Calcium and phosphate homeostasis: Concerted interplay of new regulators

Abstract

Calcium (Ca²⁺) and phosphate (P_i) are essential to many vital physiological processes. Consequently the maintenance of Ca²⁺ and P_i homeostasis is essential to a healthy existence. This occurs through the concerted action of intestinal, renal, and skeletal regulatory mechanisms. Ca²⁺ and P_i handling by these organs is under tight hormonal control. Disturbances in their homeostasis have been linked to pathophysiological disorders including chronic renal insufficiency, kidney stone formation, and bone abnormalities. Importantly, the kidneys fine‐tune the amount of Ca²⁺ and P_i retained in the body by altering their (re)absorption from the glomerular filtrate. The ion transport proteins involved in this process have been studied extensively. Recently, new key players have been identified in the regulation of the Ca²⁺ and P_i balance. Novel regulatory mechanisms and their implications were introduced for the antiaging hormone klotho and fibroblast growth factor member 23 (FGF23). Importantly, transgenic mouse models, exhibiting disturbances in Ca²⁺ and P_i balance, have been of great value in the elucidation of klotho and FGF23 functioning. This review highlights the current knowledge and ongoing research into Ca²⁺ and P_i homeostasis, emphasizing findings from several relevant knockout mouse models.

• 1,25‐Dihydroxyvitamin‐D₃
• calcium
• fibroblast growth factor 23
• klotho
• phosphate
• sodium‐phosphate cotransporter
• TRPV5
• TRPV6

Introduction

The tight control of plasma calcium (Ca²⁺) and phosphate (P_i) levels is essential to the performance of many vital physiological functions. Muscle contraction, blood clotting and neuronal excitation all require Ca²⁺, whereas P_i is vital to intracellular signaling, as a component of membrane lipids and to build the backbone of DNA. Moreover, significant elements of bone are Ca²⁺ and P_i1, 2. Several organs contribute to the exquisite regulation of Ca²⁺ and P_i homeostasis by facilitating intestinal absorption, bone (de)mineralization, and renal excretion/reabsorption of both ions. Regulation of these processes occurs by a number of hormones. The biologically active form of vitamin D (1,25‐dihydroxyvitamin D₃), parathyroid hormone (PTH), and calcitonin have been extensively studied in this regard. More recently, fibroblast growth factor member 23 (FGF23) and klotho have been identified as new players essential to the regulation of Ca²⁺ and P_i homeostasis 3–5. Remarkably, large alterations in dietary P_i and Ca²⁺ intake produce only small alterations in the circulating levels of these ions due to the combined action of these signaling molecules on bone, intestinal, and renal Ca²⁺ and P_i transport 2, 6, 7. At times these regulatory processes are overwhelmed producing disturbances in Ca²⁺ and P_i homeostasis. Patients with chronic renal insufficiency (CRI) highlight pathophysiology complicated by altered Ca²⁺ and P_i handling. These individuals have a decreased glomerular filtration rate, a decreased circulating plasma 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃) level and consequently develop secondary hyperparathyroidism 8. This review highlights the physiological mechanisms governing Ca²⁺ and P_i transport, two intimately related processes, and the clinical implications of their altered regulation.

Key messages

• Maintenance of normal phosphate (P_i) and calcium (Ca²⁺) homeostasis is crucial to many vital physiologic processes including cellular signaling, DNA structure, bone mineralization, muscle contraction, blood clotting, and neuronal excitation.

• Fibroblast growth factor member 23 (FGF23), klotho, parathyroid hormone, and 1,25‐dihydroxyvitamin‐D₃ are key regulators of Ca²⁺ and P_i homeostasis. Their concerted action alters (re)absorption from intestine, kidney, and bone via recently identified pathways.

• The concerted interplay of klotho and FGF23 contributes substantially to the regulation of renal P_i handling and may provide a therapeutic target for chronic renal insufficiency‐related hyperphosphatemia and other disorders involving disturbances of P_i balance.

Physiology of Ca²⁺ and P_i homeostasis

Ingested Ca²⁺ and P_i are absorbed by different segments of the small intestine. The active absorption of sodium (Na⁺) throughout the entire course of the intestine results in a large net water absorption. This mainly occurs in the small intestine, where Ca²⁺ and P_i are concomitantly taken up in a passive, paracellular manner, down their concentration gradient. The active transcellular absorption of P_i occurs predominately from the ileum 9, whereas active Ca²⁺ transport takes place largely from the duodenum 10.

The vast majority of whole body Ca²⁺ and P_i is stored as the mineral hydroxylapatite in the skeleton. In blood 45% of Ca²⁺ is present in a free, ionized form, 45% is bound to proteins, and a small fraction, 10%, forms complexes with anions including citrate, sulphate, and phosphate. Plasma P_i is present in its inorganic form and as a component of several organic substances including sugars, phosphoproteins, and high‐energy phosphates. The tightly regulated renal elimination of electrolytes, including Ca²⁺ and P_i, maintains a near constant plasma level of ions and significantly contributes to whole body homeostasis. Free Ca²⁺ and P_i are filtered by the glomerulus and gain entry into the renal tubule. In contrast to the gastrointestinal system, P_i is largely absorbed in a transcellular, Na⁺‐dependent manner in the proximal tubule (PT). This process is dependent on the electrochemical gradient present for Na⁺11. Renal Ca²⁺ absorption occurs paracellularly in the PT and the thick ascending limb of Henle (TAL). A small (10%–15%) highly regulated amount of Ca²⁺ is actively absorbed from the distal convoluted tubule (DCT) and the connecting tubule (CNT) 10, 11. A diverse array of ion transport proteins, localized to the apical and basolateral membranes of intestinal and renal epithelia, mediate this active transport of Ca²⁺ and P_i. The entry of these ions, from lumina into the cytosol of the specific epithelium, is tightly regulated and represents the rate‐limiting step in transcellular Ca²⁺ and P_i (re)absorption 10.

Ca²⁺ and P_i transport proteins

The ability of the small intestine to actively absorb Ca²⁺ and P_i is hormonally regulated and occurs from the duodenum and ileum, respectively. Active intestinal Ca²⁺ absorption begins with its luminal entry mediated by the epithelial Ca²⁺ channel, TRPV6 12. Ca²⁺ is subsequently shuttled to the basolateral side of the cell via the Ca²⁺‐binding protein, calbindin‐D_9K. This process is completed by transport of Ca²⁺ back into the bloodstream via the plasma membrane Ca²⁺‐adenosine triphosphatase (PMCA1b) 13.

The Na⁺‐dependent P_i cotransporter type IIb (NaPi‐IIb) is localized to the brush border of ileum. There it is responsible for active P_i transport from the intestinal lumen into the blood 9, 14. NaPi‐IIb is also present, although to a significantly decreased extent, in duodenum and jejunum, consistent with less overall active P_i reabsorption from these locales 9. The mechanism and identity of the molecules responsible for the extrusion of P_i back into the blood is not known at present.

After intestinal absorption into the blood, Ca²⁺ and P_i are filtered across the glomerulus into the tubular lumen of the nephron. Along the course of the nephron passive, paracellular Ca²⁺ absorption occurs from the PT and TAL. A three‐step process mediates the active Ca²⁺ reabsorption mechanism from the DCT and CNT. First, Ca²⁺ enters the cell via the epithelial Ca²⁺ channel TRPV5 (and to a lesser extent TRPV6). Then Ca²⁺ diffuses through the cytosol via calbindin‐D_28K, to the basolateral side of the cell. There, Ca²⁺ is extruded into the peritubular capillary by the Na⁺‐Ca²⁺‐exchanger (NCX1) and PMCA1b 10. Active P_i absorption occurs in the PT of the kidney via the Na⁺‐dependent P_i cotransporters, NaPi‐IIa and NaPi‐IIc. Both display high expression levels in the early segments (S1 and S2) of the PT 2, 11, 15–17. Figure 1 summarizes the transcellular pathways of Ca²⁺ (A) and P_i (B) transport in kidney and intestine.

Figure 1 Transcellular Ca²⁺ and P_i transport. Active Ca²⁺ and P_i transport across renal and intestinal epithelium. A: Ca²⁺ influx is mediated by TRPV5 in the renal distal convoluted and connecting tubules and TRPV6 in the duodenum. Subsequently, Ca²⁺ is transported across the cell to the basolateral membrane by Ca²⁺‐binding proteins calbindin‐D_28K (kidney) and calbindin‐D_9K (intestine). Extrusion into the blood takes place via the plasma membrane Ca²⁺‐ATPase (PMCA1b) in the intestine and sodium‐Ca²⁺ exchanger (NCX1) and PMCA1b in the kidney. B: The ileal brush border, sodium‐dependent P_i cotransporter, NaPi‐IIb, mediates entry of P_i into enterocytes. In the proximal tubules, NaPi‐IIa and NaPi‐IIc are responsible for P_i entry into the tubular epithelial cells. The transport mechanisms involved in basolateral extrusion of P_i into blood are unknown both in the intestine and kidney.

Hormonal regulation of transcellular Ca²⁺ (re)absorption

Exquisite regulation of TRPV5 and TRPV6 activity is essential to whole body Ca²⁺ homeostasis. Here, we review the regulatory effects of the classically described hormones: PTH, 1,25(OH)₂D₃, and calcitonin on active Ca²⁺ transport processes. Later we will discuss the newly identified regulators of Ca²⁺ homeostasis: tissue kallikrein (TK), the antiaging hormone klotho, and estrogen. Central to this process is the Ca²⁺‐sensing receptor (CaSR), which is expressed in the parathyroid gland where it senses blood Ca²⁺ levels 18, 19. In response to changes in blood Ca²⁺ concentrations, the CaSR regulates the release of PTH into the circulation (Figure 2). Specifically, hypocalcemia inhibits the CaSR that in turn promotes the secretion of PTH. This hormone stimulates Ca²⁺ mobilization from bone and the conversion of inactive vitamin D to 1,25(OH)₂D₃ by the renal cytochrome P450 enzyme 25‐hydroxyvitamin D₃‐1α‐hydroxylase (1αOHase) 20. Increased 1,25(OH)₂D₃ levels activate vitamin D receptor (VDR)‐mediated gene transcription, resulting in an increased transcription of Ca²⁺ transport proteins. Ultimately this results in the stimulation of Ca²⁺ (re)absorption 3, 21–24. Hypercalcemia has the opposite effect. This state activates the CaSR, thereby inhibiting PTH release and stimulating the secretion of calcitonin from the parafollicular cells of the thyroid. Calcitonin decreases osteoclast‐mediated bone resorption and promotes lowering the blood Ca²⁺ concentration 25.

Figure 2 Key players in Ca²⁺ and P_i homeostasis. The concerted interplay of intestinal uptake, reabsorption in kidney, and bone (de)mineralization establishes the maintenance of a normal Ca²⁺ and P_i balance. The calcium sensing receptor (CaSR), present in the thyroid gland, senses blood Ca²⁺ levels and triggers the secretion of the calcitropic hormones parathyroid hormone (PTH) and calcitonin. Ovarian‐produced estrogen stimulates Ca²⁺ reabsorption also. Similar hormones are involved in the regulation of P_i balance. Blood P_i levels are controlled by PTH, 1,25(OH)₂D₃, klotho, and fibroblast growth factor member 23 (FGF23). A negative feedback mechanism prevents the accumulation of FGF23 and klotho since 1αOHase‐mediated 1,25(OH)₂D₃ production is inhibited by FGF23 and klotho.

Estrogen contributes importantly to Ca²⁺ homeostasis by promoting bone mineralization and stimulating renal TRPV5 expression. This results in increased active Ca²⁺ reabsorption, independently of 1,25(OH)₂D₃26–28. PTH, 1,25(OH)₂D₃, and estrogen exert their effect on renal‐mediated Ca²⁺ handling by altering, at the transcriptional level, the expression of Ca²⁺ transporters 7. This has been confirmed, in vivo, where a direct stimulatory effect of PTH on renal Ca²⁺ transporter expression levels was demonstrated in PTH‐supplemented parathyroidectomized rats 29, and where the dietary supplementation of Ca²⁺ and the administration of estrogen to ovariectomized rats was shown to alter renal TRPV5 expression, all independently of 1,25(OH)₂D₃7, 23, 28.

The TK knockout (TK^−/−) mouse displays significant renal Ca²⁺ wasting compared to control mice, implicating an important role for TK in the regulation of active Ca²⁺ reabsorption 30. In 2006, Gkika et al. described the molecular mechanism through which TK stimulates TRPV5‐mediated active Ca²⁺ reabsorption. TK, a proteolytic enzyme that is secreted into the tubular fluid by renal epithelial cells, activates the bradykinin receptor stimulating the phospholipase C/1,2‐diacylglycerol/protein kinase C (PLC/DAG/PKC) pathway. This results in increased plasma membrane expression of TRPV5, possibly due to an inhibition of TRPV5 endocytosis. Regardless of the exact mechanism, TK increases TRPV5‐mediated Ca²⁺ transport 31.

Recently the antiaging hormone klotho has also been implicated in the regulation of the Ca²⁺ balance. Klotho knockout (klotho^−/−) mice display hypercalcemia, hyperphosphaturia, hypercalciuria, and other manifestations resembling aging 32. Chang et al. demonstrated that klotho stimulates TRPV5‐mediated Ca²⁺ transport in vitro, revealing a novel regulatory mechanism of active Ca²⁺ transport 33. Klotho is a type I transmembrane protein with β‐glucuronidase activity. This activity is contained in the extracellular domain that can be secreted into the blood, urine, and cerebrospinal fluid 32, 34. Klotho hydrolyzes extracellular N‐linked oligosaccharides present on TRPV5, entrapping the channel in the plasma membrane. This stimulatory effect of klotho on TRPV5 substantiates the important role of this antiaging hormone in Ca²⁺ homeostasis, specifically by altering the luminal membrane Ca²⁺ permeability of the DCT and the cortical collecting duct (CCD) 33. Alternatively, the mechanism of hypercalciuria observed in klotho^−/− mice has been suggested to be the result of decreased proximal Ca²⁺ absorption because of a decreased Na⁺K⁺‐ATPase activity 35.

Hormonal regulation of transcellular P_i (re)absorption

Our understanding of the hormonal control of P_i balance is rapidly evolving. Klotho and FGF23 were recently identified as new key players involved in P_i regulation, adding to what had already been delineated about the classical PTH/1,25(OH)₂D₃ regulatory pathway. PTH, 1,25(OH)₂D₃, and dietary P_i intake are known to alter P_i (re)absorption 36. An increased intestinal NaPi‐IIb expression can be stimulated by either 1,25(OH)₂D₃ and/or low P_i intake, ultimately resulting in elevated blood P_i6. Conversely, PTH decreases blood P_i levels by inhibiting P_i reabsorption from the PT. This occurs via an acute redistribution of NaPi‐IIa proteins from the brush border to intracellular lysosomes, targeting them for degradation. The overall effect of PTH is therefore to lower blood P_i levels by stimulating P_i excretion 37.

Recently, FGF23 has been labeled a phosphatonin, due to its role in P_i homeostasis 5, 38–40. Injection of FGF23 into mice results in hypophosphatemia by stimulating renal P_i excretion. This is achieved by decreasing the expression of renal NaPi‐IIa and NaPi‐IIc and inhibiting 1αOHase production. The role of FGF23 in human Ca²⁺ and P_i homeostasis has been firmly established by genetic linkage analysis. Inactivating mutations in FGF23 have been identified in patients with tumoral calcinosis causing hyperphosphatemia, and activating mutations have been shown to cause autosomal dominant hypophosphatemic rickets (ADHR) 41–44. FGF23 is a secreted, transmembrane protein produced in bone osteoblasts 45, 46, which can bind to and activate FGF receptors (FGFRs) 47, 48. Further, the involvement of klotho in FGF23 signaling was suggested as klotho^−/− mice and FGF23 knockout (FGF23^−/−) mice exhibit many common phenotypic features including hyperphosphatemia 32, 38, 49. This was confirmed in 2006, when Kurosu et al. demonstrated that klotho forms a complex with FGFRs. This new complex binds FGF23 with a higher affinity than either the FGFR or klotho alone 50. Two separate groups extended this observation by showing that direct binding of klotho to the FGFR1(IIIc) subtype converts this receptor into a specific FGF23 receptor 48, 51. In addition, Ogawa et al. demonstrated that another member of the klotho family, β‐klotho, can function as a cofactor in FGF21 signaling, through a similar interaction as with klotho and FGF23 52. FGF21 is expressed predominantly in the liver and is a metabolic regulator of glucose uptake in adipocytes, consequently it provides a potential therapeutic target in diabetes mellitus 53. Other FGF family members have been implicated in varying signal transduction pathways throughout the body 54. A recent report revealed that FGF7 is overexpressed in tumors associated with renal P_i wasting and osteomalacia 55. FGF7 inhibits P_i transport in renal epithelial cells, although the exact molecular mechanisms involved still need to be elucidated. Further investigation is necessary to determine whether other FGF superfamily members are involved in the regulation of Ca²⁺ and P_i homeostasis.

Hypercalcemia and hypocalcemia

Disturbances in both serum and whole body Ca²⁺ levels can cause severe pathological conditions, the etiology of which is both complex and variable. Hypercalcemia can result from Ca²⁺ hyperabsorption from the gastrointestinal (GI) tract, decreased urinary excretion, or an increased resorption from bone. Elevated serum PTH levels, secondary to hyperparathyroidism or a hypophosphatemic state, will cause increased Ca²⁺ absorption from the GI tract. Increased Ca²⁺ loss from bone is caused by elevated PTH and/or 1,25(OH)₂D₃ levels or skeletal metastasis, while severe dehydration will increase serum Ca²⁺ concentration without altering the total amount in blood. Symptoms and findings of hypercalcemia include fatigue, electrocardiogram abnormalities, nausea, vomiting, constipation, anorexia, abdominal pain, hypercalciuria, and consequently kidney stone formation. Treatment of hypercalcemia depends on the severity of the abnormality and ranges from dietary adaptation to the administration of calcimimetic compounds that activate the CaSR in the parathyroid glands, reducing blood PTH levels 56. Hypocalcemia can result in muscle cramping, depression, psychosis, and seizures. Causes include decreased Ca²⁺ absorption due to a poor intake, 1,25(OH)₂D₃ deficiency or resistance, lack of sunlight, decreased bone resorption, a complication of thyroid surgery (i.e. parathyroidectomy), or renal Ca²⁺ wasting. Oral Ca²⁺ and 1,25(OH)₂D₃ supplementation and ultraviolet light exposure are the current treatments for hypocalcemia.

Hypercalciuria

Hypercalciuria is a risk factor for renal Ca²⁺ stone formation and therefore contributes to this significant health and socioeconomic problem 57, 58. In the United States (US) more than 5% of the population will develop a clinically significant episode of kidney stone disease in their lifetime of which the economic impact is approximately $2 billion annually 59, 60. Hypercalciuria has been classified into at least three different forms. Absorption hypercalciuria is due to Ca²⁺ hyperabsorption from the GI tract, renal hypercalciuria is the result of a defect in renal Ca²⁺ reabsorption, whereas resorptive hypercalciuria manifests urinary Ca²⁺ wasting secondary to increased bone degradation. Other than these three types of hypercalciuria, the largest group of patients with this disorder lack an explanation for their increased Ca²⁺ excretion and have been classified as having idiopathic hypercalciuria. Individuals with a family history of nephrolithiasis are themselves prone to develop kidney stones. This strongly suggests that genetic factors are involved in the pathogenesis of idiopathic hypercalciuria 61. There is a myriad of potential disturbances in Ca²⁺ homeostasis that can cause hypercalciuria. Thus, hypercalciuria may be monogenic, polygenic, or multifactorial in its etiology. As TRPV5 as well as TRPV6 gene ablation in mice leads to severe forms of hypercalciuria, these channels were proposed as candidate genes for hypercalciuria 1, 62. To date, mutation analyses of the TRPV5‐encoding gene has not revealed a primary role for TRPV5 in autosomal dominant idiopathic hypercalciuria 63. However, the involvement of TRPV5 or TRPV6 in hypercalciuria has not been definitively excluded. Specific single nucleotide polymorphisms (SNPs) or combinations of SNPs in TRPV5/TRPV6 may modulate channel activity, and might therefore be responsible for altered renal Ca²⁺ excretion 64. Further investigation is necessary to identify mutations in TRPV5 and TRPV6 associated with disease.

Hyperphosphatemia and hypophosphatemia

Autosomal dominant hypophosphatemic rickets (ADHR) is a hereditary disorder characterized by bone malformation and renal P_i wasting. Activating mutations in the FGF23 gene have been identified in these patients 41, 65. Related diseases include X‐linked hypophosphatemia (XLH) and hereditary hypophosphatemic rickets with hypercalciuria (HHRH); both are characterized by disturbances in P_i homeostasis. Tumor‐induced osteomalacia (TIO) is characterized by hypophosphatemia secondary to renal P_i wasting and reduced blood 1,25(OH)₂D₃ levels 66, 67. Whilst the molecular identity of all the molecules responsible for these clinical disorders have yet to be identified, in a few instances genes involved in the maintenance of P_i homeostasis have been implicated other than FGF23 68, 69. Hypophosphatemia causes decreased bone mineralization and subsequently bone fragility, pain, rickets, and growth retardation 69. Treatment is aimed at the replacement of P_i and/or 1,25(OH)₂D₃.

Deactivating mutations in FGF23 cause hyperphosphatemic tumoral calcinosis, characterized by hypervitaminosis D and increased intestinal and renal P_i absorption 43. Consistent with this, FGF23^−/− mice exhibit severe hyperphosphatemia further substantiating the regulatory role of FGF23 in P_i homeostasis 49. In humans, renal insufficiency, malignancy, drug abuse, or hypoparathyroidism can lead to hyperphosphatemia. Treatment is with P_i binders or directed at the primary cause.

CRI

CRI is the progressive loss of renal function evinced by a decreasing glomerular filtration rate. Clinical symptoms and findings include hypertension, edema, hyperkalemia, hypocalcemia secondary to a decreased serum 1,25(OH)₂D₃ level, secondary hyperparathyroidism, and hyperphosphatemia. Disorders with disturbances in both Ca²⁺ and P_i homeostasis, of which CRI is just one, can lead to severe bone, cardiovascular, and other systemic diseases. CRI patients ultimately develop end stage renal disease and require renal replacement therapy, i.e. dialysis or kidney transplantation. In order to minimize the burden of this disease it is imperative to attempt normalization of the Ca²⁺ and P_i balance in these patients 70.

Elevated blood FGF23 levels are detectable in CRI patients with secondary hyperparathyroidism. This is thought to represent a compensatory mechanism in an attempt to excrete excess P_i and thus lower blood P_i levels. The klotho^−/− mice exhibit increased blood FGF23 levels as well, possibly due to their apparent hyperphosphatemia 71, 72. As klotho has been implicated in the stimulation of TRPV5‐mediated Ca²⁺ reabsorption and in the regulation of FGF23‐mediated P_i reabsorption, both klotho and FGF23 may play significant roles in the pathogenesis, treatment options and prediction of the prognosis of CRI 73–75.

Lessons from TRPV5 and TRPV6 knockout mice

The generation and characterization of TRPV5 knockout (TRPV5^−/−) mice confirmed this epithelial Ca²⁺ channel to be the gatekeeper of active Ca²⁺ reabsorption 1. TRPV5^−/− mice display severe hypercalciuria compared to control (TRPV5^+/+) mice that are normocalcemic. The knockout mice display hypervitaminosis D and upregulation of intestinal TRPV6 and calbindin‐D_9K, as a compensatory mechanism for their severe renal Ca²⁺ wasting. Cross‐breeding of TRPV5^−/− and 1αOHase knockout (1αOHase^−/−) mice was performed in order to address the role of the increased blood 1,25(OH)₂D₃ levels in these animals. TRPV5/1αOHase double knockout mice have decreased intestinal TRPV6 and calbindin‐D_9K expression, hypocalcemia, and severe bone abnormalities compared to TRPV5^−/− mice. These findings support the notion that the hypervitaminosis D observed in TRPV5^−/− mice is responsible for the upregulation of intestinal Ca²⁺ transport proteins, and the consequent intestinal Ca²⁺ hyperabsorption, likely as compensation for the renal Ca²⁺ leak 76. Notably, TRPV5^−/− mice have acidic urine and are polyuric relative to their control littermates 1. Both of these symptoms would act to promote the excretion of large amounts of Ca²⁺ without it being precipitated in the collecting ducts. TRPV5^−/− mice display significant hyperphosphaturia, predisposing them to an increased risk of Ca²⁺ phosphate precipitation. The molecular mechanism responsible for the renal P_i leak in TRPV5^−/− mice remains unknown.

The TRPV5^−/− mice show a greatly diminished expression of calbindin‐D_28K. To assess whether the absence of TRPV5 or the downregulation of calbindin‐D_28K was responsible for the phenotype observed in the TRPV5^−/− animals, double knockout mice, for both TRPV5 and calbindin‐D_28K, were generated. These mice do not display a further increase in their Ca²⁺ loss relative to TRPV5^−/− mice, confirming that TRPV5 and not calbindin‐D_28K is the rate‐limiting transporter in active Ca²⁺ reabsorption 77.

In vivo studies of specific knockout mice models significantly contribute to our knowledge of renal and intestinal Ca²⁺ handling. Recently, Bianco et al. generated the TRPV6 knockout (TRPV6^−/−) mouse in order to assess the role of this channel in intestinal Ca²⁺ absorption in vivo and its involvement in other organ systems 62. Surprisingly, these mice display skin abnormalities due to a decreased Ca²⁺ content in their epidermis and have impaired fertility. Consistent with known TRPV6 localization the knockout animals exhibit defective intestinal Ca²⁺ absorption as well as significant hypercalciuria and decreased bone mineralization compared to control littermates 62. Further, TRPV6^−/− mice have secondary hyperparathyroidism and hypervitaminosis D such that they are normocalcemic. These findings indicate that TRPV6 is central to 1,25(OH)₂D₃‐regulated Ca²⁺ absorption and overall Ca²⁺ homeostasis. Finally, the exhibition of abnormalities in multiple organs in TRPV6^−/− mice underlines the importance of this channel in other body tissues. While TRPV5 clearly functions as a gatekeeper of active Ca²⁺ reabsorption from the lumen of DCT and CNT, these recent findings suggest that TRPV6 may be an important regulator of Ca²⁺ transport during embryonic and placental development. Maintenance of normal Ca²⁺ levels is crucial for physiological functioning of the uterus and placenta, including both smooth muscle contraction and embryo implantation. Recently, Lee et al. demonstrated that estrogen regulates TRPV6 expression levels during pregnancy, which is important for normal uterine function 78. Studies of TRPV6^−/− mice during embryogenesis and development will be important to elucidate the exact role of TRPV6 and of its contribution to Ca²⁺ homeostasis during different stages of organogenesis, especially in the intestine and kidney.

Insights into P_i and Ca²⁺ homeostasis from FGF23 and klotho knockout mice

The recent generation of FGF23^−/− mice, by two independent research groups, confirmed a central role for this hormone in P_i homeostasis 38, 49. The FGF23^−/− mice display hyperphosphatemia, hypervitaminosis D, growth retardation and limb deformation. Crossing the FGF23^−/− mice with 1αOHase‐deficient animals rescued much of this phenotype 79. Specifically the atherosclerosis and ectopic skeletal and soft tissue calcifications observed in the FGF23^−/− animals are absent from the FGF23/1αOHase double knockout mice. The double knockout animals also have increased survival. Of note the phenotype is not rescued completely, the FGF23/1αOHase double knockout mice have a further reduction in bone mineral density in comparison to the FGF23^−/− mice. Together these findings strongly suggest that the aging‐like features, disturbed electrolyte levels, and impaired skeletogenesis in FGF23^−/− mice are the result of increased blood 1,25(OH)₂D₃ levels 79, 80. To further elucidate the role of 1,25(OH)₂D₃ action in FGF23^−/− mice, Hesse and co‐workers cross‐bred FGF23^−/− mice with VDR knockout (VDR^−/−) mice 81. FGF23/VDR double knockout mice display a similar phenotype to the VDR^−/− mice, suggesting that the aging‐like symptoms in FGF23^−/− mice depend upon intact signaling through the VDR 82. It is known that FGF23 expression is itself regulated by dietary P_i and 1,25(OH)₂D₃, suggesting a regulatory feedback mechanism in which 1,25(OH)₂D₃ stimulates FGF23 production, that in turn inhibits 1αOHase, thereby decreasing circulating 1,25(OH)₂D₃ levels 83, 84.

Klotho^−/− mice display a phenotype similar to FGF23^−/− mice, which includes hyperphosphatemia, hypervitaminosis D and bone abnormalities 32. The fact that the interplay of klotho and FGFRs is essential for FGF23 functioning and the finding that both klotho^−/− and FGF23^−/− mouse strains display comparable symptoms indicates that this newly identified klotho/FGF23 regulatory pathway is essential to P_i homeostasis 48, 50. Klotho^−/− mice also demonstrate other phenotypic features not described in FGF23^−/− mice, including pulmonary, neuronal, and skin disorders, implying that klotho may interact with other FGFRs throughout the body 32, 50. How klotho causes these additional phenotypic features and whether other FGF family members mediate them requires further investigation.

Outlook

The work described in this review has greatly increased our knowledge of Ca²⁺ and P_i homeostasis; however, several questions remain. Identification of the antiaging hormone klotho as a modulator of TRPV5 activity and its interaction with FGFRs permitting increased FGF23 signaling have been major breakthroughs in the understanding of both Ca²⁺ and P_i handling. An important tool in the elucidation of these research questions has been the use of knockout animal models. In this regard, it remains a challenge to determine the exact role of 1,25(OH)₂D₃ in both klotho‐ and FGF23‐mediated signaling. It will be through further investigation of these newly identified regulatory pathways, including klotho, FGF23, and 1,25(OH)₂D₃, that a clearer understanding of how the body controls Ca²⁺ and P_i balance will be achieved. This knowledge will be crucial for the manipulation of either klotho or FGF23 to a therapeutic end point.

Acknowledgements

This work was financially supported in part by grants from the Dutch Kidney Foundation (C03.6017 and C06.2170), the Netherlands Organization for Scientific Research (Zon‐Mw 016.006.001, NWO‐ALW 814.02.001, NWO‐CW 700.55.302), and the Dutch Stomach‐Intestine‐Liver foundation (MWO 03‐19). J. Hoenderop and R. T. Alexander are supported by an EURYI and a CIHR Clinician‐Scientist award, respectively. We thank the members of our laboratory for valuable discussion and advice.