fgf23 ifgf23  (Quidel)

Journal: bioRxiv

Article Title: Transgenic augmentation of erythroferrone in mice ameliorates anemia in adenine-induced chronic kidney disease

doi: 10.1101/2024.12.06.627111

Figure Lengend Snippet: WT and TG mice from (n=6-7 mice/group) were analyzed. Measured parameters include ( A ) serum phosphate, ( B ) serum intact iFGF23, and qPCR analysis of kidney tissue for ( C-G ) Slc34a1 and Slc34a3 ( encoding sodium-dependent phosphate transporters 2A and 2C), Cyp27b1 and Cyp24a1 ( encoding the enzymes that respectively generate and break down the active form of vitamin D, 1,25-dihydroxyvitamin D3) and Kl (encoding Klotho, the renal coreceptor for iFGF23). Data are mean ± SEM, analyzed by unpaired- t -test with Welch’s correction (two-tailed). *P <.05; ns = non-significant.

Article Snippet: Intact bioactive FGF23 (iFGF23) levels were assessed by ELISA (60-6800, QuidelOrtho).

Techniques: Two Tailed Test

Journal: Bone Research

Article Title: Osteocyte Egln1 /Phd2 links oxygen sensing and biomineralization via FGF23

doi: 10.1038/s41413-022-00241-w

Figure Lengend Snippet: HIF-PHI induce FGF23 in WT mice and murine osteocyte-like cells. Wild type mice (females) were injected with vehicle (PEG300/DMSO) or 50 mg·kg −1 of either FG-4592 (‘FG’, Roxadustat; red) or BAY 85-3934 (‘BAY’, Molidustat; blue) every other day for 5 days, for a total of 3 injections. a Plasma intact FGF23 and b C -terminal/total FGF23 concentrations at 4 hours after final injection. c Plasma erythropoietin (EPO) concentrations and d cortical bone (flushed of marrow) Fgf23 mRNA expression at the end of the study ( n = 6 mice per group; *** P < 0.001 versus vehicle, ### P < 0.001 versus FG treatment). e Transferrin receptor ( Tfrc ) and f Fgf23 mRNA expression in 2-week differentiated MPC2 cells treated for 24 h (blue) or 48 h (red) with 20 μmol·L −1 or 50 μmol·L −1 of the HIF-PHI FG-4592 (‘FG’; Roxadustat), AKB-6548 (‘AKB’; Vadadustat), or BAY 85-3934 (‘BAY’; Molidustat) (* P < 0.05, ** P < 0.01, *** P < 0.001 versus vehicle; # P < 0.05, ## P < 0.01 24 h versus 48 h). ( e , inset) HIF1α protein expression in MPC2 cells after 4 h of 20 μmol·L −1 or 50 μmol·L −1 HIF-PHI treatment. Flox- Fgf23 / Dmp1 -cre + and cre − mice were injected with vehicle (black) or 70 mg·kg −1 FG-4592 (red) every other day for 5 days, for a total of 3 injections. Plasma concentrations of g intact FGF23 and h EPO were measured 4 hours after final injection ( n = 4–6 mice per group; ** P < 0.01, **** P < 0.000 1)

Article Snippet: Secreted FGF23 protein was assessed using both the rodent-specific ‘intact’ FGF23 (‘iFGF23’) and ‘ C -terminal’ (or ‘total’) ‘cFGF23’ ELISAs (Quidel Laboratories, Inc.) and normalized to total protein concentration.

Techniques: Injection, Clinical Proteomics, Expressing

Journal: Bone Research

Article Title: Osteocyte Egln1 /Phd2 links oxygen sensing and biomineralization via FGF23

doi: 10.1038/s41413-022-00241-w

Figure Lengend Snippet: Osteocytes respond to HIF-PHI on the genomic level. a Transferrin receptor ( Tfrc ; red) and Fgf23 (blue) mRNA expression was measured in mesenchymal stem cells (MSC; undifferentiated MPC2 cells) and osteocytes (Ocy; 3-week differentiated MPC2 cells) treated with vehicle or 50 μmol·L −1 FG-4592 for 48 hours (** P < 0.01 vs. veh; ## P < 0.01 vs. MSC, same treatment). b RNAseq heat map of specific HIF-responsive genes and selected genes involved in mineralization and bone turnover in untreated and treated MSC and Ocy. c.i Top 15 Canonical Pathways identified in RNAseq data by Ingenuity Pathway Analysis in FG-4592-treated MSC compared to vehicle-treated MSC and c.ii FG-4592-treated Ocy compared to vehicle-treated Ocy (orange Z-score indicates predicted pathway activation and blue indicates predicted pathway inhibition). d Selected motifs, including HIF motifs, enriched in more open regions of the genome in treated Ocy compared to treated MSC or untreated Ocy by ATACseq. e Integration of RNAseq and ATACseq showed significant enrichment of HIF motifs in more open regions of the genome associated with gene upregulation compared to the entire genome. f Venn diagram of differentially expressed genes (DEG) and differentially accessible regions (DAR) that overlap in Ocy vs. MSC, identifying 337 genes that have more chromatin within −10 kb of the TSS and have upregulated gene expression. g Gene Ontology (GO) Biological Processes enriched in Ocy compared to MSC (numbers in parenthesis are gene count). h Venn diagrams of differentially expressed genes (DEG) and differentially accessible regions (DAR) in FG-4592-treated MSC vs. vehicle-treated MSC and FG-4592-treated Ocy vs. vehicle-treated Ocy, identifying 24 and 31 genes, respectively, that have more chromatin within −10 kb of the TSS and have upregulated gene expression. Of these genes in each cell type, 6 overlapped, whereas 25 genes were unique to Ocy. Among the top 3 genes in Ocy was Egln1 (encodes Phd2), a gene involved in oxygen sensing, and was thus examined further. i Representative ATACseq peaks of FG-4592-treated osteocytes (top track; crimson) compared to vehicle-treated osteocytes (bottom track; black). Highlighted in blue is the more open peak at −5 700 bp from the TSS of the Phd2 ( Egln1 ) gene. j Phd2 expression across MPC2 cell osteogenic differentiation (** P < 0.01 vs. undifferentiated cells) and in 3-week differentiated MPC2 cells treated with FG-4592 or BAY85-3934 for 48 h (** P < 0.01, *** P < 0.001, *** P < 0.000 1 vs. veh; ## P < 0.01 vs. undifferentiated cells)

Article Snippet: Secreted FGF23 protein was assessed using both the rodent-specific ‘intact’ FGF23 (‘iFGF23’) and ‘ C -terminal’ (or ‘total’) ‘cFGF23’ ELISAs (Quidel Laboratories, Inc.) and normalized to total protein concentration.

Techniques: Expressing, Activation Assay, Inhibition, Gene Expression

Journal: Bone Research

Article Title: Osteocyte Egln1 /Phd2 links oxygen sensing and biomineralization via FGF23

doi: 10.1038/s41413-022-00241-w

Figure Lengend Snippet: The role of osteocyte Phd2 in vivo. Wild type (WT) male mice were fed an adenine diet (AD) to induce CKD for 2, 4, or 6 weeks. Mice on casein diet for 8 weeks were used as controls. a Bone Fgf23 (blue) and Egln1 /Phd2 (red) mRNA expression was measured over diet duration. b Male Phd1 and c Phd3 expression in CKD bone. Numbers above timepoints indicate the % decrease in gene expression compared to the casein diet control ( n = 3–4 mice/group; ** P < 0.01; *** P < 0.001 vs. casein diet control). Fgf23 mRNA expression in d whole bone (including marrow), e osteocyte-enriched bone fractions, and f plasma total ( C -terminal) FGF23 concentrations in mice with conditional deletion of Phd2 in osteocytes (Phd2-fl/fl/Dmp1-cre + ) compared to mice without deletion (Phd2-fl/fl/Dmp1-cre − ) ( n = 6 mice/genotype; * P < 0.05, *** P < 0.001 vs. cre − mice)

Article Snippet: Secreted FGF23 protein was assessed using both the rodent-specific ‘intact’ FGF23 (‘iFGF23’) and ‘ C -terminal’ (or ‘total’) ‘cFGF23’ ELISAs (Quidel Laboratories, Inc.) and normalized to total protein concentration.

Techniques: In Vivo, Expressing, Gene Expression, Control, Clinical Proteomics

Journal: Bone Research

Article Title: Osteocyte Egln1 /Phd2 links oxygen sensing and biomineralization via FGF23

doi: 10.1038/s41413-022-00241-w

Figure Lengend Snippet: Phd2 bridges oxygen sensing mechanisms with Fgf23 production. A Phd2-KO cell line was generated in undifferentiated MPC2 cells using CRISPR/Cas9 technology and cells were differentiated for 2 or 3 weeks in osteogenic conditions. a Immunoblot of the Phd isoforms and HIF1α protein expression in 2- and 3-week differentiated WT and Phd2-KO cells. b Fgf23 expression in 3-week differentiated WT and Phd2-KO MPC2 cells (* P < 0.05 vs. WT cells). c Alizarin red stain to visualize mineral deposition in 3-week differentiated WT and Phd2-KO MPC2 cells. d Fgf23 gene expression and ( D , inset) HIF1α protein expression in 3-week differentiated WT MPC2 cells treated with the HIF-PHI FG-4592 (Roxadustat) alone or with FG-4592 and 1 μmol·L −1 or 5 μmol·L −1 of the HIF1α inhibitor BAY 87-2243 for 48 h (** P < 0.01 versus vehicle; ## P < 0.01 versus FG treatment alone). e Fgf23 expression and (inset) HIF1α protein expression in 3-week differentiated Phd2-KO cells treated with vehicle (DMSO) or with 1 μmol·L −1 or 5 μmol·L −1 of the HIF1α inhibitor BAY 87-2243 for 48 h (* P < 0.05 vs. veh). f Fgf23 expression in IDG-SW3 cells transduced with shRNA against HIF1α or HIF2α in vehicle or IOX2-treated cells (*** P < 0.001 vs. Scr control, same treatment, ### P < 0.001 vs. vehicle, same shRNA, ^^^ P < 0.001 shHIF1 vs. shHIF2). g Tfrc , ( g , inset) HIF1α protein, h Fgf23 , i Phd1, and j Phd3 gene expression in 3-week differentiated WT (solid bars) and Phd2-KO (dashed bars) MPC2 cells treated for 24 h with 20 μmol·L −1 or 50 μmol·L −1 of the HIF-PHI FG (FG-4592; Roxadustat) or BAY (BAY85-3934; Molidustat) (* P < 0.05, ** P < 0.01, *** P < 0.001 vs. veh, same genotype; # P < 0.05, ### P < 0.001 WT vs. Phd2-KO, same treatment)

Article Snippet: Secreted FGF23 protein was assessed using both the rodent-specific ‘intact’ FGF23 (‘iFGF23’) and ‘ C -terminal’ (or ‘total’) ‘cFGF23’ ELISAs (Quidel Laboratories, Inc.) and normalized to total protein concentration.

Techniques: Generated, CRISPR, Western Blot, Expressing, Staining, Gene Expression, Transduction, shRNA, Control

Journal: Bone Research

Article Title: Osteocyte Egln1 /Phd2 links oxygen sensing and biomineralization via FGF23

doi: 10.1038/s41413-022-00241-w

Figure Lengend Snippet: Phd2 is required for iron-mediated suppression of Fgf23. a Transferrin receptor ( Tfrc ; red) and Fgf23 (blue) expression in WT 3-week differentiated MPC2 cells treated for 48 h with DFO, then repleted with iron using increasing doses of Holo-transferrin (Tf) in DFO-containing media for an additional 48 h (*** P < 0.001 vs. OM ctrl; ### P < 0.001 vs. DFO alone). a inset HIF1α protein expression in MPC2 cells exposed to iron deficiency then repleted with iron using increasing doses of holo-Transferrin (Tf). b HIF1α, Phd2, Phd1, Phd3, and β-actin protein expression in 3-week differentiated WT and Phd2-KO cells exposed to iron deficiency for 48 h then iron repletion with high dose holo-Tf in DFO-containing media for an additional 48 h. c Phd2 ( Egln1 ), d Phd1 ( Egln2 ), and e Phd3 ( Egln3 ) expression in 3-week differentiated WT and Phd2-KO cells exposed to iron deficiency for 48 h then iron repletion with high dose holo-Tf in DFO-containing media for an additional 48 h (** P < 0.01, **** P < 0.000 1; # P < 0.05, ## P < 0.01 vs. genotype, same treatment). f Tfrc and g Fgf23 expression in 3-week differentiated WT (solid bars) and Phd2-KO (dashed bars) cells exposed to iron deficiency for 48 h then iron repletion with high dose holo-Tf in DFO-containing media for an additional 48 h (** P < 0.01; *** P < 0.001 vs. OM ctrl, same genotype; # P < 0.05, ### P < 0.001 vs. DFO, same genotype; ^ P < 0.05,^^^ P < 0.001 Phd2-KO vs. WT, same treatment)

Article Snippet: Secreted FGF23 protein was assessed using both the rodent-specific ‘intact’ FGF23 (‘iFGF23’) and ‘ C -terminal’ (or ‘total’) ‘cFGF23’ ELISAs (Quidel Laboratories, Inc.) and normalized to total protein concentration.

Techniques: Expressing