Development and prevention of morphologic and ultrastructural changes in uremia-induced hyperplastic parathyroid gland.

The detailed ultrastructural changes of uremia-induced hyperplastic parathyroid gland and the effects of current medical treatments for secondary hyperparathyroidism were investigated. Marked enlargement of parathyroid cell with accumulation of mitochondria and lipids and a significant increase in the thickness of the pericapillary area with increased fibrosis and appearance of fibroblast like cells were noted in the hyperplastic gland caused by uremia and phosphate retention. These ultrastructural changes and biochemical findings indicating hyperparathyroidism were significantly suppressed by all of the treatment using phosphate restriction, calcitriol, and cinacalcet. The characteristic ultrastructural changes, including the morphologic evidence of nodule formation, were indicated.

MafB interacts with Gcm2 and regulates parathyroid hormone expression and parathyroid development.

Serum calcium and phosphate homeostasis is critically regulated by parathyroid hormone (PTH) secreted by the parathyroid glands. Parathyroid glands develop from the bilateral parathyroid-thymus common primordia. In mice, the expression of transcription factor Glial cell missing 2 (Gcm2) begins in the dorsal/anterior part of the primordium on embryonic day 9.5 (E9.5), specifying the parathyroid domain. The parathyroid primordium then separates from the thymus primordium and migrates to its adult location beside the thyroid gland by E15.5. Genetic ablation of gcm2 results in parathyroid agenesis in mice, indicating that Gcm2 is essential for early parathyroid organogenesis. However, the regulation of parathyroid development at later stages is not well understood. Here we show that transcriptional activator v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (MafB) is developmentally expressed in parathyroid cells after E11.5. MafB expression was lost in the parathyroid primordium of gcm2 null mice. The parathyroid glands of mafB(+/-) mice were mislocalized between the thymus and thyroid. In mafB(-/-) mice, the parathyroid did not separate from the thymus. Furthermore, in mafB(-/-) mice, PTH expression and secretion were impaired; expression levels of renal cyp27b1, one of the target genes of PTH, was decreased; and bone mineralization was reduced. We also demonstrate that although Gcm2 alone does not stimulate the PTH gene promoter, it associates with MafB to synergistically activate PTH expression. Taken together, our results suggest that MafB regulates later steps of parathyroid development, that is, separation from the thymus and migration toward the thyroid. MafB also regulates the expression of PTH in cooperation with Gcm2.

Calcium oxalate crystal deposition in metabolic syndrome model rat kidneys.

OBJECTIVE: Although an epidemiological link between the metabolic syndrome and kidney stone formation has been reported, the mechanism by which metabolic syndrome promotes kidney stone formation has yet to be elucidated. We investigated calcium oxalate (CaOx) kidney stone formation in a rat metabolic syndrome model. METHODS: We induced hyperoxaluria in 8-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) rats, and a control strain, Long-Evans Tokushima Otsuka (LETO) rats, by administering 1.0% ethylene glycol (EG) as their drinking water for 2 weeks. Rats were divided into four groups: LETO-C (control, n = 7); LETO-SF (stone forming, n = 8); OLETF-C (n = 7); and OLETF-SF (n = 8). Urine and blood samples were collected for biochemistry testing, and the kidneys were harvested for estimation of crystal deposition and determinations of the expression of osteopontin (OPN) and monocyte chemoattractant protein-1 (MCP-1). RESULTS: Administration of EG induced hyperoxaluria to the same degree in both strains. The OLETF-SF group showed a higher grade of renal crystal deposition and significantly higher renal calcium content than the LETO-SF group. Although the OLETF-C group excreted significantly higher amounts of uric acid and more acidic urine than the LETO-C group, similar differences were not observed in rats given EG. Significant upregulation of both OPN and MCP-1 was seen in the kidneys of hyperoxaluric rats, with higher levels of expression in the OLETF-SF group than the LETO-SF group. CONCLUSIONS: The present results show for the first time that OLETF rats form more renal CaOx crystal deposits compared with control rats under EG-induced hyperoxaluric conditions. The model described here should be useful for investigating the mechanisms by which the metabolic syndrome promotes CaOx kidney stone formation.

Fibroblast growth factor 23 production in bone is directly regulated by 1{alpha},25-dihydroxyvitamin D, but not PTH.

Fibroblast growth factor 23 (FGF23), which is primarily produced by osteocytes in bone, regulates renal phosphate excretion and 1α,25-dihydroxyvitamin D [1,25(OH)(2)D(3)] metabolism. Patients with chronic kidney disease (CKD) have increased levels of circulating serum FGF23, but the direct effect on circulating FGF23 levels in renal insufficiency is still unclear. To identify the major regulator of FGF23 synthesis in renal insufficiency, we compared the effect of parathyroid hormone (PTH) and 1,25(OH)(2)D(3) on FGF23 synthesis in the calvariae of normal rats with that of uremic rats in vitro. 1,25(OH)(2)D(3) treatment significantly increased the FGF23 concentration in the medium from both groups, but the degree of increase in the uremic group was markedly higher than in the control group. A significant increase in FGF23 mRNA expression occurred as early as 4 h after treatment and reached the maximum within 8 h in the uremic group, whereas in the normal group a significant increase in FGF23 mRNA expression was observed only at 8 h. In addition, the expression of vitamin D receptor (VDR) mRNA in the calvariae of uremic rats was markedly higher than in normal rats. However, in neither group did PTH treatment affect the medium FGF23 concentration or the FGF23 mRNA levels. These results suggest that FGF23 synthesis in bone is regulated by 1,25(OH)(2)D(3) directly, not by PTH, and that increased VDR mRNA expression induced the relatively swift and strong response in the uremic group.

Development of a technique for introduction of an expressed complementary deoxyribonucleic acid into parathyroid cells by direct injection.

PTH is a major mediator of bone and mineral metabolism. However, physiological and pathological investigations of parathyroid cells (PTCs) have been limited because of the lack of available cell lines and because the organ is too small for detailed studies. Here, we describe a novel method for adenovirus-mediated cDNA transfer into PTCs, and we show the accuracy of the method in a rat model of uremia-induced secondary hyperparathyroidism. Rats underwent a 5/6-nephrectomy and were fed with a high-phosphate diet for 8 wk. The parathyroid glands were surgically exposed and adenoviruses containing LacZ or Ca-sensing receptor (CaSR) were directly injected into the glands under a zoom-stereo microscope. The parathyroid glands were analyzed for infection of adenovirus and immunohistochemically for expression of CaSR. The functional activity of exogenous CaSR in PTCs after this treatment was investigated based on changes of the calcium and PTH curve. A virus concentration of more than 10(9) plaque-forming units/ml was required for adequate infection of PTCs within 7 d after treatment. Marked increase of CaSR-positive PTCs by 2.39 +/- 0.72 times relative to control treatment, and significant colocalization of CaSR overexpression and virus labeling, were observed in glands after gene introduction. The calcium and PTH curve was shifted to the left from the basal position (set point, 1.10 +/- 0.09 to 0.76 +/- 0.12 mm; P < 0.0001), indicating successful introduction of a functionally active cDNA into the PTCs. This technique may facilitate an elucidation of biological effects through targeting and identification of specific features of PTCs, which may provide the basis for new clinical approaches.

Reduced expression of perlecan in the aorta of secondary hyperparathyroidism model rats with medial calcification.

BACKGROUND: Vascular calcification is an important complication that worsens the prognosis for dialysis patients, although its detailed molecular mechanisms are still unknown. METHODS: We produced a rat model for vascular calcification with hyperphosphatasemia and hyperparathyroidism, performing a 5/6 nephrectomy and providing a high-phosphorus, low-calcium diet for eight weeks. We examined mRNA obtained from the calcified aortae using microarray analysis, and searched for alterations in gene expression specifically in the calcified lesions. RESULTS: Medial calcification was demonstrated in the abdominal aorta of 12 out of 42 hyperparathyroidism rats. In the aortae of hyperparathyroid rats with vascular calcification, the genes for heparan sulfate proteoglycans, including perlecan, were found to be down-regulated using microarray analysis and real time PCR. Immunohistochemistry also demonstrated reduced production of perlecan in the aortae of hyperparathyroid rats. DISCUSSION: Perlecan is a major component of the vascular wall basement membrane and may play a role in protecting vascular smooth muscle cells from inflammatory cells and various toxins. It has also been reported that heparan sulfate chains may inhibit osteogenesis. Our findings indicate that perlecan may protect vascular smooth muscle cells from various factors that promote vascular calcification. CONCLUSIONS: It may be that reduced expression of perlecan in the calcified aortae of hyperparathyroid rats is a risk factor for vascular calcification.

Mechanism of phosphate-induced calcification in rat aortic tissue culture: possible involvement of Pit-1 and apoptosis.

BACKGROUND: Hyperphosphataemia is a known contributing factor in the progression of vascular calcification in dialysis patients. The cellular mechanisms underlying phosphate-induced calcification are still unclear despite intense study, so in this study, we investigated the possible involvement of the type III sodium-dependent phosphate cotransporter, Pit-1, in an aortic tissue culture model. METHODS: Aortic segments from 9-week-old male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The phosphate concentration of the medium was elevated to induce calcification, which was assessed by histology and calcium content. Phosphonoformic acid (PFA) was used to inhibit phosphate uptake. The involvement of apoptosis was examined using the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labelling (TUNEL) assay, caspase 3 activation, and inhibition of apoptosis using a general caspase inhibitor. Phenotypic changes in vascular smooth muscle cells (VSMC) were assessed using expression of osteochondrogenic differentiation markers. RESULTS: Medial vascular calcification was induced in aortas cultured in a high phosphate medium. PFA decreased the rates of calcification and apoptosis of VSMC in the media, concomitant with calcification. Caspase inhibitor reduced calcification. No phenotypic transition of VSMC was seen in this model. CONCLUSIONS: These results indicate that phosphate uptake through the type III sodium-dependent phosphate cotransporter, Pit-1, leads to induction of apoptosis and subsequent calcification of VSMC.

Improvement of impaired calcium and skeletal homeostasis in vitamin D receptor knockout mice by a high dose of calcitriol and maxacalcitol.

Vitamin D plays a major role in mineral and skeletal homeostasis through interaction with the nuclear vitamin D receptor (VDR) of target cells. Recent reports have indicated that some cellular effects of vitamin D may occur via alternative signaling pathways, but concrete evidence for mineral homeostasis has not been shown in vivo. To investigate this issue, the actions of calcitriol (1,25D) and maxacalcitol (OCT), which were developed for treatment of uremia-induced secondary hyperparathyroidism, were analyzed in VDR knockout (VDR(-/-)) mice. The VDR(-/-) mice were fed a rescue diet immediately after weaning. 1,25D, OCT or a control solution was administered intraperitoneally to these mice three times a week for eight weeks. Biological markers and bone growth were measured and bone histomorphometric analysis of the calcein-labeled tibia was performed 24 h after the final administration. Significantly higher levels of serum Ca(2+) were observed in 1,25D- and OCT-treated mice, but the serum parathyroid hormone level was unchanged by both agents. Impaired bone growth, enlarged and distorted cartilaginous growth plates, morphological abnormalities of cancellous and cortical bones; a morbid osteoid increase, lack of calcein labeling, and thinning of cortical bone, were all significantly improved by 1,25D and OCT. The significance of these effects was confirmed by bone histomorphometrical analysis. Upregulation of the calbindin D(9k) mRNA expression level in the duodenum may explain these findings, since this protein is a major modulator of Ca transport in the small intestine. We conclude that 1,25D and OCT both at a high dose exert significant effects on Ca and skeletal homeostasis with the principal improvement of Ca status in VDR(-/-) mice, and some of these effects may occur through an alternative vitamin D signaling pathway.

Tolvaptan, a selective oral vasopressin V2 receptor antagonist, ameliorates podocyte injury in puromycin aminonucleoside nephrotic rats.

BACKGROUND: Proteinuria caused by glomerular disease is characterized by podocyte injury. Vasopressin V2 receptor antagonists are effective in reducing albuminuria, although their actions on glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of tolvaptan, a selective oral V2 receptor antagonist, on podocytes in a puromycin aminonucleoside (PAN)-induced nephrosis rat model. METHODS: Rats were allocated to a control, PAN nephrosis, or tolvaptan-treated PAN nephrosis group (n = 9 per group). Urinary protein excretion and serum levels of total protein, albumin, creatinine, and total cholesterol were measured on day 10. The influence of tolvaptan on podocytes was examined in renal tissues by immunofluorescence and electron microscopy. RESULTS: PAN induced massive proteinuria and serum creatinine elevation on day 10, both of which were significantly ameliorated by tolvaptan. Immunofluorescence studies of the podocyte-associated proteins nephrin and podocin revealed granular staining patterns in PAN nephrosis rats. In tolvaptan-treated rats, nephrin and podocin expressions retained their normal linear pattern. Electron microscopy showed foot process effacement was ameliorated in tolvaptan-treated rats. CONCLUSIONS: Tolvaptan is protective against podocyte damage and proteinuria in PAN nephrosis. This study indicates that tolvaptan exerts a renoprotective effect by affecting podocyte morphology and probably function in PAN nephrosis. Tolvaptan is a promising pharmacological tool in the treatment of renal edema.

Regulation of fibroblast growth factor 23 production in bone in uremic rats.

BACKGROUND: Fibroblast growth factor 23 (FGF23) regulates renal phosphate reabsorption and 1alpha,25-dihydroxyvitamin D [1,25(OH)(2)D(3)] metabolism. Patients with chronic kidney disease (CKD) have increased levels of circulating FGF23, but the direct regulation of this elevation of FGF23 is incompletely understood. METHOD: We measured plasma parameters in uremic rats fed a high-phosphorus diet and then performed parathyroidectomy (PTX) to determine its effect. We also investigated FGF23 mRNA expression in various tissues to identify the major source of circulating FGF23. RESULT: The uremic rats displayed dramatic changes in plasma FGF23 levels, consistent with increased expression of FGF23 in bone. Elevated FGF23 was associated with phosphate and parathyroid hormone (PTH). After PTX, the elevated FGF23 had decreased, consistent with decreased expression of FGF23 in bone. Significant decreases in plasma FGF23 were associated with PTH and 1,25(OH)(2)D(3), but not phosphate. CONCLUSION: Elevated plasma FGF23 levels in uremic rats reflect the increased expression of FGF23 in bone. The expression of FGF23 in bone may be regulated by a PTH-1,25(OH)(2)D(3) axis-dependent pathway and another PTH-dependent and 1,25(OH)(2)D(3)-independent pathway in uremic rats. The pathway may be decided by the degree of renal dysfunction.

Highly concentrated calcitriol and its analogues induce apoptosis of parathyroid cells and regression of the hyperplastic gland--study in rats.

BACKGROUND: Controlling hyperplasia of the parathyroid gland (PTG) is important in the management of secondary hyperparathyroidism (SHPT). Regression of the hyperplastic PTG requires a decrease in the number of parathyroid cells (PTCs), so the present study investigated cell death caused by toxic agents or by clinically usable vitamin D metabolites. METHODS: The PTGs of Sprague-Dawley rats, which had been 5/6-nephrectomized and fed a high-phosphate diet for 12 weeks, were treated with two consecutive direct injections (DI) of calcitriol, maxacalcitol, paricalcitol, doxercalciferol or phosphate-buffered saline containing either 0.01% or 90% ethanol (0.01-ET or 90-ET, respectively). Laboratory data, including serum levels of intact parathyroid hormone (intact-PTH), were obtained before and after the treatments. The PTGs were excised 24 h after the final injection and evaluated for PTC apoptosis using light and electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) method and DNA electrophoresis. RESULTS: Treatment with any of the vitamin D metabolites and 90-ET significantly decreased the serum intact-PTH level, but only the latter significantly decreased the serum Ca level. Either treatment markedly increased the number of TUNEL-positive PTCs, but not in PTG treated with 0.01-ET. In PTGs treated with DI of any vitamin D metabolites was there ladder formation on DNA electrophoresis, as well as the characteristic morphological features of apoptosis in both the light and electron microscopic studies. CONCLUSIONS: DI of vitamin D metabolites may be effective in controlling not only the PTH level, but also PTG hyperplasia, in advanced SHPT by, at least in part, apoptosis-induced cell death. Our study was performed in rats.

Direct injection of calcitriol or its analog improves abnormal gene expression in the hyperplastic parathyroid gland in uremia.

AIMS: In this study, we investigated the effects of direct injection (DI) of calcitriol or maxacalcitol into the hyperplastic parathyroid gland (PTG) on altered gene expression related to the advanced status of secondary hyperparathyroidism (SHPT). METHODS: Sprague-Dawley rats were 5/6-nephrectomized (uremic) or sham-operated (normal). In each uremic rat, one of the bilateral PTG was treated by DI of calcitriol (PTG(CAL)) or maxacalcitol (PTG(OCT)), and the other gland was treated with control solution (PTG(CONT)). The PTG were evaluated for levels of expression of various mRNA and immunohistochemical staining of proliferating cell nuclear antigen (PCNA). RESULTS: Significant differences in levels of expression of mRNA and PCNA were confirmed between the uremic and normal groups. In PTG(CAL) and PTG(OCT), expressions of almost all mRNA and PCNA were significantly improved; both agents were able to normalize the abnormalities of the uremic PTG, in contrast to the baseline and individual PTG(CONT). However, the difference in effect between PTG(CAL) and PTG(OCT) was only small. CONCLUSION: Our results suggest that very high concentrations of calcitriol or maxacalcitol in the PTG improve abnormal gene expression and proliferation activity of parathyroid cells, and might explain the better control of SHPT using the DI technique.

Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells.

Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10(-4)M), or 10% cyclodextrin, for 6h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro.

Up-regulation of Cbfa1 and Pit-1 in calcified artery of uraemic rats with severe hyperphosphataemia and secondary hyperparathyroidism.

BACKGROUND: Cardiovascular disease is the most frequent cause of death in patients with end-stage kidney disease (ESKD). Vascular calcification is a confirmed risk factor for cardiovascular events in the general population and has a high occurrence in patients with ESKD. Despite the high prevalence of vascular calcification in ESKD, the pathogenesis of the disorder is still obscure. The present study examined the expressions of bone-associated factors in calcified arteries in subtotally nephrectomized rats with severe secondary hyperparathyroidism (SHPT). METHODS: Seven-week-old male Sprague-Dawley rats were divided into five groups as follows: sham-operated rats that received a normal diet [0.8% of phosphorus (P), 1.1% of calcium (Ca)] (Sham), sham-operated rats that received a high-phosphorus and low-calcium (HPLCa) diet (1.2% P, 0.4% Ca) (Sham+HPLCa), 5/6 nephrectomized rats that received a normal diet as the uraemic control group (Nx), and 5/6 nephrectomized rats that received a HPLCa diet to induce the development of SHPT (Nx+HPLCa), and 5/6 nephrectomized and parathyroidectomized rats that received a HPLCa diet (Nx+PTx+HPLCa). The feeding period of each group was 10 weeks. The rats were then sacrificed and their serum was examined. The upper part of the abdominal aorta was used to investigate the expression of mRNAs of core-binding factor alpha-1 (Cbfa1) and sodium-dependent phosphate cotransporter (Pit-1) by real-time reverse transcriptase polymerase chain reaction (real-time PCR) analysis. The lower part was examined for calcification by von Kossa staining. RESULTS: Serum P level and Ca x P products increased significantly in the Nx+HPLCa group compared with those of any other groups. Severe hyperparathyroidism was also observed in the Nx+HPLCa group. Vascular calcification (medial layer) was observed in the Nx+HPLCa group only. There was a significant increase in Cbfa1 and Pit-1 mRNA expression levels in the aorta of the Nx+HPLCa group compared with that of any other groups. CONCLUSIONS: These results suggest that medial layer vascular calcification in uraemic rats with severe hyperphosphataemia and SHPT may be caused in part by Cbfa1 and Pit-1.

[Apoptosis: a possible mechanism of suppressing parathyroid hyperplasia by calcimimetics].

The mechanism through which calcimimetic compounds suppress parathyroid cell growth has not been fully elucidated. We investigated the effect of the calcimimetic compound (NPS R-568:R568) on the parathyroid cell growth in vitro. Whole parathyroid glands of subtotally nephrectomized rats fed high phosphorus diet for 8 weeks were used in this study. Fresh rat parathyroid glands were incubated in a media (phosphorus concentration is 1.0 mM and calcium is 1.25 mM) with R-568 (10-4 M) or calcitriol (10-7 M) or vehicle for 6 hours. Medium PTH level was significantly decreased in both R568-treated group and calcitriol-treated group compared with either vehicle-treated group, or no-treated group. While TUNEL-positive cells were similar in calcitriol-treated group compared with both vehicle-treated and no-treated groups, they were significantly increased in R568-treated group compared with other three groups. These results suggest that calcimimetic compounds, NPS R-568 reduces PTH secretion and induces apoptosis of hyperfunctional parathyroid cell in vitro.

Biochemical and cellular effects of direct maxacalcitol injection into parathyroid gland in uremic rats.

The most important etiological factors of resistance to medical treatments for secondary hyperparathyroidism are the decreased contents of the vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in parathyroid cells and a severely swollen parathyroid gland (PTG) as a result of hyperplasia. The effects of direct maxacalcitol (OCT) injection into PTG in terms of these factors were investigated in this study. The PTG of Sprague-Dawley rats that were 5/6 nephrectomized and fed a high-phosphate diet were treated by a direct injection of OCT (DI-OCT) or vehicle (DI-vehicle). The changes in serum intact parathyroid hormone (PTH), Ca(2+), and phosphorus levels, in VDR and CaSR expression levels in parathyroid cells, and in Ca(2+)-PTH curves were examined. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA electrophoresis for PTG. DI-OCT markedly decreased serum intact PTH level, and a significant difference in this level between DI-OCT and DI-vehicle was observed. However, serum Ca(2+) and phosphorus levels did not changed markedly in both groups. The upregulations of both VDR and CaSR, the clear shift to the left downward in the Ca(2+)-PTH curve, and the induction of apoptosis after DI-OCT were observed. These findings were not observed in the DI-vehicle-treated rats. Moreover, these effects of DI-OCT were confirmed by the DI-OCT into one PTG and DI-vehicle alone into another PTG in the same rat. DI-OCT may introduce simultaneous VDR and CaSR upregulations and the regression of hyperplastic PTG, and these effects may provide a strategy for strongly suppressing PTH levels in very severe secondary hyperparathyroidism.

[Enhanced expression of EGFR, TGF-alpha, EGF in hyperplastic parathyroid glands in established stage of renal failure in rats].

It was reported that the parathyroid gland hyperplasia correlated with enhanced co-expression of TGF-alpha and its receptor EGFR at early stages of renal failure. This time, we investigated the time course for EGFR and its ligands, TGF-alpha, and EFG expression, and the influence of high-phosphorus (P) diet to EGFR and EGF expression, and the effect of EGFR-tyrosine kinase inhibitor (Gefitinib, [IRESSA; AstraZeneca]; TKI) in rat PTGs with established stage of renal failure. The levels of EGFR, EGF, TGF-alpha mRNA in rat PTGs were increased for the time periods. The serum intact PTH levels, and EGFR, EGFmRNA in rat PTGs were suppressed in normal-P diet group. Nuclei positive cells for PCNA in TKI group were suppressed. The levels of p21mRNA were increased in TKI group. These results suggested that the enhanced expression of EGFR, TGF-alpha and EGF participate in the cell proliferation of hyperplastic PTGs in established stage of renal failure.

Calcimimetic compound upregulates decreased calcium-sensing receptor expression level in parathyroid glands of rats with chronic renal insufficiency.

The reduced expression level of the calcium-sensing receptor (CaR) is attributed to the hyposensitivity of parathyroid cells to extracellular calcium concentration [Ca2+]o, which plays a crucial role in the pathogenesis of secondary hyperparathyroidism (SHPT) in patients and rats with chronic renal insufficiency (CRI). Calcimimetic compounds have been demonstrated to improve the decreased sensitivity of CaR to extracellular calcium concentration and to suppress both parathyroid hormone (PTH) oversecretion and parathyroid cell proliferation. However, the effect of calcimimetics on the reduced CaR expression level in parathyroid cells in CRI remains unclarified. The aim of this investigation was to examine the effect of the calcimimetic compound NSP R-568 (R-568) on the CaR expression in the parathyroid cells of rats with experimental CRI. Subtotally nephrectomized rats were fed a high-phosphorus diet for 8 (n = 12; Nx-8 group) or 9 wk (n = 11; Nx-9 group) to induce severe SHPT. Another group of uremic rats were fed a high-phosphorus diet for 8 wk and then orally administered R-568 (100 micromol/kg body wt) once a day for 7 d (n = 11; Nx+R-568 group). Sham-operated rats that were fed a standard diet for 9 wk were used as controls (n = 8). R-568 treatment induced a significant reduction in plasma PTH level with significant decrease in serum calcium and without change in serum phosphorus concentration. Serum 1,25(OH)2D3 level was not affected by R-568 administration. CaR mRNA and protein levels in the Nx-8 and Nx-9 groups significantly decreased compared with those in the controls; however, no significant difference in these parameters was observed between the Nx-8 and Nx-9 groups. In the Nx+R-568 group, CaR mRNA and protein levels significantly increased compared with those in either the Nx-8 or Nx-9 group. R-568 was effective in reducing the number of proliferating cell nuclear antigen-positive cells along with parathyroid gland growth suppression in the Nx+R-568 group compared with that in the Nx-9 group. The results suggest that the calcimimetic compound R-568 upregulates decreased CaR expression, and the upregulation possibly has an enhancement effect on PTH secretion and parathyroid cell hyperplasia through the improved sensitivity of CaR to [Ca2+]o.