Characterization of Phosphate Transport in Rat Vascular Smooth Muscle Cells

【摘要】  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 IC 50 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.

【关键词】  vascular calcification phosphate transport VSMC Pit Pit

Introduction

Vascular calcification (VC), the accumulation of calcium phosphate products in the wall of arteries, is a common complication of diabetes and chronic kidney disease that contributes to their morbidity and mortality. 1-3 VC is now recognized as a complex pathological process that involves cellular ossification and the participation of factors that either promote or inhibit the organized deposition of hydroxyapatite. 4,5 These factors include high phosphate concentrations, 6 uremic toxins, 7 or high glucose levels 8 among others which induce the expression of specific osteogenes. Medial calcification is primarily observed as a complication of CKD, and is mainly related, but not exclusively, to hyperphosphatemia. 6,9-10 Hyperphosphatemia appears to stimulate calcification through flux of inorganic phosphate (Pi) by transporters present in the plasma membrane of cells 11 which, in addition, may induce the formation of apoptotic bodies and matrix vesicles. 12,13

Primary and subcultures of primary aortic VSMCs are commonly used as an in vitro model to study the role of phosphate in VC. In this model, the role of Pi transport in calcification has been described, as well as the consequences of the changes in abundance of the corresponding transporters. In spite of that, the kinetic and molecular characteristic of such transport has not been analyzed fully. Consequently, we wished to determine the kinetic expression of Pi transport in VSMCs with classical biochemical approaches. We have found that VSMCs exhibit both Na-dependent and Na-independent Pi transport components with similar kinetic behavior, but that only the Na-coupled saturable uptake can be explained by the expression of both type III NaPi (NaPi-III) transporters, Pit1 and Pit2. 14 As an article has recently been published regarding the characteristics of Pi transport by Pit1 and Pit2 while preparing this manuscript, 15 this article will focus on the different and novel findings we have found.

Materials and Methods

For a detailed explanation of the methods used please see http://atvb.ahajournals.org. In short, primary cultures of VSMCs were obtained from the thoracic aortas of 2-month-old rats by microdissection and collagenase digestion. Transport assays were performed by determination of 32 P-K 2 HPO 4 /KH 2 PO 4 from uptake experiments in VSMC monolayers and in defolliculated Xenopus oocytes. Pit1 and Pit2 transporters were expressed in Xenopus oocytes by injection of the corresponding in vitro transcribed cRNAs, and Michaelian kinetic parameters were calculated by iterative nonlinear regression. The abundance of phosphate transporter transcripts in VSMCs was quantified using a iQ Sybr Green Supermix kit in an iCycler iQ Real-Time PCR Detection System (both from Bio-Rad), and calculated with the comparative C T method. Finally, RNA interference of Pit1 and Pit2 transcripts was achieved by transfection of Stealth RNAis (Invitrogen) into VSMCs.

Results

Components of Pi Transport in VSMCs

Preliminary experiments to determine the incubation time for initial velocity are explained in Methods. To determine the percentage of the uptakes in the presence and absence of Na that correspond to transport, we inhibited uptake of 0.05 mmol/L Pi with 10 mmol/L Pi itself ( Figure 1 A). Total uptake in the absence of Na + corresponds to 35% of the uptake in the presence of Na +. In the presence of 10 mmol/L P i as inhibitor, total uptake in the presence or absence of Na + was significantly inhibited to residual values of 3.0±0.2 and 3.6±0.3 pmol Pi per mg cell protein per minute, respectively. These experiments demonstrate the novel finding of an important Na-independent component of Pi transport expressed in rat VSMCs.

Figure 1. Pi transport in VSMCs. A, Pi uptake in the absence (open bars) or presence (black bars) of 10 mmol/L PO 4 as inhibitor. * P <0.0001 compared with the absence of inhibitor. B, Inhibition of Na-dependent (open) and Na-independent (black) transport by increasing pH. * P <0.0001 and # P <0.001 compared with pH 6.

To characterize the kinetics of Pi transport observed in VSMCs, Michaelian-saturating kinetics were performed from 0.01 to 5 mmol/L Pi, in the presence or absence of Na +. Pi uptake under these conditions was partially saturated as evidenced by inflection of the curves at low Pi points (supplemental Figure IB and IC, available online at http://atvb.ahajournals.org). Both experiments were fitted to equation 1 (see Methods), containing a Michaelian-saturable (transport) plus a nonsaturable (diffusion plus nonspecific binding) component. Both fits were then subtracted to assess net Na-dependent Pi transport (supplemental Figure ID). Kinetic values are summarized in Table 1 : Both total transport in the presence or absence of Na as well as net Na-dependent Pi transport exhibited the same affinity for Pi, 0.1 mmol/L, a K m value 15 times smaller than reported for human VSMCs. 16 Capacities of both Na-dependent and Na-independent Pi transport were also similar, and had an additive effect on total uptake in the presence of NaCl.

TABLE 1. Kinetic Parameters of Pi Transport

These results suggest the existence of either 2 different transport systems with similar kinetic characteristics but different dependence on Na, or alternatively the existence of a unique transport system that can couple facultatively to Na + when it is present, and has half the transport capacity in the absence of Na +. The Na dependence of Pi transport was then further analyzed with a Na-activation experiment. Fitting data to equation 2 (See Methods) produces a sigmoid curve (supplemental Figure II) and Hill coefficient of 1.6 ( Table 1 ), suggesting a stoichiometry of Na:Pi more than 1:1. This number is compatible with the expression of several transport systems with different Na:Pi stoichiometries, or alternatively to the activity of a transport system with two sodium binding sites that exhibit allosteric cooperativity.

The kinetic analysis allows us to interpret the role of Pi transport in VC. A summary of the following study is shown in Table 2. Doubling the physiological Pi concentration from 1 mmol/L 17 to a hyperphosphatemic 2 mmol/L would only increase the transport rate slightly to 1.05 times (1.22 times in the case of total uptake). The explanation for this minimal effect is that Pi transport is already saturated at 1 mmol/L (supplemental Figure IB and ID). To effectively increase the cell load of Pi there has to be an increase in the number of functional transporters (ie, increase in capacity or V max of the transport system): Doubling of V max (2 x V max ) doubles the transport rate at either 1 or 2 mmol/L Pi ( Table 2 ), and this effect is maintained with an increase in Pi concentration or increase in V max. In conclusion, the induction of VC by high Pi concentration cannot be explained exclusively as a direct effect on the transport rate of Pi as an increase in the number of functional Pi transporters has to occur to consider the Pi concentration as a key factor in the pathogenesis of VC. An increase in the number of Pi transporters has been described in response to several calcifying conditions. 16,18-20

TABLE 2. Consequences of Kinetic Parameters on Phosphate Uptake by VSMCs

pH Dependence of Pi Transport in VSMCs

A hallmark of Pi transport through the renal proximal tubules is the increase in transport activity with increasing pH of the uptake medium. 21 Because pH also affects VC, 22 we analyzed the effect of pH from 6.0 to 8.5 on VSMC Pi transport: Figure 1 B shows that Pi transport is inhibited with increasing pH in VSMCs. The effect was stronger in the presence of Na + than in its absence, because Na-independent Pi uptake (black bars) is only inhibited at pH 7.5. The dependence of pH effect on the presence of Na + points to an allosteric mechanism rather than a substitution of Na + by H + because in the latter case we would expect a stronger effect in the absence of Na + than in its presence.

Phosphonoformic Inhibition of Pi Transport in VSMCs

Phosphonoformic acid (PFA) is a well-known competitive inhibitor of the renal Pi transport. 23 It has also been successfully used to prevent calcification and to inhibit Pi transport in VSMCs. 16,19,24,25 Because the inhibition constant of PFA in kidney varies strongly with the animal species, 26 we checked whether PFA was also an inhibitor of 0.05 mmol/L Pi transport in rat VSMCs ( Figure 2 A). Increasing concentrations of PFA inhibited Pi transport with an IC 50 value of 3.9 mmol/L (from logIC 50 0.59±0.11) and 2.6 mmol/L K i (considering a K m of 0.1 mmol/L for Pi; see Methods). This suggests that PFA is very weak inhibitor of Pi transport in VSMCs compared with kidney, in which K i values ranging 0.03 to 0.21 mmol/L have been reported. 26 The inability of PFA to inhibit Pi transport can be further observed in Figure 2 B, as 1 mmol/L PFA has no effect on Pi transport and 10 mmol/L PFA only inhibits the Na-dependent component by 47%.

Figure 2. Effects of PFA on VSMC Pi transport. A, Inhibition of 0.05 mmol/L Pi transport in VSMCs with PFA. B, Inhibition by PFA of Na-dependent (white) and Na-independent uptake (black). * P <0.001 compared with Na-dependent transport with 0 mmol/L PFA.

Expression of Pi Transporters in VSMCs

VSMCs express both NaPi-III type 27 of phosphate transporters with Pit1 18,24 being more abundant than Pit2. 11 Based on our kinetic differences of Pi transport with previous reports, we determined expression of all rat Pi transporters identified today ( Figure 3 A). The accession numbers to GenBank, sequences of primers, and size of amplicons are listed in supplemental Table I. No expression was obtained for NaPi-I, NaPi-IIa, -IIb, and -IIc, as well as NPT4 and Na/PO4. The brain derived Na/Pi transporter, BNPI, was also expressed, although the expression level was only 0.3% of the main transporters. The type III NaPi transporters Pit1 and Pit2 were strongly expressed in rat VSMCs, and with similar intensity. As compared with the control Arp C T value (16.9±0.1), Pit1 C T was 21.6±0.2, and Pit2 C T was 21.7±0.09, whereas BNPI was amplified at C T 30.6±0.3, and NaPiIIa at C T 32.0±0.23. As these values only refer to mRNA levels, there is a consideration that they may not represent protein expression or biological activity.

Figure 3. Phosphate transporters in VSMCs. A, Real-time PCR showing that Pit1 and Pit2 are the major Pi transporters. BNPI only represents 0.3% of the type III transporters. B, Expression of Pit1 and Pit2 in Xenopus oocytes shows that they are strictly Na-dependent, with only partial activation by Li +. * P <0.01 compared with the corresponding water-injected level.

Expression of Pit1 and Pit2 in X laevis Oocytes

We characterized the functional expression of Pit1 and Pit2 in Xenopus laevis oocytes to ascertain whether they could explain the biochemistry of Pi transport in VSMCs. Initial velocity of Pi transport in either Pit1 or Pit2 expressing oocytes lasted more than 12 hours (data not shown). Table 1 summarizes the results of the kinetic analysis. Pit1 showed a Pi affinity of 0.1 mmol/L (similar to that of VSMCs), while Pit2 affinity was almost half of Pit1, providing a Km of 0.19 mmol/L. Both Pit1 and Pit2 follow the same Michaelian behavior, and no sign of cooperative-sigmoidal increase in Pi transport has been observed for Pit2 as has been reported for the human ortholog 15 (supplemental Figure III).

To know whether Pit1 and Pit2 are strictly Na-dependent or could be at least partially responsible for the Na-independent Pi uptake by VSMCs, Li +, K +, NH 4 +, and choline + were used to substitute for Na + in an equimolar ratio while Cl - was kept constant ( Figure 3 B). Only Li + was able to significantly increase Pi transport above the water-injected level: 5% with Pit1 and 11.7% with Pit2. However, Na-independent Pi transport was still minimal compared with the presence of Na +, and no transport was observed with K +, or with non-alkali-metal cations (NH4 + and choline + ).

Similar to the results seen in VSMCs, oocytes expressing either Pit1 or Pit2 were sensitive to pH changes of Na + -containing uptake medium ( Figure 4 A). We also determined the effect of pH in the absence of Na + ( Figure 4 B) and found that Pit1 was not functional at any of the pH values tested whereas Pit2 activity was only significant at pH of 6.0. However, Pit2 transport activity in the absence of Na + was still very small compared with the presence of Na + and therefore could not explain the Na-independent Pi transport observed in VSMCs.

Figure 4. Effect of pH and PFA on Pit-expressed transport. A, Inhibition of both Pit1 and Pit2 Na-dependent transport by increasing pH. * P <0.0001 compared with pH 6.0. B, In the absence of Na, only Pit2 expressed transport is inhibited by increasing pH. * P <0.001 compared with the corresponding water level. C and D, Inhibition of transport by PFA in Pit1- and Pit2-(squares) and water-injected (triangles) oocytes.

PFA was also a weak inhibitor of oocyte Pit1- and Pit2-expressed transport ( Figure 4C and 4 D). Net expressed transports (thick lines) provided an IC 50 and K i for Pit1 of 4.1 mmol/L and 2.7 mmol/L, respectively, whereas Pit2 appeared even more resistant than Pit1 (IC 50 5.8 mmol/L, K i 4.6 mmol/L).

Role of Pit1 and Pit2 on Pi Transport in VSMCs

The role of Pit1 and Pit2 on Pi transport measured in VSMCs was analyzed by RNA interference using Stealth RNAi. Expression of the corresponding mRNAs was inhibited within 24 hours (supplemental Figure IV). In the case of Pit1, this correlated with only a 23% inhibition of total Pi uptake (open bar), and 31% reduction in Na-coupled transport. Knock-down of Pit2, however, reduced total Pi uptake by 55% (open bar) and Na-dependent transport by 71%. Na-independent Pi transport (black bars) was not affected by RNAi treatment. Maximal effects were always observed at 24 hours after transfection decreasing substantially by 72 hours (data not shown). RNA interference was not performed on any other transporters shown in Figure 3 A as their abundance was less then 0.5% of the Type III family.

Discussion

An increasing number of articles are providing direct evidence for the role of Pi serum levels in the development of VC. The seminal investigations of Dr C.M. Giachelli focused on the role of Pi transport in the pathogenesis of VC, 11,24 but many basic characteristics of Pi transport in VSMCs are still unknown. The main aim of our work has been to clarify these characteristics, to understand the relationship between Pi transport kinetics and VC. Our work has provided several new findings, including the existence of a high-affinity Na-independent Pi transport. At this point we cannot differentiate between one transport system working facultatively with Na +, or two different systems, one being Na-dependent and the other Na-independent. Although both options are theoretically plausible as the affinity constants of Pi transport are similar, the fact that there are differences in the effects of pH in the presence and absence of Na +, as well as the fact that Pit1 and Pit2 in VSMCs only work in a Na-dependent fashion, suggests the existence of two transport systems that can be distinguished by their Na-dependence. In addition, Na-independent Pi uptake was not altered when the expression of Pit 1 and 2 were knocked down. An interesting and novel finding is that the effect on Pi transport is stronger when Pit2 rather than Pit1 is knocked down.

At a physiological serum Pi concentration (1 to 1.5 mmol/L) both Na-dependent and Na-independent components are saturated and therefore transport Pi at a rate similar to their V max. In other words, both components contribute almost equally to the total uptake of Pi by VSMCs. Our work significantly expands the understanding of the kinetics of Pi transport in VSMCs and describes a novel component, Na-independent Pi transport. This component plays a significant role in Pi transport in VSMCs and as such may play an important role in the pathogenesis of VC. Although it is speculative to describe the molecular nature of the Na-independent transport system, it clearly must be coupled to a different energy source to overcome the electrochemical gradient of the phosphate anion and therefore must be a primary active transport system or an exchanger coupled to the excretion of another anion.

An additional novel finding is that the Na-dependent Pi transport in VSMCs shows a high affinity (0.1 mmol/L) for Pi, similar to the kidney cortex. To our knowledge, previous studies only found K m values from 1.53±0.03 mmol/L 16 in primary human smooth muscle cells to 0.35±0.05 mmol/L in A-10 cells, an embryonic rat VSMC-derived permanent cell line. 18 These differences may arise from variations in the cell model used and also from the methodology used to determine the kinetic parameters (linear transformations versus non-linear regressions, equations used for the fits, weighing data to normalized distances from the curve, etc.). In addition, the high affinity for Pi in VSMCs is similar to the values of Pit1 and Pit2 K m that we have obtained in Xenopus oocytes, and are in agreement with previous studies using different expression systems. 15,27-30

Although all published works have shown a strict Na-dependent Pi transport by Pit1, several groups have also reported a partial Na-independent activity of Pit2, with a gradual selectivity for the different cations being, in general, Na + Li + K + NH 4 + =choline + =tris +. 15,27-29 We have only found a significant expression of Na-independent Pi transport when Na + was substituted with Li +, but this represented <10% of Na-coupled transport ( Figure 3 B). In addition, Pit2 is also expressed in the absence of Na + at acidic pH (6.0), which suggests that protons could mimic Na + or Li + ( Figure 4 B). Regardless, the physiological relevance of this small activation is minimal and would not explain the large Na-independent Pi uptake observed in VSMCs.

These kinetic characteristics are very relevant to vascular calcification. Our finding that at physiological Pi concentrations the transport of phosphate in VSMCs is saturated suggests that an increase in the serum phosphate from 1 mmol/L to 2 mmol/L or higher, as observed in patients with CKD, will not have immediate consequences in the cellular load of Pi. According to our analysis, Pi cell uptake in hyperphosphatemia can only be increased by expressing more active Pi transporters, as observed in an in vivo rat model of CKD. 20 Our finding might explain why diabetics who do not have hyperphosphatemia are still at increased risk of VC as alterations in transport activity alone is enough to significantly alter Pi intracellular concentrations.

Another important finding refers to the inhibition of Pi transport by PFA. In contrast to the case of NaPiIIa, PFA is a very poor inhibitor of type III Pi transporters and of VSMC Pi transport. Our results provide an IC 50 of about 3.9 mmol/L for PFA in VSMCs when the concentration of Pi is 0.05 mmol/L, and a K i of 2.6 mmol/L ( Figure 2 A). The values for PFA inhibition of Pit1 and Pit2 are in the same range, which can explain the inhibition observed in VSMCs ( Figure 4C and 4 D). At first these results are in contrast with published results, 16,24 because those authors reported half-maximal inhibition of 0.1 mmol/L Pi transport by 0.1 to 0.5 mmol/L PFA. The same authors successfully inhibited Pi- and calcium-induced 19 calcifications with only 0.05 mmol/L PFA, and Pi-mediated induction of osteocalcin and Cbfa-1 with 0.3 mmol/L PFA. PFA (1 mmol/L) is also able to prevent the ß-glycerophosphate induction of osteopontin and alkaline phosphatase in bovine VSMCs. 25 However, we do not see any effect on Pi transport at this concentration. The main difference between these reports and our work is primarily the incubation time. Whereas our experiments have been performed at initial velocity times (ie, when only significant entrance of Pi to the cells occurs), the incubation time used to inhibit Pi transport in those reports was from 90 minutes 24 to several days for inhibition of calcification and gene induction. 24,25 There is also the possibility that the effects of PFA on calcification may occur through other mechanisms including cytotoxicity, as we have observed (Villa-Bellosta, unpublished results). It may also be possible that the small amount of transport inhibition observed is enough to bring the Pi transport beneath a "threshold" such that the osteogenic signaling pathways are not activated. This would be consistent with both in vitro and in vivo works correlating increased VC with increasing levels of serum phosphate and with the theory that Pi influx works as a very precise sensor of calcifying conditions. This would explain not only the prevention of calcification with PFA but why the partial decrease in expression of just Pit1 expression with RNA interference could inhibit calcification. 11

In conclusion, we have characterized the phosphate transport of a well-established in vitro model of vascular calcification, and we have shown that its properties provide important information in the pathogenesis of ectopic osteogenesis. Additional research is necessary to characterize the molecular nature of the Na-independent transport system, as well as the regulation, respective activity, and cell biology of both Pit1 and Pit2 in VSMCs.

Acknowledgments

Sources of Funding

This work was supported by grants from the Spanish Ministry of Education and Science BFU2006-06284/BFI (to V.S.) and NIH 1 R01 DK066029-01 (to M.L.).

Disclosures

None.

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作者单位：Ricardo Villa-Bellosta; Yolanda E. Bogaert; Moshe Levi; Víctor SorribasFrom the Laboratory of Molecular Toxicology (R.V.-B., V.S.), University of Zaragoza, Spain; and the Departments of Medicine (Y.E.B., M.L.) and Physiology and Biophysics (M.L.), Division of Renal Diseases and Hypertension,