Vitamin D status in female dogs with mammary gland tumors.

BACKGROUND: Little information exists about vitamin D status in bitches with mammary tumors. OBJECTIVES: To determine whether low plasma vitamin D concentrations are found in bitches with mammary tumors. ANIMALS: Eighty-five client-owned bitches with mammary tumors (n = 21 benign, n = 64 malignant) and 39 age-matched healthy bitches. METHODS: Case-control study. Plasma ionized and total calcium, phosphorus, magnesium, urea, creatinine, albumin, total proteins, alanine aminotransferase, alkaline phosphatase, parathyroid hormone (PTH), calcitriol (1,25-dihydroxyvitamin D), and 25-hydroxyvitamin D concentrations were measured in all bitches at the time of clinical diagnosis and before any treatments. Statistical analysis was performed to compare variables among groups (control, benign, and malignant). RESULTS: No significant differences were found when plasma 25-hydroxyvitamin D concentrations in bitches with malignant (148.9 [59.9] ng/mL) and benign mammary tumors (150.1 [122.3] ng/mL) were compared with control group (129.9 [54.5] ng/mL). Parathyroid hormone was significantly higher in bitches with malignant (19.9 [20.5] pg/mL), and benign mammary tumors (14.6 [14.9] pg/mL) compared with control group (7.5 [7.5] pg/mL; P < .01). Only the presence of mammary tumors (P < .01) and age (P = .04; adjusted R2 = .22) was significant in predicting PTH. CONCLUSIONS: Bitches with mammary tumors do not have low 25-hydroxyvitamin D concentrations thus vitamin D supplementation is unlikely to be useful for prevention of mammary tumors in bitches.

Direct regulation of fibroblast growth factor 23 by energy intake through mTOR.

To test the hypothesis that fibroblast growth factor 23 (FGF23) is directly regulated by energy intake, in vivo and in vitro experiments were conducted. Three groups of rats were fed diets with high (HC), normal (NC) and low (LC) caloric content that resulted in different energy intake. In vitro, UMR106 cells were incubated in high (HG, 4.5 g/l) or low glucose (LG, 1 g/l) medium. Additional treatments included phosphorus (P), mannitol, rapamycin and everolimus. Intestinal absorption of P and plasma P concentrations were similar in the three groups of rats. As compared with NC, plasma FGF23 concentrations were increased in HC and decreased in the LC group. A significant correlation between energy intake and plasma FGF23 concentrations was observed. In vitro, mRNA FGF23 was significantly higher in UMR106 cells cultured in HG than in LG. When exposed to high P, mRNA FGF23 increased but only when cells were cultured in HG. Cells incubated with HG and mechanistic target of rapamycin (mTOR) inhibitors expressed low mRNA FGF23, similar to the values obtained in LG. In conclusion, this study shows a direct regulation of FGF23 production by energy availability and demonstrates that the mTOR signaling pathway plays a central role in this regulatory system.

Calcimimetics maintain bone turnover in uremic rats despite the concomitant decrease in parathyroid hormone concentration.

Calcimimetics decrease parathyroid hormone (PTH) secretion in patients with secondary hyperparathyroidism. The decrease in PTH should cause a reduction in bone turnover; however, the direct effect of calcimimetics on bone cells, which express the calcium-sensing receptor (CaSR), has not been defined. In this study, we evaluated the direct bone effects of CaSR activation by a calcimimetic (AMG 641) in vitro and in vivo. To create a PTH "clamp," total parathyroidectomy was performed in rats with and without uremia induced by 5/6 nephrectomy, followed by a continuous subcutaneous infusion of PTH. Animals were then treated with either the calcimimetic or vehicle. Calcimimetic administration increased osteoblast number and osteoid volume in normal rats under a PTH clamp. In uremic rats, the elevated PTH concentration led to reduced bone volume and increased bone turnover, and calcimimetic administration decreased plasma PTH. In uremic rats exposed to PTH at 6-fold the usual replacement dose, calcimimetic administration increased osteoblast number, osteoid surface, and bone formation. A 9-fold higher dose of PTH caused an increase in bone turnover that was not altered by the administration of calcimimetic. In an osteosarcoma cell line, the calcimimetic induced Erk1/2 phosphorylation and the expression of osteoblast genes. The addition of a calcilytic resulted in the opposite effect. Moreover, the calcimimetic promoted the osteogenic differentiation and mineralization of human bone marrow mesenchymal stem cells in vitro. Thus, calcimimetic administration has a direct anabolic effect on bone that counteracts the decrease in PTH levels.

Treatment of canine leishmaniasis with marbofloxacin in dogs with renal disease.

Treatment of canine leishmaniasis (CanL) represents a challenge. Due to the high prevalence of renal disease associated to CanL, it is important to find an effective drug that does not damage the kidneys. Marbofloxacin has been shown to be effective and well tolerated in non-azotemic dogs with leishmaniasis. To evaluate the safety and efficacy of marbofloxacin in dogs with leishmaniasis and decreased renal function, 28 dogs suffering from leishmaniasis and chronic kidney disease (CKD) were treated with oral marbofloxacin at 2 mg/Kg/day for 28 days. During treatment dogs were assessed by performing weekly physical exams, measuring blood pressure and evaluating blood and urine parameters. Lymph node aspirations were also obtained at days 0 and 28. The global clinical score decreased significantly, from 6.2±3.4 to 4.7±3.1 (p = 0.0001), after treatment. Marbofloxacin also decreased parasitic load in 72% of the dogs. No significant differences in plasma creatinine, urine specific gravity, urinary concentrations of cystatin C, ferritin and urinary protein loss were detected during treatment. A transient but significant decrease in blood pressure was detected up to day 14 (from 180.1±36.6 to 166.0±32.7 mmHg; p = 0.016). Moreover, dogs showed a significant increase in plasma albumin concentration (from 15.0±5.2 to 16.6±3.9 g/L; p = 0.014) and a significant decrease in globulin concentration (from 59.0±18.1 to 54.1±18.0 g/L; p = 0.005). The results demonstrate that, in addition to being effective for treatment of CanL, marbofloxacin is a very safe drug in dogs with CKD and leishmaniasis.

Chronic Vitamin D Intoxication in Captive Iberian Lynx (Lynx pardinus).

To document the biochemical and pathologic features of vitamin D intoxication in lynx and to characterize mineral metabolism in healthy lynx, blood samples were obtained from 40 captive lynx that had been receiving excessive (approximately 30 times the recommended dose) vitamin D3 in the diet, and from 29 healthy free ranging lynx. Tissue samples (kidney, stomach, lung, heart and aorta) were collected from 13 captive lynx that died as a result of renal disease and from 3 controls. Vitamin D intoxication resulted in renal failure in most lynx (n = 28), and widespread extraskeletal calcification was most severe in the kidneys and less prominent in cardiovascular tissues. Blood minerals and calciotropic hormones in healthy lynx were similar to values reported in domestic cats except for calcitriol which was higher in healthy lynx. Changes in mineral metabolism after vitamin D intoxication included hypercalcemia (12.0 ± 0.3 mg/dL), hyperphosphatemia (6.3 ± 0.4 mg/dL), increased plasma calcidiol (381.5 ± 28.2 ng/mL) and decreased plasma parathyroid hormone (1.2 ± 0.7 pg/mL). Hypercalcemia and, particularly, hyperphosphatemia were of lower magnitude that what has been previously reported in the course of vitamin D intoxication in other species. However, extraskeletal calcifications were severe. The data suggest that lynx are sensitive to excessive vitamin D and extreme care should be taken when supplementing this vitamin in captive lynx diets.

TGF-ß prevents phosphate-induced osteogenesis through inhibition of BMP and Wnt/ß-catenin pathways.

BACKGROUND: Transforming growth factor-ß (TGF-ß) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. RESULTS: Our results showed that TGF-ß induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22α, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/ß-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-ß to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-ß pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/ß-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. CONCLUSIONS: Full VSMC differentiation induced by TGF-ß may not be achieved when extracellular phosphate levels are high. Moreover, TGF-ß prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/ß-catenin pathway.

Magnesium inhibits Wnt/ß-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/ß-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/ß-catenin pathway as demonstrated by the translocation of ß-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/ß-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/ß-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/ß-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro. Inhibition of Wnt/ß-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved.

Mineral metabolism in growing cats: changes in the values of blood parameters with age.

The purpose of this study was to describe changes in calcium, phosphorus, magnesium, parathyroid hormone, calcitriol and calcidiol in cats from 3 to 15 months of age. Fourteen European shorthair healthy cats of both sexes (seven males, seven females) belonging to a research colony were studied from 3 to 15 months of age. Plasma concentrations of total calcium, ionised calcium, albumin, phosphorus, magnesium, intact parathyroid hormone (I-PTH), whole parathyroid hormone (W-PTH), calcidiol and calcitriol were measured at 3, 6, 9, 12 and 15 months of age. From 3 months of age to adulthood cats showed a decrease in calcium (both total and ionised), phosphorus and magnesium. No major changes in PTH were evident, although the ratio of W-PTH:I-PTH decreased significantly with age. A reciprocal change in vitamin D metabolites (decrease in calcitriol and increase in calcidiol) was identified during the growing process. Our results, showing changes in most parameters of mineral metabolism during growth, reinforce the need to use adequate age-related reference values for diagnostic purposes.

The effect of vitamin D derivatives on vascular calcification associated with inflammation.

BACKGROUND: Vitamin D sterols may modulate vascular response to inflammation and vascular calcification (VC). METHODS: Rat aortic rings (RARs) and human vascular smooth muscle cells (HVSMCs) were treated in vitro with phosphate (P), tumour necrosis factor alpha (TNF-α), calcitriol (CTR) and paricalcitol (PCT). Rats having undergone subtotal nephrectomy (Nx) (n = 66) on a high-phosphorus diet were treated with Escherichia coli lipopolysacharide (LPS) (40-400 µg/kg/day) or LPS plus CTR (80 ng/kg/48 h) or LPS plus PCT (240 ng/kg/48 h) for 14 days. RESULTS: In vitro, the addition of TNF-α to the medium increased the mineral content of RAR and HVSMC. Treatment with both vitamin D analogues decreased bone morphogenetic protein 2 but did not modify Runx-2. Calcification was prevented only by PCT. In vivo, treatment with LPS increased plasma levels of TNF-α, monocyte chemotactic protein-1 and interleukin-1alfa and induced calcification. The concomitant administration of LPS with either CTR or PCT led to a significant decrease in cytokine plasma levels and the decrease was more accentuated after treatment with PCT than with CTR. Rats treated with CTR showed an elevation in aortic Ca and marked Von Kossa staining; however, rats treated with PCT did not increase aortic Ca and did not show Von Kossa staining. CONCLUSION: Treatment with PCT resulted in more marked anti-inflammatory effect than treatment with CTR and, as opposed to CTR, PCT prevented VC.

High-phosphate-induced calcification is related to SM22α promoter methylation in vascular smooth muscle cells.

Hyperphosphatemia is closely related to vascular calcification in patients with chronic kidney disease. Vascular smooth muscle cells (VSMCs) exposed to high phosphate concentrations in vitro undergo phenotypic transition to osteoblast-like cells. Mechanisms underlying this transdifferentiation are not clear. In this study we used two in vitro models, human aortic smooth muscle cells and rat aortic rings, to investigate the phenotypic transition of VSMCs induced by high phosphate. We found that high phosphate concentration (3.3 mmol/L) in the medium was associated with increased DNA methyltransferase activity and methylation of the promoter region of SM22α. This was accompanied by loss of the smooth muscle cell-specific protein SM22α, gain of the osteoblast transcription factor Cbfa1, and increased alkaline phosphatase activity with the subsequent in vitro calcification. The addition of a demethylating agent (procaine) to the high-phosphate medium reduced DNA methyltransferase activity and prevented methylation of the SM22α promoter, which was accompanied by an increase in SM22α expression and less calcification. Additionally, downregulation of SM22α, either by siRNA or by a methyl group donor (S-adenosyl methionine), resulted in overexpression of Cbfa1. In conclusion, we demonstrate that methylation of SM22α promoter is an important event in vascular smooth muscle cell calcification and that high phosphate induces this epigenetic modification. These findings uncover a new insight into mechanisms by which high phosphate concentration promotes vascular calcification.

The calcimimetic AMG 641 accelerates regression of extraosseous calcification in uremic rats.

The purpose of the present study was to test the hypothesis that extraskeletal calcification regresses in uremic rats after reduction in phosphorus intake and treatment with calcimimetics. Extraosseous calcification was induced in five to six nephrectomized rats fed a high-phosphorus (1.2%) diet who received calcitriol (80 ng/kg ip) every other day for a period of 14 days. Next, dietary phosphorus was reduced to 0.6%, and rats were treated with vehicle (n = 20), calcitriol [80 ng/kg ip/48 h (n = 20)], or the calcimimetic AMG 641 [1.5 mg/kg sc/48 h (n = 20)]. Aortic and soft-tissue calcium and phosphorus content was evaluated after 14 and 28 days. At 28 days, reduction of phosphorus intake resulted in a significant decrease in tissue mineral content in vehicle- and AMG 641-treated rats but not in rats receiving calcitriol. Aortic calcium and phosphorus was lower in rats treated with AMG 641 (96.7 +/- 26.4 mg/g) than in rats receiving vehicle (178.3 +/- 38.6 mg/g). An infiltrate of phagocytic cells expressing the calcium-sensing receptor was identified in areas surrounding foci of calcification. Additional studies in parathyroidectomized rats demonstrated that AMG 641 increased the urinary excretion of calcium (6.2 +/- 0.6 vs. 3.1 +/- 0.5 mg/day, vehicle) (P < 0.001). In conclusion, experimentally induced extraosseous calcification in uremic rats can be partially resolved by reducing phosphorus intake; the addition of calcimimetics may accelerate the regression process through mechanisms potentially involving a direct stimulatory effect on mineral phagocytic cells plus an increase in urinary calcium excretion.

Effects of calcimimetics on extraskeletal calcifications in chronic kidney disease.

While the precise mechanisms of vascular calcification (VC) in chronic kidney disease (CKD) remain to be elucidated, there is a close association between VC and secondary hyperparathyroidism (HPT). The elevations in calcium, phosphorus, the Ca x P product, and parathyroid hormone (PTH) observed in patients with CKD and secondary HPT have been associated with VC and increased risk of cardiovascular morbidity and mortality. We have investigated the development of extraskeletal calcification in uremic rats with secondary HPT treated with vitamin D derivatives (calcitriol or paricalcitol), calcimimetics (R-568 or AMG 641), or the combination of both types drugs. Treatment with calcitriol resulted in a significant increase in the extraosseous calcium and phosphorus content and high mortality. By contrast, treatment with calcimimetics, which provided a better control of plasma PTH levels, did not result in extraskeletal mineral accumulation and did not cause mortality. More important, when added to calcitriol, calcimimetics prevented the development of VC and reduced mortality. Paricalcitol administration to uremic rats resulted in calcification levels and mortality rates that were lower than in rats treated with calcitriol but higher than in rats treated with calcimimetics. The mechanism(s) of action responsible for the anticalcification effect of calcimimetics are likely related to the fact that these drugs can control PTH levels without increasing the plasma Ca x P product. In addition calcimimetic activation of vascular calcium-sensing receptor may also modulate the expression of proteins that prevent the development of VC, like matrix Gla protein.

Dynamics of parathyroid hormone secretion in health and secondary hyperparathyroidism.

This review examines the dynamics of parathyroid hormone secretion in health and in various causes of secondary hyperparathyroidism. Although most studies of parathyroid hormone and calcium have focused on the modification of parathyroid hormone secretion by serum calcium, the relationship between parathyroid hormone and serum calcium is bifunctional because parathyroid hormone also modifies serum calcium. In normal animals and humans, factors such as phosphorus and vitamin D modify the basal parathyroid hormone level and the maximal parathyroid hormone response to hypocalcemia. Certain medications, such as lithium and estrogen, in normal individuals and sustained changes in the serum calcium concentration in hemodialysis patients change the set point of calcium, which reflects the serum calcium concentration at which parathyroid hormone secretion responds. Hypocalcemia increases the basal/maximal parathyroid hormone ratio, a measure of the relative degree of parathyroid hormone stimulation. The phenomenon of hysteresis, defined as a different parathyroid hormone value for the same serum calcium concentration during the induction of and recovery from hypo- and hypercalcemia, is discussed because it provides important insights into factors that affect parathyroid hormone secretion. In three causes of secondary hyperparathyroidism--chronic kidney disease, vitamin D deficiency, and aging--factors that affect the dynamics of parathyroid hormone secretion are evaluated in detail. During recovery from vitamin D deficiency, the maximal parathyroid hormone remains elevated while the basal parathyroid hormone value rapidly becomes normal because of a shift in the set point of calcium. Much remains to be learned about the dynamics of parathyroid hormone secretion in health and secondary hyperparathyroidism.

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.

Reversibility of calcitriol-induced medial artery calcification in rats with intact renal function.

UNLABELLED: VC is an important clinical entity; however, very little information is available on its resolution. Induction and regression of calcitriol-induced VC was studied in 47 rats. After calcitriol withdrawal, there was a relatively rapid regression of VC mediated by an active cellular process. INTRODUCTION: Vascular calcifications (VCs) represent an important risk factor for cardiovascular death. Although VCs are prevalent in relevant diseases (e.g., chronic kidney disease, osteoporosis, diabetes), the reversibility of extraskeletal calcifications is an unresolved issue. This study was conducted to investigate (1) the in vivo effect of calcitriol on VC and (2) whether calcitriol-induced VC would regress after suppression of calcitriol treatment. MATERIALS AND METHODS: The calcifying effect of calcitriol was studied in four groups of rats (n = 8) that received calcitriol (1 mug/kg, IP) for 2, 4, 6, and 8 days. The reversibility of VC was studied in three additional groups (n = 5) treated with 1 mug/kg of calcitriol for 8 days that were subsequently killed 1, 2, and 9 weeks after the last calcitriol dose. Aortic VC was assessed by histology and by quantification of aortic calcium and phosphorus content. The aortic wall was studied by histology and immunohistochemistry. Statistical analysis was performed by ANOVA and t-tests. RESULTS: Calcitriol administration resulted in a time-dependent induction of VC, with aortic calcium and phosphorus being significantly increased at 6 and 8 days. Treatment with calcitriol for 8 days resulted in massive medial calcification of the aorta with a 10- to 30-fold increase in the aortic Ca and P content. After suppressing calcitriol administration, a progressive decrease in von Kossa staining and aortic Ca (from 32.8 +/- 2.5 to 9.3 +/- 1.8 mg/g of tissue, p < 0.001) and P (from 11.9 +/- 1.2 to 2.7 +/- 1.8 mg/g of tissue, p = 0.001) content was evidenced. Histology of the aortic wall showed monocytes adhered to the aortic endothelium and macrophages involved in the reabsorption of calcium deposits. CONCLUSIONS: Our results show that calcitriol treatment induces time-dependent VC. After calcitriol withdrawal, VC regress rapidly with aortic calcium and phosphorus decreasing by 75% in the course of 9 weeks. An active cellular process seems to be involved in regression of VC.

Regulation of parathyroid function in chronic renal failure.

This review summarizes the factors involved in the development of hyperparathyroidism secondary (2nd-HPTH) to chronic kidney disease (CKD). Calcium and calcitriol act on their respective specific parathyroid cell receptors to inhibit parathyroid function. As well as the well-known effect of calcium and calcitriol on parathyroid cell function, there is experimental work that demonstrates that phosphate, changes in pH, PTHrP, estrogens, and some cytokines also have an effect on PTH secretion. These factors are relevant in patients with chronic kidney disease. However, low calcium, vitamin D deficiency, and an accumulation of phosphate due to the decrease in renal function are the main pathogenic factors involved in the pathogenesis of 2nd-HPTH in CKD patients.