NELL1 Antibody

What is NELL1 and how was it identified in membranous nephropathy?

NELL1 (Neural Epidermal Growth Factor-like 1) is a podocyte antigen identified by Dr. Sethi and colleagues in 2019 using laser microdissection and tandem mass spectrometry (MS/MS) . The discovery occurred during investigations of patients with membranous nephropathy who were negative for the previously established PLA2R antigen. NELL1 represents a significant breakthrough as it accounts for approximately 16% of PLA2R-negative MN cases, making it the second most common antigen associated with membranous nephropathy . The identification of this novel antigen has expanded our understanding of the immunological mechanisms underlying membranous nephropathy and improved diagnostic capabilities for previously unclassified cases.

What are the distinctive histopathological features of NELL1-associated membranous nephropathy?

NELL1-associated membranous nephropathy exhibits several distinctive histopathological features:

• Segmental to incomplete subepithelial immune deposits (55-94% of cases), rather than the global deposits typical in PLA2R-MN

• IgG1 (co)dominant staining pattern (86-96% of cases)

• Stage I-II deposits by electron microscopy (82-93% of cases)

• Presence of mesangial deposits in 7-24% of cases

• Occasional subendothelial and/or extraglomerular deposits of IgG

• Positive C3 staining in approximately 70% of cases

These distinctive features help differentiate NELL1-MN from other forms of membranous nephropathy and guide appropriate diagnostic and management approaches. The segmental pattern and IgG1 dominance are particularly useful distinguishing characteristics when evaluating kidney biopsy specimens.

What is the prevalence of NELL1-MN in different populations?

• In regions where complementary and alternative medicine (CAM) use is common, such as parts of India, the prevalence appears higher

• One study reported that approximately 35% of MN cases in India were associated with traditional indigenous medicine use, of which 88% were NELL1-positive

• The prevalence may be influenced by environmental factors, medication practices, and genetic background of different populations

Research protocols should account for these geographic variations when designing multicenter studies and interpreting results across different patient populations.

What specimen types and testing methodologies are recommended for NELL1 detection?

For optimal NELL1 detection in research settings, the following specimen requirements are recommended:

• Preferred specimen: Two unstained positively charged glass slides (25- x 75- x 1-mm) per test ordered; sections 4-microns thick

• Acceptable alternative: Formalin-fixed, paraffin-embedded (FFPE) tissue block

• Proper submission using an Immunostain Technical Only Envelope (T693)

Testing methodologies include:

• Immunohistochemistry for tissue-based detection

• Mass spectrometry for confirmatory analysis in complex cases

• Serum antibody testing (though not yet widely commercially available)

The analytic time for immunohistochemical testing is typically 1-3 days . Researchers should be aware that while tissue-based testing is well-established, serum-based anti-NELL1 antibody assays remain an area for assay development and validation.

How does NELL1-MN relate to other forms of membranous nephropathy?

NELL1-MN exists within a spectrum of antigen-associated membranous nephropathies:

• PLA2R-associated MN (70-80% of primary MN)

• NELL1-associated MN (10-15% of all MN cases)

• Other less common antigens including THSD7A, exostosin 1/2 (EXT1/2), NCAM1, and TGFBR3

Key differences between NELL1-MN and PLA2R-MN include:

• Segmental vs. global deposit distribution

• IgG1 vs. IgG4 subclass predominance

• Higher association with identifiable secondary causes in NELL1-MN

• Potentially better prognosis in NELL1-MN when the underlying trigger can be identified and removed

Rare cases of dual NELL1 and PLA2R positivity have been reported, suggesting potential overlap in pathogenic mechanisms in some patients .

What are the known secondary associations of NELL1-MN?

NELL1-MN has been associated with several secondary conditions and exposures:

• Medications:

  • Thiol-containing drugs (lipoic acid, bucillamine, tiopronin)

  • NSAIDs (noted in 30% of patients in one series)

• Traditional and complementary medicines:

  • Traditional indigenous medicines (TIM), particularly those with high mercury content

  • Complementary and alternative medicine (CAM) use (30% of patients in one case series)

• Systemic conditions:

  • Malignancies (with association rates varying from 10% to as high as 89% in some series)

  • Autoimmune diseases

  • Graft-versus-host disease (GVHD)

  • Infections

The proportion of NELL1-MN cases with identifiable secondary associations varies significantly between studies, from as low as 11% to as high as 89% . This variability likely reflects differences in patient populations, screening protocols, and regional practices.

What molecular mechanisms underlie NELL1-MN pathogenesis?

The exact molecular mechanisms of NELL1-MN remain under investigation, but current evidence suggests:

• Autoantibody formation:

  • Environmental triggers (medications, heavy metals) may modify the NELL1 antigen or disrupt immune tolerance

  • Genetic factors may predispose to autoantibody formation against NELL1

• Immune complex formation:

  • Anti-NELL1 antibodies bind to NELL1 antigen expressed on podocyte surfaces

  • These immune complexes form subepithelial deposits along the glomerular basement membrane

• Complement activation:

  • C3 positivity is observed in approximately 70% of NELL1-MN cases

  • Complement-mediated damage contributes to podocyte injury and proteinuria

• IgG subclass involvement:

  • IgG1 predominance (unlike the IgG4 dominance in PLA2R-MN) suggests different immunological mechanisms

  • The IgG1 subclass is more efficient at complement activation than IgG4

The segmental nature of deposits in NELL1-MN may reflect heterogeneous antigen expression or local factors influencing immune complex deposition.

What is the relationship between NELL1-MN and malignancy?

The association between NELL1-MN and malignancy has been observed in multiple studies:

• Prevalence: "While many cases of NELL-1 are primary and there's no disease association, there are multiple studies now that show a subset of NELL-1 positive patients have an underlying malignancy"

• Potential mechanisms:

  • Tumor expression of NELL1 leading to antibody formation

  • Paraneoplastic immune dysregulation

  • Shared genetic susceptibility factors

• Clinical implications:

  • Treatment of the underlying malignancy may lead to resolution of NELL1-MN

  • Dr. Sethi notes that physicians should "be extra vigilant in screening for hidden malignancies" in NELL1-MN patients

  • Cancer screening protocols should be incorporated into NELL1-MN research studies

• Research considerations:

  • Tissue studies examining NELL1 expression in tumor samples

  • Longitudinal monitoring of anti-NELL1 antibodies during cancer treatment

  • Correlation between tumor burden and NELL1-MN disease activity

Despite this association, it's important to note that not all studies have found high malignancy rates, suggesting geographical or population-based differences in the NELL1-MN-malignancy relationship.

How should researchers investigate thiol-related and mercury-related NELL1-MN?

When designing studies to investigate environmental triggers of NELL1-MN, researchers should consider:

• Exposure assessment protocols:

  • Comprehensive medication history focusing on thiol-containing drugs

  • Detailed CAM use questionnaires with content analysis when possible

  • Heavy metal testing in blood, urine, and hair samples

  • Analysis of products for mercury and thiol compounds

• Mechanistic investigations:

  • In vitro studies of NELL1 antigen modification by thiols or mercury

  • Animal models exploring exposure-induced autoimmunity

  • Immunological studies of T-cell responses to modified NELL1

• Clinical research design:

  • Case-control studies with appropriate matching

  • Prospective cohorts in high-risk populations

  • Withdrawal protocols with careful monitoring of clinical and immunological parameters

  • Biorepository development for future mechanistic studies

These methodologically robust approaches would help establish causality and elucidate pathogenic mechanisms in environmentally-triggered NELL1-MN.

What are the outcomes of dual NELL1 and PLA2R positive cases?

Rare cases of dual NELL1 and PLA2R positivity present unique research opportunities:

• Prevalence and detection:

  • These cases are uncommon but important to recognize

  • Comprehensive antigen profiling using immunohistochemistry for both antigens is essential

  • Mass spectrometry can confirm the presence of both antigens in glomerular deposits

• Pathophysiological considerations:

  • Potential epitope spreading mechanisms

  • Sequential or simultaneous autoimmunity development

  • Possibility of cross-reactivity between antibodies

• Clinical implications:

  • Treatment response may differ from single-antigen positive cases

  • Monitoring should include assessment of both antibodies when possible

  • Prognosis may depend on the dominant pathogenic process

Systematic characterization of dual-positive cases could provide insights into the fundamental immunological mechanisms of membranous nephropathy and inform optimized treatment approaches.

What treatment approaches have shown efficacy in NELL1-MN?

Based on current research, treatment strategies for NELL1-MN include:

• Trigger elimination:

  • Discontinuation of thiol-containing medications

  • Cessation of traditional indigenous medicines

  • Discontinuation of NSAIDs when implicated

• Conservative management:

  • ACE inhibitors or ARBs for proteinuria reduction

  • Supportive care for nephrotic syndrome

  • In one series, 30% of patients achieved complete remission with conservative management alone

• Immunosuppressive therapy:

  • Calcineurin inhibitors (CNI) with steroids for 6-12 months

  • In one case series, this approach led to complete remission in 29% and partial remission in 57% of treated patients

  • For CNI-resistant cases, cyclical cyclophosphamide-corticosteroid therapy may be effective

• Treatment of underlying conditions:

  • Cancer treatment when NELL1-MN is associated with malignancy

  • Management of associated autoimmune diseases

The data from one case series is summarized in the following table:

CNI: Calcineurin inhibitor; Data based on case series from search result

How should response to therapy be monitored in NELL1-MN research?

Comprehensive monitoring protocols for NELL1-MN research should include:

• Clinical parameters:

  • Proteinuria (24-hour collections or protein-to-creatinine ratios)

  • Serum albumin

  • Renal function (serum creatinine, eGFR)

  • Edema and other clinical manifestations

• Standardized outcome definitions:

  • Complete remission: proteinuria <500 mg/day with normal serum albumin

  • Partial remission: >50% reduction in proteinuria to <3.5 g/day with improved serum albumin

  • No response: failure to meet criteria for partial remission

  • Relapse: recurrence of nephrotic-range proteinuria after remission

• Monitoring schedule:

  • Monthly assessments during active treatment

  • Every 3 months during the first year of follow-up

  • Every 6-12 months thereafter

  • Earlier reassessment with clinical deterioration

• Special considerations:

  • Repeat kidney biopsy may be considered in non-responders

  • Development of anti-NELL1 serum assays would facilitate non-invasive monitoring

  • Cancer surveillance in cases with suspected paraneoplastic etiology

Standardized monitoring enables valid comparisons across different treatment approaches and research centers.

What prognostic factors predict outcomes in NELL1-MN?

Several factors appear to influence prognosis in NELL1-MN:

• Etiology-related factors:

  • Removable triggers (medications, CAM) associated with better outcomes

  • Absence of underlying malignancy generally predicts better prognosis

  • Non-NSAID related cases may respond better than those with chronic NSAID use

• Baseline clinical parameters:

  • Lower baseline proteinuria (<8 g/day) associated with higher spontaneous remission rates

  • Normal renal function at presentation predictive of better outcomes

  • Lower interstitial fibrosis and tubular atrophy (IFTA) on biopsy correlate with better prognosis

• Histopathological features:

  • Segmental rather than global immune deposits may predict higher remission likelihood

  • Lower intensity of C3 deposition may be associated with better outcomes

  • Lower chronicity indices on kidney biopsy correlate with better response

• Treatment-related factors:

  • Early initiation of therapy in high-risk patients

  • Achievement of initial reduction in proteinuria within 3-6 months

Understanding these prognostic factors helps guide treatment decisions and identify patients who may benefit from more aggressive therapeutic approaches.

What treatment challenges remain in NELL1-MN research?

Despite advances in understanding NELL1-MN, several research challenges remain:

• Optimization of immunosuppressive regimens:

  • Determining optimal duration of therapy

  • Identifying predictors of response to specific agents

  • Managing resistant or relapsing cases

  • Developing targeted therapies specific to NELL1-MN pathophysiology

• Biomarker development:

  • Validation of serum anti-NELL1 antibody assays

  • Correlation between antibody levels and disease activity

  • Identification of biomarkers predictive of treatment response

  • Development of non-invasive monitoring tools

• Management of secondary associations:

  • Optimal cancer screening protocols

  • Approach to patients with dual diagnoses

  • Management of recurrent disease after trigger elimination

  • Treatment of NELL1-MN in the context of multisystem autoimmune disease

Addressing these challenges requires collaborative research efforts and larger, prospective studies with standardized protocols and longer follow-up periods.

What are the optimal techniques for NELL1 detection in tissue samples?

For optimal NELL1 detection in research settings:

• Immunohistochemistry protocol:

  • Use of formalin-fixed, paraffin-embedded tissue sections (4 microns thick)

  • Heat-induced epitope retrieval

  • Validated anti-NELL1 antibodies at optimized concentrations

  • Appropriate positive and negative controls

• Interpretation guidelines:

  • Assessment of staining pattern (global vs. segmental)

  • Evaluation of staining intensity (0-3+ scale)

  • Determination of percentage of affected glomeruli

  • Documentation of deposits in other compartments (mesangial, subendothelial)

• Advanced techniques:

  • Laser microdissection coupled with mass spectrometry for confirmatory analysis

  • Multiplex immunofluorescence for co-localization studies

  • Electron microscopy correlation for ultrastructural characterization

  • Digital image analysis for quantitative assessment

Standardization of these techniques across research centers enhances reproducibility and facilitates meaningful comparison of results.

What are the key design elements for NELL1-MN clinical studies?

Rigorous NELL1-MN clinical studies should incorporate:

• Study population considerations:

  • Clear inclusion/exclusion criteria

  • Comprehensive baseline assessment including exposures

  • Systematic screening for associated conditions

  • Stratification based on clinical and histological parameters

• Intervention protocols:

  • Standardized treatment algorithms

  • Defined trigger elimination strategies when applicable

  • Protocol-driven immunosuppression adjustments

  • Management protocols for treatment failures

• Outcome measures:

  • Primary: proteinuria reduction, remission rates

  • Secondary: renal function preservation, relapse rates

  • Exploratory: biomarker responses, quality of life measures

  • Safety: adverse events, treatment complications

• Follow-up considerations:

  • Minimum follow-up period of 24-36 months

  • Standardized visit schedule and assessments

  • Protocol for managing intercurrent illnesses

  • Strategies to minimize loss to follow-up

What research gaps remain in understanding NELL1-MN?

Despite significant advances, several important knowledge gaps remain:

• Fundamental mechanisms:

  • Precise epitopes recognized by anti-NELL1 antibodies

  • Mechanisms of loss of tolerance to NELL1

  • Role of environmental factors in disease initiation

  • Genetic factors predisposing to NELL1 autoimmunity

• Diagnostic challenges:

  • Development and validation of serum-based assays

  • Standardization of tissue-based detection methods

  • Biomarkers distinguishing primary from secondary NELL1-MN

  • Non-invasive approaches to diagnosis and monitoring

• Treatment optimization:

  • Comparative effectiveness of different immunosuppressive regimens

  • Predictors of treatment response

  • Optimal duration of therapy

  • Management strategies for resistant disease

• Long-term outcomes:

  • Natural history studies with extended follow-up

  • Risk factors for progression to end-stage kidney disease

  • Long-term complications and comorbidities

  • Impact on patient survival and quality of life

Addressing these gaps requires coordinated research efforts spanning basic science, translational, and clinical domains.

How can researchers advance translational studies in NELL1-MN?

To advance translational research in NELL1-MN:

• Biorepository development:

  • Systematic collection of kidney tissue, serum, and urine samples

  • Longitudinal sampling before, during, and after treatment

  • Detailed clinical annotation of specimens

  • Standardized processing and storage protocols

• Multi-omics approaches:

  • Transcriptomic profiling of kidney tissue

  • Proteomic analysis of glomerular extracts

  • Metabolomic assessment of urine and serum

  • Integration of multi-omics data with clinical phenotypes

• Experimental models:

  • Development of animal models of NELL1-MN

  • In vitro systems using human podocytes

  • Organoid models for mechanistic studies

  • High-throughput screening platforms for therapeutic discovery

• Collaborative frameworks:

  • Establishment of international NELL1-MN research networks

  • Shared databases with harmonized data elements

  • Common biobanking protocols

  • Coordinated funding mechanisms for larger-scale studies

These translational approaches would accelerate progress from mechanistic understanding to clinical applications, ultimately improving outcomes for patients with NELL1-MN.