Dibutyl camp

NRK-52E, the rat proximal tubular cell line, was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10% fetal bovine serum. Cells were then cultured in fibronectin pre-coated flasks until they reached 70–80% confluence. IMD was over-expressed in NRK-52E by gene transfer with pcDNA3.1-IMD as we previously described [14 (link)]. Cell fibrosis model was induced by stimulating with recombinant human TGF-β1 (rhTGF-β1, 10 ng/ml, Gibco, Carlsbad, CA) for 72 h. The cells were randomly allocated into 1 of the following six groups: control (untreated cells); TGF-β1 (cells were stimulated with rhTGF-β1); TGF-β1 + empty vector (cells were transfected with the control plasmid pcDNA3.1 and stimulated with rhTGF-β1); TGF-β1 + IMD (cells were transfected with pcDNA3.1-IMD and stimulated with rhTGF-β1); TGF-β1 + dibutyl-cAMP [cells were stimulated with rhTGF-β1 plus cAMP analog dibutyl-cAMP (10⁻³mol/l, Sigma-Aldrich, St. Louis, MO)]; and TGF-β1 + IMD + H-89 [cells were transfected with pcDNA3.1-IMD and stimulated with rhTGF-β1 plus PKA inhibitor H-89 (10⁻⁵mol/l, Sigma-Aldrich, St. Louis, MO)].

Human CGRP, Human CGRP_8–37, Ang II, N‐acetyl‐L cysteine (NAC), apocynin, PP2, H‐89 and dibutyl‐cAMP were purchased from Sigma. Niclosamide was purchased from Selleck Chemicals; anti‐phospho‐Src (Tyr416), anti‐phospho‐STAT3 (Tyr705), Src and STAT3 antibodies, from Cell Signaling Technology Inc; antibodies for p47phox and GAPDH, and all secondary antibodies, from Santa Cruz Biotechnology; antibodies of RAMP1, CRLR and ATP1a1, from Santa Cruz Biotechnology; RCP antibodies, from NOVUS Institute of Biotechnology; Alzet mini‐osmotic pumps (Alzet model 1004), from DURECT Corp; and the Src (B0107) and STAT3 (F0806) ORF cDNA clones, from GeneCopoeia. Other chemicals and reagents were of analytical grade.