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

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

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

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 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

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

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

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

Impact of arteriovenous fistula blood flow on serum il-6, cardiovascular events and death: An ambispective cohort analysis of 64 Chinese hemodialysis patients

<div><p>Flows (Qa) of arteriovenous fistula (AVF) impact the dialysis adequacy in hemodialysis (HD) patients. However, data for different access flow levels on outcomes related to long-term dialysis patients, especially in Chinese patients, are limited. Herein, we performed an ambispective, mono-centric cohort study investigating the association between the AVF flows and inflammation, cardiovascular events and deaths in Chinese hemodialysis patients bearing a radio-cephalic fistula (AVF) from 2009 to 2015. Twenty-three patients (35.9%) developed at least one episode of cardiovascular disease (CVD) in two years after AVF creation. AVF Qa, IL-6 and hsCRP were significantly higher in patients with CVD than in patients without CVD. Multi-factorial binary logistic regression analysis found that the independent and strongest risk factor for CVD in HD patients was serum IL-6, which showed a positive association with AVF Qa levels in patients. Therefore, the linkage between AVF Qa tertiles and adverse clinical outcomes (cardiovascular events and mortality) was examined over a median follow-up of five years. IL-6 was significantly increased in the high AVF Qa (>1027.13 ml/min) group. Patients with median AVF Qa showed the lowest morbidity and mortality of CVD according to the AVF Qa tertiles, whereas higher Qa was associated with a higher risk of CVD, and lower AVF Qa (600 ml/min ≤AVF Qa <821.12 ml/min) had a higher risk of non-CVD death. Therefore, keeping the AVF Qa at an optimal level (821.12 to 1027.13 ml/min) would benefit HD patients, improve long-term clinical outcomes and lower AVF-induced inflammation.</p></div

Demographic and clinical characteristics of the 64 HD patients subdivided by the presence or absence of Cardiovascular Disease (CVD) after two years of hemodialysis, before the five-years follow-up.

<p>Demographic and clinical characteristics of the 64 HD patients subdivided by the presence or absence of Cardiovascular Disease (CVD) after two years of hemodialysis, before the five-years follow-up.</p