Rat Parathyroid Research Articles

Ameliorative Effect of Chitosan Against Lithium Carbonate Subacute Toxicity on Kidney and Parathyroid Gland of Adult Male Albino Rats

Ameliorative Effect of Chitosan Against Lithium Carbonate Subacute Toxicity on Kidney and Parathyroid Gland of Adult Male Albino Rats

High sodium promotes the secretion and synthesis of PTH through PiT-1-IKKβ pathway in parathyroid gland in vitro.

Parathyroid hormone (PTH) is secreted by the parathyroid glands (PTGs) and is an important hormone regulating mineral metabolism. Previous studies reported that high sodium diet will cause the increase in serum PTH, but the specific mechanism is unknown. Consequently, the present study aims to investigate the effects and mechanisms of high sodium on PTH synthesis and secretion from PTGs. We developed a tissue culture model using normal rat PTGs, discovered that sodium elicited and promoted concentration-dependent and time-dependent PTH secretion. Changes in sodium-associated transporters from PTGs incubated with high sodium were thoroughly examined. Increased expression of sodium-phosphate cotransporter Slc20a1 (also known as PiT-1) was observed. Further tests revealed that PiT-1 activated the NF-κB signaling pathway, resulting in increased IKKβ phosphorylation, IKBα degradation, and increased p65 phosphorylation followed by nuclear entry, which led to increased PTH transcription. Meanwhile, IKKβ phosphorylated SNAP23, promoting exocytosis and eventually led to increased PTH secretion. In conclusion, our findings indicate that PiT-1 plays an important role in the increased secretion and synthesis of PTH directly induced by high sodium under physiological conditions, and may provide a potential therapeutic target for secondary hyperparathyroidism (SHPT).

#6057 REDUCTION OF SUBACUTE LEAD INDUCED PARATOHORMONE LEVELS FOLLOWING ANTIOXIDANTS OR VITAMIN D/CALCIUM ADMINISTRATION IN RATS

Abstract Background and Aims Lead (Pb) is a toxic heavy metal; exposure is associated with many diseases. Accordingly, the World Health Organization considers Pb one of 10 chemicals of major public health concern. Experimental evidence suggests that Pb exposure induces both structural and functional kidney alterations. Epidemiological studies on lead exposed workers, show alteration on calcium metabolism (reduction in serum calcium, phosphorous and vitamin D and increase level of PTH) that increases with exposure length. Two of the suggested molecular mechanism of lead toxicity are: protein binding sites competition with endogenous cations such as calcium or zinc and the production of free radicals. The aim of this work is to analyze the effect of lead exposure, the therapeutic administration of antioxidants (melatonin or silymarin) or VitD +Ca and its potentially beneficial effect on calcium metabolism. Method Male Wistar rats (150–180 g body weight) of 6 weeks of age were housed in groups of three in standard cages with free access to water and food (Rodent Lab Standar Diet), under a 12/12 hour light/dark cycle, C, control: untreated rats not exposed to Pb or treatment; Pb, exposed to Pb for 1 month and assessed immediately; Pb 1NT, exposed to Pb for 1 month followed by 1 month without any treatment; Pb 1TM, exposed to Pb for 1 month followed by 1 month of melatonin treatment; Pb 1TS, exposed to Pb for 1 month followed by 1 month of silymarin treatment; Pb 1TVDCa, exposed to Pb for 1 month followed by 1 month of VitDCa treatment. M, melatonin (10 mg/kg/body weight/day dose), S silymarin (200 mg/kg/body weight/day dose) or total amount of VitD (232 IU/Kg/day) and Calcium 299 mg/Kg/day received from diet and therapeutic regimen were equal to 1.8 fold the daily requirement of VitD 800 IU/Kg/day and Calcium 1000 mg/Kg/day. Chemicals: Lead acetate trihydrate, Pb(CH3CO2)2.3H2O purity 99.99% from Sigma 215902, Melatonin Sigma M5250 98% TLC, VitaminD3 ampoules (100 000IU/mL), vitamin D oral ampoules (100 000IU/2 mL) from TRB Lab. Rat parathyroid PT Elisa Kit CUSABIO, 25-hydroxy Vitamin D3 ELISA Kit (Colorimetric) Kit NOVUS Results Blood Lead Levels There were statistical differences between the control group and all other groups P<0.0001. The group exposed to Pb for 1 month without any subsequent treatment (Pb 1NT) show differences in BLL with rats exposed to Pb and subsequently treated with antioxidant or VitD therapy and with the group immediately assessed after lead exposure. BLL were: all in μg/dL C 2.3, Pb 11, PbNT14.15, PbTM 10.28, PbTS 10.23 and Pb VDCa 10.18 Vitamin D Blood Levels Interestingly the highest mean vitamin D blood level was found in rats exposed to Pb for 1 month followed by 1 month of VitDCa therapy (Pb 1TVDCa) and the lowest mean value was found in rats exposed to Pb for 1 month and assessed immediately (Pb). PTH Blood Levels PTH blood levels were increased in rats exposed to Pb without any other treatment, whether assessed immediately after exposure or after one month. All therapies reduced blood PTH under control levels. Conversely a negative moderate correlation was found between blood levels of PTH vs. VitD r = −0.533 and r2 = 0.2843 P (two tailed) = 0.002. Conclusion All therapies using the antioxidants (melatonin or silymarin) or VitD+Ca supplementation reduced the lead induced increase in parathormone levels in rats.

Establishment and characterization of long-term human primary parathyroid tumor subclones derived from Indian PHPT.

The continuous cell line of epithelial human parathyroid cells has been proven difficult. Previously, PTH-C1 cell line was only established rat parathyroid tissue cell line known to express the parathyroid hormone-related peptide (Pthrp) gene. The paucity of continuous cell line of human parathyroid cells secreting parathyroid hormone (PTH) has imposed hurdle in in vitro assessment of the mechanisms involved in the control of parathyroid cell function and proliferation. The primary cell cultures of human parathyroid cells were derived from parathyroid adenoma tissue biopsy (n = 5). The cells were subsequently subcultured to maintained primary subclones. Karyotyping analysis was performed to analyze the genotypic identity of derived subclones. The expression of calcium-sensing receptor (CaSR) and intact parathyroid hormone (iPTH) were analyzed using immunocytochemistry and immunofluorescence. In the present study, we have used a defined condition medium to generate the continuous culture of human parathyroid cells derived from patients with parathyroid adenoma due to primary hyperparathyroidism. The subcultured primary subclones were maintained epithelial and polygonal morphology, doubling time of approximately 25h, displaying a diploid chromosome number, and secretion of PTH. This cell line produces PTH and expresses the calcium-sensing receptor (CaSR) known to be involved in parathyroid function. Altogether these findings indicate the uniqueness of the human parathyroid cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.

Kidney Failure Alters Parathyroid Pin1 Phosphorylation and Parathyroid Hormone mRNA-Binding Proteins, Leading to Secondary Hyperparathyroidism.

Secondary hyperparathyroidism (SHP) is a common complication of CKD that increases morbidity and mortality. In experimental SHP, increased parathyroid hormone (PTH) expression is due to enhanced PTH mRNA stability, mediated by changes in its interaction with stabilizing AUF1 and destabilizing KSRP. The isomerase Pin1 leads to KSRP dephosphorylation, but in SHP parathyroid Pin1 activity is decreased and hence phosphorylated KSRP fails to bind PTH mRNA, resulting in high PTH mRNA stability and levels. The up- and downstream mechanisms by which CKD stimulates the parathyroid glands remain elusive. Adenine-rich high-phosphate diets induced CKD in rats and mice. Parathyroid organ cultures and transfected cells were incubated with Pin1 inhibitors for their effect on PTH expression. Mass spectrometry was performed on both parathyroid and PTH mRNA pulled-down proteins. CKD led to changes in rat parathyroid proteome and phosphoproteome profiles, including KSRP phosphorylation at Pin1 target sites. Furthermore, both acute and chronic kidney failure led to parathyroid-specific Pin1 Ser16 and Ser71 phosphorylation, which disrupts Pin1 activity. Pharmacologic Pin1 inhibition, which mimics the decreased Pin1 activity in SHP, increased PTH expression ex vivo in parathyroid glands in culture and in transfected cells through the PTH mRNA-protein interaction element and KSRP phosphorylation. Kidney failure leads to loss of parathyroid Pin1 activity by inducing Pin1 phosphorylation. This predisposes parathyroids to increase PTH production through impaired PTH mRNA decay that is dependent on KSRP phosphorylation at Pin1-target motifs. Pin1 and KSRP phosphorylation and the Pin1-KSRP-PTH mRNA axis thus drive SHP.

Promotion of allogeneic parathyroid cell transplantation in rats with hypoparathyroidism.

This study sought to establish a rat model of hypoparathyroidism by removing the rat parathyroid glands, and compare the effects of different transplantation sites and transplantation methods using a primary culture of parathyroid cells in vitro on the hormone secretion of the model rats. Male Sprague Dawley (SD) rats were selected for in vivo parathyroid gland removal, and rats with abnormal postoperative water intake, weight gain, parathyroid hormone (PTH), and blood calcium ion concentration were selected as transplant recipients and divided into the model group, brachioradialis muscle cell transplantation group, gelatin sponge group, and subcutaneous transplantation group. The parathyroid tissue was removed and the primary cell culture was performed in vitro using homozygous SD rats as graft donors. When the parathyroid cells were able to secrete PTH, transplantation was performed, and the postoperative recovery of the PTH function of the rats with different transplantation sites and methods were observed. A recipient model with low PTH was successfully established, and parathyroid progenitor cells with obvious PTH secretion were obtained. Better secretion was observed in the brachioradialis cell group compare with other groups. The in vitro primary cell culture of the donor parathyroid cells combined with cell transplantation significantly improved the physiological function of the hypoparathyroid rats, and could potentially replace traditional clinical brachioradialis muscle tissue transplantation.

Aberrant Epigenetic Alteration of PAX1 Expression Contributes to Parathyroid Tumorigenesis.

Primary hyperparathyroidism (PHPT) results from the hypersecretion of parathyroid hormone from parathyroid tumors. A transcription factor, namely Paired box1 (PAX1), is active in parathyroid gland development. We aimed to study potential epigenetic-mediated mechanism of PAX1 gene in sporadic parathyroid adenomas. In parathyroid adenomas tissues, we analyzed the DNA methylation via bisulfite-specific polymerase chain reaction (BSP) and histone modifications via chromatin immunoprecipitation in regulating the differential expression of PAX1. The results showed that mRNA and protein expression of PAX1 was significantly reduced in parathyroid adenomas. Bisulfite sequencing demonstrated hypermethylation in the promoter region of PAX1 (35%; 14/40) and lower levels of histone 3 lysine 9 acetylation (H3K9ac) were observed on the promoter region of PAX1 (6-fold; P < .004) in parathyroid adenomas. Furthermore, upon treatment with a pharmacologic inhibitor, namely 5'aza-2 deoxycytidine, in rat parathyroid continuous cells, we found re-expression of PAX1 gene. Our study not only reveals expression of PAX1 is epigenetically deregulated but also paves a way for clinical and therapeutic implications in patients with PHPT.

Evaluation of structural and ultrastructural changes in thyroid and parathyroid glands after near infrared irradiation: study on an animal model.

Intraoperative identification of parathyroid glands is a tough task for surgeons performing thyroid or parathyroid surgery, because the small size, color and shape of these glands hinder their discrimination from other cervical tissues. In 2011, Paras described the autofluorescence of parathyroid glands, a property that could facilitate their intraoperative identification. Parathyroid glands submitted to a 785 nm laser beam emit fluorescence in the near infrared range, with a peak at 822 nm. As the intrinsic properties of secretory tissues may be affected by the exposure to the near infrared light, a situation that could preclude their intraoperative utilization, the authors compared the structural and ultra-structural patterns of rat’s thyroid and parathyroid glands submitted to irradiation replicating the conditions that allow their intraoperative identification, with those of non irradiated animals. Twenty-four Wistar rats were divided into six groups: animals of Groups 1, 3 and 5 were submitted under general anesthesia to direct irradiation of the cervical area with a 780 nm LED light for 3 minutes through a cervical incision, and animals of Groups 2, 4 and 6 were submitted to cervical dissection without irradiation. Animals of were euthanized immediately (Groups 1 and 2), at Day 30 (Groups 3 and 4) at and at Day 60 (Groups 5 and 6) and thyroid and parathyroid glands were removed: one lobe was prepared for conventional pathological examination and the other lobe for electron microscopy observed by three experienced pathological experts. Twenty-four samples were prepared for conventional histology and there were no alterations reported in any group. Due to technical problems, only 21 samples were observed by electron microscopy and there were no differences in the ultrastructure of parathyroid and thyroid glands, namely the nuclear pattern, mitochondria, endoplasmic reticulum or secretory granules, in any of the groups. These results confirm the innocuity of near infrared irradiation’, allowing its intraoperative utilization.

Ameliorative effects of jamun seed and orange peel extracts on microcystin LR induced alterations in calcitonin cells and parathyroid gland of rats.

This study investigated changes in calcitonin cells (C-cells) and parathyroid glands (PTG) induced by microcystin LR (MCLR) exposure to rats and evaluated ameliorative effects of jamun (Syzygium cumini) seed (JSE) and orange (Citrus sinensis) peel (OPE) extracts. Wistar rats were treated as-Group A (control), Group B (MCLR), Group C (MCLR + JSE), Group D (MCLT + OPE), Group E (OPE) and Group F (JSE). Microcystin dose was (10 μg/kg body wt/day whereas OPE and JSE dose was 200 mg/kg body wt/day. Thyroid and PTG were fixed on 15 and 30 days following the treatment. C-cells of treated rats for 15 days with MCLR; MCLR + JSE and MCLR + OPE exhibit degranulation, mitochondrial swelling and prominent RER. In MCLR treated rats few cells completely lack secretory granules. After 30 days MCLR treatment accumulation of secretory granules and degeneration were noticed in C-cells. C-cell nuclear volume (NV) of MCLR, MCLR + JSE and MCLT + OPE treated rats show an increase. In MCLR, MCLR + JSE and MCLR + OPE treated rats PTG exhibit hyperchromatic nuclei, nuclear elongation and increased NV after 15 days. After 30 days MCLR treatment nuclei of PTG become more hyperchromatic, more elongated, show degeneration of nuclei and increase in NV. NV is increased in Group C and Group D. PTG remain unaltered 30 days following treatment with OPE and JSE. Microcystin LR provoke physiological effects on the blood calcium and alterations in C cells and PTG, which cause serious threat to organism. These changes can be protected by JSE and OPE.

Co-transplantation with adipose-derived cells to improve parathyroid transplantation in a mice model

BackgroundAccidentally removed parathyroid glands are still challenging in neck surgery, leading to hypoparathyroidism characterized with abnormally low levels of parathyroid hormone. Parathyroid auto-transplantation is usually applied in compensation. To improve the efficiency of parathyroid transplantation, we introduced a method by co-transplanting with adipose-derived cells, including stromal vascular fractions (SVFs) and adipose-derived stem cells (ADSCs), and investigated the underlying molecular mechanisms involved in parathyroid transplantation survival.MethodsRat and human parathyroid tissues were transplanted into nude mice as parathyroid transplantation model to examine the effects of SVFs and ADSCs on grafts angiogenesis and survival rates, including blood vessel assembly and parathyroid hormone levels. Several angiogenic factors, such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF) 2, were assessed in parathyroid grafts. The effects of hypoxia were investigated on ADSCs. The modulatory roles of the eyes absent homolog 1 (EYA1), which is vital in parathyroid development, was also investigated on angiogenic factor production and secretion by ADSCs. All experimental data were statistically processed. Student’s t test was used to assess significant differences between 2 groups. For multiple comparisons with additional interventions, two-way ANOVA followed by Tukey’s post hoc test was performed. P < 0.05 was considered as significant.ResultsSVFs improve rat parathyroid transplantation survival and blood vessel assembly, as well as FGF2 and VEGF-A expression levels in parathyroid transplantation mice. Functional human parathyroid grafts have higher microvessel density and increased VEGF-A expression. The supernatant of ADSCs induced tubule formation and migration of human endothelial cells in vitro. Hypoxia had no effect on proliferation and apoptosis of human ADSCs but induced higher angiogenic factor levels of VEGF-A and FGF2, modulated by EYA1, which was confirmed by parathyroid glands transplantation in mice.ConclusionsAdipose-derived cells, including ADSCs and SVFs, improve parathyroid transplantation survival via promoting angiogenesis through EYA1-regulating angiogenetic factors in vitro and in vivo. Our studies proved an effective method to improve the parathyroid autotransplantation, which is promising for clinical patients with hypoparathyroidism when parathyroid glands were accidentally injured, removed, or devascularized.

Pharmacology of Parsabiv® (etelcalcetide, ONO-5163/AMG 416), a novel allosteric modulator of the calcium-sensing receptor, for secondary hyperparathyroidism in hemodialysis patients

Etelcalcetide hydrochloride (Parsabiv®, ONO-5163/AMG 416) is an allosteric modulator of the calcium (Ca)-sensing receptor that was originally produced by KAI Pharmaceuticals Inc. (now Amgen Inc.). It has recently been approved as the first intravenous calcimimetic agent for secondary hyperparathyroidism (SHPT) in many countries. Etelcalcetide is an intravenous injectable drug that can be administered and eliminated through the dialysis circuit in chronic kidney disease patients. In the present study, we evaluated the in vitro pharmacological profile and in vivo parathyroid hormone (PTH)- and Ca-lowering activities of etelcalcetide in a rat 5/6 nephrectomy model of chronic renal insufficiency with SHPT. Etelcalcetide increased the intracellular Ca concentration in HEK-293T cells expressing human Ca-sensing receptor with an EC50 value (95% confidence interval) of 0.53 μM (0.28–1.0 μM) and suppressed PTH secretion from rat parathyroid gland cells with 0.36 μM (0.24–0.54 μM) by activating Ca-sensing receptor. The specificity of etelcalcetide was evaluated by examining its ability to stimulate or inhibit radioligand binding to a panel of 34 off-target proteins. There were no significant changes in the presence of 10 μM etelcalcetide. Furthermore, in a rat 5/6 nephrectomy model of chronic renal insufficiency with SHPT, single intravenous administration of etelcalcetide at 0.3, 1.0, and 3.0 mg/kg decreased plasma PTH and serum Ca levels. Taken together, the present findings identify etelcalcetide as a calcimimetic with potent PTH- and Ca-lowering effects via Ca-sensing receptor agonist activity.

Ultramicroscopic changes of rat parathyroid glands after immunosuppression

Background . Parathyroid glands actively respond to exogenous and endogenous factors that confirmed by a number of fundamental researches. Currently there are no comprehensive morphological works devoted to the study of ultramicroscopic features of parathyroid glands during immune status changing. Cyclophosphamide is widely used in the clinical practice. It’s belongs to a class of drugs known as alkylating agents, which have been used to treat some types of cancer. Objective. The objective of the work was to study features of electron-microscopic structure of the rat parathyroid glands under immunosuppression caused by the administration of cyclophosphamide. Methods . The study was conducted on 24 outbred male mature rats with the weight 180±10g. Maintenance and care of animals were conducted in according to the norms of bioethics. The animals received an intramuscular injection of cyclophosphamide in a dose of 200 mg/kg body weight. The animals were withdrawn from the experiment on the 7 th and 30 th day after the injection. The ultrastructure of rat parathyroid glands in normal condition as well as after immunosuppression was studied. Results. The parenchyma of the parathyroid glands of intact animals is composed of two cell types: chief cells and oxyphil cells. Principal (chief) cells are the most numerous type of the gland’s parenchyma. They are small, polygonal cells, with a centrally located nucleus. The cytoplasm of the chief cells contains well-developed rough endoplasmic reticulum and Golgi apparatus. There are numerous dense secretory vesicles, large accumulations of glycogen, and lipid droplets in the cytoplasm. Oxyphil cells constitute a minor portion of the parenchymal cells. The cells are more rounded, considerably larger than the principal cells. Mitochondria, often with bizarre shapes and sizes, almost fill the cytoplasm and are responsible for the strong acidophilia of these cells. No secretory vesicles and little if any rough endoplasmic reticulum are present. Cytoplasmic inclusion bodies consist of occasional lysosomes, lipid droplets, and glycogen distributed among the mitochondria. Conclusion. The introduction of cyclophosphamide to rats in high doses caused marked changes in the ultrastructure of the organ. Polymorphism of cell nuclei, reducing the amount of the organelles that are participate in the protein synthesis, reducing the number of secretory granules, increasing the number of lipid and colloid droplets in the cell cytoplasm were observed in the early period of observation (7th day). On the 30th day after the administration of cyclophosphamide tendency to normalization of morphological structure of the organ revealed.

Original Research: Atorvastatin prevents rat cardiomyocyte hypertrophy induced by parathyroid hormone 1-34 associated with the Ras-ERK signaling.

We investigated the effects of atorvastatin (Ator) on cardiomyocyte hypertrophy (CMH) induced by rat parathyroid hormone 1-34 (PTH1-34) and Ras-extracellular signal regulated protein kinases 1/2 (ERK1/2) signaling. Rat cardiomyocytes were randomly divided into seven groups: normal controls (NC), PTH1-34 (10(-7) mol/L), Ator (10(-5) mol/L), farnesyl transferase inhibitors-276 (FTI-276, 4 × 10(-5) mol/L), PTH1-34 + Ator, PTH1-34 + FTI-276 and PTH1-34 + Ator + mevalonic acid (MVA, 10(-4) mol/L). After treatment, the hypertrophic responses of cardiomyocytes were assessed by measuring cell diameter, detecting protein synthesis, and single-cell protein content. The concentrations of hypertrophic markers such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured by ELISA. Protein expressions of ERK1/2, p-ERK1/2 and Ras were detected by western blotting. The results showed that compared with the PTH1-34 group, cellular diameter, 3H-leucine incorporation, single-cell protein content, ANP and BNP concentration decreased by 12.07 µm, 1622 cpm/well, 84.34 pg, 7.13 ng/L and 20.04 µg/L, respectively, and the expressions of Ras and p-ERK1/2 were downregulated in PTH1-34 + Ator group (P < 0.05). Compared to the PTH1-34 + Ator group, the corresponding hypertrophic responses and hypertrophic markers increased by 4.95 µm, 750 cpm/well, 49.08 pg, 3.12 ng/L and 9.35 µg/L, respectively, and the expressions of Ras and p-ERK1/2 were upregulated in the PTH1-34 + Ator + MVA group (P < 0.05). In conclusion, Ator prevents neonatal rat CMH induced by PTH1-34 and Ras-ERK signaling may be involved in this process.

Feasibility of injectable thermoreversible gels for use in intramuscular injection of parathyroid autotransplantation

Surgical transplantation of parathyroid tissue into the forearm muscle is one of the most commonly used surgical techniques. While simple, the procedure suffers from drawbacks. This study evaluated the feasibility of thermoreversible gel as an injectable carrier for parathyroid autotransplantation. Polyethyleneglycol-polyalanine-co-phenylalanine (PEG-PAF) thermoreversible gel (sol form at 4°C, gel form at 37°C) were manufactured. Thirty-eight Sprague-Dawley rats were divided into two groups (19 control, C group; 19 experimental, P group). The parathyroid glands of rats were excised. Parathyroid tissues were transplanted into the muscle pocket in sternocleidomastoid muscle in the C group. In the P group, the tissues were injected into the same muscle mixed with 0.3ml thermoreversible gel. The serum levels of parathyroid hormone (PTH), ionized calcium, and phosphorous were measured before surgical procedure, on 7, 21, 56, and 70days after surgery. Histology and immunohistochemistry were performed. Preoperative median PTH level of the C and the P group were 60.80 and 43.85pg/ml, respectively (p=0.641). Seventydays after surgery, median PTH level was 32.8 and 25.61pg/ml, respectively. On day 70, the PTH level was restored by 54% in the C group and 56% in the P group compared to the preoperative value (p=0.620). There were no significant intergroup differences in the ionized calcium/phosphorous level. Histology and immunohistochemistry revealed the successful transplantation of parathyroid tissues into the muscles in both groups. In conclusion, the PEG-PAF-based thermoreversible gel is a good candidate carrier material for intramuscular parathyroid autotransplantation.

Phosphorylation of Ribosomal Protein S6 Mediates Mammalian Target of Rapamycin Complex 1-Induced Parathyroid Cell Proliferation in Secondary Hyperparathyroidism.

Secondary hyperparathyroidism is characterized by increased serum parathyroid hormone (PTH) level and parathyroid cell proliferation. However, the molecular pathways mediating the increased parathyroid cell proliferation remain undefined. Here, we found that the mTOR pathway was activated in the parathyroid of rats with secondary hyperparathyroidism induced by either chronic hypocalcemia or uremia, which was measured by increased phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTOR pathway. This activation correlated with increased parathyroid cell proliferation. Inhibition of mTOR complex 1 by rapamycin decreased or prevented parathyroid cell proliferation in secondary hyperparathyroidism rats and in vitro in uremic rat parathyroid glands in organ culture. Knockin rpS6(p-/-) mice, in which rpS6 cannot be phosphorylated because of substitution of all five phosphorylatable serines with alanines, had impaired PTH secretion after experimental uremia- or folic acid-induced AKI. Uremic rpS6(p-/-) mice had no increase in parathyroid cell proliferation compared with a marked increase in uremic wild-type mice. These results underscore the importance of mTOR activation and rpS6 phosphorylation for the pathogenesis of secondary hyperparathyroidism and indicate that mTORC1 is a significant regulator of parathyroid cell proliferation through rpS6.

Differential expression and regulation of Klotho by paricalcitol in the kidney, parathyroid, and aorta of uremic rats

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 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 in Klotho in the kidney, increased expression in the parathyroid (31%), had no effect in the aortic media, but blunted the increase of Klotho in aortic adventitia. We propose that fibroblasts are responsible for 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.

PTH-C1: a rat continuous cell line expressing the parathyroid phenotype

The lack of a continuous cell line of epithelial parathyroid cells able to produce parathyroid hormone (PTH) has hampered the studies on in vitro evaluation of the mechanisms involved in the control of parathyroid cell function and proliferation. The PT-r cell line was first established from rat parathyroid tissue in 1987, but these cells were known to express the parathyroid hormone-related peptide (Pthrp) gene, but not the Pth gene. In an attempt to subclone the PT-r cell line, a rat parathyroid cell strain was isolated and named PTH-C1. During 3 years, in culture, PTH-C1 cells maintained an epithelioid morphology, displaying a diploid chromosome number, a doubling time around 15 h during the exponential phase of growth, and parathyroid functional features. PTH-C1 cell line produces PTH and expresses the calcium sensing receptor (Casr) gene and other genes known to be involved in parathyroid function. Most importantly, the PTH-C1 cells also exhibit an in vitro secretory response to calcium. Altogether these findings indicate the uniqueness of the PTH-C1 cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.

Magnesium modulates parathyroid hormone secretion and upregulates parathyroid receptor expression at moderately low calcium concentration

BackgroundThe interest on magnesium (Mg) has grown since clinical studies have shown the efficacy of Mg-containing phosphate binders. However, some concern has arisen for the potential effect of increased serum Mg on parathyroid hormone (PTH) secretion. Our objective was to evaluate the direct effect of Mg in the regulation of the parathyroid function; specifically, PTH secretion and the expression of parathyroid cell receptors: CaR, the vitamin D receptor (VDR) and FGFR1/Klotho.MethodsThe work was performed in vitro by incubating intact rat parathyroid glands in different calcium (Ca) and Mg concentrations.ResultsIncreasing Mg concentrations from 0.5 to 2 mM produced a left shift of PTH–Ca curves. With Mg 5 mM, the secretory response was practically abolished. Mg was able to reduce PTH only if parathyroid glands were exposed to moderately low Ca concentrations; with normal–high Ca concentrations, the effect of Mg on PTH inhibition was minor or absent. After 6-h incubation at a Ca concentration of 1.0 mM, the expression of parathyroid CaR, VDR, FGFR1 and Klotho (at mRNA and protein levels) was increased with a Mg concentration of 2.0 when compared with 0.5 mM.ConclusionsMg reduces PTH secretion mainly when a moderate low calcium concentration is present; Mg also modulates parathyroid glands function through upregulation of the key cellular receptors CaR, VDR and FGF23/Klotho system.

Cinacalcet Upregulates Calcium-Sensing Receptors of Parathyroid Glands in Hemodialysis Patients

Background: Cinacalcet hydrochloride (cinacalcet), a calcimimetic, has been shown to upregulate calcium-sensing receptor (CaSR) expression in parathyroid glands of rats with chronic renal insufficiency. However, the effect of cinacalcet on the reduced CaSR expression in human parathyroid glands remains to be elucidated. Methods: Four normal parathyroid glands and 71 hyperplastic parathyroid glands from 18 hemodialysis patients with refractory secondary hyperparathyroidism (SHPT) treated with (n = 10; cinacalcet group) or without (n = 8; conventional group) cinacalcet were examined immunohistochemically with a specific antibody against CaSR. The expression level of CaSR was analyzed semiquantitatively. Results: Compared with normal glands, the immunohistochemical expression of CaSR was decreased significantly in both the cinacalcet and conventional groups. In the cinacalcet group, the expression of CaSR was increased significantly compared with that in the conventional group (1.83 ± 0.14 vs. 0.87 ± 0.15, p < 0.001), even though the proportion of patients using vitamin D sterols and the mean administered dose of calcitriol equivalents were not significantly different between the two groups. The expression of CaSR was significantly decreased in the larger glands (>500 mg) compared with that in the smaller glands (<500 mg) in both groups; furthermore, it was markedly decreased in areas of nodular hyperplasia compared with diffuse hyperplasia in the cinacalcet group. Conclusions: Our results indicate that cinacalcet upregulates the depressed expression of CaSR in hemodialysis patients with SHPT, and that insufficient expression of CaSR, especially in larger glands with advanced nodular hyperplasia, underlies the pathogenesis of SHPT in patients who are resistant to cinacalcet.

Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

BackgroundHeparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction.Methodology/Principal FindingsUsing metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [35S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([35S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [35S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30–33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase.Conclusions/SignificanceBoth syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms.