Dependent Phosphate Transport Research Articles

Preferential meropenem absorption activated by 1α,25-dihydroxyvitamin D3 and shared with foscarnet, a phosphate transporter substrate, in the rat ileum

Meropenem (MEPM) is used for the treatment of serious infectious diseases solely as. InjectableTherefore, the development of an oral formulation would expand its clinical utility. To this end, an exact understanding of the absorption characteristics of MEPM is essential. In this study, MEPM absorption in the rat small intestine was investigated using an in situ loop technique and an in vitro diffusion chamber method. The disappearance ratios of MEPM (0.1 mM) were in the order of ileum > duodenum > jejunum. The extensive MEPM disappearance in the ileum was significantly reduced in the presence of foscarnet, a Na+-dependent phosphate transporter (NaPi-T) substrate, whereas glycylsarcosine, thiamine, taurocholic acid, and biapenem had no effects. The mucosal-to-serosal (M-to-S) permeation of MEPM across the rat ileal segments was very small under normal experimental conditions. However, on addition of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) to the experimental medium, the M-to-S permeation of MEPM markedly increased, showing a more than 7-fold greater apparent permeation coefficient. The present results suggest that MEPM is preferentially absorbed in the rat ileum, sharing with foscarnet, and that 1,25(OH)2D3 potentially activates the absorption of MEPM there. A likely candidate for involvement in MEPM absorption was NaPi-T or a related transporter.

A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation.

Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (Pi) from the host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires Pi from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous Pi by exploiting a gradient of Na+ ions to drive Pi uptake across the plasma membrane. The Na+-dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na+-H+-ATPase. Toxoplasma expresses one transmembrane Pi transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon Pi limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing Pi and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for Pi supply for Toxoplasma and also for protection against osmotic stresses.

New roles of the Na+/H+ exchange regulatory factor 1 scaffolding protein: a review.

Na+/H+ exchange regulatory factor 1 (NHERF1), a member of a PDZ scaffolding protein family, was first identified as an organizer of membrane-bound protein complexes composed of hormone receptors, signal transduction pathways, and electrolyte and mineral transporters and channels. NHERF1 is involved in the regulation of Na+/H+ exchanger 3, Na+-dependent phosphate transporter 2a, and Na+-K+-ATPase through its ability to scaffold these transporters to the plasma membrane, allowing regulation of these protein complexes with their associated hormone receptors. Recently, NHERF1 has received increased interest in its involvement in a variety of functions, including cell structure and trafficking, tumorigenesis and tumor behavior, inflammatory responses, and tissue injury. In this review, we highlight the evidence for the expansive role of NHERF1 in cell biology and speculate on the implications for renal physiology and pathophysiology.

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ± 0.1674 mM Pi and Vmax = 198.6 ± 10.23 Pi × h−1 × mg protein−1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.

The effect of dietary vitamin D supplementation on sodium-dependent phosphate uptake and expression of NaPi-IIb in the small intestine of weanling pigs.

Two experiments were conducted to investigate the effects of 1,25(OH)2D3 to stimulate Na+-dependent phosphate uptake in Caco-2 cells, and the effects of dietary vitamin D supplementation to vitamin D-deficient nursery pigs on Na+-dependent nutrient uptake and mRNA expression of NaPi-IIb cotransporter and calbindin D(9k) in the jejunum. In Exp. 1, 250,000 Caco-2 cells were seeded on Costar 12 mm Snapwell inserts with a 0.40 µm polycarbonate filter and a seeding density of 0.25 × 106 and studied at 15 d postconfluence. Cells were treated with 10 nM of either 1,25(OH)2D3 or vehicle for 48 h and then mounted in modified Ussing chambers for transepithelial measurements. In Exp. 2, pigs (n=32) were removed from sows at 3 d of age, placed on a vitamin D-deficient milk replacer diet and housed in a room devoid of sunlight and UV light in the range of 280 to 300nm. On d 28, serum 25(OH)D3 concentrations were measured to verify low vitamin D status. Pigs (BW 10.10 ± 0.38 kg) were then individually housed d 28 post-weaning and allotted to 1 of 2 dietary treatments. Dietary treatments consisted of corn-soybean based diets with vitamin D supplementations of 0 or 1,500 IU/kg diet for 12 d. Blood samples were taken from the brachiocephalic vein on the initial (d 0) and final day (d 10,11, or 12) of the study for analysis of serum 25(OH)D3 , P, and Ca. Pigs were euthanized and jejunal segments were harvested and used in modified Ussing chambers and for RNA isolation and subsequent quantitative RT-PCR analysis. In Exp. 1, treating Caco-2 cells with 10 nM 1,25(OH)2D3 resulted in a 52% increase (P < 0.005) in Na+-dependent phosphate uptake compared with cells treated with a vehicle. In Exp. 2, Na+-dependent phosphate and glucose transport did not differ (P > 0.10) among treatment groups. Additionally, NaPi-IIb and calbindin D(9k) mRNA expression were not different (P > 0.10) between treatment groups. No differences (P > 0.10) were detected in final serum P or 25(OH)D3 concentrations between treatments. However, serum Ca linearly increased (P < 0.05) as the concentration of supplemental vitamin D increased in the diet. Overall, while 1,25(OH)2D3 stimulated Na+-dependent phosphate uptake in Caco-2 cells, supplementing diets with 1,500 IU/kg Vitamin D3 from cholecalciferol did not increase jejunal Na+-dependent phosphate uptake or NaPi-IIb mRNA expression over that of pigs fed diets with no supplemental cholecalciferol.

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.

Analysis of opossum kidney NaPi-IIc sodium-dependent phosphate transporter to understand Pi handling in human kidney.

The role of Na+-dependent inorganic phosphate (Pi) transporters in the human kidney is not fully clarified. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by loss-of-function mutations in the IIc Na+-dependent Pi transporter (NPT2c/Npt2c/NaPi-IIc) gene. Another Na+-dependent type II transporter, (NPT2A/Npt2a/NaPi-IIa), is also important for renal Pi reabsorption in humans. In mice, Npt2c deletion does not lead to hypophosphatemia and rickets because Npt2a compensates for the impaired Pi reabsorption. To clarify the differences between mouse and human, we investigated the relation between NaPi-IIa and NaPi-IIc functions in opossum kidney (OK) cells. We cloned NaPi-IIc from OK cells and created opossum NaPi-IIc (oNaPi-IIc) antibodies. We used oNaPi-IIc small interference (si)RNA and investigated the role of NaPi-IIc in Pi transport in OK cells. We cloned opossum kidney NaPi-IIc cDNAs encoding 622 amino acid proteins (variant1) and examined their pH- and sodium-dependency. The antibodies reacted specifically with 75-kDa and 150-kDa protein bands, and the siRNA of NaPi-IIc markedly suppressed endogenous oNaPi-IIc in OK cells. Treatment with siRNA significantly suppressed the expression of NaPi-4 (NaPi-IIa) protein and mRNA. oNaPi-IIc siRNA also suppressed Na+/H+ exchanger regulatory factor 1 expression in OK cells. These findings suggest that NaPi-IIc is important for the expression of NaPi-IIa (NaPi-4) protein in OK cells. Suppression of Npt2c may downregulate Npt2a function in HHRH patients.

Renal phosphate handling and inherited disorders of phosphate reabsorption: an update.

Renal phosphate handling critically determines plasma phosphate and whole body phosphate levels. Filtered phosphate is mostly reabsorbed by Na+-dependent phosphate transporters located in the brush border membrane of the proximal tubule: NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Here we review new evidence for the role and relevance of these transporters in inherited disorders of renal phosphate handling. The importance of NaPi-IIa and NaPi-IIc for renal phosphate reabsorption and mineral homeostasis has been highlighted by the identification of mutations in these transporters in a subset of patients with infantile idiopathic hypercalcemia and patients with hereditary hypophosphatemic rickets with hypercalciuria. Both diseases are characterized by disturbed calcium homeostasis secondary to elevated 1,25-(OH)2 vitamin D3 as a consequence of hypophosphatemia. In vitro analysis of mutated NaPi-IIa or NaPi-IIc transporters suggests defective trafficking underlying disease in most cases. Monoallelic pathogenic mutations in both SLC34A1 and SLC34A3 appear to be very frequent in the general population and have been associated with kidney stones. Consistent with these findings, results from genome-wide association studies indicate that variants in SLC34A1 are associated with a higher risk to develop kidney stones and chronic kidney disease, but underlying mechanisms have not been addressed to date.

Effect of Npt2b deletion on intestinal and renal inorganic phosphate (Pi) handling.

Hyperphosphatemia is common in chronic kidney disease and is associated with morbidity and mortality. The intestinal Na+-dependent phosphate transporter Npt2b is thought to be an important molecular target for the prevention of hyperphosphatemia. The role of Npt2b in the net absorption of inorganic phosphate (Pi), however, is controversial. In the present study, we made tamoxifen-inducible Npt2b conditional knockout (CKO) mice to analyze systemic Pi metabolism, including intestinal Pi absorption. Although the Na+-dependent Pi transport in brush-border membrane vesicle uptake levels was significantly decreased in the distal intestine of Npt2b CKO mice compared with control mice, plasma Pi and fecal Pi excretion levels were not significantly different. Data obtained using the intestinal loop technique showed that Pi uptake in Npt2b CKO mice was not affected at a Pi concentration of 4mM, which is considered the typical luminal Pi concentration after meals in mice. Claudin, which may be involved in paracellular pathways, as well as claudin-2, 12, and 15 protein levels were significantly decreased in the Npt2b CKO mice. Thus, Npt2b deficiency did not affect Pi absorption within the range of Pi concentrations that normally occurs after meals. These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency.

The intestinal phosphate transporter NaPi-IIb (Slc34a2) is required to protect bone during dietary phosphate restriction

NaPi-IIb/Slc34a2 is a Na+-dependent phosphate transporter that accounts for the majority of active phosphate transport into intestinal epithelial cells. Its abundance is regulated by dietary phosphate, being high during dietary phosphate restriction. Intestinal ablation of NaPi-IIb in mice leads to increased fecal excretion of phosphate, which is compensated by enhanced renal reabsorption. Here we compared the adaptation to dietary phosphate of wild type (WT) and NaPi-IIb−/− mice. High phosphate diet (HPD) increased fecal and urinary excretion of phosphate in both groups, though NaPi-IIb−/− mice still showed lower urinary excretion than WT. In both genotypes low dietary phosphate (LDP) resulted in reduced fecal excretion and almost undetectable urinary excretion of phosphate. Consistently, the expression of renal cotransporters after prolonged LDP was similar in both groups. Plasma phosphate declined more rapidly in NaPi-IIb−/− mice upon LDP, though both genotypes had comparable levels of 1,25(OH)2vitamin D3, parathyroid hormone and fibroblast growth factor 23. Instead, NaPi-IIb−/− mice fed LDP had exacerbated hypercalciuria, higher urinary excretion of corticosterone and deoxypyridinoline, lower bone mineral density and higher number of osteoclasts. These data suggest that during dietary phosphate restriction NaPi-IIb-mediated intestinal absorption prevents excessive demineralization of bone as an alternative source of phosphate.

Effect of low levels of dietary available phosphorus on phosphorus utilization, bone mineralization, phosphorus transporter mRNA expression and performance in growing pigs

ABSTRACTA study was conducted to examine the effects of different dietary levels of available phosphorus (aP) on P excretion, bone mineralization, performance and the mRNA expression of sodium-dependent P transporters in growing pigs. Sixty-day old growing pigs (n = 54) with an average initial BW of 19.50 ± 1.11 kg were randomly allocated to a control diet (C) containing 0.23% available phosphorus (aP), T1 containing 0.17% aP and T2 containing 0.11% aP. There were 6 pens per treatment with 3 pigs per pen. Body weight and feed intake were measured weekly. At the end of each week, one pig from each pen was housed in a metabolic crate for 24 h to collect fecal and urine samples and then sacrificed to obtain third metacarpal (MC3) bones and jejunal and kidney samples. Bones were scanned by Dual Energy X-ray Absorptiometry (DEXA). Fecal and urine samples were sub-sampled and analyzed for P content. The expression of P transporter mRNA in jejunum and kidney samples was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Data were analyzed using GLM procedure of the Statistical Analysis System (SAS Institute version 9.2). Pigs fed the T2 diet had reduced (P < 0.05) average daily gain (ADG) and gain to feed (G:F) compared to those fed the C diet during week 2. Overall, ADG and G:F were also reduced (P < 0.05) in pigs fed the T2 diet compared to those fed the C and T1 diets. Bone mineral density (BMD) and bone mineral content (BMC) were reduced (P < 0.05) in pigs fed the T2 diet compared to those fed the C diet throughout the experiment. At week 1, jejunal mRNA expression of Na (+)-dependent phosphate transporter 2 (SLC34A2) was increased (P < 0.01) in pigs fed the T2 diet compared to C diet. Renal mRNA expression of Na(+)-dependent phosphate transporter 1 (SLC34A1) and SLC34A3 were increased (P < 0.05) in pigs fed the T2 diet compared to those fed the C diet at week 2 and was accompanied by lower (P < 0.05) urinary P in pigs fed the T2 diet during week 2 and week 3. In conclusion, growing pigs are highly sensitive to low dietary P as shown by reduced ADG, bone mineralization and urinary P level, but moderate reduction in dietary P up to 0.17% aP in the diet has the potential to reduce environmental pollution by reducing P concentration in swine manure and without compromising performance.

Molecular cloning and functional analysis of a H+-dependent phosphate transporter gene from the ectomycorrhizal fungus Boletus edulis in southwest China

Phosphate transporters (PTs), as entry points for phosphorus (P) in organisms, are involved in a number of P nutrition processes such as phosphate uptake, transport, and transfer. In the study, a PT gene 1632 bp long (named BePT) was cloned, identified, and functionally characterized from Boletus edulis. BePT was expected to encode a polypeptide with 543 amino acid residues. The BePT polypeptide belonged to the major facilitator superfamily and showed a high degree of sequence identity to the Pht1 family. A topology model revealed that BePT exhibited 12 transmembrane helices, divided into two halves, and connected by a large hydrophilic loop in the middle. A yeast mutant complementation analysis suggested that BePT was a functional PT which mediated orthophosphate uptake of yeast at micromolar concentrations. Green fluorescent protein–BePT fusion proteins expressed were extensively restricted to the plasma membrane in BePT transformed yeast, and its activity was dependent on electrochemical membrane potential. In vitro, quantitative PCR confirmed that the expression of BePT was significantly upregulated at lower phosphorus availability, which may enhance phosphate uptake and transport under phosphate starvation. Our results suggest that BePT plays a key role in phosphate acquisition in the ectomycorrhizal fungus B. edulis.

Assay development of inducible human renal phosphate transporter Npt2A (SLC34A1) in Flp-In-Trex-HEK293 cells

Hyperphosphatemia is associated with severe decline of renal function in chronic kidney disease and elevates cardiovascular mortality. Type II sodium dependent phosphate transporter 2A (Npt2A) plays a major role in renal phosphate reabsorption and could be explored as a target for anti-hyperphosphatemia therapy. Human Npt2A transporter activity was examined upon transfection into CHO, MDCK, HEK293, Flp-In-CHO and Flp-In-HEK293 cells. Only kidney-derived cells expressed functional Npt2A. HEK293 and Flp-In-HEK293 cell lines stably transfected with hNpt2A could be selected, but these cells were inactive in phosphate transport. This suggests that high-level, constitutive Npt2A expression has deleterious effects on the cell. By using the conditional promoter in the Flp-In-Trex vector, functional expression of Npt2A was achieved by doxycycline induction in HEK293 cells. The EGFP tagged and non-tagged, inducible stable hNpt2A-HEK293 cell lines afforded development of a robust phosphate uptake assay mediated by hNpt2A, which can be used to screen hNpt2A inhibitors and inducers of hNpt2A expression. Using this assay, the small molecule LC-1 was identified as a potent inhibitor of hNpt2A, suggesting that it is feasible to develop potent specific hNpt2A inhibitors to control phosphate overloading for hyperphosphatemia therapy.

The roles of intestinal and renal sodium dependent phosphate transporters in phosphate homeostasis

Inorganic phosphate (Pi) is an essential nutrient for several biological functions, including intracellular signal transduction, the production and function of cell membranes, and energy exchange. To achieve these functions, a transport system is required to transfer Pi across hydrophobic cell membranes. Pi (re) absorption in the small intestine and renal proximal tubules is important for Pi homeostasis. Three types of NaPi transporters (types I - III ) have been identified : solute carrier series SLC17A1 (NPT1/NaPi- I /OATv1) , SLC34 (NaPi- II a, NaPi- II b, NaPi- II c) , and SLC20 (PiT1, PiT2) , respectively. In this review, we discuss the role of NaPi transporters in Pi homeostasis.

NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

Modeling of three-dimensional structure of the H+-dependent phosphate transporter of cytoplasmic membrane from the yeast Yarrowia lipolytica

Information concerning phosphate transport systems in yeasts (specifically in Saccharomyces cerevisiae), their regulation on transcriptional and post-translational levels has been summarized. There are proofs that S. cerevisiae growing only at low pH levels cannot serve a model organism for research studies of Na+-dependent phosphate transport systems that exhibit maximum activity in alkaline media. We propose the Yarrowia lipolytica yeast as an alternative organism for such studies as it can grow at high pH values (up to 9.7) in the presence of 12% NaCl, which suggests the existence of unique systems for maintaining cell homeostasis. As far as the Y. lipolytica genome has been recently sequenced, it became possible to analyze these systems through bioinformatics approaches. A sequence of H+-dependent phosphate transporter with the same function as that of the PHO84 gene product in S. cerevisiae genome was found. Based on the sequence homology with glycerol-3-phosphate transporter from E. coli, the model of three-dimensional structure of H+-dependent phosphate transporter from Y. lipolytica (from 38 to 545 residue) was constructed, analyzed, optimized, and compared with the known models. The quality of the constructed model is better than the accepted models of the H+-dependent phosphate transporter of cytoplasmic membrane from yeast Y. lipolytica.

Abstract B189: Impact of site of conjugation on efficacy and pharmacokinetics of anti-NaPi3b antibody drug conjugate variants.

Abstract Introduction: Optimization of antibody-drug conjugates (ADCs) with therapeutic potential involves several critical parameters, including selection of the antibody, linker, cytotoxic drug, and drug-to-antibody ratio (DAR). To minimize the heterogeneity from conjugation, the antibody can be engineered at different conjugation sites to achieve a defined DAR at specific sites. Anti-NaPi3b-vc-MMAE (anti-NaPi3b-vc-E) is an ADC targeting sodium dependent phosphate transporter expressed in lung and ovarian cancers. Construction and production of the THIOMAB variant of antibody was reported previously. Briefly, a cysteine residue was engineered at Ala118 position of heavy chain (HC) or Val205 position of light chain (LC) to produce its THIOMAB HC and LC variants, respectively. The MMAE conjugates were produced with a drug to antibody ratio (DAR) of two. The purpose of this study was to evaluate the site of conjugation on ADC efficacy and antibody pharmacokinetics. Methods: The anti-tumor activity of anti-Napi3b ADC variants was evaluated in a mouse xenograft model of human ovarian cancer OVCAR3-X2.1 after single dose administration in a range of doses. The unconjugated antibodies including anti-NaPi3b (control) and anti-NaPi3b (HC and LC) and their respective ADC variants were administered intravenously to mice and rats; and the pharmacokinetic (PK) parameters measured by a total antibody assay were estimated by a two-compartmental model. Results: Based on comparisons of tumor growth inhibition, both LC and HC anti-Napi3b-vc-E showed clear dose-dependent inhibitory activity when given at 3 to 12 mg/kg versus the vehicle group. Non-NaPi3b-binding ADCs did not significantly affect tumor growth, indicating targeted anti-tumor activity. The activities of LC and HC variants were similar. Dose proportional pharmacokinetics of total antibody following administration of anti-NaPi3b-vc-E (HC and LC) were observed in mice and rats over the dose range tested. Total antibody clearance of the three unconjugated antibodies (control, thio HC, and thio LC) was comparable. Conjugation with MMAE only modestly increased the total antibody clearance in both LC and HC conjugates when compared to unconjugated antibodies. Overall, PK behavior of the HC and LC variants were similar. Conclusion: The results demonstrated that ADC variants with different sites of conjugation exhibited similar anti-tumor activities, which provided additional choice of conjugation sites for individual antibody. Cysteine mutation in either HC or LC of the unconjugated antibodies did not affect the PK when compared to the control. In addition, conjugation did not significantly change the overall clearance of the antibody, and the site of MMAE conjugations did not have a significant impact on the PK. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B189.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis

The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na+-dependent active transport of inorganic phosphate (Pi). The aim of this study was to identify amino acid residues involved in Pi binding and translocation by ANTR1 and in the Na+ dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from E. coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in E. coli and radioactive Pi transport assays and compared with bacterial cells carrying an empty plasmid. The results from Pi- and Na+-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na+ ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters.

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.