Involvement of α-klotho in growth hormone (GH) signaling

Journal of Clinical Biochemistry and Nutrition


Effects of EOS789, a novel pan-phosphate transporter inhibitor, on phosphate metabolism : Comparison with a conventional phosphate binder

Inorganic phosphate (Pi) binders are the only pharmacologic treatment approved for hyperphosphatemia. However, Pi binders induce the expression of intestinal Pi transporters and have limited effects on the inhibition of Pi transport. EOS789, a novel pan-Pi transporter inhibitor, reportedly has potent efficacy in treating hyperphosphatemia. We investigated the properties of EOS789 with comparison to a conventional Pi binder. Protein and mRNA expression levels of Pi transporters were measured in intestinal and kidney tissues from male Wistar rats fed diets supplemented with EOS789 or lanthanum carbonate (LC). 32Pi permeability was measured in intestinal tissues from normal rats using a chamber. Increased protein levels of NaPi-2b, an intestinal Pi transporter, and luminal Pi removal were observed in rats treated with LC but not in rats treated with EOS789. EOS789 but not LC suppressed intestinal protein levels of the Pi transporter Pit-1 and sodium/hydrogen exchanger isoform 3. 32Pi flux experiments using small intestine tissues from rats demonstrated that EOS789 may affect transcellular Pi transport in addition to paracellular Pi transport. EOS789 has differing regulatory effects on Pi metabolism compared to LC. The properties of EOS789 may compensate for the limitations of LC therapy. The combined or selective use of EOS789 and conventional Pi binders may allow tighter control of hyperphosphatemia. J. Med. Invest. 70 : 260-270, February, 2023.

The Journal of Medical Investigation : JMI


Tmem174, a regulator of phosphate transporter prevents hyperphosphatemia.

Renal type II sodium-dependent inorganic phosphate (Pi) transporters NaPi2a and NaPi2c cooperate with other organs to strictly regulate the plasma Pi concentration. A high Pi load induces expression and secretion of the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) that enhance urinary Pi excretion and prevent the onset of hyperphosphatemia. How FGF23 secretion from bone is increased by a high Pi load and the setpoint of the plasma Pi concentration, however, are unclear. Here, we investigated the role of Transmembrane protein 174 (Tmem174) and observed evidence for gene co-expression networks in NaPi2a and NaPi2c function. Tmem174 is localized in the renal proximal tubules and interacts with NaPi2a, but not NaPi2c. In Tmem174-knockout (KO) mice, the serum FGF23 concentration was markedly increased but increased Pi excretion and hypophosphatemia were not observed. In addition, Tmem174-KO mice exhibit reduced NaPi2a responsiveness to FGF23 and PTH administration. Furthermore, a dietary Pi load causes marked hyperphosphatemia and abnormal NaPi2a regulation in Tmem174-KO mice. Thus, Tmem174 is thought to be associated with FGF23 induction in bones and the regulation of NaPi2a to prevent an increase in the plasma Pi concentration due to a high Pi load and kidney injury.


Dietary polyphosphate has a greater effect on renal damage and FGF23 secretion than dietary monophosphate

Phosphate (Pi)-containing food additives are used in several forms. Polyphosphate (PPi) salt has more harmful effects than monophosphate (MPi) salt on bone physiology and renal function. This study aimed to analyze the levels of parathyroid hormone PTH and fibroblast growth factor 23 (FGF23) and the expression of renal / intestinal Pi transport-related molecules in mice fed with an MPi or PPi diet. There were no significant differences in plasma Pi concentration and fecal Pi excretion levels between mice fed with the high-MPi and PPi diet. However, more severe tubular dilatation, interstitial fibrosis, and calcification were observed in the kidneys of mice fed with the high PPi diet versus the MPi diet. Furthermore, there was a significant increase in serum FGF23 levels and a decrease in renal phosphate transporter protein expression in mice fed with the PPi diet versus the MPi diet. Furthermore, the high MPi diet was associated with significantly suppressed expression and activity of intestinal alkaline phosphatase protein. In summary, PPi has a more severe effect on renal damage than MPi, as well as induces more FGF23 secretion. Excess FGF23 may be more involved in inflammation, fibrosis, and calcification in the kidney. J. Med. Invest. 69 : 173-179, August, 2022.


Role of the putative PKC phosphorylation sites of the type IIc sodium-dependent phosphate transporter in parathyroid hormone regulation.


Systemic network for dietary inorganic phosphate adaptation among three organs.

Pflügers Archiv : European Journal of Physiology


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


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


Eldecalcitol Causes FGF23 Resistance for Pi Reabsorption and Improves Rachitic Bone Phenotypes in the Male Hyp Mouse.

X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D3 analog, eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.


The sodium phosphate cotransporter family and nicotinamide phosphoribosyltransferase contribute to the daily oscillation of plasma inorganic phosphate concentration.

mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD

system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels.


Effect of Osteocyte-Ablation on Inorganic Phosphate Metabolism: Analysis of Bone-Kidney-Gut Axis.

In response to kidney damage, osteocytes increase the production of several hormones critically involved in mineral metabolism. Recent studies suggest that osteocyte function is altered very early in the course of chronic kidney disease. In the present study, to clarify the role of osteocytes and the canalicular network in mineral homeostasis, we performed four experiments. In Experiment 1, we investigated renal and intestinal Pi handling in osteocyte-less (OCL) model mice [transgenic mice with the dentin matrix protein-1 promoter-driven diphtheria toxin (DT)-receptor that were injected with DT]. In Experiment 2, we administered granulocyte colony-stimulating factor to mice to disrupt the osteocyte canalicular network. In Experiment 3, we investigated the role of osteocytes in dietary Pi signaling. In Experiment 4, we analyzed gene expression level fluctuations in the intestine and liver by comparing mice fed a high Pi diet and OCL mice. Together, the findings of these experiments indicate that osteocyte ablation caused rapid renal Pi excretion (

< 0.01), thus suggesting that increased intestinal Pi absorption stimulates renal Pi excretion in OCL mice. In addition, the ablation of osteocytes and feeding of a high Pi diet affected FGF15/bile acid metabolism and controlled Npt2b expression. In conclusion, OCL mice exhibited increased renal Pi excretion due to enhanced intestinal Pi absorption. We discuss the role of FGF23-Klotho on renal and intestinal Pi metabolism in OCL mice.


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

These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency.

-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 4 mM, 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.


Regulation of renal phosphate handling: inter-organ communication in health and disease.

In this review, we focus on the interconnection of inorganic phosphate (Pi) homeostasis in the network of the bone-kidney, parathyroid-kidney, intestine-kidney, and liver-kidney axes. Such a network of organ communication is important for body Pi homeostasis. Normalization of serum Pi levels is a clinical target in patients with chronic kidney disease (CKD). Particularly, disorders of the fibroblast growth factor 23/klotho system are observed in early CKD. Identification of phosphaturic factors from the intestine and liver may enhance our understanding of body Pi homeostasis and Pi metabolism disturbances in CKD patients.


Relationship between sodium-dependent phosphate transporter (NaPi-IIc) function and cellular vacuole formation in opossum kidney cells.

NaPi-IIc/SLC34A3 is a sodium-dependent inorganic phosphate (Pi) transporter in the renal proximal tubules and its mutations cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). In the present study, we created a specific antibody for opossum SLC34A3, NaPi-IIc (oNaPi-IIc), and analyzed its localization and regulation in opossum kidney cells (a tissue culture model of proximal tubular cells). Immunoreactive oNaPi-IIc protein levels increased during the proliferative phase and decreased during differentiation. Moreover, stimulating cell growth upregulated oNaPi-IIc protein levels, whereas suppressing cell proliferation downregulated oNaPi-IIc protein levels. Immunocytochemistry revealed that endogenous and exogenous oNaPi-IIc proteins localized at the protrusion of the plasma membrane, which is a phosphatidylinositol 4,5-bisphosphate (PIP2) rich-membrane, and at the intracellular vacuolar membrane. Exogenous NaPi-IIc also induced cellular vacuoles and localized in the plasma membrane. The ability to form vacuoles is specific to electroneutral NaPi-IIc, and not electrogenic NaPi-IIa or NaPi-IIb. In addition, mutations of NaPi-IIc (S138F and R468W) in HHRH did not cause cellular PIP2-rich vacuoles. In conclusion, our data anticipate that NaPi-IIc may regulate PIP2 production at the plasma membrane and cellular vesicle formation.

The Journal of Medical Investigation : JMI


Chronic kidney disease (CKD) is an increasing problem worldwide. The number of end-stage renal disease patients requiring treatment by dialysis is estimated to be increasing by 10,000 patients per year in Japan. Furthermore, an estimated 13 million people are living with CKD in Japan. Various complications are associated with CKD, including cardiovascular disease (CVD). More than one-third of CKD patients die from CVD. Thus, prevention of CVD is a primary concern for the treatment of CKD patients. CKD-mineral and bone disorder (CKD-MBD) is a serious complication that typically leads to CVD. Hyperphosphatemia is thought to be a central-risk factor for CKD-MBD. Therefore, managing hyperphosphatemia is crucial to prevent CKD-MBD and CVD. It is difficult to achieve the target serum phosphate level through dietary modifications alone in patients with hyperphosphatemia, because most foods contain phosphate. Thus, phosphate binders such as calcium carbonate are commonly prescribed to CKD patients with hyperphosphatemia, but these have undesirable side effects. Inhibition of intestinal phosphate transport activity has also been investigated as an alternative approach for controlling serum phosphate levels in CKD patients. Nicotinamide, which is the amide of niacin, can inhibit intestinal phosphate transport. Niacin and related compounds have also been developed as drugs for hyperlipidemia conditions, especially hypertriglyceridemia with low high-density lipoprotein. This type of dyslipidemia is frequently observed in CKD patients and is a modifiable risk factor for CVD. Thus, niacin and related compounds may have utility for the treatment of both hyperphosphatemia and dyslipidemia in CKD patients to prevent CVD.

Journal of Nutritional Science and Vitaminology


Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis.

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na(+)-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na(+)-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.

Journal of the American Society of Nephrology


Molecular mechanisms of cadmium-induced fibroblast growth factor 23 upregulation in osteoblast-like cells.

Itai-itai disease is thought to be the result of chronic cadmium (Cd) intoxication. Renal proximal tubules are a major target of Cd toxicity. The whole mechanism of the adverse effects of Cd remains unresolved, especially how renal damage is related to the development of bone lesions. Fibroblast growth factor 23 (FGF23) is a bone-derived phosphaturic factor that regulates vitamin D and inorganic phosphate metabolism in the kidney. To clarify the role of FGF23 on Cd toxicity, we investigated the mechanisms of Cd-induced FGF23 production in the bone. Cd injection into mice significantly increased plasma FGF23 concentrations, but did not change FGF23 mRNA expression in bone. GalNAc-T3 is involved in secreting intact FGF23. To determine potential roles of GalNAc-T3 in Cd-induced FGF23 production, we examined the effect of Cd on GalNAc-T3 mRNA expression in vivo and in vitro. GalNAc-T3 gene expression was significantly increased in the bones of Cd-injected mice. Cd also enhanced the expression of GalNAc-T3 in cultured osteosarcoma UMR106 cells and primary osteocytes. Cd activated aryl hydrocarbon receptors (AhR) and AhR were required for GalNAc-T3 gene expression induced by Cd. In addition, Cd-dependent FGF23 production was completely inhibited by an AhR antagonist. AhR siRNA markedly suppressed the stimulation of transcriptional activity by Cd. Furthermore, Cd induced AhR activation via phosphorylation of Ser-68 by p38 kinase in the nuclear export signal of AhR. Thus, Cd stimulated GalNAc-T3 gene transcription via enhanced AhR binding to the GalNAc-T3 promoter. These findings suggest that the Cd-induced increase in GalNAc-T3 suppresses proteolytic processing of FGF23 and increases serum FGF23 concentrations.


Downregulation of renal type IIa sodium-dependent phosphate cotransporter during lipopolysaccharide-induced acute inflammation.

The type IIa sodium-dependent phosphate cotransporter (Npt2a) plays a critical role in reabsorption of inorganic phosphate (Pi) by renal proximal tubular cells. Pi abnormalities during early stages of sepsis have been reported, but the mechanisms regulating Pi homeostasis during acute inflammation are poorly understood. We examined the regulation of Pi metabolism and renal Npt2a expression during lipopolysaccharide (LPS)-induced inflammation in mice. Dose-response and time-course studies with LPS showed significant increases of plasma Pi and intact parathyroid hormone (iPTH) levels and renal Pi excretion, while renal calcium excretion was significantly decreased. There was no difference in plasma 1,25-dihydroxyvitamin D levels, but the induction of plasma intact fibroblast growth factor 23 levels peaked 3 h after LPS treatment. Western blotting, immunostaining, and quantitative real-time PCR showed that LPS administration significantly decreased Npt2a protein expression in the brush border membrane (BBM) 3 h after injection, but there was no change in renal Npt2a mRNA levels. Moreover, tumor necrosis factor- injection also increased plasma iPTH and decreased renal BBM Npt2a expression. Importantly, we revealed that parathyroidectomized rats had impaired renal Pi excretion and BBM Npt2a expression in response to LPS. These results suggest that the downregulation of Npt2a expression in renal BBM through induction of plasma iPTH levels alter Pi homeostasis during LPS-induced acute inflammation.

American Journal of Physiology, Renal Physiology


Effect of dietary components on renal inorganic phosphate (Pi) excretion induced by a Pi-depleted diet.

Dietary inorganic phosphate (Pi) is the most important factor in the regulation of renal Pi excretion. Recent studies suggest the presence of an enteric-renal signaling axis for dietary Pi as well as the existence of a mechanism by which the intestine detects changes in luminal Pi concentrations. The mechanisms of intestinal Pi sensing, however, are unknown. In the present study, we focused on Pi depletion signals and investigated the effects of dietary components on intestinal Pi sensing. After feeding rats experimental diets for 3 days, we investigated urinary Pi excretion and plasma biochemical parameters. Renal Pi excretion was suppressed in rats fed a low-Pi diet (0.02% Pi). Elimination of dietary calcium (Ca) completely blocked the suppression of Pi excretion, suggesting that the presence of Ca is essential for the Pi depletion signal. Furthermore, a minimum Ca content of more than 0.02% was necessary for the Pi depletion signal. Magnesium, lanthanum, and strontium, which are agonists of calcium sensing receptor, instead of Ca, reduced Pi excretion. Therefore, dietary Ca appears to be important for the Pi depletion-sensing mechanism in the gastrointestinal tract. In addition, the calcium sensing receptor may be involved in the Pi depletion signal.


FGF23の作用と作用機序.

FGF23の作用と作用機序.


Identification and functional analysis of a splice variant of mouse sodium-dependent phosphate transporter Npt2c.

Mutations in the SLC34A3 gene, a sodium-dependent inorganic phosphate (Pi) cotransporter, also referred to as NaPi IIc, causes hereditary hypophosphatemic rickets with hypercalciuria (HHRH), an autosomal recessive disorder. In human and rodent, NaPi IIc is mainly localized in the apical membrane of renal proximal tubular cells. In this study, we identified mouse NaPi IIc variant (Npt2c-v1) that lacks the part of the exon 3 sequence that includes the assumed translation initiation site of Npt2c. Microinjection of mouse Npt2c-v1 cRNA into Xenopus oocytes demonstrated that Npt2c-v1 showed sodium-dependent Pi cotransport activity. The characterization of pH dependency showed activation at extracellular alkaline-pH. Furthermore, Npt2c-v1 mediated Pi transport activity was significantly higher at any pH value than those of Npt2c. In an in vitro study, the localization of the Npt2c-v1 protein was detected in the apical membrane in opossum kidney cells. The expression of Npt2c-v1 mRNA was detected in the heart, spleen, testis, uterus, placenta, femur, cerebellum, hippocampus, diencephalon and brain stem of mouse. Using mouse bone primary cultured cells, we showed the expression of Npt2c-v1 mRNA. In addition, the Npt2c protein was detected in the spermatozoa head. Thus, Npt2c-v1 was expressed in extra-renal tissues such as epididymal spermatozoa and may function as a sodium-dependent phosphate transporter.


Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria.

Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.

American Journal of Physiology, Cell Physiology


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.


Thyroid hormones regulate phosphate homoeostasis through transcriptional control of the renal type IIa sodium-dependent phosphate co-transporter (Npt2a) gene.

The type IIa renal sodium-dependent phosphate (Na/Pi) co-transporter Npt2a is implicated in the control of serum phosphate levels. It has been demonstrated previously that renal Npt2a protein and its mRNA expression are both up-regulated by the thyroid hormone T3 (3,3',5-tri-iodothyronine) in rats. However, it has never been established whether the induction was mediated by a direct effect of thyroid hormones on the Npt2a promoter. To address the role of Npt2a in T3-dependent regulation of phosphate homoeostasis and to identify the molecular mechanisms by which thyroid hormones modulate Npt2a gene expression, mice were rendered pharmacologically hypo- and hyper-thyroid. Hypothyroid mice showed low levels of serum phosphate and a marked decrease in renal Npt2a protein abundance. Importantly, we also showed that Npt2a-deficient mice had impaired serum phosphate responsiveness to T3 compared with wild-type mice. Promoter analysis with a luciferase assay revealed that the transcriptional activity of a reporter gene containing the Npt2a promoter and intron 1 was dependent upon TRs (thyroid hormone receptors) and specifically increased by T3 in renal cells. Deletion analysis and EMSAs (electrophoretic mobility-shift assays) determined that there were unique TREs (thyroid-hormone-responsive elements) within intron 1 of the Npt2a gene. These results suggest that Npt2a plays a critical role as a T3-target gene, to control phosphate homoeostasis, and that T3 transcriptionally activates the Npt2a gene via TRs in a renal cell-specific manner.


L-DOPA inhibits nitric oxide-dependent vasorelaxation via production of reactive ozygen species in rat aorta

To clarify the underlying mechanisms of L-DOPA induced vasoconstriction in rat aorta. Methods: The effect of L-DOPA on phenylephrine-induced contractile force of blood vessels was examined in vitro using rat aortic ring preparations by isometric tension experiment. Involvement of nitric oxide (NO) in the effect of L-DOPA on vascular smooth muscle was studied by using N(omega)-Nitro-L-arginine (L-NNA), Sodium nitroprusside (SNP) in endothelium-intact and endothelium-denuded aortic rings. L-DOPA potentiated alpha-adrenergic receptor- and depolarization-induced vascular contraction and inhibited acetylcholine-induced vasorelaxation. This effect was diminished by pretreatment of the aortic rings with L-NNA, an inhibitor of NO synthesis, or by removing the endothelium from the ring preparations. In endothelium-denuded rings, L-DOPA inhibited exogenous NO-dependent but not cGMP-mediated vasorelaxation. Increases in cGMP levels in response to an NO donor were attenuated by L-DOPA in cultured rat aortic smooth muscle cells. L-DOPA could not contract rings (without endothelium) pretreated with 3-(5'-hydroxymethyl- 2'-furyl)-1-benzyl indazole (YC-1), an activator of guanylyl cyclase, but SOD (150 U/ml) pretreatment of rings with endothelium inhibited contraction by L-DOPA. These results suggest that L-DOPA inhibits nitric-dependent vasorelaxation on vascular smooth muscle cells via production of reactive oxygen species.

The Journal of Medical Investigation : JMI


Caveolin-1 in extracellular matrix vesicles secreted from osteoblasts.

Caveolin-1 is an essential and signature protein of caveolae, which are small invaginations of the plasma membrane enriched in cholesterol and sphingolipids. Although high levels of expression of caveolin-1 have been demonstrated in osteoblasts as well as endothelial cells, fibroblasts, and muscular cells, the role of caveolin-1 in osteoblasts has not been clarified. Here, we show that caveolin-1 is secreted from osteoblasts in the form of matrix vesicles; extracellular vesicles released from the plasma membrane of osteoblasts. In this study, caveolae and matrix vesicles were similarly enriched in cholesterol and sphingomyelin in fractions isolated from mineralizing MC3T3-E1 cells. Interestingly, in the MC3T3-E1 cells caveolin-1 was enriched in the matrix vesicle fraction as well as the caveolar membrane fraction, and the amount of caveolin-1 in the matrix vesicle fraction increased as differentiation progressed. Localization of caveolin-1 in matrix vesicles was also confirmed in murine tibia. Furthermore, overexpression of caveolin-1 enhanced matrix calcification in MC3T3-E1 cells, whereas knockdown of caveolin-1 diminished it. These results suggest that secreted caveolin-1 as a component of matrix vesicles may play an important role in osteoblast calcification.


Characterization of the molecular mechanisms involved in the increased insulin secretion in rats with acute liver failure.

To investigate the mechanism of hyperinsulinaemia in rats with acute liver failure induced by the administration of d-galactosamine (GalN), we focused on the role of polyprimidine tract-binding protein (PTB) in islet insulin synthesis. Recent reports indicate that PTB binds and stabilizes mRNA encoding insulin and insulin secretory granule proteins, including islet cell autoantigen 512 (ICA512), prohormone convertase 1/3 (PC1/3), and PC2. In the present study, glucose-stimulated insulin secretion was significantly increased in GalN-treated rats compared to controls. Levels of mRNA encoding insulin 1, ICA512, and PC1/3 were increased in the pancreatic islets of GalN-treated rats. This mRNA level elevation was not prevented by pretreatment with actinomycin D. When the PTB-binding site in insulin 1 mRNA was incubated with the islet cytosolic fraction, the RNA-protein complex level was increased in the cytosolic fraction obtained from GalN-treated rats compared to the level in control rats. The cytosolic fraction obtained from pancreatic islets obtained from GalN-treated rats had an increased PTB level compared to the levels obtained from the pancreatic islets of control rats. These findings suggest that, in rats with acute liver failure, cytosolic PTB binds and stabilizes mRNA encoding insulin and its secretory granule proteins.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease


Parathyroid hormone-dependent endocytosis of renal type IIc Na-Pi cotransporter.

Hereditary hypophosphatemic rickets with hypercalciuria results from mutations of the renal type IIc Na-P(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(i) cotransporter by parathyroid hormone (PTH). Type IIc Na-P(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(i) cotransporter in renal proximal tubules.

American Journal of Physiology, Renal Physiology


リンとビタミンDの相互作用

リンとビタミンDの相互作用


Molecular mechanism in biological transport in the kidney: Sodium-dependent glucose, phosphate, amino acid transporters.

Nihon Rinsho. Japanese Journal of Clinical Medicine


Journal of Clinical and Experimental Medicine


病態栄養専門師のための病態栄養ガイドブック

病態栄養専門師のための病態栄養ガイドブック


リン酸トランスポーターをめぐる最近の話題

リン酸トランスポーターをめぐる最近の話題


ナトリウム・グルコース，ナトリウム・リン，ナトリウム・アミノ酸共役トランスポーター


ナトリウム依存症リントランスポータ-の調節機構

ナトリウム依存症リントランスポータ-の調節機構


生体内におけるリンの役割 --- 光と影

生体内におけるリンの役割 --- 光と影


Role of the vitamin D receptor in FGF23 action on phosphate metabolism

FGF23 (fibroblast growth factor 23) is a novel phosphaturic factor that influences vitamin D metabolism and renal re-absorption of Pi. The goal of the present study was to characterize the role of the VDR (vitamin D receptor) in FGF23 action using VDR(-/-) (VDR null) mice. Injection of FGF23M (naked DNA encoding the R179Q mutant of human FGF23) into VDR(-/-) and wildtype VDR(+/+) mice resulted in an elevation in serum FGF23 levels, but had no effect on serum calcium or parathyroid hormone levels. In contrast, injection of FGF23M resulted in significant decreases in serum Pi levels, renal Na/Pi co-transport activity and type II transporter protein levels in both groups when compared with controls injected with mock vector or with FGFWT (naked DNA encoding wild-type human FGF23). Injection of FGF23M resulted in a decrease in 25-hydroxyvitamin D 1a-hydroxylase mRNA levels in VDR(-/-) and VDR(+/+) mice, while 25-hydroxyvitamin D 24-hydroxylase mRNA levels were significantly increased in FGF23M-treated animals compared with mock vector control- or FGF23WT-treated animals. The degree of 24-hydroxylase induction by FGF23M was dependent on the VDR, since FGF23M significantly reduced the levels of serum 1,25(OH)2D3 [1,25-hydroxyvitamin D3] in VDR(+/+) mice, but not in VDR(-/-) mice. We conclude that FGF23 reduces renal Pi transport and 25-hydroxyvitamin D 1a-hydroxylase levels by a mechanism that is independent of the VDR. In contrast, the induction of 25-hydroxyvitamin D 24-hydroxylase and the reduction of serum 1,25(OH)2D3 levels induced by FGF23 are dependent on the VDR.


Inhibition of Intestinal Sodium-dependent Inorganic phosphate Transport by Fibroblast Growth Factor 23

The mechanisms by which fibroblast growth factor 23 (FGF23) alters inorganic phosphate (Pi) homeostasis is not entirely clear. In the present study, we examined the effect of FGF23 on intestinal sodium-dependent Pi transport in mice. Injection of FGF23(R179Q) markedly reduced serum Pi and 1,25(OH)2D3 levels in normal mice. Those animals show the reduction of intestinal sodium-dependent Pi transport activity and the amount of type IIb sodium-dependent Pi cotransporter (type IIb NaPi) protein in the brush border membrane vesicles. In vitamin D receptor null mice (VDR-/-), FGF23(R179Q) had no effect on intestinal sodium-dependent Pi transport activity and type IIb NaPi protein levels. The present study suggests that FGF23(R179Q) reduces intestinal sodium-dependent Pi transport activity and type IIb NaPi protein levels by a mechanism that is dependent on VDR.


Inhibitory effect of JTP-59557, a new triazole derivative, on intestinal phosphate transport in vitro and in vivo

JTP-59557 [(-)-4-(2-tert-Butyl-4,5-dichlorophenyl)-5-(5-trifluoromethylpyridin-2-ylsulfanyl)-4H-[1,2,4]triazol-3-ol] showed an inhibitory effect on Na(+)-dependent inorganic phosphate (Pi) transport in intestinal brush border membrane vesicles with an IC(50) value of 0.40 microM in rabbit and with an IC(50) of 0.19 microM in rat, without affecting Na(+)-independent Pi and Na(+)-dependent d-glucose transport activities. In Chinese hamster ovary (CHO) cells expressing human type IIb Na/Pi cotransporter (type IIb), JTP-59557 decreased human type IIb-mediated Pi uptake with an IC(50) of 0.12 microM. In rabbit intestinal brush border membrane vesicles, JTP-59557 behaved as a noncompetitive inhibitor with respect to Pi. In an in vivo study, single administration of JTP-59557 significantly decreased the intestinal Pi absorption rate, when either Pi solution or laboratory chow was given to rats. In this report, we show that JTP-59557 is a potent, selective, stereospecific, noncompetitive inhibitor of intestinal Na/Pi cotransporters including type IIb, and it may represent a new class of intestinal Pi absorption inhibitor.


Vitamin D and phosphate regulate fibroblast growth factor-23 in K-562 cells

Fibroblast growth factor-23 (FGF-23) 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 D3 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.

American Journal of Physiology, Endocrinology and Metabolism


Characterization of inorganic phosphate transport in osteoclast-like cells

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

American Journal of Physiology, Cell Physiology


Posttranscriptional regulation of albumin gene expression by branched-chain amino acids in rats with acute liver injury

We previously demonstrated that the integration of albumin mRNA into functional polysomes was regulated by the supply of branched-chain amino acids (BCAA) in the liver of galactosamine-treated rats. To study the mechanism of this regulation, we investigated interaction between rat liver proteins and albumin transcripts. When albumin transcript was incubated with ribosome salt wash (RSW) extracts prepared from liver, a specific RNA-protein complex (p65) formed. Competition experiments showed that a pyrimidine-rich sequence in the coding region of albumin mRNA was required for the formation of p65. The level of p65 was increased in the RSW extracts prepared from liver of galactosamine-treated rats infused with a standard amino acid formula, compared with a BCAA-enriched amino acid formula. The protein in p65 appears to be polypyrimidine tract-binding protein (PTB), because the formation of p65 was reduced in the RSW extracts pre-incubated with anti-PTB antibody. In cell-free translation analysis, immunodepletion of PTB from rabbit reticulocyte lysate caused an increase in albumin translation. These results suggest that binding of PTB to albumin mRNA suppresses its translation. A supply of BCAA may interfere with this binding and improve the translation efficiency of albumin mRNA in injured liver.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease


Internalization of renal type IIc Na/Pi cotransporter in response to a high phosphate diet

Dietary phosphate levels regulate the renal brush-border type IIa Na-Pi cotransporter. Another Na-Pi cotransporter, type IIc, colocalizes with type IIa Na-Pi cotransporter in the apical membrane of renal proximal tubular cells. The goal of the present study was to determine whether dietary phosphate levels also rapidly regulate the type IIc Na-Pi cotransporter. Type IIa and type IIc transporter protein levels were increased in rats chronically fed a low-Pi diet compared with those fed a normal-Pi diet. Two hours after beginning a high-Pi diet, type IIa transporter levels were decreased, whereas type IIc protein levels remained unchanged. Western blot analysis of brush-border membrane prepared 4 h after beginning a high-Pi diet showed a significant reduction in type IIc transporter protein levels, and immunohistochemistry showed translocation of the type IIc-immunoreactive signal from the entire brush border to subapical membrane. Membrane fractionation studies revealed a decrease in apical membrane type IIc protein without changes in total cortical type IIc protein, which is compatible with redistribution of type IIc protein from the apical membrane to the dense membrane fraction. The microtubule-disrupting reagent colchicine prevented this reduction in apical type IIc transporter at the apical membrane but had no effect on type IIa transporter levels. These data suggest that the type IIc Na-Pi cotransporter level is rapidly regulated by rapid adaptation to dietary Pi in a microtubule-dependent manner. Furthermore, the mechanisms of the internalization of the type IIc transporter are distinct from those of the type IIa transporter.

American Journal of Physiology, Renal Physiology


Physiological Regulation of Renal Sodium-Dependent Phosphate Cotransporters

The physiological regulation of renal Pi reabsorption is mediated by renal type II Na/Pi cotransporters (type IIa and type IIc). The type IIa transporter is regulated, among other factors, by dietary Pi intake and parathyroid hormone (PTH). The PTH-induced inhibition of Pi reabsorption is mediated by endocytosis of the type IIa transporter from the brush-border membrane and subsequent lysosomal degradation. Type IIa is part of the heteromeric protein complexes organized by PDZ proteins. Furthermore, during Pi depletion the type IIc Na/Pi cotransporter is induced in the apical membrane of proximal tubular cells. The type IIc transporter is also regulated by PTH via internalization, but by a vesicular transport pathway distinct from that used by the type IIc transporter. Studying the mechanisms of type IIa and type IIc transporters has increased the understanding of the control of proximal tubular Pi handling and thus of overall Pi homeostasis.


Intestinal Na-Pi cotransporter adaptation to dietary Pi content in vitamin D receptor null mice.

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


Interaction of a farnesylated protein with rwnal type a Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate.

Treatment with PTH (parathyroid hormone) or a high-P(i) diet causes internalization of the type IIa sodium-dependent phosphate (Na/P(i) IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval. To elucidate the mechanism of internalization of Na/P(i) IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/P(i) IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/P(i) IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/P(i) IIa co-transporter.


Cloning and characterization of three PHEX homologues in Drosophila.

Inactivating mutations and/or deletions of PHEX ( Phosphate-regulating gene with Homologies to Endopeptidase on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans. In the present study, three Drosophila PHEX homologues (dPHEX-1, -2, -3) were isolated by the screening of a Drosophila cDNA library and expressed sequence tag (EST) database. The structural region involving motif II: (456)WMXXXTKXXAXXK(468) (numbered according to human PHEX), motif VI: (602)WW(603), and motif VIII: (746)CXLW(749) was conserved in the dPHEX family. Zinc-coordinating motifs (HEFTH and GENIADNGG) were also conserved in the dPHEX family. All three dPHEX genes were expressed during all stages of Drosophila development. The expression of dPHEX-1 was suppressed by dietary phosphate deprivation, but the expression of dPHEX-2 and that of dPHEX-3 were not affected. In-situ hybridization showed a ubiquitous distribution of dPHEX-1 and dPHEX-2, while dPHEX-3 was highly expressed in the larval brain. In an analysis of subcellular localization, dPHEX-1 was localized to intracellular organelles and dPHEX-3 was localized predominately in the plasma membrane of Drosophila embryonic S2 cells. Homozygosity of a dPHEX-1 mutation, a transposon insertion in the dPHEX-1 promoter region, was completely lethal at an early stage of embryonic development. The present study indicates that three homologues are likely involved in the phosphate homeostasis of Drosophila.


Effect of hydrolysis-resistant FGF23-R179Q on dietary phosphate regulation of the renal type-II Na/Pi transporter.

Fibroblast growth factor 23 (FGF23), a phosphaturic factor, is involved in the regulation of renal inorganic phosphate (Pi) reabsorption. Proteolysis-resistant FGF23 mutants expressed in rodents reduce Pi uptake in both intestine and kidney, independent of parathyroid hormone action. In the present study, we investigated whether FGF23 affects dietary regulation of Na(+)-dependent Pi (Na/Pi) cotransport in the rat kidney using wild-type FGF23 and an R179Q mutant, which disrupts a consensus proteolytic cleavage motif. Rats injected with naked human FGF23 DNA (wild-type or R179Q mutant) expressed the human FGF23 transcript in the liver. In those animals, plasma calcium and parathyroid hormone levels were not affected by FGF23 (either wild-type or R179Q mutant). FGF23-R179Q did, however, significantly decrease plasma Pi and renal Na/Pi cotransport activity and also the level of type-IIc Na/Pi cotransporter protein in brush-border membrane vesicles (BBMVs) from normal rat kidney. Western blot and immunohistochemical analyses in rats fed a low-Pi diet showed the levels of types-IIa and -IIc Na/Pi cotransporters to be markedly increased. After injection of FGF23-R179Q DNA into the rats fed a low-Pi diet, the levels of the types-IIa and -IIc transporter proteins were decreased. The FGF23 mutant thus blunts the signalling of Pi deprivation to the renal type-II Na/Pi cotransporter, suggesting that the FGF23 pathway could be involved in the signalling of dietary Pi.

Pflügers Archiv : European Journal of Physiology


肝障害モデルラットへのBCAA高含有製剤投与による低アルブミン血症改善機構の検討


Growth-related renal Type II Na/Pi cotransporter.


Human L-type amino acid transporter 1(LAT1):/characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.

Biochimica et Biophysica Acta (BBA) - Biomembranes


) Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation


Direct demonstration of humorally mediated inhibition of the transcription of phosphate transporter in XLH patients


Regulation of intestinal Na+-dependent phosphate co-transporters by a low-phosphate diet and 1,25-dihydroxyvitamin D3.


Acute regulation by dietary phosphate of the sodium-dependent phosphate transporter (NaP(i)-2) in rat kidney.


リン代謝調節機構―多臓器連関―

リン代謝調節機構―多臓器連関―


The physiological function of vitamin D which maintains phosphate homeostasis

生体内リン恒常性を維持するビタミンD作用

Inorganic phosphate (Pi) plays essential roles in many biological processes. Blood Pi level should be maintained at a constant range because hypophosphatemia causes rickets/osteomalacia, whereas hyperphosphatemia causes cardiovascular events in chronic kidney disease (CKD) or hemodialysis patients. As a phosphaturic hormone, parathyroid hormone (PTH) or fibroblast growth factor (FGF) 23 strongly suppresses Pi reabsorption. 1,25-Dihydroxyvitamin D[1,25(OH) 2D] up-regulates Pi absorption in intestines, and regulates urinary Pi excretion through PTH or FGF23.
 Activated FGF23 signal leads urinary Pi excretion, and hypophosphatemic rickets (FGF23-related hypophosphatemic rickets). Rickets characterized as vitamin D deficiency also associates with impaired renal Pi reabsorption in juvenile stage in addition to low Pi absorption in intestines. Now, burosumab (FGF23 neutralizing antibody) has a beneficent effect on FGF23-related hypophosphatemic rickets/osteomalacia. The antibody increases renal Pi reabsorption and 1,25(OH) 2D production. On the other hand, treatment with vitamin D alone in an animal model of X-linked hypophosphatemic rickets can potentially increase renal Pi reabsorption, despite of excess FGF23, and improve hypophosphatemia and bone phenotypes.
 CKD patients often accompany with mineral bone disorder (CKD-MBD) such as hyperparathyroidism, cardiovascular calcification, and osteodystrophy. Prevention and treatment for hyperphosphatemia or hypovitaminosis D would improve their QOL or mortality.
 Thus, renal Pi reabsorption is important for bone and mineral homeostasis. We focus on the metabolism of Pi and vitamin D to understand the molecular mechanism for the onset and prevention of diseases associated with Pi and vitamin D.

無機リン酸(リン)は生体内で多くの生化学反応に必要であるため，血中リン濃度は一定範囲内に維持されている．低リン血症はくる病/骨軟化症の原因となり，高リン血症は慢性腎臓病(CKD)や透析患者において心血管疾患発症のリスクファクターとなる．副甲状腺ホルモン(PTH)や線維芽細胞増殖因子23(FGF23)は，腎臓でのリン再吸収を抑制することでリン利尿ホルモンとして機能している．活性型ビタミンD[1,25(OH) 2D]は小腸でのリン吸収を促進することに加え，PTHやFGF23の産生調節も行い，生体内リン恒常性を保っている．
 FGF23シグナル活性化は，尿中リン排泄を亢進させ，低リン血症性くる病を引き起こす(FGF23関連低リン血症性くる病)．また，ビタミンD依存性くる病では，小腸でのリン吸収抑制に加えて，成長期における尿中リン排泄の亢進が低リン血症の原因となる．近年，FGF23中和抗体(Burosumab)が開発され，腎臓リン再吸収と1,25(OH) 2D産生を改善することでくる病患者に有益な効果を示し，欧米及び日本でFGF23関連低リン血症性くる病・骨軟化症の治療薬として承認されている．一方，X連鎖性低リン血症性くる病モデル動物を用いた実験では，ビタミンD製剤単独投与でも低リン血症性くる病の症状を改善できることが示された．この作用の特徴は，FGF23が高値を示すにも関わらずリン再吸収の改善がみとめられることである．
 CKD患者では，しばしば副甲状腺機能亢進や心血管疾患，骨異栄養症など骨ミネラル代謝異常(CKD-MBD: 慢性腎臓病に伴う骨ミネラル代謝異常)が出現する．CKDにおける高リン血症および1,25(OH) 2D低下を予防・治療することで患者のQOLや予後を改善することが報告されている．
 このように腎臓でのリン再吸収調節は骨ミネラル代謝に極めて重要である．我々は，リンとビタミンD代謝に注目し，疾患の発症および予防を分子レベルで理解することに努めている．


唾液腺のリン代謝における役割

唾液腺のリン代謝における役割

バイオサイエンスとインダストリー


Evidence of an intestinal phosphate transporter alternative to type IIb sodium-dependent phosphate transporter in rats with chronic kidney disease.

Phosphate is absorbed in the small intestine via passive flow and active transport.NaPi-IIb, a type II sodium-dependent phosphate transporter, is considered to mediate active phosphate transport in rodents. To study the regulation of intestinal phosphate transport in chronic kidney disease (CKD), we analyzed the expression levels of NaPi-IIb, pituitary-specific transcription factor 1 (PiT-1) and PiT-2 and the kinetics of intestinal phosphate transport using two CKD models. CKD was induced in rats via adenine orThy1 antibody injection. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the messenger RNA (mRNA) expression of NaPi-IIb, PiT-1 and PiT-2 were analyzed. The protein expression level of NaPi-IIb was measured by mass spectrometry (e.g. liquid chromatography tandem mass spectrometry). In normal rats, phosphate uptake into BBMV consisted of a single saturable component and its Michaelis constant (Km) was comparable to that of NaPi-IIb. The maximum velocity (Vmax) correlated with mRNA and protein levels of NaPi-IIb. In the CKD models, intestinal phosphate uptake consisted of two saturable components. The Vmax of the higher-affinity transport, which is thought to be responsible for NaPi-IIb, significantly decreased and the decrease correlated with reduced NaPi-IIb expression. The Km of the lower-affinity transport was comparable to that of PiT-1 and -2. PiT-1 mRNA expression was much higher than that of PiT-2, suggesting that PiT-1 was mostly responsible for phosphate transport. This study suggests that the contribution of NaPi-IIb to intestinal phosphate absorption dramatically decreases in rats with CKD and that a low-affinity alternative to NaPi-IIb, in particular PiT-1, is upregulated in a compensatory manner in CKD.


Small intestine plays an important role in the sensing of various nutrients. There is information that would imply the existence of a dietary phosphate sensing mechanism within the intestine. Recent studies suggest that intestinal factors may involve in the alteration of renal phosphate transport. The elucidation of the phosphate sensing mechanism is expected to provide molecular basis for the prevention of the hyperphosphatemia in chronic kidney disease patients.


The physiological roles of intestinal and renal phosphate transporters : an update

リン酸トランスポーターをめぐる最近の話題.


リン酸トランスポーターと疾患

リン酸トランスポーターと疾患

Bio Clinica 腎臓トランスポーター異常による疾患

Bio Clinica 腎臓トランスポーター異常による疾患


ペプチドトランスポーターの機能と生理作用

ペプチドトランスポーターの機能と生理作用

栄養・食品機能とトランスポーター

栄養・食品機能とトランスポーター


Transgenic overexpression of the extra-large Gs variant XLs enhances Gs-mediated responses in the mouse renal proximal tubule in vivo.

XLs, a variant of the stimulatory G protein -subunit (Gs), can mediate receptor-activated cAMP generation and, thus, mimic the actions of Gs in transfected cells. However, it remains unknown whether XLs can act in a similar manner in vivo. We have now generated mice with ectopic transgenic expression of rat XLs in the renal proximal tubule (rptXLs mice), where Gs mediates most actions of PTH. Western blots and quantitative RT-PCR showed that, while Gs and type-1 PTH receptor levels were unaltered, protein kinase A activity and 25-hydroxyvitamin D 1--hydroxylase (Cyp27b1) mRNA levels were significantly higher in renal proximal tubules of rptXLs mice than wild-type littermates. Immunohistochemical analysis of kidney sections showed that the sodium-phosphate cotransporter type 2a was modestly reduced in brush border membranes of male rptXLs mice compared to gender-matched controls. Serum calcium, phosphorus, and 1,25 dihydroxyvitamin D were within the normal range, but serum PTH was 30% lower in rptXLs mice than in controls (152 ± 16 vs. 222 ± 41 pg/ml; P < 0.05). After crossing the rptXLs mice to mice with ablation of maternal Gnas exon 1 (E1(m-/+)), male offspring carrying both the XLs transgene and maternal Gnas exon 1 ablation (rptXLs/E1(m-/+)) were significantly less hypocalcemic than gender-matched E1(m-/+) littermates. Both E1(m-/+) and rptXLs/E1(m-/+) offspring had higher serum PTH than wild-type littermates, but the degree of secondary hyperparathyroidism tended to be lower in rptXLs/E1(m-/+) mice. Hence, transgenic XLs expression in the proximal tubule enhanced Gs-mediated responses, indicating that XLs can mimic Gs in vivo.


腎臓と骨代謝関連ホルモン

腎臓と骨代謝関連ホルモン


Posttranscriptional Regulation of Albumin Gene Expression in Rats with Acute Liver Injury:Effect of Branched-Chain Amino Acids on Its Regulation

肝障害モデルラットのアルブミン遺伝子転写後調節機構と分岐鎖アミノ酸製剤

Reports of the Research Committee of Essential Amino Acids (Japan)