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

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.


Results
Antibody-based labelling strategy for CPP. Initially, 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). However, while CPP could be stained for both these proteins (see Supplementary Fig. S2), they also stained positively with a panel of antibodies against unrelated antigens not present in CPP, alternate subclass IgG controls and non-human species-specific targets (see Supplementary  Fig. S2). This is consistent with previous reports of non-specific adsorption of various antibodies to mineral microparticles and tissue deposits 36,37 . We therefore considered antibody-based detection of CPP unreliable and prone to false positives due to non-specific binding and adsorption of immunoglobulin. Indeed, IgG and IgM have previously been detected in proteomic studies of these particle fractions 9,35 . We therefore decided to pursue non-immunochemical means of staining using low molecular weight fluorescent probes. Background information regarding the use of light scattering and fluorescence parameters in the study of nanoparticles can be found in the Supplementary material. Application of mineral-and membrane-specific fluorescent probes to the analysis of CPP. Bisphosphonates are pyrophosphate analogues with exquisite affinity (K L > 1 × 10 6 L mol −1 ) and specificity for mineral phases 38 . Accordingly, fluorescently-conjugated bisphosphonate derivatives have already been extensively applied to in vivo imaging studies of bone 39 and ectopic calcification [40][41][42] , as well as flow cytometric-based assays of mineral-containing exosomes isolated from culture media and murine serum 43 . Here we utilise one such fluorescent derivative, OsteoSense 680EX. Since mineral deposits have also been shown to reside within, or seed from the surface of, microvesicular structures -distinct from membrane-free CPP -we further enhanced analytical specificity by the addition of a green fluorescent membrane intercalating dye, PKH67. A double staining protocol using OsteoSense 680EX and PKH67 was developed (Fig. 1a).
Gating strategy and thresholding. We set sizing gates empirically, positioning 500 nm latex beads on the fifth decade of side scatter-height (SSC-H), which is well within the acquisition window for the detector (linearity maximum <2.2 × 10 5 ). Detection of OsteoSense 680EX positive events was triggered from the 640 nm red laser using the APC-Cy7 detector. On theoretical grounds, excitation using a lower wavelength (violet or blue) laser would increase light scattering and maximize excitation of fluorochromes and the detection sensitivity of individual particle detection (although may also incur substantial photobleaching effects). However, alternative commercially-available mineral-binding dyes with lower excitation wavelengths show substantial spectral overlap with PKH67. We therefore selected the derivative with the lowest excitation wavelength (680 nm maxima) but non-overlapping spectra.
Expectedly, the scatter profile of endogenous mineral-containing nanoparticles (MNP) overlaps substantially with that generated by the buffer and electronics (Fig. 1b). For comparison, we also ran a suspension of hydroxyapatite nanoparticles (HAP), which despite their small size (<200 nm; see Supplementary Fig. S4), have very high SSC intensities. Detection thresholds were determined empirically, based on the fluorescence of buffer/ reagent blanks and unstained particles (Fig. 1c). Applying this threshold, event rates for reagent blanks were consistently <20 events/min. Lowering of the detection threshold did not appreciably increase the number of particles detected. However, we cannot exclude the possibility of missing very dimly stained particles. Lower thresholds can be applied with the use of an additional centrifugation step to remove unbound dye/dye aggregates, although this greatly impacted on recovery (15-52% of unspun levels for total CPP/μL) and increased overall imprecision due to system noise (within-run CV > 20% vs. <5% for total CPP/μL). Unstained particles appeared to have minimal autofluorescence above that of the buffer. With this setup, serum (endogenous) CPP could be detected and resolved from nonfluorescent noise and unbound dye, as a polydisperse population with SSC-H intensities less than that of 450 nm polystyrene beads (Fig. 1d). Such polydispersity is consistent with the morphological heterogeneity seen by us on imaging previously 6,27,44 . Next we applied the method to synthetically generated CPP-I and CPP-II. Both CPP-I and CPP-II stained with OsteoSense 680EX, but not PKH67, consistent with the presence of mineral and absence of a membrane (Fig. 1e). CPP-II showed more intense, but overlapping (<10%), scatter than CPP-I, consistent with size distribution analyses by other light scattering techniques and transmission electron microscopy (TEM) imaging 44,45 . The intensity of OsteoSense 680EX fluorescence was very similar for CPP-I and CPP-II and therefore offers no additional discrimination. Batches of CPP-I and CPP-II were generated using multiple pools of human serum (n = 20) as previously described 45 , and used to define gates encompassing 90 th centile of all CPP-II particles (Fig. 1f). The lack of separation between CPP-I and CPP-II also undoubtedly reflects the inability of the system to fully discriminate small differences in particle size/ composition. Therefore, CPP-II counts based on SSC-H gates only provide a measure of CPP-II abundance and not absolute numbers of crystalline particles.

Discrimination of CPP and membrane-bound particles.
We confirmed the specificity of OsteoSense 680EX staining for mineral by incubating CPP with ethylenediaminetetraacetic acid (EDTA; a mineral chelator), and monitoring the dissolution of particles over 24 hours (see Supplementary Fig. S3A). Similarly, specificity of PKH67 for membrane was demonstrated by incubating CPP with detergent (Triton X-100), resulting in the solubilisation of only the membrane-bound particle (MBP) component (see Supplementary Fig. S3B). Since detergent action only dissolves away membrane and not mineral, MBP gates could not be defined based on this treatment, and lower bounds were instead set using unstained CPP and PKH67 reagent blank (Fig. 1E). Interestingly, MBP populations consistently showed more intense scatter than CPP (presumably due to differences in optical properties given their equivalent size). TEM analysis of particles pelleted using a stepped centrifugation protocol confirmed the presence of CPP, and membrane-bound mineral-containing vesicles at higher speeds (see Supplementary Fig. S4). Importantly, vesicular structures were seldom present in pellets spun at 30,000 g for 2 h (e.g. see Supplementary Fig. S7), which we routinely use to prepare CPP from serum in vitro. Of note, while HAP nanoparticles stained strongly for OsteoSense 680EX, they remained PKH67 negative (Fig. 1c). As a final test of specificity, we demonstrated that OsteoSense 680EX -positive MNP could be sedimented using high-speed centrifugation (50,000 g, 2 h and 4 °C; see Supplementary Fig. S3C). Overall, the ability to discriminate CPP-I from CPP-II (albeit partially) and membrane-bound mineral-containing particles clearly provides an important step forward in analytical capability. Details regarding the quantitation of individual mineral-containing nanoparticles and comparison with measurements made by nanoparticle tracking analysis (NTA) and an alternative flow cytometer are provided in the Supplementary material.

Flow cytometry of serum mineral-containing nanoparticles in a cohort of dialysis patients. To
further validate the utility of the method, we analysed serum samples from 80 patients with end-stage renal disease undergoing peritoneal dialysis (PD; n = 40) or haemodialysis (HD; n = 40), who were recruited to an ongoing observational study at our centre 7,25 . Samples from individuals without CKD were also obtained to serve as controls (n = 40). Representative readouts for mineral-containing particles in human serum obtained from control, PD and HD groups are shown in Fig. 2.
Total serum CPP levels were modestly raised in the HD group compared to controls (Fig. 3a), whereas CPP-II levels were higher in the HD group compared to PD and control groups (Fig. 3b). SSC-H, on the other hand, was higher in PD and HD groups compared to controls (Fig. 3c). Overall, total CPP and CPP-II counts were strongly correlated (Fig. 3d), although CPP-II as a proportion of total CPP levels was generally greater and more variable in those on HD (Fig. 3e). Particle counts showed no relationship with SSC-H overall, or in any individual group (Fig. 3f). Again, MBP counts appeared modestly elevated in HD compared to control groups (Fig. 3g), but, while unrelated to total CPP counts (Fig. 3h), were weakly associated with CPP-II levelsin a non-linear fashion (Fig. 3i). Between group differences in serum inflammatory cytokines (interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α) are depicted in Supplementary Figure S7 and, in keeping with published data 46 , were elevated in HD and PD groups compared to controls. As shown in Table 1, in pooled analysis of HD and PD patients, both total CPP and CPP-II counts were higher in diabetics, and correlated strongly with serum phosphate, PTH, intact FGF23, IL-1β, IL-6, sclerostin (an inhibitor of Wnt/β-catenin signalling) and weakly with osteoprotegerin (a soluble decoy receptor for RANKL). Given the co-correlation of total CPP and CPP-II it is expected that both measures would associate with the same variables, however, it should be noted that coefficients were consistently greater for CPP-II than for total CPP levels. CPP-II was also weakly correlated with serum calcium and TNF-α and inversely with serum magnesium and bicarbonate concentrations. SSC-H was associated with the same biochemistries as CPP-II. Regression analyses of CPP-II and inflammatory markers are shown in Supplementary Figure S7. Interestingly, MBP levels were only weakly correlated with TNF-α and IL-6. No parameters from this analysis were associated with age, gender, serum albumin, Dickkopf-related protein 1 (DKK-1; Wnt signaling inhibitor), osteocalcin or osteopontin (both mineral-binding proteins) or the presence of residual renal function, although the numbers with the latter were small (n = 8).
In head-to-head analysis of the same samples, CPP Fet-A levels measured by ELISA were strongly associated with both CPP-II (r = 0.475, p = 0.0026) and SSC-H (r = 0.603, p = 0.0380) parameters by flow cytometry, demonstrating good agreement between methods.
Flow cytometric analysis of calciprotein particles in adenine-treated uraemic rats and the response to dietary phosphate. An important advantage of the current method is that it circumvents issues relating to the use of species-specific antisera and thus facilitates experimental modeling in animals. Indeed, attempts to measure circulating CPP in animal models have until now been restricted to qualitative and semi-quantitative analyses using Western blotting 11,47,48 . CPP (also known as fetuin-mineral complexes) have been detected in the serum of adenine-treated (0.75%) rats in as little as two weeks following dietary supplementation, even in the absence of changes in serum phosphate concentration 11 . This implied that the emergence of circulating CPP might be an early manifestation of the derangements in mineral metabolism seen in CKD. While some features of the adenine model are not representative of CKD (e.g. polyuria, weight loss), it does nonetheless recapitulate many of the features of advanced human CKD-MBD (depending on the dose and duration of administration): hyperphosphataemia, secondary hyperparathyroidism, FGF23 excess, calcitriol deficiency, high turnover osteodystrophy and ectopic calcification.
In our hands, 4 weeks of dietary adenine supplementation in rats (0.75% for 2 weeks then 0.5% for 2 weeks, n = 30) resulted in the induction of uraemia (Fig. 4a,b) and elevations in phosphate (Fig. 4d), fractional phosphate excretion (FeP; Fig. 4e), intact PTH (Fig. 4f) and FGF23 (Fig. 4g), consistent with previous reports employing the same treatment protocol 49 . Total serum CPP levels were elevated in adenine-treated animals compared to baseline (week 0) and normal renal function controls ( Fig. 4h; mean 56% increase), with CPP-II levels increased more than 4-fold from baseline (Fig. 4j). All parameters remained stable in the control animals fed standard chow. After the 4-week induction period, adenine-treated and control animals were further divided into two subgroups (n = 15/group; matched for body weight, creatinine and phosphate) and allocated to receive either standard chow (normal phosphate diet; NPD) or a high-phosphate diet (HPD) for a further 6 weeks. As expected, HPD feeding in adenine-treated animals resulted in marked elevations in phosphate (Fig. 4d), FeP (Fig. 4e), PTH (Fig. 4f) and FGF23 (Fig. 4e), as well as a reduction in serum calcium concentration (Fig. 4c) compared to NPD-fed adenine-treated animals by week 10. In HPD-fed adenine-treated rats these changes were accompanied by an increase in total CPP levels ( Fig. 4h), and a striking increase in CPP-II levels by week 10 (>14-fold over baseline; Fig. 4j). TEM analysis of the CPP-containing fraction from pooled adenine-treated/HPD-fed rat serum confirmed the presence of particles with morphologies consistent with CPP-I and CPP-II synthesised from human serum in vitro (see Supplementary Fig. S8).
Dietary phosphate supplementation in control rats had no effect on conventional serum biochemical parameters over the study period but resulted in increased FeP at 10 weeks compared to baseline and normal phosphate-fed controls (Fig. 4e). We also observed an increase in total serum CPP levels in HPD-fed controls by week 10 (Fig. 4h), equivalent in magnitude to that seen in adenine-treated rats allocated the control diet. Since high-phosphate feeding in these control animals was associated with a slight reduction in mean SSC-H (i.e. smaller particle size and/or reduced crystallinity) this is likely to reflect increased formation of CPP-I (Fig. 4i).

Flow cytometric analysis of calciprotein particles in other animal models.
To assess the broader utility of the method, we analysed archived serum samples obtained from several different animal models using the same analytical workflow (Fig. 5). As in adenine-treated rats, increased SSC-H and CPP-II levels were found in mice following subtotal nephrectomy (5/6Nx) compared to sham-operated controls (Fig. 5a). While total CPP concentrations were elevated in adenine-treated rats they were unchanged in 5/6Nx animals, suggesting increased ripening but not enhanced formation in this model. In mice with unilateral ureteric obstruction(UUO), on the other hand, flow cytometric parameters did not differ from unoperated littermates (Fig. 5b), consistent with the absence of systemic changes in mineral metabolism described in this model of experimental renal fibrosis 50 .
In an unrelated model, high-fat fed ApoE −/− mice showed increased total CPP levels compared to wild-type animals fed standard chow (Fig. 5c). Since CPP-II levels and SSC-H were unchanged, this implies that the elevation in CPP was related to increased CPP-I.

Discussion
In summary, we have developed a rapid homogeneous flow cytometric method for the detection and quantitation of individual mineral-containing nanoparticles in human and animal serum. To enhance sensitivity and specificity we have employed fluorescence-based thresholding as a signal trigger, which can reliably distinguish fluorescent particles from non-fluorescent background and free unbound dye without the need for a separation step. This method also allows the discrimination of membrane-bound from membrane-free particles and different mineral phases (amorphous CPP-I vs. crystalline CPP-II). Critically, the method has been optimised for use on a conventional benchtop instrument, without the need for manual hardware adjustments or dedicated microparticle flow cytometer. Therefore, we anticipate that the method will be readily accessible to others without the need for an experienced technician capable of complex modifications and calibration.
Using this method, we could demonstrate a consistency in findings across studies of CKD patients and in uraemic animal models, which showed that renal dysfunction is associated with enhanced ripening of CPP to the crystalline CPP-II state. In dialysis patients, circulating CPP levels were associated with markers of inflammation, bone metabolism and mineral metabolites, while in adenine-treated rats, chronic phosphate dietary loading appeared to exacerbate the changes in CPP seen with uraemia.
Links between CPP, bone and lipid metabolism. Although the current flow cytometric assay for CPP described here employs an entirely different analytical approach, it is reassuring to observe that variables associated with CPP Fet-A levels determined using the ELISA-based method, namely, phosphate, bone turnover markers (C-terminal telopeptides) and inflammatory cytokines 8, 10 , remained significantly correlated with the readout from this next generation assay in a different cohort of CKD patients. However, the new method also revealed novel associations which were not apparent using the older method; levels were found to be higher in patients with type 2 diabetes and correlated strongly with serum calcium and FGF23 concentrations. The finding of the association of CPP with FGF23 is particularly noteworthy, as the mechanism(s) driving elevations in FGF23 in CKD are poorly understood 51 . Bone-derived FGF23 has emerged as a possible 'phosphate-responsive' mediator of cardiovascular disease in CKD, but its secretion, while associated with dietary phosphate intake, paradoxically does not appear acutely or directly regulated by extracellular phosphate levels in vitro 52 or in vivo 53 . Interestingly, recent in vitro studies have revealed that CPP stimulates the secretion of FGF23 in cultured osteocytes 54 . Thus, it is tempting to speculate that CPP may at least partly mediate the stimulatory effects of dietary phosphate loading on FGF23 via increased particle formation or ripening. Further work is needed to explore this attractive hypothesis directly. Corroborating these findings, CPP Fet-A levels have recently been correlated with intact FGF23 in a diabetic cohort (with preserved renal function) and may therefore have significance beyond ureamia as described here 26 . FeP, (f) serum intact PTH, (g) serum intact FGF23 and serum flow cytometric parameters (h) total CPP, (i) CPP SSC-H and (j) CPP-II levels in control and adenine-treated rats fed normal or high-phosphate containing diets over the 10-week study period. *P < 0.05, **P < 0.01, ***P < 0.001, ‡ P < 0.0001 for adenine-treated groups vs. control groups; # P < 0.05, ## P < 0.01, ### P ≤ 0.0001 for HPD-fed adenine-treated group vs. NPD-fed adenine-treated group; § P < 0.05, § § P < 0.01 for NPD-fed adenine-treated group vs. NPD-fed control group; † P < 0.05 for HPDfed control group vs. NPD-fed control group. Data presented as mean ± SEM for each experimental group (n = 15 per group). Note logarithmic scale in (g). Ad, adenine; Ca, calcium; Cntrl, control; CPP, calciprotein particles; CrCl, creatinine clearance; FeP, fractional excretion of phosphate; FGF23, fibroblast growth factor 23; HPD, high phosphate diet; NPD, normal phosphate diet; P, phosphate; PTH, parathyroid hormone; SSC-H, side scatter-height.
Scientific RepoRts | 7: 5686 | DOI:10.1038/s41598-017-05474-y Matsui et al. reported that a high molecular weight fetuin-A-containing mineral fraction was observed in the serum of adenine-treated rats but not in animals fed a control diet 11 . Interestingly, CPP became undetectable in uraemic animals given alendronate. This is consistent with the earlier work of Price et al., who showed that administration of various bone-modulating agents had profound effects on circulating levels 47,55 , implicating the involvement of the skeleton as a source and/or sink for CPP. While the association of CPP levels with multiple bone-related cytokines (including FGF23) noted here is certainly suggestive of strong links to bone metabolism, further work is needed to evaluate the direct contribution of bone turnover to CPP formation in uraemia, and conversely, to consider the effects of circulating mineral nanoparticles on the bone itself. The purely associative nature of these findings, and the lack of adjustment for potential confounders due to the small sample size, precludes us from making more definitive comments about the interaction of CPP with bone.
Another plausible explanation for the apparent link between CPP and bone is that the interaction might be indirect, and mediated by other common factors, such as changes in inflammatory signaling 56,57 . However, counter to this argument, the strength of the association of total CPP concentrations with both FGF23 and sclerostin was only very modestly attenuated by adjustment for IL-1β (r p = 0.33; r p = 0.25, respectively) or IL-6 (r p = 0.35; r p = 0.22, respectively).
While our studies in adenine-treated rats and in 5/6 nephrectomised mice indicated a common pattern of changes in CPP ripening following induction of uraemia, substantial elevations in total CPP levels were additionally observed in the adenine model. Since the mineral and protein content of the chow fed to these animals was identical we can exclude differences in diet as a potential cause. Other possibilities included increased production (e.g. from bone) and/or impaired clearance. Clearance-related effects would appear less likely, as in normal mice, CPP clearance is mediated by Kupffer cells of the liver and marginal zone macrophages in the spleen, without involvement of the kidney 58 . While it might be anticipated that changes in bone turnover could account for the elevations in CPP-II, as suggested by our studies in human CKD, it is notable that these two uraemic animal models have quite distinct bone histomorphometry. In adenine-treated rats high-turnover bone disease occurs, but in contrast in 5/6Nx animals there are only relatively subtle changes in the mineralisation surfaces and trabecular number, without accompanying disturbances in turnover 59 . The increased levels of CPP-I seen in adenine-treated rats may therefore provide a surrogate for the increased bone turnover observed in this model. Clearly studies comparing the clearance and tissue disposition of CPP in animals with intact renal function to those with uraemia are needed to clarify this issue, in addition to longitudinal studies tracking changes in bone turnover with circulating CPP.
The finding of increased CPP-I in high-fat fed ApoE −/− mice is also noteworthy. Although this finding may partly reflect differences in feed: high-fat chow is casein-based with phosphate in a more bioavailable form than grain-based control chow; this is unlikely to fully explain this observation as CPP levels were significantly higher in ApoE −/− mice than in high-phosphate fed control rodents. Urine samples were not available to assess fractional phosphate excretion as a proxy for phosphate loading in our study, so we were unable to exclude relative phosphate loading as a contributor to the augmented CPP levels observed. As an alternative explanation, we speculate that reduced clearance of CPP may also contribute to the elevated levels in these animals, due to the competing effects of accumulating ligands, like modified LDL particles, which share the same scavenger pathways. Indeed, the same receptor (class A scavenger receptor) is involved in the endocytosis of CPP and pro-atherogenic oxidised LDL and acetylated LDL 58,60 , which are known to accumulate in high-fat fed ApoE −/− /mice 61 , and have been shown to compete for binding in vitro 58 . Oxidised LDL levels are also found to be elevated in patients with CKD, despite typically normal (unmodified) LDL cholesterol levels 8 . Whether this holds significance for atherogenic processes remains to be seen but it is interesting to note that Herrmann et al. showed that CPP are taken up avidly by lesional macrophage in the carotid arteries of ApoE −/− /Fetuin-A −/− mice 58 .

Intestinal origin of circulating CPP and responsiveness to dietary mineral loading. The in vivo
data presented here provide the first direct evidence that circulating CPP are responsive to changes in dietary mineral and highlight the disparate effects of loading on CPP ripening in animals with renal dysfunction compared to those with preserved renal function. That CPP increase with dietary phosphate content is entirely consistent with recent diurnal studies of circulating CPP in human diabetic subjects with normal renal function, where the authors noted a transient rise in levels after meals 26 . Importantly, in high-phosphate fed non-uraemic controls, we found that the rise in serum CPP-I was not accompanied by any detectable change in serum calcium and phosphate concentrations. This implies that quantitation of CPP levels may provide a better readout of dietary mineral exposure than these conventional measures.
More fundamentally this data also speaks to the likely source of at least a proportion of the circulating mineral nanoparticle pool. Indeed, if serum calcium and phosphate levels are maintained within normal limits, how can CPP formation be appreciably altered in the post-prandial state? Elegant studies by Powell and colleagues 36 may provide one plausible explanation. They delineated an endogenous intestinal nanomineral transport system in which abundant ~100 nm amorphous calcium phosphate particles, similar in appearance to CPP-I, are formed spontaneously in the distal small bowel from the high concentrations of calcium (~4 mM) and phosphate (>10 mM) present in intestinal secretions 62 . These particles bind various macromolecules (proteins and peptidoglycans) present in the gut lumen, and are subsequently transcytosed across the epithelial barrier, principally via M cells of Payer's patches, to antigen-presenting immune cells resident in the intestinal tissue. This process appears to occur constitutively, even in the fasting state, generating ~2 × 10 14 particles/day, and putatively has a role in generating immunological tolerance 36 . Indeed, it has been known for decades that both endogenous and synthetic particles (<250 nm diameter) can efficiently translocate across the small gut epithelium via small intestinal lymphoid aggregates and, to a lesser extent, villous tissue [63][64][65] . We speculate that dietary loading with mineral may overwhelm this pathway resulting in spillover of particles into the circulation via the portal system. Intriguingly, Thomas and colleagues 53 recently reported that acute oral phosphate by gavage induced elevations in PTH and sustained phosphaturia while observing no change in serum calcium, phosphate or FGF23, thus the trigger of this adaptive response was uncertain. In this regard, it is worth noting that the experimental conditions of the original in vitro studies showing the stimulation of PTH secretion in parathyroid glands by phosphate, independent of ionised calcium, would be expected to generate CPP in situ 66 . Therefore, studies investigating the role of CPP in triggering PTH secretion seem warranted. Indeed, as shown here in dialysis patients, CPP levels are strongly correlated with PTH in CKD 9 . Thomas et al. also made another highly relevant observation relating to the disposition of phosphate loads administered via either oral or intravenous routes 53 . In their study, urinary excretion at 4 h post gavage only accounted for approximately 50% of the administered load, at which point serum phosphate was unchanged (with oral loading) or normalised (with an intravenous load), and tissue phosphate levels were similar to vehicle-treated animals 53 . Since other routes of elimination (e.g. gastrointestinal) do not appear to quantitatively contribute in this situation 67 , these data are compatible with the existence of an extracellular compartment that can buffer such loads. Indeed, this is entirely consistent with much earlier intravenous loading studies by Gersh 68 , who reported the appearance of a colloidal mineral phase in the serum of dogs to which he had administered supraphysiological amounts of calcium and phosphate salts.
Overall, given the rapid clearance of CPP from the circulation 58 and the concentrations of calcium and phosphate needed to drive CPP formation in extracellular fluid in a biologically relevant timeframe 4 , the gut represents the most likely origin of the post-prandial CPP surge and circulatory pool. From a teleological perspective, we postulate that expansion of the CPP-I pool may serve as a temporary buffer for the increased mineral flux, helping to maintain ionic homeostasis, until the particle mineral load can be safely metabolised and/or renally excreted.
Scientific RepoRts | 7: 5686 | DOI:10.1038/s41598-017-05474-y While this theoretical model implies a physiological role for CPP-I formation and clearance, our data also highlights that disturbances in mineral handling found in the uraemic state (e.g. hyperphosphataemia) favour nanocrystal ripening and the accumulation of potentially pathologic CPP-II.
Multiple studies in uraemic rodent models attest to the pathological effects of dietary phosphate loading on the heart, vasculature and kidneys. Although the mechanism(s) by which phosphate imparts these effects remains to be fully elucidated at the molecular level, phosphate overload has nonetheless been implicated as a major driver of vascular calcification, endothelial dysfunction, cardiac hypertrophy, tubulointerstital fibrosis and systemic inflammation in these uraemic animals [reviewed in refs 69-71]. Due to the biological plausibility of elevated phosphate levels directly contributing to disease, and strong associative evidence, interventions aimed at restricting phosphate intake or reducing intestinal absorption with binders have formed a mainstay of therapy for patients with advanced CKD 72 . However, while the control of dietary phosphate has proven effective in attenuating cardiovascular sequelae in animals [73][74][75] , and has been associated with a survival advantage in some studies of end-stage renal disease patients [76][77][78][79] , this effect appears largely independent of effects on serum phosphate concentration itself. Indeed, serum phosphate concentrations rise relatively late in the progression of CKD 80 , despite cardiovascular disease often being evident much earlier, and at best show very weak correlations with dietary phosphate intake if at all 81 . Nonetheless, that phosphate levels remain linked to events and outcome in CKD [82][83][84] , albeit only modestly after adjustment for confounding 85 , suggests that factor(s) related to phosphate may still have pathophysiological importance as well as prognostic significance. Since substantial data from both in vitro and in vivo studies already suggests that calcium phosphate nanocrystals, rather than phosphate itself, may mediate the deleterious effects of phosphate loading on the body [13][14][15]86 , we speculate that an inability to clear CPP derived from dietary mineral intake, as appears to be the case in CKD, may result in ripening to the crystalline state and consequently drive some of the pathological processes previously attributed to 'phosphate' . Clearly, further work is needed to confirm this hypothesis before we can consider CPP as a true phosphotoxin. Moreover, it will be important to understand how uraemia might affect the absorption and clearance of CPP and whether these pathways are amenable to intervention. From a practical point of view, a further corollary of these findings is that future clinical studies will need to carefully consider the prandial status of participants.

Conclusion
Future studies are needed to evaluate the effect of dietary modification on CPP in human CKD and to assess more directly the relevance of intestinal and bone-derived CPP as a source of these circulating particles in vivo. Prospective analyses evaluating the association of flow cytometric-derived CPP measurements with vascular measures (e.g. calcification) and hard endpoints, as demonstrated using the ELISA method, are also needed to further assess the prognostic performance of this assay.
Overall, the new method described here represents an important step forward in analytical capability over the existing state-of-the-art ELISA-based techniques, allowing specific, fully quantitative and discriminative analyses of mineral-containing nanoparticles in fluids from humans and animals. From a clinical perspective, CPP testing by flow cytometric analysis may enhance our understanding of mineral handling in CKD and provide a novel indicator of therapeutic efficacy for interventions targeting the CKD-mineral bone disorder.

Methods
Human studies. Participants were enrolled (January 2014 to September 2016) in an observational study of Fetuin-A Levels in Systemic disease and Kidney Impairment (FLEKSI) conducted at the Royal Melbourne Hospital. These included 40 prevalent haemodialysis patients ("HD" group) and 40 patients undergoing peritoneal dialysis ("PD" group). All dialysis patients were stable, without evidence of inter-current illness, and were achieving small molecule clearance targets. 8 of 40 PD patients had residual renal function. PD patients were free from peritonitis for at least 6 months. All HD patients were anuric and receiving conventional maintenance HD thrice weekly (on average 4 hours per session) with a standard dialysis solution containing 1.25 mmol/L calcium, 0.5 mmol/L magnesium and 35 mmol/L bicarbonate and using a Gambro Polyflux 210 diaylser (Polyamix membrane). Dialysate was regularly monitored for impurities (<0.1 CFU/mL, <0.03 EU/mL endotoxins). PD fluids used contained 1.25 mmol/L calcium. Exclusion criteria included known pregnancy, age less than 16 years or greater than 90 years. A detailed drug history was recorded on all patients, including over the counter preparations. Medication use in these patients at the time of sampling is summarised in Supplementary Table S1. 40 healthy adult subjects were enrolled from staff and volunteers. Control subjects had no history of cardiovascular disease (exclusion criteria: previous myocardial infarction, stroke, heart failure, or receiving lipid-lowering/ antihypertensive therapy), type 2 diabetes mellitus, malignancy, recent infection or trauma, or with known renal disease. Prandial state was uncontrolled and the timing of sampling was not standardised to facilitate recruitment of patients from AM and PM haemodialysis sessions, thus dietary effects cannot be assessed.
Venous bloods were taken for standard clinical care (serum calcium, phosphate, magnesium, bicarbonate, albumin, plasma PTH, 25-hydroxy-vitamin D) and for the assessment of mineral-containing nanoparticles (CPP, MBP), inflammatory and bone markers. In HD patients, samples were taken prior to starting dialysis and circuit anticoagulation. Blood was collected into 6 mL plain (#367837) and K 2 -EDTA (#367863) tubes (both BD Biosciences, San Jose, CA, USA) using standard phlebotomy techniques. Blood samples were allowed to stand for 60 minutes and then centrifuged at 3000 g for 15 min at room temperature (Eppendorf 5430R, A-4-38 rotor or 5415 D, F45-24-11 rotor) to pellet cells. Aliquots were then stored at −80 °C until batched analysis or processed immediately for nanoparticle analysis as detailed below. All analyses were performed on samples that had not been thawed previously. Standard biochemical analysis was performed using a routine automated analyser. Serum inflammatory markers (IL-1β, IL-6 and TNF-α) were measured in technical duplicates using the human inflammatory cytokine bead array (CBA) kit (#551811), according to the manufacturer's instructions. Data were analysed using FCAP Array Software Version 3.0 (BD). Serum bone markers (DKK-1, osteocalcin, osteoprotegerin, osteopontin, sclerostin) were measured in technical duplicates using the MILLIPLEX luminex xMAP human bone magnetic bead panel (#HBNMAG-51K; EMD Millipore, Bayswater, Australia) according to the manufacturer's instructions. Serum CPP Fet-A reduction ratios were determined as described previously 7 . Patients gave written informed consent, and the study was approved by local regional ethics committee (Melbourne Health Research and Ethics Committee ref: 2012/141) and was conducted in accordance with the Declaration of Helsinki.
Animal studies. To assess the performance of the method in animals, we performed de novo studies in rats using the adenine-induced renal failure (AIRF) model as previously described 49 . Sixty male Wistar rats (8-week old; 250-325 g) were obtained from the local breeding colony. Animals were housed individually in a temperature-and humidity-controlled environment (25 °C at 25% humidity), with a 12/12 h light/dark cycle and had unlimited access to water and standard chow (Harlan-Tekland 22/5 diet 8640: 1.13% Ca, 0.94% P, 3.0 IU/g cholecalciferol, 22.6% protein). After 1 week of acclimatisation, the rats were randomly allocated into 2 groups (30/group). Uraemia was induced in one group of animals with an adenine-supplemented diet: 0.75% for 2 weeks, then 0.5% for a further 2 weeks. We employed this tapered dose to avoid severe renal failure and mortality. The remaining animals received standard chow throughout this 4 week period and served as the normal renal function Control group. Uraemic and Control groups were then further divided into 2 subgroups (15/ group) and were allocated to receive either standard chow (as above; normal phosphate diet, NPD) or a high phosphate-containing feed (0.6% Ca, 1.21% P, 18.6% protein from grain, 2.2 IU/g cholecalciferol; high phosphate diet, HPD) for a further 6 weeks. AIRF groups were balanced based on body weight (monitored weekly), serum creatinine and phosphate. Control animals were allocated according to body weight. No animals died during the study. At the beginning of week 0 (i.e. prior to the commencement of dietary adenine), and end of weeks 4 and 10, animals were placed in metabolic cages for the collection of urine. Morning blood samples were also taken at the same timepoints. Blood samples were allowed to clot for 60 min at room temperature and then centrifuged (3,000 g, 10 min) to separate serum from cells. Urine samples were collected on wet ice for 8-14 (max) hours and adjusted to estimate 24 hour clearance. Urinary sediment was removed by centrifugation (5,000 g, 15 min, 4 °C) prior to storage. Animals were not offered food during the urine collection period. Serum and urine aliquots were stored at −80 °C until analysis or processed further for nanoparticle analysis (as described elsewhere). At the end of week 10, rats were anesthetised and sacrificed by exsanguination via aortic puncture.
Calcium, creatinine and urea were measured using colorimetric kits from BioAssay Systems (DICA-500, DICT-500 and DIUR-500, respectively). Phosphate was determined using a BioVision Systems colorimetric kit (K-410). Creatinine clearance (CrCl) was estimated according to the formula: urine creatinine concentration × 24 hour urine volume/serum creatinine concentration. The fractional excretion of phosphate (FeP) was calculated according to the formula: urine phosphate concentration × serum creatinine concentration/serum phosphate concentration × urine creatinine concentration ×100%. Intact FGF23 and PTH were measured by ELISA according to the manufacturer's instructions (Immutopics Inc, San Clemente, USA). Serum IL-1β, IL-6 and TNF-α concentrations were measured by ELISA (R&D Systems, Abingdon, UK).
Additionally, we accessed archived serum samples from experiments encompassing both uraemic and non-uraemic pathologies, together with their respective controls, from which blood samples were collected at planned sacrifice. Experimental designs followed previously published reports [87][88][89][90] . In rats with subtotal nephrectomy (5/6Nx), renal insufficiency was induced by a two-step surgical procedure involving uninephrectomy followed by ligation of 2/3 branches of the renal artery supplying the remnant kidney. Control animals underwent sham surgery. Blood was taken at 6 weeks post-surgery. Animals were fed standard chow throughout as above. Unilateral ureteric obstruction (UUO) was performed in male C57BL/6J mice (8-10-week-old). Blood was taken 3-days post-surgery. A parallel control group consisted of un-operated male animals. UUO and control mice received standard chow throughout. A final group of animals consisted of male ApoE −/− mice (C57BL/6J background) that were fed a Western-style high-fat diet (21% fat, 0.21% cholesterol; #RD12079B) from 5 to 19 weeks of age. Same aged male C57BL/6J mice given standard chow were used as controls. All serum samples had been stored at −80°C and were analyzed without previous freeze/thaw cycles.
All experimental protocols were approved by the Monash University Animal Ethics Committee, and were carried out in accordance with the Australian Code of Practice for the Care and Use of Laboratory Animals for Scientific Purposes.
Electron microscopy. Cryogenic-electron microscopy was performed as previously described 44 . Briefly, particles were pelleted from diluted serum by centrifugation (30,000 to 50,000 g, 2 h, 4 °C; as indicated in the text), washed three times in TBS (pH 7.4) and re-suspended in 0.22 μm filtered MilliQ water. Samples were plunged frozen in liquid ethane before observation on a Tecnai F30 (FEI, Netherlands) operating at 300 kV.
Statistics. Variables were expressed as mean (SD) or median (25th-75th percentile) or as otherwise indicated. D' Agostino & Pearson's omnibus test was used to assess normality. Non-parametrically distributed variables were natural log-transformed before further analysis. Comparison of cohort subgroups was performed using one-way ANOVA with Bonferroni correction or Kruskal-Wallis test with Dunns post-test for multiple comparisons as appropriate. Pearson's correlation coefficients (r) were calculated to assess the association of flow cytometric parameters and other variables. Partial correlation coefficients (r p ) were determined to control for mediating factors. Unpaired Welch's t tests were used for 2 group comparisons. Deming regression and Bland-Altman plots were used to evaluate the agreement between methods. For the adenine rat study, linear mixed models were used to assess the change in measured variable from baseline. Group and time were included as fixed effects and a random intercept for each animal was included as random effects. To evaluate whether the change in measured variable from baseline differed by group, an interaction term for each group and time was introduced. Residual plots were inspected to look for deviations from normality and confirm model assumptions. Two-tailed P values of <0.05 were considered significant. Analyses were performed using Stata version 12 (StataCorp, College Station, Texas, USA).