IgA nephropathy and IgA vasculitis with nephritis have a shared feature involving galactose-deficient IgA1-oriented pathogenesis
Authors: Hitoshi Suzuki, Junichi Yasutake, Yuko Makita, Yuki Tanbo, Kohei Yamasaki, Tadashi Sofue, Toshiki Kano, Yusuke Suzuki
Journal: Kidney International (2018)

Galactose-deficient IgA1 has been proposed as an important effector molecule in IgA nephropathy (IgAN). We previously showed that the galactose-deficient IgA1-specific monoclonal antibody KM55 can detect circulating galactose-deficient IgA1 in patients with IgAN, enabling us to study the molecular roles of galactose-deficient IgA1. Herein, we further examined the pathophysiological significance of galactose-deficient IgA1 in glomerular deposits of patients with IgAN by immunohistochemistry using KM55. Immunostaining of galactose-deficient IgA1 with KM55 was performed in paraffin-embedded sections of renal biopsy specimens from 48 patients with IgAN and 49 patients with other renal diseases such as lupus nephritis, HCV-related nephropathy, IgA vasculitis with nephritis (IgA-VN), and membranous nephropathy. Glomerular galactose-deficient IgA1 was specifically detected in IgAN and IgA-VN but not in the other renal diseases. Galactose-deficient IgA1 was localized predominantly in the mesangial region as IgA deposition. However, galactose-deficient IgA1 was not detected in patients with lupus nephritis accompanied by glomerular IgA deposition. Thus, our study strongly suggests that IgAN and IgA-VN have a shared feature regarding galactose-deficient IgA1-oriented pathogenesis. Galactose-deficient IgA1 has been proposed as an important effector molecule in IgA nephropathy (IgAN). We previously showed that the galactose-deficient IgA1-specific monoclonal antibody KM55 can detect circulating galactose-deficient IgA1 in patients with IgAN, enabling us to study the molecular roles of galactose-deficient IgA1. Herein, we further examined the pathophysiological significance of galactose-deficient IgA1 in glomerular deposits of patients with IgAN by immunohistochemistry using KM55. Immunostaining of galactose-deficient IgA1 with KM55 was performed in paraffin-embedded sections of renal biopsy specimens from 48 patients with IgAN and 49 patients with other renal diseases such as lupus nephritis, HCV-related nephropathy, IgA vasculitis with nephritis (IgA-VN), and membranous nephropathy. Glomerular galactose-deficient IgA1 was specifically detected in IgAN and IgA-VN but not in the other renal diseases. Galactose-deficient IgA1 was localized predominantly in the mesangial region as IgA deposition. However, galactose-deficient IgA1 was not detected in patients with lupus nephritis accompanied by glomerular IgA deposition. Thus, our study strongly suggests that IgAN and IgA-VN have a shared feature regarding galactose-deficient IgA1-oriented pathogenesis. IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Most cases of IgAN are discovered incidentally by abnormal urinalysis (hematuria or proteinuria) and diagnosed by renal biopsy.1D'Amico G. Natural history of idiopathic IgA nephropathy: role of clinical and histological prognostic factors.Am J Kidney Dis. 2000; 36: 227-237Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 2McGrogan A. Franssen C.F. de Vries C.S. The incidence of primary glomerulonephritis worldwide: a systematic review of the literature.Nephrol Dial Transplant. 2011; 26: 414-430Crossref PubMed Scopus (316) Google Scholar In the renal biopsy specimens, IgA1, but not IgA2, is predominantly deposited in the mesangial and capillary region.3Conley M.E. Cooper M.D. Michael A.F. Selective deposition of immunoglobulin A1 in immunoglobulin A nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus.J Clin Invest. 1980; 66: 1432-1436Crossref PubMed Scopus (261) Google Scholar However, glomerular IgA is also frequently found in other types of glomerular diseases such as lupus nephritis, hepatitis C virus–related nephropathy (HCV-RN), mesangioproliferative glomerulonephritis (MPGN), and IgA vasculitis with nephritis (IgA-VN). However, the pathogenic role of glomerular IgA deposition is not clear for each glomerular disease. Galactose-deficient IgA1 (Gd-IgA1) has been identified as among the key effector molecules in the pathogenesis of IgAN, although the underlying molecular mechanisms remain under investigation.4Barratt J. Feehally J. IgA nephropathy.J Am Soc Nephrol. 2005; 16: 2088-2097Crossref PubMed Scopus (397) Google Scholar, 5Moldoveanu Z. Wyatt R.J. Lee J.Y. et al.Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney Int. 2007; 71: 1148-1154Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 6Suzuki H. Moldoveanu Z. Hall S. et al.IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1.J Clin Invest. 2008; 118: 629-639PubMed Google Scholar, 7Berthoux F. Suzuki H. Thibaudin L. et al.Autoantibodies targeting galactose-deficient IgA1 associate with progression of IgA nephropathy.J Am Soc Nephrol. 2012; 23: 1579-1587Crossref PubMed Scopus (178) Google Scholar Recently, a multihit hypothesis regarding the pathogenesis of IgAN was proposed.8Suzuki H. Kiryluk K. Novak J. et al.The pathophysiology of IgA nephropathy.J Am Soc Nephrol. 2011; 22: 1795-1803Crossref PubMed Scopus (462) Google Scholar In this hypothesis, 3 major steps are required for the onset and progression of IgAN: overproduction of Gd-IgA1 and autoantibodies against Gd-IgA1, formation of immune complexes, and deposition of those immune complexes in glomeruli.8Suzuki H. Kiryluk K. Novak J. et al.The pathophysiology of IgA nephropathy.J Am Soc Nephrol. 2011; 22: 1795-1803Crossref PubMed Scopus (462) Google Scholar Although genetic factors and secondary immune dysregulation might be involved in the production of Gd-IgA1,9Gharavi A.G. Moldoveanu Z. Wyatt R.J. et al.Aberrant IgA1 glycosylation is inherited in familial and sporadic IgA nephropathy.J Am Soc Nephrol. 2008; 19: 1008-1014Crossref PubMed Scopus (188) Google Scholar, 10Gharavi A.G. Kiryluk K. Choi M. et al.Genome-wide association study identifies susceptibility loci for IgA nephropathy.Nat Genet. 2011; 43: 321-327Crossref PubMed Scopus (443) Google Scholar, 11Suzuki H. Raska M. Yamada K. et al.Cytokines alter IgA1 O-glycosylation by dysregulating C1GalT1 and ST6GalNAc-II enzymes.J Biol Chem. 2014; 289: 5330-5339Crossref PubMed Scopus (102) Google Scholar in particular, mucosal immune dysregulation may contribute to the production of Gd-IgA1 and Gd-IgA1 immune complexes.12Suzuki H. Suzuki Y. Narita I. et al.Toll-like receptor 9 affects severity of IgA nephropathy.J Am Soc Nephrol. 2008; 19: 2384-2395Crossref PubMed Scopus (139) Google Scholar, 13Novak J. Moldoveanu Z. Julian B.A. et al.Aberrant glycosylation of IgA1 and anti-glycan antibodies in IgA nephropathy: role of mucosal immune system.Adv Otorhinolaryngol. 2011; 72: 60-63PubMed Google Scholar, 14Muto M. Manfroi B. Suzuki H. et al.Toll-Like receptor 9 stimulation induces aberrant expression of a proliferation-inducing ligand by tonsillar germinal center B cells in IgA nephropathy.J Am Soc Nephrol. 2017; 28: 1227-1238Crossref PubMed Scopus (61) Google Scholar Those findings suggest that Gd-IgA1 has a crucial role in the pathogenesis of IgAN. Previously, 2 studies revealed that glomerular IgA1 in patients with IgAN is aberrantly glycosylated.15Allen A.C. Bailey E.M. Brenchley P.E. et al.Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients.Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 16Hiki Y. Odani H. Takahashi M. et al.Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy.Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar However, no replicative study has confirmed these findings, and no other evidence for glomerular Gd-IgA1 deposition in patients with IgAN has been presented. In those studies, IgA1 was extracted from kidney biopsy specimens and the aberrant glycosylation of IgA1 was analyzed. However, it is possible that the sugar moieties on IgA1 were altered during the purification of IgA1 from biopsy specimens. In a previous study, we established an enzyme-linked immunosorbent assay system to detect circulatory Gd-IgA1 using the Gd-IgA1-specific monoclonal antibody KM55 (KM55 mAb).17Yasutake J. Suzuki Y. Suzuki H. et al.Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.Nephrol Dial Transplant. 2015; 30: 1315-1321Crossref PubMed Scopus (74) Google Scholar This novel assay revealed that circulatory Gd-IgA1 levels were elevated in patients with IgAN and are consistent with results from a Helix aspersa agglutinin lectin enzyme-linked immunosorbent assay.5Moldoveanu Z. Wyatt R.J. Lee J.Y. et al.Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney Int. 2007; 71: 1148-1154Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 17Yasutake J. Suzuki Y. Suzuki H. et al.Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.Nephrol Dial Transplant. 2015; 30: 1315-1321Crossref PubMed Scopus (74) Google Scholar However, it remains unclear whether circulatory Gd-IgA1 is involved in the glomerular deposition of IgA1 and consequent renal injury in IgAN. Therefore, we evaluated glomerular Gd-IgA1 by immunofluorescence analysis using KM55 mAb to understand the pathophysiological significance of glomerular Gd-IgA1 deposition in IgAN. We performed a neutralizing assay in renal biopsy specimens from a patient with IgAN, using recombinant glycan-intact IgA1 and recombinant aberrantly glycosylated IgA1 to test whether KM55 mAb could detect Gd-IgA1 on the tissue. The recombinant glycan-intact IgA1 and recombinant aberrantly glycosylated IgA1 were purified from the supernatants of DG44 and Lec8 cells, respectively, both transfected with a human IgA1-expressing vector. Lec8 represents a subset of Chinese hamster ovary cells with reduced transportation of uridine diphosphate galactose, whereas DG44 is a subset of Chinese hamster ovary cells with normal transportation of galactose. Galactose deficiency in both proteins was confirmed by H. aspersa agglutinin-binding. The IgA1 produced by Lec8 cells was galactose deficient. KM55 mAb was confirmed to recognize recombinant aberrantly glycosylated IgA1 but not recombinant glycan-intact IgA1 by a binding assay. Subsequently, these recombinant proteins were applied as inhibitors of KM55 mAb or an anti-IgA polyclonal antibody (anti-IgA pAb) in neutralizing assays. Recombinant aberrantly glycosylated IgA1 interfered with signals of both anti-IgA pAb and KM55 mAb, whereas only anti-IgA pAb staining was abolished by recombinant glycan-intact IgA1 (Supplementary Figure S1). These data indicated that renal deposits detected by KM55 mAb were Gd-IgA1 but not glycan-intact IgA1. Sequential thin sections were stained with anti-IgA pAb and KM55 mAb and then these images were overlaid. Both IgA and Gd-IgA1 deposited mainly in the mesangial area, indicating that anti-IgA pAb and KM55 mAb did not compete for epitopes close together as shown in Supplementary Figure S2. To analyze the localization of glomerular deposits of Gd-IgA1, we performed double staining of IgA and Gd-IgA1 using biopsy tissues from 48 patients with IgAN. In all glomeruli of the patients with IgAN, Gd-IgA1, detected by KM55 mAb, was clearly localized to a similar pattern as IgA (Figure 1). Glomerular deposits of Gd-IgA1 were not associated with pathological or clinical severity (i.e., proteinuria and low estimated glomerular filtration rate) (Supplementary Table S1). Five cases are shown as examples in Figure 1. Although the staining pattern and intensity differed among patients with IgAN, Gd-IgA1 was mainly observed in the mesangial and capillary region, and overlapped with IgA deposition. There is no correlation between the intensity of glomerular Gd-IgA1 and serum levels of Gd-IgA1. Next, we performed immunostaining with KM55 mAb to investigate whether glomerular Gd-IgA1 deposition was disease-specific in IgAN using renal biopsy specimens from various types of glomerular diseases, including lupus nephritis, membranous nephropathy (MN), HCV-RN, hepatic glomerulosclerosis, MPGN, and IgA-VN (Table 1). Glomerular Gd-IgA1 was positive in all IgAN biopsy specimens (Figure 1). In contrast, glomerular Gd-IgA1 was negative in other types of glomerular diseases, such as MPGN, idiopathic MN, secondary MN, and minimal change nephrotic syndrome, regardless of IgA positivity (Figure 2A). Furthermore, HCV-RN with secondary IgA deposition after HCV infection and secondary IgA deposition due to liver cirrhosis did not show any glomerular Gd-IgA1 (Figure 2A). Cases of lupus nephritis were then more precisely examined. Glomerular Gd-IgA1 was negative in all lupus nephritis cases. Even in cases of lupus nephritis accompanied by glomerular IgA deposition, glomerular Gd-IgA1 was negative (Figure 2B). Importantly, all cases of IgA-VN exhibited glomerular Gd-IgA1, mainly in the mesangial area, as seen in cases of IgAN (Figure 2C). Clinical data and immunofluorescence analyses of renal biopsy are indicated in Supplementary Table S1.Table 1Biopsy samplesDiseasesCasesGlomerular depositIgA (+)Gd-IgA1 (+)Anti-IgA AbKM55IgAN484848IgA-VN141414Lupus nephritis870Idiopathic MN510Secondary MN430HCV-RN330Hepatic glomerulosclerosis110ANCA-related nephropathy200Granulomatosis with polyangiitis110MPGN110MCNS300Non-IgA PGN200Nonspecific renal tubular atrophy100APSGN100Nephrosclerosis100Minor glomerular abnormalities200Total97Ab, antibody; ANCA, antineutrophil cytoplasmic antibody; APSGN, acute poststreptococcal glomerulonephritis; Gd, galactose dependent; HCV-RN, hepatitis C virus–related nephropathy; IgAN, IgA nephropathy; IgA-VN, IgA vasculitis with nephropathy; MCNS, minimal change nephrotic syndrome; MN, membranous nephropathy; MPGN, membranoproliferative glomerulonephritis; PGN, proliferative glomerulonephritis. Open table in a new tab Figure 2KM55 mAb staining. (a) KM55 mAb staining in various types of glomerular diseases. Glomerular galactose-deficient IgA1 staining was negative in cases of hepatitis C virus–related nephropathy (HCV-RN), hepatic glomerulosclerosis, idiopathic membranous nephropathy (MN), secondary MN, acute poststreptococcal glomerulonephritis (APSGN), antineutrophil cytoplasmic antibody (ANCA)–related nephropathy, and minimal change nephrotic syndrome (MCNS).(b) KM55 mAb staining in lupus nephritis. Five cases of lupus nephritis are shown as examples. In all cases of lupus nephritis that were accompanied by glomerular IgA deposition, glomerular galactose-deficient IgA1 staining was negative. (c) KM55 mAb staining in IgA vasculitis with nephritis (IgA-VN). Five cases of IgA-VN are shown as examples. All cases of IgA-VN exhibited glomerular galactose-deficient IgA1 staining, mainly in the mesangial area, as seen in cases of IgA nephropathy (IgAN). (Bars = 100 μm; original magnification X200.) To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ab, antibody; ANCA, antineutrophil cytoplasmic antibody; APSGN, acute poststreptococcal glomerulonephritis; Gd, galactose dependent; HCV-RN, hepatitis C virus–related nephropathy; IgAN, IgA nephropathy; IgA-VN, IgA vasculitis with nephropathy; MCNS, minimal change nephrotic syndrome; MN, membranous nephropathy; MPGN, membranoproliferative glomerulonephritis; PGN, proliferative glomerulonephritis. IgAN is defined as primary glomerulonephritis with predominant glomerular IgA deposition. However, glomerular IgA deposition is found not only in IgAN but also in other types of glomerular diseases such as lupus nephritis, HCV-RN, hepatic glomerulosclerosis, MPGN, and IgA-VN. Several researchers have suggested that glomerular IgA is aberrantly glycosylated in patients with IgAN.15Allen A.C. Bailey E.M. Brenchley P.E. et al.Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients.Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 16Hiki Y. Odani H. Takahashi M. et al.Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy.Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar However, no replicative study has been reported due to the complicated procedure required to analyze the glycosylation of glomerular IgA1. Another reason is that there has not been a specific antibody generated against glomerular Gd-IgA1. In addition, H. aspersa agglutinin lectin cannot be used for immunohistochemical analysis to detect glomerular Gd-IgA1.18Laitine L. Juusela H. Virtanen I. Binding of the blood group-reactive lectins to human adult kidney specimens.Anat Rec. 1990; 226: 10-17Crossref PubMed Scopus (23) Google Scholar In the present study, immunofluorescence analysis of renal biopsy specimens using KM55 mAb clearly showed glomerular deposition of Gd-IgA1. Importantly, we found that the glomerular localization of Gd-IgA1 was specific for IgAN and IgA-VN. Notably, any specimens from lupus nephritis accompanied by glomerular IgA deposition were not positive for KM55 mAb. Moreover, the KM55 mAb failed to detect Gd-IgA1 in HCV-RN, secondary IgA deposition due to liver cirrhosis, or rheumatoid arthritis, suggesting that glomerular Gd-IgA1 is more involved in primary IgA deposition than in secondary IgA deposition. IgA-VN presents with mesangioproliferative glomerulonephritis accompanied by predominant glomerular IgA deposition, in addition to other clinical manifestations such as purpura and intestinal vasculitis. Therefore, it is difficult to distinguish IgA-VN from IgAN in a renal pathological point of view. Urinary abnormalities are exacerbated after mucosal infections in both IgA-VN and IgAN. Moreover, Gd-IgA1 plays a key role in the pathogenesis of IgA-VN.19Lau K.K. Suzuki H. Novak J. Wyatt R.J. Pathogenesis of Henoch-Schönlein purpura nephritis.Pediatr Nephrol. 2010; 25: 19-26Crossref PubMed Scopus (113) Google Scholar, 20Kiryluk K. Moldoveanu Z. Sanders J.T. et al.Aberrant glycosylation of IgA1 is inherited in both pediatric IgA nephropathy and Henoch-Schönlein purpura nephritis.Kidney Int. 2011; 80: 79-87Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 21Pohl M. Henoch-Schönlein purpura nephritis.Pediatr Nephrol. 2015; 30: 245-252Crossref PubMed Scopus (89) Google Scholar Consistent with these facts, in cases of IgA-VN, glomerular Gd-IgA1 as well as predominant glomerular IgA was observed in the mesangial region. Therefore, there has been increasing discussion on whether IgA-VN is the same disease as IgAN. Gd-IgA1 can be a clear marker, which bridges these 2 independent renal diseases. Thus, the positive staining of Gd-IgA1 in IgA-VN represents evidence that supports this idea. The present study firstly provides substantial evidence that Gd-IgA1, which is an identical molecule to circulating Gd-IgA1 and is detected by KM55 mAb,17Yasutake J. Suzuki Y. Suzuki H. et al.Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.Nephrol Dial Transplant. 2015; 30: 1315-1321Crossref PubMed Scopus (74) Google Scholar has a key role in the glomerular deposition of IgA1-containing immune complexes in patients with IgAN and IgA-VN. However, serum levels of Gd-IgA1 did not correlate with the intensity of glomerular Gd-IgA1 deposition (data not shown). Genetic factors might be involved in the production of Gd-IgA1,9Gharavi A.G. Moldoveanu Z. Wyatt R.J. et al.Aberrant IgA1 glycosylation is inherited in familial and sporadic IgA nephropathy.J Am Soc Nephrol. 2008; 19: 1008-1014Crossref PubMed Scopus (188) Google Scholar, 10Gharavi A.G. Kiryluk K. Choi M. et al.Genome-wide association study identifies susceptibility loci for IgA nephropathy.Nat Genet. 2011; 43: 321-327Crossref PubMed Scopus (443) Google Scholar and the level of circulating Gd-IgA1 is not linked to the onset of IgAN. Further studies are required to clarify the mechanisms by which Gd-IgA1 is deposited in the glomerular mesangial area and induces chronic glomerular injuries in IgAN and IgA-VN. Secondary IgAN due to hepatic diseases did not show any glomerular Gd-IgA1, suggesting that it does not share its pathogenesis with IgAN and IgA-VN. We also found variability in the degree of glomerular Gd-IgA1 deposition and colocalization with IgA deposition even among patients with IgAN and IgA-VN. It is our future task to elucidate whether such variability is associated with histological features such as acute or chronic lesions, clinical markers, and prognosis. In conclusion, the KM55 mAb could be a powerful tool to detect Gd-IgA1 in circulation and glomerular deposits specifically in patients with IgAN and IgA-VN. The KM55 mAb bridges the independent status of Gd-IgA1 to support a multihit hypothesis. The present study is the first to indicate that glomerular IgA predominantly consists of Gd-IgA1, which is specific for both IgAN and IgA-VN. Thus, it is strongly suggested that IgAN and IgA-VN have a shared entity regarding the Gd-IgA1-oriented pathogenesis. Renal biopsy specimens were obtained from 2013 to 2017 at the Juntendo University Hospital with the informed consent from patients and approval of the Research Ethics Review Committees of the Juntendo University Hospital and Kyowa Hakko Kirin Co., Ltd. Immunofluorescent staining of Gd-IgA1 in clinical glomerular tissues was performed. Paraffin-embedded sections of 3-μm thickness were prepared for staining. After deparaffinization with a series of xylene and ethanol concentrations and subsequent rehydration, antigen retrieval using 0.05% bacterial protease subtilisin A (Sigma-Aldrich, Tokyo, Japan) dissolved in 5 mmol/l tris(hydroxymethyl)-aminomethane buffer (pH 7.6) was performed at room temperature for 2 hours. Samples were then rinsed with distilled water and blocked with Protein Block (Dako Japan, Tokyo, Japan) at room temperature for 30 minutes, which was followed by incubation with KM55 (100 μg/ml) at 37 °C for 60 minutes. After several washes with phosphate-buffered saline and triethanolamine-buffered saline containing 0.05% tween-20, Alexa Fluor 555-conjugated goat anti-rat IgG antibody (1:1000; Life Technologies, Carlsbad, CA) was incubated with the samples at 37 °C for 30 minutes. Samples were washed with phosphate-buffered saline and triethanolamine-buffered saline containing 0.05% tween-20 and incubated with fluorescein isiothiocyanate–conjugated polyclonal rabbit anti-human IgA antibody (100 μg/ml; Dako Japan) at 37 °C for 30 minutes. After washing with phosphate-buffered saline and triethanolamine-buffered saline containing 0.05% tween-20, slides were sealed with Fluoromount (Diagnostic BioSystems, Pleasanton, CA). The intensity of glomerular Gd-IgA1 was scored semiquantitatively (0–3 intensity). For microscopic observation of immunostained samples, a BIOREVO Fluorescence Microscope BZ-9000 (KEYENCE, Osaka, Japan), an Axioplan-2 upright fluorescent microscope (Zeiss Japan, Tokyo, Japan), and a CRi-Nuance tissue microscope (Cambridge Research and Instrumentation Inc., Hopkinton, MA) were used. Recombinant aberrantly glycosylated IgA1 and recombinant glycan-intact IgA1 were produced by human IgA1-transfected Lec8 cells (ATCC, Manassas, VA), a subset of Chinese hamster ovary cells with reduced transportation of uridine diphosphate galactose into the Golgi compartment, and human IgA1-transfected DG44 cells (ATCC), a subset of Chinese hamster ovary cells with normal galactose transportation, respectively. Recombinant proteins were purified using a HiTrap protein L column (GE Healthcare Japan, Tokyo, Japan) from the supernatant of IgA1-trasfected cells. The galactose deficiency of recombinant IgA1 from Lec8 cells was confirmed by H. aspersa lectin-binding. Specific binding of KM55 to recombinant Gd-IgA1 was confirmed by a binding assay using KM55-immobilzed plates. Either recombinant glycan-intact IgA1 (100 μg/ml) or Gd-IgA1 (100 μg/ml) was preincubated with KM55 mAb (100 μg/ml) or anti-IgA pAb (100 μg/ml) for 30 minutes at room temperature, and then the mixtures were applied to the specimen. Depositions of Gd-IgA1 and total IgA were detected according to the immunofluorescence protocol as described. This study was supported by a research grant from Kyowa Hakko Kirin Co., Ltd., based on collaborative research between Juntendo University and Kyowa Hakko Kirin Co., Ltd. All the authors declared no competing interests. The authors thank Terumi Shibata, Takako Ikegami, and Tomomi Ikeda for their fruitful scientific advice and excellent research assistance. This study was supported, in part, by the Japanese Society for the Promotion of Science Grants-in-Aid for Scientific Research Program, KAKENHI, grant 15K09274 and Practical Research Project for Renal Diseases from the Japan Agency for Medical Research and Development, AMED. Download .pptx (6.04 MB) Help with pptx files Figure S1Characterization of renal deposits detected by KM55 monoclonal antibody. Neutralization assay using recombinant glycan-intact IgA1 and recombinant galactose-deficient (Gd)-IgA1 in renal biopsy tissues from a patient with IgA nephropathy. Recombinant Gd-IgA1 interfered with signals of both Gd-IgA1 and IgA, whereas only IgA staining was abolished by recombinant glycan-intact IgA1. (Bars = 100 μm; original magnification X200.) Download .pptx (.93 MB) Help with pptx files Figure S2Glomerular deposition of IgA and galactose-deficient IgA1 in patients with IgA nephropathy. Sequential thin sections were stained with anti-IgA polyclonal antibody and KM55 monoclonal antibody and then these images were overlaid. Both IgA and galactose-deficient IgA1 deposited mainly in the mesangial area, indicating that anti-IgA polyclonal antibody and KM55 monoclonal antibody did not compete for epitopes close together. (Bars = 100 μm; original magnification X200.) Download .xlsx (.03 MB) Help with xlsx files Table S1Clinical and histological data of biopsy samples. IgA nephropathy: toward more specific diagnosis (and rescue of snails)Kidney InternationalVol. 93Issue 3PreviewThe diagnosis of IgA nephropathy relies on the histologic demonstration of glomerular mesangial IgA deposits. However, only a very small fraction of IgA, namely, galactose-deficient IgA1, seems to induce the disease. So far, this type of IgA could only be detected using mass spectrometry or lectins, which are relatively difficult to standardize. A novel monoclonal antibody, KM55, specifically recognizing galactose-deficient IgA1, may now change this. Full-Text PDF Open Archive