Method for evaluating kidney function decline risk

Abstract

To develop a new, simple method that can be an index for a kidney function decline risk in diabetes, particularly, type 2 diabetes.SOLUTION: Provided is a method for evaluating a kidney function decline risk in a subject having diabetes, the method including: (A) a step of acquiring a body fluid sample obtained from the subject; and (B) a step of measuring the concentration of cystatin A present in the body fluid sample.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method, a biomarker, and a kit for evaluating the risk of renal function decline in a subject with diabetes.

Background Art

[0002] In the typical clinical course of diabetic nephropathy (DMN), when caused by type 1 diabetes, it develops due to the appearance of microalbuminuria, and after progressing to overt albuminuria or persistent proteinuria, it follows a course leading to end-stage renal failure from a decrease in glomerular filtration rate (GFR) (Non-Patent Document 1). In type 1 diabetes, GFR transiently increases at an early stage of onset, showing glomerular hyperfiltration.

[0003] On the other hand, in type 2 diabetes, although the onset time of diabetes is unknown and hypertension is often already present before the onset of DMN compared to type 1 diabetes, if DMN develops, its clinical course is considered to be similar to that of DMN caused by type 1 diabetes.

[0004] So far, for the diagnosis of DMN, urinary albumin-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) have been used. However, in recent years, it has been pointed out that there are limitations in using ACR and eGFR for diagnosis, such as cases where albuminuria increases or progressive renal function decline is observed without proteinuria being recognized (Non-Patent Document 2).

[0005] Against this background, in addition to typical DMN, diabetic kidney disease (DKD) has been proposed as a concept including atypical diabetes-related kidney diseases. In recent years, biomarker discovery research on DKD has been conducted, but in conventional studies, cases of renal function decline are often included in the analysis target, and there has been a problem that it is difficult to accurately determine whether the reported biomarker fluctuates prior to renal function decline or fluctuates along with renal function decline.

Prior Art Documents

Non-Patent Documents

[0006] [Non-Patent Document 1] Acta Diabetol, 51: 905-15, 2014 [Non-Patent Document 2] Diabetes Care 38: 954-962, 2015 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] Therefore, an object of the present invention is to develop a novel and simple method that can serve as an indicator of the risk of renal function decline in diabetes, particularly type 2 diabetes. In particular, it is an object of the present invention to identify factors that fluctuate prior to the decline of eGFR and to provide a method for predicting the risk of future renal function decline by monitoring these factors. [Means for Solving the Problems]

[0008] As a result of intensive studies to solve the above problems, the present inventors have, for the first time, identified that certain urinary proteins are factors that fluctuate prior to the decline of eGFR in diabetic patients with normal or mildly decreased renal function (i.e., a state where the decline of renal function has not yet occurred significantly), and have newly found that the risk of renal function decline can be evaluated by measuring the concentration of such specific proteins, thus completing the present invention.

[0009] That is, in one aspect, the present invention relates to a method for measuring the concentration of a specific protein in urine for evaluating, determining, or predicting the risk of renal function decline in a subject with type 2 diabetes. More specifically, <1> A method for evaluating the risk of renal function decline in a diabetic subject, comprising: (A) a step of obtaining a body fluid sample obtained from the subject; and (B) a step of measuring the concentration of cystatin A present in the body fluid sample. <2> The method according to <1> above, wherein the subject has normal or mildly decreased renal function. <3>The method according to <1> above, wherein the estimated glomerular filtration rate (eGFR) of the subject is 60 or more; <4>The method according to <1> above, further comprising a step of comparing the measured concentration of cystatin A with a control value; <5>The method according to <1> above, comprising repeating the steps (A) and (B) a plurality of times at regular intervals and monitoring the increase or decrease in the concentration of cystatin A; <6>The method according to <1> above, wherein the body fluid sample is urine; <7>The method according to <1> above, wherein the subject is a human; <8>The method according to <1> above, wherein the measurement in the step (B) is performed by at least one of mass spectrometry, gas chromatography, liquid chromatography, gas chromatography / mass spectrometry, liquid chromatography / mass spectrometry, nuclear magnetic resonance spectroscopy, and immunoassay method; and <9>The method according to <1> above, which is used in combination with another test method for measuring at least one selected from the group consisting of estimated glomerular filtration rate (eGFR), hemoglobin A1c (HbA1c), urinary albumin / creatinine ratio (UACR), and hemoglobin (Hb). is provided.

[0010] In another aspect, the present invention also relates to a biomarker or kit for evaluating the risk of renal function decline in diabetes, and more specifically, <10>A biomarker for evaluating the risk of renal function decline in diabetes, comprising cystatin A; and <11>A kit for evaluating the risk of renal function decline in diabetes, comprising a measurement reagent for cystatin A is provided.

Advantages of the Invention

[0011] According to the present invention, for diabetic patients with normal or slightly decreased renal function (i.e., a state where the decrease in renal function has not yet occurred significantly) by existing diagnostic methods such as eGFR, the risk of future renal function decline can be predicted simply and with high accuracy by measuring the concentration of protein markers in urine. In addition, by using the information obtained by the method of the present invention in combination with general test information such as HbA1c and eGFR, it is also useful in that the risk of renal function decline can be predicted at an earlier stage.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited by these descriptions, and other than the following examples, it can be appropriately modified and implemented without departing from the spirit of the present invention.

[0014] The first aspect of the present invention is a method for evaluating the risk of renal function decline in a subject with diabetes, characterized by including the following steps (A) and (B): (A) A step of obtaining a body fluid sample obtained from the subject, and (B) A step of measuring the concentration of cystatin A present in the body fluid sample.

[0015] The kidney is an essential element of the urinary system and also provides homeostatic functions such as electrolyte control, maintenance of acid-base balance, and blood pressure control. "Renal function" as used herein includes filtering blood, secreting various toxins and metabolic waste products, and / or reabsorbing specific nutrients, amino acids, and water. For example, estimated glomerular filtration rate (eGFR), which is widely used as an indicator of chronic kidney disease (CKD), is known to indicate the degree of renal function in the kidney to excrete waste products into urine. "Risk of decreased renal function" means the risk that the function of the above-mentioned kidney may decrease after a certain period of time. More specifically, it means the possibility that the renal function represented by eGFR or the like may decrease in the future in a subject who has been diagnosed as having diabetes but whose renal function currently remains normal or slightly decreased. In the present invention, "diabetes" includes both type 1 and type 2, preferably type 2 diabetes.

[0016] The biological species to be the subject is not particularly limited, but is typically a mammal, preferably a human. In the method of the present invention, as described above, it is preferable to target a subject who has been diagnosed as having diabetes but whose renal function currently remains normal or slightly decreased. More specifically, a human with an estimated glomerular filtration rate (eGFR) of 60 or more is preferable.

[0017] In step (A), the body fluid sample obtained from the subject can be blood, serum, plasma, or urine, but is typically urine. The body fluid sample obtained from the subject can be used as it is, but in some cases, treatments such as filtration with a filter or the like can be appropriately performed.

[0018] In step (B), the concentration of the protein that serves as a biomarker for the risk of reduced renal function is measured for the body fluid sample obtained in step (A). The biomarker is cystatin A. Measuring the "concentration" of these biomarkers refers to performing detection for quantifying the biomarkers contained in the sample, and in addition, it may include cases where substances derived from the biomarkers, such as derivatives or modified products of the biomarkers, are directly detected but the biomarkers themselves are not directly detected.

[0019] As will be described later in the examples, the present inventors have for the first time found that the cystatin A, which is a biomarker, shows fluctuations in urine concentration prior to the decline of renal function in diabetic patients whose renal function remains normal or slightly reduced. Therefore, by measuring the concentration of cystatin A in the body fluid sample, the risk of future renal function decline can be evaluated, determined, or predicted. "Evaluating, determining, or predicting" the risk includes, for example, outputting by dividing the possibility of renal function decline at present or in the future into several levels or outputting numerically.

[0020] Cystatin A is a protein consisting of 98 amino acid residues with a molecular weight of approximately 11 kDa, belonging to the Cystatin superfamily, and is known to function as an inhibitor of cysteine proteases represented by cathepsin, preventing the disordered degradation of proteins inside cells (for example, V. Turk et al., FEBS Letters, 285, 213 - 219, 1991). The specific sequence can be referred to, for example, in JP-A-2007-291089. For the specific sequence, reference can be made to, for example, JP-A-2007-291089.

[0021] As will be described later in the examples, by using cystatin A in combination with the test information (HbA1c, eGFR, UACR, Hb, etc.) obtained during the general medical treatment of diabetic patients, the risk of renal function decline can be more accurately predicted at an early stage.

[0022] Therefore, in a preferred embodiment, the method of the present invention can be used in combination with another test method for measuring at least one selected from the group consisting of estimated glomerular filtration rate (eGFR), hemoglobin A1c (HbA1c), urinary albumin / creatinine ratio (UACR), and hemoglobin (Hb).

[0023] In addition, the method of the present invention can further include a step of comparing the concentration of cystatin A measured in step (B) with a control value. As used herein, "control" has the broadest meaning used in the technical field of the present invention. For example, the term "control" means an object to be compared with an object to be measured using the method of the present invention, and the protein concentration of the control can also be measured according to the method of the present invention. For example, it may include healthy subjects, subjects suffering from diseases other than diabetes, subjects who were previously diagnosed with diabetes but are in remission, subjects before or after administration of a candidate drug for treating diabetes, or the subject's own past self, etc. The control may be a sample derived from a population, and the "control value" may be the average value of those populations.

[0024] In a preferred embodiment, the method of the present invention includes repeating steps (A) and (B) a plurality of times at regular intervals over a certain period of time and monitoring the increase or decrease in the concentration of cystatin A. That is, by regularly measuring the protein concentration using the method of the present invention for the same subject, it is possible to confirm a sign of renal function decline from the trend of the concentration change. The "regular interval" can be, for example, an interval of one month, six months, one year, etc.

[0025] The measurement in step (B) can be carried out using any measuring means that is used in the relevant technical field for detecting the amount of protein. For example, mass spectrometry, gas chromatography, liquid chromatography, nuclear magnetic resonance spectroscopy, or a combination thereof, or immunological detection methods typified by ELISA or flow cytometry inspection can be used. These measurement methods can also be used in combination. From the viewpoint of suitably separating and detecting the target marker from a sample containing various types of substances, the marker contained in the sample is more preferably detected by gas chromatography / mass spectrometry (hereinafter referred to as GC / MS) or liquid chromatography / mass spectrometry (hereinafter referred to as LC / MS). Here, for mass spectrometry, it is preferable to use the multiple reaction monitoring method (MRM) or the selected reaction monitoring method (SRM). The type of the mass analyzer that performs these mass separations inside the mass spectrometer is not particularly limited, and it can appropriately include a quadrupole mass filter, an ion trap, a time-of-flight mass spectrometer, and the like.

[0026] Before being subjected to the measurement in step (B), the body fluid sample can be subjected to appropriate pretreatment. For example, when performing LC / MS, an internal standard can be added to the sample, and the internal standard can be appropriately selected according to the m / z of the marker to be detected, the type of the sample (serum, plasma, etc.), and the like.

[0027] When the above body fluid sample is measured by mass spectrometry, data corresponding to the mass spectrum and the mass chromatogram are created. Then, based on the intensity and area of the peaks in these, the detected amount of the protein is calculated, and the desired protein concentration can be calculated by multiplying the value obtained by dividing the detected amount by the detected amount of the internal standard by the known concentration of the internal standard.

[0028] As described above, by measuring the urinary concentration of cystatin A, the risk of renal function decline in diabetes can be evaluated, determined, or predicted. Therefore, cystatin A can also be used as a biomarker. Accordingly, in another aspect, the present invention also relates to a biomarker for evaluating the risk of renal function decline in diabetes, which contains cystatin A.

[0029] In a further aspect, the present invention also relates to a kit for evaluating the risk of renal function decline in diabetes, which contains a measurement reagent for cystatin A as a biomarker. Such a measurement reagent can be any compound or antibody having an affinity for cystatin A. The organic compound can be a low-molecular or high-molecular compound, for example, a dye, a fluorescent dye, a stain, a pigment, a color-developing reagent, a color-forming reagent, an immunostaining reagent, an enzyme, or a labeled compound, etc. In particular, indicators or probe molecules that specifically bind to these proteins or show absorbance changes or fluorescence responses by chemical reactions can be mentioned. Compounds labeled with radioisotopes, etc. can also be used.

[0030] The kit of the present invention can also be used in an assay for measuring and comparing the above biomarker concentration. Such assays include, for example, immunological assays (such as dot blot assays), Western blotting, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, immunoelectrophoresis, single radial immunodiffusion (SRID), radioimmunoassay (RIA), enzyme immunoassay (EIA), latex immunoassay (LIA), fluorescence immunoassay (FIA), color reaction, and colorimetry, etc.

Examples

[0031] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0032] For 593 type 2 diabetes patients (T2D), clinical information and clinical specimens were collected once a year. Collected. Among these, 403 T2D patients with eGFR ≥ 60 (mL / min / 1.73m 2 ) at registration were included in the analysis. The annual mean decline rate of eGFR (ΔeGFR) in T2D was calculated by linear regression based on eGFR from registration to the time of final clinical information collection. Patients with ΔeGFR ≥ 5% were assigned to the progressive renal function decline group, and those with ΔeGFR < 5% were assigned to the control group. The urinary protein concentration in spot urine collected in the first year of registration was measured by the multiple reaction monitoring (MRM) method. Selection of urinary proteins contributing to progressive renal function decline was performed by logistic regression analysis, and the discriminative ability of the progressive renal function decline group based on urinary protein concentration was evaluated by the area under the ROC curve (ROC-AUC).

[0033] The median observation period of the follow-up population was 5.5 years (IQR, 3.9 - 7.3), and 47 out of 403 T2D patients with eGFR ≥ 60 at registration became members of the progressive renal function decline group. In the logistic regression analysis, HDL-C (OR, 0.97; 95% CI, 0.948 - 0.996, P = 0.023), TG (OR, 1.003; 95% CI, 1.00 - 1.01, P = 0.026) , and Hb (OR, 0.74; 95% CI, 0.55 - 0.98, P = 0.033) were found to be significant factors contributing to the decline of eGFR.

[0034] Next, 44 patients in the progressive renal function decline group and 174 patients in the control group were randomly selected from 403 T2D patients with eGFR ≥ 60 at registration, and proteome analysis was performed using the spot urine samples of the subjects. Specifically, among the urinary proteins related to diabetic kidney disease (Diabetes Res Clin Pract. 2019; 147: 37 - 46), the relationship between 75 proteins for which a quantitative analysis system could be constructed and progressive eGFR decline was examined. The outline of the quantitative analysis by MRM is shown in Figure 1.

[0035] Using urine specimens collected at the time of registration from the subjects to be analyzed, the urinary concentrations of 75 proteins were measured. As a result, 11 proteins shown below exhibited significant differences in comparison between the group with gradually decreasing renal function and the control group. Among them, 7 were proteins with increased concentrations in the group with decreased renal function, and 4 were proteins with decreased concentrations in the group with decreased renal function.

[0036]

Table 1

[0037] As a result of performing a correlation analysis between the concentrations of these 11 proteins and the patient clinical information at the time of registration, these were classified into proteins correlated with HDL-c; proteins correlated with serum creatinine and eGFR; proteins correlated with UACR; and proteins correlated with the annual eGFR change rate.

[0038] Next, among the above 11 proteins, 7 proteins (cystatin A, cadherin-13, PSCA, LAMP2, ceruloplasmin, cubilin, thyroxine-binding globulin) excluding those correlated with each other were evaluated for their association with gradually decreasing renal function by logistic regression analysis (Figure 2). As a result, in particular, it was found that cystatin A is a significant factor contributing to the gradual decrease in eGFR.

[0039] In addition, regarding the concentrations of 5 proteins including cystatin A and, among others, cadherin-13, PSCA, LAMP2, and cubilin, the discriminative ability of the group with gradually decreasing renal function for the population with eGFR≧60 (218 cases) was analyzed by means of an ROC curve (Figure 3). As a result, when the outcome was ΔeGFR≧5%, the AUC when using 1 type of urinary protein was approximately 0.6 - 0.7, and it improved to 0.797 when using all 5 proteins (left in Figure 4). This exceeded the ROC-AUC by HDL-C, TG, and Hb.

[0040] Similarly, in an analysis population different from the previous analyses, for those with CKD stage 3 or above (eGFR<60), 2 Analysis was performed on the discriminative ability of the renal function decline group using protein concentrations such as cystatin A in 45 type 1 diabetic patients. As a result, the ROC-AUC was 0.759, and similar results were obtained in type 2 diabetic patients with CKD stage 3 or higher as in the group of type 2 diabetic patients with normal renal function (right in Figure 4).

[0041] From these results, by using the concentration of cystatin A in the urine as an index, a higher discriminative ability (AUC) was obtained compared to urinary ACR, which is known as an existing marker, in both the eGFR≥60 group and the eGFR<60 group.

[0042] Furthermore, the discriminative ability of cases with eGFR decline was examined by combining the urinary concentration of the above cystatin A and clinical information (Figure 5). As a result, compared with the discriminative ability ([0] in the figure) when integrating the four clinical information of HbA1c, eGFR, UACR, and Hct, when all five proteins including cystatin A ([1]), or either the concentration of Cystatin A or PSCA was added to the four clinical information ([2], [3]), the discriminative ability of cases with eGFR decline increased in both the eGFR>60 group and the eGFR<60 group. From these results, it was demonstrated that by measuring the urinary concentration of cystatin A, renal function decline in type 2 diabetic patients can be predicted from the ultra-early stage. ​​​

Claims

1. A method for evaluating the risk of renal function decline in a subject with diabetes, comprising: (A) obtaining a body fluid sample from the subject; and (B) measuring the concentration of cystatin A present in the body fluid sample. A method comprising the above steps.

2. The method according to claim 1, wherein the subject is in a state of normal or mildly decreased renal function.

3. The method according to claim 1, wherein the estimated glomerular filtration rate (eGFR) of the subject is 60 or more.

4. The method according to claim 1, further comprising comparing the measured concentration of cystatin A with a control value.

5. The method according to claim 1, comprising repeating steps (A) and (B) multiple times at regular intervals and monitoring the increase or decrease in the concentration of cystatin A.

6. The method according to claim 1, wherein the body fluid sample is urine.

7. The method according to claim 1, wherein the subject is a human.

8. The method according to claim 1, wherein the measurement in step (B) is performed by at least one of mass spectrometry, gas chromatography, liquid chromatography, gas chromatography / mass spectrometry, liquid chromatography / mass spectrometry, nuclear magnetic resonance spectroscopy, and immunological detection methods.

9. The method according to claim 1, which is used in combination with at least one other test method for measuring at least one selected from the group consisting of estimated glomerular filtration rate (eGFR), hemoglobin A1c (HbA1c), urinary albumin / creatinine ratio (UACR), and hemoglobin (Hb).

10. A biomarker for evaluating the risk of renal function decline in diabetes, comprising cystatin A. Claim 11 A kit for evaluating the risk of renal function decline in diabetes, comprising a measurement reagent for cystatin A.

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