International Journal of Rheumatic Diseases 2014
ORIGINAL ARTICLE
Human neutrophil peptide 1–3, a component of the neutrophil extracellular trap, as a potential biomarker of lupus nephritis Fa-Juan CHENG, Xu-Jie ZHOU, Yan-Feng ZHAO, Ming-Hui ZHAO and Hong ZHANG Renal Division, Department of Medicine, Peking University First Hospital, Peking University Institute of Nephrology, Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China
Abstract Objective: Human neutrophil peptides (HNP) were recently implicated in the neutrophil extracellular trap (NET) complex, the impaired degradation of which has been associated with lupus nephritis (LN). Methods: Forty LN patients, 40 SLE patients without kidney injury, 63 immunoglobulin A nephropathy (IgAN) patients, 20 minimal change disease (MCD) patients and 33 healthy controls were included in the present study. LN, IgAN and MCD patients were diagnosed with renal biopsy. LN patients were followed for a median period of 5.5 years. Clinical and laboratory data at the time of renal biopsy and follow-up were collected for each LN patient. Serum levels of HNP1–3 were measured with enzyme-linked immunosorbent assay. Results: Serum HNP1-3 levels in LN patients were significantly higher than for SLE patients without kidney injury (P < 0.001), IgAN patients (P = 0.012), MCD patients (P = 0.010) and healthy controls (P = 0.022). Serum HNP1–3 levels were an independent indicator of LN (P = 0.006, OR = 7.5, 95% CI, 1.782–31.842), were statistically correlated with urinary protein excretion (P = 0.009) and activity index (P = 0.042) and were only marginally correlated with neutrophils (P = 0.054) and white blood cell counts (P = 0.051). Serum levels of HNP1–3 were a predictor of proteinuria remission after correction for multiple parameters (multivariate hazard 0.209; 95% CI 0.046–0.951; P = 0.043). Conclusions: The data from this study indicated that HNP1–3, one component of the NET, is a potential biomarker for LN. Key words: human neutrophil peptide, lupus nephritis, systemic lupus erythematosus.
INTRODUCTION Human defensins, an important part of the innate immune system, are a family of evolutionarily closely related cationic anti-microbial peptides. The a-defensins 1–4, initially isolated from neutrophils, are also
Correspondence: Hong Zhang, Renal Division, MD, PhD, Professor of Medicine, Peking University First Hospital, Peking University Institute of Nephrology, No. 8 Xi Shi Ku Street, Xi Cheng District, Beijing 100034, China. Email: hongzh@bjmu. edu.cn
called human neutrophil peptides (HNP1–4).1 In addition to their role in anti-microbial infection, HNP1–3 have been shown to regulate inflammation in diverse ways, including serving as chemoattractants for monocytes, dendritic cells and T cells and enhancing the antigen-specific immune response.2,3 Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organs and systems. The precise etiology of SLE is currently unknown.4–6 However, in oligonucleotide microarrays, HNP was one of the most highly up-regulated molecules in SLE patients at the genome-wide level, suggest-
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
F.-J. Cheng et al.
ing an important role for HNP in the pathogenesis of SLE.7 HNP was found to be an important component of neutrophil extracellular traps (NETs), which were involved in a process called NETosis.8 NETosis refers to the extrusion of large amounts of nuclear DNA into the extracellular space by activated neutrophils, and these web-like structures called NETs (neutrophil extracellular traps) were recently associated with SLE.8 In particular, previous reports have indicated that impaired degradation of NETs was specifically associated with lupus nephritis (LN).9 It has been suggested that the inability to dismantle NETs might be a useful indicator of renal involvement in SLE patients. However, no correlation between HNP and LN has ever been identified. Therefore, in the current study, we determined HNP1–3 serum levels in patients with LN, SLE without kidney injury and glomerulonephritis caused by different diseases, and investigated the role of a-defensins in the pathogenesis of LN.
SUBJECTS AND METHODS Subjects In the present study, 40 LN patients, 40 SLE patients without kidney injury, 63 immunoglobulin A nephropathy (IgAN) patients, 20 minimal change disease (MCD) patients and 33 healthy controls matched geographically and ethnically were recruited. All SLE patients met the American College of Rheumatology (ACR) classification according to the revised ACR SLE criteria.10 For all LN patients, renal biopsies were analyzed by light microscopy, immunofluorescence and electron microscopy to confirm the LN diagnostic criteria according to the American Rheumatism Association (ARA, 1982). LN was classified according to the 2003 International Society of Nephrology (ISN)/ Renal Pathology Society (RPS) classifications.11,12 The activity index (AI) and chronic index (CI) were evaluated, and the SLE activity index was scored by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI).13 The estimated glomerular filtration rate (eGFR) was evaluated according to the abbreviated Modification of Diet in Renal Disease (MDRD) equation, which was suitably modified for the Chinese population.14 Clinical and laboratory data at the time of renal biopsy were collected for each patient. LN patients were followed for a median period of 5.5 years. We assessed the prognostic relevance of achieving proteinuria remission as defined by achieving proteinuria values < 0.3 g/day and renal function decline as defined by an increase in serum creatinine > 50%. SLE patients
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without kidney disease were enrolled from the dermatology department of Peking University. All of the SLE patients were determined to be negative for anti-neutrophil cytoplasmic antibodies (ANCA), which may be a stimulus for the release of defensins by neutrophils. IgAN and MCD patients were also verfied by renal biopsy. The diagnosis of IgAN was based upon the demonstration by immunofluorescence of IgA as the dominant or co-dominant immunoglobulin in mesangial deposits and the lack of clinical or serological evidence of other inflammatory conditions, such as SLE, vasculitis or Henoch–Schoenlein purpura. A diagnosis of MCD was confirmed by the combined determinations of no immunofluorescence findings with immunofluorescence microscopy, normal glomeruli as observed with light microscopy and diffused effacement of podocyte foot processes as observed with electronic microscopy. Clinical and laboratory data at the time of renal biopsy were collected for each IgAN and MCD patient. This study was approved by the Ethics Committee of Peking University First Hospital and all participants gave written informed consent.
Assay for serum levels of HNP1–3 Serum HNP1–3 was measured by enzyme-linked immunosorbent assay (ELISA) (Hycult Biotechnology, Uden, The Netherlands) as previously reported.15 Briefly, microtiter wells were first precoated with antibodies recognizing HNP1–3. Every sample, serially diluted standard and blank control were tested in duplicate. The volume in each well was 100 lL for all steps, and all incubations were performed at room temperature for 1 h. The plates were then washed four times with wash buffer. Sera were diluted 1 : 100 with plasma dilution buffer, and every plate contained serially diluted standard and blank controls. Binding was detected with biotinylated tracer antibody and streptavidin-peroxidase conjugate. We used the substrate tetramethylbenzidine and measured the absorbance at 450 nm (Bio-Rad 550, Tokyo, Japan). The concentration of sample HNP1–3 in the serum was calculated according to a standard curve.
Statistical analysis Continuous variables were tested in each group for a normal distribution using the Kolmogorov–Smirnov test. Normally distributed variables were expressed as the mean the standard deviation (SD) and compared using Student’s t-test or one-way analysis of variance (ANOVA). Non-parametric variables were expressed as the median and range and compared using the Mann–
International Journal of Rheumatic Diseases 2014
HNP 1–3 in lupus nephritis
Whitney U-test or Kruskal–Wallis H-test. Pearson’s chisquare was used to analyze the categorical data and a continuity correction or Fisher’s exact test was applied when necessary. For the comparison of continuous variables, two-tailed bivariate correlations and Pearson’s coefficient were calculated. Logistic regression was used to model the effect of HNP1–3 serum levels on the disease incidence. The relationship between tested parameters and renal prognosis was assessed using Cox regression. As the serum levels of HNP1–3 and urinary protein excretion were highly skewed to the right, log transformation was performed in the correlation, logistic regression and Cox regression analyses. All analyses were performed using the SPSS 16.0 program (SPSS Inc., Chicago, IL, USA). All tests were two-sided, and P < 0.05 was considered to be statistically significant.
RESULTS Patient baseline characteristics and clinical outcome As is shown in Table 1, the mean ages of LN patients, SLE patients without kidney injury, IgAN patients, MCD patients and the 33 healthy controls were 34.8 11.4, 36.3 13.2, 34.8 12.4, 32.8 10.5 and 27.9 4.8 years, respectively, while the percentages of subjects who were female were 82.5%, 85.0%, 49.2%, 65.0% and 75.8%, respectively. The median initial proteinuria of LN patients when enrolled in the study was 4.64 g/day (range 0.32– 21.04), and median serum creatinine (Scr) was 77.00 lmol/L (range 42.00–610.00). The mean eGFR was 85.62 44.73 mL/min/1.73 m2. Regarding pathological phenotypes, one (2.5%) was class II, 13 (32.5%) were class III, 20 (50.0%) were class IV and six (15%) were class V. The mean AI and CI of LN patients were 7.08 4.42 and 2.40 2.13, respectively. All patients received immunosuppressive agents, while 32.5% of patients were treated with angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (ACEI/ ARB). After a median follow-up of 5.5 years, the median of the most recent proteinuria was 0.50 g/day (range 0.02–7.40), and median Scr was 75.00 lmol/L (range 56.00–954.00). Proteinuria remission was observed in 37.5% of patients and 10% of patients experienced a decline in renal function. For IgAN patients on biopsy, median proteinuria was 1.39 g/day (range 0.17–13.72) and median Scr was 90.00 lmol/L (range 58.00–226.00). The average eGFR was 83.08 23.17 mL/min/1.73 m2. The distribution by Haas grade I, II, III, IV and V in IgAN patients was
International Journal of Rheumatic Diseases 2014
12.7, 0.0, 42.9, 34.9 and 9.5%, respectively. For MCD patients, median proteinuria was 2.66 g/day (range 0.03–12.16) and median Scr was 55.00 lmol/L (range 43.00–114.30), respectively. The average eGFR of MCD patients was 129.01 33.52 mL/min/1.73 m2.
Serum levels of HNP 1–3 in patients with different causative diseases and healthy controls We observed no differences in HNP1–3 serum levels among SLE patients without kidney injury (median 27.24 ng/mL, range 12.21–276.32), IgAN patients (median 38.99 ng/mL, range 14.95–338.57), MCD patients (median 29.75 ng/mL, range 18.31–331.72) and healthy controls (median 31.77 ng/mL, range 22.76–269.33; P > 0.05). However, as is shown in Figure 1, the median serum level of HNP1–3 in LN patients (74.79 ng/mL, range 23.59–883.63) was significantly higher than for SLE patients without kidney injury (27.24 ng/mL, range 12.21–276.32, P < 0.001) and healthy controls (P = 0.022; Fig. 1). Similarly, the median serum level of HNP1–3 in LN patients was also elevated compared to IgAN (P = 0.012) and MCD patients (P = 0.010).
Serum levels of HNP1–3 correlated with LN clinical characteristics and prognosis When adjusted for gender and age by logistical regression analysis, serum levels of HNP1–3 were found to be independent indicators of LN (P = 0.006, OR = 7.5, 95% confidence interval [CI], 1.782–31.842). When LN patients were divided into two groups according to the 90th percentile of HNP1–3 serum levels in controls, the group with higher HNP1–3 levels presented with higher proteinuria, with a median of 5.80 and a range of 2.02– 21.04 versus a median of 4.14 and a range of 0.32– 10.88, P = 0.032, respectively. In addition, HNP1–3 serum levels were significantly correlated with urinary protein excretion (r = 0.405, P = 0.009) and AI (r = 0.323, P = 0.042). A marginal correlation between HNP1–3 serum levels and neutrophil count (r = 0.307, P = 0.054) as well as white blood cell count (r = 0.310, P = 0.051) was also observed (Fig. 2). No correlation was observed between HNP1–3 serum levels and other LN clinical characteristics, such as SLEDAI, serum Scr levels, CI and anti-double stranded DNA antibody. After a median follow-up of 5.5 years, we did not observe a relationship between HNP1–3 serum level and a decline in renal function. However, we did observe that HNP1–3 serum levels were an important predictor of proteinuria remission in multivariate Cox
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Table 1 Baseline characteristics and clinical outcomes for patients and controls Parameters
LN n = 40
SLE without LN n = 40
IgAN n = 63
Demography Gender (female) 33 (82.5%) 34 (85.0%) 31 (49.2%) Age (years) 34.8 11.4 36.3 13.2 34.8 12.4 General Fever 14 (35.0%) SBP* (mmHg) 124.50 11.37 121.83 15.46 DBP* (mmHg) 78.38 8.35 78.49 12.69 Hematological system Red blood cell count 3.67 0.62 4.43 0.57 (9 1012/L) 7.00 2.78 7.14 2.61 White blood cell count (9 109/L) 192.00 62.49 230.25 59.09 Blood platelet count (9 109/L) Hemoglobin (g/L) 109.99 18.90 134.98 17.57 Kidney Urinary protein excretion 4.64 (0.32, 21.04) 1.39 (0.17, 13.72) (g/day) (median, range) Serum creatinine (lmol/L) 77.00 (42.00, 610.00) 90.00 (58.00, 226.00) (median, range) 85.62 44.73 83.08 23.17 eGFR *(mL/min/1.73 m2) Glomerulonephritis (%) Class II 1 (2.5) Class III 13 (32.5) Class IV 20 (50) Class V 6 (15) AI* 7.08 4.42 CI* 2.40 2.13 SLEDAI* 17.65 4.94 Histological grading (%) I 8 (12.7) II 0 (0) III 27 (42.9) IV 22 (34.9) V 6 (9.5) Other systems (%) Photosensitivity 9 (22.5) Oral ulcers 10 (25.0) Presence of arthritis 23 (57.5) Presence of peritonitis 9 (22.5) Presence of meningitis 3 (7.5) Immunological indicators Serum IgG (g/L) 10.83 5.57 Serum IgA (g/L) 2.89 1.04 Serum IgM (g/L) 1.09 0.62 Serum C3 (g/L) 0.52 0.29 Serum C4 (g/L) 0.12 0.82 ANAs (%) 40 (100)
4
MCD n = 20
13 (65.0%) 32.8 10.5
Healthy controls n = 33 25 (75.8%) 27.9 4.8
116.50 16.39 72.60 12.83 4.51 0.46 6.34 2.98 251.05 61.40 139.75 15.99 2.66 (0.03, 12.16) 55.00 (43.00, 114.30) 129.01 33.52
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HNP 1–3 in lupus nephritis
Table 1 (continued) Parameters
Anti-dsDNA antibodies (%) Anti-Sm (%) Anti-SSA (%) Anti-SSB (%) Anti-nRNP (%) ANCA (%) Treatment (%) ACEI or ARB Any immunosuppression Follow-up Length of follow-up (years) (median, range) Latest proteinuria (g/day) (median, range) Latest serum creatinine (lmol/L) (median, range)
LN n = 40
SLE without LN n = 40
IgAN n = 63
MCD n = 20
Healthy controls n = 33
26 (65.0) 11 (27.5) 18 (45.0) 4 (10.0) 10 (25.0) 0 (0) 13 (32.5) 40 (100) 5.50 (3.50,23.50) 0.50 (0.02, 7.40) 75.00 (56.00, 954.00)
SBP, systolic blood pressure; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; Ccr, creatinine clearance; AI, activity index; CI, chronic index; SLEDAI, systemic lupus erythematosus disease activity index; ANA, antinuclear antibodies; SSA, Sj€ ogren’s syndrome antigen A; nRNP, nuclear ribonucleoprotein; ANCA, anti-neutrophil cytoplasmic antibodies; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker.
DISCUSSION
Figure 1 Serum levels of human neutrophil peptides (HNP) 1–3 in healthy controls and patients. The horizontal bars represent the median.
regression analysis with corrections for multiple parameters, including age, sex, initial renal function and the use of ACEI/ARB and immunosuppressive agents (multivariate hazard 0.209; 95% CI, 0.046–0.951; P = 0.043).
International Journal of Rheumatic Diseases 2014
In the present study, we determined HNP1–3 serum levels in patients with LN. A significant elevation of the HNP1–3 serum levels in LN patients was observed, together with correlations with urinary protein excretion and AI, strongly suggesting an involvement of a-defensin in the disease activity of LN. To determine a possible pathogenic mechanism, SLE without kidney injury, IgAN, which is thought to be an inflammationmediated nephropathy, and MCD, a disease characterized by substantial protein excretion in the urine without deposition of immunoglobulin or immune complex, were selected as disease controls. Interestingly, only LN patients, but not SLE patients without kidney injury, IgAN patients or MCD patients, showed a significant elevation of HNP1–3 serum levels, suggesting that HNP1–3 may be a potential biomarker for LN. SLE is a prototypical autoimmune disease characterized by autoantibody production and immune complex formation/deposition in target organs such as the kidney. Recent progress has pushed the neutrophil and its unique form of cell death called the neutrophil extracellular trap (NETosis) to the forefront of the pathogenesis of SLE,8,16 and NETosis, which are composed of DNA, histones and HNPs, are a source of
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(a)
(b)
(c)
(d)
Figure 2 Two-tailed bivariate correlations between serum levels of human neutrophil peptides (HNP)1–3 and lupus nephritis (LN) clinical characteristics.
autoantigen.17–19 Impaired NET breakdown and increased NET formation have been identified in patients with SLE. These processes may contribute to the development of autoimmunity through antigenantibody complexes.9,20 It has been suggested that HNP protects DNA in this process from degradation by nucleases and thus is essential for the immunogenicity of the immune complexes.8 When the relationship between NETosis and organ damage in SLE was assessed, lupus nephritis was the most prominent feature. In the present study, no significant elevation of serum HNP1–3 levels was observed in SLE patients without kidney injury, which seemed inconsistent with previous findings that HNP levels were higher in SLE patients than in healthy controls.7,21,22 However, all of the previous studies showed a large standard deviation, suggesting significant patient heterogeneity. Therefore, we recommend further analysis of the role of HNP in patients with specific clinical characteristics. In our study, we observed that serum HNP1–3 levels in LN patients were significantly higher than in SLE patients without kidney injury and healthy controls, strongly supporting the prominent role of NETosis in LN. Recent studies have proposed two mechanisms for impaired NET breakdown in SLE: the presence of deoxyribonuvlease (DNase) I inhibitors (including DNase I gene mutations and polymorphisms)9,23–25 and anti-NET antibodies that protect NETs from degradation.8,9,26 Our results are consistent with the second hypothesis. While previous genome-wide association
6
studies have not found a genetic predisposition for DNase I in SLE, this gene may play a role in the presence of mutation and rare variations. The correlation of HNP1–3 serum levels with neutrophil count may suggest that HNP1–3 is derived from neutrophils. In addition, the correlation of serum levels of HNP1–3 with urinary protein excretion and AI, as well as its link with proteinuria non-remission, suggested that higher serum levels of HNP1–3 may act as a LN disease activity marker and indicate worse prognosis. The lack of correlation between HNP1–3 serum levels and urinary protein excretion or disease prognosis in IgAN and MCD patients (a median follow-up time of 5 years, data not shown) may also support a disease-specific role of HNP1–3 in LN. In our current study, all of the patients with LN were diagnosed in a single center and were confirmed by renal biopsy, both of which guaranteed diagnostic homogeneity. Only LN patients with a serum sample and a long follow-up period were included in the study for correlations between serum HNP1–3 and disease progression. Although this study was pivotal, it did have some limitations, including a lack of independent replicates and a lack of a mechanism to study the role of HNP1–3 in intra-kidney pathogenesis. More widespread replication and functional assays centered on renal inherent cells are needed in the future. In addition, as HNP1–3 were also observed to be significantly elevated in patients with ANCA-associated vaculitis, a detailed analysis of SLE patients with positive ANCA would be of interest in the future.
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HNP 1–3 in lupus nephritis
In conclusion, we observed elevated HNP1–3 serum levels in LN patients and correlations with urinary protein excretion and AI. The data from this study suggested that HNP1–3 may play a pathogenic role in LN and HNP1–3 could serve as a biomarker for lupus nephritis.
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ACKNOWLEDGEMENTS The authors are grateful for the participation of all of the patients and control subjects. This work was supported by grants from the Major State Basic Research Development Program of China (973 program, No. 2012CB517700), the National Natural Science Foundation of China (No. 81200524), the Foundation of Ministry of Education of China (20120001120008), the Research Fund of Beijing Municipal Science and Technology for the Outstanding PhD Program (20121000110) and the Natural Science Fund of China to the Innovation Research Group (81021004).
CONFLICT OF INTEREST
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None declared.
REFERENCES 1 Ganz T, Selsted ME, Szklarek D et al. (1985) Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 76, 1427–35. 2 Yang D, Chen Q, Chertov O, Oppenheim JJ (2000) Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol 68, 9–14. 3 Brogden KA, Heidari M, Sacco RE et al. (2003) Defensininduced adaptive immunity in mice and its potential in preventing periodontal disease. Oral Microbiol Immunol 18, 95–9. 4 Zhou XJ, Cheng FJ, Lv JC et al. (2012) Higher DEFB4 genomic copy number in SLE and ANCA-associated small vasculitis. Rheumatology (Oxford) 51, 992–5. 5 Tsokos GC (2011) Systemic lupus erythematosus. N Engl J Med 365, 2110–21. 6 Adhya Z, Borozdenkova S, Karim MY (2011) The role of cytokines as biomarkers in systemic lupus erythematosus and lupus nephritis. Nephrol Dial Transplant 26, 3273–80. 7 Bennett L, Palucka AK, Arce E et al. (2003) Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 197, 711–23. 8 Lande R, Ganguly D, Facchinetti V et al. (2011) Neutrophils activate plasmacytoid dendritic cells by releasing selfDNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med 3, 19r–73r. 9 Hakkim A, Furnrohr BG, Amann K et al. (2010) Impairment of neutrophil extracellular trap degradation is
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associated with lupus nephritis. Proc Natl Acad Sci USA 107, 9813–8. Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 40, 1725. Weening JJ, D’Agati VD, Schwartz MM et al. (2004) The classification of glomerulonephritis in systemic lupus erythematosus revisited. J Am Soc Nephrol 15, 241–50. Weening JJ, D’Agati VD, Schwartz MM et al. (2004) The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 65, 521–30. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH (1992) Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 35, 630–40. Ma YC, Zuo L, Chen JH et al. (2006) Modified glomerular filtration rate estimating equation for Chinese patients with chronic kidney disease. J Am Soc Nephrol 17, 2937– 44. Vordenbaumen S, Timm D, Bleck E et al. (2011) Altered serum levels of human neutrophil peptides (HNP) and human beta-defensin 2 (hBD2) in Wegener’s granulomatosis. Rheumatol Int 31, 1251–4. Garcia-Romo GS, Caielli S, Vega B et al. (2011) Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med 3, 20r–73r. Kessenbrock K, Krumbholz M, Schonermarck U et al. (2009) Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med 15, 623–5. Lin AM, Rubin CJ, Khandpur R et al. (2011) Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J Immunol 187, 490–500. Brinkmann V, Reichard U, Goosmann C et al. (2004) Neutrophil extracellular traps kill bacteria. Science 303, 1532–5. Villanueva E, Yalavarthi S, Berthier CC et al. (2011) Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus. J Immunol 187, 538– 52. Vordenbaumen S, Fischer-Betz R, Timm D et al. (2010) Elevated levels of human beta-defensin 2 and human neutrophil peptides in systemic lupus erythematosus. Lupus 19, 1648–53. Sthoeger ZM, Bezalel S, Chapnik N, Asher I, Froy O (2009) High alpha-defensin levels in patients with systemic lupus erythematosus. Immunology 127, 116– 22. Panneer D, Antony PT, Negi VS (2013) Q222R polymorphism in DNAse I gene is a risk factor for nephritis in South Indian SLE patients. Lupus 22, 996–1000. Yasutomo K, Horiuchi T, Kagami S et al. (2001) Mutation of DNASE1 in people with systemic lupus erythematosus. Nat Genet 28, 313–4.
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25 Shin HD, Park BL, Kim LH, Lee HS, Kim TY, Bae SC (2004) Common DNase I polymorphism associated with autoantibody production among systemic lupus erythematosus patients. Hum Mol Genet 13, 2343–50.
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26 Leffler J, Martin M, Gullstrand B et al. (2012) Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease. J Immunol 188, 3522–31.
International Journal of Rheumatic Diseases 2014
ORIGINAL ARTICLE
Human neutrophil peptide 1–3, a component of the neutrophil extracellular trap, as a potential biomarker of lupus nephritis Fa-Juan CHENG, Xu-Jie ZHOU, Yan-Feng ZHAO, Ming-Hui ZHAO and Hong ZHANG Renal Division, Department of Medicine, Peking University First Hospital, Peking University Institute of Nephrology, Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China
Abstract Objective: Human neutrophil peptides (HNP) were recently implicated in the neutrophil extracellular trap (NET) complex, the impaired degradation of which has been associated with lupus nephritis (LN). Methods: Forty LN patients, 40 SLE patients without kidney injury, 63 immunoglobulin A nephropathy (IgAN) patients, 20 minimal change disease (MCD) patients and 33 healthy controls were included in the present study. LN, IgAN and MCD patients were diagnosed with renal biopsy. LN patients were followed for a median period of 5.5 years. Clinical and laboratory data at the time of renal biopsy and follow-up were collected for each LN patient. Serum levels of HNP1–3 were measured with enzyme-linked immunosorbent assay. Results: Serum HNP1-3 levels in LN patients were significantly higher than for SLE patients without kidney injury (P < 0.001), IgAN patients (P = 0.012), MCD patients (P = 0.010) and healthy controls (P = 0.022). Serum HNP1–3 levels were an independent indicator of LN (P = 0.006, OR = 7.5, 95% CI, 1.782–31.842), were statistically correlated with urinary protein excretion (P = 0.009) and activity index (P = 0.042) and were only marginally correlated with neutrophils (P = 0.054) and white blood cell counts (P = 0.051). Serum levels of HNP1–3 were a predictor of proteinuria remission after correction for multiple parameters (multivariate hazard 0.209; 95% CI 0.046–0.951; P = 0.043). Conclusions: The data from this study indicated that HNP1–3, one component of the NET, is a potential biomarker for LN. Key words: human neutrophil peptide, lupus nephritis, systemic lupus erythematosus.
INTRODUCTION Human defensins, an important part of the innate immune system, are a family of evolutionarily closely related cationic anti-microbial peptides. The a-defensins 1–4, initially isolated from neutrophils, are also
Correspondence: Hong Zhang, Renal Division, MD, PhD, Professor of Medicine, Peking University First Hospital, Peking University Institute of Nephrology, No. 8 Xi Shi Ku Street, Xi Cheng District, Beijing 100034, China. Email: hongzh@bjmu. edu.cn
called human neutrophil peptides (HNP1–4).1 In addition to their role in anti-microbial infection, HNP1–3 have been shown to regulate inflammation in diverse ways, including serving as chemoattractants for monocytes, dendritic cells and T cells and enhancing the antigen-specific immune response.2,3 Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organs and systems. The precise etiology of SLE is currently unknown.4–6 However, in oligonucleotide microarrays, HNP was one of the most highly up-regulated molecules in SLE patients at the genome-wide level, suggest-
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
F.-J. Cheng et al.
ing an important role for HNP in the pathogenesis of SLE.7 HNP was found to be an important component of neutrophil extracellular traps (NETs), which were involved in a process called NETosis.8 NETosis refers to the extrusion of large amounts of nuclear DNA into the extracellular space by activated neutrophils, and these web-like structures called NETs (neutrophil extracellular traps) were recently associated with SLE.8 In particular, previous reports have indicated that impaired degradation of NETs was specifically associated with lupus nephritis (LN).9 It has been suggested that the inability to dismantle NETs might be a useful indicator of renal involvement in SLE patients. However, no correlation between HNP and LN has ever been identified. Therefore, in the current study, we determined HNP1–3 serum levels in patients with LN, SLE without kidney injury and glomerulonephritis caused by different diseases, and investigated the role of a-defensins in the pathogenesis of LN.
SUBJECTS AND METHODS Subjects In the present study, 40 LN patients, 40 SLE patients without kidney injury, 63 immunoglobulin A nephropathy (IgAN) patients, 20 minimal change disease (MCD) patients and 33 healthy controls matched geographically and ethnically were recruited. All SLE patients met the American College of Rheumatology (ACR) classification according to the revised ACR SLE criteria.10 For all LN patients, renal biopsies were analyzed by light microscopy, immunofluorescence and electron microscopy to confirm the LN diagnostic criteria according to the American Rheumatism Association (ARA, 1982). LN was classified according to the 2003 International Society of Nephrology (ISN)/ Renal Pathology Society (RPS) classifications.11,12 The activity index (AI) and chronic index (CI) were evaluated, and the SLE activity index was scored by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI).13 The estimated glomerular filtration rate (eGFR) was evaluated according to the abbreviated Modification of Diet in Renal Disease (MDRD) equation, which was suitably modified for the Chinese population.14 Clinical and laboratory data at the time of renal biopsy were collected for each patient. LN patients were followed for a median period of 5.5 years. We assessed the prognostic relevance of achieving proteinuria remission as defined by achieving proteinuria values < 0.3 g/day and renal function decline as defined by an increase in serum creatinine > 50%. SLE patients
2
without kidney disease were enrolled from the dermatology department of Peking University. All of the SLE patients were determined to be negative for anti-neutrophil cytoplasmic antibodies (ANCA), which may be a stimulus for the release of defensins by neutrophils. IgAN and MCD patients were also verfied by renal biopsy. The diagnosis of IgAN was based upon the demonstration by immunofluorescence of IgA as the dominant or co-dominant immunoglobulin in mesangial deposits and the lack of clinical or serological evidence of other inflammatory conditions, such as SLE, vasculitis or Henoch–Schoenlein purpura. A diagnosis of MCD was confirmed by the combined determinations of no immunofluorescence findings with immunofluorescence microscopy, normal glomeruli as observed with light microscopy and diffused effacement of podocyte foot processes as observed with electronic microscopy. Clinical and laboratory data at the time of renal biopsy were collected for each IgAN and MCD patient. This study was approved by the Ethics Committee of Peking University First Hospital and all participants gave written informed consent.
Assay for serum levels of HNP1–3 Serum HNP1–3 was measured by enzyme-linked immunosorbent assay (ELISA) (Hycult Biotechnology, Uden, The Netherlands) as previously reported.15 Briefly, microtiter wells were first precoated with antibodies recognizing HNP1–3. Every sample, serially diluted standard and blank control were tested in duplicate. The volume in each well was 100 lL for all steps, and all incubations were performed at room temperature for 1 h. The plates were then washed four times with wash buffer. Sera were diluted 1 : 100 with plasma dilution buffer, and every plate contained serially diluted standard and blank controls. Binding was detected with biotinylated tracer antibody and streptavidin-peroxidase conjugate. We used the substrate tetramethylbenzidine and measured the absorbance at 450 nm (Bio-Rad 550, Tokyo, Japan). The concentration of sample HNP1–3 in the serum was calculated according to a standard curve.
Statistical analysis Continuous variables were tested in each group for a normal distribution using the Kolmogorov–Smirnov test. Normally distributed variables were expressed as the mean the standard deviation (SD) and compared using Student’s t-test or one-way analysis of variance (ANOVA). Non-parametric variables were expressed as the median and range and compared using the Mann–
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Whitney U-test or Kruskal–Wallis H-test. Pearson’s chisquare was used to analyze the categorical data and a continuity correction or Fisher’s exact test was applied when necessary. For the comparison of continuous variables, two-tailed bivariate correlations and Pearson’s coefficient were calculated. Logistic regression was used to model the effect of HNP1–3 serum levels on the disease incidence. The relationship between tested parameters and renal prognosis was assessed using Cox regression. As the serum levels of HNP1–3 and urinary protein excretion were highly skewed to the right, log transformation was performed in the correlation, logistic regression and Cox regression analyses. All analyses were performed using the SPSS 16.0 program (SPSS Inc., Chicago, IL, USA). All tests were two-sided, and P < 0.05 was considered to be statistically significant.
RESULTS Patient baseline characteristics and clinical outcome As is shown in Table 1, the mean ages of LN patients, SLE patients without kidney injury, IgAN patients, MCD patients and the 33 healthy controls were 34.8 11.4, 36.3 13.2, 34.8 12.4, 32.8 10.5 and 27.9 4.8 years, respectively, while the percentages of subjects who were female were 82.5%, 85.0%, 49.2%, 65.0% and 75.8%, respectively. The median initial proteinuria of LN patients when enrolled in the study was 4.64 g/day (range 0.32– 21.04), and median serum creatinine (Scr) was 77.00 lmol/L (range 42.00–610.00). The mean eGFR was 85.62 44.73 mL/min/1.73 m2. Regarding pathological phenotypes, one (2.5%) was class II, 13 (32.5%) were class III, 20 (50.0%) were class IV and six (15%) were class V. The mean AI and CI of LN patients were 7.08 4.42 and 2.40 2.13, respectively. All patients received immunosuppressive agents, while 32.5% of patients were treated with angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (ACEI/ ARB). After a median follow-up of 5.5 years, the median of the most recent proteinuria was 0.50 g/day (range 0.02–7.40), and median Scr was 75.00 lmol/L (range 56.00–954.00). Proteinuria remission was observed in 37.5% of patients and 10% of patients experienced a decline in renal function. For IgAN patients on biopsy, median proteinuria was 1.39 g/day (range 0.17–13.72) and median Scr was 90.00 lmol/L (range 58.00–226.00). The average eGFR was 83.08 23.17 mL/min/1.73 m2. The distribution by Haas grade I, II, III, IV and V in IgAN patients was
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12.7, 0.0, 42.9, 34.9 and 9.5%, respectively. For MCD patients, median proteinuria was 2.66 g/day (range 0.03–12.16) and median Scr was 55.00 lmol/L (range 43.00–114.30), respectively. The average eGFR of MCD patients was 129.01 33.52 mL/min/1.73 m2.
Serum levels of HNP 1–3 in patients with different causative diseases and healthy controls We observed no differences in HNP1–3 serum levels among SLE patients without kidney injury (median 27.24 ng/mL, range 12.21–276.32), IgAN patients (median 38.99 ng/mL, range 14.95–338.57), MCD patients (median 29.75 ng/mL, range 18.31–331.72) and healthy controls (median 31.77 ng/mL, range 22.76–269.33; P > 0.05). However, as is shown in Figure 1, the median serum level of HNP1–3 in LN patients (74.79 ng/mL, range 23.59–883.63) was significantly higher than for SLE patients without kidney injury (27.24 ng/mL, range 12.21–276.32, P < 0.001) and healthy controls (P = 0.022; Fig. 1). Similarly, the median serum level of HNP1–3 in LN patients was also elevated compared to IgAN (P = 0.012) and MCD patients (P = 0.010).
Serum levels of HNP1–3 correlated with LN clinical characteristics and prognosis When adjusted for gender and age by logistical regression analysis, serum levels of HNP1–3 were found to be independent indicators of LN (P = 0.006, OR = 7.5, 95% confidence interval [CI], 1.782–31.842). When LN patients were divided into two groups according to the 90th percentile of HNP1–3 serum levels in controls, the group with higher HNP1–3 levels presented with higher proteinuria, with a median of 5.80 and a range of 2.02– 21.04 versus a median of 4.14 and a range of 0.32– 10.88, P = 0.032, respectively. In addition, HNP1–3 serum levels were significantly correlated with urinary protein excretion (r = 0.405, P = 0.009) and AI (r = 0.323, P = 0.042). A marginal correlation between HNP1–3 serum levels and neutrophil count (r = 0.307, P = 0.054) as well as white blood cell count (r = 0.310, P = 0.051) was also observed (Fig. 2). No correlation was observed between HNP1–3 serum levels and other LN clinical characteristics, such as SLEDAI, serum Scr levels, CI and anti-double stranded DNA antibody. After a median follow-up of 5.5 years, we did not observe a relationship between HNP1–3 serum level and a decline in renal function. However, we did observe that HNP1–3 serum levels were an important predictor of proteinuria remission in multivariate Cox
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Table 1 Baseline characteristics and clinical outcomes for patients and controls Parameters
LN n = 40
SLE without LN n = 40
IgAN n = 63
Demography Gender (female) 33 (82.5%) 34 (85.0%) 31 (49.2%) Age (years) 34.8 11.4 36.3 13.2 34.8 12.4 General Fever 14 (35.0%) SBP* (mmHg) 124.50 11.37 121.83 15.46 DBP* (mmHg) 78.38 8.35 78.49 12.69 Hematological system Red blood cell count 3.67 0.62 4.43 0.57 (9 1012/L) 7.00 2.78 7.14 2.61 White blood cell count (9 109/L) 192.00 62.49 230.25 59.09 Blood platelet count (9 109/L) Hemoglobin (g/L) 109.99 18.90 134.98 17.57 Kidney Urinary protein excretion 4.64 (0.32, 21.04) 1.39 (0.17, 13.72) (g/day) (median, range) Serum creatinine (lmol/L) 77.00 (42.00, 610.00) 90.00 (58.00, 226.00) (median, range) 85.62 44.73 83.08 23.17 eGFR *(mL/min/1.73 m2) Glomerulonephritis (%) Class II 1 (2.5) Class III 13 (32.5) Class IV 20 (50) Class V 6 (15) AI* 7.08 4.42 CI* 2.40 2.13 SLEDAI* 17.65 4.94 Histological grading (%) I 8 (12.7) II 0 (0) III 27 (42.9) IV 22 (34.9) V 6 (9.5) Other systems (%) Photosensitivity 9 (22.5) Oral ulcers 10 (25.0) Presence of arthritis 23 (57.5) Presence of peritonitis 9 (22.5) Presence of meningitis 3 (7.5) Immunological indicators Serum IgG (g/L) 10.83 5.57 Serum IgA (g/L) 2.89 1.04 Serum IgM (g/L) 1.09 0.62 Serum C3 (g/L) 0.52 0.29 Serum C4 (g/L) 0.12 0.82 ANAs (%) 40 (100)
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MCD n = 20
13 (65.0%) 32.8 10.5
Healthy controls n = 33 25 (75.8%) 27.9 4.8
116.50 16.39 72.60 12.83 4.51 0.46 6.34 2.98 251.05 61.40 139.75 15.99 2.66 (0.03, 12.16) 55.00 (43.00, 114.30) 129.01 33.52
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Table 1 (continued) Parameters
Anti-dsDNA antibodies (%) Anti-Sm (%) Anti-SSA (%) Anti-SSB (%) Anti-nRNP (%) ANCA (%) Treatment (%) ACEI or ARB Any immunosuppression Follow-up Length of follow-up (years) (median, range) Latest proteinuria (g/day) (median, range) Latest serum creatinine (lmol/L) (median, range)
LN n = 40
SLE without LN n = 40
IgAN n = 63
MCD n = 20
Healthy controls n = 33
26 (65.0) 11 (27.5) 18 (45.0) 4 (10.0) 10 (25.0) 0 (0) 13 (32.5) 40 (100) 5.50 (3.50,23.50) 0.50 (0.02, 7.40) 75.00 (56.00, 954.00)
SBP, systolic blood pressure; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; Ccr, creatinine clearance; AI, activity index; CI, chronic index; SLEDAI, systemic lupus erythematosus disease activity index; ANA, antinuclear antibodies; SSA, Sj€ ogren’s syndrome antigen A; nRNP, nuclear ribonucleoprotein; ANCA, anti-neutrophil cytoplasmic antibodies; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker.
DISCUSSION
Figure 1 Serum levels of human neutrophil peptides (HNP) 1–3 in healthy controls and patients. The horizontal bars represent the median.
regression analysis with corrections for multiple parameters, including age, sex, initial renal function and the use of ACEI/ARB and immunosuppressive agents (multivariate hazard 0.209; 95% CI, 0.046–0.951; P = 0.043).
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In the present study, we determined HNP1–3 serum levels in patients with LN. A significant elevation of the HNP1–3 serum levels in LN patients was observed, together with correlations with urinary protein excretion and AI, strongly suggesting an involvement of a-defensin in the disease activity of LN. To determine a possible pathogenic mechanism, SLE without kidney injury, IgAN, which is thought to be an inflammationmediated nephropathy, and MCD, a disease characterized by substantial protein excretion in the urine without deposition of immunoglobulin or immune complex, were selected as disease controls. Interestingly, only LN patients, but not SLE patients without kidney injury, IgAN patients or MCD patients, showed a significant elevation of HNP1–3 serum levels, suggesting that HNP1–3 may be a potential biomarker for LN. SLE is a prototypical autoimmune disease characterized by autoantibody production and immune complex formation/deposition in target organs such as the kidney. Recent progress has pushed the neutrophil and its unique form of cell death called the neutrophil extracellular trap (NETosis) to the forefront of the pathogenesis of SLE,8,16 and NETosis, which are composed of DNA, histones and HNPs, are a source of
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(a)
(b)
(c)
(d)
Figure 2 Two-tailed bivariate correlations between serum levels of human neutrophil peptides (HNP)1–3 and lupus nephritis (LN) clinical characteristics.
autoantigen.17–19 Impaired NET breakdown and increased NET formation have been identified in patients with SLE. These processes may contribute to the development of autoimmunity through antigenantibody complexes.9,20 It has been suggested that HNP protects DNA in this process from degradation by nucleases and thus is essential for the immunogenicity of the immune complexes.8 When the relationship between NETosis and organ damage in SLE was assessed, lupus nephritis was the most prominent feature. In the present study, no significant elevation of serum HNP1–3 levels was observed in SLE patients without kidney injury, which seemed inconsistent with previous findings that HNP levels were higher in SLE patients than in healthy controls.7,21,22 However, all of the previous studies showed a large standard deviation, suggesting significant patient heterogeneity. Therefore, we recommend further analysis of the role of HNP in patients with specific clinical characteristics. In our study, we observed that serum HNP1–3 levels in LN patients were significantly higher than in SLE patients without kidney injury and healthy controls, strongly supporting the prominent role of NETosis in LN. Recent studies have proposed two mechanisms for impaired NET breakdown in SLE: the presence of deoxyribonuvlease (DNase) I inhibitors (including DNase I gene mutations and polymorphisms)9,23–25 and anti-NET antibodies that protect NETs from degradation.8,9,26 Our results are consistent with the second hypothesis. While previous genome-wide association
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studies have not found a genetic predisposition for DNase I in SLE, this gene may play a role in the presence of mutation and rare variations. The correlation of HNP1–3 serum levels with neutrophil count may suggest that HNP1–3 is derived from neutrophils. In addition, the correlation of serum levels of HNP1–3 with urinary protein excretion and AI, as well as its link with proteinuria non-remission, suggested that higher serum levels of HNP1–3 may act as a LN disease activity marker and indicate worse prognosis. The lack of correlation between HNP1–3 serum levels and urinary protein excretion or disease prognosis in IgAN and MCD patients (a median follow-up time of 5 years, data not shown) may also support a disease-specific role of HNP1–3 in LN. In our current study, all of the patients with LN were diagnosed in a single center and were confirmed by renal biopsy, both of which guaranteed diagnostic homogeneity. Only LN patients with a serum sample and a long follow-up period were included in the study for correlations between serum HNP1–3 and disease progression. Although this study was pivotal, it did have some limitations, including a lack of independent replicates and a lack of a mechanism to study the role of HNP1–3 in intra-kidney pathogenesis. More widespread replication and functional assays centered on renal inherent cells are needed in the future. In addition, as HNP1–3 were also observed to be significantly elevated in patients with ANCA-associated vaculitis, a detailed analysis of SLE patients with positive ANCA would be of interest in the future.
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In conclusion, we observed elevated HNP1–3 serum levels in LN patients and correlations with urinary protein excretion and AI. The data from this study suggested that HNP1–3 may play a pathogenic role in LN and HNP1–3 could serve as a biomarker for lupus nephritis.
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ACKNOWLEDGEMENTS The authors are grateful for the participation of all of the patients and control subjects. This work was supported by grants from the Major State Basic Research Development Program of China (973 program, No. 2012CB517700), the National Natural Science Foundation of China (No. 81200524), the Foundation of Ministry of Education of China (20120001120008), the Research Fund of Beijing Municipal Science and Technology for the Outstanding PhD Program (20121000110) and the Natural Science Fund of China to the Innovation Research Group (81021004).
CONFLICT OF INTEREST
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None declared.
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26 Leffler J, Martin M, Gullstrand B et al. (2012) Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease. J Immunol 188, 3522–31.
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