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Proximal Tubular Handling of Phosphate
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 ).
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