Μεσαίες πόλεις : προοπτικές ανάπτυξης - διαφανείς τάσεις στο ελληνικό αστικό σύστημα

<p>HR3∶ 16 hours hypoxia followed by 3 hours reoxygenation.</p

Additional file 3: Table S3. of Questionnaire survey on nutritional supplement therapy and exercise training at hemodialysis facilities in Japan

Logistic regression analysis with conducted using per-100-patient staffing level. (PDF 111 kb

Additional file 2: Table S2. of Questionnaire survey on nutritional supplement therapy and exercise training at hemodialysis facilities in Japan

Proportion of facilities providing interventions based on lower and higher response probabilities. (PDF 88 kb

Additional file 1: Item S1. of Questionnaire survey on nutritional supplement therapy and exercise training at hemodialysis facilities in Japan

Questionnaire. (PDF 194 kb

PCR primers used for detection of mRNAs.

<p>PCR primers used for detection of mRNAs.</p

miR-205 contributes to both signalings triggered by oxidative and ER stresses in renal tubular cells.

<p>When cells are exposed to oxidative stress or ER stress, maladaptive down-regulation of miR-205 and subsequent up-regulation of EGLN2 occurs. ATF4 and HIF, which are negatively regulated by EGLN2, are supposed to contribute to the downstream suppression of anti-oxidant enzymes.</p

Change in cell survival under oxidative and ER stresses in miR-205-inhibited HK-2.

<p>(A) Inhibition of miR-205 in HK-2. Real-time RT-qPCR analysis revealed that transfection of an LNA-miR-205 inhibitor into HK-2 for 24 hours significantly inhibited miR-205 expression. The data represent the means ± S.E. of triplicate analysis from three independent experiments. **P<0.01 versus control siRNA transfected HK-2 by standard <i>t</i>-test. (B) In miR-205-inhibited HK-2, the cell number was significantly fewer at 24 and 48 hours after transfection than that of with HK-2 transfected with negative control. In contrast, up-regulation of miR-205 had no effects on the cell number. The data represent the means ± S.E. of triplicate counts from three independent experiments. *P<0.05 versus negative control transfected HK-2 at each time point by standard <i>t</i>-test. (C) Representative pictures of HK-2, 24 hours after transfection with miR-205 inhibitor or control inhibitor. Although cell growth was repressed, cell morphology of miR-205-inhibited HK-2 was not different from control siRNA transfected HK-2. (D) LDH release assay showed that modulation of miR-205 did not affect cell death, suggesting that the decrease in cell number in miR-205-inhibited HK-2 was not due to cell injury, but rather to repression of cell growth. The data represent the means ± S.E. of triplicate analysis from three independent experiments. Statistical analysises were performed by Tukey’s multiple comparison test. (E,F) Trypan blue exclusion assay showed the cell viability changes in miR-205 modulated HK-2 (▪) exposed to hypoxia-reoxygenation (0.1% O₂ for 16 hours followed by 6 hours reoxygenation), H₂O₂ (1000 µM for 6 hours) or TUN (2 µg/ml for 24 hours) compared to HK-2 transfected with negative control (□). Down-regulation of miR-205 led to significant decrease in cell viability by each stimulation (E), wheares up-regulation of miR-205 vice versa (F). Cell viabilities were not changed without any stimulations. The data are the means ± S.E. of triplicate counts from three independent experiments. *P<0.05, **P<0.01 by standard <i>t</i>-test.</p

EGLN2 as a novel downstream target of miR-205.

<p>(A) Quantitative real-time RT-PCR analysis revealed that the expression of EGLN2 was increased by miR-205 inhibition, but not significantly altered by overexpression of miR-205 compared to negative control transfected HK-2. No changes were seen in expression levels of EGLN1 (PHD2) or EGLN3 (PHD3). The data represent the means ± S.E. of triplicate analysis from three independent experiments. *P<0.05 by Dunnett’s multiple comparison test. (B) Western blot analysis showed that EGLN2 was also increased at protein level by miR-205 inhibition. The result of densitmetry represent the means ± S.E. of triplicate analysis from two independent experiments.**P<0.01 by standard <i>t</i>-test. (C) Quantitative real-time RT-PCR analysis revealed that the expression level of EGLN2 was increased in HK-2 exposed to hypoxia-reoxygenation or TUN, but these increases were supressed by miR-205 overexpression (▪) compared to negative control transfected HK-2 (□). *P<0.05 by Tukey’s multiple comparison test. (D) EGLN2 (transcript variant 1) contains the predicted binding site for miR-205 in its 3′-UTR. The predicted pairing of the mRNA target region (top) and miRNA (bottom) is as indicated, wherein solids line indicates hydrogen base pairing and the dotted line indicates Watson-Crick/wobble base pairing. The shaded sequence indicates the seed region of miR-205. (E) Mutant 3′-UTR sequence that abolished binding to miR-205. (F) HK-2 cells were transiently cotransfected with a luciferase reporters expressing EGLN2 3′-UTR or mutant EGLN2 3′-UTR miRNA target sequence and miR-205 mimic, miR-205 inhibitor, or negative control. At 24 hours posttransfection, firefly luciferase activities were measured and normalized with <i>Renilla</i> luciferase activities. Relative luciferase activity was increased by miR-205 inhibition when compared with control, while mutant EGLN2 3′-UTR had no effect on luciferase activity, indicating that miR-205 acts on the 3′-UTR of the EGLN2. Though transfection of HK-2 with EGLN2 3′-UTR vector along with miR-205 overexpression tended to decrease luciferase activity, this change was not sicnificant. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test. (G) When transiently transfected HK-2 with EGLN2 3′-UTR vector were exposed to oxidative or ER stress, the relative luciferase activities were significantly increased, and these increases were canceled with mutant EGLN2 3′-UTR vectors. Furthermore, co-transfection of EGLN2 3′-UTR vector with miR-205 mimic partially restrained these stress-induced increases of luciferase activities. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test.</p

miRNA expression change in HK-2 exposed to 10 hrs reoxygenation after hypoxia.

<p>HR10∶ 16 hours hypoxia followed by 10 hours reoxygenation.</p

Suppression of miR-205 increased oxidative stress.

<p>(A) Representative figure of the result of FACS. Formation of intracellular ROS was confirmed using CM-H2DCFDA, a dye that emits green fluorescence on reaction with ROS. miR-205-inhibited HK-2 cells were exposed to H₂O₂ in a short time as an oxidative stress inducer. The fluorescence value of miR-205-inhibited HK-2 was higher than the control both before (uncolored area) and after (gray area) induction by H₂O₂. ROS-positive expression boundaries are marked by horizontal lines (M1) on the histogram plots. (B) Changes in the proportion of ROS-positive cells by miR-205 inhibition before and after H₂O₂ treatment. Intracellular ROS was significantly increased in miR-205-inhibited HK-2 (▪) compared to negative control transfected HK-2 (□) even before the cells were exposed to H₂O₂. The data represent the means ± S.E. of duplicate analysis from three independent experiments. *P<0.05 by Tukey’s multiple comparison test. (C) Western blot analysis of CEL showed that the intensity of bands with a molecular weight corresponding to 32, 23, and 17 kDa (black arrows) were increased in miR-205-inhibited HK-2 compared with control inhibitor transfected HK-2, suggesting that intracellular ROS was increased by down-regulation of miR-205.</p