Pathogenesis of refractory secondary hyperparathyroidism

Kidney International, Vol. 61 (2002), pp. S155–S160

Pathogenesis of refractory secondary hyperparathyroidism MARIANO RODRIGUEZ, ANTONIO CANALEJO, BARTOLOME GARFIA, ESCOLASTICO AGUILERA, and YOLANDA ALMADEN Nephrology Services and Research Unit, Hospital Universitario Reina Sofia, Co´rdoba; Department of Environmental Biology and Public Health, University of Huelva, Huelva; and Department of Pathology, Veterinary Faculty, University of Co´rdoba, Co´rdoba, Spain

Pathogenesis of refractory secondary hyperparathyroidism. Calcitriol is currently used to reduce parathyroid hormone (PTH) levels in uremic patients. However, a significant number of patients fail to respond to calcitriol therapy. The data suggest that a poor response to calcitriol can be anticipated in patients with severe hyperparathyroidism (with a high basal PTH levels) and uncontrolled serum phosphate. The abnormal parathyroid response to calcitriol in uremic patients with severe parathyroid hyperplasia may be attributed, to a large extent, to the development of nodular hyperplasia as a result of clonal transformation from a diffuse polyclonal hyperplasia. The factors involved in the development of polyclonal parathyroid hyperplasia, at earlier stages of secondary hyperparathyroidism, appear to be the same factors that stimulate PTH secretion and synthesis: hypocalcemia, hyperphosphatemia and low serum calcitriol levels. Studies performed in vitro using parathyroid tissue from uremic patients who required parathyroidectomy demonstrate that in nodular hyperplasia there is an abnormal response to calcium and calcitriol, which suggests that there are factors intrinsic to the hyperplastic cell (such as decrease in calcium sensor receptors and vitamin D receptors) responsible for an abnormal regulation of parathyroid function. Accumulation of phosphate is a key factor in the pathogenesis of secondary hyperparathyroidism and a poor response to calcitriol treatment is associated with the failure to control the serum phosphorus. High phosphate stimulates PTH secretion as demonstrated by in vivo and in vitro studies. In addition, animal studies strongly suggest that phosphate increases parathyroid cell proliferation. There are growth-related genes potentially involved in uremic hyperparathyroidism; however, changes in the expression of these genes may be the consequence rather than the cause of parathyroid hyperplasia.

Calcitriol is currently used to reduce parathyroid hormone (PTH) levels in uremic patients [1], however, a significant number of patients fail to respond to calcitriol therapy [2–4]. These failures have been ascribed to intrinsic factors associated with large, hyperplastic parathyroid glands such as nodular hyperplasia [5–7] with decreased levels of vitamin D receptor [7] and calcium Key words: Uremia, calcitrol, parathyroid homone, nodular hyperplasia, vitamin D receptor.

 2002 by the International Society of Nephrology

sensor receptor [8, 9]; also, the poor response to calcitriol treatment is associated with factors such as the failure to control the serum phosphorus [2, 10, 11]. This review will analyze the characteristics and factors involved in the pathogenesis of refractory hyperparathyroidism. ABNORMAL PARATHYROID FUNCTION IN REFRACTORY HYPERPARATHYROIDISM In a recent study [12] we analyzed parathyroid function (PTH-Ca curve) in 50 hemodialysis patients with PTH greater than 300 before and after 3 months of bolus calcitriol therapy (3–6 ␮g). Patients were divided into responders and non-responders based on whether the predialysis PTH value decreased by 40% or more in response to CTR treatment; this value was selected because it represented the median for the total group of 50 patients. Before initiation of treatment, the mean basal PTH, maximal PTH, and minimal PTH were greater in non-responders than responders. Serum calcium concentration was similar in both groups and the serum phosphate was greater in non-responders than responders. The data suggest that a poor response to calcitriol can be anticipated in patients with severe hyperparathyroidism and uncontrolled serum phosphate. The probability of a response to CTR based on pre-CTR basal PTH values is shown for the model in Figure 1. A 50% probability of a response (40% reduction in basal PTH) was observed at a pre-CTR basal PTH value of 750 pg/mL. At a basal PTH of 1200 pg/mL, the probability of a response to CTR was less than 20% and at a basal PTH of 400 pg/mL, the probability of a response approached 80%. One of the parameters analyzed in this study was the ratio of basal to maximal PTH (basal PTH divided by the maximal PTH; this fraction was multiplied by 100), which in normal volunteers is 20% to 25% [13]. By correcting the actual PTH for the overall capacity to produce PTH (maximal PTH), a measure of the relative degree of PTH stimulation is obtained. When the basal calcium

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Fig. 1. Logistic regression model to predict response to calcitriol treatment. Stepwise logistic regression analysis showed that the pre-calcitriol basal PTH level was the most important predictor of the probability of a 40% reduction in basal PTH during calcitriol treatment. Using the above model, a 50% probability of a response (40% reduction in basal PTH) was observed at a pre-calcitriol basal PTH value of 750 pg/mL. At a basal PTH of 1200 pg/mL, the probability of a response to calcitriol was less than 20%, and at a basal PTH of 400 pg/mL, the probability of a response approached 80%.

is low, the basal to maximal PTH ratio should be high, indicating that the parathyroid gland is using more of its overall capacity to correct the low calcium; conversely, an increase of calcium must decrease basal PTH relative to maximal PTH. Figure 2 shows the changes in basal/ maximal PTH ratio produced by calcitriol treatment in responders and non-responders. The basal/maximal PTH ratio, which reflects the relative degree of parathyroid gland sensitivity to the serum calcium, decreased in the responders group from 52 ⫾ 3% to 33 ⫾ 3% (P ⬍ 0.001) after CTR treatment as the serum calcium concentration increased (Fig. 2A). Conversely, in the non-responders group, the basal/maximal PTH ratio did not change despite the increase in serum calcium and a similar shift to the right of the PTH-calcium curve (Fig. 2B). The magnitude of the absolute reduction in PTH in the responder group (Fig. 2C) and the lack of a change in the non responder group (Fig. 2D) can be appreciated in the PTH-calcium curves shown in these figures. The PTH response to calcitriol was also affected by high serum phosphate. In both groups, responders and non responders, patients with serum phosphate greater than 6 mg/dL showed less reduction in PTH than those with serum phosphate below 6 mg/dL. The set point was not different in responders and non responders. As reported in a previous work [14], we have observed that among patients with advanced hyperparathyroidism, a high set point is only observed in patients with high PTH and elevated serum calcium, which suggests that the PTH is driving the serum calcium; these patients are not likely to respond to calcitriol therapy. The abnormal parathyroid response to calcitriol in uremic patients with severe parathyroid hyperplasia may

be attributed, to a large extent, to the development of nodular hyperplasia as a result of clonal transformation from a diffuse hyperplasia [15, 16]. Different research groups have reported that in nodular areas there is a decrease in calcium sensor receptor expression [8, 9], which may explain the abnormal response of these parathyroid glands to the increase in calcium induced by calcitriol administration. Calcitriol acts on parathyroid glands independently of calcium, however, the decrease in vitamin D receptor density also observed in nodular hyperplasia [7] may explain the refractoriness to calcitriol treatment. Studies performed in vitro using parathyroid tissue from uremic patients that required parathyroidectomy demonstrate that in nodular hyperplasia there is an abnormal response to calcium [17, 18] and calcitriol [19], which suggests that there are factors intrinsic to the cell (such as decrease in calcium sensor and vitamin D receptors) that are responsible for an abnormal regulation of parathyroid function. We have evaluated the ability of calcium to reduce PTH secretion in vitro in parathyroid tissue from uremic patients that required parathyroidectomy [18]. As shown in Figure 3, the degree of inhibition of PTH secretion by calcium was greater in diffuse than nodular hyperplasia; in primary parathyroid hyperplasia very high calcium concentrations were necessary to produce a significant decrease in PTH secretion. In a different study we evaluated in vitro, the effect of calcitriol on parathyroid cell cycle and apoptosis in parathyroid glands from patients with severe hyperparathyroidism [19]. In these glands, parathyroid cell proliferation was not inhibited by concentrations of calcitriol ranging from 10⫺10 to 10⫺8 mol/L; a moderate decrease in proliferation was observed when calcitriol concentration in the medium reached 10⫺7 mol/L (Fig. 4). In this study, it was observed that a high concentration of calcitriol produced a decrease in the number of apoptotic cells that was parallel to the decrease in proliferation. Because calcitriol simultaneously inhibits cell proliferation and apoptosis, a reduction in the parathyroid gland mass may not occur as a direct effect of calcitriol treatment. NODULAR HYPERPLASIA The reason for the high frequency of clonal proliferation is unclear. Probably the long-standing stimulation of a tissue with a usually extremely slow growth pattern favors clonal transformation; defects in DNA repair mechanisms may play a role [20]. Mendes et al [5] first described frequent nodular formations in parathyroid glands from uremic patients with severe secondary hyperparathyroidism. Nodular formation was observed in 50% of glands weighing between 0.25 and 0.5 g parathyroid and in more than 90% of glands weighing more than 0.5 g [21]. Nodules are formed by a greater proportion of

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Fig. 2. Patterns of PTH secretion in responders and non-responders. The mean basal/maximal PTH ratio (⫻100) is shown before (solid line) and after (dashed line) calcitriol treatment in A and B and the absolute changes in basal and maximal PTH are shown in C and D. Before calcitriol treatment, the mean basal/ maximal PTH ratio was similar in the Responders (A) and Non-Responders (B). In both groups, a similar shift to the right of the PTHcalcium curve was observed during calcitriol treatment with sustained increases in the serum calcium concentration. However, only in the Responders was the rightward shift in the PTH-calcium curve associated with a decrease in the mean basal/maximal PTH ratio. In C and D, the absolute values for basal, maximal, and minimal PTH are shown. Before calcitriol treatment, the basal and maximal PTH levels were less in the Responders (C) than the NonResponders (D), and minimal PTH was not different. With calcitriol treatment basal, maximal, and minimal PTH values decreased in the Responders (C), but were essentially unchanged in the Non-Responders (D).

actively replicating cells [22–25]. Nodular hyperplasia was also associated with a greater resistance to medical suppression of PTH oversecretion [24, 25], and recurrence rates of hyperparathyroidism after PTX were significantly higher when nodular tissue instead of purely hyperplastic tissue was autografted [26]. Several authors have shown, using X chromosome inactivation analysis, that benign monoclonal tumors are present in a large proportion of hyperplastic glands [27–29] and there was no correlation between clonal development and morphology [27]. Clonal development may be caused by mutations or losses of tumor suppressor genes or activation of tumor enhancer genes [29, 30]. Losses on chromosome 11, the location of the menin

gene, have been found in only 10% of the patients [31, 32] and allelic loss of the Ha-ras gene and the tumor suppressor gene WT1 in approximately 10% of the patients [33]. The expression of calcium sensor receptor and vitamin D receptor are decreased in nodular hyperplasia, however, mutations of these two important receptors have not been identified [29].

DIFFUSE HYPERPLASIA The nodular hyperplasia of the parathyroids occurs at a late stage in the evolution of secondary hyperparathyroidism. During earlier stages of secondary hyperparathyroidism, parathyroid growth is polyclonal. The factors

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Fig. 3. Parathyroid function in adenomas, nodular and diffuse hyperplasia. The inhibition of PTH secretion by calcium in vitro in human parathyroid adenoma (䊐, N ⫽ 10), nodular hyperplasia from hemodialysis and renal transplant patients with secondary hyperparathyroidism (䊊, N ⫽ 15), and diffuse hyperplasia also from hemodialysis and renal transplant patients with secondary hyperparathyroidism (䊉, N ⫽ 21). Values of PTH are the percent (mean ⫾ SE) of maximal stimulation. Values of maximal PTH stimulation were 292 ⫾ 75, 371 ⫾ 95, and 423 ⫾ 73 pg of d/L gDNA/h, respectively. From calcium 0.8 to 1.5 mol/L, the reduction of PTH was significantly greater (P ⬍ 0.01) in diffuse hyperplasia than in adenoma.

involved in the development of polyclonal parathyroid hyperplasia appear to be the same factors that stimulate PTH secretion and synthesis: hypocalcemia, hyperphosphatemia, and low serum calcitriol levels. The precise mechanism by which each of these factors stimulates parathyroid cell proliferation is unknown. In rats on a low calcium diet with or without renal failure, there is a marked increase in parathyroid cell proliferation [34]. This effect is enhanced if rats have vitamin D deficiency. The importance of calcium on parathyroid cell proliferation is also demonstrated by the fact that in uremic rats the administration of calcimimetics prevent parathyroid cell proliferation [35]. The role of calcitriol on parathyroid cell proliferation in rats with renal failure was clearly demonstrated in a work by Szabo et al [36], in which calcitriol administration prevented parathyroid gland hyperplasia in uremic rats if calcitriol is administered from the time of induction of renal failure. However, once parathyroid hyperplasia had been established, the administration of calcitriol did not reverse the parathyroid gland hyperplasia. In vitro and in vivo studies [37, 38] indicates that calcitriol might suppress parathyroid hyperplasia by decreasing c-myc expression. Whether vitamin D receptor polymorphism plays a role in parathyroid hyperplasia secondary to renal failure is a subject of debate. While some authors have shown an association between the severity of hyperparathyroidism and vitamin D receptor polymorph-

ism [39, 40], other authors have not observed such association [41–43]. Accumulation of phosphate is a key factor in the pathogenesis of hyperparathyroidism secondary to renal failure. High phosphate stimulates PTH secretion as demonstrated by in vivo and in vitro studies [44–47]. In addition, animal studies strongly suggest that phosphate increases parathyroid cell proliferation [34, 48–50], however, possible mechanisms are not clear. In a recent work, Brown et al [51] clearly demonstrated that in renal failure rats fed a high phosphorus diet, the resulting parathyroid hyperplasia was associated with a decrease in calcium sensing receptor (CaR). This decrease in calcium sensing receptor may have been a direct effect of phosphate or a consequence of parathyroid gland hyperplasia. In a more recent study the same group shows that decrease in CaR precedes development of hyperplasia [52]. In a similar animal model Dusso et al [53] found that a low phosphorus diet may inhibit parathyroid cell proliferation by increasing the expression of the cyclin-dependent kinase inhibitor p21, whereas a high phosphate diet may stimulate parathyroid cell proliferation by enhancing the expression of transforming growth factor-␣ (TGF-␣). There are genes potentially involved in uremic hyperparathyroidism. In uremic rats the increase in parathyroid cell proliferation is associated with increased c-myc expression [38]. Acidic fibroblast growth factor autocrine system has been proposed as a mediator of calciumregulated parathyroid cell growth in a clonal cell model [54]. In hyperplastic human parathyroid glands, proliferating cells have a low expression of PTHrP [55] and an increased expression of TGF-␣. [56]. Changes in the expression of all these genes may be the consequence rather that the cause of parathyroid hyperplasia. ACKNOWLEDGMENTS This work was in part supported by grants PB93-0720 and PM980184 from Ministry of Education, PB99-0768 from Ministry of Health, Fundacio´n Reina Sofı´a-Cajasur, and the “Consejeria de Salud de la Junta de Andalucia” (JA99/190). Reprint requests to Dr. Mariano Rodriguez, Unidad de Investigacion, Hospital Reina Sofia, Avda Menendez Pidal, s/n 14004 Cordoba, Spain. E-mail: [email protected]

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Fig. 4. The effect of increasing concentrations of calcitriol on the percent of cells in the S phase of the parathyroid cell cycle and apoptosis. It was assesed by flow cyitometry in normal dog parathyroid tissue (A and B) and in human hyperplastic glands from patient with 2⬚HPT (C and D). The cell cycle and apoptosis were evaluated after 24 h incubation in two different aliquots from the same tissue sample. N ⫽ 10 for A and B; N ⫽ 30 (individual glands) for C and D. Values are mean ⫾ SE, (*) P ⬍ 0.05 vs control and (#) P ⬍ 0.05 vs. calcitriol 10⫺9 mol/L (A and B) or 10⫺8 mol/L (C and D).

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