Additional file 1: of Initial central venous pressure could be a prognostic marker for hemodynamic improvement of polymyxin B direct hemoperfusion: a retrospective cohort study

Table S1. Results of univariate regression analysis for the difference in the vasopressor dependency index between before and 24 h after PMX-DHP. (XLSX 12 kb

Primer and probe sequences for qPCR of genomic DNA and cDNA.

<p>Primer and probe sequences for qPCR of genomic DNA and cDNA.</p

EGFP expression in cultured glomeruli from Nephrin-EGFP mice.

<p>On Day 2, EGFP expression remained mainly along the periphery of glomeruli, but was markedly reduced on day 5. FBS concentration was reduced from 10% to 0.5% on day 3. Treatment with 1α,25-(OH)₂ vitamin D₃ (VD, 50 nM) and all trans-retinoic acid (RA, 1 μM) for the last 2 days induced EGFP expression. Veh, vehicle. Magnification, 10x.</p

Expression of <i>nephrin</i> and <i>podocin</i> mRNA in cultured glomeruli.

<p>Gene expression was studied on days 0–5 (D0-D5) and at 6 hours (6h). Vitamin D (VD, 50 nM) or vehicle (Veh) was added for the last two days. Expression levels were normalized by 18S ribosomal RNA and levels at D0 were defined as 100%. Mean±SEM of n = 5. Treatment with VD significantly upregulated <i>nephrin</i> expression by unpaired t test. NS, not significant. *P<0.001 vs D0 by one-way ANOVA with Dunnett’s post hoc test.</p

Schematic diagram of pronuclear injection-based targeted transgenesis (PITT).

<p>The donor vector is co-injected with Cre expression plasmid into the pronuclei of fertilized eggs obtained from seed mice. As a result of site-specific recombination between mutant loxPs (lox2272, JTZ17 and JT15) by Cre enzyme, one copy of the donor vector is integrated into <i>Rosa26</i> genomic locus to yield recombination intermediate mice. Extra sequence containing selection marker (Neo) and vector sequence is removed by flippase (FLP)–FLP recombinase target (FRT) gene rearrangement, resulting in generation of Nephrin-EGFP mice. Nephrin, <i>nephrin</i> promoter sequence; EGFP, EGFP cDNA; pA, poly A signal; Neo, neomycin resistance gene cassette; FLPe, FLPe-overexpressing deleter mouse.</p

Protein expression of EGFP and glomerular cell markers in Nephrin-EGFP mice.

<p>(<b>A-C</b>) EGFP and nephrin were co-expressed along glomerular capillaries (yellow arrowheads). Cell bodies of podocytes located at the outer surface of glomerular capillaries (green arrowheads) also expressed EGFP (<b>A</b>) but nephrin expression was spared (<b>B</b>). (<b>D</b>) Similarly, EGFP and podocin were expressed along glomerular capillaries (yellow arrowheads). Podocyte cell bodies expressed EGFP but not podocin (green arrowheads). (<b>E</b>) PDGFRβ was expressed at glomerular and peritubular capillaries (arrowheads) and walls of arterioles (arrows), and did not co-localize with EGFP. (<b>F</b>) PECAM-1 was expressed along glomerular and peritubular capillaries (arrowheads) and inner surface of arterioles (arrow), and did not merge with EGFP. Magnification, 40x.</p

An example of chemical library screening of a plate.

<p>Y axis indicates EGFP intensity/glomerular area of each well under excitation. Wells treated with vitamin D (red circles) or vehicle (blue circles) are highlighted.</p

Effects of DMSO upon EGFP signals.

<p>Vitamin D (VD) or DMSO was added for the last 2 days and fluorescence was examined as EGFP/glomerular area on day 5. Mean±SEM of n = 3. Addition of 2% DMSO significantly decreased EGFP signals both in vehicle- and VD-treated glomeruli (P<0.05). Treatment with VD significantly increased EGFP signals in the presence of all concentrations of DMSO studied (0 to 2%, P<0.001). Comparison was carried out by two-way ANOVA with Bonferroni’s multiple comparison test.</p

Quantitative evaluation of changes in EGFP, nephrin and podocin protein expression in glomeruli by vitamin D.

<p>Endogenous nephrin and podocin were visualized by immunofluorescence using primary antibodies and Alexa Fluor 568-conjugated secondary antibodies. Fluorescence intensity was measured on days 1–5 (D1-D5). Glomeruli were treated with 50 nM vitamin D (VD) or vehicle (Veh) for the last 2 days and examined on day 5 (D5), and the difference was studied by unpaired t test. For EGFP quantitation, mean background signal of wild-type glomeruli was subtracted and the level of Veh-treated Nephrin-EGFP glomeruli on D5 was defined as 1.0 unit. For nephrin and podocin, mean background signal of wild-type glomeruli incubated with secondary antibody alone was subtracted, respectively. Difference between EGFP signals among days was examined by one-way ANOVA with Bonferroni’s multiple comparison test. N = 5.</p

Quantitative evaluation of EGFP signals in glomerular areas.

<p>(<b>A</b>) Glomeruli appeared as clusters of DAPI signals. (<b>B</b>) DAPI clusters having areas larger than > 2000 μm² were recognized as glomeruli and ROI were set to such glomerular areas (yellow arrows), whereas smaller clusters were excluded from ROI (green arrows). (<b>C</b>) EGFP signals were measured only in ROI. Magnification, 10x.</p