Disorders of Calcium Metabolism

The Calcium Sensing Receptor

The maintenance of calcium homeostasis requires that a variety of cells within the body have the ability to measure the extracellular ionized calcium concentration of the ECF. This is accomplished primarily through the actions of a 7 transmembrane-spanning G protein-coupled receptor (GPCR) known as the calcium sensing receptor (CaSR). The CaSR is a member of subfamily C of GPCRs and is related to the metabotropic glutamate, pheromone and taste receptors. Evolutionarily, it is descended from periplasmic binding proteins in bacteria that serve as nutrient receptors. The human CaSR gene is located on chromosome 3q13.3–21 and consists of 7 exons and two distinct promoter regions. Expression of the CaSR gene is widespread although it is highest in organs that are involved in the regulation of calcium homeostasis, such as the parathyroid glands, the C-cells of the thyroid gland, kidney and bone. The regulation of CaSR mRNA production has not been as extensively studied as other aspects of its physiology, but gene expression has been shown to respond to calcium, vitamin D and cytokines such as interleukin 1β and interleukin 6. Regulation of the CaSR and vitamin D receptor are interrelated. Calcium and vitamin D both upregulate CaSR gene expression. Activation of the CaSR also upregulates expression of the vitamin D receptor (VDR), which, in turn, amplifies the effects of vitamin D on CaSR expression. These interactions may be particularly important in the parathyroid to allow for synergistic activity of calcium and vitamin D in regulating PTH gene expression and secretion.

In humans, the mature CaSR encompasses 1078 amino acids. It has a large extracellular domain of 612 amino acids that is heavily glycosylated and serves as the calcium-binding domain. The CaSR operates as a homodimer, which forms as the result of disulfide bonds between cysteines 129 and 131 of each monomer. The resulting extracellular portions of the complex have been suggested to approximate a Venus flytrap based on molecular modeling derived from the crystal structure of the related metabotropic glutamate receptors. Binding of calcium to clusters of acidic residues within the extracellular domain causes closure of the extracellular “flytrap” motif and this leads to conformational changes that activate intracellular signaling. The intracellular portion of the receptor has been shown to couple to G _q/11 , G _i , G _12/13 and in some instances to G _s . Most commonly, the receptor has been described to activate intracellular calcium transients and MAPK signaling cascades. The CaSR also interacts with several other cytoplasmic and/or membrane proteins, including caveolin, filamin, potassium channels (Kir4.1 and Kir4.2) and receptor activating proteins (RAMP) 1 and 3, all of which can modulate downstream receptor signaling. Finally, the receptor has been shown to heterodimerize with several metabotropic glutamate receptor isoforms and GABA receptor isoforms to generate unique hybrid receptors. Therefore, like other GPCRs, the CaSR can signal through a variety of pathways and there is likely to be considerable cell type-specific variation in its signaling.

In addition to calcium, the CaSR binds and signals in response to a variety of other cations and positively charged organic molecules such as magnesium, gadolinium, polyamines and aminoglycoside antibiotics. These molecules can activate the receptor in the absence of calcium and are known as Type 1 ligands. Other ligands, known as Type 2 agonists, have no activity independent of calcium, but instead can modify the sensitivity of the receptor to calcium. These include L-amino acids and small-molecule, allosteric activators and inhibitors known as calcimimetics or calcilytics. L-amino acids bind to the extracellular domain of the receptor in physiologic concentrations and may help to coordinate protein and calcium metabolism. Calcimimetics and calcylitics bind to the transmembrane domain of the receptor and stabilize the active or inactive conformations of the receptor respectively. These compounds have been developed as drugs; cinacalcet (Sensipar) is commercially available for the treatment of hyperparathyroidism in chronic kidney disease (CKD) while calcilytics are currently in clinical trials. The CaSR has also been shown to respond to alterations in pH, osmolality and salinity. Therefore, although its actions are critical to the regulation of calcium homeostasis, the CaSR actually is a multifunctional sensor for varied alterations in the extracellular milieu.

The CaSR is expressed widely throughout the body. It has been implicated in a variety of actions including calcium and other ion transport, the regulation of cellular proliferation and differentiation, and hormone secretion. We will limit our discussion to its functions in the three tissues of greatest importance to calcium homeostasis, the parathyroid gland, the kidney and bone. The reader is referred to other more comprehensive reviews for discussion of its many other functions.

Parathyroid Gland : The parathyroid gland expresses high levels of the CaSR, which regulates three important aspects of parathyroid physiology: parathyroid hormone ( PTH) gene expression, PTH secretion and parathyroid cell proliferation. Signaling from the CaSR leads to a reduction in PTH mRNA. This effect may, in part, be indirect and mediated by changes in the sensitivity of suppression of PTH gene expression by vitamin D. However, there is also a specific calcium response element in the PTH gene promoter located 3.6 kB upstream from the transcription start site that acts to inhibit transcription. In addition, as described in more detail in the Section on PTH, CaSR signaling destabilizes PTH mRNA and shortens its half-life. Both effects result in a reduction of PTH synthesis.

The predominant effect of CaSR activation on PTH production is inhibition of PTH secretion. Activation of the CaSR leads to a prompt suppression of PTH release, an effect that involves coupling to G _q/11 and G _i , and the generation of intracellular calcium transients after receptor activation. The control of PTH secretion by extracellular calcium is reviewed in more detail in the section on PTH, but genetic experiments in mice have shown that the coupling of PTH secretion to changes in extracellular calcium is completely dependant on the presence of the CaSR on parathyroid cells. Furthermore, the number of functional receptors defines the dose-response relationship between extracellular calcium and PTH secretion by parathyroid cells. Reduced numbers of receptors are found in some patients with familial hypocalciuric hypercalcemia as well as in chronic kidney disease (CKD) and in parathyroid adenomas. In each instance, the parathyroid cells become partially resistant to the ability of extracellular calcium to suppress PTH secretion properly.

The CaSR also regulates parathyroid gland mass, and null mutations in the CaSR gene result in parathyroid hyperplasia. Part of this effect is indirect and is mediated by the ability of CaSR signaling to enhance parathyroid sensitivity to vitamin D. However, experiments in mice deficient in the VDR or 1 α-hydroxylase (CYP27B1) have demonstrated a direct effect of CaSR signaling on parathyroid hyperplasia as well. The molecular details of how CaSR affects cell proliferation and/or hypertrophy are not yet well described.

The Kidney : Experiments performed prior to the identification of the CaSR had suggested that extracellular calcium had direct effects on renal calcium handling that were independent of any effects of PTH. However, the identification of the CaSR and the recognition of its expression throughout the renal tubule have led to the elucidation of an integrated and intrinsic response of the kidney to changes in circulating calcium levels. In fact, recent experiments in mice have suggested that CaSR-mediated alterations in renal calcium handling may be the principal defense against hypercalcemia, while PTH-mediated responses have a more potent effect on the correction of hypocalcemia. When activated by hypercalcemia, the CaSR affects different portions of the nephron to increase calcium excretion, and to acidify the urine and impair renal concentrating ability in order to protect against nephrocalcinosis or nephrolithiasis in the face of the resulting hypercalciuria.

The CaSR promotes renal calcium excretion through several actions. In the thick ascending loop of Henle (TAL), the CaSR is expressed on the basolateral surface and activation of the receptor leads to inhibition of paracellular and transcellular NaCl and divalent cation reabsorption. These effects are primarily mediated by inhibition of the activity of the renal outer medullary potassium channel (ROMK) and the apical Na ⁺ -K ⁺ -2Cl ⁻ cotransporter, NKCC2, which reduces the net lumen positive charge and lowers the electrochemical driving force for paracellular calcium, sodium and magnesium transport. In addition, CaSR signaling results in the degradation of claudin 16, which promotes paracellular transport of calcium and magnesium. Activation of the CaSR also inhibits PTH-mediated calcium reabsorption in the CTAL by inhibiting uptake of calcium across the apical membrane and in the CTAL and distal convoluted tubule by inhibiting the activity of the basolateral calcium pump, PMCA1b, which is necessary for extrusion of calcium from the cell into the ECF.

Concurrent with its effects on calcium excretion, the CaSR also increases acid and water excretion by the kidney, both of which help to maintain the solubility of calcium salts in the urine and prevent the precipitation of the excreted calcium. Activation of the CaSR in the collecting ducts leads to inhibition of the H ⁺ -ATPase, which results in increased acid secretion. Recent genetic studies have underscored the importance of urine acidification in protecting against stone formation in response to hypercalciuria, at least in mice. Hypercalcemia is also well known to inhibit renal concentrating ability. Stimulation of apical membrane CaSRs in the inner medullary collecting duct inhibits vasopressin induced aquaporin 2 (AQ2) insertion into the apical membrane and reduces the transcellular reabsorption of water. In chronic states of hypercalcemia, the CaSR also downregulates AQ2 expression via post-transcriptional mechanisms.

In addition to the direct effects of the CaSR on renal calcium handling, it also inhibits CYP27B1 activity in the proximal tubule. This results in lower levels of 1,25 (OH) ₂ -vitamin D (the active form of the hormone), which will also inhibit calcium absorption from the diet, the release of calcium from bone and calcium reabsorption in the distal tubule, all of which would help to lower circulating calcium concentrations.

The Skeleton : Studies in cell lines and in genetically altered mice suggest that the CaSR also affects bone cells independent of its effects on PTH secretion or 1,25 (OH) ₂ -vitamin D production. Osteoclasts, osteoblasts, osteocytes, growth plate chondrocytes and bone marrow immune cells have all been found to express the CaSR. Selective knockout of the receptor in osteoblasts has been shown to have profound effects on bone growth and mineralization due to impaired osteoblast activity. Studies in cell culture suggest that CaSR signaling may affect both osteoblast proliferation and differentiation. Low levels of CaSR signaling appear to promote osteoclast differentiation but high levels of calcium have been found to inhibit osteoclast function and cause osteoclast apoptosis. The regulation of bone cell function by the CaSR is an evolving issue and, at present, it is unclear whether the actions the CaSR on osteoclasts and osteoblasts are important in the acute regulation of systemic calcium metabolism. It is certainly possible that the CaSR might regulate the mobilization of calcium from the bone interstitial fluid in ways reminiscent of its actions on ion transport in other sites.

Parathyroid Hormone

The principal regulator of the minute-to-minute calcium concentration is parathyroid hormone. PTH is secreted by the four parathyroid glands, which arise from the third and fourth branchial arches during embryogenesis and normally reside near the posterior capsule of the four poles of the thyroid gland. However, extra or ectopic parathyroid glands or small rests of parathyroid tissue are commonly found from the angle of the jaw to the mediastinum, along the path of embryonic migration of the thymus and parathyroid glands. This can be an important consideration in hyperparathyroid states that require surgical intervention (see below).

Human PTH is encoded by a single gene located on chromosome 11 (11p15). The gene contains three exons; the first codes for the 5’ untranslated portion of the mRNA, the second encompasses most of the pre-pro sequences and the third encodes the actual protein sequence of the secreted hormone. The PTH gene is a member of a small gene family that also includes the parathyroid hormone-related protein (PTHrP) and the tubuloinfundibular peptide of 39 amino acids (TIP39) genes. Each of these genes has a similar exon/intron structure and all three bind related receptors with overlapping specificities (see below). The PTH and PTHrP genes likely arose from a common ancestor and lower vertebrates have multiple PTH and PTHrP genes.

PTH is initially synthesized as pre-pro-parathyroid peptide and undergoes post-translational modification to produce the active hormone. The 25-residue pre-sequence and the 6-residue pro-sequence are cleaved during the initial production of PTH, yielding the mature, 84-amino acid, full-length protein (PTH 1-84). Full-length PTH is the biologically active form of the hormone. It has a very short half-life (minutes) in the circulation and is degraded by the liver and kidney, which generates circulating carboxy-terminal (C-terminal) fragments. These fragments are cleared by the kidney but may accumulate in the circulation in renal failure.

Three factors, calcium, 1,25 dihydroxyvitamin D and phosphate, have been shown to modulate PTH mRNA levels and/or PTH secretion. Parathyroid cells express high levels of the CaSR, which allow them to respond to small changes in the circulating calcium concentration. Changes in extracellular calcium lead to reciprocal changes in PTH secretion (see below) and gene expression. The mechanisms through which CaSR signaling affects PTH gene expression are not well described. Although a negative calcium regulatory element has been identified in the PTH gene promoter region, it is currently thought that the primary effect of CaSR signaling is post transcriptional. PTH mRNA contains AU-rich elements in the 3’ untranslated region (UTR), which regulate its stability. In the setting of low extracellular calcium concentrations, specific proteins bind to these elements and protect the mRNA from degradation. Activation of CaSR signaling displaces these factors and shortens the half-life of PTH mRNA.

A steep inverse sigmoidal relationship exists between PTH secretion and the extracellular ionized calcium concentration and is defined by four parameters ( Figure 66.1 ). The first is the maximum secretory rate of the parathyroid glands. The second is the slope of the curve at the midpoint. The third is the parathyroid gland’s “set point,” or calcium concentration at which PTH secretion is half maximal. The final parameter is the basal, nonsuppressible rate of PTH secretion. The steep part of the curve encompasses the physiologic range for extracellular calcium, over which small changes in the concentration of ionized calcium elicit dramatic changes in the rate of PTH secretion. Because of this steep slope, ionized calcium concentrations are maintained in a very narrow physiologic range. A great deal of data in both genetically altered mice and in humans with genetic disorders of calcium sensing have demonstrated the primary importance of the CaSR in controlling PTH secretion. For example, mice with either global deletion or parathyroid-specific disruption of the CaSR gene have very high levels of circulating parathyroid hormone, which are not suppressed by severe hypercalcemia. The same is true of humans with null mutations in the CaSR gene. Furthermore, in both mice and humans, heterozygous disruption of the CaSR gene results in a milder form of hyperparathyroidism, demonstrating a dose effect between parathyroid CaSR levels and the sensitivity of PTH to calcium-mediated suppression. Conversely, humans with activating mutations in the CaSR gene present with hypoparathyroidism characterized by a failure to secrete PTH in response to hypocalcemia.

The molecular mechanisms by which CaSR signaling inhibits PTH secretion are not completely understood. Binding of calcium to the CaSR activates downstream signaling pathways that, in turn, suppress PTH secretion. It is clear that activation of both Gα _q and Gα ₁₁ is necessary for suppression of PTH secretion since parathyroid specific disruption of the genes for both of these G-proteins phenocopies the disruption of the CaSR gene itself. However, it is not clear how specific signaling events downstream of these G-proteins actually inhibit the secretion of PTH.

Vitamin D will be discussed in more detail below. The active form of vitamin D, 1,25 (OH) ₂ vitamin D, acts on parathyroid cells to inhibit PTH production, both by increasing circulating calcium levels and by inhibiting PTH gene transcription directly. This effect is thought to be a result of reduced transcription of the PTH gene due to the binding of 1,25 dihydroxyvitamin D to the vitamin D receptor (VDR), which in turn binds to vitamin D response elements in the 5’ flanking region of the PTH gene. Vitamin D receptors are abundant in parathyroid tissue and their levels are modulated by calcium and 1,25 (OH) ₂ vitamin D itself. Activation of the CaSR increases the expression of the VDR in parathyroid cells and 1,25 (OH) ₂ vitamin D increases the expression of the CaSR, thereby mutually sensitizing the parathyroid glands to negative feedback in states of vitamin D or calcium excess. In CKD and in parathyroid adenomas, expression of both the VDR and the CaSR is reduced, likely contributing to increased PTH production.

Phosphate stimulates PTH production and hyperphosphatemia is an important contributor to the development of secondary hyperparathyroidism in patients with chronic kidney disease. Some of these effects may be related to the drop in ionized calcium that attends any increase in circulating phosphate, but phosphate also exerts independent effects on PTH mRNA stability. Similar to the effects of low calcium, elevations in serum phosphate lead to the binding of a protein complex to the 3’UTR of PTH mRNA that inhibits its degradation. Changes in serum phosphate may also affect PTH indirectly via the actions of fibroblast growth factor 23 (FGF-23), which is secreted from osteocytes (see Chapter 68 ). The parathyroids respond to FGF-23 but, somewhat paradoxically, FGF-23 has been described to reduce PTH gene expression and secretion, even though it also lowers circulating phosphate levels. Therefore, it is possible that the actions of FGF-23 on the parathyroid cells may counterbalance the direct effects of phosphate on PTH gene expression. As discussed in Chapter 69, Chapter 91 , FGF-23 levels are elevated in CKD, although it is not known whether these elevated levels contribute to the dysregulation of PTH secretion in these patients. Our understanding of the regulation of parathyroid function by FGF-23 is an evolving area that will require further research for clarification.

PTH binds and activates the Type 1 PTH/PTHrP receptor (PTH1R), a GPCR that is shared with PTHrP. It is a member of the B subfamily of GPCRs, which also includes the secretin and calcitonin receptors. Two other PTH receptors are also included within this group. The PTH2R is primarily a receptor for TIP39. PTH can bind weakly to this receptor and activate it, but it is not known if this represents a physiological interaction. PTHrP cannot activate the PTH2R. There is also a third PTH receptor (PTH3R) documented in zebra fish, but it does not appear to be present within the human genome. Thus, as with the PTH peptide family, it appears that evolution has reduced the diversity of PTH receptors.

The amino-terminal portion of PTH [PTH(1–34)] is necessary and sufficient for full activation of the PTH1R. Amino acids in the C-terminal portion of this PTH fragment are thought to bind to the extracellular, amino-terminal portion of the PTH1R, while the amino-terminus of PTH(1–34) binds to the juxtamembrane portion of the PTH1R in order to activate downstream signaling ( Figure 66.2 ). The PTH1R has been shown to activate both the cAMP/protein kinase A (PKA) pathway and the phospholipase C/protein kinase C/calcium transient pathway. There is also evidence that the receptor activates phospholipase D and MAPK signaling. MAPK activation can occur either through a PLC/PKC-dependant pathway or via a G-protein independent pathway that involves arrestins. Many of the classic biological functions of PTH appear to be mediated to a great extent by the cAMP/PKA pathway, which requires the first two amino acids of PTH for activation. Less is known about the biological role of the other signaling pathways.

The PTH1R is highly expressed within the skeleton and kidney, the two classic target organs for PTH action. However, it is also widely expressed in many other cells throughout body. The receptor at these sites may primarily serve as a PTHrP receptor. However, circulating PTH could theoretically activate these receptors and may therefore affect physiological processes other than calcium and bone metabolism, especially in the setting of hyperparathyroidism.

PTH is the principal hormone that coordinates calcium homeostasis on a minute-to-minute basis. It does this through its actions on the kidney and on bone cells. Below, we discuss the actions of PTH on these two organs in detail.

Kidney : PTH regulates the renal handling of calcium, phosphate, sodium and hydrogen ions. It also regulates the conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. The PTH1R is expressed at different sites along the nephron, including the glomerulus, proximal tubules, the cortical ascending limbs and the distal convoluted tubules. In the proximal and thick ascending limbs the receptor is expressed on both the basolateral surface and the luminal surfaces of cells.

PTH was originally recognized for its actions to promote urinary phosphate excretion. Approximately 90% of plasma phosphate is filtered by the glomerulus and 80% is actively reabsorbed, primarily by the proximal convoluted tubules. The regulated step in phosphate reabsorption is entry of phosphate across the luminal membrane of the proximal tubule cells though two related sodium-phosphate co-transporters known as NPT2a and NPT2c. PTH inhibits phosphate uptake into these cells by triggering the withdrawal of NPT2a from the apical membrane into intracellular vesicles, which leads to proteolytic degradation of the transporter. Interestingly, activation of the luminal or basolateral PTH1R pool appears to stimulate different signaling pathways, yet both cause internalization of NPT2a. The basolateral receptors increase intracellular cAMP. At the luminal surface, the PTH1R is associated with a scaffolding protein known as NHERF1, which favors the activation of the PKC pathway over the cAMP pathway. NHERF1 has also been shown to bind to NPT2a and it has been suggested that dissociation of NHERF1 from NPT2a is necessary to allow internalization of NPT2a in response to PTH.

PTH increases calcium reabsorption by the kidney. This occurs through the stimulation of active transcellular calcium transport across epithelial cells in the cortical segment of the thick ascending loop of Henle and in the distal convoluted tubule. In distal tubule cells PTH stimulates cAMP/PKA signaling and phospholipase D, both of which are necessary to mediate calcium transport. These signaling cascades lead to hyperpolarization of the membrane potential, which stimulates the entry of calcium across the apical membrane as well as its extrusion via sodium/calcium exchange across the basolateral membrane.

PTH acts on the proximal tubule to stimulate the conversion of 25 (OH) vitamin D to 1,25 (OH) ₂ vitamin D, which is the active form of vitamin D. In this way, PTH indirectly stimulates calcium absorption from the gut and activates bone cell activity. The increase in 1,25 (OH) ₂ vitamin D is the result of stimulation of both CYP27B1 gene expression and activity (the enzyme responsible for hydroxylation of vitamin D at the C-1 position). The effects of PTH on CYP27B1 activity are opposed by the CaSR and by 1,25 (OH) ₂ vitamin D itself. PTH also inhibits the degradation of 1,25 (OH) ₂ vitamin D by decreasing CYP24A1 (24-hydroxylase) activity in the proximal tubule and thus inhibiting vitamin D catabolism.

PTH inhibits acid secretion in the proximal tubule and can produce a mild compensated hyperchlroremic acidosis. This is the result of phosphorylation of the Na ⁺ /H ⁺ exchanger (NHE3) by PKA downstream of the PTH1R. While this results in reduced bicarbonate and sodium reabsorption by the proximal tubule, the distal tubules compensate by increasing both sodium and bicarbonate absorption. As a result, there is little change in serum bicarbonate concentrations or pH, although chronic elevations in PTH can be associated with mild hyperchloremia.

The skeleton : PTH increases bone turnover although the net effect on bone depends on the pattern of PTH exposure. Continuous PTH exposure causes net bone catabolism, which is seen in states of hyperparathyroidism. In contrast, intermittent PTH exposure has an anabolic effect on the skeleton, which has been exploited pharmacologically in the treatment of osteoporosis. Below, we will discuss the actions of PTH on the three main cell types in bone, osteoclasts, osteoblasts and osteocytes.

PTH stimulates bone resorption due to an increase in the production of new osteoclasts as well as an increase in the activity of existing osteoclasts. Neither osteoclasts nor their precursors express the PTH1R, so these effects result from paracrine interactions between osteoclasts and cells within the osteoblast lineage, which do express the PTH1R. Osteoclast differentiation, activity and survival are regulated by two cytokines, colony-stimulating factor 1 (MCSF, CSF1) and receptor-activator of NFκB ligand (RANKL). CSF1 is produced by stromal/osteoblast cells and acts through its receptor c-fms to induce early monocyte/macrophage precursors to enter the osteoclast lineage. RANKL is also produced by cells in the stromal/osteoblast lineage and acts through its receptor, receptor-activator of NFκB (RANK) to promote the differentiation of osteoclast precursors into mature osteoclasts, to increase the bone-resorbing activity of mature osteoclasts and to lengthen the life-span of active osteoclasts. RANKL can also be bound by a secreted decoy receptor known as osteoprotegerin (OPG), which inhibits osteoclast formation and activity, and promotes osteoclast apoptosis. Therefore, the RANKL/OPG ratio is an important determinant of osteoclastic activity. PTH increases the secretion of CSF1 and RANKL, and suppresses the secretion of OPG raising the RANKL/OPG ratio. These alterations in local cytokines increase osteoclast numbers and activity, and stimulate bone resorption.

The direct effects of PTH on osteoblast function are complicated and vary depending on the duration of exposure to PTH as well as the state of differentiation of the cells. The reader is referred to more in-depth reviews for a detailed discussion of the specific effects of PTH on osteoblasts. Continuous and intermittent PTH both increase osteoblast numbers and rates of bone formation in vivo . This does not appear to be due to cell proliferation as most data suggest that PTH inhibits the proliferation of committed osteoblast precursors and mature osteoblasts. Intermittent exposure to PTH favors the commitment of mesenchymal precursors to the osteoblast lineage and stimulates osteoblast differentiation. In addition, intermittent PTH administration may convert previously quiescent bone lining cells into active osteoblasts. In contrast, prolonged, continuous exposure to PTH inhibits full osteoblast differentiation. These alternative effects on osteoblast differentiation may help to explain why net anabolic activity differs between continuous and intermittent PTH exposure.

The PTH1R is expressed on osteocytes and PTH inhibits osteocyte apoptosis and suppresses osteocyte production of sclerostin, an important inhibitor of Wnt signaling in bone. Experiments in mice suggest that activation of the osteocyte PTH1R influence the activity of osteoblasts and osteoclasts on bone surfaces, demonstrating that these cells may help to coordinate bone turnover in response to PTH. C-terminal fragments of PTH also affect osteocytes by binding to a specific, although as yet uncharacterized, C-terminal PTH receptor. Activation of this receptor may antagonize PTH1R actions, which could theoretically contribute to the skeletal resistance to PTH described in CKD, since decreases in GFR increase the concentration of circulating C-terminal PTH fragments.

Vitamin D

Vitamin D is a steroid hormone that contributes to the development and maintenance of cartilage and bone as well as the regulation of calcium and phosphate metabolism. It exerts a myriad of other effects, including the regulation of the immune response, the control of epithelial cell proliferation and the regulation of cell differentiation. Here we will limit our discussion to its actions on calcium homeostasis.

The biological activity of vitamin D is mediated by 1,25 (OH) ₂ vitamin D3, which circulates in pM concentrations. The production of active vitamin D is tightly regulated and is the net result of a series of photochemical and enzymatic steps that involve several tissues throughout the body. The first step in vitamin D synthesis is the formation of pre-vitamin D3 from 7-dehydrocholesterol. This reaction occurs in the plasma membrane of keratinocytes and is catalyzed by ultraviolet B light between the wavelengths of 290 and 315 nM. Previtamin D3 is thermodynamically unstable and in a temperature dependant fashion is converted into vitamin D3, which is shed into the ECF and binds to vitamin D binding protein (DBP) in the circulation. The quantity of vitamin D3 formed in the skin depends on the duration and intensity of sunlight exposure and is inhibited by sunscreen, clothing and skin pigmentation. Thus, people with darker skin and those living at latitudes farther from the equator synthesize less vitamin D, and in the Northern hemisphere, there is less vitamin D synthesis in winter months. These racial, geographical and seasonal factors all contribute to the patterns of vitamin D deficiency observed in the human population.

Vitamin D3 can be stored in fat and in the liver. As noted above, it circulates bound to DBP and it is hydroxylated at the C-25 position in order to form 25 (OH) vitamin D. This reaction occurs in the liver and is the sum of the actions of several cytochrome p450 enzymes, although the most important appears to be the microsomal enzyme, CYP2R1. Production of 25 (OH) vitamin-D is controlled by substrate availability, so the measurement of circulating 25 (OH) vitamin D is the most reliable clinical indicator of total body vitamin D stores. 25 (OH) vitamin D circulates in nM concentrations, and is also bound to DBP.

The active hormone, 1,25 (OH) ₂ vitamin D is formed in the kidney under tight regulation by PTH, phosphate, FGF-23 and calcium concentrations. 25 (OH) vitamin D is filtered into the urine and is reabsorbed together with DBP by megalin and cubulin, which are found in the apical membrane of the proximal tubule cells. Hydroxylation of 25 (OH) vitamin D is catalyzed by CYP27B1, which is a cytochrome P450 enzyme found within the inner mitochondrial membrane. The proximal tubule also expresses 24 hydroxylase activity mediated by CYP24A1 which generates 24, 25 (OH) ₂ vitamin D and calcitroic acid, the main inactive metabolites of 25 (OH) vitamin D and 1,25(OH) ₂ vitamin D, respectively. CYP24A1 antagonizes the actions of CYP27B1 both by shunting away its substrate and by inactivating its product. The synthesis of 1,25 (OH) ₂ vitamin D is regulated primarily by altering the expression of CYP27B1 and CYP24A1 in a reciprocal fashion. PTH, low calcium, low phosphate and calcitonin act to stimulate 1,25 (OH) ₂ vitamin D production, while high calcium, high phosphate, FGF23 and 1,25 (OH) ₂ vitamin D, itself, inhibit production. In tissues outside the kidney, CYP27B1 and CYP24A1 expression, and 1,25 (OH) ₂ production are also regulated by a number of growth factor and cytokine pathways as well as by toll-like receptor signaling.

Vitamin D acts as a classical steroid hormone and regulates gene expression by binding to the vitamin D receptor (VDR), a ligand-activated transcription factor that is a member of the large superfamily of steroid hormone receptors. The active receptor is a heterodimer between the VDR and the retinoic acid X (RXR) receptor. Like other steroid hormone receptors the activated VDR recruits a number of co-factors to specific sequences within target genes in order to form a multiprotein complex that includes chromatin remodeling activity and transcriptional activators or repressors. The activity of vitamin D and the VDR depends on the particular makeup of this protein complex and, like other steroid hormone/receptor complexes, is cell-type and target gene specific. We will not review the details of gene regulation by the VDR further. The interested reader is referred to more comprehensive discussions of this topic.

Vitamin D acts on all the components of calcium homeostasis and controls the acquisition of calcium from the environment as well as modulating the sensitivity of several organs to calcium and PTH. Below, we review the actions of vitamin D on calcium-regulating organs.

The Intestine : Vitamin D deficiency and genetic disruption of the vitamin D receptor cause hypocalcemia, secondary hyperparathyroidism, rickets, osteomalacia, and growth retardation. All of these disturbances can be rescued by the administration of a high calcium and lactose diet that increases vitamin D-independent gut calcium absorption or by selective, transgenic replacement of intestinal VDR expression in VDR-knockout mice. Therefore, it has become clear that the dominant actions of vitamin D on calcium and bone metabolism are to support the absorption of calcium and phosphorus from the diet. Calcium is absorbed from the intestine through a saturable, transcellular process that is prominent in the proximal small intestine and a non-saturable, paracellular process that is more prominent in the ileum and colon. Vitamin D has its greatest effects on increasing transcellular transport, although it can also increase paracellular calcium absorption. Vitamin D has traditionally been thought to increase transcellular calcium transport in enterocytes by increasing the production of proteins involved in calcium entry across the apical membrane (TRPV6), calcium translocation through the cytoplasm (calbindin D _9k , calbindin D _28k ) and calcium extrusion across the basolateral membrane (PMCA1b). However, recent knockout mouse models of TRPV6 and calbindin D _9k have suggested that the standard model of vitamin D-mediated transcellular calcium transport may be overly simplistic. Vitamin D signaling also upregulates the expression of claudin 2 and claudin 12, both of which modulate the permeability of tight junctions to calcium and, thus, likely regulate paracellular calcium transport.

It should be noted that vitamin D also increases intestinal phosphate absorption. This is caused by increases in the apical expression of NPT2b, a sodium-phosphate co-transporter related to the phosphate transporters expressed in the proximal renal tubule. However, the exact molecular mechanisms by which vitamin D regulates NPT2b expression and phosphate absorption remain to be defined.

The kidney : In addition to serving as the site of 1,25 (OH) ₂ vitamin D synthesis, the kidney is also a target of vitamin D actions. The VDR is expressed at highest levels in distal tubule cells but is also expressed at lower levels in the proximal tubule. The net effect of vitamin D on the kidney appears to be the regulation of its own synthesis in a short feedback loop in the proximal tubule and the enhancement of calcium reabsorption in the distal convoluted tubule. In the proximal tubule Vitamin D inhibits the production of CYP27B1 and enhances the production of CYP24A1 acting to inhibit further production of 1,25 (OH) ₂ vitamin D. As in the intestine, vitamin D acts on the distal tubule to regulate the production of several proteins involved in transcellular calcium reabsorption. It upregulates the expression of TRPV5 and TRPV6, the two apical calcium entry channels; Calbindin D _9k and Calbindin _28k , which are involved in cytoplasmic calcium translocation; and PMCA1b, which transports calcium out of the cell across the basolateral membrane. It also has been shown to increase PTHR1 expression in the distal tubule and to enhance the effects of PTH on distal tubular calcium reabsorption.

Bone : The vitamin D receptor is expressed in cells of the osteoblast lineage but not in mature osteoclasts. The classic presentation of vitamin D deficiency in children is growth retardation and rickets, while in adults it can result in osteomalacia. However, as noted previously, when VDR-knockout mice were treated with a high calcium “rescue” diet that normalized calcium, phosphate and PTH levels, these bone phenotypes disappeared, suggesting that the dominant effects of vitamin D on bone were indirect. However, a close examination of VDR ^-/- , CYP27B1 ^-/- and double knockout, VDR ^-/- , CYP27B1 ^-/- mice on rescue diets demonstrated that loss of vitamin D function reduced the numbers of osteoblasts and osteoblast progenitors in vivo , which is consistent with many studies in vitro that have documented effects of vitamin D on osteoblast gene expression, osteoblast proliferation and osteoblast differentiation. However, in the presence of adequate amounts of dietary calcium and phosphate, the direct effects of vitamin D on osteoblast function are subtle and may differ depending on the stage of osteoblast differentiation. The indirect effects of vitamin D on osteoclasts are more straightforward. 1,25 (OH) ₂ vitamin D increases osteoblast expression of RANKL and decreases the production of OPG by osteoblasts. By increasing the RANKL/OPG ratio, vitamin D increases the production of osteoclasts and of bone turnover. This effect of vitamin D is clinically apparent in states of 1,25 (OH) ₂ vitamin D overproduction or in the setting of excess ingestion of vitamin D, where bone resorption is excessive despite the suppression of PTH secretion.

Parathyroid gland : The parathyroid glands are rich in VDR expression and 1,25 (OH) ₂ vitamin D inhibits transcription of the PTH gene. This appears to be a direct effect of the VDR on the PTH gene promoter. Vitamin D also regulates PTH production indirectly by increasing CaSR expression in the parathyroids and by increasing systemic calcium levels. As noted previously, CaSR signaling increases VDR expression in the parathyroids, so vitamin D and calcium act synergistically to suppress PTH secretion. Therefore, states of vitamin D deficiency may sensitize the gland to low calcium levels and increase the PTH response to hypocalcemia. This may contribute to the development of parathyroid hyperplasia and secondary hyperparathyroidism in CKD. However, in mice, it appears that calcium remains the most important factor regulating parathyroid gland growth and PTH secretion. Meir and colleagues disrupted the VDR specifically in parathyroid cells and found that while CaSR levels were decreased, PTH secretion was only modestly increased and that serum calcium levels remained normal. These data suggest that the VDR may have a limited role in regulating parathyroid function at baseline.

Parathyroid Hormone-Related Protein

The PTHrP and PTH genes share structural elements and sequence homology suggesting that they arose through duplication of a common ancestor. The portion of both genes encoding the amino-terminal segments of PTH and PTHrP are highly conserved such that the peptides share 8 of the first 13 amino acids and a high degree of predicted secondary structure over the next 21 amino acids. Beyond this, however, the two genes share little similarity. PTHrP mRNA is expressed in almost every organ at some time during its development or functioning. Many different hormones and growth factors regulate PTHrP mRNA levels. As with PTH, the CaSR regulates PTHrP gene expression in many different cell types. Another common observation is that mechanical forces induce PTHrP gene expression in many sites.

Like PTH, PTHrP binds to and activates the PTH1R. Most studies in vitro suggest that this receptor binds PTHrP and PTH with equal affinity and that both peptides trigger identical signaling events. However, the human PTH1R may respond somewhat differently to PTH and PTHrP. It appears that PTH and PTHrP demonstrate differences in their ability to stabilize alternate conformational states of the receptor, so that the duration of cAMP production is shorter for PTHrP (1–36) than for PTH (1–34). This may explain why human subjects subjected to continuous infusion of the two peptides for 72 hours, were found to become hypercalcemic with lower doses of PTH 1–34 than PTHrP (1–36) and had lower 1,25 (OH) ₂ vitamin D production when infused with PTHrP. These studies may also help to explain differences in the biochemical profiles of HHM and hyperparathyroidism (see below).

In addition to being secreted, PTHrP can also be transported into the nucleus of many different cell types. The function(s) of nuclear PTHrP is unclear, but replacement of the endogenous mouse PTHrP gene with a mutant version encoding a protein that cannot enter the nucleus causes widespread cellular senescence, growth retardation and early death. This experiment suggests that nuclear PTHrP is of fundamental importance in many cell types.

PTHrP has been suggested to have many functions. The reader is referred to more comprehensive reviews for a complete discussion. Here, we will highlight two areas where PTHrP has been documented to have physiological effects in intact organisms.

Cartilage and bone : A series of genetic experiments in mice and genetic disorders in humans have demonstrated that amino-terminal PTHrP acts through the PTH1R to coordinate the rate of chondrocyte differentiation in order to maintain the orderly growth of long bones during development. PTHrP is produced by immature chondrocytes at the top of the growth plate in response to another molecule known as Indian Hedgehog (IHH) secreted by differentiating hypertrophic chondrocytes. PTHrP, in turn, acts on its receptor located on proliferating and prehypertrophic cells to slow their rate of differentiation into hypertrophic cells. Therefore, IHH and PTHrP define a local negative feedback loop that regulates the rate of chondrocyte differentiation (see Figure 66.3 ). PTHrP is also found in other cartilaginous sites such as the costal cartilage and the hyaline cartilage lining the joint space, where it appears to prevent the inappropriate encroachment of bone into these structures.

PTHrP also affects bone cells in vivo. Heterozygous PTHrP-null mice develop osteopenia with increasing age. In addition, selective deletion of the PTHrP gene from osteoblasts causes decreased bone mass, reduced bone formation, reduced mineral apposition and a reduction in the formation and survival of osteoblasts. These data suggest that PTHrP acts as an important local anabolic factor in the skeleton.

Mammary gland : PTHrP has important functions during breast development, it is involved in regulating systemic calcium metabolism during lactation and it contributes to the pathophysiology of breast cancer.

The mammary gland forms as a bud-like invagination of epidermal cells that grow into a specialized mesenchyme. The epithelial cells of the embryonic mammary bud produce PTHrP, which interacts with the PTH1R expressed on surrounding mesenchymal cells. This interaction is necessary for the outgrowth of the duct system. In both mice and humans, disruptions of PTHrP signaling cause a failure of breast development past this early bud stage.

PTHrP is also produced by breast epithelial cells during lactation and large quantities are secreted into milk. The function of PTHrP in milk is unknown, but it is secreted from the lactating breast into the circulation, where it participates in the regulation of systemic calcium metabolism. The maternal skeleton is an important source of calcium for milk production and the lactating breast secretes PTHrP into the circulation to increase bone resorption. This is the only time in which PTHrP exerts a documented systemic endocrine effect on normal calcium and bone metabolism.