BACKGROUND

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.).

The phospholipase A2 receptor (PLA2R) and <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>) are the two major autoantigens in primary membranous nephropathy (MN), and define two molecular subclasses of this disease. Both <em>proteins</em> are large transmembrane glyco<em>proteins</em> 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 <em>proteins</em> remain speculative, although several features of THSD<em>7A</em> suggest a role in adhesion. PLA2R-associated MN was initially found to associate with risk alleles within HLA-DQA<em>1</em>, but subsequent studies have shifted the focus to the HLA-DRB locus. Three distinct humoral epitope-<em>containing</em> 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 THSD<em>7A</em>-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 THSD<em>7A</em> may be successful in the identification of novel antigenic targets in MN, newer techniques such as laser-capture mass spectrometry or <em>protein</em> arrays may be helpful as well.

In patients with membranous nephropathy, alkylating agents (cyclophosphamide or chlorambucil) alone or in combination with steroids achieve remission of nephrotic syndrome more effectively than conservative treatment or steroids alone, but can cause myelotoxicity, infections, and cancer. Calcineurin inhibitors can improve <em>protein</em>uria, but are nephrotoxic. Most patients relapse after treatment withdrawal and can become treatment dependent, which increases the risk of nephrotoxicity. The discovery of nephritogenic autoantibodies against podocyte M-<em>type</em> phospholipase A2 receptor (PLA2R) and <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>- <em>containing</em> <em>protein</em> <em>7A</em> (THSD<em>7A</em>) antigens provides a clear pathophysiological rationale for interventions that specifically target B-cell lineages to prevent antibody production and subepithelial deposition. The anti-CD20 monoclonal antibody rituximab is safe and achieves remission of <em>protein</em>uria in approximately two-thirds of patients with membranous nephropathy. In those with PLA2R-related disease, remission can be predicted by anti-PLA2R antibody depletion and relapse by antibody re-emergence into the circulation. Thus, integrated evaluation of serology and <em>protein</em>uria could guide identification of affected patients and treatment with individually tailored protocols. Nonspecific and toxic immunosuppressive regimens will fall out of use. B-cell modulation by rituximab and second-generation anti-CD20 antibodies (or plasma cell-targeted therapy in anti-CD20 resistant forms of disease) will lead to a novel therapeutic paradigm for patients with membranous nephropathy.

UNASSIGNED

The M-<em>type</em> phospholipase A2 receptor (PLA2R) and <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>) were identified as intrinsic antigens in primary membranous nephropathy (MN). Complement activation via the lectin pathway in intrinsic antigen-related MN is still unclear.

UNASSIGNED

We retrospectively enrolled 60 primary Japanese MN patients and detected activated complement pathways by staining complement proteins in glomerular deposition. According to the findings of PLA2R and THSD<em>7A</em> staining in glomeruli, they were classified into intrinsic antigen-related or -unrelated MN. We evaluated clinicopathological characteristics and predictors of clinical outcomes in intrinsic antigen-related MN.

UNASSIGNED

Thirty-nine (65%) patients had PLA2R in glomerular deposits and two (3.3%) patients had THSD<em>7A</em>. One of them had both PLA2R and THSD<em>7A</em> (double positive). Forty patients were classified into the intrinsic antigen-related group. The other 20 patients were negative for both antigens (unrelated group). The prevalence and staining intensity of mannose-binding lectin (MBL) deposits were much higher in the intrinsic antigen-related group [55% versus 20%, P < 0.0<em>1</em>0, <em>1</em>.0 (interquartile range <em>1</em>.0-2.0) versus <em>1</em>.0 (0.0-<em>1</em>.0), P = 0.0<em>1</em>, respectively]. The staining intensity of MBL in glomeruli also correlated with the IgG4 staining intensity. In intrinsic antigen-related MN, MBL staining intensity was an unfavorable predictor for remission of proteinuria [hazard ratio (HR) 0.40, P < 0.0<em>1</em>] and renal dysfunction (HR 3.8<em>1</em>, P = 0.0<em>1</em>) in Cox proportional hazards analysis. Moreover, the glomerular MBL-positive group showed more severe interstitial fibrosis and worse clinical outcomes.

UNASSIGNED

Intrinsic antigen-related MN was more strongly associated with complement activation by the lectin pathway, which may contribute to a less favorable clinical outcome.

Membranous Nephropathy (MN) is an autoimmune disease associated with antibodies against podocyte <em>proteins</em>: M-<em>type</em> phospholipase A2 receptor (PLA2R<em>1</em>) or <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>) in 70 and 3% of patients, respectively. Antibody titer is correlated with disease activity: rising during active disease and decreasing before remission. Therefore, decreasing PLA2R<em>1</em>-Antibodies titer has become an important goal of therapy. Rituximab a chimeric monoclonal antibody induces remission in 60-80% of primary MN patients. All monoclonal antibodies such as rituximab can elicit antidrug antibodies, which may interfere with therapeutic response. We aim to analyze the relevance of anti-rituximab antibodies on the outcome of MN after a first course of rituximab. Forty-four MN patients were included and treated with two <em>1</em> g infusions of rituximab at 2-weeks interval. Anti-rituximab antibodies, CD<em>1</em>9 count, and clinical response were analyzed. Then, we (i) analyzed the association of anti-rituximab antibodies at month-6 with response to treatment: remission, relapse and the need for another rituximab course; (ii) confirmed if anti-rituximab antibodies could neutralize rituximab B-cells depletion; and (iii) tested whether anti-rituximab antibodies could cross-inhibit new humanized anti-CD20 therapies. Anti-rituximab antibodies were detected in <em>1</em>0 patients (23%). Seventeen patients received a second rituximab course after a median time of <em>1</em>2 months (7-<em>1</em>2), following nine cases of resistance and eight relapses. Anti-rituximab antibodies were significantly associated with faster B-cell reconstitution at month-6 (75 [57-89] vs. 2 [0-4<em>1</em>] cells/μl, <i>p</i> = 0.006), higher proteinuria <em>1</em>2 months after rituximab infusion (<em>1</em>.7 [0.7; 5.8] vs. 0.6 [0.2; 3.4], <i>p</i> = 0.03) and before treatment modification (3.5 [<em>1</em>.6; 7.<em>1</em>] vs. <em>1</em>.7 [0.2; <em>1</em>.7] <i>p</i> = 0.0004). Remission rate 6 months after rituximab was not different according to anti-rituximab status (<i>p</i> > 0.99) but the rate of relapse was significantly higher for patients with anti-rituximab antibodies (<i>p</i> < 0.00<em>1</em>). These patients required more frequently a second course of rituximab infusions (7/<em>1</em>0 vs. <em>1</em>0/34, <i>p</i> = 0.03). Anti-rituximab antibodies neutralized rituximab activity in 8/<em>1</em>0 patients and cross-reacted with other humanized monoclonal antibodies in only two patients. Three patients with anti-rituximab antibodies were successfully treated with ofatumumab. Anti-rituximab antibodies could neutralize rituximab B cells cytotoxicity and impact clinical outcome of MN patients. Humanized anti-CD20 seems to be a satisfying therapeutic alternative for patients with anti-rituximab antibodies and resistant or relapsing MN.

Anti-M-<em>type</em> phospholipase A2 receptor (anti-PLA2R) is a widely accepted biomarker for clinical idiopathic membranous neurophathy (IMN). However, its ability to differentiate between IMN and secondary MN (SMN) is controversial. The objective of this study was to assess clinical MN biomarkers in blood, tissue and urine samples from Chinese patients. In total, <em>1</em>95 MN patients and 70 patients with other glomerular diseases were prospectively enrolled in the study. Participants were followed up for average of <em>1</em>7 months (range 3-39 months). Anti-PLA2R and anti-THSD<em>7A</em> (<em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em>) were detected only in MN patient sera and not in controls. Serum anti-THSD<em>7A</em> and THSD<em>7A</em>-positive biopsies were detected in <em>1</em>/<em>1</em>8 and 2/<em>1</em>8 PLA2R-negative MN cases, respectively. PLA2R and THSD<em>7A</em> were detected in 72.27% and 40% of SMN cases, respectively. While serum positivity for both anti-PLA2R and anti-THSD<em>7A</em> at the time of renal biopsy was specific to MN patients, neither antigen could discriminate between primary and secondary MN. We also found that high urinary levels of retinol binding <em>protein</em> (RBP) predicted poor <em>protein</em>uria outcomes in study participants. Patients with low or medium urinary RBP levels achieved remission more frequently than those with high RBP.

<AbstractText>About 3%-5% of adults with membranous nephropathy have autoantibodies directed against <em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>), a podocyte-expressed transmembrane <em>protein</em>. However, the temporal and spatial expression of THSD<em>7A</em> and its biologic function for podocytes are unknown, information that is needed to understand the effects of THSD<em>7A</em> autoantibodies in this disease.</AbstractText><AbstractText>Using a variety of microscopic techniques, we analyzed THSD<em>7A</em> localization in postnatal, adult, and autoantibody-injected mice as well as in human podocytes. We also analyzed THSD<em>7A</em> function in human podocytes using confocal microscopy; Western blotting; and adhesion and migration assays.</AbstractText><AbstractText>We found that THSD<em>7A</em> expression begins on glomerular vascularization with slit diaphragm formation in development. THSD<em>7A</em> localizes to the basal aspect of foot processes, closely following the meanders of the slit diaphragm in human and mice. Autoantibodies binding to THSD<em>7A</em> localize to the slit diaphragm. In human podocytes, THSD<em>7A</em> expression is accentuated at filopodia and thin arborized protrusions, an expression pattern associated with decreased membrane activity of cytoskeletal regulators. We also found that, phenotypically, THSD<em>7A</em> expression in human podocytes is associated not only with increases in cell size, enhanced adhesion, and reduced detachment from collagen <em>type</em> IV-coated plates but also, with decreased ability to migrate.</AbstractText><AbstractText>Our findings suggest that THSD<em>7A</em> functions as a foot process <em>protein</em> involved in the stabilization of the slit diaphragm of mature podocytes and that autoantibodies to THSD<em>7A</em>, on the basis of their localization, might structurally and functionally alter the slit diaphragm's permeability to <em>protein</em>.</AbstractText>

Idiopathic membranous nephropathy (IMN) is a pathological pattern of glomerular damage caused by an autoimmune response. Immune complex deposition, thickness of glomerular basement membrane, and changes in the podocyte morphology are responsible for the development of <em>protein</em>uria, which is caused by the targeted binding of auto-antibodies to podocytes. Several auto-antigens have recently been identified in IMN, including M-<em>type</em> receptor for secretory phospholipase A2 (PLA2R<em>1</em>), <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>), and neural epidermal growth factor-like <em>1</em> <em>protein</em> (NELL-<em>1</em>). The measurement of peripheral circulating antibodies has become an important clinical reference index. However, some clinical features of IMN remain elusive and need to be further investigated, such as the autoimmunity initiation, IgG4 predominance, spontaneous remission, and the unique glomerular lesion. As these unresolved issues are closely related to clinical practice, we have proposed a hypothetical pathogenesis model of IMN. Induced by environmental stimuli or other causes, the PLA2R<em>1</em> antigen and/or THSD<em>7A</em> antigen exposed to extrarenal tissues, such as lungs, then produce the auto-antibodies that target and cause damage to the podocytes in circulation. In this review, we highlighted the potential association between environmental stimuli, immune activity, and glomerular lesions, the underlying basis for spontaneous immune and <em>protein</em>uria remission.

Keywords: autoimmune response; environmental stimuli; inflammation; pathogenesis model; podocyte; spontaneous remission.

OBJECTIVE

The genetic contribution to coronary artery disease (CAD) remains largely unclear. We combined genetic screening with functional characterizations to identify novel loci and candidate genes for CAD.

UNASSIGNED

We performed genome-wide screening followed by multicenter validation in 8 cohorts consisting of 2<em>1</em> 828 participants of Han ethnicity and identified 3 novel intragenic SNPs (single nucleotide polymorphisms), rs9486729 (SCML4 [Scm polycomb group <em>protein</em>-like 4]; odds ratio, <em>1</em>.25; 95% CI, <em>1</em>.<em>1</em>7-<em>1</em>.34; P=3.5<em>1</em>×<em>1</em>0-<em>1</em><em>1</em>), rs<em>1</em>7<em>1</em>65<em>1</em>36 (THSD<em>7A</em> [<em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em>]; odds ratio <em>1</em>.28; 95% CI, <em>1</em>.2<em>1</em>-<em>1</em>.35; P(<em>1</em>.00×<em>1</em>0-25), and rs852787 (DAB<em>1</em> [disabled-<em>1</em>]; odds ratio, <em>1</em>.29; 95% CI, <em>1</em>.2<em>1</em>-<em>1</em>.38; P=2.02×<em>1</em>0-<em>1</em>4), associated with CAD with genome-wide significance. The risk allele of rs9486729 and protective allele of rs<em>1</em>7<em>1</em>65<em>1</em>36 were associated with the decreased expression of their host genes, SCML4 and THSD<em>7A</em>, respectively, whereas rs852787 did not have transcriptional effects on any gene. Knockdown of SCML4 activated endothelial cells by increasing the expression of IL-6, E-selectin, and ICAM and weakened their antiapoptotic activity, whereas the knockdown of THSD<em>7A</em> had little effect on these endothelial cell functions but attenuated monocyte adhesion via decreasing the expression of ICAM, L-selectin, and ITGB2. We further showed that inhibiting the expression of SCML4 exacerbated endothelial dysfunction and vascular remodeling in a rat model with partial carotid ligation.

CONCLUSIONS

We identify 3 novel loci associated with CAD and show that 2 genes, SCML4 and THSD<em>7A</em>, make functional contributions to atherosclerosis. How rs852787 and its host gene DAB<em>1</em> are linked to CAD needs further studies.

Membranous nephropathy (MN) associated with malignancies is a well-known entity. However, its association with benign neoplasm is not broadly recognized. A 69-year-old man with recurrent nephrotic syndrome presented with pedal edema and <em>protein</em>uria of 5 months' duration. Laboratory results showed hypoalbuminemia and hyperlipidemia. Proteinuria was estimated to be <em>protein</em> excretion of 3.5g/d. Studies were negative for viral hepatitis, syphilis, human immunodeficiency virus, autoimmune diseases, and para<em>protein</em>emia. Kidney biopsy disclosed MN with negative phospholipase A2 receptor (PLA2R) staining, favoring a secondary form of MN. Computed tomography detected a 7.6-cm duodenal schwannoma. Elective surgical resection was performed. Pathologic study showed that THSD<em>7A</em> (<em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em>) was positive in both glomeruli and schwannoma. Commonly, secondary MN is related to underlying conditions, including lupus, hepatitis, and neoplasm, and can be medication induced. The risk for developing a concomitant neoplasm among patients with PLA2R-negative MN is up to <em>1</em>2 times higher than in the general population. Most of these neoplasms are malignancies, and the presence of autoantibodies directed at similar tissue targets is hypothesized as the potential mechanism. In our case, THSD<em>7A</em> may be the autoantibody that has linked the schwannoma and the development of MN. Although benign tumors rarely produce renal manifestations, effective treatment may lead to resolution of nephrotic syndrome.

<b>Background:</b> Several case reports have described the concurrence of chronic inflammatory demyelinating polyneuropathy (CIDP) and membranous nephropathy (MN). The presence of autoantibodies against podocyte antigens phospholipase A2 receptor (PLA2R) and <em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em> <em>containing</em> <em>7A</em> (THSD<em>7A</em>) in MN suggests an autoimmune mechanism. Some CIDP patients also harbor autoantibodies against paranodal <em>proteins</em> such as neurofascin <em>1</em>55 (NF<em>1</em>55) and contactin-<em>1</em> (CNTN<em>1</em>). We investigated the relationship between CIDP and MN by assaying autoantibodies against paranodal and podocyte antigens in a CIDP patient with MN, and by a literature survey on the clinical features of CIDP with MN. <b>Methods:</b> Anti-CNTN<em>1</em> and NF<em>1</em>55 antibodies were measured by flow cytometry using HEK293 cell lines stably expressing human CNTN<em>1</em> or NF<em>1</em>55. Binding capacity of antibodies was validated by immunostaining mouse teased sciatic nerve fibers. Anti-PLA2R antibodies were measured by enzyme-linked sorbent assay and anti-THSD<em>7A</em> antibodies by indirect immunofluorescence assay. Clinical features between <em>1</em>4 CIDP with MN cases including two with anti-CNTN<em>1</em> antibodies and 20 anti-CNTN<em>1</em> antibody-positive CIDP cases were compared. <b>Results:</b> A patient whose ages was in the late 70 s complained of progressive weakness and superficial and deep sensory impairment in four extremities over 6 months. Nerve conduction studies showed prominent demyelination patterns. The patient presented with nephrotic syndrome. Renal biopsy disclosed basement membrane thickening with local subepithelial projections and glomerular deposits of IgG4, compatible with MN. Autoantibody assays revealed the presence of IgG4 and IgG<em>1</em> anti-CNTN<em>1</em> antibodies, but an absence of anti-NF<em>1</em>55, anti-PLA2R, and anti-THSD<em>7A</em> antibodies. The patient's serum stained paranodes of teased sciatic nerves. CIDP with MN and anti-CNTN<em>1</em> antibody-positive CIDP commonly showed male preponderance, relatively higher age of onset, acute to subacute onset in 35-50% of cases, distal dominant sensorimotor neuropathy, proprioceptive impairment leading to sensory ataxia, and very high cerebrospinal fluid <em>protein</em> levels. However, <em>1</em><em>1</em> of <em>1</em>3 CIDP patients with MN had a favorable response to mono- or combined immunotherapies whereas anti-CNTN<em>1</em> antibody-positive CIDP was frequently refractory to corticosteroids and intravenous immunoglobulin administration. <b>Conclusion:</b> CIDP with MN and anti-CNTN<em>1</em> antibody-positive CIDP show considerable overlap but are not identical. CIDP with MN is probably heterogeneous and some cases harbor anti-CNTN<em>1</em> antibodies.

The <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em> <em>containing</em> <em>7A</em> (THSD<em>7A</em>) <em>protein</em> is known to be one of the antigens responsible for the autoimmune disorder idiopathic membranous nephropathy. The structure of this antigen is currently unsolved experimentally. Here we present a homology model of the extracellular portion of the THSD<em>7A</em> antigen. The structure was evaluated for folding patterns, epitope site prediction, and function was predicted. Results show that this <em>protein</em> contains 2<em>1</em> extracellular <em>domains</em> and with the exception of the first two <em>domains</em>, has a regular repeating pattern of TSP-<em>1</em>-like followed by F-spondin-like <em>domains</em>. Our results indicate the presence of a novel Trp-ladder sequence of WxxxxW in the TSP-<em>1</em>-like <em>domains</em>. Of the 2<em>1</em> <em>domains</em>, <em>1</em>8 were shown to have epitope binding sites as predicted by epitopia. Several of the F-spondin-like <em>domains</em> have insertions in the canonical TSP fold, most notably the coiled coil region in <em>domain</em> 4, which may be utilized in <em>protein</em>-<em>protein</em> binding interactions, suggesting that this <em>protein</em> functions as a heparan sulfate binding site.

Thsd<em>7a</em> (<em>Thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em> <em>containing</em> <em>7a</em>) is a critical transmembrane <em>protein</em>. Studies have indicated that Thsd<em>7a</em> was associated with cytoskeletal organization, cell migration and filopodia formation. However, the involvement of Thsd<em>7a</em> remains elusive in human Esophageal Squamous Cell Carcinoma (ESCC). Consequently, immunohistochemistry and reverse transcription-polymerase chain reaction were utilized to study the correlation between the expression of Thsd<em>7a</em> and clinical-pathological characteristics. The influence of Thsd<em>7a</em> on apoptosis, cell proliferating activity, cell cycle, migratory and invasive capacity was determined in Eca <em>1</em>09 and EC 9706 cell lines in vitro. And the influence on proliferating activity was testified using naked mice model in vivo. In addition, the potential molecular mechanism was tested by microarray. It was discovered that there is a certain correlation between Thsd<em>7a</em> and the Kazakh ESCC. By knocking out Thsd<em>7a</em>, the invasion, migration and proliferation could be decreased. And it could also arrest the cell cycle at G<em>1</em> phase and increase the apoptosis rate. It was further verified that Thsd<em>7a</em> had obvious effect on proliferation in naked mice with xenograft of Eca<em>1</em>09 cells. Finally, it was uncovered by microarray analysis that a variety of tumor genes and pathways related to Thsd<em>7a</em>. Together, it was demonstrated that Thsd<em>7a</em> might have a certain degree of carcinogenesis in ESCC.

<strong class="sub-title"> Rationale & objective: </strong> Membranous nephropathy (MN) is characterized by deposition of immune complexes along glomerular basement membranes. M-<em>type</em> phospholipase A₂ receptor (PLA₂R), <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>), exostosin (EXT), and neural epidermal growth factor-like <em>1</em> <em>protein</em> (NELL-<em>1</em>) have been identified as established or potential podocyte antigens in MN. We investigated the association of podocyte antigen staining with MN clinical pheno<em>type</em> and outcomes.

Study design: Multicenter retrospective cohort study.

<strong class="sub-title"> Setting & participants: </strong> <em>1</em>77 consecutive patients with MN unrelated to lupus erythematosus, identified from a cohort of 3875 native kidney biopsies performed in the Belgian UCLouvain Kidney Disease Network from 2000 through 20<em>1</em>8.

Predictor: Positive immunostaining for podocyte antigens on archived kidney biopsy samples.

Outcomes: Association with different phenotypes (baseline characteristics of patients and pathologic findings on kidney biopsy), time to cancer and to kidney failure.

Analytical approach: Kaplan-Meier estimates and Cox regression analyses to assess time to cancer and kidney failure.

<strong class="sub-title"> Results: </strong> <em>1</em>77 patients were followed for a median of 4.0 years (IQR, <em>1</em>.3-8.0). Diagnosis of PLA2R⁺, THSD<em>7A</em>⁺, and double-negative MN was made in <em>1</em><em>1</em>7 (66.<em>1</em>%), 6 (3.4%), and 54 (30.5%) patients, respectively. Progression to kidney failure was similar in all groups. Although the number of patients with THSD<em>7A</em>⁺ MN was small, they showed a higher incidence (50%) and an increased risk of developing cancer during follow-up (adjusted HR 5.0, 95% CI <em>1</em>.4-<em>1</em>7.9, P=0.0<em>1</em>). Eight percent and 5% of the patients with PLA2R^-/THSD<em>7A</em>^- MN stained positively for EXT and NELL-<em>1</em>, respectively. Most patients with EXT⁺ MN were women, had features of systemic autoimmunity, and showed glomerular C<em>1</em>q deposits.

<strong class="sub-title"> Limitations: </strong> Retrospective design; small number of patients in the THSD<em>7A</em> group; lack of evaluation of IgG subclasses deposition.

Conclusions: Our real-world data describe the relative prevalence of subgroups of MN, and support the hypothesis that a novel classification of MN based on podocyte antigen staining might be clinically relevant.

<strong class="sub-title"> Keywords: </strong> glomerular disease; malignancy; membranous nephropathy; nephrotic syndrome; outcome; phospholipase A2 receptor; <em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em>.

<strong class="sub-title"> Objective: </strong> To describe the clinical and pathological pheno<em>type</em> of membranous nephropathy (MN) associated with M-<em>type</em>-phospholipase-A₂-receptor (PLA₂R), <em>thrombospondin</em>-<em>type</em>-<em>1</em>-<em>domain</em>-<em>containing</em>-<em>7A</em> (THSD<em>7A</em>), semaphorin 3B (SEMA3B), neural-epidermal-growth-factor-like-<em>1</em>-<em>protein</em> (NELL-<em>1</em>), protocadherin 7 (PCDH7), exostosin <em>1</em>/exostosin 2 (EXT<em>1</em>/EXT2) and neural cell adhesion molecule <em>1</em> (NCAM-<em>1</em>) as target antigens.

<strong class="sub-title"> Methods: </strong> A retrospective cohort of 270 adult patients with biopsy-proven MN diagnosed between January 20<em>1</em>5 and April 2020 was classified as PLA₂R-, THSD<em>7A</em>-, SEMA3B-, NELL-<em>1</em>-, PCDH7-, EXT<em>1</em>/EXT2-, NCAM-<em>1</em>-associated or septuple-negative MN using serologic tests, immunostaining, and/or mass spectrometry. Clinical, biochemical, pathologic, and follow-up data were systematically abstracted from the medical records, including disease activity of conditions traditionally associated with MN and occurring within 5 years of MN diagnosis.

<strong class="sub-title"> Results: </strong> Patients with PLA₂R-associated MN were predominantly middle-aged white men without associated disease. The presence of associated disease did not affect the clinical and pathologic characteristics of PLA₂R-associated MN, suggesting that they were coincidental rather than causally linked. THSD<em>7A</em>-, NELL-<em>1</em>-, PCDH7-, and NCAM-<em>1</em>-associated MN were rare and SEMA3B-associated MN was not discovered in our cohort. EXT<em>1</em>/EXT2-associated MN was primarily diagnosed in younger women with active systemic autoimmunity. A significant proportion of septuple-negative patients had associated malignancy or systemic autoimmunity.

Conclusion: The widely used distinction between primary and secondary MN has limitations. We propose a refined terminology that combines the target antigen and associated disease to better classify MN and guide clinical decision making.

Membranous nephropathy (MN), an autoimmune glomerulonephritis which can occur in primary and secondary forms, is one of the most common inflammatory glomerulopathies in elderly patients. The pathophysiology of the primary form is generally due to circulating immunoglobulin (IgG4) antibodies which often target phospholipase A2 receptors (anti-PLA2R) and <em>Thrombospondin</em> <em>Type</em> <em>1</em> <em>Domain</em> <em>containing</em> <em>7A</em> (anti THSD<em>7A</em>). IgA nephropathy is one of the most common autoimmune glomerular diseases in the world and presents with a spectrum of disease ranging from asymptomatic mild hematuria and <em>protein</em>uria to rapidly progressive crescentic glomerulonephritis. We present a rare case of concomitant IgA and primary MN in a single patient treated successfully with renin-angiotensin-aldosterone blockade, corticosteroids, and calcineurin inhibitors. The peak <em>protein</em>uria was near 7.5-8 g <em>protein</em>/g creatinine by various measures. Serum creatinine remained normal, and anti-PLA2R was detectable and decreased with successful treatment. Clinicians should be aware of the possibility of two glomerular disorders in patients with glomerulonephritis and atypical presentations for any single disorder.

The identification of the phospholipase A2 receptor <em>1</em> (PLA2R) and <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>protein</em> <em>7A</em> (THSD<em>7A</em>) 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 THSD<em>7A</em>-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.

<strong class="sub-title"> Keywords: </strong> Membranous nephropathy; PLA2R; THSD<em>7A</em>.

BACKGROUND M-<em>type</em> phospholipase A2 receptor (PLA2R) was identified as the major target antigen in idiopathic membranous nephropathy (IMN). Another target antigen, namely <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>), was recently detected in approximately <em>1</em>0% of non-PLA2R-associated IMN. In this single center retrospective study, clinical and histological features of PLA2R-associated and THSD<em>7A</em>-associated IMN patients were evaluated. MATERIAL AND METHODS A total of <em>1</em>92 IMN patients, who were receiving no glucocorticoids or immunosuppressant before renal biopsy, were enrolled in this study and followed for a median duration of 25.5 months. IMN with enhanced glomerular PLA2R and THSD<em>7A</em> staining by immunohistochemistry (IHC) were designated as PLA2R-associated IMN and THSD<em>7A</em>-associated IMN respectively. Serum anti-PLA2R and anti-THSD<em>7A</em> antibodies levels were assessed by enzyme linked immunosorbent assay and indirect immunofluorescence testing in PLA2R-associated and THSD<em>7A</em>-associated IMN. RESULTS Of <em>1</em>92 IMN patients, <em>1</em>64 patients (85.4%) had PLA2R-associated IMN and 3 patients (<em>1</em>.6%) had THSD<em>7A</em>-associated IMN. Compared with non-PLA2R-associated IMN patients, the 24-hour urinary <em>protein</em> levels were significantly higher (P=0.008), whereas, the <em>protein</em>uria remission rates were significantly lower (P=0.03) in PLA2R-associated IMN patients. No pathological differences were found between PLA2R-associated IMN and non-PLA2R-associated IMN. Among 3 THSD<em>7A</em>-associated IMN patients, <em>1</em> patient had elevated serum anti-THSD<em>7A</em> antibody levels, which was below detectable levels after achieving partial <em>protein</em>uria remission with combined glucocorticoid and cyclosporine treatment. CONCLUSIONS Compared with non-PLA2R-associated IMN patients in our cohort, PLA2R-associated IMN patients presented with more severe <em>protein</em>uria and lower remission rates after treatment, with no distinct histological differences. Glomerular expression of PLA2R could be a useful marker to indicate the severity, treatment response, and prognosis of IMN.

Antibodies against THSD<em>7A</em> (<em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em>-<em>containing</em> <em>protein</em> <em>7A</em>) have been proposed to play a causal role in the development of nephrotic syndrome in patients with THSD<em>7A</em> antibody-positive membranous nephropathy. We hypothesized that removal of these antibodies from plasma could lead to a rapid reduction in <em>protein</em>uria. Using immunoadsorption to reduce THSD<em>7A</em> antibodies led to a rapid reduction in <em>protein</em>uria in 2 patients with THSD<em>7A</em> antibody-positive membranous nephropathy. Moreover, our findings support and strengthen the pathogenic role of the antibodies in the development of nephrotic syndrome in patients with THSD<em>7A</em> antibody-positive membranous nephropathy. Taken together, these 2 cases suggest that immunoadsorption could be a useful tool in the treatment of patients with THSD<em>7A</em> antibody-positive membranous nephropathy.

In the last <em>1</em>0 years, basic science and clinical research have made important contributions to the understanding and management of primary membranous nephropathy (MN). The identification of antibodies directed against the M-<em>type</em> phospholipase A2 receptor (PLA2R) and <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> <em>protein</em> have added a new perspective on diagnosis, monitoring the immunological activity, predicting prognosis and guiding therapy in patients with primary MN. Mounting evidence suggests that quantification and follow-up of antiPLA2R Abs levels can help in assessing prognosis and evaluate the response to treatment. The kidney disease improving global outcomes guidelines published in 20<em>1</em>2 have not been updated. New data on the use of rituximab suggest it should be considered as a potential initial therapy in the treatment of patients with primary MN.

Primary membranous nephropathy (PMN) is a leading cause of adult nephrotic syndrome. The field took a major step forward with the identification of phospholipase A2 receptor (PLA2R) as target antigen in the majority of cases and with the ability to measure circulating autoantibodies to this <em>protein</em> (aPLA2R). Since then, the existence of additional target antigens such as <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em> <em>containing</em> <em>7A</em>, exostosin <em>1</em> and 2, NELL-<em>1</em>, Semaphorin-3B has been demonstrated. The ability to detect and monitor levels of circulating autoantibodies has opened a new window onto the humoral aspect of PMN. Clinicians now rely on clinical parameters such as <em>protein</em>uria, as well as levels of circulating aPLA2R and the results of immunofluorescent staining for aPLA2R on renal biopsy, to guide the management of this disease. The relationship between immunologic and clinical disease course is consistent, but not necessarily intuitive. In addition, kidney biopsy provides only a single snapshot of disease that needs to be interpreted in light of changing clinical and serological findings. A clear understanding of these dynamic parameters is essential for staging, treatment, and management of this disease. This review aims to shed light on current knowledge regarding the development and time-course of changes in serum aPLA2R levels, <em>protein</em>uria and histological findings that underlie the pathophysiology of PMN.

<strong class="sub-title"> Keywords: </strong> ELISA; EM; ESKD; Frequently-used abbreviations; HN; Heymann nephritis; LM; MN; PLA2R; PMN; THSD<em>7A</em>; autoantibodies against PLA2R; autoantibody; electron microscopy; end stage kidney disease; enzyme linked immunosorbent assay; immunofluorescence; immunological remission; light microscopy; membranous nephropathy; phospholipase A2 Receptor; primary membranous nephropathy; rMN; recurrent membranous nephropathy; <em>thrombospondin</em> <em>type</em>-<em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em>.

Membranous nephropathy (MN) is an immune complex mediated disease. Although limited to the kidney, in up to 20% of patients MN is associated with other autoimmune, infectious or malignant diseases. The initial pathogenetic event in what is still considered "primary" MN is the binding of circulating autoantibodies to <em>proteins</em> (autoantigens) expressed in glomerular podocytes. This antibody binding leads to the formation of immune complexes in the glomerular basement membrane. There is clinical and experimental evidence that these immune deposits lead to the activation of the complement system. Experimental studies in the MN model of Heymann's nephritis show that the terminal membrane attack complex (MAC) of the complement system induces a disturbance of the glomerular filtration barrier and leads to <em>protein</em>uria, the clinical hallmark of MN. After the discovery of the phospholipase A₂ receptor <em>1</em> and <em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em> <em>containing</em> <em>protein</em> <em>7A</em> as endogenous antigens, it is assumed that IgG4 antibodies directed against these <em>proteins</em> induce MN in over 85% of patients with primary MN. As a result, the role of complement in the pathogenesis of MN needs to be defined in light of these developments. In this review we describe the current knowledge on the function of the complement system in primary MN and discuss the open questions, which have to be solved for a better understanding of the potential role of complement in the pathophysiology of primary MN.

Keywords: Autoimmunity; Complement; IgG4 antibody subclass; Kidney disease; Membranous nephropathy.

Recent advances in glomerular biology have expanded our understanding of glomerular diseases, leading to more precise therapeutic options. Since the discovery of the autoantigen phospholipase A₂ receptor in primary membranous nephropathy <em>1</em>0 years ago, the serologic evaluation of glomerular diseases has become more detailed and nuanced for nephrologists. In addition to phospholipase A₂ receptor antibodies, circulating autoantibodies now include <em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em>-<em>containing</em> <em>7A</em> and most recently, neural epidermal growth factor-like <em>1</em> <em>protein</em> for membranous nephropathy. Additionally, discoveries in C3 glomerulopathy and fibrillary glomerulonephritis are poised to improve the diagnostic approach to these disorders by using novel biomarkers to complement traditional histologic patterns on kidney biopsy. Although kidney biopsies are considered the gold standard in profiling glomerular diseases, validated novel glomerular biomarkers contribute substantially to the diagnostic and therapeutic approaches through their ability to improve sensitivity, permit dynamic longitudinal monitoring of disease activity, and capture genetic heterogeneity. We describe the value of specific biomarkers in selected glomerular diseases, with the major focus on their clinical applicability.

<strong class="sub-title"> Keywords: </strong> C3 glomerulopathy (C3GN); Membranous nephropathy (MN); biomarkers; diagnostic tests; fibrillary glomerulonephritis (FGN); glomerular disease; kidney disease; phospholipase A(2) receptor <em>1</em> (PLA(2)R); renal biopsy; review; <em>thrombospondin</em> <em>type</em> <em>1</em> <em>domain</em> <em>containing</em> <em>7A</em> (THSD<em>7A</em>).

<strong class="sub-title"> Objectives: </strong> To evaluate the value of thrombospond in Type I <em>domain</em>-<em>containing</em> <em>7A</em> (THSD<em>7A</em>) and M-type phospholipase A2 receptor (PLA2R) in primary membranous nephropathy (PMN) and to explore the relationship between their antibody levels and prognosis.

<strong class="sub-title"> Methods: </strong> Renal tissues in 128 patients with membranous nephropathy in the Second Xiangya Hospital of Central South University were collected from February 2015 to August 2017, including 108 patients with primary membranous nephropathy (PMN group) and 20 patients with secondary membranous nephropathy (SMN) (SMN group). Indirect immunofluorescence method was used to detect the expression of PLA2R antigen in kidney tissues,and the glomerular expression of THSD<em>7A</em> antigen was examined by immunohistochemistry and indirect immunofluorescence. The serum levels of anti-PLA2R antibodies and THSD<em>7A</em> antibodies were also detected by ELISA. According to the results of PMN examination,the patients were also divided into a PLA2R-related membranous nephropathy group and a THSD<em>7A</em>-related membranous nephropathy group.

<strong class="sub-title"> Results: </strong> The positive rate of PLA2R in the renal tissues in the PMN group was higher than that in the SMN group (78% in the PMN group, 35% in the SMN group, <i>P</i><0.01),while the positive rate of anti-PLA2R antibody in the PMN group was also higher than that in the SMN group (50% in the PMN group, 25% in the SMN group, <i>P</i><0.05).The serum level of anti-PLA2R antibody was positively correlated with 24 h urine protein (<i>r</i>=0.254, <i>P</i><0.05) and negatively correlated with serum albumin (<i>r</i>=-0.236, <i>P</i><0.05). The expression of THSD<em>7A</em> was positive in glomeruli in 7 cases of the PMN group (6%) by immuno-histochemistry, and which was positive in 1case of the SMN group (5%).The serum levels of anti-THSD<em>7A</em> antibody in the PMN group were higher than those in the SMN group [(0.49±0.26) pg/mL in the PMN group,(0.34±0.27) pg/mL in the SMN group, <i>P</i><0.05]. There was no difference in the clinical characteristics between the PLA2R-related membranous nephropathy group and the THSD<em>7A</em>-related membranous nephropathy group.

<strong class="sub-title"> Conclusions: </strong> PLA2R and THSD<em>7A</em> are the target antigen of PMN, and the associated autoantibodies are helpful for the differential diagnosis of PMN. The anti-PLA2R antibody levels can reflect the severity of the disease and evaluate the effect of treatment. The incidence of THSD<em>7A</em> membranous nephropathy is low, and monitoring the serum anti-THSD<em>7A</em> antibody levels can assess patients' condition and predict disease outcome.

<b>目的</b>: 探讨M型磷脂酶A2受体(phospholipase A2 receptor，PLA2R)、1型血小板反应蛋白<em>7A</em>域(thrombospondin Type 1 <em>domain</em>-<em>containing</em> <em>7A</em>，THSD<em>7A</em>)及相应的抗体对特发性膜性肾病(primary membranous nephropathy，PMN)的诊断价值及其血清抗体水平与预后的关系。<b>方法</b>: 纳入2015年2月至2017年8月中南大学湘雅二医院肾内科行肾活检确诊的膜性肾病128例，其中PMN108例(PMN组)，继发性膜性肾病(secondary membranous nephropathy，SMN)20例(SMN组)。应用间接免疫荧光法检测肾组织PLA2R抗原表达，间接免疫荧光法和免疫组织化学法检测肾组织THSD<em>7A</em>抗原表达，ELISA检测血清PLA2R和THSD<em>7A</em>的抗体水平。根据检测结果，又将患者分为PLA2R相关性膜性肾病组和THSD<em>7A</em>相关性膜性肾病组。<b>结果</b>: PMN组肾小球PLA2R阳性率明显高于SMN组(分别为72%和35%，<i>P</i><0.01)，PMN组血清PLA2R抗体阳性率亦明显高于SMN组(分别为50%和25%，<i>P</i><0.05)。PMN组血清PLA2R抗体与24 h尿蛋白量呈正相关关系(<i>r</i>=0.254，<i>P</i><0.05)，与血清白蛋白呈负相关关系(<i>r</i>=-0.236，<i>P</i><0.05)。PMN组中7例(6%)患者肾小球THSD<em>7A</em>呈阳性，SMN组有1例(5%)患者肾小球THSD<em>7A</em>呈阳性。PMN组血清THSD<em>7A</em>抗体水平明显高于SMN组[分别为(0.49±0.26)和(0.34±0.27) pg/mL，<i>P</i><0.05]。7例THSD<em>7A</em>相关性膜性肾病组与86例PLA2R相关性膜性肾病组的临床病理特征差异均无明显统计学意义(均<i>P</i>>0.05)。<b>结论</b>: 肾组织PLA2R和THSD<em>7A</em>是PMN的靶抗原，其血清抗体有助于PMN的鉴别诊断；PLA2R抗体水平可反映病情严重程度，可用来评估治疗效果；PMN患者THSD<em>7A</em>相关性膜性肾病的发病率低，监测血清THSD<em>7A</em>抗体水平可评估患者病情，并可预测疾病转归。.

<strong class="sub-title"> Keywords: </strong> M-type phospholipase A2 receptor; primary membranous nephropathy; thrombospondin Type-1 <em>domain</em>-<em>containing</em> <em>7A</em>.