Proteinuria is a key to suspect autoimmune nodopathies

Abstract Background and purpose Reports of patients who have autoimmune nodopathies concurrent with nephrotic syndrome are increasing. We investigated whether proteinuria could be a biomarker of autoimmune nodopathies. Methods Qualitative urinalysis results were retrospectively obtained from 69 patients who were diagnosed with chronic inflammatory demyelinating polyneuropathy (CIDP) at a hospital in Japan. Proteinuria was graded as mild to severe (i.e., mild, 30–99; moderate, 100–299; severe, 300 mg/dL or more) according to the results of the urine dipstick test. Autoantibodies against the paranodal proteins contactin 1 (CNTN1), neurofascin 155 (NF155), and contactin‐associated protein 1 (Caspr1) and the nodal protein neurofascin 186 (NF186) were measured, and the predominant IgG subclass was determined by enzyme‐linked immunosorbent assay in sera from the 69 patients. Results Four patients (6%), five patients (7%), and one (1%) patient were positive for anti‐CNTN1, anti‐NF155, and anti‐Caspr1 IgG4 antibodies, respectively. No patients had IgG4 antibodies against NF186. Proteinuria of mild or greater levels was found in three patients with anti‐CNTN1 IgG4 and two patients with anti‐NF155 IgG4 antibodies. The autoantibody‐positive patients more frequently had proteinuria of mild or greater levels than the seronegative patients (p = 0.01). Conclusions Proteinuria is a possible biomarker of autoimmune nodopathies associated with autoantibodies targeting CNTN1 or NF155. Urinalysis results should be carefully checked for quick differentiation of autoimmune nodopathies from CIDP. Patients who present with nephrotic syndrome should be tested for anti‐CNTN1 IgG4 antibodies, and patients who exhibit mild proteinuria should be tested for anti‐NF155 IgG4 antibodies.

autoimmune nodopathies is often impossible without measuring autoantibodies.Moreover, only a few laboratories can perform autoantibody testing, and it takes several weeks or more to obtain results.Easily detected biomarkers that can identify patients who require autoantibody testing as well as select a more appropriate choice of first-line treatment other than intravenous immunoglobulin (IVIg) are needed.
Membranous nephropathy, a major cause of nephrotic syndrome in adults, is rarely accompanied by CIDP [3].In contrast, reports of patients with both autoimmune nodopathies and membranous nephropathy associated with autoantibodies targeting CNTN1 are increasing [4][5][6][7][8].To date, CNTN1 has been recognized as a novel target in primary membranous nephropathy [9].Most of the reported patients showed heavy proteinuria or significant nephrotic syndrome and underwent renal biopsies for diagnosis.
Although the importance of both light and heavy proteinuria in disease pathophysiology might be equal, patients with asymptomatic or mild proteinuria are likely often overlooked.To elucidate the correct pathophysiology of these conditions, detection of mild proteinuria is essential.
Although high prevalence of nephrotic syndrome in autoimmune nodopathies associated with anti-CNTN1 IgG4 [5,7,10,11], antipan-neurofascin IgG1 [12], or anti-NF186 IgG [13] has increasingly been emphasized, routine urinalysis, which can be performed at every hospital, is not recommended in the diagnostic guidelines [2,14].Therefore, in our study, we tested whether proteinuria can be a biomarker of autoimmune nodopathy associated with anti-CNTN1 IgG4 antibodies in patients with suspected CIDP at a single hospital in Japan.Unexpectedly, we also found that proteinuria could be a possible biomarker of autoimmune nodopathy associated with anti-NF155 IgG4 antibodies.

ME THODS Patients
The medical records of 71 consecutive patients who visited Dokkyo Medical University Hospital between November 1997 and January 2023 and fulfilled the European Federation of Neurological Societies/Peripheral Nerve Society criteria for CIDP [14] were reviewed retrospectively.All the patients underwent systematic electrodiagnostic tests as described elsewhere [15].Results of urinalysis and blood chemistry tests were collected from 69 of the patients included in the study at their first visit.Of the 69 patients, 21 had been included in our previous studies [16,17].Blood or urinary test results suggested neither urinary tract infection nor other renal diseases in our patients.
The patients were classified into subtypes according to the existing criteria [18] as follows.The distal variant was defined by distal leg-dominant symmetric muscle weakness with fully preserved proximal limb muscles; the multifocal variant was defined by muscle weakness with sensory symptoms in a multifocal distribution; the sensory-predominant variant was defined by sensory symptoms without muscle weakness but with demyelinating motor conduction abnormalities; and the motor-predominant variant was defined by muscle weakness without sensory symptoms but with sensory nerve conduction abnormalities.Treatment response was defined as an improvement in muscle weakness (increase of 4 or more points on the Medical Research Council sum score) within 4 weeks after any of the treatments.

Urinalysis
A spot urine dipstick test was used to diagnose proteinuria.

Standard protocol approvals, registrations, and patient consents
This study was performed in accordance with the guidelines stipulated in the Declaration of Helsinki and Ethical Guidelines for Medical and Biological Research Involving Human Subjects by the Japanese government and was approved by the ethics review committee of Dokkyo Medical University (approval number R-41-5J).

Statistical analyses
Differences in proportions were examined by Fisher exact test.
Student t-test or Welch t-test was used for normally distributed continuous variables, and Mann-Whitney U-test was used for nonnormally distributed variables.A difference was considered significant at a p-value less than 0.05.Analyses were performed with SPSS 25J software (SPSS, Chicago, IL, USA).

IgG subclasses of antibodies to paranodal or nodal proteins
Table 1 shows the clinical features of the patients.Of the 69 patients, four (6%) were positive for anti-CNTN1 antibodies, five (7%) were positive for anti-NF155 antibodies, and one (1%) was positive for anti-Caspr1 IgG4 antibodies.Among the four patients positive for anti-CNTN1 IgG4, the IgG1 subclass was weakly positive in three patients.In the patient positive for anti-Caspr1 IgG4, the IgG1 subclass was also detected weakly.The five patients positive for anti-NF155 IgG4 did not have autoantibodies of the IgG1 subclass.
No patients had both anti-NF155 and anti-NF186 autoantibodies concurrently, suggesting that the anti-NF155 IgG4 antibodies did not react with NF186.The patients who did not have the IgG4 autoantibodies described above were considered "seronegative."

Anti-CNTN1 IgG4-positive patients
Patients who carried anti-CNTN1 IgG4 antibodies more frequently exhibited sensory ataxia (p = 0.009).All four patients had the typical CIDP phenotype.The cerebrospinal fluid protein level was significantly greater in patients who harbored anti-CNTN1 antibodies than in seronegative patients (p = 0.03).The four patients received both IVIg and corticosteroids.One (25%) of the patients responded to IVIg, which was less effective than it was for the seronegative patients (p = 0.02), and two (50%) patients responded to corticosteroids.Patient 1, as shown in Table 2, was described elsewhere in detail [3,16].Three of four (75%) patients who had anti-CNTN1 antibodies showed severe proteinuria, two of whom were histologically diagnosed as having membranous nephropathy.
Neither the male-to-female ratio nor the disease severity differed between the anti-NF155-positive and seronegative patients.The anti-NF155-positive patients had an earlier disease onset than the seronegative patients (p = 0.02).Patients exhibited finger tremors (p < 0.001) and sensory ataxia (p < 0.001) more frequently than seronegative patients.The cerebrospinal fluid protein level was greater in the anti-NF155-positive patients than in the seronegative patients (p = 0.01).Two of the five patients showed a relatively favorable response to IVIg (40%), but less frequently than seronegative patients (p = 0.04), and four responded to corticosteroids (80%).

Urine analysis
As shown in Table 3, proteinuria of mild or greater severity was found in five of nine patients who carried IgG4 antibodies against CNTN1 or NF155 (seropositive patients) and nine of 59 seronegative patients (odds ratio [OR] = 6.9, 95% confidence interval [CI] = 1.7-29, p = 0.01).The patient who harbored the anti-Caspr1 IgG4 antibodies did not have proteinuria.Moderate or greater proteinuria was found in three of nine seropositive patients and four of 59 seronegative patients (OR = 6.9, 95% CI = 1.4-35, p = 0.04).Severe proteinuria was found in three of nine seropositive patients and zero of 59 seronegative patients (OR = 64, 95% CI = 5.0-735, p < 0.001).
Patients who carried anti-CNTN1 IgG4 antibodies had proteinuria more frequently than those who did not.In contrast, mild or greater proteinuria was found in two of five (40%) patients who had anti-NF155 IgG4 antibodies and nine of 59 (15%) seronegative patients (p = 0.20).Moderate or greater proteinuria was found in zero of five anti-NF155-positive patients and four of 59 (7%) seronegative patients (p = 0.72).No patients in the anti-NF155-positive or seronegative groups had severe proteinuria.Urine glucose was detected in zero of nine seropositive patients and seven of 59 (12%) seronegative patients, but the difference was not significant (OR = 0.37, 95% CI = 0.04-4.2,p = 0.32).
Several nodal/paranodal molecules are recognized as target antigens for autoimmune nodopathies; for example, NF155 on the terminal loop of myelin and CNTN1 and Caspr1 on the axolemma form septatelike junctions between myelin loops and the axolemma at the paranodes [29].NF186, expressed on the axolemma at the nodes of Ranvier, forms a complex with gliomedin and neuronal cell adhesion molecule on Schwann cell microvilli and regulates sodium channel clustering on the axolemma at the nodes of Ranvier [30].These paranodal and nodal molecules play crucial roles in the generation of saltatory nerve transmission of myelinated nerve fibers [29,31].Detachment of the terminal loops of myelin and the axolemma at the paranodes was identified in patients with anti-CNTN1 and anti-NF155 nodopathies [32,33].These conditions might disrupt or alter the distributions of both sodium and potassium ion channel clusters at nodes and juxtaparanodes and impair saltatory conduction via a reduction in sodium ion influx and hyperpolarization of membrane potential [34].Autoantibodies against phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A (THSD7A) are pathogenic autoantibodies found in primary membranous nephropathy, and their predominant IgG subclass is IgG4 [35,36].PLA2R and THSD7A are expressed on the cell surface of podocytes [35,36].In membranous nephropathy, IgG4 antibodies against PLA2R or THSD7A penetrate the glomerular loop and glomerular basal membrane and induce the cytotoxicity of podocytes and leakage of proteins from vessels to renal tubules [35].Contactin is also expressed in the kidney as well as the nerves [37], although its localization in the kidney has yet to be determined.Four of 295 (1.4%) patients with isolated membranous nephropathy without neuropathy had anti-CNTN1 IgG4 antibodies [7], suggesting that contactin was expressed on the podocyte, glomerular loop, or glomerular basal membrane.NF186 is also likely expressed on glomerular podocytes as well as the nodal axolemma [38].It is unclear why patients with anti-NF155 nodopathy in our cohort exhibited mild Nerve Society, includes conditions that can cause proteinuria [2].
Diabetic polyneuropathy and uremic neuropathy often present with proteinuria, depending on the severity or stage of the primary disease.Patients with amyloid light chain amyloidosis or transthyretin familial amyloid polyneuropathy can present with amyloid nephropathy [39].Multiple myeloma or osteosclerotic myeloma can cause light chain cast nephropathy or cryoglobulinemic glomerulonephritis [40].Anti-neutrophil cytoplasmic antibody-associated vasculitis is often associated with glomerulonephritis [41].Moreover, two of five patients who harbored anti-NF140 and NF186 IgG antibodies had nephrotic syndrome caused by focal segmental glomerulosclerosis [22], and their electrodiagnosis was an axonal neuropathy.
Therefore, patients with suspected CIDP, with or without demyelinating neurophysiology, should be screened for proteinuria, followed by anti-nodal/paranodal autoantibody measurement.
Our study has several limitations.First, there was a small number of anti-NF155-and anti-CNTN1-positive patients in our study.
Second, enzyme-linked immunosorbent assay was used to determine the autoantibody positivity, which was not confirmed by cell-based assay or teased-nerve immunohistochemistry. Third, because of the retrospective nature of the study, a temporal relationship between the onset of neuropathy and the occurrence of proteinuria was uncertain.Prospective validation is required.Despite these limitations, however, we believe that the simplicity of urinalysis can be a quick method for distinguishing autoimmune nodopathies from CIDP.
In conclusion, proteinuria is a possible biomarker of autoimmune nodopathies associated with autoantibodies targeting CNTN1 or NF155.Autoantibodies against paranodal/nodal molecules, such as CNTN1, NF186, and NF155, should be investigated in patients who present with nephrotic syndrome or mild proteinuria without symptoms.Future prospective studies are needed to clarify the validity of this correlation.AUTH O R CO NTR I B UTI O N S Kei Funakoshi: Conceptualization; methodology; data curation; investigation; formal analysis; writing -original draft.Norito Kokubun: Conceptualization; investigation; writing -review and editing; project administration.Keisuke Suzuki: Writing -review and editing.Nobuhiro Yuki: Conceptualization; writing -review and editing; supervision.
Odds ratios of the grade of proteinuria for autoimmune nodopathies.