Albumin

To determine markers of inflammation, 20 µg (2 µg/µL) (or 2 µg (0.2 µg/µL) for albumin) of total plasma protein were loaded in each pocket on a 12% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, USA) and protein levels of albumin, cluster of differentiation 14 (CD14), C-reactive protein (CRP), Growth/differentiation factor-15 (GDF-15) and p16 were determined as detailed before [36 (link),37 (link)]. In brief, after blocking for 1 h at room temperature with gentle shaking, membranes were incubated with the primary antibodies (albumin, Cell signaling, Danvers, MA, USA; CDKN2A/p16, Biorbyt, UK; CD14, Santa Cruz, Dallas, TX, USA; CRP, Santa Cruz, USA; GDF-15, Santa Cruz, USA; list of antibodies used and respective dilutions see also Table S2) at 4 °C overnight with gentle shaking followed by a washing step with 1× Tris-buffered saline with Tween20 (TBST, 3 × 10 min) and incubation with the respective secondary antibody for 1.5 h shaking at room temperature (detailed information on antibodies used please also see Table S2). After a second washing step with 1 × TBST (3 × 10 min), bands were detected using Super Signal West Dura kit (Thermo Fisher Scientific, Waltham, MA, USA). Densitometric analysis of bands were performed using ImageLab Software V 6.1.0 (Bio-Rad, USA) and normalized to albumin.

FlexiGene^® DNA Kits (ID# 51206) for DNA extraction from blood were purchased from QIAGEN Inc. (Germantown, MD, USA). Calbindin, clusterin, KIM-1, GST-P1, IL-18, MCP-1, albumin, B2M, cystatin C, NGAL, osteopontin, and TFF3 assays (Bio-Plex Pro RBM human kidney toxicity assay panels 1 and 2) were purchased from Bio-Rad, Life Science (Hercules, CA, USA).