Method for testing tertiary lymphatic tissue and kit for testing tertiary lymphatic tissue

Abstract

One purpose of the present disclosure is to provide a technique for checking the presence or absence of tertiary lymphatic tissue in a subject without relying on biological tissue diagnosis. A method for testing the presence or absence of a tertiary lymphatic tissue in a subject, said method comprising a step for measuring the glutathione concentration in a bodily fluid sample collected from the subject.

Description

Technical Field

[0001] This disclosure relates to a method for examining the presence or absence of tertiary lymphoid tissue (TLT) in a test subject. Additionally, this disclosure relates to a kit for examining the presence or absence of TLT in a test subject. Background Technology

[0002] TLT, or induced ectopic lymphoid tissue, is a tissue similar to lymph nodes that forms in non-lymphoid tissue (Non-Patent Literature 1-3). In TLT, the extensive infiltration of lymphocytes, through the interaction and proliferation of T cells and B cells, becomes the starting point for an acquired immune response, amplifying the immune response. TLT is known to form in lesions of diseases with chronic inflammation, autoimmune diseases, infectious diseases, and cancers (Non-Patent Literature 1-3), and its influence on the severity and prognosis of these diseases is known.

[0003] For example, it is known that in kidney diseases such as pyelonephritis, IgA nephropathy, and lupus nephritis, the induction of TLTs leads to persistent inflammation and impaired repair (Non-Patent Literature 4). Furthermore, in recent years, the inventors of this application have demonstrated that the maturity of TLTs can be used as a biomarker reflecting the degree of tissue damage in the kidney (Non-Patent Literature 5). Specifically, as a result of tissue analysis of human pyelonephritis, it was clarified that TLTs at various maturity stages were simultaneously present in the kidney, with more mature TLTs observed in areas of severe tissue damage. On the other hand, TLT induction was not significant in areas of mild damage, and the proportion of immature TLTs formed was also higher. In addition, analysis of elderly patients confirmed that compared to non-complication cases, chronic kidney disease patients with complications had significantly more TLTs, and a higher proportion of more mature TLTs. Furthermore, it was confirmed that regardless of the type of underlying disease, TLTs are induced at the same anatomical location and undergo the same maturity stage. In addition, it has been reported that if a highly mature TLT is present in the transplanted kidney, a decrease in kidney function can be easily observed after 5 years (Non-Patent Literature 6), suggesting that TLT may be a predictive biomarker for future kidney function.

[0004] Thus, it is known that TLT may influence the severity of the disease, prognosis, and responsiveness to treatment, and confirming the presence and maturity of TLT formation is useful for determining treatment intervention strategies and assessing their effectiveness.

[0005] On the other hand, confirming the presence or absence of TLT formation requires collecting tissue from the lesion site and performing biological tissue diagnosis. However, collecting tissue from the lesion site has the following disadvantages: it requires maintaining the patient's position for a specified period of time, and there are risks such as bleeding, which places a significant burden on the patient. In addition, the risks of tissue collection are high in patients undergoing antiplatelet therapy and in the elderly, making it particularly difficult to confirm the presence or absence of TLT formation in these patients.

[0006] Existing technical documents Non-patent literature Non-patent literature 1: Sato Y. et al., Immunology of the aging kidney. Nat RevNephrol. 2019;15(10):625-40. Non-patent literature 2: Antonioli L. et al., Ectopic Lymphoid Organs and Immune-Mediated Diseases: Molecular Basis for Pharmacological Approaches. Trends MolMed. 2020;26(11):1021-1033. Non-patent literature 3: Sautes-Fridman C. et al., Tertiary lymphoid structures in theera of cancer immunotherapy. Nat Rev Cancer. 2019;19(6):307-25. Non-patent literature 4: Sato Y. et al., Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney. JCI Insight. 2016;1(11):e87680. Non-patent literature 5: Sato Y. et al., Developmental stages of tertiary lymphoid tissue reflect local injury and inflammation in mouse and human kidneys. Kidney Int. 2020;98(2):448-463. Non-patent document 6: Lee H. et al., HYPERLINK "https: / / pubmed.ncbi.nlm.nih.gov / 34725107 / "Advanced Tertiary Lymphoid Tissues in Protocol Biopsies areAssociated with Progressive Graft Dysfunction in Kidney TransplantRecipients. J Am Soc Nephrol. 2022;33(1):186-200. doi: 10.1681 / ASN.2021050715. Summary of the Invention

[0007] The problem that the invention aims to solve One objective of this disclosure is to provide a technique for examining the presence or absence of TLT in a subject without relying on biological tissue diagnostics.

[0008] Methods for solving problems The inventors of this application conducted in-depth research to address the aforementioned issues, and found that in mice with TLT formation in their kidneys, glutathione accumulates in the TLT, and the concentration of glutathione in urine samples increases. Furthermore, the inventors of this application grouped IgA nephropathy patients based on the presence or absence of TLT and analyzed the concentration of glutathione in urine samples. The results showed that in patients with TLT, the concentration of glutathione in urine samples was significantly increased compared to patients without TLT. This disclosure is based on further repeated research based on the above insights.

[0009] That is, this disclosure provides inventions in the manner listed below.

[0010] Item 1. A method for determining the presence or absence of TLT in a test subject, said method comprising the step of measuring the concentration of glutathione in a body fluid sample collected from the test subject.

[0011] Item 2. The examination method as described in Item 1, wherein the aforementioned body fluid sample is a urine sample.

[0012] Item 3. The examination method as described in Item 1 or 2, wherein the glutathione concentration is the reduced glutathione concentration.

[0013] Item 4. The examination method as described in Item 2, the examination method further comprising the step of measuring the concentration of creatinine in a urine sample.

[0014] Item 5. The examination method as described in any one of items 1 to 4, wherein the examination method is performed to examine the presence or absence of TLT in the kidney, wherein the subject is a patient with kidney disease, a subject who needs to be examined for the presence or absence of kidney disease, or a subject who has received a kidney transplant.

[0015] Item 6. A test kit for detecting the presence or absence of TLT in an organism, said test kit containing reagents for determining glutathione concentration.

[0016] Item 7. The use of reagents for determining glutathione concentration in the manufacture of a test kit for detecting the presence or absence of TLT in vivo.

[0017] Invention Effects According to one embodiment of the examination method of this disclosure, by using the concentration of glutathione in bodily fluid samples such as urine as a biomarker of TLT, the presence or absence of TLT in a subject can be examined without relying on biological tissue diagnosis. Furthermore, for example, by using the examination method of this disclosure to predict the presence or absence of TLT in the kidneys of patients with kidney disease or recipients of kidney transplants, the degree of kidney disease and the function of the transplanted kidney can be inferred, thus enabling early treatment intervention for patients at high risk of functional deterioration of the transplanted kidney. Attached Figure Description

[0018] [ Figure 1 This diagram shows the structural schematics of different maturation levels of TLTs formed in the kidney.

[0019] [ Figure 2[a] Shows the experimental route for measuring glutathione levels in kidney tissue and urine using a kidney TLT model induced by ischemia-reperfusion injury (IRI). [b] Shows images obtained by HE staining (left) and immunostaining (right) with anti-CD3e and anti-B220 antibodies on kidney tissue from mice induced by IRI. [c] Shows HE staining images (left end), oxidized glutathione (GSSG) mass spectrometry (MSI) images (second from left), reduced glutathione (GSH) MSI images (second from right), and an image obtained by fusing the HE staining image and the GSSG MSI image (right end) from kidney tissue from mice induced by IRI and control mice. In the HE staining image in c, TLT formation is observed at the arrow. [d] Shows magnified HE staining images (left), GSSG MSI images (middle), and GSH MSI images (right) of the region where TLTp forms in the kidney tissue of mice induced by IRI. In d, the image obtained by further magnifying the area circled by the dashed circle in the image shown in the upper paragraph (low) is the image shown in the lower paragraph (high). e shows the results of measuring the relative amounts of GSH, GSSG, cystine, cysteine, dipeptide Cys-Gly, and γ-glutamylcysteine ​​extracted from kidney tissue of mice with induced IRI and control mice. In e, This indicates that p < 0.05.

[0020] [ Figure 3 The results show the relative values ​​of reduced glutathione concentration in the urine of mice induced with IRI and control mice (relative values ​​with creatinine concentration in urine set to 1). Figure 3 middle, p < 0.05, ns indicates no significant difference.

[0021] [ Figure 4 The results show the results obtained by dividing IgA nephropathy patients into a TLT group (TLT+) and a non-TLT group (TLT-) and calculating the relative values ​​of urinary reduced glutathione concentration (relative values ​​with urinary creatinine concentration set to 1).

[0022] [ Figure 5 To illustrate the results of logistic regression and ROC analysis performed on IgA nephropathy patients, with patients in the TLT group set as positive (1) and patients in the non-TLT group set as negative (0), using the relative value of urinary reduced glutathione concentration (the relative value of urinary creatinine concentration set as 1) as the explanatory variable. Detailed Implementation

[0023] 1. Terminology Unless otherwise specified, the terms used in this specification have the meanings commonly understood by those skilled in the art in the fields of medicine, pharmacy, molecular biology, microbiology, and organic chemistry. Where the meaning of a term as defined in this specification differs from its commonly understood meaning, the meaning as described in this specification shall prevail.

[0024] In this disclosure, "tertiary lymphoid tissue (TLT)" refers to tissue similar to lymph nodes, specifically induced ectopic lymphoid tissue composed of aggregates of lymphocytes and formed in non-lymphoid tissue. In this disclosure, "maturity of tertiary lymphoid tissue (TLT)" refers to the degree of structural maturity of the TLT. In this disclosure, the maturity of tertiary lymphoid tissue (TLT) is based on the classification described in Non-Patent Document 5, and is classified into stages I to III as follows. Furthermore, Figure 1 The diagram shows structural illustrations of different maturity stages of TLTs formed in the kidney for reference. Figure 1 In phase 0, the lymphocytes are unorganized and are precursors to TLT, which are not TLTs as described in this disclosure.

[0025] Phase I: TLTs excluding follicular dendritic cells (FDCs) and germinal centers. Phase II: TLT includes FDC but not the hair regrowth center Phase III: TLT including FDC and hair regrowth center In this publication, the term "glutathione" encompasses both reduced glutathione and oxidized glutathione. Reduced glutathione is a tripeptide formed by the sequential linkage of glutamic acid, cysteine, and glycine via peptide bonds. In reduced glutathione, glutamic acid and cysteine ​​form an amide bond with the amino group of cysteine ​​via the γ-carboxyl group of the glutamic acid side chain. Oxidized glutathione is a molecule formed by two molecules of reduced glutathione linked by disulfide bonds.

[0026] 2. TLT Inspection Method This disclosure describes a method for examining the presence or absence of TLT in a test subject, and includes a step of measuring the glutathione concentration in a bodily fluid sample collected from the test subject. The examination method described in this disclosure is described in detail below.

[0027] [Subject] In the examination method disclosed herein, the term "subject" refers to a human or non-human animal that is the object of examination for the presence or absence of TLT formation in a living organism. Examples of non-human animals include primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cattle, horses, pigs, goats, dogs, and cats. Humans are a preferred example of a subject.

[0028] The examination method disclosed herein only requires the subject to be examined for the presence or absence of TLT formation in the organism. Preferred examples include subjects suffering from diseases where TLT formation occurs in the lesion, subjects requiring examination for the presence of such diseases, and subjects who have received organ transplants for the treatment of such diseases. Examples of diseases where TLT formation occurs in the lesion include, for example, diseases accompanied by chronic inflammation, chronic kidney disease, obesity, asthma, allergic lung disease, arteriosclerosis, autoimmune diseases, infectious diseases, and cancer.

[0029] As specific examples of test subjects in the examination method of this disclosure, patients with kidney disease or subjects who need to be examined for kidney disease can be cited. There are no particular limitations on the types of kidney disease; for example, IgA nephropathy, lupus nephritis, ANCA-associated vasculitis, diabetic nephropathy, nephrosclerosis, pyelonephritis, and chronic renal failure can be cited. Among these kidney diseases, IgA nephropathy is preferred. In kidney diseases, as kidney function progresses, there is a tendency for trace amounts of kidney tissue (TLTs) to form in the lesion site, and their maturity increases. Therefore, in the examination method of this disclosure, when subjects with kidney disease or those who need to be examined for kidney disease are used as test subjects, in addition to the presence or absence of TLTs, it is possible to infer whether or not kidney disease is present.

[0030] Furthermore, as another specific example of a subject in the examination method of this disclosure, subjects for whom future renal function needs to be predicted can be cited. Examples of subjects for whom future renal function needs to be predicted include patients with kidney disease and those who have received a kidney transplant. For example, when a kidney transplant recipient is used as a subject, the prognosis of transplanted kidney function can be predicted based on the presence or absence of TLT (transplant thrombosis).

[0031] Furthermore, as other specific examples of test subjects in the examination method of this disclosure, patients with hepatitis C or subjects who need to be examined for hepatitis C can be cited. Since TLTs tend to form in the liver and their maturity increases as hepatitis C symptoms progress, the examination method of this disclosure, when using subjects with hepatitis C or subjects who need to be examined for hepatitis C as test subjects, can, in addition to the presence or absence of TLTs, also infer the presence or absence of hepatitis C and the degree of hepatitis C symptoms.

[0032] Furthermore, cancer patients can be cited as other specific examples of test subjects in the examination method of this disclosure. There are no particular limitations on the type of cancer; for example, solid cancers such as kidney cancer, stomach cancer, lung cancer, breast cancer, liver cancer, tongue cancer, thyroid cancer, uterine cancer, ovarian cancer, and prostate cancer can be cited. Since the formation and increased maturity of TLTs in cancer tissues lead to an enhanced cancer immune response, an observed trend towards improved treatment efficacy and a better prognosis, the examination method of this disclosure, when using cancer patients, especially those treated with immune checkpoint inhibitors, as test subjects, can predict the treatment efficacy and prognosis of cancer.

[0033] [Body fluid sample] The term "body fluid sample" refers to bodily fluids such as urine, saliva, blood, sputum, and sweat collected from the aforementioned test subject, or samples prepared from bodily fluids. Among these bodily fluid samples, urine is preferred because it allows for high-precision detection of the presence or absence of TLTs and can be collected non-invasively. Furthermore, bodily fluid samples may be concentrated or filtered as needed.

[0034] [Determination of glutathione concentration] In the examination method disclosed herein, the concentration of glutathione in a body fluid sample is measured as a biomarker for TLT.

[0035] The glutathione that is to be measured can be any glutathione from reduced glutathione, oxidized glutathione, or total glutathione (both reduced glutathione and oxidized glutathione).

[0036] The determination of glutathione concentration in body fluid samples can be performed using well-known methods. For example, commercially available kits capable of quantifying glutathione concentration are available, and therefore, these kits can be used to determine the glutathione concentration in body fluid samples. Alternatively, the determination of glutathione concentration in body fluid samples can also be performed using ion chromatography-Fourier transform mass spectrometry (IC-FTMS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc.

[0037] Furthermore, when using urine samples as body fluid samples, the concentration of glutathione in urine may vary depending on the urine volume. Therefore, it is desirable to correct the concentration of glutathione in urine beforehand using the concentration of creatinine in urine, etc. For example, in one embodiment of the examination method of this disclosure, when using urine samples as body fluid samples, the value obtained by dividing the concentration of glutathione in urine by the concentration of creatinine in urine (creatinine correction value) is used as an indicator of the concentration of glutathione in urine.

[0038] [Determination of the presence or absence of TLT] The examination method disclosed herein can be applied to examine the presence or absence of TLTs in any lesion site. As a preferred example, it can be applied to examine the presence or absence of TLTs in the kidneys.

[0039] The presence or absence of TLT is correlated with the glutathione concentration in body fluid samples. When TLT is not formed in the body, the glutathione concentration in the body fluid sample is low; when TLT is formed in the body, the glutathione concentration in the body fluid sample is high. Therefore, in the examination method of this disclosure, the lower the glutathione concentration in the body fluid sample, the higher the probability that TLT has not formed in the body; the higher the glutathione concentration in the body fluid sample, the higher the probability that TLT has formed in the body.

[0040] Regarding the examination method of this disclosure, and the determination of the presence or absence of TLT based on the glutathione concentration in a body fluid sample, the determination can be made by comparing it with a reference value obtained from an object whose presence or absence of TLT is known in advance. Here, the "reference value" is a value that serves as a benchmark for determining the presence or absence of TLT; specifically, it includes the average glutathione concentration in a body fluid sample obtained from an object whose presence or absence of TLT is known in advance, and a critical value obtained from the glutathione concentration in that body fluid sample, etc.

[0041] For example, the average glutathione concentration in bodily fluid samples from subjects who have not formed TLTs in vivo can be pre-calculated and used as a reference value. If the glutathione concentration in the test subject's bodily fluid sample is below the same level as this reference value, it can be determined that the test subject is likely not to have formed TLTs in vivo. Conversely, for example, the average glutathione concentration in bodily fluid samples from subjects who have formed TLTs in vivo can be pre-calculated and used as a reference value. If the glutathione concentration in the test subject's bodily fluid sample is above the same level as this reference value, it can be determined that the test subject is likely to have formed TLTs in vivo. In addition, for example, the average glutathione concentration in body fluid samples from subjects who have not formed TLTs in the body and the average glutathione concentration in body fluid samples from subjects who have formed TLTs in the body can be calculated in advance. Based on these glutathione concentrations, a critical value for the presence or absence of TLTs can be defined, and this critical value can be used as a reference value. When the glutathione concentration in the body fluid sample of the test subject is above the critical value, it can be determined that the test subject has the potential to form TLTs in the body.

[0042] The aim is to determine the aforementioned reference values ​​for each disease being examined. For example, if the subject is a patient with kidney disease or needs to be examined for kidney disease, the average glutathione concentration in body fluid samples from patients with kidney disease who are known not to have formed TLTs in their kidneys, the average glutathione concentration in blood body fluid samples from patients with kidney disease who are known to have formed TLTs in their kidneys, and / or a cutoff value determined based on these glutathione concentrations can be used as reference values. Similarly, if the subject is a patient with hepatitis C or needs to be examined for hepatitis C, the average glutathione concentration in body fluid samples from patients with hepatitis C who are known not to have formed TLTs in their livers, the average glutathione concentration in body fluid samples from patients with hepatitis C who are known to have formed TLTs in their livers, and / or a cutoff value determined based on these glutathione concentrations can be used as reference values.

[0043] Furthermore, as shown in the Example section, 47 patients with IgA nephropathy were grouped and analyzed based on the presence or absence of TLT formation in the kidneys. The results confirmed that the average relative value of urinary glutathione concentration (the relative value of urinary glutathione concentration with urinary creatinine concentration set to 1) in the group with TLT formation in the kidneys was 0.000740; the average relative value of urinary glutathione concentration in the group without TLT formation in the kidneys was 0.000169; and the critical value of the relative value of urinary glutathione concentration that could distinguish these two groups with a sensitivity of 0.7143 and a specificity of 0.00556 was 0.0001647. Therefore, in the examination method of this disclosure, when using urine samples as body fluid samples to examine the presence or absence of TLTs in the kidneys, these average or critical values ​​of urinary glutathione concentration can also be used as reference values.

[0044] Based on the presence or absence of TLTs in an organism predicted using the examination method described in this disclosure, it is possible to infer whether the organism suffers from a disease in which TLTs are formed in the lesion, or the degree of symptoms of that disease.

[0045] For example, in the examination method of this disclosure, if the subject is a person suffering from a disease other than cancer with TLT formation and is predicted to have a possibility of forming TLT in the body, it can be inferred that the disease is progressing. Specifically, in the examination method of this disclosure, if the subject is a patient with kidney disease and is predicted to have a possibility of forming TLT in the body, it can be inferred that the kidney disease is progressing.

[0046] Furthermore, for diseases associated with TLT formation (other than cancer and infectious diseases), TLT-targeting therapies may be effective, and the development of TLT-targeting therapies is anticipated in the future. Therefore, the examination method described in this disclosure can also be used to determine whether a future TLT-targeting therapy can be administered. Specifically, in the examination method of this disclosure, if the subject is a patient suffering from a disease associated with TLT formation other than cancer and infectious diseases, and is predicted to have a high probability of TLT formation in the body, then the patient can be deemed suitable for administration of a TLT-targeting therapy. It should be noted that in this disclosure, "TLT-targeting therapies" refers to agents that stop or inhibit the progression of TLT, or cause the elimination or reduction of TLT.

[0047] Furthermore, in the examination method of this disclosure, if the subject is someone whose future kidney function needs to be predicted, and although their kidney function is normal at the time of examination, they are predicted to have a tendency to develop total kidney thrombosis (TLT), it can be inferred that there is a possibility of future kidney function decline. For example, if the subject is someone who has received a kidney transplant, and although the transplanted kidney function is normal at the time of examination, they are predicted to have a tendency to develop TLT, it can be inferred that there is a possibility of a deterioration in the prognosis of the transplanted kidney function.

[0048] In addition, in the examination method of this disclosure, when the subject is a cancer patient and is predicted to have the potential to form TLT in the body, it can be inferred that the cancer immune response is enhanced.

[0049] 3. TLT test kit Another embodiment of this disclosure provides a test kit for detecting the presence or absence of TLT in a living organism, the test kit comprising reagents for determining glutathione concentration. The test kit of this disclosure is a test kit used to perform the aforementioned test method, and the content described in the aforementioned "2. Test Method for TLT" section is also incorporated herein by reference. The test kit of this disclosure can also be provided as an in vitro diagnostic drug for detecting the presence or absence of TLT in a living organism.

[0050] The test kit disclosed herein may contain reagents for determining glutathione concentration, such as enzymes, coenzymes, buffers, substrates, and internal standards. As an example of reagents for determining glutathione concentration, a combination of glutathione reductase, NADPH, and DTNB (5-5'-dithiobis[2-nitrobenzoic acid]) may be given. Another example of reagents for determining glutathione concentration may be a combination of glutathione S-transferase, luciferin-NT (Promega), and luciferase.

[0051] When using the test kit of this disclosure to determine the concentration of glutathione in a urine sample, the test kit of this disclosure may also include a reagent for determining the concentration of creatinine in order to correct for the concentration of glutathione in the urine sample. As a reagent for determining the concentration of creatinine, for example, it may include an enzyme for detecting creatinine by an enzymatic assay, specifically including creatinine enzyme, creatine oxidase, sarcosine oxidase, and a reactive oxygen species detection agent (e.g., peroxidase, 4-aminoantipyrine, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine).

[0052] Example This disclosure is not limited in any way to the description of the foregoing embodiments of the invention and the examples shown below. Various modifications that can be readily conceived by those skilled in the art without departing from the claims are also included in this invention. The entire contents of the documents, etc., shown in this specification are incorporated herein by reference.

[0053] 1. Analysis using a kidney TLT model 1-1. Experimental Materials and Methods (1) Mice Twelve-month-old C57BL6J mice were purchased from Japan SLC, Inc. All mice were housed in an SPF (specific pathogen-free) environment at the Kyoto University Animal Laboratory. All animal experiments were conducted in accordance with the guidelines for the husbandry, management, and use of laboratory animals issued by the National Institutes of Health (NIH) after obtaining approval from the Kyoto University Animal Experimentation Committee.

[0054] (2) Kidney TLT model Mice were anesthetized with 2% isoflurane, and the skin was incised to expose the left kidney to the body surface. Vascular clamps were used to completely occlude both renal arteries for 45 minutes, followed by closure of the incision, thereby inducing ischemia-reperfusion injury (IRI). Urine was collected weekly starting from the day of IRI induction. Mice were sacrificed 45 days after IRI induction, and kidney tissue was collected (n=5). Additionally, urine and kidney tissue were collected under the same conditions as described above, except without IRI induction (control) (n=5).

[0055] (3) Kidney tissue staining (HE staining and immunostaining) Hematoxylin-eosin (HE) staining of kidney tissue was performed in the following order: Immediately after euthanasia, the kidney tissue was flash-frozen with dry ice and thinly sliced ​​into 8 μm pieces using a cryostat (CM3050, Leica) in an unembedded state. The sliced ​​tissue was fixed onto a glass slide (Matsunami Glass Ind.) and immersed in hematoxylin staining solution for 4 minutes. It was then washed with running water for 15 minutes, immersed in eosin staining solution for 2 minutes, and then subjected to HE staining.

[0056] Immunostaining of kidney tissue was performed in the following order. First, kidney tissue sections were blocked in PBS (phosphate-buffered saline) containing 5% normal goat serum at room temperature for 1 hour. Next, anti-B220 antibody (557390, BD Bioscience) and anti-CD3e antibody (55027, BD Bioscience) were reacted overnight at 4°C as primary antibodies. Then, suitable secondary antibodies were reacted at room temperature for 1 hour, followed by contrast staining with DAPI (4',6-diamidindole-2-phenylindole). Observation was performed using a confocal microscope (FV1000D; Olympus Corporation).

[0057] (4) Renal tissue mass spectrometry imaging Immediately after sacrifice, kidney tissue was flash-frozen with dry ice and thinly sliced ​​into 8 μm sections using a cryostat (CM3050, Leica) in an unembedded state. The sliced ​​tissue was fixed onto indium tin oxide (ITO) coated slides (Brucker Daltonics). Matrix-assisted laser desorption / ionization imaging (MALDI-IMS) was performed using 9-aminoacridine (10 mg / mL, dissolved in 80% ethanol) as the matrix. MALDI imaging was performed using a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometer (UltraFlextreme, Brucker Daltonics) equipped with an Nd:YAG laser. Grating scans were performed at 50 mm intervals, and data were collected based on negative reflection mode. The image was reconstructed using FlexImaging 4.1 software (Brucker Daltonics).

[0058] (5) Metabolomics analysis of kidney tissue Tissue sections embedded in SCEM (Super Cryoembedding Medium) were dissolved in 500 μL of methanol using a homogenizer (Finger Masher, AM79330, Sarstedt, Tokyo, Japan). Then, an equal volume of chloroform and 0.4 times the volume of ultrapure water were added and mixed. The mixture was centrifuged at 15,000 G at 4 °C for 15 min. The resulting supernatant was filtered using an ultrafiltration tube (Ultrafree-MC, UFC3 LCC NB; Human Metabolome Technologies, Tsuruoka, Japan). The filtrate was concentrated using a centrifugal concentrator (SpeedVac; Thermo, Yokohama, Japan). The concentrated filtrate was dissolved in 50 μL of ultrapure water for metabolomics analysis using mass spectrometry. Metabolomics analysis was performed using both anionic and cationic metabolites, as described below. It should be noted that the data analysis utilized the results from cationic metabolites, which yielded more precise values.

[0059] Metabolomics analysis of anionic metabolites was performed using ion chromatography (IC)-Fourier transform mass spectrometry (FTMS). Specifically, anionic metabolites were determined using a Q-Exactive focus mass spectrometer (Thermo Fisher Scientific) connected to a high-performance IC system (ICS-5000+). Metabolite quantification was achieved with high selectivity and sensitivity through IC separation and Fourier transform mass spectrometry. In ion chromatography, an anion electrolysis suppressor (Thermo Scientific Dionex AERS 500) was loaded, and the potassium hydroxide gradient was replaced with pure water before entering the mass spectrometer. Separation was performed using a Dionex... TM IonPac TMAS11-HC-4μm IC column (ThermoScientific) was used. The IC flow rate was 0.25 mL / min, with methanol replenishment solution added at a rate of 0.18 mL / min post-column. The concentration gradient conditions for potassium hydroxide used in IC separation were as follows: 1 mM to 100 mM (0–40 min), 100 mM (40–50 min), 1 mM (50.1–60 min), with the column temperature set at 30 °C. The orbital trap mass spectrometer was operated in ESI negative ion mode for all detections. A full mass scan (m / z 70–900) was performed at a resolution of 70,000. The automatic gain control (AGC) target was set to 3 × 10⁶ ions, and the maximum ion implantation time was set to 100 ms. Ionization parameters for the ion source were: spray voltage 3 kV, transfer temperature 320 °C, S-Lens level 50, heater temperature 300 °C, sheath gas 36, and Aux gas 10.

[0060] Metabolomics analysis of cationic metabolites was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Specifically, a triple quadrupole mass spectrometer (LCMS-8060, SHIMADZU) equipped with an orthogonal electrospray ionization (ESI) source was used in positive and negative ESI modes and multiple reaction monitoring (MRM) mode. Samples were separated using a Discovery HS F5-3 column (2.1 mm ID × 150 mm L, 3-μm particles, Sigma-Aldrich) with a stepwise gradient using mobile phase A (0.1% formic acid) and mobile phase B (0.1% acetonitrile). A step gradient was implemented with mobile phase A: mobile phase B ratios of 100:0 (0-5 min), 75:25 (5-11 min), 65:35 (11-15 min), 5:95 (15-20 min), and 100:0 (20-25 min), a flow rate of 0.25 mL / min, and a column temperature of 40 °C.

[0061] (6) Urine metabolomics analysis 50 μL of frozen urine was mixed with 500 μL of methanol and a standard (2-morpholinoethanesulfonic acid), followed by the addition of an equal volume of chloroform and 0.4 volumetric volume of ultrapure water (LC / MS grade, FUJIFILM Wako). The resulting suspension was centrifuged at 2,800 g at 4 °C for 15 min. After centrifugation, the aqueous phase was ultrafiltered using an ultrafiltration tube (Ultrafree MC-PLHCC; HumanMetabolome Technologies). The filtrate was concentrated using a vacuum concentrator (SpeedVac, Thermo Fisher Scientific). The concentrated filtrate was dissolved in 50 μL of ultrapure water for analysis based on IC-FTMS and LC-MS / MS. To correct for the loss of endogenous metabolites during sample preparation, the urinary metabolite concentrations were calculated as relative values ​​corrected for urinary creatinine concentration (with creatinine concentration set to 1). The IC-FTMS and LC-MS / MS analyses were performed using the same methods as for renal tissue metabolomics analysis.

[0062] 1-2. Results HE staining of kidney tissue from mice with induced ischemia-reperfusion injury revealed tissue presumed to be TLT in the kidney tissue. Figure 2 (b). Additionally, in tissues presumed to be TLT by HE staining, CD3e (a T cell marker) and B220 (a B cell marker) were locally present. Figure 2 (b) It was confirmed that TLT was formed by aggregates of T cells and B cells.

[0063] Furthermore, based on mass spectrometry imaging of kidney tissue in mice induced with ischemia-reperfusion injury, glutathione (oxidized and reduced forms) was confirmed to accumulate in TLT (…). Figure 2 (c, d). Furthermore, in mice induced with ischemia-reperfusion injury, the amounts of glutathione (oxidized and reduced forms) and its breakdown products (dipeptides composed of Cys-Gly) extracted from the kidneys were significantly increased compared to control mice. Figure 2 (e).

[0064] Furthermore, in mice induced with ischemia-reperfusion injury, the concentration of reduced glutathione in urine was significantly increased compared to control mice. Figure 3 ).

[0065] Based on the above results, it is clear that glutathione accumulates highly in TLTs formed in the kidneys, and an increase in urinary glutathione concentration is observed when TLTs are formed.

[0066] 2. Analysis of urine samples from patients with IgA nephropathy 2-1. Experimental Materials and Methods (1) Patient A retrospective study was conducted on 46 patients diagnosed with IgA nephropathy via renal biopsy at Kyoto University Hospital between 2014 and 2018. Renal biopsy tissue and urine samples (frozen urine) from these 46 patients were available. The 46 patients who consented to participate in this study were included in the analysis. It should be noted that patients who received immunosuppressive therapy within one year prior to their renal biopsy, patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis, patients with Henoch-Schönlein purpura nephritis, and patients with cirrhosis were excluded from the analysis.

[0067] (2) Identification of TLT In this experiment, similar to the criteria shown in Non-Patent Literature 4, aggregates of tissueed lymphocytes exhibiting signs of proliferation were defined as TLTs. TLTs vary in size; therefore, in this experiment, aggregates of 61 or more lymphocytes (T cells and B cells) containing at least one Ki67-positive cell were defined as TLTs. The quantification of the number of TLTs and the determination of their stage were performed in a blinded experiment based on the method described in Non-Patent Literature 5. In brief, firstly, mononuclear cell infiltration in the renal interstitium was confirmed based on PAS-stained sections. Then, immunofluorescence staining of 1) CD3ε and CD20, and 2) Ki67 and CD21 was performed on two consecutive sections to evaluate mononuclear cell infiltration and diagnose TLTs. Following diagnosis of TLT, TLT stages are classified based on the following criteria: i) TLTs containing neither follicular dendritic cells (FDCs) nor germinal centers are defined as stage I; ii) TLTs containing FDCs but lacking germinal centers are defined as stage II; iii) TLTs containing both FDCs and germinal centers are defined as stage III. FDCs are defined as cells strongly expressing CD21 within the TLT. Germinal centers are defined as small structures within a B-cell region containing clusters of 16 or more Ki67-positive cells.

[0068] Of the 46 patients with IgA nephropathy analyzed, transient time-to-thickness (TLT) was observed in 18 cases and not in 28 cases. For the 18 cases where TLT was observed, the TLT stage was determined: stage I in 14 cases, stage II in 4 cases, and stage III in 0 cases.

[0069] (3) Urine metabolomics analysis The concentration of reduced glutathione in urine was determined using the same method described in “1. Analysis using a renal TLT model” above.

[0070] (4) Logistic Regression Analysis and ROC Analysis Logistic regression and ROC analysis were performed using JMP Pro software (version 16). Oxidized glutathione was used as an explanatory variable in logistic regression to predict the presence or absence of tert-lactamase (TLT). ROC curves and the Youden index were used to determine the cutoff value of urinary oxidized glutathione concentration for predicting the presence of TLT; values ​​above the cutoff were considered TLT-positive.

[0071] 2-2. Results Patients with IgA nephropathy were divided into a tert-lactamase (TLT) group and a non-TLT group. The concentration of reduced glutathione in urine was measured. The results showed that the relative mean concentration of oxidized glutathione in urine was 0.000740 in the TLT group and 0.000169 in the non-TLT group. The concentration of reduced glutathione in urine was significantly increased in the TLT group compared to the non-TLT group. Figure 4 ).

[0072] In addition, patients with TLT were designated as positive (1), and patients without TLT were designated as negative (0). The relative concentration of reduced glutathione in urine was used as an explanatory variable, and a univariate analysis based on logistic regression was performed. The results showed that the AUC (Area Under Curve) was 0.893, and the relative concentration of reduced glutathione in urine with the highest sensitivity (1-specificity) was 0.0001647 (…). Figure 5 This relative value is set as the critical value, with a sensitivity of 0.7143 and a 1-specificity of 0.0556. Figure 5 ).

[0073] Based on the above results, it is clear that the concentration of glutathione in urine is significantly increased in IgA nephropathy patients with TLT. By using the concentration of glutathione in urine as an indicator, the presence or absence of TLT can be predicted.

Claims

1. A method for examining the presence or absence of tertiary lymphoid tissue in a test subject, the method comprising the step of measuring the glutathione concentration in a body fluid sample collected from the test subject.

2. The inspection method as described in claim 1, wherein, The body fluid sample is a urine sample.

3. The inspection method as described in claim 1 or 2, wherein, The glutathione concentration is the same as the reduced glutathione concentration.

4. The examination method as described in claim 2, further comprising the step of measuring the creatinine concentration in the urine sample.

5. The examination method as described in claim 1 or 2, wherein the examination method is performed to examine the presence or absence of tertiary lymphoid tissue in the kidney, wherein, The subjects were patients with kidney disease, individuals who needed to be checked for kidney disease, or individuals who had received a kidney transplant.

6. A test kit for examining the presence or absence of tertiary lymphoid tissue in an organism, said test kit containing reagents for determining glutathione concentration.

7. The reagents used to determine glutathione concentration are intended for use in the manufacture of a diagnostic kit for examining the presence or absence of tertiary lymphoid tissue in the body.

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