Sugar uptake, metabolism, and chloride secretion in the rectal gland of the spiny dogfish Squalus acanthias.

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.

Supplemental Nicotinamide Dose-Dependently Regulates Body Phosphorus Excretion via Altering Type II Sodium-Phosphate Co-Transporter Expressions in Laying Hens.

BACKGROUND: Dietary supplemental nicotinamide is used to treat hyperphosphatemia in humans. However, the mechanisms of its impact on body phosphorus homeostasis remain unclear. OBJECTIVE: This study was to determine effects and molecular mechanisms of 3 dietary nicotinamide concentrations on body phosphorus homeostasis in laying hens. METHODS: Hy-Line Brown layers (total = 21; 40 wk old; body weight: 1,876 ± 24 g) were individually housed (n = 7) and fed a corn-soybean meal-based diet supplemented with nicotinamide at 20 (N20), 140 (N140), and 1000 (N1000) mg/kg for 21 d. Serum phosphorus and fibroblast growth factor 23 (FGF23) concentrations, phosphorus and calcium excretion, and mRNA and/or protein of type II sodium-phosphate co-transporters (NPt2a, NPt2ab) and FGF23 and FGF23 receptors were measured in the intestines, calvaria, kidney, and liver. RESULTS: Hens in the N1000 group had a 16% lower serum phosphorus concentration and 22% greater phosphorus excretion than those in the N20 or N140 group (P ≤ 0.05). Compared with hens in the N20 group, hens in the N140 and N1000 groups, which did not differ, had 15-21% lower serum FGF23 concentrations, 19-22% greater calcium excretion, 43-56% lower ileum NPT2b protein production, and 1.5- to 1.6-fold greater kidney NPT2a protein production, respectively (all differences at P ≤ 0.05). CONCLUSIONS: Supplementing high concentrations of nicotinamide in diets for laying hens led to accelerated phosphorus and calcium excretions and decreased serum phosphorus and FGF23 concentrations, which were associated with downregulated intestinal NPt2b protein production. Our findings exclude kidney NPt2a protein production as a primary mechanism for the nicotinamide-induced body phosphorus loss.

Distinct functional properties of two electrogenic isoforms of the SLC34 Na-Pi cotransporter.

Inorganic phosphate (Pi ) is crucial for proper cellular function in all organisms. In mammals, type II Na-Pi cotransporters encoded by members of the Slc34 gene family play major roles in the maintenance of Pi homeostasis. However, the molecular mechanisms regulating Na-Pi cotransporter activity within the plasma membrane are largely unknown. In the present study, we used two approaches to examine the effect of changing plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) levels on the activities of two electrogenic Na-Pi cotransporters, NaPi-IIa and NaPi-IIb. To deplete plasma membrane PI(4,5)P2 in Xenopus oocytes, we utilized Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which dephosphorylates PI(4,5)P2 to phosphatidylinositol 4-phosphate (PI(4)P). Upon activation of Ci-VSP, NaPi-IIb currents were significantly decreased, whereas NaPi-IIa currents were unaffected. We also used the rapamycin-inducible Pseudojanin (PJ) system to deplete both PI(4,5)P2 and PI(4)P from the plasma membrane of cultured Neuro 2a cells. Depletion of PI(4,5)P2 and PI(4)P using PJ significantly reduced NaPi-IIb activity, but NaPi-IIa activity was unaffected, which excluded the possibility that NaPi-IIa is equally sensitive to PI(4,5)P2 and PI(4)P. These results indicate that NaPi-IIb activity is regulated by PI(4,5)P2 , whereas NaPi-IIa is not sensitive to either PI(4,5)P2 or PI(4)P. In addition, patch clamp recording of NaPi-IIa and NaPi-IIb currents in cultured mammalian cells enabled kinetic analysis with higher temporal resolution, revealing their distinct kinetic properties.

Visualizing the regulation of SLC34 proteins at the apical membrane.

The cloning of the renal NaPi-2a (SLC34A1) and NaPi-2c (SLC34A3) phosphate transporters has made it possible to characterize the molecular and biophysical regulation of renal proximal tubular reabsorption of inorganic phosphate (Pi). Dietary factors, such as Pi and K, and several hormones and phosphatonins, including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and glucocorticoids, regulate the transporters through various transcriptional, translational, and post-translational mechanisms that involve acute trafficking via endocytosis or exocytosis, interactions with PDZ domain proteins, lipid microdomains, and diffusion and clustering in the apical brush border membrane. The visualization of these trafficking events by means of novel microscopy techniques that includes fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), fluctuation correlation spectroscopy (FCS), and modulation tracking (MT), is the primary focus of this review.

The role of SLC34A2 in intestinal phosphate absorption and phosphate homeostasis.

There has recently been significant interest in the concept of directly targeting intestinal phosphate transport to control hyperphosphatemia in patients with chronic kidney disease. However, we do not have a complete understanding of the cellular mechanisms that govern dietary phosphate absorption. Studies in the 1970s documented both active and passive pathways for intestinal phosphate absorption. However, following the cloning of the intestinal SLC34 cotransporter, NaPi-IIb, much of the research focused on the role of this protein in active transcellular phosphate absorption and the factors involved in its regulation. Generation of a conditional NaPi-IIb knockout mouse has demonstrated that this protein is critical for the maintenance of skeletal integrity during periods of phosphate restriction and that under normal physiological conditions, the passive sodium-independent pathway is likely be the more dominant pathway for intestinal phosphate absorption. The review aims to summarise the most recent developments in our understanding of the role of the intestine in phosphate homeostasis, including the acute and chronic renal adaptations that occur in response to dietary phosphate intake. Evidence regarding the overall contribution of the transcellular and paracellular pathways for phosphate absorption will be discussed, together with the clinical benefit of inhibiting these pathways for the treatment of hyperphosphatemia in chronic kidney disease.

The molecular mechanism of SLC34 proteins: insights from two decades of transport assays and structure-function studies.

The expression cloning some 25 years ago of the first member of SLC34 solute carrier family, the renal sodium-coupled inorganic phosphate cotransporter (NaPi-IIa) from rat and human tissue, heralded a new era of research into renal phosphate handling by focussing on the carrier proteins that mediate phosphate transport. The cloning of NaPi-IIa was followed by that of the intestinal NaPi-IIb and renal NaPi-IIc isoforms. These three proteins constitute the main secondary-active Na+-driven pathways for apical entry of inorganic phosphate (Pi) across renal and intestinal epithelial, as well as other epithelial-like organs. The key role these proteins play in mammalian Pi homeostasis was revealed in the intervening decades by numerous in vitro and animal studies, including the development of knockout animals for each gene and the detection of naturally occurring mutations that can lead to Pi-handling dysfunction in humans. In addition to characterising their physiological regulation, research has also focused on understanding the underlying transport mechanism and identifying structure-function relationships. Over the past two decades, this research effort has used real-time electrophysiological and fluorometric assays together with novel computational biology strategies to develop a detailed, but still incomplete, understanding of the transport mechanism of SLC34 proteins at the molecular level. This review will focus on how our present understanding of their molecular mechanism has evolved in this period by highlighting the key experimental findings.

NAD metabolism and the SLC34 family: evidence for a liver-kidney axis regulating inorganic phosphate.

The solute carrier 34 (SLC34) family of membrane transporters is a major contributor to Pi homeostasis. Many factors are involved in regulating the SLC34 family. The roles of the bone mineral metabolism factors parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) in Pi homeostasis are well studied. Intracellular Pi is thought to be involved in energy metabolism, such as ATP production. Under certain conditions of altered energy metabolism, plasma Pi concentrations are affected by the regulation of a Pi shift into cells or release from the tissues. We recently investigated the mechanism of hepatectomy-related hypophosphatemia, which is thought to involve an unknown phosphaturic factor. Hepatectomy-related hypophosphatemia is due to impaired nicotinamide adenine dinucleotide (NAD) metabolism through its effects on the SLC34 family in the liver-kidney axis. The oxidized form of NAD, NAD+, is an essential cofactor in various cellular biochemical reactions. Levels of NAD+ and its reduced form NADH vary with the availability of dietary energy and nutrients. Nicotinamide phosphoribosyltransferase (Nampt) generates a key NAD+ intermediate, nicotinamide mononucleotide, from nicotinamide and 5-phosphoribosyl 1-pyrophosphate. The liver, an important organ of NAD metabolism, is thought to release metabolic products such as nicotinamide and may control NAD metabolism in other organs. Moreover, NAD is an important regulator of the circadian rhythm. Liver-specific Nampt-deficient mice and heterozygous Nampt mice have abnormal daily plasma Pi concentration oscillations. These data indicate that NAD metabolism in the intestine, liver, and kidney is closely related to Pi metabolism through the SLC34 family. Here, we review the relationship between the SLC34 family and NAD metabolism based on our recent studies.

Structural models of the NaPi-II sodium-phosphate cotransporters.

Progress towards understanding the molecular mechanisms of phosphate homeostasis through sodium-dependent transmembrane uptake has long been stymied by the absence of structural information about the NaPi-II sodium-phosphate transporters. For many other coupled transporters, even those unrelated to NaPi-II, internal repeated elements have been revealed as a key feature that is inherent to their function. Here, we review recent structure prediction studies for NaPi-II transporters. Attempts to identify structural templates for NaPi-II transporters have leveraged the structural repeat perspective to uncover an otherwise obscured relationship with the dicarboxylate-sodium symporters (DASS). This revelation allowed the prediction of three-dimensional structural models of human NaPi-IIa and flounder NaPi-IIb, whose folds were evaluated by comparison with available biochemical data outlining the transmembrane topology and solvent accessibility of various regions of the protein. Using these structural models, binding sites for sodium and phosphate were proposed. The predicted sites were tested and refined based on detailed electrophysiological and biochemical studies and were validated by comparison with subsequently reported structures of transporters belonging to the AbgT family. Comparison with the DASS transporter VcINDY suggested a conformational mechanism involving a large, two-domain structural change, known as an elevator-like mechanism. These structural models provide a foundation for further studies into substrate binding, conformational change, kinetics, and energetics of sodium-phosphate transport. We discuss future opportunities, as well as the challenges that remain.

Physiological regulation of phosphate by vitamin D, parathyroid hormone (PTH) and phosphate (Pi).

Inorganic phosphate (Pi) is an abundant element in the body and is essential for a wide variety of key biological processes. It plays an essential role in cellular energy metabolism and cell signalling, e.g. adenosine and guanosine triphosphates (ATP, GTP), and in the composition of phospholipid membranes and bone, and is an integral part of DNA and RNA. It is an important buffer in blood and urine and contributes to normal acid-base balance. Given its widespread role in almost every molecular and cellular function, changes in serum Pi levels and balance can have important and untoward effects. Pi homoeostasis is maintained by a counterbalance between dietary Pi absorption by the gut, mobilisation from bone and renal excretion. Approximately 85% of total body Pi is present in bone and only 1% is present as free Pi in extracellular fluids. In humans, extracellular concentrations of inorganic Pi vary between 0.8 and 1.2 mM, and in plasma or serum Pi exists in both its monovalent and divalent forms (H2PO4- and HPO42-). In the intestine, approximately 30% of Pi absorption is vitamin D regulated and dependent. To help maintain Pi balance, reabsorption of filtered Pi along the renal proximal tubule (PT) is via the NaPi-IIa and NaPi-IIc Na+-coupled Pi cotransporters, with a smaller contribution from the PiT-2 transporters. Endocrine factors, including, vitamin D and parathyroid hormone (PTH), as well as newer factors such as fibroblast growth factor (FGF)-23 and its coreceptor α-klotho, are intimately involved in the control of Pi homeostasis. A tight regulation of Pi is critical, since hyperphosphataemia is associated with increased cardiovascular morbidity in chronic kidney disease (CKD) and hypophosphataemia with rickets and growth retardation. This short review considers the control of Pi balance by vitamin D, PTH and Pi itself, with an emphasis on the insights gained from human genetic disorders and genetically modified mouse models.

NaPi-IIa interacting partners and their (un)known functional roles.

The sorting and stabilization of proteins at specific subcellular domains depend upon the formation of networks build up by specific protein-protein interactions. In addition, protein networks also ensure the specificity of many regulatory processes by bringing together regulatory molecules with their targets. Whereas the success on the identification of protein-protein interactions is (up to a point) technology-driven, the assignment of functional roles to specific partners remains a major challenge. This review summarizes the work that led to the identification of partners of the Na+/phosphate cotransporter NaPi-IIa as well as the effects of the interactions in the expression and/or regulation of the cotransporter.

Type II Na+-phosphate Cotransporters and Phosphate Balance in Teleost Fish.

Teleost fish are excellent models to study the phylogeny of the slc34 gene family, Slc34-mediated phosphate (Pi) transport and how Slc34 transporters contribute Pi homeostasis. Fish need to accumulate Pi from the diet to sustain growth. Much alike in mammals, intestinal uptake in fish is partly a paracellular and partly a Slc34-mediated transcellular process. Acute regulation of Pi balance is achieved in the kidney via a combination of Slc34-mediated secretion and/or reabsorption. A great plasticity is observed in how various species perform and combine the different processes of secretion and reabsorption. A reason for this diversity is found in one or two whole genome duplication events followed by potential gene loss; consequently, teleosts exhibit distinctly different repertoires of Slc34 transporters. Moreover, due to habitats with vastly different salinity, teleosts face the challenge of either preserving water in a hyperosmotic environment (seawater) or excreting water in hypoosmotic freshwater. An additional challenge in understanding teleost Pi homeostasis are the genome duplication and retention events that diversified peptide hormones such as parathyroid hormone and stanniocalcin. Dietary Pi and non-coding RNAs also regulate the expression of piscine Slc34 transporters. The adaptive responses of teleost Slc34 transporters to e.g. Pi diets and vitamin D are informative in the context of comparative physiology, but also relevant in applied physiology and aquaculture. In fact, Pi is essential for teleost fish growth but it also exerts significant adverse consequences if over-supplied. Thus, investigating Slc34 transporters helps tuning the physiology of commercially valuable teleost fish in a confined environment.

Clinical aspects of the phosphate transporters NaPi-IIa and NaPi-IIb: mutations and disease associations.

The Na+-dependent phosphate transporter NaPi-IIa (SLC34A1) is mostly expressed in kidney, whereas NaPi-IIb (SLC34A2) has a wider tissue distribution with prominent expression in the lung and small intestine. NaPi-IIa is involved in renal reabsorption of inorganic phosphate (Pi) from urine, and patients with biallelic inactivating mutations in SLC34A1 develop hypophosphatemia, hypercalcemia, hypercalciuria and nephrocalcinosis, and nephrolithiasis in early childhood. Monoallelic mutations are frequent in the general population and may impact on the risk to develop kidney stones in adulthood. SNPs in close vicinity to the SLC34A1 locus associate with the risk to develop CKD. NaPi-IIb mediates high-affinity transport of Pi from the diet and appears to be mostly important during low Pi availability. Biallelic inactivating SLC34A2 mutations are found in patients with pulmonary alveolar microlithiasis, a lung disease characterized by the deposition of microcrystals. In contrast, no evidence for disturbed systemic Pi homeostasis has been reported in these patients to date. Nevertheless, NaPi-IIb-mediated intestinal Pi absorption may be a target for pharmaceutical interventions in patients with chronic kidney disease and Pi overload.

Substrates and inhibitors of phosphate transporters: from experimental tools to pathophysiological relevance.

The control of inorganic phosphate homeostasis is mediated through the activity of sodium-coupled Pi transporters located in the intestine, kidneys, and bone. To study these transporters in either the native tissue or after heterologous expression, it is very important to use specific inhibitors of the studied transporter, in order to know the corresponding relevance in the total Pi uptake and to differentiate from the activity of other transporters. Inhibitors are also necessary as drugs for treating Pi homeostasis disorders. Under normal physiological conditions, the renal and intestinal excretion of Pi matches dietary intestinal absorption, but when the number of non-functional nephrons increase in chronic kidney disease and end-stage renal disease, the excretion of surplus Pi is progressively impaired, thereby increasing the risk of hyperphosphatemia and Pi toxicity. When the compensatory mechanisms that increase Pi excretion fail, Pi toxicity can only be prevented by reducing the intestinal absorption of Pi through phosphate binders that reduced the free Pi concentration in the lumen, and inhibitors of intestinal Pi transporters and of the paracellular absorption route. Although many potentially interesting inhibitors have been reported to date, only a few are available for experimental purposes, and even fewer have been used in independent clinical trials. In this review, we summarize the different groups of compounds reported to date as inhibitors of Pi transport. To help understand and characterize the inhibition mechanisms, we also summarize the kinetic analysis approaches and screening methods that could be applied.

Expression and function of Slc34 sodium-phosphate co-transporters in skeleton and teeth.

Under normal physiological condition, the biomineralization process is limited to skeletal tissues and teeth and occurs throughout the individual's life. Biomineralization is an actively regulated process involving the progressive mineralization of the extracellular matrix secreted by osteoblasts in bone or odontoblasts and ameloblasts in tooth. Although the detailed molecular mechanisms underlying the formation of calcium-phosphate apatite crystals are still debated, it is suggested that calcium and phosphate may need to be transported across the membrane of the mineralizing cell, suggesting a pivotal role of phosphate transporters in bone and tooth mineralization. In this context, this short review describes the current knowledge on the role of Slc34 Na+-phosphate transporters in skeletal and tooth mineralization.

Role of αKlotho and FGF23 in regulation of type II Na-dependent phosphate co-transporters.

Alpha-Klotho is a member of the Klotho family consisting of two other single-pass transmembrane proteins: ßKlotho and γKlotho; αKlotho has been shown to circulate in the blood. Fibroblast growth factor (FGF)23 is a member of the FGF superfamily of 22 genes/proteins. αKlotho serves as a co-receptor with FGF receptors (FGFRs) to provide a receptacle for physiological FGF23 signaling including regulation of phosphate metabolism. The extracellular domain of transmembrane αKlotho is shed by secretases and released into blood circulation (soluble αKlotho). Soluble αKlotho has both FGF23-independent and FGF23-dependent roles in phosphate homeostasis by modulating intestinal phosphate absorption, urinary phosphate excretion, and phosphate distribution into bone in concerted interaction with other calciophosphotropic hormones such as PTH and 1,25-(OH)2D. The direct role of αKlotho and FGF23 in the maintenance of phosphate homeostasis is partly mediated by modulation of type II Na+-dependent phosphate co-transporters in target organs. αKlotho and FGF23 are principal phosphotropic hormones, and the manipulation of the αKlotho-FGF23 axis is a novel therapeutic strategy for genetic and acquired phosphate disorders and for conditions with FGF23 excess and αKlotho deficiency such as chronic kidney disease.

LP533401 restores bone health in 5/6 nephrectomized rats by a decrease of gut-derived serotonin and regulation of serum phosphate through the inhibition of phosphate co-transporters expression in the kidneys.

LP533401 is an orally bioavailable small molecule that inhibits tryptophan hydroxylase-1, an enzyme responsible for the synthesis of gut-derived serotonin (GDS). Recently, we showed that increased GDS in rats with chronic kidney disease (CKD) affected bone strength and metabolism. We tested the hypothesis that treatment with LP533401 could reverse CKD-induced bone loss in uremia. Sixteen weeks after 5/6 nephrectomy, rats were randomized into untreated (CKD), treated with vehicle (VEH) and LP533401 at a dose of 30 or 100 mg/kg daily for 8 weeks. Treatment with LP533401 decreased serotonin turnover and restored bone mineral status, microarchitecture, and strength in CKD rats to the values observed in the controls. In parallel with the reduction of serotonin, serum phosphate levels also decreased, particularly in the LP533401, 100 mg/kg group. The mechanism underlying this phenomenon resulted from decreased expression of the renal VDR/FGF1R/Klotho/Npt2a/Npt2c axis, leading to elevated phosphate excretion in the kidneys. The elevated urinary phosphate excretion resulted in improved bone mineral status and strength in LP533401-treated rats. Unexpectedly, the standard VEH used in this model was able to reduce renal VDR/FGF1R/Klotho/Npt2a expression, leading to a compensatory increase in Npt2c mRNA levels, secondary disturbances in phosphate-regulated hormones and partial improvement in the mineral status of the trabecular bone. The decrease of serotonin synthesis together with the simultaneous reduction of renal Npt2a and Npt2c expression in rats treated with LP533401, 100 mg/kg led to an increase in 1,25(OH)2D3 levels; this mechanism seems to be particularly beneficial in relation to the mineral status of cortical bone.

Adaptation of Opossum Kidney Cells to Luminal Phosphate: Effects of Phosphonoformic Acid and Kinase Inhibitors.

BACKGROUND/AIMS: Renal reabsorption of inorganic phosphate (Pi) is mediated by SLC34 and SLC20 Na+/Pi-cotransporters the abundance of which is under hormonal control. Extracellular Pi itself also regulates the expression of cotransporters and the concentration of Pi-regulating hormones, though the signaling pathways are largely unknown. Here, we explored the mechanisms that allow renal proximal cells to adapt to changes in the concentration of Pi. METHODS: opossum kidney (OK) cells, a model of proximal epithelia, were incubated with different concentrations of Pi in the absence/presence of phosphonoformic acid (PFA), a Pi-analogue and SLC34-inhibitor, and of inhibitors of kinases involved in hormonal control of Pi-homeostasis; cells cultured in normal media were treated with uncouplers of oxidative phosphorylation. Then, the intracellular concentration of ATP and/or the Pi-transport capacity of the cultures were analyzed. RESULTS: luminal Pi regulates the Pi-transport and the intracellular ATP levels. Changes in ATP seem secondary to alterations in Pi-transport, rather than ATP acting as a signal. Adaptation of Pi-transport to high Pi was not mimicked by PFA. Transport adaptation was blocked by PFA but not by kinase inhibitors. CONCLUSIONS: in OK cells, adaptation of Pi-transport to luminal Pi does not depend on the same signaling pathways involved in hormonal regulation.

Sodium-dependent phosphate transporters in osteoclast differentiation and function.

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

Selection of a novel FGF23-binding peptide antagonizing the inhibitory effect of FGF23 on phosphate uptake.

Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine regulator of phosphate homeostasis and has been considered as a potential therapeutic target for hypophosphatemic disorders. Herein, we isolated a novel FGF23-binding peptide by screening a phage display library with FGF23180-205, the minimal epitope of FGF23 binding to the binary fibroblast growth factor receptor (FGFR)-Klotho complex. The corresponding peptide (referred to as 23-b6) showed high homology to the immunoglobulin-like (Ig-like) domain III (D3) of FGFR1c, the predominant receptor mediating the phosphaturic activity of FGF23. The 23-b6 peptide and panning target FGF23180-205 carried opposite charges and shared similar hydrophilic profiles. Functional analysis indicated that synthetic 23-b6 peptide exhibited antagonistic effect on the inhibition of phosphate uptake by FGF23 in opossum kidney cells (OK cells). The mechanisms of 23-b6 peptide impairing the bioactivity of FGF23 involved blockade of the activation of Erk cascade and up-regulation of NaPi-2a and NaPi-2c expression in OK cells. Our results demonstrate that the 23-b6 peptide is a potent FGF23 antagonist with increased effect on phosphate uptake in kidney cells and might have therapeutic potentials in hypophosphatemic disorders characterized by an abnormally high level of FGF23.

Renal phosphate transporters.

PURPOSE OF REVIEW: Phosphate homeostasis is tightly controlled by the coordinated activity of bone, kidney, intestine, and parathyroid gland. The renal phosphate transporters have emerged as key regulators of both total body phosphate homeostasis and serum phosphate concentration. This review focuses on the latest updates in phosphate transport and transporters with an emphasis on renal phosphate transporters. RECENT FINDINGS: Structure function analysis of type II sodium phosphate cotransporters has revealed motifs with significant similarity to those seen in other sodium-coupled solute transporters, identifying key amino acid residues important for solute binding and transport. Previously unidentified regulators of these transporters have been found, although their physiologic significance and interaction with more traditional regulators have not been established. Type II and type III sodium phosphate cotransporters play critical roles in bone, choroid plexus, and vascular physiology and pathophysiology. SUMMARY: Increasing knowledge of structure function relationships for sodium phosphate cotransporters, as well as greater appreciation for the complexity of their regulation and role in renal and nonrenal tissue, brings the promise of newer, more specific treatments for disorders of phosphate homeostasis. VIDEO ABSTRACT: http://links.lww.com/CONH/A10.