Calcification Propensity Research Articles

Propensity to calcification as a pathway to renal hypoxia in chronic kidney disease and in hypertension.

Propensity to calcification as a pathway to renal hypoxia in chronic kidney disease and in hypertension.

Serum calcification propensity is associated with renal tissue oxygenation and resistive index in patients with arterial hypertension or chronic kidney disease.

Arterial calcifications increase arterial stiffness and are associated with a faster decline of kidney function in patients with arterial hypertension (AH) and/or chronic kidney disease (CKD). Yet the underlying mechanisms linking arterial calcifications, vascular stiffness and renal function decline are incompletely understood. A novel in-vitro blood test evaluates the propensity of patient's serum to prevent the formation of calcifications by measuring the maturation time of calciprotein particles (CPP) [transformation time of amorphous calcium phosphate-containing primary CPP to crystalline hydroxyapatite-containing secondary CPP (T50)]. We hypothesized that a high arterial stiffness and a high propensity to calcify may be associated with high renal vascular resistance and low renal tissue oxygenation. T50 was measured in patients with AH and a preserved renal function, in CKD patients and in healthy controls, a lower T50 indicating a higher risk of calcification. Pulse wave velocity (PWV) was assessed as a measure of arterial stiffness, and renal resistive index was measured by renal Doppler ultrasound. Renal tissue oxygenation was measured by blood oxygenation level-dependent MRI using the mean R2 values of the cortex, the medulla and layers of renal parenchyma. A high R2 value corresponds to a low tissue oxygenation. Mean T50 was 246 ± 129 min in 58 CKD patients, 275 ± 111 min in 48 AH patients and 324 ± 96 min in 39 healthy controls (Panova = 0.008). In multivariable adjusted linear regression analysis, serum T50 correlated negatively with circulating calcium and phosphate levels, mean cortical and medullary R2, PWV, renal resistive index and being hypertensive. PWV was positively associated with R2 levels of outer and inner layers of renal parenchyma. The current study shows that hypertensive patients with preserved renal function as well as CKD patients have a higher risk of calcification than controls. High arterial stiffness and calcification propensity are linked to low renal tissue oxygenation and perfusion in hypertensive and CKD patients. These results provide new insights on the relationships among arterial stiffness, renal tissue oxygenation and the risk of developing CKD.

Renal Tumors of Childhood: Radiologic-Pathologic Correlation Part 2. The 2nd Decade: From the Radiologic Pathology Archives.

Malignant renal tumors account for 7% of childhood cancers, and Wilms tumors are by far the most common-but not in older children and adolescents. Among individuals in the latter half of their 2nd decade of life, renal cell carcinoma (RCC) is more common than Wilms tumor. The histopathologic spectrum of RCCs in children differs from that in adults. The most common subtype of RCC in children and adolescents is Xp11.2 translocation RCC, which is distinguished by hyperattenuation at nonenhanced computed tomography, a defined capsule, and associated retroperitoneal lymphadenopathy. Papillary RCC is the second most common histologic subtype. It enhances less intensely compared with the adjacent renal parenchyma and has a propensity for calcification. Clear cell RCC is seen in patients with von Hippel-Lindau disease and is distinguished by its relatively hypervascular nature. Medullary carcinoma affects adolescents with the sickle cell trait and is characterized by an infiltrative growth pattern and extensive metastasis at presentation. Angiomyolipoma is seen in children with tuberous sclerosis complex and is often multifocal and hypervascular, with macroscopic fat. Metanephric tumors are central, circumscribed, and typically calcified. Lymphoma usually manifests as multifocal masses, but it may involve a solitary mass or infiltrative pattern. Extensive adenopathy and involvement of the gastrointestinal tract or other organs also may be seen. Primitive neuroectodermal tumor is an aggressive neoplasm that is typically quite large at diagnosis. Knowledge of the clinical, biologic, and histopathologic features of renal tumors in older children and adolescents and their effects on the imaging appearance can help the radiologist offer a useful preoperative differential diagnosis.

Is there a role for newer biomarkers in chronic kidney disease-mineral and bone disorder management?

The current management of Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) relies largely on clinical judgement and assessment of biochemical parameters including serum calcium, phosphate and intact parathyroid hormone concentrations. In the past two decades, there has been a leap in the understanding of the pathophysiology of CKD-MBD, leading to the discovery of novel biomarkers. The potential utility of these markers in this clinical setting is an area of intense investigation. In the absence of any guidelines aiding the clinician's understanding and application of these markers, we summarise the current available literature surrounding fibroblast growth factor-23, α-Klotho, sclerostin and serum calcification propensity testing and their respective assays in the context of CKD-MBD management.

Oral Magnesium Supplementation in Chronic Kidney Disease Stages 3 and 4: Efficacy, Safety, and Effect on Serum Calcification Propensity—A Prospective Randomized Double-Blinded Placebo-Controlled Clinical Trial

IntroductionChronic kidney disease (CKD) is associated with high cardiovascular morbidity and mortality. Recent evidence suggests that increases in both serum and intracellular magnesium (Mg) can slow or even prevent the development of vascular calcification seen in CKD. Serum calcification propensity (T50) is a novel functional test, which is associated with all-cause mortality in CKD and measures the ability of serum to delay the formation of crystalline nanoparticles. Theoretically, increasing serum Mg should improve T50 and thereby reduce the propensity towards ectopic calcification.MethodsWe conducted a randomized placebo-controlled double-blinded clinical trial to investigate the safety of 2 different doses of oral Mg supplementation in subjects with CKD stages 3 and 4 as well as their effects on intracellular Mg and T50. Thirty-six subjects with CKD stages 3 and 4 were randomized to one of 3 groups (placebo, elemental Mg 15 mmol/d or elemental Mg 30 mmol/d) given as slow-release Mg hydroxide and followed for 8 weeks.ResultsThirty-four subjects completed the trial. Intracellular Mg remained stable throughout the trial despite significant increases in both serum and urine Mg. T50 increased significantly by 40 min from 256 ± 60 (mean ± SD) to 296 ± 64 minutes (95% confidence interval, 11–70, P < 0.05) in the Mg 30 mmol/d group after 8 weeks. No serious adverse events related to the study medication were reported during the study.DiscussionOral Mg supplementation was safe and well tolerated in CKD stages 3 and 4 and improved T50, but did not increase intracellular Mg. Further studies are needed to investigate the long-term effects of Mg supplementation in CKD stage 3 and 4 and whether improvement in calcification propensity is related to clinical endpoints.

Phosphocalcic Markers and Calcification Propensity for Assessment of Interstitial Fibrosis and Vascular Lesions in Kidney Allograft Recipients.

Renal interstitial fibrosis and arterial lesions predict loss of function in chronic kidney disease. Noninvasive estimation of interstitial fibrosis and vascular lesions is currently not available. The aim of the study was to determine whether phosphocalcic markers are associated with, and can predict, renal chronic histological changes. We included 129 kidney allograft recipients with an available transplant biopsy in a retrospective study. We analyzed the associations and predictive values of phosphocalcic markers and serum calcification propensity (T50) for chronic histological changes (interstitial fibrosis and vascular lesions). PTH, T50 and vitamin D levels were independently associated to interstitial fibrosis. PTH elevation was associated with increasing interstitial fibrosis severity (r = 0.29, p = 0.001), while T50 and vitamin D were protective (r = -0.20, p = 0.025 and r = -0.23, p = 0.009 respectively). On the contrary, fibroblast growth factor 23 (FGF23) and Klotho correlated only modestly with interstitial fibrosis (p = 0.045) whereas calcium and phosphate did not. PTH, vitamin D and T50 were predictors of extensive fibrosis (AUC: 0.73, 0.72 and 0.68 respectively), but did not add to renal function prediction. PTH, FGF23 and T50 were modestly predictive of low fibrosis (AUC: 0.63, 0.63 and 0.61) but did not add to renal function prediction. T50 decreased with increasing arterial lesions (r = -0.21, p = 0.038). The discriminative performance of T50 in predicting significant vascular lesions was modest (AUC 0.61). In summary, we demonstrated that PTH, vitamin D and T50 are associated to interstitial fibrosis and vascular lesions in kidney allograft recipients independently of renal function. Despite these associations, mineral metabolism indices do not show superiority or additive value to fibrosis prediction by eGFR and proteinuria in kidney allograft recipients, except for vascular lesions where T50 could be of relevance.

Blood Calcification Propensity, Cardiovascular Events, and Survival in Patients Receiving Hemodialysis in the EVOLVE Trial.

Patients receiving hemodialysis are at risk of cardiovascular events. A novel blood test (T50 test) determines the individual calcification propensity of blood. T50 was determined in 2785 baseline serum samples of patients receiving hemodialysis enrolled in the Evaluation of Cinacalcet Therapy to Lower Cardiovascular Events (EVOLVE) trial and the T50 results were related to patient outcomes. Serum albumin, bicarbonate, HDL cholesterol, and creatinine were the main factors positively/directly and phosphate was the main factor negatively/inversely associated with T50. The primary composite end point (all-cause mortality, myocardial infarction [MI], hospitalization for unstable angina, heart failure, or peripheral vascular event [PVE]) was reached in 1350 patients after a median follow-up time of 619 days. After adjustments for confounding, a lower T50 was independently associated with a higher risk of the primary composite end point as a continuous measure (hazard ratio [HR] per 1 SD lower T50, 1.15; 95% confidence interval [95% CI], 1.08 to 1.22; P<0.001). Furthermore, lower T50 was associated with a higher risk in all-cause mortality (HR per 1 SD lower T50, 1.10; 95% CI, 1.02 to 1.17; P=0.001), MI (HR per 1 SD lower T50, 1.38; 95% CI, 1.19 to 1.60; P<0.001), and PVE (HR per 1 SD lower T50, 1.22; 95% CI, 1.05 to 1.42; P=0.01). T50 improved risk prediction (integrated discrimination improvement and net reclassification improvement, P<0.001 and P=0.001) of the primary composite end point. Blood calcification propensity was independently associated with the primary composite end point, all-cause mortality, MI, and PVE in the EVOLVE study and improved risk prediction. Prospective trials should clarify whether T50-guided therapies improve outcomes.

Novel assessments of systemic calcification propensity.

Blood is a biological fluid, which controls the precipitation of calcium and phosphate and transports mineral debris. This review presents and discusses the current concepts and novel assessment methods of systemic calcification propensity in blood. Calcium and phosphate combine with calcification-inhibiting proteins, mainly fetuin-A, to form amorphous calcium phosphate-containing primary calciprotein particles (CPPs). These nanosized mineral-protein clusters undergo spontaneous transformation to secondary CPP, which contain crystalline calcium phosphate. Two recently developed methods assess complementary aspects of the calcification propensity of serum. The CPP-fetuin-A method determines the amount of sedimentable fetuin-A, whereas the T50-Test determines the transformation time point T50 from amorphous to crystalline CPPs in artificially supersaturated serum.Clinical studies in renal patients have already demonstrated close associations of the CPP-fetuin-A method with all-cause mortality, severity of coronary calcification and aortic stiffness, and of the T50-Test with cardiovascular and all-cause mortality, renal graft failure and aortic stiffening. Systemic calcification propensity can be assessed by two novel methods providing complementary information about the status and performance of the humoral calcification-regulating system in serum. These tests may help guide better patient care in the future with the use of more individualized therapies.

Serum Calcification Propensity Is a Strong and Independent Determinant of Cardiac and All-Cause Mortality in Kidney Transplant Recipients

Calcification of the vasculature is associated with cardiovascular disease and death in kidney transplant recipients. A novel functional blood test measures calcification propensity by quantifying the transformation time (T50 ) from primary to secondary calciprotein particles. Accelerated T50 indicates a diminished ability of serum to resist calcification. We measured T50 in 1435 patients 10 weeks after kidney transplantation during 2000-2003 (first era) and 2009-2012 (second era). Aortic pulse wave velocity (APWV) was measured at week 10 and after 1 year in 589 patients from the second era. Accelerated T50 was associated with diabetes, deceased donor, first transplant, rejection, stronger immunosuppression, first era, higher serum phosphate and lower albumin. T50 was not associated with progression of APWV. During a median follow-up of 5.1 years, 283 patients died, 70 from myocardial infarction, cardiac failure or sudden death. In Cox regression models, accelerated T50 was strongly and independently associated with both all-cause and cardiac mortality, low versus high T50 quartile: hazard ratio 1.60 (95% confidence interval [CI] 1.00-2.57), ptrend = 0.03, and 3.60 (95% CI 1.10-11.83), ptrend = 0.02, respectively. In conclusion, calcification propensity (T50 ) was strongly associated with all-cause and cardiac mortality of kidney transplant recipients, potentially via a cardiac nonAPWV-related pathway. Whether therapeutic improvement of T50 improves outcome awaits clarification in a randomized trial.

Acute and 3-month effects of calcium carbonate on the calcification propensity of serum and regulators of vascular calcification: secondary analysis of a randomized controlled trial.

Calcium supplements have been associated with increased cardiovascular risk, but the mechanism is unknown. We investigated the effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50). Changes in serum calcium were related to changes in T50. Calcium supplements have been associated with increased cardiovascular risk; however, it is unknown whether this is related to an increase in vascular calcification. We investigated the acute and 3-month effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50), and on three possible regulators of calcification: fetuin-A, pyrophosphate and fibroblast growth factor-23 (FGF23). We randomized 41 postmenopausal women to 1 g/day of calcium as carbonate, or to a placebo containing no calcium. Measurements were performed at baseline and then 4 and 8 h after their first dose, and after 3 months of supplementation. Fetuin-A, pyrophosphate and FGF23 were measured in the first 10 participants allocated to calcium carbonate and placebo who completed the study. T50 declined in both groups, the changes tending to be greater in the calcium group. Pyrophosphate declined from baseline in the placebo group at 4 h and was different from the calcium group at this time point (p = 0.04). There were no other significant between-groups differences. The changes in serum total calcium from baseline were significantly related to changes in T50 at 4 h (r = -0.32, p = 0.05) and 8 h (r = -0.39, p = 0.01), to fetuin-A at 3 months (r = 0.57, p = 0.01) and to pyrophosphate at 4 h (r = 0.61, p = 0.02). These correlative findings suggest that serum calcium concentrations modulate the propensity of serum to calcify (T50), and possibly produce counter-regulatory changes in pyrophosphate and fetuin-A. This provides a possible mechanism by which calcium supplements might influence vascular calcification.

Cellular Changes during Renal Failure-Induced Inflammatory Aortic Valve Disease.

BackgroundAortic valve calcification (AVC) secondary to renal failure (RF) is an inflammation-regulated process, but its pathogenesis remains unknown. We sought to assess the cellular processes that are involved in the early phases of aortic valve disease using a unique animal model of RF-associated AVC.MethodsAortic valves were obtained from rats that were fed a uremia-inducing diet exclusively for 2, 3, 4, 5, and 6 weeks as well as from controls. Pathological examination of the valves included histological characterization, von Kossa staining, and antigen expression analyses.ResultsAfter 2 weeks, we noted a significant increase in urea and creatinine levels, reflecting RF. RF parameters exacerbated until the Week 5 and plateaued. Whereas no histological changes or calcification was observed in the valves of any study group, macrophage accumulation became apparent as early as 2 weeks after the diet was started and rose after 3 weeks. By western blot, osteoblast markers were expressed after 2 weeks on the diet and decreased after 6 weeks. Collagen 3 was up-regulated after 3 weeks, plateauing at 4 weeks, whereas collagen 1 levels peaked at 2 and 4 weeks. Fibronectin levels increased gradually until Week 5 and decreased at 6 weeks. We observed early activation of the ERK pathway, whereas other pathways remained unchanged.ConclusionsWe concluded that RF induces dramatic changes at the cellular level, including macrophage accumulation, activation of cell signaling pathway and extracellular matrix modification. These changes precede valve calcification and may increase propensity for calcification, and have to be investigated further.

Living kidney donation does not adversely affect serum calcification propensity and markers of vascular stiffness.

Living kidney donors (LKDs) experience a decline in glomerular filtration rate (GFR) after donation. Calcification propensity (T50 ) can be determined by a blood test predicting all-cause mortality in patients with chronic kidney disease. We studied the impact of kidney donation on T50 and markers of arterial stiffness. We analyzed T50 prospectively before and 1year after kidney donation in 21 LKDs along with fetuin-A, mineral metabolism markers, kidney length, pulse wave velocity (PWV), augmentation index (AI), and renal resistive index (RRI) as markers of arterial stiffness. We studied the impact of kidney donation on these parameters. LKDs were 54±10years old and had a GFR of 101±18ml/min/1.73m(2) before donation, decreasing to 67±8ml/min/1.73m(2) after donation (P<0.001). Despite this, T50 improved after donation (290±53 to 312±38min, P=0.049). This change was inversely related to plasma phosphate (P=0.03), which declined after donation (P=0.002). Fetuin-A levels increased after donation (P=0.01). Upon donation, the length of the remaining kidney increased (P<0.001) while PWV, AI, and RRI remained unchanged. Calcification propensity was not adversely affected by kidney donation. This indicates that T50 is independent from GFR in LKDs and that kidney donation does neither worsen calcification propensity nor markers of vascular stiffness at 1year.

Calcification Propensity and Survival among Renal Transplant Recipients.

Calciprotein particle maturation time (T50) in serum is a novel measure of individual blood calcification propensity. To determine the clinical relevance of T50 in renal transplantation, baseline serum T50 was measured in a longitudinal cohort of 699 stable renal transplant recipients and the associations of T50 with mortality and graft failure were analyzed over a median follow-up of 3.1 years. Predictive value of T50 was assessed for patient survival with reference to traditional (Framingham) risk factors and the calcium-phosphate product. Serum magnesium, bicarbonate, albumin, and phosphate levels were the main determinants of T50, which was independent of renal function and dialysis vintage before transplant. During follow-up, 81 (12%) patients died, of which 38 (47%) died from cardiovascular causes. Furthermore, 45 (6%) patients developed graft failure. In fully adjusted models, lower T50 values were independently associated with increased all-cause mortality (hazard ratio, 1.43; 95% confidence interval, 1.11 to 1.85; P=0.006 per SD decrease) and increased cardiovascular mortality (hazard ratio, 1.55; 95% confidence interval, 1.04 to 2.29; P=0.03 per SD decrease). In addition to age, sex, and eGFR, T50 improved prognostication for all-cause mortality, whereas traditional risk factors or calcium-phosphate product did not. Lower T50 was also associated with increased graft failure risk. The associations of T50 with mortality and graft failure were confirmed in an independent replication cohort. In conclusion, reduced serum T50 was associated with increased risk of all-cause mortality, cardiovascular mortality, and graft failure and, of all tested parameters, displayed the strongest association with all-cause mortality in these transplant recipients.

Calcification propensity associated with renal graft failure

Calcification propensity associated with renal graft failure

Serum Calcification Propensity Predicts All-Cause Mortality in Predialysis CKD

Medial arterial calcification is accelerated in patients with CKD and strongly associated with increased arterial rigidity and cardiovascular mortality. Recently, a novel in vitro blood test that provides an overall measure of calcification propensity by monitoring the maturation time (T50) of calciprotein particles in serum was described. We used this test to measure serum T50 in a prospective cohort of 184 patients with stages 3 and 4 CKD, with a median of 5.3 years of follow-up. At baseline, the major determinants of serum calcification propensity included higher serum phosphate, ionized calcium, increased bone osteoclastic activity, and lower free fetuin-A, plasma pyrophosphate, and albumin concentrations, which accounted for 49% of the variation in this parameter. Increased serum calcification propensity at baseline independently associated with aortic pulse wave velocity in the complete cohort and progressive aortic stiffening over 30 months in a subgroup of 93 patients. After adjustment for demographic, renal, cardiovascular, and biochemical covariates, including serum phosphate, risk of death among patients in the lowest T50 tertile was more than two times the risk among patients in the highest T50 tertile (adjusted hazard ratio, 2.2; 95% confidence interval, 1.1 to 5.4; P=0.04). This effect was lost, however, after additional adjustment for aortic stiffness, suggesting a shared causal pathway. Longitudinally, serum calcification propensity measurements remained temporally stable (intraclass correlation=0.81). These results suggest that serum T50 may be helpful as a biomarker in designing methods to improve defenses against vascular calcification.

A tool to measure the propensity for calcification in serum

A tool to measure the propensity for calcification in serum

Nanoparticle-Based Test Measures Overall Propensity for Calcification in Serum

Vascular and soft tissue calcification contributes to cardiovascular morbidity and mortality in both the general population and CKD. Because calcium and phosphate serum concentrations are near supersaturation, the balance of inhibitors and promoters critically influences the development of calcification. An assay that measures the overall propensity for calcification to occur in serum may have clinical use. Here, we describe a nanoparticle-based assay that detects, in the presence of artificially elevated calcium and phosphate concentrations, the spontaneous transformation of spherical colloidal primary calciprotein particles (CPPs) to elongate crystalline secondary CPPs. We used characteristics of this transition to describe the intrinsic capacity of serum to inhibit the precipitation of calcium and phosphate. Using this assay, we found that both the sera of mice deficient in fetuin-A, a serum protein that inhibits calcification, and the sera of patients on hemodialysis have reduced intrinsic properties to inhibit calcification. In summary, we developed a nanoparticle-based test that measures the overall propensity for calcification in serum. The clinical use of the test requires evaluation in a prospective study.

Glutaraldehyde treatment elicits toxic response compared to decellularization in bovine pericardium

Glutaraldehyde-stabilized bovine pericardium is used for clinical application since 1970s because of its desirable features such as less immunogenicity and acceptable durability. However, a propensity for calcification is reported on account of glutaraldehyde treatment. In this study, commercially available glutaraldehyde cross-linked bovine pericardium was evaluated for its in vitro cytotoxic effect, macrophage activation, and in vivo toxic response in comparison to decellularized bovine pericardium. Glutaraldehyde-treated bovine pericardium and its extract were observed to be cytotoxic and it also caused significant inflammatory cytokine release from activated macrophages. Significant antibody response, calcification response, necrotic, and inflammatory response were noticed in glutaraldehyde-treated bovine pericardium in comparison to decellularized bovine pericardium in a rat subcutaneous implantation model. Glutaraldehyde-treated bovine pericardium also failed in acute systemic toxicity testing and intracutaneous irritation testing as per ISO 10993. With respect to healing and implant remodeling, total lack of host tissue incorporation and angiogenesis was noticed in glutaraldehyde-treated bovine pericardium compared to excellent host fibroblast incorporation and angiogenesis within the implant in decellularized bovine pericardium. In conclusion, using in vitro and in vivo techniques, this study has demonstrated that glutaraldehyde-treated bovine pericardium elicits toxic response compared to decellularized bovine pericardium which is not congenial for long-term implant performance.

Short duration gluteraldehyde cross linking of decellularized bovine pericardium improves biological response

Gluteraldehyde stabilized bovine pericardium is used for clinical application since 1970s because of its desirable features such as less immunogenicity and acceptable durability. However, a propensity for calcification and long term implant failure is reported because of gluteraldehyde treatment. There is also failure of implant to integrate into host tissue because of its resistance to tissue remodeling. Decellularized bovine pericardium, a potential alternative allows tissue remodeling but it has problems such as immunogenicity and chronic inflammatory response. In this study, decellularized bovine pericardium was subjected to short duration, low concentration gluteraldehyde cross-linking at two levels and its biological response (both in vitro and in vivo) was compared with un-crosslinked decellularized bovine pericardium and fully crosslinked normal bovine pericardium. It was observed that both un-crosslinked and partially crosslinked decellularized bovine pericardium to be non-cytotoxic and it caused significantly less inflammatory cytokine release such as TNF alpha and IL1beta from activated macrophages. Among all groups, short duration 0.2% Gluteraldehyde treated decellularized bovine pericardium showed significantly less antibody response and inflammatory response compared to un-crosslinked decellularized pericardium, short duration 0.6% gluteraldehyde treated decellularized bovine pericardium or completely cross linked bovine pericardium in juvenile rat subcutaneous implantation model. Moreover, short duration 0.2% gluteraldehyde crosslinked decellularized bovine pericardium showed minimum calcification, better host fibroblast incorporation, new collagen deposition and angiogenesis within the implant. These attributes may finally lead to better implant remodeling and sustained implant function during clinical use.

Fully phosphorylated fetuin-A forms a mineral complex in the serum of rats with adenine-induced renal failure

The serum glycoprotein fetuin-A is an important inhibitor of extra-osseous calcification, but correlations between serum fetuin-A levels and the extent of vascular calcification are controversial. In this study, we used a rat model of adenine-induced renal failure with secondary hyperparathyroidism that exhibits all characteristic features of patients with chronic kidney disease. These rats had medial vascular calcification along with reduced levels of both serum and hepatic fetuin-A. Treatment with an inhibitor of ectopic calcification, alendronate, decreased bone turnover and eliminated completely the vascular calcification in this rat model, but there was no change in the levels of hepatic and serum fetuin-A. Centrifugation of the serum of untreated rats with renal failure gave a small precipitate composed of fetuin-A, calcium, magnesium, and phosphate; this complex, absent from normal rat serum, was not found in the serum of alendronate-treated rats with renal failure. Rat serum contained three types of phosphorylated fetuin-A, as well as unphosphorylated forms, but only the fully phosphorylated fetuin-A was present in the mineral complex. The amount of this complex reflected the risk of mineral precipitation. Our results suggest that the measurement of serum fetuin-mineral complex rather than fetuin-A alone might provide a better indication of extra-osseous calcification propensity.