Journal: Kidney international

Article Title: Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease

doi: 10.1016/j.kint.2022.03.028

Figure Lengend Snippet: (a) FLAG-tagged sKL or BAPC (used as negative control) was immobilized on anti-FLAG beads, and then incubated with recombinant FGF23 protein or solvent (PBS). Protein A/G beads with immobilized anti-FGF23 antibody and incubated with FGF23 served as a positive control for FGF23 precipitation. FLAG-sKL, but not FLAG-BAPC, binds FGF23. (b) 96-well plates were coated with 10 ng of recombinant FGF1, FGF2, FGF7, FGF19, FGF21, or FGF23, washed and incubated with 40 ng of FLAG-tagged sKL or BAPC. After subsequent washes, and incubation with HRP-coupled anti-FLAG and HRP substrate, absorbance at 450 nm was measured (presented as arbitrary units; A.U.). Of all tested FGF isoforms, only FGF23 bound FLAG-sKL. (c) Fc-tagged FGFR isoforms 1c, 2c, 3c, or 4 were bound to protein A/G beads and treated with either FLAG-tagged sKL or PBS. FLAG-sKL bound to FGFR4, and to a weaker extent to FGFR4. (d) 96-well plates were coated with 40 ng of FGFR1c or FGFR4, or with 20 ng of FGF23, washed and incubated with 80 ng of FLAG-tagged sKL or BAPC, or PBS. Wells were washed again, treated with HRP-coupled anti-FLAG and HRP substrate, and analyzed for absorbance at 450 nm. FLAG-sKL bound FGFR1c and to a lower extent FGFR4 and FGF23. (e, f) 96-well plates were coated with (e) 12.5 ng of FGF2 or FGF7, or (f) 100 ng of FGF5 or FGF8b and incubated with 0.4 USP heparin or PBS (vehicle). Wells were washed, incubated with 25 ng (for FGF2 and FGF7) or 200 ng (for FGF5 and FGF8b) of Fc-tagged FGFR1c, washed again, treated with HRP-coupled anti-Fc, and HRP substrate, followed by the analysis of absorbance at 450 nm. In reactions receiving sKL, Fc-FGFR1c was pre-incubated with 50 ng (for FGF2 and FGF7) or 400 ng (for FGF5 and FGF8b) of FLAG-tagged sKL prior to the addition to wells. Heparin increased the binding of FGF2 and FGF5 to FGFR1c, but this effect did not occur in the presence of sKL. (g) Serum-starved HEK293T cells were treated with FLAG-tagged sKL or PBS for 15 minutes, followed by stimulation with FGF2, FGF5, FGF8b, or FGF23 for 10 minutes. Total protein extracts were analyzed by Western blotting. Treatment with FGF2, FGF5 and FGF8b increased levels of phosphorylated ERK (pERK) in comparison to total levels of ERK (tERK), which did not occur if cells were pre-treated with sKL. In contrast, FGF23 treatment increased pERK levels in the presence of sKL. GAPDH served as loading control. (h) 1 μg of Fc-tagged FGFR isoforms 1c, 2c, 3c, or 4 were bound to protein A/G beads and treated with either 2 μg of FLAG-tagged sKL or BAPC, or PBS, all in combination with 500 ng of FGF23. Anti-FLAG beads incubated with either FLAG-sKL or FLAG-BAPC, and anti-FGF23 antibody immobilized on protein A/G beads and treated with FGF23 served as positive controls for FLAG- and FGF23-precipitations, respectively. Co-treatments with FGF23 and sKL lead to complex formation with FGFR1c, and to a lesser extent with FGFR3c and FGFR4. (i) 96-well plates were coated with 4.5 ng of FGF23, washed and then sequentially incubated with 18 ng of FLAG-tagged sKL or PBS, 50 ng of Fc-tagged FGFR 1c, 2c, 3c, or 4, HRP-coupled anti-Fc, and HRP substrate, followed by absorbance measurement. The complex of FGF23 and sKL bound FGFR1 and FGFR4 the strongest, with weaker binding to FGFR3. (j) 96-well plates were coated with 2-fold dilutions of FGF23, ranging from 80–2.5 ng. Wells were washed, and sequentially incubated with 320–10 ng of FLAG-tagged sKL or PBS, followed by 160–5 ng of Fc-FGFR 1c, 2c, 3c, or 4, HRP-coupled anti-Fc, and HRP substrate. Binding of the sKL/FGFR1c, sKL/FGFR3c and sKL/FGFR4 complexes occurred in relation to the amount of coated FGF23. In the absence of sKL, FGF23 bound FGFR4 with weak affinity, but not FGFR1c, 2c or 3c. All graphical values are expressed as mean ± SEM; for plate-based assays n = 3 replicate wells; $ indicates that the absorbance at 450 nm surpassed plate reader limit.

Article Snippet: We used an anti-FGF23 antibody provided by Amgen (San Francisco, CA) to block FGF23 effects in vitro .

Techniques: Negative Control, Incubation, Recombinant, Solvent, Positive Control, Binding Assay, Western Blot, Comparison, IF-cells, Control

Journal: Kidney international

Article Title: Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease

doi: 10.1016/j.kint.2022.03.028

Figure Lengend Snippet: (a) NRVMs were co-treated with FGF23 (25 ng/mL) or vehicle (PBS), and with FLAG-tagged sKL (100 ng/mL), an anti-FGF23 blocking antibody (1 ug/mL) or vehicle. FGF23 treatments caused an increase in cell area, while sKL and anti-FGF23 blocked this effect. (b, e) Standard curves (linear regression) for the measurements of FLAG-tagged sKL using our plate-based assay, with FGF23 coated on 96-well plates and incubations with HRP-coupled Fc-FGFR1c for detection. sKL was applied in (b) assay buffer or (e) in rat serum as diluent. For sKL in buffer, the assay gives a linear range allowing for the calculation of sKL concentrations in samples with a limit of detection (LOD) of around 300 pg/mL. For sKL in serum, the assay is about 10x less sensitive, with an LOD of around 10 ng/mL. Absorbances measured are depicted in arbitrary units (A.U.) as individual dots, standard curves as straight lines. (d) Two commonly used commercial preparations of sKL (Com.sKL) and KL1 were analyzed in our sKL detection assay in comparison to our own FLAG-tagged sKL. Our sKL protein showed about 7x higher binding activity compared to Com.sKL, and we detected no binding of KL1. (e) Using our FGF23/Fc-FGFR1c-based detection assay, we analyzed binding affinities of different variants and isoforms of klotho, i.e. sKL and full-length klotho (KL), as well as soluble β-klotho (sβKL) and full-length β-klotho (βKL). HEK293T cells were stably transfected with FLAG-klotho constructs or a GFP vector used as negative control, and total cell lysates were analyzed. A strong signal was observed for sKL, and much weaker binding for KL. GFP, sβKL and βKL showed no measurable binding activity. (f) Rats were injected via the tail vein with FLAG-sKL (100 μg/kg) or vehicle (PBS), and serial bleeds were taken at different time points and analyzed by our FGF23/Fc-FGFR1c-based detection assay. At the 15-minute time point, high levels of sKL were detected in sKL-injected rats compared with vehicle-injected controls. sKL exhibited a half-life of less than 15 minutes and starting at 3 hours post injection we could not detect sKL anymore. (g) Rats were injected with FLAG-sKL or vehicle as before, but serial bleeds were taken at earlier time points. After about 10 minutes post injection, sKL levels were reduced by about half. Comparison between groups was performed in form of a one-way ANOVA followed by a post-hoc Tukey test (a), or in form of two-tailed t-tests (f, g). A significance level of p ≤ 0.05 was accepted as statistically significant. (a) n = 3 independent isolations, 50 cells per slide, 150 total cells per condition; *p ≤ 0.05 vs. all other groups; (c, d) n = 3; (f, g) n = 3; *p ≤ 0.05 vs. vehicle treated at same time point, #p ≤ 0.05 vs. same treatment in preceding time point; for plate-based assays n = 3 replicate wells.

Article Snippet: We used an anti-FGF23 antibody provided by Amgen (San Francisco, CA) to block FGF23 effects in vitro .

Techniques: Binding Assay, Blocking Assay, Detection Assay, Comparison, Activity Assay, Stable Transfection, Transfection, Construct, Plasmid Preparation, Negative Control, Injection, Two Tailed Test

Journal: Kidney international

Article Title: Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease

doi: 10.1016/j.kint.2022.03.028

Figure Lengend Snippet: (a) Fc-tagged FGFR isoforms 1c, 2c, 3c, or 4 were bound to protein A/G beads and treated with FGF23 in combination with either FLAG-tagged sKL, heparin or PBS. Anti-FLAG beads incubated with FLAG-sKL and anti-FGF23 antibody immobilized on protein A/G beads and treated with FGF23 served as positive controls for FLAG- and FGF23-precipitations, respectively. Co-treatments with FGF23 and sKL lead to complex formation with FGFR1c, and to a lesser extent with FGFR3c and FGFR4. Co-treatment with heparin increased binding of FGF23 to FGFR4 and to a much lesser degree to the other FGFR isoforms. (b) 96-well plates were coated with 250 ng of FGF23, washed and then sequentially incubated with 0.4 USP of heparin or vehicle (PBS), 500 ng of Fc-tagged FGFR 1c, 2c, 3c, or 4, HRP-coupled anti-Fc, and HRP substrate, followed by absorbance measurement. In the presence of heparin, FGF23 bound FGFR4 the strongest, with weaker binding to FGFR2c, and no measurable binding to FGFR1c and FGFR3c. (c) 96-well plates were coated with 2-fold dilutions of FGF23, ranging from 250 ng – 7.8125 ng. Wells were washed, and sequentially incubated with 0.4 USP of heparin or vehicle (PBS), followed by 500–15.625 ng of Fc-FGFR1c, 2c, 3c or 4, HRP-coupled anti-Fc, and HRP substrate. In the presence of heparin, FGF23 did not bind to FGFR1c or 3c, and binding of the heparin/FGFR4 and heparin/FGFR2c complexes occurred in relation to the amount of coated FGF23. In the absence of heparin, FGF23 bound FGFR4 with weak affinity, but not FGFR1c, 2c or 3c. (d) SPR sensorgrams of FGFR4-heparin interaction. Concentrations of FGFR4 (from top to bottom): 100, 500, 25, 12.5 and 6.3 nM, respectively. The black curves are the fitting curves using model from T200 Evaluation software (version 3.2). The biosensor chip response is indicated on the y-axis as a function of time (x-axis). (e) SPR sensorgrams of FGF23-heparin interaction. Concentrations of FGF23 (from top to bottom): 100, 500, 25, 12.5 and 6.3 nM, respectively. (f) SPR sensorgrams of klotho-heparin interaction. Concentrations of soluble klotho (from top to bottom): 500, 250, 125, 63 and 32 nM respectively. (g) SPR sensorgrams of FGF23-FGFR4 interaction. Concentrations of FGF23 (from top to bottom): 1000, 500, 250, 125 and 63 nM respectively. (h) Bar graphs based on triplicate experiments with standard deviation, showing normalized FGF23 binding to surface FGFR4 with addition of different concentrations of heparin in solution. (i) Bar graphs based on triplicate experiments with standard deviation, showing normalized FGF23 binding to surface FGFR4 with addition of different concentrations of soluble klotho in solution.

Article Snippet: We used an anti-FGF23 antibody provided by Amgen (San Francisco, CA) to block FGF23 effects in vitro .

Techniques: Incubation, Binding Assay, Software, Standard Deviation

Journal: Kidney international

Article Title: Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease

doi: 10.1016/j.kint.2022.03.028

Figure Lengend Snippet: (a) NRVMs were co-treated with FGF23 at indicated concentrations or vehicle (PBS), and with heparin (0.19 USP/mL) or vehicle. FGF23 treatments caused a stepwise elevation in cell area with increasing FGF23 concentrations. Heparin increased this effect at all FGF23 concentrations. (100 cells per slide, n = 3 independent isolations, 300 total cells per condition; *p ≤ 0.05 vs. PBS/PBS, &p ≤ 0.05 vs. PBS/heparin, #p ≤ 0.05 vs. FGF23 + vehicle at same concentrations). (b) ARVMs were treated with FGF23 (10 ng/mL), heparin (0.7 USP/mL) or a combination of both, and peak fluorescence [Ca 2+ ] i transients (F/F 0 ) were measured. FGF23 increased [Ca 2+ ] i , and this change significantly increased in the presence of heparin. (c) Representative line-scan images of ARVMs under 1 Hz field stimulation perfused with vehicle, heparin, FGF23, or FGF23 combined with heparin for the analysis shown in e. (d) ARVMs were treated with FGF23, heparin or a combination of both, and cell shortening after perfusion was measured. The addition of heparin significantly increased the effect of FGF23. (e) Intact hearts were isolated from adult mice, attached to a force transducer, paced, and treated with either vehicle, heparin (0.06 USP/mL), FGF23 (9 ng/mL), or a combination of FGF23 and heparin while contractile output was monitored. Waveform changes were analyzed for peak contraction force. FGF23 increased force which was further elevated in the presence of heparin. (f) FGF23 with heparin also increased the maximum slope of force development of the contractile waveform. Comparison between groups was performed by one-way ANOVA followed by a post-hoc Tukey test (a, b, c, e, f). A significance level of P<0.05 was accepted as statistically significant. (a) n = 9; *p ≤ 0.05 vs. PBS/PBS, &p ≤ 0.05 vs. PBS/Heparin; #p ≤ 0.05 vs. FGF23 + Vehicle same conc.); (b, c) (n = 5, n = 34 (Vehicle), n = 3, n = 12 (Heparin), n = 5, n = 24 (FGF23), n = 4, n = 22 (FGF23 + Heparin); *p ≤ 0.05 vs. Vehicle, &p ≤ 0.05 vs. Heparin); (e, f) n = 6 (Vehicle), n = 6 (Heparin), n = 6 (FGF23), n = 8 (FGF23 + Heparin); *p ≤ 0.05 vs. Vehicle, &p ≤ 0.05 vs. Heparin. All Values are expressed as mean ± SEM; for plate-based assays n = 3 replicate wells.

Article Snippet: We used an anti-FGF23 antibody provided by Amgen (San Francisco, CA) to block FGF23 effects in vitro .

Techniques: Cell Culture, Fluorescence, Isolation, Comparison

Journal: Kidney international

Article Title: Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease

doi: 10.1016/j.kint.2022.03.028

Figure Lengend Snippet: (a–d) Twelve-week old, male BALB/c mice received i.v. injections, 2x daily, for 5 consecutive days, with either vehicle (saline), heparin (125 IU/kg), FGF23 (40 μg/kg), or FGF23 (40 μg/kg) plus heparin at 125, 62.5 or 12.5 IU/kg, after which the mice underwent echocardiographic analysis and were sacrificed. In mice receiving FGF23 and 125 IU/kg of heparin combined, (a) T;d and (b) left ventricular (LV) mass in diastole were significantly elevated when compared to vehicle injected mice. Co-injections of FGF23 with the two lower doses of heparin did not result in significant changes. (c) Mice receiving FGF23 showed a significant increase in the ratio of heart weight to tibia length when co-injected with each heparin dose and compared to vehicle-injected mice. (d) Representative H&E stainings of cardiac cross-sections are shown (scale bar = 2 mm). (e–h) Five-week old, male BALB/c were administered a 0.2% adenine diet or control chow. After 1 week, mice were i.v. injected 3x per week with heparin (125 IU/kg) or saline. (e) The ratio of heart weight to body weight, and (f) area of individual cardiac myocytes was significantly increased in adenine mice injected with heparin. (g) Representative H&E stainings of cardiac cross-sections from all groups are shown (scale bar = 2 mm), and (h) representative images of cardiac WGA stainings for the quantification shown in g (scale bar = 100 μm). Comparison between groups was performed in form of a one-way ANOVA followed by a post-hoc Tukey test (a–c, e, f). A significance level of p ≤ 0.05 was accepted as statistically significant. (a–c) n = 4–5; *p ≤ 0.05 vs. Vehicle, &p ≤ 0.05 vs. Heparin; (e, f) n = 4–5; *p ≤ 0.05 vs. Vehicle, &p ≤ 0.05 vs. Heparin, #p ≤ 0.05 vs. Adenine. All Values are expressed as mean ± SEM.

Article Snippet: We used an anti-FGF23 antibody provided by Amgen (San Francisco, CA) to block FGF23 effects in vitro .

Techniques: Saline, Injection, Control, Comparison

Journal: American Journal of Reproductive Immunology

Article Title: Down‐Regulation of Klotho/FGF23 by Low‐Expressed VEGFR2 Inhibits the GH/IGF‐1/PI3K/AKT Signaling Pathway Inducing Intrauterine Growth Restriction in Rats

doi: 10.1111/aji.70110

Figure Lengend Snippet: Differential expression of Klotho , FGF23 , and VEGFR2 at mRNA and protein levels in placental tissue between IUGR and control groups. mRNA (A) and protein (B) expression levels of Klotho , FGF23 , and VEGFR2 in placental tissue between IUGR and control groups.

Article Snippet: Membranes were blocked with 5% (w/v) non‐fat dry milk for 1 h at room temperature, then incubated with primary antibodies: anti‐ Klotho (1:1000, #DF10309, Affinity Biosciences, Suzhou, Jiangsu, China), anti‐ FGF23 (1:1000, #DF3596, Affinity Biosciences, Suzhou, Jiangsu, China), and anti‐ VEGFR2 (1:1000, #AF6281, Affinity Biosciences, Suzhou, Jiangsu, China) overnight at 4°C.

Techniques: Quantitative Proteomics, Control, Expressing

Journal: American Journal of Reproductive Immunology

Article Title: Down‐Regulation of Klotho/FGF23 by Low‐Expressed VEGFR2 Inhibits the GH/IGF‐1/PI3K/AKT Signaling Pathway Inducing Intrauterine Growth Restriction in Rats

doi: 10.1111/aji.70110

Figure Lengend Snippet: GO and KEGG enrichment analyses of Klotho , FGF23 , and VEGFR2 . (A‐C) GO enrichment analysis of Klotho (A), FGF23 (B), and VEGFR2 (C). (D‐F) KEGG enrichment results of Klotho (D), FGF23 (E), and VEGFR2 (F).

Article Snippet: Membranes were blocked with 5% (w/v) non‐fat dry milk for 1 h at room temperature, then incubated with primary antibodies: anti‐ Klotho (1:1000, #DF10309, Affinity Biosciences, Suzhou, Jiangsu, China), anti‐ FGF23 (1:1000, #DF3596, Affinity Biosciences, Suzhou, Jiangsu, China), and anti‐ VEGFR2 (1:1000, #AF6281, Affinity Biosciences, Suzhou, Jiangsu, China) overnight at 4°C.

Techniques:

Journal: American Journal of Reproductive Immunology

Article Title: Down‐Regulation of Klotho/FGF23 by Low‐Expressed VEGFR2 Inhibits the GH/IGF‐1/PI3K/AKT Signaling Pathway Inducing Intrauterine Growth Restriction in Rats

doi: 10.1111/aji.70110

Figure Lengend Snippet: Analysis of Klotho , FGF23 , and VEGFR2 interactions, correlation between VEGFR2 and Klotho , and KEGG‐enriched PI3K/AKT signaling pathway. (A) Interaction analysis of Klotho , FGF23 , and VEGFR2 . (B) Correlation analysis of VEGFR2 with Klotho . (C) KEGG‐enriched PI3K/AKT signaling pathway.

Article Snippet: Membranes were blocked with 5% (w/v) non‐fat dry milk for 1 h at room temperature, then incubated with primary antibodies: anti‐ Klotho (1:1000, #DF10309, Affinity Biosciences, Suzhou, Jiangsu, China), anti‐ FGF23 (1:1000, #DF3596, Affinity Biosciences, Suzhou, Jiangsu, China), and anti‐ VEGFR2 (1:1000, #AF6281, Affinity Biosciences, Suzhou, Jiangsu, China) overnight at 4°C.

Techniques:

Journal: American Journal of Reproductive Immunology

Article Title: Down‐Regulation of Klotho/FGF23 by Low‐Expressed VEGFR2 Inhibits the GH/IGF‐1/PI3K/AKT Signaling Pathway Inducing Intrauterine Growth Restriction in Rats

doi: 10.1111/aji.70110

Figure Lengend Snippet: VEGFR2 downregulation reduces Klotho / FGF23 activity, leading to suppression of the GH/IGF‐1/PI3K/AKT signaling axis.

Article Snippet: Membranes were blocked with 5% (w/v) non‐fat dry milk for 1 h at room temperature, then incubated with primary antibodies: anti‐ Klotho (1:1000, #DF10309, Affinity Biosciences, Suzhou, Jiangsu, China), anti‐ FGF23 (1:1000, #DF3596, Affinity Biosciences, Suzhou, Jiangsu, China), and anti‐ VEGFR2 (1:1000, #AF6281, Affinity Biosciences, Suzhou, Jiangsu, China) overnight at 4°C.

Techniques: Activity Assay