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Cell and Transport Physiology |
* Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; and Departments of Internal Medicine and Orthopedics, Erasmus Medical Centre Rotterdam, Rotterdam, The Netherlands
Address correspondence to: Dr. Joost G.J. Hoenderop, Radboud University Nijmegen Medical Centre, 160 Cell Physiology, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Phone: +31-24-3610571; Fax: +31-24-3616413; E-mail: j.hoenderop{at}ncmls.ru.nl
Received for publication June 17, 2005. Accepted for publication July 18, 2005.
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Abstract |
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-hydroxylase double knockout (TRPV5–/–/1-OHase–/–) mice, which show undetectable serum 1,25(OH)2D3 levels, were generated. TRPV5–/–/1-OHase–/– mice displayed a significant hypocalcemia compared with wild-type mice (1.10 ± 0.02 and 2.54 ± 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D28K (7 ± 2%), calbindin-D9K (32 ± 4%), Na+/Ca2+ exchanger (12 ± 2%), and intestinal TRPV6 (40 ± 8%) and calbindin-D9K (26 ± 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5–/–/1-OHase–/– mice, more pronounced than observed in single TRPV5 or 1-OHase knockout mice. It is interesting that a renal Ca2+ leak, as demonstrated in TRPV5–/– mice, persisted in TRPV5–/–/1-OHase–/– mice, but a compensatory upregulation of intestinal Ca2+ transporters was abolished. In conclusion, the elevation of serum 1,25(OH)2D3 levels in TRPV5–/– mice is responsible for the upregulation of intestinal Ca2+ transporters and Ca2+ hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca2+ reabsorption in TRPV5–/– mice. |
Introduction |
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,25-dihydroxyvitamin D3 (1,25(OH)2D3), and parathyroid hormone (PTH) and calcitonin are the calciotropic hormones that are known to be involved in the maintenance of plasma Ca2+ concentration (2,3). Ca2+ absorption from the intestinal lumen and Ca2+ reabsorption in the renal tubules is promoted by 1,25(OH)2D3 (4–6). Synthesis of 1,25(OH)2D3 requires the renal cytochrome P450 enzyme 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) (7). In addition, PTH stimulates 1,25(OH)2D3 production by 1-OHase activation and the release of Ca2+ from bone to maintain plasma Ca2+ levels, whereas calcitonin lowers the plasma Ca2+ concentration by inhibition of bone resorption (2). 1,25(OH)2D3-stimulated transepithelial Ca2+ transport in kidney and intestine involves Ca2+ entry across the apical membrane via the epithelial Ca2+ channels TRPV5 and TRPV6, respectively. TRPV5 is localized at the luminal membrane of the late distal convoluted tubule (DCT) and connecting tubule (CNT) in kidney (8,9). TRPV6 is the homologues Ca2+ channel localized in the brush border membrane of duodenum (1,10–13). After entry of Ca2+ through TRPV5 and TRPV6, Ca2+ bound to Ca2+-binding proteins (calbindins) diffuses to the basolateral membrane of the cell. Ca2+ is finally extruded to the extracellular compartment by the Na+/Ca2+ exchanger (NCX1) in the kidney and the plasma membrane Ca2+-ATPase (PMCA1b) in intestine and kidney (9).
Recently, several animal models were designed to investigate Ca2+ absorption mechanisms and maintenance of Ca2+ balance. TRPV5 knockout (TRPV5–/–) mice were generated by targeted ablation of the TRPV5 gene. These mice displayed robust renal Ca2+ wasting as a result of impaired Ca2+ reabsorption in DCT and CNT (14). Furthermore, elevated 1,25(OH)2D3 serum levels, intestinal Ca2+ hyperabsorption, and reduced bone thickness and mineralization were demonstrated. In addition, Dardenne et al. (15) created an animal model for vitamin D deficiency rickets type 1 by genetic ablation of the 1-OHase gene. These 1-OHase knockout (1-OHase–/–) mice showed a phenotype characterized by undetectable serum levels of 1,25(OH)2D3, severe hypocalcemia, hyperparathyroidism, bone abnormalities, and retarded growth (15–17). Until now, data on detailed urine analysis of the 1-OHase–/– mice were not available. Previous studies demonstrated that 1,25(OH)2D3 supplementation rescues the severe phenotype of 1-OHase–/– mice, which is accompanied by an upregulation of TRPV5, TRPV6, calbindins, NCX1, and PMCA1b expression, resulting in the normalization of serum Ca2+ levels. It is interesting that dietary Ca2+ supplementation in 1-OHase–/– mice resulted in a similar rescue in the absence of 1,25(OH)2D3. Taken together, these studies suggest a pivotal role of the Ca2+ transport proteins in maintaining Ca2+ balance, partly independent of 1,25(OH2)D3 (18).
To investigate the contribution of elevated 1,25(OH)2D3 levels to the phenotype of TRPV5–/– mice and to elucidate the involvement of 1,25(OH)2D3 in the compensatory Ca2+ hyperabsorption, we generated a mouse model in which both TRPV5 and 1-OHase were inactivated. In this study, we describe the phenotype of TRPV5/1-OHase double knockout (TRPV5–/–/1-OHase–/–) mice, including bone analysis and semiquantification of mRNA and protein expression levels of Ca2+ transport proteins and provide an extensive comparison with TRPV5 and 1-OHase single knockout mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHase–/– mice were provided by René St-Arnaud (Shriners Hospital for Children Montreal, QU, Canada) (15). Cross-breeding of 1-OHase–/– and TRPV5–/– mice resulted in offspring that were heterozygous for both TRPV5 and 1-OHase (TRPV5+/–/1-OHase+/–). This offspring was subsequently intercrossed to obtain TRPV5–/–/1-OHase–/– mice. Genotypes were determined by PCR analysis, using specific primers as described previously (14,15). For obtaining 24-h urine samples of all mouse genotypes, 8-wk-old mice were kept in a light- and temperature-controlled room in metabolic cages that enabled 24-h urine analyses. Standard pelleted chow (0.25% [wt/vol] Na, 1.1% [wt/vol] Ca, 0.2% [wt/vol] Mg, 0.7% [wt/vol] P, and 0.9% [wt/vol] K) and drinking water were available ad libitum. After the experiment, blood was collected and mice were killed. Kidney, duodenum, tibia, and fibula were sampled. The animal ethics board of the Radboud University Nijmegen approved all animal experimental procedures.
Urine and Serum Analyses
Urine and serum Ca2+ concentrations were analyzed using a colorimetric assay kit (Roche, Mannheim, Germany). An electronic ion analyzer was used to determine urinary pH. Serum vitamin D levels were determined with a [125I]1,25(OH)2D3 RIA assay (IDS Inc., Fountain Hills, AZ). Mouse serum PTH was measured using an immunoradiometric assay (Immutopics Inc., San Clemente, CA).
Real-Time Quantitative PCR Analysis
For investigating mRNA expression levels of renal and duodenal Ca2+ transport proteins, total RNA from kidney and duodenum was isolated using TriZol Reagent (Life Technologies BRL, Life Technologies, Breda, The Netherlands) according to the manufacturer’s protocol. RNA was treated with DNase (Promega, Madison, WI) to prevent contamination with genomic DNA. Total RNA was subjected to reverse transcription using Moloney-murine leukemia virus–reverse transcriptase (Life Technologies BRL, Life Technologies, Breda, The Netherlands) (11). Renal mRNA expression levels of calbindin-D28K; calbindin-D9K; NCX1; and duodenal TRPV6, calbindin-D9K, and PMCA1b mRNA levels were quantified by real-time quantitative PCR as described previously (19,20), using the ABI Prism 7700 Sequence Detection System (PE Biosystems, Rotkreuz, Switzerland). The expression level of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase was used as an endogenous control.
Immunoblotting
Total kidney lysates of TRPV5–/– and TRPV5–/–/1-OHase–/– mice were prepared as described previously (21). The protein concentration of the homogenates was determined with the Bio-Rad protein assay (Bio-Rad, München, Germany). Samples were submitted to 16.5% (wt/vol) SDS-PAGE and blotted to polyvinyldifluoride-nitrocellulose membranes (Immobilon-P, Millipore Corp., Bedford, MA). Blots were incubated with a rabbit anti–calbindin-D28K polyclonal antibody (1:10,000) (22) or a rabbit anti–calbindin-D9K polyclonal antibody (1:5000; Swant, Bellinzona, Switzerland) at 4°C for 16 h. Subsequently, blots were incubated with a goat anti-rabbit peroxidase-labeled secondary antibody (1:10,000; Sigma, St. Louis, MO). Immunoreactive protein was detected by the chemiluminescence method (Pierce, Rockford, IL). Immunopositive bands were scanned using an imaging densitometer (Bio-Rad Gs-690) to determine pixel density (Molecular Analyst Software; BioRad Laboratories, Hercules, CA).
Bone Analyses
For evaluating the effects of TRPV5 and 1-OHase-ablation on bone homeostasis, bone thickness, mineralization, and epiphyseal growth plate development were determined in tibiae from wild-type, TRPV5–/–, 1-OHase–/–, and TRPV5–/–/1-OHase–/– mice using microcomputed tomography (Skyscan 1072; SkyScan, Antwerp, Belgium). This technique allows for high-resolution, noninvasive computed tomographic imaging in small animals and small animal organs. Tibiae and fibulae were fixed in 4% (vol/vol) PBS-buffered formaldehyde and routinely processed for plastic embedding (23). The bones were scanned using the SkyScan 1072 microtomograph, and digital cross-sections were made at the site of the diaphysis and epiphysis. X-ray imaging of tibiae and fibulae was performed to compare bone lengths and mineralization in all mouse genotypes.
Statistical Analyses
Values are expressed as means ± SEM. Statistical significance (P < 0.05) was determined by one-way ANOVA. All analyses were performed using the Statview Statistical Package software (Power PC version 4.51, Berkeley, CA) on an Apple iMac computer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHase–/– mice displayed hypocalciuria, hypocalcemia, serum 1,25(OH)2D3 levels below the detection level (<6 pmol/L), and a significant hyperparathyroidism. In comparison with wild-type mice, diuresis and urine pH were normal in 1-OHase–/– mice. The analysis of the TRPV5–/–/1-OHase–/– mice phenotype showed that net urine Ca2+ excretion does not differ from wild-type mice, although inappropriately high given the severe hypocalcemia. Serum 1,25(OH)2D3 levels were below detectable levels, and serum PTH levels were further elevated in TRPV5–/–/1-OHase–/– mice compared with 1-OHase–/– mice. In addition, diuresis and urine pH were normalized in TRPV5–/–/1-OHase–/– mice. Body weight was reduced in 1-OHase–/– and TRPV5–/–/1-OHase–/– mice compared with litter-matched wild-type mice. 1-OHase–/– and TRPV5–/–/1-OHase–/– mice had a decreased lifespan in comparison with wild-type and TRPV5–/– mice.
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-OHase single or combined gene ablation on mRNA and protein expression levels by real-time PCR analysis and semiquantitative immunoblotting. Calbindin-D28K, calbindin-D9K, and NCX1 mRNA expression levels were decreased in kidneys of TRPV5–/– and 1-OHase–/– mice compared with wild-type mice (Figure 2). It is interesting that a further significant decrease in the expression of these Ca2+ transporters was detected in TRPV5–/–/1-OHase–/– mice. Calbindin-D28K and calbindin-D9K protein abundance was significantly decreased in TRPV5–/–/1-OHase–/– mice compared with TRPV5–/– mice as detected by immunoblot (Figure 3).
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-OHase ablation resulted in a significant decrease in TRPV6 and calbindin-D9K expression in duodenum. Furthermore, 1-OHase–/– mice demonstrated a downregulation of TRPV6 and calbindin-D9K mRNA expression. PMCA1b expression levels did not differ significantly between the studied mouse genotypes.
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-OHase–/– mice (Figure 5, K and O) demonstrated reduced bone thickness in both the trabecular and the cortical compartments compared with wild-type mice (Figure 5, I and M). This reduction was more severe in 1-OHase–/– mice (Figure 5, K and O). In addition, the degree of bone mineralization was reduced in TRPV5–/– mice (Figure 5, F, J, and N) and 1-OHase–/– mice (Figure 5, G, K, and O) compared with wild-type mice (Figure 5, E, I, and M). Moreover, the epiphyseal growth plate was widened in 1-OHase–/– mice (Figure 5, G versus E), which was not observed in TRPV5–/– mice (Figure 5, F versus E). Tibial bone length was reduced in 1-OHase–/– mice (Figure 5, C versus A and B). In TRPV5–/–/1-OHase–/– mice, the thickness of cortical bone and trabeculae was decreased and mineralization was diminished compared with all other mouse genotypes (Figure 5, H, L, and P). Moreover, tibial bone length was severely reduced and epiphyseal growth plate widening was more pronounced in TRPV5–/–/1-OHase–/– mice than demonstrated in 1-OHase–/– mice (Figure 5, D and H versus C and G). Taken together, the TRPV5–/–/1-OHase–/– mice exhibited an aggravated skeletal phenotype in comparison with TRPV5–/– and 1-OHase–/– mice (Figure 5, D, H, L, and P).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHase–/– mice, which exhibit a significant hypocalcemia. The urinary Ca2+ leak demonstrated in TRPV5–/– mice persists in the TRPV5–/–/1-OHase–/– mice as an inappropriately high urinary Ca2+ excretion with respect to the hypocalcemia. Moreover, bone degradation is present in TRPV5–/–/1-OHase–/– mice, more severe than observed in TRPV5–/– mice. Therefore, the absence of Ca2+ hyperabsorption results in a more aggravated phenotype than described for TRPV5 single-ablated mice. Thus, 1,25(OH)2D3 is of crucial importance to compensate the renal Ca2+ leak and to maintain normal serum Ca2+ levels in TRPV5–/– mice.
1,25(OH)2D3 is an important regulatory hormone in Ca2+ and bone homeostasis (2). In duodenum, 1,25(OH)2D3 regulates the expression of Ca2+ transport proteins (24). Moreover, 1,25(OH)2D3 is synthesized from its precursor by the renal enzyme 1-OHase (7). 1-OHase–/– mice were previously generated as a mouse model for vitamin D deficiency rickets type I (15,16). Inactivation of 1-OHase resulted in undetectable levels of serum 1,25(OH)2D3, a significant hypocalcemia, and reduced expression levels of duodenal Ca2+ transport proteins. In contrast, TRPV5–/– mice demonstrated a normocalcemia with a significant hypervitaminosis D and increased duodenal Ca2+ transport protein expression leading to Ca2+ hyperabsorption (14). We hypothesized that the increased serum 1,25(OH)2D3 levels constitute a compensatory mechanism in an effort to correct for the significant renal Ca2+ leak. Additional gene inactivation of 1-OHase in TRPV5–/– mice was performed to investigate the effects of the elevated serum 1,25(OH)2D3 levels present in TRPV5–/– mice. This double gene ablation resulted in undetectable serum 1,25(OH)2D3 levels and downregulation of duodenal Ca2+ transport protein expression. Moreover, a hypocalcemia developed in the TRPV5–/–/1-OHase–/– mice. These data clearly showed that the increased expression of the intestinal Ca2+ transporters TRPV6 and calbindin-D9K and the resulting Ca2+ hyperabsorption in TRPV5–/– mice are due to a secondary hypervitaminosis D. These effects compensate for the renal Ca2+ leak to maintain normal serum Ca2+ levels in TRPV5–/– mice.
TRPV5–/– mice show a striking hypercalciuria. Previous micropuncture experiments demonstrated that Ca2+ reabsorption in DCT and CNT is abolished in these mice, illustrating a primary defect in renal active Ca2+ reabsorption (14). On the contrary, 1-OHase–/– mice demonstrated a significant hypocalciuria, a feature not described before, although explained by the downregulation of intestinal and renal Ca2+ transport proteins and, therefore, impairment of Ca2+ absorption and reabsorption. It is interesting that additional gene ablation of 1-OHase in TRPV5–/– mice normalized 24-h urinary Ca2+ excretion to values not significantly different from wild-type mice. Nevertheless, relative to the severe hypocalcemia, these double knockout mice still excreted inappropriately high amounts of Ca2+. Moreover, given the expression of the renal Ca2+ transporters in 1-OHase–/– mice, it can be concluded that active Ca2+ reabsorption is still present, although severely impaired despite the absence of 1,25(OH)2D3. Ca2+ reabsorption through TRPV5 therefore could be partly independent on 1,25(OH)2D3, which is in line with the results of previous studies in which the rescue of 1-OHase–/– mice by dietary Ca2+ was described, showing the regulation of Ca2+ transport proteins independent of 1,25(OH)2D3 regulation (18,25).
Hypercalciuria increases the risk for the formation of Ca2+-containing urinary crystals (26,27). In addition to hypercalciuria, TRPV5–/– mice displayed a significant polyuria and urine acidification. Polyuria facilitates the excretion of large quantities of Ca2+, and urine acidification creates an environment in which formation of crystals is restrained (28,29). It is interesting that in the double knockout mice, net calciuresis did not differ from wild-type mice and neither did urinary pH or diuresis. This indicated that the hypercalciuria in TRPV5–/– mice, which originates from the DCT and CNT, could indeed influence diuresis and pH in more distal located nephron segments. Previously, thiazide treatment leading to a decrease in Ca2+ excretion in TRPV5–/– mice was shown to normalize urinary pH and blunt polyuria (30). It was postulated that calcium-sensing receptor (CaSR) signaling in the apical membrane of the collecting duct functionally links renal Ca2+ and water metabolism (31). CaSR activation by high luminal Ca2+ concentrations might result in a decrease in water permeability of the collecting duct by downregulation of aquaporin-2 water channels and consequently increase the urine volume (32,33). We suggest that regulation of specific acid-base transport proteins, possibly as a result of CaSR activation, is involved in the observed urine acidification in TRPV5–/– mice. The exact mechanisms that are responsible for the urine acidification and polyuria in TRPV5–/– mice need further investigation.
The majority of the body Ca2+ content is stored in bone, where the balanced processes of bone formation and resorption maintain bone homeostasis (34). Detailed microcomputed tomography and x-ray analyses showed bone abnormalities in all three studied mouse models compared with wild-type mice. TRPV5–/– mice displayed reduced trabecular and cortical bone thickness (14), which could be a consequence of the negative Ca2+ balance as a result of the renal Ca2+ leak. Moreover, it was previously suggested that high serum 1,25(OH)2D3 levels reduce cortical bone thickness, bone stiffness, and strength (35). However, 1,25(OH)2D3 levels were not detectable in TRPV5–/–/1-OHase–/– mice, which demonstrated a significant decrease in bone thickness, reduced bone mineralization, and rickets, noticeably more severe than the bone abnormalities observed in TRPV5–/– and 1-OHase–/– mice (14,15). This suggested that TRPV5 gene ablation might, at least in part, be directly responsible for the detected bone abnormalities, independent of the elevated serum 1,25(OH)2D3 levels. It is interesting that the expression of TRPV5 and TRPV6 in bone tissue was previously described and these epithelial Ca2+ channels therefore could serve as direct Ca2+ providers in bone (20). However, this hypothesis is hampered by the absence of comprehensive data on the exact actions of these Ca2+ channels in bone. Alternatively, the aggravated bone phenotype demonstrated in TRPV5–/–/1-OHase–/– mice could be explained by the secondary hyperparathyroidism leading to renal osteodystrophy.
Zheng et al. (36) recently studied the vitamin D receptor (VDR)/calbindin-D28K double knockout mouse compared with VDR or calbindin-D28K single gene–ablated mice. It is interesting that the VDR/calbindin-D28K double knockout mice displayed downregulation of intestinal and renal Ca2+ transport proteins and a more aggravated phenotype, including hypercalciuria and hyperparathyroidism, a further decrease in bone mineral density and bone length, increased distortion of the growth plate, a decrease in body weight, and a decreased lifespan compared with the single gene–ablated mice. In line with our results, this study clearly revealed the critical role of vitamin D and Ca2+ transport proteins in Ca2+ homeostasis and, moreover, the crucial regulatory function of 1,25(OH)2D3 in the expression of intestinal and renal Ca2+ transport protein.
On the basis of the present data, we ascertained the important function of the renal Ca2+ transport protein TRPV5 in Ca2+ reabsorption and its possible involvement in bone Ca2+ transport, concomitant with a crucial role of 1,25(OH)2D3 in active Ca2+ (re)absorption by regulating epithelial Ca2+ transport and Ca2+ transport protein expression. The observed polyuria and urine acidification in TRPV5 knockout mice will need further investigation to obtain more insight into the responsible mechanisms.
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Acknowledgments |
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We thank Dr. R. St-Arnaud for kindly providing the 1-OHase knockout mice and the Central Animal Facility of the Radboud University Nijmegen for technical support in this study.
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