Renal tissue analysis using fluorescent imaging

Abstract

To provide a renal biopsy method having little deviation in lesion degrees of an easily-implemented and clinically-obtained test value and an actual sample tissue, without depending on a serial section method.SOLUTION: A method for analyzing a renal tissue by fluorescence imaging for detecting disorder in glomeruli, the method includes the steps of: preparing a renal tissue embedded and fixed with paraffin; removing the paraffin embedding the renal tissue by using a deparaffinizing agent; making the renal tissue from which the paraffin is removed, transparent by using a transparentization agent; fluorescently staining the glomeruli present in the transparent renal tissue; acquiring at least a part of the renal tissue containing the glomeruli as a three-dimensional image; and analyzing the glomeruli contained in the three-dimensionally-imaged renal tissue by using a cross section traversing the glomeruli and checking the presence / absence of disorder in the glomeruli.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an analytical method using fluorescent imaging of renal tissue. [Background technology]

[0002] The kidney is an organ that filters blood and discards waste products produced in the body into urine. The kidney also regulates water balance. Blood filtration in the kidney is performed by the glomeruli located in the cortex of the surface of the kidney. When the glomeruli are damaged in some way, urinary protein appears, and if the damage progresses further, the kidney's function will eventually be lost. Therefore, in order to diagnose and determine a treatment plan for patients with reduced renal function and urinary protein appearance, it is necessary to perform a renal biopsy test to analyze the condition of the glomeruli in detail. The tissue obtained by renal biopsy is sliced ​​and various stains are applied to observe under a microscope whether there are any lesions in the tissue, and a treatment plan is determined depending on the number and condition of the lesions.

[0003] Usually, renal biopsy tests are performed using thin slices and two-dimensional observations, so it is common in clinical settings for lesions to not be detected depending on the slice, especially in patients with early-stage kidney disease. In this case, there is a discrepancy between the test values ​​obtained in clinical practice and the degree of lesions in the actual specimen tissue, making it difficult to formulate an appropriate treatment plan.

[0004] As a method for overcoming the limitations of two-dimensional observation using thin sections, serial sectioning for three-dimensional tissue observation is known (Non-Patent Document 1). Serial sectioning is a technique for constructing an image of three-dimensional tissue by stacking thin slices of tissue, and is widely used around the world. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Japanese Journal of Nephrology 2005;47(7):775-782 Summary of the Invention [Problem to be solved by the invention]

[0006] However, 3D observation using serial sectioning requires time and effort to slice the entire tissue, and if a specific section is lost during the slice process, 3D reconstruction becomes difficult. For this reason, this method has not yet been applied to human pathological examinations. Therefore, the problem to be solved by the present invention is to provide a renal biopsy method that does not rely on the serial section method, can be easily performed, and has little discrepancy between the test values ​​obtained in clinical practice and the level of lesions in actual specimen tissue. [Means for solving the problem]

[0007] As a result of intensive research into solving the above-mentioned problems, the inventors discovered that the problem could be solved by making renal tissue transparent, constructing a three-dimensional image of the renal tissue, and analyzing cross-sections of the glomeruli present therein, and thus completed the present invention.

[0008] That is, the present invention includes the following inventions. [1] 1. A method for analyzing kidney tissue by fluorescence imaging to detect damage in a glomerulus, comprising: a. providing a paraffin-embedded fixed kidney tissue; b. Removing the paraffin embedding the renal tissue using a deparaffinizing agent; c. clearing the paraffin-removed kidney tissue using a clearing reagent; d. fluorescently staining glomeruli present in the cleared kidney tissue; e. obtaining a three-dimensional image of at least a portion of the renal tissue including the glomerulus; and f. A method comprising a step of analyzing a cross-section of a glomerulus contained in the 3D image of kidney tissue to confirm the presence or absence of damage in the glomerulus. [2] The method according to [1], wherein the paraffin-embedded and fixed renal tissue is a renal biopsy sample in the form of an approximately columnar or rod. [3] The method according to [1] or [2], wherein in step f, the cross sections are at least two cross sections that are substantially perpendicular to each other. [4] The method according to any of [1] to [3], wherein step d is a step of fluorescently staining a glomerulus by combining at least two antibodies, one of which is an antibody used for staining the entire interior of the glomerulus, and the other is an antibody used for staining the contour of the glomerulus. [5] The method according to [4], wherein the two antibodies are a combination of an antibody selected from the group consisting of an anti-nephrin antibody and an anti-podocin antibody, and an antibody selected from the group consisting of an anti-claudin antibody and an anti-collagen IV antibody. [6] The method according to [4], wherein the two antibodies are an anti-nephrin antibody and an anti-claudin antibody. [7] The method according to any one of [1] to [6], wherein the clarifying reagent is a clarifying reagent containing 1,3-bis(aminomethyl)cyclohexane and sodium dodecylbenzenesulfonate, and / or a clarifying reagent containing antipyrine and nicotinamide. [8] The method according to any one of [1] to [7], further comprising, prior to step c, treating the mixture with at least three types of ethanol solutions having gradually decreasing ethanol concentrations. [9] The method according to any of [1] to [8], wherein the glomerular damage is at least one selected from the group consisting of crescent formation, glomerular sclerosis, and podocyte damage.

[10] The method according to any of [1] to [8], wherein the glomerulus damage is crescent formation.

[11] A method for confirming the degree of damage to renal tissue in a subject by comparing the damage in the glomeruli detected by any of the analytical methods described in [1] to

[10] with the results of biochemical tests of the subject from which the renal tissue is derived.

[12] A method for diagnosing kidney disease, comprising: determining the degree of damage to renal tissue in a subject by comparing damage to the glomerulus detected by any of the analytical methods according to [1] to

[10] with the results of a biochemical test of the subject from which the renal tissue is derived. Effect of the Invention

[0009] According to the present invention, it is possible to provide a renal biopsy method that does not rely on the serial section method, can be easily performed, and has little discrepancy between test values ​​obtained in clinical practice and the degree of lesions in actual specimen tissue. [Brief description of the drawings]

[0010] [Figure 1] The results of tissue clearing of paraffin-embedded kidney tissues are shown in Figure 1A for mouse kidney samples, in Figure 1B for rat kidney samples, and in Figure 1C for human kidney samples. [Diagram 2] A comparison of 3D and 2D analysis protocols using rats with anti-glomerular basement membrane (GBM) nephritis is shown. [Diagram 3] The results of fluorescent immunostaining in the vehicle group and anti-GBM nephritis group are shown (Figure 3A). The left image in Figure 3A shows the results of staining with anti-nephrin antibody, the center image shows the results of staining with anti-claudin-1 antibody, and the right image shows a superimposition of the two. In addition, low-magnification images (the areas corresponding to the high-magnification images are surrounded by dotted lines in the figures) and high-magnification images in each group are shown (Figure 3B). [Figure 4] The results of the analysis by the method of the present invention using a rat sample are shown in Figures 1 to 3 on the right, which show cross-sectional images of the respective cross sections (Slices 1 to 3) in the left figures. [Diagram 5]The results of fluorescent staining targeting nephrin and claudin-1 in Slice 1 in Figure 4 are shown. The lower row shows the results of staining with DAPI (Figure 5B), anti-nephrin antibody (Figure 5C), and anti-claudin-1 antibody (Figure 5D), and the upper row shows the overlapping images (Figure 5A). [Figure 6] The results of quantifying crescent-forming glomeruli detected using the conventional detection method (two-dimensional analysis: 2D) and the detection method of the present invention (three-dimensional analysis: 3D) shown in FIG. 2 are shown. [Figure 7] The results of a conventional detection method (two-dimensional analysis) using human samples are shown. [Figure 8] 8 shows the results of analysis by the method of the present invention using human samples. The left figure (FIG. 8A) shows the detection results by a conventional method, and the right figure (FIG. 8B) shows the detection results by the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In this specification, the term "X to Y" is used to mean including X and Y as the end points. The present invention provides a method for analyzing kidney tissue by fluorescence imaging to detect damage in the glomerulus, which comprises at least the following steps a to f. a. providing a paraffin-embedded fixed kidney tissue; b. Removing the paraffin embedding the renal tissue using a deparaffinizing agent; c. clearing the paraffin-removed kidney tissue using a clearing reagent; d. fluorescently staining glomeruli present in the cleared kidney tissue; e. obtaining a three-dimensional image of at least a portion of the renal tissue including the glomerulus; and f. A process of analyzing a cross section of a glomerulus contained in the 3D image of kidney tissue to confirm the presence or absence of damage in the glomerulus. The renal tissue analysis method of the present invention makes the renal tissue transparent and constructs a three-dimensional image of the renal tissue, preferably by fluorescent staining, and then analyzes cross-sections of the glomeruli present therein, making it possible to easily perform pathological examination of the renal tissue without relying on serial sectioning methods.

[0012] In the present invention, methods known as methods for processing renal biopsy samples, as described in various guidelines and publicly known literature, can be appropriately used in each step.

[0013] (Kidney tissue preparation process) In step a, paraffin-embedded and fixed kidney tissue is prepared. The method for embedding and fixing the renal tissue in paraffin is not particularly limited, but may be any conventionally known method, and renal tissue that has been embedded and fixed in paraffin, known as a paraffin block, may be used. The renal tissue embedded and fixed in paraffin is preferably a roughly columnar or rod-shaped renal biopsy sample, and specifically, when the longitudinal direction is the length and the direction perpendicular to the longitudinal direction is the thickness or diameter, the thickness or diameter is preferably 0.3 to 1.0 mm, more preferably 0.5 to 1.0 mm, while the length is preferably 5 to 10 mm. Samples of such sizes are suitable for good three-dimensional imaging of renal tissue and cross-sectional analysis of glomeruli.

[0014] Paraffin-embedded, fixed kidney tissue is prepared and used to detect damage in the glomeruli. The kidney tissue is not particularly limited, but is preferably human kidney tissue, and may be kidney tissue derived from a human who requires a pathological examination of the kidney. Pathological examinations of the kidneys are performed on patients who are suffering from or suspected of having some kind of kidney disease, such as proteinuria, hematuria, or decreased renal function, in order to make a definitive diagnosis and determine the optimal treatment. Pathological examination of the kidney may be performed by kidney biopsy, which involves obtaining a piece of tissue from the kidney. Renal biopsy is generally performed by, but not limited to, ultrasound-guided needle renal biopsy or open renal biopsy. Renal tissue obtained by renal biopsy includes glomeruli, tubules, interstitium, and blood vessels. Renal biopsy samples may be prepared based on the Renal Biopsy Guidebook prepared by the Japanese Society of Nephrology. The patient from whom renal tissue is obtained is not particularly limited in terms of age, sex, presence or absence of other diseases, etc.

[0015] The method of the present invention contributes to confirming the presence of kidney disease by performing an analysis of kidney tissue, optionally in combination with other findings of kidney damage. The kidney disease may be a disease diagnosed according to any of the clinical guidelines prepared by the Japanese Society of Nephrology. The disease to be analyzed in the present invention may be a kidney disease, particularly a patient suffering from glomerulonephritis or glomerular damage. The glomerular damage may be at least one selected from the group consisting of crescent formation, glomerular sclerosis, and podocyte damage. The kidney tissue may be prepared for analyzing the presence or absence of kidney disease, inter alia glomerulonephritis, crescent formation, glomerulosclerosis and / or podocyte damage, preferably crescent formation. In addition, analysis of kidney tissue can help determine whether a patient is suffering from early stage kidney disease.

[0016] (Paraffin removal process) In step b, the paraffin embedding the renal tissue is removed using a deparaffinizing agent. The deparaffinizing agent used to remove paraffin from kidney tissue is not particularly limited, but may be, for example, xylene. In addition, a commercially available product known as a substitute for xylene may be used as the deparaffinizing agent. An example of a xylene substitute is a product called Hemo-De. The conditions for removing paraffin using a deparaffinizing agent are not particularly limited, and the reaction time and reaction temperature are set appropriately.

[0017] Prior to the clearing step (step c) described below, it is preferable to treat the paraffin-removed kidney tissue with at least three types of ethanol solutions with gradually decreasing ethanol concentrations. By carrying out the stepwise ethanol treatment, antigens are more easily fluorescently stained in the fluorescent staining in step d, and a three-dimensional image of the kidney tissue with higher resolution can be obtained. The ethanol treatment step preferably also serves to wash the renal tissue from which paraffin has been removed. Treating the renal tissue with ethanol can, for example, wash the renal tissue of the deparaffinizing agent or wash the renal tissue as a pretreatment for the clearing step. The concentrations of the at least three types of ethanol solutions used in the ethanol treatment can be set appropriately, but for example, ethanol solutions with at least three arbitrary concentrations within the ethanol concentration range from 100% to 60% can be prepared. The treatment with at least three types of ethanol solutions is carried out using ethanol solutions of gradually decreasing concentrations, from high to low. For example, at least three different ethanol solutions are prepared with different concentrations within the ethanol concentration range of 100% to 60%, the first treatment is performed with a 100-90% ethanol solution, the final treatment is performed with an 80-60% ethanol solution, and the concentration of the ethanol solution in between is appropriately selected as long as the concentration decreases stepwise, and at least three types of ethanol solutions may be used for the treatment. More specifically, the treatment with the ethanol solution may be carried out using ethanol solutions having at least three selected concentrations from the concentrations of 100%, 90%, 80%, and 70%. The ethanol solution is preferably an aqueous ethanol solution, and as a 100% ethanol solution, it is preferable to use absolute ethanol. The treatment with the ethanol solution may include multiple treatments using ethanol solutions of the same concentration, but since the treatment is performed using ethanol solutions of at least three different concentrations, the treatment with the ethanol solution is performed at least three times. The treatment time at each ethanol concentration is, for example, 1 to 3 hours, preferably 1.5 to 2.5 hours, 1.75 to 2.25 hours, or about 2 hours.

[0018] The method may further include a step of activating antigens before the clarification step described below. When the method of the present invention includes an ethanol treatment step and an antigen activation step, either step may be performed first between steps b and c. Alternatively, only the ethanol treatment step may be performed between steps b and c. The antigen activation is preferably performed as a pretreatment for the fluorescent staining in the subsequent step d. By activating the antigen before step c, the antigen becomes more easily fluorescently stained in the fluorescent staining in step d, and a 3D image of the kidney tissue with higher resolution can be obtained. Antigen retrieval may be carried out by techniques known for fluorescent staining of paraffin-embedded specimens, and is usually carried out using Proteinase K or by heat treatment.

[0019] (Transparency process) In step c, the renal tissue from which the paraffin has been removed is cleared using a clearing reagent. Tissue transparency refers to making biological tissue, which is an aggregate of cells, visually transparent. Tissues can be made transparent by (1) removing lipids from the tissue and (2) making the refractive index of the entire tissue uniform. By carrying out processes (1) and (2), the tissue becomes visually transparent, so the same procedure is used for the clearing of kidney tissue. The clearing of renal tissue also means making the renal tissue visually transparent, and the cleared renal tissue allows laser light to be irradiated deep into the tissue.

[0020] In order to (1) remove lipids from the tissue and (2) homogenize the refractive index throughout the tissue, known methods used for defatting and refractive index adjustment for tissue transparency can be used. Clarifying agents include 1,3-bis(aminomethyl)cyclohexane, sodium dodecylbenzenesulfonate, antipyrine, and nicotinamide. The clarifying reagent is preferably a clarifying reagent containing 1,3-bis(aminomethyl)cyclohexane and sodium dodecylbenzenesulfonate, and / or a clarifying reagent containing antipyrine and nicotinamide. Known clearing techniques include CUBIC (Clear, Unobstructed Brain and Body Imaging Cocktails and Computational analysis), ScaleS, PACT, SeeDB2, iDISCO, and BABB. From the viewpoint of ease of observation of the fine structure of renal tissue, CUBIC and ScaleS are preferred for clearing renal tissue, but it is particularly preferable to use CUBIC, which is a three-dimensional imaging technique. The clearing reagent may be any of those used in the clearing techniques described above, but CUBIC-HL or CUBIC-R is preferably used, and it is preferable to use (1) CUBIC-HL to remove lipids from tissue, and (2) CUBIC-R to homogenize the refractive index of the entire tissue. For clearing, it is preferable to use both CUBIC-HL and CUBIC-R, since it is preferable to degrease tissue with CUBIC-HL and then adjust the refractive index with CUBIC-R. The conditions for clearing using a clearing reagent are not particularly limited, and the reaction time and reaction temperature are appropriately set. In particular, when a clearing reagent in CUBIC, ScaleS, PACT, SeeDB2, iDISCO, or BABB is used as the clearing reagent, the reaction time and reaction temperature using the clearing reagent may be set according to the respective protocols.

[0021] (Fluorescent dyeing process) In step d, glomeruli present in the cleared renal tissue are fluorescently stained. In the present invention, at least a part of renal tissue including glomeruli is obtained as a three-dimensional image. For this purpose, the glomeruli are fluorescently stained. In addition, renal tissue including glomeruli may be fluorescently stained. In the fluorescent staining of glomeruli, the reagent used for the fluorescent staining is not particularly limited as long as it can image components or substances constituting the glomeruli, for example, cells present in or around the glomeruli. In the fluorescent staining of renal tissue, the reagent used for the fluorescent staining is not particularly limited as long as it can image cells. The fluorescent staining step may be a step of fluorescently staining cleared renal tissue and glomeruli present in the renal tissue. Furthermore, in the present invention, since analysis is performed using a cross section that transverses a glomerulus contained in a three-dimensionally imaged kidney tissue, it is preferable to perform fluorescent staining that allows specific imaging of the glomerulus. In the fluorescent staining of kidney tissue and / or glomeruli present in kidney tissue, a fluorescent dye or a fluorescently labeled antibody may be used. In addition, for the fluorescent staining, a fluorescently labeled primary antibody may be used, or immunohistochemical staining using a primary antibody and a fluorescently labeled secondary antibody may be used. Fluorescent staining may be performed by combining these techniques related to fluorescent staining. The fluorescent protein used for the fluorescent label is not particularly limited, and may be, for example, EGFP, FITC, Alexa, Cy3, etc. The method for producing the antibody used for the fluorescent staining may be a known method described in various protocols or known literature.

[0022] In fluorescent staining of renal tissue, it is preferable to use an antibody that is used for staining the entire renal tissue. To stain the entire kidney tissue, the target proteins to be fluorescently labeled may be aquaporin 1 and LTL (Lotus tetragonolobus lectin) for labeling kidney tubules, aquaporin 2 for collecting ducts, and CD31 for vascular endothelium. The target proteins may be one type or two or more types. In addition, to stain the entire kidney tissue, DAPI may be used to stain the nuclei of cells.

[0023] In the fluorescent staining of glomeruli present in renal tissue, it is preferable to use an antibody used for staining the entire interior of the glomerulus and / or an antibody used for staining the outline of the glomerulus. For the fluorescent staining of glomeruli present in renal tissue, one type of these antibodies may be used, or two or more types may be used. For example, one type of antibody may be used to stain the entire interior of the glomerulus, one type of antibody may be used to stain the outline of the glomerulus, or a combination of one or more types of antibodies used to stain the entire interior of the glomerulus and one or more types of antibodies used to stain the outline of the glomerulus may be used.

[0024] In order to stain the entire inside of the glomerulus, it is preferable to stain the filtration membrane formed in the capillary wall of the glomerulus. Since the filtration membrane is composed of vascular endothelial cells, basement membrane, and glomerular epithelial cells (podocytes), proteins expressed in these areas may be targeted for fluorescent labeling. In particular, proteins such as nephrin, α-actinin-4, podocin, and CD2-associated protein (CD2AP), which are known as slit diaphragm-associated molecules, are involved in maintaining the slit diaphragm structure, so it is advisable to select antibodies that recognize these proteins as antibodies used to stain the entire interior of the glomerulus. By recognizing and fluorescently labeling these slit-associated molecules, fluorescence can be observed throughout the entire glomerulus in normal glomeruli, whereas in abnormal glomeruli, the fluorescence due to these slit-associated molecules is reduced, making it possible to confirm pathological findings in the glomerulus.

[0025] In order to stain the outline of the glomerulus, it is preferable to stain proteins involved in tight junctions, etc., so that the outline (appearance) of the glomerulus can be stained. Proteins involved in tight junctions, which are intercellular adhesion mechanisms, may be targeted for fluorescent labeling, such as transmembrane proteins occludin and claudin, and intracellular lining protein Zonula occludens (ZO). When these proteins involved in tight junctions are recognized and fluorescently labeled, the fluorescence can be observed at the outline (appearance) of the glomerulus in normal glomeruli, whereas in abnormal glomeruli, the fluorescence based on these proteins increases or is confirmed in places where glomeruli should actually be located, making it possible to confirm pathological findings of the glomerulus. In order to stain the outline of the glomerulus, it is preferable to stain the extracellular matrix. Collagen (particularly collagen IV), laminin, etc., which are extracellular matrices, may be targets for fluorescent labeling.

[0026] Since it is preferable to analyze the pathology of renal tissue by the difference between the staining of normal glomeruli and the staining of abnormal glomeruli in fluorescent staining of glomeruli present in renal tissue, it is preferable to target a plurality of proteins, etc. Therefore, the fluorescent staining step is preferably a step of fluorescent staining using a combination of at least two antibodies, one of which is preferably an antibody used for staining the entire inside of the glomerulus, and the other is preferably an antibody used for staining the outline of the glomerulus. The use of a combination of an antibody used to stain the entire interior of the glomerulus and an antibody used to stain the outline of the glomerulus makes it easier to find damaged glomerular tissue. In particular, the use of a combination of an antibody used to stain the entire interior of the glomerulus and an antibody used to stain the outline of the glomerulus makes it easier to find damage in the glomerulus, including crescent formation.

[0027] As a combination of at least two antibodies, a combination of an antibody selected from the group consisting of an anti-nephrin antibody, an anti-podocin antibody, and an anti-CD2 associated protein (CD2AP) antibody, and an antibody selected from the group consisting of an anti-claudin antibody, an anti-collagen IV antibody, and an anti-laminin antibody is preferred, and a combination of an antibody selected from the group consisting of an anti-nephrin antibody and an anti-podocin antibody, and an antibody selected from the group consisting of an anti-claudin antibody and an anti-collagen IV antibody is more preferred. The combination of at least two antibodies is preferably a combination of an anti-nephrin antibody and an anti-claudin antibody. The anti-claudin antibody is preferably an anti-claudin 1 antibody.

[0028] Explaining this with reference to Figure 3, as shown in Figure 3A, in normal glomeruli shown as the vehicle group, nephrin is stained throughout the glomerulus, and the outline of the glomerulus is stained by staining for claudin 1. On the other hand, in sclerotic glomeruli shown as the anti-GBM nephritis group, nephrin is not stained in the sclerotic lesion area, but claudin 1 is stained, making it possible to detect glomerular sclerosis. Furthermore, as shown in Figure 3B, in normal glomeruli, the folded structure of the podocyte slit membrane can be confirmed as in the high-magnification image of the vehicle group. On the other hand, when damaged, the folded structure is disassembled as in the high-magnification image of the anti-GBM nephritis group, and when damaged further, the staining itself cannot be confirmed. Therefore, podocyte damage can be detected.

[0029] Fluorescent dyes known as nuclear dyes may be used for the fluorescent staining. Membrane-permeable dyes can be used to stain live cells, whereas membrane-impermeable dyes can be used to stain fixed or dead cells. These dyes generally stain nucleic acids by inserting into or binding to the double helix structure of DNA. Examples of fluorescent dyes include orange-fluorescent ethidium bromide, red-fluorescent propidium iodide, green-fluorescent acridine orange (AO), and blue-fluorescent DAPI and Hoechst. When the clearing step is performed using CUBIC, fluorescent staining with a nuclear staining dye may be performed in the order specified in the CUBIC protocol, and in this case, it is preferable to select a fluorescent reagent whose fluorescent ability does not disappear under the reaction conditions for clearing kidney tissue. As will be described later, fluorescent staining with a nuclear staining dye may be performed simultaneously with or before or after fluorescent staining of the glomeruli in the step d of fluorescent staining of the glomeruli.

[0030] The fluorescent reagents used for fluorescent staining can be selected as appropriate so long as the combination does not overlap in fluorescent wavelengths. However, since multiple proteins are targeted, it is preferable to use multiple fluorescent proteins corresponding to the targets and a fluorescent dye known as a nuclear staining dye.

[0031] In the present invention, the fluorescent staining step in step d is not particularly limited as long as it is a step of fluorescently staining glomeruli present in the cleared renal tissue, but it is preferable that it is a step of fluorescently staining glomeruli present in the cleared renal tissue by immunohistochemical staining, and it is preferable that step d further includes a step of fluorescently staining the cleared renal tissue. Note that the step of fluorescently staining the renal tissue can also be performed in step c according to the CUBIC protocol. When fluorescently staining glomeruli present in cleared renal tissue by immunohistochemical staining, it is preferable to use, as the primary antibody, an antibody used for staining the entire interior of the glomerulus or an antibody used for staining the outline of the glomerulus. When fluorescently staining cleared renal tissue and glomeruli present in the renal tissue, it is preferable to use a fluorescent dye known as a nuclear staining dye and an antibody used for staining the entire inside of the glomerulus or an antibody used for staining the outline of the glomerulus, and the combination of a fluorescent dye known as a nuclear staining dye and an antibody used for staining the entire inside of the glomerulus or an antibody used for staining the outline of the glomerulus may be a combination individually selected from the above-mentioned preferred and more preferred aspects.When fluorescently staining cleared renal tissue and glomeruli present in the renal tissue, a combination of DAPI, an anti-nephrin antibody, and an anti-claudin antibody may be used.

[0032] (Fluorescence imaging of kidney tissue) Renal tissue fluorescence imaging analysis e. obtaining a three-dimensional image of at least a portion of the renal tissue including the glomerulus; and f. A process of analyzing a cross section of a glomerulus contained in the 3D image of kidney tissue to confirm the presence or absence of damage in the glomerulus. In step e, a three-dimensional image of at least a portion of the kidney tissue including the fluorescently stained glomeruli is obtained. Next, in step f, the presence or absence of damage in the glomerulus is confirmed by analyzing a cross section transverse to the glomerulus contained in the fluorescently stained and 3D imaged kidney tissue. In the present invention, by using a method including steps a to f, it becomes possible to detect damage in the glomerulus by analyzing kidney tissue containing fluorescently labeled glomeruli by fluorescence imaging. At least a portion of renal tissue including a glomerulus that is cleared and fluorescently stained and obtained as a three-dimensional image is a sample from which images of pathological staining can be obtained from the surface to the depths of the tissue. According to the present invention, it is possible to analyze glomeruli using 3D images of renal tissue without using serial sectioning methods such as cross-sectional tomography, in which a sample is sliced ​​into thin slices, each slice is imaged, and the image data are superimposed to reconstruct the sample in 3D. Compared to three-dimensional observation using conventional serial sectioning methods, the analysis method of the present invention does not require the time and labor required to slice the entire tissue, and there is no risk of losing specific sections during slices. Therefore, it is possible to provide a renal biopsy method that is easy to perform and has little discrepancy between the test values ​​obtained in clinical practice and the level of lesions in the actual specimen tissue.

[0033] To perform analysis by fluorescent imaging of renal tissue, at least a portion of the renal tissue, including the glomeruli, is reconstructed as a 3D image based on fluorescent image data of the cleared and fluorescently stained renal tissue using high-speed, high-resolution imaging technology. In the present invention, the renal tissue embedded and fixed in paraffin is used for clearing and fluorescent staining, and the renal tissue including the glomeruli as a three-dimensional image may be a part of the renal tissue embedded and fixed in paraffin, or it may be the entire renal tissue embedded and fixed in paraffin. By performing cross-sectional analysis across the glomerulus of the constructed three-dimensional image, the glomerulus and the lesion can be observed in a two-dimensional cross section easily and accurately. As a result, the conventional two-dimensional analysis has a problem that the glomerulus is often not present in the analyzed cross section, whereas the analysis method of the present invention has an effect of being able to reliably analyze the glomerulus. Furthermore, in the conventional two-dimensional analysis, even if a glomerulus is present in the analyzed cross section, only one cross section of the glomerulus can be analyzed, so that an abnormal glomerulus such as a crescent glomerulus may be erroneously determined to be a normal glomerulus. In contrast, in the analysis method of the present invention, when no abnormality is found in one cross section of the glomerulus or when an abnormality cannot be determined but is suspected, another cross section or multiple cross sections of the glomerulus can be continuously analyzed. Therefore, the abnormal glomerulus such as a crescent glomerulus can be analyzed efficiently and accurately, and the risk of overlooking a lesion can be significantly reduced. By analyzing cross sections that cross the glomeruli in 3D images of renal tissue, it is possible to search for lesions in renal tissue in a manner that simulates the serial section method. This is thought to solve the problems posed by the limitations of conventional 3D analysis, such as false negatives in samples from patients with early-stage kidney disease, depending on the sections obtained. In this respect, in conventional examinations, lesions can only be found on thin slices, but in the case of the analysis method using the three-dimensional image of the present invention, at least a part of the renal tissue embedded and fixed in paraffin obtained by biopsy, and in some cases, the entire renal tissue, can be analyzed at once. Therefore, lesions in renal biopsy tissue can be detected with higher sensitivity. In addition, the analysis of renal tissue using the three-dimensional image of the present invention makes it possible to visualize the three-dimensional structure of the kidney, which could not be visualized in two dimensions until now. In other words, it can create a new concept of pathological tissue images that could not be found by conventional two-dimensional analysis, and greatly contribute to elucidating the pathological mechanism.

[0034] In particular, in step f, glomeruli are detected as a three-dimensional image by fluorescent staining, and then the glomeruli in the three-dimensional image are observed and analyzed in two dimensions using transverse cross sections. In step f, the 3D image is analyzed as a cross section transverse to the 2D glomerulus, making it possible to analyze deformations in the shape of the glomerulus that are difficult to detect in a 3D image by converting the 3D image into a 2D image (cross section). In step f, if even one cross-section of a glomerulus showing a damage such as a crescent is found, it is possible to confirm that there is a damage in the glomerulus at that point. From the viewpoint of accuracy, it is preferable to confirm at least two cross sections, and it is acceptable to confirm three or more cross sections. When multiple cross sections are to be analyzed, any cross section may be selected, but it is preferable that at least two cross sections that cross the glomerulus are approximately perpendicular to each other.

[0035] Renal biopsies are performed for a wide variety of diseases, and many of these cannot be diagnosed by conventional pathological diagnostic methods. Diseases that require special staining, immunopathological examination, or ultrastructural evaluation by electron microscopy require vigorous testing to ensure a definitive diagnosis. Therefore, the analysis method of the present invention makes it possible to process renal tissue collected by renal biopsy in a way that maximizes the amount of useful information available.

[0036] The analytical method of the present invention is performed as a three-dimensional renal tissue examination by tissue transparency, and more specifically, relates to a technology for obtaining a three-dimensional image of human renal tissue by making it visually transparent for renal biopsy tissue, and then analyzing it in two dimensions. This technology provides a tool for pathological diagnosis of renal tissue, and therefore also provides a method for diagnosing kidney disease. In this diagnostic method, a cross section of a glomerulus contained in a 3D image of kidney tissue is analyzed to confirm the presence or absence of damage in the glomerulus, more specifically, the presence or absence of crescent formation, glomerular sclerosis, and / or podocyte damage is analyzed, thereby providing a method for definitively diagnosing kidney disease. In addition, this diagnostic method can provide a method for definitively diagnosing early kidney disease and chronic kidney disease. That is, the diagnostic method of the present invention may be a method of diagnosing kidney disease by performing steps a to f of analyzing renal tissue by fluorescence imaging to detect damage in the glomeruli, and confirming the presence or absence of damage in the glomeruli in step f, more specifically, by analyzing the presence or absence of crescent formation, glomerular sclerosis, and / or podocyte damage. The method for diagnosing kidney disease may be an adjunct method for providing to physicians. By applying the diagnostic method of the present invention, it is possible to differentiate diseases such as, but not limited to, minimal change nephrotic syndrome and focal segmental glomerulosclerosis, and it is also possible to extract sclerotic glomeruli in glomerular diseases such as IgA nephropathy, membranous nephropathy, and lupus nephritis.

[0037] Crescent formation is a characteristic clinical finding seen when severe inflammation occurs in the glomerulus. It is believed that the higher the percentage of glomeruli that are positive for crescent formation, the more severe the condition is, and crescent formation is observed in many patients with severe glomerulonephritis. Therefore, in analysis using three-dimensional images, it is advisable to use the formation of crescents in the glomerulus as an indicator.

[0038] It is also possible to confirm the degree of damage to the renal tissue in a subject by comparing the damage in the glomerulus detected by the analysis method of the present invention with the results of a biochemical test of the subject from which the renal tissue is derived. Also, by comparing the results obtained by imaging of the damage in the glomerulus detected by the analysis method of the present invention with the results of a biochemical test of the subject from which the renal tissue is derived, it is possible to provide a method for diagnosing kidney disease by confirming the degree of damage to the renal tissue in a subject. Biochemical tests include, but are not limited to, tests for serum creatinine and urine protein. By comparing the measurement values ​​of these biochemical tests with normal values ​​and combining both with the results regarding damage in the glomeruli detected by the analytical method of the present invention, a more accurate method for diagnosing kidney disease can be provided. EXAMPLES

[0039] Example 1 <Tissue transparency of mouse kidney after paraffin replacement> From a paraffin-blocked mouse kidney sample (approximately 5 mm in diameter x 10 mm in length), a sample for tissue clearing was cut into 1 mm-thick slices using a brain slicer (MK-MS-01, Muromachi Kikai Co., Ltd.) The sample was treated at 60°C for 10 minutes in a 2 mL tube to dissolve and remove the paraffin. Subsequently, deparaffinization was performed using Hemo-De (Pharma Co., Ltd.) The operation was performed three times using 2 mL of Hemo-D, with infiltration for 1 hour, followed by overnight infiltration, and then another infiltration for 1 hour. Hemo-Desorption was performed using 100%, 90%, 80%, and 70% ethanol. The procedure consisted of 2 mL of ethanol at each concentration, with the ethanol concentration gradually decreasing, and the procedure was carried out for 2 hours. Ethanol was removed using PBS, and the procedure involved overnight penetration using 2 mL of PBS. The obtained deparaffinized samples were cleared using CUBIC-HL and CUBIC-R according to the protocol of the clearing reagent CUBIC (Tokyo Chemical Industry Co., Ltd.). The results are shown in Figure 1A (1 square is 1 mm). It was confirmed that the samples were cleared well. Similar results were obtained when a paraffin block of a rat kidney sample (approximately 10 mm in diameter x 20 mm in length) was used. The results are shown in Figure 1B. It was confirmed that the rat kidney sample was also successfully cleared.

[0040] Example 2 <Tissue clearing of human kidney biopsy samples after paraffin replacement> Using a paraffin block of a human kidney sample (approximately 1 mm in diameter x approximately 5 mm in length), the tissue was made transparent by the same method as in Example 1. The results are shown in Figure 1C. It was confirmed that the human kidney sample was also made transparent satisfactorily.

[0041] Example 3 <Comparison of 3D image analysis and 2D analysis using anti-GBM nephritis rats> For comparison, we used an anti-GBM nephritis rat model in which crescentic nephritis was induced by administering anti-type IV collagen α4 chain antibody, which is an antibody against the glomerular basement membrane (GBM). The following mouse experiments were conducted in accordance with the Animal Experimentation Implementation Guidelines of Chiba University, a national university corporation. As shown in FIG. 2, 5-week-old Wistar-Kyoto rats were divided into an anti-GBM nephritis group administered with anti-GBM antibody (anti-type IV collagen α4 chain antibody) and a vehicle group administered with physiological saline. After 14 days, sacrifice was performed, and one kidney was subjected to conventional 2D analysis by PAS staining, while the other kidney was cleared in the same manner as in Example 1 according to the method of the present invention, and then subjected to fluorescent immunostaining and 3D analysis. Specifically, the following protocol was followed. To construct a 3D image, the glomerulus and its surrounding area were stained with the following primary and secondary antibodies. Primary antibody ·Nephrin: Anti-Mouse Nephrin antibody (Polyclonal Gat IgG) (product number AF3159, manufacturer R&D SYSTEMS) ·Claudin-1: Anti-Claudin1 antibody (Rabbit antibody) (product number ab15098, manufacturer Abcam) secondary antibody Alexa Fluor-488 anti-goat IgG antibody (product number A-11055, manufacturer ThermoFisher Scientific) Alexa Fluor-555 anti-rabbit IgG antibody (product number A27039, manufacturer ThermoFihserScientific) In addition, DAPI (product number 10236276001, manufacturer MERCK) was used for staining the entire tissue.

[0042] The protocol is as follows. On day 0, 5-week-old Wistar-Kyoto rats were intraperitoneally administered a monoclonal antibody against type IV collagen α4 chain (product number 70201, manufacturer Chondrex) or saline. The antibody-administered group was the anti-GBM nephritis group, and the saline group was the control group (vehicle group). On day 14, sacrifices were made by perfusion with PBS and 4% PFA from the heart, and both kidneys were excised. After that, they were infiltrated with 4% PFA overnight at 4℃, and the next day, one of the excised kidneys was analyzed in 2D (PAS staining), and the other was cleared for 3D analysis, then fluorescent immunostained and 3D analysis was performed.

[0043] In the 3D analysis, a 3D image of the entire kidney tissue was first constructed. This allowed the location of the glomerulus in the tissue to be confirmed. Next, an image of a cross section transverse to the glomerulus was extracted and the glomerulus was analyzed. Some of the results of the 3D analysis are shown in Figure 3. Figure 3A shows a representative cross section of a glomerulus observed in the vehicle group, and Figure 3B shows a representative cross section of a glomerulus observed in the anti-GBM group. The upper left diagram of FIG. 3A shows the result of staining with an anti-nephrin antibody, and a spherical glomerulus was confirmed. The upper center diagram of FIG. 3A shows the result of staining with an anti-claudin-1 antibody, and the Bowman's capsule epithelium surrounding the glomerulus was stained, and a ring-shaped stain was confirmed surrounding the outline of the spherical glomerulus. This confirmed that the glomerulus was present in a normal shape. On the other hand, in the anti-GBM group, as shown in the lower left diagram of FIG. 3A, the glomerulus stained with the anti-nephrin antibody showed a deformed shape. In addition, as shown in the lower center diagram of FIG. 3A, the outline of the glomerulus stained with the anti-claudin-1 antibody also showed a deformation. More specifically, a glomerulus in which a crescent was formed (crescent-forming glomerulus) was observed, and it was confirmed that the Bowman's capsule epithelium had infiltrated into the place where the glomerulus was. From these results, it was found that the method of the present invention can detect abnormalities in the glomerulus with high sensitivity. As a result of the above three-dimensional analysis, only normal glomeruli were observed in the vehicle group, whereas the formation of sclerotic glomeruli and crescents was frequently observed in the anti-GBM nephritis group.

[0044] Figure 3B shows magnified images of normal glomeruli in the vehicle group and crescents in the anti-GBM group. Normal podocyte processes were observed in the vehicle group, whereas podocytes in the anti-GBM group showed abnormal morphology and lost processes.

[0045] Figure 4 shows an example of 3D analysis of a glomerulus using anti-GBM nephritis rats. The left image shows a glomerulus image acquired as a 3D image. The right image shows 2D analysis of each cross section (Slice 1, Slice 2, and Slice 3) that crosses the glomerulus. Slice 1 and Slice 2, and Slice 1 and Slice 3 are cross sections that intersect at right angles. When observing the glomeruli in the anti-GBM nephritis group, in Slice 1, the green fluorescence due to fluorescent staining of nephrin in the Bowman's capsule was negative, and the red fluorescence due to fluorescent staining of claudin-1 and the area (structure) where the nuclear staining with DAPI was positive were observed in the upper left, confirming the formation of crescents (the slice image of Slice 1 is indicated by a circle number 1. The same applies to Slice 2 and Slice 3 below). Similarly, in Slice 2, the formation of crescents was confirmed with negative green and positive red and blue. On the other hand, in Slice 3, the glomeruli were stained green all over, and red was only seen around the glomeruli, confirming the formation of normal glomeruli. In other words, if only 2D analysis was performed and the image of Slice 3 alone was analyzed, glomerular abnormalities (crescent formation) would not be detected. Thus, it was confirmed that glomerular abnormalities can be detected efficiently and accurately by performing 3D analysis using the method of the present invention, that is, by constructing a 3D image of kidney tissue to detect glomeruli and then analyzing a cross section that crosses the glomeruli. A slice image of Slice 1 is shown in FIG. 5. In FIG. 5, a cross section that crosses the glomeruli contained in the 3D imaged kidney tissue is shown, but at least a part of the kidney tissue including the glomeruli is obtained as a 3D image as a collection of each fluorescently stained image. A 3D image can be obtained as each fluorescently stained image, and a 3D image can also be obtained as a superposition image of all the fluorescents used for the fluorescent staining. FIG. 5B shows the result of staining with DAPI, and the entire tissue including the glomeruli is stained. FIG. 5C shows the result of staining with an anti-nephrin antibody, and a crescent glomerulus can be confirmed. FIG. 5D shows the result of staining with an anti-claudin-1 antibody, and the Bowman's capsule epithelium surrounding the glomerulus can be confirmed. In addition, in this image, no infiltration of Bowman's capsule-like epithelium into the missing part of the glomerulus that formed a crescent (i.e., the part where no glomerulus exists) is observed. Figure 5A is a superimposition of the respective staining results.

[0046] The results of analyzing and quantifying the glomeruli that formed crescents are shown in Table 1 and FIG. 6. The words "2D" in Table 1 and "2D" in FIG. 6 indicate data of 2D analysis performed according to the conventional method, and the words "3D" in Table 1 and "3D" in FIG. 6 indicate data of 3D analysis performed according to the method of the present invention. The detection rate of crescents in the 3D analysis was significantly higher than the detection rate of the conventional 2D analysis, which supports the above results that the analysis according to the method of the present invention was able to detect glomeruli that formed crescents more efficiently and accurately than the conventional 2D analysis. In the case of 2D analysis, only one plane of a thin slice can be observed, whereas in the case of 3D analysis, when no abnormality is found in one cross section of the glomerulus or when abnormality is suspected but cannot be determined to be abnormal, another cross section or multiple cross sections of the glomerulus can be analyzed continuously. This can significantly reduce the risk of overlooking a lesion. In other words, 3D analysis has the effect of preventing false negatives and detecting glomerular abnormalities with higher accuracy compared to 2D analysis.

[0047] [Table 1]

[0048] Example 4 <Comparison of 3D and 2D analyses using human samples> Two-dimensional and three-dimensional analyses were performed on a renal biopsy sample from a 67-year-old man who had been clinically diagnosed with rapidly progressive glomerulonephritis. For two-dimensional analysis, thin sections (2-4 μm) of the renal biopsy samples were placed on slides and stained with periodic acid Schiff (PAS). Fourteen cross-sectional images were observed under an optical microscope; however, no crescents were detected (Figure 7). On the other hand, as a result of three-dimensional analysis performed according to the method of the present invention, the formation of crescents was confirmed around the inflammatory cell infiltration area in the interstitium. The results are shown in FIG. The upper diagram of FIG. 8B shows the result of immunostaining a kidney biopsy sample using anti-nephrin antibody, anti-claudin-1 antibody, and DAPI after clearing. The entire kidney tissue is stained with DAPI. Several glomeruli stained with anti-nephrin antibody were observed. One of the observed glomeruli is shown in a square. The lower diagram of FIG. 8B shows an observation diagram of a cross section transverse to this glomerulus, in which a glomerulus forming a crescent can be confirmed. According to the method of the present invention, the existence of a glomerulus can be confirmed, and then the cross section of the glomerulus can be observed, so that the glomerulus can be analyzed efficiently without wasteful analysis of images of parts where no glomeruli are present. The result of confirming the part of kidney tissue where glomerular abnormality was confirmed in the 3D analysis of the present invention by conventional 2D analysis is shown in FIG. 8A. Glomerular abnormality was also confirmed in the conventional method.

[0049] Example 5 <Comparison of 3D and 2D analyses using human samples> The results of 2D and 3D analyses using kidney biopsy samples collected from four patients and preserved by paraffin embedding are shown in Table 2 (number of glomeruli observed per section sample) and Table 3 (detection rate (%) of crescents in glomeruli). In Tables 2 and 3, "2D" refers to data from 2D analysis according to a conventional method, and "3D" refers to data from 3D analysis according to the method of the present invention. It has been demonstrated that the method of the present invention can detect glomerular abnormalities more efficiently and accurately than conventional methods.

[0050] [Table 2]

[0051] [Table 3] [Industrial Applicability]

[0052] The present invention has industrial applicability in the field of clinical testing.

Claims

1. 1. A method for analyzing kidney tissue by fluorescence imaging to detect damage in a glomerulus, comprising: a. Providing paraffin-embedded fixed kidney tissue; b. Removing paraffin embedding the renal tissue using a deparaffinizing agent; c. clearing the paraffin-removed kidney tissue with a clearing reagent; d. fluorescently staining glomeruli present in the cleared kidney tissue; e. obtaining a three-dimensional image of at least a portion of the kidney tissue including the glomerulus; and f) analyzing a cross section of a glomerulus contained in the three-dimensionally imaged kidney tissue to confirm the presence or absence of damage in the glomerulus.

2. The method of claim 1 , wherein the paraffin-embedded and fixed renal tissue is a roughly columnar or rod-shaped renal biopsy sample.

3. The method according to claim 1 or 2, wherein in step f, the cross sections are at least two cross sections that are substantially perpendicular to each other.

4. The method according to any one of claims 1 to 3, wherein step d is a step of fluorescently staining the glomerulus by using a combination of at least two antibodies, one of which is an antibody used for staining the entire interior of the glomerulus, and the other of which is an antibody used for staining the outline of the glomerulus.

5. The method according to claim 4, wherein the two antibodies are a combination of an antibody selected from the group consisting of an anti-nephrin antibody and an anti-podocin antibody, and an antibody selected from the group consisting of an anti-claudin antibody and an anti-collagen IV antibody.

6. The method of claim 4 , wherein the two antibodies are an anti-nephrin antibody and an anti-claudin antibody.

7. The method according to any one of claims 1 to 6, wherein the clarifying reagent is a clarifying reagent containing 1,3-bis(aminomethyl)cyclohexane and sodium dodecylbenzenesulfonate, and / or a clarifying reagent containing antipyrine and nicotinamide.

8. The method according to any one of claims 1 to 7, further comprising, prior to step c, treating the mixture with at least three types of ethanol solutions having gradually decreasing ethanol concentrations.

9. The method according to any one of claims 1 to 8, wherein the glomerular damage is at least one selected from the group consisting of crescent formation, glomerular sclerosis, and podocyte damage.

10. The method according to any one of claims 1 to 8, wherein the glomerulus injury is crescent formation.

11. A method for confirming the degree of damage to renal tissue in a subject from which the renal tissue is derived, using a result regarding damage in the glomerulus detected by the analysis method according to any one of claims 1 to 10, the method further comprising comparing the measurement value of a biochemical test of the subject with a normal value.

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