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Molecular Endocrinology, doi:10.1210/me.2003-0048
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Molecular Endocrinology 17 (12): 2393-2403
Copyright © 2003 by The Endocrine Society

Klotho, a Gene Related to a Syndrome Resembling Human Premature Aging, Functions in a Negative Regulatory Circuit of Vitamin D Endocrine System

Hiroshi Tsujikawa, Yoko Kurotaki, Toshihiko Fujimori, Kazuhiko Fukuda and Yo-Ichi Nabeshima

Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; Department of Anesthesia, Kyoto University Hospital, Kyoto University, Kyoto 606-8507, Japan; and Core Research for Evolutional Science and Technology, Japan Science and Technology Corp., Saitama 332-0012, Japan

Address all correspondence and requests for reprints to: Yo-ichi Nabeshima, Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. E-mail: nabemr{at}lmls.med.kyoto-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The klotho gene encodes a novel type I membrane protein of ß-glycosidase family and is expressed principally in distal tubule cells of the kidney and choroid plexus in the brain. These mutants displayed abnormal calcium and phosphorus homeostasis together with increased serum 1,25-(OH)2D. In kl-/- mice at the age of 3 wk, elevated levels of serum calcium (10.9 ± 0.31 mg/dl vs. 10.0 ± 0.048 mg/dl in wild-type mice), phosphorus (14.7 ± 1.1 mg/dl vs. 9.7 ± 1.5 mg/dl in wild type) and most notably, 1,25-(OH)2D (403 ± 99.7 mg/dl vs. 88.0 ± 34.0 mg/dl in wild type) were observed.

Reduction of serum 1,25-(OH)2D concentrations by dietary restriction resulted in alleviation of most of the phenotypes, suggesting that they are downstream events resulting from elevated 1,25-(OH)2D. We searched for the signals that lead to up-regulation of vitamin D activating enzymes. We examined the response of 1{alpha}-hydroxylase gene expression to calcium regulating hormones, such as PTH, calcitonin, and 1,25-(OH)2D3. These pathways were intact in klotho null mutant mice, suggesting the existence of alternate regulatory circuits. We also found that the administration of 1,25-(OH)2D3 induced the expression of klotho in the kidney. These observations suggest that klotho may participate in a negative regulatory circuit of the vitamin D endocrine system, through the regulation of 1{alpha}-hydroxylase gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
WE CREATED A unique mouse strain with a shortened life span in which a single gene mutation causes multiple aging-related disorders. The responsible gene was identified as klotho. Homozygous klotho mutant mice (kl/kl mice) exhibit a variety of phenotypes and are useful as a model of human premature aging (1). On the basis of both macroscopic and histological appearance, kl/kl mice are developmentally normal until 3 wk of age, after which they display growth retardation accompanied by multiple phenotypes, and die prematurely. The mouse klotho gene encodes a type I membrane protein which consists of a N-terminal signal sequence, an extracellular domain composed of two internal repeats, a transmembrane domain and a short intracellular domain (1). Homologs are also found in human (2) and rat (3). The extracellular internal repeats show gross homology to ß-glycosidases. Klotho mRNA is predominantly expressed in the distal renal tubules and the choroid plexus in the brain. Among the phenotypes characteristic of kl/kl mice, one of the most prominent is ectopic calcification in the kidney, medial arterial layers, stomach wall, trachea, soft tissues, and bone epiphyses (1, 4). In kl/kl mice, the levels of serum calcium and phosphorus are elevated (1) accompanied by discordantly high levels of 1,25-(OH)2D (5). Because concentrations of 25-(OH)D and 24,25-(OH)2D are low, the abnormal activation of 1,25-(OH)2D is thought to be due to the elevated gene expression of 1{alpha}-hydroxylase in the mutant kidneys (5).

In this paper, we demonstrate that most of the phenotypes seen in klotho null mutants could be rescued by reduction of serum concentrations of 1,25-(OH)2D with vitamin D-deficient diets (-D.D). This indicates that the abnormal activation of vitamin D in the mutant is the major cause of the phenotypes. The normal genetic responses to administered calcium regulating hormones, such as PTH, calcitonin (CT), and 1,25-(OH)2D3 appear conserved in kl-/- mice, suggesting that the alternative circuit is defective. Similar to 25-hydroxyvitamin D 24-hyroxylase and vitamin D receptor (VDR), klotho is induced by 1,25-(OH)2D3. These data suggest that klotho plays a role in the vitamin D metabolic pathway as a negative regulator of 1,25-(OH)2D synthesis. These suggest that klotho functions in a negative regulatory circuit of vitamin D.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Effect of -D.D. on kl-/- Mice
For all experiments in this paper, we used klotho null mutants (kl-/-), which were recently established by targeted gene disruption (Fujimori, T., K. Takeshita, Y. Kurotaki, H. Honjo, H. Tsujikawa, K. Yasui, J.-H. Lee, K. Kamiya, K. Kitaichi, K. Yamamoto, M. Ito, T. Kondo, S. Iino, Y. Inden, M. Hirai, T. Murohara, I. Kodama, and Y.-i. Nabeshima, manuscript submitted) based on the observation that the phenotypes were indistinguishable from those of the original mutant (kl/kl). The original strain klotho (kl/kl) exhibited phenotypes such as growth retardation, infertility, bone malformation, ectopic calcification, skin atrophy, and a shortened life span. We compared the homozygous mutant phenotypes of the targeted disruption (kl-/-) with the reported phenotypes seen in kl/kl mutants, and found that the two strains were very similar. Growth retardation was observed at 3 wk after birth, and body weights remained under 10 g throughout the life spans of the mutants under standard nutritional and exercise conditions. Homozygous null mice had average life spans of 77 d compared with the 2-yr norm. At 7 wk, we observed reduction of white fat deposits in all organs examined. Ectopic calcification was observed in the kidney, aorta, stomach, gut, lung, and choroid plexus of the brain. Malformation of bones was also observed with abnormal calcification at the epiphysis and reduced bone density in the diaphysis. In the original klotho mutant (kl/kl), multiple transgene copies were tandemly integrated into the upstream regulatory regions of the klotho gene and resulted in severe down-regulation of klotho gene expression. Thus, the original klotho mutant (kl/kl) was not a complete null but a severe hypomorph for klotho function. Because the entire coding sequence remained intact in the klotho mutant genome, the expression of klotho could be recovered by maintaining mice on specific diets (6). When we established the original klotho mutant (kl/kl), we confirmed that the inserted transgene was not expressed, and that phenotypes in klotho mutants were not due to the expression of the transgene but rather to the disruption of the endogenous klotho gene. However, as the inserted transgene contained functional cassettes for Na+/H+ exchanger (7), it was also necessary to rule out any accidental effects of induced Na+/H+ exchanger genes on the phenotypes seen in klotho mutants. Thus, we chose to use our specifically targeted nulls rather than the original hypomorph mutants for the experiments in this paper.

Examination of serum markers revealed that in kl-/- mice at the age of 3 wk, the levels of serum calcium [10.9 ± 0.31 mg/dl vs. 10.0 ± 0.048 mg/dl of wild-type mice (wt)] and phosphorus (14.7 ± 1.1 mg/dl vs. 9.7 ± 1.5 mg/dl of wt) were significantly higher. As observed in kl/kl mice (5), the serum concentration of 1,25-(OH)2D in the null (kl-/-) mice was also significantly higher than that of wt at 3 wk (Fig. 1AGo, lanes 1 and 6) and 7 wk of age (Fig. 1AGo, lanes 2 and 7) under normal feeding conditions. We speculated that these elevated levels of 1,25-(OH)2D may lead to the phenotypes observed in kl-/- mice. To test this possibility, we fed mice with -D.D. to lower their serum levels of 1,25-(OH)2D. Heterozygous females (kl+/-) crossed with heterozygous males (kl+/-) were kept on -D.D. after they became pregnant, and pups were fed with -D.D. after weaning. This treatment did not have any significant effects on wild-type animals. 1,25-(OH)2D Serum levels were lower in 7-wk-old kl-/- mice kept on a -D.D. than in those on a normal diet (N.D.) (Fig. 1AGo, lanes 2 and 3). There were no remarkable differences in the serum 1,25-(OH)2D levels between kl-/- mice and wt mice after the restriction of vitamin D intake (-D.D.) (Fig. 1AGo, lanes 3 and 8). Wild-type animals displayed normal growth under the -D.D. (Fig. 1BGo, lane 8), although we observed slight reduction of serum 1,25-(OH)2D (Fig. 1AGo, lane 8). We examined the effects of serum 1,25-(OH)2D reduction on the growth and survival rates of the mutant mice (Fig. 1BGo, lanes 2 and 3). kl-/- mice fed N.D. did not reach body weights of more than 10 g and started to die after 7 wk. On the other hand, kl-/- mice maintained on -D.D. attained body weights of more than 20 g by 7 wk and continued to reach weights over 30 g. These mice survived longer than 15 wk, clearly demonstrating that reduction of 1,25-(OH)2D in the serum could rescue some of the phenotypes in klotho null mutants. We further analyzed other aspects of serum 1,25-(OH)2D reduction. Serum levels of calcium and phosphorus were significantly lowered to within a normal range. In mice fed with -D.D., the levels of serum calcium (7.77 ± 1.37 mg/dl vs. 8.43 ± 1.13 mg/dl in wt and kl-/- mice, respectively) and phosphorus (7.81 ± 1.66 mg/dl vs. 8.9 ± 1.14 mg/dl in wt and kl-/- mice, respectively) were significantly decreased in the kl-/- mice. Other serum markers, such as glucose, triglyceride, blood urea nitrogen, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase, etc. were within normal levels (data not shown).



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  		Fig. 1. Rescue of Klotho Phenotypes in Mice with -D.D.

A, Serum concentrations of 1,25-(OH)2D were measured in kl-/- mice on N.D. at the age of 3 or 7 wk, kl-/- mice fed with -D.D., low P.D., or low Ca.D. at the age of 7 wk. Concurrently, serum concentrations of 1,25-(OH)2D were also measured for control wt mice on the same diets as kl-/- mice. At 3 wk of age, kl-/- mice had significantly higher 1,25-(OH)2D levels compared with wt mice. This difference narrowed by 7 wk on N.D., although remaining statistically significant. In animals on -D.D., serum levels of 1,25-(OH)2D were reduced in both kl-/- and wt mice, although the difference between the two was maintained. In kl-/- mice fed with low P.D. or low Ca.D., high levels of the serum 1,25-(OH)2D were seen compared with wt counterparts. Data are expressed as mean (bars) + SD (error bars). B, Effects of dietary treatments on the growth of mutant mice. The body weights of kl-/- mice and wt mice were measured after the above treatments. kl-/- mice showed growth retardation and body weights of less than 10 g on N.D. at 7 wk [7w. (N.D)]. Mutant mice on -D.D. reached body weights over 20 g at 7 wk [7w. (-D.D)]. Wt on -D.D. had growth rates similar to animals on N.D. kl-/- mice fed with low P.D. (7w. low P.D.) as well as low Ca.D. (7w. low Ca.D.) were in the similar range of body weight at 7 wk as those fed with N.D. Data are expressed as mean (bars) + SD (error bars). C–R, Histology of kl-/- and wt animals after dietary treatments. Sections were stained with Von Kossa’s method to visualize abnormal deposit of calcium in kidneys of kl-/- mice (left panels; C, G, K, and O) and wt mice (D, H, L, and P). The accumulation of calcium, which was one of the major phenotypes in kl-/- mice was observed as dark brown spots (C). Tibia of kl-/- mice (right panels; E, I, M, and Q) and wt (F, J, N, and R) were stained with hematoxylin and eosin. Areas with abnormal calcium deposits typical to klotho mutants were observed in epiphysis as purple staining around the cartilage (E with higher magnification). These are the identical phenotypes observed in the original kl/kl mutant (1 ). C–F show the histology of animals fed with N.D., G–J with -D.D., K–N with low P.D., and O–R with low Ca.D., respectively. Ectopic calcification was not seen in the kidneys of kl-/- mutants raised on -D.D. at 7 wk of age in both kidney and bones (G and I). No histological differences were observed in kidneys of wt fed N.D. (D and F) or -D.D. (H and J). The calcium accumulation could not be alleviated in kl-/- mice fed with low P.D. in either kidneys (K) or cartilage (M) but were not seen in mutants raised on low Ca.D. in kidneys (O) and cartilages (Q). The kidneys of wt fed with low P.D. (L) or low Ca.D. (P), and cartilage based on low P.D. (N) or low Ca.D. (R) did not display abnormalities. Bar, 100 µm. The summary of phenotypes observed in the kl-/- mutants after the dietary treatment is shown on the table.

 
We then examined tissues of null mutant animals fed with -D.D. As previously described (1), significant calcification was observed in the renal cortices at 7 wk of age in kl-/- mice on N.D. (Fig. 1CGo) in contrast to wt counterparts (Fig. 1DGo). However, kl-/- mice on -D.D. had little or no calcium accumulation even after 7 wk (Fig. 1GGo). Histological examination of bones also revealed the absence of the characteristic staining corresponding to abnormal calcium deposits in kl-/- mice with reduced serum 1,25-(OH)2D (Fig. 1Go, E and I). Abnormalities were not observed in wt fed with -D.D. (Fig. 1Go, F and J). We also observed the lack of skin atrophy, arteriosclerosis and ectopic calcification in other tissues (data not shown). We searched for the remaining phenotypes after vitamin D restriction and failed to find any apparent histological abnormalities in these mice. It was possible that the low serum levels of calcium and phosphorus (8.43 ± 1.14 mg/dl and 8.9 ± 1.14 mg/dl, respectively) had an effect on the phenotypes in kl-/- mice fed with -D.D. However, we did not observe rescue of the phenotypes in kl-/- mice on basal diets used in the control experiments which contain the same levels of calcium and phosphorus as -D.D. (data not shown). We also tested the effects of low-phosphorus diets (low P.D.) (0.2% phosphorus) and low-calcium diets (low Ca.D.) (0.02% calcium). In 7-wk-old mice on low P.D., the levels of serum phosphate (7.96 ± 0.92 mg/dl vs. 8.66 ± 0.68 mg/dl in wt and kl-/- mice, respectively) were significantly decreased, but body weights were similar to that of mutants raised on N.D. (Fig. 1BGo, lanes 2 and 4), and histological abnormalities were not rescued (Fig. 1Go, K and M). kl-/-mutants fed with low Ca.D. did not exhibit the ectopic calcification in the kidney (Fig. 1OGo) nor the abnormal deposition of calcium in the cartilage (Fig. 1QGo). The levels of serum calcium (5.94 ± 0.51 mg/dl vs. 6.2 ± 0.55 mg/dl in wt and kl-/- mice, respectively) were surely decreased. However, this treatment did not have a clear effect on the growth rate of the mutants (Fig. 1BGo, lane 5). Thus reduction of dietary calcium could only partially rescue the phenotype of klotho null mutants. These suggest that the defects in calcium and phosphorus homeostasis in kl-/- mutants are mainly due to the abnormal activation of vitamin D in the absence of klotho.

Renal 1{alpha}-Hydroxylase Responds Normally to PTH, CT, and 1,25-(OH)2D3 in the Mutant
Previously, we found that the serum levels of 25-(OH)D and 24,25-(OH)2D were lower and 1,25-(OH)2D was higher in the kl/kl mutant (5). This abnormal activation of 1,25-(OH)2D may result from improper expression of enzymes involved in vitamin D metabolism. We therefore measured the levels of renal 1{alpha}-hydroxylase and 24-hydroxylase transcripts by means of Northern blot analysis. As was seen in kl/kl mutants (5), the levels of 1{alpha}-hydroxylase transcripts were also significantly increased in kl-/- mice in comparison with those of wt mice (Fig. 2AGo, lanes 1 and 8). Several factors are known to be involved in the regulation of 1{alpha}-hydroxylase gene expression (8, 9, 10, 11, 12, 13, 14). For instance, PTH and CT positively regulate the synthesis of 1,25-(OH)2D via transcriptional activation of the 1{alpha}-hydroxylase gene (15, 16, 17, 18, 19). On the other hand, 1,25-(OH)2D3 inhibits its own synthesis by the negative feedback regulation of 1{alpha}-hydroxylase activity and up-regulation of 24-hydroxylase activity (14, 16, 20). In kl-/- mice, the serum concentrations of CT were slightly higher and PTH lower, and levels of 1,25-(OH)2D were significantly higher than those of wt mice. A possible explanation for the abnormal up-regulation of 1{alpha}-hydroxylase gene expression in kl-/- mice may be that it results from an abnormal response to these known factors. We tested this possibility by comparing gene expression in mutant and wt mice in response to the administration of these hormones. Because kl-/- mutants start to exhibit morphological phenotypes with apparent damage to the kidney after the age of 4 wk, we used younger animals in which kidney cells still appeared intact to test the response to the administration of these three hormones (Fig. 2Go). In kl-/- mice that received only CT or PTH injections, a significant increase in 1{alpha}-hydroxylase mRNA level was seen (Fig. 2AGo, lanes 2 and 3). When PTH and CT were coinjected, an additive effect was observed on 1{alpha}-hydroxylase gene induction in both kl-/- (lane 4) and wt mice (lane 11). When 1,25-(OH)2D3 was given, 1{alpha}-hydroxylase transcript levels decreased slightly in kl-/- mice (lane 5). Furthermore, in kl-/- mice given 1,25-(OH)2D3 in addition to CT or PTH, an apparent decrease in 1{alpha}-hydroxylase transcript levels was seen, compared with injections of CT or PTH alone (lanes 2 and 6, and 3 and 7, respectively). These responses were similar in wt mice (lane 8–14), although the degree of change was greater in wt mice than in kl-/- mice. We also measured levels of the 24-hydroxylase transcripts (Fig. 2BGo). In kl-/- mice, basal levels were significantly higher in comparison with those of wt mice (lanes 1 and 8). The elevated levels of 24-hydroxylase were consistent with the higher levels of serum 1,25-(OH)2D (Fig. 1AGo). After 1,25-(OH)2D3 administration, 24-hydroxylase transcripts increased in both kl-/- and wt mice (lanes 5 and 12), indicating that the positive regulation of 24-hydroxylase via 1,25-(OH)2D3 was conserved in young kl-/- mice despite the higher basal levels.



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  		Fig. 2. The Conservation of Responses to Known Regulators of Renal 1{alpha}-Hydroxylase and 24-Hydroxyase Expression in the Mutant

Northern blot analysis probed with 1{alpha}-hydroxylase and 24-hydroxylase cDNA fragment (top panel) and mouse G3PDH (bottom panel). More than three experiments for each group were calculated and shown as a histogram. The relative signal intensity of 1{alpha}-hydroxylase or 24-hydroxylase vs. G3PDH in nontreated wild-type was used as the standard, which was set as one. A, The change in 1{alpha}-hydroxylase. kl-/- mice showed enhanced expression of 1{alpha}-hydroxylase without any treatment (NP.). After the injection of CT or PTH (shown as CT and PTH, respectively), 1{alpha}-hydroxylase gene expression was increased in both kl-/- and wt mice. After injection with both PTH and CT (PTH + CT), the expression was significantly elevated compared with injection of a single hormone. In both genotypes, injection of 1,25-(OH)2D3 (1,25D3) slightly reduced the expression of 1{alpha}-hydroxylase and inhibited the inductive signals of CT or PTH (shown as CT+1,25D3, PTH+1,25D3, respectively). In the absence of treatment, expression is higher in kl-/- kidneys than in wt. 1,25-(OH)2D3 enhances expression, and this induction is suppressed by CT and PTH in both genotypes. Effects of 1,25-(OH)2D3 manipulation on the expression of 1{alpha}-hydroxylase (C) and 24-hydroxylase (D) in kidney. Reducing serum concentrations of 1,25-(OH)2D with -D.D. (-D) resulted in the elevation of 1{alpha}-hydroxylase gene expression in both genotypes. After injection of 1,25-(OH)2D3 to animals on -D.D. [-D(1,25 D3)], the expression of 1{alpha}-hydroxylase was significantly decreased both in kl-/- and wt mice. Switching from a -D.D. (-D) to vitamin D-enriched (+D) diet also reduced 1{alpha}-hydroxylase expression (-D/+D). These responses were conserved in kl-/- mice although expression levels were higher than all other situations. The responses of 24-hydroxylase gene expression to the change in 1,25-(OH)2D3 were similar in kl-/- and wt (D). Data are expressed as mean (bars) + SD (error bars).

 
We further examined the effect on the vitamin D signaling pathway with mice on -D.D. (Fig. 2Go, C and D). Switching from N.D. to -D.D. resulted in a remarkable decrease in serum concentrations of 1,25-(OH)2D in kl-/- mice (Fig. 1Go). Mice were treated with -D.D. for 7 wk after birth, and kidney RNAs were examined by Northern blot analyses. The levels of 1{alpha}-hydroxylase transcripts increased in kl-/- mice (Fig. 2CGo, lanes 1 and 2) and wt mice (lanes 5 and 6). However, the levels of 24-hydroxylase transcripts decreased (Fig. 2DGo). These results revealed that kl-/- mice can respond normally to a decrease in serum 1,25-(OH)2D concentration. We then checked whether the response to 1,25-(OH)2D3 administration was conserved in mice on -D.D. Injection of 1,25-(OH)2D3 resulted in reduction of 1{alpha}-hydroxylase mRNA in both kl-/- and wt mice (Fig. 2CGo, lanes 3 and 7). Switching from a -D.D. to a vitamin D-enriched diet (-D/+D) resulted in a significant decrease in the expression of 1{alpha}-hydroxylase in kl-/- and wt mice (lanes 4 and 8). However, after the administration of 1,25-(OH)2D3, the level of 1{alpha}-hydroxylase mRNA in kl-/- mice remained similar to that in mice on N.D. The 1,25-(OH)2D3 loading resulted in elevated 24-hydroxylase gene expression in both mutant and wt mice (Fig. 2DGo). According to these results, we concluded that the positive and negative responses to serum 1,25-(OH)2D concentration changes were essentially intact in kl-/- mice. However, the 1{alpha}-hydroxylase mRNA levels in kl-/- mice were always higher than that of wt mice, suggesting the lack of a putative negative regulator of 1{alpha}-hydroxylase gene expression. Previously, we reported that the response of VDR and 24-hydroxylase gene expression to the administered 1,25-(OH)2D3 was impaired in kl/kl mice after oral administration at 7 wk (5). To resolve the apparent discrepancy in results we carried out additional experiments using kl-/- mice. Expression of VDR, 24-hydroxylase and 1{alpha}-hydroxylase was examined by Northern blotting after treatment (Fig. 3Go). kl-/- mice displayed enhanced expression of 1{alpha}-hydroxylase, 24-hydroxylase and VDR in the kidney without any treatment (NP.) at the age of 3 wk. After ip injection of 1,25-(OH)2D3 to mutants at the age of 3 wk, we observed significant down-regulation of 1{alpha}-hydroxylase transcripts, and up-regulation of VDR and 24-hydroxylase transcripts. In animals treated similarly at 7 wk of age, these responses were less than that seen at 3 wk. We also checked the genetic responses after oral administration of 1,25-(OH)2D3 (Fig. 3Go, D–F). After 1,25-(OH)2D3 was orally administered to 3-wk-old kl-/- mice, kidney 1{alpha}-hydroxylase mRNA was down-regulated (Fig. 3DGo, lane 2), and 24-hydoxylase and VDR mRNAs were up-regulated [Fig. 3Go, E (lane 2) and F (lane 2)] similar to wt mice. This was consistent with the regulation after ip administration. These results suggest that the younger klotho mutants without morphological abnormalities are more sensitive to the administration of 1,25-(OH)2D3 than older mice in which the phenotypes are manifested.



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  		Fig. 3. Changes in Gene Expression in the Kidney

Kidneys of kl-/- and wt were subjected to Northern blot analysis with 1{alpha}-hydroxylase (A), 24-hydroxyase (B), VDR (C) (top panel), and mouse G3PDH (bottom panel) probes. After the ip administration of 1,25-(OH)2D3, at 3 wk, the expression of 1{alpha}-hydroxylase gene was significantly decreased (lanes 3 and 4); however, the degree of the depression was smaller at 7 wk (lanes 1 and 2). B, Effects of 1,25-(OH)2D3 administration on the gene expression of 24-hydroxylase. Basal levels of transcript were higher in mutants both at 3 wk and 7 wk compared with wt (lanes 1 and 5, 3 and 7). After injection of 1,25-(OH)2D3 to mutants at the age of 3 wk, the induction of 24-hydroxylase gene was observed in mutants (lane 4) and wt (lane 8). On the other hand, this induction was impaired in mutants at the age of 7 wk (lane 2). C, kl-/- mice displayed enhanced expression of VDR without any treatment at 3 wk (NP., lane 3) compared with wt (lane 7). After the ip injection of 1,25-(OH)2D3 at 3 wk, the expression of VDR gene was significantly induced (lane 4). This induction was not seen at 7 wk (lane 2). D–F, Expression of 1{alpha}-hydroxylase (D), 24-hydroxylase (E), and VDR (F) after the oral administration of 1,25-(OH)2D3. After 1,25-(OH)2D3 was orally administered, 1{alpha}-hydroxylase mRNA of kidney was down-regulated, but 24-hydroxylase and VDR mRNA of kidney were up-regulated in kl-/- mice (lanes 1 and 2) similarly to wt mice (lanes 3 and 4). These were consistent with the regulation after ip administration. Data are expressed as mean (bars) + SD (error bars).

 
Judging from these results, the responses to general vitamin D signaling pathways were conserved in klotho-deficient states, but the expression levels of renal 1{alpha}-hydroxylase in kl-/- mice were severalfold higher than that of wt mice under all tested experimental conditions. This suggests the involvement of klotho in a signaling pathway that restricts the serum concentration of 1,25-(OH)2D through transcriptional regulation of vitamin D-activating enzymes.

Positive Correlation of Gene Expression Profiles among Klotho, 24-Hydroxylase, and VDR after the Administration of 1,25-(OH)2D3
To test whether klotho is regulated by vitamin D signaling, we next examined the effect of 1,25-(OH)2D3 levels on klotho gene expression in wt (Fig. 4Go). We modified serum 1,25-(OH)2D levels by various methods. When 1,25-(OH)2D3 was injected in 3-wk-old mice on N.D. (Fig. 4AGo, lane 2), the expression of klotho increased. In 7-wk-old mice kept on a -D.D., klotho expression was slightly decreased (Fig. 4AGo, lane 3). Conversely, when the diet was switched from -D.D. to vitamin D-enriched, expression levels of klotho were elevated (Fig. 4AGo, lane 4). Similar enhancement was observed in mice injected with 1,25-(OH)2D3 after treatment with -D.D. (Fig. 4AGo, lane 5). Thus, the levels of klotho transcripts changed in accordance with the serum 1,25-(OH)2D levels. Next, we examined the time course of klotho gene induction after 1,25-(OH)2D3 administration in 3-wk-old wt on N.D. (Fig. 4BGo). We killed mice and collected kidneys at 6, 8, 12, and 24 h post injection of 1,25-(OH)2D3 to analyze the expression of klotho, 24-hydroxylase, and VDR (Fig. 4Go, B–D). Klotho expression was up-regulated and reached maximal levels 8 h after administration of 1,25-(OH)2D3. The peak levels of 24-hydroxylase and VDR gene expression were also seen at 8 h post injection. The time course of klotho gene expression after 1,25-(OH)2D3 administration resembled those of the 24-hydroxylase and VDR genes, although the relative degrees of induction differed.



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  		Fig. 4. 1,25-(OH)2D3 Up-Regulates Renal Klotho Expression

The expression of klotho transcripts in kidney was analyzed by Northern blotting. A, The effects of vitamin D on the induction of klotho in wt. 1,25-(OH)2D3 (1,25D3) administration to mice on N.D. caused elevation of klotho gene expression compared with nontreated mice (NP.). Removal of 1,25-(OH)2D3 from the diet led to decreased klotho gene expression (-D). Switching from -D.D. to vitamin D-enriched diets (-D/+D) significantly increased the level of klotho transcripts. Klotho expression also increased after the injection of 1,25-(OH)2D3 in mice fed with -D.D. [-D(1,25D3)]. Klotho (B), 24-hydroxylase (C), and VDR (D) mRNAs were analyzed by Northern blotting in 3-wk-old wt on N.D. Kidneys were isolated at 6, 8, 12, and 24 h after the administration of 1,25-(OH)2D3. The levels of klotho transcripts were increased by 1,25-(OH)2D3, peaked at 8 h and returned to basal levels by 24 h. The maximum level of 24-hydroxylase expression was seen at 8 h. VDR levels stimulated by 1,25-(OH)2D3 also peaked at 8 h. Data are expressed as mean (bars) + SD (error bars).

 
The Different Localization Patterns of Cells that Express Klotho, 1{alpha}-Hydroxylase, or 24-Hydroxylase
We next examined the localization of klotho, 1{alpha}-hydroxylase, and 24-hydroxylase transcripts in kidneys to determine whether they were coexpressed (Fig. 5Go). We processed kidneys of 3-wk-old wt on N.D. for in situ hybridization. As previously reported (9, 21), 1{alpha}-hydroxylase was broadly expressed in the uriniferous tubule cells. In contrast, klotho expression was restricted to the distal convoluted tubule cells adjacent to the renal cortex. A population of cells which expressed 1{alpha}-hydroxylase overlapped with those that expressed 24-hydroxylase mRNA. The cells that expressed klotho mRNA were distinct from those expressing 1{alpha}-hydroxylase. These results suggest that the effect of klotho on the regulation of 1{alpha}-hydroxylase is not cell autonomous.



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  		Fig. 5. The Localization of Klotho, 1{alpha}-Hydroxylase, and 24-Hydroxylase mRNAs in Wild-Type Kidneys

A, In 3-wk-old mice on N.D., klotho gene expression was restricted to the distal convoluted tubule cells in the renal cortex. B, 1{alpha}-Hydroxylase was broadly expressed in the uriniferous tubule cells. C, Klotho-expressing cells were distinct from those that expressed 1{alpha}-hydroxylase and 24-hydroxylase in a partially overlapping manner. Sense probes gave rise to no staining (data not shown). Bar, 100 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The klotho mutant mice showed impaired calcium and phosphorus homeostasis together with increased serum 1,25-(OH)2D (5). As previously described, serum levels of PTH in kl/kl mice were lower than those of wt mice, and serum levels of CT in kl/kl mice were slightly higher than those of wt mice. Serum levels of PTH and CT correlate with increased levels of serum calcium in kl/kl mice (5), suggesting that the signaling pathway for PTH and CT synthesis can be normally controlled despite loss of klotho function. However, 1,25-(OH)2D levels in kl/kl mice were significantly higher than that of wt mice at all ages examined (5). This is contrary to the normal response to high serum calcium where 1,25-(OH)2D synthesis should be down-regulated when serum calcium levels are increased.

Because the original klotho mutant (kl/kl) was a hypomorph, in this paper we used a recently established klotho null mutant (kl-/-) (Fujimori, T., K. Takeshita, Y. Kurotaki, H. Honjo, H. Tsujikawa, K. Yasui, J.-H. Lee, K. Kamiya, K. Kitaichi, K. Yamamoto, M. Ito, T. Kondo, S. Iino, Y. Inden, M. Hirai, T. Murohara, I. Kodama, and Y.-i. Nabeshima, manuscript submitted) that displays identical phenotypes as the original klotho mice (kl/kl). We effectively lowered serum 1,25-(OH)2D in kl-/- mice by means of -D.D. Most of the abnormalities, including growth rate, ectopic calcification, altered serum markers, and fertility were rescued in kl-/- mice under these conditions. The life spans were also increased. Similar results were also obtained using the original mutant strain (data not shown). Restriction of only calcium in the diet also alleviated abnormal calcification in mutants, but normal growth rates were not recovered. Low P.D. showed no clear effect on the phenotypes of kl-/- mice. It is reported that the low P.D. in males and low phosphate and zinc supplement in females could rescue the phenotypes of kl/kl mutant (6). We also tested the same treatment and it did not show any changes in the null mutant (data not shown). Although we do not have clear explanation, these differences might be due to the leaky expression of klotho observed in the k//kl mice. Taken together, these suggest that high levels of 1,25-(OH)2D might be a primary cause, and may lead to increased calcium intake in the mutant. Our studies revealed that high levels of serum 1,25-(OH)2D are a major cause of the premature aging syndrome seen in klotho-deficient mice. The lowered levels of 25-(OH)D and 24,25-(OH)2D in klotho-deficient mice on N.D. suggest that the precursor is preferentially converted to an active form of vitamin D in kl-/- mice and this unbalanced activation may lead to the high levels of 1,25-(OH)2D. Loss of klotho resulted in abnormal activation of vitamin D, suggesting that klotho participates in a negative regulatory circuit of active vitamin D synthesis (Fig. 6Go). The expression of 1{alpha}-hydroxylase, the key enzyme in 1,25-(OH)2D synthesis, was enhanced in kidneys of kl-/- mice despite high levels of serum 1,25-(OH)2D. This up-regulation may lead to the abnormal activation of vitamin D. We then focused on the abnormal regulation of enzymes involved in vitamin D metabolism. We searched for the signaling pathway regulating 1,25-(OH)2D where klotho was involved.



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  		Fig. 6. Model for the Role of Klotho in the Vitamin D Feedback Loop

Loss of klotho in mice results in the abnormal elevation of 1{alpha}-hydroxylase gene expression, but normal responses to known regulators, such as 1,25-(OH)2D, PTH, and CT are intact, suggesting that signaling by these regulators are independent of klotho. Klotho expression is up-regulated by 1,25-(OH)2D, suggesting that klotho participates in a vitamin D feedback loop. We propose a novel negative regulatory circuit for the regulation of vitamin D activity that involves klotho function.

 
It is known that PTH and CT positively regulate the expression of 1{alpha}-hydroxylase (15, 16, 17, 18, 19), and that 1,25-(OH)2D3 negatively regulates the expression of 1{alpha}-hydroxylase (14, 20) and can dominantly offset the positive signals from PTH and CT. kl-/- mutants displayed normal responses to administered CT, PTH, and 1,25-(OH)2D3, indicating that the loss of Klotho function did not abolish the responses to known regulatory signals required for 1,25-(OH)2D synthesis, at least in early stages up to 3 wk. We further checked the expression of the 1{alpha}-hydroxylase gene in response to the lowered levels of 1,25-(OH)2D in mice on -D.D. The mutants had elevated expression, similar to wt and consistent with previous reports. The response of 24-hydroxylase to these calcium-regulating hormones in kl-/- mice was also in accordance with previous data (10, 15). According to these results, the signaling pathway from serum PTH, CT, and 1,25-(OH)2D for regulation of 1{alpha}-hydroxylase and 24-hydroxylase is conserved in kl-/- mutants despite their high 1,25-(OH)2D serum levels. In a previous study (5), we reported impaired induction of gene expression after the administration of 1,25-(OH)2D3 in the kl/kl mutants. The main difference between the present and our previous paper is the age of the mice used. We observed reduced expression of VDR gene/protein and the response to administered 1,25-(OH)2D3, such as VDR and 24-hydroxylase gene expression, was also reduced in 7-wk-old null (kl-/-) kidneys as observed in kl/kl mice. Normal responses to 1,25-(OH)2 D3 in both kl/kl and kl-/- mice were however observed at the age of 3 wk. In these younger animals, these responses were seen even after the oral administration of 1,25-(OH)2D3. This revealed that the pattern of impaired response is conserved between the two mutant lines. Although we do not have a clear explanation for this stage-dependent difference, it is possible that constant exposure to high levels of 1,25-(OH)2D from early stages of development may result in an increased threshold or desensitization to administered 1,25-(OH)2D3 by the age of 7 wk. VDR protein levels were significantly reduced in the mutant kidney at 7 wk (5), although the transcript was slightly reduced. This reduction of VDR protein might be a cause of impaired responses at later stages. It is also impossible to exclude the unfavorable effects of tissue damage and the loss of cells for the impaired response to administered 1,25-(OH)2D3. Indeed, both kl-/- and kl/kl homozygous mutants older than 4 wk display morphological abnormalities. Ectopic calcification becomes apparent in the kidney from 4 wk, portions of the renal tubules are progressively damaged and some tubular cell loss occurs. In this paper, we wished to focus on the primary mechanisms involving Klotho rather than more indirect downstream events. Thus, we used kl-/- mice fed on N.D. at the age of 3 wk in which the characteristic renal damage was not yet apparent. In addition, kl-/- mice were fed with -D.D. to eliminate the effects of high concentrations of 1,25-(OH)2D. The influence of the slight difference in genetic backgrounds on the response to 1,25-(OH)2D3 cannot be completely excluded, but to date, there is no evidence to support that this is the case.

Although the mutants displayed normal responses to all treatments tested, the expression of 1{alpha}-hydroxylase was persistently higher in kl-/- mutants. There is other evidence (8, 12, 13, 14) to take into consideration. That is, other endocrine signals such as estrogen, GH, IGF, and glucocorticoids up-regulate 1{alpha}-hydroxylase. However, the pituitary was atrophic in kl-/- mice, and GH was within the normal range, as is expected for IGF. The serum concentrations of glucocorticoids in kl-/- mice were also lower than that of wt mice (data not shown). Thus, we speculate that other known inducers of 1{alpha}-hydroxylase are also normally maintained in kl-/- mice. These suggest that the elevation of 1{alpha}-hydroxylase in kl-/- mice is mediated by a yet uncharacterized system. Klotho may support the function of inhibitory signals on 1{alpha}-hydroxylase expression or it may suppress activator(s) of 1{alpha}-hydroxylase expression.

Because localization patterns of cells which express klotho and 1{alpha}-hydroxylase was different, klotho-dependent changes in 1{alpha}-hydroxylase expression should not be cell autonomous, and intercellular or diffusable mechanisms are suggested to mediate Klotho function. The after two mechanisms are possible explanations for the actions of Klotho. The first is that Klotho proteins might function in modifying the structure of a ligand or a receptor that is involved in an unknown circuit for 1{alpha}-hydroxylase gene regulation. The presence of tandem repeats that share homology to ß-glycosidase (1) suggests that Klotho might possess enzymatic properties to modify the activity of a ligand or a receptor through enzymatic digestion of its sugar moiety. Importantly, the enzymatic action of Klotho has been suggested in our recent biochemical study of Klotho (Tohyama, O., and Y.-i. Nabeshima, unpublished data). Another possible mechanism is that Klotho works as a secreted signaling factor that mediates the regulatory signals. As a matter of fact, we have found that Klotho is secreted into the extracellular spaces and detectable in serum (Imura, A., and Y.-i. Nabeshima, unpublished data).

Another important finding in this report is that the expression of klotho is induced by the administration of 1,25-(OH)2D3 (Fig. 4Go). Although the induced levels were relatively lower than that of 24-hydroxylase, the time course of the induction was very similar to that of 24-hydroxylase and VDR, so-called vitamin D-responding genes (15). Thus, klotho might play an important role in the feedback loop to regulate the levels of 1,25-(OH)2D.

The next question is whether the abnormalities observed in kl-/- mice are solely dependent on the increased levels of calcium, phosphorus and 1,25-(OH)2D, or the combination of the increased serum levels and the deficiency of Klotho protein. If the latter is true, Klotho may play another role in addition to that as a regulator of calcium homeostasis. The deficiency of Klotho protein may trigger a morphological and functional deterioration of cells and tissues, which causes subsequent severe tissue damage together with the toxic action of increased calcium, phosphorus, and 1,25-(OH)2D in serum.

Because klotho homologs have been reported in rat and human (2, 3), one can speculate that the function of Klotho is also conserved between species. In view of human hypervitaminosis D, vitamin D metabolic profiles in these patients (22, 23, 24, 25, 26), especially children, have revealed massively elevated 25-(OH)D, 24,25-(OH)2D values, whereas 1,25-(OH)2D values were not elevated. These reports contrast with the situation in kl-/- mice. This predicts the existence of distinct and discrete types of vitamin D regulatory defects. Our finding suggests that a negative regulatory circuit related to klotho may exist for 1,25-(OH)2D synthesis. The molecular mechanisms underlying this pathway will be targets of future studies. Approaches analyzing the function of Klotho should contribute toward our understanding of this circuit and offer potential medical applications for the regulation of vitamin D metabolism and calcium homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals, Diets, and Administration of Hormones
A new strain of klotho null mutant mice (kl-/-) was used for all experiments in this report because the original mutant (kl/kl) was not a null but a severe hypomorph, which was reported to be affected by dietary phosphorus (6). A new knockout allele with a deletion in exon1 was established by homologous recombination in ES cells. Details will be described elsewhere (Fujimori, T., K. Takeshita, Y. Kurotaki, H. Honjo, H. Tsujikawa, K. Yasui, J.-H. Lee, K. Kamiya, K. Kitaichi, K. Yamamoto, M. Ito, T. Kondo, S. Iino, Y. Inden, M. Hirai, T. Murohara, I. Kodama, and Y.-i. Nabeshima, manuscript submitted). We could not detect Klotho protein in the homozygous mutant animal (kl-/-), indicating this is a null mutation. Although the genetic backgrounds were slightly different (heterozygous mice from chimeras derived from 129 recombinant ES cells were maintained by crossing to C57BL/6 mice compared with the original mutant (kl/kl) that had a mixed background of C57BL/6 and C3H), the phenotypes observed in mice with this new mutant allele were basically identical to those in the original klotho mutant (kl/kl) mice (1).

N.D. for both kl-/- mice and wt (NMF, Oriental Yeast Co., Tokyo, Japan), contained 1.46% calcium, 1.09% phosphorus, and 1.5 IU/g vitamin D3 with free access to food and water. The -D.D. contained 0.6% calcium and 0.4% phosphorus (no. 5826, PMI Nutrition International, Inc., St. Louis, MO). We used the basal diet (no. 5755, PMI Nutrition International, Inc.), which is identical to no. 5826 with the exception of addition of 2.2 IU/g vitamin D as control. Vitamin D-enriched diets (PMI Nutrition International, Inc.) contained 22.2 IU/g vitamin D3 and were also based on the basal diet. Low P.D., which was a gift of Kyowa Hakko Co. Ltd. (Tokyo, Japan), contained 0.2% phosphorus, and low Ca.D. (no. 5855, PMI Nutrition International, Inc.) contained 0.02% calcium. Heterozygous females (kl+/-) crossed with heterozygous males (kl+/-) were maintained on -D.D., low P.D., and low Ca.D. after they became pregnant. And these diets were all given to mice for 7 wk after the birth including nursing period. Switch from -D.D. to vitamin D-enriched diets were done at 7 wk, and mice were maintained on vitamin D-enriched diets for additional 2 wk. We killed treated mice 4 h after an ip injection of CT [2 mg/100 g body weight (BW)], 3 h after an iv injection of PTH (1 mg/10 g BW), 5 h after an ip injection of 1,25-(OH)2D3 (0.08 nmol/10 g BW), respectively (12), and 6 h after an oral administration of 1,25-(OH)2D3, Rocaltrol (Roche, Mannheim, Germany) (200 ng/100 g BW). The animal studies were conducted in accordance with the regulations and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Measurement of Active Serum 1,25-(OH)2D in Mice
Mice were anesthetized with ether. Blood samples were collected and the serum was separated by centrifugation 7500 x g for 3 min at 4 C. Serum levels of 1,25-(OH)2D were measured by RIA (SRL Inc., Tokyo, Japan).

RNA Isolation and Northern Blot Analysis
Kidneys were isolated from killed animals and total RNA was extracted from tissues according to manufacturer’s protocols (Total RNAgents and PolyA tract; Promega Corp., Madison, WI). Twenty micrograms of total RNA were used per lane, and hybridized with 32P-deoxy-CTP random primed probes (RPN 1607, Amersham Life Science, Cleveland, OH) in hybridization buffer (ExpressHyb Hybridization Solution; CLONTECH, Palo Alto, CA) at 68 C. Detection of each mRNA was performed using a 1546-bp mouse 1{alpha}-hydroxylase cDNA fragment, a 1558-bp mouse 24-hydroxylase cDNA N-terminal fragment and a 2540-bp (region encoding most of the extracellular repeats) of mouse klotho cDNA fragment. After stripping, membranes were hybridized with mouse glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probes. The membranes were exposed to imaging plate (Fujifilm, Inc., Tokyo, Japan) overnight. The bands were visualized and signal intensities were measured using an image scanner (STORM86, Molecular Dynamics, Sunnyvale, CA). The relative abundance of transcripts was judged by G3PDH.

In Situ Hybridization
In situ hybridization was performed as described (27). Anesthetized mice were perfusion-fixed with 4% paraformaldehyde and tissues were sectioned after paraffin embedding. BM purple alkaline phosphatase substrate (Roche, Mannheim, Germany) was used to visualize the signals. The corresponding sense probes used as a negative control gave no staining (data not shown). DNA templates used for the preparation of the digoxigenin-labeled riboprobes are same as those used for northern hybridization.


   ACKNOWLEDGMENTS
 
We thank T. Obata, O. Tohyama, and Dr. A. Imura for their technical support throughout the course of this work and Dr. R. Yu for critical reading of this manuscript.


   FOOTNOTES
 
This work was supported by grants of the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim.

Abbreviations: BW, Body weight; CT, calcitonin; -D.D., vitamin D-deficient diet; -D/+D, switching from a vitamin D-deficient to a vitamin D-enriched diet; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; 1{alpha}-hydroxylase, 25-hydroxyvitamin D 1{alpha}-hydroxylase; 24-hydroxylase, 25-hydroxyvitamin D 24-hydroxylase; kl-/- mice, klotho null mutant mice; kl/kl mice, klotho mice; low Ca.D., low-calcium diet; low P.D., low-phosphorus diet; N.D., normal diet; NP., no treatment; VDR, vitamin D receptor; wt, wild-type mice.

Received for publication February 10, 2003. Accepted for publication September 18, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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Nuclear Receptors:   VDR
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