Elion Hoxha

Thrombospondin Type-1 Domain-Containing 7A in Idiopathic Membranous Nephropathy

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.).

An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis

BACKGROUND The recent finding that phospholipase-A(2)-receptor antibodies (PLA(2)R-AB) may play a role in the development of primary membranous glomerulonephritis (MGN) offers the opportunity to measure a marker to help diagnose, classify and eventually monitor the course of patients with MGN. METHODS We developed an immunofluorescence test, which allows the easy and specific analysis of the presence of PLA(2)R-AB in serum. The usefulness of this test was studied in 153 healthy blood donors, 90 patients with non-membranous glomerular injuries, 17 patients with a secondary form of MGN and 100 patients with biopsy-proven primary MGN. In addition, in five patients with biopsy-proven MGN, PLA(2)R-AB levels were monitored prospectively for up to 18 months following a single dose of rituximab (RTX) (375 mg/m(2) body surface). RESULTS PLA(2)R-AB were not found in healthy controls or patients with glomerular lesions other than biopsy-proven primary MGN. Fifty-two patients with primary MGN (52%) were positive for PLA(2)R-AB. The levels ranged from 1:10 to 1:3200. In patients who had MGN and were treated with RTX the fall in PLA(2)R-AB levels was followed by a decrease in proteinuria, whereas an increase in PLA(2)R-AB levels was associated with an increase in proteinuria. CONCLUSIONS These studies show that the new test allows the monitoring of PLA(2)R-AB levels in patients with MGN and may help in making therapeutic decisions for these patients.

Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy

The M-type phospholipase A2 receptor (PLA2R) is the major target antigen in idiopathic membranous nephropathy with detectable autoantibodies in the serum of up to 70% of patients. In retrospective studies, the PLA2R-autoantibody titer in the serum was sometimes negative indicating their measurement alone may be inconclusive. In order to better differentiate between primary and secondary membranous nephropathy, we conducted a prospective study that included 88 patients with a histologic diagnosis of membranous nephropathy. Immunohistochemical analysis for PLA2R was faintly positive in kidneys from normal individuals and patients with various other glomerular injuries. In 61 of the 88 patients, PLA2R expression was strongly positive in glomeruli, and in 60 of these patients PLA2R autoantibodies were also detected in the serum. The 27 patients negative for serum PLA2R autoantibodies were faintly positive for PLA2R staining in glomeruli and in 15 of these patients a secondary cause was found. The remaining 12 patients have a yet undetected secondary cause of membranous nephropathy or have different glomerular antigens other than PLA2R. Thus, increased staining for PLA2R in glomeruli of renal biopsies tightly correlates with the presence of PLA2R autoantibodies in the serum and this may help discriminate between primary and secondary membranous nephropathy.

Phospholipase A2 Receptor Autoantibodies and Clinical Outcome in Patients with Primary Membranous Nephropathy

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, with an uncertain clinical outcome. The characterization of the phospholipase A2 receptor (PLA2R) as the major target antigen in primary MN and the detection of circulating autoantibodies in these patients is a major advance in understanding this disease. To test whether PLA2R antibody levels reflect disease activity or clinical outcome, we performed a prospective multicenter study of 133 adult patients with primary MN and detectable serum PLA2R antibodies who had not received immunosuppressive therapy. Patients were followed ≤24 months. PLA2R antibody levels associated with clinical disease activity (proteinuria) in patients with immunosuppressive therapy (n=101) or supportive care (n=32). Within 3 months, immunosuppressive therapy led to a sustained 81% reduction in PLA2R antibody levels paralleled by a 39% reduction in proteinuria. Patients who experienced remission of proteinuria after 12 months had significantly lower PLA2R antibody levels at the time of study inclusion compared with patients with no remission. Patients with high PLA2R antibody levels achieved remission of proteinuria significantly later than patients with low PLA2R antibody levels. PLA2R antibody levels fell over time in patients with spontaneous remission but remained elevated in patients who did not show a reduction in proteinuria. Multivariable Cox regression analysis confirmed PLA2R antibody level as an independent risk factor for not achieving remission of proteinuria. We conclude that a decrease in PLA2R antibody level is associated with a decrease of proteinuria in patients with primary MN.

Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy

BACKGROUND Autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) are specific markers for primary membranous nephropathy (pMN) and anti-PLA2R1 serum levels may be useful to monitor disease activity. So far, a recombinant cell-based indirect immunofluorescence assay (RC-IFA) using recombinant PLA2R1 as a substrate has been widely available but lacks a finely graduated assessment of antibody concentrations. METHODS In order to setup a standardized ELISA, the extracellular domain of human PLA2R1 was expressed in HEK293. The purified protein was used to form the solid-phase in an ELISA which was then employed to analyze sera from 200 patients with primary MN, 27 patients with secondary MN, 230 patients with other glomerular diseases, 316 patients with systemic autoimmune diseases, and from 291 healthy blood donors. RESULTS At a set specificity of 99.9% the sensitivity of the anti-PLA2R1 IgG ELISA was found to be 96.5%. A similar sensitivity (98.5%) was obtained when binding of only subclass IgG4 was analyzed. The calibrated assay showed a good class correlation with the results of the RC-IFA, was robust and could be stored for several months without any loss of quality. CONCLUSION The results demonstrate that the new test system is qualified for routine use and that it has an almost perfect agreement with both, the clinical characterization of the patients and the results generated with RC-IFA.

Renal IL-17 expression in human ANCA-associated glomerulonephritis

Interleukin-17A (IL-17) promotes inflammatory renal tissue damage in mouse models of crescentic glomerulonephritis, including murine experimental autoimmune anti-myeloperoxidase glomerulonephritis, which most likely depends on IL-17-producing Th17 cells. In human anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, however, the cellular sources of IL-17 remain to be elucidated. Therefore, we analyzed human kidney biopsies of active necrotizing and crescentic ANCA-associated glomerulonephritis by immunohistochemistry using an IL-17-specific antibody and by immunofluorescent colocalization with cell type markers. We detected numerous IL-17-expressing (IL-17(+)) cells in the glomeruli and in the tubulointerstitium. Unexpectedly, most of these IL-17(+) cells were polymorphonuclear neutrophilic granulocytes, while IL-17(+) T cells and IL-17(+) mast cells were present at significantly lower frequencies. IL-17 was not detected in other infiltrating or resident kidney cells. In those patients who had not received immunosuppressive treatment before biopsy, serum creatinine levels were positively correlated with tubulointerstitial IL-17(+) neutrophils as well as IL-17(+) T cells. Furthermore, we could demonstrate that purified human blood neutrophils expressed IL-17 protein and released it upon stimulation in vitro. In conclusion, these results support a pathogenic role for IL-17 in human ANCA-associated glomerulonephritis. Our data suggest that in the acute stage of the disease neutrophils may act as an important immediate-early innate source of IL-17 and may thereby initiate and promote ongoing renal inflammation. IL-17 may thus be a target for treating acute ANCA-associated glomerulonephritis.

A Mechanism for Cancer-Associated Membranous Nephropathy

The cause of paraneoplastic membranous nephropathy is unclear. In the current case, new expression of THSD7A in a gallbladder carcinoma and the development of membranous nephropathy may indicate a potential mechanism for the association between cancer and this nephropathy.

Autoantibodies against thrombospondin type 1 domain–containing 7A induce membranous nephropathy

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.

PLA2R antibody levels and clinical outcome in patients with membranous nephropathy and non-nephrotic range proteinuria under treatment with inhibitors of the renin-angiotensin system.

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.

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.