NaPi-IIb mRNA Expression Research Articles

Intestinal epithelial ablation of Pit-2/Slc20a2 in mice leads to sustained elevation of vitamin D3 upon dietary restriction of phosphate.

Several Na+ -dependent phosphate cotransporters, namely NaPi-IIb/SLC34A2, Pit-1/SLC20A1 and Pit-2/SLC20A2, are expressed at the apical membrane of enterocytes but their contribution to active absorption of phosphate is unclear. The aim of this study was to compare their pattern of mRNA expression along the small and large intestine and to analyse the effect of intestinal depletion of Pit-2 on phosphate homeostasis. Intestinal epithelial Pit-2-deficient mice were generated by crossing floxed Pit-2 with villin-Cre mice. Mice were fed 2weeks standard or low phosphate diets. Stool, urine, plasma and intestinal and renal tissue were collected. Concentration of electrolytes and hormones, expression of mRNAs and proteins and intestinal transport of tracers were analysed. Intestinal mRNA expression of NaPi-IIb and Pit-1 is segment-specific, whereas the abundance of Pit-2 mRNA is more homogeneous. In ileum, NaPi-IIb mRNA expression is restricted to enterocytes, whereas Pit-2 mRNA is found in epithelial and non-epithelial cells. Overall, their mRNA expression is not regulated by dietary phosphate. The absence of Pit-2 from intestinal epithelial cells does not affect systemic phosphate homeostasis under normal dietary conditions. However, in response to dietary phosphate restriction, Pit-2-deficient mice showed exacerbated hypercalciuria and sustained elevation of 1,25(OH)2 vitamin D3 . In mice, the intestinal Na+ /phosphate cotransporters are not coexpressed in all segments. NaPi-IIb but not Pit-2 mRNA is restricted to epithelial cells. Intestinal epithelial Pit-2 does not contribute significantly to absorption of phosphate under normal dietary conditions. However, it may play a more significant role upon dietary phosphate restriction.

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.

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

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

Effect of early dietary energy restriction and phosphorus level on subsequent growth performance, intestinal phosphate transport, and AMPK activity in young broilers.

We aimed to determine the effect of low dietary energy on intestinal phosphate transport and the possible underlying mechanism to explain the long-term effects of early dietary energy restriction and non-phytate phosphorus (NPP). A 2 × 3 factorial experiment, consisting of 2 energy levels and 3 NPP levels, was conducted. Broiler growth performance, intestinal morphology in 0–21 days and 22–35 days, type IIb sodium-phosphate co-transporter (NaPi-IIb) mRNA expression, adenylate purine concentrations in the duodenum, and phosphorylated adenosine monophosphate-activated protein kinase (AMPK-α) activity in 0–21 days were determined. The following results were obtained. (1) Low dietary energy (LE) induced a high feed conversion ratio (FCR) and significantly decreased body weight gain in young broilers, but LE induced significantly higher compensatory growth in low NPP (LP) groups than in the high or medium NPP groups (HP and MP). (2) LE decreased the villus height (VH) in the intestine, and LE-HP resulted in the lowest crypt depth (CD) and the highest VH:CD ratio in the initial phase. However, in the later period, the LE-LP group showed an increased VH:CD ratio and decreased CD in the intestine. (3) LE increased ATP synthesis and decreased AMP:ATP ratio in the duodenal mucosa of chickens in 0–21 days, and LP diet increased ATP synthesis and adenylate energy charges but decreased AMP production and AMP:ATP ratio. (4) LE led to weaker AMPK phosphorylation, higher mTOR phosphorylation, and higher NaPi-IIb mRNA expression. Thus, LE and LP in the early growth phase had significant compensatory and interactive effect on later growth and intestinal development in broilers. The effect might be relevant to energy status that LE leads to weaker AMPK phosphorylation, causing a lower inhibitory action toward mTOR phosphorylation. This series of events stimulates NaPi-IIb mRNA expression. Our findings provide a theoretical basis and a new perspective on intestinal phosphate transport regulation, with potential applications in broiler production.

Regulation of phosphate transport and AMPK signal pathway by lower dietary phosphorus of broilers.

Lower available P (aP) was used as a base value in nutritional strategies for mitigating P pollution by animal excreta. We hypothesized that the mechanism regulating phosphate transport under low dietary P might be related with the AMPK signal pathway. A total of 144 one-day-old Arbor Acres Plus broilers were randomly allocated to control (HP) or trial (LP) diets, containing 0.45 and 0.23% aP, respectively. Growth performance, blood, intestinal, and renal samples were tested in 21-day-old broilers. Results shown that LP decreased body weight gain and feed intake. Higher serum Ca and fructose, but lower serum P and insulin were detected in LP-fed broilers. NaPi-IIb mRNA expression in intestine and NaPi-IIa mRNA expression in kidney were higher in the LP group. AMP: ATP, p-AMPK: total AMPK, and p-ACC: total ACC ratios in the duodenal mucosa were decreased in the LP group, whereas the p-mTOR: total mTOR ratio increased. These findings suggested that the increase in phosphate transport owing to LP diet might be regulated either directly by higher mTOR activity or indirectly by the suppressive AMPK signal, with corresponding changes in blood insulin and fructose content. A novel viewpoint on the regulatory mechanism underlying phosphate transport under low dietary P conditions was revealed, which might provide theoretical guidelines for reducing P pollution by means of nutritional regulation.

Abstract 463: Evaluation of relative expression of SLC34A2/NaPi-IIb in lung cancer cell lines treated with estrogen and PKC and PKA pathway modulators

Abstract Background: Lung cancer (LC) represents a major health challenge worldwide, being the leading cause of cancer-related deaths in both men and women. SLC34A2 is a member of the solute carrier family and encodes the type IIb sodium phosphate cotransporter (NaPi-IIb). SLC34A2 expression is reduced by ten-fold in LC samples compared to normal lung. Estrogen is a known risk factor for LC in women, and a relationship between its action and SLC34A2/NaPi-IIb super-expression in certain cell lines has been proposed. Furthermore, based on the central role of PKC isoenzymes in carcinogenesis, and on studies showing that administration of cAMP stimulates the NaPi-IIb mRNA expression in adult rats alveolar type II cells, this work aimed to evaluate the correlation between PKA (cAMP mediator), PKC, estrogen signaling pathways and SLC34A2/NaPi-IIb expression levels in human LC cells. Methods: For both LC cell lines studied, A549 and H460, the effects of PKC and PKA activating and inhibitory reagents (PMA, calphostin C, dB cAMP and KT 5720) were tested in relation to SLC34A2/NaPi-IIb expression. Additionally, a dose-response curve of 17-beta-estradiol was performed for the expression of SLC34A2/NaPi-IIb in A549 cell line. SLC34A2/NaPi-IIb gene expression was analyzed by qRT-PCR, using SYBR Green PCR Master Mix as the detection system. Relative quantification of gene expression was performed by calculating the delta-delta Ct using two housekeeping genes: GAPDH and Beta-Actin. Findings: In our analysis we found that PKC and PKA did not influence the expression of SLC34A2/NaPi-IIb in LC cell line evaluated. There was, however, a considerable increase in the expression of this gene when treated with calphostin C, probably by a mechanism independent of its classic role in the PKC inhibition. There was also a trend of decreased SLC34A2/NaPi-IIb gene expression in LC cell line following the treatment with 17 beta-estradiol, and SLC34A2/NaPi-IIb - which expression is already reduced in LC - undergoes a greater reduction in its relative expression. Interpretation: We believe that maintaining the reduced expression of SLC34A2/NaPi-IIb should provide benefits to LC cells. Calphostin C induces apoptosis in several tumor cell lines by mechanisms not yet fully elucidated. Thus, we suggest a possible involvement of SLC34A2/NaPi-IIb in this pro-apoptotic pathway, which corroborates the fact that LC tumors present a reduced SLC34A2/NaPi-IIb expression compared to normal lung. Likewise, based on our findings, we believe that the inclusion of selective Estrogen receptor modulators and aromatase inhibitors in the routine clinical practice of LC might help to control the disease that is still the leading cause of cancer-related deaths worldwide. Citation Format: Murilo F. Cerri, Lucas C d Rezende, Marcela F. Paes, Klesia P. Madeira, Renata D. Daltoe, Nayara G. Tessarollo, Ian V. Silva, Leticia B a Rangel. Evaluation of relative expression of SLC34A2/NaPi-IIb in lung cancer cell lines treated with estrogen and PKC and PKA pathway modulators. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2014-463

The Influence of Dietary Calcium and Phosphorus Imbalance on Intestinal NaPi-IIb and Calbindin mRNA Expression and Tibia Parameters of Broilers.

A 2×2 factorial experiment was conducted to study the effect of dietary calcium and non-phytate phosphorus (nPP) imbalance on calbindin and NaPi-IIb mRNA levels in the small intestine and tibia parameters of broiler chicks. One hundred and forty four 1-d-old Arbor Acres male broiler chicks were divided into four treatments consisted of six replicates with six chicks each. The two dietary calcium levels were 1.10% and 0.60%, and two dietary nPP levels were 0.50% and 0.27%. Results showed that a high Ca/nPP ratio diet (4.07:1) significantly depressed feed intake and weight gain of broilers (p<0.05), but a lower Ca:nPP ratio (1.2:1) had no influence (p>0.05). Low-Ca with low-P diet resulted in low tibia minerals and tibia breaking strength of broilers, and all the tibia parameters were further decreased when the dietary ratio of Ca to P was relative higher. Low dietary Ca or P up-regulated the calbindin and NaPi-IIb mRNA expression levels. Low Ca with normal P diet up-regulated duodenal calbindin mRNA expression level to the greatest extent. Low P with a normal Ca diet significantly enhanced NaPi-IIb mRNA expression level to the highest extent. These results suggest that the calbindin and NaPi-IIb mRNA expression were enhanced by the imbalance between dietary Ca and nPP, and their expression were not only influenced by Ca or nPP level, but also the ratio of Ca:nPP.

Short communication: Phosphate transporter expression in Holstein cows

Phosphorus nutrition in cattle is increasingly becoming an important topic because excess dietary P is excreted in manure and can be washed into surface water, causing increased algal growth and eutrophication. However, little is known about the mechanism or regulation of P absorption in dairy cattle. Phosphorus transporters have been characterized in other species and homologous genes have been found to be expressed in bovine cell cultures. However, no other information is available regarding the active transport of phosphate in the digestive tract of cattle. The objective of this study was to determine the patterns of expression of a known phosphate transporter, NaPi-IIb, in 4 sections of the small intestine of Holstein cows. Ribonucleic acid was isolated from the duodenal, proximal jejunal, distal jejunal, and ileal mucosa of 20 Holstein cows. Relative amounts of NaPi-IIb mRNA expression were determined using real-time reverse-transcription PCR. Expression of NaPi-IIb was highest in the 2 distal sections and almost absent in the proximal sections. Expression did not differ between the 2 proximal sections or the 2 distal sections. These data suggest that a Na+-dependent secondary active P transport system is not responsible for P absorption in the proximal portion of the bovine small intestine, whereas it does contribute to the P absorbed in the distal sections of the bovine small intestine.

Sodium-Dependent Phosphate Uptake in the Jejunum Is Post-Transcriptionally Regulated in Pigs Fed a Low-Phosphorus Diet and Is Independent of Dietary Calcium Concentration

In rodents, severe dietary P restriction increases active phosphate absorption by the intestine. However, it remains unknown if moderate dietary P restriction has a similar effect. Weanling pigs (n = 32; body weight 7.4 ± 0.55 kg) were used in a 2 × 2 factorial design and fed dietary available P (aP) concentrations of 0.23 or 0.40% and Ca concentrations of 0.58 or 1.00% for 14 d. Diets were formulated on an aP basis instead of a total P basis, because pigs are unable to absorb phytate-P present in corn and soybean meal. Jejunal segments were mounted in modified Ussing chambers for determination of Na+-dependent nutrient transport. Intestinal mucosal scrapings were taken for RNA isolation and brush border membrane (BBM) vesicle isolation. Na+-dependent phosphate uptake and gene expression of Na-phosphate cotransporter IIb (NaPi-IIb), SGLT-1 (sodium/glucose cotransporter-1), and calbindin D(9k) and protein expression of NaPi-IIb were evaluated. Na+-dependent phosphate transport increased (P < 0.05) 46% as dietary aP concentration was decreased. However, increased Na+-dependent phosphate uptake was not accompanied by increased NaPi-IIb mRNA expression. Expression of NaPi-IIb protein in the BBM increased (P < 0.01) 84% in pigs fed low-P diets compared with pigs fed adequate-P diets. No dietary Ca effects or aP × Ca interactions were detected for Na-dependent P uptake, mRNA or protein expression of NaPi-IIb, or mRNA expression of calbindin D(9k). These data suggest that restricting dietary aP concentration by only 43% stimulates Na+-dependent phosphate uptake and expression of the NaPi-IIb protein in the BBM of the small intestine and through a post-transcriptional mechanism.

Dexamethasone and Cyclic AMP Regulate Sodium Phosphate Cotransporter (NaPi-IIb and Pit-1) mRNA and Phosphate Uptake in Rat Alveolar Type II Epithelial Cells

Alveolar epithelial type II (AT II) cells need phosphate (Pi) for surfactant synthesis. The Na-dependent (Na(d)) Pi transporters NaPi-IIb and Pit-1 are expressed in lung, but their expression, regulation, and function in AT II cells remain unclear. We studied NaPi-IIb and Pit-1 mRNA expression in cultured AT II cells isolated from adult rat lung, their regulation by agents known to enhance surfactant production, dexamethasone (dex) and dibutyryl cyclic AMP (cAMP), and the effects of dex and cAMP on Na(d) Pi uptake by this cell type. By Northern analysis, cultured AT II cells expressed both NaPi-IIb (4.8 and 4.0 kb) and Pit-1 (4.3 kb) mRNA. Treatment with 100 nmol/l dex for 24 h decreased the expression of both mRNAs (to 0.48 +/- 0.06 and 0.77 +/- 0.05, respectively, as compared to control), while 0.1 mmol/l cAMP stimulated NaPi-IIb (1.94 +/- 0.22) but not Pit-1 mRNA (0.90 +/- 0.05, compared to vehicle-treated cells). NaPi-IIb and Pit-1 proteins could not be identified by western analysis of plasma membrane preparations of cultured AT II cells. AT II cells take up Pi in a Na(d) manner. Uptake was slightly (to 0.78-fold of the control) decreased by 100 nmol/l dex but not affected by 0.1 mmol/l cAMP treatment. Although NaPi-IIb mRNA expression was maintained to some extent by AT II cells kept in primary culture, Pi uptake was more closely related to Pit-1 mRNA expression.

Targeted deletion of the tybe IIb Na+-dependent Pi-co-transporter, NaPi-IIb, results in early embryonic lethality

NaPi-IIb encodes a Na+-dependent Pi co-transporter, which is expressed in various adult tissues and mediates transport of extracellular Pi ions coupling with Na+ ion. To define the role of NaPi-IIbin vivo, NaPi-IIb gene deficient mice were generated utilizing targeted mutagenesis, yielding viable, heterozygous NaPi-IIb mice. In contrast, homozygous NaPi-IIb mice died in utero soon after implantation, indicating that NaPi-IIb was essential for early embryonic development. In situ hybridization revealed NaPi-IIb mRNA expression in the parietal endoderm, followed by the visceral endoderm, at a time point prior to establishment of a functioning chorio-allantoic placenta. At the time point of functional placenta development, the main site of NaPi-IIb production resided in the labyrinthine zone, where embryonic and maternal circulations were in closest contact. Expression patterns of NaPi-IIb suggest that NaPi-IIb plays an important role in Pi absorption from maternal circulation.

Adaptation of Epithelial Sodium-Dependent Phosphate Transport in Jejunum and Kidney of Hens to Variations in Dietary Phosphorus Intake

The objective of this study was to explore the homeostatic response of jejunal and renal epithelia regarding the inorganic phosphate (Pi) transport capacities to variations in dietary total phosphorus (tP) supply in hens. Adaptive processes were determined by quantitative measures of intake and excretion, Pi transport studies across brush border membranes, and semiquantitative detection of sodium-dependent phosphate transporters (NaPi II) based on mRNA expression in the jejunum and kidney. Twelve hens (4/group) were adapted to 3 tP feeding levels in a pair-fed manner (60 g/d): low P diet with 0.073% tP, medium P diet with 0.204% tP, and high P diet with 0.343% tP. Excretion was measured during the last 5 d of a 16-d feeding period. After slaughtering, jejunal mucosa and renal cortex were removed. Tissues were used for 32 P uptake studies in brush-border membrane vesicles by rapid filtration technique and NaPi II mRNA expression studies by northern analyses. Plasma Pi concentrations were additionally measured. The NaPi II transporter mRNA could specifically be detected in chicken jejunum and kidney. Functional parameters of Na+-dependent Pi transport indicated that these transporters were involved in chicken Pi transport across the apical membranes of jejunal and renal epithelia. Increased tP intake resulted in an increased overall tP excretion. Correlating individual data from all animals by linear regression highlighted that the adaptive decrease of renal Pi transport capacity and NaPi IIa mRNA expression was associated with an increase in plasma Pi levels and resulted in a higher tP excretion. Jejunal Pi transport capacity and NaPi IIb mRNA expression did not react to variations in dietary tP supply. In conclusion, the homeostatic response was mainly based on the adaptive capacity of the kidney in hens.

Regulation of intestinal NaPi-IIb cotransporter gene expression by estrogen.

The current experiments were designed to study the effect of beta-estradiol on type IIb sodium-coupled phosphate (NaPi-IIb) cotransporter gene expression. Uptake studies with intestinal brush-border membrane vesicles (BBMV) showed that estrogen treatment increased sodium-dependent phosphate absorption by approximately 45% in rat intestine. Northern blot analysis indicated that NaPi-IIb mRNA expression was increased by approximately 50% after estrogen treatment. Western blot analysis also detected an increase in BBMV NaPi-IIb protein expression in estrogen-treated rats. In human intestinal Caco-2 cells, NaPi-IIb mRNA abundance was increased approximately 60% after estrogen treatment, and this increase could be abolished by inhibition of gene transcription. Transfection studies with human NaPi-IIb promoter reporter constructs showed that the promoter was responsive to estrogen treatment. These studies demonstrate for the first time that estrogen stimulates intestinal sodium-dependent phosphate absorption in female rats. This stimulation is associated with increased NaPi-IIb mRNA and protein expression. Thus the effect of estrogen on intestinal Pi absorption may be partially due to activation of NaPi-IIb gene transcription.

Age-dependent regulation of rat intestinal type IIb sodium-phosphate cotransporter by 1,25-(OH)(2) vitamin D(3).

The current studies were designed to characterize type IIb sodium-inorganic phosphate (P(i)) cotransporter (NaP(i)-IIb) expression and to assess the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb gene expression during rat ontogeny. Sodium-dependent P(i) absorption by intestinal brush-border membrane vesicles (BBMVs) decreased with age, and NaP(i)-IIb gene expression also decreased proportionally with age. 1,25-(OH)(2) vitamin D(3) treatment increased intestinal BBMV P(i) absorption by approximately 2.5-fold in suckling rats and by approximately 2.1-fold in adult rats. 1,25-(OH)(2) vitamin D(3) treatment also increased NaP(i)-IIb mRNA abundance by approximately 2-fold in 14-day-old rats but had no effect on mRNA expression in adults. Furthermore, in rat intestinal epithelial (RIE) cells, 1,25-(OH)(2) vitamin D(3) increased NaP(i)-IIb mRNA abundance, an effect that was abolished by actinomycin D. Additionally, human NaP(i)-IIb gene promoter activity in transiently transfected RIE cells showed approximately 1.6-fold increase after 1,25-(OH)(2) vitamin D(3) treatment. In conclusion, we demonstrate that the age-related decrease in intestinal sodium-dependent P(i) absorption correlates with decreased NaP(i)-IIb mRNA expression. Our data also suggest that the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb expression is at least partially mediated by gene transcription in suckling rats.