Zhao Cui

Natural autoantibodies to myeloperoxidase, proteinase 3, and the glomerular basement membrane are present in normal individuals

Anti-neutrophil cytoplasmic antibodies (ANCAs) have a pathogenic role in ANCA-associated vasculitis. The origin of ANCAs and anti-glomerular basement membrane (GBM) antibodies, however, is unknown. In this study, we determined whether natural autoantibodies against myeloperoxidase (MPO), proteinase 3 (PR3), and GBM were present in each of 10 healthy Chinese and Swedish individuals, negative for all three antigens by routine ELISA. Antibodies were purified from isolated IgG by antigen-specific affinity columns. Natural anti-GBM autoantibodies gave a linear staining pattern along the GBM of human renal sections. On ethanol-fixed granulocytes, both natural anti-MPO and anti-PR3 autoantibodies gave cytoplasmic staining. The titers of natural anti-MPO/PR3 autoantibodies were significantly lower than those from patients with vasculitis. In competition ELISA, the binding of natural anti-MPO autoantibodies could be inhibited by MPO, but not by PR3 or noncollagenous domains from type IV collagen. The same specificity results were found for natural anti-PR3 and anti-GBM autoantibodies. Overall, individuals of the Chinese origin had more natural autoantibodies than did those of the Swedish origin, but no other differences were found. Hence, our study shows that healthy individuals have masked circulating, noncross-reactive, antigen-specific natural autoantibodies against MPO, PR3, and GBM in their serum and IgG fractions. Further studies are needed to determine their role if any in the etiology of ANCA-associated vasculitis and anti-GBM disease.

Antigen and Epitope Specificity of Anti–Glomerular Basement Membrane Antibodies in Patients with Goodpasture Disease with or without Anti-Neutrophil Cytoplasmic Antibodies

Goodpasture disease (GP) is defined by the presence of anti-glomerular basement membrane (anti-GBM) antibodies and rapidly progressive glomerulonephritis. Besides anti-GBM, many patients with GP produce anti-neutrophil cytoplasmic antibodies (ANCA). For elucidation of the pathophysiologic significance of ANCA in this setting, epitope and antigen specificity of the anti-GBM antibodies and antigen specificity of ANCA were studied. Bovine testis alpha(IV)NC1 (tNC1); recombinant human alpha1, alpha3, alpha4, and alpha5(IV)NC1 (ralpha1 through ralpha5); and three chimeric proteins that contain previously defined epitope regions designated E(A), E(B), and S2 were used to examine the anti-GBM antibodies by ELISA in 205 Chinese patients with GP with or without ANCA. In the 205 anti-GBM antibody-positive sera, 63 (30.7%) were also ANCA positive (61 myeloperoxidase-ANCA and six proteinase 3-ANCA, four being triple positive). All 205 sera recognized tNC1 and ralpha3(IV)NC1. In the double-positive group, 54.0, 66.7, 71.4% of the sera could recognize ralpha1, ralpha4, and ralpha5, respectively, compared with 49.3, 60.6, and 55.6% for patients with anti-GBM antibodies alone. The levels of the antibodies to ralpha3, tNC1, and the alpha3/alpha1 ratio were lower in the double-positive group than that in patients with anti-GBM antibody alone (P < 0.05). Most of the sera could recognize the epitope regions E(A), E(B), and S2, but the absorbance values to E(A), E(B), and S2 were lower in double-positive group (P < 0.05). Double-positive patients had a broader spectrum of anti-GBM antibodies and lower levels of antibodies against alpha3(IV)NC1 compared with that of patients with anti-GBM antibodies alone.

Plasma soluble urokinase receptor levels are increased but do not distinguish primary from secondary focal segmental glomerulosclerosis

In this study, we measured soluble urokinase receptor levels, a possible permeability factor, in the plasma of patients with primary focal segmental glomerulosclerosis (FSGS) and determined their association with clinical and pathological data in 74 patients with primary FSGS. Healthy donors and patients with minimal change disease, membranous nephropathy, and secondary FSGS were used as controls. The plasma-soluble urokinase receptor levels, measured by commercial ELISA kits, of patients with primary FSGS (median: 2923, interquartile range 2205-4360 pg/ml) were significantly higher than those of patients with minimal change disease (median 2050 pg/ml), membranous nephropathy (median 2029 pg/ml), and normal individuals (median 1739 pg/ml). There was no significant difference in plasma-soluble urokinase receptor levels between the 74 patients with primary and 14 patients with secondary FSGS. The soluble urokinase receptor levels increased in the order of tip variant, to a not otherwise specified variant and a cellular variant. The soluble urokinase receptor levels were significantly but negatively correlated with creatinine clearance at presentation but positively correlated with crescent formation in patients with primary FSGS. During follow-up, receptor levels decreased significantly in patients with complete remission. Thus, plasma-soluble urokinase receptor levels did not differentiate primary and secondary FSGS, and although significantly elevated in FSGS, they showed considerable overlap with other glomerular diseases.

Characteristics and prognosis of Chinese patients with anti-glomerular basement membrane disease.

Background: Patients with anti-glomerular basement membrane (GBM) disease were predominantly reported in Caucasian population and reports from Chinese were lacking. The general picture of Chinese patients with anti-GBM disease was still unclear. This study is to investigate the characteristics and prognosis of Chinese patients with anti-GBM disease. Methods: Data from 105 patients with anti-GBM disease diagnosed in our hospital, between 1997 and 2002, were analyzed retrospectively. All the 105 sera were screened by enzyme-linked immunosorbent assay (ELISA) using highly purified bovine α(IV)NC1 as solid phase ligands. Clinical and pathological data of 69 patients with complete clinical remission (n = 5), partial remission (n = 10), and treatment failure (n = 54) were compared and the prognostic factors were evaluated. Results: Patients increased chronologically and three quarters of the 105 patients were diagnosed in the last 3 years. Most of the patients were between 20 and 29 years (n = 31) and a smaller second peak was found in patients over 60 years. 25/105 (24%) were also ANCA-positive. Patients with both anti-GBM antibodies and ANCA positive were elder (50 ± 19 vs. 34 ± 15 years, p < 0.01) and female predominant (15/25 vs. 16/80, p < 0.05). 56/97 (58%) patients presented as Goodpasture syndrome, 40/97 (41%) patients presented as rapidly progressive glomerulonephritis and one patient had pulmonary hemorrhage only. The following factors predict poor prognosis: (1) serum creatinine more than 600 µmol/l on diagnosis (p < 0.01); (2) oliguria or anuria on diagnosis (p < 0.01); (3) a high percentage (>85%) of glomeruli had crescents (p < 0.01), and (4) renal involvement before pulmonary hemorrhage (p < 0.05). Patients with serum creatinine over 600 µmol/l on diagnosis had higher levels of anti-GBM antibodies (106 ± 48% vs. 73 ± 40%, p < 0.01). Intensive plasma exchange therapy predicts a better prognosis in the patients with serum creatinine less than 600 µmol/l (p < 0.05). Conclusions: Anti-GBM disease is not rare in China and behaves similarly to elsewhere. Early diagnosis and intensive plasmapheresis might be the most promising approaches to improve the outcome.

Anti-glomerular basement membrane disease: outcomes of different therapeutic regimens in a large single-center Chinese cohort study.

Anti-glomerular basement membrane (GBM) disease usually presents with rapidly progressive glomerulonephritis accompanied by pulmonary hemorrhage. The low incidence and fulminant course of disease preclude a large randomized controlled study to define the benefits of any given therapy. We conducted a retrospective survey of 221 consecutive patients seen from 1998 to 2008 in our hospital, and report here the patient and renal survival and the risk factors affecting the outcomes. Considering the similar clinical features of the patients, we could compare the effects of 3 different treatment regimens: 1) combination therapy of plasmapheresis and immunosuppression, 2) steroids and cytotoxic agents, and 3) steroids alone.The patient and renal survival rates were 72.7% and 25.0%, respectively, at 1 year after disease presentation. The serum level of anti-GBM antibodies (increased by 20 U/mL; hazard ratio [HR], 1.16; p = 0.009) and the presentation of positive antineutrophil cytoplasmic antibodies (ANCA) (HR, 2.18; p = 0.028) were independent predictors for patient death. The serum creatinine at presentation (doubling from 1.5 mg/dL; HR, 2.07; p < 0.001) was an independent predictor for renal failure.The combination therapy of plasmapheresis plus corticosteroids and cyclophosphamide had an overall beneficial effect on both patient survival (HR for patient mortality, 0.31; p = 0.001) and renal survival (HR for renal failure, 0.60; p = 0.032), particularly patient survival for those with Goodpasture syndrome (HR for patient mortality, 0.29; p = 0.004) and renal survival for those with anti-GBM nephritis with initial serum creatinine over 6.8 mg/dL (HR for renal failure, 0.52; p = 0.014). The treatment with corticosteroids plus cyclophosphamide was found not to improve the renal outcome of disease (p = 0.73).In conclusion, the combination therapy was preferred for patients with anti-GBM disease, especially those with pulmonary hemorrhage or severe renal damage. Early diagnosis was crucial to improving outcomes.Abbreviations: ANCA = antineutrophil cytoplasmic antibodies, CI = confidence interval, ELISA = enzyme-linked immunosorbent assay, ESRD = end-stage renal disease, GBM = glomerular basement membrane, HR = hazard ratio.

Copy number variation of FCGR3A rather than FCGR3B and FCGR2B is associated with susceptibility to anti-GBM disease

Anti-glomerular basement membrane antibody disease (anti-GBM disease) is a rare disorder characteristic of universally poor outcome. Fcgamma receptors (FcgammaRs) play important roles in anti-GBM disease based on evidence from animal models. Copy number variation (CNV) influences disease susceptibility. The FcgammaRs genes show CNV, and CNV of the FCGR3B gene is associated with glomerulonephritis in systemic lupus erythematosus and anti-neutrophil cytoplasmic antibody-associated small vasculitis. Here, we investigated CNV of three FCGR genes, including two (FCGR3A and FCGR3B) for activating FcgammaRs and one (FCGR2B) for inhibitory FcgammaR by duplex quantitative real-time PCR. Copy numbers were analyzed by Applied Biosystems CopyCaller Software v1.0. We first demonstrated the distribution of CNV of FCGR3A, FCGR3B and no CNV of FCGR2B in Chinese population (including 47 anti-GBM patients and 146 healthy controls). The frequency of CNV of FCGR3A was observed to be significantly higher than matched healthy controls (27.7 versus 12.3%, P = 0.013, odds ratio 1.21-6.10). Considering previous report about gene knock-out animal models and CNV effect of FCGR3A, we thus propose that CNV in members of FCGR family should have different roles in the pathogenesis of human anti-GBM disease.

Prediction of Outcomes in Crescentic IgA Nephropathy in a Multicenter Cohort Study

Crescentic IgA nephropathy (IgAN), defined as >50% crescentic glomeruli on kidney biopsy, is one of the most common causes of rapidly progressive GN. However, few studies have characterized this condition. To identify risk factors and develop a prediction model, we assessed data from patients ≥ 14 years old with crescentic IgAN who were followed ≥ 12 months. The discovery cohort comprised 52 patients from one kidney center, and the validation cohort comprised 61 patients from multiple centers. At biopsy, the mean serum creatinine (SCr) level ± SD was 4.3 ± 3.4 mg/dl, and the mean percentage of crescents was 66.4%± 15.8%. The kidney survival rates at years 1, 3, and 5 after biopsy were 57.4%± 4.7%, 45.8%± 5.1%, and 30.4%± 6.6%, respectively. Multivariate Cox regression revealed initial SCr as the only independent risk factor for ESRD (hazard ratio [HR], 1.32; 95% confidence interval [CI], 1.10 to 1.57; P=0.002). Notably, the percentage of crescents did not associate independently with ESRD. Logistic regression showed that the risk of ESRD at 1 year after biopsy increased rapidly at SCr>2.7 mg/dl and reached 90% at SCr>6.8 mg/dl (specificity=98.5%, sensitivity=64.6% for combined cohorts). In both cohorts, patients with SCr>6.8 mg/dl were less likely to recover from dialysis. Analyses in additional cohorts revealed a similar association between initial SCr and ESRD in patients with antiglomerular basement membrane disease but not ANCA-associated systemic vasculitis. In conclusion, crescentic IgAN has a poor prognosis, and initial SCr concentration may predict kidney failure in patients with this disease.

Levels of epitope-specific autoantibodies correlate with renal damage in anti-GBM disease

BACKGROUND Although the clinical importance of demonstrating the presence of anti-glomerular basement membrane (anti-GBM) antibodies is well established, less is known concerning the clinical utility of measuring the levels of autoantibodies. Two conformational epitopes of anti-GBM antibodies have been defined at residues 17-31 and 127-141 of the alpha3(IV)NC1 domain of type IV collagen [alpha3(IV)NC1], which were named as EA and EB, respectively. In order to elucidate the importance of such antibodies, we studied the levels and the epitope specificities of anti-GBM antibodies in a large cohort of Chinese patients with anti-GBM disease. METHODS All patients, with anti-GBM disease and available clinical data, diagnosed at Peking University First Hospital from 1996 to 2005 were included in the present study. Recombinant chimeric proteins containing previously defined epitope regions designated as EA and EB were used to detect anti-GBM antibodies by ELISA. Results were compared and correlated with clinical data collected at the time of diagnosis, biopsy findings and outcome after 1 year of follow-up. RESULTS A retrospective diagnosis of anti-GBM disease was made in 147 patients. Haemoptysis was recorded for 47% of these cases while 53.5% cases had oliguria or anuria at the time of diagnosis. Among these patients, the levels of anti-GBM antibodies correlated with serum creatinine at diagnosis (P < 0.05 for anti EA, EB and alpha3(IV)NC1). Oliguric patients had higher levels of autoantibodies than non-oliguric patients, however, the difference being statistically significant only for EB (P < 0.05). Renal biopsies were performed in 66 patients, and it was found that 50 (75.8%) had cresent formation in >85% of the glomeruli. There was a correlation between the percentage of crescents and levels of antibodies, but it was significant only for anti-EA antibodies (P < 0.05). Clinical data regarding the follow-up were available for 102 patients; at the end of 1 year, 88 (86.3%) were either dead or dialysis dependent. The absorbance values of anti-GBM antibodies against both EA and EB were also associated with the subsequent development, death or terminal renal insufficiency (P < 0.05). CONCLUSION In this study, patients with high levels of circulating antibodies against the specific epitopes EA and EB had a more severe renal disease at diagnosis as well as a worse prognosis.

Anti-glomerular basement membrane autoantibodies against different target antigens are associated with disease severity

Anti-glomerular basement membrane (GBM) autoantibodies are pathogenic in the development of anti-GBM disease. The main target antigen is the non-collagenous 1 domain (NC1) of collagen alpha3(IV); however, most antibodies can recognize the NC1 of collagens alpha1, alpha2, alpha4 and alpha5(IV). In this study, we analyzed the target antigens of anti-GBM autoantibodies to determine their relationship to the severity of renal damage. Natural anti-GBM autoantibodies were purified from 10 healthy individuals and from 57 patients with anti-GBM disease who were divided into groups based on the degree of renal damage defined by their serum creatinine at the time of diagnosis. We found that the sera of all 57 patients recognized alpha3(IV)NC1, while 23, 20, 28, and 48 patients also recognized the NC1 of collagens alpha1, alpha2, alpha4, and alpha5(IV), respectively. Natural anti-GBM autoantibodies recognized the NC1 of collagens alpha3 and alpha4(IV). The sera of 7 patients with the lowest level of renal damage mainly recognized the NC1 of collagens alpha3 and alpha5(IV). In the 20 patients with moderate and the 30 with severe renal damage, all five target antigens could be detected and most sera recognized three to five different alpha chains simultaneously. Regression analysis showed that only the level of autoantibodies against the NC1 of collagen alpha3(IV) was a significant independent risk factor for higher serum creatinine on diagnosis. Our study shows that autoantibodies to the NC1 of collagen alpha3(IV) were crucial in causing renal damage. Inter- and intra-molecular epitope spreading can occur during the development of human anti-GBM disease.

The role of HLA-DRB1 alleles on susceptibility of Chinese patients with anti-GBM disease.

Anti-glomerular basement membrane (GBM) disease, a rare autoimmune disorder, is associated with HLA-DR15 genotype in Caucasian and Japanese populations. But the distribution of HLA-DRB1 alleles in Chinese patients with anti-GBM disease and their association with clinical characteristics of anti-GBM disease are to be determined. The present study analyzed the HLA-DRB1 alleles by sequence based typing in 44 Chinese patients with anti-GBM disease and 200 healthy controls. The effects of DRB1 alleles on susceptibility to anti-GBM disease were examined by a relative predispositional effects (RPEs) method. The clinical and pathological data of the patients were collected and analyzed. The DRB1*1501 allele was significantly associated with anti-GBM disease (p=1.597 x 10(-7)). The RPEs test also showed a significant increased frequency of DRB1*0404 in anti-GBM disease (p=0.037). Interestingly, the patients with DRB1*1501 or 0404 had more crescent formation in glomeruli than those without the two alleles (p=0.021). But the DRB1*0404 was rare in both patients and control groups, which indicates that the importance of the *0404 allele is limited in anti-GBM disease. We conclude that the HLA-DRB1*1501 allele is a genetic marker for susceptibility to anti-GBM disease.