RESUMO
The parathyroid oxyphil cell content increases in patients with chronic kidney disease (CKD), and even more in patients treated with the calcimimetic cinacalcet and/or calcitriol for hyperparathyroidism. Oxyphil cells have significantly more calcium-sensing receptors than chief cells, suggesting that the calcium-sensing receptor and calcimimetics are involved in the transdifferentiation of a chief cell to an oxyphil cell type. Here, we compared the effect of the vitamin D analog paricalcitol (a less calcemic analog of calcitriol) and/or cinacalcet on the oxyphil cell content in patients with CKD to further investigate the genesis of these cells. Parathyroid tissue from four normal individuals and 27 patients with CKD who underwent parathyroidectomy for secondary hyperparathyroidism were analyzed. Prior to parathyroidectomy, patients had received the following treatment: seven with no treatment, seven with cinacalcet only, eight with paricalcitol only, or cinacalcet plus paricalcitol in five. Oxyphilic areas of parathyroid tissue, reported as the mean percent of total tissue area per patient, were normal, 1.03; no treatment, 5.3; cinacalcet, 26.7 (significant vs. no treatment); paricalcitol, 6.9 (significant vs. cinacalcet; not significant vs. no treatment); and cinacalcet plus paricalcitol, 12.7. Cinacalcet treatment leads to a significant increase in parathyroid oxyphil cell content but paricalcitol does not, reinforcing a role for the calcium-sensing receptor activation in the transdifferentiation of chief-to-oxyphil cell type. Thus, two conventional treatments for hyperparathyroidism have disparate effects on parathyroid composition, and perhaps function. This finding is provocative and may be useful when evaluating future drugs for hyperparathyroidism.
Assuntos
Calcimiméticos/farmacologia , Cinacalcete/farmacologia , Ergocalciferóis/farmacologia , Hiperparatireoidismo Secundário/terapia , Células Oxífilas/efeitos dos fármacos , Glândulas Paratireoides/efeitos dos fármacos , Insuficiência Renal Crônica/tratamento farmacológico , Adulto , Calcimiméticos/uso terapêutico , Calcitriol/análogos & derivados , Transdiferenciação Celular/efeitos dos fármacos , Cinacalcete/uso terapêutico , Quimioterapia Combinada/métodos , Ergocalciferóis/uso terapêutico , Feminino , Humanos , Hiperparatireoidismo Secundário/etiologia , Hiperparatireoidismo Secundário/urina , Masculino , Pessoa de Meia-Idade , Glândulas Paratireoides/citologia , Glândulas Paratireoides/metabolismo , Glândulas Paratireoides/cirurgia , Paratireoidectomia , Receptores de Detecção de Cálcio/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/urina , Uremia/complicações , Uremia/tratamento farmacológico , Uremia/urina , Vitamina D/análogos & derivadosRESUMO
Klotho plays an important role in the pathogenesis of cardiovascular disease in chronic kidney disease (CKD). Klotho is highly expressed in the kidney and parathyroid glands, but its presence in the vasculature is debated. Renal Klotho is decreased in CKD, but the effect of uremia on Klotho in other tissues is not defined. The effect of vitamin D receptor activator therapy in CKD on the expression of Klotho in various tissues is also in debate. In uremic rats (surgical 5/6th nephrectomy model), we compared 3 months of treatment with and without paricalcitol on Klotho immunostaining in the kidney, parathyroid glands, and aorta. With uremia, Klotho was unchanged in the parathyroid, significantly decreased in the kidney (66%) and the intimal-medial area of the aorta (69%), and significantly increased in the adventitial area of the aorta (67%) compared with controls. Paricalcitol prevented the decrease of Klotho in the kidney, increased expression in the parathyroid (31%), had no effect in the aortic media, but blunted the increase of Klotho in the aortic adventitia. We propose that fibroblasts are responsible for the expression of Klotho in the adventitia. In hyperplastic human parathyroid tissue from uremic patients, Klotho was higher in oxyphil compared with chief cells. Thus, under our conditions of moderate CKD and mild-to-moderate hyperphosphatemia in rats, the differential expression of Klotho and its regulation by paricalcitol in uremia is tissue-dependent.
Assuntos
Aorta/química , Conservadores da Densidade Óssea/farmacologia , Ergocalciferóis/farmacologia , Glucuronidase/análise , Rim/química , Glândulas Paratireoides/química , Uremia/metabolismo , Túnica Adventícia/química , Animais , Aorta/metabolismo , Modelos Animais de Doenças , Feminino , Fibroblastos/química , Glucuronidase/metabolismo , Humanos , Hiperparatireoidismo Secundário/metabolismo , Hiperparatireoidismo Secundário/patologia , Hiperfosfatemia/metabolismo , Rim/metabolismo , Proteínas Klotho , Nefrectomia , Células Oxífilas/química , Glândulas Paratireoides/metabolismo , Glândulas Paratireoides/patologia , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/tratamento farmacológico , Túnica Íntima/química , Uremia/etiologiaRESUMO
BACKGROUND/AIMS: The search for new therapies providing cardiorenal protection in chronic kidney disease (CKD) has led to treatments that combine conventional renin-angiotensin-aldosterone-system inhibitors with other drugs that exhibit potential in disease management. METHODS: In rats made uremic by renal ablation, we examined the effects of addition of the endothelin-A receptor antagonist atrasentan to a previously examined combination of enalapril (angiotensin converting enzyme inhibitor) and paricalcitol (vitamin D receptor activator) on cardiac and renal parameters. The effects of the individual and combined drugs were examined after a 3-month treatment. RESULTS: A decrease in systolic blood pressure, serum creatinine and proteinuria, and improvement of renal histology in uremic rats were attributed to enalapril and/or paricalcitol treatment; atrasentan alone had no effect. In heart tissue, individual treatment with the drugs blunted the increase in cardiomyocyte size, and combined treatment additively decreased cardiomyocyte size to normal levels. Perivascular fibrosis was blunted in uremic control rats with atrasentan or enalapril treatment. CONCLUSIONS: We found distinct cardiac and renal effects of atrasentan. Combination treatment with atrasentan, enalapril and paricalcitol provided positive effects on cardiac remodeling in uremic rats, whereas combination treatment did not offer further protective effects on blood pressure, proteinuria or renal histology.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Enalapril/uso terapêutico , Antagonistas dos Receptores de Endotelina/uso terapêutico , Ergocalciferóis/uso terapêutico , Coração/efeitos dos fármacos , Rim/efeitos dos fármacos , Pirrolidinas/uso terapêutico , Uremia/tratamento farmacológico , Animais , Atrasentana , Quimioterapia Combinada , Feminino , Glomerulosclerose Segmentar e Focal/patologia , Coração/fisiopatologia , Rim/fisiopatologia , Testes de Função Renal , Miocárdio/patologia , Nefrectomia , Nefrite/patologia , Ratos , Ratos Sprague-Dawley , Análise de Sobrevida , Uremia/fisiopatologiaRESUMO
The vitamin D analog ED-71 (eldecalcitol) has been shown to be superior to calcitriol and its precursor alfacalcidol in maintaining or increasing bone mass in women and animal models with osteoporosis. The mechanism for the greater effectiveness of ED-71 is unknown. In the present study, we tested the hypothesis that the higher activity of ED-71 is due to its higher affinity for the serum vitamin D binding protein (DBP) by comparing the activities of orally administered ED-71, calcitriol and 22-oxacalcitriol (OCT) in wild type (WT) and DBP-ablated (DBPko) mice. In 8-week-old male WT mice, the effects of the analogs on serum and urinary calcium and phosphate were ED-71 > calcitriol > OCT. The results in DBPko mice were identical to those of the WT mice for all parameters tested. In ovariectomized mice, ED-71 was more effective than calcitriol in increasing bone mineral density, but again, there were no differences in the responses of the WT versus DBPko OVX mice. This lack of an effect of DBP ablation on the activities of oral ED-71 occurred despite the finding that peak circulating levels of ED-71 were 100 times lower and disappeared quickly in the DBPko mice while the peak levels at 1 h in WT mice were maintained for at least 24 h. These findings indicate that although DBP has a major influence on circulating levels of vitamin D compounds, it is not responsible for the greater efficacy of ED-71 on bone and mineral metabolism.
Assuntos
Osso e Ossos/efeitos dos fármacos , Proteína de Ligação a Vitamina D/sangue , Vitamina D/análogos & derivados , Animais , Densidade Óssea , Reabsorção Óssea , Osso e Ossos/metabolismo , Calcitriol/sangue , Cálcio/sangue , Cálcio/urina , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Osteoporose/sangue , Ovariectomia , Fosfatos/sangue , Fosfatos/urina , Esteroides/sangue , Fatores de Tempo , Vitamina D/administração & dosagem , Vitamina D/sangue , Vitamina D/farmacocinéticaRESUMO
Eldecalcitol [1α,25-dihydroxy-2ß-(3-hydroxypropyloxy)vitamin D(3) ], a vitamin D analog with enhanced efficacy for treatment of osteoporosis, has been found to be less potent than 1,25-dihydroxyvitamin D(3) (calcitriol) in suppressing PTH in vivo. To define the mechanism for the latter observation, we compared the effects of eldecalcitol and calcitriol on PTH secretion by bovine parathyroid cells. While the two compounds showed similar potency when the cells were cultured in medium containing 15% newborn calf serum, eldecalcitol was 100 times more potent than calcitriol in the absence of serum. Eldecalcitol has a higher affinity for the serum vitamin D-binding protein (DBP), and therefore binding to DBP, and possibly other serum components, appears to limit the uptake and activity of eldecalcitol in parathyroid cells, providing an explanation for the lower PTH suppressing activity in vivo (100% serum). However, the 100-fold higher activity of eldecalcitol in the absence of serum was unexpected since the VDR affinity for eldecalcitol is eightfold lower than for calcitriol. The enhanced activity was not due to preferential uptake, but to a resistance to metabolism. While 1 nM [(3) H]calcitriol was completely degraded within 24 h, [(3) H]eldecalcitol was not metabolized, despite the induction of the vitamin D catabolic enzyme, 24-hydroxylase (CYP24A). The resistance to metabolism is the likely explanation for the higher potency of eldecalcitol in suppressing PTH in cell culture lacking serum. Thus, the unique properties of eldecalcitol in vivo can be attributed, at least in part, to its high-DBP affinity which increases the half-life, but limits the uptake of eldecalcitol, and to its reduced metabolism, which prolongs the activity of this analog in target tissues.
Assuntos
Hormônio Paratireóideo/antagonistas & inibidores , Hormônio Paratireóideo/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Vitamina D/análogos & derivados , Animais , Bovinos , Células Cultivadas , Osteoporose/tratamento farmacológico , Esteroide Hidroxilases/metabolismo , Vitamina D/metabolismo , Vitamina D/farmacologia , Vitamina D/uso terapêutico , Proteína de Ligação a Vitamina D/sangue , Vitamina D3 24-HidroxilaseRESUMO
The vitamin D analog 1α,25-dihydroxy-2ß-(3-hydroxypropyloxy)vitamin D(3) (ED-71 or eldecalcitol) has been developed for treatment of osteoporosis, but its effects on mineral metabolism have not been investigated in detail. In the present study, we compared the effects of eldecalcitol and calcitriol on calcium (Ca) and phosphate (Pi) handling in rats. Oral administration of eldecalcitol (0, 7.5, 20, or 50 pmol) q.o.d. for 2 weeks dose-dependently increased ionized Ca, intestinal Ca absorption, and urinary Ca excretion, while these doses of calcitriol had no significant effects. The highest dose of eldecalcitol did not alter serum Pi but stimulated both intestinal Pi absorption and urinary Pi excretion; the latter was attributable, in part, to increased serum FGF-23. The effects of high-dose eldecalcitol on Ca and Pi absorption and urinary excretion and FGF-23 persisted for several days following cessation of treatment. The higher potency of eldecalcitol on Ca and Pi handling was also observed in parathyroidectomized rats infused with PTH, excluding a role for differential regulation of PTH. Direct measurement of duodenal Ca absorption by the in situ loop method confirmed the higher potency of eldecalcitol in this segment via induction of TRPV6. These studies indicated that with chronic administration eldecalcitol is more potent than calcitriol at stimulating intestinal absorption of Ca and Pi, as well as FGF-23. The mechanisms responsible for the higher potency of eldecalcitol are speculated to be its higher vitamin D-binding protein (DBP) affinity and resistance to metabolism.
Assuntos
Cálcio/metabolismo , Fosfatos/metabolismo , Vitamina D/análogos & derivados , Animais , Calcitriol/farmacologia , Relação Dose-Resposta a Droga , Duodeno/metabolismo , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Ratos , Canais de Cátion TRPV/metabolismo , Vitamina D/farmacologiaRESUMO
AIMS: Left ventricular hypertrophy (LVH) is the most frequent cardiac complication in chronic renal disease. Previous studies implicate elevated serum phosphorus as a risk factor for LVH. METHODS: We treated 5/6 nephrectomized rats with enalapril or enalapril + sevelamer carbonate for 4 months to determine if sevelamer carbonate had an additional beneficial effect on the development of LVH and uremia-induced left ventricle (LV) remodeling. RESULTS: Uremia increased LV weight and cardiomyocyte size. Enalapril and enalapril + sevelamer blunted the increase in left ventricular weight. Only enalapril + sevelamer diminished the increase in cardiomyocyte size. Uremia increased cyclin D2 and PCNA and decreased p27 protein expression in the heart. Enalapril + sevelamer diminished the decrease in p27 expression caused by uremia. Uremia increased Ki67-positive and phosphohistone H(3)-positive interstitial cells. This was not seen in cardiomyocytes. Multivariable regression analysis showed that increased phosphorus was an independent risk factor for both increased LV weight and cardiomyocyte size. CONCLUSIONS: These data suggest left ventricular remodeling consists of cardiomyocyte hypertrophy and interstitial cell proliferation, but not cardiomyocyte proliferation. p27 and cyclin D2 may play important roles in the development of LVH. In addition, phosphorus can be an independent risk factor for the development of LVH.
Assuntos
Hipertrofia Ventricular Esquerda/etiologia , Uremia/complicações , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Animais , Proteínas de Ciclo Celular/fisiologia , Enalapril/administração & dosagem , Feminino , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Miócitos Cardíacos/patologia , Poliaminas/administração & dosagem , Ratos , Ratos Sprague-Dawley , SevelamerRESUMO
UNLABELLED: Vitamin D analogs are being developed that retain therapeutic effects but are less calcemic and phosphatemic, a concern in CKD patients who are prone to vascular calcification. We tested a new analog of vitamin D, 2MbisP, and found that it suppresses PTH at doses that do not affect serum Ca or P. INTRODUCTION: Calcitriol is used for the treatment of secondary hyperparathyroidism. However, its use is often limited by the development of hypercalcemia and hyperphosphatemia, an important consideration in patients with chronic kidney disease (CKD) because they are prone to vascular calcification. To minimize this toxicity, structural modifications in the vitamin D molecule have led to the development of calcitriol analogs with selective actions. MATERIALS AND METHODS: In this study, we compared the effects of 1,25(OH)(2)D(3) and a new analog, 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), on the development of secondary hyperparathyroidism and established secondary hyperparathyroidism in uremic rats and on mobilization of calcium and phosphorus from bone in parathyroidectomized rats. The clearance from circulation, half-life, and binding affinities to the vitamin D-binding protein and vitamin D receptor of this compound were also evaluated. RESULTS: Uremia produced a marked rise in plasma PTH, but treatment every other day for 2 wk with either 1,25(OH)(2)D(3) (4 ng) or 2MbisP (250, 750, 1500, or 3000 ng) suppressed this increase by >50%. The suppression by 1,25(OH)(2)D(3), however, was accompanied by increases in ionized calcium, phosphorus, and the calcium x phosphorus product, whereas these three parameters were unchanged by 2MbisP. The binding affinity of 2MbisP was 10-20 times less for the vitamin D receptor and 1000 times less for the serum vitamin D-binding protein compared with 1,25(OH)(2)D(3). Also, 2MbisP was cleared more rapidly from the circulation (t1/2 = 10 min) than 1,25-(OH)(2)D(3) (t1/2=7-9 h). In parathyroidectomized rats fed calcium-or phosphorus-deficient diets, daily injections of 2MbisP (1500 or 3000 ng), unlike 1,25(OH)(2)D(3) (50 ng), had no effect on calcium or phosphorus mobilization from bone. CONCLUSIONS: In uremic rats, 2MbisP can suppress PTH at doses that do not affect plasma calcium, phosphorus, and calcium x phosphorus product. This new vitamin D analog may represent an important tool in the treatment of secondary hyperparathyroidism in patients with CKD.
Assuntos
Calcitriol/farmacocinética , Di-Hidroxicolecalciferóis/farmacologia , Hiperparatireoidismo Secundário/tratamento farmacológico , Uremia/tratamento farmacológico , Vitaminas/farmacocinética , Animais , Calcinose/induzido quimicamente , Calcinose/metabolismo , Calcinose/patologia , Calcitriol/efeitos adversos , Calcitriol/análogos & derivados , Cálcio/metabolismo , Doença Crônica , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Hiperparatireoidismo Secundário/etiologia , Hiperparatireoidismo Secundário/metabolismo , Hiperparatireoidismo Secundário/patologia , Fósforo/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Calcitriol/metabolismo , Uremia/complicações , Uremia/metabolismo , Uremia/patologia , Doenças Vasculares/induzido quimicamente , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Proteína de Ligação a Vitamina D/metabolismo , Vitaminas/efeitos adversosRESUMO
BACKGROUND: The phosphatonins fibroblast growth factor-23 (FGF-23) and FRP-4 are inhibitors of tubular phosphate reabsorption that may play a role in the hyperphosphatemia associated with chronic kidney disease (CKD) or in the hypophosphatemia associated with renal transplants. METHODS: Plasma FGF-23, FRP-4, phosphorus and parathyroid hormone were measured in patients at all stages of CKD. Phosphate regulation of FGF-23 and secreted frizzled related protein-4 (sFRP-4) was examined in end-stage renal disease patients in the presence and absence of therapeutic phosphate binder usage. In renal transplant patients, plasma FGF-23, sFRP-4 and phosphorus concentrations were determined before and 4-5 days after transplantation. RESULTS: Plasma FGF-23 correlated with creatinine clearance (r2 = -0.584, p < 0.0001) and plasma phosphorus (r2 = 0.347, p < 0.001) in CKD patients and with plasma phosphorus (r2 = 0.448, p < 0.001) in end-stage renal disease patients. Phosphate binder withdrawal increased FGF-23 levels. In kidney transplant patients, dramatic decreases in FGF-23 (-88.8 +/- 5.4%) and phosphorus (-64 +/- 10.2%) were observed by 4-5 days post-transplantation. In patients with post-transplant hypophosphatemia, FGF-23 levels correlated inversely with plasma phosphorus (r2 = 0.661, p < 0.05). sFRP-4 levels did not change with creatinine clearance or hyperphosphatemia in CKD or end-stage renal disease patients, and no relation was noted between post-transplant sFRP-4 levels and hypophosphatemia. CONCLUSIONS: In CKD, FGF-23 levels rose with decreasing creatinine clearance rates and increasing plasma phosphorus levels, and rapidly decreased post-transplantation suggesting FGF-23 is cleared by the kidney. Residual FGF-23 may contribute to the hypophosphatemia in post-transplant patients.
Assuntos
Fatores de Crescimento de Fibroblastos/sangue , Hipofosfatemia/metabolismo , Falência Renal Crônica/metabolismo , Transplante de Rim , Proteínas Proto-Oncogênicas/sangue , Feminino , Fator de Crescimento de Fibroblastos 23 , Humanos , Hipofosfatemia/sangue , Falência Renal Crônica/sangue , Túbulos Renais/metabolismo , Masculino , Hormônio Paratireóideo/sangue , Fosfatos/sangue , Fosfatos/metabolismo , Complicações Pós-Operatórias/sangueRESUMO
Maintenance of a normal serum phosphate level depends on absorption in the gut, reabsorption and excretion by the kidney, and the flux between the extracellular and skeletal pools. Phosphate homeostasis is a coordinated, complex system of crosstalk between the bone, intestine, kidney, and parathyroid gland. Dysfunction of this system has serious clinical consequences in healthy individuals and those with conditions, such as CKD, in which hyperphosphatemia is associated with increased risks of cardiovascular morbidity and mortality. The last half-century of renal research has helped define the contribution of the parathyroid hormone, calcitriol, fibroblast growth factor 23, and Klotho in the regulation of phosphate. However, despite new discoveries and insights gained during this time, what remains unchanged is the recognition that phosphate retention is the initiating factor for the development of many of the complications observed in CKD, namely secondary hyperparathyroidism and bone and cardiovascular diseases. Controlling phosphate load remains the primary goal in the treatment of CKD. This review discusses the clinical effects of dysregulated phosphate metabolism, particularly in CKD, and its association with cardiovascular disease. The importance of early control of phosphate load in the treatment of CKD is emphasized, and the latest research in the treatment of phosphate retention is discussed.
Assuntos
Doenças Cardiovasculares/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/metabolismo , Hiperfosfatemia/terapia , Fosfatos/metabolismo , Insuficiência Renal Crônica/metabolismo , Doenças Ósseas Metabólicas/etiologia , Doenças Cardiovasculares/etiologia , Fator de Crescimento de Fibroblastos 23 , Homeostase , Humanos , Hiperparatireoidismo Secundário/etiologia , Proteínas Klotho , Fosfatos/sangue , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/complicaçõesRESUMO
UNLABELLED: Primary cultures of bovine parathyroid cells rapidly lose calcium responsiveness. Here, we show that bovine parathyroid cells grown in collagen coalesce into an organoid ("pseudogland") with stable calcium responsiveness. These findings also illustrate the importance of 3-D cellular architecture in parathyroid gland function. INTRODUCTION: The ability of extracellular calcium to suppress parathyroid hormone (PTH) secretion is quickly lost in primary monolayer cultures of bovine parathyroid cells. This has been attributed to a decrease in the expression of the cell surface calcium-sensing receptor (CaR), but other factors, including normal cell-to-cell interaction, may be critical. Here we describe a novel system for culturing bovine parathyroid cells that promotes re-formation of a three-dimensional (3-D) cellular architecture and re-establishment of calcium responsiveness. MATERIALS AND METHODS: Dispersed bovine parathyroid cells were cultured as monolayers or were mixed with type I collagen and placed in culture plates. CaR mRNA and the calcium regulation of PTH secretion were measured over a period of several weeks in parathyroid cells cultured both in collagen matrix and as monolayers. Calcium regulation of PTH mRNA was also investigated. RESULTS AND CONCLUSIONS: Within 1-2 weeks in collagen culture, parathyroid cells coalesced into a small mass approximately 1-2 mm in size (referred to as a pseudogland). Suppression of PTH secretion by high calcium was blunted at 1 day in collagen, but returned within 1 week, and was retained through 3 weeks; the calcium set point (1.05 +/- 0.04 mM) was similar to that reported for freshly dispersed cells. PTH mRNA was also suppressed by increasing extracellular calcium. CaR mRNA expression was decreased at 1 day in collagen and increased with time in culture, although never reaching the level found in dispersed cells. In bovine parathyroid cells cultured as monolayers, however, suppression of PTH by calcium was observed only at day 1 in culture. CaR mRNA content fell by 70% at day 1 but remained stable thereafter. Thus, a total loss of calcium responsiveness in monolayers was observed despite significant residual expression of CaR, suggesting that loss of the calcium response cannot be attributed solely to decreased CaR. In summary, the pseudogland model illustrates the importance of the 3-D cellular architecture in parathyroid gland function and provides a useful model in which to investigate calcium-mediated control of parathyroid gland functions, especially those requiring extended treatment.
Assuntos
Sinalização do Cálcio , Colágeno/fisiologia , Glândulas Paratireoides/citologia , Animais , Reatores Biológicos , Matriz Óssea , Bovinos , Células Cultivadas , Colágeno/ultraestrutura , Glândulas Paratireoides/anatomia & histologia , RNA Mensageiro/metabolismo , Ratos , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Fatores de TempoRESUMO
Secondary hyperparathyroidism (secondary HPT), a common disorder in chronic renal failure (CRF) patients, is characterized by hypersecretion of parathyroid hormone (PTH), parathyroid hyperplasia, and decreased expression of the calcium-sensing receptor (CaR). Dietary phosphate loading promotes secondary HPT, and phosphate restriction prevents and arrests secondary HPT in CRF. This study examined the ability of phosphate restriction to restore parathyroid CaR expression and function. Uremic rats fed a 1.2% P diet for 2 weeks developed secondary HPT with down-regulated CaR expression. Continuation on the 1.2% P diet for 2 more weeks worsened the secondary HPT and further decreased CaR, but switching the rats to a 0.2% P diet for 2 weeks normalized PTH, arrested parathyroid hyperplasia, and restored CaR expression to normal. The calcium-PTH relationship was abnormal in uremic rats fed a high phosphate (HP) diet with a right-shifted calcium set point but was corrected by 2 weeks of phosphate restriction. A time course revealed that following the switch to a low phosphate diet, PTH levels were normalized by day 1, and growth was arrested by day 2, but CaR expression was restored between days 7 and 14. We conclude that although phosphate restriction restores CaR expression and function in parathyroid glands of uremic rats, it is a late event and not involved in the arrest of secondary HPT.
Assuntos
Hiperparatireoidismo Secundário/prevenção & controle , Fosfatos/administração & dosagem , Receptores de Superfície Celular/metabolismo , Receptores de Hormônios Paratireóideos/metabolismo , Animais , Feminino , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de CálcioRESUMO
The active vitamin D compound 1alpha,24(S)-dihydroxyvitamin D(2) (1,24(OH)(2)D(2)) is under development as a therapy for disorders including cancer and secondary hyperparathyroidism. 1,24(OH)(2)D(2) is a potent inhibitor of cell proliferation in vitro and, relative to calcitriol (1,25(OH)(2)D(3)), has reduced calcemic activity in vivo. To examine the mechanisms underlying this reduced calcemic activity, we studied the tissue distribution in rats of radiolabeled 1,24(OH)(2)D(2) or 1,25(OH)(2)D(3) over 24h. Serum levels of 1,24(OH)(2)D(2) were lower than those of 1,25(OH)(2)D(3) at all time points; however, tissue levels of radiolabeled compounds followed different patterns. In duodenum and kidney, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) rose to similar levels at early time points; 1,24(OH)(2)D(2) levels then declined more rapidly. In bone marrow, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were present at similar levels at all time points. In liver, 1,24(OH)(2)D(2) levels were two-fold higher than 1,25(OH)(2)D(3) at 1h post-injection, declining to similar levels by 8h. In vitamin D-deficient rats, doses of 1,24(OH)(2)D(2) 30-fold higher than 1,25(OH)(2)D(3) were required to produce equal stimulation of intestinal calcium absorption. In the same deficient animals, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were nearly equipotent at stimulating bone calcium mobilization. In cultured bone cells, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were equipotent at stimulating osteoclast formation and bone resorption. In summary, the reduced calcemic activity of 1,24(OH)(2)D(2) may result from altered pharmacokinetics relative to 1,25(OH)(2)D(3), resulting in relatively rapid decreases in 1,24(OH)(2)D(2) levels and activity in target organs such as intestine. Further studies will be necessary to confirm these findings and to confirm the clinical utility of 1,24(OH)(2)D(2).
Assuntos
Cálcio/metabolismo , Ergocalciferóis/metabolismo , Animais , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
The vitamin D analog ED-71 [1α,25-dihydroxy-2ß-(3-hydroxypropyloxy)vitamin D(3)] has been approved for treatment of osteoporosis in Japan, but its effects on mineral metabolism have not been fully explored. We investigated the actions of ED-71 on phosphate (Pi) absorption and induction of the intestinal sodium/phosphate cotransporters. Oral treatment of vitamin D-deficient rats with ED-71 (20 pmol every other day for 8 d) produced a maximal 8-fold increase in duodenal Pi absorption, measured by the in situ loop method, whereas 1,25-dihyroxyvitamin D(3) [1,25(OH)(2)D(3]), at doses up to 150 pmol, had no effect. This action of ED-71 was attributable to a dramatic 24-fold induction of sodium-dependent Pi transporter type IIb (NaPi-IIb) mRNA in the duodenum; Pit-1 and Pit-2 mRNA levels were not increased. In vitamin D-replete rats, ED-71 treatment (50 pmol) at 72 and 24 h before death increased NaPi-IIb mRNA in the duodenum and jejunum, but not the ileum, whereas 1,25(OH)(2)D(3) at 1000 pmol was ineffective in all segments. Single oral doses of ED-71 increased mouse intestinal NaPi-IIb mRNA and protein between 6 and 24 h. Surprisingly, rat lung NaPi-IIb was not increased by ED-71, despite its coexpression with the vitamin D receptor in alveolar type II cells. However, ED-71 did not induce intestinal NaPi-IIb in vitamin D receptor-ablated mice. The greater potency of ED-71 than 1,25(OH)(2)D(3) on NaPi-IIb appears to be due to much higher and more prolonged levels of ED-71 in the circulation. In summary, ED-71, due to its disparate pharmacokinetics, is a much more potent inducer of intestinal Pi absorption and NaPi-IIb than 1,25(OH)(2)D(3), suggesting a role for this analog in the treatment of Pi-wasting disorders.
Assuntos
Calcitriol/análogos & derivados , Intestinos/efeitos dos fármacos , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Deficiência de Vitamina D/metabolismo , Absorção/efeitos dos fármacos , Animais , Calcitriol/farmacologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Vitamina D/análogos & derivadosRESUMO
CONTEXT: Parathyroid oxyphil cells, whose function is unknown, are thought to be derived from chief cells. Oxyphil cells increase in number in parathyroid glands of patients with chronic kidney disease (CKD) and are even more abundant in patients receiving treatment for hyperparathyroidism with calcitriol and/or the calcimimetic cinacalcet. OBJECTIVE: We examined oxyphil and chief cells of parathyroid glands of CKD patients for differential expression of genes important to parathyroid function. DESIGN/SETTING/PARTICIPANTS: Parathyroid tissue from CKD patients with refractory hyperparathyroidism was immunostained for gene expression studies. MAIN OUTCOME MEASURE: Immunostaining for PTH, PTHrP, calcium-sensing receptor, glial cells missing 2, vitamin D receptor, 25-hydroxyvitamin D-1α-hydroxylase, and cytochrome c was quantified and expression reported for oxyphil and chief cells. RESULTS: Expression of all proteins analyzed, except for the vitamin D receptor, was higher in oxyphil cells than in chief cells. CONCLUSION: Human parathyroid oxyphil cells express parathyroid-relevant genes found in the chief cells and have the potential to produce additional autocrine/paracrine factors, such as PTHrP and calcitriol. Additional studies are warranted to define the secretory properties of these cells and clarify their role in parathyroid pathophysiology.
Assuntos
Perfilação da Expressão Gênica , Células Oxífilas/metabolismo , Glândulas Paratireoides/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/fisiologia , Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/sangue , Receptores de Calcitriol/fisiologia , Receptores de Detecção de Cálcio/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Parathyroid glands express the 25-hydroxyvitamin D(3) 1α-hydroxylase (1αOHase). 1,25-dihydroxyvitamin D(3) (calcitriol) synthesized by extrarenal tissues generally does not enter the circulation, but plays an autocrine/paracrine role specific to the cell type, and is regulated by the needs of that particular cell. While the role of calcitriol produced in the parathyroid glands presumably is to suppress PTH and cell growth, its regulation in this cell type has not been defined. In the present study, we found that regulation of the human parathyroid 1αOHase differs from the renal enzyme in that it is induced by FGF-23 and extracellular calcium. Hyperplastic parathyroid glands from patients with chronic kidney failure normally display a heterogeneous cellularity. We found that the 1αOHase is expressed at much higher levels in oxyphil cells than in chief cells in these patients. Recent findings indicate that oxyphil cell content is increased by treatment with calcium receptor activators (calcimimetics). Here, we demonstrate that the calcimimetic cinacalcet increases the expression of 1αOHase in human parathyroid cultures. Additionally, we found that the 1αOHase in human parathyroid cultures is functionally active, as evidenced by the ability of the enzyme to 1-hydroxylate 25(OH)D(3) in parathyroid monolayers. Calcium, as well as cinacalcet, also induced expression of the degradation enzyme 24-hydroxylase, indicating the presence of a negative feedback system in the parathyroid cells. Therefore, local production of 1αOHase suggests an autocrine/paracrine role in regulating parathyroid function and may mediate, in part, the suppression of PTH by calcium and FGF-23.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Glândulas Paratireoides/enzimologia , Calcitriol/metabolismo , Cálcio/metabolismo , Células Cultivadas , Fator de Crescimento de Fibroblastos 23 , Humanos , Glândulas Paratireoides/metabolismo , Glândulas Paratireoides/patologia , RNA Mensageiro/metabolismoRESUMO
Vitamin D compounds regulate PTH at the transcriptional level, presumably via binding to the vitamin D receptor (VDR), but the exact mechanism is presently unclear. We recently reported that the several vitamin D prohormones with low VDR affinity suppressed PTH, even when their activation was inhibited, raising the possibility that their actions may be VDR independent. To test this hypothesis, we developed a novel organ culture that allowed the assessment of activities of the prohormones on PTH release from wild-type and VDR-null thyroparathyroid explants. The cultures remained viable with respect to PTH release for at least 2 weeks. Full suppression of PTH by the native vitamin D hormone, 1α,25-dihydroxyvitamin D(3) [1α,25 (OH)(2)D(3)], required 2 days, consistent with a transcriptional mechanism, and was reversible, indicating that reduced PTH was not attributable to cell death. Inhibition of PTH release by 1α,25 (OH)(2)D(3) and two prohormones, 25-hydroxyvitamin D(3) and 1α-hydroxyvitamin D(2), was observed in explants from wild-type mice but not in those from VDR-null mice. These findings 1) are the first direct demonstration of the role of the VDR in regulation of PTH by 1α,25(OH)(2)D(3), 2) confirm that the suppressive actions of the vitamin D prohormones are mediated by the VDR, and 3) introduce a novel organ culture model that allows the ex vivo study of the function of parathyroid glands from transgenic animals.
Assuntos
Calcifediol/farmacologia , Ergocalciferóis/farmacologia , Glândulas Paratireoides/efeitos dos fármacos , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo , Receptores de Calcitriol/deficiência , Animais , Calcifediol/metabolismo , Calcitriol/metabolismo , Calcitriol/farmacologia , Ergocalciferóis/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos/métodos , Glândulas Paratireoides/citologia , Hormônio Paratireóideo/antagonistas & inibidores , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Receptores de Calcitriol/genética , Transcrição GênicaRESUMO
Glial cells missing-2 (Gcm2) is the key regulating transcription factor for parathyroid gland development. The continued expression of high levels of Gcm2 in mature parathyroid glands suggests that it is required for maintenance of parathyroid cell differentiation. The role of Gcm2 in parathyroid cell physiology, however, has not been fully studied. In this study, we examined the effects of Gcm2 silencing on cultured human parathyroid cells. Collagenase-dispersed human parathyroid cells from patients with chronic kidney disease were placed in monolayer cultures and infected with lentivirus expressing shRNA for human Gcm2. Seventy-two hours after infection, mRNA was processed and analyzed for Gcm2, PTH, vitamin D receptor (VDR), calcium-sensing receptor (CaR), 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (1-OHase), and proliferating cell nuclear antigen (PCNA) by real-time PCR (qPCR). Protein expression of affected genes was analyzed by immunoblot 72 h after infection. Gcm2 mRNA and protein were decreased by 74.2 +/- 12.2% (SD; n = 3 experiments; p < 0.01) and 67.5 +/- 15.7% (n = 2; p < 0.01), respectively. CaR mRNA and protein were reduced by 47.8 +/- 21.1% (n = 3; p < 0.01) and 48.1 +/- 4.3% (n = 3; p < 0.01), respectively. However, VDR, PTH, 1-OHase, and PCNA were not significantly affected by Gcm2 silencing. Further analysis of CaR mRNA indicated that transcripts containing exon 1B, derived by transcription from CaR promoter 2, were downregulated (58.8 +/- 19.27%; n = 3; p < 0.05) by Gcm2 silencing. Exon 1A-containing transcripts from promoter 1 were expressed at very low levels in the cultures. These results indicate that one function of Gcm2 is to maintain high levels of CaR expression in parathyroid cells.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/metabolismo , Glândulas Paratireoides/metabolismo , Receptores de Detecção de Cálcio/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Éxons/fisiologia , Inativação Gênica , Humanos , Proteínas Nucleares/genética , Glândulas Paratireoides/citologia , Hormônio Paratireóideo/biossíntese , Antígeno Nuclear de Célula em Proliferação/biossíntese , Regiões Promotoras Genéticas/fisiologia , Receptores de Calcitriol/biossíntese , Receptores de Detecção de Cálcio/genética , Esteroide Hidroxilases/biossíntese , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Extracellular Ca reduces parathyroid hormone (PTH) levels through several mechanisms, but many details of the intracellular steps involved have been difficult to elucidate because of the lack of a suitable parathyroid cell model. The present studies utilized our Ca-responsive bovine parathyroid organoid culture system (pseudoglands) to examine PTH mRNA in intact parathyroid cells. Increasing medium calcium from 0.4 to 3.0 mM reduced PTH mRNA to 20-30% of basal by 16 h. Reducing medium Ca from 3.0 to 0.4 mM restored PTH mRNA levels over a 24-h period. PTH mRNA was also reduced by the calcimimetic R-568, confirming the role of the calcium-sensing receptor. PTH decay rates were determined by placing pseudoglands in either 0.4 or 3.0 mM Ca for 2 h and then blocking gene transcription. PTH mRNA remained stable for at least 24 h in pseudoglands incubated in 0.4 mM Ca, but fell gradually by 62% in the presence of 3.0 mM Ca. Blocking transcription prior to the addition of high-Ca medium dramatically blunted the Ca-induced degradation of PTH mRNA, indicating that acceleration of PTH mRNA decay by Ca requires gene transcription. Pharmacologic investigation of the signaling pathways involved indicated that the Ca-induced reduction of PTH mRNA did not involve MAP kinase, phospholipase D, or cyclic AMP. However, increasing cytosolic Ca with thapsigargin or the Ca ionophore A23187 decreased PTH mRNA levels. In summary, Ca-mediated destabilization of PTH mRNA requires gene transcription and involves increases in cytosolic Ca.
Assuntos
Compostos de Anilina/metabolismo , Cálcio/metabolismo , Glândulas Paratireoides/citologia , Hormônio Paratireóideo , RNA Mensageiro/metabolismo , Transcrição Gênica , Animais , Cálcio/agonistas , Bovinos , Células Cultivadas , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Fenetilaminas , Propilaminas , Estabilidade de RNA , Transdução de Sinais/fisiologia , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Active vitamin D compounds are widely used in the treatment of secondary hyperparathyroidism associated with renal failure. These compounds reduce PTH secretion through vitamin D receptor (VDR)-dependent repression of PTH gene transcription. In previous studies, 1alpha(OH)D3, a vitamin D prodrug, inhibited PTH secretion in cultured bovine parathyroid cells, but it was unclear whether 1alpha(OH)D3 itself or an active metabolite produced this inhibition. METHODS: We determined the effectiveness of the vitamin D prodrugs 1alpha(OH)D3, 1alpha(OH)D2 and 1alpha(OH)-24(R)-methyl-25-ene-D2 (BCI-210) at inhibiting PTH secretion in bovine parathyroid cell cultures, and examined the metabolism of [3H]1alpha(OH)D2 in these cells. RESULTS: All three prodrugs suppressed PTH secretion with approximately 10% of the activity of 1,25(OH)2D3; much higher activity than expected based on the VDR affinities of these prodrugs (0.25% of 1,25(OH)2D3). Parathyroid cells activated [3H]1alpha(OH)D2 to both 1,25(OH)2D2 and 1,24(OH)2D2. 1,24(OH)2D2 was detectable at 4 h, increased to a maximum at 8 h, and then decreased. In contrast, 1,25(OH)2D2 levels increased linearly with time, suggesting the presence of constitutively active vitamin D-25-hydroxylase not previously reported in parathyroid cells. The cytochrome P-450 inhibitor ketoconazole (50 microM) reduced 1alpha(OH)D2 metabolism to below detectable levels, but did not significantly affect suppression of PTH by 1alpha(OH)D2. CONCLUSIONS: The vitamin D prodrugs 1alpha(OH)D3, 1alpha(OH)D2 and BCI-210 suppressed PTH production by cultured parathyroid cells. The ability of 1alpha(OH)D2 to reduce PTH despite inhibition of its metabolism suggests a direct action of this 'prodrug' on the parathyroid gland, but the mechanism underlying this activity is not yet known.