2), a cofactor required for TLR4 to bind to lipopolysaccharide or fetuin A. This result suggests that calcium phosphate particles, which are components of calciprotein particles, can specifically bind to TLR4, a Toll-like receptor.

[0038] <Test II: Resistance of TLR4-deficient mice to high-phosphate diet-induced renal tubular injury and inflammation> To generate mice lacking TLR4 in renal tubular cells, Tlr4 floxed mice were crossed with recombinant mice expressing Cre recombinase in renal epithelial cells under the control of the mouse Cdh16 promoter. Tlr4-Cre mice are homozygous for the Tlr4 floxed allele and harbor the Cre transgene, while Cre mice harbor only the Cre transgene. At 4 weeks of age, male mice were placed on a high-phosphate diet containing 2.0% inorganic phosphate for 4 weeks. Relative mRNA levels of selected markers in the kidney were determined by quantitative RT-PCR. There was no difference in urinary phosphate excretion between Tlr4-Cre mice (N = 13) and Cre mice (N = 9), suggesting no difference in dietary phosphate intake. Relative mRNA levels of various markers in the kidney are shown in Figure 2. Data in the figure represent the mean ± standard deviation. ** indicates P < 0.01 vs. Cre by Mann-Whitney U test. αSMA is α-smooth muscle actin, IL6 is interleukin-6, MCP1 is monocyte chemotactic protein-1, MMP2 is matrix metalloproteinase-2, TGFβ1 is transforming growth factor-β1, IL1β is interleukin-1β, OPN is osteopontin, Kim1 is kidney injury molecule-1, and Ngal is neutrophil gelatinase-associated lipocalin.

[0039] As shown in Figure 2, TLR4 expressed in renal tubules was required for a high-phosphate diet to induce renal injury.

[0040] Wild-type mice (4-week-old C57BL / 6 males, WT) and TLR4-deficient mice (4-week-old males, TLR4 lps-del ) were placed on a normal diet containing 0.35% inorganic phosphate (NP) or a high-phosphate diet containing 2.0% inorganic phosphate (HP) for 4 weeks. The relative mRNA levels of the indicated markers were determined by quantitative RT-PCR. The relative mRNA levels of various markers are shown in Figure 3. The data in the figure represent the mean ± standard deviation (WT; NP, WT; HP, Tlr4). lps-del NP and Tlr4 lps-del(N=10, 8, 8, and 13, respectively, for WT and HP. * and ** indicate P<0.05 and 0.01, respectively, for WT and NP by Mann-Whitney U test. # and ## indicate P<0.05 and 0.01, respectively, for WT and HP by Mann-Whitney U test. IL6 is interleukin-6, MCP1 is monocyte chemoattractant protein-1, MMP2 is matrix metalloproteinase-2, Kim1 is kidney injury molecule-1, TNFα is tumor necrosis factor α, and NLRP3 is nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3.

[0041] As shown in Figure 3, TLR4-deficient mice were resistant to phosphate-induced renal fibrosis.

[0042] These results demonstrate that TLR4-deficient mice are resistant to high-phosphate diet-induced renal tubule injury and inflammation. Furthermore, deletion of the renal tubule-specific TLR4 gene attenuated renal injury. These results suggest that TLR4 expression is essential for the high-phosphate diet-induced renal injury.

[0043] <Test III: Effect of fetuin A on human proximal tubule cell line in high-phosphate medium> Human proximal tubule cell line (HK-2) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) in 96-well plates. The medium was then replaced with control medium (DMEM containing 0.1% FBS with calcium and phosphorus concentrations adjusted to 3 mM and 1 mM, respectively) or high-phosphorus medium (DMEM containing 0.1% FBS with calcium and phosphorus concentrations adjusted to 3 mM and 7 mM, respectively). Simultaneously with the medium replacement, bovine fetuin A was added to each medium to a final concentration of 0, 0.5, or 5 mg / mL. As a positive control, alendronate was added to each medium to a final concentration of 0.1 mg / mL instead of fetuin A. After 24 hours, cell viability was quantified using the MTT assay. We have previously reported that calciprotein particles do not form in control medium, but do form in high-phosphate medium, and that alendronate inhibits calciprotein particle formation (Akiyama, K., et al. Calciprotein particles regulate fibroblast growth factor-23 expression in osteoblasts. Kidney Int 97, 702-712, 2019). Figure 4 shows the cell viability of human proximal tubule cell lines cultured in control medium or high-phosphate medium when fetuin A or alendronate was added. In the figure, the horizontal axis represents the concentration (mg / mL) of fetuin A or alendronate added, and the vertical axis represents relative cell viability.

[0044] As shown in Figure 4, the addition of fetuin A was confirmed to suppress renal tubular cell injury caused by calciprotein particles in a high-phosphate medium. Fetuin A is known to bind to Toll-like receptors (Pal, D., et al. Nat Med 18, 1279-1285, 2012). Therefore, the results of this study suggest that fetuin A competitively inhibits the binding of calciprotein particles to Toll-like receptor 4 (TLR4) expressed in the proximal tubule, thereby suppressing calciprotein particle-induced cell injury.

[0045] The present invention is not limited to the above-described embodiments, but includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to add, delete, and / or replace part of the configuration of each embodiment with other configurations.

Claims

[Claim 1] A pharmaceutical agent for preventing or treating renal tubular disorders, comprising fetuin A or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof as an active ingredient, which inhibits the binding of Toll-like receptor 4 expressed in the proximal tubules to calciprotein particles in urine.

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