Autoantibodies Associated with Renal Involvement in Patients with Systemic Lupus Erythematosus

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1 H. P. Kuei, Y. L. Shih, C. C. Lee, et al Autoantibodies Associated with Renal Involvement in Patients with Systemic Lupus Erythematosus Hsiu-Ping Kuei, Yung-Luen Shih, Chin-Cheng Lee and Chih-Cheng Chuang 2 Department of Pathology and Laboratory Medicine, 2 Department of Rheumatology, Allergy and Clinical Immunology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan Lupus nephritis has often been associated with anti-ds DNA. The aim of this study was to investigate whether renal involvement of systemic lupus erythematosus (SLE) was associated with any autoantibody patterns. Medical records of patients with SLE (57 patients) seen from 999 through 2002 by the rheumatology service at the Shin Kong Wu Ho-Su Memorial Hospital were retrospectively analyzed. Persistent proteinuria was used as the evidence of renal involvement among the SLE patients. Results of autoantibody tests at diagnosis were identified retrospectively. Twenty (35%) of the 57 SLE patients had renal involvement. Anti-Ro/SS-A antibodies were present in the serum of 6 of the 20 (80%) patients with renal involvement and 9 of the 37 (5%) without renal involvement (p=0.034). IgG anti-cardiolipin antibodies (IgG acl) were present in the serum of 5 of the 20 (75%) patients with renal involvement and 3 of the 37 (35%) without renal involvement (p=0.004). In addition, low levels of complement C3 were present in the serum of 3 of the 20 (65%) patients with renal involvement. Anti-ds DNA, anti-la/ss-b, anti-sm, and anti-nrnp antibodies showed no correlation with renal involvement. Renal involvement in our SLE population was frequently found and the results correlated significantly with the presence of anti-ro/ss-a, IgG acl autoantibodies. Key words: Autoantibody, renal involvement, systemic lupus erythematosus (SLE) Introduction Systemic lupus erythematosus (SLE) is the most clinically and serologically diverse of the autoimmune connective tissue diseases because it may affect any organ of the body and displays a broad spectrum of clinical and immunological manifestations. The clinical features include articular and mucocutaneous involvement, renal diseases, hematologic abnormalities, and central nervous system diseases []. As a result of immunological abnormalities, renal disorders occur frequently in patients with SLE [2-4]. The prognosis of patients with SLE is determined by the severity of renal involvement [5,6]. In most large series, one-third to one-half of SLE patients show evidence of clinical nephritis. While the renal manifestations of SLE vary with the severity of the renal lesion, proteinuria is present in the vast majority of cases. Much effort has been expended to identify serologic markers that correlate with clinical nephritis in SLE patients. There is abundant evidence that DNA-anti-DNA complexes participate in nephritis [7], but not all patients with antibodies to DNA develop nephritis. When other serologic subgroups were investigated, patients with precipitating antibodies to Ro/SS-A alone showed a 37-42% frequency of nephritis [8,9], while SLE patients with both anti-ro/ss-a and anti-la/ss-b precipitins had nephritis at the very low frequency of 4% [8]. SLE patients with antibodies to nuclear RNP (nrnp) alone also had a low prevalence of nephritis (3%), while those with anti-nrnp and anti-ro/ss-a, anti-nrnp and anti- Received: June, 2006 Revised: October 3, 2006 Accepted: November 20, 2006 Address correspondence to: Dr. Chin-Cheng Lee, Department of Pathology and Laboratory Medicine, Shin Kong Wu Ho-Su Memorial Hospital, No. 95, Wen-Chan Road, Shih-Lin District, Taipei, Taiwan. Tel: (02) ext 220 Fax: (02) M002058@ms.skh.org.tw J Biomed Lab Sci 2006 Vol 8 No ~4 23

2 Autoantibody and renal involvement in SLE patients Sm, or anti-sm alone had nephritis frequencies of 57%, 53%, and 63%, respectively [9]. In the study by Hopkinson et al. [0], a further interesting finding was that a positive result for IgG anti-cardiolipin antibodies was an independent risk factor in the development of lupus nephritis. We found that anti-ro/ss-a, and IgG anti-cardiolipin antibodies (acl) were associated with renal involvement in patients with SLE. The significance of laboratory data in a group of patients with SLE at our hospital was studied. Patients Materials and Methods A total of 57 patients (54 women and 3 men) with SLE were studied. The median age at diagnosis was 37 years (range, 3-77 years). All patients fulfilled at least four of the revised American College of Rheumatology (ACR) criteria for the diagnosis of SLE []. This study was carried out in rheumatology clinics at the Shin Kong Wu Ho-Su Memorial Hospital from 999 through Each patient underwent extensive serologic and immunological studies. Laboratory Evaluation of Renal Involvement Parameters Renal involvement was considered if at least one of the following features were present: persistent proteinuria of > 0.5 g/24 h by a turbidimetric method (Roche Diagnostics GmbH) or 3+ proteinuria by a dipstick method, cellular casts, serum creatinine.4 mg/dl by a Jaffe method (Roche Diagnostics GmbH), or abnormal results of renal biopsy (classes Ⅱ, Ⅲ, Ⅳ, or Ⅴ). Clinical and Serological Parameters The hospital charts were reviewed to obtain age, sex, clinical manifestations, blood pressure, hematography, urinalysis, complement titer, serum creatinine, and 24- hour urine protein levels. A diagnosis of anemia was made when the hemogloblin level was less than g/dl. In addition to prednisone treatment that was used by most patients in the study, a substantial proportion of patients had exposures to immunosuppressive treatment. In each patient the following serological parameters were determined. C3 and C4 components of complement were determined by nephelometry (Beckman, Array Protein System) and normal ranges were mg/dl and 9-33 mg/dl, respectively. Antinuclear antibodies (ANA) were determined by immunofluorescence in Hep-2 substrates (DiaSorin Inc. Stillwater, Minn, U.S.A.) and regarded as positive if the ratio was greater than :80. Antibodies to the cellular antigens Ro/SSA, La/SSB, Sm, and RNP by a double immunodiffusion method (MBL Co, LTD, Nagoya, Japan). Antibodies to double stranded DNA (ds DNA) were determined by an indirect immunofluorescent assay using Crithidia luciliae as the substrate and the results were considered as positive if the ratio was greater than :6. Antibodies to IgG cardiolipin (acl) were detected using an enzyme-linked immunosorbent assay (ELISA) kit (The Binding Site LTD, Birmingham, U.K.) and regarded as positive if the result was more than GPL-U/ml (reference value). Statistical Analysis Statistical significance was analyzed using the Fisher s exact test. A p value of less than 0.05 was considered statistically significant. Analysis was done using the SPSS 9.0 software package (SPSS, Inc., Chicago, Ill, U.S.A.). Results Renal involvement was found in 20/57 (35%) patients (8 women and two men). Mean ages of the patients with and without renal involvement were 33.6±2.2 and 38.±9.7 years, respectively (p = 0.40). Fifteen (26.3%) patients had undergone renal biopsies in this study. Of them, two (3.3%) had class Ⅲ, 0 (66.6%) had class Ⅳ, three (20.0%) had class Ⅴ diseases. However, possibly due to the small case number, autoantibodies among the groups with different pathological findings did not reach statistical significance. Table shows the distribution of various factors with regard to the presence of renal involvement. Patients with renal involvement had a greater prevalence of anti-ro/ss-a antibodies and IgG anti-cardiolipin antibodies compared to those without renal involvement. In addition, low levels of complement C3 were more frequently found in patients with renal involvement than those without. The presence of other autoantibodies showed no correlation with renal involvement. Positive anti-dsdna was more frequently found in patients with renal involvement (95%), yet it did not reach statistical significance. No significant difference was found in clinical findings of the SLE patients, no matter whether they had renal involvement or not (Table ). As regard to treat- 24 J Biomed Lab Sci 2006 Vol 8 No~4

3 H. P. Kuei, Y. L. Shih, C. C. Lee, et al ment, the use of immunosuppressive medicine was significantly higher among patients with renal involvement. However, no association was found between the presence of autoantibodies and the use of immunosuppressive treatment (Table 2). Table. Comparison of clinical and laboratory findings between the 57 SLE patients with and without renal Involvement Variable Clinical findings Renal involvement (n = 20) No. (%) of patients No Renal involvement (n = 37) p value Hemolytic anemia 7 (35) (30) NS Leukopenia < 4000/mm 3 6 (30) 4 (38) NS Thrombocytopenia <00000/mm 3 (5) 3 (8) NS Malar rash 6 (30) 8 (49 ) NS Alopecia 5 (25) 8 (22) NS Photosensitivity 9 (45) 5 (4) NS Oral ulcer 4 (20) 7 (9) NS Raynaud s phenomenon 5 (25) 8 (22) NS Arthritis 4 (20) 3 (8) NS Serositis 0 (50) 2 (32) NS Neuropsychiatic 4 (20) 6 (6) NS Hypertension 6 (30) 7 (9) NS Treatment Prednisone 9 (95) 30 (8) NS Immunosuppressive 4 (70) 5 (4) <0.00 Laboratory findings Anti-ds DNA 9 (95) 3 (83.7) NS Anti-Ro/SSA antibody 6 (80) 9 (5) < 0.05 Anti-La/SSB antibody 5 (25) 7 (8.9) NS Anti-Sm antibody 3 (5) 3 (8) NS Anti-nRNP antibody 3 (5) 8 (2.6) NS Anti-cardiolipin antibody 5 (75) 3 (35) < Low complement (C3) 3 (65) (29.7) < 0.0 NS = not significant (p 0.05). Table 2. Comparison of the presence of autoantibodies between SLE patients with or without immunosuppressive treatment Autoantibody With immunosuppressive treatment (n = 9) No. (%) of patients Without immunosuppressive treatment (n = 38) p value Anti-ds DNA 8 (95) 32 (84) NS Anti-Ro/SSA antibody 3 (68) 22 (58) NS Anti-La/SSB antibody 4 (2) 8 (2) NS Anti-Sm antibody 2 () 4 () NS Anti-nRNP antibody 2 () 9 (24) NS Anti-cardiolipin antibody 9 (47) 9 (50) NS NS = not significant (p 0.05). J Biomed Lab Sci 2006 Vol 8 No ~4 25

4 Autoantibody and renal involvement in SLE patients Discussion The overall prevalence of renal involvement in this cohort was approximately 35%, which is comparable with the incidence found in previous studies [2]. As described, 6 (80%) patients with renal involvement were positive for anti-ro/ss-a, and 5 (75%) for IgG anticardiolipin antibodies (IgG acl). However, the investigations on the relationship between anti-ro/ss-a antibody and lupus nephritis gave conflicting results. Some researchers observed an association of anti-ro/ss-a antibody with nephritis [8,3]. Comparison of the autoantibody profiles of patients with and without renal involvement showed a similar frequency of positive anti-ds DNA in both groups. Although there was a general correlation between the presence of anti-ds DNA antibodies and SLE, several researchers have demonstrated a discrepancy with lupus nephritis. In a recent study, Alba et al. [4] retrospectively studied different populations of autoantibodies in SLE patients with biopsy confirmed lupus nephritis. Sixty-eight percent of patients with lupus nephritis had positive results of anti-dsdna antibodies. In patients without lupus nephritis, approximately half of the patients had anti-ds DNA antibodies. However, one of the reasons for the discordance between anti-ds DNA titers and the association with lupus nephritis in different studies might be attributable to the use of different DNA substrates for the immunoassays [5]. Since different substrates were used (i.e., bacterial DNA in Farr assay, mammalian DNA, such as calf thymus, in ELISA, and protozoan DNA within the kinetoplast organelle in Crithidia lucilae assay), patients might be negative for one but positive for another assay. One of the primary requisites for autoantibodies to induce nephritis depends on their abilities to deposit in kidney and initiate inflammation [6]. Three mechanisms have been proposed to explain the ability of anti-ds DNA antibodies to localize in kidney [7]. Occasionally lupus patients who are genuinely anti-dna negative develop clinically significant nephritis. This may occur because of antigen excess and the inability to detect circulating anti-dna deposited in kidney. The positive rate of autoantibodies in our SLE patients with renal involvement was however lower than those in other reports for anti-nrnp and anti-sm [9,8]. Several factors may explain the lower prevalence in our study. First, the serum sample used to determine renal involvement prevalence was taken routinely during the study period and was therefore not necessarily obtained during an active phase of the disease. Second, we determined renal involvement only by renal function assays in the laboratory, and we did not look for other histological abnormalities. Third, most SLE patients in this study had histories of long-term steroid and/or immunosuppressive therapy, which may have decreased the risk of renal diseases, a possibility also supported by Masaaki et al. [9]. It should be reemphasized that these observations indicated the autoantibodies (anti-ro/ss-a and IgG acl) exhibited uniform association with nephritis in our study. A significant difference in the prevalence of acl between the two groups was found in our study (Table ). However, the increased risk may be accounted for by anti-cardiolipin antibodies that were not specifically examined. In our study, the presence of IgG anti-cardiolipin antibodies, rather than anti-dsdna antibodies, were a strong risk factor in the development of renal SLE, which was comparable with previous studies [20], even though in this series of patients the presence of positive anti-cardiolipin antibodies correlated strongly with the presence of antibodies against dsdna [2] and anti-dsdna antibodies which have been implicated in the pathogenesis of renal SLE [22]. In addition, low complement C3 levels were found in 3 of the 20 (65%) patients with renal involvement in our study. Hypocomplementemia is felt to be a distinguishing feature in high risk populations [23]. Multiple low complement determinations were significantly predictives of renal insufficiency [24] and patients with SLE with nephritis were more frequently found to have markedly low complement component levels [25]. In summary, 57 patients with SLE were studied to evaluate whether autoantibodies were associated with the development of renal involvement in such patients. Of the 57 patients, 20 had renal involvement and had an increased frequency of anti-ro/ss-a and IgG acl compared with the patients without renal involvement. Our data suggest that anti-ro/ss-a and IgG acl autoantibodies may be useful predictors for the development of renal diseases in SLE patients and may provide important clues for understanding lupus nephritis. References. Cervera R, Khamashta MA, Font J, et al: Systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 000 patients. Medicine (Baltimore) 993; 72: Laitman RS, Glicklich D, Sablay LB, et al: Effect of long-term normalization of serum complement levels on 26 J Biomed Lab Sci 2006 Vol 8 No~4

5 H. P. Kuei, Y. L. Shih, C. C. Lee, et al the course of lupus nephritis. Am J Med 989; 87: Smeenk R, Brinkman K, van den Brink H, et al: Antibody to DNA in patients with systemic lupus erythematosus: their role in the diagnosis, the follow-up and the pathogenesis of the disease. Clin Rheumatol 990; 9 (suppl ): Esdaile JM, Abrahamowicz M, MacKenzie T, et al: The time dependence of long-term prediction in lupus nephritis. Arthritis Rheum 994; 37: Derksen RH, Hene RJ and Kater L: The long-term clinical outcome of 56 patients with biopsy-proven lupus nephritis followed at a single center. Lupus 992; : Anonymous. Lupus nephritis: prognostic factors and probability of maintaining life-supporting renal function 0 years after the diagnosis. Gruppo Italiano per lo Studio della Nefrite Lupica (GISNEL). Am J Kidney Dis 992; 9: Tan EM, Schur PH, Carr Ri, et al: Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J Clin Invest 966; 45: Wasicek CA and Reichlin M: Clinical and serological differences between systemic lupus erythematosus patients with antibodies to Ro versus patients with antibodies to Ro and La. J Clin Invest 982; 69: Maddison PJ, Mogavero H, Provost TT, et al: The clinical significance of autoantibodies to a soluble cytoplasmic antigen in systemic lupus erythematosus and other connective tissue diseases. J Rheumatol 979; 6: Hopkinson ND, Jenkinson C, Muir KR, et al: Racial group, socioeconomic status, and the development of persistent proteinuria in systemic lupus erythematosus. Ann Rheum Dis 2000; 59: Tan EM, Cohen AS, Fries JF, et al: The 982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 982; 25: Estes D and Christian CL: The natural history of systemic lupus erythematosus by prospective analysis. Medicine 97; 50: Nossent JC, Henzen-Logman SC, Vroom TM, et al: Relation between serological data at the time of biopsy and renal histology in lupus nephritis. Rheumatol Int 99; : Alba P, Bento L, Cuadrado MJ, et al: Anti-ds DNA, anti-sm antibodies, and the lupus anticoagulant: significant factors associated with lupus nephritis. Ann Rheum Dis 2003; 62: Haugbro K, Nossent JC, Winkler T, et al: Anti-ds DNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 2004; 63: Waldman M and Madaio MP: Pathogenic autoantibodies in lupus nephritis. Lupus 2005; 4: Rekvig OP, Kalaaji M and Nossent H: Anti-DNA antibody subpopulations and lupus nephritis. Autoimmun Rev 2004; 3: Hochberg MC, Dorsch CA, Feinglass EJ, et al: Survivorship in systemic lupus erythematosus: Effect of antibody to extractable nuclear protein. Arthritis Rheum 98; 24: Nakano M, Ueno M, Hasegawa H, et al: Renal haemodynamic characteristics in patients with lupus nephritis. Ann Rheum Dis 998; 57: McAlindon T, Giannotta L, Taub N, et al: Environmental factors predicting nephritis in systemic lupus erythematosus. Ann Rheum Dis 993; 52: Hopkinson N: Systemic lupus erythematosus: a clinical and epidemiological study. (Thesis DM). Nottingham, Winfield J, Faiferman I and Koffler D: Avidity of anti-dna antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus: Association of high avidity antinative DNA antibody with glomerulonephritis. J Clin Invest 977; 59: Lange K, Wasserman E and Slobody LB: Significance of serum complement levels for diagnosis and prognosis of acute and subacute glomerulonephritis and lupus erythematosus disseminatus. Ann Intern Med 960; 53: Sullivan KE, Wisnieski JJ, Winkelstein JA, et al: Serum complement determinations in patients with quiescent systemic lupus erythematosus. J Rheumatol 996; 23: Swaak AJG, Aarden LA, Statious van Eps LW, et al: Anti-ds DNA and complement profiles as prognostic guides in systemic lupus erythematosus. Arthritis Rheum 979; 22: J Biomed Lab Sci 2006 Vol 8 No ~4 27

6 Autoantibody and renal involvement in SLE patients 紅斑性狼瘡病人腎臟侵犯與否與自體抗體間之相關性 桂秀平 施勇綸 李進成 莊志誠 2 新光吳火獅紀念醫院 病理檢驗科 2 過敏免疫風濕科 狼瘡腎炎通常與抗雙股去氧核醣核苷酸抗體有關 此篇研究的目的, 即在於探討國人紅斑性狼瘡病例中, 這些自體抗體與腎臟侵犯的相關性 我們回溯性研究了 999 年到 2002 年間, 於本院過敏免疫風濕科接受治療的 57 位紅斑性狼瘡病人的病歷資料 根據持續性蛋白尿出現與否來判定紅斑性狼瘡病人是否有腎臟侵犯, 以回溯性呈現診斷時的抗體 具腎臟侵犯的紅斑性狼瘡病人占 35%, 有腎臟侵犯的病人比沒有腎臟侵犯的病人較常出現抗 Ro 抗體 (6/20 比 9/37,p=0.034) 或抗牛心脂素抗體 (5/20 比 3/37,p=0.004) 另外, 有腎臟侵犯的病人具有較低的補體 (C3) 值占 65% 腎臟侵犯與否和抗雙股去氧核醣核苷酸 抗 La 抗體 抗 Sm 抗體及抗 nrnp 抗體較無相關, 腎臟侵犯的紅斑性狼瘡病人與抗 Ro 抗體或抗牛心脂素抗體具有意義的相關 關鍵詞 : 自體抗體 腎臟侵犯及紅斑性狼瘡 接稿日期 :95 年 6 月 日修稿日期 :95 年 0 月 3 日接受日期 :95 年 月 20 日通訊作者 : 李進成新光醫院病理檢驗科台北市士林區文昌路 95 號電話 :(02) 轉 220 傳真 :(02) M002058@ms.skh.org.tw 28 J Biomed Lab Sci 2006 Vol 8 No~4