Antineutrophil Cytoplasmic Autoantibody–Negative Pauci-Immune Crescentic Glomerulonephritis

Antineutrophil Cytoplasmic Autoantibody–Negative Pauci-immune Crescentic Glomerulonephritis

Min Chen, Feng Yu, Su-Xia Wang, Wan-Zhong Zou, Ming-Hui Zhao, and Hai-Yan Wang Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing, People’s Republic of China

Pauci-immune crescentic glomerulonephritis (CrGN) is one of the most common causes of rapidly progressive glomerulonephritis. The majority of patients with pauci-immune CrGN had circulating antineutrophil cytoplasmic autoantibody (ANCA). However, patients with ANCA-negative pauci-immune CrGN were not investigated fully. This study aimed to analyze the characteristics of this subgroup of patients. Patients whose pauci-immune CrGN was diagnosed from 1997 to 2006 in one center were studied retrospectively. The criteria of pauci-immune was defined as “the intensity of glomerular immunoglobulins staining by direct immunofluorescence assay in renal sections was negative to 1؉ staining on a scale of 0 to 4؉.” Clinical and pathologic characteristics were compared between patients with and without ANCA. Among the 85 patients with pauci-immune CrGN, 28 (32.9%) were ANCA negative. Compared with the 57 ANCA-positive patients, the ANCA-negative patients were much younger (39.7 ؎ 17.0 versus 57.6 ؎ 14.0 yr; P < 0.001). The level of urinary protein and the prevalence of nephrotic syndrome were significantly higher in ANCA-negative patients than that in ANCA-positive patients (P < 0.01 and P < 0.001, respectively). However, the prevalence of extrarenal involvement was significantly lower in ANCA-negative patients than that in ANCA-positive patients. The renal survival was poorer in ANCA-negative patients than that in ANCA-positive ones (P < 0.05). ANCA-negative pauci-immune CrGN was not rare and might represent an independent disease entity from ANCA-positive vasculitis. J Am Soc Nephrol 18: 599–605, 2007. doi: 10.1681/ASN.2006091021

auci-immune crescentic glomerulonephritis (CrGN) is patients with negative ANCA constituted a minority of patients one of the most common causes of rapidly progressive with pauci-immune CrGN, this subgroup of patients had not P glomerulonephritis. The characteristic feature of pauci- been investigated fully except for a few studies (7,8). This study immune CrGN is focal necrotizing and crescentic glomerulo- investigated the clinical and pathologic characteristics of pa- nephritis with little or no glomerular staining for Ig by immu- tients with ANCA-negative pauci-immune CrGN, and a com- nofluorescence microscopy examination. The majority of parison was made in detail between patients with ANCA- patients with pauci-immune CrGN have glomerular diseases as negative and ANCA-positive pauci-immune CrGN. a part of a systemic small vessel vasculitis, including Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss Materials and Methods syndrome, or as a part of renal-limited vasculitis (1–3). Patients Antineutrophil cytoplasmic autoantibody (ANCA) was a se- Patients whose pauci-immune CrGN was diagnosed from 1997 to rologic marker for primary systemic small vessel vasculitis. 2006 in the Renal Division and Institute of Nephrology, Peking Uni- Most patients with a cytoplasmic ANCA (cANCA) obtained by versity First Hospital, were enrolled in this retrospective study. Renal biopsy was performed at the time of diagnosis. “Pauci-immune” was indirect immunofluorescence (IIF) assay have ANCA directed defined as “the intensity of glomerular immunoglobulins staining by against proteinase-3 (PR3), as determined by antigen-specific direct immunofluorescence assay in renal sections was negative to 1ϩ ELISA. Patients with a perinuclear ANCA (pANCA) mostly staining on a scale of 0 to 4ϩ.” “Crescentic glomerulonephritis” was have ANCA directed against one of a variety of antigens, such defined as “over 50% of the glomeruli had crescents formation in the as myeloperoxidase (MPO). It was reported that approximately renal specimen.” Patients with secondary vasculitis or with anti–glo- 80 to 90% of patients with pauci-immune CrGN had circulating merular basement membrane antibodies were excluded. The clinical ANCA (4,5), and ANCA might play a major role in the patho- and pathologic data were extracted for analysis. For extrarenal involve- genesis of pauci-immune CrGN (6). ment, only manifestations that were both strongly suggestive of vas- It was noted in a number of patients with pauci-immune culitis and included in the Birmingham Vasculitis Activity Score (9) or CrGN, however, that circulating ANCA was negative. Because the Vasculitis Damage Index (10) were counted when analyzed. The clinical manifestations were recorded at the time of presentation as well as during follow-up. Informed consent was obtained from each patient when renal biopsy was performed. Received September 19, 2006. Accepted November 24, 2006.

Published online ahead of print. Publication date available at www.jasn.org. Detection of ANCA ANCA tests were performed by both IIF assay and antigen-specific Address correspondence to: Dr. Ming-Hui Zhao, Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing 100034, P.R. China. Phone: ELISA for all patients at the time of presentation before immunosup- ϩ86-10-66551736; Fax: ϩ86-10-66551055; E-mail: [email protected] pressive treatment was instituted.

IIF Assay as renal survival. P Ͻ 0.05 was considered significant. Analysis was Standard IIF assays were performed according to the manufacturer’s performed with SPSS statistical software package (version 13.0; Chi- instructions (EUROIMMUN, Lu¨beck, Germany). Ethanol-fixed human cago, IL). polymorphonuclear leukocytes were used to detect ANCA, and mon- key liver sections were used to exclude antinuclear antibodies. cANCA Results and pANCA were distinguished according to staining patterns by two Demographic Features and ANCA Specificity experienced technicians. Eighty-five patients received a diagnosis of pauci-immune CrGN. Twenty-eight (32.9%) of the 85 were ANCA negative Antigen-Specific ELISA both in IIF assay and ELISA. Nine (10.6%) of the 85 were Two highly purified known ANCA antigens, PR3 and MPO, purified cANCA positive, and all of the sera could recognize PR3. as previously reported (11), were used as solid-phase ligands in ELISA. Forty-eight (56.5%) of the 85 were pANCA positive, and all of ␮ Antigens were diluted to 1 to 2 g/ml with 0.05 M bicarbonate buffer the sera could recognize MPO. Among the 28 patients with (pH 9.6) and were coated onto the wells of one half of a Costar ANCA-negative pauci-immune CrGN, 16 were male and 12 microtiter plate; the wells in the other half were coated with coating were female. The average age at onset of the disease (defined as buffer alone and acted as antigen-free wells. The volumes of this step and subsequent steps were 100 ␮l, all incubations were carried out at “patients had signs, symptoms, or laboratory findings closely 37°C for 1 h, and plates were washed three times with PBS that related to the disease [including both renal and extrarenal]”) contained 0.1% Tween-20 (PBST) between stages. Test serum samples was 39.7 yr (range 15 to 65 yr). The median interval between were diluted 1:50 with PBST and coated in duplicate on both antigen- onset of the disease and renal biopsy was 1 mo (range 0.2 to 30 coated wells and antigen-free wells; every plate contained positive, mo). negative, and blank (PBST) controls. The binding was detected with Compared with the 57 patients with ANCA-positive pauci- horseradish peroxidase–conjugated goat anti-human IgG Gibco (BRL, immune CrGN, patients with negative ANCA were signifi- Grand Island, NY) 1:5000 in PBST. The horseradish peroxidase sub- cantly younger at onset of the disease (39.7 Ϯ 17.0 versus 57.6 Ϯ strate o-phenylenediamine was used at 0.4 mg/ml in 0.1 mol/L citrate 14.0 yr; P Ͻ 0.001). The interval from onset of the disease to phosphate buffer (pH 5.0). The reaction was stopped by 2.0 mol/L renal biopsy was significantly shorter in ANCA-negative group H SO , and the results were recorded as the net A490 nm (average 2 4 than that in ANCA-positive group (P Ͻ 0.01; Table 1). value of antigen wells minus average value of antigen-free wells) and expressed as percentage of the known positive controls. Samples were considered positive when they exceeded the mean ϩ 3 SD from 100 Constitutional Symptoms normal blood donors. Of the 28 patients with ANCA-negative pauci-immune CrGN, six (21.4%) had fever, eight (28.6%) had fatigue, two Renal Histopathology (7.1%) had significant weight loss (Ͼ4 kg in 1 mo), two (7.1%) Renal specimens were evaluated using direct immunofluorescence had arthralgia, two (7.1%) had muscle pain, and five (17.9%) (for Ig and complement components) and light and electron micros- had skin rash. The prevalences of fever, weight loss, muscle copy. For light microscopy, paraffin sections were stained with silver, pain, and arthralgia were significantly lower in patients with periodic acid-Schiff, hematoxylin & eosin, and trichrome and were ANCA-negative pauci-immune CrGN than those in patients forwarded to two pathologists. Both pathologists scored the biopsies with ANCA-positive pauci-immune CrGN (21.4 versus 66.7% separately, blinded to patients’ data and the scores of the other ob- [␹2 ϭ 15.39, P Ͻ 0.001]; 7.1 versus 35.1% [␹2 ϭ 7.64, P Ͻ 0.01]; 7.1 server, according to a previously standardized protocol for scoring versus 26.3% [␹2 ϭ 4.31, P Ͻ 0.05]; 7.1 versus 40.4% [␹2 ϭ 9.97, renal biopsies of patients with ANCA-associated vasculitis (12–14). In P Ͻ 0.01]; respectively; Table 1). short, each glomerulus was scored separately on the presence of fibrinoid necrosis, crescents (cellular/fibrous and segmental/circumferen- tial), glomerulosclerosis (local/segmental/global), granulomatous re- Renal Manifestations actions, and a number of other lesions. The presence of glomerular Of the 28 patients with ANCA-negative pauci-immune lesions was calculated as the percentage of the total number of glomer- CrGN, all had hematuria and proteinuria. The level of urinary uli in a biopsy. Interstitial and tubular lesions were scored semiquan- protein was 5.47 Ϯ 3.32 g/24 h. Thirteen patients (46.4%) had titatively on the basis of the percentage of the tubulointerstitial com- nephrotic syndrome. The level of initial serum creatinine was partment that was affected: Interstitial infiltrates (Ϫ, 0%; ϩ, 0 to 20%; 625.6 Ϯ 374.5 ␮mol/L (range 99.3 to 1402 ␮mol/L). Twenty- ϩϩ ϩϩϩ Ͼ Ϫ ϩ , 20 to 50%; and , 50%), interstitial fibrosis ( , 0%; ,0to four patients (85.7%) had elevated initial serum creatinine, and ϩϩ Ͼ Ϫ ϩ 50%; and , 50%), and tubular atrophy ( , 0%; , 0 to 50%; and 15 patients (53.6%) were dialysis dependent on diagnosis. Com- ϩϩ, Ͼ50%). Vascular lesions were scored as presence or absence. pared with the 57 patients with ANCA-positive pauci-immune Differences in scoring between the two pathologists were resolved by CrGN, patients with negative ANCA had higher level of uri- re-reviewing the biopsies and coming to a consensus. nary protein (5.47 Ϯ 3.32 versus 2.23 Ϯ 2.27 g/24 h; P Ͻ 0.01) and higher prevalence of nephrotic syndrome (46.4 versus 8.8%; Statistical Analyses ␹2 ϭ 16.0, P Ͻ 0.001; Table 1). Differences of quantitative parameters between groups were as- sessed using the t test (for data that were normally distributed) or nonparametric test (for data that were not normally distributed). Dif- Renal Histology Ϯ ferences of semiquantitative results were tested using the Mann-Whit- In each renal biopsy specimen, an average of 23.7 13.2 ney U test. Differences of qualitative results were compared using ␹2 glomeruli could be seen. Of the 28 patients with ANCA-nega- test. Kaplan-Meier curves were used to analyze patient survival as well tive pauci-immune CrGN, 9.6 Ϯ 12.2% (0 to 41.7%) of the J Am Soc Nephrol 18: 599–605, 2007 ANCA-Negative Pauci-Immune CrGN 601

Table 1. Comparison of clinical and laboratory features of patients with pauci-immune CrGN with and without ANCAa Ϫ ϩ ANCA( ) ANCA( ) P Feature (n ϭ 28) (n ϭ 57)

Male/female 16/12 30/27 Ͼ0.05 Age of onset of disease 39.7 Ϯ 17.0 57.6 Ϯ 14.0 Ͻ0.001 Interval between onset of the disease and 1 (0.2, 30) 3 (0.25, 60) Ͻ0.01 diagnosis (mo; median ͓min, max͔) Fever 6 (21.4%) 38 (66.7%) Ͻ0.001 Fatigue 8 (28.6%) 30 (52.6%) Ͼ0.05 Weight loss 2 (7.1%) 20 (35.1%) Ͻ0.01 Muscle pain 2 (7.1%) 15 (26.3%) Ͻ0.05 Arthralgia 2 (7.1%) 23 (40.4%) Ͻ0.01 Skin rash 5 (17.9%) 9 (15.8%) Ͼ0.05 Urinary protein (g/24 h) 5.47 Ϯ 3.32 2.23 Ϯ 2.27 Ͻ0.001 Nephrotic syndrome 13 (46.4%) 5 (8.8%) Ͻ0.001 Initial Scr (␮mol/L) 625.6 Ϯ 374.5 574.1 Ϯ 359.7 Ͼ0.05 Pulmonary 3 (10.7%) 21 (36.8%) Ͻ0.05 Hemoptysis 2 (7.1%) 12 (21.1%) Ͼ0.05 Pulmonary interstitial fibrosis 0 (0%) 9 (15.8%) Ͼ0.05 Ophthalmic 1 (3.6%) 14 (24.6%) Ͻ0.05 Otic 0 (0%) 18 (31.6%) Ͻ0.001 Nasal 0 (0%) 10 (17.5%) Ͻ0.05 Gastrointestinal 2 (7.1%) 4 (7.0%) Ͼ0.05 Anemia 23 (82.1%) 54 (94.7%) Ͼ0.05 Leukocytosis 4 (14.3%) 21 (36.8%) Ͻ0.05 Thrombocytosis 8 (28.6%) 19 (33.3%) Ͼ0.05 Elevated ESR 15 (53.6%) 43 (75.4%) Ͼ0.05 BVAS 13.9 Ϯ 2.4 20.1 Ϯ 5.0 Ͻ0.001

aBVAS, Birmingham Vasculitis Activity Score; CrGN, crescentic glomerulonephritis; ESR, erythrocyte sedimentation rate; Scr, serum creatinine.

glomeruli were normal, and 76.0 Ϯ 17.1% (50.0 to 100%) of the age of cellular crescent tended to be higher in patients with glomeruli had crescents. The mean and median percentage of negative ANCA than that in patients with positive ANCA global sclerosis was 5.9 and 0.0% (0 to 50.0%), respectively. The (61.6 Ϯ 35.3 versus 45.9 Ϯ 34.1%; P ϭ 0.05; Table 2). mean and median percentage of fibrinoid necrosis of glomeruli Of the 28 patients with ANCA-negative pauci-immune tuft was 2.8 and 0.0% (0 to 25.0%), respectively. Compared with CrGN, 25 (89.3%) had interstitial infiltrates, and four (14.3%), 13 the 57 patients with ANCA-positive pauci-immune CrGN, pa- (46.4%), and eight (28.6%) scored as mild (ϩ), dense (ϩϩ), and tients with negative ANCA had a significantly lower percent- very dense (ϩϩϩ), respectively. Seven of the 28 (25.0%) pa- age of normal glomeruli (9.6 Ϯ 12.2 versus 16.3 Ϯ 14.7%; P Ͻ tients had interstitial fibrosis, and all were scored as diffuse 0.05). Among glomeruli with crescent formation, the percent- (ϩϩ). Tubular atrophy was present in 26 (92.8%) of 28 biopsies,

Table 2. Percentages of various glomerular lesions Ϫ ϩ ANCA( ) ANCA( ) P Lesion (n ϭ 28) (n ϭ 57)

Normal glomeruli 9.6 Ϯ 12.2 16.3 Ϯ 14.7 Ͻ0.05 Crescent 76.0 Ϯ 17.1 72.7 Ϯ 14.3 Ͼ0.05 cellulara 61.6 Ϯ 35.3 45.9 Ϯ 34.1 0.05 fibrousa 20.8 Ϯ 28.8 27.6 Ϯ 28.8 Ͼ0.05 Fibrinoid necrosis 0.0 (0 to 25.0) 0.0 (0 to 37.5) Ͼ0.05 Glomerular sclerosis 0.0 (0 to 50.0) 0.0 (0 to 40.0) Ͼ0.05

aThe ЈintermediateЈ type of crescents (fibrocellular crescents) were not included for statistics. 602 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 599–605, 2007 and three (10.7%) of 28 and 23 (82.1%) of 28 scored as focal (ϩ) Other Clinical Manifestations and diffuse (ϩϩ), respectively. Interstitial granulomatous in- Of the 28 patients with ANCA-negative pauci-immune flammation was present in one (3.6%) 28 biopsies. Interstitial CrGN, two (7.1%) had gastrointestinal involvement manifested fibrosis was more prevalent and severe in patients with nega- as abdominal pain or bloody diarrhea. Only one (3.6%) patient tive ANCA than that in patients with positive pANCA (P Ͻ had ophthalmic involvement manifested as conjunctivitis and 0.05; Figure 1). Arteriolosclerosis was present in 19 (67.9%) of 28 uveitis. None had otic or nasal involvement. Compared with patients, and necrotizing lesions of arteriole was present in two the 57 patients with ANCA-positive pauci-immune CrGN, the (7.1%) of 28 patients with ANCA-negative pauci-immune prevalences of ophthalmic, otic, and nasal involvement in pa- CrGN. tients with negative ANCA were significantly lower (3.6 versus 24.6% [␹2 ϭ 4.34, P Ͻ 0.05]; 0 versus 31.6% [␹2 ϭ 11.2, P Ͻ 0.001]; 0 versus 17.5% [␹2 ϭ 4.01, P Ͻ 0.05], respectively). When pa- Pulmonary Manifestations tients with pauci-immune CrGN were considered as part of Of the 28 patients with ANCA-negative pauci-immune systemic vasculitis, the level of Birmingham Vasculitis Activity CrGN, three (10.7%) had pulmonary involvement; two (7.1%) Score (9) at diagnosis was significantly lower in patients with had hemoptysis, and one (3.6%) had multiple nodules. None ANCA-negative pauci-immune CrGN than that in patients had pulmonary interstitial fibrosis revealed by chest radiogra- with ANCA-positive pauci-immune CrGN (13.9 Ϯ 2.4 versus phy or computerized tomography scan. Compared with the 57 20.1 Ϯ 5.0; P Ͻ 0.001; Table 1). patients with ANCA-positive pauci-immune CrGN, the prevalence of pulmonary involvement in patients with negative ANCA was significantly lower (10.7 versus 36.8%; ␹2 ϭ 6.33, Laboratory Findings P Ͻ 0.05; Table 1). Of the 28 patients with ANCA-negative pauci-immune CrGN, 23 (82.1%) had anemia, four (14.3%) had leukocytosis, eight (28.6%) had thrombocytosis and 15 (53.6%) had elevated erythrocyte sedimentation rate.

Treatment and Outcomes Treatment protocols were comparable between patients with ANCA-positive and ANCA-negative pauci-immune CrGN. The majority of patients (25 of 28) received corticosteroid com- bined with daily oral or monthly intravenous cyclophospha- mide. Nineteen of the 28 patients received intravenous meth- ylprednisolone pulse therapy. After the induction therapy, 18 (72.0%) of 25 patients achieved complete or partial remission. Seven (25.0%) of the 28 patients died, and 12 (42.9%) of 28 patients progressed to ESRD and received renal replacement therapy. The median time of survival and renal survival was 96.0 and 12.7 mo, respectively. Six (21.4%) and 11 (39.3%) of 28 patients died and progressed to ESRD within 1 yr after diagnosis, respectively. Compared with the patients with positive ANCA, patients with negative ANCA had poorer renal outcome (P Ͻ 0.05; Figure 2). No significant difference was found between the survivals of these two subgroups of patients (Figure 3). The causes of death of the patients are listed in Table 3.

Discussion There has been a strong association between ANCA and pauci-immune CrGN, and ANCA was used to be an important tool in distinguishing pauci-immune CrGN from other types of CrGN, such as anti–glomerular basement membrane antibody– associated nephritis, IgA nephropathy, and lupus nephritis. It was reported that the likelihood of a positive ANCA was inversely proportional to the intensity of glomerular Ig staining by direct immunofluorescence assay in renal specimens with ϩ Figure 1. Differences of tubulointerstitial lesion. (A) I versus II: CrGN. When the staining of immunoglobulins was 1 or less P Ͼ 0.05; I versus III: P Ͼ 0.05. (B) I versus II: P Ͼ 0.05; I versus (on a scale of 0 to 4ϩ), the likelihood of a positive ANCA was III: P Ͼ 0.05. (C) I versus II: P Ͼ 0.05; I versus III: P Ͻ 0.05. approximately 80 to 90% (4,5). However, in some patients with J Am Soc Nephrol 18: 599–605, 2007 ANCA-Negative Pauci-Immune CrGN 603

Table 3. Causes of death in the two patient subgroups

ANCA(Ϫ) ANCA(ϩ) Cause of Death (n ϭ 7) (n ϭ 17)

Complications of ESRD 4 1 Severe infections 2 9 Massive pulmonary hemorrhage 0 2 Cardiac events 0 1 Unknown 1 4

immune CrGN, and this proportion was higher than that in most other studies (7,13,18,19). It was found in our study that among the patients with pauci-immune CrGN, there was significant difference between ANCA-negative group and ANCA-positive group. Compared with ANCA-positive patients, ANCA-negative patients had greater degree of proteinuria as well as higher prevalence of Figure 2. Renal survivals of patients with pauci-immune cres- nephrotic syndrome; in renal histology, ANCA-negative pa- centic glomerulonephritis (CrGN) with and without antineu- tients tended to have more severe lesions on glomeruli (fewer trophil cytoplasmic autoantibody (ANCA). The solid line rep- normal glomeruli). It was speculated that these differences resents ANCA-negative patients; the dashed line represents might be contributing factors to poorer renal outcome of ANCA-positive patients. ANCA-negative patients than ANCA-positive ones in our study. The level of initial serum creatinine was comparable between these two subgroups of patients. However, ANCA- negative patients had a shorter interval from onset of the disease to diagnosis. Therefore, the renal injury seemed to be a more “fulminant” process in ANCA-negative patients than in ANCA-positive ones. The renal histology of the patients in our study was different from the study of Eisenberger et al. (7), which investigated 20 patients with ANCA-negative pauci-immune CrGN and found that active lesions were similar between ANCA-positive and ANCA-negative patients, whereas chronic lesions, including interstitial fibrosis and glomerulosclerosis, were more promi- nent in ANCA-negative patients. Because the numbers of patients in both studies were limited, sampling error could not be excluded. However, the proportion of fibrinoid necrosis in this study was much lower than others (20). This might be due to the limited number of sections of specimens in our study. Regarding extrarenal involvement, Hedger et al. (8) investigated 35 patients with ANCA-negative rapidly progressive glomerulonephritis and found that they had fewer airway Figure 3. Patient survivals of patients with pauci-immune CrGN symptoms than ANCA-positive ones but failed to find any with and without ANCA. The solid line represents ANCA-nega- other difference between these two subgroups of patients. Sim- tive patients; the dashed line represents ANCA-positive patients. ilar results were confirmed by the study of Eisenberger et al. (7). However, the latter study did not compare the extrarenal involvement of ANCA-negative patients with ANCA-positive ones. In our study, it was found that ANCA-negative patients pauci-immune CrGN, serum ANCA was negative. Studies on had many fewer constitutional symptoms and much less extra- this subgroup of patients were not sufficient as a result of the renal involvement, including lung, eye, ear, nose, and throat, limited number of patients (7,8,15–17). than ANCA-positive patients. ANCA-negative patients also This study retrospectively analyzed 28 patients with ANCA- were much younger at onset of disease than ANCA-positive negative pauci-immune CrGN in our single center, which was ones. In another words, ANCA-negative patients seemed to one of the largest series to date focused on this subgroup of have many fewer “vasculitic” manifestations than ANCA-pos- patients. It constituted nearly one third of patients with pauci- itive ones. 604 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 599–605, 2007

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