Prediagnostic Appearance of Thrombospondin Type-1 Domain 7A Autoantibodies in Membranous Nephropathy

Abstract

Key Points

• The entire extracellular domain of thrombospondin type-1 domain 7A (THSD7A) in the luciferase immunoprecipitation system immunoassay was required to detect autoantibodies with high sensitivity in membranous nephropathy (MN).

• In THSD7A-seropositive MN patients, changes in antibody levels precede changes in clinical status.

• Seropositive THSD7A antibodies were detected in some patients with MN considered to be secondary to autoimmunity or cancer.

Background

Pathogenic autoantibodies against thrombospondin type-1 domain 7A (THSD7A) are present in approximately 3% of patients with membranous nephropathy (MN). Compared with PLA2R antibodies, less is known about THSD7A autoantibodies (ABs) because of the relative rarity and the lack of a commercially available quantitative immunoassay.

Methods

In this study, we describe the development and validation of a highly quantitative luciferase immunoprecipitation system (LIPS) assay for detecting THSD7A ABs and used it to study dominant THSD7A epitopes, disease associations, and monitoring disease activity. The Department of Defense Serum Repository (DODSR) was then used to analyze THSD7A AB in 371 longitudinal serum samples collected before clinical diagnosis of MN from 110 PLA2R-negative MN subjects.

Results

LIPS analysis demonstrated that a near full-length THSD7A (amino acids 1–1656) detected robust autoantibody levels in all known seropositive MN patients with 100% sensitivity and specificity compared with ELISA and/or Western blotting. Most of the THSD7A-seropositive subjects in our pilot cohort had evidence of coexisting autoimmunity or cancer. Moreover, three THSD7A-seropositive patients undergoing immunosuppressive therapy showed longitudinal autoantibody levels that tracked clinical status. Additional epitope analysis of two smaller protein THSD7A fragments spanning amino acids 1-416 and 1-671 demonstrated lower sensitivity of 32% and 44%, respectively. In the DODSR cohort, THSD7A seropositivity was detected in 4.5% of PLA2R-negative MN patients. In one primary and in one secondary MN-associated with cancer, THSD7A ABs were detectable <1 month before biopsy-proven diagnosis. In addition, three patients with lupus membranous nephropathy had detectable THSD7A ABs years before hypoalbuminemia and biopsy-proven diagnosis.

Conclusions

Although further studies are needed to explore the significance of THSD7A ABs in lupus membranous nephropathy, this study describes a novel, highly sensitive LIPS immunoassay for detecting THSD7A ABs and adds to the existing literature on THSD7A-associated MN.

Clinical Trial registry name and registration number:

NCT00977977; registration date: September 16, 2009.

Introduction

Membranous nephropathy (MN) is an autoimmune disease affecting the kidneys and a leading cause of adult nephrotic syndrome.¹ In MN, autoantibodies directed against extracellular proteins expressed by podocytes form electron-dense immune deposits in the kidney.² Before the discovery of MN antigens, MN was classified as primary or idiopathic when an etiology could not be identified and secondary when another condition (e.g., systemic autoimmune disease, malignancy, infection) was identified as the cause. The discovery of numerous target autoantigens has transformed how we classify patients with MN.³ The first major antigenic target, M-type phospholipase A₂ receptor (PLA2R), to which 70%–80% of patients with primary MN develop autoantibodies, was discovered in 2009.⁴ Autoantibodies against a second podocyte protein, thrombospondin type-1 domain-containing 7A (THSD7A), were identified in 2014⁵ and are present in 2% to 10% of patients with MN.^6,7 Owing to widely available PLA2R immunoassays, autoantibody titers and trajectory have been well described before and at MN diagnosis, as well as after treatment and relapse and now inform diagnosis, management, and prognosis in PLA2R-associated MN.⁸ By contrast, fewer data exist for clinical applications of THSD7A AB testing. This is due to a combination of low seroprevalence and lack of a widely available commercial assay, in part due to the relatively large size of the THSD7A protein required for autoantibody screening. Current THSD7A AB assays including Western blot and the semiquantitative indirect immunofluorescence assay have limitations.^5,9,10 A quantitative ELISA for detecting THSD7A ABs is currently not commercially available and in limited use.⁶

We previously developed highly sensitive and specific luciferase immunoprecipitation system (LIPS) assays, based on recombinant luciferase-tagged antigen, to detect PLA2R ABs.^11,12 In this study, a LIPS immunoassay was developed for quantitative measurements of THSD7A ABs and used to describe the trajectory and dynamics of autoantibody levels before MN diagnosis in the preclinical phase, at diagnosis, after treatment, and before relapse.

Material and Methods

Patients and Samples

This study screened 502 patient sera for THSD7A ABs from three different cohorts (Figure 1). A pilot cohort consisted of deidentified patient samples collected at the National Institutes of Health, Bethesda, MD, under protocols approved by the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases. This cohort established the optimal THSD7A AB assay to maximize assay sensitivity and specificity. The pilot cohort included a total of 96 samples, from 34 untreated PLA2R-seropositive and 35 PLA2R-seronegative MN subjects and 27 disease controls with other glomerular diseases. THSD7A AB status was assigned for each sample before unblinding. After unblinding, samples identified as THSD7A-seropositive by LIPS were confirmed by Western blot for THSD7A AB and/or by THSD7A tissue staining of renal biopsy. LIPS was also used for quantification of serial autoantibody levels in THSD7A-seropositive subjects in the pilot cohort undergoing immunosuppressive treatment.

Figure 1.

Characteristics of the three cohorts screened for THSD7A AB by LIPS. This study screened 502 patient serum samples for THSD7A AB from three different cohorts. The characteristics of each cohort are shown above. THSD7A AB status was assigned for each sample before unblinding. Three THSD7A-seropositive patients from the pilot cohort who were treated were also monitored longitudinally for changes in autoantibody levels and clinical response. LIPS, luciferase immunoprecipitation systems; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

A validation cohort consisting of samples provided by L.H. Beck Jr. (Boston University) was used to assess the diagnostic performance of the assay and contained 35 blinded serum samples from subjects with MN previously classified by Western blot as THSD7A-seropositive (n=25) or seronegative (n=10). Antibody levels based on LIPS in 24 THSD7A-seropositive samples were also compared with the IgG4 anti-THSD7A ELISA.⁶

The third cohort was from the Department of Defense Serum Repository (DoDSR) (protocol #0042, Walter Reed National Military Medical Center). We previously reported the emergence of prediagnostic PLA2R antibody in longitudinal, DODSR biobanked serum samples collected before MN diagnosis.¹² For this study, samples previously identified as PLA2R AB-negative were analyzed for THSD7A autoantibodies before the diagnosis of MN based on renal biopsy. In total, 371 longitudinal samples from 75 patients classified as primary membranous nephropathy and 35 with secondary membranous nephropathy were evaluated in a single-blinded manner. For the DODSR cohort, the terms primary and secondary were used because sample collection and classification in this database predates identification of MN antigens. Seropositive and seronegative status was assigned for each sample before unblinding. The measurements of serum albumin were also evaluated as previously described.¹²

THSD7A Luciferase Fusion Proteins for LIPS Autoantibody Testing

To study THSD7A ABs, a near full-length protein (1656/1657 amino acids) along with two protein fragments were developed as Gaussia luciferase fusion proteins for LIPS. For plasmid construction, a clone containing the entire human THSD7A cDNA clone¹³ was used as template for PCR and the cDNA was subcloned as an N-terminal fusion protein with Gaussia luciferase. Two THSD7A fragments encompassing amino acid residues 1-416 and 1-671 were also generated in a similar fashion. Although a general protocol describing LIPS testing is available,¹⁴ a detailed LIPS methodology used to study THSD7A antibodies is provided in Supplemental Information 1. A small number of additional LIPS immunoassays for measuring autoantibodies against known lupus and other autoimmune targets, including Ro52, Ro60, RNP-A, Smith-D3, GAD65, and IFN-α, were tested in selected patients and have been described previously.¹⁵

Statistics

GraphPad Prism software (San Diego, CA) was used to analyze and plot autoantibody measurement data. Cutoff values for determining THSD7A seropositivity was based on optimal separation using receiver operator characteristic (ROC) analysis. The heatmap was constructed based on Z-scores of THSD7A-seropositive antibody levels based on the THSD7A-seronegative controls as described.¹⁵

Results

Detection of THSD7A ABs by LIPS in a Pilot Cohort

A near full-length THSD7A chimeric fusion protein with Gaussia luciferase was used in LIPS to evaluate autoantibodies in a pilot cohort. After decoding, the control group (n=27) all had low THSD7A AB levels with an average value of approximately 10,000 LU (Figure 2). Similarly, low autoantibody levels were found in 34 (of 34) known PLA2R-seropositive and 30 (of 35) PLA2R-seronegative MN subjects (Figure 2). Five PLA2R-seronegative subjects showed higher seropositive THSD7A AB levels (Figure 2). Three of these samples showed approximate values of 215,000 LU, 220,000 LU, and 313,000 LU; one had modest autoantibody levels (125,000 LU); and one had a lower value of 35,000 LU. Western blot and/or tissue staining of the renal biopsy from the five subjects confirmed THSD7A seropositivity. Using ROC analysis, a tentative cutoff value of 30,000 LU was chosen and yielded 100% sensitivity and specificity for detecting all five subjects.

Figure 2.

LIPS detection of THSD7A AB in subjects with MN and kidney disease controls. Kidney disease controls (Kidney CTRLS) and PLA2R-seropositive and seronegative MN subjects were tested by LIPS with the near full-length THSD7A-Gaussia fusion protein. Each circle represents the antibody level in light units (LU) in individual serum sample and is plotted on the y-axis using a log₁₀ scale. The cutoff value for the assay for determining THSD7A seropositive and seronegative status is shown by the dotted line. THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Clinical data for the five THSD7A-seropositive subjects revealed three women and two men with a mean age at presentation of 42±8 years (Table 1). Mean proteinuria at diagnosis was 11.6±4 g, and eGFR was 82±30 ml/min per 1.73 m². Subject 1 had onset of nephrotic syndrome during the third trimester of pregnancy and underwent renal biopsy after delivery. Subject 4 was diagnosed with MN shortly after delivering a baby complicated by eclampsia, but the timing of kidney disease onset was unknown. In addition, three subjects had coexisting autoimmune or inflammatory disorders: lupus, heterozygous loss-of-function TNFAIP3 gene mutation causing A20 haploinsufficiency (HA20), and refractory pustular psoriasis.

Table 1.

Characteristics of THSD7A AB-seropositive subjects

Subject	Age (yr)	Sex	Race	SCr (mg/dl)	eGFR (ml/min per 1.73 m2)	Proteinuria (g/day)	THSD7A Ab Titer LU (LIPS)a	Coexisting Disorders	Selected Serologies (LIPS)b	Treatments/Outcome
1	29	F	White	0.7	120	4.2	35,036	HA20c	Ro52 Ro60 RNP-A IFNα	Anakinra, tofacitinib PR; relapse after self-discontinuation of medications
2	48	M	Black	1.1	83	16.9	215,884	Lupus	Ro52 Ro60 RNP-A GAD65	Cytoxan, rituximab, corticosteroids; PR
3	46	M	White	0.8	111	13.4	221,519	Refractory Generalized Pustular Psoriasis	Negative	Cyclosporine, anakinra NR, died
4	36	F	Black	1.3	55	13.7	135,806	Eclampsia	Negative	Rituximab CR, sustained
5	51	F	Asian	1.5	42	9.8	313,858	—	Negative	Rituximab + cyclosporine; CR, followed by relapse

F, female; M, male; SCr, serum creatinine; eGFR, CKD-EPI Creatinine (2021); PR: partial remission defined as >50% reduction in peak proteinuria and <3.5 g/d; CR: complete remission defined as proteinuria <300 mg/d; NR, no response.

^aCutoff value for LIPS assay seropositivity: 30,000 LU.

^bSelected serologies performed by LIPS: Ro52, Ro60, RNP-A, GAD65, and IFN-α.

^cHA20: haploinsufficiency of A20 due to heterozygous loss-of-function mutation in TNFAIP.

All five THSD7A-seropositive subjects were PLA2R-seronegative. To explore the coexistence of other antibodies, these subjects were tested for autoantibodies against a limited panel of other autoantigens associated with systemic lupus erythematosus and other autoimmune diseases.¹⁵ Subject 2 harbored Ro52, Ro60, RNP-A, and high-level GAD65 autoantibodies, and subject 1 with HA20 harbored Ro52, Ro60, RNP-A, and IFN-α1 autoantibodies. These findings highlight the diversity of other autoantigen targets seen in these subjects (Table 1). Treatment and outcomes of these patients are provided in Table 1.

THSD7A AB LIPS Testing in a Validation Cohort

The LIPS assay was next used to assess an independent, validation MN cohort. Based on the cutoff value above 30,000 LU, 25 patients were assigned as THSD7A-seropositive and 10 patients as seronegative (Figure 3). After unblinding, the THSD7A serological status in these samples demonstrated 100% sensitivity and specificity compared with Western blot analysis. Additional comparisons revealed that LIPS closely tracked (Pearson R=0.58, P=0.017) the THSD7A IgG4 levels determined by ELISA (data not shown).

Figure 3.

Validation of the LIPS assay for detecting THSD7A AB in an independent cohort. LIPS detection of THSD7A AB was evaluated in a validation cohort of blinded samples from known seronegative and seropositive MN patients. Classification status of seronegative (−) or seropositive (+) was based on THSD7A ELISA antibody testing and/or Western blot analysis. Each circle represents antibody levels in light units (LU) derived from individual subjects plotted on the y-axis using a log₁₀ scale. The cutoff value is shown by the dotted line, and LIPS testing showed 100% sensitivity and specificity. LIPS, luciferase immunoprecipitation systems; MN, membranous nephropathy; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Immunoreactive Regions of THSD7A Protein

A previous study identified an important antigenic region located in the N terminus of the THSD7A molecule.¹⁴ To investigate immunoreactivity of different regions of THSD7A, two additional smaller fragments, THSD7A-∆1 (amino acids 1-416) and THSD7A-∆2 (amino acids 1-671), were tested with the validation cohort, and the results were compared with full-length THSD7A protein (Figure 4A). A heatmap normalized to the 10 seronegative patients was used to visualize immunoreactivity in the 25 subjects with MN (Figure 4B). The smallest THSD7A-∆1 fragment detected only 33% (8/25) of the MN samples as seropositive. The larger THSD7A-∆2 fragment was slightly more informative detecting 45% (11/25) as seropositive. Although many of the THSD7A-∆1 and THSD7A-∆2–seropositive patients overlapped, three seropositive subjects detected by the smaller THSD7A-∆1 protein fragment were seronegative with the larger THSD7A-∆2 fragment. Overall, these results suggest that there are multiple antigenic regions within the protein, yet the near full-length THSD7A is the most informative, showing 100% sensitivity, because it likely captures important immunoreactive targets in the C-terminal part of the molecule.

Figure 4.

Detection of patient seropositivity against multiple epitopes of THSD7A. (A) Schematic of THSD7A-Δ1, THSD7A-Δ2, and near full-length THSD7A (FL) chimeric Gaussia luciferase (Luc) fusion proteins used for testing the different MN serum samples. The location of the transmembrane region (amino acids 1608-1628) of THSD7A is shown in THSD7A-FL by the blue box. (B) A heat map is shown of the autoantibody responses to the three different THSD7A proteins from the validation cohort, in which each row represents a different MN sample. To construct the heatmap, the autoantibody values for the 10 seronegative controls (not shown) and the THSD7A-seropositive MN subjects were color-coded based on a Z-score scale shown on the right representing the number of standard deviations above the mean of the controls for that antigen. Coloring in the heat map indicates that the relative antibody levels are at least greater than the mean of the controls plus three standard deviations. The signal intensities range from white to dark red indicating low and high autoantibody levels, respectively. MN, membranous nephropathy; THSD7A, thrombospondin type-1 domain 7A.

Monitoring Treatment Response by THSD7A AB Detection

THSD7A ABs were next evaluated in longitudinal samples from three subjects with MN (2, 4 and 5) in the pilot cohort who were receiving treatment. Before treatment, all three patients had sustained high levels of THSD7A antibody and proteinuria. Treatment of subject 2 with cyclophosphamide and rituximab led to reduced THSD7A autoantibodies over 12 months paralleling the decrease in proteinuria (Figure 5A). By 8 months, THSD7A AB levels decreased below the cutoff value, corresponding to partial remission of proteinuria and proteinuria nadired at 0.355 g/d by 12 months. Subject 4 responded to rituximab monotherapy and showed a similar pattern of antibody depletion in advance of proteinuria improvement (Figure 5B). In subject 5, THSD7A AB was fully depleted below the cutoff value by 6 months after rituximab and cyclosporine, which preceded complete remission (based on proteinuria) by 3 months (Figure 5C). Autoantibody re-emergence occurred at 48 months, and re-treatment with rituximab led to clinical and immunologic remission. Two additional relapses occurred in subject 5 with similar patterns in THSD7A AB and proteinuria (Figure 5C). Immunologic relapse was apparent 5 months in advance of clinical relapse. It is notable that seropositivity at the earliest time of presentation was detectable with large THSD7A protein, as well as the two fragments, but with relapse, autoantibodies were only detectable with near full-length protein (data not shown). Overall, these results demonstrate that the LIPS THSD7A immunoassay is a sensitive method for monitoring clinically informative changes in autoantibodies.

Figure 5.

Association between THSD7A AB and clinical disease activity in three MN subjects receiving immunosuppressive treatment. THSD7A AB levels (blue circles and lines) and proteinuria (black squares and lines) are shown over time in three different MN patients (A-C). The red arrow denotes the start of treatment, in which all three patients had sustained high levels of THSD7A AB and proteinuria for months before treatment. The x axis is the time in months after initiation of immunosuppressive treatment. The left y axis represents the scale of the autoantibody levels in LU determined by LIPS with the near full-length protein. The right y axis denotes the level of urinary protein excretion expressed as grams per day. The blue dotted line represents the seropositive cutoff value for LIPS; the black dotted line represents complete remission status (proteinuria <300 mg/d). MN, membranous nephropathy; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

THSD7A AB before Disease Diagnosis

The emergence of PLA2R AB was previously studied in retrospective, biobanked serum MN samples from DODSR.¹² In this study, previously identified PLA2R-seropositive patients from the DODSR cohort were excluded, and the remainder were evaluated for THSD7A AB. Of 371 PLA2R-seronegative samples, 15 sera were THSD7A-seropositive and after decoding, were found to comprise five subjects from screening 75 primary MN and 35 secondary MN. Thus, 4.5% (5/110) of the PLA2R-negative MN cohort were THSD7A-seropositive. One patient was classified as primary MN and another was classified as cancer-associated secondary MN, and three (2.7%) had lupus membranous nephropathy (LMN) and had been classified as secondary MN. Serum albumin levels in serial samples were determined and plotted over time with the THSD7A AB levels (Figure 6). The subjects with primary (Figure 6A) and cancer-associated (Figure 6B) MN were seropositive for THSD7A AB only in the last longitudinal sample (also confirmed positive by THSD7A IgG4 ELISA) less than 1 month before histologic diagnosis and showed co-occurrence of hypoalbuminemia. By contrast, seropositive THSD7A ABs were detected in three subjects with LMN up to 5 years before histologic diagnosis (Figure 6, C–E). Appearance of THSD7A AB preceded hypoalbuminemia in LMN subjects 8 and 10. In LMN subject 9, THSD7A seropositivity fluctuated over time (Figure 6D) appearing to show a subclinical presentation of LMN manifesting as emergence of THSD7A AB with hypoalbuminemia, followed by immunologic and clinical remission and then relapse before biopsy diagnosis of LMN. Autoantibodies against three lupus-associated autoantigens were assessed in subjects 8–10 (Figure 7). In subject 8, Ro52, Ro60, and THSD7A ABs were present at the first time point and remained positive. Subject 9 was seronegative for Ro52 and Ro60 autoantibodies at the first measured time point, but seropositive in the second and last time point concurrent with the appearance of THSD7A AB (Figure 7). Subject 10 was seronegative for the three circulating lupus autoantibodies.

Figure 6.

Prediagnostic appearance of THSD7A AB before diagnosis. Autoantibody analysis of the DODSR cohort identified five THSD7A-seropositive subjects. The five patients included one with primary MN (A), one cancer-associated MN (B), and three with lupus membranous nephropathy (C–E). THSD7A autoantibody levels in LU derived from serial samples of the seropositive patients are plotted in blue. The blue, horizontal dotted line is the cutoff for determining THSD7A seropositivity. The right y-axis denotes the serum albumin levels, and the cutoff value for determining hypoalbuminemia is shown by the black dotted line. Time zero represents biopsy-proven diagnosis of MN. MN, membranous nephropathy; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Figure 7.

Prediagnostic appearance of THSD7A AB relative to appearance of Ro52 and Ro60 ABs. From testing the three subjects with lupus membranous nephropathy, two (Pt 8 and Pt 9) were also seropositive for Ro52 and Ro60 ABs. THSD7A autoantibody levels are plotted in blue. The Ro52 and Ro60 ABs are shown in black, and the approximate cutoff value for seropositivity is shown by the red dotted line. The cutoff value for THSD7A AB seropositivity is shown with the dotted blue line. THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Discussion

In this study, we developed and validated a LIPS THSD7A AB immunoassay based on luciferase fusion proteins. A near full-length THSD7A recombinant protein was chosen for the immunoassay after detailed investigation. While the exact form, intact versus proteolyzed, and immunoreactive regions of THSD7A are not well established, mapping revealed that multiple regions are targeted by autoantibodies. An N-terminal THSD7A fragment containing the first 416 amino acids showed only limited sensitivity (33%) for detecting seropositivity, and a larger N-terminal fragment only improved sensitivity marginally to 45%. The highest sensitivity of 100% required the entire extracellular domain. These findings are partly consistent with Seifert et al.¹⁶ who used Western blotting and found that 87% of subjects had autoantibodies that recognized amino acids 48-192 of THSD7A. One potential explanation for this discrepancy is that more denaturing methods such as Western blot or ELISA may expose epitopes not available to antibody binding to the native THSD7A molecule. Further evidence for the importance of the C-terminal region in LIPS testing was detected in a THSD7A-treated patient, in which autoantibodies were present early in the course of the disease against near full-length and two protein fragments, but later in the course of disease, immunoreactivity was only seen against the full-length protein. These results suggest that epitope spreading may occur during disease progression and/or response to treatment.

THSD7A AB showed similar dynamics to PLA2R AB, in which levels declined with immunosuppressive treatment, disappeared with clinical remission, and reappearance preceded proteinuria and predicted relapse indicating that THSD7A AB levels are a good clinical marker of disease activity consistent with other studies.^6,17,18 By analyzing longitudinal DODSR samples, we provide the first description of the natural history of THSD7A AB emergence before diagnosis of MN. THSD7A AB was detected only within 1 month of the diagnosis for both primary MN and malignancy-associated MN, but the limited patient number and timing of samples preclude firm conclusions. By contrast, THSD7A ABs were detected in three patients with LMN years before diagnosis and were elevated before prediagnostic hypoalbuminemia. In two patients with LMN, the appearance of THSD7A AB was concurrent with other lupus-associated antibodies. In one subject, subclinical disease flare manifesting as transient THSD7A seropositivity and hypoalbuminemia was evident years before formal diagnosis. We previously reported similar episodes of undiagnosed subclinical flares in PLA2R-associated MN.¹²

THSD7A seropositivity was 5.2% in the pilot cohort, and 4.5% of the PLA2R-seronegative patients in the DODSR cohort. Among these ten THSD7A-seropositive MN patients, 70% had evidence of a secondary comorbidity or systemic autoimmunity. There was one patient with MN (0.9%) associated with malignancy in the DODSR cohort. THSD7A MN has been associated with an increased risk of cancer compared with PLA2R-associated MN.¹⁹ It is notable that THSD7A ABs were not detected earlier in this case of cancer-associated MN, which might have been expected if a link existed between a THSD7A-expressing tumor and autoantibody generation.

THSD7A-positive MN has been associated with a variety of other diseases.^20,21 In the pilot cohort, several patients with MN were considered secondary to another disease. We report the first patient with THSD7A-associated MN potentially related to haploinsufficiency in A20 (HA20), an autoinflammatory condition manifesting as genital, oral, and/or gastrointestinal ulcers²² related to enhanced NFκB.²³ Treatment of the underlying disease led to remission of proteinuria supporting a link between HA20 and development of MN. One subject also had psoriasis, which has been associated with MN. Treatment of the underlying skin disease did not lead to improvement in proteinuria. This may reflect the severe, refractory nature of the skin disease or suggests coincidental occurrence of two diseases. In support of the latter, a study of 24 MN patients with psoriasis did not show evidence of glomerular expression of PLA2R or THSD7A or circulating autoantibodies.²⁴ Interestingly in the pilot cohort, two subjects with THSD7A-associated MN were diagnosed shortly after pregnancy. THSD7A-associated MN during pregnancy has been reported.²⁵ Because THSD7A is expressed in placental vasculature and umbilical vein endothelial cells,¹³ these sites could be a source of extrarenal expression leading to autoantibody production, but this is purely speculation.

Four of the ten THSDA-seropositive cases were LMN. THSD7A antibodies have only rarely been associated with class V LN,⁵ whereas exostosins 1 and 2, NCAM1, and TGF-β receptor 3 are well-recognized target autoantigens in LMN.^26–28 Reasons for the higher-than-expected prevalence of THSD7A AB in LMN in our study are unclear but may be because of misclassification of patients in the DODSR cohort or differences in the detection method (THSD7A renal antigen expression versus LIPS) and assay error. Nevertheless, the simultaneous appearance of lupus autoantibodies with THSD7A AB supports the validity of our findings.

There are several limitations of this study mainly because of inherent drawbacks of the DODSR cohort.¹² As it relies on historical charting, there is potential for misclassification of patients with MN as primary versus secondary and misattribution to the secondary cause. The time intervals between samples and diagnosis were variable for each case and limited, which may have led to underestimation of seropositive cases. Another limitation is the small percentage of detectable THSD7A-seropositive patients, similar to previous reports. Further studies are needed to better understand THSD7A AB contribution to primary MN, LMN, and other secondary forms of MN, as well as describe how genetics and the dynamics of the autoantibody may affect disease presentation and outcome. Overall, the LIPS technology appears well-suited for the development of a panel of MN autoantigens in a high-throughput format that could serve as noninvasive biomarkers to facilitate diagnosis without a kidney biopsy, an approach currently supported by the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines for PLA2R-associated MN.

Disclosures

P.D. Burbelo reports the following: Patents or Royalties: NIH and HHS. L.H. Beck reports the following: Consultancy: Alexion, Novartis, Visterra, and Ionis; Research Funding: Pfizer; Honoraria: UpToDate, Inc.; Patents or Royalties: I am a coinventor on and receive royalties related to the US patent “Diagnostics for Membranous Nephropathy”; and Advisory or Leadership Role: Kidney Medicine editorial board. J.M. Keller reports the following: Employer: BioNTech SE. G. Lambeau reports the following: Patents or Royalties: Euroimmun DE. S.W. Olson reports the following: Employer: Novartis employee (April 2021). D.M. Schwartz reports the following: Other Interests or Relationships: I have an agreement to consult through Guidepoint Consulting on intracellular signal transduction and cytokines. I have not consulted through this company or other companies in the last 12 months. The remaining authors have nothing to disclose.

Funding

This work was supported by the intramural research programs of the National Institute of Dental and Craniofacial Research and National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Z99 DE999999), as well as an extramural grant DK097053 to L.H. Beck. This work was supported by the Agence Nationale de la Recherche (ANR-20-CE14-0024-01 to G.L.) and the Fondation de la Recherche Médicale (DEQ20180339193 to G.L.).

Author Contributions

S.W. Olson and M. Waldman conceptualized the study, provided supervision, and were responsible for data curation; L.H. Beck, P.D. Burbelo, M. Joshi, G. Lambeau, and M. Waldman were responsible for investigation; P.D. Burbelo and J.M. Keller were responsible for methodology; Y.-J. Chuang, M. Joshi, and D.M. Schwartz were responsible for resources; M. Waldman was responsible for funding acquisition and project administration; P.D. Burbelo and M. Waldman were responsible for formal analysis and wrote the original draft; and L.H. Beck, P.D. Burbelo, Y.-J. Chuang, J.M. Keller, G. Lambeau, S.W. Olson, D.M. Schwartz, and M. Waldman reviewed and edited the manuscript.

Data Sharing Statement

All data are included in the manuscript and/or supporting information.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/KN9/A254.

Luciferase Immunoprecitation System (LIPS) Methodology.

Supplemental Figure 1A. Schematic of the construction and expression for generating the THSD7A-luciferase recombinant protein.

Supplemental Figure 1B. Schematic overview of the LIPS assay.

Supplemental references.