Renal Ca²⁺ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5

Ca²⁺ ions play a fundamental role in many cellular processes, and the extracellular concentration of Ca²⁺ is kept under strict control to allow the proper physiological functions to take place. The kidney, small intestine, and bone determine the Ca²⁺ flux to the extracellular Ca²⁺ pool in a concerted fashion. Transient receptor potential (TRP) cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have recently been postulated to be the molecular gatekeepers facilitating Ca²⁺ influx in these tissues and are members of the TRP family, which mediates diverse biological effects ranging from pain perception to male aggression. Genetic ablation of TRPV5 in the mouse allowed us to investigate the function of this novel Ca²⁺ channel in maintaining the Ca²⁺ balance. Here, we demonstrate that mice lacking TRPV5 display diminished active Ca²⁺ reabsorption despite enhanced vitamin D levels, causing severe hypercalciuria. In vivo micropuncture experiments demonstrated that Ca²⁺ reabsorption was malfunctioning within the early part of the distal convolution, exactly where TRPV5 is localized. In addition, compensatory hyperabsorption of dietary Ca²⁺ was measured in TRPV5 knockout mice. Furthermore, the knockout mice exhibited significant disturbances in bone structure, including reduced trabecular and cortical bone thickness. These data demonstrate the key function of TRPV5 in active Ca²⁺ reabsorption and its essential role in the Ca²⁺ homeostasis.

Ca²⁺ is the most abundant cation in the human body and serves a number of important physiological functions, including fertilization, synaptic transmission, muscle contraction, blood clotting, and bone mineralization. The extracellular Ca²⁺ concentration is controlled by the kidney, intestine, and bone through the action of the calciotropic hormones, including parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃). In humans, the daily dietary Ca²⁺ intake is less than 1,000 mg, of which only 30% is absorbed in the intestinal tract. This percentage is significantly enhanced during growth, pregnancy, and lactation by increased levels of circulating 1,25-(OH)₂D₃. Although there is continuous turnover of bone mass, there is no net gain or loss of Ca²⁺ from bone in a young and healthy individual. This indicates that healthy adults excrete a maximum of 300 mg Ca²⁺ in the urine to balance the intestinal Ca²⁺ uptake and that the remaining 98% of the Ca²⁺ filtered in the glomeruli is reabsorbed along the nephron. The molecular mechanism responsible for Ca²⁺ absorption in the small intestine and the kidney was elusive for a long time.

The cloning of transient receptor potential (TRP) cation channel subfamily V, member 5 (TRPV5; originally called ECaC1) from vitamin D–responsive rabbit renal epithelial cells (1) and TRP cation channel subfamily V, member 6 (TRPV6; originally called CaT1) from rat duodenum (2) has ignited research into transcellular Ca²⁺ (re)absorption at the molecular level (1). Mammals harbor at least 21 genes of the so-called TRP channels, whose functions remain mostly unknown (3). TRPV5 has been implicated as the Ca²⁺ influx channel in the process of vitamin D–responsive active Ca²⁺ reabsorption in the kidney (1, 4). In comparison, the TRPV5 homolog TRPV6, which displays an amino acid sequence identity of about 75% to TRPV5, has been postulated to be the Ca²⁺ influx channel facilitating Ca²⁺ absorption in enterocytes (2). TRPV6 is ubiquitously expressed and has been implicated as part of the capacitative Ca²⁺ entry mechanism and, therefore, intracellular Ca²⁺ signaling (5). TRPV5 and TRPV6 are Ca²⁺-selective channels that belong, together with the temperature-activated vanilloid receptors, to the TRPV subfamily (3, 4). Genomic cloning has demonstrated that TRPV5 and TRPV6 form a unique pair of novel Ca²⁺ channels that are transcribed from distinct genes juxtaposed on human chromosome 7q35 (4).

The present study investigated the physiological function of TRPV5 by the generation and characterization of TRPV5 knockout mice. Here, we demonstrate that the new TRP member TRPV5 is the gatekeeper in active Ca²⁺ reabsorption in the kidney. Ablation of the TRPV5 gene seriously disturbs renal Ca²⁺ handling, resulting in compensatory intestinal hyperabsorption and bone abnormalities.

Genetic ablation of TRPV5 in the mouse allowed us to investigate the requirement of TRPV5 functioning in maintaining the Ca²⁺ balance. The conditional targeting strategy was aimed at deleting the functionally critical region of the protein that encodes exon 13, comprising the entire pore region, using Cre-loxP–mediated recombination (Figure 1a). This procedure will allow the engineering of tissue-specific TRPV5 knockout mice strains in the near future, but in the present study TRPV5 null allele mutants were generated to address the overall function of this channel. To this end, TRPV5^loxneo/+ and EIIa-cre transgenic mice were crossed (8). The murine TRPV5 gene is encoded by 15 exons and spans about 15 kb (GenBank accession number, AF336378). Southern blot together with PCR analysis demonstrated the deletion of pore-encoding exon 13, as shown in Figure 1, b and c. Immunohistochemical costaining of kidney cortex using antibodies against TRPV5 and kallikrein, a specific maker for distal convolution that comprises distal convoluted and connecting tubules (20), confirmed that the mutant TRPV5 allele was inactivated (Figure 1d). Importantly, ablation of the TRPV5 locus did not affect renal TRPV6 expression, confirming the gene-specific inactivation of TRPV5 (Figure 1e).

Gross observation revealed no obvious phenotype in the TRPV5-null mice. TRPV5^–/– and TRPV5^+/– mice were fertile and had similar average litter sizes (Figure 2a). TRPV5^–/– pups nursed without aid and thrived to adulthood at a rate similar to that of wild-type mice, as assessed by size and weight at sexual maturity. TRPV5^–/– females displayed normal maternal behaviors such as crouching and nursing, and transfer of a nursing female to a new cage initiated nest-building activity and efficient pup retrieval.

Figure 2

Phenotypic characterization of TRPV5 knockout mice. (a) Body weights at an age of 8 weeks of TRPV5^+/+, TRPV5^+/–, and TRPV5^–/– mice (n = 29–43 mice). (b) The 24-hour urine volumes obtained from mice maintained in metabolic cages (n = 9 mice, three mice per cage). (c) Analysis of pH of urine obtained from TRPV5^+/+, TRPV5^+/–, and TRPV5^–/– mice (n = 9 mice, three mice per cage). (d) Total excretion of Ca²⁺, Na⁺, and K⁺ in TRPV5^+/+, TRPV5^+/–, and TRPV5^–/– mice (n = 9 mice, three mice per cage). Of note, TRPV5^+/– mice were similar to TRPV5^+/+ mice for the parameters measured. Data are averaged values ± SEM from mice at an age of 8 weeks. *P < 0.05, significant difference from TRPV5^+/+ mice.

TRPV5 ^+/+, TRPV5 ^+/–, and TRPV5 ^–/– mice were housed for 48 hours in metabolic cages (three mice per cage), and urine and serum samples were collected to investigate whether the electrolyte metabolism was disturbed. Polyuria was consistently observed in TRPV5 ^–/– mice compared with heterozygous mutants and wild-type littermates (Figure 2b). Importantly, TRPV5^–/– mice displayed a significant calciuresis, as they excreted about six times more Ca²⁺ in their urine than did their TRPV5^+/+ and TRPV5^+/– littermates (Figure 2d, left panel). The urinary Ca²⁺ concentration of the knockout mice reached values as high as 20 mM, compared with 6 mM for TRPV5^+/+ littermates. In addition, urinary phosphate excretion was significantly increased in TRPV5^–/– mice compared with that of TRPV5^+/+ mice (512 ± 30 μmol and 327 ± 25 μmol per 24 hours per three mice, respectively; n = 6, P < 0.05). Polyuria facilitates the excretion of large quantities of Ca²⁺ by reducing the potential risk of Ca²⁺ precipitation. It has been demonstrated that a high luminal Ca²⁺ concentration activates the calcium-sensing receptor in the apical membrane of the inner medullary collecting duct. As a consequence, the arginine vasopressin–elicited (AVP-elicited) water permeability is blunted to prevent a further rise in urinary Ca²⁺ concentration and possibly stone formation (21). Hypercalciuria-induced polyuria has been observed in humans and animal models (22–24). Notably, TRPV5^–/– mice produced urine that was significantly more acidic than that of their TRPV5^+/+ and TRPV5^+/– littermates (Figure 2c). Acidification of the urine is known to prevent renal stone formation in hypercalciuria, as Ca²⁺ oxalate stones cannot be formed at a pH of 5–6 (25). Neutralization of the acidic urine obtained from TRPV5^–/– mice resulted in the development of large crystals, confirming that acidification of the urine of the knockout mice is crucial for the prevention of kidney stone formation. A better understanding of the molecular mechanism by which hypercalciuria occurs will be important for the development of clinical tools that may lead to screening of families of “stone formers” before the onset of overt disease and to early treatment.

A primary renal origin of the observed calciuresis was supported by the fact that hypercalciuria persisted when TRPV5^–/– mice consumed a Ca²⁺-deficient diet for 1 week (data not shown). Furthermore, the GFR was not different between TRPV5^–/– and TRPV5^+/+ mice (0.31 ± 0.01 ml/min and 0.30 ± 0.07 ml/min, respectively; n = 6), indicating a primary defect in renal Ca²⁺ reabsorption as the cause of calciuresis in TRPV5^–/– mice. To localize the site of defective Ca²⁺ reabsorption along the nephron, we performed micropuncture studies (9–11) in anesthetized TRPV5^–/– and TRPV5^+/+ mice (Figure 3a). Confirming the results of the experiments in the metabolic cages, TRPV5^–/– mice showed calciuresis as a consequence of reduced renal Ca²⁺ reabsorption. Quantitative free-flow collection of tubular fluid revealed unaffected Ca²⁺ reabsorption in TRPV5^–/– mice up to the LPT. In contrast, mean Ca²⁺ delivery to puncturing sites within the distal convolution was significantly enhanced in TRPV5^–/– mice.

Because K⁺ secretion occurs along the distal nephron sites accessible to micropuncture (together with water reabsorption in the connecting tubule and cortical collecting duct), the distal luminal [K⁺] was used as an indicator of the distal collection site (Figure 3b). Based on the shape of the relationship between distal luminal [K⁺] and fractional Ca²⁺ delivery, it is evident that, in TRPV5^–/– mice, in contrast to TRPV5^+/+ mice, fractional Ca²⁺ delivery increases with higher [K⁺], indicating a defect in Ca²⁺ reabsorption along the distal convolution, which is consistent with the expression site of TRPV5. Given the potential internephron variations, the observation that some nephrons of TRPV5^–/– mice with low luminal [K⁺] exhibit low and some high fractional Ca²⁺ delivery is consistent with a rapid increase in luminal Ca²⁺ delivery along the early distal convolutions of mice lacking TRPV5. The increase in mean fractional Ca²⁺ delivery with increasing luminal [K⁺] in TRPV5^–/– mice cannot be explained by a simple lack of Ca²⁺ reabsorption, but would be consistent with Ca²⁺ back-leak into the lumen in the absence of transcellular reabsorption. In comparison, if the primary Ca²⁺ reabsorption defect were upstream of the distal convolution, for example, in the thick limb of Henle’s loop, then fractional Ca²⁺ delivery should be enhanced in TRPV5^–/– mice distal nephrons irrespective of the luminal [K⁺]. This contrasts with the observation made in the TRPV5^–/– mice, indicating that the transport defect is localized primarily in the distal convolution. The fractional delivery of Na⁺ to the distal convolution was not significantly different between TRPV5^–/– and TRPV5^+/+ mice (4 ± 1% vs. 5 ± 1%, respectively; n = 6, P > 0.2), which argues against the idea that a primary decrease in Na⁺ reabsorption upstream of the distal convolution indirectly influences Ca²⁺ reabsorption.

In the Ca²⁺-reabsorbing epithelial cells of the distal convolution, the influx of Ca²⁺ is rapidly buffered by cytosolic calcium-binding proteins, that is, calbindin-D_28K and calbindin-D_9K, and the bound Ca²⁺ diffuses to the basolateral membrane where a Na⁺-Ca²⁺ exchanger (NCX1) and, to a lesser extent, an ATP-dependent Ca²⁺ pump extrude Ca²⁺ to the blood compartment, completing the process of transcellular Ca²⁺ transport (4). The expression of the Ca²⁺ transport proteins in the kidney was investigated by quantitative real-time PCR as described previously (13). Inactivation of the TRPV5 gene was accompanied by a decrease in calbindin-D_28K and NCX1 mRNA levels (Figure 3d). As transcellular Ca²⁺ reabsorption in the distal convolution was abolished in TRPV5^–/– mice, the concomitant decrease in calbindin-D_28K and NCX1 mRNA levels in the presence of elevated 1,25-(OH)₂D₃ levels suggests a regulatory mechanism controlled primarily by TRPV5. Thus, TRPV5 or the Ca²⁺ influx through TRPV5 possibly controls the transcription of the other Ca²⁺ transport genes, including calbindin-D_28K and NCX1. These findings emphasize the gatekeeper function of TRPV5. Although downregulation of renal calbindin-D_28K is secondary to TRPV5 ablation in the TRPV5^–/– mice, the reduced calbindin-D_28K level may further augment the severity of the hypercalciuria. Interestingly, calbindin-D_28K knockout mice fed a Ca²⁺ diet (1% weight/volume) displayed an approximately twofold increase in urinary Ca²⁺ excretion compared with that of their wild-type littermates (26), whereas TRPV5^–/– mice fed the same Ca²⁺ diet excreted about six times more Ca²⁺ in their urine than did their littermates.

In contrast to the excessive renal Ca²⁺ wasting noted, natriuresis and kaliuresis were not observed in the knockout mice, demonstrating the specific effect of TRPV5 inactivation on Ca²⁺ reabsorption (Figure 2d). Importantly, despite renal Ca²⁺ wasting, the serum Ca²⁺ concentration remained normal, suggesting successful compensatory mechanisms (Figure 4d). We investigated whether the impairment of transcellular Ca²⁺ reabsorption indirectly affected intestinal Ca²⁺ absorption. Active Ca²⁺ uptake by the small intestine is favored as compensation for renal Ca²⁺ wasting. To provide functional evidence for intestinal hyperabsorption in TRPV5^–/– mice, we assessed Ca²⁺ absorption by measuring serum ⁴⁵Ca²⁺ at early time points after oral gavage, as described (14). A significant increase in the rate ⁴⁵Ca²⁺ absorption was observed in TRPV5^–/– mice compared with their wild-type littermates, indicating a compensatory role for the small intestine (Figure 3c). Of note, this may actually represent an underestimation due to the applied single-⁴⁵Ca²⁺-isotope technique used instead of a dual-isotope technique (27). Whereas TRPV5 is expressed predominantly in the kidney, TRPV6 is highly expressed in small intestine and has been implicated as the Ca²⁺ influx mechanism in enterocytes (1, 2). To test this hypothesis, duodenal RNA was extracted from knockout and wild-type littermates and analyzed for expression of TRPV6 and calbindin-D_9K by quantitative real-time PCR analysis. TRPV6 and calbindin-D_9K expression levels were significantly upregulated in TRPV5^–/– mice, consistent with an increased Ca²⁺ absorption (Figure 3, c and e).

Figure 4

Bone phenotypes of female and male TRPV5^+/+ and TRPV5^–/– mice. (a) Representative cross-sectional x-ray images of the femoral head (I), the lesser trochanter (II), and the diaphysis (III) in TRPV5^+/+ and TRPV5^–/– mice. Note the decreased cortical bone width in the trochanter and diaphysis (arrows). (b) MOI as a parameter of bone quality. (c) Three-dimensional reconstruction of femurs from TRPV5^+/+ and TRPV5^–/– mice. Note the reduced cortical and Tb.Th in TRPV5^–/– mice. (d) Changes in serum levels of 1,25-(OH)₂D₃, PTH, and Ca²⁺ in TRPV5^+/+, TRPV5^+/–, and TRPV5^–/– mice. Values are expressed as percentage of TRPV5^+/+ values ± SEM. The absolute values for PTH are TRPV5^+/+, 55 ± 15 pg/ml; TRPV5^+/–, 59 ± 14 pg/ml; and TRPV5^–/–, 60 ± 16 pg/ml; for 1,25-(OH)₂D₃, TRPV5^+/+, 239 ± 38 pmol/l; TRPV5^+/–, 225 ± 32 pmol/l; TRPV5^–/–, 691 ± 88 pmol/l; and for free Ca²⁺ concentration, TRPV5^+/+, 1.20 ± 0.02 mM; TRPV5^+/–, 1.25 ± 0.03 mM; and TRPV5^–/–, 1.20 ± 0.03 mM. *P < 0.05, significant difference from wild-type mice.

Serum 1,25-(OH)₂D₃ levels were significantly elevated in TRPV5^–/– mice (Figure 4d), which explains the compensatory increased intestinal TRPV6 and calbindin-D_9K mRNA expression and Ca²⁺ absorption. The TRPV5^–/– mouse is the first model to our knowledge to have sustained highly elevated 1,25-(OH)₂D₃ levels while remaining normocalcemic (Figure 4d) and normophosphatemic, as serum phosphate was not different between the TRPV5^+/+ and TRPV5^–/– mice (2.68 ± 0.29 and 3.04 ± 0.18 mM, respectively, n = 6, P > 0.2). The elevated 1,25-(OH)₂D₃ levels observed in vitamin D receptor (VDR)-null mice (14) and in rickets type II are normally accompanied by hypocalcemia and increased serum PTH levels. In contrast, in TRPV5^–/– mice with normal functioning VDRs, PTH was not significantly changed (Figure 4d). The elevated 1,25-(OH)₂D₃ levels in TRPV5^–/– mice were not due to increased expression of renal 25-hydroxyvitamin D₃-1α-hydroxylase mRNA, since no differences were measured in the expression of this enzyme between TRPV5^+/+ and TRPV5^–/– mice (100 ± 13% and 139 ± 29%, respectively; n = 4, P > 0.2). Studies to clarify the role of 1,25-(OH)₂D₃ in bone have been hampered by the complexity of distinguishing between disturbances in Ca²⁺ homeostasis and changes in serum 1,25-(OH)₂D₃ levels. Normal PTH plus normocalcemia make the TRPV5^–/– mouse a unique model to study the effect of 1,25-(OH)₂D₃ on bone.

The homeostasis of bone tissue is maintained by the balanced processes of bone resorption and formation. The bone formation marker alkaline phosphatase was not different between the groups (Table 1), while osteocalcin was slightly but significantly higher in sera of TRPV5^–/– versus TRPV5^+/+ mice. Bone resorption was studied by TRAP staining of femoral bone sections, by measurement of osteoclast number and surface, and by measurement of urinary deoxypyridinoline. Both N.Oc/BS and Oc.S/BS were significantly increased in female TRPV5^–/– mice, with males showing a similar trend (Table 1). Urinary deoxypyridinoline showed large variations and was not significantly different between TRPV5^–/– and TRPV5^+/+ mice (66 ± 8 nmol/nmol creatinine and 92 ± 21 nmol/nmol creatinine, respectively; n = 5–9, P > 0.2).

Table 1

Bone analysis of TRPV5^+/+ and TRPV5^–/– mice

Detailed microcomputed tomography analyses of the femur demonstrated that Tb.Th in the femoral head of TRPV5^–/– mice was significantly reduced compared with that of TRPV5^+/+ mice (Table 1 and Figure 4, a and c). BV/TV was not different between the genotypes. This could not be explained by a difference in Tb.N (Table 1). Alternatively, it is possible that the trabeculae are longer, that is, they protrude further into the bone marrow cavity, and thereby compensate for reduced Tb.Th. CD and SMI were not altered in the femoral head, irrespective of phenotype and sex. Analyses of the diaphysis showed that Ct.V, Ct.V/Dp.V, and Ct.Th were significantly reduced in TRPV5^–/– versus TRPV5^+/+ mice (Table 1 and Figure 4, a and c). Finally, the polar MOI was significantly reduced in TRPV5^–/– mice, indicative of an altered geometrical distribution and reduced mechanical properties of the diaphyseal bone (Table 1 and Figure 4b). The observed changes in bone structure cannot be attributed to differences in body weight (Figure 2a). Also, the bone lengths of TRPV5^–/– and TRPV5^+/+ mice were similar. In summary, the unchanged alkaline phosphatase together with the elevated N.Oc/BS and Oc.S/BS in the TRPV5^–/– mice may explain the observed reduced trabecular and cortical thickness.

It is unclear whether the reduced Tb.Th and Ct.Th found in the TRPV5^–/– mice is a direct effect of the genotype or results from prolonged elevated 1,25-(OH)₂D₃ levels. Interestingly, a recent study of long-term treatment of mice with 1,25-(OH)₂D₃ demonstrated a decrease in cortical thickness and cross-sectional area and a 50% reduction in stiffness (28), which is in line with the current observations. We hypothesize that the bone phenotype of the TRPV5^–/– mice, due to prolonged elevated 1,25-(OH)₂D₃ levels, develops and progresses throughout life. However, at present it is premature to draw definitive conclusions about bone metabolism, and future experiments will certainly address this point in detail.

The epithelial Ca²⁺ channels TRPV5 and TRPV6 have been studied extensively in epithelial tissues controlling the Ca²⁺ homeostasis and exhibit a range of distinctive properties that distinguish them from other TRP channels (3, 4). TRP channels display an extraordinary assortment of selectivities and activation mechanisms, some of which represent previously unrecognized modes for regulating ion channels (3). Moreover, there is only limited information about the biological functions of TRP channels, which appear to be equally diverse and range from pain perception to male aggression. The present study demonstrated that the new TRP member TRPV5 is the gatekeeper in active Ca²⁺ reabsorption in the kidney. Ablation of the TRPV5 gene seriously disturbs renal Ca²⁺ handling, resulting in compensatory intestinal hyperabsorption and bone abnormalities. These deficiencies in Ca²⁺ handling have been reported frequently in patients with idiopathic hypercalciuria, although the molecular basis for this disorder remains unknown. Dysfunction of TRPV5 may contribute to such disturbances of Ca²⁺ homeostasis, and the TRPV5 knockout mouse is, therefore, a powerful animal model for elucidating the molecular mechanism underlying idiopathic hypercalciuria. Finally, the increased 1,25-(OH)₂D₃ levels and effects on bone structure indicate that alterations in TRPV5 may have implications for age-related bone disorders, including osteoporosis.

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This work was supported by the Dutch Organization for Scientific Research (Zon-Mw 016.006.001), an EMBO fellowship to J.G.J. Hoenderop, and National Science Foundation grants to E. Hummler and B.C. Rossier. The authors thank C.H. van Os, H.A.P. Pols, and P.M.T. Deen for stimulating discussions; K. Richter, D.Y. Huang, M. van Abel, and C.J. Buurman for technical assistance; R. Bouillon (Catholic University Leuven, Leuven, Belgium) for providing the osteocalcin antibody; and H. Westphal (National Institute of Child Health and Human Development, Bethesda, Maryland, USA) and J. van Es (Hubrecht Laboratory, Utrecht, The Netherlands) for providing the Ella-cre-deletor mice.

Johannes P.T.M. van Leeuwen, Edith Hummler, and René J.M. Bindels contributed equally to this work.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: parathyroid hormone (PTH); 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃); transient receptor potential (TRP); TRP cation channel subfamily V, member 5 (TRPV5); TRP cation channel subfamily V, member 6 (TRPV6); last loop of proximal tubules (LPT); hypoxanthine-guanine phosphoribosyl transferase (HPRT); bone volume (BV); total bone marrow volume including trabeculae (TV); trabecular bone volume fraction (BV/TV); trabecular thickness (Tb.Th); trabecular number (Tb.N); connectivity density (CD); structure model index (SMI); cortical volume (Ct.V); endocortical volume (Ec.V); total diaphyseal volume (Dp.V), cortical thickness (Ct.Th); cortical bone volume fraction (Ct.V/Dp.V); moment of inertia (MOI); Tartrate-resistant acid phosphatase (TRAP); number of osteoclasts per bone surface area (N.Oc/BS); surface area of osteoclasts per bone surface area (Oc.S/BS); arginine vasopressin (AVP); Na⁺-Ca²⁺ exchanger (NCX1); vitamin D receptor (VDR).

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