A 30-year-old woman in steroid therapy for eosinophilia presented with nephrotic syndrome during steroid tapering. cell count, hemoglobin, platelet, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactic dehydrogenase, total protein, albumin, blood urea nitrogen, creatinine, uric acid, C-reactive protein, match component 3, match component 4, immunoglobulin, rheumatoid element, antinuclear antibodies, antineutrophil cytoplasmic antibody, myeloperoxidase-, protease 3-, N-acetyl–D-glucosamidase Open in a separate windowpane Fig. 1 Micrographs showing glomeruli in the kidney. a Periodic acid-Schiff (PAS) staining of the renal biopsy specimen showing a minor glomerular abnormality. Initial magnification??200. b Periodic acid methenamine metallic (PAM) staining showing focal segmental spike formations (reddish arrow). Initial magnification??400. c Masson trichrome staining showing ENMD-2076 granular immune complex along the glomerular basement membrane. Initial magnification??400 ENMD-2076 Open in a separate windowpane Fig. 2 Immunofluorescence microscopy (IF). IF image of the renal specimen showing granular 2?+?capillary SKP1 wall staining for IgG, 1?+?capillary wall staining for IgM. No staining for IgA, C1q, C3c. Initial magnification??200 Open in a separate window Fig. 3 IgG subclass staining in IF IgG4 and IgG1 IF image of the renal specimen showing granular 2?+?capillary wall structure staining for IgG4 and IgG1. No staining was observed for IgG2 and IgG3. Initial magnification??200 Open in a separate window Fig. 4 Electron microscopy (EM). EM image of the renal specimen exposed subepithelial deposits in the glomerular basement membrane. Initial magnification??5000 and??10,000 Next, we used an anti-PLA2R antibody to screen for the PLA2R antigen responsible for MN. Immunofluorescence imaging of a renal specimen did not show enhanced granular manifestation of PLA2R along the glomerular basement membrane (Fig.?5a). PLA2R was not recognized in the individuals serum by western blotting (Fig.?6a remaining). Based on the above results, we in the beginning diagnosed the condition as IMN However, a protein having a molecular excess weight higher than that of PLA2R was recognized by western blot (Fig.?6a right). Open in a separate windowpane Fig. 5 Immunofluorescence staining in the glomeruli. a Immunofluorescence imaging of a renal specimen from the patient did not show any enhanced granular manifestation of M-type phospholipase A2 receptor (PLA2R) along the glomerular basement membrane. b Immunofluorescence image of renal specimen from the patient showing granular staining for thrombospondin type-1 domain-containing 7 A (THSD 7 A) along the glomerular basement membrane. Initial magnification??200 Open in a separate window Fig. 6 Detection of circulating anti-THSD7A IgG. Anti-THSD7A antibody was measured by western blot analysis and commercial indirect immunofluorescence test (IFT). Western blot analyses of human being glomerular draw out (HGE) and recombinant PLA2R protein were performed under non-reducing conditions with individuals serum, using a commercial anti-PLA2R rabbit polyclonal antibody as the primary antibody. The bands were recognized by chemical luminescence imaging with HRP-labeled anti-human IgG mouse monoclonal antibody and HRP-labeled anti-rabbit IgG goat polyclonal antibody. A commercial indirect IFT for anti-THSD7A IgG (Euroimmun) was performed with the individuals serum according to the manufacturers protocol. a The picture over the still left displays the full total consequence of traditional western blotting of recombinant PLA2R, portrayed in HEK293 cells, under nonreducing condition with sufferers serum and anti-PLA2R rabbit polyclonal antibody. The sufferers serum didn’t acknowledge the recombinant PLA2R proteins. The picture on the proper shows the consequence of traditional western blotting from the HGE under nonreducing condition with sufferers serum and anti-PLA2R rabbit polyclonal antibody. The sufferers serum recognized an individual music group (white arrowhead) whose molecular weight was ENMD-2076 greater than that of PLA2R (dark arrowhead) that was subsequently defined as THSD7A. b Fluorescence microscopy pictures obtained utilizing a industrial indirect IFT. The sufferers serum sure to the recombinant THSD7A proteins over the cell surface area As the proteinuria spontaneously improved, PSL dosage was preserved at 4?mg. An imperfect remission (urine proteins?