Method and kit for testing the progression of chronic kidney disease in a subject.

Abstract

The present invention provides a simple examination means for promptly identifying the progression of chronic kidney disease. One aspect of the present invention relates to a method for examining the progression of chronic kidney disease in a subject, said method including the following steps: a measurement step for measuring the creatinine concentration in the blood and urine of the subject and the phosphorous concentration in the urine; a calculation step for calculating an estimate for the phosphorous concentration in the primary urine on the basis of formula (II); and a phosphorous concentration comparison step for comparing the estimate for the phosphorous concentration in the primary urine, as obtained in the calculation step, with the phosphorous concentration in the primary urine of a normal subject. Another aspect of the present invention relates to a method for suppressing the progression of chronic kidney disease in a non-human mammal subject.

Description

Technical Field

[0001] One aspect of the present invention relates to a method and a kit for examining the progression of chronic kidney disease in a subject. Another aspect of the present invention relates to a method for suppressing the progression of chronic kidney disease in a subject which is a non-human mammal.

Background Art

[0002] Chronic kidney disease, which is a type of kidney disorder, often occurs not only as a kidney disease such as chronic glomerulonephritis but also as a renal complication of lifestyle-related diseases such as diabetes and hypertension. In order to suppress the progression of chronic kidney disease, there is no way other than identifying the disease that caused the kidney disorder in each patient and thoroughly treating the disease. When chronic kidney disease progresses to end-stage renal failure, renal replacement therapy (for example, dialysis or kidney transplantation) becomes necessary. In such cases, not only does the patient's activities of daily living (ADL) and / or prognosis deteriorate, but it also places a great burden on medical economics. Therefore, the development of a test method for early detection of the progression of chronic kidney disease and a method for suppressing the progression of chronic kidney disease is desired.

[0003] A pathological condition commonly observed in the progression process of chronic kidney disease is a decrease in the number of functional nephrons. A nephron is a functional unit of the kidney consisting of a renal tubule and a glomerulus. When the number of nephrons decreases, a compensatory mechanism works to increase the excretion amount per nephron of substances to be excreted in urine. The problem at this time is phosphorus. When the phosphorus excretion amount per nephron increases, the phosphorus concentration in the primary urine rises, and fine particles containing calcium phosphate crystals (hereinafter, also referred to as "calciprotein particles") are formed in the renal tubule lumen. Calciprotein particles are composite nanoparticles of calcium phosphate crystals and fetuin A, which is a serum protein. Calciprotein particles are known to have an activity of damaging renal tubular cells. Therefore, the formation of calciprotein particles causes kidney damage.

[0004] Furthermore, calciprotein particles are known to appear in the bloodstream. Therefore, the formation of calciprotein particles can cause vascular diseases such as endothelial damage and vascular calcification, as well as non-infectious inflammation.

[0005] For example, Patent Document 1 describes a method for measuring calciprotein particles and a method for assisting in the examination of chronic kidney disease. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 6566697 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] As mentioned above, there is a need for diagnostic tests to detect the progression of chronic kidney disease at an early stage. However, no clinical indicators for the early detection of chronic kidney disease progression have been identified to date.

[0008] Therefore, the present invention aims to provide a simple diagnostic method for the early detection of the progression of chronic kidney disease. [Means for solving the problem]

[0009] The inventors investigated various means to solve the above-mentioned problems. The inventors found that an estimated value of the phosphorus concentration in the glomerular filtrate, which is related to the formation of calciprotein particles that cause kidney damage, can be calculated based on the phosphorus concentration and creatinine concentration measured by ordinary blood and urine tests. The inventors found that the progression of chronic kidney disease can be easily tested by comparing the estimated value of the phosphorus concentration in the glomerular filtrate in a subject with the phosphorus concentration in the glomerular filtrate in a normal subject. Based on the above findings, the inventors completed the present invention.

[0010] In other words, the present invention encompasses the following aspects and embodiments. (1) A method for examining the progression of chronic kidney disease in a subject, comprising the following steps: A measurement process for measuring the creatinine concentration in the target blood and urine, and the phosphorus concentration in the urine. The following equation (II):

number

[0011] The present invention makes it possible to provide a simple diagnostic method for the early detection of the progression of chronic kidney disease.

[0012] This specification includes the content described in the specification and / or drawings of Japanese Patent Application No. 2021-075828, which forms the basis of the priority claim of this application. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows an overview of phosphorus excretion in kidney nephrons. [Figure 2] Figure 2 shows the results of analyzing the relationship between the estimated phosphorus concentration in the glomerular filtrate and the relative mRNA level of the marker substance gene or the serum marker substance concentration in Experiment II, using a log-log plot. In the figure, A is the relationship between the estimated phosphorus concentration in the glomerular filtrate (horizontal axis, mg / dL) and the relative mRNA level of osteopontin, a marker of renal tubular injury (vertical axis), and B is the relationship between the estimated phosphorus concentration in the glomerular filtrate (horizontal axis, mg / dL) and the serum fibroblast growth factor 23 (FGF23) concentration (vertical axis, pg / mL). [Figure 3]Figure 3 shows the results of analyzing the relationship between the estimated phosphorus concentration in the glomerular filtrate and the concentration of marker substances in urine or serum in Experiment III, using a log-log plot. In the figure, A shows the relationship between the estimated phosphorus concentration in the glomerular filtrate (horizontal axis, mg / dL) and the L-type fatty acid-binding protein (L-FABP) concentration in urine (vertical axis, μg / gCre), and B shows the relationship between the estimated phosphorus concentration in the glomerular filtrate (horizontal axis, mg / dL) and the FGF23 concentration in serum (horizontal axis, pg / mL). [Figure 4] Figure 4 shows the progression of renal events in patients in the low-FGF23 group and the high-FGF23 group during the 5-year follow-up period in Study III. In the figure, the horizontal axis represents the follow-up period (days), and the vertical axis represents the probability of no renal events. [Figure 5] Figure 5 shows one embodiment of a method for examining the progression of chronic kidney disease in a subject according to one aspect of the present invention. [Figure 6] Figure 6 shows a flowchart of one embodiment of the present invention, in which the subject is a cat, illustrating a method for examining the progression of chronic kidney disease and a method for suppressing the progression of chronic kidney disease in a subject. [Modes for carrying out the invention]

[0014] <1. Methods for testing the progression of chronic kidney disease> The inventors have found that an estimated value of the phosphorus concentration in the glomerular filtrate, which is related to the formation of calciprotein particles that cause kidney damage, can be calculated based on the phosphorus and creatinine concentrations measured in routine blood and urine tests. The inventors have found that the progression of chronic kidney disease can be easily examined by comparing the estimated value of the phosphorus concentration in the glomerular filtrate in a subject with the phosphorus concentration in the glomerular filtrate in a normal subject. Therefore, one aspect of the present invention relates to a method for examining the progression of chronic kidney disease in a subject (hereinafter also referred to as the "examination method").

[0015] In each embodiment of the present invention, "progression of chronic kidney disease" means the worsening of symptoms of chronic kidney disease, which is a type of kidney damage. Typically, this is determined by findings of kidney damage from urine tests, blood tests, or imaging studies, etc., and / or normal values ​​(usually 60 mL / min 1.73 m³). 2 Chronic kidney disease (CKD) is diagnosed when the glomerular filtration rate (GFR) remains below 50% for several months or longer. The progression of chronic kidney disease can also be determined by a staging system based on indicators such as creatinine concentration, symmetric dimethylarginine (SDMA) concentration, urine condition, and blood pressure (Japanese Society of Nephrology, CKD Treatment Guidelines 2012, Tokyo Medical Publishing).

[0016] In the testing method of this embodiment, the subject is preferably a human or non-human mammal (for example, a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, baboon, or chimpanzee) subject or patient, more preferably a human patient or a feline, and even more preferably a human patient or a cat. Chronic kidney disease is increasing not only in humans but also in non-human mammals. Felines, especially cats, have a higher incidence of chronic kidney disease compared to humans and other mammals such as dogs, and it is known that many cases progress from chronic kidney disease to chronic renal failure. One of the reasons for this is that felines, especially cats, have a smaller total number of nephrons in a single kidney compared to other mammals (cats: 200,000, dogs: 400,000, humans: 1,000,000 or more). The incidence of chronic renal failure across all age groups has been reported to be 0.9% for dogs and 1.6% for cats, while the incidence of chronic renal failure in animals aged 15 years and older has been reported to be 5.7% for dogs and 15.3% for cats (DiBartola, 1995). Therefore, by applying the examination method of this embodiment to the aforementioned subjects, the progression of chronic kidney disease can be detected early with a simple examination.

[0017] The inspection method according to this embodiment includes a measurement step, a calculation step, and a phosphorus concentration comparison step. Furthermore, the inspection method according to this embodiment may optionally include a normal value determination step and a marker substance concentration comparison step. Each step will be described in detail below.

[0018] [1-1. Measurement process] This process includes measuring the creatinine concentration in the target blood and urine, as well as the phosphorus concentration in the urine.

[0019] In this process, the creatinine concentration in the blood and urine, and the phosphorus concentration in the urine, can be measured by blood and urine tests that are commonly performed in the art. The creatinine concentration in the blood (e.g., serum) is preferably quantified by an enzymatic method. The creatinine concentration in the urine is preferably quantified by an enzymatic method or a colorimetric method. The phosphorus concentration in the urine is preferably quantified by an enzymatic method, a colorimetric method, or a direct molybdate method, etc.

[0020] This step may further include measuring the concentration of a marker substance, which is fibroblast growth factor 23 (FGF23) in the blood of the subject or L-type fatty acid-binding protein (L-FABP) in the urine. In this embodiment, it is preferable to quantify the FGF23 concentration in the blood by ELISA. It is preferable to quantify the L-FABP concentration in the urine by ELISA.

[0021] [1-2. Calculation process] Figure 1 shows an overview of phosphorus excretion in renal nephrons. As will be explained in the following example (Test I), if the formation of calcium phosphate particles in the renal tubular fluid is required for tubular damage, then there should be a threshold for phosphorus concentration in the proximal tubular glomerular filtrate (PTFp) above which calcium phosphate precipitation and tubular damage occur. However, it is technically difficult to collect glomerular filtrate from living subjects and directly measure the phosphorus concentration in the glomerular filtrate. The inventors have found that it is possible to estimate the phosphorus concentration in the glomerular filtrate based on creatinine concentrations in the blood and urine, as well as phosphorus concentration in the urine.

[0022] This process is represented by the following equation (II):

number

[0023] In equation (II), ePTFp is an estimate of the phosphorus concentration in the glomerular filtrate. Up is the phosphorus concentration in urine. Ucr is the creatinine concentration in urine. Scr is the concentration of creatinine in the blood.

[0024] As explained above, the creatinine concentration in the blood and urine of the subject, as well as the phosphorus concentration in the urine, can be measured by blood and urine tests that are commonly performed in this field. Therefore, by performing this step using the concentrations of each substance obtained in the measurement step, it is possible to calculate an estimated value of the phosphorus concentration in the glomerular filtrate, which is an indicator of the progression of chronic kidney disease, with a simple test.

[0025] [1-3. Normal Value Determination Process] The testing method of this embodiment may optionally include a normal value determination step for determining normal values ​​of phosphorus concentration in glomerular filtrate and / or marker substance concentrations in blood or urine (e.g., FGF23 in blood and L-FABP in urine) in a normal subject. In the testing method of this embodiment, if normal values ​​of phosphorus concentration in glomerular filtrate and / or marker substance concentrations in blood or urine in a normal subject have already been determined, the steps described below can be carried out using these normal values. However, if these normal values ​​have not been determined in a normal subject, these normal values ​​can be obtained by carrying out this step.

[0026] This process can be carried out by analyzing the relationship between estimated phosphorus concentrations in glomerular filtrate obtained from multiple subjects and the concentrations of marker substances (e.g., FGF23 in blood and L-FABP in urine) using a log-log plot. The relationship between estimated phosphorus concentrations in glomerular filtrate and marker substance concentrations usually fits a two-phase linear regression. The concentrations of marker substances (e.g., FGF23 in blood and L-FABP in urine) are nearly constant in the low range of estimated phosphorus concentrations in glomerular filtrate (ePTFp), but begin to increase when the estimated phosphorus concentrations in glomerular filtrate (ePTFp) exceed a certain threshold (Figure 5). In this case, the nearly constant values ​​of marker substance concentrations (B and D in Figure 5) and the specific thresholds of estimated phosphorus concentrations (ePTFp) (A and C in Figure 5) can be determined as normal values ​​for marker substance concentrations and estimated phosphorus concentrations in normal subjects.

[0027] In this process, the phosphorus concentration in the glomerular filtrate of normal subjects, determined by log-log plot analysis with different marker substances, is usually the same value. For example, the phosphorus concentration in the glomerular filtrate corresponding to the FGF23 concentration in the blood of normal subjects, determined by log-log plot analysis of the estimated phosphorus concentration in the glomerular filtrate (ePTFp) and the FGF23 concentration in the blood, is usually the same value as the phosphorus concentration in the glomerular filtrate corresponding to the L-FABP concentration in the urine of normal subjects, determined by log-log plot analysis of the estimated phosphorus concentration in the glomerular filtrate (ePTFp) and the L-FABP concentration in the urine (A=C in Figure 5).

[0028] Normal values ​​for phosphorus concentration in glomerular filtrate and / or marker substance concentrations in blood or urine (e.g., FGF23 in blood and L-FABP in urine) in normal subjects can vary depending on the type of subject. For example, in human subjects, the phosphorus concentration in glomerular filtrate in normal subjects is usually 5 mg / dL or less, for example, in the range of 0.1 to 3 mg / dL, and particularly 2.3 mg / dL. For example, in human subjects, the FGF23 concentration in blood (e.g., serum) in normal subjects is usually 100 pg / mL or less, for example, in the range of 10 to 80 pg / mL, and particularly 53 pg / mL. For example, in human subjects, the L-FABP concentration in urine in normal subjects is usually 10 μg / gCre or less, for example, in the range of 1 to 10 μg / gCre, and particularly 8.4 μg / gCre. For example, in felines, especially cats, the phosphorus concentration in the glomerular filtrate of healthy animals is usually below 700 mg / dL, typically in the range of 50–700 mg / dL (Geddes, RF et al., The effect of feeding a renal diet on plasma fibroblast growth factor 23 concentrations in cats with stable azotemic chronic kidney disease. J Vet Intern Med, 27, 1354-1361, 2013).

[0029] [1-4. Phosphorus concentration comparison process] This step includes comparing the estimated phosphorus concentration in the glomerular filtrate obtained in the calculation step with the phosphorus concentration in the glomerular filtrate of a normal subject.

[0030] In this process, the normal value for phosphorus concentration in the glomerular filtrate of a normal subject may be a predetermined value, or it may be a value obtained by performing the normal value determination process described above each time the test method of this embodiment is performed.

[0031] In this step, the estimated phosphorus concentration in the glomerular filtrate obtained in the calculation step is compared with the phosphorus concentration in the glomerular filtrate of a normal subject. If the estimated phosphorus concentration in the glomerular filtrate of a subject exceeds that of a normal subject, it can be determined that the subject is at high risk of progression of chronic kidney disease. Therefore, by performing this step, the progression of chronic kidney disease can be detected early based on the estimated phosphorus concentration in the glomerular filtrate calculated based on phosphorus concentration and creatinine concentration obtained from a simple test.

[0032] [1-5. Marker substance concentration comparison process] The testing method of this embodiment may further include, if desired, a marker substance concentration comparison step of comparing the concentration of the marker substance in the blood or urine of a subject with the concentration of the marker substance in the blood or urine of a normal subject.

[0033] In this process, the marker substances in the blood or urine are, for example, FGF23 in the blood and L-FABP in the urine.

[0034] In this process, the normal values ​​for the concentration of marker substances in the blood or urine of a normal subject may be predetermined values, or the values ​​obtained by performing the normal value determination process described above each time the test method of this embodiment is performed may be used.

[0035] In this step, the concentration of the marker substance in the blood or urine obtained in the measurement step is compared with the concentration of the marker substance in the blood or urine of a normal subject. If the result of the comparison shows that the concentration of the marker substance in the blood or urine of the subject exceeds that of a normal subject, it can be determined that the subject is at high risk of progression of chronic kidney disease. As explained above, the normal value of the concentration of the marker substance in the blood or urine of a normal subject can be determined by analyzing the relationship between the estimated phosphorus concentration in the glomerular filtrate and the concentration of the marker substance in the blood or urine using a log-log plot. Therefore, by performing this step, the progression of chronic kidney disease can be detected early based on the concentration of the marker substance in the blood or urine obtained by a simple test and the estimated phosphorus concentration in the glomerular filtrate calculated based on the phosphorus concentration and creatinine concentration, which are also obtained by a simple test.

[0036] In the testing method of this embodiment, each step may be performed only once or multiple times. When each step is performed multiple times, it is preferable to perform the measurement step, calculation step and phosphorus concentration comparison step (and optionally marker substance concentration comparison step) again after the previous phosphorus concentration comparison step (and optionally marker substance concentration comparison step) to determine the change in the risk of progression of chronic kidney disease. In the testing method of this embodiment, by performing each step multiple times, changes in the risk of progression of chronic kidney disease can be detected early with a simple testing method.

[0037] <2. A program to implement methods for testing the progression of chronic kidney disease> Another aspect of the present invention relates to a program (hereinafter also referred to as the "test program") for performing a method for testing the progression of chronic kidney disease in a subject.

[0038] The inspection program of this embodiment can be used to execute the inspection method of one embodiment of the present invention on an analysis device (e.g., a computer, smartphone, or tablet terminal). The inspection program of this embodiment includes a measurement step, a calculation step, and a phosphorus concentration comparison step. The inspection program of this embodiment may also optionally include a normal value determination step and a marker substance concentration comparison step. Each step corresponds to each step of the inspection method of one embodiment of the present invention described above.

[0039] The inspection program of this embodiment is typically used in a form stored on a storage medium such as memory or a hard drive. Therefore, the inspection program of this embodiment may be provided in the form of a storage medium (e.g., memory or a hard drive) that stores the inspection program.

[0040] <3. Kit for testing the progression of chronic kidney disease> Another aspect of the present invention relates to a kit for testing the progression of chronic kidney disease in a subject (hereinafter also referred to as the "test kit"). The test kit of this aspect includes at least a measuring member for measuring the creatinine concentration and phosphorus concentration in the urine of the subject, and an instruction manual.

[0041] In the test kit of this embodiment, the measuring member is used to measure the creatinine concentration and phosphorus concentration in the urine of the subject. Preferably, the measuring member is one that quantifies the creatinine concentration and phosphorus concentration in the urine by the means described above. Preferably, the measuring member is a test strip that quantifies the creatinine concentration and phosphorus concentration in the urine by a colorimetric method. In this embodiment, the creatinine concentration and phosphorus concentration in the urine of the subject can be easily measured by comparing the color of the colored test strip with the color of a color sample. For example, the creatinine concentration and phosphorus concentration in the urine can be determined by visually comparing the color of the colored test strip with the color of the color sample. Alternatively, the colored test strip and color sample may be photographed with a camera, the color of the colored test strip may be quantified from the obtained image data, and this value may be substituted into a calibration curve created in advance by quantifying the color of the color sample to calculate the creatinine concentration and phosphorus concentration in the urine. The color of the colored test strip can be quantified using commercially available image analysis software, or a program can be prepared to capture image data, quantify the color, and calculate the density.

[0042] In the test kit of this embodiment, the instructions describe the procedure for the test method of one embodiment of the present invention as described above. By measuring the creatinine concentration and phosphorus concentration in the urine of the subject using the measuring member included in the test kit of this embodiment, and performing the test method of one embodiment of the present invention based on the instructions using the obtained values, it is possible to calculate an estimated value of the phosphorus concentration in the glomerular filtrate, which is an indicator of the progression of chronic kidney disease.

[0043] In the testing method according to one aspect of the present invention described above, the creatinine concentration in the blood (Scr) used in the calculation step does not usually fluctuate significantly in a short period of time, whereas the creatinine concentration in the urine (Ucr) and the phosphorus concentration in the urine (Up) are known to fluctuate depending on the daily diet and / or the amount of water consumed. Therefore, if the creatinine concentration in the blood (Scr) of the subject is obtained in advance, the creatinine concentration in the urine (Ucr) and the phosphorus concentration in the urine (Up) of the subject can be measured periodically using the measuring element included in the testing kit of this aspect. By performing the testing method according to one aspect of the present invention based on the instructions using the obtained creatinine concentration in the urine (Ucr) and phosphorus concentration in the urine (Up) and the creatinine concentration in the blood (Scr) obtained in advance, an estimated value of the phosphorus concentration in the glomerular filtrate, which is an indicator of the progression of chronic kidney disease, can be calculated, and the progression of chronic kidney disease can be easily tested.

[0044] In one embodiment, the test kit of this embodiment is preferably used in combination with a test program of one embodiment of the present invention, and more preferably in combination with an analysis device (e.g., a computer, smartphone, or tablet terminal) having a camera and a storage medium (e.g., memory or hard drive, etc.) that stores the test program of one embodiment of the present invention and a program for performing image data capture, color quantification, and density calculation of the measuring member (e.g., test paper) described above. In this embodiment, for example, the subject can use the analysis device to capture image data of the colored measuring member (e.g., test paper), perform color quantification, and density calculation, and further execute the test program of one embodiment of the present invention. This allows the subject to easily check the progression of chronic kidney disease themselves.

[0045] <4. Methods to suppress the progression of chronic kidney disease> Another aspect of the present invention relates to a method for suppressing the progression of chronic kidney disease in a subject (hereinafter also referred to as the "suppression method").

[0046] In each embodiment of the present invention, "suppression of the progression of chronic kidney disease" means slowing the progression of chronic kidney disease by treating the symptoms of chronic kidney disease.

[0047] In the suppression method of this embodiment, the subject is preferably a human or non-human mammal (for example, a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, baboon, or chimpanzee) subject or patient, more preferably a human patient or a feline, and even more preferably a human patient or a cat. In a particular embodiment, the subject is a non-human mammal as exemplified above, preferably a feline, and more preferably a cat. By applying the suppression method of this embodiment to the subject, the progression of chronic kidney disease can be detected early with a simple examination, and the progression of chronic kidney disease can be suppressed.

[0048] The suppression method according to this embodiment includes a measurement step, a calculation step, a phosphorus concentration comparison step, and a treatment step. Furthermore, the suppression method according to this embodiment may also include a normal value determination step and a marker substance concentration comparison step. Each step will be described in detail below.

[0049] In the suppression method of this embodiment, the measurement step, calculation step, phosphorus concentration comparison step, normal value determination step, and marker substance concentration comparison step can be carried out in the same manner as the steps in the inspection method of one embodiment of the present invention described above.

[0050] [4-1. Treatment process] This process includes providing therapeutic intervention for the progression of chronic kidney disease based on the comparison results of the phosphorus concentration comparison process.

[0051] In this process, therapeutic intervention is preferably carried out by dietary therapy or drug therapy. Dietary therapy may include, for example, restricting phosphorus intake through a low-phosphorus diet. For example, if the subject is a human, the low-phosphorus diet is preferably a plant-based food or a food with a low content of food additives containing phosphorus. For example, if the subject is a feline, especially a cat, the low-phosphorus diet is preferably a low-phosphorus pet food, a plant-based food, or a food with a low content of food additives containing phosphorus. Drug therapy may include, for example, the administration of drugs such as phosphorus binders and calciprotein (CPP) formation inhibitors. Phosphorus binders include calcium carbonate, non-calcium-containing phosphorus binders, and Na + / H + It is preferable that the inhibitor is an exchange transporter 3 (NHE3) inhibitor or a sodium-dependent phosphate transporter 2b (Npt2b) inhibitor. The CPP formation inhibitor is preferably a bisphosphonate, magnesium, or zinc.

[0052] In the suppression method of this embodiment, each step may be performed only once or multiple times. If each step is performed multiple times, the same therapeutic intervention may be performed in each treatment step, or different therapeutic interventions may be performed. After the previous treatment step, it is preferable to perform the measurement step, calculation step, and phosphorus concentration comparison step (and optionally a marker substance concentration comparison step) again to determine the change in the risk of progression of chronic kidney disease, and based on that determination, decide whether to perform the same therapeutic intervention as the previous time or a different therapeutic intervention in the current treatment step. In the suppression method of this embodiment, by performing each step multiple times, it is possible to detect changes in the risk of progression of chronic kidney disease early using simple testing methods and to implement therapeutic intervention early. This makes it possible to suppress the progression of chronic kidney disease, for example, to end-stage renal failure. [Examples]

[0053] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0054] <Test I: Calculation of estimated phosphorus concentration in glomerular filtrate> Figure 1 shows an overview of phosphate excretion in renal nephrons. If the formation of calcium phosphate particles in the renal tubular fluid is required for tubular injury, then there must be a threshold for the phosphate concentration in the proximal tubular glomerular filtrate (PTFp) above which calcium phosphate precipitation and tubular injury occur. Since it is technically difficult to collect glomerular filtrate from the corticomedullary junction of living mice, we decided to estimate PTFp from the phosphate and creatinine concentrations in the blood and urine based on the following assumptions: First, the phosphate concentration in glomerular filtration is equal to the phosphate concentration in the blood. Second, the proximal tubule reabsorbs 70% of the filtered water under standard conditions (Figure 1) (Baum M, and Quigley R. Proximal tubule water transport-lessons from aquaporin knockout mice. Am J Physiol Renal Physiol. 2005;289(6):F1193-4). This results in a 3.33-fold increase in solute concentration. Thirdly, phosphate reabsorption occurs exclusively in the proximal tubule (Figure 1) (Blaine J, Chonchol M, and Levi M. Renal control of calcium, phosphate, and magnesium homeostasis. Clinical journal of the American Society of Nephrology: CJASN. 2015;10(7):1257-72). Finally, the phosphate excretion rate (FEp), defined as the ratio of phosphate excretion to creatinine excretion, indicates the phosphate fraction that was not reabsorbed in the proximal tubule. Therefore, the blood phosphate concentration (Sp) and the value obtained by multiplying FEp by 3.33 are estimated to reflect PTFp. This value is taken as the estimated value of PTFp.

[0055]

number

[0056] As is clear from equation (I), the phosphorus concentration in the blood, Sp, cancels out in the equation and therefore does not affect the estimated phosphorus concentration in the glomerular filtrate. In other words, the estimated phosphorus concentration in the glomerular filtrate, ePTFp, is expressed by the following equation (II).

[0057]

number

[0058] To verify the estimated phosphorus concentration in the glomerular filtrate represented by equation (II), it was compared with measured values ​​of phosphorus concentration in the glomerular filtrate. Table 1 shows the measured values ​​of phosphorus concentration in the glomerular filtrate collected from the proximal tubules of living SD rats by micropuncture, and the estimated values ​​of phosphorus concentration in the glomerular filtrate calculated based on equation (II). The measured values ​​of Sp, FEp, and phosphorus concentration in the glomerular filtrate in the table are described in the literature (Bank, N., et al. Micropuncture study of renal phosphate transport in rats with chronic renal failure and secondary hyperparathyroidism. J Clin Invest 61, 884-894, 1978).

[0059] [Table 1]

[0060] As shown in Table 1, the estimated phosphorus concentration in the glomerular filtrate calculated based on equation (II) was in good agreement with the measured phosphorus concentration in the glomerular filtrate.

[0061] <Experiment II: Relationship between phosphorus concentration in the glomerular filtrate and tubular injury in model mice> Normal mice and unilateral nephrectomized mice were fed a diet containing 0.35%, 1.0%, 1.5%, or 2.0% inorganic phosphate for 12 weeks. The relative mRNA levels of designated marker substance genes were determined by quantitative RT-PCR. The concentration of serum fibroblast growth factor 23 (FGF23) was quantified by ELISA. Serum creatinine concentration was quantified by enzymatic methods. Urinary creatinine concentration was quantified by enzymatic methods. In addition, urinary phosphorus concentration was quantified by the molybdate direct method. An estimated value of phosphorus concentration in the glomerular filtrate was calculated based on equation (II). Figure 2 shows the results of analyzing the relationship between the estimated phosphorus concentration in the glomerular filtrate and the relative mRNA levels of marker substance genes or serum marker substance concentrations using a log-log plot. In the figure, A shows the relationship between the estimated phosphorus concentration in the glomerular filtrate (horizontal axis, mg / dL) and the relative mRNA level of osteopontin, a marker of renal tubular injury (vertical axis), while B shows the relationship between the estimated phosphorus concentration in the glomerular filtrate (horizontal axis, mg / dL) and the serum FGF23 concentration (vertical axis, pg / mL).

[0062] As shown in Figure 2, in a log-log plot, the relationship between estimated phosphorus concentration in the glomerular filtrate, the expression levels of marker substance genes, and serum FGF23 concentration fit a two-phase linear regression with a zero slope in the first phase. Specifically, serum FGF23 concentration remained nearly constant in the low range of estimated phosphorus concentration in the glomerular filtrate (ePTFp), but began to increase when the estimated phosphorus concentration in the glomerular filtrate (ePTFp) exceeded 5.18 mg / dL (Figure 2B). Consistent with the increase in serum FGF23 concentration, the expression level of osteopontin, a marker of tubular injury, also began to increase (Figure 2A). This was followed by increases in the expression levels of inflammatory markers such as monocyte chemotactic protein-1 (MCP1) and transforming growth factor-β1 (TGFβ1) (data not shown). Increased expression of fibrosis markers and decreased renal function (decreased creatinine clearance) became clearly apparent when the estimated phosphorus concentration in the glomerular filtrate reached approximately 10 mg / mL (data not shown).

[0063] The mechanism of renal tubular damage associated with increased FGF23 concentration is thought to be as follows: Increased phosphorus intake and / or a decrease in nephron number leads to an increase in phosphorus excretion per nephron to maintain phosphorus homeostasis. This requirement aligns with an increase in FGF23 concentration, a hormone that increases phosphorus excretion per nephron. However, increased FGF23 concentration increases phosphorus concentration in the glomerular filtrate, raising the risk of calcium phosphate particle formation in the renal tubules. Calcium phosphate particles in the renal tubules bind to Toll-like receptor 4 (TLR4) expressed in tubular cells, inducing tubular damage. When nephrons are damaged due to tubular damage, FGF23 further increases to compensate for the decrease in nephron number unless phosphorus intake decreases, thereby triggering a vicious cycle of deterioration leading to progressive nephron loss.

[0064] <Study III: Relationship between phosphorus concentration in the glomerular filtrate and tubular injury in human patients with chronic kidney disease> We investigated whether the relationship between estimated phosphorus concentration in glomerular filtrate and FGF23 concentration observed in mice in Experiment II could also be observed in humans. 148 non-dialysis patients with chronic kidney disease (CKD) at various stages were selected to measure estimated phosphorus concentration in glomerular filtrate and serum FGF23 levels. L-type fatty acid-binding protein (L-FABP) concentration in urine was quantified by ELISA. Serum FGF23 concentration was quantified by ELISA. Figure 3 shows the results of analyzing the relationship between estimated phosphorus concentration in glomerular filtrate, urinary L-FABP concentration, and serum FGF23 concentration using log-log plots. In the figure, A shows the relationship between estimated phosphorus concentration in glomerular filtrate (horizontal axis, mg / dL) and urinary L-FABP concentration (vertical axis, μg / gCre), and B shows the relationship between estimated phosphorus concentration in glomerular filtrate (horizontal axis, mg / dL) and serum FGF23 concentration (horizontal axis, pg / mL).

[0065] As shown in Figure 3, in this cross-sectional study, the relationship between estimated phosphorus concentration in the glomerular filtrate and FGF23 concentration fit a biphase linear regression similar to that of Study II. Serum FGF23 concentration remained nearly constant in the low range of estimated phosphorus concentration in the glomerular filtrate (ePTFp), but began to increase when the estimated phosphorus concentration in the glomerular filtrate (ePTFp) exceeded 2.32 mg / dL (Figure 3B). Similarly, urinary L-FABP concentration remained nearly constant in the low range of estimated phosphorus concentration in the glomerular filtrate (ePTFp), but began to increase when the estimated phosphorus concentration in the glomerular filtrate (ePTFp) exceeded 2.32 mg / dL (Figure 3A). The results suggest that patients with an estimated phosphorus concentration in the glomerular filtrate exceeding 2.32 mg / dL and a serum FGF23 concentration exceeding 53 pg / mL have tubulointerstitial damage and progressive CKD (dotted and solid arrows in Figures 3A and 3B).

[0066] To investigate this possibility, a planned study was conducted using blood samples from preserved specimens of the EMPATHY study at Jichi Medical University to explore the relationship between serum FGF concentration and incidental renal events. These preserved specimens were from patients with hyperlipidemia and diabetic retinopathy, but also with progressive CKD (eGFR < 30 mL / min / 1.73 m²). 2The data was obtained from patients who did not have [specific condition]. The median FGF23 concentration was 54.7 pg / mL, with an interquartile range (IQR) of 44.0 pg / mL to 69.0 pg / mL. 5039 patients were stratified into two groups, the low-FGF23 group and the high-FGF23 group, based on a reference value of 53 pg / mL serum FGF concentration. During the 5-year follow-up period, renal events were defined as the initiation of renal replacement therapy or at least a twofold increase in serum creatinine concentration in 100 patients. This was 0.6% (14 out of 2336 patients) in the low-FGF23 group and 3.2% (86 out of 2703 patients) in the high-FGF23 group. Figure 4 shows the progression of renal events in the low-FGF23 group and the high-FGF23 group during the 5-year follow-up period. In the figure, the horizontal axis represents the follow-up period (days), and the vertical axis represents the probability of no renal events.

[0067] As shown in Figure 4, an increased risk of renal events was observed in the high-FGF23 group. Cox regression analysis showed that the high-FGF23 group had a significantly higher risk of renal events compared to the low-FGF23 group (hazard ratio (HR) 5.18, 95% confidence interval (CI) 2.94–9.11, P<0.001 by log-rank test). This relationship was maintained even after adjusting for age, sex, body mass index, and importantly, serum creatinine (HR 2.84, 95% CI 1.57–5.13, P<0.001). The results of this study clearly indicate that human patients with serum FGF23 concentrations exceeding 53 pg / mL are at high risk of chronic kidney disease progression.

[0068] <Study IV: Examination and treatment of the progression of chronic kidney disease in the subjects> Figure 5 shows one embodiment of a method for examining the progression of chronic kidney disease in a subject according to one aspect of the present invention. As shown in Figure 5, the creatinine concentration in the subject's blood (Scr), creatinine concentration in the urine (Ucr), and phosphorus concentration in the urine (Up) are measured (measurement step). Optionally, marker substances, such as the FGF23 concentration in the blood and the L-FABP concentration in the urine, are also measured. The following formula (II):

number

[0069] The method of the present invention can be applied not only to humans but also to various non-human mammals. Figure 6 shows a flowchart of one embodiment of a method for examining the progression of chronic kidney disease and a method for suppressing the progression of chronic kidney disease in a subject according to one aspect of the present invention, in which the subject is a cat. As shown in Figure 6, the measurement step and calculation step are performed on the cat subject. If the normal value A or C for phosphorus concentration in the glomerular filtrate and the concentration of marker substances, for example, the FGF23 concentration B in the blood and the L-FABP concentration D in the urine, have not been determined in a normal cat subject, the normal value determination step should be performed. The estimated value of phosphorus concentration in the glomerular filtrate (ePTFp) obtained in the calculation step is compared with the phosphorus concentration A or C in the glomerular filtrate of a normal subject (phosphorus concentration comparison step). In addition, the concentration of marker substances in the subject, for example, the FGF23 concentration in the blood or the L-FABP concentration in the urine, is compared with the concentration of marker substances in a normal subject, for example, the FGF23 concentration B in the blood or the L-FABP concentration D in the urine (marker substance concentration comparison step). In the phosphorus concentration comparison step, if the estimated phosphorus concentration in the glomerular filtrate of a cat (ePTFp) exceeds phosphorus concentration A or C (A and C are usually the same value) in the glomerular filtrate of a normal cat, it can be determined that the cat is at high risk of progression of chronic kidney disease. Similarly, in the marker substance concentration comparison step, if the FGF23 concentration in the blood or L-FABP concentration in the urine of a cat exceeds FGF23 concentration B or L-FABP concentration D in the blood or urine of a normal cat, it can be determined that the cat is at high risk of progression of chronic kidney disease.

[0070] Based on the comparison results of the phosphorus concentration comparison step or the marker substance concentration comparison step, if it is determined that the risk of progression of chronic kidney disease is high in cats, therapeutic intervention will be implemented to prevent the progression of chronic kidney disease (treatment step). Therapeutic intervention can be carried out, for example, by restricting phosphorus intake through feeding low-phosphorus cat food (treatment (1)). On the other hand, if it is determined that the risk of progression of chronic kidney disease is low in cats, no special treatment is necessary.

[0071] After a certain period has elapsed since the therapeutic intervention by treatment (1), the measurement process, calculation process, phosphorus concentration comparison process, and marker substance concentration comparison process are carried out. If the results indicate that the risk of progression of chronic kidney disease in cats remains high, further therapeutic intervention is performed to prevent the progression of chronic kidney disease (treatment process). Further therapeutic intervention may be carried out, for example, by restricting phosphorus intake through feeding low-phosphorus cat food in addition to administering phosphate binders (treatment (2)). On the other hand, if the therapeutic intervention by treatment (1) indicates that the risk of progression of chronic kidney disease in cats has decreased, the therapeutic intervention by treatment (1) should be continued.

[0072] As described above, by determining the risk of chronic kidney disease progression based on the method for examining the progression of chronic kidney disease in the subjects of the present invention, the progression of chronic kidney disease can be predicted. Therefore, by providing early therapeutic intervention to subjects for whom the progression of chronic kidney disease is predicted by the method of the present invention, the progression of chronic kidney disease, such as the transition to end-stage renal failure, can be suppressed.

[0073] <Study V: A kit to test the progression of chronic kidney disease in the subjects> Test strips were prepared to quantify creatinine and phosphorus concentrations in urine using a colorimetric method. Color samples of the creatinine and phosphorus test strips were photographed with a camera. Using image analysis software (Image J), ​​the colors of the creatinine and phosphorus color samples were quantified from the obtained image data to create calibration curves for creatinine and phosphorus concentrations. Urine samples 1-3 were dropped onto the test strips, and photographs were taken after 60 seconds. Using Image J, the colors of the test strips were quantified from the obtained image data. The numerical values ​​(signal intensity) of the test strip colors were substituted into the calibration curve to calculate the creatinine and phosphorus concentrations in the urine. The results are shown in Tables 2 and 3.

[0074] [Table 2]

[0075] [Table 3]

[0076] It should be noted that the present invention is not limited to the embodiments described above, and various modifications are included. For example, the embodiments described above are described in detail for the purpose of clearly illustrating the present invention, and are not necessarily limited to those having all the configurations described. Furthermore, it is possible to add, delete, and / or replace some of the configurations in each embodiment with other configurations.

[0077] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

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