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BackgroundIn chronic kidney disease, serum phosphorus (P) elevations stimulate parathyroid hormone (PTH) production, causing severe alterations in the bone–vasculature axis. PTH is the main regulator of the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, which is essential for bone maintenance and also plays an important role in vascular smooth muscle cell (VSMC) calcification. The discovery of a new RANKL receptor, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), which is important for osteoblast differentiation but with an unknown role in vascular calcification (VC), led us to examine the contribution of LGR4 in high P/high PTH–driven VC.MethodsIn vivo studies were conducted in subtotally nephrectomized rats fed a normal or high P diet, with and without parathyroidectomy (PTX). PTX rats were supplemented with PTH(1–34) to achieve physiological serum PTH levels. In vitro studies were performed in rat aortic VSMCs cultured in control medium, calcifying medium (CM) or CM plus 10−7 versus 10−9 M PTH.ResultsRats fed a high P diet had a significantly increased aortic calcium (Ca) content. Similarly, Ca deposition was higher in VSMCs exposed to CM. Both conditions were associated with increased RANKL and LGR4 and decreased OPG aorta expression and were exacerbated by high PTH. Silencing of LGR4 or parathyroid hormone receptor 1 (PTH1R) attenuated the high PTH–driven increases in Ca deposition. Furthermore, PTH1R silencing and pharmacological inhibition of protein kinase A (PKA), but not protein kinase C, prevented the increases in RANKL and LGR4 and decreased OPG. Treatment with PKA agonist corroborated that LGR4 regulation is a PTH/PKA-driven process.ConclusionsHigh PTH increases LGR4 and RANKL and decreases OPG expression in the aorta, thereby favouring VC. The hormone’s direct pro-calcifying actions involve PTH1R binding and PKA activation.

Background: Mineral and bone metabolism disorders are common complications in hemodialysis patients that present significant geographical variability. Objectives: The objective of this study was to assess these disorders for the first time in hemodialysis patients from Peru. Methods: The study included 1551 hemodialysis patients from 55 centers affiliated with the Social Health System of Peru in the city of Lima. Demographic data, comorbidities, treatments and biochemical parameters were collected from each patient. Serum calcium, phosphorus and PTH levels were categorized according to the recommended ranges in the KDOQI and KDIGO guidelines. Results: The mean age of the patients was 59.5 ± 15.6 years, with a mean time on hemodialysis of 58.0 ± 54.2 months. All patients were dialyzed with a calcium concentration in the dialysis fluid of 3.5 mEq/l and 68.9% of patients were prescribed phosphate-binding agents (98.4% of them calcium carbonate). A high percentage of patients showed serum calcium above, and serum phosphorus below, the recommended ranges in the KDOQI guidelines (32.8% and 37.3%, respectively). More than half of the patients had serum PTH values below the recommended ranges of both the KDOQI and KDIGO guidelines (56.4% and 51.6%, respectively). Conclusions: Patients included in this study were younger than those from other studies and showed both hypophosphataemia and suppressed PTH, probably due to an excessive calcium overload through dialysis fluid and the use of calcium-containing phosphate binding agents. Resumen: Antecedentes: Las alteraciones del metabolismo óseo y mineral son complicaciones frecuentes de los pacientes de hemodiálisis que presentan una gran variabilidad geográfica. Objetivos: El objetivo del presente estudio fue evaluar por primera vez dichas alteraciones en pacientes de hemodiálisis de Perú. Métodos: El estudio incluyó 1.551 pacientes de hemodiálisis de 55 centros concertados con el seguro social de salud de Perú, pertenecientes a la ciudad de Lima. De cada paciente se recogieron datos demográficos, comorbilidades, tratamientos y parámetros bioquímicos. Los valores de calcio, fósforo y PTH fueron categorizados según los rangos recomendados en las guías KDOQI y KDIGO. Resultados: La edad media de los pacientes fue de 59,5 ± 15,6, con tiempo medio en hemodiálisis de 58,0 ± 54,2 meses. Todos los pacientes se dializaban con una concentración de calcio en el líquido de diálisis de 3,5 mEq/l y el 68,9% recibían captores de fósforo (98,4% carbonato de calcio). Se observó un alto porcentaje de pacientes con calcio sérico por encima y fósforo sérico por debajo de los rangos recomendados en las guías KDOQI (32,8% y 37,3% respectivamente). Más de la mitad de los pacientes tenían valores de PTH por debajo de los rangos recomendados, tanto en KDOQI como en KDIGO (56,4% y 51,6% respectivamente). Conclusiones: Los pacientes incluidos en el presente estudio se caracterizaron por ser más jóvenes que los de otros estudios y por tener hipofosforemia y PTH suprimida, probablemente debido a una excesiva sobrecarga de calcio a través del líquido de diálisis y el empleo de captores de fósforo con calcio. Keywords: Chronic kidney disease, Hemodialysis, Calcium, Phosphorus, Parathyroid hormone, Palabras clave: Enfermedad renal crónica, Hemodiálisis, Calcio, Fósforo, Hormona paratiroidea

Vascular calcification (VC) is a complication of chronic kidney disease that predicts morbidity and mortality. Uremic serum promotes VC, but the mechanism involved is unknown. A role for 1,25(OH)2 D3 in VC has been proposed, but the mechanism is unclear because both low and high levels have been shown to increase it. In this work we investigate the role of 1,25(OH)2 D3 produced in vascular smooth muscle cells (VSMCs) in VC. Rats with subtotal nephrectomy and kidney recipient patients showed increased arterial expression of 1α-hydroxylase in vivo. VSMCs exposed in vitro to serum obtained from uremic rats also showed increased 1α-hydroxylase expression. Those increases were parallel to an increase in VC. After 6 days with high phosphate media, VSMCs overexpressing 1α-hydroxylase show significantly higher calcium content and RUNX2 expression than control cells. 1α-hydroxylase null mice (KO) with subtotal nephrectomy and treated with calcitriol (400 ng/kg) for 2 weeks showed significantly lower levels of vascular calcium content, Alizarin red staining, and RUNX2 expression than wild-type (WT) littermates. Serum calcium, phosphorus, blood urea nitrogen (BUN), PTH, and 1,25(OH)2 D3 levels were similar in both calcitriol-treated groups. In vitro, WT VSMCs treated with uremic serum also showed a significant increase in 1α-hydroxylase expression and higher calcification that was not observed in KO cells. We conclude that local activation of 1α-hydroxylase in the artery mediates VC observed in uremia. © 2016 American Society for Bone and Mineral Research.

Background. Serum phosphate is a key parameter in the management of chronic kidney disease-mineral and bone disorder (CKD-MBD). The timing of phosphate measurement is not standardized in the current guidelines. Since the optimal range of these biomarkers may vary depending on the duration of the interdialytic interval, in this analysis of the Current management of secondary hyperparathyroidism: a multicentre observational study (COSMOS), we assessed the influence of a 2- (midweek) or 3-day (post-weekend) dialysis interval for blood withdrawal on serum levels of CKD-MBD biomarkers and their association with mortality risk. Methods. The COSMOS cohort (6797 patients, CKD Stage 5D) was divided into two groups depending upon midweek or post-weekend blood collection. Univariate and multivariate Cox's models adjusted hazard ratios (HRs) by demographics and comorbidities, treatments and biochemical parameters from a patient/centre database collected at baseline and every 6 months for 3 years. Results. There were no differences in serum calcium or parathyroid hormone levels between midweek and post-weekend patients. However, in post-weekend patients, the mean serum phosphate levels were higher compared with midweek patients (5.5 ± 1.4 versus 5.2 ± 1.4 mg/dL, P < 0.001). Also, the range of serum phosphate with the lowest mortality risk [HR ≤ 1.1; midweek: 3.5-4.9 mg/dL (95% confidence interval, CI: 2.9-5.2 mg/dL); post-weekend: 3.8-5.7 mg/dL (95% CI: 3.0-6.4 mg/dL)] showed significant differences in the upper limit (P = 0.021). Conclusion. Midweek and post-weekend serum phosphate levels and their target ranges associated with the lowest mortality risk differ. Thus, clinical guidelines should consider the timing of blood withdrawal when recommending optimal target ranges for serum phosphate and therapeutic strategies for phosphate control. © 2018 The Author(s).

Background The aim of this study was to investigate whether nanomolar concentrations of lanthanum could influence the calcium-sensing receptor (CaSR) response. Methods Embryonic kidney (HEK-293) cells transiently transfected with the human CaSR were used to test the ability of lanthanum to activate the CaSR, either alone or in combination with calcium. CaSR activation was measured by flow cytometry. Parathyroid glands from 4-month-old male Wistar rats with normal renal function (n = 60) were also cultured ex vivo with different concentrations of lanthanum to measure parathyroid hormone (PTH) secreted to the medium and PTH mRNA. Results The maximal CaSR activation induced by 1 muM lanthanum chloride (LaCl(3)) was similar to that induced by 16 mM calcium chloride (CaCl(2) 16 mM: 294 +/- 14%; LaCl(3) 1 muM: 303 +/- 11%). Lanthanum half effective concentration (EC(50)) was 77.28 nM, lower than the 2.30 mM obtained for calcium, supporting the concept that this metal is a strong agonist of the CaSR. Moreover, lanthanum was also able to enhance CaSR sensitivity to calcium. The presence of 1 nM LaCl(3) significantly left-shifted the CaSR response curve, changing the EC(50) value for calcium from 2.30 mM (calcium alone) to 1.26 mM (calcium + 1 nM lanthanum). The parathyroid glands cultured with lanthanum showed a trend to secrete less PTH compared to the control glands: 1.51 +/- 0.23 (control), 0.91 +/- 0.17 (La 100 nM) and 1.04 +/- 0.18 (La 400 nM) [(pg/h)/(pg/h), mean +/- SEM] (ANOVA P = 0.0145). A similar trend was also observed in PTH synthesis measured by PTH mRNA levels. Conclusions These in vitro findings demonstrate that lanthanum, at nanomolar concentrations, is an agonist of the CaSR able to activate it in the absence of calcium. In addition, it can also enhance CaSR sensitivity to calcium, modulating PTH synthesis and secretion.

Estrogen deficiency is the main factor implicated in bone loss in postmenopausal osteoporosis.1 As a consequence of the lack of estrogens, bone turnover increases, leading to an imbalance between bone formation and bone resorption, favoring the latter.2,3 This imbalance affects calcium–phosphate metabolism and may increase serum parathyroid hormone (PTH) levels.4 Estrogen replacement therapy prevents bone loss and fractures,5,6 acting directly on bone cells through their specific estrogen receptors (ERs): α and β.7,8 In addition, in postmenopausal women, estrogens can also reduce PTH serum levels4,9 through an as of yet poorly understood mechanism. A possible direct effect of estrogens reducing PTH acting through ERα and ERβ located in the parathyroid cells has been suggested, but the existence of ERα and ERβ in parathyroid tissue is still a controversial issue.10–13 Estrogens may also decrease PTH secretion by acting on other factors such as calcium,14,15 1,25(OH)2 D3 (calcitriol),15 and phosphorus,16,17 among others. Recently, fibroblast growth factor 23 (FGF23), involved in phosphorus and vitamin D metabolism,18 has been suggested to influence PTH synthesis and secretion.19 In women with chronic kidney disease (CKD), little is known about the role that estrogen deficiency plays in the pathogenesis and progression of bone disease.20,21 Understanding the mechanism through which estrogens act on PTH is also a subject of interest in these patients, because of the high prevalence of secondary parathyroid disorders.22 Because several aspects of the effects of estrogens on PTH remain unclear, the objective of this study was to investigate the factors and mechanisms involved in the likely effect of estrogens on the parathyroid gland.

Vascular calcification is a frequent cause of morbidity and mortality in patients with CKD and the general population. The common association between vascular calcification and osteoporosis suggests a link between bone and vascular disorders. Because microRNAs (miRs) are involved in the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells, we investigated whether miRs implicated in osteoblast differentiation and bone formation are involved in vascular calcification. Different levels of uremia, hyperphosphatemia, and aortic calcification were induced by feeding nephrectomized rats a normal or high-phosphorus diet for 12 or 20 weeks, at which times the levels of eight miRs (miR-29b, miR-125, miR-133b, miR-135, miR-141, miR-200a, miR-204, and miR-211) in the aorta were analyzed. Compared with controls and uremic rats fed a normal diet, uremic rats fed a high-phosphorous diet had lower levels of miR-133b and miR-211 and higher levels of miR-29b that correlated respectively with greater expression of osteogenic RUNX2 and with lower expression of several inhibitors of osteoblastic differentiation. Uremia per se mildly reduced miR-133b levels only. Similar results were obtained in two in vitro models of vascular calcification (uremic serum and high-calcium and -phosphorus medium), and experiments using antagomirs and mimics to modify miR-29b, miR-133b, and miR-211 expression levels in these models confirmed that these miRs regulate the calcification process. We conclude that miR-29b, miR-133b, and miR-211 have direct roles in the vascular smooth muscle calcification induced by high phosphorus and may be new therapeutic targets in the management of vascular calcification.

Background: Abnormalities in serum phosphorus, calcium and parathyroid hormone (PTH) have been associated with poor survival in haemodialysis patients. This COSMOS (Current management Of Secondary hyperparathyroidism: a Multicentre Observational Study) analysis assesses the association of high and low serum phosphorus, calcium and PTH with a relative risk of mortality. Furthermore, the impact of changes in these parameters on the relative risk of mortality throughout the 3-year follow-up has been investigated. Methods: COSMOS is a 3-year, multicentre, open-cohort, prospective study carried out in 6797 adult chronic haemodialysis patients randomly selected from 20 European countries. Results: Using Cox proportional hazard regression models and penalized splines analysis, it was found that both high and low serum phosphorus, calcium and PTH were associated with a higher risk of mortality. The serum values associated with the minimum relative risk of mortality were 4.4 mg/dL for serum phosphorus, 8.8 mg/dL for serum calcium and 398 pg/mL for serum PTH. The lowest mortality risk ranges obtained using as base the previous values were 3.6-5.2 mg/dL for serum phosphorus, 7.9-9.5 mg/dL for serum calcium and 168-674 pg/mL for serum PTH. Decreases in serum phosphorus and calcium and increases in serum PTH in patients with baseline values of >5.2 mg/dL (phosphorus), >9.5 mg/dL (calcium) and

The search for biomarkers of hypertension and diabetes-induced damage to multiple target organs is a priority. We analyzed the correlation between plasma cardiotrophin-1 (CT-1), a chemokine that participates in cardiovascular remodeling and organ fibrosis, and a wide range of parameters currently used to diagnose morphological and functional progressive injury in left ventricle, arteries, and kidneys of diabetic and hypertensive patients, in order to validate plasma levels of CT-1 as clinical biomarker. This is an observational study with 93 type 2-diabetic patients, 209 hypertensive patients, and 82 healthy controls in which we assessed the following parameters: plasma CT-1, basal glycaemia, systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP), left ventricular hypertrophy (LVH by electrocardiographic indexes), peripheral vascular disease (by pulse wave velocity—PWV, carotid intima-media thickness—C-IMT, and ankle-brachial index—ABI), and renal impairment (by microalbuminuria, albumin/creatinine urinary ratio, plasma creatinine concentrations, and glomerular filtration rate). Hypertensive or diabetic patients have higher plasma CT-1 than control patients. CT-1 positively correlates with basal glycaemia, SBP, DBP, PP, LVH, arterial damage (increased IMT, decreased ABI), and early renal damage (microalbuminuria, elevated albumin/creatinine ratio). CT-1 also correlates with increased 10-year cardiovascular risk. Multiple linear regression analysis confirmed that CT-1 was associated with arterial injury assessed by PWV, IMT, ABI, and cardiac damage evaluated by Cornell voltage duration product. Increases in plasma CT-1 are strongly related to the intensity of several parameters associated to target organ damage supporting further investigation of its diagnostic capacity as single biomarker of cardiovascular injury and risk and, possibly, of subclinical renal damage.

Vascular calcification remains one of the main factors associated to morbidity and mortality in both ageing and chronic kidney disease. Both hyperphosphataemia, a well-known promoter of vascular calcification, and abnormal processing defects of lamin A/C have been associated to ageing. The main aim of this study was to analyse the effect of phosphorus load in the differential expression pattern of genes and proteins, particularly of lamin A/C, which are involved in phenotypic change of the vascular smooth muscle cells to osteoblast-like cells. The in vivo study of the calcified abdominal aortas from nephrectomized rats receiving a high phosphorus diet showed among others, a repression of muscle related proteins and overexpression of lamin A/C. Similar results were observed in vitro, where primary vascular smooth muscle cells cultured in calcifying medium showed increased expression of prelamin A and lamin A and abnormalities in the nuclear morphology. Co-immunoprecipitation assays showed novel and important physical interactions between lamin A and RUNX2 during the process of calcification. In fact, the knockdown of prelamin A and lamin A inhibited the increase of Runx2, osteocalcin and osteopontin gene expression, calcium deposition, nuclear abnormalities and the RUNX2 protein translocation into the nucleus of the cell. These in vivo and in vitro results highlight the important role played by lamin A in the process of vascular calcification.

Effect of aluminium on calcium-sensing receptor expression, proliferation, and apoptosis of parathyroid glands from rats with chronic renal failure. Background To assess the effect of aluminium on the calcium-sensing receptor expression, proliferation, and apoptosis in parathyroid glands from rats with chronic renal failure, 2 1 / 2 -month-old male Wistar rats were 7/8 nephrectomized. Methods Eight weeks after surgery the rats were divided into two groups, one receiving intraperitoneal AlCl 3 for 8 weeks and the other receiving intraperitoneal placebo. Serum Al, Ca, P, creatinine, and PTH were measured. Parathyroid glands were removed, formaldehyde-fixed, and paraffin-embedded. Calcium-sensing receptor and proliferation were detected by immunohistochemistry and apoptosis by TUNEL and propidium iodide uptake. Results At the end of the study, despite higher levels of serum P in the aluminium group (6.27 ± 0.63 vs. 5.56 ± 0.58 mg/dL; P = 0.045), serum PTH was lower (89.6 ± 57.7 vs. 183.1 ± 123.8 pg/mL; P = 0.059). No significant differences were found in the calcium-sensing receptor expression between groups (aluminium: 27.1 ± 7.6; placebo: 25.4 ± 3.5 RU). Rats receiving aluminium showed a significantly lower cell proliferation rate than the control rats (0.54 ± 0.69 vs. 4.43 ± 3.10 cells/mm 2 ; P = 0.003). No apoptotic events were detected. Conclusion Aluminium was able to reduce the cell proliferation of the parathyroid glands. Due to the low apoptosis rate, however, it was not possible to find any change. Aluminium had no effect on the calcium-sensing receptor expression.

Effect of VDR gene polymorphisms on osteocalcin secretion in calcitriol-stimulated human osteoblasts. Background The impact of vitamin D receptor (VDR) gene polymorphisms in bone metabolism remains controversial. Some authors have found a beneficial effect of some VDR gene polymorphisms, while others found no differences, or even a lower bone mass in subjects with the same type of polymorphisms. The aim of this study was to assess if the VDR gene polymorphisms could have an effect on the calcitriol-stimulated osteocalcin in human osteoblasts. Methods Osteoblasts were obtained from human femoral necks replaced because of osteoarthritis. Bones were cut into pieces of 1 to 2mm and placed in a nylon mesh. After the migration of osteoblasts, the pieces were collected and cultured with different concentrations of calcitriol (10 −8 , 10 −9 , and 10 −10 mol/L). After 48 hours of incubation with calcitriol, the osteocalcin secreted into the medium (corrected by either total proteins or total DNA content) was measured. The DNA was extracted from the osteoblasts, amplified by polymerase chain reaction (PCR), and analyzed for target sequences sites of the Bsm I, Apa I, Taq I, and Fok I restriction enzymes. Results The response observed in osteocalcin secretion in the bb or TT genotypes doubled the response observed in the BB or tt genotypes (calcitriol 10 −8 and 10 −9 mol/L). A slight trend was also observed with the aa genotype. Men showed higher levels of osteocalcin secretion than women. Age did not show any influence in osteocalcin secretion. Conclusion VDR alleles and gender demonstrated an effect on the osteocalcin secretion. BB or tt genotypes, and also the "A" allele, showed the lowest calcitriol-stimulated osteocalcin secretion.

Background. Aluminium overload leads to parathyroid hormone (PTH) suppression. However, it is unclear whether a decrease in synthesis or release of the hormone is mainly involved. The aim of this study was to assess the effect of an acute administration of aluminium on PTH synthesis and release in rats with chronic renal failure and secondary hyperparathyroidism. Methods. The study was performed using 100 adult male Wistar rats (body weight 443 ± 54 g). 7/8 nephrectomy was performed and the rats were maintained on a high dietary phosphorous intake. Five weeks after surgery, the rats were randomly divided into two groups, one loaded with aluminium (AlCl 3 ) and the other given placebo. Aluminium or placebo were administered i.p. for two consecutive days. The placebo group received saline at the same pH as the aluminium solution. After 2 weeks, serum calcium, phosphorous, creatinine, PTH, and aluminium were measured. The parathyroid glands were removed and PTH messenger RNA (mRNA) was measured by northern blot. Intact PTH was measured by IRMA (Rat PTH R , Nichols Institute). Results. No differences in serum PTH levels were found between the two groups after 5 weeks of renal failure. At the end of the study the rats given aluminium had higher aluminium levels than the placebo group and lower PTH levels. No significant differences were found for calcium, phosphorous, renal function, or body weight. PTH mRNA expression was lower in the aluminium group than in the placebo group. Conclusion. The administration of aluminium in rats with chronic renal failure resulted in reductions in serum PTH and PTH mRNA. Thus far, previous studies had demonstrated that aluminium suppressed PTH release. The present findings suggest that PTH synthesis is also reduced.

To assess the effect of concomitant iron and aluminum loads on bone aluminum accumulation and on the response to the deferoxamine test in rats with the same aluminum surcharge, Wistar rats with chronic renal failure were divided into three groups: iron-overloaded rats (N = 6) (intraperitoneal iron); iron-depleted rats (N = 6) (blood withdrawal two to three times per week); control rats (N = 4) (no manipulation). All groups received intraperitoneal aluminum simultaneously. After 6 wk, a deferoxamine challenge test was performed. Thereafter, bone aluminum and iron were measured. The iron-overloaded rats showed higher bone iron content (iron overloaded: 147.7+/-55.4 microg/g; iron depleted: 7.9+/-1.0, and controls 13.3+/-9.9 microg/g, p0.010) and lower bone aluminum content (iron overloaded: 14.2+/-4.0 microg/g; iron depleted: 70.9+/-35.1 microg/g; controls: 72.7+/-28.3 microg/g p0.005). No differences were found between the iron-depleted and control rats. After the deferoxamine infusion, the iron-depleted rats tended to have higher serum aluminum increments (p = NS) and higher urinary aluminum excretion (p0.012, p0.020) than control rats despite similar amounts of aluminum in bone of the two groups. Aluminum bone accumulation was minor if iron and aluminum loads were given concomitantly. The iron depletion influenced the results of the deferoxamine challenge test in rats with similar bone aluminum burden.

is based on the prevention of aluminium exposure [1] Background. The recommended dose of desferriox- and increased removal of aluminium by dialysis [2,3]. amine for the treatment of aluminium intoxication is Nevertheless, aluminium transfer during dialysis is 5m g/kg/week. However, there are no data about the limited because serum aluminium is bound to high eYciency of lower doses. The objective of this study molecular weight proteins, mainly transferrin, and, was to investigate the capacity of very low doses of therefore, it is not dialysable. desferrioxamine in the generation of ultrafiltrable The most eVective way of removing aluminium from aluminium. serum is to increase the amount of ultrafiltrable alumiMethods. Five patients undergoing haemodialysis with nium by using desferrioxamine. This drug removes a similar biochemical profile and serum aluminium aluminium from tissues [3] and increases serum alumilevels >40 mg/l were studied. The three diVerent doses nium levels, and may displace the aluminium bound of desferrioxamine used (0.5, 2.5 and 5.0 mg/kg) were to transferrin, forming a low molecular weight aluminiadministered randomly to each patient at 1 week um‐desferrioxamine complex which is dialysable intervals. Total and ultrafiltrable serum aluminium was [4‐6 ]. The percentage of ultrafiltrable aluminium after measured before and 44 h after the administration of desferrioxamine administration has been reported to desferrioxamine. be >60% [7‐11], with doses of desferrioxamine ranResults. All doses of desferrioxamine significantly ging between 10 and 100 mg/kg [7‐9]. Other authors increased the total serum aluminium; no diVerences have found similar results using 5‐20 mg/kg doses were found between 2.5 and 5.0 mg/kg. The total [12]. However, there are no data using doses lower serum aluminium levels doubled with the 2.5 and than this. 5.0 mg/kg doses, while the increase with 0.5 mg/kg Previous in vitro studies performed by our group was lower (32.6%, P 5m g/kg) increases the ultrafiltrable (potentially dialysable) aluminium. Patients and methods

Several dialysis techniques have been used to improve aluminium removal. So far there are no data available using paired filtration-dialysis (PFD). In this study, we evaluated the aluminium removed by PFD in two phases. Bovine plasma with known concentrations of aluminium and desferrioxamine was used in both experiments. In phase I, the aluminium removal was investigated using the PFD system (single pass) in its usual configuration, modifying the order of the convective and diffusive processes, dialysis with high permeability membranes and dialysis with low permeability membranes. During the second phase, the experiment lasted longer using recirculation, and the PFD was compared with conventional dialysis using high permeability membranes. Changes in the PFD configuration did not alter the aluminium removal; the efficiency of PFD for aluminium removal was very close to that of dialysis with high permeability membranes and much greater than with low permeability membranes. The aluminium is removed mainly in the first part of the dialysis. Aluminium mobilization using the double chamber technique (PFD) was efficient and might be of value for those patients with aluminium overload who needs high depurative techniques and are unable to tolerate high-flux techniques.

Background: Microparticles are present in low concentrations in normal plasma, but increase in several diseases including atherosclerosis, mainly through membrane activation processes or during apoptosis. The number of circulating microparticles has been proposed as a marker of subclinical atherosclerosis, but their potential role in its progression has not been fully characterized. Methods: Microparticles released by vascular smooth muscle cells treated with lysophosphatidylcholine were isolated and their composition characterized by proteomic techniques. Some hits were confirmed by western blot in this and other cell lines involved in atherosclerosis. Platelet poor plasma was obtained from patients suffering from carotid stenosis (>70%) by venipuncture and subsequential centrifugation. Platelet poor plasma was analyzed by flow cytometry and the microparticle fraction was analyzed by SDS-PAGE. Results: Among the proteins identified as components of vascular smooth muscle cells-derived microparticles, caveolin-1 was confirmed by western blot. Caveolin-1 is detected in endothelial cell and fibroblast-derived microparticles but not in those released by other cell types involved in atherosclerosis progression. Caveolin-1 was detected in the microparticle fraction in 73.33% of patients compared to 15.38% of controls. The presence of Caveolin-1 did not correlate with the counts of circulating endothelial-derived microparticles. Conclusions: Pro-atherogenic stimuli induce the release of microparticles containing Caveolin-1 by vascular smooth muscle cells and endothelial cells. Caveolin-1 is detected in microparticles isolated from plasma obtained from patients suffering from atherosclerosis. Due to its role in the regulation of smooth muscle cell proliferation we hypothesize that the release of microparticles in response to pro-atherogenic stimuli may be involved in the progression of the disease through the regulation of caveolin-1 levels in vascular smooth muscle cells.

Hyperphosphatemia has been associated with higher mortality risk in CKD 5 patients receiving dialysis. Here, we determined the association between the use of single and combined phosphate- binding agents and survival in 6797 patients of the COSMOS study: a 3-year follow-up, multicenter, open-cohort, observational prospective study carried out in 227 dialysis centers from 20 European countries. Patient phosphate-binding agent prescriptions (time-varying) and the case- mix–adjusted facility percentage of phosphate- binding agent prescriptions (instrumental variable) were used as predictors of the relative all-cause and cardiovascular mortality using Cox proportional hazard regression models. Three different multivariate models that included up to 24 variables were used for adjustments. After multivariate analysis, patients prescribed phosphate-binding agents showed a 29 and 22% lower all-cause and cardiovascular mortality risk, respectively. The survival advantage of phosphate- binding agent prescription remained statistically significant after propensity score matching analysis. A decrease of 8% in the relative risk of mortality was found for every 10% increase in the case-mix–adjusted facility prescription of phosphate-binding agents. All single and combined therapies with phosphate-binding agents, except aluminum salts, showed a beneficial association with survival. The findings made in the present association study need to be confirmed by randomized controlled trials to prove the observed beneficial effect of phosphate-binding agents on mortality.

The aim of this study was to compare the sensitivity and specificity of the two histochemical stains most commonly used as indirect markers of the aluminium bone content. The clinical study was made in 28 biopsies from patients undergoing haemodialysis and the experimental study in 17 tibias from Wistar rats aluminium overloaded and with different deposits of iron. All samples were stained with aluminon, solochrome azurine and Perls and aluminium bone content was also measured. When the positive cases with Perls were excluded in the clinical study (without iron interference), the trabecular surface stained with solochrome azurine correlated with the aluminium bone content (r = 0.71, P0.001). With aluminon, on the other hand, no correlation was found. Solochrome azurine was always positive with aluminium contents greater than 8 micrograms/g. Aluminon was positive over 17 micrograms/g. In the experimental study, the iron concentration, in addition to other parameters, was also measured. As in the clinical study, the trabecular surface stained with solochrome azurine correlated with the aluminium content. If the positive Perls cases were excluded, the trabecular surface stained with solochrome azurine doubled the trabecular surface stained with aluminon (P0.001). No intratrabecular aluminon staining was observed while the intratrabecular solochrome staining correlated with the aluminium content (P0.001). Solochrome azurine was more sensitive than the aluminon and its lack of specificity can be easily corrected by employing Perls staining to exclude the iron interference.

Background. Chronic kidney disease–mineral and bone disorders (CKD–MBD) are important complications of CKD5D patients that are associated with mortality. Methods. COSMOS is a multicentre, open cohort, prospective, observational 3-year study carried out in haemodialysis patients from 20 European countries during 2005–07. The present article describes the main characteristics of the European dialysis population, the current practice for the prevention, diagnosis and treatment of secondary hyperparathyroidism and the differences across different European regions. Results. The haemodialysis population in Europe is an aged population (mean age 64.8 ± 14.2 years) with a high prevalence of diabetes (29.5%) and cardiovascular disease (76.0%), and 28.7% of patients have been on haemodialysis more than 5 years. Patients from the former Eastern countries are younger (59.3 ± 14.3 versus 66.0 ± 13.9), having a lower proportion of diabetics (24.1 versus 30.7%). There were relevant differences in the frequency of measurement of the main CKD–MBD biochemical parameters [Ca, P and parathyroid hormone (PTH)] and the Eastern countries showed a poorer control of these biochemical parameters (K/DOQI and K/DIGO targets). Overall, 48.0% of the haemodialysis patients received active vitamin D treatment. Calcitriol use doubled that of alfacalcidiol in the Mediterranean countries, whereas the opposite was found in the non-Mediterranean countries. The criteria followed to perform parathyroidectomy were different across Europe. In the Mediterranean countries, the level of serum PTH considered to perform parathyroidectomy was higher than in non-Mediterranean countries; as a result, in the latter, more parathyroidectomies were performed in the year previous to inclusion to COSMOS. Conclusions. The COSMOS baseline results show important differences across Europe in the management of CKD–MBD.

Summary Background and Objectives A high body mass index (BMI) is associated with lower mortality in patients undergoing hemodialysis. Short-term weight gains and losses are also related to lower and higher mortality risk, respectively. The implications of weight gain or loss may, however, differ between obese individuals and their nonobese counterparts. Design, Setting, Participants, & Measurements The Current Management of Secondary Hyperparathyroidism: A Multicenter Observational Study (COSMOS) is an observational study including 6797 European hemodialysis patients recruited between February 2005 and July 2007, with prospective data collection every 6 months for 3 years. Time-dependent Cox proportional hazard regressions assessed the effect of BMI and weight changes on mortality. Analyses were performed after patient stratification according to their starting BMI. ResultsAmong 6296 patients with complete data, 1643 died. At study entry, 42% of patients had a normal weight (BMI, 20–25 kg/m2), 11% were underweight, 31% were overweight, and 16% were obese (BMI $30 kg/m2). Weight loss or gain (,1% or .1% of body weight) was strongly associated with higher rates of mortality or survival, respectively. After stratification by BMI categories, this was true in nonobese categories and especially in underweight patients. In obese patients, however, the association between weight loss and mortality was attenuated (hazard ratio, 1.28 [95% confidence interval (CI), 0.74 to 2.14]), and no survival benefi to f gaining weight was seen (hazard ratio, 0.98 [95% CI, 0.59 to 1.62]). ConclusionsAssuming that these weight changes were unintentional, our study brings attention to rapid weight variations as a clinical sign of health monitoring in hemodialysis patients. In addition, a patient’ sB MI modifies the strength of the association between weight changes with mortality. Clin J Am Soc Nephrol 8: 1725–1733, 2013. doi: 10.2215/CJN.10951012

The current regulation of parathyroid hormone (PTH) and the development of parathyroid disorders in chronic kidney disease involve complex mechanisms. Factors such as calcium, phosphorous, calcitriol, vitamin D receptor, calcium-sensing receptor and fibroblast growth factor 23 play a key role in the regulatory process in the pathogenesis of secondary hyperparathyroidism.This review provides an analysis of published results related to the different models and approaches used to study the mechanisms involved in the pathogenesis of secondary hyperparathyroidism. The review includes clinical studies, animal and ex vivo/in vitro models which have been extensively used in this area.Readers will have an overview of the main findings and progress achieved in the knowledge of the parathyroid function combining the results obtained from the different models used to understand the parathyroid gland regulation.Each of the available models used to study the complex system of parathyroid regulation has advantages and limitations; therefore, it is necessary to combine the information obtained from more than one model in order to have a more complete knowledge of the mechanisms involved in PTH regulation.

Mineral metabolism parameters may play a role in the survival of patients with chronic kidney disease (CKD).In the CORES Study, we analysed the association between calcium, phosphorus and PTH and mortality (all-cause and cardiovascular) in 16 173 haemodialysis (HD) patients over 18 years from six Latin American countries, who underwent haemodialysis up to 54 months. Unadjusted, case-mix-adjusted and time-dependent multivariable-adjusted hazard ratio (HR) of death were calculated for categories of serum albumin-corrected calcium (Ca(Alb)), phosphorus and PTH using as 'reference values' the range in which the lowest death rate was observed. Age, gender, vitamin D treatment, diabetes, vintage, vascular access, weight, blood pressure and laboratory variables (serum albumin, haemoglobin, creatinine, ferritin and Kt/V) were used as confounding variables.Low (9.5 mg/dL) and high (10.5 mg/dL) Ca(Alb) increased the HR for all-cause mortality. Low (9.0 mg/dL) Ca(Alb) increased the HR for cardiovascular mortality. High phosphorus (5.5 mg/dL) increased the HR for both all-cause and cardiovascular mortality. Low phosphorus (4.0 and3.0 mg/dL) increased the HR for both all-cause and cardiovascular mortality. Furthermore, low (150 pg/mL) and high (500 and300 pg/mL) PTH increased the HR for both all-cause and cardiovascular mortality. In addition, only phosphorus6.0 mg/dL increased the HR for cardiovascular hospitalizations. No effect was observed with Ca(Alb) or PTH.In summary, in 16,173 HD patients, elevated and reduced serum levels of albumin-corrected calcium, phosphorus and PTH levels were associated with increments in all-cause mortality. Similar results were obtained when only cardiovascular mortality was analysed.

El objetivo del estudio fue analizar el riesgo de fractura calculado mediante la herramienta FRAX® y sus determinantes, en pacientes con trasplante renal remitidos a nuestra consulta para control de las repercusiones óseas. Se estudiaron 113 pacientes, 42 hombres y 71 mujeres portadores de trasplante renal funcionante a los que se les realizó una encuesta general. A todos los pacientes se les hizo una densitometría ósea en columna lumbar PA (L2 - L4) y cadera (cuello femoral y cadera total) con un densitómetro Hologic QDR 1000®. Con los datos recogidos de la encuesta se calculó el riesgo de fractura mediante la herramienta FRAX®. La edad media de los pacientes fue 57±9 años y un tiempo medio trasplantados de 4,6±4,4 años. El riesgo de presentar fractura osteoporótica mayor a los 10 años fue del 6,54±4,9% y de fractura de cadera de 2,06±2,67%. Cuando analizamos el riesgo según el sexo observamos como en las mujeres la probabilidad de presentar fractura osteoporótica mayor a los 10 años fue significativamente superior que en los hombres (7,6±5,2% vs 4,8±3,7%, p=0,001). El riesgo de fractura de cadera a los 10 años fue también ligeramente superior en las mujeres que en los hombres, pero no de forma significativa (2,3±2,8% vs1,6±2,4%).

Within the extracellular loops of the seven-transmembrane domain of the calcium-sensing receptor (CaR) there is a region (I819-E837) relevant for calcimimetic activity. As the naturally occurring variant Ala826Thr is within this important region, it may be postulated that this change may influence the CaR response to calcium and R-568. Human embryonic kidney (HEK-293) cells transiently transfected with three different human CaRs (wild-type [A826], variant allele [T826], and artificial mutant [W826]) were used to test the ability of calcium alone or in combination with the calcimimetic R-568 to modulate CaR activity. CaR activation was detected by flow cytometry using a fluorescent probe. Intracellular calcium changes were measured in response to changes in extracellular calcium alone or with different R-568 concentrations. The change of the alanine in the 826 position (A826) for threonine (T826) worsened calcium sensitivity, increasing the EC(50) value from 2.34 +/- 0.48 mM (A826, wild-type) to 2.96 +/- 0.75 mM (T826) (P < 0.05). The T826 receptor reached a similar response with 1 muM R-568 compared with the wild-type receptor. On the contrary, the artificial introduction of a tryptophan in the same position (W826) did not affect calcium sensitivity (EC(50) = 2.64 +/- 0.81 mM) but reduced the ability of the receptor to respond to R-568. The results demonstrate the importance of the 826 residue in the CaR response to calcium and calcimimetics. Since the A826T change was described as a natural variant, the differences in the calcium and calcimimetic responses observed between the alleles could have potential clinical impact.

Background The regulatory mechanisms of parathyroid hormone (PTH) synthesis are complex, involving calcium, calcitriol, the calcium-sensing receptor (CaR) and the vitamin D receptor (VDR). In this study, the effects of calcium and calcitriol on the simultaneous expression of CaR and VDR mRNA and protein levels were assessed in parathyroid glands cultured in vitro. Methods Parathyroid glands (N = 424) were removed and cultured for 24 h to study the effect of calcium on the CaR, VDR and PTH. In addition, the effect of calcitriol at low calcium concentrations (0.6 mM) on CaR and VDR levels was studied after 48 h of incubation. CaR, VDR and PTH mRNAs were measured by quantitative real-time PCR (qRT-PCR), and CaR and VDR protein levels were measured by immunohistochemistry. Results PTH gene expression was reduced by high calcium concentration. No differences were found in the CaR mRNA levels among the different calcium concentrations tested (0.6 mM calcium: 100%; 1.2 mM calcium: 120%; 2.0 mM calcium: 112%; median values), but VDR gene expression rose when calcium increased (0.6 mM calcium: 100%; 1.2 mM calcium: 164%; 2.0 mM calcium: 195%; median values). Calcitriol increased both CaR (control: 100%; 10(-8) M calcitriol: 196%; median values) and VDR genes expression (control: 100%; 10(-8) M calcitriol: 176%; median values). The same findings were corroborated at protein levels for both CaR and VDR. Conclusions In parathyroid glands cultured in vitro, calcium up-regulates VDR but not CaR. Conversely, calcitriol up-regulates both VDR and CaR mRNAs and protein levels, even at low calcium concentrations.

Advantages of adjusting the initial dose of intravenous calcitriol according to PTH levels.BackgroundTo assess the usefulness of starting calcitriol therapy with a dose proportional to the degree of hyperparathyroidism, 141 patients from 28 centers were treated with intravenous calcitriol for 6 months. The aim was to achieve a final PTH between 125 and 250 pg/mL. Patients with serum PTH>250 pg/mL were included in the study and divided into 4 groups according to baseline PTH levels.MethodsThe study was completed by 100 patients, a third of which were treated strictly according to the protocol, labeled “compliants”; thus, calcitriol was started according to baseline PTH levels. Two thirds of patients, labeled “noncompliants,” showed one or more violation in the dosage regimen.ResultsAfter 2 months of treatment with calcitriol, 59% of the “compliants” and 35% of the “noncompliants” decreased their PTH levels>40% (P = 0.022), 70%, and 49%, respectively after 3 months of treatment. After 3 months of treatment, 67% of the “compliants” reached the target (PTH 125 to 250 pg/mL) in contrast with 23% of the “noncompliants” (P < 0.001). The number of hypercalcemic and hyperphosphatemic episodes was significantly lower in the “compliants” group (P < 0.006).ConclusionThese results demonstrate several advantages when calcitriol therapy is started with a dose proportional to the severity of hyperparathyroidism.

Vitamin D status and secondary hyperparathyroidism: The importance of 25-hydroxyvitamin D cut-off levels.BackgroundSerum 25-hydroxyvitamin D is the best indicator of vitamin D status. However, some controversy remains regarding “normal” and “abnormal” values. This study's aim was to assess vitamin D status and prevalence of secondary hyperparathyroidism.MethodsA random sample of 326 subjects (164 women and 162 men, aged 68 ± 9; range, 54 to 89) participating in the European Vertebral Osteoporosis Study (EVOS) was used to assess vitamin D status and secondary hyperparathyroidism. Only those subjects who had never received any kind of treatment for osteoporosis were included in this analysis.ResultsSerum 25-hydroxyvitamin D levels were “deficient” (18 ng/mL) in 33% of subjects. The prevalence of secondary hyperparathyroidism (PTH>65 pg/mL) according to 25-hydroxyvitamin D levels was 33% (18 ng/mL), respectively. There were no cases of secondary hyperparathyroidism with 25-hydroxyvitamin D levels>40ng/mL. The independent predictors for PTH were 25-hydroxyvitamin D and serum creatinine in both sexes, but age was a predictor only in men.ConclusionThese remarkable findings demonstrate the importance of maintaining higher 25-hydroxyvitamin D levels to avoid stimulation of the parathyroid gland.

Chronic aluminium exposure and toxicity related to aluminium absorption and contaminated dialysis fluid continue to be a problem for many patients with renal failure, particularly in South America and in some developing countries. The two most prevalent sources of aluminium in this population are water used to prepare dialysate and aluminium-containing phosphate binders. Of particular concern is the effect of aluminium at the level of the bone, the haematopoietic system and the brain. Here we focus mainly on the adverse effects of aluminium on bone, the preferred organ of aluminium accumulation in the body. Unfortunately, aluminium has a cumulative effect, thus even short-term exposure to aluminium in phosphate binders adds to the total load and may contribute to the risk of aluminium-related bone disease. Even a bone biopsy does not allow a precise determination of total bone aluminium content. We examine the mechanisms by which aluminium contributes to abnormal bone remodelling. Studies indicate that aluminium has a direct effect, inhibiting bone formation and resorption. There is also evidence for an indirect effect through the action of aluminium on parathyroid hormone synthesis and by its modulation of calcium activity. We discuss commonly used techniques for identifying aluminium load and review studies of chelation therapy as a method to lower aluminium load. It is apparent that aluminium overload has serious consequences for patients with chronic renal failure, yet this problem can be largely prevented by the use of aluminium-free phosphate binders. The deleterious effects on bone remodelling caused by chronic exposure to aluminium suggest that caution should be observed when using other metals as phosphate binders.

Cinacalcet is an effective treatment for secondary hyperparathyroidism in patients on dialysis. Until now, no randomized, placebo-controlled, long-term trial has tested this drug in individuals with chronic kidney disease who are not receiving dialysis.

Previous studies have shown a different effect of aluminum (Al) on bone metabolism in animals with chronic renal failure and conversely, positive osteogenic effects in animals with normal renal function. The aim of this study was to evaluate the effect of aluminum on bone metabolism in osteopenic rats. We studied male Wistar rats with severe osteopenia induced by adding NH4Cl (2%) to the drinking water over a 6-month period. The rats were divided into two groups and followed for 4 months. The Aluminum group (G1) received AlC13 intraperitoneally (10 mg/kg/5 days/week) (n = 8); the Control group (G2) did not receive any treatment after stopping the administration of NH4Cl (n = 5). In all animals we measured biochemical markers (serum Ca, P, Cr, Al, osteocalcin, hydroxyproline) as well as bone mineral density and bone histomorphometry (BV/TV, CTh, ObS/BS, OTh, and NOc/TV). Bone aluminum content, measured by atomic absorption spectrometry, was 101.6 +/- 13 microg/g in the Al overloaded group and 1.31 +/- 0.14 in controls. Bone mineral density, evaluated by dual X-ray absorptiometry (DXA) at the proximal extremity of the tibia was significantly higher in G1 (0.292 +/- 0.01 g/cm2 versus 0. 267 +/- 0.02 g/cm2). No significant differences were found between the biochemical markers. In the histomorphometric parameters we observed significant differences in G1 compared with G2: an increase in BV/TV (18.59 +/- 5.6 versus 7.69 +/- 3.08%) and in CTh (0.52 +/- 0.06 versus 0.36 +/- 0.07 mm) with a moderate increment of the osteoid thickness (14.05 +/- 4.72 versus 5.25 +/- 0.9 microm) (P0. 05). Changes in others parameters and the relationship between biochemical parameters of bone remodeling, Al, and histology were analyzed. These findings indicate that in rats with normal renal function, Al is able to induce bone formation even when osteopenia is present.

Mechanisms of aluminum-induced microcytosis: Lessons from accidental aluminum intoxication. Twenty-three hemodialysis patients exposed to an accidental aluminum overload, showed increased erythropoietin requirements and decreased erythrocyte mean corpuscular volume (MCV). At the peak of the intoxication, MCV and plasma aluminum levels changed from unrelated (r = 0.02) to strongly related (r = 0.425) variables. The molar proportion of plasma aluminum to plasma iron increased dramatically (from 1:13.8 to 1:2.4). This significant increment in the aluminum/iron ratio made higher the relative offer of aluminum with respect to iron to the erythroid precursor cells. Accordingly, in a subset of 13 randomly selected aluminum-intoxicated patients we found increased intraerythrocytic aluminum, which paralleled the increase in plasma aluminum. Furthermore, in the aluminum-intoxicated group, intraerythrocytic ferritin, a marker of iron content, and the ratio between erythrocyte and plasma ferritin were lower (P < 0.01 and

Role of iron metabolism in absorption and cellular uptake of aluminum. The effect of iron status on aluminum (Al) absorption was investigated in this study in vivo using an animal model and in vitro using an intestinal mucosal cell line. In the in vivo model rats were rendered iron overloaded by intraperitoneal injection of iron dextran (5 mg/48 hr) or iron deficient by phlebotomy (2.5 to 3 ml blood/week). These rats, and normal controls, were then dosed with Al(OH) 3 (40 mg/day) for 30 days. Urinary excretion of Al was significantly greater in the iron deficient group than in the other two groups throughout the study period, and brain Al at the end of the experiment was significantly increased in the iron depleted group (1.93 µg/g) and decreased in the iron overloaded group (0.73 µg/g) compared with controls (1.42 µg/g). The brain Al levels in iron overloaded rats were no higher than those in normal rats that had not been dosed with Al(OH) 3 (0.61 Mg/g). No significant differences were found in serum Al levels. In the in vitro experiments cultures of the rat intestinal cell line RIE1 were iron overloaded by addition of iron nitrilotriacetate (0.1mM) or iron depleted with desfer-rioxamine (5 µg/ml) for 20 days prior to pulsing with Al transferrin (0.5 mg/ml) for 24 hours. Uptake of Al was significantly greater in the iron depleted cells (2.3 ng/µg cell DNA) than in iron overloaded (0.81 ng) or untreated (0.83 µg) cells. These studies show that iron depletion markedly increases absorption and cellular uptake and suggest that susceptible individuals, such as renal failure patients, run an increased risk of toxicity if they are iron deficient.

Throughout recent years, one of the most dynamic and quickly growing areas in the field of nephrology has been renal osteodystrophy. Despite the fact that bone metabolic disorders have always been one of the most important complications associated with chronic renal failure, they have, until recently, only been linked to morbidity in chronic renal failure patients, and never to mortality.

HPLC was used to study the effectiveness of two different desferrioxamine (DFO) administration strategies (15 mg/kg DFO, 1 h or 44 h before dialysis) on generation of aluminoxamine and ferrioxamine in five hemodialysis patients. The percentage of ultrafilterable aluminum and iron in these patients was also investigated by electrothermal atomic absorption spectrometry. The administration of DFO in both schemes increased the ultrafilterable serum aluminum concentrations from a mean of 17.1 ± 1.6% to a mean of 75.7 ± 14.1%. However, 1 h after DFO infusion, only 38.8 ± 7.7% of the total serum aluminum was bound to DFO; 44 h after DFO infusion, only 15.8 ± 8.0% was bound. Similar results were obtained for ferrioxamine. These results suggest that the ultrafilterable serum fraction contains aluminum and iron chelated by DFO and by DFO metabolites, which retain similar metal-chelating abilities.