Effect Of Nutritional Calcium And Phosphate Loading On Calciprotein Particle Kinetics in Adults With Normal And Impaired Kidney Function

Abstract

Plasma approaches metastability concerning 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 minimize 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 is 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.

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Introduction

Calcium and phosphate form highly insoluble salts in aqueous solution and while metabolism in all living organisms is dependent on these essential nutrients, their coexistence in biological fluids creates an inherent mineralization risk and the need for tight regulation1. Most extracellular fluids like plasma, are considered metastable or approaching metastability concerning their calcium and phosphate ionic activities2, readily sustaining crystal growth if nucleated. This tendency for calcium and phosphate to precipitate has been harnessed by vertebrates for their unique biomechanical properties during the evolution of the skeletal and dental tissues. However, at the same time, this mandated mechanisms of rapid mobilization and efficient bulk transport of mineral precursors to meet the demands of growth and repair of these hard tissues, as well as strategies of restricting mineralization to these sites and preventing unwanted calcification in soft tissues3. Indeed, even modest elevations in plasma calcium and phosphate concentrations are associated with an increased risk of pathological calcification of the arteries and tissues4. Since excess calcium and phosphate are almost exclusively excreted by the kidney, this risk, along with its associated cardiovascular sequelae, is substantially higher in those with Chronic Kidney Disease (CKD), where disturbances in mineral metabolism become evident with relatively small decrements in glomerular filtration rate (GFR)5,6. Given the abundance of calcium and phosphate in the diet to which mammals are intermittently exposed, one might suspect the risk of ectopic mineralization to be highest following a meal due to the anticipated surge in mineral ions absorbed from the intestine. Yet, contrary to this expectation, plasma calcium and phosphate concentrations change minimally in the post-prandial period and while increased urinary excretion helps to maintain homeostasis7, this response is not instantaneous and lags by some hours after ingestion of millimolar amounts of calcium and phosphate8. As a solution to this physiological challenge, a blood-borne system for buffering and transporting minerals has long been suspected9, and recent attention has turned to colloidal mineral-protein complexes that could fulfill this function and safely chaperone the intermittent fux of minerals through the extracellular fluid to sites of utilization or disposal10.

In plasma, the most potent and abundant protein mineralization regulator is the liver-derived glycoprotein fetuin-A11, which interacts with nascent colloidal mineral-protein complexes to form calciprotein particles (CPP)12,13. Analogous to how apolipoproteins solubilize their lipid cargo for transport, fetuinA stabilizes poorly soluble calcium phosphates, preventing crystal growth and precipitation, while facilitating their uptake in tissues for utilization or clearance14–16. An inability to make or sufficiently stabilize CPP, as seen in fetuin-A knockout mice, results in one of the most severe phenotypes of ectopic calcification known17, where mineral-containing complexes precipitate directly in the lumen of the microvasculature leading to occlusion, ischemia, necrosis, and fbrosis18. CPP is generated through a series of ordered steps, initially with the binding of spontaneously formed clusters of calcium and phosphate ions to fetuin-A, forming calciprotein monomers (CPM)19, which then serve as the building blocks for larger polymeric structures; first, coalescing to form spherical primary CPP (CPP-I) containing amorphous calcium phosphate, before transforming into larger and denser ellipsoidal secondary CPP (CPP-II) containing crystalline hydroxyapatite12.

Beyond the purported physiological role of CPP formation in sequestering and dispersing excess minerals, elevated levels are also observed in states of impaired mineral metabolism, including in CKD20. In this setting, higher serum levels of CPM and CPP have been linked to an increased risk of cardiovascular events21–25 and mortality22. Pre-clinical studies suggest that CPP may directly induce vascular smooth muscle cell calcifcation26,27, activation of cellular inflammatory and cytotoxic pathways28,29, as well as vascular luminal and endothelial lesions30. Taken together, it has been proposed that prolonged exposure to chronically elevated levels of serum CPP may help explain the links between excess dietary minerals and poor patient outcomes in CKD14,31. Intriguingly, recent data also suggest that circulating CPM is filtered at the glomerulus29, implying that kidney impairment may affect CPP metabolism via multiple mechanisms.

Recent methodological advancements permit direct quantification of CPM32, CPP-I, and CPP-II33, with these assays now applied across several observational and interventional clinical studies24,25,34–39. Another novel complementary method for assessing this system is the T50 test, a functional assessment of the capacity of serum to resist ex vivo formation of CPP-II when challenged with supersaturating amounts of calcium and phosphate40. A lower T50 has been consistently associated with an increased risk of vascular pathology and mortality in individuals with normal kidney function41, as well as in various CKD cohorts, including both non-dialysis22,42,43 and also dialysis-dependent CKD44. In addition to the kinetics of CPP-II formation, the size (hydrodynamic radius) of the CPP-II molecules generated in the T50 assay can also be measured40,45, and may provide additional prognostic information about vascular risk46,47.

The notion of a dietary origin of CPP is strongly supported by the observation that high-phosphate feeding is associated with increased ambient levels of CPM48 and CPP33 in animal studies, and the reduction in serum CPM34 and CPP37 in hemodialysis patients treated with intestinal phosphate binders. However, definitive observation of the effect of nutritional mineral intake on acute CPP kinetics in humans is lacking. Furthermore, the impact of CKD on post-prandial CPP metabolism has yet to be documented. To address these key evidence gaps, we conducted a controlled study of the effect of standardized food intake on serum CPM, CPP-I, CPP-II, T50, and CPP-II size, in fasted adults with normal or impaired kidney function.

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Methodology

1. Study population.

We studied 14 individuals with CKD and 16 age- and gender-matched healthy controls. Each participant had to be at least 18 years of age to be eligible. Participants were excluded if they: (i) had a history of mineral or bone disease, other than related to CKD; (ii) were being treated with an intestinal phosphate binder or calcitriol; or (iii) had a gastrointestinal disorder, history of lactose intolerance or were unwilling to consume the study meal. For the CKD group, we recruited seven individuals with an estimated glomerular filtration rate (eGFR) between 30 and 60 mL/min/1.73 m2 and seven with an eGFR<30 mL/min/1.73 m2, excluding participants who required dialysis or with a previous kidney transplant. Healthy controls had no history of chronic medical conditions and had normal kidney function (eGFR>60 mL/min/1.73 m2 ). The study was conducted by the Declaration of Helsinki.

All individuals provided written informed consent, and the study was approved by the local ethics committee (Melbourne Health Human Research Ethics Committee MH2018.363).

2. Procedure.

Each participant was studied after an overnight fast and sample collection commenced between 7.30 and 9.30 am. An intravenous cannula was inserted at the start of the study period. Before each blood sample was collected, an initial 5 mL draw from the cannula was discarded. Two initial fasting blood samples were taken 30 min apart to account for baseline variation. The mean values of these two-time points were used as “time 0”. Immediately after the collection of the second fasting sample, participants consumed a standardized meal (Sanitarium Up&Go liquid breakfast; 250 mL, vanilla flavor) containing 815 kJ energy, 300 mg calcium, and 188 mg phosphate (Table 1). Participants were instructed to consume the entirety of the drink within 5 min. Serial blood samples were collected at five post-prandial time points (30, 60, 120, 180, and 240 min) from the commencement of the meal. During the study period, participants were allowed to drink water but were not allowed to consume any other food or drink.

3. Outcome measures.

Blood was collected for repeated measurement of novel markers of mineral metabolism (CPM, CPP-I, CPP-II, T50, and CPP-II size) at each time point. Fetuin-A was measured at each time point, given its role as the principal mineral-binding protein present in CPM and CPP. We also measured serum phosphate, total calcium, magnesium, albumin, and bicarbonate at each time point, and serum intact parathyroid hormone (PTH) and intact fibroblast growth factor-23 (iFGF23) at three-time points (0, 120, and 240 min). Serum citrate was measured at 0, 30, and 60 min. Serum urea, creatinine, and 1,25 dihydroxy vitamin D were measured once at fasting baseline (0 min). Blood samples for novel markers of mineral metabolism, PTH, iFGF23, 1,25 dihydroxy vitamin D, and serum citrate, were allowed to clot for 30 min before centrifugation, and then serum aliquots were stored at −80 °C until batch analysis. All other biochemical measurements were performed at the time of sample collection using standard laboratory methods.

4. Gel‑fltration and fow cytometric assays for CPM and CPP.

We employed two complementary assays to quantitate different fractions of the circulating CPP pool49. Both assays use the fluorescently-labeled bisphosphonate derivative, OsteoSense 680EX (Perkin Elmer), which binds specifically to solid-phase calcium phosphate and preferentially to crystalline phases (e.g. hydroxyapatite). The ‘gel-fltration’ method of Miura et al.32 was used to measure small (<50 nm diameter), low-density mineral-laden fetuin-A colloids. Briefly, frozen serum samples were thawed for 24 h at 25 °C to induce aggregation of CPM and phase transition to crystalline calcium phosphate. Samples were then centrifuged for 30,000g for 2 h at 4 °C to remove larger CPP-I and CPP-II, leaving less dense crystal-laden fetuin-A monomer and multimers for staining with OsteoSense (0.5 µM) in HEPES-buffered DMEM (pH 8.0). Unbound dye was subsequently removed by gel filtration (Micro Bio-Spin® Columns with Bio-Gel® P-30, Bio-Rad) and the resultant fluorescence was measured using an infrared scanner (Odyssey CLx, LI-COR; EX 685 nm, EM 700 nm). Miura and colleagues32 referred to the mineral detected as low-density (L)-CPP, however, here we refer to them as CPM to reflect the origin of this mineral fraction in vivo. In our hands, the mean interassay analytical coefficient of variation (CVA) for the CPM assay was 4.9%.

For flow cytometric analysis, aliquots of frozen serum were processed using the standardized procedures described previously50. CPP-I and CPP-II were measured as previously described using a BD FACSVerse flow cytometer setup to resolve particles<200 nm from the background and operate with fluorescence triggering on OsteoSense-positive events33,37. In this assay, CPP is distinguished from membrane-delimited mineral-containing extracellular vesicles using phosphatidyl serine–binding cadherin-FITC (Haematologic Technologies Inc., Essex Junction, VT). CPP-I and CPP-II were distinguished by differences in side scatter (SSC) intensity (related to particle size) and OsteoSense fluorescence intensity. Interassay CVA for CPP-I and CPP-II were<15% and<10%, respectively.

5. Other mineral markers.

Serum T50 was measured by Calciscon AG, Biel, Switzerland, as previously described using a Nephelostar nephelometer (BMG Labtech, Ortenberg, Germany)40. The mean interassay CVA for T50 was 3.4%. CPP-II hydrodynamic radius was measured by dynamic light scattering using a DynaPro Plate Reader II (Wyatt Technology, Santa Barbara, CA, USA) as described by Chen et al.47 Te interassay CVA for CPP-II size was 4%. Commercial immunoassays were used to measure iFGF23 (Kainos Laboratories, Tokyo, Japan), 1,25 dihydroxy vitamin D Immunodiagnostic Systems, Boldon, UK), and fetuin-A (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Mean interassay CVA were 3.8%, 5.5%, and 3.2%, respectively. Serum citrate was measured using a colorimetric assay (Sigma-Aldrich, Darmstadt, Germany) with a mean interassay CVA of 3.5%.

6. Statistical analysis.

Using GLIMPSE, a validated linear mixed model power and sample size calculator51, we estimated that 10 participants would provide>90% power to detect a doubling of CPP in the post-prandial period with a type I error rate of 0.05. The study was not powered to detect a difference between groups.

Demographic and fasting biochemical data were compared between groups using an unpaired t-test or Kruskal–Wallis test for normal and skewed continuous variables respectively, and a chi-squared test for categorical variables.

We aimed to describe the within and between-group post-prandial response for each repeated parameter. To do this, we fitted linear mixed-effects models (LMM) for each parameter, using a restricted maximum likelihood approach and with an unstructured covariance matrix52. For each LMM we modeled group, categorical time, and group-by-time interaction as fixed effects, and a random intercept was included for each participant to account for the correlation of repeated measures. The control group and time ‘0’ were used as the reference values for the group and time, respectively. Coefficient estimates for group-by-time interaction terms were used to test for differences in post-prandial response between the CKD and control groups. After fitting each LMM, we also performed post hoc pairwise comparisons to test for differences in mean values between groups at each time point, and to test for deviation from the fasting baseline within each group. For these pairwise comparisons, we used the Bonferroni correction method to adjust for multiple comparisons. CPM, CPP-I, CPP-II, CPP-II size, PTH, and iFGF23 were naturally log-transformed before fitting LMMs to ensure normal distribution of residuals. For ease of interpretation, coefficient estimates for interaction terms from these models were then exponentiated to derive estimates of the percentage change. For the LMM of CPM, group-by-time interaction terms suggested a significant difference in post-prandial response between groups. To further explore the effect of kidney function on post-prandial levels of CPM we also calculated the area under the curve (AUC) for CPM using the cubic spline method (time 0 to 240 min) and examined the relationship between eGFR, AUC, and maximum concentration of CPM (using Spearman rank correlation) as well as between CKD and time of maximum CPM concentration (using chi-squared test).

Several samples had undetectable levels of CPP-I (2 samples in the control group, and 6 in the CKD group) or CPP-II (5 in the control group and 10 in the CKD group). For the main analyses, the lower limit of quantification for the assay (133 particles/mL) was used for these lef-censored values. To assess for potential bias from this approach, we performed a sensitivity analysis where LMMs for CPP-I and CPP-II were refitted after imputing lefcensored values using multi-level Tobit regression, where time and group were entered as independent variables 53.

Two-tailed p values<0.05 were considered significant. All data were analyzed using Stata MP version 17.0 (StataCorp, College Station, USA) and figures were produced using GraphPad Prism version 9.2.0 (GraphPad Sofware, San Diego, USA).

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Discussion

To the best of our knowledge, this is the first study to report a significant post-prandial effect of nutritional intake on serum levels of CPM, CPP-I, and CPP-II in humans. These effects were common to individuals with normal and impaired kidney function; however, the post-prandial excursion of serum CPM was much more pronounced in CKD participants. We also found an early and transient post-prandial effect on T50, which was present regardless of kidney function and accompanied by a concomitant increase in serum fetuin-A.

Our findings are consistent with the notion that intestinal absorption of a dietary mineral load can directly lead to the formation of circulating CPM, CPP-I, and CPP-II. This has previously been proposed10,15,16 but is based largely on animal data33,48. In contrast, evidence in humans has been indirect, coming from studies of intestinal phosphate binders in hemodialysis-dependent CKD patients34,37,55. A small study by Yamada et al. suggested diurnal variation in serum CPP with post-prandial peaks, however, this study used an older assay technique that was unable to separately quantify CPM and CPP sub-species, and participants were all hospitalized for the management of unstable diabetes56. In contrast, all participants in this study were clinically stable, and as far as we are aware, for the first time, we have demonstrated that post-prandial excursions are seen not only in individuals with CKD but also in healthy adults, substantiating the role of CPM and CPP formation in the normal physiological response to the ingestion of food.

Surges of serum CPM and CPP were seen even after relatively modest, and physiologically relevant, nutritional mineral loads (Table 1). In contrast, there was minimal post-prandial variation in the more conventional markers of mineral metabolism. Previous studies have similarly shown limited post-prandial deviation in phosphate7,57, except when subjects are challenged with large pharmacological loads58,59. In health, total body phosphate is regulated, such that net intestinal absorption is matched by urinary excretion8. However, this response is not instantaneous, and a lag of several hours may be seen before augmentation of urinary phosphate excretion occurs, even when the phosphate load is given intravenously8. Instead, animal models have demonstrated that other, non-renal, mechanisms serve to maintain serum ionic concentrations more acutely, via distribution to bone and other tissues59–61. It is plausible that the formation of CPM and CPP may be an important additional temporary depot of phosphate (and calcium), that can acutely buffer local mineral loads, such as from the gastrointestinal system14. The physicochemical properties of CPM and CPP mean that potentially large quantities of otherwise insoluble minerals can exist in the circulation without risk of precipitation, which ostensibly facilitates minerals to be safely transported in bulk to sites of use or clearance.

We observed a transient increase in serum fetuin-A in the CKD group. In contrast, fetuin-A appeared stable in controls, however, when pairwise comparisons were repeated without correction for multiple comparisons, there were significant increases at 30 and 60 min in controls (Supplementary Table S8), indicating that we may have been underpowered to detect an underlying effect. Beyond being a negative acute phase reactant, with levels strongly suppressed in response to acute and chronic infammation62, little is known about other mechanisms that directly regulate the hepatic synthesis and secretion of fetuin-A, and there is a paucity of previous data about diurnal, or acute post-prandial variation in any species. Given the observed rise in serum fetuin-A after feeding, especially in the CKD group, and the requisite role of fetuin-A in CPM and CPP formation, it is intriguing to consider whether feeding may be “sensed” via a yet unknown mechanism, leading to the hepatic release of fetuinA to coincide with an influx of mineral from the intestines. Indeed, Uedono et al. recently suggested that CPP itself may be a trigger for fetuin-A expression in cultured hepatocytes63. Thus, mechanisms controlling fetuin-A release and their response to mineral loading warrant further investigation.

T50 is a functional assessment of the serum’s ability to resist ex vivo CPP-II formation, representing a composite of various potentiating (including calcium and phosphate) and inhibiting (including fetuin-A, albumin, magnesium, citrate, and bicarbonate) calcification factors40. We observed an early but transient increase in T50. This was seen in both groups, but in the CKD group, the peak was coincident with increases in serum fetuin-A. Further, among all participants, change in T50 from baseline was very closely correlated with change in serum fetuin-A (Supplementary Fig. S2), suggesting that fetuin-A may be the main factor underlying the observed changes in T50. Indeed, we did not find corresponding early post-prandial changes in other known modulators of T50 (serum phosphate, calcium, bicarbonate, magnesium, or albumin), although this does not exclude more subtle changes in one, or a combination, of these factors. For instance, a positive group-by-time interaction was present for serum bicarbonate in the CKD group at 120 min, which overlapped with the increase in T50. As for fetuin-A, there was also a correlation between the change in bicarbonate and T50 from baseline (Supplementary Fig. S4), albeit this was relatively weak compared to the correlation with the former (r=0.385 vs. r=0.839). Nevertheless, the post-prandial “alkaline tide” is a recognized phenomenon, and it is plausible that multiple factors contributed to the observed post-prandial changes in T5064. The study meal also contained a surprising amount of citrate (~16 mM), a known potent inhibitor of CPP formation, however, although there was a small increase in serum citrate in the CKD group, this change did not coincide with the observed changes in T50, nor was there any overall correlation between change in T50 and serum citrate across both groups. Other previously described modulators of T50, such as pyrophosphate and zinc, were not measured in this study. Although these, or other yet unknown, factors may also have contributed to the observed post-prandial increase in T50, we note that quite substantial changes in concentration of these small inorganic molecules are generally required to significantly impact T5065. In contrast to the post-prandial effects on T50, the hydrodynamic radius of CPP-II remained ostensibly unchanged in both groups.

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While post-prandial excursions of CPM were observed in both groups, CPM levels were consistently higher in the CKD group at each of the time points, and group-by-time interaction terms indicated that the magnitude of the post-prandial response was significantly larger in CKD participants than in controls. Accordingly, in exploratory analysis, there was a strong correlation between eGFR and CPM AUC and maximum concentration. Peak levels of CPM also tended to occur later in the CKD group. Theoretically, these differences could indicate increased production of CPM following nutritional intake and/or an impaired capacity to clear CPM in those with CKD. The latter is supported by recent data by Koeppert et al., who used live two-photon microscopy to show that circulating CPM is predominantly cleared by glomerular filtration in mice29. A reasonable prediction, therefore, may be that the capacity to clear CPM becomes impaired as GFR declines. However, alterations in other aspects of mineral homeostasis, including bone turnover66, and delayed renal excretion of acute phosphate loads7,59 are also commonly seen in CKD and may well have contributed to elevations in post-prandial levels. Recent evidence has questioned the long-held notion that CKD affects net intestinal phosphate absorption57,67,68. We did however note that six of the 14 individuals in the CKD group were taking cholecalciferol. Considering the potential for stimulatory effects of vitamin D on intestinal mineral absorption, we performed an ad hoc exploratory analysis to test whether the use of nutritional vitamin D impacted the acute response of CPM, CPP-I, and CPP-II to feeding (Supplementary Tables S9–S11). Apart from a single positive group-by-time interaction for CPP-I at 30 min in those taking cholecalciferol, all other interaction terms and post hoc pairwise comparisons for CPM, CPP-I, and CPP-II were not significant, suggesting that cholecalciferol use was unlikely to have had a substantial effect on post-prandial CPM/CPP kinetics. While the small sample size and potential for confounding may preclude definitive conclusions, the lack of effect may also reflect the predominance of phosphate absorption via the paracellular pathway when the mineral is abundant, which is not actively regulated by the vitamin D axis69.

In contrast to CPM, we did not observe a strong effect on kidney function in either CPP-I or CPP-II. Unlike CPM, circulating CPP is primarily cleared from circulation by non-renal mechanisms. Animal and in vitro models have suggested rapid clearance of CPP-I predominantly by liver sinusoidal endothelial cells28, and of CPP-II by resident macrophages of the liver and spleen70. It is plausible that while participants in our CKD group displayed evidence of altered CPM metabolism, that these discrete CPP clearance pathways were sufficient to maintain normal post-prandial CPP profiles. We had anticipated that the CKD group would have discerningly higher levels of CPP compared to controls based on previous studies, albeit these studies enrolled patients with more advanced CKD who were dialysis dependent33, or used the older and indirect method of CPP measurement20,23. Individuals with more advanced CKD than those studied here may exhibit greater differences in fasting and post-prandial CPP levels. The lack of separation for CPP between our CKD and control group may also be due to our limited participant numbers, which also limited our ability to formally test for an effect of the CKD stage (Supplementary Fig. S6). Indeed, our study was powered-based on examining post-prandial responses rather than between-group differences. Of note, CPP-I appeared to return to fasting levels earlier in controls than CKD participants, and there was a positive group-by-time interaction for CPP-II in CKD participants at 120 min. Both findings potentially signal that we may have observed more pronounced between-group effects in a larger cohort. Higher analytical imprecision for flow cytometry-based CPP measurements may have also contributed to the null findings.

In addition to providing new insights into the physiology of CPM and CPP metabolism, our study also has direct relevance for optimizing the use of these novel markers of mineral metabolism in future studies. While each of these novel assays has shown promise in early clinical work, very few studies have reported43 or controlled for fasting/absorptive status46,56. In this study, several individuals had undetectable levels of CPP-I or CPP-II in the fasting and early post-prandial period, and this profound effect of fasting on serum CPP is in itself previously unreported and notable funding. In contrast, fasting CPM levels were measurable and significantly higher in the CKD group than in controls, which suggests that sustained elevations in CPM may not necessarily manifest in elevated CPP levels in those with non-dialysis-dependent CKD.

Previous epidemiological studies have suggested links between elevated levels of CPM and CPP with a range of surrogate markers of vascular disease21,23,24, as well as with cardiovascular events25 and all-cause mortality22. Further, lab-based studies have provided plausible mechanisms by which CPP may mediate these pathological vascular outcomes26–28. However, while in vitro studies have suggested that many of these toxicities are induced in a dose-dependent manner if CPP does have a normal physiological role in health then it is unclear at what point these particles may become injurious. Knowledge of the effect of nutritional intake on CPP kinetics as revealed here may prove crucial to further understanding this process. A threshold concentration for the onset of pathological effects of CPM and CPP may (at least initially) only be reached in the post-prandial state in vivo. If so, dynamic testing of the post-prandial response may provide additional opportunities to evaluate earlier manifestations of dysregulated mineral metabolism, as well as the risk of associated vascular disease. Another important possibility is that the composition and thus the intrinsic toxicity of CPP-I and CPP-II differs among health and CKD45.

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Limitations

We acknowledge that this study has several limitations, including limited patient numbers, as already discussed. Notably, we also used a standardized meal given after an overnight fast, so cannot comment on the effect of varying meal composition or the effect of subsequent meals given throughout the rest of the day. We chose the meal based on its commercial availability, allowing for standardization between participants, and because it represented physiologically relevant nutritional loads (Table 1). It is however possible that if participants were challenged with larger mineral loads, further separation between those with normal and impaired kidney function may have been apparent. Similarly, given we only observed individuals for four hours post-meal, it is conceivable that the cumulative effect of subsequent meals may have also distinguished groups further.

We did not recover feces or urine and so cannot comment on the total minerals absorbed or excreted during the study. We assume that changes in each measure seen after feeding are directly related to nutritional intake. This assumption is supported indirectly by detailed imaging studies showing fux of calcium and phosphate ions following food intake57, as well as direct evidence from animal studies showing that acute oral gavage of mice with a buffered phosphate solution results in serum spikes in CPM/CPP48. Nevertheless, we did not observe participants over extended fasting conditions, and so cannot conclusively account for non-dietary related diurnal fluctuations in any of the studied parameters. We did however have two fasting samples, the average of which was used as “time 0”, and variability between these fasting samples was trivial compared to the magnitude of changes seen in the post-prandial period.

We used the Bonferroni method to account for the multiple post hoc pairwise comparisons between and within groups for each parameter. This is undoubtedly a conservative approach, and to our knowledge not one adopted by no other similar feedings studies of post-prandial mineral metabolism. We chose this approach given the large number of time points and comparisons that were made and reasoned that it was preferable to focus on the most robust and significant signals. However, as a result, we may have missed smaller, but potentially relevant effects. We did not correct for multiple testing across different mineral parameters given the likelihood of interdependent physiologically linked changes.

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Conclusion

Our study has revealed for the first time that nutritional mineral intake leads to the formation of CPM and CPP in blood as a normal physiological response to feeding. These findings corroborate the hypothesis that CPM/ CPP formation helps to build post-prandial mineral loads, functioning as a temporary circulating store of bulk calcium phosphates ultimately destined for utilization/storage (e.g., a mineral precursor for bone mineralization) or elimination. We also observed higher fasting levels of serum CPM and a larger post-prandial response in those with impaired kidney function, suggesting that CPM metabolism is manifestly altered in CKD. Analysis of post-prandial CPM/CPP handling may provide new insights into the mechanisms linking excessive dietary calcium and phosphate intake to increased risks of cardiovascular disease in patients with impaired mineral excretion. More broadly, these novel findings underscore the important, but often neglected, contribution of colloidal biochemistry to mineral homeostasis.

References

1. Magalhães, M. C. F., Marques, P. A. A. P. & Correia, R. N. Biomineralization—Medical Aspects of Solubility (eds. Königsberger, E. & Königsberger, L.). 71–123. (Wiley, 2006).

2. Holt, C., Lenton, S., Nylander, T., Sorensen, E. S. & Teixeira, S. C. Mineralisation of soft and hard tissues and the stability of biofluids. J. Struct. Biol. 185, 383–396 (2014).

3. Reznikov, N., Steele, J. A. M., Fratzl, P. & Stevens, M. M. A materials science vision of extracellular matrix mineralization. Nat. Rev. Mater. 1, 16041 (2016).

4. Smith, E. R. Vascular calcification in uremia: New-age concepts about an old-age problem. Methods Mol. Biol. 1397, 175–208 (2016).

5. Chen, J. et al. Coronary artery calcification and risk of cardiovascular disease and death among patients with chronic kidney disease. JAMA Cardiol. 2, 635–643 (2017).

6. Kestenbaum, B. R. et al. Incidence and progression of coronary calcification in chronic kidney disease: The Multi-Ethnic Study of Atherosclerosis. Kidney Int. 76, 991–998 (2009).

7. Isakova, T. et al. Postprandial mineral metabolism and secondary hyperparathyroidism in early CKD. J. Am. Soc. Nephrol. 19, 615–623 (2008).

8. Scanni, R., vonRotz, M., Jehle, S., Hunter, H. N. & Krapf, R. Te human response to acute enteral and parenteral phosphate loads. J. Am. Soc. Nephrol. 25, 2730–2739 (2014).

9. Pasch, A., Jahnen-Dechent, W. & Smith, E. R. Phosphate, calcification in blood, and mineral stress: Te physiologic blood mineral buffering system and its association with cardiovascular risk. Int. J. Nephrol. 2018, 9182078 (2018).

10. Jahnen-Dechent, W. et al. Mud in the blood: The role of protein-mineral complexes and extracellular vesicles in biomineralization and calcification. J. Struct. Biol. 212, 107577 (2020).

11. Schinke, T. et al. The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis. J. Biol. Chem. 271, 20789–20796 (1996).

12. Heiss, A. et al. Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J. Biol. Chem. 278, 13333–13341 (2003).

13. Cai, M. M., Smith, E. R. & Holt, S. G. Te role of fetuin-A in mineral trafficking and deposition. Bonekey Rep. 4, 672 (2015).

14. Smith, E. R., Hewitson, T. D. & Jahnen-Dechent, W. Calciprotein particles: Mineral behaving badly? Curr. Opin. Nephrol. Hypertens. 29, 378–386 (2020).

15. Jahnen-Dechent, W., Schäfer, C., Ketteler, M. & McKee, M. D. Mineral chaperones: A role for fetuin-A and osteopontin in the inhibition and regression of pathologic calcification. J. Mol. Med. (Berl.) 86, 379–389 (2008).

16. Jahnen-Dechent, W. & Smith, E. R. Nature’s remedy to phosphate woes: Calciprotein particles regulate systemic mineral metabolism. Kidney Int. 97, 648–651 (2020).

17. Schafer, C. et al. The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J. Clin. Invest. 112, 357–366 (2003).

18. Herrmann, M. et al. Lumenal calcification and micro vasculopathy in fetuin-A-deficient mice lead to multiple organ morbidity. PLoS ONE 15, e0228503 (2020).

19. Heiss, A., Pipich, V., Jahnen-Dechent, W. & Schwahn, D. Fetuin-A is a mineral carrier protein: Small angle neutron scattering provides new insight on Fetuin-A controlled calcification inhibition. Biophys. J. 99, 3986–3995 (2010).

20. Smith, E. R. et al. Serum fetuin-A concentration and fetuin-A-containing calciprotein particles in patients with chronic inflammatory disease and renal failure. Nephrology (Carlton) 18, 215–221 (2013).

21. Smith, E. R. et al. Phosphorylated fetuin-A-containing calciprotein particles are associated with aortic stiffness and a procyclic milieu in patients with pre-dialysis CKD. Nephrol. Dial. Transplant. 27, 1957–1966 (2012).

22. Smith, E. R. et al. Serum calcification propensity predicts all-cause mortality in predialysis CKD. J. Am. Soc. Nephrol. 25, 339–348 (2014).

23. Hamano, T. et al. The fetuin-mineral complex reflects extraosseous calcification stress in CKD. J. Am. Soc. Nephrol. 21, 1998–2007 (2010).

24. Nakazato, J. et al. Association of calciprotein particles measured by a new method with coronary artery plaque in patients with coronary artery disease: A cross-sectional study. J. Cardiol. 74, 428–435 (2019).

25. Gatate, Y. et al. Mid-term predictive value of calciprotein particles in maintenance hemodialysis patients based on a gel-filtration assay. Atherosclerosis 303, 46–52 (2020).

26. Cai, M. M. X., Smith, E. R., Tan, S. J., Hewitson, T. D. & Holt, S. G. Te role of secondary calciprotein particles in the mineralization paradox of chronic kidney disease. Calcif. Tissue. Int. 101, 570–580 (2017).

27. Aghagolzadeh, P. et al. The calcification of vascular smooth muscle cells is induced by secondary calciprotein particles and enhanced by tumor necrosis factor alpha. Atherosclerosis 251, 404–414 (2016).

28. Koppert, S. et al. Cellular clearance and biological activity of calciprotein particles depend on their maturation state and crystallinity. Front. Immunol. 9, 1991 (2018).

29. Koeppert, S. et al. Live imaging of calciprotein particle clearance and receptor-mediated uptake: role of calciprotein monomers. Front. Cell. Dev. Biol. 9, 633925 (2021).

30. Shishkova, D. et al. Calcium phosphate bions cause intimal hyperplasia in intact aortas of normolipidemic rats through endothelial injury. Int. J. Mol. Sci. 20, 5728 (2019).

31. Kuro-o, M. A phosphate-centric paradigm for pathophysiology and therapy of chronic kidney disease. Kidney Int. Suppl. 2011(3), 420–426 (2013).

32. Miura, Y. et al. Identification and quantification of plasma calciprotein particles with distinct physical properties in patients with chronic kidney disease. Sci. Rep. 8, 1256 (2018).

33. Smith, E. R. et al. A novel fluorescent probe-based flow cytometric assay for mineral-containing nanoparticles in serum. Sci. Rep. 7, 5686 (2017).

34. Nakamura, K. et al. The effect of lanthanum carbonate on calciprotein particles in hemodialysis patients. Clin. Exp. Nephrol. 24, 323–329 (2020).

35. Tiong, M. K. et al. Effect of a medium cut-of dialyzer on protein-bound uremic toxins and mineral metabolism markers in patients on hemodialysis. Hemodial. Int. https://doi.org/10.1111/hdi.12924 (2021).

36. Tiong, M. K., Smith, E. R., Toussaint, N. D., Al-Khayyat, H. F. & Holt, S. G. Reduction of calciprotein particles in adults receiving infliximab for chronic inflammatory disease. JBMR Plus 5, e10497 (2021).

37. Smith, E. R., Pan, F. F. M., Hewitson, T. D., Toussaint, N. D. & Holt, S. G. Effect of sevelamer on calciprotein particles in hemodialysis patients: Te sevelamer versus calcium to reduce fetuin-A-containing calciprotein particles in dialysis (SCaRF) randomized controlled trial. Kidney Int. Rep. 5, 1432–1447 (2020).

38. Ruderman, I., Smith, E. R., Toussaint, N. D., Hewitson, T. D. & Holt, S. G. Longitudinal changes in bone and mineral metabolism after cessation of cinacalcet in dialysis patients with secondary hyperparathyroidism. BMC Nephrol. 19, 113 (2018).

39. Bressendorf, I. et al. The effect of increasing dialysate magnesium on calciprotein particles, inflammation, and bone markers: Post hoc analysis from a randomized controlled clinical trial. Nephrol. Dial. Transplant. 36, 713–721 (2021).

40. Pasch, A. et al. The nanoparticle-based test measures the overall propensity for calcification in serum. J. Am. Soc. Nephrol. 23, 1744–1752 (2012).

41. Eelderink, C. et al. Serum calcification propensity and the risk of cardiovascular and all-cause mortality in the general population: The PREVENT Study. Arterioscler. Tromb. Vasc. Biol. 40, 1942–1951 (2020).

42. Bundy, J. D. et al. Serum calcification propensity and coronary artery calcification among patients with CKD: Te CRIC (Chronic Renal Insufficiency Cohort) study. Am. J. Kidney. Dis. 73, 806–814 (2019).

43. Bundy, J. D. et al. Serum calcification propensity and clinical events in CKD. Clin. J. Am. Soc. Nephrol. 14, 1562–1571 (2019).

44. Pasch, A. et al. Blood calcification propensity, cardiovascular events, and survival in patients receiving hemodialysis in the EVOLVE trial. Clin. J. Am. Soc. Nephrol. 12, 315–322 (2017).

45. Smith, E. R., Hewitson, T. D., Hanssen, E. & Holt, S. G. Biochemical transformation of calciprotein particles in uremia. Bone 110, 355–367 (2018).

46. Chen, W. et al. Associations of serum calciprotein particle size and transformation time with arterial calcification, arterial stiffness, and mortality in incident hemodialysis patients. Am. J. Kidney Dis. 77, 346–354 (2021).

47. Chen, W. et al. Patients with advanced chronic kidney disease and vascular calcification have a large hydrodynamic radius of secondary calciprotein particles. Nephrol. Dial. Transplant. 34, 992–1000 (2019).

48. Akiyama, K. I. et al. Calciprotein particles regulate fibroblast growth factor-23 expression in osteoblasts. Kidney Int. 97, 702–712 (2020).

49. Smith, E. R. Calciprotein particles: A mineral biomarker in need of better measurement. Atherosclerosis 303, 43–45 (2020).

50. Smith, E. R. Te isolation and quantitation of fetuin-A-containing calciprotein particles from biological fluids. Methods Mol. Biol. 1397, 221–240 (2016).

51. Guo, Y., Logan, H. L., Glueck, D. H. & Muller, K. E. Selecting a sample size for studies with repeated measures. BMC Med. Res. Methodol. 13, 100 (2013).

52. Liu, C., Cripe, T. P. & Kim, M. O. Statistical issues in longitudinal data analysis for treatment efficacy studies in the biomedical sciences. Mol. Ter. 18, 1724–1730 (2010).

53. Tobin, J. Estimation of relationships for limited dependent variables. Econometrica 26, 24–36 (1958).

54. Ter Meulen, K. J. et al. Citric-acid dialysate improves the calcification propensity of hemodialysis patients: A multicenter prospective randomized cross-over trial. PLoS ONE 14, e0225824 (2019).

55. Tiem, U. et al. The effect of phosphate binder therapy with sucroferric oxyhydroxide on calcification propensity in chronic hemodialysis patients: a randomized, controlled, crossover trial. Clin. Kidney J. 14, 631–638 (2021).

56. Yamada, H. et al. Daily variability in serum levels of calciprotein particles and their association with mineral metabolism parameters: A cross-sectional pilot study. Nephrology (Carlton) 23, 226–230 (2018).

57. Stremke, E. R. et al. Intestinal phosphorus absorption in moderate CKD and healthy adults was determined using a radioisotopic tracer. J. Am. Soc. Nephrol. 32, 2057–2069 (2021).

58. Volk, C. et al. Acute effects of an inorganic phosphorus additive on mineral metabolism and cardiometabolic risk factors in healthy subjects. J. Clin. Endocrinol. Metab. 107, e852–e864 (2022).

59. Turner, M. E. et al. Impaired phosphate tolerance was revealed with an acute oral challenge. J. Bone Miner. Res. 33, 113–122 (2018).

60. Tomas, L. et al. Acute adaption to oral or intravenous phosphate requires parathyroid hormone. J. Am. Soc. Nephrol. 28, 903–914 (2017).

61. Zelt, J. G. et al. Acute tissue mineral deposition in response to a phosphate pulse in experimental CKD. J. Bone Miner. Res. 34, 270–281 (2019).

62. Lebreton, J. P. et al. The serum concentration of human alpha 2 HS glycoprotein during the inflammatory process: evidence that alpha 2 HS glycoprotein is a negative acute-phase reactant. J. Clin. Invest. 64, 1118–1129 (1979).

63. Uedono, H. et al. Effects of fetuin-A-containing calciprotein particles on posttranslational modifications of fetuin-A in HepG2 cells. Sci. Rep. 11, 7486 (2021).

64. Niv, Y. & Fraser, G. M. Te alkaline tide phenomenon. J. Clin. Gastroenterol. 35, 5–8 (2002).

65. Smith, E. R., Hewitson, T. D. & Holt, S. G. Diagnostic tests for vascular calcification. Adv. Chronic Kidney Dis. 26, 445–463 (2019).

66. Sprague, S. M. et al. Diagnostic accuracy of bone turnover markers and bone histology in patients with CKD treated by dialysis. Am. J. Kidney Dis. 67, 559–566 (2016).

67. Vorland, C. J. et al. Kidney disease progression does not decrease intestinal phosphorus absorption in a rat model of chronic kidney disease-mineral bone disorder. J. Bone Miner. Res. 35, 333–342 (2020).

68. Marks, J. et al. Intestinal phosphate absorption in a model of chronic renal failure. Kidney Int. 72, 166–173 (2007).

69. Hill Gallant, K. M. & Vorland, C. J. Intestinal phosphorus absorption: Recent findings in translational and clinical research. Curr. Opin. Nephrol. Hypertens. 30, 404–410 (2021).

70. Herrmann, M. et al. Clearance of fetuin-A-containing calciprotein particles is mediated by scavenger receptor-A. Circ. Res. 111, 575–584 (2012).

71. Sanitarium Health Food Company. UP&GO™ Vanilla Ice Flavour. https://www.sanitarium.com.au/products/up-and-go/up-and-go/ vanilla-ice-favor (2021).

72. Australian Government National Health and Medical Research Council. Nutrient Reference Values for Australia and New Zealand. https://www.nrv.gov.au/nutrients (2021).

Mark K.Tiong1,2, Michael M. X. Cai1 , Nigel D.Toussaint1,2, Sven‑JeanTan1,2, Andreas Pasch3,4,5 & Edward R. Smith1,2

1 Department of Nephrology, The Royal Melbourne Hospital, Grattan Street, Parkville, VIC 3052, Australia.

2 Department of Medicine (RMH), University of Melbourne, Parkville, Australia.

3 Calciscon AG, Biel, Switzerland.

4 Lindenhofspital Bern, Bern, Switzerland.

5 Department of Physiology and Pathophysiology, Johannes Kepler University, Linz, Austria.