A Translational Approach to Micro-Inflammation in End Stage Renal Disease: Molecular Effects of Low Levels of Interleukin 6

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

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

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

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

enhancing CVD risk. This hypothesis is supported by Ridker et al [28] who observed that very small increases in IL-6 levels (1.81 vs 1.46 pg/ml) was associated, in apparently healthy subjects, with an increased risk of future myocardial infarction. No data are available in ESRD patients demonstrating that small increases in circulating levels of IL-6 are sufficient to activate, in hepatocytes, the gp130-STAT3 cascade, as well as to regulate the gene expression of some acute phase proteins involved in the cardiovascular risk. This issue was the main object of the present study. In particular, we have investigated whether low (< 2 pg/ml) serum concentrations of IL-6, obtained by incubating serum fractions from ESRD patients in culture medium of primary human hepatocytes, are able to induce in these cells significant IL-6-dependent molecular modifications (i.e. gp130 and STAT3 activation), as well as to modulate a different gene expression of fetuin-A and hepcidin.

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

METHODS Patient selection In order to obtain sufficient amount of serum we selected 12 ESRD patients undergoing regular dialysis treatment (RDT) in our renal unit. All patients were undergoing standard dialysis for at least 1 year before the study. No patient presented fever or any clinical symptom of inflammation and none assumed steroid or immunosuppressive therapy. 6 healthy, age-matched, laboratory staff volunteers were also included in the study, as healthy control group. The study was approved by the local Ethic Committee and all enrolled subjects (healthy and ESRD patients) gave their informed consent before the study. In Table 1 are reported the clinical features of both healthy subjects (as control group) and ESRD patients included in the study. The ESRD patients were divided in two subgroups on the basis of C-reactive protein (C-RP) circulating levels. First subgroup (C-RP ≤ 3 mg/L) included 6 patients (non- inflamed ESRD); even the second group (C-RP > 3 mg/L) included 6 patients (micro-inflamed ESRD). Despite the small number of patients, the resulted serum amount necessary to perform our cell experiments was sufficient and adequate. Sera fractions (10 ml from each serum sample), obtained from all subjects of the same group (Healthy subjects, non-inflamed ESRD and micro-inflamed ESRD, respectively) were, indeed, pooled together in order to obtain larger amounts of serum with the same characteristics (in particular, with the same concentrations of IL-6 and its soluble receptors). C-RP and Cytokine Assays C-RP circulating levels were determined by a high-sensitivity ELISA assay (Bender MedSystems, Vienna, Austria) on serum aliquots of all subjects included in the study. The lower detection limit was 3 pg/ml, and the overall intra-assay coefficient of variation has been calculated to be 6.9%. A circulating value of C-RP ≤ 3.0 mg/L was considered within the normal range [29]. The concentrations of IL-6, sIL-6R and sgp130 in serum pools were evaluated by using three different ELISA kits (Quantikine, R&D Systems, Minneapolis, MN). The lower detection limit of IL-6, sIL-6R and sgp130 assays was respectively 0.1 pg/ml, 1.5 pg/ml and 0.05 pg/ml. Inter- and intra-assay variation coefficient resulted < 5% for all assays. IL-6 blocking In order to confirm the specific role of IL-6 in the study, we also performed experiments after the neutralization of this cytokine in the serum by using a specific blocking antibody, the anti-human IL-6. This antibody produced in goats immunized with purified recombinant human interleukin-6 (rIL- 6) was selected for its ability to neutralize IL-6 activity of both natural and recombinant human IL-6 (AB-206-NA, R&D System, Inc, Minneapolis, MN). Additionally in direct ELISA this antibody shows no cross-reactivity with other cytokines tested. Optimal concentration of antibody required to neutralize rIL-6 was determined using 3 samples with different concentration of rIL-6 prepared in our laboratory: Std 1 (5 pg/ml of rIL-6); Std 2 (10 pg/ml of rIL-6); Std 3 (20 pg/ml of rIL-6). To measure the ability of the antibody to neutralize rIL-6 in these standards, rIL-6 was incubated with various concentrations of the antibody at 4°C over night and evaluated at room temperature by IL-6 ELISA assay previously described. Design of the study on hepatocytes THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 We performed our study on human hepatocytes incubated for 24 hours with low (< 2.0 pg/ml) IL-6 concentrations obtained by the addition of human serum drawn from both ESRD patients and healthy subjects. Human hepatocyte cultures CryopreservedAccepted primary human hepatocytes (BDManuscript Bioscience, Milan, Italy) were thawed and cultured in batch system. Human hepatocytes were seeded at a final concentration of 2.5x105 cells/cm2 on modified polyetheretherketone (PEEK-WC) semipermeable membrane. We prepared these

membranes by inverse phase technique by using the direct immersion-precipitation method [30]. Cell cultures were stimulated with three different low (< 2 pg/ml) IL-6 concentrations obtained by adding serum fractions from healthy subjects (medium IL-6 concentration = 0.1 pg/ml), non- inflamed ESRD patients (medium IL-6 concentration = 0.6 pg/ml) and micro-inflamed ESRD patients (medium IL-6 concentration = 1.8 pg/ml). Since cell culture medium cannot contain more than 20% as serum, the IL-6 concentrations in the cultures resulted five times lower than IL-6 concentrations in whole serum (as reported in Table 2). It is important to outline that 1.8 pg/ml is an IL-6 concentration uncommon in inflamed ESRD patients and also lower than in non-inflamed (Table 2). Therefore, to avoid any confusion, we will report in the rest of the paper only the final IL-6 concentration in the medium (with no indication about the origin group). In order to evaluate the specific effect of IL-6 contained in serum samples, human hepatocytes were also treated with 20% serum from micro-inflamed ESRD patients (8.88 pg/ml IL-6) and neutralized 12 hours before with anti-human IL-6 antibody. Similarly, the effects of IL-6 were differentiated from the effects of other components contained in the sera, stimulating hepatocytes with human recombinant IL-6 (rIL-6, Sigma, Milan, Italy) at the same concentrations of the 20% serum (0.1, 0.6, and 1.8 pg/ml, respectively). In addition, we also utilized a rIL-6 concentration of 2.97 pg/ml (corresponding to the IL-6 concentration observed in the whole serum pool of non-inflamed ESRD patients, Table 2). Each group of cultures, constituted by 12 samples, was stimulated for 24 hours and then utilized for the immuno-staining of phosphorylated-STAT3 (p-STAT3), gp130 and gp80. Cells were incubated at 37°C in a 5% CO2; 20% O2 atmosphere (v/v) with 95% relative humidity for the duration of the experiments. Hepatocyte staining for Laser Confocal Scanning Microscopy After 24 hours of culture, hepatocytes were washed with PBS, fixed for 15 min in 3% paraformaldheyde in PBS at room temperature and dehydrated for 5 min with cold methanol and 5 min with cold acetone. For double immunostaining of p-STAT3 and gp130 were utilized a rabbit polyclonal affinity purified antibody raised against p-STAT3 of human origin and a mouse monoclonal antibody raised against gp130 of human origin (Santa Cruz Biotechnology, Santa Cruz, CA). For double immunostaining of gp80 and gp130 were utilized a rabbit polyclonal affinity purified antibody raised against a peptide mapping at the C-terminus of IL-6Rα/gp80 of human origin and the mouse monoclonal antibody raised against gp130 of human origin (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Primary antibodies were incubated 1:200 for 2 hours at room temperature. As secondary antibodies were utilized a CyTM2-conjugated AffiniPure donkey anti-rabbit IgG and a CyTM3- conjugated AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch Europe Ltd, Cambridge, UK) incubated 1:100 for 1.5 hours at room temperature. To visualize nucleic acid the samples were washed twice in PBS and incubated for 20 min with DAPI 0.2 µg/ml (Molecular Probes Inc, Eugene, OR). Finally samples were washed, mounted and viewed with Laser Confocal Scanning Microscope THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 (Fluoview FV300, Olympus, Italy). Imaging and Quantitative Analysis Imaging of labelled human hepatocytes were obtained by using an Olympus Fluoview FV300 Laser Confocal Scanning Microscope (Olympus, Italy). Quantitative analysis was performed on different area ofAccepted 3 samples of each investigated treatmentManuscript using Fluoview 5.0 software (Olympus Corporation) and calculating the average intensity per cell vs the axis z of acquired images of 0.5 µm optical thickness. Calibration curves of fluorescence average intensity for p-STAT3, gp130 and gp80

were also obtained by incubating cells with rIL-6 at the same concentrations obtained in the 20% serum (0.1, 0.6 and 1.8 pg/ml, respectively) and a rIL-6 concentration of 2.97 pg/ml corresponding to the IL-6 concentration observed in the whole serum pool of non-inflamed ESRD patients (Table 2). Western blotting analysis The results obtained with confocal analysis were also compared with western blotting analysis. For western blotting analysis the proteins were extracted according the following procedure. After 24 hours of stimulation with serum IL-6 (0.6, 1.8 pg/ml) and under serum-free condition (Control), human hepatocytes were washed once with cold PBS and then pelletted by centrifugation. The cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100) supplemented with protease and and phosphatase inhibitor cocktails, vortexed and incubated for 40 min at 4°C. During the incubation time, the samples have been sonicated for 30 s and centrifuged at 10,000 rpm for 20 min at 4 °C. The supernatants were transferred in new tubes and the protein concentration was determined by using the QBIT fluorometer (Invitrogen, Paisley, UK). Western blotting was performed as previously described [31]. Equal amounts of protein (30 µg) were boiled for 5 minutes, separated under denaturing conditions by SDS-PAGE on 6% polyacrylamide Tris-glycine gels and electroblotted to nitrocellulose membrane. Non-specific sites were blocked with 5% non fat dry milk in 0.1% Tween-20 in Tris-buffered saline (TBS-T) for 1 hour at room temperature and incubated overnight with anti-human IL-6Rα/gp80 (1:200), anti-human gp130 (1:200), anti-human p-STAT3 (1:200), anti-human β-actin (1:500) primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The antigen–antibody complex was detected by incubation of the membranes for 1 h at room temperature with a peroxidase-coupled anti-IgG antibody (1:3000) (Santa Cruz Biotechnology, Santa Cruz, CA) and revealed using the ECL Plus Western blotting detection system (Amersham, USA) according to the manufacturer's instruction. Each membrane was exposed to the film for 1 minutes. Quantitative analysis by RealTime-PCR of fetuin-A and hepcidin RealTime-PCR analysis was performed on human hepatocytes after 24 hours of stimulation with 20% serum from patients containing IL-6 at concentration of 0.1, 0.6 and 1.8 pg/ml, 20% serum with blocked IL-6 and under serum free condition (Control). Cells were detached from PEEK-WC membrane by the addition of 0.05% collagenase in PBS (Sigma, Milan, Italy). Hepatocytes were then lysed in guanidinium isothiocyanate. Total RNA was extracted by the single-step method, using phenol and chloroform/isoamylalcohol. 4 µg of total RNA were subjected to cDNA synthesis for 1 h at 37°C using the ‘‘Ready to go You-Primer First-Stand Beads’’ kit (Amersham Pharmacia Biotech Little Chalfont Buckinghamshire, UK) in a reaction mixture containing 0.5 microgr oligo-dT (Amersham Pharmacia Biotech Little Chalfont Buckinghamshire, UK). Real-time quantitative PCR analysis for fetuin-A and hepcidin genes was performed using ABI prism 7500 (Applied Biosystem, Foster City, CA) and the 50 exonuclease assay (Taq-Man technology), as described elsewhere [18]. The results were analyzed using a comparative method, and THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 the values were normalized to the β-actin expression and converted into fold change based on a doubling of PCR product in each PCR cycle, according to the manufacturer’s guidelines. Statistical analysis Statistical analysis was performed using the ANOVA (followed by Bonferroni, as post hoc test) and linear regressionAccepted analysis. Results are expressed Manuscriptas mean ± SD; statistical significance was defined as p<0.05.

RESULTS Serum cytokine concentrations As shown in Table 2, the whole serum pool obtained from micro-inflamed ESRD patients showed circulating values of IL-6 (8.88 pg/ml) higher than the pool from non-inflamed ESRD patients (2.97 pg/ml); the last, in turn, resulted higher than that from healthy subjects (0.53 pg/ml). These data confirm previous results of our group and other authors [25-28, 32, 33]. Similar differences were obtained with the concentrations of “agonistic” IL-6 soluble receptor (sIL-6R). On the contrary, no difference was observed among the concentrations of soluble “antagonistic” IL-6 receptor (sgp130) (Table 2). Human hepatocytes were incubated with serum fractions pooled together according to the three groups of enrolled subjects; since hepatocyte culture medium contain only 20% of serum, IL-6 cultures concentrations resulted five fold lower than whole serum pools (0.1, 0.6 and 1.8 pg/ml of IL-6, respectively, as reported in Table 2). The specific effects of IL-6 on hepatocytes were also evaluated by treatment with serum containing blocked IL-6. The concentration of antibody required to neutralize rIL-6 activity was evaluated by using different concentrations of anti human IL-6 antibody. As shown in Figure 1, the rIL- 6 (10 pg/ml) was completely neutralized by using 2.00 µg/ml of antibody. This concentration was therefore chosen to ensure the complete neutralization of IL-6 contained in the serum from micro- inflamed ESRD patients.

Effects of low levels of IL-6 on gp130 and STAT3 activation We explored, by confocal microscopy, the expression of gp130 and the activation of the STAT3 (that occur via phosphorylation at tyrosine residue 705) on human hepatocytes cultured for 24 hours. In Fig. 2 we report gp130 (red) and p-STAT3 (yellow), detected by immunofluorescence after 24 hours of culture with serum fractions; gp130 and p-STAT3 fluorescence signals were similar in Control (untreated cells) and in hepatocytes incubated with an IL-6 concentration of both 0.1 and 0.6 pg/ml. No remarkable differences were found among the groups. On the contrary, the stimulation of hepatocytes with an IL-6 concentration of 1.8 pg/ml resulted in a noticeable increase of the gp130 expression and in a strong activation of the STAT3. Hepatocytes treated with serum containing neutralized IL-6 showed a fluorescence signal similar to the untreated cells and to hepatocytes incubated with serum containing 0.1 pg/ml IL-6. Similar results were obtained already after 3 hours of incubation (data not reported). Besides the gp130 membrane receptor we also explored the expression of the gp80 and its localization on the cell membrane. In Fig. 3 we can see also the co-localization of gp80 (or IL-6R, green) and gp130 (red) on the hepatocyte membrane and their different expression after 24 h of different treatments. The fluorescence signal was high for both receptors only in the samples treated with an IL-6 concentration of 1.8 pg/ml. Conversely, the gp80 and gp130 expression decreased to a large extent in the other cultures and in the treatment with serum containing blocked IL-6. To confirm these observations we also performed a quantitative analysis of the fluorescence THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 average intensity for immuno-stained gp80, gp130 and p-STAT3 (Fig. 4). In Control (untreated cells), in hepatocytes treated with IL-6 at concentrations of 0.1 and 0.6 pg/ml and in hepatocytes treated with serum containing neutralized IL-6 the fluorescence signals for gp80, gp130 and p-STAT3 were similar. On the contrary, a strong STAT3 activation was found in hepatocytes treated with serum IL-6 concentration of 1.8 pg/ml; the average intensity in this group reached a peak value of 8463±3335 significantly (p<0.05)Accepted higher with respect to the ot herManuscript cultures. Also the average intensity for gp80 and for gp130, that were 11955±4670 and 10061±3710, respectively, resulted to be significantly higher (p<0.05) than those found in the other cultures. A decrease of cytokine concentration from 1.8 pg/ml to

0.6 pg/ml resulted in a decrease of the fluorescence average intensity of both IL-6 membrane-bound receptors and p-STAT3. The blocking of IL-6 abrogated the induction effect on STAT3 and on IL-6 membrane-bound receptors, as demonstrated by the fluorescence average intensity values. Afterwards, in order to confirm the role of serum IL-6 in STAT3 activation, we also cultured human hepatocytes with recombinant IL-6 (rIL-6) at the same concentrations of serum fractions. Fig. 5 shows the linear increase of fluorescence average intensity for gp80, gp130 and p-STAT3 with increasing the concentration of rIL-6 from 0 to 2.97 pg/ml (the last concentration was identical to the IL-6 concentration found in the whole serum of non-inflamed ESRD patients): the R-squared values of the straight lines through the axis resulted 0.975 for gp80, 0.988 for gp130 and 0.975 for p-STAT3, respectively. Also in this case the stimulation of cells with rIL-6 recombinant produced an increase of the expression IL-6 membrane-bound receptors with the consequent strong activation of p-STAT3 at higher concentrations of rIL-6. However, it is interesting to note the different effect of rIL-6 with respect to the serum IL-6 from patients Fig. 6 shows the comparison of the fluorescence average intensity values of gp80, gp130 and p-STAT3 among control, cells treated with rIL-6 and cells treated with the same cytokine concentration from patient’s serum. A significant difference in the fluorescence average intensity values for cells treated with serum IL-6 (0.6 pg/ml) was found only for stained gp80 (p<0.05). No remarkable differences were observed between the groups treated with rIL-6 and the control (Fig. 6a). On the contrary striking differences were found at increasing concentration of serum IL-6: all the fluorescence average intensity of the immunostained proteins for hepatocytes treated with serum IL-6 at 1.8 pg/ml resulted significantly higher (p<0.01) with respect to those incubated with the rIL-6 at the same concentration (Fig. 6b). Finally, when hepatocytes were incubated with rIL-6 at concentration of 2.97 pg/ml, a value found in the serum of non-inflamed ESRD patients, a further increase of the fluorescence signal for gp80, gp130 and p-STAT3 was observed (data not shown). Western blotting results confirmed the significant increase of STAT3 activation, gp80 and gp130 IL-6 receptor expression in the presence of serum containing 1.8 pg/ml of IL-6, as observed by confocal microscope quantitative analysis. Marked bands were observed in the expression of pSTAT3, gp80 and gp130 in the hepatocyte samples treated serum IL-6 with 20% serum from micro-inflamed ESRD patients (1.8 pg/ml of IL-6) with respect to those treated with 20% serum from non-inflamed ESRD patients (0.6 pg/ml of IL-6) and control (Fig. 7).

Effects of inflammation on fetuin-A and hepcidin gene expression In Figure 8 we report the results of RealTime-PCR analysis for both hepcidin and fetuin-A expressed in human hepatocytes treated with 20% serum containing IL-6 at concentration of 0.1, 0.6 and 1.8 pg/ml, 20% serum with blocked IL-6 and under serum free condition (Control). It is evident that the treatment of cells with serum containing 1.8 pg/ml of IL-6 up-regulated (31.6-fold) the hepcidin and down-regulated the fetuin-A (8.9 fold) gene expression with respect to the Control. As a result, fetuin-A gene expression was modulated to a less extent with respect to the hepcidin. The treatment of cells with serum containing blocked IL-6 abrogated the up- and down- THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 regulation of hepcidin and fetuin-A gene expression respectively reaching values similar to untreated cultures (Control).

Accepted Manuscript

DISCUSSION

The results of this study clearly demonstrate that low concentrations of IL-6 (1.8 pg/ml) present in the culture medium of primary human hepatocytes and arising from the addition of serum fractions from micro-inflamed ESRD patients, were necessary and sufficient to activate, in these cells, gp80, gp130 and STAT3; this phenomenon, observed by confocal microscopy and western blotting analysis after 24 hours of incubation (Figs. 2, 4, 7), was associated with a noticeable (over 30 times) up- regulation of hepcidin gene expression (Fig. 8). Lower effects on STAT3 and IL-6 receptors activation were obtained with the same concentrations of recombinant IL-6 (Figs. 5 and 6). No significant effect was obtained incubating human hepatocytes with both 0.1 and 0.6 pg/ml of IL-6. Importantly, when IL- 6 activity was completely blocked in the serum containing 1.8 pg/ml of this cytokine, no effect was observed in STAT3 activation. It is important to underline that these inflammatory effects were obtained by incubating hepatocytes cultures with IL-6 concentrations five fold lower than those circulating in apparently healthy ESRD patients. On the basis of these results, therefore, we can speculate that circulating levels of IL-6, even in non-inflamed ESRD patients (serum IL-6 = 2.97 pg/ml, C-RP ≤ 3.0 mg/L), are sufficient to activate the acute phase response in human hepatocytes. Therefore, our data suggest that the control of inflammation in our patients represents still a great problem and the target value of 1 pg/ml of IL-6, considered as normal, is quite far to reach, despite the evident improvement of RRT quality. STAT3, the major signal transducer downstream of gp130-like receptors, was first described as a DNA-binding activity from IL-6-stimulated hepatocytes, able of selectively interacting with an enhancer element in the promoter of acute-phase genes [14]. Recent studies have demonstrated that IL- 6 induced hepcidin expression through STAT3 activation and subsequent hepcidin promoter binding, and that STAT3 was necessary and sufficient for the IL-6 responsiveness of the hepcidin promoter [22, 34]. In our study, a significant activation of STAT3, associated with a gp130 over-expression, was observed after 24 hours of incubation with 1.8 pg/ml of serum IL-6 in the medium. The results of our study also demonstrate, although at lesser extent, a serum-induced down- regulation of fetuin-A gene expression. This phenomenon is not secondary to STAT3 activation since it is well known that fetuin-A encoding gene is controlled by a series of binding sites for different C/EBP (enhancer binding protein) isoforms [35, 36]. IL-1, IL-6, and TNF-α activate, through specific cell receptors, three major transcription factor families: NFkB, C/EBPs, and STATs [37]. These factors differently regulate acute phase protein genes expression at the transcriptional level. IL-1 and TNF-α activate NFkB while IL-6 specifically induces the tyrosine phosphorylation and DNA binding activity of STAT3. CCAAT/enhancer binding proteins (C/EBPs) are a family of six proteins, the most widely expressed, and most well studied, being the C/EBP-α and C/EBP-β isoforms. These isoforms, regulated by both IL-1 and IL-6, display contrasting functions in gene activation and cell proliferation [38, 39]. Our results, therefore, suggest that low circulating levels of IL-6 may also play a significant role in activating the C/EBPs transcription factor.

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 The second important result of our study is the significant difference between the effects obtained in human hepatocytes incubated with serum fractions with 1.8 pg/ml of IL-6, as compared with those observed with the same concentration of recombinant IL-6 (Fig. 6). This serum enhancement of IL-6 effects could be explained by at least two different factors: a) the presence in the inflamed serum of other pro-inflammatory cytokines (i.e. IL-1 and TNF-α) and factors, and b) the presence in the Acceptedserum of the soluble IL-6 receptor (sIL-6R). Manuscript The first factor is less reasonable, since the axis gp130-STAT3 is specifically and exclusively activated by IL-6-type cytokines (mainly IL-6) [14, 40], while IL-1-type cytokines (like IL-1 and TNF−α) stimulate acute phase response by NFkB and/or

C/EPBs activation [40, 41]. In addition, the specific IL-6 neutralization completely abolished any effect induced by the serum. Besides the membrane-bound gp80 (or IL-6R), a naturally occurring soluble form, named sIL- 6R, is generated by two independent mechanisms, limited proteolysis of the membrane protein and translation from an alternatively spliced mRNA [33, 34, 42]. Interestingly, the sIL-6R together with IL- 6 stimulates cells that only express gp130, a process named trans-signaling [12]. This process implicates that cells which were originally unresponsive to IL-6 (because of the lack of membrane- bound IL-6R) become now responsive via the soluble sIL-6R/IL-6 complex. About 70% of the circulating IL-6 forms a complex with the sIL-6R in the blood and binds directly to membrane gp130. The other 30% is supposed to have only a transient existence in the blood, or binds to the membrane- bound receptor (IL-6R). sIL-6R functions, therefore, as a carrier protein for its ligand, thereby markedly prolonging the plasma half-life of IL-6 and indicating that IL-6 signaling is increased by sIL- 6R [12]. sIL-6R is largely present in our serum samples, particularly in the serum from micro-inflamed ESRD patients (Table 2). Taken together, these results allow us to define more accurately the micro-inflammation and demonstrate that even small amounts of IL-6 (< 2 pg/ml), in presence of sufficient concentrations of sIL-6R, are able to effectively modulate (up- and down-regulate, respectively) different genes of acute phase proteins, such as hepcidin and fetuin-A, that are emerging as contributors to cardiovascular mortality in ESRD patients [43, 44]. The specific role of IL-6 in the activation of STAT3 pathway and in the modulation of hepcidin and fetuin-A gene expression was proven by blocking IL-6 present in the serum, which, as expected, did not induce any increase in the STAT3 activation and IL-6 cell membrane receptors expression. These results are consistent with those obtained by Ridker et al [28], who observed an increased risk of future myocardial infarction associated with a small increase in IL-6 circulating levels ([IL-6]: 1.81 vs 1.46 pg/ml) in apparently healthy subjects. In conclusion, our results for the first time demonstrate, at molecular level, the chain of events underlying the incidence of some risk factors involved in cardiovascular events in apparently healthy ESRD patients and open a new scenario in the study and relevance of micro-inflammation. THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

ACKNOWLEDGMENTS This work was supported by the “Fondo per gli Investimenti della Ricerca di Base” (RBNE012B2K-2002) from “Ministero dell’Istruzione, dell’Universita` e della Ricerca, MIUR” and by the “PRIN 2007” (2007SCPN4C), also from MIUR. This study was presented in part at the Annual Meeting of the American Society of Nephrology, Philadelphia, PA, November 4 - 9, 2008 and published in abstract form.

DISCLOSURE No author declares any potential Conflict of Interest in relationship with pharmaceutical companies, biomedical device manufacturers, or other corporations whose products or services are related to the subject matter of the article. .

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

REFERENCES

1) Schiffrin, E.L., Lipman, M.L., Mann, F.E. (2007) Chronic kidney disease: effects on the cardiovascular system. Circulation 116, 85-97. 2) Kaysen, G.A. (2001) The microinflammatory state in uremia: causes and potential consequences. J. Am. Soc. Nephrol. 12, 1549-1557. 3) Cozzolino, M., Brancaccio, D., Gallieni, M., Slatopolsky, E. (2005) Pathogenesis of vascular calcification in chronic kidney disease. Kidney Int. 68, 429-436. 4) Levin, A. (2002) Anemia and left ventricular hypertrophy in chronic kidney disease populations: a review of the current state of knowledge. Kidney Int. 61(S80), S35-S38. 5) Memoli, B., Libetta, C., Rampino, T., Dal Canton, A., Conte, G., Scala, G., Ruocco, M.R., Andreucci, V.E. (1992) Hemodialysis related induction of interleukin 6 production by peripheral blood mononuclear cells. Kidney Int. 42, 320-326. 6) Memoli, B., Minutolo, R., Bisesti, V., Postiglione, L., Conti, A., Marzano, L., Capuano, A., Andreucci, M., Balletta, M.M., Guida, B., Tetta, C. (2002) Changes of serum albumin and C-reactive protein are related to changes of interleukin 6 release by peripheral blood mononuclear cells in hemodialysis patients treated with different membranes. Am. J. Kidney Diseases 39, 266-273. 7) Stenvinkel, P., Barany, P., Heimburger, O., Pecoits-Filho, R., Lindholm, B. (2002) Mortality, malnutrition, and atherosclerosis in ESRD: what is the role of interleukin 6? Kidney Int. 61(S80), S103-S108. 8) Bologa, R.M., Levine, D.M., Parker, T.S., Cheigh, J.S., Serur, D., Stenzel, K.H., Rubin, A.L. (1998) Interleukin 6 predicts hypoalbuminemia, hypocholesterolemia, and mortality in hemodialysis patients. Am. J. Kidney Dis. 32, 107-114. 9) Yeun, J.Y., Levine, R.A., Mantadilok, V., Kaysen, G.A. (2000) C-reactive protein predicts all-cause and cardiovascular mortality in hemodialysis patients. Am. J. Kidney Dis. 35, 469- 476. 10) Rao, M., Guo, D., Perianayagam, M.C., Tighiouart, H., Jaber, B.L., Pereira, B.J., Balakrishnan,V.S. (2005) Plasma interleukin-6 predicts cardiovascular mortality in hemodialysis patients. Am. J. Kidney Dis. 45, 324-333. 11) Knupfer, H., Preiss, R. (2008) sIL-6R: more than an agonist? Immunology and Cell Biology 86, 87-91. 12) Heinrich, P.C., Graeve, L., Rose-John, S,. Schneider-Mergener, J., Dittrich, E., Erren, A., Gerhartz, C., Hemmann, U., Lutticken, C., Wegenka, U., Weiergraber, O., Horn, F. (1995) Membrane-bound and soluble interleukin 6 receptor: Studies on structure, regulation of expression and signal transduction. Ann. N. Y. Acad. Sci. 762, 222-236. 13) Heinrich, P.C., Behrmann, I., Muller-Newen, G., Schaper, F., Graeve, L. (1998) Interleukin- 6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem. J. 334, 297- 314. 14) Levy, D.E., Lee, C.K. (2002) What does Stat3 do? J. Clin. Invest. 109, 1143-1148. THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 15) Ketteler, M., Bongartz, P., Westenfeld, R., Wildberger, J.E., Mahnken, A.H., Bohm, R., Metzger, T., Wanner, C., Jahnen-Dechent, W., Floege, J. (2003) Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study. Lancet 361, 827-833. 16) Ashby, D.R., Gale, D.P., Busbridge, M., Murphy, K.G., Duncan, N.D., Cairns, T.D., Taube, D.H.,Accepted Bloom, S.R., Tam, F.W., Chapman, Manuscript R.S., Maxwell, P.H., Choi, P. (2009) Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease. Kidney Int. 75, 976-981.

17) Schafer, C., Heiss, A., Schwarz, A., Westenfeld, R., Ketteler, M., Floege, J., Muller-Esterl, W., Schinke, T., Jahnen-Dechent, W. (2003) The serum protein α2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J. Clin. Invest. 112, 357-366. 18) Memoli, B., De Bartolo, L., Favia, P., Morelli, S., Lopez, L.C., Procino, A., Barbieri, G., Curcio, E., Giorno, L., Esposito, P., Cozzolino, M., Brancaccio, D., Andreucci, V.E., d'Agostino, R., Drioli, E. (2007) Fetuin-A gene expression, synthesis and release in primary human hepatocytes cultured in a galactosylated membrane bioreactor. Biomaterials 28, 4836-4844. 19) Ganz, T. (2007) Molecular control of iron transport. J. Am. Soc. Nephrol. 18, 394-400. 20) Nemeth, E., Valore, E.V., Territo, M., Schiller, G., Lichtenstein, A., Ganz, T. (2003) Hepcidin, a putative mediator of inflammation, is a type II acute phase protein. Blood 101, 2461-2463. 21) Nemeth, E., Rivera, S., Gabayan, V., Keller, C., Taudorf, S., Pedersen, B.K., Ganz, T. (2004) IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J. Clin. Invest. 113, 1271-1276. 22) Wrighting, D.M., Andrews, N.C. (2006) Interleukin 6 induces hepcidin expression through STAT3. Blood 108, 3204-3209. 23) Zalunardo, N., Levin, A. (2006) Anemia and the hearth in chronic kidney disease. Semin. Nephrol. 26, 290-295. 24) Yildiz, A., Oflaz, H., Pusuroglu, H., Mercanoglu, F., Genchallac, H., Akkaya, V., Iklizer, T.A., Sever, M.S. (2003) Left ventricular hypertrophy and endothelial dysfunction in chronic hemodialysis patients. Am. J. Kidney Diseases 41, 616-623. 25) Tsirpanlis, G., Bagos, P., Ioannou, D., Bleta, A., Marinou, I., Lagouranis, A., Chatzipanagiotou, S., Nicolaou, C. (2004) The variability and accurate assessment of microinflammation in hemodialysis patients. Nephrol. Dial. Transplant. 19, 150-157. 26) Liu, Y., Coresh, J., Eustace, J.A., Longenecker, J.C., Jaar, B., Fink, N.E., Tracy, R.P., Power, N.R., Klag, M.J. (2004) Association between cholesterol level and mortality in dialysis patients. JAMA 291, 451-459. 27) Preti, H.A., Cabanillas, F., Talpaz, M., Tucker, S.L., Seymour, J.F., Kurzrock, R. (1997) Prognostic value of serum interleukin 6 in diffuse large cell lymphoma. Ann. Intern. Med. 127, 186-194. 28) Ridker, P.M., Rifai, N., Stampfer, M.J., Hennekens, C.H. (2000) Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation 101, 1767-1772. 29) Pearson, T.A., Mensah, G.A., Wayne, A.R., Anderson, J.L., Cannon, R.O., Criqui, M., Fadl, Y.Y., Fortmann, S.P., Hong, Y., Myers, G.L., Rifai, N., Smith, C., Taubert, K., Tracy, R.P., Vinicor, F. (2003) Markers of inflammation and cardiovascular disease: application to clinical and public health practice. Circulation 107, 499–511. 30) De Bartolo, L., Morelli, S., Rende, M., Gordano, A., Drioli, E. (2004) New modified THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes. Biomaterials 25, 3621-3629. 31) Panno, M.L., Giordano, F., Mastroianni, F., Morelli, C., Brunelli, E., Palma, M.G., Pellegrino, M., Aquila, S., Miglietta, A., Mauro, L., Bonofiglio, D., Andò, S. (2006) Evidence that low doses of Taxol enhance the functional transactivatory properties of p53 on p21Accepted waf promoter in MCF-7 breast cancer Manuscript cells. FEBS Letters 580, 2371-2380. 32) Memoli, B., Postiglione, L., Cianciaruso, B., Bisesti, V., Cimmaruta, C., Marzano, L., Minutolo, R., Cuomo, V., Guida, B., Andreucci, M., Rossi, G. (2000) Role of different

dialysis membranes in the release of interleukin 6 soluble receptor in uremic patients. Kidney Int. 58, 417-424. 33) Memoli, B., Grandaliano, G., Soccio, M., Postiglione, L., Guida, B., Bisesti, V., Esposito, P., Procino, A., Marrone, D., Michael, A., Andreucci, M., Schena, F.P., Pertosa, G. (2005) In vivo modulation of soluble "antagonist" IL-6 receptor synthesis and release in end stage renal disease. J. Am. Soc. Nephrol. 16, 1099-1107. 34) Pietrangelo, A., Dierssen, U., Valli, L., Garuti, C., Rump, A., Corradini, E., Ernst, M., Klein, C., Trautwein, C. (2007) STAT3 is required for IL-6-gp130-dependent activation of hepcidin in vivo. Gastroenterology 132, 294-300. 35) Gangneux, C., Daveau, M., Hiron, M., Derambure, C., Papaconstantinou, J., Salier, J.P. (2003) The inflammation-induced down-regulation of plasma fetuin-A (α2HS- Glycoprotein) in liver results from the loss of interaction between long C/EBP isoforms at two neighboring binding sites. Nucleic Acids Research 31, 5957-5970. 36) Wöltje, M., Tschöke, B., von Bülow, V., Westenfeld, R., Denecke, B., Gräber, S., Jahnen- Dechent, W. (2006) CCAAT enhancer binding protein and hepatocyte nuclear factor 3 are necessary and sufficient to mediate dexamethasone-induced up-regulation of alpha2HS- glycoprotein/fetuin-A gene expression. Journal. Mol. Endocrinol. 36, 261-277. 37) Calkhoven, C.F., Muller, C., Leutz, A. (2000) Translational control of C/EBP-α and C/EBP- β isoform expression. Genes & Development, 14, 1920-1932. 38) Mackey, S.L., Darlington, G.J. (2004) CCAAT Enhancer-binding protein is required for interleukin-6 receptor signaling in newborn hepatocytes. The Journal of Biological Chemistry 279, 16206-16213. 39) Nerlov, C. (2007) The C/EBP family of transcription factors: a paradigm for interaction between gene expression and proliferation control. Trends in Cell Biology 17, 318-324. 40) Moshage, H. (1997) Cytokines and the hepatic acute phase response. J. Pathol. 181, 257- 266. 41) Kramer, F., Torzewski, J., Kamenz, J., Veit, K., Hombach, V., Dedio, J., Ivashchneko, Y. (2008) Interleukin-1beta stimulates acute phase response and C-reactive protein synthesis by inducing an NFKB- and C/EBPbeta-dependent autocrine interleukin-6 loop. Mol. Immunol. 45, 2678-2689. 42) Jones, S.A., Horiuchi, S., Topley, N., Yamamoto, N., Fuller, G.M. (2001) The soluble interleukin 6 receptor: mechanisms of production and implications in disease. Faseb J. 15, 43-58. 43) Mehrotra, R. (2007) Emerging role for fetuin-A as contributor to morbidity and mortality in chronic kidney disease. Kidney Int. 72, 137-140. 44) Pertosa, G., Simone, S., Ciccone. M., Porreca, S., Zaza, G., Dalfino, G., Memoli, B., Procino, A., Bonomini, M., Sirolli, V., Castellano, G., Gesualdo, L., Ktena, M., Schena, F.P., Grandaliano, G. (2009) Serum fetuin a in hemodialysis: a link between derangement of calcium-phosphorus homeostasis and progression of atherosclerosis? Am. J. Kidney Dis. 53,

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 467-474.

Accepted Manuscript

FIGURE LEGENDS

Figure 1. Decrease of recombinant human IL-6 (rIL-6) activity at different concentrations of anti human IL-6 antibody. Figure 2. Confocal images of hepatocytes after 24 hours of stimulation with serum containing different concentrations of IL-6, serum with blocked IL-6 and under IL-6-free condition (Control). Hepatocytes were double immunostained for p-STAT3 (yellow) and gp130 (red). Merge represents the co- localization of two fluorescence signals. DAPI counterstaining was used to detect nuclei (blue). Image is representative of 12 experiments. Figure 3. Confocal images of hepatocytes after 24 hours of stimulation with serum containing different concentrations of IL-6, serum with blocked IL-6 and under IL-6-free condition (Control). Hepatocytes were double immunostained for gp80 (green) and gp130 (red). Merge represents the co-localization of two fluorescence signals. DAPI counterstaining was used to detect nuclei (blue). Image is representative of 12 experiments. Figure 4. Fluorescence average intensity expression of gp 80 (full bar), gp130 (empty bar) and p- STAT3 (dashed bar), in hepatocytes incubated for 24 hours with serum containing different concentrations of IL-6, serum with blocked IL-6 and under IL-6-free condition (Control). Data are expressed as mean ± SD of 12 experiments. § p<0.05 versus all. Figure 5. Fluorescence average intensity expression of gp 80 (a), gp130 (b) and p-STAT3 (c) after 24 hours of stimulation with increasing concentrations of recombinant IL-6 (rIL-6). Data are expressed as mean ± SD of 12 experiments. *p<0.05 vs rIL-6 = 0 pg/ml; †p<0.05 vs rIL-6 = 0.6 pg/ml; ‡p<0.05 vs rIL-6 = 1.8 pg/ml; § p<0.05 vs all. Figure 6. Comparison of fluorescence average intensity expression of gp 80 (full bar), gp130 (empty bar) and p-STAT3 (dashed bar) after 24 hours of stimulation with: a) recombinant IL-6 (rIL-6), serum with the same IL-6 concentration (0.6 pg/ml) and under serum-free condition (Control); b) rIL-6, serum with the same IL-6 concentration (1.8 pg/ml) and under serum-free condition (Control). Data are expressed as mean ± SD of 12 experiments. *p<0.05 vs gp80 under rIL-6 (0.6 pg/ml) stimulation; § p<0.001 with respect to rIL-6 (1.8 pg/ml). Figure 7. Western blotting of gp80, gp130 and p-STAT3 expression in human hepatocytes treated with serum IL-6 at concentrations of 0.6 and 1.8 pg/ml and under IL-6-free condition (Control). The same blot was probed for β-actin as control. Figure 8. Hepcidin and fetuin-A gene expressions in human hepatocytes cultured in batch system. Bar graph shows the relative expression of hepcidin (black bar) and fetuin-A (white bar) mRNA abundance, as determined by Real-Time PCR, in hepatocytes cultured for 24 hours with serum containing different concentrations of IL-6, serum with blocked IL-6 and under serum-free condition (Control). The values on the Y-axis represent arbitrary units derived from the mean expression of both genes compared with the lowest value for each gene set at 1. Each bar represents the mean ± SD of 6 experiments. * p<0.05 vs other gene expressions of hepcidin; ** p<0.05 vs other gene expressions of fetuin-A.

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

TABLES 1 & 2

Table 1. Clinical features of healthy subjects and ESRD patients, non-inflamed and micro- inflamed, included in the study. Data are expressed as mean ± SD.

Healthy Non-inflamed ESRD Micro-inflamed ESRD Gender (M/F) 4/6 3/6 4/6 Age (years) 48.8 ±6.4 52±8.2 53.6±4.2 Time on HD (months) ---- 28.64±6.12 26.12±5.34 BW (Kg) 62.8±3.4 61.8±2.8 62.2±3.2 BMI (Kg/m2) 22.2±0.8 21.8±1.4 22.6±1.3 Cause of ESRD: GN, ADPKD, Renal Vascular ---- 1, 1, 3, 1 1, 1, 2, 2 Disease/Hypertension, Other/Unknown Smoking 2/6 3/6 2/6 Kt/V ---- 1.36±0.04 1.32±0.04 nPCR (g/Kg/day) ---- 1.18±0.3 1.22±0.4 C-RP (mg/L) 2.30±0.4 2.60±0.2 6.98±0.6 * Serum Albumin (g/dl) 4.02±0.6 3.96±0.4 3.16±0.2 *

* p<0.01 vs Healthy and non-inflamed

Abbreviations: M/F: male/female, BW: Body Weight, BMI: Body Mass Index, GN: Glomerulonephritis, ADPKD: Adult Dominant Polycystic Kidney Disease, nPCR: normalized protein catabolic rate, C-RP: C-reactive protein.

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

Table 2. IL-6 (both in whole serum pools and culture media), sIL-6R (whole serum pools) and sgp130 (whole serum pools) concentrations, measured in 3 different pools obtained from serum samples of 6 healthy control subjects (Healthy), 6 non-inflamed ESRD patients (ESRD, C-reactive protein ≤ 3 mg/L) and 6 micro-inflamed ESRD patients (micro-inflamed-ESRD, C-reactive protein > 3 mg/L), respectively. Healthy ESRD micro-inflamed-ESRD

IL-6 (pg/ml) 0.53 2.97 8.88 (whole serum) IL-6 (pg/ml) 0.1 0.6 1.8 (culture medium) sIL-6R (ng/ml) 18.2 50 67 (whole serum) sgp130 (ng/ml) 306 273 290 (whole serum)

Data are expressed as a mean of 3 determinations for all assays; variation coefficient intra-assay ranged between 2.0-2.5 % for all assays performed.

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

Figures 1-8

12.00

10.00

8.00

6.00

rIL-6 [pg/ml] 4.00

2.00

0.00 0.00 1.00 2.00 3.00 4.00 5.00

anti-IL-6 neutralizing antibody [µg/ml]

FIGURE 1 THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

gp130 p-STAT3 Merge

Control

Serum IL-6 = 0.1 pg/ml

Serum

IL-6 = 0.6 pg/ml

Serum IL-6 = 1.8 pg/ml

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634

Serum with blocked IL-6 Accepted Manuscript

FIGURE 2 20

gp130 gp80 Merge

Control

Serum IL-6 = 0.1 pg/ml

Serum

IL-6 = 0.6 pg/ml

Serum IL-6 = 1.8 pg/ml THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634

Serum with blocked IL-6 Accepted Manuscript

FIGURE 3 21

18000 §

15000 § § 12000

9000

6000

3000 Fluorescence average intensity

0 Control 0.1 0.6 1.8 Serum with [______] blocked IL-6 serum IL-6 (pg/ml)

FIGURE 4 THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

15000

y = 1187.6x + 5342.3 ‡ R2 = 0.9755 † 10000 *

5000

Fluorescence average intensity 0 00.511.522.53 (a) rIL-6 concentration [pg/ml] 15000

y = 830.74x + 4693.1 2 R = 0.9883 § 10000 *

5000

Fluorescence average intensity 0 0 0.5 1 1.5 2 2.5 3

rIL-6 concentration [pg/ml] (b)

15000

y = 912.87x + 5236.3 § R2 = 0.9774 10000 * THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 5000

Fluorescence average intensity average Fluorescence Accepted0 Manuscript 00.511.522.53

18000

15000

12000 age intensity * 9000

6000

3000 Fluorescence aver

0 Control rIL-6 (0.6 pg/ml) serum IL-6 (0.6 pg/ml) (a)

18000 § § 15000 § 12000

9000

6000

3000 Fluorescence average intensity 0

THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Control rIL-6 (1.8 pg/ml) serum IL-6 (1.8 pg/ml)

(b) Accepted Manuscript

FIGURE 6 24

Serum IL-6 (pg/ml)

Control 0.6 1.8

gp80

gp130

p-STAT3

β-actin

FIGURE 7 THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript

35 *

15 [fold difference] mRNA expression 10

5 ** 0 Control 0.1 0.6 1.8 Serum with [______] blocked IL-6 IL-6 (pg/ml)

FIGURE 8 THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/CS20090634 Accepted Manuscript