Table_1_Cardiovascular diseases morbidity and mortality among children, adolescents and young adults with dialysis therapy.DOCX

BackgroundThe age-specific burden of cardiovascular disease (CVD) and mortality in pediatric and young adult patients with end-stage kidney disease (ESKD) remains unclear. We aimed to examine the prevalence and incidence of CVD and all-cause mortality in children and adolescents compared with adults with dialysis in Taiwan.MethodsThis retrospective observational cohort study comprised 3,910 patients with more than 2 time point receipts of dialysis therapy in a year, including 156 aged ResultsAmong patients receiving dialysis treatment, the risk of composite CVD events [HR, 1.63 (1.22–2.19)] and mortality [HR, 1.76 (1.38–2.25)] was greater in children than the dialysis initiated in older patients. Non-atherosclerotic CVD was more prevalent, especially in younger patients, within the first 6 months after the initiation of dialysis. After 6 months of initial dialysis, the risk of atherosclerotic CVD was higher in adults than those for adolescents and children. The magnitude of CVD risk in adolescents who initiated dialysis therapy was higher in females [HR, 2.08 (1.50–2.88)] than in males [HR, 0.75 (0.52–1.10)].ConclusionYounger patients undergoing chronic dialysis with a higher risk of CVD events than older patients are associated with a faster onset of non-atherosclerotic CVD and a higher risk of both CVD- and non-CVD-related mortality.</p

Effect of inflammatory cytokines and Golgi glycosylation enzyme inhibitors on protein translocation of SCAP from ER to Golgi in THP-1 macrophages.

<p>THP-1 macrophages plated in chamber slides were cultured in serum-free experimental medium or experimental medium with 40 ng/ml IL-6 or 50 ng/ml TNF-α in the absence or presence of 25 µg/ml LDL or glycosylation inhibitors (2.5 ng/ml kifunensine and 2.5 ng/ml swainsonine) for 24 h at 37°C. The translocation of SCAP from the ER to the Golgi was investigated using confocal microscopy after dual-staining as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075650#s2" target="_blank">Materials and Methods</a> section (A). The co-localization efficiency of SCAP with Golgi was quantified by Carl Zeiss Aim software. Results represent mean±SD from 4 separate fields (B). ^*P<0.05 vs control; ^**P<0.01 vs control; ^***P<0.01 vs IL-6; ^#P<0.05 vs LDL; ^##P<0.01 vs LDL; ^### P<0.05 vs TNF-α.</p

The primers for real-time PCR.

<p>The primers for real-time PCR.</p

Effects of inflammation on SCAP stability in THP-1 macrophages.

<p>The cells were treated with or without 40/ml IL-6 or 50 ng/ml TNF-α for 24 h, and then chased in the presence of 50 µmol/l CHX for 0, 2, 4, 8, 16 and 24 h. The cells were lysed in equal volumes of buffer and protein levels plotted as a percentage of the SCAP remaining compared with the amount at 0 h. The histogram represents mean± SD of the densitometric scans of SCAP protein bands from 4 experiments, normalized to β- Actin and expressed as a percentage of control. ^*P<0.05 vs 0 h; ^**P<0.01 vs 0 h.</p

Effects of inflammation on LDLr, HMGCoAR and SREBP2 expression in THP-1 macrophages.

<p>THP-1 macrophages were incubated in serum free medium for 24 h at 37°C. The medium was then replaced by fresh serum-free medium in the absence (control) or presence of 40 ng/ml IL-6 or 50 ng/ml TNF-α or 25 µg/ml LDL alone or 40 ng/ml IL-6 plus 25 µg/ml LDL or 50 ng/ml TNF-α plus 25 µg/ml LDL for 24 h at 37°C. The mRNA levels were determined following the ΔΔ threshold cycle (Ct) protocol for real time RT-PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075650#s2" target="_blank">Materials and Methods</a> section. β-Actin served as a reference gene. Results represent the mean± SD from 4 experiments (A). The protein levels were examined by Western blotting (B). The histogram represents mean± SD of the densitometric scans of N-SREBP2, HMGCoAR and LDLr protein bands from four experiments, normalized by comparison with β-Actin and expressed as a percentage of control (C). ^*P<0.05 vs control; ^**P<0.01 vs control; ^#P<0.05 vs LDL; ^## P<0.01 vs LDL.</p

Effects of inflammation and Golgi glycosylation enzyme inhibitor on SCAP expression in THP-1 macrophages.

<p>THP-1 macrophages were incubated in serum free medium for 24 h at 37°C. The medium was then replaced by fresh serum-free medium in the absence (control) or presence of 40 ng/ml IL-6 or 50 ng/ml TNF-α or 25 µg/ml LDL alone or cytokine plus 25 µg/ml LDL or cytokine plus inhibitors for 24 h at 37°C. The mRNA levels were determined following the ΔΔ threshold cycle (Ct) protocol for real time RT-PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075650#s2" target="_blank">Materials and Methods</a> section. β-Actin served as a reference gene. Results represent the mean± SD from 4 experiments (A and D). The protein levels were examined by Western blotting (B). The histogram represents mean± SD of the densitometric scans of SCAP protein bands from four experiments, normalized by comparison with β-Actin and expressed as a percentage of control (C). ^*P<0.05 vs control; ^#P<0.05 vs LDL.</p

Effects of inflammation and Golgi glycosylation enzyme inhibitors on intracellular cholesterol accumulation in THP-1 macrophages.

<p>THP-1 macrophages were incubated in serum free medium for 24 h at 37°C. The medium was then replaced by fresh serum-free medium in the absence (control) (I) or presence of 40 ng/ml IL-6 (II) or 50 ng/ml TNF-α (III) or 25 µg/ml LDL (IV) or 40 ng/ml IL-6 plus 25 µg/ml LDL(V) or 50 ng/ml TNF-α plus 25 µg/ml LDL (VI) or glycosylation inhibitors (VII), or 40 ng/ml IL-6 plus inhibitors (VIII), or 50 ng/ml TNF-α plus inhibitors (IX) for 24 h at 37°C. Cell viability was determined using the MTT assay as described in Methods. Cell viability is expressed as the percentage of absorbance from treated cells compared to untreated cells. Graphs represent the average values (M±SD) of three independent experiments with triplicate biological measurements for each experiment (A). The cells were examined for lipid inclusions by Oil Red O (ORO) staining. The results are typical of those observed in four separate experiments (×400). Semi-quantitative analysis of ORO positive staining was performed by the Image-J software. Results represent mean± SD from 4 separate fields (B). Intracellular free cholesterol and total cholesterol were assayed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075650#s2" target="_blank">Materials and Methods</a> section. Values are means ± SD of duplicate wells from 4 experiments (C and D). ^*P<0.05 vs control; ^**P<0.01 vs control; ^***P<0.01 vs IL-6; ^#P<0.05 vs LDL; ^##P<0.01 vs LDL; ^###P<0.01 vs TNF-α.</p