PMC6868084_01
(A) Kidney lesions in post-HSCT patient showing characteristic features of crescentic glomerulonephritis, with >90% of the 32 glomeruli sampled displaying cellular or fibrocellular crescents, with segmental fibrinoid necrosis and with extensive acute tubular injury and focal, 10-20% interstitial fibrosis and tubular atrophy (Jones' silver stain). (B-E) Binding of patient serum antibodies to frozen sections from normal human kidney (immunofluorescent staining). (B) Distinct linear staining of GBM observed on intact kidney section, which is strongly increased after pre-treatment with acidic urea (C). (D) There is no staining with normal human serum (1:50). (E) GBM staining was abolished by adsorption of patient serum on alpha3NC1-coated magnetic beads (E), which removed 95% of alpha3-antibody as demonstrated by testing of original (GP) and absorbed (MB) serum using indirect ELISA of on alpha3NC1-coated plate (F).
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The biopsy showed characteristic features of crescentic glomerulonephritis, with >90% of the 32 glomeruli sampled (8 globally sclerosed) displaying cellular or fibrocellular crescents, with segmental fibrinoid necrosis and with extensive acute tubular injury and focal, 10-20% interstitial fibrosis and tubular atrophy (Figure 1A). When applied to frozen sections of normal human kidney, the patient's serum at 1:50 dilution demonstrated strong linear anti-GBM staining, which was greatly enhanced by acidic urea treatment (Figures 1B,C). The specificity of the staining and the nature of deposited antibody were established by immunoadsorbtion of serum on alpha3NC1-coated magnetic beads, which nearly abolished staining in parallel with removal of alpha3NC1 antibody (Figures 1E,F). This closely resembled the enhanced immunofluorescent staining of human kidney sections after acidic urea (Figure 1C).
(A) Testing of NC1 domain specificity at the onset of the disease by ELISA showed that antibodies targeted predominantly alpha3NC1 domain with a lesser reactivity to alpha1 and alpha5NC1 domains. (B) Patient serum was pre-incubated with increasing concentrations of the alpha1, alpha3, and alpha5NC1 monomers and binding to corresponding immobilized monomers was measured by ELISA. Differential sensitivity to inhibition by the three NC1 monomers indicates that alpha3 antibodies has the highest affinity. (C) Pre-adsorption of patient serum with alpha3NC1-coated magnetic beads (MBser) significantly reduced immunoreactivity to alpha1 and alpha5NC1 monomers compared to original serum (GPser). (D) alpha3NC1-specific antibodies predominantly target the EA epitope with lower reactivity to EB epitope. (E) Binding of purified GP antibody labeled with biotin to alpha3NC1 monomer was competed by patient serum (Pt), as well as control serum mix from 8 GP patients (GP), but not by the normal human serum (N). (F) Patient's antibodies strongly react with the denatured NC1 hexamers from bovine (bGBM) or human (hGBM) glomerular basement membrane, while minimally react with native NC1 hexamers. (G) ELISA analysis of serial serum samples demonstrated the absence of anti-GBM antibodies 5 weeks prior to presentation (12/30/2013) by comaprison with normal human serum (NHS), with a rapid and strong development of immunoreactivity in 5 weeks. (H) There was no IgM binding to any of the six human recombinant NC1 domains of collagen IV in serum samples collected before or at the onset of the disease. The dashed line indicates threshold determined as mean +- 3x SEM for normal human serum.
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Serum collected at initial presentation showed that a majority of antibody targeting the alpha3NC1 monomer of collagen IV with weaker reactivity against alpha1 and alpha5NC1 monomers, indicating that alpha3NC1 is the primary autoantigen (Figure 2A). This was further supported by measuring the affinity of circulating antibodies toward human alpha1, alpha3, and alpha5NC1 domains (Figure 2B). Furthermore, adsorption of patient serum on alpha3NC1-coated magnetic beads not only removes alpha3 specific antibodies, but significantly reduces immunoreactivity to alpha1 and alpha5NC1 (Figure 2C), indicating at least partial cross-reactivity of the major alpha3-specific antibody with the alpha1 and alpha5NC1 domains. Circulating antibodies from the HSCT patient targeted predominantly the EA epitope of alpha3NC1 monomer with lower reactivity toward EB epitope (Figure 2D) suggesting a key role of the EA region in the initiation of anti-GBM disease in this case. Patient serum antibodies efficiently competed with binding of biotinylated GP antibody to coated alpha3NC1 in a dose-dependent manner and was undistinguishable from the control mix of GP sera (Figure 2E), showing that current patient's circulating antibody targets the same epitopes as in patients with classical GP disease. Finally, although there was minimal antibody reactivity with native alpha345NC1 hexamers from bovine or human GBM, binding of antibody was significantly increased upon hexamer dissociation by a protein denaturant (Figure 2F). ELISA analysis revealed the rapid development of antibodies to the alpha3NC1 monomer of collagen IV in patient serum over a period of 5 weeks (Figure 2G). Surprisingly, we did not detect IgM reactivity to any of collagen IV NC1 domains either prior or or at the onset of the anti-GBM disease (Figure 2H).
Patient-restricted coding variants within type IV collagen proteins. Domain architecture and relative locations of missense variants within COL4A3 (Uniprot: Q01955) and COL4A4 (Uniprot: P53420) proteins that differed between the patient and the cord-blood donor. No patient-restricted variants were observed in COL4A5. The black bars represent anti-GBM disease-associated EA-alpha3 and EB-alpha3 epitopes.
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NC1 domain (Figure 3) and would not be predicted to directly influence the quaternary structure of the native alpha345NC1 hexamer. No variants were detected which differed between donor and recipient, in the alpha3, alpha4 or alpha5NC1 domains.