PPARα siRNA accelerated high glucose-induced mesangial cell proliferations via ERK1/2 and PI3K-AKT pathways.

<p>PPARα siRNA was transfected into mesangial cells for 5h, followed by stimulating with high glucose for 24h, or high glucose plus Ly294002 (10µmol/L), or high glucose plus U0126 (10µmol/L). Cells were analyzed by flow cytometry after PI staining. Data in the bar graphs represent the average values ± SE of experiments performed in triplicate. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose; HG, high glucose.</p

MTT assay for cell proliferation analysis.

<p>Fenofibrate inhibited mesangial cell proliferation induced by high glucose for 12h, 24 h or 48h, at a final concentration 1, 10, 50, 100µM, respectively. The maximum inhibition was reversed by the administration of MK886 (1 or 10 µmol/L). The result is average values ± SE of representative data from experiments in triplicate. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose (5mM); HG, high glucose (40 mM); FN, fenofibrate; MK, MK886.</p

Expression and activity of PPARα in rat mesangial cells (MCs) treated with high glucose and/or fenofibrate.

<p>Mesangial cells were stimulated with high glucose 40mmol/L, in the absence or presence of fenofibrate (10–100 µM), or 100 µM fenofibrate plus MK886 (1 or 10 µM) for 24 h. (A) After incubation, PPARα mRNA and protein levels were determined by real time RT-PCR and Western Blot, normalized to β-actin or GAPDH. (B) For detection of PPARα activity, MCs were transfected with a PPRE-luciferase reporter vector for 5 h before treatment. Its activity was measured using the luciferase assay system. Data in the bar graphs represent the average values ± SE of experiments performed in triplicate. *P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose (5mM); HG, high glucose (40 mM); FN, fenofibrate; MK, MK886.</p

Effect of fenofibrate on the phosphorylation of ERK1/2 and PI3K-AKT in rat mesangial cells.

<p>(A) Representative western blot of p-AKT. (B) Representative western blot of p-ERK1/2. The figure shows the average volume density of phosphorylated ERK1/2 and AKT corrected for the loading control, total ERK1/2 and AKT. Values are given as mean±S.D. from 3 separate experiments. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose (5mM); HG, high glucose (40 mM); FN, fenofibrate; MK, MK886.</p

Fenofibrate reduced mesangial cell proliferations stimulated by high glucose.

<p>MCs were preincubated for 2h with various concentrations of fenofibrate, and then stimulated with high glucose for 24h. Fenofibrate inhibits mesangial cell proliferation induced by high glucose at a final concentration 1, 10, 50, 100µM, respectively. Cells were analyzed by flow cytometry after PI staining and the relative percentage of cells in different cell cycle phases are reported, while the percentage of apoptotic events was ignored. Data in the bar graphs represent the average values ± SE of experiments performed in triplicate. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose (5mM); HG, high glucose (40 mM); FN, fenofibrate; MK, MK886.</p

Effect of fenofibrate on the expression and secretions of extracellular matrix component in high glucose treated mesangial cells.

<p>(A) Representative western blot of Collagen-IV. (B) Collagen-IV secretion in culture media by ELISA assay. Protein levels were determined by Western Blot, normalised to GAPDH. Values are given as mean ±S.D. from 3 independent experiments in triplicate and p<0.05 is considered statistically significant. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose (5mM); HG, high glucose (40 mM); FN, fenofibrate; MK, MK886.</p

Effect of PPARα siRNA on ERK1/2 and PI3K-AKT pathways, and the Collagen-IV expression in high glucose treated mesangial cells.

<p>(A) Representative western blot of PPARα. (B) Representative western blot of p-AKT. (C) Representative western blot of p-ERK1/2. (D) Representative western blot of Collagen-IV. Protein levels were determined by western Blot, normalised to GAPDH. Phosphorylated ERK1/2 and AKT were corrected for the loading control, total ERK1/2 and AKT. Values are given as mean ±S.D. from 3 independent experiments in triplicate and p<0.05 is considered statistically significant. ^*P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose; HG, high glucose.</p

Expression CDK4 in rat mesangial cells (MCs) treated with high glucose.

<p>Mesangial cells were stimulated with high glucose 40mmol/L, in the absence or presence of fenofibrate (100 µM), or PPARα siRNA plus Ly294002 (10µmol/L), or high glucose plus U0126 (10µmol/L) for 24 h. CDK4 mRNA levels were determined by real time RT-PCR, normalised to β-actin. Data in the bar graphs represent the average values ± SE of experiments performed in triplicate. *P<0.05 vs NG; ^# p<0.05 vs HG. NG, normal glucose; HG, high glucose; FN, fenofibrate.</p

AMD3100 increased inflammatory cytokine secretion, and promoted CD3-positive cell infiltration following UUO.

<p>A. Expression of renal inflammatory cytokines (IL-6, TNF-α, IL-10 and IFN-γ) was detected by RT-PCR. B. Percentages of different inflammatory cell types (cells positive for CD45, CD3, CD11b and F4/80) in the kidney were measured by flow cytometry. C. Immunofluorescence was used to further determine CD3-positive cell infiltration into the kidney, and the number of infiltrated CD3-positive cells was determined. (***<i>p</i><0.001, **<i>p</i><0.005, *<i>p</i><0.05, n.s. means no significant difference, scale = 100μm, magnification×400)</p

AMD3100 promoted T cell chemotaxis but not proliferation.

<p>A. Fold changes in T cell-related chemokines were measured by RT-PCR. B. Double staining of CD3 (red) and PCNA (green) was examined by immunofluorescence, and the number of double-positive cells (white arrows) was analyzed. (***<i>p</i><0.001, **<i>p</i><0.005, *<i>p</i><0.05, n.s. means no significant difference, scale = 100μm, magnification×400)</p