Toward a working definition of C3 glomerulopathy by immunofluorescence
2013; Elsevier BV; Volume: 85; Issue: 2 Linguagem: Inglês
10.1038/ki.2013.340
ISSN1523-1755
AutoresJean Hou, Glen S. Markowitz, Andrew S. Bomback, Gerald B. Appel, Leal C. Herlitz, Michael B. Stokes, Vivette D. D’Agati,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoPrecise immunofluorescence criteria for C3 glomerulopathy remain to be defined. Here we tested hierarchical immunofluorescence criteria with varying stringency for C3 glomerulopathy in a cohort with dense deposit disease as the gold standard and then applied these criteria to analyze the incidence of C3 glomerulopathy in membranoproliferative glomerulonephritis (MPGN) types 1 and 3. Among 319 archived cases of primary MPGN types 1–3, immunofluorescence reports were retrospectively coded as glomerular deposits of the following: C3 only; C3 dominant with trace or 1+ immunoglobulin (Ig)M only; and C3 dominant and at least two orders of intensity stronger than any combination of IgG, IgM, IgA, and C1q. The most restrictive criteria of ‘C3 only’ captured only half of the cases with dense deposit disease (compared with 8% of type 1 and 10% of type 3). Adding the most liberal definition identified 88% of those with dense deposit disease (compared with 31% of type 1 and 39% of type 3). The unaccounted 12% had stronger intensity of Ig staining, but it never exceeded the intensity of C3. Among MPGN type 3, 90% of C3 glomerulopathy cases were the Strife and Anders variant. Repeat biopsies in C3 glomerulopathy revealed a change in immunofluorescence pattern in 10 of 23 biopsies. The prevalence of low serum C3 and/or low C4 did not significantly differ among the three immunofluorescence criteria. Thus, ‘C3 only’ is an impractical definition of C3 glomerulopathy, and we propose a definition of C3 dominant and at least two orders of magnitude more intense than any other immune reactant, which requires validation by alternative pathway evaluation. These criteria provide a framework for identifying patients most likely to benefit from investigations of alternative complement pathway dysregulation. Precise immunofluorescence criteria for C3 glomerulopathy remain to be defined. Here we tested hierarchical immunofluorescence criteria with varying stringency for C3 glomerulopathy in a cohort with dense deposit disease as the gold standard and then applied these criteria to analyze the incidence of C3 glomerulopathy in membranoproliferative glomerulonephritis (MPGN) types 1 and 3. Among 319 archived cases of primary MPGN types 1–3, immunofluorescence reports were retrospectively coded as glomerular deposits of the following: C3 only; C3 dominant with trace or 1+ immunoglobulin (Ig)M only; and C3 dominant and at least two orders of intensity stronger than any combination of IgG, IgM, IgA, and C1q. The most restrictive criteria of ‘C3 only’ captured only half of the cases with dense deposit disease (compared with 8% of type 1 and 10% of type 3). Adding the most liberal definition identified 88% of those with dense deposit disease (compared with 31% of type 1 and 39% of type 3). The unaccounted 12% had stronger intensity of Ig staining, but it never exceeded the intensity of C3. Among MPGN type 3, 90% of C3 glomerulopathy cases were the Strife and Anders variant. Repeat biopsies in C3 glomerulopathy revealed a change in immunofluorescence pattern in 10 of 23 biopsies. The prevalence of low serum C3 and/or low C4 did not significantly differ among the three immunofluorescence criteria. Thus, ‘C3 only’ is an impractical definition of C3 glomerulopathy, and we propose a definition of C3 dominant and at least two orders of magnitude more intense than any other immune reactant, which requires validation by alternative pathway evaluation. These criteria provide a framework for identifying patients most likely to benefit from investigations of alternative complement pathway dysregulation. The clinical and pathologic approach to membranoproliferative glomerulonephritis (MPGN) has evolved markedly in recent years. The term MPGN was originally introduced in the 1970s to describe a morphologic pattern of glomerular proliferation with mesangial interposition and duplication of glomerular basement membranes as defined by light and electron microscopy.1.Mandalenakis N. Mendoza N. Pirani C.L. et al.Lobular glomerulonephritis and membranoproliferative glomerulonephritis: a clinical and pathologic study based on renal biopsies.Medicine. 1971; 50: 319-355Crossref PubMed Scopus (65) Google Scholar This proliferation occurs in response to deposition of immunoglobulins (Igs) and/or complement in the glomerular capillary walls. Historically, three major forms of MPGN have been recognized. In type 1, there is mesangial proliferation with regular circumferential mesangial interposition and double contouring of glomerular basement membranes in response to subendothelial electron-dense deposits.2.Levy M. Gubler M.C. Sich M. et al.Immunopathology of membranoroliferative glomerulonephritis with subendothelial deposits (Type I MPGN).Clin Immunol Immunopathol. 1978; 10: 477-492Crossref PubMed Scopus (40) Google Scholar Type 2, also known as dense deposit disease (DDD), is characterized by distinctive, highly electron-dense, ribbon-like intramembranous deposits that transform the lamina densa, often associated with ring-shaped mesangial deposits and variable hump-shaped subepithelial deposits.3.Habib R. Gubler M.C. Loirat C. et al.Dense deposit disease: a variant of membranoproliferative glomerulonephritis.Kidney Int. 1975; 7: 204-215Abstract Full Text PDF PubMed Scopus (168) Google Scholar In type 3 of Strife and Anders, there are more complex and ill-defined intramembranous deposits associated with subendothelial and subepithelial deposits, causing disruption and fraying of the lamina densa.4.Strife C.F. McEnery P.T. McAdams A.J. et al.Membranoproliferative glomerulonephritis with disruption of the glomerular basement membrane.Clin Nephrol. 1977; 7: 65-72PubMed Google Scholar By contrast, type 3 of Burkholder is characterized by more discrete subendothelial, intramembranous, and subepithelial deposits, often with overlapping features of MPGN type 1 and membranous glomerulopathy4.Strife C.F. McEnery P.T. McAdams A.J. et al.Membranoproliferative glomerulonephritis with disruption of the glomerular basement membrane.Clin Nephrol. 1977; 7: 65-72PubMed Google Scholar,5.Burkholder P.M. Hyman L.R. Krueger R.P. Characterization of mixed membranous and proliferative glomerulonephritis: recognition of three varieties.Perspect Nephrol Hypertens. 1973; 1: 557-589PubMed Google Scholar (Figure 1). These categories were cumbersome to apply because of their complexity, the frequent occurrence of morphologic heterogeneity in a given case, and the lack of pathogenetic specificity. For example, many cases of DDD lack membranoproliferative features altogether, causing the term MPGN type 2 to be discarded.6.Walker P.D. Ferrario F. Joh K. et al.Dense deposit disease is not a membranoproliferative glomerulonephritis.Mod Pathol. 2007; 20: 605-616Crossref PubMed Scopus (101) Google Scholar With greater understanding of etiology over the past decade, there has been a major shift away from morphologic patterns to focus on composition of the deposits as defined by immunofluorescence (IF). Those cases with substantial deposits of Ig are considered immune complex mediated and should prompt an investigation of underlying autoimmune, infectious, or paraprotein-related disease.7.Sethi S. Fervenza F.C. Zhang Y. et al.C3 glomerulonephritis: clinicopathological findings, complement abnormalities, glomerular proteomic profile, treatment, and follow-up.Kidney Int. 2012; 82: 465-473Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar The term C3 glomerulopathy (C3G), proposed to encompass those cases with deposition of C3 only, includes DDD and those forms of MPGN type 1 and type 3 with predominant deposits of C3.8.Sethi S. Fervenza F.C. Membranoproliferative glomerulonephritis: pathogenetic heterogeneity and proposal for a new classification.Semin Nephrol. 2011; 31: 341-348Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar,9.Fakhouri F. Fremeaux-Bacchi V. Noel L.H. et al.C3 glomerulopathy: a new classification.Nat Rev Nephrol. 2010; 6: 494-499Crossref PubMed Scopus (268) Google Scholar A major breakthrough was the recognition that abnormal control of the alternative pathway (AP) of complement underlies C3G, irrespective of morphologic appearance. Potential abnormalities include genetic deficiencies in regulators of the alternative complement pathway (such as complement factor (CF)H, CFHR1–5, CFI, and CD46)10.Habbig S. Mihatsch M.J. Heinen S. et al.C3 deposition glomerulopathy due to a functional factor H defect.Kidney Int. 2009; 75: 1230-1234Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,11.Abrera-Abeleda M.A. Nishimura C. Frees K. et al.Allelic variants of complement genes associated with dense deposit disease.J Am Soc Nephrol. 2011; 22: 1551-1559Crossref PubMed Scopus (78) Google Scholar and autoantibodies to factor H, factor B, and the C3 convertase (known as C3 nephritic factors).12.Strobel S. Zimmering M. Papp K. et al.Anti-factor B autoantibody in dense deposit disease.Mol Immunol. 2010; 47: 1476-1483Crossref PubMed Scopus (85) Google Scholar, 13.Goodship T.H. Pappworth I.Y. Toth T. et al.Factor H autoantibodies in membranoproliferative glomerulonephritis.Mol Immunol. 2012; 52: 200-206Crossref PubMed Scopus (59) Google Scholar, 14.Ohi H. Watanabe S. Fujita T. et al.Significance of C3 nephritic factor (C3NeF) in non-hypocomplementaemic serum with membranoproliferative glomerulonephritis (MPGN).Clin Exp Immunol. 1992; 89: 479-484Crossref PubMed Scopus (28) Google Scholar Proteomic analysis by mass spectrometry performed on laser-captured glomeruli has identified complement debris (including C5, C6, C7, C8, and C9) in the glomerular deposits, consistent with activation of the AP in the fluid phase.15.Sethi S. Gamez J.D. Vrana J.A. et al.Glomeruli of dense deposit disease contain components of the alternative and terminal complement pathway.Kidney Int. 2009; 75: 952-960Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar With the advent of specific clinical testing for abnormalities of the AP and specific therapies targeting the AP,16.Zuber J. Fakhouri F. Roumenina L.T. et al.Use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies.Nat Rev Nephrol. 2012; 8: 643-657Crossref PubMed Scopus (391) Google Scholar these distinctions are gaining greater clinical relevance. An impediment in the diagnosis of C3G is the lack of a working definition of IF criteria that has been developed and validated in a well-defined disease cohort. Although the term ‘C3 only’ is often applied as a theoretical construct, it is not clear whether such a strict definition is practicable. For example, pathologists recognize that IgM is frequently trapped in areas of sclerosis and in thickened glomerular capillary walls in diverse glomerular diseases.17.Bolton W.K. Benton F.R. Maclay J.G. et al.Spontaneous glomerular sclerosis in aging Sprague-Dawley rats. I. Lesions associated with mesangial IgM deposits.Am J Pathol. 1976; 85: 277-302PubMed Google Scholar A major question is whether C3 dominance might occur on a background of less intense deposition or trapping of other immune reactants, including IgG, IgA, and C1q. We aimed to examine these questions by testing different set points for IF definition of C3G in a cohort of DDD as the gold standard. Once optimal criteria were found, we applied these criteria to a group of primary MPGN type 1 and type 3 to determine the incidence of C3G, define the relationship of C3G to the Strife and Anders versus Burkholder subgroups of primary MPGN type 3, and explore clinical and demographic correlates. A total of 796 biopsies with diagnoses of MPGN 1–3 were identified from 1999 to 2012. After exclusion of 396 cases with clear underlying etiology (secondary MPGN due to hepatitis C infection, cryoglobulinemia, paraprotein deposition, and so on), 58 cases with inadequate tissue for IF or previous treatment with eculizumab, and 23 repeat biopsies, a total of 319 cases of primary MPGN were recruited into the study. As shown in Table 1, the largest number of primary cases was from MPGN1 (n=200), followed by MPGN3 (n=77) and MPGN2/DDD (n=42). Using MPGN2/DDD as the prototype for C3G, application of the strictest definition of C3-only staining (criterion 1) captured only 50% of the total cases. Allowing for low-level IgM deposition (criterion 2) increased the case recovery to 71.4%. Further broadening the criteria to permit low-level IgG, IgM, IgA, and/or C1q staining (criterion 3) identified 88.1% of total MPGN2/DDD cases. Broadening to criterion 4 added only 4.8% of DDD cases, while this change added 26.5% of MPGN1 cases and 13% of MPGN3 cases, suggesting loss of specificity. Therefore, the first three criteria were chosen as cutoffs for the gold-standard group of DDD and used as optimal ‘defining criteria’ for C3G. We then applied these criteria to cases of MPGN1 and MPGN3 to determine the incidence of C3G in these groups. Sixty-one cases (30.5%) of MPGN1 and 30 cases (39%) of MPGN3 successfully met the defining criteria for C3G (Table 1). Of the 30 C3G cases recovered from MPGN3, 90% were Strife and Anders variant and 10% were Burkholder variant.Table 1Primary membranoproliferative glomerulonephritis (MPGN) cases meeting criteria 1–3DiagnosisNumber of primary casesCriterion 1: C3 only (%)Criterion 2: C3 dominant and up to 1+ IgM only (%)Criterion 3: C3 dominant and ≥2 orders of intensity greater than any combination of IgG, IgM, IgA, and C1q (%)Criteria 1, 2, or 3 (%)MPGN 120016 (8%)13 (6.5%)32 (16%)61 (30.5%)MPGN2/DDD4221 (50%)9 (21.4%)7 (16.7%)aSeven cases that fulfilled criterion 3 displayed C3-dominant staining with the following immunofluorescence patterns: two cases with IgG only; one case with IgM only; two cases with C1 only; and two cases with IgG, IgM, IgA, and C1.37 (88.1%)MPGN 3778 (10.4%)11 (14.3%)11(14.3%)30 (39%)Total31945 (14.1%)33 (10.3%)50 (15.7%)128 (40.1%)Abbreviations: DDD, dense deposit disease; Ig, immunoglobulin.a Seven cases that fulfilled criterion 3 displayed C3-dominant staining with the following immunofluorescence patterns: two cases with IgG only; one case with IgM only; two cases with C1 only; and two cases with IgG, IgM, IgA, and C1. Open table in a new tab Abbreviations: DDD, dense deposit disease; Ig, immunoglobulin. Cases of MPGN 1–3 that did not meet the third cutoff for C3G are outlined in Table 2. Five MPGN2/DDD cases failed to meet the defining C3G IF criteria: two cases with C3 dominance and >1+ IgG or IgM, two cases with C3 co-dominance with IgG or IgM, and one case (an outside consult) reported as negative for C3 and other immune reactants. Of note, no DDD case exhibited stronger staining intensity for Ig than C3. A significant subset of MPGN1 (26.5%) and MPGN3 (13%) cases also exhibited C3-dominant staining that failed to meet the C3G criteria. Co-dominant staining of C3 with IgG or IgM was observed more frequently in primary MPGN1 and MPGN3 than in MPGN2/DDD. The incidence of IgG-dominant staining, which was never observed in MPGN2/DDD, was higher in cases of MPGN3 than in those of MPGN1.Table 2Primary membranoproliferative glomerulonephritis (MPGN) cases not meeting criteria 1–3Criterion 5: C3 co-dominantDiagnosisNumber of cases NOT meeting criteria 1–3 (%)Criterion 4: C3 dominant and >1+any Ig/C1q (%)5a: C3/IgM co-dominant (%)5b: C3/IgG co-dominant (%)Criterion 6: IgM dominant IgM>C3 (%)Criterion 7: IgG dominant IgG>C3 (%)Criterion 8: C3 negative (%)MPGN1139 (69.5%)53 (26.5%)18 (9%)25 (18%)14 (7%)16 (8%)13 (6.5%)MPGN2/DDD5 (11.9%)2 (4.8%)1 (2.4%)1 (2.4%)001 (2.4%)MPGN347 (61%)10 (13%)4 (5.2%)15 (19.5%)1 (1.3%)16 (20.8%)1 (1.3%)Total191 (59.9%)65 (20.4%)23 (7.2%)41 (12.9%)15 (4.7%)32 (10%)15 (4.7%)Abbreviations: DDD, dense deposit disease; Ig, immunoglobulin. Open table in a new tab Abbreviations: DDD, dense deposit disease; Ig, immunoglobulin. Twenty-three patients underwent repeat biopsies, including 12 with MPGN1, 8 with DDD, and 3 with MPGN3. Subsequent biopsies for each patient were then separately categorized and summarized. Overall, 13 of the 23 patients maintained the same IF staining patterns on initial and follow-up biopsies (Table 3). On repeat biopsy, 2 of the 23 patients met a more stringent criterion for C3G, and 4 of the 23 met a more liberal criterion for C3G. Only two C3G patients had follow-up biopsies not meeting the C3G criteria, although in both cases C3 staining was stronger than IgG. Two patients showed the opposite trend, with initial biopsies that showed C3-dominant staining not meeting the criteria but with repeat biopsy meeting the C3G criteria. Of the three patients initially diagnosed with acute post-infectious glomerulonephritis who had repeat biopsies diagnosed as C3G, two had MPGN1 and one had MPGN3 on repeat biopsy.Table 3Primary membranoproliferative glomerulonephritis (MPGN) cases with serial biopsiesIF criterion of initial biopsyIF criterion of repeat biopsy12345a5b14122611aTwo patients (8.7%) had an initial biopsy that failed to meet criteria 1–3 and a subsequent biopsy that did meet criteria.31131aTwo patients (8.7%) had an initial biopsy that failed to meet criteria 1–3 and a subsequent biopsy that did meet criteria.41bTwo patients (8.7%) had an initial biopsy that did meet criteria 1–3 and a subsequent biopsy that did not.5a1bTwo patients (8.7%) had an initial biopsy that did meet criteria 1–3 and a subsequent biopsy that did not.5bAbbreviation: IF, immunofluorescence.Thirteen of 23 patients (57%) had no change in immunofluorescence criteria (bolded).a Two patients (8.7%) had an initial biopsy that failed to meet criteria 1–3 and a subsequent biopsy that did meet criteria.b Two patients (8.7%) had an initial biopsy that did meet criteria 1–3 and a subsequent biopsy that did not. Open table in a new tab Abbreviation: IF, immunofluorescence. Thirteen of 23 patients (57%) had no change in immunofluorescence criteria (bolded). Of the 128 cases that met classification criteria as C3G, 72 (56%) were male with a mean age of 34.4 years (Table 4). In this cohort, 65 of the 98 patients (66%) with reported data on race/ethnicity were white. On average, these patients presented with renal insufficiency (mean serum creatinine 2.6; median 1.6mg/dl and mean estimated glomerular filtration rate 59.3; median 48.8ml/min per 1.73m2) and nephrotic-range proteinuria (mean 24-hour proteinuria 4.5; median 3.6g/day). Serum complement levels were low in ∼75% of patients, primarily in the pattern of low C3 with normal-range C4 levels.Table 4Clinical and biological features of C3G patientsAll C3G (n=128)C3G in MPGN1 (n=61)C3G in MPGN2 (n=37)C3G in MPGN3 (n=30)P-valueaP-value from analysis of variance, testing null hypothesis that mean is equal among all three categories.IF criterion 1 (n=45)IF criterion 2 (n=33)IF criterion 3 (n=50)P-valueaP-value from analysis of variance, testing null hypothesis that mean is equal among all three categories.Mean age (years)34.435.735.930.10.438.126.936.10.06Female (%)43.852.529.743.30.0933.330.362.00.003Race/ethnicity (%) White50.854.148.746.70.857.854.642.00.3 Hispanic16.49.816.230.00.0517.824.210.00.2 Black6.36.65.46.71.00.09.110.00.1 Asian3.13.32.73.31.00.00.08.00.04 Unknown23.426.227.013.30.324.412.130.00.2Mean disease duration (months)bExcludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36).41.529.748.453.50.241.153.628.40.2Median serum creatinine (mg/dl)bExcludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36).1.61.91.61.00.051.91.31.50.04Median eGFR (ml/min per 1.73m2)bExcludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36).48.835.848.886.70.0139.065.051.50.09Median 24-h proteinuria (g/day)bExcludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36).3.63.62.64.20.61.54.44.90.004Median serum albumin (g/dl)bExcludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36).3.22.83.93.40.023.83.42.40.0001Serum complements (%)bExcludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36). Normal24.521.817.437.50.227.626.720.90.8 Low C3 and normal C460.854.682.654.20.0562.163.358.10.9 Low C3 and low C414.723.60.08.30.0210.310.020.90.3 Low C3 overall75.578.282.662.50.272.473.379.10.8Hematuria (%)bExcludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36). None16.34.625.928.60.0123.312.013.50.4 Microscopic68.586.462.371.40.0766.780.081.10.3 Gross15.29.111.10.00.0233.38.05.40.003Hypertension (%)46.147.551.436.70.542.239.454.00.4Abbreviations: C3G, C3 glomerulopathy; eGFR, estimated glomerular filtration rate; IF, immunofluorescence; MPGN, membranoproliferative glomerulonephritis.a P-value from analysis of variance, testing null hypothesis that mean is equal among all three categories.b Excludes missing data for subjects on disease duration (n=51), serum creatinine (n=11), proteinuria (n=49), serum albumin (n=59), serum complements (n=26), and hematuria (n=36). Open table in a new tab Abbreviations: C3G, C3 glomerulopathy; eGFR, estimated glomerular filtration rate; IF, immunofluorescence; MPGN, membranoproliferative glomerulonephritis. These presenting historical and clinical data were examined according to original histopathologic diagnoses—MPGN1, MPGN2, or MPGN3—as well as by our proposed IF criteria schemes for diagnosing C3G. Age and sex distribution were relatively similar among the C3G patients originally classified as MPGN1, MPGN2/DDD, and MPGN3. In contrast, Hispanic patients were more likely to demonstrate the MPGN2 and MPGN3 pattern than MPGN1. Patients with MPGN3 were also marked by significantly lower presenting serum creatinine (and correspondingly higher estimated glomerular filtration rate) than patients with MPGN1 or MPGN2. Although the presence of low C3 levels overall did not differ between the MPGN subtypes, interestingly, no patients with MPGN2 demonstrated low C3 and low C4 in contrast to a small proportion of those patients with MPGN1 and MPGN3 with this complement profile. Women were overrepresented in the subgroup of C3G patients who were included based on IF criterion 3. This subgroup also included the only Asian patients in the cohort. Patients who met the strictest IF criterion 1 for diagnosis of C3G presented with the highest incidence of gross hematuria and the most severe renal dysfunction, with mean serum creatinine 3.5; median 1.9mg/dl and mean estimated glomerular filtration rate 50.2; median 39.0ml/min per 1.73m2 (which, in turn, likely influenced their significantly lower level of proteinuria than IF criteria 2 and 3 subgroups). Notably, the prevalence of low C3 and/or low C4 serum levels did not significantly differ among these IF criteria subgroups. We retrospectively reviewed a random subset of 125 biopsies of secondary MPGN, including 45 hepatitis C–associated MPGN (of which 18 also had cryoglobulinemia), 30 cryoglobulinemic glomerulonephritis, 30 lupus nephritis with membranoproliferative pattern, 10 hepatitis B–associated MPGN, 5 combined hepatitis B and HIV–associated MPGN, and 5 combined hepatitis B and C–associated MPGN. Only one case of hepatitis C–associated MPGN (without cryoglobulinemia) met the IF criterion 2; one case of hepatitis B–associated MPGN met IF criterion 3; and no case met IF criterion 1. These data indicate that our proposed IF criteria for C3G are rarely found in biopsies with secondary MPGN. C3G is a histopathologic diagnosis that aims to be both descriptive, by relaying the findings of IF microscopy, and physiologic, by implicating a complement-mediated (rather than immune complex–mediated) glomerulonephritis. Although C3G is defined by IF findings in the proper clinical and morphologic context, no prior studies have investigated what the precise IF criteria for diagnosis should be in pragmatic terms. We applied a hierarchical set of IF criteria to define the optimal cutoff for the diagnosis of C3G using MPGN2/DDD as a gold standard. A definition of ‘C3 only’ identified only half of the DDD patients, whereas a definition of C3 dominance of at least two orders of magnitude stronger than any other immune reactant captured 88.1% of DDD patients. Further relaxing the defining criteria captured a few additional DDD patients but many more MPGN1 and MPGN3 patients, suggesting reduced specificity. Our results indicate that the theoretical definition of C3G as ‘C3 only’ staining is too stringent if the goal of diagnosing C3G is to identify suitable candidates for AP evaluation. Allowing for IgM staining increased the retrieval to 71.4% of MPGN2/DDD. The presence of IgM staining in other glomerular diseases has been attributed to nonspecific trapping in areas of sclerosis or glomerular capillary wall thickening, without necessarily contributing to disease pathogenesis.17.Bolton W.K. Benton F.R. Maclay J.G. et al.Spontaneous glomerular sclerosis in aging Sprague-Dawley rats. I. Lesions associated with mesangial IgM deposits.Am J Pathol. 1976; 85: 277-302PubMed Google Scholar The potential for low-level IgG staining also may exist in C3G. Serologic evidence of AP dysregulation has been recently identified in more than half of a single cohort of patients with MPGN1,18.Servais A. Noel L.H. Roumenina L.T. et al.Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies.Kidney Int. 2012; 82: 454-464Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar with C3NeF, an acquired autoantibody of C3 convertase, identified in MPGN1 as frequently as it was in C3G. In that study, IF revealed concomitant C3 and IgG deposits in a subset of patients who had clear serologic evidence of AP abnormalities. Our findings agree that the presence of IgG does not exclude C3G. Our proposed working definition of C3G as C3 dominance of at least two orders of magnitude more intense than any other immune reactant, beyond identifying 88.1% of MPGN2/DDD cases, qualified 30.5% of cases of MPGN1 and 39% of cases of MPGN3 as C3G. Among cases of MPGN3, the vast majority was Strife and Anders (90%) variant, which is consistent with the previously reported finding of C3-dominant deposits in Strife and Anders variant in a smaller cohort.19.Bomback A.S. Appel G.B. Pathogenesis of the C3 glomerulopathies and reclassification of MPGN.Nat Rev Nephrol. 2012; 8: 634-642Crossref PubMed Scopus (117) Google Scholar Although our biopsy database archived cases according to historical MPGN categories, it should be emphasized that C3G is not restricted to a membranoproliferative pattern and may exhibit other histological phenotypes. A diagnosis of C3G should prompt an investigation of the alternative complement pathway, specifically testing for mutations in regulators (e.g. factor B) and inhibitors (e.g. factor H) of the AP, autoantibodies directed at such regulators and inhibitors, and a functional assessment of AP activity. These investigations are noninvasive and, in most cases, can influence therapeutic decisions, allowing for the opportunity to tailor therapies to detected defects in the AP.20.Nester C.M. Smith R.J. Treatment options for C3 glomerulopathy.Curr Opin Nephrol Hypertens. 2013; 22: 231-237Crossref PubMed Scopus (46) Google Scholar Given the importance of such testing, any diagnostic criteria for C3G should veer toward being more inclusive rather than exclusive. In the largest study of 56 cases of C3G to date, for example, acquired and genetic complement abnormalities were found in patients with phenotypes of MPGN1 and MPGN2/DDD.18.Servais A. Noel L.H. Roumenina L.T. et al.Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies.Kidney Int. 2012; 82: 454-464Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar Clinical features of C3G have also been reported in a pediatric cohort21.He R.J. Xiao H.J. Wang S.X. et al.[Characteristics of pediatric C3 glomerulopathy with decreased factor H in 3 cases].Zhonghua Er Ke Za Zhi. 2012; 50: 939-943PubMed Google Scholar and familial C3G.22.Athanasiou Y. Voskarides K. Gale D.P. et al.Familial C3 glomerulopathy associated with CFHR5 mutations: clinical characteristics of 91 patients in 16 pedigrees.Clin J Am Soc Nephrol. 2011; 6: 1436-1446Crossref PubMed Scopus (109) Google Scholar,23.Gale D.P. de Jorge E.G. Cook H.T. et al.Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis.Lancet. 2010; 376: 794-801Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar Analysis of a subset of patients with diverse causes of secondary MPGN found only 2 of 125 (1.6%) cases meeting these proposed IF criteria for C3G. Our data now provide a practicable, nonconstrictive, pathologic definition of C3G that identifies those patients most likely to benefit from genetic and functional studies of AP dysregulation. Analysis of repeat biopsies in 23 patients provides further support for our definition of C3G. After initial biopsy diagnosis of C3G by meeting IF criteria 1–3 in 23 patients, repeat biopsies in the majority (13/23) met the identical IF criterion as the first biopsy. On the other hand, 2/23 C3G patients met a more stringent criterion and 4/23 met a more liberal criterion, while still fulfilling the definition for C3G on repeat biopsy. Further, two patients meeting criterion 4 on initial biopsy met C3G criteria 2 and 3, respectively, on repeat biopsy. Conversely, only two patients fulfilling C3G criterion 1 on initial biopsy met criteria 4 and 5a, respectively, on repeat biopsy. These data support fluidity between these subgroups and the ability to transform from one criterion class to another over time. These data also support the importance of a less constrictive definition that allows for such fluidity. It is not clear as to why some patients had a repeat biopsy that transformed to a more liberal criterion beyond criterion 3. This finding is consistent with the small percentage of MPGN2/DDD patients who manifested variable intensities of Ig staining on initial biopsy while never exceeding the staining intensity for C3. Conceivably, glomeruli may be subjected to other forms of immunologic injury, and harboring an abnormality in AP regulation does not exclude the possibility of other immune assaults that could promote IgG or other Ig deposition, including classical pathway activation. More studies are needed to address the potential for multiple immunologic ‘hits’ in this population. Whether glomerular staining for IgG represents inconsequential passive ‘trapping’ of a circulating plasma protein or superimposed active immune-mediated injury capable of promoting glomerular leukocyte infiltration and proliferation requires further study. Clinical analysis of this C3G cohort revealed that patients with MPGN2/DDD were more likely to have isolated reduction in serum C3 (82.6%) compared with those with MPGN1 and MPGN3 (54.6% and 54.2%, respectively). No patient with MPGN2/DDD had low serum C3 and C4, as compared with a small percentage (23.6% and 8.3%, respectively) of MPGN1 and MPGN3 with this complement profile. These findings support the concept that classical complement activation may contribute to glomerular injury in some C3G patients with MPGN1 and MPGN3 phenotypes.24.D'Agati V.D. Bomback A.S. C3 glomerulopathy: what’s in a name?.Kidney Int. 2012; 82: 379-381Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar In other respects, those patients meeting defining criteria of C3G within the three major pathologic subcategories of MPGN had similar demographic and clinical presenting features, supporting the thesis that the IF pattern is a more important diagnostic criterion than the light and electron microscopic patterns. Interestingly, C3G patients who met the strictest IF criterion 1 for diagnosis of C3G presented with the highest incidence of gross hematuria, more severe renal dysfunction, and lower-level proteinuria than those meeting criteria 2 and 3. This tendency to an acute nephritic (as opposed to nephrotic) presentation resembles the typical clinical presentation of postinfectious glomerulonephritis. Further study is needed to determine differences in the incidence of preceding infections among these IF criteria subgroups. Notably, the prevalence of low C3 and/or low C4 levels did not significantly differ among these IF criteria subgroups. The most liberal criterion (criterion 3) for diagnosing C3G demonstrated a trend toward greater prevalence of low C4 than stricter criteria (1 and 2); however, that again raises the issue of whether a defect in AP may heighten susceptibility to or amplify injury from immune complex deposition in some C3G cases. The finding of several cases initially diagnosed as postinfectious glomerulonephritis but fulfilling criteria for C3G on subsequent biopsy underscores the potential difficulty in diagnosing C3G in such patients without clinical follow-up and repeat biopsy.25.Sandhu G. Bansal A. Ranade A. et al.C3 glomerulopathy masquerading as acute postinfectious glomerulonephritis.Am J Kidney Dis. 2012; 60: 1039-1043Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,26.Zand L. Kattah A. Fervenza F.C. et al.C3 glomerulonephritis associated with monoclonal gammopathy: a case series.Am J Kidney Dis. 2013; 62: 506-514Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar Our study has a number of limitations. First, as a retrospective study, it is based on IF recordings in the biopsy report rather than reevaluation of the original IF glass slides. Thus, the precise character and distribution of the staining for each immune reactant and in each glomerulus cannot be evaluated. Because the IF findings are reported as glomerular intensity of staining, it is not clear as to how the distribution of the staining for C3 and the staining for other immune reactants compare. In particular, whether there is complete or partial colocalization in individual glomeruli cannot be assessed. Second, the relationship of IgM staining with the presence of sclerosing features also cannot be defined without examining the same glomerulus by light microscopy and IF using serial sections. Finally, although we provide presenting clinical data on our cohort of C3G, our study does not include workup of the AP system, treatment, or follow-up information, which will be the subject of future investigations. Thus, we cannot speak to the true ‘sensitivity’ or ‘specificity’ of the proposed criteria. Nevertheless, using the cases of DDD (diagnosed by EM) as a gold standard or true positive, we have clearly shown that a ‘C3 only’ criterion has far too low sensitivity (50%) and that a more inclusive criterion, allowing for some low-level Ig staining, increases this sensitivity. In conclusion, we performed the first study to assess IF criteria for C3G in a large, well-defined biopsy cohort. By using unbiased hierarchical definitions to find an optimal working definition of C3G for practicing pathologists, we provide a framework for triage of patients who are most likely to benefit from molecular and functional assays of AP dysregulation. An optimal definition of C3G, one that is both descriptive and physiologic, should cast a relatively wide net to maximize opportunities to identify AP abnormalities. The Columbia Renal Pathology Laboratory database was queried for cases diagnosed as MPGN1, 2/DDD, or 3 from the years 1999 to 2012. Detailed pathology reports were retrospectively reviewed. We excluded the following cases: (1) all cases with a clear etiology (such as hepatitis C infection, cryoglobulinemia, and dysproteinemia); (2) cases where complete IF findings were not detailed; (3) cases inadequate for IF on frozen tissue; and (4) cases treated with eculizumab, because of potential confounding IF features.27.Herlitz L.C. Bomback A.S. Markowitz G.S. et al.Pathology after eculizumab in dense deposit disease and C3 GN.J Am Soc Nephrol. 2012; 23: 1229-1237Crossref PubMed Scopus (131) Google Scholar A total of 319 biopsies of primary MPGN types 1–3 were identified, including 23 cases with multiple biopsies. For patients with repeat biopsies, only the first biopsy diagnostic of MPGN was analyzed in the initial criteria study. Three additional patients with initial biopsy diagnosis of postinfectious glomerulonephritis and subsequent biopsy diagnosis of MPGN were included among the repeat biopsy analysis. All biopsies were stained at the time of initial biopsy diagnosis according to standard techniques applied to frozen sections using fluorescein isothiocyanate–conjugated antisera to IgG, IgM, IgA, C3c, C1q, fibrin, albumin, kappa, and lambda light chains (Dako, Carpinteria, CA). IF findings were graded on a scale of 0 to 3 by one of four renal pathologists as follows: 0; trace; 1+; 2+; and 3+. For this study, the glomerular IF findings recorded in the biopsy reports were retrospectively reviewed. Using DDD as the validation group, we proposed hierarchical IF criteria with decreasing stringency for diagnosis of C3G: criterion 1, glomerular deposits of C3 only; criterion 2, C3-dominant deposits and ≤1+ IgM only; and criterion 3, C3-dominant deposits and ≥2 orders of intensity stronger than any combination of IgG, IgM, IgA, and C1q. Illustrative examples are shown in Figure 2. These criteria were then applied to cases of primary MPGN1 and MPGN3 to determine the incidence of C3G so defined and the relationship to the Strife and Anders versus Burkholder variants of MPGN3. Cases not meeting the third cutoff were further classified as fulfilling criterion 4: C3-dominant deposits and C3; criterion 7, IgG>C3; and criterion 8, no C3 staining. For those cases defined by IF criteria as C3G, the medical record was reviewed for presenting demographic and clinical features including age, gender, race/ethnicity, duration of proteinuria or renal dysfunction, significant comorbidities (e.g. diabetes and hypertension), and laboratory and serologic findings (including serum creatinine, serum albumin, 24-hour urine protein excretion, urinalysis, and serum C3 and C4 levels). For demographic, clinical, and laboratory findings, continuous variables were expressed as mean values and categorical variables were expressed as prevalence rates. Comparisons were made using analysis of variance (STATA version 11.0, StataCorp, College Station, TX) between three subgroups: MPGN1 versus MPGN2 versus MPGN3 by original diagnostic criteria, and IF criterion 1 versus IF criterion 2 versus IF criterion 3 by the proposed inclusion criteria for C3G detailed above.
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