Tgf β1

HK-2 cells (ATCC, American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) medium supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in a 95% air, 5% CO₂ atmosphere. HK-2 cells were divided into the normal group (NG), TGF-β1 (5 ng/mL) (Abgent, San Diego, CA, USA) stimulation group, and TGF-β1 (5 ng/mL) plus CTGF siRNA group. Meantime, to examine the direct effect of CTGF on interstitial fibrosis, HK-2 cells were randomly divided into two groups: the NG group and the CTGF (10 ng/mL) stimulation group. Furthermore, we transfected cells with Sirt1 overexpression plasmid20 (link) (addgene.com, plasmid 1791) to observe the effect of Sirt1 overexpression on interstitial fibrosis and CTGF in the presence of rhTGF-β1. HK-2 cells were randomly divided into three groups: NG, the TGF-β1 (5 ng/mL) stimulation group, and the TGF-β1 (5 ng/mL) plus Sirt1 overexpression group. Transfections of HK-2 cells with CTGF siRNA plasmid or Sirt1 overexpression plasmid were performed with Lipofectamine 2000 according to the manufacturer’s instructions. A negative control plasmid was transfected at the same time.