Application of polypeptide or its fragment in preparing kit for detecting vascular endothelial cell damage

Abstract

The present invention relates to the use of a polypeptide or its antibody binding fragment that can form an antigen-antibody complex in contact with a biological sample obtained from the patient in the preparation of a vascular endothelial cell damage detection reagent or kit; wherein the polypeptide or its antibody binding fragment includes at least two selected from the following groups: alpha enolase protein (ENO1), talin 1 (TLN1), filament protein-A (FLNA), desmosome nexin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), membrane protrusion protein (MSN). The vascular endothelial cell autoantibodies of the present application can be used to diagnose vascular endothelial cell damage by simply detecting the titer in serum, and have very great clinical application value.

Description

[0001] This application is a divisional application with application number 202310216552.5, application date March 2, 2023, and invention name: Use of polypeptides or fragments thereof in the preparation of a kit for detecting vascular endothelial cell damage. Technical Field

[0002] The present invention relates to the field of diagnostic kits, in particular to the use of polypeptides or fragments thereof in preparing a kit for detecting vascular endothelial cell damage. Background Art

[0003] Idiopathic nephrotic syndrome (INS) is one of the most common primary glomerular diseases in children, with approximately 28,000 to 56,000 new cases reported annually in China. Steroid therapy is effective for most patients, but approximately 10% to 20% of patients are resistant to steroids, a condition known as steroid-resistant nephrotic syndrome (SRNS). Among patients with INS, 8% to 35% develop end-stage renal disease (ESRD) 5 years after diagnosis, and 24% to 66% develop ESRD 15 years after diagnosis. However, the pathogenesis of INS has not been fully elucidated to date.

[0004] Minimal change disease (MCD) is the leading cause of nephrotic syndrome in children and accounts for 10-15% of nephrotic syndrome in adults. In patients with minimal change disease, glomeruli appear essentially normal under light microscopy, and the only histopathological abnormality visible under electron microscopy is diffuse effacement of podocyte foot processes. Therefore, a common view is that the pathogenesis of INS may be related to systemic dysfunction of T cells, and selective T cell inhibitors such as cyclosporine / tacrolimus are effective in some patients. In addition to T cells, circulating permeability factors such as soluble urokinase-type plasminogen activator receptor are also possible pathogenic factors. However, current clinical treatments based on the above theory do not help all patients achieve clinical remission. Therefore, researchers have been exploring other possible pathological mechanisms. Summary of the Invention

[0005] In response to the technical problems existing in the prior art, the present invention proposes the use of a polypeptide or its antibody binding fragment that can form an antigen-antibody complex in contact with a biological sample obtained from the patient in the preparation of a vascular endothelial cell injury detection reagent or kit; wherein the polypeptide or its antibody binding fragment includes at least two selected from the following groups: alpha enolase protein (ENO1), talin 1 (TLN1), filamin-A (FLNA), desmosome nexin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane protrusion protein (MSN).

[0006] In some embodiments, the polypeptide or antibody binding fragment thereof is a combination of heat shock protein 90β (HSP90AB1) and cytoskeleton-associated protein 4 (CKAP4), or a combination of talin 1 (TLN1) and desmoglein 1 (DSG1), or a combination of talin 1 (TLN1) and moesin (MSN).

[0007] In some embodiments, the vascular endothelial cell damage is indicative of nephrotic syndrome.

[0008] In some embodiments, the nephrotic syndrome is idiopathic nephrotic syndrome.

[0009] In some embodiments, the idiopathic nephrotic syndrome is childhood idiopathic nephrotic syndrome.

[0010] In some embodiments, the biological sample is serum.

[0011] In some embodiments, the biological sample is a biological sample of a patient before immunotherapy.

[0012] In some embodiments, the present invention also proposes the use of a polypeptide or an antibody binding fragment thereof that can form an antigen-antibody complex in contact with a biological sample obtained from the patient in the preparation of a vascular endothelial cell injury detection reagent or kit; wherein the polypeptide or antibody binding fragment thereof includes at least three selected from the following groups: alpha enolase protein (ENO1), talin 1 (TLN1), filament protein-A (FLNA), desmosome nexin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane protrusion protein (MSN).

[0013] In some embodiments, the polypeptide or antibody binding fragment thereof is a combination of desmoglein 1 (DSG1), moesin (MSN) and talin 1 (TLN1), or a combination of moesin (MSN), myosin light chain 1 (MYL1) and talin 1 (TLN1), or a combination of alpha enolase ENO1, talin 1 (TLN1) and heat shock protein 90β (HSP90AB1).

[0014] In some embodiments, the present invention also proposes the use of a polypeptide or antibody binding fragment capable of forming any antigen-antibody complex in contact with a biological sample obtained from the patient in the preparation of a reagent or kit for specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schönlein purpura, and Kawasaki disease; wherein the polypeptide or its antibody binding fragment includes at least two selected from the following groups: alpha enolase protein (ENO1), talin 1 (TLN1), filament protein-A (FLNA), desmosome nephrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane protrusion protein (MSN).

[0015] In some embodiments, the present invention also proposes a reagent or kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schönlein purpura, and Kawasaki disease, comprising: a polypeptide or an antibody-binding fragment thereof, which is capable of contacting with a biological sample obtained from the patient to form any antigen-antibody complex; wherein the polypeptide or antibody-binding fragment thereof includes at least two selected from the following groups: alpha enolase protein (ENO1), talin 1 (TLN1), filament protein-A (FLNA), desmosome nephrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane protrusion protein (MSN).

[0016] The gold standard for diagnosing endothelial cell injury is electron microscopy. However, electron microscopy requires renal puncture, which is damaging, expensive, and time-consuming, making it unsuitable for routine use. The endothelial cell autoantibodies described in this application can be used to diagnose endothelial cell injury by simply measuring their titer in serum, thus possessing significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Below, the preferred embodiments of the present invention will be further described in detail with reference to the accompanying drawings, in which:

[0018] FIG1 is a diagram of endothelial cell autoantibody detection according to an embodiment of the present invention; Figures 1A-1IDetection of endothelial cell autoantibodies in INS patients and other control group participants according to one embodiment of the present application; NC represents a normal control group; HSP represents Henoch-Schonlein purpura; HSPN represents Henoch-Schonlein purpura nephritis; KD represents Kawasaki disease; NS represents nephrotic syndrome; Figure 1J-1K Shows the changing pattern of endothelial cell autoantibodies in INS patients according to one embodiment of the present application;

[0019] FIG2 shows the diagnostic value of 9 endothelial cell autoantibodies in INS according to one embodiment of the present invention; wherein, Figure 2A is a ROC curve of an anti-ENO1 antibody according to one embodiment of the present application; Figure 2B is a ROC curve of an anti-TLN1 antibody according to one embodiment of the present application; Figure 2C is a ROC curve of an anti-FLNA antibody according to one embodiment of the present application; Figure 2D : is the ROC curve of the anti-AHNAK antibody according to one embodiment of the present application; Figure 2E is a ROC curve of an anti-DSG1 antibody according to one embodiment of the present application; Figure 2F is a ROC curve of an anti-MYL1 antibody according to one embodiment of the present application; Figure 2G is a ROC curve of the anti-HSP90AB1 antibody according to one embodiment of the present application; Figure 2H is a ROC curve of an anti-CKAP4 antibody according to one embodiment of the present application; Figure 2I is a ROC curve of an anti-MSN antibody according to one embodiment of the present application;

[0020] FIG3 is a graph showing the correlation between the grayscale value of vascular endothelial cell autoantibodies and the clinical condition of INS patients according to an embodiment of the present invention; wherein, Figure 3A According to one embodiment of the present application, compared with before remission, all nine vascular endothelial cell autoantibodies were significantly reduced after remission; Figure 3B For patients with INS according to one embodiment of the present application, the first seven vascular endothelial cell autoantibodies are significantly negatively correlated with serum albumin; Figure 3C According to one embodiment of the present application, eight vascular endothelial cell autoantibodies are significantly negatively correlated with the international normalized ratio (INR) of INS patients before remission; Figure 3D According to one embodiment of the present application, 7 vascular endothelial cell autoantibodies are significantly negatively correlated with the platelet count of INS patients before remission;

[0021] FIG4 is a diagram of renal endothelial injury in patients with INS according to an embodiment of the present invention; wherein, Figure 4A- Figure 4G The present invention provides seven typical signs of endothelial injury observed in renal biopsy specimens of INS patients under an electron microscope according to one embodiment of the present application; Figure 4HThe following is a pattern of different signs of endothelial injury in 20 patients with INS according to one embodiment of the present application; Figure 4I This is an immunofluorescence image of human kidney showing upregulation of caveolin-1 (green) and colocalization with EHD3 (red) according to one embodiment of the present application;

[0022] FIG5 shows the effect of anti-ENO1 antibodies in vitro according to one embodiment of the present invention; wherein, Figures 5A-5C The actin skeleton changes and separation of Eahy926 cells after pretreatment with ENO1 protein by isotype control antibody and anti-ENO1 antibody, isotype control antibody and normal rabbit serum and anti-ENO1 antibody and normal rabbit serum, anti-ENO1 antibody positive serum and anti-ENO1 antibody positive serum according to one embodiment of the present application; Figure 5D-5F The difference between the level of thrombomodulin in the supernatant of Eahy926 cells treated with the isotype control antibody according to one embodiment of the present application and the anti-ENO1 antibody, the isotype control antibody normal rabbit serum and the anti-ENO1 antibody normal rabbit serum, the anti-ENO1 antibody serum and the anti-ENO1 serum pretreated with the ENO1 protein;

[0023] Figure 6 The figure is the histology (HE staining, PAS staining and Masson staining) of mouse kidney injected with antibodies according to one embodiment of the present invention;

[0024] Figure 7 The damage to endothelial cells and podocytes of mice injected with anti-ENO1 antibodies can be seen under an electron microscope according to one embodiment of the present invention; wherein, Figure 7 A shows that autophagosomes can be observed in the glomerular endothelial cells of mice injected with the anti-ENO1 antibody according to one embodiment of the present application; Figure 7 B shows that mitochondrial damage can be observed in the podocytes of mice treated with the anti-ENO1 antibody according to one embodiment of the present application; Figure 7 C- Figure 7 D represents that foot process fusion of podocytes can be observed in podocytes of mice injected with anti-ENO1 antibodies according to one embodiment of the present application, but not in mice injected with isotype antibodies; and

[0025] FIG8 is a graph showing the sensitivity and specificity of one or more antibodies used in combination according to an embodiment of the present invention; Figures 8A-8I This is the sensitivity and specificity profile of the antibody when used alone; Figure 8J-8M This is the sensitivity and specificity profile when two antibodies are used in combination; Figure 8N-Figure 8Q Figure 2 shows the sensitivity and specificity of the three antibodies when used in combination. DETAILED DESCRIPTION

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0027] In the detailed description that follows, reference may be made to the various drawings that form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Each specific embodiment of the present application is described below in sufficient detail to enable a person of ordinary skill in the art to implement the technical solutions of the present application. It should be understood that other embodiments may be utilized or that structural, logical, or electrical changes may be made to the embodiments of the present application.

[0028] The nouns used in this article have the following meanings:

[0029] The "nephrotic syndrome" or "NS" mentioned in this article refers to a clinical syndrome that is caused by a series of pathophysiological changes due to the increased permeability of the glomerular filtration membrane to plasma proteins and the loss of a large amount of plasma proteins in the urine. Its main clinical features are massive proteinuria, hypoproteinemia, hyperlipidemia and edema.

[0030] The "idiopathic nephrotic syndrome," or "INS," referred to in this article, is a condition of unknown etiology. Increased permeability of the glomerular filtration membrane leads to increased plasma protein filtration, causing massive proteinuria and, in turn, a series of clinical syndromes characterized by podocyte lesions. Children with this condition often present with massive proteinuria, hypoproteinemia, edema, and hyperlipidemia.

[0031] The "polypeptide or its fragment" mentioned herein refers to a combination of two or more polypeptide indicators used to detect the same disease. Among them, the polypeptide or its antibody-binding fragment is selected from: alpha enolase protein (ENO1), talin 1 (TLN1), filamin-A (FLNA), desmosome nexin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and moesin (MSN).

[0032] Among them, the sequence of the antibody binding fragment of α-enolase protein (ENO1) is shown in SEQ ID NO.1; the sequence of the antibody binding fragment of talin 1 (TLN1) is shown in SEQ ID NO.2; the sequence of the antibody binding fragment of filamin-A (FLNA) is shown in SEQ ID NO.3; the sequence of the antibody binding fragment of desmosome nexin (AHNAK) is shown in SEQ ID NO.4; the sequence of the antibody binding fragment of desmoglein-1 (DSG1) is shown in SEQ ID NO.5; the sequence of the antibody binding fragment of myosin light chain 1 (MYL1) is shown in SEQ ID NO.6; the sequence of the antibody binding fragment of heat shock protein 90β (HSP90AB1) is shown in SEQ ID NO.7; the sequence of the antibody binding fragment of cytoskeleton-associated protein 4 (CKAP4) is shown in SEQ ID NO.8; and the sequence of the antibody binding fragment of membrane protrusion protein (MSN) is shown in SEQ ID NO.9.

[0033] When the above polypeptide or its antibody-binding fragment is used to detect vascular endothelial cell damage, it can be contacted with a biological sample obtained from a patient to form an antigen-antibody complex. The antigen-antibody complex includes an anti-ENO1-IgG antibody complex, an anti-TLN1-IgG antibody complex, an anti-FLNA-IgG antibody complex, an anti-AHNAK-IgG antibody complex, an anti-DSG1-IgG antibody complex, an anti-MYL1-IgG antibody complex, an anti-HSP90AB1-IgG antibody complex, an anti-CKAP4-IgG antibody complex, and an anti-MSN-IgG antibody complex.

[0034] The "isotype control antibody" mentioned in this article refers to the IgG antibody extracted from the serum of rabbits of the same species and age that have not been immunized with protein, which is used as the control group.

[0035] The term "HE staining" used in this article refers to hematoxylin-eosin staining, a commonly used staining method in paraffin sectioning. Hematoxylin, an alkaline stain, primarily stains chromatin within the cell nucleus and nucleic acids within the cytoplasm purplish-blue; eosin, an acidic dye, primarily stains components of the cytoplasm and extracellular matrix red.

[0036] The "Masson stain," also known as Masson's stain, mentioned in this article, is one of the primary methods for visualizing fibers in tissue and is a definitive and classic technique for staining collagen fibers. The staining principle is related to the size of the anionic dye molecule and its tissue penetration. Molecular size is reflected by molecular weight: small molecules easily penetrate dense, low-permeability tissues, while large molecules are limited to loose, high-permeability tissues. However, light green or aniline blue have high molecular weights, so Masson staining results in muscle fibers appearing red and collagen fibers appearing green or blue. This method is primarily used to distinguish collagen fibers from muscle fibers.

[0037] The "PAS stain" mentioned in this article refers to Periodic Acid-Schiff stain. In histology, it is primarily used to detect carbohydrates in tissues. Periodic acid oxidizes the hydroxyl groups on two adjacent carbon atoms of carbohydrates into aldehyde groups, which are then reacted with Schiff's reagent to produce a purple-red color. PAS staining results in red color, while cell nuclei appear blue.

[0038] In 2021, the inventor team first proposed the concept of autoimmune podocytopathy, which has been widely recognized. Recently, Watts et al. found anti-glomerular cell adhesion molecule receptor (Nephrin) autoantibodies in the serum of children and adult MCD patients, which also provided strong evidence for the inventor's innovative theory. In addition, the presence of anti-renin autoantibodies in the serum of patients with focal segmental glomerulosclerosis (FSGS) after transplantation further supports the inventor's point of view. A new idiopathic nephrotic syndrome mouse model induced by immunization with the podocyte protein Crb2 provides direct evidence for the role of podocyte autoantibodies in the pathogenesis of INS.

[0039] Podocyte autoantibodies are considered a major cause of glomerular podocyte injury. However, from an anatomical perspective, a glomerular endothelial barrier exists between circulating podocyte autoantibodies and podocytes. Unless glomerular endothelial integrity is compromised, podocyte autoantibodies cannot reach podocytes.

[0040] Through a series of experiments, the inventors discovered that multiple anti-endothelial autoantibodies are present in the serum of children with INS. These autoantibodies cause glomerular endothelial cell damage, disrupting the endothelial barrier between circulating podocyte autoantibodies and podocytes. This is a key cause of glomerular endothelial cell damage. The multiple anti-endothelial autoantibodies discovered by the inventors, and their combinations, can be used to diagnose endothelial cell damage and, by extension, INS.

[0041] The present application proposes the use of a polypeptide or its antibody binding fragment that can form an antigen-antibody complex in contact with a biological sample obtained from the patient in the preparation of a vascular endothelial cell injury detection reagent or kit; wherein the polypeptide or its antibody binding fragment is selected from the following groups: α-enolase protein (ENO1), talin 1 (TLN1), filament protein-A (FLNA), desmosome nexin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane protrusion protein (MSN).

[0042] In some embodiments, any one of the nine polypeptides or antibody binding fragments can be used alone to detect vascular endothelial cell damage. In some embodiments, at least two of the nine polypeptides or antibody binding fragments can also be used in combination.

[0043] Furthermore, in some embodiments, when two of the above-mentioned 9 polypeptides or antibody binding fragments are used in combination, the sensitivity of the combination of talin 1 (TLN1) and desmoglein 1 (DSG1) is the highest, reaching 88.2; the sensitivity of the combination of filament protein A (FLNA) and desmoglein 1 (DSG1) is slightly lower, at 70.6, and the sensitivity of any two of the other 9 polypeptides or antibody binding fragments is between the two, which are not listed here. In addition, in some embodiments, when talin 1 (TLN1) and membrane protrusion protein (MSN) are used in combination, the specificity is the highest, reaching 85.7, and the specificity of any other two combinations is also above 57.1.

[0044] In some embodiments, when three of the above-mentioned nine polypeptides or antibody binding fragments are used in combination, the sensitivity of the combination of desmoglein 1 (DSG1), moesin (MSN) and talin 1 (TLN1), or the combination of moesin (MSN), myosin light chain 1 (MYL1) and talin 1 (TLN1) is relatively high, at 76.5; the sensitivity of the combination of cytoskeleton-associated protein 4 (CKAP4), desmoglein 1 (DSG1) and filamin A (FLNA) is slightly lower, at 64.7; when any three of the other nine polypeptides or antibody binding fragments are used in combination, the sensitivity is between the two, which are not listed here. In addition, in some embodiments, the combination of desmoglein 1 (DSG1), moesin (MSN) and talin 1 (TLN1), or the combination of moesin (MSN), myosin light chain 1 (MYL1) and talin 1 (TLN1) has the highest specificity, reaching 85.7, and the specificity of any other three combinations is also above 64.3.

[0045] In some embodiments, when a combination of at least four of the above nine polypeptides or antibody binding fragments is used, the combination basically includes the two-by-two or three-way combinations listed in this application and will not be listed here.

[0046] The present application further proposes the use of a polypeptide or antibody binding fragment capable of forming any antigen-antibody complex in contact with a biological sample obtained from the patient in the preparation of a reagent or kit for specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schönlein purpura, and Kawasaki disease; wherein the polypeptide or its antibody binding fragment includes at least two selected from the following groups: alpha enolase protein (ENO1), talin 1 (TLN1), filament protein-A (FLNA), desmosome nephrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane protrusion protein (MSN).

[0047] The present application further proposes a reagent or kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schönlein purpura, and Kawasaki disease, comprising: a polypeptide or an antibody-binding fragment thereof, which is capable of contacting with a biological sample obtained from the patient to form any antigen-antibody complex; wherein the polypeptide or antibody-binding fragment thereof includes at least two selected from the following groups: alpha enolase protein (ENO1), talin 1 (TLN1), filament protein-A (FLNA), desmosome nephrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane-spiked protein (MSN).

[0048] In some embodiments, the polypeptides or antibody-binding fragments described herein are expressed in vascular endothelial cells, and the titer of the antibody complex can indicate vascular endothelial cell damage. Furthermore, this indication of vascular endothelial cell damage can indicate nephrotic syndrome. In some embodiments, the nephrotic syndrome is idiopathic nephrotic syndrome. Furthermore, the idiopathic nephrotic syndrome is pediatric idiopathic nephrotic syndrome.

[0049] In some embodiments, when using one or more of the nine polypeptides or antibody-binding fragments thereof to detect vascular endothelial cell damage, a biological sample is first obtained from the patient. This biological sample may be serum, and the biological sample is obtained from the patient before immunotherapy. In some embodiments, the titer of the antigen-antibody complex in the biological sample can be measured to detect vascular endothelial cell damage and further to detect nephrotic syndrome.

[0050] This application will illustrate the invention through the following examples. A total of 149 INS patients were recruited. The control group was divided into four groups: one group consisted of 100 healthy children undergoing physical examinations (normal controls, NCs); and the other three groups consisted of 100 children each with other vasculitis-related diseases, such as Henoch-Schönlein purpura (HSP), Henoch-Schönlein purpura nephritis (HSPN), and Kawasaki disease (KD). Serum was collected from the children and stored at -20°C until use.

[0051] Example 1: Detection of serum endothelial autoantibodies in children with INS and control group

[0052] Protein microarrays were used to measure endothelial cell autoantibody titers in the serum of INS patients and control subjects. The protein microarrays were produced and used as described below:

[0053] Cell culture and protein extraction: HUVEC-derived cell line EAhy926 was purchased from the Cell Bank of the Chinese Academy of Sciences. Eahy926 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Eahy926 cells were then washed with sterile PBS, harvested, and lysed in 30 mm Tris-HCl, 8 M urea, 4% CHAPS, and protease inhibitor cocktail (#ab65621; Abcam, diluted 1:200) on ice. Next, the sample was centrifuged at 12,000 g for 30 minutes at 4 degrees Celsius. The supernatant was then collected for later use. The total protein concentration was determined using a bicinchoninic acid (BCA) protein concentration assay kit (Pierce TM , Thermo) determination.

[0054] Two-dimensional electrophoresis and immunoblotting: Two-dimensional electrophoresis was performed using a 2D electrophoresis system (etan TM IPGphor 3 TM , GE Healthcare) were subjected to two-dimensional electrophoresis of EAhy926 protein extracts. 25 μl of total protein (100 μg) was loaded into 13 cm, pH 3-10 IPG strips (GE Healthcare) for isoelectric focusing as the first-dimensional electrophoresis. The gel strips obtained from the first-dimensional electrophoresis were further separated by SDS electrophoresis in the second dimension. The separated proteins were electrophoretically transferred to PVDF membranes. The blocked cell membranes were incubated overnight at 4°C with 1:200 diluted serum, 20 of which were from patients with nephrotic syndrome and 10 from healthy controls. The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, followed by enhanced chemiluminescence detection reagents. The membranes were finally imaged using a Bio-Rad imager and analyzed using PD Quest software.

[0055] Briefly, autoantigens were labeled on a nitrocellulose membrane and blocked with a bovine serum albumin sealing solution. After being fixed in the wells of a polyvinyl chloride assay plate, serum was added to the plate. After washing, detection was performed using biotin-conjugated anti-human IgG. The array was cleaned and developed using a 5-bromo-4-chloro-3-indoleyl phosphate (BCIP) / nitro blue tetrazolium chloride (NBT) substrate and scanned at 2400 dpi using a flatbed scanner (Canscan 9000 Mark II, Canon).

[0056] like Figure 1A-Figure 1I As shown in Figure 2, the concentrations of nine endothelial cell autoantibodies in INS patients were higher than those in the control group. Figure 1J The results showed that in patients with INS, the positive rate of anti-ENO1 antibodies was the highest (51.01%), and the positive rates of other identified endothelial cell autoantibodies were as follows: talin-1 endothelial cell autoantibodies (anti-TLN1 antibodies), 50.34%; filamin-A endothelial cell autoantibodies (anti-FLNA antibodies), 48.32%; desmosome nexin (anti-AHNAK antibodies), 45.64%; desmoglein-1 endothelial cell autoantibodies (anti-FLNA antibodies), 48.32%; desmoglein-2 endothelial cell autoantibodies (anti-AHNAK antibodies), 45.64%; desmoglein-3 endothelial cell autoantibodies (anti-FLNA antibodies), 48.32%; desmoglein-4 endothelial cell autoantibodies (anti- Autoantibodies (anti-DSG1 antibodies), 42.95%; myosin light chain 1 vascular endothelial cell autoantibodies (anti-MYL1 antibodies), 42.28%; heat shock protein 90-β vascular endothelial cell autoantibodies (anti-HSP90AB1 antibodies), 40.27%; cytoskeleton-associated protein 4 vascular endothelial cell autoantibodies (anti-CKAP4 antibodies), 38.93%; moesin vascular endothelial cell autoantibodies (anti-MSN antibodies), 31.54%. Figure 1K As shown in Figure 2, only 11% of all INS patients were negative for all nine endothelial autoantibodies. In other words, 89% of INS patients in this study were positive for at least one endothelial autoantibody.

[0057] The inventors then performed receiver operating characteristic (ROC) analysis on the grayscale values ​​of endothelial cell autoantibodies in the serum of INS patients and control subjects, and calculated the area under the ROC curve (ROC-AUC). The results showed that all nine identified endothelial autoantibodies could easily distinguish INS patients from controls, with AUCs greater than 0.7 (Figure 2). Among them, the ROC curve for α-enolase endothelial cell autoantibodies (anti-ENO1 antibodies) showed the strongest discrimination between INS patients and controls (AUC of 0.806).

[0058] Example 2: Circulating endothelial autoantibodies are associated with the condition and coagulation function of INS patients

[0059] In vivo pathogenicity testing of endothelial cell autoantibodies: The inventors investigated the pathogenicity of anti-ENO1 antibodies by tail vein injection into BALB / c mice (n=5). To control for the short half-life of the injected isotype, 200 μg of each anti-ENO1 antibody was injected twice, 24 hours apart. The development of proteinuria was monitored every 24 hours for 7 days using metabolic cages. Five BALB / c mice were injected with isotype control IgG at the same dose as the control group. Kidneys were harvested on day 7. Tissue sections (5 μm thick) were prepared and histologically assessed for tissue morphology, damage, and fibrosis using hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), or Masson's trichrome staining. Electron microscopy analysis was performed according to standard procedures. Kidneys were fixed in 4% buffered paraformaldehyde, followed by 1% osmium hydroxide in 0.1 M sodium carbonate buffer, stained with 1% uranyl acetate, and embedded in epoxy resin. Ultrathin sections were cut and contrasted with uranyl acetate in methanol and then with lead citrate. Micrographs were generated using a transmission electron microscope.

[0060] In vitro pathogenicity testing of endothelial cell autoantibodies: The inventors tested the in vitro pathogenicity of endothelial cell autoantibodies using three comparative methods. In the first comparison, EAhy926 cells were treated with anti-ENO1 antibodies or rabbit isotype IgG for 2 hours, with untreated cells serving as blank controls. Eahy926 cell morphology was observed under a light microscope. Intracellular penile staining revealed the actin cytoskeleton. Apoptosis was detected using a commercially available kit (#556547, BD Biosciences). For comparison, EAhy926 cells were treated with rabbit serum or rabbit serum and anti-ENO1 antibodies. For comparison, anti-ENO1 antibody-positive serum from patients with INS was first mixed with the corresponding recombinant protein in a humidified incubator at 37°C for 30 minutes. EAhy926 cells were then treated with either endothelial cell autoantibody-positive serum from patients with INS or the serum-recombinant protein complex. The second two comparative methods examined post-treatment cell morphology, structural changes in the cytoskeletal protein F-actin, and levels of thrombomodulin in the culture supernatant.

[0061] Statistical Analysis: Normally distributed data are presented as mean ± standard deviation, while non-normally distributed data are presented as median and interquartile range. Paired comparisons were performed using paired t-tests or Wilcoxon matched-pairs signed-rank tests. Linear regression and Pearson's coefficients were used to characterize the correlations between endothelial cell autoantibodies and other variables. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 9.0 software.

[0062] Comparison of endothelial cell autoantibody levels in INS patients at different disease states (before and after remission) to confirm endothelial damage in INS patients. Figure 3A As shown in Table 1, all nine endothelial cell autoantibodies decreased significantly after remission. Hypercoagulation is often observed in INS patients. Therefore, the inventors conducted a correlation analysis between the titers of circulating endothelial cell autoantibodies and coagulation function-related laboratory test results in INS patients before remission. Figure 3B As shown in Figure 2, in patients with INS, seven vascular endothelial cell autoantibodies were significantly negatively correlated with serum albumin and INR, among which anti-CKAP4 antibodies had the best correlation with albumin (R=-0.42, p<0.001); Figure 3C As shown in Figure 2, anti-MYL1 antibodies were most correlated with INR (R = -0.30, p = 0.004). Figure 3D As shown in the results, 8 vascular endothelial cell autoantibodies were significantly positively correlated with the platelet count in patients with INS, among which anti-DSG1 antibody had the best correlation with the platelet count (R=0.37, p<0.001).

[0063] Table 1 Characteristics of the subjects

[0064]

[0065]

[0066] Example 3: Kidneys of INS patients show signs of glomerular endothelial damage

[0067] To confirm whether glomerular endothelial damage exists in patients with INS, the inventors performed electron microscopy focused on the endothelium in renal biopsy samples from 20 patients. Figure 4A- Figure 4G As shown in Figure 4A, a total of seven signs of endothelial injury were observed, including endothelial cell proliferation (Figure 4A), immune complex deposition (under the endothelium) (Figure 4B), endothelial cell swelling or vacuolar degeneration (Figure 4C), widening of the intercellular spaces between endothelial cells (Figure 4D), endothelial cell mitochondrial swelling (Figure 4E), honeycomb structure (Figure 4F), and loss of endothelial cell permeability (Figure 4C). Figure 4G In the present invention, endothelial cell swelling or vacuolar degeneration, mitochondrial swelling are the most common signs of glomerular endothelial injury, and the positive rate of each sign is 85% (eg Figure 4H It is worth noting that Figure 4I As shown, in addition to endothelial fenestration fusion, all 20 patients with INS also showed at least one other sign of glomerular endothelial injury. Immunofluorescence of human renal tissue also showed signs of endothelial injury, including upregulation of caveolin-1 expression.

[0068] Example 4: Effect of anti-ENO1 antibodies on EAhy926 cells

[0069] In order to determine whether endothelial cell autoantibodies have a pathogenic effect, the inventors tested the effect of anti-ENO1 antibodies on EAhy926 cells, because the anti-ENO1 antibody in the present invention has the highest positive rate in INS patients. Figures 5A-5D As shown, EAhy926 cells treated with anti-ENO1 antibody had a higher detachment rate than cells treated with isotype control antibody, and cytoskeletal disruption was manifested by the disappearance of actin-stress fibers rather than higher levels of thrombomodulin. Figure 5E As shown in Figure 3, the level of thrombomodulin in the supernatant of EAhy926 cells treated with normal rabbit serum and anti-ENO1 antibody was significantly higher than that in normal rabbit serum and isotype control antibody. Figure 5F As shown, the concentration of thrombomodulin in the cell culture supernatant was significantly lower in the presence of anti-ENO1 antibody plus serum treated with ENO1 recombinant protein than in the absence of serum. This antibody-induced damage was enhanced in the presence of serum and attenuated by preincubating the antibody with the corresponding recombinant protein.

[0070] Example 5: Injection of identified endothelial autoantibodies can cause endothelial cell and podocyte damage in BALB / c mice

[0071] like Figure 6 As shown in Figure 2, BALB / c mice were injected with anti-ENO1 antibodies. HE staining, Masson staining, and PAS staining showed no obvious pathological changes in the kidneys of the antibody-injected mice. However, autophagosomes were observed in the glomerular endothelial cells of the mice, indicating that the recognized vascular endothelial autoantibodies may cause damage to the glomerular endothelial cells (e.g., Figure 7 A- Figure 7 D). Figure 7 A- Figure 7 As shown in Figure 2 (D), in addition to the autophagosomes observed in glomerular endothelial cells, podocytes also showed signs of damage, with both SEM and TEM showing partial fusion of foot processes; in addition, some mitochondrial changes were also observed, including irregular shape, vacuolization, and cristae fragmentation.

[0072] Example 6: Diagnostic efficiency of endothelial autoantibodies in serum of INS patients for endothelial cell damage

[0073] The applicant immobilized the potential autoantigen proteins identified by screening onto a protein chip. Serum from patients with nephrotic syndrome was used as the primary antibody. After incubation with a secondary antibody, enzyme working solution, and substrate solution, the chip was read using a scanner to obtain the grayscale value corresponding to each antigen protein on the protein chip. The grayscale value represents the relative concentration of autoantibodies in the serum. INS patients were divided into an endothelial cell injury group and a non-endothelial cell injury group based on the presence or absence of endothelial cell damage under electron microscopy in renal tissue sections. Receiver-operating characteristic (ROC) analysis was performed based on the serum endothelial cell autoantibody concentration to obtain area under the curve (AUC), sensitivity, and specificity.

[0074] like Figures 8A-8Q As shown in Table 2, the inventors performed ROC analysis on the grayscale values ​​of vascular endothelial cell autoantibodies in the serum of INS patients and control subjects, and calculated the area under the ROC curve (ROC-auc). Figures 8A-8Q As shown, all nine identified peptides, whether used alone or in combination of two or more, can easily diagnose INS patients, with an AUC greater than 0.5. Among them, the ROC curves of the combination of desmoglein 1 + moesin + talin 1, α-enolase + talin 1 + heat shock protein 90β, moesin + myosin light chain 1 + talin 1, or talin 1 + desmoglein 1 showed higher diagnostic efficiency among INS patients, with the highest AUC being 0.811.

[0075] Table 2

[0076]

[0077] The above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

1. Use of a polypeptide or antibody-binding fragment thereof that can form an antigen-antibody complex in contact with a biological sample obtained from a patient in the preparation of a reagent or kit for detecting vascular endothelial cell damage; wherein the polypeptide or antibody binding fragment thereof is a combination of talin 1 (TLN1) and moesin (MSN); wherein the vascular endothelial cell damage is indicative of childhood idiopathic nephrotic syndrome; Wherein the biological sample is serum.

2. The use according to claim 1, wherein the biological sample is a biological sample of a patient before immunotherapy.

3. Use of a polypeptide or antibody-binding fragment thereof that is capable of forming any antigen-antibody complex in contact with a biological sample obtained from a patient in the preparation of a reagent or kit for specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or Kawasaki disease; wherein the polypeptide or antibody binding fragment thereof is a combination of talin 1 (TLN1) and moesin (MSN); wherein the nephrotic syndrome is idiopathic nephrotic syndrome in children; Wherein the biological sample is serum.

4. A reagent or kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or Kawasaki disease, comprising: A polypeptide or an antibody-binding fragment thereof that is capable of forming any antigen-antibody complex in contact with a biological sample obtained from a patient; wherein the polypeptide or antibody binding fragment thereof is a combination of talin 1 (TLN1) and moesin (MSN); wherein the nephrotic syndrome is idiopathic nephrotic syndrome in children; Wherein the biological sample is serum.

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