recombinant human fgf23  (R&D Systems)

Journal: Journal of Cell Communication and Signaling

Article Title: The renal response to FGF23 shifts from phosphaturia toward inflammation in kidney disease

doi: 10.1002/ccs3.70061

Figure Lengend Snippet: Anti‐GBM disease causes tubular damage and partial renal resistance to FGF23. (A) Depicts the experimental workflow in male C57BL/6 mice undergoing induction of anti‐GBM disease using nephrotoxic serum followed by 6 days of intravenous (IV) FGF23 or vehicle treatment. (B) Shows the glomerular filtration rate of different experimental groups at days 0 and 7. (C) Shows urinary albumin/creatinine ratio at day 7. (D) Shows the example renal sections negative or positive for renal tubular casts (arrows) and quantitative tubular cast scores. (E) Shows plasma phosphate and fractional excretion of phosphate of healthy mice and mice with anti‐GBM treated with vehicle or FGF23. Analyses in panel (B): paired t ‐test. Analyses in panels (C–E): two‐way ANOVA. Anti‐GBM, anti‐glomerular basement membrane; d, disease state; t, treatment.

Article Snippet: Recombinant human FGF23 was obtained from R&D Systems, Minneapolis, MN, USA, and distributed via Thermo Fisher (Cat. #100‐52).

Techniques: Filtration, Clinical Proteomics, Membrane

Journal: Journal of Cell Communication and Signaling

Article Title: The renal response to FGF23 shifts from phosphaturia toward inflammation in kidney disease

doi: 10.1002/ccs3.70061

Figure Lengend Snippet: Six‐day course of FGF23 treatment induces renal transcriptional signatures of inflammatory responses and injury. (A) Indicates the number of differentially expressed genes according to experimental comparison in renal bulk RNA‐Seq. (B, C) Depict volcano plots of DEGs above a cutoff of adjusted p < 0.05 and log2‐fold change >1, in the comparison of FGF23 versus vehicle effect in mice with anti‐GBM (B) and the interaction between treatment and disease effect (C). (D–F) Depict significant Reactome gene set enrichment analyses of FGF23 effects in healthy mice (D), mice with anti‐GBM disease (E), and the interaction between treatment and disease effect (F). (G) Depicts a network of ligand–receptor interaction pairs that were significant for FGF23 versus vehicle comparisons in mice with anti‐GBM disease by bulk RNA‐Seq. The ligand–receptor interactions were inferred using R/BulkSignalR. Anti‐GBM, anti‐glomerular basement membrane disease. N = 3 for anti‐GBM groups and n = 4 for healthy groups.

Article Snippet: Recombinant human FGF23 was obtained from R&D Systems, Minneapolis, MN, USA, and distributed via Thermo Fisher (Cat. #100‐52).

Techniques: Comparison, RNA Sequencing, Membrane

Journal: Journal of Cell Communication and Signaling

Article Title: The renal response to FGF23 shifts from phosphaturia toward inflammation in kidney disease

doi: 10.1002/ccs3.70061

Figure Lengend Snippet: Bulk RNA‐Seq deconvolution and immunofluorescence staining reveal an FGF23‐driven increase in overall immune cell and macrophage abundance in the kidneys of mice with anti‐GBM disease. (A, B) Depict the annotation of renal cell clusters in the reanalysis of the single‐cell RNA‐Seq dataset GSE107585 of murine kidney from 7 sex‐mixed healthy C57BL/6 mice; see also Supporting Information Figure . (C) Shows a wedding pie plot of the bulk deconvolution of the renal cellular composition according to FGF23 treatment and anti‐GBM disease state, as indicated by labels. Overall renal immune cells and macrophage‐like cells are displayed by bulk deconvolution (D, G) and by immunofluorescence with automated quantification for CD45 (E, F) and F4/80 (H, I). RNA‐Seq: N = 3 for anti‐GBM groups and n = 4 for healthy groups. Immunofluorescence: n = 4 per group. Statistical analysis: two‐way ANOVA. anti‐GBM, anti‐glomerular basement membrane disease; Baso, basophil; CD, collecting duct; d, disease state; DCT, distal convoluted tubule; DLOH, descending limb of Henle; EC, endothelial cell; Granul, granulocyte; i, interaction; IC, intercalated cells; Ly, lymphocyte; Mono, monocyte; Mph, macrophage; NK, natural killer cell; PC, principal cells; PT, proximal tubule; S, segment; t, treatment.

Article Snippet: Recombinant human FGF23 was obtained from R&D Systems, Minneapolis, MN, USA, and distributed via Thermo Fisher (Cat. #100‐52).

Techniques: RNA Sequencing, Immunofluorescence, Staining, Single Cell, Membrane

Journal: Journal of Cell Communication and Signaling

Article Title: The renal response to FGF23 shifts from phosphaturia toward inflammation in kidney disease

doi: 10.1002/ccs3.70061

Figure Lengend Snippet: Immune protein profiling highlights an increase in circulating soluble tumor necrosis factor receptors induced by FGF23 and anti‐GBM disease in mice. A plasma cytokine protein array shows FGF23 effects in healthy male C57BL/6 mice (A) and in mice treated with nephrotoxic serum to induce anti‐GBM disease (B). The interaction between treatment and disease state (C) and the overall disease effect (D) are shown. (E–F) Depict analyses of soluble TNF receptors 1 and 2 by two‐way ANOVA. d, disease state; t, treatment. N = 4 biologically independent replicates per group. Anti‐GBM, anti‐glomerular basement membrane.

Article Snippet: Recombinant human FGF23 was obtained from R&D Systems, Minneapolis, MN, USA, and distributed via Thermo Fisher (Cat. #100‐52).

Techniques: Clinical Proteomics, Protein Array, Membrane

Journal: Journal of Cell Communication and Signaling

Article Title: The renal response to FGF23 shifts from phosphaturia toward inflammation in kidney disease

doi: 10.1002/ccs3.70061

Figure Lengend Snippet: Renal immune cell recruitment driven by FGF23 excess is exposure time dependent. Renal microarray transcriptome datasets GDS3361 of male Fgf23 transgenic and control mice or sex‐matched Hyp mice of dataset GDS879 (B) underwent bulk deconvolution with reference to single‐cell RNA‐Seq dataset GSE107585 of murine kidney from 7 sex‐mixed healthy C57BL/6 mice and, subsequently, visualization of overall fractions of inferred immune cells and macrophage‐like cells, as indicated. (C) Shows the experimental workflow of experiments with female BALB/c mice undergoing Adriamycin (doxorubicin) nephropathy followed by a single intravenous (IV) injection of FGF23 or vehicle. (D) Shows the urinary albumin/creatinine ratio 7 days after induction of Adriamycin nephropathy. Statistical analysis: two‐way ANOVA. d, disease state. (E–G) Show significant Reactome gene set enrichment analyses of renal FGF23 effects in healthy mice (E), mice with Adriamycin nephropathy (F), and the interaction between treatment and disease effect (G). (H) Depicts the log‐fold change of 8 transcripts with lowest adjusted p ‐value in the interaction analysis of FGF23 effect in diseased versus FGF23 effect in healthy mice in a 2 × 2 factorial design. N = 5 (A), 10 (B), or 4 (C–H) biologically independent replicates per group.

Article Snippet: Recombinant human FGF23 was obtained from R&D Systems, Minneapolis, MN, USA, and distributed via Thermo Fisher (Cat. #100‐52).

Techniques: Microarray, Transgenic Assay, Control, Single Cell, RNA Sequencing, IV Injection

Journal: Journal of Cell Communication and Signaling

Article Title: The renal response to FGF23 shifts from phosphaturia toward inflammation in kidney disease

doi: 10.1002/ccs3.70061

Figure Lengend Snippet: Intrarenal proinflammatory effects of FGF23 applied ex vivo in PCKS. (A) Male DBA/2J mice underwent dietary treatment with 0.2% adenine for 15 weeks, followed by organ collection and preparation of 300 µm PCKS for a 24‐h treatment with FGF23 or vehicle ex vivo. No disease‐free controls were used for this substudy. (B) Depicts the fibrotic changes in a representative 4 µm section of PCKS stained with hematoxylin and eosin (left scale bar, 500 µm; right, 100 µm). (C) Shows the log‐fold change of upregulated transcripts with the lowest adjusted p ‐value in the FGF23 versus vehicle comparison. (D–F) Show gene set enrichment analyses of FGF23 effects in Reactome (D), WikiPathways (E), and Pathway Interaction Database (F) gene sets. N = 4 biologically independent replicates per group. PCKS, precision‐cut kidney slices.

Article Snippet: Recombinant human FGF23 was obtained from R&D Systems, Minneapolis, MN, USA, and distributed via Thermo Fisher (Cat. #100‐52).

Techniques: Ex Vivo, Staining, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: The renal response to FGF23 shifts from phosphaturia toward inflammation in kidney disease

doi: 10.1002/ccs3.70061

Figure Lengend Snippet: FGF23 is associated with renal immune cell content in human patients with IgA nephropathy. As a reference, single‐nucleus RNA‐Seq data from 5 human kidney biopsies ( GSE199711 ) of 2 healthy controls and 3 patients with chronic kidney disease (CKD) were reanalyzed and annotated (A); see also Supporting Information Figure . 35 patients with IgA nephropathy from the Karolinska Kidney Biopsy Cohort showed an inverse univariable association between circulating FGF23 and measured glomerular filtration rate (GFR) (B). The associations between transcriptome‐inferred renal fibroblasts and immune cells (C–D) or macrophages (E–F) and circulating FGF23 are shown, with adjustment for GFR, 25OH‐vitamin D and parathyroid hormone. (D, F) Show disaggregated data stratified by GFR in CKD stages I–II and III–V.

Article Snippet: Recombinant human FGF23 was obtained from R&D Systems, Minneapolis, MN, USA, and distributed via Thermo Fisher (Cat. #100‐52).

Techniques: RNA Sequencing, Filtration

Journal: Biochemical Journal

Article Title: Noncanonical RGS14 structural determinants control hormone-sensitive NPT2A-mediated phosphate transport

doi: 10.1042/BCJ20240122

Figure Lengend Snippet: RGS14 truncation mutants were generated to identify functional domains suppressing hormone-regulated phosphate transport. ( A ) Serial deletions from the N-terminus of RGS14 were generated, as outlined in Experimental procedures. Previously characterized functional domains are depicted in the full-length (WT) RGS14 and the truncation mutants (1- – 4). Regulator of G protein Signaling (RGS) domain, Ras-binding domains 1 and 2 ( R1, R2 ), G protein regulatory (Go-Loco) motif, and -DSAL PDZ ligand are depicted. ( B ) Transfected WT-RGS14 and truncation mutants (constructs 1–4) expressed in HEK293 cells. Molecular weight markers (kDa) are presented for reference. Empty vector-transfected cell lysate (V) served as a negative control. ( C ) Hormone-sensitive phosphate uptake was measured in OK cells transfected with WT-RGS14 or the indicated truncation mutant. Phosphate transport was normalized to baseline phosphate uptake under control conditions (100%). Where indicated, cells were treated with 100 nM FGF23 or PTH before phosphate uptake measurements. n = 6. **** P < 0.001 vs . control.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25-251 (herein referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (2604-FG-025).

Techniques: Generated, Functional Assay, Binding Assay, Transfection, Construct, Molecular Weight, Plasmid Preparation, Negative Control, Mutagenesis, Control

Journal: Biochemical Journal

Article Title: Noncanonical RGS14 structural determinants control hormone-sensitive NPT2A-mediated phosphate transport

doi: 10.1042/BCJ20240122

Figure Lengend Snippet: As indicated, OK cells were seeded on 12-well plates and transfected with RGS14 (WT or mutants) or empty vector (-). Cells were treated with 100 nM FGF23 or PTH for 2 hr, and Pi uptake was measured as detailed in Experimental procedures. Data were normalized to vector control (100%). ** P < 0.01 vs . control, *** P < 0.001 vs . control, **** P < 0.0001 vs control. n = 4.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25-251 (herein referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (2604-FG-025).

Techniques: Transfection, Plasmid Preparation, Control

Journal: Biochemical Journal

Article Title: Noncanonical RGS14 structural determinants control hormone-sensitive NPT2A-mediated phosphate transport

doi: 10.1042/BCJ20240122

Figure Lengend Snippet: ( A ) RGS14 Ser 266 Ala and Ser 269 Ala phosphorylation. HPCT cells expressing the indicated HA-RGS14 linker were treated with 100 nM PTH or FGF23 for 30 min or vehicle control ( C ). Cell lysates were prepared, and the linker peptide was immunoprecipitated with anti-HA agarose (Methods). The phosphorylated linker was assessed with an anti-phosphoserine antibody. Cell lysates exhibited comparable probe expression. Molecular weight markers (kDa) are shown on the right. Illustrative of n = 4. ( B ) Paired analysis of PTH and FGF23 effects on phosphorylation of the wild-typeWT linker peptide or the mutated Ser 266,269 Ala probe. ** P < 0.01. ( C ) Hormone-induced phosphorylation of full-length RGS14. HK-2 cells were transfected with FLAG-RGS14 and HA-NHERF1. After 24 h, cells were serum-starved overnight and then treated with 100 nM FGF23 or PTH for 30 min. FLAG-RGS14 was immunoprecipitated as detailed in Experimentalexperimental Proceduresprocedures. Molecular weight markers are shown to the right. Representative of n = 6. ( D ) Quantification of hormone-stimulated phosphorylation of full-length RGS14. ** P < 0.01. ( E ) PTH and FGF23 failed to promote phosphorylation in HK-2 cells transfected with FLAG-RGS14 but not HA-NHERF1.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25-251 (herein referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (2604-FG-025).

Techniques: Phospho-proteomics, Expressing, Control, Immunoprecipitation, Molecular Weight, Transfection

Journal: Biochemical Journal

Article Title: Noncanonical RGS14 structural determinants control hormone-sensitive NPT2A-mediated phosphate transport

doi: 10.1042/BCJ20240122

Figure Lengend Snippet: Working model of the role of RGS14 in controlling NPT2A-mediated phosphate uptake under resting conditions ( A ) and following stimulation with PTH or FGF23 ( B ). ( A ) NPT2A binds NHERF1 PDZ1 and human RGS14 binds PDZ2, stabilizing the [NHERF1:NPT2A] complex at the membrane surface permitting constitutive phosphate uptake. ( B ) PTH, acting through the PTH receptor (PTHR) or FGF23 through FGFR1, promotes RGS14 phosphorylation and its dissociation from NHERF1 leading to sequestration of NPT2A and cessation of phosphate uptake [ , ].

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25-251 (herein referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (2604-FG-025).

Techniques: Membrane, Phospho-proteomics

Journal: Cells

Article Title: Asparagine614 Determines the Transport and Function of the Murine Anti-Aging Protein Klotho

doi: 10.3390/cells13201743

Figure Lengend Snippet: Interaction of Klotho-EGFP N614Q with the proteasome and ER-chaperones. ( A ) Scheme of the GFP-TRAP immunoprecipitation followed by mass spectroscopy (MS). HEK239T cells expressing GFP or Klotho-EGFP variants were used. Created with BioRender.com. ( B ) Bar graph illustrating KEGG pathway analysis of proteins exhibiting increased interaction with Klotho-EGFP N614Q compared to Klotho-EGFP WT . ( C ) Heatmap plot of proteins that exhibit enhanced interaction with Klotho-EGFP N614Q associated with protein processing in the ER . The color code indicates a log2-fold change. ( D ) Western blot analysis of proteins co-IPed with Klotho-EGFP variants from lysates of HEK293T cells stably expressing Klotho-EGFP WT or N614Q after GFP-TRAP IP. Samples were probed with antibodies against Klotho, HSPA5, and ERGIC53. Where indicated, cells were treated with 2.5 nM FGF23 for 5 min. ( E ) Quantification of the interaction of Klotho-EGFP variants with HSPA5 and ERGIC53; displayed is the relative intensity of HSPA5 or ERGIC53 to KL in %. (n = 4 experiments, mean ± SD, two-sided Student’s t -test. For full-size blots, see .

Article Snippet: Dulbecco’s modified Eagle Medium + GlutaMax (Gibco, Waltham, MS, USA, 61965-026), Hygromycin B (50 mg/mL) (Thermo Fischer Scientific, 10687010, Waltham, MS, USA), penicillin/streptomycin (Merck, P0781, Rahway, NJ, USA), Fetal Bovine Serum (F7524 Sigma-Aldrich, Burlington, MA), ROTI ® Histofix 4% paraformaldehyde (Carl Roth, No. P087.1, Karlsruhe, Germany), Hoechst 33342 (Invitrogen, H1399, Waltham, MS, USA), Human FGF23 protein (R&D Systems, 2604, Minneapolis, MN, USA), LipofectamineTM 2000 Transfection Reagent (Thermo Fischer Scientific, 11668019), Protease Inhibitor Cocktail (Sigma P8340, Burlington, MA, USA), phosphatase inhibitors (PhosSTOP, Roche, #4906837001, Basel, Switzerland), CHAPSO (Carl Roth), I-BlockTM powder (T2015, Applied Biosystems, Thermo Fisher Scientific).

Techniques: Immunoprecipitation, Mass Spectrometry, Expressing, Western Blot, Stable Transfection

Journal: Cells

Article Title: Asparagine614 Determines the Transport and Function of the Murine Anti-Aging Protein Klotho

doi: 10.3390/cells13201743

Figure Lengend Snippet: N-glycosylation is not essential for Klotho-FGFR interaction. HEK293T cells stably expressing Klotho-EGFP variants were serum-starved for 16 h and incubated for 5 min with FGF23, followed by lysis and processing for Western blotting with the indicated antibodies. ( B ) Quantification of n = 4 independent experiments, as shown in ( A ). The ratio of the phospho (p)-ERK to total (t)-ERK in the presence and absence of FGF23 is displayed as mean ± SD, with statistical analysis performed using a two-sided Student’s t -test (ns: non-significant) Vertical black lines indicate splicing; for full-size blots, see .

Article Snippet: Dulbecco’s modified Eagle Medium + GlutaMax (Gibco, Waltham, MS, USA, 61965-026), Hygromycin B (50 mg/mL) (Thermo Fischer Scientific, 10687010, Waltham, MS, USA), penicillin/streptomycin (Merck, P0781, Rahway, NJ, USA), Fetal Bovine Serum (F7524 Sigma-Aldrich, Burlington, MA), ROTI ® Histofix 4% paraformaldehyde (Carl Roth, No. P087.1, Karlsruhe, Germany), Hoechst 33342 (Invitrogen, H1399, Waltham, MS, USA), Human FGF23 protein (R&D Systems, 2604, Minneapolis, MN, USA), LipofectamineTM 2000 Transfection Reagent (Thermo Fischer Scientific, 11668019), Protease Inhibitor Cocktail (Sigma P8340, Burlington, MA, USA), phosphatase inhibitors (PhosSTOP, Roche, #4906837001, Basel, Switzerland), CHAPSO (Carl Roth), I-BlockTM powder (T2015, Applied Biosystems, Thermo Fisher Scientific).

Techniques: Glycoproteomics, Stable Transfection, Expressing, Incubation, Lysis, Western Blot

Journal: Cells

Article Title: Asparagine614 Determines the Transport and Function of the Murine Anti-Aging Protein Klotho

doi: 10.3390/cells13201743

Figure Lengend Snippet: FGF23 does not activate ER-resident Klotho-EGFP-EGFR3 complexes. ( A , C ) Western blot of HEK293T cell lysates stably expressing Klotho-EGFP WT or N614Q after treatment with different FGF23 concentrations for 5 min ( A ) or with 2.5 nM FGF23 for the indicated times ( B ), probed with the indicated antibodies. p, phospho;t, total. ( B ) Quantification of n = 4 independent experiments from ( A ). Displayed is the ratio of phospho(p)-ERK normalized to total (t)-ERK. ( C ) A representative blot from n = 2 independent experiments. ( D ) HEK293T cells stably expressing EGFP, Klotho-EGFP WT, or N614Q-treated with 2.5 nM FGF23 for 5 min were subjected to IP with GFP-TRAP. IP lysates (Inp., input) after IP were separated by SDS-PAGE, blotted, and probed with antibodies against FGFR3 or Klotho. ( E ) Quantification of n = 3 independent experiments from ( D ). Displayed is the ratio of FGFR3 to Klotho-EGFP variant. A two-sided Student’s t -test was used to assess statistical significance * p < 0.05. Error bars indicate SD. Vertical black lines indicate splicing; for full-size blots, see .

Article Snippet: Dulbecco’s modified Eagle Medium + GlutaMax (Gibco, Waltham, MS, USA, 61965-026), Hygromycin B (50 mg/mL) (Thermo Fischer Scientific, 10687010, Waltham, MS, USA), penicillin/streptomycin (Merck, P0781, Rahway, NJ, USA), Fetal Bovine Serum (F7524 Sigma-Aldrich, Burlington, MA), ROTI ® Histofix 4% paraformaldehyde (Carl Roth, No. P087.1, Karlsruhe, Germany), Hoechst 33342 (Invitrogen, H1399, Waltham, MS, USA), Human FGF23 protein (R&D Systems, 2604, Minneapolis, MN, USA), LipofectamineTM 2000 Transfection Reagent (Thermo Fischer Scientific, 11668019), Protease Inhibitor Cocktail (Sigma P8340, Burlington, MA, USA), phosphatase inhibitors (PhosSTOP, Roche, #4906837001, Basel, Switzerland), CHAPSO (Carl Roth), I-BlockTM powder (T2015, Applied Biosystems, Thermo Fisher Scientific).

Techniques: Western Blot, Stable Transfection, Expressing, SDS Page, Variant Assay

Journal: Cells

Article Title: Asparagine614 Determines the Transport and Function of the Murine Anti-Aging Protein Klotho

doi: 10.3390/cells13201743

Figure Lengend Snippet: Low amounts of Klotho-EGFP at the plasma membrane are sufficient for ERK activation. ( A ) HEK293T cells stably expressing Klotho-EGFP at low (l), median (m), and high (h) levels were incubated with 2.5 nM FGF23 for 5 min, lysed, separated on SDS-PAGE, and subjected to Western Blotting with the indicated antibodies. ( B ) Quantification of n = 4 independent experiments from A. The displayed ratio is p-ERK/t-ERK. A Two-sided Student’s t -test was used to assess statistical significance, (ns: non-significant). Error bars indicate SD. For full-size blots, see .

Article Snippet: Dulbecco’s modified Eagle Medium + GlutaMax (Gibco, Waltham, MS, USA, 61965-026), Hygromycin B (50 mg/mL) (Thermo Fischer Scientific, 10687010, Waltham, MS, USA), penicillin/streptomycin (Merck, P0781, Rahway, NJ, USA), Fetal Bovine Serum (F7524 Sigma-Aldrich, Burlington, MA), ROTI ® Histofix 4% paraformaldehyde (Carl Roth, No. P087.1, Karlsruhe, Germany), Hoechst 33342 (Invitrogen, H1399, Waltham, MS, USA), Human FGF23 protein (R&D Systems, 2604, Minneapolis, MN, USA), LipofectamineTM 2000 Transfection Reagent (Thermo Fischer Scientific, 11668019), Protease Inhibitor Cocktail (Sigma P8340, Burlington, MA, USA), phosphatase inhibitors (PhosSTOP, Roche, #4906837001, Basel, Switzerland), CHAPSO (Carl Roth), I-BlockTM powder (T2015, Applied Biosystems, Thermo Fisher Scientific).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Stable Transfection, Expressing, Incubation, SDS Page, Western Blot

Journal: Cells

Article Title: Asparagine614 Determines the Transport and Function of the Murine Anti-Aging Protein Klotho

doi: 10.3390/cells13201743

Figure Lengend Snippet: ( A ) Venn diagram illustrating the overlap and distinctions between differentially up-regulated phosphorylated proteins in Klotho-EGFP WT and N614Q mutant after FGF23 treatment. ( B ) Pathway enrichment analysis depicting the differentially up-regulated phosphorylated proteins between Klotho-EGFP WT and N614Q after FGF23 treatment, highlighting the activation of key signaling pathways. ( C , D ) Heatmap illustrating the differentially expressed phosphosites and the predicted kinases responsible for their phosphorylation (the rows) in cells expressing Klotho-EGFP WT ( C ) or Klotho-EGFP N614Q ( D ). The red arrows on the right side of each heatmap indicate the presence of the ERK downstream target ELK1 and MAPK1 itself. The arrows at the bottom of the heatmap denote the predicted MAPKs responsible for phosphorylating the phosphosites depicted in the heatmap.

Article Snippet: Dulbecco’s modified Eagle Medium + GlutaMax (Gibco, Waltham, MS, USA, 61965-026), Hygromycin B (50 mg/mL) (Thermo Fischer Scientific, 10687010, Waltham, MS, USA), penicillin/streptomycin (Merck, P0781, Rahway, NJ, USA), Fetal Bovine Serum (F7524 Sigma-Aldrich, Burlington, MA), ROTI ® Histofix 4% paraformaldehyde (Carl Roth, No. P087.1, Karlsruhe, Germany), Hoechst 33342 (Invitrogen, H1399, Waltham, MS, USA), Human FGF23 protein (R&D Systems, 2604, Minneapolis, MN, USA), LipofectamineTM 2000 Transfection Reagent (Thermo Fischer Scientific, 11668019), Protease Inhibitor Cocktail (Sigma P8340, Burlington, MA, USA), phosphatase inhibitors (PhosSTOP, Roche, #4906837001, Basel, Switzerland), CHAPSO (Carl Roth), I-BlockTM powder (T2015, Applied Biosystems, Thermo Fisher Scientific).

Techniques: Mutagenesis, Activation Assay, Protein-Protein interactions, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: a. Experimental Overview. Pooled male and female kidneys from FGF23-injected mice were dissociated into single-cell suspensions followed by scRNAseq and downstream computational and molecular analysis. b. Unsupervised UMAP clustering identified various renal cell populations divided into 21 distinct cell types. c. Known sex-specific markers (described in Results) were used to identify male and female cell populations. d. Dot plot of representative genes show sex-specific cell types. e. Stacked bar plot displays the relative proportions of each cell-type after FGF23 treatment. The different cell types identified are color-coded and annotated for clustering as in .

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: Injection

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: a. Ridgeplots show KL mRNA expression in select renal cells. b. snATACseq detected a differential chromatin accessibility between PT, LOH, DCT, and CNT versus Stromal and Endothelial cells at the KL promoter region. The red bracket shows differential chromatin accessibility in a region located between exon 1 and exon 2 when comparing PT cells types versus LOH, DCT, CNT, and CD-IC. c. Cell states in PT and DCT cells in response to FGF23 with a time-dependent clustering in DCT versus PT. d. Ridgeplot analysis shows KL expression in PT(S1-S3) and DCT. The vertical red line demarcates cells with low KL expression (KL low ) or high KL expression (KL high ). e. The Monocle program was used to cluster PT-S1 and PT-S2_S3, to separate cells into KL high and KL low groups, and display cells by treatment condition (0, 1, 4, and 12h). f. Overview of nephron segment specific pathways activated in proximal tubule and distal tubule cells in response to FGF23. Ingenuity Pathway Analysis (IPA) was used to predict the statistically significant canonical pathways upregulated in PT and DT in response to FGF23 at 1, 4, and 12h. The z-score represents the ratio of the number of upregulated genes found in each pathway over the total number of genes known to be involved in that pathway; the size of the dot represents the p value.

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: Expressing

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: a-j. The mRNA expression of FGF23 target genes Kl , Egr1, Cyp24a1, Cyp27b1, and Slc34a1, respectively, was tested in the kidney scRNAseq dataset and by qPCR analysis. Feature plots show the expression of FGF23 targets in the different renal cell populations. The kinetic curves represent the qPCR analysis in total kidney with male data shown in blue and female data represented in red. The qPCR data are expressed as fold change (2-ΔΔCt) relative to the housekeeping gene β-actin. Data are shown as mean +/- standard deviation. k. Dot plot of representative genes that define FGF23 bioactivity in PT(S1-S3), DCT, and CNT.

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: a-Top. SCENIC analysis was performed on PT-S1 cell data derived from FGF23-treated mice at different time points, and the cells dimensionally reduced in a t-distributed stochastic neighbor embedding (t-SNE) plot. a-Bottom. The heatmap shows regulon activities in PT-S1 at 0h, 1h, 4h, and 12h after FGF23 treatment. The red coloring represents the highest regulon activity. b-d. t-SNE plots illustrate regulon activities of Egr1 , Fos , and Bhlhe40 (blue) of PT-S1 cells in parallel with the transcription factor mRNA expression detected in the SCENIC-generated dataset (orange color in t-SNE plot). The violin plots show the corresponding mRNA expression in the original scRNAseq dataset. The insets illustrate the AUCell score distribution for a respective regulon and the predicted binding motifs. e-f. The mRNA expression of EGR1, FOS, and BHLHE40 obtained from in vitro studies were measured by qPCR. The red kinetic curve represents the HEK293-mKL cell studies whereas the black kinetic curve shows the data from native HEK293 cell experiments. h-Top. SCENIC analysis was performed in DCT cells derived from the FGF23-treated mice at different time points. h-Bottom. The heatmap graphic shows regulon activities in DCT at 0h, 1h, 4h, and 12h following FGF23 administration. The red color represents the highest regulon activity. i-k. t-SNE plots show the regulon activity of Atf4 , Ddit3 , and Stat3 in DCT in blue in parallel with the transcription factor mRNA expression (t-SNE plot with orange dots) in the SCENIC dataset. The violin plots display mRNA expression in the scRNAseq dataset. The insets illustrate the AUCell score distribution for the regulon and the predicted binding motifs. l-m. The kinetic curves show the mRNA expression of ATF4, and DDIT3 . The red kinetic curve represents the mKL-HEK293 cell line, and the black kinetic curve highlights the HEK293 parent cells. n. The violin plot shows the mRNA expression of Cdkn1a in the scRNAseq dataset.

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: Derivative Assay, Activity Assay, Expressing, Generated, Binding Assay, In Vitro

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: a. Schematic representing HEK293-mKL cell treatment with FGF23. b-c. Following the treatment, RNAseq and ATACseq were performed, and violin plots were generated to isolate differentially expressed genes and chromatin accessibility. The pie charts under the ATAC violin plots show the repartition of peaks along the genome. d. Chromatin accessibility at the EGR1 genomic region shows increased accessibility with FGF23 treatment. e. An enlargement at the distal EGR1 enhancer region isolated the genomic coordinates within the regions of FGF23-induced chromatin accessibility increases. f. Integration of ATACseq and RNAseq data displays selected genes that showed both increased chromatin accessibility (x-axis) with gene expression alterations (y-axis). g. Functional enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) on upregulated DEGs with FGF23 treatment (numbers in parenthesis are gene counts). h. Heatmap displays the genes associated with NF-κB pathway activation. i. HEK293-mKL cells were pretreated with BMS-34554 (10 μM) for 1h followed by FGF23 (50 ng/ml) for 4h. Immunoblots were then performed to assess the phosphorylation of ERK, cJUN, and cFOS. j. HEK293-mKL cells were pretreated with BMS-34554 (10 μM) for 1h followed by FGF23 (50 ng/ml) for 4h and subsequent EGR1 and FOS mRNA expression analysis by qPCR. k. Experimental design shows the workflow of the treatment performed by treating C57BL/6 or Hyp mice with BMS and/or FGF23. l-m. qPCR analysis of mouse Egr1, Fos, Cyp27b1, and Cyp24a1 following treatments.

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: Generated, Isolation, Gene Expression, Functional Assay, Activation Assay, Western Blot, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: a. Violin plots display the expression of Tnfrsf12a and Tnfrsf1a in PT-S1 and DCT. The kinetic curves show Tnfrsf12a mRNA expression in total kidney samples in male (blue) and female (red) mice treated with FGF23 for 0, 1, 4, 12, and 24h. b. In vitro analysis highlights the expression of TNFRSF12A, EGR1 , and TNFRSF1A in HEK293 (black line) and HEK293-mKL (red line) cells treated with FGF23 for different times. c. Correlation analysis shows the positive interrelationship between EGR1 and TNFRSF12A. d. HEK293-mKL cells were treated with FGF23 (50 ng/ml) for four hours and RNAseq was performed. The heatmap graphic displays differential gene expression of TNFRs genes that change with FGF23 treatment. e. HEK293-mKL cells were treated with FGF23 (50 ng/ml) for 4 hours and ATACseq was performed. Representative ATACseq peaks of cells treated with vehicle (top track) compared to cells treated with FGF23 (lower track) highlights the increase of chromatin accessibility across the TNFRSF12A gene body. f. HEK293-mKL cells were pretreated with the MEK inhibitor U0126 (5, or 10 μM) for 1h prior to FGF23 administration (50 ng/ml) for 10 minutes; p-ERK immunoblot. g. HEK293-mKL cells were pretreated with U0126 (5, or 10 μM) for 1h prior to FGF23 (50 ng/ml) for 4 hours followed by RNA extraction and assessment of EGR1 and TNFRSF12A mRNAs by qPCR. h. HEK293-mKL cells were pretreated with BMS-34554 (10 μM) for 1h followed by FGF23 (50 ng/ml) for 4h then TNFRSF12A and CXCL8 mRNA expression analysis. i. HEK293-mKL cells were treated with 50 ng/ml of FGF23 for 0, 1, 2, 4, 8, and 24h. The mRNA expression of CCL5 , CXCL8, and IL6 obtained from in vitro studies were measured by qPCR. The red kinetic curve represents the HEK293-mKL cell studies whereas the black kinetic curve shows the data from HEK293 cell line experiments. j. HEK293-mKL cells were treated with the ligand of TNFRSF12A (TNFSF12, TWEAK 100 ng/ml) for 1h followed by FGF23 treatment for 4h and pERK immunoblot. k. HEK293-mKL cells were pretreated with TWEAK (100 ng/ml) for 1h followed by FGF23 (50 ng/ml) for 16h. EGR1 expression was then tested by qPCR to assess FGF23 bioactivity. l. In vivo studies were performed by treating C57BL/6 mice with recombinant mouse TWEAK for 4 days at the rate of one injection per day. On the fourth day mice received FGF23 treatment for 4h and Tnfrsf12a was assessed by qPCR. m-p. HEK293-mKL cells were pretreated with TNF (100 ng/ml) for 16 h followed with FGF23 (50 ng/ml) for 4 h. pNF-κB, total NF-κB, and β-actin were evaluated by immunoblot ( m ). The mRNA expression of NFKB ( n ), CXCL8 ( o ), and EGR1 ( p ) were assessed by qPCR.

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: Expressing, In Vitro, Gene Expression, Western Blot, RNA Extraction, In Vivo, Recombinant, Injection

Journal: bioRxiv

Article Title: Dynamic Single Cell Transcriptomics Defines Kidney FGF23/KL Bioactivity and Novel Segment-Specific Inflammatory Targets

doi: 10.1101/2024.05.24.595014

Figure Lengend Snippet: The graphic shows a proposed model of crosstalk between KL-dependent FGF23 bioactivity and inflammation-induced NF-κB signaling.

Article Snippet: In some experiments, cells were pretreated with BMS-345541 (BMS; Selleck Chemicals, Houston, TX), U0126 (MAPK inhibitor), rTWEAK (1090-TW; R&D Systems), rTNFα (210-TA; R&D Systems) prior to FGF23 (2604-FG; R&D Systems) treatment.

Techniques: