Victor Sorribas

The Na+-Pi cotransporter PiT-2 (SLC20A2) is expressed in the apical membrane of rat renal proximal tubules and regulated by dietary Pi

The principal mediators of renal phosphate (P(i)) reabsorption are the SLC34 family proteins NaPi-IIa and NaPi-IIc, localized to the proximal tubule (PT) apical membrane. Their abundance is regulated by circulatory factors and dietary P(i). Although their physiological importance has been confirmed in knockout animal studies, significant P(i) reabsorptive capacity remains, which suggests the involvement of other secondary-active P(i) transporters along the nephron. Here we show that a member of the SLC20 gene family (PiT-2) is localized to the brush-border membrane (BBM) of the PT epithelia and that its abundance, confirmed by Western blot and immunohistochemistry of rat kidney slices, is regulated by dietary P(i). In rats treated chronically on a high-P(i) (1.2%) diet, there was a marked decrease in the apparent abundance of PiT-2 protein in kidney slices compared with those from rats kept on a chronic low-P(i) (0.1%) diet. In Western blots of BBM from rats that were switched from a chronic low- to high-P(i) diet, NaPi-IIa showed rapid downregulation after 2 h; PiT-2 was also significantly downregulated at 24 h and NaPi-IIc after 48 h. For the converse dietary regime, NaPi-IIa showed adaptation within 8 h, whereas PiT-2 and NaPi-IIc showed a slower adaptive trend. Our findings suggest that PiT-2, until now considered as a ubiquitously expressed P(i) housekeeping transporter, is a novel mediator of P(i) reabsorption in the PT under conditions of acute P(i) deprivation, but with a different adaptive time course from NaPi-IIa and NaPi-IIc.

Characterization of Phosphate Transport in Rat Vascular Smooth Muscle Cells Implications for Vascular Calcification

Objective—Hyperphosphatemia and inorganic phosphate (Pi) transport by vascular smooth muscle cells (VSMCs) have been implicated in the pathogenesis of vascular calcification. The aim of this work has been to characterize Pi transport in VSMCs. Methods and Results—Primary cultures of VSMCs express both high affinity Na-dependent and Na-independent components of Pi transport. Under physiological conditions both transport systems are saturated, show similar activity, and are inhibited by increasing pH. The Na-dependent transport is also weakly inhibited by phosphonoformic acid (PFA) (3.9 mmol/L IC50 at 0.05 mmol/L Pi). Real-time polymerase chain reaction shows that Pit1 and Pit2 are expressed to the same degree, and no other Pi transporters are significantly expressed. When expressed in Xenopus oocytes they are strictly Na-dependent, with high affinities for Pi, and are inhibited by increasing pH, but only weakly inhibited by PFA. We have used RNA interference to demonstrate that Pit1 and Pit2 are the transporters responsible for Na-dependent Pi transport in VSMCs. Conclusions—Taken together these novel findings suggest new roles of Pi transport in the pathogenesis of VC and have implications as potential future clinical targets.

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca²(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM² but not with 2 × 1 or 1 × 4 Ca × P(i) mM². Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.

Role of thyroid hormone in regulation of renal phosphate transport in young and aged rats.

In the present study, we have examined the cellular mechanisms mediating the regulation of renal proximal tubular sodium-coupled inorganic phosphate (Na/Pi) transport by thyroid hormone (T3) in young and aged rats. Young hypothyroid rats showed a marked decrease in Na/Pi cotransport activity, which was associated with parallel decreases in type II Na/Pi cotransporter (NaPi-2) protein and messenger RNA (mRNA) abundance. In contrast, administration of long-term physiological and supraphysiological doses of T3 resulted in significant increases in Na/Pi cotransport activity, protein, and mRNA levels. Nuclear run-on experiments indicated that thyroid hormone regulates NaPi-2 mRNA levels by a transcriptional mechanism. In aged rats, although there were no changes in T3 serum levels (when compared with young animals), there were significant decreases in serum Pi concentration, renal Na/Pi cotransport activity, and NaPi-2 protein and mRNA abundance. These effects were mediated, at least in part, by a reduction in...

Phosphonoformic Acid Prevents Vascular Smooth Muscle Cell Calcification by Inhibiting Calcium-Phosphate Deposition

Objective—The role of inorganic phosphate in the pathogenesis of vascular calcification (VC) has been studied extensively in recent years. Phosphonoformic acid (PFA), an inhibitor of type II Pi transporters, has been traditionally used to study the involvement of Pi transport in VC, because PFA also prevents calcium deposition in vitro. However, aortic vascular smooth muscle cells (VSMCs) only express PFA-resistant, type III transporters (Pit-1 and Pit-2). Therefore, in this article we have studied the mechanism of VC prevention by PFA. Methods and Results—Radiotracer Pi uptake in rat VSMCs was not inhibited at the concentrations at which PFA prevents calcification. Alternative mechanisms whereby PFA could prevent calcification, such as cytotoxicity or phosphodiesterase inhibition, have also been excluded. The progression of calcification also took place in fixed cells. The kinetics of VC prevention by PFA, pyrophosphate, phosphonoacetate, and bisphosphonates was similar in live and fixed cells, showing mean effective concentrations in the micromolar range. Conclusions—PFA mainly prevents VC through a physicochemical mechanism that is independent of any cellular metabolic activity, including Pi transport. Conversely, PFA seems to act similarly to its chemical analogues, inorganic pyrophosphate, and bisphosphonates, as suggested decades ago.

Role of rat sodium/phosphate cotransporters in the cell membrane transport of arsenate

Inorganic arsenate (As(V)) is a common contaminant of underground water. Following oral exposure, it is assumed that As(V) is distributed and crosses cell membranes through inorganic phosphate (Pi) transporters. We have tested this hypothesis by studying the inhibition of rat Na/Pi cotransporters by As(V) in Xenopus laevis oocytes and in several rat tissues. The ubiquitously expressed type III Pi transporters (PiT-1 and PiT-2) showed a low affinity for As(V) (K(i) approximately 3.8 mM), similar to the Pi transport system in aortic vascular smooth muscle cells (K(i) 1.5 mM). The type II renal isoforms, NaPi-IIa and NaPi-IIc, were also poorly inhibited by As(V) (K(i) approximately 1 mM), similar to the Pi transport from kidney cortex brush-border membrane (BBM) vesicles. Conversely, the high-affinity intestinal transporter, NaPi-IIb, was very efficiently inhibited with a K(i) of 51 microM, similar to the Pi transport from intestinal BBM vesicles. Taking into account the 1.1 mM Pi in blood and renal ultrafiltrate, and the nanomolar range of As(V) exposures, we have determined that the contribution by Na/Pi cotransporters to As(V) membrane transport is negligible, given that 10-15 mM As(V) would be necessary in these fluids to be significantly transported. Intestinal transport is an exception, because Pi competition is weak, thereby considering that its concentration in lumen mainly depends on low Pi levels from ingested fresh water, and because As(V) very efficiently inhibits Pi intestinal transport. Our data agree with current toxicokinetic knowledge, and they explain the asymmetric excretion of trivalent and pentavalent arsenic species into bile and urine.

Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability

Renal proximal tubular Na-dependent phosphate transport (Na/Pi cotransport) has been studied extensively in the opossum kidney (OK) cell line. Recently, we cloned a complementary deoxyribonucleic acid (cDNA) (NaPi-4) from OK cells encoding an apical NaPi cotransport system. OK cells exposed to a low-Pi medium, as compared to high-Pi media, responded with an increase in Na/Pi cotransport, which was followed by an increase in NaPi-4 messenger ribonucleic acid (mRNA) abundance; maximal stimulation of Na/Pi cotransport was reached in 2 h, with no further increase for up to 16 h. NAPi-4 mRNA abundance was unaltered for 2 h, then increased to a maximum after 6–16 h in cells treated with low Pi medium. NaPi-4 mRNA decay rate was lowered by low-Pi media when compared to high-Pi media, with no increase in the NaPi-4 mRNA transcription rate. These data suggest that the upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after 2 h) in the expression of Na/Pi cotransport, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4–6 h), resulting in an increase in NaPi-4 mRNA abundance, due to an increased stability.

Arsenate transport by sodium/phosphate cotransporter type IIb.

Arsenic is a metalloid that causes the dysfunction of critical enzymes, oxidative stress, and malignancies. In recent years several transporters of As(III) have been identified, including aquaglyceroporins (AQP) and multidrug resistance proteins (MRP). As(V) transport, however, has not been sufficiently studied because it has been assumed that arsenate is taken up by mammalian cells through inorganic phosphate (Pi) transporters. In this paper we have analyzed the role of Pi transporters in the uptake of arsenate by directly using (73)As(V) as a radiotracer in phosphate transporter-expressing Xenopus laevis oocytes. The affinities of Pi transporters for H(3)AsO(4) were lower than the affinities for Pi. NaPiIIa, NaPiIIc, Pit1, and Pit2 showed a K(m) for arsenate that was >1mM (i.e., at least ten times lower than the affinities for Pi). The NaPiIIb isoform showed the highest affinity for As(V) in mouse (57 microM), rat (51 microM), and human (9.7 microM), which are very similar to the affinities for Pi. Therefore, NaPiIIb can have a prominent role in the toxicokinetics of arsenic following oral exposure to freshwater or food contaminated with As(V).

Thyroid hormone stimulation of Na/Pi-cotransport in opossum kidney cells.

Thyroid hormone (T3), a known stimulator of renal proximal tubular brush border membrane Na-dependent phosphate (Pi) uptake (Na/Pi-cotransport), stimulated Na-dependent Pi transport in opossum kidney (OK) cells. Na/Pi-cotransport was stimulated in a time- and dose-dependent manner with maximal effects (57%) at 24 h and 10−10 M T3. This stimulation was related to an increase in the apparent capacity (Vmax) of Na/Pi-cotransport. Treatment with T3 had no effect on Na-independent transport of Pi or of l-arginine. The stimulation of Na/Pi-cotransport was paralleled by an increase in the messenger ribonucleic acid (mRNA) encoding the OK cell apical Na/Pi-cotransporter (termed NaPi 4); the mRNA levels related to the activity of Na-independent l-arginine transport (rBAT) were unaffected by T3. Actinomycin D (10−7M) completely prevented the stimulatory effect of T3 on OK cell Na/Pi-cotrransport and on NaPi-4 mRNA content. In conclusion, T3 stimulates apical Na/Pi-cotransport in OK cells most likely by enhancing its transcription.

Phosphate Transporters in Renal, Gastrointestinal, and Other Tissues

Inorganic phosphate (Pi) is essential for all living organisms. Bound to organic molecules, Pi fulfills structural, metabolic, and signaling tasks. Therefore, cell growth and maintenance depends on efficient transport of Pi across cellular membranes into the intracellular space. Uptake of Pi requires energy because the substrate is transported against its electrochemical gradient. Till recently, 2 major families of physiologically relevant Pi-specific transporters have been identified: the solute carrier families Slc34 and Slc20. Interestingly, phylogenetic links can be detected between prokaryotic and eukaryotic transporters in both families. Because less complex model organisms are often instrumental in establishing paradigms for protein function in human beings, a brief assessment of Slc34 and Slc20 phylogeny is of interest.