Journal: Journal of the American Society of Nephrology

Article Title: Targeted Disruption of a Proximal Tubule–Specific TMEM174 Gene in Mice Causes Hyperphosphatemia and Vascular Calcification

doi: 10.1681/asn.2021121578

Figure Lengend Snippet: Figure 1. A novel kidney-specific protein regulates Pi homeostasis. siRNA library screening identified that the TMEM174 gene regulates Pi uptake in cells expressing NPT2A. (A) siRNA knockdown efficiency, (B) total Pi uptake, and (C) levels of NPT2A protein. Human renal tubular cells expressing NPT2A were treated with 20 nM siRNA and then with FGF23 (10 nM) in the presence of 100 ng/ml secretory KL and 2 mg/ml heparin for 1 hour. (D) TMEM174 and NPT2A protein expression in human proximal tubular cells treated with 20 nM TMEM174 siRNA and then treated with FGF23 (10 nM) for 1–4 hours in the presence of 100 ng/ml secretory KL, 2 mg/ml heparin, and 0.1% BSA. Flarebio and Novus TMEM174 antibodies were used for human proximal tubular cells. The asterisk (*) represents nonspecific protein signal. (E) TMEM174 and NPT2A protein expression in OK-P cells treated with 20 nM TMEM174 siRNA and then with PTH (10 nM) for 1–4 hours in the presence of 0.1% BSA. Biomatik TMEM174 antibody was used for OK-P cells. Statistical analysis was performed using a one-way ANOVA. The experiments were repeated three times. ***P,0.001, **P,0.01 (ver- sus Scr1FGF23), #P,0.05 (versus Scr-FGF23).

Article Snippet: Primary renal proximal tubular cells (PCS400-010) were purchased from ATCC and were grown and maintained at 37 C, in an atmosphere of 5% carbon dioxide, in a growth media (PCS- 400-030) in the absence/presence of 10–100 nM FGF23 (2604-FG; R&D System) and 100 ng/ml of a secretory form of KL, which was synthesized and purified from HEK293- expressing pcDNA3.1-KL-His cells at the Cell Technologies Core Facility at the University of Colorado Denver.

Techniques: Library Screening, Expressing, Knockdown

Journal: Journal of the American Society of Nephrology

Article Title: Targeted Disruption of a Proximal Tubule–Specific TMEM174 Gene in Mice Causes Hyperphosphatemia and Vascular Calcification

doi: 10.1681/asn.2021121578

Figure Lengend Snippet: Figure 3. Disruption of Pi homeostasis by TMEM174 deficiency. Serum (A) Pi, (B) FGF23, (C) PTH, and (D) 1,25(OH)2D in 8-week- old TMEM174 KO male mice fed a chow diet. (E) TMEM174, renal Pi cotransporters, and NHEFR1 in the renal BBM of TMEM174 KO mice. Flarebio TMEM174 antibody was used to detect mouse TMEM174. (F) Immunofluorescence analysis of NPT2A in the kidneys of TMEM174 KO mice.25–27 (G) Total Pi uptake and (H) sodium-independent Pi uptake in the renal BBM of TMEM174 KO mice. Urinary (I) Pi and (J) Ca excretion of TMEM174 KO mice. Urine was collected in metabolic cages for 24 hours. (K) Alizarin red staining, (L) calcified lesions, and (M) aortic Ca content in the aortic sinus of TMEM174 KO mice. Statistical analysis was performed using a two-tailed t test. *P,0.05, **P,0.01, ***P,0.001. WT, wild type.

Article Snippet: Primary renal proximal tubular cells (PCS400-010) were purchased from ATCC and were grown and maintained at 37 C, in an atmosphere of 5% carbon dioxide, in a growth media (PCS- 400-030) in the absence/presence of 10–100 nM FGF23 (2604-FG; R&D System) and 100 ng/ml of a secretory form of KL, which was synthesized and purified from HEK293- expressing pcDNA3.1-KL-His cells at the Cell Technologies Core Facility at the University of Colorado Denver.

Techniques: Disruption, Staining, Two Tailed Test

Journal: Acta physiologica (Oxford, England)

Article Title: Short-term fasting of mice elevates circulating fibroblast growth factor 23 (FGF23).

doi: 10.1111/apha.14049

Figure Lengend Snippet: FIGURE 9 Effects of short-term fasting (16 h) of mice on fibroblast growth factor 23 (FGF23) formation in thymus, spleen, bone marrow, pancreas, and liver. Arithmetic means ± SEM (n = 5) of relative Fgf23 mRNA abundance normalized to hypoxanthine guanine phosphoribosyl transferase (Hprt) expression in thymus (A), spleen (C), bone marrow (D), pancreas (F), and liver (H) from fed mice and mice fasted for 16 h. Representative Western Blots and arithmetic means ± SEM (n = 5) of the ratio of FGF23 (⁓30 to 32 kDa) over β-tubulin (⁓55 kDa) or β-actin (⁓45 kDa) in thymus (B), bone marrow (E), and pancreas (G) from fed mice and mice fasted for 16 h (Fast). For all panels, n denotes the number of specimens included. There were no dropouts in any analysis. *p < 0.05, ‡p < 0.001 indicate significant differences compared to fed mice. ns, non-significant. n.d., not detectable. (A, D, E, G: unpaired t-test; B, C, F: Mann–Whitney U test).

Article Snippet: Proteins from UMR106 cells, NRVM, kidney, heart, thymus, bone marrow, pancreas (each 30 μg), or bone (10 μg) were subjected to standard Western Blot procedure using 10% or 12% SDS- PAGE gels or 4%– 20% precast SDS- PAGE gels (for FGF23 detection in NRVM), respectively, and the following primary antibodies: anti- NFκB- p65 (#8242, Cell Signaling Technology), anti- phospho- NFκB- p65 (#3033, Cell Signaling Technology), anti- FGF23 (MAB26291, R&D Systems), anti- FGF23 (#6320, Immutopics), anti- NaPiIIa (NBP2- 13328, Novus Biologicals, Bio- Techne), anti- Klotho (AF1819, R&D Systems), anti- β- actin ((#3700 (for lysates from UMR106 cells) and #8457 (for mouse tissue lysates), Cell Signaling Technology)), anti- GAPDH (#2118, Cell Signaling Technology), or anti- β- tubulin (#2146, Cell Signaling Technology).

Techniques: Expressing, Western Blot, MANN-WHITNEY

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: Scientific Reports

Article Title: FGF23, a novel muscle biomarker detected in the early stages of ALS

doi: 10.1038/s41598-021-91496-6

Figure Lengend Snippet: FGF23 mRNA levels are increased in ALS muscle tissue. FGF23 mRNA levels were assessed in human muscle samples by qPCR using GAPDH as an internal housekeeping control. Disease samples were expressed as a fold-change (mean ± SEM) compared to normal control tissue (set at 1). *** P = 0.0001. BI, biceps brachii; DL, deltoid; GC, gastrocnemius; Myo, myopathy disease control; neuro, neuropathy disease control, TA, tibialis anterior; VL, vastus lateralis.

Article Snippet: Human plasma FGF-23 was analyzed using U-PLEX Human FGF-23 Assay (K1516EK, MSD), and mouse plasma FGF23 was measure using mouse/rat FGF23 (intact) ELISA kit (60-6800, Quidel) according to the manufacturer’s instruction.

Techniques: Control

Journal: Scientific Reports

Article Title: FGF23, a novel muscle biomarker detected in the early stages of ALS

doi: 10.1038/s41598-021-91496-6

Figure Lengend Snippet: FGF23 protein is increased in ALS muscle tissue. Sections from 6 ALS and 4 normal muscle biopsy samples were immunostained with an anti-FGF23 antibody and counterstained with Hoechst and wheat germ agglutin (WGA). All 6 ALS patient samples but no control samples showed positive staining. Three of the ALS and two of the normal control sections are shown here. ALSp1 (deltoid), ALSp2 (vastus lateralis), ALSp3 (vastus lateralis), Ctrl1 (deltoid), Ctrl 2 (vastus lateralis). Asterisks highlight areas of grouped atrophy and arrowheads highlight several of the loci where FGF23 and WGA immunostaining colocalizes. Scale bars, 100 μm.

Article Snippet: Human plasma FGF-23 was analyzed using U-PLEX Human FGF-23 Assay (K1516EK, MSD), and mouse plasma FGF23 was measure using mouse/rat FGF23 (intact) ELISA kit (60-6800, Quidel) according to the manufacturer’s instruction.

Techniques: Control, Staining, Immunostaining

Journal: Scientific Reports

Article Title: FGF23, a novel muscle biomarker detected in the early stages of ALS

doi: 10.1038/s41598-021-91496-6

Figure Lengend Snippet: FGF23 immunoreactivity is higher in areas of grouped atrophy in human ALS muscle tissue. Using ImageJ Fluorescence Intensity (FI) Analysis function, we compared the FGF23 FI (per μM 2 ) of 37 atrophic fibers (< 25 μM minimal feret’s diameter) sampled from areas of grouped atrophy seen in 5 human ALS muscle samples with 37 non-atrophic fibers (> 25 μM minimal feret’s diameter) from the same sections. Representative photomicrographs of patient ALSp1 are shown with regions of interest highlighted for 6 grouped atrophic fibers (AF) and 6 non-atrophic fibers (NAF). An FGF23 intensity ratio was calculated by dividing the FI in atrophic fibers by the FI in non-atrophic factors for each patient. A ratio was also calculated between a similar number of non-atrophic fibers in the same section to the NAF region of interest as a control. The FI ratio was nearly sixfold higher in areas of grouped atrophy versus non-atrophic fibers. ** P = 0.006. Scale bar, 100 μm.

Article Snippet: Human plasma FGF-23 was analyzed using U-PLEX Human FGF-23 Assay (K1516EK, MSD), and mouse plasma FGF23 was measure using mouse/rat FGF23 (intact) ELISA kit (60-6800, Quidel) according to the manufacturer’s instruction.

Techniques: Fluorescence, Control

Journal: Scientific Reports

Article Title: FGF23, a novel muscle biomarker detected in the early stages of ALS

doi: 10.1038/s41598-021-91496-6

Figure Lengend Snippet: FGF23 is increased in SOD1 G93A muscle. ( A ) The clinical timeline of disease progression in the SOD1 G93A mouse is shown above . Below is a qPCR analysis of gastrocnemius muscle samples from littermate controls (WT) and SOD1 G93A mice at different ages as indicated. Data points are the mean ± SEM of 6–8 mice. * P < 0.05, *** P < 0.0005. ( B ) Photomicrographs of gastrocnemius muscle sections from a WT and SOD1 G93A mouse (60 d) immunostained with an anti-FGF23 antibody and counterstained with Hoechst and WGA. Scale bar, 100 μm. Arrowheads highlight several areas of merged FGF23 and WGA staining. ( C ) ELISA analysis of FGF23 in plasma samples obtained at the ages indicated. Data points are the mean ± SEM of 3 mice per group. ** P < 0.01.

Article Snippet: Human plasma FGF-23 was analyzed using U-PLEX Human FGF-23 Assay (K1516EK, MSD), and mouse plasma FGF23 was measure using mouse/rat FGF23 (intact) ELISA kit (60-6800, Quidel) according to the manufacturer’s instruction.

Techniques: Biomarker Discovery, Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Scientific Reports

Article Title: FGF23, a novel muscle biomarker detected in the early stages of ALS

doi: 10.1038/s41598-021-91496-6

Figure Lengend Snippet: Plasma FGF23 concentration.

Article Snippet: Human plasma FGF-23 was analyzed using U-PLEX Human FGF-23 Assay (K1516EK, MSD), and mouse plasma FGF23 was measure using mouse/rat FGF23 (intact) ELISA kit (60-6800, Quidel) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Concentration Assay, Control

Journal: Scientific Reports

Article Title: FGF23, a novel muscle biomarker detected in the early stages of ALS

doi: 10.1038/s41598-021-91496-6

Figure Lengend Snippet: FGF23 in human plasma samples from ALS patients and healthy controls. ( A ) Baseline levels of log-transformed plasma FGF23 concentration (pg/ml) among controls, and faster and slower progressing ALS patients. Boxes show median (middle line), and 25th and 75th percentiles (lower and upper border, respectively); whiskers extend to a maximum of 1.5 × interquartile range (IQR), or to the most extreme value if it is less than 1.5 × IQR from the 25th or 75th percentile. ( B ) Longitudinal changes in log-transformed plasma FGF23 among controls. ( C ) Longitudinal changes in log-transformed plasma FGF23 among ALS slower progressors (ALSFRS-R decline < 0.8 point/month). ( D ) Longitudinal changes in log-transformed plasma FGF23 among ALS faster progressors (ALSFRS-R decline > 1.2 points/month).

Article Snippet: Human plasma FGF-23 was analyzed using U-PLEX Human FGF-23 Assay (K1516EK, MSD), and mouse plasma FGF23 was measure using mouse/rat FGF23 (intact) ELISA kit (60-6800, Quidel) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Transformation Assay, Concentration Assay

Journal: BMC Medicine

Article Title: The anti-aging factor Klotho protects against acquired long QT syndrome induced by uremia and promoted by fibroblast growth factor 23

doi: 10.1186/s12916-021-02209-9

Figure Lengend Snippet: Demographic data of patients with dialysis-dependent CKD by quartile of circulating FGF23 levels

Article Snippet: Adult 14-week-old male C57BL/6 J mice (Charles River Laboratories International Inc.) were used to study the effect of FGF23 on heart rhythm by a single-dose intraperitoneal injection of recombinant mouse FGF23 (40 μg/kg) (R&D Systems) during ECG recording.

Techniques:

Journal: BMC Medicine

Article Title: The anti-aging factor Klotho protects against acquired long QT syndrome induced by uremia and promoted by fibroblast growth factor 23

doi: 10.1186/s12916-021-02209-9

Figure Lengend Snippet: FGF23 induces prolonged ventricular repolarization in healthy mice. A ECG waveforms in wild-type healthy mice before (black line) and after (red line) FGF23 administration (40 μg/kg). B–F Time course of QRS interval ( B ), QT interval ( C ), QTc interval ( D ), JT interval ( E ), and T peak to T end (TpTe) interval ( F ) measures from ECG recordings after vehicle ( N = 5 mice) or FGF23 ( N = 9 mice) administration. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus baseline

Article Snippet: Adult 14-week-old male C57BL/6 J mice (Charles River Laboratories International Inc.) were used to study the effect of FGF23 on heart rhythm by a single-dose intraperitoneal injection of recombinant mouse FGF23 (40 μg/kg) (R&D Systems) during ECG recording.

Techniques:

Journal: BMC Medicine

Article Title: The anti-aging factor Klotho protects against acquired long QT syndrome induced by uremia and promoted by fibroblast growth factor 23

doi: 10.1186/s12916-021-02209-9

Figure Lengend Snippet: Macroscopic and biochemical parameters in the experimental CKD model and after treatment with recombinant Klotho

Article Snippet: Adult 14-week-old male C57BL/6 J mice (Charles River Laboratories International Inc.) were used to study the effect of FGF23 on heart rhythm by a single-dose intraperitoneal injection of recombinant mouse FGF23 (40 μg/kg) (R&D Systems) during ECG recording.

Techniques: Recombinant

Journal: BMC Medicine

Article Title: The anti-aging factor Klotho protects against acquired long QT syndrome induced by uremia and promoted by fibroblast growth factor 23

doi: 10.1186/s12916-021-02209-9

Figure Lengend Snippet: Klotho prevents Kv4.2 α-subunit downregulation in ventricular myocytes from Nfx and after FGF23 incubation. A mRNA expression of Kv4.2 channel subunit in Sham ( N = 9 mice), Nfx ( N = 8 mice), Sham+rKL ( N = 9 mice), and Nfx+rKL ( N = 7 mice) mice. B mRNA expression of Kv4.2 in neonatal ventricular cardiomyocytes incubated with vehicle ( N = 6 replicate wells), FGF23 ( N = 9 replicate wells), rKL ( N = 9 replicate wells), and FGF23 + rKL ( N = 11 replicate wells). Data are presented as mean ± SEM. * P < 0.05 vs. the corresponding control (Sham or vehicle-neonatal cardiomyocytes) and # P < 0.05 vs. FGF23-neonatal cardiomyocytes

Article Snippet: Adult 14-week-old male C57BL/6 J mice (Charles River Laboratories International Inc.) were used to study the effect of FGF23 on heart rhythm by a single-dose intraperitoneal injection of recombinant mouse FGF23 (40 μg/kg) (R&D Systems) during ECG recording.

Techniques: Incubation, Expressing, Control

Journal: BMC Medicine

Article Title: The anti-aging factor Klotho protects against acquired long QT syndrome induced by uremia and promoted by fibroblast growth factor 23

doi: 10.1186/s12916-021-02209-9

Figure Lengend Snippet: Diagram representing the involvement of FGF23-Klotho axis in acquired long QT syndrome. CKD, chronic kidney disease; FGF23, fibroblast growth factor 23; I tof , fast transient outward potassium current; kl/kl , Klotho hypomorphic mice; Kv4.2, K + channel 4.2 subunit; Nfx, nephrectomy; sKL, soluble Klotho; Tg-kl, Klotho overexpression mice

Article Snippet: Adult 14-week-old male C57BL/6 J mice (Charles River Laboratories International Inc.) were used to study the effect of FGF23 on heart rhythm by a single-dose intraperitoneal injection of recombinant mouse FGF23 (40 μg/kg) (R&D Systems) during ECG recording.

Techniques: Over Expression

Journal: Pharmacognosy Magazine

Article Title: Fucoidan Attenuated Kidney and Bone Damage Caused by CKD-MBD in Mice by Upregulating Klotho

doi: 10.1177/09731296231172549

Figure Lengend Snippet: Figure 1. Fucoidan Treatment Reversed the Changes of Serum Biochemical Indexes Caused by the Modeling. A–D, the Levels of BUN, Creatinine, ALP, and Pi in the Serum of Mice in the Five Groups were Detected by an Automatic Biochemical Analyzer. E–G, the Levels of iPTH, FGF23, and 1,25 (OH)2D3 in the Serum of Mice in the Five Groups Were Determined by ELISA. H, the Relative BMD of Mice in the Five Groups Was Measured by X-Ray Densitometers.

Article Snippet: Enzyme-linked Immunosorbent Assay (ELISA) The contents of intact parathyroid hormone (iPTH), fibroblast growth factor 23 (FGF23), and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, DHVD3) in the serum were measured by the Mouse iPTH ELISA Kit (E-EL-M0709, Elabscience, China), Mouse FGF23 ELISA Kit (E-EL-M2415C, Elabscience, China), and Mouse DHVD3 ELISA Kit (E-EL-0016C, Elabscience, China), respectively.

Techniques: Enzyme-linked Immunosorbent Assay