Saudi Journal of Kidney Diseases and Transplantation

RENAL DATA FROM THE ARAB WORLD
Year
: 2013  |  Volume : 24  |  Issue : 6  |  Page : 1271--1279

Immunological aspects of biopsy-proven lupus nephritis in Bahraini patients with systemic lupus erythematosus


Eman M Farid1, Adla B Hassan2, Ali A Abalkhail3, Amgad E El-Agroudy4, Sameer Al-M Arrayed4, Sumaya M Al-Ghareeb4,  
1 Department of Pathology (Immunology) Salmaniya Medical Complex, Manama, Kingdom of Bahrain
2 Department of Internal Medicine (Rheumatology), College of Medicine, Arabian Gulf University, Salmaniya Medical Complex, Manama, Kingdom of Bahrain
3 Department of Pathology, College of Medicine, Arabian Gulf University, Salmaniya Medical Complex, Manama, Kingdom of Bahrain
4 Department of Nephrology, Salmaniya Medical Complex, Manama, Kingdom of Bahrain

Correspondence Address:
Eman M Farid
Department of Pathology, College of Medicine, Arabian Gulf University, Salmaniya Medical Complex, P. O. Box 12, Manama
Kingdom of Bahrain

Abstract

Lupus nephritis (LN) is a frequent and potentially serious complication of systemic lupus erythematosus (SLE) that may influence morbidity and mortality. Immunological investigations are aiding tools to the kidney biopsy findings in early diagnosis, in addition to monitoring the effect of therapy. The aim of the present study is to highlight the role of these investigations in a group of Bahraini patients and to determine whether there is any positive association between these findings and the outcome of LN. The current study is a retrospective case-control study of randomly selected 88 SLE patients, 44 with biopsyproven LN and 44 without, acting as controls. All renal biopsies performed during the period from 1996 to 2012 were classified according to the World Health Organization classification. Immunological investigations analyzed are: Antinuclear antibodies (ANA), anti-ds DNA, anti-ENA, anti-cardiolipin antibodies (abs) and complement components C3, C4. Human leukocyte antigen (HLA) typing class II was performed on selected cases. All patients had positive ANA (100%). A significantly high frequency of anti-Smith abs among the non-LN group (43.18%) compared with the LN group (18.18%) was found (P <0.001). On the other hand, the anti-Ro/SSA abs in the non-LN group was also found at a statistically higher frequency (20.45%) compared with that in the LN group (4.54%) (P <0.01). Anti-ds-DNA abs were found to be higher in the LN group (84.09%) compared with the non-LN group (70.45%), but the difference was not statistically significant (P = 0.082). There was a positive association of ANA positivity and low C3 and or C4 in the studied group. In our study, 88.2% of the HLA typed patients had HLADR2, DR3 or both. In conclusion, in our Arabic Bahraini SLE patients, the presence of anti-Smith, anti-Ro/SSA and anti-RNP antibodies and the absence of anti-dsDNA antibodies are independent predictive markers for renal involvement. However, more prospective studies with a larger number of patients are essential to ascertain those findings.



How to cite this article:
Farid EM, Hassan AB, Abalkhail AA, El-Agroudy AE, Arrayed SA, Al-Ghareeb SM. Immunological aspects of biopsy-proven lupus nephritis in Bahraini patients with systemic lupus erythematosus.Saudi J Kidney Dis Transpl 2013;24:1271-1279


How to cite this URL:
Farid EM, Hassan AB, Abalkhail AA, El-Agroudy AE, Arrayed SA, Al-Ghareeb SM. Immunological aspects of biopsy-proven lupus nephritis in Bahraini patients with systemic lupus erythematosus. Saudi J Kidney Dis Transpl [serial online] 2013 [cited 2019 Sep 21 ];24:1271-1279
Available from: http://www.sjkdt.org/text.asp?2013/24/6/1271/121286


Full Text

 Introduction



Lupus nephritis (LN) is a frequent and potentially serious complication of systemic lupus erythematosus (SLE) that may influence both morbidity and mortality directly and indirectly. Immunological investigations, namely anti-nuclear antibodies (ANA), anti-double-stranded-DNA (anti-ds DNA) antibodies, anti-Smith (anti-Sm) antibodies and complement components (C3 and C4) are aiding tools to the kidney biopsy findings in early diagnosis. Moreover, these antibodies are also useful in monitoring the effect of therapy in LN. Renal disease occurs in 40-75% of patients, with a mean of 50% in patients with SLE, some time in the course of their illness, most often within 5 years of the onset of disease. [1] On the other hand, 25- 50% of SLE patients will have kidney involvement at the time of presentation. [1] The pathogenic role of anti-dsDNA antibodies in LN was established long ago, and it was indicated by in situ formation of immune complexes at the renal level. [2],[3],[4] Extractable nuclear antigens (ENA) such as anti-Ro/SSA, anti-La/SSB, anti-Sm, anti-RNP and anti-phospholipid antibodies may also contribute to the pathogenesis of LN, but the evidence is still controversial. [5]

Autoantibodies and ethnic and demographic factors have controversial effects on the development of renal disease. [6],[7] The presentation is highly variable, ranging from mild asymptomatic proteinuria to rapidly progressive glomerulonephritis with hematuria and red cell casts. In order to provide a real and effective high-standard care to patients with LN, the clinician must be aware of the diverse clinical and pathological manifestations of the same, especially in the early stages of the disease when prompt and optimal management is needed to prevent irreversible damage. The optimal management in patients with LN includes early diagnosis and appropriate therapy. Early diagnosis of LN could be achieved only by renal biopsy, which is essential in establishing diagnosis and prognosis, and guiding the management.

Autoantibodies and autoantibody-producing B cells are crucial for the initiation of LN. Their precise role in the development of the nephritic lesions is incompletely understood. However, an earlier study has reported that autoantibody-producing B cells influence the activation of autoreactive T cells that infiltrate the kidney to produce vasculitis and interstitial nephritis. [8]

Another study revealed that among a wide range of anti-ribosomal P antibody concentrations, the mutual occurrence of anti-P antibodies with anti-dsDNA antibodies was strongly associated with LN. [9] The presence of LN has been found to be uncommon in patients with both anti-Ro/SSA and anti-La/SSB antibodies as well as in those with anti-La/SSB antibodies alone. [10] In a longitudinal study, the Ro/SSA and La/SSB antibody profile was very high and fixed at an early stage of SLE disease, including renal disease, and in most patients this hardly changes over time, which could indicate its implication of disease and renal development. [11]

The strongest SLE associations with major histocompatibility complex (MHC) are those with human leukocyte antigen (HLA) class II genes. The HLA-D is the susceptibility gene to end organ damage in SLE. [12] The associations of SLE with HLA are inconsistent in different ethnic groups. On the other hand, reports about the associations between HLA and antinuclear antibodies in patients with SLE have been consistent across ethnic groups. These include the association between HLA-DR3 with anti-Ro and anti-La antibodies, HLA-DR2 with anti-Sm antibodies and HLA-DR2 with anti-dsDNA antibodies. [13],[14],[15] Another study in Jamaican patients with SLE has demonstrated that the HLA-DR6 allele is associated with the absence of ANAs and HLA-DR3 with the presence of anti-Ro/La antibodies. [16]

The purpose of the current study was to evaluate the presence or absence of any association with the development of nephritis within SLE patients regarding immunological, serological and demographic data obtained at the time of the first kidney biopsy.

 Patients and Methods



This was a retrospective case-control study performed on 88 randomly selected patients with SLE who were being followed-up at the Nephrology and Rheumatology Outpatient clinics of Salmaniya Medical Complex, Kingdom of Bahrain, a governmental hospital. Of these, 44 patients were with biopsy-proven LN and 44 SLE patients without LN served as controls. All LN patients had renal biopsy, which proved the diagnosis during the period from 1996 to 2012. The age of the patients ranged from 15 to 50 years (mean = 30.1 years) in the LN group while it was 13-52 years (mean = 32.5 years) in the non-LN group. The female to male ratio was 35:5 (7:1) in the LN group and 43:1 in the non-LN group.

The immunological investigations analyzed were ANA, anti-ds DNA, anti-ENA (anti-Ro/ SSA, La/SSB, Smith, anti-RNP, anti-histones), complement components C3 C4 and anti-cardiolipin antibodies. HLA typing class II was performed on selected cases (24 patients).

ANAs were detected in human sera by an indirect immunofluorescence test - HEp-2 cells by an immunofluorescence assay (IFA) (Bio-Rad, Hercules, CA, USA). Details of the method are described elsewhere. [17] Anti-ds-DNA antibodies were detected by an automated enzyme fluoroimmunoassay, the EliA system (Phadia AB, Uppsala, Sweden). Serum complement components C3 and C4 and rheumatoid factor (RF) were measured by the nephlometry method using a BN Systems nephlometer (Siemens, Munich, Germany). Details of the method are described elsewhere. [18] Serum anti-cardiolipin antibodies were detected by an enzyme immunoassay (EIA) for IgG and IgM antibodies using anti-cardiolipin Varelisa kits (Pharmacia Diagnostics, Freiburg, Germany). Details of the method are described elsewhere. [19]

Anti-ENA (anti-SSA, SSB, Smith, anti-RNP, anti-histones) was performed by a line immunoassay INNO-LIA™ ANA from Immunogenetics company, New Jersey, USA. Details of the method are described elsewhere. [20] HLA class II was performed serologically by the microcytotoxicity technique using lymphobeads (Biotest Co., Dreieich, Germany) and fluorescence stains using Terasaki plates (Biotest Co, Dreieich, Germany). [21]

 Statistical Analysis



Associations between demographic and immunological profiles and the presence or absence of nephritis were analyzed by the tf test. To verify whether an association existed between the different immunological profiles, Fisher's exact test was used. The results were expressed as percentages and as .P-value. A .P-value of <0.05 (two tailed) was considered significant. All analyses were performed with the SPSS statistical software.

 Results



Frequency of autoantibodies

All results are shown in [Table 1]. Within all our Bahraini SLE patients (n = 88), (six male and 82 female), we had two groups of patients; 44 SLE patients with biopsy-proven LN (LN group) and 44 SLE patients without LN (non-LN group). A comparison was made between the presences of autoantibodies in both groups.{Table 1}

All SLE patients, both LN and non-LN, had positive ANA (100%). ENA was found to be high in the non-LN group (>50%) compared with the LN group (<35%), but this did not reach statistical significance. In our current study, we found a high frequency of anti-Sm abs among our non-LN group patients (43.18%) compared with the LN group patients (18.18%), and this was statistically significant(P <0.001). On the other hand, the anti-Ro/SSA abs in our non-LN group patients was also found at a high frequency 9/44 (20.45%) compared with only 2/44 (4.54%) in the LN group, and this reached statistical significance (P <0.01). In contrast to anti-Sm abs, the frequency of anti-ds-DNA abs was found to be higher in the LN group [37/44 (84.09%)] compared with the non-LN group [31/44 (70.45%)], but the difference was mild and not statistically significant.

The serum levels of C3 and C4 were equally low in both patient groups: 37/44 (84.09%) for each complement in the LN group and 42/44 (95.45%) in the non-LN group for each of C3 and C4.

Anti-phospholipid (APL) antibody was found equally 7/44 (15.91%) in both the LN and non-LN patients, respectively. All the seven non-LN patients were females with anti-ds-DNA antibodies positive except one patient, who was negative for ds-DNA, but positive for anti-Sm antibodies. While in LN patients, six were females and one was male out of a total of five males in this LN group (5/44). Moreover, all patients were anti-ds-DNA positive except one patient who was negative for ds-DNA and positive for anti-His, anti-Ro and anti-La antibodies, as displayed in [Table 2]. In LN group with positive APL antibodies, end-stage renal disease (ESRD) has been demonstrated in four patients of ten patients (40%) in the study, as depicted in [Table 3].{Table 2}{Table 3}

The other investigated antibodies were found in a few patients in both groups. Anti-La/SSB and anti-Histone antibodies were equally found in LN patients 2/44 (4.55%), but in 5/44 (11.36%) in the non-LN group for each antibody. Concerning the anti-RNP antibody levels, these were not different in both groups as illustrated in [Table 1].

Association between different autoantibodies The associations between different auto-antibodies are demonstrated in [Table 3]. There is a negative association between positive ds-DNA (68.18%) and positive anti-Sm antibodies (14.77%) (P <0.001), i.e. those who were positive for one antibody were negative for the other. Only seven patients were positive for both antibodies together, and six of these patients were in the non-LN group.

Among all SLE patients, there is a strong positive association between anti-Sm and anti-Ro/SSA antibodies (P <0.01), i.e. the patients are either negative for both antibodies (64/88) or positive for both (6/88). However, those six patients who were positive for both antibodies were in the non-LN group. In the non-LN patients, there were only nine patients (9/44) who were positive for Ro/SSA antibodies. However, six of those nine patients were also positive for anti-Sm antibodies, while there was no patient in the LN group who was positive for both anti-Sm and anti-Ro/SSA. Moreover, in the LN group, the only two patients who were positive for anti-Ro/SSA were negative for anti-Sm. Thus,there is an association between the presence of both anti-Ro/SSA and anti-Sm antibodies in our non-LN group, but this did not reach statistical significance (P = 0.062).

There is also an association between anti-Sm (30.6%) and anti-RNP (11%) antibodies in the whole group (P <0.01). There were 67/88 patients who were negative for both antibodies, and only 5/88 patients were positive for both antibodies. However, those five patients were all in the non-LN group, and none of the patients in the LN group was positive for both antibodies.

HLA typing

In our current retrospective study, the total number of patients for whom HLA typing was performed was only 24 patients (17 in the LN group and seven in the non-LN group), as shown in [Table 4]. HLA DR2 was found in nine LN patients (52.9%) and in four non-LN patients (57.1%). HLA DR3 was found in 11 LN patients (64.7%) and in five non-LN patients (71.4%). HLA DR2 and DR3 were found in six patients with LN (35%) and in two non-LN patients (28.6%). HLA DR4 was found in three patients with LN (17.6%) and in two non-LN patients (28.6%). HLA DR7 was found in four patients with LN (23.5 %), while none of the seven patients without LN has this HLA type.{Table 4}

Our LN patients were diagnosed as having LN based on the results of their biopsies and were classified histologically into different classes according to the World Health Organization classification. [24] The results of our biopsy-proven LN patients are shown in [Table 5]. [Table 5] also demonstrates the clinico-pathological outcome with long-term follow-up of the patients. Deaths were considered by the presence of death certificates in the medical record or as the last time the patient was seen at the hospital. The HLA frequency versus LN biopsy/WHO classification and autoantibodies, age and the living/expired status are summarized in [Table 6].{Table 5}{Table 6}

 Discussion



In the Kingdom of Bahrain, secondary glome rular disease comprised 33.6% of all diseases, of which LN was the most common lesion (38.9%). [25] Immunological, serological and demographic data were obtained at the time of the first kidney biopsy; thus, the results would not be affected by the therapy.

Anti-dsDNA antibodies are reported to be more prevalent in patients with SLE who have renal disease. Moreover, earlier studies have shown evidence supporting a pathogenic role for DNA - anti-DNA immune complexes in LN. [26],[27] In our current study, the prevalence of anti-dsDNA antibodies is consistent with previous reports, although the frequency of anti-ds-DNA abs was noted to be higher in the LN group compared with the non-LN group, but the difference was mild and not statistically significant.

Alba et al have reported that young, Black patients with anti-dsDNA, anti-Sm antibodies and positive LA appear to have a higher risk of renal involvement. [28] On the contrary, in our current Arabic Bahraini population, the frequency of anti-Sm was significantly higher in the non-LN group compared with the LN group. The question that may be asked is whether its presence was protective in nature and absence correspondingly indicates an increased risk for renal involvement. The same is true for anti-Ro/SSA antibodies, which is not only found in a high frequency in our non-LN group but also in strong association with anti-Sm antibodies. Therefore, in our current Arabic Bahraini population, the presence of both anti-Sm and anti-Ro/SSA antibodies seemed to be probably protective in nature. Similarly, in our cohort, statistically significant association between the anti-Sm and anti-RNP antibodies was found and, furthermore, the patients who proved to be positive for anti-Sm and anti-RNP antibodies were all in the non-LN group. Thus, the presence of anti-RNP antibodies also seemed to be a protective marker for renal involvement. Our results have been supported by the study done by Al Attia et al, who could not demonstrate any positive association between anti-Ro/SSA, anti-Sm, anti-RNP and anti-ds-DNA antibodies and renal involvement in Arab patients with LN. [5] The occurrence of high ENA antibodies in our non-LN group could be due to the high frequency of anti-Sm, anti-Ro/SSA and anti-RNP antibodies in this group compared with the LN group.

Given that APL antibodies were equally detected in both LN and non-LN patients (15.91%), we cannot predict its association with renal involvement, as reported previously. [29] Nevertheless, based on the implication of APL antibodies in renal prognosis, we noted that in our present study 40% of those with ESRD were APL-positive patients; however, this observation needs to be clarified in a larger number of patients.

Although an earlier study has shown an increased prevalence of renal disease in male patients with SLE, [30] in the Bahraini population, females were more frequently affected than males with LN (75% vs. 25%). [25] Our current study also revealed the same evidence, as the ratio between females and males is 35:5 (7:1). HLA types were performed only for one-quarter (27%) of our cohort, although our results about HLA types are consistent with both the previous reports [22] as well as with a recent study [23] in the Bahraini population, as shown in [Table 3]. As seen in the table, the frequencies of all the above-mentioned HLA types are higher in Bahraini SLE patients compared with healthy controls.

In our study, 88.2% (15/17) of our HLA typed patients had HLADR2, DR3 or both. Regarding HLADR2 (DR15 and DR16), nine of our patients (52.9%) had this allele. Recently, Marchini et al found that the DR15 allele is associated with class IV. [31] This evaluation could not be made in our study due to the small sample size. We noticed that four (23.5%) of our HLA typed patients had HLADR7, an allele not known to be associated with SLE. Moreover, we observed that the patients having this allele had a quick deterioration, reaching class V ESRD very rapidly and thus raising the possibility that this allele may play a role in the severity of LN, an observation that needs to be studied in a larger group of patients.

In conclusion, in our opinion, among Arabic Bahraini SLE patients the presence of anti-Sm, anti-Ro/SSA and anti-RNP antibodies and absence of anti-dsDNA antibodies may be considered as independent markers which probably indicates lesser renal involvement. However, prospective and large studies are essential to ascertain the actual incidence and etiology of LN in Arabic Bahraini SLE patients.

References

1Walker RJ, Bailey RR, Swainson CP, Lynn KL. Lupus nephritis: A 13-year experience. N Z Med J 1986;99:894-6.
2Garin EH, Donnelly WH, Shulman ST, et al. The significance of serial measurements of serum complement C3 and C4 components and DNA binding capacity in patients with lupus nephritis. Clin Nephrol 1979;12:148-55.
3Asero R, Banfi G, Radelli L, et al. Relationship between antibodies to dsDNA and to soluble cellular antigens and histologically defined glomerulonephritis in patients with SLE. Auto-immunity 1990;7:13-21.
4Rajagopalan S, Zordan T, Tsokos GC, Datta SK. Pathogenic anti-DNA autoantibody-inducing T helper cell lines from patients with active lupus nephritis: Isolation of CD4-8- T helper cell lines that express the gamma delta T-cell antigen receptor. Proc Natl Acad Sci U S A 1990;87:7020-4.
5Al Attia HM. Lupus Nephritis among Arabs -Differences with other Races; Emphasis on clinicopathological and serological perspectives. Saudi J Kidney Dis Transpl 2000;11:370-80.
6Fiehn C, Hajjar Y, Mueller K, Waldherr R, Ho AD, Andrassy K. Improved clinical outcome of lupus nephritis during the past decade: Importance of early diagnosis and treatment. Ann Rheum Dis 2003;62:435-9.
7Bastian HM, Alarcon GS, Roseman JM, et al. Systemic lupus erythematosus in a multiethnic US cohort (LUMINA) XL II: Factors predictive of new or worsening proteinuria. Rheumatology (Oxford) 2007;46:683-9.
8Madaio MP. B cells and autoantibodies in the pathogenesis of lupus nephritis. Immunol Res 1998;17:123-32.
9Reichlin M, Wolfson-Reichlin M. Correlations of anti-dsDNA and anti-ribosomal P autoanti-bodies with lupus nephritis. Clin Immunol 2003;108:69-72.
10Wasicek CA, Reichlin M. Clinical and serological differences between systemic lupus ery-thematosus patients with antibodies to Ro versus patients with antibodies to Ro and La. J Clin Invest 1982;69:835-43.
11Hassan AB, Lundberg IE, Isenberg D, Wahren-Herlenius M. Serial analysis of Ro/SSA and La/SSB antibody levels and correlation with clinical disease activity in patients with systemic lupus erythematosus. Scand J Rheumatol 2002;31:133-9.
12Fu SM, Deshmukh US, Gaskin F. Pathogenesis of systemic lupus erythematosus revisited 2011: End organ resistance to damage, autoantibody initiation and diversification, and HLA-DR. J Autoimmun 2011;37:104-12.
13Alvarellos A, Ahearn JM, Provost TT, et al. Relationships of HLA-DR and MT antigens to autoantibody expression in systemic lupus erythematosus. Arthritis Rheum 1983;26:1533-5.
14Reveille JD, Schrohenloher RE, Acton RT, Barger BO. DNA analysis of HLA-DR and DQ genes in American blacks with systemic lupus erythematosus. Arthritis Rheum 1989;32:1243-51.
15Olsen ML, Arnett FC, Reveille JD. Contrasting molecular patterns of MHC class II alleles associated with the anti-Sm and anti-RNP precipitin autoantibodies in systemic lupus erythe-matosus. Arthritis Rheum 1993;36:94-104.
16Christian N, Smikle MF, DeCeulaer K, Daniels L, Walravens MJ, Barton EN. Antinuclear antibodies and HLA class II alleles in Jamaican patients with systemic lupus erythematosus. West Indian Med J 2007;56:130-3.
17van Venrooij WJ, Charles P, Maini RN. The consensus workshops for the detection of auto-antibodies to intracellular antigens in rheumatic diseases. J Immunol Methods 1991;140:181-9.
18Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol 2000;53:424-32.
19Harris EN. Special report. The Second International Anti-cardiolipin Standardization Workshop/ the Kingston Anti-Phospholipid Antibody Study (KAPS) group. Am J Clin Pathol 1990;94:476-84.
20Pottel H, Wiik A, Locht H, et al. Clinical optimization and multicenter validation of antigen-specific cut-off values on the INNO-LIA ANA update for the detection of autoantibodies in connective tissue disorders. Clin Exp Rheumatol 2004;22:579-88.
21Terasaki PI, Cho Y, Takemoto S, Cecka M, Gjertson D. Twenty-year follow-up on the effect of HLA matching on kidney transplant survival and prediction of future twenty-year survival. Transplant Proc 1996;28:1144-5.
22Eman Farid RA. Immunological disorders and serological characteristics in SLE: Correlation with clinical findings. Egypt J Microbiol 2000; 9:669-74.
23Farid E. Distribution of clinically important HLA alleles among healthy Bahraini population, In Press.
24Weening JJ, D'Agati VD, Schwartz MM, et al. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 2004;65:521-30.
25Al Arrayed A, George SM, Malik AK, et al. Renal biopsy findings in the kingdom of bahrain: A 13-year retrospective study. Saudi J Kidney Dis Transpl 2004;15:503-7.
26Dang H, Harbeck RJ. The in vivo and in vitro glomerular deposition of isolated anti-double-stranded-DNA antibodies in NZB/W mice. Clin Immunol Immunopathol 1984;30:265-78.
27Koffler D, Schur PH, Kunkel HG. Immunological studies concerning the nephritis of systemic lupus erythematosus. J Exp Med 1967; 126:607-24.
28Alba P, Bento L, Cuadrado MJ, et al. Anti-dsDNA, anti-Sm antibodies, and the lupus anti-coagulant: Significant factors associated with lupus nephritis. Ann Rheum Dis 2003;62:556-60.
29Tektonidou MG, Sotsiou F, Nakopoulou L, Vlachoyiannopoulos PG, Moutsopoulos HM. Antiphospholipid syndrome nephropathy in patients with systemic lupus erythematosus and antiphospholipid antibodies: Prevalence, clinical associations, and long-term outcome. Arthritis Rheum 2004;50:2569-79.
30Blum A, Rubinow A, Galun E. Predominance of renal involvement in male patients with systemic lupus erythematosus. Clin Exp Rheumatol 1991;9:206-7.
31Marchini M, Antonioli R, Lleo A, et al. HLA class II antigens associated with lupus nephritis in Italian SLE patients. Hum Immunol 2003; 64:462-8.