IgG subclasses and complement pathway in segmental and global membranous nephropathy

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1 Pediatr Nephrol (2010) 25: DOI /s ORIGINAL ARTICLE IgG subclasses and complement pathway in segmental and global membranous nephropathy Yoshie Segawa & Satoshi Hisano & Misao Matsushita & Teizo Fujita & Shinichi Hirose & Morishige Takeshita & Hiroshi Iwasaki Received: 27 September 2009 / Revised: 28 December 2009 / Accepted: 30 December 2009 / Published online: 12 February 2010 # IPNA 2010 Abstract The aim of our study was to clarify the association between immunoglobulin G(IgG) subclasses and the complement pathway in patients with idiopathic membranous nephropathy (MN). Immunofluorescence (IF) was performed in 16 MN patients and 20 controls using antibodies against IgG, IgA, IgM, C1q, C3c, C4d, IgG1, IgG2, IgG3, IgG4, mannose binding lectin (MBL), C4-binding protein (C4-bp), factor B, C5b-9, and CD59. MN was classified into two types, segmental MN (S-MN; six patients) and global MN (G-MN; ten patients), according to the distribution of IgG deposits along the glomerular capillary wall. No deposition of any antibody was found in the controls. IF revealed IgG1, IgG3, C1q, C3c, C4d, C4-bp, C5b-9, and CD59 deposits in patients with S-MN, whereas IgG1, IgG2, IgG3, IgG4, C3c, C4d, MBL, factor B, C4-bp, C5b-9, and CD59 deposits were detected in those with G-MN. There was a higher deposition of IG1, IgG2, Y. Segawa : S. Hisano (*) : M. Takeshita : H. Iwasaki Department of Pathology, Faculty of Medicine, Fukuoka University, Nanakuma , Jonan-ku, Fukuoka City , Japan hisanos1@cis.fukuoka-u.ac.jp Y. Segawa : S. Hirose Department of Pediatrics, Faculty of Medicine, Fukuoka University, Fukuoka City, Japan M. Matsushita Institute of Glycotechnology and Department of Applied Biochemistry, Tokai University, Hiratsuka, Japan T. Fujita Department of Immunology, Fukushima Medical University, Fukushima, Japan and IgG4 in patients with G-MN than in those with S-MN, whereas the intensity of C1q deposits was higher in S-MN than in G-MN patients. In contrast, the intensity of factor B and MBL was higher in G-MN than in S-MN patients. This is the first report of S-MN patients showing complement activation of the classical pathway associated with IgG1 and IgG3 and G-MN patients showing complement activation of both the alternative and lectin pathways associated with IgG2 and IgG4. Keywords Complement pathway. IgG subclasses. Lectin pathway. Membranous nephropathy Introduction Idiopathic membranous nephropathy (MN) is a chronic kidney disease [1, 2]. It is characterized histologically by thickening of the glomerular basement membrane with spike formation, as observed by light microscopy (LM), fine granular deposits of immunoglobulin G (IgG) along the glomerular capillary wall, as observed by immunofluorescence microscopy (IF), and subepithelial electron-dense deposits (EDD) that are visibly in the electron microscope (EM) [1, 2]. IF usually reveals deposits of IgG globally along the glomerular capillary wall [1, 2], but patients with idiopathic segmental MN (S-MN) in whom IgG was deposited in a portion of the glomerular capillary wall have also been reported [3]. In this study, Obana et al. observed that the frequency of C1q deposits in S-MN was higher than that in global MN (G-MN), and they detected a higher prevalence of mesangial EDD in patients with S-MN [3]. The clinical outcome was not different between the two groups [3]. C1q is the first component of the complement pathway and,

2 1092 Pediatr Nephrol (2010) 25: consequently, the presence of C1q deposits indicate activation of the classical complement pathway. Idiopathic MN is reported to activate the alternative complement pathway based on findings of the combined deposition of IgG and C3 on IF. Idiopathic MN appears to be characterized by glomerular capillary deposits of predominantly IgG1 and IgG4, whereas glomerular capillary deposits of predominantly IgG1 and IgG3 are found in lupus MN [4, 5]. The aim of our study was to elucidate the association between the different IgG subclasses and the complement pathway in patients with S-MN and G-MN. Materials and methods Kidney tissue was obtained by means of percutaneous renal biopsy from 16 patients with idiopathic MN, aged 2 23 years (mean age 11.8±5.6 years), between 1994 and The percutaneous renal biopsy was performed on these patients due to persistent proteinuria or nephrotic syndrome. Patients with systemic lupus erythematosus, mixed connective tissue disease, viral hepatitis B and C, and malignant tumor were excluded from this study. The following patients were used as controls: ten patients with minimal change nephrotic syndrome (NS) and ten with thin basement membrane disease. Informed consent was obtained from patients and/or their parents before the renal biopsy was performed. The study protocol was implemented following approval of the Human Ethics Review Committee of Fukuoka University. Sections stained with periodic acid and Schiff and periodic acid methenamine silver were observed by LM. Idiopathic MN was divided into two groups according to the distribution of IgG deposits along the glomerular capillary wall as observed by IF: S-MN and G-MN. Segmental and global findings were determined based on the classification of lupus nephritis [6] as follows: the global lesion was identified to be the lesion occupying 50% of the glomerular tuft; the segmental lesion was determined as the lesion occupying <50% of the glomerular tuft. Mesangial hypercellularity was semiquantitatively determined on the basis of the Oxford classification of IgA nephropathy [7] on a score of 0 3+ as follows: 0, normal (<4, mesangial cells/mesangial area); 1, mild (4 5 mesangial cells/mesangial area); 2, moderate (6 7 mesangial cells/mesangial area); 3, severe (>8 mesangial cells/mesangial area). For quantification of the mesangial hypercellularity, the total score of mesangial hypercellularity in all glomeruli was divided by the total glomeruli, and the mean score was expressed as the average score of mesangial hypercellularity per glomerulus in each patient. The degree of subepithelial EDD and mesangial EDD was determined by EM and assessed on a scale of 0 3 according to the percentage of the glomerular basement membrane per total glomerular basement membrane or the percentage of total mesangial area, respectively, as follows: 0, no deposits; 1, occupying <25% of total glomerular basement membrane and total mesangial area, respectively; 2, occupying 25 50% of total glomerular basement membrane and total mesangial area, respectively; 3, occupying >50% of total glomerular basement membrane and total mesangial area, respectively. The clinical records examined at the time of the biopsy and the latest follow-up, including blood pressure, urinalysis, serum protein, creatinine clearance, and serum C3 and C4 concentrations, were obtained from the pediatricians and the internal medicine physicians who followed-up these 16 patients. Hematuria and proteinuria were determined as previously reported [8, 9]. Proteinuria was expressed semiquantitatively as milligram per deciliter. Hypertension in children was defined as blood pressure higher than the 95 percentile for age, as indicated by the Task Force on Blood Pressure Control in Children [10]. Hypertension in adults was defined according to the National High Blood Pressure Education Program Coordinating Committee [11]. Immunohistological staining Immunohistological staining was performed according to our previous reports [12, 13]. Fluorescein isothiocyanate (FITC)-labeled anti-igg, -IgA, -IgM, -C1q, and -C3c, and fibrinogen polyclonal antibodies (Dako, Copenhagen, Denmark), monoclonal anti-human C4d antibody (Quidel, San Diego, CA), and anti-factor B polyclonal antibody (Biogenesis, Poole, UK) were used. Monoclonal anti-igg1, -IgG2, and -IgG4 (Chemicon, Temecula, CA) and monoclonal antibodies against IgG3 (Zymed Laboratories, San Francisco, CA) were used. Monoclonal antibodies against human C4-binding protein (C4-bp; 11-2D3; Fuji yakuhin, Toyama, Japan), C5b-9 (Dako), and CD59 (Cosmobio, Tokyo, Japan) were used. Monoclonal antibodies against human mannose binding lectin (MBL; 3E7) was provided from T.F., as previously reported [14, 15]. Sections (thickness 4μm) cut from frozen tissues were immersed into Tris-buffered saline (TBS) three times for 5 min each time. Diluted FITC-labeled polyclonal antibodies (IgG, IgA, IgM, C3c, C1q, and fibrinogen), C4d monoclonal antibody, IgG1, IgG2, IgG3 and IgG4 monoclonal antibodies, MBL monoclonal antibody, C4-bp monoclonal antibody, C5b-9 monoclonal antibody, CD59 monoclonal antibody, and factor B polyclonal antibody were overlaid on the sections, following which the sections were incubated at 4 C overnight. Sections on which FITC-labeled polyclonal antibodies were overlaid were observed by IF. Immunohistochemical studies for monoclonal antibodies (IgG subclasses, C4d, MBL, C4-bp, CD5b-9, and CD59)

3 Pediatr Nephrol (2010) 25: and factor B polyclonal antibody were performed using the avidin biotin method as previously reported [12, 13]. Sections overlaid by monoclonal antibodies were incubated with biotin-labeled antibody against mouse IgG (Dako), and those overlaid by factor B polyclonal antibody were incubated with biotin-labeled antibody against goat IgG (Dako) at room temperature for 30 min. After three washes in TBS, the sections were incubated with FITC-labeled avidin for 30 min and then observed by IF. The area of the immunohistological deposits in glomeruli was based semiquantitatively on a grade of 0 to 3+ as follows: 0, negativity of glomerular area; 1+, 10 50% positivity of glomerular area; 2+, 51 75% positivity of glomerular area; 3+, >76% positivity of glomerular area. Immunohistochemical specificity was confirmed by an absorption test with antibodies and replacement of the primary antibodies to nonimmunized sera in accordance with the protocol described in our previous reports [12, 13]. As a negative staining control, nonimmune normal serum or TBS was substituted for the primary antibodies in accordance with previous reports [12, 13, 16]. Statistical analysis The data were expressed as mean ± standard deviation (SD). The difference in the mean values between groups was examined for statistical significance using the Mann Whitney U test. The association of categorical variables was examined by the chi-square test. A P value <5% was considered to be statistically significant difference. Results Immunohistochemical characterization of IgG, IgG subclasses, complement components, and complement regulatory factors Global and segmental deposits of IgG and the IgG subclasses along the glomerular capillary wall are shown in Fig.1. Glomerular deposits of complement components and complement regulatory factors are shown in Fig. 2. There was no deposition of any antibody in the controls. Profiles of patients with membranous nephropathy The profiles of 16 patients with MN are listed in Table 1. The patients were classified into two groups according to the distribution of IgG as shown in Table 1. Twopatients (patient nos. 3 and 10) had hypertension, and six (patient nos. 7 10, 12, and 14) in the G-MN group showed NS at the time of the biopsy. None of patients, with the exception of patient no. 3, had renal impairment at the time of renal biopsy. There was no difference in the stage classification based on EM observations between the two groups. Mesangial EDD was evident in five patients (patient nos. 1 3, 5, and 6) in the S-MN group and in two patients (patient nos. 14 and 15) in the G-MN group. Patients in the S-MN group were observed to have deposits of IgG1, IgG3, C1q, C3c, C4d, C4-bp, C5b-9, and CD59 along the glomerular capillary wall, whereas glomerular capillary deposits of IgG1, IgG2, IgG3, IgG4, C3c, C4d, MBL, factor B, C4-bp, C5b-9, and CD59 were detected in those with G-MN. No IgG2 and IgG4 deposits were detected in any patient in the S-MN group. In the S-MN group, the deposition of both IgG1 and IgG occurred segmentally along the glomerular capillary wall in all patients but one (patient no. 2), and IgG3 deposition was shown segmentally along the glomerular capillary wall in three patients (patients nos. 1, 2, and 5). IgG1 and IgG4 deposition as well as IgG deposition occurred globally along the glomerular capillary wall in all patients in the G-MN group. The deposits of complement components and complement regulatory factors were not consistent with the distribution of IgG deposition in the G-MN and S-MN groups. Comparison of clinical and histopathological indicators in patients with S-MN and G-MN Our comparison of clinical and pathological findings between the two patient groups is shown in Table 2. The mean age at onset of the disease and the mean duration between onset and biopsy were not different between S-MN and G-MN patients. There were more patients with NS in the G-MN group than in the S-MN group; consequently, the degree of proteinuria at the biopsy was higher in the G-MN group than in the S-MN group. The mesangial hypercellularity index was higher in the S-MN patients than in the G-MN patients. The mean intensity of IgG1, IgG2, and IgG4 deposits was greater in the G-MN group than in the S-MN group, that of C1q deposits was greater in the S-MN group than in the G-MN group, and that of MBL and factor B deposits was significantly stronger in the G-MN group than in the S-MN group. The degree of mesangial EDD was greater in the S-MN group than in the G-MN group, but the degree of subepithelial EDD and stage classification of MN were not different between the two groups (Table 1). Blood pressure and the degree of hematuria at the time of both the biopsy and the latest follow-up were not different between the two groups. Hematuria and proteinuria improved through the period of the follow-up. Creatinine clearance was not different between the two groups. Eight patients (patient nos. 2, 5, 6, 10, 12 15) showed normal urinalysis and normal renal function at the latest follow-up, as shown in Table 1. Renal failure was evident in one patient (patient

4 1094 Pediatr Nephrol (2010) 25: Fig. 1 Immunoglobulin G (IgG) and IgG subclasses are deposited along the glomerular capillary wall. a IgG in patients with global membranous nephropathy (G-MN), b IgG in patients with segmental MN (S-MN), c IgG1 in G-MN, d, IgG1inS-MN,e IgG2 in G-MN, f IgG3 in G-MN, g IgG4 in G-MN. Magnification 400 a b c d e f g no. 3) through the follow-up; the remaining patients had persistent urinary abnormalities with normal renal function. The clinical outcome was not different between the two groups through the mean follow-up of 70.5 months. None of patients with MN showed any clinical manifestation of collagen disease and infectious diseases through the followup period. Discussion The results of our study show that patients with idiopathic MN can be classified into those with S-MN and G-MN, respectively, thereby confirming the observations of Obana et al. [3]. Both our results and those of Obana et al. [3] demonstrate that the frequency of C1q deposits and

5 Pediatr Nephrol (2010) 25: Fig. 2 Complement components and complement regulatory factors are deposited along the glomerular capillary wall. a C1q in S-MN, b C3c in G-MN, c C4d in G-MN, d factor B in G-MN, e C4bp in G-MN, f C5b- 9 in G-MN, g CD59 in S-MN, h mannose binding lectin (MBL) in G-MN. Magnification 400 a b c d e f g h mesangial EDD was higher in S-MN patients than in G-MN patients. However, our study is the first to clarify the association between IgG subclasses and the complement pathway. Patients with S-MN showed complement activation of the classical pathway associated with IgG1 and IgG3, whereas those with G-MN showed complement activation of both the alternative and lectin pathways associated with IgG2 and IgG4. S-MN may be an early stage of idiopathic MN or it may be an incomplete or atypical form. The size of the immune complex may be larger in S-MN than in G-MN patients and, therefore, a large immune complex may be trapped in the

6 1096 Pediatr Nephrol (2010) 25: Table 1 Profiles of patients with membranous nephropathy at the time of biopsy Patient cohort Clinical findings Light microscopy Electron microscopy Immunofluorescence findings Treatment a Follow-up (months) Outcome b Patient no Age (years) BP H P Mode Ccr Mes Stage Subepi Mesa IgG1/ IgG2 IgG3/ IgG4 C1q/ C3 C4d/ C4b-p C5b-9/ CD59 Factor B/MBL Segmental / Asymptomatic /0 1/0 1/0 1/2 3/1 0/0 ARB 14 P+H / Asymptomatic /0 1/0 1/0 3/2 2/1 0/0 34 N / Asymptomatic /0 0/0 1/0 2/1 2/1 0/0 ACEI 62 RF / Asymptomatic /0 2/0 2/0 3/1 2/1 1/0 Unkown / Asymptomatic /0 1/0 3/3 3/1 3/3 0/0 Pr+CyA+D 51 N / Asymptomatic /0 2/0 3/1 N.D./2 3/1 0/0 ACEI 27 N Global / Nephrotic / Nephrotic / Nephrotic / Nephrotic /3 2/3 0/0 3/2 3/1 1/1 Pr+CyA+D 171 P /3 2/2 0/0 3/1 2/1 1/1 Pr+CyA+D 168 P+H /2 2/3 0/3 3/2 3/1 1/2 Pr+CyA 161 RF /1 1/3 0/3 3/2 2/1 1/1 Pr+CyA+D 24 N / Asymptomatic /1 3/3 1/2 3/2 2/1 1/1 Unknown / Nephrotic /1 3/2 1/3 N.D./2 1/1 1/0 Pr+CyA+ACEI 36 N / Asymptomatic /1 3/3 0/0 3/1 1/1 2/1 Pr+D 119 N / Nephrotic /3 3/3 0/3 3/1 2/1 1/1 Pr+CPA+ACEI 32 N / Asymptomatic /0 1/3 0/0 N.D./1 3/1 1/0 ACEI 18 N / Asymptomatic /1 0/3 0/0 3/1 2/1 1/1 Unkown BP, Blood pressure (mmhg); H, hematuria (red blood cells/high-powered field); P, proteinuria (mg/dl); Ccr, creatinine clearance (ml/min/1.73 m 2 ); Mes, mesangial hypercellularity; subepi, subepithelial electron-dense deposits; mesa, mesangial electron-dense deposits; MBL, mannose binding lectin; N.D., not determined a ARB, Angiotensin II receptor blocker; ACEI, angiotensin converting enzyme inhibitor; Pr, prednisolone; CyA, cyclosporin A; D, dipyridamole; CPA, cyclophosphamide b N, Normal examination; RF, renal failure; P+H, proteinuria + hematuria

7 Pediatr Nephrol (2010) 25: Table 2 Clinical and histopathological indicators in patients with membranous nephropathy Clinical and histopathological indicators Segmental Global P value Number of patients 6 10 Age at onset (years) 12.0± ±5.2 n.s. Time between onset and biopsy (months) 23.8± ±8.0 n.s. Mode of onset Nephrotic syndrome 0 6 < Asymptomatic 6 4 Mesangial hypercellularity index 0.24± ± Intensity of deposits IgG1 1.2± ± IgG2 0.0± ± IgG3 1.2± ±1.1 n.s. IgG4 0.0± ± C1q 2.0± ± C3c 0.7± ±1.5 n.s. C4d 2.4± ±0.0 n.s. Factor B 0.2± ± C4bp 1.5± ±0.5 n.s. C5b-9 2.5± ±0.7 n.s. CD59 1.3± ±0.8 n.s. MBL 0.0± ± Mesangial EDD 1.8± ± Time between biopsy and the latest follow-up (months) 37.6± ±70.0 n.s. Blood pressure (mmhg) At biopsy 118.3± ±16.5 n.s. At latest follow-up 112.2± ±14.3 n.s. Hematuria (RBC/hpf) At biopsy 17.1±15.7* 21.3±23.2** n.s. At latest follow-up 1.5± ±2.4 n.s. Proteinuria (mg/dl) At biopsy 76.0± ±323.2*** n.s. At latest follow-up 17.0± ±49.0 n.s. Creatinine clearance (ml/min/1.73 m 2 ) At biopsy 126.3± ±39.2 n.s. At latest follow-up 113.1± ±52.6 n.s. Values are expressed as mean ± SD. Data were analyzed by using the Mann Whitney U test *P= at biopsy vs. latest follow-up, **P= at biopsy vs. at latest follow-up, ***P= at biopsy vs. latest follow-up; n.s. not significant mesangium of the former. However, the degree of subepithelial EDD and the stage classification of MN were not different between the two groups and, consequently, the difference in the distribution of IgG deposits along the glomerular capillary wall between the two groups cannot be explained by the size of the immune complex. S-MN patients showed notable mesangial hypercellularity, C1q deposits, and mesangial EDD, indicating that S-MN may be secondary MN, although none of our patients showed any clinical manifestations of collagen diseases, any history of specific treatment, and any evidence of hepatitis viruses before the biopsy and during the period of the follow-up. Deposits of IgG subclasses and the complement pathway were apparently different between the two groups, suggesting the possibility that S-MN may be different from G-MN. A different antigenic stimulation or a different host immune response to the antigen may characterize the two groups. Based on the results of our study, we were unable to clarify mechanisms that can explain the difference in the association of IgG subclasses with the complement pathway between the two groups.

8 1098 Pediatr Nephrol (2010) 25: C1q is the first component of the classical pathway, and MBL is an activating factor of the lectin complement pathway [17 19]. Factor B activates the alternative complement pathway in the presence of factor D and properdin [20]. C4-bp is a sensitive indicator of C4 consumption and inactivates C4b as a cofactor for factor I [21]. C5b-9, a membrane attack complex, is an end product of complement activation [22]. CD59 is the sole inhibitor of the formation of the membrane attack complex [22]. Glomerular deposits of MBL, C1q, C3c, factor B, C4-bp, C5b-9, and CD59 indicate the occurrence of ongoing complement activation and complement regulation in situ in the glomerulus. On the basis of the combined presence of IgG and C3 deposits, as evidenced by IF, idiopathic MN in general seems to be activated through the alternative complement pathway. Haas [4] and Imai et al. [5] described a predominance of IgG4 in idiopathic MN. Doi et al. [23] reported that there was no demonstrable deposits of C1q and C4, as determined by IF, in patients with idiopathic MN, which was consistent with the lack of classical pathway-activating capacity of IgG4. In contrast, deposits of IgG1 and IgG3 have been found in patients with lupus MN [4, 5]. IgG1 and IgG3 appear to have the potential activity of the classical complement pathway [24, 25]. In our study, deposits of IgG1, IgG3, and C1q were found in the S-MN group. These findings are consistent with the activation of the classical pathway associated with IgG1 and IgG3. The lower intensity of IgG1 in the S-GN group than in the G-MN group is consistent with the segmental distribution of IgG shown by IF. We observed the deposition of MBL, C3c, C4d, and factor B in G-MN patients. In contrast, there were fewer patients with C1q deposits and the intensity of the C1q deposits was weaker in G-MN patients than in S-MN patients. The intensity of factor B and MBL deposits was greater in G-MN than in S-GM patients. These results indicate complement activation through both the alternative pathway and lectin pathways in G-MN patients. It is likely that IgG2 has the potentiality for activating the alternative pathway [26]. Therefore, the alternative pathway may be promoted to being associated with IgG2 subclass in the G-MN group. We reported previously that activation through the lectin pathway was detected in some patients with IgA glomerulonephritis, Schönlein-Henoch purpura nephritis, and poststreptococcal glomerulonephritis [12, 13, 27]. Therefore, it is not surprising that there are some patients with idiopathic MN showing activation through the lectin pathway. Deposits of IgG1, IgG2, IgG3, and IgG4 were detected in the G-MN group and the deposition of IG1, IgG2, and IgG4 was greater in G-MN patients than in S-MN patients. In the G-MN group, the intensity of IgG1 and IgG4 was stronger than that of IgG2 and IgG3. The predominance of IgG4 in our study is consistent with results reported previously [4, 5, 23]. Neither IgG2 nor IgG4 was detected in S-MN patients. To the best of our knowledge, the function of IgG4 is unknown in the complement pathway. It may play a pivotal role in the activation through the lectin pathway. The complement activation in G-MN patients is considered to occur through both the alternative pathway and lectin pathways associated with IgG2 and IgG4. The clinical outcome was not different between the two groups through the mean follow-up of 70.5 months. Two limitations to our study are the small number of subjects and the short follow-up. Future studies with a larger number of patients and a longer follow-up are needed in order to clarify whether S-MN is a distinct clinical entity or systemic manifestations will be shown in the future in patients with S-MN. In conclusion, two types of idiopathic MN can be differentiated according to the distribution of IgG deposits along the glomerular capillary wall: S-MN and G-MN. S-MN patients show complement activation of the classical pathway associated with IgG1 and IgG3, whereas G-MN patients show complement activation of both the alternative and lectin pathways associated with IgG2 and IgG4. References 1. Glassock RJ (2003) Diagnosis and natural course of membranous nephropathy. Semin Nephrol 23: Jennette JC, Olson JL, Schwartz MM, Silva FG (2007) Heptinstall s pathology of the kidney, 6th edn. Lippincott Williams & Wilkins, Philadelphia 3. Obana M, Nakanishi K, Sako M, Yata N, Nozu K, Tanaka R, Iijima K, Yoshikawa N (2006) Segmental membranous glomerulonephritis in children: comparison with global membranous glomerulonephritis. Clin J Am Soc Nephrol 1: Haas M (1994) IgG subclasses deposits in glomeruli of lupus and nonlupus membranous nephropathies. Am J Kidney Dis 23: Imai H, Hamai K, Komatsuda A, Ohtani H, Miura AB (1997) IgG subclasses in patients with membranoproliferative glomerulonephritis, membranous nephropathy, and lupus nephritis. Kidney Int 51: Weening JJ, D Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, Balow JE, Bruijin JA, Cook T, Ferrario F, Fogo AB, Ginzler EM, Hebert L, Hill G, Hill P, Jennette JC, Kong NC, Lesavre P, Lockshin M, Looi L-M, Makino H, Moura L, Nagata M (2004) The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 65: Working group of the International IgA Nephropathy Network and the Renal Pathology Society: Roberts ISD, Cook HT, Troyanov S, Alpers CE, Amore A, Barratt J, Berthoux F, Bonsib S, Bruijn JA, Cattran DC, Coppo R, D'Agati V, D'Amico G, Emancipator S, Emma F, Feehally J, Ferrario F, Fervenza FC, Florquin S, Fogo A, Geddes CC, Groene H-J, Haas M, Herzenberg AM, Hill PA, Hogg RJ, Hsu SI, Jennette JC, Joh K, Julian BA, Kawamura T, Lai FA, Li L-S, Li PKT, Liu Z-H, Mackinnon B, Mezzano S, Schena FP, TominoY, Walker PD, Wang H, Weening JJ, Yoshikawa N, Zhang

9 Pediatr Nephrol (2010) 25: H (2009) The Oxford classification of IgA nephropathy: pathology definitions, correlations, and reproducibility. Kidney Int 76: Hisano S, Ueda K (1989) Asymptomatic haematuria and proteinuria: pathology and clinical outcome in 54 children. Pediatr Nephrol 3: Hisano S, Kawano M, Hatae K, Kaku Y, Yamane I, Ueda K, Uragoh K, Honda S (1991) Asymptomatic isolated microhematuria: natural history of 136 children. Pediatr Nephrol 5: Task Force on Blood Pressure Control in Children (1977) Report of the task force on blood pressure control in children. Pediatrics 59[Suppl 5]: Chobanian AV, Bakris GL, Black HR, Cushman WG, Green LA, Izzo JL, Kones DW, Materson BJ, Oparil S, Wright JK Jr, Rocella EJ, the National High Blood Pressure Education Program Coordinating Committee (2003) The seventh report of the joint national committee on prevention, detection, and treatment of high blood pressure: The JNC 7 report. JAMA 289: Hisano S, Matsushita M, Fujita T, Endo Y, Takebayashi S (2001) Mesangial IgA2 deposits and lectin pathway-mediated complement activation in IgA glomerulonephritis. Am J Kidney Dis 38: Hisano S, Matsushita M, Fujita T, Iwasaki H (2005) Activation of the lectin complement pathway in Henoch-Schönlein purpura nephritis. Am J Kidney Dis 45: Matsushita M, Takahashi A, Hatsuse H, Kawakami M, Fujita T (1992) Human mannose-binding protein is identical to a component of Ra-reactive factor. Biochem Biophys Res Commun 183: Terai I, Kobayashi K, Matsushita M, Fujita T, Matsuno K (1995) α2-macroglobulin binds to and inhibits mannose-binding proteinassociated serine protease. Int Immunol 7: Nagata M, Akioka Y, Tsunoda Y, Kamatsu Y, Kawaguchi H, Yamaguchi Y, Ito K (1995) Macrophage in childhood IgA nephropathy. Kidney Int 48: Matsushita M, Fujita T (1992) (1992) Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J Exp Med 176: Thiel S, Vorup-Jensen T, Stover CM, Schwaeble W, Laursen SB, Poulsen K, Willis AC, Eggleton P, Hansen S, Holmskov U, Reid KB, Jensenius JC (1997) A second serine protease associated with mannose-binding lectin that activates complement. Nature 386: Takahashi M, Endo Y, Fujita T, Matsushita M (1999) A truncated form of mannose-binding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway. Int Immunol 11: Huang Y, Krein PM, Muruve DA, Winston BW (2002) Complement factor B gene regulation: Synthesis effects of TNF-alpha and IFN-gamma in macrophages. J Immunol 169: Endo M, Ohi H, Ohsawa I, Fujita T, Matsushita M, Fujita T (2000) Complement activation through the lectin pathway in patients with Henoch-Schönlein purpura nephritis. Am J Kidney Dis 35: Harris CL, Hanna SM, Mizuno M, Holt DS, Marchbank KJ, Morgan BP (2003) Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: tissue distribution and functional comparison. Immunology 109: Doi T, Kanatsu K, Nagai H, Suehiro S, Kuwahara T, Hamashima Y (1984) Demonstration of C3d deposits in membranous nephropathy. Nephron 37: Cunningham PN, Quigg RJ (2005) Contrasting roles of complement activation and its regulation in membranous nephropathy. J Am Soc Nephrol 16: Bindon CI, Hale G, Bruggemann M, Waldman H (1988) Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as C1q. J Exp Med 168: Bjornson AB, Lobel JS (1987) Direct evidence that decreased serum opsonization of streptococcus pneumonia via the alternative complement pathway in sickle cell disease is related to antibody deficiency. J Clin Invest 79: Hisano S, Matsushita M, Fujita T, Takeshita M, Iwasaki H (2007) Activation of the lectin complement pathway in post-streptococcal acute glomerulonephritis. Pathol Int 57: