Calciprotein particle counts associate with vascular remodelling in chronic kidney disease.

AIMS: Calciprotein particles (CPPs) are circulating calcium and phosphate nanoparticles associated with development of vascular calcification (VC) in chronic kidney disease (CKD). Although recent studies have been focusing on associations of CPPs with presence of VC in CKD, insights in the underlying processes and mechanisms by which CPPs might aggravate VC and vascular dysfunction in vivo are currently lacking. Here, we assessed overall burden of abdominal VC in healthy kidney donors and CKD patients, and subsequently performed transcriptome profiling in vascular tissue obtained from these subjects, linking outcome to CPP counts and calcification propensity. METHODS AND RESULTS: Calcification scores were quantified in renal arteries, iliac arteries and abdominal aorta, using computed tomography (CT) scans of kidney donors and CKD patients. Vascular tissue was collected from kidney donors (renal artery) and CKD patients (iliac artery), after which bulk RNA sequencing and gene set enrichment analysis (GSEA) was performed on a subset of patients. Calcification propensity (crystallization time, T50) was measured using nephelometry, and CPP counts with microparticle flow cytometric analysis. Increased calcification scores (based on CT) were found in CKD patients compared to kidney donors. Transcriptome profiling revealed enrichment for processes related to endothelial activation, inflammation, extracellular matrix (ECM) remodelling and ossification in CKD vascular biopsies compared to kidney donors. Calcification propensity was increased in CKD, as well as CPP counts, of which the latter significantly associated with markers of vascular remodelling. CONCLUSIONS: Our findings reveal that CKD is characterized by systemic VC with increased calcification propensity and CPP counts. Transcriptome profiling showed altered vascular gene expression with enrichment for endothelial activation, inflammation, ECM remodelling and ossification. Moreover, we demonstrate for the first time that vascular remodelling processes are associated with increased circulating CPP counts. Interventions targeting CPPs are promising avenues for alleviating vascular remodelling and VC in CKD.

The effect of parathyroid hormone lowering by etelcalcetide therapy on calcification propensity and calciprotein particles in hemodialysis patients.

Background: This study investigated whether parathyroid hormone (PTH) lowering with etelcalcetide, and the consequent effects on mineral and bone metabolism, could improve serum calcification propensity (T50 time) and decrease calciprotein particle (CPP) load in hemodialysis patients with secondary hyperparathyroidism. Methods: In this single-arm, prospective, dose-escalation proof-of-principle study, hemodialysis patients received etelcalcetide at 2.5 mg/dialysis session with increments of 2.5 mg every 4 weeks to a maximum dose of 15 mg three times a week or until a pre-specified safety endpoint was reached, followed by an 8-week wash-out phase. Results: Out of 36 patients recruited (81% male, 62 ± 13 years), 16 patients completed the study per protocol with a mean maximum tolerated dose of etelcalcetide of 9.5 ± 2.9 mg/dialysis session. With escalating doses of etelcalcetide, PTH and serum calcium levels significantly decreased (P < 0.0001). While there was no significant change in T50 times or serum phosphate levels, etelcalcetide did yield significant and consistent reductions in serum levels of endogenous calciprotein monomers [-35.4 (-44.4 to -26.5)%, P < 0.0001], primary [-22.4 (-34.5 to -10.3)%, P < 0.01] and secondary CPP [-29.1 (-45.7 to -12.4)%, P < 0.01], an effect that was reversed after therapy withdrawal. Serum levels of osteoclastic markers significantly decreased with escalating doses of etelcalcetide, while levels of the osteoblastic marker remained stable. Conclusions: Lowering of PTH with etelcalcetide did not result in statistically significant changes in T50. By contrast, homogenous reductions in serum levels of calciprotein monomers, primary and secondary CPP were observed.

Serum T50 predicts cardiovascular mortality in individuals with type 2 diabetes: A prospective cohort study.

BACKGROUND AND AIMS: Individuals with type 2 diabetes (T2D) have a higher risk of cardiovascular disease, compared with those without T2D. The serum T50 test captures the transformation time of calciprotein particles in serum. We aimed to assess whether serum T50 predicts cardiovascular mortality in T2D patients, independent of traditional risk factors. METHODS: We analyzed 621 individuals with T2D in this prospective cohort study. Cox regression models were performed to test the association between serum T50 and cardiovascular and all-cause mortality. Causes of death were categorized according to ICD-10 codes. Risk prediction improvement was assessed by comparing Harrell's C for models without and with T50. RESULTS: The mean age was 64.2 ± 9.8 years, and 61% were male. The average serum T50 time was 323 ± 63 min. Higher age, alcohol use, high-sensitive C-reactive protein, and plasma phosphate were associated with lower serum T50 levels. Higher plasma triglycerides, venous bicarbonate, sodium, magnesium, and alanine aminotransferase were associated with higher serum T50 levels. After a follow-up of 7.5[5.4-10.7] years, each 60 min decrease in serum T50 was associated with an increased risk of cardiovascular (fully adjusted HR 1.32, 95% CI 1.08-1.50, and p = 0.01) and all-cause mortality (HR 1.15, 95%CI 1.00-1.38, and p = 0.04). Results were consistent in sensitivity analyses after exclusion of individuals with estimated glomerular filtration rate <45 or <60 mL/min/1.73 m2 and higher plasma phosphate levels. CONCLUSIONS: Serum T50 improves prediction of cardiovascular and all-cause mortality risk in individuals with T2D. Serum T50 may be useful for risk stratification and to guide therapeutic strategies aiming to reduce cardiovascular mortality in T2D.

Liver and spleen predominantly mediate calciprotein particle clearance in a rat model of chronic kidney disease.

Calciprotein particles (CPPs) provide an efficient mineral buffering system to prevent the complexation of phosphate and calcium in the circulation. However, in chronic kidney disease (CKD), the phosphate load exceeds the mineral buffering capacity, resulting in the formation of crystalline CPP2 particles. CPP2 have been associated with cardiovascular events and mortality. Moreover, CPP2 have been demonstrated to induce calcification in vitro. In this study, we examined the fate of CPP2 in a rat model of CKD. Calcification was induced in Sprague-Dawley rats by 5/6 nephrectomy (5/6-Nx) combined with a high-phosphate diet. Control rats received sham surgery and high-phosphate diet. Twelve weeks after surgery, kidney failure was significantly induced in 5/6-Nx rats as determined by enhanced creatinine and urea plasma levels and abnormal kidney histological architecture. Subsequently, radioactive and fluorescent (FITC)-labeled CPP2 ([89Zr]Zr-CPP2-FITC) were injected intravenously to determine clearance in vivo. Using positron emission tomography scans and radioactive biodistribution measurements, it was demonstrated that [89Zr]Zr-CPP2-FITC are mainly present in the liver and spleen in both 5/6-Nx and sham rats. Immunohistochemistry showed that [89Zr]Zr-CPP2-FITC are predominantly taken up by Kupffer cells and macrophages. However, [89Zr]Zr-CPP2-FITC could also be detected in hepatocytes. In the different parts of the aorta and in the blood, low values of [89Zr]Zr-CPP2-FITC were detectable, independent of the presence of calcification. CPP2 are cleared rapidly from the circulation by the liver and spleen in a rat model of CKD. In the liver, Kupffer cells, macrophages, and hepatocytes contribute to CPP2 clearance.NEW & NOTEWORTHY Calciprotein particles (CPPs) buffer calcium and phosphate in the blood to prevent formation of crystals. In CKD, increased phosphate levels may exceed the buffering capacity of CPPs, resulting in crystalline CPPs that induce calcification. This study demonstrates that labeled CPPs are predominantly cleared from the circulation in the liver by Kupffer cells, macrophages, and hepatocytes. Our results suggest that targeting liver CPP clearance may reduce the burden of crystalline CPP in the development of vascular calcification.

Magnesium and Vascular Calcification in Chronic Kidney Disease: Current Insights.

Magnesium (Mg) plays crucial roles in multiple essential biological processes. As the kidneys are the primary organ responsible for maintaining the blood concentration of Mg, people with chronic kidney disease (CKD) may develop disturbances in Mg. While both hyper- and hypomagnesemia may lead to adverse effects, the consequences associated with hypomagnesemia are often more severe and lasting. Importantly, observational studies have shown that CKD patients with hypomagnesemia have greater vascular calcification. Vascular calcification is accelerated and contributes to a high mortality rate in the CKD population. Both in vitro and animal studies have demonstrated that Mg protects against vascular calcification via several potential mechanisms, such as inhibiting the formation of both hydroxyapatite and pathogenic calciprotein particles as well as limiting osteogenic differentiation, a process in which vascular smooth muscle cells in the media layer of the arteries transform into bone-like cells. These preclinical findings have led to several important clinical trials that have investigated the effects of Mg supplementation on vascular calcification in people with CKD. Interestingly, two major clinical studies produced contradictory findings, resulting in a state of equipoise. This narrative review provides an overview of our current knowledge in the renal handling of Mg in health and CKD and the underlying mechanisms by which Mg may protect against vascular calcification. Lastly, we evaluate the strength of evidence from clinical studies on the efficacy of Mg supplementation and discuss future research directions.

Quantification of Calciprotein Particles (CPPs) in Serum/Plasma Samples Using a Fluorescent Bisphosphonate.

Calciprotein particles (CPPs) are mineral-protein complexes containing solid-phase calcium-phosphate and the serum protein fetuin-A. CPPs are dispersed in the blood as colloids. Previous clinical studies revealed that circulating levels of CPPs were correlated with inflammation and vascular calcification/stiffness in patients with chronic kidney disease (CKD). Measurement of blood CPP levels is challenging because CPPs are unstable and change their physical and chemical properties spontaneously over time in vitro. Several different methods have been developed for quantification of blood CPP levels with different advantages and limitations. We have developed a simple and sensitive assay using a fluorescent probe that bound to calcium-phosphate crystals. This assay may be useful as a clinical test to evaluate the cardiovascular risk and prognosis in CKD patients.

Reconsidering the role of albumin towards amorphous calcium phosphate-based calciprotein particles formation and stability from a physico-chemical perspective.

The formation of calciprotein particles (CPPs) in serum is a physiological phenomenon fundamental to prevent the rise of ectopic calcifications. CPPs are colloidal hybrid particles made of amorphous calcium phosphate stabilized by a protein, fetuin-A. Since albumin is the most abundant protein present in serum, we aimed at understanding if it plays a synergic action together with fetuin-A towards CPPs formation and stability. CPPs were prepared using a constant fetuin-A concentration (5 µM) and different concentrations of albumin (0-606 µM). The stability of CPPs, their crystallization and sedimentation were followed in situ by combining turbidimetry, precipitation analysis and dynamic light scattering. The morphology was investigated by scanning electron microscopy and cryo-transmission electron microscopy, while crystallinity was inspected by infrared spectroscopy. The effect of albumin on the amount of formed CPPs was also studied, as well as the amount of protein adsorbed on CPPs. We found that albumin is not able to prolong the lifetime of the amorphous phase, but it is very effective in delaying the sedimentation of CPPs after crystallization. Albumin also significantly decreases the amount and size of CPPs when present in their synthetic medium, likely playing a fundamental role in our organism together with fetuin-A towards the stabilization of CPPs.

Secondary calciprotein particle size is associated with patient mortality in peripheral artery disease.

BACKGROUND AND AIMS: Secondary calciprotein particles (CPP-II) induce inflammation and contribute to vascular calcification. CPP-II size is associated with vascular calcification in patients with chronic kidney disease (CKD) and all-cause mortality in hemodialysis patients. Here, we investigate for the first time a possible role of CPP-II size in patients with peripheral artery disease (PAD) without severe CKD. METHODS: We measured the hydrodynamic radius (Rh) of CPP-II by using dynamic light scattering in a cohort of 281 PAD patients. Mortality was evaluated over a period of ten years by central death registry queries. 35% of patients died during the observation period (median of 8.8 (6.2-9.0) years). Cox-regression analyses were performed to estimate hazard ratios (HR) and 95% confidence intervals (CI) and to allow for multivariable adjustment. RESULTS: The mean CPP-II size was 188 (162-218) nm. Older patients, patients with reduced kidney function, and those with media sclerosis had larger CPP-II (p < 0.001, p = 0.008, and p = 0.043, retrospectively). There was no association between CPP-II size and overall atherosclerotic disease burden (p = 0.551). CPP-II size was independently significantly associated with all-cause (HR 1.33 (CI 1.01-1.74), p = 0.039) and cardiovascular mortality (HR 1.52 (CI 1.05-2.20), p = 0.026) in multivariable regression analyses. CONCLUSIONS: Large CPP-II size is associated with mortality in PAD patients and might be a new feasible biomarker for the presence of media sclerosis in this patient population.

Calciprotein Particles Induce Endothelial Dysfunction by Impairing Endothelial Nitric Oxide Metabolism.

BACKGROUND: Calciprotein particles (CPPs) are associated with the development of vascular calcifications in chronic kidney disease. The role of endothelial cells (ECs) in this process is unknown. Here, we investigated the interaction of CPPs and ECs, thereby focusing on endothelial nitric oxide metabolism and oxidative stress. METHODS: CPPs were generated in calcium- and phosphate-enriched medium. Human umbilical vein endothelial cells were exposed to different concentrations of CPPs (0-100 µg/mL) for 24 or 72 hours. Ex vivo porcine coronary artery rings were used to measure endothelial cell-dependent vascular smooth muscle cell relaxation after CPP exposure. Serum samples from an early chronic kidney disease cohort (n=245) were analyzed for calcification propensity (measure for CPP formation) and nitrate and nitrite levels (NOx). RESULTS: CPP exposure for 24 hours reduced eNOS (endothelial nitric oxide synthase) mRNA expression and decreased nitrite production, indicating reduced nitric oxide bioavailability. Also, 24-hour CPP exposure caused increased mitochondria-derived superoxide generation, together with nitrotyrosine protein residue formation. Long-term (72 hours) exposure of human umbilical vein endothelial cells to CPPs induced eNOS uncoupling and decreased eNOS protein expression, indicating further impairment of the nitric oxide pathway. The ex vivo porcine coronary artery model showed a significant reduction in endothelial-dependent vascular smooth muscle cell relaxation after CPP exposure. A negative association was observed between NOx levels and calcification propensity (r=-0.136; P=0.049) in sera of (early) chronic kidney disease patients. CONCLUSIONS: CPPs cause endothelial cell dysfunction by impairing nitric oxide metabolism and generating oxidative stress. Our findings provide new evidence for direct effects of CPPs on ECs and pathways involved.

Effect of lanthanum carbonate on serum calciprotein particles in patients with stage 3-4 CKD-results from a placebo-controlled randomized trial.

BACKGROUND: Calciprotein particles (CPP) are colloidal aggregates of calcium phosphate and the mineral-binding protein fetuin-A, and are potential mediators of cardiovascular disease in chronic kidney disease (CKD). Emerging evidence suggests non-calcium-containing phosphate binders may reduce serum CPP in patients with kidney failure who require dialysis; however, it is unclear whether similar interventions are effective in patients with earlier stages of CKD. METHODS: The IMpact of Phosphate Reduction On Vascular End-points in CKD (IMPROVE-CKD) was a multi-centre, placebo-controlled, randomized trial of lanthanum carbonate on cardiovascular markers in 278 participants with stage 3b/4 CKD. In this pre-specified exploratory analysis, primary (CPP-I) and secondary CPP (CPP-II) were measured in a sub-cohort of participants over 96 weeks. Treatment groups were compared using linear mixed-effects models and the relationship between serum CPP and pulse wave velocity (PWV) and abdominal aortic calcification (AAC) was examined. RESULTS: A total of 253 participants had CPP data for baseline and at least one follow-up timepoint and were included in this analysis. The mean age was 62.4 ± 12.6 years, 32.0% were female and the mean estimated glomerular filtration rate (eGFR) was 26.6 ± 8.3 mL/min/1.73 m2. Baseline median serum CPP-I was 14.9 × 104 particles/mL [interquartile range (IQR) 4.6-49.3] and median CPP-II was 3.3 × 103 particles/mL (IQR 1.4-5.4). There was no significant difference between treatment groups at 96 weeks in CPP-I [22.8% (95% confidence interval -39.2, 36.4), P = 0.65] or CPP-II [-18.3% (95% confidence interval -40.0, 11.2), P = 0.20] compared with a placebo. Serum CPP were not correlated with baseline PWV or AAC, or with the progression of either marker. CONCLUSIONS: Lanthanum carbonate was not associated with a reduction of CPP at 96 weeks when compared with a placebo in a CKD cohort.

Vascular calcification maladaptively participates in acute phosphate homeostasis.

AIMS: Non-renal extravasation of phosphate from the circulation and transient accumulation into tissues and extracellular fluid is a regulated process of acute phosphate homeostasis that is not well understood. This process is especially relevant in the setting of chronic kidney disease (CKD), where exposure to increased phosphate is prolonged due to inefficient kidney excretion. Furthermore, CKD-associated mineral dysregulation induces pathological accumulation of phosphate causing vascular calcification (VC). Our objective was to determine whether the systemic response to acute phosphate challenges is altered by VC. METHODS AND RESULTS: After bolus phosphate administration, circulating and tissue deposition of this challenge was assessed in two rat models of VC using a radiolabelled phosphate tracer. In an adenine-induced model of CKD (N = 70), animals with VC had a blunted elevation of circulating 33PO4 following oral phosphate administration (P < 0.01), and the discordant deposition could be traced to the calcified arteries (11.4 [7.5-13.1] vs.43.0 [35.5-53.7] pmol/ng tissue, P < 0.001). In a non-CKD model of VC, calcification was induced with 0.5 ug/kg calcitriol and then withdrawn (N = 24). New phosphate uptake by the calcified vasculature correlated to the pre-existing burden of calcification (r = 38, P < 0.001) and was substantially attenuated in the absence of calcification stimulus (P < 0.01). Phosphate accrual was stimulated by the phosphate challenge and not present to the same degree during passive disposition of circulating phosphate. Further, the form of phosphate that deposited to the vasculature was predominately amorphous inorganic phosphate and not that which was bound in matured calciprotein particles. CONCLUSIONS: In the process of calcification, arteries acutely deposit substantial amorphous phosphate while blunting the elevation in the circulation, thereby altering the systemic disposition of phosphate and identifying VC as a participatory mineral homeostatic organ. This study demonstrates the negative vascular consequence of acute fluctuations in circulating phosphate, and supports the importance of phosphate bioavailability and diet management in CKD patients as a mediator of cardiovascular risk.

Effect of the phosphate binder sucroferric oxyhydroxide in dialysis patients on endogenous calciprotein particles, inflammation, and vascular cells.

BACKGROUND: Calciprotein particles (CPPs), colloidal mineral-protein nanoparticles, have emerged as potential mediators of phosphate toxicity in dialysis patients, with putative links to vascular calcification, endothelial dysfunction and inflammation. We hypothesized that phosphate binder therapy with sucroferric oxyhydroxide (SO) would reduce endogenous CPP levels and attenuate pro-calcific and pro-inflammatory effects of patient serum towards human vascular cells in vitro. METHODS: This secondary analysis of a randomised controlled crossover study compared the effect of 2-week phosphate binder washout with high-dose (2000 mg/day) and low-dose (250 mg/day) SO therapy in 28 haemodialysis patients on serum CPP levels, inflammatory cytokine/chemokine arrays and human aortic smooth muscle cell (HASMC) and coronary artery endothelial cell (HCAEC) bioassays. RESULTS: In our cohort (75% male, 62 ± 12 years) high-dose SO reduced primary (amorphous) and secondary (crystalline) CPP levels {-62% [95% confidence interval (CI) -76 to -44], P < .0001 and -38% [-62 to -0.14], P < .001, respectively} compared with washout. Nine of 14 plasma cytokines/chemokines significantly decreased with high-dose SO, with consistent reductions in interleukin-6 (IL-6) and IL-8. Exposure of HASMC and HCAEC cultures to serum of SO-treated patients reduced calcification and markers of activation (IL-6, IL-8 and vascular cell adhesion protein 1) compared with washout. Serum-induced HASMC calcification and HCAEC activation was ameliorated by removal of the CPP-containing fraction from patient sera. Effects of CPP removal were confirmed in an independent cohort of chronic kidney disease patients. CONCLUSIONS: High-dose SO reduced endogenous CPP formation in dialysis patients and yielded serum with attenuated pro-calcific and inflammatory effects in vitro.

Calciprotein Particles Induce Cellular Compartment-Specific Proteome Alterations in Human Arterial Endothelial Cells.

Calciprotein particles (CPPs) are indispensable scavengers of excessive Ca2+ and PO43- ions in blood, being internalised and recycled by liver and spleen macrophages, monocytes, and endothelial cells (ECs). Here, we performed a pathway enrichment analysis of cellular compartment-specific proteomes in primary human coronary artery ECs (HCAEC) and human internal thoracic artery ECs (HITAEC) treated with primary (amorphous) or secondary (crystalline) CPPs (CPP-P and CPPs, respectively). Exposure to CPP-P and CPP-S induced notable upregulation of: (1) cytokine- and chemokine-mediated signaling, Ca2+-dependent events, and apoptosis in cytosolic and nuclear proteomes; (2) H+ and Ca2+ transmembrane transport, generation of reactive oxygen species, mitochondrial outer membrane permeabilisation, and intrinsic apoptosis in the mitochondrial proteome; (3) oxidative, calcium, and endoplasmic reticulum (ER) stress, unfolded protein binding, and apoptosis in the ER proteome. In contrast, transcription, post-transcriptional regulation, translation, cell cycle, and cell-cell adhesion pathways were underrepresented in cytosol and nuclear compartments, whilst biosynthesis of amino acids, mitochondrial translation, fatty acid oxidation, pyruvate dehydrogenase activity, and energy generation were downregulated in the mitochondrial proteome of CPP-treated ECs. Differentially expressed organelle-specific pathways were coherent in HCAEC and HITAEC and between ECs treated with CPP-P or CPP-S. Proteomic analysis of mitochondrial and nuclear lysates from CPP-treated ECs confirmed bioinformatic filtration findings.

Serum Calciprotein Monomers and Chronic Kidney Disease Progression.

INTRODUCTION: Elevated levels of fibroblast growth factor-23 (FGF23) in chronic kidney disease (CKD) are associated with progression of CKD. FGF23 inhibits proximal tubular phosphate reabsorption, raising phosphate concentrations in the tubular fluid of functioning nephrons, predisposing to spontaneous precipitation of calcium phosphate crystals and resultant tubular injury. Calciprotein monomers (CPM) form spontaneously in biological fluids when clusters of calcium phosphate ions are bound by the liver-derived glycoprotein fetuin-A. Serum CPM are elevated in CKD and are postulated to trigger FGF23 secretion. CPM are also readily filtered at the glomerulus into the tubular fluid, suggesting that higher CPM levels could be associated with progression of CKD via FGF23-mediated increased phosphate load but also through direct effects in the proximal tubule. METHODS: ACADEMIC was a prospective observational study of 200 stable outpatients with CKD stages 3 and 4. Participants were followed until commencement of dialysis or death. In this study, we examined a sub-cohort of 189 participants who had baseline serum available for measurement of CPM. Cox proportionate hazard regression models were used to examine the association between CPM and a composite kidney disease outcome (commencement of dialysis or reduction in estimated glomerular filtration rate [eGFR] >30%). Linear regression models were used to examine the association between CPM and annualized eGFR slope. RESULTS: Relative to the lowest tertile, the highest tertile of CPM was associated with increased risk of the composite kidney disease outcome in univariate models and after sequential adjustment for conventional risk factors for progression of CKD (adjusted hazard ratio 4.22; 95% confidence interval [CI] 1.91, 9.33, p < 0.001). Natural log-transformed CPM was also inversely associated with eGFR slope in univariate and multivariate adjusted models (adjusted ß-coefficient -1.66, 95% CI: -3.10, -0.22, p = 0.024). In exploratory mediation analysis, the association between serum CPM and eGFR slope was partially mediated by iFGF23; however, the majority of the association was direct and independent of the iFGF23 pathway. CONCLUSION: Elevated levels of CPM are associated with the progression of CKD. This association was partially mediated via FGF23, consistent with recent evidence that FGF23 predisposes to spontaneous precipitation of calcium phosphate crystals leading to tubular injury. However, serum CPM also appeared to have a direct association with eGFR slope, raising the possibility that CPM may also be associated with progression of CKD through additional pathways.

Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation.

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca2+ and PO43- ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca2+ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies.

Correction of Vascular Calcification and Hyperphosphatemia in CKD Rats Treated with ASARM Peptide.

Background: Abnormalities in calcium, phosphorus, PTH, vitamin D metabolism, bone, and vascular calcification occur in chronic kidney disease mineral bone disorder (CKD-MBD). Calciphylaxis, involving painful, ulcerative skin lesions, is also a major problem associated with CKD-MBD. There are no quality medical interventions to address these clinical issues. Bone ASARM peptides are strong inhibitors of mineralization and induce hypophosphatemia by inhibiting phosphate uptake from the gut. We hypothesize treatment of CKD-MBD rats with ASARM peptides will reverse hyperphosphatemia, reduce soft-tissue calcification, and prevent calciphylaxis. Methods: To test our hypothesis, we assessed the effects of synthetic ASARM peptide in rats that had undergone a subtotal 5/6th nephrectomy (56NEPHREX), a rodent model of CKD-MBD. All rats were fed a high phosphate diet (2% Pi) to worsen mineral metabolism defects. Changes in serum potassium, phosphate, BUN, creatinine, PTH, FGF23, and calcium were assessed in response to 28 days of ASARM peptide infusion. Also, changes in bone quality, soft-tissue calcification, and expression of gut Npt2b (Slc34a2) were studied following ASARM peptide treatment. Results: Rats that had undergone 56NEPHREX treated with ASARM peptide showed major improvements in hyperphosphatemia, blood urea nitrogen (BUN), and bone quality compared with vehicle controls. Also, ASARM-infused 56NEPHREX rats displayed improved renal, brain, and cardiovascular calcification. Notably, ASARM peptide infusion prevented the genesis of subdermal medial blood vessel calcification and calciphylaxis-like lesions in 56NEPHREX rats compared with vehicle controls. Conclusions: ASARM peptide infusion corrects hyperphosphatemia and improves vascular calcification, renal calcification, brain calcification, bone quality, renal function, and skin mineralization abnormalities in 56NEPHREX rats. These findings confirm our hypothesis and support the utility of ASARM peptide treatment in patients with CKD-MBD.

Small particles with large impact: Insights into the unresolved roles of innate immunity in extracellular vesicle-mediated cardiovascular calcification.

Extracellular vesicles (EVs) are critical in the initiation and progression of cardiovascular calcification, and immune cell infiltration and inflammation have a central role in this process. EVs egress from various cardiovascular cell types, which when acquiring specific properties, become calcifying. These calcifying EVs form nidi for microcalcification, which can progress to the macrocalcification lesions that are visualized clinically. We make the distinction between inflammatory-driven and mineral dysregulation-driven calcification, which both share EVs as a central initiator. In inflammation-mediated calcification, inflammation precedes microcalcification and results from EV release from macrophages. Local cellular crosstalk mediated by EVs as well as circulating EVs and other inflammatory nanoparticles, such as calciprotein particles and lipoproteins, are also critical in the progression of cardiovascular calcification. It is imperative that future work links the already established and to be discovered roles of inflammation and innate immunity in cardiovascular calcification to these key signaling and functional roles of these nanoparticles. It remains an understudied area with high potential to unravel mechanistic roles and has important implications in drug target research.

Impact of etelcalcetide on fibroblast growth factor-23 and calciprotein particles in patients with secondary hyperparathyroidism undergoing haemodialysis.

AIM: Recently, we demonstrated the efficacy of etelcalcetide in the control of secondary hyperparathyroidism (SHPT). This post hoc analysis aimed to evaluate changes in fibroblast growth factor-23 (FGF23) and calciprotein particles (CPPs) after treatment with calcimimetics. METHODS: The DUET trial was a 12-week multicenter, open-label, parallel-group, randomized (1:1:1) study with patients treated with etelcalcetide plus active vitamin D (E + D group; n = 41), etelcalcetide plus oral calcium (E + Ca group; n = 41), or control (C group; n = 42) under maintenance haemodialysis. Serum levels of FGF23 and CPPs were measured at baseline, and 6 and 12 weeks after the start. RESULTS: In the linear mixed model, serum levels of FGF23 in etelcalcetide users were significantly lower than those in non-users at week 6 (p < .001) and week 12 (p < .001). When compared the difference between the E + Ca group and the E + D group, serum levels of FGF23 in the E + Ca group were significantly lower than those in the E + D group at week 12 (p = .017). There were no significant differences in the serum levels of CPPs between etelcalcetide users and non-users at week 6 and week 12, while CPPs in the E + Ca group were significantly lower than those in the E + D group (p < .001) at week 12. CONCLUSION: Etelcalcetide may be useful through suppression of FGF23 levels among haemodialysis patients with SHPT. When correcting hypocalcaemia, loading oral calcium preparations could be more advantageous than active vitamin D for the suppression of both FGF23 and CPPs.

Reduced hip bone mineral density is associated with high levels of calciprotein particles in patients with Fabry disease.

Calciprotein particles (CPP) are nanoscale mineralo-protein aggregates that help stabilize excess mineral in the circulation. We examined the relationship between CPP and bone mineral density in Fabry disease patients. We found an inverse correlation with total hip and femoral neck density, but none with lumbar spine. PURPOSE: Calciprotein particles (CPP) are colloidal mineral-protein complexes made up primarily of the circulating glycoprotein fetuin-A, calcium, and phosphate. They form in extracellular fluid and facilitate the stabilization, transport, and clearance of excess minerals from the circulation. While most are monomers, they also exist in larger primary (CPP-I) and secondary (CPP-II) form, both of which are reported to be raised in pathological states. This study sought to investigate CPP levels in the serum of patients with Fabry disease, an X-linked systemic lysosomal storage disorder that is associated with generalized inflammation and low bone mineral density (BMD). METHODS: We compared serum CPP-I and CPP-II levels in 59 patients with Fabry disease (37 female) with levels in an age-matched healthy adult cohort (n=28) and evaluated their association with BMD and biochemical data obtained from routine clinical review. RESULTS: CPP-I and CPP-II levels were higher in male Fabry disease patients than female sufferers as well as their corresponding sex- and age-matched controls. CPP-II levels were inversely correlated with BMD at the total hip and femoral neck, but not the lumbar spine. Regression analyses revealed that these associations were independent of common determinants of BMD, but at the femoral neck, a significant association was only found in female patients. CONCLUSION: Low hip BMD was associated with high CPP-II in patients with Fabry disease, but further work is needed to investigate the relevance of sex-related differences and to establish whether CPP measurement may aid assessment of bone disease in this setting.