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

Edward R Smith,Stephen G Holt,Andreas Pasch,Michael M X Cai,Tim D Hewitson,Matthias Bachtler,Parisa Aghagolzadeh

doi:10.1038/s41598-017-05474-y

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.

Highlights

The plasma concentrations of calcium and phosphate required to generate and maintain our apatite-based endoskeleton are close to that which would precipitate in aqueous solution[1]
We have used this method extensively[8] and it performs reproducibly in our hands[7, 8, 10, 24, 25] and others[9, 26], several theoretical and technical shortcomings were apparent[27]. These relate to: (1) the use of fetuin-A as a surrogate for mineral-containing particles given its presence in various particulates that may be sedimented with Calciprotein particles (CPP); (2) failure to detect fetuin-A-poor CPP in states of fetuin-A deficiency; (3) substantial analytical error encountered when deriving estimates of CPP Fet-A levels based on the small numerical differences between total fetuin-A in serum and after centrifugation following substantial dilution of each (1 in 10,000); (4) that measurements provide a readout of fetuin-A protein enrichment rather than particle number; and (5) that such measurements do not allow the discrimination of CPP-I from CPP-II, which may have profoundly different biological effects[32]
We developed an antibody labelling strategy for CPP based on previously published proteomic analyses[9, 35], staining for two protein targets ubiquitously present in CPP: fetuin-A (FetA) and apolipoprotein A1 (ApoA1)

Summary

Introduction

The plasma concentrations of calcium and phosphate required to generate and maintain our apatite-based endoskeleton are close to that which would precipitate in aqueous solution[1]. We have used this method extensively (after minor modifications)[8] and it performs reproducibly in our hands[7, 8, 10, 24, 25] and others[9, 26], several theoretical and technical shortcomings were apparent[27] These relate to: (1) the use of fetuin-A as a surrogate for mineral-containing particles given its presence in various particulates that may be sedimented with CPP (apoptotic bodies[28], exosomes[29], opsonised cell debris30, 31); (2) failure to detect fetuin-A-poor CPP in states of fetuin-A deficiency (i.e. patients with end-stage renal disease19); (3) substantial analytical error encountered when deriving estimates of CPP Fet-A levels based on the small numerical differences between total fetuin-A in serum and after centrifugation following substantial dilution of each (1 in 10,000); (4) that measurements provide a readout of fetuin-A protein enrichment rather than particle number; and (5) that such measurements do not allow the discrimination of CPP-I from CPP-II, which may have profoundly different biological effects[32]. Further we illustrate how application of this new method provides insight into dysregulated mineral handling in CKD and the origin of these particles in vivo

Results

Discussion

Conclusion