In comparison, the control animal showed no proteinuria over the course of the experiment (Figure ?(Number1G1G and Supplemental Number 7). in which immune complexes are deposited beneath the epithelium in the glomeruli. The condition introduces a high risk for end-stage kidney disease. Seventy percent to 80% of individuals with MN have circulating antibodies against phospholipase A2 receptor 1 (PLA2R1), and levels correlate with treatment response and prognosis. However, experimental evidence that human being anti-PLA2R1 antibodies induce MN has been elusive. Methods In passive transfer experiments, minipigs received plasma or purified IgG from individuals with PLA2R1-connected MN or from healthy controls. Anti-PLA2R1 antibodies and proteinuria were monitored using Western blot, ELISA, and Coomassie staining. Kidney cells were analyzed using immunohistochemistry, UMI-77 immunofluorescence, electron microscopy, and proteomic analyses. Results Minipigs, like humans, communicate PLA2R1 on podocytes. Human being anti-PLA2R1 antibodies bound to minipig PLA2R1 and in vivo. Passive transfer of human being anti-PLA2R1 antibodies from individuals with PLA2R1-connected MN to minipigs led to histological characteristics of human being early-stage MN, activation of components of the match cascade, and low levels of proteinuria. We observed development of an autologous, later phase of disease. Conclusions A translational approach from humans to minipigs showed that human being anti-PLA2R1 antibodies are pathogenic in MN, although in the heterologous phase of disease UMI-77 only low-level proteinuria developed. Main autoimmune membranous nephropathy (MN) is initiated by binding of autoantibodies to antigens indicated on podocytes.1 Antibodies against PLA2R1 and Thrombospondin type-1 domain-containing protein 7A (THSD7A) are found in 70%C80% and 2%C3% of patients with MN, respectively.2,3 In individuals with PLA2R1- or THSD7A-associated MN, subepithelial immune deposits in glomeruli contain antibodies against the respective molecule, suggesting that PLA2R1 and THSD7A may serve as endogenous antigens.2,3 Transfer of human being UMI-77 anti-THSD7A antibodies to mice resulted in a disease, which mimics human being MN and proven the pathogenicity of human being anti-THSD7A antibodies.4 In contrast to THSD7A, PLA2R1 is not expressed on rodent podocytes and the pathogenicity of human being anti-PLA2R1 antibodies has so far not been shown. We used minipigs, which display a PLA2R1 manifestation pattern on podocytes identical to that found in human being glomeruli, to study the pathogenicity of human being anti-PLA2R1 antibodies. Methods Experiments were performed on healthy, full-grown female G?ttingen minipigs (authorities body approval quantity N108/2018). Details about the animal housing conditions and all experimental methods are defined in the Supplemental Methods. Anonymized samples from individuals with biopsy-proven PLA2R1-connected MN or healthy controls were utilized for the transfer experiments. Human material was collected as part of a prospective study, which was authorized by the local ethics committee of the chamber of physicians in Hamburg and carried out in accordance with the ethical principles stated from the Declaration of Helsinki. Written educated consent was received from all participants before inclusion in the study. The Supplemental Methods describe the selection criteria and preparation of samples in detail. This section also identifies the anti-PLA2R1 antibody measurements, Western blot experiments, and the immunohistochemical, immunofluorescence, and electron microscopy investigations. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium using the Proteomics Identifications Database partner repository with the dataset identifier PXD039024. Details of the sample preparation and mass spectrometric analysis are available as Supplemental Methods. Results and Conversation Sera from individuals with PLA2R1-connected MN showed an IgG4-dominating reactivity to conformation-dependent epitopes of the porcine PLA2R1 (pPLA2R1), as typically demonstrated for the human being PLA2R1 (hPLA2R1) UMI-77 antigen (Supplemental Numbers 1 and 2). PLA2R1 antibodyCpositive plasma from individuals with PLA2R1-connected MN was transferred into minipig A at four time points Mouse monoclonal to cMyc Tag. Myc Tag antibody is part of the Tag series of antibodies, the best quality in the research. The immunogen of cMyc Tag antibody is a synthetic peptide corresponding to residues 410419 of the human p62 cmyc protein conjugated to KLH. cMyc Tag antibody is suitable for detecting the expression level of cMyc or its fusion proteins where the cMyc Tag is terminal or internal. (Supplemental Number 3). As a negative control, plasma from healthy donors was transferred into the control minipig B. Seven days before the 1st plasma transfer, the renal mass of each minipig was reduced by removal of one kidney.
