Citrate anion improves chronic dialysis efficacy, reduces systemic inflammation and prevents Chemerin-mediated microvascular injury

Systemic inflammation and uremic toxins (UT) determine the increased cardiovascular mortality observed in chronic hemodialysis (HD) patients. Among UT, the adipokine Chemerin induces vascular dysfunction by targeting both endothelial and vascular smooth muscular cells (EC and VSMC). As Citrate anion modulates oxidative metabolism, systemic inflammation and vascular function, we evaluated whether citrate-buffered dialysis improves HD efficiency, inflammatory parameters and chemerin-mediated microvascular injury. 45 patients were treated in sequence with acetate, citrate and, again, acetate-buffered dialysis solution (3 months per interval). At study admission and after each treatment switch, we evaluated dialysis efficacy and circulating levels of chemerin and different inflammatory biomarkers. In vitro, we stimulated EC and VSMC with patients’ plasma and we investigated the role of chemerin as UT. Citrate dialysis increased HD efficacy and reduced plasma levels of CRP, fibrinogen, IL6 and chemerin. In vitro, patients’ plasma induced EC and VSMC dysfunction. These effects were reduced by citrate-buffered solutions and paralleled by the decrease of chemerin levels. Consistently, chemerin receptor knockdown reduced EC and VSMC dysfunction. In conclusion, Switching from acetate to citrate improved dialysis efficacy and inflammatory parameters; in vitro, chemerin-induced EC and VSMC injury were decreased by using citrate as dialysis buffer.


Cell isolation and culture
Human umbilical vein-derived endothelial cells (EC) were obtained by ATCC (PCS-100-010-ATCC, Manassas VA). EC were plated with EBM medium supplemented with 10% fetal calf serum Experiments were performed without FCS and after 24h (EC except angiogenesis), 72h (angiogenesis assay) or 96h (VSMC) incubation with patients' plasma diluted 1:10. Peripheral Blood Mononuclear Cells (PBMC) were obtained from healthy volunteer after isolation with Ficoll-Hypaque (GE Health Care) density gradient and added to cell cultures in selected experiments.

Functional assays
Cell death: EC were cultured in 96-well plates, incubated with appropriate stimuli, subjected to TUNEL assay (Chemicon Int. Temecula, CA) and analyzed under a fluorescence microscope to detect stained cells in 10 non-consecutive fields.
Nitric Oxide and Reactive Oxygen Species production: Nitric Oxide Synthase (NOS) activity was assessed in EC by 4,5-diaminofluorescein diacetate assay (DAF-2 DA -Enzo Life Sciences, Inc, Farmingdale, NY). DAF-2 DA is a precursor of the fluorescent dye triazolofluorescein that is activated by NOS. Reactive Oxygen Species (ROS) production was assessed by Image-iT® Detection Kit (Life Technologies, Carlsbad, CA, USA). The assay is based on 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxyH2 DCFDA), a compound that releases fluorescence after binding ROS. Experiments were conducted according to manufacturers' instructions. After incubation with selected stimuli, cells were analyzed under a fluorescence microscope or re-suspended with EDTA and analyzed by FACS.
Cell adhesion: PBMC were labeled for 15 minutes with 10 µm of fluorescent Vybrant Cell Tracer kit (Life Technologies) in RPMI. Fluorescent cells were re-suspended in EBM without FCS (50 × 10 6 /ml) and added to confluent monolayer of EC on six-well plates. After 1hr at 37°C of slight agitation, samples were washed, fixed with 4% paraformaldehyde and observed by UV light microscopy. Fluorescent cells were quantified in 10 different fields at x200 magnification.
Angiogenesis on Matrigel: EC were cultured on growth factor reduced Matrigel (Becton Dickinson, San Jose, CA) in EBM enriched by selected stimuli. After 12h, EC were observed under a Nikoninverted microscope (Nikon, Kanagawa, Japan). Image analysis was performed with the MicroImage analysis system (Casti Imaging, Venice, Italy).
Cell calcification: intracellular calcium deposits were quantified by red-alizarin staining. VSMC were cultured in 24-well plates and fixed with 4% paraformaldehyde in PBS for 45 min at 4°C.
Cells were then washed in distilled water, exposed to Alizarin Red (2% aqueous, Sigma Aldrich, St Louis, MO) for 5 min, washed again and analyzed by inverted light microscopy. Cell lysates were analyzed in an automatized spectrophotometer at a wavelength of 570 nm.
Quantitative RT-PCR for RUNX2: total RNA was extracted by VSMC after appropriate stimulation using mirVana RNA isolation kit (Life Technologies). RNA concentration and purity were detected by the NanoDrop1000 spectrophotometer. We evaluated RUNX2 mRNA expression by using High Analysis of RUNX2 protein expression in VSMC: For immunofluorescence, VSMC cultured on chamber slides (Thermo Scientific, Waltham, MA, USA) were fixed in ethanol/acetic acid 2:1 and incubated with primary rabbit polyclonal antibodies directed to human RUNX2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Slides were washed with PBS and then incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 30 min at 4°C. All samples were counterstained by 2.5 µg/ml Hoechst (Sigma Aldrich) for 5 minutes, mounted with anti-fade mounting medium (Sigma Aldrich), and examined by confocal microscopy (LSM5 PASCAL; Zeiss, Jena, Germany). For FACS analysis, cells cultured in 12-well plates were detached with EDTA and stained for 30m at 4°C with primary antibodies directed to human RUNX2 (Santa Cruz Biotechnologies). After washing, VSMC were incubated with FITC-conjugated secondary