Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: Calvarial bone

Objectives: Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods: Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-κβ ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results: The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P≤0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions: These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. © 2009 John Wiley & Sons A/S.

Cell Therapy with Bone Marrow Mononuclear Cells in Elastase-Induced Pulmonary Emphysema

Emphysema is characterized by destruction of alveolar walls with loss of gas exchange surface and consequent progressive dyspnea. This study aimed to evaluate the efficiency of cell therapy with bone marrow mononuclear cells (BMMC) in an animal model of elastase-induced pulmonary emphysema. Emphysema was induced in C57Bl/J6 female mice by intranasal instillation of elastase. After 21 days, the mice received bone marrow mononuclear cells from EGFP male mice with C57Bl/J6 background. The groups were assessed by comparison and statistically significant differences (p & 0. 05) were observed among the groups treated with BMMC and evaluated after 7, 14 and 21 days. Analysis of the mean linear intercept (Lm) values for the different groups allowed to observe that the group treated with BMMC and evaluated after 21 days showed the most significant result. The group that received no treatment showed a statistically significant difference when compared to other groups, except the group treated and evaluated after 21 days, evidencing the efficacy of cell therapy with BMMC in pulmonary emphysema. © 2012 Springer Science+Business Media New York.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Peritoneal dialysis per se is a risk factor for sclerostin-associated adynamic bone disease

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mineral metabolism in singleton and twin-pregnant dairy goats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Association of apolipoprotein B and adiponectin receptor 1 genes with carcass, bone integrity and performance traits in a paternal broiler line

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Homogenous demineralized dentin matrix and platelet-rich plasma for bone tissue engineering in cranioplasty of diabetic rabbits: biochemical, radiographic, and histological analysis

This study evaluated the effects of homogenous demineralized dentin matrix (HDDM) slices and platelet-rich plasma (PRP) in surgical defects created in the parietal bones of alloxan-induced diabetic rabbits, treated with a guided bone regeneration technique. Biochemical, radiographic, and histological analyses were performed. Sixty adult New Zealand rabbits were divided into five groups of 12: normoglycaemic (control, C), diabetic (D), diabetic with a PTFE membrane (DM), diabetic with a PTFE membrane and HDDM slices (DM-HDDM), and diabetic with PTFE membrane and PRP (DM-PRP). The quantity and quality of bone mass was greatest in the DM-HDDM group (respective radiographic and histological analyses: at 15 days, 71.70±16.50 and 50.80±1.52; 30 days, 62.73±16.51 and 54.20±1.23; 60 days, 63.03±11.04 and 59.91±3.32; 90 days, 103.60±24.86 and 78.99±1.34), followed by the DM-PRP group (respective radiographic and histological analyses: at 15 days 23.00±2.74 and 20.66±7.45; 30 days 31.92±6.06 and 25.31±5.59; 60 days 25.29±16.30 and 46.73±2.07; 90 days 38.10±14.04 and 53.38±9.20). PRP greatly enhanced vascularization during the bone repair process. Abnormal calcium metabolism was statistically significant in the DM-PRP group (P<0.001) for all four time intervals studied, especially when compared to the DM-HDDM group. Alkaline phosphatase activity was significantly higher in the DM-HDDM group (P<0.001) in comparison to the C, D, and DM-PRP groups, confirming the findings of intense osteoblastic activity and increased bone mineralization. Thus, HDDM promoted superior bone architectural microstructure in bone defects in diabetic rabbits due to its effective osteoinductive and osteoconductive activity, whereas PRP stimulated angiogenesis and red bone marrow formation.

Uranium metabolism associated with ontogenetic growth of birds: case studies with broilers and ducks

Cobb broilers and domestic ducks, both one-day-old, were treated using ration doped with 20 ppm of uranyl nitrate. Uranium concentrations in the tibia (μg-U/g-bone) were measured by neutron activation analysis as function of the animals’ age, from the neonatal period to maturity. Results show that Uranium and Calcium qualitatively follow the same metabolic pathway, and that adult ducks incorporate on average ten times more Uranium than broilers. Data interpretation shows that the Uranium clearance rate in broilers is substantially higher than that in ducks, suggesting that metabolic characteristics favoring Calcium retention in bone may hinder the elimination of Uranium in ducks. The need for further comparative biochemistry studies between Galliformes and Anseriformes is addressed.

Mandibular cortical bone evaluation on cone beam computed tomography images of patients with bisphosphonate related osteonecrosis of the jaws

Objectives The objective of this study was to develop a technique for detecting cortical bone dimensional changes in patients with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Study Design Subjects with BRONJ who had cone-beam computed tomography imaging were selected, with age- and gender-matched controls. Mandibular cortical bone measurements to detect bisphosphonate-related cortical bone changes were made inferior to mental foramen, in 3 different ways: within a fixed sized rectangle, in a rectangle varying with the cortical height, and a ratio between area and height. Results Twelve BRONJ cases and 66 controls were evaluated. The cortical bone measurements were significantly higher in cases than controls for all 3 techniques. The bone measurements were strongly associated with BRONJ case status (odds ratio 3.36-7.84). The inter-rater reliability coefficients were high for all techniques (0.71-0.90). Conclusions Mandibular cortical bone measurement is a potentially useful tool in the detection of bone dimensional changes caused by bisphosphonates. Long-term administration of bisphosphonates (BPs) affects bone quality and metabolism following accumulation in bone.1 Since the first cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) were published in 2003,2 there has been a search for factors that can predict the onset of the condition. Oral and intravenous BPs reduce bone resorption, increase mineral content of bone, and alter bony architecture.3, 4, 5 and 6 Previous studies have demonstrated these changes both radiographically and following histologic analysis.1, 3, 7, 8, 9 and 10 The BP-related jaw changes may present radiological features, such as thickening of lamina dura and cortical borders, diffuse sclerosis, and narrowing of the mandibular canal3 and 11; however, oral radiographs of patients taking BPs do not consistently show radiographic changes to the jaws.11 and 12 The challenge is to find imaging tools that could improve the detection of changes in the bone associated with BP use. Various skeletal radiographic features associated with BRONJ in conventional periapical and panoramic radiographs, computed tomography, magnetic resonance imaging, and nuclear bone scanning have been described.3, 8, 9, 10 and 11 There has also been a search for BP-related quantitative methods for the evaluation of radiographic images, to avoid observer subjectivity in interpretation. Factors thought to be important include trabecular and cortical structure, and bone mineralization.4 Consequently, measurable bone data have been reported in subjects taking BPs through many techniques, including bone density, architecture, and cortical bone thickness.1, 4, 7 and 13 Trabecular microarchitecture of postmenopausal women has been evaluated with noninvasive techniques, such as high-resolution magnetic resonance images showing less deterioration of the bone 1 year after initiation of oral BP therapy.4 A decrease in bone turnover and a trend for an increase in the bone wall thickness has been detected by histomorphometry in subjects taking BPs.1 Alterations in the cortical structure of the second metacarpal have been detected in digital x-ray radiogrammetry of postmenopausal women treated with BPs.7 Mandibular cortical width may be measured on dental panoramic radiographs, and it has been suggested as a screening tool for referring patients for bone densitometry for osteoporosis investigation.14 and 15 Inhibition of the intracortical bone remodeling in the mandible of mice taking BPs has been reported.16 Thus, imaging evaluation of the mandibular cortical bone could be a biologically plausible way to detect BP bone alterations. Computed tomography can assess both cortical and trabecular bone characteristics. Cone-beam computed tomography (CBCT) can provide 3-dimensional information, while using lower doses and costing less than conventional CT. The CBCT images have been studied as a tool for the measurement of trabecular bone in patients with BRONJ.13 Therefore, cortical bone measurements on CBCT of the jaws might also help to understand bone changes in patients with BRONJ. There is no standard in quantifying dimensional changes of mandibular cortical bone. We explored several different approaches to take into consideration possible changes in length, area, and volume. These led to the 3 techniques developed in this study. This article reports a matched case-control study in which mandibular cortical bone was measured on CBCT images of subjects with BRONJ and controls. The aim of the study was to explore the usefulness of 3 techniques for detecting mandibular cortical bone dimensional changes caused by BP.

Effects of neonatal castration and androgenization on sexual dimorphism in bone, leptin and corticosterone secretion

This study investigated the role of neonatal sex steroids in rats on sexual dimorphism in bone, as well as on leptin and corticosterone concentrations throughout the lifespan. Castration of males and androgenization of females were used as models to investigate the role of sex steroids shortly after birth. Newborn Wistar rats were divided into four groups, two male groups and two female groups. Male pups were cryoanesthetized and submitted to castration or sham-operation procedures within 24 h after birth. Female pups received a subcutaneous dose of testosterone propionate (100 mu g) or vehicle. Rats were euthanized at 20, 40, or 120 postnatal days. Body weight was also measured at 20, 40, and 120 days of age, and blood samples and femurs were collected. The length and thickness of the femurs were measured and the areal bone mineral density (areal BMD) was determined by dual-energy X-ray absorptiometry (DEXA). Biomechanical three-point bending testing was used to evaluate bone breaking strength, energy to fracture, and extrinsic stiffness. Blood samples were submitted to a biochemical assay to estimate calcium, phosphorus, alkaline phosphatase, leptin, and corticosterone levels. Weight gain, areal BMD and bone biomechanical properties increased rapidly with respect to age in all groups. In control animals, skeletal sexual dimorphism, leptin concentration, and dimorphic corticosterone concentration patterns were evident after puberty. However, androgen treatment induced changes in growth, areal BMD, and bone mass properties in neonatal animals. In addition, neonatally-castrated males had bone development and mechanical properties similar to those of control females. These results suggest that the exposure to neonatal androgens may represent at least one covariate that mediates dimorphic variation in leptin and corticosterone secretions. The study indicates that manipulation of the androgen environment during the critical period of sexual differentiation of the brain causes long-lasting changes in bone development, as well as serum leptin and corticosterone concentrations. In addition, this study provides useful models for the investigation of bone disorders induced by hypothalamic hypogonadism. (C) 2011 Elsevier Inc. All rights reserved.

Trabecular bone loss after administration of the second-generation antipsychotic risperidone is independent of weight gain

Second generation antipsychotics (SGAs) have been linked to metabolic and bone disorders in clinical studies, but the mechanisms of these side effects remain unclear. Additionally, no studies have examined whether SGAs cause bone loss in mice. Using in vivo and in vitro modeling we examined the effects of risperidone, the most commonly prescribed SGA, on bone in C57BL6/J (B6) mice. Mice were treated with risperidone orally by food supplementation at a dose of 1.25 mg/kg daily for 5 and 8 weeks, starting at 3.5 weeks of age. Risperidone reduced trabecular BV/TV, trabecular number and percent cortical area. Trabecular histomorphometry demonstrated increased resorption parameters, with no change in osteoblast number or function. Risperidone also altered adipose tissue distribution such that white adipose tissue mass was reduced and liver had significantly higher lipid infiltration. Next, in order to tightly control risperidone exposure, we administered risperidone by chronic subcutaneous infusion with osmotic minipumps (0.5 mg/kg daily for 4 weeks) in 7 week old female B6 mice. Similar trabecular and cortical bone differences were observed compared to the orally treated groups (reduced trabecular BV/TV, and connectivity density, and reduced percent cortical area) with no change in body mass, percent body fat, glucose tolerance or insulin sensitivity. Unlike in orally treated mice, risperidone infusion reduced bone formation parameters (serum P1NP, MAR and BFR/BV). Resorption parameters were elevated, but this increase did not reach statistical significance. To determine if risperidone could directly affect bone cells, primary bone marrow cells were cultured with osteoclast or osteoblast differentiation media. Risperidone was added to culture medium in clinically relevant doses of 0, 2.5 or 25 ng/ml. The number of osteoclasts was significantly increased by addition in vitro of risperidone while osteoblast differentiation was not altered. These studies indicate that risperidone treatment can have negative skeletal consequences by direct activation of osteoclast activity and by indirect non-cell autonomous mechanisms. Our findings further support the tenet that the negative side effects of SGAs on bone mass should be considered when weighing potential risks and benefits, especially in children and adolescents who have not yet reached peak bone mass. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism. (C) 2011 Elsevier Inc. All rights reserved.

The consequences of growth hormone-releasing hormone receptor haploinsufficiency for bone quality and insulin resistance

Objective Growth hormone (GH)/insulin-like growth factor (IGF) axis and insulin are key determinants of bone remodelling. Homozygous mutations in the GH-releasing hormone receptor (GHRHR) gene (GHRHR) are a frequent cause of genetic isolated GH deficiency (IGHD). Heterozygosity for GHRHR mutation causes changes in body composition and possibly an increase in insulin sensitivity, but its effects on bone quality are still unknown. The objective of this study was to assess the bone quality and metabolism and its correlation with insulin sensitivity in subjects heterozygous for a null mutation in the GHRHR. Patients and methods A cross-sectional study was performed on 76 normal subjects (68.4% females) (N/N) and 64 individuals (64.1% females) heterozygous for a mutation in the GHRHR (MUT/N). Anthropometric features, quantitative ultrasound (QUS) of the heel, bone markers [osteocalcin (OC) and CrossLaps], IGF-I, glucose and insulin were measured, and homeostasis model assessment of insulin resistance (HOMAIR) was calculated. Results There were no differences in age or height between the two groups, but weight (P = 0.007) and BMI (P = 0.001) were lower in MUT/N. There were no differences in serum levels of IGF-I, glucose, T-score or absolute values of stiffness and OC, but insulin (P = 0.01), HOMAIR (P = 0.01) and CrossLaps (P = 0.01) were lower in MUT/N. There was no correlation between OC and glucose, OC and HOMAIR in the 140 individuals as a whole or in the separate MUT/N or N/N groups. Conclusions This study suggests that one allele mutation in the GHRHR gene has a greater impact on energy metabolism than on bone quality.

Peripheral arterial disease and diabetes: effects on endothelial progenitor cell differentiation and nitric oxide metabolism

In the recent years it is emerged that peripheral arterial disease (PAD) has become a growing health problem in Western countries. This is a progressive manifestation of atherothrombotic vascular disease, which results into the narrowing of the blood vessels of the lower limbs and, as final consequence, in critical leg ischemia. PAD often occurs along with other cardiovascular risk factors, including diabetes mellitus (DM), low-grade inflammation, hypertension, and lipid disorders. Patients with DM have an increased risk of developing PAD, and that risk increases with the duration of DM. Moreover, there is a growing population of patients identified with insulin resistance (IR), impaired glucose tolerance, and obesity, a pathological condition known as “metabolic syndrome”, which presents increased cardiovascular risk. Atherosclerosis is the earliest symptom of PAD and is a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation. Endothelial dysfunction is a broad term that implies diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing factors. The secretion of these agents is considerably reduced in association with the major risks of atherosclerosis, especially hyperglycaemia and diabetes, and a reduced vascular repair has been observed in response to wound healing and to ischemia. Neovascularization does not only rely on the proliferation of local endothelial cells, but also involves bone marrow-derived stem cells, referred to as endothelial progenitor cells (EPCs), since they exhibit endothelial surface markers and properties. They can promote postnatal vasculogenesis by homing to, differentiating into an endothelial phenotype, proliferating and incorporating into new vessels. Consequently, EPCs are critical to endothelium maintenance and repair and their dysfunction contributes to vascular disease. The aim of this study has been the characterization of EPCs from healthy peripheral blood, in terms of proliferation, differentiation and function. Given the importance of NO in neovascularization and homing process, it has been investigated the expression of NO synthase (NOS) isoforms, eNOS, nNOS and iNOS, and the effects of their inhibition on EPC function. Moreover, it has been examined the expression of NADPH oxidase (Nox) isoforms which are the principal source of ROS in the cell. In fact, a number of evidences showed the correlation between ROS and NO metabolism, since oxidative stress causes NOS inactivation via enzyme uncoupling. In particular, it has been studied the expression of Nox2 and Nox4, constitutively expressed in endothelium, and Nox1. The second part of this research was focused on the study of EPCs under pathological conditions. Firstly, EPCs isolated from healthy subject were cultured in a hyperglycaemic medium, in order to evaluate the effects of high glucose concentration on EPCs. Secondly, EPCs were isolated from the peripheral blood of patients affected with PAD, both diabetic or not, and it was assessed their capacity to proliferate, differentiate, and to participate to neovasculogenesis. Furthermore, it was investigated the expression of NOS and Nox in these cells. Mononuclear cells isolated from peripheral blood of healthy patients, if cultured under differentiating conditions, differentiate into EPCs. These cells are not able to form capillary-like structures ex novo, but participate to vasculogenesis by incorporation into the new vessels formed by mature endothelial cells, such as HUVECs. With respect to NOS expression, these cells have high levels of iNOS, the inducible isoform of NOS, 3-4 fold higher than in HUVECs. While the endothelial isoform, eNOS, is poorly expressed in EPCs. The higher iNOS expression could be a form of compensation of lower eNOS levels. Under hyperglycaemic conditions, both iNOS and eNOS expression are enhanced compared to control EPCs, as resulted from experimental studies in animal models. In patients affected with PAD, the EPCs may act in different ways. Non-diabetic patients and diabetic patients with a higher vascular damage, evidenced by a higher number of circulating endothelial cells (CECs), show a reduced proliferation and ability to participate to vasculogenesis. On the other hand, diabetic patients with lower CEC number have proliferative and vasculogenic capacity more similar to healthy EPCs. eNOS levels in both patient types are equivalent to those of control, while iNOS expression is enhanced. Interestingly, nNOS is not detected in diabetic patients, analogously to other cell types in diabetics, which show a reduced or no nNOS expression. Concerning Nox expression, EPCs present higher levels of both Nox1 and Nox2, in comparison with HUVECs, while Nox4 is poorly expressed, probably because of uncompleted differentiation into an endothelial phenotype. Nox1 is more expressed in PAD patients, diabetic or not, than in controls, suggesting an increased ROS production. Nox2, instead, is lower in patients than in controls. Being Nox2 involved in cellular response to VEGF, its reduced expression can be referable to impaired vasculogenic potential of PAD patients.

Progenitori endoteliali nei pazienti con Chronic Kidney Disease - Mineral and Bone Disorder (CKD-MBD) in fase uremica: effetti del trattamento con vitamina D.

L'insufficienza renale cronica (CKD) è associata ad un rischio cardiovascolare più elevato rispetto alla popolazione generale: fattori come uremia, stress ossidativo, età dialitica, infiammazione, alterazioni del metabolismo minerale e presenza di calcificazioni vascolari incidono fortemente sulla morbosità e mortalità per cause cardiovascolari nel paziente uremico. Diversi studi hanno verificato il coinvolgimento dei progenitori endoteliali (EPC) nella malattia aterosclerotica ed è stato dimostrato che esprimono osteocalcina, marcatore di calcificazione. Inoltre, nella CKD è presente una disfunzione in numero e funzionalità delle EPC. Attualmente, il ruolo delle EPC nella formazione delle calcificazioni vascolari nei pazienti in dialisi non è stato ancora chiarito. Lo scopo della tesi è quello di studiare le EPC prelevate da pazienti con CKD, al fine di determinarne numero e fenotipo. È stato anche valutato l'effetto del trattamento in vitro e in vivo con calcitriolo e paracalcitolo sulle EPC, dato il deficit di vitamina D dei pazienti con CKD: il trattamento con vitamina D sembra avere effetti positivi sul sistema cardiovascolare. Sono stati valutati: numero di EPC circolanti e la relativa espressione di osteocalcina e del recettore della vitamina D; morfologia e fenotipo EPC in vitro; effetti di calcitriolo e paracalcitolo sull’espressione di osteocalcina e sui depositi di calcio. I risultati dello studio suggeriscono che il trattamento con vitamina D abbia un effetto positivo sulle EPC, aumentando il numero di EPC circolanti e normalizzandone la morfologia. Sia calcitriolo che paracalcitolo sono in grado di ridurre notevolmente l’espressione di OC, mentre solo il paracalcitolo ha un effetto significativo sulla riduzione dei depositi di calcio in coltura. In conclusione, il trattamento con vitamina D sembra ridurre il potenziale calcifico delle EPC nell’uremia, aprendo nuove strade per la gestione del rischio cardiovascolare nei pazienti affetti da CKD.