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 (klothohm) 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, klothohm 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 klothohmD+ mice than in klotho+/+D+ mice, klothohmD− mice, and klothohmD−/+ mice. Plasma concentrations of 1,25(OH)2D3, adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klothohmD+ mice than in klotho+/+D+ mice. Plasma volume was significantly smaller in klothohmD−/+ mice, and plasma urea, Ca2+, phosphate and Na+, but not K+ concentrations were significantly higher in klothohmD+ 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 klothohmD+ 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.
- cell volume
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 (klothohm) 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 klothohm 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, klothohm 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 klothohm mice. Surprisingly, the excessive formation of 1,25(OH)2D3 in klothohm mice was paralleled by hyperaldosteronism, which was similarly reversed by a vitamin D-deficient diet.
MATERIALS AND METHODS
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 (klothohm) 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).
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 klothohmD+ 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 klothohmD+ mice, and thus plasma volume cannot be determined in those mice. Instead, plasma volume was determined in the more robust klothohmD−/+ mice, which had a similar hyperaldosteronism as the klothohmD+ mice.
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 (Vt) was determined continuously and the transepithelial resistance (Rt) was estimated from the voltage deflections (ΔVt) elicited by imposing test currents (It). The resulting Rt was calculated according to Ohm's law.
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.
At the age of 8 wk, the body size was smaller for klothohmD+ 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 klothohmD− and klothohmD−/+ mice was not significantly different from the body weight of klotho+/+D+ mice (Fig. 1B).
The plasma volume per gram body weight was smaller in klothohmD−/+ 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 klothohmD+ mice than of klothohmD−/+ mice (Fig. 2B). The plasma volume could not be determined in klothohmD+ mice.
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 klothohmD+ than in klotho+/+D+ mice. Sustained vitamin D deficiency led to a significant decrease in plasma urea concentration (Fig. 3).
The plasma Na+, Ca2+, phosphate, but not K+ concentrations were significantly higher in klothohmD+ mice than in klotho+/+D+ mice (Fig. 4) and were not significantly different between klothohmD−/+ mice and klotho+/+D+ mice or betweeen klothohmD− 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).
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 klothohmD+ 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 klothohmD− mice than in klothohmD+ mice (Fig. 5A).
The plasma PTH concentration tended to be lower in klothohmD+ 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 klothohmD+ 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 klothohmD− mice than in klothohmD+ mice, but again tended to be higher in klothohmD−/+ mice than in klotho+/+D+ mice (Fig. 5C).
The plasma aldosterone concentration was significantly higher in klothohmD+ 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 klothohmD− mice than in klothohmD+ mice, but was again significantly higher in klothohmD−/+ mice than in klotho+/+D+ mice (Fig. 5D).
The plasma ACTH concentration was significantly higher in klothohmD+ 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 klothohmD−/+ 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 klothohmD+ mice significantly declined following treatment of the mice with a Ca2+-deficient diet. Nevertheless, the plasma aldosterone concentration remained significantly higher in klothohmD+ 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.
Further experiments were performed to study whether urine salt wasting contributes to the hyperaldosteronism of klothohm mice. To this end, the urinary Na+ and creatinine concentrations of klotho+/+D+ and klothohmD+ 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 klothohmD+ mice.
The volume depletion was expected to decrease blood pressure. Due to the small size of klothohmD+ mice, it was technically impossible to determine their blood pressure. Blood pressure was, however, significantly lower in klothohmD−/+ mice than in klotho+/+D+ mice (Fig. 7). Blood pressure in klothohmD− mice was between levels in klothohmD−/+ mice and klotho+/+D+ mice (Fig. 7).
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 klothohmD+ mice and klotho+/+D+ mice (Fig. 8). As a result, the amiloride-sensitive current was significantly larger in klothohmD+ mice than in klotho+/+D+ mice (Fig. 8). The observations revealed enhanced ENaC activity in Klotho-deficient mice (Fig. 8).
Additional experiments were performed to elucidate whether salt intake modified the survival of klothohmD+ mice. Dietary salt diet indeed modified the life span of Klotho-deficient mice. When treated with a low-salt diet, the klothohmD+ mice tended to die earlier (at an age of 62.9 ± 8.9 days, n = 14) than klothohmD+ mice on a normal-salt diet (at an age of 66.8 ± 5.6 days, n = 26). In contrast, the life span of klothohmD+ 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).
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 klothohmD+ mice than in klotho+/+D+ mice. Thus additional mechanisms may contribute to the volume depletion of klothohmD+ 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.
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.
No conflicts of interest, financial or otherwise, are declared by the authors.
The authors acknowledge the technical assistance of Elfriede Faber and Ganesh Pathare and the meticulous preparation of the manuscript by Lejla Subasic and Tanja Loch.
- Copyright © 2010 the American Physiological Society