THSD7A-associated Membranous Nephropathy Research Articles

Prevalence and prognosis of malignancy in THSD7A-associated membranous nephropathy: a systematic literature review and clinical case study

Background This study aims to investigate the incidence and prognosis of malignancy in individuals with thrombospondin type-1 domain-containing 7A (THSD7A)-associated membranous nephropathy (MN). Methods First, we performed a systematic literature review of prevalence of malignancy in THSD7A-associated MN. Then, we conducted a retrospective analysis of 454 patients diagnosed with MN through renal biopsy at our hospital between January 2016 and December 2020. We assessed the presence of serum anti-THSD7A antibodies and performed immunohistochemical staining of renal tissue for THSD7A. Subsequently, we followed patients with THSD7A-associated MN for a minimum of 3–5 years, collecting their clinical, pathological characteristics, and prognosis. Additionally, we conducted a literature review on patients with THSD7A-associated MN in conjunction with malignancy. Results We identified a total of nine articles containing comprehensive data on THSD7A-associated MN and malignancy. Among 235 patients with THSD7A-positive MN, 36 individuals had concurrent malignancies, resulting in a malignancy prevalence of 13.3% (95% CI: 8.9–17.7%). In our center, we followed up with 15 patients diagnosed with THSD7A-associated MN and observed three cases of concomitant tumors: two cases of lung adenocarcinoma and one case of small cell lung cancer with multiple metastases. The prevalence of malignancy in our cohort was 20%. Notably, we detected positive THSD7A staining in both renal and lung cancer tissues in one patient with small cell lung cancer. Conclusions Patients with THSD7A-associated MN should undergo vigilant follow-up assessments, with a particular focus on actively seeking potential tumorigenic lesions to prevent misdiagnosis or oversight.

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

Key PointsThe 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.BackgroundPathogenic 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.MethodsIn 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.ResultsLIPS 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.ConclusionsAlthough 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.

The Alternative Pathway Is Necessary and Sufficient for Complement Activation by Anti-THSD7A Autoantibodies, Which Are Predominantly IgG4 in Membranous Nephropathy.

Membranous nephropathy (MN) is an immune kidney disease characterized by glomerular subepithelial immune complexes (ICs) containing antigen, IgG, and products of complement activation. Whereas proteinuria is caused by complement-mediated podocyte injury, the pathways of complement activation remain controversial due to the predominance of IgG4 in ICs, an IgG subclass considered unable to activate complement. THSD7A, a transmembrane protein expressed on podocytes, is the target autoantigen in ~3% of cases of primary MN. In this study, we analyzed sera from 16 patients with THSD7A-associated MN with regard to the anti-THSD7A IgG subclasses and their ability to fix complement in vitro. The serum concentration of anti-THSD7A IgG varied over two orders of magnitude (1.3-243 μg/mL). As a relative proportion of all IgG anti-THSD7A, IgG4 was by far the most abundant subclass (median 79%), followed by IgG1 (median 11%). IgG4 was the dominant subclass of anti-THSD7A antibodies in 14 sera, while IgG1 was dominant in one and co-dominant in another. One quarter of MN sera additionally contained low levels of anti-THSD7A IgA1. ICs formed by predominantly IgG4 anti-THSD7A autoantibodies with immobilized THSD7A were relatively weak activators of complement in vitro, compared to human IgG1 and IgG3 mAbs used as positive control. Complement deposition on THSD7A ICs was dose-dependent and occurred to a significant extent only at relatively high concentration of anti-THSD7A IgG. C3b fixation by THSD7A ICs was completely abolished in factor B-depleted sera, partially inhibited in C4-depleted sera, unchanged in C1q-depleted sera, and also occurred in Mg-EGTA buffer. These results imply that THSD7A ICs predominantly containing IgG4 activate complement at high IgG4 density, which strictly requires a functional alternative pathway, whereas the classical and lectin pathways are dispensable. These findings advance our understanding of how IgG4 antibodies activate complement.

Characterization of THSD7A-antibodies not binding to glomerular THSD7A in a patient with diabetes mellitus but no membranous nephropathy

Membranous nephropathy (MN) is an autoimmune disease caused by autoantibodies against the podocyte antigens phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type 1 domain containing protein 7A (THSD7A) in 80% and 2–3% of patients, respectively. THSD7A antibodies are considered to be pathogenic and highly specific for MN patients. Using an indirect immunofluorescence test (IIFT) we detected THSD7A-antibodies (titre 1:10) in the serum of a patient with high proteinuria who, however, in the kidney biopsy was diagnosed with diabetic nephropathy and MN was excluded as a possible cause of proteinuria. Different immunofluorescence assays and Western blot techniques using recombinant THSD7A (rTHSD7A) or THSD7A from different human tissues revealed that the circulating THSD7A-autoantibodies were only of the IgG3 subclass. The patient serum reacted exclusively with rTHSD7A and only when the antigen was present in reducing Western blot conditions, or on formaldehyde-fixed cells for the IIFT. Our findings show for the first time the existence of circulating THSD7A-antibodies recognizing denatured/reduced rTHSD7A, which do not react with glomerular THSD7A in vivo and are thus presumptively non-pathogenic. As a consequence, kidney biopsy or Western blot analyses of THSD7A under non-reducing conditions should be performed to confirm the diagnosis of THSD7A-associated MN, especially in cases with low THSD7A-antibody levels in the IIFT.

Recurrent membranous nephropathy with a possible alteration in the etiology: a case report

BackgroundPhospholipase A2 receptor 1 (PLA2R1) and thrombospondin type-1 domain-containing 7A (THSD7A) are the two major pathogenic antigens for membranous nephropathy (MN). It has been reported that THSD7A-associated MN has a higher prevalence of comorbid malignancy than PLA2R1-associated MN. Here we present a case of MN whose etiology might change from idiopathic to malignancy-associated MN during the patient’s clinical course.Case presentationA 68-year-old man with nephrotic syndrome was diagnosed with MN by renal biopsy. Immunohistochemistry showed that the kidney specimen was negative for THSD7A. The first course of corticosteroid therapy achieved partial remission; however, nephrotic syndrome recurred 1 year later. Two years later, his abdominal echography revealed a urinary bladder tumor, but he did not wish to undergo additional diagnostic examinations. Because his proteinuria increased consecutively, corticosteroid therapy was resumed, but it failed to achieve remission. Another kidney biopsy was performed and revealed MN with positive staining for THSD7A. PLA2R1 staining levels were negative for both first and second biopsies. Because his bladder tumor had gradually enlarged, he agreed to undergo bladder tumor resection. Pathological examination indicated that the tumor was THDS7A-positive bladder cancer. Subsequently, his proteinuria decreased and remained in remission.ConclusionsThis case suggests that the etiology of MN might be altered during the therapeutic course. Intensive screening for malignancy may be preferable in patients with unexpected recurrence of proteinuria and/or change in therapy response.

Update membranous nephropathy

Membranous nephropathy is an autoimmune disease caused in most cases by binding of circulating antibodies to antigens on podocytes. PLA2R1 and THSD7A have been identified as target antigens in 70-80 % and 2-3 % of patients, respectively. The detection of PLA2R1- and THSD7A-antibodies in the blood and staining of renal biopsies for the respective antigens allow the correct diagnosis of PLA2R1- and THSD7A-associated membranous nephropathy, respectively, in practically 100 % of cases. Measurement of PLA2R1- and THSD7A-antibodies is helpful in order to further individualize the decision whether to perform a renal biopsy in patients with suspected membranous nephropathy. PLA2R1-antibody level is a strong predictor for remission of proteinuria, loss of renal function and relapse of disease. Moreover, treatment decisions in patients with PLA2R1-associated MN are increasingly based on the PLA2R1-antibody levels. Rituximab was shown to be non-inferior to ciclosporine A to induce remission of proteinuria after 12 months. After 24 months rituximab was superior to ciclosporine A for the same endpoint. Development of novel treatment strategies, focusing on disease pathogenesis, remains highly relevant for these patients.

Membranous nephropathy associated with thrombospondin type-1 domain-containing 7A (THSD7A) in an adult woman with eosinophilia

A 30-year-old woman on steroid therapy for eosinophilia presented with nephrotic syndrome during steroid tapering. She was diagnosed with membranous nephropathy (MN) stage II–III (positive for IgG1 and IgG4) by renal biopsy. There was no evidence of secondary MN. Her urinary protein level was controlled to 0.5 g/day or less, and her eosinophil count in white blood cell differential was stabilized at less than 10% without increasing the steroid dosage. The renal specimen did not show any enhanced granular expression of PLA2R along the glomerular basement membrane, and PLA2R was not detected in the patient’s serum. On retrospective analysis, enhanced granular staining for thrombospondin type-1 domain-containing 7A (THSD7A) in the glomeruli was detected in the biopsy, and anti-THSD7A IgG was detected in the serum using a commercial indirect immunofluorescence test (IFT). Based on these, the case was considered as THSD7A-associated MN with comorbid eosinophilia. The causal relationship between THSD7A-related MN and eosinophilia was unclear. However, a few cases of THSD7A-associated MN with eosinophilia have been reported, and further clarification on the relationship between THSD7A-related MN and eosinophilia is warranted.

SUN-030 PHOSPHOLIPASE A2 RECEPTOR AND THROMBOSPONDIN TYPE-1 DOMAIN-CONTAINING 7A IN MALIGNANCY MEMBRANOUS NEPHROPATHY

Phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) have recently been identified as pathogenic antigens in patients with membranous nephropathy (MN). Notably, PLA2R is detected in a few patients with malignancy-associated MN. Furthermore, a high incidence of cancer is reported in patients with THSD7A-associated MN. It is thus necessary to clarify the usefulness of PLA2R and THSD7A in malignancy-associated MN. Serum anti-PLA2R antibodies and glomerular PLA2R expression were first screened in 36 patients with malignancy-associated MN by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence, respectively, followed by examination of serum anti-THSD7A antibodies and glomerular THSD7A. THSD7A expression in cancer tissues was also assessed in 9 of the 36 patients. Among the 36 patients, 12 (33%) were positive for both glomerular PLA2R staining and serum anti-PLA2R antibodies, and 2 were positive for either glomerular PLA2R staining or serum anti-PLA2R antibodies. IgG4 was the dominant IgG subclass in PLA2R-related patients. One patient with positve glomerular PLA2R staining was also positive for THSD7A staining. Among the remaining 22 (61%) patients who were double negative for glomerular PLA2R staining and serum anti-PLA2R antibodies, 17 of 20 (85%) had IgG1 dominant deposits in glomeruli, and 2 (9%) were positive for glomerular THSD7A staining. Serum anti-THSD7A antibody was not detected in any of the 36 patients. Among the 9 patients with available cancer tissues, enhanced expression of THSD7A in the cancer tissues was observed for 5 (56%), and one also showed enhanced glomerular expression of THSD7A. Screening of glomerular PLA2R antigen and serum anti-PLA2R antibodies is necessary in patients with malignancy-associated MN, whereas the incidence of glomerular THSD7A antigen or circulating anti-THSD7A antibodies is uncommon.

Features of phospholipase A2 receptor and thrombospondin type-1 domain-containing 7A in malignancy-associated membranous nephropathy

AimsPhospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) were identified as pathogenic antigens in patients with membranous nephropathy (MN). Notably, PLA2R is detected in few patients with malignancy-associated...

Novel ELISA for thrombospondin type 1 domain-containing 7A autoantibodies in membranous nephropathy

Autoantibodies against phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type 1 domain-containing 7A (THSD7A) are emerging as biomarkers to classify membranous nephropathy (MN) and to predict outcome or response to treatment. Anti-THSD7A autoantibodies are detected by Western blot and indirect immunofluorescence test (IIFT). Here, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) optimized for quantitative detection of anti-THSD7A autoantibodies. Among 1012 biopsy-proven MN patients from 6 cohorts, 28 THSD7A-positive patients were identified by ELISA, indicating a prevalence of 2.8%. By screening additional patients, mostly referred because of PLA2R1-unrelated MN, we identified 21 more cases, establishing a cohort of 49 THSD7A-positive patients. Twenty-eight patients (57%) were male, and male patients were older than female patients (67 versus 49 years). Eight patients had a history of malignancy, but only 3 were diagnosed with malignancy within 2 years of MN diagnosis. We compared the results of ELISA, IIFT, Western blot, and biopsy staining, and found a significant correlation between ELISA and IIFT titers. Anti-THSD7A autoantibodies were predominantly IgG4 in all patients. Eight patients were double positive for THSD7A and PLA2R1. Levels of anti-THSD7A autoantibodies correlated with disease activity and with response to treatment. Patients with high titer at baseline had poor clinical outcome. In a subgroup of patients with serial titers, persistently elevated anti-THSD7A autoantibodies were observed in patients who did not respond to treatment or did not achieve remission. We conclude that the novel anti-THSD7A ELISA can be used to identify patients with THSD7A-associated MN and to monitor autoantibody titers during treatment.

Thrombospondin type-1 domain-containing 7A-associated membranous nephropathy after resection of rectal cancer: a case report

BackgroundThrombospondin type-1 domain-containing 7A (THSD7A) is a target antigen in idiopathic membranous nephropathy (MN). Patients with THSD7A-associated MN are known to have a high possibility of developing malignancy. However, there are few case reports on THSD7A-associated MN with malignancy, and details of its characteristics have not been clarified thoroughly. Here, we report the case of a 77-year-old male patient who was diagnosed with THSD7A-associated MN after resection of rectal cancer.Case presentationA 77-year-old man who had developed bilateral peripheral edema, persistent proteinuria, and nephrotic syndrome was admitted to our hospital. He was diagnosed with MN based on a renal biopsy 3 years after resection of rectal cancer, and positive staining for THSD7A in both kidney and rectal cancer tissues suggested that these two diseases were related. Furthermore, THSD7A staining of metastatic lymph nodes revealed deposition of THSD7A in the secondary lymph follicles and sinus. Recurrence of rectal cancer was suspected; however, tumor recurrence was not observed on chest and abdominal computed tomography (CT) and colonoscopy. There was no lymph node enlargement. The patient was kept on observation with supportive therapy. Consequently, although nephrotic syndrome persisted, obvious recurrence and metastasis of the primary tumor were not observed.ConclusionThis is the first case in which pathological examination results suggested that THSD7A-associated MN was caused by rectal cancer. Based on the reports of THSD7A-associated MN with malignancy and the pathogenesis of MN, lymph node metastasis may be a risk for cancer-related MN.

Antigen-Specific IgG Subclasses in Primary and Malignancy-Associated Membranous Nephropathy.

Membranous nephropathy (MN) is an autoimmune disease caused by binding of circulating antibodies to podocyte antigens in the kidney. For decades and still today primary MN has been considered to have an unspecified IgG4-driven autoimmune genesis, while secondary MN has been associated with other diseases, most notably cancer, and not linked to IgG4. Immunologic mechanisms of primary and malignancy-associated MN are assumed to be different, however, this has never been systematically evaluated. The identification of Phospholipase A2 Receptor 1 (PLA2R1) and Thrombospondin Type-1 Domain-Containing 7A (THSD7A) as target antigens in MN allows a pathogenesis-driven differential diagnosis. Recent data showing a molecular link between increased THSD7A-expression in tumors and THSD7A-antibody positive MN suggest a similar pathogenesis of malignancy-associated and primary MN. In order to better define the underlying immunologic processes, we systematically analyzed circulating antigen-specific IgG subclasses in the serum of 76 patients with PLA2R1-associated MN and 41 patients with THSD7A-associated MN in relationship to concurrent malignancy and disease outcome. Twenty-three patients in the study had malignancy-associated MN. We analyzed antigen-specific IgG subclasses in the serum of all patients at baseline and in 55 patients during follow-up by Western blot applying antigens derived from human kidney and lung. At baseline all 117 patients were positive for IgG4-antibodies against either PLA2R1 or THSD7A, while IgG3, IgG1, and IgG2-antibodies were found in 87, 72, and 26% of patients, respectively. There were no differences in the IgG subclass distribution between patients with primary vs. cancer-associated MN and no association with disease outcome. Moreover, levels of antigen-specific IgG4-antibodies were not different between primary and malignancy-associated MN and levels of all IgG subclasses did not differ between these groups. Both podocytes and lung bronchioles showed expression of both PLA2R1 and THSD7A when analyzed by immunofluorescence and Western blot. Every antigen-specific IgG subclass showed identical binding in both organs and autoantibodies bound the respective antigen only under non-reducing conditions. We conclude that antigen-specific IgG subclasses do not differentiate primary from malignancy-associated MN or predict disease prognosis. These data support the view that one common pathway may lead to primary and cancer-associated MN induced by PLA2R1- or THSD7A-antibodies.

The Most N-Terminal Region of THSD7A Is the Predominant Target for Autoimmunity in THSD7A-Associated Membranous Nephropathy.

Background Thrombospondin type 1 domain-containing 7A (THSD7A) has been identified as a pathogenic autoantigen in membranous nephropathy (MN). However, the THSD7A epitopes targeted by patient autoantibodies are unknown.Methods We performed an in silico analysis of the THSD7A multidomain structure, expressed the folded domains in HEK293 cells, and tested for domain reactivity with 31 serum samples from patients with THSD7A-associated MN using Western and native blotting. Immunogenicity of the antigen domains was further investigated by cDNA immunization of rabbits and mice.Results We characterized the extracellular topology of THSD7A as a tandem string of 21 thrombospondin type 1 domains. Overall, 28 serum samples (90%) recognized multiple epitope domains along the molecule. Detailed epitope mapping revealed that the complex consisting of the first and second N-terminal domains (amino acids 48-192) was recognized by 27 of 31 patient serum samples (87%). Serum recognizing one or two epitope domains showed lower anti-THSD7A antibody levels than serum recognizing three or more epitope domains. During follow-up, a loss of epitope recognition was observed in seven of 16 patients, and it was accompanied by decreasing antibody levels and remission of proteinuria. In four of 16 patients, epitope recognition patterns changed during follow-up. Notably, immunization experiments in rabbits and mice revealed that induced antibodies, like patient autoantibodies, preferentially bound to the most N-terminal domains of THSD7A.Conclusions Our data show that the immune response in THSD7A-associated MN is polyreactive and that autoantibodies predominantly target the most N-terminal part of THSD7A.

Tissue staining for THSD7A in glomeruli correlates with serum antibodies in primary membranous nephropathy: a clinicopathological study

Thrombospondin type-1 domain-containing 7A (THSD7A) is the most recently recognized target antigen in patients with membranous nephropathy. We stained membranous nephropathy biopsies processed in our laboratory for phospholipase A2 receptor and THSD7A over an 18-month period and selected all THSD7A-positive cases for study. Serum samples from most patients were tested by an indirect immunofluorescence assay for the presence of THSD7A antibodies (Euroimmun). A total of 31 patients were diagnosed with THSD7A-associated membranous nephropathy for a prevalence of 2.4% among patients with membranous nephropathy. The patients were most often male (male-to-female ratio of 1.6) with a mean age of 62 years and a mean proteinuria of 9.6 g per day (range 1.1–15.9). Two of the 31 patients had a history of cancer and none were diagnosed with malignancy on follow-up. Serum samples were available at the time of biopsy from 24 patients and all tested positive for antibodies against THSD7A. Conversely, all 20 serum samples from patients with membranous nephropathy who had negative staining for THSD7A were negative for serum reactivity to THSD7A. We conclude that THSD7A tissue staining of kidney biopsies with membranous nephropathy is a sensitive and specific method for the diagnosis of THSD7A-associated membranous nephropathy and it correlates strongly with the serum antibody testing. We also present the clinicopathologic details of the largest cohort to date of THSD7A-associated membranous nephropathy from a single institution.

THSD7A-associated membranous nephropathy in a patient with neurofibromatosis type 1

Target antigens in idiopathic membranous nephropathy (MN) include the phospholipase A2 receptor (PLA2R), and in some cases, the thrombospondin type 1 domain-containing 7A (THSD7A). A notable phenomenon is the high rate of cancer (reported to be as high as 20%) in patients with THSD7A-associated MN. Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by NF1 gene mutation, and clinically characterized by multiple cutaneous neurofibromas and café-au-lait spots. In this article, we report a patient with NF1 who developed THSD7A-associated MN when the NF1 skin lesions deteriorated. The patient, a 62-year-old male, was referred to us for nephrotic syndrome for 6 months. Physical examination revealed multiple cutaneous nodules throughout the entire body, and the patient noted recent increase in the numbers of these skin lesions. Cutaneous nodules excisional biopsy suggested NF1 and Sanger sequencing using genomic DNA extracted from peripheral blood revealed a previously reported heterozygous frameshift NF1 mutation (c.1541_1542delAG, p. Gln514fs). Renal biopsy revealed MN and immunohistochemistry (IHC) showed enhanced staining of THSD7A as well as PLA2R along the glomerular basement membrane whereas the serum level of THSD7A and PLA2R were both within normal range. The neurofibroma tissues were positive for THSD7A but not for PLA2R on IHC. The patient did not respond to 6-month treatment with glucocorticosteroid and cyclophosphamide. In this exceptional case, strong positive staining of THSD7A in both skin and renal biopsy samples, together with the temporal association between nephrotic syndrome and skin lesions and lack of treatment response, suggested the possibility that MN could be the result of immune response to THSD7A in NF1. This report may improve understanding of the mechanistic link between MN and cancer.

A Heterologous Model of Thrombospondin Type 1 Domain-Containing 7A-Associated Membranous Nephropathy.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target for autoimmunity in patients with membranous nephropathy (MN). Circulating autoantibodies from patients with THSD7A-associated MN have been demonstrated to cause MN in mice. However, THSD7A-associated MN is a rare disease, preventing the use of patient antibodies for larger experimental procedures. Therefore, we generated antibodies against the human and mouse orthologs of THSD7A in rabbits by coimmunization with the respective cDNAs. Injection of these anti-THSD7A antibodies into mice induced a severe nephrotic syndrome with proteinuria, weight gain, and hyperlipidemia. Immunofluorescence analyses revealed granular antigen-antibody complexes in a subepithelial location along the glomerular filtration barrier 14 days after antibody injection, and immunohistochemistry for rabbit IgG and THSD7A as well as ultrastructural analyses showed the typical characteristics of human MN. Mice injected with purified IgG from rabbit serum that was taken before immunization failed to develop any of these changes. Notably, MN developed in the absence of detectable complement activation, and disease was strain dependent. In vitro, anti-THSD7A antibodies caused cytoskeletal rearrangement and activation of focal adhesion signaling. Knockdown of the THSD7A ortholog, thsd7aa, in zebrafish larvae resulted in altered podocyte differentiation and impaired glomerular filtration barrier function, with development of pericardial edema, suggesting an important role of THSD7A in glomerular filtration barrier integrity. In summary, our study introduces a heterologous mouse model that allows further investigation of the molecular events that underlie MN.

PLA2R and THSD7A: Disparate Paths to the Same Disease?

The phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) are the two major autoantigens in primary membranous nephropathy (MN), and define two molecular subclasses of this disease. Both proteins are large transmembrane glycoproteins expressed by the podocyte, and both induce IgG4-predominant humoral immune responses that produce circulating autoantibodies that can be used clinically for diagnostic and monitoring purposes. The biologic roles of these proteins remain speculative, although several features of THSD7A suggest a role in adhesion. PLA2R-associated MN was initially found to associate with risk alleles within HLA-DQA1, but subsequent studies have shifted the focus to the HLA-DRB locus. Three distinct humoral epitope-containing regions have been defined within the extracellular portion of PLA2R, and it appears that the number of targeted epitopes may determine disease severity. Although similar information is not yet available for THSD7A-associated MN, this form of MN may have a unique association with malignancy. Finally, it appears likely that other autoantigens in primary MN exist. Although protocols similar to those that identified PLA2R and THSD7A may be successful in the identification of novel antigenic targets in MN, newer techniques such as laser-capture mass spectrometry or protein arrays may be helpful as well.

An Indirect Immunofluorescence Method Facilitates Detection of Thrombospondin Type 1 Domain-Containing 7A-Specific Antibodies in Membranous Nephropathy.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target antigen identified in adult membranous nephropathy (MN) along with the major antigen phospholipase A2 receptor 1 (PLA2R1). The prevalence of THSD7A-Ab-positive patients is unknown, and it is unclear whether the clinical presentation differs between patients positive for PLA2R1-Ab or THSD7A-Ab. We screened serum samples of 1276 patients with MN from three different cohorts for the presence of THSD7A-Ab by Western blot analysis and a newly developed indirect immunofluorescence test (IFT). Compared with Western blot analysis, the IFT had a 92% sensitivity and a 100% specificity. The prevalence of THSD7A-associated MN in a prospective cohort of 345 patients with MN was 2.6%, and most were women. In this cohort, the percentage of patients with THSD7A-associated MN and malignant disease significantly exceeded that of patients with PLA2R1-associated MN and malignant disease. In all cohorts, we identified 40 patients with THSD7A-associated MN, eight of whom developed a malignancy within a median time of 3 months from diagnosis of MN. In one patient with THSD7A-associated MN and metastases of an endometrial carcinoma, immunohistochemistry showed THSD7A expression on the metastatic cells and within follicular dendritic cells of the metastasis-infiltrated lymph node. We conclude that the IFT allows sensitive and specific measurement of circulating THSD7A-Ab in patients with MN. Patients with THSD7A-associated MN differ in their clinical characteristics from patients with PLA2R1-associated MN, and more intensive screening for the presence of malignancies may be warranted in those with THSD7A-associated MN.