Improvement of renal hypofunction by substance having ammonia excretion promoting action

Abstract

An object of the present invention is to provide a novel means for suppressing renal hypofunction, which can fundamentally improve diseases accompanied by renal hypofunction including chronic kidney disease (CKD).SOLUTION: The inventors of the present invention have solved the above-mentioned problems by providing a novel means for suppressing renal hypofunction, which suppresses and improves renal hypofunction in vivo by a substance having an ammonia excretion promoting action. More specifically, the present invention has solved the above problems by providing a renal hypofunction inhibitor comprising a substance having an ammonia excretion promoting action in vivo. The substance having an ammonia excretion promoting action in a living body is lactulose, ornithine, sodium benzoate, or a derivative thereof, or any combination thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to providing a new means for suppressing renal dysfunction in vivo and improving renal function using a substance having an ammonia excretion-promoting effect. More specifically, the present invention relates to providing a new means for removing senescent cells from a living body using a substance having an ammonia excretion-promoting effect in vivo, thereby suppressing renal dysfunction. [Background technology]

[0002] It is known that "senescent cells," which are cells that can no longer proliferate with age, accumulate in the human body and cause diseases such as chronic kidney disease (CKD) and aging phenomena. Furthermore, recent research has reported that removing senescent cells can prevent or delay bodily dysfunction and extend healthy lifespan (Non-Patent Document 1).

[0003] Chronic kidney disease (CKD), one of the serious diseases associated with decreased kidney function, is thought to affect 13.3 million people in Japan (one in eight adults aged 20 and over).Although it has been called a new national disease, only symptomatic treatment is available and no fundamental cure exists.

[0004] Lactulose has been shown to reduce Bacteroides and Clostridium, improving the intestinal environment, and has been suggested to significantly reduce serum creatinine and blood urea nitrogen (BUN) levels compared to before lactulose administration, and to improve renal dysfunction through a reduction in indoxyl sulfate, a uremic toxin (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] DJ Baker, et al., Nature. 2011 Nov 2; 479(7372): 232-236 [Non-patent document 2] Grant-in-Aid for Scientific Research Research Results Report 18K06774 [Non-patent document 3] Johmura Y., SCIENCE, Vol 371, Issue 6526, pp. 265-270, 2021 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a new means for suppressing renal dysfunction, which can fundamentally improve diseases accompanied by renal dysfunction, including chronic kidney disease (CKD). [Means for solving the problem]

[0007] The inventors of the present invention have solved the above-mentioned problems by providing a new means for suppressing renal function decline in vivo, that is, by using a substance having an ammonia excretion-promoting effect to suppress and improve renal function decline in vivo. More specifically, the present invention has solved the above-mentioned problems by providing an agent for suppressing renal function decline, which comprises a substance having an ammonia excretion-promoting effect in vivo.

[0008] More specifically, the present application provides the following aspects to solve the above-mentioned problems: [1]: A renal function inhibitor containing a substance that promotes ammonia excretion in the body; [2]: The agent for suppressing kidney function decline according to claim 1, wherein the substance having an ammonia excretion promoting effect in vivo is lactulose, ornithine, sodium benzoate, or a derivative thereof, or any combination thereof; [3]: The agent for suppressing renal function decline according to claim 1 or 2, wherein the renal function decline is selected from renal function decline in the early nephropathy stage (stage 2), renal function decline in the overt nephropathy stage (stage 3A), and renal failure stage (stage 4); [4]: Renal function decline is classified as G2, G3a, G3b, or G4 (GFR value of 15 mL / min / 1.73 m 2 or more, 90 mL / min / 1.73 m 2 The agent for suppressing renal function decline according to claim 1 or 2, wherein the renal function decline is in a state of less than 100%. [5]: A pharmaceutical composition for treating diseases accompanied by decreased kidney function, comprising a substance having an ammonia excretion promoting effect in vivo; [6]: The pharmaceutical composition according to claim 5, wherein the substance having an ammonia excretion promoting effect in vivo is lactulose, ornithine, sodium benzoate, or a derivative thereof, or any combination thereof; [7]: The pharmaceutical composition according to claim 5 or 6, wherein the renal function decline is selected from renal function decline in the early nephropathy stage (stage 2), renal function decline in the overt nephropathy stage (stage 3A), and renal failure stage (stage 4); [8]: Renal function decline is classified as G2, G3a, G3b, or G4 (GFR value of 15 mL / min / 1.73 m 2 or more, 90 mL / min / 1.73 m 2 The pharmaceutical composition according to claim 5 or 6, wherein the pharmaceutical composition is in a state of less than 100 mg / kg. [Effects of the Invention]

[0009] The present invention provides an agent for suppressing renal function decline, which contains a substance that has the effect of promoting ammonia excretion in the body, thereby providing a new means for suppressing renal function decline in the body and improving renal function through the excretion of ammonia. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the results of measuring the mRNA expression level of p21, a marker for senescent cells, in the kidney. [Figure 2] FIG. 2 shows the results of measuring the amount of urea nitrogen (BUN) in plasma. [Figure 3] FIG. 3 shows the results of measuring the amount of cystatin C in plasma. DETAILED DESCRIPTION OF THE INVENTION

[0011] The inventors of the present invention focused on the mechanism by which excess ammonia produced by metabolism maintains the survival of senescent cells, and revealed that a substance that has the effect of promoting ammonia excretion in the body can provide a new means for removing senescent cells from the body and preventing aging (Patent Application No. 2022-49514, filed March 25, 2022).

[0012] The inventors of the present invention conducted further research and development based on the above patent application, and hypothesized that ingesting lactulose, a component that promotes the excretion of ammonia in the body, may be able to remove senescent cells and inhibit or improve the decline in kidney function. They decided to use aged animals to verify the effects on senescent kidney cells and markers of kidney function.

[0013] As a result, the inventors of the present invention have found that a substance having an ammonia excretion-promoting effect can suppress and improve renal function decline in the body, and have demonstrated that a substance having an ammonia excretion-promoting effect can provide a new means for suppressing renal function decline. More specifically, the present invention provides an agent for suppressing renal function decline, which comprises a substance having an ammonia excretion-promoting effect in the body.

[0014] In the present invention, a substance having an ammonia excretion-promoting effect can efficiently remove senescent cells in the kidney, which are markers for p21 expression, and can also suppress and improve renal dysfunction in vivo. From this, it is predicted that the mechanism by which a substance having an ammonia excretion-promoting effect can suppress and improve renal dysfunction in vivo is via the efficient removal of senescent cells.

[0015] Senescent cells are generally defined as cells that have irreversibly stopped cell division due to damage to chromosomal DNA caused by various stressors, such as telomere abnormalities, oxidative stress, DNA damage, oncogene activation, mitochondrial dysfunction, viral infection, and inflammation. Senescent cells are known to express p16 and p21 genes at high levels, and these genes are considered representative markers of senescent cells. Furthermore, functionally, the accumulation of these senescent cells in the body leads to the overexpression and extracellular secretion of various inflammatory proteins, such as cytokines and chemokines, which can lead to the decline of various tissue functions, chronic inflammation, and carcinogenesis.

[0016] Based on these characteristics, it has been proposed that aging and geriatric diseases can be improved by removing senescent cells with senolytic drugs (Non-Patent Document 3).

[0017] A common characteristic of senescent cells is that the accumulation of defective proteins causes damage to the intracellular lysosomal membrane, resulting in leakage of the contents of the lysosomal lumen, which has a pH of around 5, into the cell, causing a decrease in intracellular pH.In such cells, the production of glutaminase 1 (GLS1), a glutamine metabolic enzyme, is activated, resulting in the excessive production of glutamate and ammonia from glutamine.It is thought that this excessive production of ammonia regulates the homeostasis of intracellular pH and maintains survival.

[0018] Therefore, it is believed that removing ammonia from within cells and acidifying the intracellular pH of senescent cells, thereby making them unable to survive, can promote the removal of senescent cells and improve pathologies caused by their accumulation. In the present invention, the researchers focused particularly on pathologies caused by the accumulation of senescent cells in the kidneys.

[0019] There are several pathways for ammonia excretion within cells. Specifically, The urea cycle (ornithine cycle) in which ornithine or citrulline reacts with ammonia to ultimately produce urea, which is then detoxified and excreted. Acid amide formation from the reaction of glutamic acid (Glu) or aspartic acid (Asp) with ammonia to form glutamine (Gln) or asparagine (Asn) Reaction of α-ketoglutaric acid with ammonia to form glutamic acid (Glu) Creatine production · Activation of the urea cycle to reduce blood ammonia levels A pathway that acts on intestinal bacteria to reduce ammonia production and thereby reduce blood ammonia levels ・Pathway where it binds with ammonia and is excreted from the body as hippuric acid The following ammonia excretion pathways are known:

[0020] The substance that can be used as an active ingredient in the present invention and has the effect of promoting ammonia excretion in vivo refers to a substance that acts on one or more of the above-mentioned ammonia excretion pathways to promote ammonia excretion.For example, substances that react with ammonia, such as ornithine or citrulline, which are components of the ornithine cycle, glutamic acid or aspartic acid in acid amide production, and α-ketoglutaric acid in reaction with keto acid, can be used for this purpose, but are not limited to these.

[0021] More specifically, for example, ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAA), acetylglutamic acid, sodium benzoate, lactulose, lactitol, sodium phenylbutyrate, sodium phenylacetate, rifaximin, carglumic acid, or derivatives thereof can be used, but are not limited to these. For example, substances currently being studied for treating hyperammonemia can also be used in the future.

[0022] The derivative of the above-mentioned compound means a compound obtained by changing a small part of the molecular structure of the above-mentioned compound, and refers to a compound that maintains the basic structure and properties of the above-mentioned compound while undergoing modifications such as the introduction of a functional group, oxidation, reduction, or atom substitution in part of the structure.

[0023] In a preferred embodiment of the present invention, lactulose, ornithine, sodium benzoate, or a derivative thereof, or any combination thereof can be used as the substance having an ammonia excretion promoting effect in the body.

[0024] The amount of the active ingredient in the present invention varies depending on the substance that can be used as the active ingredient, and can be the amount commonly used for each substance. The intake schedule for each active ingredient may be any, for example, once a day, three times a day, or less frequently, such as once a week or once a month.

[0025] The agent for suppressing renal function decline of the present invention may contain, in addition to the above-mentioned active ingredients, various additives that are commonly used in foods or medicines.

[0026] In the present invention, the suppression of decline in renal function can include bringing the values ​​of renal function markers in plasma closer to the normal range and reducing the values ​​of senescent cell markers in the kidney. -Albumin, type IV collagen, and ceruloplasmin as glomerular injury markers Markers of renal tubular damage include NGAL, α1-MG, KIM-1, L-FABP, angiotensinogen, and NAG Inflammatory markers include inflammatory cytokines (IL-6, IL-8, IL-18, IP-10, TNF-α, TNF-α receptor), growth factors (TGF-β, CTGF), adhesion molecules (ICAM-1, VCAM-1), fetuin-A, soluble CD40 ligand, human α1 acid glycoprotein, - 8-OHdG and pentosidine as oxidative stress markers CKD markers: glomerular filtration rate (GFR), cystatin C, and uric acid By measuring these and comparing the values ​​with the normal range, it is possible to determine whether or not kidney function is declining or being prevented from declining.

[0027] The agent for suppressing renal function decline of the present invention can be applied to any disease in which renal function declines, such as glomerulonephritis, chronic kidney disease (CKD), etc. In particular, the agent for suppressing renal function decline of the present invention is particularly effective against CKD, and is even more effective against renal function decline selected from early nephropathy stage (stage 2), overt nephropathy stage (stage 3A), and renal failure stage (stage 4) of CKD.

[0028] CKD, which is the target of the renal function decline inhibitor of the present invention, has various pathological conditions ranging from a state in which there is no decline in renal function at all with only abnormal urinary findings to end-stage renal failure requiring renal replacement therapy (dialysis or kidney transplant). Therefore, CKD is characterized by a decline in renal function due to the glomerular filtration rate (GFR (ml / min / 1.73m 2 )) are divided into six stages: G1, G2, G3a, G3b, G4, and G5. The characteristics of these stages are summarized in Table 1.

[0029] [Table 1]

[0030] The agent for suppressing renal function decline of the present invention is effective when renal function decline is in the GFR category of G2, G3a, G3b, or G4 (i.e., when the GFR value is 15 mL / min / 1.73 m 2or more, 90 mL / min / 1.73 m 2 It is preferred to administer to individuals who are in a state where:

[0031] The present invention can treat or prevent diseases accompanied by renal function decline by administering the aforementioned agent for suppressing renal function decline to an individual with renal decline. Therefore, in a second aspect, the present invention can provide a composition having an effect of suppressing renal function decline, which comprises a substance having an effect of promoting ammonia excretion in the body.

[0032] The composition in this embodiment may refer to a food composition (a composition for a general food, or a composition for a food having a bioregulatory function (e.g., a food for specified health uses, a food with nutrient function claims, or a food with functional claims)) or a pharmaceutical composition. In one preferred embodiment, the present invention can provide a pharmaceutical composition for treating a disease accompanied by decreased kidney function, which comprises a substance having an ammonia excretion promoting effect in the body.

[0033] The composition of the present invention may contain, in addition to the above-mentioned active ingredient (a substance having the effect of promoting ammonia excretion in the body), other ingredients or various additives commonly used in foods or medicines, and any type of these other ingredients or various additives is acceptable.

[0034] As described above, the substance having the ammonia excretion-promoting effect that can be used in the composition of the present invention is one that can efficiently remove senescent cells in the kidney, as indicated by the expression of p21, and can also inhibit and improve renal dysfunction in vivo. Specific examples of such substances include, but are not limited to, ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, lactulose, lactitol, sodium phenylbutyrate, sodium phenylacetate, rifaximin, carglumic acid, and derivatives thereof. For example, substances currently being used to treat hyperammonemia in clinical studies may also be used in the future.

[0035] In a preferred embodiment of the composition of the present invention, lactulose, ornithine, sodium benzoate, or a derivative thereof, or any combination thereof can be used as the substance having the effect of promoting ammonia excretion in the body.

[0036] The content of the active ingredient in the composition of the present invention varies depending on the substance that can be used as the active ingredient, and each substance can be contained in an amount generally used for that substance.

[0037] The composition of the present invention can be applied to any disease in which kidney function declines, such as glomerulonephritis, chronic kidney disease (CKD), etc. In particular, the agent for suppressing kidney function decline of the present invention is particularly effective against CKD, and is even more effective against kidney function decline in the early nephropathy stage (stage 2), overt nephropathy stage (stage 3A), and renal failure stage (stage 4) of CKD.

[0038] The composition of the present invention is effective in treating patients with reduced renal function who are in the GFR category of G2, G3a, G3b, or G4 (GFR value of 15 mL / min / 1.73 m 2 or more, 90 mL / min / 1.73 m 2It is preferred to administer to individuals who are in a state where:

[0039] The present invention will be specifically illustrated by the following examples, which are not intended to limit the present invention in any way. [Example]

[0040] Example 1: Examination of the effect of removing senescent cells In this example, lactulose, which has the effect of promoting ammonia excretion, was administered to animals to confirm the effect of lactulose on removing senescent cells.

[0041] (1-1) Sample preparation The experiment involved 14 male C57BL / 6J mice (C57BL / 6J-Aged mice) aged 78 weeks. Each animal was housed in a SPF environment with a temperature of 20-26°C, humidity of 30-70%, and a 12-hour lighting schedule (lights on at 7:00 AM, lights off at 7:00 PM). Temperature and humidity were recorded daily and stored as raw data.

[0042] The animals were housed individually in polycarbonate mouse cages (170 W × 240 D × 120 H mm, manufactured by CLEA Japan, Inc.) and covered with paper chips, Peperclean (manufactured by Japan SLC Co., Ltd.), autoclaved (121°C, sterilization for 40 minutes, drying for 30 minutes).

[0043] The mice were given free access to autoclave-sterilized (121°C, sterilization for 15 minutes, drying for 5 minutes) solid mouse / rat feed MF (manufactured by Oriental Yeast Co., Ltd.).

[0044] Water was provided ad libitum in 100 mL polycarbonate bottles containing 0.025% sodium hypochlorite (Tsurukuron, Tsurumi Soda Co., Ltd.). The Aged+L group, described below, received oral administration of lactulose-containing lactulose water for one month. The lactulose water was prepared by adding lactulose syrup (60%, Kowa Pharmaceuticals) to water to make a 7.2% lactulose solution.

[0045] 78-week-old mice were divided into two groups of seven mice each (Group 1 and Group 2), and the experimental conditions were assigned as shown in the table below.

[0046] [Table 2]

[0047] Each group was given either normal water or lactulose water ad libitum for one month. One month after the start of the experiment, all animals were sacrificed, dissected, and kidneys were collected from each animal for measurement of senescent cell markers.

[0048] (1-2) Senescent cell marker measurement (1-2-1) RNA extraction Tissues collected from mice at autopsy were immersed in RNA later (Ambion) overnight and then stored at -80°C. Total RNA was extracted from kidney tissue using the TRIzol-chloroform method.

[0049] 50-100 mg of tissue extracted from RNA late was placed in 1 ml of TRIzol Reagent (Invitrogen Life Technologies) and crushed. After leaving the mixture at room temperature for 5 minutes, 0.2 ml of chloroform was added and vortexed. The mixture was then left at room temperature for 2-3 minutes and centrifuged at 12,000 x g for 15 minutes at 4°C. 250 μl of the upper layer was collected and 0.5 ml of isopropanol was added. The mixture was left at room temperature for 10 minutes and then centrifuged again at 12,000 x g for 10 minutes at 4°C.

[0050] The RNA was purified with 75% ethanol, evaporated to dryness under reduced pressure, and dissolved in 50 μl of RNA-free water. The absorbance ratio at 260 nm and 280 nm (A260 / 280 nm) was measured using a NanoDrop ND-1000 (NanoDrop Technologies). The RNA purity was confirmed by confirming that the ratio was within the range of 1.9–2.1.

[0051] (1-2-2) Real-time RT-PCR Using total RNA extracted from the tissues, mRNA expression analysis was performed by quantitative real-time PCR for p21, which is generally known in the art as a senescence marker.

[0052] cDNA was synthesized from total RNA using the PrimeScript® RT reagent Kit (Perfect Real Time) according to the manufacturer's protocol. The cDNA was amplified using the Thermal Cycler Dice Real Time System TP800 (Takara Bio) and purified with SYBR® Premix Ex Taq TM Fluorescence was detected using a Perfect Real Time (Perfect Real Time) and real-time PCR detection system (Takara Bio).

[0053] The PCR reaction consisted of one cycle of 95°C for 10 seconds, followed by 40 cycles of [95°C for 5 seconds, 60°C for 30 seconds], and then one cycle of [95°C for 15 seconds, 60°C for 30 seconds, 95°C for 15 seconds].

[0054] The mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard for normalization, and the relative mRNA levels of the target mRNA were calculated. Primers were designed using the web application PRIMER3 and synthesized by Invitrogen. The primer sequences for amplifying the target gene, p21, a marker of aging, are shown below.

[0055] [ka]

[0056] The results are shown in Figure 1. The mRNA expression level of p21, a marker of senescent cells, in the kidneys of the lactulose-fed group ("Aged+L" group) was significantly lower than that of the unfed old mice ("Aged" group) (*p<0.05). These results indicate that senescent cells were efficiently removed in the lactulose-fed group ("Aged+L" group).

[0057] Example 2: Examination of effects on kidney function markers In this example, lactulose, which has the effect of promoting ammonia excretion, was administered to animals to confirm the effect of lactulose on kidney function.

[0058] (2-1) Measurement of blood urea nitrogen (BUN) levels In (1-1), plasma samples were collected from mice at the time of autopsy and BUN, a marker of kidney function, was measured for each group. A DetectX Urea Nitrogen Colorimetric Detection Kit (Arbor Assays) was used to measure BUN colorimetrically. Plasma samples and the urea nitrogen standard solution included in the kit were mixed with Color Reagent A / B, incubated at room temperature for 30 minutes, and then quantified by measuring the color development at OD 450 nm.

[0059] The results are shown in Figure 2. Measurement of BUN levels, a marker of kidney function, showed that plasma BUN levels were significantly lower in the lactulose-intake group ("Aged+L" group) than in the untreated old mice ("Aged" group) (*p<0.05). These results indicate that kidney function was improved in the lactulose-intake group ("Aged+L" group).

[0060] (2-2) Measurement of cystatin C levels Cystatin C, a kidney function marker, was measured for each group using plasma collected from mice at autopsy in (1-1). For the measurement, an ELISA kit, Cystatin C Mouse ELISA Kit (BioVendor Laboratory Medicine, Inc.), which quantifies cystatin C using an antibody-based sandwich method, was used. Plasma samples were reacted with the samples included in the kit, and the color development was measured at OD 450 nm to quantify the amount.

[0061] The results are shown in Figure 3. Measurement of cystatin C levels, a marker of kidney function, showed a significant decrease in plasma cystatin C levels in the lactulose-intake group ("Aged+L" group) compared to the unintaken old mice ("Aged" group) (*p<0.05). These results indicate that kidney function was improved in the lactulose-intake group ("Aged+L" group). In other words, it was revealed that lactulose intake improves chronic kidney disease (CKD) in old mice through the removal of senescent cells. [Industrial Applicability]

[0062] The present invention provides an agent for suppressing renal function decline, which contains a substance that has the effect of promoting ammonia excretion in the body, thereby providing a new means for suppressing renal function decline in the body and improving renal function using a substance that has the effect of promoting ammonia excretion.

Claims

1. An agent for suppressing decline in kidney function, comprising a substance that promotes ammonia excretion in the body.

2. 2. The agent for suppressing renal function decline according to claim 1, wherein the substance having an ammonia excretion promoting effect in vivo is lactulose, ornithine, sodium benzoate, or a derivative thereof, or any combination thereof.

3. 3. The renal function decline suppressant according to claim 1 or 2, wherein the renal function decline is selected from renal function decline in the early nephropathy stage (stage 2), renal function decline in the overt nephropathy stage (stage 3A), and renal function decline in the renal failure stage (stage 4).

4. Renal function decline is classified as G2, G3a, G3b, or G4 (GFR value of 15 mL / min / 1.73 m 2 or more, 90 mL / min / 1.73 m 2 3. The agent for suppressing renal function decline according to claim 1 or 2, wherein the amount of renal function decline is less than 100%.

5. A pharmaceutical composition for treating a disease accompanied by decreased renal function, comprising a substance having an ammonia excretion promoting effect in vivo.

6. 6. The pharmaceutical composition according to claim 5, wherein the substance having an ammonia excretion promoting effect in vivo is lactulose, ornithine, sodium benzoate, or a derivative thereof, or any combination thereof.

7. 7. The pharmaceutical composition of claim 5 or 6, wherein the renal function decline is selected from renal function decline in the early nephropathy stage (stage 2), renal function decline in the overt nephropathy stage (stage 3A), and renal failure stage (stage 4).

8. Renal function decline is classified as G2, G3a, G3b, or G4 (GFR value of 15 mL / min / 1.73 m 2 or more, 90 mL / min / 1.73 m 2 The pharmaceutical composition according to claim 5 or 6, wherein the pharmaceutical composition is in a state of less than 100 mg / kg.

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