Fluorofenidone reduces PKC-ζ expression in the kidney of <i>db/db</i> mice.

<p>The expression of PKC-ζ and GAPDH in the indicated groups of animals was examined by western blot. Typical results are shown (A). Western blot results of individual animals were quantified, and expressed as fold change compared to PKC-ζ in <i>db/m</i> kidneys. Means±SD (n = 3 per group) are graphed. *: <i>p</i><0.05 in comparison to <i>db/m</i>; # <i>p</i><0.05 in comparison to the respective Ctrl mice (B). C) PKC-ζ in mice (n = 3 per group) treated with FD and LOS was expressed as the percentages of PKC-ζ in the kidneys of mock-treated <i>db/db</i> mice. The indicated comparisons were also analyzed.</p

Fluorofenidone inhibits TGF-β1 upregulation in the kidneys of <i>db/db</i> mice in a DN progression dependent manner.

<p>A) RNA was purified from the indicated mice (n = 3 per group). TGF-β1 mRNA abundance was subsequently determined by real-time PCR following our published methodology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111242#pone.0111242-Peng1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111242#pone.0111242-Wang2" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111242#pone.0111242-Wang1" target="_blank">[16]</a>. Real-time PCR was performed 3 times in triplicate. TGF-β1 mRNA levels were normalized to actin, and expressed as the fold change in reference to TGF-β1 mRNA in <i>db/m</i> mice. *: <i>p</i><0.05 in comparison to <i>db/m</i>; #: <i>p</i><0.05 in comparison to the respective Ctrl mice; other comparison are also presented. B) TGF-β1 protein in the indicated mice (n = 6 per group) was quantified using ELISA. Assay was repeated three times in triplicate. TGF-β1 protein was expressed as ng per mg of total protein (ng/mg total protein). Statistical analysis was performed as detailed above.</p

Fluorofenidone offers greater protection of glomerular expansion at earlier than later stages of DN pathogenesis.

<p>A) Experimental schedule. Six <i>db/db</i> or <i>db/m</i> mice were mock-treated with vehicle at the indicated time points; six <i>db/db</i> mice were given either fluorofenidone (FD) or losartan daily, starting at 5, 8, or 12-week old; all groups were treated until the age of 24-weeks old. Duration of treatments for all schedules is indicated. B) Typical PAS staining images of glomeruli for the respective animal groups indicated in panel A. Ctrl (control): mock treatment. Scale bars represent 20 µm. C) Glomerular expansion index was derived by quantification of glomerular expansion based on PAS staining; means±SD are graphed. *: <i>p</i><0.05 in comparison to <i>db/m</i>; #: <i>p</i><0.05 in comparison to the respective Ctrl (diabetic) mice; $: <i>p</i><0.05 in the indicated comparison. C: Ctrl, F: fluorofenidone. L: losartan. D) Urinary albumin excretion was determined using 24-hour urine collections (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111242#pone-0111242-t002" target="_blank">Table 2</a> for details). As comparable levels of UAE were obtained in 5, 8, and 12-week old mock-treated animals, the average UAE for mock-treated <i>db/db</i> mice as a single entity was calculated; this was used to derive the percentages of UAE in fluorofenidone and losartan-treated <i>db/db</i> mice. Six mice were used in individual groups.</p

Fluorofenidone and losartan are without effects on diabetic pathogenesis of <i>db/db</i> mice.

<p>Fluorofenidone and losartan are without effects on diabetic pathogenesis of <i>db/db</i> mice.</p

Fluorofenidone and losartan protect the progression of diabetic nephropathy in <i>db/db</i> mice.

<p>Fluorofenidone and losartan protect the progression of diabetic nephropathy in <i>db/db</i> mice.</p

Fluorofenidone inhibits AKT activation in the diabetic kidneys of <i>db/db</i> mice.

<p>AKT activation (phosphorylation of AKT at serine 473, p-AKT), total AKT, and GAPDH in individual groups were determined by western blot; typical results from the 5-week old animals are shown (A). Quantification of western blot results is expressed as fold change compared to that in <i>db/m</i> mice. The symbols * and # represent <i>p</i><0.05 in comparison to <i>db/m</i> and the individual Ctrl mice, respectively (B). Three mice were used in individual groups.</p

The kinetics of renal function in early stages of diabetic pathogenesis in <i>db/db</i> mice.

<p>The kinetics of renal function in early stages of diabetic pathogenesis in <i>db/db</i> mice.</p

Fluorofenidone inhibits ERK activation in the diabetic kidneys of <i>db/db</i> mice.

<p>Phosphorylated ERK (p-ERK), indicative of ERK activation, and total ERK protein levels in the indicated groups were examined by western blot. Typical results for animals in the individual groups are shown (A). Quantification of western blot results is included in panel B. ERK activation was expressed as fold change compared to that in <i>db/m</i> mice. The symbols * and # are for <i>p</i><0.05 in comparison to <i>db/m</i> and the individual Ctrl mice, respectively (B). Three mice were used in individual groups.</p

Fluorofenidone decreased p22^phox expression in the kidney of <i>db/db</i> mice at earlier stages of DN development.

<p>A) The expression of p22^phox and GAPDH in the indicated groups of animals was determined by western blot. Typical results are included. B) Western blot results of individual animals were quantified, and expressed as fold change compared to p22^phox in <i>db/m</i> mice. Means±SD are graphed. The symbols * and # represent <i>p</i><0.05 in comparison to <i>db/m</i> and the individual Ctrl mice, respectively. C) The p22^phox expression in mice treated with FD and LOS was expressed as the percentages of p22^phox in the kidneys of mock-treated <i>db/db</i> mice. The indicated comparison (FD 5-week vs FD 12-week) was analyzed by 2-tailed Student t-test. Three mice were used in individual groups.</p