Androgen therapy does not prevent bone loss and arterial calcifications in male rats with chronic kidney disease.

Patients suffering from chronic kidney disease (CKD) often experience bone loss and arterial calcifications. It is unclear if hypogonadism contributes to the development of these complications and whether androgen therapy might prevent them. Male adult rats were randomized into four groups. The first group received standard chow (control), while three other groups were fed a 0.25% adenine/low vitamin K diet (CKD). Two CKD groups were treated with testosterone or dihydrotestosterone (DHT), whereas the control group and one CKD group received vehicle (VEH). CKD animals had 10-fold higher serum creatinine and more than 15-fold higher parathyroid hormone levels compared to controls. Serum testosterone levels were more than two-fold lower in the CKDVEH group compared to control + VEH and CKD + testosterone groups. Seminal vesicle weight was reduced by 50% in CKDVEH animals and restored by testosterone and DHT. CKD animals showed a low bone mass phenotype with decreased trabecular bone volume fraction and increased cortical porosity, which was not rescued by androgen treatment. Aortic calcification was much more prominent in CKD animals and not unequivocally prevented by androgens. Messenger RNA expression of the androgen receptor-responsive genes Acta1 and Col1a1 was reduced by CKD and stimulated by androgen treatment in levator ani muscle but not in the bone or aortic tissue. We conclude that adenine-induced CKD results in the development of hypogonadism in male rats. Androgen therapy is effective in restoring serum testosterone levels and androgen-sensitive organ weights but does not prevent bone loss or arterial calcifications, at least not in the presence of severe hyperparathyroidism.

Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency.

Bone turnover requires the interaction of several proteases during the resorption phase. Indirect evidence suggests that the plasminogen activator/plasmin pathway is involved in bone resorption and turnover, and recently we have shown that this cascade plays a role in the degradation of nonmineralized bone matrix in vitro. To elucidate the role of the plasminogen activator inhibitor 1 (PAI-1) in bone turnover in vivo, bone metabolism was analyzed in mice deficient in the expression of PAI-1 gene (PAI-1-/-) at baseline (8-week-old mice) and 4 weeks after ovariectomy (OVX) or sham operation (Sham) and compared with wild-type (WT) mice. PAI-1 inactivation was without any effect on bone metabolism at baseline or in Sham mice. However, significant differences were observed in the response of WT and PAI-1-/- mice to ovariectomy. The OVX WT mice showed, as expected, decreased trabecular bone volume (BV/TV) and increased osteoid surface (OS/BS) and bone formation rate (BFR), as assessed by histomorphometric analysis of the proximal tibial metaphysis. In contrast, no significant change in any of the histomorphometric variables studied was detected in PAI-1-/- mice after ovariectomy. As a result, the OVX PAI-1-/- had a significantly higher BV/TV, lower OS/BS, lower mineral apposition rate (MAR) and BFR when compared with the OVX WT mice. However, a comparable decrease in the cortical thickness was observed in OVX PAI-1-/- and WT mice. In addition, the cortical mineral content and density assessed in the distal femoral metaphysis by peripheral quantitative computed tomography (pQCT), decreased significantly after ovariectomy, without difference between PAI-1-/- mice and WT mice. In conclusion, basal bone turnover and bone mass are only minimally affected by PAI-1 inactivation. In conditions of estrogen deficiency, PAI-1 inactivation protects against trabecular bone loss but does not affect cortical bone loss, suggesting a site-specific role for PAI-1 in bone turnover.

Bone resorption induced by 1 alpha,25 dihydroxyvitamin D(3) in vivo is not altered by inactivation of the plasminogen activator inhibitor 1.

One of the proteolytic systems produced by bone cells is the plasminogen activator/plasmin pathway, which involves the two plasminogen activators and the type 1 plasminogen activator inhibitor (PAI-1) and results in plasmin generation. We have recently demonstrated that this pathway plays a specific role in the degradation of the nonmineralized matrix of bone in vitro. To evaluate whether PAI-1 is required during bone resorption in vivo, we studied the effects of PAI-1 inactivation on bone metabolism using systemic administration of 1alpha,25 dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] as model. PAI-1-deficient (PAI-1-/-) and wild-type (WT) mice were injected intraperitoneally with 1,25(OH)(2)D(3) (2 microg/kg) or vehicle every other day during 4 weeks and analyzed using biochemical parameters of bone turnover, histomorphometric analysis of the proximal tibial metaphysis, and pQCT analysis of the distal femoral metaphysis. PAI-1 inactivation did not affect bone metabolism in vehicle-treated mice. Treatment with 1,25(OH)(2)D(3) induced bone resorption similarly in PAI-1-/- and WT mice, as assessed by the increase in the urinary excretion of calcium (2. 2-fold and 2.3-fold, respectively) and of pyridinoline crosslinks (by 24% and 22%, respectively). In addition, a comparable reduction in bone mass was observed in PAI-1-/- and WT mice after treatment with 1,25(OH)(2)D(3), as evidenced by the decrease in the femoral calcium content (by 25% and 32%, respectively), in the trabecular bone volume (by 50% and 40%, respectively), in the trabecular mineral content (by 17% and 15%, respectively), and in the cortical mineral content (by 45% and 52%, respectively). The parameters of bone turnover also increased after 1,25(OH)(2)D(3) treatment. Serum osteocalcin was, respectively, 25% and 28% higher in PAI-1-/- and WT mice treated with 1,25(OH)(2)D(3) compared with the mice injected with vehicle. Similarly, the osteoid surface increased in 1, 25(OH)(2)D(3)-treated PAI-1-/- and WT mice by 40% and 51%, respectively, the mineral apposition rate increased by 15% and 8%, respectively, and the bone formation rate by 54% and 48%, respectively. These data indicate that PAI-1 is not critical during bone resorption induced by 1,25(OH)(2)D(3) in vivo.

Skeletal effects of estrogen deficiency as induced by an aromatase inhibitor in an aged male rat model.

Aromatization of androgens into estrogens may be important for maintenance of the male skeleton. To address this hypothesis, we evaluated the skeletal effects of selective estrogen deficiency as induced by the aromatase inhibitor vorozole (Vor), with or without 17beta-estradiol (E(2)) administration (1.35 microg/day), in aged (12-month-old) male rats. A baseline group was killed at the start of the experiment (Base). The control group (Control), the group treated with vorozole alone (Vor), the group treated with E(2) alone (E(2)), or the group with a combination of both (Vor + E(2)) were killed 15 weeks later. Vorozole significantly increased serum testosterone (T) and reduced serum E(2) compared with Control. Body weight gain and serum insulin-like growth factor-I (IGF-I) were also lower in Vor, whereas significant weight loss and decrease of serum IGF-I occurred as a result of E(2) administration. Bone formation as assessed by serum osteocalcin was unaffected but osteoid surface in the proximal metaphysis of the tibia was increased in Vor-treated rats. Bone resorption as evaluated by urinary deoxypyridinoline excretion was increased in Vor. Biochemical parameters of bone turnover were reduced significantly in all E(2) treated rats. Premature closure of the growth plates and decreased osteoid and mineralizing surfaces were also observed in E(2) and Vor + E(2). Apparent bone density of lumbar vertebrae and femur, as measured by dual-energy X-ray absorptiometry (DXA), was significantly reduced in Vor. Vorozole decreased femoral bone density mainly in the distal femur (trabecular and cortical region). This decrease of bone density was not present in E(2) and Vor + E(2). Similar findings were observed when bone density was assessed by peripheral quantitative computed tomography (pQCT); that is, trabecular density of the distal femur, the proximal tibia, and the distal lumbar vertebra were all lower in Vor. This decrease in density was not observed in all E(2)-treated animals. In conclusion, administration of the aromatase inhibitor, vorozole, to aged male rats induces net trabecular bone loss in both the appendicular and axial skeleton, despite a concomitant increase in serum testosterone. E(2) administration is able to prevent this trabecular bone loss in vorozole-treated animals.

Maternal hypervitaminosis D reduces fetal bone mass and mineral acquisition and leads to neonatal lethality.

Pregnancy challenges maternal calcium handling because sufficient calcium has to be transferred to the fetus to ensure fetal bone mass acquisition. 1,25(OH)2 vitamin D [1,25(OH)2D] is an important regulator of calcium homeostasis during adulthood, yet its role seems redundant for the maternal adaptations to pregnancy as well as during fetal development. However, not only deficiency but also excess of 1,25(OH)2D can be harmful and we therefore questioned whether high maternal 1,25(OH)2D levels may injure fetal development or neonatal outcome, as maternal-fetal transport of 1,25(OH)2D has been largely disputed. To this end, vitamin D receptor (VDR) null (Vdr(-/-)) females, displaying high 1,25(OH)2D levels, were mated with Vdr(+/-) males to obtain pregnancies with fetuses that are responsive (Vdr(+/-)) or resistant (Vdr(-/-)) to 1,25(OH)2D. Surprisingly, most of the Vdr(+/-) neonates died shortly after birth, whereas none of the Vdr(-/-). Mechanistically, we noticed that in Vdr(+/-) embryos, serum calcium levels were normal, but that skeletal calcium storage was reduced as evidenced by decreased mineralized bone mass as well as bone mineral content. More precisely, bone formation was decreased and the level of bone mineralization inhibitors was increased. This decreased fetal skeletal calcium storage may severely compromise calcium balance and survival at birth. In conclusion, these data indicate that high maternal 1,25(OH)2D levels are transferred across the placental barrier and adversely affect the total amount of calcium stored in fetal bones which is accompanied by neonatal death.

Development of micro-CT protocols for in vivo follow-up of mouse bone architecture without major radiation side effects.

In vivo micro-computed tomography (micro-CT) will offer unique information on the time-related changes in bone mass and structure of living mice, provided that radiation-induced side effects are prevented. Lowering the radiation dose, however, inevitably decreases the image quality. In this study we developed and validated a protocol for in vivo micro-CT imaging of mouse bone architecture that retains high quality images but avoids radiation-induced side effects on bone structure and hematological parameters. The left hindlimb of male C57Bl/6 mice was scanned in vivo at 3 consecutive time points, separated each time by a 2-week interval. Two protocols for in vivo micro-CT imaging were evaluated, with pixel sizes of 9 and 18 µm and administered radiation doses of 434 mGy and 166 mGy per scan, respectively. These radiation doses were found not to influence trabecular or cortical bone architecture in pre-pubertal or adult mice. In addition, there was no evidence for hematological side effects as peripheral blood cell counts and the colony-forming capacity of hematopoietic progenitor cells from bone marrow and spleen were not altered. Although the images obtained with these in vivo micro-CT protocols were more blurred than those obtained with high resolution (5 µm) ex vivo CT imaging, longitudinal follow-up of trabecular bone architecture in an orchidectomy model proved to be feasible using the 9 µm pixel size protocol in combination with a suitable bone segmentation technique (i.e. local thresholding). The image quality of the 18 µm pixel size protocol was too degraded for accurate bone segmentation and the use of this protocol is therefore restricted to monitor marked changes in bone structure such as bone metastatic lesions or fracture healing. In conclusion, we developed two micro-CT protocols which are appropriate for detailed as well as global longitudinal studies of mouse bone architecture and lack noticeable radiation-induced side effects.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis.

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

Optimal discrete wavelet design for cardiac signal processing.

The question of designing the best wavelet for a given signal is discussed from the perspective of orthogonal filter banks. Two performance criteria are proposed to measure the quality of a wavelet, based on the principle of maximization of variance. The method is illustrated and evaluated by means of a worked example from biomedicine in the area of cardiac signal processing. The experimental results show the potential of the approach.

Time-related increase of biochemical markers of bone turnover in androgen-deficient male rats.

Bone loss during androgen deficiency has been associated with accelerated bone turnover and imbalance between bone formation and resorption but the relative increase of both phenomena is not well described. Serum osteocalcin as marker of bone formation and urinary excretion of pyridinoline (PYD) and deoxypyridinoline (DPD) as markers of bone resorption were measured in both orchidectomized (ORCH, n = 8) and sham-operated (SHAM, n = 8) aged (12-month-old) male rats from 2 days before until 66 days after surgery. PYD and DPD were significantly higher in the ORCH group compared to the SHAM group starting from 21 days after surgery until the end of the experiment. Serum osteocalcin was only significantly increased in the ORCH group at 30 and 40 days. The maximal increase of serum osteocalcin was also smaller than the increase in PYD and DPD (30% versus 74% and 112%, respectively). The two markers of bone resorption were correlated with osteocalcin (r = 0.63 for PYD and r = 0.71 for DPD). Based on these results, we conclude that (1) bone resorption, as measured by PYD and DPD, increased during androgen deficiency; (2) moreover, the increase of bone resorption, as measured by DPD and PYD, was followed by a more moderate increase in bone formation as measured by serum osteocalcin, supporting the hypothesis that androgen deficiency causes accelerated bone turnover and imbalance between bone resorption and bone formation.

Decreased osteoblast activity in spontaneously diabetic rats. In vivo studies on the pathogenesis.

Diabetes in both humans and rats is accompanied by low bone formation, which is presumably caused by serum-borne factors. To explore its pathogenesis, we carried out experiments in diabetic and nondiabetic BB rats, using plasma osteocalcin concentrations (OC) as a marker for osteoblast activity. In nondiabetic rats, the i.v. infusion of glucose (30%, 4 d) did not change OC; s.c. insulin infusion (4 U/d, 14 d) reduced OC by 27% (p < 0.01). In diabetic rats, OC were decreased from the first day of glycosuria (71 +/- 5% of paired controls), declining exponentially to 24 +/- 3% after 5 wk. Insulin infusion (1, 2, and 3 U/d, 14 d) produced gradual restoration of OC. OC were better correlated with insulin-like growth factor-I (IGF-I) than with insulin levels in these experiments. OC were dramatically increased 4 d after adrenalectomy (ADX) in all diabetic rats (73 +/- 8 vs 22 +/- 4 micrograms/L before ADX; p < 0.001), but not if corticosterone was administered. Ligand blotting of IGF binding proteins showed a marked decrease in two bands (44-49 and 32-35 kDa) 10-14 d after diabetes onset; the density of these bands was increased, but not normalized after ADX. Thus, decreased osteoblast activity is present from the onset of diabetes, is dependent on endogenous corticosterone, and cannot be reproduced by hyperglycemia in nondiabetic rats.