CaSR Agonists

The CaSR can be activated by Ca ²⁺ , Mg ²⁺ and certain other polycations (e.g., Gd ³⁺ , polylysine, polyarginine, and neomycin ). These agonists are referred to as type I agonists as they can directly and independently activate the receptor ( Fig. 63.1 ). CaSR activity can also be modulated by other substances or conditions that function by modifying the EC ₅₀ for extracellular Ca ²⁺ (up or down); allosteric activators of the CaSR of this type are referred to as type II allosteric activators ( Fig.63.1 ). Thus, type II activators require the presence of extracellular Ca ²⁺ and function as allosteric modifiers of Ca ²⁺ affinity. Currently identified physiological type II agonists include polyamines (e.g., spermine), L-amino acids (especially aromatic amino acids), extracellular pH, and extracellular ionic strength, primarily changes in NaCl concentration in a physiological context. Increases in polyamine or amino acid concentrations, or isosmotic reductions in ionic strength reduce the EC ₅₀ (increase the affinity) for extracellular Ca ²⁺ . At a constant ionized Ca ²⁺ concentration, these changes in the concentrations of type II agonists will increase activation of the CaSR. As a consequence, CaSR responses to changes in concentrations of divalent minerals or type I agonists must be viewed in the setting of a specific “extracellular environment” (i.e., presence or absence of polyamines and L-amino acids, pH, and ionic strength). Interestingly, the CaSR appears to have initially evolved as a salinity sensor in marine species where these organisms used the effects of ionic strength (salinity) on activation of the receptor by the Ca ²⁺ /Mg ²⁺ present in salt water to regulate tissue responses for salinity adaptation. With the evolution of land-based tetrapods and the loss of the ocean reservoir of Ca ²⁺ /Mg ²⁺ , we see the first appearance of the parathyroid glands and parathyroid hormone (PTH), which are required for divalent mineral regulation of the internal “ocean” represented by the extracellular fluids (ECFs) of terrestrial organisms. Currently identified CaSR-active small molecules (calcimimetics and calcilytics) are used in the treatment of certain disorders of calcium homeostasis. They function as allosteric activators and antagonists, respectively, of the CaSR via the type II mechanism and therefore, require extracellular Ca ²⁺ to act ( Fig. 63.1 ).

Schematic representation of the dimeric calcium-sensing receptor (CaSR) localized to caveolin-rich membrane domains. ECD, extracellular domain; TMD, transmembrane domain containing 7 α-helical membrane-spanning segments. Activators/agonists of the receptor can either be type I, which don’t require the presence of Ca ²⁺ to activate the CaSR, or as type II allosteric modifiers that potentiate the action of Ca ²⁺ , which must be present at a concentration of ~1 mM for a type II agent to activate the receptor. Polyamines, such as spermine, act primarily in the type II mode, but can also serve as weak type I agonists. See text for discussion. See color insert.

Ligand Binding to the CaSR

Class C GPCR receptors have a large (hundreds of residues) N-terminal extracellular domain (ECD) that is joined to the canonical 7-transmembrane (7-TM) domain typical of GPCRs. The ECDs of the class C-related metabotropic glutamate receptors or the distantly related bacterial periplasmic nutrient-binding proteins (e.g., the maltose-binding protein (MBP)) form a bilobed structure that has a ligand binding region within the central cleft between these lobes ( Fig. 63.2A ). Following ligand binding, there is a conformational change in the ECD, which results in the two lobes moving toward one another to enclose the ligand ( Fig. 63.2A , Venus-flytrap model). This molecular motion in the ECD is thought to be translated into conformational changes in the 7-TM domain, leading to G-protein activation.

The Venus flytrap model of the extracellular domain of one monomer of the calcium-sensing receptor (CaSR). A: Structures of the bacterial periplasmic maltose-binding protein in the absence and present of ligand (yellow structure). Note the closing of the lobes around the ligand upon ligand binding (dashed arrows). B: Structural model of the human CaSR made by threading the human sequence through the extracellular domain of mGluR. Note the bilobed structure that is similar to the bacterial maltose-binding protein. Potential calcium interacting sites are shown. The best characterized of these is in the crevice between the two lobes of the VFT, which faces upward and to the left at about 10 o’clock. Structure colors: red=α helices; blue=β strands. See color insert.

The ligand-binding sites on class-C receptors, including the CaSR, are located on the ECD and, to a lesser extent, in the 7-TM domains. While an x-ray crystal structure of the ECD of the CaSR has not been obtained, the ECD can be modeled on the basis of the known structure of several metabotropic glutamate receptors. This structural model is shown in Fig. 63.2B with the location of potential Ca ²⁺ -interacting regions composed of negatively charged amino acid residues. In addition, three serine residues (S-147, S-169, and S-170) and proline-823 in the ECD are crucial for maximal responsiveness to extracellular Ca ²⁺ . Receptor activation by Ca ²⁺ is greatly reduced by removal of the ECD, emphasizing the importance of negatively charged acidic (and other) residues in the ECD for the binding of polyvalent cations. Additional acidic residues in the second and third extracellular loops in the 7-TM domain have also been suggested to participate in Ca ²⁺ -binding interactions. In contrast, the response to calcimimetics is retained when the ECD is removed from the CaSR, indicating that these type II activators bind at a different site than does Ca ²⁺ . Observations indicate that the critical sites for interaction of calcimimetics with the CaSR are located in the 7-TM domain, primarily the TM6–TM7 region, with Glu837, at the outer end of TM7, serving as an “anchor” by binding the amino group in the aliphatic linker between the two hydrophobic ends of the molecule. In addition, negative allosteric modulators of the CaSR (calcilytics) have been identified that exhibit a binding site in the 7-TM domain of the CaSR that overlaps with but is not identical to that for the calcimimetics.

The Concept of Superagonism for Agonist Binding to the Class C Receptors

As described previously, type II agonists (including calcimimetics) of the CaSR can act as allosteric enhancers or positive allosteric modulators. It is clear that most GPCRs possess allosteric binding sites that can be recognized by small-molecule ligands. GPCR class-C receptors, including the CaSR, form dimers (or even multimers), and this intermolecular interaction is believed to play an important role in allosteric activation. Type I ligand binding enhances dimerization of the CaSR associated with formation of intermolecular disulfide linkages. Thus, most of the receptors on plasma membranes of cells are in the dimeric (and multimeric) form, since cell surface CaSRs are exposed to millimolar concentrations of extracellular Ca ²⁺ . Although the intermolecular disulfide links between ECDs are not essential for dimerization, they play an important structural role and help to maintain the receptor in its inactive state in the absence of agonist. Initial dimerization takes place in the endoplasmic reticulum, and this interaction may be necessary for the receptor’s transport to the cell surface. Once on the cell surface, intermolecular interactions between CaSR monomers are essential for normal function of this receptor. Specifically, in the presence of agonist, dimerization of the CaSR and other class C receptors appears to enhance downstream cellular signaling, providing “superagonism”. This dimer-driven superagonism, which can also be thought of a positive cooperativity, likely accounts for the ability of type II agonists (e.g., L-amino acids) to activate the receptor by an allosteric mechanism and the remarkable ability of this receptor to detect small changes in ionized Ca ²⁺ from the normal plasma Ca ²⁺ of 1.1-1.3 mM (Ca ²⁺ -Ca ²⁺ allosteric enhancing effect). Given that the Hill coefficient of the dimeric CaSR is 3–4, it is likely that there are at least 2 binding sites for calcium on each of the two monomers, resulting in the substantial degree of positive cooperativity needed for the receptor’s exquisite sensitivity to small changes in Ca ²⁺ . An intact COOH-terminus on the CaSR is also required for cell surface expression.

Complex Signaling: the Receptor is Promiscuous

CaSR coupling to G-protein has been referred to as promiscuous, since type I ligands activate one or more of several G proteins (e.g., Gα _q11 , Gα _i2–i3 ). As with most GPCRs, the COOH-terminal tail of the CaSR and one or more intracellular loops are crucial for signal transduction. The CaSR-generated cytosolic signal is a complex of phospholipase activation (PLC, cPLA ₂ , PLD) and the generation of diverse cellular second messengers (see for reviews), Gα _q⧸⧸11− mediated PLC activation →↑IP ₃ →↑cytosolic Ca ²⁺ concentration (Ca ²⁺ _i ) and Ca ²⁺ _i oscillations as well as →↑DAG→↑PKC; Gα _i -mediated→↓cAMP; ↑intracellular Ca ²⁺ →↑phosphodiesterase (PDE)→↓cAMP; cytosolic (c)PLA ₂ activation→↑arachidonic acid (AA)→↑P ₄₅₀ →↑20-HETE; phosphatidylinositol 3-kinase (PI3K)→ PIP ₃ ; MAP kinases, such as ERK1/2, c-Jun activated N-terminal kinase (JNK), and p38 MAPK; filamin scaffolding of Gα _q →↑Lbc RhoGEF→↑Rho A GTPase leading to activation of a serum response element (SRE) and Gα _12/13 →↑PLD→↑phosphatidic acid (PA). It remains unclear in many cases how this second messenger “soup” and related intracellular signaling pathways integrate to modulate cellular functions, such as PTH secretion or renal responses to extracellular Ca ²⁺ . The ability of the CaSR to be modulated by such a wide variety of agonists and extracellular conditions likely accounts for its multifunctional nature in regulating divalent mineral balance as well as in modulating diverse cellular functions seemingly unrelated to mineral homeostasis. Examples of the latter include CaSR effects on salt and water transport by the kidney and gastrointestinal epithelia. The CaSR also provides proliferation-differentiation-apoptosis signals to certain epithelial cells (e.g., keratinocytes in the skin, mammary gland cells and colonocytes).

CaSR Regulates PTH Secretion

The level and constancy of plasma ionized Ca ²⁺ are set in large part by regulating the release of Ca ²⁺ from bone via the actions of secreted PTH and 1,25(OH) ₂ D ₃ as well as by modulation of the Ca ²⁺ excretion by the kidney, and, indirectly via the action of 1,25(OH) ₂ D ₃ , intestinal calcium absorption, as noted above. Figure 63.3 shows the typical inverse relationship between PTH secretion and plasma Ca ²⁺ . In contrast there is a direct relationship between urinary Ca ²⁺ excretion and plasma Ca ²⁺ ( Fig. 63.4 ). These effects of plasma ionized Ca ²⁺ on PTH secretion and renal Ca ²⁺ excretion are mediated by the CaSR. The role of the CaSR in divalent mineral homeostasis has been established by the identification and characterization of inherited hyper- or hypocalcemic disorders that result from CASR gene mutations (the official designation of the gene for the human CaSR is in all capital letters and italicized) (chromosome 3q13.3–21; Fig. 63.5 ). In vitro studies in mammalian cells expressing normal or mutant CaSR proteins have confirmed that these receptor mutations alter the Ca ²⁺ EC ₅₀ of the CaSR (CaSR refers to the CaSR protein). Loss-of-function mutations in one or both of the CASR alleles result in hypercalcemic disorders due to upward resetting of the CaSR EC ₅₀ for ionized Ca ²⁺ in both the parathyroid gland and kidney. ^48,83,404 Autosomal dominant hypercalcemia, in which one CASR allele has an inactivating mutation, typically present s as a benign hypercalcemic disorder called familial hypocalciuric hypercalcemia (type 1 FHH; OMIM 145980, known as HHC1) or as symptomatic hypercalcemia in neonatal hyperparathyroidism (NHPT). Two phenotypically similar conditions (HHC2, OMIM 145981; and HHC3, OMIM 600740) are linked to the long and short arms of chromosome 19, respectively, but the responsible genes have not yet been identified. Neonatal severe hyperparathyroidism (NSHPT) results from consanguineous unions in FHH families in which both alleles have inactivating mutations or, occasionally, in families in which the two parents have different inactivating mutations (i.e., producing a compound heterozygous infant). Infants with NSHPT exhibit severe hypercalcemia frequently necessitating total parathyroidectomy. The hypercalcemia in these loss-of-function CaSR disorders is usually associated with reduced renal Ca ²⁺ excretion, rather than the increased Ca ²⁺ excretion that is observed in other hypercalcemic states (e.g., 1° HPT and malignant hypercalcemia). The abnormal renal response in FHH demonstrates the important role of the CaSR in the hypercalciuric response to hypercalcemia.

Inverse relationship between ionized serum calcium and parathyroid hormone (PTH) secretion. PTH secretion is maximal at low ionized calcium; raising calcium reduces secretion via several intracellular signaling pathways, including decreased levels of cAMP and increases in the products of PLA ₂ and PLC action. Ionized calcium sensing by the parathyroid gland is mediated by the calcium-sensing receptor (CaSR). The EC ₅₀ for calcium-sensing by the gland is shown (1.2 mM Ca ²⁺ _o ); at normal ambient levels of Ca ²⁺ _o , the gland is about 70–75% suppressed. See color insert.

Direct relationship between serum calcium and renal calcium excretion demonstrating the calcium-sensing ability of the nephron (solid black curve). Note the steep relationship after reaching a threshold serum calcium concentration. In individuals with calcium-sensing receptor (CaSR)–inactivating mutations (FHH, familial hypocalciuric hypercalcemia), the relationship (solid gray line) is flatter than normal. This defect in the relationship is not due to a disturbance in calcium transport function of the thick ascending limb since loop diuretics can markedly increase calcium excretion in FHH. In the absence of parathyroid hormone and vitamin D, the threshold of serum calcium at which calcium excretion begins is reduced but the steep relationship between serum calcium and renal calcium excretion remains. The latter is due to the action of the CaSR.

A: Mutations of the calcium-sensing receptor (CaSR) on chromosome 3q13.3-21 alter the steady-state serum ionized calcium concentration and the calcium-sensing ability of the kidney (serum calcium–urine calcium excretion relationship; Fig. 63.4 ). Receptor inactivating mutations give rise to hypercalcemia and hypocalciuria while activating mutations result in the opposite phenotype. B: In mice, knocking out the Casr gene results in a lethal phenotype from severe hypercalcemia and hyperparathyroidism within a few days after birth. The severe phenotype can be rescued by deleting the Gcm2 gene, which is required for development of the parathyroid gland. These double-knockout mice have low circulating levels of PTH that is secreted from thymus but is not under the control of the CaSR. See text for discussion.

Homozygous CaSR knockout mice exhibit marked parathyroid hyperplasia and hyperparathyroidism and die soon after birth due to severe hypercalcemia ( Fig. 63.5 ). Support for the role of PTH in causing the severe and lethal hypercalcemia came form the observations that the lethal mouse phenotype can be rescued by knocking out the PTH gene or by deletion of the Gcm2 gene, which is the “master gene” needed for development of the parathyroid glands. Studies carried out in the mice with knockout of PTH and/or the CaSR have documented the crucial role of CaSR-regulated PTH secretion as a “floor” preventing hypocalcemia, while the CaSR-mediated upregulation of renal Ca ²⁺ excretion and stimulation of calcitonin secretion are an effective “ceiling” limiting increases in serum calcium in response to dietary or other forms of calcium load. Indeed, the PTH knockout mice defend against hypercalcemia just as well as the wild type mice, even though they lack PTH and the ability to suppress it while hypercalcemic.

Autosomal dominant, gain-of-function (activating) mutations in the CaSR result in an opposite shift in plasma ionized calcium (i.e., hypocalcemia) due to downward resetting of the receptor EC ₅₀ (autosomal dominant hypocalcemia (ADH); OMIM 146200). In some individuals with severe activating mutations, a renal salt wasting disorder with hypercalciuria has been observed that mimics the hyperprostaglandin E ₂ syndrome (type V Bartter syndrome). The latter confirms the important role of the CaSR in regulating renal Ca ²⁺ handling and clearly demonstrates the importance of the CaSR in regulating salt transport in the thick ascending limb (TAL). CASR gene polymorphisms also appear to contribute to the normal variation in steady-state plasma ionized Ca ²⁺ concentration, at least in certain populations. CaSR-activating or -inhibiting autoantibodies can result in autoimmune hypoparathyroidism or an acquired syndrome mimicking FHH, called autoimmune hypocalciuric hypercalcemia, respectively. Mice with an activating mutation in the CaSR exhibit a phenotype similar to that in patients with ADH. All these various lines of evidence convincingly document the key role of the CaSR in mediating the effects of Ca ²⁺ on PTH secretion.

In addition to reducing the secretion of PTH, activation of the CaSR increases the degradation of full length, biologically active PTH1-84 to PTH7-84 and smaller carboxyterminal fragments, thereby decreasing the secretion of intact PTH further still during hypercalcemia and, conversely, increasing it in the setting of hypocalcemia.

CaSR Regulates Expression of the preproPTH Gene

Elevated levels of Ca ²⁺ _o and calcimimetics not only inhibit PTH secretion but also decrease the levels of the mRNA encoding preproPTH; this action of the calcimimetics proves the mediatory role of the CaSR in regulating preproPTH gene expression. The CaSR-mediated alteration in the level of preproPTH mRNA is the result of a change in preproPTH mRNA stability rather than in gene transcription per se. 1,25(OH) ₂ D ₃ , in contrast, acts by a direct inhibitory action on the transcription of this gene. Naveh-Many, Silver and coworkers have clarified the molecular mechanisms by which Ca ²⁺ _o and the CaSR control the stability of preproPTH mRNA. Exposing parathyroid cells to elevated levels of Ca ²⁺ activates the CaSR and, through a pathway that involves stimulation of calmodulin (CaM) and protein phosphatase 2B, post-translationally modifies and reduces the binding of the preproPTH mRNA stabilizing factor, AU-rich factor (AUF-1), to an AU-rich element in the 3’ untranslated region (UTR) of the preproPTH mRNA. The loss of AUF-1 from this binding site permits a second, destablizing protein, K-homology splicing regulator protein (KSRP), to bind to the same site. KSRP subsequently interacts with and is activated by the peptidyl-prolyl isomerase, Pin-1, and, pari passu, recruits the endoribonuclease, PMR1, which is part of the RNA-cleaving exosome. PMR-1 then degrades prepro-PTH mRNA by cleaving it internally.

CaSR Regulates Parathyroid Cellular Proliferation

Studies in humans with NSHPT or in mice homozygous for knock out of the Casr gene (the symbols for mouse genes are italicized with only the first letter capitalized) have proven the CaSR’s importance in regulating parathyroid cellular proliferation. In both cases, marked parathyroid cellular proliferation and glandular enlargement ensue despite severe hypercalcemia, documenting that the CaSR has an essential role in tonically inhibiting parathyroid cellular proliferation. Studies in uremic rat models have illuminated the mechanisms by which high dietary intake of Ca ²⁺ , acting via the CaSR, controls parathyroid proliferation. Induction of the cyclin dependent kinase inhibitor, p21 ^WAF1 , and downregulation of the growth factor, TGF-α, and its receptor, the epidermal growth factor receptor (EGFR), both of which are upregulated in this setting, are key components of this mechanism. 1,25(OH) ₂ D ₃ appears to act in a similar way to inhibit parathyroid cellular proliferation in similar experimental models. An additional mechanism that may participate in stimulation of parathyroid growth during hypocalcemia is an endothelin-1-mediated stimulation of parathyroid cellular growth. Similar studies are difficult to perform in non-uremic animals owing to their much slower rate of parathyroid proliferation, but it seems likely that similar mechanisms participate. Reduced expression of p21 and another cyclin dependent kinase inhibitor, p27, may also participate in the dysregulation of parathyroid growth in both primary (1°) and secondary (2°) hyperparathyroidism (HPT). The second messenger pathway(s) that link the CaSR to the regulation of parathyroid proliferation have not yet been clarified.

Interactions of Vitamin D and the CaSR in the Regulation of Parathyroid Function

A large body of data has stressed the importance of vitamin D in reducing expression of the preproPTH gene and parathyroid proliferation. What is the relative importance of the VDR and CaSR in controlling parathyroid function? The CaSR clearly regulates the secretion of PTH over a time frame from seconds to minutes or longer and is the dominant regulator of acute changes in secretory rate. Over a longer time frame of three weeks, vitamin D deficiency and hypocalcemia both modulate preproPTH mRNA levels in the rat, although hypocalcemia of ~6 mg/dl more powerfully stimulates preproPTH gene expression than does vitamin D deficiency.

Recent studies utilizing mouse knockout models, however, have provided surprising results regarding the relative importance of the VDR and CaSR in controlling parathyroid gland function in vivo . As noted before, homozygous knock out of exon 5 of the CaSR causes striking hyperparathyroidism with marked elevations in both PTH and parathyroid gland size, which clearly cannot be compensated by the remaining VDR gene. This mouse model, if anything, likely underestimates the consequences of loss of the CaSR on parathyroid function, since knock out of exon 5 of the CaSR produces, in some tissues, an alternatively spliced CaSR that lacks exon 5 (which encodes part of the CaSR ECD) and can seemingly still signal. Thus the VDR apparently has limited ability to offset loss of the CaSR in this animal model. In contrast, studies of mice with knockout of the VDR have demonstrated that the CaSR effectively compensates for loss of the vitamin D receptor with regard to the control of parathyroid function. That is, while VDR-/- mice develop strikingly elevated levels of PTH and marked parathyroid enlargement on a standard diet, administering a calcium-rich “rescue” diet normalizes both serum Ca ²⁺ and PTH levels. Thus hypocalcemia per se rather than vitamin D deficiency is seemingly the dominant contributor to the elevated PTH levels. In addition, if the rescue diet is begun early in life, it completely prevents the parathyroid enlargement in the VDR-/- mice, showing that hypocalcemia per se rather than loss of the VDR was also a critical contributor to parathyroid growth in this setting. Subsequently, Meir, et al. created mice with knockout of the VDR only in the parathyroid glands. In this way, the actions of the VDR on the parathyroid could be separated from systemic alterations in mineral ion homeostasis, e.g., owing to loss of the VDR in kidney and intestine. The mice with parathyroid-specific VDR ablation manifest only modest (~30%) elevations in serum PTH but do not exhibit any change in the number of proliferating parathyroid cells and have normal serum calcium concentrations. Thus, while administering exogenous 1,25(OH) ₂ D ₃ clearly suppresses preproPTH gene transcription and parathyroid proliferation in vivo and in vitro , vitamin D seemingly has only a limited role in regulating parathyroid function in vivo under normal physiological conditions. These results should not be taken, however, to mean that 1,25(OH) ₂ D ₃ has no useful therapeutic role, especially in the 2° HPT of renal insufficiency (see below and elsewhere in this volume). Finally, it would be interesting to investigate CaSR signaling efficiency in the parathyroid in mice with deficient VDR signaling to determine to what extent compensatory alterations in the CaSR and/or its downstream signaling components contribute to the phenotypes observed with global or parathyroid-specific knockout of the VDR.

CaSR in the Proximal Tubule

For some time, there has been both in vivo and in vitro evidence that Ca ²⁺ _o directly modulates the 1-hydroxylation of 25-hydroxyvitamin D ₃ in the PT. To avoid the confounding impact of Ca ²⁺ _o -induced alterations in circulating PTH levels in vivo , Treschel et al. and Weisinger et al. used thyroparathyroidectomized rats infused with PTH to “clamp” the circulating PTH level. Changes in 1,25(OH) ₂ D ₃ levels were then assessed during alterations in serum Ca ²⁺ . The steep inverse sigmoidal relationship between Ca ²⁺ _o and 1,25(OH) ₂ D ₃ levels that was observed was reminiscent of the relationship of PTH to Ca ²⁺ _o in vivo and in vitro . Similar results were obtained in vitro utilizing an SV40-transformed human PT cell line, that is, enhanced 1,25(OH) ₂ D ₃ synthesis at low Ca ²⁺ _o and inhibition at high Ca ²⁺ _o . Recent data supports the CaSR’s role in mediating the direct effects of Ca ²⁺ _o on the 1-hydroxylation of 25-hydroxyvitamin D ₃ . Maiti and Beckman utilized the PT cell line, HK-2G, in which high Ca ²⁺ _o inhibits CYP27B1 expression, to show that high Ca ²⁺ upregulates VDR expression by a p38 MAPK-dependent mechanism. Knocking down the CaSR with siRNA obviated the high Ca ²⁺ _o -elicited increase in VDR, showing the latter was CaSR-mediated. However, use of siRNA to document that the CaSR mediated the concomitant inhibition of CYP27B1 expression was not reported. It has also not yet been shown that the high Ca ²⁺ _o -induced, CaSR-mediated increase in VDR expression in the PT by itself can account for the reduced expression of CYP27B1.

Furthermore, 1,25(OH)2D ₃ regulates CaSR expression in the kidney: One study in vitamin D–deficient rats found a 90% increase in CaSR expression in the kidneys of 1,25(OH)2D ₃ -replete animals, and vitamin D replete rats showed a similar increase in response to 1,25(OH)2D ₃ . Vitamin D-responsive elements (VDREs) in the CASR and Casr genes and provide a mechanism whereby 1,25(OH)2D ₃ upregulates CaSR expression in parathyroid, thyroid C-cell, and kidney. As noted earlier, since 1,25(OH)2D ₃ upregulates its own expression as well as that of the CaSR, and the CaSR upregulates its own expression and that of the VDR, there could be synergistic interactions between the effects of Ca ²⁺ _o , acting via the CaSR, and 1,25(OH) ₂ D ₃ on the PT and elsewhere in the kidney. CaSR expression in the PT is reduced by PTH and dietary phosphate loading.

Transcripts for the CaSR are present in the regions where PTH/PTHrP mRNA is expressed in the proximal convoluted (PCT) and straight tubule (PST) (as well as in glomerulus, CTAL, DCT and cortical CD). High Ca ²⁺ _o inhibits PTH-induced cAMP accumulation in the PCT and CTAL. This action could explain, at least in part, the inhibition by the CaSR of PTH-stimulated phosphate excretion. Thus the CaSR directly modulates the action of PTH on the PT (and of renal Ca ²⁺ reabsorption in the TAL) in addition to its central, inhibitory effect on PTH secretion. The CaSR protein is in the subapical compartments of the PT, where it mediates some of the effects of high Ca ²⁺ on PTH-induced cAMP production. We have hypothesized a role for the receptor in the transport of phosphate and/or in the regulation of a local ionic homeostasis, bypassing systemic levels of calciotropic hormones (see following paragraphs).

McKinney and coworkers reported that luminal and peritubular perfusion of rabbit PCT with high Ca ²⁺ _o (5 mM) modulated water and HCO ₃ ⁻ reabsorption. Because a large fraction of Na ⁺ transport in PT depends on basolateral Na ⁺ , K ⁺ -ATPase (NKA), NKA activity provides an index of solute reabsorption. Preliminary studies by Hebert’s group have suggested that increases in Ca ²⁺ _o reduce NKA activity by 20 to 35% in a rabbit PT cell line. This magnitude of NKA inhibition is similar to that observed with dopamine, a known regulator of PT solute and volume transport. Modulation of PT NKA by CaSR activation would enable filtered (i.e., luminal) Ca ²⁺ to regulate Ca ²⁺ delivery to distal nephron segments. In contrast, since only 15% of filtered Mg ²⁺ is reabsorbed in the PT (in contrast to the 50–60% of Ca ²⁺ reabsorbed proximally), activation of the CaSR in the PT would be expected to have little effect of Mg ²⁺ homeostasis.

CaSR in the Thick Ascending Limb of Henle

About 25% of filtered calcium is reabsorbed along the TAL ( Fig. 63.6 ). It is well established that increases in extracellular Ca ²⁺ and Mg ²⁺ inhibit Cl ⁻ transport in TAL. Increases in plasma, but not urinary, Ca ²⁺ (or Mg ²⁺ ) concentrations directly modulate NaCl and mineral ion transport in the rat loop of Henle, consistent with CaSR expression on basolateral membranes of the TAL. Since most divalent mineral reabsorption mediated by the TAL occurs in CTAL, the effect of plasma Ca ²⁺ on renal Ca ²⁺ (Mg ²⁺ ) handling relates to changes in interstitial ionized Ca ²⁺ and Mg ²⁺ surrounding the CTAL. In medullary thick ascending limb (MTAL) and CTAL the lumen-positive transepithelial voltage generated by NaCl reabsorption drives most divalent mineral reabsorption via the paracellular route in mouse, rat, and rabbit. In addition, a portion of Ca ²⁺ absorption in the CTAL (at least in the mouse) may also traverse the transcellular route (see Chapter 65 ). A number of hormones (e.g., vasopressin, parathyroid hormone, glucagon, calcitonin), coupled to Gαs-linked receptors, increase cellular cAMP accumulation thereby stimulating NaCl and divalent mineral reabsorption through both paracellular and transcellular routes. As we shall see in the following paragraphs, the CaSR uses a variety of mechanisms and second-messenger pathways to modulate NaCl reabsorption, and thereby divalent mineral absorption, in the TAL.

Both genetic experiments-of-nature and acquired disorders have established that the CaSR is a significant regulator of functionally coupled salt and divalent mineral transport in the TAL. Activating mutations of the CaSR are generally associated with autosomal dominant hypocalcemia (see preceding sections) ; however, certain activating mutations of the CaSR have been found in individuals presenting with the phenotypic picture of Bartter syndrome combined with the typical hypocalcemic hypercalciuria present with activating CaSR mutations. Bartter syndrome is a set of renal salt- and Ca ²⁺ – and Mg ²⁺ -wasting disorders due to low or absent ion transport in the TAL. Mutations in several salt transporter genes or in genes encoding transporter regulatory proteins in the TAL cause Bartter syndrome. It is now clear that the CaSR is one of these regulatory proteins. In addition, an acquired Bartter-like phenotype has also been observed in certain individuals receiving the aminoglycosides, gentamicin or amikacin, likely reflecting aminoglycoside antibiotic-induced activation the CaSR in the TAL.

At least three second-messenger pathways appear to be involved in CaSR-mediated regulation of transport in the TAL: (1) the CaSR→↑PLA ₂ →↑AA →↑P ₄₅₀ →↑20-HETE pathway; (2) the CaSR→↓AC/↑PDE→↓cAMP pathway; and (3) the CaSR→↑TNF→↑COX-2→↑PGE ₂ pathway. A second-messenger pathway commonly coupled to CaSR stimulation is Gα _q activation of phospholipase C, release of IP ₃ and a rise in Ca ²⁺ _i via release of Ca ²⁺ _o from internal stores (CaSR→↑PLC→↑IP ₃ →↑Ca ²⁺ _i pathway). Increasing Ca ²⁺ _o does lead to rises in Ca ²⁺ _i in the rabbit, rat, and mouse TAL but there are divergent data regarding whether this increase in Ca ²⁺ _i is functionally coupled to the Gα _q/11 →↑PLC pathway. The rise in intracellular Ca ²⁺ appears to take place via extracellular entry in the rabbit, but in the mouse CTAL CaSR agonists do induce a rise in Ca ²⁺ _i from a thapsigargin-sensitive pool. In the rat, increases in Ca ²⁺ _o produce a concentration-dependent increase in IP ₃ implicating the classical CaSR→↑PLC→↑IP ₃ →↑Ca ²⁺ _i pathway. Regardless of the mechanism, an important conclusion is that CaSR activation in the TAL gives rise to an increase in Ca ²⁺ _i .

The CaSR→↑PLA ₂ →↑AA→↑P ₄₅₀ →↑20-HETE pathway couples CaSR activation to regulation of apical K ⁺ channels in the TAL ( Fig. 63.7 ). Since the apical 30-pS and 70-pS recycling K ⁺ channels are crucial for maintaining NaCl transport by both the CTAL and MTAL, inhibition of this channel would decrease NaCl, Ca ²⁺ and Mg ²⁺ transport. Ca ²⁺ _o and the other CaSR agonists, activate phospholipase A2 (PLA ₂ ) releasing arachidonic acid (AA), which can be metabolized by a CYP4A P ₄₅₀ -hydroxylase to generate 20-HETE (the CaSR→↑PLA ₂ →↑AA→↑P ₄₅₀ →↑20-HETE pathway) . 20-HETE inhibits the 70-pS (but not the 30-pS) apical K ⁺ channel in inside-out membrane patches. In contrast, AA can inhibit the 30-pS (ROMK1) K ⁺ channel activity independently of the generation of AA metabolites. Thus, both the 30-pS and 70-pS K ⁺ channels in the apical membranes of TAL can be inhibited by components of the CaSR→↑PLA ₂ →↑AA→↑P ₄₅₀ →↑20-HETE pathway. This pathway has also been implicated in the inhibition of salt transport in the CTAL by angiotensin II (Ang II) and bradykinin. This effect of Ang II is mediated, at least in part, by P ₄₅₀ →↑20-HETE–mediated inhibition of the 70-pS K ⁺ channel. In addition, this pathway has been implicated in the effect of a K ⁺ -deficient diet to reduce apical 70-pS K ⁺ channel activity. In fact, K ⁺ depletion enhances the Ca ²⁺ _o -induced inhibition of the 70-pS K ⁺ channel. Finally, exogenous AA can inhibit adenylate cyclase activity in CTAL by a pertussis toxin–sensitive mechanism ; this would modulate the effects of Gαs-coupled hormones on NaCl transport, and thereby divalent mineral absorption.

The PLA ₂ →↑AA→↑P ₄₅₀ →↑20-HETE and the CaSR→↑TNF→↑COX-2→↑PGE ₂ pathways couple calcium-sensing receptor (CaSR) activation to regulation of salt and divalent mineral handling in the thick ascending limb. See text for discussion.

The CaSR→↓AC/↑PDE→↓cAMP pathway also couples CaSR activation to regulation of salt transport in the TAL ( Fig. 63.8 ). Increases in Ca ²⁺ _o reduce vasopressin-stimulated cAMP production in isolated mouse and rat TAL segments by inhibiting adenylate cyclase (AC) activity and increasing nucleotide degradation in the TAL. Small increases in Ca ²⁺ _o (0.5 to>1.5 mM) produce a modest decrease in cAMP production but a major increase in phosphodiesterase (PDE)-dependent cAMP degradation. Studies on cyclic nucleotide-generating secretagogues in rat and mouse colon support the potent effect of the CaSR to increase PDE activity and suggest that a rise in Ca ²⁺ _o is crucial for activation of PDE. Larger (non-physiological) increases in Ca ²⁺ _o (> 1.5 mM) produce an even greater decrease in cAMP production in the TAL. The CaSR agonist-induced rise in Ca ²⁺ _i is also crucial for the latter, probably by modulating a Ca ²⁺ -inhibitable adenylate cyclase isoform in the TAL (AC types 5 and 6) ^. The mechanism for CaSR-mediated modulation of cAMP has been studied in detail in HEK cells, where CaSR stimulation reversed or prevented cAMP accumulation by activating a pertussis-sensitive Gα _i and by increasing Ca ²⁺ _i . Low-frequency, but not high-frequency, Ca ²⁺ _i oscillations correlated with CaSR-mediated reductions in cAMP. Certain hormones, like PTH, also may enhance the paracellular permeability for Ca ²⁺ and Mg ²⁺ , at least in mouse CTAL . The CaSR regulates both the active transcellular and passive paracellular reabsorption in mouse CTAL. It should be noted that in bovine parathyroid cells extracellular Ca ²⁺ decreases cAMP accumulation by a pertussis toxin-sensitive mechanism, implicating functional coupling of the parathyroid CaSR to Gα _i2 . However, in rat TAL, pertussis toxin has no effect on cAMP accumulation induced by increasing Ca ²⁺ _o to 2.5 mM.

The CaSR→↓cAMP/↑PDE→↓cAMP pathway couples CaSR activation to regulation of salt and divalent mineral transport in the thick ascending limb (TAL). See text for discussion.

The CaSR is also expressed in MTAL where it regulates NaCl reabsorption and alters the countercurrent mechanism crucial for urinary concentrating ability. This CaSR effect in MTAL could be important for regulating the concentration of divalent minerals in the final urine, ensuring that divalent minerals are excreted in a less concentrated urine, and thereby, reducing the risk of stone formation or nephrocalcinosis. Information on the effects of CaSR agonists on transport processes in MTAL is inferential. It seems likely that the CaSR in MTAL also functions via the CaSR→↑PLA ₂ →↑AA→↑P ₄₅₀ →↑20-HETE pathway as well as by reducing cAMP, based on a number of studies in MTAL showing that 20-HETE inhibits salt transport by reducing Na ⁺ -K ⁺ -2Cl ⁻ cotransport, Na ⁺ -K ⁺ -ATPase, and apical 70-pS K ⁺ channel activities.

The CaSR→↑TNF→↑COX-2→↑PGE ₂ pathway also participates in CaSR-mediated regulation of salt transport by the TAL ( Fig. 63.7 ). CaSR agonists increase cyclooxygenase-2 (COX-2) expression and COX-2–dependent synthesis of prostaglandin E ₂ (PGE ₂ ) in primary cultured MTAL cells. CaSR-mediated increases in TNF production in rat MTAL cells depend on activation of PLC and a downstream calcineurin- and NFAT-(nuclear factor of activated T cells) dependent pathway. The increase in COX-2 associated with CaSR stimulation depends, at least in part, on a rise in TNF levels and a TNF autocrine feedback on MTAL cells. Since PGE ₂ reduces NaCl transport by TAL, this CaSR-linked, cytokine-mediated mechanism may contribute to regulating NaCl and divalent mineral handling by CTAL and MTAL. PGE ₂ also inhibits the 70-pSK ⁺ channel in TAL by reducing vasopressin-stimulated cAMP accumulation and by a PKC-dependent pathway. It has been suggested that variations in the local interstitial ionic Ca ²⁺ concentration could influence COX-2 activity and PGE ₂ production along the TAL from outer medulla to cortex A clinical consequence of the CaSR→↑TNF→↑COX-2→↑PGE ₂ pathway can be seen in chronic hypercalcemia where high urinary PGE ₂ excretion results from upregulated COX-2 expression. The influence of Ca ²⁺ _o on salt transport in TAL via the CaSR→↑TNF→↑COX-2→↑PGE ₂ pathway also provides one mechanism for the PGE ₂ -dependent renal polyuria and salt loss occurring in chronic hypercalcemia.

The integrated effect of stimulation of the CaSR→↑TNF→↑COX-2→↑PGE ₂ , CaSR→↑PLA ₂ →↑AA→↑P ₄₅₀ →↑20-HETE and CaSR→↓AC/↑PDE→↓cAMP pathways in TAL is a “loop diuretic”–like effect of activation of the CaSR by its agonists. This diuretic effect likely accounts for the observation that increasing serum ionized Ca ²⁺ concentration in healthy men by ~25% results in about a 1.5-fold increase in Na ⁺ excretion and the diuresis and natriuresis that accompanies hypercalcemic states. Chronic hypercalcemia, induced in rats either by PTH to vitamin D, also downregulates several Na ⁺ transporters, including the Na ⁺ -K ⁺ -2Cl ⁻ cotransporter. While it seems likely that the CaSR mediates these effects, at least in part, this has not been studied experimentally. These latter two studies also point out that reduced expression of Na ⁺ transporters in several nephron segments, in addition to TAL, may contribute to the solute and volume depletion observed in some hypercalcemic patients. In addition, this integrated effect of CaSR activity on the TAL and its consequent loop diuretic-like effect may participate in the influence of calcium intake on blood pressure. The possible impact of molecular variations in the CaSR in Na ⁺ balance, blood pressure and other processes, such as colonic neoplasia, are currently under investigation.

Reabsorption of Ca ²⁺ in MTAL and CTAL through the paracellular pathway is accompanied by little water flow since this tubular epithelium has a low water permeability due to the lack of apical aquaporins. Thus, we might anticipate that the resultant rise in Ca ²⁺ concentration at the basolateral surface of the TAL would activate the CaSR and immediately reduce further NaCl and Ca ²⁺ reabsorption. The CaSR, however, has a built in mechanism ( Fig. 63.9 ), whereby increasing ionic strength reduces the receptor’s sensitivity (increases the EC ₅₀ ) to extracellular Ca ²⁺ by an allosteric mechanism. The reduction in CaSR activation at higher ionic strength (NaCl concentration) translates to less inhibition of ion reabsorption. The obvious advantage is that both Na ⁺ and Ca ²⁺ contribute toward their final reabsorption and accumulation at the basolateral membrane of this nephron segment via regulated activity of the CaSR.

Role of the allosteric modifying effect of ionic strength in modulating calcium-sensing receptor (CaSR) function in the medullary thick ascending limb. Both Na ⁺ and Ca ²⁺ contribute to their final reabsorption and accumulation at the basolateral membrane of this nephron segment via regulated activity of the CaSR. See text for discussion.

As discussed previously, Ca ²⁺ and Mg ²⁺ reabsorption by the TAL are coupled. This might seem contradictory since normal plasma concentrations of Mg ²⁺ are well below the EC ₅₀ values for CaSR activation by these divalent cations in vitro . However, genetic diseases in humans in which the CaSR gene is mutated indicate that the CaSR is also involved in Mg ²⁺ _o homeostasis (i.e., serum Mg ²⁺ is high-normal or mildly elevated in FHH). While it is still unclear how this is achieved, Mg ²⁺ _o might enhance receptor sensitivity to Ca ²⁺ _o (a calcimimetic-like effect). However, the tubule Mg ²⁺ concentration at TAL can be twice that in the initial glomerular filtrate, owing to the limited reabsorption of Mg ²⁺ in the PT compared with other ions such as Na ⁺ , Cl ⁻ , and Ca ²⁺ . This increase in Mg ²⁺ concentration and delivery to the TAL would enhance Mg ²⁺ reabsorption via the paracellular pathway and give rise to higher interstitial Mg ²⁺ concentrations near the basolateral membrane. The high basolateral Mg ²⁺ concentration may be sufficient to activate the basolateral CaSR in CTAL and suppress reabsorption of further magnesium.

The EC ₅₀ for Ca ²⁺ stimulation of the CaSR can be modified by extracellular pH, suggesting that the receptor might also act as a pH sensor along the nephron. An analogous role for the CaSR has recently be documented in the stomach. Thus, not only Ca ²⁺ _o , but also the state of acid–base balance could influence salt and water transport along the nephron by modulating the CaSR’s activity. Although direct evidence is not available, several mechanisms responsible for NH ₄ ⁺ or HCO ₃ ⁻ transport could be affected by the CaSR, which could, therefore, alter acid-base balance. The TAL actively reabsorbs NH ₄ ⁺ by transcellular and paracellular pathways. The bulk of transcellular absorption depends on Na ⁺ -K ⁺ (NH ₄ ⁺ )-2Cl ⁻ cotransporter activity where NH ₄ ⁺ instead of K ⁺ is transported with Na ⁺ and Cl ⁻ . Paracellular NH ₄ ⁺ transport depends on the transepithelial voltage just like the transport of Ca ²⁺ or Mg ²⁺ . Similar to the influence of the CaSR on divalent mineral absorption, stimulating CaSR (high Ca ²⁺ _o and/or pH) may reduce NH ₄ ⁺ absorption by the TAL. Therefore, the activity of the CaSR could influence net urinary acid excretion by modulating the accumulation of ammonium/ammonia in the renal medulla. The TAL also reabsorbs HCO ₃ ⁻ and this transport can be inhibited by angiotensin II via the PLA ₂ →↑AA →↑P ₄₅₀ →↑20-HETE pathway. Similarly, activation of this same pathway by the CaSR would likely reduce HCO ₃ ⁻ absorption.

Finally, macula densa cells express substantial CaSR immunoreactivity on their basolateral side. Although the receptor’s function in this region has not been investigated, indirect evidence suggests that the CaSR could sense changes in Ca ²⁺ _o and, accordingly, regulate tubuloglomerular feedback.

CaSR in the DCT

The DCT reabsorbs about 10% of filtered Ca ²⁺ by an active transcellular mechanism, as Ca ²⁺ movement through the intercellular spaces is negligible because of the markedly low permeability of the tight junctional membranes to Ca ²⁺ (see Chapter 65 ). In this nephron segment, Ca ²⁺ absorption is inversely proportional to Na ⁺ absorption and strongly regulated by calciotropic factors (PTH, vitamin D). Ca ²⁺ absorption is a three-step process. First, apical Ca ²⁺ entry occurs down its electrochemical gradient through the Ca ²⁺ -selective channel, TRPV5 (see Chapter 65 ). Second, Ca ²⁺ must diffuse across the cell, presumably bound to the Ca ²⁺ -binding proteins, calbindins D _9K and D _28K . Finally, basolateral efflux of Ca ²⁺ into the bloodstream is uphill and requires energy achieved by means of a plasma membrane Ca ²⁺ -ATPase (PMCA) and the Na+/Ca ²⁺ exchanger (NCX1). 1,25(OH) ₂ D ₃ acts in the DCT to increase active Ca ²⁺ reabsorption by upregulating the expression of the key molecules participating in transcellular Ca ²⁺ transport, including TRPV5, calbindins-D _9K and D _28K , NCX1, and PMCA1B. The role of vitamin D in regulating this pathway was shown unequivocally using 1 α(OH)ase-/- mice, which lack any endogenous 1,25(OH) ₂ D ₃ . Repleting these mice with 1,25(OH) ₂ D ₃ increases the expression of TRPV5, calbindin-D _28K , calbindin-D _9K , NCX1 and PMCA1B.

The CaSR is expressed basolaterally, intracellularly, and occasionally apically in a punctate, vesicle-like pattern in DCT. What role does it play in modulating Ca ²⁺ transport in this nephron segment? Interestingly, restoring normocalcemia and normophosphatemia in the 1 α(OH)ase ^-/- mice with a high Ca ²⁺ high phosphate, lactose-containing “rescue” diet upregulated the expression of TRPV5, calbindin D _28K , NCX1 and PMCA1b. These effects of Ca ²⁺ supplementation likely reflect direct actions of Ca ²⁺ on the same cell type(s) upon which 1,25(OH) ₂ D ₃ acts in the DCT. In fact, earlier studies had shown that 1,25(OH) ₂ D ₃ and/or elevated medium Ca ²⁺ upregulated the expression of calbindin-D _28K in primary chicken kidney cells, likely by acting upon the same calbindin-D _28K -containing cells. The rescue diet, however, did not fully normalize renal Ca ²⁺ handling in the VDR-/- mice, as urinary Ca ²⁺ excretion in the VDR-/- mice was twice that in normal mice ingesting the same rescue diet. Therefore, dietary Ca ²⁺ -induced upregulation of several components of the transcellular pathway for Ca ²⁺ absorption does not completely compensate for loss of the VDR.

A recent study demonstrated that raising Ca ²⁺ _o enhanced Ca ²⁺ reabsorption in DCT, in contrast to the CaSR-mediated inhibition of Ca ²⁺ reabsorption in CTAL. In this study, the CaSR and TRPV5 were co-expressed in the same cells, and activating the CaSR on the apical membrane increased TRPV5 activity, with a resultant rise in Ca ²⁺ _i by means of a PKC-dependent phosphorylation of amino acid residues S299 and S654 in TRPV5. This stimulation of the activity of TRPV5 was suppressed by a dominant negative CaSR, proving the receptor’s involvement. What is the purpose of a CaSR-dependent stimulation of Ca ²⁺ reabsorption in DCT? Topala, et al. suggested that this provides a local feedback mechanism for adjusting Ca ²⁺ reabsorption in DCT as a function of the prevailing urinary Ca ²⁺ concentration, perhaps mitigating the risk of Ca ²⁺ -containing stones when the urine reaching the DCT has an excessively high Ca ²⁺ concentration.

The DCT, along with the CTAL, is also an important nephron segment for Mg ²⁺ conservation, which occurs through TRPM6, an apical Mg ²⁺ channel homologous to TRPV5. The CaSR is expressed in the mouse DCT cell line, MDCT, and cAMP production stimulated by hormones signaling through Gαs activation (PTH, AVP, calcitonin and glucagon) is inhibited by CaSR agonists. Moreover, aminoglycosides can inhibit PTH-stimulated Mg ²⁺ uptake in these cells likely by activating the CaSR. Additionally, the ability of 1,25(OH) ₂ D ₃ to stimulate Mg ²⁺ uptake into MDCT cells is abrogated by elevated Ca ²⁺ _o and Mg ²⁺ _o . This latter effect of divalent minerals is abolished by pretreatment with CaSR antibodies or antisense CaSR mRNA oligonucleotides.

CaSR in Collecting Duct

Inner Medullary Collecting Duct

In the IMCD, the CaSR is expressed in the same endosomes in which the vasopressin-activated AQP2 water channels reside ( Fig. 63.10 ). Vasopressin-activated V2 receptors on the basolateral membrane of IMCD cells produce cyclic AMP accumulation in these cells. Cyclic AMP activates protein kinase A (PKA), which, in turn, phosphorylates AQP2 (and certainly other proteins) leading to movement of these endosomes to the apical plasma membrane. The increase in water permeability of the apical membrane enhances transtubular movement of water driven by the interstitium-to-luminal osmotic gradient. The result of these events is an increase in final urine osmolality. Since the AQP2-containing endosomes also contain CaSR protein, insertion of these endosomes into the apical membrane increases surface expression of both AQP2 and CaSR. Activation of the CaSR by increases in tubular Ca ²⁺ concentrations reduces vasopressin-stimulated increases in water permeability in the in vitro -perfused IMCD, presumably due to reduced trafficking of AQP2 to the luminal membrane during CaSR activation. Since CaSR stimulation can reduce cAMP accumulation in TAL cells, a similar CaSR-mediated reduction in cAMP in IMCD might account for the reduction in water permeability. Vasopressin-enhanced water permeability in the IMCD is also virtually absent in dihydrotachysterol-induced chronic hypercalcemia. A reduction in AQP2 protein expression and activation of the CaSR contribute to this effect.

In the inner medullary collecting duct (IMCD), the calcium-sensing receptor (CaSR) is expressed in the same endosomes in which the vasopressin-activated aquaporin-2 (AQP2) water channels are expressed. Activation of the CaSR by increases in tubular fluid Ca ²⁺ concentrations reduce vasopressin-stimulated increases in water permeability by modulating the trafficking of AQP2. Since the EC ₅₀ for Ca ²⁺ _ο stimulation of the CaSR is modified by pH, ionic strength, polyamines, and amino acids, many constituents of the tubule fluid in the terminal collecting duct can influence receptor activity and its consequences on IMCD water permeability. See text for discussion.

A consequence of these effects of CaSR stimulation in the IMCD is regulation of the concentration of ionized Ca ²⁺ in final urine. Since the EC ₅₀ for Ca ²⁺ _o stimulation of the CaSR is modified by pH, ionic strength, polyamines, and amino acids (a calcimimetic-like effect, see for review), many tubule fluid constituents in the IMCD can influence receptor activity and its consequences on IMCD water permeability. Ca ²⁺ solubility in the tubule fluid or crystal growth may be influenced by many factors altering the CaSR’s EC ₅₀ for Ca ²⁺ (see Chapter 68 ). Moreover, Ca ²⁺ delivery to the IMCD is increased whenever the CaSR is stimulated in the TAL. If luminal Ca ²⁺ increases in the terminal IMCD above a certain level determined by the integration of factors influencing the CaSR, the enhanced CaSR stimulation in IMCD will alter reabsorption to limit further increases in Ca ²⁺ concentration. This influence of Ca ²⁺ on water transport in IMCD provides an example of physiological “trade-off” where water conservation is sacrificed to ensure uneventful excretion of divalent cations in a soluble form in terrestrial vertebrates.

CaSR and Renin Secretion

Renin plays important roles in sodium, volume and blood pressure homeostasis: Hypovolemia is sensed by the juxtaglomerular (JG) cells of the afferent arteriole in the kidney, which, in turn, increases renin secretion. Renin converts circulating angiotensinogen to angiotensin II, a potent endogenous pressor that also increases production of the sodium-retaining hormone, aldosterone, by the adrenal zona glomerulosa. The elevation in blood pressure stimulated by angiotensin II combined with the sodium retention promoted by aldosterone tends to normalize sodium and volume homeostasis. It has been known for several decades that elevating Ca ²⁺ _o inhibits renin release by the JG cells. Only more recently, however, has it been demonstrated convincingly that the CaSR is expressed by JG cells and mediates the inhibition of renin release by high Ca ²⁺ _o . The role that CaSR-mediated regulation of renin secretion plays in normal blood pressure and volume homeostasis is not currently understood. The capacity to modulate renin secretion pharmacologically, e.g., using a CaSR antagonist, could potentially be of therapeutic benefit in high renin forms of hypertension.

Developmental Regulation of Renal CaSR Expression

In the newborn, reduced responsiveness of the kidney to vasopressin and PTH could potentially reflect effects of the CaSR on these aspects of renal function. In the developing rat kidney, there is little prenatal expression of the CaSR, except in large tubules and branching ureteric buds of developing nephrons. CaSR mRNA and protein increase markedly during the first postnatal week, principally due to expression of the receptor in the developing TAL and, to a lesser extent, in the CD. The receptor’s level of expression remains nearly constant after postnatal day 14. Thus it is possible that some of the previously described changes in renal handling of divalent cations and water in the perinatal and immediate postnatal periods are related, in part, to the increasing levels of CaSR expression and its resultant inhibitory effects on the actions of PTH and antidiuretic hormone on the developing nephron.

The CaSR in Cartilage

The chondrocytic cell line, RCJ3.1C5.18, expresses easily detectable levels of CaSR mRNA and protein. When these cells are incubated with elevated levels of Ca ²⁺ _o , there is suppression of the early differentiation marker, aggrecan, and enhanced expression of several markers of terminal differentiation, namely osteopontin, osteonectin and osteocalcin, as well as increased synthesis of cartilagenous matrix, another indication of enhanced differentiation. Several of these effects of the CaSR were potentiated by overexpressing the wild type CaSR or inhibited by transfecting the cells with a CaSR with defective signaling capacity, suggesting a mediatory role for the receptor. Growth plate cartilage also expresses the CaSR. Initial studies of cartilage in mice with homozygous knock out of exon 5 of the CaSR revealed rickets, suggesting that the receptor might be required for normal cartilage development. However, when these severely hyperparathyroid mice were “rescued” by knock out of the PTH gene (CaSR-/-PTH-/-) or of the key parathyroid transcription factor, Gcm-2 (CaSR-/-Gcm-/-), there was no apparent cartilage phenotype, and the CaSR’s role in cartilage, if any, was questioned.

Additional studies at about the same time, however, demonstrated that keratinocytes from CaSR-/- mice could generate a variant CaSR with exon 5 spliced out. This finding raised the possibility that skeletal expression of the “exon 5-less” CaSR in the original CaSR-/- mice might, in fact, possess biological activity capable of rescuing the CaSR-/-PTH-/- and CaSR ^-/- Gcm-/- mice from any skeletal consequences of losing the full length CaSR. In fact, chondrocytes from CaSR-/- mice had the same cellular responses to Ca ²⁺ _o seen in wild type chondrocytes, presumably owing to the presence of a biologically active CaSR lacking exon 5. However, it has not yet been possible to directly document biological activity of the exon 5-less CaSR when it is expressed in heterologous cell systems.

To study further the biological actions of the CaSR in bone using knock out mouse models, Chang, et al. developed mice in which exon 7 of the CaSR was “floxed” by inserting loxP sites flanking this exon. Mating of these mice with mice expressing the Cre recombinase only in a particular tissue of interest owing to the recombinase being driven by a suitable promoter specific for that tissue, results in recombinase-mediated excision of the floxed exon and recombination of the ends of the remaining gene. As a result, transcription and translation now produces a truncated CaSR protein lacking exon 7. Mice with exon 7 of the CaSR floxed were mated with mice expressing the Cre recombinase only in chondrocytes to examine the consequences of knocking out the CaSR in cartilage. Exon 7 encodes the entire CaSR transmembrane domain and C-tail, and when the CaSR gene lacks exon 7, it can only generate the CaSR’s ECD in a soluble form, which would be released extracellularly and presumably be incapable of signaling. Chondrocyte-specific CaSR deletion resulted in death of embryos by day 13, an unexpectedly severe phenotype in view of the multiple hormonal or other factors regulating chondrocyte development and function. Subsequent use of an inducible Cre recombinase made it possible to delete exon 7 of the CaSR on days 16–18 of embryonic life, i.e., subsequent to when the embryos died in the previous model. This maneuver produced viable embryos, which nevertheless displayed delayed development of their growth plates. These data suggested, therefore, a critical, non-redundant role(s) for the CaSR in cartilage development.

The CaSR in Osteoblasts

Initially, some, but not all, studies found the CaSR in intact bone, primary osteoblasts in culture and osteoblastic cell lines (for review, see ). In osteoblastic cells expressing the CaSR, high Ca ²⁺ _o has actions that would be expected to stimulate bone formation. These include promoting proliferation of pre-osteoblasts, enhancing expression of the mRNAs that encode osteoblast differentiation markers, such as Cbfa-1, osteocalcin, osteopontin, and collagen 1, and stimulating mineralized nodule formation.

Studies in the mice with global knock out of exon 5 of the CaSR revealed, in addition to the rickets alluded to above, severe hyperparathyroid bone disease resulting from loss of the CaSR in the parathyroid, which complicated interpretation of the impact of losing the CaSR on osteoblast function per se. Studies in “rescued” CaSR-/-PTH-/- and CaSR-/-Gcm-/- mice, however, showed little or no difference in their bone histology and histomorphometry from that observed in control mice, suggesting that the CaSR does not have an important role in the formation and turnover of the skeleton. However, conditional knock out of exon 7 of the CaSR in osteoblasts utilizing osteoblast-specific Cre’s produced mice exhibiting poor postnatal growth and skeletal development, with small poorly mineralized skeletons. Most of these mice experienced long bone and rib fractures and died by 3 weeks of age. Their bones had reduced levels of both early and late markers of osteoblast differentiation, including type 1 collagen, insulin-like growth factor-1 (IGF-1, a key osteoblast growth factor), alkaline phosphatase, and osteocalcin. There was also a higher than normal rate of osteoblast apoptosis. These results suggest key roles for the CaSR in promoting proliferation, differentiation and survival of osteoblasts as well as in enhancing mineralization of the skeleton. They also have the important implication that a calcimimetic with some specificity for osteoblasts might have potential utility as a bone anabolic agent.

The CaSR in Osteoclasts

As with osteoblasts, some studies failed to detect the CaSR in osteoclasts, while others found it in cell lines considered to be models of osteoclast precursors (e.g., RAW 264.7), in multinucleated osteoclasts differentiated from these precursors in vitro and in at least some mature osteoclasts in bone sections. Available evidence suggests that while the CaSR serves a permissive role in osteoclastogenesis in vitro , high Ca ²⁺ _o concentrations also directly suppress osteoclast activity and enhance their apoptosis. Therefore, if the CaSR has similar actions in vivo , a calcimimetic targeting both osteoblasts and osteoclasts specifically might not only stimulate bone formation but also inhibit bone resorption, although an in vitro investigation that did not detect CaSR transcripts in osteoclasts or their precursors did not observe any functional effect of a calcimimetic on these cells. Clearly more work is necessary to determine the CaSR’s role, if any, in osteoclasts in vivo . The effects of Ca ²⁺ _o on osteoclast function have also been suggested to result from an entirely different Ca ²⁺ _o -sensing mechanism (for review, ).

Roles of the CaSR in the Stomach

In the stomach, the CaSR is expressed in the mucus-secreting surface epithelium, the parietal cells in the gastric crypts, and in the gastrin-secreting G-cells. It directly stimulates acid secretion and also does so indirectly by stimulating gastrin secretion, which, in turn, promotes secretion of histamine from gastric ECL cells. The latter acts directly on parietal cells to stimulate gastric acid secretion. Although gastric acid secretion might not seem relevant to Ca ²⁺ homeostasis, both calcium carbonate and calcium phosphate salts are more soluble under acidic conditions (CaCO ₃ will not dissolve above pH 5.0). Therefore, an acidic environment in the stomach will promote the availability and, subsequently in the small intestine, the absorption of free luminal Ca ²⁺ . Recent work has suggested that the CaSR in the stomach senses not only Ca ²⁺ but also amino acids and pH. Thus the receptor likely integrates several types of signals that impact its functions in the stomach. The response to amino acids may promote the secretion of acid needed to stimulate the gastric phase of protein digestion, while the response of the CaSR to pH could represent, at least in part, the long-sought pH sensor needed for the feedback regulation of acid secretion, stimulating it when pH is high and inhibiting it when pH is low.

Roles of the CaSR in the Small Intestine

The CaSR is present in the small intestine, likely in the same cells expressing the VDR (although this has not yet been formally proven) as well as in cell lines of intestinal origin. However, its functions in the intestine are incompletely characterized. Available data suggest that it can act together with vitamin D to enhance the expression of the various components of the transcellular calcium transport system or substitute, at least in part, for the actions of 1,25(OH) ₂ D ₃ in certain instances. For example, in organ cultures of fetal rat duodenum, not only 1,25(OH) ₂ D ₃ but also elevated levels of Ca ²⁺ _o increase the expression of the mRNA for calbindin-D _9K . Subsequently, van Abel, et al. demonstrated that administering the rescue diet described earlier to 1α(OH)ase-/- mice normalized serum Ca ²⁺ concentration concomitant with statistically significant,>10-fold increases in the intestinal expression of TRPV6 and calbindin-D _9K , similar to the effect of dietary rescue in the kidneys of these mice. While not formally proven, these actions of calcium are likely to be CaSR-mediated. Thus elevated levels of Ca ²⁺ can apparently substitute, at least partially, for a lack of vitamin D in maintaining sufficient levels of expression of key elements of the intestinal transcellular Ca ²⁺ transport system, suggesting a vitamin D-independent role for Ca ²⁺ _o in regulating its own absorption in the intestine. An additional function of the CaSR in the small intestine may be to promote digestion via the capacity of cholecystokinin (CCK)-secreting endocrine cells to secrete CCK in response to calcium or upon exposure to peptides or aromatic amino acids. The secreted cholecystokinin, in turn, would stimulate pancreatic enzyme secretion and enhance small intestinal digestion of ingested nutrients.

Roles of the CaSR in the Colon

Although the proximal small intestine is often thought of as the predominant site for intestinal calcium absorption, substantial absorption also occurs in the cecum of the large intestine by a vitamin D-responsive mechanism. The cloning of the apical intestinal calcium uptake channel, TRPV6, provided a molecular basis for regulated calcium absorption in the cecum, as this channel was expressed at robust levels in this segment of the large bowel. An additional function of the CaSR in the large intestine, which is apparently uninvolved in calcium homeostasis per se, is to inhibit the secretion of fluid by the crypts, particularly when stimulated by cAMP. This action has been suggested to represent a potential target for the treatment of diarrheal disease, such as cholera (e.g., with a calcimimetic). Finally, the CaSR expressed in the colon regulates the growth and differentiation of cells within the colonic crypt, promoting cellular differentiation and inhibiting cell growth. It does so by mechanisms that include: (1) upregulation of cyclin-dependent kinase inhibitors such as p21, (2) promotion of E-cadherin expression and suppression of beta-catenin expression, (3) increased local levels of 1,25(OH) ₂ D ₃ (which also enhances differentiation and inhibits proliferation), and (4) reduction in c-myc expression. These actions of the CaSR may contribute to the reduction in the risk of colonic adenomas or colon cancer observed with increased dietary intake of calcium in many but not all studies (for reviews, see ).

Calcimimetics are Type II Allosteric Enhancers: Role in 2° HPT

Calcimimetics are small, lipophilic organic molecules that function as allosteric enhancers (superagonists) of the CaSR. Thus, calcimimetics are type II agonists that left-shift the ionized Ca ²⁺ -PTH secretion curve in parathyroid glands (lower the EC ₅₀ for ionized Ca ²⁺ ) without affecting the minimal or maximal responses ( Fig. 63.1 ). As type II agonists, calcimimetics have no effect on PTH secretion in the absence of extracellular Ca ²⁺ . In 5/6 nephrectomized and normal rats, calcimimetics cause a dose-dependent decrease in serum PTH and ionized Ca ²⁺ . A first-generation calcimimetic, NPS R-568, was shown to reduce PTH secretion in vitro and in vivo , but it was entirely metabolized by the hepatic P ₄₅₀ system and exhibited suboptimal pharmacokinetics in certain individuals. Second-generation calcimimetics (e.g., cinacalcet HCl) overcame this problem in metabolism of the drug (see following sections). Calcimimetics have been shown to reduce PTH secretion in 1° HPT, 2° HPT, and parathyroid carcinoma. While calcimimetics can produce a transient increase in calcitonin secretion from thyroidal C cells, the dose of calcimimetic that suppresses PTH secretion is at least 10-fold lower than required to alter calcitonin secretion. This apparent specificity of calcimimetics for parathyroid as opposed to other cell types examined to date is an important factor enabling targeted therapy of various forms of hyperparathyroidism.

Calcimimetics reduce plasma Ca ²⁺ . The fall in plasma Ca ²⁺ is clearly not due to increased renal excretion of Ca ²⁺ , given that it occurs in nephrectomized rats, but instead results from the left shift in the relationship between serum Ca ²⁺ and PTH. In mild 1° HPT calcimimetics acutely and chronically lower serum PTH and normalize serum Ca ²⁺ without increasing urine Ca ²⁺ excretion in most patients. Activation of the kidney CaSR would be expected to increase urinary Ca ²⁺ excretion at any given level of serum Ca ²⁺ , and thus the lack of effect of calcimimetics on renal Ca ²⁺ excretion in 1° HPT suggests that at doses that reduce the parathyroid set-point, these agents do not exert a calciuric effect owing to their direct renal action. In 1° HPT, a reduced filtered load of Ca ²⁺ , decreased bioavailability of the drug in the kidney, or lower calcimimetic sensitivity of the renal, relative to the parathyroid, CaSR have been suggested as the causes of any apparent effect of calcimimetics on the kidney. However, like parathyroidectomy, the calcimimetic NPS R-467 prevents furosemide-induced nephrocalcinosis in rats, suggesting that the reduction in PTH is an important indirect factor in the modulation of renal Ca ²⁺ handling in this model. The calcimimetic, cinacalcet HCl (also known as AM 073, KRN 1493, NPS 1493; Cinacalcet HCl, Sensipar in the United States; Mimpara in the European Union), which exhibits good bioavailability and pharmacodynamics, lowers serum PTH and serum Ca ²⁺ in hemodialysis and peritoneal dialysis patients with 2° HPT. On the basis of findings from randomized, double-blind studies, cinacalcet HCl was approved for the treatment of secondary HPT.

In the 5/6 nephrectomy model of chronic renal failure in the rat, calcimimetics prevent parathyroid hyperplasia when given at the time of renal mass reduction and reduce gland mass when given after the development of diffuse hyperplasia. These changes in parathyroid gland mass are associated with an increase CaSR and VDR expression as well as a reduction in parathyroid cell proliferation. The effects of extracellular Ca ²⁺ on parathyroid cell proliferation in culture have been variable ; however, parathyroid cells in culture can exhibit a rapid decrease in CaSR expression complicating interpretation of these studies. R-467 suppressed DNA synthesis by 35% in one study, while Ca ²⁺ had the opposite effect in cultured parathyroid cells derived from uremic patients that continue to express the CaSR. While the mechanisms for the disparate effects of extracellular Ca ²⁺ and calcimimetics remain to be defined, the downstream mediators of CaSR action on parathyroid gland proliferation–differentiation are under investigation. The CaSR-mediated activation of the mitogen-activated protein kinases (MAPKs; ERK1/2) appears to be linked to the inhibition of PTH secretion.

Calcimimetics, the Ca×Pi Product, and Cardiovascular Risk in Dialysis Patients

The elevation of the serum calcium×phosphate product (Ca×Pi) commonly occurring in dialysis patients predisposes them to vascular and tissue calcification and contributes importantly to their increased risk of cardiovascular events. Also, plasma PTH levels, as well as those of Pi and calcium, impact on patient survival. As a “uremic toxin”, the effects of PTH are believed to occur through an alteration in myocardial function and of microvessel thickness. In addition, high Pi concentrations increase vascular calcification, an effect that is exacerbated by an elevation in serum calcium concentration. These events are of relevance if one considers that, until the calcimimetics became available, all maneuvers aimed at reducing PTH levels yielded an increase in plasma ionized calcium and of the Ca×Pi product. Indeed, treatment of 2° HPT with 1,25(OH) ₂ D ₃ or its analogues, while generally initially effective in reducing serum PTH, the calcemic and the phosphatemic effects of vitamin D can ultimately enhance the abnormal mineral metabolism and exacerbate vascular calcification, although retrospective studies have suggested a reduction in overall mortality with vitamin D therapy. In contrast, cinacalcet HCl generally lowers serum Ca ²⁺ , serum phosphate, and the serum Ca×Pi product modestly (by ~15%) and decreases both vascular and soft tissue calcification in uremic rats treated with calcitriol, suggesting that this calcimimetic could potentially lower morbidity and mortality due to cardiovascular complications. In the rat uremic model, NPS R-568 treatment has been shown to diminish cardiovascular changes associated with chronic renal failure (cardiac interstitial fibrosis, capillary length density, and arteriolar wall thickness). It is unclear whether calcimimetics reduce cardiovascular risk in animal models by lowering plasma PTH and/or through a direct effect on blood vessels. Evidence that the CaSR is present in rat and porcine blood vessels and that activation of the receptor by high calcium or calcimimetics results in vasodilatation has been demonstrated. Also, in uremic rats, but not in sham-operated animals, the calcimimetic R-568 causes a marked and sustained antihypertensive effect. While these animal studies support the potential for a beneficial effect of calcimimetics on cardiovascular risk in chronic renal failure in humans, this latter possibility is currently under investigation. A small meta analysis of four randomized, double-blind, placebo-controlled clinical trials utilizing cinacalcet or placebo in patients who were already being treated with vitamin D and phosphate binders assessed the impact of cinacalcet therapy on end-points other than serum mineral ions and PTH. The results indicated that, relative to patients not receiving cinacalcet who were receiving standard care, patients treated with cinacalcet had a >90% decrease in the rate of parathyroidectomy and about a 40% reduction in hospitalizations for cardiovascular events. The study was not sufficiently powered, however, to determine the effect of the drug on “hard” cardiovascular end-points, such as myocardial infarction or death. Two randomized controlled studies have recently been completed that address this latter point, the ADVANCE (“A randomized study to evaluate the effects of cinacalcet plus low dose vitamin D on vascular calcification in subjects with chronic kidney disease (CKD) receiving hemodialysis”) and EVOLVE studies (EValuation Of Cinacalcet HCl Therapy to Lower cardioVascular Events). The results of the ADVANCE trial showed that Cinacalcet produced modest but statistically significant reductions in some indices of vascular (e.g., volume of coronary artery calcification) and valvular (i.e., aortic valve) calcification. ^350a The second of these has as its primary end-points all-cause mortality and first nonfatal cardiovascular event in 3800 chronic dialysis patients treated with a flexible regimen of traditional therapies, and, in addition, receiving either cinacalcet or placebo. While not yet published, the initial results from the EVOLVE trial did not show any reduction in overall cardiovascular mortality in the Cinacalcet-treated patients ( www.amgen.com/media/media_pr_detail.jsp?releaseID=1703773 ).

The severity of osteitis fibrosa is directly proportional to the severity of the 2° HPT and the magnitude of PTH overproduction. While high and sustained elevations in serum PTH in hyperparathyroidism cause osteitis fibrosa, smaller and transient increases in PTH, e.g., those produced by a subcutaneous injection of PTH1-34 or PTH1-84 for the treatment of osteoporosis, have a net anabolic effect on bone. PTH secretion in healthy individuals exhibits complex fluctuations—a circadian rhythm as well as an ultradian rhythm of several PTH pulses per hour. These serum PTH rhythms are generally preserved in chronic renal failure and correlate with serum Ca ²⁺ , serum phosphate, and bone metabolism. Chronic suppression of PTH secretion with vitamin D analogues in 2° HPT is associated with inhibition of the ultradian and circadian rhythms of PTH, which may contribute to the low bone turnover disease of chronic renal failure. However, while calcimimetics appear to induce a circadian rhythm in serum PTH, interestingly, they do not increase bone mass in ovariectomized rats. In the rat 5/6 nephrectomy model of 2° HPT, NPS R-568 halts the progression of, and reverses osteitis fibrosa cystica. This response of bone is consistent with the PTH-lowering effect of calcimimetics. In the rat uremic model, daily intermittent NPS R-568 induces an exaggerated circadian fluctuation in PTH and stabilizes cortical and cancellous bone mass.