Hyperaldosteronism in Klotho-deficient mice

Abstract

Klotho is a membrane protein participating in the inhibitory effect of FGF23 on the formation of 1,25-dihydroxyvitamin-D(3) [1,25(OH)(2)D(3)]. It participates in the regulation of renal tubular phosphate reabsorption and stimulates renal tubular Ca(2+) reabsorption. Klotho hypomorphic mice (klotho(hm)) suffer from severe growth deficit, rapid aging, and early death, events largely reversed by a vitamin D-deficient diet. The present study explored the role of Klotho deficiency in mineral and electrolyte metabolism. To this end, klotho(hm) mice and wild-type mice (klotho(+/+)) were subjected to a normal (D(+)) or vitamin D-deficient (D(-)) diet or to a vitamin D-deficient diet for 4 wk and then to a normal diet (D(-/+)). At the age of 8 wk, body weight was significantly lower in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice, klotho(hm)D(-) mice, and klotho(hm)D(-/+) mice. Plasma concentrations of 1,25(OH)(2)D(3,) adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. Plasma volume was significantly smaller in klotho(hm)D(-/+) mice, and plasma urea, Ca(2+), phosphate and Na(+), but not K(+) concentrations were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. The differences were partially abrogated by a vitamin D-deficient diet. Moreover, the hyperaldosteronism was partially reversed by Ca(2+)-deficient diet. Ussing chamber experiments revealed a marked increase in amiloride-sensitive current across the colonic epithelium, pointing to enhanced epithelial sodium channel (ENaC) activity. A salt-deficient diet tended to decrease and a salt-rich diet significantly increased the life span of klotho(hm)D(+) mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)(2)D(3) in Klotho-deficient mice results in extracellular volume depletion, which significantly contributes to the shortening of life span.

Fig. 1.

Body weight of Klotho wild-type (klotho^+/+) and Klotho hypomorphic mice (klotho^hm) with and without a vitamin D-deficient diet. A: photograph of 8-wk-old klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺) as well as klotho^hm mice maintained on a continued vitamin D-deficient diet (klotho^hmD⁻) or on an initial vitamin D-deficient diet for 4 wk followed by a control diet for a further 4 wk (klotho^hmD^−/+). B: arithmetic means ± SE (n = 7–12) of the body weight of klotho^hmD⁺, klotho^+/+D⁺, klotho^hmD⁻, and klotho^hmD^−/+ mice. *,#Significant difference from klotho^+/+D⁺ mice and from klotho^hmD⁺ mice (ANOVA, P < 0.001).

Fig. 2.

Plasma volume and fluid intake of klotho^+/+ mice and klotho^hm mice. A: arithmetic means ± SE of the plasma volume (n = 9–14) of klotho^hm mice on an initial vitamin D-deficient diet for 4 wk followed by a control diet for a further 4 wk (klotho^hmD^−/+) and klotho^+/+ mice on a vitamin D-containing control diet (klotho^+/+D⁺). *Significant difference from klotho^+/+D⁺ mice (Student's t-test, P < 0.01). B: arithmetic means ± SE of the fluid intake (n = 4–9) of klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺) or on an initial vitamin D-deficient diet for 4 wk followed by a control diet for a further 4 wk (klotho^hmD^−/+). *Significant difference from klotho^+/+D⁺ mice (ANOVA, P < 0.05).

Fig. 3.

Plasma urea concentration of klotho^+/+ mice and klotho^hm mice with and without a vitamin D-deficient diet. Shown are arithmetic means ± SE (n = 3–8) of the plasma urea concentrations of klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺) as well as klotho^hm mice maintained on a continued vitamin D-deficient diet (klotho^hmD⁻) or on initial vitamin D-deficient diet for 4 wk followed by a control diet for a further 4 wk (klotho^hmD^−/+). *Significant difference from klotho^+/+D⁺ mice. #Significant difference from klotho^hmD⁺ mice (ANOVA, P < 0.05).

Fig. 4.

Plasma Na⁺, K⁺, Ca²⁺, and phosphate concentrations of klotho^+/+ mice and klotho^hm mice with and without vitamin D-deficient diet. Shown are arithmetic means ± SE (n = 6 -16) of the plasma Na⁺, K⁺, Ca²⁺, and phosphate concentrations of klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺) as well as klotho^hm mice maintained on a continued vitamin D-deficient diet (klotho^hmD⁻) or on an initial vitamin D-deficient diet for 4 wk followed by a control diet for further 4 wk (klotho^hmD^−/+). *Significant difference from klotho^+/+D⁺ mice (ANOVA, P < 0.05).

Fig. 5.

Plasma 1,25(OH)₂D₃, parathyroid hormone (PTH), antidiuretic hormone (ADH), and aldosterone concentrations of klotho^+/+ and klotho^hm mice with and without a vitamin D-deficient diet. Shown are arithmetic means ± SE of the plasma 1,25(OH)₂D₃ (n = 12–20), PTH (n = 5–14), ADH (n = 12–17), and aldosterone concentrations (n = 10–18) of klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺) as well as klotho^hm mice maintained on a continued vitamin D-deficient diet (klotho^hmD⁻) or on initial vitamin D-deficient diet for 4 wk followed by a control diet for a further 4 wk (klotho^hmD^−/+). *Significant difference from klotho^+/+D⁺ mice. #Significant difference from klotho^hmD⁺ mice (ANOVA, P < 0.05).

Fig. 6.

Plasma aldosterone concentrations of klotho^+/+ mice and klotho^hm mice with and without a low-Ca²⁺ diet. A: arithmetic means ± SE (n = 4–7) of the plasma aldosterone concentrations of klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺) before (left, +Ca²⁺) and after (right, −Ca²⁺) administration of a Ca²⁺-deficient diet for 1 wk. *Significant difference from klotho^+/+D⁺ mice (Student's t-test, P < 0.05). #Significant difference from klotho^hmD⁺ before treatment with Ca²⁺-deficient diet (Student's paired t-test, P < 0.05). B: arithmetic means ± SE (n = 5–6) of the urinary Na⁺ over creatinine concentration of klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺) before (left bars) and after (right bars) administration of a low-salt diet. The values are the means of the individual values determined for every mouse 3–4 days under a normal diet and 3–4 days under a low-salt diet.

Fig. 7.

Blood pressure of klotho^+/+ mice and klotho^hm mice with and without a vitamin D-deficient diet. Shown are arithmetic means ± SE (n = 12–22) of the systolic blood pressure of klotho^hm mice maintained on a continued vitamin D-deficient diet (klotho^hmD⁻) or on an initial vitamin D-deficient diet for 4 wk followed by a control diet for a further 4 wk (klotho^hmD^−/+) and of klotho^+/+ mice on a vitamin D-containing control diet (klotho^+/+D⁺). *Significant difference from klotho^+/+D⁺ mice (ANOVA, P < 0.01).

Fig. 8.

Amiloride-sensitive transepithelial current in distal colon of klotho^+/+ mice and klotho^hm mice with and without a vitamin D-deficient diet. A: representative original tracings of the transepithelial colonic potential difference (V_te) of klotho^hm and klotho^+/+ mice on a vitamin D-containing control diet (klotho^hmD⁺ and klotho^+/+D⁺). Addition of the epithelial Na channel (ENaC) blocker amiloride is indicated by arrows and results in a decrease in V_te. B: arithmetic means ± SE (n = 4–5) of the amiloride-sensitive equivalent short-circuit current in distal colonic epithelium of klotho^hmD⁺ mice and klotho^+/+D⁺ mice. *Significant difference from klotho^+/+D⁺ mice (Student's t-test, P < 0.05).

Fig. 9.

Survival of klotho^hm mice without vitamin D-deficient diet on a control, low-, and high-salt diet. The percentage of surviving klotho^hm mice maintained on a vitamin D-containing control diet (x), salt-deficient diet (○), and salt-rich diet (●) as a function of age. Survival tended to be shorter in animals receiving a salt-deficient diet and was significantly extended in animals receiving a salt-rich diet compared with the survival of animals receiving a control diet (ANOVA, P < 0.01).