Patients and blood samples
Ten CKD patients with end-stage renal disease enrolled at the HD unit of “Fondazione IRCCS Policlinico San Matteo”, were included in this proof-of-concept study. Exclusion criteria included recent illness (within the previous 2 months), significant anemia (Hb < 10 g/dl), autoimmune disease, but also systemic diseases as vasculitis, amyloidosis, rheumatic disease; HBV, HCV, HIV positivity or other active viral positivity, active bacterial infection, active or previous cancer remission, previous transplantation.
CKD patients were on HD, 4 hours thrice weekly, with non-reused standard low-flux polysulfone dialyzers. The dialytic procedure was a standard bicarbonate dialysis, and the Kt/V index ranged from 1.24 to 1.35. The surface of the filters was tailored to the patients’ needs, and the patients were clinically stable; more in detail, no relevant changes occurred in the plasma levels of urea, phosphate, calcium, and creatinine, or in hematocrit, Kt/V, and leukocyte counts. No patient was under treatment with drugs acting directly on the immune system, such as steroids or other immunosuppressants. Blood samples was collected from patients just before starting the dialysis session at the end of the long interdialytic interval.
Teen age and sex-matched hospital staff healthy individuals were also included in the study as controls. Participant characteristics are summarized in Table 1.
PBL isolation and treatments
Blood was collected form CKD patients and healthy control subjects in Vacutainer® tubes containing sodium heparin as anticoagulant and PBL were isolated by density gradient centrifugation using Lympholyte-H (Cedarlane, Burlington, Ontario) according with the manufacturer’s protocol. Briefly, whole blood was diluted 1:1 in sterile saline, layered onto Lympholyte-H cell separation medium and centrifuged at 870 x g for 30 min at room temperature without brakes. The layer containing PBL between the upper (plasma, thrombocytes) and the lower phase (Lympholyte-H) was carefully collected using a Pasteur pipette, transferred to a fresh tube, and washed twice with phosphate-buffered saline (PBS). The PBL pellets were suspended in ammonium chloride-potassium lysing buffer (Invitrogen, Thermo-Fisher Scientific, Milan, Italy) and incubated for 10 min at room temperature with gentle mixing to lyse contaminating RBC. This was followed by a wash of the purified cells with PBS-EDTA.
In some experiments, the effects of uremic plasma (u-Pl) and its HMW fraction, prepared as described later, were investigated in control PBL (c-PBL) obtained from the local blood bank. Outdated blood bags containing buffy coat intended for disposal were obtained at the day of the expiry date and treated for PBL isolation using the same Lympholyte-H density gradient isolation procedure described earlier for fresh PBL.
Uremic and healthy control PBL were maintained at 37 °C in a humidified atmosphere containing 5% CO2 suspended in RPMI-1640 (GIBCO, Life Technologies) containing 1% L-glutamine and 10% vol/vol foetal bovine serum (FBS, GIBCO, Life Technologies). For the treatments, uremic or healthy control plasma or their HMW fractions were used at 10 % vol/vol instead of FBS.
A hemocytometer was used to count the total cell number and cell viability was assessed by Trypan Blue exclusion test using a solution 0.4% w/vol of this dye that was obtained from Sigma-Aldrich, St. Louis, Missouri, United States.
Plasma fractionation
To study the effects of uremic retention solutes on PBL viability, a fraction enriched of these solutes was prepared starting from u-Pl and healthy control plasma (c-Pl) by in vitro ultrafiltration (u-HMW and c-HMW, respectively). Micro-concentrator membranes (Vivaspin 6mL, Sartorius Stedim Biotech GmbH, Germany) with 50 kDa nominal cutoff were used for this solute enrichment procedure according to manufacturer instructions. The 50 kDa MW cutoff was selected for this procedure considering the limited efficacy of dialyzer membranes used in both diffusive and convective hemodialysis methods (nominal cutoffs ranging between 10 and 30 kDa) to remove cell-death promoting solutes (33, 34). At the same time, preliminary data suggested that plasma protein fractions enriched of large solutes prepared by ultrafiltration carried out either in vivo in patients treated with protein-leaking (PL) dialysers (nominal cut-off > 50 kDa) or in vitro using 50 kDa cutoff micro-concentrators, contain most of the proteinaceous solutes that cause DNA damage and cell death in mononuclear cell lines (35); on the other way around, middle molecules and small solutes obtained by in vitro ultrafiltration using 10 kDa cutoff micro-concentrators are responsible for a minor part of the apoptotic activity of u-Pl studied in mononuclear cell lines (33).
Protein carbonyl analysis
Protein carbonyls were investigated by immunoblot and spectrophotometric methods as described in ((36) and references therein) using the Brady's reagent 2,4-dinitrophenylhydrazine (2,4-DNPH), according to Levine et al. (37). More in detail, the immunoblot analysis of 2,4-DNPH-reactive carbonyls was performed in plasma proteins separated by two-dimension electrophoresis (2D-PAGE) performed under denaturation conditions and transferred by electroblotting onto a nitrocellulose membrane for identification after derivatization using an anti-2,4-DNPH antibody (Abcam, Cambridge, United Kingdom). For the first-dimension of 2D gradient PAGE analyses, from 100 to 300 mg of total proteins were loaded onto immobilized pH gradient gel strips (17 cm, pH 3–10 NL) and isoelectric focusing was performed using a Protean IEF cell system (Bio-Rad, Hercules, California, United States). The second dimension was performed on 9–16% polyacrylamide linear gradient electrophoresis gels using a Protean II xi 2-D System (Bio-Rad). The gels were stained with silver stain and image analysis was performed using the PD-Quest 2D gel software (Bio-Rad, Hercules, California, United States). Reference 2D maps of human plasma proteins were obtained from SWISS-PROT database (https://world-2dpage.expasy.org/swiss-2dpage) and protein identity was confirmed by LC-MS analysis after protein isolation by manual spot picking and in-gel digestion using MS grade trypsin as described in (26, 38).
Mononuclear cell lines and macrophage differentiation of THP-1 cells
THP-1 human monocytic leukemia cells and human K562 erythroleukemic cell line (American Type Culture Collection, ATCC, Manassas, VA, USA) maintained in RPMI as described earlier for human PBL, were used at intervals of passage in culture between 10 to 25.
For macrophage differentiation, THP-1 cells seeded in 6-well plates (106 cells/mL) were treated with 5 ng phorbol-12-myristate-13-acetate (PMA)/ml for 48 h. After differentiation, the cells were washed three times with PBS and used for the incubation with plasma samples and their HMW fractions. These were added to the cell culture media of mononuclear cell lines and differentiated macrophages instead of FBS.
Cell viability and apoptotic cell death determination
Peripheral blood mononuclear leukocytes from healthy controls or CKD patients were seeded (15,000 cells/well) for the incubation (2 hrs) with autologous or heterologous plasma or HMW fractions and then the cells were stained with 0.4% trypan blue dye to assess in triplicate viable and non-viable cells.
Cell viability of THP1 and K562 cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, Missouri, United States) assay performed according with the procedure previously published in (39) or using a Via1-Cassette™ (Chemometec) kit with NucleoCounter ® NC-3000™. The cassettes were loaded with immobilized acridine orange (AO) and DAPI for labeling of total cell and dead cell number, respectively.
Apoptosis was assessed by cytofluorimetric analysis using an Attune NxT Acustic Focusing Cytometer (Thermo Fisher Scientific); after incubation for 24h with plasma samples or their HMW fractions, THP1 cells (2 × 105/well) were harvested and stained with Annexin V, Alexa Fluor™ 488 conjugate (Thermo Fisher Scientific) and propidium iodide (PI, Clontech), and then apoptosis was determined according with manufacturers’ instructions. Staurosporin (50 µM, Sigma-Adrich, St. Louis, Missouri, United States) was used for positive control tests.
Autophagy assay
Autophagy was studied in THP1 cells by flow cytometry analysis (Attune NxT Acousting Focusing Cytometer; Thermo Fisher Scientific) and microplate immunofluorescence microscopy (Operetta CLS, Perkin Elmer). Autophagic vacuoles were identified utilizing a Cyto-ID Autophagy Green Detection Kit (Enzo Life Sciences, Inc.) and Rapamycin and overnight starvation (cell culture in the absence of FBS) were used as positive controls.
DNA fragmentation assay
THP-1 cells were grown in 6 well plates and treated with whole plasma or HMW fractions.
The cells (1 × 106/well) were suspended in 200 μl PBS and fixed by adding 800 μl of iced 100% ethanol and then incubated overnight at 4°C. The cell pellets were collected by centrifugation and were resuspended in hypotonic buffer containing 5 μg/ml RNase and 50 μg/ml propidium iodide and incubated for 1 h at 37 °C. Fluorescence emitted from the propidium iodide-DNA complex was quantified after excitation of the fluorescent dye by NucleoCounter ® NC-3000™.
Cellular and extracellular Reactive Oxygen Species (ROS)
PBL, THP-1 cells and THP-1 macrophages were treated with plasma or HMW fractions for 3 hrs and then washed twice before incubation in 50 μM DCFH-DA solution (Sigma-Aldrich, St. Louis, Missouri, United States) for 30 min at 37˚C in the dark. Then the cells were washed with PBS and the fluorescence was recorded using a DTX880 Multimode Detector microplate reader (Beckman Coulter).
Extracellular ROS were determined using the Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen). Briefly, 100 μL of cell culture medium were collected in 96-well plates for the incubation (10 min, at 37°C) in a humidified atmosphere with 5% CO2 in the presence of 25 μL of HRP (1 U/mL) and 25 μL of Amplex red reagent. Then, the fluorescence was measured at λexcitation= 560 ± 20 nm and λemission= 585 ± 20 nm in a DTX880 Multimode Detector microplate reader (Beckman Coulter). The assay was calibrated with authentic H2O2.
Cellular protein extraction and immunoblot
Cells were resuspended in 200 μl of ice-cold Cell Lysis Buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM ethyleneglycol-tetra-acetic acid (EGTA), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/ml leupeptin (Cell Signaling Technology), and 20 μl/ml protease inhibitor cocktail (Pierce, Thermo-Fisher Scientific). After centrifugation at 14,000 g for 20 min at 4 °C, the supernatant containing cellular proteins was collected and the BCA protein assay (Pierce, Thermo-Fisher Scientific) was used to measure protein concentrations; bovine serum albumin (BSA) was used as an external calibration standard.
After extraction, the protein samples were first resolved by 4-12% SDS-PAGE and then transferred to nitrocellulose membrane for immunoblot analysis as described in (40). Primary antibodies from Cell Signaling Technology utilized in this study were anti-GAPDH (#5174) and β-actin (#3700) that were used to normalize immunoblot data, and anti-c-Jun (#9165,), anti-phospho-ERK1/2 (#4377), anti-ERK1/2 (#4695), anti-phospho-SAPK/JNK (#4671), anti-JNK (#9258), anti-PERK (C33E10) (#3192; 1:1000), anti-IRE1α (#3294, 1:1000). Anti-Transferrin (YIF-LF_MA0242; 1:500), anti-Fibrinogen (YIF-LF_MA0108; 1:2000) and anti-human serum albumin (1:1000) were from Adipogen Life Sciences.
Peroxidase-conjugated secondary antibodies were Anti-rabbit (#7074) or anti-mouse (#7076) IgG HRP-linked Antibody (Cell Signaling Technology).
The signals were visualized by enhanced chemiluminescence reagent (ECL Plus, Pierce or ECL Clarity Max, Bio-Rad) and band quantification was performed by Gel-Pro Analyzer.
Statistics
Study variables (presented as mean ± SD) were processed for analysis of variance by ANOVA test and differences between mean values in the experimental groups were assessed using the t-test of Student (two-sided test for normally distributed data) or Mann-Whitney test (for nonparametric data), when appropriate; p values < 0.05 were accepted. Correlations between continuous dependent variables were assessed using the Pearson's correlation test.
Ethical statement
The present study was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of “Fondazione IRCCS Policlinico «San Matteo»” of Pavia, Italy. All patients provided written informed consent. This clinical trial is registered at www.clinicaltrials.gov (NCT02981992).