TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

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 (Pi) 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 Pi 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 Pi-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 Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-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.

Acanthamoeba castellanii phosphate transporter (AcPHS) is important to maintain inorganic phosphate influx and is related to trophozoite metabolic processes.

Acanthamoeba castellanii is a free-living amoeba and the etiological agent of granulomatous amoebic encephalitis and amoebic keratitis. A. castellanii can be present as trophozoites or cysts. The trophozoite is the vegetative form of the cell and has great infective capacity compared to the cysts, which are the dormant form that protect the cell from environmental changes. Phosphate transporters are a group of proteins that are able to internalize inorganic phosphate from the extracellular to intracellular medium. Plasma membrane phosphate transporters are responsible for maintaining phosphate homeostasis, and in some organisms, regulating cellular growth. The aim of this work was to biochemically characterize the plasma membrane phosphate transporter in A. castellanii and its role in cellular growth and metabolism. To measure inorganic phosphate (Pi) uptake, trophozoites were grown in liquid PYG medium at 28 °C for 2 days. The phosphate uptake was measured by the rapid filtration of intact cells incubated with 0.5 µCi of 32Pi for 1 h. The Pi transport was linear as a function of time and exhibited Michaelis-Menten kinetics with a Km = 88.78 ± 6.86 µM Pi and Vmax = 547.5 ± 16.9 Pi × h-1 × 10-6 cells. A. castellanii presented linear phosphate uptake up to 1 h with a cell density ranging from 1 × 105 to 2 × 106 amoeba × ml-1. The Pi uptake was higher in the acidic pH range than in the alkaline range. The oxygen consumption of living trophozoites increased according to Pi addition to the extracellular medium. When the cells were treated with FCCP, no effect from Pi on the oxygen flow was observed. The addition of increasing Pi concentrations not only increased oxygen consumption but also increased the intracellular ATP pool. These phenomena were abolished when the cells were treated with FCCP or exposed to hypoxia. Together, these results reinforce the hypothesis that Pi is a key nutrient for Acanthamoeba castellanii metabolism.

Phosphorus absorption and gene expression levels of related transporters in the small intestine of broilers.

To investigate the P absorption and gene expression levels of related co-transporters, type IIb sodium-dependent phosphate co-transporter (NaPi-IIb), inorganic phosphate transporter 1 (PiT-1) and inorganic phosphate transporter 2 (PiT-2) in the small intestine of broilers, 450 1-d-old Arbor Acres male broilers were randomly allocated to one of three treatments with ten replicate cages of fifteen birds per cage for each treatment in a completely randomised design. Chickens were fed a diet with no added inorganic P (containing 0·06 % non-phytate P (NPP)) or with either 0·21 or 0·44 % NPP for 21 d. Plasma P concentration in the hepatic portal vein, mRNA and protein expression levels of NaPi-IIb, PiT-1 and PiT-2 were determined at 7, 14 and 21 d of age. The results showed that the concentration of P in plasma in the hepatic portal vein increased as dietary NPP increased (P<0·0001). At 14 and 21 d of age, the increase in dietary NPP inhibited (P<0·003) NaPi-IIb mRNA expression level in the duodenum, as well as PiT-1 mRNA and protein expression levels in the ileum, but promoted NaPi-IIb protein expression level (P<0·002) and PiT-2 mRNA and protein expression levels (P<0·04) in the duodenum. These results suggest that NaPi-IIb, PiT-1 and PiT-2 might be important P transporters in the small intestine of broilers. Higher intestinal P absorption may be achieved by up-regulating the protein expression levels of NaPi-IIb and PiT-2 and down-regulating the protein expression of PiT-1.

Active removal of inorganic phosphate from cerebrospinal fluid by the choroid plexus.

The P(i) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF P(i) concentration. The large sheet-like fourth CP of the shark was mounted in Ussing chambers where unidirectional (33)P(i) fluxes revealed potent active transport from CSF to the blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 Ω·cm(2) and electrical potential difference of +0.9 ± 0.17 mV (CSF side positive). Active P(i) absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na(+)-free medium, and increasing the K(+) concentration from 5 to 100 mM. Li(+) stimulated transport twofold compared with Na(+)-free medium. Phosphonoformic acid (1 mM) had no effect on active P(i) transport. RT-PCR revealed both P(i) transporter (PiT)1 and PiT2 (SLC20 family) gene expression, but no Na(+)-P(i) cotransporter II (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot analysis and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both the shark and rat. PiT1 appeared to be localized primarily to vascular endothelial cells. Taken together, these data indicate that the CP actively removes P(i) from CSF. This process has transport properties consistent with a PiT2, Na(+)-dependent transporter that is located in the apical region of the CP epithelium.

Identification and expression analysis of pathogenicity-related genes of Rhizoctonia solani anastomosis groups infecting rice.

Sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph; Thanatephorus cucumeris) is a major constraint in rice production. Among the different anastomosis groups (AGs) of Rhizoctonia solani, AG1-IA causes sheath blight of rice, which induce necrotic lesions on leaf sheaths of the infected plants. Several reports contradict the host specificity of anastomosis groups in Rhizoctonia solani. There is lack of information on the pathogenicity genes of these Rhizoctonia solani anastomosis groups during sheath blight infection in rice. In the present study, Rhizoctonia solani isolates collected from diverse rice growing regions of India were screened for anastomosis groups and two groups namely, AG1-IA, AG2-2 were identified. Accordingly, comparative studies were made with AG1-IA (GenBank ID: 16,395) and AG2-2 (GenBank ID: 2,318,768) group sequences, which enabled the identification of specific gene clusters (119 in AG1-IA and 604 in AG2-2) belonging to these groups. Pathogen Host Interaction (PHI) blast with these specific gene clusters could further identify genes involved in host pathogen interaction (38 in AG1_IA and 150 in AG2-2), which were shortlisted for qRT-PCR validation based on qcov cutoff values representing different phenotypic categories of PHI blast. Expression analysis-based validation in sheath blight susceptible (Pusa Basmati 1) and resistant (Pusa 1908-13-12-5) rice genotypes showed that most of the genes expressed significantly higher in the susceptible variety Pusa Basmati 1. The genes like inorganic phosphate transporter (AG1_IPT), Bromodomain containing protein (AG1_BrD), Aldehyde dehydrogenase (AG1_AldD), AMP binding domain (AG1_AMP) and Heme peroxidase (AG1_HmPr) were upregulated in the susceptible genotype, PB 1 at 72hpi compared to Pusa 1908-13-12-5. Among these, inorganic phosphate transporter (AG1_IPT), Bromodomain containing protein (AG1_BrD) and Heme peroxidase (AG1_HmPr) were specific to Rhizoctonia solani AG1-IA. Through the present study, we could demonstrate the AG1-IA-specific interactions of Rhizoctonia solani causing sheath blight disease of rice, which is a step forward in understanding the specificity of Rhizoctonia solani with reference to sheath blight disease of rice. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02934-1.

Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter.

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (Pi). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔµH+ with inversely directed Pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and Pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔµH+-dependent fashion, with glutamate preferred over Pi. When facing the extracellular space, Pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons.

Characterization of six PHT1 members in Lycium barbarum and their response to arbuscular mycorrhiza and water stress.

Phosphorus (P) is vitally important for most plant processes. However, the P available to plants is present in the soil in the form of inorganic phosphate (Pi), and is often present in only limited amounts. Water stress further reduces Pi availability. Previous studies have highlighted the important roles of members of the PHOSPHATE TRANSPORTER 1 (PHT1) family and arbuscular mycorrhizal (AM) associations for Pi acquisition by plants growing in various environments. In order to understand the Pi uptake of Lycium barbarumL., a drought-tolerant ligneous species belonging to the Solanaceae family, we cloned and characterized six L. barbarum genes encoding transporter proteins belonging to the PHT1 family, and investigated their transcriptional response to AM associations and water stress. The six cloned PHT1 genes of L. barbarum had a similar evolutionary history to that of PHT1 genes found in other Solanaceae species. Three of these genes (LbPT3, LbPT4 and LbPT5) were AM-induced; the other three genes (LbPT1, LbPT2 and LbPT7) played distinct roles in Pi acquisition, translocation and remobilization in roots and leaves. AM-induced PHT1 genes maintained their function under water stress, while moderate and severe water stress upregulated non-AM-induced PHT1 genes in roots and leaves, respectively. Moreover, although LbPT1 was upregulated in AM roots under water stress, LbPT2 and LbPT7 were inhibited in AM roots, which suggested that an AM association satisfied the demand for Pi in roots under water stress and that LbPT1 may play a role in translocating Pi from roots to shoots in this situation.

Activation of peroxisome proliferator-activated receptor γ inhibits vascular calcification by upregulating Klotho.

Cardiovascular diseases are common in patients with chronic kidney disease. One of the key symptoms is the calcification of the vascular smooth muscle cells (VSMCs), which is induced by dysregulated mineral metabolism with high circulating levels of inorganic phosphate (Pi) and calcium. Klotho, which was originally identified as an aging suppressor gene, has been shown to be associated with vascular calcification. Since Klotho was recently identified as a target for nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ, the present study aimed to determine whether PPARγ regulates VSMC calcification through modulating the expression levels of Klotho. It was demonstrated that the expression of PPARγ was downregulated during Pi-induced VSMC calcification. In addition, treatment with PPARγ agonists inhibited the calcification and enhanced the expression of Klotho in VSMCs in a PPARγ-dependent manner. Of note, loss of Klotho expression by RNA interference abolished the ability of PPARγ activation to inhibit VSMC calcification. Furthermore, activation of Klotho as well as PPARγ inhibited the expression of Pi transporter 1/2 and reduced Pi influx into VSMCs. To the best of our knowledge, the present study was the first to demonstrate that PPARγ regulates VSMC calcification through activating Klotho.

Molecular cloning and characterization of an inorganic phosphate transporter protein encoding gene in Polyporus umbellatus / 中草药

Objective To clone an inorganic phosphate transporter (PiT) gene from Polyporus umbellatus and perform the bioinformatics and expression mode analysis. Methods Using RT-PCR.to clone the full-length cDNA of PiT. The characteristics of physiochemical properties, conserved domains, signal peptide and transmembrane domain of the predicted PiT protein were determined by using bioinformatic tools. Results A inorganic phosphate transporter (PiT) gene (NCBI: KU179154), designated as PuPiT, was cloned from Polyporus umbellatus sclerotia by RT-PCR. The full open reading frame cDNA sequence of PuPiT was 1 590 bp, encoding a putative PiT protein with 530 amino acids with a molecular weight of 57 552, and a theoretical pI of 6.82. The amino acids possess 12 membrane-spanning domains. Phylogenetic tree analysis indicated that PuPiT had the highest similarity with PiT from Moniliophthora rorer, and had high similarity with Moniliophthora roreri, Laccaria bicolor, and Heterobasidion irregulare. Quantitative real-time PCR showed that PuPiT expressed in both the symbiotic part and non-symbiotic part. Meanwhile, the expression of PuPiT in the symbiotic part was significantly up-regulated, about 12 times more than that in the non-symbiotic part. This result showed that PuPiT might play an important role in the Pi accumulating. Conclusion Molecular cloning and characterization of the novel PuPiT gene will be useful for further functional determination of the gene involving in phosphorus translocation regulation and symbiotic process.