Generally speaking, there are at least two different mechanisms which determine the magnitude of 2° hyperparathyroidism. The first results in an increase in PTH synthesis and secretion, and the second in an increase in parathyroid gland mass, mostly due to enhanced cell proliferation (schematic representation in Figure 2). Whereas acute stimulation of PTH synthesis and/or release almost generally occurs in the absence of cell growth stimulation, these two processes appear to be tightly linked whenever there is longstanding stimulation. The main factors involved in the control of both of them are again calcitriol, calcium, and phosphate. In the following, the disturbances of the mechanisms controlling parathyroid function will be discussed subsequently for each of these three factors, although there are numerous interactions between them. Thereafter, the influence of other factors and comorbid conditions related to renal failure will be discussed.
Calcitriol. The above mentioned decrease in plasma calcitriol aggravates hyperparathyroidism via several mechanisms. The first is direct and results from an insufficient inhibition of PTH synthesis due to low circulating calcitriol levels and a disturbed action of calcitriol at the level of the preproPTH gene. It is well established that calcitriol, after forming a complex with its receptor, vitamin D receptor (VDR) and heterodimerizing with the retinoic acid receptor (RXR), directly inhibits preproPTH gene transcription by binding to a specific DNA response element (VDRE) located in the 5’-flanking region of the gene. In CKD, in addition to low extracellular concentrations of calcitriol, at least two other factors interfere with calcitriol’s action on the preproPTH gene . The first factor is a reduced expression of the VDR gene product in hyperplastic parathyroid tissue of CKD patients . This reduction is particularly marked in nodular parathyroid tissue, as compared to diffusely hyperplastic tissue. The second factor is reduced binding of calcitriol to VDR, a slowed nuclear migration of the calcitriol–VDR complex and a less efficient action on the preproPTH gene, in association with the uremic state . Of note, extracellular calcium appears to play a role in the regulation of VDR expression. Thus, Garfia et al. showed that low extracellular Ca reduced VDR expression in rat parathyroid glands independently of calcitriol, whereas high extracellular Ca enhanced VDR expression . Hypocalcemia may prevent by this mechanism the feedback of increased plasma calcitriol concentrations on the parathyroids.
The second level at which calcitriol regulates PTH gene expression involves calreticulin. Calreticulin is a calcium binding protein which is present in the endoplasmic reticulum of the cell, and also may have a nuclear function. It regulates gene transcription via its ability to bind a protein motif in the DNA-binding domain of nuclear hormone receptors of sterol hormones. Sela et al. proposed that calreticulin might inhibit vitamin D's action on the PTH gene, based on in vitro and in vivo experiments . They fed rats either a control diet or a low calcium diet, which led to increased PTH mRNA levels despite high serum calcitriol levels that would be expected to inhibit PTH gene transcription. Their postulate that high calreticulin levels in the nuclear fraction might prevent the effect of calcitriol on the PTH gene was strongly supported by the observation that hypocalcemic rats had increased levels of calreticulin protein in their parathyroid nuclear fraction. This could explain why hypocalcemia leads to increased PTH gene expression despite high serum calcitriol levels, and might also be relevant for the refractoriness of 2° hyperparathyroidism to calcitriol treatment observed in many CRF patients.
The third mechanism of calcitriol action could be indirect, via an effect on CaR expression. Brown et al showed in a recent study that calcitriol, but not calcium itself, regulated the expression of CaR mRNA. They found that rats fed a vitamin D-deficient diet had a low expression of CaR mRNA and that it could be upregulated by the administration of a calcitriol bolus. These results are however in contrast to those published by Rogers et al who found no effect of calcitriol on CaR mRNA expression. If calcitriol levels influence the expression of CaR, calcitriol deficiency in CRF may indirectly affect the control of PTH secretion via abnormal extracellular calcium sensing by the parathyroid cell.
The fourth mechanism by which calcitriol acts on parathyroid tissue is again a direct one. It concerns the well-known inhibitory effect of vitamin D on cell proliferation and the induction of differentiation towards mature, slowly growing cells. A decrease in plasma calcitriol and a perturbed action at its molecular targets favors abnormal cell growth. This is the case with parathyroid tissue as well, and secondary parathyroid hyperplasia ensues . The importance of vitamin D in the parathyroid hyperplasia of experimental uremia has first been shown by Szabo et al . These authors administered increasing doses of calcitriol to rats either at the time of inducing chronic renal failure or at a later time point, when uremia was already well established. They were able to prevent parathyroid cell proliferation entirely when calcitriol was given at start of uremia, but not when given later on. In contrast, Taniguchi et al were able to induce regression of both parathyroid hyperplasia and hypertrophy in uremic rats by administering calcitriol only 8 weeks after the creation of chronic renal failure . Fukagawa et al showed that pharmacologic doses of calcitriol repressed c-myc expression in the parathyroid tissue of uremic rats and suggested that the hormone might suppress parathyroid hyperplasia by this pathway.
To answer the question of a possible direct calcitriol action on parathyroid cells, several studies were performed in experimental models in vitro. Nygren et al. showed in primary cultures of bovine parathyroid cells maintained in short-term culture, that these cells underwent significant increases both in number and size in response to fetal calf serum, and that the addition of 10-100 ng/ml calcitriol almost completely inhibited cell proliferation whereas hypertrophy was unaffected. Kremer et al subsequently confirmed in same parathyroid cell model that calcitriol exerted an anti-proliferative action. They further suggested that this inhibition occurred via a reduction of c-myc mRNA expression. There has been only one previous report under long-term culture conditions (up to 5 passages) of the effect of calcitriol on bovine parathyroid cell proliferation, also showing an inhibitory action . We subsequently confirmed this direct antiproliferative effect of calcitriol in a human parathyroid cell culture system derived from hyperplastic parathyroid tissue of patients with severe 2° uremic hyperparathyroidism (Figure 3).
A final mechanism is the potential association between parathyroid function and vitamin D receptor (VDR) polymorphism. Fernandez et al separated hemodialysis patients with same serum calcium and time on hemodialysis treatment into two groups, according to their serum intact PTH levels, namely low PTH (<12 pmol/L) or high PTH (>60 pmol/L). They found that the BB genotype and the B allele were significantly more frequent in the low PTH than in the high PTH group (32.3 % vs 12.5 %, and 58.8% vs 39.1%, respectively). This information suggests that VDR gene polymorphism influences parathyroid function in chronic renal failure. Similar results have been reported by two different Italian groups and in a large sample of Japanese hemodialysis patients . In this latter study, after excluding diabetic patients and patients with less than ten years on dialysis treatment, the authors observed lower plasma intact PTH levels in patients with BB than with Bb or bb alleles. A relationship between Apa I polymorphism (A/a alleles) and the severity of hyperparathyroidism has also been sought in Japanese hemodialysis patients . Plasma PTH levels in AA and Aa groups were approximately half that of the aa group. However, other groups found no difference in PTH levels for various VDR polymorphisms . Moreover, although in some clinical conditions VDR polymorphism may be associated with variations of the half life of the VDR gene transcript or of VDR function , there has been no report so far showing that in uremic patients with 2° hyperparathyroidism the density of parathyroid cell VDR varies with different VDR genotypes. In addition, although VDR genotypes may have some influence on the degree of 2° parathyroid hyperplasia, the mechanism by which this may occur remains unknown at present.
Calcium. It has long been known that extracellular Ca2+ is the major regulator of PTH secretion. Small changes in Ca2+ concentration result in immediate changes of PTH release which are short-lived or long-lived, depending on the velocity of the restoration of Ca2+ towards its normal level. Hypercalcemia inhibits the secretion of human PTH (1–84) [(1–84)hPTH] from the parathyroid gland to a greater extent than secretion of C-PTH fragments, resulting in a high C-PTH/(1–84)hPTH ratio in the circulation. PTH degradation occurs within the parathyroid glands before secretion, although peripheral metabolism is also observed after secretion. N-terminal truncation to generate several different large carboxyl-terminal fragments or non-(1–84)hPTH has also been reported to occur in both normal and uremic parathyroid tissue }. In the circulation, there is an inverse relation between Ca2+ and PTH . It obeys a sigmoidal curve. While the majority of in vitro studies have reported a decreased responsiveness of hyperplastic parathyroid cells to calcium, in vivo studies have not always led to the same conclusion. This is likely due to different methods used to assess the dynamics of PTH secretion.
Several in vitro studies have shown that the set point of calcium for PTH secretion (that is the calcium concentration required to produce half maximal PTH secretion) is greater in parathyroid cells from 1° (adenomas) and 2° (uremic) hyperplastic parathyroid glands than in normal parathyroid cells . Such a relatively poor response to calcium should contribute to the increased PTH levels observed in uremic patients with 2° hyperparathyroidism.
We and others have demonstrated that both 1° parathyroid adenoma and 2° uremic, hyperplastic parathyroid gland tissue exhibit a decrease in the expression of CaR protein . In 2° uremic hyperparathyroidism, there is a significant CaR decrease in diffusely growing hyperplastic tissue, although the decrease is even more marked in nodular areas (characteristic of advanced hyperplasia) (Figure 4). Since changes in intracellular calcium elicited by hyper or hypocalcemia depend on the CaR, its decreased expression explains, at least in part, the impaired intracellular calcium response to extracellular calcium and hence a reduced inhibitory effect of calcium on PTH secretion.
Almaden et al studied the calcium-regulated PTH response in vitro, using respectively primary parathyroid adenoma and uremic hyperplastic tissue, the latter both of the nodular and the diffuse type . They found that in parathyroid adenomas PTH secretion was less responsive to an increase in extracellular calcium than in 2° hyperplasia; among the latter, nodular tissue was less responsive than diffusely hyperplastic tissue. The decreased secretory response to calcium observed in nodular hyperplasia could be explained by the markedly reduced CaR expression in CKD, as demonstrated by Gogusev et al . Cañadillas et al subsequently showed that this was indeed the case. The lower the CaR expression in uremic parathyroid tissue, the weaker was the secretory response to low medium calcium concentration . The decreased response can be overcome, at least partially, by PTHrp, as recently shown by Lewin et al. These authors observed that the administration of PTHrp significantly stimulated the impaired secretory capacity of the parathyroid glands of uremic rats in response to hypocalcemia . Of note, this observation also implies that the PTH/PTHrp receptor is expressed on the parathyroid cell.
The shift of the calcium set point to the right in dialysis patients in vivo has been a much less constant finding than the right shift observed in the above mentioned studies in uremic parathyroid tissue in vitro. While in CKD patients with a mild to moderate degree of hyperparathyroidism the set point was most often found to be normal, an altered set point was observed in presence of severe parathyroid overfunction with hypercalcemia . This anomaly could at least in part be due to CaR down-regulation . In CKD patients with less severe parathyroid overfunction, there was actually a considerable controversy regarding the results of in vivo assessments of parathyroid gland function . In part, disparities among reports reflected technical differences in experimental methods and/or variations in the mathematical modeling of PTH secretion in vivo . Another difficulty in interpreting the results of in vivo dynamic tests of parathyroid gland function relates to the issue of parathyroid gland size. Because there is a basal, or non-suppressible, component of PTH release from the parathyroid cell even at high extracellular calcium concentrations, excess PTH secretion may result solely from increases in parathyroid gland mass . This can occur in the absence of a defect in calcium-sensing at the level of the parathyroid cell. Since parathyroid gland hyperplasia is present to some extent in nearly all patients with chronic renal failure, alterations in PTH secretion due to increases in parathyroid gland mass cannot readily be distinguished from those attributable to changes in calcium-sensing by the parathyroid cell using the four parameter model for in vivo studies.
The role of calcium in parathyroid cell proliferation is less clear than is generally assumed. Calcium deficiency, in the presence or absence of hypocalcemia, together with vitamin D deficiency or reduced generation of calcitriol, probably is a major stimulus of parathyroid hyperplasia. Thus Naveh-Many et al showed that calcium deprivation, together with vitamin D deficiency, greatly enhanced the rate of parathyroid cell proliferation in normal rats and also in rats with chronic renal failure, using the cell cycle-linked antigen, PCNA . The concomitant decrease in CaR expression in CRF, as observed in parathyroid glands of both dialysis patients and uremic rats , should theoretically enhance parathyroid tissue hyperplasia further. Indirect support for this contention came from the observation that the administration of the calcimimetic compound NPS R-568, a calcium-sensing receptor agonist, led to the suppression of parathyroid cell proliferation in rats with renal insufficiency . However, in the study by Naveh-Many et al the dietary regimen was poor in both calcium and vitamin D. In contrast, when feeding normal rats on a calcium-deficient diet alone, in the absence of concomitant vitamin D deficiency, Wernerson et al observed parathyroid cell hypertrophy, not hyperplasia.
The question whether the effect of calcium is direct or indirect remains therefore unsolved at present. It can only be answered by in vitro studies. Unfortunately, until recently none of the available culture systems using normal parathyroid cells allowed the maintainance of functionally active cells for prolonged time periods. They were all characterized by a rapid and significant loss of PTH secretion, within 3 to 4 days . One culture model has been described, using bovine parathyroid cell organoids, which maintained the ability to modulate PTH secretion in response to extracellular Ca2+ [Ca2+e] and tissue-like morphology for 2 weeks in culture . However, only one long-term study of bovine parathyroid cells demonstrated a release of bioactive bovine PTH but with reduced sensitivity to calcium . Other reports showed that the rapid decrease in PTH responsiveness of cultured bovine parathyroid cells to changes in [Ca2+e] was associated with a marked reduction in CaR expression . Yet other parathyroid cell-derived culture models proposed in the literature were in fact devoid of any PTH secretory capacity.
To study direct effects of [Ca2+e] on the parathyroid cell in vitro, we developed a functional human parathyroid cell culture system capable of maintaining regulation of its secretory activity and the expression of extracellular CaR mRNA and protein for several weeks. For this purpose, we used parathyroid cells derived from hyperplastic parathyroid tissue of dialysis patients with severe 2° hyperparathyroidism . In a subsequent study, we obtained evidence with this experimental model that parathyroid cell proliferation index, as estimated by [3H]-thymidine incorporation into an acid-precipitable fraction as a measure of DNA synthesis, could be directly stimulated by high [Ca2+e] in the incubation medium, compared with low [Ca2+e] (Figure 5). We confirmed this finding in independent experiments using the cell cycle-linked antigen Ki-67 to determine parathyroid cell proliferation. However, the addition of the calcimimetic NPS R-467 to the incubation medium led to a decrease in proliferation (Figure 6). Of interest, calcimimetics have been subsequently shown to upregulate the expression of the CaR in parathyroid glands of uremic rats . In an attempt to unify our apparently contradictory in-vitro observations with respect to findings made in vivo, we propose the following hypothesis. The effect of calcium on parathyroid cell proliferation could occur along two different pathways, via two distinct mechanisms. Inhibition of proliferation would occur via the well-known parathyroid CaR-dependent pathway, whereas stimulation of proliferation would occur via a second pathway (Figure 7). It must be noted that all parathyroid tissue samples used in our study stemmed from uremic patients with long-term renal failure and severe secondary hyperparathyroidism. Since such parathyroid tissues generally exhibit decreased CaR expression, it is possible that the number of CaR expressed in the parathyroid cell membranes of our culture model was insufficient to inhibit cell proliferation. A recent report showed that the human CaR gene has two promoters and two 5’ untranslated exons and that the alternative usage of these exons leads to production of multiple CaR mRNAs in parathyroid . The expression of CaR mRNA produced by one of the two promoters of CaR gene is specifically reduced in PT adenomas, suggesting a role in PTH hypersecretion and proliferation. Moreover, the membrane-bound 550-kD Ca2+-binding glycoprotein megalin, belonging to the low-density lipoprotein receptor superfamily, has been identified in parathyroid chief cells as another putative calcium-sensing molecule which could be involved in calcium-regulated cellular signalling processes as well . Based on these observations, one can postulate that parathyroid cells express multiple CaR-like molecules. Consequently, if the well-known parathyroid CaR is down-regulated, parathyroid cell proliferation by calcium may occur via a different CaR isoform. Another possibility is an alteration in post-receptor signal transduction that could occur in hyperparathyroid states or under cell culture conditions. Our observations are in line with findings by Ishimi et al. which were incompatible with a direct effect of low [Ca2+e] in the pathogenesis of parathyroid hyperplasia . However, the extrapolation from such in vitro observations to the in vivo setting should be done with caution, and further work is needed to define the precise pathway(s) by which calcium regulates parathyroid tissue growth.
Phosphate. Hyperphosphatemia is associated with increased PTH secretion. This effect may occur within minutes after an increase of phosphate in the intestinal lumen . The stimulation occurs via both direct and indirect mechanisms. The initially proposed indirect mechanism, which remains true according to present knowledge, is via a decrease in plasma Ca2+ concentration (see above). Hyperphosphatemia also leads, at least theoretically, to an inhibition of the renal synthesis of calcitriol. It must be noted, however, that in general calcitriol production is markedly reduced due to the loss of nephron mass. This leaves little room for an additional inhibitory role of plasma phosphate in the uremic state.
A direct action of phosphate on PTH secretion by the parathyroid cell has long been suspected. However, it has been formally demonstrated in vitro only in 1996 . This demonstration required the use of either intact parathyroid glands (from rats) (Figure 8) or parathyroid tissue slices (from cows) whereas it had not been possible to obtain such a direct stimulation using the classic model of isolated bovine parathyroid cells. The elevation of plasma phosphate concentration in the incubation milieu of the experimental models using intact (or partially intact) parathyroid tissue leads to a stimulation of PTH secretion within some hours, in the absence of any change in extracellular ionized calcium concentration. It can however be abrogated by an increase in cytosolic calcium.
Silver’s group reported subsequently that phosphate, like calcium, regulates pre-pro-PTH gene expression post-transcriptionally by changes in protein-PTH mRNA interactions at the 3'-UTR which determine PTH mRNA stability. They identified the minimal sequence for protein binding in the PTH mRNA 3'-UTR and determined its functionality. They found that the conserved PTH RNA protein-binding region conferred responsiveness to calcium and phosphate and determined PTH mRNA stability and levels . Thus a low calcium diet increased stability, whereas a low phosphate diet decreased stability of PTH mRNA (Figure 9). The PTH mRNA 3’-untranslated region-binding protein was subsequently identified by this research group as AUF1.
In addition to its stimulatory effect on PTH secretion a high phosphate diet also rapidly induces parathyroid hyperplasia. It has long been shown in experimental animal models that a phosphate-rich diet induced an increase in parathyroid gland function and volume . Subsequently, studies showed that phosphate-rich diets, when fed to animals with chronic renal failure leading to high plasma phosphate levels, induced parathyroid hyperplasia even when changes in plasma calcium and calcitriol concentration were carefully avoided, pointing to a direct effect of phosphate on cell proliferation . Conversely, early dietary phosphate restriction was capable of preventing both PTH oversecretion and parathyroid hyperplasia . Interestingly, dietary phosphate restriction following phosphate overload in rats also led to an immediate decrease in PTH secretion, in the absence of a regression of parathyroid gland size.
Our group wished to know whether the stimulatory effect of phosphate on parathyroid cell proliferation was direct or indirect. To answer this question, we used the above described in vitro model of human parathyroid cell maintained in long-term culture . We could show that the cell proliferation index was directly stimulated by high phosphate concentrations in the incubation medium, compared with low phosphate concentration (Figure 10). These experiments demonstrate that phosphate is capable to stimulate not only PTH secretion, but also to induce parathyroid tissue hyperplasia by a direct mode of action.
Since FGF-23 plays an important role in the control of plasma phosphorus, its plasma concentration is elevated in chronic renal failure and the increase is associated with plasma PTH as well, it is probable that in turn FGF-23 influences parathyroid function, either indirectly or directly.
Other factors and conditions. As already pointed out above the uremic state is another long suspected, albeit still ill defined factor in the pathogenesis of 2° hyperparathyroidism. Recently, several pieces of evidence have been provided in favor of a role of uremic toxins which interfere with the binding of calcitriol to VDR and with the nuclear uptake of the hormone-receptor complex . This should have consequences not only for PTH synthesis and secretion, but also for parathyroid growth.
Diabetic patients on dialysis have relatively low plasma PTH levels, compared with non diabetic dialysis patients. The high incidence of low bone turnover in uremic diabetic patients has been attributed to low PTH levels, possibly via an inhibition of PTH secretion or a modification of the PTH peptide by the accumulation of advanced glycation end-products such as pentosidine . However, experimental studies have demonstrated that the metabolic abnormalities associated with diabetes can also directly decrease bone turnover, independent of PTH . In general, patients with low bone turnover tend to develop hypercalcemia when on a normal or high calcium intake, probably due to a diminished skeletal capacity to take up calcium. This in turn tends to reduce plasma PTH. Thus low bone turnover favors the occurrence of hypoparathyroidism. Another question is whether in diabetic patients abnormalities such as hyperglycemia and insulin deficiency or resistance may directly affect parathyroid function. In an in vitro study using dispersed bovine parathyroid cells, high glucose and low insulin concentrations suppressed the PTH response to low calcium . These results are compatible with the view that diabetes directly inhibits parathyroid function. However, in experiments in renal failure rats fed a high phosphorus diet to induce 2° hyperparathyroidism, diabetes did not affect the development of parathyroid overfunction.
Aluminum bone disease is generally associated with low serum PTH levels and a decreased PTH response to stimulation with hypocalcemia . In such patients, high amounts of aluminum are also found in parathyroid glands . The relatively low PTH levels may reflect either an inhibition by the hypercalcaemia commonly observed in this condition or a direct inhibitory effect of aluminum on parathyroid cell function . Direct toxic effects of the tace element have also been demonstrated in studies in vitro.
Data obtained in experimental animals and results of clinical studies have been more controversial. Whereas some experiments indicated that aluminium did not decrease plasma PTH levels in vivo , other experiments were affirmative . Whatever the mechanisms involved, clinical data clearly showed that the introduction of an aluminum-free dialysis fluid and the discontinuation of aluminum contamination of the dialysate or aluminum removal with deferoxamine resulted in an increase in plasma PTH levels and in PTH response to hypocalcemia . Thus, although there appears to be an association between aluminum toxicity and parathyroid gland function, the interaction is complex.
Figure 2 provides a schematic view of the main mechanisms involved in the abnormal synthesis and secretion of PTH and in the hyperplasia of parathyroid tissue.