Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice

Abstract

Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [1,25(OH)2D3] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyperphosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on 1,25(OH)2D3, FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.

Figures

Figure 1. Impact of spironolactone treatment on plasma biochemical parameters and on survival of kl/kl mice.

Arithmetic mean ± SEM (n = 4–17) of (A) plasma calcium concentration (mg/dl), (B) plasma inorganic phosphate concentration (mg/dl), (C) plasma FGF23 concentration (pg/ml, (D) plasma calcitriol concentration (pmol/l), (E) body weight (g), (F) systolic blood pressure (mmHg), (G) plasma urea nitrogen concentration (mg/dl), and (H) plasma cystatin C concentration (ng/ml) in WT mice (white bars) and kl/kl mice (kl/kl; black bars), treated with control solution (Ctr) or spironolactone (Spr). ##P < 0.01, compared with kl/kl mice; *P < 0.05, **P < 0.01, ***P < 0.001, compared with WT control-treated mice. (I) Kaplan-Meier blot showing survival of kl/kl mice (black line; n = 13) and kl/kl mice treated with spironolactone (kl/klspr; gray line; n = 12; P < 0.01). Four mice were censored due to end of observational period.

Figure 2. Influence of spironolactone treatment on soft tissue calcification in kl/kl mice.

(A) H&E and von Kossa staining of lung (top panels) and kidney (bottom panels) tissue sections (original magnification, ×400) from WT mice, kl/kl mice, and kl/kl mice treated with spironolactone. (B) Von Kossa staining of thoracic aorta sections (original magnification, ×100) from WT mice, kl/kl mice, and kl/kl mice treated with spironolactone. (C) Arithmetic mean ± SEM (n = 7) of calcified aortic area/total aortic area (%) in thoracic aorta sections from kl/kl mice (black bar) and kl/kl mice treated with spironolactone (gray bar). *P < 0.05, compared with kl/kl mice.

Figure 3. Spironolactone sensitivity of aortic Pit1, Tnfa, and Alpl gene expression.

Arithmetic mean ± SEM (n = 5–9; arbitrary units) of aortic (A) Pit1, (B) Tnfa, and (C) Alpl mRNA levels in WT mice (white bars) and kl/kl mice (black bars), treated with control solution or spironolactone. exp., expression. #P < 0.05 compared with kl/kl mice; *P < 0.05, **P < 0.01 compared with WT control-treated mice.

Figure 4. Effect of spironolactone treatment on aortic osteoinductive signaling.

Arithmetic mean ± SEM (n = 5–9; arbitrary units) of mRNA levels encoding (A) Msx2, (B) Cbfa1, and (C) Osx in aortic tissue of WT mice (white bars) and kl/kl mice (black bars), treated with control solution (left columns) or spironolactone (right columns). ##P < 0.01, compared with kl/kl mice; *P < 0.05, ***P < 0.001, compared with WT control-treated mice. (D) Immunohistochemical analysis and confocal microscopy (original magnification, ×400) of Msx2, Cbfa1, and osterix expression in thoracic aortic tissue of WT mice, kl/kl mice, and kl/kl mice treated with spironolactone. Osteoblastic marker expression is represented by green labeling, nuclei are labeled in blue, and actin staining is labeled in red. Scale bar: 20 μm.

Figure 5. Influence of spironolactone treatment on renal osteoinductive signaling in kl/kl mice.

Arithmetic mean ± SEM (n = 7–8; arbitrary units) of mRNA levels encoding (A) Tnfa, (B) Msx2, (C) Cbfa1, and (D) Osx in renal tissue of WT mice (white bars) and kl/kl mice (black bars), treated with control solution or spironolactone. #P < 0.05, ##P < 0.01, compared with kl/kl mice; **P < 0.01, ***P < 0.001, compared with WT control-treated mice. (E) Immunohistochemical analysis and confocal microscopy (original magnification, ×400) of Cbfa1 and osterix expression in renal tissue of WT mice, kl/kl mice, and kl/kl mice treated with spironolactone. Osteoblastic marker expression is represented by green labeling, nuclei are labeled in blue, and actin staining is labeled in red. Scale bar: 20 μm.

Figure 6. Aldosterone sensitivity of PIT1 gene expression in HAoSMCs.

Arithmetic mean ± SEM (n = 6; arbitrary units) of PIT1 mRNA levels in HAoSMCs after 24-hour treatments with vehicle alone (white bar), with aldosterone (Aldo, 1–100 nM, black bars), or with spironolactone (Spiro, 0–10 μM, gray bars). #P < 0.05, compared with HAoSMCs treated with 100 nM aldosterone alone; *P < 0.05, compared with HAoSMCs treated with vehicle alone.

Figure 7. Influence of aldosterone on TNFA expression and osteoinductive signaling in HAoSMCs.

Arithmetic mean ± SEM (n = 6; arbitrary units) of mRNA levels encoding (A) TNFA, (B) MSX2, (C) CBFA1, and (D) ALPL in HAoSMCs after 24-hour treatments with vehicle alone (Control, white bars), with 100 nM aldosterone alone (Aldo, black bars), with 100 nM aldosterone and 10 μM spironolactone (Aldo+Spiro, dark gray bars), or with 10 μM spironolactone alone (Spiro, light gray bars). (E) Arithmetic mean ± SEM (n = 4; U/mg protein) of ALP activity of whole cell extracts from HAoSMCs after 7-day treatments with vehicle alone (white bar), with 100 nM aldosterone alone (black bar), with 100 nM aldosterone and 10 μM spironolactone (dark gray bar), or with 10 μM spironolactone alone (light gray bar). #P < 0.05, ##P < 0.01, compared with HAoSMCs treated with 100 nM aldosterone alone; *P < 0.05, **P < 0.01, compared with HAoSMCs treated with vehicle alone.

Figure 8. Effects of aldosterone/spironolactone in high phosphate conditions and of FGF23 during aldosterone treatment on PIT1 and CBFA1 expression in HAoSMCs.

Arithmetic mean ± SEM (n = 6–9; arbitrary units) of mRNA levels encoding (A) PIT1 and (B) CBFA1 in HAoSMCs after 24-hour treatments with vehicle alone (Control, white bars), with 2 mM β-glycerophosphate (Pi, dark gray bars), or with cotreatment with 100 nM aldosterone (Pi+Aldo, black bars), 100 nM aldosterone/10 μM spironolactone (Pi+Aldo+Spiro, light gray bars), or 10 μM spironolactone (Pi+Spiro, light gray bars). (C) Representative original bands of KLOTHO (KL) and calibrator/control GAPDH mRNA expression. (D) Arithmetic mean ± SEM (n = 6; arbitrary units) of KL mRNA levels in HAoSMCs after 48-hour silencing with 10 nM of negative control siRNA (Neg. siRNA, white bar) or with 10 nM klotho siRNA (KL siRNA, black bar). Arithmetic mean ± SEM (n = 8–9; arbitrary units) of mRNA levels encoding (E) PIT1 and (F) CBFA1 in HAoSMCs after 48-hour silencing with 10 nM negative control siRNA (white bars) or with 10 nM klotho siRNA (black bars), without or with 100 nM aldosterone and 5 ng/ml FGF23 (Aldo+FGF23) treatment for 24 hours. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control-treated HAoSMCs. †P < 0.05, †††P < 0.001, compared with HAoSMCs treated with 2 mM β-glycerophosphate or aldosterone alone. ##P < 0.01, ###P < 0.001, compared with HAoSMCs treated with 2 mM β-glycerophosphate and 100 nM aldosterone or HAoSMCs silenced with negative control siRNA and treated with 100 nM aldosterone and 5 ng/ml FGF23.

Figure 9. PIT1 dependence of aldosterone-induced TNFA expression and osteoinductive signaling in HAoSMCs.

(A) Representative original bands of PIT1 and calibrator/control GAPDH mRNA expression in HAoSMCs after 48-hour silencing with 10 nM of negative control siRNA or with 10 nM PIT1 siRNA. (B) Arithmetic mean ± SEM (n = 6; arbitrary units) of PIT1 mRNA levels in HAoSMCs after 48-hour silencing with 10 nM of negative control siRNA (white bar) or with 10 nM PIT1 siRNA (black bar). Arithmetic mean ± SEM (n = 6; arbitrary units) of mRNA levels encoding (C) TNFA, (D) MSX2, (E) CBFA1, and (F) ALPL in HAoSMCs after 48-hour silencing with 10 nM of negative control siRNA (white bars) or with 10 nM PIT1 siRNA (black bars), without or with treatment for 24 hours with 100 nM aldosterone. (G) Arithmetic mean ± SEM (n = 4; U/mg protein) of ALP activity of whole cell extracts from HAoSMCs after 7-day silencing with 10 nM of negative control siRNA (white bars) or with 10 nM PIT1 siRNA (black bars), without or with treatment with 100 nM aldosterone. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with HAoSMCs silenced with negative control siRNA and treated with 100 nM aldosterone; *P < 0.05, **P < 0.01, ***P < 0.001, compared with HAoSMCs silenced with negative control siRNA.