Influence of parathyroid mass on the regulation of PTH secretion

http://www.kidney-international.org & 26 International Society of Nephrology Influence of parathyroid mass on the regulation of PTH secretion E Lewin 1,2 and K Olgaard 2 1 Nephrological Department B, The Copenhagen County Hospital in Herlev, Denmark and 2 Nephrological Department P, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark In advanced uremia, parathyroid hormone (PTH) levels should be controlled at a moderately elevated level in order to promote normal bone turnover. As such, a certain degree of parathyroid gland (PG) hyperplasia has to be accepted. No convincing evidence of apoptosis or of involution of PG hyperplasia exists. However, even considerable parathyroid hyperplasia can be controlled when the functional demand for increased PTH levels is abolished. When 2 isogenic PG were implanted into one parathyroidectomized (PTX) rat normalization of Ca 2 þ and PTH levels and normal suppressibility of PTH secretion by high Ca 2þ was obtained. Similarly, normal levels of Ca 2 þ and PTH and suppressibility of PTH secretion were obtained when Eight isogenic PG from uremic rats were implanted into normal rats or when long-term uremia and severe secondary hyperparathyroidism (sec. HPT) was reversed by an isogenic kidney transplantation. Normalization of PTH levels after experimental kidney transplantation took place despite a persistent decrease of vitamin D receptor (VDR) mrna and calcium sensing receptor (CaR) mrna in PG. Thus, in experimental models PTH levels are determined by the functional demand and not by parathyroid mass, per se. When non-suppressible sec. HPT is present in patients referred to PTX, nodular hyperplasia with differences in gene expression between different nodules has been observed in most cases. An altered expression of some autocrine/ paracrine factors has been demonstrated in the nodules. Enhanced expression of PTH-related peptide (PTHrP) has been demonstrated in PG from patients with severe secondary HPT. PTHrP has been shown to stimulate PTH secretion in vivo and in vitro. PTH/PTHrP receptor was demonstrated in the parathyroids. The low Ca 2þ stimulated PTH secretion was enhanced by 3% by PTHrP 1 4. The altered quality of the parathyroid mass and not only the increased parathyroid mass, per se, might be responsible for non-controllable hyperparathyroidism in uremia and after kidney transplantation.. doi:1.138/sj.ki.51597 KEYWORDS: PTH; secondary hyperparathyroidism; uremia; experimental hyperparathyroidism Correspondence: K Olgaard, Nephrological Department P 2132, University of Copenhagen, Rigshospitalet, 9 Blegdamsvej, Copenhagen DK-21, Denmark. E-mail: olgaard@rh.dk HYPERPLASIA AND THE POSSIBILITY OF REGRESSION OF HYPERPLASIA OF THE PARATHYROID GLANDS Parathyroid tissue is a discontinuous replicator tissue, which is characterized by a low cell turnover, a low rate of mitosis, and no separate stem cells. 1,2 As estimated by Parfitt the mean life span of normal parathyroid cells is 2 years in humans and 2 years in rats. 2,3 Mitosis can be stimulated by functional demand. 4 In human subjects parathyroid growth progresses in response to chronic renal failure through several stages from diffuse hyperplasia to nodular hyperplasia and to formation of adenomas. Hyperplasia is initiated by hypocalcemia and phosphorus (P) retention and becomes more severe as the result of calcitriol deficiency. 5 Similarity exists in the gene expression between the cells in each nodule, while differences in gene expressions may be found between nodules. 6,7 Monoclonal growth of the parathyroid cells has been found in a majority of the uremic patients with refractory hyperparathyroidism. 8 The genes responsible for monoclonality have not been identified. Apparently, somatic mutations confer a growth advantage to clones of parathyroid cells, that are causing monoclonal growth and nodular parathyroid hyperplasia, although these two phenomena are not strictly linked. It seems that at the initial stages, development of parathyroid hyperplasia is a regulatory phenomenon, but that during the progression it escapes from normal growth control. The main factors responsible for parathyroid hyperplasia appear to be the same as those responsible for the enhanced parathyroid hormone (PTH) biosynthesis and secretion. The precise molecular mechanisms involved in parathyroid hyperplasia are sparsely clarified. The potential role of transforming growth factor-a, known to promote cell growth, in the parathyroid hyperplasia has been addressed. 9 12 Enhanced expression of transforming growth factor-a and its receptor, the epidermal growth factor receptor, occurs early after the onset of uremia in rats and is aggravated by high P or low calcium diets. 12 Furthermore, there is enhanced transforming growth factor-a expression in human hyperplastic parathyroid tissue. 13 Conversely, high dietary calcium, P restriction, and vitamin D therapy of uremic rats prevent the increase in transforming growth factor-a and induce expression of the cyclin-dependent S16

kinase inhibitor, p21. 9,1 Finally, female gender may favor parathyroid cell proliferation. 14 An important question is whether regression of parathyroid hyperplasia can be induced. Can the increased glandular mass seen in uremia be reduced? Such a reduction would call for massive apoptosis to take place in the parathyroids. This question has not yet been resolved. Examination for apoptosis in the parathyroids is not an easy task. 1 The parathyroids have an extremely low cell turnover, and, probably, a poorly developed program for cell deletion. The number of apoptotic cells in normal human parathyroids is very low, 1/1, parathyroid cells. 15 In rat models, several research groups have been unable to find evidence for programmed parathyroid cell death in normal or in hyperplastic parathyroid tissue. 3,16,17 Experimental studies documented that parathyroid hyperplasia is easily prevented, but poorly reversible. 18 2 If reversal by apoptosis takes place at all, it is probably an extremely slow process. 3 There are no known stimuli for apoptosis in the parathyroid cell, 1 although an effect of vitamin D is a possibility has not been completely excluded as such a stimulus. 21 23 CLINICAL VERSUS EXPERIMENTAL STUDIES OF PARATHYROID GROWTH In human studies, in general as well as in daily clinical practice evaluating parathyroid growth and glandular hyperplasia is not possible nor is it possible to obtain a biopsy of the parathyroid glands. Histological, immunohistochemical and molecular examination of parathyroid tissues from uremic patients is only available in the relatively rare situations, in which parathyroidectomy is performed. These are usually cases where hyperparathyroidism, resistant to treatment, is present in patients after years on dialysis treatment. Attempts to estimate the glandular size and nodularity, using ultrasonography, scintigraphy, and positron emission tomography scans, are also usually limited to this minority of the uremic population, in which severly increased glandular mass is present. Thus, the different stages in the development of parathyroid growth from normal glands to severe hyperplasia are not available for direct studies. We, therefore, depend upon knowledge obtained from experimental studies. In experimental models of uremia parathyroid glands are easily available and manipulation of the different factors of relevance for parathyroid growth is possible, either by dietary or pharmacological means. However, the degree of experimental uremia is usually relatively mild since more severe uremia is relatively short lasting as the rats will not survive. Nodular parathyroid hyperplasia has never been induced in animal uremic models. As such, parathyroid glands in experimental uremic animals represent diffuse chief cell hyperplasia with the exception of in a single study, where a heterogenous pattern of proliferation and expression of CaR was observed in female uremic rats kept on a high P diet. 24 This means that there is a huge gap between our ability to evaluate parathyroid hyperplasia in experimental studies and the human clinical situation. We might assume that the experimental models represent an early phase in the development of parathyroid hyperplasia in uremia, while parathyroid tissue from parathyroidectomized (PTX) patients represents the very late stage of parathyroid hyperplasia. Accepting this assumption, experimental data might provide us with important information. Parathyroid hyperplasia can be prevented by phosphate restriction, early initiation of treatment with active vitamin D analogs, or calcimimetics. 11,12,18,2,25 Established parathyroid hyperplasia can be arrested by similar initiatives, 11,12,18,2,25 and it can also be controlled by reversal of uremia. 26,27 Clinical experience has largely confirmed this assumption. Today, secondary hyperparathyroidism (sec. HPT) can be controlled in patients with long-term uremia in whom considerable parathyroid hyperplasia is to be expected. Several clinical studies have documented that PTH levels can be suppressed in most uremic patients and this suppression can be maintained by continuous treatment with phosphate binders, vitamin D analogs or calcimimetics. 28 38 PTH levels return, however, to pre-treatment values when the treatment is stopped. 28,29,33 This rebound of PTH levels suggest that even with good control of parathyroid function under maintainance treatment, no involution of hyperplastic glands takes place. Nevertheless, even severe HPT can be controlled in patients that previously needed PTX. 38 However, some patients remain unresponsive to treatment. 23,39,4 The long-term response to calcitriol has been reported to depend upon the size of the largest parathyroid gland. 23 This, however, does not prove that the amount of parathyroid mass by itself is decisive for the response to treatment. The quality of the increased mass might still be of significant importance for whether it is controllable by calcitriol treatment or not. Following kidney transplantation, plasma PTH will fall in most patients with sec. HPT despite previous long-term uremia. Part of this fall might be due to clearance of C-terminal PTH fragments as a result of the improvement in glomerular filtration rate (GFR), as most assays which have been used until recently co-measure some long C-terminal PTH fragments. 41 43 In most cases plasma PTH returns to near normal over time, although not all studies are confirmative. 44 The normalization of GFR seems, however, to be decisive for the normalization of the PTH levels. 45 48 In transplanted patients with reduced GFR, elevated PTH levels are found consistent with the degree of uremia and independent of the transplanted condition. Only a minority of transplanted patients requires The risk of developing post-transplant HPT increases with the duration of dialysis 49,52,53 and with the severity of the pre-transplant HPT. 46,52,54 As such, the degree of parathyroid hyperplasia is believed to determine the ability of the parathyroid glands to involute after transplantation. 55 One would expect patients requiring parathyroidectomy to PTX. 45,49 51 S17

have the most severe changes of the parathyroids, such as monoclonal nodular hyperplasia and formation of adenomas. Again in this situation the quality of the parathyroid mass and not only increased parathyroid mass per se might be responsible for persistent hyperparathyroidism after kidney transplantation. 56 Histological analysis of surgically removed parathyroids revealed that 9% of larger glands weighing more than.5 g were characterized by nodular hyperplasia. 57 This type of hyperplasia was shown to differ in gene expressions between nodules. 6 For example, in the same patient some nodules expressed only PTH, some both PTH and PTH-related peptide (PTHrP), while some other nodules only expressed PTHrP. 7 PTHrP has been shown by our group to be a peptide that dramatically enhances PTH secretion in response to hypocalcemia in normal as well as in uremic rats, 58,59 (Figure 1). As such, altered expression and regulation of paracrine/autocrine factors in different nodules might be responsible for the finding that the PTH secretion from the increased parathyroid mass cannot be controlled. Another important aspect in evaluating parathyroid hyperplasia in animal models and in human parathyroid glands is the different cellular composition of the glands. Normal human glands contain different cell types, chief cells, and oxyphilic cells, while rat glands consist only of chief cells. 6 Traditionally, parathyroid oxyphilic cells have been regarded as having no specific function, at least no known function. However, oxyphilic adenomas have been shown to be functional and the cells contained PTH and/or PTHrP. 61 64 In normal human glands oxyphilic cells have a sparse diffuse distribution, while multiple nodules consisting of oxyphilic cells have been found in parathyroid hyperplasia. These nodules expressed enhanced cyclooxygenase-2 reactivity, besides expressing PTHrP. 6,64 As such, nodular hyperplasia in human parathyroid glands might have additional regulatory mechanisms, as compared to rodent glands. CONTROL OF INCREASED PARATHYROID MASS An important observation from several experimental studies on sec. HPT is, that the increased proliferation of parathyroid cells, which is induced by uremia, can be arrested. Administration of calcium-sensing receptor agonists (calcimimetics), calcitriol, or low P diet have led to suppression of parathyroid cell proliferation in uremic rats. 11,12,18,2,25 This creates a possibility for controlling the degree or severity of the increase in parathyroid mass in uremia. Furthermore, it has been shown that PTH secretion from increased parathyroid mass could be controlled, according to the functional demand. In two models of parathyroid hyperplasia, 2 normal, isogenic parathyroid glands or eight isogenic parathyroid glands from uremic rats were implanted into one normal recipient rat after removal of its own two parathyroid glands 27,65 (the rat only has two glands) (Figure 2). A transient period of hypercalcemia occurred initially after the increase of the parathyroid mass. Within 2 weeks, however, plasma calcium returned to normal levels and remained normal for the following 6 weeks of observation. PTH levels became normal from the 3rd day after implantation of the 2 parathyroid glands and remained normal for the following 6 weeks. 27 The important observations from these two models of parathyroid hyperplasia are that both calcium and PTH levels eventually became normal and that this normalization took place whether the increased parathyroid mass consisted of normal or uremic parathyroid glands. 27 Subsequently, parathyroid function was examined and normal suppressibility of PTH secretion by calcium was demonstrated in the rats with 2 parathyroid glands implanted (1 times increased parathyroid mass). 27 In another model we showed that the functional demand determines PTH levels in uremic animals whether they have two or eight parathyroid glands present. Thus, PTH levels of 5/6 nephrectomized rats, uremic for 8 weeks and kept on a high P diet, were severely increased. Then the rat s two parathyroid glands were removed and eight isogenic parathyroid glands were implanted into each rat. After implantation of eight glands, and during the same uremic condition, the rat developed exactly the same level of sec. HPT with PTH levels at around 8 pg/ml. Thus, persisting functional demand reintroduced the same degree of hyperparathyroidism in rats with eight glands as in rats with two glands. 27 In another set of experiments two uremic hyperplastic glands were implanted into a normal PTX rat. One week later these rats had normal PTH levels, similar to that of normal control rats. In the following 4 weeks the rats were challenged with a diet of high P and low calcium. This change in the functional demand resulted in completely the same increase in PTH secretion from uremic hyperplastic glands, as seen from normal glands of control rats. 27 a PTH, pg/ml 8 6 4 2 PTHrP 1-4 PTHrP 1-86 Control 1 2 3 4 5 6 Minutes b Ca 2+ mmol/l 1.5 1.3 1.1.9.7.5 1 2 3 4 5 6 Minutes Figure 1 Expression of autocrine/paracrine factors might modulate parathyroid function. (a b) Enhanced expression of PTHrP has been demonstrated in glands from patients with severe sec. HPT due to uremia. PTH/PTHrP receptor was demonstrated in the parathyroids. This figure shows the effect of N-terminal PTHrP on the secretory response of PTH to an acute induction of hypocalcemia in the rat. The rate of reduction of plasma-ca 2 þ by an ethyleneglycol-bis(b-aminoethyl ether) N,N,N,N -tetraacetic acid (EGTA) infusion was similar in all groups of rats. A bolus of PTHrP 1 4, PTHrP 1 86, or vehicle was injected at time. PTHrP significantly enhanced by 3% (Po.1) the low-ca 2 þ -stimulated PTH secretion in vivo. N ¼ 6 in each group, mean7s.e.m. Modified from Lewin et al. 58 S18

In another model, 27 severe sec. HPT, due to long-term uremia and associated with hypocalcemia and hyperphosphatemia, was rapidly reversed after reversal of uremia by an experimental, isogenic kidney transplantation (Figure 3). The circulating levels of PTH became normal within 1 week after normalization of GFR, plasma calcium, and plasma P levels. The precise mechanism behind this rapid reversal of sec. HPT after normalization of GFR is not completely clarified. CaR and VDR mrna were reduced in parathyroid glands obtained from uremic rats with severe sec. HPT. The dramatic decrease of PTH secretion following reversal of uremia by kidney transplantation, occurred however with unchanged and significantly diminished expression of the CaR and VDR genes in the parathyroid glands. 26 Circulating levels of PTH increased 2-fold in uremic rats given a high P diet, while the CaR mrna levels decreased by approximately 6%. Four to 8 days after kidney transplantation PTH levels became normal, despite a persistence of very low expression of CaR mrna, similar to that of uremic rats. Thus, to our surprise, the rapid normalization of circulating PTH levels after transplantation was not associated with normalization of the parathyroid CaR gene expression. This might indicate the existence of a secretory mechanism in parathyroid cells that is not coupled to CaR and which responds to reversal of uremia or to the simultaneous normalization of plasma Ca 2 þ and P levels. Similarly, results of Tagahashi from Dr Slatopolsky s group pointed towards a potent mechanism controlling exocytosis of PTH from hyperplastic parathyroid glands. They studied the PTH levels in uremic rats after 2 weeks of high dietary P and then examined the conditions after 1 further week on a low P intake. The glandular mass was significantly increased and PTH levels were significantly elevated when the rats were on a high P diet. After switching to a low P diet, parathyroid mass was still increased, but despite that the PTH levels were significantly reduced. Then, they looked upon the cytosolic Plasma Ca 2+, mmol/l 1.6 1.5 1.4 1.3 1.2 1.1 1. * * * * *.9 1 2 3 4 5 6 7 14 21 28 35 42 Days after implantation of 2 parathyroid glands Figure 2 Even considerable parathyroid hyperplasia can be controlled in the normal organism. When 2 isogenic parathyroid glands were implanted into one PTX rat a short-lasting hypercalcemia was induced, soon followed, however, by normalization of Ca 2 þ and PTH levels. This figure shows plasma Ca 2 þ and plasma PTH levels in rats with 2 isogenic parathyroid glands implanted. Parathyroidectomy of their own parathyroid glands was performed on day, before the implantation of the 2 glands. The line ( ) depicts plasma Ca 2 þ levels, while the bars depict PTH levels. N ¼ 6, mean7s.e.m. *Po.5 versus the level at day, before PTX. Modified from Lewin et al. 27 1 8 6 4 2 Plasma PTH, pg/ml PTH content and found that PTH content in these huge parathyroid glands was still elevated. As such normalization of PTH secretion in response to low P occured, despite persistently increased glandular mass, and despite increased amount of PTH in the secretory granules. This observation indicates, that there is a potent secretory mechanism controlling increased glandular mass. 66 Plasma urea, mmol/l 3 25 2 15 1 5 2 14 12345 15 TX Weeks of uremia and after transplantation (TX) 1 Figure 3 Severe parathyroid hyperplasia can be controlled when the functional demand for increased PTH levels is abolished by normalization of the kidney function. This figure shows kidney function and basal PTH levels before introduction of uremia by 5/6 nephrectomy ( 2 weeks), during uremia ( 2 to weeks), and after reversal of uremia by an isogenic kidney transplantation (TX). The lines ( ) depict kidney function expressed as plasma urea and the bars depict PTH levels. The uremic rats were kept on a high P diet. N ¼ 12, mean7s.e.m. Modified from Lewin et al. 27 PTH, pg/ml 125 1 75 5 25 Uremia-P TX Controls 75 5 25.75 1. 1.25 1.5 1.75 2. Ca 2+, mmol/l Figure 4 Suppressibility of PTH secretion by Ca 2 þ is not influenced by the parathyroid mass, itself. Parathyroid function was investigated by the Ca 2 þ /PTH relationship during acute inductions of hypocalcemia and hypercalcemia in uremic rats kept on high P diet (Uremia-P), kidney transplanted, 3 weeks after transplantation (TX), and in normal control rats (controls). The PTH secretory response to hypocalcemia was significantly higher in uremic rats (Po.1) than in kidney transplanted and normal control rats. The PTH secretion was suppressed to same extend in kidney transplanted rats and normal control rats, while uremic hyperphosphatemic rats had a significantly higher (Po.5) PTH secretion at high Ca 2 þ. N ¼ 6, mean7s.e.m. Modified from Lewin et al. 27 Plasma PTH, pg/ml S19

CaR mrna/ actin mrna % of control 3 * 2 1 * Control CRF hp TX 1 w In uremic hyperphosphatemic rats high plasma calcium did not suppress PTH secretion to the same extent as seen in normal and uremic, normophosphatemic rats. Three weeks after an experimental kidney transplantation normal suppressibility of PTH secretion by calcium was, however, restored 27 (Figure 4). When the parathyroid CaR gene expression was examined at 4 weeks after experimental kidney transplantation, the expression was upregulated (Figure 5), indicating that CaR expression is regulated according to the functional demand for PTH secretion, although this regulation is rather slow. These results clearly demonstrate that even considerable parathyroid hyperplasia can be controlled when the functional demand for increased PTH levels is abbolished by normalization of GFR, plasma calcium, and plasma P levels. CONCLUSION PTH secretion can be controlled in normal rats despite considerably increased parathyroid mass, consisting of normal glands or uremic glands. In uremia, increased PTH secretion is regulated by increased functional demand for PTH due to hypocalcemia, P retention, decreased 1ahydroxylase activity, skeletal resistance, and other factors. PTH secretion depends upon and responds to changes in functional demand, independently of the parathyroid mass. In patients with long-term severe uremia extreme heterogenicity of gene and protein expression develops in nodular parathyroid hyperplasia. PTH secretion might be affected by autocrine/paracrine factors, for example PTHrP. The expression of such factors in the different noduli might be of importance for the increased PTH secretion in nodular parathyroid hyperplasia. The parathyroid mass is to a large extent of less importance for PTH secretion than the quality of parathyroid mass. The content of the different noduli in severe parathyroid hyperplasia is determining the PTH secretion. * TX 4 w Figure 5 Upregulation of the parathyroid CaR gene expression after reversal of uremia by kidney transplantation (TX) does not occur immediately. The expression of CaR was still diminished 1 week after TX, at a time when circulating PTH levels already were normalized. The figure shows the CaR gene expression in the parathyroid glands of normal control rats, uremic rats on a high P diet (CRF hp), and kidney transplanted (TX), 1 and 4 weeks after kidney transplantation. N ¼ 5 8, mean7s.e.m. *Po.1 versus control rats. REFERENCES 1. Drueke TB. Cell biology of parathyroid gland hyperplasia in chronic renal failure. J Am Soc Nephrol 2; 11: 1141 1152. 2. Parfitt AM. The hyperparathyroidism of chronic renal failure: a disorder of growth. Kidney Int 1997; 52: 3 9. 3. Wang Q, Palnitkar S, Parfitt AM. 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