Macromolecular IgA1 taken from patients with familial IgA Nephropathy or their asymptomatic relatives have higher reactivity to mesangial cells in vitro
2009; Elsevier BV; Volume: 75; Issue: 12 Linguagem: Inglês
10.1038/ki.2009.71
ISSN1523-1755
AutoresKa Ying Tam, Joseph C.K. Leung, Loretta Y.Y. Chan, Man Fai Lam, Sydney C.W. Tang, Kar Neng Lai,
Tópico(s)Celiac Disease Research and Management
ResumoMultiple cases of IgA nephropathy (IgAN) may occur in families; we compared their prognosis to sporadic cases of this disease. We isolated macromolecular IgA1 from 60 patients with familial IgAN, 91 of their asymptomatic relatives, 43 patients with sporadic IgAN (SpIgAN), 90 of their asymptomatic relatives, and 43 healthy subjects. Compared with SpIgAN patients, those with multiplex familial IgAN (MpIgAN) had more advanced renal histopathology and more galactose-deficient macromolecular IgA1 in their serum. Further, when we tested the effects of the macromolecular IgA1 on human mesangial cells in culture, we found that the macromolecular IgA1 taken from familial clusters had enhanced binding to mesangial cells and caused increased expression of interleukin-6, tumor necrosis factor-α, and monocyte chemotactic peptide-1. The macromolecular IgA1 isolated from asymptomatic relatives caused increased cytokine expression in the mesangial cells when derived from MpIgAN compared with SpIgAN or healthy controls. While these studies suggest that macromolecular IgA1 isolated from patients with MpIgAN is more pathogenic than that from patients with SpIgAN, long term follow-up will be needed to clarify the risk in asymptomatic relatives of the patients with multiplex familial disease. Multiple cases of IgA nephropathy (IgAN) may occur in families; we compared their prognosis to sporadic cases of this disease. We isolated macromolecular IgA1 from 60 patients with familial IgAN, 91 of their asymptomatic relatives, 43 patients with sporadic IgAN (SpIgAN), 90 of their asymptomatic relatives, and 43 healthy subjects. Compared with SpIgAN patients, those with multiplex familial IgAN (MpIgAN) had more advanced renal histopathology and more galactose-deficient macromolecular IgA1 in their serum. Further, when we tested the effects of the macromolecular IgA1 on human mesangial cells in culture, we found that the macromolecular IgA1 taken from familial clusters had enhanced binding to mesangial cells and caused increased expression of interleukin-6, tumor necrosis factor-α, and monocyte chemotactic peptide-1. The macromolecular IgA1 isolated from asymptomatic relatives caused increased cytokine expression in the mesangial cells when derived from MpIgAN compared with SpIgAN or healthy controls. While these studies suggest that macromolecular IgA1 isolated from patients with MpIgAN is more pathogenic than that from patients with SpIgAN, long term follow-up will be needed to clarify the risk in asymptomatic relatives of the patients with multiplex familial disease. IgA Nephropathy (IgAN), the most common primary glomerular disease, is a major cause of end-stage renal disease (ESRD).1.D'Amico G. The commonest glomerulonephritis in the world: IgA nephropathy.Q J Med. 1987; 64: 709-727PubMed Google Scholar IgAN exhibits a complex trait with the involvement of both genetic and environmental factors in the pathogenesis. Mucosal pathogens interacting with the mucosal IgA immune system play an important role in the pathogenesis of IgAN.2.Pouria S. Barratt J. Secondary IgA nephropathy.Semin Nephrol. 2008; 28: 27-37Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Deposition of polymeric IgA1 (pIgA1) and IgA1-contained immune complexes in mesangium is a hallmark in IgAN. Among the abnormalities that have been reported in IgAN, galactose-deficient (Gal-deficient) O-linked glycans at the hinge region of the IgA1 heavy chain is the most consistent finding and has been implicated in the pathogenesis of IgAN.3.Novak J. Julian B.A. Tomana M. et al.IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy.Semin Nephrol. 2008; 28: 78-87Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar IgA Nephropathy may occur in sporadic and familial forms.4.Egido J. Garcia-Hoyo R. Lozano L. et al.Immunological studies in familial and sporadic IgA nephropathy.Semin Nephrol. 1987; 7: 311-314PubMed Google Scholar Familial aggregation has been reported in Caucasian, Latin-American, and Asian populations with the incident rate accounting up to 10–15% of all cases in some regions.5.Wakai K. Kawamura T. Matsuo S. et al.Risk factors for IgA nephropathy: a case–control study in Japan.Am J Kidney Dis. 1999; 33: 738-745Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 6.Hsu S.I. Ramirez S.B. Winn M.P. et al.Evidence for genetic factors in the development and progression of IgA nephropathy.Kidney Int. 2000; 57: 1818-1835Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7.Izzi C. Sanna-Cherchi S. Prati E. et al.Familial aggregation of primary glomerulonephritis in an Italian population isolate: Valtrompia study.Kidney Int. 2006; 69: 1033-1040Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 8.Beerman I. Novak J. Wyatt R.J. et al.The genetics of IgA nephropathy.Nat Clin Pract Nephrol. 2007; 3: 325-338Crossref PubMed Scopus (59) Google Scholar The relationship between patients in familial IgAN varies greatly from first and second degree to more distant relationships.8.Beerman I. Novak J. Wyatt R.J. et al.The genetics of IgA nephropathy.Nat Clin Pract Nephrol. 2007; 3: 325-338Crossref PubMed Scopus (59) Google Scholar Familial clustering suggests an inheritable genetic predisposition of IgAN, although it does so with incomplete penetrance, nor does it follow the Mendelian transmission pattern.9.Frimat L. Kessler M. Controversies concerning the importance of genetic polymorphism in IgA nephropathy.Nephrol Dial Transplant. 2002; 17: 542-545Crossref PubMed Scopus (10) Google Scholar Recent family-based genome-wide scan linkage studies identified several susceptibility loci, including 6q22–23 [IGAN-1], 3p24–23, 4q26–31, 17q12–22, and 2q36, from different cohorts, yet no single locus is responsible for disease linkage in these studies.10.Hsu S.I. Racial and genetic factors in IgA nephropathy.Semin Nephrol. 2008; 28: 48-57Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Examination of possible candidate genes, including C4 null allele, galactosyl transferase, and major histocompatibility complex antigens, failed to show an association with the pathogenesis of IgAN.6.Hsu S.I. Ramirez S.B. Winn M.P. et al.Evidence for genetic factors in the development and progression of IgA nephropathy.Kidney Int. 2000; 57: 1818-1835Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar The issue that familial IgAN confers an increased risk of progression to ESRD with a poor prognosis remains controversial.11.Schena F.P. Cerullo G. Rossini M. et al.Increased risk of end-stage renal disease in familial IgA nephropathy.J Am Soc Nephrol. 2002; 13: 453-460PubMed Google Scholar Data on the nature of IgA1 molecules from familial IgAN are limited. Recently, Gharavi et al.12.Gharavi 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 (171) Google Scholar reported the heritability of the aberrant IgA1 glycoform. Higher serum level of Gal-deficient IgA1 is observed in multiplex familial IgAN (MpIgAN) patients when compared with that in sporadic form. Moreover, elevated Gal-deficient IgA1 level is also found among a significant portion of at-risk asymptomatic blood-related members as compared with that among married-in individuals, raising the notion of genetic transmission of Gal-deficient IgA1 production. Herein, we examined circulating macromolecular IgA1 from IgAN patients and from relatives of patients with MpIgAN and compared it with that isolated from patients with sporadic IgAN (SpIgAN) and from their relatives. We further examined whether these macromolecular IgA1 exhibited active binding to human mesangial cells (HMCs), thus promoting glomerular damage after mesangial deposition. The clinical characteristics of the MpIgAN (also abbreviated as FA) and SpIgAN (also abbreviated as SA) are summarized in Table 1. The duration of follow-up for these patients varied from 2 to 10 years. The histological grading at the time of biopsy was higher in MpIgAN than that in SpIgAN (P=0.037). Elevation of urinary protein was detected in MpIgAN at the time of latest follow-up as compared with that in SpIgAN (P=0.013), although the rate of changes in urinary protein was comparable between the two groups of IgAN patients. No significant difference was observed in serum creatinine and glomerular filtration rate between MpIgAN and SpIgAN.Table 1Clinical data of multiplex familial or sporadic IgANSporadic IgAN patients (SA)Multiplex familial IgAN patients (FA)P-valueNumber of patients4360Number of families4330Gender (male/female)13/3020/40NSGradeaGrade: histological grading determined by the classification published by Lai et al.45.1.35±0.571.63±0.740.037Follow-up (year)5.67±2.274.97±2.15NSCreatinine (mmol/l) Initial97.57±30.7189.78±21.67NS Follow-up123.43±53.05112.00±38.41NS Change per year4.73±7.825.82±10.27NSProteinuria (g/day) Initial0.91±0.701.37±0.76NS Follow-up0.97±1.042.25±1.370.013 Change per year0.04±0.170.21±0.31NSGFR (ml/min per 1.73 mm 2 body surface area) Initial102.64±26.7787.07±18.50NS Follow-up76.91±23.7162.13±17.34NS Change per year-4.37±4.81-5.44±4.04NSValues are mean±s.d. Data were compared by unpaired t-test. GFR, glomerular filtration rate; NS, not significant.a Grade: histological grading determined by the classification published by Lai et al.45.Lai K.N. Ho C.P. Chan K.W. et al.Nephrotic range proteinuria—a good predictive index of disease in IgA nephropathy?.Q J Med. 1985; 57: 677-688PubMed Google Scholar. Open table in a new tab Values are mean±s.d. Data were compared by unpaired t-test. GFR, glomerular filtration rate; NS, not significant. The serum IgA1 level of both groups of IgAN patients was significantly higher than that in healthy controls (CL) (FA/SA vs CL, P<0.05), despite no significant difference between these two groups of IgAN patients (FA vs SA, NS) (Figure 1a). The serum level of macromolecular IgA1 was higher in IgAN patients and also in relatives of MpIgAN (FR) when compared with that in controls (FA/SA/FR vs CL, P<0.05). In contrast to serum IgA1, MpIgAN had a higher serum macromolecular IgA1 level than did SpIgAN (FA vs SA, P<0.05) (Figure 1b). The serum level of Gal-deficient macromolecular IgA1 (Gal-deficient IgA1) is shown in Figure 2a. Patients with MpIgAN had a higher serum level of Gal-deficient macromolecular IgA1 than did those with SpIgAN (FA vs SA, P<0.05). The serum Gal-deficient macromolecular IgA1 level in both groups of IgAN patients was also significantly higher than that in their corresponding family relatives or in the healthy controls (FA vs FR and SA vs relatives of SpIgAN (SR), P<0.001; and FA/SA vs CL, P<0.001). Most intriguingly, asymptomatic relatives of patients with MpIgAN showed a higher serum Gal-deficient macromolecular IgA1 level than did the controls or relatives of those with SpIgAN (FR vs CL/SR, P<0.001), with no difference in the serum Gal-deficient macromolecular IgA1 level between controls and relatives of SpIgAN (CL vs SR, NS). A similar finding was observed for HMCs binding of macromolecular IgA1 (Figure 2b). There was higher HMCs binding of macromolecular IgA1 from MpIgAN (FA vs SA, P<0.05). The HMCs binding of macromolecular IgA1 for both groups of IgAN patient was significantly higher than their that in corresponding family relatives or controls (FA vs FR and SA vs SR, P<0.001; FA/SA vs CL, P<0.001). Relatives of MpIgAN patients exhibited higher macromolecular IgA1 binding to HMCs when compared with controls or relatives of SpIgAN patients (FR vs CL/SR, P<0.01). Figure 3 depicts the gene expression and protein synthesis of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemotactic peptide-1 (MCP-1) in HMCs cultured with macromolecular IgA1. Compared with SpIgAN, macromolecular IgA1 from MpIgAN induced a higher cytokine production by cultured HMCs (FA vs SA, P<0.05). Macromolecular IgA1 from MpIgAN induced twofold increases in the mRNA expression of MCP-1 and TNF-α by HMCs. Protein synthesis of MCP-1 and TNF-α was increased by two- and 25-fold, respectively, under similar experimental conditions. More strikingly, macromolecular IgA1 from MpIgAN induced 18- and 390-fold increases in the mRNA expression (FA, median=1965, range: 832–5257; SA, median=106, range: 18–1060) and protein synthesis of IL-6, respectively (FA, median=1426, range: 420–2798; SA, median=3.66, range: 2–11.64) as compared with that from SpIgAN. Macromolecular IgA1 from relatives of the MpIgAN family also induced greater synthesis of IL-6 and MCP-1 when compared with that from controls or relatives of the SpIgAN family (FR vs CL/SR, P<0.01). Similarly, the expression of these cytokines by HMCs cultured with macromolecular IgA1 from controls did not differ from that of relatives of SpIgAN (SR vs CL, NS). The increased expression of IL-6, TNF-α, and MCP-1 by HMCs cultured with macromolecular IgA1 was abolished after the fractions were depleted of IgA1 (Supplementary Table 1), excluding the potential presence of other biologically active components in the macromolecular IgA1 fractions. Download .doc (.04 MB) Help with doc files Supplementary Table 1 Data of cytokine production by HMCs were further confirmed by time- and dose–response studies. Patients with MpIgAN induced greater production of IL-6, TNF-α, and MCP-1 by HMCs for all time points examined as compared with those with SpIgAN (FA vs SA, P 250 ng/ml) was required to induce a comparable level of protein synthesis of MCP-1 and TNF-α.Figure 5Gene expression and protein release of IL-6, MCP-1, and TNF-α in HMCs cultured with a different dose of macromolecular IgA1 for 4 or 48 h. Results were obtained from experiments using three independent samples (each sample was pooled from 10 randomly selected cases in each group) and are expressed as mean±s.e.m. The gene expression is expressed as fold change related to the value of medium control. Data were analyzed by one-way analysis of variance with Bonferroni's correction. *P<0.05, #P<0.01, δP<0.001 versus dose zero in the corresponding group.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The correlations between serum level of Gal-deficient IgA1, HMCs binding of macromolecular IgA1, and the cytokine production by HMCs after binding to macromolecular IgA1 were examined. There was no correlation between HMCs binding of macromolecular IgA1 and serum level of Gal-deficient macromolecular IgA1, or the cytokine production by HMCs (Tables 2 and 3). In contrast, the gene expression of IL-6 and MCP-1 in HMCs cultured with macromolecular IgA1 correlated with the serum level of Gal-deficient macromolecular IgA1 in patients with IgAN and in their relatives (P<0.05), but not in healthy controls (Table 2). The synthesis of IL-6 and MCP-1 by HMCs cultured with macromolecular IgA1 correlated well in all studied subjects (P<0.05) (Table 4).Table 2Correlation between serum level of Gal-deficient macromolecular IgA1and the HMCs binding of macromolecular IgA1 or macromolecular IgA1-induced cytokine production by HMCsIL-6MCP-1TNF-αHMCs bindingmRNAProteinmRNAProteinmRNAProteinCLr=0.1345r=0.2983r=0.0034r=0.0643r=0.1167r=-0.0083r=0.0715NSNSNSNSNSNSNSSRr=0.3124r=0.2179r=0.3157r=0.3124r=0.0461r=0.1012r=0.0332NSP=0.0479P=0.0037P=0.0040NSNSNSSAr=0.1439r=0.3181r=0.3482r=0.4242r=-0.0368r=0.2497r=-0.2625NSP=0.0427P=0.0471P=0.0057NSNSNSFRr=0.0450r=0.2390r=0.4974r=0.3807r=0.1943r=-0.0778r=-0.1307NSP=0.0296P<0.0001P=0.0002NSNSNSFAr=-0.1980r=0.3398r=0.7601r=0.4512r=0.1150r=0.6726r=-0.0188NSP=0.0128P<0.0001P=0.0006NSP<0.0001NSHMCs, human mesangial cells; IL, interleukin; MCP, monocyte chemotactic peptide; NS, not significant; r, spearman's correlation r; TNF, tumor necrosis factor. Open table in a new tab Table 3Correlation between the HMCs binding of macromolecular IgA1 and macromolecular IgA1-induced cytokine production by HMCsIL-6MCP-1TNF-αmRNAProteinmRNAProteinmRNAProteinCLr=-0.2058r=-0.2885r=-0.2930r=-0.2604r=-0.1540r=0.2570NSNSNSNSNSNSSRr=0.0629r=0.2177r=0.0766r=0.0887r=0.1307r=-0.1005NSNSNSNSNSNSSAr=0.1017r=0.1361r=-0.0746r=-0.0269r=0.2093r=-0.0250NSNSNSNSNSNSFRr=-0.0017r=0.1443r=-0.0769r=-0.1044r=-0.0844r=-0.2461NSNSNSNSNSNSFAr=0.1777r=-0.2434r=0.0451r=-0.0786r=-0.1024r=-0.1761NSNSNSNSNSNSHMCs, human mesangial cells; IL, interleukin; MCP, monocyte chemotactic peptide; NS, not significant; r, spearman's correlation r; TNF, tumor necrosis factor. Open table in a new tab Table 4Correlation between different cytokines production by HMCs following incubation with macromolecular IgA1IL-6 vs MCP-1IL-6 vs TNF-αMCP-1 vs TNF-αmRNAProteinmRNAProteinmRNAProteinCLr=0.9686r=0.4843r=0.8980r=0.0824r=0.9408r=0.1836P<0.0001P=0.0012P<0.0001NSP<0.0001NSSRr=0.8812r=0.2955r=0.5160r=0.0307r=0.5112r=-0.1384P<0.0001P=0.0052P<0.0001NSP<0.0001NSSAr=0.5125r=0.5184r=0.4988r=0.0626r=0.3953r=0.2964P=0.0004P=0.0010P=0.0007NSP=0.0087NSFRr=0.6566r=0.4614r=0.4757r=-0.2123r=0.5946r=-0.1698P<0.0001P<0.0001P<0.0001NSP<0.0001NSFAr=0.4424r=0.2772r=0.4584r=0.1785r=0.5362r=-0.0042P=0.0006P=0.0386P=0.0003NSP<0.0001NSHMCs, human mesangial cells; IL, interleukin; MCP, monocyte chemotactic peptide; NS, not significant; r, spearman's correlation r; TNF, tumor necrosis factor. Open table in a new tab HMCs, human mesangial cells; IL, interleukin; MCP, monocyte chemotactic peptide; NS, not significant; r, spearman's correlation r; TNF, tumor necrosis factor. HMCs, human mesangial cells; IL, interleukin; MCP, monocyte chemotactic peptide; NS, not significant; r, spearman's correlation r; TNF, tumor necrosis factor. HMCs, human mesangial cells; IL, interleukin; MCP, monocyte chemotactic peptide; NS, not significant; r, spearman's correlation r; TNF, tumor necrosis factor. In this study, we present our novel finding that there is an enhanced reactivity of HMCs toward macromolecular IgA1 isolated from MpIgAN as compared with that toward macromolecular IgA1 from SpIgAN. Such enhanced reactivity may be relevant to the disease progression in MpIgAN. Interestingly, this increased HMCs reactivity was also evidenced, although to a lesser extent, in asymptomatic family relatives of MpIgAN. Familial IgAN was initially reported in the late 1970s,13.Tolkoff-Rubin N.E. Cosimi A.B. Fuller T. et al.IGA nephropathy in HLA-identical siblings.Transplantation. 1978; 26: 430-433Crossref PubMed Scopus (86) Google Scholar, 14.Sabatier J.C. Genin C. Assenat H. et al.Mesangial IgA glomerulonephritis in HLA-identical brothers.Clin Nephrol. 1979; 11: 35-38PubMed Google Scholar with an increasing recognition in the past two decades mainly in European population.8.Beerman I. Novak J. Wyatt R.J. et al.The genetics of IgA nephropathy.Nat Clin Pract Nephrol. 2007; 3: 325-338Crossref PubMed Scopus (59) Google Scholar, 15.Levy M. Familial cases of Berger's disease and anaphylactoid purpura: more frequent than previously thought.Am J Med. 1989; 87: 246-248Abstract Full Text PDF PubMed Scopus (32) Google Scholar IgAN is the most common primary glomerular disease in China,16.Xie Y. Chen X. Epidemiology, major outcomes, risk factors, prevention and management of chronic kidney disease in China.Am J Nephrol. 2008; 28: 1-7Crossref PubMed Scopus (59) Google Scholar but data of familial IgAN in the Chinese population are limited.17.Li P.K. Burns A.P. So A.K. et al.Familial IgA nephropathy: a study of HLA class II allogenotypes in a Chinese kindred.Am J Kidney Dis. 1992; 20: 458-462Abstract Full Text PDF PubMed Scopus (14) Google Scholar, 18.Li Y.J. Du Y. Li C.X. et al.Family-based association study showing that immunoglobulin A nephropathy is associated with the polymorphisms 2093C and 2180T in the 3′ untranslated region of the Megsin gene.J Am Soc Nephrol. 2004; 15: 1739-1743Crossref PubMed Scopus (40) Google Scholar MpIgAN is reportedly associated with a poorer renal prognosis, with 64% developing into ESRD whereas only 8% does so in SpIgAN.11.Schena F.P. Cerullo G. Rossini M. et al.Increased risk of end-stage renal disease in familial IgA nephropathy.J Am Soc Nephrol. 2002; 13: 453-460PubMed Google Scholar However, a recent study suggests that the presence of familial disease does not confer an increased risk for disease progression.19.Izzi C. Ravani P. Torres D. et al.IgA nephropathy: the presence of familial disease does not confer an increased risk for progression.Am J Kidney Dis. 2006; 47: 761-769Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar In this study, we have adopted a functional approach by comparing the HMCs reactivity toward Gal-deficient macromolecular IgA1 from multiplex familial or SpIgAN, thereby providing additional pathogenetic data to address these controversial findings. Neither clinical data nor the renal histological staining pattern is useful in differentiating multiplex familial from SpIgAN.19.Izzi C. Ravani P. Torres D. et al.IgA nephropathy: the presence of familial disease does not confer an increased risk for progression.Am J Kidney Dis. 2006; 47: 761-769Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 20.Julian B.A. Woodford S.Y. Baehler R.W. et al.Familial clustering and immunogenetic aspects of IgA nephropathy.Am J Kidney Dis. 1988; 12: 366-370Abstract Full Text PDF PubMed Scopus (20) Google Scholar Our current data also show that there was no difference in serum creatinine or glomerular filtration rate between MpIgAN and SpIgAN, either at the time of first presentation or latest follow-up, for an intermediate period averaging 5 years. However for proteinuria, a proven prognostic marker in IgAN, a significant difference was observed between two groups of patients at the latest follow-up, although just failing to reach a statistical difference when the data were normalized with the duration of follow-up. This may not be surprising with the variable progression rate for IgAN patients reaching ESRD within a few years to more than 50 years.21.Geddes C.C. Rauta V. Gronhagen-Riska C. et al.A tricontinental view of IgA nephropathy.Nephrol Dial Transplant. 2003; 18: 1541-1548Crossref PubMed Scopus (146) Google Scholar Contrary to the Italian data,19.Izzi C. Ravani P. Torres D. et al.IgA nephropathy: the presence of familial disease does not confer an increased risk for progression.Am J Kidney Dis. 2006; 47: 761-769Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar we observed a severer histological grading in MpIgAN patients at first biopsy. It may suggest that familial aggregation is associated with a more advanced renal lesion. As the serum creatinine, proteinuria, and glomerular filtration rate were comparable between MpIgAN and SpIgAN at the time of first biopsy, the histological findings were less likely to be because of sampling bias. With the variable, relentless, and slowly progressive course of IgAN, our study acknowledged the drawback of including patients with different follow-up durations or treatment protocols. Instead, we examined the possible inherent role of Gal-deficient macromolecular IgA1 on HMCs binding and reactivity. These macromolecular IgA1s were isolated from MpIgAN and SpIgAN during clinical quiescence. Our study of the intrinsic reactivity of macromolecular IgA1 to HMCs should unlikely be influenced by the rate of progression and treatment protocols of the disease. Gharavi et al.12.Gharavi 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 (171) Google Scholar reported the presence of elevated circulating Gal-deficient IgA in MpIgAN. The current consensus supports a key role of Gal-deficient pIgA1 in the pathogenesis of IgAN, as Gal-deficient pIgA1 can self-aggregate or bind to soluble IgA Fc receptor or other circulating proteins, including C3, IgG, IgM, and fibronectin, to form circulating IgA1-immune complexes with an aberrant glycosylation pattern22.Mestecky J. Tomana M. Moldoveanu Z. et al.Role of aberrant glycosylation of IgA1 molecules in the pathogenesis of IgA nephropathy.Kidney Blood Press Res. 2008; 31: 29-37Crossref PubMed Scopus (67) Google Scholar that may favor mesangial deposition of these complexes.3.Novak J. Julian B.A. Tomana M. et al.IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy.Semin Nephrol. 2008; 28: 78-87Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 22.Mestecky J. Tomana M. Moldoveanu Z. et al.Role of aberrant glycosylation of IgA1 molecules in the pathogenesis of IgA nephropathy.Kidney Blood Press Res. 2008; 31: 29-37Crossref PubMed Scopus (67) Google Scholar, 23.Tomana M. Matousovic K. Julian B.A. et al.Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 24.Allen 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 (244) Google Scholar However, the exact mechanism leading to mesangial deposition of pIgA1 is not yet satisfactorily defined. Novak et al.25.Novak J. Vu H.L. Novak L. et al.Interactions of human mesangial cells with IgA and IgA-containing immune complexes.Kidney Int. 2002; 62: 465-475Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar observed that HMCs bound better to the Gal-deficient pIgA1 myeloma protein than to the normal galactosylated IgA1 myeloma protein. Moreover, immune complexes containing Gal-deficient pIgA1 isolated from IgAN patients bound better to HMCs than did the Gal-deficient pIgA1 myeloma protein. This suggests that aberrant glycosylation of macromolecular IgA1 is not the only determinant controlling mesangial binding; and other factors are also involved. We had shown earlier that mesangial binding of macromolecular IgA1 could be size- and charge-dependent.26.Leung J.C. Chan L.Y. Tang S.C. et al.Glycosylation profile of differently charged IgA1 and their binding characteristics to cultured mesangial cells in IgA nephropathy.Nephron Exp Nephrol. 2007; 107: e107-e118Crossref PubMed Scopus (7) Google Scholar, 27.Leung J.C. Tsang A.W. Chan L.Y. et al.Size-dependent binding of IgA to HepG2, U937, and human mesangial cells.J Lab Clin Med. 2002; 140: 398-406Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Our present results show no significant correlations between the amounts of Gal-deficient macromolecular IgA1 with their HMCs binding or with their induction of protein expression of TNF-α and MCP-1 by HMCs. Our data implicate that both the quantity and quality of macromolecular IgA1 from SpIgAN and MpIgAN may influence the binding of macromolecular IgA1 and their activation of HMCs. Heterogeneity of the size of macromolecular IgA1 has been shown earlier28.Czerkinsky C. Koopman W.J. Jackson S. et al.Circulating immune complexes and immunoglobulin A rheumatoid factor in patients with mesangial immunoglobulin A nephropathies.J Clin Invest. 1986; 77: 1931-1938Crossref PubMed Scopus (157) Google Scholar and induction of HMCs proliferation has been restricted to the macromolecular IgA1 of size 800–900 kDa.29.Novak J. Tomana M. Matousovic K. et al.IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells.Kidney Int. 2005; 67: 504-513Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar Other than the size, the number and sites of attachment of the Gal-deficient O-linked glycans of macromolecular IgA1 may also have an effect on their activity on HMCs. It has been suggested that not all the terminal Gal-deficient GalNAc residues of IgA1-immune complexes are occupied by anti-GalNAc antibodies.22.Mestecky J. Tomana M. Moldoveanu Z. et al.Role of aberrant glycosylation of IgA1 molecules in the pathogenesis of IgA nephropathy.Kidney Blood Press Res. 2008; 31: 29-37Crossref PubMed Scopus (67) Google Scholar The HAA reactivity with the exposed GalNAc can be affected by the sites of attachment by anti-GalNAc antibodies, which in turn may alter the conformation and size of the IgA1-immune complexes and affect the HMCs binding characteristics.22.Mestecky J. Tomana M. Moldoveanu Z. et al.Role of aberrant glycosylation of IgA
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