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] Summary of the invention

[0012] In a first aspect, the present invention relates to 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.

[0013] 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. 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.

[0014] In some embodiments, the at least one biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

[0015] In some embodiments, the detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

[0016] 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.

[0017] A second aspect, relates to 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.

[0018] A third aspect relates to 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.

[0019] In some embodiments, the diagnostic kit is an enzyme-linked immunosorbent assay (ELISA).

[0020] 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.

[0021] Brief description of drawings

[0022] Figure 1 : Detection of Antibodies against-Podocin, -Kirrell and -PTPRO in kidney biopsy and serum of patients with proteinuria or nephrotic syndrome.

[0023] 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).

[0024] Detailed description of the invention

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In some embodiments, the detection of said antibodies in the biological sample indicates the presence of a kidney disease in the subject.

[0030] 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.

[0031] Preferably, the method comprises detecting in the at least one biological sample antibodies capable of recognizing and binding Podocin and Kirrell .

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In some embodiment, the kidney disease is selected from the group consisting of: a podocytopathy, proteinuria and nephrotic syndrome.

[0038] 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.

[0039] Kidney diseases associated with the presence of these autoantibodies may encompass a range of conditions, including podocytopathies, proteinuria, and nephrotic syndrome.

[0040] In some aspects, the presence of autoantibodies against at least one protein above disclosed in the biological sample may suggest the presence of podocytopathies

[0041] 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.

[0042] 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.

[0043] Preferably, the kidney disease is a podocytopathy.

[0044] 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.

[0045] In some embodiments, the biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

[0046] 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.

[0047] In some embodiments, serum provides a rich and accessible source of autoantibodies, making it a commonly used sample type in diagnostic assays.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] Preferably, detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Preferably, the method comprises detecting in the at least one biological sample antibodies capable of recognizing and binding Podocin and Kirrell .

[0068] 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.

[0069] 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.

[0070] In some embodiments, the biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

[0071] 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.

[0072] Preferably, detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

[0073] 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.

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

[0076] A third aspect of the present invention reflates to a diagnostic kit for detecting antibodies, preferably autoantibodies in a subject.

[0077] 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.

[0078] Preferably, the kit is an enzyme-linked immunosorbent assay (ELISA).

[0079] Preferably, the kit comprises Podocin and Kirrell recombinant proteins and / or fragments therefor. Preferably, the kit comprises Podocin, Kirrell and PTPRO recombinant proteins and / or fragments therefor.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In some cases, the diagnostic kit may also include reagents necessary for performing the enzyme-linked immunosorbent assay (ELISA). These 5 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 10 detectable signal, such as a colour change, indicating the presence of the autoantibodies.

[0089] 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 15 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.

[0090] In some embodiments, the diagnostic kit may also include instructions for 20 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.

[0091] 25

[0092] Table 1.

[0093] EXAMPLE

[0094] Identification of Autoantibodies against Podocin, Kirrell and PTPRO

[0095] 5 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- 10 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.

[0096] 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. The results obtained in biopsies negative for IgG-nephrin colabeling, suggested a possible IgG co-labeling for Podocin, Kirrell and PTPRO (Figure 1A-D).

[0097] 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.

[0098] Diagnostic Assay Development

[0099] 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).

[0100] 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.

[0101] 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), or recombinant human Kirrell (amino acid residues 17-493) or recombinant human PTPRO (amino acid residues 1-593) and incubated ON at 4°C. 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).

[0102] Plates were washed, blocked, washed again and incubated with biotin- conjugated goat anti-human IgG Fc 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. After washing, substrate was added, and the absorbance was measured at 450 nm.

[0103] Therapeutic Implications

[0104] 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.

[0105] References

[0106] 1. Kopp JB, Anders HJ, Susztak K, Podesta MA, Remuzzi G, Hildebrandt F, Romagnani P. Podocytopathies. Nat Rev Dis Primers. 2020; 6:68.

[0107] 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.

[0108] 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

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: 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.

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 Podocin, Kirrell and PTPRO.

3. The method according to claim 2, comprising detecting in the at least one biological sample the presence antibodies capable of recognizing and binding Podocin and Kirrell .

4. The method according to anyone of claims 1-3, comprising detecting in the at least one biological sample the presence antibodies capable of recognizing and binding Podocin, Kirrell and PTPRO.

5. The method according to anyone of claims 1-4, wherein the kidney disease is selected from the group consisting of: a podocytopathy, proteinuria and nephrotic syndrome.

6. The method according to claim 5, wherein the kidney disease is a podocytopathy.

7. The method according to anyone of claims 1-6, wherein the at least onebiological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

8. The method according to anyone of claims 1-7, wherein the detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

9. The method according to anyone of claims 1-8, further comprising selecting a treatment strategy for the subject based on the detection of autoantibodies 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.

10. 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.

11. The method according to claim 10, comprising detecting in the at least one biological sample the presence antibodies capable of recognizing and binding at least two proteins selected from Podocin, Kirrell and PTPRO.

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

13. The method according to anyone of claims 10-12, comprising detecting in the at least one biological sample the presence antibodies capable of recognizing and binding Podocin, Kirrell and PTPRO.

14. The method according to anyone of claims 10-13, wherein the at least one biological sample is selected from serum, plasma, urine, or kidney biopsy tissue.

15. The method according to anyone of claims 10-14, wherein the detecting step comprises performing an enzyme-linked immunosorbent assay (ELISA).

16. The method according to anyone of claims 10-15, further comprising selecting a treatment strategy for the subject based on the detection of antibodies against Podocin, Kirrell , or PTPRO.

17. The method according to claim 16, wherein the treatment strategy comprises administering immunosuppressive therapy to the subject when antibodies against Podocin, Kirrell , or PTPRO are detected.

18. A diagnostic kit for detecting autoantibodies 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.

19. The diagnostic kit according to claim 18, wherein kit is an enzyme- linked immunosorbent assay (ELISA).

20. The diagnostic kit according to claim 18 or 19, 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 IDNO: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 IDNO:4-6.

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