Possible Implication of Intermolecular Epitope Spreading in the Production of Anti-Glomerular Basement Membrane Antibody in Anti-Neutrophil Cytoplasmic Antibody-associated Vasculitis

Objective Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is sometimes complicated by anti-glomerular basement membrane (GBM) disease. Proteases, including elastase, released from neutrophils activated by ANCA are implicated in the pathogenesis of AAV. Epitopes of anti-GBM antibody exist in the α3-subunit non-collagenous (NC1) domain of collagen type IV [Col (IV)]. This region, called α3(IV)NC1, is structurally cryptic. This study aimed to determine the production mechanism of anti-GBM antibody in AAV. Methods We rst examined whether α3(IV)NC1 could be revealed by the digestion of formalin-xed, paran-embedded (FFPE) normal kidney sections and Col (IV) by proteases, including neutrophil elastase, using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Next, the reveal of α3(IV)NC1 and the inltration of CD11c + macrophages in the affected kidneys were evaluated by IHC and immunouorescent staining using FFPE sections. Finally, the production of anti-GBM antibody in AAV rats was determined by ELISA. GBM epitopes, and then the host’s immune system produce anti-GBM antibody. inactivate the proteases. Recombinant human α3(IV)NC1 (1.25 µg/ml) as a positive control (PC) and the samples were subjected to ELISA to detect α3(IV)NC1. The optical density of the PC was set as 100, and the relative values of samples (% reactivity) were calculated. Experiments were repeated three times independently. Each experiment was conducted in duplicate. *p<0.05; n.s., not signicant.


Introduction
Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic small vessel vasculitis accompanied by the presence of ANCA in the serum [1]. This disease entity includes microscopic polyangiitis (MPA), granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis. The major antigens of ANCA are myeloperoxidase (MPO) and proteinase 3. The ANCAmediated activation of neutrophils is critically implicated in the pathogenesis of AAV [2], and the neutrophils activated by ANCA can release proteases, including elastase. Antigen retrieval from FFPE normal kidney sections FFPE normal kidney sections were cut into 4 µm sections, mounted on silane-coated glass slides, depara nized with xylene, and then autoclaved at 121℃ under acidic condition (0.2 M HCl, pH 0.9, 6 min), neutral condition (citrate buffer, pH 6.0, 10 min), or alkaline condition (Tris-EDTA, pH 9.0, 20 min). Sections that did not undergo heating were used as controls. In other experiments, FFPE normal kidney sections were depara nized with xylene, heated under neutral condition, and then exposed to proteases derived from S. griseus (0.5 mg/ml) for 30 min at 37℃. After rinsing in running tap water for 5 min, the sections were subjected to IHC for α3(IV)NC1.
IHC for α3(IV)NC1 using FFPE normal kidney sections After antigen retrieval, the sections were exposed to 3% hydrogen peroxide (H 2 O 2 )-methanol to inactivate endogenous peroxidase in the tissues followed by blocking the non-speci c binding of antibodies and then allowed to react with rat anti-α3(IV)NC1 mAb (1:1000) for 1 h at room temperature (RT). After rinsing with phosphate-buffered saline (PBS), the sections were next made to react with the HRP-conjugated goat anti-rat IgG2a antibody (1:1000) for 1 h at RT. After rinsing with PBS, the HRP activity on tissue sections was detected by 3,3¢-diaminobenzidine (DAB) substrates followed by counterstaining with Mayer's hematoxylin dye. Detection of α3(IV)NC1 and macrophages in FFPE kidney sections of patients with renal disease by IHC FFPE kidney sections of patients with renal disease were autoclaved for 20 min at 121℃ in Tris-EDTA (pH 9.0) to retrieve the antigens. Thereafter, the sections were exposed to 3% H 2 O 2 -methanol to inactivate endogenous peroxidase in the tissues followed by blocking the non-speci c binding of antibodies and then allowed to react with rat anti-α3(IV)NC1 mAb (1:1000) for 1 h at RT. As a control, isotype rat IgG2a (MBL, Nagoya, Japan) was used. After rinsing with PBS, the sections were next made to react with the HRP-conjugated goat anti-rat IgG2a antibody (1:1000) for 1 h at RT. After rinsing with PBS, the HRP activity on tissue sections was detected by DAB substrates followed by counterstaining with Mayer's hematoxylin dye.
In other experiments, the sections stained for α3(IV)NC1 were further allowed to react with rabbit anti-CD11c pAb (1:500) for 1 h at RT before counterstaining. After rinsing with PBS, the sections were next made to react with alkaline phosphatase (AP)-polymer reagents (Dako) for 1 h at RT. After rinsing with PBS, the AP activity on tissue sections was detected by fuchsin chromogen followed by counterstaining with Mayer's hematoxylin dye.
Alternatively, FFPE kidney sections of patients with renal disease were autoclaved for 20 min at 121℃ in citrate buffer (pH 6.0) to retrieve the antigens. Thereafter, the sections were exposed to 3% H 2 O 2 -methanol to inactivate endogenous peroxidase in the tissues followed by blocking the non-speci c binding of antibodies and then allowed to react with mouse anti-CD68 mAb (1:50) for 1 h at RT. After rinsing with PBS, the sections were next made to react with the HRP-conjugated goat anti-mouse IgG antibody (1:100) for 1 h at RT. After rinsing with PBS, the HRP activity on tissue sections was detected by DAB substrates followed by counterstaining with Mayer's hematoxylin dye.
Detection of α3(IV)NC1 and macrophages in FFPE kidney sections of patients with renal disease by immuno uorescence staining FFPE kidney sections of patients were autoclaved for 20 min at 121℃ in Tris-EDTA (pH 9) to retrieve the antigens. Thereafter, the sections were subjected to blocking of the non-speci c antibody binding and then allowed to react with rabbit anti-CD11c pAb (1:500) overnight at 4℃. After rinsing with PBS, the sections were subsequently made to react with Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:1000) for 1 h at RT. The sections were next allowed to react with mouse anti-CD68 mAb (1:50) for 1 h at RT. After rinsing with PBS, the sections were subsequently made to react with Alexa Fluor 488conjugated goat anti-mouse IgG antibody (1:500) for 1 h at RT followed by mounting using a solution containing 4¢,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).

Making of AAV model rats
According to Little's protocol [13], WKY rats (4 weeks old, male) were immunized with human native MPO (1600 μg/kg) on day 0 and given an intraperitoneal injection of pertussis toxin (800 ng) on days 0 and 2 (Group 1, original protocol, n=11). Because the administration of PMA has been shown to exacerbate renal disorder in other AAV model rats [14], PMA was employed to modify the original protocol. Materially, WKY rats immunized with human MPO were further given an intraperitoneal injection of PMA (1 μg) on days 7, 14, 21, 28, and 35 (Group 2, modi ed protocol, n=12). For controls, rats were similarly immunized with or without recombinant human MPO light chain, which has been shown to induce non-pathogenic ANCA [15].
Urinalysis and serological and histopathological examinations of AAV model rats All rats were euthanized on day 42 for blood and tissue sampling. Chronological blood sampling by tail cut was carried out on days 0, 14, and 28. Urine samples were collected using a metabolic cage on day 40. Hematuria was assessed at urine sampling immediately by a dipstick (Siemens Healthineers, Erlangen, Germany). The serum was separated by centrifugation. ANCA was detected quantitatively using ow cytometry (FCM). In brief, human peripheral blood neutrophils were xed with 4% paraformaldehyde, and then the plasma membrane of neutrophils was penetrated using Permeabilization Wash Buffer (BioLegend, San Diego, CA, USA). Cells (1×10 6 /ml) were allowed to react with 1:500 diluted rat sera for 30 min at RT. After washing with the buffer, cells were next made to react with 4 μg/ml FITC-conjugated rabbit anti-rat IgG antibody for 30 min at RT. After washing with the buffer, cells were subjected to FCM using an Attune Acoustic Focusing Cytometer (Applied Biosystems, Foster City, CA, USA). Serum Creactive protein (CRP) levels at the time of death were determined by a commercial laboratory service (Daiichi Kishimoto Clinical Laboratory, Sapporo, Japan).
FFPE tissues of the systemic organs were cut into 4 μm sections, depara nized with xylene, and then subjected to hematoxylin and eosin (HE) staining. Erythrocyte casts were counted in the maximum longitudinal section of the kidney specimen.

Detection of anti-GBM antibody in rat sera
Wells of 96-well plates were coated with 2.5 µg/ml recombinant human α3(IV)NC1 that exhibits a high homology with the rat counterpart (50 µl/well) overnight at 4℃ and then washed four times with PBS-T and soaked in PBS-T containing 1% skim milk (150 µl/well) for 1 h at RT to block the non-speci c antibody binding. After the removal of the blocking solution, 1:10 diluted rat sera (50 µl/well) were added into the wells and then incubated for 1 h at RT. After washing with PBS-T, the wells were next incubated with 1:10,000 diluted HRP-conjugated goat anti-rat IgG antibody (50 µl/well) for 1 h at RT. After washing with PBS-T, TMB was added into the wells. After 30 min incubation at RT, 1 M H 2 SO 4 was added to stop the reaction. Absorbance at 450 nm was measured using Multiskan FC. To determine IgG subclass of anti-GBM antibody, 1:20,000 diluted HRP-conjugated anti-rat IgG1, IgG2a, and IgG2b antibodies were used as secondary antibodies.
IHC for rat tissues FFPE kidney sections of normal rats were cut into 4 µm sections, mounted on silane-coated glass slides, depara nized with xylene, and then heated using a water bath for 20 min at 95℃ in citrate buffer (pH 6.0) or autoclaved for 6 min at 105℃ in 0.2 M HCl (pH 0.9). These sections were subjected to IHC for α3(IV)NC1. FFPE kidney sections of AAV rats were depara nized with xylene and then heated using a water bath for 20 min at 95℃ in citrate buffer (pH 6.0) to retrieve α3(IV)NC1 and CD11c or Tris-EDTA (pH 9.0) to retrieve CD68. Thereafter, the sections were exposed to 3% H 2 O 2 -methanol to inactivate endogenous peroxidase in the tissues followed by blocking the non-speci c binding of antibodies and then allowed to react with mouse anti-tumstatin, α3(IV)NC1 mAb (1 µg/ml), rabbit anti-CD11c pAb (1:100), or mouse anti-CD68 mAb (1:50) for 1 h at RT. These anti-human antibodies have been con rmed to react with each rat counterpart. After rinsing with PBS, the sections were next made to react with the HRP-conjugated goat anti-mouse IgG antibody (1:100) or the HRP-conjugated goat anti-rabbit IgG antibody (1:500) for 1 h at RT. After rinsing with PBS, the HRP activity on tissue sections was detected by DAB substrates followed by counterstaining with Mayer's hematoxylin dye.

Statistics
Student's t-test and Mann-Whitney U-test were applied for comparison between parametric and nonparametric groups, respectively. p<0.05 was regarded as statistically signi cant.

Results
Retrieval of α3(IV)NC1 from FFPE normal kidney sections by protease digestion in IHC First, the retrieval of α3(IV)NC1 from FFPE normal kidney sections in IHC was examined using anti-α3(IV)NC1 mAb. This mAb recognizes a cryptic IPSTVKA amino acid sequence in the α3(IV)NC1 region.
Although the anti-α3(IV)NC1 mAb did not bind to the epitope without antigen retrieval (Fig. 1A), a clear reaction with GBM and the basement membrane of Bowman's capsule that connects to the glomerulus was observed when the sections were heated under acidic condition (pH 0.9; Fig. 1B). This linear staining pattern was consistent with the characteristic distribution of GBM antigens that was recognized by the sera of patients with anti-GBM disease [16]. In contrast, no reaction appeared when the sections were heated under neutral condition (pH 6.0; Fig. 1C) or alkaline condition (pH 9.0; Fig. 1D). Interestingly, the anti-α3(IV)NC1 mAb bound to GBM in FFPE normal kidney sections when the sections were exposed to proteases derived from S. griseus after heating under neutral condition (Fig. 1E). Although the nuclear staining by hematoxylin in Fig. 1B was weaker than that in Fig. 1E, Col (IV) was stained equivalently between the two. These ndings corresponded to previous reports [10,12] and suggested that proteases can digest Col (IV), resulting in the reveal of the cryptic α3(IV)NC1.

Reveal of α3(IV)NC1 by protease digestion of Col (IV)
Protease treatment that aims to digest formalin-mediated protein bridges is one of the common methods for antigen retrieval in IHC. To determine that the proteases derived from S. griseus digested not only formalin-mediated protein bridges but also Col (IV) in FFPE normal kidney sections and consequently revealed α3(IV)NC1, whether α3(IV)NC1 could be revealed by the protease digestion of Col (IV) was assessed by ELISA. Materially, Col (IV) was digested by diverse concentrations of proteases, and then the samples were subjected to ELISA for α3(IV)NC1 detection. Results demonstrated that α3(IV)NC1 was detected when Col (IV) was digested by an appropriate concentration (125 μg/ml) of the proteases derived from S. griseus (Fig. 1F). Because the proteases derived from S. griseus possess at least three caseinolytic activities and one aminopeptidase activity, it seems likely that α3(IV)NC1 itself was digested at concentrations higher than 125 μg/ml. Next, a similar examination was carried out using a neutrophilspeci c protease, NE, instead of the S. griseus-derived general proteases. The reveal of α3(IV)NC1 was also observed when Col (IV) was digested by NE in a dose-dependent manner (at a concentration of ³500 μU/ml; Fig. 1G). These ndings suggested that proteases, including NE, can digest Col (IV) and result in the reveal of α3(IV)NC1.
Reactivity of anti-α3(IV)NC1 antibody with GBM in FFPE kidney sections of patients It was hypothesized that, in AAV, proteases, including NE, derived from neutrophils activated by ANCA could digest Col (IV) of GBM. To elucidate this hypothesis, whether the anti-α3(IV)NC1 mAb could react with GBM in FFPE kidney sections of patients with MPA was assessed by IHC. As a histopathological feature of MPA, glomeruli showed diverse remarks from unaffected to global sclerosis. As shown in Fig.  2A, any speci c binding of the mAb to unaffected glomeruli did not appear when the sections were heated under alkaline condition and not treated by proteases. In contrast, weak binding of the mAb to GBM was observed in crescentic glomeruli of MPA without any particular antigen retrieval (Fig. 2B). Weak binding of the mAb to GBM was also observed in crescentic glomeruli of IgA nephritis (IgAN; Fig. 2C). Moreover, an obvious binding of the anti-α3(IV)NC1 mAb was observed in the sclerotic glomeruli of MPA (Fig. 2D), IgAN (Fig. 2E), and hypertensive arteriosclerosis (Fig. 2F). Although the typical linear staining of GBM was blurred on the destructed glomeruli, the speci city of the antibody binding was guaranteed because there was no background staining when isotype control rat IgG2a was used as the primary antibody (Figs. 2B and 2D, inset). Although these ndings are consistent with the authors' hypothesis in part, the reveal of α3(IV)NC1 in affected glomeruli appears not only in MPA but also in other renal disorders.

Distribution of CD11c + macrophages
Although the reveal of α3(IV)NC1 in destructed glomeruli was not speci c to AAV, the secondary production of anti-GBM antibody rarely occurs in diseases other than AAV. Therefore, the focus was next set on antigen-presenting cells (APCs). It has been shown that tissue-in ltrating CD11c + macrophages play roles in antigen presentation [17]. IF staining demonstrated the presence of cell-surface CD11c + and cytoplasmic CD68 + macrophages in glomeruli, Bowman's capsules, and renal tubules in MPA (Fig. 3A). In contrast, in ltration of CD11c + macrophages was not apparent in kidney tissues of patients with IgAN and hypertensive arteriosclerosis (Figs. 3B-3D), although there were a certain amount of CD68 + macrophages, which indicates active state of the diseases (Figs. 3B, 3C, and 3E).
Establishment of AAV rat model with exacerbation of renal tissue damage Although WKY rats immunized with human native MPO are useful models of AAV [13], the renal manifestation is mild compared to typical MPA in humans. To make the renal disorder worse in this model, the original protocol was modi ed using PMA based on the authors' previous study, demonstrating that PMA administration exacerbated renal disorders in the drug-induced AAV rat model [14]. WKY rats (n=23) were immunized with human MPO according to the original protocol [13] and then divided into two groups, namely, Group 1 without PMA administration (original protocol, n=11) and Group 2 with PMA administration (modi ed protocol, n=12). Group 2 rats were given an intraperitoneal injection of PMA (1 μg) on days 7, 14, 21, 28, and 35.
All rats in Groups 1 and 2 produced ANCA, and the titer was comparable between the two (Figs. 5A and 5B). Severe hematuria was detected in both groups of rats (Fig. 5C, left), whereas CRP levels were signi cantly higher in Group 2 than in Group 1 (Fig. 5C, middle). Correspondingly, the degree of renal tissue destruction represented by the number of erythrocyte casts in the kidney sections tended to be larger in Group 2 compared to Group 1 (Figs. 5C, right, and 5D). In ltration of neutrophils into the upstream renal tubules of the erythrocyte casts was observed (Fig. 5D, inset). Although both groups of rats did not develop overt crescentic glomerulonephritis, the collective ndings indicated that renal tubular disorder was exacerbated in the AAV rat model by PMA.

Production of anti-GBM antibody in exacerbated AAV model rats
To determine the production of anti-GBM antibody in AAV models, serum samples on days 0 and 42 were subjected to ELISA (Fig. 6A). Anti-GBM antibody was not detected in all day 0 sera (n=23). Whereas neither rats in Group 1 nor rats given intraperitoneal PMA injection with mock-immunization produced anti-GBM antibody, two rats in Group 2 (#6 and #9) produced anti-GBM antibody. Interestingly, chronological analysis of autoantibody production revealed that anti-GBM antibody production followed ANCA production in these rats (Fig. 6B). Also, the subclass of anti-GBM antibody produced in Group 2 AAV rats was determined as IgG2a, one of the pathogenic subclasses [18] (Fig. 6C).
Interstitial tubular rather than glomerular damage was exacerbated in AAV rats by PMA administration.
Contrary to humans, Col (IV), including α3(IV)NC1, was distributed in the basement membrane of tubules as well as in glomeruli in rats (Fig. 6D). This nding corresponded to the literature reporting that the α3subunit of Col (IV) is distributed in the basement membrane of renal tubules in rats [19]. IHC demonstrated the reveal of α3(IV)NC1 in the affected renal interstitial tissues of Group 2 rats with anti-GBM antibody production and in ltration of CD11c + macrophages around the sites but not in Groups 1 and 2 rats without anti-GBM antibody production (Fig. 6E).

Discussion
Anti-GBM disease is an organ-speci c autoimmune disorder characterized by anti-GBM antibodymediated diffuse necrotizing crescentic glomerulonephritis and pulmonary hemorrhage [3]. A linear IgG deposition along the autologous GBM-usually detected by IF staining using frozen sections of a biopsied kidney-is a diagnostic hallmark of this disease. However, anti-GBM antibody in patients' serum cannot bind to GBM in FFPE normal kidney sections because α3(IV)NC1, which covers the major epitopes of anti-GBM antibody, is structurally cryptic [5,6,10]. Although a contribution of the anti-peroxidasin antibody to the misfolding of the sulphilimine linkage of 3-4-5 α-subunit trimers of NC1 domain, which conceals α3(IV)NC1, has been suggested [20], the mechanism of the reveal of the cryptic epitopes in patients with anti-GBM disease remains elusive.
Based on the clinical observation of the close association between anti-GBM disease and AAV [7,8], it was hypothesized that proteases released from neutrophils activated by ANCA could digest Col (IV) and consequently reveal cryptic epitopes. In this study, it was successfully demonstrated that (1) α3(IV)NC1 was revealed by the digestion of Col (IV) by proteases, including NE; (2) although the reveal of α3(IV)NC1 was observed in affected glomeruli regardless of causative diseases, that accompanied by CD11c + macrophage in ltration was a characteristic of MPA (AAV); and (3) anti-GBM antibody was produced subsequent to ANCA in some AAV model rats.
The results of ELISA have demonstrated that 500 μU/ml or higher concentrations of NE are required to reveal α3(IV)NC1 from Col (IV). Concerning this issue, we have assumed that NE released from ANCAactivated neutrophils digests GBM in the glomerular microenvironment.
The reveal of α3(IV)NC1 was observed in affected glomeruli regardless of causative diseases, including IgAN and hypertensive arteriosclerosis. It has been shown that matrix metalloproteinase-9 is involved in the formation of mesangial lesions in IgAN [21] and that mast cell-derived tryptase and chymase are implicated in the development of hypertensive nephropathy [22]. Similarly to the neutrophil-derived proteases in AAV, these proteases may contribute to reveal α3(IV)NC1 due to the destruction of glomeruli while sclerosis occurs. However, this study has demonstrated that the reveal of α3(IV)NC1 in affected glomeruli accompanied by CD11c + macrophage in ltration was a characteristic of MPA (AAV). In addition, it was noted that some CD11c + macrophages, which in ltrated into the lesions of MPA, have CD11c + macrophages can act as APCs [17]. Although the pathway that leads to the anti-GBM antibody production has not been uncovered, it is assumed that the α3(IV)NC1-incorporated CD11c + macrophages ow into the regional lymph nodes, present the GBM epitopes on class II major histocompatibility complex (MHC), and then helper T-cells control the subsequent immune reaction. Discovery of the mechanism is a critical future subject.
Interestingly, anti-GBM antibody was produced in some AAV model rats made by the modi ed protocol with additional injections of PMA. PMA administration exacerbated the destruction of renal tubules in AAV model rats. When the recombinant human MPO light chain, instead of native MPO, was used as an immunogen, ANCA was not detected by FCM [15], and renal tissue destruction was minimal regardless of PMA administration (unpublished results). Serum CRP levels were not elevated signi cantly in the rats immunized with the non-pathogenic recombinant human MPO light chain followed by PMA administration. These ndings suggested that PMA itself cannot induce renal tissue destruction if it is not accompanied by ANCA, which can bind neutrophil MPO, and that the elevated CRP levels in Group 2 rats can be re ected by renal interstitial in ammation. PMA could induce the priming of neutrophils [23], enhance the ANCA-mediated activation of these cells, and contribute to the exacerbation of renal tissue destruction. As Col (IV), including α3(IV)NC1, is distributed in the basement membrane of renal tubules in rats, the reveal of α3(IV)NC1 in the affected interstitial space of renal tubules is reasonable.
Based on the collective ndings, it was suggested that, in AAV, proteases released from neutrophils activated by ANCA digest Col (IV) and result in the reveal of α3(IV)NC1, AAV-speci c CD11c + macrophages catch and present the epitopes in the region, and then the host's immune system produces anti-GBM antibody (Fig. 7). This corresponded to the concept of intermolecular epitope spreading.
Epitope spreading was rst described in autoimmune models of multiple sclerosis (MS) [24]. This phenomenon involves the following steps: tissue destruction by a primary immune response against a certain epitope, phagocytosis of tissue debris by APCs, and induction of secondary immune response against neo-epitopes [25]. Epitope spreading occurs both in a single antigen and among diverse antigens; the former is called intramolecular epitope spreading and the latter is called intermolecular epitope spreading. Intramolecular epitope spreading is associated with the step-by-step progression of the disease, such as MS. In contrast, intermolecular epitope spreading is related to disease conversion and complication. For example, the implication of intermolecular epitope spreading has been discussed in a conversion from pemphigus vulgaris to pemphigus foliaceus [26] and in a complication of Hashimoto's thyroiditis with Graves' disease [27]. To the authors' knowledge, this is the rst report suggesting that intermolecular epitope spreading is involved in the production of anti-GBM antibody in AAV.
All AAV patients do not produce an anti-GBM antibody regardless of the reveal of α3(IV)NC1 with a close association with CD11c + macrophages in the kidneys. Other factors, e.g., genetic background such as MHC haplotypes, might be involved in the mechanism of anti-GBM antibody production in AAV patients.
In contrast, in AAV induced in inbred WKY rats, the severity of renal tissue destruction could be critically implicated in the production of anti-GBM antibody because the reveal of α3(IV)NC1 in affected renal tissues was detected only in rats with anti-GBM antibody production.
It has been shown that the complication of anti-GBM disease is linked to the poor prognosis of AAV [3]. In fact, 38% of anti-GBM antibody + AAV patients developed alveolar hemorrhage, while 23% of anti-GBM antibody -AAV patients did. It is necessary to determine the pathogenicity of anti-GBM antibody following ANCA in AAV rats in future studies. Because the subclass of IgG has been reported to be associated with the severity of anti-GBM disease [18], the subclass of anti-GBM antibody in AAV rats was examined by ELISA, and it was determined as IgG2a, one of the pathogenic subclasses. Also, one of the two AAV rats with anti-GBM antibody production developed severer pulmonary hemorrhage than other rats (unpublished results). Although further investigation is needed, this study paves the way for anti-GBM antibody production in AAV, which can be linked to the poor prognosis of AAV.

Declarations
Ethical approval and consent to participate The use of human materials was permitted by the Ethics Committee of the Faculty of Health Sciences, Hokkaido University (permission nos. 17-85, 18-7, and 18-34). Experiments using rats were permitted by the Animal Experimentation Committee of Hokkaido University (permission no. 15-0034) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hokkaido University.

Consent for publication
Not applicable.

Availability of data and materials
The data sheets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
None.  Reveal of α3(IV)NC1 in patients' kidneys HE staining and IHC for α3(IV)NC1 without any particular antigen retrieval using FFPE kidney sections of patients with MPA (A, B, and D), IgAN (C and E), and hypertensive arteriosclerosis (F). Unaffected glomerulus (A), crescentic glomerulus (B and C), and sclerotic glomerulus (D-F). Representative microphotographs are shown. Insets in B and D represent background staining when rat IgG2a was used as isotype control.

Figure 3
In ltration of CD11c+ macrophages into kidneys IF staining for CD11c (red) and CD68 (green) using FFPE kidney sections of patients with MPA (A), IgAN (B), and hypertensive arteriosclerosis (C). Arrows indicate cell-surface CD11c+ and cytoplasmic CD68+ macrophages. Arrowheads indicate CD68+ macrophages. Representative microphotographs are shown. The number of CD11c+ macrophages (D) and CD68+ macrophages (E) in 10 high-power elds focusing on glomeruli was counted and then compared between MPA and non-MPA (IgAN and hypertensive arteriosclerosis).
White arrowheads indicate CD11c+ macrophages, and yellow arrowheads indicate those that take α3(IV)NC1 into their cytoplasm (D). Representative microphotographs are shown.  Production of anti-GBM antibody in AAV model rats Anti-GBM antibody in sera was measured by ELISA (A). Anti-GBM antibody was not detected in all day 0 sera. Whereas no rat in Group 1 produced anti-GBM antibody, two rats in Group 2 (#6 and #9) produced anti-GBM antibody. Chronological production of ANCA and anti-GBM antibody in #6 and #9 rats in Group 2 (B). ANCA titer was determined as the mean uorescent intensity of FCM. The IgG subclass of anti-GBM antibody was determined by ELISA (C).