Serologic Studies in Membranous Nephropathy: Novel Strategies and Strengthened Associations

Introduction

Serologic studies have transformed clinical practice for patients with phospholipase A2 receptor (PLA2R)-positive membranous nephropathy (MN) by enabling noninvasive therapeutic disease monitoring. Although it is well known that anti-PLA2R antibodies correlate with disease activity and disease onset, this is less established for circulating antibodies against minor MN autoantigens because of a lower frequency of positivity in disease, including thrombospondin type-1 domain-containing 7A (THSD7A). In this issue, Burbelo et al. characterize the temporality of anti-THSD7A antibodies with disease activity and demonstrate evidence of seropositivity before disease onset in their original research article “Prediagnostic Appearance of THSD7A Autoantibodies in Membranous Nephropathy.”¹ The authors also use a highly sensitive and novel technique of serologic detection through use of the luciferase immunoprecipitation system (LIPS), which may hold promise for the development of future serologic assays in MN and other autoimmune diseases.

THSD7A and PLA2R Antibodies as Ideal Biomarkers of Disease

Serologic testing for THSD7A and PLA2R antibodies in MN have proven to have many characteristics of optimal serologic biomarkers for autoimmune diseases (Figure 1). Positive anti-THSD7A or anti-PLA2R serology shows near perfect correlation with tissue staining for THSD7A or PLA2R on kidney biopsy.^2–4 For both of these antigens, the presence of circulating autoantibodies precedes clinical recognition of disease.^1,5,6 This is challenging to establish because serum samples are not often available before clinical presentation. Burbelo et al. identified the presence of prediagnostic antibodies through screening of a large biorepository of blood samples donated by veterans with the Department of Defense serum repository.¹ The presence of circulating antibodies before transplantation in patients developing early disease recurrence provides further evidence of anti-THSD7A and PLA2R antibodies preceding disease manifestations.^7–9

Figure 1.

Characteristics of serologic testing for THSD7A and PLA2R antibodies in membranous nephropathy. THSD7A and PLA2R antibodies are identified before disease activity and correlate with the degree of proteinuria. Antibody titers fall in response to immunosuppressive therapy and rise before a clinical disease flare, providing usefulness in guiding treatment. Autoantibodies are identified in patients with MN driven by “secondary” disease triggers, where they may serve as a biomarker for both diseases (such as autoimmunity or malignancy). Serologic testing needs to be reliable with high sensitivity and specificity for use in clinical practice, as seen with the measurement of both anti-THSD7A and anti-PLA2R antibodies. MN, membranous nephropathy; PLA2R, phospholipase A2 receptor; THSD7A, thrombospondin type-1 domain-containing 7A.

Antibody titers for THSD7A and PLA2R correlate with disease activity, increasing with worsened proteinuria and falling with disease remission.^3,10,S1-2 Antibody levels decrease before a decline in proteinuria and in response to immunosuppression, which allows for therapeutic monitoring in practice and clinical trials.^1,S3 Finally, the presence of anti-THSD7A or anti-PLA2R autoantibodies in patients with remission can predict disease recurrence, predict prognosis, and allow early initiation of immunosuppressive therapy.^S4,S5 This is critical because it can prevent disease flares and reduce resulting end-organ damage by early initiation of treatment. Autoantibody titers may identify patients expected to have spontaneous remission without immunosuppression, although only established for PLA2R at this time.^S6 In patients in whom MN is “secondary” to autoimmunity or cancer, antibody titers may correlate with the underlying disease state in addition to the patient's kidney disease. In this study, anti-THSD7A antibodies were identified in patients with a history of systemic lupus erythematosus and malignancy.¹ Anti-THSD7A antibodies were previously found to disappear on clinical remission of an underlying cancer,^S7 indicating that serology can be used to monitor both the patient's kidney disease and the triggering malignancy.

Serologic studies make a noninvasive diagnosis possible when there is sufficiently high sensitivity and specificity of testing. According to the 2021 Kidney Disease Improving Global Outcomes glomerular disease guidelines, patients with positive serology for anti-PLA2R antibodies with normal kidney function and a lack of comorbid conditions that can induce proteinuria (such as diabetes mellitus) have a presumptive diagnosis of PLA2R-positive MN and may forgo kidney biopsy.^S8 Because immunosuppressive therapies are not without risk, near perfect specificity is required if initiating immunosuppression on the basis of serologic studies alone. This may be achieved with assay redundancy, where it would be less likely to have false positives with two separate assay platforms. For PLA2R-positive MN, this includes testing by both indirect immunofluorescence assay and ELISA. However, not all antigens are amenable to ELISA-based testing due to requirement of proteins being in a native conformation or other factors, and therefore, additional testing platforms are needed for development or diagnostic and/or confirmatory tests.

Challenges in Autoantibody Detection May Require Use of Novel Assay Platforms

Serologic detection of autoantibodies is influenced by multiple variables. Sufficient sensitivity is required for detection, and adequate avidity is needed for antibodies to react to target antigen. The source of antigen must be in an appropriate conformation for antibody binding. The antigen source may include peptides, protein fragments, or full-length protein or require binding under native conditions. Epitopes can be dependent on the presence of particular post-translational modifications that may not be recapitulated with the use of synthetic recombinant proteins.

Several of these challenges to antibody detection may be overcome through the use of new technologies, including the LIPS assay as used in this study by Burbelo et al.^1,6 for detection of THSD7A and PLA2R antibodies. LIPS assays use expression of native protein in a mammalian cell line (Chinese hamster ovary cells) and can be adapted to partial or full-length proteins of an antigen of interest. There is the capacity to clone a large target sequence in the plasmid vectors used in the assay. As such, the entire extracellular domain of THSD7A was used as a source of antigen with adequate expression and high sensitivity for antibody detection.¹ LIPS technology has been used for detection of antibodies of multiple autoimmune and infectious diseases.^S9-12 It also provides the capability for multiplexing for testing panels of autoantibodies, such as antinuclear antibodies specificities.^S13 The LIPS assay can also be adapted to a point-of-care platform using magnetic sticks containing neodymium to capture immune complexes bound to paramagnetic protein A/G beads, which can be detected rapidly with a luminometer, known as the LIPSTICKS (luciferase immunoprecipitation systems-based rapid immunoassay) assay.^S14

Future Outlook for Serologic Testing in MN

In addition to THSD7A and PLA2R, circulating autoantibodies have also been identified for most of the MN antigens, including MN associated with neural epidermal growth factor-1,^S15 serine protease HTRA1 (HTRA1),^S16 semaphorin 3B,^S17 neural cell adhesion molecule-1,^S18 contactin-1,^S19 and netrin G1.^S20 Identification of these antibodies in patient sera opens the door for serologic assay development for disease monitoring, as is available for THSD7A and PLA2R. Antibodies have not been identified at this time for some MN types, including exostosin 1/2–positive MN^S21 and TGF-β receptor 3–positive MN,^S22 despite strong supporting evidence that these proteins are true antigens because these proteins can be immunoprecipitated from immune complexes within kidney disease and demonstrate colocalization with immunoglobulin. It is possible, and likely, that the protein characteristics of these antigens and availability of epitopes may require new approaches for antibody detection.

It is uncertain that serologic assays for THSD7A and other minor antigens will be able to be used for primary diagnosis, such as PLA2R. MN is the leading cause of idiopathic nephrotic syndrome in nondiabetic patients, for which approximately 70% of patients are PLA2R-positive.^S23 For other MN antigens, there is a much lower disease frequency, often in the <1%–5% range. Therefore, the pretest probability of PLA2R-positive MN in a patient with nephrotic syndrome and normal renal function is fairly high, equating to >20%, while the pretest probability of other “idiopathic” MN antigens, such as semaphorin 3B, HTRA1, or netrin G1-associated MN would be significantly lower. With a lower prevalence within a population of patients with nephrotic syndrome, there is a higher risk of false-positive results. Nonetheless, there is clear value in serologic assay development to enable monitoring of disease activity for minor antigens.

Development of multiplex serologic testing for MN autoantigens may also be a viable alternative to immunostaining for antigen type on kidney biopsies. Currently, the workflow of evaluating MN biopsies requires multiple immunostains on serial kidney biopsy sections for antigen typing. Replacing immunostaining with serologic testing of blood samples collected at the time of biopsy might conserve tissue and reduce costs. However, there may be reduced sensitivity because a subset of patients could be in serologic remission at the time of biopsy or have delayed detection of circulating antibodies that are preferentially bound to target antigen within podocytes, as in the “kidney as a sink” hypothesis.^S24

A critical need exists for the development of additional serologic testing methods for detection of autoantibodies in MN and other autoimmune diseases. Circulating antibodies have yet to be identified for some types of MNs (such as exostosin 1/2–positive MN and TGF-β receptor 3–positive MN)^S21,S22; highly sensitive assays are needed for optimal clinical management; and confirmatory methods are critical to reduce the rate of false-positive results. The LIPS assay described by Burbelo et al. provides a novel platform for improved sensitivity, high specificity, and potential capabilities for multiplexing.

Disclosures

T.N. Caza reports the following: Advisory or Leadership Role: Editorial board member, Kidney International Reports. C.P. Larsen reports the following: Ownership Interest: Arkana Laboratories. The authors receive research funding to study membranous nephropathy from the National Institutes of Health (grants 1R41DK130602, 2R44DK130702-02, and 1R43AR081720-01).

Funding

None.

Author Contributions

T.N. Caza and C.P. Larsen wrote the original draft and edited the manuscript.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/KN9/A312.

Supplemental References.