Method for assisting evaluation of renal pathology, renal pathology evaluation system, and renal pathology evaluation program

Abstract

The present invention provides a method for assisting in the evaluation of the condition of kidneys, a system for evaluating the condition of kidneys, and a program for evaluating the condition of kidneys, using, as an index, the ratio of reabsorption of D-serine and / or D-asparagine in target kidneys and excretion of the D-serine and / or D-asparagine. The present invention also provides a method for monitoring the condition of kidneys and a method for monitoring the effect of treatment on kidney disease.

Description

Technical Field

[0001] The present invention relates to a method for assisting in the evaluation of kidney disease states, a kidney disease state evaluation system, and a kidney disease state evaluation program.

Background Art

[0002] The kidney is an important organ that maintains the homeostasis of the living body environment through the excretion and absorption of body components, and is responsible for functions such as the excretion of waste products, the regulation of blood pressure, the adjustment of body fluid volume and ions, and the production of blood and bones. A typical index representing kidney function is the glomerular filtration rate (GFR). The glomerular filtration rate represents the volume of fluid filtered from the blood by the glomerulus per minute, and the measurement of inulin clearance is regarded as the international standard (gold standard). However, the measurement of inulin clearance requires a continuous infusion of inulin over 2 hours, as well as multiple urine and blood samplings, which places a heavy burden on the subject and the implementer. Therefore, in daily clinical practice, the measurement of inulin clearance is only carried out in limited situations such as donors during living kidney transplantation, and is replaced by the measurement of other markers such as creatinine. In addition, inulin clearance is difficult to apply when the kidney disease state changes in a short time, such as in acute kidney injury. The values of many markers deviate greatly from the actual glomerular filtration rate such as inulin clearance, which is the gold standard, and hinder the accurate diagnosis of kidney diseases.

[0003] Creatinine is widely measured in clinical practice as an indicator of kidney 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, and a part of it is metabolized into creatinine, which is transported to the kidneys via the blood, filtered in the glomeruli, and then excreted into the urine without being reabsorbed in the renal tubules. When the glomerular filtration capacity decreases, excretion is impaired, and it accumulates in the blood, causing the value to increase, making it a useful indicator of uremic toxin accumulation, so it is used to evaluate kidney function. However, the amount of creatinine in the blood does not show an obvious abnormal value unless the GFR decreases by 50% or more, so it cannot be said to be a sensitive marker.

[0004] Cystatin C is a protein with a molecular weight of 13.36 kDa produced at a certain rate from nucleated cells throughout the body. Since all of it is filtered in the glomeruli and then decomposed in the kidneys after reabsorption in the renal tubules, it is considered to be removed from the blood according to the filtration volume, and the amount in the blood becomes an indicator of GFR. However, when kidney function deteriorates severely, the increase in the amount of cystatin C in the blood becomes blunted, and it is difficult to accurately evaluate kidney function in end-stage kidney disease.

[0005] As described above, there has been no biomarker that can fully meet the clinical requirement of measuring the accurate kidney disease state of individual patients in a wide range from early to late stage with only non-invasive specimens or blood that can be collected without imposing a large burden on the subjects and patients.

[0006] Previously, D-amino acids, which were thought not to exist in the mammalian body, have been shown to be present in various tissues and play physiological functions. The amounts of D-serine, D-alanine, D-proline, D-glutamic acid, and D-aspartic acid in the blood vary in patients with renal insufficiency and correlate with creatinine, indicating that they may serve as markers for renal insufficiency (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). Furthermore, it has been disclosed that an amino acid selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, D-allothreonine, D-glutamine, D-proline, and D-phenylalanine can be used as a pathological index value for kidney disease (Patent Document 1). In addition, 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 in urine vary acutely due to kidney damage, and it has been disclosed that parameters related to these amino acids can be used as pathological index values for kidney disease (Patent Document 2). In recent years, urinary LFABP, blood NGAL, urinary KIM-1, etc. have been developed as markers for kidney disease, but they are not related to glomerular filtration ability.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0009] Compared with the known kidney disease markers to date, a method for more accurately evaluating and determining the kidney disease state of a subject over a wider range is desired.

Means for Solving the Problems

[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 kidney disease states. As a result, they found that new pathological information useful for evaluating and determining kidney disease states can be obtained, leading to the present invention.

[0011] Therefore, the present invention relates to the following: [1] A method for assisting in the evaluation of kidney disease states, using the ratio of reabsorption to excretion of D - serine and / or D - asparagine in the target kidney as an index. [2] The method according to item 1, wherein the ratio is the excretion rate of D - serine into the urine of the subject (subject D - serine excretion rate) and / or the excretion rate of D - asparagine (subject D - asparagine excretion rate). [3] The method according to item 2, wherein the excretion rate of D - serine and / or the excretion rate of D - asparagine is calculated by correction using a correction factor derived from blood and / or urine. [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 volume 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 amount and L - amino acid amount. [7] The method according to item 3, wherein the correction factor is L - serine and / or L - asparagine. [8] The excretion rate of the D - serine is determined by the following formula:

Number

Number

[10] The step of evaluating the renal condition is to evaluate the kidney disease or its risk of the subject, or to predict the induction or prognosis of kidney disease when the target D-serine excretion rate and / or target D-asparagine excretion rate are not included in the first criterion. The method according to item 9.

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

[12] The method according to any one of items 9 to 11, wherein the first criterion is in the range of the mean value of the non-renal disease target D-serine excretion rate and / or non-renal disease target D-asparagine excretion rate ± 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] The method according to any one of items 9 to 14, wherein the first criterion is in the range of 0.4 to 0.9.

[16] The logarithmically transformed target D-serine excretion rate (target D-serine LN excretion rate) and / or logarithmically transformed target D-asparagine excretion rate (target D-asparagine LN excretion rate), and The second criterion calculated from the logarithmically transformed value of the D-serine excretion rate in urine in a plurality of non-renal disease subjects (non-renal disease target D-serine LN excretion rate) and / or the logarithmically transformed value of the D-asparagine excretion rate (non-renal disease target D-asparagine LN excretion rate), and Comparing and evaluating the renal condition based on the relationship between the target D-serine LN excretion rate and / or target D-asparagine LN excretion rate and the second criterion; The method according to any one of items 2 to 8, including this.

[17] The method according to item 16, wherein the step of evaluating the renal condition is to evaluate the kidney disease or the risk of suffering therefrom of the subject, or to predict the induction or prognosis of kidney disease, when the subject D-serine LN excretion rate and / or the subject D-asparagine LN excretion rate are not included in the second criterion.

[18] The method according to item 17, wherein the kidney disease is chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis or polycystic kidney, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, 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 in the range of the mean value ± standard deviation × coefficient Z of the non-kidney disease subject D-serine LN excretion rate and / or the non-kidney disease subject D-asparagine LN excretion rate.

[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 any one of items 16 to 21, wherein the second criterion is in the range of 3.5 to 5.0.

[23] A method for monitoring a renal condition by 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 over time, and using the variation as an index.

[24] The method according to item 23, for monitoring the renal condition caused by kidney disease such as chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis or polycystic kidney, or kidney disease caused by systemic lupus erythematosus, primary aldosteronism, prostatic hyperplasia, Fabry disease or minimal change nephrotic syndrome.

[25] A method for monitoring the treatment effect of kidney disease by 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 with kidney disease before and after treatment intervention over time, and using the variation as an index.

[26] The method according to item 25, wherein the kidney disease is a kidney disease caused by chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis or polycystic kidney, or systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[27] A renal disease evaluation system including a memory unit, an input unit, an analysis and measurement unit, a data processing unit, and an output unit, wherein the memory unit stores a threshold value input from the input unit and a calculation formula for the excretion rate of D-serine and / or a calculation formula for the excretion rate of D-asparagine into urine, the analysis and measurement unit quantifies the amount of D-serine and / or the amount of D-asparagine in a blood sample and / or a urine sample, the data processing unit calculates the excretion rate of D-serine and / or the excretion rate of D-asparagine into urine generated from elements including the quantified amount of D-serine and / or the amount of D-asparagine in the blood sample and / or urine sample and the calculation formula for the excretion rate of D-serine and / or the calculation formula for the excretion rate of D-asparagine stored in the memory unit, the data processing unit evaluates the renal disease state based on the threshold value stored in the memory unit and the excretion rate of D-serine and / or the excretion rate of D-asparagine into urine, and the output unit outputs an evaluation result of the renal disease state of the subject. An evaluation system characterized by the above.

[28] The calculation formula for the excretion rate of D-serine is the following formula: [Number] [wherein, U D-Ser represents the amount of D-serine in urine, P D-Ser represents the amount of D-serine in blood, U cre represents the amount of creatinine in urine, P cre represents the amount of creatinine in blood. ] and / or, the calculation formula for the excretion rate of D-asparagine is the following formula: [Number] [wherein, U D-Asn represents the amount of D-asparagine in urine, P D-Asn represents the amount of D-asparagine in blood, U cre represents the amount of creatinine in urine, P cre represents the amount of creatinine in blood.] The evaluation system according to item 27, which is as described above.

[29] A program for causing an information processing apparatus including an input unit, an output unit, a data processing unit, and a storage unit to evaluate a renal condition, the program including the following: Storing in the storage unit a threshold value for evaluating a renal condition input from the input unit, a calculation formula for the excretion rate of D-serine and / or D-asparagine into urine, and variables necessary for the calculation; Storing in the storage unit variables necessary for calculating the amount of D-serine and / or D-asparagine in a blood sample and / or a urine sample input from the input unit and the excretion rate of D-serine and / or D-asparagine into urine; Causing the data processing unit to call the calculation formula for the excretion rate of D-serine and / or D-asparagine into urine and the amount of D-serine and / or D-asparagine in the blood sample and / or urine sample stored in the storage unit and the variables, substituting them into the calculation formula for the excretion rate of D-serine and / or D-asparagine into urine, and calculating the excretion rate of D-serine and / or D-asparagine; Causing the data processing unit to compare the threshold value stored in the storage unit with the excretion rate of D-serine and / or D-asparagine and evaluate the renal condition; Causing the output unit to output an evaluation result of the renal condition of the subject A program including instructions for causing the information processing apparatus to perform the above.

[30] The calculation formula for the excretion rate of D-serine is the following formula: [Number] [In the formula, U D-Ser represents the amount of D-serine in urine, P D-Ser represents the amount of D-serine in blood, U cre represents the amount of creatinine in urine, P cre represents the amount of creatinine in blood. ] and / or, The calculation formula for the D-asparagine excretion rate is the following formula:

Equation

Advantages of the Invention

[0012] Analysis of the renal dynamics (reabsorption, excretion rate) of D-serine and / or D-asparagine according to the present invention provides a method for more accurately determining the renal condition of a subject over a wider range compared to currently known kidney disease markers.

Brief Description of the Drawings

[0013]

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Embodiments for Carrying Out the Invention

[0014] The present invention relates to a method for determining kidney diseases by analyzing the dynamics (reabsorption, excretion) of D-serine and / or D-asparagine in the kidney. The inventors have found that the dynamics (reabsorption, excretion) of D-serine and D-asparagine in the kidney reflect kidney diseases respectively and can be used for determining kidney diseases in a subject. Therefore, the present invention may be a method for determining kidney diseases by analyzing the dynamics (reabsorption, excretion) of D-serine in the kidney, or a method for determining kidney diseases by analyzing the dynamics (reabsorption, excretion) of D-asparagine in the kidney, or a method for determining kidney diseases by analyzing the dynamics (reabsorption, excretion) of both D-serine and D-asparagine in the kidney. Kidney diseases can also be determined by using the respective analysis results of the dynamics (reabsorption, excretion) of D-serine or D-asparagine in the kidney. However, by using the analysis results of both the dynamics (reabsorption, excretion) of D-serine and D-asparagine in the kidney, the evaluation accuracy can be improved, and false negative and false positive determinations are also possible.

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

[0016] In this specification, the "excretion rate of D-serine into the urine of a subject" may be referred to as the "subject D-serine excretion rate", and the "excretion rate of D-serine into the urine in a non-kidney disease subject" may be referred to as the "non-kidney disease subject D-serine excretion rate", and they have the same meaning even if they are interchanged with each other. Also, in this specification, the "excretion rate of D-asparagine into the urine of a subject" may be referred to as the "subject D-asparagine excretion rate", and the "excretion rate of D-asparagine into the urine in a non-kidney disease subject" may be referred to as the "non-kidney disease subject D-asparagine excretion rate", and they have the same meaning even if they are interchanged with each other.

[0017] In this specification, the "log-transformed D-serine excretion rate of the subject" may be referred to as the "D-serine LN excretion rate of the subject", and the "value obtained by log-transforming the excretion rate of D-serine into urine in a non-renal disease subject" may be referred to as the "D-serine LN excretion rate of non-renal disease subject", and they indicate the same meaning even if they are interchanged with each other. Also, in this specification, the "log-transformed D-asparagine excretion rate of the subject" may be referred to as the "D-asparagine LN excretion rate of the subject", and the "value obtained by log-transforming the excretion rate of D-asparagine into urine in a non-renal disease subject" may be referred to as the "D-asparagine LN excretion rate of non-renal disease subject", and they indicate the same meaning even if they are interchanged with each other.

[0018] In this specification, when simply referred to as "subject", it refers to all mammals, preferably humans, regardless of the presence or absence of kidney disease. In this specification, the "non-renal disease subject" refers to a subject who does not have kidney disease or has not been diagnosed with kidney disease. For example, a subject who does not suffer from kidney disease and other diseases that induce kidney damage is preferred.

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

[0020] In the present invention, excretion is an index indicating the proportion of the amount of a target component filtered by the glomerulus that is excreted into urine through the regulatory function of the renal tubules such as reabsorption and secretion. In addition to ratios and percentages, it is expressed in any unit. Further, a value excluding the effects of water reabsorption and concentration can be calculated by correction with a correction factor, and it may be represented as fractional excretion (FE). Since the urine concentration rate may not be constant, the ratio of reabsorption and excretion of D-serine and / or D-asparagine in the target kidney may be corrected using a "correction factor" for correcting the urine concentration rate. For example, in one embodiment of the present invention, the target D-serine excretion rate and / or the target D-asparagine excretion rate may be corrected with a correction factor derived from blood and / or urine. The excretion rate is most simply expressed as the ratio of the amount of the target component in urine to the glomerular filtration amount of the target component, and the glomerular filtration amount obtained from inulin clearance or the like in the calculation, the actually measured urine volume, and the amount of the target component in blood and / or urine may be used. For calculating the D-amino acid excretion rate, the amount of L-amino acid in urine (preferably, the amount of L-serine and / or L-asparagine) can be used as a urine volume correction factor. Creatinine clearance calculated using the amount of creatinine in urine or the amount of creatinine in blood as a correction factor can be used. For example, the excretion rate of D-serine is represented by the following formula. This may be multiplied by 100 and expressed as a percentage (%).

Number

[0021] Further, for example, the excretion rate of D-asparagine is represented by the following formula. This may be multiplied by 100 and expressed as a percentage (%).

Number

[0022] In kidney diseases, the fractional excretion rate of sodium is used to distinguish whether it is due to dehydration or kidney damage. In addition, the fractional excretion rate of potassium and the fractional excretion rate of urea nitrogen are also clinically used as indicators for disease state determination. Generally, the excretion rate is understood according to the principle of homeostasis that if the intake or biosynthesis amount of the target component is large, the excretion amount in urine increases, and if the intake is small and the biodegradation amount is large, it decreases. Therefore, disorders and pathological changes in the kidneys, which are responsible for the main homeostasis of in-vivo components, can affect the change in the excretion rate. Unlike creatinine, a conventional kidney disease marker that is all excreted, and cystatin C that is all reabsorbed, D-serine and D-asparagine are considered to be strictly regulated for excretion and reabsorption in the renal tubules like electrolytes, and it was conceived that they might become more sensitive and accurate pathological markers.

[0023] In the present invention, D-serine and D-asparagine used for analysis are optical isomers of L-serine and D-asparagine, which are amino acids constituting proteins. The amounts of D-serine and D-asparagine are strictly controlled in each tissue and body fluid by metabolic enzymes and transporters such as serine racemase and D-amino acid oxidase. On the other hand, when kidney 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 and urine" may refer to the amount of D-serine and / or D-asparagine in a specific blood volume or urine volume, or may be expressed as a concentration. The amount of D-serine and / or D-asparagine in blood and urine is measured as the amount in a sample obtained by centrifugation, sedimentation separation, or pretreatment for analysis in the collected blood and urine. Therefore, the amount of D-serine and / or D-asparagine in blood and urine can be measured as the amount in a blood sample derived from blood such as whole blood, serum, and plasma, or as the amount in a urine sample derived from urine excluding total urine, solid components, proteins, etc. As an example, in the case of analysis using HPLC, the amount of D-serine contained in a predetermined amount of blood and urine is represented by a chromatogram and can be quantified by comparison with a standard product and analysis by calibration regarding the height, area, shape, and size of the peak. By comparison with a sample with a known D-serine and / or D-asparagine concentration, it is possible to measure the D-serine and / or D-asparagine concentration in blood and urine, and the D-serine and / or D-asparagine concentration in blood and urine can be used as the amount of D-serine and / or D-asparagine in blood and urine. Also, in the enzyme method, the amino acid concentration 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, for example, chiral column chromatography, measurement using an enzymatic method, or even quantification by an immunological technique using monoclonal antibodies that discriminate optical isomers of amino acids. The measurement of the amounts of D-serine and L-serine in the sample in the present invention may be carried out using any method well known to those skilled in the art. For example, chromatography methods, 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.), antibody methods (T. Ohgusu et al., Analytical Biochemistry, 357 (2006), 15., etc.), gas chromatography (GC) (H. Hasegawa et al., Journal of Mass Spectrometry, 46 (2011), 502., M. C. 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 in the present invention may combine a plurality of separation and analysis methods. More specifically, a sample containing a component having an optical isomer is passed through a first column packing material as a stationary phase together with a first liquid as a mobile phase to separate the component of the sample. Each of the components of the sample is individually held in a multi-loop unit. Each of the components of the sample individually held in the multi-loop unit is supplied through a flow path to a second column packing material having an optically active center as a stationary phase together with a second liquid as a mobile phase to resolve the optical isomers contained in each of the components of the sample. By using an optical isomer analysis method characterized by including a step of detecting the optical isomers contained in each of the components of the sample, the amounts of D- / L-amino acids in a sample can be measured (Japanese Patent No. 4,291,628). In HPLC analysis, D- and L-amino acids are sometimes derivatized with a fluorescent reagent such as o-phthalaldehyde (OPA) or 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in advance, or diastereomerized using N-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys), etc. (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, Vol. 53, pp. 677-690 (2004)). Alternatively, D-amino acids can be measured by an immunological method using a monoclonal antibody that discriminates optical isomers of amino acids, for example, a monoclonal antibody that specifically binds to D-serine, L-serine, D-asparagine, or L-asparagine, etc. Also, when the total amount of the D-form and the L-form is used as an index, it is not necessary to separate and analyze the D-form and the L-form, and amino acids can be analyzed without distinguishing between the D-form and the L-form. In that case as well, separation and quantification can be performed by an enzymatic method, an antibody method, GC, CE, or HPLC.

[0027] The amounts of D-serine and D-asparagine in the blood strongly correlate with the glomerular filtration rate as compared with creatinine, which is a conventional marker. This is because the amount of creatinine in the blood is strongly affected by muscle mass, showing high values in athletes, patients with acromegaly, and during high meat intake, and low values in patients with neuromuscular diseases (such as muscular dystrophy), emaciation, long-term bedridden, frailty, sarcopenia, locomotive syndrome, with amputation, and during protein intake restriction, thus it cannot accurately reflect renal function. In contrast to the fact that the amount of D-serine in the blood is maintained within a very small range of about 1-2% of the total serine amount in healthy individuals without pathological findings, an amount ranging from 30-60% is present in urine. Interestingly, unlike approximately 99% of L-serine being reabsorbed in the renal tubules, about 50-80% of D-serine is excreted. Also, in healthy individuals without pathological findings, in contrast to the fact that the amount of D-asparagine in the blood is maintained within a very small range of about 0.1-0.6% of the total asparagine amount, an amount ranging from 20-50% is present in urine. Interestingly, unlike approximately 99% of L-asparagine being reabsorbed in the renal tubules, about 50-80% of D-asparagine is also excreted.

[0028] In the present invention, the excretion rates of D-serine and D-asparagine proposed are shown by multivariate analysis (OPLS) of chiral amino acid metabolomics and related parameters not to correlate with the glomerular filtration rate, unlike the amounts of D-serine and D-asparagine in the blood. Since it was suggested that the reabsorption of optical isomers of serine and asparagine in the renal tubules of the kidney is strictly controlled respectively, for the purpose of examining the physiological significance of D-serine and D-asparagine, 15 healthy volunteers were recruited as the study population to analyze the excretion rates of D-serine and D-asparagine in non-renal disease subjects. The test protocol was approved by the Ethics Committee at the National Institute of Biomedical Innovation, Health and Nutrition, and written informed consent was obtained from all subjects. The non-renal disease subjects had an average age of 44, a male ratio of 80%, an average height of 1.70 m, an average weight of 68.9 kg, and an average BSA of 1.80 m 2, with an average BMI of 22.6 kg / m 2 It was a group with an average serum creatinine of 0.75 mg / dL.

[0029] Using the following formula from the quantitative analysis values of D-serine and D-asparagine in the blood and urine of the subjects, the average excretion rate of D-serine was calculated to be 62.76% and its average logarithmic value was 4.12, and the average excretion rate of D-asparagine was 64.12% and its average logarithmic value was 4.16 (Figure 1). [Equation] [In the formula, U D-Ser represents the amount of D-serine in urine, P D-Ser represents the amount of D-serine in blood, U cre represents the amount of creatinine in urine, P cre represents the amount of creatinine in blood.] [Equation] [In the formula, U D-Asn represents the amount of D-asparagine in urine, P D-Asn represents the amount of D-asparagine in blood, U cre represents the amount of creatinine in urine, P cre represents the amount of creatinine in blood.]

[0030] Regarding D-serine, when a histogram was created for the obtained logarithmic data at the 6th percentile, a normal distribution-like shape was observed (Figure 3). Therefore, a Shapiro-Wilk Normality Test was performed on this data, and a value of P = 0.395 was obtained. Thus, the null hypothesis was not rejected, and the possibility of a normal distribution was supported. Therefore, the reference values for these non-renal disease subjects were calculated to be 42.46 - 89.66% from the mean ± 1.96 standard deviations, and their logarithmic values were 3.75 - 4.50. Subjects outside this range can assist in predicting the disease state, risk, or prognosis of kidney disease and kidney impairment.

[0031] Regarding D-asparagine, when a histogram was created for the obtained logarithmic data in six-digit classes, a normal distribution-like shape was observed (Figure 5). Therefore, the Shapiro-Wilk Normality Test was performed on this data, and since a value of P = 0.243 was obtained, the null hypothesis was not rejected, supporting the possibility of a normal distribution. Thus, the reference values for these non-renal disease subjects were calculated to be 51.65 - 78.74% from the mean value ± 1.96 standard deviations, and their logarithmic values were 3.95 - 4.37. Subjects outside this range can assist in predicting kidney disease, the condition, risk, or prognosis of kidney disease.

[0032] Since the amount of D-serine and D-asparagine in the blood strongly correlates with the glomerular filtration rate, it has been shown that their analysis can be applied to the severity classification (G1 - 5) of chronic kidney disease (CKD) defined in the Japanese Society of Nephrology Guidelines. However, the D-serine excretion rate, which analyzes by adding the amount of D-serine in urine, and the D-asparagine excretion rate, which analyzes by adding the amount of D-asparagine in urine, can assist in evaluating kidney disease states from a completely different mechanism that does not correlate with the glomerular filtration rate. Therefore, they have high clinical utility in differential diagnosis, disease state, and prognosis diagnosis, which were difficult with conventional markers.

[0033] In one embodiment, the present invention is as follows: said target D-serine excretion rate and / or target D-asparagine excretion rate, and a first criterion calculated from the excretion rate of D-serine into urine (non-renal disease subject D-serine excretion rate) and / or the excretion rate of D-asparagine (non-renal disease subject D-asparagine excretion rate) in a plurality of non-renal disease subjects, and comparing them, and evaluating a kidney disease state from the relationship between said target D-serine excretion rate and / or target D-asparagine excretion rate and said first criterion, and may provide a method including the step.

[0034] Thus, for example, in the first embodiment of the above invention, the present invention said target D-serine excretion rate, and A first criterion calculated from the excretion rate of D-serine into urine in a plurality of non-renal disease subjects (non-renal disease subject D-serine excretion rate) and comparing, and evaluating a renal condition from the relationship between the subject D-serine excretion rate and the first criterion. A method may be provided that includes the step of

[0035] Also, for example, in the second embodiment of the above invention, the present invention the subject D-asparagine excretion rate and a first criterion calculated from the excretion rate of D-asparagine into urine in a plurality of non-renal disease subjects (non-renal disease subject D-asparagine excretion rate) and comparing, and evaluating a renal condition from the relationship between the subject D-asparagine excretion rate and the first criterion. A method may be provided that includes the step of

[0036] Also, for evaluating a renal condition, the method of the first embodiment and the method of the second embodiment described above may be used in combination. In this case, not only is the accuracy of evaluating a renal condition improved, but false positives and false negatives can also be determined.

[0037] As used herein, the "first criterion" refers to a criterion calculated from the excretion rate of D-serine into urine (non-renal disease subject D-serine excretion rate) and / or the excretion rate of D-asparagine (non-renal disease subject D-asparagine excretion rate) in a plurality of non-renal disease subjects and used for evaluating the renal condition of a subject. That is, by comparing the subject D-serine excretion rate and / or the subject D-asparagine excretion rate with the first criterion, the renal condition in the subject can be evaluated. The number of "non-renal disease subjects" employed for calculating the first criterion is preferably a number sufficient to calculate a statistically significant criterion. For example, numbers such as 3, 5, 10, 15, 20, 30, 50, 100 or more can be employed in the present invention.

[0038] In one embodiment, the first criterion in the present invention may be in the range of the average value ± standard deviation × coefficient Z of the D-serine excretion rate and / or D-asparagine excretion rate in non-renal disease subjects. In the present specification, the "coefficient Z" is a coefficient used to calculate a confidence interval used in statistics. For example, a value between 1.0 and 3.0 is preferable, and 1.96 is more preferable. Also, in one embodiment, the first criterion is preferably in the range of 0.4 to 0.9.

[0039] In one embodiment, the step of evaluating a renal condition may be to evaluate the renal disease or the risk of suffering therefrom of the subject, or to predict the induction or prognosis of renal disease, when the subject D-serine excretion rate and / or subject D-asparagine excretion rate are not included in the first criterion.

[0040] In other embodiments, the present invention provides the following: The logarithmically transformed subject D-serine excretion rate (subject D-serine LN excretion rate) and / or the logarithmically transformed subject D-asparagine excretion rate (subject D-asparagine LN excretion rate), A second criterion calculated from the logarithmically transformed value of the excretion rate of D-serine into urine (non-renal disease subject D-serine LN excretion rate) and / or the logarithmically transformed value of the excretion rate of D-asparagine (non-renal disease subject D-asparagine LN excretion rate) in a plurality of non-renal disease subjects, And comparing, and evaluating a renal condition from the relationship between the subject D-serine LN excretion rate and / or subject D-asparagine LN excretion rate and the second criterion, may provide a method including the step.

[0041] Therefore, for example, in the first embodiment of the above invention, the present invention provides The logarithmically transformed subject D-serine excretion rate (subject D-serine LN excretion rate), A second criterion calculated from the logarithmically transformed value of the excretion rate of D-serine into urine (non-renal disease subject D-serine LN excretion rate) in a plurality of non-renal disease subjects, Comparing the excretion rate of the target D-serine LN with the second criterion to evaluate the renal condition, a method including this step may be provided.

[0042] Also, for example, in the second embodiment of the above invention, the present invention The logarithmically transformed excretion rate of the target D-asparagine (target D-asparagine LN excretion rate) and A second criterion calculated from the logarithmically transformed excretion rate of D-asparagine into urine in a plurality of non-renal disease subjects (non-renal disease subject D-asparagine LN excretion rate), and Comparing them, and evaluating the renal condition from the relationship between the target D-asparagine LN excretion rate and the second criterion, a method including this step may be provided.

[0043] Also, for evaluating the renal condition, the method of the first embodiment and the method of the second embodiment described above may be used in combination. In this case, not only the accuracy of evaluating the renal condition is improved, but false positives and false negatives can also be determined.

[0044] In this specification, the "logarithmically transformed value" refers to a value obtained by transforming the target value into a logarithm. For example, it may be a value obtained by transforming the target value into a natural logarithm, or a value obtained by transforming the target value using an arbitrary base such as a common logarithm.

[0045] As used herein, the "second criterion" refers to a criterion calculated from the logarithm-transformed value of the excretion rate of D-serine into urine in a plurality of non-renal disease subjects (non-renal disease subject D-serine LN excretion rate) and / or the logarithm-transformed value of the excretion rate of D-asparagine (non-renal disease subject D-asparagine LN excretion rate), and is used to evaluate the renal condition of a subject. That is, by comparing the subject D-serine LN excretion rate and / or the subject D-asparagine LN excretion rate with the second criterion, the renal condition of the subject can be evaluated. The number of "non-renal disease subjects" employed to calculate the second criterion is preferably a number sufficient to calculate a statistically significant criterion. For example, numbers such as 3, 5, 10, 15, 20, 30, 50, 100 or more can be employed in the present invention.

[0046] In one embodiment, the second criterion may be in the range of the average value of the non-renal disease subject D-serine LN excretion rate and / or the non-renal disease subject D-asparagine LN excretion rate ± standard deviation × coefficient Z. In this case, the coefficient Z preferably has a value between 1.0 and 3.0, and more preferably 1.96. Also, in one embodiment, the second criterion is preferably in the range of 3.5 to 5.0.

[0047] In one embodiment, the step of evaluating the renal condition may be to evaluate the renal disease or its risk of onset of the subject, or to predict the induction or prognosis of renal disease, when the subject D-serine LN excretion rate and / or the subject D-asparagine LN excretion rate are not included in the second criterion.

[0048] When the disease state is determined by the method of the present invention, a treatment policy can be determined based thereon. Depending on each disease state, the treatment method can be appropriately selected, and the excretion rate may be controlled while monitoring the excretion rate of D-serine and / or D-asparagine over time so as to fall within the reference range for non-renal subjects. Treatment interventions include lifestyle improvement, dietary guidance, blood pressure management, anemia management, electrolyte management, uremotoxin management, blood glucose management, immune management, and lipid management, etc., which are guided independently or in combination. As for lifestyle improvement, smoking cessation and weight loss to a BMI value of less than 25 are recommended. As for dietary guidance, salt reduction and protein restriction are carried out. Among these, in particular, for blood pressure management, anemia management, electrolyte management, uremotoxin management, blood glucose management, immune management, and lipid management, treatment by medication can be performed. As for blood pressure management, it is managed to be 130 / 80 mmHg or less, and in some cases, antihypertensive drugs may be administered.As antihypertensive drugs, diuretics (thiazide diuretics, such as trichlormethiazide, benzylhydrochlorothiazide, hydrochlorothiazide, thiazide-like diuretics, such as meclofenamic acid, indapamide, tripamide, mefruside, loop diuretics, such as furosemide, potassium-sparing diuretics·aldosterone antagonists, such as triamterene, spironolactone, eplerenone, etc.), calcium antagonists (dihydropyridine-based, such as nifedipine, amlodipine, efonidipine, cilnidipine, nicardipine, nisoldipine, nitrendipine, nilvadipine, barnidipine, felodipine, benidipine, manidipine, azelnidipine, aranidipine, benzothiazepine-based, diltiazem, etc.), angiotensin-converting enzyme inhibitors (captopril, enalapril, acepril, delapril, cilazapril, lisinopril, benazepril, imidapril, temocapril, quinapril,trandolapril, berindopril erbumin, etc.), angiotensin receptor antagonists (angiotensin II receptor antagonists, such as losartan, candesartan, valsartan, telmisartan, olmesartan, irbesartan, azilsartan, etc.), sympathetic nerve blockers (β-blockers, such as atenolol, bisoprolol, betaxolol, metoprolol, aceprolol, celiprolol, propranolol, nadolol, carteolol, pindolol, nipradilol, amosulalol, alotiolol, carvedilol, labetalol, bevantolol, urapidil, terazosin, prazosin, doxazosin, bunazosin, etc.) and the like can be used. As antianemic drugs, erythropoietin preparations, iron agents, HIF-1 inhibitors and the like are used. As electrolyte regulators, calcium receptor agonists (such as cinacalcet, etelcalcetide, etc.) and phosphorus adsorbents are used. As uremic toxin adsorbents, activated carbon and the like are used. The blood glucose level is controlled to be less than 6.9% of Hba1c, and hypoglycemic drugs may be administered as the case may be.As hypoglycemic agents, SGLT2 inhibitors (ipragliflozin, dapagliflozin, luseogliflozin, tofogliflozin, canagliflozin, empagliflozin, etc.), DPP4 inhibitors (sitagliptin phosphate, vildagliptin, saxagliptin, alogliptin, linagliptin, teneligliptin, treagliptin, anagliptin, omarigliptin, etc.), sulfonylurea agents (tolbutamide, acetohexamide, chlorpropamide, glycopyramide, glibenclamide, glibenzide, glimepiride, etc.), thiazolidine agents (pioglitazone, etc.), biguanide agents (metformin, buformin, etc.), α-glucosidase inhibitors (acarbose, voglibose, miglitol, etc.), glinide agents (nateglinide, mitiglinide, repaglinide, etc.), insulin preparations, NRF2 activators (bardoxolone methyl, etc.), etc. are used. As immunomodulation, immunosuppressants (steroids, tacrolimus, anti-CD20 antibody, cyclohexamide, mycophenolate mofetil (MMF), etc.) are used. For lipid management, it is managed to be less than 120 mg / dL of LDL-C, and in some cases, lipid-lowering drugs such as statin drugs (rosuvastatin, pitavastatin, atorvastatin, cerivastatin, fluvastatin, simvastatin, pravastatin, lovastatin, mevastatin, etc.), fibrate drugs (clofibrate, bezafibrate, fenofibrate, clinofibrate, etc.), nicotinic acid derivatives (nicotinic acid tocolerol, nicomol, nicertol, etc.), cholesterol transporter inhibitors (ezetimibe, etc.), PCSK9 inhibitors (evolocumab, etc.), EPA preparations, etc. are used. Any of these drugs can be in the form of a single agent or a combination. Depending on the degree of renal function decline, renal replacement therapies such as peritoneal dialysis, hemodialysis, continuous hemodiafiltration, hemofiltration (plasma exchange, plasma adsorption, etc.), and kidney transplantation are performed.

[0049] Therefore, in one embodiment, the present invention may be a method for monitoring a kidney disorder, which measures 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 over time, and uses the variation thereof as an index. Further, in another embodiment, the present invention may be a method for monitoring the treatment effect of a kidney disease, which measures 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 with a kidney disease before and after treatment intervention over time, and uses the variation thereof as an index.

[0050] By using the method of the present invention, it is possible to evaluate kidney diseases in a subject, such as chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis or polycystic kidney, or kidney diseases caused by systemic lupus erythematosus, primary aldosteronism, prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[0051] In another aspect of the present invention, it may relate to a system and a program for executing a method for assisting the evaluation of the above kidney disorder. FIG. 11 is a configuration diagram of the kidney disorder evaluation system of the present invention. The sample analysis system 10 shown in FIG. 11 is configured to be able to implement the method for assisting the evaluation of the kidney disorder of the present invention. Such a sample analysis system 10 includes a storage unit 11, an input unit 12, an analysis measurement unit 13, a data processing unit 14, and an output unit 15, and can analyze a blood sample and / or a urine sample and output the calculated excretion rate and the disease state information.

[0052] More specifically, in the sample analysis system 10 of the present invention, the storage unit 11 stores the excretion rate calculated from the amount of D-serine and / or D-asparagine in the blood sample and urine sample input from the input unit 12, the reference value, the disease state information correspondence table or graph. The analysis measurement unit 13 separates and quantifies D-serine and / or D-asparagine in the blood sample and / or urine sample. The data processing unit 14 determines the disease state by substituting the excretion rate calculated from the amount of D-serine and / or D-asparagine into the formula obtained from the reference value and disease state information, or by reading it from the correspondence table or graph, and the output unit 15 can output the disease state information.

[0053] In a more preferred embodiment, the kidney disease state evaluation system of the present invention may further include a step in which the storage unit 11 stores the reference value input from the input unit 12, and a step in which the data processing unit 14 compares the excretion rate calculated from the separated and quantified amount of D-serine and / or D-asparagine with the reference value. In this case, when the D-serine excretion rate and / or D-asparagine excretion rate is outside the reference range, the output unit 15 outputs a suspicion of kidney disease.

[0054] The storage unit 11 includes a memory device such as a RAM, ROM, 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 the data measured by the analysis measurement unit, the data and instructions input from the input unit, the calculation processing results performed by the data processing unit, and also stores various computer programs, databases, etc. used for various processes of the information processing device. The computer program may be installed via a computer-readable recording medium such as a CD-ROM, DVD-ROM, or via the Internet. The computer program is installed in the storage unit using a known setup program or the like. The storage unit stores data on a formula, correspondence table, or graph calculated from the relationship between the D-serine excretion rate input from the input unit 12 in advance and the disease state. It can also store the kidney disease state classification according to the excretion rate. [[ID=IO]]

[0055] The input unit 12 is an interface or the like, and also includes an operation unit such as a keyboard and a mouse. Thus, the input unit can input the data measured by the analysis measurement unit 13, instructions for arithmetic processing performed by the data processing unit 14, and the like. Further, when the analysis measurement unit 13 is external, for example, the input unit 12 may include an interface unit that can input measured data and the like via a network or a storage medium separately from the operation unit.

[0056] The analysis measurement unit 13 performs a measurement process of D-serine and / or D-asparagine in a blood sample and / or a urine sample. Therefore, the analysis measurement unit 13 has a configuration that enables separation and measurement of the D-form and L-form of amino acids. Amino acids may be analyzed one by one, but some or all types of amino acids can be analyzed together. The analysis measurement unit 13 is not intended to be limited to the following, but may be, for example, a chiral chromatography system including a sample introduction unit, an optical splitting column, and a detection unit, preferably a high performance liquid chromatography system. From the viewpoint of detecting only a specific amino acid amount, quantification by an enzymatic method or an immunological method may be performed. The analysis measurement unit 13 may be configured separately from the kidney disease evaluation system, and the measured data and the like may be input via the input unit 12 using a network or a storage medium.

[0057] The data processing unit 14 can evaluate and determine the renal condition by calculating the excretion rate from the measured amount of D - serine and / or D - asparagine, substituting it into an equation calculated from the relationship with the excretion amount, or reading it from a correspondence table or graph. When an equation, correspondence table, or graph calculated from the relationship with the D - serine excretion rate and / or D - asparagine excretion rate requires additional correction values, such as age, weight, gender, height, etc., such information is input from the input unit in advance and stored in the storage unit. When calculating the excretion rate and pathological condition information, the data processing unit can call such information, substitute it into the equation, 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 diseases and renal condition classifications from the determined excretion rate and pathological condition information. The data processing unit 14 performs various arithmetic operations on the data measured by the analysis measurement unit 13 and stored in the storage unit 11 according to the program stored in the storage unit. The arithmetic operations are performed by the CPU included in the data processing unit. This CPU includes functional modules for controlling the analysis measurement unit 13, input unit 12, storage unit 11, and output unit 15, and can perform various controls. Each of these units may be composed of independent integrated circuits, microprocessors, firmware, etc.

[0058] The output unit 15 is configured to output the excretion rate and pathological condition information, which are the results of the arithmetic operations performed by the data processing unit. The output unit 15 may be a display device such as a liquid crystal display that directly displays the results of the arithmetic operations, an output means such as a printer, or an interface unit for outputting to an external storage device or via a network. It is also possible to output the D - serine excretion rate and / or and / or L - asparagine excretion rate, and / or renal condition classification, either in combination with or independently of the glomerular filtration ability.

[0059] FIG. 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 apparatus including an input unit, an output unit, a data processing unit, and a storage unit to evaluate the renal condition. The program of the present invention is as follows: The storage unit stores a threshold value for evaluating a renal condition input from the input unit, a calculation formula for the excretion rate of D-serine into urine and / or a calculation formula for the excretion rate of D-asparagine, and variables necessary for the calculation. The storage unit stores the amount of D-serine and / or D-asparagine in a blood sample and / or a urine sample input from the input unit, and variables necessary for calculating the excretion rate of D-serine and / or D-asparagine into urine. The data processing unit calls the calculation formula for the excretion rate of D-serine into urine and / or the calculation formula for the excretion rate of D-asparagine pre-stored in the storage unit, the amount of D-serine and / or D-asparagine in the blood sample and / or urine sample stored in the storage unit, and the variables, substitutes them into the calculation formula for the excretion rate of D-serine into urine and / or the calculation formula for the excretion rate of D-asparagine, and calculates the excretion rate of D-serine and / or D-asparagine. The data processing unit compares the threshold value stored in the storage unit with the excretion rate of D-serine and / or D-asparagine to evaluate the renal condition. The output unit outputs the evaluation result of the target renal condition. It includes a command for causing the information processing apparatus to execute the above. The program of the present invention may be stored in a storage medium or provided via an electric communication line such as the Internet or a LAN.

[0060] When the information processing apparatus includes an analysis measurement unit, instead of inputting the value of the amount of D-serine and / or D-asparagine from the input unit, the analysis measurement unit may measure the value from a blood sample and / or a urine sample and store it in the storage unit, and it may include a command for causing the information processing apparatus to execute this.

[0061] All documents mentioned in this specification are hereby incorporated by reference in their entirety.

[0062] The embodiments of the present invention described below 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 description in the claims. Changes to the present invention, such as addition, deletion, and substitution of the constituent elements of the present invention, can be made on the condition that the gist of the present invention is not deviated from.

Example

[0063] Investigation population For the purpose of diagnosis and / or treatment, a retrospective study was conducted on patients with primary aldosteronism (PA), myeloma kidney (IGAN), diabetic nephropathy (DM), and IgA nephropathy (IGAN) from a cohort of kidney disease patients who were admitted to the Department of Nephrology, Osaka University Hospital between 2016 and 2017. Since the subjects with IgA nephropathy had blood pressure exceeding the reference range, an angiotensin II receptor blocker (ARB) was administered as an antihypertensive agent. Separately, 15 healthy volunteers were recruited as non-kidney disease subjects at the National Institute of Biomedical Innovation, Health and Nutrition described above. The test protocol was approved by the ethics committee at each facility, and written informed consent was obtained from all subjects.

[0064] Measurement of D-serine and D-asparagine in blood and urine Sample preparation Sample preparation from human plasma and urine was performed as follows: Twenty volumes of methanol were added to 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. To the residue, 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, and then heated at 60 °C for 2 minutes. 75 μL of 0.1% TFA aqueous solution (v / v) was added to stop the reaction, and 2 μL of the reaction mixture was subjected to two-dimensional HPLC.

[0065] Quantification of Amino Acid Enantiomers by Two-Dimensional HPLC Amino acid enantiomers were quantified using the following two-dimensional HPLC system. The NBD derivative of amino acid was separated and eluted with a mobile phase (5 - 35% MeCN, 0 - 20% THF, and 0.05% TFA) using a reversed-phase column (KSAA RP, 1.0 mm i.d. × 400 mm; Shiseido Co., Ltd.). The column temperature was set at 45 °C and the flow rate of the mobile phase was 25 μL / min. The fraction of the separated amino acid was collected using a multi-loop valve and continuously optically resolved with a chiral column (KSAACSP-001S, 1.5 mm i.d. × 250 mm; Shiseido). As the mobile phase, a mixed solution of MeOH - MeCN containing citric acid (0 - 10 mM) or formic acid (0 - 4%) was used according to the retention of amino acids. NBD-amino acid was detected by fluorescence at 530 nm using an excitation light of 470 nm. The retention time of NBD-amino acid was identified using a standard product of amino acid enantiomers and quantified using a calibration curve.

[0066] Calculation of D-Serine Excretion Rate and D-Asparagine Excretion Rate The amount of D-serine in blood and urine, the amount of D-asparagine in blood and urine, and the amount of creatinine were substituted into the following formula for calculation.

Equation

Equation

[0067] Evaluation and determination of disease state As described above, the reference values of D-serine excretion rate in non-renal disease subjects were calculated to be 42.46 - 89.66% from the mean ± 1.96 standard deviations, and the logarithmic values were 3.75 - 4.50. Also, the reference values of D-asparagine excretion rate in non-renal disease subjects were calculated to be 51.65 - 78.74% from the mean ± 1.96 standard deviations, and the logarithmic values were 3.95 - 4.37. For D-serine excretion rate in renal disease subjects, for PA it was 117.24% exceeding the standard, for IgAN it was 64.56% within the standard, for DM it was 42.58% at the lower limit of the standard (lower than the measured value of non-renal disease subjects), and for MGRS it was 107.01% exceeding the standard, and there were clearly disease states deviating from the reference values (Figure 2). Also, for D-asparagine excretion rate in renal disease subjects, for PA it was 150.78% exceeding the standard, for IgAN it was 45.71% within the standard, for DM it was 53.75% at the lower limit of the standard (lower than the measured value of non-renal disease subjects), and for MGRS it was 105.25% exceeding the standard, and there were clearly disease states deviating from the reference values (Figure 2).

[0068] In the future, it is necessary to increase the variations of disease states and the number of subjects to improve the analysis accuracy. However, the usefulness of D-serine excretion rate and D-asparagine excretion rate in elucidating the pathological mechanism exceeding the theoretically impossible excretion rate of 100% without the biosynthesis and secretion of D-serine and D-asparagine, for research aiming at drug discovery and treatment, and further for assisting in the differential diagnosis of disease states in clinical practice has been confirmed. Equivalent results could also be obtained for the logarithmically transformed values.

[0069] Monitoring of treatment effect The D-serine excretion rate of IgAN patients administered with ARB for hypertension decreased from 64.56% to 25.73%, falling below the reference value (Figure 2). The D-asparagine excretion rate of IgAN patients administered with ARB also decreased from 45.71% to 35.39%, falling below the reference value (Figure 2). It is suggested that pathological changes such as blood pressure reduction affect the excretion rate during therapeutic interventions such as drug administration. The usefulness of D-serine excretion rate and D-asparagine excretion rate in assisting in making decisions such as continuing or discontinuing treatment was shown in research aimed at elucidating the pharmacological mechanism and developing new drugs, as well as in monitoring the effects during therapeutic interventions.

Example

[0070] Subject Information A 36-year-old woman with systemic lupus erythematosus was admitted to Osaka University Hospital. After obtaining written informed consent under the ethical approval of the university, blood and urine were collected over time. The serum creatinine level increased rapidly from 0.57 mg / dL 90 days before admission to 11.68 mg / dL, and the urinary protein concentration increased from 0.5 g / gCre to 4.0 g / gCre. Blood pressure was 122 / 65 mmHg, heart rate was 64 bpm, percutaneous arterial oxygen saturation was 100% (room air), and body temperature was 36.5°C. Mouse ulcers, hair loss, and retinal hemorrhage were noted, but abnormal lung sounds, heart sounds, and lower limb edema were not observed. In clinical examinations, blood hemoglobin was 4.6 g / dL, normal complement levels were C3: 88 mg / dL, C4: 21 mg / dL, positive anti-dsDNA antibody was 13.0 IU / mL1, and P-ANCA was 182.0 U / mL. As rapidly progressive glomerulonephritis was suspected, plasma exchange (PE) sessions were continued and a renal biopsy was performed. 79% of the glomeruli showed cellular crescents, 13% showed cellular fibrous crescents, glomerular capillaries were thickened with bulging and spikes, but glomerulosclerosis was not observed. The interstitial area showed moderate diffuse infiltration of inflammatory cells, but there were only a few arterioles. Tubular atrophy was localized and mild. Immunofluorescent staining was positive for granular overall glomerular capillary walls for IgG, IgA, IgM, C3, C4, and C1q. Crescentic glomerulonephritis potentially associated with ANCA and lupus nephritis class V were diagnosed. Pulse methylprednisolone therapy (1 g for 3 days), followed by oral prednisone (40 mg / day), intermittent pulse intravenous cyclophosphamide therapy (500 mg / m 2 ) and mycophenolate mofetil (MMF, treated at 500 mg / day) were administered. Additionally, 8 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. Follow-up renal biopsy showed regression of glomerular cellular crescents, but 30% of the glomeruli were globally sclerosed and capillary thickening persisted.

[0071] D-serine excretion rate The collected blood and urine samples were prepared as in Example 1, and the D-serine excretion rate was calculated together with the D-serine quantification.

[0072] Evaluation and determination of disease states, monitoring of treatment effects The D-serine excretion rate was 0 (below the standard) immediately after the start of treatment, 0 (below the standard) after 8 days, 0 (below the standard) after 12 days, 0 (below the reference value) after 16 days, 0 (below the reference value) after 22 days, 58.9% (within the standard) after 29 days, 87.6% (exceeding the standard) after 34 days, and 41.7% (within the standard) after 48 hours. As the creatinine level returned to the normal range with treatment, the D-serine excretion rate transiently increased and remained within the standard calculated in Example 1.

[0073] In the treatment process of kidney damage caused by systemic lupus erythematosus, an increase in the D-serine excretion rate was observed. This suggests that the kidney is controlling the excretion rate in response to risks and damages caused by some reason to defend the living body. From the above, the usefulness of D-serine excretion rate monitoring was confirmed in research aimed at elucidating pathological and pharmacological mechanisms, drug discovery and treatment, and further for assisting in pathological and differential diagnosis and treatment policy determination in clinical practice.

Example

[0074] For the purpose of diagnosis and / or treatment, a retrospective study was conducted on patients with interstitial nephritis (TIN), benign prostatic hyperplasia (BPH), Fabry disease (Fabry), and minimal change nephrotic syndrome (MCNS) from a cohort of kidney disease patients admitted to the Department of Nephrology, Osaka University Hospital between 2016 and 2017. The test protocol was approved by the Ethics Committee at Osaka University, and written informed consent was obtained from all subjects.

[0075] D-serine excretion rate The collected blood and urine samples were adjusted and quantified in the same manner as in Example 1, and then the D-serine excretion rate was calculated.

[0076] Evaluation, determination, and differentiation of disease states The amount of D-serine and the D-serine excretion rate in the blood of each subject were plotted on two-axis coordinates together with the kidney disease subjects in Example 1 (Figure 13). It was shown that the separation ability of the plot was higher than that based only on the information on the amount of D-serine and the D-serine excretion rate in the blood of each pathological condition, and it was useful for differentiating the cause and assisting in the evaluation and determination of the disease state.

Claims

1. A method for providing an index for differentiating kidney diseases in a subject having kidney diseases, comprising: the index being the excretion rate and / or reabsorption rate of D-serine and / or D-asparagine filtered by glomeruli in the kidney of the subject into urine, wherein the fact that the index of the subject is within the normal range of a healthy person indicates that the kidney disease is IgA nephropathy, and the fact that the index of the subject is outside the normal range of a healthy person indicates that the kidney disease is any one of primary aldosteronism (PA), myeloma kidney (MGRS), and diabetic nephropathy (DM).

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

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

4. The method according to 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. The method according to claim 2, wherein the correction factor is L-serine and / or L-asparagine.

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

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