[Calcimimetics, mechanisms of action and therapeutic applications]. / Les calcimimétiques, mécanismes d'action et applications thérapeutiques.

The extracellular calcium-sensing receptor (CaR) on the parathyroid cell surface negatively regulates secretion of parathyroid hormone (PTH). Its activation by small changes in the extracellular concentration of ionized calcium (ec[Ca2+]) decreases PTH secretion and secondarily bone turnover. CaR is an ideal target for compounds that may be developed to modulate its activity - activating calcimimetics and inhibiting calcilytics. Calcimimetics can amplify the sensitivity of the CaR to ec(Ca2+), thereby suppressing PTH levels and in turn reducing blood Ca++. They dose-dependently reduce the secretion of PTH in cultured parathyroid cells, in animal models and in humans. In uremic animals, these compounds prevent parathyroid cell hyperplasia when given at the onset of the disease and stop cell proliferation if they are administered afterwards, when the hyperplasia already exists. They normalize plasma PTH levels and bone remodeling. In uremic patients undergoing hemodialysis, calcimimetics reduce plasma PTH concentrations in the short (12 weeks) and long (2 years) terms. They also reduce serum levels of calcium-phosphorus product. Calcimimetics are therefore an alternative for the treatment of secondary hyperparathyroidism, particularly in dialysis patients, when increased serum levels of calcium-phosphorus product, the attendant risk of cardiovascular calcification, and its lack of efficacy limit use of the standard treatment.

[The PTH/PTHrP receptor: biological implications]. / El receptor PTH/PTHrP: implicaciones biológicas.

Since its discovery in 1923, the parathyroid hormone (PTH), was thought to be the sole hormone capable of stimulating bone resorption, renal tubular calcium reabsorption, calcitriol synthesis, and urinary excretion of phosphate. However, in 1987, the PTHrP (PTH-related peptide), was demonstrated to share most of the biological actions of PTH through the activation of the same receptor. This receptor was cloned in 1992 and named PTH/PTHrP receptor or PTH-R1. Both, PTH and PTHrP bind with great affinity to PTH-R1 and stimulate a signal transduction system involving different G-proteins, phospholipase C, and adenylate cyclase. A third member of the PTH family, the TIP-39 (tuberoinfundibular peptide), binds and activates another PTH receptor (PTH-R2). There is evidence for other PTH receptors, a PTH-R3, probably specific for PTHrP in keratinocytes, kidney, placenta and a PTH-R4 specific for C-terminal PTH fragments. Activating mutations in the PTH-R1 gene cause Jansen type metaphyseal chondrodysplasia, whereas inactivating mutations are responsible for Blomstrand type rare chondrodysplasia and enchondromatosis. The renal and bone PTH-R1 expression is upregulated in vitamin D deficient rats and by endotoxin, interleukin-2, dexamethasone, T3, and TGF beta. On the contrary, PTH, PTHrP, angiotensin-II, IGF-1, PGE2, vitamin D, and chronic renal failure decrease its expression. In conclusions, the biological implications of the identification and cloning of different PTH receptors are at their beginning. The almost ubiquitous distribution of PTHrP and PTH-R1, the numerous PTHrP and PTH fragments, let us suppose the existence of other PTH-related receptors, and a great complexity of the bone and mineral metabolism.

Plasma bone-specific alkaline phosphatase changes in hemodialysis patients treated by alfacalcidol.

Vitamin D derivatives correct high bone remodeling by decreasing plasma iPTH concentration in uremic patients with secondary hyperparathyroidism. However, without bone biopsy, plasma iPTH alone might not provide sufficient information regarding vitamin D-induced bone changes. Plasma bone-specific alkaline phosphatase (bAP) seems more sensitive than iPTH in assessing the degree of bone remodeling. We prospectively studied the evolution of iPTH and bAP in 14 adult hemodialysis patients treated for 1 year by i.v. alfacalcidol pulses. The mean total alfacalcidol dose was 0.08 +/- 0.02 g/kg/week. Ten patients completed the study, 2 patients had to be parathyroidectomized before week 24 because of hypercalcemia and uncontrolled hyperphosphatemia, and 2 other patients died before week 36. Mean iPTH levels diminished from 826 +/- 300 pg/ml (range 507 - 1,500 pg/ml) at baseline to 436 +/- 371 pg/ml (range 18 - 1,095 pg/ml) after 52 weeks of treatment (48% of decrease). Only 2 patients normalized plasma iPTH levels while 8/10 normalized bAP. Five patients remained with plasma iPTH concentrations higher than 5-fold the normal value. In contrast, plasma bAP levels declined from 47.6 +/- 32.2 ng/ml (range 15.4 - 130.0 ng/ml) at baseline to 17.8 +/- 9.9 ng/ml (range 8.0 +/- 38.0 ng/ml) at week 52 (63% of decrease). Bone histomorphometry was available in 6 patients after 15.8 +/- 5.1 months of alfacalcidol treatment. None of them met the criteria of adynamic bone disease as they had increased bone resorption and marrow bone fibrosis. Bone formation rate was normal in 2 patients and unmeasurable in the other 4. Two patients showed signs of osteomalacia. In conclusion, alfacalcidol preferentially reduced bone formation rate rather than the other histological parameters of secondary hyperparathyroidism. It reduced plasma bAP more efficiently than iPTH.

Unilateral surgery for primary hyperparathyroidism on the basis of technetium Tc 99m sestamibi and iodine 123 subtraction scanning.

HYPOTHESIS: Parathyroid scanning, based on simultaneous recording of technetium Tc 99m sestamibi and iodine 123 images, is able to identify patients with multiple parathyroid gland disease and is a safe imaging technique for unilateral parathyroid surgery. DESIGN: Scintigraphic criteria of eligibility for unilateral surgery were prospectively tested against findings of conventional bilateral surgery. SETTING: Patients referred to an endocrine surgeon in a university hospital. PATIENTS: Seventy consecutive patients with primary hyperparathyroidism had dual-isotope scanning before conventional surgery. Forty-one patients had scan findings compatible with unilateral surgery, with a single focus of high intensity seen on the anterior and lateral views. The remaining 29 patients had 1 or more criteria of ineligibility: (1) scan findings pointing to multiple gland disease, (2) no well-identified focus, (3) contralateral thyroid nodule requiring surgical management, or (4) family history of hyperparathyroidism or multiple endocrine disease. MAIN OUTCOME MEASURES: Number of enlarged parathyroid glands at surgical inspection and calcemia follow-up. RESULTS: None of the 41 patients, with a single well-defined focus on the scan image, showed evidence of multiple parathyroid involvement. Each parathyroid adenoma was resected from the precise site predicted by the subtraction scan. Nine patients (13%) had surgical findings of multiple parathyroid gland disease. All 9 were ineligible based on preoperative image findings. CONCLUSIONS: Unilateral surgery can be safely offered to 60% of patients with primary hyperparathyroidism, on the basis of simultaneous (99m)Tc-sestamibi and (123)I scanning. This may reduce the length of the operation, anesthesia requirements, and hospital stay, and the risks of hypoparathyroidism and injury to the recurrent laryngeal nerve.

Vitamin D receptor as a candidate tumor-suppressor gene in severe hyperparathyroidism of uremia.

Most chronic renal failure patients with severe refractory hyperparathyroidism harbor at least one monoclonal parathyroid tumor, but the specific acquired genetic defects that confer this clonal selective advantage remain poorly understood. Somatic inactivation of the vitamin D receptor (VDR) gene could contribute to clonal outgrowth, because a parathyroid cell containing this lesion would have an impaired response to the antiproliferative influence of 1,25-dihydroxyvitamin D3. Furthermore, diminished expression of VDR protein has been described in uremia-associated parathyroid tumors. Therefore, to assess VDR gene inactivation's potential pathogenetic role in this disease, we rigorously analyzed the VDR gene in 59 parathyroid tumors surgically resected from uremic patients. First, Southern blotting and/or PCR analyses of 29 tumor samples from 14 genetically informative patients revealed no allelic losses at the VDR locus. Next, direct DNA sequencing of all VDR splice junctions, associated intronic sequences, and virtually the entire VDR-coding region for all 59 tumors revealed no acquired mutations. Last, 37 tumor DNA samples were subjected to comparative genomic hybridization, and no chromosomal losses in the VDR region (12cen-q12) were observed. These observations suggest that inactivating defects within the VDR gene do not commonly contribute to the primary pathogenesis of severe refractory hyperparathyroidism in uremia.

Preoperative imaging of parathyroid glands with technetium-99m-labelled sestamibi and iodine-123 subtraction scanning in secondary hyperparathyroidism.

BACKGROUND: Parathyroidectomy is unsuccessful in 10-30% of uraemic patients operated on for secondary hyperparathyroidism. We investigated the usefulness of preoperative radionuclide imaging, with simultaneous recording of the distribution images of iodine-123 and technetium-99m-labelled sestamibi. METHODS: 11 patients with secondary hyperparathyroidism underwent prospective imaging and parathyroidectomy. Plasma concentrations of intact parathyroid hormone (PTH) were measured in all patients before and 6 months after subtotal parathyroidectomy. FINDINGS: Preoperative scanning showed 42 hot-spots suggesting enlarged parathyroid glands. 45 glands were discovered at surgery, and the parathyroidectomy was deemed successful in ten patients. Among the latter, one patient had a supernumerary parathyroid gland detected by scanning and resected from the left thymus. Another patient showed ectopic uptake corresponding to a large parathyroid gland in the upper mediastinum, and another had a parathyroid gland well above the thyroid. No false-positive scan findings were documented. In the patient for whom parathyroidectomy failed, preoperative scanning suggested five enlarged parathyroid glands, though the surgeon found only four glands, in their normal positions. Hyperparathyroidism persisted (intact PTH 527 ng/L, 6 months after surgery). A second scan confirmed the preoperative scan, showing a fifth parathyroid gland in the middle of the right thyroid lobe. INTERPRETATION: Simultaneous recording of 99mTc-sestamibi and 123I improved the imaging of parathyroid glands in secondary hyperparathyroidism. The technique can identify ectopic and supernumerary parathyroid glands.

Circulating biochemical markers of bone remodeling in uremic patients.

Chronic renal failure is often associated with bone disorders, including secondary hyperparathyroidism, aluminum-related low-turnover bone disease, osteomalacia, adynamic osteopathy, osteoporosis, and skeletal beta2-microglobulin amyloid deposits. In spite of the enormous progress made during the last few years in the search of noninvasive methods to assess bone metabolism, the distinction between high- and low-turnover bone diseases in these patients still frequently requires invasive and/or costly procedures such as bone biopsy after double tetracycline labeling, scintigraphic-scan studies, computed tomography, and densitometry. This review is focused on the diagnostic value of several new serum markers of bone metabolism, including bone-specific alkaline phosphatase (bAP), procollagen type I carboxy-terminal extension peptide (PICP), procollagen type I cross-linked carboxy-terminal telopeptide (ICTP), pyridinoline (PYD), osteocalcin, and tartrate-resistant acid phosphatase (TRAP) in patients with chronic renal failure. Most of the observations made by several groups converge to the conclusion that serum bAP is the most sensitive and specific marker to evaluate the degree of bone remodeling in uremic patients. Nonetheless, PYD and osteocalcin, in spite of their retention and accumulation in the serum of renal insufficient patients, are also excellent markers of bone turnover. The future generalized use of these markers, individually or in combination with other methods, will undoubtedly improve the diagnosis and the treatment of the complex renal osteodystrophy.

Pamidronate corrects the down-regulation of the renal parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor mRNA in rats bearing Walker tumors.

Human hypercalcemia of malignancy (HHM) is generally due to the release into the circulation of parathyroid hormone-related peptide (PTHrP). PTHrP stimulates osteoclastic bone resorption and renal calcium reabsorption through the activation of a receptor similar to that of PTH (PTH-R). However, there is scarce information about the PTH-R regulation in the setting of the hypercalcemia. In the present study, we assessed the molecular basis of renal PTH-R regulation in Walker tumor-bearing rats either treated or not by a bisphosphonate, pamidronate. Twenty-seven 6-week-old rats were randomly divided into three experimental groups: WC- APD- (9 control rats), WC+ APD- (9 Walker tumor-bearing rats), and WC+ APD+ (9 Walker tumor-bearing rats receiving 15 mg/kg/day of sodium pamidronate every day for seven days). Pamidronate induced a significant decrease in the mean tumor weight (9.3+/-0.8 vs 6.3+/-0.6 g). Seven days after the subcutaneous implantation of the Walker cells, plasma total calcium was 10.8+/-0.4, 16.8+/-0.6, and 12.9+/-0.6 mg/dl in WC- APD-, WC+ APD-, and WC+ APD+, respectively. Plasma PTHrP concentration was undetectable, 15.9+/-2.6, and 7.2+/-1.4 pmol/l, respectively. Bone histomorphometric results showed high resorption in WC+ APD-, which returned below the basal level of the WC- APD- with pamidronate treatment. Densitometric analysis of Northern blots revealed that the renal PTH-R mRNA expression in WC+ WPD- rats was a quarter of the levels in the WC- APD- and WC+ APD+ groups. WC+ APD- also had a decreased PTH-stimulated cAMP production in renal membranes. The PTH-R was expressed in the Walker tumor and it was not modified by pamidronate treatment. In conclusion, the expression of PTH-R receptor mRNA is significantly reduced in the kidney of rats bearing Walker carcinoma tumor. Its regulation is tissue-specific: pamidronate, which partially corrected the hypercalcemia and elevated circulating PTHrP, normalized the PTH-R mRNA expression in the kidney but not in the tumor.

Estrogen stimulates PTHrP but not PTH/PTHrP receptor gene expression in the kidney of ovariectomized rat.

The aim of the present study was to test the hypothesis that the decreased renal tubular reabsorption of calcium observed in estrogen deficiency is associated with a local regulation of either PTHrP or PTH/PTHrP receptor genes in the kidney. Rats were randomly sham-operated (S) or ovariectomized receiving either vehicle (OVX) or 4 microg E2/kg/day (OVX+E4) or 40 microg E2/kg/d (OVX+E40) during 14 days using alzet minipumps. Plasma PTH and calcium levels were lower in untreated OVX animals than in all other groups (P < 0.01). Plasma PTH was higher in OVX+E40 than in OVX+E4 (P < 0.05). PTHrP mRNA expression in the kidney was unaffected by ovariectomy but was increased in OVX+E40 (0.984 +/- 0.452 for PTHrP/GAPDH mRNAs expression vs. 0.213 +/- 0.078 in sham, P < 0.01). PTH/PTHrP receptor mRNA expression and the cAMP response of renal membranes to PTH were unaffected by ovariectomy and estrogen substitution. In conclusion, renal PTHrP and PTH/PTHrP receptor mRNAs are not modified by ovariectomy. However, 17beta-estradiol increases renal expression of PTHrP mRNA without evident changes in its receptor expression and function. This may help to explain the pharmacological action of estrogen in the kidney, especially how it prevents the renal leak of calcium in postmenopausal women.

Secondary hyperparathyroidism: detection with I-123-Tc-99m-Sestamibi subtraction scintigraphy versus US.

PURPOSE: To compare iodine-123-technetium-99m-sestamibi subtraction scintigraphy with ultrasonography (US) for detection of parathyroid abnormalities in uremic patients with secondary hyperparathyroidism. MATERIALS AND METHODS: Fourteen adult uremic patients with severe secondary hyperparathyroidism underwent subtraction scintigraphy before total or subtotal parathyroidectomy. Subtraction scintigrams were acquired with a double-energy-window technique. US was performed with high-frequency transducers and standard methods. RESULTS: 1-123-Tc-99m-sestamibi subtraction scintigraphy correctly demonstrated 41 of 50 surgically confirmed enlarged parathyroids; US demonstrated 27. Sensitivity for detection of hyperplastic glands was 82% for scintigraphy and 54% for US. Scintigrams were correct in seven patients, and US scans were correct in five. Scintigraphy demonstrated all four enlarged parathyroids in six of 12 patients who were to undergo first surgery, whereas US demonstrated all four enlarged glands in three of the 12 patients. Gland weight was correlated with likelihood of detection with either method. Glands undetected at scintigraphy were significantly (P < .01) smaller (mean weight, 257 mg) than those undetected at US (mean weight, 467 mg). CONCLUSION: I-123-Tc-99m-sestamibi subtraction scintigraphy is efficient for detection of enlarged parathyroid glands in uremic patients with secondary hyperparathyroidism and is more sensitive than US.

[Incidence and development of aortic stenosis in chronic hemodialysis. An ultrasonographic and biological study of 112 patients]. / Incidence et évolutivité des rétrécissements aortiques chez l'hémodialysé chronique. Une étude échographique et biologique sur une population de 112 patients.

Valvular calcification in chronic haemodialysis patients has already been reported in the literature, particularly the abnormally high incidence of aortic stenosis. In this study, 112 haemodialysis patients were followed up by Doppler echocardiography for a period of 36 months. Sixteen patients developed aortic valvular calcification with aortic stenosis over an 18.7 +/- 7.5 months period. The indexed aortic valve surface area decreased from 1.24 +/- 0.9 cm2/m2 to 0.66 +/- 0.21 cm2/m2 with abnormally rapid progression. Eight patients with aortic stenosis died during the 3 year study period. These results reflect the abnormal extra-skeletal calcification of chronic haemodialysis patients. Several predisposing factors were demonstrated: age (68.5 +/- 11.1 years versus 57.1 +/- 16.3 years in patients without calcifications), male gender, a longer period of dialysis than the patients without aortic stenosis (8.1 +/- 5.3 versus 5.9 +/- 5.7 years), abnormalities of calcium and phosphate metabolism, increased of the phosphocalcic product by hyperphosphoraemia and not by hypercalcaemia, hypoparathyroidism in 62% and hyperparathyroidism in 38% an increase in vitamin D 3 (19.7 +/- 14 ng/ml versus 9.6 +/- 6.3 ng/ml) biological signs of adynamic osteodystrophy. Calcific aortic stenosis is a commonly observed valvular lesion in haemodialysis patients: its progression may be very rapid, associated with a poor prognosis. Old age, male gender, duration of haemodialysis, hyperphosphataemia associated with hypoparathyroidism and raised Vitamin D3 are predisposing factors.

[Alkaline phosphatase of bone origin in hemodialyzed patients. 110 assays]. / Phosphatase alcaline d'origine osseuse chez les patients hémodialysés. 100 dosages.

OBJECTIVES: To evaluate the value of plasma bone-specific alkaline phosphatase (bAP) in the diagnosis and the prediction of the type of renal osteodystrophy in hemodialysis patients. To examine whether bAP correlated with the two classical biochemical markers of bone turnover in dialysis patients which are total alkaline phosphatases (tAP) and intact PTH (iPTH). METHODS: One hundred adult uremic patients undergoing regular hemodialysis therapy were included in the study. Plasma bAP was determined using a radioimmunometric assay. Intact PTH (1-84) was measured using a radioimmunometric assay, Allegro Intact PTH, Nichols Institute, CA, USA. Other laboratory tests including calcium, phosphorus, and tAP were performed by automated methods. RESULTS: Based on previous biochemical and histological data obtained in the same type of patients, we arbitrarily divided plasma bAP levels in normal (10-20 ng/ml), low (< 10 ng/ml), and high (> 20 ng/ml). We found here that bAP levels were normal in 27 patients, high in 36, and low in 37. In the group with normal bAP, 8/27 patients had iPTH levels higher than 200 pg/ml, the other 19 patients had an iPTH within the normal range. In the 36 patients with high bAP, 10 showed markedly high iPTH and clinical signs of severe hyperparathyroidism leading to the indication of a surgical parathyroidectomy in 7 patients and i.v. calcitriol therapy in the other 3. However, 9 of these 36 patients had an iPTH lower than 200 pg/ml and 17 between 200-500 pg/ml. In the 37 patients with low bAP, iPTH levels were also low in 15 of them, but 12 patients had iPTH levels higher than 200 pg/ml. There was a good correlation between bAP, tAP, and iPTH. However, bAP correlated better with iPTH than tAP. A bAP > 20 ng/ml had a sensitivity of 56%, specificity of 92% and positive predictive value of 80% in the prediction of an PTH higher than > 400 pg/ml and therefore of the biological diagnosis of hyperparathyroidism. A bAP < 10 ng/ml had a sensitivity of 70%, specificity of 56% and a positive predictive value of 48% in predicting a normal-low iPTH (< 100 pg/ml). CONCLUSION: In the absence of bone biopsy, plasma bAP self provides useful information about bone remodeling in hemodialysis patients. The combination of bAP and iPTH measurements further define the type of bone turnover. either elevated plasma bAP or iPTH alone are no always anonymous with secondary hyperparathyroidism.

Effects of new vitamin D analogues on parathyroid function in chronically uraemic rats with secondary hyperparathyroidism.

BACKGROUND: Treatment with calcitriol or its analogue, alfacalcidol often leads to hypercalcaemia, hyperphosphataemia or both in patients which chronic renal failure and advanced secondary hyperparathyroidism. We tested three new vitamin D analogues (CB 1093, EB 1213, GS 1725) in an attempt to identify potentially non hypercalcaemic compounds, capable of decreasing plasma parathyroid hormone (PTH) concentration. METHODS: Male Wistar AF rats aged 12-14 weeks were fed a synthetic, phosphate-rich diet and underwent either sham surgery (control) or a standard two-step 5/6th nephrectomy. Four weeks later, renal function was mildly decreased in the latter. Chronic renal failure rats were then divided into six groups, with 8-10 rats in each group. They received daily 1.p. injections, from days 0 to 4, of either placebo, calcitriol, or one of the following three active vitamin D analogues: CB1093, 0.25 micrograms; EB1213, 0.25 or 1.25 micrograms; and GS1725, 0.025 micrograms/kg body weight per day, respectively. Sham-operated rats received no drug. On day 5, arterial blood was sampled and rats were sacrificed. RESULTS: At predefined dosage schedules, all three compounds significantly decreased plasma immunoreactive PTH levels (except EB1213 at low dose). The decrement was somewhat less marked than that obtained with calcitriol, at the dose of 0.25 micrograms/kg b.w. per day. However, calcitriol induced a marked increase in plasma calcium and phosphate concentrations at that dose, whereas vitamin D analogues led to a more modest increase in plasma calcium level, and none to a worsening of hyperphosphataemia. CB1093 treatment was even associated with a significant decrease in plasma phosphate level. CONCLUSION: All three calcitriol analogues tested are promising as non-hypercalcaemic agents in the treatment of uraemic secondary hyperparathyroidism. However, more prolonged administration to uraemic rats of calcitriol analogues with slightly modified dosage schedules and of calcitriol with lower non-hypercalcaemic dose is required for an optimal comparison before considering clinical trials.

Plasma total versus bone alkaline phosphatase as markers of bone turnover in hemodialysis patients.

Plasma total versus bone alkaline phosphatase as markers of bone turnover in hemodialysis patients. Plasma bone-specific alkaline phosphatase (bAP) has been demonstrated to be more reliable than total alkaline phosphatases (tAP) in providing information about bone turnover in patients with metabolic bone diseases. This study surveyed 42 hemodialysis patients who underwent a systematic transiliac bone biopsy for histomorphometry study. Plasma bAP was determined by using a new immunoassay (Tandem-R Ostase, Hybritech, Liège, Belgium). Plasma bAP values were compared with those of two other plasma markers of bone metabolism, namely tAP and intact parathyroid hormone (iPTH), for the correlations with bone histomorphometric parameters. Patients with high-turnover bone disease (HTBD) (N = 32) had significantly higher plasma bAP levels than patients with normal or low bone turnover (N/LTBD) (N = 10) (66.9 +/- 63.5 ng/mL versus 10.8 +/- 4.2 ng/mL, respectively). Bone formation and resorption were highly correlated in these patients, and plasma bAP levels were positively correlated with bone resorption parameters, including osteoclast surface (r = 0.39, P < 0.0001) and osteoclast number/mm2 (r = 0.36, P < 0.001), and with bone formation parameters, osteoblast surface (r = 0.50, P < 0.005), and bone formation rate (r = 0.91, P < 0.0001). The bone formation rate was better correlated with plasma bAP levels than with either plasma tAP or iPTH concentrations. Plasma bAP level equal or higher than 20 ng/mL, either alone or combined with plasma iPTH of 200 pg/mL, had the highest sensitivity, specificity, and predictability values for the diagnosis of high-turnover bone disease, and formally excluded patients with normal or LTBD. In conclusion, plasma bAP can be measured with a reliable immunoassay in hemodialysis patients. It represents a highly sensitive and specific biochemical marker of skeletal remodeling in these patients. Therefore, both serum iPTH and bAP are complementary in diagnoses of the type of renal osteodystrophy.

Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines.

PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum pyridinoline as a specific marker of collagen breakdown and bone metabolism in hemodialysis patients.

Type I collagen represents more than 90% of bone matrix. Quantitative analysis of collagen cross-link molecules such as pyridinoline (PYD) provides valuable information on bone resorption rate. We have studied 37 hemodialysis patients who underwent a systematic transiliac bone biopsy for histomorphometry study. Eighteen of them had tetracycline double labeling, allowing to determine dynamic, in addition to static bone parameters. Measurement of serum-free PYD was performed using a new competitive enzyme immunoassay. Serum PYD values were compared with those of three other serum markers of bone metabolism, namely intact PTH (iPTH), bone-specific alkaline phosphatase (bAP), and osteocalcin, for the correlations with bone histomorphometric parameters. Serum PYD levels (mean +/- SD) were significantly higher in dialysis patients than in normal individuals, 90.6 +/- 99.6 nM versus 1.9 +/- 0.4 nM, respectively. Patients with high turnover bone disease had significantly higher serum PYD levels than patients with normal or low bone turnover, 108.8 +/- 108.0 nM versus 34.1 +/- 12.8 nM, respectively. Serum PYD levels were positively correlated with bone resorption parameters including osteoclast surface (r = 0.59, p < 0.0001) and osteoclast number/mm2 (r = 0.61, p < 0.0001), and also with bone formation parameters, osteoblast surface (r = 0.43, p < 0.008), double-labeled surface (r = 0.81, p < 0.001), and BFR (r = 0.91, p < 0.0001). The BFR was better correlated with serum PYD levels than with either serum iPTH or osteocalcin concentrations. However, correlation with serum bAP was comparable.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia.

The pathogeneses of parathyroid disease in patients with uremia and nonfamilial primary parathyroid hyperplasia are poorly understood. Because of multigland involvement, it has been assumed that these common diseases predominantly involve polyclonal (non-neoplastic) cellular proliferations, but an overall assessment of their clonality has not been done. We examined the clonality of these hyperplastic parathyroid tumors using X-chromosome inactivation analysis with the M27 beta (DXS255) DNA polymorphism and by searching for monoclonal allelic losses at M27 beta and at loci on chromosome band 11q13. Fully 7 of 11 informative hemodialysis patients (64%) with uremic refractory hyperparathyroidism harbored at least one monoclonal parathyroid tumor (with a minimum of 12 of their 19 available glands being monoclonal). Tumor monoclonality was demonstrable in 6 of 16 informative patients (38%) with primary parathyroid hyperplasia. Histopathologic categories of nodular versus generalized hyperplasia were not useful predictors of clonal status. These observations indicate that monoclonal parathyroid neoplasms are common in patients with uremic refractory hyperparathyroidism and also develop in a substantial group of patients with sporadic primary parathyroid hyperplasia, thereby changing our concept of the pathogenesis of these diseases. Neoplastic transformation of preexisting polyclonal hyperplasia, apparently due in large part to genes not yet implicated in parathyroid tumorigenesis and possibly including a novel X-chromosome tumor suppressor gene, is likely to play a central role in these disorders.

Plasma kinetics of 125I-labelled amyloid P component in beta 2M amyloidosis: a possible approach to quantitate disease activity.

Dialysis amyloidosis is one of the most incapacitating complications of long-term dialysis treatment. Quantitative assessment of amyloid deposition using radiolabelled tracers has been recently proposed but convincing evidence of its validity in uraemic patients remains to be provided. We studied the plasma kinetics of i.v. administered 125I-labelled serum amyloid P component (125I-SAP) in 20 chronic haemodialysis patients compared with those of nine healthy volunteers and three non-dialysed patients with systemic amyloidosis. Plasma clearance of the tracer was abnormal in 17 of 20 dialysis patients in whom plasma radioactivity declined in a bi-exponential mode, in contrast to the single-exponential slope observed in all healthy controls. 125I-SAP plasma half-life of the second component, probably reflecting metabolic clearance, was significantly prolonged in these dialysis patients compared with the healthy controls (35.3 versus 24.6 h, P < 0.001). Among the long-term haemodialysis patients the calculated extravascular distribution of 125I-SAP was significantly greater in those with severe arthropathy than in asymptomatic patients. These findings demonstrate for the first time that SAP clearance is disturbed in haemodialysis patients due to both failing renal elimination and retention in extravascular sites. The extravascular diffusion is greatly enhanced in patients with clinical evidence of amyloidosis. Therefore the study of plasma 125I-SAP kinetics promises to be a valuable tool to quantitate the extent of amyloidosis.

[Mode of action of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) in target organs]. / Modes d'action de l'hormone parathyroïdienne (PTH) et du peptide apparenté à la PTH (PTHrP) au niveau des organes cibles.

Calcium and phosphorus metabolism is mainly regulated by PTH through its actions on kidney and bone. PTHrP, which is associated with the hypercalcemia of malignancy syndrome, binds to and activates the same receptor that PTH does. cDNA clones of PTH/PTHrP receptors from rat osteosarcoma (ROS 17/2.8) and opossum kidney (OK) cells are highly homologous and are members of a novel G protein-linked receptor family that includes calcitonin, glucagon, GLP-1, GHRH, VIP, and secretin receptors. Analysis of the protein sequence predicts a receptor with 7 transmembrane domains, a 155 amino acids (aa) extracellular (EC) N-terminal, and 130aa intracellular C-terminal domaina. The extracellular domain has 6 conserved cysteines and 4 potential glycosylation sites. When transfected in COS cells, both receptors are able to bind PTH and PTHrP active fragments with equal affinity. Likewise, agonists activate both adenylate cyclase and phospholipase C efficiently. The N-terminal EC domain and the first EC loop seem to determine the receptor binding capacity with the agonists. Activation of adenylate cyclase and phospholipase C might involve multiple sites between the 3rd helix and the C-terminal tail. Partial characterization of the rat PTH/PTHrP receptor gene demonstrates the existence of at least 15 exons. The first six transmembrane domains are encoded by separated exons. The PTH/PTHrP receptor mRNA is expressed mainly in kidney and bone, and also is widely expressed in many tissues, but not all. A major 2.3-2.5 kb transcript is observed in all these tissues. Nevertheless, 2 larger transcripts are observed in kidney and liver, and multiple smaller mRNA species are observed in kidney, skin, and testis.(ABSTRACT TRUNCATED AT 250 WORDS)