High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro

Phosphate retention plays an important role in the pathogenesis of secondary hyperparathyroidism in patients with renal failure. In in vitro studies, high extracellular phosphate levels directly stimulate PTH secretion in rat and bovine parathyroid tissue. The present study evaluates the effect of high phosphate levels on the secretion of PTH and the production of prepro PTH mRNA in human hyperplastic parathyroid glands. The study includes parathyroid glands obtained from patients with primary adenomas and from hemodialysis and kidney-transplant patients with diffuse and nodular secondary hyperplasia. The experiments were performed in vitro using small pieces of parathyroid tissue. The ability of high calcium levels to decrease PTH secretion was less in adenomas than in secondary hyperplasia; among the secondary hyperplasia, nodular was less responsive to an increase in calcium than diffuse hyperplasia. In diffuse hyperplasia, PTH secretion was increased in response to 3 and 4 mM phosphate compared with 2 mM phosphate, despite a high calcium concentration in the medium; prepro PTH mRNA levels increased after incubation in 4 mM phosphate. Similar results were obtained with nodular hyperplasia, except that the elevation of PTH secretion in response to 3 mM phosphate did not attain statistical significance. In adenomas, high calcium concentrations (1.5 mM) did not result in inhibition of PTH secretion, independent of the phosphate concentration, and the prepro PTH mRNA was not significantly increased by high phosphate levels. In conclusion, first, the PTH secretory response to an increase in calcium concentration is less in nodular than diffuse hyperplasia; second, high phosphate levels directly affect PTH secretion and gene expression in patients with advanced secondary hyperparathyroidism.     

Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice

1,25-Dihydroxyvitamin D3 plays a major role in intestinal calcium transport. To determine what phenotypic abnormalities observed in vitamin D receptor (VDR)-ablated mice are secondary to impaired intestinal calcium absorption rather than receptor deficiency, mineral ion levels were normalized by dietary means. VDR-ablated mice and control littermates were fed a diet that has been shown to prevent secondary hyperparathyroidism in vitamin D-deficient rats. This diet normalized growth and random serum ionized calcium levels in the VDR-ablated mice. The correction of ionized calcium levels prevented the development of parathyroid hyperplasia and the increases in PTH messenger RNA synthesis and in serum PTH levels. VDR-ablated animals fed this diet did not develop rickets or osteomalacia. However, alopecia was still observed in the VDR-ablated mice with normal mineral ions, suggesting that the VDR is required for normal hair growth. This study demonstrates that normalization of mineral ion homeostasis can prevent the development of hyperparathyroidism, osteomalacia, and rickets in the absence of the genomic actions of 1,25-dihydroxyvitamin D3.     

Cell-specific signal transduction of parathyroid hormone (PTH)-related protein through stably expressed recombinant PTH/PTHrP receptors in vascular smooth muscle cells

PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.     

In vivo regulation of chromogranin A messenger ribonucleic acid in the parathyroid by 1,25-dihydroxyvitamin D: studies in normal rats and in chronic renal insufficiency

Chromogranin-A (CgA) and PTH are the two major secretory products of the parathyroid gland. In vitro, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases CgA, but decreases PTH messenger RNA (mRNA) levels. We investigated the physiological significance of the induced changes in CgA expression by examining the effects of 1,25-(OH)2D3 on parathyroid CgA mRNA levels in vivo. Normal rats were injected with 1,25-(OH)2D3 at 48 and 24 h before blood sampling and isolation of both parathyroid glands. Parathyroid total RNA was extracted and CgA and PTH mRNA quantified by Northern blot analysis. CgA mRNA levels increased 1.6-, 3.2- and 5.6-fold, whereas PTH mRNA levels decreased by 37, 63 and 97%, respectively, with 1,25-(OH)2D3 doses of 10, 50, and 250 pmol/100 g BW. Parathyroid gland CgA expression also was examined in rats with mild chronic renal insufficiency, induced by a 5/6 nephrectomy 5 weeks earlier. Chronic renal insufficiency rats, fed normal chow, had elevated serum urea, creatinine, and PTH levels and reduced 1,25-(OH)2D3 but normal serum levels of calcium and phosphate. PTH mRNA levels were elevated 4-fold and CgA mRNA levels were 50% lower in the uremic animals. This indicates that the regulation of CgA expression in normocalcemic rats occurs at physiological 1,25-(OH)2D3 concentrations. In summary, increases and decreases in serum 1,25-(OH)2D3 levels are associated with corresponding increases and decreases in CgA mRNA levels in the parathyroid glands of rats. Therefore, this study is the first to demonstrate the physiological relevance of the earlier in vitro observations.     

Dietary gamma-linolenic acid enhances mouse macrophage-derived prostaglandin E1 which inhibits vascular smooth muscle cell proliferation

We previously demonstrated that macrophages isolated from mice fed gamma-linolenic acid (GLA)-enriched diets reduce vascular smooth muscle cell (SMC) proliferation in a cyclooxygenase-dependent fashion and may therefore favorably modulate the atherogenic process. The present study was conducted to elucidate the mechanism(s) by which dietary GLA influences the ability of macrophages to modulate SMC growth programs. Resident peritoneal macrophages were isolated from C57BL/6 female mice fed diets containing variable GLA compositions at 10% (wt/wt), treated with various antibodies and co-cultured with cycling naive vascular SMC isolated from nonpurified diet-fed mice. Smooth muscle cell proliferation and intracellular cAMP levels were measured after co-culture. In parallel experiments, cycling naive vascular SMC isolated from nonpurified diet-fed mice were dosed with exogenous prostaglandin E1 (PGE1 ) for various periods and challenged with cycloheximide for 4 h (8-12 h after PGE1 addition), and intracellular cAMP levels were measured at various time points. Macrophages isolated from mice fed GLA-enriched dietary oils significantly reduced SMC proliferation in co-culture compared with controls (macrophages from mice fed a corn oil diet containing no GLA). Anti-PGE1 antiserum treatment (1:50 or 1:100) blocked the ability of GLA-enriched macrophages to down-regulate SMC proliferation, a response reversed by exogenous PGE1 treatment. Macrophages isolated from mice fed GLA-enriched dietary oils elevated SMC intracellular cAMP levels in a biphasic fashion. In addition, exogenous PGE1 (1 nmol/L to 10 micromol/L) exerted a similar biphasic cAMP response in SMC, and the second phase of cAMP elevation was antagonized by cycloheximide. In conclusion, dietary GLA enhances mouse macrophage-derived prostaglandin E1, which inhibits vascular SMC proliferation.     

Recovery of free ADP, Pi, and free energy of ATP hydrolysis in human skeletal muscle

BACKGROUND: Recent studies have demonstrated that a high concentration of phosphate directly stimulates parathyroid hormone (PTH) secretion. High serum levels of phosphate are usually observed in patients with end-stage renal disease. The aim of the present study was to evaluate whether serum phosphate concentration had an acute effect on PTH secretion in hemodialysis patients. The levels of serum phosphate were manipulated during the hemodialysis session by using a phosphate free dialysate or a dialysate with a high content of phosphate. METHODS: Ten stable hemodialysis patients with PTH values above 300 pg/ml were included in the study. A PTH-calcium curve was obtained during both high phosphate and phosphate free hemodialysis. RESULTS: The serum phosphate concentration remained high (2.17 +/- 0.18 mM) throughout the high phosphate hemodialysis and decreased progressively to normal levels (1.02 +/- 0.06 mM) during the phosphate free hemodialysis. The serum PTH levels at maximal inhibition by hypercalcemia (minimal PTH) were greater during the high phosphate than the phosphate free hemodialysis (413 +/- 79 vs. 318 +/- 76 pg/ml, P < 0.003). In all patients the values of minimum PTH were greater during the high phosphorus than the phosphorus free hemodialysis. The values of maximally stimulated PTH during hypocalcemia and the set point of the PTH-calcium curve were similar during the high phosphate and the phosphate free hemodialysis. CONCLUSION: The maintenance of high serum phosphorus levels during hemodialysis prevented, in part, the inhibition of PTH secretion by calcium, which strongly suggests that in hemodialysis patients high serum phosphate contributes directly to the elevation of PTH levels despite normal or high serum calcium concentration.     

Lack of effect of hypomagnesemia on elevated plasma 1,25-dihydroxycholecalciferol in acutely phosphate-deprived rats

The effect of hypomagnesemia occurring in phosphate deprivation on elevated levels of plasma 1,25-dihydroxycholecalciferol (1,25(OH)2D3) was evaluated in rats. Phosphate deprivation was associated with an elevated plasma 1,25(OH)2D3, hypophosphatemia, and hypomagnesemia. With oral magnesium supplement during phosphate deprivation, plasma magnesium did not fall, but plasma inorganic phosphate fell and 1,25(OH)2D3 rose to comparable levels seen in phosphate-deprived rats lacking magnesium supplement. These results indicate that a rise in plasma 1,25(OH)2D3 during phosphate deprivation is not due to concomitant hypomagnesemia and support the postulate that phosphate deprivation per se is responsible for the elevated plasma 1,25(OH)2D3.     

The effects of a high fat diet on leptin mRNA, serum leptin and the response to leptin are not altered in a rat strain susceptible to high fat diet-induced obesity

Osborne-Mendel (OM) and S5B/Pl rats differ in their sensitivity to develop obesity when fed a high fat (HF) diet; OM rats become obese, whereas S5B/Pl rats remain thin. We have investigated the possibilities that either an impaired leptin response or resistance to leptin action underlies the sensitivity to this form of obesity in OM rats. In Experiment 1, OM and S5B/Pl rats fed a nonpurified diet were killed at d 0 or were fed either a HF (56% fat energy) or a low fat (LF, 10% fat energy) diet for 2 or 7 d. The HF diet increased serum leptin significantly by d 2 to levels that were similar in both rat strains. At 7 d, leptin levels were lower than at d 2 but remained higher than levels in the d 0 control groups. The leptin mRNA:18S RNA ratio in epididymal adipose tissue increased to higher levels in HF-fed OM rats than in S5B/Pl rats fed that diet. However, although the LF diet had no effect in S5B/Pl rats, it increased leptin mRNA levels in epididymal adipose tissue of OM rats compared with the controls fed the nonpurified diet. In Experiment 2, OM and S5B/Pl rats were fed HF or LF diets for 5 wk. At that time, their feeding response to a range of leptin doses (0, 1, 5 or 10 microgram) given intracerebroventricularly was tested after overnight food deprivation. There was a similar dose-dependent reduction in energy intake in response to leptin in both OM and S5B/Pl rats. These responses were independent of the diet. The data suggest that the susceptibility of OM rats to HF diet-induced obesity is not related to either a loss of central sensitivity to leptin or a failure to enhance leptin production acutely, although the failure to maintain chronically increased levels of serum leptin could contribute to the obesity.     

Three-dimensional triple-quantum-filtered 23Na imaging of the dog head in vivo

Uninephrectomized rats with diet-induced hypercholesterolemia develop interstitial inflammation and fibrosis after 8 to 12 weeks. Fibrosis has been associated with the accumulation of lipid peroxidation products within the tubulointerstitium, along with increased renal mRNA levels for transforming growth factor beta-1 (TCF-beta 1), some matrix proteins, and the tissue inhibitor of metalloproteinases (TIMP-1). However, mRNA levels for urokinase-type plasminogen activator (uPA) have been found to be decreased. The purpose of the present study was to determine whether antioxidant therapy could attenuate interstitial fibrosis in hypercholesterolemic rats and to determine changes in the pattern of renal gene expression induced by antioxidant therapy. Three groups of uninephrectomized rats were studied after 12 weeks of feeding standard rat chow, an atherogenic diet (standard chow plus 4% cholesterol/1% cholic acid), or an atherogenic diet supplemented with high doses of the antioxidants probucol and vitamin E. Rats fed the atherogenic diet developed hypercholesterolemia and a 56% increase in total kidney collagen compared with rats fed standard chow. In comparison, the hypercholesterolemic rats treated with antioxidants had normal levels of renal lipid peroxidation products and a normal kidney collagen content. In contrast, there were no significant differences in urinary albumin excretion rates or the number of interstitial macrophages between the two hypercholesterolemic groups. Compared with the untreated hypercholesterolemic group, antioxidant therapy induced significant reductions in renal mRNA levels for procollagen III (to 60% of untreated levels), collagen IV (60%), and TIMP-1 (20%), while uPA levels were significantly increased (to 210%). Paradoxically, antioxidant therapy was associated with a significant increase in renal TGF-beta 1 mRNA levels (to 150%), although TGF-beta 1 protein expression shifted from interstitial to tubular epithelial cells in predominance. The results of the present study demonstrate the efficiency of antioxidant therapy in preventing renal interstitial fibrosis in hypercholesterolemic rats with a single kidney. Based on changes in renal gene expression at the mRNA level, impaired matrix protein synthesis and increased intrarenal activity of the metalloproteinases and uPA/plasmin may play a role in the attenuation of fibrosis.     

Normal phosphate transport in cells from the S2 and S3 segments of Hyp-mouse proximal renal tubules

An intrinsic phosphate (Pi) transport defect in the proximal tubule (PT) presumably underlies X-linked hypophosphatemic rickets. We recently reported normal Pi transport in the S1 segment of the Hyp mouse PT. Whether Pi wasting results from an abnormality in the S2 or S3 segment remains unknown. Thus, we compared Pi transport in S2 and S3 immortalized cells from transgenic (simian virus 40) normal and Hyp mice. These cells display biochemical features of PT cells, including alkaline phosphatase- and hormone- stimulated cAMP activity as well as gluconeogenesis. Moreover, kinetic studies in S2 cells reveal a similar Km[0.26 +/- 0.03 (+/-SEM) vs. 0.22 +/- 0.03 mM] and maximum velocity (Vmax; 5.5 +/- 0.66 vs. 5.9 +/- 0.72 nmol/mg x 5 min) in normal and Hyp mice, respectively. Km and Vmax were also similar in cells from the S3 segment; however, the Vmax values in S3 cells in normal and Hyp mice (2.8 +/- 0.45 and 3.0 +/- 0.56 nmol/mg x 5 min) were reduced in both animal models compared to those in S2 cells (P < 0.001), whereas the Km values in S3 cells from normal and Hyp mice (0.10 +/- 0.02 and 0.11 +/- 0.04 mM) were increased relative to those in S2 cells (P < 0.001). These data indicate that Pi transport throughout the PT of Hyp mice is intrinsically normal. Such observations exclude the presence of a nascent defect in renal Pi transport in the kidneys of Hyp mice and support the hypothesis that a humoral abnormality underlies X-linked hypophosphatemic rickets.     

Troglitazone action is independent of adipose tissue

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.     

Human hydrometry: comparison of multifrequency bioelectrical impedance with 2H2O and bromine dilution

The renal transport and fractional reabsorption of inorganic sulfate is altered under conditions of sulfate deficiency or excess. The objective of this study was to examine the cellular mechanisms of adaptation of renal sodium/sulfate cotransport after varying dietary intakes of a sulfur containing amino acid, methionine. Female Lewis rats were divided into four groups and fed diets containing various concentrations of methionine (0, 0.3, 0.82 and 2.46%) for 8 days. Urinary excretion rates and renal clearance of sulfate were significantly decreased in the animals fed a 0% methionine diet or a 0.3% methionine diet, and significantly increased in the animals fed a 2.46% methionine diet when evaluated on days 4 and 7. Serum sulfate concentrations were unchanged by diet treatment in all animals. The fractional reabsorption of sulfate was significantly increased in the animals fed the 0% methionine diet and the 0.3% methionine diets, and decreased in the animals fed the 2.46% methionine diet. Increased mRNA and protein levels for the sodium/sulfate transporter (NaSi-1) were found in the kidney cortex following treatment with the 0 and 0.3% methionine diet groups. Sulfate homeostasis by renal reabsorption is maintained by an up-regulation of steady state levels of NaSi-1 mRNA and protein when the diet is low in methionine.     

Molecular cloning of a novel putative Ca2+ channel protein (TRPC7) highly expressed in brain

BACKGROUND: Skeletal muscle abnormalities contribute considerably to the clinical expression of heart failure. Deconditioning, underperfusion and an increased number of type IIb glycolytical fibres lead to early lactate production and muscle fatigue at low exercise levels. Aerobic muscle metabolism may also be impaired, as suggested by biopsy studies. Thus far, no data are available from non-invasive studies to indicate the extent of aerobic muscle dysfunction during low-grade exercise which does not induce acidosis. METHODS AND RESULTS: Mitochondrial function of skeletal muscle during fibre type I activation was studied in 22 patients with chronic heart failure [NYHA class III, left ventricular ejection fraction 28 +/- 2%, (patients)] on ACE inhibitors, diuretics and digoxin, and in 20 normal subjects, using 31P NMR spectroscopy of a single right forearm flexor muscle during three mild intermittent exercise levels (0-40% of maximum voluntary contraction) and recovery time. At rest, the inorganic phosphate/phosphocreatine ratio was different [0.13 +/- 0.005 (patients) vs 0.09 +/- 0.002 (normal subjects), P = 0.0001]. However, intracellular pH was comparable. Local acidosis (tissue pH < 6.9) was avoided to prevent fibre type IIb activation. Calculated resting phosphate potential levels were comparable, but the slope and intercept of the linear relationship of phosphate potential and workload were significantly lower in patients than in normal subjects (11.7 +/- 0.7 vs 15.8 +/- 0.6 and 139 +/- 7 vs 196 +/- 7, patients vs normal subjects, indicating early exhaustion of intracellular energy at lower exercise levels. Also, maximum calculated workload at which tissue ADP stabilized was lower in patients than in normal subjects (88 +/- 7% vs 120 +/- 4% of maximum voluntary workload, patients vs normal subjects, P < 0.05). Time to recovery to pre-test phosphocreatine levels was prolonged by 46% in patients compared to normal subjects (P < 0.05). CONCLUSIONS: In heart failure, oxidative fibre mitochondrial function in skeletal muscle is impaired, as reflected by the reduced phosphate potential and oxidative phosphorylation rate, early exhaustion and slowed recovery of intracellular energy reserve at workloads, which do not affect intracellular pH.     

Femoral abnormalities and vitamin D metabolism in X-linked hypophosphatemic (Hyp and Gy) mice

X-linked hypophosphatemia is a genetic bone disease in humans and mice. Two closely linked mutations in mice, Hyp and Gy, cause low plasma phosphate and a rachitic and osteomalacic bone disease. Because of the controversy as to whether Gy is a good model for X-linked hypophosphatemia, the phenotypic severity of these two mutations was compared in both sexes and on two genetic backgrounds. The depression in plasma levels of phosphate was similar in all 10-week-old mutant mice. Male Hyp mice and heterozygous female Hyp mice were affected with similar severity in terms of reduced tail growth, shortened femora, reduced femoral mineral content, and abnormal mineral composition of the femoral matrix. In contrast, male Gy mice did not survive on the C57BL/6J background and were more severely affected than female Gy mice on the B6C3H background. The hybrid B6C3H background ameliorated the bone disease compared with the inbred C57BL/6J background for both mutant strains. There was no evidence of change in the plasma levels of 1,25-dihydroxyvitamin D, duodenal level of vitamin D-dependent calcium-binding protein, or urinary level of calcium in these adult mutant mice. In summary, Gy mice have a sexual dimorphism not present in Hyp mice. These two genes may indicate the presence of multiple gene loci in the human disease, with multiple proteins involved in the pathophysiology of the bone disease.