Fig. 5: TMAO promotes myofibroblast differentiation and exacerbates renal fibrotic injury.
A Representative ratiometric traces (340/380 nm) from Human Renal Fibroblasts (HRFs) loaded with the Ca2+ indicator Fura-2 and stimulated with 100 µM TMAO. B Serum-starved HRFs were preincubated with the MEK inhibitor trametinib (10 nM) for 30 min prior to stimulation with 100 µM TMAO for the indicated times. Phospho-ERK1/2 levels were probed by Western blot; membranes were stripped and re-probed for total ERK1/2. C Serum-starved HRFs were stimulated with the indicated concentrations of TMAO and phospho-ERK1/2 and total ERK1/2 levels were determined as in (B). HRFs in complete medium were preincubated with the indicated concentrations of TMAO for 30 min and stimulated with TGF-β1 (5 nM) or vehicle for 24 h. Phospho-ERK1/2, phospho-SMAD3 (D) and alpha-smooth muscle actin (αSMA) (E) levels were probed with western blot. Β-actin levels for (D, E) were probed in parallel western blots. For B–E a representative image from three independent biological repeats is shown. F Immunostaining with αSMA of kidney sections (×20 magnification) from obstructed (UUO; 5 days post-surgery) or contralateral sham-operated (control) kidneys. Animals were fed normal chow (control), a diet containing 0.12% w/w TMAO (TMAO) or 1% choline w/w (Choline) for 6 weeks prior to surgery, as indicated. n = 6 per group. G Quantification of positive αSMA staining as (%) of stained area/field of view averaged from 5 images per animal. H Western blot of whole-kidney lysates for αSMA and vimentin expression. Tubulin, as loading control, was probed in parallel western blots. A representative photomicrograph from n = 2 Western blots with n = 1 animals for control (non-ligated kidneys, chow diet; C) n = 6 animals for all other groups is shown. OD of the (I) αSMA and (J) vimentin bands in (H) normalized against tubulin. The normalized density of the sham-control samples was arbitrarily set to 1. For all graphs, error bars represent the mean ± SEM of data from n = 4–6 animals per group. *P < 0.05 versus the UUO control. Source data are provided as a Source Data file.
