In vertebrates, the level of inorganic phosphate (Pi) is tightly balanced both inside the cell and in the whole organism. A number of different Na+ -dependent Pi cotransport systems involved in Pi homeostasis have been identified and characterized at the molecular level in the past 7 years. The transporters constitute three different protein families denoted NaPi-I, NaPi-II and NaPi-III. NaPi-I from the rabbit was the first member of this family to be cloned. However, it still resists efforts to unravel its physiological role and a clear-cut functional identity: is it a Cl channel, a Na+ /Pi cotransporter, a regulator, or does it perform a combination of these functions? These questions provide a slight taste of the problems associated with orphan genes derived from sequencing projects. The members of the NaPi-II protein family are crucially involved in tightly controlled renal Pi excretion and, as recently discovered, intestinal Pi absorption. The expression and the cellular distribution of NaPi-II in the proximal tubular epithelium are affected by hormonal and metabolic factors known to influence extracellular fluid Pi homeostasis. Recently, the expression of NaPi-II has been demonstrated in osteoclasts and brain; however, the physiological roles of NaPi-II in these tissues remain to be established. The members of the third protein family, NaPi-III, have been identified on the basis of their function as viral receptors. The widespread expression of this family suggests that NaPi-III is involved in supplying the basic cellular metabolic needs for Pi.

Inorganic phosphate (Pi) is involved in a large number of biochemical processes and contributes to the structure of DNA, RNA, proteins and phospholipids. Because of its limited solubility in the presence of divalent cations, the concentration of free Pi is balanced in the millimolar range in both intra-and extracellular fluids. Bacteria, yeast, plants and vertebrates have developed their own strategies to control Pi homeostasis, with different membrane transporters being involved. Since Pi translocation does not primarily contribute to acute cellular responses to stress (changes in pH or osmolarity, hormonal stimuli, alterations in membrane potential), there is a limit, at least theoretically, to the number of differently regulated Pi transport systems.

Several organs play a crucial role in vertebrate Pi homeostasis. The daily need for Pi is covered by intestinal absorption from the diet in the proximal intestine. Bones represent the major storage compartment of Pi, and Pi is needed in the intracellular space for metabolic and structural purposes. The extracellular concentration of Pi is controlled via tightly regulated renal excretion. Hormones and metabolic factors influencing Pi homeostasis operate primarily at this step (Berndt and Knox, 1992). For this reason, the molecular components involved in renal Pi transport, especially the Na+ /Pi cotransport system, have attracted scientific interest. In a series of elegant studies, the physiological, regulatory and functional aspects of this membrane transport system have been unravelled (Murer et al. 1991). However, the isolation of the first cDNA encoding a Na+ /Pi cotransporter was achieved as late as 1991 by expression cloning using the Xenopus laevis oocyte system (Werner et al. 1991). It soon became clear that the protein, then termed NaPi-1, did not match the physiological and functional characteristics reported for renal Na+ /Pi cotransport (Biber et al. 1993). The search was reinitiated, and 2 years later the first two members of a protein family denoted NaPi-II were reported (Magagnin et al. 1993). These proteins expressed in oocytes mimicked the hallmarks of mammalian renal Pi reabsorption, i.e. pH-dependency and regulation by parathyroid hormone and by Pi availability (Murer and Biber, 1996: Biber et al. 1996). The protein family NaPi-III includes viral receptors which were shown to mediate Na+ -dependent Pi transport after expression in Xenopus laevis oocytes (Kavanaugh et al. 1994). This review summarizes the overwhelming impact of the molecular approach on our understanding of Pi homeostasis and tries to integrate the details of Pi handling at the cellular and whole-body levels.

The first success in the search for the regulated renal Na+ /Pi cotransport system was a cDNA clone derived from rabbit kidney cortex (Werner et al. 1991). It encoded a membrane protein of 465 amino acid residues which stimulated Na+ -dependent Pi uptake after expression in Xenopus laevis oocytes. Experimental inconsistencies (the cDNA did not match the mRNA size-fraction that stimulated Na+ /Pi uptake in Xenopus oocytes) indicated the presence of an additional transport protein. Furthermore, the transport activity expressed in oocytes could not be correlated with the functional characteristics of renal Pi reabsorption, e.g. pH-dependency and regulation of transport activity in response to Pi availability (Biber et al. 1993).

Members of the NaPi-I protein family have now been cloned from human (Chong et al. 1993; Miyamoto et al. 1995; Ni et al. 1996), rat (Li and Xie, 1996; Ni et al. 1994), rabbit (Werner et al. 1991) and mouse (Chong et al. 1995) tissue and are predominantly expressed in the kidney, liver and brain. Similar proteins have been identified in Caenorhabditis elegans in a large sequencing project (Wilson et al. 1994b), but the sequences have not been further characterized. The different proteins range in size from 465 to 576 amino acid residues. They are proposed to span the membrane 6–10 times. However, the topology of NaPi-I has never been addressed experimentally.

The functional properties of NaPi-I isolated from rabbit kidney and from human kidney have been characterized after expression in Xenopus oocytes, with somewhat ambiguous results. Werner et al. (1991) and later Bröer et al. (1998) reported rabbit NaPi-I-induced Na+ /Pi uptake in Xenopus oocytes, as determined by radiotracer flux measurements. However, the transport activity could not be linearly correlated with the amount of RNA injected or with the incubation time. Electrophysiological measurements revealed that Pi transport was paralleled by a Cl conductance sensitive to Cl channel blockers but also to organic anions such as uric acid, benzylpenicillin, Phenol Red and probenecid (Busch et al. 1996). In these studies, a Pi-induced current was observed only at Pi concentrations greater than 1 mmol l−1 in the superfusate. Because of the predominant Cl conductance, it was concluded that rabbit NaPi-I does not itself represent a Na+ /Pi cotransporter, but that its overexpression stimulates an intrinsic transport activity. The increased Na+ /Pi transport activity after the expression of rabbit NaPi-I in MDCK (Madin-Darby-canine-kidney) and LLC-PK1 (porcine kidney) cells could also involve an intrinsic system (Quabius et al. 1995). The hypothesis that NaPi-I is a modulator was challenged by Miyamoto et al. (1995), who cloned human kidney NaPi-I and expressed the protein in Xenopus oocytes. In contrast to the rabbit clone, human NaPi-I cRNA stimulated Na+ /Pi cotransport activity dramatically. The increase in Na+ /Pi cotransport activity was consistent with the data obtained after expressing the rat brain isoform in oocytes (Li and Xie, 1996).

It may be an extremely difficult task to establish a clear functional picture of NaPi-I-related proteins. A bifunctional protein is conceivable in view of recent reports of ‘multifunctional’ systems such as SGLT1 (Na+ /glucose transporter 1), which mediates glucose uptake and water permeability (Loo et al. 1996), and EAAT 5 (excitatory amino acid transporter 5)/ASCT 1 (ASC-type amino acid transporter), which exhibit both glutamate/amino acid transport activity and a Cl conductance (Zerangue and Kavanaugh, 1996a,b). It is also possible that there is a protein network with different clustered permeabilities (i.e. a Pi transporter and a Cl conductance) that interfere with each other at a functional level. Such an assumption is promoted by the C-terminal motif (–T/HTRL) found in renal and liver isoforms of NaPi-I. Related short C-terminal peptides are known to bind to so-called PDZ-domains mediating the protein–protein interactions which are involved in the clustering of membrane proteins (Staub and Rotin, 1997).

The physiological relevance of the NaPi-I proteins is not clear. Several reports have ruled out a prominent role for NaPi-I proteins in regulating body Pi homeostasis. Li et al. (1996) have suggested a link between rat liver NaPi-I and glucose metabolism. They showed that insulin stimulated NaPi-I expression and Na+ /Pi uptake in rat hepatocytes. The effect was reversed by glucagon. An increase in the level of NaPi-I mRNA in response to insulin could also be measured in the kidney. Other Pi-transporting systems (NaPi-II, NaPi-III) were not directly affected (Li et al. 1996). Unfortunately, brain was not included in the study. These intriguing data suggest the hypothesis that NaPi-I serves to supply the great demand of Pi in the liver, kidney and brain as a result of their high level of glucose metabolism.

Soon after its identification, convincing functional and physiological evidence demonstrated the dominant role of NaPi-II-related proteins in renal Pi reabsorption. Nucleic acid probes and/or peptide-derived antisera were used to quantify the level of NaPi-II-related mRNA and protein in response to physiological changes (Levi et al. 1994; Kempson et al. 1995). Immunohistochemical approaches demonstrated that the Na+ /Pi cotransporter is confined to the brush border of proximal tubular cells (Custer et al. 1994). In a series of elegant experiments, Lötscher et al. (1996, 1997) established a link between the action of parathyroid hormone and Pi availability and brush-border membrane integration/retrieval of NaPi-II protein. The issue of regulation has been addressed in recent review articles and will not be discussed here in detail (Levi et al. 1996; Murer et al. 1998).

Aspects of the structure and function of NaPi-II

The functional properties of NaPi-II-related proteins expressed in Xenopus oocytes were found to match those of experiments using brush-border membrane vesicles and cell culture models (Amstutz et al. 1985; Hoffmann et al. 1976; Malmström et al. 1987). The binding of Pi shows Michaelis–Menten characteristics with a Km in the range 20–100 μmol l−1 depending on species (at 100 mmol l−1 Na+ ). Na+ interacts in a cooperative way with the transporter with a stoichiometry of 3Na+ to 1Pi. The Km was found to be approximately 50 mmol l−1 at resting potential and neutral pH. Protons decreased the affinity of the transporter for Na+, resulting in the characteristic pH-dependency of Na+ /Pi reabsorption (Busch et al. 1994). The kinetic properties of NaPi-II have been investigated in detail by electrophysiological means, including pre-steady-state analysis with flounder and rat NaPi-II (Busch et al. 1994; Forster et al. 1997, 1998). The closely related functional characteristics within the protein family are paralleled by highly conserved hydrophobic domains. Consequently, the members of the NaPi-II family identified to date probably share a common three-dimensional structure.

The model for NaPi-II proposes eight membrane-spanning domains (Fig. 2). The topology of membrane proteins is notoriously difficult to investigate and, according to our own findings, the NaPi-II proteins are particularly intractable to analysis, because modifications that are tolerated in other proteins (insertion of epitopes or C-terminal truncations) interfere with protein trafficking and Na+ /Pi transport activity. Three regions of NaPi-II have been experimentally assigned to either cytoplasmic or extracellular domains. The large loop between hydrophobic regions three and four was found to be glycosylated and therefore extracellular (Hayes et al. 1994). The N and C termini were shown to face the cytoplasm by immunohistochemical means using oocytes expressing epitope-tagged NaPi-II from flounder (Kohl et al. 1998). They were assayed either without permeabilizing the membrane or in thin sections. Only the transporters carrying an epitope in the glycosylated loop gave rise to increased fluorescence in the whole-cell assay. In oocyte sections, all the tagged functional transporters were detected (Fig. 3; Kohl et al. 1998). These findings are of particular importance since the two termini and the extracellular loop represent the poorly conserved parts of the protein family. Any differences observed in the cellular handling of NaPi-II isoforms are probably due to these parts of the protein.

Fig. 1.

Evolutionary tree of the different members of the NaPi-I protein family. The values on the right indicate the degree of identity relative to the human kidney clone. The database accession numbers are as follows: mouse kidney, X77241; rat liver, U28504; human kidney, X71355 and D28532; rabbit kidney, M76466; human brain, I73259; rat brain, U07609; Caenorhabditis elegans, Z22177.

Fig. 1.

Evolutionary tree of the different members of the NaPi-I protein family. The values on the right indicate the degree of identity relative to the human kidney clone. The database accession numbers are as follows: mouse kidney, X77241; rat liver, U28504; human kidney, X71355 and D28532; rabbit kidney, M76466; human brain, I73259; rat brain, U07609; Caenorhabditis elegans, Z22177.

Fig. 2.

Topological model of a NaPi-II protein showing eight membrane-spanning domains with both the N and C terminus inside the membrane. Flag epitopes for immunodetection of the protein are indicated (Kohl et al. 1998). The number of N-glycosylation sites varies among species; however, they are consistently localized within the large extracellular loop. V/ATXL represents the potential PDZ-interacting motif at the C terminus, which may influence protein sorting.

Fig. 2.

Topological model of a NaPi-II protein showing eight membrane-spanning domains with both the N and C terminus inside the membrane. Flag epitopes for immunodetection of the protein are indicated (Kohl et al. 1998). The number of N-glycosylation sites varies among species; however, they are consistently localized within the large extracellular loop. V/ATXL represents the potential PDZ-interacting motif at the C terminus, which may influence protein sorting.

Fig. 3.

Topological analysis of the flounder NaPi-II protein after expression in Xenopus laevis oocytes. The left-hand panels (A,C) show experiments using intact oocytes including pictograms of the proposed model with the relevant Flag epitopes. The transporters carrying an epitope in the glycosylated loop (C) give rise to increased levels of fluorescence in the whole-cell assay. The right-hand panels (B,D) represent cryosections of oocytes from the same batch. All the tagged functional transporters were detected. The oocytes in A and B express a NaPi-II construct carrying Flag epitopes (stars) at both termini. The expressed proteins in C and D carry an epitope in the glycosylated loop.

Fig. 3.

Topological analysis of the flounder NaPi-II protein after expression in Xenopus laevis oocytes. The left-hand panels (A,C) show experiments using intact oocytes including pictograms of the proposed model with the relevant Flag epitopes. The transporters carrying an epitope in the glycosylated loop (C) give rise to increased levels of fluorescence in the whole-cell assay. The right-hand panels (B,D) represent cryosections of oocytes from the same batch. All the tagged functional transporters were detected. The oocytes in A and B express a NaPi-II construct carrying Flag epitopes (stars) at both termini. The expressed proteins in C and D carry an epitope in the glycosylated loop.

An integrative approach

Under non-pathological conditions, the mammalian kidney exclusively reabsorbs Pi, whereas the kidney in fish and birds reabsorbs or secretes Pi according to the physiological requirements (Renfro and Gupta, 1990). To investigate the role of NaPi-II under these circumstances, a homologous cDNA clone was isolated from flounder kidney (Werner et al. 1994). The localization of NaPi-II within the renal structures of the flounder was of particular interest. The euryhaline winter flounder (Pleuronectes americanus) exhibits low rates of glomerular filtration in order to minimize water loss. A short reabsorbing segment, PI, follows the glomerulus (this part shows functional and morphological similarities to the mammalian proximal tubule). A long adjacent segment, PII, presumably mediates secretion of solutes into the urine (Beyenbach and Liu, 1996; Elger et al. 1998). The collecting tubule connects to the bladder (homologous to the mammalian distal tubule). Interestingly, NaPi-II was absent in segment PI but expressed at a high level in PII and the collecting tubule. In the secreting segment PII, the transporter was confined to the basolateral membrane, but in the collecting tubule it was detected apically (Fig. 4; Kohl et al. 1996). NaPi-II is hypothesized to mediate Pi secretion in segment PII, whereas the same transporter is thought to fine-tune the excretion of Pi by modulating reabsorption in the collecting tubule (Elger et al. 1998).

Fig. 4.

Thin section of a flounder kidney showing a glomerulus, reabsorbing proximal tubule (PI), secreting tubule (PII) and collecting tubule. The blue coloration (Dapi-staining) indicates nuclear structures. The green fluorescence arises from lens culinaris lectin and is localized to the brush border of the reabsorbing segment PI. The flounder NaPi-II-derived red fluorescence stains the basolateral side of the secreting segment PII and, less intensely, the apical side of the brush border of the collecting tubule. Scale bar, 20 μm.

Fig. 4.

Thin section of a flounder kidney showing a glomerulus, reabsorbing proximal tubule (PI), secreting tubule (PII) and collecting tubule. The blue coloration (Dapi-staining) indicates nuclear structures. The green fluorescence arises from lens culinaris lectin and is localized to the brush border of the reabsorbing segment PI. The flounder NaPi-II-derived red fluorescence stains the basolateral side of the secreting segment PII and, less intensely, the apical side of the brush border of the collecting tubule. Scale bar, 20 μm.

The cellular and tubular distribution of NaPi-II in the flounder is not reflected in the mammalian kidney. Consistent with a unidirectional Pi flux from the lumen to the blood, NaPi-II is present exclusively in the apical cell compartment. However, the situation is reversed when the Na+ /Pi cotransporter from both rat and flounder is expressed in MDCK cells (Quabius et al. 1996). Whereas the rat cotransporter showed no preference in sorting, the flounder homologue was confined specifically to the apical membrane (B. Kohl, B. Huelseweh and A. Werner, in preparation). The production of rat/flounder transporter chimeras followed by heterologous expression in MDCK cells should clarify this issue. There is a structural motif (V/ATXL) (see Fig. 2) located at the very end of the C terminus which may influence protein sorting. However, these four amino acids are conserved in all NaPi-II proteins and are therefore unlikely to contribute to the specific localization of rat and flounder NaPi-II. Recent results from a two-hybrid screen established the C-terminal interaction of mouse NaPi-II with a protein containing the PDZ domain (L. Dehmelt and A. Werner, in preparation). Such an interaction might be important for the clustering of NaPi-II with other transport proteins and/or for anchoring it to the cytoskeleton. Most interestingly, a comparable PDZ-interacting motif is also found in the renal forms of NaPi-I protein.

The kidney and beyond

To date, the primary structures of the renal NaPi-II proteins from human, rat (Magagnin et al. 1993), rabbit (Verri et al. 1995), mouse (Collins and Ghishan, 1994), bovine (Helps et al. 1995), opossum (Sorribas et al. 1994), carp, flounder (Werner et al. 1994) and zebrafish (P. Nalbant, C. Boehmer, L. Dehmelt, F. Werner and A. Werner, manuscript submitted) tissues have been determined (Fig. 5). In the flounder, it has been demonstrated that the same Na+ /Pi cotransporter is expressed in the kidney and intestine (Kohl et al. 1996). This is in contrast to mammalian systems where no intestinal NaPi-II homologue has yet been reported. Interestingly, such an intestinal isoform was recently cloned from zebrafish, predicting a NaPi-II homologue in mammalian intestine as well (P. Nalbant and A. Werner, in preparation). A comparison of the different renal isoforms revealed that two subfamilies can be distinguished. The first group comprises all mammalian species, with the exception of the bovine transporter which, together with the fish transporters, constitutes the second group. The reason for this peculiar marriage is not clear at present. In ruminants, Pi is secreted very efficiently in the salivary glands and later reabsorbed in the intestine. It supports the microorganisms in the rumen and serves as a buffering system against volatile fatty acids (Shirazi-Beechey, 1996). Whether the ability to secrete Pi, i.e. the potential to direct the Na+ /Pi cotransporter to the basolateral membrane, correlates with the expression of this special NaPi-II isoform needs to be tested.

Fig. 5.

Evolutionary tree of the different members of the NaPi-II protein family. The values on the right indicate the degree of identity relative to the human kidney clone. The database accession numbers are as follows: mouse kidney, L33878 and U22465; rat kidney, L13257; opossum, L26308; carp (A. Werner, unpublished results); flounder kidney and intestine, U13963; bovine kidney, X81699; human kidney, L13258; rabbit kidney, U20793.

Fig. 5.

Evolutionary tree of the different members of the NaPi-II protein family. The values on the right indicate the degree of identity relative to the human kidney clone. The database accession numbers are as follows: mouse kidney, L33878 and U22465; rat kidney, L13257; opossum, L26308; carp (A. Werner, unpublished results); flounder kidney and intestine, U13963; bovine kidney, X81699; human kidney, L13258; rabbit kidney, U20793.

Recent reports by Gupta et al. (1997) and Hisano et al. (1997) have added a new component to the current view of NaPi-II-mediated Pi transport. Gupta et al. (1997) established a link between NaPi-II-mediated Pi transport and bone resorption by osteoclasts from chick and rabbit. In active osteoclasts, solute flux is directed from the site of bone resorption, the acidic compartment between the bone matrix and the ruffled border, to the blood. However, NaPi-II activity is inhibited at high proton concentrations (Amstutz et al. 1985; Hoffmann et al. 1976). Consequently, NaPi-II is unlikely to be involved in generating a transcellular Pi flux as observed in renal epithelia. This assumption was corroborated by Gupta et al. (1997), who confined the transporter to the basolateral compartment in osteoclasts. Transcytosis is involved in the vectorial transport of bone degradation products across the osteoclasts (Salo et al. 1997; Nesbitt and Horton, 1997). If Pi is translocated via the same pathway, then a basolateral Pi supply for the high metabolic needs may be necessary. A similar line of argument (increased Pi supply for metabolically active cells) was followed to explain the expression of NaPi-II in brain (Hisano et al. 1997).

The most recent family of Na+ /Pi cotransporters to be described was originally identified as a retroviral receptor for gibbon ape leukaemia virus (Glvr-1; O’Hara et al. 1990) and for rat amphotropic virus (Ram-1; van Zeijl et al. 1994; Miller et al. 1994). A slight similarity with a putative phosphate permease from Neurospora crassa suggested that the membrane proteins might exhibit Pi transport activity (Kavanaugh et al. 1994). Both receptors, human Glvr-1 and rat Ram-1, were found to induce Pi transport in a Na+ -dependent manner after expression in Xenopus oocytes. The affinity for Pi (at pH 7.5) obeyed the Michaelis–Menten equation with a Km of approximately 25 μmol l−1. The dependence on Na+ concentration was sigmoidal, with a Km between 40 and 50 mmol l−1 and a Hill coefficient of 1.5–2. In contrast to the NaPi-II-related proteins, these novel Pi transporters showed decreased activity at alkaline pH. This was explained by a preference for monovalent phosphate as a substrate. Referring to the newly discovered functional characteristics of the viral receptors, Glvr-1 and Ram-1 were denoted Pit-1 and Pit-2, respectively (Kavanaugh and Kabat, 1996).

The members of the NaPi-III protein family are almost ubiquitously expressed in rat tissues. Only spleen was entirely negative in northern blots with both specific probes. Interestingly, the level of Pit-related mRNA increased three-to fivefold in response to a 24 h incubation in Pi-free medium (long-term adaptation). This effect was not dependent on transcription, but could be related to enhanced mRNA stability (Chien et al. 1998). cDNA sequences related to Pit-1 and Pit-2 have been cloned from human (O’Hara et al. 1990; van Zeijl et al. 1994), rat (Miller et al. 1994), Chinese hamster (Wilson et al. 1994a) and mouse (B. O’Hara, unpublished results; Fig. 6) tissue. Both NaPi-III-related subfamilies show a similar topology with two hydrophobic domains, each spanning the membrane 5–6 times. The major structural differences are found in a hydrophilic loop between the two hydrophobic domains.

Fig. 6.

Evolutionary tree of the different members of the NaPi-III protein family. The values on the right indicate the degree of identity relative to the human clone denoted Pit-1. The database accession numbers are as follows: Human Pit-1, L20859; human Pit-2, L20852; chinese hamster Pit-1, U13946; chinese hamster Pit-2, U13945; mouse Pit-1, M73696; rat Pit-2, L19931.

Fig. 6.

Evolutionary tree of the different members of the NaPi-III protein family. The values on the right indicate the degree of identity relative to the human clone denoted Pit-1. The database accession numbers are as follows: Human Pit-1, L20859; human Pit-2, L20852; chinese hamster Pit-1, U13946; chinese hamster Pit-2, U13945; mouse Pit-1, M73696; rat Pit-2, L19931.

The broad expression range and the ability to adapt to changes in extracellular Pi concentration suggest a housekeeping role for NaPi-III at the cellular level. Whether it is responsible for other functions is still an open question, but recent reports have demonstrated that the expression of NaPi-III is compatible with the presence of other NaPi-related Pi transporters (Boyer et al. 1998; Tenenhouse et al. 1998). Since housekeeping proteins are located basolaterally, determining the cellular distribution of NaPi-III would be of great interest. The fact that the two viruses, GALV and RAM, mediate wide-range blood-borne infections suggests a basolateral occurrence of NaPi-III in epithelia. Upon infection, a virus interferes with the surface expression of its receptor. In the case of GALV and RAM, this leads to a downregulation of Na+ /Pi uptake, leading to severe Pi deprivation. This effect of viral infection is not only of (patho)physiological importance but represents a tool with which to study cellular Pi homeostasis. Furthermore, the dual function of NaPi-III as a Na+ /Pi cotransporter and a viral receptor opens up new strategies for investigating the extracellular structures of the transporter and its topology in general.

Three different families of Na+ /Pi cotransporter have been identified. The members of the NaPi-I family have been linked with insulin-stimulated glucose metabolism in liver, kidney and brain. NaPi-II proteins in the kidney and intestine are responsible for intracellular Pi accumulation in order to establish a transepithelial flux of Pi. The third protein family, NaPi-III, exhibits the characteristics of a housekeeping system. However, attempts to establish a correlation between the structure and a physiological function, such as an ‘accumulating system’ (NaPi-II) or ‘housekeeping system’ (NaPi-III), seem to oversimplify the situation. Job-sharing was obviously invented long ago and holds for Pi transporters. This flexibility complicates a detailed description of Pi homeostasis and the possibility of interfering in pathophysiological situations.

Amstutz
,
M.
,
Mohrmann
,
M.
,
Gmaj
,
P.
and
Murer
,
H.
(
1985
).
Effect of pH on phosphate transport in rat renal brush border membrane vesicles
.
Am. J. Physiol
.
248
,
F705
F710
.
Berndt
,
T. J.
and
Knox
,
F. G.
(
1992
).
Renal regulation of phosphate excretion
.
In The Kidney: Physiology and Pathophysiology
(ed.
D. W.
Seldin
and
G.
Giebisch
), pp.
2511
2532
.
New York
:
Raven Press
.
Beyenbach
,
K. W.
and
Liu
,
P. L.
(
1996
).
Mechanism of fluid secretion common to aglomerular and glomerular kidneys
.
Kidney Int
.
49
,
1543
1548
.
Biber
,
J.
,
Caderas
,
G.
,
Stange
,
G.
,
Werner
,
A.
and
Murer
,
H.
(
1993
).
Effect of low-phosphate diet on sodium/phosphate cotransport, mRNA and protein content and on oocyte expression of phosphate transport
.
Pediatr. Nephrol
.
7
,
823
826
.
Biber
,
J.
,
Custer
,
M.
,
Magagnin
,
S.
,
Hayes
,
G.
,
Werner
,
A.
,
Lötscher
,
M.
,
Kaissling
,
B.
and
Murer
,
H.
(
1996
).
Renal Na/Pi-cotransporters
.
Kidney Int
.
49
,
981
985
.
Boyer
,
C. J.
,
Baines
,
A. D.
,
Beaulieu
,
E.
and
Beliveau
,
R.
(
1998
).
Immunodetection of a type III sodium-dependent phosphate cotransporter in tissues and OK cells
.
Biochim. biophys. Acta
1368
,
73
83
.
Bröer
,
S.
,
Schuster
,
A.
,
Wagner
,
C. A.
,
Bröer
,
A.
,
Forster
,
I.
,
Biber
,
J.
,
Murer
,
H.
,
Werner
,
A.
,
Lang
,
F.
and
Busch
,
A. E.
(
1998
).
Chloride conductance and Pi transport are separate functions induced by the expression of NaPi-1 in Xenopus oocytes
.
J. Membr. Biol
.
164
,
71
77
.
Busch
,
A. E.
,
Schuster
,
A.
,
Waldegger
,
S.
,
Wagner
,
C. A.
,
Zempel
,
G.
,
Broer
,
S.
,
Biber
,
J.
,
Murer
,
H.
and
Lang
,
F.
(
1996
).
Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions
.
Proc. natn. Acad. Sci. U.S.A
.
93
,
5347
5351
.
Busch
,
A. E.
,
Waldegger
,
S.
,
Herzer
,
T.
,
Biber
,
J.
,
Markovich
,
D.
,
Hayes
,
G.
,
Murer
,
H.
and
Lang
,
F.
(
1994
).
Electrophysiological analysis of Na+/Pi cotransport mediated by a transporter cloned from rat kidney and expressed in Xenopus oocytes
.
Proc. natn. Acad. Sci. U.S.A
.
91
,
8205
8208
.
Chien
,
M. L.
,
O’neill
,
E.
and
Garcia
,
J. V.
(
1998
).
Phosphate depletion enhances the stability of the amphotropic murine leukemia virus receptor mRNA
.
Virology
240
,
109
117
.
Chong
,
S. S.
,
Kozak
,
C. A.
,
Liu
,
L.
,
Kristjansson
,
K.
,
Dunn
,
S. T.
,
Bourdeau
,
J. E.
and
Hughes
,
M. R.
(
1995
).
Cloning, genetic mapping and expression analysis of a mouse renal sodium-dependent phosphate cotransporter
.
Am. J. Physiol
.
268
,
F1038
F1045
.
Chong
,
S. S.
,
Kristjansson
,
K.
,
Zoghbi
,
H. Y.
and
Hughes
,
M. R.
(
1993
).
Molecular cloning of the cDNA encoding a human renal sodium phosphate transport protein and its assignment to chromosome 6p21.-p23
.
Genomics
18
,
355
359
.
Collins
,
J. F.
and
Ghishan
,
F. K.
(
1994
).
Molecular cloning, functional expression, tissue distribution and in situ hybridization of the renal sodium phosphate (Na+ /Pi) transporter in the control and hypophosphatemic mouse
.
FASEB J
.
8
,
862
868
.
Custer
,
M.
,
Lotscher
,
M.
,
Biber
,
J.
,
Murer
,
H.
and
Kaissling
,
B.
(
1994
).
Expression of Na–P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry
.
Am. J. Physiol
.
266
,
F767
F774
.
Elger
,
M.
,
Werner
,
A.
,
Herter
,
P.
,
Kohl
,
B.
,
Kinne
,
R. K.
and
Hentschel
,
H.
(
1998
).
Na–P(i) cotransport sites in proximal tubule and collecting tubule of winter flounder (Pleuronectes americanus
).
Am. J. Physiol
.
274
,
F374
F383
.
Forster
,
I. C.
,
Hernando
,
N.
,
Biber
,
J.
and
Murer
,
H.
(
1998
).
The voltage dependence of a cloned mammalian renal type II Na+ /Pi cotransporter
.
J. gen. Physiol
.
112
,
1
18
.
Forster
,
I. C.
,
Wagner
,
C. A.
,
Busch
,
A. E.
,
Lang
,
F.
,
Biber
,
J.
,
Hernando
,
N.
,
Murer
,
H.
and
Werner
,
A.
(
1997
).
Electrophysiological characterization of the flounder type II Na+ /Pi cotransporter (NaPi-5) expressed in Xenopus laevis oocytes
.
J. Membr. Biol
.
160
,
9
25
.
Gupta
,
A.
,
Guo
,
X. L.
,
Alvarez
,
U. M.
and
Hruska
,
K. A.
(
1997
).
Regulation of sodium-dependent phosphate transport in osteoclasts
.
J. clin. Invest
.
100
,
538
549
.
Hayes
,
G.
,
Busch
,
A. E.
,
Lotscher
,
M.
,
Waldegger
,
S.
,
Lang
,
F.
,
Verrey
,
F.
,
Biber
,
J.
and
Murer
,
H.
(
1994
).
Role of N-linked glycosylation in rat renal Na/Pi-cotransport
.
J. biol. Chem
.
269
,
24143
24149
.
Helps
,
C.
,
Murer
,
H.
and
Mcgivan
,
J.
(
1995
).
Cloning, sequence analysis and expression of the cDNA encoding a sodium-dependent phosphate transporter from the bovine renal epithelial cell line NBL-1
.
Eur. J. Biochem
.
228
,
927
930
.
Hisano
,
S.
,
Haga
,
H.
,
Li
,
Z.
,
Tatsumi
,
S.
,
Miyamoto
,
K. I.
,
Takeda
,
E.
and
Fukui
,
Y.
(
1997
).
Immunohistochemical and RT-PCR detection of Na+-dependent inorganic phosphate cotransporter (NaPi-2) in rat brain
.
Brain Res
.
772
,
149
155
.
Hoffmann
,
N.
,
Thees
,
M.
and
Kinne
,
R.
(
1976
).
Phosphate transport by isolated renal brush border vesicles
.
Pflügers Arch
.
362
,
147
156
.
Kavanaugh
,
M. P.
and
Kabat
,
D.
(
1996
).
Identification and characterization of a widely expressed phosphate transporter/retrovirus receptor family
.
Kidney Int
.
49
,
959
963
.
Kavanaugh
,
M. P.
,
Miller
,
D. G.
,
Zhang
,
W.
,
Law
,
W.
,
Kozak
,
S. L.
,
Kabat
,
D.
and
Miller
,
A. D.
(
1994
).
Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters
.
Proc. natn. Acad. Sci. U.S.A
.
91
,
7071
7075
.
Kempson
,
S. A.
,
Lotscher
,
M.
,
Kaissling
,
B.
,
Biber
,
J.
,
Murer
,
H.
and
Levi
,
M.
(
1995
).
Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules
.
Am. J. Physiol
.
268
,
F784
F791
.
Kohl
,
B.
,
Herter
,
P.
,
Hulseweh
,
B.
,
Elger
,
M.
,
Hentschel
,
H.
,
Kinne
,
R. K.
and
Werner
,
A.
(
1996
).
Na–Pi cotransport in flounder: same transport system in kidney and intestine
.
Am. J. Physiol
.
270
,
F937
F944
.
Kohl
,
B.
,
Wagner
,
C. A.
,
Huelseweh
,
B.
,
Busch
,
A. E.
and
Werner
,
A.
(
1998
).
The Na+–phosphate cotransport system (NaPi-II) with a cleaved protein backbone: implications on function and membrane insertion
.
J. Physiol., Lond
.
508
,
341
350
.
Levi
,
M.
,
Kempson
,
S. A.
,
Lotscher
,
M.
,
Biber
,
J.
and
Murer
,
H.
(
1996
).
Molecular regulation of renal phosphate transport
.
J. Membr. Biol
.
154
,
1
9
.
Levi
,
M.
,
Lotscher
,
M.
,
Sorribas
,
V.
,
Custer
,
M.
,
Arar
,
M.
,
Kaissling
,
B.
,
Murer
,
H.
and
Biber
,
J.
(
1994
).
Cellular mechanisms of acute and chronic adaptation of rat renal P(i) transporter to alterations in dietary P(i
).
Am. J. Physiol
.
267
,
F900
F908
.
Li
,
H.
,
Ren
,
P.
,
Onwochei
,
M.
,
Ruch
,
R. J.
and
Xie
,
Z.
(
1996
).
Regulation of rat Na+ /Pi cotransporter-1 gene expression: the roles of glucose and insulin
.
Am. J. Physiol
.
271
,
E1021
E1028
.
Li
,
H.
and
Xie
,
Z.
(
1996
).
Molecular cloning of two rat Na+ /Pi cotransporters: Evidence for differential tissue expression of transcripts
.
Cell. molec. Biol. Res
.
41
,
451
460
.
Loo
,
D. D.
,
Zeuthen
,
T.
,
Chandy
,
G.
and
Wright
,
E. M.
(
1996
).
Cotransport of water by the Na+/glucose cotransporter
.
Proc. natn. Acad. Sci. U.S.A
.
93
,
13367
13370
.
Lötscher
,
M.
,
Kaissling
,
B.
,
Biber
,
J.
,
Murer
,
H.
and
Levi
,
M.
(
1997
).
Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content
.
J. clin. Invest
.
99
,
1302
1312
.
Lötscher
,
M.
,
Wilson
,
P.
,
Nguyen
,
S.
,
Kaissling
,
B.
,
Biber
,
J.
,
Murer
,
H.
and
Levi
,
M.
(
1996
).
New aspects of adaptation of rat renal Na–Pi cotransporter to alterations in dietary phosphate
.
Kidney Int
.
49
,
1012
1018
.
Magagnin
,
S.
,
Werner
,
A.
,
Markovich
,
D.
,
Sorribas
,
V.
,
Stange
,
G.
,
Biber
,
J.
and
Murer
,
H.
(
1993
).
Expression cloning of human and rat renal cortex Na/Pi cotransport
.
Proc. natn. Acad. Sci. U.S.A
.
90
,
5979
5983
.
Malmström
,
K.
,
Stange
,
G.
and
Murer
,
H.
(
1987
).
Identification of proximal tubular transport functions in the established kidney cell line, OK
.
Biochim. biophys. Acta
902
,
269
277
.
Miller
,
D. G.
,
Edwards
,
R. H.
and
Miller
,
A. D.
(
1994
).
Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus
.
Proc. natn. Acad. Sci. U.S.A
.
91
,
78
82
.
Miyamoto
,
K.
,
Tatsumi
,
S.
,
Sonoda
,
T.
,
Yamamoto
,
H.
,
Minami
,
H.
,
Taketani
,
Y.
and
Takeda
,
E.
(
1995
).
Cloning and functional expression of a Na+ -dependent phosphate co-transporter from human kidney: cDNA cloning and functional expression
.
Biochem. J
.
305
,
81
85
.
Murer
,
H.
and
Biber
,
J.
(
1996
).
Molecular mechanisms of renal apical Na/phosphate cotransport
.
A. Rev. Physiol
.
58
,
607
618
.
Murer
,
H.
,
Forster
,
I.
,
Hilfiker
,
H.
,
Pfister
,
M.
,
Kaissling
,
B.
,
Lotscher
,
M.
and
Biber
,
J.
(
1998
).
Cellular/molecular control of renal Na/Pi-cotransport
.
Kidney Int. (Suppl
.)
65
,
S2
S10
.
Murer
,
H.
,
Werner
,
A.
,
Reshkin
,
S.
,
Wuarin
,
F.
and
Biber
,
J.
(
1991
).
Cellular mechanisms in proximal tubular reabsorption of inorganic phosphate
.
Am. J. Physiol
.
260
,
C885
C899
.
Nesbitt
,
S. A.
and
Horton
,
M. A.
(
1997
).
Trafficking of matrix collagens through bone-resorbing osteoclasts
.
Science
276
,
266
269
.
Ni
,
B.
,
Du
,
Y.
,
Wu
,
X.
,
Dehoff
,
B. S.
,
Rosteck
,
P. R.
and
Paul
,
S. M.
(
1996
).
Molecular cloning, expression and chromosomal localization of a human brain-specific Na+ -dependent inorganic phosphate cotransporter
.
J. Neurochem
.
66
,
2227
2238
.
Ni
,
B.
,
Rosteck
,
P. R.
,
Nadi
,
N. S.
and
Paul
,
S. M.
(
1994
).
Cloning and expression of a cDNA encoding a brain-specific Na+ -dependent inorganic phosphate cotransporter
.
Proc. natn. Acad. Sci. U.S.A
.
91
,
5607
5611
.
O’hara
,
B.
,
Johann
,
S. V.
,
Klinger
,
H. P.
,
Blair
,
D. G.
,
Rubinson
,
H.
,
Dunn
,
K. J.
,
Sass
,
P.
,
Vitek
,
S. M.
and
Robins
,
T.
(
1990
).
Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus
.
Cell. Growth Differ
.
1
,
119
127
.
Quabius
,
E. S.
,
Murer
,
H.
and
Biber
,
J.
(
1995
).
Expression of a renal Na/Pi cotransporter (NaPi-1) in MDCK and LLC-PK1 cells
.
Pflügers Arch
.
430
,
132
136
.
Quabius
,
E. S.
,
Murer
,
H.
and
Biber
,
J.
(
1996
).
Expression of proximal tubular Na–Pi and Na–SO4 cotransporters in MDCK and LLC-PK1 cells by transfection
.
Am. J. Physiol
.
270
,
F220
F228
.
Renfro
,
J. L.
and
Gupta
,
A.
(
1990
).
Comparative physiology of phosphate transport across renal plasma membranes
.
In Comparative Aspects of Sodium Cotransport Systems
(ed.
R. K. H.
Kinne
), pp.
216
240
.
Basel
:
Karger
.
Salo
,
J.
,
Lehenkari
,
P.
,
Mulari
,
M.
,
Metsikko
,
K.
and
Vaananen
,
H. K.
(
1997
).
Removal of osteoclast bone resorption products by transcytosis
.
Science
276
,
270
273
.
Shirazi-Beechey
,
S. P.
,
Penny
,
J. I.
,
Dyer
,
J.
,
Wood
,
I. S.
,
Tarpey
,
P. S.
,
Scott
,
D.
and
Buchan
,
W.
(
1996
).
Epithelial phosphate transport in ruminants, mechanisms and regulation
.
Kidney Int
.
49
,
992
996
.
Sorribas
,
V.
,
Markovich
,
D.
,
Hayes
,
G.
,
Stange
,
G.
,
Forgo
,
J.
,
Biber
,
J.
and
Murer
,
H.
(
1994
).
Cloning of a Na/Pi cotransporter from opossum kidney cells
.
J. biol. Chem
.
269
,
6615
6621
.
Staub
,
O.
and
Rotin
,
D.
(
1997
).
Regulation of ion transport by protein–protein interaction domains
.
Curr. Opin. Nephrol. Hypertens
.
6
,
447
454
.
Tenenhouse
,
H. S.
,
Gauthier
,
C.
,
Martel
,
J.
,
Gesek
,
F. A.
,
Coutermarsh
,
B. A.
and
Friedman
,
P. A.
(
1998
).
Na+ –phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression
.
J. Bone Mineral Res
.
13
,
590
597
.
Van Zeijl
,
M.
,
Johann
,
S. V.
,
Closs
,
E.
,
Cunningham
,
J.
,
Eddy
,
R.
,
Shows
,
T. B.
and
O’hara
,
B.
(
1994
).
A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family
.
Proc. natn. Acad. Sci. U.S.A
.
91
,
1168
1172
.
Verri
,
T.
,
Markovich
,
D.
,
Perego
,
C.
,
Norbis
,
F.
,
Stange
,
G.
,
Sorribas
,
V.
,
Biber
,
J.
and
Murer
,
H.
(
1995
).
Cloning of a rabbit renal Na–Pi cotransporter, which is regulated by dietary phosphate
.
Am. J. Physiol
.
268
,
F626
F633
.
Werner
,
A.
,
Moore
,
M. L.
,
Mantei
,
N.
,
Biber
,
J.
,
Semenza
,
G.
and
Murer
,
H.
(
1991
).
Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex
.
Proc. natn. Acad. Sci. U.S.A
.
88
,
9608
9612
.
Werner
,
A.
,
Murer
,
H.
and
Kinne
,
R. K.
(
1994
).
Cloning and expression of a renal Na–Pi cotransport system from flounder
.
Am. J. Physiol
.
267
,
F311
F317
.
Wilson
,
C. A.
,
Farrell
,
K. B.
and
Eiden
,
M. V.
(
1994a
).
Properties of a unique form of the murine amphotropic leukemia virus receptor expressed on hamster cells
.
J. Virol
.
68
,
7697
7703
.
Wilson
,
R.
,
Ainscough
,
R.
,
Anderson
,
K.
,
Baynes
,
C.
,
Berks
,
M.
,
Bonfield
,
J.
,
Burton
,
J.
,
Connell
,
M.
,
Copsey
,
T.
,
Cooper
,
J.
et al. 
. (
1994b
).
2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans
.
Nature
368
,
32
38
.
Zerangue
,
N.
and
Kavanaugh
,
M. P.
(
1996a
).
ASCT-1 is a neutral amino acid exchanger with chloride channel activity
.
J. biol. Chem
.
271
,
27991
27994
.
Zerangue
,
N.
and
Kavanaugh
,
M. P.
(
1996b
).
Flux coupling in a neuronal glutamate transporter
.
Nature
383
,
634
637
.