Systemic lupus erythematosus (SLE) is an autoimmune disease that can affect essentially any organ or tissue. Lupus nephritis (LN) is one of the most serious complications of SLE because it is the major predictor of poor prognosis. The presence of autoantibodies directed against several cytoplasmic and nuclear antigens is a characteristic feature of SLE and plays a very important role in the pathogenesis of LN.1 Antineutrophil cytoplasmic autoantibodies (ANCA) constitute a distinct class of autoantibodies that are directed against cytoplasmic constituents of human neutrophils, and they are mainly associated with small vessel vasculitides (SVVs), such as microscopic polyangiitis, granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis.2 According to the pattern of indirect immunofluorescence (IIF) staining, ANCA can be divided into the cytoplasmic staining pattern (c-ANCA) which representative target antigen is proteinase-3 (PR3) and the perinuclear staining pattern (p-ANCA) which representative target antigen is myeloperoxidase (MPO). The subtype of ANCA was determined by antigen-specific enzyme-limked immunosorbent assay (ELISA).3 ANCA were first described in patients with necrotizing glomerulonephritis.4 Until now, ANCA have been detected in infectious, neoplastic, and inflammatory diseases.5,6 Circulating ANCA in serum is also present in connective tissue diseases such as SLE.7 However, because of there are a broad range of autoantibodies in the SLE, and some are closely associated with specific clinical manifestations and disease activity, the presence of ANCA has not received the same attention as in SVV.8
Recently, a number of studies concerning ANCA prevalence in SLE have appeared. However, these reports have been controversial about the clinical relevance and disease activity of ANCA,9–14 and the role of ANCA in SLE remains unclear. Moreover, only rare investigations have addressed the renal outcomes of LN patients with ANCA. Therefore, we retrospectively analyzed the baseline characteristics and outcomes of 154 patients with LN and evaluated the risk factors for adverse renal outcomes.
A total of 154 Chinese patients, from January 2011 to December 2013, who fulfilled at least 4 of the 2012 American College of Rheumatology revised criteria for the diagnosis of SLE15 and had renal involvement confirmed by renal biopsy, were analyzed retrospectively. Renal biopsy was performed at the time of diagnosis and before immunosuppressive therapy was initiated. Drug-induced LN and patients with other autoimmune diseases were excluded. None of the patients had a history of propylthiouracil, isoniazide, or hydralazine use, which could cause ANCA positivity. The details of the recruitment process are shown in Figure 1.
This study was in compliance with the Declaration of Helsinki and was approved by the Medical Ethical Committee of the Xinqiao Hospital, Third Military Medical University. The requirement for patient consent was waived by the Medical Ethical Committee because this study involved no more than minimal risk as a retrospective study, and no identifiable information was used.
The patients’ demographic and clinical data were reviewed retrospectively for age, sex, medical history, medications, and follow-up duration by medical records. Clinical features included butterfly erythema, fever, oral ulcer, arthritis, nervous system disorder, serositis, alopecia, and photosensitivity. Laboratory data consisted of levels of erythrocytes, leukocytes, platelets, hemoglobin, albumin, erythrocyte sedimentation rate, serum creatinine (SCr), uric acid, complement C3, serum IgG, 24-h proteinuria, and autoantibodies. The neutrophil to lymphocyte ratio (NLR) and the estimated glomerular filtration rate (eGFR) were also included in the study. eGFR was estimated according to the Chronic Kidney Disease Epidemiology Collaboration 2-level race equation16 Laboratory data regarding the presence of autoantibodies, including anti-dsDNA, anti-Sm, anti-SSA, anti-SSB, anti-Ro-52, antiribosomal P, anti-U1RNP, antihistone, antinucleosome, anticardiolipin, antimitochondrial antibody M2 and ANCA, were collected from the Department of Clinical Laboratory. Serum antinuclear antibodies were detected using the indirect immunofluorescence assay (EUROIMMUN; Lübeck, Germany) and anti-dsDNA antibodies were detected using the Crithidia luciliae indirect immunofluorescence test (EUROIMMUN; Lübeck, Germany). Antiextractable nuclear antigen antibodies, including anti-Sm, anti-SSA, anti-SSB, anti-Ro-52, antiribosomal P, anti-U1RNP, antihistone, antinucleosome, and antimitochondrial antibody M2 antibodies, were detected using the immunodotting assay (EUROIMMUN; Lübeck, Germany). Anticardiolipin antibodies were detected using ELISA (EUROIMMUN; Lübeck, Germany) PR3-ANCA and MPO-ANCA were evaluated using EUROBlot (EUROIMMUN; Lübeck, Germany) and IIF (EUROIMMUN; Lübeck, Germany), according to the protocols provided by the manufacturer.
The renal biopsies were processed according to standard techniques for light microscopy, immunofluorescence (IF), and electron microscopy. For light microscopy, paraffin sections were stained with hematoxylin and eosin, Masson's trichrome, periodic acid-Schiff, and silver methenamine. Glomerular sclerosis, crescent formation, fibrinoid necrosis, microthrombosis, tubular atrophy, and interstitial fibrosis were observed. For renal tissue with IF staining, the presence of IgA, IgG, IgM, and C3 was documented (estimated semiquantitatively on a scale of 0–4 [0 for absence, 1 for mild, 2 for moderate, 3 for severe, 4 for very severe]). Localization of deposits was verified using electron microscopy. LN was classified according to the International Society of Nephrology/Renal Pathology Society classes.17 Pathological parameters, such as the activity index (AI), chronic index (CI), and tubular interstitial lesion (TIL), were assessed using a modification of the criteria18 involving semiquantitative scoring of specific renal pathological features by 2 renal pathologists. AI included endocapillary hypercellularity, leukocyte infiltration, cellular crescents, karyorrhexis, fibrinoid necrosis, formation of wire loops, and interstitial inflammatory cell infiltration. CI included glomerular sclerosis, fibrous crescents, interstitial fibrosis, and tubular atrophy. TIL included tubule degeneration and necrosis, tubular atrophy, interstitial inflammatory cell infiltration, and interstitial fibrosis. Differences in scoring between the 2 pathologists were resolved by re-reviewing the biopsies and reaching a consensus.
The patients were followed up in an outpatient clinic specified for patients with LN. The primary end point was defined as death, and the secondary end point was defined as end-stage renal disease (ESRD) or the doubling of SCr. The combined end point was defined as a composite outcome of death, ESRD or the doubling of SCr. ESRD was defined as maintenance hemodialysis, renal transplantation and eGFR ≤15 mL/min/1.73 m2. Remission of LN was defined as protein-negative urine and normal or near-normal (within 10% of normal eGFR if previously abnormal) eGFR.19 If patients were lost to follow-up during the study, they were followed until the last recorded visit.
All statistical analyses were performed using SPSS, version 20.0 (SPSS, Chicago, IL). For continuous variables, the results are expressed as the mean ± standard deviation, median (range), or median (interquartile range, IQR), as appropriate. Categorical variables are reported as the number and frequency. The 1-sample K-S test was used to determine the normality of the data distribution. According to their normality, continuous variables were compared with the independent samples t test, the paired samples t test or the Mann–Whitney U test. Comparisons were based on the Chi-square test or Fisher's exact test for categorical variables. Kaplan–Meier analysis was used to compare survival in the ANCA-positive and ANCA-negative groups. A multivariate Cox regression model was used to evaluate risk factors for poor outcomes. Relevant variables that were significantly associated with poor outcomes by univariate analysis were included in multivariate models. All tests were 2-sided, and P < 0.05 was considered significant.
General Patient Data
Among the 154 patients with LN enrolled in this study, 26 (16.88%) were seropositive for ANCA, with MPO-ANCA presenting in 24 (92.31%) patients, whereas 2 (7.69%) patients showed PR3-ANCA. In the ANCA-positive group, 4 patients (15.38%) were men, and 22 (84.62%) were women, with a median age of 35 (IQR, 23–51) years old at diagnosis. There were no significant differences in sex, age, or disease duration between the ANCA-positive and ANCA-negative groups (Table 1).
Clinical and Laboratory Parameters
The clinical and laboratory features of the patients in the 2 groups are shown in Table 1. The incidences of alopecia, oral ulcers, photosensitivity and skin lesions, and psychosomatic manifestations in the ANCA-positive group were significantly higher than in the ANCA-negative group (19.23% vs 3.13%, P = 0.007; 15.38% vs 2.34%, P = 0.02; 19.23% vs 7.03%, P = 0.02; and 7.69% vs 0.00%, P = 0.03, respectively). Compared with the ANCA-negative group, the ANCA-positive group had significantly lower levels of complement C3 (0.3 [IQR, 0.2–0.5] vs 0.4 [IQR, 0.3–0.6] g/L; P = 0.03). Additionally, the positive rates of antinucleosome antibodies, antihistone antibodies, antimitochondrial antibody M2, and anticardiolipin antibodies were significantly higher in the ANCA-positive patients than in the ANCA-negative patients (53.84% vs 17.97%, P < 0.001; 53.84% vs 20.31%, P < 0.001; 19.23% vs 5.47%, P = 0.03; 30.77% vs 8.59%, P = 0.005, respectively).
The parameters of renal histopathology of the LN patients with and without ANCA are listed in Table 2. We observed that the distributions of LN classifications were similar in the 2 groups. The incidence and proportion of glomerular sclerosis were higher in the ANCA-positive group than in the ANCA-negative group (53.85% vs 25.00%, P = 0.003; 6.27% vs 0.00%, P = 0.004, respectively). There were no significant differences in the SLEDAI, AI, CI, or TIL scores between the 2 groups, whereas the ANCA-positive group had a notably higher chronic index score than the ANCA-negative group (1 [IQR, 0–2.5] vs 0 [IQR, 0–1], respectively, P = 0.01).
Treatment and Outcome
The therapies for LN patients with and without ANCA were compared, and there were no significant differences in therapy between the 2 groups (Table 3).
All 26 of the ANCA-positive patients were followed up for 1 to 38 months (mean 15.0 ± 10.6 months). At the end of the study, 1 patient died, 2 patients underwent maintenance hemodialysis (one of whom received renal transplantation after 11 months), 2 patients developed the fifth stage of chronic kidney disease (CKD), another 2 patients experienced SCr doubling, and 6 patients achieved remission. In the ANCA-negative group, 9 patients were lost to follow-up, and the remaining 119 patients were followed up for 1 to 40 months (mean 17.9 ± 9.8 months). Four patients died, 3 patients underwent maintenance hemodialysis, 3 patients developed the fifth stage of CKD, and 59 patients achieved remission. The mortality rates between the 2 groups had no significant difference (P > 0.99), but the complete remission rate in the ANCA-negative group was higher than that in the ANCA-positive group (49.58% vs 23.08%, respectively, P
= 0.01). The cumulative renal survival rate in the ANCA-positive group was significantly lower than that in the ANCA-negative group (71.26% vs 91.48%, log-rank = 6.59, P = 0.01; Figure 2).
Univariate Cox regression analysis showed that reduced eGFR (hazard ratio [HR], 1.02; 95% confidence interval, 1.01 to 1.04; P < 0.001), NLR (HR, 1.29; 95% confidence interval, 1.12 to 1.47; P < 0.001), ANCA (HR, 3.25; 95% confidence interval, 1.24 to 8.55; P = 0.02), crescent formation (HR, 2.89; 95% confidence interval, 1.00 to 7.82; P = 0.04), and glomerulosclerosis (HR, 2.72; 95% confidence interval, 1.05 to 7.05; P = 0.04) were risk factors for LN renal survival. Multivariate Cox analysis revealed that reduced eGFR (HR, 1.02; 95% confidence interval, 1.01 to 1.03; P = 0.005), NLR (HR, 1.20; 95% confidence interval, 1.02 to 1.40; P = 0.03), and ANCA (HR, 3.37; 95% confidence interval, 1.12 to 10.09; P = 0.03) remained independent risk factors for the patients’ renal survival after adjusting for age, sex, crescent formation, and glomerulosclerosis (Table 4).
Our study found that the prevalence of ANCA in LN was 16.9% (26 of 154 patients). The predominant ANCA pattern observed was p-ANCA, of which 92.31% of cases had anti-MPO antibodies. Regarding clinical features, this study demonstrated that more multisystem damage occurred in ANCA-positive LN patients than in ANCA-negative LN patients. Among the studied laboratory parameters, the frequencies of antinucleosome antibody, antihistone antibody, antimitochondrial antibody M2, and anticardiolipin antibody were significantly higher in the ANCA-positive group than in the ANCA-negative group, whereas the level of complement C3 was lower in the ANCA-positive group. Moreover, ANCA-positive LN patients showed high scores on the pathological chronic index.
Several previous studies have investigated the role of ANCA in SLE.8–14,20–25 Some reported no associations between ANCA positivity and disease activity, and the clinical picture and antibody profile were similar in ANCA-positive and ANCA-negative SLE patients. These studies also found no association of ANCA with renal involvement. However, other studies have reported a correlation. Lee et al9 reported a significant correlation between the presence of ANCA and crescent formation in patients with World Health Organization (WHO) class IV glomerulonephritis but not with the overall presence of renal involvement. Chin et al23 also studied the relationship between ANCA and nephritis. They found that p-ANCA was significantly associated with the presence of nephritis, particularly with WHO class IV, and with the presence of anti-dsDNA antibody. In addition, they reported an association between the presence of ANCA and deterioration of renal function. Pan et al13 conducted a study among patients with new-onset SLE and identified correlations between ANCA positivity and the presence of various clinical manifestations of SLE, anti-dsDNA antibody, and anti-Sm antibody. Many factors could be involved in these disparities, such as demographic characteristics, the application of different classifications, and the patient selection criteria. Our study found some correlations of ANCA with severe clinicopathologic features, which suggests that ANCA could play a role in the pathogenesis of LN.
Previous studies have focused on the clinical manifestations of LN patients with ANCA, but few have investigated the outcomes of these patients. Yu et al14 reported that the mortality rate in the ANCA-positive group was higher than in the ANCA-negative group and that ANCA was a risk factor for mortality, but the renal outcomes of the ANCA-positive LN patients were not mentioned. In our study, the remission rate in the ANCA-positive group was lower than in the ANCA-negative group, and ANCA was an independent risk factor for poor renal outcomes. In addition, we found that 2 additional factors, reduced eGFR and NLR, were also independent risk factors for poor renal outcomes. Decreases in eGFR are strongly and consistently associated with the risks of ESRD and mortality,26 so eGFR could predict the prognosis of LN patients. NLR is an important measurement of systemic inflammation because it is cost effective and readily available, and it can be calculated easily.27 Many recent studies have suggested that an elevated NLR is associated with poor survival of subjects with cancer,28 but its value in LN has been uncertain. The contribution of elevated NLR in LN patients must be confirmed by prospective studies.
We found that ANCA was associated with poor renal outcomes of LN patients, but the pathogenesis of ANCA in LN patients is far from clear. Hervier et al29 found that ANCA, usually MPO-ANCA, occurs in a small minority of patients with SLE who seemed to have vasculitic features of ANCA-associated disease, which is considered to be an overlapping syndrome. This finding could constitute proof that ANCA participates in the pathogenesis of LN. In the ANCA-positive group, we observed that other autoantibodies appeared more frequently, perhaps due to neutrophil extracellular trap (NET) formation. NETs, which are released via a unique form of cell death called NETosis, consist of decondensed chromatin DNA in association with histones, granular proteins, and a few cytoplasmic proteins.30–32 These NETs might provide a novel source of autoantigens. Autoantigens can be recognized by the immune system, producing pathogenic antibodies to accelerate the progression of LN. In addition to NETs, ANCA might activate the complement pathway to participate in the pathogenesis of LN. Recent studies have provided strong evidence that complement system activation, particularly through an alternative pathway, is involved in the pathogenesis of ANCA-associated vasculitides,33 and complement system activation also plays an important role in the pathogenesis of LN.34 In addition, our research found that LN patients with ANCA had lower levels of complement C3 than those without ANCA. Collectively, we hypothesize that the occurrence of ANCA in LN is a multifactorial process. The exact mechanism is of great interest for further investigations.
However, healthy individuals also have circulating autoantibodies against MPO and PR3.33 Compared with pathogenic MPO-ANCA, natural MPO-ANCA has lower titers, lower avidity, less subclass diversity, and less capability to activate neutrophils in vitro.35 In addition, Roth et al36 found that certain MPO epitopes were specific for active disease, and others remained present during remission or were also present in healthy individuals. The high sensitivity and specificity of ANCA detection should be examined in further studies, and the epitopes should be determined.
Our study has several limitations. The major limitation of this study was that the follow-up time was limited. Therefore, we could not effectively evaluate the renal outcomes. In addition, due to limitations based on the relatively small sample size in each subgroup of LN WHO classifications, the results of the renal outcomes of the LN patients among different categories must be validated in larger cohorts. Finally, although we speculate that ANCA could induce NETs and active complement system to participate in the pathogenesis of LN, such experiments were not performed because this study was retrospective.
In conclusion, ANCA in LN patients is not rare, and LN patients with ANCA present with more severe clinicopathologic injuries. ANCA is an independent risk factor for poor renal outcomes in LN patients.
The authors thank Prof. Changjun Cai of the Department of Mathematical Statistics at Third Military Medical University and Prof. Rufu Xu of the Evidence-based Medicine Center of Xinqiao Hospital for their advice on the statistical analyses.
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