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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

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.

Role of sodium-dependent Pi transporter/Npt2c on Pi homeostasis in klotho knockout mice different properties between juvenile and adult stages.

SLC34A3/NPT2c/NaPi-2c/Npt2c is a growth-related NaPi cotransporter that mediates the uptake of renal sodium-dependent phosphate (Pi). Mutation of human NPT2c causes hereditary hypophosphatemic rickets with hypercalciuria. Mice with Npt2c knockout, however, exhibit normal Pi metabolism. To investigate the role of Npt2c in Pi homeostasis, we generated α-klotho-/- /Npt2c-/- (KL2cDKO) mice and analyzed Pi homeostasis. α-Klotho-/- (KLKO) mice exhibit hyperphosphatemia and markedly increased kidney Npt2c protein levels. Genetic disruption of Npt2c extended the lifespan of KLKO mice similar to that of α-Klotho-/- /Npt2a-/- mice. Adult KL2cDKO mice had hyperphosphatemia, but analysis of Pi metabolism revealed significantly decreased intestinal and renal Pi (re)absorption compared with KLKO mice. The 1,25-dihydroxy vitamin D3 concentration was not reduced in KL2cDKO mice compared with that in KLKO mice. The KL2cDKO mice had less severe soft tissue and vascular calcification compared with KLKO mice. Juvenile KL2cDKO mice had significantly reduced plasma Pi levels, but Pi metabolism was not changed. In Npt2cKO mice, plasma Pi levels began to decrease around the age of 15 days and significant hypophosphatemia developed within 21 days. The findings of the present study suggest that Npt2c contributes to regulating plasma Pi levels in the juvenile stage and affects Pi retention in the soft and vascular tissues in KLKO mice.

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

BACKGROUND: Injection of parathyroid hormone (PTH) rapidly stimulates renal Pi excretion, in part by downregulating NaPi-IIa (Npt2a/SLC34A1) and NaPi-IIc (Npt2c/SLC34A3) transporters. The mechanisms underlying the effects of PTH on NaPi-IIc are not fully elucidated. METHODS: We analyzed the effect of PTH on inorganic phosphate (Pi) reabsorption in Npt2a-/- mice to eliminate the influence of Npt2a on renal Pi reabsorption. In opossum kidney (OK) cells and Xenopus oocytes, we investigated the effect of NaPi-IIc transporter phosphorylation. Studies of mice with mutations of NaPi-IIc protein in which serine and threonine were replaced with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated NaPi-IIc, were also performed to evaluate the involvement of phosphorylation in the regulation of transport function. RESULTS: The Npt2a-/- experiments showed that PTH administration rapidly inactivated NaPi-IIc function in the apical membrane of proximal tubular cells. Analysis of mutant proteins (S71, S138, T151, S174, T583) at putative protein kinase C sites, revealed that S138 markedly suppressed the function and cellular expression of mouse NaPi-IIc in Xenopus oocytes and OK cells. In addition, 138D had a short half-life compared with wild-type protein. CONCLUSIONS: The present study suggests that acute regulation of NaPi-IIc protein by PTH is involved in the inactivation of Na+-dependent Pi cotransporter activity and that phosphorylation of the transporter is involved in the rapid modification.

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

BACKGROUND: The role of Na+-dependent inorganic phosphate (Pi) transporters in the human kidney is not fully clarified. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by loss-of-function mutations in the IIc Na+-dependent Pi transporter (NPT2c/Npt2c/NaPi-IIc) gene. Another Na+-dependent type II transporter, (NPT2A/Npt2a/NaPi-IIa), is also important for renal Pi reabsorption in humans. In mice, Npt2c deletion does not lead to hypophosphatemia and rickets because Npt2a compensates for the impaired Pi reabsorption. To clarify the differences between mouse and human, we investigated the relation between NaPi-IIa and NaPi-IIc functions in opossum kidney (OK) cells. METHODS: We cloned NaPi-IIc from OK cells and created opossum NaPi-IIc (oNaPi-IIc) antibodies. We used oNaPi-IIc small interference (si)RNA and investigated the role of NaPi-IIc in Pi transport in OK cells. RESULTS: We cloned opossum kidney NaPi-IIc cDNAs encoding 622 amino acid proteins (variant1) and examined their pH- and sodium-dependency. The antibodies reacted specifically with 75-kDa and 150-kDa protein bands, and the siRNA of NaPi-IIc markedly suppressed endogenous oNaPi-IIc in OK cells. Treatment with siRNA significantly suppressed the expression of NaPi-4 (NaPi-IIa) protein and mRNA. oNaPi-IIc siRNA also suppressed Na+/H+ exchanger regulatory factor 1 expression in OK cells. CONCLUSION: These findings suggest that NaPi-IIc is important for the expression of NaPi-IIa (NaPi-4) protein in OK cells. Suppression of Npt2c may downregulate Npt2a function in HHRH patients.

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

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