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Tsukuba Research Laboratories, Kyowa Hakko Kogyo Company Limited, Tsukuba-shi, Ibaraki 305-0841, Japan;
*
Tokyo Research Laboratories, Kyowa Hakko Kogyo Company Limited, Machida-shi, Tokyo 194-8533, Japan; and
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
1To whom correspondence should be addressed. E-mail: tkamiya{at}kyowa.co.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • klotho • phosphorus • zinc orotate • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
), and 1012 amino acid residues in humans (3
). The existence of a soluble form of the klotho protein has also been proposed (1
,4
). In klotho mutant mice, the coding region of the klotho gene is preserved but its expression is markedly decreased by the insertion of an exogenously introduced nonfunctional gene within its promoter region (1
).
Within their short life span of 
9 wk, klotho mutant homozygous mice show many characteristics similar to the senescence-related diseases of humans. These include ectopic calcification in the arterial wall and stomach, osteoporosis and abnormal bone formation, atrophy of the thymus, disruption of prukinje cells, gonad atrophy (sterility), thinning of the skin, ataxia and abnormality of the pituitary gland, as well as hypoglycemia and hyperphosphatemia (1
). Although the data obtained to date support the idea that klotho plays an important role in aging and senescence-related diseases, little is known about the exact function and the regulation of this gene.
The aim of our study was to determine whether any dietary substance might modulate the phenotypes of the klotho mutant mice. If it were possible to manage klotho-related diseases using a nutritional approach, it would be a first choice for the prevention or treatment of such diseases until a superior gene therapy is established.
We fed the mice >70 kinds of laboratory diets, each of which included sufficient nutrients with some additives, such as pure compounds or crude natural products. Among the diets tested, we found one that evidently supported the growth of the male homyzygotes and prolonged their life span. To identify components that would play a key role in this "klotho-remedy" diet, the contents of minerals, amino acids, lipids and carbohydrates in the diet were compared with those of other diets. Through extensive testing, we found that the klotho-remedy effect was not brought on by the presence of a certain compound in the diet but was caused by the restriction of a nutritionally indispensable component, i.e., phosphorous. This led us to investigate the regulation of klotho gene expression and to look for a substance that could rescue female klotho homozygotes. These new findings are reported in this paper.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Each genotype of newborn klotho mutant mice from the mating male and female heterozygotes was obtained through reproduction by many pairs of heterozygous parents. Genotyping was done using the polymerase chain reaction (PCR) procedure with the primers: 5'-TGGAGATTGGAAGTGGACG-3', 5'-CAAGGACCAGTTCATCATCG-3' and 5'-TTAAGGACTCCTGCATCTGC-3', which discriminate the homozygotes of klotho mutant mice from other genotypes. The DNA separated from the tail of each mouse was amplified with LA-Tag DNA polymerase (TaKaRa Shuzo, Tokyo, Japan). Amplification cycles were 30 times at 94°C for 30 s, 56°C for 30 s and 72°C for 90 s. Mutated and wild alleles were visualized as signals at 920 and 458 bp, respectively.
All of the diets used in each experiment (Table 1
) had a modified AIN-76 composition (5
). Potassium dihydrogen phosphate (KH2PO4) was used to control the phosphorus content in each diet. Both potassium dihydrogen phosphate and zinc orotate (Kyowa Hakko Kogyo, Tokyo, Japan) were added in place of cornstarch. A nonpurified diet, CE-2 (CLEA Japan, Tokyo, Japan) was used for the maintenance of heterozygous parents and for the induction of homozygous phenotypes. OA-2, another nonpurified diet (CLEA Japan) was used for pregnant and nursing mice. All mice were housed in a humidity- (55 ± 10%) and temperature- (23 ± 1°C) controlled room with a 12-h light:dark cycle. Water and food were consumed ad libitum. Leftover food was replaced with fresh food and the appearance of the mice was checked twice a week.
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Effect of phosphorus.
Male and female homozygotes (n = 4) were fed a 0.4 g/100 g phosphorus diet (Table 1)
beginning just after weaning at wk 3 of age. They were fed until their death or killed at wk 29 of age for pathohistological observation of the testis.
Some male homozygotes fed the 0.4 g/100 g phosphorus diet were killed for histopathological observation, for comparison with age-matched male homozygotes fed CE-2 (n = 4). Other male homozygotes fed phosphorus-controlled diets (Table 1)
were killed for analysis of klotho protein expression in the kidneys, and serum glucose and inorganic phosphorus concentrations (n = 4). The body weight gain rate during the feeding period was calculated as the body weight change (g) per feeding period (d).
To determine the effect of phosphorus on wild-type mice, 3-wk-old C3H/He mice (SLC, Shizuoka, Japan) were fed phosphorus-controlled diets as shown in Table 1
(n = 4), and then killed for analysis of klotho protein expression in the kidneys, and for serum glucose and inorganic phosphorus concentrations.
Effect of zinc orotate.
Female homozygotes (n = 4) were fed diets with or without zinc orotate (Table 1)
beginning just after weaning. At 9 wk of age, they were coupled with male homozygotes raised according to the procedure described above. Genotypes of the pups born from a homozygous male and female couple were determined by PCR as described above. Some other female homozygotes were killed for analysis of klotho protein expression in the kidneys.
Serum chemical analysis.
Serum glucose was measured enzymatically with Determiner L Glu II (Kyowa Medex, Tokyo, Japan). Serum inorganic phosphorus was measured with the Phospher C test WAKO (Wako Pure Chemical, Osaka, Japan). The serum zinc level was measured with the Zn-test WAKO (Wako Pure Chemical), in which the reacted product of 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol sodium salt and zinc was quantified colorimetrically.
Western blotting.
A fresh kidney from each mouse was homogenized in a lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 10 g/L Triton-X 100) on ice, and the protein concentration of the centrifuged supernatant (1000 x g, for 10 min) was measured with an assay kit (DC protein assay; Bio-Rad Laboratories, Hercules, CA). A quarter volume of the sample buffer (25 mmol/L Tris-HCl buffer, pH 6.5, 5% glycerol, 1% SDS, 1% 2-mercaptoethanol, 0.5% bromophenol blue) was added to the kidney lysate whose protein concentration had been adjusted to 1 g/L with distilled water, and was heated at 100°C for 5 min. Each sample (25 µL) was applied to a 5–20% gradient polyacrylamide-gel (PAGEL NPG-520L; Atto, Tokyo, Japan) and electrophoresed in a running buffer (25 mmol/L Tris-HCl, pH 8.3, 192 mmol/L glycine, 0.1% SDS).
Proteins in a polyacrylamide-gel plate were electrophoretically transferred to a membrane (Immobilon; Millipore, Bedford, MA). The first antibody, anti-klotho protein monoclonal antibody (KM2119) (6
), was diluted to 200 µg protein/L, and was reacted for 60 min. The second antibody, horseradish peroxidase–conjugated anti-rat immunoglobulins sheep antibody (Amersham Pharmacia Biotech, Uppsala, Sweden), was diluted to 1:2000 and reacted for 60 min. The reacted klotho protein was visualized by the enhanced chemiluminescence method (Hyperfilm; Amersham Pharmacia Biotech), and the signal intensity was determined densitometrically (CS-9000; Shimadzu, Tokyo, Japan). The relative value of the signal intensity against the standard kidney lysate was calculated.
Statistical analysis.
Statistical analyses were performed with two-way ANOVA (SAS preclinical package Version 4; SAS Institute Japan, Tokyo, Japan), one-way ANOVA, Student’s t test or Kruskal-Wallis test (StatFlex Version 5.0; Artec, Osaka, Japan). Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The average life span of the klotho homozygotes bred in our colony was 9.6 ± 1.1 wk and their average body weight did not exceed 10 g when they were fed the nonpurified diet, CE-2. However, when the klotho homozygous males were fed the 0.4 g/100 g phosphorus diet starting at wk 3 of age, they continued to gain body weight until reaching 20 g or more at wk 20 of age (Fig. 1A
). Four males examined survived to the end of the experiment at wk 29 of age (data not shown). Females did not show such a response. Spermatogenesis was observed in the male mice fed the 0.4 g/100 g phosphorus diet (Fig. 1B
).
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) than the mice fed CE-2 (Fig. 2A
). The mice fed CE2 had thinner skin, more severely curved vertebrae and less mobility than the mice fed the 0.4 g/100 g phosphorus diet (data not shown). Pathohistologically, the mice fed the 0.4 g/100 g phosphorus diet showed only slight ectopic calcification in the kidneys (Fig. 2C
), whereas the mice fed CE2 showed severe kidney calcification (Fig. 2D
). In the cartilage of the knee, the mice fed the 0.4 g/100 g phosphorus diet had few of the hypertrophic chondrocyte-like cells that were abundant in the mice fed CE2 (Fig. 2E
,F
).
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. All of these phosphorus levels are within nutritionally normal levels for wild-type mice (CE-2 contains 1.03 g/100 g phosphorus). Compared with the mice fed the 0.4 g/100 g phosphorus diet, the mice fed 0.6 or 0.8 g/100 g phosphorus diets had significantly lower body weight gains (Fig. 3A
). The body weight gain rates of the mice fed phosphorus-supplemented diets were 138.1 ± 114.5 mg/d in those fed 0.8 g/100 g; and 153.1 ± 133.7 mg/d in those fed 0.6 g/100 g. Both were significantly lower than that of mice fed the 0.4 g/100 g phosphorus diet (421.4 ± 100.2 mg/d, P < 0.01). The mice fed the 0.4 g/100 g phosphorus diet had higher serum glucose levels (9.66 ± 0.07 mmol/L) than those fed the 0.6 or 0.8 g/100 g phosphorus diets (6.78 ± 0.72 and 6.83 ± 0.19 mmol/L, respectively; P < 0.001). Significantly lower serum inorganic phosphorus levels were also seen in mice fed the 0.4 g/100 g phosphorus diet (4.06 ± 0.52, 5.16 ± 0.29 and 5.16 ± 0.39 mmol/L for 0.4, 0.6 and 0.8 g/100 g phosphorus diets, respectively, P < 0.001).
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In female homozygotes, the 0.4 g/100 g phosphorus diet had little effect on klotho phenotypes and all these mice, except one, died by the age of 10 wk (Fig. 1A
). We subsequently tried to find a substance to attenuate the symptoms of the female homozygotes, and found an effective compound, zinc orotate. At wk 6 of age, females fed a diet containing 0.25 g/100 g zinc orotate had a significantly higher body weight (16.5 ± 1.5 g) than females fed the diet without zinc orotate (12.1 ± 5.4 g, P < 0.05, Fig. 3B
). All of the females fed the 0.25 g/100 g zinc orotate diet survived to wk 8 of age and the average body weight was 17.8 g.
Zinc chloride, but not orotic acid, had similar effects on female homozygotes (data not shown), suggesting that zinc was the key constituent of zinc orotate in the favorable action observed (CE-2 contains 0.00638 zinc g/100 g).
There were no significant differences in serum glucose (with 0.25 g/100 g zinc orotate, 9.69 ± 1.28 mmol/L; without zinc orotate, 7.01 ± 1.41 mmol/L, P = 0.078) or serum inorganic phosphorus (with 0.25 g/100 g zinc orotate, 4.39 ± 0.16 mmol/L; without zinc orotate, 4.87 ± 0.55 mmol/L, P > 0.1).
At wk 9 of age, the homozygous females fed the 0.25 g/100 g zinc orotate diet were mated with male adult homozygotes fed the 0.4 g/100 g phosphorus diet. Seven pups were born from one pair of homozygotes and were confirmed as homozygotes by genotyping (Fig. 4
). A total of 45 pups were born from 6 pairs of homozygotes by wk 25 of age (data not shown).
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To determine whether the effect of zinc orotate might reflect a difference in serum zinc levels in klotho homozygotes and normal mice, the body weight and the serum zinc levels of 6-wk-old mice fed the nonpurified diet were measured (Table 2
). Serum zinc levels differed between homozygous and wild-type mice (P = 0.0001, Table 2
).
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Klotho protein expression in the kidneys of the male homozygotes fed phosphorus-restricted diets was analyzed by Western blotting. The mice fed the diets containing 0.6 or 0.8 g/100 g phosphorus had undetectable levels of klotho protein (Fig. 5A
). In contrast, the mice fed the 0.4 g/100 g phosphorus diet had clearly detectable amounts of klotho protein (Fig. 5A
). In the case of female mice fed the 0.4 g/100 g phosphorus diet (without zinc orotate), no klotho protein expression in the kidneys was observed (Fig. 5B
). When female mice were given the 0.4 g/100 g phosphorus diet supplemented with 0.25 g/100 g zinc orotate, slight signals in the klotho protein expression were detected (Fig. 5B
).
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The time-dependent profile of the klotho up-regulation by phosphorus restriction was measured on klotho homozygous males. Two mice were killed just after weaning, and some were fed the 0.25 g/100 g zinc orotate, 0.4 g/100 g phosphorous diet and killed after 2, 4 or 7 d. On d 2, neither of the mice showed any clear signals in klotho protein expression (Fig. 6A
, B). Although some variance in the expression levels was seen among the mice, it took 4–7 d before the klotho protein in the kidneys reached detectable levels (Fig. 6A
, B
).
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Wild-type C3H/He mice and klotho heterozygotes grew equally well when fed any of the diets used above (CE2, 0.4, 0.6 or 0.8 g/100 g phosphorus diets with or without zinc orotate; data not shown), suggesting that the effects of dietary phosphorus at normal levels and of zinc supplementation were specific to klotho homozygotes. To determine whether excessive dietary phosphorus affects the expression of the klotho protein in wild-type mice, male C3H/He mice were fed a diet containing 1.5 g/100 g phosphorus. In these mice, ectopic calcification in the kidneys occurred (data not shown). No significant changes occurred in serum glucose, serum inorganic phosphorus or body weight with the 1.5 g/100 g phosphorus diet. Western blot analysis revealed that the klotho protein expression in the kidneys had an inverse relation to the level of dietary phosphorus (0.4 g/100 g phosphorus, 77.1 ± 22.6; 1.0 g/100 g phosphorus, 38.7 ± 13.5; 1.5 g/100 g phosphorus, 32.2 ± 30.2, n = 4, P < 0.01, Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In considering the relationship of klotho expression to the dietary phosphorus level, the klotho protein seemed to be negatively controlled by dietary phosphorus. In male homozygotes, klotho protein expression was greatly activated by the phosphorus-restricted diet. In wild-type mice, kidney calcification and growth inhibition occur when mice are fed excessive amounts of phosphate (7
–11
). Our results showed that klotho protein expression in wild-type mice was diminished by dietary phosphorus at a concentration that caused kidney calcification. Therefore, klotho mutant and wild-type mice seem to share common characteristics (but with different sensitivities) in the dietary phosphorus/klotho gene expression relationship. It is likely that a mutation in the promoter region of the klotho gene made it hypersensitive to dietary phosphorus compared with normal mice.
Some molecules respond to the level of dietary phosphorus. The sodium/phosphate-cotransporters enhance the reabsorption of phosphate in the kidneys and absorption of dietary phosphate in the intestines, when dietary phosphate is restricted (12
–14
). The regulatory region of the klotho gene might include an unknown element that functions similarly to the promoter of sodium/phosphate-cotransporters. A delay in the activation of the klotho gene upon the feeding of a phosphorus-restricted diet suggests the possibility of the regulation of this gene by phosphorus-related molecules such as parathyroid hormone, serum calcium or serum glucose. The difference in the susceptibility to phosphorus restriction between males and females suggests that some sex-specific metabolic activity or organ function may be involved in the regulation of the klotho gene.
We found that the zinc in zinc orotate was responsible for its favorable effect, by comparing the average body weight of female homozygotes fed phosphorus-restricted diets supplemented with either zinc chloride, orotic acid or zinc orotate on the same molar basis. The average body weights of the mice fed these three diets were 15.8 ± 3.0, 12.4 ± 3.0 and 17.3 ± 0.8 g, respectively, at 6 wk of age. However, zinc orotate improved the reproductive capability of homozygotes more than zinc chloride (data not shown), suggesting that orotate may play some part in the action of zinc orotate in these mice.
The serum zinc levels in homozygotes were lower than in wild-type mice. This suggests that there is some impairment in the zinc-dependent metabolism in klotho mutant homozygotes that can lead to some klotho phenotypes. In fact, there are some similarities between the symptoms of a zinc-deficient status and klotho phenotypes. For example, zinc deficiency causes growth retardation and hypogonadism (15
,16
). The nuclear receptors for steroid hormones are zinc finger proteins (16
). Collagenase and alkaline phosphatase, which are involved in the metabolism in the skin and bone, are zinc enzymes (17
–19
). Cultured osteoblastic cells derived from klotho homozygous mice have also been shown to have lower alkaline phosphatase activity (20
). Because the rescued females had a low level of klotho expression, it is likely that zinc orotate exerted its effect not through the activation of the klotho gene but mainly through inhibiting the subsequent events caused by the insufficient expression of the klotho gene. With a sufficient intake of zinc, the minimum requirement of klotho expression for healthy growth seems lower in females than in males. Otherwise, the weaker ability of the klotho gene to be activated by phosphorus restriction caused the higher zinc requirement in female homozygotes, whereas male homozygotes maintained enough zinc through sufficient recovery of klotho expression.
We have shown a procedure to rescue klotho homozygotes by continuously feeding a specific diet beginning at weaning. When this diet was fed from 2 wk after weaning in place of CE-2, the mice could not be rescued (data not shown), suggesting that klotho expression is particularly important in the neonatal period for healthy growth. In addition, our results on adult wild-type mice using excessive dietary phosphorus suggest the importance of the klotho gene in adult mice for maintaining homeostasis, whose impairment may cause some pathological states.
The dietary protocol we developed is a simple, noninvasive and reproducible procedure to modulate klotho expression in vivo. Though the mechanisms of klotho gene regulation have yet to be investigated, this system would be a valuable tool to achieve the following: 1) identify nutritional factors that affect klotho gene expression; 2) evaluate substances that affect the utilization or absorption of phosphorus and zinc; 3) study the role of zinc under conditions of limited klotho expression; and 4) study male- or female-specific metabolism in which klotho gene expression is involved.
Manuscript received July 10, 2001. Initial review completed July 21, 2001. Revision accepted September 9, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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