Elsevier

Bone

Volume 110, May 2018, Pages 355-367
Bone

Full Length Article
Biochemical transformation of calciprotein particles in uraemia

https://doi.org/10.1016/j.bone.2018.02.023Get rights and content

Highlights

  • Circulating CPP accumulate in uraemia and have emerged as possible nanoscale mediators of phosphotoxicity.

  • CPP are enriched for apolipoproteins, complement, lipid and nucleic acid during ripening in uraemic serum.

  • The accretion of bacterial-derived products may evoke innate immune responses and partly account for proinflammatory effects.

  • CPP-II synthesised using uraemic serum can be used to model the effect of endogenous CPP.

Abstract

Calciprotein particles (CPP) have emerged as nanoscale mediators of phosphate-induced toxicity in Chronic Kidney Disease (CKD). Uraemia favors ripening of the particle mineral content from the amorphous (CPP-I) to the crystalline state (CPP-II) but the pathophysiological significance of this transformation is uncertain. Clinical studies suggest an association between CPP ripening and inflammation, vascular dysfunction and mortality. Although ripening has been modelled in vitro, it is unknown whether particles synthesised in serum resemble their in vivo counterparts. Here we show that in vitro formation and ripening of CPP in uraemic serum is characterised by extensive physiochemical rearrangements involving the accretion of mineral, loss of surface charge and transformation of the mineral phase from a spherical arrangement of diffuse domains of amorphous calcium phosphate to densely-packed lamellar aggregates of crystalline hydroxyapatite. These physiochemical changes were paralleled by enrichment with small soluble apolipoproteins, complement factors and the binding of fatty acids. In comparison, endogenous CPP represent a highly heterogeneous mixture of particles with characteristics mostly intermediate to synthetic CPP-I and CPP-II, but are also uniquely enriched for carbonate-substituted apatite, DNA fragments, small RNA and microbe-derived components. Pathway analysis of protein enrichment predicted the activation of cell death and pro-inflammatory processes by endogenous CPP and synthetic CPP-II alike. This comprehensive characterisation validates the use of CPP-II generated in uraemic serum as in vitro equivalents of their endogenous counterparts and provides insight into the nature and pathological significance of CPP in CKD, which may act as vehicles for various bioactive ligands.

Introduction

Calcium and phosphate do not precipitate directly from extracellular fluid as naked apatite crystals, but, as in bone, through an ordered process of crystallisation, directed by various mineral-binding proteins like fetuin-A [1]. This interaction results in the self-assembly of submicron aggregates of calcium phosphate and acidic proteins, called calciprotein particles (CPP), which are dispersed in biological fluids as colloids - akin to the way apolipoproteins solubilise lipids as lipoprotein particles [2,3]. Notionally, this humoral homeostatic system provides a buffer against procalcific disturbances in mineral metabolism, retarding crystal growth whilst facilitating clearance and metabolism of insoluble calcium phosphates [[4], [5], [6]].

In vitro, protein-bound calcium phosphate ion clusters associate to form spherical structures containing mineral as amorphous calcium phosphate (primary CPP; CPP-I). CPP-I can subsequently undergo “ripening” to larger particles containing mineral in more crystalline phases like hydroxyapatite (secondary CPP; CPP-II) [2,3]. While CPP formation may limit the toxicity of insoluble mineral, the accumulation and ripening of particles in response to sustained disturbances in mineral metabolism, as seen in patients and animals with Chronic Kidney Disease (CKD) [[7], [8], [9]], may be injurious [4,10]. Serum CPP strongly correlate with inflammatory markers and are associated with structural and functional manifestations of cardiovascular disease such as the extent of coronary artery calcification and aortic stiffening [7,8,[11], [12], [13]]. Importantly, higher serum CPP also portends an increased mortality risk in an inflammation-related manner [11].

In the context of CKD, phosphate overload is viewed as a principal driver of ectopic vascular calcification and downstream cardiovascular sequelae, although the mechanism is not fully understood. At the molecular level, compelling data points to the fact that it is nanocrystals formed in mineral stress that induce vascular smooth muscle cell (VSMC) calcification and proinflammatory pathways rather than the direct effects of phosphate itself [[14], [15], [16]]. The accumulation of CPP and ripening to the crystalline state may therefore be an important step in phosphate-driven toxicity [4,17]. Consistent with this notion, preliminary in vitro studies suggest that CPP ripening has a profound influence on biological effect, with only crystalline CPP-II able to induce inflammation and mineralisation in VSMC [[18], [19], [20]]. However, it is not clear whether synthetically-generated CPP accurately model the effects of their endogenous counterparts, of which no systematic characterisation has been undertaken. Here we address some of these important knowledge gaps with detailed physiochemical and biochemical comparisons of endogenous CPP isolated from uraemic serum with synthetic CPP derived from the same sera.

Section snippets

Study population

Participants were enrolled (Jan 2014 to Sept 2016) in a prospective observational study conducted at the Royal Melbourne Hospital. Two groups of participants were enrolled: patients with end-stage renal failure undergoing conventional maintenance haemodialysis thrice weekly and healthy volunteers as controls. Details of study design, inclusion and exclusion criteria have been described elsewhere [9]. Healthy adult subjects had no history of cardiovascular disease or its risk factors (exclusion

Physiochemical characterisation of calciprotein particles in uraemic human serum

Although the process of in vitro CPP formation and transformation in serum is well defined [31], the physiochemical characteristics of endogenous particles isolated from uraemic patients have not been systematically described. Here, we used the pooled serum of patients undergoing haemodialysis (n = 60) to isolate endogenous CPP (referred to subsequently as uraemic human serum CPP – UHS-CPPENDOG), and the same sera to synthesise CPP-I or CPP-II (UHS-CPP-ISYNTH; UHS-CPP-IISYNTH) ex vivo as

Discussion

This is the first systematic characterisation of endogenous CPP isolated from uraemic human serum, and extends our earlier semi-quantitative analyses of particles in serum from patients on dialysis and in the peritoneal effluent collected from a patient undergoing peritoneal dialysis [23,37]. Cryo-TEM analysis of serum from high-phosphate-fed rats with adenine-induced renal failure has also revealed the presence of circulating CPP in these animals [9]. Critically no study has directly compared

Conclusion

Our detailed characterisation of the physiochemical properties of endogenous and synthetic CPP demonstrate subtle differences in mineral, protein and lipid composition, but suggest an equivalence of synthetic crystalline CPP-II derived from uraemic serum and endogenous CPP isolated from dialysis patients in terms of their predicted effects on cell viability and inflammatory pathways. The existence of subtle structural and compositional differences in CPP generated from non-uraemic and uraemic

Disclosure statement

ERS holds stock in Calciscon AG and has received grant support from Amgen, Baxter and Sanofi. SGH has received grant support/honoraria/consultancy fees from Amgen, Baxter, Otsuka, AstraZeneca, Gilead and Sanofi. TDH and EH report no relevant conflicts of interest.

Authorship roles

Study concept and design: ERS; Study conduct: ERS and SGH. Data collection: ERS, TDH, EH. Data analysis: ERS, EH; Data interpretation: ERS, TDH, EH, SGH. Drafting manuscript: ERS. Revising content: ERS, TDH, EH, SGH. Approval of final version of manuscript submitted: ERS, TDH, EH, SGH.

Acknowledgements

The proteomic work was undertaken at Australian Proteome Analysis Facility (Macquarie University, NSW, Australia), infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS). This work was generously supported by funding from a Grant-in-aid (GIA-015-2015) from Melbourne Health to ERS, TDH and SGH.

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