Sodium-dependent Pi Transport Research Articles

Fibroblast Growth Factor 23 Neutralizing Antibody Ameliorates Abnormal Renal Phosphate Handling in Sickle Cell Disease Mice.

We assessed the involvement of fibroblast growth factor 23 (FGF23) in phosphaturia in sickle cell disease (SCD) mice. Control and SCD mice were treated with FGF23 neutralizing antibody (FGF23Ab) for 24 hours. Serum ferritin was significantly increased in SCD mice and was significantly reduced in female but not male SCD mice by FGF23Ab. FGF23Ab significantly reduced increased erythropoietin in SCD kidneys. Serum intact FGF23 was significantly increased in SCD female mice and was markedly increased in SCD male mice; however, FGF23Ab significantly reduced serum intact FGF23 in both genotypes and sexes. Serum carboxy-terminal-fragment FGF23 (cFGF23) was significantly reduced in SCD IgG male mice and was markedly but not significantly reduced in SCD IgG female mice. FGF23Ab significantly increased cFGF23 in both sexes and genotypes. Serum 1,25-dihydroxyvitamin D3 was significantly increased in SCD IgG and was further significantly increased by FGF23Ab in both sexes and genotypes. Significantly increased blood urea nitrogen in SCD was not reduced by FGF23Ab. The urine phosphate (Pi)/creatinine ratio was significantly increased in SCD in both sexes and was significantly reduced by FGF23Ab. Increased SCD kidney damage marker kidney injury molecule 1 was rescued, but sclerotic glomeruli, increased macrophages, and lymphocytes were not rescued by short-term FGF23Ab. FGF23Ab significantly reduced increased phospho-fibroblast growth factor receptor 1, αKlotho, phosphorylated extracellular signal-regulated kinase, phosphorylated serum/glucocorticoid-regulated kinase 1, phosphorylated sodium-hydrogen exchanger regulatory factor-1, phosphorylated janus kinase 3, and phosphorylated transducer and activator of transcription-3 in SCD kidneys. The type II sodium Pi cotransporter (NPT2a) and sodium-dependent Pi transporter PiT-2 proteins were significantly reduced in SCD kidneys and were increased by FGF23Ab. We conclude that increased FGF23/FGF receptor 1/αKlotho signaling promotes Pi wasting in SCD by downregulating NPT2a and PIT2 via modulation of multiple signaling pathways that could be rescued by FGF23Ab.

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

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

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

Dietary protein and calcium modulate parathyroid vitamin D receptor expression in young ruminants

For economic reasons and in order to minimize nitrogen excretion and thus pollution, the crude protein content in the diet of livestock animals should be as low as possible without negatively affecting the animals´ health and performance. As ruminants can efficiently use dietary protein because of the ruminohepatic circulation of urea, they are considered to cope more easily with such a feeding regime than monogastric animals. However, despite unaltered daily weight gain, massive changes in mineral homeostasis and vitamin D metabolism were observed with dietary protein reduction (N-) in young, growing goats. Serum concentrations of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) were decreased with a low N intake, even if calcium (Ca) was also restricted (Ca-). Interestingly, concentrations of cyclic adenosine monophosphate (cAMP) measured as an indirect assessment for the parathyroid hormone (PTH) activity were not affected by low protein. Therefore, it was hypothesized that the sensitivity of the parathyroid gland is modulated during these dietary interventions. Four groups of male German colored goats received a control (N+/Ca+), a reduced protein (N-/Ca+), a reduced Ca (N+/Ca-) or a reduced protein and Ca (N-/Ca-) diet. After six weeks we determined the expression of PTH, PTH receptor, Ca sensing receptor (CASR), vitamin D receptor (VDR), retinoid X receptor (RXRα), Klotho, fibroblast growth factor receptor 1c-splicing form, and the sodium-dependent Pi transporter (PiT1) in the parathyroid glands. Concentrations of cAMP were not affected, while those of Ca and 1,25-(OH)2D3were diminished and that of 25-hydroxyvitamin D3 was increased with N- feeding. The expression patterns of the described target genes were not altered. In contrast, animals fed the Ca- rations showed enhanced serum 1,25-(OH)2D3 and cAMP levels with no changes in blood Ca concentrations demonstrating an efficient adaptation. The mRNA expression of expression of VDR and CASR in the parathyroid gland was significantly diminished and RXRα, PTHR and PiT1 expression was elevated. Instead of the assumed desensitization of the parathyroid gland with N-, our results indicate elevated responsiveness to decreased blood Ca with feeding Ca-.

Paracellular transport of phosphate along the intestine.

Inorganic phosphate (Pi) is crucial for many biological functions, such as energy metabolism, signal transduction, and pH buffering. Efficient systems must exist to ensure sufficient supply for the body of Pi from diet. Previous experiments in humans and rodents suggest that two pathways for the absorption of Pi exist, an active transcellular Pi transport and a second paracellular pathway. Whereas the identity, role, and regulation of active Pi transport have been extensively studied, much less is known about the properties of the paracellular pathway. In Ussing chamber experiments, we characterized paracellular intestinal Pi permeabilities and fluxes. Dilution potential measurements in intestinal cell culture models demonstrated that the tight junction is permeable to Pi, with monovalent Pi having a higher permeability than divalent Pi. These findings were confirmed in rat and mouse intestinal segments by use of Ussing chambers and a combination of dilution potential measurements and fluxes of radiolabeled 32Pi. Both techniques yielded very similar results, showing that paracellular Pi fluxes were bidirectional and that Pi permeability was ~50% of the permeability for Na+ or Cl-. Pi fluxes were a function of the concentration gradient and Pi species (mono- vs. divalent Pi). In mice lacking the active transcellular Pi transport component sodium-dependent Pi transporter NaPi-IIb, the paracellular pathway was not upregulated. In summary, the small and large intestines have a very high paracellular Pi permeability, which may favor monovalent Pi fluxes and allow efficient uptake of Pi even in the absence of active transcellular Pi uptake.NEW & NOTEWORTHY The paracellular permeability for phosphate is high along the entire axis of the small and large intestine. There is a slight preference for monovalent phosphate. Paracellular phosphate fluxes do not increase when transcellular phosphate transport is genetically abolished. Paracellular phosphate transport may be an important target for therapies aiming to reduce intestinal phosphate absorption.

Nephronectin Expression is Inhibited by Inorganic Phosphate in Osteoblasts.

Nephronectin (Npnt), an extracellular matrix protein, is known to be a ligand of integrin α8β1, and it has also been known to play critical roles as various organs. In the present study, elevated extracellular inorganic phosphate (Pi) strongly inhibited the expression of Npnt in MC3T3-E1 cells, while the existence of extracellular calcium (Ca) was indispensable for its effect. Furthermore, Pi-induced inhibition of Npnt gene expression was recovered by inhibitors of both sodium-dependent Pi transporter (Pit) and fibroblast growth factor receptors (Fgfrs). These results demonstrated that Npnt gene expression is regulated by extracellular Pi via Pit and Fgfrs.

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

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

A Role of Intestinal Alkaline Phosphatase 3 (Akp3) in Inorganic Phosphate Homeostasis

Background/Aims: Hyperphosphatemia is a serious complication of late-stage chronic kidney disease (CKD). Intestinal inorganic phosphate (Pi) handling plays an important role in Pi homeostasis in CKD. We investigated whether intestinal alkaline phosphatase 3 (Akp3), the enzyme that hydrolyzes dietary Pi compounds, is a target for the treatment of hyperphosphatemia in CKD. Methods: We investigated Pi homeostasis in Akp3 knockout mice (Akp3^-/-). We also studied the progression of renal failure in an Akp3^-/- mouse adenine treated renal failure model. Plasma, fecal, and urinary Pi and Ca concentration were measured with commercially available kit, and plasma fibroblast growth factor 23, parathyroid hormone, and 1,25(OH)₂D₃ concentration were measured with ELISA. Brush border membrane vesicles were prepared from mouse intestine using the Ca²⁺ precipitation method and used for Pi transport activity and alkaline phosphatase activity. In vivo intestinal Pi absorption was measured with oral ³²P administration. Results: Akp3^-/- mice exhibited reduced intestinal type II sodium-dependent Pi transporter (Npt2b) protein levels and Na-dependent Pi co-transport activity. In addition, plasma active vitamin D levels were significantly increased in Akp3^-/- mice compared with wild-type animals. In the adenine-induced renal failure model, Akp3 gene deletion suppressed hyperphosphatemia. Conclusion: The present findings indicate that intestinal Akp3 deletion affects Na⁺-dependent Pi transport in the small intestine. In the adenine-induced renal failure model, Akp3 is predicted to be a factor contributing to suppression of the plasma Pi concentration.

Hypophosphatemia promotes lower rates of muscle ATP synthesis

Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [31P]-magnetic resonance spectroscopy. By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent Pi transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a−/−) mice compared with their wild-type littermate controls. Rates of VATP normalized in both hypophosphatemic groups after restoring plasma Pi concentrations. Furthermore, VATP was directly related to cellular and mitochondrial Pi uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum Pi content and muscle VATP. After oral Pi repletion and normalization of serum Pi levels, muscle VATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP synthesis, in part, may be caused by low blood Pi concentrations, which may explain some aspects of muscle weakness observed in patients with hypophosphatemia.—Pesta, D. H., Tsirigotis, D. N., Befroy, D. E., Caballero, D., Jurczak, M. J., Rahimi, Y., Cline, G. W., Dufour, S., Birkenfeld, A. L., Rothman, D. L., Carpenter, T. O., Insogna, K., Petersen, K. F., Bergwitz, C., Shulman, G. I. Hypophosphatemia promotes lower rates of muscle ATP synthesis.

Inorganic Phosphate Activates the AKT/mTORC1 Pathway and Shortens the Life Span of an α‑Klotho-Deficient Model.

Inorganic phosphate (Pi) has been implicated in the pathogenesis of accelerated aging; however, the underlying mechanisms remain elusive. Herein, we demonstrated in cultured cells and invivo that increased levels of extracellular Pi activated the AKT/mammalian target of rapamycin complex1 (mTORC1) pathway by suppressing membrane-bound phosphatase and tensin homolog (PTEN) levels in a manner requiring the sodium-dependent Pi transporter PiT‑1. High levels of extracellular Pi also led to phosphorylation of Ser/Thr clusters in the C‑terminal tail of PTEN, which has been shown to dissociate PTEN from the membrane. Notably, blockade of mTORC1 activity by rapamycin treatment prolonged the life span of hyperphosphatemic α‑Klotho-deficient (Kl(-/-)) mice. Dietary correction of hyperphosphatemia or treatment with rapamycin also rescued the brown adipose tissue dysfunction and oxidative damage observed in Kl(-/-) mice. Furthermore, rapamycin treatment partially rescued these effects and extended the life span when Kl(-/-) mice were maintained on a high-phosphate diet. Finally, rapamycin reduced circulating Pi levels in Kl(-/-) mice, apparently by decreasing the localization of sodium-dependent Pi transport protein2a at the renal brush border membrane. Therefore, the activation of mTORC1 may create a vicious loop that exacerbates the retention of Pi, which in turn may enhance oxidative damage and ultimately shorten the life span of Kl(-/-) mice. These results demonstrate that Pi has important roles in the aging process, and the blockade of mTORC1 may have therapeutic potential for premature aging-like symptoms associated with hyperphosphatemia.

Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure.

Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca2+ and Pi regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL) expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca2+ excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D3 were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin-D28k, and sodium-dependent Pi transporter type 2b (NaPi2b), whereas the renal expression of sodium-dependent Pi transporter type 2a (NaPi2a) and type 3 (PIT2) were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca2+/Pi homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.

Adaptive responses of calcium and phosphate homeostasis in goats to low nitrogen intake: renal aspects

In a previous study, in goats, we showed that apart from variations in dietary calcium (Ca) and phosphorus intake, also low dietary nitrogen (N) intake altered plasma concentrations of hormones, which regulate Ca and phosphate (Pi ) homeostasis. These hormonal responses in goats were in accordance with findings in monogastric animals and humans with low protein intake. In the aforementioned studies, alterations of electrolyte transport in the kidneys were also observed. However, whether renal electrolyte transport in goats is also involved in the adaptation of Ca and Pi homeostasis to low N intake remains unknown. Thus, the aim of the present study was to investigate whether in addition to the hormonal changes, as observed in our former study, renal Ca transport and renal Pi transport were also altered by low N intake in goats. Therefore, in kidney samples from the goats used in our former study, the protein expression of Ca and Pi transporters and of related regulatory proteins was examined. Furthermore, the uptake of Pi into isolated brush border membrane vesicles (BBMV) was detected. The results showed that the protein amount of the renal sodium-dependent Pi transporter NaPi IIa was elevated, and concomitantly, protein expression of its upstream regulators, the parathyroid hormone receptor and the extracellular signal-regulated kinases 1 and 2 was decreased. However, Pi uptake into renal BBMV was not enhanced. Furthermore, protein expression of the renal Ca channel, the transient receptor potential cation channel subfamily V member 5 (TRPV5) and of the vitamin D receptor was not influenced by dietary N reduction. We conclude that regulation of renal Pi transporter expression in goats is involved in the adaptation of electrolyte homeostasis to low N intake.

Adaptation of electrolyte handling to low crude protein intake in growing goats and consequences for in vivo electrolyte excretion

In a previous study it was shown that low crude protein (LCP) intake affected the systemic and renal electrolyte handling (EH) in growing goats. The present study aimed to evaluate the consequences of these changes on the electrolyte excretion and its in vivo regulation by functional studies. Growing male Saanen-type goats were fed diets high or low in crude protein content, and nitrogen (N), phosphorus (P) and calcium (Ca) balance trials were performed to investigate electrolyte excretion. Additionally, plasma urea, Ca, phosphate (Pi) and endocrine regulators of electrolyte homeostasis including insulin-like growth factor-1 (IGF-1), calcitriol, cross-linked N-telopeptide of type I collagen and parathyroid hormone (PTH) concentrations were determined. Renal adaptation to LCP intake was investigated by determining the protein expression of the sodium-dependent Pi transporter types IIa and IIc (NaPi IIa and IIc) and the PTH receptor (PTHR) by Western blotting. To demonstrate associations between the measured variables and thereby to identify the possible pathways of electrolyte handling in goats, linear regression analyses were performed. While N metabolism was adapted to LCP intake, the plasma IGF-1 and, by trend, calcitriol concentration decreased and also the renal PTHR protein expression declined. However, Ca and P excretion and retention remained unaffected. Linear regression analyses showed that calcitriol and IGF-1 concentrations were associated with further key factors of systemic and renal EH. In summary, LCP intake in growing goats led to an adaptation of the systemic and renal EH, and IGF-1 and calcitriol appeared to play significant roles. However, these adaptations seemed to be counterbalanced by the whole systemic network regulating the electrolyte handling and therefore did not have any impact on the functional electrolyte excretion.

The Vitamin D Analog ED-71 Is a Potent Regulator of Intestinal Phosphate Absorption and NaPi-IIb

The vitamin D analog ED-71 [1α,25-dihydroxy-2β-(3-hydroxypropyloxy)vitamin D(3)] has been approved for treatment of osteoporosis in Japan, but its effects on mineral metabolism have not been fully explored. We investigated the actions of ED-71 on phosphate (Pi) absorption and induction of the intestinal sodium/phosphate cotransporters. Oral treatment of vitamin D-deficient rats with ED-71 (20 pmol every other day for 8 d) produced a maximal 8-fold increase in duodenal Pi absorption, measured by the in situ loop method, whereas 1,25-dihyroxyvitamin D(3) [1,25(OH)(2)D(3]), at doses up to 150 pmol, had no effect. This action of ED-71 was attributable to a dramatic 24-fold induction of sodium-dependent Pi transporter type IIb (NaPi-IIb) mRNA in the duodenum; Pit-1 and Pit-2 mRNA levels were not increased. In vitamin D-replete rats, ED-71 treatment (50 pmol) at 72 and 24 h before death increased NaPi-IIb mRNA in the duodenum and jejunum, but not the ileum, whereas 1,25(OH)(2)D(3) at 1000 pmol was ineffective in all segments. Single oral doses of ED-71 increased mouse intestinal NaPi-IIb mRNA and protein between 6 and 24 h. Surprisingly, rat lung NaPi-IIb was not increased by ED-71, despite its coexpression with the vitamin D receptor in alveolar type II cells. However, ED-71 did not induce intestinal NaPi-IIb in vitamin D receptor-ablated mice. The greater potency of ED-71 than 1,25(OH)(2)D(3) on NaPi-IIb appears to be due to much higher and more prolonged levels of ED-71 in the circulation. In summary, ED-71, due to its disparate pharmacokinetics, is a much more potent inducer of intestinal Pi absorption and NaPi-IIb than 1,25(OH)(2)D(3), suggesting a role for this analog in the treatment of Pi-wasting disorders.

The Role Of Salivary Glands In Phosphate Homeostasis

Hyperphosphatemia is recognized as a contributor to vascular calcification in patients with chronic kidney disease (CKD) and hemodialysis (HD) patients and is independently associated with cardiac mortality. Dietary inorganic phosphorus (Pi) restriction, and the Pi binders are important therapy for dialysis patients with hyperphosphatemia. Recent study reported that salivary secretion of Pi to be an important determinant of hyperphosphatemia in patients with CKD and in those with ESRD under chronic dialysis. In the present study, we investigated the role of type IIb sodium-dependent Pi transporter (Npt2b) on salivary Pi excretion in mice. The expression of Npt2b protein was detected at the apical side of duct cells in the salivary glands, suggesting that ductal cells appears to be able to reabsorb Pi, thereby modifying the Pi concentration in the final saliva. In wild-type mice (Wt mice) fed a high Pi diet, the levels of plasma and salivary Pi are significantly higher than those in mice fed a low Pi diet. In Npt2b+/- mice, the salivary Pi concentrations were significantly increased compared with those in Npt2b+/+ mice. Npt2b+/- mice with adenine-induced renal failure had low plasma and salivary Pi levels, and plasma creatinine and BUN levels compared with Npt2b+/+ mice treated with adenine. In conclusion, Npt2b is involved in Pi secretion by salivary glands.

Ex vivo intestinal studies on calcium and phosphate transport in growing goats fed a reduced nitrogen diet

In ruminant feeding, the reduction of dietary protein is an effective approach for decreasing the excretion of N. In non-ruminant species, the intestinal absorption of Ca was affected when dietary protein was reduced. Therefore, it was the aim of the present study to characterise the intestinal absorption of Ca and inorganic phosphate (P(i)) in goats fed different N and Ca diets. Intestinal flux rates of Ca and P(i) were determined in goats fed a reduced N and Ca diet by Ussing chamber experiments. For a more mechanistic approach, the uptake of Ca and P(i) in intestinal brush-border membrane vesicles (BBMV), the expression levels of the epithelial Ca channel transient receptor potential vanilloid channel type 6 (TRPV6), the sodium-dependent P(i) transporter (NaPi) IIb and the vitamin D receptor (VDR) were measured. In goats fed a reduced N and Ca diet, the intestinal flux rates of Ca and P(i) were elevated. However, the reduced N and Ca diet had no effect on the uptake of Ca and P(i) in intestinal BBMV, while the expression of TRPV6 and NaPi IIb protein in the corresponding intestinal segments was even decreased. The mRNA expression of NaPi IIb and VDR was not affected. Therefore, a post-transcriptional regulation of TRPV6 and NaPi IIb protein was suggested in goats fed a reduced N and Ca diet. From these data, it can be concluded that the intestinal absorption of Ca and P(i) in growing goats was affected by changes in dietary N and Ca intake like those in single-stomached animals but differently modulated.

Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b+/−mice

An inorganic phosphate (P(i))-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P(i) (Na/P(i)) transport system is involved in intestinal P(i) absorption and is regulated by several factors. The type II sodium-dependent P(i) transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P(i). In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P(i) excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)(2)D(3) levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At 20 wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P(i) cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma P(i) levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na(+)/P(i) transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.

Phosphate increases bone morphogenetic protein-2 expression through cAMP-dependent protein kinase and ERK1/2 pathways in human dental pulp cells

Extracellular phosphate (Pi) is known to play a key role in promoting osteoblastic differentiation by altering gene expression and cellular function. Importantly, it may be possible to use this knowledge as a means to deliver Pi to local sites to regenerate mineralized tissues associated with the oral cavity. Therefore, we determined the ability of Pi to regulate differentiation of pulp cells toward an odontoblast phenotype and further determined if this was in part due to an increase in the expression of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization. Results showed that Pi increased BMP-2 expression at both mRNA and protein level and BMP-2 promoter activity. Signaling inhibitors revealed that increased BMP-2 expression was dependent on cAMP/protein kinase A but not the protein kinase C signaling pathway. Treatment with 8-Br-cAMP, a cell-permeable analog of cAMP, enhanced Pi-mediated BMP-2 expression, but treatment with 8-Br-cAMP alone did not increase BMP-2, suggesting that cAMP is indispensable but not sufficient for Pi-mediated BMP-2 expression. Pi activated ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating a requirement for activation of ERK1/2. ERK1/2 pathway may operate independently of cAMP-dependent signaling because MDL12,330A, an adenylate cyclase inhibitor, did not inhibit phosphorylation of ERK1/2 in response to Pi. Pulp cells expressed the sodium-dependent Pi transporter (NaPi) III type, but not NaPi-I type or NaPi-II type. Pi-mediated BMP-2 increase was inhibited in the presence of phosphonoformic acid, an inhibitor not only of NaPi transport but also of crystal nucleation. Furthermore, a similar inhibition was observed in the presence of pyrophosphate, a mineralization inhibitor. These findings demonstrate, for the first time, that Pi regulates BMP-2 expression via cAMP/protein kinase A and ERK1/2 pathways in human dental pulp cells.

Distribution of vesicular glutamate transporters in rat and human retina

Central nervous system neurons have traditionally been thought to express exclusively membrane transporters and/or vesicular transporters for their transmitter. Three vesicular glutamate transporters have recently been cloned: BNPI/VGLUT1 (a brain-specific sodium-dependent inorganic phosphate (Pi) transporter), and its homologs DNPI/VGLUT2 (differentiation-associated sodium-dependent Pi transporter) and VGLUT3. We investigated the subcellular distributions of these three vesicular transporters in rat and human retina. VGLUT1 was present in the outer and inner plexiform layers (OPL and IPL), as shown by punctate staining in both human and rat retina. In the OPL, it was colocalized with synaptophysin, consistent with its expression in glutamatergic photoreceptor terminals, and it was present in PKC-α-labeled glutamatergic bipolar cell terminals in the IPL. By contrast, VGLUT2 was present in horizontal cells and ganglion cells in rat and human retina. In human retina, VGLUT2 was also found in some amacrine cells, including GAD-immunopositive amacrine cells. VGLUT3 was present in glycine-releasing amacrine cells in rat retina but was restricted to a few ganglion cells in human retina. The distribution of VGLUT1 in excitatory synaptic terminal was consistent with its involvement in glutamate release at excitatory synapses, whereas the cellular distributions of VGLUT2 and VGLUT3 suggested that these molecules may be involved in functions other than glutamate release, such as glutamate storage for GABA synthesis in non-glutamatergic neurons.

Effect of potassium dichromate on renal brush border membrane enzymes and phosphate transport in rats

Chromium is widely used in industry but exposure to chromium compounds in the workplace can result in nephrotoxicity. Various nephrotoxicants affect the brush border membrane (BBM) lining the epithelial cells of the proximal tubule, but there have been no studies regarding the effect of potassium dichromate (K2Cr2O7), a hexavalent chromium compound, on renal BBM. In the present work, the effect of administering a single intraperitoneal dose (15 mg/kg body weight) of K2Cr2O7 on rat renal BBM enzymes and inorganic phosphate (Pi) transport was studied. The animals were administered normal saline (control) or K2Cr2O7 and sacrificed 1, 2, 4 and 8 days after treatment. K2Cr2O7 induced reversible damage to the rat kidney function as indicated by serum creatinine (Scr) and urea nitrogen levels. The activities of BBM marker enzymes were significantly decreased in isolated BBM vesicles (BBMV) and homogenates of cortex and medulla on 1, 2 and 4 days after administration of K2Cr2O7 with complete recovery to control values after 8 days. The decrease in the activities of the enzymes was mainly due to changes in maximum velocity (V(max)) values, while the Michaelis constant (Km) remained unchanged. The sodium dependent Pi transport across BBMV was reduced by 50% after treatment with K2Cr2O7. Thus, the administration of a single dose of K2Cr2O7 leads to impairment in the functions of renal BBM. These results suggest that the nephrotoxicity of K2Cr2O7 may be mediated, at least in part, by its effect on renal BBM.