High Phosphate Treatment Research Articles

Abstract 13254: Klotho Suppresses Endothelial-to-Mesenchymal Transition in the Aorta of Mice With Chronic Kidney Disease by Inhibition of Endothelial Senescent Activity

Background: Klotho is an anti-aging protein expressed primarily in the kidney and vasculature. Chronic kidney disease (CKD) results in hyperphosphatemia. We reported that high levels of phosphate downregulate Klotho expression in vascular cells. Endothelial-to-mesenchymal transition (EndMT) in arteries promotes the development of atherosclerosis, and CKD is a significant risk factor for atherosclerosis. However, the impact of CKD on EndMT in arteries is unclear and whether Klotho insufficiency plays a role in the mechanism underlying vascular EndMT remains unknown. This study tested the hypothesis that Klotho insufficiency associated with CKD induces senescence in arterial endothelial cells to upregulate EndMT. Methods and Results: We developed a CKD model in C57/BL6 mice (5-6 months old) on AIN-76A diet and treated with adenine and calcitriol. CKD was confirmed by markedly elevated blood levels of creatinine and phosphate, as well as by renal tubular damage. En face immunofluorescence staining of aorta and image quantification revealed that the levels of biomarkers of senescence (P21) and EndMT (vimentin) were greater, while Klotho levels were lower, in the aortic arch endothelial cells of CKD mice compared to sham control mice. P21 and vimentin were co-localized in a greater number of endothelial cells in the aortic arch of CKD mice. High phosphate treatment upregulated the levels of P21 and vimentin and reduced the levels of Klotho in both primary human and mouse aortic endothelial cells, and knockdown P21 attenuated EndMT induced by high phosphate. Recombinant Klotho suppressed both senescence and EndMT in primary human aortic endothelial cells. Further, prolonged treatment of CKD mice with recombinant Klotho attenuated endothelial senescence and EndMT in the aortic arch. Conclusion: Klotho insufficiency associated with CKD promotes endothelial senescence and EndMT in aorta. High phosphate is capable of inducing endothelial senescent change that provokes EndMT. Administration of recombinant Klotho or upregulation of Klotho expression may have therapeutic potential for suppressing pro-atherosclerosis activity in large arteries by prevention of endothelial senescence and resultant EndMT.

Intermedin1‑47 inhibits high phosphate‑induced vascular smooth muscle cell calcification by regulating Wnt/β‑catenin signaling.

Vascular calcification is a major risk factor for cardiovascular disease and accounts for a large proportion of deaths from cardiovascular disease in patients with chronic kidney disease. The high incidence, rapid progression and irreversibility of vascular smooth muscle cell (VSMC) calcification in patients has attracted attention. In the present study, the effect of intermedin1‑47 (IMD1‑47), an important isoform of intermedin, was investigated on the calcification of rat cardiovascular VSMCs induced by high phosphate (HP). To stimulate osteoblast‑like differentiation and calcification in rat VSMCs, 10mM β‑sodium glycerophosphate was used. The VSMCs were then treated with three doses of IMD1‑47 and the effects of IMD1‑47 on VSMC calcification, on the expression of osteogenic markers [osteoprotegerin, Runt‑related transcription factor2 (Runx2) and osteopontin (OPN)] and on alkaline phosphatase (ALP) activity were assessed. HP treatment significantly enhanced the cellular calcium content of VSMCs, the expression of osteogenic markers, and ALP activity, while IMD1‑47 significantly reversed these effects in a dose‑dependent manner. The protein expression levels of Wnt1, Wnt3a and active β‑catenin were determined and it was found that IMD1‑47 significantly inhibited their expression. Following β‑catenin silencing, the protein expression levels Runx2 and OPN were increased compared with the IMD1‑47 treatment alone, indicating a role for the Wnt/β‑catenin pathway in the effects of IMD1‑47 on osteogenic markers. The present study suggested that IMD1‑47 inhibited HP‑induced VSMC calcification by regulating the Wnt/β‑catenin signaling pathway.

High phosphate impairs arterial endothelial function through AMPK-related pathways in mouse resistance arteries.

In patients with renal disease, high serum phosphate shows a relationship with cardiovascular risk. We speculate that high phosphate (HP) impairs arterial vasodilation via the endothelium and explore potential underlying mechanisms. Isolated vessel relaxation, endothelial function, glomerular filtration rate (GFR), oxidative stress status and protein expression were assessed in HP diet mice. Mitochondrial function and protein expression were assessed in HP-treated human umbilical vein endothelial cells (HUVECs). High phosphate (1.3%) diet for 12weeks impaired endothelium-dependent relaxation in mesenteric arteries, kidney interlobar arteries and afferent arterioles; reduced GFR and the blood pressure responses to acute administration of acetylcholine. The PPARα/LKB1/AMPK/eNOS pathway was attenuated in the endothelium of mesenteric arteries from HP diet mice. The observed vasodilatory impairment of mesenteric arteries was ameliorated by PPARα agonist WY-14643. The phosphate transporter PiT-1 knockdown prevented HP-mediated suppression of eNOS activity by impeding phosphorus influx in HUVECs. Endothelium cytoplasmic and mitochondrial reactive oxygen species (ROS) were increased in HP diet mice. Moreover HP decreased the expression of mitochondrial-related antioxidant genes. Finally, mitochondrial membrane potential and PGC-1α expression were reduced by HP treatment in HUVECs, which was partly restored by AMPKα agonist. HP impairs endothelial function by reducing NO bioavailability via decreasing eNOS activity and increasing mitochondrial ROS, in which the AMPK-related signalling pathways may play a key role.

Hdac9 inhibits medial artery calcification through down-regulation of Osterix

BackgroundsMedial artery calcification (MAC) significantly contributes to the increased cardiovascular death in patients with chronic kidney disease (CKD). Previous genome-wide association studies have shown that various genetic variants of the histone deacetylase Hdac9 are associated with cardiovascular disease, but the role of Hdac9 in MAC under CKD conditions remains unclear. MethodsHigh phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD) were used in the present study. Alizarin red staining, calcium quantitative assay, qPCR, western blotting and histology were performed. ResultsHdac9 expression was significantly down-regulated during high phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD). Furthermore, high phosphate treatment inhibited phosphorylation of Akt, and pharmacological inhibition of Akt signaling reduced Hdac9 expression in cultured VSMCs. Knockdown of Hdac9 significantly enhanced calcium deposition in VSMCs. Conversely, adenovirus mediated-overexpression of Hdac9 inhibited high phosphate induced VSMC in vitro calcification. Our subsequent mechanistic studies revealed that the anti-calcific effect of Hdac9 was mediated through down-regulation of osteoblast-specific transcription factor Osterix. ConclusionThese data suggest that Hdac9 is a novel inhibitor of MAC and may represent a potential therapeutic target for MAC in CKD patients.

OGT knockdown counteracts high phosphate-induced vascular calcification in chronic kidney disease through autophagy activation by downregulating YAP

AimsPathological vascular calcification (VC), a major risk factor for cardiovascular mortality, is a highly prevalent finding in patients with chronic kidney disease (CKD). We previously analyzed several pathways protecting against high phosphate-induced VC through induction of autophagy. Here, we explored how O-GlcNAc transferase (OGT) affected high phosphate-induced VC of CKD though mediation of autophagy. Main methodsIn the rats with CKD induced by 5/6 nephrectomy, the VC process was accelerated by a high phosphate diet. The calcification of vascular smooth muscle cells (VSMCs) was induced by high phosphate treatment. We then experimentally tested the effect of OGT on high phosphate-induced VC by conducting loss-of-function experiments. Co-immunoprecipitation and GST pull-down assays were performed to evaluate interaction between OGT and Yes-associated protein (YAP). In mechanistic studies of this pathway, we measured autophagy protein expression and autophagosome formation, as well as calcium deposition and calcium content in VSMCs and in vivo in response to altered expression of OGT and/or YAP. Key findingsOGT was up-regulated in high phosphate-induced VC models in vitro and in vivo. High phosphate-induced calcification in the rat aorta and VSMCs were suppressed by OGT silencing. OGT promoted the glycosylation of YAP to enhance its stability. Importantly, over-expressing YAP reduced autophagy and OGT expedited high phosphate-induced VC by inhibiting autophagy through upregulation of YAP. SignificanceOGT silencing downregulated YAP to induce autophagy activation, thus suppressing high phosphate-induced VC, which highlighted a promising preventive target against high phosphate-induced VC in CKD.

Klotho/FGF23 axis mediates high phosphate-induced vascular calcification in vascular smooth muscle cells via Wnt7b/β-catenin pathway.

Vascular calcification (VC) plays as a critical role on cardiovascular disease (CVD) and acts as a notable risk factor in cardiovascular system. Vascular smooth muscle cells (VSMCs) calcification can be triggered by high phosphate treatment; however, the explicit mechanism remains unclear. In the present study, we isolated VSMCs from primary rat artery, applied β-GP (β-glycerophosphate) for inducing VSMCs calcification in vitro to explore the mechanism of phosphate-induced calcification in VSMCs. Alizarin red staining was performed to assess the mineralization in VSMCs. Calcium deposition experiment was taken to evaluate the calcium content. ALP staining was determined to assess the ALP activity. The recombinant adenoviruses were constructed for the overexpression of Klotho and FGF23, respectively. qRT-PCR and western blot analysis were subjected to measure the expression of Klotho/FGF23 and correlated genes among Wnt7b/β-catenin pathway. We found that the calcium content was obviously increased and Alizarin red staining was positive in calcification group exposure with high phosphate in a time-dependent manner. The expression of Klotho and FGF23 was significantly decreased in the calcification group. However, overexpression of Klotho and FGF23 markedly reversed VSMCs calcification stimulating with high phosphate treatment. Moreover, Wnt7b/β-catenin inhibitor DKK1 could partly attenuate the effect of high phosphate on calcified VSMCs. These findings demonstrated that Klotho/FGF23 axis could modulate high phosphate-induced VSMCs calcification via Wnt7b/β-catenin signaling pathway. Our findings unravel that Klotho/FGF23- Wnt7b/β-catenin axis functions as a crucial role in the VSMCs calcification.

Phospholipase D: A new mediator during high phosphate-induced vascular calcification associated with chronic kidney disease.

Vascular calcification (VC) is the pathological accumulation of calcium phosphate crystals in one of the layers of blood vessels, leading to loss of elasticity and causing severe calcification in vessels. Medial calcification is mostly seen in patients with chronic kidney disease (CKD) and diabetes. Identification of key enzymes and their actions during calcification will contribute to understand the onset of pathological calcification. Phospholipase D (PLD1, PLD2) is active at the earlier steps of mineralization in osteoblasts and chondrocytes. In this study, we aimed to determine their effects during high-phosphate treatment in mouse vascular smooth muscle cell line MOVAS, in the ex vivo model of the rat aorta, and in the in vivo model of adenine-induced CKD. We observed an early increase in PLD1 gene and protein expression along with the increase in the PLD activity in vascular muscle cell line, during calcification induced by ascorbic acid and β-glycerophosphate. Inhibition of PLD1 by the selective inhibitor VU0155069, or the pan-PLD inhibitor, halopemide, prevented calcification. The mechanism of PLD activation is likely to be protein kinase C (PKC)-independent since bisindolylmaleimide X hydrochloride, a pan-PKC inhibitor, did not affect the PLD activity. In agreement, we found an increase in Pld1 gene expression and PLD activity in aortic explant cultures treated with high phosphate, whereas PLD inhibition by halopemide decreased calcification. Finally, an increase in both Pld1 and Pld2 expression occurred simultaneously with the appearance of VC in a rat model of CKD. Thus, PLD, especially PLD1, promotes VC in the context of CKD and could be an important target for preventing onset or progression of VC.

High phosphate-induced downregulation of PPARγ contributes to CKD-associated vascular calcification

Medial arterial calcification associated with hyperphosphatemia is a main cause of cardiovascular mortality in patients with chronic kidney disease (CKD), but the mechanisms underlying high phosphate-induced vascular calcification remain largely unknown. Here, we observed a significant decrease in the expression of peroxisome proliferator-activated receptor-gamma (PPARγ) in calcified arteries both in CKD patients and in a mouse model of CKD with hyperphosphatemia. In vitro, high phosphate treatment led to a decreased expression of PPARγ in mouse vascular smooth muscle cells (VMSCs), accompanied by apparent osteogenic differentiation and calcification. Pretreatment with PPARγ agonist rosiglitazone significantly reversed high phosphate-induced VSMCs calcification. Further investigation showed that methyl-CpG binding protein 2 (Mecp2)-mediated epigenetic repression was involved in high phosphate-induced PPARγ downregulation. Moreover, the expression of Klotho that has the ability to inhibit vascular calcification by regulating phosphate uptake decreased with the PPARγ reduction in VSMCs after high phosphate treatment, and rosiglitazone failed to inhibit high phosphate-induced calcification in VSMCs with knockdown of Klotho or in aortic rings from Klotho-deficient (kl/kl) mice. Finally, an in vivo study demonstrated that oral administration of rosiglitazone could increase Klotho expression and protect against high phosphate-induced vascular calcification in CKD mice. These findings suggest that the inhibition of PPARγ expression may contribute to the pathogenesis of high phosphate-induced vascular calcification, which may provide a new therapeutic target for vascular calcification in CKD patients.

α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

Endothelial cells (ECs) express fibroblast growth factor (FGF) receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs) were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS) expression, nitric oxide (NO) production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

Bioaccumulation of silver nanoparticles into Daphnia magna from a freshwater algal diet and the impact of phosphate availability

This study assessed the bioavailability, toxicity, and transfer of silver nanoparticles (AgNPs) in comparison with AgNO3 in two model food chain organisms: the alga Chlamydomonas reinhardtii and the grazing crustacean Daphnia magna. The effects of phosphate, a potential Ag+-binding ligand and a determinant of phytoplankton productivity, were evaluated. Nano Ag derived from coated AgNPs and AgNO3 was accumulated at similar concentrations into microalgae during high phosphate treatment, but AgNO3 accumulation was increased by low phosphate availability. After feeding on Ag-containing algae, D. magna equally accumulated AgNO3 and nano-derived Ag. There were significant reductions in feeding when D. magna were fed Ag-contaminated algae, with the AgNO3, low phosphate-exposed cells being ingested the least. Nutritional quality characteristics including fatty acid and trace nutrient content were similar in all algal samples, indicating that feeding reduction is specifically due to the presence of Ag, with AgNO3 being more toxic than nano Ag.

Effects of phosphate on growth and skeletal density in the scleractinian coral Acropora muricata: A controlled experimental approach

Phosphate contamination can negatively affect corals, modifying growth rates, skeletal density, reproduction, mortality, and zooxanthellae. We determined the effects of elevated phosphate on coral growth and density. Genetically distinct colonies of Acropora muricata were sub-divided and distributed among three 110-L aquaria, and exposed to phosphate levels of 0.09, 0.20, and 0.50mgL−1 for four months. Total skeletal length, living tissue length, weight, branch production, and polyp extension were measured. Linear extension and tissue growth increased under all conditions. Growth rates were highest at a phosphate concentration of 0.50mgL−1. Weight increased through time, graded from low to high with phosphate concentration. Density decreased through time, and was significantly lowest in the high phosphate treatment. Phosphate concentration produced no visible effects of stress on the corals, as indicated by polyp extension and lack of mortality. It is suggested that the phosphate enhanced growth was due to increased zooxanthellar populations and photosynthetic production within the coral. Skeletal density reduction may be due to phosphate binding at the calcifying surface and the creation of a porous and structurally weaker calcium carbonate/calcium phosphate skeleton. Increased phosphate concentrations, often characteristic of eutrophic conditions, caused increased coral growth but also a more brittle skeleton. The latter is likely more susceptible to breakage and damage from other destructive forces (e.g., bioerosion) and makes increased coral growth a poor indicator of reef health.

The CPPDD-Associated ANKH M48T Mutation Interrupts the Interaction of ANKH with the Sodium/Phosphate Cotransporter PiT-1

Numerous dominant human homolog of progressive ankylosis (ANKH) mutations have been identified in familial calcium pyrophosphate dihydrate crystal deposition disease (CPPDD). Due to the dominant nature of these mutations, we investigated whether ANKH interacts with other proteins; and if so, whether any CPPDD-associated ANKH mutation might disrupt such protein interactions. Stable ATDC5 ANKH wt- and ANKH M48T-transfectants were generated. Lysates from these transfectants were used to identify candidate protein interaction with ANKH by coimmunoprecipitation followed by Western blot analysis. The effect of high phosphate on the expression of genes involved in modulating Pi (inorganic phosphate)/PPi (inorganic pyrophosphate) homeostasis in these transfectants was assessed. We showed that ANKH protein associates with the sodium/phosphate cotransporter PiT-1, and that ANKH M48T mutant protein failed to interact with PiT-1. We also showed that upon high phosphate treatment, the normally coordinated upregulation of endogenous Ank and PiT1 transcript expression was disrupted in ANKH M48T transfectants. Our results suggested that there is a coordinated interrelationship between 2 key participants of Pi and PPi metabolism, ANKH and PiT-1.

The identification and quantification of arsenic-induced phytochelatins—comparison between plants with varying As sensitivities

The results presented in this paper provide evidence for the role of phytochelatins (PCs) in the detoxification of arsenic in six nonhyperaccumulating plant species, Agropyron repens, Glecoma hederacea, Leonurus marrubiastrum, Lolium perenne, Urtica dioica and Zea mays, in a pot experiment with high phosphate treatment. These plants differed in their arsenic sensitivities and were selected to investigate whether PCs with longer chains in roots of arsenic-tolerant species are synthesized. Raised concentrations of total PCs were measured in plant species with a range of sensitivities to arsenic at equivalent levels of arsenic exposure, determined as the inhibition of root biomass. In addition, the production of PCs as a function of accumulated arsenic was studied. Long-term PC synthesis (over a 5-week period) was positively, but non-linearly correlated with arsenic, suggesting that probably not all As is bound by PCs. Moreover, it could be shown that the synthesis of different chain lengths of PCs is associated with differences in As tolerance. In the more tolerant grasses A. repens and L. perenne it was chiefly the dithiol PC2 which was measured. In contrast, the dominant PC species in the less tolerant plants U. dioica, G. hederacea, L. marrubiastrum and Z. mays was PC3, while PC2 and PC3 were detected as well.

Effects of soil phosphorus on spore germination and hyphal growth of arbuscular mycorrhizal fungi.

The direct effects Of soil phosphorus on sport germination and hyphal growth of the arbuscular mycorrhizal fungi (AM), Glomus etunicatum Beck &Beck &Gerd., E3 and Scutellospora heterogama (Nicol &Gerd) Walker &Sanders, were investigated by means of two soil-agar plate experiments. In the first experiment, the percentage spore germination and hyphal length of G. etunicatum, measured at ten different P levels and three periods of incubation, were affected by the P status of the soil. The best correslation with P level in the soil occurred at 7 d of incubation for spore germination and at 14 d far hyphal growth and hyphal growth increment. Spore germination was stimulated when 12.5 μ PO g-1 was applied to the soil and decreased with further soil P increments. Hyphal growth decreased significantly with soil P amendments above 37.5 μg P g-1 In the second experiment including three different P levels and the three AM endophytes, soil P affected spore germination and hyphal growth, and both decreased at the high phosphate treatment for G. etunicatum and S. heterogama. The K3 spare germination was similar to the other two species, but the hyphal growth was poorer. S. heterogama was more sensitive to P fertilization than the Glomus spp. and for the three species the hyphal production was not proportional to spore size.

EVIDENCE THAT ARSENIC TOLERANCE IN HOLCUS LANATUS L. IS CAUSED BY AN ALTERED PHOSPHATE UPTAKE SYSTEM

The uptake of arsenate by arsenic tolerant and non-tolerant plants of Holcus lanatus L., and the interaction of this uptake with phosphate level, was studied. The tolerance of the different plants, as measured by root growth, was also investigated. Arsenate inhibited the root growth of non-tolerant plants much more than that of tolerant ones, but at low levels of arsenate, the high phosphate treatment reduced the toxicity of arsenate in non-tolerants but not in tolerants. Tolerant plants took up less arsenate than non-tolerants. The effect of phosphate on arsenate uptake was very small in the tolerant plants, but in non-tolerants phosphate inhibited arsenate uptake when the phosphate was at a higher concentration than arsenate. These results are compatible with the hypothesis that arsenate is taken up by the phosphate uptake system in non-tolerant plants, but in tolerant plants this system has become altered so that it no longer recognizes arsenate.

Effects of different levels of zinc and phosphorus on the growth of subterranean clover (Trifolium subterraneum L.)

Four water culture experiments are described in which the effects of the addition of all combinations of graduated amounts of phosphorus and zinc on the growth of subterranean clover were studied.In the first experiment, the variety Dwalganup was used, and in the other experiments the varieties Clare and Edenhope. Varietal differences in the symptoms of zinc deficiency are described. Phosphorus toxicity symptoms occurred in the variety Clare but not in Edenhope. The effects of increases in zinc or phosphorus level, or both, on growth of top + root (T + R) and on percentage top are described. A highly significant variety x phosphorus interaction effect on T + R was recorded at adequate zinc levels, which indicated that the phosphorus requirement of Edenhope was considerably greater than that of Clare. By contrast, tolerance to high levels of zinc in the substrate was greater in Clare than in Edenhope. In the presence of added phosphate, the initial T + R response to small, but still insufficient, additions of zinc to the substrate occurred in root growth more than in top growth, whereas when a level of zinc adequate for normal growth was supplied, top growth of the plants benefited more than root growth. Highly significant zinc x phosphorus interaction effects on T + R occurred. No relationship existed between zinc concentration and the occurrence of zinc deficiency symptoms. The critical zinc level was not a point of narrow range, but included a wide range of values, depending on the age of the plants and the phosphate level. The depression in T + R induced by high phosphate when the zinc level was insufficient was not due to a decrease in zinc concentration resulting from the high phosphate treatment, but such plants gave a high value for the phosphorus/zinc ratio. The results suggest that zinc is essential to phosphate utilization by the plant. Evidence of redistribution of phosphorus from tops to roots, and of decreases in the total amount of phosphorus in phosphorus-deficient plants, was obtained in one experiment between days 27 and 60.