Elevated Phosphate Levels Research Articles

The inositol hexakisphosphate kinases IP6K1 and -2 regulate human cellular phosphate homeostasis, including XPR1-mediated phosphate export

Phosphate's central role in most biochemical reactions in a living organism requires carefully maintained homeostasis. Although phosphate homeostasis in mammals has long been studied at the organismal level, the intracellular mechanisms controlling phosphate metabolism are not well-understood. Inositol pyrophosphates have emerged as important regulatory elements controlling yeast phosphate homeostasis. To verify whether inositol pyrophosphates also regulate mammalian cellular phosphate homeostasis, here we knocked out inositol hexakisphosphate kinase (IP6K) 1 and IP6K2 to generate human HCT116 cells devoid of any inositol pyrophosphates. Using PAGE and HPLC analysis, we observed that the IP6K1/2-knockout cells have nondetectable levels of the IP6-derived IP7 and IP8 and also exhibit reduced synthesis of the IP5-derived PP-IP4. Nucleotide analysis showed that the knockout cells contain increased amounts of ATP, whereas the Malachite green assay found elevated levels of free intracellular phosphate. Furthermore, [32Pi] pulse labeling experiments uncovered alterations in phosphate flux, with both import and export of phosphate being decreased in the knockout cells. Functional analysis of the phosphate exporter xenotropic and polytropic retrovirus receptor 1 (XPR1) revealed that it is regulated by inositol pyrophosphates, which can bind to its SPX domain. We conclude that IP6K1 and -2 together control inositol pyrophosphate metabolism and thereby physiologically regulate phosphate export and other aspects of mammalian cellular phosphate homeostasis.

Nutrition and phosphate awareness in patients with end stage renal failure (ESRF) at an in-centre haemodialysis unit in western australia (WA)

Rationale: When compared with other renal dietary restrictions, phosphate is commonly not adhered to in patients with ESRF. This, along with poor understanding of medical consequences, is associated with non-compliance. It was hypothesised that patients’ knowledge of phosphate and nutrition was likely to be poor resulting in elevated phosphate levels.

Acidosis affects muscle contraction by slowing the rates myosin attaches to and detaches from actin.

The loss of muscle force and power during fatigue from intense contractile activity is associated with, and likely caused by, elevated levels of phosphate ([Formula: see text]) and hydrogen ions (decreased pH). To understand how these deficits in muscle performance occur at the molecular level, we used direct measurements of mini-ensembles of myosin generating force in the laser trap assay at pH 7.4 and 6.5. The data are consistent with a mechanochemical model in which a decrease in pH reduces myosin's detachment from actin (by slowing ADP release), increases non-productive myosin binding (by detached myosin rebinding without a powerstroke), and reduces myosin's attachment to actin (by slowing the weak-to-strong binding transition). Additional support of this mechanism is found by incorporating it into a branched pathway model for the effects of [Formula: see text] on myosin's interaction with actin. Including pH-dependence in one additional parameter (acceleration of [Formula: see text]-induced detachment), the model reproduces experimental measurements at high and low pH, and variable [Formula: see text], from the single molecule to large ensemble levels. Furthermore, when scaled up, the model predicts force-velocity relationships that are consistent with muscle fiber measurements. The model suggests that reducing pH has two opposing effects, a decrease in attachment favoring a decrease in muscle force and a decrease in detachment favoring an increase in muscle force. Depending on experimental details, the addition of [Formula: see text] can strengthen one or the other effect, resulting in either synergistic or antagonistic effects. This detailed molecular description suggests a molecular basis for contractile failure during muscle fatigue.

A study on prevalence of cardiac valvular calcification and its correlation with serum phosphate levels in chronic kidney disease

Background: Higher cardiovascular morbidity found to be more in early stages of chronic kidney disease patients. Present study was done to study the prevalence of cardiac valvular calcification correlated with serum phosphate levels in chronic kidney disease.Methods: A total of 153 (78 chronic kidney disease patients, 75 controls with age and sex matched) coming to ESIC Hospital, Chennai were utilized to conduct the present study. Patients of stages 3 to 5 with matched controls of age and sex were considered for this study. Cases were classified in to different stages of chronic kidney disease based on estimated Glomerular Filtration Rate which was calculated using Cockcroft-Gault equation using age, body weight, and serum creatinine. The blood samples of patients belonging to both the groups were tested for serum creatinine and serum phosphate. The Echocardiogram was done for the patient with chronic kidney disease to assess cardiac valvular calcification.Results: 51% of the subjects in the study group were detected as having chronic kidney disease in ultrasonogram. 20% of the patients in the study group were having coronary artery disease compared to 4% in the control group. Mean serum phosphate levels between subjects with Valvular calcification in chronic kidney disease and without calcification was statistically significant among the study group was noted in the present study.Conclusions: Elevated levels of serum phosphate correlated with cardiac valvular calcification showed significant role in chronic kidney disease.

Proteomic profiling of extracellular vesicles released from vascular smooth muscle cells during initiation of phosphate-induced mineralization

ABSTRACTPurpose/Aim: Elevated serum phosphate is one of the major factors contributing to vascular calcification. Studies suggested that extracellular vesicles released from vascular smooth muscle cells significantly contribute to the initiation and progression of this pathology. Recently, we have demonstrated that elevated phosphate stimulates release of extracellular vesicles from osteogenic cells at the initiation of the mineralization process. Here, we used MOVAS cell line as an in vitro model of vascular calcification to examine whether vascular smooth muscle cells respond to high phosphate levels in a similar way and increase formation of extracellular vesicles. Materials and Methods: Vesicles residing in extracellular matrix as well as vesicles released to culture medium were evaluated by nanoparticle tracking analyses. In addition, using mass spectrometry and protein profiling, protein composition of extracellular vesicles released by MOVAS cells under standard growth conditions and upon exposure to high phosphate was compared. Results: Significant increase of the number of extracellular vesicles was detected after 72 h of exposure of cells to high phosphate. Elevated phosphate levels also affected protein composition of extracellular vesicles released from MOVAS cells. Finally, the comparative analyses of proteins in extracellular vesicles isolated from extracellular matrix and from conditioned medium identified significant differences in protein composition in these two groups of extracellular vesicles. Conclusions: Results of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.

Central role of the proximal tubular \u03b1Klotho/FGF receptor complex in FGF23-regulated phosphate and vitamin D metabolism

Fibroblast growth factor 23 (FGF23) plays critical roles in phosphate handling and vitamin D metabolism in the kidney. However, the effector cells of FGF23 in the kidney remain unclear. αKlotho, a putative enzyme possessing β-glucuronidase activity and also a permissive co-receptor for FGF23 to bind to FGF receptors (FGFRs), is expressed most abundantly in distal convoluted tubules, whereas it is expressed modestly in proximal tubules. Key molecular players of phosphate homeostasis and vitamin D-metabolizing enzymes are known to localize in proximal tubules. To clarify the direct function of FGF23 on proximal tubules, we ablated αKlotho or Fgfr1–4 genes specifically from these tubules using the Cre-loxP-mediated genetic recombination. Both conditional knockout mouse lines showed similar phenotypes that resembled those of systemic αKlotho or Fgf23 knockout mice. Compared with control mice, they showed significantly elevated levels of plasma phosphate, FGF23 and 1,25-dihydroxyvitamin D, ectopic calcification in the kidney and aging-related phenotypes like growth retardation, osteoporosis and shortened lifespan. These findings suggest that the primary function of FGF23 on mineral metabolism is mediated through αKlotho/FGFR co-receptors expressed in proximal tubular cells, and that the putative enzymatic function of αKlotho in the proximal tubule has a minor role in systemic mineral metabolism.

Short communication: Development of a rapid laboratory method to polymerize lactose to nondigestible carbohydrates

Nondigestible carbohydrates with a degree of polymerization between 3 and 10 (oligosaccharides) are commonly used as dietary fiber ingredients in the food industry, once they have been confirmed to have positive effects on human health by regulatory authorities. These carbohydrates are produced through chemical or enzymatic synthesis. Polylactose, a polymerization product of lactose and glucose, has been produced by reactive extrusion using a twin-screw extruder, with citric acid as the catalyst. Trials using powdered cheese whey permeate as the lactose source for this reaction were unsuccessful. The development of a laboratory method was necessary to investigate the effect of ingredients present in permeate powder that could be inhibiting polymerization. A Mars 6 Microwave Digestion System (CEM Corp., Matthews, NC) was used to heat and polymerize the sugars. The temperatures had to be lowered from extrusion conditions to produce a caramel-like product and not decompose the sugars. Small amounts of water had to be added to the reaction vessels to allow consistent heating of sugars between vessels. Elevated levels of water (22.86 and 28.57%, vol/wt) and calcium phosphate (0.928 and 1.856%, wt/wt) reduced the oligosaccharide yield in the laboratory method. Increasing the citric acid (catalyst) concentration increased the oligosaccharide yield for the pure sugar blend and when permeate powder was used. The utility of the laboratory method to predict oligosaccharide yields was confirmed during extrusion trials of permeate when this increased acid catalyst concentration resulted in similar oligosaccharide concentrations.

Evaluation of Antiurolithiatic Potentials of Hydro-Alcoholic Extract of Cucumis sativus L.

Abstract: Introduction: Cucumis sativus fruits are claimed for their antiurolithiatic activity in traditional system of medicine. Materials and Methods: Present study was undertaken to evaluate lithotryptic effect using hydro-alcoholic extract of Cucumis sativus (HCS). Ethylene glycol (0.75% v/v) was used to induce calculi in Wistar albino rats. Assessed various parameters like, Biochemical, Histopathological and routine urine analysis. Results: Treatment with preventive and curative doses of HCS was found to exert dose dependent antiurolithiatic action. Increased urine volume in HCS treated groups as compared to diseased group was indicative of diuretic property. Elevated calcium, phosphate and oxalate levels in diseased group animal were found to be decreased in animals treated with HCS. Increased levels of serum creatinine, BUN and uric acid were considerably brought down towards normal values in proportion to HCS doses administered. Histopathology of kidney reveals severe renal damage due toperformed crystals. Animals treated with HCS showed remarkable recovery, suggestive of prevention of nucleation and aggregation of stone forming components. Conclusion: It can be concluded that, results are supportive to claims made. The exact mechanism of antiurolithiatic effect may further needs to be studied. Key words: Cucumis sativus, Antiurolithiatic, Ethylene glycol (0.75 % v/v), Diuretic, Cystone, Kidney stone.

Elevated Phosphate Levels Trigger Autophagy-Mediated Cellular Apoptosis in H9c2 Cardiomyoblasts

Background/Aim: In chronic kidney disease (CKD), kidneys fail to maintain phosphorus homeostasis in serum. Elevated phosphorus levels in serum have been associated with cardiovascular diseases in CKD patients and in normal individuals. In this study, we evaluated the level of autophagy- and apoptosis-related markers under different concentrations of hyperphosphate in myocardial cells. Methods: Modulation inflicted on the levels of various survival-, autophagy-, and apoptosis-related markers were determined by Western blotting analysis using total protein extract. FITC-annexin V staining was performed to quantify the apoptotic cells in all groups. Results: Hyperphosphate treatments showed to induce autophagy-related proteins beclin-1, ATG7, and LC3 II through the pAMPK-ULK1 pathway in Western blotting analysis. Further, apoptosis-associated proteins such as Bax, Bid, cytochrome c, and c-caspase-9 were also upregulated with hyperphosphate treatment. 3-Methyladenine, an autophagy inhibitor, inhibited apoptosis significantly in FITC-annexin V staining, and the inhibition of Bax, cytochrome c, and c-caspase-3 was shown by Western blotting. Conclusion: The results suggest that hyperphosphate in H9c2 cardiomyoblasts would lead to cellular apoptosis via autophagy, which is mediated by the pAMPK signaling pathway. Our findings revealed the possible mechanism responsible for the heart damage under hyperphosphatemia.

Cerebrospinal Fluid Phosphate in Delirium after Hip Fracture

Aims: Phosphate is essential for neuronal activity. We aimed to investigate whether delirium is associated with altered phosphate concentrations in cerebrospinal fluid (CSF) and serum. Methods: Seventy-seven patients with hip fracture were assessed for delirium before and after acute surgery. Prefracture dementia was diagnosed by an expert panel. Phosphate was measured in CSF obtained immediately before spinal anesthesia (n = 77) and in serum (n = 47). CSF from 23 cognitively healthy elderly patients undergoing spinal anesthesia was also analyzed. Results: Hip fracture patients with prevalent delirium had higher CSF phosphate concentrations than those without delirium (median 0.63 vs. 0.55 mmol/L, p = 0.001). In analyses stratified on dementia status, this difference was only significant in patients with dementia. Serum phosphate was ∼1 mmol/L; there was no association between serum phosphate concentration and delirium status. CSF phosphate did not correlate with serum levels. Conclusion: Patients with delirium superimposed on dementia have elevated phosphate levels.

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

Maintenance of cellular phosphate homeostasis is essential for cellular life. The PhoU protein has emerged as a key regulator of this process in bacteria, and it is suggested to modulate phosphate import by PstSCAB and control activation of the phosphate limitation response by the PhoR-PhoB two-component system. However, a proper understanding of PhoU has remained elusive due to numerous complications of mutating phoU, including loss of viability and the genetic instability of the mutants. Here, we developed two sets of strains of Sinorhizobium meliloti that overcame these limitations and allowed a more detailed and comprehensive analysis of the biological and molecular activities of PhoU. The data showed that phoU cannot be deleted in the presence of phosphate unless PstSCAB is inactivated also. However, phoU deletions were readily recovered in phosphate-free media, and characterization of these mutants revealed that addition of phosphate to the environment resulted in toxic levels of PstSCAB-mediated phosphate accumulation. Phosphate uptake experiments indicated that PhoU significantly decreased the PstSCAB transport rate specifically in phosphate-replete cells but not in phosphate-starved cells and that PhoU could rapidly respond to elevated environmental phosphate concentrations and decrease the PstSCAB transport rate. Site-directed mutagenesis results suggested that the ability of PhoU to respond to phosphate levels was independent of the conformation of the PstSCAB transporter. Additionally, PhoU-PhoU and PhoU-PhoR interactions were detected using a bacterial two-hybrid screen. We propose that PhoU modulates PstSCAB and PhoR-PhoB in response to local, internal fluctuations in phosphate concentrations resulting from PstSCAB-mediated phosphate import.IMPORTANCE Correct maintenance of cellular phosphate homeostasis is critical in all kingdoms of life and in bacteria involves the PhoU protein. This work provides novel insights into the role of the Sinorhizobium meliloti PhoU protein, which plays a key role in rapid adaptation to elevated phosphate concentrations. It is shown that PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death. Additionally, a new model for phosphate sensing in bacterial species which involves the PhoR-PhoB two-component system is presented. This work provides new insights into the bacterial response to changing environmental conditions and into regulation of the phosphate limitation response that influences numerous bacterial processes, including antibiotic production and virulence.

SgrT, a Small Protein That Packs a Sweet Punch.

The SgrS small RNA (sRNA) has been shown to protect against elevated levels of glucose phosphate by regulating the stability and translation of mRNAs encoding proteins involved in sugar transport and catabolism. The sRNA also was known to encode a protective 43-amino-acid protein, SgrT, but little was known about its mechanism of action. Lloyd et al. (J Bacteriol 199:e00869-16, 2017, https://doi.org/10.1128/JB.00869-16) use cell biological and genetic approaches to demonstrate that the small protein interacts with the PtsG importer to block glucose transport by this phosphotransferase system and promote utilization of nonpreferred carbon sources to maintain growth during glucose-phosphate stress.

Abstract 242: MMP-3 Promotes SMC Transformation During Medial Artery Calcification

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality. It occurs with diabetes and chronic kidney disease. We have previously demonstrated that the matrix metallopeptidase 3 (MMP-3) is strongly induced in arterial calcification. We also showed that MMP-3 inhibition decreases calcium accumulation in vascular SMCs and that MMP-3 deficient mice develop less medial calcification than wildtype controls. In this series of continuing experiments, we evaluate the effects of MMP-3 on SMCs phenotypic transformation in vitro and in vivo. Methods and Results: Confluent rat aortic smooth muscle cells (RASMCs) cultured in calcification medium containing elevated calcium and phosphate levels for 7 days showed increased MMP-3 activity, decreased expression of the SMC markers SM-actin and SM-MHC, and increased expression of the bone markers alkaline phosphatase (ALP) and osterix (Osx). Cells were next exposed a selective MMP-3 inhibitor 2(EMD Millipore). In the presence of inhibitor, MMP-3 activity (MMP-3 activity assay kit, Abcam) was significantly decreased. Additionally, SMC osteogenic transformation was prevented as demonstrated by maintenance of SM22α and SM-MHC expression with reduction of ALP and Osx expression. In confirmatory experiments, MMP-3 reduction with siRNA inhibited the calcification of SMC that were exposed to calcification medium. We next evaluated phenotypic marker expression in MMP-3 knockout and wild-type mice injected with vitamin D 3 , a model of medial artery calcification. At 7 days after injection, expression of the SMC marker gene SM-MHC was significantly greater in MMP-3 KO mice than controls, while bone cell marker genes (Runx2, ALP, Osx) were decreased as measured by qPCR. Deletion of MMP-3 thus inhibited the osteogenic transformation of medial SMCs in vitamin D 3 -treated mice suggesting that it may control calcification via local effects within the arterial wall. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification through local effects on the phenotypic state of vascular SMCs, and further, that it may serve as a therapeutic target to reduce calcification and improve outcomes in our PAD patient population.

Protective effect of ethyl acetate fraction of Biophytum sensitivum extract against sodium oxalate-induced urolithiasis in rats

The methanolic whole plant extract of Biophytum sensitivum (感应草gǎnyìng cǎo) has been found to possess antiurolithiatic effect. The present study was undertaken to evaluate the antiurolithiatic effect of some fractions of methanolic whole plant extract of B. sensitivum (MBS) in rats as a step toward activity-directed isolation of antiurolithiatic component. The MBS was successively extracted with dichloromethane, ethyl acetate, ethanol and water to obtain fractions. Sodium oxalate (70 mg/kg, i.p.) was administered to rats for seven days to develop calcium oxalate urolithiasis. These rats were treated with two doses (20 and 40 mg/kg, p.o.) of the fractions, 1 h before sodium oxalate injections. Antiurolithiatic activity was assessed by estimating biochemical changes in urine, serum and kidney homogenate along with histological changes in kidney tissue. Sodium oxalate administration caused biochemical alterations in urine which was found to be prevented significantly by the ethyl acetate fraction. Supplementation with ethyl acetate fraction prevented the elevation of serum creatinine, uric acid and blood urea nitrogen levels. The elevated calcium, oxalate and phosphate levels in the kidney tissue homogenate of lithiatic rats were significantly reduced by the treatment with ethyl acetate fraction. The ethyl acetate fraction also caused significant decrease in lipid peroxidation activity, accumulation of calcium oxalate deposits and histological changes in the kidney tissue. The results showed that the antiurolithiatic component of the methanolic whole plant extract of the plant is contained in the ethyl acetate fraction. The effect is attributed to its diuretic, antioxidant, nephroprotective properties and effect on lowering the concentration of urinary stone-forming constituents.

Progression and outcomes of non-dialysis dependent chronic kidney disease patients: A single center longitudinal follow-up study.

Despite increase global prevalence of End stage renal disease (ESRD) and subsequent need for renal replacement therapy (RRT), relatively little is known about disease progression and prognosis of earlier stages of CKD. Current study was conducted to examine rate of CKD progression, predictors of ESRD and death. A total 621 patients with estimated glomerular filtration rate (eGFR) of 15-59ml/min/1.73m2 (CKD stage 3 & 4) were selected and followed up for 10 years or until ESRD or death, whichever occurred first. Subjects who did not meet inclusion criteria were excluded (n=1474). Annual cumulative decline in eGFR was 3.01±0.40 ml/min/1.73m2 . Overall disease progression was observed in 60% patients while 18% died. Among patients with CKD stage 3, 21% progressed to stage 4, 10% to stage 5ND (non-dialysis) and 31% to RRT while mortality was observed in 16% patients. On the other hand, 8% patients with CKD stage 4 progressed to stage 5ND, 31% to RRT and mortality was observed in 24% cases. Patients with CVD, higher systolic blood pressure, elevated phosphate levels, heavy proteinuria, microscopic hematuria and use of diuretics were more likely to develop ESRD. Advancing age, low eGFR, low systolic blood pressure, low hemoglobin and baseline diabetes were found to be significant predictors of mortality while being female reduced risk of mortality. Our data suggest that, in this CKD cohort, patients were more likely to develop ESRD than death. Prime importance should be given to mild forms of CKD to retard and even reverse CKD progression.

Bone Histo-Morphology in Chronic Kidney Disease Mineral Bone Disorder.

A trephine biopsy helps us in understanding the skeletal symptoms of the CKD when the clinical and biochemical parameters are not conclusive. A biopsy in unexplained bone pain/fractures, unexplained hypercalcemia and elevated phosphate levels helps in guiding the proper management of the patient.

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Case Presentation: A 50-year-old female with severe diffuse bone/muscle pain, polyarthralgia, and multiple spontaneous fractures was admitted with suicidal ideation, saying she would rather die than continue living with this pain. Her primary care physician had diagnosed osteoporosis and fibromyalgia and started bisphosphonate, calcium/vitamin D, and gabapentin. Endocrinology evaluation revealed hypophosphatemia; lower normal total calcium; low 1,25-dihydroxy-vitamin D, normal creatinine; and elevated alkaline phosphatase, parathyroid hormone, and urine phosphate levels. She was treated with intravenous phosphate supplementation. Lower extremity radiographs were normal. Indium-111 pentetreotide scan showed focal uptake (Fig. 1, arrow) below her right knee; the focus on single-photon emission computed tomography/computed tomography (SPECT/CT) included a lytic proximal right fibular lesion. Right knee magnetic resonance imaging showed a T1-hypointense, T2-hyperintense, gadolinium-enhancing mass (Fig. 2, arrowhead) in the posterior tibialis muscle, eroding into the medial fibular cortex (arrow). CT showed widespread lytic bone lesions, fractures of the bilateral ribs, right scapula, and pelvis; no metastasis to lungs, organs, or soft tissues; and no blastic bone lesions. What is the diagnosis? Fig. 2 View Large Image Figure Viewer

The oxygen isotopic composition of phosphate in river water and its potential sources in the Upper River Taw catchment, UK

The need to reduce both point and diffuse phosphorus pollution to aquatic ecosystems is widely recognised and in order to achieve this, identification of the different pollutant sources is essential. Recently, a stable isotope approach using oxygen isotopes within phosphate (δ18OPO4) has been used in phosphorus source tracing studies. This approach was applied in a one-off survey in September 2013 to the River Taw catchment in south-west England where elevated levels of phosphate have been reported. River water δ18OPO4 along the main channel varied little, ranging from +17.1 to +18.8‰. This was no >0.3‰ different to that of the isotopic equilibrium with water (Eδ18OPO4). The δ18OPO4 in the tributaries was more variable (+17.1 to +18.8‰), but only deviated from Eδ18OPO4 by between 0.4 and 0.9‰. Several potential phosphate sources within the catchment were sampled and most had a narrow range of δ18OPO4 values similar to that of river Eδ18OPO4. Discharge from two waste water treatment plants had different and distinct δ18OPO4 from one another ranging between +16.4 and +19.6‰ and similar values to that of a dairy factory final effluent (+16.5 to +17.8‰), mains tap water (+17.8 to +18.4‰), and that of the phosphate extracted from river channel bed sediment (+16.7 to +17.6‰). Inorganic fertilizers had a wide range of values (+13.3 to +25.9‰) while stored animal wastes were consistently lower (+12.0 to +15.0‰) than most other sources and Eδ18OPO4. The distinct signals from the waste water treatment plants were lost within the river over a short distance suggesting that rapid microbial cycling of phosphate was occurring, because microbial cycling shifts the isotopic signal towards Eδ18OPO4. This study has added to the global inventory of phosphate source δ18OPO4 values, but also demonstrated the limitations of this approach to identifying phosphate sources, especially at times when microbial cycling is high.

Elevated serum phosphate levels are associated with decreased amputation-free survival after interventions for critical limb ischemia

ObjectiveElevated serum phosphate levels have been associated with increased risks of cardiovascular events and death in several patient populations. The effects of serum phosphate on outcomes in patients with critical limb ischemia (CLI) have not been evaluated. In this study, we assessed the effect of abnormal phosphate levels on mortality and major limb events after surgical intervention for CLI. MethodsA retrospective review was undertaken to identify all patients at a single institution who underwent a first-time open or endovascular intervention for CLI between 2005 and 2014. Patients without recorded postoperative phosphate levels were excluded. Postoperative phosphate levels ≤30 days of the initial operation were recorded, and the mean was calculated. Patients were stratified according to mean phosphate levels (low: <2.5 mg/dL, normal: 2.5-4.5 mg/dL, and high: >4.5 mg/dL). Patient demographics, comorbidities, and operative details were compared in univariate analysis. Multivariable regression and Cox proportional hazard modeling were used to account for patient demographics and comorbid conditions. ResultsWe identified 941 patients, including 42 (5%) with low phosphate, 768 (82%) with normal phosphate, and 131 (14%) with high phosphate. Patients with elevated phosphate were younger and had higher rates of congestive heart failure, diabetes, and dialysis dependence. Bypass was more common among patients with normal phosphate compared with high or low phosphate levels. There was no difference in the Wound, ischemia, and Foot infection (WiFi) classification or TransAtlantic Inter-Society Consensus classification among the cohorts. There were significant differences in 1-year mortality (low: 19%, normal: 17%, high: 33%; P < .01) and 3-year mortality (low: 38%, normal: 34%, high: 56%; P < .01) between phosphate cohorts. Major amputation (low: 12%, normal: 12%, high: 15%) and restenosis (low: 21%, normal: 24%, high: 28%) tended toward worse outcomes among patients with elevated phosphate levels but did not reach statistical significance. After adjustment for baseline characteristics, mortality was higher (hazard ratio [HR], 1.7; 95% confidence interval [CI], 1.3-2.2) and amputation-free survival was lower (HR, 1.5; 95% CI, 1.2-1.9) among patients with elevated compared with normal phosphate levels. A subgroup analysis was then performed to assess dialysis and nondialysis patients separately. Patients with elevated serum phosphate levels maintained a significantly higher risk of mortality in each group (dialysis: HR, 1.8; 95% CI, 1.2-2.6; nondialysis: HR, 1.5; 95% CI, 1.04-2.10). ConclusionsElevated phosphate levels are associated with increased mortality and decreased amputation-free survival after interventions for CLI. Future studies evaluating the effects of phosphate reduction in patients with CLI are warranted.

Intracellular alkalinization by phosphate uptakeviatype III sodium–phosphate cotransporter participates in high‐phosphate‐induced mitochondrial oxidative stress and defective insulin secretion

Elevated plasma levels of inorganic phosphate (Pi) are harmful, causing, among other complications, vascular calcification and defective insulin secretion. The underlying molecular mechanisms of these complications remain poorly understood. We demonstrated the role of Pi transport across the plasmalemma on Pi toxicity in INS-1E rat clonal β cells and rat pancreatic islet cells. Type III sodium-phosphate cotransporters (NaPis) are the predominant Pi transporters expressed in insulin-secreting cells. Transcript and protein levels of sodium-dependent phosphate transporter 1 and 2 (PiT-1 and -2), isotypes of type III NaPi, were up-regulated by high-Pi incubation. In patch-clamp experiments, extracellular Pi elicited a Na+-dependent, inwardly rectifying current, which was markedly reduced under acidic extracellular conditions. Cellular uptake of Pi elicited cytosolic alkalinization; intriguingly, this pH change facilitated Pi transport into the mitochondrial matrix. Increased mitochondrial Pi uptake accelerated superoxide generation, mitochondrial permeability transition (mPT), and endoplasmic reticulum stress-mediated translational attenuation, leading to reduced insulin content and impaired glucose-stimulated insulin secretion. Silencing of PiT-1/2 prevented Pi-induced superoxide generation and mPT, and restored insulin secretion. We propose that Pi transport across the plasma membrane and consequent cytosolic alkalinization could be a therapeutic target for protection from Pi toxicity in insulin-secreting cells, as well as in other cell types.-Nguyen, T. T., Quan, X., Xu, S., Das, R., Cha, S.-K., Kong, I. D., Shong, M., Wollheim, C. B., Park, K.-S. Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high-phosphate-induced mitochondrial oxidative stress and defective insulin secretion.