Mcp 1

Cells were lysed and total protein was estimated by using DC™ Protein Assay Kit (Bio‐Rad). Mice plasma was diluted with PBS in a ratio of 1:10 before gel separation. Proteins were resolved in 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane (Millipore). Membranes were incubated in 5% nonfat dry milk, followed by incubation with a primary and secondary antibody with three alternate wash. Blots were finally incubated with NBT and BCIP in alkaline phosphatase buffer until the bands were visible. The primary antibodies used for the study were anti‐vitronectin (1:1,000) (cat no. 45139; Abcam), anti‐transferrin (1:10,000) (cat no. Ab82411; Abcam), anti‐β‐actin (1:10,000) (cat. no. A5441; Sigma), anti‐ApoA2 (1:1000) (cat. no. A14690; Ab clonal), anti‐ApoA1 (1:1000) (cat no. sc‐376818; Santa Cruz), IL‐1β (1:1000) (cat no. sc‐52012; Santa Cruz), MCP‐1 (1:2000) (cat no. A7277; Ab clonal). Quantitative analysis was done by Image J software. The ratio of levels of experimental protein/loading control was used for the evaluation of relative levels of the protein.

The kidney and lung tissues that were preserved in aqueous formaldehyde solution for 24 h were dehydrated and embedded in paraffin blocks. The paraffin blocks were sliced at 4-μm thickness with a sliding microtome (SM2125, Leica Biosystems, Nussloch, Germany). For histochemical examinations, the specimens were deparaffinized in xylene and sequentially rehydrated using 100% ethanol, 90% ethanol, 70% ethanol, and water. To investigate pathological features of the kidneys, periodic acid Schiff (PAS) (Sigma/Merck, Darmstadt, Germany) staining was carried out to colorize brush borders and glomerular capillaries. To examine fibrosis, the specimens were subjected to Pico Sirius-red (Merck, Darmstadt, Germany) staining. The kidney specimens were immunohistochemically stained with TGF-β1 (Arigo Laboratories, Hsinchu, Taiwan), MCP-1 (ABclonal, Woburn, MA, USA), and IL-6 (ABclonal, Woburn, MA, USA) antibodies. The distributions of TGF-β1, IL-6, and MCP-1 in the kidneys were detected using commercially available peroxidase IHC kits, and developed with diaminobenzidine (DAB) (ThermoFisher, Waltham, MA, USA). The histochemical and immunohistochemical specimens were microscopically scanned and digitalized with a Pannoramic MIDI digital scanner (3DHISTECH Ltd., Budapest, Hungary). Microscopic photos were acquired using Pannoramic Viewer 1.14 (3DHISTECH Ltd., Budapest, Hungary).