Icam 1

Cell culture supernatants were harvested after stimulation for 48 h (described in Section 4.2), centrifuged at 300× g for 5 min, and used for the quantification of IL-6 and ICAM-1, or stored at −20 °C for later measurement. Both proteins were quantified using commercially available EIA kits (IL-6: Immunotools, Friesoythe, Germany; No. 31670069; ICAM-1 R&DSystems, Wiesbaden, Germany; No. DY720-05), as described by the manufacturer. In brief, the wells were coated with the capture antibody overnight at room temperature. Then, nonspecific binding sites were blocked with a blocking buffer for 1 h and washed with PBS/0.05 % Tween. The standard (IL-6 assay: 8–500 pg/mL, ICAM-1: 31.1-2000 pg/mL) or the samples were added for 2 h at room temperature. All samples were diluted in assay buffer (IL-6 1:25; ICAM-1 1:10) and run in duplicate. After incubation, the plates were washed and the biotinylated detection antibody was added for 2 h at room temperature, washed again, and incubated with horseradish-peroxidase-streptavidin for an additional 30 min. After washing, the substrate Tetramethylbenzidine was added for 5–20 min and the reaction was stopped and measured (450 vs. 620 nm). The data are calculated as ng/mL in the supernatant.

On admission blood samples were taken from all patients (fasting for 12 hours). Three samples were collected and centrifuged and plasma and serum were frozen at −80°C. Biochemical analyses necessary to determine kidney function were performed and urea, creatinine, cystatin C, electrolytes, albumin, aldosterone, and lipids concentrations were measured; plasma renin activity (PRA) was assessed. On the basis of serum creatinine and cystatin C, an estimated glomerular filtration rate (eGFR) with the Schwartz [8 (link)] and Filler [9 (link)] formulas was calculated. Patients were divided into groups depending on the stage of CKD [group 1: CKD stages 1 + 2 (GFR > 60), group 2: CKD stage 3 (GFR = 30–59), group 3: CKD stage 4 (GFR = 15–29 mL/min/1.73 m²), group 4: dialyzed children].
To assess oxidative stress the concentration of oxidized LDL particles, as an effect of lipid oxidation, and the concentration of carbonyl groups resulting from oxidation of proteins were used. Concentrations of serum high sensitive C-reactive protein (hsCRP) (R&D Systems, USA); oxLDL (Mercodia Inc., Sweden); protein carbonyl groups (Cayman Chemical Company, USA); and intercellular adhesion molecule-1 (ICAM-1) (R&D Systems, USA) were determined with enzyme-linked immunosorbent assay (ELISA). Insulin levels (BioSource, Belgium) were measured using the IRMA method.

Biomarkers of neutrophil activation included L-selectin (CD62L), intercellular adhesion molecule-1 (ICAM-1, CD54), and matrix metalloproteinase-9 (MMP-9, gelatinase B) (R&D Systems, Minneapolis, MN, USA), as well as IL-8 (as mentioned above), the levels of which were determined by conventional ELISA kits.

Imaging plates (384-well plates CellCarrierUltra, Perkin Elmer) were pre-coated overnight with crossed concentrations of ICAM-1 (0; 0.5; 1; 2; 4 µg/ml, R&D systems) and anti-CD3 Ab (0: 0.1; 0.5; 1; 5; 10 µg/ml, OKT3 clone, Invitrogen). CD8⁺ T cells were seeded in the plate at 37 °C and stained 5 min later with conformation-specific anti-LFA-1 Ab: m24 (coupled to AlexaFluor647, Biolegend) and Hi-111 (coupled to AlexaFluor488, Biolegend), each used at 1 µg/ml during 15 min. Cells were fixed with 4% paraformaldehyde for 15 min at 37 °C and stained overnight by AlexaFluor546-coupled phalloïdin (Thermo Fisher) at 0.4 µg/ml. Nuclei were stained with DAPI at 10 µg/ml for 5 min. Pictures were acquired with an automated high-content confocal microscope (Opera Phenix, Perkin Elmer) and analyzed by the Harmony software (Perkin Elmer) as described earlier⁵⁹ .

Cell pellets were resuspended in Pierce IP lysis buffer (Thermo Fisher Scientific, Munich, Germany) containing protease inhibitors. Ten milligram of liver tissue was homogenized in lysis buffer (PBS + 0.1% Triton). Protein quantification was performed using the BCA kit from Thermo Fisher. Between 5 and 35 μg of protein samples were loaded into each lane of an any kD Mini-PROTEAN TGX Precast Protein Gel and subsequently transferred to a PVDF membrane. After blocking (5% skim milk in PBS/Tween 20 0.05%) the membrane was probed with antibodies against Icam1 (R&D Systems, Abingdon, UK) and αSMA (Abcam, Cambridge, UK). β-actin (Cell signaling, Frankfurt, Germany) served as a loading control.