Dietary inorganic phosphorus regulates the intestinal peptide transporter PepT1.

BACKGROUND: Both organic and inorganic phosphorus (Pi) are present in regularly consumed foods, such as meats, eggs, and dairy products. Pi is often included in foods as an additive (as hidden phosphorus). The intestinal peptide transporter PepT1 mediates protein absorption, which is disturbed in renal insufficiency. Our aim was to determine the effects of dietary Pi content on the peptide transport activity and expression of PepT1. METHODS: The following animal models were used: (1) 7-week-old male Wistar rats; and (2) rats that underwent 3/4 nephrectomy to induce chronic kidney disease (CKD). The rats were fed a normal-protein (20%) diet containing low (0.02%), normal (0.6%), or high (1.2%) Pi levels. They were also fed diets containing varying amounts of protein and either low or normal Pi levels as follows: (1) low Pi/normal protein, (2) low Pi/high (50%) protein, (3) normal Pi/normal protein, and (4) normal Pi/high protein. RESULTS: Intestinal peptide transport activity and PepT1 expression levels were significantly higher in the CKD rats than in sham-operated control ones. Compared with the normal-protein diet, the high-protein diet increased PepT1 expression in the CKD rats. Intestinal dipeptide transport activity and PepT1 protein levels did not increase in the rats fed the low-Pi/high-protein diet. In contrast, intestinal dipeptide transport activity and PepT1 protein expression were markedly increased in the rats fed the normal-Pi/high-protein diet. CONCLUSION: Dietary Pi levels regulate intestinal peptide transport activity through PepT1.

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.

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.

Hypophosphatemia in vitamin D receptor null mice: effect of rescue diet on the developmental changes in renal Na+ -dependent phosphate cotransporters.

We analyzed vitamin D receptor (VDR) (-/-) mice fed either a normal diet or a rescue diet. Weanling VDR (-/-) mice had hypophosphatemia and hyperphosphaturia. Renal Na(+)-dependent inorganic phosphate (Pi) cotransport activity was significantly decreased in weanling VDR (-/-) mice. In VDR (+/+) mice, renal Npt2a/Npt2c/PiT-2 protein levels were significantly increased at 21 and 28 days of age compared with that at 1 day of age. Npt2c and PiT-2 protein levels were maximally expressed at 28 days of age. Npt2a protein levels were significantly decreased in mice at 28 days of age compared with 21 and 60 days of age. In VDR (-/-) mice, Npt2a/Npt2c/PiT-2 protein levels were considerably lower than those in age-matched VDR (+/+) mice at 21 and 28 days of age. The reduced Npt2a/Npt2c/PiT-2 protein recovered completely in VDR-null mice fed the rescue diet. Although Pi transport activity and Npt2b were reduced in the proximal intestine in VDR (-/-) mice, Npt2b protein levels were not reduced in the distal intestine in VDR (-/-) mice. The rescue diet did not affect intestinal Npt2b protein levels in VDR (-/-) mice. Thus, reduced intestinal Pi absorption in VDR (-/-) mice does not seem to be the only factor that causes hypophosphatemia; reduced Npt2a, Npt2c, or PiT-2 protein levels during development might also cause hypophosphatemia and rickets in VDR (-/-) mice. Furthermore, dietary intervention completely normalized the expression of the renal phosphate transporters (Npt2a/Npt2c/PiT-2) in VDR (-/-) mice, suggesting that the lack of VDR activity is not the cause of impaired renal phosphate reabsorption.

Phosphaturic action of fibroblast growth factor 23 in Npt2 null mice.

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

Fibroblast growth factor 23 mediates the phosphaturic actions of cadmium.

Phosphaturia has been documented following cadmium (Cd) exposure in both humans and experimental animals. The fibroblast growth factor 23 (FGF23)/klotho axis serves as an essential phosphate homeostasis pathway in the bone-kidney axis. In the present study, we investigated the effects of Cd on phosphate (Pi) homeostasis in mice. Following Cd injection into WT mice, plasma FGF23 concentration was significantly increased. Urinary Pi excretion levels were significantly higher in Cd-injected WT mice than in control group. Plasma Pi concentration decreased only slightly compared with control group. No change was observed in plasma parathyroid hormone and 1,25-dihydroxy vitamin D(3) in both group of mice. We observed a decrease in phosphate transport activity and also decrease in expression of renal phosphate transporter SLC34A3 [NaPi-IIc/NPT2c], but not SLC34A1 [NaPi-IIa/NPT2a]. Furthermore, we examined the effect of Cd on Npt2c in Npt2a-knockout (KO) mice which expresses Npt2c as a major NaPi co-transporter. Injecting Cd to Npt2aKO mice induced significant increase in plasma FGF23 concentration and urinary Pi excretion levels. Furthermore, we observed a decrease in phosphate transport activity and renal Npt2c expression in Cd-injected Npt2a KO mice. The present study suggests that hypophosphatemia induced by Cd may be closely associated with the FGF23/klotho axis.

Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development.

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

The roles of Na/Pi-II transporters in phosphate metabolism.

The renal type II Na/Pi cotransporters, Na/Pi-IIa and Na/Pi-IIc, are expressed in the brush border membrane (BBM) of the renal proximal tubule cells. Because it has long been thought that Na/Pi-IIa alone can regulate the reabsorption of phosphate in the proximal renal tubules, Na/Pi-IIc has not been paid much attention by the renal research community. Recent studies, however, have identified Na/Pi-IIc mutations as the defective cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). This finding indicates that Na/Pi-IIc has a rather important role in renal Pi reabsorption and bone mineralization, and that it may be a key determinant of plasma Pi concentrations in humans. Studies of Na/Pi-IIc mice indicate that Na/Pi-IIc is necessary for normal calcium homeostasis, but its role in the regulation of Pi metabolism and bone physiology may be different from that in HHRH patients. Of note, Na/Pi-IIc KO mice display abnormal vitamin D regulation without hypophosphatemia or hyperphosphaturia. Thus, Na/Pi-IIc may be involved in regulating renal vitamin D synthesis in the proximal tubular cells. The identification of proteins that interact with Na/Pi-IIc is an important area of future research. The physiologic roles of Na/Pi-IIa and Na/Pi-IIc require future elucidation.

Type IIc sodium-dependent phosphate transporter regulates calcium metabolism.

Primary renal inorganic phosphate (Pi) wasting leads to hypophosphatemia, which is associated with skeletal mineralization defects. In humans, mutations in the gene encoding the type IIc sodium-dependent phosphate transporter lead to hereditary hypophophatemic rickets with hypercalciuria, but whether Pi wasting directly causes the bone disorder is unknown. Here, we generated Npt2c-null mice to define the contribution of Npt2c to Pi homeostasis and to bone abnormalities. Homozygous mutants (Npt2c(-/-)) exhibited hypercalcemia, hypercalciuria, and elevated plasma 1,25-dihydroxyvitamin D(3) levels, but they did not develop hypophosphatemia, hyperphosphaturia, renal calcification, rickets, or osteomalacia. The increased levels of 1,25-dihydroxyvitamin D(3) in Npt2c(-/-) mice compared with age-matched Npt2c(+/+) mice may be the result of reduced catabolism, because we observed significantly reduced expression of renal 25-hydroxyvitamin D-24-hydroxylase mRNA but no change in 1alpha-hydroxylase mRNA levels. Enhanced intestinal absorption of calcium (Ca) contributed to the hypercalcemia and increased urinary Ca excretion. Furthermore, plasma levels of the phosphaturic protein fibroblast growth factor 23 were significantly decreased in Npt2c(-/-) mice. Sodium-dependent Pi co-transport at the renal brush border membrane, however, was not different among Npt2c(+/+), Npt2c(+/-), and Npt2c(-/-) mice. In summary, these data suggest that Npt2c maintains normal Ca metabolism, in part by modulating the vitamin D/fibroblast growth factor 23 axis.

Hereditary hypophosphatemic rickets with hypercalciuria: a study for the phosphate transporter gene type IIc and osteoblastic function.

Two cases of hereditary hypophosphatemic rickets with hypercalciuria (HHRH) were reported in Japanese female siblings. Both of them manifested short stature and bowed legs, and biochemical examination revealed hypophosphatemia, phosphaturia, and hypercalciuria. The serum concentrations of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) were elevated. In the oral phosphate loading test, serum phosphate levels were markedly increased in the HHRH patients, and the elevation was much higher than that in patients affected with X-linked hypophosphatemic rickets (XLH), suggesting the increased gastrointestinal absorption of phosphate in HHRH. Bone histology studies showed increased osteoid surface and width in HHRH, which was compatible with osteomalacia. In the HHRH patients, there were no hypomineralized periosteocytic lesions, which was a hallmark of XLH in bone histology. In one of the HHRH patients, phosphate administration alone almost completely cured the osteomalacia within a year, although pharmacological doses of 1,25(OH)(2)D(3) had little effect. In osteoblasts isolated from a HHRH patient, basal alkaline phosphatase (ALP) activities and osteocalcin syntheses by a physiological concentration of 1,25(OH)(2)D(3) were not stimulated by the increased medium phosphate concentrations from 0.5 to 4 mM. In contrast, these two parameters were stimulated by the increased medium phosphate concentrations both in normal and XLH osteoblasts, although the regulatory patterns of increased osteocalcin syntheses were different from normal to XLH osteoblasts; 2 and 4 mM of phosphate concentrations at least were necessary for normal and XLH osteoblasts, respectively. The gene analysis of phosphate transporter revealed a novel heterozygous mutation (R564C) in the exon of phosphate transporter NPT type IIc. These lines of evidence suggested that the pathogenesis of osteomalacia in HHRH was different from XLH in terms of the utility of phosphate in osteoblasts. These abnormalities were speculated to be associated with the abnormal functions of phosphate transporter gene type IIc, although the exact roles of this phosphate transporter in the human osteoblast are still unknown.

Correlation between hyperphosphatemia and type II Na-Pi cotransporter activity in klotho mice.

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