Method for assisting in the diagnosis of hypertension and kit for detecting ENaCγ fragments in urine-derived extracellular vesicles

Abstract

A diagnostic support method for hypertension using a biomarker of mineralocorticoid receptor signaling activity and a kit for detecting ENaCγ fragments in urine-derived extracellular vesicles are provided. [Solution] A method for assisting in the diagnosis of hypertension in a subject animal, comprising: a recovery step for recovering urine-derived extracellular vesicles from the urine of the subject animal; a detection step for detecting ENaCγ fragments contained in the urine-derived extracellular vesicles during the recovery step; and an evaluation step for evaluating that there is a high need for treatment or a high need for optimization of treatment if the detected amount of the ENaCγ fragments in the detection step is equal to or greater than a predetermined standard value.

Description

[Technical Field]

[0001] The present invention relates to a method for assisting in the diagnosis of hypertension in an animal subject and a kit for detecting ENaCγ fragments in urine-derived extracellular vesicles. [Background technology]

[0002] Aldosterone and mineralocorticoid receptors (MRs) play important roles in hypertension and target organ damage. For example, primary aldosteronism (PA), which accounts for 5-20% of hypertensive patients, is caused by activation of the MR due to excessive secretion of aldosterone from the adrenal gland. PA is treated by adrenalectomy or with MR antagonists. In addition to treating hypertension, MR antagonists are known to improve the prognosis of patients with chronic heart failure and diabetic kidney disease. Aldosterone and MRs also play important roles in preventing the progression of cardiovascular and renal diseases.

[0003] The release of exosome-containing extracellular vesicles (uEVs: urinary extracellular vesicles) is known to provide information about active tissue signaling pathways. For example, Non-Patent Document 1 discloses that uEVs are secreted into urine, and Non-Patent Documents 1 to 3 disclose that urinary extracellular vesicles have potential as biomarkers. Furthermore, Non-Patent Document 4 discloses that proteomic analysis of urine can provide information for predicting response to diabetes and hypertension treatment. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Patricia A. Gonzales et al., “Large-scale proteomics and phosphoproteomics of urinary exosomes.” Journal of the American Society of Nephrology, Vol. 20, pp.363-379, 2009.

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

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[0005] Aldosterone and MR are pathologically important in hypertension, kidney disease, etc., and biomarkers of MR signaling activity are expected to be useful for diagnosing and supporting these diseases. However, useful biomarkers of MR signaling activity have not yet been established.

[0006] The present invention has been made in consideration of the above circumstances, and aims to provide a diagnostic support method for hypertension using a biomarker for MR signaling activity, and a kit for detecting ENaCγ (epithelial sodium channel γ) fragments in uEV. [Means for solving the problem]

[0007] The present invention includes the following aspects. (1) A method for assisting in the diagnosis of hypertension in a test animal, comprising: A recovery step of recovering urine-derived extracellular vesicles from the urine of the subject animal; The recovery step includes a detection step of detecting ENaCγ fragments contained in the urine-derived extracellular vesicles. The method comprises an evaluation step of evaluating that there is a high need for treatment or a high need for optimization of treatment if the detected amount of the ENaCγ fragment in the detection step is equal to or greater than a predetermined standard value. (2) The method described in (1), wherein the hypertension is primary aldosteronism. (3) The method according to (1) or (2), wherein the detection step is carried out by an immunological technique. (4) The method according to any one of (1) to (3), wherein the ENaCγ fragment is a fragment obtained by cleaving ENaCγ with a protease that recognizes an RKRR motif or an RKRK motif. (5) The method according to any one of (1) to (4), wherein the treatment is treatment with a mineralocorticoid receptor antagonist. (6) A kit for detecting ENaCγ fragments in urine-derived extracellular vesicles, comprising: A kit comprising a substance that specifically binds to the ENaCγ fragment. (7) The kit according to (6), which is used to assist in the diagnosis of hypertension in a test animal. (8) The kit according to (7), wherein the hypertension is primary aldosteronism. [Effects of the Invention]

[0008] The present invention uses the ENaCγ fragment in uEVs as a biomarker for the pathology of hypertension. Therefore, the diagnostic support method for hypertension and the kit for detecting the ENaCγ fragment in uEVs according to the present invention can noninvasively provide information useful for diagnosing hypertension, particularly information regarding the efficacy of MR antagonists. [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1(A) shows the results of pathway enrichment analysis in Example 1. FIG. 1(B) is a Venn diagram showing the results of comparing the results of pathway enrichment analysis in Example 1 with the ESBL (Epithelial Systems Biology Laboratory) database. [Figure 2] (A) is a Venn diagram showing the results of comparing the list obtained from pathway enrichment analysis of the ESBL database with the list obtained from uEVs of PA patients in Example 1. (B) is a dot plot of 62 pathways specific to urinary extracellular vesicles (uEVs) of PA patients. [Figure 3] (A) is a diagram showing the MS / MS spectrum of the peptide of SEQ ID NO: 3 in Example 1. (B) is a diagram showing the MS / MS spectrum of the peptide of SEQ ID NO: 4 in Example 1. (C) is a diagram showing the peptides (SEQ ID NOs: 3 and 4) identified in Example 1, and a part of the ENaCγ sequence (SEQ ID NO: 5) containing the furin cleavage site (SEQ ID NO: 1). [Figure 4] The left side of (A) is a representative example of Western blot analysis using uEVs isolated from PA patients (PA in the figure) and healthy subjects (HC in the figure) in Example 2. The right side of (A) is a representative example of Western blot analysis using uEVs and the plasma-enriched fraction from aldosterone-injected rats in Example 2. (B) shows the results of quantification of ENaCγ fragments in PA patients and healthy subjects in Example 2. [Figure 5] 1 is a representative example of Western blot analysis using uEVs isolated from PA patients in Example 3. [Figure 6](A) is a scatter plot showing the relationship between the amount of ENaCγ fragment and the aldosterone-renin ratio (ARR) in Example 3. (B) is a scatter plot showing the relationship between the amount of ENaCγ and serum potassium ion concentration in Example 3. (C) is a scatter plot showing the relationship between the amount of ENaCγ and the fractional excretion of potassium ion (FEK) rate in Example 3. (D) is a scatter plot showing the relationship between the amount of ENaCγ fragment and the ratio of urinary sodium ion concentration to urinary potassium ion concentration in Example 3. [Figure 7] FIG. 10 shows the results of comparing the amount of ENaCγ fragment in uEV between unilateral PA patients and bilateral PA patients in Example 3. [Figure 8] (A) is a representative example of Western blot analysis of PA patients before and after PA treatment in Example 4. (B) is a diagram showing the change in ENaCγ fragment levels from baseline in PA patients after treatment in Example 4. [Figure 9] FIG. 10 shows the change in ENaCγ fragment levels from baseline in patients who underwent adrenalectomy and patients who underwent targeted therapy using an MR antagonist in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Method for supporting the diagnosis of hypertension in test animals> The diagnostic support method for hypertension in a subject animal according to this embodiment (hereinafter, sometimes simply referred to as the "diagnostic support method") includes: 1. A method for aiding in the diagnosis of hypertension in an animal subject, comprising: A recovery step of recovering uEVs from the urine of the test animal; The recovery step includes a detection step of detecting an ENaCγ fragment contained in the uEV. The method further comprises an evaluation step of evaluating that there is a high need for treatment or a high need for optimization of treatment when the amount of the ENaCγ fragment detected in the detection step is equal to or greater than a predetermined reference value.

[0011] In patients with PA, the aldosterone-to-renin ratio (ARR) is used to screen for aldosterone hypersecretion. The ARR has also been used to address postoperative biochemical outcomes in unilateral PA (aldosterone-producing adenoma or unilateral adrenal hyperplasia). However, uncertainty remains regarding the optimization of MR antagonist treatment in other hypertensive conditions, including bilateral PA, and currently, there is no standard approach to guide MR blockade in hypertension. Furthermore, MR signaling activity may be regulated by multiple mechanisms.

[0012] The inventors focused on the ENaCγ fragment in uEVs and discovered that MR signaling activity in the kidney can be accurately and noninvasively detected by using the ENaCγ fragment as a biomarker for MR signaling activity in the kidney, leading to the completion of the present invention.

[0013] ENaC regulates apical sodium reabsorption in the distal nephron and plays a central role in aldosterone-mediated sodium transport. Aldosterone-induced increases in ENaC-mediated sodium reabsorption are mediated by multiple mechanisms, with an increase in the open probability of single channels playing a dominant role. The key process controlling the open probability of the channel is proteolytic activation by furin and other proteases. The increase in the ENaCγ fragment in the kidney is biochemically detected as a band shift from 93 kDa to 75 kDa in humans (Non-Patent Document 5).

[0014] The "ENaCγ fragment" used in the diagnostic support method of this embodiment refers to a fragment obtained by cleavage of ENaCγ, one of the subunits constituting ENaC. The ENaCγ fragment is not particularly limited as long as it is a partial fragment of ENaCγ, and examples thereof include fragments cleaved by proteases. The proteases preferably recognize an RKRX (X: basic amino acid) motif, and more preferably proteases that recognize an RKRR (SEQ ID NO: 1) motif or a RKRK (SEQ ID NO: 2) motif. An example of a protease that recognizes an RKRR (SEQ ID NO: 1) motif is furin, and an example of a protease that recognizes an RKRK (SEQ ID NO: 2) motif is prostasin. It is known that ENaCγ is primarily cleaved by furin or prostasin (Non-Patent Documents 5 and 6). The ENaCγ fragment is preferably a fragment cleaved by furin or a fragment cleaved by prostasin, or both.

[0015] As shown in the Examples below, the amount of ENaCγ fragments was determined by the ARR and fractional excretion of potassium ions (FEK) in the uEV of PA patients. + ) rate and negatively correlates with serum potassium ion concentration. In other words, the amount of ENaCγ fragment in uEV increases in PA patients and decreases with the inhibition of MR signaling activity. The diagnostic support method of this embodiment is advantageous in that it can accurately assess the strength of MR signaling activity by using the amount of ENaCγ fragment in uEV as a biomarker. Here, strong MR signaling activity refers to a state in which the transcription of target molecules whose transcription is controlled by MR is activated and their expression levels are increased.

[0016] <Recovery process> The recovery step in the diagnostic support method of this embodiment is a step of recovering uEVs from the urine of a test animal.

[0017] Urine from test animals may be collected by conventional methods, and may be pretreated by adding a proteolytic inhibitor or by centrifugation.

[0018] uEVs can be collected from the urine of test animals by conventional methods. For example, uEVs can be concentrated and isolated using centrifugation, ultracentrifugation, density gradient centrifugation, filtration, size exclusion chromatography, etc. Furthermore, the diagnostic support method of this embodiment can be carried out using reagents, devices, commercially available kits, etc. for these collection methods.

[0019] In the present specification, the "subject animal" is not particularly limited as long as it is an animal that excretes urine, but is preferably a mammal. The mammal may be a human or a non-human mammal. Examples of non-human mammals include pet animals such as dogs and cats, livestock animals such as cows, pigs, and horses, and laboratory animals such as mice and rats. The subject animal may be a hypertensive patient, a person suspected of having hypertension, or a healthy individual. Hypertension may be essential hypertension or secondary hypertension, with primary aldosteronism (PA) being particularly preferred as secondary hypertension.

[0020] <Detection process> The detection step in the diagnostic support method of this embodiment is a step of detecting the ENaCγ fragment contained in the uEV after the recovery step.

[0021] The method for detecting ENaCγ is not particularly limited, but it is preferable to use an immunological method, as it allows for simple, rapid and highly sensitive detection. The immunological technique is not particularly limited as long as it allows simple, rapid, and highly sensitive detection, and examples include Western blotting, enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), immunochromatography, fluorescent antibody technique, latex agglutination, immunoturbidimetry, immunonephelometry, radioimmunoassay, etc. The diagnostic support method of this embodiment may be carried out using reagents, devices, kits, etc. for these immunological techniques.

[0022] In immunological techniques, it is preferable to use a substance that specifically binds to the ENaCγ fragment. The substance in question is not particularly limited in type or origin as long as it can specifically bind to the ENaCγ fragment, and antibodies, nucleic acid aptamers, low-molecular-weight compounds, etc. can be used. Among these, it is preferable to use the anti-ENaCγ antibody described in the Examples below, since the commonly used Western blotting method or ELISA method can be used.

[0023] <Evaluation process> The evaluation process in the diagnostic support method of this embodiment is a process of evaluating that there is a high need for treatment or a high need for optimization of treatment when the amount of ENaCγ fragment detected in the detection process is above a predetermined standard value.

[0024] If the detected amount of ENaCγ fragment in a patient undergoing treatment is equal to or greater than a predetermined reference value, it is highly likely that the treatment is not providing a sufficient therapeutic effect, and therefore, it is possible to provide information that the treatment needs to be optimized, such as by increasing the dosage of the therapeutic drug, optimizing the administration interval, or switching to another therapeutic drug. Alternatively, if the detected amount is less than a predetermined reference value, it can be evaluated that there is little need for treatment or that the treatment is likely to be appropriate. More specifically, if the subject animal is a hypertensive patient not receiving MR antagonist treatment and the detected amount of ENaCγ fragment is equal to or greater than a predetermined reference value, it can be determined that the subject animal is likely to achieve a sufficient therapeutic effect and is therefore in need of MR antagonist treatment. If the subject animal is a hypertensive patient receiving MR antagonist treatment and the detected amount of ENaCγ fragment is equal to or greater than a predetermined reference value, it can be determined that the subject animal is not achieving a sufficient therapeutic effect with the current MR antagonist treatment and that treatment optimization is necessary, for example, an increase in the MR antagonist dosage should be considered. If the subject animal is a hypertensive patient receiving MR antagonist treatment and the detected amount of ENaCγ fragment is less than a predetermined reference value, it can be determined that the MR signaling activity is sufficiently low and that a treatment other than MR antagonist treatment is likely to be necessary to achieve a more sufficient therapeutic effect.

[0025] The predetermined reference value can be determined in advance based on data on the amount of ENaCγ fragment in uEVs of healthy individuals and hypertensive patients, or data on the amount of ENaCγ fragment in uEVs of the same subject animal at a certain time point (before treatment, during treatment), etc. For example, a threshold value that can distinguish the amount of ENaCγ fragment in uEVs of healthy individuals from the amount of ENaCγ fragment in uEVs of PA patients can be set as the "predetermined reference value."

[0026] The amount of ENaCγ fragment in uEV and its reference value do not need to be an absolute amount, but may be a relative value that allows for comparison of the amount. Furthermore, in the evaluation step, it is not essential to strictly quantify the ENaCγ fragment detected in the detection step, as long as it is possible to determine whether it is equal to or greater than a predetermined reference value.

[0027] The recovery step, detection step, and evaluation step are steps carried out in vitro.

[0028] In one aspect of the present invention, the diagnostic support method of this embodiment may further include another step after the evaluation step or in parallel with the evaluation step. Such another step may include a step of providing treatment to the hypertensive patient based on the evaluation results. When the diagnostic support method of this embodiment further includes a treatment step in addition to the evaluation step, it may be a method of treating the hypertensive patient.

[0029] In the treatment step, a treatment policy for hypertension can be determined based on the evaluation results of the diagnostic support method of this embodiment. As a treatment method for hypertension patients, a method conventionally performed in this technical field can be adopted, such as adrenalectomy or treatment with an MR antagonist, with treatment with an MR antagonist being preferred. In treatment with an MR antagonist, the type or dosage of the MR antagonist can be determined based on the evaluation results of the diagnostic support method of this embodiment.

[0030] Examples of MR antagonists include spironolactone (CAS number: 52-01-7), eplerenone (CAS number: 107724-20-9), osedurenone (KBP-5074, CAS number: 1359969-24-6), balcinrenone (AZD9977, CAS number: 1850385-64-6), apararenone (MT-3995, CAS number: 945966-46-1), esaserenone (CS-3150, CAS number: 1632006-28-0), finerenone (BAY 94-8862, CAS number: 1050477-31-0), and modified compounds thereof.

[0031] MR signaling activity is known to correlate with diseases such as chronic kidney disease, chronic heart failure, and diabetic kidney disease (Non-Patent Document 7, Non-Patent Document 8). Therefore, the amount of ENaCγ fragment in uEV is also useful as a biomarker for diseases associated with MR signaling activity such as chronic kidney disease, chronic heart failure, and diabetic kidney disease, and the diagnostic support method of the present embodiment can also be applied to the diagnosis support of these diseases. For example, although some MR antagonists have been approved as therapeutic agents for chronic kidney disease, the diagnostic support method of the present embodiment is also useful for evaluating the efficacy of the MR antagonist in chronic kidney disease. <C

[0032] <Kit for detecting ENaCγ fragment in uEV> The kit of the present embodiment is a kit for detecting an ENaCγ fragment in uEV, and contains a substance that specifically binds to the ENaCγ fragment.

[0033] As the substance that specifically binds to the ENaCγ fragment, as long as it can specifically bind to the ENaCγ fragment, its type, origin, etc. are not particularly limited. For example, the same substances as those that specifically bind to the ENaCγ fragment in the above diagnostic support method can be mentioned, and it is preferable to use an anti-ENaCγ antibody.

[0034] The kit of the present embodiment may contain substances other than the substance that specifically binds to the ENaCγ fragment as long as the effects of the present invention are not impaired. Substances other than the substance that specifically binds to the ENaCγ fragment include, for example, a buffer solution, and reagents and devices used in immunological techniques, etc.

[0035] The kit of the present embodiment is preferably used to support the diagnosis of hypertension in a test animal, and it is more preferable that the hypertension is PA. Examples of the test animal include the same ones as those in the test animal in the above diagnostic support method.

[0036] In one embodiment, the present invention provides an agent that specifically binds to an ENaCγ fragment to aid in the diagnosis of hypertension in an animal subject.

[0037] In one embodiment, the present invention provides use of a substance that specifically binds to an ENaCγ fragment for producing a kit for detecting an ENaCγ fragment in uEVs. [Example]

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0039] [material and method] (patient) This study included 63 PA patients who were diagnosed with PA and agreed to donate urine to Yokohama Rosai Hospital and Teikyo University Hospital between 2017 and 2022. Of these PA patients, 26 participated in our previous analysis (Non-Patent Document 9). The diagnosis of PA was made according to the Japanese Society of Hypertension's Guidelines for Hypertension Treatment (JSH2019). All patients underwent adrenal vein sampling to determine laterality (bilateral or unilateral PA). This study also included 11 healthy volunteers who agreed to donate urine for EV isolation. This study was approved by the Institutional Review Boards of Yokohama Rosai Hospital and Teikyo University Hospital (17-149-3 and 19-207-2). This study was conducted in accordance with the Declaration of Helsinki, and informed consent was obtained from all participants included in this study.

[0040] (Isolation of uEV-enriched fraction) The isolation of the uEV-enriched fraction was performed according to our previous study (Non-Patent Document 9) and the method of Knepper's laboratory (Non-Patent Document 10). Specifically, an aliquot (50 mL) of urine sample was immediately placed in a sterile container containing a protease inhibitor cocktail (Roche Diagnostics, Switzerland). The sample was centrifuged at 1,500 × g for 10 minutes at 4°C to remove insoluble components, followed by a further centrifugation at 17,000 × g for 15 minutes. The resulting supernatant was stored until combined with the supernatant described below. The pellet was resuspended in a pH 7.6 separation solution containing 10 mM triethanolamine and 250 mM sucrose. Subsequently, the sample was incubated with 200 mg / mL dithiothreitol to disrupt the polymer network. The incubated sample was then centrifuged at 17,000 × g for 10 minutes. The resulting supernatant and the above supernatant, i.e., the two supernatants obtained by centrifugation at 17,000 × g, were mixed and further centrifuged at 200,000 × g for 60 minutes. Isolation of the uEV-enriched fraction, i.e., enrichment of exosome-like particles (20-100 size), was confirmed by electron microscopy.

[0041] (Liquid chromatography tandem mass spectrometry (LC-MS / MS)) The uEV pellet after the centrifugation was suspended in ultrapure water, and the protein concentration in the uEV pellet was measured using a 660 nm protein assay (Pierce, USA). Sample preparation for LC-MS / MS analysis was performed similarly to the study by Qi Y et al. (Non-Patent Document 11). Specifically, 50 μg of protein sample was dried using a centrifugal concentrator and then reduced in volume by incubation at 56°C for 45 minutes in a solution containing 50 mM NH4HCO3, 50% trifluoroethanol (TFE), and 20 mM DTT (dithiothreitol). The sample was then alkalized with iodoacetamide to a final concentration of 40 mM at room temperature for 1 hour. Excess iodoacetamide was quenched by adding 40 mM DTT, and the solution was incubated for another hour in the dark. The TFE concentration in the solution was reduced to less than 5% by adding 50 mM NH4HCO3.

[0042] Next, trypsin solution was added to the solution (trypsin:uEV protein = 1:50 (mass ratio)) and incubated at 37°C for 15 hours. The digestion was terminated by adding formic acid. The resulting peptide solution was then centrifuged at 20,000 × g for 20 minutes at 4°C, and the supernatant was collected. The pellet was extracted twice with a 0.1% formic acid solution in 50% acetonitrile and centrifuged. The resulting extract and supernatant were combined and dried using a centrifugal concentrator. The pellet was then dissolved in a 0.1% formic acid solution and centrifuged at 215,000 × g for 20 minutes at 4°C. The resulting supernatant was subjected to GL-TipSDB-SCX (GL Sciences, Japan) in the same manner as described in Non-Patent Document 12. The resulting eight peptide fractions were each concentrated using a centrifugal concentrator.

[0043] The peptide fraction was applied to an Ultimate 3000 capillary nano LC system (Thermo Fisher Scientific, USA) equipped with a C18 capillary column (particle size: 3 μm, inner diameter: 75 μm, packing length: 125 mm, Nikkyo Technos, Japan). The peptides were eluted with Solution A (5% acetonitrile / 0.1% formic acid) and Solution B (90% acetonitrile / 0.1% formic acid) at a flow rate of 300 nl / min over 120 min. Mass spectrometry was performed using a Q-Exactive orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Precursor ions between m / z 400 and 2,000 were selected and analyzed using the Top 10 method. MS / MS spectra were searched against the SwissProt Homo sapiens database (version 2017-10-25) using the Sequest HT search engine integrated into Proteome Discoverer 2.4 (Thermo Fishier Scientific, USA).

[0044] The mass tolerance for precursor ions was ±10 ppm, and for fragment ions ±0.02 Da. A maximum of two missed cleavages was allowed during trypsin digestion. The list of identified proteins was compared with the uEV protein database at the Epithelial Systems Biology Laboratory (ESBL) of the National Heart, Lung, and Blood Institute (NHLBII) using Draw Venn Diagram (Ghent University, http: / / bioinformatics.psb.ugent.be / ). UniProtKB accession numbers were converted to gene IDs using SynGO34 (NPL 13), and different isoforms were grouped into a single ID.

[0045] (Comparison of ENaCγ fragment amounts in uEVs from PA patients) In Example 1, 11 PA patients before targeted treatment (adrenalectomy or administration of an MR antagonist) and 11 healthy volunteers were included to compare the amount of EnaCγ. Example 2 includes a separate cohort of 51 PA patients, of which 5 were excluded due to lack of material. In Example 3, 15 patients (14 of whom also participated in the study in the Example) from whom post-treatment urine samples were obtained were included, and changes in ENaCγ abundance before and after treatment were compared.

[0046] The sample size was calculated based on a t-test to detect a difference between the two groups. Based on our previous study (Non-Patent Document 9), we estimated the effect size of the target protein to be 1.2 and calculated that at least 10 subjects per group were required to detect a difference with 80% statistical power.

[0047] Serum aldosterone levels and plasma renin activity data were available for all PA patients. Serum aldosterone levels were measured using radioimmunoassay (RIA) in all but one patient. In one patient, aldosterone CLEIA values ​​measured using chemiluminescence enzyme immunoassay (CLEIA, Fujirebio, Tokyo; reference range 4.0–82.1 pg / mL) were converted to aldosterone (RIA) using the following formula: RIA(pg / ml)=CLEIA(pg / ml)×1.174+42.3

[0048] Estimated GFR (eGFR) was calculated based on the Pancreas Society formula (Non-patent Document 14).

[0049] (Animal samples) EVs derived from 24-hour urine collections from control and aldosterone-injected rats were obtained in our previous study (Non-Patent Document 9) and were used to validate the ENaCγ fragment obtained in human uEV samples.

[0050] Male Sprague-Dawley rats (6 weeks old) were purchased from Tokyo Experimental Animals Co., Ltd. and used. Animal experiments were approved by the Teikyo University Animal Experiment Ethics Committee (#20-007) and conducted in accordance with the guidelines of Teikyo University.

[0051] (Western blotting) The uEV pellet was resuspended in isolation solution and added to Laemmli sample buffer. The quality of the sample was confirmed by assessing the Alix content. The sample was separated on a polyacrylamide gel and transferred to a PVDF membrane. The PVDF membrane was blotted with 5% milk and then incubated with an anti-Alix antibody (Proteintech, USA). It was then incubated with a peroxidase-conjugated anti-rabbit antibody (GE, USA).

[0052] Signals were visualized using ECL reagents (Perkin Elmer, USA). The amount of ENaCγ used for Western blotting was adjusted according to the Alix level in the sample. ENaCγ fragments in uEVs were detected using a previously validated antibody (Non-Patent Document 15) (StressMarq, USA). In the multiple Western blotting in Example 3, a common uEV sample collected from a PA patient was included in each blot for normalization. After ENaCγ detection, the PVDF membrane was stripped and reprobed with Alix to normalize the ENaCγ signal.

[0053] (statistics) In this study, all statistical analyses were performed using JMP version 17.0.0 (SAS Institute, Inc.) and GraphPad Prism software, version 10.2.3 (GraphPad Software, Inc., USA), and two-sided significance was set at P<0.05.

[0054] Comparisons between two groups were performed using unpaired t-tests. For correlation analyses, ARR, FEK rate, urinary sodium ion to potassium ion ratio, and ENaCγ fragment levels were transformed into natural logarithms, and correlation coefficients were calculated using the Pearson test. Sensitivity analyses were performed excluding patients receiving angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs). Unpaired samples t-tests were used to compare ENaCγ fragment levels between patients with bilateral PA and those with unilateral PA. Multiple regression analysis was used to assess the independent association between ENaCγ and ARR after adjusting for potential confounders. Paired t-tests were used to assess changes in ENaCγ fragment levels over time. All statistical tests were two-sided, and a significance level of less than 0.05 was considered statistically significant.

[0055] [Example 1] Comprehensive analysis of proteins in uEVs in PA patients EVs isolated from the urine of PA patients before targeted treatment (adrenalectomy or pharmacological MR blockade) were digested with trypsin. The resulting peptides were analyzed by LC-MS / MS, resulting in the identification of a total of 1,940 proteins (false discovery rate <0.05). Representative exosome markers identified included the tetraspanin family, CD9 (UniProtKB accession number: P21926), CD63 (UniProtKB accession number: P08962), CD81 (UniProtKB accession number: P60033), and CD82 (UniProtKB accession number: P27701).

[0056] For pathway enrichment analysis, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG). To do this, we converted UniProtKB accession numbers into gene symbols and classified different isoforms into single identities. The resulting 1,850 IDs were subjected to enrichment analysis.

[0057] The most prominent pathway was endocytosis (Figure 1(A)), which was classified into 102 pathways. These proteins included tumor susceptibility 101 (Tsg101), programmed cell death 6-interacting protein (ALG-2-interacting protein X; Alix), and other specific molecules associated with the endosomal sorting complex required for transport (ESCRT), such as vacuolar protein sorting-associated protein (VPS) and charged multivesicular body protein (CHMP). These findings indicate that the samples are enriched for exosome-associated proteins. In addition to endocytosis, other pathways that were highly expressed included those related to vesicle formation, such as lysosomes, actin cytoskeleton regulation, and phagocytosis (Figure 1(A)).

[0058] Next, we compared the results of the above analysis with the ESBL database to obtain information on the proteins abundant in uEVs from PA patients. As a result, 1194 IDs (65%) were specific to uEVs from PA patients, while 433 IDs were not detected in the ESBL database (Fig. 1(B)).

[0059] To obtain more detailed information, we performed pathway enrichment analysis in the ESBL database and compared the resulting list with the list obtained from uEVs in PA patients. KEGG pathway analysis revealed that 59 pathways were common, 18 pathways were unique to the ESBL database, and 62 pathways were unique to the list obtained from uEVs of PA patients ( Figure 2(A) ).

[0060] As shown in Figure 2(B), among the 62 pathways mentioned above, the high expression of the AGE-RAGE signaling pathway (04933 in Figure 2(B)) in diabetic complications was consistent with multiple evidences indicating the role of aldosterone and MR in the pathophysiology of diabetic complications. Other highly expressed pathways included cell adhesion molecules (04514 in Figure 2(B)), glycosaminoglycan degradation (00531 in Figure 2(B)), and insulin signaling (04910 in Figure 2(B)), which were involved in the pathophysiology induced by aldosterone and MR.

[0061] Mechanistically, insulin signaling and aldosterone signaling may converge at multiple levels, including sgk1 and its downstream targets. Rac1 GTPase, which is involved in MR and promotes signal transduction, was included in 22 of the 62 pathways (35%), including the Ras signaling pathway (04014 in Figure 2(B)), the chemokine signaling pathway (04062 in Figure 2(B)), and the axon guidance pathway (04360 in Figure 2(B)). This highlights the close relationship between the two signaling mechanisms.

[0062] In addition to the above pathways, the aldosterone-regulated sodium reabsorption pathway (04960, aldosterone-regulated sodium reabsorption in Figure 2(B)) was significantly expressed in uEVs from PA patients (Figure 2(B)). As shown in Table 1, of the 12 proteins detected in this pathway, 8 (75%) were specific to uEVs from PA patients, specifically NEDD4-2, NHERF2, ROMK, and ENaCγ.

[0063] [Table 1]

[0064] In contrast, other major salt transport proteins in the distal nephron, such as Na-K-2Cl cotransporter 2 (NKCC2), Na-Cl cotransporter (NCC), and pendrin, were all detected both in uEVs from PA patients and in the ESBL database.

[0065] Detailed investigation of the ENaCγ peptides identified in uEVs from PA patients (Figure 3(A) and Figure 3(B)) revealed that the two peptides (SEQ ID NOs: 3 and 4) were derived from the N- and C-termini of the RKRR (SEQ ID NO: 1) motif in human ENaCγ (hENaCγ), respectively (Figure 3(C) and SEQ ID NO: 5). This motif is a proteolytic activator of ENaCγ that is recognized and cleaved by furin. The detection of these peptides suggests that ENaCγ fragments in uEVs could be potential markers of renal MR signaling activity.

[0066] [Example 2] ENaCγ in uEVs exists as a 75-kDa cleaved ENaCγ fragment and is increased in PA patients. To confirm the results of the above proteomic analysis, we analyzed the amount of ENaCγ fragments in uEVs from 11 PA patients and 11 healthy volunteers. Western blot analysis of the ENaCγ fragment in human uEVs using a previously validated antibody (Non-Patent Document 15) detected a signal at approximately 75 kDa, which coincided with the molecular weight of the ENaCγ fragment (Figure 4(A), left).

[0067] Western blotting was also performed using uEVs isolated from aldosterone-injected rats to verify the specific signal in uEVs (Fig. 4(A) right). Comparison of the ENaCγ signal in uEVs and kidneys from aldosterone-injected rats confirmed that ENaCγ in uEVs exists primarily as a 75 kDa ENaCγ fragment (Fig. 4(A) right). Quantification of the ENaCγ fragment signal in uEVs from healthy volunteers and PA patients revealed that the amount of ENaCγ fragment in PA patients (PA in Figure 4(B)) was 4.8-fold increased compared to that in healthy volunteers (HC in Figure 4(B)) (Figure 4(B)). These findings demonstrate that the amount of ENaCγ fragments in uEVs increases in PA patients.

[0068] [Example 3] Relationship between ENaCγ fragment amount and clinical indicators in PA patients We then evaluated the relationship between baseline ENaCγ fragment levels and clinical indicators in PA patients. Alix signals were not detected in uEVs in 5 of the 51 PA patients, likely due to insufficient material. Therefore, we analyzed the remaining 46 PA patients. Of the 46 PA patients, 23 (50%) had unilateral PA, which was associated with systolic hypertension, hypokalemia, and a high ARR.

[0069] Western blotting was performed using uEVs from PA patients in the same manner as described in Example 2 (Figure 5). Correlation analysis based on the amount of ENaCγ fragment quantified by Western blotting revealed a positive correlation between the amount of ENaCγ fragment corrected by Alix and ARR (r = 0.31, P = 0.035) (Figure 6(A)).

[0070] We also evaluated the relationship between the amount of ENaCγ fragments in uEVs of PA patients and other clinical indicators, including serum potassium ion concentration, FEK ratio, and urinary sodium-to-potassium ratio. The results revealed that the amount of ENaCγ fragments in uEVs of PA patients was negatively correlated with serum potassium ion concentration (Figure 6(B)) and positively correlated with FEK ratio (Figure 6(C)). This is consistent with the idea that increased ENaC activation in PA patients promotes potassium ion secretion in the kidney and decreases serum potassium ion concentration.

[0071] On the other hand, the ratio of sodium to potassium in urine, another potential indicator of MR signaling activity, did not correlate with the amount of ENaCγ fragments in uEVs of PA patients (Fig. 6(D)), possibly because this ratio is easily affected by dietary intake.

[0072] When patients were classified according to the type of PA, the amount of ENaCγ fragment was significantly higher in patients with unilateral PA (Fig. 7, Unilateral PA) than in patients with bilateral PA (Fig. 7, Bilateral PA), which was consistent with the higher ARR in the former patients in our cohort. Of the 46 PA patients, three were treated with angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs). However, the relationship between the ENaCγ fragment levels and clinical indices was not affected even if these three patients were excluded.

[0073] Furthermore, after adjusting for clinical factors such as age, sex, smoking status, alcohol intake, and eGFR, we performed multiple regression analysis to examine the correlation between the amount of ENaCγ fragment in uEVs and ARR in PA patients (Table 2). Model 1 included age and sex as clinical factors. Model 2 included the same factors as Model 1, as well as smoking status, alcohol intake, and eGFR as clinical factors. These models revealed a correlation between the amount of ENaCγ fragment in uEVs and ARR in PA patients (Table 2).

[0074] [Table 2]

[0075] [Example 4] Changes in ENaCγ fragment levels before and after treatment We evaluated changes in the amount of ENaCγ fragments in uEVs of 15 PA patients who provided urine samples at follow-up visits (at least 3 months, mean 7.8 months, after MR antagonist treatment or adrenalectomy). Specifically, Western blotting was performed using uEVs from PA patients as described in Example 2 (Figure 8(A)). The results showed that the amount of ENaCγ fragments in uEVs from PA patients after treatment decreased by 37% (Figure 8(B), P<0.01). Furthermore, when patients were divided according to the treatment they received (adrenalectomy or targeted therapy with an MR antagonist), the decrease in the amount of ENaCγ fragments in uEVs in each PA patient was more pronounced after adrenalectomy (ADX in Figure 9) than after targeted therapy with an MR antagonist (MRA in Figure 9) (Figure 9). These findings demonstrate that the ENaCγ fragment in uEVs from PA patients decreases with the inhibition of MR signaling activity. [Industrial Applicability]

[0076] According to the present invention, information useful for diagnosing hypertension, particularly information regarding the effectiveness of MR antagonists, can be provided non-invasively.

Claims

1. 1. A method for aiding in the diagnosis of hypertension in an animal subject, comprising: A recovery step of recovering urine-derived extracellular vesicles from the urine of the subject animal; The recovery step includes a detection step of detecting ENaCγ fragments contained in the urine-derived extracellular vesicles. and an evaluation step of evaluating that there is a high need for treatment or a high need for optimization of treatment when the amount of the ENaCγ fragment detected in the detection step is equal to or greater than a predetermined reference value.

2. The method of claim 1, wherein the hypertension is primary aldosteronism.

3. The method according to claim 1 or 2, wherein the detecting step is carried out by an immunological technique.

4. The method according to claim 1 or 2, wherein the ENaCγ fragment is a fragment obtained by cleaving ENaCγ with a protease that recognizes a RKRR motif or a RKRK motif.

5. 3. The method of claim 1 or 2, wherein the treatment is treatment with a mineralocorticoid receptor antagonist.

6. A kit for detecting ENaCγ fragments in urine-derived extracellular vesicles, comprising: A kit comprising a substance that specifically binds to the ENaCγ fragment.

7. 7. The kit of claim 6, for use in aiding in the diagnosis of hypertension in an animal subject.

8. The kit of claim 7, wherein the hypertension is primary aldosteronism.

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