Idiopathic membranous nephropathy is an autoimmune disease. In approximately 70% of patients, it is associated with autoantibodies against the phospholipase A2 receptor 1 (PLA2R1). Antigenic targets ...in the remaining patients are unknown.
Using Western blotting, we screened serum samples from patients with idiopathic membranous nephropathy, patients with other glomerular diseases, and healthy controls for antibodies against human native glomerular proteins. We partially purified a putative new antigen, identified this protein by means of mass spectrometry of digested peptides, and validated the results by analysis of recombinant protein expression, immunoprecipitation, and immunohistochemical analysis.
Serum samples from 6 of 44 patients in a European cohort and 9 of 110 patients in a Boston cohort with anti-PLA2R1-negative idiopathic membranous nephropathy recognized a glomerular protein that was 250 kD in size. None of the serum samples from the 74 patients with idiopathic membranous nephropathy who were seropositive for anti-PLA2R1 antibodies, from the 76 patients with other glomerular diseases, and from the 44 healthy controls reacted against this antigen. Although this newly identified antigen is clearly different from PLA2R1, it shares some biochemical features, such as N-glycosylation, membranous location, and reactivity with serum only under nonreducing conditions. Mass spectrometry identified this antigen as thrombospondin type-1 domain-containing 7A (THSD7A). All reactive serum samples recognized recombinant THSD7A and immunoprecipitated THSD7A from glomerular lysates. Moreover, immunohistochemical analyses of biopsy samples from patients revealed localization of THSD7A to podocytes, and IgG eluted from one of these samples was specific for THSD7A.
In our cohort, 15 of 154 patients with idiopathic membranous nephropathy had circulating autoantibodies to THSD7A but not to PLA2R1, a finding that suggests a distinct subgroup of patients with this condition. (Funded by the French National Center for Scientific Research and others.).
Perspectives in membranous nephropathy Tomas, Nicola M.; Huber, Tobias B.; Hoxha, Elion
Cell and tissue research,
08/2021, Letnik:
385, Številka:
2
Journal Article
Recenzirano
Odprti dostop
The identification of the phospholipase A2 receptor 1 (PLA2R) and thrombospondin type-1 domain-containing protein 7A (THSD7A) as podocyte antigens in adult patients with membranous nephropathy (MN) ...has strongly impacted both experimental and clinical research on this disease. Evidence has been furnished that podocyte-directed autoantibodies can cause MN, and novel PLA2R- and THSD7A-specific animal models have been developed. Today, measurement of serum autoantibody levels and staining of kidney biopsies for the target antigens guides MN diagnosis and treatment worldwide. Additionally, anti-PLA2R antibodies have been proven to be valuable prognostic biomarkers in MN. Despite these impressive advances, a variety of questions regarding the disease pathomechanisms, clinical use of antibody measurement, and future treatments remain unanswered. In this review, we will outline recent advances made in the field of MN and discuss open questions and perspectives with a focus on novel antigen identification, mechanisms of podocyte injury, clinical use of antibody measurement to guide diagnosis and treatment, and the potential of innovative, pathogenesis-based treatment strategies.
Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, and one-third of patients develop end-stage renal disease (ESRD). Circulating autoantibodies against the podocyte ...surface antigens phospholipase A2 receptor 1 (PLA2R1) and the recently identified thrombospondin type 1 domain-containing 7A (THSD7A) are assumed to cause the disease in the majority of patients. The pathogenicity of these antibodies, however, has not been directly proven. Here, we have reported the analysis and characterization of a male patient with THSD7A-associated MN who progressed to ESRD and subsequently underwent renal transplantation. MN rapidly recurred after transplantation. Enhanced staining for THSD7A was observed in the kidney allograft, and detectable anti-THSD7A antibodies were present in the serum before and after transplantation, suggesting that these antibodies induced a recurrence of MN in the renal transplant. In contrast to PLA2R1, THSD7A was expressed on both human and murine podocytes, enabling the evaluation of whether anti-THSD7A antibodies cause MN in mice. We demonstrated that human anti-THSD7A antibodies specifically bind to murine THSD7A on podocyte foot processes, induce proteinuria, and initiate a histopathological pattern that is typical of MN. Furthermore, anti-THSD7A antibodies induced marked cytoskeletal rearrangement in primary murine glomerular epithelial cells as well as in human embryonic kidney 293 cells. Our findings support a causative role of anti-THSD7A antibodies in the development of MN.
Thrombospondin type 1 domain-containing 7A (THSD7A) is a target antigen identified in adult membranous nephropathy (MN) along with the major antigen phospholipase A
receptor 1 (PLA
R1). The ...prevalence of THSD7A-Ab-positive patients is unknown, and it is unclear whether the clinical presentation differs between patients positive for PLA
R1-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 PLA
R1-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 PLA
R1-associated MN, and more intensive screening for the presence of malignancies may be warranted in those with THSD7A-associated MN.
Abstract
Membranous nephropathy (MN) is an autoimmune disease caused by autoantibodies against the podocyte antigens phospholipase A
2
receptor 1 (PLA
2
R1) 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.
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.
, 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.
Patients with primary membranous nephropathy (MN) who experience spontaneous remission of proteinuria generally have an excellent outcome without need of immunosuppressive therapy. It is, however, ...unclear whether non-nephrotic proteinuria at the time of diagnosis is also associated with good prognosis since a reasonable number of these patients develop nephrotic syndrome despite blockade of the renin-angiotensin system. No clinical or laboratory parameters are available, which allow the assessment of risk for development of nephrotic proteinuria. Phospholipase A2 Receptor antibodies (PLA2R-Ab) play a prominent role in the pathogenesis of primary MN and are associated with persistence of nephrotic proteinuria. In this study we analysed whether PLA2R-Ab levels might predict development of nephrotic syndrome and the clinical outcome in 33 patients with biopsy-proven primary MN and non-nephrotic proteinuria under treatment with blockers of the renin-angiotensin system. PLA2R-Ab levels, proteinuria and serum creatinine were measured every three months. Nephrotic-range proteinuria developed in 18 (55%) patients. At study start (1.2±1.5 months after renal biopsy and time of diagnosis), 16 (48%) patients were positive for PLA2R-Ab. A multivariate analysis showed that PLA2R-Ab levels were associated with an increased risk for development of nephrotic proteinuria (HR = 3.66; 95%CI: 1.39-9.64; p = 0.009). Immunosuppressive therapy was initiated more frequently in PLA2R-Ab positive patients (13 of 16 patients, 81%) compared to PLA2R-Ab negative patients (2 of 17 patients, 12%). PLA2R-Ab levels are associated with higher risk for development of nephrotic-range proteinuria in this cohort of non-nephrotic patients at the time of diagnosis and should be closely monitored in the clinical management.
Membranous nephropathy (MN) is an autoimmune disease induced by circulating antibodies against the podocyte protein phospholipase A2 receptor 1 (PLA2R1-ab) in 80% of patients and represents the ...leading cause of nephrotic syndrome in adults. PLA2R1-ab levels correlate with disease activity and treatment response. However, their predictive role for long-term renal outcome is not clear.
The aim of this prospective observational multicenter study was to investigate the predictive role of PLA2R1-ab levels at the time of diagnosis for long-term outcome in a cohort of 243 patients with newly diagnosed biopsy-proven PLA2R1-associated MN. Statistical analyses included Cox proportional hazard models. The primary study endpoint was defined prior to data collection as doubling of serum creatinine or development of end-stage renal disease.
During the median follow-up time of 48 months, 36 (15%) patients reached the study endpoint. Independent predictors for reaching the study endpoint were baseline PLA2R1-ab levels (HR = 1.36, 95%CI 1.11-1.66, p = 0.01), percentage of tubular atrophy and interstitial fibrosis (HR = 1.32, 95%CI 1.03-1.68, p = 0.03), PLA2R1-ab relapse during follow-up (HR = 3.22, 95%CI 1.36-7.60, p = 0.01), and relapse of proteinuria (HR = 2.60, 95%CI 1.17-5.79, p = 0.02). Fifty-four (22%) patients received no immunosuppressive treatment during the study, in 41 (76%) of them PLA2R1-ab spontaneously disappeared during follow-up, 29 (54%) patients had a complete remission of proteinuria, and 19 (35%) had a partial remission. Patients not treated with immunosuppression were more often females and had lower PLA2R1-ab levels, proteinuria, and serum creatinine at baseline compared to patients receiving immunosuppression. However, no conclusion on the efficacy of immunosuppressive therapies can be made, since this was not a randomized controlled study and treatment decisions were not made per-protocol.
PLA2R1-ab levels are, in addition to pre-existing renal damage, predictive factors for long-term outcome and should therefore be considered when deciding the treatment of patients with MN.
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