Method for assisting diagnosis of CUBN-related nephropathy and diagnostic kit for CUBN-related nephropathy

Abstract

The main purpose set by the present inventors is to provide a technology capable of noninvasively assessing, at an early stage, the likelihood that a subject has CUBN-related nephropathy. The present inventors have found that at least one protein selected from the group consisting of myoglobin in a urine sample, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin-A receptor 5, ephrin-B2:extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphilin-2, lithostatine-1β, adhesion G protein-coupled receptor E2, α-amylase 1, extracellular ADP ribosyl transferase 3, type VI collagen α3 chain, aminopeptidase N, heart‑type fatty acid‑binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-associated protein 12, and prostate-related microseminoprotein can be a biomarker for CUBN-related nephropathy. Further improvements have been made to complete the present disclosure.

Description

Methods for assisting in the diagnosis of CUBN-associated nephropathy and diagnostic kits for CUBN-associated nephropathy

[0001] This disclosure relates to a method for assisting in the diagnosis of CUBN-associated nephropathy and a diagnostic kit for CUBN-associated nephropathy, etc.

[0002] Most proteins that pass through the glomerular capillary wall are reabsorbed (endocytosis) in the proximal tubule, so in healthy individuals, only a very small amount of protein is excreted in the urine. Megalin and cubilin are known proteins involved in this reabsorption mechanism. Recently, it has been reported that abnormalities in the CUBN gene, which codes for cubilin located in the proximal tubule, cause asymptomatic tubular proteinuria (CUBN-associated nephropathy) (Non-patent Literature 1). CUBN-associated nephropathy is sometimes called chronic benign proteinuria (PROCHOB). This disease is not rare, and recent reports have shown that it does not require treatment because the renal function prognosis is extremely good.

[0003] Boger CA, Chen MH, Tin A, et al. CUBN is a gene locus for albuminuria. J Am Soc Nephrol. 2011;22(3):555-570. doi:10.1681 / ASN.2010060598

[0004] On the other hand, since the urinary findings and pathological findings of CUBN-related nephropathy are very similar to those of glomerular diseases, it cannot be differentiated from glomerular diseases unless genetic testing is performed. However, genetic testing is costly and time-consuming, so it is not necessarily suitable as a diagnostic method in clinical settings. Since glomerular diseases are progressive and have a poor renal function prognosis, in many cases that are later definitively diagnosed as CUBN-related nephropathy by genetic testing, invasive renal biopsy has been performed, or treatment with renin-angiotensin system inhibitors (RAS inhibitors) has been initiated. In fact, as will be described later in the Examples of the present specification, 75% of CUBN-related nephropathy patients evaluated by the present inventors underwent unnecessary renal biopsy before a definitive diagnosis of CUBN-related nephropathy was made. Furthermore, 45% were prescribed RAS inhibitors (ACE inhibitors and / or ARBs), but no clinically significant decrease in proteinuria was observed, and the treatment was discontinued after the definitive diagnosis. That is, as described above, although CUBN-related nephropathy has an extremely good renal function prognosis and does not require treatment, it cannot be distinguished from glomerular diseases without genetic testing, so unnecessary treatment and renal biopsy that imposes a heavy burden on patients are performed. It is also expected that there are a considerable number of cases in which genetic testing is not performed, CUBN-related nephropathy is overlooked, and unnecessary treatment is continued indiscriminately.

[0005] The present inventors aimed mainly to provide a technique that can non-invasively evaluate the possibility that a subject has CUBN-related nephropathy at an early stage.

[0006] The present inventors have found that a specific protein in a urine sample can be used as a biomarker for CUBN-related nephropathy. Further repeated improvements were made, and the present disclosure was finally completed.

[0007] This disclosure includes, for example, the following subject matter: Section 1. A method for assisting in the diagnosis of CUBN-associated nephropathy, comprising the step of measuring the amount of at least one protein selected from the group consisting of myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein in a urine sample taken from a subject, A method wherein, if the amount of the protein is above a reference value, it is suggested that the subject is likely to have CUBN-associated nephropathy, and if the amount of the protein is below the reference value, it is suggested that the subject is unlikely to have CUBN-associated nephropathy. Item 2. A method for assisting in the diagnosis of CUBN-associated nephropathy, comprising the step of measuring the amount of myoglobin in a urine sample taken from a subject, wherein, if the amount of myoglobin is above a reference value, it is suggested that the subject is likely to have CUBN-associated nephropathy, and if the amount of myoglobin is below the reference value, it is suggested that the subject is unlikely to have CUBN-associated nephropathy. Item 3. The method according to item 1 or 2, further comprising the step of measuring the amount of β2-microglobulin (β2MG) in a urine sample taken from the subject, wherein, if the amount of β2MG is below the reference value, it is suggested that the subject is likely to have CUBN-associated nephropathy. Item 4. The method according to any one of items 1 to 3, wherein the reference value for the amount of myoglobin is 20 to 40 ng (20 to 40 ng / mL) per 1 mL of urine sample. Item 5. The method according to any one of items 1 to 3, wherein the reference value for the amount of myoglobin is 15 to 30 ng (15 to 30 ng / mgCr) per 1 mg of urine creatinine.Item 6. The method according to any one of items 3 to 5, wherein the reference value for the amount of β2MG is 150 to 300 μg per liter of urine sample (150 to 300 μg / L). Item 7. The method according to any one of items 3 to 5, wherein the reference value for the amount of β2MG is 0.1 to 20 μg per 1 mg of urine creatinine (0.1 to 20 μg / mgCr). Item 8. The method according to any one of items 1 to 7, further comprising the step of evaluating the presence or absence of CUBN gene mutations in the subject who is suggested to have a high probability of having CUBN-associated nephropathy. Item 9. The method according to any one of items 1 to 8, performed on subjects who meet at least one of the following criteria: total protein content of 10–20 mg (10–20 mg / dL) or more per 1 dL of urine sample, total protein-creatinine ratio (TP / Cr ratio) of 0.1–0.5 g / gCr or more, and albumin-creatinine ratio (Alb / Cr ratio) of 20–40 mg (20–40 mg / gCr) or more in urine. Item 10. A diagnostic kit for CUBN-associated nephropathy, comprising reagents for measuring the amount of at least one protein selected from the group consisting of myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein in a urine sample collected from a subject.Item 11. A diagnostic kit for CUBN-associated nephropathy, used to measure the amount of a target protein in a urine sample collected from a subject, comprising an antibody that specifically binds to the target protein, wherein the target protein is at least one protein selected from the group consisting of myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein. Item 12. A diagnostic kit for CUBN-associated nephropathy, used to measure the amount of myoglobin in a urine sample collected from a subject, and comprising an anti-myoglobin antibody. Item 13. The kit according to any one of items 10 to 12, further comprising an anti-β2-microglobulin (β2MG) antibody. Item 14. A diagnostic kit for CUBN-associated nephropathy, used to measure the amounts of myoglobin and β2-microglobulin (β2MG) in a urine sample collected from a subject, and comprising an anti-myoglobin antibody and an anti-β2-microglobulin (β2MG) antibody.

[0008] This disclosure provides a technology that allows for the early, non-invasive assessment of whether a subject has CUBN-associated nephropathy.

[0009] The following figures show the results of quantification of myoglobin in proteomics analysis. In the figures, "CUBN" refers to the group of patients with CUBN-associated nephropathy, "Neph" refers to the group of patients with nephrotic syndrome, and "Alport" refers to the group of patients with Alport syndrome (the same applies hereafter). The following figures show the results of quantification of folate receptor β in proteomics analysis. The following figures show the results of quantification of vascular cell adhesion protein 1 in proteomics analysis. The following figures show the results of quantification of cathepsin Z in proteomics analysis. The following figures show the results of quantification of ephrin type A receptor 5 in proteomics analysis. The following figures show the results of quantification of ephrin-B2: extracellular domain in proteomics analysis. The following figures show the results of quantification of lipocalin-1 in proteomics analysis. The following figures show the results of quantification of inosine triphosphate pyrophosphatase in proteomics analysis. The following figures show the results of quantification of dipeptidyl peptidase 4 in proteomics analysis. The following figures show the results of quantification of twinphyllin-2 in proteomics analysis. This figure shows the results of comparing urinary myoglobin concentrations (MB, ng / mL) in five groups (CUBN-associated nephropathy group, moderate proteinuric glomerular disease group, severe proteinuric glomerular disease group, Dent disease type 1 group, and proteinuria-negative control group). This figure shows the results of comparing myoglobin / creatinine ratios (MB / Cr, ng / mgCr) in the five groups. This figure shows the results of comparing urinary protein / creatinine ratios (uTP / Cr, g / gCr) in the five groups. This figure shows the results of comparing urinary myoglobin concentrations (MB, ng / mL) between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group. This figure shows the results of comparing myoglobin / creatinine ratios (MB / Cr, ng / mgCr) between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group. This figure shows the results of comparing the urinary protein / creatinine ratio (uTP / Cr, g / gCr) between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group. This figure shows the results of comparing the urinary β2-microglobulin concentration (uβ2MG) in the five groups. This figure shows the results of comparing the urinary β2-microglobulin concentration (uβ2MG) between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group. This figure shows the results of comparing the β2-microglobulin / creatinine ratio (β2MG / Cr) in the five groups.This figure shows the results of comparing the β2-microglobulin / creatinine ratio (β2MG / Cr) between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group. This figure shows receiver operating characteristic (ROC) curves created to distinguish between patients with CUBN-associated nephropathy and patients with moderate proteinuric glomerular disease using urinary myoglobin concentration (MB) as an indicator. This figure shows receiver operating characteristic (ROC) curves created to distinguish between patients with CUBN-associated nephropathy and patients with moderate proteinuric glomerular disease using myoglobin / creatinine ratio (MB / Cr) as an indicator. This figure shows an example of a screening method for CUBN-associated nephropathy using urinary β2-microglobulin and myoglobin.

[0010] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, methods for assisting in the diagnosis of CUBN-associated nephropathy and diagnostic kits for CUBN-associated nephropathy, and this disclosure includes everything disclosed herein and recognizable to those skilled in the art.

[0011] The method for assisting the diagnosis of CUBN-associated nephropathy as encompassed in this disclosure includes the following components in a urine sample taken from a subject: myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular The method includes a step of measuring the amount of at least one protein selected from the group consisting of the glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-related microseminoprotein, wherein if the amount of the protein is above a reference value, it is suggested that the subject is likely to have CUBN-associated nephropathy, and if the amount of the protein is below the reference value, it is suggested that the subject is unlikely to have CUBN-associated nephropathy. Hereinafter, the method included in this disclosure may be referred to as the "method of this disclosure".

[0012] CUBN-associated nephropathy refers to a kidney disease caused by mutations in the CUBN (Cubilin) ​​gene. Cubilin is mainly expressed in the proximal tubules and works in conjunction with megalin to reabsorb protein in the proximal tubules. A clinical characteristic of CUBN-associated nephropathy is proteinuria. If a mutation in the CUBN gene is found through genetic analysis, a definitive diagnosis of CUBN-associated nephropathy can be made.

[0013] The timing and method of urine sample collection are not particularly limited as long as the desired effect is achieved. For example, it may be a random urine sample collected at any time without specifying a particular time, an early morning urine sample collected immediately after waking up, or a timed urine sample collected from urine excreted within a certain period of time (e.g., 2 hours, 4 hours, 24 hours, etc.). It may also be a midstream urine sample collected from the middle portion of urine after the first part of urination has been discarded, a catheterized urine sample collected directly from the bladder using a urethral catheter, or a cystopuncture urine sample collected directly from the bladder using a needle.

[0014] The storage container for the collected urine sample is not particularly limited as long as the desired effect is achieved. For example, it may be a sterile, airtight container made of resin or glass. Although not particularly limited, the storage container for the collected urine sample in the art of this disclosure preferably contains a stabilizer. The stabilizer preferably contains at least one selected from the group consisting of antioxidants, preservatives, and chelating agents.

[0015] Specific examples of antioxidants include sulfites such as sodium sulfite, sodium disulfite, sodium bisulfite, potassium sulfite, and potassium disulfite, as well as ascorbic acid and erythorbic acid. Antioxidants can be used individually or in combination of two or more. While not particularly limited, in the technology of this disclosure, the antioxidant preferably contains at least one type of sulfite, and more preferably contains sodium disulfite.

[0016] Specific examples of preservatives include isothiazolinones such as methylisothiazolinone and methylchloroisothiazolinone; parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben; benzoic acid and its salts; and phenoxyethanol. Antioxidants can be used individually or in combination of two or more. While not particularly limited, in the technology of this disclosure, the preservative preferably contains at least one isothiazolinone, and more preferably contains methylisothiazolinone and / or methylchloroisothiazolinone. A mixture of methylisothiazolinone and methylchloroisothiazolinone is commercially available under names such as "Proclin™".

[0017] Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts, nitrilotriacetic acid (NTA) and its salts, diethylenetriaminepentaacetic acid (DTPA) and its salts, and citric acid and its salts. Chelating agents can be used individually or in combination of two or more. While not particularly limited, in the art of this disclosure, the chelating agent preferably contains at least one selected from the group consisting of EDTA and its salts, and more preferably contains EDTA-3Na.

[0018] While not particularly limited, the storage container for urine samples collected in the art of this disclosure preferably contains antioxidants, preservatives, and chelating agents, more preferably contains at least one selected from the group consisting of at least one sulfite, at least one isothiazolinone, and EDTA and its salts, and even more preferably contains sodium disulfite, methylisothiazolinone, methylchloroisothiazolinone, and EDTA-3Na.

[0019] The method for storing the collected urine sample is not particularly limited as long as the desired effect is obtained. For example, it may be stored at room temperature (around 15-25°C), refrigerated at around 0-10°C, or frozen below freezing. Although not particularly limited, refrigerated storage at around 0-10°C is preferred in the technology of this disclosure.

[0020] As described above, the method disclosed herein involves collecting myoglobin, folate receptor beta, vascular cell adhesion protein 1, cathepsin Z, ephrin type-A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinfilin-2, lithostathine-1-beta, adhesion G protein-coupled receptor E2, and alpha-amylase 1 from a urine sample taken from a subject. 1) The method includes measuring the amount of at least one protein selected from the group consisting of extracellular ADP-ribosyltransferase 3, collagen alpha-3(VI) chain, aminopeptidase N, heart-type fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibulin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein.The method of the present disclosure may include a step of measuring the amount of two or more proteins (2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more) selected from the group. The method of the present disclosure more preferably includes a step of measuring the amount of myoglobin.

[0021] In the technology of this disclosure, the method for measuring the amount of protein is not particularly limited as long as the desired effect is obtained. Examples include methods for detecting specific binding between nucleic acids and proteins using nucleic acid aptamers, methods for detecting specific binding between antibodies and antigens using antibodies, and mass spectrometry. More specifically, antibody-based methods include chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), latex agglutination, and Western blotting. Although not particularly limited, in the technology of this disclosure, the amount of protein is preferably measured by an antibody-based method, and more preferably by CLEIA.

[0022] In the technology of this disclosure, the amount of a specific protein in a urine sample is compared to a threshold value and can serve as an indicator of whether the subject from which the urine sample originates is likely to have CUBN-associated nephropathy. Specifically, if the amount of a specific protein in a urine sample is equal to or greater than the threshold value, it is suggested that the subject from which the urine sample originates is likely to have CUBN-associated nephropathy. Conversely, if the amount of a specific protein in a urine sample is less than the threshold value, it is suggested that the subject from which the urine sample originates is unlikely to have CUBN-associated nephropathy. In other words, if the amount of a specific protein in a urine sample is equal to or greater than the threshold value, it is suggested that the urine sample is likely to originate from a subject with CUBN-associated nephropathy, and if the amount of a specific protein in a urine sample is less than the threshold value, it is suggested that the urine sample is unlikely to originate from a subject with CUBN-associated nephropathy.

[0023] Insofar as the effects of this disclosure are obtained, the reference value is not particularly limited and can be appropriately set by those skilled in the art. The reference value can be set, for example, by the following methods: collecting urine samples from multiple subjects with CUBN-associated nephropathy and measuring the amount of a specific protein in the urine samples to calculate the amount of the specific protein per unit volume; similarly, collecting urine samples from multiple healthy individuals (subjects without CUBN-associated nephropathy) and measuring the amount of a specific protein in the urine samples to calculate the amount of the specific protein per unit volume; comparing the data from the group of subjects with CUBN-associated nephropathy and the group of healthy individuals to find the value that can distinguish the two groups with the highest accuracy, and setting that value as the reference value. When setting the reference value, sensitivity and specificity may be taken into consideration. Furthermore, the reference value may vary depending on the method used to measure the amount of the specific protein.

[0024] More specifically, reference values ​​may be set based, for example, on ROC curve (Receiver Operating Characteristic curve) analysis. An ROC curve is created by determining the probability (%) of a positive result in subjects with CUBN-associated nephropathy (True Position Fraction, sensitivity) and the probability (%) of a negative result in healthy individuals (Specificity) for a specific amount of protein per unit volume, and plotting the sensitivity against [100 - Specificity] (False Position Fraction (FPF)).

[0025] While not particularly limited, if the protein is myoglobin, the specific reference value may be, for example, 1.0 to 100 ng per 1 mL of urine sample (1.0 to 100 ng / mL). The upper or lower limits of the above range are 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0. The reference values ​​for myoglobin levels may be 5, 10, 15, 20, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL. The reference values ​​for myoglobin levels are more preferably 10 to 50 ng / mL, more preferably 20 to 40 ng / mL, even more preferably 25 to 35 ng / mL, and particularly preferably 31.5 ng / mL.

[0026] Furthermore, although not particularly limited, the amount of a specific protein in the urine sample may be corrected using the urinary creatinine (Cr) value. Correcting with the urinary creatinine value can reduce the effect of urine concentration. Although not particularly limited, if the protein is myoglobin, the specific reference value may be, for example, 5 to 100 ng / mgCr for urinary myoglobin / Cr. The upper or lower limits of the above range may be 5, 10, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 ng / mgCr. The reference range for urinary myoglobin / Cr is preferably 10-40 ng / mgCr, more preferably 15-30 ng / mgCr, even more preferably 20-25 ng / mgCr, and particularly preferably 22.2 ng / mgCr.

[0027] While not particularly limited, the method of this disclosure preferably further includes a step of measuring the amount of β2-microglobulin (β2MG) in the urine sample. If the method of this disclosure further includes a step of measuring the amount of β2MG, CUBN-associated nephropathy can be detected with even greater accuracy. The measured amount of β2MG is compared to a reference value and, together with the amount of the above-mentioned protein, can serve as an indicator of whether the subject from which the urine sample originates is likely to have CUBN-associated nephropathy. Specifically, if the amount of β2MG in the urine sample is less than the reference value, it is suggested that the subject from which the urine sample originates is very unlikely to have Dent's disease, which can cause high levels of urinary myoglobin in addition to CUBN-associated nephropathy. In other words, if the myoglobin level in the urine sample is high and the amount of β2MG is less than the reference value, it is suggested that the urine sample is likely to originate from a subject with CUBN-associated nephropathy.

[0028] While not particularly limited, the specific reference value for β2MG may be, for example, 100 to 400 μg (100 to 400 μg / L) per liter of urine sample. The upper or lower limit of the above range may be 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 μg / L. The reference value for β2MG is preferably 120 to 350 μg / L, more preferably 150 to 300 μg / L, even more preferably 180 to 220 μg / L, and particularly preferably 200 μg / L.

[0029] Furthermore, although not particularly limited, specific reference values ​​for β2MG amounts may be, for example, 0.1 to 20 μg (β2MG / Cr) per 1 mg of urinary creatinine (0.1 to 20 μg / mgCr). The upper or lower limits of the above range may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg / mgCr. The reference range for β2MG / Cr is preferably 0.2 to 18 μg / mgCr, more preferably 0.3 to 15 μg / mgCr, and even more preferably 0.3 to 10 μg / mgCr.

[0030] While not particularly limited, the method disclosed herein preferably includes a step of further evaluating the presence or absence of CUBN gene mutations in subjects suggested to have a high probability of having CUBN-associated nephropathy. If a CUBN gene mutation is detected in such subjects, a definitive diagnosis of CUBN-associated nephropathy can be made. The method for evaluating the presence or absence of CUBN gene mutations is not particularly limited as long as the desired effect is obtained, and examples include methods using next-generation sequencing or DNA microarrays.

[0031] While not particularly limited, the method disclosed herein is preferably applied to subjects who meet at least one of the following criteria: a total protein content of 10–20 mg (10–20 mg / dL) or more in a 1 dL urine sample, a total protein-to-creatinine ratio (TP / Cr ratio) of 0.1–0.5 g / gCr or more in urine, and an albumin-creatinine ratio (Alb / Cr ratio) of 20–40 mg / gCr or more in urine. Because such subjects have a higher-than-normal amount of protein in their urine, renal impairment is suspected, and treatment or renal biopsy may be considered. However, if such subjects have CUBN-associated nephropathy, treatment or renal biopsy is unnecessary. Therefore, from the perspective of avoiding unnecessary treatment or renal biopsy, these subjects can be said to benefit particularly greatly from applying the method disclosed herein.

[0032] The method disclosed herein is more preferably applied to subjects in which the total protein content in the urine sample is 12 to 18 mg / dL or more, and particularly preferably applied to subjects in which it is 15 mg / dL or more. The upper or lower limit of the range may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / dL.

[0033] The method of this disclosure is more preferably applied to subjects with an Alb / Cr of 22 to 38 mg / gCr or higher, even more preferably to subjects with an Alb / Cr of 25 to 35 mg / gCr or higher, particularly preferably to subjects with an Alb / Cr of 27 to 33 mg / gCr or higher, and most preferably to subjects with an Alb / Cr of 30 mg / gCr or higher. The upper or lower limit of the range may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mg / gCr.

[0034] The method of this disclosure is more preferably applied to subjects having a TP / Cr ratio of 0.12 to 0.4 g / gCr or higher, even more preferably to subjects having a TP / Cr ratio of 0.12 to 0.3 g / gCr or higher, particularly preferably to subjects having a TP / Cr ratio of 0.15 to 0.2 g / gCr or higher, and most preferably to subjects having a TP / Cr ratio of 0.15 g / gCr or higher. The upper or lower limit of the range may be 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, or 0.5 g / gCr.

[0035] This disclosure also includes a diagnostic kit for CUBN-associated nephropathy, which includes reagents for measuring the amount of at least one protein selected from the group consisting of myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein (sometimes referred to as the "target protein") in a urine sample taken from a subject. The kit included in this disclosure may be referred to as the "Kit of this Disclosure." The information described in relation to the method of this disclosure shall be incorporated into the Kit of this Disclosure as appropriate. Similarly, the information described in relation to the Kit of this Disclosure shall be incorporated into the method of this disclosure as appropriate.

[0036] The kits of this disclosure can be used to measure the amount of a target protein in a urine sample collected from a subject. The reagents for measuring the amount of the target protein are not limited as long as the desired effect is achieved. Specific reagents include, for example, antibodies that specifically bind to the target protein. While not particularly limited, the kits of this disclosure preferably contain an anti-myoglobin antibody, and more preferably contain both an anti-myoglobin antibody and an anti-β2-microglobulin (β2MG) antibody.

[0037] The kit of this disclosure may be used to compare the amount of the measured target protein to a reference value. If the amount of the target protein is above the reference value, it may be suggested that the subject from which the urine sample originates is likely to have CUBN-associated nephropathy. If the amount of the target protein is below the reference value, it may be suggested that the subject from which the urine sample originates is unlikely to have CUBN-associated nephropathy.

[0038] In this specification, the term “comprising” includes not only “containing” but also “essentially consisting of” and “consisting of.” Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0039] Furthermore, the various characteristics (properties, numerical values, structure, function, etc.) described for each embodiment of this disclosure described above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein.

[0040] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0041] I. Proteomics Analysis Urine samples from seven patients with CUBN-associated nephropathy diagnosed by comprehensive genetic analysis conducted by the inventors, along with urine samples from seven patients with nephrotic syndrome and six patients with Alport syndrome (glomerular diseases), were used as controls. Proteomics analysis was commissioned to Soma Logic. In this proteomics analysis, a modified nucleic acid aptamer "SOMAmer®," which has the property of being slow to dissociate after binding to a protein (slow off-rate), was bound to the target protein, and more than 7,000 types of proteins were quantitatively measured by microarray analysis.

[0042] Proteomic analysis revealed that the 23 proteins listed in the table below were significantly increased in urinary excretion in patients with CUBN-associated nephropathy.

[0043]

[0044] The quantification results of the proteins myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type-A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, and twinfilin-2 are shown in Figures 1 to 10. In the figures, "CUBN" indicates the CUBN-related nephropathy patient group, "Neph" indicates the nephrotic syndrome patient group, and "Alport" indicates the Alport syndrome patient group, respectively. These proteins were significantly higher in the CUBN-related nephropathy patient group compared to both the nephrotic syndrome patient group and the Alport syndrome patient group (fold change ≥ 2.5-fold and P<0.05 by Student's t-test). One case showing an outlier in the nephrotic syndrome patient group developed secondary proximal tubular damage associated with nephrotic syndrome, so it can be judged to have presented the same laboratory findings as patients with CUBN-related nephropathy.

[0045] From the above results, it is suggested that the 23 types of proteins described above can serve as urinary biomarkers for CUBN-related nephropathy.

[0046] II. Analysis of urinary myoglobin level by CLEIA 1 Among the proteins identified by proteomics analysis, the present inventors further increased the number of specimens and performed analysis by chemiluminescent enzyme immunoassay (CLEIA) for myoglobin. Specifically, urinary myoglobin levels were measured by CLEIA for 8 cases of CUBN-related nephropathy, 8 cases of Dent disease, 2 cases of Alport syndrome, 1 case of orthostatic proteinuria, 1 case of ANCA-associated vasculitis, 1 case of IgA nephropathy, 1 case of Henoch-Schönlein purpura nephritis, 1 case of nephrotic syndrome, and 2 normal specimens. This analysis was commissioned to LSI Medience Corporation.

[0047] Generally, in the measurement of urinary myoglobin levels, it is considered desirable to place collected specimens promptly into a dedicated container containing a stabilizer. If a specimen is placed in a non-dedicated container that does not contain a stabilizer, myoglobin in the specimen may degrade before measurement, potentially resulting in a measured value lower than the actual value.

[0048] In this study, both samples placed in dedicated containers containing stabilizers (sodium disulfite, Proclin®, and EDTA-3Na) and samples placed in non-dedicated containers without stabilizers were used. As mentioned above, the presence or absence of stabilizers may affect the measurement of urinary myoglobin, so the results below are presented separately for samples placed in dedicated containers and non-dedicated containers.

[0049] The table below shows the urinary myoglobin (urinary MB), urinary β2-microglobulin (urinary β2MG), urinary albumin (urinary Alb), and urinary creatinine (urinary Cre) levels for each patient.

[0050]

[0051] Generally, a cutoff value of 2.0 ng / mL is used for urinary myoglobin levels in urine tests. In patients with CUBN-associated nephropathy, 6 out of 7 cases showed urinary myoglobin levels of 2.0 ng / mL or higher (positive) even when samples were collected in non-dedicated containers. Furthermore, 1 out of 1 case showed positive urinary myoglobin levels when samples were collected in dedicated containers.

[0052] In patients with Dent's disease, 4 out of 8 samples collected in non-dedicated containers were positive. Furthermore, one patient (patient ID 14) who tested negative in a non-dedicated container tested positive when re-tested using a sample collected in a dedicated container. In all patients with Dent's disease, urinary β2-microglobulin levels were elevated above the normal range of 200 μg / L. On the other hand, in patients with CUBN-associated nephropathy, β2-microglobulin levels were below 200 μg / L in 7 out of 8 cases.

[0053] In all samples from patients with glomerular kidney diseases such as Alport syndrome, orthostatic proteinuria, ANCA-associated vasculitis, IgA nephropathy, Henoch-Schönlein purpura nephritis, and nephrotic syndrome, as well as from healthy children, urinary myoglobin levels were less than 2.0 ng / mL.

[0054] These results suggest that urinary myoglobin levels may serve as a urinary biomarker for CUBN-associated nephropathy. Furthermore, it was suggested that combining urinary myoglobin levels with urinary β2-microglobulin levels could enable more accurate detection of CUBN-associated nephropathy.

[0055] III. Analysis of Urinary Myoglobin Levels using CLEIA 2 The inventors conducted an analysis with an increased number of samples to further verify the usefulness of urinary myoglobin levels as a urinary biomarker for CUBN-associated nephropathy. Specifically, 20 patients with genetically confirmed CUBN-associated nephropathy were included. Seven of these patients also participated in "II. Analysis of Urinary Myoglobin Levels using CLEIA 1" described above, and some of the data overlaps.

[0056] For comparison, 31 patients with glomerular disease presenting with moderate proteinuria, 11 patients with glomerular disease presenting with severe proteinuria, 5 patients with Dent disease type 1, and 9 proteinuria-negative controls were enrolled. The 31 patients with glomerular disease presenting with moderate proteinuria consisted of 10 asymptomatic proteinuria, 7 IgA nephropathy, 6 IgA vasculitis nephritis, 3 Alport syndrome, 2 hereditary nephrotic syndrome, 1 ANCA-associated vasculitis, and 1 C3 nephritis. The 11 patients with glomerular disease presenting with severe proteinuria consisted of 7 idiopathic nephrotic syndrome, 3 IgA vasculitis nephritis, and 1 hereditary nephrotic syndrome. Note that the data partially overlaps with "II. Analysis of urinary myoglobin levels by CLEIA 1" described above.

[0057] Moderate proteinuria was defined as a urinary protein / creatinine ratio (uTP / Cr) of 0.15 < uTP / Cr ≤ 4.0 g / gCr in early morning random urine samples, while severe proteinuria was defined as uTP / Cr > 4.0 g / gCr. Excluding patients with asymptomatic hematuria and non-renal disease, all patients with proteinuria had a uTP / Cr > 0.15. At the time of urine collection, none of the patients had a history or clinical evidence of conditions known to increase myoglobin production, such as recent strenuous exercise, muscle injury, or rhabdomyolysis.

[0058] In this analysis, random urine samples were promptly collected in a dedicated collection tube containing sodium disulfite, Proclin (trademark), and EDTA-3Na. The required collection volume was 6 mL. Samples were stored at room temperature according to the manufacturer's instructions. Urinary myoglobin concentration was measured using CLEIA. In addition, the urinary myoglobin / creatinine ratio (MB / Cr) was calculated.

[0059] Statistical analysis was performed using JMP 14.0. Continuous variables are shown as median and interquartile range (IQR), and categorical variables are shown as number and percentage. Exploratory comparisons of continuous variables among the five groups were performed using the Kruskal-Wallis test, and if the overall test was significant, pairwise post-hoc comparisons were performed using Dunn's test with Bonferroni correction. Categorical variables were compared using Fisher's exact test. The primary comparison was between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group, and continuous variables were compared using the Mann-Whitney U test. ROC curve analysis was performed for urinary myoglobin (MB) and MB / Cr, and the area under the curve (AUC) was calculated. The optimal cutoff value was determined using the Youden index, and sensitivity, specificity, positive predictive value, and negative predictive value were calculated. A two-sided p-value < 0.05 was considered statistically significant.

[0060] The baseline characteristics of the subjects are shown in the table below. Data are expressed as median [interquartile range] or n (%). Cr-eGFR represents the estimated glomerular filtration rate based on creatinine. The median age of onset in the CUBN-associated nephropathy group was 3.0 years, and the median age at examination was 8.1 years. The proportion of males was similar in the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group. Creatinine-based eGFR was preserved in both groups, while serum albumin was low in the severe proteinuric glomerular disease group.

[0061] Renal biopsies were performed in 75.0% (15 / 20) of the CUBN-associated nephropathy group and 64.5% (20 / 31) of the moderate proteinuria glomerular disease group. Glomerular abnormalities were minimal in CUBN-associated nephropathy patients who underwent renal biopsies. Prior to a definitive genetic diagnosis, 45.0% (9 / 20) of the CUBN-associated nephropathy group received ACE inhibitors and / or ARBs, but no clinically significant reduction in proteinuria was observed, and treatment was discontinued after genetic confirmation.

[0062]

[0063] The results of comparing urinary myoglobin and related laboratory findings between groups are shown in the table and Figures 11A to 13D below. Unless otherwise noted, data are shown as median [interquartile range]. A positive urinary MB test was defined as urinary myoglobin > 2.0 ng / mL, which corresponds to the lower limit of quantification (LLOQ) of the assay. Values ​​below LLOQ were substituted as 1.0 ng / mL for analysis. MB represents myoglobin, MB / Cr represents the myoglobin / creatinine ratio, uβ2MG represents urinary β2-microglobulin, and uTP / Cr represents the urinary total protein / creatinine ratio.

[0064]

[0065] uTP / Cr showed differences among the groups, with the highest value observed in the group with severe proteinuric glomerular disease (Figure 11C). On the other hand, uTP / Cr was similar between the CUBN-associated nephropathy group and the group with moderate proteinuric glomerular disease (Figure 12C).

[0066] uβ2MG levels differed between groups, with a significant increase observed in the Dent disease type 1 group (Figure 13A). In contrast, uβ2MG levels were similar between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group (Figure 13B). The β2MG / Cr ratio differed between groups, with a significant increase observed in the Dent disease type 1 group (Figure 13C). CUBN-associated nephropathy levels were slightly higher than those of moderate proteinuric glomerular disease (Figure 13D).

[0067] Urinary MB levels differed among the groups (Figure 11A). The median MB levels were 180.5 ng / mL in the CUBN-associated nephropathy group, 2.2 ng / mL in the moderate proteinuric glomerular disease group, 78.7 ng / mL in the severe proteinuric glomerular disease group, 45.3 ng / mL in the Dent disease type 1 group, and 1.0 ng / mL in the proteinuria-negative control group. Urinary MB levels were higher in the CUBN-associated nephropathy group, the severe proteinuric glomerular disease group, and the Dent disease type 1 group than in the moderate proteinuric glomerular disease group. Urinary MB levels were significantly higher in the CUBN-associated nephropathy group than in the moderate proteinuric glomerular disease group (Figure 12A). MB / Cr also differed among the groups (Figure 11B). The median MB / Cr was 216.8 ng / mgCr in the CUBN-associated nephropathy group, 2.35 ng / mgCr in the moderate proteinuric glomerular disease group, 45.79 ng / mgCr in the severe proteinuric glomerular disease group, 89.7 ng / mgCr in the Dent disease type 1 group, and 1.11 ng / mgCr in the proteinuria-negative control group. MB / Cr was higher in the CUBN-associated nephropathy group, the severe proteinuric glomerular disease group, and the Dent disease type 1 group than in the moderate proteinuric glomerular disease group. In a direct comparison between the CUBN-associated nephropathy group and the moderate proteinuric glomerular disease group, MB / Cr was significantly higher in the CUBN-associated nephropathy group (Figure 12B).

[0068] ROC curve analysis was performed to compare patients with CUBN-associated nephropathy and patients with moderate proteinuric glomerular disease using urinary MB concentration and MB / Cr ratio. Both indicators showed high discriminative ability, with AUCs of 0.979 for urinary MB and 0.972 for MB / Cr (Figures 14A and 14B). Using the optimal cutoff values ​​determined by the Youden index, a cutoff value of 31.5 ng / mL for urinary MB showed a sensitivity of 94.7%, specificity of 97.0%, positive predictive value of 94.7%, and negative predictive value of 97.0%. A cutoff value of 22.2 ng / mgCr for MB / Cr showed a sensitivity of 100%, specificity of 93.8%, positive predictive value of 90.5%, and negative predictive value of 100%.

[0069] Figure 15 shows an example of a screening method for CUBN-associated nephropathy using urinary β2-microglobulin and urinary myoglobin, as suggested by this disclosure. In patients with mild proteinuria and preserved renal function, urinary β2-microglobulin (uβ2MG) and urinary myoglobin (MB) are measured. Based on the combination of uβ2MG and MB values, patients are classified into the following three categories: (1) high uβ2MG and high MB suggesting Dent's disease, (2) low uβ2MG and high MB suggesting CUBN-associated nephropathy, and (3) low uβ2MG and low MB suggesting glomerular disease.

Claims

1. A method for assisting in the diagnosis of CUBN-associated nephropathy, comprising the step of measuring the amount of at least one protein selected from the group consisting of myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion-type G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein in a urine sample collected from a subject. A method wherein if the amount of the protein is above a reference value, it is suggested that the subject is likely to have CUBN-associated nephropathy, and if the amount of the protein is below the reference value, it is suggested that the subject is unlikely to have CUBN-associated nephropathy.

2. A method for assisting in the diagnosis of CUBN-associated nephropathy, comprising the step of measuring the amount of myoglobin in a urine sample taken from a subject, wherein if the amount of myoglobin is equal to or greater than a reference value, it is suggested that the subject is likely to have CUBN-associated nephropathy, and if the amount of myoglobin is less than the reference value, it is suggested that the subject is unlikely to have CUBN-associated nephropathy.

3. The method according to claim 1 or 2, further comprising the step of measuring the amount of β2-microglobulin (β2MG) in a urine sample taken from the subject, wherein if the amount of β2MG is below a reference value, it is suggested that the subject is likely to have CUBN-associated nephropathy.

4. The method according to claim 1 or 2, wherein the reference value for the amount of myoglobin is 20 to 40 ng (20 to 40 ng / mL) per 1 mL of urine sample.

5. The method according to claim 1 or 2, wherein the reference value for the amount of myoglobin is 15 to 30 ng per 1 mg of urinary creatinine (15 to 30 ng / mgCr).

6. The method according to claim 3, wherein the reference value for the amount of β2MG is 150 to 300 μg per liter of urine sample (150 to 300 μg / L).

7. The method according to claim 3, wherein the reference value for the amount of β2MG is 0.1 to 20 μg per 1 mg of urinary creatinine (0.1 to 20 μg / mgCr).

8. The method according to claim 1 or 2, further comprising the step of evaluating the presence or absence of a CUBN gene mutation in the subject who is suggested to have a high probability of having CUBN-associated nephropathy.

9. The method according to claim 1 or 2, performed on a subject that falls under at least one of the groups selected from the group consisting of a total protein content of 10 to 20 mg (10 to 20 mg / dL) or more in 1 dL of urine sample, a total protein-to-creatinine ratio (TP / Cr ratio) of 0.1 to 0.5 g / gCr or more, and an albumin-to-creatinine ratio (Alb / Cr ratio) of 20 to 40 mg (20 to 40 mg / gCr) or more in urine.

10. A diagnostic kit for CUBN-associated nephropathy, comprising reagents for measuring the amount of at least one protein selected from the group consisting of myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein in a urine sample collected from a subject.

11. A diagnostic kit for CUBN-associated nephropathy, used to measure the amount of a target protein in a urine sample collected from a subject, comprising an antibody that specifically binds to the target protein, wherein the target protein is at least one protein selected from the group consisting of myoglobin, folate receptor β, vascular cell adhesion protein 1, cathepsin Z, ephrin type A receptor 5, ephrin-B2: extracellular domain, lipocalin-1, inosine triphosphate pyrophosphatase, dipeptidyl peptidase 4, twinphyllin-2, lysostatin-1β, adhesion-type G protein-coupled receptor E2, α-amylase 1, extracellular ADP-ribosyltransferase 3, type VI collagen α3 chain, aminopeptidase N, cardiac fatty acid-binding protein, extracellular glycoprotein lacritin, MANSC domain-containing protein 1, neuronal pentraxin receptor, fibrin-7, low-density lipoprotein receptor-related protein 12, and prostate-associated microseminoprotein.

12. A diagnostic kit for CUBN-associated nephropathy, which is used to measure the amount of myoglobin in a urine sample collected from a subject, and which contains an anti-myoglobin antibody.

13. The kit according to any one of claims 10 to 12, further comprising an anti-β2-microglobulin (β2MG) antibody.

14. A diagnostic kit for CUBN-associated nephropathy, used to measure the amount of myoglobin and β2-microglobulin (β2MG) in a urine sample collected from a subject, and comprising an anti-myoglobin antibody and an anti-β2-microglobulin (β2MG) antibody.

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