Application of ADAM10 or ADAM17 in preparation of medicine for preventing / treating membranous nephropathy

Abstract

The invention discloses application of ADAM10 or ADAM17 serving as a target spot in preparation of a medicine for preventing / treating membranous nephropathy and application of an ADAM10 or ADAM17 inhibitor in preparation of the medicine for preventing / treating membranous nephropathy. The invention finds that ADAMs mediate the deposition process of immune complexes of autoantigens such as PLA2R1 of membranous nephropathy, and enzyme digestion of podocytes to transmembrane autoantigen proteins such as PLA2R can be inhibited by performing targeted intervention on the activity of ADAMs, especially the activity of ADAM10 and ADAM17, so that immune complexes deposited in glomerulus are reduced, the podocyte damage of membranous nephropathy is improved, and the therapeutic effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology. More specifically, the present invention relates to the use of ADAM10 or ADAM17 as a target for the prevention / treatment of membranous nephropathy and the use of an inhibitor of ADAM10 or ADAM17 in the preparation of a drug for the prevention / treatment of membranous nephropathy. Background Art

[0002] Membranous nephropathy is a common glomerular disease. It is a kidney disease caused by autoantibodies targeting glomerular podocytes. Its glomerular pathological damage is characterized by the deposition of a large number of immune complexes on the basement membrane under the glomerular podocytes, accompanied by extensive thickening of the basement membrane. PLA2R and other antigens are the main antigens that mediate the autoimmune response of membranous nephropathy. They are expressed on podocytes, thus mediating autoantibody-targeted podocyte damage. Immunosuppressive therapy is often used clinically to treat membranous nephropathy, the purpose of which is to inhibit the autoimmune response and reduce the level of autoantibodies represented by anti-PLA2R antibodies, thereby delaying podocyte damage.

[0003] Existing studies have shown that disintegrin-metalloproteinases play a key role in kidney disease, but the expression level and mechanism of action of ADAM10 or ADAM17 in patients with membranous nephropathy are not clear, and there have been no reports on the regulation of PLA2R by ADAM10 or ADAM17 in membranous nephropathy. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these purposes and other advantages according to the present invention, there is provided a use of ADAM10 or ADAM17 as a target in the preparation of a drug for preventing / treating membranous nephropathy.

[0006] The present invention also provides use of a reagent for regulating the expression level of ADAM10 or ADAM17 in preparing a drug for preventing / treating membranous nephropathy.

[0007] Preferably, in the application, the expression level of ADAM10 or ADAM17 is the expression level of ADAM10 or ADAM17 in renal cortical tissue.

[0008] The present invention also provides use of an inhibitor of ADAM10 or ADAM17 in preparing a drug for preventing / treating membranous nephropathy.

[0009] Preferably, in the application, the inhibitor of ADAM10 or ADAM17 is used to inhibit the enzymatic cleavage of antigen proteins by podocytes.

[0010] Preferably, in the application, the inhibitor of ADAM10 or ADAM17 is selected from one or more of Batimastat (BB-94), GI254023X, GW280264X, or Aderbasib.

[0011] Preferably, in the above application, the antigen protein is PLA2R protein.

[0012] The present invention has at least the following beneficial effects: The present invention finds that ADAMs mediate the immune complex deposition process of autoantigens such as PLA2R1 in membranous nephropathy. By targeted intervention of ADAMs activity, especially the activity of ADAM10 and ADAM17, the enzymatic cleavage of transmembrane autoantigen proteins such as PLA2R by podocytes can be inhibited, thereby reducing the immune complexes deposited in the glomeruli, thereby improving the podocyte damage in membranous nephropathy and exerting a therapeutic effect.

[0013] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The protein structures of ADAM10 and ADAM17; Figure 2 This is an immunofluorescence staining image of the extracellular segment of PLA2R1 in human kidney tissue in Experiment 1 of the present invention; Figure 3 It is an enlarged view of the immunofluorescence staining of the extracellular segment and intracellular segment of PLA2R1 in human kidney tissue corresponding to membranous nephropathy (MN) in Experiment 1 of the present invention; Figure 4 The extracellular segment of PLA2R1 in Experiment 1 of the present invention ( Figure 4 a) Intracellular segment ( Figure 4 b) The fluorescence intensity of the extracellular segment is statistically plotted, and the ratio of the fluorescence intensity of the extracellular segment to the intracellular segment is displayed ( Figure 4 c); Figure 5 This is the immunoelectron microscopic staining image corresponding to membranous nephropathy in Experiment 2 of the present invention, wherein Figure 5 a is a double-labeled immunoelectron microscopic image of the extracellular and intracellular segments of PLA2R1 corresponding to a patient with membranous nephropathy; Figure 5 b is the double-labeled immunoelectron microscopic image of the extracellular and intracellular segments of PLA2R1 corresponding to the second patient with membranous nephropathy; Figure 6 This is a graph showing the results of an in vitro cell experiment in Experiment 3 of the present invention, wherein Figure 6a is the western blot band diagram corresponding to different inhibitors under the condition of using PMA as stimulation, Figure 6 b is Figure 6 Statistical graph of the bands in a, Figure 6 c is the western blot band diagram corresponding to different inhibitors under stimulation conditions using anti-PLA2R1 IgG in the serum of patients with membranous nephropathy. Figure 6 d is Figure 6 Statistical graph of the bands in c. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments so that those skilled in the art can implement the invention with reference to the description.

[0016] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0017] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0018] In the description of the present invention, the terms "lateral", "longitudinal", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc. to indicate directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings and are only for the convenience of describing the present invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as a limitation on the present invention.

[0019] ADAMs is a family of single-pass transmembrane, secreted metalloendopeptidases. All ADAMs have specific domains, consisting of a propeptide domain, a metalloproteinase domain, a disintegrin domain, a cysteine-rich domain, an epidermal growth factor-like domain, a transmembrane domain, and an intracellular domain. ADAMs with protease activity are called shedding enzymes. For example, ADAM10 and ADAM17 have functional protease activity domains. Their enzymatic activity depends mainly on protein-protein interactions. The disintegrin and cysteine-rich domains in ADAM10 and ADAM17 have important regulatory effects on their enzymatic activity. The binding of this domain to the specific region of the enzyme substrate is the key reason why ADAM10 and ADAM17 have enzyme substrate specificity. The enzyme activity sequence of ADAM10 and ADAM17 is HEXGHNLGXXHD, and its catalytic element is composed of 3 histidines bound to Zn2+, and glutamic acid is the catalytic residue; the protein structure of ADAM10 is shown in the figure below. Figure 1 As shown in A, the protein structure of ADAM10 is shown in Figure 1 As shown in B.

[0020] Based on the understanding of the pathogenesis of membranous nephropathy, the present invention found through in vitro experiments that PLA2R autoantibodies can activate the activity of ADAMs, thereby enzymatically cleaving the extracellular fragments of PLA2R, causing it to change from transmembrane expression on podocytes to a free state; the present invention further studies and clarifies that after PLA2R is cleaved by ADAMs, it can be bound by PLA2R autoantibodies and interact with the glomerular basement membrane, thereby depositing on the glomerular basement membrane, which is the key mechanism for the formation of a large amount of subepithelial immune complex deposition in the glomerulus of membranous nephropathy. Furthermore, the present invention achieves the effect of inhibiting the enzymatic cleavage-shedding-deposition of PLA2R by intervening in the activity of ADAMs, especially by intervening in the enzyme activity of ADAM10 and ADAM17, and has a therapeutic effect on reducing the deposition of immune complexes in membranous nephropathy.

[0021] The present invention uses a variety of commercial inhibitors of ADAMs to intervene in in vitro cell models and homologous mouse models of membranous nephropathy, and finds that intervening in the activity of ADAMs can reduce podocyte enzymatic cleavage of PLA2R and glomerular immune complex deposition levels to varying degrees. It can be inferred that ADAM10 and ADAM17 among ADAMs can be used as targets for the prevention / treatment of membranous nephropathy.

[0022] The following specific test examples are used to illustrate the argument.

[0023] <Test example> 1. Immunofluorescence experiment The specific experimental steps are as follows: 1. Dewaxing and hydration of paraffin sections: bake the renal tissue sections in a 65℃ oven for 1 hour, and then quickly place them in xylene, xylene I, II, and III for 5 minutes each; hydrate with gradient alcohol, immerse in anhydrous ethanol I-anhydrous ethanol II-95% ethanol-85% ethanol-75% ethanol-distilled water for 10 minutes in sequence; 2. Antigen repair: Wash once with distilled water for 5 minutes, immerse the sections in antigen repair solution, heat in a microwave until the liquid boils, turn to medium-low heat to maintain boiling for 15 minutes, and cool to room temperature; 3. Blocking: Wash with PBS (phosphate buffered saline) 3 times, 5 minutes each time, wipe off the liquid on the slice, circle the tissue with a tissue pen to prevent the liquid from drying up, add 10% goat serum to the slice, and block at room temperature for 1 hour; 4. Aspirate the blocking solution, add the diluted mixed primary antibody [mouse antibody against the extracellular segment of PLA2R1 (ab211490, Abcam) and rabbit antibody against the intracellular segment of PLA2R1 (the preparation process of the rabbit antibody against the intracellular segment of PLA2R1 is: synthesizing the selected antigen epitope peptide; immunizing rabbits with a mixture of antigen and adjuvant subcutaneously; separating serum to purify rabbit anti-PLA2R1 extracellular segment immunoglobulin)), the volume ratio of mouse antibody against the extracellular segment of PLA2R1 to the blocking solution is 1:400; the volume ratio of rabbit antibody against the intracellular segment of PLA2R1 to the blocking solution is 1:200] to cover the tissue surface, and incubate in a refrigerator at 4°C overnight; 5. The next day, the primary antibody was recovered and washed with PBST (phosphate-tween buffer) three times, 5 minutes each time; 6. Wipe off the liquid on the slices, add the diluted mixed fluorescent secondary antibody [fluorescent conjugated goat anti-mouse IgG antibody (STORANGE-1001, abberior) and fluorescent conjugated goat anti-rabbit IgG antibody (STRED-1001, abberior) with a volume ratio of 1:200 for both antibodies and solvent] to cover the tissue, and incubate at room temperature in the dark for 30 minutes; 7. Discard the secondary antibody and wash with PBST 3 times, 5 min each time; 8. Wipe the liquid off the slices, add DAPI (4',6-diamidino-2-phenylindole) to cover the tissue, and incubate at room temperature in the dark for 5 minutes; 9. Discard DAPI and wash with PBST 3 times, 5 min each time; 10. Wipe off the tissue traces and excess liquid, add a drop of anti-fluorescence quenching mounting medium on the tissue, seal the slide, and seal with nail polish; 11. Observation and photography using laser confocal fluorescence microscope.

[0024] Human podocytes from various glomerular diseases were obtained according to the above experimental steps to obtain the corresponding immunofluorescence staining images of the extracellular and intracellular segments of PLA2R1 in human kidney tissues, as shown in FIG. Figure 2 Shown, from left to right are membranous nephropathy (MN), focal segmental glomerulosclerosis (FSGS), minimal change nephropathy (MCD), diabetic nephropathy (DKD), lupus nephritis (LN), IgA nephropathy (IgAN), and neutrophil cytoplasmic antibody-associated glomerulonephritis (ANAC); Figure 3 for Figure 2 Enlarged image of immunofluorescence staining of the extracellular and intracellular segments of PLA2R1 in human kidney tissue corresponding to membranous nephropathy (MN); Figure 2~3 As shown, the fluorescent staining of the extracellular segment and the intracellular segment corresponding to membranous nephropathy are not co-localized, which indicates that PLA2R1 on the podocytes of the glomeruli of patients with membranous nephropathy is sheared, and the distance between the extracellular segment and the podocyte becomes larger, which means that the sheared extracellular segment is separated from the cell.

[0025] Further analysis of the extracellular segment ( Figure 4 a) Intracellular segment ( Figure 4 b) The fluorescence intensity is statistically analyzed, and the results are as follows Figure 4 As shown, Figure 4 c is the ratio of the fluorescence intensity of the extracellular segment to that of the intracellular segment; Figure 4 The results showed that only membranous nephropathy had obvious extracellular segment fluorescence, which also indicated that only PLA2R1 in membranous nephropathy was cleaved.

[0026] 2. Immunoelectron microscopy experiment: The specific experimental steps are as follows: 1. Renal cortical tissue was dehydrated with a graded ethanol series at -20°C and then embedded in epoxy resin at 4°C.

[0027] 2. The tissue was then cut into 80 nm ultrathin sections and stored at 4°C.

[0028] 3. Sections were rehydrated for immunoelectron microscopy staining, blocked with 1% BSA / Tris buffer, and incubated with primary antibodies overnight at 4°C.

[0029] 4. After washing, apply secondary antibody and incubate at room temperature for 20 minutes, then at 37°C for 1 hour, and finally at room temperature for 30 minutes.

[0030] 5. Double immunolabeling Immunostaining was performed using primary antibodies against the extracellular domain (ab211490, 1:50, Abcam) and intracellular domain (1:200, homemade) of PLA2R1, as well as goat anti-mouse IgG secondary antibodies conjugated to 4 nm colloidal gold (115-185-146, 1:25, Jackson Immunoresearch Laboratories) and goat anti-rabbit IgG secondary antibodies conjugated to 12 nm colloidal gold (111-205-144, 1:25, Jackson Immunoresearch Laboratories).

[0031] 6. Immunostaining was followed by uranyl acetate staining to enhance contrast.

[0032] 7. Use a JEM-1400 transmission electron microscope to photograph the sections.

[0033] Renal cortical tissues from two different membranous nephropathy patients were used for the above experiment 2. The results are as follows: Figure 5 As shown, Figure 5 a is a double-labeled immunoelectron microscopic image of the extracellular and intracellular segments of PLA2R1 in a patient with membranous nephropathy. Figure 5 b is a double-labeled immunoelectron micrograph of the extracellular and intracellular segments of PLA2R1 in patients with membranous nephropathy. The results show that the PLA2R1 deposited on the glomerular basement membrane of membranous nephropathy is the extracellular segment, while the intracellular segment is mainly on podocytes. This more clearly shows that after PLA2R1 in membranous nephropathy is sheared, its extracellular segment is deposited on the glomerular basement membrane.

[0034] The results of experiments one and two showed that through in vivo double immunofluorescence and immunoelectron microscopy labeling of the extracellular domain and intracellular domain of the main antigen protein of membranous nephropathy (phospholipase A2 receptor, PLA2R1), the present application has discovered that the formation mechanism of the pathological characteristic of membranous nephropathy - subglomerular epithelial immune complex deposition is that PLA2R1 is cleaved by podocytes and then shed and deposited on the glomerular basement membrane.

[0035] 3. In vitro cell experimental methods: The specific experimental steps are as follows: 1. The differentiated human podocytes were uniformly seeded in 6-well or 12-well culture plates.

[0036] 2. Treated with PMA (P8139, Sigma-Aldrich) (200nM, 5 hours) or patient IgG (100ug / ml, 5 hours) or serum (10% of culture medium, 5 hours), and / or pretreated with different shearing enzyme inhibitors (BB-94 10μM, ARP-100 10μM, MMP9-IN-1 10μM, GI254023X 10μM, GW280264X 10μM, Verubecestat 10μM, DAPT 50μM, Belnacasan 10μM) for 30 minutes.

[0037] 3. Collect the cell culture medium and centrifuge for 30 minutes at 12,000 rpm and 4°C.

[0038] 4. Cell pellets were lysed in RIPA lysis buffer (PC101, Epizyme) containing a protease inhibitor cocktail for 10 min (4693132001, Roche Diagnostics) and then centrifuged for 10 min at 12,000 rpm at 4°C.

[0039] 5. The supernatant was used as cell lysate for further Western blot analysis.

[0040] 6. Cell lysate samples were loaded on 4-20% SDS-PAGE gels and separated under reducing or non-reducing conditions. Proteins were then transferred to polyvinylidene difluoride membranes (IPVH0010, Sigma-Aldrich) at 400 mA for 30 minutes.

[0041] 7. After transfer, the membrane was blocked with blocking buffer (PS108P, Epizyme) at room temperature for 20 minutes and incubated with primary antibody at 4°C overnight.

[0042] 8. Incubate with goat anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (HRP) as secondary antibody.

[0043] 9. The protein expression was displayed using ECL (SQ201, Epizyme), and the protein expression signal was detected using the Tanon 5200 chemiluminescence imaging analysis system.

[0044] The results of Experiment 3 are as follows Figure 6 As shown, Figure 6 a is the western blot band diagram corresponding to different inhibitors under the condition of using PMA as stimulation, Figure 6 b is Figure 6 Statistical graph of the bands in a, Figure 6c is the western blot band diagram corresponding to different inhibitors under stimulation conditions using anti-PLA2R1 IgG in the serum of patients with membranous nephropathy. Figure 6 d is Figure 6 The statistical graph of the bands in c shows that when podocytes are stimulated by PMA, PLA2R1 will be cleaved, leaving intracellular fragments in the cells, and only inhibitors of ADAM10 and 17 (BB94, GI254023X, GW280264X) can reduce the amount of intracellular fragments, indicating that inhibition of ADAM10 / 17 can reduce the cleavage of PLA2R1 by podocytes. Anti-PLA2R1 IgG in the serum of patients with membranous nephropathy was used as a stimulation condition, and it was also shown that inhibitors of ADAM10 and 17 (BB94, GI254023X, GW280264X) can reduce the amount of intracellular fragments, indicating that inhibition of ADAM10 / 17 can reduce the cleavage of PLA2R1 caused by autoantibodies in patients with membranous nephropathy on podocytes. In summary, PMA and anti-PLA2R1 antibodies of patients with membranous nephropathy can induce human podocytes to cleave PLA2R1, among which ADAM10 / 17 is the key enzyme causing cleavage.

[0045] Experiment 3 found through in vitro inhibition experiments that ADAM10 and ADAM17 in the disintegrin-metalloproteinase family (ADAM) mediated the podocyte enzymatic cleavage of PLA2R1 mediated by membranous nephropathy autoantibodies. Inhibiting the activity of ADAM10 and ADAM17 can effectively reduce the enzymatic cleavage of PLA2R1 by podocytes, thereby reducing the subepithelial immune complex deposition in membranous nephropathy and achieving the effect of improving kidney damage.

[0046] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0047] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. Application of ADAM10 or ADAM17 as a target in the preparation of drugs for the prevention / treatment of membranous nephropathy.

2. Use of reagents for regulating the expression level of ADAM10 or ADAM17 in the preparation of drugs for preventing / treating membranous nephropathy.

3. The use according to claim 2, characterized in that The expression level of ADAM10 or ADAM17 is the expression level of ADAM10 or ADAM17 in renal cortical tissue.

4. Use of inhibitors of ADAM10 or ADAM17 in the preparation of drugs for preventing / treating membranous nephropathy.

5. The use according to claim 4, characterized in that The inhibitor of ADAM10 or ADAM17 is used to inhibit the enzymatic cleavage of antigen proteins by podocytes.

6. The use according to claim 5, characterized in that The inhibitor of ADAM10 or ADAM17 is selected from one or more of batimastat, GI254023X, GW280264X or Aderbasib.

7. The use according to claim 5, characterized in that The antigen protein is PLA2R protein.

Images