Figure 1.

The antifibrotic actions of RLX were blocked by the AT₁R antagonist irbesartan in vitro. (A) Representative Western blots of renal phosphorylated (phospho)-p44 and p42 MAPK (phospho-ERK1/2), total p44 and p42 MAPK (ERK1/2), nNOS, phospho-nNOS, and α-tubulin; (B) TGF-β1, phospho-Smad2, total Smad2, α-SMA, and α-tubulin; and (C) representative gelatin zymographs of latent (L) and active (A) MMP-9 and MMP-2 levels and representative Western blots of L-MMP-13 and α-tubulin expression from untreated (control) RRMFs and cells treated with RLX (16.8 nM) alone, or in the presence of irbesartan (0.1, 1, and 10 μM) after 72 hours in culture. The total p44 and p42 MAPK (ERK1/2), unphosphorylated Smad2, and α-tubulin blots (A) were included to demonstrate the quality and equivalent loading of protein samples. (B) Also shown are the relative mean±SEM OD levels of phospho-ERK1/2 (corrected for total ERK1/2 levels); nNOS and phospho-nNOS (both corrected for α-tubulin levels); TGF-β1, α-SMA (both corrected for α-tubulin levels), and phospho-Smad2 (corrected for total Smad2 levels); and MMP-9, MMP-2, and MMP-13 (corrected for α-tubulin levels) from each of the groups studied, as determined by densitometry scanning (from n=3–4 separate experiments conducted in duplicate), to that of the untreated group, which was expressed as 1 in each case. *P<0.01 versus untreated cells; ^#P<0.01 versus RLX alone-treated cells.