A novel fluorescent probe-based flow cytometric assay for mineral-containing nanoparticles in serum

Abstract

Calciprotein particles, nanoscale aggregates of insoluble mineral and binding proteins, have emerged as potential mediators of phosphate toxicity in patients with Chronic Kidney Disease. Although existing immunochemical methods for their detection have provided compelling data, these approaches are indirect, lack specificity and are subject to a number of other technical and theoretical shortcomings. Here we have developed a rapid homogeneous fluorescent probe-based flow cytometric method for the detection and quantitation of individual mineral-containing nanoparticles in human and animal serum. This method allows the discrimination of membrane-bound from membrane-free particles and different mineral phases (amorphous vs. crystalline). Critically, the method has been optimised for use on a conventional instrument, without the need for manual hardware adjustments. Using this method, we demonstrate a consistency in findings across studies of Chronic Kidney Disease patients and commonly used uraemic animal models. These studies demonstrate that renal dysfunction is associated with the ripening of calciprotein particles to the crystalline state and reveal bone metabolism and dietary mineral as important modulators of circulating levels. Flow cytometric analysis of calciprotein particles may enhance our understanding of mineral handling in kidney disease and provide a novel indicator of therapeutic efficacy for interventions targeting Chronic Kidney Disease-Mineral Bone Disorder.

Inhibition of vascular calcification by inositol phosphates derivatized with ethylene glycol oligomers

“…Chronic exposure to CPPs, e.g., when clearance becomes overwhelmed as in CKD, is a likely driver of VC 24 . CPPs were found to be present in serum from high-phosphate-diet-fed rats with adenine-induced renal failure 72 . By stabilizing CPPs, (OEG 2 ) 2 -IP4 may aid mineral clearance from the circulation before VC can ensue.…”

Cellular Clearance and Biological Activity of Calciprotein Particles Depend on Their Maturation State and Crystallinity

“…Indeed, we reported finding only copious amounts of secondary CPP in post-mortem ascites of a peritoneal dialysis patient who had died of sclerosing peritonitis ( 24 ). Other studies indicate that secondary CPP, while occasionally observed in serum and peritoneal effluent of dialysis patients, are present in much lower amounts than primary CPP ( 25 ). Thus, it remained of critical importance to understand the handling and effects of primary CPP in comparison with their more stable crystalline counterparts.…”

Lumenal calcification and microvasculopathy in fetuin-A-deficient mice lead to multiple organ morbidity

“…Furthermore, the accumulation of fetuin-A-containing mineral particles was noted in the peritoneal dialysate of a patient suffering from calcifying peritonitis [10] and in patients suffering from chronic kidney disease (CKD) [11]. Recent work has shown that both the abundance of CPP [12][13][14] and the kinetics of CPP formation [15][16][17][18] in serum of CKD patients can be used to monitor the propensity for calcification of these patients. The function of fetuin-A as an inhibitor of calcification is underscored by clinical studies showing that low fetuin-A serum levels are associated with calcification disease [19][20][21][22][23][24][25].…”