Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice (original) (raw)
Spironolactone treatment did not exhibit a profound effect on the phenotype of kl/kl mice. Serum calcium and phosphate concentrations were significantly higher in klotho-deficient (kl/kl) mice than in WT mice, a difference not significantly affected by life-long treatment with the MR receptor antagonist spironolactone (Figure 1, A and B). Serum FGF23 levels were significantly higher in kl/kl mice and insensitive to spironolactone treatment (Figure 1C). Similarly, the serum levels of 1,25(OH)2D3 were significantly higher in kl/kl mice than in WT mice, a difference again not significantly modified by spironolactone treatment (Figure 1D). As illustrated in Figure 1E, body weight of kl/kl mice was significantly lower than body weight of WT mice. Despite a tendency toward increased body weight following spironolactone treatment, no significant difference was observed between spironolactone-treated and untreated kl/kl mice. Blood pressure was slightly reduced in the kl/kl mice but was not significantly affected by the spironolactone treatment (Figure 1F). The elevated plasma urea nitrogen levels in kl/kl mice were reduced by the spironolactone treatment (Figure 1G), but plasma cystatin C levels were elevated by the spironolactone treatment (Figure 1H).
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/kl_spr; gray line; n = 12; P < 0.01). Four mice were censored due to end of observational period.
Even though spironolactone treatment did not significantly modify hypercalcemia, hyperphosphatemia, or excessive 1,25(OH)2D3 and FGF23 plasma concentrations, and even though spironolactone treatment did not appreciably modify the growth deficit of kl/kl mice, spironolactone treatment was followed by a slight but statistically significant advantage in survival of kl/kl mice (Figure 1I).
Spironolactone reduced tissue calcification in kl/kl mice. In search of the underlying mechanisms contributing to or accounting for the effect of spironolactone on the life span of kl/kl mice, animals were subjected to histological evaluation. As illustrated in Figure 2A, extensive soft tissue calcifications were observed in lungs and kidneys of kl/kl mice but not of WT mice. The calcifications in kl/kl mice were substantially decreased following spironolactone treatment. As von Kossa–positive staining was most abundant in vascular tissue of the organ sections, additional histology was used to analyze calcification in aortic tissue. As a result, calcification in aorta was significantly less pronounced in spironolactone-treated kl/kl mice than in control-treated kl/kl mice. No calcification was found in WT mice (Figure 2, B and C).
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.
Spironolactone treatment reduced aortic osteoinductive signaling in kl/kl mice. As spironolactone treatment reduced calcification in kl/kl mice without affecting serum phosphate levels, the effect of spironolactone may have been the result of an inhibitory effect on aldosterone-dependent active procalcification reprogramming of vascular tissue. As described earlier, kl/kl mice express high levels of Pit1, which is considered essential in procalcification reprogramming (6). As shown for aortic tissue (Figure 3), Pit1 transcript levels were significantly higher in kl/kl mice than in WT mice. The increased Pit1 transcript levels were significantly decreased following spironolactone treatment of kl/kl mice (Figure 3A). Similarly, the transcript levels of Tnfa, an important initiator of osteoblastic differentiation, were significantly higher in aortic tissue of kl/kl mice than in WT mice, a difference again significantly blunted by spironolactone treatment (Figure 3B). Moreover, the mRNA levels of alkaline phosphatase (Alpl) were significantly higher in kl/kl mice than in WT mice, a difference again blunted following treatment with spironolactone (Figure 3C). Further experiments addressed Tnf-α–dependent signaling. In aortic tissue (Figure 4A), Msx2 mRNA levels were increased in kl/kl mice as compared with those in WT mice, a difference significantly blunted by spironolactone treatment. As shown in Figure 4, B and C, the mRNA expression of both the chondrogenic/osteogenic Cbfa1 and the osteogenic osterix (Osx) transcription factors was upregulated in kl/kl mice as compared with WT mice, a difference again blunted by spironolactone treatment. A similar regulation of the osteogenic proteins was found in immunostaining with subsequent confocal microscopy of aortic tissue, showing increased expression of Msx2, Cbfa1, and osterix in kl/kl mice, which was reduced by spironolactone treatment. In addition, the reduced expression of endothelial nitric oxide synthase in aortas of kl/kl mice was mitigated by spironolactone treatment (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI64093DS1). This finding was accompanied by a reduction of Pai1 expression in the kl/kl mice following treatment with spironolactone (Supplemental Figure 1B).
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.
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.
Spironolactone reduced osteoinductive signaling in calcified soft tissues of kl/kl mice. In view of the strong calcification in kidney and lung tissue of kl/kl mice (Figure 2A), osteoinductive signaling was further analyzed in those tissues. Similar to what has been observed in aortic tissue, renal Tnfa transcript levels were elevated in kl/kl mice as compared with those in WT mice, a difference again significantly blunted by spironolactone treatment (Figure 5A). Similarly, renal Msx2, Cbfa1, and Osx gene transcript levels and Cbfa1 and osterix protein abundance were increased in kl/kl mice as compared with WT mice, a difference again partially prevented by spironolactone treatment (Figure 5, B–E). As shown in Supplemental Figure 2, NF-κB p65, as Tnf-α effector, and Wnt signaling, as effector of Msx2 procalcification signaling, were both upregulated in kl/kl mice and reduced by spironolactone treatment.
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.
Similar observations were made in lung tissue. As shown in Supplemental Figure 3A. Pit1 expression was increased in lung tissue from kl/kl mice. Again, Pit1 expression was significantly reduced by spironolactone treatment. Furthermore, mRNA expression of Tnfa and Alpl (Supplemental Figure 3, B and C) as well as Msx2, Cbfa1, and Osx (Supplemental Figure 4, A–C) was significantly higher in kl/kl mice as compared with WT mice, a difference again significantly blunted by spironolactone treatment. These findings were also reflected by an increased protein abundance of Msx2, Cbfa1, and osterix in lung tissue of kl/kl mice, which was reduced by spironolactone treatment (Supplemental Figure 4D).
Aldosterone enhanced osteoinductive signaling in HAoSMCs via PIT1. In order to further define causal relationships in osteoinductive signaling, primary HAoSMCs were treated for 24 hours with aldosterone and with the MR antagonist spironolactone. To avoid potential effects of endogenous ligands in the medium, the experiments were performed using charcoal-stripped FBS media. In a concentration-dependent manner, aldosterone induced the transcription of type III sodium-dependent phosphate transporter PIT1 (Figure 6). At the concentration of 100 nM, aldosterone significantly increased PIT1 transcript levels. At concentrations as low as 0.1 nM, aldosterone increased PIT1 mRNA expression (statistically significant in t test as compared with control, P < 0.05). Aldosterone-induced PIT1 mRNA expression was significantly suppressed by cotreatment with 10 μM spironolactone. As illustrated in Figure 7, aldosterone (100 nM) significantly increased the mRNA expression of TNFA, MSX2, CBFA1, and ALPL in HAoSMCs, an effect abrogated by cotreatment with spironolactone (10 μM). Moreover, aldosterone treatment significantly enhanced ALP activity in HAoSMCs, an effect abrogated by spironolactone cotreatment (Figure 7E).
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.
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.
Further experiments addressed the role of aldosterone and the protective effects of spironolactone on HAoSMCs at high phosphate concentrations (Figure 8, A and B). Treatment of HAoSMCs with 2 mM β-glycerophosphate increased PIT1 and CBFA1 mRNA expression. Addition of aldosterone to the high phosphate medium significantly increased the expression of PIT1 and CBFA1 as compared with high phosphate medium alone. Interestingly, spironolactone exerted a profound effect on PIT1 and CBFA1 expression induced by high phosphate medium beyond counteracting the effects of added aldosterone.
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.
Additional experiments addressed the role of FGF23 in aldosterone-induced osteoblastic differentiation. To this end, HAoSMCs were treated with 100 nM aldosterone and/or 5 ng/ml human FGF23 with or without silencing of klotho (Figure 8, C–F). Treatment of HAoSMCs with FGF23 blunted the effects of aldosterone treatment on PIT1 and CBFA1 mRNA expression. The protective effects of FGF23 were abrogated by silencing of klotho in HAoSMCs.
Additional experiments addressed the role of PIT1 in aldosterone-induced osteoblastic differentiation. To this end, RNA interference was used to suppress endogenous PIT1 mRNA levels in HAoSMCs. To confirm the effects of silencing on HAoSMCs, PIT1 expression levels were examined by quantitative RT-PCR. As shown in Figure 9, PIT1 mRNA levels were significantly reduced in HAoSMCs silenced with PIT1 siRNA but not in HAoSMCs transfected with the negative control siRNA. Treatment with 100 nM aldosterone significantly upregulated the transcript levels of TNFA, MSX2, CBFA1, and ALPL in HAoSMCs pretreated with negative control siRNA. In contrast, TNFA, MSX2, CBFA1, and ALPL mRNA levels were not increased in response to aldosterone in the HAoSMCs silenced with PIT1 siRNA (Figure 9, C–F). These results suggest that PIT1 contributed to or even accounted for induction of osteogenic gene expression in HAoSMCs by aldosterone. Moreover, ALP activity was increased by aldosterone, an effect again abrogated in HAoSMCs silenced with PIT1 siRNA (Figure 9G).
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.