Cinc 1

Concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine in plasma samples were determined with a Cobas c111 reader (Roche, Rotkreuz, Switzerland) according to manufacturer's instructions. Plasma samples were also assayed for C-reactive protein (CRP; Biovendor, Brno, Czech Republic), interleukin-6 (IL-6; R&D Systems, MN, USA), cytokine-induced neutrophil chemoattractant-1 (CINC-1; R&D Systems, MN, USA), and prostaglandin E2 (PGE2, Cayman Chemical, MI, USA) according to manufacturers' instructions.

High-sensitivity ELISA kits were used according to the manufacturer’s instructions to measure tissue Hsp72 (Enzo Life Sciences, intra-assay precision <5%, inter-assay precision <13%), CINC-1, IL-1β, IL-6, and IL-10 (R&D Systems, Minneapolis, MN). Optical densities were measured using a SpectraMax Plus 354 plate reader. MLN lysates were diluted 1:4 for Hsp72, neat for CINC-1, and 1:2 for IL-1β, IL-6, and IL-10. AT lysates were diluted 1:4 for Hsp72 and 1:2 for CINC-1, IL-1β, IL-6, and IL-10. Spleen lysates were diluted 1:4 for Hsp72; 1:2 for CINC-1; 1:8 for IL-1β; 1:2 for IL-6; and 1:4 for IL-10. Liver lysates were diluted 1:4 for Hsp72; 1:25 for the CINC-1 stress group, and CINC-1 controls were run neat.

Commercial ELISA kits were used to measure the concentrations of cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1; R&D Systems, Minneapolis, MN, USA) and SCF (R&D Systems) in rat sera and CdM from MSCs. The procedures were based on instructions provided by each manufacturer. Serum 8‐isoprostane levels were measured by 8‐isoprostane express enzyme immune assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to manufacturer's protocol. As 8‐isoprostane might be esterified in lipids, all samples were pre‐hydrolysed by incubating with the same volume of 15% (w/v) KOH at 40°C for 60 min. and neutralized with 1 M potassium phosphate to get total 8‐isoprostane levels.

Culture media was collected from each group including, K562 cell line, BMSCs and co-culture of K562 cell line with BMSCs. ELISA was performed according to the manufacturer’s instructions (ExCell Biology, Shanghai, China; CINC-1 from R&D Systems). Briefly, a 96-well plate was coated with detection Reagent A and stored overnight at 4°C. Then, 50 μL of cell culture media from each groups and standard solution were added into the 96-well plate, which had been coated with human interleukin (IL)-6, IL-8 and transforming growth factor beta (TGF-β) antibodies, and detected via the ELISA sandwich technique. After terminating the reaction, the optical density at a wavelength of 450 nm in each well was determined using a spectrophotometer.

The serum levels of BUN and creatinine were measured using automated, standardized procedures (Roche Hitachi 917/747, Mannheim, Germany). The levels of kidney injury molecule-1 (KIM-1; R&D Systems, Minneapolis, MN, USA), cytokine-induced neutrophil chemoattractant-1 (CINC-1; R&D Systems, Minneapolis, MN, USA), neutrophil gelatinase-associated lipocalin (NGAL, R&D Systems, Minneapolis, MN, USA), corticosterone (BioVendor, Germany), norepinephrine (LDN, Nordhorn, Germany), epinephrine (LDN, Nordhorn, Germany), and angiotensin II (Ang II, R&D Systems, Minneapolis, MN, USA) were determined through enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

Serum levels of cytokine-induced neutrophil chemoattractant-1 (CINC-1) (R&D Inc. Minneapolis, MN) and total adiponectin (Invitrogen, Carlsbad, USA) were analyzed using. ELISA kits according to the manufacturer’s instructions.