Abrogation of FGFR signaling blocks β-catenin-induced adrenocortical hyperplasia and aldosterone production

Abstract

Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors critical for organogenesis and tissue maintenance, including in the adrenal gland. Here we delineate the role of FGFR2 in the morphogenesis, maintenance, and function of the adrenal cortex with a focus on the zona glomerulosa (zG). zG-specific Fgfr2 deletion (Fgfr2-cKO) resulted in impaired zG cell identity, proliferation, and transdifferentiation into zona fasciculata (zF) cells during postnatal development. In adult mice, induced deletion of Fgfr2 led to loss of mature zG cell identity, highlighting the importance of FGFR2 for the maintenance of a differentiated zG state. Strikingly, Fgfr2-cKO was sufficient to fully abrogate β-catenin-induced zG hyperplasia and to reduce aldosterone levels. Finally, short-term treatment with pan-FGFR small molecule inhibitors suppressed aldosterone production in both WT and β-catenin gain-of-function mice. These results demonstrate a critical role for FGFR signaling in adrenal morphogenesis, maintenance, and function and suggest that targeting FGFR signaling may benefit patients with aldosterone excess and/or adrenal hyperplasia.

Keywords: Cardiology; Endocrinology; Homeostasis; Hypertension; Mouse models.

Figure 1. Tissue-specific Fgfr2 KO (Fgfr2 cKO) impairs zG cell identity and transdifferentiation.

(A) Lineage-tracing by immunofluorescence for GFP⁺ cells in adrenal glands from 20-week-old female Fgfr2-cKO and control mice (Ctrl, AS^Cre/+ R26R^mTmG/+). White dashed box demarcates region enlarged to the right. (B) Quantification of GFP⁺ cells within the zG and zF from female Ctrl and Fgfr2-cKO (cKO) mice (20–24 weeks). One-way ANOVA with post hoc Tukey’s test, ****P < 0.0001, n = 5, 6 mice, respectively. (C) Representative images of DAB2 immunostaining in adrenal glands from adult female Ctrl and Fgfr2-cKO mice (10–24 weeks). (D) Quantification of DAB2⁺ cells in female Ctrl and Fgfr2-cKO mice (10–24 weeks). Student’s t test, ****P < 0.0001, n = 6, 5, respectively. (E) Representative pictures of Aldosterone Synthase (AS; Cyp11b2) immunostaining in adrenal glands from female Ctrl and Fgfr2-cKO mice (10–24 weeks). (F) Quantification of AS⁺ cells in female Ctrl and Fgfr2-cKO mice (10–24 weeks). Student’s t test, *P < 0.05, n = 7, 5, respectively. (G) Representative images of coimmunostaining of GFP (green) and Ki67 (magenta) in Ctrl and Fgfr2-cKO adrenal glands from female mice (20–24 weeks). White arrowheads point to GFP and Ki67–copositive cells. (H) Quantification of GFP and Ki67–copositive cells as a proportion of total GFP⁺ cells in the zG and zF of female Ctrl and Fgfr2-cKO mice (20–24 weeks). Student’s t test, *P < 0.05. n = 5, 3 mice, respectively. Scale bars: 50 μm. DAPI (blue), nuclei. Dashed white lines correspond to the zG-zF boundary.

Figure 2. Inducible KO of Fgfr2 (Fgfr2-icKO) in adults leads to a loss of zG cell identity.

(A) Schematic of AS^CreER induction with tamoxifen. (B) Representative images of GFP and DAB2 coimmunostaining in adrenal glands from adult AS^CreER/+ R26R^mTmG/+ (Ctrl) and AS^CreER/+ Fgfr2^fl/fl R26R^mTmG/+ (Fgfr2-icKO) mice at 10 weeks of age (4 weeks after tamoxifen). White arrowheads point to GFP and DAB2–copositive cells. (C) Quantification of DAB2 and GFP–copositive cells as a proportion of total GFP⁺ zG cells in male and female Ctrl and Fgfr2-icKO (icKO) mice. Adrenal glands from male mice are represented with black dots; adrenal glands from female mice are represented with magenta dots. Student’s t test, ***P < 0.001, n = 7, 5, respectively. (D) Representative images of GFP and AS coimmunostaining in adrenal glands from adult Ctrl and Fgfr2-icKO mice at 10 weeks of age (4 weeks post tamoxifen). White arrowheads point to GFP and AS–copositive cells. (E) Quantification of GFP and AS–copositive cells as a proportion of total GFP⁺ zG cells in male and female Ctrl and Fgfr2-icKO mice. Adrenal glands from male mice are represented with black dots; adrenal glands from female mice are represented with magenta dots. Student’s t test, ***P < 0.001, n = 6, 6, respectively. Scale bars: 50 μm. DAPI (blue), nuclei. Dashed white lines correspond to the zG-zF boundary.

Figure 3. Fgfr2 deletion abrogates β-catenin–induced zG hyperplasia.

(A) Representative images of GFP (green) and DAB2 (red) immunostaining of Ctrl (AS^Cre/+ R26R^mTmG/+), βCat-GOF, and βCat-GOF Fgfr2-cKO adrenal glands from adult mice (6–10 weeks). (B) Quantification of GFP⁺ cells as a proportion of total cells in the zG (left) and the zF (right) in adult male and female mice (6–10 weeks). One-way ANOVA, ***P < 0.001, ****P < 0.0001, n = 5–7 per group. (C) Quantification of DAB2⁺ cells in the zG of adult male and female mice (6–10 weeks). One-way ANOVA with post hoc Tukey’s test, ****P < 0.0001, n = 5–7 per group. (D) Representative images of GFP (green) and AS (red) immunostaining of Ctrl, βCat-GOF, and βCat-GOF Fgfr2-cKO adrenal glands from adult mice (6–10 weeks). (E) Quantification of AS⁺ cells in the zG of adult male and female mice. One-way ANOVA with post hoc Tukey’s test, **P < 0.01, ****P < 0.0001, n = 5–7 per group. Adrenal glands from male mice are represented with black dots; adrenal glands from female mice are represented with magenta dots (B, C, and E). (F) Mean 24-hour urine aldosterone corrected for creatinine in male Ctrl, βCat-GOF, and βCat-GOF Fgfr2-cKO mice (6–20 weeks). Mice were fed with normal chow for 4 days, followed by low-sodium chow for 6 days. Urine was collected daily for days 1–4 (regular diet, normal sodium) and 8–11 (low sodium). One-way ANOVA with post hoc Tukey’s test, *P < 0.05, **P < 0.01, ****P < 0.0001, n = 5–7 per group. Scale bars: 50 μm. DAPI (blue), nuclei. Dashed white lines correspond to the zG-zF boundary.

Figure 4. Pharmacological inhibition of FGFR lowers aldosterone secretion and zG proliferation.

(A) Urinary aldosterone excretion (corrected for creatinine) of adult male C57BL/6J (WT) mice (12 weeks) treated with 30 mg/kg/day AZD4547 or vehicle per os starting on day 4 (red arrow). Two-way ANOVA followed by Fisher’s least significant difference test, ****P < 0.0001 (for comparison of posttreatment AZD4547 versus vehicle), n = 6, 5 mice, respectively. P values for all comparisons are shown in the table. Pretreatment: days 1–4; posttreatment: days 6–10. (B) Average urine aldosterone excretion (corrected for creatinine) from A starting 48 hours after AZD4547 (days 6–10). Student’s t test, n = 6, 5 mice, respectively. (C) Renin mRNA expression levels (qPCR) in kidneys from male C57BL/6J mice (7–8 weeks) treated with 10 mg/kg/day AZD4547 or vehicle by oral gavage for 7 days. Student’s t test, n = 6, 6 mice, respectively. (D and E) Representative images of Ki67 and β-Catenin immunostaining (D) and quantifications (E) in adrenal glands from male C57BL/6J mice (7–8 weeks) treated with 10 mg/kg/day AZD4547 or vehicle. Yellow arrowheads indicate Ki67⁺ zG cells. Student’s t test, n = 3, 4 mice, respectively. Scale bars: 50 μm. DAPI (blue), nuclei. Dashed white lines correspond to the zG-zF boundary. (F) Urinary aldosterone excretion (corrected for creatinine) of adult female βCat-GOF mice (36–39 weeks) treated with 30 mg/kg/day AZD4547 or vehicle per os starting on day 4 (red arrow). Two-way ANOVA followed by Fisher’s least significant difference test, *P < 0.05 (for comparison of posttreatment AZD4547 versus vehicle), n = 5, 5 mice, respectively. P values for all comparisons are shown in the table. Pretreatment: days 1–4; posttreatment: days 6–10. (G) Average urine aldosterone excretion (corrected for creatinine) from F, starting 48 hours after AZD4547 (days 6–10). Student’s t test, n = 5, 5 mice, respectively. All comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.