Renal markers and their use

Abstract

This invention provides biomarkers for diagnosing kidney function in cats or dogs, and diagnostic methods utilizing these biomarkers. [Solution] A method for diagnosing the renal function of a cat or dog, wherein the diagnosis is based on the analysis of metabolites in the urine, the analysis of which is a decrease in at least one selected from the group consisting of 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, or an increase in at least one selected from the group consisting of dimethyl sulfone, dimethylamine, methanol, taurine, 2-oxoglutarate, indole-3-acetate, N-phenylacetylglycine, and urocanate.

Description

Technical Field

[0001] The present invention relates to a kidney disease marker and its use.

Background Art

[0002] Kidney diseases in cats are diseases that occupy the upper ranks of the causes of death. Therefore, it is important to accurately diagnose with a test method that can evaluate renal dysfunction at an early stage and understand the disease state. However, there are few reports on research in this field in veterinary clinical practice. Currently, in actual clinical settings, it is classified based on plasma creatinine and SDMA (symmetric dimethylarginine) according to the criteria for staging chronic kidney disease by the International Renal Interest Society (IRIS) (Non-Patent Document 1). At a stage where the kidney disease has progressed to a certain extent, breeders often visit the hospital due to polydipsia and polyuria.

[0003] Regarding the diagnosis of kidney disease, Patent Documents 1 and 2 describe a method for determining whether an animal is suffering from kidney disease, which includes measuring β-aminoisobutyric acid (BAIB) in a urine or blood sample from the animal, and determining the kidney disease based on the concentration of BAIB in the sample. Patent Document 3 describes a method for analyzing the urine of a subject suspected of having renal failure, which includes D-serine and L-serine, D-histidine and L-histidine, D-asparagine and L-asparagine, D-arginine and L-arginine, D-allo-threonine and L-threonine, D-glutamic acid and L-glutamic acid, D-alanine and L-alanine, D-proline and L-proline, D-valine and L-valine, and D-allo-isoleucose. The description includes the steps of: measuring the concentrations of a pair of D- and L-forms of at least one amino acid from the amino acid group consisting of phenyl and L-isoleucine, D-phenylalanine and L-phenylalanine, and D-lysine and L-lysine; calculating a disease state index value indicating the proportion of the D-form from the concentrations of the pair of D- and L-forms of the at least one amino acid; and relating a significant decrease in the disease state index value to the renal failure of the subject.

[0004] Meanwhile, pharmaceutical or food compositions for administration or ingestion to subjects with impaired renal function or those at risk have been investigated. In this regard, Patent Document 4 describes a composition for regulating URAT1 (urate transporter 1) activity, characterized by containing taurine as an active ingredient. This document explains that URAT1, which is expressed in the kidneys, is known to be involved in the transport of factors that play important roles in the body, such as uric acid, nicotinic acid, and succinic acid, as well as drugs such as salicylic acid and indomethacin, and that it is suggested that regulating its activity may allow for good regulation of bodily functions. Patent Document 5 also describes an amino acid composition or amino acid solution for improving renal dysfunction, characterized by comprising threonine, proline, glycine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, aspartic acid, serine, glutamic acid, alanine, methionine, tryptophan, histidine, and arginine. Furthermore, Patent Document 6 describes a composition suitable for improving the kidney function of animals, which contains one or more antioxidants in amounts that improve kidney function, and a smaller amount of protein and / or phosphorus compared to the maximum amounts of protein and phosphorus typically recommended for healthy animals of the same species or lineage. Preferred antioxidants described include β-carotene, selenium, coenzyme Q10 (ubiquinone), luetin, tocotrienol, soy isoflavone, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine, vitamin E, vitamin C, α-lipoic acid, and L-carnitine.

[0005] On the other hand, based on experimental results in spontaneously hypertensive, stroke-prone rats, the present inventors have developed a renal function maintenance and protection agent characterized by containing a triglyceride of docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA), or a mixture of a triglyceride of docosahexaenoic acid (DHA) and a triglyceride of eicosapentaenoic acid (EPA), as an active ingredient for maintaining and protecting renal function (Patent Document 7). In addition, they have developed a feline renal function protection agent containing an ester of docosahexaenoic acid (DHA) (Patent Document 8). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication WO2016 / 176691 (Japanese Patent Publication 2018-515761, Patent No. 6923449) [Patent Document 2] Japanese Patent Publication No. 2021-181994 (Divisional application of Patent Document 1) [Patent Document 3] Patent No. 5740523 [Patent Document 4] International release WO2013 / 018706 [Patent Document 5] Japanese Patent Publication No. 2002-275059 [Patent Document 6] Japanese Patent Publication No. 2015-7093 [Patent Document 7] Japanese Patent Publication No. 2020-2125 [Patent Document 8] Japanese Patent Publication No. 2022-26979 [Non-patent literature]

[0007] [Non-Patent Document 1] IRIS Kidney - Guidelines - IRIS Staging of CKD (http: / / www.iris-kidney.com / guidelines / staging.html) [Overview of the project] [Problems that the invention aims to solve]

[0008] Plasma creatinine levels are influenced by factors other than kidney function, such as sex, age, and muscle mass, and are known to have a so-called "blind area" where mild renal impairment may not show abnormal values. Conversely, if plasma creatinine levels are elevated due to muscle mass or exercise, renal function may be underestimated. On the other hand, urea nitrogen levels are not specific to renal function and are influenced by many factors, including protein intake.

[0009] While urinary protein and urinary albumin tests are performed, urinary protein can be positive regardless of the presence or absence of kidney disease. Furthermore, although homology is observed in urinary albumin testing because it uses a method for measuring human urinary albumin, its measurement sensitivity is low. Therefore, a biomarker with high sensitivity and specificity that can evaluate renal function abnormalities at an early stage is desired. [Means for solving the problem]

[0010] Metabolomics is a method for comprehensively measuring the metabolome, which is the totality of metabolites, as one of the biomarker discovery methods. While it is widely used in preclinical and clinical settings for human drug development, metabolome analysis in veterinary medicine is still relatively rare. In this study, we attempted to predict metabolites specific to renal disease and plasma creatinine levels from urinary metabolites by measuring urinary metabolites using nuclear magnetic resonance (NMR) in the urine of healthy and renal disease-affected cats.

[0011] The present invention provides the following: [1] A method for diagnosing kidney function in cats or dogs: This is a diagnostic method based on the analysis of metabolites in urine. Analysis of metabolites A reduction of at least one selected from the group consisting of 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, or A method comprising increasing at least one substance selected from the group consisting of dimethyl sulfone, dimethylamine, methanol, taurine, 2-oxoglutarate, indole-3-acetate, N-phenylacetylglycine, and urocanate. [2] The method according to 1, wherein the analysis of metabolites includes the analysis of at least one selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, and urocanate. [3] The method described in 1 or 2 for the diagnosis of cats or dogs with chronic kidney disease of stages 1 to 4 according to the International Renal Interest Society (IRIS) diagnostic criteria. [4] A method for predicting the plasma creatinine level of a subject: This method predicts based on the analysis of metabolites in urine. A method for analyzing metabolites, comprising two or more analyses selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate. [5] The prediction method described in 4, wherein the analysis of metabolites is 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate. [6] A method for diagnosing kidney function in cats or dogs: This is a diagnostic method based on the analysis of metabolites in plasma. Analysis of metabolites N,N-Dimethylglycine, Proline, Urea, 5-Hydroxylysine, Asparagine, 5-Oxo-2-tetrahydrofurancarboxylic acid, Azelaic acid, Carboxymethyllysine, Cysteine, Cystine, Glucaric acid, Hydroxyproline, Methionine sulfoxide, N6-Acetyllysine, Pelargonic acid, Pimelic acid, Pipecolic acid A decrease in at least one selected from the group consisting of (acid), stachydrine, sulfotyrosine, taurocholic acid, terephthalic acid, and γ-glutamylornithine, or 1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 2-Hydroxyoctanoic acid, 3-Methoxytyrosine, 3-Ureidoisobutyric acid, 3-Ureidopropionic acid, 7-Methylguanine, Allantoic acid, Argininosuccinic acid, Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Cystathionine, Ethanolamine phosphate Phosphate, gluconic acid, gluconolactone, glycylaspartic acid, indole-3-acetic acid, isethionic acid, isocitric acid, isovalerylcarnitine, N-acetylaspartic acid, N-acetylglycine, N-acetylputrescine, N1-methylguanosine, N6,N6,N6-trimethyllysine, nalidixic acid, phenaceturic acid A method comprising increasing at least one substance selected from the group consisting of acid, putrescine, pyridoxal, and uridine. [7] A method for diagnosing the kidney function of a cat or dog: A method for diagnosis based on the analysis of metabolites in plasma, at least one decrease selected from the group consisting of proline and γ-glutamyl ornithine, or at least one increase selected from the group consisting of asymmetric dimethyl-L-arginine (ADMA), isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, and 3-ureidopropionic acid. [8] The method according to 6 or 7, wherein the metabolite analysis includes at least one analysis selected from the group consisting of isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, 3-ureidopropionic acid, and proline. [9] The method according to any one of 6 to 8 for the diagnosis of cats or dogs at stage 1 or 2 of the diagnostic criteria for chronic kidney disease of IRIS.

[10] A pharmaceutical or food composition containing any one selected from the group consisting of taurine, proline, and γ-glutamyl ornithine for ingestion by a subject with reduced renal function or at risk thereof.

[11] The composition according to 10, wherein the subject is a cat or a dog.

[12] The composition according to 10 or 11, wherein the subject is a cat or a dog at stage 1 or 2 of the diagnostic criteria for chronic kidney disease of IRIS.

[13] The composition according to any one of 10 to 12, wherein the composition further includes at least one selected from the group consisting of docosahexaenoic acid (DHA)-bound lipids and eicosapentaenoic acid (EPA)-bound lipids.

[14] The composition according to any one of 10 to 13 for ingestion by a subject diagnosed with renal function by the method according to any one of 1 to 9.

[15] A pharmaceutical or food composition to be administered to a subject diagnosed with renal function by any one of the methods described in items 1 to 9, comprising at least one selected from the group consisting of docosahexaenoic acid (DHA) bound lipids and eicosapentaenoic acid (EPA) bound lipids.

[16] The composition according to any one of claims 13 to 15, comprising at least one selected from the group consisting of DHA-binding lipids and EPA-binding lipids as fish oil. [Brief explanation of the drawing]

[0012] [Figure 1] Prediction of plasma creatinine from urinary metabolites using orthogonal partial least squares regression (full dataset and variable importance (VIP) for prediction) (n=45, 40 metabolites) R2X = 0.383, R2Y = 0.935, Q2Y = 0.51 [Figure 2] Relationship between observed and predicted values ​​in multiple regression analysis using 5 metabolites: Multiple R-squared: 0.7806, Adjusted R-squared: 0.7525, F-statistic: 27.75, p-value: 7.14E-12 [Modes for carrying out the invention]

[0013] [Analysis of metabolites in urine] This invention relates to a method for diagnosing renal function in cats or dogs, and a marker used therefor. The method of this invention uses metabolites in the urine as markers and diagnoses the renal function of the subject based on the analysis of the markers. Analysis includes detection of the target substance, quantification, and judgment by comparison with reference values ​​(such as decrease or increase). A decrease includes values ​​lower than those shown in the healthy control group, and an increase includes values ​​higher than those shown in the healthy control group.

[0014] In one preferred embodiment, the analysis of metabolites is a reduction of at least one selected from the group consisting of 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline.

[0015] In relation to the present invention, when the substance to be analyzed is represented as an anion (salt, ester) (represented by replacing -ic acid with -ate), that substance may be measured as an acid depending on the measurement conditions. That is, in relation to the present invention, anionic substances listed as targets for analysis may be measured as acids depending on the analysis conditions. For example, 2-hydroxyisobutyrate, 2-phenylpropionate, and 3-aminoisobutyrate may be measured as 2-hydroxyisobutyric acid, 2-phenylpropionic acid, and 3-aminoisobutyric acid, respectively. Furthermore, in relation to the present invention, when analyzing 2-hydroxyisobutyrate, it also includes analyzing 2-hydroxyisobutyric acid. The same applies to other substances.

[0016] In another preferred embodiment, the analysis of metabolites is an elevation of at least one selected from the group consisting of dimethyl sulfone, dimethylamine, methanol, taurine, 2-oxoglutarate or 2-oxoglutaric acid, indole-3-acetate or indole-3-acetic acid, N-phenylacetylglycine, and urocanate.

[0017] More specifically, when targeting chronic kidney disease (polycystic kidney disease (PKD) + other chronic kidney disease (CKD)), the analysis of metabolites preferably shows a decrease in any of the following selected from the group consisting of 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, or an increase in any of the following selected from the group consisting of dimethyl sulfone, dimethylamine, methanol, and taurine.

[0018] Furthermore, when targeting PKD, it is preferable that the analysis of metabolites shows a decrease in any of the group selected from 2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline, or an increase in any of the group selected from 2-oxoglutarate, dimethyl sulfone, dimethylamine, indole-3-acetate, methanol, N-phenylacetylglycine, taurine, and urocanate.

[0019] In a particularly preferred embodiment, the analysis of metabolites is at least one analysis selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, and urocanate.

[0020] The method of analyzing the target substance is not particularly limited. Those skilled in the art can collect urine using an appropriate method, remove insoluble matter by centrifugation as needed, and add buffer to prepare a sample for measurement. The sample can be analyzed, for example, by nuclear magnetic resonance (NMR), gas chromatography (GC), high-performance chromatography (HPLC), mass spectrometry (MS), or time-of-flight mass spectrometry (TOF-MS). Furthermore, if appropriate for the specific substance, it can be analyzed by immunological methods. For reference, various biochemical tests, such as urine specific gravity, pH, and UPC (urine protein: creatinine), may be performed on the collected urine sample.

[0021] [Methods for predicting plasma creatinine levels] The present invention also relates to a method for predicting a target plasma creatinine level based on the analysis of metabolites in urine.

[0022] For the purpose of predicting the plasma creatinine level of the target, analysis of metabolites in urine is preferably performed using any of the following selected substances: methanol, 2-hydroxyisobutyrate, N-acetylglycine, tyrosine, indole-3-acetate, formate, urocanate, trigonelline, and lactate, and more preferably using any of the following selected substances: 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate.

[0023] To predict plasma creatinine levels, the inventors selected nine metabolites with the highest variable importance (VIP) from orthogonal partial least squares regression using all experimental data (45 samples, 40 metabolites): Methanol, 2-Hydroxyisobutyrate, N-Acetylglycine, Tyrosine, indole-3-acetate, Formate, Urocanaate, Trigonelline, and Lactate. They then performed multiple regression analysis using stepwise variable selection (decrease method) with p-values. As a result, they obtained the following regression equation using five metabolites: Lactate, Methanol, Urocanaate, 2-Hydroxyisobutyrate, and Trigonelline.

[0024]

number

[0025] In the formula, [Lactate], [Methanol], [Urocanate], [2-Hydroxyisobutyrate], and [Trigonelline] represent the urinary concentration ( / Cre) of each substance. The calculated [Plasma Creatinine] represents the plasma creatinine concentration (mg / dL).

[0026] The method for analyzing the target substance in urine to predict plasma creatinine levels is not particularly limited. Those skilled in the art can perform the analysis as appropriate. An example of such an analysis method is the aforementioned nuclear magnetic resonance (NMR) spectrometer.

[0027] Traditionally, CKD grades have been classified based on plasma creatinine levels obtained from blood samples, but frequent blood sampling is difficult. However, according to the present invention, plasma creatinine levels can be predicted from urinary metabolites. This facilitates diagnosis by pet owners and is useful for selecting therapeutic or complete nutritional diets based on the diagnostic results.

[0028] [Analysis of metabolites in plasma] The present invention also relates to a method for diagnosing the renal function of a subject, which is a method for diagnosing based on the analysis of metabolites in plasma. The analysis of metabolites in plasma can be performed as an analysis of metabolites in blood or serum. The following explanation will use the case of analyzing metabolites in plasma as an example.

[0029] In a preferred embodiment, for the purpose of diagnosing the renal function of the subject, analysis of metabolites in plasma is performed, including N,N-dimethylglycine, proline, urea, 5-hydroxylysine, asparagine, 5-oxo-2-tetrahydrofurancarboxylic acid, azelaic acid, carboxymethyllysine, cysteine, cystine, glucaric acid, hydroxyproline, methionine sulfoxide, N6-acetyllysine, pelargonic acid, pimelic acid, and pipecolic acid. This is a reduction of at least one substance selected from the group consisting of (1) acid, stachydrine, sulfotyrosine, taurocholic acid, terephthalic acid, and γ-glutamylornithine.

[0030] In another preferred embodiment, analysis of metabolites in plasma includes 1-methyladenosine, 2-aminobutyric acid, 2-hydroxybutyric acid, 2-hydroxyoctanoic acid, 3-methoxytyrosine, 3-ureidoisobutyric acid, 3-ureidopropionic acid, 7-methylguanine, allantoic acid, argininosuccinic acid, ascorbate 2-sulfate, carnosine, and cis-aconitic acid. (acid), cystathionine, ethanolamine phosphate, gluconic acid, gluconolactone, glycylaspartic acid, indole-3-acetic acid, isethionic acid, isocitric acid, isovalerylcarnitine, N-acetylaspartic acid, N-acetylglycine, N-acetylputrescine, N1-methylguanosine, N6,N6,N6-trimethyllysine, nalidixic acid An increase in at least one substance selected from the group consisting of (acid), phenaceturic acid, putrescine, pyridoxal, and uridine.

[0031] In another preferred embodiment, analysis of metabolites in plasma is a reduction of at least one selected from the group consisting of proline and γ-glutamylornithine.

[0032] In another preferred embodiment, analysis of metabolites in plasma is performed to identify an elevation of at least one selected from the group consisting of asymmetric dimethyl-L-arginine (ADMA), isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, and 3-ureidopropionic acid.

[0033] In one particularly preferred embodiment, the analysis of metabolites in plasma is at least one analysis selected from the group consisting of isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, 3-ureidopropionic acid, and proline.

[0034] The method of analyzing the target substance is not particularly limited. A person skilled in the art can collect blood using an appropriate method, add anticoagulants or buffers as needed, and centrifuge to remove blood cell components, etc., to obtain plasma, which can then be used for measurement. As mentioned above, the measurement can be performed using nuclear magnetic resonance (NMR), gas chromatography (GC), high-performance chromatography (HPLC), mass spectrometry (MS), or time-of-flight mass spectrometry (TOFMS). Furthermore, if appropriate for the specific substance, it can be analyzed using immunological methods.

[0035] [Pharmaceutical or food composition] The present invention also relates to pharmaceutical or food compositions for administration to subjects with impaired renal function or at risk thereof. Pharmaceuticals include veterinary pharmaceuticals, and food compositions include pet food, and pet food includes complete and balanced diets, snacks, therapeutic diets, and other purpose diets (nutritional supplements, calorie supplements, animal supplements).

[0036] <Active ingredients> The active ingredients of the composition are selected from the group consisting of taurine, proline, gamma-glutamyl ornithine, docosahexaenoic acid (DHA) or its binding lipids, and eicosapentaenoic acid (EPA) or its binding lipids. There may be one active ingredient or two or more.

[0037] In one preferred embodiment, the active ingredient is selected from the group consisting of taurine, proline, and γ-glutamylornithine.

[0038] In another preferred embodiment, the active ingredient is selected from the group consisting of DHA-binding lipids and EPA-binding lipids, with DHA-binding lipids being more preferred. If the DHA-binding lipid is a glyceride ester, it is sufficient that DHA is bound to any of the α, β, or γ positions of the glyceride. That is, it may be mono-, di-, or tri-glycerides of DHA, or a mixed glyceride ester of DHA and other fatty acids. In this case, there are no particular restrictions on the DHA content in the glyceride ester, but for example, the ratio of DHA to the total fatty acids constituting the glyceride ester can be, for example, 20-99% by mass, 50-99% by mass, 60-95% by mass, or 65-90% by mass. The same applies to EPA-binding lipids.

[0039] The raw materials for DHA-binding lipids and EPA-binding lipids are not particularly limited and can be obtained from fish oil, marine mammal oil, algal-derived lipids, and crustacean-derived lipids. In a preferred embodiment, the composition contains DHA-binding lipids and EPA-binding lipids as fish oil. The type of fish is not particularly limited, but it is preferably a fish of the Salmonidae, Herringidae, or Gadidae families. The Salmonidae family includes the genera Salmonidae, Salmonidae, Salmonidae, and Sakhalin, but it is more preferable that the fish be of the Salmonidae or Salmonidae genera. Examples of Salmonidae fish include chum salmon, coho salmon (silver salmon), pink salmon, cherry salmon, masu salmon, Taiwanese trout, Satsuki trout, amago trout, Biwa trout (steelhead trout), Chinook salmon (king salmon), sockeye salmon, kokanee salmon, and kunimasu trout. Examples of fish in the genus Salmon include Atlantic salmon and brown trout. The family Clupeidae includes the subfamilies Clupeinae, Gizzard Shadinae, and Ehiravinae, but it is preferable to select fish from the subfamily Clupeinae and the genus Clupeinae. Examples of fish in the genus Clupeinae include herring and Atlantic herring. The family Gadidae includes the genera Gadus, Micromesistius, Gadiculus, Trisopterus, Microgadus, Eleginus, Merlangius, Melanogrammus, Pollachius, Boreogadus, and Arctogadus, but it is preferable to select fish from the genus Cod. Examples of fish in the genus Cod include Alaska pollock, Pacific cod, Pacific pollock, and Greenland cod. The fish roe may be wild-caught or farmed.

[0040] The fish oil is preferably derived from fish roe. Examples of fish roe include salmon roe, ikura, herring roe, and cod roe.

[0041] <Application> The composition can be used for the treatment of kidney disease. It can also be used to improve the kidney function of the target patient. Treatment includes reducing the risk of onset, prevention, therapy, and suppression or cessation of progression. Therapy includes symptomatic treatment and curative treatment. Kidney disease includes chronic kidney disease and polycystic kidney disease.

[0042] The composition can be administered to subjects diagnosed with impaired renal function using the diagnostic method described above.

[0043] Furthermore, the composition can be used to improve conditions in subjects where at least one of the substances selected from the group consisting of isethionic acid, 2-aminobutyric acid, 2-hydroxybutyric acid, and 3-ureidopropionic acid is present in low levels in the plasma.

[0044] <Dose, content> The amount of the composition of the present invention ingested should be any amount that produces the desired effect. The amount ingested can be appropriately determined considering various factors such as the age, weight, and symptoms of the target individual.

[0045] The amount of active ingredients in a composition can be appropriately determined depending on the form of the composition. For example, if the active ingredients are any of the group consisting of taurine, proline, and γ-glutamylornithine, the amount of active ingredients per composition (if there are multiple active ingredients, this refers to the total amount of multiple ingredients) can be 0.1% or more on a dry weight basis, preferably 0.3% or more, and may be, for example, 0.6% or more or 1.1% or more. The upper limit of the active ingredients can be 10% or less on a dry weight basis, 9% or less, 8% or less, 7% or less, or 6% or less, regardless of the lower limit. In relation to the present invention, % means mass % unless otherwise specified.

[0046] If the active ingredient is one of the group consisting of taurine, proline, and γ-glutamylornithine, the daily intake of the composition may be 10 mg or more, preferably 30 mg or more, more preferably 60 mg or more, and even more preferably 100 mg or more, as the amount of the active ingredient (if there are multiple active ingredients, this refers to the total amount of multiple ingredients). The upper limit of the daily intake of the active ingredient may be 10 g or less, 5 g or less, 1 g or less, 500 mg or less, 400 mg or less, 300 mg or less, or 200 mg or less, regardless of the lower limit.

[0047] Intake may be once a day, or multiple times a day, for example, 2 to 10 times. The amount of active ingredient per dose may be, for example, 1 mg or more, preferably 3 mg or more, more preferably 6 mg or more, and even more preferably 11 mg or more. The upper limit of the amount of active ingredient per dose may be 10 g or less, 10 g or less, 5 g or less, 1 g or less, 500 mg or less, 400 mg or less, 300 mg or less, or 200 mg or less, regardless of the lower limit.

[0048] When the active ingredient is a DHA-bound lipid, the amount of DHA can be 25 mg / kg or more, preferably 50 mg / kg or more, more preferably 100 mg / kg or more, and even more preferably 200 mg / kg or more. The upper limit of the active ingredient per day can be 5000 mg / kg or less, 2500 mg / kg or less, 2000 mg or less, 1000 mg / kg or less, 750 mg / kg or less, 500 mg / kg or less, or 300 mg / kg or less, regardless of the lower limit.

[0049] <Other ingredients, additives> The composition may contain other nutrients and functional ingredients that are acceptable as pharmaceuticals or foods. Examples of such ingredients include lipids (e.g., milk fat, vegetable oils, medium-chain fatty acid-containing oils), proteins, amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), carbohydrates, vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid, and nicotinic acid), electrolytes (e.g., sodium, potassium, calcium, magnesium), minerals (e.g., copper, zinc, iron, cobalt, manganese), dietary fiber, antibiotics, etc.

[0050] The composition may also further contain additives that are permitted as pharmaceuticals or food products. Examples of such additives include inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coatings, colorants, preservatives, buffers, pH adjusters, emulsifiers, stabilizers, sweeteners, antioxidants, flavorings, and acidulants.

[0051] <Dosage form / Form> The composition may be prepared in any form, such as solid, liquid, mixture, suspension, powder, granules, paste, jelly, gel, or capsule. It can also be in the form of granules, powder, paste, or concentrated liquid for administration mixed with beverages or food. Furthermore, it can be in any dosage form suitable for oral administration, such as solid preparations like tablets, granules, powders, pills, or capsules; liquid preparations like liquids, suspensions, or syrups; gels, aerosols, etc. It can also be in any form, such as dry, soft-dry, semi-moist, or wet, as well as canned, aluminum tray, or retort pouch.

[0052] <Manufacturing method, display, etc.> In the manufacture of a composition, the stage at which the active ingredient is added can be selected as appropriate. The stage of addition is not particularly limited as long as it does not significantly impair the properties of the active ingredient. For example, the active ingredient can be mixed with the raw materials. Alternatively, the active ingredient can be added at the final stage of manufacturing to produce a composition containing the active ingredient.

[0053] The composition of the present invention may be labeled with its intended use (purpose) and may also be labeled as recommended for administration to a specific target. The labeling may be direct or indirect. Examples of direct labeling include inscriptions on tangible objects such as the product itself, packaging, containers, labels, and tags. Examples of indirect labeling include advertising and promotional activities by place or means, such as websites, in-store displays, brochures, exhibitions, seminars such as media seminars, books, newspapers, magazines, television, radio, mailings, emails, and audio recordings.

[0054] [others] In relation to the present invention, the term "subjects" includes cats and dogs, preferably cats, unless otherwise specified. The breeds of cats and dogs are not limited. The subjects include healthy individuals at risk of kidney disease. The present invention is suitable for cats or dogs with IRIS chronic kidney disease at stages 1, 2, 3, or 4, and may be suitable for cats or dogs at stage 1 or 2.

[0055] In relation to the present invention, when referring to amino acids, they may be L-forms or D-forms, but unless otherwise specified, L-forms are preferred.

[0056] In relation to the present invention, the term "ingestion" is used not only to mean ingesting a food composition, but also to mean administering a pharmaceutical composition, and the term "administration" is used not only when administering a pharmaceutical composition, but also when ingesting a food composition. [Examples]

[0057] [Analysis of cat urine] I. Materials and Methods I-1. Subject The study included 10 healthy cats diagnosed by a veterinary nephrologist, 19 cats with polycystic kidney disease (PKD), 13 cats with other chronic kidney disease (CKD), and 3 cats with other illnesses. The PKD and CKD groups were combined to form a total of 32 cats with chronic kidney disease (CKD+PKD). Healthy cats No. 7 and 9 both showed glomerular filtration rates within the reference range and were individuals with high muscle mass.

[0058] I-2. Sample Preparation Blood samples were collected using vacuum blood collection tubes containing EDTA-2Na, and plasma was obtained by centrifugation (2,000 × g, 4 °C, 10 minutes). The obtained plasma was then subjected to biochemical tests (creatinine). Urine samples were collected by cystopuncture, and biochemical tests (urine specific gravity, pH, UPC (urine protein: creatinine)) were performed. Some samples were frozen and stored at -80°C until NMR measurement. The frozen urine samples were thawed at 0-4°C and centrifuged (2,000 × g, 4°C, 10 minutes). To the resulting supernatant (200 μL), 400 μL of 0.2 M phosphate buffer (adjusted to pH 7.4 with sodium dihydrogen phosphate and disodium hydrogen phosphate, containing 10% (v / v) heavy water and 3 mM 3-trimethylsilyl[2,2,3,3,-2 d4] sodium propionate (TSP) as an internal standard (final concentration: 1 mM)) was added and mixed, followed by centrifugation (2,000 × g, 4°C, 10 minutes). The resulting supernatant (550 μL) was transferred to an NMR tube and used for measurement.

[0059] I-3. NMR measurement A nuclear magnetic resonance (NMR) spectrometer, JNM-ECZ (500 MHz, JEOL Ltd., Akishima, Tokyo), was used. The measurement conditions were as follows: Measurement temperature: 298 K, Observation center: 4.665 ppm, Measurement range: 15 ppm, Data points: 32,768, Number of integrations: 128, Delay time: 5 seconds / scan, Pulse flip angle: 90°. In addition, the light water signal was eliminated by pre-saturation.

[0060] I-4. Analysis Each measured 1 The 1H-NMR spectrum was analyzed using NMR Suite (ver. 7.4) (Chenomx, Toronto, Canada) to estimate urinary metabolites. Metabolite concentrations were then determined based on the concentration of the internal standard sample (TSP). Since the sample was a spot urine sample obtained by bladder puncture, values ​​corrected for urinary creatinine were used.

[0061] I-5. Statistical analysis For comparisons between groups, an independent Student's t-test was performed. Orthogonal partial least square regression (OPLSR) was analyzed using R (ver.3.6.1) and RStudio (ver.1.1.463). As a preprocessing step, normalization was performed by converting the mean to 0 and the variance to 1. Furthermore, the OPLSR model equation was validated using leave-one-out. Multiple regression analysis was performed using R (ver.3.4.1) and EZR (ver.1.37).

[0062] II. Results and Discussion II-1. Background information of the cats used in the study Table 1 shows background information regarding the renal function of the cats included in the study.

[0063] [Table 1]

[0064] 1 Seventy metabolites were estimated as feline urinary metabolites using 1H-NMR. Of these, 40 metabolites with high identification confidence (Tables 2 and 4) were selected for analysis. Table 2 compares metabolites from healthy cats (n=10) and cats with chronic kidney disease (CKD+PKD) (n=32), and Table 3 lists metabolites that showed significant changes compared to healthy cats. Similarly, Table 4 compares healthy cats and PKD cats, and Table 5 lists metabolites that showed significant changes.

[0065] II-2. Urinary metabolites that distinguish healthy cats from cats with kidney disease In cats with chronic kidney disease (CKD+PKD), six metabolites—2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline—were found to have significantly lower urinary excretion levels compared to healthy cats, while four metabolites—dimethyl sulfone, dimethylamine, methanol, and taurine—were found to have significantly higher urinary excretion levels.

[0066] Furthermore, in PKD, six metabolites—2-hydroxyisobutyrate, 2-phenylpropionate, 3-aminoisobutyrate, adenosine, glycine, and trigonelline—were excreted at significantly low levels, while eight metabolites—2-oxoglutarate, dimethyl sulfone, dimethylamine, indole-3-acetate, methanol, N-phenylacetylglycine, taurine, and urocanate—were excreted at significantly high levels.

[0067] Previous studies (Non-Patent Literature 2) involving humans and rodents have reported significant changes in tryptophan, valine, 3-methyl histidine, glutamine, glycine, homocysteine, phenylalanine, and trigonelline in CKD, and significant changes in allantoin, 2-oxoglutaric acid, citrate, hippuric acid, malic acid, uric acid, fumaric acid, glutamic acid, glutamine, and hypoxanthine in PKD.

[0068] In comparison with these, trigonelline showed consistent behavior across species in cats, while differences in behavior were observed for glycine and 2-oxoglutarate. Furthermore, among the metabolites identified in this study, taurine was excreted at 10 times the rate in cats with chronic kidney disease (PKD and CKD+PKD) compared to healthy cats.

[0069] [Table 2]

[0070] [Table 3]

[0071] [Table 4]

[0072] [Table 5]

[0073] [Table 6]

[0074] II-3. Model equation for predicting renal function from urinary metabolites Next, we attempted to predict plasma creatinine levels from urinary metabolites. Currently, CKD grades are classified based on plasma creatinine levels, but frequent blood sampling is difficult due to cats' agitation. Therefore, if prediction from urinary metabolites is possible, it is expected to lead to self-medication by pet owners and allow them to select appropriate therapeutic or complete nutritional diets.

[0075] Using data from all 45 cats listed in Table 1, we attempted to construct a regression model to predict plasma creatinine levels from urinary metabolites, encompassing a wide range of cats from healthy to those with kidney disease.

[0076] Regression analysis is a method for predicting and explaining a response variable Y using explanatory variables X. PLS regression analysis, a type of regression analysis, allows for a reduction in the number of variables by determining latent variables, which are contracted data, instead of using the explanatory variables directly. For example, in multiple regression analysis, if there are strong correlations between explanatory variables, the variables are likely to influence each other, such as canceling out the predictions made by one variable, making it difficult to create a reliable predictive model. This phenomenon is called multicollinearity, but PLS regression analysis reduces the number of variables used in regression by using latent variables, thereby reducing the risk of multicollinearity. Therefore, it is possible to create a good predictive model using more explanatory variables, making it useful for analyzing multivariate correlations (Non-Patent Literature 3).

[0077] In this study, we constructed a predictive model equation using OPLS, which transforms the statistical space into a Cartesian coordinate system for easier visual understanding. To objectively evaluate the quality of the predictive model, we used the R2Y value, an indicator of the model's linearity, and the Q2Y value, an indicator of its predictive power. A better predictive model is one in which R2Y and Q2Y values ​​are closer to 1. Specifically, an R2Y value of 0.65 or higher is considered sufficient for approximate quantitative prediction, and a Q2Y value of 0.5 or higher is considered a good predictive model (Non-Patent Literature 4).

[0078] Furthermore, by using the VIP value, it is possible to understand the contribution of each explanatory variable to the predictive performance of the predictive model. The VIP value is calculated for each explanatory variable, and the larger the value, the greater its contribution to the predictive performance of the predictive model. Therefore, by using the VIP value as an indicator, it is expected that we can estimate the urinary metabolite components that are considered important for plasma creatinine levels.

[0079] In the model (Figure 1) using all data (45 samples, 40 metabolites), a relatively good model was constructed with R2Y=0.935 and Q2Y=0.51. In the graph, the vertical axis (actual) shows the actual plasma creatinine value, and the horizontal axis (predicted) shows the plasma creatinine value estimated from urinary metabolites, plotted for each sample. If the predicted value and the measured value match, the result will lie on the y=x line.

[0080] From a practical standpoint, a predictive model with fewer variables and easier calculation is required. From the 40 metabolites in the previous model, nine metabolites were selected that had high VIP values ​​while maintaining OPLS predictive ability: Methanol, 2-Hydroxyisobutyrate, N-Acetylglycine, Tyrosine, indole-3-acetate, Formate, Urocanate, Trigonelline, and Lactate. Furthermore, a multiple regression analysis was performed using stepwise variable selection (decrease method) with p-values ​​(Figure 2 and Table 7), resulting in five metabolites remaining and obtaining the following regression equation.

[0081]

number

[0082] The squared multiple correlation coefficient (R²) between predicted and observed values ​​was R² = 0.7806, indicating that the model can explain approximately 78% of plasma creatinine fluctuations. An F-test evaluation of the overall model showed P < 0.001, demonstrating statistically significant validity. Furthermore, the variance expansion factor (VIF) values ​​were less than 5 for each variable, suggesting a low probability of multicollinearity. Therefore, this study demonstrates the construction of a good model equation for predicting plasma creatinine from five urinary metabolites.

[0083] Therefore, by targeting these metabolites, it becomes possible to predict kidney function from urine collected at home using NMR, mass spectrometry, or test strips or pet sheets that exhibit specific color reactions, thus eliminating the need for blood sampling.

[0084] [Table 7]

[0085] III. DHA administration study III-1. Administration of DHA-rich fish oil to healthy cats and cats with PKD Five healthy cats and five cats with PKD were administered 0.45 mL / kg (250 mg / kg as DHA) of DHA-rich fish oil (DHA-RS: manufactured by Maruha Nichiro) directly or mixed with commercially available pet food (paste form) before evening feeding for 28 days. Three of the PKD cats were also administered 3-4 mg / kg of a vasopressin receptor antagonist. During the administration period, water was available at will. Healthy cats were fed 25 g / cat of general complete nutrition food in the morning and evening, while PKD cats were fed 20 g / cat of renal disease therapeutic food. General physical examinations (body weight, BCS, TPR, CRT, tent test), blood tests (CBC, blood smear, biochemical tests, α1AG, SDMA), and urinalysis (urine specific gravity, urine stick, urine sediment, UPC, FE, urinary NAG index) were performed before the start of administration and 28 days after administration.

[0086] General hematological and blood biochemical tests showed normal values. Indicators of renal function, such as serum SDMA (symmetric dimethylarginine), UPC (urine protein: creatinine), and proximal tubular injury marker (urinary NAG index), decreased in all cases without exception, and a significant overall decrease was observed before and after administration (Table 8).

[0087] III-2. Taurine in urine Sample preparation method, 1 For H-NMR measurement and estimation and quantification of urinary metabolites, see 0 [Analysis of cat urine]. See section I. Materials and Methods. Quantitative data were corrected for creatinine. Statistical analysis was performed using R (ver. 3.4.1) and EZR (ver. 1.37), including repeated-measures ANOVA and Tukey-Kramer multiple comparison tests.

[0088] Repeated measures ANOVA in two levels—test animals (healthy and PKD) and DHA administration (at the start of administration and 28 days after administration)—showed a significant interaction. Multiple comparisons across all groups revealed that urinary taurine levels were significantly higher in PKD cats than in healthy cats before administration, but decreased to levels comparable to healthy cats after DHA administration (Table 9). Since taurine is an essential amino acid for cats, we inferred that creating taurine-fortified feed, in addition to the renal protective effect of DHA mentioned above, would be effective.

[0089] [Table 8]

[0090] JPEG2026123244000011.jpg95170

[0091] [Metabolome analysis of feline plasma using CE-TOFMS] I. Materials and Methods I-1. Subject We used 5 healthy cats and 5 cats with polycystic kidney disease (PKD).

[0092] I-2. Sample Preparation Blood was collected using a vacuum blood collection tube containing EDTA-2Na, and the plasma was obtained by centrifugation (2,000 × g, 4 °C, 10 minutes). 30 μL of feline plasma was added to 120 μL of methanol solution prepared to a concentration of 20 μM of internal standard and mixed. 90 μL of Milli-Q water was added and mixed, and the mixture was transferred to an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5 kDa). This was centrifuged (9,100 × g, 4 °C, 120 minutes) and ultrafiltration was performed. The filtrate was allowed to dry, and then dissolved again in Milli-Q water for measurement.

[0093] I-3. CE-TOFMS measurement In this test, measurements in cation mode and anion mode were performed under the following conditions.

[0094] Cationic metabolite (cation mode) device Agilent CE-TOFMS system (Agilent Technologies) Unit 6 Capillary: Fused silica capillary id 50 μm × 80 cm Measurement conditions Run buffer: Cation Buffer Solution (p / n : H3301-1001) Rinse buffer: Cation Buffer Solution (p / n : H3301-1001) Sample injection: Pressure injection 50 mbar, 10 seconds CE voltage: Positive, 30 kV MS ionization: ESI Positive MS capillary voltage: 4,000 V MS scan range: m / z 50-1,000 Sheath liquid: HMT Sheath Liquid (p / n : H3301-1020)

[0095] Anionic metabolite (anion mode) device Agilent CE-TOFMS system (Agilent Technologies), Unit 14. Capillary: Fused silica capillary id 50 μm × 80 cm². Measurement conditions Run buffer: Anion Buffer Solution (p / n : I3302-1023) Rinse buffer: Anion Buffer Solution (p / n : I3302-1023) Sample injection: Pressure injection 50 mbar, 10 seconds CE voltage: Positive, 30 kV MS ionization: ESI Negative MS capillary voltage: 3,500 V MS scan range: m / z 50-1,000 Sheath liquid: HMT Sheath Liquid (p / n : H3301-1020)

[0096] I-4. Analysis Peaks detected by CE-TOFMS were automatically extracted using MasterHands ver.2.19.0.2 (developed by Keio University), an automated integration software, to obtain peaks with a signal-to-noise (S / N) ratio of 3 or higher. Mass-to-charge ratio (m / z), peak area value, and migration time (MT) were then obtained. Metabolites were estimated based on the m / z and MT values ​​of the detected peaks, and relative area values ​​were calculated based on internal standard samples. Unpaired Student's t-tests were performed for comparisons between groups.

[0097] II. Results II-1. Background information of the cats used in the study Table 10 shows background information regarding the renal function of the cats studied.

[0098] [Table 10]

[0099] II-2. Plasma metabolites (relative area values) used to distinguish between healthy cats and cats with kidney disease. Metabolic analysis was performed on feline plasma using CE-TOFMS. Candidate compounds were identified at the 248 (cation 148, anion 100) peak based on the m / z and MT values ​​of substances registered in the HMT metabolite library and the Known-Unknown library (Human Metabolome Technologies, Yamagata). Of the 248 metabolites estimated, those significantly higher in PKD cats compared to healthy cats were: 1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 2-Hydroxyoctanoic acid, 3-Methoxytyrosine, 3-Ureidoisobutyric acid, 3-Ureidopropionic acid, 7-Methylguanine, ADMA, Allantoic acid, Argininosuccinic acid, Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Citrulline, Cystathionine, Ethanolamine phosphate, Gluconic acid, Gluconolactone, Gly-Asp, Indole-3-acetic acid, Isethionic acid, Isocitric acid, Isovalerylcarnitine, Lys, N-Acetylaspartic acid, N-Acetylglycine, N-Acetylputrescine. Thirty-seven metabolites were identified for N1-Methylguanosine, N6,N6,N6-Trimethyllysine, Nalidixic acid, Phenaceturic acid, Putrescine, Pyridoxal, SDMA, Threonic acid, and Uridine (Table 11). Metabolites that showed significantly lower levels included 5-Hydroxylysine, 5-Oxo-2-tetrahydrofurancarboxylic acid, Asn, Azelaic acid, Carboxymethyllysine, Cys, Cystine, Glucaric acid, and Hydroxyproline.Twenty-five metabolites were observed: Methionine sulfoxide, N,N-Dimethylglycine, N6-Acetyllysine, Ornithine, Pelargonic acid, Pimelic acid, Pipecolic acid, Pro, Stachydrine, Sulfotyrosine, Taurocholic acid, Terephthalic acid, Thr, Trigonelline, Urea, and γ-Glu-Ornithine (Table 12). ,

[0100] Previous studies in humans and rodents have reported that in CKD, ADMA, Citrulline, Lysine, N,N-Dimethylglycine, Proline, SDMA, Threonic acid, and Urea levels increase, while Ornithine, Threonine, and Trigonelline levels decrease (Table 13). In comparison, in cats, the behavior of ADMA, Citrulline, Lysine, Ornithine, SDMA, Threonine, Threonic acid, and Trigonelline was consistent across species, while differences were observed in the behavior of N,N-Dimethylglycine, Proline, and Urea.

[0101] [Table 11]

[0102] [Table 12]

[0103] [Table 13]

[0104] III. DHA administration study III-1. Administration of DHA-rich fish oil to healthy cats and cats with PKD Five healthy cats and five cats with PKD were administered 0.45 mL / kg (250 mg / kg as DHA) of DHA-rich fish oil (DHA-RS: Maruha Nichiro) directly or mixed with commercially available pet food (paste form) before their evening feeding for 28 days. Three of the PKD cats were also administered 3-4 mg / kg of a vasopressin receptor antagonist. During the administration period, the cats were allowed free access to water. Healthy cats were fed 25 g / cat of general complete nutrition food in the morning and evening, while PKD cats were fed 20 g / cat of renal disease therapeutic food. General physical examinations (body weight, BCS, TPR, CRT, tent test), blood tests (CBC, blood smear, biochemical tests, α1AG, SDMA), and urinalysis (urine specific gravity, urine stick, urine sediment, UPC, FE, urinary NAG index) were performed before the start of administration and 28 days after administration.

[0105] General hematology tests and blood biochemistry tests showed normal values. Indicators of renal function, such as serum SDMA (symmetric dimethylarginine), UPC (urine protein: creatinine), and proximal tubular injury marker (urinary NAG index), decreased in all cases without exception, and a significant overall decrease was observed before and after administration (Table 14).

[0106] III-2. Plasma metabolites (relative area value) For sample preparation methods, CE-TOFMS measurement, and estimation and semi-quantification of plasma metabolites, see 0 [Analysis of cat urine]. See section I. Materials and Methods. Statistical analysis was performed using R (ver. 3.4.1) and EZR (ver. 1.37), including repeated-measures ANOVA and Tukey-Kramer multiple comparison tests.

[0107] Repeated measures ANOVA was performed on two levels of test animals (healthy and PKD) and DHA administration (at the start of administration and 28 days after administration). Metabolites showing significant interaction were extracted, and multiple comparisons were conducted across all groups. Plasma levels of ADMA, Isethionic acid, SDMA, 2-Aminobutyric acid, 2-Hydroxybutyric acid, and 3-Ureidopropionic acid were significantly higher in PKD cats compared to healthy cats before administration, but decreased to levels comparable to healthy cats after DHA administration (Table 15). Conversely, plasma levels of Proline and γ-Glu-Ornithine were significantly lower in PKD cats compared to healthy cats before administration, but increased to levels comparable to healthy cats after DHA administration (Table 16). In addition to SDMA, a conventional indicator of renal function, the above seven metabolites show promise as novel indicators of renal function. Furthermore, it was inferred that creating feed fortified with Proline and γ-Glu-Ornithine would be effective.

[0108] [Table 14]

[0109] [Table 15]

[0110] IV. Results (Quantitative Values) IV-1. Plasma metabolites (relative area values) that distinguish healthy cats from cats with kidney disease. Quantitative analysis was performed on the target metabolites. The calibration curve was corrected using the peak area of ​​an internal standard substance, and the concentration of each substance was calculated using a 100 μM single calibration dose (internal standard substance 200 μM).

[0111] Quantitative values ​​were calculated for the peaks of 153 substances (94 cations, 59 anions) detected among the target metabolites. Of the 153 estimated metabolites, 26 were found to be significantly higher in PKD cats compared to healthy cats: 1-Methyladenosine, 2-Aminobutyric acid, 2-Hydroxybutyric acid, 3-Ureidopropionic acid, 7-Methylguanine, Allantoic acid, Ascorbate 2-sulfate, Carnosine, cis-Aconitic acid, Citrulline, Cystathionine, Ethanolamine phosphate, Gluconic acid, Gly-Asp, Indole-3-acetic acid, Isethionic acid, Isocitric acid, Lys, N-Acetylaspartic acid, N-Acetylglycine, N-Acetylputrescine, Phenaceturic acid, Putrescine, Pyridoxal, Threonic acid, and Uridine (Table 16). Metabolites that showed significantly lower levels included 5-Hydroxylysine and Asn. Seventeen metabolites were identified: azelaic acid, Cys, Cystine, Hydroxyproline, Methionine sulfoxide, N,N-Dimethylglycine, N6-Acetyllysine, Ornithine, Pelargonic acid, Pimelic acid, Pipecolic acid, Pro, Thr, Trigonelline, and Urea (Table 17).

[0112] [Table 16]

[0113] [Table 17]

[0114] IV-2. DHA administration test (quantitative value) Repeated measures ANOVA in test animals (healthy and PKD) and two levels of DHA administration (at the start of administration and 28 days after administration) revealed that metabolites with significant interaction effects were extracted, and multiple comparisons were performed across all groups. Plasma isethionic acid, 2-Aminobutyric acid, 2-Hydroxybutyric acid, and 3-Ureidopropionic acid were significantly higher in PKD cats compared to healthy cats before administration, but decreased to levels comparable to healthy cats after DHA administration (Table 18). Conversely, plasma proline was significantly lower in PKD cats compared to healthy cats before administration, but increased to levels comparable to healthy cats after DHA administration (Table 18). In addition to SDMA, a conventional indicator of renal function, the above four metabolites are expected to be novel indicators of renal function. Furthermore, it was inferred that creating a proline-fortified feed would be effective.

[0115] [Table 18]

[0116] [Example of product manufactured: Complete and balanced cat food] Tuna 10.00g Soy protein 1.00g Fish extract 1.00g Yeast extract 0.30g Vegetable oil 1.30g Fish oil 1.00g Starches 0.50g Thickener 0.10g Vitamins and minerals 0.70g Amino acids (taurine) 0.03g Water 24.07g Total 40.0g

[0117] The mixture, formulated according to the above composition, is filled into containers, retort-sterilized, and a complete and balanced cat food containing 0.3% taurine on a dry matter basis is produced.

[0118] [References cited in the Examples section] Non-patent document 2: Abbiss H, Maker GL, Trengove RD. Metabolomics Approaches for the Diagnosis and Understanding of Kidney Diseases. Metabolites. 2019 Feb 14;9(2):34. doi: 10.3390 / metabo9020034. PMID: 30769897; PMCID: PMC6410198. Non-patent document 3: Jonsson, P., Gullberg, J., Nordstrom, A., Kusano, M., Kowalczyk, M., Sjostrom, M., Moritz, T.: A strategy for identifying differences in large series of metabolomic samples analyzed by GC / MS, Anal. Chem., 2004, 76, 1738-1745. Non-patent document 4: Eriksson, L. and Johansson, E.: Multi- and megavariate data analysis principles and applications, p. 43-70, 94-97, 105-107, 489-491, Umetrics AB, Umea (2001).

Claims

1. A method for predicting the plasma creatinine level of a subject: This is a prediction method based on the analysis of metabolites in urine. A method for analyzing metabolites, comprising two or more analyses selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate.

2. The prediction method according to claim 1, wherein the analysis of metabolites is the analysis of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate.

3. Analysis of metabolites A step of measuring the urinary concentrations of two or more substances selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate, as well as the urinary concentration of creatinine; A step of calculating the urinary concentration ( / Cre) normalized by the urinary creatinine concentration by dividing two or more urinary concentrations selected from the group consisting of 2-hydroxyisobutyrate, trigonelline, methanol, urocanate, and lactate by the urinary concentration of creatinine; and The process of calculating the target plasma creatinine concentration from the normalized urinary concentration ( / Cre) and the regression equation. A prediction method according to claim 1 or 2, including the following:

4. The aforementioned regression equation is, The prediction method according to claim 3, wherein multiple regression analysis is performed using stepwise variable selection (decrease method) with p-values ​​for the urinary concentrations ( / Cre) of nine metabolites: methanol, 2-hydroxyisobutyrate, N-acetylglycine, tyrosine, indole-3-acetate, formate, urocanate, trigonelline, and lactate, normalized by urinary creatinine concentration, and the resulting regression equation is obtained.

5. The prediction method according to claim 3, wherein the regression equation is the following equation; [Math 1] In the formula, [Lactate], [Methanol], [Urocanate], [2-Hydroxyisobutyrate], and [Trigonelline] represent the urinary concentrations ( / Cre) of lactate, methanol, urocanate, 2-hydroxyisobutyrate, and trigonelline, normalized by urinary creatinine concentration, and [Plasma Creatinine] represents the plasma creatinine concentration (mg / dL).

6. The prediction method according to any one of claims 1 to 5, wherein the subject is a cat or a dog.

7. A prediction method according to claim 6, used for diagnosing the kidney function of a target.

8. A pharmaceutical or food composition comprising any one selected from the group consisting of taurine, proline, and γ-glutamylornithine, for administration to a subject diagnosed with renal function who has impaired renal function or is at risk of developing it, according to the method of claim 7.

9. The composition according to claim 8, wherein the composition further comprises at least one selected from the group consisting of docosahexaenoic acid (DHA)-binding lipids and eicosapentaenoic acid (EPA)-binding lipids.

10. A pharmaceutical or food composition comprising at least one selected from the group consisting of docosahexaenoic acid (DHA)-binding lipids and eicosapentaenoic acid (EPA)-binding lipids, for administration to a subject diagnosed with renal function according to the method of claim 7.

11. The composition according to claim 9 or 10, comprising at least one selected from the group consisting of DHA-binding lipids and EPA-binding lipids as fish oil.

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