Marie-Claude Monier-Faugere

The Phytoestrogen Genistein Reduces Bone Loss in Short-Term Ovariectomized Rats

Abstract: The incidence of fractures and of osteoporosis differs between Oriental and Western Caucasian women. This may depend, at least in part, on nutritional factors, including dissimilarities in dietary intake of phytoestrogens. To investigate this possibility, 2-month-old female rats were ovariectomized (OVX) or sham-operated (SHAM), fed a casein-based diet, injected daily with subcutaneous genistein (GEN), the most abundant and best characterized phytoestrogen, or vehicle (Veh) and killed 21 days after surgery. As expected, ovariectomy resulted in loss of bone mineral density (BMD) and in uterine atrophy. However, administration of 5 mg GEN per gram body weight (b.w.) ameliorated the ovariectomy-induced loss of BMD (189 ± 2 mg/cm2 in OVX and 192 ± 2 in OVX with 5 mg GEN/g b.w. per day; p<0.05). One microgram GEN per gram body weight did not affect the BMD loss and the effect of the 5 mg and 25 mg GEN per gram body weight were statistically not different. A trend toward reduced uterine atrophy (21% reduction) was noted with the 25 mg GEN dose, but not with the 1 mg and 5 mg doses. A separate experiment with 2 x 2 factorial design was conducted to elucidate the mechanism by which GEN ameliorates ovariectomy-induced bone loss. In this experiment, histomorphometry demonstrated a dramatic reduction in trabecular bone volume after ovariectomy (7.6 ± 0.7% of total bone volume in SHAM-Veh vs 3.3 ± 0.2% in OVX-Veh; p<0.01) and less bone loss in OVX rats injected with 5 mg GEN per gram per day (3.3 ± 0.2% of total bone volume in OVX-Veh vs 5.2 ± 0.4% in OVX-GEN; p<0.01). Administration of GEN was associated with higher bone formation rate per tissue volume and with a trend toward a higher number of osteoblasts per bone perimeter. The parameters of bone resorption were not affected by GEN. The concentration of serum osteocalcin and the urinary excretion of deoxypyridinoline provided corroborating results. Since production of proinflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis, the effect of GEN on lipopolysaccharide-induced in vitro production of Tumor necrosis factor-alpha (TNFa) was tested in monocytic cells from the same four rat groups. Production of TNFa was markedly elevated in OVX-Veh as compared with the SHAM-Veh rats, but this was blocked by GEN in the OVX rats. This study shows that GEN reduces both trabecular and compact bone loss after ovariectomy and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption. Lack of action of GEN on uterine atrophy supports the possibility that this GEN dose affects target tissues via non-estrogenic mechanisms. Modulation of cytokine production may be involved in the effect of GEN on bone.

Predictive value of serum parathyroid hormone levels for bone turnover in patients on chronic maintenance dialysis

With the increasing occurrence of adynamic bone disease, it is essential to determine the level of bone turnover in chronically dialyzed patients before instituting vitamin D therapy. To assess the value of serum parathyroid hormone (PTH) levels for prediction of bone turnover, we determined sensitivity, specificity, and predictive value positive of serum PTH, alone or in combination with other variables, in 79 patients who underwent one or two bone biopsies. Serum PTH levels were determined by a radioimmunometric assay and were obtained at the time of bone biopsies. Patients were classified into (1) low or normal and (2) high bone turnover according to the value of activation frequency of bone. There were 57 biopsy specimens taken from hemodialysis patients and 39 specimens from continuous ambulatory peritoneal dialysis patients (CAPD). All patients with serum PTH levels within or below the normal range had low or normal bone turnover. Values of serum PTH above 450 pg/mL were 100% and 95.5% specific for high bone turnover in hemodialysis and CAPD patients, respectively. Values of serum PTH between 65 and 450 pg/mL had worse predictive value positive in CAPD patients (48.6% to 78.6%) than in hemodialysis patients (67.3% to 87.1%). When other characteristics of the patients were taken into consideration, only age in hemodialysis patients and serum ionized calcium in CAPD patients improved the predictive value of serum PTH. All hemodialysis patients younger than 45 years of age with serum PTH levels above 65 pg/mL (n = 15) had high bone turnover, and CAPD patients with low or normal bone turnover had higher serum ionized calcium. However, overall, bone turnover could not be predicted by serum PTH measurements in 30% of hemodialysis and 51.3% of CAPD patients. The data suggest that for patients with serum PTH levels between 65 and 450 pg/mL, bone biopsies are indicated to precisely assess bone turnover prior to initiation of vitamin D therapy.

Renal Osteodystrophy in the First Decade of the New Millennium: Analysis of 630 Bone Biopsies in Black and White Patients

Renal osteodystrophy occurs early during loss of kidney function. There are 26 million American patients with chronic kidney disease (CKD), and almost all patients with CKD stage 5 have abnormal bone histology. Six hundred and thirty bone biopsies from adult CKD‐5 patients on dialysis were evaluated by histomorphometry and analyzed using the turnover (T), mineralization (M), and volume (V) classification. There were racial differences; whites exhibited predominantly low turnover (62%), whereas blacks showed mostly normal or high turnover (68%). A mineralization defect was observed in only 3% of patients. In whites, cancellous bone volume was low, normal, or high in approximately the same number of patients, whereas in blacks, cancellous bone volume was high in two‐thirds of the patients. More than 80% of blacks and whites with low cancellous bone volume had thin trabeculae owing to low bone formation. Cortical thickness was low in half the whites, whereas it was normal in three‐quarters of blacks. Cortical porosity was high in 50% of whites, whereas three‐quarters of blacks had high porosity. In summary, the TMV system gives relevant information. It should be expanded to include the architecture of cancellous and cortical bone. There are racial differences. Low bone volume and low bone turnover are more frequent than heretofore appreciated, whereas mineralization defects nowadays are observed rarely in adults. These findings call for an adjustment of the current therapeutic paradigm that takes into consideration race and risk of low bone volume and turnover. The latter have been shown to be associated with increased vascular calcifications.

Sclerostin and Dickkopf-1 in Renal Osteodystrophy

BACKGROUND AND OBJECTIVES The serum proteins sclerostin and Dickkopf-1 (Dkk-1) are soluble inhibitors of canonical wnt signaling and were recently identified as components of parathyroid hormone (PTH) signal transduction. This study investigated the associations between sclerostin and Dkk-1 with histomorphometric parameters of bone turnover, mineralization, and volume in stage 5 chronic kidney disease patients on dialysis (CKD-5D). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In a cross-sectional study, 60 CKD-5D patients underwent bone biopsies followed by histomorphometry. Levels of sclerostin, Dkk-1, and intact PTH (iPTH) were determined in blood. RESULTS Serum levels of sclerostin and iPTH correlated negatively. In unadjusted analyses, sclerostin correlated negatively with histomorphometric parameters of turnover, osteoblastic number, and function. In adjusted analyses, sclerostin remained a strong predictor of parameters of bone turnover and osteoblast number. An observed correlation between sclerostin and cancellous bone volume was lost in regression analyses. Sclerostin was superior to iPTH for the positive prediction of high bone turnover and number of osteoblasts. In contrast, iPTH was superior to sclerostin for the negative prediction for high bone turnover and had similar predictive values than sclerostin for the number of osteoblasts. Serum levels of Dkk-1 did not correlate with iPTH or with any histomorphometric parameter. CONCLUSIONS Our data describe a promising role for serum measurements of sclerostin in addition to iPTH in the diagnosis of high bone turnover in CKD-5D patients, whereas measurements of Dkk-1 do not seem to be useful for this purpose.

Effects of Sevelamer Hydrochloride and Calcium Carbonate on Renal Osteodystrophy in Hemodialysis Patients

Disturbances in mineral metabolism play a central role in the development of renal bone disease. In a 54-wk, randomized, open-label study, 119 hemodialysis patients were enrolled to compare the effects of sevelamer hydrochloride and calcium carbonate on bone. Biopsy-proven adynamic bone disease was the most frequent bone abnormality at baseline (59%). Serum phosphorus, calcium, and intact parathyroid hormone were well controlled in both groups, although calcium was consistently lower and intact parathyroid hormone higher among patients who were randomly assigned to sevelamer. Compared with baseline values, there were no changes in mineralization lag time or measures of bone turnover (e.g., activation frequency) after 1 yr in either group. Osteoid thickness significantly increased in both groups, but there was no significant difference between them. Bone formation rate per bone surface, however, significantly increased from baseline only in the sevelamer group (P = 0.019). In addition, of those with abnormal microarchitecture at baseline (i.e., trabecular separation), seven of 10 in the sevelamer group normalized after 1 yr compared with zero of three in the calcium group. In summary, sevelamer resulted in no statistically significant changes in bone turnover or mineralization compared with calcium carbonate, but bone formation increased and trabecular architecture improved with sevelamer. Further studies are required to assess whether these changes affect clinical outcomes, such as rates of fracture.

Early chronic kidney disease-mineral bone disorder stimulates vascular calcification

The chronic kidney disease-mineral and bone disorder (CKD-MBD) syndrome is an extremely important complication of kidney diseases. Here we tested whether CKD-MBD causes vascular calcification in early kidney failure by developing a mouse model of early CKD in a background of atherosclerosis stimulated arterial calcification. CKD equivalent in glomerular filtration reduction to human CKD stage 2 stimulated early vascular calcification and inhibited the tissue expression of α-klotho (klotho) in the aorta. In addition, osteoblast transition in the aorta was stimulated by early CKD as shown by the expression of the critical transcription factor, RUNX2. The ligand associated with the klotho-fibroblast growth factor receptor complex, FGF23, was found to be expressed in the vascular media of sham operated mice. Its expression was decreased in early CKD. Increased circulating levels of the osteocyte secreted proteins, FGF23, and sclerostin may have been related to increased circulating klotho levels. Finally, we observed low turnover bone disease with a reduction in bone formation rates more than bone resorption. Thus, the CKD-MBD, characterized by cardiovascular risk factors, vascular calcification, increased circulating klotho, FGF23 and sclerostin levels, and low turnover renal osteodystrophy, was established in early CKD. Early CKD caused a reduction of vascular klotho, stimulated vascular osteoblastic transition, increased osteocytic secreted proteins, and inhibited skeletal modeling producing the CKD-MBD.

Intermittent and Continuous Administration of the Bisphosphonate Ibandronate in Ovariohysterectomized Beagle Dogs: Effects on Bone Morphometry and Mineral Properties

Bisphosphonates have emerged as a valuable treatment for postmenopausal osteoporosis. Bisphosphonate treatment is usually accompanied by a 3–6% gain in bone mineral density (BMD) during the first year of treatment and by a decrease in bone turnover. Despite low bone turnover, BMD continues to increase slowly beyond the first year of treatment. There is evidence that bisphosphonates not only increase bone volume but also enhance secondary mineralization. The present study was conducted to address this issue and to compare the effects of continuous and intermittent bisphosphonate therapy on static and dynamic parameters of bone structure, formation, and resorption and on mineral properties of bone. Sixty dogs were ovariohysterectomized (OHX) and 10 animals were sham‐operated (Sham). Four months after surgery, OHX dogs were divided in six groups (n = 10 each). They received for 1 year ibandronate daily (5 out of 7 days) at a dose of 0, 0.8, 1.2, 4.1, and 14 μg/kg/day or intermittently (65 μg/kg/day, 2 weeks on, 11 weeks off). Sham dogs received vehicle daily. At month 4, there was a significant decrease in bone volume in OHX animals (p < 0.05). Doses of ibandronate ≥ 4.1 μg/kg/day stopped or completely reversed bone loss. Bone turnover (activation frequency) was significantly depressed in OHX dogs given ibandronate at the dose of 14 μg/kg/day. This was accompanied by significantly higher crystal size, a higher mineral‐to‐matrix ratio, and a more uniformly mineralized bone matrix than in control dogs. This finding lends support to the hypothesis that an increase in secondary mineralization plays a role in gain in BMD associated with bisphosphonate treatment. Moreover, intermittent and continuous therapies had a similar effect on bone volume. However, intermittent therapy was more sparing on bone turnover and bone mineral properties. Intermittent therapy could therefore represent an attractive alternative approach to continuous therapy.

Effect of bisphosphonates on vascular calcification and bone metabolism in experimental renal failure

Although it is known that bisphosphonates prevent medial vascular calcification in vivo, their mechanism of action remains unknown and, in particular, whether they act directly on the blood vessels or indirectly through inhibition of bone resorption. To determine this, we studied the effects of two bisphosphonates on calcification of rat aortas in vitro and on in vivo aortic calcification and bone metabolism in rats with renal failure. We produced vascular calcification in rats with adenine-induced renal failure fed a high-phosphate diet. Daily treatment with either etidronate or pamidronate prevented aortic calcification, with the latter being 100-fold more potent. Both aortic calcification and bone formation were reduced in parallel; however, bone resorption was not significantly affected. In all uremic rats, aortic calcium content correlated with bone formation but not with bone resorption. Bisphosphonates also inhibited calcification of rat aortas in culture and arrested further calcification of precalcified vessels but did not reverse their calcification. Expression of osteogenic factors or calcification inhibitors was not altered by etidronate in vitro. Hence, these studies show that bisphosphonates can directly inhibit uremic vascular calcification independent of bone resorption. The correlation between inhibition of aortic calcification and bone mineralization is consistent with a common mechanism such as the prevention of hydroxyapatite formation and suggests that bisphosphonates may not be able to prevent vascular calcification without inhibiting bone formation in uremic rats.

CKD-Induced Wingless/Integration1 Inhibitors and Phosphorus Cause the CKD–Mineral and Bone Disorder

In chronic kidney disease, vascular calcification, renal osteodystrophy, and phosphate contribute substantially to cardiovascular risk and are components of CKD-mineral and bone disorder (CKD-MBD). The cause of this syndrome is unknown. Additionally, no therapy addresses cardiovascular risk in CKD. In its inception, CKD-MBD is characterized by osteodystrophy, vascular calcification, and stimulation of osteocyte secretion. We tested the hypothesis that increased production of circulating factors by diseased kidneys causes the CKD-MBD in diabetic mice subjected to renal injury to induce stage 2 CKD (CKD-2 mice). Compared with non-CKD diabetic controls, CKD-2 mice showed increased renal production of Wnt inhibitor family members and higher levels of circulating Dickkopf-1 (Dkk1), sclerostin, and secreted klotho. Neutralization of Dkk1 in CKD-2 mice by administration of a monoclonal antibody after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification. Mechanistically, neutralization of Dkk1 suppressed aortic expression of the osteoblastic transcription factor Runx2, increased expression of vascular smooth muscle protein 22-α, and restored aortic expression of klotho. Neutralization of Dkk1 did not affect the elevated plasma levels of osteocytic fibroblast growth factor 23 but decreased the elevated levels of sclerostin. Phosphate binder therapy restored plasma fibroblast growth factor 23 levels but had no effect on vascular calcification or osteodystrophy. The combination of the Dkk1 antibody and phosphate binder therapy completely treated the CKD-MBD. These results show that circulating Wnt inhibitors are involved in the pathogenesis of CKD-MBD and that the combination of Dkk1 neutralization and phosphate binding may have therapeutic potential for this disorder.

Low Bone Volume—A Risk Factor for Coronary Calcifications in Hemodialysis Patients

BACKGROUND AND OBJECTIVES There is increasing evidence that altered bone metabolism is associated with cardiovascular calcifications in patients with stage 5 chronic kidney disease on hemodialysis (HD). This study was conducted to evaluate the association between bone volume, turnover, and coronary calcifications in HD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In a cross-sectional study, bone biopsies and multislice computed tomography were performed in 38 HD patients. Bone volume/total volume, activation frequency, and bone formation rate/bone surface were determined by histomorphometry and coronary calcifications were quantified by Agatston scores. RESULTS Prevalence of low bone turnover was 50% and of low bone volume was 16%. Among the studied traditional cardiovascular risk factors, only age was found to be associated with coronary calcifications. Lower bone volume was a significant risk factor for coronary calcifications during early years of HD, whereas this effect was not observed in patients with dialysis duration >6 yr. Histomorphometric parameters of bone turnover were not associated with coronary calcifications. CONCLUSIONS Low bone volume is associated with increased coronary calcifications in patients on HD.