Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2010 Oct;207(1):67-75.
doi: 10.1677/JOE-10-0181. Epub 2010 Jul 30.

Altered renal FGF23-mediated activity involving MAPK and Wnt: effects of the Hyp mutation

Emily G Farrow  1 , Lelia J Summers, Susan C Schiavi, James A McCormick, David H Ellison, Kenneth E White
Affiliations

Altered renal FGF23-mediated activity involving MAPK and Wnt: effects of the Hyp mutation

Emily G Farrow et al. J Endocrinol. 2010 Oct.

Abstract

Fibroblast growth factor-23 (FGF23), a hormone central to renal phosphate handling, is elevated in multiple hypophosphatemic disorders. Initial FGF23-dependent Erk1/2 activity in the kidney localizes to the distal convoluted tubule (DCT) with the co-receptor α-Klotho (KL), distinct from Npt2a in proximal tubules (PT). The Hyp mouse model of X-linked hypophosphatemic rickets (XLH) is characterized by hypophosphatemia with increased Fgf23, and patients with XLH elevate FGF23 following combination therapy of phosphate and calcitriol. The molecular signaling underlying renal FGF23 activity, and whether these pathways are altered in hypophosphatemic disorders, is unknown. To examine Npt2a in vivo, mice were injected with FGF23. Initial p-Erk1/2 activity in the DCT occurred within 10 min; however, Npt2a protein was latently reduced in the PT at 30-60 min, and was independent of Npt2a mRNA changes. KL-null mice had no DCT p-Erk1/2 staining following FGF23 delivery. Under basal conditions in Hyp mice, c-Fos and Egr1, markers of renal Fgf23 activity, were increased; however, KL mRNA was reduced 60% (P<0 VSports手机版. 05). Despite the prevailing hypophosphatemia and elevated Fgf23, FGF23 injections into Hyp mice activated p-Erk1/2 in the DCT. FGF23 injection also resulted in phospho-β-catenin (p-β-cat) co-localization with KL in wild-type mice, and Hyp mice demonstrated strong p-β-cat staining under basal conditions, indicating potential crosstalk between mitogen-activated protein kinase and Wnt signaling. Collectively, these studies refine the mechanisms for FGF23 bioactivity, and demonstrate novel suppression of Wnt signaling in a KL-dependent DCT-PT axis, which is likely altered in XLH. Finally, the current treatment of phosphate and calcitriol for hypophosphatemic disorders may increase FGF23 activity. .

PubMed Disclaimer

Conflict of interest statement

Declaration of Interest

EF, LS, SD, JM, and DE, have nothing to declare V体育安卓版. SS is employed by Genzyme, Corp. KW receives research funding from Genzyme, and royalties for licensing the FGF23 gene to Kyowa Hakko Kirin, Ltd.

Figures

Figure 1
Figure 1. Control of Npt2a by FGF23
a) Mice were injected with FGF23 or vehicle for 10, 30, or 60 minutes. Under standard staining conditions Npt2a expression was tested. Npt2a was reduced 30–60 minutes following injection with FGF23 (top row). Sections were co-stained with actin (red), to mark the apical membrane, and co-localization of Npt2a in the apical membrane was detected (yellow). By 60 minutes Npt2a was not detected in the apical membrane (middle row). Higher magnification of the proximal tubule shows the progressive removal of Npt2a from the apical membrane (bottom row); b) Sections were co-stained for Npt2a and p ERK1/2. p-ERK1/2 activity was absent from the vehicle injected animals. p-ERK1/2 was confirmed in the 10 min animals and localization was distinct from Npt2a expression in the PT; no p-ERK1/2 activity was detected in the PT at any time point tested. At the 30 and 60 min time points, Npt2a staining was not detected as reduced (as in Figure 1a) due to the effects of signal amplification for Npt2a and pERK1/2. c) The reduction of Npt2a at 60 min was independent of transcription, as no change in Npt2a mRNA was detected (P=0.7). d) WT or KL-null mice were injected with vehicle or 10 μg of FGF23 (WT vehicle, left panel; WT FGF23, middle panel) to test for renal MAPK activity. In the KL-null animals, the LacZ gene is inserted into the Klotho locus, which enabledβ-galactosidase (βgal) immunofluorescence to be used as a surrogate marker for KL (green, right panel). Following FGF23 delivery to KL-null mice, p-ERK1/2 activity was absent from the DCT labeled with β-gal (KL) (right panel). As confirmed, strong p-ERK1/2 activity co-localized with KL in WT animals injected with FGF23 (middle).
Figure 1
Figure 1. Control of Npt2a by FGF23
a) Mice were injected with FGF23 or vehicle for 10, 30, or 60 minutes. Under standard staining conditions Npt2a expression was tested. Npt2a was reduced 30–60 minutes following injection with FGF23 (top row). Sections were co-stained with actin (red), to mark the apical membrane, and co-localization of Npt2a in the apical membrane was detected (yellow). By 60 minutes Npt2a was not detected in the apical membrane (middle row). Higher magnification of the proximal tubule shows the progressive removal of Npt2a from the apical membrane (bottom row); b) Sections were co-stained for Npt2a and p ERK1/2. p-ERK1/2 activity was absent from the vehicle injected animals. p-ERK1/2 was confirmed in the 10 min animals and localization was distinct from Npt2a expression in the PT; no p-ERK1/2 activity was detected in the PT at any time point tested. At the 30 and 60 min time points, Npt2a staining was not detected as reduced (as in Figure 1a) due to the effects of signal amplification for Npt2a and pERK1/2. c) The reduction of Npt2a at 60 min was independent of transcription, as no change in Npt2a mRNA was detected (P=0.7). d) WT or KL-null mice were injected with vehicle or 10 μg of FGF23 (WT vehicle, left panel; WT FGF23, middle panel) to test for renal MAPK activity. In the KL-null animals, the LacZ gene is inserted into the Klotho locus, which enabledβ-galactosidase (βgal) immunofluorescence to be used as a surrogate marker for KL (green, right panel). Following FGF23 delivery to KL-null mice, p-ERK1/2 activity was absent from the DCT labeled with β-gal (KL) (right panel). As confirmed, strong p-ERK1/2 activity co-localized with KL in WT animals injected with FGF23 (middle).
Figure 1
Figure 1. Control of Npt2a by FGF23
a) Mice were injected with FGF23 or vehicle for 10, 30, or 60 minutes. Under standard staining conditions Npt2a expression was tested. Npt2a was reduced 30–60 minutes following injection with FGF23 (top row). Sections were co-stained with actin (red), to mark the apical membrane, and co-localization of Npt2a in the apical membrane was detected (yellow). By 60 minutes Npt2a was not detected in the apical membrane (middle row). Higher magnification of the proximal tubule shows the progressive removal of Npt2a from the apical membrane (bottom row); b) Sections were co-stained for Npt2a and p ERK1/2. p-ERK1/2 activity was absent from the vehicle injected animals. p-ERK1/2 was confirmed in the 10 min animals and localization was distinct from Npt2a expression in the PT; no p-ERK1/2 activity was detected in the PT at any time point tested. At the 30 and 60 min time points, Npt2a staining was not detected as reduced (as in Figure 1a) due to the effects of signal amplification for Npt2a and pERK1/2. c) The reduction of Npt2a at 60 min was independent of transcription, as no change in Npt2a mRNA was detected (P=0.7). d) WT or KL-null mice were injected with vehicle or 10 μg of FGF23 (WT vehicle, left panel; WT FGF23, middle panel) to test for renal MAPK activity. In the KL-null animals, the LacZ gene is inserted into the Klotho locus, which enabledβ-galactosidase (βgal) immunofluorescence to be used as a surrogate marker for KL (green, right panel). Following FGF23 delivery to KL-null mice, p-ERK1/2 activity was absent from the DCT labeled with β-gal (KL) (right panel). As confirmed, strong p-ERK1/2 activity co-localized with KL in WT animals injected with FGF23 (middle).
Figure 1
Figure 1. Control of Npt2a by FGF23
a) Mice were injected with FGF23 or vehicle for 10, 30, or 60 minutes. Under standard staining conditions Npt2a expression was tested. Npt2a was reduced 30–60 minutes following injection with FGF23 (top row). Sections were co-stained with actin (red), to mark the apical membrane, and co-localization of Npt2a in the apical membrane was detected (yellow). By 60 minutes Npt2a was not detected in the apical membrane (middle row). Higher magnification of the proximal tubule shows the progressive removal of Npt2a from the apical membrane (bottom row); b) Sections were co-stained for Npt2a and p ERK1/2. p-ERK1/2 activity was absent from the vehicle injected animals. p-ERK1/2 was confirmed in the 10 min animals and localization was distinct from Npt2a expression in the PT; no p-ERK1/2 activity was detected in the PT at any time point tested. At the 30 and 60 min time points, Npt2a staining was not detected as reduced (as in Figure 1a) due to the effects of signal amplification for Npt2a and pERK1/2. c) The reduction of Npt2a at 60 min was independent of transcription, as no change in Npt2a mRNA was detected (P=0.7). d) WT or KL-null mice were injected with vehicle or 10 μg of FGF23 (WT vehicle, left panel; WT FGF23, middle panel) to test for renal MAPK activity. In the KL-null animals, the LacZ gene is inserted into the Klotho locus, which enabledβ-galactosidase (βgal) immunofluorescence to be used as a surrogate marker for KL (green, right panel). Following FGF23 delivery to KL-null mice, p-ERK1/2 activity was absent from the DCT labeled with β-gal (KL) (right panel). As confirmed, strong p-ERK1/2 activity co-localized with KL in WT animals injected with FGF23 (middle).
Figure 2
Figure 2. In vivo and in vitro MAPK signaling
a) To test for novel markers of renal activity, mice were injected with 10 μg of FGF23 i.p. for 1 hour and the kidneys harvested for RNA preparation and qPCR. Egr1, measured as a positive control, was increased 70-fold over vehicle injected mice. c-Fos, was increased 13-fold (P<0.05). b) HEK293 cells stably expressing mKL were treated with 100 ng/mL of FGF23 for 30 and 60 min. EGR1 mRNA increased 80- and 200-fold at 30 and 60 min, respectively in the FGF23-treated mKL cells compared to vehicle control (P<0.05). c-Fos increased 20- and 30-fold at 30 and 60 min, respectively (P<0.05).
Figure 3
Figure 3. Mapk signaling in Hyp mice
a) Hyp mice had intact serum FGF23 levels approximately 12-fold higher than WT controls; b) Hyp mice had a 200% increase in Egr1 and c-Fos mRNA, and Klotho expression was reduced by 60% compared to wild type controls (P<0.05).
Figure 4
Figure 4. Response of Hyp to FGF23 delivery
Results from vehicle-injected Hyp mice are shown in the upper panels, and FGF23-injected Hyp mice in the lower panels at 10 min post-injection. Phospho-ERK1/2 staining was only observed in the FGF23-injected animals; Klotho (KL) was positive in both vehicle- and FGF23-injected animals. p-ERK1/2 staining (red) localized to the nucleus in the same nephron segment as KL (green) in FGF23-injected mice as shown by p-ERK1/2 and KL co-staining (‘Merge’ column; arrows show positive nuclear p-ERK1/2 co-localized with KL). Nuclei were stained blue using DAPI in the Merge column.
Figure 5
Figure 5. Wnt pathway in renal FGF23 signaling
a) Strong p-β-catenin signaling was detected in the glomeruli of all animals (green), separate from the DCT, which was demarcated using anti-NCC IF (red). b) p-β-catenin signaling was tested by IF in vehicle and FGF23-injected mice at 10 min following injection. p-β-catenin signaling was not detected in the vehicle injected animals. In the FGF23-injected animals, p-β-catenin (red) was detected in the distal tubule, co-localized with Klotho (green). This activity did not co-localize with Npt2a in the renal PT (right column).
Figure 5
Figure 5. Wnt pathway in renal FGF23 signaling
a) Strong p-β-catenin signaling was detected in the glomeruli of all animals (green), separate from the DCT, which was demarcated using anti-NCC IF (red). b) p-β-catenin signaling was tested by IF in vehicle and FGF23-injected mice at 10 min following injection. p-β-catenin signaling was not detected in the vehicle injected animals. In the FGF23-injected animals, p-β-catenin (red) was detected in the distal tubule, co-localized with Klotho (green). This activity did not co-localize with Npt2a in the renal PT (right column).
Figure 6
Figure 6. Renal p-β-catenin expression in Hyp mice
a) p-β-catenin (red) was readily detectable in Hyp kidney in the basal state, localized to the renal DCT with KL (green staining; left panel); b) p-β-catenin (red) was spatially separate from Npt2a (green; middle panel). c) In the Hyp basal state, strong p-β-catenin signal (green) was co-localized with NCC, a marker for the renal DCT (red; right panel).
See this image and copyright information in PMC

References

    1. Azam N, Zhang MY, Wang X, Tenenhouse HS, Portale AA. Disordered regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase gene expression by phosphorus in X-linked hypophosphatemic (hyp) mice. Endocrinology. 2003;144:3463–3468. - "VSports最新版本" PubMed
    1. Bachar-Dahan L, Goltzmann J, Yaniv A, Gazit A. Engrailed-1 negatively regulates beta-catenin transcriptional activity by destabilizing beta-catenin via a glycogen synthase kinase-3beta-independent pathway. Mol Biol Cell. 2006;17:2572–2580. - V体育ios版 - PMC - PubMed
    1. Bacic D, Schulz N, Biber J, Kaissling B, Murer H, Wagner CA. Involvement of the MAPK-kinase pathway in the PTH-mediated regulation of the proximal tubule type IIa Na+/Pi cotransporter in mouse kidney. Pflugers Arch. 2003;446:52–60. - PubMed (V体育安卓版)
    1. Bostanjoglo M, Reeves WB, Reilly RF, Velazquez H, Robertson N, Litwack G, Morsing P, Dorup J, Bachmann S, Ellison DH. 11Beta-hydroxysteroid dehydrogenase, mineralocorticoid receptor, and thiazide-sensitive Na-Cl cotransporter expression by distal tubules. J Am Soc Nephrol. 1998;9:1347–1358. - PubMed
    1. Custer M, Lotscher M, Biber J, Murer H, Kaissling B. Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry. Am J Physiol. 1994;266:F767–774. - PubMed

Publication types

MeSH terms