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Erythrocyte and glomerular C4d deposits as a biomarker for active lupus nephritis
The Egyptian Rheumatologist xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
The Egyptian Rheumatologist journal homepage: www.elsevier.com/locate/erhe
Original Article
Erythrocyte and glomerular C4d deposits as a biomarker for active lupus nephritis Hanan Abd El Halima, Mona Salahb, Wesam Ismailc, Ahmed Fathya, Hala Ramadana, a b c
⁎
Internal Medicine Department, Faculty of Medicine, Cairo University, Cairo, Egypt Clinical Pathology Department, Faculty of Medicine, Cairo University, Cairo, Egypt Pathology Department, Faculty of Medicine, Bani Sweif University, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: SLE C4d LN Renal biopsy SLEDAI
Introduction: Lupus nephritis (LN) is a serious manifestation of systemic lupus erythematosus (SLE) and histologically evident even in those without clinical manifestations of renal disease. Aim of the work: To assess C4d on erythrocytes (E-C4d) and glomerular deposits (G-C4d) in SLE patients and study its association to LN and disease activity. Patients and methods: 61 subjects were enrolled including 15 with LN (study group); 15 with renal disease not due to SLE (control A group); 16 SLE patients with no renal affection (control B group) and 15 healthy individuals (control C group). Flow cytometry system was used for C4d immunohistochemical staining. SLE disease activity index (SLEDAI) was assessed for SLE patients. Results: The age was comparable among groups; for LN patients was 28.3 ± 8.2 years; group A (35.9 ± 13.3); group B (27.1 ± 8.8) and group C (29.4 ± 7.1) (p = .06). Patients were mostly females. The disease duration of LN patients was 1–2 years; group A (3–5 years) and group B (5–10 years). E-C4d and G-C4d deposits were significantly higher in LN patients (8.08 ± 2.93 and 2.3 ± 0.97) in comparison to the control groups (A/B/C) (A: 3.78 ± 0.38 and 0.6 ± 1.12; B: 3.72 ± 0.32; C: 3.55 ± 0.44 p < .001, p < .001, p < .001 MFI respectively). E-C4d and G-C4d significantly correlated with LN activity (r = 0.8, p < .001 and r = 0.7, p = .005) and with SLEDAI (r = 0.9, p = .005 and r = 0.8, p = .002, respectively). Conclusion: Erythrocytic C4d correlated significantly with the LN activity which might serve as a potential biomarker for renal activity in the future instead of biopsy and may further help in the optimum follow up of LN patients.
1. Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that chiefly influences women. It has a myriad of clinical manifestations, yet the disease outcome and organ involvements remain unpredictable. In spite of the advances in the survival of SLE patients, the pathogenesis is still unknown. Comorbidities due to both disease and treatment, and the many faces of SLE require intensive investigations [1]. Lupus nephritis (LN) is one of the most frequent and serious complications in SLE patients. Autoimmune-mediated inflammation in both renal glomerular and tubulointerstitial tissues is the major pathological finding of LN. In clinical practice, increased anti-double stranded deoxyribonucleic acid (anti-dsDNA) antibody titer and consumed complement C3 and C4 levels are potential disease-activity surrogate biomarkers in LN [2]. LN usually arises within 5 years of diagnosis and is histologically evident in most SLE patients, even in
those without clinical manifestations of renal disease [3]. Renal biopsy is still the gold standard for deciding therapy in LN but its invasive nature prevents it from being used repetitively. Since histopathology remains the cornerstone for deciding treatment in LN, the ultimate goal of ‘the quest for a biomarker of LN’ is to find a marker that could reflect the in vivo events obviating the need for renal biopsy [4]. More evidences suggest that autoantibodies other than anti-double stranded deoxyribonucleic acid (anti-dsDNA) antibodies, as anti-nucleosome, anti-C1q, anti-C3b, anti-cardiolipin (aCL), anti-ribonuclear proteins, and anti-glomerular matrix antibodies, may also be involved in LN [2]. An ideal biomarker for LN would be the one which reflects renal disease activity better, correlates with renal histology, predicts flares, is easily measurable, not affected by age, gender and ethnicity, is specific to SLE and renal involvement for making an early diagnosis of LN. However, no biomarker seems to have all the desired qualities [5]. Several biomarkers have been proposed in studies on Egyptian SLE
Peer review under responsibility of Egyptian Society of Rheumatic Diseases. ⁎ Corresponding author. E-mail address: [email protected] (H. Ramadan). http://dx.doi.org/10.1016/j.ejr.2017.10.002 Received 13 October 2017; Accepted 21 October 2017 1110-1164/ © 2017 Egyptian Society of Rheumatic Diseases. Publishing services provided by Elsevier B.V. All rights reserved. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: El Halim, H.A., The Egyptian Rheumatologist (2017), http://dx.doi.org/10.1016/j.ejr.2017.10.002
The Egyptian Rheumatologist xxx (xxxx) xxx–xxx
H.A.E. Halim et al.
patients [6–10] with preference to LN [11–15]. Systemic lupus erythematosus is characterized by a vicious cycle maintaining systemic inflammation. It starts by autoantibody production, immune complex formation and complement activation that contribute to inflammation, tissue damage and further autoantibody production [16]. Future in-depth studies on the association of oxidative stress to complement pathways in SLE pathogenesis are encouraged [17]. Complement factors (C3 and C4) are traditional markers of disease activity. Their low levels signify consumption in the clearing process and correlate with disease activity. Apart from C3 and C4, their degradation products also have been tested as biomarkers in LN [5]. C4d is now increasingly recognized as a potential biomarker in fields where antibodies can cause tissue damage, as systemic autoimmune diseases and pregnancy. C4d holds promise to detect patients at risk for the consequences of antibody-mediated disease. Moreover, the emergence of new therapeutics that inhibits complement activation makes C4d a marker with potential to identify patients who may benefit from these drugs [18]. Serum C4d detected by quantitative micro-assay plate enzyme immunoassay is more sensitive than C3, C4 and C5b-9 in SLE patients with moderate-to-severe disease activity [19]. In LN, Glomerular-C4d (G-C4d) deposition can be detected in the majority of cases with an immune-fluorescence pattern, as a result of immune complex deposition. Biopsies of LN patients with prominent diffuse GC4d staining had detectable glomerular microthrombi significantly more than in patients with focal/mild C4d staining. The extent of G-C4d deposition correlates with LN activity and with thrombotic microangiopathy (TMA). Also TMA correlate with LN activity [20]. The aim of the present work was to assess C4d on circulating erythrocytes (E-C4d) and deposits in renal glomerular tissues (G-C4d) and study their correlation with LN activity.
Table 1 Demographic, laboratory and activity features and medications received by lupus nephritis patients. Variable mean ± SD or n (%)
LN patients (n = 15)
Age (years) Sex F:M
28.3 ± 8.2 12 (80): 3 (20)
Hemoglobin (g/dl) TLC (×103/mm3) Platelets (×103/mm3) sCreatinine (mg/dl) ESR (mm/1st hr) A/C sAlbumin (mg/dl) E-C4d (MFI) Complement C3 (mg/dl) C4 (mg/dl)
9.9 ± 1.9 5.8 ± 1.9 218 ± 71.5 1.4 ± 0.61 100.2 ± 30.6 2.04 ± 1.1 2.6 ± 0.5 8.1 ± 2.9 32.5 ± 10.3 12.9 ± 4.5
SLEDAI score Renal activity score
13.9 ± 0.9 4.3 ± 3.7
Medications Steroids Azathioprine Mycophenolate mofetil Cyclophosphamide
15 (100) 15 (100) 9 (60) 6 (40)
LN: lupus nephritis, TLC: total leucocytic count, ESR: erythrocyte sedimentation rate, A/C: albumin-creatinine ratio, E-C4d: C4d on erythrocytes, MFI: mean fluorescence intensity, SLEDAI: systemic lupus erythematosus disease activity index.
2.1. Statistical analysis Data were coded and entered using the statistical package SPSS version 23. Data was summarized using mean ± standard deviation for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparisons among groups were done using analysis of variance (ANOVA) with multiple comparisons post hoc test. For comparing categorical data, Chi square (χ2) test was performed. Exact test was used instead when the expected frequency is < 5. Spearmans correlation test was used. P-values < .05 was considered significant.
2. Patients and methods A total of 61 subjects were enrolled in this study and included 15 patients with LN (study group); 15 with renal disease not due to SLE (control A group); 16 SLE patients without renal affection (control B group) and 15 healthy individuals (control C group). All SLE patients met the systemic lupus international collaborating clinics (SLICC) classification criteria [21]. This study was conducted at Internal Medicine Hospital Kasr Al-Ainy, Faculty of Medicine, Cairo University. The study conforms to the 1995 Helsinki declaration and was approved by Cairo University Hospitals’ ethical committee. Informed consent was obtained from all patients. Patients were subjected to history taking, thorough clinical examination and the following laboratory investigations: complete blood count (CBC), erythrocyte sedimentation rate (ESR), serum albumin, serum creatinine, urine analysis, anti-nuclear antibody (ANA), antidsDNA, serum C3 and C4, aCL (IgG and IgM) and urine albumin/ creatinine ratio. E-C4d was assayed by flow cytometry in all subjects. SLE disease activity index (SLEDAI) was assessed in SLE patients [22]. Renal biopsy was done for LN patients and control A group and histopathological evaluation by immunohistochemistry staining for G-C4d. Renal specimens were processed for light microscopy and classified according to the 1982 modified world health organization (WHO) morphologic classification of LN and renal activity and chronicity were considered [23]. Flow cytometry system (EPICS XL, Bickman Coulter) was used (Primary anti body: Anti C4d Ab (ab36075). Vial 50 micro Lat 0.2 mg/ ml. Secondary Antibody: Goat Anti Rabbit IgG H & L (AleXa Flour ®488) (ab150077) vial 500 µg at 2 mg/ml). Venous blood was taken using sterile tubes containing an EDTA salt as the anti coagulant. The samples were kept at room temperature (18–25 °C) and not shaken. The samples were homogenized by gentle agitation and were analyzed within 24 h of venipuncture. Surface expression of C4d on gated cells was expressed as specific mean fluorescence intensity (MFI) (C4d-specific mean the isotype control mean fluorescence).
3. Results The age was comparable among groups; for LN patients was 28.3 ± 8.2 years; group A (35.9 ± 13.3 years); group B (27.1 ± 8.8 years) and group C (29.4 ± 7.1 years) (p = .06). The frequency of female patients was significantly higher in LN patients (80%) and controls B (100%) and C (73.3%) compared to control A (40%) (p = .001). The disease duration of the patients with LN was 1–2 years, of group A was 3–5 years and of group B was 5–10 years. Table 1 shows the demographic, clinical, laboratory and activity features of the study groups as well as the medications received. The SLEDAI score was significantly higher in SLE patients with LN patients compared to those without patients (p = .03). Serum creatinine levels of LN patients were significantly higher than control B and C (p < .001) but lower than control A (p = .7). The ESR levels were significantly higher in LN patients than control (A/B/C) (p < .001). A/ C ratio in LN patients was significantly higher than control B and C (p < .001) but lower than control A (p = .9). Serum C3 and C4 were significantly lower in LN patients (p < .001) compared with the 3 control groups (A/B/C). MFI was significantly higher in LN patients (8.08 ± 2.93) in comparison to the control groups (A/B/C) (A: 3.78 ± 0.38; B: 3.72 ± 0.32; C: 3.55 ± .0.44 p < .001, p < .001, p < .001 respectively). (Fig. 1). Renal biopsy results for LN and renal-non nephritis groups showed that the activity and chronicity indices in LN group were 4.27 ± 3.7 and 1.73 ± 2.3, respectively, while in renal group they were 2
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Fig. 3. Correlation between erythrocytic C4d (E-C4d) and systemic lupus erythematosus disease activity index (SLEDAI) in lupus nephritis patients.
Fig. 1. Erythrocyte C4d (EC4d) in the lupus nephritis, renal non-lupus and lupus nonrenal patient compared to healthy controls. (green color represents the median of MFI and purple color represents the 3rd percentile of MFI).
0.87 ± 1.6 and 5.6 ± 2.5 respectively. In LN activity index was significantly higher (p < .001) than in the renal group. Intense positive G-C4d deposits were found in almost all LN patients (14/15) and were found in 4 patients of renal disease group (4/15) (p < .001). All G-C4d deposits were associated with the presence of granular glomerular immune deposits. In LN patients, E-C4d significantly correlated with G-C4d deposits (r = 0.7, p = .007), with LN activity index (r = 0.8, p < .001) and with SLEDAI (r = 0.9, p = .005). G-C4d deposits significantly correlated with activity index and SLEDAI (r = 0.7, p = .005 and r = 0.8, p = .002, respectively). Both E-C4d and G-C4d did not significantly correlate with the chronicity index or with serum C3 and C4. Serum C3 and C4 significantly inversely correlated with activity index (r = −0.53, p = .04, r = −0.3, p = .4) and SLEDAI (r = −0.6, p = .02, r = −0.4, p = .2 respectively). Significant correlations are presented in Figs. 2–5. In renal disease patients (control A group), E-C4d did not
Fig. 4. Correlation between erythrocytic C4d (E-C4d) and C3 in lupus nephritis patients.
Fig. 5. Correlation between erythrocytic C4d (E-C4d) and C4 in lupus nephritis patients.
significantly correlate with G-C4d deposits and both E-C4d and G-C4d did not correlate with the renal activity or chronicity indices and with the serum C3 and C4. Serum C3 and C4 did not significantly correlate with the renal activity and chronicity indices. In SLE patients with no renal affection (control B group), E-C4d did not significantly correlate with SLEDAI score or with serum C3 and C4. 4. Discussion Fig. 2. Correlation between erythrocytic C4d (E-C4d) and renal activity index in lupus nephritis patients.
Lupus nephritis is an important manifestation of SLE that affects treatment decisions, as well as long-term outcomes. Effective treatment 3
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Conflict of interest
of LN requires correct diagnosis, timely intervention and early treatment of any disease relapse. All these are possible only with a good biomarker that reflects the disease activity better than existing markers [5]. The complement system consists of a tightly regulated network of proteins that play an important role in host defense and inflammation. Inappropriate complement activation and complement deficiencies are the underlying cause of the pathophysiology of many diseases including SLE [24]. In the present study E-C4d were significantly higher in LN patients compared to SLE patients without renal involvement, to patients with renal disease not due to SLE and to healthy control. E-C4d in LN patients correlated significantly with G-C4d deposits, LN activity index and SLEDAI but not with the complement factors or chronicity index. The results are in concur with Batal et al. [25] who observed that E-C4d was elevated in LN patients and also correlated significantly with the renal activity index but not with serum C3, C4 and chronicity index. Furthermore, Kao et al. [26] found that levels of E-C4d were greater in LN patients than in either renal or lupus non-renal patients and that EC4d correlated with kidney disease activity index and with the SLEDAI score but was not associated with serum C3 and C4. The current findings also go with the results of Mora et al. [27] who observed a significant correlation between the E-C4d and SLEDAI. As regard Glomerular-C4d deposits they were found to be significantly higher in LN patients compared to renal non-lupus patients and were associated with granular glomerular immune deposits and significantly correlated with the activity index and SLEDAI score. This goes with the results of Sahin et al. [18] who observed a relationship between G-C4d staining and LN activity. They found a significant correlation between the G-C4d deposits with the activity index and granular glomerular immune deposits. In accordance, Sato et al. [28] found that G-C4d deposition correlated with immune complex deposition showing different patterns according to the LN subtypes. However, they reported no significant correlation with the disease activity of SLE and histological LN activity. In disagreement, Kim et al. [29] found that GC4d deposits did not correlate with SLEDAI score and real activity index in LN patients. Serum complement (C3 and C4) were significantly consumed in LN patients compared to the SLE patients without LN, patients with renal (non-lupus) disease and healthy control. Both C3 and C4 correlated negatively with activity index and SLEDAI score in LN patients. This was in harmony to the findings of Narayanan, et al. [30] who observed a significant negative correlation of C3 and C4 with the SLEDAI. On subgroup analysis it was noticed that this correlation is stronger for renal lupus. Serum C3 and C4 were not correlated significantly with EC4d in all study groups and were not correlated with G-C4d and chronicity index in LN and renal non-lupus patients and did not correlate with activity index in renal non-lupus patients or with SLEDAI score in lupus non-renal patients. This agrees with the results of Villegas-Zambrano et al. [31] who found that C3 and C4 evaluation is of limited utility in the monitoring of LN patients and patients with active kidney disease (non-lupus) as C3 and C4 did not correlate with the chronicity index, but in contrast in the present work, C3 and C4 did not correlate with activity index in LN patients which agrees with the results of Hasni Mahayidin et al. [32] who found in SLE patients there was no correlation between the levels of C3 and C4 with the E-C4d and SLEDAI score. In conclusion, C4d on circulating RBCs were found to be correlated significantly with LN activity which might help in using E-C4d as a biomarker in detection of LN activity in the future instead of renal biopsy which is considered an invasive method and may help in follow up of lupus nephritis patients. Erythrocyte-C4d might help also in follow up of LN patients with active disease so we need more work in this field on big number of patients. A larger scale, longitudinal study is recommended to verify the present results and determine the impact of erythrocyte and glomerular C4d on the response to the disease damage, medications received and prognosis.
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Contents lists available at ScienceDirect
The Egyptian Rheumatologist journal homepage: www.elsevier.com/locate/erhe
Original Article
Erythrocyte and glomerular C4d deposits as a biomarker for active lupus nephritis Hanan Abd El Halima, Mona Salahb, Wesam Ismailc, Ahmed Fathya, Hala Ramadana, a b c
⁎
Internal Medicine Department, Faculty of Medicine, Cairo University, Cairo, Egypt Clinical Pathology Department, Faculty of Medicine, Cairo University, Cairo, Egypt Pathology Department, Faculty of Medicine, Bani Sweif University, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: SLE C4d LN Renal biopsy SLEDAI
Introduction: Lupus nephritis (LN) is a serious manifestation of systemic lupus erythematosus (SLE) and histologically evident even in those without clinical manifestations of renal disease. Aim of the work: To assess C4d on erythrocytes (E-C4d) and glomerular deposits (G-C4d) in SLE patients and study its association to LN and disease activity. Patients and methods: 61 subjects were enrolled including 15 with LN (study group); 15 with renal disease not due to SLE (control A group); 16 SLE patients with no renal affection (control B group) and 15 healthy individuals (control C group). Flow cytometry system was used for C4d immunohistochemical staining. SLE disease activity index (SLEDAI) was assessed for SLE patients. Results: The age was comparable among groups; for LN patients was 28.3 ± 8.2 years; group A (35.9 ± 13.3); group B (27.1 ± 8.8) and group C (29.4 ± 7.1) (p = .06). Patients were mostly females. The disease duration of LN patients was 1–2 years; group A (3–5 years) and group B (5–10 years). E-C4d and G-C4d deposits were significantly higher in LN patients (8.08 ± 2.93 and 2.3 ± 0.97) in comparison to the control groups (A/B/C) (A: 3.78 ± 0.38 and 0.6 ± 1.12; B: 3.72 ± 0.32; C: 3.55 ± 0.44 p < .001, p < .001, p < .001 MFI respectively). E-C4d and G-C4d significantly correlated with LN activity (r = 0.8, p < .001 and r = 0.7, p = .005) and with SLEDAI (r = 0.9, p = .005 and r = 0.8, p = .002, respectively). Conclusion: Erythrocytic C4d correlated significantly with the LN activity which might serve as a potential biomarker for renal activity in the future instead of biopsy and may further help in the optimum follow up of LN patients.
1. Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that chiefly influences women. It has a myriad of clinical manifestations, yet the disease outcome and organ involvements remain unpredictable. In spite of the advances in the survival of SLE patients, the pathogenesis is still unknown. Comorbidities due to both disease and treatment, and the many faces of SLE require intensive investigations [1]. Lupus nephritis (LN) is one of the most frequent and serious complications in SLE patients. Autoimmune-mediated inflammation in both renal glomerular and tubulointerstitial tissues is the major pathological finding of LN. In clinical practice, increased anti-double stranded deoxyribonucleic acid (anti-dsDNA) antibody titer and consumed complement C3 and C4 levels are potential disease-activity surrogate biomarkers in LN [2]. LN usually arises within 5 years of diagnosis and is histologically evident in most SLE patients, even in
those without clinical manifestations of renal disease [3]. Renal biopsy is still the gold standard for deciding therapy in LN but its invasive nature prevents it from being used repetitively. Since histopathology remains the cornerstone for deciding treatment in LN, the ultimate goal of ‘the quest for a biomarker of LN’ is to find a marker that could reflect the in vivo events obviating the need for renal biopsy [4]. More evidences suggest that autoantibodies other than anti-double stranded deoxyribonucleic acid (anti-dsDNA) antibodies, as anti-nucleosome, anti-C1q, anti-C3b, anti-cardiolipin (aCL), anti-ribonuclear proteins, and anti-glomerular matrix antibodies, may also be involved in LN [2]. An ideal biomarker for LN would be the one which reflects renal disease activity better, correlates with renal histology, predicts flares, is easily measurable, not affected by age, gender and ethnicity, is specific to SLE and renal involvement for making an early diagnosis of LN. However, no biomarker seems to have all the desired qualities [5]. Several biomarkers have been proposed in studies on Egyptian SLE
Peer review under responsibility of Egyptian Society of Rheumatic Diseases. ⁎ Corresponding author. E-mail address: [email protected] (H. Ramadan). http://dx.doi.org/10.1016/j.ejr.2017.10.002 Received 13 October 2017; Accepted 21 October 2017 1110-1164/ © 2017 Egyptian Society of Rheumatic Diseases. Publishing services provided by Elsevier B.V. All rights reserved. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: El Halim, H.A., The Egyptian Rheumatologist (2017), http://dx.doi.org/10.1016/j.ejr.2017.10.002
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patients [6–10] with preference to LN [11–15]. Systemic lupus erythematosus is characterized by a vicious cycle maintaining systemic inflammation. It starts by autoantibody production, immune complex formation and complement activation that contribute to inflammation, tissue damage and further autoantibody production [16]. Future in-depth studies on the association of oxidative stress to complement pathways in SLE pathogenesis are encouraged [17]. Complement factors (C3 and C4) are traditional markers of disease activity. Their low levels signify consumption in the clearing process and correlate with disease activity. Apart from C3 and C4, their degradation products also have been tested as biomarkers in LN [5]. C4d is now increasingly recognized as a potential biomarker in fields where antibodies can cause tissue damage, as systemic autoimmune diseases and pregnancy. C4d holds promise to detect patients at risk for the consequences of antibody-mediated disease. Moreover, the emergence of new therapeutics that inhibits complement activation makes C4d a marker with potential to identify patients who may benefit from these drugs [18]. Serum C4d detected by quantitative micro-assay plate enzyme immunoassay is more sensitive than C3, C4 and C5b-9 in SLE patients with moderate-to-severe disease activity [19]. In LN, Glomerular-C4d (G-C4d) deposition can be detected in the majority of cases with an immune-fluorescence pattern, as a result of immune complex deposition. Biopsies of LN patients with prominent diffuse GC4d staining had detectable glomerular microthrombi significantly more than in patients with focal/mild C4d staining. The extent of G-C4d deposition correlates with LN activity and with thrombotic microangiopathy (TMA). Also TMA correlate with LN activity [20]. The aim of the present work was to assess C4d on circulating erythrocytes (E-C4d) and deposits in renal glomerular tissues (G-C4d) and study their correlation with LN activity.
Table 1 Demographic, laboratory and activity features and medications received by lupus nephritis patients. Variable mean ± SD or n (%)
LN patients (n = 15)
Age (years) Sex F:M
28.3 ± 8.2 12 (80): 3 (20)
Hemoglobin (g/dl) TLC (×103/mm3) Platelets (×103/mm3) sCreatinine (mg/dl) ESR (mm/1st hr) A/C sAlbumin (mg/dl) E-C4d (MFI) Complement C3 (mg/dl) C4 (mg/dl)
9.9 ± 1.9 5.8 ± 1.9 218 ± 71.5 1.4 ± 0.61 100.2 ± 30.6 2.04 ± 1.1 2.6 ± 0.5 8.1 ± 2.9 32.5 ± 10.3 12.9 ± 4.5
SLEDAI score Renal activity score
13.9 ± 0.9 4.3 ± 3.7
Medications Steroids Azathioprine Mycophenolate mofetil Cyclophosphamide
15 (100) 15 (100) 9 (60) 6 (40)
LN: lupus nephritis, TLC: total leucocytic count, ESR: erythrocyte sedimentation rate, A/C: albumin-creatinine ratio, E-C4d: C4d on erythrocytes, MFI: mean fluorescence intensity, SLEDAI: systemic lupus erythematosus disease activity index.
2.1. Statistical analysis Data were coded and entered using the statistical package SPSS version 23. Data was summarized using mean ± standard deviation for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparisons among groups were done using analysis of variance (ANOVA) with multiple comparisons post hoc test. For comparing categorical data, Chi square (χ2) test was performed. Exact test was used instead when the expected frequency is < 5. Spearmans correlation test was used. P-values < .05 was considered significant.
2. Patients and methods A total of 61 subjects were enrolled in this study and included 15 patients with LN (study group); 15 with renal disease not due to SLE (control A group); 16 SLE patients without renal affection (control B group) and 15 healthy individuals (control C group). All SLE patients met the systemic lupus international collaborating clinics (SLICC) classification criteria [21]. This study was conducted at Internal Medicine Hospital Kasr Al-Ainy, Faculty of Medicine, Cairo University. The study conforms to the 1995 Helsinki declaration and was approved by Cairo University Hospitals’ ethical committee. Informed consent was obtained from all patients. Patients were subjected to history taking, thorough clinical examination and the following laboratory investigations: complete blood count (CBC), erythrocyte sedimentation rate (ESR), serum albumin, serum creatinine, urine analysis, anti-nuclear antibody (ANA), antidsDNA, serum C3 and C4, aCL (IgG and IgM) and urine albumin/ creatinine ratio. E-C4d was assayed by flow cytometry in all subjects. SLE disease activity index (SLEDAI) was assessed in SLE patients [22]. Renal biopsy was done for LN patients and control A group and histopathological evaluation by immunohistochemistry staining for G-C4d. Renal specimens were processed for light microscopy and classified according to the 1982 modified world health organization (WHO) morphologic classification of LN and renal activity and chronicity were considered [23]. Flow cytometry system (EPICS XL, Bickman Coulter) was used (Primary anti body: Anti C4d Ab (ab36075). Vial 50 micro Lat 0.2 mg/ ml. Secondary Antibody: Goat Anti Rabbit IgG H & L (AleXa Flour ®488) (ab150077) vial 500 µg at 2 mg/ml). Venous blood was taken using sterile tubes containing an EDTA salt as the anti coagulant. The samples were kept at room temperature (18–25 °C) and not shaken. The samples were homogenized by gentle agitation and were analyzed within 24 h of venipuncture. Surface expression of C4d on gated cells was expressed as specific mean fluorescence intensity (MFI) (C4d-specific mean the isotype control mean fluorescence).
3. Results The age was comparable among groups; for LN patients was 28.3 ± 8.2 years; group A (35.9 ± 13.3 years); group B (27.1 ± 8.8 years) and group C (29.4 ± 7.1 years) (p = .06). The frequency of female patients was significantly higher in LN patients (80%) and controls B (100%) and C (73.3%) compared to control A (40%) (p = .001). The disease duration of the patients with LN was 1–2 years, of group A was 3–5 years and of group B was 5–10 years. Table 1 shows the demographic, clinical, laboratory and activity features of the study groups as well as the medications received. The SLEDAI score was significantly higher in SLE patients with LN patients compared to those without patients (p = .03). Serum creatinine levels of LN patients were significantly higher than control B and C (p < .001) but lower than control A (p = .7). The ESR levels were significantly higher in LN patients than control (A/B/C) (p < .001). A/ C ratio in LN patients was significantly higher than control B and C (p < .001) but lower than control A (p = .9). Serum C3 and C4 were significantly lower in LN patients (p < .001) compared with the 3 control groups (A/B/C). MFI was significantly higher in LN patients (8.08 ± 2.93) in comparison to the control groups (A/B/C) (A: 3.78 ± 0.38; B: 3.72 ± 0.32; C: 3.55 ± .0.44 p < .001, p < .001, p < .001 respectively). (Fig. 1). Renal biopsy results for LN and renal-non nephritis groups showed that the activity and chronicity indices in LN group were 4.27 ± 3.7 and 1.73 ± 2.3, respectively, while in renal group they were 2
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Fig. 3. Correlation between erythrocytic C4d (E-C4d) and systemic lupus erythematosus disease activity index (SLEDAI) in lupus nephritis patients.
Fig. 1. Erythrocyte C4d (EC4d) in the lupus nephritis, renal non-lupus and lupus nonrenal patient compared to healthy controls. (green color represents the median of MFI and purple color represents the 3rd percentile of MFI).
0.87 ± 1.6 and 5.6 ± 2.5 respectively. In LN activity index was significantly higher (p < .001) than in the renal group. Intense positive G-C4d deposits were found in almost all LN patients (14/15) and were found in 4 patients of renal disease group (4/15) (p < .001). All G-C4d deposits were associated with the presence of granular glomerular immune deposits. In LN patients, E-C4d significantly correlated with G-C4d deposits (r = 0.7, p = .007), with LN activity index (r = 0.8, p < .001) and with SLEDAI (r = 0.9, p = .005). G-C4d deposits significantly correlated with activity index and SLEDAI (r = 0.7, p = .005 and r = 0.8, p = .002, respectively). Both E-C4d and G-C4d did not significantly correlate with the chronicity index or with serum C3 and C4. Serum C3 and C4 significantly inversely correlated with activity index (r = −0.53, p = .04, r = −0.3, p = .4) and SLEDAI (r = −0.6, p = .02, r = −0.4, p = .2 respectively). Significant correlations are presented in Figs. 2–5. In renal disease patients (control A group), E-C4d did not
Fig. 4. Correlation between erythrocytic C4d (E-C4d) and C3 in lupus nephritis patients.
Fig. 5. Correlation between erythrocytic C4d (E-C4d) and C4 in lupus nephritis patients.
significantly correlate with G-C4d deposits and both E-C4d and G-C4d did not correlate with the renal activity or chronicity indices and with the serum C3 and C4. Serum C3 and C4 did not significantly correlate with the renal activity and chronicity indices. In SLE patients with no renal affection (control B group), E-C4d did not significantly correlate with SLEDAI score or with serum C3 and C4. 4. Discussion Fig. 2. Correlation between erythrocytic C4d (E-C4d) and renal activity index in lupus nephritis patients.
Lupus nephritis is an important manifestation of SLE that affects treatment decisions, as well as long-term outcomes. Effective treatment 3
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Conflict of interest
of LN requires correct diagnosis, timely intervention and early treatment of any disease relapse. All these are possible only with a good biomarker that reflects the disease activity better than existing markers [5]. The complement system consists of a tightly regulated network of proteins that play an important role in host defense and inflammation. Inappropriate complement activation and complement deficiencies are the underlying cause of the pathophysiology of many diseases including SLE [24]. In the present study E-C4d were significantly higher in LN patients compared to SLE patients without renal involvement, to patients with renal disease not due to SLE and to healthy control. E-C4d in LN patients correlated significantly with G-C4d deposits, LN activity index and SLEDAI but not with the complement factors or chronicity index. The results are in concur with Batal et al. [25] who observed that E-C4d was elevated in LN patients and also correlated significantly with the renal activity index but not with serum C3, C4 and chronicity index. Furthermore, Kao et al. [26] found that levels of E-C4d were greater in LN patients than in either renal or lupus non-renal patients and that EC4d correlated with kidney disease activity index and with the SLEDAI score but was not associated with serum C3 and C4. The current findings also go with the results of Mora et al. [27] who observed a significant correlation between the E-C4d and SLEDAI. As regard Glomerular-C4d deposits they were found to be significantly higher in LN patients compared to renal non-lupus patients and were associated with granular glomerular immune deposits and significantly correlated with the activity index and SLEDAI score. This goes with the results of Sahin et al. [18] who observed a relationship between G-C4d staining and LN activity. They found a significant correlation between the G-C4d deposits with the activity index and granular glomerular immune deposits. In accordance, Sato et al. [28] found that G-C4d deposition correlated with immune complex deposition showing different patterns according to the LN subtypes. However, they reported no significant correlation with the disease activity of SLE and histological LN activity. In disagreement, Kim et al. [29] found that GC4d deposits did not correlate with SLEDAI score and real activity index in LN patients. Serum complement (C3 and C4) were significantly consumed in LN patients compared to the SLE patients without LN, patients with renal (non-lupus) disease and healthy control. Both C3 and C4 correlated negatively with activity index and SLEDAI score in LN patients. This was in harmony to the findings of Narayanan, et al. [30] who observed a significant negative correlation of C3 and C4 with the SLEDAI. On subgroup analysis it was noticed that this correlation is stronger for renal lupus. Serum C3 and C4 were not correlated significantly with EC4d in all study groups and were not correlated with G-C4d and chronicity index in LN and renal non-lupus patients and did not correlate with activity index in renal non-lupus patients or with SLEDAI score in lupus non-renal patients. This agrees with the results of Villegas-Zambrano et al. [31] who found that C3 and C4 evaluation is of limited utility in the monitoring of LN patients and patients with active kidney disease (non-lupus) as C3 and C4 did not correlate with the chronicity index, but in contrast in the present work, C3 and C4 did not correlate with activity index in LN patients which agrees with the results of Hasni Mahayidin et al. [32] who found in SLE patients there was no correlation between the levels of C3 and C4 with the E-C4d and SLEDAI score. In conclusion, C4d on circulating RBCs were found to be correlated significantly with LN activity which might help in using E-C4d as a biomarker in detection of LN activity in the future instead of renal biopsy which is considered an invasive method and may help in follow up of lupus nephritis patients. Erythrocyte-C4d might help also in follow up of LN patients with active disease so we need more work in this field on big number of patients. A larger scale, longitudinal study is recommended to verify the present results and determine the impact of erythrocyte and glomerular C4d on the response to the disease damage, medications received and prognosis.
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