Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Blood urea nitrogen (BUN), serum creatinine ( A ), serum FGF23 and serum phosphate (Pi) levels ( B ). Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , Saa1 ( C, D ) and Hamp ( E ) expression levels in liver tissue. ( F ) Complete blood count (CBC) analysis. ( G ) Representative gross pathology of Perls’ Prussian blue-stained spleen sections (scale bar, 50 μm). Larger magnification is shown in supplementary figure and legends. ( H ) Liver Pi levels. All values are mean ± standard error of the mean (SEM; n = 8–9 mice/group; *p ≤ 0.05 vs. Fgfr4 +/+ + control diet, # p ≤ 0.05 vs. Fgfr4 −/− + control diet, $ p ≤ 0.05 vs. Fgfr4 +/+ + adenine diet) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median Fgfr4 +/+ + control diet measurements.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Control, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: ( A ) Serum FGF23 and serum Pi levels. ( B, C ) Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , and Saa1 expression levels in liver tissue. ( D ) Scatter plots showing correlations between liver Pi and serum Pi levels. ( E ) Scatter plots showing correlations between liver Hamp expression and liver Pi levels (a = slopes are significantly different from each other). ( F ) CBC analysis. ( G ) Representative gross pathology of Perls’ Prussian blue-stained spleen sections (scale bar, 50 μm). Larger magnification is shown in supplementary figure and legends. All values are mean ± standard error of the mean (SEM; n = 8 mice/group; *p ≤ 0.05 vs. Fgfr4 +/+ + 0.7% Pi diet, # p ≤ 0.05 vs. Fgfr4 −/− + 0.7% Pi diet, $ p ≤ 0.05 vs. Fgfr4 +/+ + 2% Pi diet, @ p ≤ 0.05 vs. Fgfr4 −/− + 2% Pi diet, & p ≤ 0.05 vs. Fgfr4 +/+ + 3% Pi diet) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median Fgfr4 +/+ + 0.7% Pi diet measurements. Scatter plot shadows indicate 95% confidence interval.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: BUN, serum creatinine ( A ), serum FGF23 and serum Pi levels ( B ). Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , and Saa1 ( C, D ) and Hamp ( E ) expression levels in liver tissue. ( F ) CBC analysis. ( G ) Representative gross pathology of Perls’ Prussian blue-stained spleen sections (scale bar, 50 μm). Larger magnification is shown in supplementary figure and legends. ( H ) Liver Pi levels. All values are mean ± standard error of the mean (SEM; n = 7–9 mice/group; *p ≤ 0.05 vs. Col4a3 +/+ + 0.6% Pi diet, # p ≤ 0.05 vs. Col4a3 −/− + 0.6% Pi diet) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median Col4a3 +/+ + 0.6% Pi diet measurements.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: ( A ) Immunoblot analysis of total protein extracts from primary hepatocytes ( n = 5 independent isolations). β-Actin serves as loading control. Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 , Saa1 ( B, C ), Hamp ( D ), and Slc20a1 ( E ) expression levels in primary hepatocytes; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. control [Ctrl]). Dotted lines indicate median Ctrl measurements. ( F ) qPCR analysis of Slc20a1 expression levels in primary hepatocytes following stimuli, with or without phosphonoformic acid (PFA); values are mean ± standard error of the mean (SEM; n = 6 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. 1 mM PFA Ctrl). Dotted lines indicate median vehicle Ctrl measurements. ( G ) Immunoblot analysis of total and phosphorylated p65 (NFκB) protein levels from primary hepatocytes following stimuli, with or without PFA ( n = 5 independent isolations). β-Actin serves as loading control. ( H–J ) qPCR analysis of Il1b , Il6 , Saa1 ( H–I ) and Hamp ( J ) expression levels in primary hepatocytes following stimuli, with or without PFA; values are mean ± standard error of the mean (SEM; n = 6 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. 1 mM PFA Ctrl) where statistical analyses were calculated by one-way analysis of variance (ANOVA; B–E ) or by two-way ANOVA ( F, H–J ) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median vehicle Ctrl measurements. Figure 6—source data 1. Original western blots. Original uncropped western blots of the cropped western blots shown in . The molecular weight is indicated on the right in kDa.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing, Comparison, Molecular Weight

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Quantitative polymerase chain reaction (qPCR) analysis of Il1b , Il6 ( A ) and Hamp ( B ) expression levels in primary hepatocytes following stimuli, with or without BAY 11-7082; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. 20 μM BAY 11-7082 Ctrl). Dotted lines indicate median vehicle Ctrl measurements. ( C ) qPCR analysis of Hamp expression levels in primary hepatocytes following stimuli with or without anti-IL1β, anti-IL6, or both antibodies in combination; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. anti-IL1β Ctrl, $ p ≤ 0.05 vs. anti-IL6 Ctrl, @ p ≤ 0.05 vs. anti-IL1β + anti-IL6 Ctrl) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate median vehicle Ctrl measurements.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Quantitative polymerase chain reaction (qPCR) analysis of primary hepatocytes shows expression levels of Saa1 ( A ), Hp ( B ), and Slc20a1 ( C ) following lipopolysaccharide (LPS) or Pi stimulation, with or without BAY 11-7082; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs 20 μM BAY 11-7082 Ctrl). Dotted lines indicate corresponding median measurements from vehicle Ctrl. ( D, E ) qPCR analysis of primary hepatocytes shows Saa1 and Hp expression levels following LPS or Pi stimulation, with or without anti-IL1β, anti-IL6, or both antibodies in combination; values are mean ± standard error of the mean (SEM; n = 4 independent isolations; *p ≤ 0.05 vs. vehicle control [Ctrl], # p ≤ 0.05 vs. anti-IL1β Ctrl, $ p ≤ 0.05 vs. anti-IL6 Ctrl, @ p ≤ 0.05 vs. anti-IL1β + anti-IL6 Ctrl) where statistical analyses were calculated by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. Dotted lines indicate corresponding median measurements from vehicle Ctrl.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Comparison

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet: Oligonucleotides used as sequence specific primers in quantitative polymerase chain reaction (qPCR) analyses.

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Sequencing, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

doi: 10.7554/eLife.74782

Figure Lengend Snippet:

Article Snippet: Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems).

Techniques: Knock-Out, Cell Culture, Recombinant, Iron Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Software, Control

Journal: The Journal of Neuroscience

Article Title: CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

doi: 10.1523/JNEUROSCI.2695-17.2017

Figure Lengend Snippet: Antihyperalgesic effect of HMWH. A, LMWH (1 μg) was injected intradermally on the dorsum of the hindpaw. Ten minutes later, HMWH (1 μg, black circles) or vehicle (open circles) was injected at the same site and the mechanical nociceptive threshold evaluated over time. Significant reversal of LMWH-induced hyperalgesia was observed in the group treated with HMWH (F(1.16) = 83.33, ****p < 0.0001, when both groups are compared, two-way repeated-measures ANOVA, followed by Bonferroni post hoc test). B, Four pronociceptive mediators, PGE2 (100 ng), epinephrine (epi, 100 ng), TNFα (100 ng), or IL-6 (10 ng), were injected intradermally on the dorsum of the hindpaw. Ten minutes after PGE2 and epinephrine, or 30 min after TNFα and IL-6, HMWH (1 μg, black bars) or vehicle (white bars) was injected at the same site. Measurement of the mechanical nociceptive threshold after an additional 30 min showed a significant attenuation of the hyperalgesia induced by all four pronociceptive mediators, in the groups treated with HMWH (PGE2 groups: t(10) = 5.676, ***p = 0.0001; epi groups: t(10) = 4.150, **p = 0.0010; TNFα groups: t(10) = 6.365, ****p < 0.0001; IL-6 groups: t(10) = 5.461, ***p = 0.0001, when the vehicle and HMWH groups are compared, unpaired Student's t test). C, Rats received four intraperitoneal injections of the neurotoxic chemotherapeutic drug paclitaxel (1 mg/kg), once every other day. Evaluation of mechanical nociceptive threshold 24 h after the last injection of paclitaxel showed robust mechanical hyperalgesia. Then HMWH (1 μg, black bar) or vehicle (white bar) was injected intradermally at the site of nociceptive testing on the dorsum of the hindpaw. Mechanical nociceptive threshold was again evaluated 30 min later. Whereas hyperalgesia was still observed in the vehicle-treated group, in the group that received HMWH, it was markedly attenuated (t(10) = 4.677, ###p = 0.0004, when control and HMWH groups are compared, unpaired Student's t test). A, Control group, n = 12 paws; HMWH group, n = 6. B, C, All groups, n = 6 paws.

Article Snippet: The following drugs were used in this study: hyaluronic acid sodium salt from Streptococcus pyrogenes (HMWH), from Calbiochem; epinephrine, prostaglandin E 2 (PGE 2 ), hyaluronic acid sodium salt from Streptococcus equi (LMWH), and the cancer chemotherapeutic agent paclitaxel, from Sigma-Aldrich; a peptide CD44 receptor agonist A6 ( Piotrowicz et al., 2011 ; Finlayson, 2015 ) from GenScript; rat recombinant interleukin-6 (IL-6) and rat recombinant TNFα, from R&D Systems; the selective activator of PKCε, psi ε Receptor for Activated C Kinase (ψεRACK), from Biomatik; and the potent membrane-permeable cAMP analog 8-bromo cAMP sodium salt (Tocris Bioscience).

Techniques: Injection

Journal: The Journal of Neuroscience

Article Title: CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

doi: 10.1523/JNEUROSCI.2695-17.2017

Figure Lengend Snippet: Antihyperalgesic effect of HMWH is CD44-dependent. A, Rats were treated daily with a spinal intrathecal injection of ODN sense or antisense for CD44 mRNA (120 μg) for 3 consecutive days. On the fourth day, TNFα (100 ng, left) or IL-6 (10 ng, right) was injected intradermally on the dorsum of the hindpaw. Thirty minutes later, vehicle (white bars) or HMWH (1 μg, black bars) was injected at the same site. Evaluation of the mechanical nociceptive threshold 30 min later showed a significant reversal of the hyperalgesia induced by TNFα and IL-6 in the groups that had been treated with antisense, compared with those treated with sense (TNFα, sense groups: t(10) = 7.306, ****p < 0.0001; antisense groups: t(10) = 1.007, p = 0.1688, nonsignificant; IL-6, sense groups: t(10) = 9.077, ****p < 0.0001; antisense groups: t(10) = 0.8891, p = 0.1974, nonsignificant, when the vehicle and HMWH groups are compared, unpaired Student's t test). B, Rats received four intraperitoneal injections of the chemotherapeutic drug paclitaxel (1 mg/kg, every other day). Intrathecal injections of ODN sense or antisense were performed, once a day, from days 5–7 of paclitaxel treatment. At 24 h after the last injections of paclitaxel and ODNs, vehicle (white bars) or HMWH (1 μg, black bars) was injected intradermally on the dorsum of the hindpaw. Mechanical nociceptive threshold was evaluated before treatment with paclitaxel and 30 min after the injection of vehicle or HMWH. Significant attenuation of paclitaxel-induced hyperalgesia was observed only in the ODN sense-treated group (t(10) = 4.781, ***p = 0.0004, compared with the antisense-treated group, unpaired Student's t test). Together, these results indicate that HMWH acts at CD44 to produce antihyperalgesia. n = 6 paws, all groups.

Article Snippet: The following drugs were used in this study: hyaluronic acid sodium salt from Streptococcus pyrogenes (HMWH), from Calbiochem; epinephrine, prostaglandin E 2 (PGE 2 ), hyaluronic acid sodium salt from Streptococcus equi (LMWH), and the cancer chemotherapeutic agent paclitaxel, from Sigma-Aldrich; a peptide CD44 receptor agonist A6 ( Piotrowicz et al., 2011 ; Finlayson, 2015 ) from GenScript; rat recombinant interleukin-6 (IL-6) and rat recombinant TNFα, from R&D Systems; the selective activator of PKCε, psi ε Receptor for Activated C Kinase (ψεRACK), from Biomatik; and the potent membrane-permeable cAMP analog 8-bromo cAMP sodium salt (Tocris Bioscience).

Techniques: Injection