BMJ Open. Treatment and outcome of lupus nephritis with antineutrophil cytoplasmic antibody positivity

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1 Treatment and outcome of lupus nephritis with antineutrophil cytoplasmic antibody positivity Journal: Manuscript ID bmjopen-0-0 Article Type: Research Date Submitted by the Author: -Dec-0 Complete List of Authors: Li, Cui; National Clinical Research Center of Kidney Diseases, Jinling Hospital, 0 East Zhongshan Road, Nanjing 00, Jiangsu, China. Zhou, Min-Lin Liang, Dan-Dan Wang, Jing-Jing Yang, Jing Zeng, Cai-Hong Zhang, Hai-Tao <b>primary Subject Heading</b>: Renal medicine Secondary Subject Heading: Pathology Keywords: antineutrophil cytoplasmic autoantibody, clinical features, lupus nephritis, outcome, pathological presentations, treatment : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

2 Page of TITLE PAGE Title: Treatment and outcome of lupus nephritis with antineutrophil cytoplasmic antibody positivity Name and address of the corresponding author:hai-tao Zhang, National Clinical Research Center of Kidney Diseases, Jinling Hospital, 0 East Zhongshan Road, Nanjing 00, Jiangsu, China. htzhang@.com Detail of all co-authors: Cui Li, doctor, MD; Min-Lin Zhou, statisticians, MD; Dan-Dan Liang, pathologist, MD; Jing-Jing Wang, doctor, MD; Jing Yang, doctor, PhD; Cai-Hong Zeng, professor of pathology PhD; Hai-Tao Zhang, professor of medicine, MD. All co-authors from National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China. Note: Cui Li is the first author and Hai-Tao Zhang is the corresponding author of this study. Word count: words. - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

3 Page of ABSTRACT Objective: To assess the clinical features, pathological presentations, treatment and outcome of lupus nephritis (LN) with antineutrophil cytoplasmic antibody(anca) positivity. Design: A retrospective cohort. Methods: Patients (n = ) between and 00 were retrospectively included from Jinling Hospital in China if presenting with biopsy-proven LN with ANCA positivity. Clinicopathological characteristics and outcome were analyzed and compared with a control group (n = ). We further compared treatment response and outcome of ANCA-positive LN patients based on the treatment issued. Results: The cohort consisted of 0 women and men with a median age of years at biopsy, including MPO-ANCA, PR-ANCA and with double positivity. LN with ANCA positivity exhibited higher hematuria, serum creatinine levels and SLEDAI scores. On pathological evaluation, Class IV LN predominance was observed, accounting for.%. Light microscopy revealed significantly higher scores of AI and CI, including cellular crescents, interstitial inflammation, tubular atrophy, and interstitial fibrosis. ANCA-positive LN patients receiving mycophenolate mofetil as induction therapy had higher complete remission and better renal outcome than those requiring cyclophosphamide. During follow-up, end-stage renal disease developed in (.%) ANCA-positive LN patients, all of whom were MPO-ANCA positive. Conclusions: LN with ANCA positivity was characterized by massive hematuria, advanced renal insufficiency. Higher complete remission and better prognosis were observed when using mycophenolate mofetil than cyclophosphamide as induction therapy. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

4 Page of Strengths and limitations of this study: In this study, clinicopathological characteristics and outcome were compared between all LN patients with ANCA positivity (n = ) and ANCA negativity (n = ) at Jinling Hospital in China between and 00 in retrospect. It is the first study which analyses treatment response and outcome of LN patients with ANCA positivity based on the treatment issued (mycophenolate mofetil versus cyclophosphamide). One of the limitations is that the study is restricted to only the biopsy-proven LN patients and those without biopsy have not been included. A larger cohort is needed to study the renal prognosis of this population. - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

5 Page of INTRODUCTION Lupus nephritis (LN) is an immune complex glomerular nephritis that develops as a frequent complication of systemic lupus erythematous (SLE). Autoantibody production in SLE patients is a hallmark of the disease entity as well as its activity and prognosis. The pathogenesis of LN involves a variety of mechanisms, including the loss of tolerance, innate and adaptive immunity responses activated by nucleic acids released from netting or apoptotic neutrophils, antibody binding to multiple intrarenal autoantigens, and intrarenal complement activation, leading to glomerular injury and immune complex deposition. Antineutrophil cytoplasmic autoantibody (ANCA) is the probable cause of a distinct form of vasculitis that can be accompanied by necrotizing granulomatosis. Based on enzyme-linked immunoadsordent assay (ELISA), the major target antigens of ANCA are proteinase (PR) and myeloperoxidase (MPO). LN patients with ANCA positivity have been described in case reports or small series over the last years. - However, due to the relatively small amount of research to date, the purpose of this study was to assess the clinical features, pathological presentations, treatment, remission and outcome of LN with ANCA positivity. METHODS Patients A cohort of Chinese patients with biopsy-proven LN at Jinling Hospital from January to December 00 were retrospectively reviewed. 0 Patients who fulfilled the following criteria were included in this study: () age years; () patients who met the American Rheumatologic Association criteria for the diagnosis of SLE; () biopsy-proven LN; () presence of ANCA - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

6 Page of positivity; () duration of follow-up months; () complete baseline and follow-up data. Simultaneously, LN patients with ANCA negativity were included to the control group during the same period and compared with the ANCA-positive LN patients. Renal morphology For light microscopy, biopsy specimens were processed for hematoxylin-eosin, periodic acid-schiff, Masson trichrome, and Jones methenamine silver staining. Pathological parameters such as activity index (AI) and chronic index (CI) were determined using a modification of an earlier reported system involving semi-quantitative scoring of specific biopsy features. Vasculopathy was defined according to renal vascular complications of SLE. Biopsy specimens were reviewed and reclassified according to the 00 International Society of Nephrology (ISN)/Renal Pathology Society (RPS). All biopsy specimens were examined by two renal pathologists. Differences in classification and scores between the two were resolved by reviewing the biopsies. Data collection The following data were collected retrospectively at biopsy: gender, age, duration of LN, SLE disease activity index (SLEDAI), hypertension, extrarenal manifestations, hour urinary protein excretion, urinary sediment, serum albumin (SAlb), serum creatinine (SCr), estimated glomerular filtration rate (egfr), C, C, and ANCA specificity. Based on the treatment issued during the first months, patients were assigned to the mycophenolate mofetil (MMF) group if they received a minimum cumulative dose of 0 g MMF by oral; patients were assigned to the cyclophosphamide (CYC) group if they received a minimum cumulative dose of g intravenous CYC. Patients who were given less CYC or MMF, tripterygium - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

7 Page of glycosides or other immunosuppressors were assigned to the other regimens group. All patients received glucocorticoids. Information about treatment response and outcome was collected after induction therapy. The following definitions were used: duration of LN, the time from LN diagnosis to first-time renal biopsy; hypertension, blood pressure > 0/0 mmhg; complete remission (CR), proteinuria 0. g/ h with a normal SAlb level ( g/l) and SCr level (< 0. μmol/l); partial remission (PR), decline in proteinuria excretion by > 0% of the baseline value and proteinuria > 0. g/ h but <. g/ h, with a normal SCr level or an elevation by < % of the baseline value, without extrarenal activity; total remission (TR), CR or PR; End-stage renal disease (ESRD), egfr < ml/min/per. m, initiation of dialysis therapy that continued for > months, or transplantation. egfr was calculated using the Chronic Kidney Disease Epidemiology Collaboration 00 creatinine equation (CKD-EPI). Disease activity was assessed by SLEDAI. Antigen-specific ELISA was used to detect ANCA. Statistical analysis All data were analyzed using the statistical software SPSS, version.0 (IBM). Quantitative data are expressed as the mean ± standard deviation or median with the interquartile range (IQR). All parameters were compared using the χ test or Fisher test for categorical data and the t test, Man-Whitney test, Kruskal-Wallis test, or One-Way ANOVA test for continuous data. Kaplan-Meier curves and the log rank test were used to analyze remission and renal survival. P < 0.0 was considered to be statistically significant. RESULTS - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

8 Page of Patient information Among the cases, had ANCA specificity untested, showed ANCA negativity, and (.%) exhibited ANCA positivity, including with MPO-ANCA positivity, with PR-ANCA positivity, and with double positivity. Their clinical and laboratory features at biopsy are shown in Table. Of LN patients with ANCA positivity, 0 (.%) were women and (.%) men. The median age was (IQR, - 0) years. The median duration of LN was. (IQR,..) months. (0.%) patients had hypertension. LN patients with ANCA positivity presented with higher hematuria (median 0 [IQR, - ] versus [IQR, - ] 0 /ml; P = 0.00), higher levels of SCr (median.0 [IQR,..] versus. [IQR,. 0.] μmol/l; P < 0.00), and higher SLEDAI scores (median [IQR, - ] versus [IQR, 0 - ] scores; P < 0.00) than the controls. Conversely, there was no significant difference in the incidence of hypertension or the duration of LN, sex, age, SAlb, and serum C and C between the two groups. - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

9 Page of Table. Clinical and laboratory findings at biopsy according to ANCA. ANCA (+)(n = ) ANCA (-)(n = ) P-value Men (.) (.) 0.0 Age, yrs ( - 0) 0( - ) 0. Duration of LN, mo.(. -.).(.0.0) 0. SLEDAI, score ( ) (0 - ) < 0.00 Hypertension (0.) (.) 0. Proteinuria, g/ h.(..0).(..) 0. Urinary RBC count, 0 /ml 0( - ) ( - ) 0.00 SAlb, g/l.0(..).0(.0.0) 0. SCr, μmol/l.0(..).(. 0.) < 0.00 egfr, ml/min/per. m.(.0.).(..0) <0.00 < 0 ml/min/per. m (.) (.0) 0.00 < 0 ml/min/per. m (.) (.) 0.00 C, g/l 0.(0. 0.) 0.(0. 0.) 0. C, g/l 0.0(0.0 0.) 0.(0.0 0.) 0. Values are given as n (%) or median (interquartile range). LN, lupus nephritis; SLEDAI, systemic lupus erythematous disease activity index; RBC, red blood cell; SAlb, serum albumin; SCr, serum creatinine; egfr, estimated glomerular filtration rate; C, complement ; C, complement. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

10 Page of The observed extrarenal manifestations are listed in Table. We did not note any differences when comparing LN patients with and without ANCA positivity. Table. Extrarenal manifestations at biopsy according to ANCA ANCA (+)(n = ) ANCA (-)(n = ) P-value Fever (non-infectious) (.) (.) 0.0 Malar rash (.) (.) 0.0 Photosensitivity (.) (.0) 0. Mouth ulcer (.) (0.0) 0. Alopecia (.) (.) 0. Arthritis (.0) (.) 0. Serositis (.) (.) 0. Neurological disorder (.) (.0) 0. Raynaud phenomenon (.) (.0) 0. Anemia (.) (0.) 0.00 Leukopenia (.) (.) 0.0 Thrombocytopenia (.) (.) 0.0 Values are given as n (%). : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

11 Page 0 of Renal morphology The renal biopsy findings are shown in Table. Among the LN patients with ANCA positivity, 0 (.%) presented with class Ⅳ (including 0 [0.%] Ⅳ-G and 0 [0.%] Ⅳ-S), (.%) with class Ⅲ, (.%) with class Ⅳ + Ⅴ, (.%) with class Ⅴ, (.0%) with class Ⅲ +Ⅴ, and only (.0%) with class Ⅱ. On renal pathological evaluation, the scores of cellular crescents, interstitial inflammation, tubular atrophy and interstitial fibrosis among the ANCA-positive LN patients were higher than those in the control group (P = 0.00, P = 0.00, P = 0.00, P = 0.00, respectively). Furthermore, the LN patients with ANCA positivity had higher scores of AI and CI (P =0.0, P = 0.00, respectively) : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

12 Page of Table. Pathologic findings according to ANCA. ANCA (+)(n = ) ANCA (-)(n = ) P-value Number of glomeruli (0 - ) ( - 0) 0.0 AI, score. ±.. ±. 0.0 Endocapillary hypercellualrity. ±.. ±. 0. Karyorrhexis/fibrinoid necrosis 0. ± ± Cellular crescents. ±.0 0. ± Subendothelial hyaline deposits 0. ± ± Glomerular leukocyte infiltration.00 ±.0 0. ± Interstitial inflammation. ± ± CI, score. ±.. ± Glomerular sclerosis 0. ± ± Tubular atrophy.00 ± ± Fibrous crescents 0. ± ± Interstitial fibrosis.00 ± ± Vasculopathy Hyaline degeneration of artery (0.0) (.) 0.00 Noninflammatory necrotizing (.0) (.) 0.00 vasculopathy Inflammatory (.0) (0.) 0.0 necrotizingvasculopathy Thrombotic microanglopathy (.0) (.) 0. Atherosclerosis (.) (.) 0.0 Values are given as n (%) or mean ± SD. AI, activity index; CI, chronicity index. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

13 Page of Treatment regimens and response Among the LN patients with ANCA positivity, 0 required MMF as induction therapy; received CYC, and were assigned to the other regimens group. We further compared data of treatment response between MMF group and CYC group, as shown in Table. The patients receiving MMF had a higher CR rate ( [0.00%] versus 0 [.%]; P = 0.0) than those requiring CYC. In addition, the median onset time of CR was shorter in MMF group (median. [IQR,..] versus. [IQR,. 0.] month; P = 0.00). Furthermore, LN patients with ANCA positivity exhibited lower levels of proteinuria (P = 0.00), hematuria (P = 0.0), and SCr (P = 0.0), and also had lower scores of SLEDAI (P = 0.0) and higher levels of SAlb (P = 0.0) at the last follow-up. - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

14 Page of Table. Treatment response of LN patients with ANCA positivity according to induction therapy. Total (N=) MMF (n=0) CYC (n=) P-value Clinical data at biopsy Men (.0) (.00) (.) 0. Age, yrs ( - 0) ( - ) ( - ) 0. SLEDAI, score ( - ) ( -) ( - ) 0. Proteinuria, g/ h Urinary RBC count, 0 /ml SAlb, g/l SCr, μmol/l MPO-ANCA positivity Follow-up time Treatment response Complete remission.(.0.).(..).(..0) 0. (0-0) ( - ) (0 - ) 0..(..0).(..).0(..0) 0.(..).(. 0.0) 0.0.(.0.0) 0. (.) (.00) (.) (..).(..).(..0) 0. (.) (0.00) 0(.) 0.0 Partial remission (.) (0.00) (.) 0. No response (.) 0(0.00) (.) 0.0 Onset time of CR, mo.0(..).(..).(. 0.) 0.00 Onset time of PR, mo.(. 0.).0(..).(..) 0.00 Clinical data at the last follow-up SLEDAI, score (0 - ) (0 - ) ( - ) 0.0 Proteinuria, g/ h Urinary RBC count, 0 /ml SAlb, g/l SCr, μmol/l 0.(0..) 0.(0. 0.).0(0..) 0.00 ( - ) ( - ) ( ) 0.0.0(.0.0(..(..0).0).0) 0.0.(.0.(. 0.(..).) 0.) 0.0 Values are given as n (%) or median (interquartile range). SLEDAI, systemic lupus erythematous disease activity index; RBC, red blood cell; SAlb, serum : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

15 Page of albumin; SCr, serum creatinine; MPO-ANCA, myeloperoxidase antineutrophil cytoplasmic antibody. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

16 Page of outcome During follow-up (average for. [IQR,..] months), ESRD developed in (.%) ANCA-positive LN patients, all of whom were MPO-ANCA positive, including with class Ⅳ and with class Ⅲ. The renal survival rates for LN with ANCA positivity were.0% at year,.0% at years and.0% at 0 years; these values were similar to the control group, at.0%,.0% and.00%, respectively (Figure A). The renal survival rates for ANCA-positive LN patients receiving MMF as induction therapy were 00% at year, 00% at years and 00% at 0 years; these values were higher than those requiring CYC, at.0%,.0%,.0%, respectively (P = 0.0) (Figure B). DISCUSSION ANCA positivity rate ranges from 0% to 0% in LN. - In this study, we found that LN with ANCA positivity was rare in LN, which accounted for.% of total biopsy-proven LN, lower than the literatures. It can be explained by the fact, that only the biopsy-proven LN patients are included and those without biopsy have been excluded. This study showed that LN with ANCA positivity presents with unique features. First, a predominance of childbearing women was observed, lower than and the disease was characterized by massive hematuria and renal functional lesion, similar to the literature. - Second, it was not surprising that LN patients with ANCA positivity had significantly higher proportions of crescents, higher AI and CI scores. This result was partly accordance with a previous report. Nasr found that all biopsies from 0 patients of LN with ANCA positivity exhibited prominent necrosis and crescents, with absent or rare subendothelial deposits. In our study, LN with ANCA positivity included all types of proliferative LN, and class Ⅳ and Ⅲ were - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

17 Page of predominant, similar to the literature. 0 Opinions are divided about whether ANCA positivity has any association with SLEDAI. - In our study, LN patients with ANCA positivity had higher SLEDAI scores, which might be associated with higher hematuria caused of kidney injury aggravated by ANCA. However, we did not observe any differences in extrarenal manifestations between LN with and without ANCA positivity. Because of the infrequence of ANCA-positive LN, there are no well-established treatment methods for these patients, and CYC is largely used as induction therapy. Nasr showed that six of ten patients reached complete or near-complete remission with CYC. In patients who did not respond well to CYC, rituximab was administered, and with satisfactory results. Most authors, including us, have suggested aggressive immunosuppressive therapy. In our study, patients receiving MMF as induction therapy had higher remission rates than those requiring CYC. The relative specificity for activated lymphocytes as well as antiproliferative and antifibrotic actions may be responsible for the beneficial effects of MMF in LN with ANCA positivity. 0- This result should be further studied in LN with ANCA positivity. ESRD developed in seven ANCA-positive LN patients, all of whom were MPO-ANCA positive, a phenomenon that might be explained by the following two reasons. First, MPO-ANCA is more common in Asia, including China. Second, several recent studies have reported worse renal survival in AAV patients with MPO-ANCA, - and MPO-ANCA is most likely related to smoldering disease, which causes ESRD. Overall, LN patients with MPO-ANCA positivity should be given greater attention. We believed that LN patients with ANCA positivity would have worse renal outcome, however we could not draw the conclusion in this study. To further research the renal - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

18 Page of prognosis of this population, a larger cohort and longer following-up are needed. Further detailed analysis of LN patients with ANCA positivity revealed only four having a history of taking PTU. It has been reported that - % of patients treated with PTU develop serum ANCA positivity, of whom -.% have clinical symptoms of vasculitis. 0 Of note, one patient in our study had a history of silicon exposure, which has been associated with AAV. Hogan proposed that high levels of exposure to silica dust are associated with ANCA-associated small-vessel vasculitis. However, ANCA-positive LN patients were rare induced by silica exposure or PTU. A putative pathogenic mechanism for vascular inflammation begins with ANCA-induced activation of primed neutrophils and monocytes, leading to activation of the alternative complement pathway. Based on a deeper understanding of the pathogenesis of SLE, the increased cell death and enhanced extracellular trap formation observed in SLE-derived neutrophils might have key roles in the induction of autoimmunity and the development of organ damage. It has been suggested that LN might facilitate the process of ANCA formation by promoting neutrophil degranulation and priming neutrophils, increasing the surface expression of ANCA, including MPO-ANCA and PR-ANCA. We observed that.% LN patients with ANCA positivity did not display crescents. This might be explained by the fact that ANCA might participate in the development of crescents in LN as a major pathogenic mechanism and as a gradual process, and that the ANCA titer and duration of ANCA positivity would affect the formation of crescents. There are some limitations to the findings of this report. First, all patients included in this study were of Chinese Han ethnicity; therefore, our results may not completely apply to non-asian - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

19 Page of individuals. Second, due to the limitation of a retrospective study, our conclusions require further validation. In conclusion, LN with ANCA positivity was characterized by massive hematuria, advanced renal insufficiency. Higher CR and better prognosis were observed when using MMF than CYC as induction therapy. Author affiliations All co-authors from National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China. Contributors All authors contributed significantly to this manuscript, including contributions in the study design (C L, HT Z), chart reviews (C L, ML Z, DD L, HT Z), interpretation of the data (C L, ML Z, DD L, HT Z), and preparation of the manuscript (C L, ML Z, DD L, JJ W, J Y, CH Z, HT Z). Funding This work was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (No. 0BAI0B0, No. 0BAIB0) and the Jiangsu Provincial clinical science and technology project (No. BL000). Competing interests None declared. Ethics approval We received approval from the Ethics Committee of Jinling Hospital, Nanjing University School of Medicine, China (The ethics approval number: 0NZKYKS-00-0). Data sharing statement no additional data are available - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

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22 Page of Hogan SL, Satterly KK, Dooley MA, et al. Silica exposure in anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and lupus nephritis. J Am Soc Nephrol 00;:-.. Jennette JC, Falk RJ. Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat Rev Nephrol 0;0:-.. Kaplan MJ. Neutrophils in the pathogenesis and manifestations of SLE. Nat Rev Nephrol 0;: : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

23 Page of Legends to figures Figure : Comparison of renal survival based on (A) serum ANCA and (B) induction treatment. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

24 Page of Figure xmm (00 x 00 DPI) - : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

25 Page of Reporting checklist Section &Topic No Item Reported on page TITLE PAGE a Title P b Name and address of the corresponding author P c Detail of all co-authors P d Word count P ABSTRACT a Objective P b Design c Methods P d Results e Conclusions f Strengths and limitations of this study P INTRODUCTION P METHODS a Patients P b Renal morphology c Data collection P d Statistical analysis RESULTS a Patient information P b Renal morphology P P P P P P0 c Treatment regimens and response P d Outcome DISCUSSION P Author affiliations P Contributors P Funding P Competing interests 0 P Ethics approval P Data sharing statement P REFERENCES P Legends to figures P - P : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

26 Treatment and clinicopathological characteristics of lupus nephritis with antineutrophil cytoplasmic antibody positivity: a case-control study Journal: Manuscript ID bmjopen-0-0.r Article Type: Research Date Submitted by the Author: 0-Apr-0 Complete List of Authors: Li, Cui; National Clinical Research Center of Kidney Diseases, Jinling Hospital, 0 East Zhongshan Road, Nanjing 00, Jiangsu, China. Zhou, Min-Lin Liang, Dan-Dan Wang, Jing-Jing Yang, Jing Zeng, Cai-Hong Liu, Zhi-Hong; National Clinical Research Center of Kidney Diseases, Jinling Hospital, 0 East Zhongshan Road, Nanjing 00, Jiangsu, China. Zhang, Hai-Tao; National Clinical Research Center of Kidney Diseases, Jinling Hospital, 0 East Zhongshan Road, Nanjing 00, Jiangsu, China. <b>primary Subject Heading</b>: Renal medicine Secondary Subject Heading: Pathology Keywords: antineutrophil cytoplasmic autoantibody, clinical features, lupus nephritis, outcome, pathological presentations, treatment : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright.

27 Page of TITLE PAGE Title: Treatment and clinicopathological characteristics of lupus nephritis with antineutrophil cytoplasmic antibody positivity: a case-control study Name and address of the corresponding author:hai-tao Zhang, National Clinical Research Center of Kidney Diseases, Jinling Hospital, 0 East Zhongshan Road, Nanjing 00, Jiangsu, China. htzhang@.com Detail of all co-authors: Cui Li, doctor, MD; Min-Lin Zhou, statisticians, MD; Dan-Dan Liang, pathologist, MD; Jing-Jing Wang, doctor, MD; Jing Yang, doctor, PhD; Cai-Hong Zeng, professor of pathology, PhD; Zhi-Hong Liu, professor of medicine, MD; Hai-Tao Zhang, professor of medicine, MD. All co-authors from National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China. Note: Cui Li is the first author and Hai-Tao Zhang is the corresponding author of this study. Word count: words : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

28 Page of ABSTRACT Objective: To assess the clinical features, pathological presentations, treatment and outcome of lupus nephritis (LN) with antineutrophil cytoplasmic anti-body (ANCA) positivity Design: A case-control study Methods: Patients (n = ) between and 00 were retrospectively included from Jinling Hospital in China if presenting with biopsy-proven LN with ANCA positivity. Clinicopathological characteristics and outcome were analyzed and compared with a control group (n = ). We further compared treatment response and outcome of ANCA-positive LN patients based on the treatment issued. Results: The study consisted of 0 women and men with a median age of years at biopsy, including MPO-ANCA, PR-ANCA and patients with double positivity. LN with ANCA positivity exhibited higher hematuria, serum creatinine levels and SLEDAI scores. On pathological evaluation, Class IV LN was predominant, accounting for.%. Light microscopy revealed significantly higher scores of AI and CI, including cellular crescents, interstitial inflammation, tubular atrophy, and interstitial fibrosis. ANCA-positive LN patients receiving mycophenolate mofetil as induction therapy had higher remission rate and better renal outcome than those requiring cyclophosphamide. During follow-up, end-stage renal disease developed in (.%) patients of positive ANCA, all of whom were MPO-ANCA positive. Conclusions: Characteristics of LN with ANCA positivity were massive hematuria, advanced renal insufficiency. We observed higher remission rate and better prognosis when using mycophenolate mofetil than cyclophosphamide as induction therapy. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

29 Page of Strengths and limitations of this study: A case-control study This study is the largest case-control study to summarize retrospectively clinicopathological characteristics and outcome of lupus nephritis (LN) patients with ANCA positivity (n = ) and ANCA negativity (n = ) in China. Comparison of intravenous cyclophosphamide with oral mycophenolate mofetil in the treatment of LN with ANCA positivity for the first time LN patients without biopsy were not included. A larger study was in need to describe the difference between MPO-ANCA positive and PR-ANCA positive LN patients. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

30 Page of INTRODUCTION Lupus nephritis (LN) is an immune complex glomerular nephritis that develops as a frequent complication of systemic lupus erythematous (SLE). Autoantibody production in SLE patients is a hallmark of the disease entity as well as its activity and prognosis. Intravenous cyclophosphamide (CYC) has been the traditional regimen for treating LN. However, CYC is frequently associated with severe side effects, and infections contribute to the overall mortality. Recent studies have established mycophenolate mofetil (MMF), a selective lymphocyte anti-proliferative agent, as a safe and an effective alternative to CYC for LN. Until now, steroids, CYC, and MMF remain first-line therapeutics for treatment of LN. Antineutrophil cytoplasmic antibody (ANCA) is the probable cause of a distinct form of vasculitis accompanied by necrotizing granulomatosis. Based on enzyme-linked immunoadsordent assay (ELISA), the major target antigens of ANCA are proteinase (PR) and myeloperoxidase (MPO). LN patients with ANCA positivity existed in case reports or small series over the last years. - However, due to the relatively small amount of research to date, the clinical features, pathological presentations, and outcome of LN with ANCA positivity were not clear. Moreover, only rare investigations have addressed the treatment of this population. Therefore, we summarized retrospectively clinicopathological characteristics and outcome of LN patients with ANCA positivity and ANCA negativity. Furthermore, we compare the efficacy, renal relapse rate, adverse events, and outcome between MMF and CYC as induction therapy in LN patients with ANCA positivity. METHODS : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

31 Page of Patients Chinese patients with biopsy-proven LN at Jinling Hospital from January to December 00 were retrospectively reviewed. Patients who fulfilled the following criteria were included in this study: () age years; () patients who met the American Rheumatologic Association criteria for the diagnosis of SLE; () biopsy-proven LN; () presence of ANCA positivity; () duration of follow-up months; () complete baseline and follow-up data. Simultaneously, LN patients with ANCA negativity were included to the control group during the same period and compared with the ANCA-positive LN patients. Renal morphology For light microscopy, we processed biopsy specimens for hematoxylin-eosin, periodic acid-schiff, Masson trichrome, and Jones methenamine silver staining. Pathological parameters such as activity index (AI) and chronic index (CI) were determined using a modification of an earlier reported system involving semi-quantitative scoring of specific biopsy features. Vasculopathy was defined according to renal vascular complications of SLE. Biopsy specimens were reviewed and reclassified according to the 00 International Society of Nephrology (ISN)/Renal Pathology Society (RPS). Two renal pathologists examined biopsy specimens. Differences in classification and scores between the two were resolved by reviewing the biopsies. Data collection The following data were collected retrospectively at biopsy: gender, age, duration of LN, SLE disease activity index (SLEDAI), hypertension, extrarenal manifestations, hour urinary protein excretion, urinary sediment, serum albumin (SAlb), serum creatinine (SCr), estimated glomerular filtration rate (egfr), C, C, and ANCA specificity (Tested by ELISA). : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

32 Page of MMF was prescribed at doses of g/d for months, while CYC was administered at g/m body surface area once a month for months. The total dose of CYC was less than g. Patients who were given less CYC or MMF, tripterygium glycosides (TW) or other immunosuppressors were assigned to the other regimens group. All patients received three methylprednisolone injections of 00 mg per day for days followed by oral corticosteroids at doses of mg per kilogram of body weight per day. Maintenance therapy was started at end of induction therapy with low doses prednisone and immunosuppressors, including azathioprine, leflunomide, and TW. The primary efficacy endpoint was total remission (TR) rate at months, and the endpoint for renal outcome was end-stage renal disease (ESRD). When patients were at the end of follow-up, we recorded their laboratory findings at the last visit. The following definitions were used: duration of LN, the time from LN diagnosis to the first-time renal biopsy; hypertension, blood pressure > 0/0 mmhg; complete remission (CR), proteinuria 0. g/ h with a normal SAlb level ( g/l) and SCr level (< 0. μmol/l); partial remission (PR), decline in proteinuria excretion by > 0% of the baseline value and proteinuria > 0. g/ h but <. g/ h, with a normal SCr level or an elevation by < % of the baseline value, without extrarenal activity; TR, CR or PR; renal relapse, proteinuria.0 g/ h in patients with CR or proteinuria.0 g/ h in patients with PR, with or without active urine sediment or increase in SCr by 0%; ESRD, egfr < ml/min/per. m, initiation of dialysis therapy continued for > months, or transplantation. egfr was calculated using the Chronic Kidney Disease Epidemiology Collaboration 00 creatinine equation (CKD-EPI). 0 Disease activity was assessed by SLEDAI. Statistical analysis : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

33 Page of All data were analyzed using the statistical software SPSS, version.0 (IBM). Quantitative data were presented as mean ± standard deviation or median with the interquartile range (IQR). All parameters were compared using the χ test or Fisher test for categorical data and the t test, Man-Whitney test, Kruskal-Wallis test, or One-Way ANOVA test for continuous data. Kaplan-Meier curves and the log rank test were used analyzing renal survival. P < 0.0 was considered significant. RESULTS Patient information Among the cases, had ANCA specificity untested, showed ANCA negativity, and (.%) exhibited ANCA positivity, including with MPO-ANCA positivity, with PR-ANCA positivity, and with double positivity. Of LN patients with ANCA positivity, 0 (.%) were women and (.%) were men. The median age was (IQR, - 0) years. The median duration of LN was. (IQR,..) months. (0.%) patients had hypertension. (Table ) LN patients with ANCA positivity had higher hematuria (median 0 [IQR, - ] versus [IQR, - ] 0 /ml; P = 0.00), higher levels of SCr (median.0 [IQR,..] versus. [IQR,. 0.] μmol/l; P < 0.00), and higher SLEDAI scores (median [IQR, - ] versus [IQR, 0 - ] scores; P < 0.00) than the controls. Conversely, there was no significant difference in the incidence of hypertension or the duration of LN, sex, age, SAlb, and serum C and C between the two groups. (Table ) : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

34 Page of Table Clinical and laboratory findings at biopsy according to ANCA ANCA (+)(n = ) ANCA (-)(n = ) P-value Men (.) (.) 0.0 Age, yrs ( - 0) 0( - ) 0. Duration of LN, mo.(. -.).(.0.0) 0. SLEDAI, score ( ) (0 - ) < 0.00 Hypertension (0.) (.) 0. Proteinuria, g/ h.(..0).(..) 0. Urinary RBC count, 0 /ml 0( - ) ( - ) 0.00 SAlb, g/l.0(..).0(.0.0) 0. SCr, μmol/l.0(..).(. 0.) < 0.00 egfr, ml/min/per. m.(.0.).(..0) <0.00 < 0 ml/min/per. m (.) (.0) 0.00 < 0 ml/min/per. m (.) (.) 0.00 C, g/l 0.(0. 0.) 0.(0. 0.) 0. C, g/l 0.0(0.0 0.) 0.(0.0 0.) 0. Values are given as n (%) or median (interquartile range). LN, lupus nephritis; SLEDAI, systemic lupus erythematous disease activity index; RBC, red blood cell; SAlb, serum albumin; SCr, serum creatinine; egfr, estimated glomerular filtration rate; C, complement ; C, complement. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

35 Page of The observed extrarenal manifestations are summrized in Table. We did not note any differences when comparing LN patients with and without ANCA positivity. Table Extrarenal manifestations at biopsy according to ANCA ANCA (+)(n = ) ANCA (-)(n = ) P-value Fever (non-infectious) (.) (.) 0.0 Malar rash (.) (.) 0.0 Photosensitivity (.) (.0) 0. Mouth ulcer (.) (0.0) 0. Alopecia (.) (.) 0. Arthritis (.0) (.) 0. Serositis (.) (.) 0. Neurological disorder (.) (.0) 0. Raynaud phenomenon (.) (.0) 0. Anemia (.) (0.) 0.00 Leukopenia (.) (.) 0.0 Thrombocytopenia (.) (.) 0.0 Values are given as n (%). : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

36 Page 0 of Renal morphology The renal biopsy findings are shown in Table. Among the LN patients with ANCA positivity, 0 (.%) presented with class Ⅳ (including 0 [0.%] Ⅳ-G and 0 [0.%] Ⅳ-S), (.%) with class Ⅲ, (.%) with class Ⅳ + Ⅴ, (.%) with class Ⅴ, (.0%) with class Ⅲ +Ⅴ, and only (.0%) with class Ⅱ. On renal pathological evaluation, the scores of cellular crescents, interstitial inflammation, tubular atrophy and interstitial fibrosis among the ANCA-positive LN patients were higher than those in the control group (P = 0.00, P = 0.00, P = 0.00, P = 0.00, respectively). Furthermore, the LN patients with ANCA positivity had higher scores of AI and CI (P =0.0, P = 0.00, respectively). 0 : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

37 Page of Table Pathologic findings according to ANCA ANCA (+)(n = ) ANCA (-)(n = ) P-value Number of glomeruli (0 - ) ( - 0) 0.0 AI, score. ±.. ±. 0.0 Endocapillary hypercellualrity. ±.. ±. 0. Karyorrhexis/fibrinoid necrosis 0. ± ± Cellular crescents. ±.0 0. ± Subendothelial hyaline deposits 0. ± ± Glomerular leukocyte infiltration.00 ±.0 0. ± Interstitial inflammation. ± ± CI, score. ±.. ± Glomerular sclerosis 0. ± ± Tubular atrophy.00 ± ± Fibrous crescents 0. ± ± Interstitial fibrosis.00 ± ± Vasculopathy Hyaline degeneration of artery (0.0) (.) 0.00 Noninflammatory necrotizing (.0) (.) 0.00 vasculopathy Inflammatory necrotizing (.0) (0.) 0.0 vasculopathy Thrombotic microanglopathy (.0) (.) 0. Atherosclerosis (.) (.) 0.0 Values are given as n (%) or mean ± SD. AI, activity index; CI, chronic index. : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -

38 Page of Treatment regimens and response Among the LN patients with ANCA positivity, 0 required MMF as induction therapy; received CYC, and were in the other regimens group (Detail therapies in Supplementary File). We further compared data of baseline characteristics, efficacy, relapse rate, and adverse events between MMF and CYC group. The baseline characteristics of the study population are summrized in Supplementary Table. All the baseline characteristics were comparable between the two groups, with the exception of cellular crescents, which was significantly higher in the MMF group (P= 0.00). Patients receiving MMF had a higher TR rate ([.00%] versus [.%]; P = 0.0) and CR rate ([.00%] versus [.%]; P = 0.00) than those requiring CYC, as shown in Table. Furthermore, patients receiving MMF exhibited lower levels of proteinuria (P = 0.00), hematuria (P = 0.0), and SCr (P = 0.0), and had lower scores of SLEDAI (P = 0.0) and higher levels of SAlb (P = 0.0) at the last follow-up. We noticed two groups had similar renal relapse rate. During follow-up, (0%) subjects in the MMF group and (.%) in the CYC group reported at least one adverse event (P = 0.). Both the groups had an equal number of infection, arthralgia, arthrosis necrosis, menstrual disorder or menolipsis, cardiac vascular disease, tumor, and epilepsy. (Table ) : first published as 0./bmjopen-0-0 on July 0. Downloaded from on January 0 by guest. Protected by copyright. -