Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: Models of the regulation of apical membrane TRPV5 in renal distal tubules by Klotho and FGF23. Model based on previous studies for the regulation of apical membrane TRPV5 by secreted Klotho. TRPV5 is necessary for apical entry of calcium, which is then transported through the cell bound to calbindin D9k and D28k, and extruded at the basolateral side via PMCA1 and NCX. Secreted Klotho is thought to specifically hydrolyze sugar residues from the glycan chains on TRPV5 which in turn stabilizes TRPV5 in the membrane through interaction of the sugar residues with extracellular galectin (Chang et al , ; Cha et al , ). The cellular secretion process of Klotho in this model is unclear. Adapted from Cha et al . Our proposed model of Fgf23-αKlotho signaling in renal distal tubular cells. Fgf23 binds to the basolateral FGFR1c-Klotho complex and activates ERK1/2 leading to SGK1 phosphorylation. SGK1 in turn activates WNK4, stimulating WNK4-TRPV5 complex formation, and increasing intracellular transport of fully glycosylated TRPV5 from the Golgi apparatus to the plasma membrane. PTH signaling activates membrane-anchored TRPV5 by protein kinase A (PKA)-mediated phosphorylation.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Membrane, Glycoproteomics, Phospho-proteomics, Clinical Proteomics

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: Renal calcium reabsorption and TRPV5 plasma membrane abundance in Fgf23 and Klotho deficient mouse models. A–D Urinary excretion of calcium corrected for creatinine (UrCa/Crea) (A), Western blotting quantification of core (75 kDa) and complex (92 kDa) glycosylated TRPV5 protein expression in renal cortical total membrane fractions (B), and Western blot analysis of membrane-bound Klotho in renal total protein extracts (C) in 9-month-old male WT, VDR Δ/Δ , Kl −/− /VDR Δ/Δ , and Fgf23 −/− /VDR Δ/Δ on rescue diet. Antibody specificity and glycosylation of TRPV5 and Klotho was controlled by a glycosylation assay (pink staining) followed by Western blot analysis of TRPV5 and Klotho, respectively (D). Anti-Klotho antibodies detecting the membrane-bound (anti-cytoplasmic domain, upper panel) or the membrane-bound and ectodomain shed (anti KL2 domain, lower panel) forms of the protein were used. Protein extracts from lung and spleen, as well as kidney extracts from Kl −/− /VDR Δ/Δ mice served as negative controls for TRPV5 and Klotho protein expression, respectively. Data information: * P < 0.05 vs. WT, # P < 0.05 vs. VDR Δ/Δ mice. Only the 135 kDa transmembrane isoform of Klotho was quantified in (C). Data in (A–C) represent mean ± s.e.m. of 4–9 animals each. Frames in Western blot images shown in (B) and (D) indicate splicing events. Source data are available online for this figure.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Membrane, Western Blot, Expressing, Glycoproteomics, Staining

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: Membrane-bound αKlotho and TRPV5 do not co-localize in renal distal tubule cells. Immunohistochemical co-staining with anti-transmembrane αKlotho (red or green) antibody, anti-TRPV5 (green), anti-β-actin (red), and DAPI (blue) of paraffin sections from kidneys of 9-month-old WT, VDR Δ/Δ , and Fgf23 −/− /VDR Δ/Δ mice on rescue diet. Right panels show H&E-stained paraffin sections for comparison of subcellular localization. Scale bar, 20 μm. Immuno-electron microscopic staining using anti-Klotho antibodies against transmembrane, and transmembrane and shed forms in kidneys of WT (left panels) and Klotho −/− (right panels) mice. Upper panels show the apical cell area, lower panels show the basolateral area with the basal labyrinth. Arrows mark the apical cell membrane, asterisks mark mitochondria, and the symbol # marks the nucleus. Scale bar, 500 nm.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Membrane, Immunohistochemical staining, Staining, Comparison

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: FGF23 increases urinary calcium reabsorption, TRPV5 plasma membrane abundance and activity in the kidney in gain-of-function mouse models. A, B Urinary calcium excretion (A) and serum PTH (B) in 4-month-old WT mice injected i.p. with vehicle or a single dose of 10 μg rFGF23 per mouse at time 0. C Urinary calcium excretion in 4-month-old WT, VDR Δ/Δ , and Kl −/− /VDR Δ/Δ mice on rescue diet injected i.p. with vehicle or a single dose of 10 μg rFGF23 per mouse, 8 h post-injection. D Immunohistochemical co-staining of kidney paraffin sections with anti-transmembrane αKlotho (red) antibody, anti-TRPV5 (green) and DAPI (blue). Original magnification ×630. E Western blot analysis of membrane-bound Klotho in renal total protein extracts from 4-month-old WT, VDR Δ/Δ , and Kl −/− /VDR Δ/Δ mice treated with vehicle or rFGF23 (10 μg/mouse) 8 h before necropsy. F Complex glycosylated TRPV5 protein expression in isolated distal tubular segments treated for 2 h in vitro with rFGF23 alone or in combination with a specific FGFR inhibitor (iFGFR). G Quantification and original images of intracellular Ca 2+ levels in renal distal tubular cells in 300-μm-thick kidney slices of 3-month-old WT mice treated with vehicle or rFGF23 (10 μg/mouse) 8 h before necropsy. Images are overlays of fluorescent with phase contrast images. Kidney slices were stained with the calcium-sensitive dye Fluo-4. Ruthenium red (10 μM) was used as TRPV inhibitor. H Time-dependent changes in distal tubular fluorescence in Fluo-4-loaded, 300-μm-thick kidney slices of 3-month-old WT mice treated at time 0 with rFGF23 (100 ng/ml) or vehicle. After 120, 135, and 150 min, 1, 10, and 50 μM of the TRPV inhibitor ruthenium red (RR) was added, respectively. Data information: Data in (A–C) represent mean ± s.e.m. of 3–5 animals each. * P < 0.05 vs. vehicle-treated control in (A–C). Data in (E) represent mean ± s.e.m. of 3–5 animals each. In (G), * P < 0.05 vs. vehicle-treated, # P < 0.05 vs. rFGF23-treated WT mice. In (H), * P < 0.05 vs. vehicle, # P < 0.05 vs. fluorescence at 120 min in rFGF23-treated slices. Fluorescence intensity in (G) and (H) was quantified in 7–9 regions of interest per experimental group and time point from three independent experiments. Scale bar, 20 μm in (D) and (G). Source data are available online for this figure.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Membrane, Activity Assay, Injection, Immunohistochemical staining, Staining, Western Blot, Expressing, Isolation, In Vitro, Fluorescence, Control

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: FGF23 increases apical plasma membrane abundance of TRPV5 and causes re-distribution of WNK4. Immunohistochemical co-staining with anti-WNK4 (red) antibody, anti-TRPV5 (green) and DAPI (blue) of kidney paraffin sections from 4-month-old WT, VDR Δ/Δ and Klotho −/− /VDR Δ/Δ mice treated with vehicle or rFGF23 (10 μg/mouse) 8 h before necropsy. Scale bar, 20 μm. Right panel shows quantification of distal tubular TRPV5 and WNK4 co-localization performed in four animals per group with 3–6 images per animal and 4–6 different regions of interest per image. Immuno-electron microscopic staining using anti-TRPV5 (left panels) and anti-WNK4 (right panels) antibodies in kidneys from 3 to 4-month-old WT mice treated with vehicle (Veh) or rFGF23 (10 μg/mouse) 8 h before necropsy. Lower panels show higher magnification of the apical cell area from representative sections. Scale bar, 500 nm. Quantification of the relative grey levels in the apical area of the distal tubular cells between nucleus and apical cell membrane in the anti-TRPV5-stained immuno-electron microscopic pictures performed in four animals per group with 4–5 images per animal and 4–6 different regions of interest per image. Data information: * P < 0.05 vs. vehicle-treated controls in (A) and (C).

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Membrane, Immunohistochemical staining, Staining

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: FGF23 increases TRPV5 protein abundance and channel activity in MDCK cells transfected with murine TRPV5, SGK1 and WNK4. Complex glycosylated TRPV5 protein expression in total protein homogenates of MDCK cells transiently transfected with murine TRPV5 (T), SGK1 (S) and WNK4 (W) constructs, alone or in combination, and treated for 12 h with vehicle, rFGF23 or recombinant Klotho (rKlotho) alone or in combination. Mock-transfected cells were used as a negative control (Co). Quantification and original images of intracellular Ca 2+ levels in MDCK cells transiently transfected with murine TRPV5, SGK1 and WNK4 constructs after treatment with vehicle, rFGF23 or recombinant Klotho (rKlotho) for 12 h. Ruthenium red (10 μM) was used as TRPV inhibitor. Time-dependent changes and original images of intracellular Ca 2+ levels in MDCK cells transiently transfected with murine TRPV5, SGK1 and WNK4 constructs, and treated at time 0 with rFGF23 (100 ng/ml) or vehicle in the presence or absence of 1.5 mM EGTA added to the culture medium. After 105 min, 1 μM of ruthenium red (RR) was added. MDCK cells were stained with the calcium-sensitive dye Fluo-4 in (B) and (C). Data information: Data in (A) represent mean ± s.e.m. of 6–9 samples each from three independent experiments. Data in (B) and (C) represent mean ± s.e.m. of 3–4 samples each from three independent experiments. Scale bar, 20 μm in (B), 7 μm in (C). * P < 0.05 vs. vehicle in (A) and (B), and vs. EGTA + rFGF23 in (C), # P < 0.05 vs. fluorescence at 105 min in (C). Source data are available online for this figure.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Quantitative Proteomics, Activity Assay, Transfection, Expressing, Construct, Recombinant, Negative Control, Staining, Fluorescence