Additional file 1 of Impact of HIF prolyl hydroxylase inhibitors in heart failure patients with renal anemia

Additional file 1. Receiver operating characteristic curve for (Figure S1) ferritin level at baseline and (Figure S2) TSAT value at 1 month after the start of HIF-PH inhibitor treatment

β-CG prevents loss of spiral ganglion cells.

<p>(A) Representative photomicrographs of spiral ganglion cells in the cochlear basal turns of control (arrow) and βCG-fed mice (white arrow) at 12 months of age (scale bar: 50 μm). Spiral ganglion cells were lost in control mice and preserved in βCG-fed mice. (B) Quantitative analysis revealed that the spiral ganglion cell density was significantly lower in the control than in the βCG-fed mice (<i>n</i> = 4, <i>P</i> < 0.05). (C) Transmission electron microscopy revealed that the numbers of lipofuscin granules in spiral ganglion cells was significantly higher in the control mice (long arrow) than in the βCG-fed mice (scale bar: 5 μm). Moreover, morphological damage such as nuclear invaginations (white arrow) or vacuolation in the myelin sheath (arrowheads) were also observed in control mice but were virtually absent in β-CG-fed WT mice (Fig 5C, right panel).</p

Composition of experimental diets.

<p>Composition of experimental diets.</p

Consumption of β-CG prevents hearing impairment in mice.

<p>At 6 and 8 months, control mice showed no threshold shifts at any frequencies. β-CG consumption did not affect ABR thresholds at these ages. At the age of 12 months, control mice showed a significant increase in ABR thresholds at 8, 16, and 32 kHz. Consumption of β-CG prohibited the increase in ABR thresholds (<i>n</i> = 7 in each group).</p

β-CG increases cochlear blood flow in WT mice.

<p>Quantitative analysis revealed that casein-fed mice showed a marked reduction in cochlear blood flow of 27.6% ± 8.5% versus βCG-fed mice (<i>P</i> < 0.05).</p

β-CG increases eNOS expression, reduces oxidative damage, and maintains microstructure in the stria vascularis.

<p>(A) Representative photomicrographs of tissue immunostained for eNOS. eNOS expression in the stria vascularis of the cochlear basal turn was higher in βCG-fed mice than in the control mice (scale bar: 50 μm). (B) Representative photographs of 4-HNE staining in the SV. Expression of 4-HNE was abundant in control mice and was reduced by β-CG consumption (scale bar: 50 μm). (C) Transmission electron microscopy revealed greater numbers of lipofuscin granules in control mice (upper-left panel, arrow) than in βCG-fed mice (scale bar: 5 μm). Vacuolar degeneration in microvascular pericytes was frequent in control mice (lower-left panel, arrowheads) (scale bar: 2 μm).</p

Overexpression of KLF15 rescues the reduction of adipolin expression caused by TNFα.

<p>Quantitative RT-PCR method was used for measurement of mRNA levels. <b>A</b>, Adipolin mRNA levels treated with adenovirus expressing KLF9 (Ad-KLF9), KLF15 (Ad-KLF15) or β-galactosidase (Ad-β-gal) at 150 moi for 24 h in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with TNFα (10 ng/ml) or vehicle for 24 h. N = 3 in each group. <b>B</b>, Adiponectin mRNA levels treated with Ad-KLF15 or Ad-β-gal at 150 moi for 24 h in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with TNFα (10 ng/ml) or vehicle for 24 h. N = 3 in each group.</p

Ablation of KLF15 by siRNA reduces adipolin expression in adipocytes.

<p>KLF15, adipolin (APL) and adiponectin (APN) mRNA levels were determined by quantitative RT-PCR method. <b>A</b>, KLF15 mRNA levels in 3T3-L1 adipocytes at 48 h after transfection with siRNA targeting KLF15 (si-KLF15) (20 nM) or non-targeting control siRNA (si-Control) (20 nM). N = 3 in each group. <b>B</b>, mRNA levels of APL and APN in 3T3-L1 adipocytes transfected with si-KLF15 (20 nM) or si-Control (20 nM). N = 3 in each group.</p

Expression of KLF15 augments the promoter activity of adipolin.

<p><b>A and B</b>, Effect of KLF15 and KLF9 on the promoter activity of adipolin. Protein levels of KLF15 (A) and KLF9 (B) in HEK293 cells transfected with pShuttle vector expressing KLF15 tagged with FLAG, KLF9 tagged with FLAG or empty vector (MOCK). Expression of KLF15 and KLF9 was evaluated by Western blot analyses using anti-FLAG antibody. HEK293 cells were transfected with pShuttle vector expressing KLF15, KLF9 or MOCK, along with pGL3-basic vectors containing adipolin promoter region (−66/−1 or −111/−1) or empty pGL3 vector in the presence of pRL-SV40. Promoter activity was assessed by luciferase reporter assay. Results are normalized relative to the values of empty pShuttle vectors (MOCK). N = 6 in each group. <b>C</b>, Luciferase assay for determination of adipolin promoter activity in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with pGL3-basic vectors containing adipolin promoter (−66/−1 or −111/−1) or empty pGL3 vector in the presence of pRL-SV40. N = 6 in each group.</p

Inhibition of JNK restores TNFα-mediated reduction of adipolin expression in 3T3-L1 adipocytes.

<p>The mRNA levels of adipolin (APL) and KLF15 were measured by quantitative RT-PCR method. <b>A and B</b>, Effect of JNK inhibitor on mRNA levels of APL (A) and KLF15 (B) in adipocytes. 3T3-L1 adipocytes were cultured in the presence or absence of JNK inhibitor, SP600125 (10 µM) for 1 h followed by treatment with TNFα (10 ng/ml) or vehicle for 24 h. N = 3 in each group. <b>C</b>, Effect of p38 MAPK inhibitor on APL mRNA levels in adipocytes. 3T3-L1 adipocytes were treated with or without p38 MAPK inhibitor, SB203580 (10 µM) for 1 h followed by stimulation with TNFα (10 ng/ml) or vehicle for 24 h. N = 3 in each group.</p