Pharmacology of Mammalian Na+-Dependent Transporters of Inorganic Phosphate.

Inorganic phosphate (Pi) is an essential component of many biologically important molecules such as DNA, RNA, ATP, phospholipids, or apatite. It is required for intracellular phosphorylation signaling events and acts as pH buffer in intra- and extracellular compartments. Intestinal absorption, uptake into cells, and renal reabsorption depend on a set of different phosphate transporters from the SLC20 (PiT transporters) and SLC34 (NaPi transporters) gene families. The physiological relevance of these transporters is evident from rare monogenic disorders in humans affecting SLC20A2 (Fahr's disease, basal ganglia calcification), SLC34A1 (idiopathic infantile hypercalcemia), SLC34A2 (pulmonary alveolar microlithiasis), and SLC34A3 (hereditary hypophosphatemic rickets with hypercalciuria). SLC34 transporters are inhibited by millimolar concentrations of phosphonoformic acid or arsenate while SLC20 are relatively resistant to these compounds. More recently, a series of more specific and potent drugs have been developed to target SLC34A2 to reduce intestinal Pi absorption and to inhibit SLC34A1 and/or SLC34A3 to increase renal Pi excretion in patients with renal disease and incipient hyperphosphatemia. Also, SLC20 inhibitors have been developed with the same intention. Some of these substances are currently undergoing preclinical and clinical testing. Tenapanor, a non-absorbable Na+/H+-exchanger isoform 3 inhibitor, reduces intestinal Pi absorption likely by indirectly acting on the paracellular pathway for Pi and has been tested in several phase III trials for reducing Pi overload in patients with renal insufficiency and dialysis.

Pit 1 transporter (SLC20A1) as a key factor in the NPP1-mediated inhibition of insulin signaling in human podocytes.

Podocytes are crucially involved in blood filtration in the glomerulus. Their proper function relies on efficient insulin responsiveness. The insulin resistance of podocytes, defined as a reduction of cell sensitivity to this hormone, is the earliest pathomechanism of microalbuminuria that is observed in metabolic syndrome and diabetic nephropathy. In many tissues, this alteration is mediated by the phosphate homeostasis-controlling enzyme nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). By binding to the insulin receptor (IR), NPP1 inhibits downstream cellular signaling. Our previous research found that hyperglycemic conditions affect another protein that is involved in phosphate balance, type III sodium-dependent phosphate transporter 1 (Pit 1). In the present study, we evaluated the insulin resistance of podocytes after 24 h of incubation under hyperinsulinemic conditions. Thereafter, insulin signaling was inhibited. The formation of NPP1/IR complexes was observed at that time. A novel finding in the present study was our observation of an interaction between NPP1 and Pit 1 after the 24 h stimulation of podocytes with insulin. After downregulation of the SLC20A1 gene, which encodes Pit 1, we established insulin resistance in podocytes that were cultured under native conditions, manifested as a lack of intracellular insulin signaling and the inhibition of glucose uptake via the glucose transporter type 4. These findings suggest that Pit 1 might be a major factor that participates in the NPP1-mediated inhibition of insulin signaling.

Golgi damage caused by dysfunction of PiT-2 in primary familial brain calcification.

The Golgi apparatus is vital for protein modification and molecular trafficking. It is essential for nerve development and activity, and damage thereof is implicated in many neurological diseases. Primary familial brain calcification (PFBC) is a rare inherited neurodegenerative disease characterized by multiple brain calcifications. SLC20A2, which encodes the inorganic phosphate transporter 2 (PiT-2) protein, is the main pathogenic gene in PFBC. The PiT-2 protein is a sodium-dependent phosphate type III transporter, and dysfunction leads to a deficit in the cellular intake of inorganic phosphate (Pi) and calcium deposits. Whether the impaired Golgi apparatus is involved in the PFBC procession requires elucidation. In this study, we constructed induced pluripotent stem cells (iPSCs) derived from two PFBC patients with different SLC20A2 gene mutations (c.613G > A or del exon10) and two healthy volunteers as dependable cell models for research on pathogenic mechanism. To study the mechanism, we differentiated iPSCs into neurons and astrocytes in vitro. Our study found disruptive Golgi structure and damaged autophagy in PFBC neurons with increased activity of mTOR. We also found damaged mitochondria and increased apoptosis in the PFBC dopaminergic neurons and astrocytes. In this study, we prove that dysfunctional PiT-2 leads to an imbalance of cellular Pi, which may disrupt the Golgi apparatus with impaired autophagy, mitochondria and apoptosis in PFBC. Our study provides a new avenue for understanding nerve damage and pathogenic mechanism in brain calcifications.

Role of phosphate transporter PiT-2 in the pathogenesis of primary brain calcification.

Primary brain calcification (PBC), also known as idiopathic basal ganglia calcification (IBGC), primary familial brain calcification (PFBC) and so on, is a rare intractable disease characterized by abnormal mineral deposits, including mostly calcium in the basal ganglia, thalamus, and cerebellum. The causative gene of familial PBC is SLC20A2, which encodes the phosphate transporter PiT-2. Despite this knowledge, the molecular mechanism underlying SLC20A2-associated PBC remains unclear. In the present study, we investigated whether haploinsufficiency or a dominant-negative mechanism reduced Pi uptake in two PiT-2 variants (T115 M and R467X). We demonstrated that the presence of T115 M or R467X had no dominant-negative effect on Pi transport activity of wild-type (WT). In addition, the subcellular localization of R467X completely differed from that of WT, indicating that there is no interaction between R467X and WT. Conversely, T115 M and WT showed almost the same localization. Therefore, we examined the interaction between T115 M and WT using the bioluminescence resonance energy transfer (BRET) method. Although WT and T115 M interact with each other, T115 M does not inhibit WT's Pi transport activity. These results suggest that the role of SLC20A2 in the pathogenesis of PBC may involve decreased intracellular Pi uptake by a haploinsufficiency mechanism rather than a dominant-negative mechanism; agents promoting PiT-2 dimerization may be promising potential therapeutic agents for PBC.

Hyperglycemic environment disrupts phosphate transporter function and promotes calcification processes in podocytes and isolated glomeruli.

Soft tissue calcification is a pathological phenomenon that often occurs in end-stage chronic kidney disease (CKD), which is caused by diabetic nephropathy, among other factors. Hyperphosphatemia present during course of CKD contributes to impairments in kidney function, particularly damages in the glomerular filtration barrier (GFB). Essential elements of the GFB include glomerular epithelial cells, called podocytes. In the present study, we found that human immortalized podocytes express messenger RNA and protein of phosphate transporters, including NaPi 2c (SLC34A3), Pit 1 (SLC20A1), and Pit 2 (SLC20A2), which are sodium-dependent and mediate intracellular phosphate (Pi) transport, and XPR1, which is responsible for extracellular Pi transport. We found that cells that were grown in a medium with a high glucose (HG) concentration (30 mM) expressed less Pit 1 and Pit 2 protein than podocytes that were cultured in a standard glucose medium (11 mM). We found that exposure of the analyzed transporters in the cell membrane of the podocyte is altered by HG conditions. We also found that the activity of tissue nonspecific alkaline phosphatase increased in HG, causing a rise in Pi generation. Additionally, HG led to a reduction of the amount of ectonucleotide pyrophosphatase/phosphodiesterase 1 in the cell membrane of podocytes. The extracellular concentration of pyrophosphate also decreased under HG conditions. These data suggest that a hyperglycemic environment enhances the production of Pi in podocytes and its retention in the extracellular space, which may induce glomerular calcification.

PDGF-BB is involved in phosphate regulation via the phosphate transporters in human neuroblastoma SH-SY5Y cells.

Inorganic phosphate (Pi) is the second most abundant inorganic ion in the body. Since abnormalities in Pi metabolism are risk factors for various diseases, serum Pi levels are strictly controlled. Type-III sodium-dependent Pi transporters, PiT-1 (encoded by SLC20A1) and PiT-2 (encoded by SLC20A2), are distributed throughout the tissues of the body, including the central nervous system, and are known to be responsible for extracellular to intracellular Pi transport. Platelet-derived growth factor (PDGF) is a major growth factor of mesenchymal cells. PDGF-BB, a homodimer of PDGF-B, regulates intracellular Pi by increasing PiT-1 expression in vascular smooth muscle cells. However, the effects of PDGF-BB on Pi transporters in neurons have yet to be reported. Here, we investigated the effect of PDGF-BB on Pi transporters in human neuroblastoma SH-SY5Y cells. PDGF-BB did not induce SLC20A1 mRNA expression, but it increased the intracellular uptake of Pi via PiT-1 in SH-SY5Y cells. Among the signaling pathways associated with PDGF-BB, AKT signaling was shown to be involved in the increase in Pi transport. In addition, the PDGF-BB-induced increase in Pi mediated neuroprotective effects in SLC20A2-suppressed cells, in an in vitro model of the pathological condition found in idiopathic basal ganglia calcification. Moreover, the increase in Pi uptake was found to occur through promotion of intracellular PiT-1 translocation to the plasma membrane. Overall, these results indicate that PDGF-BB exerts neuroprotective effects via Pi transport, and they demonstrate the potential utility of PDGF-BB against abnormal Pi metabolism in neurons.

Characteristics and therapeutic potential of sodium-dependent phosphate cotransporters in relation to idiopathic basal ganglia calcification.

Type-III sodium-dependent phosphate transporters 1 and 2 (PiT 1 and PiT 2, respectively) are proteins encoded by SLC20A1 and SLC20A2, respectively. The ubiquitous distribution of SLC20A1 and SLC20A2 mRNAs in mammalian tissues supports the housekeeping maintenance and homeostasis of intracellular inorganic phosphate (Pi), which is absorbed from interstitial fluid for normal cellular functions. SLC20A2 variants have been found in patients with idiopathic basal ganglia calcification (IBGC), also known as Fahr's disease or primary familial brain calcification (PFBC). Thus, disrupted Pi homeostasis is considered one of the major factors in the pathogenic mechanism of IBGC. In this paper, among the causative genes of IBGC, we focused specifically on PiT2, and its potential for a therapeutic target of IBGC.

Phosphate Overload Stimulates Inflammatory Reaction via PiT-1 and Induces Vascular Calcification in Uremia.

OBJECTIVE: Vascular calcification (VC) is an important risk factor for cardiovascular disease in maintenance hemodialysis (MHD) patients. Hyperphosphatemia and microinflammation statement are known major contributors to the development of VC; however, the mechanisms are unknown. The aims of this study were to explore the risk factors of VC in MHD patients and to explore whether high phosphate could increase the secretion of inflammatory cytokines via PiT-1 in monocytes. METHODS: A cross-sectional study was conducted on 65 MHD patients to assess the relevance of coronary artery calcification (CAC), inflammatory factors, serum phosphate, and sodium-dependent phosphate cotransporter (NPT) mRNA expression of peripheral blood mononuclear cells (PBMCs). Multivariate logistic regression analysis was used to analyze the predictors of CAC. The calcification effects of high phosphate (HP), TNF-α, and supernatants of healthy human monocytes treated with HP were further evaluated in cultured HASMCs. RESULTS: Diabetes, longer dialysis vintage, higher serum TNF-α levels, and PiT-1 mRNA expression of PBMCs) were independent risk factors of CAC in MHD patients. The mRNA levels of PiT-1 in PBMCs were positively correlated with serum phosphate, CAC scores, and Pit-2 mRNA levels of PBMCs. The expressions of TNF-α, IL-6, and PiT-1 in human monocytes were significantly increased in a dose-dependent manner after treatment with HP, which was subsequently inhibited by NPT antagonist phosphonoformic acid. Neither TNF-α alone nor supernatants of monocytes stimulated with HP promoted the expression of osteopontin and Runt-related transcription factor 2 (Runx2) or caused mineralization in human aortic smooth muscle cells, but combined with HP intervention, the calcification effects were markedly increased in human aortic smooth muscle cells and ameliorated by phosphonoformic acid treatment. CONCLUSION: Hyperphosphatemia directly increased the synthesis and secretion of TNF-α by monocytes may via PiT-1 pathway, resulting in elevated systemic inflammatory response, which may further aggravate VC induced by phosphate overload in MHD patients.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein−umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

Interplay between primary familial brain calcification-associated SLC20A2 and XPR1 phosphate transporters requires inositol polyphosphates for control of cellular phosphate homeostasis.

Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.

The impact of type III sodium-dependent phosphate transporters (Pit 1 and Pit 2) on podocyte and kidney function.

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 H2 PO4- . 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.

Adapter Protein RapGEF1 Is Required for ERK1/2 Signaling in Response to Elevated Phosphate in Vascular Smooth Muscle Cells.

The sodium-dependent phosphate transporter, SLC20A1, is required for elevated inorganic phosphate (Pi) induced vascular smooth muscle cell (VSMC) matrix mineralization and phenotype transdifferentiation. Recently, elevated Pi was shown to induce ERK1/2 phosphorylation through SLC20A1 by Pi uptake-independent functions in VSMCs, suggesting a cell signaling response to elevated Pi. Previous studies identified Rap1 guanine nucleotide exchange factor (RapGEF1) as an SLC20A1-interacting protein and RapGEF1 promotes ERK1/2 phosphorylation through Rap1 activation. In this study, we tested the hypothesis that RapGEF1 is a critical component of the SLC20A1-mediated Pi-induced ERK1/2 phosphorylation pathway. Co-localization of SLC20A1 and RapGEF1, knockdown of RapGEF1 with siRNA, and small molecule inhibitors of Rap1, B-Raf, and Mek1/2 were investigated. SLC20A1 and RapGEF1 were co-localized in peri-membranous structures in VSMCs. Knockdown of RapGEF1 and small molecule inhibitors against Rap1, B-Raf, and Mek1/2 eliminated elevated Pi-induced ERK1/2 phosphorylation. Knockdown of RapGEF1 inhibited SM22α mRNA expression and blocked elevated Pi-induced downregulation of SM22α mRNA. Together, these data suggest that RapGEF1 is required for SLC20A1-mediated elevated Pi signaling through a Rap1/B-Raf/Mek1/2 cell signaling pathway, thereby promoting ERK1/2 phosphorylation and inhibiting SM22α gene expression in VSMCs.

Phosphate as a Signaling Molecule.

Phosphorus plays a vital role in diverse biological processes including intracellular signaling, membrane integrity, and skeletal biomineralization; therefore, the regulation of phosphorus homeostasis is essential to the well-being of the organism. Cells and whole organisms respond to changes in inorganic phosphorus (Pi) concentrations in their environment by adjusting Pi uptake and altering biochemical processes in cells (local effects) and distant organs (endocrine effects). Unicellular organisms, such as bacteria and yeast, express specific Pi-binding proteins on the plasma membrane that respond to changes in ambient Pi availability and transduce intracellular signals that regulate the expression of genes involved in cellular Pi uptake. Multicellular organisms, including humans, respond at a cellular level to adapt to changes in extracellular Pi concentrations and also have endocrine pathways which integrate signals from various organs (e.g., intestine, kidneys, parathyroid glands, bone) to regulate serum Pi concentrations and whole-body phosphorus balance. In mammals, alterations in the concentrations of extracellular Pi modulate type III sodium-phosphate cotransporter activity on the plasma membrane, and trigger changes in cellular function. In addition, elevated extracellular Pi induces activation of fibroblast growth factor receptor, Raf/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) and Akt pathways, which modulate gene expression in various mammalian cell types. Excessive Pi exposure, especially in patients with chronic kidney disease, leads to endothelial dysfunction, accelerated vascular calcification, and impaired insulin secretion.

Evidence of an intestinal phosphate transporter alternative to type IIb sodium-dependent phosphate transporter in rats with chronic kidney disease.

BACKGROUND: Phosphate is absorbed in the small intestine via passive flow and active transport.NaPi-IIb, a type II sodium-dependent phosphate transporter, is considered to mediate active phosphate transport in rodents. To study the regulation of intestinal phosphate transport in chronic kidney disease (CKD), we analyzed the expression levels of NaPi-IIb, pituitary-specific transcription factor 1 (PiT-1) and PiT-2 and the kinetics of intestinal phosphate transport using two CKD models. METHODS: CKD was induced in rats via adenine orThy1 antibody injection. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the messenger RNA (mRNA) expression of NaPi-IIb, PiT-1 and PiT-2 were analyzed. The protein expression level of NaPi-IIb was measured by mass spectrometry (e.g. liquid chromatography tandem mass spectrometry). RESULTS: In normal rats, phosphate uptake into BBMV consisted of a single saturable component and its Michaelis constant (Km) was comparable to that of NaPi-IIb. The maximum velocity (Vmax) correlated with mRNA and protein levels of NaPi-IIb. In the CKD models, intestinal phosphate uptake consisted of two saturable components. The Vmax of the higher-affinity transport, which is thought to be responsible for NaPi-IIb, significantly decreased and the decrease correlated with reduced NaPi-IIb expression. The Km of the lower-affinity transport was comparable to that of PiT-1 and -2. PiT-1 mRNA expression was much higher than that of PiT-2, suggesting that PiT-1 was mostly responsible for phosphate transport. CONCLUSIONS: This study suggests that the contribution of NaPi-IIb to intestinal phosphate absorption dramatically decreases in rats with CKD and that a low-affinity alternative to NaPi-IIb, in particular PiT-1, is upregulated in a compensatory manner in CKD.

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.

Several phosphate transport processes are present in vascular smooth muscle cells.

We have studied inorganic phosphate (Pi) handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting of 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under nonprecipitating conditions. This work not only shows that the Pi handling by cells is highly complex but also that the transport systems are shared with other ions such as bicarbonate or sulfate.NEW & NOTEWORTHY In addition to the inorganic phosphate (Pi) transporters PiT1 and PiT2, rat vascular smooth muscle cells show a sodium-dependent Pi transport system that is inhibited by DIDS and SITS. A sodium-independent Pi uptake system of high affinity is also expressed, which is inhibited by sulfate, bicarbonate, and arsenate. The exit of excess Pi is through an exchange with extracellular chloride. Whereas the metabolic effects of the inhibitors, if any, cannot be discarded, kinetic analysis during initial velocity suggests competitive inhibition.

Oxidative stress by Ca2+ overload is critical for phosphate-induced vascular calcification.

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 Ca2+ ([Ca2+]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 [Ca2+]i exclusively dependent on extracellular Na+ and Ca2+ 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 Ca2+ channel (VGCC) blocker, inhibited Pi-induced [Ca2+]i elevation, oxidative stress, ERK activation, and osteogenic differentiation. These protective effects were reiterated by extracellular Ca2+-free condition, intracellular Ca2+ 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 Ca2+ influx via VGCC, and subsequent [Ca2+]i increase elicits oxidative stress and osteogenic differentiation. PiT-1/2 mediates Pi-induced [Ca2+]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 Ca2+ entry via voltage-gated Ca2+ channels in vascular smooth muscle cells. Cytosolic Ca2+ increase and subsequent oxidative stress are indispensable for osteogenic differentiation and calcification. In addition, plasmalemmal abundance of PiT-1/2 relies on Ca2+ 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.

Isolation and Characterization of Three Sodium-Phosphate Cotransporter Genes and Their Transcriptional Regulation in the Grass Carp Ctenopharyngodon idella.

It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members (slc20a1a, slc20a1b and slc20a2) from grass carp Ctenopharyngodon idella, and explored their responses to inorganic phosphorus (Pi). Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors (SREBP1, NRF2 and VDR) were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (-783/-771 bp) negatively but VDR (-260/-253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (-1187/-1178 bp), NRF2 (-572/-561 bp) and VDR(615/-609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (-987/-977 bp), NRF2 (-1469/-1459 bp) and VDR (-1124/-1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake.

Extracellular phosphate (Pi) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular Pi levels implies the existence of a Pi-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in Pi sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent Pi transporters PiT1 and PiT2 in mediating Pi signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the Pi-dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for Pi signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing Pi transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and Pi-regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent Pi transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular Pi levels. Of note, when two putative Pi-binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular Pi These observations suggested that Pi binding rather than Pi uptake may be the key factor in mediating Pi signaling through the PiT proteins. Taken together, these results demonstrate that Pi-regulated PiT1-PiT2 heterodimerization mediates Pi sensing independently of Pi uptake.

SLC20A2 variants cause dysfunctional phosphate transport activity in endothelial cells induced from Idiopathic Basal Ganglia Calcification patients-derived iPSCs.

Idiopathic Basal Ganglia Calcification (IBGC) is a rare neuropsychiatric illness also known as Fahr's disease or Primary Familial Brain Calcification (PFBC). IBGC is caused by SLC20A2 variants, which encodes the inorganic phosphate (Pi) transporter PiT-2, a transmembrane protein associated with Pi homeostasis. We have reported novel SLC20A2 variants in the Japanese population and established an induced pluripotent stem cells (iPSCs) from an IBGC patient carrying a SLC20A2 variant. To investigate the effect of these SLC20A2 variants identified in our previous study, we used Chinese hamster ovary (CHO) cells expressing these variant proteins using the Flp-In system (Flp-In CHO cells), and showed that variant SLC20A2 proteins significantly disrupted the Pi transport activity in Flp-In CHO cells. Endothelial cells (ECs) represent important target cells for elucidating the pathology of IBGC. Using patient-derived iPSCs in this study, we differentiated these cells into ECs and found no significant difference in their differentiation capacity into ECs compared with control iPSCs. However, the Pi transport activity of IBGC patient-derived iPS-ECs was significantly decreased compared with that of control iPS-ECs without changing the gene expression of the other SLC 20 family members. We confirmed that SLC20A2 variants caused the loss of function of the Pi transport activity in both Flp-In CHO cells and disease-specific iPSCs. This is the first report to show an in vitro model of iPSCs in IBGC with patient-identified SLC20A2 variants. These useful tools will help in elucidating IBGC pathogenesis and can be used for screening drug candidates.