Epstein Barr Virus in Patients with Nephropathy ...

THE EGYPTIAN JOURNAL OF IMMUNOLOGY

Vol. 25 (1), 2018 Page: 01-08

Epstein Barr Virus in Patients with Nephropathy Associated with Systemic Lupus Erythematous, Pilot Study in Egyptian Patients Maysaa E Zaki1, Noha T Abou El-Khier2, Nashwa M. Al-Kasaby2, Mostafa Abdelsalam3, Mohammed K Nassar3, Ahmed M Abdelwahab3 Departments of 1Clinical Pathology, 2Medical Microbiology & Immunology and 3 Nephrology Unit, Faculty of Medicine, Mansoura University, Mansoura, Egypt. Systemic lupus erythematosus (SLE) is an autoimmune disease affecting young age adults especially females. Infection with Epstein Barr virus (EBV) represents a common pathogen associated with SLE activity. This study investigates the occurrence of EBV in SLE patients with renal complications by serological markers and molecular detection of EBV genome in renal biopsies and examine the association of EBV with the pathological grades in renal diseases. The study included nineteen patients with systemic lupus nephropathy and thirteen patients with non-lupus nephropathy. Renal biopsies were subjected to detection of EBV by PCR. Serum autoantibodies (anti- dsDNA, anti-Sm and anti-RNP) and EBV-IgM and IgG antibodies were detected by ELISA. The commonest autoantibody was anti- dsDNA (73.7%) followed by anti-Sm (57.8%) and anti-RNP (31.6%). The EBV-PCR revealed that 31.6% of patients with lupus nephropathy showed positive LMP1 gene expression in renal biopsies On the other hand, serological markers for EBV showed no significant difference between both groups; IgM for EBV was positive in 26.3% of patients with lupus nephropathy and 7.7% in non-lupus nephropathy, while IgG was positive in 26.3% and 15.4 % respectively. Positive LMPI-PCR was demonstrated in all (3/3) patients with severe degree of nephropathy as compared to 23.1% of patients with moderate degree of nephropathy. A significant association was found between EBV-PCR and anti-Sm, (P=0.01), anti- dsDNA (P=0.001), and IgG for EBV and anti- dsDNA (P=0.03). In conclusion, Molecular detection of EBV DNA in renal biopsies can be applied for laboratory diagnosis in SLE nephropathy. The severity of nephropathy associated with SLE seems to be aggravated by the presence of EBV. Further extended studies are required to elucidate this association.

S

ystemic lupus erythematosus (SLE) is an autoimmune disease that affects mainly women in the young age with incidence up to 35 cases per 100.000 per year [1-3]. The etiology and pathogenesis of SLE are attributed to many factors such as genetic and hormonal susceptibility with triggering factors like viral infections and ultraviolet light exposure [4]. Viral infections are claimed to induce autoantibodies production [5, 6]. Among the common viruses associated with SLE, Epstein Barr virus (EBV), a member of herpes virus family known as human herpesvirus 4. It is composed of a 172 kb

linear dsDNA genome inside an envelopeenclosed icosahedral capsid [7]. EBV infection is common viral infectious pathogen affecting mainly 95% of the world population with a latent infection [8]. EBV is transmitted through respiratory tract and replicates at mucosal surfaces in nasopharyngeal epithelial cells then the virus infects B lymphocytes through the binding of envelope glycoprotein 350 to the B-cell type 2 complement receptor (CD21) [9,10]. The association of EBV with SLE has been continuously investigated with considerable evidence suggesting involvement of EBV with the pathogenesis

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EBV in Patients with Nephropathy Associated with SLE, Pilot Study in Egyptian Patients

of SLE. A link between Epstein-Barr virus (EBV) and lupus has been suggested through serologic, molecular, and experimental studies [8, 11-13]. The most dramatic complication of SLE is the affection of kidneys with subsequent renal failure the condition collectively known as lupus nephritis (LN) [14]. There are various reports about the involvement of EBV with the pathogenesis of LN [8, 11-14]. However, to best of our knowledge, there are no reports about this condition in Egypt, nor the study of such association with polymerase chain reaction for EBV in renal biopsies. Our aim was to study the occurrence of EBV in SLE patients with renal complications by serological markers and by detection of EBV genome in renal biopsies by molecular method and to correlate the association of EBV with the pathological grades in renal diseases.

Material and Method This cross-sectional study was carried out in Mansoura University hospital, Egypt. The study comprised nineteen patients with lupus nephropathy and thirteen patients with non-lupus nephropathy. The study was performed according to the principles of Declaration of Helsinki, and the procedures were approved by the local institutional review board (IRB). Written informed consent were obtained from all participants. Patients with SLE met the diagnostic criteria for SLE published by the American College of Rheumatology in 1997 [15]. The diagnosis and classification of stages nephritis was confirmed by histopathology examination of renal biopsies and the nephritis was graded according to the criteria of International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification [16]. Patients more than 18 years with systemic lupus associated nephritis, and patients with nephritis not associated with SLE fully diagnosed by renal biopsies were enrolled in this study.

Patients with morbidities such as malignancies and liver diseases were excluded from the study. Participants were subjected to thorough history taking and clinical examination. Renal biopsy from each participant was divided in two aliquots one in sterile tube for molecular detection of EBV by PCR that was kept frozen at -200C and the other sample in formalin for histopathological examination. Blood samples were collected for serological determination of anti- dsDNA antibodies by enzyme linked immuosorbant assay (ELISA) (DIASTAT, Malmö SWEDEN), anti-Sm, anti-RNP by ELISA (EUROIMMUN AG, Luebeck, Germany), EBV specific immunoglobulin M (IgM) and immunoglobulin G (IgG) for viral capsid antigens by Euro immune enzyme linked immunosorbant assay (ELISA- EUROIMMUN AG, Luebeck, Germany). Polymerase Chain Reaction for EBV in Renal Biopsies 

DNA Extraction

DNA was extracted from renal biopsies. DNA was digested with RNAase (10 mg/ml) and protein K enzymes (1 mg/ml) for 16 hours at 37°C [18] followed by extraction by the use of QIAamp® DNA Mini kits (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions [17]. 

Amplification for EBV DNA

The amplification of extracted DNA was performed by the use of ready to use amplification kit End Point PCR kit (V-9-100R, Sacace Biotechnologies Srl, Italy), that uses specific primers for amplification of the latent membrane protein 1 (LMP1) coding region and visualization of the amplified products by agarose gel electrophoresis. The kit uses human Betaglobin gene as internal control primer. The amplification mixtures were prepared by adding of 10 μL of extracted DNA over 30 μL ready to use amplification tubes. For negative control amplification tubes 10 μL of buffer is added and for positive control tubes 10 μL of ready to use positive control is added. Amplification programs used was as the following 95°С for 5 min, 42 cycles (95°С 10 sec 65°С 10 sec 72°С 10 sec) followed by one minute at 72°С. The amplified product was analyzed by gel electrophoresis at 2% for 30 minutes. Positive control and positive samples have two bands at 500bp for EBV and at 723bp for detection of the amplified


product of human β-globin gene. Negative control and negative samples have only one band at 723bp. Statistical Analysis Data was analyzed using Statistical Package for Social Science software computer program version 22 (SPSS, Inc., Chicago, IL, USA). Quantitative parametric data were presented in mean and standard deviation. Inter-group comparison of categorical data was performed by using chi square test (X2-value). P value less than 0.05 was considered stastistically significant.

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Results The present study comprised 32 patients; 19 patients with nephropathy associated with SLE (lupus nephropathy), and 13 patients with nephropathy not associated with SLE (non-lupus nephropathy). The patients with lupus nephropathy were significantly (P=0.003) younger in age compared to patients with non-lupus nephropathy (mean ±SD 27.3± 9.7 & 43.5 ±18.6, respectively), table (1).

Table 1. Demographic data of the studied patients. Patients with Lupus Nephropathy

Patients with non- lupus Nephropathy

(N=19)

(N=13)

27.3±9.7

43.5± 18.6

0.003

Male

5 (36.8%)

7 (53.8%)

NS

Female

14(73.7%)

6 (46.2%)

Age

P value

Gender

P >0.05 is not significant (NS).

The majority of patients with lupus nephropathy had moderate stages [3,4] of nephropathy while patients with non-lupus

nephropathy had mild stages [1, 2], (64.4%, 84.6% respectively, P=0.001), table (2).

Table 2. Classification of stages of nephropathy among studied patients. Mild stage

Moderate stage

Severe stage

(1,2)

(3,4)

(5,6)

Patients with lupus nephropathy (N=19)

3(15.8%)

13(64.4%)

3(15.8%)

Patients with non- lupusNephropathy (N=13)

11(84.6%)

0(0%)

2(15.4%)

P value=0.001 between different stages of nephropathy by Chi-Square test

The distribution of autoantibodies among the studied patients revealed that patients with non-lupus nephropathy had no autoantibodies. The majority of patients with

lupus nephropathy were positive for antidsDNA (73.7%) followed by anti-Sm (57.8%) and anti-RNP (31.6%), table (3).

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Table 3. Distribution of autoantibodies among studied patients Patients with Lupus Nephropathy

Patients with non- lupus Nephropathy

(N=19)

(N=13)

anti-Sm

11(57.8%)

0(0%)

anti-RNP

6(31.6%)

0(0%)

anti- dsDNA

14(73.7%)

0(0%)

The study of EBV markers in the patients revealed that 31.6% of patients with lupus nephropathy had positive PCR for LMP1 gene in renal biopsies and patients with non lupus nephropathy were all negative by PCR for EBV LMP1 gene. On the other hand, serological markers for EBV had no

significant difference between both groups; IgM for EBV was positive in 26.3% of patients with lupus nephropathy and in 7.7% in non-lupus nephropathy and IgG was positive in 26.3% of lupus nephropathy patients and in 15.4% of non-lupus nephropathy patients, table (4).

Table 4. Distribution of EBV markers among the studied patients.

Patients with lupus nephropathy n=19

PCR for EBV gene

IgM

IgG

6(31.6%)

5(26.3%)

5(26.3%)

0

1(7.7%)

2(15.4%)

0.03

NS

NS

Patients with non-lupus Nephropathy n=13 P value P >0.05 is not significant (NS).

The positive PCR for LMP1 gene were mainly among patients with severe degree of nephropathy; all patients with severe nephropathy (3 patients) had positive PCR and 23.1% patients with moderate degree of nephropathy were positive for EBV LMP1 gene, table (5). On the other hand, the

presence of EBV virus DNA in renal biopsies detected by PCR was significantly associated with anti-Sm, (P=0.01), and antidsDNA (P=0.001). In addition, IgG for EBV was significantly associated with antidsDNA (P=0.03), table (6).

Table 5. Distribution of Positive PCR for LMP1 gene among stages of nephropathy in patients with lupus nephropathy stage of nephropathy

Positive PCR for EBV

Mild (stages 1, 2)

Moderate (stages 3,4)

Severe (stages 5-6)

n=3

n=13

n=3

0

3 (23.1%)

3 (100%)


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Table 6. Association of autoantibodies with EB markers among the studied patients. PCR

IgG

IgM

Positive

Negative

OR

Positive

Negative

OR

Positive

Negative

OR

-Positive

6

5

0-0.1

4

1

0.010.24

1

10

0.30.70

-Negative

0

21

*P=0.01

7

20

5

16

-Positive

3

3

0.5-0.3

2

3

0.2-0.5

1

5

0.9-1.0

-Negative

3

23

P=0.2

8

23

P=0.4

5

21

P=0.7

-Positive

6

8

0-0.1

5

9

0.0-.01

4

10

0.3-0.6

-Negative

0

18

*P=001

0

18

P=0.03*

2

16

P=0.4

anti-Sm

P=0.1

P=0.5

anti-RNP

antidsDNA

P >0.05 is not significant (NS).

Discussion Various environmental factors can trigger the development of SLE. Among these factors, EBV plays a crucial role. Several reports established correlation between EBV infection and SLE [7,8,11,18]. Among EBV antigens, LMP1 association with diseases was described in nasopharyngeal carcinoma [19], and in nephropathy associated with SLE [20]. However, to best of our knowledge, there are no reports about this topic among Egyptian patients. In the present study, 31.6% of patients with lupus nephropathy had positive PCR for LMP1 gene in renal biopsies, EBV IgM was positive in 26.3% and IgG in 26.3% of patients with lupus nephropathy. The result was statistically significant for LMP1 gene by PCR in the patients with lupus nephropathy compared to patients with nonlupus nephropathy.

Previous studies reported higher positive rates 58.7% [14] and 78.4% [20]. The difference can be explained by the difference in age and number of the studied patients. Moreover, the difference of the used technique to diagnose EBV associated nephropathy. In other reports, they used immunohistochemical stains while in this work we used a more specific detection method for EBV DNA by PCR in renal biopsies. In agreement with other studies [14, 20] positive PCR for LMP1 gene was mainly among patients with severe degree of nephropathy. In our study, all patients with severe nephropathy (3 patients) had positive PCR for LMP1 gene and two patients with moderate degree of nephropathy (15.4%) were positive for EBV DNA. EBV infection is likely to be a promoting factor in the development of higher grade of lupus nephropathy. More work is required to further determine this correlation.

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Serological markers for EBV had no significant difference between both groups; IgM for EBV was positive in 26.3% of patients with lupus nephropathy and in 7.7% in non-lupus nephropathy and IgG was positive in 26.3% of patients with lupus nephropathy and in 15.4% of non-lupus patients. The comparison of serological markers for EBV infection in adults yield usually non significant difference as the majority of adults have positive markers for EBV[13,22,23]. In the present study of autoantibodies among lupus nephropathy patients, the majority had positive anti- dsDNA (73.7%) followed by anti-Sm (57.8%) and anti-RNP (31.6%). The positive rates for anti-Sm in previous studies had ranges from 35.3% up to 62.5% [20, 21]. The positive rates of autoantibodies in SLE depends upon several factors among which is the age of the patients. Previous reports had explained the association of anti-Sm autoantibodies in SLE patients with EBV infection as a crossreaction with a peptide from EBV nuclear antigen 1 [24-26]. Supporting the hypothesis of molecular mimicry mechanism that leads to the development of anti-Sm and anti-RNP autoantibodies in SLE patients [26,27]. The interesting finding of the present study was the significant association of LMP1 gene by PCR in renal biopsies and the presence of anti-Sm, (P=0.01), and antidsDNA (P=0.001). Also, IgG for EBV was significantly associated with anti- dsDNA (P=0.03), Similar results were reported by Ding et al. [20]. It is hypothesized that LMP1 is another triggering gene for development of SLE [28,29]. The aberrant production of autoantibodies in SLE can target various nuclear antigens leading to the development of lupus

nephropathy [30,31]. Among various autoantibodies, Anti-Sm antibody had the most significant association with renal involvement [32]. The present study had some limitations. First, low number of included patients. More patients should be included in expanded longitudinal study. Second, association between various techniques in detecting of EBV by immunohistochemical stain and by PCR in renal biopsies can aid in detecting patients infected with EBV as serological markers have limited value in diagnosis of EBV in adults. However, the results of the present study is promising in detecting of EBV LMP1 gene in renal biopsies by PCR. From this study, we can conclude that EBV is a common pathogen associated with lupus nephropathy. Molecular detection of EBV DNA in renal biopsies can be applied for laboratory diagnosis in adults as serological markers are of limited value. The severity of nephropathy associated with SLE seems to be aggravated by the presence of EBV. Further extended studies are required to elucidate this association.

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