USE OF ANTI-UCHL1 IGG PLASMA CONCENTRATION FOR DIAGNOSING IDIOPATHIC STEROID SENSITIVE NEPHROTIC SYNDROME

Abstract

The present invention relates to methods and kits for diagnosing Idiopathic steroid sensitive nephrotic syndrome. The inventors showed that idiopathic nephrotic syndrome (INS) patients display a significant plasma level of anti-UCHL1 IgG that target the podocytes. Based on the correlation between the plasma level of anti UCHL1 IgG and proteinuria, they suggested that anti UCHL1 IgG plays a central role in the development of massive proteinuria. In particular, the present invention relates to a method of determining whether a subject suffers from idiopathic steroid sensitive nephrotic syndrome (INS) comprising i) determining the concentration of plasma anti-UCHL1 IgG in a sample obtained from the subject ii) comparing the concentration determined at step i) with a predetermined reference value and iii) concluding that the subject suffer from idiopathic steroid sensitive nephrotic syndrome when the concentration determined at step i) is higher than the predetermined reference value.

Claims

1. A method of determining whether a subject suffers from idiopathic steroid sensitive nephrotic syndrome (INS) or of predicting the risk of relapse in a subject suffering from INS, comprising i) determining the concentration of plasma anti-UCHL1 IgG in a sample obtained from the subject ii) comparing the concentration determined at step i) with a predetermined reference value and iii) concluding that the subject suffers from idiopathic steroid sensitive nephrotic syndrome when the concentration determined at step i) is higher than the predetermined reference value.

2. (canceled)

3. The method of claim 1 wherein the predetermined reference value is the concentration of plasma anti-UCHL1 IgG determined in a population of healthy individuals.

4. The method of claim 3 wherein it is concluded that the patient suffers from INS or is at risk of relapse when the concentration of plasma anti-UCHL1 IgG is at least 1.5 fold higher than the concentration determined in a population of healthy individuals.

5. The method of claim 1 wherein the sample is a blood sample.

6. A method of determining whether the subject achieves a response with an immunosuppressive treatment comprising i) determining the concentration of plasma anti-UCHL1 IgG in a sample obtained from the subject before the treatment ii) determining the concentration of plasma anti-UCHL1 IgG in a sample obtained from the subject after the treatment, iii) comparing the concentration determined at step i) with the concentration determined at step ii) and iv) concluding that the subject achieves a response when the concentration determined at step ii) lower than the concentration determined at step i).

7. The method of claim 6 wherein the immunosuppressive agent is selected from the group consisting of 6-mercaptopurine (6-MP), cyclophosphamide, mycophenolate, prednisolone, sirolimus, dexamethasone, rapamycin, FK506, mizoribine, azothioprine and tacrolimus.

8. The method of claim 6 wherein the immunosuppressive agent is selected from the group consisting of calcineurin inhibitors, corticosteroids, and B cell depleting agents.

9. The method of claim 8 wherein the B cell depleting agent is ristuximab.

10. The method of claim 6 wherein a decrease in the concentration after the treatment compared to the concentration before the treatment of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% indicates that the subject achieves a response.

11. The method of claim 6 wherein a concentration which returns to the basal concentration indicates that the subject has achieved a response.

12. The method of claim 1 wherein the detection and quantification of anti-UCHL1 IgG in the sample is performed by ELISA.

13. The method of claim 1 wherein the detection and quantification of anti-UCHL1 IgG is performed with a kit or device comprising at least a UCHL1 protein or fragments thereof; at least one solid support wherein the UCHL1 protein or fragments thereof is deposited on the support and a detection antibody, wherein the detection antibody is specific for the anti-UCHL1 IgG in the sample of the subject and the detection antibody produces a detectable signal.

14. A method of treating idiopathic steroid sensitive nephrotic syndrome (INS) in a subject in need thereof comprising i) diagnosing the subject by the method of claim 1 and ii) administering to the subject a therapeutically effective amount of least one immunosuppressive agent.

15. A method of treating a subject suffering from idiopathic steroid sensitive nephrotic syndrome (INS) by removing anti-UCHL1 IgG from extracellular body fluid from the subject comprising the steps of a) providing the extracellular body fluid which has been obtained from the subject, b) contacting the extracellular body fluid with a biocompatible solid support capable of capturing the anti-UCHL1 IgG, and c) reinfusing the extracellular body fluid from step b) into the subject.

16. The method of claim 15, wherein the extracellular body fluid is blood.

Description

FIGURES

[0042] FIG. 1. Effect of plasma exposure on morphology of podocytes and neutralization with monoclonal antibodies. (A) Adherent cultured podocytes, (B) retracted podocytes incubated for 2 h with 9% plasma and (C) detached podocytes incubated for 2 h with 13% plasma, magnification 10. Adherent podocytes incubated for 2 h with (D) 20% PBS and (E) 20% FCS, magnification 10. (F) Neutralization of plasma able to detach podocytes with monoclonal anti-IgG for 30 min at 37 C. before adding 13% of this treated plasma on podocytes avoid cell detachment, magnification 10. (G) Neutralization efficacy of 13% plasma exposure for 2 h on podocytes after plasma neutralization with monoclonal anti-IgG or isotype control for 30 min at 37 C. (H) Neutralization efficacy of 13% plasma exposure for 2 h on podocytes after plasma neutralization with monoclonal anti-IgM or isotype control for 30 min at 37 C. Neutralizations with monoclonal anti-IgG, monoclonal anti-IgM and their isotype controls were performed in plasma samples able to detach podocytes from 17 controls, 23 INS proteinuria and 19 INS remission Neutralization efficacy between the 3 groups was not different (.sup.2 test). Differences in neutralization efficacy with antibodies and their isotype controls were tested using the Fisher exact test, *p<0.05, **p<0.01, ***p<0.001.

[0043] FIG. 2. Effect of plasma fraction exposure on podocytes. (A) Measurement of IgGlevels by ELISA in pools of plasma fractions in 27 controls children, 45 INS proteinuria and 17 INS remission; differences between groups were compared with the Kruskal-Wallis test for each pool of fractions. Pools of fractions from INS proteinuria had lower levels of IgG than those from INS remission (a) p<0.01, (b) p<0.05 and than those from controls (c) p<0.01 and (d) p<0.05. (B) Effect of pooled fractions 19-25 from 25 controls, 34 INS proteinuria and 11 INS remission after 2 h of incubation on podocytes. Differences between groups were compared by .sup.2 test; * p<0.05, *** p<0.001. (C) Neutralization efficacy of 11 pooled fractions 19-25 exposure on podocytes for 2 h after co-incubation of these pooled fractions 19-25 from INS proteinuria able to detach podocytes with monoclonal anti-IgG or isotype control for 30 min at 37 C. Fisher exact test was performed to compare differences in neutralization efficacy with anti-IgG and it isotype control, **p<0.01. (D) Viability of podocytes was evaluated by flow cytometry in pooled fractions 19-25 from 6 controls and 4 INS proteinuria inducing no effect on podocytes (adherence) and from 6 INS proteinuria inducing detachment of podocytes after 2 h of incubation and then treatment of all conditions with EDTA. No difference in cell viability was found between groups using .sup.2 test.

[0044] FIG. 3. Plasma anti-UCHL1 IgG level in INS patients and correlation with proteinuria. (A) Plasma anti-UCHL1 IgG were measured by ELISA in 38 control children, 42 INS patients including 58 samples of INS proteinuria and 75 samples of INS remission, 9 proteinuric children with Henoch Schonlein Purpura (HSP), 8 healthy adults and 8 proteinuric adults with minimal change disease (MCD). Differences between groups were evaluated using the non-parametric Kruskal-Wallis test; *p<0.05, **p<0.01, ***p<0.001; p<0.01 between proteinuric patients with INS and HSP; p<0.01 between MCD and healthy adults; p<0.05 between all groups of children compared to healthy adults. (D) Correlation between proteinuria and anti-UCHL1 IgG in 43 samples from the 18 INS patients who had one sample with anti-UCHL1 IgG per g of IgG>0.85 (upper limit of controls) at various stages of the disease (Spearman test). The regression curve intercepted Y axis at 0.690.13.

[0045] FIG. 4. Changes of proteinuria and serum albumin after intravenous injection of anti-UCHL1 Abs in mice.

(a) A plasma sample from a patient with high concentration of circulating anti-UCHL1 IgG Abs was submitted to affinity chromatography with a column substituted with recombinant UCHL1. Dosages of anti-UCHL1 IgG Abs showed that the purified fraction retained most of the anti-UCHL1 IgG Abs while the depleted plasma fraction contained an insignificant residual level of anti-UCHL1 IgG Abs.
(b) The effect of intravenous injections of the purified fraction of anti-UCHL1 IgG Abs () and of the depleted plasma (O) in mice were compared. A significant proteinuria was observed at 48 and 72 h after intravenous injection (n=5 mice). *p<0.05
(c) The development of proteinuria was associated with hypoalbuminemia by 72 h (C) in mice injected with the purified anti-UCHL1 IgG Abs () compared to those injected with the depleted plasma (); n=5 mice. *p<0.05

[0046] FIG. 5. Renal morphology after intravenous injection of anti-UCHL1 Abs in mice. [0047] (a,b) Kidney slides stained with Masson trichrome: the glomerular morphology was similar in mice injected with depleted plasma (a) and purified anti UGHL1 Abs (b) [0048] (c,d) Kidney slides with anti-human IgG staining: No IgG deposit was observed in glomeruli from mice injected with depleted plasma (c) and purified anti UGHL1 Abs (d). [0049] (e-h) Ultramicroscopy revealed major changes in podocyte morphology at different magnification. Mice injected with anti UCHL1 IgG abs exhibited foot process effacement compared to mice injected with depleted plasma where foot process remained perfectly delineated.

EXAMPLE 1

[0050] Material & Methods

[0051] Patients and Samples

[0052] Eighty-five INS patients sampled at various stages of the disease, i.e. in relapse and/or in remission and 79 control subjects were prospectively recruited at the Robert DebrHospital in Paris between 2010 and 2016. The study protocol was approved by the local ethics committee. Pediatric patients were enrolled following written informed consent of the parents. INS children were sampled if they were <18 year-old, at least 1 month from any steroid therapy and either 3 months from immunosuppressive therapy. INS relapse and remission were defined by the French National Authority for Health, respectively a proteinuria/creatininuria ratio >0.25 g/mmol and a hypoalbuminemia <30 g/l, and a proteinuria/creatininuria ratio <0.02 g/mmol and albuminemia >30 g/l. Due to ethical reasons in pediatric, biopsies were not performed in children <11 year-old and in adolescents when they were steroid sensitive and had no complication of INS. It is universally admitted that patients with steroid sensitive nephrotic syndrome commonly display minimal change disease at renal histology. In our series of 85 INS patients with steroid sensitive nephrotic syndrome, only 7 patients had renal histology that consistently showed minimal change disease (MCD). Control subjects were <18 year-old, had neither proteinuria nor a history of nephrotic syndrome, and they had never received steroids or immunosuppressive therapy. Additional patients with others nephropathies and healthy adults were included to validate the identified antibody in the last experiment of our study. Nine Henoch Scholein Purpura (HSP) proteinuric children were included at the Robert Debr Hospital, 8 proteinuric adults suffering from MCD and 8 healthy adult were included at the Xavier Bichat Hospital. Plasma from EDTA venous peripheral blood were collected from patients and controls matched by age and gender and were stored at 80 C. until experiments.

[0053] Plasma Fractionation

[0054] Some plasma aliquots of 500 L were fractionated by size-exclusion chromatography through a Superdex 200 HR10/30 column (GE Healthcare Life Sciences, Velizy-Villacoublay, France) connected to a High Performance Liquid Chromatography (HPLC) AKTA-basic automated liquid chromatography system (GE Healthcare Life Sciences) and plasma fractions eluted in phosphate buffer saline (PBS) were stored at 20 C. until use.

[0055] Podocyte Cell Culture

[0056] The adherent immortalized human podocyte cell line AB8/13 transfected with SV40 retrovirus was proliferated under permissive conditions (33 C., 5% CO2) and was then differentiated under non-permissive conditions (37 C., 5% CO2) as previously described. Podocytes were routinely cultured in RPMI 1640 Glutamax culture medium (Life technologies, Saint-Aubin, France) supplemented with 10% fetal calf serum (FCS, HyClone, GE Healthcare Life Sciences, Velizy-Villacoublay, France), 1% penicillin-streptomycin (Sigma-Aldrich) and 1% insulin transferrin selenium (Life Technologies, Villebon sur Yvette, France). Podocytes were used for experimentations after 14 days at 37 C. and 1 day of FCS starvation.

[0057] Incubation of Total Plasma and Plasma Fractions with Podocytes

[0058] Human podocytes cultured on uncoated 24-well plates (CytoOne, Starlab, Orsay, France) were exposed to 2%, 5%, 9%, 13%, 16% and 20% of filtered plasma or PBS or FCS diluted in culture medium during 2 h. Plasma were also neutralized with monoclonal antibodies against IgG (human anti-human IgG Fc, clone 6864, Bio-Rad AbD Serotec Ltd, Oxford, UK) or IgM (mouse anti-human IgM, clone M15/8, Bio-Rad AbD Serotec Ltd), or their irrelevant isotype controls (HuCAL Fab-dHLX-MH negative control antibody from Bio-Rad AbD Serotec Ltd and murine IgG1, clone 320 produced in our lab, respectively) for 30 min at 37 C. and then exposed at 13% in culture medium ofpodocytes during 2 h. Human podocytes cultured on collagen type 1 (0.2 mg/mL, Institut de Biotechnologies Jacques Boy, Reims, France) coated Nunc Lab-Tek 4-chamber slides (ThermoFisher Scientific, Villebon sur Yvette, France) were exposed to 50% ofplasma fractions diluted in culture medium during 2 h. Plasma fractions were neutralized with monoclonal antibodies against IgG (human anti-human IgG Fc, clone 6864, Bio-Rad AbD Serotec Ltd) or it irrelevant isotype control (HuCAL Fab-dHLX-MH negative control antibody, Bio-Rad AbD Serotec Ltd) for 30 min at 37 C. and then exposed at 50% in culture medium of podocytes during 2 h. After 2 h of total plasma or plasma fraction incubation, podocyte morphology was evaluated by light microscopy (Leica DM-IRB, Leica Microsystems, Nanterre, France).

[0059] Podocyte Viability after Plasma Fraction Incubation

[0060] After stimulation with plasma fractions, detached and adherent podocytes were incubated with 0.01 M EDTA for 3 min at 37 C., 5% CO2, then washed twice with cold buffer from fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences, Le Pont de Claix, France), followed by an incubation with annexin V coupled to FITC and propidium iodide for 15 min at room temperature. Then podocytes were collected using BD FACSDiva Software by flow cytometry (FACSCantoII, BD Biosciences). Results were analyzed using the FlowJo software (Ashland, Oreg., USA) to determine the podocyte viability.

[0061] Double Immunoprecipitation and Mass Spectrometry

[0062] Plasma fractions of interest from 3 patients sampled both in relapse and in remission and from 3 controls matched in age and sex were immunoprecipitated for 1 h at room temperature with monoclonal human anti-human IgG Fc, (clone 6864, Bio-Rad AbD Serotec Ltd) or with it irrelevant isotype control (HuCAL Fab-dHLX-MH negative control antibody, Bio-Rad AbD Serotec Ltd), both coupled to CNBr-activated Sepharose 4B beads (GE Healthcare Life Sciences), then washed 6 times with PBS-Tween 20 0.05%, and incubated with podocyte solubilized in radio-immunoprecipitation assay lysis buffer containing protease and phosphatase inhibitor cocktails (all from Sigma-Aldrich) for 1 h at room temperature before 2 last washing with PBS-Tween 20 0.05%. Beads coupled with IgG or with irrelevant isotype control from plasma fractions that have targeted podocyte proteins during double immunoprecipation were digested overnight with 500 ng of trypsin (Sequence Grade Trypsin, Promega, Charbonnires-les-Bains, France) for 16 h at room temperature. Enzymatic activity was stopped by addition of formic acid (Sigma Aldrich) to a final concentration of 3%. The resulting peptides were separated by a method that couples liquid chromatography with tandem mass spectrometry. Data were first processed with Proteome Discoverer 1.4 software (Thermo Fisher Scientific) coupled to an inhouse Mascot search server (Matrix Science, 2.4.1. version, Boston, Mass.) using UniProtKB/Swiss-Prot database and the following parameters: human species, mass tolerance of 7 ppm for precursor ions or 0.5 Da for fragments, and two missed cleavage sites. Partial chemical modifications such as oxidation, phosphorylation, acetylation, deamidation and glutathionylation, were taken into consideration for the queries. False discovery rate was estimated using a reversed database search approach. The relevant podocyte proteins targeted by IgG from plasma fractions of patients and controls were identified after exclusion of proteins also immunoprecipated by isotype control in all groups. A qualitative analysis of these results with X.sup.2 test (p-value 0.05) was performed to show if identified podocyte proteins were the target or not of IgG from the plasma fractions by comparison of the three groups. Then the relative abundance of each protein identified was estimated by label-free quantification using the Progenesis QI software (Nonlinear Dynamics Ltd, 4.1 version, Newcastle, UK) using the following procedure: chromatogram alignment, peptide abundance normalization, statistical analyses of features, and peptides identification using the Mascot server. A decoy search was performed and the significance threshold was fixed to 0.05. Peptides with an ion score less than 15 were rejected. Conflicts for the identification of some peptides were resolved manually. Peptide features with ANOVA (analysis of variance) p-value 0.05 between groups were selected as identified podocyte proteins targeted by IgG from plasma fractions of the different groups.

[0063] Identification of One Relevant IgG Directed Against Identified Targeted Podocyte Protein

[0064] Human podocytes cultured on uncoated 24-well plates (CytoOne, Starlab, Orsay, France) were exposed during 2 h to increasing concentrations (2.5 to 40 g/mL) of commercial IgG directed against 5 relevant podocyte proteins identified after mass spectrometry analysis including rabbit IgG anti-human UCHL1 (Cusabio, Wuhan, P.R. China), or rabbit IgG anti-human ERLIN2 (LSBIO, Seattle, Wash., USA), or rabbit anti-human NAMPT (Raybiotech, Norcross, Ga., USA) or rabbit IgG anti-human SND1 (LSBio), or mouse IgG anti-TCP1 alpha (Novusbio, Littleton Colo., USA). Localization of UCHL1 in podocytes was performed in adherent podocytes incubated with 0.01 M EDTA for 3 min at 37 C., 5% CO2, then washed with PBS, bovine serum albumin (BSA) 2%, Azide 0.01%. Extracellular UCHL1 staining of podocytes was performed with a rabbit anti-UCHL1 coupled to FITC (1/50 dilution, Cusabio) or isotype control coupled to Alexa Fluor 488 (Life technologies) and washed twice before analysis. Intracellular UCHL1 staining was performed with the Cytofix/Cytoperm kit (BD Biosciences) using antibodies described above. Stained podocytes were collected with BD FACSDiva Software by flow cytometry (FACSCantoII). Results were analyzed using the FlowJo software. Podocytes were allowed to adhere on fibronectin-coated coverslips. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at 37 C., then permeabilized in PBS with 0.2% saponin and 0.1% Bovin Serum Albumin (BSA) for 20 min at room temperature, stained with rabbit IgG anti-UCHL1 (1/15 dilution) vs mouse anti-ERAP2 (1/200 dilution, clone 3F5, Bio-Techne Ltd., Abingdon, UK) or mouse anti-IRAP (1/100 dilution, clone 3E1, Cell Signaling Technology) or mouse anti-gm130 (1/200 dilution, clone 35/GM130, BD Biosciences) for 1 h. After washing in PBS with 0.2% saponin and 0 0.1% BSA podocytes were stained with goat anti-rabbit coupled with Alexa Fluor 488, anti-mouse IgG coupled with Alexa Fluor 568, phalloidin coupled with Alexa Fluor 647 (all diluted at 1/50) and Dapi at 1 g/mL (all reagents from Life Technologies) for 1 h. After washing and mounting, coverslips were observed on a Leica SP8 confocal microscope (Leica Microsystems, Nanterre, France) and analyzed with Image J software.

[0065] Then podocytes were incubated with commercial IgG anti-UCHL1 at 40 g/mL preincubated for 30 min at 37 C. with increasing concentrations (60, 125, 250 and 500 g/mL) of albumin and recombinant UCHL1 produced and purified in our lab. Briefly, human UCHL1 DNA sequence was inserted in plasmid pET-28b (+) (Novagen, Millipore SAS, Molsheim, France), then recombinant UCHL1 was produced in E. coli T7 express LysY system (New England Biolabs, Evry France) and purified in HiTrap TALON crude column, 5 mL Superflow connected to an AKTA start chromatography system (both from GE Healthcare Life Sciences). Plasma fractions of interest were also preincubated with 250 and 500 g/mL of recombinant UCHL1 during 30 min at 37 C. then incubated on podocytes. After 2 h of incubation, podocytes morphology was evaluated by light microscopy (Leica DM-IRB).

[0066] Measurement of Immunoglobulins and Autoantibodies by ELISA

[0067] Concentrations of IgG and IgM in total plasma and in plasma fractions were assessed by ELISA using Bethyl kits (Bethyl Laboratories, Montgomery, Tex., USA). Concentrations of IgG and IgM anti-UCHL1 in plasma were assessed by ELISA. Microtitration plates were coated with 5 g/mL of recombinant UCHL1 (Cusabio) in PBS for 2 h at room temperature. After washing with PBS-Tween 20 0.05%, the blocking solution (PBS-BSA 1%) was added for 1 h at room temperature. Diluted plasma (1/100 and 1/200 in PBS-BSA 1% for measurement of IgG and IgM respectively) and undiluted urines were incubated over night at 4 C. After washing with PBS-Tween 20 0.05%, the detection of antibodies against UCHL1 was performed using a rabbit anti-human IgG (H+L) or a goat anti-human IgM, both coupled with alkaline phosphatase (1/2000, Southern Biotech, Birmingham, Ala., USA) and substrate (SIGMAFAST p-nitrophenyl phosphate tablets; Sigma-Aldrich, Saint-Louis, Mo., USA). Positive control was a sheep IgG anti-human/mouse/rat anti-UCHL1 (R&D Systems Europe, Lille, France) detected using a rabbit anti-sheep IgG (H+L) coupled with alkaline phosphatase (Southern Biotech, Birmingham, Ala., USA). Plates were read at 405 nm in an Infinite M200 (Tecan, Manndorf, Switzerland).

[0068] Statistical Analyses

[0069] For all the data except those of proteomics, qualitative data were first analyzed using the Chi2-test and if significant, by Fisher's exact test. Spearman's rank correlation test was used to assess the correlation between 2 variables. Quantitative data were first analyzed by the non parametric Kruskal-Wallis test and if significant, the Mann-Whitney test was applied for between-group analyses. P<0.05 was considered statistically significant. Results are presented as means.e.m. Statistical analyses were performed using the GraphPad Prism 5.0 software (La Jolla, Calif., USA).

[0070] Results:

[0071] Population Characteristics

[0072] Table 1 reports characteristics of the 85 INS patients and the 76 controls. The 2 groups were not different for age and gender distributions. Controls consisted of patients with non proteinuric renal diseases. Patients were systematically sampled at distance of any treatment by prednisone or immunosuppressant drugs (Table 2). Biological findings of patients at sampling during proteinuria (n=84 samples) and remission (n=86) are provided in Table 2.

[0073] Retraction and Detachment of Cultured Podocytes Upon Total Plasma Exposure

[0074] Adherent cultured podocytes (FIG. 1A) were exposed to increasing concentrations of plasma from controls and INS patients in relapse and remission that induced significant morphological changes in a dose dependent manner in all groups (Table 3). With concentrations below 10% of plasma, podocytes remained attached on the plastic surface but displayed morphological changes marked by a foot process retraction (FIG. 1B) in 20 to 40% of controls and below 20% of INS patients. Detachment of podocytes was observed when the plasma concentration was increased over 13% (FIG. 1C). Detachment occurred immediately after exposure with a majority of plasma. A delay of 15 minutes was observed with the remaining plasma and detachment was systematically preceded by foot process retraction. Control experiments showed that either phosphate buffer saline (PBS) 20% or foetal calf serum (FCS) 20% in culture medium had no effect on podocytes (FIGS. 1D and 1E). After washing in fresh culture medium with 10% FCS, detached podocytes were also able to stick again on the plastic surface. Plasma that detached podocytes were previously incubated with either monoclonal anti-IgG or anti-IgM antibodies or irrelevant isotype controls to test the hypothesis of the involvement of immunoglobulins in detachment. Anti-IgG antibodies neutralized the detachment of podocytes (FIG. 1F) by the plasma from 50 to 60% of patients and controls, opposite to inefficient neutralization with isotype control (p<0.0001, FIG. 1G). By contrast, anti-IgM antibodies and it isotype control had no significant effect on detachment by the plasma (p=0.18, FIG. 1H). The direct contact of total plasma had finally a dose-dependent deleterious effect on adhesion of podocytes that was prevented by anti-IgG neutralization.

[0075] Detachment of Cultured Podocytes by Fractions of Plasma

[0076] While the effect of total plasma on podocytes did not showed any statistical differences between controls and INS patients, plasma were fractionated using high performance liquid chromatography by size exclusion. Thirty five plasma fractions (1 to 35) were collected. IgG were detected at various levels in all tested pooled fractions (FIG. 2A). Lowest levels of IgG were detected in many pools of fractions (13 to 31) from INS proteinuria compared to INS remission and controls (FIG. 2A), as also shown in total plasma of our cohort (Table 2). Neither pools of fractions below 18 or over 26 had no detectable effect on podocytes adhesion. By contrast, a significant detachment of podocyte was observed with 47% of pooled fractions 19-25 from INS proteinuria whereas the same fractions from 25 controls and 91% from those of INS patients in remission had no effect on podocyte adhesion (p<0.0001, FIG. 2B). After neutralization with monoclonal anti-IgG of fractions from patients with proteinuria that previously detach the cells, podocytes detachment was avoided in 88% of them (FIG. 2C). Previous incubation of the same plasma fractions with an irrelevant isotype control neutralized only 13% of them (p<0.0001). After washing in fresh culture media, podocytes detached by fraction 19-25 were still able to stick again on the plastic surface. Consistently, 90% of detached podocytes by plasma fraction 19-25 from INS patients with proteinuria have a normal viability, as they displayed few features of apoptosis or necrosis (FIG. 2D).

[0077] Identification of a Target Podocyte Protein to Patient Antibodies

[0078] The neutralization of plasma fractions 19-25 from INS patients with proteinuria by anti-IgG antibodies suggested that those plasma fractions contained antibodies targeting a specific podocyte protein. Consequently, we performed sequential immunoprecipitations, firstly for the capture of IgG contained in the plasma fractions 19-25 from 3 control children and 3 INS patients at proteinuria and remission, and secondly by the immunoadsorption of podocyte lysates on those captured IgG. The obtained IgG-podocyte protein complexes were digested by trypsin and analyzed using mass spectrometry. Two working lists of candidate podocyte proteins targeted by IgG from plasma fractions were generated.

[0079] Among the 1450 different proteins identified by qualitative analysis using Proteome Discoverer, only 29 podocyte proteins were differentially targeted by IgG of plasma fractions from the 3 control and the 3 INS patients (p<0.05). Three proteins including 2 ubiquitous proteins, i.e. cyclin-dependent kinase 1 and eukaryotic translation initiation factor 3 subunit E, and one specific protein of the podocyte, the ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), were only targeted by IgG from patients with proteinuria (p<0.01). Consequently, UCHL1 was then explored as a potential candidate podocyte target in INS proteinuria.

[0080] Moreover, among the 453 different proteins identified by Progenesis QI using quantitative analysis, only 17 podocyte proteins were differentially targeted by IgG of plasma fractions from the 3 controls and the 3 INS patients (p0.05). Eleven podocyte proteins were highly targeted by IgG from INS proteinuria compared to others groups and more investigations were conducted in the 4 proteins showing the higher fold change between groups, including erlin-2, T-complex protein 1 subunit alpha (TCP1 alpha), staphylococcal nuclease domain-containing protein 1 (SND1, all these proteins were also significantly different when targeted by IgG of the 3 groups in qualitative analysis in Supplemental Table 1, p<0.043) and nicotinamide phosphoribosyltransferase (NAMPT). Polyclonal commercial antibodies directed against the 5 candidate proteins were tested on podocytes and only one commercial anti-UCHL1 IgG was able to detach podocytes. Consistently UCHL1 showed a minor expression of the protein at the podocyte surface, but the main expression was inside the podocyte and was precisely localized in the endoplasmic reticulum of podocytes. The specificity of anti-UCHL1 IgG was also controlled by the neutralization of podocyte detachment when the antibody was previously incubated with the recombinant protein UCHL1, whereas albumin was not able to neutralize this antibody. As a control experiment, recombinant protein UCHL1 and albumin had no effect on podocyte morphology. Preincubation with recombinant protein UCHL1 can neutralize the plasma fraction of interest (19-25) from INS proteinuria, which were able to detach podocytes.

[0081] Increase of Plasma Anti-UCHL1 IgG in INS Patients with Proteinuria

[0082] A specific homemade ELISA was elaborated to detect anti-UCHL1 IgG in the plasma of different controls and patients. The concentration of anti-UCHL1 IgG was significantly increased in INS patients during proteinuria compared to control children and INS patients in remission (FIG. 3A). In addition, 9 proteinuric children with IgA nephropathy related to Henoch-Schnlein Purpura, 8 healthy adults and 8 adults with minimal change disease (MCD) showed very low plasma level of anti-UCHL1 IgG. Then, we selected the 18 INS patients with proteinuria, i.e. 42% of the tested patients that had a plasma level of anti-UCHL1 IgG over the highest level of controls. For those patients, 43 samples at various stages of the disease were available during other flares and remission phases. In those samples, the level of anti-UCHL1 IgG was significantly correlated to the level of proteinuria (FIG. 3B).

DISCUSSION

[0083] While numerous clinical facts including the efficacy of drugs targeting the B cells and the association of memory B cells with relapses, the involvement of IgG might be reconsidered in INS. Based on a cellular assay of podocyte detachment in vitro and a proteomic approach, we were able to identify an autoantibody of IgG type in the plasma of patients that targets podocytes. It turns out that the targeted protein is UCHL1, a protein mainly expressed in brain, pancreas and kidney, which controls the degradation of proteins in the proteasome through the regulation of ubiquitination.

[0084] A first step of our work was to study the effect of different concentrations of total plasma on cultured podocytes. A previous study had showed that the podocyte morphology was disrupted in the presence of 10% plasma from nephrotic patients in culture medium with a retraction of foot process formation..sup.12 Accordingly, we observed the same morphological changes with plasma concentration below 10%, but with a minority of plasma from INS patients. Paradoxically, up to 50% of control plasma were able to retract the podocytes at a concentration below 10%. Over the concentration of 10%, plasma from INS patients and controls led to podocyte detachment with a prevalence that was proportional to the increase of plasma concentration, suggesting that cultured podocytes are highly sensitive to circulating plasma proteins. Consistently, in vivo podocytes are highly protected from high concentration of plasma proteins due to the retention of plasma proteins in the glycocalyx and the inner layer of the basement membrane..sup.13 Interestingly, podocyte retraction in vitro might be understood as a model of minimal change disease while podocyte detachment is already known to be the first cellular step of focal segmental glomerulosclerosis (FSGS). Following the observation that anti-IgG antibodies were able to block the detachment of podocytes by total plasma, we found that cultured podocytes were specifically detached by specific plasma fractions obtained from INS patients with proteinuria. The fact that this fraction contained IgG and that anti-IgG were able to block the detachment of podocyte supported the involvement of specific antibodies. In a continuous effort to identify the specificity of this antibody, we were able to point UCHL1 as a podocyte target. UCHL1 was previously identified in a study of podocyte exosome from urine of healthy individuals..sup.14

[0085] Anti-UCHL1 IgG were effectively increased in 42% of the nephrotic patients of our series during proteinuria and their plasma level were correlated with intensity of proteinuria. UCHL1 is involved both in the processing of ubiquitin precursors and of ubiquitinated proteins. UCHL1 is a deubiquitination enzyme that is consequently supposed to protect intracellular proteins from the degradation in the proteasome. UCHL1 is primarily expressed in brain nerve cells and is recognized as a neuronal iconic protein..sup.15 As a matter of fact, numerous neuronal proteins are expressed in the podocytes. Consistently, Nestin, Neuron-specific enolase, Synaptopodin and UCHL1 play important roles in maintaining the formation of processes in podocytes.15 The damage of podocytes exposed to adriamycin, a toxic that gives experimental nephrotic syndrome in rats, correlated in a time manner with a down expression of those neuronal proteins resulting in the retraction of the cytoskeleton and changes in cell morphology. UCH-L1 has been associated with irreversible FSGS..sup.16

[0086] Expression of UCHL1 in podocytes has been reported in renal diseases with massive proteinuria.17 UCHL1 was uniformly highly expressed in primary membranous nephritis, while mostly absent in MCD and highly heterogeneous in FSGS which is known to result from different pathophysio logical causes..sup.17 Interestingly, the balance of the proteasome is critical for the stability of podocytes. Rac1 activation in podocytes induces rapid foot process effacement and proteinuria via podocine and nephrin degradation in the proteasomal pathway..sup.18-catenin plays a critical role in mediating podocyte injury/dysfunction in proteinuric kidney disease via the loss of podocyte-specific nephrin, podocalyxin, and synaptopodin in the podocyte due to protein degradation through the ubiquitin proteasomal pathway..sup.19

[0087] This is undoubtful that circulating anti-UCHL1 IgG can be physiologically internalized into the podocyte through the neonatal Fc receptor as any antibodies that cross the endothelial layer and the glomerular basement membrane. The transcytosis of antibodies to the urinary chamber ogf the glomerula has been shown to avoid the accumulation of antibodies in the subpodocyte space..sup.20 One appealing hypothesis is that intracellular UCHL1 might be accidentally redirected in the urinary space with anti-UCHL1 IgG leading to a podocyte depletion in UCHL1. Consistently, it is supposed that podocyte depletion in UCHL1 activate cytoskeleton protein degradation in the podocyte due to the lack of de-ubiquitination and lead to proteinuria and foot process effacement. As a matter of fact, a lot of podocyte specific proteins (podocin, synaptopodin, nephrin and galectin-1) have a decreased expression during the phase of proteinuria..sup.12, 21 Moreover, the level of proteinuria has been inversely correlated to the level of nephrin expression in the podocyte..sup.22 This mechanism also fits with the complete reversibility of the disease via a rapid restoration of podocyte when IgG anti-UCHL1 are cleared from the plasma after a treatment with steroids or immunosuppressive drugs.

[0088] In conclusion, a set of INS patients display a significant plasma level of anti-UCHL1 IgG that target the podocytes. Based on the correlation between the plasma level of anti UCHL1 IgG and proteinuria, we suggest that anti UCHL1 IgG plays a central role in the development of massive proteinuria.

[0089] Tables:

TABLE-US-00001 TABLE 1 General characteristics of the patients and controls. Controls* INS patients P Demographics Number of patients (n) 76 85 N/A Gender ratio (male/female) 49/27 57/29 0.52 Age (years) 8.9 (6.4-12.9) 9.5 (6.3-13.1) 0.51 Disease characteristics Age at onset (years) N/A 3.7 (2.3-6.2) 0.99 First flare (n) N/A 25 <0.0001 Steroid sensitivity (n) N/A 12 0.25 Steroid dependency (n) N/A 48 <0.0001 History of previous therapies None (n) 76 25 Prednisone (n) 0 60 Mycophenolate mofetil (n) 0 29 Anti-calcineurin inhibitors (n) 0 23 Rituximab (n) 0 26 *Controls included 41 congenital anomalies of the kidney and the urinary tract, 13 tubular diseases, 6 isolated hematuria, 5 ciliopathies, 5 lithiasis, 5 miscellaneous kidney diseases and 4 healthy controls after systematic family investigation. INS = idiopathic nephritic syndrome; n.a. = not applicable. Data are expressed as median (interquartile range). The 2 groups of patients were equal in term of gender (p = 0.86) and age (p = 0.92).

TABLE-US-00002 TABLE 2 Clinical and biological characteristics of patients at sampling INS INS controls proteinuria remission p Number of samples 76 84 86 Number of relapses N/A 4 (0-7) 4 (2-7) 0.17 since INS onset Relapse-free period in INS (months) Steroid free period N/A 14.5 (6.3-26.6) 11.2 (5.6-19.8) 0.15 in INS (months) Immunosuppressant N/A 10.9 (6.0-22.2) 9.2 (4.8-23.8) 0.86 free period in INS (months) Proteinuria (g/mmol 0.01 (0.00-0.016).sup.a 0.365 (0.196-0.971).sup.c 0.009 (0.006-0.165).sup.a <0.0001 creatinine) Serum albumin (g/L) 44 (42-45).sup.a 26 (12-34).sup.c 42 (41-46).sup.a <0.0001 eGFR 105 (91-126)a 128 (114-147)bd 122 (103-135)b <0.0001 (mL/min/1.73 m.sup.2) Serum creatinine 46 (33-61)a 37 (29-44)bd 39 (35-54)ab 0.005 (mol/L) Plasma IgG (g/L) 6.9 (4.4-10.2)a 4.2 (2.0-6.6)c 6.5 (4.8-9.5)a <0.0001 Plasma IgM (g/L) 0.7 (0.5-1.0)a 1.0 (0.6-1.5)bc 0.8 (0.5-1.3)a 0.01 Data are expressed as median (interquartile range). INS = idiopathic nephritic syndrome including 85 patients with one or more samples at 2 stages of the disease: proteinuria and remission. eGFR = estimated glomerular filtration rate for the children using the revised Schwartz formula (k-value 50413) updated in 2009 (ref); N/A = not applicable. When superscript letters are different (a, b, c, d), medians showed significant difference between groups, P < 0.01.

EXAMPLE 2

[0090] Material & Methods

[0091] Injections of Anti-UCHL1 Abs to Mice

[0092] Anti-UCHL1 Abs were purified from 8 ml plasma from a patient with high concentration of plasma anti-UCHL1 IgG Abs using the AminoLink Plus Immobilization Kit (Thermo Scientific) and concentrated using Amicon Ultra-15 centrifugal filters with a molecular cut-off of 100 kDa to a final volume of 1 ml. Five male BALB/c mice were then intravenously injected once with an adjusted volume containing 200 g of affinity-purified anti-UCHL1 Abs. The remaining depleted serum was concentrated using Amicon Ultra-15 centrifugal filters with a molecular cut-off of 100 kDa. Five male BALB/c mice were then injected intravenously with an adjusted volume containing 200 g of total IgG. Development of proteinuria was monitored using metabolic cages every 24 h for 72 h. Kidneys at 72 h were collected, one fixed with 4% paraformaldehyde for Masson Trichrome staining and one frozen in OCT compound for immunohistochemistry with anti-human IgG coupled with biotin (Southern Biotech).

[0093] Results

[0094] Induction of Proteinuria in Mice by Injection of Patient Anti-UCHL1 IgG Abs

[0095] Human anti-UCHL1 IgG Abs purified by affinity chromatography was prepared from an INS patient at relapse with high plasma levels of anti-UCHL1 IgG Abs (FIG. 4a). Compared to mice injected with the depleted plasma from the same patient, mice injected with anti-UCHL1 IgG Abs exhibited a significantly higher proteinuria and lower serum albumin after 72 h (FIG. 4b,c). Histology examination of the kidneys harvested in sacrificed animals at 72 h showed minor morphological changes and no IgG deposits in animals injected with anti-UCHL1 IgG Abs compared to those injected with PBS and depleted plasma (FIG. 5 a-d). Ultramicroscopy showed foot process effacement in mice injected with patient's anti-UCHL1 IgG Abs while mice injected with the depleted plasma displayed foot processes with a normal shape.

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