FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway

Abstract

Fibroblast growth factor-23 (FGF23) is a bone-derived endocrine regulator of phosphate homeostasis which inhibits renal tubular phosphate reabsorption. Binding of circulating FGF23 to FGF receptors in the cell membrane requires the concurrent presence of the co-receptor αKlotho. It is still controversial whether αKlotho is expressed in the kidney proximal tubule, the principal site of phosphate reabsorption. Hence, it has remained an enigma as to how FGF23 downregulates renal phosphate reabsorption. Here, we show that renal proximal tubular cells do express the co-receptor αKlotho together with cognate FGF receptors, and that FGF23 directly downregulates membrane expression of the sodium-phosphate cotransporter NaPi-2a by serine phosphorylation of the scaffolding protein Na(+)/H(+) exchange regulatory cofactor (NHERF)-1 through ERK1/2 and serum/glucocorticoid-regulated kinase-1 signaling.

Fig. 1

αKlotho is expressed in renal proximal tubules. (A) mRNA expression of αKlotho, SGK1, calbindin D28k (Cal28k), TRPV5, and FGFR1–4 normalized to β2-microglobulin expression in renal proximal tubules (PT) and distal tubules (DT) harvested from 4 mice by laser capture microdissection (LCM). For the purpose of comparison, mRNA expression levels in PT were plotted relative to the expression levels in DT. Data are mean ± SD. *Denotes P < 0.05 vs. DT by t-test. (B) Co-staining of paraffin sections from murine kidneys with anti-αKlotho (red) antibody and DAPI (blue), showing basolateral αKlotho staining pattern in distal and proximal tubules. Proximal tubule from inset in the merged image is shown at higher magnification in the lower right panel. Lower left and middle panels show H&E-stained paraffin sections for comparison of subcellular localization. Lower middle panel (inset in lower left panel) shows H&E-stained proximal tubule at higher magnification. Original magnification × 630. (C) Western blot analysis of Klotho protein expression normalized to β‐actin expression in isolated proximal and distal tubular segments from wild-type mice. PT, proximal tubule; DT, distal tubule.

Fig. 2

FGF23 directly activates ERK1/2–SGK1 signaling in proximal tubule epithelial cells. Western blot analysis of phosphorylated ERK1/2 (pERK1/2) and SGK1 (pSGK1) and total (phosphorylated and non-phosphorylated) ERK1/2 (tERK1/2) and SGK1 (tSGK1) in samples of total protein isolated from cultured primary proximal tubular cells that had been treated for 0.5, 1, 2 and 4 h either with vehicle (Co) or 100 ng/ml recombinant FGF23 (rFGF23) (A), or for 2 h either with vehicle (Co) or with 1, 2.5, 10 or 100 ng/ml of rFGF23 (B). (C) Western blot analysis of pSGK1/tSGK1 ratio in proximal tubular segments treated for 2 h with vehicle (Co) or 100 ng/ml rFGF23 alone or in combination with an ERK1/2 inhibitor (iERK1/2). The quantitative data are from 4 independent experiments each. Data are mean ± SD. *Denotes P < 0.05 vs. control by ANOVA followed by SNK test.

Fig. 3

FGF23 regulates NaPi-2a membrane expression in the proximal tubular segments through SGK1 activation. Western blot analysis of NaPi-2a expression in isolated proximal tubular segments treated with recombinant FGF23 (rFGF23), an SGK1 inhibitor (iSGK1), or a mixture of rFGF23 and iSGK1. PTH was used as a positive control. The quantitative data are from at least 4 independent experiments each. Data are mean ± SD. *Denotes P < 0.05 vs. control for the same time point by ANOVA followed by SNK test.

Fig. 4

FGF23 regulates renal proximal tubular membrane abundance of the NaPi-2a/NHERF-1 complex in vivo and leads to phosphorylation of NHERF-1 in vitro. (A) Reciprocal co-immunoprecipitation (co-IP) of the NaPi-2a/NHERF-1 complex in BBMV preparations from recombinant FGF23- (rFGF23) and vehicle-treated mice (Veh, n = 4 animals each). BBMVs were isolated 8 h post-injection. WB: Western blot. Data are mean ± SD. *Denotes P < 0.05 vs. vehicle control by t-test. (B) Co-staining of kidney paraffin sections with anti-phosphoserine (green) and anti-NHERF-1 antibodies (red) from rFGF23- and vehicle-treated mice, 8 h post-injection. Original magnification × 630. (C) Immunoprecipitation (IP) of serine-phosphorylated (P-Ser) proteins, followed by Western blot analysis of NHERF-1, from isolated proximal tubular segments treated for 2 h with rFGF23, PTH, or iSGK1, or mixtures of either rFGF23 or PTH plus iSGK1. Co, control; Veh, vehicle.

Fig. 5

FGF23-induced regulation of NaPi-2a membrane expression in proximal tubular segments is Klotho dependent at lower concentrations. Western blot analysis of NaPi-2a expression in proximal tubular segments isolated from 3-month-old wild-type, VDR^∆/∆, and Kl^−/−/VDR^∆/∆ mice. Segments were treated for 2 h with 1–100 ng/ml recombinant FGF23 (rFGF23) in vitro. The quantitative data are from at least 4 independent experiments each. Data are mean ± SD. *Denotes P < 0.05 vs. control within the same genotype by ANOVA followed by SNK test.

Fig. 6

Proposed model of FGF23 signaling in proximal tubular cells. FGF23 binds to the FGFR1c-αKlotho complex and activates ERK1/2 kinase leading to SGK1 phosphorylation. SGK1 in turn phosphorylates NHERF-1, leading to internalization and degradation of NaPi-2a. PTH signals through activation of PKA and PKC, which also leads to phosphorylation of NHERF-1. KL, αKlotho; PKA, protein kinase A; PKC, protein kinase C.