Ass.Prof.Dr.Laila Rashed Assistant Professor of Medical Biochemistry

Transcription

1 A Study of Hepcidin and Monocyte Chemoattractant Protein -1 in Patients with Systemic Lupus Erythematosus Presented by Moataz Fatthy Mohammed M.Sc. Internal Medicine for partial fullfilment requirements of M.D degree in internal medicine Under Supervision of Prof.Dr. Dawlat Belal Professor of Internal Medicine Cairo University Ass.Prof.Dr.Seham Bakry Assistant Professor of Internal Medicine Cairo University Ass.Prof.Dr.Laila Rashed Assistant Professor of Medical Biochemistry Cairo University Faculty of medicine Cairo University 2012

2 ACKNOWLEDGEMENT First and foremost thanks to (ALLAH) who is the most beneficial and most merciful. It is a great pleasure to express my profound gratitude and deep thanks to Prof. Dr. Dawlat Belal Professor of Internal Medicine, Faculty of Medicine, Cairo University for her keen supervision, generous cooperation, great help and encouragement to finish this work. I am thankful to Prof. Dr. Seham Bakry Professor of Internal Medicine, Faculty of Medicine, Cairo University for her great concern and kind supervision. I wish to express my gratitude to Dr. Laila Rashed professor of Medical Biochemistry, Faculty of Medicine, Cairo University, for her careful supervision, valuable cooperation and encouragement. I am thankful to Prof. Dr. Esmat Sheeba, Head of Internal Medicine Department, Faculty of Medicine, Cairo University, for his support and encouragement to finish this work. My special thanks to my mother for her care, love and generosity that can never be sufficiently acknowledged.

3 Abstract Kidney disease is one of the most serious manifestations of systemic lupus erythematosus (SLE). Despite the improvement in the medical care of SLE in the past two decades, the prognosis of lupus nephritis remains unsatisfactory. Besides exploring more effective but less toxic treatment modalities that will further improve the remission rate, early detection and treatment of renal activity may spare patients from intensive immunosuppressive therapies and reduce renal damage. Conventional clinical parameters such as creatinine clearance, proteinuria, urine sediments, anti-dsdna and complement levels are not sensitive or specific enough for detecting ongoing disease activity in the lupus kidneys and early relapse of nephritis. Thus, novel biomarkers are necessary to enhance the diagnostic accuracy and sensitivity of lupus renal disease, prognostic stratification, monitoring of treatment response and detection of early renal flares. This study reviews promising biomarkers that have recently been evaluated in longitudinal studies of lupus nephritis. Our study included sixty systemic lupus erythematosis patients divided into 2 groups nephritic and non-nephritic patients where urinary monocyte chemoattractant protein-1 and hepcidin was evaluated, compared and correlated in both groups and in control group, with each other and with other routine variables and concluded that Urinary hepcidin and urinary MCP-1 can be accepted as diagnostic predictors of lupus nephritis in patients with systemic lupus erythematosis. Also may be significant and reliable factors for predicting pathological changes of renal biopsy in lupus nephritis patients. Key words: Lupus nephritis Urinary biomarkers Monocyte chemoattractant protein-1 Hepcidin

4 Contents List of Abbreviations I List of Tables IV List of Figures V Introduction and Aim of work 1 Review of Literature Systemic Lupus Erythematosis 5 Lupus Nephritis 33 Urinary Biomarkers in Lupus Nephritis 51 Subjects and Methods 77 Results 90 Discussion 111 Conclusion 124 Summary 127 Recommendations 128 References 129 Arabic summary 180

5 List of abbreviations List of Abbreviations ACR American college of rheumatology AGP α1-acid-glycoprotein AKI Acute kidney injury ANA Anti nuclear antibody ANOVA Analysis of variance Anti-RNP Anti-Ribonucleoprotein Anti-sm Anti-smith apl antiphospholipid antibodies APS Anti phospholipid syndrome BILAG British Isles Lupus Assessment Group BMPs Bone Morphogenetic Proteins BNP Brain natriuretic peptide C3 Complement 3 C4 Complement 4 C-C Chemokine CCL2 Chemokine ligand 2 CHD Coronary heart disease CKD Chronic kidney disease CNS Central nervous system CP Ceruloplasmin CPB cardiopulmonary bypass Cr Creatinine CREBH Cyclic AMP response element-binding protein H CRP C-reactive protein CSF Cerebrospinal fluid CVA Cerebrovascular accident DHEA Di hydro epiandrosterone DNA Deoxyribonucleic acid EAE Experimental autoimmune encephalomyelitis ECG Electrocardiogram ECLAS European Consensus Lupus Activity Measurement ELISA Enzyme linked immunosorbant assay I

6 List of abbreviations ER ESA GBM G-CSF GP GWAS HAMP HLA IC ICAM-1 IFNγ IgG IL ITGAM JAK kda LA LAI LDL LEAP-1 LFA-1 LN L-PGDS MCP-1 mepcr MMF MMP MRI MS NFκB NGAL NMR NSAID OPG endoplasmic reticulum Erythropoiesis stimulating agents Glomerular basement membrane Granulocyte colony stimulating factor Glycoprotein Genome-wide association studies Hepcidin antimicrobial peptide Human leucocytic antigen Immune complex intercellular adhesion molecule Interferon gamma Immunoglobulin G Interleukin integrin alpha M janus kinase kilodaltons lupus anticoagulant Lupus Activity Index Loe density lipoprptein Liver-Expressed Antimicrobial Protein-1 leukocyte function-associated molecule-1 Lupus nephritis lipocalin-type prostaglandin D-synthetase Monocyte chemoattractant protein-1 Membrane expression of endothelial protein C receptor Mycophenolate mofetil metalloproteinase Magnetic resonance imaging Multiple sclerosis nuclear factor κb Neutrophil Gelatinase-Associated Lipocalin Nuclear magnetic resonance Non steroid anti inflammatory drug Osteoprotegerin II

7 List of abbreviations RA Rheumatoid artheritis RANKL Receptor activator of nuclear factor κb ligand RANTES Regulated on activation normal T cell expressed and secreted ROC Receiver Operating Characteristic SLAM Systemic Lupus Activity Measure SLE systemic lupus erythematosus SLEDAI Systemic Lupus Erythematosus Disease Activity Index SNP Single nucleotide pleomorphism STAT4 signal transducer and activator of transcription 4 gene TF transferrin TGF-β Tumor growth factor TH2 T helper 2 TNF Tumor necrosis factor TRAPS Tumor necrosis factor receptor-1 associated periodic syndrome TWEAK Tumor Necrosis Factor-Like Inducer of Apoptosis VCAM Vascular cell adhesion molecule VEGF Vascular endothelium growth factor III

8 List of Tables List of Tables Table No. Subject Page No. 1 Modified 1997 classification criteria for SLE 11 2 Commonly abnormal tests on body fluids in SLE 22 3 SLE Disease Activity Index domains 29 4 Urine biomarkers that correlate with lupus nephritis activity in cross-sectional studies Biomarkers that correlate with histological findings in lupus nephritis Biomarkers that correlate with prognosis in lupus nephritis Comparison between mean ± SD of albumin/creatinine ratio in all the studied groups Comparison between mean ± SD of creatinine in all the studied groups Comparison between mean ± SD of GFR in all the studied groups. 10 Comparison between mean ± SD of C3 in all the studied groups. 11 Comparison between mean ± SD of C4 in all the studied groups. 12 Comparison between mean ± SD of ESR in all the studied groups. 13 Comparison between mean ± SD of ADNA in all the studied groups. 14 Comparison between mean ± SD of Urinary MCP-1 in all the studied groups. 15 Comparison between mean ± SD of Urinary Hepcidin in all the studied groups. 16 Comparison between mean ± SD of Urinary MCP-1 and Hepcidin in lupus nephritis group (III) according to renal biopsy result. 17 Correlations between Urinary MCP-1 with creatinine, GFR and albumin/creatinine ratio in patients groups II and III. 18 Correlations between Urinary hepcidin with creatinine, GFR and albumin/creatinine ratio in patients groups II and III. 19 Correlations between Urinary MCP-1 with C3 and C4 in patients groups II and III. 20 Correlations between Urinary Hepcidin with C3 and C4 in patients groups II and III. 21 Correlation between MCP-1 and hepcidin in groups I, II and III IV

9 List of Figures List of Figures Figure No. Subject Page 1 Tubuloreticular structures in the glomerular endothelial cells 40 2 Mesangial proliferative lupus nephritis 41 3 Membranoproliferative glomerulonephritis 44 4 Membranous nephropathy 46 5 Chemokine (C-C motif) 53 6 Solution structure of hepcidin Mechanism of action of AntiC1q antibodies 70 8 Anti-α-actinin antibodies 73 9 Method of dilution of the marker Urinary MCP-1 (pg/ml) in all the studied groups Urinary hepcidin (ng/ml) in all the studied groups Urinary MCP-1 (pg/ml) in lupus nephritis groups according to 99 renal biopsy result. 13 Urinary hepcidin (ng/ml) in lupus nephritis groups according to 99 renal biopsy result. 14 Correlation between urinary MCP albumin/creatinine ratio in group II and III. 15 Correlation between urinary MCP-1 and GFR in group II and 101 group III 16 Correlation between urinary hepcidin and albumin/creatinine 103 ratio in group II and III VI

10 List of Figures 17 Correlation between urinary hepcidin and GFR in group II and 103 group III 18 Correlation between urinary MCP-1 and C Correlation between urinary MCP-1 and C Correlation between urinary hepcidin and C Correlation between urinary hepcidin and C Correlation between urinary MCP-1 and urinary hepcidin in 108 group III 23 Urinary hepcidin ROC curve for SLE and lupus nephritis 109 patients. 24 Urinary MCP-1 ROC curve for SLE and lupus nephritis patients. 110 VI

11 Introduction & Aim of work Introduction Kidney disease is one of the most serious manifestations of systemic lupus erythematosus (SLE). Glomerulonephritis is one of the commonest and most serious manifestations of systemic lupus erythematosus (Mok CC et al, 2005).Renal involvement in SLE carries significant morbidity and mortality. The 5- and 10-year renal survival rates of lupus nephritis in the 1990s range between 83-92% and 74-84%, respectively ( Dooley MA et al, 1997). The prognosis of lupus nephritis is particularly bad in certain ethnic groups such as the Africans and Hispanics (Mok CC et al, 2006).Despite the overall improvement in the care of SLE in the past two decades, the prognosis of lupus nephritis remains unsatisfactory. Up to 25% of patients still develop end stage renal failure 10 years after onset of renal disease (Mok CC et al, 2010). Besides exploring more effective but less toxic treatment modalities that will further improve the remission rate, early detection and treatment of renal activity may spare patients from intensive immunosuppressive therapies and reduce renal damage. Conventional clinical parameters such as creatinine clearance, proteinuria, urine sediments, anti-dsdna and complement levels are not sensitive or specific enough for detecting ongoing disease activity in the lupus kidneys and early relapse of nephritis. 1

12 Introduction & Aim of work Thus, novel biomarkers are necessary to enhance the diagnostic accuracy and sensitivity of lupus renal disease, prognostic stratification, monitoring of treatment response and detection of early renal flares. There have been promising biomarkers that have recently been evaluated in longitudinal studies of lupus nephritis. (Rovin BH et al, 2007) Although a large number of novel biomarkers have been studied in lupus nephritis, none of them have been rigorously validated in large-scale longitudinal cohorts of patients with different ethnic background. It is unlikely at this juncture that a candidate biomarker standalone can replace conventional clinical parameters to monitor disease progress and detect early renal flares. Urine biomarkers appear to be more encouraging than serum biomarkers possibly because they are the direct products or consequences of kidney inflammation or injury. Future directions in SLE biomarker research should focus on a combination of novel markers with conventional clinical parameters to enhance the sensitivity and specificity for the prediction of renal flares and prognosis in lupus nephritis. (Mok CC et al, 2010). 2

13 Introduction & Aim of work Aim of the work In order to improve the prognosis of lupus nephritis further, newer strategies with better efficacy but lower toxicities are necessary. More sensitive and specific clinical markers for the onset or relapse of renal disease activity in patients with SLE may allow earlier institution of treatment and even preventive strategies so that the efficacy of existing therapies can be enhanced while treatment-related complications can be minimized. In addition to the refinement of outcome assessment tools in SLE,inclusion of novel biomarkers as surrogate end-points in future lupus nephritis clinical trials may increase the feasibility of identification of subsets of patients who would benefit most from the newer regimens. Our study will include sixty patients with SLE subdivided into two groups. Group (I) Twenty healthy subjects will be recruited as controls.group (II) SLE patients wihout evidence of kidney affection. Group (III) SLE patients with lupus nephritis proved by renal biopsy. Urinary samples of both groups and control group will be examined for hepcidin (A low molecular weight peptide hormone mainly produced by the liver) (Malyszko J. et al, 2007 ). 3

14 Introduction & Aim of work Urinary excretion of hepcidin is greatly enhanced in patients with iron overload, infections, or inflammatory diseases) (Nemeth E et al, 2003). and Monocyte chemoattractant Protein-1 (A leukocyte chemotactic factor that is involved in mediating inflammation and injury in lupus nephritis) (Rovin BH et al, 2007). The markers will be compared with each other and other usual laboratory investigations: complement levels (C3, C4), antibodies to double-stranded DNA, ESR and albumin/creatinine ratio in urine using proper statistical methods. 4

15 Systemic lupus erythematosus CHAPTER ( I ) Systemic lupus erythematosus SLE is a systemic inflammatory disease characterized by autoantibody production and IC deposition (Yuan et al., 2011). It is a complex disease characterized by unpredictable flares of disease activity and irreversible damage to multiple organ systems (Gillespie, 2010). Lupus is characterized by a variety of clinical and laboratory abnormalities, including rash, arthritis, leucopenia, thrombocytopenia, alopecia, fever, nephritis, and neurologic disease. Most or all of the symptoms of acute lupus are attributable to immunologic attack on the affected organs. Many complications of long-term disease are attributable both to the disease and to its treatment (Zonana-Nacach et al., 2000). Epidemiology: SLE is primarily a disease of young women. Women between 15 and 45 years of age are the most commonly affected; the female-to-male ratio in this age group is between 6:1 and 9:1. African Americans are four times as likely to develop lupus as are whites (Gladman, 2004). The disease incidence in Asians, Hispanics, and Native Americans falls between that of blacks and whites. No explanation suggests why African Americans are more frequently affected; racial differences in SLE incidence persist when socioeconomic differences have been controlled. Overall survival is lower in African Americans (Karlson et al., 1997). The familial aspects of lupus are striking: approximately 10% of persons with lupus have family members with lupus or other autoimmune disease. Susceptibility to lupus is higher in persons with specific genetic deficiencies (Gladman, 2004) 5

16 Systemic lupus erythematosus In the United States, the annual incidence of SLE averages 5.1 per 100,000 population. The reported prevalence is 52 cases per 100,000 population. (Danchenko N et al, 2006) According to a 2008 report from the National Arthritis Data Working Group, approximately 250,000 Americans have SLE.The frequency of SLE could be increasing due to milder forms of the disease that are now being recognized. (Helmick CG et al, 2008) The frequency of SLE varies by race and ethnicity, with higher rates reported among black and Hispanic people. The prevalence of SLE is approximately 40 per 100,000 whites in Rochester, Minnesota, versus 100 per 100,000 Hispanic persons in Nogales, Arizona. The incidence of SLE in black women is approximately 4 times higher than in white women. SLE is also more frequent in Asian women than in white women. (Balluz L et al, 2001) In women, prevalence rates vary from 164 (white) to 406 (African American) per 100,000 (Chakravarty EF et al, 2007). Estimated incidence rates are 1 to 25 per 100,000 in North America, South America, Europe and Asia ( Pons-Estel GJ et al, 2010) The disease appears to be more common in urban than rural areas (Petri M.et al, 2002).The prevalence of SLE is higher among Asians, Afro- Americans, Afro-Caribbeans, and Hispanic Americans compared with Americans of European decent in the United States, and among Asian Indians compared with Caucasians in Great Britain (Peschken CA et al, 1999) Discoid skin lesions may be more frequent clinical manifestations in patients with Northern European than those with Southern European ancestry; the former group is, however, less likely to have anti-cardiolipin and anti-dsdna antibodies (Chung SA et al, 2009) 6

17 Systemic lupus erythematosus Blacks and Mexican Hispanics in the United States have a poorer renal prognosis than Caucasians, a finding not entirely independent of socioeconomic status. Blacks are more likely to have anti-sm, anti-rnp, discoid skin lesions, proteinuria, psychosis, and serositis (Fernández M et al, 2007) Blacks with lupus nephritis are also less likely to respond to cyclophosphamide treatment than Whites (Appel GB et al, 2009) The incidence of lupus is dramatically higher in women than in men. During the childbearing years, the female-to-male ratio is about 12:1. In patients with SLE that begins during childhood or later, the female-tomale ratio is approximately 2:1. The race- and sex-specific incidence rates of definite SLE per 100,000 persons were 0.4 in white males, 3.5 in white females, 0.7 in African American males, and 9.2 in African American females (Motha MB et al, 2009) In children, in whom sex hormonal effects are presumably minimal, the female-to-male ratio is 3:1.In adults, especially in women of childbearing years, the ratio ranges from 7:1 to 15:1.In "older" individuals, especially post-menopausal women, the ratio is approximately 8:1 ( Lahita RG. Et al, 1999) In support of the potential role of estrogens in predisposing to SLE, the Nurse's Health study showed that women with early menarche, or treated with estrogen-containing regimens, such as oral contraceptives or postmenopausal hormone replacement therapies, have a significantly increased risk for SLE (hazard ratios of 1.5 to 2.1) (Costenbader KH. Et al, 2007) Factors related to the X chromosome may also be important in predisposing women to SLE. (Moser KL et al, 2009 ). 7

18 Systemic lupus erythematosus There is also evidence for a gene dose effect, since the prevalence of XXY (Klinefelter's syndrome) is increased 14-fold in men with SLE when compared with the general population of men, whereas XO (Turner's syndrome) is underrepresented in women (Scofield RH et al, 2008 ) Other possibilities for female predisposition include: X-inactivation, imprinting, X or Y chromosome genetic modulators, differential methylation of DNA and acetylation of histones bound to DNA, intrauterine influences, chronobiologic differences, pregnancy, and menstruation (Ballestar E. et al, 2006) Men with lupus tend to have higher frequencies of renal disease, skin manifestations, cytopenias, serositis, neurologic involvement, thrombosis, cardiovascular disease, hypertension, and vasculitis than women (Lu LJ et al, 2010) In contrast, Raynaud phenomenon, photosensitivity, and mucosal ulceration are less frequent manifestations in men than women. Most, but not all studies suggest that men have a higher one-year mortality rate (Lu LJ et al, 2010) Median ages at diagnosis for white females range from 37 to 50 years, in white males from 50 to 59, in black females from 15 to 44 and in black males from 45 to 64 (Rus, V et al, 2002) The clinical status is poorer in those with less education (Fernández M et al, 2007) Clinical status is also poorer in those with lower socioeconomic status and with inadequate access to medical care.the extent and degree of activity of SLE varies in different countries and in different ethnic groups (Pons-Estel BA. Et al, 2004) Lupus tends to be milder in the elderly, who often have a presentation more similar to that of drug-induced lupus. Clinical features of lupus in 8

19 Systemic lupus erythematosus older patients include the following : A lower ratio of affected women to men than for younger patients Lower incidence of malar rash, photosensitivity, purpura, alopecia, Raynaud phenomenon, renal, central nervous system, and hematologic involvement, Lower prevalence of anti- La, anti-sm, and anti-rnp antibodies and of hypocomplementemia. Greater prevalence of sicca symptoms, serositis, pulmonary involvement, and musculoskeletal manifestationsgreater prevalence of rheumatoid factor. (Lalani S et al, 2010) Pathophysiology and Pathogenesis: Autoantibodies Circulating antibodies to a broad list of autoantigens characterize SLE. Antinuclear antibodies, usually defined by IF, are present in almost all lupus patients; tests for ANA constitute a screening test (sensitive, but not specific) for the illness. Autoantibodies to nuclear constituents (double-stranded DNA, single-stranded DNA, histones, ribonuclear proteins, and other nuclear antigens, such as the Smith (Sm) antigen confirm the diagnosis and are likely pathogenic. For example, these autoantibodies cause glomerulonephritis by inciting inflammation when deposited as complement-fixing ICs on GBMs or by binding directly to the GBM (Ippolito et al, 2011) Abnormal Innate and Adaptive Immunity Genetic defects of IC processing are unusually frequent in lupus patients, suggesting that SLE arises because of incomplete or improper disposal of exogenous material (Walport, 2000). Such defects include abnormalities in complement (e.g., deficiency of C1q), apoptotic pathways and phagocytic cells (Blanco et al., 2001). 9

20 Systemic lupus erythematosus Defective clearance of ICs may result in their persistence in large quantities (Davies et al., 2002). Other theories of pathogenesis argue that genetic predispositions that promote T helper type 2 cell (Th2) responses or cytokine dysregulation are the underlying defects leading to the development of SLE (Marrack et al., 2001). Gene array data strongly indicate that interferon genes are markedly upregulated in patients with active SLE (Crow and Kirou, 2004). Genetic Susceptibilities Twin and family studies of SLE make it abundantly clear that the illness is highly heritable. HLA types DR 3 and DR 4 predominate in SLE patients (Reveille, 1999). Persons with genetic deficiencies of complement appear to be more susceptible to the development of lupus (Abel and Agnello, 2004). Infections Autoantibodies can be identified in serum specimens up to a decade before the earliest symptoms of SLE. Autoantibodies first appear as one specific antibody, and then generalize just before clinical onset. Whether this progression reflects response to infection or autoimmunity is unknown (Arbuckle et al., 2003). Estrogen Some investigators attribute the female predominance of SLE and its occurrence in childbearing years to the upregulating effect of estrogen on the immune system, a phenomenon demonstrable largely in vitro. However, this argument applies to autoimmunity in general, not specifically to lupus, and it fails to explain why other autoimmune diseases have much less striking female-to-male ratios. Furthermore, 10