FGF23 regulates renal sodium handling and blood pressure

Abstract

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone regulating renal phosphate reabsorption and vitamin D synthesis in renal proximal tubules. Here, we show that FGF23 directly regulates the membrane abundance of the Na(+):Cl(-) co-transporter NCC in distal renal tubules by a signaling mechanism involving the FGF receptor/αKlotho complex, extracellular signal-regulated kinase 1/2 (ERK1/2), serum/glucocorticoid-regulated kinase 1 (SGK1), and with-no lysine kinase-4 (WNK4). Renal sodium (Na(+)) reabsorption and distal tubular membrane expression of NCC are reduced in mouse models of Fgf23 and αKlotho deficiency. Conversely, gain of FGF23 function by injection of wild-type mice with recombinant FGF23 or by elevated circulating levels of endogenous Fgf23 in Hyp mice increases distal tubular Na(+) uptake and membrane abundance of NCC, leading to volume expansion, hypertension, and heart hypertrophy in a αKlotho and dietary Na(+)-dependent fashion. The NCC inhibitor chlorothiazide abrogates FGF23-induced volume expansion and heart hypertrophy. Our findings suggest that FGF23 is a key regulator of renal Na(+) reabsorption and plasma volume, and may explain the association of FGF23 with cardiovascular risk in chronic kidney disease patients.

Keywords: aldosterone; blood pressure; fibroblast growth factor‐23; heart hypertrophy; sodium homeostasis.

© 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Figures

Figure 1. Fgf23 or Klotho deficiency induces renal sodium wasting caused by reduced expression of the Na+:Cl− co-transporter NCC

1. A, B (A) Urinary Na+ excretion corrected by urinary creatinine (Crea) (n = 10–12, one-way ANOVA followed by SNK test, *P = 0.0114 versus WT, #P = 0.0325 versus VDRΔ/Δ for Fgf23−/−/VDRΔ/Δ, *P = 0.0218 versus WT, #P = 0.0185 versus VDRΔ/Δ for Kl−/−/VDRΔ/Δ) and (B) urinary aldosterone concentration corrected by urinary creatinine and serum aldosterone concentration measured by ELISA (n = 8–10, one-way ANOVA followed by SNK test, *P < 0.05 versus WT, #P < 0.05 versus VDRΔ/Δ), in 9-month-old male wild-type (WT), VDRΔ/Δ, Fgf23−/−/VDRΔ/Δ, or Kl−/−/VDRΔ/Δ compound mutant mice on the rescue diet.

2. C, D Western blotting analysis of NCC and α-ENaC protein expression in renal cortical total membrane fractions (n = 7–9, one-way ANOVA followed by SNK test, *P < 0.005 versus WT, #P < 0.005 versus VDRΔ/Δ), and immunohistochemical detection of NCC and α-ENaC protein expression in paraffin sections of paraformaldehyde-fixed kidneys (n = 3–5) in 9-month-old male wild-type (WT), VDRΔ/Δ, Fgf23−/−/VDRΔ/Δ, or Kl−/−/VDRΔ/Δ compound mutant mice on the rescue diet. Data represent mean ± s.e.m.

Source data are available for this figure.

Figure 2. Decreased mean arterial pressure and blood volume in Fgf23- or Klotho-deficient mice

1. A, B Mean arterial pressure (A) and blood volume (B) (n = 6–8, one-way ANOVA followed by SNK test, *P < 0.05 versus WT, #P < 0.05 versus VDRΔ/Δ) in 9-month-old male wild-type (WT), VDRΔ/Δ, Fgf23−/−/VDRΔ/Δ, or Kl−/−/VDRΔ/Δ compound mutant mice on the rescue diet. Data represent mean ± s.e.m.

Figure 3. Gain of FGF23 function induces renal Na+ retention through increased renal NCC expression and channel activation

1. A Urine volume (n = 15–17), urinary Na+ excretion per 12 h (n = 15–17, Student's t-test, *P = 0.0085), urinary Na+ excretion corrected by urinary creatinine (Crea) (n = 15–17, Student's t-test, *P = 0.0251), serum Na+ concentration (n = 15–17, Student's t-test, *P = 0.0308), serum and urinary aldosterone concentrations corrected by urinary creatinine (n = 4–5, Student's t-test, * serum P = 0.0040, urine P = 0.0156), and plasma renin activity (RPA) (n = 4–5) after 5 days of treatment of 3-month-old male wild-type mice with vehicle (Veh) or recombinant FGF23 (10 μg per mouse per day).

2. B, C Western blotting quantification (B) of NCC, α-ENaC, β-ENaC, and γ-ENaC protein expression in renal cortical total membrane fractions (n = 4–5, Student's t-test, *NCC P = 0.0014, α-ENaC P = 0.0007, β-ENaC P = 0.0251, γ-ENaC P = 0.0344), and immunohistochemical detection (C) of NCC and α-ENaC protein expression in kidney sections of 3-month-old wild-type mice treated for 5 days with vehicle or rFGF23 (n = 3–4).

3. D Western blotting quantification of NCC phosphorylation at Ser71, Ser91, and Thr58 (pNCC S71, pNCC S91, pNCC T55) in total kidney homogenates of 3-month-old wild-type mice treated for 5 days with vehicle or rFGF23 (n = 4–5, Student's t-test, *pNCC S71 P = 0.0001, pNCC S91 P = 0.0182, pNCC T55 P = 0.0056).

4. E Reciprocal immunoprecipitation (IP) of serine-phosphorylated (P-Ser) proteins, followed by Western blot (WB) analysis of WNK4 or vice versa from homogenized renal cortex protein samples of 3-month-old male wild-type mice treated for 5 days with vehicle or rFGF23 (n = 5–6, Student's t-test, *WNK4-P-Ser P = 0.0057). For co-immunoprecipitation of NCC/WNK4 complexes, WNK4 or NCC were immunoprecipitated with specific antibodies (anti-NCC and anti-WNK4) from homogenized renal cortex protein samples of 3-month-old wild-type mice treated for 5 days with vehicle or rFGF23. Western blot analysis was performed with corresponding anti-NCC or anti-WNK4 antibodies to identify co-precipitated NCC and WNK4 protein, respectively (n = 4–6, Student's t-test, WNK4-P-NCC *P = 0.0116).

5. F Quantification and original images of intracellular Na+ levels in renal distal tubular cells in live 300-μm-thick kidney slices of 3-month-old WT mice treated with vehicle or rFGF23 (10 μg/mouse), 8 h before necropsy (n = 4, one-way ANOVA followed by SNK test, *P = 0.0026 versus vehicle-treated mice, #P = 0.0175 versus rFGF23-treated mice). Kidney slices were stained with the sodium-sensitive dye SBFI. Chlorothiazide (CTZ, 10 μM) was used as NCC inhibitor.

6. G Time-dependent changes in intracellular Na+ levels in renal distal tubules in SBFI-loaded, 300-μm-thick, live kidney slices of 3-month-old WT mice treated in vitro at time 0 with rFGF23 (100 ng/ml) or vehicle (n = 3–6). After 105 min, 10 μM CTZ or vehicle was added. Fluorescence intensity in G and H was quantified in 4–9 regions of interest per image, sample, and time point from 2–3 independent experiments. Student's t-test, *P < 0.05 versus vehicle-treated or versus vehicle- + CTZ vehicle-treated (past 105 min). Data represent mean ± s.e.m.

Source data are available for this figure.

Figure 4. FGF23 administration induces hypertension and heart hypertrophy in a Klotho-dependent fashion

1. Representative aortic blood pressure curves, heart weight/body weight ratios, and H&E-stained paraffin cross-sections of hearts from 3-month-old male wild-type mice treated for 5 days with vehicle or 10 μg rFGF23 per mouse per day (n = 5–6, Student's t-test, *P = 0.0216). Insets show systolic (BPs), diastolic (BPd), and mean arterial pressure (MAP).

2. Urinary Na+ excretion per 12 h and heart weight/body weight ratio in 3-month-old male wild-type, VDRΔ/Δ, and Kl−/−/VDRΔ/Δ compound mutant mice treated for 5 days with vehicle or 10 μg rFGF23 per mouse per day (n = 4–6, Student's t-test, *P < 0.05 versus vehicle). Data represent mean ± s.e.m.

Figure 5. FGF23 regulates NCC expression and phosphorylation in isolated distal tubular segments in a Klotho-dependent manner

1. Western blotting quantification of NCC expression in isolated distal tubular segments from wild-type (WT) and Fgf23−/− mice treated for 2 h in vitro with vehicle or rFGF23 alone or in combination with specific ERK1/2 (iERK1/2) or SGK1 inhibitors (iSGK1) (n = 4–6, one-way ANOVA followed by SNK test, *P < 0.05 versus vehicle, #P < 0.05 versus rFGF23 alone).

2. Western blotting quantification of NCC phosphorylation at Ser71 (pNCC S71) in isolated distal tubular segments from WT and Kl−/− mice treated for 2 h in vitro with vehicle or rFGF23 alone or in combination with specific ERK1/2 or SGK1 inhibitors (n = 4, one-way ANOVA followed by SNK test, *P = 0.0002 versus vehicle, #P < 0.005 versus rFGF23 alone).

3. Western blotting quantification of α-ENaC protein expression in isolated distal tubular segments from WT mice treated for 2 h in vitro with vehicle (Veh) or rFGF23 (n = 3–4). Data represent mean ± s.e.m.

Source data are available for this figure.

Figure 6. Co-treatment of mice with rFGF23 and chlorothiazide abrogates the untoward cardiovascular effects of rFGF23

Urinary Na+ excretion per 12 h, urine volume, blood volume, central venous pressure, mean arterial pressure, and heart/body weight ratio in 3-month-old male wild-type mice treated for 5 days with vehicle (Veh), recombinant FGF23 (10 μg per mouse per day), or chlorothiazide (CTZ, 25 mg/kg) alone or in combination (n = 8–10). One-way ANOVA followed by SNK test, *P < 0.05 versus vehicle, #P < 0.05 versus rFGF23. Data represent mean ± s.e.m.

Figure 7. Dietary Na+ modulates the effects of rFGF23 on blood pressure and renal Na+ handling

1. A Mean arterial blood pressure (MAP) of 3-month-old male wild-type mice treated for 5 days with vehicle (Veh) or 10 μg rFGF23 per mouse per day on high (High Na), normal (Normal Na), and low (Low Na) sodium diets (n = 6–7, Student's t-test, * P < 0.05 versus vehicle). Inset shows results of two-way ANOVA.

2. B, C Urinary Na+ excretion corrected by urinary creatinine (Crea), serum Na+ concentration (n = 6–7, Student's t-test * P < 0.05 versus vehicle) and urinary aldosterone corrected by urinary creatinine (Crea) and serum aldosterone concentrations (n = 6–7, Student's t-test, * urine P < 0.0005, serum P < 0.05 versus vehicle) after 5 days of treatment of 3-month-old male wild-type mice with vehicle or rFGF23 (10 μg per mouse per day) on high, normal and low sodium diets.

3. D, E Western blotting quantification of NCC, α-ENaC, β-ENaC and γ-ENaC protein expression in renal cortical total membrane fractions (n = 4–5, Student's t-test, *P < 0.05 versus vehicle), and ratio of phospho-SGK1 versus total-SGK1 protein expression in kidney total homogenates of 3-month-old wild-type mice on high, normal, and low sodium diets treated for 5 days with vehicle or rFGF23 (n = 4–5, Student's t-test, *P < 0.01 versus vehicle). Data represent mean ± s.e.m.

Source data are available for this figure.

Figure 8. Hyp mice show hypertension and increased NCC expression and phosphorylation

1. A–E Serum intact Fgf23 concentration (A, n = 8–9, Student's t-test, *P = 0.0001 versus vehicle); heart-to-body weight ratio, serum Na+ and urinary Na+ excretion corrected by urinary creatinine (Crea) (B); mean arterial blood pressure (C, n = 8–9, Student's t-test, *P < 0.05 versus vehicle); Western blotting quantification of renal NCC membrane expression and NCC phosphorylation at Ser71, Ser91, and Thr55 (pNCC S71, pNCC S91, pNCC T55) (n = 8–9) (D); and urinary aldosterone corrected by urinary creatinine and serum aldosterone concentrations in 3-month-old male wild-type (WT) and Hyp mice (n = 8–9, Student's t-test, *P < 0.05 versus vehicle) (E). Data represent mean ± s.e.m.

Source data are available for this figure.

Figure 9. Proposed model of FGF23-mediated bone-kidney-heart axis

Increased circulating FGF23 augments distal renal tubular NCC expression and activity which leads to renal Na+ retention, volume expansion, hypertension, and heart hypertrophy. As a counter-regulatory mechanism, hypernatremia and increased blood volume decrease aldosterone secretion from adrenal glands, leading to a downregulation of renal α-ENaC expression. A low sodium diet augments the hypertensive effect of increased FGF23 signaling in this model, because it interferes with the counter-regulatory downregulation of aldosterone. Similarly, in chronic kidney failure FGF23 and aldosterone signaling pathways are concurrently activated, potentially leading to a stimulation of both NCC and α-ENaC-driven Na+ reabsorption mechanisms in renal distal tubules, and subsequent augmentation of the FGF23-induced volume expansion, hypertension, and heart hypertrophy.