Fig. 4.

Chronic NIC exposure augments TGF-β1-mediated induction of profibrotic genes as well as activation of α-SMA promoter in cultured renal proximal tubule cells. A: LLC-PK₁ cells were treated with 10 ng/ml TGF-β1 for 3 days. In a set of experiments, cells were pretreated with 200 μM NIC for 24 h before treatment with TGF-β1. Expression of α-SMA, vimentin (vim), fibronectin, and E-cadherin was determined by Western blotting in cell lysates as described in materials and methods. B: densitometry results of blots shown in A; n = 3 independent experiments. Expression of those proteins were normalized to actin expression *P < 0.05 compared with none. #P < 0.05 compared with the TGF-β1 group. C: LLC-PK₁ cells were transfected with an α-SMA promoter-luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h. Some cells were treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1 for 24 h while other cells were treated with TGF-β1 only. Firefly (α-SMA promoter) and Renilla luciferase activities were determined as described in materials and methods. Values represent relative luciferase activities (firefly/Renilla) expressed as a percentage of controls. Values are means ± SD; n = 3 independent experiments. *P < 0.05 compared with none or as indicated.