Method for assisting in the evaluation of renal pathology, system for evaluating renal pathology, and program for evaluating renal pathology

Abstract

The present invention provides: a method for assisting the evaluation of renal pathological conditions, said method comprising using, as an index, a combination of renal reabsorption and excretion ratios of D-serine and / or D-asparagine in a subject with the blood D-serine and / or D-asparagine levels; a system for evaluating renal pathological conditions; and a program for evaluating renal pathological conditions. The present invention also provides: a method for monitoring renal pathological conditions; and a method for monitoring an effect of treating a renal disease.

Description

[Technical Field]

[0001] The present invention relates to a method for assisting in the evaluation of renal pathology, a system for evaluating renal pathology, and a program for evaluating renal pathology. [Background technology]

[0002] The kidneys are important organs that maintain homeostasis through the excretion and absorption of bodily components. They excrete waste products, regulate blood pressure, and regulate body fluid volume and ions, as well as produce blood and bones. A representative index of renal function is the glomerular filtration rate (GFR). GFR represents the amount of fluid filtered from the blood by the glomerulus per minute, and measurement of inulin clearance is considered the international gold standard. However, measurement of inulin clearance requires continuous infusion of inulin over a two-hour period and multiple urine and blood samples, placing a significant burden on both the subject and the operator. Therefore, in clinical practice, measurement of inulin clearance is limited to limited situations, such as in donors for living-donor kidney transplants, and is substituted by measurement of other markers such as creatinine. Furthermore, inulin clearance is difficult to apply in cases where renal pathology changes rapidly, such as acute kidney injury. The values ​​of many markers are significantly different from the actual glomerular filtration rate, such as the gold standard inulin clearance, which hinders the accurate diagnosis of kidney disease.

[0003] Creatinine is commonly measured in clinical settings as an indicator of renal function. Creatinine is the final metabolite of creatine, which is necessary for muscle contraction. Creatine produced in the liver is taken up by muscle cells, where a portion is metabolized to form creatinine. This creatinine is then transported to the kidney via the blood, filtered by the glomerulus, and excreted in the urine without being reabsorbed by the renal tubules. When glomerular filtration capacity declines, excretion is impaired, and creatinine remains in the blood, resulting in an increase in its value, making it a useful indicator of uremic toxin accumulation and therefore used to evaluate renal function. However, blood creatinine levels do not show clearly abnormal values ​​unless the GFR is reduced by 50% or more, making it an insensitive marker.

[0004] Cystatin C is a protein with a molecular weight of 13.36 kDa that is produced at a constant rate by nucleated cells throughout the body. It is all filtered by the glomerulus, then reabsorbed by the renal tubules and broken down in the kidneys. It is thought to be removed from the blood according to the amount of filtration, and the amount in the blood serves as an indicator of GFR. However, when renal function is severely impaired, the increase in blood cystatin C levels slows, making it difficult to accurately assess renal function in end-stage renal disease.

[0005] As described above, there have been no biomarkers that can adequately meet the clinical demand for accurate measurement of individual patients' renal pathology over a wide range from early to late stages using only blood or samples that can be collected non-invasively, without placing a heavy burden on subjects or patients.

[0006] D-amino acids, which were previously thought to be absent in mammalian organisms, have been found to be present in various tissues and to play a role in physiological functions. It has been shown that the amounts of D-serine, D-alanine, D-proline, D-glutamic acid, and D-aspartic acid in the blood fluctuate in patients with renal failure and correlate with creatinine, making them potential markers of renal failure (Non-Patent Documents 1, 2, 3, and 4). Furthermore, it has been disclosed that amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, D-allothreonine, D-glutamine, D-proline, and D-phenylalanine are used as indicators of the pathological state of kidney disease (Patent Document 1). It has also been disclosed that urinary D-serine, D-histidine, D-asparagine, D-arginine, D-allothreonine, D-glutamic acid, D-alanine, D-proline, D-valine, D-alloisoleucine, D-phenylalanine, and D-lysine fluctuate sharply due to renal damage, and that parameters related to these amino acids are used as pathological indicators of kidney disease (Patent Document 2).In recent years, urinary LFABP, blood NGAL, urinary KIM-1, and the like have been developed as markers for kidney disease, but they are not related to glomerular filtration capacity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2013 / 140785 [Patent Document 2] Patent No. 5740523 [Non-patent literature]

[0008] [Non-Patent Document 1] Fukushima, T. et al., Biol. Pharm. Bull. 18: 1130(1995) [Non-patent document 2] Nagata.Y Viva Origino Vol.18(No.2) (1990) Abstracts of the 15th Academic Conference [Non-patent document 3] Ishida et al., Kitasato Medicine 23:51-62 (1993) [Non-patent document 4] Yong Huang et al. Biol. Pharm. Bull. 21:(2)156-162(1998) Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need for a method to evaluate and determine a subject's renal pathology more broadly and accurately than currently known kidney disease markers. [Means for solving the problem]

[0010] The present inventors focused on the dynamics of filtration, reabsorption, and excretion of D-serine and D-asparagine in the kidney and analyzed the relationship between their excretion rates and renal pathology. They found that new pathological information useful for the evaluation and assessment of renal pathology could be obtained, leading to the present invention.

[0011] Thus, the present invention relates to: [1] A method for assisting in the evaluation of renal pathology using a combination of the reabsorption and excretion rate of D-serine and / or D-asparagine in a subject's kidney and the amount of D-serine and / or D-asparagine in the blood as an indicator. [2] The method of Item 1, wherein the rate is the excretion rate of D-serine (subject D-serine excretion rate) and / or the excretion rate of D-asparagine (subject D-asparagine excretion rate) into the urine of the subject. [3] The method according to Item 2, wherein the D-serine excretion rate and / or the D-asparagine excretion rate are calculated by correcting using a blood- and / or urine-derived correction factor. [4] The method according to Item 3, wherein the correction factor is one or more correction factors selected from the group consisting of glomerular filtration rate and urine volume. [5] The method according to Item 3, wherein the correction factor is one or more correction factors selected from the group consisting of inulin clearance and creatinine clearance. [6] The method according to Item 3, wherein the correction factor is one or more correction factors selected from the group consisting of creatinine levels and L-amino acid levels. [7] The method of claim 3, wherein the correction factor is L-serine and / or L-asparagine. [8] The excretion rate of the D-serine is calculated by the following formula:

number

number

[10] The method according to Item 9, wherein the step of evaluating the renal pathology is to evaluate the subject's kidney disease or risk of developing kidney disease, or to predict the induction or prognosis of kidney disease, when the first subject coordinates are not included in the first criteria.

[11] The method according to Item 10, wherein the kidney disease is chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis, polycystic kidney disease, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[12] The method according to any one of Items 9 to 11, wherein the first criterion is a range of the mean value of the plot of the non-renal disease coordinates ± the standard deviation × coefficient Z.

[13] The method according to Item 12, wherein the coefficient Z is a value between 1.0 and 3.0.

[14] The method according to item 12 or 13, wherein the coefficient Z is 1.96.

[15] A method for assisting in the evaluation of renal pathology based on the relationship between the regression equation calculated by regression analysis of the plot of the non-renal disease coordinates and the target coordinates.

[16] a second coordinate system on which the logarithmically transformed target D-serine excretion rate (target D-serine LN excretion rate) and / or the logarithmically transformed target D-asparagine excretion rate (target D-asparagine LN excretion rate) and the logarithmically transformed blood D-serine amount and / or D-asparagine amount are plotted; a second criterion calculated from the non-renal disease coordinates plotting the logarithmically transformed urinary D-serine excretion rate (D-serine LN excretion rate in non-renal disease subjects) and / or D-asparagine excretion rate (D-asparagine LN excretion rate in non-renal disease subjects) and the logarithmically transformed blood D-serine amount and / or D-asparagine amount in multiple non-renal disease subjects; and evaluating a renal pathology based on the relationship between the second object coordinates and the second standard. The method according to any one of items 2 to 8, comprising:

[17] The method according to Item 16, wherein the step of evaluating the renal pathology is to evaluate the subject's kidney disease or risk of developing kidney disease, or to predict the induction or prognosis of kidney disease, when the second subject coordinates are not included in the second criteria.

[18] The method according to Item 17, wherein the kidney disease is chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis, polycystic kidney disease, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[19] The method according to any one of Items 16 to 18, wherein the second criterion is a range of the mean value of the plot of the non-renal disease coordinates ± the standard deviation × coefficient Z.

[20] The method according to Item 19, wherein the coefficient Z is a value between 1.0 and 3.0.

[21] The method according to item 19 or 20, wherein the coefficient Z is 1.96.

[22] The method according to Item 16, wherein the second criterion is that the distance from the mean value of the plot of the non-renal disease coordinates is 0.6 or less.

[23] A method for assisting in the evaluation of renal pathology based on the relationship between a regression equation calculated from the regression line of a plot of non-renal disease coordinates based on the logarithmically transformed values ​​and target coordinates based on the logarithmically transformed values.

[24] A method for monitoring renal pathology, which comprises measuring the excretion rate of D-serine (target D-serine excretion rate) and / or the excretion rate of D-asparagine (target D-asparagine excretion rate) into a subject's urine and the amount of D-serine and / or D-asparagine in the blood over time, and using the fluctuations in the target D-serine excretion rate and / or the target D-asparagine excretion rate and the amount of D-serine and / or D-asparagine in the blood as indicators.

[25] The method according to Item 24, wherein renal pathology due to chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis, polycystic kidney disease, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome is monitored.

[26] A method for monitoring the therapeutic effect of a renal pathology, comprising measuring the urinary excretion rate of D-serine (target D-serine excretion rate) and / or the excretion rate of D-asparagine (target D-asparagine excretion rate) in urine of a subject with kidney disease before and after therapeutic intervention and the amount of D-serine and / or D-asparagine in the blood over time, and using the fluctuations in the target D-serine excretion rate and / or the target D-asparagine excretion rate and the amount of D-serine and / or D-asparagine in the blood as indicators.

[27] The method according to Item 26, wherein the kidney disease is chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis, polycystic kidney disease, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[28] A method for assisting in the evaluation of renal pathology using the amount of D-serine and / or D-asparagine in the blood of subjects from whom urine cannot be collected as an indicator.

[29] The method according to Item 28, which aids in the evaluation of renal pathology due to chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis, polycystic kidney disease, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[30] A method for assisting in determining whether a subject has systemic lupus erythematosus when the subject's blood D-serine level is 9 nmol / mL or higher.

[31] A system for evaluating a renal pathology, comprising: a memory unit, an input unit, an analysis and measurement unit, a data processing unit, and an output unit, the memory unit stores the threshold value and the formula for calculating the urinary D-serine excretion rate and / or the formula for calculating the D-asparagine excretion rate input from the input unit; the analytical measurement unit quantifies the amount of D-serine and / or D-asparagine in the blood sample and / or urine sample; a data processing unit that calculates the urinary D-serine excretion rate and / or D-asparagine excretion rate based on elements including the quantified D-serine amount and / or D-asparagine amount in the blood sample and / or urine sample and a calculation formula for the D-serine excretion rate and / or a calculation formula for the D-asparagine excretion rate stored in the memory unit; a data processing unit that evaluates a renal pathology based on a comparison between a threshold stored in the storage unit and a combination of the urinary D-serine excretion rate and / or D-asparagine excretion rate and the blood D-serine amount and / or D-asparagine amount; the output unit outputs the evaluation result of the target renal pathology; An evaluation system comprising:

[32] The D-serine excretion rate is calculated by the following formula:

number

number

[33] A program for causing an information processing device including an input unit, an output unit, a data processing unit, and a storage unit to evaluate a renal pathology, the program comprising: a threshold value for evaluating a renal pathology input from an input unit, a calculation formula for the urinary D-serine excretion rate and / or a calculation formula for the D-asparagine excretion rate, and variables required for the calculation are stored in a storage unit; The variables input from the input unit and required for calculating the amount of D-serine and / or D-asparagine in the blood sample and / or urine sample and the D-serine excretion rate and / or D-asparagine excretion rate into urine are stored in a memory unit; causing the data processing unit to call up the formula for calculating the urinary D-serine excretion rate and / or the formula for calculating the D-asparagine excretion rate, which are stored in advance in the storage unit, the amounts of D-serine and / or D-asparagine in the blood sample and / or urine sample, and the variables, which are stored in the storage unit, and to substitute the variables into the formula for calculating the urinary D-serine excretion rate and / or the formula for calculating the D-asparagine excretion rate, thereby calculating the D-serine excretion rate and / or the D-asparagine excretion rate; causing a data processing unit to evaluate a renal pathology based on a comparison of the threshold value stored in a memory unit with a combination of the urinary D-serine excretion rate and / or D-asparagine excretion rate and the blood D-serine amount and / or D-asparagine amount; The output unit outputs the evaluation results of the target kidney pathology. A program including instructions for causing the information processing device to execute the above.

[34] The D-serine excretion rate is calculated using the following formula:

number

number

[0012] The analysis of the renal kinetics (reabsorption and excretion rates) of D-serine and / or D-asparagine of the present invention provides a method for determining the renal pathology of a subject more broadly and accurately than currently known renal disease markers. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the D-serine excretion rate and D-asparagine excretion rate in non-renal disease subjects and their logarithmic values. [Figure 2] FIG. 2 shows the D-serine excretion rate and D-asparagine excretion rate in subjects with renal disease, along with their logarithmic values. [Figure 3] FIG. 3 is a histogram of the logarithm of the D-serine excretion rate calculated from the D-serine and creatinine levels measured in the blood and urine of a subject. [Figure 4]FIG. 4 is a plot of the logarithm of the D-serine amount in blood measured in subjects without renal disease and patients with renal disease and the D-serine excretion rate. [Figure 5] FIG. 5 is a histogram of the logarithm of the D-asparagine excretion rate calculated from the D-asparagine and creatinine levels measured in the blood and urine of subjects. [Figure 6] FIG. 6 is a plot of the logarithm of the D-asparagine levels in blood measured in non-renal subjects and renal disease patients and the D-asparagine excretion rate. [Figure 7] FIG. 7 is a plot of the logarithm of the D-serine amount in blood measured in subjects without renal disease and patients with renal disease and the D-serine excretion rate. [Figure 8] FIG. 8 is a plot of the logarithm of the D-asparagine levels in blood measured in non-renal subjects and renal disease patients and the D-asparagine excretion rate. [Figure 9] Figure 9 is a chart showing the treatment and medication details and progress of a patient with systemic lupus erythematosus. [Figure 10] FIG. 10 is a plot of the amount of D-serine in the blood and the D-serine excretion rate measured over time before and after therapeutic intervention in a patient with systemic lupus erythematosus. [Figure 11] FIG. 11 shows a configuration diagram of the renal pathological condition evaluation system of the present invention. [Figure 12] FIG. 12 is a flowchart showing an example of the operation for evaluating a renal pathological condition according to the program of the present invention. [Figure 13] FIG. 13 is a plot of the amount of D-serine in the blood and the D-serine excretion rate measured in patients diagnosed with kidney disease. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a method for assessing renal pathology by analyzing the kinetics (reabsorption, excretion) of D-serine and / or D-asparagine in the kidney. The present inventors have found that the kinetics (reabsorption, excretion) of D-serine and D-asparagine in the kidney each reflect renal pathology and can be used to assess renal pathology in a subject. Therefore, the present invention may be a method for assessing renal pathology by analyzing the kinetics (reabsorption, excretion) of D-serine in the kidney, a method for assessing renal pathology by analyzing the kinetics (reabsorption, excretion) of D-asparagine in the kidney, or a method for assessing renal pathology by analyzing the kinetics (reabsorption, excretion) of D-serine and D-asparagine in the kidney. Renal pathology can be diagnosed using the results of analyzing the kinetics (reabsorption, excretion) of D-serine or D-asparagine in the kidney, respectively. However, using the results of analyzing the kinetics (reabsorption, excretion) of both D-serine and D-asparagine in the kidney increases the accuracy of the evaluation and also makes it possible to determine false negatives and false positives.

[0015] In this specification, terms such as "first," "second," etc. are used to distinguish one element from another; for example, a first element may be expressed as a second element, and similarly, a second element may be expressed as a first element, without departing from the scope of the present invention.

[0016] In this specification, "urinary D-serine excretion rate of a subject" may be expressed as "subject D-serine excretion rate," and "urinary D-serine excretion rate in a non-renal disease subject" may be expressed as "non-renal disease subject D-serine excretion rate," and these terms have the same meaning when used interchangeably. In this specification, "urinary D-asparagine excretion rate of a subject" may be expressed as "subject D-asparagine excretion rate," and "urinary D-asparagine excretion rate in a non-renal disease subject" may be expressed as "non-renal disease subject D-asparagine excretion rate," and these terms have the same meaning when used interchangeably.

[0017] In this specification, "logarithmically transformed D-serine excretion rate in a subject" may be referred to as "subject D-serine LN excretion rate," and "logarithmically transformed value of urinary D-serine excretion rate in non-renal disease subjects" may be referred to as "non-renal disease subject D-serine LN excretion rate," and these terms have the same meaning when used interchangeably. In this specification, "logarithmically transformed D-asparagine excretion rate in a subject" may be referred to as "subject D-asparagine LN excretion rate," and "logarithmically transformed value of urinary D-asparagine excretion rate in non-renal disease subjects" may be referred to as "non-renal disease subject D-asparagine LN excretion rate," and these terms have the same meaning when used interchangeably.

[0018] As used herein, the term "subject" refers to all mammals, preferably humans, regardless of whether they have kidney disease. As used herein, a "non-renal disease subject" refers to a subject who does not have kidney disease or has never been diagnosed with kidney disease, and is preferably a subject who does not suffer from kidney disease or other diseases that induce kidney damage.

[0019] In one embodiment, the present invention provides a method for assisting in the evaluation of renal pathology, using as an index a combination of the reabsorption and excretion rates of D-serine and / or D-asparagine in a subject's kidneys and the amount of D-serine and / or D-asparagine in blood. The reabsorption and excretion rates of D-serine and D-asparagine can be calculated by quantifying the amounts of D-serine and D-asparagine in blood and urine, respectively. Therefore, in one embodiment, the "reabsorption and excretion rates of D-serine and / or D-asparagine in a subject's kidneys" in the present invention may be the "excretion rate of D-serine into the subject's urine" ("subject's D-serine excretion rate") and / or the "excretion rate of D-asparagine into the subject's urine" ("subject's D-asparagine excretion rate").

[0020] In the present invention, the excretion rate is an index showing the proportion of a target component excreted in urine after passing through the renal tubule's regulatory functions of reabsorption and secretion, relative to the amount filtered through the glomerulus. It can be expressed as a ratio, percentage, or any other unit. Furthermore, a value that eliminates the effects of water reabsorption and concentration can be calculated by correction with a correction factor, and this value is sometimes expressed as fractional excretion (FE). Because urine concentration rates may not be constant, a "correction factor" that corrects for the urinary concentration rate may be used to correct the reabsorption and excretion rates of D-serine and / or D-asparagine in the subject's kidneys. For example, in one embodiment of the present invention, the target D-serine excretion rate and / or target D-asparagine excretion rate may be corrected with a blood- and / or urine-derived correction factor. The excretion rate is most simply expressed as the ratio of the amount of the target component in urine divided by the glomerular filtration rate of the target component, and the calculation may use the glomerular filtration rate obtained from inulin clearance or the like, the actually measured urine volume, or the amount of the target component in the blood and / or urine. The amount of L-amino acids in urine (preferably L-serine and / or L-asparagine) can be used as a urine volume correction factor to calculate the D-amino acid excretion rate. Creatinine clearance calculated from the amount of creatinine in urine or the amount of creatinine in blood can be used as a correction factor; for example, the excretion rate of D-serine is expressed by the following formula. This may also be expressed as a percentage (%) by multiplying it by 100.

number

[0021] Furthermore, for example, the excretion rate of D-asparagine can be expressed as the following formula: This may be multiplied by 100 to express it as a percentage (%).

number

[0022] In kidney disease, the fractional excretion rate of sodium is used to distinguish between dehydration and renal damage. The fractional excretion rate of potassium and urea nitrogen are also used clinically as indicators of pathological condition. Generally, excretion rates are understood based on the principle of homeostasis: a high intake or biosynthesis of a target component increases its urinary excretion rate, whereas a low intake and high biodegradation rate decrease its excretion rate. Therefore, kidney damage and pathological changes, which are responsible for the primary homeostasis of in vivo components, can affect excretion rates. While traditional kidney disease markers, creatinine, are entirely excreted and cystatin C, are entirely reabsorbed, D-serine and D-asparagine are thought to be tightly regulated in the renal tubules, similar to electrolytes, and thus may serve as more sensitive and accurate pathological markers.

[0023] In the present invention, D-serine and D-asparagine used in the analysis are optical isomers of L-serine and D-asparagine, which are proteinogenic amino acids. The amounts of D-serine and D-asparagine are strictly regulated in each tissue and body fluid by metabolic enzymes such as serine racemase and D-amino acid oxidase, as well as transporters. However, when renal damage occurs, the amounts of D-serine and D-asparagine in blood and urine fluctuate.

[0024] In the present invention, the "amount of D-serine and / or D-asparagine in blood or urine" may refer to the amount of D-serine and / or D-asparagine in a specific volume of blood or urine, and may be expressed as a concentration. The amount of D-serine and / or D-asparagine in blood or urine is measured as the amount in a sample obtained by centrifuging, sedimenting, or otherwise pretreating the collected blood or urine for analysis. Therefore, the amount of D-serine and / or D-asparagine in blood or urine can be measured as the amount in a blood sample derived from collected whole blood, serum, plasma, or the like, or in a urine sample derived from whole urine or urine from which solid components, proteins, etc. have been removed. For example, in the case of analysis using HPLC, the amount of D-serine contained in a given volume of blood or urine is represented by a chromatogram, and can be quantified by comparison with a standard or by calibration analysis of peak height, area, shape, and size. The concentrations of D-serine and / or D-asparagine in blood and urine can be measured by comparing them with samples with known concentrations of D-serine and / or D-asparagine, and the concentrations of D-serine and / or D-asparagine in blood and urine can be used as the amounts of D-serine and / or D-asparagine in blood and urine. In addition, in the enzymatic method, the amino acid concentrations can be calculated by quantitative analysis using a calibration curve of a standard product.

[0025] The amounts of D- and L-amino acids, such as D-serine and / or D-asparagine and L-serine and / or L-asparagine, can be measured by any method, such as chiral column chromatography, enzymatic assays, or immunological techniques using monoclonal antibodies that distinguish optical isomers of amino acids. The amounts of D-serine and L-serine in a sample according to the present invention can be measured by any method known to those skilled in the art. For example, chromatographic methods and enzymatic methods (Y. Nagata et al., Clinical Science, 73 (1987), 105. Analytical Biochemistry, 150 (1985), 238., A. D'Aniello et al., Comparative Biochemistry and Physiology Part B, 66 (1980), 319. Journal of Neurochemistry, 29 (1977), 1053., A. Berneman et al., Journal of Microbial & Biochemical Technology, 2 (2010), 139., W. G. Gutheil et al., Analytical Biochemistry, 287 (2000), 196., G. Molla et al., Methods in Molecular Biology, 794 (2012), 273., T. Ito et al., Analytical Biochemistry, 371 (2007), 167., etc.) are used. Ohgusu et al., Analytical Biochemistry, 357 (2006), 15., etc.), gas chromatography (GC) (H. Hasegawa et al., Journal of Mass Spectrometry, 46 (2011), 502., MC Waldhier et al., Analytical and Bioanalytical Chemistry, 394 (2009), 695., A. Hashimoto, T. Nishikawa et al., FEBS Letters, 296 (1992), 33., H. Bruckner and A. Schieber, Biomedical Chromatography, 15 (2001), 166., M. Junge et al., Chirality, 19 (2007), 228., M. C. Waldhier et al., Journal of Chromatography A, 1218 (2011), 4537., etc.), capillary electrophoresis (CE) (H. Miao et al., Analytical Chemistry, 77 (2005), 7190., D. L. Kirschner et al., Analytical Chemistry, 79 (2007), 736., F. Kitagawa, K. Otsuka, Journal of Chromatography B, 879 (2011), 3078., G. Thorsen and J. Bergquist, Journal of Chromatography B, 745 (2000), 389., etc.), high performance liquid chromatography (HPLC) (N. Nimura and T. Kinoshita, Journal of Chromatography, 352 (1986), 169., A. Hashimoto et al., Journal of Chromatography, 582 (1992), 41., H. Bruckner et al., Journal of Chromatography A, 666 (1994), 259., N. Nimura et al., Analytical Biochemistry, 315(2003), 262., C. Muller et al., Journal of Chromatography A, 1324 (2014), 109.,S. Einarsson et al., Analytical Chemistry, 59 (1987), 1191., E. Okuma and H. Abe, Journal of Chromatography B, 660 (1994), 243., Y.Gogami et al., Journal of Chromatography B, 879 (2011), 3259., Y. Nagata et al., Journal of Chromatography, 575 (1992), 147., S. A. Fuchs et al., Clinical Chemistry, 54 (2008), 1443., D. Gordes et al., Amino Acids, 40 (2011), 553., D. Jin et al., Analytical Biochemistry, 269 (1999), 124., J. Z. Min et al., Journal of Chromatography B, 879 (2011), 3220., T. Sakamoto et al., Analytical and Bioanalytical Chemistry, 408 (2016), 517., W. F. Visser et al., Journal of Chromatography A, 1218 (2011), 7130., Y. Xing et al., Analytical and Bioanalytical Chemistry, 408 (2016), 141., K. Imai et al., Biomedical Chromatography, 9 (1995), 106., T. Fukushima et al., Biomedical Chromatography, 9 (1995), 10., R. J. Reischl et al., Journal of Chromatography A, 1218 (2011), 8379., R. J. Reischl and W. Lindner, Journal of Chromatography A, 1269 (2012), 262., S. Karakawa et al., Journal of Pharmaceutical and Biomedical Analysis, 115 (2015), 123., etc.).

[0026] The optical isomer separation and analysis system of the present invention may combine multiple separation and analysis steps. More specifically, the amount of D- / L-amino acids in a sample can be measured by using a method for analyzing optical isomers, which comprises the steps of: passing a sample containing components having optical isomers together with a first liquid as a mobile phase through a first column packing as a stationary phase to separate the components of the sample; individually retaining each of the components of the sample in a multi-loop unit; supplying each of the components of the sample individually retained in the multi-loop unit together with a second liquid as a mobile phase through a flow path to a second column packing having an optically active center as a stationary phase to separate the optical isomers contained in each of the components of the sample; and detecting the optical isomers contained in each of the components of the sample (Japanese Patent No. 4291628). In HPLC analysis, D- and L-amino acids may be derivatized in advance with fluorescent reagents such as o-phthalaldehyde (OPA) or 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), or diastereomerized using N-tert-butyloxycarbonyl-L-cysteine ​​(Boc-L-Cys) (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, Vol. 53, pp. 677-690 (2004)). Alternatively, D-amino acids can be measured by immunological methods using monoclonal antibodies that distinguish optical isomers of amino acids, such as monoclonal antibodies that specifically bind to D-serine, L-serine, D-asparagine, or L-asparagine. Furthermore, when the total amount of D- and L-isomers is used as an indicator, it is not necessary to separate and analyze the D- and L-isomers; amino acids can also be analyzed without distinguishing between D- and L-isomers. In this case, separation and quantification can be performed using enzymatic methods, antibody techniques, GC, CE, or HPLC.

[0027] Blood D-serine and D-asparagine levels correlate more strongly with glomerular filtration rate than the traditional marker creatinine. This is because blood creatinine levels are strongly influenced by muscle mass; they are elevated in athletes, patients with acromegaly, and individuals consuming large amounts of meat, while they are low in patients with neuromuscular diseases (e.g., muscular dystrophy), emaciation, prolonged bed rest, frailty, sarcopenia, locomotive syndrome, anputation, and individuals with protein restriction. Therefore, they do not accurately reflect renal function. In healthy individuals without pathological findings, blood D-serine levels are maintained within a very small range of approximately 1–2% of total serine, whereas levels in urine range from 30–60%. Interestingly, unlike L-serine, which is reabsorbed approximately 99% by the renal tubules, approximately 50–80% of D-serine is excreted. In healthy individuals without pathological findings, the amount of D-asparagine in the blood is maintained within a very small range of approximately 0.1-0.6% of the total asparagine, whereas the amount in the urine ranges from 20-50%. Interestingly, while approximately 99% of L-asparagine is reabsorbed in the renal tubules, approximately 50-80% of D-asparagine is excreted.

[0028] Chiral amino acid metabolomics and multivariate analysis of related parameters (OPLS) have shown that the excretion rates of D-serine and D-asparagine proposed in this study, unlike the amounts of D-serine and D-asparagine in blood, are not correlated with glomerular filtration rate. Because it has been suggested that the reabsorption of the optical isomers of serine and D-asparagine in the renal tubules is strictly controlled, 15 healthy volunteers were recruited as a study population to analyze the excretion rates of D-serine and D-asparagine in non-renal disease subjects to investigate the physiological significance of D-serine and D-asparagine. The study protocol was approved by the ethics committee of the National Institutes of Biomedical Innovation, Health and Nutrition, and written informed consent was obtained from all subjects. The non-renal disease subjects had a mean age of 44, 80% males, a mean height of 1.70 m, a mean weight of 68.9 kg, and a mean BSA of 1.80 m. 2, average BMI22.6kg / m 2 , the mean serum creatinine was 0.75 mg / dL.

[0029] Using the following formulas based on the quantitative analysis of D-serine and D-asparagine in the subjects' blood and urine, the average excretion rate of D-serine was calculated to be 62.76%, with an average logarithm of 4.12. The average excretion rate of D-asparagine was calculated to be 64.12%, with an average logarithm of 4.16 (Figure 1).

number

number

[0030] When the logarithmic data for D-serine were plotted in 6 quantiles, a normal distribution-like shape was observed (Figure 3). A Shapiro-Wilk normality test was performed on this data, yielding a P value of 0.395, supporting the null hypothesis and suggesting a normal distribution. Therefore, the reference values ​​for these non-renal disease subjects were calculated as 42.46-89.66% of the mean ± 1.96 standard deviation, with the logarithmic values ​​ranging from 3.75-4.50. Subjects falling outside this range may be useful for predicting the pathology, risk, or prognosis of kidney disease and renal impairment.

[0031] Furthermore, when the logarithmic data for D-asparagine were plotted in 6 quantiles, a normal distribution-like shape was observed (Figure 5). A Shapiro-Wilk normality test was performed on this data, yielding a value of P = 0.243, supporting the null hypothesis and suggesting a normal distribution. Therefore, the reference values ​​for these non-renal disease subjects were calculated to be 51.65-78.74% of the mean ± 1.96 standard deviation, with logarithmic values ​​ranging from 3.95-4.37. Subjects falling outside this range may be useful for predicting kidney disease, its pathology, risk, or prognosis.

[0032] Because the amounts of D-serine and D-asparagine in the blood are strongly correlated with the glomerular filtration rate, analysis of these levels has been shown to be applicable to the severity classification (G1-5) of chronic kidney disease (CKD) defined in the guidelines of the Japanese Society of Nephrology. However, the D-serine excretion rate, which is analyzed by adding the amount of D-serine in urine, and the D-asparagine excretion rate, which is analyzed by adding the amount of D-asparagine in urine, can assist in the evaluation of renal pathology through completely different mechanisms that are not correlated with the glomerular filtration rate, and are therefore highly clinically useful in differential diagnosis, pathology, and prognosis, which were difficult with conventional markers.

[0033] In one embodiment, the present invention provides a first object coordinate system obtained by plotting the object D-serine excretion rate and / or object D-asparagine excretion rate and the blood D-serine amount and / or D-asparagine amount in the object; The first criterion is calculated from non-renal disease coordinates obtained by plotting the urinary excretion rates of D-serine (D-serine excretion rate in non-renal disease subjects) and / or D-asparagine (D-asparagine excretion rate in non-renal disease subjects) and the blood D-serine and / or D-asparagine levels in multiple non-renal disease subjects. and evaluating a renal pathology based on the relationship between the first object coordinates and the first standard. The present invention may provide a method comprising:

[0034] Therefore, for example, in the first embodiment of the invention, a first object coordinate system on which the object D-serine excretion rate and the blood D-serine level in the object are plotted; The first criterion is calculated from the non-renal disease coordinates plotting the urinary D-serine excretion rate (D-serine excretion rate in non-renal disease subjects) and the blood D-serine level in multiple non-renal disease subjects. and evaluating a renal pathology based on the relationship between the first object coordinates and the first standard. The present invention may provide a method comprising:

[0035] Furthermore, for example, in the second embodiment of the invention, a first object coordinate system obtained by plotting the subject D-asparagine excretion rate and the blood D-asparagine level in the subject; The first criterion is calculated from the non-renal disease coordinates plotting the urinary D-asparagine excretion rate (D-asparagine excretion rate in non-renal disease subjects) and the blood D-asparagine level in multiple non-renal disease subjects. and evaluating a renal pathology based on the relationship between the first object coordinates and the first standard. The present invention may provide a method comprising:

[0036] In addition, the method of the first embodiment and the method of the second embodiment described above may be used in combination to evaluate renal pathology. In this case, not only is the accuracy of the evaluation of renal pathology improved, but false positives and false negatives can also be determined.

[0037] As used herein, the term "first criterion" refers to a criterion calculated from coordinates (referred to as "non-renal disease coordinates") plotting the urinary D-serine excretion rates (D-serine excretion rates in non-renal disease subjects) and / or D-asparagine excretion rates (D-asparagine excretion rates in non-renal disease subjects) and the blood D-serine and / or D-asparagine levels in multiple non-renal disease subjects, and is used to evaluate the renal pathology of a subject. In one embodiment, the first criterion usable in the present invention may be calculated from non-renal disease coordinates plotting the urinary D-serine excretion rates (D-serine excretion rates in non-renal disease subjects) and the blood D-serine levels in multiple non-renal disease subjects. In another embodiment, the first criterion usable in the present invention may be calculated from non-renal disease coordinates plotting the urinary D-asparagine excretion rates (D-asparagine excretion rates in non-renal disease subjects) and the blood D-asparagine levels in multiple non-renal disease subjects. The number of "non-renal disease subjects" used to calculate the first criterion is preferably a number sufficient to calculate a statistically significant criterion, and for example, a number of 3, 5, 10, 15, 20, 30, 50, 100 or more can be used in the present invention.

[0038] As used herein, the term "first object coordinates" refers to coordinates obtained by plotting a target D-serine excretion rate and / or a target D-asparagine excretion rate and a blood D-serine amount and / or a blood D-asparagine amount in a subject for which a renal pathology is being evaluated. For example, in one embodiment, the first object coordinates that can be used in the present invention may be coordinates obtained by plotting a target D-serine excretion rate and a blood D-serine amount in a subject for which a renal pathology is being evaluated. Furthermore, for example, in one embodiment, the first object coordinates that can be used in the present invention may be coordinates obtained by plotting a target D-asparagine excretion rate and a blood D-asparagine amount in a subject for which a renal pathology is being evaluated. In the present invention, the renal pathology in a subject can be evaluated by comparing the first object coordinates with a first standard.

[0039] In one embodiment, the first criterion in the present invention may be the range of the mean value of the plot of non-renal disease coordinates ± standard deviation × coefficient Z. As used herein, "coefficient Z" is a coefficient used to calculate confidence intervals used in statistics, and is preferably, for example, a value between 1.0 and 3.0, more preferably 1.96. In another embodiment, the first criterion is preferably in the range of 0.4 to 0.9.

[0040] In one embodiment, the step of evaluating a renal pathology included in the present invention may be to evaluate a subject's renal disease or risk of developing renal disease, or to predict the induction or prognosis of renal disease, when the first subject coordinate is not included in the first criterion.

[0041] In one embodiment, the kidney disease that can be evaluated in the present invention may be, for example, chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis or polycystic kidney disease, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease or minimal change nephrotic syndrome.

[0042] In another embodiment, the present invention may provide a method for assisting in the evaluation of renal pathology based on the relationship between the regression equation calculated by regression analysis of the plot of non-renal disease coordinates and the target coordinates. The coordinate position and distance between the plot of the analysis target and the regression equation enable evaluation of changes in D-serine and / or D-asparagine kinetics in non-renal disease patients. For example, a shift in the excretion rate axis to the positive side indicates enhanced excretion, whereas a shift to the negative side indicates enhanced reabsorption, and the greater the distance, the greater the degree of renal pathology.

[0043] In another embodiment, the present invention provides a method for producing a composition comprising: a second object coordinate system on which the logarithmically transformed target D-serine excretion rate (target D-serine LN excretion rate) and / or the logarithmically transformed target D-asparagine excretion rate (target D-asparagine LN excretion rate) and the logarithmically transformed blood D-serine amount and / or D-asparagine amount are plotted; a second criterion calculated from the non-renal disease coordinates plotting the logarithmically transformed urinary D-serine excretion rate (D-serine LN excretion rate in non-renal disease subjects) and / or D-asparagine excretion rate (D-asparagine LN excretion rate in non-renal disease subjects) and the logarithmically transformed blood D-serine amount and / or D-asparagine amount in multiple non-renal disease subjects; and evaluating a renal pathology based on the relationship between the second object coordinates and the second standard. The present invention may provide a method comprising:

[0044] Therefore, for example, in the first embodiment of the invention, a second coordinate system on which the logarithmically transformed target D-serine excretion rate (target D-serine LN excretion rate) and the logarithmically transformed blood D-serine amount are plotted; The second criterion was calculated from the non-renal disease coordinates plotting the logarithmically transformed urinary D-serine excretion rate (D-serine LN excretion rate in non-renal disease subjects) and the logarithmically transformed blood D-serine level in multiple non-renal disease subjects. and evaluating a renal pathology based on the relationship between the second object coordinates and the second standard. The present invention may provide a method comprising:

[0045] Furthermore, for example, in a second embodiment of the present invention, a second coordinate system plotting the logarithmically transformed target D-asparagine excretion rate (target D-asparagine LN excretion rate) and the logarithmically transformed blood D-asparagine level; The second criterion is calculated from the non-renal disease coordinates plotting the logarithmically transformed urinary D-asparagine excretion rate (D-asparagine LN excretion rate in non-renal disease subjects) and the logarithmically transformed blood D-asparagine level in multiple non-renal disease subjects. and evaluating a renal pathology based on the relationship between the second object coordinates and the second standard. The present invention may provide a method comprising:

[0046] In addition, the method of the first embodiment and the method of the second embodiment described above may be used in combination to evaluate renal pathology. In this case, not only is the accuracy of the evaluation of renal pathology improved, but false positives and false negatives can also be determined.

[0047] In this specification, the term "logarithmically transformed value" refers to a value obtained by converting a target value into a logarithm. For example, the term may be a value obtained by converting a target value into a natural logarithm, or a value obtained by converting a target value using any base such as common logarithm.

[0048] As used herein, the term "second criterion" refers to a criterion calculated from coordinates (referred to as "non-renal disease coordinates") plotting the logarithmically transformed target D-serine excretion rate (target D-serine LN excretion rate) and / or the logarithmically transformed target D-asparagine excretion rate (target D-asparagine LN excretion rate) and the logarithmically transformed blood D-serine and / or D-asparagine levels, and is used to evaluate the renal pathology of a subject. In one embodiment, the second criterion that can be used in the present invention may be calculated from non-renal disease coordinates plotting the logarithmically transformed target D-serine excretion rate (target D-serine LN excretion rate) and the logarithmically transformed blood D-serine level. In another embodiment, the second criterion that can be used in the present invention may be calculated from non-renal disease coordinates plotting the logarithmically transformed target D-asparagine excretion rate (target D-asparagine LN excretion rate) and the logarithmically transformed blood D-asparagine level. The number of "non-renal disease subjects" used to calculate the second criterion is preferably a number sufficient to calculate a statistically significant criterion, and for example, a number of 3, 5, 10, 15, 20, 30, 50, 100 or more can be used in the present invention.

[0049] In one embodiment, the second criterion that can be used in the present invention may be the range of the mean value of the plot of non-renal disease coordinates ± standard deviation × coefficient Z. In this case, coefficient Z is preferably a value between 1.0 and 3.0, more preferably 1.96. In another embodiment, the second criterion is preferably in the range of 3.5 to 5.0.

[0050] In one embodiment, the second criterion that can be used in the present invention can be a distance of 0.6 or less from the mean value of the plot of non-renal disease coordinates.

[0051] In one embodiment, the step of assessing renal pathology of the present invention may be to assess the subject's renal disease or risk of developing renal disease, or to predict the induction or prognosis of renal disease, when the second subject coordinate is not included in the second criterion.

[0052] In another embodiment, the present invention may be a method for assisting in the evaluation of renal pathology based on the relationship between a regression equation calculated from the regression line of a plot of non-renal disease coordinates based on logarithmically transformed values ​​and target coordinates based on logarithmically transformed values. The coordinate position and distance between the plot of the analysis target and the regression equation enable evaluation of changes in D-serine and / or D-asparagine kinetics in non-renal disease patients. For example, a shift in the excretion rate axis to the positive side indicates increased excretion, whereas a shift to the negative side indicates increased reabsorption, and the greater the distance, the greater the degree of renal pathology.

[0053] Once the pathological condition is determined using the method of the present invention, a treatment plan can be determined based on the diagnosis. Treatment methods can be appropriately selected depending on the pathological condition. For example, the first or second target coordinates may be monitored over time to ensure they fall within the reference range for non-renal subjects (e.g., the range of the first or second standard). Therapeutic interventions include lifestyle improvement, dietary guidance, blood pressure management, anemia management, electrolyte management, uremic toxin management, blood glucose level management, immune management, and lipid management, either independently or in combination. Lifestyle improvement measures include smoking cessation and weight loss to a BMI of less than 25. Dietary guidance includes reducing salt intake and restricting protein intake. Medication may be used to manage blood pressure, anemia, electrolyte management, uremic toxin management, blood glucose level management, immune management, and lipid management. Blood pressure management is typically maintained at 130 / 80 mmHg or less, and antihypertensive drugs may be administered as needed.Examples of antihypertensive drugs include diuretics (thiazide diuretics, e.g., trichlormethiazide, benzylhydrochlorothiazide, hydrochlorothiazide; thiazide-like diuretics, e.g., meticrane, indabamide, tolvamide, mefruside; loop diuretics, e.g., furosemide; potassium-sparing diuretics / aldosterone antagonists, e.g., triamterene, spironolactone, eplerenone, etc.); calcium channel blockers (dihydropyridines, e.g., nifedipine, amlodipine, efonidipine, cilnidipine, nicardipine, nisoldipine, nitrendipine, nilvadipine, barnidipine, felodipine, benidipine, manidipine, azelnidipine, aranidipine; benzothiazepines, diltiazem, etc.); angiotensin-converting enzyme inhibitors (captopril, enalapril, Acela); Pril, delapril, cilazapril, lisinopril, benazepril, imidapril, temocapril, quinapril, trandolapril, belindopril erbumine, etc.), angiotensin receptor antagonists (angiotensin II receptor antagonists, for example, losartan, candesartan, valsartan, telmisartan, olmesartan, irbesartan, azilsartan, etc.), sympatholytic agents (β-blockers, for example, atenolol, bisoprolol, betaxolol, metoprolol, aceptolol, celiprolol, propranolol, nadolol, carteolol, pindolol, nipradilol, amosulalol, arotinolol, carvedilol, labetalol, bevantolol, urapidil, terazosin, brazosin, doxazosin, bunazosin, etc.), etc. may be used. Erythropoietin preparations, iron supplements, HIF-1 inhibitors, etc. are used to treat anemia. Calcium receptor agonists (cinacalcet, etelcalcetide, etc.) and phosphate binders are used as electrolyte regulators. Activated charcoal is used as a uremic toxin binder. Blood glucose levels are controlled to keep Hba1c below 6.9%, and hypoglycemic drugs are administered in some cases.Hypoglycemic drugs include SGLT2 inhibitors (ipragliflozin, dapagliflozin, luseogliflozin, tofogliflozin, canagliflozin, empagliflozin, etc.), DPP4 inhibitors (sitagliptin phosphate, vildagliptin, saxagliptin, alogliptin, linagliptin, teneligliptin, trelagliptin, anagliptin, omarigliptin, etc.), sulfonylureas (tolbutamide, acetohexamide, etc.), and acetaminophen. Treatment options include thiazolidinediones (e.g., thiazolidinedione, thiazolidinedione ... Lipid management involves maintaining LDL-C levels below 120 mg / dL. Medications for treating dyslipidemia, such as statins (rosuvastatin, pitavastatin, atorvastatin, cerivastatin, fluvastatin, simvastatin, pravastatin, lovastatin, mevastatin, etc.), fibrates (clofibrate, bezafibrate, fenofibrate, clinofibrate, etc.), nicotinic acid derivatives (tocholerol nicotinate, nicomol, niceritrol, etc.), cholesterol transporter inhibitors (ezetimibe, etc.), PCSK9 inhibitors (evolocumab, etc.), and EPA preparations, may be used. These medications may be administered as single agents or in combination. Depending on the severity of renal dysfunction, renal replacement therapy, such as peritoneal dialysis, hemodialysis, continuous hemodiafiltration, hemopheresis (plasma exchange, plasma adsorption, etc.), or kidney transplantation, may be used.

[0054] Therefore, in one embodiment, the present invention may be a method for monitoring a renal pathology, which comprises measuring the excretion rate of D-serine (subject D-serine excretion rate) and / or the excretion rate of D-asparagine (subject D-asparagine excretion rate) into the urine of a subject and the amount of D-serine and / or D-asparagine in the blood over time, and using fluctuations in the target D-serine excretion rate and / or the target D-asparagine excretion rate and the amount of D-serine and / or D-asparagine in the blood as indicators. For example, in one embodiment, the present invention may be a method for monitoring a renal pathology by measuring the excretion rate of D-serine into a subject's urine (subject D-serine excretion rate) and the amount of D-serine in the blood over time, and using fluctuations in the target D-serine excretion rate and the amount of D-serine in the blood as indicators. For example, in one embodiment, the present invention may be a method for monitoring a renal pathology by measuring the excretion rate of D-asparagine into a subject's urine (subject D-asparagine excretion rate) and the amount of D-asparagine in the blood over time, and using fluctuations in the target D-asparagine excretion rate and the amount of D-asparagine in the blood as indicators, or may be a method for monitoring a renal pathology by combining both.

[0055] In another embodiment, the present invention may be a method for monitoring the therapeutic effect of a renal pathology, in which the urinary excretion rate of D-serine (target D-serine excretion rate) and / or the excretion rate of D-asparagine (target D-asparagine excretion rate) and the blood D-serine and / or D-asparagine levels of a subject with kidney disease are measured over time before and after therapeutic intervention, and the changes in the target D-serine excretion rate and / or the target D-asparagine excretion rate and the blood D-serine and / or D-asparagine levels are used as indicators. For example, in one embodiment, the present invention may be a method for monitoring the therapeutic effect of a renal pathology by measuring the urinary excretion rate of D-serine (target D-serine excretion rate) and the amount of D-serine in the blood of a subject with renal disease over time before and after therapeutic intervention, and using the fluctuations in the target D-serine excretion rate and the amount of D-serine in the blood as indicators. For example, in one embodiment, the present invention may be a method for monitoring the therapeutic effect of a renal pathology by measuring the urinary excretion rate of D-asparagine (target D-asparagine excretion rate) and the amount of D-asparagine in the blood of a subject with renal disease over time before and after therapeutic intervention, and using the fluctuations in the target D-asparagine excretion rate and the amount of D-asparagine in the blood as indicators, or may be a method for monitoring the therapeutic effect of a renal pathology by combining both.

[0056] The methods of the present invention can be used to evaluate kidney disease in a subject, for example, chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis or polycystic kidney disease, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[0057] In another embodiment, the present invention provides a method for assisting in the evaluation of renal pathology using the blood D-serine and / or D-asparagine levels of a subject from whom urine cannot be collected as an index. As used herein, "a subject from whom urine cannot be collected" refers to, for example, a subject with chronic renal failure or acute renal failure whose renal function is extremely impaired and who is eligible for renal replacement therapy (dialysis, plasma exchange, kidney transplantation, etc.).

[0058] In another embodiment, the present invention provides a method for assisting in determining whether a subject has systemic lupus erythematosus when the D-serine level in the subject's blood is 9 nmol / mL or higher.

[0059] Another aspect of the present invention may relate to a system and a program for executing the above-described method for assisting in the evaluation of renal pathology. FIG. 11 is a configuration diagram of a system for evaluating renal pathology of the present invention. The sample analysis system 10 shown in FIG. 11 is configured to be able to implement the method for assisting in the evaluation of renal pathology of the present invention. Such a sample analysis system 10 includes a memory unit 11, an input unit 12, an analysis / measurement unit 13, a data processing unit 14, and an output unit 15, and is capable of analyzing blood samples and / or urine samples and outputting calculated excretion rates and pathology information.

[0060] More specifically, in the sample analysis system 10 of the present invention, the memory unit 11 stores a combination of the excretion rate and the amount of D-serine and / or D-asparagine in blood samples and urine samples input from the input unit 12, as well as a reference value / pathological condition information correspondence table or graph. The analysis and measurement unit 13 separates and quantifies the D-serine and / or D-asparagine in the blood samples and / or urine samples. The data processing unit 14 determines the pathological condition by substituting the excretion rate and the amount of D-serine and / or D-asparagine in blood calculated from the amount of D-serine and / or D-asparagine into a formula obtained from the reference value / pathological condition information or by reading them out from the correspondence table or graph. The output unit 15 outputs the pathological condition information.

[0061] In a more preferred embodiment, the system for evaluating a renal pathology of the present invention may further include a step in which the memory unit 11 stores a reference value input from the input unit 12, and a step in which the data processing unit 14 compares a combination of the excretion rate calculated from the separated and quantified amounts of D-serine and / or D-asparagine and the blood D-serine and / or D-asparagine amount with the reference value. In this case, if the combination of the D-serine excretion rate and / or D-asparagine excretion rate and the blood D-serine and / or D-asparagine amount is outside the reference range, the output unit 15 outputs an indication of suspected kidney disease.

[0062] The storage unit 11 includes a memory device such as RAM, ROM, or flash memory, a fixed disk device such as a hard disk drive, or a portable storage device such as a flexible disk or optical disk. The storage unit stores data measured by the analysis / measurement unit, data and instructions input from the input unit, results of calculations performed by the data processing unit, computer programs used for various processes in the information processing device, databases, etc. The computer programs may be installed from a computer-readable recording medium such as a CD-ROM or DVD-ROM, or via the Internet. The computer programs are installed in the storage unit using a known setup program or the like. The storage unit stores data on formulas, correspondence tables, or graphs calculated from the relationship between the combination of D-serine excretion rate and blood D-serine amount input in advance from the input unit 12 and the pathological condition. It can also store renal pathological condition classifications based on the excretion rate.

[0063] The input unit 12 is an interface or the like, and also includes an operation unit such as a keyboard, mouse, etc. This allows the input unit to input data measured by the analysis and measurement unit 13, instructions for arithmetic processing to be performed by the data processing unit 14, etc. Furthermore, if the analysis and measurement unit 13 is external, for example, the input unit 12 may include an interface unit, separate from the operation unit, that can input measured data, etc. via a network or storage medium.

[0064] The analytical measurement unit 13 performs a measurement step of D-serine and / or D-asparagine in blood samples and / or urine samples. Therefore, the analytical measurement unit 13 has a configuration that enables separation and measurement of D- and L-isomers of amino acids. Amino acids may be analyzed one by one, or some or all types of amino acids may be analyzed simultaneously. The analytical measurement unit 13 is not intended to be limited to the following, but may be, for example, a chiral chromatography system, preferably a high-performance liquid chromatography system, equipped with a sample introduction unit, an optical resolution column, and a detection unit. To detect only the amount of a specific amino acid, quantification may be performed using an enzymatic method or an immunological method. The analytical measurement unit 13 may be configured separately from the renal pathology evaluation system, and measured data, etc., may be input via the input unit 12 using a network or a storage medium.

[0065] The data processing unit 14 calculates the excretion rate from the measured D-serine and / or D-asparagine amounts, and substitutes the calculated excretion rate into a formula calculated from the relationship between the excretion rate and the blood D-serine and / or D-asparagine levels, or reads the calculated value from a correspondence table or graph, thereby evaluating and determining renal pathology. If the formula, correspondence table, or graph calculated from the relationship between the D-serine and / or D-asparagine excretion rate and the blood D-serine and / or D-asparagine levels requires additional correction values, such as age, weight, sex, and height, such information is input in advance via the input unit and stored in the memory unit. When calculating the excretion rate and pathological condition information, the data processing unit can call up such information and substitute it into the formula or read it from the correspondence table or graph to calculate the excretion rate and pathological condition information. The data processing unit 14 can also determine kidney disease and renal pathology classification from the determined excretion rate, blood D-serine and / or D-asparagine levels, and pathology information. The data processing unit 14 performs various arithmetic operations on the data measured by the analysis and measurement unit 13 and stored in the memory unit 11, in accordance with a program stored in the memory unit. The arithmetic operations are performed by a CPU included in the data processing unit. This CPU includes functional modules that control the analysis and measurement unit 13, input unit 12, memory unit 11, and output unit 15, and can perform various controls. Each of these units may be composed of an independent integrated circuit, microprocessor, firmware, etc.

[0066] The output unit 15 is configured to output a combination of the excretion rate and the blood D-serine and / or D-asparagine levels, which are the results of the arithmetic processing performed by the data processing unit, as well as pathological condition information. The output unit 15 may be an output means such as a display device, such as a liquid crystal display, that directly displays the results of the arithmetic processing, or a printer, or it may be an interface unit for outputting to an external storage device or via a network. The D-serine excretion rate and / or D-asparagine excretion rate, the blood D-serine and / or D-asparagine levels, and / or renal pathological condition classification may also be output together with or independently of the glomerular filtration capacity.

[0067] 12 is a flowchart showing an example of the operation for determining the excretion rate and pathological condition information by the program of the present invention. Specifically, the program of the present invention is a program for causing an information processing device including an input unit, an output unit, a data processing unit, and a storage unit to evaluate a renal pathological condition. The program of the present invention includes the following: a threshold value for evaluating a renal pathology input from an input unit, a calculation formula for the urinary D-serine excretion rate and / or a calculation formula for the D-asparagine excretion rate, and variables required for the calculation are stored in a storage unit; The variables input from the input unit and required for calculating the amount of D-serine and / or D-asparagine in the blood sample and / or urine sample and the D-serine excretion rate and / or D-asparagine excretion rate into urine are stored in a memory unit; causing the data processing unit to call up the formula for calculating the urinary D-serine excretion rate and / or the formula for calculating the D-asparagine excretion rate, which are stored in advance in the storage unit, the amounts of D-serine and / or D-asparagine in the blood sample and / or urine sample, and the variables, which are stored in the storage unit, and to substitute the variables into the formula for calculating the urinary D-serine excretion rate and / or the formula for calculating the D-asparagine excretion rate, thereby calculating the D-serine excretion rate and / or the D-asparagine excretion rate; causing a data processing unit to evaluate a renal pathology based on a comparison of the threshold value stored in a memory unit with a combination of the urinary D-serine excretion rate and / or D-asparagine excretion rate and the blood D-serine amount and / or D-asparagine amount; The output unit outputs the evaluation results of the target kidney pathology. The program of the present invention may be stored on a storage medium or provided via a telecommunications line such as the Internet or a LAN.

[0068] If the information processing device is equipped with an analytical measurement unit, instead of inputting values ​​of the D-serine amount and / or D-asparagine amount from the input unit, the analytical measurement unit may include instructions to cause the information processing device to measure the values ​​from a blood sample and / or a urine sample and store them in the memory unit.

[0069] All documents mentioned herein are incorporated by reference in their entirety.

[0070] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0071] Study population For diagnostic and / or therapeutic purposes, nt of Nephrology,Osaka University Hospit A retrospective study was conducted on patients with primary aldosteronism (PA), myeloma nephropathy (IGAN), diabetic nephropathy (DM), and IgA nephropathy (IGAN) from a cohort of kidney disease patients admitted to the National Institutes of Biomedical Innovation, Health, and Nutrition (NIH) between 2016 and 2017. Because the blood pressure of the IgA nephropathy subjects was above the reference range, they were administered angiotensin II receptor blockers (ARBs) as antihypertensive agents. Separately, 15 healthy volunteers without kidney disease were recruited from the National Institutes of Biomedical Innovation, Health, and Nutrition (NIH). The study protocol was approved by the ethics committees of each institution, and written informed consent was obtained from all subjects.

[0072] Measurement of D-serine and D-asparagine in blood and urine Sample preparation Sample preparation from human plasma and urine was performed as follows: A 20-fold volume of methanol was added to the plasma and mixed thoroughly. After centrifugation, 10 μL of the supernatant obtained from the methanol homogenate was transferred to a brown tube and dried under reduced pressure. 20 μL of 200 mM sodium borate buffer (pH 8.0) and 5 μL of a fluorescent labeling reagent (40 mM 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in anhydrous MeCN) were added to the residue, followed by heating at 60°C for 2 minutes. 75 μL of a 0.1% TFA aqueous solution (v / v) was added to quench the reaction, and 2 μL of the reaction mixture was subjected to two-dimensional HPLC.

[0073] Quantitative determination of amino acid optical isomers by two-dimensional HPLC The amino acid optical isomers were quantified using the following two-dimensional HPLC system. The NBD derivatives were purified using a reversed-phase column (KSAA RP, 1.0 mm i.d. × 400 mm; The mobile phase (5-35% MeCN, 0-20% THF, and 0.05% The amino acids were separated and eluted with TFA. The column temperature was set at 45°C, and the mobile phase flow rate was set at 25 μL / min. The separated amino acid fractions were collected using a multi-loop valve and subsequently optically resolved on a chiral column (KSAACSP-001S, 1.5 mm i.d. × 250 mm; Shiseido). The mobile phase consisted of a mixture of MeOH-MeCN containing citric acid (0-10 mM) or formic acid (0-4%), depending on the retention of the amino acid. NBD-amino acids were detected at 530 nm with excitation at 470 nm. The retention time of the NBD-amino acids was determined by the amino acid. The acid was identified using standards of optical isomers and quantified using a calibration curve.

[0074] Calculation of D-serine excretion rate and D-asparagine excretion rate The amounts of D-serine, D-asparagine, and creatinine in blood and urine were calculated by substituting them into the following formula.

number

number

[0075] Evaluation and assessment of pathological condition The logarithmic transformation values ​​of blood D-serine levels and D-serine excretion rates for subjects with kidney disease and those without kidney disease were plotted on a two-axis coordinate. The non-renal disease group formed a cluster, with the logarithmic mean value of blood D-serine levels being 0.40 and the logarithmic mean value of D-serine excretion rates being 4.12. In this case, the reference range for the distance from the mean can be calculated as 0.558, which is the mean ± 1.96 standard deviations. In the kidney disease patient group, IGAN was within the reference range, but PA, MGRS, and DM were outside the reference range. DM was well separated from the reference range for blood D-serine levels, demonstrating its usefulness in differentiation (Figure 4). Furthermore, in this two-axis plot, the non-renal disease group showed a correlation coefficient R 2 The linearity was high, with a ρ = 0.601, demonstrating that regression analysis can be used for pathological analysis (Figure 7). Although it will be necessary to increase the number of subjects and the variety of pathological conditions in the future to improve analytical accuracy, the combination of blood D-serine levels and the renal reabsorption and excretion rates of D-serine as an index has been confirmed to be useful in research aimed at elucidating pathological mechanisms, drug discovery and treatment, and as an aid to pathological and differential diagnosis in clinical practice. Equivalent results were also obtained with logarithmically transformed values.

[0076] The logarithmic transformation values ​​of blood D-asparagine levels and logarithmic transformation values ​​of D-asparagine excretion rates in subjects with renal disease and those without renal disease were also plotted on a two-axis coordinate (Figure 6). The non-renal disease group formed a cluster, with the logarithmic mean value of blood D-asparagine levels being -1.95 and the logarithmic mean value of D-asparagine excretion rate being 4.16. In this case, the reference range for the distance from the mean can be calculated as 0.515, which is the mean ± 1.96 standard deviations. In the renal disease patient group, IGAN was within the reference range, but PA, MGRS, and DM were outside the reference range. DM was well separated from the reference range of blood D-asparagine levels, demonstrating its usefulness in differentiation (Figure 6). Furthermore, in this two-axis plot, the non-renal disease group had a correlation coefficient R 2 = 0.0002, demonstrating that regression analysis can be used for pathological analysis (Figure 8). Although it will be necessary to increase the variety of pathological conditions and the number of subjects to improve analytical accuracy, the combination of blood D-asparagine levels and the renal reabsorption and excretion rates of D-asparagine as an index has been confirmed to be useful in research aimed at elucidating pathological mechanisms, drug discovery and treatment, and as an aid to pathological and differential diagnosis in clinical practice. Equivalent results were also obtained with logarithmically transformed values.

[0077] Monitoring treatment effectiveness The D-serine excretion rate in IGAN administered an ARB for hypertension fluctuated from 64.56% to 25.73%, a value below the reference value (Figure 2). Furthermore, the D-asparagine excretion rate in IGAN administered an ARB also fluctuated from 45.71% to 35.39%, a value below the reference value (Figure 2). These results suggest that pathological changes, such as a decrease in blood pressure, affect excretion rates during therapeutic interventions such as drug administration. These findings demonstrate the usefulness of D-serine and D-asparagine excretion rates in aiding decision-making regarding the continuation or discontinuation of treatment in research aimed at elucidating pharmacological mechanisms and drug discovery, as well as in monitoring the effects of therapeutic interventions. [Example]

[0078] Subject information A 36-year-old woman was admitted to Osaka University Hospital with systemic lupus erythematosus. After written informed consent was obtained under the university's ethical approval, blood and urine samples were collected serially. Ninety days before admission, her serum creatinine level had rapidly deteriorated from 0.57 mg / dL to 11.68 mg / dL, and her urinary protein concentration had rapidly deteriorated from 0.5 g / g Cre to 4.0 g / g Cre. Her blood pressure was 122 / 65 mmHg, her heart rate was 64 bpm, her percutaneous arterial oxygen saturation was 100% (room air), and her body temperature was 36.5°C. Mouse ulcers, hair loss, and retinal hemorrhage were noted, but no abnormal lung sounds, heart sounds, or lower limb edema were observed. Laboratory tests revealed a blood hemoglobin level of 4.6 g / dL, normal complement levels of C3: 88 mg / dL, C4: 21 mg / dL, positive anti-dsDNA antibodies (13.0 IU / mL), and P-ANCA (182.0 U / mL). Rapidly progressive glomerulonephritis was suspected, leading to a plasma exchange (PE) session and a renal biopsy. Seventy-nine percent of the glomeruli exhibited cellular crescents, and 13% exhibited cellular fibrous crescents. Glomerular capillaries were thickened with bubbling and spikes, but glomerular sclerosis was not observed. The interstitial areas showed moderate, diffuse infiltration of inflammatory cells, but only slight fibrillation. Tubular atrophy was focal and mild. Immunofluorescence staining was positive for IgG, IgA, IgM, C3, C4, and C1q, with a granular, global glomerular capillary wall positivity. A diagnosis of crescentic glomerulonephritis potentially associated with ANCA and lupus nephritis class V was made. Prednisolone pulse therapy (1 g for 3 days) was initiated, followed by oral prednisolone (40 mg / day) and intermittent pulse intravenous cyclophosphamide therapy (500 mg / m 2 The patient was treated with erythropoietin (MMF) and mycophenolate mofetil (MMF, 500 mg / day). Additionally, eight series of plasma exchanges were performed. In response to these treatments, the serum creatinine level decreased to 0.72 mg / dL, but the urinary protein level persisted. A follow-up kidney biopsy showed regression of the glomerular cellular crescents, but 30% of the glomeruli were grossly sclerotic, and capillary thickening persisted.

[0079] D-serine excretion rate The collected blood and urine samples were prepared and quantified in the same manner as in Example 1, and the D-serine excretion rate was calculated.

[0080] Evaluation and assessment of disease state, monitoring of treatment effects The blood D-serine concentration of SLE patients immediately after admission was 17.06 nmol / mL, an order of magnitude higher than that of the non-renal group, and this value alone can determine the difference in pathological condition. The blood D-serine concentration was 0 (below reference) immediately after the start of treatment, 0 (below reference) after 8 days, 0 (below reference) after 12 days, 0 (below reference) after 16 days, 0 (below reference) after 22 days, 58.9% (within reference) after 29 days, 87.6% (above reference) after 34 days, and 41.7% (within reference) after 48 hours. As creatinine levels returned to the normal range with treatment, the D-serine excretion rate transiently increased and fell within the reference range calculated in Example 1.

[0081] In renal damage caused by systemic lupus erythematosus, we observed a phenomenon in which D-serine excretion rates rose above the reference range during the progression and regression of acute renal damage (Figure 10). This suggests that the kidneys are regulating excretion rates as a biological defense against risk or damage caused by some cause. To further improve the accuracy of assessing the pathology, progression, and improvement / deterioration of the disease, we referenced information on blood D-serine levels. This confirmed the usefulness of monitoring a two-axis plot of D-serine excretion rate and blood D-serine levels in assisting in pathology, differential diagnosis, evaluation, and treatment decision-making, as it determines whether D-serine excretion represents a state of combating disease risk or damage or a state of sedation (Figure 4). This information can also be used in research aimed at elucidating pathological and pharmacological mechanisms and drug discovery and treatment. [Example]

[0082] A retrospective study was conducted on patients with interstitial nephritis (TIN), benign prostatic hyperplasia (BPH), Fabry disease, and minimal change nephrotic syndrome (MCNS) from a cohort of kidney disease patients admitted for diagnostic and / or therapeutic purposes to the Department of Nephrology, Osaka University Hospital between 2016 and 2017. The study protocol was approved by the ethics committee at Osaka University, and written informed consent was obtained from all subjects.

[0083] D-serine excretion rate The collected blood and urine samples were prepared and quantified in the same manner as in Example 1, and the D-serine excretion rate was calculated.

[0084] Evaluation, assessment, and differentiation of pathological conditions The blood D-serine levels and D-serine excretion rates of each subject were plotted on a two-axis coordinate system (Figure 13) along with those of the renal disease subjects in Example 1. The plots provided higher resolution than the blood D-serine levels and D-serine excretion rates for each pathological condition, demonstrating their usefulness in assisting in the differentiation of causes and the evaluation and assessment of disease progression.

Claims

1. A method for providing information for the differentiation of kidney disease, comprising: The provision of such information: A combination of the reabsorption and excretion rate of glomerular filtered D-serine in the kidney of a subject with kidney disease and the amount of D-serine in the blood; or Combination of the reabsorption and excretion rate of glomerular filtered D-asparagine in the kidneys of subjects with kidney disease and the amount of D-asparagine in the blood As an indicator, A method, wherein the subject's indicator indicates whether the kidney disease is any of IgA nephropathy, primary aldosteronism (PA), myeloma-related renal syndrome (MGRS), and diabetic nephropathy (DM).

2. The method according to claim 1, wherein the D-serine excretion rate and / or the D-asparagine excretion rate are calculated by correcting using a correction factor derived from blood and / or urine.

3. The method of claim 2, wherein the correction factor is one or more correction factors selected from the group consisting of glomerular filtration rate and urine volume.

4. 3. The method of claim 2, wherein the correction factor is one or more correction factors selected from the group consisting of inulin clearance and creatinine clearance.

5. The method according to claim 2, wherein the correction factor is one or more correction factors selected from the group consisting of creatinine amount and L-amino acid amount.

6. 3. The method of claim 2, wherein the correction factor is L-serine and / or L-asparagine.

7. The excretion rate of the D-serine is calculated by the following formula: [Equation 1] [In the formula, U Ｄ－Ｓｅｒ represents the amount of D-serine in urine, P Ｄ－Ｓｅｒ represents the amount of D-serine in the blood, U ｃｒｅ represents the amount of creatinine in the urine, P ｃｒｅ represents the amount of creatinine in the blood. and / or The excretion rate of the D-asparagine is calculated by the following formula: [Equation 2] [In the formula, U Ｄ－Ａｓｎ represents the amount of D-asparagine in urine, P Ｄ－Ａｓｎ represents the amount of D-asparagine in the blood, U ｃｒｅ represents the amount of creatinine in the urine, P ｃｒｅ The method according to claim 1 or 2, wherein creatinine is calculated from the above formula:

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