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Am J Physiol Renal Physiol 291: F1177-F1183, 2006. First published May 16, 2006; doi:10.1152/ajprenal.00038.2006
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Age-dependent alterations in Ca2+ homeostasis: role of TRPV5 and TRPV6

Monique van Abel,1 Sylvie Huybers,1 Joost G. J. Hoenderop,1 Annemiete W. C. M. van der Kemp,1 Johannes P. T. M. van Leeuwen,2 and René J. M. Bindels1

1Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen; and 2Department of Internal Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands

Submitted 1 February 2006 ; accepted in final form 8 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aging is associated with alterations in Ca2+ homeostasis, which predisposes elder people to hyperparathyroidism and osteoporosis. Intestinal Ca2+ absorption decreases with aging and, in particular, active transport of Ca2+ by the duodenum. In addition, there are age-related changes in renal Ca2+ handling. To examine age-related changes in expression of the renal and intestinal epithelial Ca2+ channels, control (TRPV5+/+) and TRPV5 knockout (TRPV5–/–) mice aged 10, 30, and 52 wk were studied. Aging of TRPV5+/+ mice resulted in a tendency toward increased renal Ca2+ excretion and significantly decreased intestinal Ca2+ absorption, which was accompanied by reduced expression of TRPV5 and TRPV6, respectively, despite increased serum 1,25(OH)2D3 levels. Similarly, in TRPV5–/– mice the existing renal Ca2+ loss was more pronounced in elder animals, whereas the compensatory intestinal Ca2+ absorption and TRPV6 expression declined with aging. In both mice strains, aging resulted in a resistance to 1,25(OH)2D3 and diminished renal vitamin D receptor mRNA levels, whereas serum Ca2+ levels remained constant. Furthermore, 52-wk-old TRPV5–/– mice showed severe hyperparathyroidism, whereas PTH levels in elder TRPV5+/+ mice remained normal. In 52-wk-old TRPV5–/– mice, serum osteocalcin levels were increased in accordance with the elevated PTH levels, suggesting an increased bone turnover in these mice. In conclusion, downregulation of TRPV5 and TRPV6 is likely involved in the impaired Ca2+ (re)absorption during aging. Moreover, TRPV5–/– mice likely develop age-related hyperparathyroidism and osteoporotic characteristics before TRPV5+/+ mice, demonstrating the importance of the epithelial Ca2+ channels in Ca2+ homeostasis.

ECaC; CaT1; VDR; PTH; 1,25(OH)2D3; aging; osteoporosis

REGULATION OF THE CA2+ ABSORPTIVE activity of the intestine and kidney is crucial for the maintenance of normal extracellular Ca2+ levels. Intestinal Ca2+ absorption and renal tubular reabsorption of filtered Ca2+ are modulated on the basis of the body's overall need for Ca2+ gain or loss. Part of the Ca2+ (re)absorption occurs through the active transcellular pathway, which can be envisaged as a three-step process. In the distal convoluted tubule (DCT) and connecting tubule (CNT) cells of the kidney, Ca2+ from the pro-urine enters the epithelial cells via the luminal Ca2+ channel TRPV5. Subsequently, Ca2+ is transported across the cell in association with the Ca2+-binding protein calbindin-D28k and is finally extruded into the bloodstream via the Na+/Ca2+-exchanger (NCX1) and the plasma membrane Ca2+-ATPase (PMCA1b) in the basolateral membrane. In enterocytes, the main proteins involved in active Ca2+ absorption are the homologous TRPV6 channel at the luminal membrane, calbindin-D9k as the Ca2+-binding protein, and only PMCA1b at the basolateral side. In general, TRPV5 seems to be the major isoform in kidney, whereas TRPV6 is ubiquitously expressed with the highest concentrations in the prostate, stomach, brain, lung, and small intestine (21, 22, 32, 38). Moreover, active Ca2+ (re)absorption is the primary target for regulation by the calciotropic hormones 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and parathyroid hormone (PTH). Also, estrogens can stimulate active Ca2+ (re)absorption in both kidney and duodenum (46).

It is well known that aging is accompanied by alterations in Ca2+ homeostasis, which predisposes older patients to certain Ca2+-related disorders, including hyperparathyroidism and osteoporosis. For instance, intestinal Ca2+ absorption decreases with aging and, in particular, active transport of Ca2+ by the duodenum (6, 14). Furthermore, there are age-related changes in renal Ca2+ handling, like reduced renal tubular function and a decline in response of the kidney to PTH during aging (7, 17, 35, 36). Renal and intestinal calbindins play an important role in active Ca2+ transport, and their expression decreases with age in parallel with the age-related decline in Ca2+ (re)absorption (2, 6, 26). In addition, the capacity of 1,25-dihydroxyvitamin D3 to stimulate Ca2+ absorption also reduces with age, whereas circulating levels of PTH rise in rats and humans (5, 12). Moreover, age-related increases in PTH levels may play an important role in bone remodeling. Bone loss occurs with aging, leading to reduced bone strength and, therefore, of osteoporotic fracture risk in the elderly (31, 36, 41).

Interestingly, TRPV5 knockout (TRPV5–/–) mice display several alterations in Ca2+ homeostasis. For instance, they are impaired in active Ca2+ reabsorption, despite enhanced vitamin D levels, causing severe hypercalciuria (23). In contrast to the excessive renal Ca2+ wasting, intestinal TRPV6 and calbindin-D9K expression and Ca2+ absorption are increased, which could function as a compensatory mechanism to maintain a normal serum Ca2+ concentration. Furthermore, TRPV5–/– mice exhibited significant disturbances in bone structure, including reduced trabecular and cortical bone thickness (23). These findings suggest an essential role for both TRPV5 and TRPV6 in Ca2+ homeostasis. Moreover, dysfunction of these channels may contribute to disturbances in Ca2+ homeostasis and have implications for age-related changes in Ca2+ metabolism.

The present study investigated the physiological role of TRPV5 and TRPV6 in the aging kidney and intestine. Our results suggest that downregulation of the renal and duodenal proteins involved in transcellular Ca2+ transport, including TRPV5 and TRPV6, are responsible for the impaired renal Ca2+ reabsorption and duodenal Ca2+ absorption in aging. Moreover, TRPV5–/– mice develop age-related hyperparathyroidism and osteoporotic characteristics earlier compared with control mice, demonstrating the importance of these epithelial Ca2+ channels in Ca2+ homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. TRPV5–/– mice were generated and genotyped as described previously (23). TRPV5–/– and wild-type mice (TRPV5+/+) were fed standard chow and given water ad libitum. Seven to nine littermates of both genotypes of different ages were used. At the age of 10, 30, and 52 wk, mice were placed in metabolic cages (Techniplast, Buguggiate, Italy) for 48 h. Mice were allowed to adapt to these cages for 1 day, and then 24-h urine samples were collected. Mice were killed, and blood, kidney, and duodenum samples were collected. The animal ethics board of the Radboud University Nijmegen approved all animal experimental procedures.

Analytic procedures. Serum and urine Ca2+ concentrations were analyzed using a colorimetric assay kit as described previously (10). Serum phosphorus levels were measured on a Hitachi autoanalyzer (Hitachi, Tokyo, Japan). Serum PTH levels were measured using mouse intact PTH ELISA kit (Immunotopics, San Clemente, CA). Serum osteocalcin and 1,25(OH)2D3 concentrations were determined by a radioimmunoassay (RIA) (50) and immunoextraction followed by quantitation by 125I-RIA (IDS, Boldon, UK) (13), respectively.

In vivo 45Ca2+ absorption assay. Ca2+ absorption was assessed by measuring serum 45Ca2+ at early time points after oral gavage. Mice were fasted overnight (12 h) before the test. Animals were hemodynamically stable under anesthesia (urethane, 1.4 mg/g body wt) during the experiment. The solution used to measure Ca2+ absorption contained 0.1 mM CaCl2, 125 mM NaCl, 17 mM Tris, and 1.8 g/l fructose and was enriched with 20 µCi 45CaCl2/ml (18 Ci/g; New England Nuclear, Newton, MA). For the oral tests, 15 µl/g body wt of this solution were administrated by gavage as described previously (49). Blood samples were obtained at 2, 4, 8, and 12 min after oral gavage, and serum (10 µl) was analyzed by liquid scintillation counting. The change in the serum Ca2+ concentration was calculated from the 45Ca2+ content of the serum samples and the specific activity of the administrated 45Ca2+.

RNA isolation and quantitative PCR. Total RNA from kidney and duodenal mucosa was isolated using TRIzol reagent (GIBCO BRL, Life Technologies, Breda, The Netherlands) according to the manufacturer's protocol. Total DNAse-treated RNA (2 µg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (GIBCO BRL) as described previously (19). Expression of TRPV5, TRPV6, calbindin-D28K, calbindin-D9K, NCX1, PMCA1b, and the vitamin D receptor (VDR) as well as mRNA levels of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT), as an endogenous control, were determined by quantitative real-time PCR on an ABI Prism 7700 Sequence Detection System (PE Biosystems, Rotkreuz, Switzerland). The following sequences for mouse VDR primers and probe were used: forward, 5'-AATGGAGATTGCCGCATCAC-3'; reverse, 5'-TGTCCACGCAGCGTTTGA-3'; probe, 5'-AGGACAACCGGCGACACT GCCA-3'. The sequences of other target genes used are as described previously (45, 46).

Immunohistochemistry. Kidney tissue was cut into pieces, placed in 1% (wt/vol) periodate-lysine-paraformaldehyde fixative for 2 h at room temperature, and incubated overnight at 4°C in PBS containing 15% (wt/vol) sucrose. Subsequently, kidney tissue was frozen in liquid nitrogen and 7-µm sections were cut for the staining procedure. For detection of TRPV5 abundance, kidney sections were stained with guinea pig anti-TRPV5 antiserum (1:50) (19). To visualize TRPV5, sections were stained with goat anti-guinea pig Alexa 488-conjugated anti-IgG (1:300, Sigma). To quantify TRPV5 protein expression, digital images of the kidney sections were taken with a Zeiss Axioskop microscope (Thornwood, NY), and the integrated optical density was measured by computer analysis with Image-Pro Plus version 3.0 software (Media Cybernetics, Silver Spring, MD).

Immunoblotting. For protein analysis, frozen kidney tissue was homogenized in ice-cold solubilization buffer as previously described (48). Total protein fractions (10 µg) were separated on 12% (wt/vol) SDS-PAGE gels and blotted to polyvinylidene difluoride-nitrocellulose membranes (Immobilon-P, Millipore, Bedford, MA). Blots were incubated with rabbit anti-calbindin-D28K (1:10,000) (9) or mouse anti-beta-actin (1:25,000, Sigma) and thereafter with peroxidase-conjugated goat anti-rabbit/mouse antibody (1:2,000, Sigma). Immunoreactive protein was detected using the enhanced chemiluminescence method as described by the manufacturer (Amersham, Buckinghamshire, UK). Protein expression was quantified by computer-assisted densitometry with the use of Image-Pro Plus version 3.0 software (Media Cybernetics).

Statistical analysis. Values are expressed as means ± SE. Statistical significance of differences between groups was determined by ANOVA followed by pairwise comparisons using the method of least significant difference. Differences in means with P < 0.05 were considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Serum parameters. No significant differences were observed in body weight between TRPV5–/– and TRPV5+/+ mice, except at the age of 52 wk TRPV5+/+ mice have an increased body weight compared with TRPV5–/– mice. With advancing age, body weight increased significantly (Table 1). Serum Ca2+ concentrations were slightly elevated in TRPV5–/– mice compared with TRPV5+/+ mice but did not significantly change with increasing age in either strain. In addition, serum PTH levels were not different between TRPV5–/– and TRPV5+/+ mice, except for a substantial increase in 52-wk-old TRPV5–/– mice. Levels of serum phosphorus were higher in 30-wk-old TRPV5–/– mice compared with all other groups (Table 1). Circulating levels of 1,25(OH)2D3 were significantly elevated in TRPV5–/– mice compared with TRPV5+/+ mice at 10 wk of age. Interestingly, in TRPV5+/+ mice, serum 1,25(OH)2D3 concentrations increased with aging, whereas in TRPV5–/– mice, levels remained constantly elevated (Table 1). Serum concentrations of the bone turnover marker osteocalcin were significantly higher in TRPV5–/– mice compared with TRPV5+/+ mice and decreased in both mice strains with age. Moreover, osteocalcin levels rose again in 52-wk-old TRPV5–/– mice (Table 1).


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  		Table 1. Effect of age on serum parameters in TRPV5+/+ and TRPV5–/– mice

 
Aging results in increased renal Ca2+ excretion and decreased expression of Ca2+ transport proteins. To investigate the effect of aging on renal Ca2+ handling, mice were placed in metabolic cages and 24-h urine samples were collected to determine the amount of Ca2+ excretion. Urinary Ca2+ loss in TRPV5–/– mice was significantly higher compared with TRPV5+/+ mice. Moreover, Ca2+ excretion increased during aging. Similarly, in TRPV5+/+ mice there was a tendency toward elevated Ca2+ excretion with increasing age (Fig. 1A). Furthermore, the expression of genes encoding the Ca2+ transport proteins involved in transcellular Ca2+ reabsorption was examined using quantitative real-time PCR. In TRPV5+/+ mice, a decline in TRPV5 mRNA expression was observed with increasing age (Fig. 1B). In addition, a similar reduction in calbindin-D28K expression was detected in TRPV5+/+ mice during aging, whereas the expression was significantly decreased in TRPV5–/– mice independent of age (Fig. 1D). Expression of the extrusion protein NCX1 was also lower in TRPV5–/– mice, but no changes were measured in either TRPV5–/– or TRPV5+/+ mice with increasing age (Fig. 1E). No differences in TRPV6 mRNA expression were observed between TRPV5–/– and TRPV5+/+ mice with aging, except for a remarkable upregulation in 52-wk-old TRPV5–/– mice (Fig. 1C). Furthermore, VDR mRNA levels were significantly lower in TRPV5–/– mice compared with TRPV5+/+ mice. During aging, expression of VDR was decreased in both groups of mice (Fig. 1F).


Figure 1
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  		Fig. 1. Effect of aging on renal Ca2+ excretion and mRNA expression levels of genes encoding renal Ca2+ transport proteins. A: total excretion of Ca2+ in 24-h urine samples from 10-, 30-, and 52-wk-old TRPV5+/+ (filled bars) and TRPV5–/– (open bars) mice. Using real-time quantitative PCR, renal mRNA expression of TRPV5 (B), TRPV6 (C), calbindin-D28K (D), Na+/Ca2+-exchanger (NCX1; E), and vitamin D receptor (VDR; F) of the different experimental groups was measured and presented as a ratio to hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression (n = 7–9). Values are means ± SE; n = 9 mice, 3 mice/cage. *P < 0.05 vs. all. #P < 0.05 vs. TRPV5+/+. {dagger}P < 0.05 vs. TRPV5–/–. {ddagger}P < 0.05 vs. 10-wk-old TRPV5+/+.

 
Subsequently, examination of renal Ca2+ transport protein expression by immunohistochemistry revealed a marked decrease in TRPV5 protein abundance with increasing age, as indicated by the reduced immunopositive staining (Fig. 2A). The corresponding integrated optical density analysis confirmed a significant decline in TRPV5 protein abundance (Fig. 2B). Furthermore, Western blot analysis of calbindin-D28K demonstrated decreased protein abundance in TRPV5–/– mice compared with the TRPV5+/+ mice. In addition, a downregulation of calbindin-D28K abundance in 52-wk-old TRPV5+/+ mice was observed compared with younger wild-type animals (Fig. 2C). Densitometrical analysis of the intensity of the immunocomplexes confirmed this decrease in calbindin-D28K protein expression with age in TRPV5+/+ mice (Fig. 2D). The corresponding beta-actin bands did not vary significantly in density, which precludes unequal loading as an explanation for the differences.


Figure 2
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  		Fig. 2. Effect of aging on protein expression levels of TRPV5 and calbindin-D28K in the kidney. Immunofluorescence staining of kidney cortex sections of TRPV5+/+ mice of different ages (in wk) showing the abundance of TRPV5 (A), which was quantified by computer analysis of the integrated optical density and expressed in relative percentages (B), is shown. Immunoblots of samples (10 µg protein each) from homogenates of kidney tissue were labeled with antibodies against calbindin-D28K and beta-actin (C). Abundance of calbindin-D28K protein was quantified by computer-assisted densitometric analysis and expressed in relative percentages (D). Values are means ± SE; n = 7–9. *P < 0.05 vs. all. {dagger}P < 0.05 vs. TRPV5–/–.

 
Aging results in reduced Ca2+ absorption and duodenal expression of Ca2+ transport proteins. Following renal analysis, the effect of aging on intestinal Ca2+ absorption was examined. Although the 45Ca2+ absorption rate in TRPV5–/– mice was significantly higher compared with TRPV5+/+ littermates (Fig. 3A), aging reduced the amount of 45Ca2+ absorbed in both mice strains as shown by the peak 45Ca2+ absorption values at 4 min after oral gavage of mice from different age groups (Fig. 3B). To address the molecular mechanism responsible for the decreased absorption, the expression of genes encoding proteins involved in intestinal Ca2+ absorption was examined. The expression of TRPV6 and calbindin-D9K mRNA was significantly upregulated in TRPV5–/– mice (Fig. 3, C and D). Moreover, aging resulted in a decline in TRPV6 levels in both mice strains (Fig. 3C), whereas no changes were observed in the expression of calbindin-D9K (Fig. 3D). PMCA1b mRNA levels decreased with age in TRPV5+/+ mice, whereas no significant changes were present in the expression of PMCA1b in TRPV5–/– mice with increasing age (Fig. 3E). In addition, no significant changes in the expression of duodenal VDR were observed during aging in TRPV5+/+ or TRPV5–/– mice (Fig. 3F). Finally, aging did not affect the expression of the housekeeping gene HPRT in both kidney and duodenum (data not shown).


Figure 3
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  		Fig. 3. Effect of aging on intestinal Ca2+ absorption and mRNA expression levels of genes encoding duodenal Ca2+ transport proteins. A: changes in serum Ca2+ within 12 min after 45Ca2+ administration by oral gavage in TRPV5+/+ ({blacksquare}) and 10-wk-old TRPV5–/– ({blacktriangleup}) mice. The change in the serum Ca2+ concentration ({Delta}µM) is obtained by the equation (cpm 10 µl serum/cpm 10 µl stock solution) x 102 µM. B: comparison of {Delta}µM at 4 min after 45Ca2+ administration by oral gavage between 10-, 30-, and 52-wk-old TRPV5+/+ (filled bars) and TRPV5–/– (open bars) mice. Using real-time quantitative PCR, duodenal mRNA expression of TRPV6 (C), calbindin-D9K (D), PMCA1b (E), and VDR (F) of the different experimental groups was measured and presented as a ratio to HPRT expression. Values are means ± SE; n = 7–9. *P < 0.05 vs. all. #P < 0.05 vs. TRPV5+/+. {dagger}P < 0.05 vs. 30-wk-old TRPV5+/+. {ddagger}P < 0.05 vs. 52-wk-old TRPV5+/+. §P < 0.05 vs. 10- and 30-wk-old TRPV5–/–.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrated that an age-related decrease in the expression of the key players responsible for active Ca2+ transport, including TRPV5 and TRPV6, contributes to the decline in intestinal and renal Ca2+ (re)absorption with aging. Furthermore, our results indicated that mice lacking TRPV5 develop severe hyperparathyroidism with aging, suggesting that these mice are more susceptible to age-related osteoporosis.

A significant increase in the rate of Ca2+ absorption was observed in TRPV5–/– mice compared with their wild-type littermates, which was accompanied by an upregulation of duodenal TRPV6 and calbindin-D9K expression. Importantly, despite the renal Ca2+ wasting in TRPV5–/– mice, serum Ca2+ levels remained normal and were even slightly increased compared with TRPV5+/+ mice. As suggested previously, the increased duodenal expression of TRPV6 and calbindin-D9K, thereby increasing Ca2+ absorption, could act as a compensatory mechanism triggered by the elevated 1,25(OH)2D3 levels in TRPV5–/– mice (23). This was confirmed recently in a study by Renkema et al. (40) demonstrating that the compensatory upregulation of intestinal Ca2+ transporters and Ca2+ hyperabsorption were abolished in TRPV5/25-hydroxyvitamin-D3-1{alpha}-hydroxylase double knockout mice, which have undetectable serum 1,25(OH)2D3 levels. The slightly elevated serum Ca2+ levels in these knockout mice could be due to an overcompensation by 1,25(OH)2D3 and intestinal Ca2+ absorption to correct renal Ca2+ loss (8, 27). Moreover, aging was associated with a decline in duodenal Ca2+ absorption in both mice strains. In addition, the expression of duodenal TRPV6 in TRPV5–/– and TRPV5+/+ mice as well as PMCA1b in TRPV5+/+ mice was significantly reduced with age. Previous studies in rats and humans also showed a reduction in intestinal Ca2+ absorption as well as a decline in the expression of the Ca2+-transporting proteins (3, 4, 11). Our results imply that TRPV6 is crucial for duodenal Ca2+ absorption and that the decline in expression of this protein is the main cause of the decrease in duodenal Ca2+ absorption with aging.

In the kidneys of TRPV5+/+ mice, expression of TRPV5 and calbindin-D28K mRNA decreased during aging. Previous studies demonstrated a reduction in renal calbindin-D28K expression with increasing age (2, 26). Decreased expression of TRPV5 and calbindin-D28K could lead to a reduced Ca2+ reabsorption ability, resulting in hypercalciuria, as shown in the TRPV5–/– mice and described previously (23, 47). These results suggest that TRPV5 is a key component in renal Ca2+ reabsorption. In accordance with the decreased expression of TRPV5, a tendency toward increased urinary Ca2+ excretion was observed in the aging TRPV5+/+ mice. Although the hypercalciuria is even more pronounced in elder TRPV5–/– mice, no further decline in the expression of the Ca2+ transporting proteins is observed with increasing age. Interestingly, TRPV6 expression was increased in the 1-yr-old TRPV5–/– mice. TRPV6 is present in DCT and CNT as well as other tubular segments (34). However, the presence of TRPV6 does not rescue the renal Ca2+ loss in TRPV5–/– mice (23) and, therefore, its role in active Ca2+ reabsorption is questionable. Future investigations should unravel the role of TRPV6 in the various renal tubules and the possible involvement during aging.

Aging in TRPV5+/+ mice was accompanied by increasing serum 1,25(OH)2D3 levels, whereas no differences were observed in aging TRPV5–/– mice. This latter finding could be explained by the fact that 1,25(OH)2D3 in these knockout mice is already maximally elevated. 1,25(OH)2D3 is a known stimulator of intestinal and renal Ca2+ (re)absorption and upregulates the expression of proteins involved in active Ca2+ transport, including TRPV5 and TRPV6 (20, 24, 45). Therefore, the decline in the expression of the Ca2+-transporting proteins shown in our study suggests that there is a decrease in 1,25(OH)2D3 sensitivity. Indeed, VDR mRNA levels in kidneys of both TRPV5+/+ and TRPV5–/– mice gradually decrease with age, suggesting that the kidney develops a refractoriness to 1,25(OH)2D3 levels. Evidently, Li et al. (28) showed that VDR knockout mice have impaired Ca2+ conservation capability in the kidney, despite increased serum 1,25(OH)2D3 levels. Furthermore, intestinal Ca2+ absorption as well as expression of duodenal TRPV6 are decreased in VDR knockout mice (49). However, no significant differences were found in duodenal VDR mRNA levels during aging in both TRPV5+/+ and TRPV5–/– mice. Although several reports described a decrease in intestinal VDR expression (25, 29), other studies showed that receptor occupancy was reduced in elder rats (43). Our findings indicate an age-related intestinal resistance to the action of 1,25(OH)2D3, which is apparently not due to a reduction in duodenal VDR mRNA levels, thereby resulting in the decline in TRPV6 expression and Ca2+ absorption with aging.

Interestingly, aging of TRPV5+/+ mice was not associated with a change in serum PTH levels, whereas 52-wk-old TRPV5–/– mice showed severe hyperparathyroidism. Also of interest, serum phosphorus levels were elevated in 30-wk-old TRPV5–/–. Hyperphosphatemia leads to parathyroid cell proliferation (33), which could result in the observed hyperparathyroidism in 52-wk-old TRPV5–/– mice, thereby reducing the serum phosphorus levels back to normal. Furthermore, PTH, in addition to 1,25(OH)2D3, is involved in renal and intestinal Ca2+ reabsorption (1, 39, 51). Recent findings by our group show that PTH stimulates the expression of renal TRPV5, calbindin-D28K, and NCX1 (47). Although serum PTH levels remained constant or increased, renal Ca2+ reabsorption and intestinal Ca2+ absorption declined with age. This would be compatible with a mechanism of desensitization as has been described in previous reports, which demonstrated the blunting of PTH-mediated signal transduction pathways as well as loss of renal and intestinal PTH receptors (15, 16, 18, 30). Moreover, the elevated PTH levels in elder TRPV5–/– mice have also been found in humans and may play an important role in age-related bone loss or senile osteoporosis (31, 36, 42). Indeed, the relatively constant levels of serum Ca2+ and the decline in renal and intestinal Ca2+ (re)absorption in aged animals observed in the present study may suggest that bone resorption increases with aging. Osteocalcin levels in TRPV5–/– mice are significantly elevated compared with TRPV5+/+ mice, suggestive of increased bone turnover, which could be the result of the reduced bone thickness in TRPV5–/– mice described previously (23). After the decline in osteocalcin levels in both mice strains, which is probably due to the lower need of bone formation and turnover to build the skeleton in adult animals, serum osteocalcin levels increased in 52-wk-old TRPV5–/– mice in accordance with the elevation of serum PTH. PTH indirectly activates osteoclasts and modifies the phenotype of the osteoblast from a cell involved in bone formation to a cell directing bone resorption (37, 44). Thus the age-associated hyperparathyroidism in TRPV5–/– could lead to increased bone turnover, thereby resulting in net bone loss and aggravating the existing reduction in bone volume.

Previous studies indicated that estrogens can upregulate active Ca2+ (re)absorption in both kidney and duodenum (46). This was accompanied by an increased expression level of the Ca2+ transport proteins in female compared with male mice in both the kidney and duodenum. However, our data do not allow us to conclude that the age-dependent regulation of Ca2+ transporters is gender (in)dependent.

In summary, the present findings demonstrated a decline in renal and intestinal Ca2+ (re)absorption through TRPV5 and TRPV6 during aging, which is of physiological significance in understanding the imbalance in mineral metabolism associated with aging. Moreover, TRPV5–/– mice likely develop age-related hyperparathyroidism and osteoporotic characteristics earlier compared with TRPV5+/+ mice, corroborating the importance of the epithelial Ca2+ channels in Ca2+ homeostasis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by grants of the Dutch Organization of Scientific Research (Zon-Mw 902.18.298, Zon-Mw 016.006.001) and the Dutch Kidney Foundation (C03.6017).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. M. Bindels, 286 Cell Physiology, Radboud Univ. Nijmegen Medical Centre, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands (e-mail: r.bindels{at}ncmls.ru.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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