Method and kit for detecting antibodies against podocin, kirrel1, and ptpro for kidney disease diagnosis

Abstract

The present invention relates to a method for assessing the presence of a kidney disease in a subject, comprising at least one step of detecting in at least one biological sample isolated from said subject the presence of antibodies capable of recognising and binding at least one protein selected from the group consisting of: Podocin, Kirrel1 and PTPRO. Further, the invention relates to a method and a kit for detecting said antibodies.

Description

[0001] Method and Kit for Detecting Antibodies Against Podocin, Kirrell, and PTPRO for Kidney Disease Diagnosis

[0002] DESCRIPTION

[0003] Technical field

[0004] The present invention relates to a method for assessing the presence of a kidney disease in a subject comprising at least one step of detecting in at least one biological sample isolated from said subject the presence of antibodies capable of recognising and binding at least one protein selected from the group consisting of: Podocin / NPHS2, kirre like nephrin family adhesion molecule 1 (Kirrell ) / NEPH1 , and protein tyrosine phosphatase recettore di tipo 0 (PTPRO) / Glepp-1 .

[0005] Background art

[0006] Podocytopathies are a group of kidney disorders characterized by dysfunction or injury to podocytes, specialized cells in the glomerulus that play a crucial role in maintaining the filtration barrier of the kidney. These disorders typically manifest as proteinuria, where abnormal amounts of protein are excreted in the urine, and can progress to nephrotic syndrome, which is characterized by severe proteinuria, low serum albumin levels, and edema. Podocytopathies can result from various causes, including genetic mutations, immune-mediated mechanisms, and environmental factors such as toxins or infections (1 ).

[0007] Current diagnostic methods for podocytopathies primarily rely on kidney biopsy and subsequent analysis using immunofluorescence and light microscopy (1 ). These techniques focus on identifying pathological patterns, such as minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS). However, these methods often fail to provide insights into the specific underlying causes of podocyte injury, particularly in cases where autoimmune mechanisms may be involved (1 ).

[0008] The limitations of current diagnostic approaches present several challenges in the management of podocytopathies. First, the invasive nature of kidney biopsies carries risks and may not be suitable for all patients, especially those with contraindications or in cases where repeated assessments are necessary. Second, the inability to identify specific autoantibodies that may contribute to the pathogenesis of podocytopathies can lead to non-targeted treatment approaches. This often results in the use of broad-spectrum immunosuppressants or steroids, which may cause unnecessary side effects in patients who do not have an autoimmune component to their disease.

[0009] Furthermore, the lack of specific biomarkers for podocytopathies makes it difficult to monitor disease progression and treatment response accurately. This can lead to suboptimal management strategies and potentially worse outcomes for patients. Recently, Watts et al. described the existence of anti-nephrin antibodies in some patients with podocytopathy (2).

[0010] It has been appreciated that a non-invasive diagnostic method is needed that overcomes one or more of these problems.

[0011] However, the reliability of these assays has been met with skepticism. A significant number of patients, among them those which appear to suffer from autoimmune-related podocytopathies, cannot be diagnosed reliably using the methods described by Watts et al. Specific assays have been developed to address shortcomings (EP25162358.3).

[0012] However, it has been appreciated that additional non-invasive diagnostic methods are needed that overcome one or more of these problems. Furthermore, there is a need to improve the existing methods with regard to diagnostic reliability, in particular increase sensitivity and / or specificity.

[0013] Summary of the invention

[0014] In a first aspect, the problem underlying the present invention is solved by a method for assessing the presence of a kidney disease in a subject, comprising detecting in at least one biological sample isolated from said subject the presence of antibodies capable of recognising and binding at least one protein selected from the group consisting of: Podocin, Kirrell and PTPRO, or at least one epitope thereof, wherein the presence of said antibodies indicates the presence of a kidney disease in the subject.

[0015] Preferably, the method comprises detecting in the at least one biological sample the presence of antibodies capable of recognizing and binding at least two proteins selected from Podocin, Kirrell and PTPRO, more preferably, antibodies capable of recognizing and binding Podocin and Kirrell , even more preferably, antibodies capable of recognizing and binding Podocin, Kirrell and PTPRO.

[0016] In some embodiments, the kidney disease is selected from the group consisting of: a podocytopathy, proteinuria and nephrotic syndrome, preferably, the kidney disease is a podocytopathy.

[0017] In some embodiments, the at least one biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

[0018] In some embodiments, the detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

[0019] In some embodiments, the method further comprises a step of selecting a treatment strategy for the subject based on the detection of antibodies against Podocin, Kirrell , or PTPRO, preferably the treatment strategy comprises administering immunosuppressive therapy to the subject when antibodies against Podocin, Kirrell , or PTPRO are detected.

[0020] In a second aspect, the problem underlying the present invention is solved by a method of detecting antibodies capable of recognising and binding at least one protein selected from the group consisting of: Podocin, Kirrell and PTPRO in a patient, said method comprising: a. obtaining a biological sample from the patient; and b. detecting whether the antibodies are present in the biological sample by contacting the biological sample with at least one protein selected from the group consisting of: Podocin, Kirrell and PTPRO or fragment thereof and detecting the binding between the antibodies and the at least one protein. In a third aspect the problem underlying the present invention is solved by a diagnostic kit for detecting antibodies in a subject, comprising: at least one recombinant protein selected from Podocin, Kirrell and PTPRO; and a secondary antibody conjugated to a detectable label capable of recognising and binding at least one immunoglobulin domain.

[0021] In some embodiments, the diagnostic kit is an enzyme-linked immunosorbent assay (ELISA).

[0022] In some embodiments, the diagnostic kit comprises at least one protein having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with at least one sequence selected from: SEQ ID NO: 1-3 or at least one protein having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with at least one sequence selected from: SEQ ID NO:4-6.

[0023] Brief description of drawings

[0024] Figure 1 shows the detection of Antibodies against-Podocin, -Kirrell and - PTPRO in kidney biopsy and serum of patients with proteinuria or nephrotic syndrome.

[0025] A-D. Combined immunofluorescent labelling for IgG (green) and different slit antigens (red) detected by high resolution confocal microscopy in four different patients with proteinuria or nephrotic syndrome. A shows colocalization of IgG with Podocin (red), B shows colocalization of IgG with Kirrell , C shows colocalization of IgG with PTPRO and D shows absence of colocalization between IgG and ACTN4. E-H: Combined immunofluorescent labelling for IgG (green) and different slit antigens (red) detected by STED microscopy in four different patients with proteinuria or nephrotic syndrome. E shows colocalization of IgG with Podocin (red), F shows colocalization IgG with Kirrell , G shows colocalization of IgG with PTPRO and H shows absence of colocalization between IgG and ACTN4. I-K Results of ELISA assay assessment in the serum of patients with proteinuria or nephrotic syndrome and non-proteinuric controls of autoantibodies against Podocin (I), Kirrell (J) or PTPRO (J).

[0026] Figure 2 shows the results of an indirect immunofluorescence analysis of a sample from a patient and a healthy subject.

[0027] Figure 3 Shows the results of an integrated assessment of clinical progression, autoimmune profile and renal biopsy results of a 5 year old patient suffering from an autoimmune podocytopathy associated with the presence of autoantibodies to Podocin.

[0028] Figure 4 shows a representative FACS assay performed on kidney progenitor-derived cultured podocytes. Figure 4A displays the FACS plot obtained from podocytes incubated with serum from a patient in disease remission, whereas Figure 4B shows the plot obtained from a patient sample containing circulating anti-podocin autoantibodies during relapse.

[0029] Detailed description of the invention

[0030] A first aspect of the present invention relates to a method for assessing the presence of a kidney disease in a subject. Preferably, the method is an in vitro method. Preferably, the subject is a mammalian subject, preferably from the group comprising a dog, cat, donkey, horse, sheep, camel, primate, mouse, rabbit, rat, monkey or human, more preferably a primate, most preferably a human.

[0031] In some embodiments, the method comprises at least one step of detecting in at least one biological sample isolated from said subject antibodies, preferably autoantibodies. In a preferred embodiment, the presence of antibodies capable of recognising and binding to Podocin or at least one epitope thereof is detected in a sample from a subject. In another preferred embodiment, the presence of antibodies capable of recognising and binding to Kirrell or at least one epitope thereof is detected in a sample from a subject. In another preferred embodiment, the presence of antibodies capable of recognising and binding to PTPRO, or at least one epitope thereof is detected in a sample from a subject. In a preferred embodiment, an epitope comprises a stretch of 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more consecutive amino acid residues from an autoantigen, preferably from the group comprising Podocin, Kirrell and PTPRO, more preferably Podocin. The epitope may be a conformational epitope comprising 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more consecutive amino acid residues from an autoantigen, preferably from the group comprising Podocin, Kirrell and PTPRO, more preferably Podocin.

[0032] Preferably, said antibodies are capable of recognising and binding at least one protein selected from the group consisting of: Podocin, Kirrell and PTPRO, or at least one epitope thereof.

[0033] More preferably, said antibodies are capable of specifically recognising and binding at least one protein selected from the group consisting of: Podocin, Kirrell and PTPRO, or at least one epitope thereof.

[0034] In some embodiments, the detection of said antibodies in the biological sample indicates the presence of a kidney disease in the subject.

[0035] Preferably, the method comprises detecting in the at least one biological sample antibodies capable of recognizing and binding at least two proteins selected from Podocin, Kirrell and PTPRO.

[0036] Preferably, the method comprises detecting in the at least one biological sample antibodies capable of recognizing and binding Podocin and Kirrell .

[0037] In a preferred embodiment, the method comprises detecting in the at least one biological sample antibodies capable of recognizing and binding Podocin, Kirrell and PTPRO.

[0038] In some embodiments, the antibodies are capable of recognizing and binding at least one protein having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with at least one sequence selected from: SEQ ID NO:1- 3.

[0039] In some embodiments, the antibodies are capable of recognizing and binding at least two proteins having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with at least one sequence selected from: SEQ ID NO:1- 3.

[0040] In some embodiments, the antibodies are capable of recognizing and binding two proteins having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO:1 and 2.

[0041] In some embodiments, the antibodies are capable of recognizing and binding the protein having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO:1 , 2 and 3.

[0042] In some embodiment, the kidney disease is selected from the group consisting of: a podocytopathy, proteinuria and nephrotic syndrome.

[0043] In some cases, the method provides a valuable tool for the interpretation of results in the context of kidney diseases, including podocytopathies. The interpretation of results may provide valuable information for the diagnosis, prognosis, and management of these conditions.

[0044] In a preferred embodiment, the methods, uses, mammalian antibodies, carriers and kits described herein are for diagnosing a disease and / or for aiding in the diagnosis of a disease. In a preferred embodiment, the term “diagnosis", as used herein, is to be understood in its broadest possible sense and may refer to any kind of procedure aiming to obtain information instrumental in the assessment whether a patient, known or an anonymous subject from a cohort, suffers or is likely or more likely than the average or a comparative subject, the latter preferably having similar symptoms, to suffer from a certain disease or disorder in the past, at the time of the diagnosis or in the future, to find out how the disease is progressing or is likely to progress in the future or to evaluate the responsiveness of a patient or patients in general with regard to a certain treatment, for example the administration of immunosuppressive drugs, or to find out whether a sample is from such a patient. Such information may be used for a clinical diagnosis but may also be obtained by an experimental and / or research laboratory for the purpose of general research, for example to determine the proportion of subjects suffering from the disease in a patient cohort or in a population. Such information may also be used to determine whether a sample is or is not a sample from a subject who is more likely than an average healthy subject to suffer, have suffered or suffer in the future from a nephrological autoimmune disease. Alternatively, a person who or a sample of whom is the subject of research may be diagnosed, for example to find out whether this patient is a suitable subject for the study, for example as a part of a cohort of subjects suffering from a disease or as a test subject, for example for testing the efficacy of a candidate drug. In a preferred embodiment, patients suffering from an autoimmune disease, preferably a nephrological autoimmune disease such as a podocytopathy are selected for inclusion in a cohort for a clinical study aiming to test the efficacy of immunosuppressive drugs, preferably systemic immunomodulators. For example, if a patient has such autoantibodies, they and their samples may be included into the cohort, because there is the potential that the autoimmune disease may be ameliorated by administration of immunosuppressive drugs, preferably systemic immunomodulators. In other words, the term “diagnosis” comprises not only diagnosing, but also prognosticating and / or monitoring the course of a disease or disorder, including monitoring the response of one or more patients to the administration of a drug or candidate drug, for example to determine its efficacy. While the result may be assigned to a specific patient for clinical diagnostic applications and may be communicated to a medical doctor or institution treating said patient, this is not necessarily the case for other applications, for example in diagnostics for research purposes, where it may be sufficient to assign the results to a sample from an anonymized patient. Thus, in some embodiments, the person to be diagnosed, i.e. , the “subject” or “patient”, is an anonymous blood donor whose blood may be donated or used to obtain therapeutically or diagnostically useful antibodies. The term “diagnosis” also refers to negative diagnosis, i.e., the case where a mammalian antibody to a mammalian, preferably human nephrin are absent in a sample from a patient. In these embodiments, the absence indicates that the patient may suffer from a disease other than a disease associated with the presence of said antibodies. In another preferred embodiment, the detection of a mammalian, preferably human antibody to mammalian, preferably human nephrin is considered to imply a definitive diagnosis of a nephrological autoimmune disease because of the presence of the antibody.

[0045] Thus, in a preferred embodiment, the term "diagnosis", "diagnosing" or "diagnostic" or similar terms encompasses diagnosis, prognosis, theragnosis and monitoring in a nephrological autoimmune disease, preferably a podocytopathy. As used herein, the term “theragnosis” refers to the identification, for example by diagnostic methods, of patients who might benefit from a particular therapy and, optionally, the subsequent treatment of said patients, for example by administration of an immunosuppressive drug.

[0046] In a preferred embodiment, the term "diagnosis" does not imply that the diagnostic methods or products according to the present invention will be definitive and sufficient to finalize the diagnosis on the basis of a single test or a limited number of autoimmune parameters, but may refer to a contribution to what is referred to as a "differential diagnosis", i.e. a systematic diagnostic procedure considering the likelihood of a range of possible conditions on the basis of a range of diagnostic parameters. This may include an indirect diagnosis, including the exclusion of conditions based on negative diagnostic results, which may point a clinician to diseases other than those that have been diagnosed. See Baenkler H. W. (2012), General aspects of autoimmune diagnostics, in Renz, H., Autoimmune diagnostics, 2012, de Gruyter, page 3. In a preferred embodiment, the term "diagnosis" means that the method or product or use may be used for aiding in the diagnosis of an autoimmune disease, more preferably a nephrological autoimmune disease, more preferably a podocytopathy. In a preferred embodiment, the samples of a patient are may be considered for examination using the methods, uses or products according to the present invention following determining one or more parameters from the group comprising urinary protein-to-creatinine ratio, more preferably if it is increased, levels of serum albumin, more preferably reduced levels and low-density lipoprotein cholesterol, more preferably if it is reduced. In a preferred embodiment, if membranous nephropathy is excluded, more preferably based on negative results when methods for detecting an autoantibody related to membranous nephropathy show negative results, more preferably an autoantibody from the group comprising anti-PLA2R, anti-THSd7a, Exostosin and anti-NELL1. Methods for detecting such autoantibodies have been disclosed in the state of the art, for example in US10670597 BB, US2022187320 AA, WO21023836 A1 , EP4010703 A1 and US2022389108 AA.

[0047] Preferably, the disease is associated with the presence of an autoantibody to podocin. Preferably, the disease is associated with the presence of an autoantibody to Kirrell . Preferably, the disease is associated with the presence of an autoantibody to PTPRO.

[0048] In a preferred embodiment, immunosuppressive treatment administered following the detection of autoantibodies to Podocin, Kirrell and PTPRO include an immunosuppressive drug, preferably selected from the group comprising prednisone, eculizumab, a calcineurin inhibitor such as cyclosporine or tacrolimus, mycophenolate mofetil a B-cell targeting therapy, preferably selected from the group comprising Rituximab (RTX) cyclophosphamide and Obinutuzumab, Daratumumab or other B cell / plasma cell blockers under investigation or plasmapheresis [See KDIGO Clinical Practice Guideline for the Management of Glomerular Diseases, 2021 and 2025 and Example 4], Kidney diseases associated with the presence of these autoantibodies may encompass a range of conditions, including podocytopathies, proteinuria, and nephrotic syndrome.

[0049] In some aspects, the presence of autoantibodies against at least one protein above disclosed in the biological sample may suggest the presence of podocytopathies

[0050] Proteinuria, another condition associated with these autoantibodies, is characterized by an abnormal amount of protein in the urine. This condition may be indicative of damage to the glomeruli, the tiny blood vessels in the kidneys where blood is filtered.

[0051] Nephrotic syndrome, a more severe kidney disorder, may also be associated with the presence of these autoantibodies. This syndrome is characterized by high levels of protein in the urine, low levels of protein in the blood, and swelling in the legs and ankles.

[0052] Preferably, the kidney disease is a podocytopathy, more preferably associated with the presence of one or more antibodies selected from the group comprising anti-Podocin, anti-Kirrel1 and anti-PTPRO, preferably anti-Podocin. Preferably the kidney disease is a podocytopathy not associated with the presence of anti-nephrin antibodies.

[0053] In some embodiments, the presence of antibodies against these proteins in a biological sample may indicate the presence of kidney diseases, preferably of a podocytopathy. The disclosed method may detect these autoantibodies, providing valuable information for the early detection and treatment strategy selection for these conditions. Early detection may allow for a more timely intervention and selection of an appropriate treatment strategy, potentially improving patient outcomes.

[0054] In some embodiments, the biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

[0055] Biological samples suitable for antibodies detection may vary. Serum, a component of blood obtained after coagulation and removal of blood cells, may serve as a source of autoantibodies. In a preferred embodiment, the sample is selected from the group comprising kidney tissue or blood more preferably serum, plasma and whole blood. The blood sample may be a dried blood spot.

[0056] In some embodiments, serum provides a rich and accessible source of autoantibodies, making it a commonly used sample type in diagnostic assays.

[0057] Plasma, another component of blood, may also be used for antibodies detection. In some cases, plasma may be preferred over serum due to its ability to provide a more comprehensive profile of the patient's immune response.

[0058] Urine may also serve as a biological sample for antibodies detection. In some aspects, urine may contain antibodies that have been filtered out of the blood by the kidneys. The presence of these autoantibodies in urine may indicate kidney damage or disease.

[0059] Kidney biopsy tissue may also be used for antibodies detection. In some cases, a small piece of kidney tissue is removed and examined for the presence of autoantibodies. This method may provide direct evidence of autoantibody activity within the kidney, potentially offering more specific information about the nature and extent of the disease.

[0060] In some embodiments, the choice of biological sample may depend on various factors, including the nature of the disease, the patient's condition, and the specific requirements of the detection assay. The disclosed methods and kits may accommodate a variety of biological samples, providing flexibility in the detection of autoantibodies associated with kidney diseases.

[0061] The process of obtaining and preparing biological samples for antibodies detection may vary depending on the type of sample. In some aspects, the blood is allowed to clot at room temperature to obtain serum, while for plasma, an anticoagulant may be added to the blood immediately after collection to prevent clotting. The clotted blood or anticoagulated blood is then centrifuged to separate the serum or plasma from the blood cells. The serum or plasma is then collected for further analysis.

[0062] In the case of kidney biopsy tissue, a small piece of kidney tissue may be obtained from a patient through a biopsy procedure. The tissue sample may then be processed for autoantibody detection. In some embodiments, the tissue is homogenized to break down the tissue structure and release the proteins. The homogenate may then be centrifuged to separate the soluble proteins from the insoluble material. The soluble proteins, which may include the antibodies, are then collected for further analysis.

[0063] In some embodiments, the biological samples may be subjected to additional processing steps, such as purification or concentration, to enhance the detection of the autoantibodies. These steps may involve techniques such as precipitation, chromatography, or filtration.

[0064] In some embodiments, the detection of antibodies in the biological sample is achieve through techniques known to a skilled person. Preferably, said technique is select form: an immunoassay, preferably an enzyme linked immunosorbent assay (ELISA) or a blotting assay, preferably a western blot, flow cytometry, immunofluorescence and immunohistochemistry.

[0065] Preferably, detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

[0066] The ELISA involves the use of an enzyme that reacts with a substrate to produce a detectable signal, thereby indicating the presence of the autoantibodies. The biological samples suitable for testing may include, but are not limited to, serum, plasma or urine.

[0067] In some embodiments, an intensity of colour change observed in the enzyme-linked immunosorbent assay (ELISA) is proportional to the amount of autoantibodies present in the sample. This allows for not only the detection of the autoantibodies, but also the quantification of their levels. The colour change may be measured using a spectrophotometer or other suitable detection device. The measured intensity may then be compared to a standard curve or reference values to determine the concentration of the autoantibodies in the sample.

[0068] In another embodiment, immunofluorescence is used to detect the presence of an autoantibody to Podocin, Kirrell , or PTPRO, preferably Podocin. A secondary antibody comprising a detectable fluorescent label, more preferably FITC (fluorescein isothiocyanate), may be used. Commercially available equipment including mounting solutions, washing solutions, cover glasses and slides with eukaryotic cells overexpressing the proteins Podocin, Kirrell , or PTPRO, preferably Podocin, or an epitope thereof, may be used (for example, FB 112d-1005-1 or ZZ 3000-0112, EUROIMMUN Medizinische Labordiagnostika AG). The person skilled in the art is familiar with protocols and vectors for transiently overexpressing a polypeptide in a eukaryotic cell, for example the pTriEx system from Novagen and with protocols and vectors for stably transfecting a eukaryotic cell, for example the pcDNATM4 / TO vector system from Invitrogen. In a preferred embodiment, a fixed mammalian cell may be used. In a preferred embodiment, the term “fixed” cell, as used herein, refers to a cell that has been treated with a reactive chemical compound to the effect that the cell is no longer metabolically active, but still presents its epitopes for immunostaining with antibodies and their subsequent detection, for example by fluorescence. More preferably, the reactive chemical compound is selected from the group comprising acetone, formalin, methanol and ethanol or mixtures thereof, preferably all of them. The person skilled in the art is familiar with protocols that may be used to prepare fixed cells. Essentially, the cell which is attached to a solid support is washed by using washing buffer, followed by contacting with the reactive compound, for example immersion. Pure acetone or formalin or aqueous dilutions of the reactive chemical compound may be used.

[0069] According to the present invention, the presence of an antibody may be determined in a qualitative or a quantitative manner. In a preferred embodiment, the term “detecting in a quantitative manner”, as used herein, means that not only the presence of an antibody is detected, but that a result is obtained that includes information regarding the absolute or relative amount of the antibody in the sample. In a more preferred embodiment, a value representing absolute concentration is obtained. In another mor preferred embodiment, a value representing a relative concentration or change of concentration is obtained. In another preferred embodiment, also referred to as “semi-quantitative” approach, the concentration of the antibody is placed in one of several groups, most preferably a concentration window meaning that it is virtually absent, a concentration window meaning that a borderline result is obtained and a concentration window meaning that the antibody is present. A further distinction into categories such as “weak positive” or “strong positive” signal is possible.

[0070] In a preferred embodiment, the term “detecting” an antibody means that a detection signal indicates the presence of an antibody binding to a specific autoantigen, preferably from the group comprising Podocin, Kirrell , or PTPRO, preferably Podocin. In other words, such signal can be distinguished from a signal relating to an antibody binding to another autoantigen. This type of detection is performed if, for example, an ELISA with a purified Podocin protein is performed. In another preferred embodiment, the term “detecting” as used herein, means that a detection signal indicates the presence of an antibody which may bind to two or more autoantigens, for example two from the group comprising Podocin, Kirrell , or PTPRO. In other words, it is known that the signal relates to an antibody from a group of antibodies binding to a group of autoantigens, but it is unclear which one or more than one from this group of antibodies is present in the sample. This type of detection is performed if, for example, an ELISA with a mixture of antigens, for example Podocin and Kirrell , is performed.

[0071] In some embodiments, the concentration of the antibodies is correlated with the severity or stage of the kidney disease. Higher concentrations of antibodies may indicate a more advanced stage of the disease or a more severe form of the disease. Conversely, lower concentrations of antibodies may indicate an early stage of the disease or a less severe form of the disease.

[0072] In some embodiments, the results of the antibodies detection assay may be used in conjunction with other diagnostic tests or clinical findings to confirm the diagnosis of a kidney disease. For example, the results may be used in combination with kidney function tests, imaging studies, or histological examination of kidney biopsy tissue.

[0073] In some embodiments, the results of the antibodies detection assay may be used to monitor the progress of the disease or the response to treatment. Changes in the concentration of the antibodies over time may reflect changes in the disease activity or the effectiveness of the treatment. For example, a decrease in the concentration of the antibodies may indicate an improvement in the disease condition or a positive response to treatment.

[0074] In some embodiments, the method further comprises at least a step of selecting a treatment strategy for the subject based on the detection of antibodies against Podocin, Kirrell , or PTPRO.

[0075] Preferably, the choice of immunosuppressive therapy may depend on the specific autoantibodies detected. For example, if autoantibodies against Podocin are detected, a certain type of immunosuppressive therapy may be selected. Similarly, if autoantibodies against Kirrell or PTPRO are detected, a different type of immunosuppressive therapy may be chosen.

[0076] In some embodiments, the effectiveness of the selected treatment strategy is monitored by periodically testing for the presence and levels of the autoantibodies. A decrease in the concentration of the autoantibodies may indicate a positive response to the treatment, while an increase in the concentration of the autoantibodies may suggest a need to adjust the treatment strategy. The disclosed methods and kits may thus provide a valuable tool for the ongoing management of kidney diseases, including podocytopathies, proteinuria, and nephrotic syndrome.

[0077] A second aspect of the present invention relates to a method for detecting antibodies capable of recognising and binding at least one protein selected from the group consisting of: Podocin, Kirrell and PTPRO or fragments thereof in a patient, said method comprising: a. optionally, but preferably, obtaining a biological sample from the patient; and b. detecting whether the antibodies are present in the biological sample by contacting the biological sample with at least one protein selected from the group consisting of: Podocin, Kirrell and PTPRO or fragment thereof and detecting the binding between the antibodies and the at least one protein. Preferably, the method comprises detecting in the at least one biological sample autoantibodies capable of recognizing and binding at least two proteins selected from Podocin, Kirrell and PTPRO.

[0078] Preferably, the method comprises detecting in the at least one biological sample antibodies capable of recognizing and binding Podocin and Kirrell .

[0079] In a preferred embodiment, the method comprises detecting in the at least one biological sample antibodies capable of recognizing and binding Podocin, Kirrell and PTPRO.

[0080] Preferably, Podocin, Kirrell and PTPRO sequences are as above defined. In some embodiments, the presence of antibodies against these proteins in a biological sample may indicate the presence of kidney diseases, preferably of a podocytopathy. The disclosed method may detect these autoantibodies, providing valuable information for the early detection and treatment strategy selection for these conditions.

[0081] In some embodiments, the biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

[0082] In some embodiments, the detection of antibodies in the biological sample is achieve through techniques known to a skilled person. Preferably, said technique is select form: an immunoassay, preferably an enzyme linked immunosorbent assay (ELISA) or a blotting assay, preferably a western blot, flow cytometry, immunofluorescence and immunohistochemistry.

[0083] Preferably, detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

[0084] In some embodiments, the results of the antibody detection assay may be used to monitor the progress of the disease or the response to treatment. Changes in the concentration of the antibodies over time may reflect changes in the disease activity or the effectiveness of the treatment. For example, a decrease in the concentration of the antibodies may indicate an improvement in the disease condition or a positive response to treatment.

[0085] In some embodiments, the method further comprises at least a step of selecting a treatment strategy for the subject based on the detection of antibodies against Podocin, Kirrell , or PTPRO.

[0086] Preferably, the treatment strategy comprises administering an effective amount of an immunosuppressive therapy to the subject when autoantibodies against Podocin, Kirrell , or PTPRO are detected.

[0087] A third aspect of the present invention reflates to a diagnostic kit for detecting antibodies, preferably autoantibodies in a subject.

[0088] In some embodiments, the kit comprises: at least one recombinant protein selected from Podocin, Kirrell and PTPRO or a fragment thereof, and a secondar antibody conjugated to a detectable label capable of recognising and binding at least one immunoglobulin domain.

[0089] Preferably, the kit is an enzyme-linked immunosorbent assay (ELISA).

[0090] Preferably, the kit comprises Podocin and Kirrell recombinant proteins and / or fragments thereof.

[0091] Preferably, the kit comprises Podocin, Kirrell and PTPRO recombinant proteins and / or fragments thereof.

[0092] A protein or fragment thereof may be fused to one or more than one linker. Suitable linkers are part of vectors, for example pET vector series (Novagen). They are flexible stretches of amino acids, typically comprising glycine, serine or theorine residues. For example, the peptide GGGGS is a known linker. In a preferred embodiment, the linkers has a length of 1-50, preferably 2-40, more preferably 3-20 amino acids. A protein or fragment thereof may be fused to one or more affinity tag. The affinity tag may be selected from the group of tags comprising His, immobilized nickel, glutathione, chitin, 18A, ACP, Aldehyde, Avi, BCCP, Calmodulin, Chitin binding protein, E-Tag, ELK16, FLAG, flash, poly glutamate, poly aspartate, GST, GFP, HA, Isope, maltose binding protein, myc, nus, NE, ProtA, ProtC, Tho1d4, S-Tag, SnoopTag, SpyTag, SofTag, Streptavidin, Strep-tag II, T7 Epitope Tag, TAP, TC, Thioredoxin, Ty, V5, VSV, biotin, Xpress Tag and a recombinant antibody binding to the ligand to an affinity tag. Useful proteases include, but are not limited to TEV, Thrombin, Factor Xa or Enteropeptidase. The protein or fragment may be fused to one or more linker and / or one or more affinity tag via its C-terminus. The protein or fragment may be fused to one or more linker and / or one or more affinity tag via its N-terminus. The protein or fragment may be fused to one or more linker and / or one or more affinity tag via its C-terminus. Linkers, affinity tags and protease cleavage sites are described in the state of the art, for example in Chen, X., Zaro, J. L., & Shen, W. C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357-1369, Li, G., Huang, Z., Zhang, C., Dong, B.-J., Guo, R.-H., Yue, H.-W., Yan, L.-T., & Xing, X.-H. (2015). Construction of a linker library with widely controllable flexibility for fusion protein design. Applied Microbiology and Biotechnology, 100, 215-225 and Arai, R., Ueda, H., Kitayama, A., Kamiya, N., & Nagamune, T. (2001 ). Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Engineering, Design & Selection, 14(8), 529-532, Arnau, J., Lauritzen, C., Petersen, G. E., & Pedersen, J. (2006). Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expression and Purification, 48(1 ), 1-13 and augh, D. S. (2011 ). An overview of enzymatic reagents for the removal of affinity tags. Protein Expression and Purification, 80(2), 283-293.

[0093] In some embodiments, the kit comprises at least one protein having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with at least one sequence selected from: SEQ ID NO: 1-3, preferably S EQ ID NO:1. Preferably, the kit comprises at least a sequence having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 1 , more preferably the kit comprises at least a sequence having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 4.

[0094] Preferably, the kit comprises at least a sequence having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 2 or its portion, more preferably the kit comprises at least a sequence having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO:5.

[0095] Preferably, the kit comprises at least a sequence having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 3 or its portion, more preferably the kit comprises at least a sequence having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 6.

[0096] In a preferred embodiment, the kit comprises at least sequences having 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 4 and 5.

[0097] In a preferred embodiment, the kit comprises at least sequences having 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 4 and 6.

[0098] In yet other preferred embodiment, the kit comprises at least a sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 4-6.

[0099] Any protein comprised by a kit or used for practicing a use or method according to the present invention has biological activity. In a preferred embodiment, such biological activity is the ability to bind specifically to an autoantibody binding specifically to the autoantigen of interest, preferably from the group comprising Podocin, Kirrell and PTPRO, preferably Podocin, as found in a patient suffering from an autoimmune disease associated with such autoantibody, preferably a podocytopathy. For example, whether or not a protein has such biological activity may be checked by determining whether or not it binds specifically to an autoantibody from a sample of a patient suffering from the disease comprising an autoantibody binding specifically to wild type autoantigen, preferably as determined by ELISA as described in Example 2 of this application.

[0100] In a preferred embodiment, the protein, comprised by a kit or not, is associated, preferably as a coating, with a carrier, which is preferably selected from the group comprising a glass slide, preferably for microscopy, a biochip, a microtiter plate, a lateral flow device, a test strip, a membrane, preferably a line blot, a chromatography column and a bead, preferably a magnetic or fluorescent bead.

[0101] In some embodiments, the disclosed diagnostic kits comprise recombinant proteins and other necessary reagents for performing the detection assay. These recombinant proteins may be produced through genetic engineering techniques and may mimic the proteins to which the autoantibodies bind. The reagents included in the kits may facilitate the reaction between the antibodies and the recombinant proteins, thereby enabling the detection of the antibodies.

[0102] In a preferred embodiment, a kit is provided which may comprise a cell expressing the protein or a carrier coated or configured for coating with the protein. In a preferred embodiment, the kit comprises one or more, preferably all reagents from the group comprising a secondary antibody, preferably labeled with a detectable label, a washing solution, a positive control, a negative control, a detergent, a cover glass, a mounting medium and a physiological salt solution, preferably PBS, or salt required to prepare it. In a preferred embodiment, the positive control is a diluted sample, preferably serum, from a patient suffering from a neurological autoimmune disease or a monoclonal antibody binding specifically to an autoantigen, preferably from the group comprising Podocin, Kirrell and PTPRO, preferably Podocin. The negative control may be a diluted sample from a healthy subject, for example a blood donor.

[0103] I may be a diluted sample from a healthy subject, for example a blood donor. In a more preferred embodiment, a protein provided or used according to the present invention such as a polypeptide is an isolated polypeptide, wherein the term “isolated” means that the polypeptide has been enriched compared to its state upon production using a biotechnological or synthetic approach and is preferably pure, i.e. at least 60, 70, 80, 90, 95 or 99 percent of the polypeptide in the respective liquid consists of said polypeptide as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection. The person skilled in the art is familiar with methods for engineering nucleic acids and polypeptides encoded (for example, described in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning, CSH or in Brown T. A. (1986), Gene Cloning - an introduction, Chapman & Hall) and for producing and purifying native or recombinant polypeptides (for example Handbooks ..Strategies for Protein Purification", ..Antibody Purification", published by GE Healthcare Life Sciences, and in Burgess. R. R.. Deutscher, M. P. (2009): Guide to Protein Purification).

[0104] In a preferred embodiment, the antibody is detected using a method from the group comprising immunodiffusion, immunoelectrophoresis, light scattering immunoassays, agglutination, labeled immunoassays such as those from the group comprising radiolabeled immunoassays, enzyme immunoassays such as colorimetric assays, and immunofluorescence, more preferably immunofluorescence. Such detection methods are disclosed in the state of the art. for example in Wild. The Immunoassay Handbook. 3rd edition. Elsevier. 2006, ISBN 0080445268.

[0105] In some cases, the diagnostic kit may also include reagents necessary for performing the enzyme-linked immunosorbent assay (ELISA). These reagents may include, but are not limited to. an enzyme-linked antibody that is specific for the autoantibodies, a substrate for the enzyme, and a buffer solution for diluting the biological sample. The enzyme-linked antibody binds to the autoantibodies that are attached to the recombinant proteins, and the substrate reacts with the enzyme to produce a detectable signal, such as a colour change, indicating the presence of the autoantibodies.

[0106] In some embodiments, the diagnostic kit may further include a microtiter plate for performing the assay. The microtiter plate may have multiple wells, allowing for the simultaneous testing of multiple samples or the testing of a single sample under different conditions. The microtiter plate may be made of a material, such as polystyrene, that allows for the immobilization of the recombinant proteins and the subsequent binding of the autoantibodies.

[0107] In some embodiments, the diagnostic kit may also include instructions for performing the assay. These instructions may provide guidance on the preparation of the biological sample, the coating of the microtiter plate with the recombinant proteins, the addition of the biological sample and the enzyme-linked antibody, the washing steps to remove unbound material, the addition of the substrate, and the interpretation of the results.

[0108] The diagnostic kit may comprise a secondary antibody. In a preferred embodiment, a secondary antibody is an antibody binding specifically to all antibodies from an antibody class, preferably a mammalian antibody class, more preferably human antibody class such as IgG. Secondary antibodies typically recognize the constant domain of said class but may also recognize other epitopes shared by antibodies from the class of interest, for example a conformational epitope across the 3D structure. A wide range of them is commercially available, for example from Thermo Fisher. It may be a monoclonal or a polyclonal antibody. In a preferred embodiment, the term “recognized”, as used herein, means that the secondary antibody binds specifically to the antibody or antibodies to be detected. A secondary antibody may bind specifically to all isotypes from the antibody class. For example a secondary antibody to IgG class antibodies may bind to lgG1 , lgG2, lgG3 and lgG4 isotypes. This may be achieved by using as a secondary antibody to the class, preferably to IgG class antibodies, a mixture comprising an antibody binding specifically to each IgG isotype or a single antibody which reacts with all isotypes of interest. The use of secondary antibodies is explained in Kruger, N. J., Detection of Polypeptides on Blots Using Secondary Antibodies, in The Protein Protocols Handbook (ed. J. M. Walker), page 967, volume 1996, Springer. Briefly, such secondary antibodies may be generated by immunizing a laboratory animal with the antibody to be recognized or a mixture of the antibodies to be recognized.

[0109] In a preferred embodiment, the term „autoantibody“, used interchangeably with the term “anti-X”, wherein “X” represents an autoantigen, as used herein, refers to an antibody binding specifically to an endogenous molecule of the animal, preferably mammal, more preferably human, which produces said autoantibody, wherein the level of such antibody is more preferably elevated compared to the average healthy subject. The autoantibody may have the sequence of an antibody’s constant regions from the animal, preferably human, making it, but the variable region is able to bind specifically to the endogenous molecule of the animal, more specifically an autoantigen or an epitope thereof, preferably from the group comprising Podocin, Kirrell and PTPRO, preferably Podocin. In a preferred embodiment, the autoantibody is isolated and / or purified from a sample, preferably tissue, serum, plasma, blood or CSF from the animal, preferably human. The autoantibody is a polyclonal, native antibody from the animal rather than a synthetic or recombinant antibody. The autoantibody may be part of a composition which comprises a preservative such as azide or a protease inhibitor. The autoantibody may be in a diluted blood sample, preferably diluted using an aqueous buffer. The autoantibody may serve as a positive control to develop a diagnostic kit or reagent or confirm the quality or diagnostic kit or may be included in the kit as a positive control or as a reagent, for example as a ligand competing with an autoantibody to be detected. It may be labeled with a detectable label. In a preferred embodiment, the autoantibody is an autoantibody binding to Podocin. In a preferred embodiment, the autoantibody is an autoantibody binding to Kirrell . In a preferred embodiment, the autoantibody is an autoantibody binding to PTPRO.

[0110] The autoantibody to be detected or a secondary antibody used binds preferably specifically to the autoantigen or antibody to be detected, 5 respectively, preferably Podocin, Kirrell and PTPRO, more preferably

[0111] Podocin in the case of an autoantibody to be detected. In a preferred embodiment, the term “binding specifically”, as used herein, preferably means that the binding reaction is stronger than a binding reaction characterized by a dissociation constant of 1 x 10’5M, more preferably 1 x 10 10’7M, more preferably 1 x 10’8M, more preferably 1 x 10’9M, more preferably 1 x 10’1° M, more preferably 1 x 10’11M, more preferably 1 x 10’ 12M, as determined by surface plasmon resonance using Biacore equipment at 25 °C in PBS buffer at pH 7.

[0112] In a preferred embodiment, any information or data demonstrating the 15 presence of absence of the autoantibody may be communicated to the patient or a medical doctor treating the patient, preferably by telephone, by fax, in a written form or via the internet, for example as an email or text message.

[0113]

[0114]

[0115] EXAMPLES

[0116] Example 1 : Identification of Autoantibodies against Podocin, Kirrell and

[0117] 5 PTPRO

[0118] By applying high-resolution microscopy and immunolabeling for IgG and Nephrin and high-resolution confocal microscopy to a large cohort of kidney biopsies, the inventors recently identified IgG deposits on the slit diaphragm in patients with podocytopathies and showed that in many cases these did not colocalize with Nephrin (3). Patients without IgG- Nephrin colocalization on kidney biopsy also tested negative for anti- Nephrin antibodies in serum (3), suggesting they may have autoantibodies recognizing other antigens of the slit diaphragm.

[0119] Based on this evidence, the inventors screened 42 kidney biopsies positive for anti-slit antibodies for different slit antigens (Nephrin, Podocin, Kirrell , Kirrel2, Actinin alpha4, PTPRO, FAT1 ) by high resolution confocal microscopy. For high resolution confocal microscopy, 10 pm cryosections were stained, as previously reported (Raglianti V et al, Kidney Int. 2024, PMID 39368741 ). Immunolabeling experiments were done in a blinded fashion by two independent investigators. Briefly, sections were fixed in 95% cold ethanol for 10 minutes and subsequently blocked for one hour at room temperature with phosphate buffer saline supplemented with 2% bovine serum albumin and 2% fetal bovine serum. All antibodies were diluted in this blocking solution and incubated for one hour at room temperature. Nephrin was detected using 4 pg / ml primary polyclonal sheep anti-human nephrin (R&D Systems; AF4269), followed by a secondary Alexa Fluor 555-conjugated donkey anti-sheep IgG (Invitrogen; A-21436) diluted 1 :100. Podocin was detected using 20 pg / ml primary polyclonal rabbit anti-human Podocin (Merck; P0372), followed by a secondary Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Invitrogen; A-31572) diluted 1 :100. Kirrel 1 was detected using a primary polyclonal rabbit anti-human Kirrel 1 (Merck; ABS1511 ) diluted 1 :50, followed by a secondary Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Invitrogen; A-31572) diluted 1 :100. Kirrel 2 was detected using a primary polyclonal rabbit anti-human Kirrel 2 (Merck; HPA071587) diluted 1 :100, followed by a secondary Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Invitrogen; A-31572) diluted 1 :100. Actinin alpha4 was detected using 2.5 pg / ml primary polyclonal rabbit anti-human Actinin alpha4 (BiCell Scientific; #00174), followed by a secondary Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Invitrogen; A-31572) diluted 1 :100. PTPRO was detected using 1.5 pg / ml primary polyclonal rabbit anti-human PTPRO (Thermofisher; PA5-56964), followed by a secondary Alexa Fluor 555- conjugated donkey anti-rabbit IgG (Invitrogen; A-31572) diluted 1 :100. FAT1 was detected using 4 pg / ml primary polyclonal rabbit anti-human FAT1 (Merck; HPA023882), followed by a secondary Alexa Fluor 555- conjugated donkey anti-rabbit IgG (Invitrogen; A-31572) diluted 1 :100. IgG immune deposits were detected using 1.1 pg / ml primary monoclonal mouse anti-human IgG antibody (Abeam; ab200699), followed by a secondary AlexaFluor 488-conjugated goat anti-mouse lgG2a (Invitrogen; A-21131 ) diluted 1 :500. Images were acquired on a Leica Stellaris 5 confocal microscope, with Leica HC PL APO CS2 100x / 1.40 oil objective and highly sensitive and low noise hybrid detectors (HyD). In order to obtain a high resolution (i.e. 140 nm), we used sub-airy unit pinhole size (0.5 AU) and deconvolution algorithms (Huygens Professional software, Scientific Volume Imaging B.V.) to benefit from a superior performance of the confocal device. The results obtained in biopsies negative for IgG- nephrin colabeling, suggested a possible IgG co-labeling for Podocin, Kirrell and PTPRO (Figure 1A-D).

[0120] Subsequent screening of biopsies for co-labeling with slit diaphragm proteins using STED microscopy identified IgG colocalization with Podocin in 26% of cases, Kirrell in 7%, and PTPRO in 23%, which demonstrated that IgG deposits were specific to Podocin, Kirrell , and PTPRO, respectively (Figure 1 E-H). Overall, 22% of cases showed IgG colabelling with more than one antigen, including Nephrin. Super-resolution microscopy techniques, such as STED microscopy, achieve a high spatial resolution, typically around 60 nm. At this nanometer-scale precision, the distances measured between two fluorescent signals approach the dimensions of macromolecular complexes. Consequently, the observation of colocalization under STED conditions provides evidence for a binding event between the two proteins. Therefore, colocalization observed with STED microscopy can be regarded as a strong indication of true proteinprotein interaction.

[0121] Example 2: Diagnostic ELISA Assay Development

[0122] Based on these findings, the inventors developed an ELISA to detect anti- Podocin antibodies in serum samples. The ELISA successfully identified anti-Podocin antibodies in all patients who had shown Podocin-IgG colocalization in kidney biopsies (Figure 11). The inventors also extended serum analysis for Podocin autoantibodies to a cohort including 66 patients with a diagnosis of podocytopathy based on availability of a serum sample (41 with also kidney biopsy available, 27 positive and 14 negative for anti-slit antibodies, and 25 idiopathic nephrotic syndrome). Overall anti-Podocin antibodies were observed in 18% of 66 cases. Additionally, an ELISA for detecting anti-Kirrel1 antibodies was developed, identifying the presence of these antibodies in serum in 4.5% of the same 66 cases, including patients with Kirrell antibodies found in the biopsy, suggesting high specificity (Figure 1J). One hundred controls affected by non-proteinuric kidney diseases all tested negative for Podocin or Kirrell antibodies (Figure 11, J). Finally, the inventors developed an ELISA assay for detecting anti-PTPRO antibodies that identified the presence of these antibodies in 18% of 17 cases, including patients with PTPRO antibodies found in the biopsy, confirming the high specificity (Figure 1 K). By contrast, 15 controls affected by non-proteinuric kidney diseases all tested negative (Figure 1 K).

[0123] The ELISA tests offer a reliable and non-invasive alternative to kidney biopsy, which can be used to guide treatment decisions. The ability to detect these autoantibodies enables more accurate diagnosis and allows for the personalization of treatments, especially in cases where immunosuppressive therapy may be beneficial. Protocol to detect anti Podocin, anti-Kirrel1 and anti-PTPRO Antibodies ELISA plates were coated with 100 ng per well of recombinant human Podocin (amino acid residues 259-383 KVERIEIKDVRLPAGLQHSLAVEAEAQRQAKVRMIAAEAEKAASESLRMA AEILSGTPAAVQLRYLHTLQSLSTEKPSTWLPLPFDLLNCLSSPSNRTQ GSLPFPSPSKPVEPLNPKKKDSPML, SEQ ID NO. 8), or recombinant human Kirrell (amino acid residues 17- 493QGTQTRFSQEPADQTWAGQRAVLPCVLLNYSGIVQWTKDGLALG MGQGLKAWPRYRWGSADAGQYNLEITDAELSDDASYECQATEAALRS RRAKLTVLIPPEDTRIDGGPVILLQAGTPHNLTCRAFNAKPAATIIWFRDG TQQEGAVASTELLKDGKRETTVSQLLINPTDLDIGRVFTCRSMNEAIPSG KETSIELDVHHPPTVTLSIEPQTVQEGERWFTCQATANPEILGYRWAKG GFLIEDAHESRYETNVDYSFFTEPVSCEVHNKVGSTNVSTLVNVHFAPRI VVDPKPTTTDIGSDVTLTCV WGNPPLTLTWTKKDSNMVLSNSNQLLLK SVTQADAGTYTCRAIVPRIGVAEREVPLYVNGPPIISSEAVQYAVRGDGG KVECFIGSTPPPDRIAWAWKENFLEVGTLERYTVERTNSGSGVLSTLTIN NVMEADFQTHYNCTAWNSFGPGTAIIQLEEREVL, SEQ ID NO. 9) or recombinant human PTPRO (amino acid residues 1- 593MGHLPTGIHGARRLLPLLWLFVLFKNATAFHVTVQDDNNIWSLEAS DVISPASVYWKITGESKNYFFEFEEFNSTLPPPVIFKASYHGLYYIITLWV NGNVVTKPSRSITVLTKPLPVTSVSIDYKPSPETGVLFEIHYPEKYNVFTR VNISYWEGKDFRTMLYKDFFKGKTVFNHWLPGMCYSNITFQLVSEATFN KSTLVEYSGVSHEPKQHRTAPYPPQNISVRIVNLNKNNWEEQSGNFPEE SFMRSQDTIGKEKLFHFTEETPEIPSGNISSGWPDFNSSDYETTSQPYW WDSASAAPESEDEFVSVLPMEYENNSTLSETEKSTSGSFSFFPVQMILT WLPPKPPTAFDGFHIHIEREENFTEYLMVDEEAHEFVAELKEPGKYKLSV TTFSSSGSCETRKSQSAKSLSFYISPSGEWIEELTEKPQHVSVHVLSSTT ALMSWTSSQENYNSTIVSWSLTCQKQKESQRLEKQYCTQVNSSKPIIE NLVPGAQYQWIYLRKGPLIGPPSDPVTFAIVPTGIKDLMLYPLGPTAWL S WTRPYLGVFRKYWEMFYFN PATMTSEWTTYYE IAATVS LTAS Wl FP , SEQ ID NO. 10) and incubated ON at 4°C(Podocin: PO 9287, R&D Biotechne; Kirrell : 10165 K1 , R&D Biotechne; PTPRO: ab114815, abeam). Briefly, 100 ng / well of recombinant human protein was diluted in carbonate coating Buffer (Thermo Fisher catalog number CB01100) and incubated overnight at 4°C. Plates were then washed 3x with PBS (Catalog number 14190-094 Gibco) with 0.05% Tween 20 (catalog number: P1379 Merck) and blocked with SuperBlock (Thermo Fisher Catalog number 37515). (Raglianti V et al, JASN 2025, PMID 39883528). All recombinant proteins were diluted with coating buffer. Control wells incubated ON at 4°C with coating buffer in the absence of recombinant proteins were used to determine nonspecific binding (in the absence of antigen) for each patient sample, and this allowed for background subtraction. Sample concentration was determined based on a double dilution series of an antibody of known concentration (anti-Podocin corresponding to amino acid residues 367-383; anti-Kirrel1 corresponding to amino acid residues 17-200, anti-PTPRO corresponding to amino acid residues 151-284) Commercially available antibodies (Podocin: product number P0372, Merck; Kirrell : product number ABIN7268117, Antibodies Online; PTPRO: product number abPA56964 Invitrogen) were used..

[0124] Plates were washed, blocked, washed again with PBS (Catalog number 14190-094 Gibco) with 0.05% Tween 20 (catalog number: P1379 Merck) and incubated with biotin-conjugated goat anti-human IgG Fc (Secondary antobody: goat anti-human IgG Fc highly cross-adsorbed biotin conjugated A18833 Thermo Fisher) for the patient sample and with goat anti-rabbit IgG Fc for the antibodies employed to determine sample concentration. Plates were then washed again and incubated with HRP- conjugated avidin (HRP AVIDIN: catalog number 405103 BioLegend). After washing, substrate was added, and the absorbance was measured at 450 nm.

[0125] Therapeutic Implications

[0126] Patients identified as having autoantibodies against Podocin or Kirrell or PTPRO benefit from immunosuppressive therapy, which can mitigate the autoimmune-driven podocyte damage. Indeed, we observed reduction of the titer of anti-Podocin antibodies after treatment with steroids in parallel to reduction and disappearance of proteinuria. In contrast, patients without these autoantibodies may not respond well to such treatments, and alternative therapeutic strategies may be more appropriate. The identification of these autoantibodies allows for tailored treatment protocols that can improve patient outcomes and minimize unnecessary exposure to medications with potential side effects.

[0127] Example 3: Diagnostic immunofluorescence assay

[0128] HEK 293 cells transiently transfected with an empty pTriEx-1 vector expressing a His-tagged Podocin (SEQ ID NO. 7) were grown in DMEM medium comprising 10% heat inactivated fetal calf serum and 1 % antibiotic-antimycotic (Invitrogen # 15240) at 37 °C and 5% CO2. For transfections the ExGen500 protocol (Catalog number 12783652, Thermo Fisher) was used. Fixed cells were prepared by contacting cells grown on microscopy slides with formalin by washing the cells in PBS followed by brief incubation in formalin (1 ,825 % in PBD).

[0129] The methodology and reagents used were according to the manufacturer’s instruction in the IIFT Neurology Mosaics (EUROIMMUN Medizinische Labordiagnostika AG, product number FA112d-1 ). The test system centers around the incubation of combinations of substrates with diluted patient sample. If the reaction is positive, specific antibodies of classes IgA, IgG and IgM attach to the antigens. In a second step, the attached antibodies are stained with FITC-labelled anti-human antibodies and made visible with a fluorescence microscope.

[0130] Briefly, human serum samples were diluted in PBS-Tween, followed by vortexing for 2 seconds. 30 pl sample per field was incubated for 30 minutes using the TITERPLANE technology, followed by washing in PBS- Tween for 1 s, followed by incubation in PBS-Tween for 5 minutes in a cuvette for thorough washing. 25 pl of secondary antibody conjugate was then applied and incubated for 30 minutes using the TITERPLANE technology, followed by washing in PBS-Tween for 1 s, followed by incubation in PBS-Tween for 5 minutes in a cuvette for thorough washing.

[0131] After the incubations, the carrier with the fields was covered with up to 10 pl of mounting medium per field and a glass cover slide, followed by fluorescence microscopy analysis using a EUROSTAR microscope (EUROIMMUN Medizinische Labordiagnostika AG, Lubeck).

[0132] Reactivity with a commercial antibody recognizing the C-terminal His tag confirmed that the full-length protein was expressed (Fig. 2A, left panel). As expected, no fluorescence could be observed if an empty vector was used as a control (Fig. 2A, right panel).

[0133] Fig. 2B shows cells expressing Podocin following incubation with a sample from a healthy subject and Fig. 2C with a patient sample. It is clear that a detectable immunofluorescence activity can be observed, demonstrating that immunofluorescence is another method that may be used to detect the autoantibodies.

[0134] Example 4: Anti-podocin Enzyme-Linked Immunosorbent Assay Guides Immunotherapy in Steroid-Resistant Nephrotic Syndrome

[0135] Here, we report a case where the novel anti-podocin (AP) IgG ELISA could: (i) clarify autoimmunity as a cause of SRNS, (ii) identify lack of immunological response to steroids underlying the incomplete clinical response, and c) identify immediate relapse after an initial immunological response to retuximab (RTX). In addition, monitoring serum AP IgG levels was key in guiding the decision for deep B-cell depletion with obinutuzumab and in confirming the immunological response to the latter.

[0136] Fig. 3 shows the integrated assessment of clinical progression, autoimmune profile, and renal biopsy results. In the first kidney biopsy: (a) representative image of routine immunofluorescence revealing the absence of glomerular deposition of IgG. Bar = 25 pm. (b) Representative image of high-resolution confocal microscopy detecting IgG (green) deposition along the slit diaphragm, colocalized (yellow) with podocin (red). Bar = 25pm. (c) Representative super resolution stimulated emission depletion (STED) microscopy image confirming IgG (green) colocalization (yellow) with podocin (red) and the details split by channel (C’,C”) Bar = 2pm. Representative fluorescence intensity profile plot (inlet) showing the complete overlap between IgG (green) and podocin (red) signals, (d) Representative super resolution STED microscopy showing no IgG (green) colocalization with nephrin (red) and the details split by channel (D’,D”). Bar = 2pm. Representative fluorescence intensity profile plot (inlet) showing the lack of overlap between IgG (green) and nephrin (red) signals. In the second biopsy: (e) representative image of routine immunofluorescence showing a mild deposition of IgG. (f) Representative image of high-resolution confocal microscopy showing that a part of IgG (green) colocalized (yellow) with podocin (red). Bar = 25pm. A detail in (g). (h) Representative image of immunofluorescence staining showing colocalization (yellow) of IgG (green) and C1q (red). Bar = 25pm. (i) Time course showing the consistently aligned trends of proteinuria (black triangle) and serum anti-podocin IgG levels (red dot), which move in line. In contrast, serum antinephrin IgG levels (empty dot) do not follow the same pattern. Values are expressed in relative units (Rll / rnl) due to the patient’s severe persistent hypogammaglobulinemia. RTX, rituximab; STED, stimulated emission depletion; uPCR, urinary protein-to-creatinine ratio. *kidney biopsy.

[0137] A 5-year-old white female presented with sudden onset nephrotic syndrome (NS) shortly after an upper respiratory tract infection. Initial laboratory evaluation showed an increased urinary protein-to-creatinine ratio of 18.5 mg / mg, reduced levels of serum albumin (1.39 g / dl) and IgG (139 mg / dl), but an elevated low-density lipoprotein cholesterol of 295 mg / dl. The serum creatinine of 0.39 mg / dl was normal for her age and body mass. After clinical exclusion of secondary causes, treatment with prednisone 60 mg / m2 / d was initiated. Concurrently, the patient developed symptoms and laboratory findings consistent with de novo type 1 diabetes mellitus controlled well with insulin. Over 4 weeks, proteinuria did not change, so the patient was classified as steroid-resistant nephrotic syndrom (SRNS). Antiproteinuric therapy with ramipril was initiated and a kidney biopsy was performed (Fig. 3 a-d). Routine light microscopy revealed minimal changes and standard immunofluorescence was negative (Fig. 3 a).

[0138] However, high-resolution microscopy localized IgG deposits along the filtration slit (Fig. 3 b). Superresolution stimulated emission depletion microscopy further revealed that IgG colocalized with podocin and not with nephrin (Fig. 3 c-d). Subsequent serum ELISA testing identified the presence of AP IgG, while anti-nephrin (AN) and anti-Kirrel1 (AK) IgG were absent, supporting the diagnosis of a steroid-resistant anti-podocin autoimmune podocytopathy (APAuP) and excluding anti-nephrin autoimmune podocytopathy (ANAuP) or anti-Kirrel1 autoimmune podocytopathy (AKAuP). Whole-exome sequencing excluded a monogenic podocytopathy or phenocopies. After having confirmed the diagnosis of an autoimmune podocytopathy underlying the severe SRNS (Fig. 3 c-d), 2 doses of RTX (375 mg / m2) were administered 2 weeks apart (Fig. 3 i) followed by complete depletion of circulating CD19+ B cells. RTX therapy resulted in a decline of urinary protein-to-creatinine ratio from 11 to 5.9 mg / mg (Fig. 3 i) and edema, hypoalbuminemia, as well as hypogammaglobulinemia improved. Two weeks after the second RTX dose, the patient developed a respiratory infection with a throat swab positive for Chlamydia pneumoniae. Thoracic ultrasound and x-rays confirmed bilateral pneumonia, thus antibiotic treatment with macrolides was started. The infection triggered a sudden relapse of severe NS with diuretic-resistant edema and a corresponding increase in the urinary protein-to-creatinine ratio (11-98 mg / mg), new onset of microhematuria and leukocyturia, and serum creatinine increase from 0.33 mg / dl to 1.3 mg / dl (Fig. 3 i). This relapse of NS was accompanied with serum AP IgG levels increasing up to 3 times the levels detected at onset, whereas AN IgG levels remained negative (Fig. 3 i and Table 1 ). In addition, functional complement assays indicated classical complement pathway activation (Wieslab test, increased sC5b-9, increased c3d). With the clinical suspect of an infection-triggered rapidly progressive glomerulonephritis (RPGN) superimposed on SRNS, we performed a second kidney biopsy which confirmed a proliferative glomerulonephritis (GN) with few crescents, as well as IgG and C3 deposition along the glomerular basement membrane (Fig. 3 e, Table 1 ). High-resolution microscopy revealed extensive and severe disruption of the slit diaphragm, with a granular pattern of IgG and podocin colocalization (Fig. 3 f and g). Furthermore, a part of IgG deposits showed colocalization with C1q, confirming activation of the classical complement pathway (Fig. 3 h). Given the rapidly progressive glomerulonephritis (RPGN), evidence of classical complement pathway activation, and aiming to avoid further corticosteroid use for the concomitant type 1 diabetes mellitus, we used the complement C5 blocker eculizumab (Fig. 3 i). This therapy induced a rapid decline in serum creatinine from 1.3 mg / dl to 0.7 mg / dl, but no improvement in NS, suggesting a good treatment effect of eculizumab on the infection-related RPGN (Fig. 3 i). However, serum creatinine did not decrease further below 0.7 mg / dl, and severe NS persisted along with high serum levels of AP IgG (Fig. 3 i). In this constellation of RTX-resistant APAuP, we did not consider readministering eculizumab or using a calcineurin inhibitor, mycophenolate mofetil, or cyclophosphamide (Table 1 ).

[0139] AKAuP, anti-Kirrel1 AuP; ANAuP, antinephrin AuP; APAuP, antipodocin AuP; AuP, autoimmune podocytopathy; CF, cyclophosphamide; CNI, calcineurin inhibitors; ELISA, enzyme-linked immunoadsorbent assay; MMF, mycophenolate mofetil; RPGN, rapidly progressive glomerulonephritis; RTX, rituximab; SRNS, steroid-resistant nephrotic syndrome.

[0140] Instead, we opted to treat this proven form of an autoimmune NS like other RTX-resistant autoimmune diseases with the second-generation B-cell- depleting drug, obinutuzumab (Fig. 3 i, Table 1 ). Upon treatment with 1 g / m2obinutuzumab, serum AP IgG levels rapidly declined as did serum creatinine levels to 0.3 mg / dl. This was followed by a gradual improvement in proteinuria up to urinary protein-to-creatinine ratio of 1 mg / mg and a complete clinical remission of NS (Fig. 3 i). i, Table 1 ). Upon treatment with 1 g / m2obinutuzumab, serum AP IgG levels rapidly declined as did serum creatinine levels to 0.3 mg / dl. This was followed by a gradual improvement in proteinuria up to urinary protein-to-creatinine ratio of 1 mg / mg and a complete clinical remission of NS (Fig. 3 i).

[0141] This case illustrates the complexity of RTX-resistant SRNS in a pediatric patient, complicated by RPGN triggered by pneumonia. This case underscores 3 key points as follows:

[0142] (i) Diagnostic utility of AP ELISA: serum AP IgG provided a rapid, noninvasive diagnosis of AuP (i.e., APAuP), distinguishing it from other forms such as ANAuP and AKAuP. In our case, only AP IgG was detected, whereas AN and AK antibodies were absent. Given the technical challenges associated with AN ELISA, all 3 assays were previously validated in a large idiopathic NS cohort by comparing serum results with IgG presence and colocalization with nephrin, podocin, or Kirrell in kidney biopsies of the same patients, assessed by using stimulated emission depletion microscopy. Notably, the AP ELISA showed high diagnostic accuracy, with 100% sensitivity and 97% specificity, as previously detailed. This ELISA works similarly to anti-phospholipase A2 receptor antibody testing and allows repeated measurement to guide treatment.

[0143] (ii). Monitoring disease activity: beyond diagnosis, AP IgG ELISA enabled real-time monitoring of disease activity, and outperformed CD19-based 13- cell tracking. In APAuP, like ANAuP, proteinuria improved in parallel with antibody decline; unlike phospholipase A2 receptor antibody-related disease, where immune deposits delay remission. This correlation helps explain varying clinicopathological presentations in antibody-mediated podocytopathies.

[0144] (iii). Therapeutic guidance: the identification of AP antibodies directly influenced treatment, avoiding a trial-and-error approach. Conventional secondline therapies (calcineurin inhibitor, mycophenolate mofetil, and cyclophosphamide) were bypassed, because the ELISA pointed to autoreactive B cells as the pathogenic driver. Obinutuzumab was selected for its superior B-cell depleting efficacy, supported by experience in RTX- resistant phospholipase A2 receptor antibody-positive NS and lupus nephritis.

[0145] This case also offers broader clinical insights. First, APAuP may coexist with type 1 diabetes. This overlap could reflect shared autoimmune predisposition or cross-reactivity, because podocin is expressed in pancreatic tissue. Second, APAuP may evolve into RPGN with hematuria and complement activation, particularly in the setting of Chlamydia pneumoniae infection. In this case, eculizumab led to transient improvement; however, NS persisted until AP IgG was cleared, suggesting that extremely high AP IgG levels — amplified during pneumonia — may have contributed to this severe course. Indeed, AP antibodies levels mirrored not only NS progression, but also renal function decline and hematuria onset.

[0146] In summary, assays for the detection of anti-slit diaphragm antibodies, autoantibodies such as anti-podocin, anti-Kirrel-1 and anti-PTPRO offer a promising tool for diagnosing, monitoring and guiding treatment of AuPs.

[0147] Example 5: Flow Cytometry (FACS) Analysis of Podocyte Autoantibody Binding

[0148] In this study, flow cytometry (FACS) was performed to evaluate the presence of circulating autoantibodies directed against podocyte slit diaphragm proteins in patient sera. Specifically, sera from patients presenting anti-slit autoantibodies (e.g., recognizing podocin or nephrin) were tested and compared with sera collected from the same patients during disease remission. The rationale was to assess whether patient- derived antibodies can bind to podocytes in vitro, thereby supporting the hypothesis of an autoimmune component targeting podocyte-specific proteins. Although this assay does not identify which specific podocyte antigen is recognized, it provides a valuable complementary method to support findings obtained by ELISA and super-resolution microscopy. For this analysis, podocytes derived from differentiated renal progenitor cells (RPCs) were used (4). Cells were detached at 60-80% confluency using a 5 mM EDTA solution and then divided into 60.000 cell per tubes. The cells were first washed with phosphate-buffered saline (PBS) and subsequently fixed in cold 95% ethanol for 10 minutes. After fixation, cells were washed twice with PBS containing 0.5% bovine serum albumin and 0.02% sodium azide to minimize nonspecific binding. To block Fc receptors and prevent background signal, 5 pL of FcR blocking reagent human (Miltenyi Biotec; cat. 130-059-901 ) were added to each cell pellet, eceptors and prevent background signal, 5 pL of FcR blocking reagent human (Miltenyi Biotec; cat. 130-059-901 ) were added to each cell pellet. For antibody binding, 25 pL of patient serum were added directly to the blocked cell suspension and incubated for 30 minutes at room temperature. Following incubation, the cells were washed thoroughly to remove unbound antibodies. Samples were then incubated for an additional 30 minutes on ice with a fluorophore-conjugated secondary antibody, specifically Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Invitrogen; cat. A-21445) diluted 1 :300 in PBS, allowing visualization of serum-derived antibodies bound to the podocyte surface. After the secondary antibody incubation, 500 pL of FACS buffer were added to each sample prior to acquisition. Samples were analyzed using a MACSQuant Analyzer (Miltenyi Biotec), and data were processed using FlowLogic software (Miltenyi Biotec). erum-derived antibodies bound to the podocyte surface. After the secondary antibody incubation, 500 pL of FACS buffer were added to each sample prior to acquisition. Samples were analyzed using a MACSQuant Analyzer (Miltenyi Biotec), and data were processed using FlowLogic software (Miltenyi Biotec).

[0149] Binding of the secondary antibody confirmed the presence of circulating autoantibodies in a patient who was confirmed by ELISA and high- resolution microscopy to carry anti-podocin antibodies, but not anti- nephrin, anti-Kirrel1 , or anti-PTPRO. rin, anti-Kirrel1 , or anti-PTPRO.

[0150] Figure 4 shows a representative FACS assay performed on kidney progenitor-derived cultured podocytes. Figure 4A displays the FACS plot obtained from podocytes incubated with serum from a patient in disease remission, whereas Figure 4B shows the plot obtained from a patient sample containing circulating anti-podocin autoantibodies during relapse. A clear FACS signal is detectable during relapse but not during remission, demonstrating that this technique provides an additional method to identify and confirm the presence of podocyte-targeting autoantibodies.

[0151] References

[0152] 1. Kopp JB, Anders HJ, Susztak K, Podesta MA, Remuzzi G, Hildebrandt F, Romagnani P. Podocytopathies. Nat Rev Dis Primers. 2020; 6:68.

[0153] 2. Watts AJB, Keller KH, Lerner G, Rosales I, Collins AB, Sekulic M, Waikar SS, Chandraker A, Riella LV, Alexander MP, Troost JP, Chen J, Fermin D, Yee JL, Sampson MG, Beck LH Jr, Henderson JM, Greka A, Rennke HG, Weins A. Discovery of Autoantibodies Targeting Nephrin in Minimal Change Disease Supports a Novel Autoimmune Etiology.J Am Soc Nephrol. 2022; 33:238-252.

[0154] 3. Raglianti V, Angelotti ML, Cirillo L, Ravaglia F, Landini S, Palazzo V, Melica ME, Antonelli G, Conte C, Buti E, Errichiello C, De Chiara L, Peired AJ, Lasagni L, Buccoliero AM, Marco Allinovi M, Manonelles Montero A, Cruzado JM, Bruschi M, Ghiggeri GM, Angeletti A, Anders HJ, Lazzeri E, Mazzinghi B, Becherucci F, Romagnani P. Anti-slit antibodies on kidney biopsy identify pediatric patients with steroid-resistant nephrotic syndrome responsive to second-line immunosuppressants. Kidney Int. 2024 Oct 3:S0085-2538(24)00694-X

[0155] 4. Melica ME, Angelotti ML, Antonelli G, Peired AJ, Conte C, De Chiara L, Mazzinghi B, Lazzeri E, Lasagni L, Romagnani P. Preparation of Human Kidney Progenitor Cultures and Their Differentiation into

[0156] Podocytes. Bio Protoc. 2023; 13:e4757.

Claims

CLAIMS1 . A method for assessing the presence of a kidney disease in a subject, comprising detecting in at least one biological sample isolated from said subject the presence of antibodies capable of recognising and binding at least one protein selected from the group consisting of: Kirrell , PTPRO and Podocin, or at least one epitope thereof, wherein the presence of said antibodies indicates the presence of a kidney disease in the subject.

2. The method according to claim 1 , comprising detecting in the at least one biological sample the presence antibodies capable of recognizing and binding at least two proteins selected from Kirrell , PTPRO and Podocin.

3. The method according to claim 1 or 2, wherein the kidney disease is selected from the group consisting of: a podocytopathy, proteinuria and nephrotic syndrome.

4. The method according to claim 3, wherein the kidney disease is a podocytopathy.

5. The method according to anyone of claims 1-4, wherein the at least one biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

6. The method according to anyone of claims 1-5, wherein the detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

7. The method according to anyone of claims 1-6, further comprisingselecting a treatment strategy for the subject based on the detection of autoantibodies against Kirrell , PTPRO or Podocin,, preferably the treatment strategy comprises administering immunosuppressive therapy to the subject when antibodies against Kirrell , PTPRO or Podocin are detected.

8. A method of detecting antibodies capable of recognising and binding at least one protein selected from the group consisting of: Kirrell , PTPRO and Podocin in a patient, said method comprising: a. obtaining a biological sample from the patient; and b. detecting whether the antibodies are present in the biological sample by contacting the biological sample with at least one protein selected from the group consisting of: Kirrell , PTPRO and Podocin or fragment thereof and detecting the binding between the antibodies and the at least one protein.

9. The method according to claim 8, comprising detecting in the at least one biological sample the presence antibodies capable of recognizing and binding at least two proteins selected from Kirrell , PTPRO and Podocin.

10. The method according to claim 8 or 9, comprising detecting in the at least one biological sample the presence antibodies capable of recognizing and binding Kirrell , PTPRO and Podocin.

11. The method according to anyone of claims 8-10, wherein the at least one biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

12. The method according to anyone of claims 8-11 , wherein the detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

13. The method according to anyone of claims 8-12, further comprising selecting a treatment strategy for the subject based on the detection of antibodies against Kirrell , PTPRO or Podocin.

14. The method according to claim 13, wherein the treatment strategy comprises administering immunosuppressive therapy to the subject when antibodies against Kirrell , PTPRO and Podocin are detected.

15. A diagnostic kit for detecting autoantibodies in a subject, comprising: at least one recombinant protein selected from Kirrell , PTPRO and Podocin; and a secondary antibody conjugated to a detectable label capable of recognising and binding at least one immunoglobulin domain.

16. The diagnostic kit according to claim 15, wherein kit is an enzyme- linked immunosorbent assay (ELISA).

17. The diagnostic kit according to claim 15 or 16, wherein the kit comprises at least one protein having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with at least one sequence selected from: SEQ ID NO: 1-3 or at least one protein having 85, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with at least one sequence selected from: SEQ ID NO:4-6.

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