Disorders of Renal Tubular Phosphate Transport

The kidney plays a major role in the maintenance of inorganic phosphate (Pi) homeostasis and, as such, ensures that an adequate supply of this ubiquitous anion is available for proper cellular function and skeletal mineralization. Physiologic studies have revealed that the bulk of filtered Pi is reabsorbed in the proximal tubule, with higher rates of reabsorption in the early segments and in deep nephrons. This review will summarize the progress that has been made in the molecular identification and regulation of renal Pi transporters. In addition, it will focus on the pathophysiology of inherited (X-linked hypophosphatemia [XLH], autosomal dominant hypophosphatemic rickets [ADHR], and hereditary hypophosphatemic rickets with hypercalciuria [HHRH]) and acquired (oncogenic hypophosphatemic osteomalacia [OHO]) renal Pi wasting disorders, and discuss the role of two novel Pi-regulating genes, PHEX and FGF-23, in the regulation of Pi homeostasis. For recent reviews see references 1–5. Cellular Mechanism of Proximal Tubular Pi Reabsorption Pi reabsorption in the proximal tubule is mediated by Na+-dependent, secondary-active transport mechanisms. Influx of Pi from the tubular lumen into the epithelial cell involves brush border membrane-associated Na/Pi cotransporter(s) which are rate-limiting and targets for physiologic/pathophysiologic regulation. Efflux of Pi across the basolateral membrane may involve an anion exchange mechanism and/or a “Pi leak” to complete transcellular reabsorptive flux, and a Na+-dependent Pi uptake mechanism to guarantee Pi uptake from the interstitium if apical influx is insufficient to maintain cellular metabolism [for review see (1)]. Na/Pi Cotransporters Three distinct and unrelated families of mammalian Na/Pi cotransporters have been identified: type I, type II, and type III (solute carrier series SLC17, SLC34, and SLC20, respectively, in the human gene nomenclature database [http://www.gene.ucl.ac.uk/nomenclature]). All three types are expressed in proximal tubular cells and have the capacity to induce an increase in Na-dependent Pi uptake in heterologous expression systems (e.g. Xenopus laevis oocytes) (for review, see references 1 and 6). The type I Na/Pi cotransporter is localized in the brush border membrane of proximal tubular cells (7). It is unlikely, however, that this transporter is an important player in proximal tubular Pi flux. Heterologous expression studies demonstrated that type I-mediated Na/Pi cotransport does not have the characteristics and regulatory features of brush border membrane Na/Pi cotransport and suggested a channel function, mediating the flux of chloride and organic anionic compounds (1,8,9). Three closely related type II Na/Pi cotransporters are expressed in the apical membrane of absorptive/reabsorptive epithelia: type IIa (10), type IIb (11), and type IIc (12). Type IIa is more abundant in kidneys of adult animals and type IIc in kidneys of young animals and neither is detected in the intestine (12). Type IIb is expressed in small intestine and type II alveolar cells, but not in kidney (11). A key role for the type IIa transporter in renal Pi handling in adult animals is supported by the following observations: (1) exclusive proximal tubular brush border membrane localization (13); (2) disruption of the gene encoding type IIa in mice (Npt2−/−) leads to impaired renal Pi reabsorption and a 70 to 80% loss of brush border membrane Na/Pi cotransport (14); (3) abundance of type IIa protein in the brush border membrane correlates with Na/Pi cotransport activity under a variety of physiologic/pathophysiologic conditions (for review, see reference 1); (4) residual brush border membrane Na/Pi cotransport in Npt2−/− mice is not responsive to regulatory phenomena (e.g., parathyroid hormone [PTH] and dietary Pi intake [15,16]; see below). A candidate for residual Na/Pi cotransport activity in brush border membranes of type IIa knockout mice is the type IIc cotransporter (12). In favor of this interpretation is the similarity between type IIa and type IIc with regard to inter-/intra-nephron expression, namely higher in deep/juxtamedullary nephrons as compared with superficial/cortical nephrons, and proximal tubular segment localization, namely higher in S1/S2 segments as compared with S3 segments of proximal tubules ([12,13]; unpublished observations). In addition, the demonstration that type IIc protein abundance is 2.7-fold higher in renal brush border membranes of Npt2−/− mice, relative to wild-type littermates, provides support for this hypothesis (17). The type III Na/Pi cotransporters are cell-surface viral receptors (18) and appear to exhibit ubiquitous renal (and extrarenal) expression. Type III mRNA expression is detected in all nephron segments (19). To date, precise information on membrane localization of type III protein (e.g., apical versus basolateral) is not available. It has been suggested that type III Na/Pi cotransporters are responsible for basolateral Pi influx in all tubular cells to maintain cell metabolism as well as in proximal tubular cells under conditions of limited apical influx (for review, see reference 1). The Type IIa Na/Pi Cotransporter: Structure and Function Type IIa-mediated Na/Pi cotransport is electrogenic — inward flux of a positive charge —and involves the cotransport of three Na+-ions and one Pi-anion (preferentially divalent) (20). The binding of one Na+-ion to the negatively charged carrier is followed by the interaction of Pi and two Na+-ions with the carrier (20,21). The transfer of the fully-loaded carrier is electroneutral and electrogenicity is achieved by the reorientation of the empty carrier after the discharge of Na+ and Pi ions to the cell interior (21). Similar to other Na/solute cotransporters, there is a significant Na+ leak after interaction with the first Na+-ion (20). The latter is of minimal physiologic significance because the transporter is preferentially in its fully loaded transport cycle in the presence of Pi (20). Considerable information is available on the structure of the type IIa Na/Pi cotransporter. This is summarized in Figure 1 and is derived from different analytical approaches: (1) hydrophobicity predictions (10); (2) antibody accessibility together with epitope insertion (22); (3) cysteine insertion and accessibility of permeant/impermeant reagents (23,24); and (4) glycosylation studies (25). These studies demonstrated several important features of the type IIa transporter (Figure 1): (1) the transporter has a large extracellular loop, which separates it into two domains (10,25); (2) there is intramolecular homology within two domains (ICL1 and ECL3) (24); (3) the NH2- and COOH-termini are oriented intracellularly (22). Figure 1. : Structure/function relationship of the type IIa Na/Pi cotransporter. See text for details.The functional significance of specific domains of the type IIa Na/Pi cotransporter has also been addressed (Figure 1): (1) cysteine-insertion studies suggested that ICL1 and ECL3 comprise an important part of a “permeation pore” participating in both “cotransport” and “Na+ leak” function (23,24); (2) chimera construction — based on different transport properties of type IIa and IIb cotransporters (10,11) — suggested the involvement of three amino acid residues in determining the pH-dependence of type IIa (increased transport at higher pH) (26,27); (3) the chimera approach also suggested the importance of two basic amino acid residues in ICL3 for PTH-dependent internalization (28) (see below); (4) deletion studies documented that the COOH-terminus contains information for brush border membrane expression, i.e., a terminal PDZ-binding motif and an internal signal (29). Both the NH2- and COOH-termini portions of type IIa protein are required for transport activity. However, functional studies revealed that cleavage of the type IIa protein backbone, between the two glycosylation sites in the large extracellular loop, does not interfere with transport function (30,31). It is assumed that under this condition a disulfide bridge within this large extracellular loop stabilizes the transporter (Figure 1). Although the type IIa transporter might be part of a multimeric complex (see below), one transporter unit mediates Na/Pi cotransport (32). The Type IIa Na/Pi Cotransporter as a Target for Physiologic/Pathophysiologic Regulation Proximal tubular Na/Pi cotransport is regulated by a variety of conditions/factors, which elicit either an increase (e.g., increased Pi demand, Pi deprivation) or a decrease (e.g., PTH) in Pi reabsorption. Regulation is achieved primarily by an alteration in the amount of type IIa protein in the brush border membrane (for review, see reference 1). Accordingly, increased and decreased Pi reabsorption are, respectively, the result of increased and decreased abundance of type IIa protein in the membrane. Changes in Na/Pi cotransport and type IIa protein expression occur primarily in the absence of changes in type IIa mRNA levels, except perhaps after prolonged treatment with 1,25-dihydroxyvitamin D3 (1,25(OH)2D), thyroid hormone (T3), PTH, or prolonged changes in dietary Pi. Thus, changes in Na/Pi cotransport are attributable to either membrane insertion of type IIa protein, which may be preceded by de novo synthesis of the protein, or to membrane retrieval of type IIa protein, followed by its lysosomal degradation (for review, see reference 1). Thus, membrane trafficking of type IIa protein is an important element in the regulation of Na/Pi cotransport. The microtubular network does not participate in the initial internalization step, but it is crucial for the delivery of type IIa protein to the lysosomes (33). In contrast, (rapid) insertion of the type IIa transporter depends on an intact microtubular network (33). The intracellular signaling mechanism involved in insertion/retrieval of type IIa protein is not completely understood. We postulate that synthesis/insertion of type IIa protein is always high and “driven” by the high renal content of type IIa mRNA (34) and that the internalization of type IIa protein is the regulated process. Several studies have examined the signals for type IIa internalization. PTH action is initiated by binding to receptors that activate protein kinase A (PK-A) and/or protein kinase C (PK-C) signaling pathways, depending on their membrane localization (basolateral PTH receptors activate PK-A and PK-C; apical PTH receptors activate PK-C [35]). For atrial natriuretic protein (and nitrous oxide), type IIa protein internalization is mediated by PK-G activation (36). Studies in opossum kidney (OK) cells demonstrated that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway also participates in PTH-induced signaling (37). In addition, FGF-23, a factor contributing to renal Pi wasting in autosomal dominant hypophosphatemic rickets and oncogenic hypophosphatemic osteomalacia (see below), inhibits type IIa-mediated Na/Pi cotransport in OK cells via activation of MAPK (38). Moreover, it was recently documented in isolated perfused cortical slices that the different signaling pathways (PK-A, PK-C, PK-G) converge on the ERK/MAPK pathway to internalize type IIa protein (D. Bacic, C. Wagner, J. Biber, H. Murer; submitted). The downstream targets for ERK/MAPK-mediated phosphorylation remain unknown. Although the type IIa Na/Pi cotransporter is a phosphoprotein, changes in its phosphorylation state are not associated with PTH-induced internalization, thereby excluding it as a downstream target (39). As an alternative, phosphorylation of proteins that associate with type IIa may play a role in its regulation (see below). Endocytosis/retrieval of type IIa Na/Pi cotransporters occurs at intermicrovillar clefts and seems to involve clathrin (40). Internalized transporters are then routed to the lysosomes for degradation (41,42). There is no evidence for recycling of the type IIa protein, although it cannot be excluded as a fine-tuning system in response to small fluctuations in PTH levels. Although retrieval of type IIa protein appears to occur for most phosphaturic conditions (1,43), it is unclear whether internalization is preceded by transporter inhibition, as is the case for PTH-dependent inhibition of the Na+/H+-exchanger (NHE-3) (44) (for review, see 2). The internalization of the type IIa protein in response to phosphaturic signals is a highly specific process. Other brush border membrane proteins, e.g., the Na/sulfate cotransporter, are not internalized, whereas others that are internalized are recycled back to the brush border membrane (e.g., NHE-3 [2,44]). Specificity of retrieval can be achieved at the level of the transporter and/or at the level of the cellular machinery involved in recognition and internalization of the transporter (see below). One of these processes has to be under regulatory control. Internalization of the type IIa transporter is independent of “endocytosis motifs.” e.g., tyrosine, dileucine, or diacidic motifs (45). A chimera approach IIa versus type a amino acid motif in (Figure that participates in the of the type IIa Na/Pi cotransporter The Type IIa as of a Specificity in apical (e.g., and retrieval of type IIa protein a role for other proteins that with this Na/Pi cotransporter. several type proteins and localized in the proximal tubular epithelial cell and are and with the three amino at the of the type IIa and are both associated with the brush border membrane and may be part of a that is of several transporters and regulatory proteins, as kinase and may be involved in the interaction of type IIa protein with the H. J. in OK cells have suggested a role for and in apical of the type IIa Na/Pi cotransporter and a was from studies in mice for the gene Thus, these type IIa proteins might a regulatory complex and regulatory The interaction of the type IIa transporter with or which occurs via one of in two in may be (and under a regulatory condition (e.g., by and result in the of the transporter from the apical and available for internalization. In the binding of the type IIa transporter to via a specific interaction with one of the may the cotransporter from recycling and its lysosomal delivery and might also participate in tubular Pi reabsorption. In support of this hypothesis is the demonstration that Pi wasting in knockout mice is associated with a decrease in type IIa protein abundance in the renal brush border membrane of Tubular Pi is abundant in the Pi is to except under (for review, see reference The will focus on hypophosphatemic associated with renal Pi hypophosphatemia autosomal dominant hypophosphatemic rickets oncogenic hypophosphatemic osteomalacia and hereditary hypophosphatemic rickets with hypercalciuria All are by and, with the of regulation of renal metabolism 1). Although recent have to an of the skeletal in these (for see references will focus on the renal Pi transport and studies in have to of its of renal phosphate wasting in and is by and and renal in the reabsorption of filtered Pi and the metabolism of The features that from other hypophosphatemic are its dominant of its higher in and the of two and which have as to the mechanism for renal Pi wasting in The in Pi reabsorption in and mice in the proximal tubule, after is specific for and involves a decrease in the of a high capacity Na/Pi cotransport system in the The decrease is to a in the renal abundance of type IIa Na/Pi cotransporter mRNA and protein in kidneys of and mice with the role of the type IIa transporter in renal Pi reabsorption In addition, type IIc protein abundance is decreased in renal brush border membranes of mice of (17). and renal studies that the renal Pi transport in mice is not to the kidney but is mediated by a factor that is in and/or by The gene responsible for was by and PHEX to a gene with homology to on the The PHEX gene a amino acid protein that significant homology to the of which the These are type II membrane with a large extracellular highly cysteine residues and a for and The in the PHEX gene to are in a database available The are the gene and and as well as and and and are with loss of PHEX studies that in to the wild-type PHEX protein, which is to the in the PHEX gene result in proteins that remain in the are is the that proteins can be from the to the cell Thus, for trafficking these a mechanism for loss of PHEX function and a for the of novel in the gene have also been in the and of the human mice a large deletion in the gene whereas mice have a deletion in the that the is a gene deletion which may these exhibit features that are not in mice (see reference Several demonstrated that PHEX is expressed in and but not in kidney (see reference The of PHEX expression is with the skeletal and in and and the that the renal Pi leak in mice is on a factor Although it is not loss of PHEX function leads to a decrease in renal Pi it has been suggested that PHEX is involved in the of a phosphaturic hormone or the activation of a Pi hormone and that loss of PHEX function is associated with either an of phosphaturic hormone or a in a Pi In either renal type type and perhaps other Na/Pi cotransporters be and Pi wasting However, PHEX have not been The of activity is by its membrane To by membrane and its a and of PHEX was The for this approach was based on studies that a and of activity with that to that of the the as was by the However, the precise function of and its role in the regulation of brush border membrane Na/Pi cotransporters and renal Pi and the pathophysiology of remain to be are with Pi in to and This of is from is complete of rickets and is to a of Pi and Pi leads to and the of the skeletal and an of to and based on a of the mechanism of action of the PHEX gene are In this the will as an to their The features of are to of 1). However, is it with autosomal dominant and is by and of The gene responsible for was by and a of the factor FGF-23, a that is and to and at a in FGF-23, in unrelated involve the two residues in this cleavage and expression is not in with the of and However, it is expressed in from with an acquired renal Pi wasting with features of and (see below). To the mechanism in the gene elicit Pi the of on renal Pi handling was examined in intact of cells, either wild-type or the in the cleavage into mice to the of significant hypophosphatemia and In addition, the of both wild-type and of in mice a significant decrease in Pi mice also a decrease in renal Pi reabsorption that was associated with a decrease in type IIa protein expression in the brush border membrane that this major renal Na/Pi cotransporter is a target for regulation by In contrast, of the and no on Pi homeostasis in mice Although these studies the phosphaturic action of to the intact protein, in the of the intact and may have to these The recent demonstration that of the gene in mice provides evidence for a physiologic role for in the maintenance of Pi homeostasis In studies of the of on renal Na/Pi cotransport are not as either no or Na/Pi cotransport in OK Moreover, the inhibition that was was either or on the presence of in the In the latter it was demonstrated that to and that inhibition of Na/Pi cotransport was mediated by a MAPK signaling pathway (38). the are with the that FGF-23, at in high can renal Pi reabsorption and that to renal Pi wasting in is to the physiologic role of FGF-23, to the relative phosphaturic of the and and to whether is a PHEX Although there are with regard to cleavage by PHEX it is to that and are by in autosomal and genes, respectively, that function in the pathway (Figure 2). A for in and below). Figure : A hypothesis for renal phosphate wasting in hypophosphatemia autosomal dominant hypophosphatemic rickets and oncogenic hypophosphatemic osteomalacia See text for of with from reference for is to that of and of a of Pi and However, the mechanism of and degradation are well novel will be also as is an acquired and of renal Pi with and features of and 1). In to and also exhibit and Several types of have been associated with this but the appears to from that the features of result from the of a of the in complete of the and with this hypothesis is the demonstration that from also cotransport in OK cells There is the or signaling of the phosphaturic (see reference from to to both and and as well as to Moreover, the to either or have no on the of a for inhibition of Na/Pi in target renal These that several phosphaturic may be by studies on the molecular of and expressed in to the identification of protein and extracellular As (see in the gene that of the are responsible for Moreover, can elicit the renal and skeletal features of and to mice there is no information on the of or on renal Pi Thus, although there is evidence to that to the of the role of and in this to be is also to whether the factor is related to the phosphaturic factor responsible for the renal Pi wasting in the of (see The for with is of the the of the phosphaturic However, the are in to and the cannot be or treatment with in with can be initiated and the is for Pi in and Figure a that a pathway to the for renal Pi wasting in and The hypothesis that is either or that and/or its and are by and that FGF-23, which phosphaturic is not by The PHEX can amino residues the PHEX cleavage is most distinct from the motif in In the case of that loss of PHEX function in the of in the and that this in leads to the inhibition of renal Pi reabsorption. In the case of that in the cleavage which the of into and and their by to the of a of the which also inhibits renal Pi reabsorption. In the case of that of its and degradation by and respectively, to its in the and inhibition of renal Pi reabsorption. with this is the recent demonstration that the of is in the of with and compared with with of the features of and renal Pi and but it can be from the latter by the increased of and associated hypercalciuria 1). was first in a large and a have been (see reference Pi will all the and in with the of the renal Pi and it was suggested on the of these that is a of renal Pi reabsorption that the renal type IIa Na/Pi cotransporter is an important of Pi homeostasis and a target for its regulation (see reference and that mice for the type IIa gene exhibit a that namely renal Pi and an increase in the of with associated hypercalciuria it was suggested that in the human may be responsible for To this the and a of the in two from the and in unrelated from small families a in and a in neither with in the demonstrated that these as well as in the not to in the These excluded as a candidate gene for and that in renal Na/Pi cotransporter, or a is responsible for the A is to and the gene that is in the with It is of that in the gene as have recently been in two with renal Pi wasting and associated with either or Although the that one of the gene is for the expression of the other or conditions may to the and a different is in case In contrast, mice for the type IIa gene exhibit neither hypercalciuria renal features that are in (Npt2−/−) in the was supported by from the of and and the This not have been with the of and and

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.