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Osteopontin ( OPN ) and fetuin-A immunohistochemistry on decalcified human cortical bone. a Immunolabeling for OPN in bone shows intense staining at cement lines at the interfaces between osteons ( arrows ) and weak to moderate staining throughout the lamellar bone. b Immunolabeling for fetuin-A shows strong staining at cement lines ( arrows ) and throughout the lamellar bone ( asterisk )
Source publication
Clinical nephrologists are well aware of the consequences of pathologic mineralization (calcification). Several studies have found a strong association between vascular and valvular mineralization and advanced or end-stage chronic kidney disease (CKD), with shorter survival times and increased morbidity. In the cardiology community, until quite rec...
Similar publications
Vascular calcification, which contributes to cardiovascular disease in patients with uremic hyperphosphatemia, is associated with vascular cell expression of osteogenic genes, including bone morphogenetic protein (BMP)-2 and osteopontin (OPN). High inorganic phosphate levels in vitro stimulate the osteogenic conversion of smooth muscle cells; howev...
Citations
... Based on this obtained from RP biopsies may help us create in vitro models of stone pathogenesis and/or animal models that mimic the human physiologic condition. We know that pathologic calcification of certain soft tissues can be reversed through mineral dissolution and phagocytosis [44], so perhaps the same may be true for plaque formation and their associated kidney stones. Future studies are also needed to further elucidate the role of laser-based removal of RP papillary tissue and how this may prevent the formation of future stones. ...
Alexander Randall first published renal papillary tip findings from stone formers in 1937, paving the way for endoscopic assessment to study stone pathogenesis. We performed a literature search to evaluate the safety of papillary tip biopsy and clinical insights gained from modern renal papillary investigations. A search on the topic of renal papillary biopsy provided an overview of Randall’s plaques (RP), classification systems for renal papillary grading, and a summary of procedure type, complications, and outcomes. Within 26 identified manuscripts, 660 individuals underwent papillary tip biopsy percutaneously (n = 562), endoscopically (n = 37), or unspecified (n = 23). Post-operative hemoglobin changes were similar to controls. One individual (0.2%) reported fever > 38°, and long-term mean serum creatinine post-biopsy (n = 32) was unchanged. Biopsies during ureteroscopy or PCNL added ~20–30 min of procedure time. Compared to controls, papillary plaque-containing tissue had upregulation in pro-inflammatory genes, immune cells, and cellular apoptosis. Urinary calcium and papillary plaque coverage were found to differ between RP and non-RP stone formers, suggesting differing underlying pathophysiology for these groups. Two renal papillary scoring systems have been externally validated and are used to classify stone formers. Overall, this review shows that renal papillary biopsies have a low complication profile with high potential for further research. Systematic adaption of a papillary grading scale, newer tissue analysis techniques, and the development of animal models of Randall’s plaque may allow further exploration of plaque pathogenesis and identify targets for prevention therapies in patients with nephrolithiasis.
... Current treatments for the removal of calcified plaque include mechanical removal using rotary blade ablation or chemically dissolving in situ with acid via a catheter device [199][200][201] . In addition to mechanical and chemical debridement, reduced mineral ion intake from the diet or ions from bone stores have also been considered potential treatments for pathological/ectopic calcification in clinical practices [201] . ...
Biomineralization is a process that leads to the formation of hierarchically arranged structures in mineralized tissues, such as bone and teeth. Extensive research has been conducted on the crystals in bones and teeth, with the aim of understanding the underlying mechanisms of the mineralization process. Pathological/ectopic mineralization, such as kidney stones, calcific tendinitis, and skeletal fluorosis, shares some similar features but different mechanisms to physiological mineralization. A better understanding will provide new perspectives for treating pathological/ectopic mineralization-related diseases. This review provides an overview of the mechanisms of the crystallization and growth of crystals in physiological and pathological conditions from a chemistry perspective. By linking the microstructures and functions of crystals formed in both conditions, potential approaches are proposed to treat pathological/ectopic mineralization-related diseases.
... This biomineralization, namely vascular ossification process [4] is strongly regulated by promoters and inhibitors of calcification. In human blood, the formation of so called primary calciprotein particles (CPPs) prevents the precipitation of supersaturated calcium and phosphate [5][6][7][8]. These CPPs mainly consist of non-crystalline calcium and phosphate, bound to fetuin-A and other proteins, and are part of the mineral buffering system inherent in blood [8]. ...
Background
Vascular calcification is a major contributor to the high cardiac burden among hemodialysis patients. A novel in vitro T50-test, which determines calcification propensity of human serum, may identify patients at high risk for cardiovascular (CV) disease and mortality. We evaluated whether T50 predicts mortality and hospitalizations among an unselected cohort of hemodialysis patients.
Methods
This prospective clinical study included 776 incident and prevalent hemodialysis patients from 8 dialysis centers in Spain. T50 and fetuin-A were determined at Calciscon AG, all other clinical data were retrieved from the European Clinical Database. After their baseline T50 measurement, patients were followed for two years for the occurrence of all-cause mortality, CV-related mortality, all-cause and CV-related hospitalizations. Outcome assessment was performed with proportional subdistribution hazards regression modelling.
Results
Patients who died during follow-up had a significantly lower T50 at baseline as compared to those who survived (269.6 vs. 287.7 min, p = 0.001). A cross-validated model (mean c statistic: 0.5767) identified T50 as a linear predictor of all-cause-mortality (subdistribution hazard ratio (per min): 0.9957, 95% CI [0.9933;0.9981]). T50 remained significant after inclusion of known predictors. There was no evidence for prediction of CV-related outcomes, but for all-cause hospitalizations (mean c statistic: 0.5284).
Conclusion
T50 was identified as an independent predictor of all-cause mortality among an unselected cohort of hemodialysis patients. However, the additional predictive value of T50 added to known mortality predictors was limited. Future studies are needed to assess the predictive value of T50 for CV-related events in unselected hemodialysis patients.
... Dysregulated mineral metabolism, cell and tissue damage and lack of calcification inhibitors are prime causes of calcification. Proteins inhibiting calcification also called mineral chaperones avidly bind mineral and co-localize with established calcifications [8]. The mineral chaperone fetuin-A is particularly interesting in this regard because it binds and stabilizes calcium phosphate precursor phases as colloids, thus allowing their transport and removal mitigating the risk of mineral precipitation [9]. ...
Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable.
Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice.
Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity.
Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.
... It is thought that OPN's regulation of pathological calcification is related to several functions. 37 Firstly, it is an effective hydroxyapatite growth inhibitor by binding to crystal surfaces and, by this way blocking further addition of mineral ions to crystal growth sites. The combination of electronegative glutamic and aspartic acid residues, serine / threonine kinase substrate sites, and putative calcium binding motifs give OPN the ability to bind a significant amount of Ca + 2 (50 moles of calcium to 1 mol of osteopontin). ...
Background & Objective: The choroid plexus (CP) is a tissue plexus located in the ventricles that produces hormones and nutrients, primarily cerebrospinal fluid (CSF). CP is also an important component of the blood CSF barrier. Osteopontin (OPN) is an extracellular matrix protein that plays a role in various physiological and pathological conditions such as bone reshaping, wound healing, vascular disorders, and inflammatory diseases. It is considered that OPN physiologically regulate bio- mineralization in bone tissue and reduce the growth and accumulation of calcium crystals in epithelial tissues. In our study, the role of OPN in CP calcification was studied. Methods: A total of 90 people, without any disease but with CP calcification (45 people) and age and gender matched control without CP calcification (45 people) were studied. Calcified and normal CP tissue was identified in brain computed tomography (CT) images. Serum OPN levels were measured in individuals with and without CP calcification using a human OPN enzyme-linked immunosorbent test kit (ELISA) from morning fasting venous blood samples. Results: Serum OPN level was found to be statistically significantly lower in patients with CP calcification than those without CP calcification (p=0.007). Conclusions: Our results show that OPN may have a significant role in the calcification process of CP.
... Dysregulated mineral metabolism, cell and tissue damage and lack of calcification inhibitors are prime causes of calcification. Proteins inhibiting calcification also called mineral chaperones avidly bind mineral and co-localize with established calcifications [8]. The mineral chaperone fetuin-A is particularly interesting in this regard because it binds and stabilizes calcium phosphate precursor phases as colloids, thus allowing their transport and removal mitigating the risk of mineral precipitation [9]. ...
Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.
... In plasma, the most potent and abundant protein mineralisation regulator is the liver-derived glycoprotein fetuin-A 11 , which interacts with nascent colloidal mineral-protein complexes to form calciprotein particles (CPP) 12,13 . Analogous to the manner in which apolipoproteins solubilise their lipid cargo for transport, fetuin-A stabilises poorly soluble calcium phosphates, preventing crystal growth and precipitation, while facilitating their uptake in tissues for utilisation or clearance [14][15][16] . An inability to make or sufficiently stabilise CPP, as seen in fetuin-A knockout mice, results in one of the most severe phenotypes of ectopic calcification known 17 , where mineral-containing complexes precipitate directly in the lumen of the microvasculature leading to occlusion, ischaemia, necrosis and fibrosis 18 . ...
... Our findings are consistent with the notion that intestinal absorption of a dietary mineral load can directly lead to formation of circulating CPM, CPP-I and CPP-II. This has previously been proposed 10,15,16 , but based largely on animal data 33,48 . In contrast, evidence in humans has been indirect, coming from studies of intestinal phosphate binders in haemodialysis dependent CKD patients 34,37,55 . ...
Plasma approaches metastability with respect to its calcium and phosphate content, with only minor perturbations in ionic activity needed to sustain crystal growth once nucleated. Physiologically, calcium and phosphate are intermittently absorbed from the diet each day, yet plasma concentrations of these ions deviate minimally post-prandially. This implies the existence of a blood-borne mineral buffer system to sequester calcium phosphates and minimise the risk of deposition in the soft tissues. Calciprotein particles (CPP), endogenous mineral-protein colloids containing the plasma protein fetuin-A, may fulfill this function but definitive evidence linking dietary mineral loading with their formation is lacking. Here we demonstrate that CPP are formed as a normal physiological response to feeding in healthy adults and that this occurs despite minimal change in conventional serum mineral markers. Further, in individuals with Chronic Kidney Disease (CKD), in whom mineral handling is impaired, we show that both fasting and post-prandial levels of CPP precursors are markedly augmented and strongly inversely correlated with kidney function. This study highlights the important, but often neglected, contribution of colloidal biochemistry to mineral homeostasis and provides novel insight into the dysregulation of mineral metabolism in CKD.
... Extracellular polyphosphate (polyP i ) also participates in phosphates transport and compartmentalization. Poly-P i granules chelate calcium ions to form neutrally charged amorphous complexes [83,84]. Fetuin A is a systemic inhibitor of mineralization which prevents growth of nascent crystal nuclei in blood and facilitates its recycling by macrophages [85][86][87][88]. One fetuin is known to sequester 54-72 phosphate ions and 90-120 calcium ions. ...
Tissue-nonspecific alkaline phosphatase (TNAP) is a key enzyme responsible for skeletal tissue mineralization. It is involved in the dephosphorylation of various physiological substrates, and has vital physiological functions, including extra-skeletal functions, such as neuronal development, detoxification of lipopolysaccharide (LPS), an anti-inflammatory role, bile pH regulation, and the maintenance of the blood brain barrier (BBB). TNAP is also implicated in ectopic pathological calcification of soft tissues, especially the vasculature. Although it is the crucial enzyme in mineralization of skeletal and dental tissues, it is a logical clinical target to attenuate vascular calcification. Various tools and studies have been developed to inhibit its activity to arrest soft tissue mineralization. However, we should not neglect its other physiological functions prior to therapies targeting TNAP. Therefore, a better understanding into the mechanisms mediated by TNAP is needed for minimizing off targeted effects and aid in the betterment of various pathological scenarios. In this review, we have discussed the mechanism of mineralization and functions of TNAP beyond its primary role of hard tissue mineralization.
... Fetuin-A is a multifunctional glycoprotein produced by the liver. Through the formation of soluble colloidal troponin particles, it strongly inhibits vascular calcification in the circulatory system [4]. Fetuin-A, a soluble transforming growth factor β (TGF-β) antagonist, regulates cytokinedependent osteogenesis and fibrosis and inhibits cell proliferation [5]. ...
... Ketteler et al. also reported a lower level of fetuin-A in dialysis patients than in the general population, and it is related to all-cause and cardiovascular mortality [12]. Calcium and phosphorus overload and chronic microinflammation in patients with ESRD may lead to excessive consumption of fetuin-A and reduce its levels [4]. Moreover, Talib et al. observed a strong negative correlation between the level of fetuin-A and E/E′ [6]. ...
... The following mechanism may lead to LVDD: a low fetuin-A level in dialysis patients leads to increased left ventricular afterload and coronary artery insufficiency through vascular calcification, eventually causing left ventricular hypertrophy and thus, decreasing diastolic function [13][14][15]. Fetuin-A is also a soluble TGF-β antagonist [4], and continuously low levels of fetuin-A reduce the inhibitory effect of TGF-β, which indirectly increases myocardial fibrosis and leads to increased myocardial hypertrophy and stiffness. Excess calcium and phosphorus levels, which cause ectopic and soft tissue calcification, also promote the development of diastolic dysfunction [16,17]. ...
Objective
To identify the relationship between serum fetuin-A levels and left ventricular diastolic dysfunction (LVDD) among maintenance haemodialysis patients.
Methods
In a cross-sectional study, 75 dialysis patients with end-stage renal disease (ESRD) were recruited, and fetuin-A levels were detected using an enzyme-linked immunosorbent assay (ELISA). Echocardiography measurements were recorded according to the recommendations of the American Society of Echocardiography. The ratio of early diastolic transmitral inflow velocity (E) to early diastolic annular velocity (E′) was measured using tissue Doppler imaging and E/E′ > 15 was defined as diastolic dysfunction. The association of serum fetuin-A concentrations with echocardiographic parameters was analysed by calculating the bivariate linear correlation. A binary logistic regression analysis was conducted to determine the variables associated with LVDD.
Results
Compared to patients without diastolic dysfunction, patients with diastolic dysfunction were older, a higher percentage had a history of coronary artery disease, and presented with a high systolic pressure, high parathyroid hormone level, high N-terminal pro-brain natriuretic peptide (NT-proBNP) level, high LV mass index, high left atrium diameter, and low serum creatinine and fetuin-A levels. Serum fetuin-A levels showed a negative correlation with E/E′ (r = − 0.299, P = 0.009). Fetuin-A levels were considered an independent predictor of diastolic dysfunction.
Conclusion
A decrease in the serum fetuin-A level is associated with an increased risk of LVDD in patients on haemodialysis.
... Interestingly, the present study showed that the caudate nucleus also expressed several molecules with an antiosteogenic action, including osteopontin, CA-II, MGP, sclerostin, ISG15, ENPP1, and USP18 (20,39,46). Dense connective tissue or skeletal dysplasia is observed in patients with an inactivating mutation of several of these molecules, including sclerostin, klotho, MGP, ENPP1, and osteopontin (21,41,(47)(48)(49). ...
Context:
Basal-ganglia calcification (BGC) is common (70%) in patients with chronic-hypoparathyroidism. Interestingly, cortical-gray matter is spared from calcification. The mechanism of BGC, role of hyperphosphatemia and modulation of osteogenic-molecules by PTH in its pathogenesis is not clear.
Objective:
We assessed expression of large repertoire of molecules with pro or anti-osteogenic effects including neuroprogenitor-cells in caudate, dentate and cortical-gray matter from normal autopsy tissues. Effect of high-phosphate and PTH was assessed in an ex-vivo model of BGC using striatum tissue culture of Sprague-Dawley rat.
Methods:
The mRNA and protein expression of 39 molecules involved in multiple osteogenic pathways were assessed in 25 autopsy-tissues using RT-PCR, western-blot and immunofluorescence. The striatal-culture was maintained in hypoparathyroid milieu for 24-days with and without (a) high-phosphate (10 mm/β-glycerophosphate) and (b) PTH (1-34) (50 ng/ml DMEM-F12 media) for their effect on striatal-calcification and osteogenic-molecules.
Results:
Pro-calcification molecules (OSTEONECTIN, β-CATENIN, KLOTHO, FZD4, NT5E, LRP5, WNT3A, COLLAGEN 1-α, and SOX2-positive neuroprogenitor stem cells) had significantly higher expression in the caudate than gray-matter. Caudate-nuclei also had higher expression of anti-osteogenic-molecules (OSTEOPONTIN, CARBONIC ANHYDRASE-II, MGP, SCLEROSTIN, ISG15, ENPP1 and USP18). In ex-vivo model striatum-culture showed increased propensity for calcified nodules with mineral deposition similar to that of bone tissue on fourier-transform infrared spectroscopy, alizarin and Von-Kossa stain. Mineralization in striatal-culture was enhanced by high-phosphate and decreased by exogenous PTH through increased expression of CA-II.
Conclusion:
This study provides a conceptual advance on the molecular mechanisms of BGC and possibility of PTH therapy to prevent this complication in hypoparathyroid milieu.
