PMC8185035_01
Histological features of the first renal biopsy. (A) Neither proliferative changes nor thickening of glomerular capillary walls was observed in the sections (periodic acid-methenamine-silver stain) on light microscopy. (B) Granular deposition of immunoglobulin (Ig)M on glomerular capillary walls was shown on immunofluorescence (IF) staining. (C) On the other hand, IF staining for IgG was negative. (D) Deposition of IgM on glomerular capillary walls was further confirmed by immunoperoxidase staining on formalin-fixed, paraffin-embedded tissue sections using an automatic staining machine by a clinical laboratory testing company (SRL, Inc., Tokyo, Japan). (E) Electron microscopy sections identified electron-dense subepithelial deposits. The effacement of podocyte foot processes is also shown. IF light-chain staining showed that whereas there was strong deposition of lambda chains on the glomerular capillary walls (F), there was no deposition of kappa chains (G).
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F1
There were no proliferative changes, and the glomerular capillary walls were not thickened (Figure 1A). IF staining on frozen tissue sections showed granular 2+ deposition of IgM (Figure 1B) on the capillary walls, without deposition of IgG (Figure 1C), IgA, complement C1q, or C3 (data not shown). Immunoperoxidase staining for IgM on formalin-fixed, paraffin-embedded tissue (FFPE) sections were performed using an automatic staining machine by a clinical laboratory testing company (SRL, Inc., Tokyo, Japan). The stained slides further confirmed the deposition of IgM on the glomerular capillary walls (Figure 1D). Subepithelial electron-dense deposits were found on electron microscopy, but spike formation of the glomerular basement membrane was obscure (Figure 1E). Although stage I MN was suggested, IF staining for light chains demonstrated the deposition of only lambda chains but not kappa chains (Figures 1F,G). To rule out the existence of masked Ig deposits, we further performed IF staining for IgG, IgA, IgM, and light chains on FFPE sections after protease digestion as previously described, which also showed the granular deposition of IgM and lambda chains on the capillary walls, but with no deposition of IgG or kappa chains (Supplementary Figure 1).
Histological features of the second renal biopsy. (A) Thickening of the glomerular capillary walls accompanied with spike formation and a bubbly appearance (see inset) was observed in the sections (periodic acid-methenamine-silver stain) on light microscopy. (B) IF staining for IgM demonstrated weak reactivity on the glomerular capillary walls. (C) IF staining for IgG was extensively positive on the glomerular capillary walls. (D) IF staining for IgG subclasses demonstrated dominant deposition of IgG2. Deposition of IgG4 was faintly observed. IF staining for light chains showed extensive deposition of both lambda chains (E) and kappa chains (F). (G) Unlike IF staining, immunoperoxidase staining for IgM of formalin-fixed, paraffin-embedded tissue (FFPE) sections after antigen retrieval, using an automatic staining machine by a clinical laboratory testing company (SRL, Inc.) showed strong IgM deposition on the glomerular capillary walls. (H) Immunoperoxidase staining for thrombospondin-type-1-domain-containing-7A on FFPE sections showed diffuse global-positive staining on the glomerular capillary walls.
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F2
Thickening of the glomerular capillary walls accompanied by spike formation and a bubbly appearance was also observed, suggesting disease progression of MN (Figure 2A). IF staining of frozen tissue sections demonstrated weakened IgM staining (Figure 2B) and augmented IgG staining (Figure 2C), in which IgG2 was dominant with weaker deposition of IgG4 (Figure 2D). Light chain restriction was not observed by IF staining this time, and both lambda and kappa light-chain deposits were observed at almost equal levels (Figures 2E,F). Thus, in the second renal biopsy, routine IF staining on a frozen tissue section showed weak IgM staining in glomeruli; however, immunoperoxidase staining for IgM on a FFPE section after antigen retrieval showed strong IgM deposition on the glomerular capillary walls (Figure 2G). IgG was augmented in the renal tissue of the second renal biopsy, serum anti-PLA2R antibody level measured by enzyme-linked immunosorbent assay, and IF staining for PLA2R on FFPE sections were both negative (Supplementary Figure 2). Immunoperoxidase staining for neural epidermal growth factor-like 1 protein (NELL-1) on FFPE sections was also negative (Supplementary Figure 2), whereas that for THSD7A on FFPE sections showed a diffuse positive staining pattern on the glomerular capillary walls (Figure 2H). Immunoperoxidase staining for THSD7A on FFPE sections of the first renal biopsy was therefore performed and the staining result was compared with that of the second renal biopsy. Immunoperoxidase staining for amyloid P, a recently identified marker of membranous-like glomerulopathy with masked IgG deposits, was negative in the FFPE sections of both renal biopsy tissues (Supplementary Figure 2).
Clinical course of the present patient. Although the therapeutic effects of an angiotensin II type 1 receptor blocker (ARB) and a steroid (prednisolone; PSL) were insufficient, a substantial reduction in proteinuria and improvement of renal function as well as hematological responses were achieved by rituximab treatment.
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F3
Compared to the second renal biopsy, which showed diffuse and global strong staining, the staining result of the first renal biopsy showed focal and segmental granular immunoreactivity on the glomerular capillary walls (Supplementary Figure 3). THSD7A was negative in cells of the bone marrow by immunoperoxidase staining of FFPE sections (Supplementary Figure 3). Her clinical course is shown in Figure 3.