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PTHrP receptor ligands in normal and uremic rats
Kidney International, Vol. 64 (2003), pp. 63–70
Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats EWA LEWIN, BARTOLOME GARFIA, YOLANDA ALMADEN, MARIANO RODRIGUEZ, and KLAUS OLGAARD Nephrological Department B, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark; Nephrological Department P, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; and Unidad de Investigacio´n, Hospital Universitario Reina Sofia, Cordoba, Spain
were significantly increased. The CRF rats on high phosphorus diet had significant hypocalcemia (Ca⫹⫹, 1.04 ⫾ 0.02 mmol/L vs. 1.28 ⫾ 0.03 mmol/L, P ⬍ 0.001), hyperphosphatemia (3.48 ⫾ 0.3 mmol/L vs. 2.25 ⫾ 0.1 mmol/L, P ⬍ 0.001) and severe secondary HPT, PTH (984 ⫾ 52 pg/mL vs. 226 ⫾ 32 pg/mL, P ⬍ 0.001) compared to CRF rats on a standard phosphorus diet. The maximal PTH response to hypocalcemia was enhanced in CRF rats (maximum PTH 382 ⫾ 58 pg/mL vs. 196 ⫾ 29 pg/mL in normal rats, P ⬍ 0.01) and further enhanced by PTHrP 1-40 to 826 ⫾ 184 pg/mL (P ⬍ 0.01). The secretory capacity of the parathyroid glands in response to low Ca⫹⫹ was severely diminished in uremia. In CRF rats given a high phosphorus diet, the basal PTH levels were at the upper part of the calcium/PTH curve, and the induction of more marked hypocalcemia did not stimulate PTH secretion further (maximum PTH 1475 ⫾ 208 pg/mL vs. basal 1097 ⫾ 69 pg/mL, NS). PTHrP, however, further enhanced the maximal PTH levels significantly (maximum PTH 3142 ⫾ 296 pg/mL, P ⬍ 0.01). The presence of the PTH/PTHrP receptor in the rat parathyroid glands was confirmed by RT-PCR technique. Conclusion. PTHrP enhanced significantly, in a dose-related manner, the low Ca⫹⫹-stimulated PTH secretion in normal rats. The PTH/PTHrP receptor is present in rat parathyroid glands. The impaired secretory capacity of the parathyroid glands in uremic rats is significantly enhanced by PTHrP. An autocrine/paracrine role in the parathyroid glands of the PTH/ PTHrP receptor targeting peptides, PTHrP and PTH, is suggested. Thus, it is hypothesized that PTH during hypocalcemia might have a positive auto-feedback regulatory role on its own secretion.
Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats. Background. The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/ paracrine factors. We have previously shown that N-terminal parathyroid hormone-related protein (PTHrP) enhanced the secretory PTH response to low calcium in vivo and in vitro in rat parathyroid glands. N-terminal PTHrP fragments are equipotent to N-terminal PTH as ligands for the PTH/PTHrP receptor that is demonstrated in parathyroid tissue. This supports the possibility that the parathyroid cells respond to PTH/ PTHrP receptor ligands and as such are target for an autoregulatory action of PTH and PTHrP. Our aim was to search for the PTH/PTHrP receptor in rat parathyroid glands and to examine the effects of PTHrP 1-40 on PTH secretion in in vivo models of secondary hyperparathyroidism (HPT) in uremic rats. Methods. PTH secretion was examined during ethyleneglycol tetraacetic acid (EGTA)-induced hypocalcemia both with and without PTHrP. Five groups, each of six normal rats, received a bolus of increasing doses of 0.1, 1.0, 10, and 100 g of PTHrP 1-40, or vehicle only. Chronic renal failure (CRF) was induced by 5/6 nephrectomy. One group of 12 CRF rats received a standard diet, while another CRF group of 18 rats received a high phosphorus diet to induce more severe HPT. After 8 weeks of uremia, the rats were infused with EGTA and PTHrP 1-40 or vehicle. The presence of the PTH/PTHrP receptor in the rat parathyroid glands was examined by reverse transcription-polymerase chain reaction (RT-PCR) technique. PTH was measured by a rat PTH assay that does not crossreact with PTHrP. Results. In a dose-related manner, PTHrP enhanced the PTH response to hypocalcemia in normal rats. A similar rate of decrease of plasma Ca⫹⫹ was induced by EGTA in all experimental groups. In CRF rats, plasma creatinine (0.99 ⫾ 0.10 mmol/L vs. 0.33 ⫾ 0.01 mmol/L, P ⬍ 0.001) and plasma PTH (226 ⫾ 32 pg/mL vs. 69 ⫾ 16 pg/mL, P ⬍ 0.001) levels
It has previously been shown by our group, that N-terminal parathyroid hormone-related protein (PTHrP) 1-40 and 1-86, used as surrogate for parathyroid hormone (PTH), enhance the secretory response of PTH to hypocalcemia in rats in vivo [1]. This effect of PTHrP was immediate, taking place within 5 minutes. No effect of PTHrP was found in vivo during normo- and hypercalcemia. In that investigation, it was ensured that the peripheral metabolism of PTH was not affected by PTHrP and that there was no cross-reactivity of PTHrP in the rat PTH assay [1]. A direct effect of PTHrP on the rat
Key words: secondary hyperparathyroidism, calcium/PTH curve, autocrine-paracrine factor, PTHrP, PTH/PTHrP receptor, uremia. Received for publication October 18, 2002 and in revised form December 16, 2002, and January 28, 2003 Accepted for publication February 24, 2003
2003 by the International Society of Nephrology
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parathyroids was confirmed by the demonstration in vitro that low Ca⫹⫹-induced PTH secretion was augmented by PTHrP [1]. These results support the possibility that the parathyroid cells respond to PTH/PTHrP receptor ligands. PTH and PTHrP might as equipotent ligands for the receptor have regulatory functions on the secretion of PTH. The mature PTHrP protein contains a limited region of sequence homology with PTH in its immediate N-terminal region. PTH and PTHrP activate a common G-protein coupled receptor, the PTH/PTHrP type 1 receptor [2–4] and PTHrP was used as a surrogate for PTH because it mimics the action of PTH by acting through the PTH/ PTHrP receptor. PTHrP is expressed in a wide variety of normal fetal and adult tissues [5–13]. The PTH/PTHrP receptor is frequently expressed in cells that produce PTHrP or in cells immediately adjacent to PTHrP-producing cells. This spatial proximity, together with the fact that little, if any, PTHrP circulates under normal physiologic conditions have led to the view that PTHrP is a locally acting autocrine/paracrine factor [3, 14]. PTHrP mRNA is expressed in normal human and bovine parathyroid tissues [13, 15, 16]. The expression of PTHrP in the parathyroid glands is not only a feature of neoplastic transformation, although abnormal expression of PTHrP mRNA and protein has been found in human parathyroid adenomas, hyperplasia, and carcinomas [13, 17–19], but it could be a feature of normal physiology, as well. PTH and PTHrP have been demonstrated to be colocalized in the same secretory granules and to be secreted simultaneously from parathyroid adenomas [19]. The PTH/PTHrP receptor has been demonstrated in human parathyroid tissue [18]. However, the physiologic role of PTHrP in the parathyroid glands remains to be established [3]. N-terminal PTHrP fragments are equipotent to N-terminal PTH fragments as ligands for the PTH/PTHrP receptor [20]. Therefore, in the present study, N-terminal PTHrP was used as a surrogate for N-terminal PTH due to the properties of the rat PTH assay, which comeasures the N-terminal PTH fragments (i.e., rat, human, and bovine PTH), but not PTHrP. The uremic condition is associated with disturbances in calcium, phosphorus, and PTH metabolism leading to development of secondary hyperparathyroidism (HPT). The Ca⫹⫹/PTH relationship is abnormal in uremia; the PTH secretion, at the basal condition, is toward the upper part of the Ca⫹⫹/ PTH curve, and the maximal secretory response to hypocalcemia is often increased [21]. The aim of present investigation therefore was to search for the PTH/PTHrP receptor in the parathyroid glands and to examine the effects of PTHrP 1-40 on PTH secretion in in vivo models of secondary HPT in uremic rats.
METHODS Animals Inbred male Dark Aguti (DA) rats (Moellegaarden, Denmark) were used, weighing 200 to 225 g at the start of the study. The experimental studies on the rats were performed in accordance with the NIH Guidelines for Care and Use of Laboratory Animals and was approved by our institution. The experimental procedures were performed in rats under anesthesia with 50 g/kg intraperitoneal injection of pentobarbital (Mebumal). Uremia Chronic renal failure (CRF) was induced by one-step 5/6 nephrectomy. A group of 12 CRF rats was given a standard diet containing 0.9% calcium, 0.7% phosphorus, and 1000 IU vitamin D/kg. To induce more severe HPT, another group of 18 CRF rats was given a high phosphorus diet containing 0,9% calcium, 1.5% phosphorus, and 1000 IU vitamin D/kg. The duration of uremia was 8 weeks. A group of 12 normal control rats was given a standard diet. Rats were allowed free access to food and water. Peptide PTHrP 1-40 was obtained from Saxon Biochemicals, GMBH, Hannover, Germany. PTHrP was first dissolved in deionized water to a concentration of 1 mg/mL and then diluted in saline to the concentration needed. Induction of hypocalcemia Hypocalcemia was induced by infusion of 30 mmol/L ethyleneglycol tetraacetic acid (EGTA), 3 mL/hour through a catheter inserted in the femoral vein. Samples for determination of plasma PTH and plasma Ca⫹⫹ were taken at 0, 5, 10, 20, 30, 40, and 50 minutes from a corresponding catheter in the femoral artery. The sample volume of 0.8 mL was replaced by 0.8 mL of saline, resulting in the same weight of the rats before and after the experiments. Maximum PTH is the highest PTH value obtained during induction of hypocalcemia. The relative secretory capacity is defined as the relative increase in PTH secretion from baseline in response to an acute induction of hypocalcemia, expressed in percent of the basal PTH level. Experimental protocols Dose-response study. At time 0, one of five groups, each with six normal rats, received vehicle or an intravenously bolus dose of 0.1, 1.0, 10, or 100 g of PTHrP 1-40. Hypocalcemia was then induced by an infusion of 30 mmol/L EGTA and samples for determination of plasma PTH and plasma Ca⫹⫹ were taken as described above.
Lewin et al: PTHrP and PTH secretion in uremic rats
The effect of PTHrP on low Ca⫹⫹-stimulated PTH secretion in vivo in uremic rats kept on standard diet. A group of uremic rats received a bolus of 1 mL of 100 g/mL of PTHrP 1-40 at time 0 (N ⫽ 6). Another group of uremic rats received 1 mL of vehicle only (N ⫽ 6). Hypocalcemia was then induced and samples for determination of plasma PTH and plasma Ca⫹⫹ were taken as described above. The effect of PTHrP on low Ca⫹⫹-stimulated PTH secretion in vivo in uremic rats kept on a high phosphorus diet. A group of uremic hyperphosphatemic rats received a bolus of 1 mL of 100 g/mL of PTHrP 1-40 at time 0 (N ⫽ 9). Another group of uremic hyperphosphatemic rats received 1 mL of vehicle only (N ⫽ 9). Then, hypocalcemia was induced and samples for determination of plasma PTH and plasma Ca⫹⫹ were taken as described above. Parathyroid PTH/PTHrP receptor—RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Two rat parathyroid glands and a small slice of kidney cortex (1 mm3) were obtained for total RNA extraction. Tissue samples were separately placed in nuclease-free 1.5 mL microcentrifuge tubes. Then, 400 L of a lysis/ binding buffer from a commercial kit for tissue total RNA extraction (High Pure RNA Tissue Kit; Roche, Mannheim; Germany) were added to the samples. The tissues (parathyroid and kidney cortex) were ultrasonicated for 5 minutes at 4⬚C to allow for complete cell rupture. Subsequently, tissues were homogenized by passing the lysate 10 to 20 times through a 20 gauge needle attached to a sterile plastic syringe until a homogeneous lysate was achieved. Then, total RNA was extracted using the High Pure RNA Tissue Kit. Total RNA was quantified by spectrophotometry. The PTH/PTHrP receptor in parathyroid glands and kidney cortex were amplified using the kit, Access RT-PCR System (Promega, Madison WI, USA) using a specific primer and 100 ng of kidney cortex total RNA and 200 ng of parathyroid total RNA per sample. Kidney cortex and parathyroid PTH/PTHrP receptor-mRNA were amplified at 24 and 40 cycles, respectively. DNA amplifications were processed by a Genetic Analyzer Abi Prism 310 (Perkin Elmer, Foster City, CA, USA). Data were analyzed using specific software Gene Scan version 3.1/1998 (Perkin Elmer). The PTH/PTHrP receptor primers were: PTH/PTHrP receptor sense CGGAAGCTGCTCAAATCC PTH/PTHrP receptor antisense AAAAATCCCTGGAAG
5⬘-FAM-TAC 5⬘-GCGACA
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Plasma measurements Plasma creatinine, urea, phosphorus, and total calcium were measured by an ETACHEM 250 Analyzer (Kodak, Rochester, NY, USA). Plasma PTH was measured by a rat PTH immunoradiometric assay (IRMA) from Immunotopics, San Clemente, CA, USA. The intra-assay coefficient of variance in our laboratory was 4% and interassay coefficient of variance 5%. PTHrP 1-40 did not cross-react with the rat PTH assay. No cross-reactivity with C-terminal PTH fragments was found in our laboratory [1, 22]. Plasma Ca⫹⫹ at actual pH was measured by a calcium-selective electrode (Radiometer, Copenhagen, DK). Statistics The results are expressed as mean ⫾ SEM. MannWhitney test or t test were used for comparison between groups. P ⬍ 0.05 is considered significant. RESULTS In uremic rats, plasma creatinine and urea levels were significantly increased (P ⬍ 0.001), as shown in Table 1. The uremic rats kept on a high phosphorus diet developed significant hypocalcemia (P ⬍ 0.001), hyperphosphatemia (P ⬍ 0.001), and severe secondary HPT (P ⬍ 0.001), as compared to normal rats and uremic rats given a standard diet (Table 1). The effect in vivo of increasing doses of PTHrP 1-40 on the low calcium–induced PTH secretion in the rat is shown in Figure 1A. This effect was clearly dose-related with increasing doses of PTHrP resulting in an enhanced PTH response to hypocalcemia (P ⬍ 0.01). The rates of reduction of plasma Ca⫹⫹ levels were not different between the experimental groups (Fig. 1B). In order to ensure the presence of the PTH/PTHrP receptor in the parathyroid glands, three sets of RTPCR measurements were performed in parathyroid glands, using normal kidney tissue as control. These investigations clearly demonstrated the presence of the PTH/PTHrP receptor in the parathyroid glands as well as in the kidneys, as shown in Figure 2. The relative secretory capacity of the parathyroid glands in normal rats, uremic rats kept on a standard diet, and uremic rats kept on a high phosphorus diet is shown in Figure 3. The relative secretory capacity was significantly decreased (P ⬍ 0.05) in uremia and practically zero in uremic rats kept on a high phosphorus diet. The PTH secretory response to PTHrP and hypocalcemia in normal rats and in uremic rats kept on a standard diet is shown in Figure 4. In uremic rats, the PTH levels induced by hypocalcemia were significantly enhanced compared to that of normal rats and this response was further significantly enhanced by PTHrP (P ⬍ 0.01). In normal control rats, PTH increased from a basal PTH
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Lewin et al: PTHrP and PTH secretion in uremic rats Table 1. Plasma parameters of normal and uremic rats
Normal N ⫽ 12 Chronic renal failure standard diet N ⫽ 12 Chronic renal failure high phosphorus diet N ⫽ 18 a
Creatinine lmol/L
Urea mmol/L
Ca⫹⫹ mmol/L
Phosphorus mmol/L
Parathyroid hormone pg/mL
33 ⫾ 1 99 ⫾ 10a 86 ⫾ 5a
6⫾1 26 ⫾ 9a 16 ⫾ 2a
1.26 ⫾ 0.04 1.28 ⫾ 0.03a 1.04 ⫾ 0.02a,b
1.85 ⫾ 0.1 2.25 ⫾ 0.1 3.48 ⫾ 0.3a,b
69 ⫾ 16 226 ⫾ 32a 984 ⫾ 52a,b
P ⬍ 0.001 versus normal rats; b P ⬍ 0.001 versus chronic renal failure standard diet rats
Fig. 1. Effect of increasing doses of N-terminal parathyroid hormonerelated protein (PTHrP) 1-40 on the secretory response of PTH to an acute induction of hypocalcemiain normal rats. Hypocalcemia (A ) was induced by an ethyleneglycol tetraacetic acid (EGTA) infusion. The rate of reduction of plasma Ca⫹⫹ was similar in all groups of rats (B ). A bolus of PTHrP 1-40 or vehicle was injected at time 0. PTHrP 1-40 enhanced significantly (P ⬍ 0.01) in a dose-related manner the low Ca⫹⫹-stimulated PTH secretion. N ⫽ 6 in each group, mean ⫾ SEM.
level of 61 ⫾ 22 pg/mL to a maximum PTH level of 196 ⫾ 29 pg/mL in the vehicle-treated group and from a basal PTH level of 78 ⫾ 25 pg/mL to a maximum PTH level of 488 ⫾ 68 pg/mL in the PTHrP-treated group. In uremic rats, PTH increased from a basal PTH level of 242 ⫾ 45 pg/mL to a maximum PTH level of 382 ⫾ 58 pg/mL in the vehicle-treated group and from a basal PTH level of 209 ⫾ 50 pg/mL to a maximum PTH level of 826 ⫾ 184 pg/mL in the PTHrP-treated group. The proportion of the increment in PTH secretion in re-
Fig. 2. Representative electropherograms of parathyroid hormone/ parathyroid hormone-related protein (PTH/PTHrP) receptor mRNAs in three different samples of normal rat kidney cortex and parathyroid glands.
sponse to PTHrP compared to that of hypocalcemia alone was, however, the same in normal and uremic rats. Thus, in normal rats maximal PTH was increased by 249% by PTHrP, while in uremic rats PTHrP increased the PTH response by 216% (NS).
Lewin et al: PTHrP and PTH secretion in uremic rats
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Fig. 3. The relative secretory capacity of the parathyroid glands in normal rats, uremic rats kept on normal diet [chronic renal failure (CRF)], and uremic rats kept on high phosphorus diet [CRF high phosphorus (P)]. The relative secretory capacity is defined as the relative increase in parathyroid hormone (PTH) secretion from baseline in response to an acute induction of hypocalcemia, and expressed in percent of basal PTH levels. The relative secretory capacity is significantly decreased in uremia (P ⬍ 0.05) and practically 0 in uremic rats kept on high phosphorus diet. N ⫽ 6 to 9, mean ⫾ SEM.
The PTH secretory response to PTHrP and hypocalcemia in uremic rats kept on a high phosphorus diet is shown in Figure 5. In uremic rats given a high phosphorus diet, the PTH levels, at the basal condition, were already at the upper part of the calcium/PTH curve. Induction of even severe hypocalcemia was not able to stimulate PTH secretion further. PTHrP administration, however, enhanced the PTH levels significantly (P ⬍ 0.001) and the continuous increase strongly suggests that a maximum was not even obtained in the present experimental design. The relative secretory capacity of PTH secretion was significantly increased by PTHrP administration to uremic rats kept on a high phosphorus diet (P ⬍ 0.01), as shown in Figure 6. DISCUSSION In the present investigation it is shown that N-terminal PTHrP administered in vivo in a dose-related manner enhanced the secretory response of PTH to hypocalcemia in rats. In uremic rats, the maximal PTH secretory response to low calcium was significantly enhanced compared to that of normal rats, and this response was further increased several fold by the administration of PTHrP. It has previously been demonstrated that the maximal PTH secretory response is dependent upon the rate of fall of plasma Ca⫹⫹ [23, 24]. In the present investigation, the rate of decrease in plasma Ca⫹⫹ was well controlled by the infusion of the calcium chelator and
Fig. 4. The effect of parathyroid hormone-related protein (PTHrP) 1-40 on the secretory response of parathyroid hormone (PTH) to an acute induction of hypocalcemia in uremic rats kept on a standard diet (- - -) and in normal rats (______) (A ). Hypocalcemia was induced by an ethyleneglycol tetraacetic acid (EGTA) infusion. The rate of reduction of plasma Ca⫹⫹ was similar in all groups of rats (B). A bolus of PTHrP 1-40 or vehicle was injected at time 0. PTHrP significantly enhanced the low Ca⫹⫹-stimulated PTH secretion in both group of rats (P ⬍ 0.01). N ⫽ 6 in each group, mean ⫾ SEM. CRF is chronic renal failure.
the rate of decrease was similar in the group of rats infused with PTHrP and in the control group of rats. Therefore, differences in the degree of hypocalcemia cannot explain the difference obtained in the response of PTH secretion to hypocalcemia. We have previously shown in normal rats that the peripheral metabolism of PTH was not affected by PTHrP. In uremia, however, an influence of PTHrP on the peripheral metabolism of PTH cannot be completely excluded. In general, elevated circulating levels of PTH in uremic HPT might be an expression of both a marked increase in glandular secretion and of a reduced elimination of PTH [25], which is of importance for the interpretation of the results obtained in vivo. The parathyroid glands are not controlled by a superior hypothalamic-pituitary axis, as it most often is observed in other endocrine glands [26]. Therefore, the parathyroid glands are likely to use autocrine/paracrine regulatory mechanisms. The PTH/PTHrP receptor was in the present investigation demonstrated in rat parathy-
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Lewin et al: PTHrP and PTH secretion in uremic rats
Fig. 5. The Caⴙⴙ/parathyroid hormone (PTH) relationship during ethyleneglycol tetraacetic acid (EGTA)-induced hypocalcemia in uremic rats kept on a high phosphorus diet and receiving a bolus injection of parathyroid hormone-related protein (PTHrP) 1-40 or vehicle. The arrow depicts the direction of change of plasma Ca⫹⫹. In the vehicletreated rats, induction of severe hypocalcemia did not further enhance PTH levels. Administration of PTHrP 1-40 did, however, enhance the PTH levels significantly (P ⬍ 0.001). N ⫽ 9 in each group, mean ⫾ SEM.
Fig. 6. The relative secretory capacity of the parathyroids in uremic rats kept on a high phosphorus diet after administration of parathyroid hormone-related protein (PTHrP) 1-40. The relative secretory capacity is defined as the relative increase in parathyroid hormone (PTH) secretion from baseline in response to an acute induction of hypocalcemia, and expressed in percent of basal PTH levels. The relative secretory capacity is significantly increased by PTHrP (P ⬍ 0.01). N ⫽ 9 in each group, mean ⫾ SEM. EGTA is ethyleneglycol tetraacetic acid.
roid glands and has previously been demonstrated in human parathyroid tissue [18]. This supports the possibility that PTHrP, as a ligand for the receptor, might regulate PTH secretion, but also that PTH-producing parathyroid cells are target for an autoregulatory action of N-terminal PTH. On the basis of the present results, which showed that N-terminal PTHrP very fast, within 5 minutes, and in a dose-related manner enhanced the PTH secretory response to hypocalcemia, we would like to propose a model for the existence of a positive autofeedback regulatory mechanism of N-terminal PTH on its own secretion. PTH is secreted by parathyroid glands, there are PTH/PTHrP receptors on the parathyroid cells [18] and PTH is an equipotent ligand to PTHrP for this receptor [20]. Thus, our hypothesis is that PTH in a hypocalcemic condition enhances its own secretion by a paracrine/autocrine mechanism. It has, however, to be stated that the in vivo models will not exactly reflect the events at the paracrine/autocrine level. The possible existence of such a positive feedback will allow for explanation of several phenomena in the parathyroid glands. The suppression of PTH secretion by Ca⫹⫹ is mediated via activation of the calcium-sensing receptor on the parathyroid cell surface [27]. How low Ca⫹⫹ stimulates PTH release from the parathyroid cells remains obscure.
Our observation, that activation of the PTH/PTHrP receptor increases PTH secretion by several fold, but only during hypocalcemia, suggests a possibility for amplification of PTH release by PTH itself, when an increased level is needed. Cultured parathyroid cells in contact with others parathyroid cells have been shown to secrete more PTH than isolated parathyroid cells [28]. This result has not been explained, but could be mediated by PTH from one cell stimulating further release from the neighboring cells. Finally, a transient nature of the initial increase of PTH levels in response to a reduction in plasma Ca⫹⫹ has been described in human subjects and in rats [1, 29]. A rapid and pronounced decrease in plasma Ca⫹⫹ is followed by an initial rapid increase in plasma PTH level, which soon declines to a new lower level, despite a continuous fall in plasma Ca⫹⫹. It is reasonable to speculate that a positive feedback would cause augmented secretion during the initial stage of elevated PTH levels giving rise to a greater bolus of PTH release. Then, other inhibitory factors must eventually take over to suppress the PTH release. As such, several inhibitory paracrine/autocrine factors have been proposed (e.g., chromogranin A–related peptides and endothelin-1) [30–32]. Autoreceptor effects on hormone or neurotransmitter
Lewin et al: PTHrP and PTH secretion in uremic rats
secretion are well known. Most autoreceptors mediate a negative feedback on the secretion. We are, however, not alone with the theory of the existence of a positive autoregulatory feedback mechanism relating to PTH, as the beta cell insulin system appears to be a rare example of such a positive feedback of insulin on the secretion of insulin [33–37]. A possible link between impaired insulin secretion and insulin resistance at the level of the pancreas has been suggested as a mechanism involved in the development of type 2 diabetes [34, 35]. We speculated that an eventual disturbed response to the PTH/PTHrP receptor ligand might be an additional factor in the increased PTH secretion in secondary HPT due to uremia. The present results, however, contradict such a possibility as the increase in PTH secretion in response to PTHrP was proportionally the same in uremic rats on a standard diet and normal rats. The pattern of the continued and progressive increase in the high phosphorus uremic rats given PTHrP seems to be different. The explanation for this continued rise of PTH in that group is not clear from the present investigation. The relative secretory capacity of the parathyroid glands is in the present investigation shown to be severely diminished in uremic animals. The physiologic importance of this observation is at present not clear. The fluctuation of plasma PTH levels is possibly of importance for maintaining the sensitivity of bone cells to PTH and for maintaining normal bone metabolism [38–40]. An abnormal fluctuation of plasma PTH levels has been described in patients with metabolic bone diseases, as such diminished circadian fluctuations of PTH have been reported in patients with age-related osteoporosis [38] and osteoporotic subjects exhibited a disturbed pulse amplitude and frequency modulation of PTH secretion with smaller and less pulses [41]. Altered calcium-modulated oscillatory PTH secretion pattern has also been shown in patients with secondary HPT with increased spontaneous PTH burst frequency, but with blunted responsiveness to changes in Ca⫹⫹[42]. Treatment of osteoporosis with intermittent administration of PTH has been shown to have an anabolic effect on the skeleton [43, 44]. We have demonstrated, that the relative secretory capacity is practically 0 in uremic, severely hyperparathyroid rats. In uremic rats kept on a high phosphorus diet, PTH levels were already at the basal condition at the upper part of the calcium/PTH curve, and induction of even severe hypocalcemia was not able further to stimulate PTH secretion. In the present study it is, however, clearly shown that even in these rats with severe HPT it was possible further to stimulate PTH levels, as PTHrP enhanced the levels of PTH significantly. Thus, the impaired secretory capacity can be expanded, and there is a potential for induction of fluctuation of circulating PTH even in severe secondary HPT. The present results furthermore clearly showed that
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also in the case of uremia is the level of PTH, which has been considered as the maximal secretory activity of the parathyroid cells, in fact, not the maximum, and that the maximal PTH secretion can be enhanced even further by several fold. As such, the present results oppose the common assumption, that the maximal PTH secretion is an expression of the parathyroid mass [45]. The secretory response of the parathyroid glands to hypocalcemia is a tightly regulated phenomenon, which is mediated via a combined influence of extracellular Ca⫹⫹ and a spectrum of paracrine and autocrine factors [45], which might include peptides targeting the PTH/PTHrP receptor. The hypercalcemia of malignancy in human subjects arises from an inappropriate activation of the renal and bone PTH/PTHrP receptors by PTHrP, which is generated by the malignant cells. In this condition the PTH secretion from the parathyroid glands is suppressed. When plasma PTH levels are elevated, a concomitant primary HPT is considered. In a previous study, we have shown that PTH levels were suppressed during hypercalcemia despite PTHrP administration [1]. This is in agreement with the clinical findings with the hypercalcemia of malignancy. CONCLUSION PTHrP enhanced significantly the low Ca⫹⫹-stimulated PTH secretion in vivo in a dose-related manner in normal rats. The impaired secretory capacity of the parathyroid glands in uremic rats was significantly enhanced by PTHrP. The presence of the PTH/PTHrP receptor is demonstrated in rat parathyroid glands in the present investigation. An autocrine/paracrine role of PTH/PTHrP receptor targeting peptides in the parathyroid glands is suggested. Thus, PTH might during hypocalcemia have a positive auto-feedback regulatory role on its own secretion. ACKNOWLEDGMENTS This study was supported in part by the Danish Research Council with grant no. 28809/9601595 and in part by Danish Kidney Association. We would like to express our thanks to Kirsten Bang for her technical assistance. Reprint requests to Ewa Lewin, M.D., Nephrological Department B, University of Copenhagen, Herlev hospital, 75 Herlev Ringvej, DK2730 Herlev, Denmark. E-mail: [email protected]
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Lewin et al: PTHrP and PTH secretion in uremic rats roles of parathyroid hormone-related protein in normal physiology. Physiol Rev 76:127–173, 1996 Orloff JJ, Reddy D, Papp AE de, et al: Parathyroid hormonerelated protein as a prohormone: Posttranslational processing and receptor interactions. Endocr Rev 15:40–60, 1994 Kronenberg HM, Lee K, Lanske B, Segre GV: Parathyroid hormone-related protein and Indian hedgehog control the pace of cartilage differentiation. J Endocrinol 154:S39–S45, 1997 Rodda CP, Kubota M, Heath JA, et al: Evidence for a novel parathyroid hormone-related protein in fetal lamb parathyroid glands and sheep placenta: Comparison with a similar protein implicated in humoral hypercalcemia of malignancy. J Endocrinol 117:261–271, 1988 Karaplis AC, Vautour L: Parathyroid hormone-related peptide and the parathyroid hormone-related peptide receptor in skeletal development. Curr Opin Nephrol Hypertens 6:308–313, 1997 Robinson NR, Sibley CP, Mughal MZ, Boyd RD: Fetal control of calcium transport across the rat placenta. Pediatr Res 26:109–115, 1989 MacIssac RJ, Heath JA, Rodda CP, et al: Role of the fetal parathyroid glands and parathyroid hormone-related protein in the regulation of placental transport of calcium, magnesium and inorganic phosphate. Reprod Fertil Dev 3:447–457, 1991 Moseley JM, Hayman JA, Danks JA, et al: Immunohistochemical detection of parathyroid hormone-related protein in human fetal epithelia. J Clin Endocrinol Metab 73:478–484, 1991 Massfelder T, Fiaschi-Taesch N, Stewart AF, Helwig JJ: Parathyroid hormone-related peptide—A smooth muscle tone and proliferation regulatory protein. Curr Opin Nephrol Hypert 7:27–32, 1998 Aya K, Tanaka H, Ichinose Y, et al: Expression of parathyroid hormone-related peptide messenger ribonucleic acid in developing kidney. Kidney Int 55:1696–1703, 1999 Ikeda K, Weir EC, Mangin M, et al: Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas. Mol Endocrinol 2:1230–1236, 1988 Martin TJ, Moseley JM, Williams ED: Parathyroid hormonerelated protein: Hormone and cytokine. J Endocrinol 154:S23–S37, 1997 Connor CS, Drees BM, Thurston A, et al: Bovine parathyroid issue: A model to compare the biosynthesis and secretion of parathyroid hormone and parathyroid hormone-related peptide. Surgery 106:1057–1062, 1989 Connor C, Drees B, Hamilton J: Parathyroid hormone-like peptide and parathyroid hormone are secreted from bovine parathyroid via different pathways. Biochim Biophys Acta 1178:81–86, 1993 Danks JA, Ebeling PR, Hayman JA, et al: Immunohistochemical localisation of parathyroid hormone-related protein in parathyroid adenoma and hyperplasia. J Pathol 161:27–33, 1990 Matsushita H, Hara M, Endo Y, et al: Proliferation of parathyroid cells negatively correlates with expression of parathyroid hormonerelated protein in secondary parathyroid hyperplasia. Kidney Int 55:130–138, 1997 Matsushita H, Usui M, Hara M, et al: Co-secretion of parathyroid hormone and parathyroid-hormone-related protein via a regulated pathway in human parathyroid adenoma cells. Am J Pathol 150:861–871, 1997 Mannstadt M, Juppner H, Gardella TJ: Receptors for PTH and PTHrP: Their biological importance and functional properties. Am J Physiol 277:F665–F675, 1999 Felsenfeld AF, Llach F: Parathyroid gland function in chronic renal failure. Kidney Int 43:771–789, 1993 Lewin E, Wang W, Olgaard K: Reversibility of experimental secondary hyperparathyroidism. Kidney Int 52:1232–1241, 1997 Grant FD, Conlin PR, Brown EM: Rate and concentration dependence of parathyroid hormone dynamics during stepwise changes in serum ionised calcium in normal humans. J Clin Endocrinol Metab 71:370–377, 1990 Schwarz P: Dose response dependency in regulation of acute PTH (1–84) release and suppression in normal humans: A citrate and calcium infusion study. Scand J Clin Lab Invest 53:601–605, 1993
25. Schmitt CP, Huber D, Mehls O, et al: Altered instantaneous and calcium-modulated oscillatory PTH secretion patterns in patients with secondary hyperparathyroidism. J Am Soc Nephrol 9:1832– 1844, 1998 26. Rosenblatt M, Kronenberg HM, Pitts JT: Parathyroid hormone, in Endocrinology (vol 2), edited by DeGroot LJ, Philadelphia, WB Saunders Company, 1989, pp 848–891 27. Brown EM, Gamba G, Riccardi D, et al: Cloning and characterization of an extracellular Ca(2⫹)-sensing receptor from bovine parathyroid. Nature 366:575–580, 1993 28. Nielsen PK, Feldt-Rasmussen U, Olgaard K: A direct effect in vitro of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells. Nephrol Dial Transplant 11:1762–1768, 1996 29. Schwarz P, Sorensen HA, Transbol I, McNair P: Regulation of acute parathyroid hormone release in normal humans: Combined calcium and citrate clamp study. Am J Physiol 263:E195–E198, 1992 30. Fascitto BH, Gorr S-U, Bourdeau AM, Cohn DV: Autocrine regulation of parathyriod secretion: inhibition of secretion by chromogranin-A (secretory protein-1) and potentiation of secretion by chromogranin-A and pancreastatin antibodies. Endocrinology 127:1329–1335, 1990 31. Fascitto BH, Gorr S-U, DeFranco DJ, et al: Pancreastatin, a presumed product of chromogranin-A (secretory protein-1) processing, inhibits secretion from porcine parathyroid cells in culture. Endocrinology 125:1617–1622, 1989 32. Fujii Y, Moreira JE, Orlando C, et al: Endothelin as autocrine factor in the regulation of parathyroid cells. Proc Natl Acad Sci USA 88:4235–4239, 1991 33. Xu GG, Rothenberg PL: Insulin receptor signaling in the beta-cell influences insulin gene expression and insulin content: Evidence for autocrine beta-cell regulation. Diabetes 47:1243–1252, 1998 34. Aspinwall CA, Lakey JRT, Kennedy RT: Insulin-stimulated insulin secretion in single pancreatic beta cells. J Biol Chem 274:6360– 6365, 1999 35. Saltiel AR, Kahn CR: Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414:799–806, 2001 36. Jackerott M, Baudry A, Lamothe B, et al: Endocrine pancreas in insulin receptor-deficient mouse pups. Diabetes 50:S146–S149, 2001 37. Leibiger B, Leibiger IB, Moede T, et al: Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic beta cells. Mol Cell 7:559–570, 2001 38. Hesch RD, Brabant G, Rittinghaus EF, et al: Pulse amplitude and frequency modulation of PTH and its modulation of PTH receptors—Osteoporosis as an example of dynamic disease, in New Actions of Parathyroid Hormone, edited by Massry SG, Fugita T, New York, Plenum Press, 1989, p 51 39. Harms HM, Kaptaina U, Kulpmann WR, et al: Pulse amplitude and frequency modulation of parathyroid hormone in plasma. J Clin Endocrinol Metab 69:843–851, 1989 40. Schmitt CP, Schaefer F, Bruch A, et al: Control of pulsatile and tonic parathyroid hormone secretion by ionized calcium. J Clin Endocrinol Metab 81:4236–4243, 1996 41. Fraser WD, Logue FC, Christie JP, et al: Alteration of the circadian rhythm of intact parathyroid hormone and serum phosphate in women with established postmenopausal osteoporosis. Osteoporos Int 8:121–126, 1998 42. Schmitt CP, Huber D, Mehls O, et al: Altered instantaneous and calcium-modulated oscillatory PTH secretion patterns in patients with secondary hyperparathyroidism. J Am Soc Nephrol 9:1832– 1844, 1998 43. Fujita T: Parathyroid hormone in the treatment of osteoporosis. BioDrugs 15:721–728, 2001 44. Dempster DW, Cosman F, Kurland ES, et al: Effects of daily treatment with parathyroid hormone on bone microarchitecture and turnover in patients with osteoporosis: A paired biopsy study. J Bone Miner Res 16:1846–1853, 2001 45. Lewin E, Nielsen PK, Olgaard K: The calcium/parathyroid hormone concept of the parathyroid glands. Curr Opin Nephrol Hypertens 4:324–333, 1995
Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats EWA LEWIN, BARTOLOME GARFIA, YOLANDA ALMADEN, MARIANO RODRIGUEZ, and KLAUS OLGAARD Nephrological Department B, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark; Nephrological Department P, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; and Unidad de Investigacio´n, Hospital Universitario Reina Sofia, Cordoba, Spain
were significantly increased. The CRF rats on high phosphorus diet had significant hypocalcemia (Ca⫹⫹, 1.04 ⫾ 0.02 mmol/L vs. 1.28 ⫾ 0.03 mmol/L, P ⬍ 0.001), hyperphosphatemia (3.48 ⫾ 0.3 mmol/L vs. 2.25 ⫾ 0.1 mmol/L, P ⬍ 0.001) and severe secondary HPT, PTH (984 ⫾ 52 pg/mL vs. 226 ⫾ 32 pg/mL, P ⬍ 0.001) compared to CRF rats on a standard phosphorus diet. The maximal PTH response to hypocalcemia was enhanced in CRF rats (maximum PTH 382 ⫾ 58 pg/mL vs. 196 ⫾ 29 pg/mL in normal rats, P ⬍ 0.01) and further enhanced by PTHrP 1-40 to 826 ⫾ 184 pg/mL (P ⬍ 0.01). The secretory capacity of the parathyroid glands in response to low Ca⫹⫹ was severely diminished in uremia. In CRF rats given a high phosphorus diet, the basal PTH levels were at the upper part of the calcium/PTH curve, and the induction of more marked hypocalcemia did not stimulate PTH secretion further (maximum PTH 1475 ⫾ 208 pg/mL vs. basal 1097 ⫾ 69 pg/mL, NS). PTHrP, however, further enhanced the maximal PTH levels significantly (maximum PTH 3142 ⫾ 296 pg/mL, P ⬍ 0.01). The presence of the PTH/PTHrP receptor in the rat parathyroid glands was confirmed by RT-PCR technique. Conclusion. PTHrP enhanced significantly, in a dose-related manner, the low Ca⫹⫹-stimulated PTH secretion in normal rats. The PTH/PTHrP receptor is present in rat parathyroid glands. The impaired secretory capacity of the parathyroid glands in uremic rats is significantly enhanced by PTHrP. An autocrine/paracrine role in the parathyroid glands of the PTH/ PTHrP receptor targeting peptides, PTHrP and PTH, is suggested. Thus, it is hypothesized that PTH during hypocalcemia might have a positive auto-feedback regulatory role on its own secretion.
Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats. Background. The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/ paracrine factors. We have previously shown that N-terminal parathyroid hormone-related protein (PTHrP) enhanced the secretory PTH response to low calcium in vivo and in vitro in rat parathyroid glands. N-terminal PTHrP fragments are equipotent to N-terminal PTH as ligands for the PTH/PTHrP receptor that is demonstrated in parathyroid tissue. This supports the possibility that the parathyroid cells respond to PTH/ PTHrP receptor ligands and as such are target for an autoregulatory action of PTH and PTHrP. Our aim was to search for the PTH/PTHrP receptor in rat parathyroid glands and to examine the effects of PTHrP 1-40 on PTH secretion in in vivo models of secondary hyperparathyroidism (HPT) in uremic rats. Methods. PTH secretion was examined during ethyleneglycol tetraacetic acid (EGTA)-induced hypocalcemia both with and without PTHrP. Five groups, each of six normal rats, received a bolus of increasing doses of 0.1, 1.0, 10, and 100 g of PTHrP 1-40, or vehicle only. Chronic renal failure (CRF) was induced by 5/6 nephrectomy. One group of 12 CRF rats received a standard diet, while another CRF group of 18 rats received a high phosphorus diet to induce more severe HPT. After 8 weeks of uremia, the rats were infused with EGTA and PTHrP 1-40 or vehicle. The presence of the PTH/PTHrP receptor in the rat parathyroid glands was examined by reverse transcription-polymerase chain reaction (RT-PCR) technique. PTH was measured by a rat PTH assay that does not crossreact with PTHrP. Results. In a dose-related manner, PTHrP enhanced the PTH response to hypocalcemia in normal rats. A similar rate of decrease of plasma Ca⫹⫹ was induced by EGTA in all experimental groups. In CRF rats, plasma creatinine (0.99 ⫾ 0.10 mmol/L vs. 0.33 ⫾ 0.01 mmol/L, P ⬍ 0.001) and plasma PTH (226 ⫾ 32 pg/mL vs. 69 ⫾ 16 pg/mL, P ⬍ 0.001) levels
It has previously been shown by our group, that N-terminal parathyroid hormone-related protein (PTHrP) 1-40 and 1-86, used as surrogate for parathyroid hormone (PTH), enhance the secretory response of PTH to hypocalcemia in rats in vivo [1]. This effect of PTHrP was immediate, taking place within 5 minutes. No effect of PTHrP was found in vivo during normo- and hypercalcemia. In that investigation, it was ensured that the peripheral metabolism of PTH was not affected by PTHrP and that there was no cross-reactivity of PTHrP in the rat PTH assay [1]. A direct effect of PTHrP on the rat
Key words: secondary hyperparathyroidism, calcium/PTH curve, autocrine-paracrine factor, PTHrP, PTH/PTHrP receptor, uremia. Received for publication October 18, 2002 and in revised form December 16, 2002, and January 28, 2003 Accepted for publication February 24, 2003
2003 by the International Society of Nephrology
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parathyroids was confirmed by the demonstration in vitro that low Ca⫹⫹-induced PTH secretion was augmented by PTHrP [1]. These results support the possibility that the parathyroid cells respond to PTH/PTHrP receptor ligands. PTH and PTHrP might as equipotent ligands for the receptor have regulatory functions on the secretion of PTH. The mature PTHrP protein contains a limited region of sequence homology with PTH in its immediate N-terminal region. PTH and PTHrP activate a common G-protein coupled receptor, the PTH/PTHrP type 1 receptor [2–4] and PTHrP was used as a surrogate for PTH because it mimics the action of PTH by acting through the PTH/ PTHrP receptor. PTHrP is expressed in a wide variety of normal fetal and adult tissues [5–13]. The PTH/PTHrP receptor is frequently expressed in cells that produce PTHrP or in cells immediately adjacent to PTHrP-producing cells. This spatial proximity, together with the fact that little, if any, PTHrP circulates under normal physiologic conditions have led to the view that PTHrP is a locally acting autocrine/paracrine factor [3, 14]. PTHrP mRNA is expressed in normal human and bovine parathyroid tissues [13, 15, 16]. The expression of PTHrP in the parathyroid glands is not only a feature of neoplastic transformation, although abnormal expression of PTHrP mRNA and protein has been found in human parathyroid adenomas, hyperplasia, and carcinomas [13, 17–19], but it could be a feature of normal physiology, as well. PTH and PTHrP have been demonstrated to be colocalized in the same secretory granules and to be secreted simultaneously from parathyroid adenomas [19]. The PTH/PTHrP receptor has been demonstrated in human parathyroid tissue [18]. However, the physiologic role of PTHrP in the parathyroid glands remains to be established [3]. N-terminal PTHrP fragments are equipotent to N-terminal PTH fragments as ligands for the PTH/PTHrP receptor [20]. Therefore, in the present study, N-terminal PTHrP was used as a surrogate for N-terminal PTH due to the properties of the rat PTH assay, which comeasures the N-terminal PTH fragments (i.e., rat, human, and bovine PTH), but not PTHrP. The uremic condition is associated with disturbances in calcium, phosphorus, and PTH metabolism leading to development of secondary hyperparathyroidism (HPT). The Ca⫹⫹/PTH relationship is abnormal in uremia; the PTH secretion, at the basal condition, is toward the upper part of the Ca⫹⫹/ PTH curve, and the maximal secretory response to hypocalcemia is often increased [21]. The aim of present investigation therefore was to search for the PTH/PTHrP receptor in the parathyroid glands and to examine the effects of PTHrP 1-40 on PTH secretion in in vivo models of secondary HPT in uremic rats.
METHODS Animals Inbred male Dark Aguti (DA) rats (Moellegaarden, Denmark) were used, weighing 200 to 225 g at the start of the study. The experimental studies on the rats were performed in accordance with the NIH Guidelines for Care and Use of Laboratory Animals and was approved by our institution. The experimental procedures were performed in rats under anesthesia with 50 g/kg intraperitoneal injection of pentobarbital (Mebumal). Uremia Chronic renal failure (CRF) was induced by one-step 5/6 nephrectomy. A group of 12 CRF rats was given a standard diet containing 0.9% calcium, 0.7% phosphorus, and 1000 IU vitamin D/kg. To induce more severe HPT, another group of 18 CRF rats was given a high phosphorus diet containing 0,9% calcium, 1.5% phosphorus, and 1000 IU vitamin D/kg. The duration of uremia was 8 weeks. A group of 12 normal control rats was given a standard diet. Rats were allowed free access to food and water. Peptide PTHrP 1-40 was obtained from Saxon Biochemicals, GMBH, Hannover, Germany. PTHrP was first dissolved in deionized water to a concentration of 1 mg/mL and then diluted in saline to the concentration needed. Induction of hypocalcemia Hypocalcemia was induced by infusion of 30 mmol/L ethyleneglycol tetraacetic acid (EGTA), 3 mL/hour through a catheter inserted in the femoral vein. Samples for determination of plasma PTH and plasma Ca⫹⫹ were taken at 0, 5, 10, 20, 30, 40, and 50 minutes from a corresponding catheter in the femoral artery. The sample volume of 0.8 mL was replaced by 0.8 mL of saline, resulting in the same weight of the rats before and after the experiments. Maximum PTH is the highest PTH value obtained during induction of hypocalcemia. The relative secretory capacity is defined as the relative increase in PTH secretion from baseline in response to an acute induction of hypocalcemia, expressed in percent of the basal PTH level. Experimental protocols Dose-response study. At time 0, one of five groups, each with six normal rats, received vehicle or an intravenously bolus dose of 0.1, 1.0, 10, or 100 g of PTHrP 1-40. Hypocalcemia was then induced by an infusion of 30 mmol/L EGTA and samples for determination of plasma PTH and plasma Ca⫹⫹ were taken as described above.
Lewin et al: PTHrP and PTH secretion in uremic rats
The effect of PTHrP on low Ca⫹⫹-stimulated PTH secretion in vivo in uremic rats kept on standard diet. A group of uremic rats received a bolus of 1 mL of 100 g/mL of PTHrP 1-40 at time 0 (N ⫽ 6). Another group of uremic rats received 1 mL of vehicle only (N ⫽ 6). Hypocalcemia was then induced and samples for determination of plasma PTH and plasma Ca⫹⫹ were taken as described above. The effect of PTHrP on low Ca⫹⫹-stimulated PTH secretion in vivo in uremic rats kept on a high phosphorus diet. A group of uremic hyperphosphatemic rats received a bolus of 1 mL of 100 g/mL of PTHrP 1-40 at time 0 (N ⫽ 9). Another group of uremic hyperphosphatemic rats received 1 mL of vehicle only (N ⫽ 9). Then, hypocalcemia was induced and samples for determination of plasma PTH and plasma Ca⫹⫹ were taken as described above. Parathyroid PTH/PTHrP receptor—RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Two rat parathyroid glands and a small slice of kidney cortex (1 mm3) were obtained for total RNA extraction. Tissue samples were separately placed in nuclease-free 1.5 mL microcentrifuge tubes. Then, 400 L of a lysis/ binding buffer from a commercial kit for tissue total RNA extraction (High Pure RNA Tissue Kit; Roche, Mannheim; Germany) were added to the samples. The tissues (parathyroid and kidney cortex) were ultrasonicated for 5 minutes at 4⬚C to allow for complete cell rupture. Subsequently, tissues were homogenized by passing the lysate 10 to 20 times through a 20 gauge needle attached to a sterile plastic syringe until a homogeneous lysate was achieved. Then, total RNA was extracted using the High Pure RNA Tissue Kit. Total RNA was quantified by spectrophotometry. The PTH/PTHrP receptor in parathyroid glands and kidney cortex were amplified using the kit, Access RT-PCR System (Promega, Madison WI, USA) using a specific primer and 100 ng of kidney cortex total RNA and 200 ng of parathyroid total RNA per sample. Kidney cortex and parathyroid PTH/PTHrP receptor-mRNA were amplified at 24 and 40 cycles, respectively. DNA amplifications were processed by a Genetic Analyzer Abi Prism 310 (Perkin Elmer, Foster City, CA, USA). Data were analyzed using specific software Gene Scan version 3.1/1998 (Perkin Elmer). The PTH/PTHrP receptor primers were: PTH/PTHrP receptor sense CGGAAGCTGCTCAAATCC PTH/PTHrP receptor antisense AAAAATCCCTGGAAG
5⬘-FAM-TAC 5⬘-GCGACA
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Plasma measurements Plasma creatinine, urea, phosphorus, and total calcium were measured by an ETACHEM 250 Analyzer (Kodak, Rochester, NY, USA). Plasma PTH was measured by a rat PTH immunoradiometric assay (IRMA) from Immunotopics, San Clemente, CA, USA. The intra-assay coefficient of variance in our laboratory was 4% and interassay coefficient of variance 5%. PTHrP 1-40 did not cross-react with the rat PTH assay. No cross-reactivity with C-terminal PTH fragments was found in our laboratory [1, 22]. Plasma Ca⫹⫹ at actual pH was measured by a calcium-selective electrode (Radiometer, Copenhagen, DK). Statistics The results are expressed as mean ⫾ SEM. MannWhitney test or t test were used for comparison between groups. P ⬍ 0.05 is considered significant. RESULTS In uremic rats, plasma creatinine and urea levels were significantly increased (P ⬍ 0.001), as shown in Table 1. The uremic rats kept on a high phosphorus diet developed significant hypocalcemia (P ⬍ 0.001), hyperphosphatemia (P ⬍ 0.001), and severe secondary HPT (P ⬍ 0.001), as compared to normal rats and uremic rats given a standard diet (Table 1). The effect in vivo of increasing doses of PTHrP 1-40 on the low calcium–induced PTH secretion in the rat is shown in Figure 1A. This effect was clearly dose-related with increasing doses of PTHrP resulting in an enhanced PTH response to hypocalcemia (P ⬍ 0.01). The rates of reduction of plasma Ca⫹⫹ levels were not different between the experimental groups (Fig. 1B). In order to ensure the presence of the PTH/PTHrP receptor in the parathyroid glands, three sets of RTPCR measurements were performed in parathyroid glands, using normal kidney tissue as control. These investigations clearly demonstrated the presence of the PTH/PTHrP receptor in the parathyroid glands as well as in the kidneys, as shown in Figure 2. The relative secretory capacity of the parathyroid glands in normal rats, uremic rats kept on a standard diet, and uremic rats kept on a high phosphorus diet is shown in Figure 3. The relative secretory capacity was significantly decreased (P ⬍ 0.05) in uremia and practically zero in uremic rats kept on a high phosphorus diet. The PTH secretory response to PTHrP and hypocalcemia in normal rats and in uremic rats kept on a standard diet is shown in Figure 4. In uremic rats, the PTH levels induced by hypocalcemia were significantly enhanced compared to that of normal rats and this response was further significantly enhanced by PTHrP (P ⬍ 0.01). In normal control rats, PTH increased from a basal PTH
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Lewin et al: PTHrP and PTH secretion in uremic rats Table 1. Plasma parameters of normal and uremic rats
Normal N ⫽ 12 Chronic renal failure standard diet N ⫽ 12 Chronic renal failure high phosphorus diet N ⫽ 18 a
Creatinine lmol/L
Urea mmol/L
Ca⫹⫹ mmol/L
Phosphorus mmol/L
Parathyroid hormone pg/mL
33 ⫾ 1 99 ⫾ 10a 86 ⫾ 5a
6⫾1 26 ⫾ 9a 16 ⫾ 2a
1.26 ⫾ 0.04 1.28 ⫾ 0.03a 1.04 ⫾ 0.02a,b
1.85 ⫾ 0.1 2.25 ⫾ 0.1 3.48 ⫾ 0.3a,b
69 ⫾ 16 226 ⫾ 32a 984 ⫾ 52a,b
P ⬍ 0.001 versus normal rats; b P ⬍ 0.001 versus chronic renal failure standard diet rats
Fig. 1. Effect of increasing doses of N-terminal parathyroid hormonerelated protein (PTHrP) 1-40 on the secretory response of PTH to an acute induction of hypocalcemiain normal rats. Hypocalcemia (A ) was induced by an ethyleneglycol tetraacetic acid (EGTA) infusion. The rate of reduction of plasma Ca⫹⫹ was similar in all groups of rats (B ). A bolus of PTHrP 1-40 or vehicle was injected at time 0. PTHrP 1-40 enhanced significantly (P ⬍ 0.01) in a dose-related manner the low Ca⫹⫹-stimulated PTH secretion. N ⫽ 6 in each group, mean ⫾ SEM.
level of 61 ⫾ 22 pg/mL to a maximum PTH level of 196 ⫾ 29 pg/mL in the vehicle-treated group and from a basal PTH level of 78 ⫾ 25 pg/mL to a maximum PTH level of 488 ⫾ 68 pg/mL in the PTHrP-treated group. In uremic rats, PTH increased from a basal PTH level of 242 ⫾ 45 pg/mL to a maximum PTH level of 382 ⫾ 58 pg/mL in the vehicle-treated group and from a basal PTH level of 209 ⫾ 50 pg/mL to a maximum PTH level of 826 ⫾ 184 pg/mL in the PTHrP-treated group. The proportion of the increment in PTH secretion in re-
Fig. 2. Representative electropherograms of parathyroid hormone/ parathyroid hormone-related protein (PTH/PTHrP) receptor mRNAs in three different samples of normal rat kidney cortex and parathyroid glands.
sponse to PTHrP compared to that of hypocalcemia alone was, however, the same in normal and uremic rats. Thus, in normal rats maximal PTH was increased by 249% by PTHrP, while in uremic rats PTHrP increased the PTH response by 216% (NS).
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Fig. 3. The relative secretory capacity of the parathyroid glands in normal rats, uremic rats kept on normal diet [chronic renal failure (CRF)], and uremic rats kept on high phosphorus diet [CRF high phosphorus (P)]. The relative secretory capacity is defined as the relative increase in parathyroid hormone (PTH) secretion from baseline in response to an acute induction of hypocalcemia, and expressed in percent of basal PTH levels. The relative secretory capacity is significantly decreased in uremia (P ⬍ 0.05) and practically 0 in uremic rats kept on high phosphorus diet. N ⫽ 6 to 9, mean ⫾ SEM.
The PTH secretory response to PTHrP and hypocalcemia in uremic rats kept on a high phosphorus diet is shown in Figure 5. In uremic rats given a high phosphorus diet, the PTH levels, at the basal condition, were already at the upper part of the calcium/PTH curve. Induction of even severe hypocalcemia was not able to stimulate PTH secretion further. PTHrP administration, however, enhanced the PTH levels significantly (P ⬍ 0.001) and the continuous increase strongly suggests that a maximum was not even obtained in the present experimental design. The relative secretory capacity of PTH secretion was significantly increased by PTHrP administration to uremic rats kept on a high phosphorus diet (P ⬍ 0.01), as shown in Figure 6. DISCUSSION In the present investigation it is shown that N-terminal PTHrP administered in vivo in a dose-related manner enhanced the secretory response of PTH to hypocalcemia in rats. In uremic rats, the maximal PTH secretory response to low calcium was significantly enhanced compared to that of normal rats, and this response was further increased several fold by the administration of PTHrP. It has previously been demonstrated that the maximal PTH secretory response is dependent upon the rate of fall of plasma Ca⫹⫹ [23, 24]. In the present investigation, the rate of decrease in plasma Ca⫹⫹ was well controlled by the infusion of the calcium chelator and
Fig. 4. The effect of parathyroid hormone-related protein (PTHrP) 1-40 on the secretory response of parathyroid hormone (PTH) to an acute induction of hypocalcemia in uremic rats kept on a standard diet (- - -) and in normal rats (______) (A ). Hypocalcemia was induced by an ethyleneglycol tetraacetic acid (EGTA) infusion. The rate of reduction of plasma Ca⫹⫹ was similar in all groups of rats (B). A bolus of PTHrP 1-40 or vehicle was injected at time 0. PTHrP significantly enhanced the low Ca⫹⫹-stimulated PTH secretion in both group of rats (P ⬍ 0.01). N ⫽ 6 in each group, mean ⫾ SEM. CRF is chronic renal failure.
the rate of decrease was similar in the group of rats infused with PTHrP and in the control group of rats. Therefore, differences in the degree of hypocalcemia cannot explain the difference obtained in the response of PTH secretion to hypocalcemia. We have previously shown in normal rats that the peripheral metabolism of PTH was not affected by PTHrP. In uremia, however, an influence of PTHrP on the peripheral metabolism of PTH cannot be completely excluded. In general, elevated circulating levels of PTH in uremic HPT might be an expression of both a marked increase in glandular secretion and of a reduced elimination of PTH [25], which is of importance for the interpretation of the results obtained in vivo. The parathyroid glands are not controlled by a superior hypothalamic-pituitary axis, as it most often is observed in other endocrine glands [26]. Therefore, the parathyroid glands are likely to use autocrine/paracrine regulatory mechanisms. The PTH/PTHrP receptor was in the present investigation demonstrated in rat parathy-
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Fig. 5. The Caⴙⴙ/parathyroid hormone (PTH) relationship during ethyleneglycol tetraacetic acid (EGTA)-induced hypocalcemia in uremic rats kept on a high phosphorus diet and receiving a bolus injection of parathyroid hormone-related protein (PTHrP) 1-40 or vehicle. The arrow depicts the direction of change of plasma Ca⫹⫹. In the vehicletreated rats, induction of severe hypocalcemia did not further enhance PTH levels. Administration of PTHrP 1-40 did, however, enhance the PTH levels significantly (P ⬍ 0.001). N ⫽ 9 in each group, mean ⫾ SEM.
Fig. 6. The relative secretory capacity of the parathyroids in uremic rats kept on a high phosphorus diet after administration of parathyroid hormone-related protein (PTHrP) 1-40. The relative secretory capacity is defined as the relative increase in parathyroid hormone (PTH) secretion from baseline in response to an acute induction of hypocalcemia, and expressed in percent of basal PTH levels. The relative secretory capacity is significantly increased by PTHrP (P ⬍ 0.01). N ⫽ 9 in each group, mean ⫾ SEM. EGTA is ethyleneglycol tetraacetic acid.
roid glands and has previously been demonstrated in human parathyroid tissue [18]. This supports the possibility that PTHrP, as a ligand for the receptor, might regulate PTH secretion, but also that PTH-producing parathyroid cells are target for an autoregulatory action of N-terminal PTH. On the basis of the present results, which showed that N-terminal PTHrP very fast, within 5 minutes, and in a dose-related manner enhanced the PTH secretory response to hypocalcemia, we would like to propose a model for the existence of a positive autofeedback regulatory mechanism of N-terminal PTH on its own secretion. PTH is secreted by parathyroid glands, there are PTH/PTHrP receptors on the parathyroid cells [18] and PTH is an equipotent ligand to PTHrP for this receptor [20]. Thus, our hypothesis is that PTH in a hypocalcemic condition enhances its own secretion by a paracrine/autocrine mechanism. It has, however, to be stated that the in vivo models will not exactly reflect the events at the paracrine/autocrine level. The possible existence of such a positive feedback will allow for explanation of several phenomena in the parathyroid glands. The suppression of PTH secretion by Ca⫹⫹ is mediated via activation of the calcium-sensing receptor on the parathyroid cell surface [27]. How low Ca⫹⫹ stimulates PTH release from the parathyroid cells remains obscure.
Our observation, that activation of the PTH/PTHrP receptor increases PTH secretion by several fold, but only during hypocalcemia, suggests a possibility for amplification of PTH release by PTH itself, when an increased level is needed. Cultured parathyroid cells in contact with others parathyroid cells have been shown to secrete more PTH than isolated parathyroid cells [28]. This result has not been explained, but could be mediated by PTH from one cell stimulating further release from the neighboring cells. Finally, a transient nature of the initial increase of PTH levels in response to a reduction in plasma Ca⫹⫹ has been described in human subjects and in rats [1, 29]. A rapid and pronounced decrease in plasma Ca⫹⫹ is followed by an initial rapid increase in plasma PTH level, which soon declines to a new lower level, despite a continuous fall in plasma Ca⫹⫹. It is reasonable to speculate that a positive feedback would cause augmented secretion during the initial stage of elevated PTH levels giving rise to a greater bolus of PTH release. Then, other inhibitory factors must eventually take over to suppress the PTH release. As such, several inhibitory paracrine/autocrine factors have been proposed (e.g., chromogranin A–related peptides and endothelin-1) [30–32]. Autoreceptor effects on hormone or neurotransmitter
Lewin et al: PTHrP and PTH secretion in uremic rats
secretion are well known. Most autoreceptors mediate a negative feedback on the secretion. We are, however, not alone with the theory of the existence of a positive autoregulatory feedback mechanism relating to PTH, as the beta cell insulin system appears to be a rare example of such a positive feedback of insulin on the secretion of insulin [33–37]. A possible link between impaired insulin secretion and insulin resistance at the level of the pancreas has been suggested as a mechanism involved in the development of type 2 diabetes [34, 35]. We speculated that an eventual disturbed response to the PTH/PTHrP receptor ligand might be an additional factor in the increased PTH secretion in secondary HPT due to uremia. The present results, however, contradict such a possibility as the increase in PTH secretion in response to PTHrP was proportionally the same in uremic rats on a standard diet and normal rats. The pattern of the continued and progressive increase in the high phosphorus uremic rats given PTHrP seems to be different. The explanation for this continued rise of PTH in that group is not clear from the present investigation. The relative secretory capacity of the parathyroid glands is in the present investigation shown to be severely diminished in uremic animals. The physiologic importance of this observation is at present not clear. The fluctuation of plasma PTH levels is possibly of importance for maintaining the sensitivity of bone cells to PTH and for maintaining normal bone metabolism [38–40]. An abnormal fluctuation of plasma PTH levels has been described in patients with metabolic bone diseases, as such diminished circadian fluctuations of PTH have been reported in patients with age-related osteoporosis [38] and osteoporotic subjects exhibited a disturbed pulse amplitude and frequency modulation of PTH secretion with smaller and less pulses [41]. Altered calcium-modulated oscillatory PTH secretion pattern has also been shown in patients with secondary HPT with increased spontaneous PTH burst frequency, but with blunted responsiveness to changes in Ca⫹⫹[42]. Treatment of osteoporosis with intermittent administration of PTH has been shown to have an anabolic effect on the skeleton [43, 44]. We have demonstrated, that the relative secretory capacity is practically 0 in uremic, severely hyperparathyroid rats. In uremic rats kept on a high phosphorus diet, PTH levels were already at the basal condition at the upper part of the calcium/PTH curve, and induction of even severe hypocalcemia was not able further to stimulate PTH secretion. In the present study it is, however, clearly shown that even in these rats with severe HPT it was possible further to stimulate PTH levels, as PTHrP enhanced the levels of PTH significantly. Thus, the impaired secretory capacity can be expanded, and there is a potential for induction of fluctuation of circulating PTH even in severe secondary HPT. The present results furthermore clearly showed that
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also in the case of uremia is the level of PTH, which has been considered as the maximal secretory activity of the parathyroid cells, in fact, not the maximum, and that the maximal PTH secretion can be enhanced even further by several fold. As such, the present results oppose the common assumption, that the maximal PTH secretion is an expression of the parathyroid mass [45]. The secretory response of the parathyroid glands to hypocalcemia is a tightly regulated phenomenon, which is mediated via a combined influence of extracellular Ca⫹⫹ and a spectrum of paracrine and autocrine factors [45], which might include peptides targeting the PTH/PTHrP receptor. The hypercalcemia of malignancy in human subjects arises from an inappropriate activation of the renal and bone PTH/PTHrP receptors by PTHrP, which is generated by the malignant cells. In this condition the PTH secretion from the parathyroid glands is suppressed. When plasma PTH levels are elevated, a concomitant primary HPT is considered. In a previous study, we have shown that PTH levels were suppressed during hypercalcemia despite PTHrP administration [1]. This is in agreement with the clinical findings with the hypercalcemia of malignancy. CONCLUSION PTHrP enhanced significantly the low Ca⫹⫹-stimulated PTH secretion in vivo in a dose-related manner in normal rats. The impaired secretory capacity of the parathyroid glands in uremic rats was significantly enhanced by PTHrP. The presence of the PTH/PTHrP receptor is demonstrated in rat parathyroid glands in the present investigation. An autocrine/paracrine role of PTH/PTHrP receptor targeting peptides in the parathyroid glands is suggested. Thus, PTH might during hypocalcemia have a positive auto-feedback regulatory role on its own secretion. ACKNOWLEDGMENTS This study was supported in part by the Danish Research Council with grant no. 28809/9601595 and in part by Danish Kidney Association. We would like to express our thanks to Kirsten Bang for her technical assistance. Reprint requests to Ewa Lewin, M.D., Nephrological Department B, University of Copenhagen, Herlev hospital, 75 Herlev Ringvej, DK2730 Herlev, Denmark. E-mail: [email protected]
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