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-D₃ [1,25(OH)₂D₃]. It participates in the regulation of renal tubular phosphate reabsorption and stimulates renal tubular Ca²⁺ 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^hmD⁺ mice than in klotho^+/+D⁺ mice, klotho^hmD⁻ mice, and klotho^hmD^−/+ mice. Plasma concentrations of 1,25(OH)₂D_3, adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice. Plasma volume was significantly smaller in klotho^hmD^−/+ mice, and plasma urea, Ca²⁺, phosphate and Na⁺, but not K⁺ concentrations were significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice. The differences were partially abrogated by a vitamin D-deficient diet. Moreover, the hyperaldosteronism was partially reversed by Ca²⁺-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^hmD⁺ mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)₂D₃ in Klotho-deficient mice results in extracellular volume depletion, which significantly contributes to the shortening of life span.

klotho is a protein expressed and residing in the cell membrane of the kidney, parathyroid glands, and choroid plexus (12, 14). The extracellular domain of the protein may be cleaved and released into the cerebrospinal fluid and blood (3). The expression of Klotho is a powerful determinant of life span (7). Klotho hypomorphic (klotho^hm) mice suffer from severe growth retardation and rapid aging (7), leading to premature death within <5 mo (7). Conversely, overexpression of Klotho increases the life span substantially (8).

Klotho increases the cell membrane protein abundance and activity of the renal epithelial Ca²⁺ channel TRPV5, thus favoring renal tubular Ca²⁺ reabsorption (13). Klotho enhances the Na⁺-K⁺-ATPase activity at a decreased extracellular Ca²⁺ concentration in renal epithelial cells and parathyroid glands (4). Moreover, Klotho contributes to the downregulation of the 1α-hydroxylase and thus limits the production of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] (10, 14, 17). Dietary vitamin D restriction reverses the growth deficit and increases the life span of klotho^hm mice (14).

The present study explored the influence of Klotho and Klotho-dependent 1,25(OH)₂D₃ formation on mineral and electrolyte metabolism. To this end, klotho^hm mice were compared with wild-type mice (klotho^+/+). The mice were fed either a normal (D⁺) or a vitamin D-deficient (D⁻) diet, or the first 4 wk a vitamin D-deficient diet followed by a normal diet (D^−/+). As expected, a vitamin D-deficient diet was followed by almost normal growth of klotho^hm mice. Surprisingly, the excessive formation of 1,25(OH)₂D₃ in klotho^hm mice was paralleled by hyperaldosteronism, which was similarly reversed by a vitamin D-deficient diet.

MATERIALS AND METHODS

Mice.

All animal experiments were conducted according to the guidelines of the American Physiological Society as well as the German law for the welfare of animals and were approved by local authorities. Klotho hypomorphic mice (klotho^hm) were compared with wild-type mice (klotho^+/+). The origin of the mice, breeding, and genotying were described previously (7). Congenic strains of Klotho-hypomorphic mice were produced by repeated backcrosses (>9 generations) to the 129 inbred strain and used in this study. The mice had access to water ad libitum and to either control food (Altromin C1000), a vitamin D-deficient diet (Altromin C1017), a low-salt diet (Altromin C1036), a high-salt diet (Altromin C1051), or a low-Ca²⁺ diet (Altromin C1031).

Blood chemistry.

To obtain blood specimens, animals were lightly anesthetized with diethylether (Roth, Karlsruhe, Germany), and ∼50–200 μl of blood was withdrawn into heparinized capillaries by puncturing the retroorbital plexus. The plasma concentrations of Na⁺ and K⁺ were determined using a flame photometer or by a photometric method (FUJI FDC 3500i, Sysmex, Norsted, Germany); the urea, phosphate, and Ca²⁺ concentrations were measured by the same photometric method. The plasma aldosterone concentrations were determined by using a commercial RIA kit (Demeditec, Kiel, Germany). The plasma intact parathormone concentrations were measured using an ELISA kit (Immunotopics, San Clemente, CA). A RIA kit was employed to determine plasma concentrations of 1,25(OH)-vitamin D₃ (IDS, Boldon, UK). The plasma ADH concentrations were determined utilizing a commercial EIA-Kit (AVP EIA Kit, Phoenix Europe, Karlsruhe, Germany), and the plasma ACTH concentrations were determined utilizing a commercial ELISA-Kit (ACTH_ELISA, MD Bioproducts, Zurich, Switzerland). As the extremely fragile klotho^hmD⁺ mice cannot be maintained in metabolic cages, spontaneously voided urine was collected for the determination of Na⁺ and creatinine concentrations. The urinary Na⁺ concentrations were determined by flame photometry and the urinary creatinine concentrations by utilizing the Jaffé reaction.

Determination of plasma volume.

The plasma volume was assessed by dye dilution using Evans Blue (Sigma, Taufkirchen, Germany). Mice were anesthetized with diethylether, and 30–50 μl of an Evans Blue stock solution (3 mg/ml in 0.9% NaCl) were injected intravenously into the left retroorbital plexus using a 30-gauge microfine insulin syringe (BD, Heidelberg, Germany). The exact applied volume was determined by weighing the syringe before and after injection. Repeated blood samples (20–25 μl) were drawn from the right retroorbital plexus during superficial diethylether anesthesia after 30, 60, and 100 min, which yielded a volume of 10 μl plasma after centrifugation. Absorbance was measured at 620 nm against blank mouse serum after recovery in 90 μl PBS (PBS tablets, Invitrogen, Karlsruhe, Germany). The plasma concentrations of Evans Blue were calculated using the stock solution dissolved in mouse serum as a standard. To correct for the clearance of Evans Blue during distribution time, linear regression of the log-transformed concentrations was applied to calculate the y-intercept, which represents the imaginary concentration of Evans Blue in its final distribution volume (2). Division of the applied dose of Evans Blue (in mg) by the y-intercept (in mg/ml) resulted in the distribution volume of Evans Blue, which was normalized for body weight. The procedure cannot be tolerated by the fragile klotho^hmD⁺ mice, and thus plasma volume cannot be determined in those mice. Instead, plasma volume was determined in the more robust klotho^hmD^−/+ mice, which had a similar hyperaldosteronism as the klotho^hmD⁺ mice.

Blood pressure.

Systolic arterial blood pressure was determined by the tail-cuff method (model 179, IITC). Application of this method requires certain precautions to reduce the stress of the animals, including appropriate training of the mice over multiple days and adequate prewarming to dilate the tail artery. The animals were placed in a heated chamber at an ambient temperature of 30–32°C, and from each animal blood pressure traces were recorded in one session until the deviation of a minimum of five traces was <5 mmHg. The readings from 3 days were then averaged to obtain a mean blood pressure under the respective treatment. All recordings and data analysis were done using a computerized data-acquisition system and software (PowerLab 400 and Chart 4, AdInstruments, Colorado Springs, CO). All measurements were performed by the same person during a defined time (between 2 and 4 PM).

Ussing chamber experiments.

ENaC activity was estimated from the amiloride-sensitive potential difference across the colonic epithelium. After removal of the outer serosal and the muscular layer of late distal colon under a microscope, tissues were mounted onto a custom-made mini-Ussing chamber with an opening diameter of 0.99 mm and an opening area of 0.00769 cm². The serosal and luminal perfusate contained (in mM) 145 NaCl, 1 MgCl₂, 2.6 Ca-gluconate, 0.4 KH₂PO₄, 1.6 K₂HPO₄, and 5 glucose. To assess ENaC-mediated transport, 50 μM amiloride (in ethanol; Sigma, Schnelldorf, Germany) were added to the luminal perfusate. In all Ussing chamber experiments, the transepithelial potential difference (V_t) was determined continuously and the transepithelial resistance (R_t) was estimated from the voltage deflections (ΔV_t) elicited by imposing test currents (I_t). The resulting R_t was calculated according to Ohm's law.

Statistics.

Data are provided as means ± SE; n represents the number of independent experiments. All data were tested for significance using ANOVA or a paired or unpaired Student t-test. Where applicable, GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA) or SAS Jmp version 8.0.1 (SAS Institute, Cary, NC) was used. Only results with P < 0.05 were considered statistically significant.

RESULTS

At the age of 8 wk, the body size was smaller for klotho^hmD⁺ mice than for klotho^+/+D⁺ mice (Fig. 1A). Both transient and sustained vitamin D deficiency fully reversed the growth retardation of Klotho-hypomorphic mice; i.e., the body weight of klotho^hmD⁻ and klotho^hmD^−/+ mice was not significantly different from the body weight of klotho^+/+D⁺ mice (Fig. 1B).

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).

The plasma volume per gram body weight was smaller in klotho^hmD^−/+ than in klotho^+/+D⁺ mice, pointing to extracellular volume depletion of Klotho-deficient mice (Fig. 2A). The volume depletion occurred despite a significantly larger fluid intake of klotho^hmD⁺ mice than of klotho^hmD^−/+ mice (Fig. 2B). The plasma volume could not be determined in klotho^hmD⁺ mice.

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).

Extracellular volume depletion is expected to decrease renal urea clearance and thus to increase plasma urea concentration. As illustrated in Fig. 3, the plasma urea concentration was indeed markedly higher in klotho^hmD⁺ than in klotho^+/+D⁺ mice. Sustained vitamin D deficiency led to a significant decrease in plasma urea concentration (Fig. 3).

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).

The plasma Na⁺, Ca²⁺, phosphate, but not K⁺ concentrations were significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice (Fig. 4) and were not significantly different between klotho^hmD^−/+ mice and klotho^+/+D⁺ mice or betweeen klotho^hmD⁻ mice and klotho^+/+D⁺ mice (Fig. 4). Thus lack of Klotho increased the plasma Na⁺, Ca²⁺, and phosphate concentrations, effects partially or fully reversed by transient or sustained vitamin D restriction. The plasma K⁺ concentration was not significantly different between the genotypes and was not significantly influenced by dietary vitamin D in Klotho hypomorphic mice (Fig. 4).

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).

The hypercalcemia and hyperphosphatemia could have resulted from enhanced formation of 1,25(OH)₂D₃, with subsequent stimulation of intestinal Ca²⁺ and phosphate absorption. The plasma concentration of 1,25(OH)₂D₃ was indeed significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice (Fig. 5A). A vitamin D-restricted diet significantly decreased 1,25(OH)₂D₃ formation. Accordingly, the plasma 1,25(OH)₂D₃ concentration was significantly lower in klotho^hmD⁻ mice than in klotho^hmD⁺ mice (Fig. 5A).

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).

The plasma PTH concentration tended to be lower in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice, a difference, however, not reaching statistical significance (Fig. 5B).

The extracellular volume depletion is expected to stimulate ADH release. The plasma ADH concentration was significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice (Fig. 5C). Sustained, but not transient, dietary vitamin D restriction decreased the plasma ADH concentration to levels similar to control animals. The plasma ADH concentration tended to be lower in klotho^hmD⁻ mice than in klotho^hmD⁺ mice, but again tended to be higher in klotho^hmD^−/+ mice than in klotho^+/+D⁺ mice (Fig. 5C).

The plasma aldosterone concentration was significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice (Fig. 5D). Again, sustained, but not transient, dietary vitamin D restriction reversed the hyperaldosteronism. Accordingly, the plasma aldosterone concentration was significantly lower in klotho^hmD⁻ mice than in klotho^hmD⁺ mice, but was again significantly higher in klotho^hmD^−/+ mice than in klotho^+/+D⁺ mice (Fig. 5D).

The plasma ACTH concentration was significantly higher in klotho^hmD⁺ mice (149 ± 17 nM, n = 4) than in klotho^+/+D⁺ mice (75 ± 12 nM, n = 5) but was not significantly affected by transient or sustained vitamin D depletion; i.e., it was similarly high in klotho^hmD^−/+ mice (157 ± 27 nM, n = 4).

The extracellular volume depletion and subsequent stimulation of aldosterone release could have resulted from hypercalcemia. To explore this possibility, additional experiments were performed with a Ca²⁺-deficient diet. As illustrated in Fig. 6A, the plasma aldosterone concentration in klotho^hmD⁺ mice significantly declined following treatment of the mice with a Ca²⁺-deficient diet. Nevertheless, the plasma aldosterone concentration remained significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice even under a Ca²⁺-deficient diet (Fig. 6A). Plasma aldosterone levels in klotho^+/+D⁺ mice were unaffected by a Ca²⁺-deficient diet.

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.

Further experiments were performed to study whether urine salt wasting contributes to the hyperaldosteronism of klotho^hm mice. To this end, the urinary Na⁺ and creatinine concentrations of klotho^+/+D⁺ and klotho^hmD⁺ mice were determined under a normal diet and a low-salt diet for 3 days each. As shown in Fig. 6B, salt depletion dramatically reduced the urinary Na⁺/creatinine ratio in klotho^+/+D⁺ mice but not in klotho^hmD⁺ mice.

The volume depletion was expected to decrease blood pressure. Due to the small size of klotho^hmD⁺ mice, it was technically impossible to determine their blood pressure. Blood pressure was, however, significantly lower in klotho^hmD^−/+ mice than in klotho^+/+D⁺ mice (Fig. 7). Blood pressure in klotho^hmD⁻ mice was between levels in klotho^hmD^−/+ mice and klotho^+/+D⁺ mice (Fig. 7).

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).

The volume depletion and lowered blood pressure in view of the hyperaldosteronism may have resulted from decreased aldosterone sensitivity of mineralocorticoid target tissues. To explore this possibility, Ussing chamber experiments were performed in the terminal colon of klotho^hmD⁺ mice and klotho^+/+D⁺ mice (Fig. 8). As a result, the amiloride-sensitive current was significantly larger in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice (Fig. 8). The observations revealed enhanced ENaC activity in Klotho-deficient mice (Fig. 8).

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).

Additional experiments were performed to elucidate whether salt intake modified the survival of klotho^hmD⁺ mice. Dietary salt diet indeed modified the life span of Klotho-deficient mice. When treated with a low-salt diet, the klotho^hmD⁺ mice tended to die earlier (at an age of 62.9 ± 8.9 days, n = 14) than klotho^hmD⁺ mice on a normal-salt diet (at an age of 66.8 ± 5.6 days, n = 26). In contrast, the life span of klotho^hmD⁺ mice on a salt-rich diet was significantly more extended (age of 214.0 ± 19.1 days, n = 4) than the life span of animals on either a control diet or salt-deficient diet. (Fig. 9).

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).

DISCUSSION

The present observations confirm the marked influence of Klotho deficiency on 1,25(OH)₂D₃ formation (10, 14, 17) as well as plasma Ca²⁺ (6) and phosphate (11) concentration. Klotho participates in the inhibition of 1α-hydroxylase and thus decreases 1,25(OH)₂D₃ production (10, 14, 17). As 1,25(OH)₂D₃ stimulates intestinal and renal Ca²⁺ and phosphate transport (9, 11), the unrestrained formation of 1,25(OH)₂D₃ presumably accounts for the hypercalcemia and hyperphosphatemia in Klotho hypomorphic mice (10, 14, 17). In view of the scatter of the present data, however, other mechanisms contributing to the deranged Ca²⁺ and phosphate metabolism cannot be excluded.

More importantly, the present observations reveal a novel functional consequence of Klotho deficiency, i.e., extracellular volume depletion with subsequent increase in ADH release and hyperaldosteronism. The volume depletion further leads to decreased blood pressure. At least in theory, the volume depletion of Klotho hypomorphic mice could be due to hypercalcemia and subsequent activation of the Ca²⁺-sensing receptor CasR. CasR regulates the renal tubular Na⁺ reabsorption; i.e., stimulation of the receptor inhibits renal tubular Na⁺ transport, leading to subsequent renal salt loss (15). Vitamin D-induced hypercalcemia has previously been shown to downregulate Na-K-2Cl cotransporter expression in the thick ascending limb of Henle's loop, which is expected to foster renal salt wasting and extracellular volume contraction (16). Accordingly, treatment of the mice with a Ca²⁺-deficient diet significantly and substantially blunted the hyperaldosteronism. The plasma volume was decreased and plasma aldosterone levels were enhanced in animals receiving a transiently vitamin D-deficient diet. In those mice, the unrestrained 1α-hydroxylase activity is expected to result in excessive 1,25(OH)₂D₃ formation as soon as vitamin D is added to the diet.

During a Ca²⁺-deficient diet, the plasma aldosterone level still remained significantly higher in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice. Thus additional mechanisms may contribute to the volume depletion of klotho^hmD⁺ mice, and more than a single disorder contributes to the derangement of electrolyte metabolism in Klotho hypomorphic mice. Klotho has previously been shown to upregulate the Na⁺-K⁺-ATPase (4) and, at least in theory, Klotho deficiency could result in decreased renal tubular Na⁺-K⁺-ATPase activity, thus compromising renal tubular salt reabsorption. The defect is particularly apparent under a salt-deficient diet, which leads to a rapid decrease of urinary Na⁺ output in wild-type mice but not in Klotho hypomorphic mice.

Klotho deficiency led to a significant increase in ACTH release. Unlike the aldosterone plasma level, the increased ACTH level could not be reversed by vitamin D deficiency and is presumably caused by mechanisms other than 1,25(OH)₂D₃ excess. It should be kept in mind that Klotho is not only expressed in parathyroid glands and the kidney, but as well in the choroid plexus (12, 14) and is released into cerebrospinal fluid (3). Thus Klotho deficiency could modify cerebral functions and hypothalamic control of hormone release more directly.

Klotho deficiency may not only affect the endocrine system but similarly compromise the function of the autonomous nerve system. In an earlier study, Klotho deficiency has been shown to impair the increase in catecholamine release during stress (12).

The hyperaldosteronism leads to an increase in electrogenic Na⁺ transport in the terminal colon, an epithelium similarly mineralocorticoid sensitive as the renal collecting duct (1). The observation illustrates that aldosterone is effective in Klotho hypomorphic mice and that extracellular volume as well as blood pressure are decreased despite increased aldosterone release and action.

In view of the hyperaldosteronism, the possibility was explored of whether the volume depletion may contribute to the decreased life span of Klotho-deficient mice. The results indeed demonstrate that a salt-rich diet significantly and substantially extended the life span of Klotho-deficient mice. These observations do not contradict the role of other pathophysiological mechanisms leading to the dramatic decrease in the life span of Klotho hypomorphic mice but clearly demonstrate a strong impact of extracellular volume depletion on the survival of those mice.

Similar to what has been shown for the life span (14), growth deficit (14) and erythrocyte survival (5), vitamin D restriction reverses the effect of Klotho deficiency on the plasma mineral and electrolyte concentrations as well as on hyperaldosteronism. The observations reveal that excessive formation of 1,25(OH)₂D₃ substantially contributes to or even accounts for the hyperaldosteronism in Klotho hypomorphic mice.

In conclusion, lack of Klotho leads to profound derangements not only of mineral but as well of electrolyte metabolism, resulting in a decrease in blood pressure and hyperaldosteronism. The effect could be largely reversed by vitamin D deficiency and is thus at least in part secondary to excessive formation of 1,25(OH)₂D₃. Salt repletion significantly enhances the life span of Klotho hypomorphic mice, and salt deficiency thus significantly contributes to the early death of those animals.

GRANTS

This study was supported by the Deutsche Forschungsgemeinschaft and by the National Institutes of Health, National Heart, Lung, and Blood Institute's Proteomics Initiative NO1-HV-28184 (K. P. Rosenblatt), the Welch Foundation Endowment in Chemistry and Related Science, Grant L-AU-0002, and by the Center for Clinical and Translational Sciences, which is funded by National Institutes of Health Clinical and Translational Award UL1 RR024148-05 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Welch Foundation, the National Center for Research Resources, or the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.