Timp 1

Antibodies for Bax, Bcl-2, caspase-3, and tissue inhibitor of metalloproteinases 1 (TIMP1) were obtained from Santa Cruz Biotechnology, Inc. Antibodies for MMP-9 and MMP-2 were ordered from Cell Signaling Technology, Inc (Shanghai, China). Triphenyltetrazolium (TTC) was ordered from Sigma. Acetonitrile 99.9% was of high-performance liquid chromatography (HPLC) grade purchased from TEDIA (Lisbon, USA). Reference compounds HSYA and safflower yellow B (purity ≥99%) were obtained from National Standards Center of China.

For protein analysis, cells were washed with 1× PBS and lysed in a buffer containing 1% sodium dodecyl sulfate (SDS) and 60 mM Tris-Cl, pH 6.8, and tissues were lysed in PRO-PREP^TM (iNtRON Biotechnology). The lysate was mixed briefly using a vortex, boiled for 10 min, and centrifuged at 13,000 g for 10 min at 4°C. Protein concentrations were assessed using the BCA assay kit (Pierce). Protein samples of equal amount were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblot analysis were performed using the following antibodies: polyclonal antibodies against GPx7 (Proteintech), Col1α1 (Abcam), TNFα (Cell Signaling), αSMA, TIMP1, MMP13, ADRP, and GAPDH (Santa Cruz Biotechnology).

MDA-MB-231 cells were treated with various irisin doses for 24 h. Proteins were extracted with a RIPA lysis buffer and quantified with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). The quantified protein was then loaded onto SDS-PAGE gel and subjected to electrophoresis. The protein loaded on the SDS-PAGE gel was transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) in a cold room, blocked with 5% BSA or 5% skim milk, and reacted with primary antibodies detecting MMP-2, MMP-9, TIMP-1, TIMP-2 (Santa Cruz Biotechnology, Dallas, TX, USA), vimentin (Abcam, Cambridge, MA, USA), and HIF-1 (Cell Signaling Technology, Danver, MA, USA) at 4 °C overnight. The membrane was washed and reacted with secondary antibodies. After 30 min of washing, an enhanced chemiluminescence reagent (Animal Genetics Inc., Suwon, Korea) was used to detect bound antibodies.

Whole kidneys were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer, then centrifuged at 14,000× g for 25 minutes at 4°C prior to collection of the supernatants. The protein concentration was measured by Bradford’s method, and the supernatants were stored at −80°C. The cell lysates (50 µg of protein/lane) or whole HK-2 cell extracts (40 µg of protein/lane) were loaded, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to polyvinylidenedifluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with primary antibodies for CTGF (Abcam, Cambridge, UK), TGF-β1, collagen type I (Col1), α-SMA, TIMP-1, PAI-1, E-cad, or β-actin (Santa Cruz Biotechnology). Subsequently, the membranes were incubated with goat antirabbit IgG or goat antimouse IgG HRP conjugate, and then immersed in ECL Plus Western Blotting Detection Reagent (Amersham, Piscataway, NJ, USA) and exposed to Hyperfilm ECL (Amersham). The intensity of the bands was measured using Lab Works 4.5 software (UVP, Upland, CA, USA).

Kidneys were homogenized using a standard technique 21. Specific antibodies against Bax (1:200, Santa Cruz), Bcl‐2 (1:200, Abcam), transforming growth‐factor‐β (TGF‐β) (1:200, Santa Cruz), connective tissue growth factor (CTGF) (1:200, Bio Vision), PAI‐1 (1:200, Abcam), TIMP‐1 (1:200, Santa Cruz) were used with Western blotting protocols. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as loading controls and protein expression quantified with image‐pro plus 6.0 software. The expression of KIM1 in the STK was assessed by immunofluorescence staining. Kidney fibrosis was tested by Masson's Trichrome staining, assessed in 5‐µm sections of each kidney using a computer‐aided image‐analysis program (AxioVision, Carl Zeiss Micro Imaging, Thornwood, NY). Fibrosis area was quantified randomly in 10–15 fields each section. Apoptosis was assessed in renal sections stained with terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL, Promega) and F4/80 (1:200, Abcam) macrophages stained by immunohistochemistry. In 10–15 random fields sampled in each section, positive cells were manually counted. Renal oxidative damage was evaluated by 8‐hydroxy‐2′‐deoxyguanosine (8‐OHDG, 1:200, Abcam) immunohistochemistry, quantified from 10 random fields as percent of positive cells.