Autoantibodies against laminin-521 are pathogenic in anti-glomerular basement membrane disease

Anti-glomerular basement membrane (anti-GBM) disease is an organ-specific autoimmune disorder characterized by autoantibodies against GBM components. Evidence from human inherited kidney diseases and animal models suggests that the α, β, and γ chains of laminin-521 are all essential for maintaining the glomerular filtration barrier. We previously demonstrated that laminin-521 is a novel autoantigen within the GBM and that autoantibodies to laminin-521 are present in about one-third of patients. In the present study, we investigated the pathogenicity of autoantibodies against laminin-521 with clinical and animal studies. Herein, a rare case of anti-GBM disease was reported with circulating autoantibodies binding to laminin-521 but not to the NC1 domains of α1-α5(IV) collagen. Immunoblot identified circulating IgG from this patient bound laminin α5 and γ1 chains. A decrease in antibody levels was associated with improved clinical presentation after plasmapheresis and immunosuppressive treatments. Furthermore, immunization with laminin-521 in female Wistar-Kyoto rats induced crescentic glomerulonephritis with linear IgG deposits along the GBM, complement activation along with infiltration of T cells and macrophages. Lung hemorrhage occurred in 75.0% of the rats and was identified by the presence of erythrocyte infiltrates and hemosiderin-laden macrophages in the lung tissue. Sera and kidney-eluted antibodies from rats immunized with laminin-521 demonstrated specific IgG binding to laminin-521 but not to human α3(IV)NC1, while the opposite was observed in human α3(IV)NC1-immunized rats. Thus, our patient data and animal studies imply a possible independent pathogenic role of autoantibodies against laminin-521 in the development of anti-GBM disease. Anti-glomerular basement membrane (anti-GBM) disease is an organ-specific autoimmune disorder characterized by autoantibodies against GBM components. Evidence from human inherited kidney diseases and animal models suggests that the α, β, and γ chains of laminin-521 are all essential for maintaining the glomerular filtration barrier. We previously demonstrated that laminin-521 is a novel autoantigen within the GBM and that autoantibodies to laminin-521 are present in about one-third of patients. In the present study, we investigated the pathogenicity of autoantibodies against laminin-521 with clinical and animal studies. Herein, a rare case of anti-GBM disease was reported with circulating autoantibodies binding to laminin-521 but not to the NC1 domains of α1-α5(IV) collagen. Immunoblot identified circulating IgG from this patient bound laminin α5 and γ1 chains. A decrease in antibody levels was associated with improved clinical presentation after plasmapheresis and immunosuppressive treatments. Furthermore, immunization with laminin-521 in female Wistar-Kyoto rats induced crescentic glomerulonephritis with linear IgG deposits along the GBM, complement activation along with infiltration of T cells and macrophages. Lung hemorrhage occurred in 75.0% of the rats and was identified by the presence of erythrocyte infiltrates and hemosiderin-laden macrophages in the lung tissue. Sera and kidney-eluted antibodies from rats immunized with laminin-521 demonstrated specific IgG binding to laminin-521 but not to human α3(IV)NC1, while the opposite was observed in human α3(IV)NC1-immunized rats. Thus, our patient data and animal studies imply a possible independent pathogenic role of autoantibodies against laminin-521 in the development of anti-GBM disease. Translational StatementLaminin-521 (LM521) has recently been identified as a novel autoantigen of anti–glomerular basement membrane (anti-GBM) disease. However, the pathogenicity of these antibodies has not been unequivocally proven because of co-occurrence of pathogenic antibodies against the noncollagenous domain of the α3 chain of type IV collagen (α3[IV]NC1). Herein, we reported a rare case of anti-GBM disease with circulating antibodies binding to LM521 but not to the NC1 domains of α1 to α5 chains of type IV collagen. We further demonstrated that immunization of LM521 in Wistar-Kyoto rats could induce anti-LM521 antibodies, resulting in anti-GBM nephritis and pulmonary hemorrhage without the involvement of anti-α3(IV)NC1 antibodies. Together, these findings demonstrate a pathogenic role of antibodies against LM521 in the development of anti-GBM disease. Future work is required to understand the underlying mechanism eliciting anti-LM521 antibody and to define the critical pathogenic epitopes. Laminin-521 (LM521) has recently been identified as a novel autoantigen of anti–glomerular basement membrane (anti-GBM) disease. However, the pathogenicity of these antibodies has not been unequivocally proven because of co-occurrence of pathogenic antibodies against the noncollagenous domain of the α3 chain of type IV collagen (α3[IV]NC1). Herein, we reported a rare case of anti-GBM disease with circulating antibodies binding to LM521 but not to the NC1 domains of α1 to α5 chains of type IV collagen. We further demonstrated that immunization of LM521 in Wistar-Kyoto rats could induce anti-LM521 antibodies, resulting in anti-GBM nephritis and pulmonary hemorrhage without the involvement of anti-α3(IV)NC1 antibodies. Together, these findings demonstrate a pathogenic role of antibodies against LM521 in the development of anti-GBM disease. Future work is required to understand the underlying mechanism eliciting anti-LM521 antibody and to define the critical pathogenic epitopes. Anti–glomerular basement membrane (anti-GBM) disease is an organ-specific autoimmune disease clinically characterized by rapidly progressive glomerulonephritis and an increased risk of pulmonary hemorrhage.1Cui Z. Zhao M.H. Advances in human antiglomerular basement membrane disease.Nat Rev Nephrol. 2011; 7: 697-705Crossref PubMed Scopus (67) Google Scholar In most patients, it is associated with anti-GBM antibodies against the noncollagenous domain of the α3 chain of type IV collagen, hereafter designated α3(IV)NC1. The diagnosis of anti-GBM disease was determined on serologic testing for the presence of anti-GBM antibodies and a kidney biopsy revealing linear IgG deposition along the GBM. Compelling evidence that circulating autoantibodies against α3(IV)NC1 are directly pathogenic has been confirmed in passive transfer studies,2Lerner R.A. Glassock R.J. Dixon F.J. The role of anti-glomerular basement membrane antibody in the pathogenesis of human glomerulonephritis.J Exp Med. 1967; 126: 989-1004Crossref PubMed Scopus (556) Google Scholar,3Kohda T. Okada S. Hayashi A. et al.High nephritogenicity of monoclonal antibodies belonging to IgG2a and IgG2b subclasses in rat anti-GBM nephritis.Kidney Int. 2004; 66: 177-186Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar and autoimmunity against α3(IV)NC1 is sufficient to induce anti-GBM nephritis in experimental models.4Sado Y. Boutaud A. Kagawa M. et al.Induction of anti-GBM nephritis in rats by recombinant alpha 3(IV)NC1 and alpha 4(IV)NC1 of type IV collagen.Kidney Int. 1998; 53: 664-671Abstract Full Text PDF PubMed Scopus (83) Google Scholar However, anti-GBM antibodies specific for α3(IV)NC1 cannot be detected by routine immunoassays in up to 10% of patients, for whom the diagnosis is established only by kidney biopsy.5Kidney Disease: Improving Global Outcomes (KDIGO) Glomerular Diseases Work GroupKDIGO 2021 clinical practice guideline for the management of glomerular diseases.Kidney Int. 2021; 100: S1-S276PubMed Scopus (654) Google Scholar The autoantigens targeted in these patients with anti-GBM disease remain largely unknown. Laminin-521 (LM521) is a newly identified target antigen in anti-GBM disease. We have previously demonstrated that autoantibodies against LM521 were detected in one-third of patients with anti-GBM disease, and they were associated with lung involvement.6Shen C.R. Jia X.Y. Luo W. et al.Laminin-521 is a novel target of autoantibodies associated with lung hemorrhage in anti-GBM disease.J Am Soc Nephrol. 2021; 32: 1887-1897Crossref PubMed Scopus (8) Google Scholar Besides type IV collagen, LM521 is another major component of mature GBM, composed of 3 chains, named α5, β2, and γ1. Evidence from human inherited kidney diseases and animal models suggests that the α, β, and γ chains of LM521 are all essential for maintaining the glomerular filtration barrier.7Miner J.H. Glomerular basement membrane composition and the filtration barrier.Pediatr Nephrol. 2011; 26: 1413-1417Crossref PubMed Scopus (99) Google Scholar In addition, passive transfer of maternal IgG alloantibodies against human laminin α5 chain induces perinatal anti-GBM disease in newborn mice transgenically expressing human LAMA5, but not in wild-type littermates.8Abrahamson D.R. Steenhard B.M. Stroganova L. et al.Maternal alloimmune IgG causes anti-glomerular basement membrane disease in perinatal transgenic mice that express human laminin α5.Kidney Int. 2019; 96: 1320-1331Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar However, whether an autoimmune response targeting LM521 could elicit anti-LM521 autoantibodies mediating anti-GBM nephritis is still unknown. In the present study, we identified a rare case of biopsy-proven anti-GBM nephritis in which serum antibodies bound to LM521, but not antibodies against the NC1 domains of α1 to α5 chains of type IV collagen, were detected. Nephritogenicity of anti-LM521 autoantibodies was further confirmed in an experimental rat model of anti-GBM disease induced by immunization with LM521. Taken together, these results provide the first evidence that autoantibodies against LM521 may be sufficient to mediate anti-GBM nephritis in vivo. Serum samples were obtained from a patient showing typical linear IgG deposition along GBM on kidney biopsy, but without anti-GBM antibodies detected by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Euroimmun). The absence of anti-α3(IV)NC1 antibodies was further confirmed by immunoblot analysis. The in-house ELISA for human α1(IV)NC1 to α5(IV)NC1 was also performed, as described previously, to exclude the presence of antibodies against other α chains of type IV collagen.9Zhao J. Cui Z. Yang R. et al.Anti-glomerular basement membrane autoantibodies against different target antigens are associated with disease severity.Kidney Int. 2009; 76: 1108-1115Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Serum anti-LM521 autoantibodies of the patient were detected by ELISA and immunoblot, as described previously.6Shen C.R. Jia X.Y. Luo W. et al.Laminin-521 is a novel target of autoantibodies associated with lung hemorrhage in anti-GBM disease.J Am Soc Nephrol. 2021; 32: 1887-1897Crossref PubMed Scopus (8) Google Scholar Briefly, recombinant human LM521 (rhLM521; BioLamina) was coated at 2 μg/ml for ELISA, and serum samples were diluted at 1:100. Serum samples from 30 healthy individuals were used as normal controls to build up a cutoff absorbance value as mean + 3 SDs. Serum samples from 60 patients with anti-GBM disease with previously reported undetectable anti-GBM antibodies were also analyzed.10Shen C.R. Jia X.Y. Cui Z. et al.Clinical-pathological features and outcome of atypical anti-glomerular basement membrane disease in a large single cohort.Front Immunol. 2020; 11: 2035Crossref PubMed Scopus (14) Google Scholar For immunoblot analysis, rhLM521 loaded in sample buffer at 4 μg/lane was electrophoresed in 6% sodium dodecylsulfate–polyacrylamide gel under reducing conditions and transferred to polyvinylidene fluoride membranes, which were then incubated with serum samples diluted at 1:50. Alkaline phosphatase–conjugated antihuman IgG (1:5000; Sigma Aldrich) was incubated for 60 minutes at room temperature, and IgG bound to the membrane was detected using the nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as a substrate. The recombinant proteins were produced as described previously.11Cui Z. Zhao M.H. Jia X.Y. et al.Antibodies to α5 chain of collagen IV are pathogenic in Goodpasture's disease.J Autoimmun. 2016; 70: 1-11Crossref PubMed Scopus (19) Google Scholar In brief, cDNA from the NC1 domain of human type IV collagen α1, α2, α3, α4, and α5 was ligated to a type X collagen triple-helix leader sequence and subcloned into the pcDNA3 vector. The constructs were then stably transfected into a human embryonic kidney cell line (HEK 293), and recombinant proteins were harvested and purified from the medium. Female Wistar-Kyoto (WKY) rats, aged 4–5 weeks, were purchased from Vital River Laboratories. Eight rats were immunized with either 0.4 μg/g of rhLM521 (BioLamina) or 0.4 μg/g of recombinant human α3(IV)NC1, both emulsified in complete Freund adjuvant (CFA; Sigma-Aldrich) by single footpad injection. Negative controls were immunized with CFA with phosphate-buffered saline (PBS). Experimental rats were killed at the end of week 7 after immunization. Blood, kidney, and lung tissues were collected from killed rats. All animal experiments were approved by the Experimental Animal Ethics Committee of Peking University First Hospital. Before and after immunization, 24-hour urine and blood samples were collected at each week. Urinary protein, serum creatinine, and blood urea nitrogen were analyzed by an automatic biochemical analyzer (UniCel DxC 600 Synchron; Beckman Coulter, Inc.). Kidney tissues were examined by immunofluorescence and light and electron microscopy. For immunofluorescence, frozen kidney sections (5 μm thick) were fixed in acetone and incubated with fluorescein isothiocyanate–conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories Inc.) at 1:50. For light microscopy, kidney tissues were fixed in 10% buffered formalin and embedded in paraffin. Kidney sections (3 μm thick) were stained with periodic acid–Schiff. At least 100 glomeruli per section were assessed. For electron microscopy, kidney tissues were fixed in 3% glutaraldehyde and then 1% osmium tetroxide, followed by dehydration in graded ethanol, and embedded in Epon 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (JEM-1230; JEOL).6Shen C.R. Jia X.Y. Luo W. et al.Laminin-521 is a novel target of autoantibodies associated with lung hemorrhage in anti-GBM disease.J Am Soc Nephrol. 2021; 32: 1887-1897Crossref PubMed Scopus (8) Google Scholar,12Shi Y. Jia X.Y. Gu Q.H. et al.A modified peptide derived from Goodpasture autoantigen arrested and attenuated kidney injuries in a rat model of anti-GBM glomerulonephritis.J Am Soc Nephrol. 2020; 31: 40-53Crossref PubMed Scopus (6) Google Scholar Lung tissues were examined by light microscopy. Lung sections (3 μm thick) were stained with hematoxylin and eosin to observe lung hemorrhage. Perls stain was used to identify hemosiderin-laden macrophages in the lung. At least 200 cells were counted at a magnification of ×400, and hemosiderin-laden macrophage index was reported as a percentage. Intrakidney CD4+ T cells, CD8+ T cells, forkhead box protein 3–expressing (foxp3)+ T cells, macrophage infiltrates, and C3d, C4d, and fibrin deposition were detected by immunohistochemistry, as previously described.12Shi Y. Jia X.Y. Gu Q.H. et al.A modified peptide derived from Goodpasture autoantigen arrested and attenuated kidney injuries in a rat model of anti-GBM glomerulonephritis.J Am Soc Nephrol. 2020; 31: 40-53Crossref PubMed Scopus (6) Google Scholar Paraffin-embedded, formalin-fixed kidney sections (3 μm thick) underwent either heat-mediated (for CD4, CD8, C4d, and foxp3) or enzyme-mediated (CD68 with 0.04% pepsin or C3d and fibrin with 0.4 mg/ml proteinase K) antigen retrieval. All sections were subjected to 3% H2O2 to block endogenous peroxidase and 3% bovine serum albumin to block unspecific binding sites. The incubation of monoclonal antibodies (anti-CD4 [Cell Signaling Technology] and anti-foxp3 [Abcam]) or polyclonal antibodies (anti-CD8 [Santa Cruz Biotechnology]; anti-CD68 [Abcam]; anti-fibrin [DAKO]; anti-C3d [R&D Systems]; and anti-C4d [Biomedica]) was performed overnight at 4 °C. Then, the staining was developed with 3,3΄-diaminobenzidine. At least 20 consecutive glomeruli per sample were assessed by Image-Pro Plus, version 6.0 (Media Cybernetics). CD4+ T cells, CD8+ T cells, foxp3+ cells, and macrophage (CD68+) infiltration were shown as cells per glomerular cross-section. C3d, C4d, and fibrin were displayed as integrated optical density per glomerular cross-section. For lung sections, cell infiltrates of CD4+ T cells, CD8+ T cells, and macrophages were detected by immunohistochemistry, as described above. Positive cells in the lung sections were quantified as the mean positive cells of 5 high-power fields per sample, using Image-Pro Plus. IgG deposited in kidneys was eluted at acidic pH, as previously described.13Hu S.Y. Gu Q.H. Wang J. et al.The pathogenicity of T cell epitopes on human Goodpasture antigen and its critical amino acid motif.J Cell Mol Med. 2017; 21: 2117-2128Crossref PubMed Scopus (8) Google Scholar In brief, the cortical portion of the kidney was incised with a scalpel, mixed with cold PBS, and homogenized with a homogenizer. The homogenate was spun at 2500g for 10 minutes at 4 °C. The sediments were washed 5 times by centrifugation with cold PBS. After the last wash, the precipitate was suspended in 5 times the volume of 0.1 mol/l glycine at pH 2.8 and incubated for 2 hours at 4 °C with constant shaking. The supernatant was removed and immediately adjusted to pH 7.0 with 2 mol/l Tris-HCl (pH 9.0) and dialyzed against several changes of PBS. The specificity of serum- and kidney-eluted antibodies toward rhLM521 and α3(IV)NC1 was analyzed by ELISA. rhLM521 (2 μg/ml) and α3(IV)NC1 (2 μg/ml) were coated overnight on the 96-well plates. Serum samples and kidney-eluted antibodies diluted at 1:100 in PBS with 0.1% Tween-20 were incubated in the plate for 1 hour at 37 °C. Then, alkaline phosphatase–conjugated goat anti-rat IgG (Sigma) diluted in PBS with 0.1% Tween-20 at 1:5000 was added at 37 °C for 1 hour. The p-nitrophenyl phosphate (1 mg/ml; Sigma) in substrate buffer (1 mol/L diethanolamine and 0.5 mmol/l MgCl2, pH 9.8) was used as a substrate, and color development was measured spectrophotometrically at 405 nm (Bio-Rad). Rat IgG subclasses against rhLM521 were detected by using goat anti-rat IgG1, IgG2a, IgG2b, and IgG2c monoclonal antibodies, and followed by horseradish peroxidase–labeled donkey anti-goat IgG, diluted 1:5000 in 0.1% PBS with 0.1% Tween-20. Horseradish peroxidase substrate 3,3΄,5,5΄-tetramethylbenzidine was added for color development. The plates were measured at 450 nm, as described above. Immunoblot analysis of serum antibodies against rhLM521 in WKY rats was performed as described above. Briefly, rhLM521 was electrophoresed on a 6% sodium dodecylsulfate–polyacrylamide gel under reducing conditions, and serum samples were diluted at 1:50. Data in the text were expressed as mean ± SEM or median (interquartile range), according to the distribution. Two-tailed unpaired 1-way analysis of variance was used to compare variables among 3 groups. For nonparametric variables, the Kruskal-Wallis test was used to compare variables among 3 groups. Differences were considered statistically significant when P < 0.05. SPSS, version 26.0 (SPSS Inc.), and Prism 8.0 software (GraphPad Software) were used for statistical analysis. A 37-year-old man was admitted to our hospital with bilateral lower limb edema for 3 weeks. He was a long-term cigarette smoker and did not quit before admission. Estimated glomerular filtration rate was 51.2 ml/min per 1.73 m2 on diagnosis. Serum creatinine was 134.3 μmol/l (normal range, 44–133 μmol/l) on diagnosis and progressed rapidly to 157.2 μmol/l in 1 week. Urinary sediment showed heavy microscopic hematuria (red blood cell count, 120–150 per high-power field). Serum albumin was 28.9 g/l, and 24-hour urinary protein was 11.13 g/24 h. Serologic tests were negative for anti-GBM antibodies against α3(IV)NC1 (using a commercial ELISA kit), anti-neutrophil cytoplasmic autoantibody, anti-nuclear antibodies, and anti–double-stranded DNA antibodies. A kidney biopsy was performed the day after admission. Kidney pathology showed crescentic glomerulonephritis with bright linear IgG deposition along the GBM, indicating anti-GBM nephritis (Figure 1a–e). Anti-GBM antibodies were further investigated using recombinant human α1-5(IV)NC1 as solid phase antigens by ELISA (Figure 1f), but came back all negative. However, serum anti-LM521 antibodies were tested positive (Figure 1h). The existence of autoantibodies against rhLM521 but not against α3(IV)NC1 was validated by immunoblot. Circulating IgG bound 2 subunits of ≈400 kDa (α5) and 200 kDa (γ1) of rhLM521 that are separated under reducing conditions (Figure 1g). No anti-LM521 antibodies were found in 60 patients with anti-GBM disease without detectable anti-α3(IV)NC1 antibodies by both immunoblot and ELISA (data not shown). Plasmapheresis was initiated on day 9 after admission and continued for 6 sessions, until the serum anti-LM521 IgG became undetectable. The patient was treated with oral prednisone (1 mg/kg per day and tapered gradually) and cyclophosphamide (1 mg/kg, orally once a day for 9 months). He has been followed up for 1 year on discharge. Kidney function has returned to normal range, and proteinuria has subsided. The clinical course and laboratory findings are summarized in Figure 1h. The development and nephritogenicity of anti-LM521 antibodies were investigated in WKY rats immunized with a single footpad injection of rhLM521 at 0.4 μg/g in CFA. All rhLM521-immunized rats (8/8 [100%]) developed proteinuria starting from week 3 after immunization (Figure 2a) and peaked at week 7 (145.7 ± 30.5 vs. 3.5 ± 0.6 mg/24 h in CFA-immunized control rats; P = 0.006; Figure 2d). Serum creatinine and blood urea nitrogen increased in rats immunized with rhLM521 (Figure 2b and c), also peaking at week 7 (blood urea nitrogen: 27.0 [interquartile range, 23.0–34.0] vs. 18.5 [interquartile range, 17.0–21.5] mmol/l; P = 0.038; serum creatinine: 31.5 [interquartile range, 23.4–38.5] vs. 26.1 [interquartile range, 19.4–28.5] μmol/l; P = 0.270; Figure 2e and f), compared with CFA-immunized control rats. Kidney injury was evaluated by immunofluorescence, light microscopy, and electron microscopy in rats killed at week 7 (Figure 3a). All rats immunized with rhLM521 exhibited linear IgG deposits along the GBM by direct immunofluorescence. Crescent formation was observed in all rhLM521-immunized rats, with an average percentage of 64.8% ± 21.3% in all glomeruli (Figure 3b), predominantly cellular crescents. Similar pathologic lesions were observed in recombinant human α3(IV)NC1-immunized rats, but none of the CFA-immunized rats showed any kind of kidney injury. The lung lesions were investigated by macroscopic pathologic observation, hematoxylin and eosin stain, and Perls stain (Figure 4a). Scattered bleeding spots were shown in the lungs of 6 to 8 rats (75%) immunized with rhLM521. Alveolar hemorrhage was identified by the presence of erythrocyte infiltrates (Figure 4a) and hemosiderin-laden macrophages by Perls stain in the lung tissues (Figure 4a and b). Lung hemorrhage was also observed in the recombinant human α3(IV)NC1-immunized rats, but not in CFA-immunized control rats. The intralung infiltrates of CD4+ T cells (29.7 ± 2.2 vs. 21.1 ± 1.8; P = 0.022; Figure 4c), CD8+ T cells (36.7 ± 1.8 vs. 24.2 ± 1.3; P < 0.001; Figure 4d), and macrophages (29.4 ± 2.1 vs. 19.1 ± 1.9; P = 0.007; Figure 4e) were significantly elevated in the rats immunized with rhLM521, compared with CFA-immunized control rats. Antibody responses in serum samples and kidney eluates of rhLM521-immunized rats were detected by ELISA using rhLM521 and recombinant human α3(IV)NC1 as solid-phase ligands (Figure 5a–f). Rats immunized with rhLM521 developed a high level of serum antibodies against rhLM521 by week 1, peaked at week 2, and remained at high levels thereafter. No serum samples recognized α3(IV)NC1 in rats immunized with rhLM521. IgG of kidney elutes from rhLM521-immunized rats bound to rhLM521, but not to human α3(IV)NC1, and vice versa. Antigen specificity of serum antibodies in rhLM521-immunized rats was confirmed by immunoblot analyses. Serum IgG from rats immunized with rhLM521 bound all 3 bands (α5, β2, and γ1 chains) of LM521 under reducing conditions (Figure 5j). The IgG subclass distribution against rhLM521 from serum samples and kidney elutes was further analyzed by ELISA. In rhLM521-immunized rats, the anti-rhLM521 IgG subclass includes IgG1, IgG2a, IgG2b, and IgG2c, with the lowest level of IgG2c in both serum samples and kidney elutes (Figure 5g–i). No IgG subclass recognized α3(IV)NC1 in rhLM521 rats. The intrakidney inflammatory responses were assessed by immunohistopathologic analyses (Figure 6a). The significant infiltrates of CD4+ T cells (2.2 ± 0.2 vs. 0.3 ± 0.1; P < 0.001; Figure 6b), CD8+ T cells (4.0 ± 0.4 vs. 0.2 ± 0.1; P < 0.001; Figure 6c), and macrophages (5.4 ± 0.3 vs. 0.04 ± 0.03; P < 0.001; Figure 6e) were seen in rhLM521-immunized rats, compared with those of CFA-immunized rats. Foxp3+ T cells were decreased in the kidney tissues of rhLM521-immunized rats, compared with CFA controls (0.30 [interquartile range, 0.1–0.9] vs. 2.9 [interquartile range, 2.6–3.2]; P = 0.001; Figure 6d). Complement deposition of C3d and C4d along the GBM was also significantly higher in rhLM521-immunized rats (Figure 6f and g). The discovery of pathogenic anti-α3(IV)NC1 antibodies was a milestone in delineation of disease cause and diagnosis of anti-GBM disease. However, the GBM is composed of numerous other components besides type IV collagen, potentially autoantigenic. Previous studies reported autoantibodies targeting α5(IV)NC1,11Cui Z. Zhao M.H. Jia X.Y. et al.Antibodies to α5 chain of collagen IV are pathogenic in Goodpasture's disease.J Autoimmun. 2016; 70: 1-11Crossref PubMed Scopus (19) Google Scholar peroxidasin,14McCall A.S. Bhave G. Pedchenko V. et al.Inhibitory anti-peroxidasin antibodies in pulmonary-renal syndromes.J Am Soc Nephrol. 2018; 29: 2619-2625Crossref PubMed Scopus (20) Google Scholar and other molecules, such as α1/α2(IV) collagen chains15Borza D.B. Chedid M.F. Colon S. et al.Recurrent Goodpasture's disease secondary to a monoclonal IgA1-kappa antibody autoreactive with the alpha1/alpha2 chains of type IV collagen.Am J Kidney Dis. 2005; 45: 397-406Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and entactin16Saxena R. Bygren P. Arvastson B. et al.Circulating autoantibodies as serological markers in the differential diagnosis of pulmonary renal syndrome.J Intern Med. 1995; 238: 143-152Crossref PubMed Scopus (64) Google Scholar in sporadic cases. Whether autoantibodies against GBM antigens other than collagen IV could mediate anti-GBM disease remains an enigma. We have recently identified LM521 as a novel GBM autoantigen in patients with anti-GBM disease. Distinguished from the above “atypical” antigens, anti-LM521 antibodies are present in about one-third of patients with anti-GBM disease and significantly associated with lung involvement. However, the pathogenicity of anti-LM521 antibodies could not be unequivocally proven because of the coexistence of anti-α3(IV)NC1 antibodies in our previous cohort.6Shen C.R. Jia X.Y. Luo W. et al.Laminin-521 is a novel target of autoantibodies associated with lung hemorrhage in anti-GBM disease.J Am Soc Nephrol. 2021; 32: 1887-1897Crossref PubMed Scopus (8) Google Scholar To the best of our knowledge, the current study presented the first case of anti-GBM nephritis whose serum contained anti-LM521 antibodies without the concomitant presence of anti-α3(IV)NC1 antibodies. Moreover, immunization with LM521 was sufficient to induce crescentic nephritis and lung hemorrhage in WKY rats without the production of antibodies against α3(IV)NC1, providing further independent evidence of the nephritogenicity of anti-LM521 antibodies. The patient described in this study presented with typical crescentic nephritis with bright linear IgG deposition along the GBM. A thorough immunologic investigation confirmed the serum autoantibodies were directed to LM521 in the absence of antibodies against α3(IV)NC1 or any other α chains of type IV collagen. After plasma exchange and immunosuppressive treatments, serum creatinine decreased in parallel with the levels of anti-LM521 antibody titers, indicating a possible pathogenic role of anti-LM521 antibodies without the involvement of anti-α3(IV)NC1 antibodies. The nephritogenicity of anti-LM521 antibodies was further demonstrated by immunizing WKY rats with rhLM521. All rats immunized with rhLM521 developed kidney injury, including proteinuria and elevated levels of blood urea nitrogen and serum creatinine, as well as concurrent lung hemorrhage. Kidney pathology showed typical linear IgG deposits and crescentic glomerulonephritis. The clinical spectrum and pathologic features were in accordance with the phenotype of human anti-GBM disease. In parallel, antibodies elicited by the immunization of rhLM521 were directed exclusively against LM521. No antibodies against α3(IV)NC1 were detected in either the circulation or the kidney tissues of immunized rats. These findings provide the first direct evidence that autoimmune responses to LM521 could induce anti-GBM glomerulonephritis. In line with our previous findings that anti-LM521 antibodies comprised mainly IgG1 and IgG4 subclasses in human anti-GBM disease,6Shen C.R. Jia X.Y. Luo W. et al.Laminin-521 is a novel target of autoantibodies associated with lung hemorrhage in anti-GBM disease.J Am Soc Nephrol. 2021; 32: 1887-1897Crossref PubMed Scopus (8) Google Scholar the current studies showed that rats immunized with rhLM521 had high levels of IgG1, IgG2a, and IgG2b and low levels of IgG2c against rhLM521 in both serum samples and kidney elutes. Human IgG1 and rat IgG1, IgG2a, and IgG2b could cause inflammation through complement activation and binding of IgG Fcγ receptors on macrophages. Both effector mechanisms appear to be essential for the development of anti-GBM disease.17Ma R. Cui Z. Liao Y.H. et al.Complement activation contributes to the injury and outcome of kidney in human anti-glomerular basement membrane disease.J Clin Immunol. 2013; 33: 172-178Crossref PubMed Scopus (38) Google Scholar, 18Suzuki Y. Shirato I. Okumura K. et al.Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 19Otten M.A. Groeneveld T.W. Flierman R. et al.Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.J Immunol. 2009; 183: 3980-3988Crossref PubMed Scopus (37) Google Scholar In contrast, human IgG4 and rat Ig2c are probably noninflammatory.20Vidarsson G. Dekkers G. Rispens T. IgG subclasses and allotypes: from structure to effector functions.Front Immunol. 2014; 5: 520Crossref PubMed Scopus (1583) Google Scholar, 21Boltz-Nitulescu G. Bazin H. Spiegelberg H.L. Specificity of fc receptors for IgG2a, IgG1/IgG2b, and IgE on rat macrophages.J Exp Med. 1981; 154: 374-384Crossref PubMed Scopus (61) Google Scholar, 22Medgyesi G.A. Miklós K. Kulics J. et al.Classes and subclasses of rat antibodies: reaction with the antigen and interaction of the complex with the complement system.Immunology. 1981; 43: 171-176PubMed Google Scholar Our current results identified complement deposits along GBM and severe infiltration of macrophages in glomeruli. These findings suggest that anti-LM521 antibodies may exert a pathogenic role in glomeruli by both antibody-dependent cell-mediated cytotoxicity and a complement-dependent cytotoxicity mechanism, leading to severe inflammatory reactions, fracture of GBM, and crescent formation. As an important extracellular matrix component, laminin has been implicated in a broad spectrum of inherited and acquired human diseases.23Florea F. Koch M. Hashimoto T. et al.Autoimmunity against laminins.Clin Immunol. 2016; 170: 39-52Crossref PubMed Scopus (12) Google Scholar For example, a point mutation in LAMB5, which encodes the laminin α5 chain, resulted in the development of severe proteinuria, also revealing the pathogenic role of LM521 dysfunction.24Falcone S. Nicol T. Blease A. et al.A novel model of nephrotic syndrome results from a point mutation in Lama5 and is modified by genetic background.Kidney Int. 2022; 101: 527-540Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Similarly, mutations in LAMB2 that encodes the β2 chain within LM521 subunits trigger Pierson syndrome, clinically characterized by nephrotic syndrome and eventually end-stage kidney disease.25Funk S.D. Lin M.H. Miner J.H. Alport syndrome and Pierson syndrome: diseases of the glomerular basement membrane.Matrix Biol. 2018; 71–72: 250-261Crossref PubMed Scopus (76) Google Scholar Treatment with exogenous LM521 successfully ameliorates the kidney function in an animal model of Pierson syndrome, emphasizing the importance of LM521 for maintaining the stability of the glomerular filtration barrier.26Lin M.H. Miller J.B. Kikkawa Y. et al.Laminin-521 protein therapy for glomerular basement membrane and podocyte abnormalities in a model of Pierson syndrome.J Am Soc Nephrol. 2018; 29: 1426-1436Crossref PubMed Scopus (27) Google Scholar The laminin 511-E8 fragment has been identified as a target autoantigen in human autoimmune pancreatitis.27Shiokawa M. Kodama Y. Sekiguchi K. et al.Laminin 511 is a target antigen in autoimmune pancreatitis.Sci Transl Med. 2018; 10eaaq0997Crossref PubMed Scopus (135) Google Scholar The laminin γ1 chain is 1 of the autoantigens recognized by the patients with anti-p200 pemphigoid, a subepidermal blistering skin autoimmune disease.28Dainichi T. Kurono S. Ohyama B. et al.Anti-laminin gamma-1 pemphigoid.Proc Natl Acad Sci U S A. 2009; 106: 2800-2805Crossref PubMed Scopus (143) Google Scholar We note that circulating antibodies recognizing laminin γ1 chain were also present in our current case. Intriguingly, concurrence of anti-GBM disease and pemphigoid has been reported previously.29Plaisier E. Borradori L. Hellmark T. et al.Anti-glomerular basement membrane nephritis and bullous pemphigoid caused by distinct anti-alpha 3(IV)NC1 and anti-BP180 antibodies in a patient with Crohn's disease.Am J Kidney Dis. 2002; 40: 649-654Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,30Hoorn E.J. Taams N.E. Hurskainen T. et al.Bullous pemphigoid with a dual pattern of glomerular immune complex disease.Am J Kidney Dis. 2016; 67: 302-306Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar It remains unclear whether such a complication is due to the presence of autoantibodies targeting a single autoantigen in different basement membranes. However, our case may hint at the possibility of concurrent anti-GBM disease when kidney injury occurs in patients with pemphigoid, and vice versa. Furthermore, the critical nephritogenic subunits of LM521 merit future investigation. In contrast to classic anti-GBM disease, a spectrum of patients with anti-GBM disease with undetectable anti-GBM antibodies by classic immunoassays have been well documented in recent years.10Shen C.R. Jia X.Y. Cui Z. et al.Clinical-pathological features and outcome of atypical anti-glomerular basement membrane disease in a large single cohort.Front Immunol. 2020; 11: 2035Crossref PubMed Scopus (14) Google Scholar,31Nasr S.H. Collins A.B. Alexander M.P. et al.The clinicopathologic characteristics and outcome of atypical anti-glomerular basement membrane nephritis.Kidney Int. 2016; 89: 897-908Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar In our rat model, rats immunized by LM521 developed crescentic nephritis without the involvement of anti-α3(IV)NC1 antibodies, indicating a possible role of independent nephrogenicity of anti-LM521 antibodies. Thus, the investigation of anti-LM521 antibodies in patients with anti-GBM disease without anti-α3 antibodies would provide further support. However, we did not find more patients positive for anti-LM521 antibodies in our previous cohort.10Shen C.R. Jia X.Y. Cui Z. et al.Clinical-pathological features and outcome of atypical anti-glomerular basement membrane disease in a large single cohort.Front Immunol. 2020; 11: 2035Crossref PubMed Scopus (14) Google Scholar LM521 exerts a crucial role in maintaining the intact structure of the glomerular barrier, as described above. Coincidently, the patient described here also presented prominent nephrotic syndrome atypical for rapidly progressive glomerulonephritis. Nephrotic syndrome is not a common feature among patients with anti-GBM disease without combined membranous nephropathy.32Jia X.Y. Hu S.Y. Chen J.L. et al.The clinical and immunological features of patients with combined anti-glomerular basement membrane disease and membranous nephropathy.Kidney Int. 2014; 85: 945-952Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar,33Ahmad S.B. Santoriello D. Canetta P. et al.Concurrent anti-glomerular basement membrane antibody disease and membranous nephropathy: a case series.Am J Kidney Dis. 2021; 78: 219-225.e1Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Therefore, we speculated that anti-LM521 antibodies might cause injury to GBM structure, resulting in nephrotic syndrome in this patient. The clinical associations of anti-LM521 antibodies in patients fitting this profile merit future work. Although our previous study reported an association between anti-LM521 antibodies and pulmonary hemorrhage,6Shen C.R. Jia X.Y. Luo W. et al.Laminin-521 is a novel target of autoantibodies associated with lung hemorrhage in anti-GBM disease.J Am Soc Nephrol. 2021; 32: 1887-1897Crossref PubMed Scopus (8) Google Scholar the current patient did not present with that clinical manifestation despite a long history of smoking. There might be several reasons why the current patient did not develop pulmonary hemorrhage. First, the patient was diagnosed early, and treatment was initiated immediately on an increase in serum creatinine, which may have preempted the development of pulmonary hemorrhage. Second, our previous study indicated that patients with anti-GBM disease with dual positivity of anti-LM521 antibodies and anti-α3(IV)NC1 antibodies were more inclined to develop pulmonary hemorrhage. Although the current patient presented antibodies against LM521 alone, there was a probability that pulmonary hemorrhage might be diminished without the copresence of anti-α3(IV)NC1 antibodies. In summary, the present study documented a case of rapidly progressive glomerulonephritis possessing anti-GBM antibodies targeting LM521, but not the α3(IV)NC1 domain of type IV collagen. The immunization of LM521 could induce crescentic glomerulonephritis and lung hemorrhage in WKY rats, fully resembling the clinical profile of anti-GBM disease. Taken together, these results indicate a possible pathogenic role of anti-LM521 antibodies for anti-GBM disease. D-BB reports receiving honorarium fees from Novo-Nordisk. All the other authors declared no competing interests. There are no data to declare in our article. The technical support by Guo-sheng Yang and Miao Wang was greatly appreciated. This work was supported by the National Natural Science Foundation of China (82090020 to M-hZ, 82070732 to ZC, 82270763 to X-yJ, 82000666 to MT, and 82200789 to C-rS), the Beijing Municipal Science and Technology Commission Foundation (Z221100007922041 to ZC), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-046), and the National Heart, Lung, and Blood Institute of the National Institutes of Health (R21HL168484 to D-BB). In This IssueKidney InternationalVol. 104Issue 6PreviewHorne et al. examined the progression of acute kidney injury (AKI) to chronic kidney disease (CKD) by prospectively following >800 hospitalized individuals who either never had AKI or had ≥1 episode of AKI. These patients were matched for age, kidney function, and diabetes. After 5 years, kidney disease progression occurred in 30% of the AKI group compared with 7% of the no-AKI group (adjusted odds ratio, 2.49). After an initial episode of AKI, future episodes were more common, with an odds ratio of 2.71, and contributed additively to the risk of kidney function decline. Full-Text PDF Laminin-521: a novel target for pathogenic autoantibodies in anti–glomerular basement membrane diseaseKidney InternationalVol. 104Issue 6PreviewAnti–glomerular basement membrane (anti-GBM) disease is typically characterized by autoimmunity against the α3 chain of type IV collagen. Rarely, circulating autoantibodies are not detected. These atypical cases follow a more indolent clinical course, and underlying mechanisms, including alternative target antigens, require investigation. In this issue of Kidney International, Kuang et al. describe a case of anti-GBM disease with autoantibodies against the GBM component laminin-521 and demonstrate that laminin-521 is pathogenic in a rat model of anti-GBM glomerulonephritis. Full-Text PDF