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Determination of Severity of Murine IgA Nephropathy by Glomerular Complement Activation by Aberrantly Glycosylated IgA and Immune Complexes

2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2012.06.038

1525-2191

Azusa Hashimoto, Yusuke Suzuki, Hitoshi Suzuki, Isao Ohsawa, Rhubell Brown, Stacy Hall, Yuichi Tanaka, Jan Novák, Hiroyuki Ohi, Yasuhiko Tomino,

Platelet Disorders and Treatments

The pathogenic roles of glomerular deposition of components of the complement cascade in IgA nephropathy (IgAN) are not completely clarified. To investigate the pathologic role of complement pathways in IgAN, two IgAN-prone mouse models were examined. Grouped ddY (gddY) mice showed significant high proteinuria, severe glomerular lesions, and extracellular matrix expansion compared with high serum IgA (HIGA) mice but with similar intensity of glomerular IgA deposition. Glomerular activation of the classical, lectin, and alternative pathways was demonstrated by significantly stronger staining for complement (C)3, C5b-9, C1q, C4, mannose-binding lectin (MBL)-A/C, MBL-associated serine protease-2, and factor B and properdin in gddY mice than in HIGA mice. Similarly, the serum levels of IgA-IgG2a/IgM and IgA–MBL-A/C immune complexes and polymeric IgA were significantly higher in gddY mice than in HIGA mice. Moreover, the serum levels of aberrantly glycosylated IgA characterized by the binding of Sambucus nigra bark lectin and Ricinus communis agglutinin I were significantly higher in gddY mice than in HIGA mice. This aberrancy in glycosylation was confirmed by monosaccharide compositional analysis of purified IgA using gas-liquid chromatography. This study is the first to demonstrate that aberrantly glycosylated IgA may influence the formation of macromolecular IgA including IgA-IgG immune complexes and subsequent complement activation, leading to full progression of IgAN. The pathogenic roles of glomerular deposition of components of the complement cascade in IgA nephropathy (IgAN) are not completely clarified. To investigate the pathologic role of complement pathways in IgAN, two IgAN-prone mouse models were examined. Grouped ddY (gddY) mice showed significant high proteinuria, severe glomerular lesions, and extracellular matrix expansion compared with high serum IgA (HIGA) mice but with similar intensity of glomerular IgA deposition. Glomerular activation of the classical, lectin, and alternative pathways was demonstrated by significantly stronger staining for complement (C)3, C5b-9, C1q, C4, mannose-binding lectin (MBL)-A/C, MBL-associated serine protease-2, and factor B and properdin in gddY mice than in HIGA mice. Similarly, the serum levels of IgA-IgG2a/IgM and IgA–MBL-A/C immune complexes and polymeric IgA were significantly higher in gddY mice than in HIGA mice. Moreover, the serum levels of aberrantly glycosylated IgA characterized by the binding of Sambucus nigra bark lectin and Ricinus communis agglutinin I were significantly higher in gddY mice than in HIGA mice. This aberrancy in glycosylation was confirmed by monosaccharide compositional analysis of purified IgA using gas-liquid chromatography. This study is the first to demonstrate that aberrantly glycosylated IgA may influence the formation of macromolecular IgA including IgA-IgG immune complexes and subsequent complement activation, leading to full progression of IgAN. IgA nephropathy (IgAN) was first reported in 1968 by Berger and Hinglais1Berger J. Hinglais N. Intercapillary deposits of IgA-IgG.J Urol Nephrol. 1968; 74: 694-695PubMed Google Scholar and is a common form of progressive primary glomerulonephritis. The major histologic characteristics of IgAN are mesangial cell proliferation and matrix expansion with granular deposition of IgA, predominantly polymeric IgA1 (pIgA1),2Tomino Y. Sakai H. Miura M. Endoh M. Nomoto Y. Detection of polymeric IgA in glomeruli from patients with IgA nephropathy.Clin Exp Immunol. 1982; 49: 419-425PubMed Google Scholar and complement (C)3 in the glomerular mesangial areas. This deposition in glomeruli may provide insights into IgAN pathogenesis. In IgAN, IgA1 has aberrant glycosylation of O-glycans in the hinge region3Allen A.C. Bailey E.M. Brenchley P.E. Buck K.S. Barratt J. Feehally J. 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 (272) Google Scholar, 4Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. 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 (302) Google Scholar and is predominantly in the polymeric form (pIgA).2Tomino Y. Sakai H. Miura M. Endoh M. Nomoto Y. Detection of polymeric IgA in glomeruli from patients with IgA nephropathy.Clin Exp Immunol. 1982; 49: 419-425PubMed Google Scholar, 5Novak J. Julian B.A. Tomana M. Mestecky J. Progress in molecular and genetic studies of IgA nephropathy.J Clin Immunol. 2000; 21: 310-327Crossref Scopus (100) Google Scholar, 6Floege J. Feehally J. IgA nephropathy: recent developments.J Am Soc Nephrol. 2000; 11: 2395-2403Crossref PubMed Google Scholar, 7Van der Boog P.J. Van Kooten C. Fijter J.W. Daha M.R. Role of macromolecular IgA in IgA nephropathy.Kidney Int. 2005; 67: 813-821Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar However, the pathologic interaction between the complement cascade and pIgA has not been completely clarified in IgAN. Three different pathways of complement activation have been described. The classical pathway is triggered by activation of the C1 complex (composed of one molecule of C1q, two molecules of C1r, and two molecules of C1s, thus forming C1qr2s2).This activation occurs when C1q binds to IgM- or IgG-antigen complexes or when C1q binds directly to the surface of a pathogen. The lectin pathway is homologous to the classical pathway but contains opsonin, mannose-binding lectin (MBL), and ficolins instead of C1q. This pathway is activated by binding of MBL to mannose residues on the pathogen surface, thereby leading to activation of MBL-associated serine proteases (MASP-1 and MASP-2). These proteases split C4 into C4a and C4b and C2 into C2a and C2b. Similar to that in the classical pathway, C4b and C2a then bind together to form C3 convertase. The alternative pathway is triggered by spontaneous C3 hydrolysis [C3(H2O)] directly due to the breakdown of the thioester bond. This change in shape allows the binding of plasma protein factor B [C3(H2O)Bb, C3 convertase]. This convertase cleaves C3 proteins into C3a and C3b, which are then capable of covalently binding to a pathogenic membrane surface. Properdin is a component of the alternative pathway. It forms complexes with C3b to stabilize the alternative C3 convertase that further cleaves more C3. In all three pathways, C3 convertase cleaves and activates component C3 by forming C3a and C3b. It also cleaves C5 into C5a and C5b. C5b initiates the membrane attack pathway by forming a membrane attack complex with other recruited complement components (C6, C7, C7, C8, and multiple C9 molecules). Membrane attack complex is the cytolytic end product of the complement cascade. It forms a transmembrane channel that causes osmotic lysis of the target cell. We used IgAN animal models to investigate IgAN pathogenesis. The ddY mouse is an animal model of spontaneous IgAN. Glomerular lesions in this mouse include mesangial proliferation and extracellular matrix expansion with paramesangial IgA deposition that closely resemble those found in human IgAN.8Imai H. Nakamoto Y. Asakura K. Miki K. Yasuda T. Miura A.B. Spontaneous glomerular IgA deposition in ddY mice: an animal model of IgA nephritis.Kidney Int. 1985; 27: 756-761Abstract Full Text PDF PubMed Scopus (128) Google Scholar In pooled serum and glomerular elutes of 16-, 40-, and 60-week-old ddY mice, the ratio of dimeric IgA and pIgA in total IgA increases markedly with age.9Muso E. Yoshida H. Takeuchi E. Shimada T. Yashiro M. Sugiyama T. Kawai C. Pathogenic role of polyclonal and polymeric IgA in a murine model of mesangial proliferative glomerulonephritis with IgA deposition.Clin Exp Immunol. 1991; 84: 459-465PubMed Google Scholar However, the IgAN incidence in commercially available ddY mice is highly variable.10Kawaguchi S. Immunopathological study of glomerular IgA deposition in ddY mice.Acta Pathol Jpn. 1988; 38: 1-10PubMed Google Scholar The high serum IgA (HIGA) mouse is one of the IgAN models derived from the ddY mouse.11Miyawaki S. Muso E. Takeuchi E. Matsushima H. Shibata Y. Sasayama S. Yoshida H. Selective breeding for high serum IgA levels from noninbred ddY mice: isolation of a strain with an early onset of glomerular IgA deposition.Nephron. 1997; 76: 201-207Crossref PubMed Scopus (56) Google Scholar This mouse is an inbred strain derived from ddY mice by selective mating of animals with high serum IgA levels. No renal abnormalities manifest in the HIGA mouse until approximately 10 weeks of age, with serum IgA levels increasing dramatically thereafter and becoming elevated by 25 weeks of age. Although this mouse shows high serum IgA levels, the increase is not associated with the severity of glomerular injury and the incidence of IgAN. We have evaluated commercially available ddY mice using serial renal biopsies obtained at 20, 40, and 60 weeks of age. These mice were divided into three groups: early-onset (approximately 20 weeks), late-onset (approximately 40 weeks), and quiescent (even at 60 weeks) mice.12Suzuki H. Suzuki Y. Yamanaka T. Hirose S. Nishimura H. Toei J. Horikoshi S. Tomino Y. Genome-wide scan in a novel IgA nephropathy model identifies a susceptibility locus on murine chromosome 10, in a region syntenic to human IGAN1 on chromosome 6q22-23.J Am Soc Nephrol. 2005; 16: 1289-1299Crossref PubMed Scopus (69) Google Scholar In ddY mice with disease onset, the levels of serum IgA-IgG2a immune complex (IC) correlated strongly with the severity of glomerular lesions,13Suzuki H. Suzuki Y. Aizawa M. Yamanaka T. Kihara M. Pang H. Horikoshi S. Tomino Y. Th1 polarization in murine IgA nephropathy directed by bone marrow-derived cells.Kidney Int. 2007; 72: 319-327Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar although no significant correlation was observed between serum IgA levels and the incidence of glomerular injury among the groups. We bred early-onset mice in a specific pathogen–free room and established a mouse IgAN model with an almost 100% incidence of the disorder.14Okazaki K, Suzuki Y, Otsuji M, Suzuki H, Kihara M, Kajiyama T, Hashimoto A, Nishimura H, Brown R, Hall S, Novak J, Izui S, Hirose S, Tomino Y: Development of a model of early-onset IgA nephropathy [published online July 12, 2012]. J Am Soc NephrolGoogle Scholar This mouse strain was called grouped ddY (gddY). Although the HIGA and gddY models are derived from ddY mice, their ratios of disease onset, phenotype, and disease severity are very different. In this study, we examined these differences from the aspect of complement activation. Eleven female gddY mice were maintained at the animal facility of Juntendo University, Tokyo, Japan. The mice were fed regular chow (Oriental Yeast Co. Ltd., Tokyo, Japan) and water ad libitum and were housed in a specific pathogen–free room. Age-matched female HIGA and BALB/c mice were maintained in an identical manner. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Juntendo University Faculty of Medicine. Urinary albumin levels were measured using an enzyme-linked immunosorbent assay kit (Exocell Inc., Philadelphia, PA). Using a commercial kit (Exocell Inc.), urinary creatinine levels were measured based on Jaffe's reaction with alkaline picrate. Urinary albumin was normalized to urinary creatinine. Kidneys were obtained after perfusion with normal saline. Renal tissue specimens for microscopic evaluation were fixed in 15% formaldehyde, embedded in paraffin, cut into 3-μm-thick sections, and then stained with H&E and PAS. The mesangial area in the renal specimen of each mouse was determined by calculating the mean nucleus-free, PAS-positive area in >20 glomeruli located in the glomerular hilus, as described previously.15Kobayashi I. Nogaki F. Kusano H. Ono T. Miyawaki S. Yoshida H. Muso E. Interleukin-12 alters the physicochemical characteristics of serum and glomerular IgA and modifies glycosylation in a ddY mouse strain having high IgA levels.Nephrol Dial Transplant. 2002; 17: 2108-2116Crossref PubMed Scopus (34) Google Scholar Renal specimens for immunofluorescence (IF) were mounted in OCT compound (Sakura Finetek Japan Co. Ltd., Tokyo, Japan) and stored at −80°C. The specimens were then cut into 3-μm-thick sections, fixed with acetone at −20°C for 4 minutes, and stained (IF staining) for IgA, IgG2a, C3, C4, C1q, C5b-9, MBL-A, MBL-C, MASP-2, and factor B and properdin. In brief, the sections were washed with PBS, blocked with a blocking agent (DS Pharma Biomedical Co. Ltd., Osaka, Japan) at room temperature for 30 minutes, and then incubated at room temperature for 60 minutes with the following primary antibodies: goat anti-IgA, goat anti-IgG2a (Bethyl Laboratories Inc., Montgomery, TX), rat anti-C3, rat anti-C4, rat anti-C1q, rat anti-MBL-A, rat anti-MBL-C (Hycult Biotech, Uden, The Netherlands), anti-MASP-2, anti-factor B and anti-properdin (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-C5b-9 (Abcam Inc., Cambridge, MA). After three washes with PBS, the samples were incubated for 30 minutes with secondary antibodies compatible with the primary antibody to some degree, washed, and then mounted in mounting medium (Dako, Tokyo, Japan). Images were taken using a confocal laser microscope (Olympus Corp., Tokyo, Japan) with the same detector sensitivity for the same target antigen. The fluorescence-positive area was determined using ImageJ software (National Institutes of Health, Bethesda, MD) as described previously.15Kobayashi I. Nogaki F. Kusano H. Ono T. Miyawaki S. Yoshida H. Muso E. Interleukin-12 alters the physicochemical characteristics of serum and glomerular IgA and modifies glycosylation in a ddY mouse strain having high IgA levels.Nephrol Dial Transplant. 2002; 17: 2108-2116Crossref PubMed Scopus (34) Google Scholar For double IF staining (IgA-IgG2a, IgA–MBL-A, IgA–MBL-C, and IgA-C1q), the primary and secondary antibodies were added to the incubation mixture. Rabbit anti-mouse IgG2a was detected using Cy5-conjugated donkey anti-rabbit IgG antibody. The other primary antibodies were detected using the same secondary antibodies that were used for single IF staining. An enzyme-labeled antibody method was used to assess type IV collagen expression in the paraffin-embedded sections. After deparaffinization and rehydration, the renal sections were washed twice for 5 minutes with PBS. The sections were treated with 0.3% H2O2 for 30 minutes to block endogenous peroxide activity. They were then incubated at room temperature for 60 minutes with anti-type IV collagen antibody (AbD Serotec MorphoSys, Oxford, UK). After reaction with the primary antibody, the sections were reacted with horseradish peroxidase–conjugated goat anti-rabbit antibody (Nichirei Biosciences, Tokyo, Japan) and then visualized by treatment with diaminobenzidine (Dako). Quantitative analysis of type IV collagen expression in the glomerular areas was performed using minor modifications of a previously described method.16Lu T.C. Wang Z.H. Feng X. Chuang P.Y. Fang W. Shen Y. Levy D.E. Xiong H. Chen N. He J.C. Knockdown of Stat3 activity in vivo prevents diabetic glomerulopathy.Kidney Int. 2009; 76: 63-71Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar In brief, 20 random glomerular cross sections were chosen from each tissue section and examined. The examined areas were outlined, the positive staining patterns were identified, and the percentage of positive staining area in each glomerular cross section was then measured. Three nephrologists and pathologists quantified them in a blinded manner. To check the specificity of antibodies used, we examined kidneys of NOD SCID mice and MBL knockout mice as negative controls (data not shown). Glomeruli were isolated by a sieving technique. Total RNA was purified from the isolated glomeruli using an RNA extraction kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. Aliquots (500 ng) of total glomerular RNA were reverse transcribed (Life Technologies, Tokyo, Japan) and were analyzed using a real-time PCR system (Life Technologies) and Power SYBR green PCR master mix (Life Technologies). The primer sequence for type IV collagen has been published previously.17Herbach N. Schairer I. Blutke A. Kautz S. Siebert A. Goke B. Wolf E. Wanke R. Diabetic kidney lesions of GIPRdn transgenic mice: podocyte hypertrophy and thickening of the GBM precede glomerular hypertrophy and glomerulosclerosis.Am J Physiol Renal Physiol. 2009; 296: 819-829Crossref PubMed Scopus (60) Google Scholar Mouse glyceraldehyde-3-phosphate dehydrogenase was chosen as the housekeeping gene. All real-time PCR measurements were performed in triplicate and included no template controls. The glomerular transcript abundance of type IV collagen was calculated by the 2−ΔΔCT method.18Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (127155) Google Scholar Serum IgA levels were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Bethyl Laboratories Inc.). Aberrantly glycosylated bound serum IgA levels were measured using biotinylated Sambucus nigra bark lectin (SNA; Vector Laboratories, Burlingame, CA) and Ricinus communis agglutinin I (RCA-I; Vector Laboratories).19Chintalacharuvu S.R. Emancipator S.N. The glycosylation of IgA produced by murine B cells is altered by Th2 cytokines.J Immunol. 1997; 159: 2327-2333Crossref PubMed Google Scholar Microtiter plates coated with goat anti-mouse IgA used for quantification of serum IgA were incubated with these samples, and biotinylated SNA or RCA-I was then added. Horseradish peroxidase–avidin D (Vector Laboratories) was applied, and color developed after 15 minutes of exposure to 3,3′,5,5′-tetramethylbenzidine (Becton Dickinson, Tokyo, Japan). The color development reaction was stopped by adding 2 N of H2SO4, and absorbance at 450 nm was read using a microplate reader (Nihon Molecular Devices, Tokyo, Japan). A standard curve was produced for each of the two lectins using serial dilutions of the reference serum (Bethyl Laboratories Inc.). The quantity of lectin bound to 1 g of IgA was defined as 1 U. All the samples were reduced by 9% 2-mercaptoethanol (Wako Pure Chemical Industries Ltd., Tokyo, Japan) to avoid the influence of lectin binding by other constituent molecules in IgA-IC. Western blot analysis using serum samples under the reducing condition confirmed that the IgA-IgG IC was resolved (data not shown). IgA was purified from sera of gddY and HIGA mice, and the purity of the preparations was assessed by SDS-PAGE. Trichloroacetic acid-precipitated 10-μg protein for each IgA sample was analyzed by gas-liquid chromatography with sorbitol as the internal standard. The analyses were performed using a gas chromatograph (model 5890; Hewlett-Packard, Sacramento, CA) equipped with a 25-m fused silica (0.22-mm inner diameter) OV-1701 wall-coated open tubular column (Chrompack Inc., Bridgewater, NJ) and an electron capture detector. Standard sugars were used for quantification. The results are expressed relative to the internal standard and specific sugar standard, using the area under the peak in chromatograms.20Renfrow M.B. Cooper H.J. Tomana M. Kulhavy R. Hiki Y. Toma K. Emmett M.R. Mestecky J. Marshall A.G. Novak J. determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry.J Biol Chem. 2005; 280: 19136-19145Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 21Raska M. Takahashi K. Czernekova L. Zachova K. Hall S. Moldoveanu Z. Elliott M.C. Wilson L. Brown R. Jancova D. Barnes S. Vrbkova J. Tomana M. Smith P.D. Mestecky J. Renfrow M.B. Novak J. Glycosylation patterns of HIV-1 gp120 are cell-producing type dependent and affect antibody recognition.J Biol Chem. 2010; 285: 20860-20869Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar Serum IC (IgA-IgG2a, IgA–MBL-A, and IgA–MBL-C) levels were measured by sandwich ELISA using a modification of a method described previously.13Suzuki H. Suzuki Y. Aizawa M. Yamanaka T. Kihara M. Pang H. Horikoshi S. Tomino Y. Th1 polarization in murine IgA nephropathy directed by bone marrow-derived cells.Kidney Int. 2007; 72: 319-327Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar In brief, microtiter plates (Nalge Nunc International, Tokyo, Japan) were coated with anti-mouse IgA antibody. After incubation, the microtiter plates were washed with PBS containing 0.05% Tween 20 and then were blocked at room temperature for 60 minutes with 250 μL per well of blocking solution (1% blocking agent with PBS containing 0.05% Tween 20). The plates were then stored at 4°C until use. The microtiter plates coated with antibody were loaded in duplicate with diluted serum and were incubated at room temperature for 60 minutes. In the assay for the measurement of IgA-IgG2a complexes, the sample and antibody were diluted with blocking solution. For other complex assays, the samples were diluted with PBS containing 1 mmol/L CaCl2 and the antibody was diluted with blocking solution containing 1 mmol/L CaCl2. After washing with PBS containing 0.05% Tween 20, 90 μL per well of anti-IgG2a–horseradish peroxidase was used in the IgA-IgG2a complex assays. After incubation at room temperature for 60 minutes, 90 μL of 3,3′,5,5′-tetramethylbenzidine was added to each well. In the other assays, rat anti-MBL-A or rat anti-MBL-C was added to the wells and was incubated at room temperature for 60 minutes. After the plates were washed, biotinylated donkey anti-rat IgG was applied. After 60-minute incubation at room temperature, the microplates were washed five times with PBS containing 0.05% Tween 20, and horseradish peroxidase–avidin D was applied. After 60-minute incubation and further washing, the color was developed by 3,3′,5,5′-tetramethylbenzidine using the same method described for IgA-IgG2a IC. In all the assays, the color development reaction was stopped by adding 90 μL of 2 N H2SO4 to each well, and absorbance at 450 nm was read using the microplate reader. For comparison, each absorbance was adjusted for the serum IgA level. The molecular weight of IgA was determined by Western blot analysis using a modification of a method described previously.22Oida E. Nogaki F. Kobayashi I. Kamata T. Ono T. Miyawaki S. Serikawa T. Yoshida H. Kita T. Muso E. Quantitative trait loci (QTL) analysis reveals a close linkage between the hinge region and trimeric IgA dominancy in a high IgA strain (HIGA) of ddY mice.Eur J Immunol. 2004; 34: 2200-2208Crossref PubMed Scopus (11) Google Scholar The serum was resolved by 5% to 20% gradient SDS-PAGE and was transferred onto polyvinylidene difluoride membranes (Nihon Millipore KK, Tokyo, Japan). The membranes were blocked with 4% blocking solution and then were incubated with diluted goat anti-mouse IgA at room temperature for 60 minutes. After washing in PBS, the sections were incubated with the secondary antibody that corresponded to the primary antibody used. IgA was detected by chemiluminescence using ECL (GE Healthcare, Tokyo, Japan) and radiography (Konica Minolta Healthcare, Tokyo, Japan). The samples were analyzed under nonreducing conditions. The data are expressed as mean ± SD. The statistical significance of the experimental observations was determined by analysis of variance, with the level of significance set at P < 0.05. Descriptive statistical analysis was performed using statistical software (IBM Japan, Tokyo, Japan). The severity of glomerular injury was examined in both IgAN-prone strains of mice. The urinary albumin level in gddY mice at 11 weeks of age was considerably higher than that in age-matched HIGA mice (Figure 1A). Mesangial matrix expansion in gddY mice was also more severe than that in HIGA mice (Figure 1, B and C). Furthermore, expression of type IV collagen mRNA and protein in gddY mice was higher than that in HIGA mice (Figure 1, D–F). Although the degree of glomerular injury was very different in the two strains of mice, glomerular IgA deposition was identical in both IgAN-prone mouse strains (Figure 1, G and H). The degree of activation of complement pathways was investigated in murine IgAN. IF staining showed that glomerular deposition of C3 and C5b-9 was more abundant in gddY mice than in HIGA mice (Figure 2A). This finding indicated greater complement activation in gddY mice. To identify which complement pathways were involved, the key components of the three pathways were measured. The positive areas of glomerular C1q and C4 staining in gddY mice were significantly higher than those in HIGA mice (Figure 2C), indicating that the classical pathway in gddY mice was considerably more activated than that in HIGA mice. Next, the components of the lectin pathway, such as MBL-A/C and MASP-2, were analyzed in the glomeruli of both IgAN-prone strains of mice (Figure 2B). The amount of MBL-A/C deposition in gddY mice was significantly higher than that in HIGA mice. Similarly, the positive area of MASP-2 staining in gddY mice was also significantly higher than that in HIGA mice. These findings indicated that activation of the lectin pathway in glomeruli of gddY mice was higher than that in glomeruli of HIGA mice. Furthermore, the positive area of glomerular factor B and properdin staining in gddY mice was significantly higher than that in HIGA mice (Figure 2D). This finding indicated that the alternative pathway in gddY mice was also markedly more activated than that in HIGA mice. To assess why the classical pathway was more activated in gddY mice, the characteristics of glomerular ICs and their serum levels were investigated. Although serum IgA levels in HIGA mice were significantly higher than those in gddY mice (Figure 3A), the serum level of IgA-IgG2a IC per unit of IgA in gddY mice was significantly higher than that in HIGA mice (Figure 3B). Double IF staining showed that C1q was co-deposited with IgG2a and IgM (Figure 3, C and D). However, the positive area of glomerular IgG2a and IgM staining in gddY mice was significantly higher than that in HIGA mice (Figure 3, C and D). To assess the underlying mechanism that caused greater activation of the lectin pathway in gddY mice, MBL binding to IgA-IC was investigated (Figure 4). MBL-A and MBL-C were co-localized with IgA (Figure 4, A and B). Although serum IgA levels were significantly higher in HIGA mice than in gddY mice, serum levels of MBL bound to IgA-IC in gddY mice were significantly increased compared with those in HIGA mice (Figure 4, C and D). The molecular forms of serum IgA were analyzed in two IgAN-prone strains of mice (Figure 5). The amount of high-molecular-weight IgA in HIGA mice was higher than that in gddY mice (Figure 5A), similar to the situation observed for serum IgA levels. After compensation by serum IgA levels, the amount of high-molecular-weight IgA in gddY mice was not different from that in HIGA mice (Figure 5A). Next, the lectin-binding assays were used19Chintalacharuvu S.R. Emancipator S.N. The glycosylation of IgA produced by murine B cells is altered by Th2 cytokines.J Immunol. 1997; 159: 2327-2333Crossref PubMed Google Scholar to access the glycoform of IgA in both IgAN-prone strains of mice. This assay was designed using biotinylated SNA and RCA-I that recognize terminal sialic acid (particularly Neu5Aca2-6Gal) and galactose residues, respectively. Serum IgA from gddY mice showed a significantly lower affinity to lectin SNA and RCA-I than that from HIGA mice (Figure 5B). These findings suggest that the content of terminal sialic acid (amount of binding lectin SNA) and terminal galactose (amount of binding RCA-I) on IgA in gddY mice was significantly less than that on IgA in HIGA mice. Monosaccharide compositional analysis of purified IgA from gddY and HIGA mice was determined by gas-liquid chromatography. Sugar composition was consistent with the result of a lectin ELISA using SNA and RCA-I. Specifically, monosaccharide composition revealed that IgA purified from sera of gddY mice had a lower content of galactose and sialic acid compared with that of IgA from sera of HIGA mice (Table 1).Table 1Monosaccharide Composition of Murine IgA Determined by Gas-Liquid ChromatographySugarHIGA micegddY miceMannose0.4850.204GlcNAc0.2740.141GalNAcNDNDGalactose0.3020.148SA0.2570.135IS1.0001.000Data are expressed as the average of two experiments relative to standard sugars and normalized to the IS, based on the area under the peak in the chromatograms. Ten micrograms of IgA was used per sample.GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; IS, internal standard; ND, not detected; SA, sialic acid. Open table in a new tab Data are expressed as the average of two experiments relative to standard sugars and normalized to the IS, based on the area under the peak in the chromatograms. Ten micrograms of IgA was used per sample. GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; IS, internal standard; ND, not detected; SA, sialic acid. The degree of glomerular injury was more severe in gddY mice than in HIGA mice. gddY mice showed marked glomerular deposition of C5b-9 and C3 compared with HIGA mice, suggesting that in situ activation of the complement cascade was greater in gddY mice. This finding is compatible with the report that C5b-9 staining is more intense in human IgAN cases with advanced glomerular lesions than in those with minimal glomerular lesions.23Rauterberg E.W. Lieberknecht H.M. Wingen A.M. Ritz E.