Journal: Endocrine Connections

Article Title: Cholecalciferol supplementation effectively improved tertiary hyperparathyroidism, FGF23 resistance and lowered coronary calcification score: a prospective study

doi: 10.1530/EC-22-0123

Figure Lengend Snippet: Change of laboratory parameters in KT group at four time points ( n = 209).

Article Snippet: Serum concentrations of intact FGF23 (iFGF23) (Boster Biological Technology., Ltd., Wuhan, China) and KLA (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) were measured using ELISA methods with the available commercial kits.

Techniques:

Journal: Endocrine Connections

Article Title: Cholecalciferol supplementation effectively improved tertiary hyperparathyroidism, FGF23 resistance and lowered coronary calcification score: a prospective study

doi: 10.1530/EC-22-0123

Figure Lengend Snippet: Comparison of laboratory parameters between vitamin D-treated group and -untreated group at month 6 ( n = 209).

Article Snippet: Serum concentrations of intact FGF23 (iFGF23) (Boster Biological Technology., Ltd., Wuhan, China) and KLA (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) were measured using ELISA methods with the available commercial kits.

Techniques: Comparison

Journal: Endocrine Connections

Article Title: Cholecalciferol supplementation effectively improved tertiary hyperparathyroidism, FGF23 resistance and lowered coronary calcification score: a prospective study

doi: 10.1530/EC-22-0123

Figure Lengend Snippet: Comparison of laboratory parameters and CACS between vitamin D-treated group and -untreated group at month 12 ( n = 209).

Article Snippet: Serum concentrations of intact FGF23 (iFGF23) (Boster Biological Technology., Ltd., Wuhan, China) and KLA (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) were measured using ELISA methods with the available commercial kits.

Techniques: Comparison

Journal: Endocrine Connections

Article Title: Cholecalciferol supplementation effectively improved tertiary hyperparathyroidism, FGF23 resistance and lowered coronary calcification score: a prospective study

doi: 10.1530/EC-22-0123

Figure Lengend Snippet: Change of (A) iFGF23 (logarithmic scale; Ln iFGF23), (B) KLA, (C) BCTX and (D) P1NP in treated and untreated group within 12 months posttransplant. * P < 0.05 compared between groups. ** P < 0.01 compared between groups. # P < 0.05 compared with HC group. † P < 0.05 compared with CKD group.

Article Snippet: Serum concentrations of intact FGF23 (iFGF23) (Boster Biological Technology., Ltd., Wuhan, China) and KLA (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) were measured using ELISA methods with the available commercial kits.

Techniques:

Journal: Endocrine Connections

Article Title: Cholecalciferol supplementation effectively improved tertiary hyperparathyroidism, FGF23 resistance and lowered coronary calcification score: a prospective study

doi: 10.1530/EC-22-0123

Figure Lengend Snippet: Multivariate regression analysis of risk factors for THPT and CACS at month 12.

Article Snippet: Serum concentrations of intact FGF23 (iFGF23) (Boster Biological Technology., Ltd., Wuhan, China) and KLA (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) were measured using ELISA methods with the available commercial kits.

Techniques:

Journal: PLOS ONE

Article Title: Phosphorus-independent role of FGF23 in erythropoiesis and iron homeostasis

doi: 10.1371/journal.pone.0315228

Figure Lengend Snippet: C57BL/6J male mice were fed a diet containing 0.6% inorganic phosphorus (Pi), 1.2% Pi, or 1.65% Pi for 2 weeks. (A) Body weight after 2 weeks of phosphorus supplementation. (B-C) Serum concentration of (B) intact and (C) C-terminal FGF23 measured by ELISA. (D-E) Phosphorus levels measured in (D) urine as ratio of phosphorus to creatinine and (E) serum. (F-H) Quantitative real-time RT-PCR for renal (F) α-Klotho , (G) Napi2a , and (H) Napi2c expression. Data are expressed as fold change (2 -ΔΔCt ) relative to housekeeping gene Hprt . Samples were measured in duplicates. Data are represented as mean ± SD. All data were analyzed for normality with Shapiro-Wilk test and homogeneity of variance by Brown-Forsythe test. For samples with normal distribution, one-way ANOVA was performed compared to 0.6% Pi with Dunnett’s multiple comparison test (B, C, E, F, G, H). When the samples were in normal distribution but not in homogeneity of variance, Welch’s ANOVA was performed (D). The samples not in normal distribution were analyzed with non-parametric Kruskal-Wallis test (A). (n = 7–10 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 compared to 0.6% Pi.

Article Snippet: Serum FGF23 levels were measured using mouse FGF23 Intact and C-terminal ELISA assays (Quidel Corporation/Immutopics International, San Clemente, CA, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Comparison

Journal: PLOS ONE

Article Title: Phosphorus-independent role of FGF23 in erythropoiesis and iron homeostasis

doi: 10.1371/journal.pone.0315228

Figure Lengend Snippet: C57BL/6J male mice were fed a diet containing 0.6% inorganic phosphorus (Pi), 1.2% Pi, or 1.65% Pi for 2 weeks. (A) Serum erythropoietin (EPO) levels measured by ELISA. (B-E) Quantitative real-time RT-PCR for expression of (B) renal Epo , (C) bone marrow Epo receptor (EpoR), (D) renal Hif-2α , and (E) bone marrow erythroferrone ( Erfe ). (F) Regression curve for correlation between bone marrow Erfe expression levels and serum FGF23 levels. (G) Quantitative real-time RT-PCR for splenic expression of heme oxygenase-1 ( Hmox-1 ). Data are expressed as fold change (2 -ΔΔCt ) relative to housekeeping gene Hprt . Data are represented as mean ± SD. All data were analyzed for normality with Shapiro-Wilk test and homogeneity of variance by Brown-Forsythe test. For samples with normal distribution and equal variance, one-way ANOVA with Dunnett’s multiple comparison test was performed (C, D, G). Samples with unequal variance were analyzed with Welch’s ANOVA (A, B, E). For the correlation curve, we applied simple linear and simple logistic regression (F). (n = 7–10 per group). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Serum FGF23 levels were measured using mouse FGF23 Intact and C-terminal ELISA assays (Quidel Corporation/Immutopics International, San Clemente, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Comparison

Journal: The FASEB Journal

Article Title: Lysophosphatidic Acid Synergizes With 1,25‐Dihydroxyvitamin D to Promote Fibroblast Growth Factor‐23 Synthesis via MAPK Signaling and Induction of the IL12A Gene

doi: 10.1096/fj.202502235R

Figure Lengend Snippet: Stimulation of FGF23 production by LPA and 1,25D and the whole transcriptome analysis of Ocy454 cells. (A) Secreted total FGF23 (cFGF23) concentrations in the culture medium of mouse long bone explants in response to vehicle (VEH), LPA alone, 1,25D alone, or LPA/1,25D after 24 h of stimulation. (B) Serum intact FGF23 (iFGF23) levels following the intraperitoneal LPA injection into the wild‐type and VDR KO littermates. (C) Experiment design for whole transcriptome analysis in Ocy454 cells. (D) Ven diagrams of differentially expressed genes (DEGs) in response to LPA, 1,25D, or LPA/1,25D treatment 2 and 8 h after stimulation. (E) Volcano plots of LPA and 1,25D responsive genes demonstrating LPA and 1,25D act on Ocy454 cells. (F) Expression profile of osteocyte genes in response to LPA, 1,25D, or the LPA/1,25D treatment after 2‐ and 8‐h stimulation. One‐way ANOVA followed by Tukey's (A) or two‐way ANOVA followed by Bonferroni (B) post hoc tests for multiple comparisons. Mean ± SEM, n = 3–4 experiments and n = 7–10 mice/group, respectively. DEGs and adjusted p values obtained by Benjamini‐Hochberg (BH) method using Limma package on R studio (D–F). Blue: 1,25D and Red: LPA responsive genes (E). Asterisks represent * p < 0.05, ** p < 0.01, *** p < 0.001 compared to vehicle‐treated group (F).

Article Snippet: Serum was collected 24 h later, and intact FGF23 (iFGF23) concentrations were measured using the iFGF23 (60–6800, Quidel) kit according to the manufacturer's instructions.

Techniques: Injection, Expressing

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: Representative bone section showing immunofluorescence for A) FGF23 (red); DMP1 (green); and their co-expression (yellow). Live nuclei stain blue (DAPI). Solid arrows indicate FGF23 immunoreactivity and dashed arrows signify DMP1 immunoreactivity; arrowheads identify osteocytes with co-expression. TB: trabecular bone; BM: bone marrow. The diffuse, low-intensity marrow auto-fluorescence in each section contrasts with the bright, distinct staining of individual osteocytes in trabecular bone.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Immunofluorescence, Expressing, Staining, Fluorescence

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: FGF23 (red); the early osteocyte marker e11/gp38 (green); and their co-expression (yellow) in cancellous bone of a pediatric dialysis patient. Live nuclei stain blue (DAPI). Solid arrows indicate FGF23 immunoreactivity and dashed arrows signify e11/gp38 immunoreactivity; arrowheads identify osteocytes with co-expression. TB: trabecular bone; BM: bone marrow. The diffuse, low-intensity marrow auto-fluorescence in each section contrasts with the bright, distinct staining of individual osteocytes in trabecular bone.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Marker, Expressing, Staining, Fluorescence

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: FGF23 (red); MEPE (green); and their co-expression (yellow) in cancellous bone of a pediatric dialysis patient. Live nuclei stain blue (DAPI). Solid arrows indicate FGF23 immunoreactivity and dashed arrows signify MEPE immunoreactivity; arrowheads identify osteocytes with co-expression. TB: trabecular bone; BM: bone marrow. The diffuse, low-intensity marrow auto-fluorescence in each section contrasts with the bright, distinct staining of individual osteocytes in trabecular bone.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Staining, Fluorescence

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: (A) The total number of osteocytes in bone, as determined by the number of osteocytes staining for DAPI normalized by bone area, in trabecular bone of 5 pediatric healthy controls, 12 pediatric pre-dialysis CKD patients, and 10 pediatric dialysis patients not receiving active vitamin D sterol therapy. (B) The relationship between numbers of FGF23-expressing osteocytes expressed as a percentage of total, DAPI-positive, osteocytes (X axis) (FGF23-expressing osteocytes/osteocyte number) and the number of FGF23-expressing osteocytes per trabecular bone area (Y axis) (FGF23-expressing osteocytes/B.Ar). Closed (black) circles show represent healthy controls; open circles represent pre-dialysis CKD patients; closed grey circles represent dialysis patients.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Staining, Expressing

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: Biochemical values and trabecular bone histomorphometric indices of turnover, mineralization, and volume in a cross-section of 56 dialysis patients, not receiving active vitamin D sterol therapy, whose bone biopsy samples were evaluated for FGF23 staining.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Staining

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: (A) Osteoid volume (displayed as mean ± standard error) in 28 pediatric dialysis patients with normal numbers of early (FGF23-expressing) osteocytes (“Nl bone FGF23”) as compared to values in 28 pediatric dialysis patients with numbers of FGF23-expressing osteocytes above the upper range of normal (“High bone FGF23”). (B) Number of FGF23-expressing osteocytes (FGF/B.Ar) in 42 pediatric dialysis patients with normal mineralization parameters on bone histomorphometry (Nl min) as compared to numbers of FGF23-expressing osteocytes in 14 pediatric dialysis patients with a mineralization defect (min defect). The asterisk indicates a significant (p<0.05) difference between groups. Grey shading in each panel represents the normal range.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Expressing

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: (A) Schematic depiction of phases of mineral accumulation in newly formed bone packets. The dotted line marks the transition between primary and secondary mineralization. (B) Trabecular bone section demonstrating immunohistochemical staining for FGF23. The red arrow points to area of bone with a cluster of FGF23 expressing osteocytes; the blue arrow points to an area of peripheral bone with active bone formation. (C) Backscattered electron image of bone core adjacent to the FGF23-stained section shown in (B). Heterogeneously mineralized bone matrix is observed with dark gray areas corresponding to low and bright gray to high mineral contents. Bone packets are circled in white; calcium content of each (weight % Ca) is written adjacent to each circumscribed packet. Red font is used to indicate weight % Ca of the FGF23-containing packet. (D) Image (C) with red pixels depicting areas of bone with mineral content below 16 weight % calcium and with bone packets numbered to correspond to mineral accumulation phases depicted in (A). (E) An enlarged image of bone packet #1 (i.e. an area of active bone formation). (F) Confocal laser scanning microscopic fluorescence image from the area of bone packet #1 demonstrating tetracycline incorporation at the mineralization front.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Immunohistochemical staining, Staining, Expressing, Fluorescence

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: Quantification of extracted Alizarin Red S dye from healthy control (closed bars) and pediatric dialysis patient (open bars) primary osteoblasts grown to confluence and cultured under standard pro-mineralizing conditions in the presence or absence of exogenous human FGF23 for 2 weeks. Values represent the mean ± standard error values for the 3 controls and 6 patients. The asterisk indicates a between-group difference in mineral accumulation.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture

Journal: Kidney international

Article Title: Impaired Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

doi: 10.1016/j.kint.2018.08.011

Figure Lengend Snippet: A) Ethidium bromide (Et.Br)/acridine orange (Ac.O) staining of serum deprived MLO-Y4 cells after 48 hours in 100 nM FGF23. B) The percentage of dead cells, as defined by the percentage of cells staining with Et.Br (orange) relative to the total number of live cells staining with Ac.O (green), after 48 hours in culture under control (3mM phosphate) and high phosphate (10mM phosphate) conditions with varying (0 and 100 ng/mL) FGF23 concentrations. Values represent the average ± standard error for n=8 replicates. The asterisk indicates a difference (p<0.01) from cells treated with 10 mM Pi.

Article Snippet: Primary osteoblasts were grown to confluence in the presence of DMEM, 10% fetal bovine serum, and 100 μg/ml ascorbic acid and then stimulated to mineralize by adding 10 mM β-glycero-phosphate and 10 −8 M dexamethasone and varying (0 mM, 2 mM, 5mM, and 10 mM) concentrations of β-glycero-phosphate or recombinant human FGF23 (0 and 100 ng/mL) (R&D Systems, Minneapolis, MN).

Techniques: Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Intact Fibroblast Growth Factor 23 Regulates Chronic Kidney Disease–Induced Myocardial Fibrosis by Activating the Sonic Hedgehog Signaling Pathway

doi: 10.1161/JAHA.122.026365

Figure Lengend Snippet: Blood samples were collected 16 weeks after surgery. A , Blood urea nitrogen (BUN) levels (n=6 rats/group, * P <0.05 and ** P <0.01). B , Serum creatinine (SCR) levels (n=6 rats/group, * P <0.05 and ** P <0.01). C and D , Serum transforming growth factor (TGF)‐β1 and fibroblast growth factor (FGF) 2 levels (n=6 rats/group, not significant [ns]). E , FGF23 levels in the chronic kidney disease (CKD)+vehicle group and CKD+GANT‐61 group were higher than those in the sham operation group (n=6 rats/group, * P <0.05 and ** P <0.01). F , Urinary albumin/creatinine ratio (n=6 rats/group, * P <0.05 and ** P <0.01).

Article Snippet: The serum iFGF23 concentration in the SD rats was determined using an FGF23 ELISA kit (Cloud‐Clone Corp, Wuhan, China) for the detection of full‐length FGF23.

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Intact Fibroblast Growth Factor 23 Regulates Chronic Kidney Disease–Induced Myocardial Fibrosis by Activating the Sonic Hedgehog Signaling Pathway

doi: 10.1161/JAHA.122.026365

Figure Lengend Snippet: A through F, The mRNA levels of α‐smooth muscle actin (α‐SMA), collagen‐1, collagen‐3, Shh, Patch 1 (Ptch‐1), and glioma 1 (Gli‐1) in the FGF23 group were significantly increased compared with those in the control group. No significant differences were observed between the negative control (NC)+FGF23 group and the FGF23 group. The mRNA levels of α‐SMA, collagen‐1, collagen‐3, Shh, Ptch‐1, and Gli‐1 in the plasmid carrying the Shh gene (Shhp)+FGF23 group were significantly higher than those in the NC+FGF23 group. The mRNA levels of α‐SMA, collagen‐1, collagen‐3, Shh, Ptch‐1, and Gli‐1 in the plasmid carrying an RNA interference sequence targeting the Shh gene (Shh‐RNAi)+FGF23 group were significantly decreased compared with those in the NC+FGF23 group (n=3 samples/group, ** P <0.01 and *** P <0.001). Cells were primary myocardial fibroblasts.

Article Snippet: The serum iFGF23 concentration in the SD rats was determined using an FGF23 ELISA kit (Cloud‐Clone Corp, Wuhan, China) for the detection of full‐length FGF23.

Techniques: Negative Control, Plasmid Preparation, Sequencing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Intact Fibroblast Growth Factor 23 Regulates Chronic Kidney Disease–Induced Myocardial Fibrosis by Activating the Sonic Hedgehog Signaling Pathway

doi: 10.1161/JAHA.122.026365

Figure Lengend Snippet: A through F , The protein levels of α‐smooth muscle actin (α‐SMA), collagen‐1, collagen‐3, Shh, Smoothened (Smo), and glioma 1 (Gli‐1) in the FGF23 group were significantly increased compared with those in the control group. No significant differences were observed between the negative control (NC)+FGF23 group and the FGF23 group. Levels of the α‐SMA, collagen‐1, collagen‐3, Shh, Smo, and Gli‐1 proteins in the plasmid carrying the Shh gene (Shhp)+FGF23 group were significantly higher than those in the NC+FGF23 group. α‐SMA, collagen‐1, collagen‐3, Shh, Smo, and Gli‐1 protein levels in the plasmid carrying an RNA interference sequence targeting the Shh gene (Shh‐RNAi)+FGF23 group were significantly decreased compared with those in the NC+FGF23 group (n=3 samples/group, * P <0.05, ** P <0.01, and *** P <0.001). Cells were primary myocardial fibroblasts.

Article Snippet: The serum iFGF23 concentration in the SD rats was determined using an FGF23 ELISA kit (Cloud‐Clone Corp, Wuhan, China) for the detection of full‐length FGF23.

Techniques: Negative Control, Plasmid Preparation, Sequencing