Can calcimimetics inhibit parathyroid hyperplasia? Evidence from preclinical studies

The cells of the parathyroid gland secrete parathyroid hormone (PTH), which plays a pivotal role in maintaining circulating levels of ionized calcium (Ca 2+ ) within a narrow physiological range. The main actions of PTH include (i) releasing calcium and phosphorus from bone, (ii) decreasing renal calcium excretion, (iii) increasing urinary phosphorus excretion and (iv) stimulating renal production of calcitriol (1,25- dihydroxy vitamin D3), the active form of vitamin D. Vitamin D and its receptors (VDRs) also play key roles in calcium homeostasis: vitamin D acts on VDRs in the intestine to increase calcium absorption, and on VDRs in parathyroid cells to inhibit PTH mRNA synthesis [ 1 ]. Secondary hyperparathyroidism (SHPT) represents an adaptive response to the progressively impaired control of calcium, phosphorus and vitamin D in chronic kidney disease (CKD). It is characterized by parathyroid hyperplasia and excessive synthesis and secretion of PTH, resulting in excessive bone resorption, soft-tissue and vascular calcification and significantly increased risk for cardiovascular morbidity and mortality [ 2 , 3 ]. Extracellular calcium is the primary physiological stimulus regulating secretion of PTH and there is an inverse, sigmoidal relationship between the levels of plasma PTH and calcium. A cell surface receptor located on parathyroid cells, the calcium-sensing receptor (CaR), has been recognized as the primary mechanism that mediates the effects of Ca 2+ on PTH secretion [ 4 , 5 ]. The CaR also appears to play a key role in the excessive cell proliferation that occurs in parathyroid hyperplasia [ 5 ]. Drugs that mimic or potentiate the action of Ca 2+ at this receptor, calcimimetics, have become available for treatment of dialysis patients (CKD stage 5) with insufficient control of PTH and calcium and/or phosphate levels on traditional therapies [ 6 ]. This article overviews the key pathophysiological mechanisms that drive parathyroid hyperplasia in SHPT and examines the potential of calcimimetics for attenuating this condition, based on emerging data from animal models. CKD is associated with disturbed calcium and phosphorus homeostasis and decreased calcitriol production. PTH secretion is increased in an attempt to correct serum calcium and phosphate: however, as renal failure progresses, higher PTH levels are required to maintain calcium homeostasis, in association with an increased phosphate burden resulting from decreased glomerular filtration and insufficient renal production of calcitriol. Phosphate accumulation and calcitriol deficiency decrease serum calcium, which stimulates the parathyroid to produce PTH ( Figure 1 ). Accumulation of phosphorus also stimulates parathyroid cell function directly and the decrease in circulating calcitriol leads to disinhibition of PTH synthesis [ 7–10 ]. Schematic representation of the key factors involved in secondary hyperparathyroidism and parathyroid (PT) hyperplasia. PTG, parathyroid gland; PTH, parathyroid hormone. Parathyroid cells are generally quiescent and rarely divide under normal physiological conditions [ 11 , 12 ], but can proliferate in response to mitogenic stimuli such as low levels of calcium and calcitriol and elevated phosphorus. Indeed, these are key factors in the development of parathyroid hyperplasia, as well as in excessive PTH synthesis and secretion, as summarized in Table 1 . Although the parathyroid glands initially respond to increased demand by increasing PTH secretion and synthesis, parathyroid cells subsequently begin to proliferate, leading to a diffuse hyperplasia [ 13 ]. Subsequently, transformation from a polyclonal to a more aggressive monoclonal or multiclonal growth pattern occurs [ 14 ]. The glands become grossly enlarged and exhibit a nodular hyperplasia [ 13 , 14 ] ( Figure 2 ). Such nodules are composed of more tightly packed cells featuring larger nuclei and a greater prevalence of cell cycle markers, oxyphil cells and acinar cell arrangements compared with those seen in diffuse hyperplasia [ 12 , 15 ]. Nodules may eventually coalesce to form a single large tumour, which may in rare cases ultimately undergo malignant transformation [ 16 ]. Increased cell volume (cell hypertrophy) appears to play only a minor role in parathyroid gland enlargement caused by uraemia, in contrast with that induced by hypocalcaemia or hyperphosphataemia in the presence of normal renal function, where parathyroid cell hypertrophy prevails over cell proliferation [ 12 ]. Postulated evolution of parathyroid hyperplasia in renal hyperparathyroidism. Adapted from Tominaga et al . [ 131 ] with permission. Both CaR [ 17–20 ] and VDR [ 15,,21–24 ] are progressively down-regulated in the course of parathyroid hyperplasia. Nodular hyperplasia in patients with CKD is associated with a lower density of both CaR [ 17 ] and VDR [ 21 , 22 ] than diffuse hyperplasia, and VDR density was reported to be negatively correlated with both the weight and proliferative activity of the glands [ 21 ]. Overview of the roles of calcium, calcitriol and phosphorus in secondary hyperparathyroidism Overview of the roles of calcium, calcitriol and phosphorus in secondary hyperparathyroidism Enlargement of the parathyroid glands markedly increases the capacity for PTH production. Indeed, basal calcium-independent (non-suppressible) PTH secretion, that parallels the increased gland size [ 25 ], becomes an important factor in elevated PTH levels when the parathyroid glands are 50–100 times their normal size. Moreover, as CaR and VDR expression are reduced in the course of hyperplasia, the parathyroid glands become increasingly resistant to regulation by calcium and calcitriol [ 15 , 26 , 27 ]. Thus, PTH becomes sufficiently elevated to overcome skeletal resistance and mobilize calcium and phosphorus from bone. A vicious cycle ensues, whereby hypersecretion of PTH increases serum calcium and phosphorus levels, but resistance of the parathyroids to calcium regulation allows PTH secretion to continue unabated (‘tertiary hyperparathyroidism’) ( Figure 1 ). Surgical parathyroidectomy may be required if PTH levels cannot be controlled by pharmacological means. The key factors that mediate the transformation of diffuse parathyroid hyperplasia to aggressive tumour-like growth remain to be elucidated [ 28 ]. Changes in the expression of various growth factors/growth factor receptors and tumour enhancer/suppressor genes have been observed in hyperplastic parathyroid tissue, as summarized in Table 2 . It is not clear whether these changes are a cause or a consequence of parathyroid hyperplasia, but such factors may act as autocrine or paracrine regulators of parathyroid cell proliferation in response to Ca 2+ , phosphate and/or active vitamin D. For instance, the three main modulators of parathyroid cell proliferation, namely calcium, phosphate and vitamin D, all modulate signalling via the highly mitogenic transforming growth factor-α/epidermal growth factor receptor (TGF-α/EGFR) growth loop and also regulate p21 expression [ 29 ]. Reduced expression of VDR-dependent p21/p27 may play a key role in nodular parathyroid gland growth, as these genes regulate progression from the G 1 to the S phase of the cell cycle, via inhibition of cyclin-dependent kinase [ 30 ]. Summary of changes in expression of various receptors, genes, growth factors and other molecules observed in hyperplastic parathyroid tissue ↑, enhanced or de novo expression; ↓ , decreased expression; CaR, calcium-sensing receptor; EGFR, epidermal growth factor receptor; TGF, transforming growth factor; VDR, vitamin D receptor. Summary of changes in expression of various receptors, genes, growth factors and other molecules observed in hyperplastic parathyroid tissue ↑, enhanced or de novo expression; ↓ , decreased expression; CaR, calcium-sensing receptor; EGFR, epidermal growth factor receptor; TGF, transforming growth factor; VDR, vitamin D receptor. Parathyroid cells are extremely sensitive to minute alterations in extracellular Ca 2+ , rapidly producing large changes in PTH production and release, and therefore, plasma PTH levels. The CaR is an evolutionarily conserved G protein-coupled cell surface receptor cloned by Hebert and Brown in 1993 [ 4 ] and identified as the sensor for extracellular calcium-mediated regulation of PTH secretion. The CaR has three major domains: a large (612-amino-acid) extracellular ligand-binding N-terminal; a smaller hydrophobic core with 7 membrane-spanning domains (250 amino acids) and an intracellular C-terminal (approximately 250 amino acids). Stimulation of the CaR by elevated extracellular Ca 2+ levels in turn activates the mitogen-activating protein kinase C pathway, via both G-protein-linked phospholipase C and tyrosine phosphorylation of Shc (Src homolog and collagen) [ 31 , 32 ], resulting in activation of phospholipase A2 and production of arachidonic acid [ 31 ]. Arachidonic acid and its metabolites suppress PTH secretion [ 32–35 ]. CaRs are expressed in many tissues, with the highest density being found in the chief cells of the parathyroid gland. CaRs in the kidney also participate in calcium homeostasis [ 5 ]. There are several lines of evidence to support the involvement of the CaR in both excessive PTH secretion and synthesis and parathyroid hyperplasia: (i) The relationship between calcium sensing and abnormalities of the CaR gene. Loss-of-function mutations of the CaR gene are associated with an increased calcium set-point, as shown by an increase in the concentration of Ca 2+ required to inhibit PTH release [ 36–38 ]. Moreover, the presence of two, rather than one, abnormal alleles for the CaR gene is associated with more marked elevation of the calcium set-point, higher serum PTH and calcium levels, and parathyroid hyperplasia. Thus, patients with familial hypocalciuric hypercalcaemia (FHH), who are heterozygotes, have a slight increase in the calcium set-point, with mild asymptomatic hypercalcaemia and normal or slightly elevated PTH levels [ 39 ]. Patients with neonatal severe hyperparathyroidism (NSHPT), who are homozygotes, exhibit a more pronounced increase in the set-point, with more severe hypercalcaemia and PTH elevation, resulting in parathyroid hyperplasia and bone disease [ 39 ]. Knockout mouse models have confirmed these observations, with animals heterozygous for inactivating mutations of the CaR gene exhibiting signs consistent with FHH and those homozygous for such mutations showing NSHPT-like symptomatology [ 40 ]. The development of parathyroid hyperplasia in these models, despite elevated serum calcitriol levels, supports the key role of calcium-dependent signalling, rather than vitamin D-mediated pathways, in parathyroid hyperplasia. Conversely, activating mutations of the CaR are associated with autosomal dominant hypocalcaemia, characterized by hypocalcaemia with inappropriately normal or low PTH levels [ 41 , 42 ]. An increased set-point for Ca 2+ may also be present in patients with SHPT [ 43 , 44 ], although this is not a consistent observation [ 45 , 46 ]. It appears to be more evident in patients with advanced SHPT, autonomous (tertiary) hyperparathyroidism or primary hyperparathyroidism [ 47 , 48 ]. (ii) The association of parathyroid hyperplasia with down-regulation of the CaR in uraemic animals [ 19 , 20 ] and humans [ 17 , 18 ], as already discussed. (iii) The activity of calcimimetics in SHPT. In both animal models and patients with hyperparathyroidism, calcimimetics are able to reduce plasma PTH, calcium and/or phosphorus levels, as discussed in subsequent sections. Calcium, the endogenous ligand for the CaR, negatively regulates transcription of the PTH gene [ 5 ]. Sustained hypercalcaemia results in reduced proliferation of parathyroid cells, as shown in uraemic rats ( Figure 3 ). Calcium loading significantly decreased both the weight of parathyroid glands and the number of proliferating cells [ 49 ]. Although low calcium intake has been found to be associated with markedly enhanced parathyroid cell proliferation in rats [ 50 ], low Ca 2+ concentration in vitro did not directly stimulate proliferation of cultured parathyroid cells isolated from glands from haemodialysis patients with severe SHPT [ 51 ]. Based on these in vitro observations, it has been suggested that increasing extracellular calcium may inhibit parathyroid cell proliferation under conditions of normal or high CaR expression, but stimulate it under low CaR expression [ 51 ]. Effect of dietary calcium on parathyroid weight ( a ) and cell proliferation, as assessed by PCNA ( b ) in 5/6 nephrectomized rats [ 49 ]. Male Sprague–Dawley rats (300–350 g) were anaesthetized (3% isoflurane in O 2 ) and the upper bifurcation of the left renal artery ligated. Animals were allowed to recover for 1 week, after which they were anaesthetized and the right kidney removed. After recovery, animals received for 40 days: calcium gluconate 3% in chow, calcium gluconate 3% in water, control diet (standard chow), or deionized (DI) water and standard chow. Animals were sacrificed on day 40 and the parathyroids removed, weighed and processed for PCNA staining, as described previously by Colloton et al . [ 56 ]. The number of PCNA-positive cells was counted by a treatment-blinded observer. Data shown as mean ± SEM * P = 0.0003 vs DI water control; ** P = 0.0002 vs diet control. Calcium also appears to regulate VDR expression by parathyroid cells independently of calcitriol [ 52 ]. VDR mRNA and protein levels were lower in hypocalcaemic, than in normocalcaemic, rats and this prevented the inhibitory effect of calcitriol on PTH mRNA. Thus, hypocalcaemia may increase PTH mRNA directly via a or by VDR are that mimic or potentiate the effects of extracellular Ca 2+ at the calcimimetics are that directly stimulate the CaR by with its extracellular and include and [ ]. calcimimetics are modulators of the to with the membrane-spanning of the receptor, a that increasing to extracellular Ca 2+ . include and [ ]. The include the and the and calcimimetics increase the of parathyroid cells to extracellular calcium, PTH release and the response to the left in vitro [ , ]. In uraemic calcimimetics in serum PTH and increases in serum levels [ ]. In calcimimetics are at times more in serum PTH levels than in increasing levels [ ], although the for this are not The mechanism and of action of calcimimetics from those of vitamin D which reduce PTH gene transcription and hormone synthesis over a of several or [ ]. PTH secretion within with a decrease within 2 in patients with SHPT [ ]. by a number of results in a pattern of serum PTH [ ] that may have effects on bone [ ]. In data that activation of the CaR by calcimimetics PTH mRNA by of the protein [ ]. The of calcimimetics in PTH levels in dialysis patients [ , ] may their mechanism of action as rather than receptor at the The PTH in patients with primary parathyroid [ ], as well as in of primary [ ], that CaR signalling is in advanced parathyroid hyperplasia and parathyroid despite the marked in CaR expression that these Table 3 the effects of calcimimetics and vitamin D on serum calcium and phosphorus and It be that calcimimetics are in with vitamin D and/or phosphate in dialysis patients and may have or effects on PTH, as well as the and actions of vitamin D of the effects of calcimimetics and vitamin D on serum Ca and P and parathyroid hormone a In patients with b In models ↑, For of the effects of calcimimetics and vitamin D on serum Ca and P and parathyroid hormone a In patients with b In models ↑, For The effects of calcimimetics on parathyroid hyperplasia have been in vitro , in parathyroid cells from uraemic patients and in uraemic rats ( Table 4 in the 5/6 nephrectomized which of of the three of the left renal artery and of the right This represents an stage in parathyroid hyperplasia: it has not been to hyperplasia in uraemic in uraemic models in uraemic models have also been in the of SHPT. in rats chronic renal failure within a of only 4 as of its and in kidney results in and This is by extremely elevated PTH and severe parathyroid hyperplasia, as well as severe bone and calcification of [ ]. [ , ] and [ 56 , ] inhibit parathyroid cell proliferation in the 5/6 as by marked in the of cells [ ], PCNA-positive cells [ 56 , , ] and parathyroid cell [ , ]. Indeed, parathyroid cell proliferation was reduced to control levels by treatment ( Figure 4 ) [ 56 ]. also effects on parathyroid cells in [ 51 ]. of 4 of on parathyroid weight and proliferation in 5/6 nephrectomized Colloton et al . [ 56 ]. Animals were or for 4 6 animals were sacrificed and parathyroid glands for of parathyroid weight and parathyroid proliferation by proliferating cell Data shown as mean ± SEM 5/6 vs control; * P vs control; P vs control. The decrease in parathyroid cell proliferation induced by calcimimetics appears to be to an increase in cells the p21 [ ]. In the 5/6 of more than the number of cells compared to control ( P ( Figure 5 ) [ ]. In contrast to vitamin D, which proliferation of many cell [ 1 , ], calcimimetics not have effects on cells [ 56 , ] or C cells [ ]. of on p21 expression in parathyroid Sprague–Dawley rats 5/6 as described Colloton et al . [ 56 ]. after rats were in by for 6 at 6 or by for 6 by for 3 at After parathyroids were for p21 cell expression, in a mouse p21 monoclonal Data shown as mean ± SEM ( = 13 et al . [ ]. * P vs control. after from the of the uraemic calcimimetics prevented the increase in parathyroid gland weight and/or volume that in animals [ , ]. of hyperplasia was within after of the [ ]. were also active in models of severe SHPT with a diet or the For instance, an prevented development of parathyroid hyperplasia, as by both parathyroid weight and the number of proliferating cells, in rats [ ]. Parathyroid weight increased to after only 4 in the compared to a weight of in the to that seen in rats a normal diet ( Figure 6 ). was not active in this although it did reduce serum effects of a and calcitriol in chronic renal failure with parathyroid hyperplasia. Male Sprague–Dawley rats (300–350 g) were a standard diet and received or received a diet for and received calcitriol calcitriol or 3 the of the treatment animals were sacrificed and the parathyroids and weighed ( = at Data shown as mean ± et al . [ ]. to animals with parathyroid hyperplasia, after calcimimetics progression of hyperplasia [ 56 , ], under conditions of phosphate loading [ ]. Indeed, parathyroid weight in animals with was that in nephrectomized ( Figure 4 ). Based on data from rats and it was that this to a of in the [ 56 ]. this did not of whether of parathyroid hyperplasia et al . [ ] this effect by of animals at 4 or 11 after treatment reduced parathyroid gland volume for to the size seen in the a standard diet was of for parathyroid gland such that mean gland volume was to that in This was found to be to a decrease in volume of the parathyroid of gland also via of parathyroid This be to the extremely of these cells [ 12 ]. Indeed, et al . [ ] were not able to as by in or uraemic or in of parathyroid hyperplasia by calcimimetics was of serum levels, that this effect was not by renal function [ 56 , ]. have also been shown to decreased parathyroid CaR in uraemic rats [ ], with both mRNA and protein being to control levels [ ], and to VDR both in vitro [ ] and in [ , ]. also the effects of calcitriol on VDR mRNA and VDR protein in normal rats [ ]. of parathyroid proliferation the of both CaR and VDR expression [ ]. The key of excessive PTH secretion and synthesis and parathyroid cell proliferation in SHPT are hypocalcaemia, hyperphosphataemia and low serum calcitriol levels. therapies such as vitamin D and phosphate not these Indeed, the and actions of vitamin D to of hypercalcaemia and hyperphosphataemia in Thus, in are and can to disease progression [ ]. Moreover, response to vitamin D is parathyroid hyperplasia has to the advanced nodular form [ ]. The of to reduce parathyroid proliferation is in the high of the dialysis [ , ]. In to the increased risk of vascular calcification by elevated Ca [ , ] animal and in vitro data that calcitriol may vascular by decreasing in vascular [ , ]. Although calcitriol parathyroid cell proliferation in vitro and in [ , ], cultured parathyroid cells [ 51 ] and parathyroid tissue [ ] from patients with SHPT only at high calcitriol and tissue from patients with primary hyperparathyroidism did not respond [ ]. The effects of active vitamin D on parathyroid hyperplasia in uraemic animal models to be on the and as well as the [ , , ]. did not parathyroid hyperplasia at a to that shown to vascular calcification [ , ]. It is whether vitamin D can of parathyroid hyperplasia associated with SHPT, with animal data [ ] and [ , ] been of vitamin D directly the parathyroid gland over can reduce gland size [ ] and parathyroid cell [ , , ], but this may a effect high rather than a pharmacological calcitriol did not in parathyroid cells in models of SHPT [ 50 , ]. The activity of vitamin D in parathyroid hyperplasia may several decreased VDR expression [ 15 , 21 , ], and/or a in [ , ]. to control serum phosphate also to resistance to calcitriol [ , ], as in the [ ]. data that the CaR is a more important of parathyroid hyperplasia than the In a diet with to calcium was able to parathyroid gland hyperplasia [ ]. In in the severe SHPT be prevented only by of the PTH [ ] or [ ] gene. Accumulation of phosphate SHPT directly and via several mechanisms [ , ] ( Table 1 ). of the phosphate was reported to reduce parathyroid cell proliferation [ ] and reduce parathyroid gland hypertrophy [ ] in uraemic this represents a effect via control of or an by increased serum calcium levels, or to be development of parathyroid hyperplasia an extremely of signalling the CaR appears to be the important of the disease of the CaR by calcimimetics in with allows control of PTH in dialysis patients increasing plasma levels of Ca 2+ , and/or vitamin D. Indeed, serum calcium, phosphorus and are reduced [ , ]. Data from uraemic that calcimimetics can inhibit the development and progression of parathyroid hyperplasia, shown activity in models of severe and/or hyperplasia. of parathyroid hyperplasia has also been with these observations, data from a a in the risk for as well as a significantly reduced risk of and cardiovascular in dialysis patients with [ ]. also the role of parathyroid hyperplasia in increasing the capacity for PTH these that calcimimetics may be able to disease progression in SHPT and be if in the of CKD This is being with [ ] and and data are with animal data that calcimimetics can also the decreased expression of CaR and VDR that parathyroid hyperplasia, the response to calcitriol. may also inhibit development of renal and cardiovascular changes associated with SHPT and vascular calcification [ , ]. is to whether calcimimetics are in attenuating parathyroid hyperplasia in This was with the of of of . The in this was by It has not been in with the of the Colloton et al . which has been in and is as has received and from and and a from both and has received and from is by and in the