A Translational Approach to Micro-Inflammation in End Stage Renal Disease: Molecular Effects of Low Levels of Interleukin 6
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A translational approach to micro-inflammation in End Stage Renal Disease: molecular effects of low levels of Interleukin 6 Bruno Memoli, Simona Salerno, Alfredo Procino, Loredana Postiglione, Sabrina Morelli, Maria Luisa Sirico, Francesca Giordano, Margherita Ricciardone, Enrico Drioli, Vittorio E. Andreucci, et al. To cite this version: Bruno Memoli, Simona Salerno, Alfredo Procino, Loredana Postiglione, Sabrina Morelli, et al.. A translational approach to micro-inflammation in End Stage Renal Disease: molecular effects oflowlev- els of Interleukin 6. Clinical Science, Portland Press, 2010, 119 (4), pp.163-174. 10.1042/CS20090634. hal-00593329 HAL Id: hal-00593329 https://hal.archives-ouvertes.fr/hal-00593329 Submitted on 14 May 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Clinical Science Immediate Publication. Published on 09 Apr 2010 as manuscript CS20090634 A translational approach to micro-inflammation in End Stage Renal Disease: molecular effects of low levels of Interleukin 6 by Bruno Memoli MD, **Simona Salerno PhD, Alfredo Procino PhD, *Loredana Postiglione PhD, **Sabrina Morelli PhD, Maria Luisa Sirico MD, *** Francesca Giordano PhD, *Margherita Ricciardone PhD, **Enrico Drioli PhD, Vittorio E. Andreucci MD, and **Loredana De Bartolo PhD from Depts of Nephrology and *Cellular and Molecular Biology and Pathology, University Federico II of Naples, **Institute of Membrane Technology, National Research Council of Italy, ITM-CNR, ***Department of Cellular Biology, University of Calabria, Rende, Italy Running Title: Effects of low levels of IL-6 Corresponding author: Prof. Bruno Memoli MD, Department of Nephrology, University Federico II of Naples, Via S. Pansini 5, 80131 Napoli, Italy. THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Phone & Fax: +390817462641; e-mail: [email protected] Accepted Manuscript 1 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited Clinical Science Immediate Publication. Published on 09 Apr 2010 as manuscript CS20090634 ABSTRACT Inflammation plays a key role in the progression of cardiovascular disease, the leading cause of mortality in End Stage Renal Disease (ESRD). In the last years inflammation has been greatly reduced but mortality remains high. This study was addressed to assess if low (< 2 pg/ml) circulating levels of interleukin 6 (IL-6) are necessary and sufficient to activate STAT3 transcription factor in human hepatocytes, and if this micro-inflammatory state can be associated with changes in gene expression of some acute phase proteins involved in cardiovascular mortality in ESRD. Human hepatocytes were treated for 24 hours in presence and absence of serum fractions from ESRD patients and healthy subjects with different concentration of IL-6. The specific role of the cytokine was also evaluated by cell experiments with serum containing blocked IL-6. Furthermore a comparison of the effects of IL-6 from patient serum and recombinant (rIL-6) at increasing concentrations was performed. Confocal microscopy and western blotting demonstrated a STAT3 activation associated with IL- 6 cell membrane-bound receptors over-expression only in hepatocytes cultured with 1.8 pg/ml of serum IL-6. A linear STAT3 activation and IL-6 receptors expression was also observed after incubation with rIL-6. The hepatocyte treatment with 1.8 pg/ml of serum IL-6 was also associated with 31.6-fold hepcidin gene expression up-regulation and 8.9-fold fetuin-A gene expression down-regulation. In conclusion these results demonstrated that low (<2 pg/ml) circulating levels of IL-6, as present in non-inflamed ESRD patients, are sufficient to activate some inflammatory pathways and can differently regulate hepcidin and fetuin-A gene expression. Key Words: End Stage Renal Disease, micro-inflammation, IL-6, STAT3, Hepcidin, Fetuin-A THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript 2 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited Clinical Science Immediate Publication. Published on 09 Apr 2010 as manuscript CS20090634 INTRODUCTION Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in end stage renal disease (ESRD); approximately 50% of ESRD patients die from CVD and cardiovascular mortality is 15 to 30 times higher than in age-adjusted general population [1]. In ESRD, in addition to traditional Framingham risk factors, a considerable number of non- classical factors are known to play a role in the CVD progression, such as inflammation [2], vascular calcifications [3] and left ventricular hypertrophy [4]. The presence of inflammation, mainly induced by the poor biocompatibility of renal replacement therapy (RRT) [5-7], is evidenced by elevated circulating levels of C-reactive protein (C-RP) and interleukin 6 (IL-6), both considered strong predictors of poor outcome [8-10]. Bologa et al [8], in particular, have reported that the relative risk associated with each 1 pg/ml increase in IL-6 concentration was enhanced by 4.4% in ESRD patients. IL-6 is a cytokine that provokes a broad range of cellular and physiological responses, including the immune response, inflammation, hematopoiesis, and oncogenesis by regulating cell growth, gene activation, proliferation, survival, and differentiation. IL-6 signals through a receptor composed of two different subunits, an alpha subunit (glycoprotein 80 or gp80 or IL-6R) that produces ligand specificity and gp130, a receptor subunit shared in common with other cytokines in the IL-6 family [11, 12]. Binding of IL-6 to its receptor initiates cellular events including activation of JAK (Janus Kinases) and Ras-mediated signaling. Activated JAK phosphorylate and activate STAT transcription factors, particularly STAT3 (Signal Transducers and Activators of Transcription-3) [13]. Phosphorylated STAT3 then forms a dimer and translocates into the nucleus to activate transcription of genes containing STAT3 response elements [14]. IL-6 is also the major inductor of acute phase protein synthesis in liver cells. A typical pattern of acute phase protein in ESRD is represented by lower plasma concentrations of fetuin-A [15] and higher concentrations of hepcidin [16]. Vascular calcifications have been associated in ESRD patients to low circulating levels of fetuin-A [15]. Fetuin-A, in fact, is an important circulating inhibitor of calcification in vivo [17]; it is produced by hepatocytes and down regulated by IL-6 [18]. Low concentrations of this glycoprotein were associated with raised amounts of C-reactive protein and enhanced cardiovascular mortality [15]. Hepcidin is a circulating peptide hormone mainly synthesized in the liver, which has been recently proposed as a factor regulating the iron homeostasis through the interaction with the main iron export protein, ferroportin [19]. When hepcidin concentration increases, it binds to ferroportin molecules and induces their internalization and degradation, and iron release is decreased. Inflammation causes an increase of hepcidin production, which is, therefore, a potent mediator of anemia in chronic diseases [19-22]; anemia, in turn, plays a key role in left ventricular hypertrophy (LVH) occurrence and progression [23]. LVH has long been known as an important and independent risk factor for death and cardiovascular events, in both dialysis patients and general population [24]. In the last years a great improvement in the quality of RRT has been obtained; but, despite the reduction of inflammation, cardiovascular mortality remains still high. In ESRD the concept of micro-inflammation (i.e. small increases in circulating levels of IL-6 THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 and C-reactive protein, with no signs or clinical symptoms of inflammation) is still object of more accurate evaluation [25]. In particular, it is not clear which concentration of IL-6 is necessary and sufficient to trigger a micro-inflammatory condition and how the last may be more accurately defined. A mean level of 4.4 pg/ml of IL-6 was observed in ESRD patients free of inflammatory clinical events, with a large intra-individual variation [25]. In another study values between 1.6 and 2.5 pg/ml were observed in dialysisAccepted patients with no inflammation/malnutrition Manuscript [26]. These values are markedly different from those found in healthy population, where circulating levels are detected around 1.0 pg/ml or less [27]. This could suggest that even small raises in IL-6 circulating values could play a role in Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited Clinical Science Immediate Publication. Published on 09 Apr 2010 as manuscript CS20090634 enhancing CVD risk. This hypothesis is supported by Ridker et al [28] who observed that very small increases