Kanako Katai

Hypophosphatemic rickets accompanying McCune-Albright syndrome : evidence that a humoral factor causes hypophosphatemia

Abstract. McCune–Albright syndrome (MAS) is sometimes complicated by hypophosphatemia. However, it remains unclear whether a humoral factor is associated with the cause of hypophosphatemia. We isolated cells with mutations of the Gsα gene from fibrous bone dysplasia tissues of two MAS patients (MAS cells). Severe combined immunodeficiency (SCID) mice were subjected to experiments using from one of these cells patients. Effects of conditioned media (CM) isolated from MAS cells (MAS-CM) on phosphate transport were investigated by using rat renal slices, the renal cell line OK-B, rat intestinal rings and the human intestinal cell line Caco-2. In addition, the effects of MAS-CM on human sodium-dependent phosphate transporter (NPT2) gene promoter activity expression were investigated in the renal cell line OK-B2400 and were compared with the effects of CM isolated from a patient with oncogenic hypophosphatemic osteomalacia (OHO). MAS cells caused significant hypophosphatemia (P < 0.05) and elevated serum alkaline phosphatase activity (P < 0.05) in SCID mice. The MAS-CM significantly inhibited phosphate uptake in everted intestinal rings (P < 0.01), whereas it had no effect on glucose uptake. The MAS-CM had no effect on either phosphate uptake in the kidney or NPT2 gene promoter activity. In contrast, the CM of the OHO patient significantly inhibited phosphate uptake and NPT2 gene promoter activity. These results indicate that the humoral factor derived from fibrous dysplasia cells of the MAS patient is different to that from OHO patients, because the humoral factor from the MAS patient inhibited phosphate transport not in the kidney but in the intestine.

Identification of Three Isoforms for the Na+-dependent Phosphate Cotransporter (NaPi-2) in Rat Kidney

We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2α, NaPi-2β, and NaPi-2γ) from a rat kidney library. NaPi-2α cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2β encodes 327 amino acids which are identical to the N-terminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2γ encodes 268 amino acids which are identical to the C-terminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 α, β, and γ were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2α and NaPi-2γ were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms. A functional analysis demonstrated that NaPi-2γ and -2α markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the full-length transporter.

Effects of Truncation of the COOH-terminal Region of a Na+-independent Neutral and Basic Amino Acid Transporter on Amino Acid Transport in Xenopus Oocytes

To determine the role of a neutral and basic amino acid transporter (NBAT) in amino acid transport, we microinjected several COOH-terminal deletion mutants of NBAT cRNA into Xenopus oocytes and measured transport activity for arginine, leucine, and cystine in the presence and absence of sodium. Wild-type NBAT significantly stimulated the uptake of all three amino acids 10-20-fold compared with controls. On the other hand, no mutant, except a Δ511-685 mutant, stimulated the uptake of these amino acids. The Δ511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Δ511-685 mutant also increased the uptake of leucine. The Δ511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Δ511-685 mutant was inhibited by a 100-fold excess of unlabeled leucine in the presence of sodium. Inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium, and an increase in the inhibition of L-arginine uptake by L-histidine was seen when the extracellular pH was decreased. Furthermore, an inward current in oocytes injected with the Δ511-685 mutant was recorded electrophysiologically when basic amino acids were applied. Homoserine was also taken up, but sodium was necessary for their transport. These properties of the Δ511-685 mutant correspond to those of the y+ amino acid transporter. If NBAT is a component of the b0,+-like amino acid transport system, it is unlikely that a mutant protein (Δ511-685) is able to stimulate an endogenous y+-like transport system. These results suggest that NBAT functions as a activator of the amino acid transport system in Xenopus oocytes.

Effect of dietary phosphate on Na+-dependent phosphate cotransporters function and expression in the rat kidney

BackgroundThree types (typeI, II, andIII) of sodium-dependent phosphate cotransporters have recently been isolated and shown to be expressed in the mammalian kidney. Understanding of the functional roles and regulation of each transporter is still fragmented, however.MethodsWe analyzed the functional roles of each transporter by using antisense oligonucleotides in theXenopus oocytes expression system, and by localization in the proximal tubules of rat kidney using immunohistochemistry.ResultsPhosphate uptake in brush border membranes was increased by about 2 times in rats fed a low-phosphate, as compared with a high-phosphate, diet. Expression of typeI, II, andIII transporter mRNAs was observed in renal poly(A)+RNA, isolated from the rats fed a low-phosphate diet. Phosphate uptake increased about 2.5-fold inXenopus oocytes injected with the poly(A)+RNA, compared with those given RNA from rats fed a high-phosphate diet. Hybrid depletion of the typeII sodium-dependent phosphate transporter (NaPi-2), but not of the typeI (rNaPi-1) or typeIII transporters (PiT-1 and PiT-2), significantly decreased phosphate transport activity in oocytes injected with the poly(A)+RNA from each experimental group rat kidney. In rats fed the lowphosphate diet, NaPi-2 immunoreactivity increased markedly in the brush border membranes of renal proximal tubular cells, whereas rNaPi-1 protein was not changed.ConclusionThis study suggests that the typeII transporter functions mainly as a sodium-dependent phosphate cotransporter, and is regulated by dietary phosphate in the rat kidney.

Molecular and cellular regulation of renal phosphate transporters in X-linked hypophosphatemia

ConclusionRecent investigations of X-linked hypophosphatemia support the concept that the kidney is intrinsically normal in this disorder, and that the characteristic phosphaturia is caused by a humoral factor. The mechanisms involved in the pathophysiology of X-linked hypophosphatemia, Hyp, and oncogenic hypophosphatemic osteomalacia are complex and are the results of mutations in a putative zinc metalloprotease. Inactivation inPHEX gene function initiates a series of events that result in severe perturbations in renal Pi transport and metabolism of vitamin D. There are a number of possible working models that could explain the experimental observations. However, our studies clearly show that a humoral factor (phosphatonin) inhibits the transcription of the type II Na+/Pi cotransporter gene (Fig. 4). Phosphatonin may be a key modulator of phosphate homeostasis.

Role of rBAT gene products in cystinuria.

To investigate whether rBAT gene products function as a crystine transporter component or as a transport activator, we microinjected several C–terminal deletion mutants of rBAT cRNA into Xenopus oocytes, and measured transport activity for arginine, leucine and cystine in the presence and absence of sodium. Wild type rBAT significantly stimulated the uptake of all 3 amino acids 10–20 fold compared to control mutants. On the other hand, no mutant, except a Δ511–685 mutant, stimulated the uptake of these amino acids. However, the Δ511–685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Δ511–685 mutant also increased the uptake of leucine. The Δ511–685 mutant did not stimulate crystine uptake in the presence and absence of sodium. Furthermore, inhibition of L–arginine uptake by L–homoserine was seen only in the presence of sodium. These results suggest that mutant rBAT stimulates the endogenous amino acid transport system y+ in oocytes. Finally, rBAT gene products, as the primary cause of cystinuria, may function as activators of the amino acid transport system in renal brush border membrane.