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The type IIc [Na.sup.+]-dependent phosphate cotransporter (NaPi-IIc) is specifically targeted to, and expressed on, the apical membrane of renal proximal tubular cells and mediates phosphate transport. In the present study, we investigated the signals that determine apical expression of NaPi-IIc with a focus on the role of the N- and the C-terminal tails of mouse NaPi-IIc in renal epithelial cells [opossum kidney (OK) and Madin-Darby canine kidney cells]. Wild-type NaPi-IIc, the cotransporter NaPi-IIa, as well as several IIa-IIc chimeras and deletion mutants, were fused to enhanced green fluorescent protein (EGFP), and their cellular localization was analyzed in polarized renal epithelial cells by confocal microscopy and by cell-surface biotinylation. Fluorescent EGFP-fused NaPi-IIc transporter proteins are correctly expressed in the apical membrane of OK cells. The apical expression of N-terminal deletion mutants (deletion of N-terminal 25, 50, or 69 amino acids) was not affected by truncation. In contrast, C-terminal deletion mutants (deletion of C-terminal 45, 50, or 62 amino acids) did not have correct apical expression. A more detailed mutational analysis indicated that a domain (amino acids WLHSL) in the cytoplasmic C terminus is required for apical expression of NaPi-IIc in renal epithelial cells. We conclude that targeting of NaPi-IIc to the apical cell surface is regulated by a unique amino acid motif in the cytoplasmic C-terminal domain. Npt2c; SLC34A3; localization; OK cell; hereditary hypophosphatemic rickets with hypercalciuria doi: 10.1152/ajprenal.00189.2009.

In the present study, we evaluated the roles of type II and type III sodium-dependent [P.sub.i] cotransporters in fibroblast growth factor 23 (FGF23) activity by administering a vector encoding FGF23 with the R179Q mutation (FGF23M) to wild-type (WT) mice, Npt2a knockout (KO) mice, Npt2c KO mice, and [Npt2a.sup.-/-][Npt2c.sup.-/-] mice (DKO mice). In Npt2a KO mice, FGF23M induced severe hypophosphatemia and markedly decreased the levels of Npt2c, type III Na-dependent [P.sub.i] transporter (PiT2) protein, and renal Na/[P.sub.i] transport activity. In contrast, in Npt2c KO mice, FGF23M decreased plasma phosphate levels comparable to those in FGF23M-injected WT mice. In DKO mice with severe hypophosphatemia, FGF23M administration did not induce an additional increase in urinary phosphate excretion. FGF23 administration significantly decreased intestinal Npt2b protein levels in WT mice but had no effect in Npt2a, Npt2c, and DKO mice, despite marked suppression of plasma 1,25[(OH).sub.2][D.sub.3] levels in all the mutant mice. The main findings were as follow: 1) FGF23-dependent phosphaturic activity in Npt2a KO mice is dependent on renal Npt2c and PIT-2 protein; 2) in DKO mice, renal [P.sub.i] reabsorption is not further decreased by FGF23M, but renal vitamin D synthesis is suppressed; and 3) downregulation of intestinal Npt2b may be mediated by a factor(s) other than 1,25[(OH).sub.2][D.sub.3]. These findings suggest that Npt2a, Npt2c, and PIT-2 are necessary for the phosphaturic activity of FGF23. Thus complementary regulation of Npt2 family proteins may be involved in systemic [P.sub.i] homeostasis. phosphate; transporter doi: 10.1152/ajprenal.00375.2009.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare autosomal recessively inherited disorder, characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH is caused by a defect in the sodiumdependent phosphate transporter (NaPi-IIc/Npt2c/NPT2c), which was thought to have only a minor role in renal phosphate (Pi) reabsorption in adult mice. In fact, mice that are null for Npt2c ([Npt2c.sup.-/-]) show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. To obtain insights and the relative importance of Npt2a and Npt2c, we now studied [Npt2a.sup.-/-] [Npt2a.sup.+/+], [Npt2a.sup.+/-] [Npt2c.sup.-/-], and [Npt2a.sup.-/-] [Npt2c.sup.-/-] double-knockout (DKO). DKO mice exhibited severe hypophosphatemia, hypercalciuria, and rickets. These findings are different from those in Npt2a KO mice that show only a mild phosphate and bone phenotype that improve over time and from the findings in Npt2c KO mice that show no apparent abnormality in the regulation of phosphate homeostasis. Because of the nonreddundant roles of Npt2a and Npt2c, DKO animals showed a more pronounced reduction in [P.sub.i] transport activity in the brush-border membrane of renal tubular cells than that in the mice with the single-gene ablations. A high-[P.sub.i] diet after weaning rescued plasma phosphate levels and the bone phenotype in DKO mice. Our findings thus showed in mice that Npt2a and Npt2c have independent roles in the regulation of plasma [P.sub.i] and bone mineralization. hereditary hypophosphatemic rickets; bone mineralization

Background. Gender differences in spousal caregivers and their relationship to care experiences, social demographics, and caregivers' depression were examined. Methods. A stratified random sample of 2,020 users of public long-term care insurance in a city of Osaka prefecture, Japan, participated in this study. Responses from 308 spouses (56.2% wives, 43.8% husbands) were analyzed. Variables relating to care experiences, social demographics, and caregivers' depression were compared by conducting simultaneous analyses of multiple populations. Results. Wives caring for husbands had higher depression scores than husbands caring for wives. Wives tended to adopt 'emotional support seeking' and 'willing commitment' as coping strategies for their caregiving experience. Husband caregivers used more home-care services; however, increased service use had no effect on husbands' depression. The availability of secondary caregivers reduced depression for caregivers, regardless of gender. Conclusions. The effects on depression differed related to the caregiver's gender. Husbands relied more on family or home-care services when caring for their wives, whereas wives provided higher levels of care, positively accepted their role, and did not seek to share caregiving, but sought emotional support. Key Words: Gender differences--Long-term care insurance--Spousal caregivers

Improved spatial resolution without sacrificing sensitivity is one of the most challenging developmental goals for small animal PET scanners. The 3-layer configuration that we propose here utilizes relative offsets of haft a crystal pitch in x-and y-directions, and pulse shape discrimination to obtain depth of interaction (DOI). Three layers of crystals with a dimension 1.5 x 1.5 x 7.0 [mm.sup.3] were composed of a [L.sub.0.2] GSO ([Lu.sub.0.4] [Gd.sub.1.6] Si[O.sub.4]: Ce) crystal layer and a [L.sub.0.9] GSO ([Lu.sub.1.8] [Gd.sub.0.2] Si[O.sub.4]: Ce) crystal layer aligned with each other, and a [L.sub.0.9] GSO crystal layer offset at haft a crystal pitch in x- and y-directions. The [L.sub.0.9] GSO crystal layer was attached to a Hamamatsu 1-19500 fiat-panel PMT. The devised small animal PET scanner has a diameter of 84 mm with one detector ring, and can be upgraded to two detector rings. GEANT4 Monte-Carlo simulation was used to estimate sensitivities of ~12% and ~20%, respectively, at the center of one and two PMT ring system with an energy window of 350 ~ 750 keV. We present flood images with peak-to-valley ratios of about 5-6 obtained using (22) Na and layer identification capability of ~99% with pulse shape analysis, and verified the basic concepts of multi-layer small animal PET. Index Terms--Animal PET, depth of interaction (DOI), lutetium gadolinium oxyorthosilicate (LGSO), three-layer crystal.

Photon detecting Geiger-mode solid-state devices are being actively researched and developed because, unlike photomultiplier tubes (PMT), they can be used in high-magnetic-field and radio-frequency environments, such as in magnetic resonance imaging (MRI) scanners. In addition, some Geiger-mode solid-state devices have higher photon detection efficiencies than PMTs and higher gains than avalanche photo-diodes (APD). We tested Geiger-mode solid-state photomultipliers (SSPM) inside a 3 T MRI to study the possibility of using them in combined PET/MRI scanners. Approximately 16% energy resolutions and ~1.3 ns coincidence time resolutions with [sup.22]Na and lutetium yttrium oxyorthosilicate (LYSO) were obtained for full-width at half maximum (FWHM) for T1, T2, and gradient echo [T2.sup.*] MRI pulse sequences with little MR image degradation. The study shows that SSPMs have excellent potential for use in combined PET/MRI scanners. Index Terms--MRI, multimodal imaging, PET, PET/MRI.

Recent studies have demonstrated that klotho protein plays a role in calcium/phosphate homeostasis. The goal of the present study was to investigate the regulation of Na-[P.sub.i] cotransporters in klotho mutant (kl/kl) mice. The kl/kl mice displayed hyperphosphatemia, high plasma 1,25[(OH).sub.2][D.sub.3] levels, increased activity of the renal and intestinal sodium-dependent [P.sub.i] cotransporters, and increased levels of the type IIa, type IIb, and type IIc transporter proteins compared with wild-type mice. Interestingly, transcript levels of the type IIa/type IIc transporter mRNA abundance, but not transcripts levels of type lib transporter mRNA, were markedly decreased in kl/kl mice compared with wild-type mice. Furthermore, plasma fibroblast growth factor 23 (FGF23) levels were 150-fold higher in kl/kl mice than in wild-type mice. Feeding of a low-[P.sub.i] diet induced the expression of klotho protein and decreased plasma FGF23 levels in kl/kl mice, whereas colchicine treatment experiments revealed evidence of abnormal membrane trafficking of the type IIa transporter in kl/kl mice. Finally, feeding of a low-[P.sub.i] diet resulted in increased type IIa Na-[P.sub.i] cotransporter protein in the apical membrane in the wild-type mice, but not in kl/kl mice. These results indicate that hyperphosphatemia in klotho mice is due to dysregulation of expression and trafficking of the renal type IIa/IIc transporters rather than to intestinal [P.sub.i] uptake. phosphate transporter; klotho; fibroblast growth factor 23

Hereditary hypophosphatemic rickets with hypercalciuria results from mutations of the renal type IIc Na-[P.sub.i] cotransporter gene, suggesting that the type IIc transporter plays a prominent role in renal phosphate handling. The goal of the present study was to investigate the regulation of the type IIc Na-[P.sub.i] cotransporter by parathyroid hormone (PTH). Type IIc Na-[P.sub.i] cotransporter levels were markedly increased in thyroparathyroidectomized (TPTX) rats. Four hours after administration of PTH, type IIc transporter protein levels were markedly decreased in the apical membrane fraction but recovered to baseline levels at 24 h. Immunohistochemical analyses demonstrated the presence of the type IIc transporter in the apical membrane and subapical compartments in the proximal tubular cells in TPTX animals. After administration of PTH, the intensity of immunoreactive signals in apical and subapical type IIc transporter decreased in the renal proximal tubular cells in TPTX rats. Colchicine completely blocked the internalization of the type IIc transporter. In addition, leupeptin prevented the PTH-mediated degradation of the type IIa transporter in lysosomes but had no effect on PTH-mediated degradation of the lysosomal type IIc transporter. In PTH-treated TPTX rats, the internalization of the type IIc transporter occurred after administration of PTH(1-34) (PKA and PKC activator) or PTH(3-34) (PKC activator). Thus the present study demonstrated that PTH is a major hormonal regulator of the type IIc Na-[P.sub.i] cotransporter in renal proximal tubules. phosphate transporter; proximal tubule; regulation

During bone resorption, a large amount of inorganic phosphate ([P.sub.i]) is generated within the osteoclast hemivacuole. The mechanisms involved in the disposal of this [P.sub.i] are not clear. In the present study, we investigated the efflux of [P.sub.i] from osteoclast-like cells. [P.sub.i] efflux was activated by acidic conditions in osteoclast-like cells derived by the treatment of RAW264.7 cells with receptor activator of nuclear factor-[kappa]B ligand. Acid-induced [P.sub.i] influx was not observed in renal proximal tubule-like opossum kidney cells, osteoblast-like MC3T3-E1 cells, or untreated RAW264.7 cells. Furthermore, [P.sub.i] efflux was stimulated by extracellular [P.sub.i] and several [P.sub.i] analogs [phosphonoformic acid (PFA), phosphonoacetic acid, arsenate, and pyrophosphate]. [P.sub.i] efflux was time dependent, with 50% released into the medium after 10 min. The efflux of [P.sub.i] was increased by various inhibitors that block [P.sub.i] uptake, and extracellular [P.sub.i] did not affect the transport of [sup.14]C]PFA into the osteoclast-like cells. Preloading of cells with [P.sub.i] did not stimulate [P.sub.i] efflux by PFA, indicating that the effect of [P.sub.i] was not due to transstimulation of [P.sub.i] transport. [P.sub.i] uptake was also enhanced under acidic conditions. Agents that prevent increases in cytosolic free [Ca.sup.2+] concentration, including acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 2-aminoethoxydiphenyl borate, and bongkrekic acid, significantly inhibited [P.sub.i] uptake in the osteoclast-like cells, suggesting that [P.sub.i] uptake is regulated by [Ca.sup.2+] signaling in the endoplasmic reticulum and mitochondria of osteoclast-like cells. These results suggest that osteoclast-like cells have a unique [P.sub.i] uptake/efflux system and can prevent [P.sub.i] accumulation within osteoclast hemivacuoles. phosphate transporter; RAW264.7; proton dependent; acidification

Fibroblast growth factor-23 (FGF23) has been recently identified as playing an important pathophysiological role in phosphate homeostasis and vitamin D metabolism. To elucidate the precise physiological regulation of FGF-23, we characterized the mouse FGF-23 5'-flanking region and analyzed its promoter activity. The 5'-flanking region of the mouse FGF-23 gene contained a TFIID site (TATA box) and several putative transcription factor binding sites, including MZF1, GATA-1 and c-Ets-1 motifs, but it did not contain the typical sequences of the vitamin D response element. Plasmids encoding 554-bp (pGL/-0.6), 364-bp (pGL/-0.4) and 200-bp (pGL/-0.13) promoter regions containing the TFIID element and + 1-bp fragments drove the downstream expression of a luciferase reporter gene in transfection assays. We also found that FGF-23 mRNA was expressed in K-562 erythroleukemia cell lines but not in MC3T3-E1, Raji, or Hep G2 human carcinoma cells. Treatment with 1,25-dihydroxyvitamin [D.sub.3] in the presence of high phosphate markedly stimulated pGL/-0.6 activity, but calcium had no effect. In addition, the plasma FGF-23 levels were affected by the dietary and plasma inorganic phosphate concentrations. Finally, the levels of plasma FGF-23 in vitamin D receptor-null mice were significantly lower than in wild-type mice. The presents study demonstrated that vitamin D and the plasma phosphate level are important regulators of the transcription of the mouse FGF-23 gene. gene regulation; vitamin D receptor; phosphate homeostasis

Osteoclasts possess inorganic phosphate ([P.sub.i]) transport systems to take up external [P.sub.i] during bone resorption. In the present study, we characterized [P.sub.i] transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-[kappa]B ligand (RANKL). In undifferentiated RAW264.7 cells, [P.sub.i] transport into the cells was [Na.sup.+] dependent, but after treatment with RANKL, [Na.sup.+]-independent [P.sub.i] transport was significantly increased. In addition, compared with neutral pH, the activity of the [Na.sup.+]-independent [P.sub.i] transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The [Na.sup.+]-independent system consisted of two components with [K.sub.m] of 0.35 mM and 7.5 mM. The inhibitors of [P.sub.i] transport, phosphonoformic acid, and arsenate substantially decreased [P.sub.i] transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a [K.sup.+] ionophore, valinomycin, significantly suppressed [P.sub.i] transport activity. Analysis of BCECF fluorescence indicated that [P.sub.i] transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular [K.sup.+] ion stimulated [P.sub.i] transport, suggesting that membrane voltage is involved in the regulation of [P.sub.i] transport activity. Finally, bone particles significantly increased [Na.sup.+]-independent [P.sub.i] transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a [P.sub.i] transport system with characteristics that are different from those of other [Na.sup.+]-dependent [P.sub.i] transporters. We conclude that stimulation of [P.sub.i] transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. [Na.sup.+]-dependent phosphate cotransporter; RAW264.7; phosphate uptake

Recent studies suggest that vitamin D may play a role in intestinal [Na.sup.+]-dependent phosphate transport adaptation to variable levels of dietary [P.sub.i]. Therefore, the goal of the current study was to assess [Na.sup.+]-dependent [P.sub.i] cotransport activity in transgenic mice to determine whether vitamin D is an essential mediator of this process. Intestinal brush-border membrane (BBM), [Na.sup.+]-dependent [P.sub.i] cotransport activity was significantly decreased in vitamin D receptor (VDR) null [VDR (-/-)] mice compared with wild-type (VDR+/+) mice. While intestinal Na-[P.sub.i] cotransporter (type IIb) mRNA levels were similar in VDR (-/-) and VDR (+/+) mice, type IIb Na-[P.sub.i] cotransporter protein expression was markedly suppressed in VDR (-/-) mice compared with VDR (+/+) mice. Furthermore, Na-[P.sub.i] cotransport activity in renal BBM was similar in VDR (-/-) and VDR (+/+) mice, but type IIa Na-[P.sub.i] cotransporter protein expression was decreased in VDR (-/-) mice. After administration of a low-[P.sub.i] diet, type IIb protein expression was significantly increased in VDR (+/+) and VDR (-/-) mice, and type IIb protein expression was present in the intestinal BBM of VDR (-/-) mice. These data demonstrate that intestinal Na-[P.sub.i] cotransport adaptation to a low-[P.sub.i] diet occurs independently of vitamin D. dietary phosphate; phosphate transport; intestine