Fumito Aranami

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.

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.

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.

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.

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.

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)2D) 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)2D3 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)2D3 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.

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.