Use of polypeptides or fragments thereof in the preparation of a kit for detecting vascular endothelial cell damage

Abstract

The present application relates to the use of a polypeptide or antibody binding fragment thereof capable of forming an antigen-antibody complex with a biological sample obtained from the patient in the preparation of a reagent or kit for detecting vascular endothelial cell damage; wherein the polypeptide or antibody binding fragment thereof comprises at least two selected from the following group: alpha enolase protein (ENO1), ankle protein 1 (TLN1), filamin-A (FLNA), desmosomal linker protein (AHNAK), desmosomal core glycoprotein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90 beta (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), membrane spike protein (MSN). The vascular endothelial cell autoantibody of the present application can be used to diagnose vascular endothelial cell damage by simply detecting the titer in serum, which has very great clinical application value.

Description

[0001] This application is a divisional application of the application number 202310216552.5, the application date is March 2, 2023, and the invention name is the application of polypeptide or its fragment in the preparation of a kit for detecting vascular endothelial cell damage. TECHNICAL FIELD

[0002] The present application relates to the field of diagnostic kits, in particular to the application of polypeptide or its fragment in the preparation of a kit for detecting vascular endothelial cell damage. BACKGROUND

[0003] Idiopathic nephrotic syndrome (INS) is one of the most common primary glomerular diseases in children, with about 28,000-56,000 new cases per year in China. Steroid treatment is effective for most patients, but about 10% to 20% of patients do not respond to steroid treatment, which is called steroid-resistant nephrotic syndrome (SRNS). Among INS patients, 8% to 35% of patients develop end-stage renal disease (ESRD) 5 years after diagnosis, and 24% to 66% of patients develop end-stage renal disease 15 years after diagnosis. However, to date, the pathogenesis of INS has not been fully elucidated.

[0004] Minimal change disease (MCD) is the main cause of nephrotic syndrome in children, accounting for 10-15% of nephrotic syndrome in adults. The glomeruli of patients with minimal change disease appear essentially normal under light microscopy, and the only histopathological abnormality visible under electron microscopy is diffuse foot process effacement of podocytes. Therefore, a prevailing 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 for 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 theories do not help all patients achieve clinical remission. Therefore, researchers have been exploring other possible pathological mechanisms. SUMMARY

[0005] To solve the technical problems in the prior art, the present application provides a use of a polypeptide or an antibody binding fragment thereof capable of forming an antigen-antibody complex with a biological sample obtained from the patient in the preparation of a reagent or kit for detecting vascular endothelial cell damage; wherein the polypeptide or the antibody binding fragment thereof comprises at least two selected from the following group: alpha enolase protein (ENO1), ankle protein 1 (TLN1), filamin-A (FLNA), desmosomal ankyrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane spike protein (MSN).

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

[0007] In some embodiments, the vascular endothelial cell damage indicates nephrotic syndrome.

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

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

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

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

[0012] In some embodiments, the present application also provides a use of a polypeptide or an antibody binding fragment thereof capable of forming an antigen-antibody complex with a biological sample obtained from the patient in the preparation of a reagent or kit for detecting vascular endothelial cell damage; wherein the polypeptide or the antibody binding fragment thereof comprises at least three selected from the following group: alpha enolase protein (ENO1), ankle protein 1 (TLN1), filamin-A (FLNA), desmosomal ankyrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90β (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane spike protein (MSN).

[0013] In some embodiments, wherein 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 90 beta (HSP90AB1).

[0014] In some embodiments, the present application also provides a use of a polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex with a biological sample obtained from the patient in the manufacture of a reagent or kit for specifically detecting nephrotic syndrome relative to purpura nephritis, allergic purpura, Kawasaki disease; wherein the polypeptide or antibody binding fragment thereof comprises at least two selected from the following group: alpha enolase protein (ENO1), talin 1 (TLN1), filamin-A (FLNA), AHNAK, desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90 beta (HSP90AB1), CKAP4, moesin (MSN).

[0015] In some embodiments, the present application also provides a reagent or kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, allergic purpura, Kawasaki disease, comprising: a polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex with a biological sample obtained from the patient; wherein the polypeptide or antibody binding fragment thereof comprises at least two selected from the following group: alpha enolase protein (ENO1), talin 1 (TLN1), filamin-A (FLNA), AHNAK, desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90 beta (HSP90AB1), CKAP4, moesin (MSN).

[0016] The gold standard for vascular endothelial cell damage is electron microscopy. However, electron microscopy requires kidney biopsy, is invasive and expensive, and has a long detection time, which is not suitable for routine application. The vascular endothelial cell autoantibody of the present application can be used for diagnosing vascular endothelial cell damage by simply detecting the titer in serum, which has great clinical application value. BRIEF DESCRIPTION OF DRAWINGS

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

[0018] FIG. 1 is an endothelial cell autoantibody detection according to an embodiment of the present application; wherein Figures 1A-1IDetection of endothelial cell autoantibodies in INS patients and other control participants according to an embodiment of the present application; NC represents normal control; HSP represents Henoch-Schonlein purpura; HSPN represents Henoch-Schonlein purpura nephritis; KD represents Kawasaki disease; NS represents nephrotic syndrome; Figures 1J-1K Variation of endothelial cell autoantibodies in INS patients according to an embodiment of the present application;

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

[0020] Figure 3 is correlation between gray value of vascular endothelial cell autoantibody and clinical condition of INS patients according to an embodiment of the present application; wherein, Figure 3A 9 vascular endothelial cell autoantibodies are significantly reduced after remission compared with before remission according to an embodiment of the present application; Figure 3B 7 vascular endothelial cell autoantibodies in INS patients before remission are significantly negatively correlated with serum albumin according to an embodiment of the present application; Figure 3C 8 vascular endothelial cell autoantibodies are significantly negatively correlated with international normalized ratio (INR) before remission of INS patients according to an embodiment of the present application; Figure 3D 7 vascular endothelial cell autoantibodies are significantly negatively correlated with platelet count before remission of INS patients according to an embodiment of the present application;

[0021] Figure 4 is kidney endothelial injury in INS patients according to an embodiment of the present application; wherein, Figure 4A- Figure 4G 7 typical signs of endothelial injury in kidney biopsy specimens of INS patients observed under electron microscope according to an embodiment of the present application; Figure 4HFigure 2 is a pattern of different signs of endothelial injury in 20 INS patients according to one embodiment of the present application; Figure 4I Figure 3 is an immunofluorescence of human kidney showing up-regulation of caveolin-1 (green) co-localized with EHD3 (red) according to one embodiment of the present application;

[0022] Figure 5 is the effect of anti-ENOl antibody in vitro according to one embodiment of the present application; wherein, Figures 5A-5C Figure 6 is the actin cytoskeleton changes and isolation of Eahy926 cells after pre-treatment of ENOl protein with isotype control antibody, anti-ENOl antibody, isotype control antibody and normal rabbit serum, anti-ENOl antibody and normal rabbit serum, anti-ENOl antibody positive serum and anti-ENOl antibody positive serum according to one embodiment of the present application; Figures 5D-5F Figure 7 is the difference in thrombomodulin levels in supernatant of Eahy926 cells treated with isotype control antibody, anti-ENOl antibody, isotype control antibody and normal rabbit serum, anti-ENOl antibody and normal rabbit serum, anti-ENOl antibody serum and anti-ENOl serum pre-treated with ENOl protein according to one embodiment of the present application;

[0023] Figure 6 Figure 8 is the histology of kidney of mice injected with antibody (HE staining, PAS staining and Masson staining) according to one embodiment of the present application;

[0024] Figure 7 Figure 9 is the damage of endothelial cells and podocytes of mice injected with anti-ENOl antibody under electron microscope according to one embodiment of the present application; wherein, Figure 7 A is autophagosome observed in glomerular endothelial cells of mice injected with anti-ENOl antibody according to one embodiment of the present application; Figure 7 B is mitochondrial damage observed in podocytes of mice treated with anti-ENOl antibody according to one embodiment of the present application; Figure 7 C- Figure 7 D is the fusion of podocyte foot processes observed in podocytes of mice injected with anti-ENOl antibody, but not observed in mice injected with isotype antibody according to one embodiment of the present application; and

[0025] Figure 8 is a sensitivity and specificity map when one or several antibodies are used in combination according to one embodiment of the present application; wherein Figures 8A-8I is a sensitivity and specificity map when the antibodies are used individually; Figures 8J-8M is a sensitivity and specificity map when two antibodies are used in combination; Figures 8N-8Q is a sensitivity and specificity map when three antibodies are used in combination. DETAILED DESCRIPTION

[0026] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0027] In the following detailed description, reference will be made to the accompanying drawings, which form a part of this description. In the drawings, similar symbols in different drawings represent similar components. Various specific embodiments of the present application will be described in sufficient detail to enable one of ordinary skill in the art to make and use the technical solutions of the present application. It is to be understood that other embodiments can be utilized and structural, logical, and electrical changes can be made without departing from the scope of the present application.

[0028] The terms appearing herein have the following meanings:

[0029] The term "nephrotic syndrome" or "NS" as used herein refers to a clinical syndrome caused by increased permeability of the glomerular filtration membrane to plasma proteins and massive loss of plasma proteins from the urine, resulting in a series of pathophysiological changes, with massive proteinuria, hypoproteinemia, hyperlipidemia and edema as the main clinical features.

[0030] The term "idiopathic nephrotic syndrome" or "INS" as used herein refers to a group of diseases of unknown etiology caused by increased permeability of the glomerular filtration membrane, resulting in increased filtration of plasma proteins, leading to massive proteinuria and a series of clinical syndromes characterized by podocyte lesions. Children with this type of disease often show symptoms such as massive proteinuria, hypoproteinemia, edema, hyperlipidemia, etc.

[0031] The term "polypeptide or fragment thereof" as used herein refers to a combination of two or more polypeptide indicators for detecting the same disease. The polypeptide or antibody binding fragment thereof is selected from the group consisting of alpha enolase protein (ENO1), ankle protein 1 (TLN1), filamin-A (FLNA), desmosomal ankyrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90 beta (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), and membrane spike protein (MSN).

[0032] The sequence of the antibody binding fragment of alpha enolase protein (ENO1) is shown as SEQ ID NO. 1; the sequence of the antibody binding fragment of ankle protein 1 (TLN1) is shown as SEQ ID NO. 2; the sequence of the antibody binding fragment of filamin-A (FLNA) is shown as SEQ ID NO. 3; the sequence of the antibody binding fragment of desmoglein (AHNAK) is shown as SEQ ID NO. 4; the sequence of the antibody binding fragment of desmoglein-1 (DSG1) is shown as SEQ ID NO. 5; the sequence of the antibody binding fragment of myosin light chain 1 (MYL1) is shown as SEQ ID NO. 6; the sequence of the antibody binding fragment of heat shock protein 90 beta (HSP90AB1) is shown as SEQ ID NO. 7; the sequence of the antibody binding fragment of cytoskeleton-associated protein 4 (CKAP4) is shown as SEQ ID NO. 8; and the sequence of the antibody binding fragment of moesin (MSN) is shown as SEQ ID NO. 9.

[0033] When the polypeptide or the antibody binding fragment thereof described above 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" as used herein refers to an IgG antibody extracted from the serum of a rabbit of the same breed and age without protein immunization, which is used as a control group.

[0035] The "HE staining" as used herein refers to hematoxylin-eosin staining, which is one of the commonly used staining methods in paraffin section technology. Hematoxylin staining solution is alkaline, which mainly makes the chromatin in the nucleus and the nucleic acid in the cytoplasm purple blue; eosin is an acid dye, which mainly makes the components in the cytoplasm and extracellular matrix red.

[0036] Masson staining, also known as Masson's staining, is one of the main methods for showing fibers in tissues, and is a classic and authoritative technique for staining collagen fibers. The staining principle of this method is related to the size of the anionic dye molecule and the permeability of the tissue. The size of the molecule is reflected by the molecular weight, and small molecular weight can easily penetrate the structure of dense and low permeability tissue; while large molecular weight can only enter the structure of loose and high permeability tissue. However, the molecular weight of light green or aniline blue is very large, so after Masson staining, muscle fibers appear red, collagen fibers appear green or blue, and it is mainly used to distinguish collagen fibers and muscle fibers.

[0037] PAS staining, also known as Periodic Acid-Schiff stain, is mainly used to detect sugars in tissues in histology. Periodic acid oxidizes the hydroxyl groups on the adjacent two carbons of the sugar to aldehyde groups, and then reacts with the aldehyde groups using Schiff's reagent to produce a purple red color. PAS positive is red, and the cell nucleus is blue.

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

[0039] Podocyte autoantibodies are considered an important cause of glomerular podocyte damage. However, from an anatomical perspective, there is a glomerular endothelial barrier between circulating podocyte autoantibodies and podocytes. Unless the integrity of the glomerular endothelial cells is damaged, podocyte autoantibodies cannot reach the podocytes.

[0040] The inventor found through a series of experiments that there are multiple anti-vascular endothelial cell autoantibodies in the serum of children with INS. Anti-vascular endothelial cell autoantibodies cause damage to glomerular endothelial cells, thereby causing damage to the endothelial barrier between circulating podocyte autoantibodies and podocytes. This is an important cause of glomerular endothelial cell damage. The multiple anti-vascular endothelial cell autoantibodies and their combinations found by the inventor can be used to diagnose endothelial cell damage and further diagnose INS.

[0041] The present application provides the use of polypeptides or antibody binding fragments thereof capable of forming antigen-antibody complexes with biological samples obtained from the patient in the preparation of a reagent or kit for detecting vascular endothelial cell damage; wherein the polypeptides or antibody binding fragments thereof are selected from the following group: alpha enolase protein (ENO1), ankle protein 1 (TLN1), filamin-A (FLNA), desmosomal ankyrin (AHNAK), desmoglein-1 (DSG1), myosin light chain 1 (MYL1), heat shock protein 90 beta (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), membrane spike protein (MSN).

[0042] In some embodiments, any one of the above 9 polypeptides or antibody binding fragments can be used alone to detect vascular endothelial cell damage. In some embodiments, at least two of the above 9 polypeptides or antibody binding fragments can also be used in combination.

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

[0044] In some embodiments, when three of the above 9 polypeptides or antibody binding fragments are used in combination, desmoglein-1 (DSG1), membrane spike protein (MSN), and ankle protein 1 (TLN1) are used in combination, or membrane spike protein (MSN), myosin light chain 1 (MYL1), and ankle protein 1 (TLN1) are used in combination, the sensitivity is relatively high, reaching 76.5; cytoskeleton-associated protein 4 (CKAP4), desmoglein-1 (DSG1), and filamin-A (FLNA) are used in combination, the sensitivity is slightly lower, reaching 64.7; the sensitivity of any other combination of the 9 polypeptides or antibody binding fragments is between the two, which will not be listed here. In addition, in some embodiments, desmoglein-1 (DSG1), membrane spike protein (MSN), and ankle protein 1 (TLN1) are used in combination, or membrane spike protein (MSN), myosin light chain 1 (MYL1), and ankle protein 1 (TLN1) are used in combination, the specificity is the highest, reaching 85.7, and the specificity of any other combination is also above 64.3.

[0045] In some embodiments, when at least four of the above-mentioned nine polypeptides or antibody binding fragments are used in combination, the combination thereof substantially comprises all of the two-by-two combinations or three-by-three combinations listed in the present application, which are not listed here.

[0046] The present application further provides use of a polypeptide or antibody binding fragment capable of forming any antigen-antibody complex with a biological sample obtained from the patient in the manufacture of a reagent or kit for specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, Kawasaki disease; wherein the polypeptide or antibody binding fragment thereof comprises at least two selected from the group consisting of alpha-enolase protein (ENOl), ankle protein 1 (TLN1), filamin-A (FLNA), desmoglein associated protein (AHNAK), desmoglein core glycoprotein-1 (DSGl), myosin light chain 1 (MYL1), heat shock protein 90 beta (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), membrane spur protein (MSN).

[0047] The present application further provides a reagent or kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, Kawasaki disease, comprising: a polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex with a biological sample obtained from the patient; wherein the polypeptide or antibody binding fragment thereof comprises at least two selected from the group consisting of alpha-enolase protein (ENOl), ankle protein 1 (TLN1), filamin-A (FLNA), desmoglein associated protein (AHNAK), desmoglein core glycoprotein-1 (DSGl), myosin light chain 1 (MYL1), heat shock protein 90 beta (HSP90AB1), cytoskeleton-associated protein 4 (CKAP4), membrane spur protein (MSN).

[0048] In some embodiments, the above-mentioned polypeptide or antibody binding fragment expressed in the vascular endothelial cells, the titer of the antibody complex thereof can indicate vascular endothelial cell damage. Further, the indication of vascular endothelial cell damage can indicate nephrotic syndrome. In some embodiments, the nephrotic syndrome is idiopathic nephrotic syndrome. Further, the idiopathic nephrotic syndrome is idiopathic nephrotic syndrome in children.

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

[0050] The application will be illustrated by the following examples. In the present application, 149 INS patients were recruited, and the control group was divided into 4 groups: 1 group was 100 healthy children (normal control, NC) who underwent physical examination, and the other 3 groups were 100 children with other diseases related to vasculitis, such as allergic purpura (HSP), allergic purpura nephritis (HSPN), Kawasaki disease (KD), etc. The serum of children was collected and stored at -20℃ until use.

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

[0052] The titer of vascular endothelial cell autoantibodies in the serum of INS patients and control participants was detected by protein chip. The production and use of protein chip followed the description as follows:

[0053] Cell culture and protein extraction: huvec-derived cell strain EAhy926 was purchased from the Chinese Academy of Sciences Cell Bank. Eahy926 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Then the Eahy926 cells were washed with sterile PBS, harvested, and lysed on ice in a buffer containing 30 mm Tris-HCl, 8 M urea, 4% CHAPS and protease inhibitor cocktail (#ab65621; Abeam, diluted 1:200). Next, the samples were centrifuged at 4 degrees Celsius at 12000g for 30 minutes. Then the supernatant was collected for use. The total protein concentration was determined using the bicinchoninic acid (BCA) protein concentration assay kit (Pierce TM , Thermo).

[0054] Two-dimensional electrophoresis and immunoblotting: two-dimensional electrophoresis system (etan TM IPGphor3 TM , GE Healthcare) was used for two-dimensional electrophoresis of EAhy926 protein extract according to the manufacturer's instructions. 25 μl of total protein (100 μg) was loaded into 13 cm, pH 3-10 IPG strips (GE Healthcare) for isoelectric focusing as a one-dimensional electrophoresis. The gel strip obtained from one-dimensional electrophoresis was further separated by sds electrophoresis in two dimensions. The separated proteins were electrophoretically transferred to a PVDF membrane. The blocked cell membrane was incubated with 1:200 diluted serum overnight at 4℃, including 20 cases from nephrotic syndrome patients and 10 cases from healthy controls. Incubated with horseradish peroxidase (HRP) conjugated secondary antibody, then incubated with enhanced chemiluminescence detection reagent. Finally, the membrane was imaged using a Bio-Rad imager, and analyzed using PD Quest software.

[0055] Briefly, autoantigens were blotted on nitrocellulose membranes and blocked with a solution of bovine serum albumin. After immobilization in the wells of a polyvinyl chloride assay plate, serum was added to the plate. After washing, a biotin-conjugated anti-human IgG was used for detection. The array was washed and developed using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) / nitro blue tetrazolium chloride (NBT) substrate and scanned using a flatbed scanner (canScan 9000 Mark II, Canon) at 2400 dpi.

[0056] As shown in Figures 1A-1I , the concentrations of the 9 autoantibodies against vascular endothelial cells in INS patients were higher than those in the control group. As shown in Figure 1J , the positive rate of anti-ENO1 antibody was the highest (51.01%) in INS patients, and the positive rates of other identified autoantibodies against vascular endothelial cells were as follows: anti-TLN1 antibody, 50.34%; anti-FLNA antibody, 48.32%; anti-AHNAK antibody, 45.64%; anti-DSG1 antibody, 42.95%; anti-MYL1 antibody, 42.28%; anti-HSP90AB1 antibody, 40.27%; anti-CKAP4 antibody, 38.93%; and anti-MSN antibody, 31.54%. Figure 1K As shown in , only 11% of the 9 autoantibodies against vascular endothelial cells were negative in all INS patients. In other words, 89% of the INS patients in this study had at least one positive autoantibody against vascular endothelial cells.

[0057] Then, the inventors performed ROC analysis on the gray values of autoantibodies against vascular endothelial cells in the serum of INS patients and control subjects, and calculated the area under the ROC curve (ROC-auc). The results showed that all 9 identified autoantibodies against vascular endothelial cells could easily distinguish INS patients from controls, with an AUC greater than 0.7 (Figure 2). Among them, the ROC curve of anti-ENO1 antibody had the strongest distinction between INS patients and controls (AUC of 0.806).

[0058] Example 2: Circulating autoantibodies against vascular endothelial cells are related to the condition and coagulation function of INS patients

[0059] In vivo pathogenicity test of vascular endothelial cell autoantibodies: The inventors investigated the pathogenicity of anti-ENOl antibodies by injecting anti-ENOl antibodies into BALB / c mice (n=5) via the tail vein. To control the short half-life of the injected isotype antibodies, 200 pg of each antibody was injected twice at 24-hour intervals. Proteinuria development was monitored every 24 hours using metabolic cages for 7 days. Five BALB / c mice were injected with isotype control IgG at the same dose as the control group. Kidneys were collected on day 7. Tissue sections (5 pm thick) were prepared for histological evaluation of tissue morphology, injury, and fibrosis using hematoxylin and eosin (H&E), periodic acid-Schiff staining (PAS), or Masson’s trichrome staining. Electron microscopy analysis was performed according to standard procedures. Kidneys were fixed in 4% buffered paraformaldehyde, followed by the addition of 1% osmium tetroxide in 0.1 M sodium carbonate buffer, then stained with 1% uranyl acetate and embedded in epoxy resin. Ultrathin sections were cut and counterstained with uranyl acetate in methanol, followed by lead citrate counterstaining. Micrographs were generated using a transmission electron microscope.

[0060] In vitro pathogenicity test of vascular endothelial cell autoantibodies: The inventors investigated the in vitro pathogenicity of vascular endothelial cell autoantibodies by three comparative approaches. The first comparison was to treat EAhy926 cells with anti-ENOl antibodies or rabbit isotype IgG for 2 hours, with untreated cells as a blank control. Cell morphology was observed under light microscopy. Intracellular phalloidin staining visualized the actin cytoskeleton. Apoptosis detection was performed using a commercially available kit (#556547, BD Biosciences). For comparison, EAhy926 cells were treated with rabbit serum or rabbit serum and anti-ENOl antibodies, respectively. For comparison, INS patient anti-ENOl antibody-positive serum was first mixed with the corresponding recombinant protein for 30 minutes at 37°C in a humidified incubator. EAhy926 cells were then treated with INS patient vascular endothelial cell autoantibody-positive serum or serum-recombinant protein complexes. The latter two comparative approaches detected changes in cell morphology, cytoskeletal protein F-actin structure, and thrombomodulin levels in the culture medium supernatant after treatment.

[0061] Statistical analysis: Measurement data with normal distribution were expressed as mean ± standard deviation, and measurement data without normal distribution were expressed as median and interquartile range. For paired comparisons, we used the paired t-test or Wilcoxon paired signed-rank test. Linear regression and Pearson’s coefficient were used to characterize the correlation between vascular endothelial cell autoantibodies and other variables. P<0.05 was defined as statistically significant. All statistical analyses were performed using GraphPad Prism version 9.0 software.

[0062] To confirm the endothelial damage in INS patients, the levels of vascular endothelial cell autoantibodies were compared between pre-remission and post-remission INS patients. As shown in Figure 3A Table 1, all 9 vascular endothelial cell autoantibodies were significantly decreased in post-remission INS patients. Hypercoagulability is often observed in INS patients. Therefore, the inventors performed correlation analysis between the titers of circulating vascular endothelial autoantibodies and coagulation laboratory test results in pre-remission INS patients. As shown in Figure 3B Table 2, 7 of the 9 vascular endothelial cell autoantibodies were significantly negatively correlated with serum albumin and INR in INS patients, with the anti-CKAP4 antibody showing the best correlation with albumin (R = -0.42, p < 0.001); as shown in Figure 3C Table 3, the anti-MYL1 antibody showed the best correlation with INR (R = -0.30, p = 0.004). In addition, as shown in Figure 3D Table 4, 8 of the 9 vascular endothelial cell autoantibodies were significantly positively correlated with platelet counts in INS patients, with the anti-DSG1 antibody showing the best correlation with platelet counts (R = 0.37, p < 0.001).

[0063] Table 1. Subject characteristics

[0064]

[0065]

[0066] Example 3: Renal glomerular endothelial damage in INS patients

[0067] To confirm whether glomerular endothelial damage exists in INS patients, the inventors performed focused endothelial electron microscopy on renal biopsy samples from 20 patients. As shown in Figure 4A- Figure 4G Table 5, a total of 7 endothelial damage signs were observed, including endothelial cell proliferation (Figure 4A), immune complex deposition (subendothelial) (Figure 4B), endothelial cell swelling or vacuolar degeneration (Figure 4C), widening of the endothelial cell gap (Figure 4D), endothelial cell mitochondrial swelling (Figure 4E), honeycomb structure (Figure 4F), and loss of endothelial cell permeability Figure 4G In the present invention, endothelial cell swelling or vacuolar degeneration and mitochondrial swelling were the most common signs of glomerular endothelial damage, with a positive rate of 85% for each (as shown in Figure 4H Table 5). Notably, as shown in Figure 4I Table 5, all 20 INS patients exhibited at least one other sign of glomerular endothelial damage in addition to endothelial fenestrations. Human kidney tissue immunofluorescence also showed signs of endothelial damage, with up-regulated expression of microcyst protein-1 (Caveolin-1).

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

[0069] To determine whether the vascular endothelial cell autoantibody patient has a pathogenic effect, the inventors examined the effect of anti-ENOl antibodies on EAhy926 cells, as anti-ENOl antibodies had the highest positive rate in INS patients in the present application. As shown in Figures 5A-5D , the detachment rate of EAhy926 cells treated with anti-ENOl antibodies was higher than that of cells treated with isotype control antibodies, and cytoskeletal damage was manifested as the disappearance of actin-stress fibers, rather than higher levels of thrombomodulin. As shown in Figure 5E , the level of thrombomodulin in the supernatant of EAhy926 cells treated with normal rabbit serum and anti-ENOl antibodies was significantly higher than that of normal rabbit serum and isotype control antibodies. As shown in Figure 5F , the concentration of thrombomodulin in the cell culture supernatant was significantly lower in anti-ENOl antibody+ serum treated with ENOl recombinant protein than in untreated serum. This damage caused by the antibody can be enhanced in the presence of serum, and can be attenuated by preincubating the antibody with the corresponding recombinant protein.

[0070] Example 5: Injection of identified vascular endothelial autoantibodies can cause damage to endothelial cells and podocytes in BALB / c mice

[0071] As shown in Figure 6 , anti-ENOl antibodies were injected into BALB / c mice. No obvious pathological changes were observed in the kidneys of antibody-injected mice by HE staining, Masson staining, and PAS staining. However, autophagosomes were observed in the glomerular endothelial cells of the mice, indicating that the identified vascular endothelial autoantibodies can cause damage to the glomerular endothelial cells (as shown in Figure 7 A- Figure 7 D). As shown in Figure 7 A- Figure 7 D, in addition to the observation of autophagosomes in the glomerular endothelial cells, there were also signs of damage to the podocytes, with partial fusion of the foot processes shown by SEM and TEM; in addition, some changes in mitochondria were also observed, including irregular shape, vacuolization, and ridge rupture.

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

[0073] The applicant immobilized the screened potential autoantigen proteins onto a protein chip. Using serum from patients with nephrotic syndrome (INS) as the primary antibody, after incubation with secondary antibody, enzyme working solution, and substrate solution, the grayscale value corresponding to each antigen protein on the protein chip was obtained using a scanner. The grayscale value represents the relative content of the autoantibody in the serum. Based on the presence or absence of endothelial cell damage in kidney tissue sections from INS patients under electron microscopy, INS patients were divided into an endothelial cell damage group and an endothelial cell non-damage group. ROC analysis was performed based on the concentration of endothelial cell autoantibodies in the serum to obtain the AUC value, sensitivity, and specificity.

[0074] like Figures 8A-8Q As shown in Table 2, the inventors performed ROC analysis on the gray 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). The results are as follows. 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 combinations of desmosome core glycoprotein 1 + membrane spike protein + ankle protein 1, α-enolase + ankle protein 1 + heat shock protein 90β, membrane spike protein + myosin light chain 1 + ankle protein 1, or ankle protein 1 + desmosome core glycoprotein 1 show higher diagnostic efficiency among INS patients, with the highest AUC being 0.811.

[0075] Table 2

[0076]

[0077] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. Use of a reagent for detecting HSP90AB1 autoantibody and a reagent for detecting CKAP4 autoantibody in the preparation of a kit for detecting vascular endothelial cell damage; wherein the vascular endothelial cell damage indicates idiopathic nephrotic syndrome in children.

2. Use of a reagent for detecting HSP90AB1 autoantibody and a reagent for detecting CKAP4 autoantibody in the preparation of a kit for distinguishing nephrotic syndrome from purpura nephritis, Henoch-Schonlein purpura, Kawasaki disease; wherein the nephrotic syndrome is idiopathic nephrotic syndrome in children.

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