Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate

The present experiments investigate medial artery calcification in adult rats made uremic by feeding a synthetic diet containing 0.75% adenine for 4 weeks. Calcification was assessed by Alizarin red staining of intact aortas, by von Kossa staining of carotid artery sections, and by calcium and phosphate incorporated into the thoracic aorta. The major conclusions are as follows: Lowering the protein content of the diet from 25 to 2.5% dramatically increases the frequency and extent of medial artery calcification in uremic rats without significantly affecting the elevation in serum creatinine, phosphate, or parathyroid hormone. This observation suggests that low dietary protein intake could be a risk factor for medial artery calcification in uremic patients. Medial artery calcification in uremic rats is prevented by a dose of ibandronate that inhibits bone resorption. The observation suggests that bone resorption inhibitors could prevent artery calcification in uremic patients. Medial artery calcification in uremic rats correlates with increased serum bone Gla protein (BGP; osteocalcin), but not with serum matrix Gla protein or fetuin. This finding indicates that it could be of interest to examine the relation between serum BGP and artery calcification in uremic patients. Each of these conclusions lends support for our hypothesis that medial artery calcification is linked to bone resorption. Future investigations of the as yet unknown biochemical basis for this link will be facilitated by the present discovery that a synthetic, 2.5% protein diet containing 0.75% adenine produces consistent and dramatic medial calcification in adult rats within just 4 weeks.     

Sevelamer hydrochloride reverses parathyroid gland enlargement via regression of cell hypertrophy but not apoptosis in rats with chronic renal insufficiency.

BACKGROUND: Dietary phosphate restriction suppresses parathyroid hormone (PTH) secretion, synthesis, and parathyroid cell proliferation in experimental animals with chronic renal insufficiency (CRI), independently of serum calcium and 1,25(OH)2D3 levels. This study was conducted to examine whether sevelamer hydrochloride (sevelamer), a metal-free phosphate binder, could regress an advanced parathyroid gland (PTG) hyperplasia and enlargement in rats with CRI. METHODS: Male Sprague-Dawley rats were fed a diet containing adenine for 6 weeks to establish CRI. Normal rats and adenine-treated rats were sacrificed to obtain the PTG (baseline group). The adenine diet was changed to a normal diet or diet containing 1 or 3% sevelamer for another 4 weeks. Time course changes of serum levels of calcium, phosphorus, and PTH were measured. At the end of the study, the PTG was weighed and examined histologically. RESULTS: Adenine-treated rats developed severe CRI with marked elevation of serum phosphorus and PTH. The PTG weight markedly increased with enlarged cell volume (i.e. cell hypertrophy) at baseline. Sevelamer treatment rapidly lowered serum phosphorus and PTH levels within 6 days, and after 4 weeks, reduced the PTG weight by 38% compared to adenine-treated rats at baseline. The reduction in PTG weight was due to regression of cell hypertrophy, but not to decreased cell number by apoptosis. Decreased expression of calcium receptor in the PTG at baseline was partially recovered by the sevelamer treatment. CONCLUSIONS: The sevelamer treatment can reduce the PTG weight with a reduction in serum PTH levels via regression of cell hypertrophy but not apoptosis in rats with CRI. Reduced PTG function might contribute to the regression of cell hypertrophy.     

Sevelamer hydrochloride prevents ectopic calcification and renal osteodystrophy in chronic renal failure rats

BACKGROUND: Hyperphosphatemia is associated with severe complications, including ectopic calcification of soft tissues, secondary hyperparathyroidism, and renal osteodystrophy (ROD). Sevelamer hydrochloride is a nonabsorbed calcium- and metal-free phosphate binder that lowers serum phosphorus levels in hemodialysis patients. This study examined the efficacy of sevelamer in preventing ectopic calcification of soft tissues and ROD in adenine-induced renal failure rats. METHODS: Male, 12-week-old Wistar-Jcl rats were freely fed an adenine diet (0.75 g adenine in 100 g normal diet) for four weeks. After three weeks of the adenine diet, when serum phosphorus levels had significantly increased, the rats were freely fed a normal diet that contained 1% or 2% of sevelamer for another five weeks. Time course changes of serum levels of phosphorus, calcium, and parathyroid hormone (PTH) were measured. At the end of the study, calcium and phosphorus levels in the heart and aorta were measured, and the calcification of kidney, heart, aorta, and stomach were histopathologically examined. The severity of ROD was evaluated by a histopathologic and morphometric analysis of the femurs. RESULTS: Compared with the adenine controls (N = 10), the sevelamer-treated (1%, N = 6; and 2%, N = 10) groups of adenine-induced renal failure rats had reduced serum phosphorus, serum calcium x phosphorus product, and serum PTH levels. Moreover, in the treatment groups, sevelamer suppressed calcification of the aorta media, and also the osteoid volume, fibrosis volume, and porosity ratio of femurs. CONCLUSION: These results suggest that sevelamer treatment might contribute to the suppression of ectopic calcification and ROD.     

Hyperplasia of the parathyroid gland without secondary hyperparathyroidism

BACKGROUND: Low dietary phosphorus (P) prevents parathyroid gland (PTG) hyperplasia and the development of secondary hyperparathyroidism (SH) in uremic rats. The present study explores the effects of P restriction on parathyroid hormone (PTH) synthesis and secretion and PT cell growth in rats with established SH and PTG hyperplasia. METHODS: Normal and 5/6 nephrectomized rats were fed a high P (0.8%) diet. After two weeks, the normal rats and half of the uremic rats were sacrificed (U-HP) while the remaining uremic rats were switched to a low P (0.2%) diet (U-HP-LP). RESULTS: High dietary P induced a significant increase in serum P, PTH, and PTG weight, but not ionized calcium compared to normal animals fed the same diet (N-HP). P restriction returned serum P and PTH to normal levels by one week. In contrast, PTG size did not regress and glands remained enlarged for up to eight weeks with no evidence of apoptosis. Ribonuclease protection assay and metabolic labeling studies demonstrated similar PTH/actin mRNA ratios and 35S-labeled PTH among the three groups. Intracellular intact PTH was higher in U-HP and U-HP-LP rats compared to N-HP animals with no differences between the two uremic groups. PTG-PTH content correlated only with PTG weight, and serum PTH only with serum P. The PTG secretory response to calcium remained intact. CONCLUSIONS: In established chief-cell hyperplasia, P restriction restores normal serum PTH levels without affecting PTG hyperplasia, PTH synthesis, PTG cytosolic PTH or the PTH secretory response to calcium, suggesting an impaired exocytosis of PTH.     

Lanthanum carbonate decreases PTH gene expression with no hepatotoxicity in uraemic rats.

BACKGROUND: Lanthanum (La) carbonate is an effective phosphate-binder, used to reverse hyperphosphataemia due to chronic kidney disease. Some recent studies in rodents have cast safety uncertainties. The aims of this study were to examine the effects of La on parathyroid hormone (PTH) gene expression and hepatic toxicity. METHODS: Uraemia was induced in rats by a 0.3% adenine-containing diet for 2 weeks. Thereafter, rats were maintained for 4 weeks on an adenine diet with 1.5 or 3% La. Normal, uraemic, and uraemic rats fed a low-phosphorus (P) diet served as controls. RESULTS: Azotaemia developed in all adenine-fed rats. La of 3%, but not 1.5% La, decreased serum P to normal in uraemic rats. Both La 1.5 and 3% decreased urine P excretion. Plasma PTH was tripled in uraemic compared with normal rats. Both the 3.0% La and the low-P diet decreased PTH to normal. PTH mRNA content was increased 2-fold in uraemic compared with normal rats. The 3% La or the low-P diet decreased PTH mRNA levels to normal in uraemic rats. Liver enzymes were normal in all groups. Adenine-induced uraemia decreased liver weight with no additional effect of La. Liver weights corrected for body-weights were the same in all study groups, including the La group. Therefore, La had no effect upon absolute or corrected liver weight. Liver magnetic resonance imaging and microscopy did not reveal toxic changes due to La. CONCLUSIONS: These findings confirm that in rats with adenine-induced uraemia, the P-binder La reverses the hyperphosphataemia and hyperparathyroidism. They are the first demonstration that La decreases PTH gene expression. Importantly, we found no evidence of drug-induced liver toxicity.     

Disturbances in Bone Largely Predict Aortic Calcification in an Alternative Rat Model Developed to Study Both Vascular and Bone Pathology in Chronic Kidney Disease

Because current rat models used to study chronic kidney disease (CKD)‐related vascular calcification show consistent but excessive vascular calcification and chaotic, immeasurable, bone mineralization due to excessive bone turnover, they are not suited to study the bone‐vascular axis in one and the same animal. Because vascular calcification and bone mineralization are closely related to each other, an animal model in which both pathologies can be studied concomitantly is highly needed. CKD‐related vascular calcification in rats was induced by a 0.25% adenine/low vitamin K diet. To follow vascular calcification and bone pathology over time, rats were killed at weeks 4, 8, 10, 11, and 12. Both static and dynamic bone parameters were measured. Vascular calcification was quantified by histomorphometry and measurement of the arterial calcium content. Stable, severe CKD was induced along with hyperphosphatemia, hypocalcemia as well as increased serum PTH and FGF23. Calcification in the aorta and peripheral arteries was present from week 8 of CKD onward. Four and 8 weeks after CKD, static and dynamic bone parameters were measurable in all animals, thereby presenting typical features of hyperparathyroid bone disease. Multiple regression analysis showed that the eroded perimeter and mineral apposition rate in the bone were strong predictors for aortic calcification. This rat model presents a stable CKD, moderate vascular calcification, and quantifiable bone pathology after 8 weeks of CKD and is the first model that lends itself to study these main complications simultaneously in CKD in mechanistic and intervention studies. © 2015 American Society for Bone and Mineral Research.     

Systemic disorders of calcium dynamics in rats with adenine-induced renal failure: Implication for chronic kidney disease-related complications

Aim:   Chronic kidney disease (CKD) causes the dysregulation of systemic mineral metabolism. A major issue in CKD patients is the emergence of ectopic calcification in soft tissues, presumably due to increased levels of calcium (Ca) or inorganic phosphorus (Pi); however, the precise mechanisms have not been fully elucidated. Therefore, this study aims to evaluate Ca dynamics in an animal model of CKD.
Methods:   Renal failure was produced in rats by feeding an adenine-containing diet for 4 weeks, and time-course changes in biochemical parameters, including Ca, Pi, creatinine (Cr), blood urea nitrogen (BUN), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D₃, and N-telopeptide and cross-linked collagen type I (NTx), were monitored once a week during the feeding period. Intestinal absorption, tissue contents, and urinary excretion of Ca were monitored using radioisotope (RI) ⁴⁵Ca.
Results:   Adenine-fed rats exhibited renal failure, ectopic calcification and altered serum parameters, including elevated levels of serum Pi, Cr, PTH and BUN. Serum Ca levels were not increased in rats with renal failure. RI-based experiments revealed that abnormal Ca dynamics including attenuated intestinal absorption, increased incorporation into soft tissues, particularly aortic tissue, in which it was increased threefold, and enhanced urinary excretion occurred in renal failure rats.
Conclusion:   Rats with renal failure induced by an adenine diet exhibited severe abnormality of Ca dynamics, including Ca shortage and ectopic accumulation of Ca. These findings would provide useful information to research CKD-related complications.     

Vascular calcification: contribution of parathyroid hormone in renal failure

Hyperphosphatemia is a driving force in the pathogenesis of vascular calcification (VC) and secondary hyperparathyroidism associated with renal failure. To test for the possible contribution of parathyroid hormone (PTH) to cardiovascular calcification, we removed the parathyroid glands from rats but infused synthetic hormone at a supraphysiologic rate. All rats were pair-fed low, normal, or high phosphorus diets and subjected to a sham or 5/6 nephrectomy (remnant kidney). Control rats were given a normal diet and underwent both sham parathyroidectomy and 5/6 nephrectomy. Heart weight/body weight ratios and serum creatinine levels were higher in remnant kidney rats than in the sham-operated rats. Remnant kidney rats on the high phosphorus diet and PTH replacement developed hyperphosphatemia and hypocalcemia along with low bone trabecular volume. Remnant kidney rats on the low phosphorus diet or intact kidney rats on a normal phosphorus diet, each with hormone replacement, developed hypercalcemia. All rats on PTH replacement developed intense aortic medial calcification, and some animals presented coronary calcification. We suggest that high PTH levels induce high bone turnover and medial calcification resembling M?mckeberg's sclerosis independent of uremia. This model may be useful in defining mechanisms underlying VC.     

Calcimimetic R-568 decreases extraosseous calcifications in uremic rats treated with calcitriol

Calcimimetics decrease parathyroid hormone (PTH) levels in uremic patients with secondary hyperparathyroidism without increasing serum calcium (Ca). The aim of this study was to evaluate the effect of calcimimetic R-568 alone or in combination with calcitriol on vascular and other soft tissue calcifications in uremic rats with secondary hyperparathyroidism. Sham-operated and 5/6 nephrectomized Wistar rats were studied. 5/6 Nephrectomized rats were treated with vehicle, calcitriol (80 ng/kg every other day), R-568 (1.5 and 3 mg/kg per d), and both calcitriol and R-568 1.5 mg/kg, as above. Rats were killed after 14 or 56 d of treatment. Blood was drawn for biochemical measurements. Aortic, heart, kidney, lung, and stomach tissue samples were processed for histopathology and measurement of tissue Ca and phosphorus content. PTH concentrations were significantly reduced by all treatments. Treatment with calcitriol induced significant vascular calcification (aortic Ca increased to 4.2+/-1.2 mg/g at day 14 and to 11.4+/-0.7 mg/g at day 56; P<0.05 versus vehicle). Treatment with R-568 did not induce vascular calcification. Concurrent administration of R-568 with calcitriol reduced the aortic Ca (1.9+/-0.2 mg/g at day 14 and 7.5+/-1.4 mg/g at day 56) in relation to calcitriol alone. Soft tissue calcifications mirrored aortic mineralizations. Survival was significantly (P<0.001) reduced in calcitriol-treated rats, and mortality was attenuated (P=0.01) by concurrent treatment with R-568. In uremic rats, R-568 reduces elevated PTH levels without inducing vascular calcification, prevents calcitriol-induced vascular calcification, and decreases mortality.     

Comparative effects of vitamin K and vitamin D supplementation on prevention of osteopenia in calcium-deficient young rats

The aim of this study was to clarify the difference in the effects of vitamin K and vitamin D supplementation on the development of osteopenia in young rats under mild calcium deficiency. Sixty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into six groups with 10 rats in each group: baseline control, 0.5% (normal) calcium diet, 0.1% (low) calcium diet, 0.1% calcium diet + vitamin K (30 mg/100 g, food intake), 0.1% calcium diet + vitamin D (25 microg/100 g, food intake), and 0.1% calcium diet + K + D. After 10 weeks of feeding, serum calcium, 25-hydroxyvitamin D(3) [25 (OH) D(3)], 1,25-dihydroxyvitamin D(3) [1,25 (OH)(2) D(3)], and parathyroid hormone (PTH) levels were measured, and intestinal calcium absorption and renal calcium reabsorption were evaluated. Bone histomorphometric analyses were performed on cortical bone of the tibial shaft and cancellous bone of the proximal tibia. Calcium deficiency induced hypocalcemia, increased serum PTH and 1,25 (OH)(2) D(3) levels with decreased serum 25 (OH) D(3) level, stimulated intestinal calcium absorption and renal calcium reabsorption, and reduced maturation-related cortical bone gain as a result of decreased periosteal bone gain and enlarged marrow cavity but did not significantly influence maturation-related cancellous bone gain. Vitamin K supplementation in calcium-deficient rats stimulated renal calcium reabsorption, retarded the abnormal elevation of serum PTH level, increased maturation-related cancellous bone gain, and retarded the reduction in maturation-related cortical bone gain. On the other hand, vitamin D supplementation in calcium-deficient rats stimulated intestinal calcium absorption via increased serum 1,25 (OH)(2) D(3) level with prevention of the abnormal elevation of serum PTH level, prevented hypocalcemia, reduced the maturation-related cancellous bone gain, and prevented the reduction in periosteal bone gain and enhanced enlargement of the marrow cavity with no significant effect on the reduction in maturation-related cortical bone gain. However, no synergistic effect of vitamin K and vitamin D on intestinal calcium absorption, renal calcium reabsorption, and cancellous and cortical bone mass was found. This study shows the differential effects of vitamin K and vitamin D supplementation on the development of osteopenia in young rats under mild calcium deficiency. Vitamin K supplementation stimulates renal calcium reabsorption, increases maturation-related cancellous bone gain, and retards the reduction in maturation-related cortical bone gain, whereas vitamin D supplementation stimulates intestinal calcium absorption and prevents the reduction in maturation-related periosteal bone gain by inducing accumulation of calcium from cancellous and endocortical bone.     

Adverse effects of hyperphosphatemia on myocardial hypertrophy, renal function, and bone in rats with renal failure

BACKGROUND: Hyperphosphatemia and disturbances in calcium or parathyroid hormone (PTH) metabolism contribute to the high incidence of cardiovascular disease and renal osteodystrophy in chronic renal failure (CRF). We evaluated the effect of hyperphosphatemia on the cardiovascular system, on renal function, and on bone in experimental uremia. METHODS: Wistar rats were submitted to parathyroidectomy (PTx) and 5/6 nephrectomy (Nx) with minipump implantation, delivering 1-34 rat PTH (physiologic rate), or were sham-operated and received vehicle. Only phosphorus content (low-phosphorus (LP) 0.2%; high-phosphorus (HP) 1.2%) differentiated diets. We divided the groups as follows: PTx +Nx +LP; sham + LP; PTx + Nx + HP; and sham + HP. Tail-cuff pressure and weight were measured weekly. After 2 months, biochemical, arterial, and myocardial histology and bone histomorphometry were analyzed. RESULTS: Heart weight normalized to body weight (heart weight/100 g body weight) was higher in PTx + Nx + HP rats (PTx + Nx + HP = 0.36 +/- 0.01 vs. sham + HP = 0.29 +/- 0.01, PTx + Nx + LP = 0.32 +/- 0.01, sham + LP = 0.28 +/- 0.01) (P < 0.05). Serum creatinine levels were higher in PTx + Nx + HP rats than in PTx + Nx + LP rats (1.09 +/- 0.13 vs. 0.59 +/- 0.03 mg/dL) (P < 0.05). Levels of PTH did not differ significantly between the groups. Myocardial and arterial histology detected no vascular calcification or fibrosis. Bone histomorphometry revealed an association, unrelated to uremia, between HP diets and decreased trabecular connectivity. CONCLUSION: Myocardial hypertrophy, impaired renal function, and adverse effects on bone remodeling were associated with hyperphosphatemia and were not corrected by PTH replacement. Although no vascular calcification was observed in this model, we cannot rule out an adverse effect of hyperphosphatemia on the vascular bed. Our finding underscores the importance of phosphorus control in reducing morbidity and mortality in CRF patients.     

Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism

Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. BACKGROUND: Secondary hyperparathyroidism (secondary HPT) in chronic renal insufficiency (CRI) is characterized by multiglandular hyperplasia. METHODS: In this study, we investigated the effects of the calcimimetic NPS R-568 on the parathyroid gland in rats with CRI induced by ligation of the renal arteries and severe secondary HPT induced by dietary phosphorus loading. Six days after surgery, high-phosphorus diet feeding was started, and NPS R-568 was administered to the rats for 56 days either by daily gavage (30 or 100 micromol/kg) or by continuous subcutaneous infusion (20 micromol/kg. day). RESULTS: After 54 days, serum PTH levels in vehicle-treated CRI rats were 1019 vs. 104 pg/mL in sham-operated controls. Infusion of NPS R-568 maintained serum PTH at levels comparable with those of sham-operated controls, whereas daily gavage also prevented much of the increase in CRI controls and decreased PTH levels intermittently in a dose-dependent fashion. Parathyroid gland enlargement was caused predominantly by hyperplasia. Total cell number per kg body wt was 3.5-fold higher in vehicle-treated CRI rats than in sham-operated controls. Both infusion and high-dose gavage of NPS R-568 completely prevented the increase in parathyroid cell number. CONCLUSION: These results demonstrate that the calcimimetic compound NPS R-568 can prevent both the increase in serum PTH levels and parathyroid hyperplasia in rats with CRI and severe secondary HPT. Moreover, these changes occurred despite decreases in serum 1, 25(OH)2D3 and increases in serum phosphate, suggesting a dominant role for the calcium receptor in regulating parathyroid cell proliferation.     

Paricalcitol aggravates perivascular fibrosis in rats with renal insufficiency and low calcitriol

Cardiovascular complications are a major problem in chronic renal failure. We examined the effects of plasma calcium, phosphate, parathyroid hormone (PTH), and calcitriol on cardiac morphology in 5/6 nephrectomized rats. Fifteen weeks after nephrectomy rats were given a control diet, high-calcium or -phosphorus diet, or given paricalcitol treatment for 12 weeks. Sham-operated rats were on a control diet. Blood pressure, plasma phosphate, and PTH were increased, while the creatinine clearance was reduced in remnant kidney rats. Phosphate and PTH were further elevated by the high-phosphate diet but suppressed by the high-calcium diet, while paricalcitol reduced PTH without influencing phosphate or calcium. The high-calcium diet increased, while the high-phosphate diet reduced plasma calcium. Plasma calcitriol was significantly reduced in other remnant kidney groups, but further decreased after paricalcitol. Cardiac perivascular fibrosis and connective tissue growth factor were significantly increased in the remnant kidney groups, and further increased in paricalcitol-treated rats. Hence, regardless of the calcium, phosphate, or PTH levels, cardiac perivascular fibrosis and connective tissue growth factor increase in rats with renal insufficiency in association with low calcitriol. Possible explanations are that aggravated perivascular fibrosis after paricalcitol in renal insufficiency may be due to further suppression of calcitriol, or to a direct effect of the vitamin D analog.     

Reversal of secondary hyperparathyroidism by phosphate restriction restores parathyroid calcium-sensing receptor expression and function.

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.     

Phosphate depletion in the rat: effect of bisphosphonates and the calcemic response to PTH

BACKGROUND: The removal of phosphate from the diet of the growing rat rapidly produces hypercalcemia, hypophosphatemia, hypercalciuria, and hypophosphaturia. Increased calcium efflux from bone has been shown to be the important cause of the hypercalcemia and hypercalciuria. It has been proposed that the increased calcium efflux from bone is osteoclast mediated. Because bisphosphonates have been shown to inhibit osteoclast-mediated bone resorption, this study was performed to determine whether bisphosphonate-induced inhibition of osteoclast function changed the biochemical and bone effects induced by phosphate depletion. METHODS: Four groups of pair-fed rats were studied: (a) low-phosphate diet (LPD; phosphate less than 0.05%), (b) LPD plus the administration of the bisphosphonate Pamidronate (APD; LPD + APD), (c) normal diet (ND, 0.6% phosphate), and (d) ND + APD. All diets contained 0.6% calcium. A high dose of APD was administered subcutaneously (0.8 mg/kg) two days before the start of the study diet and on days 2, 6, and 9 during the 11 days of the study diet. On day 10, a 24-hour urine was collected, and on day 11, rats were either sacrificed or received an additional APD dose before a 48-hour parathyroid hormone (PTH) infusion (0.066 microgram/100 g/hr) via a subcutaneously implanted miniosmotic pump. RESULTS: Serum and urinary calcium were greater in the LPD and LPD + APD groups than in the ND and ND + APD groups [serum, 11.12 +/- 0.34 and 11.57 +/- 0.45 vs. 9.49 +/- 0.17 and 9.48 +/- 0.15 mg/dl (mean +/- SE), P < 0.05; and urine, 8.78 +/- 2.74 and 16.30 +/- 4.68 vs. 0.32 +/- 0.09 and 0.67 +/- 0.28 mg/24 hr, P < 0.05]. Serum PTH and serum and urinary phosphorus were less in the LPD and LPD + APD than in the ND and ND + APD groups (P < 0.05). The calcemic response to PTH was less (P < 0.05) in the LPD and LPD + APD groups than in the ND group and was less (P = 0.05) in the LPD + APD than in the ND + APD group. Bone histology showed that phosphate depletion increased the osteoblast and osteoclast surface, and treatment with APD reduced the osteoblast surface (LPD vs. LPD + APD, 38 +/- 4 vs. 4 +/- 2%, P < 0.05, and ND vs. ND + APD, 20 +/- 2 vs. 5 +/- 2%, P < 0.05) and markedly altered osteoclast morphology by inducing cytoplasmic vacuoles. CONCLUSIONS: (a) Phosphate depletion induced hypercalcemia and hypercalciuria that were not reduced by APD administration. (b) The calcemic response to PTH was reduced in phosphate-depleted rats and was unaffected by APD administration in normal and phosphate-depleted rats, and (c) APD administration markedly changed bone histology without affecting the biochemical changes induced by phosphate depletion.     

Secondary hyperparathyroidism: Pathophysiology, histopathology, and medical and surgical management

It is generally accepted that morphological changes of the parathyroid glands appear early in renal failure. When diffuse hyperplasia develops into a nodular type, the cells grow monoclonally and proliferate aggressively, with abnormal suppression of parathyroid hormone (PTH) secretion under high extracellular calcium. Based on histopathological and pathophysiological findings, patients with nodular hyperplasia in renal hyperparathyroidism might be refractory to medical treatment, including calcitriol pulse therapy. Thus, parathyroid surgery is indicated for individuals developing hypercalcemia, elevated PTH levels, and/or bone disease, who cannot be effectively treated medically. The detection of enlarged parathyroid glands by image diagnosis is another criterion for surgery. In our experience, parathyroidectomy is an effective treatment; however, the timing of the operation is important, because skeletal deformity and vessel calcification cannot be expected to diminish even after successful surgery. Technically, it is important to identify all parathyroid glands and, in autotransplantation, to use an adequate amount of suitable, tissue, namely, a diffuse type of hyperplastic tissue, to guarantee satisfactory postoperative function.     

Failure of high doses of calcitriol and hypercalcaemia to induce apoptosis in hyperplastic parathyroid glands of azotaemic rats.

BACKGROUND: Whether calcitriol administration, which is used to treat secondary hyperparathyroidism in dialysis patients, induces regression of parathyroid-gland hyperplasia remains a subject of interest and debate. If regression of the parathyroid gland were to occur, the presumed mechanism would be apoptosis. However, information on whether high doses of calcitriol can induce apoptosis of parathyroid cells in hyperplastic parathyroid glands is lacking. Consequently, high doses of calcitriol were given to azotaemic rats and the parathyroid glands were evaluated for apoptosis. METHODS: Rats were either sham-operated (two groups) or underwent a two-stage 5/6 nephrectomy (three groups). For the first 4 weeks, all rats were given a high (1.2%) phosphorus (P) diet to stimulate parathyroid gland growth and then were changed to a normal (0.6%) P diet for 2 weeks. At week 7, three of the five groups were given high doses of calcitriol (500 pmol/100 g body weight) intraperitoneally every 24 h during 72 h before sacrifice. The five groups during week 7 were: (i) normal renal function (NRF)+0.6% P diet; (ii) NRF+0.6% P+calcitriol; (iii) renal failure (RF)+0.6% P; (iv) RF+1.2% P+calcitriol; and (v) RF+0.6% P+calcitriol. Parathyroid glands were removed at sacrifice and the TUNEL stain was performed to detect apoptosis. RESULTS: At sacrifice, the respective serum calcium values in calcitriol-treated groups (groups 2, 4, and 5) were 15.52+/-0.26, 13.41+/-0.39 and 15.12+/-0.32 mg/dl. In group 3, PTH was 178+/-42 pg/ml, but in calcitriol-treated groups, PTH values were suppressed, 8+/-1 (group 2), 12+/-2 (group 4), and 7+/-1 pg/ml (group 5). Despite, the severe hypercalcaemia and marked PTH suppression in calcitriol-treated groups, the percentage of apoptotic cells in the parathyroid glands was very low (range 0.08+/-0.04 to 0.25+/-0.20%) and not different among the five groups. CONCLUSIONS: We found no evidence in hyperplastic parathyroid glands that apoptosis could be induced in azotaemic rats by the combination of high doses of calcitriol and severe hypercalcaemia despite the marked reduction in PTH levels that was observed.     

Calcium homeostasis and bone pathology in magnesium deficient rats

Summary Calcium homeostasis and bone pathology were studied in weanling rats fed a low (70 ppm) magnesium diet for 2–21 days. The rats developed significant, progressive hypercalcemia after 6 days on the diet. The increase in blood calcium was accompanied by progressive hypoactivity of the parathyroid gland (PTG), as determined by histologic and morphometric analyses. Thus hyperactivity of the PTG could not have been responsible for the hypercalcemia observed. Histologic examination of femora and humeri from magnesium-deficient rats showed progressive subperiosteal hyperplasia, consisting of undifferentiated osteoprogenitor cells and fibrous tissue, after 7 days of deficiency. The presence of unmineralized osteoid tissue in the metaphyses indicated that mineralization was not proceeding normally. The alterations in differentiation of osteoprogenitor cells, together with the failure of mineralization, resulted in significantly lower rates of bone formation (as measured by fluorochrome labeling) in the magnesium-deficient rats. Basophilic cementing lines and inactive osteocytes in the cortices of bones from magnesium-deficient rats indicated that bone resorption was also severely reduced in magnesium deficiency. We postulate that bone magnesium depletion (66% by day 21) has a direct negative effect on osteoblastic and osteocytic activity, and may explain, in part, the decreased responsiveness of bone to parathyroid hormone (PTH) that has been observed in magnesium-deficient animals.