Medial artery calcification of uremic patients: a histological, histochemical and ultrastructural study.

Recent findings suggest that vascular calcification (VC) is an active process similar to bone mineralization, the vascular smooth muscle cells (VSMCs) undergoing phenotypic differentiation into osteoblastic cells and synthesizing calcification-regulating proteins found in bone. This study has investigated the VC process of uremic patients, with a morphologic approach. Epigastric artery samples from 49 uremic, non-diabetic patients were taken during kidney transplantation. Sections from paraffin-embedded samples were stained with hematoxylin/eosin and von Kossa. CD68 was immunohistochemically detected, and sections from frozen samples were stained with Oil Red O. Deeply calcified samples were stained with Picrosirius Red, PAS, and Alcian blue. Specimens from one patient with moderate and one with severe VC were examined under the electron microscope. None of the samples had atherosclerosis. Calcifications were found in the media of 38 patients. In 23, dot-like calcifications were irregularly scattered near the adventitia (light VC); in 11, granular calcifications formed concentric rings near the adventitia (moderate-advanced VC); in 4, zones of consolidated calcifications were found (severe VC). These zones were poor in collagen, glycoproteins and proteoglycans. In cases with moderate or severe VC, VSCMs showed necrotic changes. Matrix vesicles could be recognized in the extracellular spaces. In cases with severe VC, uncalcified or partially calcified membranous bodies were found, together with Liesegang rings. Patches of fibrin were also found. These findings point to a mainly degenerative mechanism of VC, which proceeds from the outer portion of the media. An active mechanism, however, cannot be excluded. A unifying hypothesis is suggested.

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone.

Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm(2) show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.

Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint.

During embryogenesis the bone tissue of craniomandibular joint (CMJ) is formed through two pathways: intramembranous ossification and endochondral ossification. The development process is under the control of regulatory factors. The osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kappaB ligand are key regulators of osteoclastogenesis. The aim of this study is the localization of OPG and RANKL mRNA and protein in the foetal CMJ by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: OPG and RANKL mRNA and protein were co-localized in the same cell types; OPG and RANKL were specially immunolocated in osteogenic cells; immunolabeling was often seen in the nucleus and cytoplasm of otherwise negative hypertrophic chondrocytes; IHC and ISH labeling decreased from proliferative to hypertrophic chondrocytes; early osteocytes showed dual protein expression and some of the mature osteocytes were ISH-negative; periosteal osteoclasts and chondroclasts were mostly stained by IHC and variably labeled by ISH; the new bone matrix and trabecular borders showed intense immunolabeling. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism in the CMJ development and their extracellular presence in the new bone matrix and trabecular borders suggests a local regulatory role.

Calcified tissue histochemistry: from microstructures to nanoparticles.

It has long been recognized that histochemistry and cytochemistry offer the only ways of gathering information about the biochemical composition of tissues and cells without disrupting their microscopic architecture. A variety of methods have been put forward for studying nuclei acids, proteins, carbohydrates, lipids, enzymes and other components of intact tissues and cells. By now, many of these have only a historical interest. Some do, however, survive in microscopic and ultramicroscopic applications, and have become incorporated in the most refined and precise techniques that are currently available. Histochemical reactions range from the classic procedures carried out on histological sections to yield final stained products recognizable under the light microscope, to those which are applied on ultrathin sections, using heavy metals or other electron-dense compounds to reveal specific components under the electron microscope; others range from procedures based on the antigen-antibody reaction that are capable of revealing the presence of specific biological molecules, to the biophysical techniques which permit the qualitative and quantitative analysis of elements; lastly, there are the recently proposed ultra-high resolution methods that allow nanoparticles to be recognized. This brief review, which is based on personal experience and on the data in the literature, will discuss the most important methods now being used.

Localization of the glucocorticoid receptor mRNA in cartilage and bone cells of the rat. An in situ hybridization study.

The in vivo localization of glucocorticoid receptor (GR) mRNA expression was studied in the cartilage and bone cells of the femur of young adult rats to compare its distribution with that of the GR protein, which had previously been shown histochemically in the same areas. To achieve this, we used a synthetic oligodeoxynucleotide as a probe, in line with the published human GR (hGR) cDNA sequence. The probe was coupled to fluorescein (FL), applying a rapid Fast-Tag TM FL nucleic acid labeling method. Negative controls were achieved by using sense sequences of the hGR oligoprobe, similarly coupled by using the Fast-Tag TM FL labeling kit. Dewaxed sections were treated for in situ hybridization (ISH) histochemistry with the antisense and sense oligoprobes. The ISH reaction product was more intense in the cytoplasm of proliferative and maturative chondrocytes of the growth plate cartilage than in that shown in the hypertrophic ones. In the metaphyseal secondary ossification zone, osteoblasts (OBs) and osteocytes (OCs) were variably labeled, whereas osteoclasts (OCLs) were always intensely stained. The labeling was also visible in some bone marrow cells, in articular chondrocytes, in the cells of tendon-bone junctions, and in the perichondrium and periosteal cells. Our results confirm a cellular co-location of GR protein and mRNA. In agreement with GR immunolocalization, the variability of labeling appeared to be related to the cell cycle, the stage of differentiation and cell-type differences.

Alkaline phosphatase and tartrate-resistant acid phosphatase in osteoblasts of normal and pathologic bone.

A review of histochemical and immunohistochemical studies on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) in osteoblasts leads to the following conclusions: 1) AP is a typical (non-specific) marker of osteoblasts, and TRAP is a typical (non-specific) marker of osteoclasts; 2) both enzymes may be used to identify the precursors of these cells and are found in young osteocytes; 3) both are released into the extracellular space; 4) both are expressed by metaphyseal osteoblast-like cells whose ultrastructural characteristics are similar to those of post-osteoclastic cells of the basic multicellular unit (BMU) and are also exhibited by osteoblasts and macrophages; 5) the increased numbers of these cells in hypocalcemic animals suggests that the local calcium ion concentration may trigger the transition of the reversal into the formation phase of the BMU.

Renal cortical calcification in syngeneic intact rats and those receiving an infrarenal thoracic aortic graft: possible etiological roles of endothelin, nitrate and minerals, and different preventive effects of long-term oral treatment with magnesium, citrate and alkali-containing preparations.

Renal cortical nephrocalcinosis (C-NC) is a rare disorder of uncertain etiology. Using highly inbred (syngeneic) male Lewis rats, we describe the spontaneous occurrence of histologically detectable C-NC in sham operated control rats (Sham; n=12), its aggravation following grafting of the ascending thoracic aorta from a donor rat to the infrarenal aorta of a recipient (ATx; n=12), and differences in C-NC inhibition after 12 weeks of oral administration of magnesium (Mg), citrate and alkali. C-NC is characterized by Kossa-positive areas located in cells of the proximal tubule close to blood vessels and also, to a lesser extent, within glomeruli. After ATx there was vascular overproduction of endothelin (ET-1) but decreased production of nitrate; in renal cortical tissue there was an excess of calcium over Mg and phosphorus and oxalate over citrate. In plasma there was an increase in calcium and creatinine within the normal range. Calcification of tubular cells was eliminated by a preparation containing potassium, sodium and bases (from citrate degradation and bicarbonate) in addition to Mg. Less effective than the latter was Mg-potassium citrate and least effective, Mg citrate. The former treatment also normalized calcemia and urinary nitrate, but only incompletely suppressed ET-1 and had no significant effect on glomerular calcification or tissue and urinary oxalate. Urinary ET-1 excess appeared directly related to the cortical tissue calcium/Mg ratio, and urinary excretion of Mg, citrate and total protein appeared to be inversely related to the severity of C-NC. It was concluded that (1) the highly inbred rat is prone to precipitation of calcium phosphate in the renal cortex; (2) this type of C-NC occurs in close proximity to and within renal vascular tissue and is associated with an imbalance of vasoconstrictors and vasodilators of endothelial origin; (3) effective inhibition of C-NC can be achieved by an alkalinizing combination of Mg, potassium, sodium and citrate, underscoring its utility in the prophylaxis of pathological calcium phosphate deposition. The significance of these findings for the etiology and treatment of clinical disorders with renal and vascular calcification is uncertain and requires further investigation.

The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study.

In a previous report we showed that young rats fed a calcium-free diet for 28 days developed severe hypocalcaemia and showed a significant increase in serum alkaline phosphatase activity. The main histological and cytochemical changes exhibited by these animals in bone of the metaphyseal primary spongiosa were: (1) hyperplasia of osteoblasts, (2) an increase in the frequency of tartrate-resistant acid phosphatase (TRAP)-positive osteoblasts apposed to osteoid, and (3) an excessive amount of osteoid tissue. In addition to typical osteoblasts, there was a subpopulation of osteoblast-like cells with coated pits, lysosome-like bodies and large cytoplasmic processes. In the present study, we investigated how the above parameters change when calcium-depleted rats are placed on a normal diet for 7 days. Such a regimen normalized calcium concentration and alkaline phosphatase activity in the serum. The osteoid thickness returned to normal and, in some areas, was fully calcified. Most osteoblasts no longer showed TRAP activity and their ultrastructure was similar to that found in controls. Despite an intense alkaline phosphatase activity, some of them still exhibited a number of macrophagic characteristics. They were TRAP-positive, and showed electron-dense bodies in the cytoplasm facing bone, an abundance of coated pits, calcified spicules impinging on the cell membrane and large processes extending into the mineralized matrix. We concluded that calcium deficiency causes hyperplasia of osteoblasts in primary spongiosa and an increase in expression of TRAP. It also induces changes in their phenotype characterized by the acquisition of macrophagic cellular features. While TRAP activity is normalized by calcium repletion, macrophagic characteristics persist. These results suggest that the osteoblast can modulate its phenotype according to its physiological status.

Histomorphometric assessment of bone turnover in uraemic patients: comparison between activation frequency and bone formation rate.

AIMS: The histomorphometric assessment of bone formation rate (BFR/BS) in bone biopsies from uraemic patients is of crucial importance in differentiating low from high turnover types of renal osteodystrophy. However, since BFR/BS relies on osteoblasts, activation frequency (Ac.f), encompassing all remodelling phases, has recently been preferred to BFR/BS. This study was carried out to consider whether estimation of Ac.f is superior, in practical terms, to that of BFR/BS in distinguishing between different rates of bone turnover in uraemic patients. METHODS AND RESULTS: Bone biopsies from 27 patients in predialysis (20 men and seven women; mean age 53 +/- 12 years) and 37 in haemodialysis (22 men and 15 women; mean age 53 +/- 12 years) were examined. The types of renal osteodystrophy were classified on the basis of morphology. Bone formation rate and Ac.f were evaluated according to standardized procedures. The Ac.f was calculated both as a ratio between BFR/BS and wall thickness (W.Th) and as a reciprocal of erosion, formation and quiescent periods (EP, FP and QP). Patients were affected by renal osteodystrophy with predominant hyperparathyroidism (two predialysis and 16 dialysis), predominant osteomalacia (three predialysis and seven dialysis) or that of advanced (nine predialysis and five dialysis) or mild (seven predialysis and four dialysis) mixed type or adynamic type (six predialysis and five dialysis). Activation frequency, which with either formula requires the measurement of W.Th, i.e. the thickness of bone structural units (BSUs), was not calculated in three dialysis patients with severe hyperparathyroidism and in one predialysis and four dialysis patients with severe osteomalacia, because only incomplete BSUs were found. In dialysis, EP was higher in the adynamic than in the other types of osteodystrophy. During both predialysis and dialysis, FP was higher in osteomalacia than in the other forms of osteodystrophy, and in adynamic osteopathy than in hyperparathyroidism or in advanced and mild mixed osteodystrophy. During predialysis and dialysis, QP was higher in the adynamic than in the other forms of osteodystrophy. Correlations were found between BFR/BS and Ac.f, during predialysis (r=0.97) and dialysis (r=0.95). CONCLUSIONS: The superiority of Ac.f in assessing bone turnover, in comparison to BFR/BS, is conceptual rather than practical. The highest values for FP in osteomalacia and for QP in adynamic bone allow a clearer characterization of these low turnover conditions.

Bcl-2 and Bax expression in cartilage and bone cells after high-dose corticosterone treatment in rats.

The expression of Bcl-2 and Bax has been evaluated by immunohistochemistry in normal rats, and in rats after treatment with high-dose corticosterone (CORT). Proliferative (PC) and maturative/hypertrophic (MaHC) chondrocytes of the growth plate have been examined, as well as osteoblasts (Obs), osteocytes (Ots) and osteoclasts (Ocs) of the metaphyseal secondary spongiosa. For each cell type, the Bcl-2 and Bax immunopositive cells were expressed as a percentage of the total number of cells. Bcl-2 and Bax expression was considered to be enhanced when the percentage of positive cells rose. Bcl-2 and Bax were expressed in all cell types, and two main kinds of labeling distribution, both suggestive of association with intracellular organelles, were observed in the cytoplasm: scarce and spotty labeling (type 1) or abundant, granular and diffuse labeling (type 2). In some cases, nuclear membranes could also be seen to be positive. Positive PCs and Obs generally showed a labeling of type 1, MaHCs and Ocs of type 2, while Ots varied with labeling of type 1 or type 2. CORT administration induced a fall in the percentage of Bcl-2 immunopositive cells, and a rise in that of Bax immunopositive cells, in PCs and Ots. The same trend was observed in MaHCs, although the Bcl-2 decrease was not significant. The percentage of Bcl-2 and Bax immunopositive Obs rose, and their labeling distribution shifted from type 1- to type 2-labeled cells. Ocs showed the highest immunopositivity for both Bcl-2 and Bax, which did not change after CORT administration. These data suggest that CORT treatment, by lowering Bcl-2, and raising Bax expression, may promote the apoptotic process in PCs, MaHCs and Ots. Obs, however, do not undergo the same variations. This finding, together with the results of a previous study showing that CORT administration raises the frequency of apoptotic Obs, does not support a direct relationship between apoptosis and Bax overexpression, at least in Obs. The CORT effect might be related to cell types and their state of differentiation, so that Bcl-2 and Bax might regulate not only the machinery of cell death, but also cell proliferation and differentiation.

Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff- and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.

A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats.

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.

A morphological study of bone and articular cartilage in ochronosis.

An ochronotic femoral head has been studied morphologically under the light and the electron microscope. Its articular cartilage showed the alterations already reported in the literature, mainly consisting of erosions of the surface, pigment accumulation in chondrocytes and intercellular matrix, chondrocyte degeneration, the formation of pigmented, calcified and uncalcified microshards, and the presence of granulation tissue with macrophagic cells. The changes in bone were less severe than those in cartilage. Pigment was present in the calcified matrix. This did not seem to disturb the organization of the bone tissue, although it was diffusely osteoporotic, perhaps because of limb disuse. The preservation of calcified matrix might depend on the fact that its collagen fibrils are encrusted by mineral substance, which avoids the dangerous effects that the deposition of ochronotic pigment induces in the fibrils of soft connective tissues. On the other hand, the newly formed osteoid matrix remains uncalcified for too short a time to be modified by the pigment. Diffuse or granular pigmentation was found in a few osteocytes, while several of them were condensed or reduced to cellular fragments. Bone resorption often occurred near these osteocytes. However, this did not seem to alter the degree of bone remodelling, possibly because of the relatively low numbers of degenerated or dead osteocytes. Pigment was also contained in the cytoplasmic vacuoles of otherwise active osteoclasts, whereas it was not found in osteoblasts. On the whole, ochronosis in bone seems to induce the same changes as in other connective tissues. However, their severity appears to be limited by calcification, which prevents modifications in collagen fibrils, and by bone remodelling, which to some extent eliminates the oldest, pigment-richest parts of the tissue.

Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone.

A connection has been suggested between glucocorticoid-induced osteopenia and an increase in the apoptosis of bone cells, and between the dimerization of the glucocorticoid receptor (GR) and the development of apoptosis. On this basis, a study has been carried out on the relationships between the occurrence of apoptotic cells and their detectable GR content, and between apoptosis frequency and changes in histomorphometric variables, in the growth plate and secondary spongiosa of rat long bones after the high-dose (10 mg/day) administration of corticosterone (CORT) and after recovery. The main results of the CORT treatment were: a significant increase in apoptotic osteoblasts, and a concomitant decrease in the histomorphometric variables of bone formation, with a reversal of both values during recovery; a nonsignificant increase in the apoptosis of osteoclasts, without changes in the histomorphometric variables of bone resorption; a significant increase in apoptotic terminal hypertrophic chondrocytes; the presence of GR in all types of skeletal cells in control rats, with different (cytoplasmic and/or nuclear) immunohistochemical detection in the same type of cell; a decrease in GR detection in proliferative chondrocytes and osteocytes in CORT and recovery groups, and in the maturative/hypertrophic chondrocytes of the recovery group; a fall in growth cartilage width, possibly due to the reduced proliferation of proliferative chondrocytes and increased apoptosis in terminal hypertrophic chondrocytes. In conclusion, pharmacological doses of CORT reduce bone formation by increasing osteoblast apoptosis; they reduce growth cartilage width, probably by inhibiting chondrocyte proliferation and increasing the apoptosis of terminal hypertrophic chondrocytes, and they reduce osteocyte GR. Although these effects appear to be mediated by the presence of GR in all skeletal cells, no precise correlation between GR immunohistochemical detection and apoptosis induction has been found.

Nephrocalcinosis and hyperlipidemia in rats fed a cholesterol- and fat-rich diet: association with hyperoxaluria, altered kidney and bone minerals, and renal tissue phospholipid-calcium interaction.

To determine whether an "atherogenic" diet (excess of cholesterol and neutral fat) induces pathological calcification in various organs, including the kidney, and abnormal oxalate metabolism, 24 male Sprague-Dawley rats were fed either normal lab chow (controls, n = 12) or the cholesterol- and fat-rich experimental diet (CH-F, n = 12) for 111 +/- 3 days. CH-F rats developed dyslipidemia [high blood levels of triglycerides, total, low-density lipoprotein (LDL)-, very low-density lipoprotein (VLDL)-, high-density lipoprotein (HDL)-bound cholesterol, total phospholipids], elevated serum total alkaline phosphatase and lactate dehydrogenase (LDH) levels, in the absence of changes in overall renal function, extracellular mineral homeostasis [serum protein-corrected total calcium, magnesium, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D)], plasma glycolate and oxalate levels. There was a redistribution of bone calcium and enhanced exchange of this within the extraosseous space, which was accompanied by significant bone calcium loss, but normal bone histomorphometry. Liver oxalate levels, if expressed per unit of defatted (DF) dry liver, were three times higher than in the controls. Urinary glycolate, oxalate, calcium and total protein excretion levels were elevated, the latter showing an excess of proteins > 100 kD and a deficit of proteins > 30-50 kD. Urinary calcium oxalate supersaturation was increased, and calcium phosphate supersaturation was unchanged. There were dramatically increased (by number, circumference, and area) renal calcium phosphate calcifications in the cortico-medullary region, but calcium oxalate deposits were not detectable. Electron microscopy (EM) and elemental analysis revealed intratubular calcium phosphate, apparently needle-like hydroxyapatite. Immunohistochemistry of renal tissue calcifications revealed co-localization of phospholipids and calcium phosphate. It is concluded that rats fed the CH-F diet exhibited: (1) a spectrum of metabolic abnormalities, the more prominent being dyslipidemia, hyperoxaluria, hypercalciuria, dysproteinuria, loss of bone calcium, and calcium phosphate nephrocalcinosis (NC); and (2) an interaction between calcium phosphate and phospholipids at the kidney level. The biological significance of these findings for the etiology of idiopathic calcium urolithiasis in humans is uncertain, but the presented animal model may be helpful when designing clinical studies.

Zinc mapping in bone tissues by histochemistry and synchrotron radiation-induced X-ray emission: correlation with the distribution of alkaline phosphatase.

Zinc distribution in osteons was mapped by synchrotron radiation-induced X-ray emission analysis in both human and porcine adult bone, as well as in porcine bone by histochemistry using Timm's method. Both procedures showed that zinc is not uniformly distributed, being in its highest concentration on haversian bone surfaces. When Timm's method was applied in conjunction with a procedure leading to partial zinc extraction, three zinc pools were specifically detected: a loose one, found in the mineralizable osteoid; a mineral one, bound to the bone mineral; and a tenacious one, firmly bound to an organic component located in the osteoid and mineralizing organic matrix. The alkaline phosphatase distribution was also mapped in porcine adult bone by histochemistry and immunohistochemistry and it was found codistributed with tenacious zinc mainly at the calcification front. The data suggest that alkaline phosphatase is buried as a bone matrix protein during initial mineralization.

Cytochemical demonstration of the glucocorticoid receptor in skeletal cells of the rat.

The in vivo localization of the glucocorticoid receptor (GR) was studied in cartilage and bone cells of femurs of young adult rats. Deparaffinized sections were treated with a polyclonal antibody raised against the amino-terminus of human GR; the immunoreaction was detected with the streptavidin-biotin amplification method. Histomorphometric, computer-assisted analysis of GR-positive cells was performed by counting the percentage of GR-immunostained cells in the proliferative and maturative/hypertrophic zone of the epiphyseal growth plate cartilage, and of the percentage of positive osteoblasts (OBs), osteoclasts (OCLS) and osteocytes (OCs) in the metaphyseal secondary ossification zone. Numbers of OBs and OCLs per mm of metaphyseal endosteal perimeter, and numbers of OCs per mm2 of trabecular area were also counted. Immunopositive cells were found both in cartilage and bone, with variable degree of nuclear and/or cytoplasmic immunostaining; immunonegative cells were present among the positive ones. Histomorphometry showed that about 54% of chondrocytes in the proliferative zone, and 55% of chondrocytes in the maturative/hypertrophic zone of the growth plate were labeled; in metaphyseal bone, 68% of OBs, 65% of OCs, and 98% of OCLs were GR-positive. The density of positive cells was 12.06 OBs/mm, 3.32 OCLs/mm, and 520.40 OCs/mm2. These results, for the first time obtained in vivo, show that GR is present in cartilage and bone cells, and that the degree of GR-immunostaining is variable in the same type of cell. This may be dependent on the cell cycle and stage of differentiation, and may reflect a variable cellular sensitivity to the stimulation of the glucocorticoid hormone.

Bone markers in the diagnosis of low turnover osteodystrophy in haemodialysis patients.

BACKGROUND: Renal osteodystrophy includes a number of low and high turnover bone histologic patterns which require a bone biopsy for their full identification. The role of intact PTH and several classical and more recent bone markers in the non-invasive diagnosis of renal bone disease in patients with CRF in HD requires further definition since available published data are limited. METHODS: In addition to intact PTH, alkaline phosphatase (AP) and osteocalcin (BGP), bone alkaline phosphatase isoenzyme (BALP), tartrate resistant acid phosphatase (TRAP), C-terminal cross-linked peptide of collagen type 1 (ICTP) and deoxypyridinoline (DPD) were measured in the serum of 41 patients on haemodialysis, subjected at the same time to transiliac bone biopsy for histomorphometric, histodynamic and aluminium histochemical examination. Histodynamic evaluation following double tetracycline label, was carried out in 37 patients. The patients had no evidence of active cytolytic and cholestatic liver disease and a history of very limited aluminium exposure. RESULTS: The patients had differing degrees of hyper-parathyroidism, with intact PTH ranging from normal to very elevated levels. Serum values of the markers BGP, ICTP and DPD, normally excreted through the kidneys, were on average very high. The correlation coefficients of the humoral parameters vs dynamic variables, such as BFR/BS, were high. The highest values were: intact PTH 0.798, AP 0.900, BALP 0.891, ICTP 0.807. The patients, grouped in low turnover osteodystrophy (LTO; 9), mixed osteodystrophy (MO; 9) and prevalent hyperparathyroidism (HP; 23), showed significant difference in the levels of most humoral and static and dynamic parameters (ANOVA). Bone aluminium histochemistry was negative in all cases. Discrimination of LTO patients from the other groups by humoral parameters, at the highest value of accuracy, showed 100% sensitivity and 93.7% specificity with a cut-off of 12.9 ng/ml for BALP; 88.9% sensitivity and 93.7% specificity with a cut-off of 21.5 ng/ml for DPD, and 88.9% sensitivity and 90.6% specificity with a cut-off of 79.7 pg/ml for intact PTH. The other markers had lower values. A standardized z-score approach for evaluation of all humoral parameters was also carried out. Using all variables, a correct classification of MO/HP and of LTO was possible in 93.8 and 88.9% of the cases, respectively. Predictive power was 96.8 and 80%, respectively for MO/HP and LTO. When the only variables used were intact PTH and BALP, a correct classification of MO/HP and LTO was possible in 90.6% and 88.9%, respectively. Predictive value of MO/HP was 96.7% and for LTO 72.7%. Predictive values using PTH and AP were 96.3% and 57.2%, respectively. CONCLUSION: Intact PTH and several relatively new bone markers are of certain value in the non-invasive diagnosis of renal osteodystrophy. However some of the humoral markers carry the same quality of information and the use of intact PTH and BALP may be adequate in the discrimination of bone histologic patterns. In cases exempt from liver disease, PTH and AP may be used as a less costly alternative. Bone biopsy could be chiefly limited to cases with borderline humoral values and to all those with a suspected aluminium overload.

A histomorphometric long-term longitudinal study of trabecular bone loss in glucocorticoid-treated patients: prednisone versus deflazacort.

Administration of a corticosteroid with minor osteopenic effects is considered an effective prevention of glucocorticoid osteoporosis. Deflazacort, an oxazolinic derivative of prednisolone, is reported to be less harmful to cancellous bone mass than other equally effective corticosteroids. However, comparative long-term studies, particularly on trabecular bone, are needed before a smaller detrimental effect on bone of deflazacort can be unequivocally confirmed. We conducted such a prospective long-term study using histomorphometric analysis of iliac bone. For the study, 18 pairs of nonimmobilized patients, matched for age, sex, menopausal state, corticosteroid dose, and type and severity of the disease, were randomly submitted to treatment with therapeutically equivalent doses of prednisone or deflazacort. Bone biopsies from iliac crest were taken before and at various times during treatment. In order to represent the time-related trabecular bone loss and find out possible differences between patients on prednisone or deflazacort, a previously described model of bone loss kinetics was applied. No significant differences in biochemical indices of bone turnover or in histomorphometric variables between prednisone- and deflazacort-treated patients were recorded before treatment. The mean duration of treatment at the final biopsy was similar for prednisone and deflazacort (15.8 and 15.2 months, respectively). Patients showed evident clinical improvement with both treatments. Osteoid and resorption surfaces showed no significant differences throughout the observation period in any of the 18 pairs. On the contrary, both steroids induced a significant decrease in trabecular bone, although the bone loss rate induced by prednisone was significantly higher than that induced by deflazacort (P < 0.05). The kinetics of bone loss and the different osteopenic effects of the two drugs are described by the negative exponential function fitted to data from patients never previously given glucocorticoids; the model yields highly significant difference (P approximately equal to 0.01) between the two drugs and allows estimation of the difference even 3 years after the beginning of treatment (-3.0%/year versus -1.1%/year for prednisone and deflazacort, respectively). This prospective long-term study confirms that an exponential model accurately describes the trabecular bone loss induced by long-term corticosteroid treatment and demonstrates that deflazacort, at therapeutically effective doses, induces less trabecular bone loss than prednisone.