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Chronic kidney disease (CKD) is associated with systemic phosphate elevations, called hyperphosphatemia. Translational studies have shown that hyperphosphatemia contributes to CKD-associated inflammation and injury in various tissues, including the kidney, heart, liver, and parathyroid gland. Mechanisms underlying pathologic actions of elevated phosphate on cells are not well understood but seem to involve uptake of phosphate through sodium phosphate cotransporters and phosphate-induced signaling via FGFR1 (fibroblast growth factor receptor 1). Clinical studies indicate patients with CKD are more likely to develop inflammatory and restrictive lung diseases, such as fibrotic interstitial lung diseases, and here we aimed to determine whether hyperphosphatemia can cause lung injury. We found that a mouse model of CKD and hyperphosphatemia, induced by an adenine-rich diet, develops lung fibrosis and inflammation. Elevation of systemic phosphate concentration by administration of a high-phosphate diet in a mouse model of primary lung inflammation and fibrosis, induced by bleomycin, exacerbated lung injury in the absence of kidney damage. Our in vitro studies identified increases of proinflammatory cytokines in human lung fibroblasts exposed to phosphate elevations. Phosphate activated ERK 1/2 (extracellular signal-related kinase 1/2) and PKB/AKT (protein kinase B) signaling, and pharmacological inhibition of ERK, AKT, FGFR1, or sodium phosphate cotransporters prevented phosphate-induced proinflammatory cytokine upregulation. In addition, inhibition of FGFR1 or sodium phosphate cotransporters decreased the phosphate-induced activation of ERK and AKT. Our study suggests that phosphate can directly target lung fibroblasts and induce an inflammatory response and that hyperphosphatemia in CKD and non-CKD models contributes to lung injury. Phosphate-lowering strategies might protect from CKD-associated lung injury.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disorder caused by deficient FGF23 signaling and resultant ectopic calcification. Here, we systematically characterized and quantified macro- and micro-calcification in a HFTC cohort using CT and 18F-sodium fluoride PET/CT (18F-NaF PET/CT). Fourier-transform infrared (FTIR) spectroscopy was performed on 4 phenotypically different calcifications from a patient with HFTC, showing the dominant component to be hydroxyapatite. Eleven patients with HFTC were studied with CT and/or 18F-NaF PET/CT. Qualitative review was done to describe the spectrum of imaging findings on both modalities. CT-based measures of volume (eg, total calcific burden and lesion volume) and density (Hounsfield units) were quantified and compared to PET-based measures of mineralization activity (eg, mean standardized uptake values-SUVs). Microcalcification scores were calculated for the vasculature of 6 patients using 18F-NaF PET/CT and visualized on a standardized vascular atlas. Ectopic calcifications were present in 82% of patients, predominantly near joints and the distal extremities. Considerable heterogeneity was observed in total calcific burden per patient (823.0 ± 670.1 cm3, n = 9) and lesion volume (282.5 ± 414.8 cm3, n = 27). The largest lesions were found at the hips and shoulders. 18F-NaF PET offered the ability to differentiate active vs quiescent calcifications. Calcifications were also noted in multiple anatomic locations, including brain parenchyma (50%). Vascular calcification was seen in the abdominal aorta, carotid, and coronaries in 50%, 73%, and 50%, respectively. 18F-NaF-avid, but CT-negative calcification was seen in a 17-year-old patient, implicating early onset vascular calcification. This first systematic assessment of calcifications in a cohort of patients with HFTC has identified the early onset, prevalence, and extent of calcification. It supports 18F-NaF PET/CT as a clinical tool for distinguishing between active and inactive calcification, informing disease progression, and quantification of ectopic and vascular disease burden., (Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research 2024.)

Chronic kidney disease (CKD) is associated with various pathologic changes, including elevations in serum phosphate levels (hyperphosphatemia), vascular calcification, and skeletal muscle atrophy. Elevated phosphate can damage vascular smooth muscle cells and cause vascular calcification. Here, we determined whether high phosphate can also affect skeletal muscle cells and whether hyperphosphatemia, in the context of CKD or by itself, is associated with skeletal muscle atrophy. As models of hyperphosphatemia with CKD, we studied mice receiving an adenine-rich diet for 14 weeks and mice with deletion of Collagen 4a3 ( Col4a3 -/- ). As models of hyperphosphatemia without CKD, we analyzed mice receiving a high-phosphate diet for three and six months as well as a genetic model for klotho deficiency ( kl / kl ). We found that adenine, Col4a3 -/- , and kl / kl mice have reduced skeletal muscle mass and function and develop atrophy. Mice on a high-phosphate diet for six months also had lower skeletal muscle mass and function but no significant signs of atrophy, indicating less severe damage compared with the other three models. To determine the potential direct actions of phosphate on skeletal muscle, we cultured primary mouse myotubes in high phosphate concentrations, and we detected the induction of atrophy. We conclude that in experimental mouse models, hyperphosphatemia is sufficient to induce skeletal muscle atrophy and that, among various other factors, elevated phosphate levels might contribute to skeletal muscle injury in CKD.

Purpose: Vascular calcification is a frequently occurring complication of end-stage renal disease (ESRD). This study focused on the significance of long non-coding RNA Fas cell surface death receptor-antisense 1(lncRNA FAS-AS1) in ESRD-related vascular calcification aiming to explore a potential biomarker for the detection., Methods: The study enrolled 65 healthy individuals, 79 ESRD patients (48 patients with vascular calcification), and 93 early-stage (I-IV) chronic kidney disease (CKD) patients. The expression of FAS-AS1 in serum was evaluated by real-time quantitative polymerase chain reaction (PCR). The diagnostic potential of FAS-AS1 was assessed in discriminating ESRD patients, vascular calcification, and the severity of vascular calcification. In vitro, the vascular smooth muscle cells (VSMCs) were treated with a hyperphosphatemia medium to evaluate the effect of FAS-AS1 on VSMCs calcification., Results: Elevated serum FAS-AS1 was observed in ESRD patients, which could discriminate from healthy individuals and early-stage CKD patients. FAS-AS1 was associated with the development of ESRD and the occurrence of vascular calcification. FAS-AS1 was also upregulated in vascular calcification patients, especially the patients with severe calcification, which showed diagnostic significance in evaluating vascular calcification degrees. Calcified VSMCs showed significantly increased levels of Ca 2+ , reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), which was attenuated by silencing FAS-AS1., Conclusions: FAS-AS1 discriminated ERSD patients and was associated with the occurrence of vascular calcification. The knockdown of FAS-AS1 suppressed hyperphosphatemia-induced vascular calcification via alleviating oxidative stress and inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cardiovascular calcification can occur in vascular and valvular structures and is commonly associated with calcium deposition and tissue mineralization leading to stiffness and dysfunction. Patients with chronic kidney disease and associated hyperphosphatemia have an elevated risk for coronary artery calcification (CAC) and calcific aortic valve disease (CAVD). However, there is mounting evidence to suggest that the susceptibility and pathobiology of calcification in these two cardiovascular structures may be different, yet clinically they are similarly treated. To better understand diversity in molecular and cellular processes that underlie hyperphosphatemia-induced calcification in vascular and valvular structures, we exposed aortic vascular smooth muscle cells (AVSMCs) and aortic valve interstitial cells (AVICs) to high (2.5 mM) phosphate (Ph) conditions in vitro, and examined cell-specific responses. To further identify hyperphosphatemic-specific responses, parallel studies were performed using osteogenic media (OM) as an alternative calcific stimulus. Consistent with clinical observations made by others, we show that AVSMCs are more susceptible to calcification than AVICs. In addition, bulk RNA-sequencing reveals that AVSMCs and AVICs activate robust ossification-programs in response to high phosphate or OM treatments, however, the signaling pathways, cellular processes and osteogenic-associated markers involved are cell- and treatment-specific. For example, compared to VSMCs, VIC-mediated calcification involves biological processes related to osteo-chondro differentiation and down regulation of 'actin cytoskeleton'-related genes, that are not observed in VSMCs. Furthermore, hyperphosphatemic-induced calcification in AVICs and AVSMCs is independent of P13K signaling, which plays a role in OM-treated cells. Together, this study provides a wealth of information suggesting that the pathogenesis of cardiovascular calcifications is significantly more diverse than previously appreciated., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vascular calcification is a serious complication of hyperphosphatemia that causes cardiovascular morbidity and mortality. Previous studies have reported that plasmalemmal phosphate (Pi) transporters, such as PiT-1/2, mediate depolarization, Ca 2+ influx, oxidative stress, and calcific changes in vascular smooth muscle cells (VSMCs). However, the pathogenic mechanism of mitochondrial Pi uptake in vascular calcification associated with hyperphosphatemia has not been elucidated. We demonstrated that the phosphate carrier (PiC) is the dominant mitochondrial Pi transporter responsible for high Pi-induced superoxide generation, osteogenic gene upregulation, and calcific changes in primary VSMCs isolated from rat aortas. Notably, acute incubation with high Pi markedly increased the protein abundance of PiC via ERK1/2- and mTOR-dependent translational upregulation. Genetic suppression of PiC prevented Pi-induced ERK1/2 activation, superoxide production, osteogenic differentiation, and vascular calcification of VSMCs in vitro and aortic rings ex vivo. Pharmacological inhibition of mitochondrial Pi transport using butyl malonate (BMA) or mersalyl abolished all pathologic changes involved in high Pi-induced vascular calcification. BMA or mersalyl also effectively prevented osteogenic gene upregulation and calcification of aortas from 5/6 subtotal nephrectomized mice fed a high-Pi diet. Our results suggest that mitochondrial Pi uptake via PiC is a critical molecular mechanism mediating mitochondrial superoxide generation and pathogenic calcific changes, which could be a novel therapeutic target for treating vascular calcification associated with hyperphosphatemia., (© 2023. The Author(s).)

Background: Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disease characterized by hyperphosphatemia and ectopic calcification, predominantly at periarticular locations. This study was performed to characterize the clinical profile of tumoral calcinosis and to identify gene mutations associated with HFTC and elucidated its pathogenic role., Methods: The three subjects (two male and one female) were aged 30, 25 and 15 years, respectively. The clinical features, histopathological findings, and outcomes of three subjects with HFTC were retrospectively reviewed. The three subjects were analyzed for FGF23 , GALNT3 and KL mutations. Function of mutant gene was analyzed by western blotting and wheat germ agglutinin affinity chromatography., Results: All subjects had hyperphosphatemia and elevated calcium-phosphorus product. Calcinosis positions included the left shoulder, left index finger, and right hip. Bone and joint damage were present in two cases and multiple foci influenced body growth in one case. The histopathological features were firm, rubbery masses comprising multiple nodules of calcified material bordered by the proliferation of mononuclear or multinuclear macrophages, osteoclastic-like giant cells, fibroblasts, and chronic inflammatory cells. The novel mutation c.484A>G (p.N162D) in exon 3 of FGF23 was identified in one subject and his family members. Measurement of circulating FGF23 in the subject confirmed low intact FGF23 and increased C-terminal fragment. In vitro experiments showed that the mutant FGF23 proteins had defective O-glycosylation and impaired protein proteolysis protection., Conclusion: We identified a novel FGF23 missense mutation, and confirmed its damaging role in FGF23 protein O-glycosylation. Our findings expand the current spectrum of FGF23 variations that influence phosphorus metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zuo, Yang, Liu, Yan, Wang, Wang, Deng, Cao and Yang.)

Hyperphosphatemia associated with chronic kidney disease (CKD) not only dysregulates mineral metabolism and bone diseases, but also strongly contributes to the progression of kidney disease itself. We have identified a novel drug for hyperphosphatemia, EOS789, that interacts with several sodium-dependent phosphate transporters (NaPi-IIb, PiT-1, and PiT-2) known to contribute to intestinal phosphate absorption. In this study, we investigated whether EOS789 could ameliorate kidney disease progression in glomerulonephritis rats. Anti-glomerular basement membrane (GBM) nephritis was induced in rats by intravenously administering two types of anti-rat GBM antibodies. We evaluated the effect of EOS789 administered in food admixture on hyperphosphatemia and kidney disease progression. In an anti-GBM nephritis rats, which exhibit a significant increase in serum phosphate and a decline in renal function, EOS789 dose-dependently improved hyperphosphatemia and EOS789 at 0.3% food admixture significantly ameliorated kidney dysfunction as shown in the decline of serum creatinine and BUN. Renal histopathology analysis showed that EOS789 significantly decreased crescent formation in glomeruli. To elucidate the mechanism underlying glomerular disease progression, human mesangial cells were used. High phosphate concentration in media significantly increased the expression of Collagen 1A1, 3A1, and αSMA mRNA in human mesangial cells and EOS789 dose-dependently suppressed these fibrotic markers. These results indicate that EOS789 prevented glomerular crescent formation caused by mesangial fibrosis by ameliorating hyperphosphatemia. In conclusion, EOS789 would not only be useful against hyperphosphatemia but may also have the potential to relieve mesangial proliferative glomerulonephritis with crescent formation., (© 2022 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

The sodium-dependent phosphate transporters Pit 1 and Pit 2 belong to the solute carrier 20 (SLC20) family of membrane proteins. They are ubiquitously distributed in the human body. Their crucial function is the intracellular transport of inorganic phosphate (Pi) in the form of H 2 PO 4 - . They are one of the main elements in maintaining physiological phosphate homeostasis. Recent data have emerged that indicate novel roles of Pit 1 and Pit 2 proteins besides the well-known function of Pi transporters. These membrane proteins are believed to be precise phosphate sensors that mediate Pi-dependent intracellular signaling. They are also involved in insulin signaling and influence cellular insulin sensitivity. In diseases that are associated with hyperphosphatemia, such as diabetes and chronic kidney disease (CKD), disturbances in the function of Pit 1 and Pit 2 are observed. Phosphate transporters from the SLC20 family participate in the calcification of soft tissues, mainly blood vessels, during the course of CKD. The glomerulus and podocytes therein can also be a target of pathological calcification that damages these structures. A few studies have demonstrated the development of Pi-dependent podocyte injury that is mediated by Pit 1 and Pit 2. This paper discusses the role of Pit 1 and Pit 2 proteins in podocyte function, mainly in the context of the development of pathological calcification that disrupts permeability of the renal filtration barrier. We also describe the mechanisms that may contribute to podocyte damage by Pit 1 and Pit 2., (© 2021 Wiley Periodicals LLC.)

Nanocellulose has gained much attention because of its excellent properties. Cationic cellulose nanocrystals (cCNC) shows good adsorptivity toward negative ions and molecules. Phosphate binders are most used to treat hyperphosphatemia and it is significant to develop its alternatives with high specific and low cost in the clinic. Herein, we prepared cCNC and characterized it by FTIR, TEM, dynamic light scattering, and viscosity method. We simulated the binding process of cationic cellulose for phosphate and used it as phosphate binder for hyperphosphatemia therapy to study the phosphate binding effect and evaluate the oral toxicity. Cationic cellulose improved the conditions of mice models and efficiently decreased the level of phosphate in the serum. cCNC had a better binding effect than cationic microcrystalline cellulose both in vitro and in vivo. cCNC could be used as alternatives to phosphate binder for therapy of chronic renal failure and hyperphosphatemia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chronic kidney disease (CKD) is common in geriatric cats, and is characterised in the majority of cases by tubulointerstitial inflammation and fibrosis. Hyperphosphataemia is a frequent complication of CKD and is independently associated with severity of renal fibrosis and disease progression. Transforming growth factor-beta 1 (TGF-β1) signalling is thought to be a convergent pathway which mediates the progression of renal fibrosis in CKD. The aims of this study were to explore the interaction between increased extracellular phosphate and the TGF-β1 signalling pathway by investigating: (a) the effect of a commercially available, phosphate-restricted, diet on urinary TGF-β1 excretion in cats with CKD; and (b) the role of increased extracellular phosphate in regulating proliferation, apoptosis, and expression of genes related to TGF-β1 signalling and extracellular matrix (ECM) production in feline proximal tubular epithelial cells (FPTEC) and cortical fibroblasts from cats with azotaemic CKD (CKD-FCF). The dietary intervention study revealed no effect of dietary phosphate restriction on urinary active TGF-β1 excretion after 4-8 weeks (P=0.98), despite significantly decreasing serum phosphate (P<0.001). There was no effect of increased growth media phosphate concentration (from 0.95mM to 2mM and 3.5mM) on proliferation (P=0.99) and apoptotic activity in FPTEC (P=0.22), or expression of genes related to ECM production and the TGF-β1 signalling pathway in FPTEC and CKD-FCF (P>0.05). These findings suggest the beneficial effects of dietary phosphate restriction on progression of feline CKD may not occur through modulation of renal TGF-β1 production, and do not support a direct pro-fibrotic effect of increased extracellular phosphate on feline renal cells., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The sodium-phosphate cotransporter NPT2a plays a key role in the reabsorption of filtered phosphate in proximal renal tubules, thereby critically contributing to phosphate homeostasis. Inadequate urinary phosphate excretion can lead to severe hyperphosphatemia as in tumoral calcinosis and chronic kidney disease (CKD). Pharmacological inhibition of NPT2a may therefore represent an attractive approach for treating hyperphosphatemic conditions. The NPT2a-selective small-molecule inhibitor PF-06869206 was previously shown to reduce phosphate uptake in human proximal tubular cells in vitro. Here, we investigated the acute and chronic effects of the inhibitor in rodents and report that administration of PF-06869206 was well tolerated and elicited a dose-dependent increase in fractional phosphate excretion. This phosphaturic effect lowered plasma phosphate levels in WT mice and in rats with CKD due to subtotal nephrectomy. PF-06869206 had no effect on Npt2a-null mice, but promoted phosphate excretion and reduced phosphate levels in normophophatemic mice lacking Npt2c and in hyperphosphatemic mice lacking Fgf23 or Galnt3. In CKD rats, once-daily administration of PF-06869206 for 8 weeks induced an unabated acute phosphaturic and hypophosphatemic effect, but had no statistically significant effect on FGF23 or PTH levels. Selective pharmacological inhibition of NPT2a thus holds promise as a therapeutic option for genetic and acquired hyperphosphatemic disorders.

Hyperphosphatemia is the primary risk factor for vascular calcification, which is closely associated with cardiovascular morbidity and mortality. Recent evidence showed that oxidative stress by high inorganic phosphate (Pi) mediates calcific changes in vascular smooth muscle cells (VSMCs). However, intracellular signaling responsible for Pi-induced oxidative stress remains unclear. Here, we investigated molecular mechanisms of Pi-induced oxidative stress related with intracellular Ca 2+ ([Ca 2+ ] i ) disturbance, which is critical for calcification of VSMCs. VSMCs isolated from rat thoracic aorta or A7r5 cells were incubated with high Pi-containing medium. Extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin were activated by high Pi that was required for vascular calcification. High Pi upregulated expressions of type III sodium-phosphate cotransporters PiT-1 and -2 and stimulated their trafficking to the plasma membrane. Interestingly, high Pi increased [Ca 2+ ] i exclusively dependent on extracellular Na + and Ca 2+ as well as PiT-1/2 abundance. Furthermore, high-Pi induced plasma membrane depolarization mediated by PiT-1/2. Pretreatment with verapamil, as a voltage-gated Ca 2+ channel (VGCC) blocker, inhibited Pi-induced [Ca 2+ ] i elevation, oxidative stress, ERK activation, and osteogenic differentiation. These protective effects were reiterated by extracellular Ca 2+ -free condition, intracellular Ca 2+ chelation, or suppression of oxidative stress. Mitochondrial superoxide scavenger also effectively abrogated ERK activation and osteogenic differentiation of VSMCs by high Pi. Taking all these together, we suggest that high Pi activates depolarization-triggered Ca 2+ influx via VGCC, and subsequent [Ca 2+ ] i increase elicits oxidative stress and osteogenic differentiation. PiT-1/2 mediates Pi-induced [Ca 2+ ] i overload and oxidative stress but in turn, PiT-1/2 is upregulated by consequences of these alterations. NEW & NOTEWORTHY The novel findings of this study are type III sodium-phosphate cotransporters PiT-1 and -2-dependent depolarization by high Pi, leading to Ca 2+ entry via voltage-gated Ca 2+ channels in vascular smooth muscle cells. Cytosolic Ca 2+ increase and subsequent oxidative stress are indispensable for osteogenic differentiation and calcification. In addition, plasmalemmal abundance of PiT-1/2 relies on Ca 2+ overload and oxidative stress, establishing a positive feedback loop. Identification of mechanistic components of a vicious cycle could provide novel therapeutic strategies against vascular calcification in hyperphosphatemic patients.

Hyperphosphataemia increases cardiovascular mortality in patients with kidney disease. Direct effects of high inorganic phosphate (Pi) concentrations have previously been demonstrated on endothelial cells (ECs), including generation of procoagulant endothelial microvesicles (MVs). However, no mechanism directly sensing elevated intracellular Pi has ever been described in mammalian cells. Here, we investigated the hypothesis that direct inhibition by Pi of the phosphoprotein phosphatase PP2A fulfils this sensing role in ECs, culminating in cytoskeleton disruption and MV generation. ECs were treated with control (1 mM [Pi]) vs. high (2.5 mM [Pi]), a condition that drives actin stress fibre depletion and MV generation demonstrated by confocal microscopy of F-actin and NanoSight Nanoparticle tracking, respectively. Immuno-blotting demonstrated that high Pi increased p-Src, p-PP2A-C and p-DAPK-1 and decreased p-TPM-3. Pi at 100 μM directly inhibited PP2A catalytic activity. Inhibition of PP2A enhanced inhibitory phosphorylation of DAPK-1, leading to hypophosphorylation of Tropomyosin-3 at S284 and MV generation. p-Src is known to perform inhibitory phosphorylation on DAPK-1 but also on PP2A-C. However, PP2A-C can itself dephosphorylate (and therefore inhibit) p-Src. The direct inhibition of PP2A-C by Pi is, therefore, amplified by the feedback loop between PP2A-C and p-Src, resulting in further PP2A-C inhibition. These data demonstrated that PP2A/Src acts as a potent sensor and amplifier of Pi signals which can further signal through DAPK-1/Tropomyosin-3 to generate cytoskeleton disruption and generation of potentially pathological MVs.

Aim: Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare endocrine disorder caused by autosomal recessive variants in GALNT3, FGF23, and KL leading to progressive calcification of soft tissues and subsequent clinical effects. The aim of this was to study the cause of HFTC in an Iranian family., Patients and Methods: Four generations of a family with HFTC were studied for understanding the genetic pattern of the disease. Whole exome sequencing was applied on genomic DNA of the proband. Based on its result, genetically altered sequences were checked in his family through sanger sequencing. Then bioinformatics approaches as well as co-segregation analysis were applied to validate the genetic alteration., Results: A novel homozygous variant in exon four of GALNT3, namely p.R261Q was found. The parents and sister were carriers., Conclusion: To our knowledge, it is the first-reported Iranian family with GALNT3-CDG novel variant.

Objective: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (P i ) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC., Methods and Results: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by P i and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of P i -mediated VC, is reduced in TDAG51 -/- VSMCs. To explain these observations, we identified that TDAG51 -/- VSMCs express reduced levels of the type III sodium-dependent P i transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular P i uptake. Significantly, in response to hyperphosphatemia induced by vitamin D 3 , medial VC was attenuated in TDAG51 -/- mice., Conclusions: Our studies highlight TDAG51 as an important mediator of P i -induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare and disabling disorder of fibroblast growth factor 23 (FGF23) deficiency or resistance. The disorder is manifest by hyperphosphatemia, inappropriately increased tubular reabsorption of phosphate and 1,25-dihydroxy-Vitamin D, and ectopic calcifications. HFTC has been associated with autosomal recessive pathogenic variants in: (1) the gene encoding FGF23; (2) GALNT3 , which encodes a protein responsible for FGF23 glycosylation; and (3) KL , the gene encoding KLOTHO, a critical co-receptor for FGF23 signaling. An acquired autoimmune form of hyperphosphatemic tumoral calcinosis has also been reported. Periarticular tumoral calcinosis is the primary cause of disability in HFTC, leading to pain, reduced range-of-motion, and impaired physical function. Inflammatory disease is also prominent, including diaphysitis with cortical hyperostosis. Multiple treatment strategies have attempted to manage blood phosphate, reduce pain and inflammation, and address calcifications and their complications. Unfortunately, efficacy data are limited to case reports and small cohorts, and no clearly effective therapies have been identified. The purpose of this review is to provide a background on pathogenesis and clinical presentation in HFTC, discuss current approaches to clinical management, and outline critical areas of need for future research., (Copyright © 2020 Boyce, Lee, Roszko and Gafni.)

Background: Phosphate binders are commonly used in the treatment of patients with hyperphosphatemia. While phosphate binders are used to lower phosphate, the effects of specific phosphate binder types on vitamin D metabolism are unknown., Methods: We performed a secondary analysis of the Phosphate Normalization Trial in which patients with moderate to advanced chronic kidney disease were randomized to receive either placebo, sevelamer carbonate, lanthanum carbonate or calcium acetate for 9 months. We evaluated changes in serum concentrations of vitamin D metabolites including 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the ratio of 24,25(OH)2D3 to 25-hydroxyvitamin D [the vitamin D metabolite ratio (VMR)] and the ratio of serum 1,25(OH)2D to 25-hydroxyvitamin D., Results: Compared with placebo, randomization to the calcium acetate arm was associated with a 0.6 ng/mL (95% CI 0.2, 1) and 13.5 pg/ng (95% CI 5.5, 21.5) increase in 24,25(OH)2D and VMR, respectively, and a 5.2 pg/mL (95% CI 1.1, 9.4) reduction in 1,25(OH)2D. Randomization to sevelamer carbonate was associated with a 0.5 ng/mL (95% CI -0.9, -0.1) and 11.8 pg/ng (95% CI -20, -3.5) reduction in 24,25(OH)2D3 and VMR, respectively. There was no association of the sevelamer arm with the change in 1,25(OH)2D3, and randomization to lanthanum carbonate was not associated with a change in any of the vitamin D metabolites., Conclusion: Administration of different phosphate binders to patients with moderate to severe CKD results in unique changes in vitamin D metabolism., (© The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background: High serum phosphate (Pi) levels strongly associate with cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients with vascular calcification playing a major role in the pathogenesis of related cardiovascular disease. High-Pi challenged vascular smooth muscle cells (VSMCs) undergo simil-osteoblastic transformation and actively deposit calcium-phosphate crystals. Iron-based Pi-binders are used to treat hyperphosphatemia in CKD patients., Methods: In this study, we investigated the direct effect of iron citrate on extracellular matrix (ECM) modification induced by high-Pi, following either prophylactic or therapeutic approach., Results: Iron prophylactically prevents and therapeutically blocks high-Pi induced calcification. Masson's staining highlights the changes of muscular ECM that after high-Pi stimulation becomes fibrotic and which modifications are prevented or partially reverted by iron. Interestingly, iron preserves glycogen granules and either prevents or partially reverts the formation of non-glycogen granules induced by high-Pi. In parallel, iron addition is able to either prevent or block the high-Pi induced acid mucin deposition. Iron inhibited calcification also by preventing exosome osteo-chondrogenic shift by reducing phosphate load (0,61 ± 0.04vs0,45 ± 0.05, PivsPi + Fe, p < 0,05, nmol Pi/mg protein) and inducing miRNA 30c (0.62 ± 0.05vs3.07 ± 0.62; PivsPi + Fe, p < 0.01, relative expression). Studying aortic rings, we found that iron significantly either prevents or reverts the high-Pi induced collagen deposition and the elastin decrease, preserving elastin structure (0.7 ± 0.1 vs 1.2 ± 0.1; Pi vs Pi + Fe, p < 0.05, elastin mRNA relative expression)., Conclusions: Iron directly either prevents or partially reverts the high-Pi induced osteo-chondrocytic shift of ECM. The protection of muscular nature of VSMC ECM may be one of the mechanisms elucidating the anti-calcific effect of iron., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Hypophosphatemia was previously shown to affect the duration of admission, mechanical ventilator requirements, mortality and morbidity during pediatric intensive care. Different from previous studies, our study was planned with the aim of showing whether hyperphosphatemia affects morbidity and mortality in pediatric intensive care patients as much as hypophosphatemia., Method: Patients' ages, genders, reason for admission, underlying diseases, phosphorus levels examined on admission and on the 1-4th and 5-10th-days, duration on mechanical ventilation, duration of admission, final status and PRISM and PELOD scores calculated in the first 24 hours of admission were recorded., Results: Mortality was distinctly higher for those who were hypophosphatemic and hyperphosphatemic compared to those who were normophosphatemic. The highest mortality was identified in those who were hyperphosphatemic on the 5-10th-days. PELOD scores were only significantly different according to admission phosphorus levels (p:0.04)., Conclusion: In our study, we identified that hyperphosphatemia is a serious problem as hypophosphatemia for patients who admitted to the PICU. Patients identified to be hyperphosphatemic on admission had a significantly higher PELOD score. The significant difference of hyperphosphatemia in terms of PELOD score is one of the important points shown in our study. It should not be forgotten that like hypophosphatemia, hyperphosphatemia may cause serious problems in pediatric intensive care patients., (Copyright© of YS Medical Media ltd.)

How inorganic phosphate (Pi) homeostasis is regulated in Drosophila is currently unknown. We here identify MFS2 as a key Pi transporter in fly renal (Malpighian) tubules. Consistent with its role in Pi excretion, we found that dietary Pi induces MFS2 expression. This results in the formation of Malpighian calcium-Pi stones, while RNAi-mediated knockdown of MFS2 increases blood (hemolymph) Pi and decreases formation of Malpighian tubule stones in flies cultured on high Pi medium. Conversely, microinjection of adults with the phosphaturic human hormone fibroblast growth factor 23 (FGF23) induces tubule expression of MFS2 and decreases blood Pi. This action of FGF23 is blocked by genetic ablation of MFS2. Furthermore, genetic overexpression of the fly FGF branchless (bnl) in the tubules induces expression of MFS2 and increases Malpighian tubule stones suggesting that bnl is the endogenous phosphaturic hormone in adult flies. Finally, genetic ablation of MFS2 increased fly life span, suggesting that Malpighian tubule stones are a key element whereby high Pi diet reduces fly longevity previously reported by us. In conclusion, MFS2 mediates excretion of Pi in Drosophila, which is as in higher species under the hormonal control of FGF-signaling.

Medial vascular calcification has emerged as a putative key factor contributing to the excessive cardiovascular mortality of patients with chronic kidney disease (CKD). Hyperphosphatemia is considered a decisive determinant of vascular calcification in CKD. A critical role in initiation and progression of vascular calcification during elevated phosphate conditions is attributed to vascular smooth muscle cells (VSMCs), which are able to change their phenotype into osteo-/chondroblasts-like cells. These transdifferentiated VSMCs actively promote calcification in the medial layer of the arteries by producing a local pro-calcifying environment as well as nidus sites for precipitation of calcium and phosphate and growth of calcium phosphate crystals. Elevated extracellular phosphate induces osteo-/chondrogenic transdifferentiation of VSMCs through complex intracellular signaling pathways, which are still incompletely understood. The present review addresses critical intracellular pathways controlling osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification during hyperphosphatemia. Elucidating these pathways holds a significant promise to open novel therapeutic opportunities counteracting the progression of vascular calcification in CKD.

Phosphate binders and dialysis can have harmful side-effects during the treatments of hyperphosphatemia. Therefore, we evaluated the capability of intestinal bacteria (lactic acid bacteria and bifidobacteria) as phosphate-accumulating organisms (PAOs) for phosphate accumulation, with the aim of determining whether PAO-formulated food can prevent hyperphosphatemia in the early stages. However, methods for estimating microbial phosphate-accumulation capacities require significant improvements regarding specificity, cost, and simplicity. The presented method analyzed cell-free broth to assess the phosphate accumulation capability of cells. Active cells and the constructed phosphate-deficient cells were incubated in assay salt media. After incubation, phosphate-deficient cell-free broth was taken as sample and the blank was the active cell-free broth. Therefore, effects of interfering agents and other metabolites were avoided and enhanced the specificity remarkably. Phosphate contents were assessed by reactions with toluidine blue O. In contrast to the case in previous studies, the shift in the first absorbance peak was found to be inversely proportional to the phosphate concentration. The minimum detectable phosphate concentrations for the 11th isolate of Lactobacillus casei JCM 1134 and 8th isolate of Bifidobacterium adolescentis JCM 1275 were determined to be 1.24 and 0.4 mg/L, respectively. Further, the validation results were found to be significant (p-value < 0.05).

Hyperphosphatemic familial tumoral calcinosis (HFTC)/hyperostosis-hyperphosphatemia syndrome (HHS) is an autosomal recessive disorder of ectopic calcification due to deficiency of or resistance to intact fibroblast growth factor 23 (iFGF23). Inactivating mutations in FGF23, N-acetylgalactosaminyltransferase 3 (GALNT3), or KLOTHO (KL) have been reported as causing HFTC/HHS. We present what we believe is the first identified case of autoimmune hyperphosphatemic tumoral calcinosis in an 8-year-old boy. In addition to the classical clinical and biochemical features of hyperphosphatemic tumoral calcinosis, the patient exhibited markedly elevated intact and C-terminal FGF23 levels, suggestive of FGF23 resistance. However, no mutations in FGF23, KL, or FGF receptor 1 (FGFR1) were identified. He subsequently developed type 1 diabetes mellitus, which raised the possibility of an autoimmune cause for hyperphosphatemic tumoral calcinosis. Luciferase immunoprecipitation systems revealed markedly elevated FGF23 autoantibodies without detectable FGFR1 or Klotho autoantibodies. Using an in vitro FGF23 functional assay, we found that the FGF23 autoantibodies in the patient's plasma blocked downstream signaling via the MAPK/ERK signaling pathway in a dose-dependent manner. Thus, this report describes the first case, to our knowledge, of autoimmune hyperphosphatemic tumoral calcinosis with pathogenic autoantibodies targeting FGF23. Identification of this pathophysiology extends the etiologic spectrum of hyperphosphatemic tumoral calcinosis and suggests that immunomodulatory therapy may be an effective treatment.

Hyperphosphatemia, the elevated level of inorganic phosphate (Pi) in serum, is associated with increased cardiovascular morbidities and mortality. The effects of high Pi on endothelial cells are not well studied. This study investigated high Pi-induced endothelial cell apoptosis and the role of microRNA-21. Mouse myocardial endothelial cells (MEC) were cultured in normal (1 mM) and high (5 mM) Pi conditions. Apoptosis was detected by TUNEL staining and flow cytometry. MicroRNA profiles of MEC response to changes in Pi concentration were obtained using gene expression arrays. Expression levels of the microRNA-21 target genes, programmed cell death gene 4 ( PDCD4), poly(ADP-ribose) polymerase ( PARP), and phosphatase and tensin homolog ( PTEN), as well as NF-κB were measured by Western blotting and RT-PCR. MicroRNA-21-specific inhibitors and mimics were used to study effects of microRNA-21 on MEC apoptosis and gene expression regulations. High Pi induced MEC apoptosis and upregulated microRNA-21 expression. MicroRNA-21-specific mimics reproduced high Pi-induced apoptosis in normal Pi medium, and microRNA-21 inhibitors ameliorated the high Pi induction of apoptosis, suggesting that microRNA-21 mediated high Pi-induced MEC apoptosis. The microRNA-21 targets PDCD4, PTEN, PARP, and NF-κB were significantly downregulated in high Pi conditions. High Pi-induced downregulation of PDCD4 was abolished by microRNA-21 inhibitors and selective ERK inhibitor (selumetinib) and was reproduced by microRNA-21 mimics. Inhibitors and mimics of microRNA-21 did not have effects on high Pi-induced NF-κB downregulation. Selumetinib blocked high Pi-induced NF-κB downregulation. MicroRNA-21 mediates high Pi-induced endothelial cell apoptosis, which involves an ERK1/2/microRNA-21/PDCD4 pathway. High Pi-induced downregulation of NF-κB expression is mediated by an ERK1/2 signaling-dependent but microRNA-21-independent mechanism.

Mineral bone disease (MBD) is a common complication of chronic kidney disease (CKD) characterized by disruption of normal mineral homeostasis within the body. One or more of the following may occur: hypocalcemia, hyperphosphatemia, secondary hyperparathyroidism (SHPT), decreased vitamin D and vascular calcification (VC). The greater the decrease in renal function, the worse the progression of CKD-MBD. These abnormalities may lead to bone loss, osteoporosis and fractures. CKD-MBD is a major contributor to the high morbidity and mortality among patients with CKD. Another well-known complication of CKD is cardiovascular disease (CVD) caused by increased atherosclerosis and VC. CVD is the leading cause of morbidity and mortality in CKD patients. VC is linked to reduced arterial compliance that may lead to widened pulse pressure and impaired cardiovascular function. VC is a strong predicator of cardiovascular mortality among patients with CKD. Elevated phosphorus levels and increased calcium-phosphorus product promote VC. Controlling mineral disturbances such as hyperphosphatemia and SHPT is still considered among the current strategies for treatment of VC in CKD through restriction of calcium based phosphate binders in hyperphosphatemic patients across all severities of CKD along with dietary phosphate restriction and use of calciminetics. Additionally, Vitamin D insufficiency is common in CKD and dialysis patients. The causes are multifactorial and a serious consequence is SHPT. Vitamin D compounds remain the first-line therapy for prevention and treatment of SHPT in CKD. Vitamin D may also have atheroprotective effects on the arterial wall, but clinical studies do not show clear evidence of reduced cardiovascular mortality with vitamin D administration. This review discusses the issues surrounding CKD-MBD, cardiovascular disease and approaches to treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hyperphosphatemic familial tumoral calcinosis (HFTC), secondary to fibroblast growth factor 23 (FGF23) gene mutation, is a rare genetic disorder characterized by recurrent calcified masses. We describe young Lebanese cousins presenting with HFTC, based on a retrospective chart review and a prospective case study. In addition, we present a comprehensive review on the topic, based on a literature search conducted in PubMed and Google Scholar, in 2014 and updated in December 2017. While the patients had the same previously reported FGF23 gene mutation (homozygous c.G367T variant in exon 3 leading to a missense mutation), they presented with variable severity and age of disease onset (at 4 years in patient 1 and at 23 years in patient 2). A review of the literature revealed several potential patho-physiologic pathways of HFTC clinical manifestations, some of which may be independent of hyperphosphatemia. Most available treatment options aim at reducing serum phosphate level, by stimulating renal excretion or by inhibiting intestinal absorption. HFTC is a challenging disease. While the available medical treatment has a limited and inconsistent effect on disease symptomatology, surgical resection of calcified masses remains the last resort. Research is needed to determine the safety and efficacy of FGF23 replacement or molecular therapy, targeting the specific genetic aberration. Hyperphosphatemic familial tumoral calcinosis is a rare genetic disorder characterized by recurrent calcified masses, in addition to other visceral, skeletal, and vascular manifestations. It remains a very challenging disease.

Hyperphosphatemia is a common complication of chronic kidney disease (CKD) and is associated with cardiovascular disease (CVD), which has contributed to an increase in mortality of CKD patients. The onset of CVD often varies by time-of-day. Acute myocardial infarction or ventricular arrhythmia occurs most frequently during early morning. Blood pressure (BP) and heart rate circadian rhythms account for the diurnal variations in CVD. Preservation of normal circadian time structure from the cardiomyocyte level to the whole organ system is essential for cardiovascular health and CVD prevention. Independent risk factors, such as reduced heart rate variability (HRV) and increased BP variability (BPV), are particularly prevalent in patients with CKD. Analysis of HRV is an important clinical tool for characterizing cardiac autonomic status, and reduced HRV has prognostic significance for various types of CVD. Circadian BP rhythms are classified as extreme dipper, dipper, non-dipper or riser. It has been reported that nocturnal riser BP pattern contributes to cardiovascular threats. Previous studies have indicated that the circadian rhythm of serum phosphate in CKD patients is consistent with the general population, with the highest diurnal value observed in the early morning hours, followed by a progressive decrease to the lowest value of the day, which occurs around 11:00 am. Rhythm abnormalities have become the main therapeutic target for treating CVD in CKD patients. It has been reported that high levels of serum phosphate are associated with reduced HRV and increased BPV in CKD patients. However, the mechanisms related to interactions between hyperphosphatemia, HRV and BPV have not been fully elucidated. This review focuses on the evidence and discusses the potential mechanisms related to the effects of hyperphosphatemia on HRV and BPV.

Background: Accelerated muscle atrophy is associated with a three-fold increase in mortality in chronic kidney disease (CKD) patients. It is suggested that hyperphosphatemia might contribute to muscle wasting, but the underlying mechanisms remain unclear. Although evidence indicates that autophagy is involved in the maintenance of muscle homeostasis, it is not known if high phosphate levels can result in activation of autophagy, leading to muscle protein loss., Methods: Immortalized rat L6 myotubes were exposed to a high concentration of phosphate, with or without autophagy inhibition. Myotube atrophy was examined by phase contrast microscopy. Autophagic activity was assessed by measuring the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 using quantitative real-time polymerase chain reaction and western blot., Results: Phosphate induced cell atrophy in L6 myotubes in a dose- and time-dependent manner, and these responses were not associated with calcification or osteogenesis. Phosphate also dose- and time-dependently increased the LC3-II/LC3-I ratio. Inhibition of autophagy with wortmannin or knockdown of Atg5 significantly suppressed myotube atrophy caused by high phosphate concentration., Conclusions: High phosphate concentration induces muscle cell atrophy through the activation of autophagy. Targeting autophagy could be a therapeutic strategy for preventing muscle wasting caused by hyperphosphatemia.

Hyperphosphatemia is related to some pathologies, affecting vascular cell behavior. This work analyzes whether high concentration of extracellular phosphate induces endothelial senescence through up-regulation of endothelin-1 (ET-1), exploring the mechanisms involved. The phosphate donor β-glycerophosphate (BGP) in human endothelial cells increased ET-1 production, endothelin-converting enzyme-1 (ECE-1) protein, and mRNA expression, which depend on the AP-1 activation through ROS production. In parallel, BGP also induced endothelial senescence by increasing p16 expression and the senescence-associated β-galactosidase (SA-ß-GAL) activity. ET-1 itself was able to induce endothelial senescence, increasing p16 expression and SA-ß-GAL activity. In addition, senescence induced by BGP was blocked when different ET-1 system antagonists were used. BGP increased ROS production at short times, and the presence of antioxidants prevented the effect of BGP on AP1 activation, ECE-1 expression, and endothelial senescence. These findings were confirmed in vivo with two animal models in which phosphate serum levels were increased: seven/eight nephrectomized rats as chronic kidney disease models fed on a high phosphate diet and aged mice. Both models showed hyperphosphatemia, higher levels of ET-1, and up-regulation in aortic ECE-1, suggesting a direct relationship between hyperphosphatemia and ET-1. Present results point to a new and relevant role of hyperphosphatemia on the regulation of ET-1 system and senescence induction at endothelial level, both in endothelial cells and aorta from two animal models. The mechanism involved showed a higher ROS production, which probably activates AP-1 transcription factor and, as a result, ECE-1 expression, increasing ET-1 synthesis, which in consequence induces endothelial senescence., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

This study investigates the effect of nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) signaling pathway in vascular calcification (VC) via inducing Autophagy in renal vascular smooth muscle cells (VSMCs). VSMCs were assigned into six experimental groups: the normal control, high phosphorus, si-negative control (si-NC), Nrf2-siRNA, over-expressed Nrf2, and negative control (NC) groups. RT-PCR was applied to detect the mRNA expressions of the desired Nrf2-ARE signaling pathway-related genes (Nrf2, NQO-1, HO-1, γ-GCS). The protein products of these genes: apoptosis-related genes (LC3I and LC3II), osteogenic marker proetins (Runt-related transcription factor 2) Runx2 and BMP2 were all detected by Western blotting. Autophagosomes in VSMCs were observed under a transmission electron microscope. We discovered an increased calcium ion concentration and upregulated Runx2, BMP2, Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions in the high phosphorous, si-NC and Nrf2-siRNA, and NC groups, compared with the normal control group. Compared to the high phosphorus and si-NC groups, higher levels of Runx2 and BMP2 but decreased Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions were detected in the Nrf2-siRNA group. The high phosphorus, si-NC and over-expressed Nrf2 experimental groups all had increased Nrf2, NQO-1, HO-1, γ-GCS, and LC3II/LC3I expressions as well as high numbers of autophagosomes compared with the normal control group. Finally, we detected a lower amount of autophagosomes presence and Nrf2, NQO-1, HO-1 γ-GCS, and LC3II/LC3 protein expression of Nrf2-siRNA group than that of the high phosphorus and si-NC groups. Activation of Nrf2-ARE signaling pathway may prevent hyperphosphatemia-induced VC by inducing autophagy in VSMCs. J. Cell. Biochem. 118: 4708-4715, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: Hyperphosphataemia is an independent risk factor for accelerated cardiovascular disease in chronic kidney disease (CKD), although the mechanism for this is poorly understood. We investigated the effects of sustained exposure to a high-phosphate environment on endothelial function in cellular and preclinical models, as well as in human subjects., Methods: Resistance vessels from rats and humans (± CKD) were incubated in a normal (1.18 mM) or high (2.5 mM) phosphate concentration solution and cells were cultured in normal- (0.5 mM) or high-phosphate (3 mM) concentration media. A single-blind crossover study was performed in healthy volunteers, receiving phosphate supplements or a phosphate binder (lanthanum), and endothelial function measured was by flow-mediated dilatation., Results: Endothelium-dependent vasodilatation was impaired when resistance vessels were exposed to high phosphate; this could be reversed in the presence of a phosphodiesterase-5-inhibitor. Vessels from patients with CKD relaxed normally when incubated in normal-phosphate conditions, suggesting that the detrimental effects of phosphate may be reversible. Exposure to high-phosphate disrupted the whole nitric oxide pathway with reduced nitric oxide and cyclic guanosine monophosphate production and total and phospho endothelial nitric oxide synthase expression. In humans, endothelial function was reduced by chronic phosphate loading independent of serum phosphate, but was associated with higher urinary phosphate excretion and serum fibroblast growth factor 23., Conclusions: These directly detrimental effects of phosphate, independent of other factors in the uraemic environment, may explain the increased cardiovascular risk associated with phosphate in CKD., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Objective To analyze the clinical characteristics and endoscopic features of patients with lanthanum deposition in the stomach. Patients We retrospectively reviewed seven patients with lanthanum deposition in the stomach who were diagnosed at Okayama Saiseikai General Hospital. We investigated the patient sex, age at diagnosis, medical and medication histories, gastrointestinal symptoms, complications, presence or absence of gastric atrophy, and outcome. We also investigated any changes in the endoscopic features if previous endoscopic images were available. Results Seven patients (six males and one female) had lanthanum deposition. The median age was 65 years (range, 50-79 years). All patients had been undergoing dialysis (continuous ambulatory peritoneal dialysis in one patient, hemodialysis in six patients). The dialysis period ranged from 16 to 73 months (median, 52 months). The patients had all been taking lanthanum carbonate for a period ranging from 5 to 45 months (median, 27 months). Gastric atrophy was noted in 6 patients (85.7%). One patient had difficulty swallowing, and 1 other patient had appetite loss. The other 5 patients were asymptomatic. Endoscopic features included annular whitish mucosa (n = 4), diffuse whitish mucosa (n = 3), and whitish spots (n = 2). Five patients underwent multiple esophagogastroduodenoscopy. The endoscopic features were unchanged in 2 patients, whereas the whitish mucosa became apparent and spread during the course in 3 patients. Conclusion We identified 7 patients with lanthanum deposition in the stomach. All patients showed whitish lesions macroscopically. Although the pathogenicity of gastric lanthanum deposition is uncertain, lanthanum-related lesions in the stomach progressed during continuous lanthanum phosphate intake in several patients.

Background: Sucroferric oxyhydroxide is a non-calcium, iron-based phosphate binder indicated for the treatment of hyperphosphataemia in adult dialysis patients. This post hoc analysis of a randomized, 24-week Phase 3 study and its 28-week extension was performed to evaluate the long-term effect of sucroferric oxyhydroxide on iron parameters., Methods: A total of 1059 patients were randomized to sucroferric oxyhydroxide 1.0-3.0 g/day (n = 710) or sevelamer carbonate ('sevelamer') 2.4-14.4 g/day (n = 349) for up to 52 weeks. The current analysis only included patients who completed 52 weeks of continuous treatment (n = 549). Changes in iron-related parameters and anti-anaemic product use during the study were measured., Results: Some changes in iron-related parameters across both treatment groups were observed during the first 24 weeks of the study, and to a lesser extent with longer-term treatment. There were small, but significantly greater increases in mean transferrin saturation (TSAT) and haemoglobin levels with sucroferric oxyhydroxide versus sevelamer during the first 24 weeks (change in TSAT: +4.6% versus +0.6%, P = 0.003; change in haemoglobin: +1.6 g/L versus -1.1 g/L, P = 0.037). Mean serum ferritin concentrations also increased from Weeks 0 to 24 with sucroferric oxyhydroxide and sevelamer (+119 ng/mL and +56.2 ng/mL respectively; no statistically significant difference between groups). In both treatment groups, ferritin concentrations increased to a greater extent in the overall study population [>70% of whom received concomitant intravenous (IV) iron], compared with the subset of patients who did not receive IV iron therapy during the study. The pattern of anti-anaemic product use was similar in both treatment groups, with a trend towards higher use of IV iron and erythropoiesis-stimulating agents with sevelamer., Conclusions: Initial increases in some iron-related parameters were observed in both treatment groups but were more pronounced with sucroferric oxyhydroxide. These differences between treatment groups with respect to changes in iron parameters are likely due to minimal iron absorption from sucroferric oxyhydroxide., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA.)

Familial tumoral calcinosis (FTC)/hyperostosis-hyperphosphatemia syndrome (HHS) is a rare disorder caused by mutations in the genes encoding fibroblast growth factor-23 (FGF23), N-acetylgalactosaminyltransferase 3 (GALNT3), or KLOTHO. The result is functional deficiency of, or resistance to, intact FGF23 (iFGF23), causing hyperphosphatemia, increased renal tubular reabsorption of phosphorus (TRP), elevated or inappropriately normal 1,25-dihydroxyvitamin D 3 (1,25D), ectopic calcifications, and/or diaphyseal hyperostosis. Eight subjects with FTC/HHS were studied and treated. Clinical manifestations varied, even within families, ranging from asymptomatic to large, disabling calcifications. All subjects had hyperphosphatemia, increased TRP, and elevated or inappropriately normal 1,25D. C-terminal FGF23 was markedly elevated whereas iFGF23 was comparatively low, consistent with increased FGF23 cleavage. Radiographs ranged from diaphyseal hyperostosis to massive calcification. Two subjects with severe calcifications also had overwhelming systemic inflammation and elevated C-reactive protein (CRP). GALNT3 mutations were identified in seven subjects; no causative mutation was found in the eighth. Biopsies from four subjects showed ectopic calcification and chronic inflammation, with areas of heterotopic ossification observed in one subject. Treatment with low phosphate diet, phosphate binders, and phosphaturia-inducing therapies was prescribed with variable response. One subject experienced complete resolution of a calcific mass after 13 months of medical treatment. In the two subjects with systemic inflammation, interleukin-1 (IL-1) antagonists significantly decreased CRP levels with resolution of calcinosis cutis and perilesional inflammation in one subject and improvement of overall well-being in both subjects. This cohort expands the phenotype and genotype of FTC/HHS and demonstrates the range of clinical manifestations despite similar biochemical profiles and genetic mutations. Overwhelming systemic inflammation has not been described previously in FTC/HHS; the response to IL-1 antagonists suggests that anti-inflammatory drugs may be useful adjuvants. In addition, this is the first description of heterotopic ossification reported in FTC/HHS, possibly mediated by the adjacent inflammation. © 2016 American Society for Bone and Mineral Research., Competing Interests: Statement: All other authors have no conflicts of interest., (© 2016 American Society for Bone and Mineral Research.)

Aims: Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification., Methods and Results: Experiments were performed in hyperphosphatemic klotho-hypomorphic mice (kl/kl), in subtotal nephrectomy and vitamin D3-overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 (Pai1) and of osteogenic markers Msx2, Cbfa1, and Alpl. Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1, MSX2, CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine., Conclusion: Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Aims: Hyperphosphatemia is associated with accelerated vascular endothelial dysfunction in patients with chronic kidney disease (CKD). The purpose of this study is to investigate the molecular mechanisms underlying hyperphosphatemia-caused endothelial dysfunction., Main Methods: The metabolic fingerprinting of human umbilical vein endothelial cells (HUVECs) subjected to hyperphosphatemia was characterized using an integrated metabolomics approach. HUVECs cultured in physiologically simulated hyperphosphatemia with or without phosphonoformic acid, a sodium-dependent phosphate transporter inhibitor (N=6) were collected for metabolomics analysis. Multivariate principle component analysis and partial least squares discriminant analysis were applied to analyze the metabolic data. The key metabolites were confirmed by quantitative analysis using liquid chromatography coupled with tandem mass spectrometer (LC-MS/MS)., Key Findings: 36 metabolites were significantly altered in HUVECs following the challenges of hyperphosphatemia mimic, involving several metabolic pathways (all P<0.05). Among them, ornithine increased significantly in the HUVECs mediated by hyperphosphatemia mimic, and its levels positively correlated with cell apoptosis rate (r=0.674, P=0.002), and several additional metabolites in multiple metabolic pathways. The changes in the levels of ornithine and other several metabolites were supported by subsequent quantitative analyses using LC-MS/MS. Further study demonstrated that the increase in ornithine level may result from the increased expression of arginase 2 in HUVECs, which mediates the hydrolysis of arginine to form ornithine., Significance: This is the first study demonstrating ornithine a key molecule mediating hyperphosphatemia-induced apoptosis of ECs. Arginase 2 may be a therapeutic target for hyperphosphatemia-associated cardiovascular events., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aging is conditioned by genetic and environmental factors. Hyperphosphatemia is related to some pathologies, affecting to vascular cells behavior. This work analyze whether high concentration of extracellular phosphate induces vascular smooth muscle cells senescence, exploring the intracellular mechanisms and highlighting the in vivo relevance of this phenomenon. Human aortic smooth muscle cells treated with β-Glycerophosphate (BGP, 10mM) suffered cellular senescence by increasing p53, p21 and p16 expression and the senescence associated β-galactosidase activity. In parallel, BGP induced ILK overexpression, dependent on the IGF-1 receptor activation, and oxidative stress. Down-regulating ILK expression prevented BGP-induced senescence and oxidative stress. Aortic rings from young rats treated with 10mM BGP for 48h, showed increased p53, p16 and ILK expression and SA-β-gal activity. Seven/eight nephrectomized rats feeding a hyperphosphatemic diet and fifteenth- month old mice showed hyperphosphatemia and aortic ILK, p53 and p16 expression. In conclusion, we demonstrated that high extracellular concentration of phosphate induced senescence in cultured smooth muscle through the activation of IGF-1 receptor and ILK overexpression and provided solid evidences for the in vivo relevance of these results since aged animals showed high levels of serum phosphate linked to increased expression of ILK and senescence genes., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction., Methods: The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry., Results: The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells., Conclusions: Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis., (Copyright © 2015 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Hypocalcemia is a frequent abnormality that has been associated with disease severity and outcome in hospitalized foals. However, the pathogenesis of equine neonatal hypocalcemia is poorly understood. Hypovitaminosis D in critically ill people has been linked to hypocalcemia and mortality; however, information on vitamin D metabolites and their association with clinical findings and outcome in critically ill foals is lacking. The goal of this study was to determine the prevalence of vitamin D deficiency (hypovitaminosis D) and its association with serum calcium, phosphorus, and parathyroid hormone (PTH) concentrations, disease severity, and mortality in hospitalized newborn foals., Methods and Results: One hundred newborn foals ≤72 hours old divided into hospitalized (n = 83; 59 septic, 24 sick non-septic [SNS]) and healthy (n = 17) groups were included. Blood samples were collected on admission to measure serum 25-hydroxyvitamin D3 [25(OH)D3], 1,25-dihydroxyvitamin D3 [1,25(OH) 2D3], and PTH concentrations. Data were analyzed by nonparametric methods and univariate logistic regression. The prevalence of hypovitaminosis D [defined as 25(OH)D3 <9.51 ng/mL] was 63% for hospitalized, 64% for septic, and 63% for SNS foals. Serum 25(OH)D3 and 1,25(OH) 2D3 concentrations were significantly lower in septic and SNS compared to healthy foals (P<0.0001; P = 0.037). Septic foals had significantly lower calcium and higher phosphorus and PTH concentrations than healthy and SNS foals (P<0.05). In hospitalized and septic foals, low 1,25(OH)2D3 concentrations were associated with increased PTH but not with calcium or phosphorus concentrations. Septic foals with 25(OH)D3 <9.51 ng/mL and 1,25(OH) 2D3 <7.09 pmol/L were more likely to die (OR=3.62; 95% CI = 1.1-12.40; OR = 5.41; 95% CI = 1.19-24.52, respectively)., Conclusions: Low 25(OH)D3 and 1,25(OH)2D3 concentrations are associated with disease severity and mortality in hospitalized foals. Vitamin D deficiency may contribute to a pro-inflammatory state in equine perinatal diseases. Hypocalcemia and hyperphosphatemia together with decreased 1,25(OH)2D3 but increased PTH concentrations in septic foals indicates that PTH resistance may be associated with the development of these abnormalities.

Background: Hyperphosphatemic familial tumoral calcinosis (HFTC) is to a rare autosomal recessive disorder characterized by cutaneous and sub-cutaneous calcified masses, usually adjacent to large joints. The aim of the current study was to report on the clinico-pathological features of a patient with HFCT, with emphasis on alterations in the jawbones and teeth and the subsequent therapeutic interventions., Case Report: A 13-year-old male patient with HFTC diagnosis came to our attention for dental anomalies and maxillary and mandibular hypoplasia. OPT highlighted multiple impacted teeth, short and bulbous teeth, and pulp chamber and canal obliterations. Lateral cephalometric radiograms pointed out retrusion of both jaws, skeletal class II malocclusion, and deep-bite. He underwent orthopedic, orthodontic, conservative, and surgical treatments, allowing the correction of maxillo-facial and dental abnormalities and dysmorphisms without adverse effects. The surgical samples were sent for conventional and confocal laser scanning microscope (CLSM) histopathological examination, which highlighted several metaplastic micro- and macro-calcifications in the soft tissues, and typical islands of homogenous, non-tubular, dentino-osteoid calcified structures in dentinal tissues., Conclusions: The management of maxillo-facial abnormalities in patients affected by HFTC is very difficult and, requires a combined therapeutic approach. To date, very few indications have been published in the literature.

Background: The mechanisms responsible for the hyperphosphatemia in patients with sickle cell disease (SCD) and preserved glomerular filtration rate (GFR) are not fully understood. The role of fibroblast growth factor 23 (FGF23), a phosphaturic hormone has not been investigated in SCD. Hence, we evaluated parameters of renal tubular phosphorus handling and their relation to prevailing FGF23 levels in a cohort of young SCD patients., Methods: Renal tubular phosphate handling and circulating levels of various analytes including FGF23 and parathyroid hormone (PTH) were measured in 24 children with SCD and normal estimated GFR in a cross sectional study. Correlation and regression analysis were employed to derive relationships between serum phosphorus and several variables., Results: Most children showed elevated age- adjusted serum phosphorus (5.1 ± 0.7 mg/dl) levels. Tubular re-absorption of phosphorus(TRP) (96.3 ± 2.1%) and tubular maximum re-absorption of phosphorus per unit volume of GFR (TMP/GFR) (4.9 ± 0.6 mg/dl) were both elevated. Plasma intact FGF23 concentrations were elevated (81 ± 38 pg/ml) while the average PTH values were normal in most patients (50 ± 27 pg/ml). Univariate analysis showed significant correlations of serum phosphorus with TMP/GFR, alkaline phosphatase, age, lactate dehydrogenase (LDH), and log intact FGF23. TMP/GFR correlated with log intact FGF23 (r = 0.5, P< or = 0.01) but not with PTH. Multiple regression analysis yielded an independent relationship of serum phosphorus with TMP/GFR., Conclusion: The elevated serum phosphorus concentrations with simultaneously increased TMP/GFR and elevated FGF23 levels collectively suggest that patients with SCD display proximal tubular resistance to the action of FGF23 before any decline in GFR., (© 2014 Wiley Periodicals, Inc.)

The concept of chronic kidney disease-mineral bone disorder (CKD-MBD) does not appear to fulfil the requirements for a syndrome at first glance, but its definition has brought some clear-cut benefits for clinicians and patients, including wider and more complex diagnostic and therapeutic approaches to the management of this challenging set of issues. Admittedly, not all components of CKD-MBD are present in all patients at all times, but these are highly interrelated, involving mineral and bone laboratory abnormalities, clinical and histological bone disease and finally, cardiovascular disease. The presence of typical biological bone ossification processes in an ectopic anatomical location in CKD has helped to define the existence of an unprecedented bone-vascular relationship, extending its interest even to other medical specialities. For now, we believe that CKD-MBD does not reach full criteria to be defined as a syndrome. However, this novel concept has clearly influenced current clinical guidelines. The National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF/KDOQI™) guidelines in 2003 for instance recommended that calcium-based phosphate binders should be avoided to treat hyperphosphataemia in the presence of cardiovascular calcifications. In 2009, the KDIGO and other guidelines reinforced and extended this recommendation by stating that it is reasonable to choose oral phosphate binder therapy by taking into consideration other components of CKD-MBD. Similarly, it is also considered reasonable to use information on vascular/valvular calcification to guide the management of CKD-MBD. Our current assumption as a working group 'CKD-MBD' is that CKD-MBD has the potential to be defined a true syndrome, such as a constellation of concurrent signs and symptoms that suggest a common underlying mechanism for these components as opposed to the term disease. The term 'syndrome' also implies that in any patient at risk due to the presence of one or a few components of the entire syndrome, the screening for additional components is highly recommended. However, it has not currently been demonstrated that there is an additive predictive value, which can be derived from identifying individual components. Despite all we have learned about this putative syndrome, we have been left with only a hypothetical framework about how to treat patients. So while we agree that the concept of CKD-MBD has influenced, and continues to influence, our current clinical hypotheses, definitive proof of a benefit of interventions in CKD-MBD is still lacking and a global-multiple therapeutic approach to treat simultaneously several components of CKD-MBD should be tested by well-designed new randomized controlled trials., (© The Author 2014. Published by Oxford University Press onbehalf of ERA-EDTA. All rights reserved.)

Disorders of calcium and phosphorus homeostasis present both acute and chronic clinical consequences for newborns. The etiologies responsible range from iatrogenic, idiopathic, and inherited metabolic abnormalities. Maintenance of physiologically normal serum calcium and phosphorus requires complex interactions between the kidneys, gastrointestinal tract, and bone. Calciotropic hormones such as vitamin D and parathyroid hormone, as well as hormones controlling phosphorus homeostasis, such as fibroblast growth factor-23 (FGF-23), are essential in controlling these interactions. In newborns, calcium and phosphorus balance must necessarily be positive in order to provide the requisite building blocks for growth and maturation. Renal tubular handling of these minerals is a key control point in regulating the overall body balance in calcium and phosphorus. Adaptive changes in renal calcium and phosphorus reabsorption in newborns explain how a net positive total body balance of these minerals is achieved. Monogenetic disorders leading to abnormal renal handling of calcium and/or phosphorus have immediate clinical consequences in terms of complications associated with high or low levels of these minerals. Perhaps more importantly, chronic abnormalities of calcium and/or phosphorus, without treatment, may have serious consequences for growth and development of the growing skeleton. This article serves to review calcium and phosphorus regulation in the human body, describe differences in handling of these minerals by the newborn, and review the conditions, both acquired and congenital, that may present with abnormalities in calcium and/or phosphorus in the newborn period.

The role of hyperphosphatemia in the pathogenesis of secondary hyperparathyroidism, cardiovascular disease, and progression of renal failure is widely known. Here we studied effects of dietary phosphate restriction on mortality and vascular calcification in uremic rats. Control and uremic rats were fed a high-phosphate diet and at 3 months a portion of rats of each group were killed. Serum phosphate and the calcium phosphate product increased in uremic rats, as did aortic calcium. Of the rats, 56% had positive aortic staining for calcium (von Kossa), RUNX2, and osteopontin. The remaining uremic rats were continued on diets containing high phosphate without and with sevelamer, or low phosphate, and after 3 more months they were killed. Serum phosphate was highest in uremic rats on high phosphate. Serum PTH and FGF-23 were markedly lower in rats on low phosphate. Mortality on high phosphate was 71.4%, with sevelamer reducing this to 37.5% and phosphate restriction to 5.9%. Positive aortic staining for von Kossa, RUNX2, and osteopontin was increased, but phosphate restriction inhibited this. Kidneys from low-phosphate and sevelamer-treated uremic rats had less interstitial fibrosis, glomerulosclerosis, and inflammation than those of uremic rats on high phosphate. Importantly, kidneys from rats on low phosphate showed improvement over kidneys from high-phosphate rats at 3 months. Left ventricles from rats on low phosphate had less perivascular fibrosis and smaller cardiomyocyte size compared to rats on high phosphate. Thus, intensive phosphate restriction significantly reduces mortality in uremic rats with severe vascular calcification.

Ectopic mineralization has been linked to several common clinical conditions with considerable morbidity and mortality. The mineralization processes, both metastatic and dystrophic, affect the skin and vascular connective tissues. There are several contributing metabolic and environmental factors that make uncovering of the precise pathomechanisms of these acquired disorders exceedingly difficult. Several relatively rare heritable disorders share phenotypic manifestations similar to those in common conditions, and, consequently, they serve as genetically controlled model systems to study the details of the mineralization process in peripheral tissues. This overview will highlight diseases with mineral deposition in the skin and vascular connective tissues, as exemplified by familial tumoral calcinosis, pseudoxanthoma elasticum, generalized arterial calcification of infancy, and arterial calcification due to CD73 deficiency. These diseases, and their corresponding mouse models, provide insight into the pathomechanisms of soft tissue mineralization and point to the existence of intricate mineralization/anti-mineralization networks in these tissues. This information is critical for understanding the pathomechanistic details of different mineralization disorders, and it has provided the perspective to develop pharmacological approaches to counteract the consequences of ectopic mineralization., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)