PLOS ONE RESEARCH ARTICLE Contactin-1 links autoimmune neuropathy and membranous glomerulonephritis Janev Fehmi ID1☯*, Alexander J. Davies1☯, Marilina Antonelou2, Stephen Keddie3, Sonja Pikkupeura1, Luis Querol4, Emilien Delmont5, Andrea Cortese3,6, Diego Franciotta7, Staffan Persson8, Jonathan Barratt9, Ruth Pepper2, Filipa Farinha ID10, Anisur Rahman10, Diana Canetti11, Janet A. Gilbertson11, Nigel B. Rendell11, Aleksandar Radunovic12, Thomas Minton13, Geraint Fuller14, Sinead M. Murphy15, Aisling S. Carr3, Mary R. Reilly3, Filip Eftimov16, Luuk Wieske16, Charlotte E. Teunissen16, Ian S. D. Roberts17, Neil Ashman18, Alan D. Salama2‡, Simon Rinaldi1‡ a1111111111 a1111111111 1 Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, a1111111111 United Kingdom, 2 University College London Department of Renal Medicine, Royal Free Hospital, London, a1111111111 United Kingdom, 3 Centre for Neuromuscular Disease, National Hospital of Neurology and Neurosurgery and Department of Neuromuscular Diseases, UCL Institute of Neurology, Queen Square, London, United a1111111111 Kingdom, 4 Neuromuscular Diseases Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain, 5 Referral Centre for ALS and Neuromuscular Diseases, Hospital La Timone, Marseille, France, 6 Department of Brain and Behaviour sciences, University of Pavia, Pavia, Italy, 7 IRCCS, Mondino Foundation, Pavia, Italy, 8 Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund University, Lund, Sweden, 9 Department of Cardiovascular OPEN ACCESS Sciences, University of Leicester, Leicester, United Kingdom, 10 Centre for Rheumatology and Bloomsbury Rheumatology Unit, Division of Medicine, University College London, London, United Kingdom, 11 Wolfson Citation: Fehmi J, Davies AJ, Antonelou M, Keddie Drug Discovery Unit and National Amyloidosis Centre, Centre for Amyloidosis and Acute Phase Proteins, S, Pikkupeura S, Querol L, et al. (2023) Contactin-1 Division of Medicine, University College London, London, United Kingdom, 12 Barts Neuromuscular links autoimmune neuropathy and membranous Diseases Centre, Royal London Hospital, London, United Kingdom, 13 Institute of Clinical Neurosciences, glomerulonephritis. PLoS ONE 18(3): e0281156. University of Bristol, Bristol, United Kingdom, 14 Department of Neurology, Gloucestershire Royal Hospital, https://doi.org/10.1371/journal.pone.0281156 Gloucester, United Kingdom, 15 Department of Neurology, Tallaght University Hospital & Academic Unit of Neurology, Trinity College, Dublin, Ireland, 16 Department of Neurology and Neurophysiology, Amsterdam Editor: Zhanjun Jia, University of Utah School of Neuroscience, Amsterdam UMC, Location AMC, Amsterdam, The Netherlands, 17 Department of Cellular Medicine, UNITED STATES Pathology, John Radcliffe Hospital, Oxford, United Kingdom, 18 Barts Renal Unit, The Royal London Hospital, London, United Kingdom Received: November 29, 2022 ☯ These authors contributed equally to this work. Accepted: January 11, 2023 ‡ ADS and SR also contributed equally to this work. Published: March 9, 2023 * Janev.fehmi@ndcn.ox.ac.uk Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of Abstract all of the content of peer review and author responses alongside final, published articles. The Membranous glomerulonephritis (MGN) is a common cause of nephrotic syndrome in editorial history of this article is available here: adults, mediated by glomerular antibody deposition to an increasing number of newly recog- https://doi.org/10.1371/journal.pone.0281156 nised antigens. Previous case reports have suggested an association between patients with Copyright: © 2023 Fehmi et al. This is an open anti-contactin-1 (CNTN1)-mediated neuropathies and MGN. In an observational study we access article distributed under the terms of the investigated the pathobiology and extent of this potential cause of MGN by examining the Creative Commons Attribution License, which permits unrestricted use, distribution, and association of antibodies against CNTN1 with the clinical features of a cohort of 468 patients reproduction in any medium, provided the original with suspected immune-mediated neuropathies, 295 with idiopathic MGN, and 256 controls. author and source are credited. Neuronal and glomerular binding of patient IgG, serum CNTN1 antibody and protein levels, Data Availability Statement: All relevant data are as well as immune-complex deposition were determined. We identified 15 patients with within the paper and its Supporting Information immune-mediated neuropathy and concurrent nephrotic syndrome (biopsy proven MGN in files. 12/12), and 4 patients with isolated MGN from an idiopathic MGN cohort, all seropositive for Funding: This work was supported by a GBS|CIDP IgG4 CNTN1 antibodies. CNTN1-containing immune complexes were found in the renal Foundation International Benson Fellowship PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 1 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy awarded to JF (1709HM001/SB17) (https://www. glomeruli of patients with CNTN1 antibodies, but not in control kidneys. CNTN1 peptides gbs-cidp.org/) and a Medical Research Council UK were identified in glomeruli by mass spectroscopy. CNTN1 seropositive patients were Clinician Scientist Fellowship (MR/P008399/1) (https://www.ukri.org/councils/mrc/) awarded to largely resistant to first-line neuropathy treatments but achieved a good outcome with esca- SR. AJD is supported by a UKRI Future Leaders lation therapies. Neurological and renal function improved in parallel with suppressed anti- Fellowship (MR/V02552X/1) and a Human body titres. The reason for isolated MGN without clinical neuropathy is unclear. We show Immune Discovery Initiative award funded by the that CNTN1, found in peripheral nerves and kidney glomeruli, is a common target for auto- NIHR Oxford Biomedical Research Centre. CT received support from the EU/EFPIA Innovative antibody-mediated pathology and may account for between 1 and 2% of idiopathic MGN Medicines Initiative Joint Undertaking (EMIF grant cases. Greater awareness of this cross-system syndrome should facilitate earlier diagnosis 115372) (https://european-union.europa.eu/ and more timely use of effective treatment. institutions-law-budget/institutions-and-bodies/ institutions-and-bodies-profiles/innovative-health- initiative-joint-undertaking-ihi-ju_en). AC thanks the Medical Research Council (MR/T001712/1), Fondazione CARIPLO (2019-1836), Italian Ministry of Health (Ricerca Corrente 2020), and the Introduction Inherited Neuropathy Consortium (INC) for grant support. ASC received funding from NIHR Peripheral neuropathy and renal disease commonly co-occur. In some cases, neuropathy may University College London Hospitals Biomedical be secondary to uraemia, micronutrient deficiencies or the imbalanced metabolic milieu of research centre. MA is a clinical fellow with grant renal failure [1]. Other causes include diabetes, haematological disorders such as lymphoma or reference number MR/P001777/1. FF was myeloma [2], and drugs or metabolites that are both nephro- and neuro-toxic [3, 4]. Genetic supported by LUPUS UK. The funders had no role in study design, data collection and analysis, neuropathies, such as those associated with Fabry disease [5] and Charcot-Marie-Tooth domi- decision to publish, or preparation of the nant-intermediate type E [6], can also be complicated by proteinuria and progressive renal fail- manuscript. ure. Accurate identification of the underlying cause is crucial for guiding management. Competing interests: SR runs a not-for-profit Membranous glomerulonephritis (MGN) is one of the most common causes of nephrotic diagnostic testing service for nodal/paranodal syndrome in adults and is strongly associated with autoantibodies to kidney antigens [7–9]. antibodies. All other authors have declared that no Previous small series and case reports have suggested an association between nephrotic syn- competing interests exist. This does not alter our drome and inflammatory neuropathies, namely Guillain-Barré syndrome (GBS) or chronic adherence to PLOS ONE policies on sharing data inflammatory demyelinating polyneuropathy (CIDP). However, the mechanisms linking these and materials. conditions have remained unclear. More recently, this combined presentation has been described in some patients with nodal or paranodal antibodies [10–13]. It has been speculated that this is due to a common autoimmune process involving both the peripheral nerve and kid- ney. Here, we demonstrate that antibodies targeting contactin-1 (CNTN1), a neuronal mem- brane protein anchoring paranodal myelin to the underlying axon, mechanistically connect these pathologies, in the largest cohort to date and identify a distinct and treatable neuro-renal syndrome. In addition, we show that a small percentage of idiopathic MGN may be caused by anti-CNTN1 antibodies, without overt neuropathy, and confirm that CNTN1 peptides are expressed in the affected glomeruli while RNA expression has been demonstrated in normal glomeruli, adding CNTN1 to the list of other important MGN antigens. Materials and methods Patient cohort and samples From January 2015 to August 2019 we prospectively recruited patients attending the periph- eral nerve clinic in the John Radcliffe Hospital (Oxford, UK) with either confirmed or sus- pected inflammatory neuropathy to an observational study (Research Ethics Committee approval number 14/SC/0280). These patients provided informed written consent. Serum samples from these patients, and patients with suspected inflammatory neuropathies, received by our laboratory for diagnostic testing between August 2017 and August 2019. were screened for antibodies against paranodal (CNTN1, contactin-associated protein 1—Caspr1, neurofas- cin 155—NF155) and nodal (NF140/186) antigens. PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 2 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy To investigate whether CNTN1 antibodies might be more widely associated with nephrotic syndrome caused by idiopathic MGN itself, we examined 295 serum samples from patients with idiopathic membranous nephropathy, collected as part of the MRC Glomerulonephritis bank [14]. Serum samples from 70 patients with other antibody-mediated CNS neurological disorders, 20 with multiple sclerosis, 120 individuals without neurological disease, and 46 patients with lupus nephritis, including pure class V membranous lupus nephritis, were also obtained as controls (S1 Fig). Information that could identify individual participants was stored confidentially and only accessible at the time of data collection to treating physicians, or those with necessary ethical approval and Good Clinical Practice (GCP) training. Data was subsequently de-identified. Nodal/paranodal antibody testing and binding to peripheral nerve. All sera were ini- tially screened using a live, transiently transfected cell-based assay (CBA), as previously described with slight modification [12]. Positive results, as well as titre and IgG subclass, were confirmed using ELISA. Cell based assay (CBA). Human embryonic kidney 293T (HEK) cells were either co- transfected with human DNA plasmid constructs for CNTN1 and Caspr1, NF155, or NF186 followed by incubation with patient sera. Fluorescently tagged secondary and tertiary antibod- ies against human IgG or human IgG subclasses 1–4 were used to visualise cell membrane binding by an investigator blind to the sample identity. Myelinating co-cultures. Sera were assessed for topographical binding of IgG using mye- linated co-cultures, generated using sensory neurons derived from human induced pluripotent stem cells (iPSC), according to a previously published protocol [15, 16]. For immunolabelling, live co-cultures were incubated with patient sera then fixed prior to labelling with secondary antibodies against human IgG. Teased nerve fibres. Single fibres from 4% paraformaldehyde-fixed mouse sciatic nerve were teased on glass sides, air-dried and permeabilised with 100% acetone (10 min at –20˚C), and blocked with 5% fish gelatin and 0.1% triton-X in PBS. They were incubated overnight at 4˚C with guinea pig contactin-associated protein 1 (Caspr1) antibodies (1:1000) to label the paranodes, and patient sera (1:200). Immunolabelling was visualised with fluorescent-conju- gated secondary antibodies. Human kidney CNTN1 immunohistochemistry. Tissue sections of kidney biopsies from patients 1, 2, 12 and 13 (S1 Table in S1 File), as well as healthy human kidney, were deparaffi- nised, rehydrated and underwent antigen retrieval by boiling in citrate buffer. After permeabi- lisation and blocking (0.3% triton-X 100, 10% normal donkey serum in PBS), sections were incubated overnight at 4˚C with goat anti-CNTN1 (1:800; 2.5 μg/ml) (AF904, R&D systems), followed by a biotinylated anti-rabbit secondary antibody. Endogenous peroxidase was quenched with 3% hydrogen peroxide prior to labelling with a horseradish peroxidase-strepta- vidin complex (Vectorstain Elite ABC HRP kit, Vector, PK-6100) and developed with diami- nobenzidine (DAB) reagent (SK-4100, Vector) followed by haematoxylin counterstain. Sections of healthy human cortex were used as positive control. Laser microdissection and proteomics Formalin-fixed paraffin embedded renal or human brain tissues, were laser micro-dissected and captured using the Leica LDM7 system, and analysed in a proteomics approach [17] by LC-MS/MS using a Thermo Scientific Q-Exactive Plus mass spectrometer. MS raw data were analysed by Mascot software (Matrix Science, London, UK) using the Swiss-Prot human database. PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 3 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy Pre-adsorption of sera with CNTN1 protein Sera available locally (from patients 1–3 and 12, 13 and 15 S1 Table in S1 File) were pre- adsorbed at 1:100 dilution with 1μg of CNTN1 protein overnight at 4˚C and reapplied for test- ing in the CBA and against myelinated co-cultures to confirm CNTN1 as the specific antibody target. Serum CNTN1 protein measurement Serum CNTN-1 protein levels were measured on the Luminex1 platform according to the manufacturer’s instructions (Human Magnetic Luminex Assay, R&D systems) as previously described [18]. Samples were coded randomly and analysed in duplicate. Measurements with a coefficient of variation >15% and outliers were repeated. Serum immune complex precipitation Whole sera were treated with polyethylene glycol (PEG) 6000 to a final concentration of 2.5% overnight at 4˚C. Immune complexes were precipitated as previously described [19], by centri- fugation at 2000g for 30 minutes at 4˚C, resuspending pellets in 30μl of prewarmed PBS, and analysed using SDS-PAGE and western blotting. Statistics Statistical analysis was performed using Prism 8 (version 8.0.2). Statistical significance for con- tingency data was assessed by Fishers exact test. Differences between CNTN1 antibody titres in remission versus in active disease, and in serum CNTN1 levels in the presence or absence of nephrotic syndrome, were assessed by the Mann-Whitney test. A p-value of <0.05 was consid- ered significant. Results Contactin-1 antibodies serologically link immune-mediated neuropathies to membranous glomerulonephritis causing nephrotic syndrome We prospectively screened 468 patients with suspected inflammatory neuropathies for anti- bodies against nodal and paranodal targets (CNTN1, Caspr1, neurofascin 155 –NF155, neuro- fascin 186 –NF186) using a live, cell-based assay (see methods online). Thirty-six (7.7%) patients were seropositive for antibodies targeting one of the nodal/paranodal proteins. Ten patients (2.1% of the total cohort and 27.8% of the seropositive group) were monospecific for CNTN1 antibodies (S1 Fig), and eight of these additionally had nephrotic syndrome. Among the 432 seronegative patients, 147 (34%) underwent investigation of renal function; in contrast with the seropositive group, only 5/147 (3.4%) of these had features supporting a diagnosis of nephrotic syndrome; (p<0.001, Fisher’s exact test, OR for nephrotic syndrome with CNTN1 antibodies versus seronegative patients = 113.6, 95% CI 11.2 to 169.1). Two patients with anti- bodies against other nodal/paranodal targets were diagnosed with nephrotic syndrome. Subsequently, a further seven patients with CNTN1 antibodies, an immune-mediated neu- ropathy, and confirmed nephrotic syndrome or suspected nephrotic syndrome with MGN confirmed on biopsy, were retrospectively identified from international collaborating centres. IgG4 was the predominant subclass, except in four patients, where IgG1 was equally reactive (S2 Fig). Thus a total of 15 patients, summarised here (S1 Table in S1 File) had a CNTN1 anti- body-associated neuropathy and nephropathy. PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 4 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy We also tested for CNTN1 antibodies in 295 patients with idiopathic MGN without neu- ropathy. Four (1.4%) were seropositive (S1 Fig). PLA2R antibody testing was performed where possible, and found to be negative in these four, and 10/15 patients with CNTN1 antibodies, nephrotic syndrome and a neuropathy (14/19 in total)—the remaining sera unavailable for further testing. Testing for other newly described MGN antigens was not done due to lack of clinical assay availability. CNTN1 antibodies were not detected in 256 control sera (90 from patients with other neu- rological diseases, 46 from patients with lupus nephritis, 12 of which had a pure class V mem- branous lupus nephritis, and 120 from individuals with other non-neurological, non-renal diseases). CNTN1-containing immune complexes are found in the glomeruli of patients with CNTN1 antibodies Renal biopsies were performed in 12 of the 15 patients. In all cases biopsies were characteristic of membranous glomerulonephritis, with diffuse thickening of glomerular capillary walls, and basement membrane spikes and lucencies evident on silver stain. Where available, immunos- taining revealed glomerular capillary wall IgG (Fig 1A) and complement C3 (not shown), with subepithelial electron dense deposits, representative of immune complexes, on electron microscopy (Fig 1B). Granular deposition of CNTN1 protein was confirmed along glomerular basement membranes by immunohistochemistry (Fig 1C and S5 Fig). Furthermore, laser dis- section of glomeruli followed by LC-MS/MS proteomic analysis revealed a 10 amino acid pep- tide (p.617-626, ATSVALTWSR) in CNTN1-immunopositive renal tissue, which matched with recombinant CNTN1 protein (S6 Fig). In contrast, no CNTN1 staining was observed in glomeruli from healthy donor kidney tis- sue (not shown) or PLA2R-associated MGN patient kidney tissue (Fig 1D). However, CNTN1 mRNA was detectable in healthy human kidney by polymerase chain reaction (PCR), and low- level CNTN1-protein expression could be observed by western blot (S7 Fig). Low serum CNTN1 protein levels correlated with immune complex formation We have recently shown that serum levels of CNTN1 protein (sCNTN1) are lower in anti- body-positive neuropathy patients compared with seronegative and healthy controls [18]. Consistent with this, we found sCNTN1 levels were significantly lower at presentation in CNTN1-antibody positive patients (median 1.02 ng/ml, range 0.02 to 36.37) compared to 25 randomly selected seronegative patients with CIDP (four of whom had concurrent nephrotic syndrome) (median 10.67 ng/ml, range 3.96 to 30.06) and 25 seronegative patients with nephrotic syndrome only (median 13.91 ng/ml, range 4.37 to 26.72) (p = 0.003 and p<0.001, Kruskal-Wallis) (Fig 2A). Overall, there was a non-significant trend for sCNTN1 and CNTN1 antibody levels to be inversely correlated (p = 0.099, R2 = 0.1088, simple linear regression) (Fig 2B). Finally, using western blotting we observed CNTN1 within the PEG precipitates iso- lated from the serum of a patient with active renal and neurological disease (patient 14 in S1 Table in S1 File). CNTN1 was not detected within the PEG precipitates isolated from the serum of the same patient during remission, or in that of a healthy control donor (Fig 2C and S8A and S8B Fig) suggesting the association of CNTN1 with immune complexes at disease peak. Conversely, CNTN1 was undetectable in the soluble fraction of PEG-treated serum of P14 during active disease (S8A and S8B Fig), consistent with the low levels of serum CNTN1 in the other anti-CNTN1 antibody patient samples (Fig 2A). PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 5 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy Fig 1. Contactin-1 immunoreactivity in kidney immune deposits and peripheral nerve tissue. A) Immunofluorescence of IgG deposition along glomerular capillary wall in anti-CNTN1 positive MGN patient kidney (P12 from S1 Table in S1 File). B) Electron microscopy reveals immune complex deposits on the extracapillary side of the glomerular basement membrane, visible as electron dense material (white arrows). C) Diaminobenzidine immunohistochemistry for CNTN1 is observed along the basement membrane of glomeruli from the same anti-CNTN1 positive MGN patient. Higher magnification image of a glomerulus demonstrating enrichment of CNTN1 in deposits lining the thickened basement membrane, characteristic of primary MGN (inset, black arrows). Equivalent results were obtained from biopsies of three further patients (S5 Fig). D) No CNTN1 labelling was observed in biopsy sections from an anti-PLA2R-positive patient with MGN. Scale bars in C-D, 20 μm. E-G) Labelling of the paranode in teased fibres from mouse sciatic nerve with IgG (green) from pre-treatment anti-CNTN1-positive human sera (E) is abrogated post-treatment (F) and absent in healthy control sera (G). Bound human IgG co-localises with CASPR at the paranode (magenta). Scale bars, 10μm. H) Human IgG binding (green) to myelinated co-cultures treated with patient sera sampled before steroid treatment. Cultures were permeabilised with methanol prior to sera incubation. Note diffuse axonal pattern and paranodal localisation of human IgG (green) (arrowheads in inset). Myelin basic protein (MBP, red) indicates myelin, and neurofilament (NF200, blue) indicates axons. Scale bar, 25μm (5μm in inset) I) Loss of human IgG binding (green) to myelinated co-cultures treated with patient sera sampled after steroid treatment. Scale bar 50 μm. J, K) CNTN1-transfected HEK293 cells (CBA) treated with patient sera sampled before (J) and after (K) steroid treatment. Serum titration in CNTN1 CBA shows a fall of anti-CNTN1 titre from 1:6400 to negative subsequent to steroid treatment. Scale bars, 50 μm. https://doi.org/10.1371/journal.pone.0281156.g001 CNTN1 antibodies from seropositive patients target peripheral nerves and paranodes Patient serum showed IgG reactivity with teased mouse nerve fibres, colocalising with Caspr1 at the paranodes (Fig 1E–1G). IgG bound to unmyelinated axons in live human stem cell- derived sensory neuron co-cultures (Fig 1H), as well as paranodal regions of myelinated axons (Fig 1H, inset). Following immunosuppressive treatment which led to complete suppression of CNTN1 antibody titres on the transient transfection cell-based assay (Fig 1J and 1K), all IgG labelling was lost from neuronal axons (Fig 1I). Further, locally available sera from patients found to have CNTN1 antibodies, neuropathy and nephrotic syndrome were pre-adsorbed with recombinant CNTN1 protein and re-applied to live CBA or neuronal co-cultures (S3 and S4 Figs). Immunolabelling was markedly reduced for all pre-absorbed sera tested in both assays, confirming CNTN1 as the antigen for serum IgG binding in the live cell culture systems. PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 6 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy Fig 2. Fall is serum CNTN1 protein is associated with immune complex formation in active disease. A) Serum CNTN1 (sCNTN1) protein levels are significantly lower in CNTN1 antibody seropositive CIDP (n = 11; sera available for testing from 10 patients in prospective group and 1 in retrospective group) vs seronegative CIDP patients (n = 29), and those with nephrotic syndrome but without CIDP (n = 25). Red diamonds and black dots indicate patients with and without nephrotic syndrome, respectively. Although 9/11 patients from the CNTN1 Ab+ CIDP group developed MGN, at the time of testing for sCNTN1 only 8/ 11 had nephrotic syndrome. Kruskal-Wallis p<0.001 overall, with post-test pairwise comparisons (CNTN1 Ab+ vs. seronegative CIDP, p = 0.003, vs. nephrotic syndrome alone p<0.001, seronegative CIDP vs. nephrotic syndrome ns, p = 0.19). B) Overall, there was a non-significant trend for sCNTN1 and CNTN1 antibody levels to be negatively correlated (p = 0.099, R2 = 0.1088, simple linear regression). However in 5/8 patients where CNTN1 antibodies later became undetectable, sCNTN1 remained low. C) Western blot for CNTN1 in the insoluble fraction of polyethylene glycol (PEG) treated healthy control normal human serum (NHS) and serum of patient 14 at nadir of active disease compared to remission. Recombinant CNTN1 protein (0.003 μg) loading; 5μl immune complexes purified from 300μl of patient serum. A repeat blot reveals reciprocal CNTN1 bands detectable only from the soluble fraction of the NHS and P14 serum in remission, but not during nadir of active disease (5μl loaded). https://doi.org/10.1371/journal.pone.0281156.g002 Patients with CNTN1 antibodies have a distinct clinical syndrome The majority of patients with CNTN1 antibodies presented with a neuro-renal syndrome char- acterised by a rapidly progressive, disabling, sensory-motor neuropathy accompanied by typi- cal nephrotic syndrome, with oedema, low serum albumin, and elevated levels of urinary proteinuria. Additional features, including pain, tremor and autonomic dysfunction, were common, and over one-quarter had a prior diagnosis of diabetes. A summary of clinical char- acteristics is provided in Table 1, and further detail in S1 Table in S1 File. Smaller numbers of patients had isolated kidney or nerve involvement. PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 7 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy Table 1. Summary of patients with neuropathy, nephrotic syndrome and CNTN1 antibodies. SUMMARY OF KEY CHARACTERISTICS Characteristic Patients % (No.) CLINICAL Age (median, range) 59 (39–79) Male 80 (12/15) Diabetes 26.7 (4/15) Acute/Subacute onset 86.7 (13/15) Additional clinical features (inc ataxia, tremor, cranial nerve palsy, autonomic 93.3 (14/15) dysfunction and abdominal weakness) Pain 53.3 (8/15) Median nadir disability—mRS (range) 5 (2–6) INVESTIGATIONS CSF Median protein g/l (range) 1.99 (0.24–5.93) Normal protein 21.4 (3/14) WCC >5 28.6 (4/14) Meets electrodiagnostic criteria for CIDP A 93.3 (14/15) Median serum albumin g/l (range) 22 (11–36) Median urinary PCR mg/mmol (range) 1032 (633–4638) Median urinary protein g/24hr (range) 3.35 (2.2–10) Clinical criteria met for nephrotic syndromeB 92.3 (12/13)C MGN on renal biopsy 100 (12/12) Complement (C3 or C1q) deposition noted 58 (7/12) PLA2R negative (IHC or serum antibodies) 100 (10/10) TREATMENT RESPONSE D First line therapy Responders 20 (3/15) (Steroids +/-IVIg +/- PE) Non-responders 66.7 (10/15) Escalation therapyE Responders 73 (8/11) (inc. RTX) Non-responders 27 (3/11) OUTCOME mRS (median, range) 1 (0–6) Complete remission (of both neuropathy/nephropathy) 31 (5/15) Significant improvement (� 2 points mRS) 38 (6/15) Minimal improvement/stabilisation (of both neuropathy/nephropathy) 6 (1/15) Death 25 (3/15) A As per EFNS/PNS [20] B One or more of urinary PCR >300mg/mmol, proteinuria >3g/24 hours, serum albumin <35g/l C 1 patient did not meet clinical criteria for nephrotic syndrome, and 2 did not have nephrotic investigations available. All 3 of these had biopsy proven MGN. D Definition of treatment response Responder: Complete remission, Sustained functional improvement > = 2 points mRS Non-responder: Death, no response, transient response or improvement by only 1 point on the mRS E Escalation therapy includes (in order of frequency used): Rituximab, Cyclophosphamide, Azathioprine, Tacrolimus, Autologous stem cell transplant, Mycophenolate Mofetil, Immunoadsorption Abbreviations: CIDP–chronic inflammatory demyelinating polyneuropathy, CSF–Cerebrospinal fluid, IHC–Immunohistochemistry, IVIg–Intravenous immunoglobulin, mRS–Modified Rankin score, PE–Plasma exchange, PCR–Protein:Creatinine ratio, RTX–Rituximab, WCC–White cell count. https://doi.org/10.1371/journal.pone.0281156.t001 The nephropathy and neuropathy are immunotherapy responsive and improve in parallel with a reduction in CNTN1 antibody titres Overall, 75% of patients achieved either complete remission, significant improvement, or sta- bilisation of their neuropathy and nephropathy in parallel. However, only 20% (3/15) responded well to the first-line treatments typically used for inflammatory neuropathies (ste- roids, IVIg, plasmapheresis) (Table 1). Four different treatment modalities, ultimately PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 8 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy including escalation immunotherapies such as rituximab (RTX), were typically required to achieve a response. Two patients (noticeably the eldest of the cohort) died from multi-organ or respiratory failure whilst on the intensive care unit. A third patient died due to complica- tions from coronavirus disease-19 (COVID-19) following prior treatment with IVIg, steroids and rituximab. CNTN1 antibody titres were high prior to immunosuppressive treatment, and lower in remission of both neuropathy and nephropathy (Fig 3A) (p = 0.001). In all 6 patients for whom both pre- and post-treatment serum samples were available, anti-CNTN1 titres fell in parallel with a reduction on the CIDP Disease Activity Scale (CDAS, Fig 3B). Serial assess- ments in one patient showed that improvements in strength and disability paralleled the fall in CNTN1 antibody titres, and a fall in urinary protein levels followed shortly after (Fig 3C). Discussion This study identifies CNTN1 antibodies as the pathological link between immune-mediated neuropathies and nephrotic syndrome caused by MGN, providing a mechanistic explanation for numerous previous clinical observations [10–13, 21]. We also demonstrate a significant association between CNTN1 antibody titres and immunotherapy responsiveness in this neuro-renal syndrome, and establish CNTN1 as another new antigenic target in a small pro- portion of “idiopathic” membranous glomerulonephritis. A previous report found non-con- cordant presentation of nephrotic syndrome and neuropathy in a patient with anti-CNTN1 antibodies—separated by a year, implying that some patients may further develop the com- bined neuro-renal syndrome at a later stage [22]. Without treatment, up to half of patients with MGN develop end-stage renal failure [23]. In keeping with recent data, patients with CNTN1 antibodies were poorly responsive to the typi- cal first line therapies used for inflammatory neuropathies [24]. However, escalation therapies substantially reduced disability overall, and the associated renal disease invariably improved in parallel, as has recently been described in one further case report [25]. Around 70% of patients with idiopathic MGN have detectable serum IgG4 autoantibodies against the glomerular podocyte antigen phospholipase A2 receptor (PLA2R) [7]. As such, test- ing for PLA2R antibodies has transformed the management of MGN, in many cases potentially avoiding the need for diagnostic biopsy. Similarly, the early detection of CNTN1 antibodies may negate the need for further renal investigation in anti-PLA2R negative patients, and pro- vide an opportunity for earlier, therapeutic intervention. In addition, falling anti-CNTN1 titres mirrored clinical improvement, suggesting their potential as a prognostic marker. Indeed, high titre PLA2R antibodies predict a worse prognosis, [26] with reduced titres found to pre- cede the onset of remission [27]. As with anti-PLA2R MGN [28], we found the detection of serum CNTN1 antibodies often preceded the onset of proteinuria- confirming their utility as biomarkers. Renal biopsy still needs to be considered for seronegative patients in whom antibody-medi- ated nephrotic syndrome is suspected, as evidenced by the fact that we observed one patient to have glomerular CNTN1-immune-complex deposition (patient 2 in S5 Fig) despite having become CNTN1 seronegative (following steroid treatment) by the time of renal biopsy. We confirm the presence of CNTN1 in both renal and peripheral nerve tissue, and provide evidence that CNTN1 antibodies identify an aberrant immune response targeting both periph- eral nerve and kidney. Our observation of low-level expression of CNTN1 protein in healthy kidney is supported by recent single cell transcriptomic profiling of human kidney, which identified CNTN1 in podocytes [29], while others have recently described CNTN1 expression in normal kidney glomeruli and co-localisation of CNTN1 and IgG4 in the glomerular PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 9 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy Fig 3. CNTN1 antibody titres correlate with disease activity. A) CNTN1 antibody titres were significantly higher in active disease (on or pre-treatment) (n = 15), compared to remission off treatment) (n = 6, p = 0.001, Mann-Whitney). B) In all 6 patients for whom serial samples were available, CNTN1 antibody titres fell in parallel with a reduction in neuropathy disease activity (measured using the CIDP Disease Activity Scale–CDAS) and became undetectable in 5 patients who achieved treatment-free remission. C) In one patient, where this level of detail was available, serial assessments showed that improvements in strength (Medical Research Council sum score, MRC-SS), functional abilities (inflammatory neuropathy Rasch-built Overall Disability Scale, iR-ODS) and normalisation of renal function (Protein Creatinine Ratio, PCR) correlated with falling CNTN1 antibody titres. Grey arrows indicate IVIg administration (2g/kg over 5 days), and black arrows pulsed dexamethasone (40mg daily for 4 days, repeated after 4 weeks). https://doi.org/10.1371/journal.pone.0281156.g003 basement membrane of an affected patient with MGN [30]. This suggests that, similar to what is seen in anti-PLA2R MGN, circulating CNTN1 antibodies bind directly to the antigen in situ, which is then shed and accumulates in the glomerular space [31]. Emerging data also reveals the presence of CNTN1 protein in serum, and show levels reflect antibody status and neurological disease activity [18]. In contrast to PLA2R, which is PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 10 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy undetectable in the serum of either controls or anti-PLA2R MGN patients [7], we detected CNTN1 protein in the serum of healthy controls, as well as seronegative CIDP and other forms of nephrotic syndrome, but levels were significantly lower in CNTN1 antibody-positive patients (Fig 2). The detection of CNTN1 protein within the PEG-precipitated fraction of patient serum sampled during a period of high anti-CNTN1 antibody titre suggests that CNTN1 may exist as circulating immune complexes within the blood in CNTN1 antibody- positive patients. The lack of mesangial and/or capillary immune complexes in patient biopsies may argue against these being the cause of nephrotic syndrome [32], though it remains possi- ble that smaller circulating immune complexes can cause a subepithelial pattern of deposition [33]. Our findings encourage a fresh look at the potential for deposition of circulating immune complexes in human renal disease [34]. Recent studies provide insight into the pathogenic mechanisms of CNTN1 antibody-medi- ated peripheral neuropathy [35, 36]. Why some CNTN1 antibody patients preferentially develop nerve or renal injury is unclear. One or other disease may simply remain sub-clinical. Alternatively, pre-existing kidney injury may facilitate subsequent immune-mediated glomer- ular pathology, as can be seen in mice, where CNTN1 expression is upregulated within the kid- ney by glomerular injury [37]. The prior diagnosis of diabetes in over a quarter of patients in our cohort may be relevant in this regard. Further, antibodies against glomerular antigen THSD7A can be associated with an increased risk of cancer-associated MGN [38]. Although CNTN1 has well established roles in association with numerous solid organ cancers [39], malignancy was not detected as a potential source of CNTN1 antigen in the 6 patients in our cohort who were radiologically screened (S1 Table in S1 File). However, it is possible some patients may have subsequently developed cancer following diagnosis, as has been previously described [40]. In summary, the improvement of neuropathy and renal disease following immunotherapy highlights the utility of CNTN1 antibodies in the identification of a clinically reversible disorder. Supporting information S1 Checklist. (DOCX) S1 Fig. Flowchart summarising detection of patients with CIDP, nephrotic syndrome and CNTN1 antibodies through prospective nodal/paranodal autoantibody screening. (TIFF) S2 Fig. Titre and subclass specificity comparison between CBA and ELISA. (TIFF) S3 Fig. Immunoreactivity of IgG from CNTN1-positive patients on CBA show they are specific for Contactin-1 (CNTN1). (TIFF) S4 Fig. Axonal pattern of IgG binding from CNTN1-positive patients in myelinated neuro- nal co-cultures is specific to the CNTN1 antigen. (TIFF) S5 Fig. Contactin-1 immunoreactivity in kidney immune deposits for patents 1, 2 and 13. (TIFF) PLOS ONE | https://doi.org/10.1371/journal.pone.0281156 March 9, 2023 11 / 15 PLOS ONE Contactin-1 links neuropathy and nephropathy S6 Fig. LC-MS/MS of CNTN1 peptides. (TIFF) S7 Fig. CNTN1 is expressed in healthy human kidney. (TIFF) S8 Fig. CNTN1-containing immune complexes are present in serum at disease nadir. (TIFF) S1 File. (DOCX) S1 Raw images. (PDF) Acknowledgments We thank Prof. Moin Saleem (University of Bristol) for the kind gift of the human podocyte cultures, and Dr Lucy McDermott for the YWHAZ PCR primers. We thank Hannah Brooks and acknowledge the Oxford Brain Bank, supported by the Medical Research Council (MRC), Brains for Dementia Research (BDR) (Alzheimer Society and Alzheimer Research UK), Autis- tica UK and the NIHR Oxford Biomedical Research Centre for frozen human brain tissue. We also thank Dr David Maldonado-Perez and the team at the Oxford Centre for Histopathology Research (OCHRe) and Oxford Radcliffe Biobank (ORB) supported by the University of Oxford, the Oxford Biomedical Research Centre (BRC) Digital Pathology theme, Cancer Research UK (CRUK) Oxford Cancer Centre, National Institute for Health Research Thames Valley and South Midlands Clinical Research Network (NIHR CRN) and Research Capability Funding (NIHR RCF) for the frozen human kidney samples. The MRC GN bank was sup- ported by the MRC and Kidney Research UK. Author Contributions Conceptualization: Alexander J. Davies, Alan D. Salama, Simon Rinaldi. Data curation: Alexander J. Davies, Simon Rinaldi. Formal analysis: Janev Fehmi, Alexander J. Davies. Funding acquisition: Simon Rinaldi. Investigation: Janev Fehmi, Alexander J. Davies, Sonja Pikkupeura, Ruth Pepper, Filipa Fari- nha, Anisur Rahman, Diana Canetti, Janet A. Gilbertson, Nigel B. Rendell, Filip Eftimov, Luuk Wieske, Charlotte E. Teunissen, Ian S. D. Roberts, Neil Ashman, Alan D. Salama, Simon Rinaldi. Methodology: Janev Fehmi, Alexander J. Davies, Alan D. Salama, Simon Rinaldi. Project administration: Janev Fehmi, Alan D. Salama, Simon Rinaldi. Resources: Marilina Antonelou, Luis Querol, Emilien Delmont, Andrea Cortese, Diego Fran- ciotta, Staffan Persson, Jonathan Barratt, Aleksandar Radunovic, Thomas Minton, Geraint Fuller, Sinead M. Murphy, Aisling S. Carr, Mary R. Reilly, Ian S. D. Roberts, Neil Ashman, Alan D. Salama, Simon Rinaldi. Supervision: Alan D. Salama, Simon Rinaldi. Validation: Janev Fehmi, Alexander J. Davies, Simon Rinaldi. 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