In the kidney, filtered inorganic phosphate ions are reabsorbed along the proximal tubules. This transepithelial process involves sodium-dependent phosphate transporters that are localized at the apical (brush border) membrane. Currently, three Na/Pi-cotransporters that belong to the SLC 20 (Pit-2) and SLC 34 (NaPi-lla and NaPi-llc) families have been assigned to proximal tubular phosphate reabsorption, whereby SLC34 proteins play the major role. The primary functional difference between SLC34 and SLC20 proteins is that the former preferentially transport divalent Pi whereas the latter prefer monovalent Pi.
Renal excretion of phosphate is controlled by the number of Na/Pi-cotransporters residing in the apical membrane. The abundance of Na/Pi-cotransporters is controlled by a multitude of hormones and metabolic factors, Genetic diseases associated with disturbed phosphate homeostasis affect either Na/Pi-cotransporters directly or the stability and production of regulatory factors.
Keywords
proximal tubule, phosphate, NaPi-cotransporters, regulation, NHERF1
Introduction
The renal capacity to reabsorb inorganic phosphate ions (H 2 PO 4 − /HPO 4 2− ; abbreviated as Pi) is a major determinant of whole-body Pi homeostasis, which is required for normal cellular functions (e.g., energy metabolism and signaling mechanisms) and bone growth and remodeling. Consequently, urinary excretion of Pi is tightly controlled according to the body needs by a variety of hormones, diets, and metabolic factors. Diverse pathophysiological conditions and genetic diseases that are manifested by an altered renal reabsorption of Pi have been described (see also Chapter 69 ).
The basic concepts of the renal handling of Pi and the regulation of renal Pi excretion have been reviewed in a number of articles to which the reader is referred for original publications not cited in this chapter. Here we will focus on sodium-dependent Pi cotransporters that are responsible for renal reabsorption of Pi and we will address the cellular mechanisms involved in the regulation of these cotransporter proteins.
Proximal Tubular Reabsorption of Phosphate
Under normal physiological and dietary conditions, approximately 80% of the filtered Pi is reabsorbed in the proximal tubules. Studies on isolated perfused proximal tubules and brush border membrane vesicles (BBMVs) isolated from the superficial or the deep kidney cortex indicated that the rates of Na-dependent Pi transport in convoluted tubules are approximately threefold higher than rates observed in straight tubules, which suggests intranephronal heterogeneity along the proximal segments S1, S2, and S3. In addition, there is evidence of an internephronal heterogeneity as the fractional delivery of Pi to the early distal tubules of juxtamedullary nephrons is smaller compared with that of superficial nephrons.
There is no Pi reabsorption in the segments of the loop of Henle and Pi reabsorption along the distal part of the nephron remains controversial. Taking different methodological approaches and species differences into account, it appears that up to 10% of the filtered load may be handled by distal tubular segments, yet the molecular mechanisms are still unknown.
Apical Entry Step
Studies performed with isolated proximal tubules and BBMVs showed that the apical uptake of Pi is strictly dependent on the presence of sodium ions (i.e., occurs via secondary active, sodium-dependent transport mechanism(s): Na/Pi-cotransport). Furthermore, in all experimental systems, Na/Pi-cotransport activity is modulated by the extracellular pH, with higher uptake rates observed at more alkaline pH.
Assuming a membrane potential of ≈–65 mV, a stochiometry of overall Na/Pi cotransport of at least two sodium ions per one Pi ion and a concentration gradient for sodium of 10:1, the intracellular accumulation of Pi is found to be >100:1 (intra- versus extracellular). However, as indicated by nuclear magnetic resonance (NMR) studies, the intracellular Pi concentration may be assumed to be in the range of 0.7 to 1.8 mM. As this concentration of Pi is far below the thermodynamic equilibrium, it follows that the driving forces for the apical entry step(s) of Pi are always in excess. Therefore, changes of the transport rates (due to changes on either the number of transport units or their kinetic properties. e.g., K m ) offer possibilities for an efficient regulation of the apical uptake of Pi. In fact, there is ample evidence showing that most regulatory factors that influence the overall proximal tubular Pi transport capacity (TmPi/GFR-tubular maximum for Pi reabsorption per unit of glomerular filtration rate) alter the abundance of apical Na/Pi-cotransporters.
Basolateral Exit of Phosphate
Exit of Pi at the basolateral side of the proximal tubular cell occurs down the electrochemical gradient of Pi. Transport of Pi through the basolateral membrane is not well understood and the corresponding Pi transporter proteins have not been identified. To maintain the intracellular Pi concentration high enough to sustain the intracellular metabolism, basolateral exit mechanisms are regarded as “controlled leak” pathways. Different mechanisms for the basolateral exit of Pi have been proposed, such as a phosphate/anion (bicarbonate) exchange or a sodium-independent, pathway.
Gene Products Involved in Proximal Tubular Phosphate Reabsorption
Several mammalian membrane proteins have been cloned, which, after expression in oocytes of Xenopus laevis , mediate Na/Pi cotransport. These Na/Pi-cotransporters have been grouped into type I, type II, and type III Na/Pi-cotransporters and more recently were assigned to the solute carrier (SLC) families 17 (type I), 20 (type III), and 34 (type II). Studies with Npt2 knockout mice and analysis of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) indicated that the type II Na/Pi-cotransporters are of major importance for proximal tubular reabsorption of Pi. One member of the SLC20 family, PiT-2, has been localized at the BBM of proximal tubules as well, however its role in renal handling of Pi is however less clear. Na/Pi-cotransporters localized at the apical membrane of proximal tubular cells are indicated in Figure 68.1 .
The type I Na/Pi-cotransporter (NaPi-I, SLC17A1) was originally identified on the basis of its Na/Pi cotransport activity after expression in oocytes. Moreover, a number of findings have questioned its role in the renal handling of Pi-I besides acting as a Na/Pi-cotransporter: i) NaPi-1 also exhibits anion channel activity; ii) Na/Pi-cotransport mediated by NaPi-I is not dependent on the pH; and iii) alterations of the renal handling of Pi could not be correlated with changes of NaPi-I protein content.
The Type ll Na/Pi-Cotransporter Family SLC34
The Na/Pi-cotransporter family SLC34 comprises three members: type IIa/NaPi-IIa (SLC34A1), type IIb/NaPi-IIb (SLC34A2), and type IIc/NaPi-IIc (SLC34A3) NaPi-IIa and NaPi-IIc are expressed in the kidneys. Expression of NaPi-IIb has not been reported in the kidney but in a number of other epithelial and epithelial-like tissues, for example in small intestine, liver, testes and lung. In the small intestine NaPi-IIb protein is localized at the apical membrane of enterocytes and, as demonstrated with a conditional mouse knock-out model, is involved in reabsorption of dietary Pi.
NaPi-IIa (SLC34A1) has been cloned from renal tissues of different species. The human (NPT2; chromosome 5q35) and mouse genes (Npt2; chromosome 13) are approximately 16 kb in length and are arranged into 13 exons and 12 introns. In promoter constructs analyzed so far, elements for transcriptional regulations by bicarbonate/CO 2 tension, vitamin D, and Pi deficiency have been detected. However, the physiological significance of transcriptional regulation of NaPi-IIa gene expression has not been established unequivocally.
By reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridizations, expression of NaPi-IIa mRNA has been detected exclusively in the proximal tubules. This was confirmed by immunohistochemical analysis, which demonstrated that NaPi-IIa protein is restricted to the BBM of proximal tubular cells. By immunogold electron microscopy it was shown that NaPi-IIa cotransporters are evenly distributed along the whole length of the microvilli. Under Pi balanced dietary conditions the abundance of the NaPi-IIa protein is usually stronger in proximal tubules of juxtamedullary nephrons and decreases gradually along the tubular axes.
NaPi-IIa consists of approximately 635 amino acids. On immunoblots this protein is detected between 80 and 100 kDa. Interestingly, after denaturation in the presence of reducing agents, two bands of approximately 45 and 50 kDa are observed, which is likely due to a proteolytic cleavage between the N-glycosylation sites contained within the extracellular loop between the transmembrane regions TM5 and TM6 (see Figure 68.2 ). Separate expression of the cleavage products in oocytes of X. laevis oocytes did not result in Na/Pi cotransport activity, whereas after coexpression of both parts, Na/Pi cotransport could be restored indicating that proteolytically cleaved NaPi-IIa-cotransporters are functional. Whether a fraction of the NaPi-IIa protein content in apical membranes is proteolytically processed in vivo or if cleavage occurs during the isolation of proximal tubular BBMV, is currently not known.
Expression of NaPi-IIc (SLC34A3) has been detected in kidney, heart, spleen, and placenta. In kidneys, the largest amount of NaPi-IIc protein was detected in weaning animals; in adult mice, the abundance of this cotransporter is markedly decreased. On the basis of these observations, NaPi-IIc has been referred as a “growth related Na/Pi-cotransporter”. In mouse and rat kidneys, NaPi-IIc was localized at the BBM of S1 and S2 segments but was not observed at the luminal membrane of S3 segments.
Type III Na/Pi-Cotransporters Family SLC20
The retroviral receptors Glvr-1 (PiT-1) and Ram-1 (PiT-2) have been shown to induce Na/Pi-cotransport in oocytes of X. laevis and were assigned to the SLC20 Na/Pi-cotransporter protein family.
Transcripts of PiT-1 and PiT-2 have been detected in many tissues, including kidney, small intestine, liver, lung, striated muscle, heart, and brain. In kidneys, expression of PiT-1/2 mRNA was observed in the cortex and medulla. It has been estimated that SLC20 mRNA accounts for only approximately 0.5% of total renal Na/Pi-cotransporter mRNA.
In addition to NaPi-IIa and NaPi-IIc, PiT-2 (SLC20A2) has also been localized at the proximal tubular apical membrane as well ( Figure 68.1 ). No other nephron localization for PiT-2 is known so far. Although, by in-situ hybridization, PiT-1 mRNA has been detected throughout the entire renal tissue, the localization of the PiT-1 protein remains to be determined.
Relative Roles of Na/Pi-Cotransporters in Renal Handling of Pi
Studies performed with knock-out mouse models and analysis of hereditary human diseases with hypophosphatemia suggested that the relative roles of NaPi-IIa and NaPi-IIc in renal reabsorption of Pi in rodents and humans might differ. In mice (and possibly all rodents) NaPi-lla appears to be of major importance whereas in humans the relative roles of NaPi-IIa and NaPi-IIc are less clear.
Mouse Studies
A central role of NaPi-IIa in the renal handling of Pi was demonstrated with mice in which the Npt2 (NaPi-IIa) gene was ablated. Npt2 deficient mice show massive renal loss of Pi and consequently hypophosphatemia, as well as hypercalciuria due to elevated levels of 1,25-(OH) 2 VitD3. Flux measurements performed with isolated BBMVs demonstrated that, compared with BBMV’s of control mice, Na/Pi uptake was reduced by 70 to 80%. The remaining 20 to 30% of Na/Pi-cotransport activity was attributed to an up-regulation of NaPi-IIc. In contrast, NaPi-IIc deficient mice did not develop hypophosphatemia or phosphaturia, and Na/Pi-cotransport in isolated BBMVs was unchanged. NaPi-IIc −/− mice showed hypercalcemia and hypercalciuria due to elevated levels of 1,25-(OH) 2 VitD3 suggesting a role of NaPi-IIc in calcium metabolism. In BBMVs isolated from kidneys of NaPi-IIa/NaPi-IIc double knock out mice still residual Na/Pi-cotransport was observed that is likely due to PiT-2. However, the overall role of PiT-2 in renal handling of Pi remains to be determined.
Human Studies
In a genome-wide study of a large prospective cohort NaPi-IIa has been found to be associated with serum concentration of Pi. In fact, several mutations in the NaPi-IIa gene have been described in patients with hypercalciuria and elevated urinary Pi excretion. However, in all carriers, NaPi-IIa mutations were heterozygous and could not be correlated with hyperphosphaturia. On the other hand, a homozygous duplication in the NaPi-IIa gene (I154_V160 dup) causes autosomal recessive Fanconi’s syndrome and hypophosphytemic rickets. Complete loss of function of this mutant due to the missorting of the mutated NaPi-IIa was observed in in-vitro studies. Compared to NaPi-IIa, a more critical role of NaPi-IIc in Pi homeostasis in humans has been become evident. In patients with HHRH several mutations have been mapped in the SLC34A3 gene. Thus, these findings indicate that the function of NaPi-IIc in humans likely remains more important during adulthood compared to mice and may not be strongly dependent on growth.
Transport Mechanisms
SLC 34
Once correctly targeted to the membrane, NaPi-IIa/c transport characteristics are influenced only by changes in membrane potential (for NaPi-IIa) and external pH (for both NaPi-IIa and NaPi-IIc). So far, no evidence exists of post-translational modification of their kinetic properties. With constant pH and membrane potential, the transport capacity is therefore a direct function of the number of proteins present in the membrane. SLC34 protein membrane abundance is influenced by a variety of physiologically important regulatory factors (see below). NaPi-IIa and NaPi-IIc Na/Pi cotransport kinetics have been characterized in Sf9 cells and in Xenopus oocytes (by tracer uptake and electrophysiology). Transport is Na + -dependent and displays an apparent affinity constant for Pi typically <0.1 mM and an apparent affinity constant for Na + in the range 40–60 mM. Arsenate is the only other substrate known to be transported by the type IIa Na + /Pi-cotransporters.
It was established by means of heterologous expression in Xenopus oocytes that NaPi-IIa and NaPi-IIc preferentially transport divalent Pi (HPO 4 2− ). An important mechanistic difference between NaPi-IIa and NaPi-IIc is that transport activity for NaPi-IIa is electrogenic, whereas for NaPi-IIc it is electroneutral. Electrogenic NaPi-IIa translocates one net positive charge per transport cycle and the transport rate increases with membrane hyperpolarization, whereas electroneutral NaPi-IIc is insensitive to membrane potential and no net charge is translocated. This functional distinction is reflected in their respective Na + :Pi stoichiometries- 3:1 for NaPi-IIa and 2:1 for NaPi-IIc. It also follows that the theoretical Pi concentrating capacity is approximately 100-fold higher for NaPi-IIa, which, however implies a greater energetic cost to the cell resulting from Na + and charge accumulation. The preference for divalent Pi explains, in part, the strong dependence on external pH that would result from the titration of Pi species. In addition, protons can act directly on the transporter protein by competing with Na + binding and modulating conformational changes associated with the empty carrier states.
The loading of NaPi-IIa and NaPi-IIc proteins with substrates is proposed to be ordered. Biophysical studies (presteady-state analysis and voltage clamp fluorometry) have established that two Na + ions bind sequentially and cooperatively before phosphate. A third Na + binding transition precedes a rate-limiting reorientation of the fully loaded carrier. The order of substrate release at the cytosol is unknown. For NaPi-IIc, one of the two Na + ions, which confers electrogenicity to NaPi-IIa, can still interact with the protein but is not cotransported.
Transport by NaPi-IIa and NaPi-IIc is blocked by the competitive inhibitor phosphonoformic acid or foscarnet (PFA) with a reported inhibition constant ≈0.4–0.6 mM. PFA itself is not transported. Several other inhibitors of type IIb Na/Pi-cotransporters with significantly lower inhibitory constants than PFA have been reported. These possibly act in a non-competitive manner on NaPi-II proteins and a phosphophloretin compound was reported to exhibit inhibition at micromolar concentrations although its efficacy on heterologously expressed NaPi-II-cotransporters is unknown.
In addition to the cotransport-related current, two other currents related to the expressed protein can be observed for the electrogenic NaPi-IIa under voltage clamp: a cation leak that is active in the absence of Pi, and presteady-state currents. The leak is proposed to be mediated by the translocation of a single Na + ion per cycle at a rate <10% of the cotransport mode. It is unlikely to have physiological consequences, because Pi is normally present at sufficiently high concentrations to ensure that cotransport mode dominates and it is not observed in the electro-neutral NaPi-IIc. However, naturally occurring mutations in NaPi-IIc are reported to result in a significant Na + -leak, which illustrates how minor changes in the amino acid composition can have profound effects on function and important clinical consequences for phosphate homeostasis. Presteady-state current relaxations, induced by rapid changes in membrane voltage reflect non-linear charge movements associated with the electrogenic NaPi-IIa. Detailed study of their properties led to the identification and quantification of voltage-dependent steps in the transport cycle (namely the voltage-dependent reorientation of the empty carrier and the first Na + binding step) and estimations of the transport or turnover rate (number of Pi molecules translocated per second per protein), to be ≈10 s −1 (e.g., see Table 2 in Ref ).
SLC20
The two known mammalian isoforms of SLC20 proteins (PiT-1, SLC20A1 and PiT-2, SLC20A2) were originally identified as retroviral receptors Glvr-1 and Ram-1, respectively. They were later shown to be Na + -coupled Pi transporters and the electrogenicity and basic kinetics of transport characterized. It became apparent from these and other studies, e.g., that SLC20 proteins were functionally distinct from SLC34 transporters and the differences were underscored in an electrophysiological characterization of PiT-1 transport kinetics. The most important kinetic parameter that distinguishes SLC20 from SLC34 proteins is that the former preferentially transport monovalent Pi (H 2 PO 4 – ) with a 2:1 Na + :Pi stoichiometry. Like SLC34 proteins, the apparent affinities for Pi and Na + are also typically ≤100 μM and ≈50 mM, respectively. Another kinetic property that distinguishes SLC34 and SLC20 proteins is the weaker sensitivity to pH in the latter case: for PiT-1 the maximum transport rate is relatively constant over a 3 pH units and the apparent Pi affinity decreases only at pH <6. The preference for monovalent Pi and insensitivity of transport kinetics to reduced pH would allow SLC20 proteins to transport Pi under conditions where SLC34 proteins are functionally compromised. Unlike SLC34 proteins that are considered exclusively Na + driven, it was shown that Li + can replace Na + as the driving cation for PiT-1, albeit with a reduced transport rate. Furthermore, in the absence of Na + , lowering pH from 7.5 to 6.0 induces significant Pi uptake in Xenopus oocytes that expressed PiT-2. It has been proposed that H + may also substitute for Na + for this isoform. PFA is a weak inhibitor of PiT-1,2 mediated transport activity and to date no specific inhibitors for SLC20 have been reported. Kinetic studies suggest that Na + is the first ion to interact followed by a random binding of Na + and Pi. Finally, in contrast to SLC34 proteins in which the final translocation of substrates is assumed electroneutral, this partial reaction of the transport cycle may confer electrogenicity to PiT-1,2.
Structure-Function Relationships
SLC 34
The current model of the secondary structure of the type II Na/Pi-cotransporter is depicted in Figure 68.2 and predicts 12 transmembrane domains (TMDs) and cytoplasmically oriented N- and C-termini, the intracellular localization of which has been verified by epitope tagging and in vitro glycosylation assays. In this model, TMDs 1 to 5 are separated, from TMDs 6 to 12 by a large extracellular loop that contains two N-glycosylation sites. This loop also contains one essential disulfide bridge; a second bridge has also been proposed that possible links TMD 5 to TMD 11. Both bridges are thought to be important for defining final folding of the membrane-bound protein. The large extracellular loop and the N- and C-termini represent the greatest source of variation between species. The C-termini is important for targeting and protein-protein interactions. For example, the TRL motif in the C-terminus plays a role as a PDZ binding motif and a KR motif located in an intracellular linker region ( Figures 68.2 and 68.3 ) is critical for PTH sensitivity (see below). In contrast, the central “core” of TMDs shows a high degree of residue similarity or identity, and is largely conserved even in bacterial homologs, which suggests its importance in defining the transport characteristics. Given that the electrogenic (NaPi-IIa) and electroneutral (NaPi-IIc) isoforms share the same substrate specificities and preference for divalent Pi, this suggests that structural features that determine substrate recognition and translocation are similar. Substituted cysteine accessibility mutagenesis assays have provided additional insight into the membrane topology and strongly suggest that the transport pathway is likely formed by two inverted repeat segments comprising TMDs 3 and 4 and TMDs 7 and 8. It was also established that the NaPi-IIa functional unit is a monomer although evidence from biochemical studies and using the split ubiquitin approach and freeze fracture of Xenopus oocyte membranes containing the expressed transporter suggest that dimerization is likely. Close comparison of the NaPi-IIa and NaPi-IIc sequences has revealed one region between the predicted TMD3 and TMD4 that contains specific residues important for conferring electrogenicity and defining the transport stoichiometry. The functional importance of these residues was confirmed experimentally.
SLC20
Like SLC34 proteins, their current topology is predicted to comprise 12 TMDs but with extracellular N- and C-terminal tails ( Figure 68.2 ). Also like SLC34 cotransporters, the structure of SLC20 proteins contains an inverted repeat architecture. This topology is suggested by bioinformatic predictions, epitope tagging, cysteine scanning and in vitro glycosylation studies, e.g., Several studies have focussed on identifying the viral receptor domains, which has led to further insight into the secondary structure of PiT’s. For example, the large intracellular domain and associated TMDs have been removed and shown not to be required for retroviral recognition, although it is unknown if the transport function was compromised. PiT-1 and PiT-2 differ in the location of their virus binding sites: for PiT-2 this has been identified in the first extracellular loop, whereas that for PiT-1, has been suggested to be in the fourth extracellular loop ( Figure 68.2 ).
Protein-Protein Interactions of Type II Na/Pi-co-Transporters
Intracellular sorting, hormonal regulation and stability at the plasma membrane of a given protein may be controlled via its association with interacting partners. By Yeast-two hybrid screens several proteins have been identified that interact with NaPi-IIa and NaPi-IIc. No proteins that may interact with the PiT-2 transporter have been identified so far.
NaPi-IIa Associated Proteins
NaPi-IIa interacts, among others, with several PDZ domain-containing proteins, namely the four members of the NHERF family and Shank2E ( Figure 68.3 ). Importantly, these five proteins are located either at the renal proximal BBM (NHERF1, NHERF3 and Shank2E) or in its close proximity (NHERF2, NHERF4).
PDZ domains are modules involved in protein-protein interactions, and are widely expressed across philia (for review see ). They consist of 80 to 100 amino acids distributed in six β strands (βA–βF) and two α helices (αA–αB). PDZ domains typically bind to the C-terminus (PDZ-binding motif) of their interacting partners, via a conserved motif (R/K-XXX-G-ϕ-G-ϕ, where X is any amino acid and ϕ is a hydrophobic residue) located between helix αB and strand βB. NHERF1 and NHERF2 consist of two PDZ domains and a C-terminal sequence that interacts with actin-binding proteins from the Ezrin-Radisin-Moesin (ERM) family ( Figure 68.3 ). NHERF3 and NHERF4 contain 4 PDZ domains but lack the ERM-binding domain, whereas Shank2E contains six ankyrin repeats, one SH3 domain and a single PDZ domain ( Figure 68.3 ). The presence of multiple interacting domains within each of these proteins, together with their ability to form homo or heterodimers, opens the possibility for the formation of multiprotein complexes that can bring together regulatory and target proteins. Indeed, in addition to NaPi-IIa and several other renal transporters, NHERF1 and NHERF2 also interact with other proteins involved in Pi homeostasis, such as the parathyroid hormone receptor (PTHR), phospholipase Cβ and small G-proteins. Similarly, NHERF3 interacts with several renal transporters as well as with the Ste20-related serine/threonine protein kinase (SLK) and the Dual-specific A-kinase anchoring protein 2 (D-AKAP2). Shank2 associates with several renal transporters and with the F-binding proteins cortactin, α-fodrin, and Abp1.
Biochemical studies indicated that the interaction of NaPi-IIa with PDZ-proteins requires the presence of the last three amino acid residues (TRL) and involves particular PDZ domains within the NHERFs. NaPi-IIa interacts with the first PDZ domain of NHERF1, and this interaction is hormonally regulated by a mechanism that involves phosphorylation of NHERF1 (see below). The absence of NHERF1, or preventing its association with NaPi-IIa, leads to mislocalization of the cotransporter. Thus, NHERF1 deficient mice are characterized by hyperphosphaturia due to reduced expression of NaPi-IIa in the BBM. As the proximal BBM is heavily enriched in actin, the molecular mechanism by which NHERF1 controls the apical expression of NaPi-IIa may rely on the ability of NHERF1 to bind to the actin cytoskeleton via its ERM-binding domain. Despite being phosphaturic, NHERF1 deficient mice are normophosphatemic, suggesting a compensatory extra-renal mechanism. Normal phosphaturia and normal apical expression of NaPi-IIa has been observed in NHERF2 −/− mice.
NHERF3, previously known as PDZK1, also colocalizes with NaPi-IIa at the BBM of proximal tubules. Unlike NHERF1, NHERF3 does not have a domain able to bind to the actin cytoskeleton. However, its presence at the proximal BBM may be explained by its ability to heterodimerize with NHERF1. Nevertheless, the physiological role of the interaction of NHERF3 with NaPi-IIa remains unknown, since the absence of NHERF3 does not affect neither the expression nor the regulation of NaPi-IIa.
NaPi-IIc Associated Proteins
NaPi-IIc interacts with NHERF1 and NHERF3, but not with the other PDZ-proteins that associate with NaPi-IIa. However, NaPi-IIc does not contain a typical C-terminal PDZ-binding motif, and the molecular domains involved in the interactions are different from those of NaPi-IIa. In contrast to NaPi-lla, chronic adaptation of NaPi-llc to a low Pi-diet is impaired in PDZK1 deficient mice.
Regulation of Proximal Tubular Reabsorption of Pi
The extent of the renal proximal tubular reabsorption of Pi is regulated by a variety of hormones, diets, and metabolic factors and several hereditary and acquired disorders have been shown to result in disturbances of renal clearance of Pi. Most physiological and pathophysiological alterations of proximal Pi reabsorption can be explained by changes of apical Na/Pi-cotransport activity that is due to an alteration of abundances of Na/Pi-cotransporters NaPi-IIa, NaPi-IIc and PiT-2. Alternative modes for the regulation of Na/Pi-cotransporters, such as by changes of the membrane lipid composition (see below), have been proposed as well.
A reduction of the number of type II Na/Pi-cotransporters is achieved by endocytic retrieval at the intermicrovillar clefts ( Figure 68.3 ). Endocytosis of NaPi-IIa occurs by receptor-mediated endocytosis involving clathrin-coated vesicles. Results obtained with kidney-specific megalin knockout mice are also in accord with this notion. In proximal tubules of these mice, the endocytotic machinery was severely impaired and the apical abundance and rate of endocytosis of NaPi-IIa were reduced. In contrast to NaPi-IIa, NaPi-IIb (transfected into OK cells) is not regulated by PTH. On the basis of chimera constructs made between the NaPi-IIa and the NaPi-IIb proteins, a dibasic amino-acid motif (RK or RR) contained in an intracellular loop ( Figure 68.2 ) was identified and demonstrated to be required for the PTH responsiveness of NaPi-IIa. A yeast two-hybrid screen performed against this intracellular loop identified PEX19, a peroxisomal farnesylated protein, as an additional NaPi-IIa associated protein. Although PEX19 interacts with AP2, it remains to be shown if PEX19 provides a link between NaPi-IIa and the endocytotic machinery.
After its internalization, NaPi-IIa is associated with early endosomes and subsequently routed to lysosomes and degraded. In contrast, although initially associated with clathrin-coated vesicles, internalized NaPi-IIc has not been detected in the early endosomes and was not degraded in the lysosomes. Therefore, it was proposed that, under acute regulatory conditions, internalized NaPi-IIc, in contrast to the NaPi-IIa isoform, may be able to recycle back to the membrane.
Little is known about the recovery of NaPi-IIa proteins after PTH-induced internalization and lysosomal degradation. In a study performed with rats, it was shown that after PTH stimulation, the recovery of urinary excretion of Pi and the apical abundance of the NaPi-IIa did not parallel in time. Three hours after PTH injection, renal Pi excretion was normalized, whereas the abundance of the NaPi-IIa protein was still reduced. Based on in vitro experiments, de novo protein synthesis is required for the recovery of NaPi-IIa.
Parathyroid Hormone
Although the main signal activating parathyroid hormone (PTH) secretion is a reduction in the circulating levels of calcium, there is also evidence suggesting that the parathyroid gland responds to changes in circulating Pi. PTH reduces renal reabsorption of Pi by inhibiting proximal Na/Pi-cotransport. PTH receptors (PTHR) belong to the family of G-protein coupled receptors, and within the renal proximal tubule are localized at the apical and basolateral membranes. Binding of PTH to the apical PTHR results predominantly in an activation of PLC and subsequent stimulation of protein kinase C (PKC) pathways. In contrast, binding of PTH to basolateral receptors leads preferentially to activation of adenyl cyclase, and via cyclic AMP (cAMP), to stimulation of protein kinase A (PKA). The effect of basolateral PTHR on Pi transport may be modulated by the calcium sensing receptor.
The phosphaturic effect of PTH is achieved by promoting endocytosis of NaPi-IIa followed by its lysosomal degradation, thus resulting in a reduced amount of cotransporters at the BBM. This effect is detected within minutes upon PTH administration, and was reproducible in different animal and cellular models. In isolated proximal tubules, agonist binding to apical and basolateral PTHR results in internalization and degradation of NaPi-IIa, which suggests that activation of both PKC and PKA pathways trigger endocytosis of NaPi-IIa. However, recent studies using modified PTH analogs indicate that PKA signaling represents the predominant pathway. Both signaling cascades seem to involve MAP kinases ERK1/2. PTH also seems to reduce the apical abundance of NaPi-IIc and PiT-2. However, both effects are considerably slower than the regulation of NaPi-IIa. Although endocytosed NaPi-IIc undergoes degradation, this process does not take place in lysosomes. Thus, it appears that upon removal from the BBM NaPi-IIa and NaPi-IIc are not targeted to the same intracellular compartment.
PTH does not induce internalization of the NaPi-IIa interacting proteins NHERF1 and NHERF3. This suggests that the association of NaPi-IIa with these partners may be hormonally regulated by an on/off mechanism. NHERF1 and NHERF3 are phosphorylated in basal conditions. Several kinases, including GRK6A, PKC, PKA and Cdc2, are known to phosphorylate NHERF1. Moreover, incubation with PTH or pharmacological stimulation of PKA and PKC results in NHERF1 hyperphosphorylation. Serine-77 and threonine-95 were identified as the residues phosphorylated in response to PTH. Both amino acids are located within the first PDZ domain of NHERF1, i.e., the domain engaged in interaction with NaPi-IIa. This modification destabilizes the association of NaPi-IIa with NHERF1, allowing the retrieval of the cotransporter from the BBM. Current data suggest that NaPi-IIa is not a phosphoprotein, either in basal conditions or upon PTH administration.
NHERF1 interacts with apical PTHR as well as with PLCβ. The presence of an apical complex consisting of NHERF1, PTHR and PLCβ plays a critical role on the regulation of NaPi-IIa. Thus, endocytosis of NaPi-IIa after stimulation of apical PTHR is impaired in proximal tubules of NHERF1-deficient mice. This effect could not be explained by a general impairment of the endocytic machinery, since the internalization of the NaPi-IIa in response to pharmacological activation of PKC or PKA was normal. Instead, it seems to be due to the inability of the NHERF1-deficient tubules to properly activate PLC in response to PTH administration.
NHERF1 and NHERF3 also interact with the PKA anchoring proteins ezrin and D-AKAP2, respectively. However, such a close spatial arrangement of PKA with NaPi-IIa appears not to be required for the regulation of the cotransporter, since the endocytosis of NaPi-IIa induced via the PKA signaling pathway is normal in NHERF1 as well as PDZK1-deficient mice.
Dopamine
The cells of the proximal tubule have the ability to produce dopamine (DA), and this intrarenal production is stimulated in response to high dietary Pi. The DA receptors are G-protein coupled receptors that act by stimulating (D1-like) or inhibiting (D2-like) adenylyl cyclase. DA exerts a phosphaturic effect which, as in the case of PTH, is achieved by reducing the expression of NaPi-IIa at the BBM. Pharmacological experiments in isolated proximal tubules indicated that agonist binding to apical (but not to basolateral) D1-like receptors results in internalization of NaPi-IIa. Furthermore, this effect was blunted by PKA inhibitors but not by inhibitors of PKC or ERK1/2. This suggests that PTH and dopamine down-regulate NaPi-IIa via independent pathways.
Similar to the PTHR, D1-like receptors interact with NHERF1 and this interaction seems to be crucial for the DA-induced downregulation of NaPi-IIa. Indeed DA failed to downregulate NaPi-IIa in NHERF1-deficient mice, an observation that associates with the inability of DA to stimulate either cAMP accumulation or PKC activity. Furthermore, DA administration results in phosphorylation of NHERF1 at the same sites than PTH does (serine-77 and threonine-95). Thus, endocytosis of NaPi-IIa in response to either PTH or DA requires previous destabilization of the association between the cotransporter and NHERF1.
Phosphatonins
Hereditary and acquired disorders such as X-linked hypophosphatemia (XHL), autosomal hypophosphatemic rickets [ADHR), and oncogenic hypophosphatemic osteomalacia (OHO) show hypophosphatemia due to increased renal wasting of Pi. These disorders led to the identification of several factors (phosphatonins) such as fibroblast growth factor-23 (FGF23), secreted frizzled related protein-4 (sFRP4), and matrix extracellular phosphoglycoprotein (MEPE).
FGF23 is produced and secreted in bones by osteoblasts/osteocytes. Both serum concentrations of Pi and 1.25-[OH] 2 VitD3 stimulate secretion of FGF23. The phosphaturic effect of FGF23 is explained by reduction of the apical abundances of NaPi-IIa and NaPi-IIc. Accordingly, FGF23 deficient mice are hyperphosphatemic and show elevated serum levels of 1,25-[OH] 2 VitD3. As expected, FGF23 transgenic mice show phosphaturia that correlates with reduced amounts of both NaPi-IIa and NaPi-IIc proteins. Similarly, overexpression of FGF23 and in vitro treatment of isolated proximal tubules reduce the abundances of NaPi-IIa, NaPi-IIc and PiT2. Cellular mechanisms leading to altered abundances of apical Na/Pi-cotransporters by FGF23 are not known. Klotho is required as a cofactor for the cellular effect of FGF23 via its receptor FGFR1c. Additionally to its function as a cofactor, Klotho may act directly on Na/Pi-cotransporters by modifying sugar residues and thereby altering transport rates.
Injection of sFRP-4 elicits a decrease of NaPi-IIa abundance and an increase of Pi excretion. Observed altered phosphorylation of beta-catenin indicates a mechanism involving Wnt-signaling pathway up-stream to down-regulation of NaPi-IIa by sFRP-4.
MEPE, a member of the SIBLING (short integrin-binding ligand interacting glycoprotein) protein family, is primarily expressed in bone cells. The phosphaturic action of MEPE is paralleled by a decrease of NaPi-IIa protein abundance.
Atrial Natriuretic Factor/Nitric Oxide
The atrial natriuretic factor (ANF) and nitric oxide (NO) have been described to regulate proximal tubular Na/Pi-cotransport. Studies on isolated tubules demonstrated that these factors lead to a reduction (via endocytosis) of the content of NaPi-IIa in the apical membrane, via an activation of protein kinase G.
Dietary Phosphate Content
The dietary content of Pi is a potent regulator of proximal tubular Pi reabsorption and its effect has been extensively studied by micropuncture/microperfusion experiments and using isolated BBMVs. The content of Pi in the diet provokes altered rates of apical Na/Pi-cotransport; no changes of the apparent K m value for Pi have been described. To a large extent, this adaptive phenomenon is independent of PTH, 1.25-[OH] 2 VitD3, growth hormone, or plasma calcium. The effects of changes in the dietary content of Pi on the rate of apical Na/Pi-cotransport and the abundances of Na/Pi-cotransporters are observed within two to four hours (acute adaptation). The physiological signaling mechanisms involved in this adaptive phenomenon are poorly understood. It remains to be shown if hypophosphatemia is sensed directly by the proximal tubules or eventually at other sites such as in the central nervous system or in the small intestine. Of interest, after gavage of Pi to the upper small intestine of rats, urinary excretion of Pi increased within 15 minutes and was independent of PTH, FGF23 and altered serum Pi concentration. Based on these observations it has been postulated that a (yet unknown) phosphaturic factor is released from small intestinal mucosa due to an activation of one or more phosphate sensing mechanisms.
Intake of diets of different contents of Pi alters the abundances of NaPi-IIa, NaPi-IIc and PiT-2, yet with different half-times of responses. The fastest response is observed for NaPi-IIa (t 1/2 ≈1 hr), followed by PiT-2 (t 1/2 ≈8 hrs) and NaPi-IIc (t 1/2 2 ≈24 hrs). The acute (within two hours) upregulation of NaPi-IIa was shown to be independent of protein synthesis, and an important role of the microtubules has been suggested on the basis of the blockage of the adaptive response by colchicine. High-Pi diet leads to an internalization of NaPi-IIa, which is followed by degradation in the lysosomes. High-Pi diet also promotes an internalization of the NaPi-IIc, however no lysosomal degradation of the NaPi-IIc protein was observed.
In agreement with the role of NHERF1 in the apical positioning of NaPi-IIa, upregulation of this cotransporter by low-Pi diet was slightly impaired in NHERF1-deficient mice. Although NaPi-IIa strongly interacts with NHERF3 (PDZK1), its adaptation to dietary Pi is not impaired in NHERF3 −/− mice. In contrast, dietary regulation of NaPi-IIc is blunted in the absence of NHERF3.
In adult mice, adaptation of the NaPi-IIa protein to a low-Pi diet was not paralleled by a change of the NaPi-IIa mRNA, whereas in weaning animals, an increase of NaPi-IIa mRNA has been reported. In contrast, NaPi-IIc mRNA has been shown to be upregulated by a low-Pi diet in all development stages. Enhanced transcription of type II Na/Pi-cotransporter mRNA induced by a low-Pi diet may be explained by an enhanced binding of the transcription factor TFE3 to a phosphate response element contained within the promoter regions of the NaPi-IIa and NaPi-IIc gene. In a mouse model deficient in the phosphatase calcineurin Aα, the level of NaPi-IIa mRNA was reduced and the adaptive effect of a low-Pi diet was blunted.
Regulation by Lipids/Potassium Depletion
In addition to changes in the number of NaPi-IIa proteins, alterations in the proximal tubular Pi reabsorption, such as those observed after adaptation by a low-Pi diet or potassium depletion, have been proposed to correlate with changes in the membrane lipid composition including cholesterol, shingomyelin, and glycoshingolipids. Furthermore, thyroid hormone status was shown to modulate Na/Pi-cotransport and the cholesterol/phospholipids ratio in proximal tubular apical membranes.
Potassium depletion provokes increased urinary excretion of Pi, and BBMVs isolated from potassium-depleted rats exhibited decreased Na/Pi-cotransport. Decreased Na/Pi-cotransport in BBMV’s was explained by a reduction of the abundance of NaPi-IIc and PiT-2, but not of NaPi-IIa protiens. Paradoxically, the abundance of NaPi-IIa is upregulated by potassium depletion.
Fasting/Diabetes
Fasting increases urinary Pi excretion, which is due to a decrease of tubular reabsorption of Pi. Northern blot analysis indicated that fasting for 48 hours did not result in a decrease of renal expression of NaPi-IIa or NaPi III mRNA.
Insulin stimulates BBM Na/Pi-cotransport. In agreement, proximal Pi reabsorption was reduced in streptotocin-induced diabetes, though without changes of NaPi-IIa or type III Na/Pi-cotransporters mRNAs. Moreover, no change of the NaPi-IIa protein abundance in BBMV was detected. In rats treated with streptotocin, the adaptive response to a low-Pi diet on the amount of NaPi-IIa was blunted indicating that insulin may have a permissive effect on the response of NaPi-IIa to changes of the dietary content.
Ontogeny/Aging
During ontogenesis and aging, transport rates in renal BBMv and the relative abundances of NaPi-IIa and NaPi-IIc are altered. In kidneys of newborn rats, expression of the NaPi-IIa was only observed in functional, juxtamedullary nephrons and was absent in the undifferentiated structures (S-shaped bodies) of the outer cortex. During suckling, the distribution of NaPi-IIa is uniform throughout the cortex and correlates with the formation of differentiated brush borders. In kidneys of weaning rats, the expression pattern changed to a pattern similar to that observed in adults. Since during these transitions no changes of NaPi-IIa mRNA have been detected, it was suggested that the ontogenic changes on the expression of NaPi-IIa are due to posttranscriptional mechanisms. Expression of NaPi-IIc is not observed in suckling animals; instead it is expressed predominantly in weaning animals and is markedly reduced in adults.
Acidosis
Metabolic acidosis leads to an increased urinary Pi excretion, which is related to a reduction of proximal tubular apical Na/Pi-cotransport. As this effect was dependent on intact adrenal glands, an involvement of corticosteroids has been suggested.
In rats fed a normal-Pi diet, metabolic acidosis provoked a decrease of the content of the NaPi-IIa protein as well as of mRNA. In mice, fed a normal-Pi diet, metabolic acidosis results in a decrease of the content of NaPi-IIa and NaPi-IIc mRNA. However, both transporters were upregulated at the protein level. Increased urinary excretion under metabolic acidotic conditions may be explained, at least in part, by the sensitivity of type II Na/Pi-cotransporters to luminal pH -values.
Intoxication by Heavy Metals
In kidneys of rats injected with cadmium for two or more weeks, reduced Na/Pi cotransport correlated with a decrease of NaPi-IIa. Despite morphological changes of the microvillar structures, the abundance of aquaporin-1 and the Na/sulfate cotransporter NaSi-1 were not affected, indicating that exposure to cadmium results in a selective loss of NaPi-IIa. On the other hand, a direct effect of heavy metals, including cisplatin, on NaPi-IIa cannot be excluded as SH groups may play an important role in the Na/Pi-cotransport.
Mice that were injected with cadmium, showed increased levels of FGF23 and increased urinary excretion of Pi that correlated with a decreased abundance of NaPi-IIc, but, interestingly, not of NaPi-IIa proteins.
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