Characterisation of exogenous folate transport in Plasmodium falciparum

Folate salvage by Plasmodium falciparum is an important source of key cofactors, but little is known about the underlying mechanism. Using synchronised parasite cultures, we observed that uptake of this dianionic species against the negative-inward electrochemical gradient is highly dependent upon cell-cycle stage, temperature and pH, but not on mono- or divalent metal ions. Energy dependence was tested with different sugars; glucose was necessary for folate import, although fructose was also able to function in this role, unlike sugars that cannot be processed through the glycolytic pathway. Import into both infected erythrocytes and free parasites was strongly inhibited by the anion-channel blockers probenecid and furosemide, which are likely to be acting predominantly on specific folate transporters in both cases. Import was not affected by high concentrations of the antifolate drugs pyrimethamine and sulfadoxine, but was inhibited by the close folate analogue methotrexate. The pH optimum for folate uptake into infected erythrocytes was 6.5–7.0. Dinitrophenol and nigericin, which strongly facilitate the equilibration of H+ ions across biological membranes and thus abolish or substantially reduce the proton gradient, inhibited folate uptake profoundly. The ATPase inhibitor concanamycin A also greatly reduced folate uptake, further demonstrating a link to ATP-powered proton transport. These data strongly suggest that the principal folate uptake pathway in P. falciparum is specific, highly regulated, dependent upon the proton gradient across the parasite plasma membrane, and is likely to be mediated by one or more proton symporters.
1
Introduction
Reduced folates are essential cofactors for one-carbon transfer reactions, including the conversion of dUMP to dTMP, which is a prerequisite for DNA synthesis. Because of this, the folate pathway has long been a target for drugs deployed against rapidly reproducing cells such as cancers and a range of microbial pathogens. Whereas most microorganisms can synthesise the folates they need from the simple precursors GTP, p-aminobenzoic acid (pABA) and glutamate, higher animals (but not plants) have lost the ability to do this and depend on dietary intake of pre-formed folate as an essential nutrient. The human malaria parasite Plasmodium falciparum is able to exploit both of these routes [1–4]. Thus, it can utilise folate provided in culture medium in vitro or salvaged from the host plasma in vivo on the one hand, or convert the above precursors de novo into folate derivatives on the other, a characteristic also shared by the related apicomplexan parasite Toxoplasma gondii
[5,6]. The relative importance of the biosynthetic and salvage pathways across the complete life cycle in vivo and the interplay between them is poorly understood, although existing data support the view that both are necessary for healthy propagation of the parasite, at least in the erythrocytic stages [7].
An important aspect of exogenous folate utilisation is the machinery and mechanism(s) by which folate is imported into the parasite. The highly polar nature of folate derivatives suggests that salvage must employ some kind of mediated transport process, as diffusion alone across the membrane is likely to be far too inefficient. Moreover, folate molecules are dianionic at physiological pH and must be imported into the parasite against an inwardly negative electropotential that has been measured as ca. −95 mV in P. falciparum
[8]. Although the transport of other key molecules, such as pantothenate, lactate, glucose and choline, has been investigated [9–13], there has been no detailed study to date of this aspect of folate metabolism in P. falciparum, a better understanding of which might lead to new ways of inhibiting parasite growth. A preliminary approach to this end has been taken by demonstrating that the anti-gout drug, probenecid, which among other things inhibits folate transport in mammalian cells [14], can increase the sensitivity of P. falciparum to antifolate inhibitors [15]. Here, we demonstrate that folate uptake by this parasite is a regulated process that is critically dependent upon provision of glucose or another sugar that can proceed through the glycolytic pathway, that the existence of a pH gradient across the plasma membrane is also required for efficient transport, and that folate is transported principally by a proton-symport mechanism.
2
Materials and methods
2.1
Chemicals
[3′,5′,7,9-3H]folic acid, 24 Ci mmol−1, 1 mCi ml−1 was from Amersham, UK, [3′,5′,7,9-3H]folinic acid (26 Ci mmol−1, 1 mCi ml−1) and [3′,5′,7,9-3H]5-methyltetrahydrofolic acid (44 Ci mmol−1, 1 mCi ml−1) were both from Moravek, California. Folic acid, folinic acid, 5-methyltetrahydrofolic acid, 2,4-dinitrophenol, concanamycin A, d- and l-glucose, d-fructose, d-xylose, d-galactose, 6-deoxy-d-glucose, probenecid and furosemide were all purchased from Sigma, UK. Note that we use the term ‘folate’ generically to indicate derivatives of the folate family of molecules regardless of their oxidation state, modifications at the 5 and 10 positions or polyglutamation status.
2.2
Parasite culture
P. falciparum was routinely cultured under 1% O2, 3% CO2, 96% N2 in RPMI 1640 medium, supplemented with d-glucose (22 mM final concentration), hypoxanthine (36 mM), HEPES (25 mM), gentamicin sulfate (50 μg/ml) and 0.5% Albumax II (Invitrogen). The cultures were synchronised by haemolysis of mature, late trophozoite-stage parasitised erythrocytes by suspension in 9 volumes of a 5% sorbitol solution at room temperature for 5 min. Cells surviving the treatment were used to set up new cultures and the process repeated where necessary to achieve a tighter synchronisation [16]. Given that synchrony is never perfect, time zero for the erythrocytic cycle was taken as the point where ca. 90% of infected erythrocytes were ring form, with the remainder as very late schizonts. To obtain free parasites, synchronised cultures, normally in the late (mature) trophozoite stages (ca. 30 h into the cycle), were quickly lysed with 0.05% (w/v) saponin at room temperature, permeabilising the red cell and parasitophorous membranes and leading to haemolysis [17,18]. Any remaining unlysed red cells were further treated with a PBS wash containing the same amount of saponin. The freed parasites were then resuspended in the appropriate buffer with or without glucose, depending upon the purpose of the assays. Saponin-freed parasites that are glucose-replete maintain intracellular levels of ATP for periods of at least 30 min and do not show evidence of leakage [19].
2.3
Uptake assay of radiolabelled folates
Folate uptake/export assays were performed on free parasites, parasitised red cells or uninfected red cells, as appropriate. Cell numbers were estimated with a haemocytometer. Normally preparations containing 107–108 parasites were used for each assay point (or ca. 107–108 uninfected red cells). Prior to uptake assays by naked parasites, infected cells were lysed in 0.05% saponin and the freed parasites washed either in PBS or folate/pABA depleted RPMI 1640, depending upon the experiment. Wash steps were also performed with different buffered or non-buffered isotonic salt solutions where necessary as indicated in the relevant text.
All cells, washed extensively to remove folate present in the culture medium, were mixed with or without glucose (20 mM final concentration) and inhibitors as appropriate, together with radiolabel (normally 1 μCi/ml, equivalent to 38 nM, unless otherwise stated) to make up a total volume of 100 μl with PBS or alternative buffer. All the components were prewarmed to 37 °C before the start of the assay, the reaction mix incubated at 37 °C normally for 30 min, and stopped by addition of 1 ml ice-cold PBS.
When assaying for folate uptake by whole parasitised red cells, these were spun down after the uptake period and the pellet washed with 1 ml ice-cold PBS at least three times to bring extracellular label down to the background level. The washed parasite pellet obtained by subsequent saponin treatment was then lysed with 0.02% SDS before counting. Uptake by naked parasites was determined in the same way. Unless otherwise indicated, the uptake values represent labelled folate that has been imported into synchronised trophozoite stage parasites. All assays were routinely performed in triplicate on different batches of parasites unless otherwise indicated, and the data expressed as the mean ± S.D.
2.4
Extraction and affinity purification of folate derivatives
For characterisation of labelled folates, parasite pellets were washed three times in 1 ml PBS to remove any extracellular label and resuspended in 1 ml extraction buffer (0.1 M Tris–HCl, 2% ascorbic acid, pH 7.5), heated in a boiling water bath for 10 min, centrifuged at 10,000 × g for 10 min and supernatants stored at −20 °C until affinity purification and HPLC analysis, performed as described [20]. Unlabelled folic acid, folinic acid and PteG2 to PteG5 of the polyglutamated forms of folic acid were added into the extracted folates as internal standards. An identical aliquot of sample was also spiked with 3H-folinic acid in a second run to confirm the identity of the radioactive peak of folinic acid that had been extracted from the parasites.
2.5
pH measurements
3H-folinic acid uptake as a function of pH was measured in a combination HEPES-MES buffer system containing 20 mM of each component, and the salt concentration made up to 154 mM with NaCl. By taking the temperature coefficients of the buffers into account, pH values were adjusted at room temperature such that the apparent pH value at the assay temperature (37 °C) was exactly the pH required. Glucose was added to 20 mM as required. The internal pH of isolated parasites was measured as described [21] using the pH-sensitive fluorescent indicator BCECF-AM. Loading was achieved by incubating isolated parasites suspended at a cell density of 0.9–2.1 × 108 cells/ml in folate and pABA depleted RPMI 1640 without Albumax II, and containing 10 μM BCECF-AM, for 10 min at 37 °C. The cells were then washed by centrifugation and resuspended in culture medium and used immediately. An aliquot of cells was transferred to an appropriate buffer in a 2 ml cuvette in the temperature-controlled chamber of a spectrofluorometer, maintained at 37 °C. The sample was measured at 520 nm after successive excitations at 440 and 495 nm. The ratio of the fluorescence emissions (excitation at 495 nm/excitation at 440 nm) was used to calculate the internal pH of the cells. A standard curve was prepared with parasites resuspended in 130 mM KCl, 10 mM NaCl, 1 mM MgCl2 and 40 mM of MES-HEPES buffer from pH 5.5 to pH 8 and 30 μM nigericin [21].
3
Results
3.1
Uptake of folates over the asexual erythrocytic cycle
To monitor the ability of P. falciparum to salvage folate at different stages of the erythrocytic cycle, synchronised parasite cultures were incubated with different forms of radiolabelled folate. 5-Methyltetrahydrofolate (5-MeTHF) is the fully reduced form most abundant in human plasma [22] and thus the principal derivative available to the parasite in vivo. Folinic acid (5-formylTHF), although not known to be a normal physiological substrate for the parasite, is a more stable member of the fully reduced (active) forms that closely resembles 5-MeTHF and is used clinically (leucovorin). The capacity of ring stages to take up both of these compounds was very low but increased markedly ca. 15 h into the cycle as the parasites entered the trophozoite stages, peaking at around 30–35 h, before falling to very low levels again in mature schizonts (Fig. 1a). The numbers of parasites at the different stages were monitored throughout such time-courses and showed that uptake of folate increased as the parasites proceeded through the trophozoite stages against a near-constant number of infected cells, as expected for a synchronous culture. At around 40 h, some schizonts began to release merozoites, leading to a sharp rise in the total number of infected cells, but the uptake from this population continued to diminish, consistent with lower levels of uptake for both rings and schizonts, relative to trophozoites (Fig. 1a). To check that the marked reduction of uptake in the latter stages of the cell-cycle (beyond 40 h) was not due to parasites becoming unhealthy or inviable, the phenomenon was also monitored through a subsequent cycle, where the same pattern was repeated (data not shown). Only minor differences were seen between parasite clones of different lineages, and labelled folic acid, the stable oxidised form extensively used experimentally to study folate metabolism and a common dietary supplement, gave qualitatively similar results to the reduced folates. This pattern strongly suggested that folate uptake is a process regulated by P. falciparum, likely to involve a transport mechanism more complex than facilitated diffusion. On the basis of the data above, parasites at the late trophozoite stage of synchronous cultures were used to investigate the characteristics of uptake in subsequent experiments, using 3H-folinic acid as the label, as this is considerably more stable than 5-MeTHF, is more readily available in labelled form, and has been shown in earlier studies to provide a better source of cofactor than equimolar amounts of folic acid [23].
Uptake of folinic acid into the parasite was also markedly dependent upon temperature. Only a very low level of transport was observed between 10 and 25 °C, over which the parasites are viable, but metabolically retarded. The rate of uptake increased dramatically above ca. 30 °C, peaking quite sharply between 37 and 39 °C, but was abolished entirely above 45 °C, a temperature at which the parasites are no longer viable. Data for free parasites are shown in Fig. 1b; very similar profiles were obtained for infected erythrocytes with the same optimum temperature (data not shown). This demonstrated that active parasite metabolism in live organisms at their normal growth temperature is required for folate to be imported to a significant degree.
3.2
Kinetics of folate uptake
Assays of the time-dependence of folate uptake into trophozoites showed that it was linear (R2 ≥ 0.98) over a period of at least 80 min (Fig. 2a). In subsequent experiments, unless otherwise indicated, a standard incubation time of 30 min was therefore adopted to ensure that adequate counts for accurate measurements were obtained, whilst remaining well within the window of linearity. Uptake rates of folinic acid into parallel samples of free parasites and infected red cells in equal numbers were comparable, with somewhat higher levels measured in the latter (Fig. 2b), indicating that the rate-limiting step is not passage across the erythrocytic membrane, but rather across the parasite plasma membrane. In free parasite cells, the folate uptake process exhibited saturable and non-saturable components (Fig. 2c), indicative of more than one mechanism. In low concentrations of the substrate (in the region of 0 to 15 μM, which encompasses the normal range of human plasma folate, 30–40 nM [24]), folinic acid uptake is non-linear, consistent with the operation of a carrier-mediated process (Fig. 2c). At higher concentrations up to mM levels, uptake became linear, a phenomenon also observed in other folate transport systems [25,26], including that of Toxoplasma
[6]. Subtraction of the linear component yielded Km values for the saturable process of 3.4 ± 0.8 and 2.4 ± 0.3 μM, respectively, for FCB and K1 parasite strains (Fig. 2d). Interestingly, while their Km values were closely similar, the  observed for K1 (34.7 ± 2.6 pmol/107 parasites/h) was ca. 10-fold higher than that for FCB (3.3 ± 0.3 pmol/107 parasites/h).
3.3
Export of folinic acid
Export of folinic acid from free parasites was compared with that from uninfected cells. Uptake was first allowed to proceed into either infected or uninfected erythrocytes for 1 h in PBS plus 20 mM glucose at 37 °C with labelled folinic acid. After incubation and washing, cells were resuspended in the same buffer without label and incubated for different times (Fig. 3). Cells were then spun down and the radioactivity in the supernatant measured. To measure label exported from free parasites, they were first released from erythrocytes with 0.05% saponin and then washed and treated in a similar manner as for the uninfected red cells. The level of label in the supernatant of the red cell aliquots tripled over the 80 min incubation time, indicative of facile export. However, export of the label from free parasites was much less rapid, only increasing by ∼20% over the same period.
In the case of Lactobacillus salivarius, folate internalised over a similar period cannot be released from the cells and is recovered in cell extracts primarily in polyglutamated forms that play an important role in the retention and accumulation of intracellular folate [27,28], and previous studies using P. falciparum have demonstrated the presence of polyglutamated forms of folate produced from labelled monoglutamated precursors [2,23,20]. To examine whether the slow rate of export seen above was due to trapping by polyglutamation, the folinic acid internalised after 1 h was then extracted and analysed by HPLC on a C18 column [23,20]. The elution profiles indicated that the imported folinic acid had not been significantly modified by this stage, as no labelled polyglutamated folates could be detected in the cell extracts after this relatively short period of incubation, nor was there evidence of any conversion of label to another form of folate (data not shown). These results, together with our earlier data, where overnight incubations were required to detect polyglutamated forms [23,20], suggest that although these forms of folate probably play an important role in ultimately retaining the folates in malaria parasites and modulating the affinity of different enzymes for them, conversion to such forms is not rapid and appears not to play any immediate role in the salvage process. The low rate of net export from the parasite is however consistent with the operation of a transport system that actively imports folates into the parasite, where they are efficiently retained (see below).
3.4
The dependence of folate uptake on carbon source
To investigate possible metabolic requirements for folate uptake, its dependence on glucose and other sugars was studied in parasitised red cells. In the absence of glucose or at concentrations <1 mM, only a very low level of import was measurable. As the glucose concentration was raised, a marked increase in imported folinic acid was observed, peaking around 20 mM (Fig. 4a). This corresponds closely to the level of glucose in standard P. falciparum culture medium (22 mM), empirically determined historically to give optimal levels of growth. Time course experiments also showed that the higher the glucose concentration, the longer the uptake process could be sustained by the parasites (Fig. 4b).
d-Glucose could be replaced by the alternative hexose sugar d-fructose, which can also be utilised in the glycolytic pathway of P. falciparum
[29,30], but substitution by sugars that cannot, i.e. l-glucose, d-galactose, d-xylose or 6-deoxy-d-glucose, almost completely abolished uptake (Fig. 4a). Cellular energy in P. falciparum is predominantly provided by glycolysis, with the parasite using ca. 100 times more glucose than uninfected erythrocytes to supply their considerable energy needs [31], and uptake of folate is thus clearly dependent upon the operation of normal levels of intracellular energy metabolism.
3.5
Effect of pH on folate uptake
In the presence of glucose or other sugar susceptible to glycolysis, as in the above experiments, the malaria parasite has a higher cytoplasmic pH (7.2–7.4) and a lower parasitophorous vacuole pH (6.9), produced by proton pumping activity on their plasma membrane [21,32]. Such pumping is necessary to prevent the build-up of intracellular acidity, and the higher proton concentration thus created outside the cell can be utilised for the purposes of nutrient transport [21]. This prompted us to investigate the effect of pH on the uptake of folinic acid. For this, a HEPES-MES buffer system was used that provided a wider range of buffering capacity than either buffer alone. The HEPES-MES system contained 20 mM of each component and the salt concentration was made up to 154 mM with NaCl. Initially, glucose (20 mM) was included in all wash steps and assay reactions. For parasitised red cells under these conditions, the optimal pH was 6.5–7.0, uptake then decreasing linearly with increasing pH between pH 7.0 and 8.0 (Fig. 5a), with a similar pattern being observed for free parasites over this range (Fig. 5b). However, the transmembrane pH gradient generated in this way is dynamic, subject to change by normal cellular metabolism. To avoid this potentially confounding effect, similar experiments were also conducted in the absence of glucose, thus cutting off the energy supply for the proton pump(s), with the desired buffering and osmotic requirements maintained by NaCl, HEPES and MES. A series of artificially created, constant transmembrane proton gradients were established in this way for the uptake assay. Saponin-released parasites were washed thrice with 154 mM NaCl to remove folate and other nutrients and to effect exhaustion of the glucose, thereby bringing all parasites in a given sample to the same internal pH. The results showed that under these conditions, the uptake of folinic acid is positively correlated with the transmembrane pH gradient over a wider range (Fig. 5c). The lower the pH of the resuspension buffer, the greater the pH gradient thereby provided, which in turn yielded a higher uptake of folinic acid. Above pH 6.5, the pH response curve flattened out and exhibited a relatively constant, low level of uptake. This inflection point may correlate with the internal pH of the parasites when depleted of glucose under the above conditions [21]. In some cases this was confirmed by measuring the internal pH value with BCECF-AM as described in Section 2. Attempts to directly measure a change in pH as a result of folate import in the absence of glucose and using a minimal buffer concentration were unsuccessful, possibly because of the very small quantities that are taken up and the relatively slow rates involved.
3.6
Effect of perturbing the plasma membrane proton gradient on folate uptake
2,4-Dinitrophenol (DNP) is a widely used ionophore in the uncoupling of oxidative phosphorylation from electron transport on the mitochondrial membrane, as well as in studies of transport across the plasma membrane. Mechanistically, it provides an efficient by-pass route for protons that dissipates their gradient and hence the proton motive force. Here it was found that it also inhibits folinic acid uptake into parasite cells, with an IC50 of ca. 26 μM (Fig. 6a and Table 1), again suggesting that this uptake is intimately coupled with the energy metabolism of the parasites. Similarly, the K+/H+ antiporter nigericin had a profound effect on the uptake of label, which decreased in a dose-dependent manner, with a marked drop in the 100 pM range. Interestingly, this effect levelled off at around 1 nM to about 75% inhibition and showed little further drop up to 1 μM nigericin (Fig. 6b), suggesting that the major, but possibly not sole, factor determining the uptake of folinic acid is the magnitude of the pH gradient. A similar phenomenon was observed when the V-type ATPase inhibitor concanamycin A [33] was employed. Such an enzyme has been identified as the principal source of proton pumping in P. falciparum
[21,32]. This inhibitor was effective at sub-micromolar concentrations (IC50 ca. 16 nM; Fig. 6c), but only abolished ca. 60% of folate uptake, with no further effect beyond about 100 nM.
3.7
Metal ion dependency of folate uptake
To investigate whether folate uptake was affected by other cations, in addition to its dependence on the proton gradient demonstrated above, parasitised cells were first washed with 100 volumes of buffer containing either (i) PBS (the buffer in which other uptake experiments were conducted), (ii) a near-physiological concentration of Na+ (130 mM NaCl, 20 mM Tris–HCl, pH 7.0), (iii) an equivalent concentration of K+ (130 mM KCl, 20 mM Tris–HCl), (iv) the non-ionic polyol mannitol (130 mM mannitol, 20 mM Tris–HCl), or (v) Tris alone (150 mM Tris–HCl). Washes were repeated thrice to reduce the contribution of pre-existing salts to an insignificant level compared to those in the assay buffer. Uptake rates of folinic acid from all five of these solutions were similar (<±20% from the mean). Also, the presence of EDTA up to 5 mM or Mg2+ up to 10 mM had only a minor effect (data not shown). It thus appears unlikely that sodium, potassium or divalent metal ions are critically involved in the co-transport of folate into the malaria parasite.
3.8
Effect of antifolate drugs and other inhibitors on folate uptake
A range of other potential inhibitors and competitors were tested for their ability to affect transport of folate into the parasite (Table 1). Antifolate drugs exert their effect principally by binding to target enzymes (DHFR and DHPS) in the folate pathway, lowering pools of reduced folate and thus compromising key metabolic steps, especially DNA synthesis. However, in principle, they might also exhibit an inhibitory capability by interfering with the uptake of exogenous folate [4]. To investigate this possibility, uptake of labelled folinic acid into free parasites was monitored in the presence of the DHFR inhibitors pyrimethamine (PYR) and methotrexate (MTX), and the DHPS inhibitor sulfadoxine (SDX). Over a range of 0.4 nM to 40 μM for PYR and 0.3 nM to 322 μM for SDX, no inhibitory effect on folate uptake could be detected (the highest concentrations are ca. 900 and 500 times greater than the respective IC50 values for these drugs measured for growth inhibition). However, MTX did show inhibition of folinic acid uptake, consistent with its much closer structural relationship to folate than either Pyr or SDX. This was seen clearly when MTX (IC50 of ca. 13 μM) was compared to unlabelled folinic acid (2.6 μM), 5-MeTHF (3.5 μM) and folic acid (19.8 μM), where the drug competed with uptake of the label to a similar degree. The specificity of the transport process across the parasite plasma membrane suggested by the above results is consistent with a complete lack of competition with pantothenate (Table 1), the precursor of coenzyme A and an essential parasite nutrient [21], which is also a small but unrelated organic acid with a molecular weight about half that of folate.
To further explore the nature of the transporters involved in folate uptake, we employed two known channel blockers, probenecid and furosemide. Probenecid is known to inhibit folate uptake via an anion carrier in mammalian cells at mM levels [34,35], and is able to sensitise parasites to antifolate drugs [15]. Furosemide has been extensively used in nutrient transport studies of the malaria parasite, as it efficiently inhibits the so-called new permeability pathways (NPP) [36–38], which first appear in the erythrocyte membrane of parasitised red cells about 12–15 h into their intraerythrocytic development. The NPP show characteristics of anion-selective channels, but are of broad specificity and increase erythrocyte permeability to a range of small molecular weight anions, cations and other species [39–42], including essential nutrients such as pantothenate [11]. In experiments conducted on free parasites, where the NPP are not relevant, probenecid strongly inhibited folinic acid uptake with an IC50 of ca. 46 μM, and interestingly, furosemide was just as effective (IC50 of ca. 39 μM) (Fig. 7a and Table 1). When parasitised erythrocytes were used in the assay, furosemide blocked uptake effectively with an IC50 value of ca. 21 μM, similar to that for the free parasite, whereas probenecid was now less effective (IC50 of ca. 187 μM). The two drugs were assayed individually and also in combination under identical conditions, but no synergy was apparent between them (Fig. 7b). Indeed, the apparent absence even of an additive effect showed that when both drugs are present in equimolar amounts, the overall effect is equivalent to that seen with the drug of higher potency, i.e. furosemide. This suggests that the two drugs are likely to be acting on the same target. Given this and the above results with free parasites, where the target involved is shown by our prior experiments to behave as a specific folate transporter, it seems probable that the major effect of these two drugs on infected erythrocytes is also at folate transporters on the erythrocyte membrane, rather than the NPP, although we cannot exclude transport via the latter acting to supplement the endogenous capacity of the erythrocyte to import the higher levels of folate required by the parasite.
4
Discussion
Although P. falciparum possesses the biosynthetic machinery required to produce folate derivatives de novo, folate salvage is also an important aspect of the parasite's metabolism [7]. We present several lines of evidence to demonstrate that transport of exogenous folate into P. falciparum is primarily a carrier-mediated process dependent upon cellular energy and the trans-plasma membrane proton gradient. Uptake over the erythrocytic cycle is clearly regulated, with the highest capacity occurring at the late trophozoite stage. This coincides with a period of intense metabolic activity and the onset of multiple rounds of DNA replication, for which folate cofactors are essential, and resembles the pattern seen in the folate auxotroph Leishmania, a parasite that also imports folates maximally in the logarithmic growth phase [43–45]. The exogenous folate requirement of P. falciparum can be satisfied in vitro by folic acid, 5-formylTHF (folinic acid) and 5-MeTHF, similar to that of the folate-requiring bacterium Lactobacillus casei, but not L. salivarius, which cannot use the last of these [27]. The process also exhibits a marked temperature dependence, characteristic of a membrane carrier system, with significant uptake only occurring close to the parasite's optimum growth temperature of 37 °C. Once imported, net loss in the reverse direction appears to be a slow process, but over the short periods monitored here, did not involve a detectable level of polyglutamation that would trap the folate within the cell, although a significant portion of imported folate is ultimately modified in this way [2,23,20]. This may imply that the role of polyglutamation in this case may be more related to enzyme-substrate recognition, rather than intracellular trapping.
The Km values of ca. 2–3 μM that we measure for the saturable binding component for folinic acid of two strains of P. falciparum are higher than those of 0.4 and 42 nM, respectively, for folic acid uptake into Lactobacillus
[27], Xenopus oocytes [46], and of 250–700 nM for such uptake into Leishmania parasites, depending upon the species [47]. This may reflect the absolute dependence of these three organisms on folate salvage, whereas P. falciparum also possesses an active folate biosynthesis pathway. The  values we observed are of the same order as the rate of uptake of the vitamin pantothenic acid into the parasite [11] but considerably slower than that of glucose [48,49,10], presumably reflecting the great difference between reusable cofactors required in limited quantities and the enormous consumption of the latter as the principal source of energy. The more rapid uptake of folate into the antifolate-resistant strain of K1 compared to the sensitive FCB strain, as indicated by their quite different  values, could result from the selection of parasites better able to cope with drug-mediated inhibition of folate metabolism by increasing their salvage efficiency, in addition to the familiar mutation events in genes encoding folate pathway enzymes that reduce drug binding to the latter [50]. Moreover, if the mutant enzymes (DHFR-TS and PPPK-DHPS) from such parasites process their substrates in vivo with a lower efficiency than in the wild-type, compensation by an increased flux through the salvage pathway might be important for healthy growth, even in the absence of drug. These observations merit extension to other parasite strains with differing responses to antifolate drugs, to further test this hypothesis.
In principle, import of folates into the infected erythrocyte could occur via the new permeation pathway(s) (NPP) induced in the host cell membrane by the growing parasite [39,41,42]. However, mammalian cells also exhibit two types of endogenous folate transport system, the reduced folate carriers (RFC), which possess multiple transmembrane domains and have a greater affinity for the reduced folates than for folic acid, and the folate receptors (FR), which are GPI-anchored external folate binding proteins that mediate transport of both folic acid and 5-MeTHF with high specificity [51–53]. In mature erythrocytes, the FRs lose functionality with age [51] and the RFC is thought then to be the major transporter type [25,53]. Whatever the exact nature of the relevant transporters on the erythrocyte membrane, they appear not to represent a bottleneck for folate uptake into the parasite, as indicated by our rate experiments. Nor would the parasitophorous vacuolar membrane, which is highly porous and freely permeable to small molecules [54]. The rate-limiting step is therefore passage through the parasite plasma membrane, a process that is strongly inhibited in free parasites both by probenecid, a proven inhibitor of folate transport in mammalian cells [34] and furosemide. Given the similarity in these anion channel blockers (both are benzoic acid derivatives of comparable size with aminosulfonyl substituents on the benzene ring) and the evidence that they target the same sites on infected erythrocytes, we conclude that their major effect with respect to folate uptake at the erythrocyte membrane is likely to be at endogenous folate transporters rather than the NPP. Consistent with the view that the transporters involved in the parasite plasma membrane also have a high degree of specificity for folate, we note that the antifolate drugs PYR and SDX, which have some structural features in common with folate, do not compete with uptake of folinic acid even at very high concentrations. In contrast, the anticancer antifolate MTX, which is a much closer structural analogue to folic acid than either PYR or SDX, does inhibit uptake to a similar extent to the unlabelled folates themselves (μM range) in competition experiments. We note that the lack of inhibition of folate uptake by PYR that we report here rules out our earlier hypothesis [4] that such inhibition might contribute to the synergy observed between PYR and SDX.
We have demonstrated that folate uptake into P. falciparum is dependent upon the presence of d-glucose or other sugar that can be metabolised via glycolysis, a process that is essential to maintain the proton and electrochemical gradients across the parasite plasma membrane [21,8]. d-Fructose can be phosphorylated by hexokinase to fructose 6-phosphate, the substrate of the third glycolytic enzyme, phosphofructokinase-1. Interestingly, although fructose is only about half as efficient as glucose in maintaining overall growth of the parasite [30], it was as efficient as glucose in supporting folate import on a molar basis in our experiments. This may reflect the fact that only a small fraction of the glycolytic flux is likely to be necessary to support import of the relatively low amounts of exogenous folate required by the parasite. Of the other sugars we tested, which were all unable to support uptake, l-glucose is stereochemically excluded, while 6-deoxy-d-glucose and d-xylose can bind to hexokinase but cannot be phosphorylated. d-Galactose, derived principally from lactose hydrolysis, can be converted to glucose 6-phosphate in mammals via a four-step mechanism involving uridine diphosphate derivatives, but its inability to support folate import here would be consistent with an apparent lack of galactokinase and the other enzymes required for this conversion in the predicted proteome of P. falciparum.
The dramatic reduction of folate transport in the absence of a metabolisable sugar mirrors the rapid decline of intracellular ATP concentration and drop in the internal pH of the parasite seen in the same circumstances [21]. This suggested that inward movement of folates depends upon maintenance of a pH (interior alkaline) or electrochemical gradient (interior negative), or both, across the plasma membrane. Such gradients can be generated by membrane-bound ATPases, and a range of organic anions move into bacterial cells using proton symport. To differentiate between movement of folate via symport with protons or another cationic species, we examined the influence of the latter, but saw only minor changes in uptake rates in the presence or absence of Na+, K+, Mg2+ or EDTA. This differs from the folate transport system in Lactobacillus, which although similarly dependent upon glucose, also has a requirement for divalent cations [55,27].
The pH optimum that we observe for folate import (6.5–7.0) corresponds well with that (pH 6.9) measured just outside of the parasite in infected erythrocytes [32], 0.4 units lower than the pH of 7.3 in the parasite cytoplasm [21,32]. The primary mechanism for exporting protons in P. falciparum is thought to be via a V-type H+ ATPase on the plasma membrane [21,32], and consistent with this, we found that concanamycin A, a specific inhibitor of such ATPases [56], inhibited folate uptake in the low nM range. Genes encoding the A and B subunits of a V-ATPase have been identified in P. falciparum
[57,58] and immunological studies indicate that this enzyme is expressed throughout the erythrocytic cycle [32]. Interestingly though, a significant acidification of the extracellular space just outside of the parasitophorous vacuole could only be measured in trophozoites [32], the stage of the life-cycle at which we observe the highest levels of exogenous folate uptake. The substantial proton gradient that is generated via the V-ATPase is the major contributor to an inwardly negative membrane potential of ca. −95 mV [8], but protons are also extruded in a symport system together with the large amounts of lactate generated by parasite glycolysis [12]. This may explain why we did not observe 100% inhibition of uptake with concanamycin, even at μM concentrations, as it blocks only the major portion of the total proton export.
The importance of the proton gradient across the plasma membrane was emphasised by the sensitivity of folate uptake to the ionophores DNP and nigericin, both of which strongly perturb this gradient. In particular, we find that folate uptake is exquisitely sensitive to nigericin, with substantial inhibition at sub-nM levels. Whereas DNP collapses the pH gradient completely by enabling free passage of protons in both directions, the K+/H+ antiporter mechanism of nigericin is presumably limited by the degree to which the K+ gradient can be perturbed, which may explain why we also did not observe 100% inhibition of uptake with this compound. Although the profiles observed for nigericin and concanamycin A can be explained in principle by assuming that neither abolishes the pH gradient completely, we cannot exclude the possibility that there is also a component of folate transport independent of this gradient. This scenario is suggested by the fact that a low level of transport persists even when the external pH is set at the same, or a higher, level than the internal pH of parasites deprived of glucose, and would also be consistent with the two-phase nature of the concentration dependence of uptake. At first sight, this picture is difficult to reconcile with the data for DNP, which by insertion into the membrane, permits rapid equilibration of proton concentrations on each side, but we note that complete abolition of uptake only occurs at ca. 1 mM, a concentration at which quite profound changes to the membrane might be expected, possibly impacting on any proton gradient-independent component.
A plausible model encompassing our observations would be that folate uptake at the parasite plasma membrane is mediated by a transport complex or multi-binding site protein possessing a binding domain specific for folate recognition and a probenecid/furosemide-sensitive proton symport channel through which the folate is internalised. To date, no folate transporters have been functionally identified in P. falciparum, but bioinformatics studies [59] predict the existence of 3 candidate proteins (gene loci MAL8P1.13, PF11_0172 and PF10_0215 in PlasmoDB; http://www.plasmodb.org) that are related to biochemically verified molecules in Leishmania
[47,45], Synechocystis and Arabidopsis
[60]. Related putative candidates have also been identified in Toxoplasma gondii
[6]. This sub-family of folate-biopterin transporters (FBT) are thought on bioinformatic grounds to function as proton symporters [59], and on this basis, the data we present here would be consistent with one or more of these proteins providing the machinery for folate uptake in P. falciparum, which might ultimately present a novel target type for parasite inhibition. We are currently investigating the genes encoding these candidate molecules in knockout studies to better understand the relationship between the phenomena reported here and the parasite proteins involved.

bcecf-am, 2′,7′-bis-(2-carboxyethyl)-5,6-carboxylfluorescein acetoxymethyl ester


Mrc1 protects uncapped budding yeast telomeres from exonuclease EXO1

Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork machinery and is necessary for checkpoint activation after replication stress. In this study, we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1 contributes to the vitality of both cdc13-1 and yku70Δ telomere capping mutants. Cells with telomere capping defects containing MRC1 or mrc1AQ, a checkpoint defective allele, exhibit similar growth, suggesting growth defects of cdc13-1 mrc1Δ are not due to checkpoint defects. This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping. Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding a nuclease that contributes to single stranded DNA accumulation after telomere uncapping, improves growth of cdc13-1 mrc1Δ strains and decreases ssDNA production. Our observations show that Mrc1, a core component of the replication fork, plays an important role in telomere capping, protecting from nucleases and checkpoint pathways.
1
Introduction
Telomeres are specialized DNA–protein complexes at the end of eukaryotic chromosomes. Proper telomere structure is essential for chromosome integrity and genome stability because telomeres protect natural chromosome ends from degradation and end-to-end fusion and because they ensure complete genome replication. Telomeres differ from Double Strand Breaks (DSBs) in that they normally fail to activate DNA repair or DNA damage checkpoint pathways. If that was the case, then they would undergo recombination and chromosomal fusions and dicentric chromosomes would be generated [1–4].
Many proteins associate with telomeric DNA. These proteins ensure that telomeres behave differently from DSB ends and help maintain chromosomal stability. Some telomeric proteins bind specifically to dsDNA and others show higher affinity to ssDNA. In budding yeast, there are numerous proteins with affinity for telomeric dsDNA, such as Rap1, Sir2, Sir3, Sir4, Rif1 and Yku70/Yku80 [5]. The budding yeast telomeric ssDNA ends are thought to be protected by three essential proteins, Cdc13, Stn1 and Ten1 [6–11].
If telomeres become uncapped, they activate a DNA damage response pathway leading to cell cycle arrest [12–14]. Moreover, recently it has been suggested that telomeres trigger a transient DNA damage response in each S phase in order to complete DNA replication and cap chromosome ends [15]. DNA damage response pathways are complex networks which include – among other components – mediators. Mediators facilitate the transmission of the DNA damage signal from sensors to downstream effectors; activation of the latter affecting cell cycle progression [16]. Mrc1 (Mediator of Replication Checkpoint 1) appears to take the mediator role in Rad53 activation under replication stress [17]. However, parallel pathways exist because in mrc1Δ mutants Rad53 activation occurs through Rad9 (another mediator protein), presumably because the accumulation of “DNA damage” rather than “replication defects” in mrc1Δ mutants leads to activation of Rad9 and thereby activation of Rad53 [17].
Mrc1 also appears to be directly involved in DNA replication and, because of this, mrc1Δ cells display a slow S phase [18]. Mrc1 is an active component of the replication machinery, loaded onto DNA shortly after replication initiates, and moving with other components of replication forks [19–21]. In the presence of hydroxyurea, a type of replication stress, Mrc1 appears to form a stable replication-pausing complex preventing the uncoupling of the replication machinery from DNA synthesis [20–22]. According to this model, Mrc1 mediates activation of Rad53 under conditions of replication stress so that subsequent DNA repair events occur and cell replication resumes normal function [22]. However, recent experiments suggest that the role of Mrc1 at stalled replication forks is more than activating Rad53, since mrc1AQ cells, defective in Rad53 activation, are not defective in replication fork initiation or progression [18,20,21]. mrc1AQ is a mutant allele in which SQ/TQ residues have been substituted with AQ, resulting in its inability to mediate phosphorylation and activation of Rad53 [18].
Although Mrc1 is involved in the DNA replication checkpoint, it has been shown that it is not required for the DNA damage checkpoint, since cdc13-1 mrc1Δ double mutants arrest in G2 at non-permissive temperatures [17]. It has been reported that activation of Rad53 in response to telomere shortening still occurs in the absence of Rad9 and that Mrc1 is responsible for this activation in telomerase-deficient cells, in which telomeres continually shorten until they activate a checkpoint [23]. Surprisingly, though, tlc1Δ mrc1Δ double mutants arrest cell division, suggesting that Mrc1 is not required for cell cycle arrest in telomerase negative cells. In contrast, after cdc13-1 induced telomere uncapping, Rad53 activation is entirely Rad9-dependent and Mrc1-independent [23].
Here we investigated the role of Mrc1 at uncapped telomeres, using the temperature sensitive cdc13-1 and yku70Δ mutations to uncap telomeres. Our experiments indicate that Mrc1 protects telomeres from the DNA damage response and that the role of Mrc1 in DNA replication forks, rather than in checkpoint activation, is important for protection of telomeres.
2
Materials and methods
2.1
Yeast strains and plasmids
All strains in the W303 background are RAD5 and they contain an ade2-1 mutation (Supplemental Table 1); therefore yeast extract/peptone/dextrose (YEPD) was supplemented with adenine at 50 mg/l. Strains 3393–3402 are in the S288C background (Supplemental Table 1) and they were generated by mating a single gene deletion mutant array [24] with a cdc13-1 query strain [25]. To construct strains, standard genetic procedures of transformation and tetrad analysis were used [26]. pMRC1 and pmrc1AQ, also carrying the URA3 gene were a gift from Steven Elledge [17,18].
MRC1 was disrupted in two different ways. Firstly, the MRC1 ORF was substituted with KanMX6, with a PCR based method [27]. Primers 5′-tcgttattcgcttttgaacttatcaccaaatattttagtgCGGATCCCCGGGTTAATTAA-3′ (#878) and 5′-ctggagttcaatcaacttcttcggaaaagataaaaaaccaGAATTCGAGCTCGTTTAAAC-3′ (#881), which contain homology to upstream and downstream sequences of MRC1 (bases in lowercase), were used to amplify a 1559 bp KanMX6 sequence (pFA6a-kanMX6; [27]). The PCR product was transformed into yeast and candidate colonies were selected for G418 resistance. Integration of the KanMX6 marker into the MRC1 locus was confirmed by PCR, using two sets of primers: (i) forward 5′-CCAAGAACAGACAAACAACTAAGGA-3′ (#876) with reverse primer 5′-TCAGCATCCATGTTGGAATT-3′ (#81) and (ii) forward 5′-CCATCCTATGGAACTGCCTC-3′(#82) with reverse 5′-CCTAGACTCGGGTGCCATCT-3′ (#880). Disruption of MRC1 was also confirmed by Southern blot (data not shown). Alternatively, MRC1 was substituted with URA3 using a restriction enzyme digest approach. First, pMRC1 was digested with XhoI and a 5008 bp fragment containing the full MRC1 gene was cloned into XhoI digested pIC19H vector (2.7 kb) to create pAT1065. A correct clone was identified by restriction digests. pAT1065 was digested with SpeI to remove a 2.31 kb DNA fragment containing the bulk of MRC1, which was replaced with a 1.3 kb BamHI URA3 gene fragment from pDL349 (pBSB + KS containing a BamHI fragment containing the URA3 gene) by blunt cloning following treatment with DNA polymerase I Large (Klenow) fragment (New England Biolabs). Positive clones were selected by restriction enzyme digests to identify the disruption of the bulk of MRC1 with URA3 (pAT1066). Disruption of MRC1 was also confirmed by Southern blot (data not shown). To disrupt MRC1, pAT1066 was digested with XhoI prior to transformation of yeast.
2.2
Spot tests
Single colonies were inoculated into 2 ml YEPDextrose (YEPD) and incubated overnight, with aeration, at 23 °C. The following day, 200 μl of each culture was inoculated into 2 ml of fresh YEPD and returned to 23 °C. Cells were grown for three more hours, and cell numbers were determined in a haemocytometer. The cells were then centrifuged (13,000 rpm for 10 s in a microcentrifuge), washed twice with sterile water and resuspended in water to a final concentration of 1.5 × 107 cells/ml. A five-fold dilution series of each of the cultures was prepared using sterile water in a 96 well plate and 3–5 μl spotted onto plates using a 48-prong replica plating device. Plates were incubated at various temperatures for 2–3 days before being photographed. For spot tests with strains containing pMRC1, pmrc1AQ or pRS416 the steps were as described above, but strains were grown on selective medium (-URA). All strains shown as if on a single agar plate were grown on the same plate, although in some cases their positions were moved using Adobe Photoshop and Adobe Illustrator CS. Unless otherwise stated, at least two different strains of the same genotype were spot tested and representative examples are shown.
2.3
Yeast transformation
High efficiency transformations needed for gene disruptions were performed according to Gietz et al. [28]. For plasmid transformations a more rapid method was used [29].
2.4
Western blots
Protein extracts were prepared by glass bead breakage in TCA, essentially as previously described [30,31]. Bio-Rad gels (7.5% Tris–HCl), Schleicher and Schuell Protan Nitrocellulose membranes and the Pierce Supersignal West Pico Chemiluminescent Substrate detection kit were used in a standard Western blot procedure. Rabbit anti-Rad53 polyclonal antibody (AbDL50, 1:1000 dilution, a gift from Dan Durocher [32]) was a primary antibody used with an anti-rabbit-HRP (AbDL7, 1:10,000 dilution, Dako P0448) as a secondary antibody used. Mouse anti-tubulin (TAT-1, AbDL42, 1:2000 dilution, a gift from Keith Gull [33] and anti-mouse-HRP (AbDL6, 1:10,000 dilution, Dako P0447) were used for tubulin loading controls.
2.5
Telomere length measurement by Southern blot
Strains were grown to saturation in liquid YEPD at 23 °C. DNA from each strain was subjected to XhoI cut. The digested DNA was loaded on a 0.8% agarose gel, run at low voltage overnight, transferred to a Magna nylon membrane and UV cross-linked. The membrane was then hybridised with a Y′-TG probe [34]. A non-radioactive detection kit was used for the detection of the hybridisation (Amersham, Arlington Heights, IL).
2.6
Synchronous cultures
cdc13-1 cdc15-2 bar1Δ strains with additional mutations (see Supplemental Table 1) were grown in YEPD at 23 °C overnight. In the morning, cells were adjusted to a concentration of 8 × 106 buds/ml in 250 ml. Cultures were grown for three more hours, then arrested with 20 nM α-factor for a further 2.5 h. The cultures were then released from G1 arrest by centrifugation and washed twice in YEPD and placed at 36 °C, 40 min after the culture was first centrifuged. Cell cycle position was monitored as previously described [13]. DNA was prepared and the fraction of single stranded DNA (ssDNA) was measured as previously described [35].
2.7
Asynchronous cultures
cdc13-1 strains with additional mutations indicated were grown in YEPD at 23 °C overnight. In the morning, cells were adjusted to a concentration of 1 × 107 cells/ml and temperature was raised to 27.3 °C. Every 90 min samples were taken for cell cycle position and cell density was re-adjusted to 1 × 107 cells/ml. Cell numbers were determined with a haemocytometer. Samples for Western blots were collected from exponentially growing cultures 2 h after the temperature was raised from 23 °C to 36 °C.
2.8
Cell cycle position determination
A 1 ml sample of culture was centrifuged for 8–10 s at high speed, the supernatant was aspirated, and cells were fixed at 70% ethanol overnight. The fixed cells were washed twice in water before being resuspended in 4,6-diamidino-2-phenylindole (DAPI, 0.2 μg/ml). Cell cycle distribution was monitored by DAPI staining of nuclei and fluorescence microscopy. For DAPI staining, 100 cells for each sample were counted and classified as: (1) unbudded, single DAPI-stained body; (2) small budded, single DAPI-stained body, with the bud <50% of the diameter of the mother cell; (3) medial nuclear division, single DAPI-stained body, with bud >50% diameter of mother cell, the cdc13-1 arrest point; (4) late nuclear division, two buds, and two DAPI-stained bodies, the cdc15-2 arrest point [36].
3
Results
3.1
Mrc1 contributes to the vitality of cdc13-1 and yku70Δ mutants
Since Mrc1 plays a role in the checkpoint response to stalled replication, we wondered if it also plays a role at uncapped telomeres. The temperature sensitive cdc13-1 mutation causes a defect in Cdc13, a telomere binding protein, and cells containing this mutation accumulate large amounts of ssDNA at telomeres at non-permissive temperatures [12,37,38]. Interestingly, deletion of checkpoint proteins, like Chk1, Mec1, Mec3, Rad9, Rad17, Rad24 and Rad53 improves growth of cdc13-1 strains at semi-permissive temperatures [39–41]. This is presumably because checkpoint pathways inhibit cell division by responding to low levels of ssDNA that accumulates at telomeres at semi-permissive temperatures. Deletion of other checkpoint proteins, like the MRX complex, which appears to play a role in telomere capping, worsens the growth of cdc13-1 strains [42]. Therefore, we wanted to investigate whether Mrc1 plays a role at uncapped telomeres and, if so, whether it behaved like Rad9 or MRX. Fig. 1A shows that deletion of MRC1 dramatically reduces the growth of cdc13-1 mutant strains at 25 °C. The effect of Mrc1 is not as profound as that of the MRX complex, as cdc13-1 mre11Δ and cdc13-1 rad50Δ display more severe growth defects than cdc13-1 mrc1Δ even at 23 °C (Supplementary Fig. 1). Thus, Mrc1, like MRX, but unlike the majority of checkpoint proteins, contributes to the vitality of cdc13-1 strains.
We next wanted to investigate whether analogous growth defects of cdc13-1 mrc1Δ strains occur in yku70Δ strains. Yku70 is a telomere capping protein which is also involved in dsDNA damage repair and in Non-Homologous End Joining (reviewed in Ref. [43]). Deletion of YKU70 results in a temperature sensitive phenotype at 37 °C, due to telomere uncapping, which activates a Chk1-dependent cell cycle arrest [36]. Fig. 1B demonstrates that deletion of MRC1 results in a severe growth defect of yku70Δ mutant strains at 35 °C and 36 °C. Thus, Mrc1 contributes to the vitality of yku70Δ strains. We conclude that Mrc1 contributes to the vitality of two cell types defective in telomere capping.
Mrc1, Tof1 and Csm3 are three proteins that play similar, although distinct roles in DNA replication [19–21,44]. Therefore, we wished to address whether Tof1 and Csm3, like Mrc1, contributed to the vitality of cdc13-1 mutants. Spot test analysis showed that although deletion of TOF1 or CSM3 also confers some growth defects on cdc13-1 mutants, deletion of MRC1 has a stronger phenotype (Fig. 1C). Therefore, we decided to focus on understanding the role of MRC1 at uncapped telomeres.
3.2
Growth defects of cdc13-1 mrc1Δ cells are not due to checkpoint defects
In budding yeast, two independent roles have been previously reported for Mrc1. One role implicates Mrc1 as a mediator of checkpoint activation under replication stress and the other role is as part of the replication machinery [17–21]. Therefore, we investigated whether the heightened temperature sensitivity phenotype of cdc13-1 mrc1Δ mutant strains is a result of a replication defect, a checkpoint defect or both.
cdc13-1 mrc1Δ mutants were complemented with either wild type pMRC1, pmrc1AQ or an empty vector (pRS416) and strains were grown at various temperatures. Fig. 2 shows that at 26.2 °C, complementation of mrc1Δ cdc13-1 mutant strains, with either pMRC1 or pmrc1AQ allele improves growth compared to the empty vector control (compare rows 1–3). Thus, we conclude that the checkpoint role of Mrc1 is not important for the vitality of cdc13-1 strains.
At higher temperature we noticed an increased growth of cdc13-1 mrc1Δ cells carrying the mrc1AQ allele, compared to pMRC1. However, this phenotype was observed even in the presence of the wild type MRC1 allele, suggesting that this effect is due to some type of dominant effect of mrc1AQ (Fig. 2, compare rows 2 and 11).
3.3
Exo1 inhibits growth of cdc13-1 mrc1Δ and yku70Δmrc1Δ mutants
EXO1 encodes a nuclease known to contribute to ssDNA production at uncapped telomeres of cdc13-1 and yku70Δ strains [36,39]. If Exo1-dependent ssDNA production at uncapped telomeres is responsible for the poor growth of cdc13-1 mrc1Δ and yku70Δ mrc1Δ mutants, then removing Exo1 should suppress their poor growth. Fig. 3A demonstrates that at 25 °C, cdc13-1 mrc1Δ exo1Δ triple mutants exhibit better growth than cdc13-1 mrc1Δ strains, showing that Exo1 contributes to the growth defects observed in cdc13-1 mrc1Δ strains. Importantly, deleting EXO1 also reverses the growth defect of mrc1Δ yku70Δ mutants (Fig. 3B). These data suggest that Mrc1 protects uncapped telomeres from Exo1.
3.4
Effects of checkpoint mutations on cdc13-1 mrc1Δ and yku70Δ mrc1Δ growth
To understand if checkpoint pathways are activated in mrc1Δ strains after telomere uncapping, we wanted to combine cdc13-1 mrc1Δ and yku70Δ mrc1Δ strains with checkpoint mutations. A genetic screen has revealed that mrc1Δ is synthetically lethal with rad9Δ, rad17Δ or rad24Δ checkpoint mutations [24]. Consistent with these results we were unable to recover viable offspring carrying mrc1Δ in combinations of any of these checkpoint genes (data not shown). However, we were able to combine mrc1Δ with rad53Δ and chk1Δ, encoding two downstream checkpoint kinases (analogues of human Chk2 and Chk1, respectively).
When cdc13-1 mutants grow at non-permissive temperatures Rad53 and Chk1 dependent parallel pathways are activated [36,45,46]. We deleted RAD53 or CHK1 and examined the effects in cdc13-1 mrc1Δ mutants. Deletion of RAD53 requires simultaneous deletion of SML1 to obtain viable spores. Sml1 is a small protein that inhibits the activity of ribonucleotide reductase (RNR) which catalyzes the rate-limiting step of de novo dNTP synthesis [47]. Normally Sml1 is degraded in a Rad53-dependent manner during S phase [48]. We found that removal of RAD53 and SML1 improved the growth of cdc13-1 mrc1Δ strains (Fig. 4A). However, deletion of SML1 alone (cdc13-1 mrc1Δ sml1Δ) also rescued the growth defects associated with MRC1 deletion (Fig. 4A). Thus, we were unable to observe any strong role for Rad53, in maintaining vitality of cdc13-1 mrc1Δ mutants, other than in degrading Sml1. We suggest that the reason that deleting Sml1 improves the growth of cdc13-1 mrc1Δ strains is that increased ribonucleotide reductase activity may stabilise the replication forks.
Removal of Chk1, like removal of Rad53, does not rescue growth of cdc13-1 mrc1Δ strains, indicating that Chk1 does not inhibit growth of these mutants (Fig. 4B). Therefore, we find no evidence that inactivating DNA damage checkpoint pathways improves growth of cdc13-1 mrc1Δ mutants.
A CHK1 deletion strongly rescues growth of yku70Δ mrc1Δ mutants at restrictive temperatures (Fig. 4C) similarly to its effect in yku70Δ mutants [36]. Thus, mrc1Δ yku70Δ uncapped telomeres qualitatively behave like yku70Δ uncapped mutants. The effects of chk1Δ in yku70Δ mrc1Δ and cdc13-1 mrc1Δ strains are consistent with earlier findings showing that the Chk1-dependent pathway is more important in yku70Δ mutants [36,45].
3.5
Mrc1 is not required for the cell cycle arrest after cdc13-1 uncapping
To directly test whether Mrc1 plays a checkpoint role in cdc13-1 strains, cdc13-1 strains with additional mutations were first grown at the semi-permissive temperature 27.3 °C. Strains defective in telomere capping (cdc13-1) arrest at medial nuclear division before entry to anaphase after 3 h at 27.3 °C (Fig. 5A). As expected, when the checkpoint is compromised in cdc13-1 chk1Δ, cdc13-1 rad9Δ or cdc13-1 rad53Δ cells, no accumulation at medial nuclear division is observed over 9 h (Fig. 5A). In contrast, cdc13-1 and cdc13-1 mrc1Δ strains rapidly accumulate at medial nuclear division and within 3 h more than 90% of cells are arrested at this point. Consistent with these conclusions cell numbers stopped increasing in cdc13-1 and cdc13-1 mrc1Δ strains, but continued to increase in the other strains (Fig. 5B). We conclude that Mrc1 is not required for the checkpoint response to cdc13-1 dependent telomere uncapping.
Rad53 and Chk1 are components of parallel checkpoint pathways that respond to cdc13-1 induced telomere uncapping [45,46]. It appears that the Rad53 pathway is more important for arrest of cdc13-1 mrc1Δ mutants because 80% of cdc13-1 mrc1Δ chk1Δ cells have arrested at medial nuclear division by 7.5 h, whereas there is no arrest of cdc13-1 mrc1Δ rad53Δ cells.
To test the role of Mrc1 in checkpoint control in a single cell cycle we combined mrc1Δ with cdc13-1 cdc15-2 and bar1 mutations. Over many years we and others have used these mutations to determine the effects of checkpoint proteins in responding to telomere uncapping [13,39,40,45]. Bar1 encodes a protease that degrades the mating pheromone α-factor. Cells bearing the bar1 mutation can efficiently arrest in G1 phase of the cell cycle with low levels of α-factor. Cdc15 is necessary for mitotic exit. At 36 °C, cdc13-1 cdc15-2 bar1 control strains, released from alpha factor arrest, accumulate at medial nuclear division due to cdc13-1-dependent telomere uncapping. However, if cells have escaped the G2/M checkpoint, like cdc13-1 rad9Δ cdc15-2 bar1 strains, they arrest at late nuclear division due to cdc15-2 and they are unable to proceed to the next cycle. The cdc15-2 dependent cell cycle arrest helps in two ways, it ensures that DNA damage checkpoint defects are easily quantified because cells with checkpoint defects accumulate at a later stage of the cell cycle and that DNA damage caused by cdc13-1 is not amplified during new rounds of DNA replication.
cdc13-1 cdc15-2 bar1 strains with additional mutations were arrested with α-factor, then released from G1 and transferred to a non-permissive temperature to induce telomere uncapping and the cell cycle position was monitored. Fig. 5C shows that in contrast to cdc13-1 rad9Δ strains cdc13-1 and cdc13-1 mrc1Δ strains arrest at medial nuclear division with similar kinetics at 36 °C, supporting the idea that Mrc1 does not play a role in checkpoint activation after cdc13-1 telomere uncapping. Interestingly, cdc13-1 mrc1Δ exo1Δ remain arrested at medial nuclear division (Fig. 5C and D) which contrasts to the behaviour of cdc13-1 exo1Δ that begin to escape arrest during the 4-h period in analogous experiments [39]. This difference most likely reflects the fact that cdc13-1 mrc1Δ exo1Δ mutants have more severe telomere capping defect than cdc13-1 exo1Δ mutants and therefore activate a stronger checkpoint signal.
In response to both replication stress and DNA damage, activation of the checkpoint machinery induces phosphorylation and activation of Rad53 kinase. Therefore, we addressed the role of Mrc1 in Rad53 phosphorylation after telomere uncapping. cdc13-1 strains were exposed to 36 °C for 2 h and Rad53 phosphorylation was measured by Western blot. Rad53 phosphorylation is observed in cdc13-1 mrc1Δ strains but not in cdc13-1 rad9Δ mutants (Fig. 5F), confirming a previous study [23]. We conclude that activation of Rad53 after cdc13-1 dependent uncapping at non-permissive temperatures is Rad9-dependent but Mrc1-independent.
3.6
Mrc1 contributes to telomere length regulation
If Mrc1 plays a protective role at telomeres, this predicts that strains lacking Mrc1 may have short telomeres. Fig. 6A shows that absence of Mrc1 results in shorter telomeres, compared to the wild type. However, the telomere length defects of mrc1Δ mutants are not as severe as in rad50Δ or yku70Δ mutants, and this may help explain why cdc13-1 mrc1Δ cells grow better than cdc13-1 rad50Δ cells (Supplementary Fig. 1). Our experiment is consistent with replication proteins having an important role in telomere length regulation [49–52].
3.7
Mrc1 protects telomeres from extended ssDNA accumulation
All our data suggest an important role of Mrc1 in telomere capping but no role in cell cycle arrest. To directly test the role of Mrc1 in telomere capping, we measured ssDNA accumulation on the 3′ TG strand, at PDA1, a single copy locus approximately 30 kb away from the right end of chromosome V in cdc13-1 strains (Fig. 6B). cdc13-1 strains were synchronised, as in Fig. 5C and D in order to follow the effects of Mrc1 on ssDNA accumulation at non-permissive temperatures within a single cell cycle. We find that cdc13-1 mrc1Δ mutants, like cdc13-1 rad9Δ strains, accumulate more 3′ TG ssDNA at PDA1, 30 kb from the uncapped telomere, compared to cdc13-1 strains (Fig. 6C) [39]. This shows that Mrc1, like Rad9, protects cdc13-1 mutants from ssDNA production. Consistent with our conclusion, increased ssDNA accumulation, closer to the telomere, in telomere repeats, was recently reported in cdc13-1 mrc1Δ and yku70Δ mrc1Δ mutants using both in gel and dot blot analyses [53]. Importantly, ssDNA production is reduced in cdc13-1 mrc1Δ exo1Δ strains in comparison to cdc13-1 mrc1Δ strains showing that Mrc1 protects uncapped telomeres from Exo1-dependent nuclease action. This ssDNA data is consistent with our finding that Exo1 contributes to the poor growth of cdc13-1 mrc1Δ and yku70Δ mrc1Δ mutants (Fig. 3). Taken together, we conclude that Mrc1 inhibits accumulation of Exo1-dependent ssDNA accumulation after telomere uncapping and, by this criterion, contributes to telomere capping.
4
Discussion
Our experiments demonstrate that Mrc1 contributes to the vitality of budding yeast cells with uncapped telomeres. Therefore, Mrc1 behaves differently from many other known checkpoint proteins such as Chk1, Mec1, Rad9, Rad17, Rad24 or Rad53, deletion of which improves the growth of cdc13-1 mutants at semi-permissive temperatures [39,40]. The effect of Mrc1 is more similar to that of the MRX complex, another checkpoint complex with roles in telomere capping [36,42,54,55]. It seems that it is the role of Mrc1 at the replication fork that contributes to the vitality of telomere capping mutants, rather than its role in checkpoint activation. Our findings are in accordance with recent work that demonstrated a protective role of Mrc1 in cells with cdc13-1 or yku70Δ uncapped telomeres or in telomerase deficient cells [53]. Additionally, our work demonstrates that the growth defects of cdc13-1 mrc1Δ and yku70Δ mrc1Δ mutants and enhanced ssDNA levels of cdc13-1 mrc1Δ strains are suppressed when the nuclease encoded by EXO1 is deleted. Therefore, Mrc1 protects uncapped telomeres from Exo1.
Mrc1 is recruited to the replication machinery as DNA replication initiates and is required for normal rates of replication fork progression [17,18,22]. The biochemical role of Mrc1 in replication fork progression is unclear which makes it difficult to know its precise role in telomere capping. Mrc1 is also part of a replication-pausing complex formed when replication is arrested by the S phase poison hydroxyurea (HU), and required for replication fork restart after HU. However, this restart role for Mrc1 is not universal, since Mrc1 plays no role in replication restart after cells are treated with the alkylating agent MMS [22].
It is interesting that there is evidence from budding yeast, fission yeast and human cells that telomeric sequences contain DNA regions that slow or stall replication forks [15,56,57]. From this it seems plausible that telomeric DNA may be more dependent on proteins like Mrc1, which contribute to fork stability and restart, than other chromosomal regions. That is because the replication fork struggles to reach the end of the chromosome in mrc1Δ mutants where a telomere capping defect is observed.
Numerous studies on budding yeast mutants with DNA replication defects have demonstrated interactions between DNA replication and telomere structure. For example both cdc17/pol1 and cdc44/rfc1 (large subunit of replication factor C) mutants affect telomere length [49]. Here we show that budding yeast mrc1Δ mutants have short telomeres. In S. cerevisiae, cdc17/pol1 mutants, encoding temperature sensitive DNA polymerase α, exhibit very long telomeres, high levels of telomeric ssDNA and elevated recombination at telomeres [50,51]. Interestingly, the B subunit of DNA polymerase α physically interacts with Stn1, which in turn interacts with Cdc13 [10,52]. This shows there is a very direct interaction between budding yeast telomere capping proteins and the replication fork machinery, and suggests that telomere capping is intimately linked with DNA replication. In this regard it is, perhaps, relevant that the 5′–3′ exonuclease Exo1 is involved in generating single stranded DNA at uncapped telomeres [36,39] and in processing stalled replication forks [58] and highlights the similarities between uncapped telomeres and stalled replication forks.
cdc13-1 cells maintain a functional telomere cap (low levels of telomeric ssDNA), when released from G1 arrest into the S phase poison hydroxyurea. HU stalls replication forks and stops late origins of replication from firing. However, if the same cdc13-1 cells are permitted to complete DNA replication, by removing the S phase poison HU, telomere uncapping occurs and high levels of ssDNA are observed [59]. Therefore, Cdc13-dependent telomere capping may depend on a coordinated interaction between the chromosome end, Cdc13/Stn1/Ten1 and the DNA replication fork. Further studies examining the interactions between telomeric DNA, the telomere cap and the replication fork will be necessary to better understand these interactions.


Gadolinium and nephrogenic systemic fibrosis: time to tighten practice

Nephrogenic systemic fibrosis (NSF) is a relatively new entity, first described in 1997. Few cases have been reported, but the disease has high morbidity and mortality. To date it has been seen exclusively in patients with renal dysfunction. There is an emerging link with intravenous injection of gadolinium contrast agents, which has been suggested as a main triggering factor, with a lag time of days to weeks. Risk factors include the severity of renal impairment, major surgery, vascular events and other proinflammatory conditions. There is no reason to believe that children have an altered risk compared to the adult population. It is important that the paediatric radiologist acknowledges emerging information on NSF but at the same time considers the risk:benefit ratio prior to embarking on alternative investigations, as children with chronic kidney disease require high-quality diagnostic imaging.
Introduction
Nephrogenic systemic fibrosis (NSF; formerly named nephrogenic fibrosing dermopathy) is a systemic disease that was first described in 1997 and recognized as a separate clinical entity in 2000 [1–4]. Until now, all confirmed cases of NSF have been reported in patients with chronic kidney disease (CKD) with estimated glomerular filtration rates (eGFR) <60 ml/min per 1.73 m2 or end-stage renal failure requiring renal replacement therapy. NSF affects both sexes with a reported age range of 8–87 years and has no race predilection [5, 6]. Relatively few paediatric cases have been reported, which may reflect the lower incidence of CKD in children.
Patients present with swelling, induration and tightening of the skin, often with a peau d’orange appearance or textured plaques, papules or nodules, usually limited to the extremities (from ankles to mid-thighs and from wrists to mid-upper arms). The skin lesions sometimes involve the trunk, but spare the neck and face. The condition develops over days to weeks and may ultimately result in severe contractures and disability. Patients may experience burning, itching or sharp pains in involved areas. Deep pain has also been described in the hips and ribs. Radiography may reveal calcifications of the soft tissue [1, 5–9]. Systemic involvement may be fatal with fibrosis of the lungs, skeletal muscles, pleura, pericardium, myocardium, kidneys, muscle, bone, testes and dura [2–4, 10]. Clinical differential diagnoses include scleroderma, scleromyxoedema, systemic sclerosis/morphoea, eosinophilic fasciitis and eosinophilia-myalgia syndrome, and the condition may mimic cellulitis, panniculitis or drug reactions [6–8, 11, 12].
The severity of CKD appears to be the main determinant of risk. Also, several contributing risk factors have been suggested: hypercoagulability and thrombotic events; cardiomyopathy; hepatic disease; idiopathic pulmonary fibrosis; systemic lupus erythematosus; brain tumours; recent surgical procedures, especially those with a major vascular component; and other proinflammatory conditions, including liver transplantation [5, 6, 8, 13, 14].
A specific aetiology is still unknown. The only common factor in all patients is the underlying kidney disease, with most patients dialysis-dependent. However, NSF has also been reported in patients who have never been dialysed [1, 5, 6, 8]. The physiopathological mechanism for fibrosis is not yet known. Histopathologically, biopsies from the skin lesions show a proliferation of fibrocytes (which may extend into subcutaneous tissue), together with thick collagen bundles and surrounding clefts with a variable amount of mucin and elastic fibres. Fibrocytes have a specific immunophenotype and express CD34, CD11b, CD45, HLA-DR, CD71, CD80, CD86, which implies that they are recruited from the circulation and do not arise as a result of proliferation in situ. Inflammatory cells are not usually evident, although small clusters of perivascular mononuclear cells may be seen [11, 15, 16].
Therapeutic options in NSF are limited. Improvements have been reported with extracorporeal photopheresis, plasmapheresis, ultraviolet phototherapy, intravenous immunoglobulin and renal transplantation [17–21].
Link with gadolinium-based contrast agents
The main concern for the radiologist is a growing body of evidence that clinical use of gadolinium-based contrast agents may trigger the disease. A number of patients with NSF seem not to have had any previous gadolinium exposure, but these are still under investigation. Grobner [22] was the first to report this association in January 2006. Grobner described nine patients with end-stage renal failure on dialysis who underwent MR angiography (MRA) with gadodiamide (Omniscan; GE Healthcare, Chalfont St Giles, UK); five (mean age 57.2±10.7 years) developed NSF after 2–4 weeks. All patients who developed NSF had confirmed metabolic acidosis, whilst all patients who did not develop NSF had normal pH and bicarbonate level at the time of the MRA. The average contrast agent volume used was 35 ml.
Marckmann et al. [23] reviewed case notes from their own nephrology department from August 2005 to May 2006 and found 13 patients with confirmed NSF, all exposed to gadodiamide. All patients were adults (33–66 years of age, mean 50 years), developed NSF 2–75 days (mean 25 days) after gadolinium exposure and had an eGFR <8 ml/min. From this cohort of renal patients the authors estimated an odds ratio between 32.5 and 47.6 for developing NSF following gadolinium exposure.
The link between gadolinium exposure and NSF has been further strengthened by two reports of gadolinium deposits in tissue samples of patients with NSF. High et al. [24] detected gadolinium in 4 out of 13 skin and soft-tissue biopsies from seven patients with documented NSF. All patients with NSF received gadolinium-based contrast medium prior to disease and the interval between dosing and biopsy could be determined with certainty only for two of four patients (age range 4–11 months). The same group reported in a research letter following quantitative analysis of the four tissue samples in which gadolinium was detected that the amount of gadolinium in the affected tissue of patients with NSF was approximately 35- to 150-fold the level of gadolinium in the bone of healthy volunteers with normal renal function. In a case report, Boyd et al. [25] found gadolinium deposits in areas of calcium phosphate deposition in blood vessels in a 68-year-old patient with NSF who had been previously exposed to gadodiamide.
Sadowski et al. [26] retrospectively reviewed data from 13 patients with confirmed NSF (eight males, five females, age 17–69 years), all exposed to gadodiamide. The median time between onset of NSF symptoms and last contrast-enhanced MRI examination was 11.5 days. The dose of contrast agent administered varied from 0.1 to 0.31 mmol/kg (actual) body weight. At the time of the contrast-enhanced MRI all 13 patients had CKD (eGFR <60 ml/min per 1.73 m2) and were hospitalized for a proinflammatory event (major surgery, infection or vascular event). The group with NSF had significantly lower eGFR, more proinflammatory events, and more contrast-enhanced MR examinations per patient (P<0.002) than did the control group. The incidence of NSF in this study and institution was 4.6% per year.
A review of six further NSF cases that were also related in time to gadodiamide administration has recently been published [27]. Patients were aged 23–71 years and the onset of symptoms consistent with NSF was between 19 days and 2 months after gadodiamide exposure. All patients had severe renal failure and were exposed to a gadodiamide dose ranging from 0.11 to 0.36 mmol/kg body weight. Despite having normal serum bicarbonate, five of the six patients had an elevated anion gap metabolic acidosis.
Broome et al. [28] have recently reported a retrospective study of 12 patients with NSF, all of whom received a double dose (0.2 mmol/kg body weight) of gadodiamide 2–11 weeks before the development of skin fibrosis. This group included eight men and four women (age 26–64 years) and all had renal insufficiency at the time of gadodiamide administration. The odds ratio for development of NSF in the gadodiamide-exposed patients compared with the gadodiamide-unexposed patients was 22.3 and the prevalence of NSF among gadodiamide-exposed dialysis patients was 4.0%. The odds ratio for development of NSF with double-dose compared to single-dose gadodiamide administration was 12.1. Ten patients were dialysed within 2 days of gadodiamide administration, but this failed to prevent the development of NSF.
Although the overwhelming majority (around 90%) of patients with gadolinium-associated NSF had been exposed to gadodiamide, there are now several reports of exposure to other gadolinium contrast agents. In a recent editorial in Radiology, Kuo et al. [29] reported 57 patients with NSF investigated by the United States Food and Drug Administration (FDA). In 43 of these the NSF was linked to gadodiamide; in 6 to gadopentetate dimeglumine (Magnevist; Berlex Imaging, Montville, N.J.); in 2 to gadoversetamide (OptiMARK; Mallinckrodt, St. Louis, Mo.); and in 3 to gadodiamide plus gadoversetamide; in 3 other patients, the associated specific gadolinium-based MR contrast agent was not definitively identified. Sadowski et al. [26] also reported one patient with confirmed NSF associated with both gadodiamide and gadobenate dimeglumine (MultiHance, Bracco Diagnostics, Milan, Italy). NSF related to specific gadolinium-based contrast agents is also monitored by the manufacturers, who have recently published several reports [30–32]. In a retrospective study in Scotland of 1,826 haemodialysis patients, Collidge et al. [33] found a positive association between the cumulative dose of gadodiamide and the development of NSF. Patients who developed NSF had received a higher median cumulative dose of gadodiamide (0.39 mmol/kg vs. 0.23 mmol/kg in patients without NSF).
A case-control study by Marckmann et al. [34] conducted in 19 patients with histologically verified NSF and 19 matched controls found that increasing cumulative gadodiamide exposure, high-dose epoietin-b treatment and higher serum concentrations of ionized calcium and phosphate increase the risk of gadodiamide-related NSF in patients with renal failure.
Pathophysiology
Although not completely understood, several factors are known to contribute to the physiopathological mechanism in NSF. Gadolinium chelates are mostly eliminated through the kidneys. Renal impairment will therefore prolong the presence of these agents in the body, facilitating the release of the toxic free gadolinium ion (Gd3+) by dissociation from its chelate (chemical imbalance, acidosis, anion gap, inflammation) and its subsequent binding to endogenous ions. This chemical process, known as transmetallation, may be promoted by acidosis and anion gap, e.g. under inflammatory conditions [35–40]. It has therefore been suggested that both the chemical stability of a particular chelate (cyclic compounds are more stable than linear ones in vitro) and the dose administered are risk factors for triggering NSF [28, 41]. Once released Gd3+ forms hydroxides and phosphates (insoluble at a pH >6.2) and these are probably engulfed by phagocytic cells [42]. This might affect the reticuloendothelial system, inhibiting the activity of certain enzymes with consequent foreign body and fibrous reactions [41, 43–45]. Gadodiamide, the agent involved in NSF in most patients to date, is the least stable in terms of susceptibility to transmetallation in vitro, being an open-chain compound [46]. Free gadolinium ions may remain in the body for weeks to months, allowing more time for toxic effects [47]. Gadolinium is a well-known inorganic calcium channel blocker and its acute toxicity may also partly be explained via this mechanism [48, 49]. A review of the properties of commercial gadolinium contrast agents and their relation to NSF is presented in Table 1.
Table 1Properties and approval status of extracellular gadolinium contrast agents (CNS central nervous system, MRA magnetic resonance angiography, N/A not available)Chemical nameTrade nameManufacturerCharge and chemical structureConditional stability at pH 7.4 (log)Excess chelate (mg/ml)Thermodynamic stability constant (log)Kinetic stabilityaBody region(s) approvedApproval statusApproved doses for imaging (mmol/kg)Approved doses for children (mmol/kg)NSF related to gadoliniumGadodiamideOmniscanGE HealthcareNonionic linear14.91216.935 sCNS, whole bodyUSA, EU, JapanBody 0.1–0.3, CNS 0.1–0.3, MRA 0.1–0.3From 6 months: 0.1Yes (180 cases worldwide)Gadopentetate dimeglumineMagnevistBayer Schering PharmaIonic linear17.70.422.110 minCNS, whole bodyUSA, EU, JapanBody 0.1, CNS 0.1–0.2, MRA 0.1–0.30.1 (doses of 0.2 may be used if necessary for children older than 2 years)Yes (78 cases worldwide)Gadobenate dimeglumineMultiHanceBraccoIonic linear16.9None22.6N/ACNS, liverUSA, EULiver 0.05, CNS 0.1, MRA not approvedNot approved <18 yearsYes (1 case in a patient coadministered Omniscan)GadoversetamideOptiMARKTycoNonionic linear15.028.416.6N/ACNS, liverUSABody 0.1, CNS 0.1, MRA not approvedNot approved <18 yearsYesGadoterate meglumineDotaremGuerbetIonic cyclic18.8None25.8>1 monthCNS, whole bodyEUBody 0.1, CNS 0.1–0.3, MRA 0.20.1NoGadoteridolProHanceBraccoNonionic cyclic17.10.2323.83 hCNS, whole bodyUSA, EU, JapanBody 0.1–0.3, CNS 0.1–0.3, MRA not approved0.1 from 2 years, caution 6 months to 2 years, contraindicated <6 monthsNoGadobutrolGadovistBayer Schering PharmaNon-ionic cyclicN/AN/A21.85 minCNS, MRAEU, CanadaBody not approved, CNS 0.1–0.3, MRA (imaging 1 FOV) 0.1–0.15, MRA (imaging >1 FOV) 0.2–0.3Not approved <18 yearsNoGadoxetic acidPrimovistBayer Schering PharmaIonic linearN/AN/A23.5N/ALiverUSA, EUBody 25 μmol/kg or 0.1 ml/kg, CNS not approved, MRA not approvedNot approved <18 yearsNoGadofosvesetVasovistBayer Schering PharmaIonic linearN/AN/AN/AN/AAbdominal and limb vesselsEUMRA 0.03Not approved <18 yearsNoaKinetic stability: dissociation half-life at pH 1.0.Table modified from Bellin MF (2006) Eur J Radiol 60:314–323.
NSF in children
NSF in children has also been reported. Five patients under 18 years of age have thus far been reported (discussed below) out of a total of ten in the central database of the International Center for Nephrogenic Fibrosing Dermopathy Research (Dr. S. Cowper, personal communication). However, 13 possible patients have been reported to an online paediatric nephrology forum in June 2007 (https://listhost.uchicago.edu/mailman/private/pedneph/) and these are still being investigated in relation to previous gadolinium exposure.
In 2003 Jan et al. [50] reported two paediatric patients with nephrogenic fibrosing dermopathy: a 16-year-old female patient with a life-long history of kidney failure and an 8-year-old boy with a 9-month history of membranoproliferative glomerulonephritis type II. Both patients had the clinical and histopathological features of NSF and no contrast-enhanced MRI procedures were known to be associated with these patients. However, this was before the publication of a possible link with gadolinium.
Jain et al. [51] reported two more patients with NSF in 2004: a 9-year-old boy on peritoneal dialysis and a 19-year-old male on haemodialysis with a 10-year history of previous failed renal transplants, both diagnosed with NSF on skin biopsies. The 9-year-old boy had a solitary MRA performed in June 2002, approximately 2 months prior to development of NFD. The history of the 19-year-old male was more complicated as he underwent 13 separate MRA procedures, all with gadolinium contrast agents, between April 2000 and August 2004. At least nine of the MRAs were performed prior to the NFD diagnosis in January 2003 [52]. Also, both patients had persistent metabolic acidosis in the months preceding the development of NSF, which may have been a contributing factor [22].
DiCarlo et al. [53] described another patient with histopathologically proven NSF. This patient was a 17-year-old male renal transplant recipient (15 years following transplant) who had recently begun peritoneal dialysis due to graft failure. No relationship to MRI investigations was mentioned.
Recently, Auron et al. [54] reported two patients with NSF. One of the patients was a 13-year-old boy previously reported at the age of 8 years by Jan et al. [50] in 2003 (discussed above). The second patient was a 20-year-old male who at the age 14 years was diagnosed with acute monoblastic leukaemia and 2 years later underwent bone marrow transplant and later developed kidney failure. No references to contrast-enhanced MRI are available from these reports.
Our own hospital has a large paediatric renal unit with up to 30 renal transplants per year. We have actively used MRI both for concurrent conditions and complications to CKD, as well as for providing a presurgical vascular road-map, using contrast-enhanced MRA (Gd-DTPA, Magnevist, Schering, Germany, 0.1–0.3 mmol/kg, i.v.). A review of all contrast-enhanced scans in children referred via our nephrology service (March 2002 to March 2007) showed that 75 nephrology patients (neonate to 18 years of age; median 9.6 years) had had 93 MRI scans with a follow-up of at least 6 months. There were no patients with NSF in this high-risk cohort.
Regulatory advice
Regulatory advice can be obtained from the websites listed in Table 2. The FDA has issued a general warning for all gadolinium-based contrast agents and advise against their use in patients with eGFR <30 ml/min per 1.73 m2 and in all patients with acute renal insufficiency “unless the diagnostic information is essential and not available with non-contrast enhanced magnetic resonance imaging”. At the FDA’s request the manufacturers of gadolinium-based contrast agents have revised the product labels, which now include a new boxed warning and a new warnings section that describes the risk of NSF. The European Medicines Agency has so far not published any update of general contraindications, and is not in a position to do so since all but one gadolinium-based agent are licensed by individual member states’ regulatory bodies. However, the issue was discussed at a meeting of its pharmacovigilance group in June and May 2007, and issued specific advice as follows (http://www.mhra.gov.uk):The use of Omniscan® and Magnevist® is contraindicated in patients with severe renal impairment (i.e. GFR or eGFR <30 mL/min/1·73 m2). Omniscan is also contraindicated in patients with renal dysfunction who have had, or who are awaiting, liver transplantation. For patients with moderate renal impairment (GFR or eGFR 30–59 ml/min/1·73 m2) or neonates and infants up to 1 year of age, Omniscan and Magnevist should be used only after careful consideration. All patients should be screened for renal dysfunction by obtaining a history and/or laboratory tests, especially before Omniscan or Magnevist are used. Haemodialysis shortly after administration of a gadolinium-containing MRI contrast agent in patients currently under haemodialysis may be useful for removal of contrast agent from the body. However, there is no evidence to suggest that haemodialysis can prevent or treat development of NSF. Careful consideration should also be given to the use of the other gadolinium-containing MRI contrast agents in patients with severe renal impairment.Table 2Websites for regulatory updates and for information and registration of cases (accessed on 20 September 2007)OrganizationInformation providedURLEuropean Medicines AgencyRegulatory informationhttp://www.emea.europa.eu/US Food and Drug AdministrationRegulatory informationhttp://www.fda.gov/cder/drug/infopage/gcca/default.htmInformation for healthcare professionalshttp://www.fda.gov/cder/drug/InfoSheets/HCP/gcca_200705.htmInternational Center for Nephrogenic Fibrosing Dermopathy ResearchUpdated information and contact details for registration of caseshttp://www.icnfdr.org/
Consequences for paediatric practice
There is an associated high morbidity and mortality with CKD, and imaging aids clinical management, such as assessment of secondary vasculopathies, infections etc, and surgical planning prior to renal transplantation. Patients at risk also include children with known or suspected CKD, as well as newborns and infants with renal immaturity or congenital cardiopathy. It seems plausible that impaired excretion, dissociation of the gadolinium chelate, and prolonged tissue retention of gadolinium is a trigger for NSF in patients with CKD. This risk increases with cumulative dose of gadolinium, with decreasing GFR <60 ml/min per 1.73 m2, and in the presence of acidosis and proinflammatory conditions. The scarcity of reported children with NSF may well be due to the lower prevalence of CKD and secondary conditions in children. It is therefore imperative that paediatric practitioners are prudent and immediately tighten their local policies for use of intravenous gadolinium-based contrast agents.
It is difficult at present to suggest general guidelines, because:
The absolute risk of gadolinium exposure, as well as the risk associated with the suspected contributing clinical and biochemical factors, is unknown. Each case must therefore be assessed individually.The risk attributable to the kinetic stability of different gadolinium-based agents is not clear. The majority of cases have been reported in association with a less stable agent (gadodiamide); however, we are still in an early stage of recording cases, and also the market share and the actual use of different agents in this patient population is not fully known. Thus, it seems reasonable to discontinue the use of gadodiamide in at-risk patients.The relationship to dose is not completely known. Since there is no indication that we are dealing with a side effect not related to dose, it seems reasonable to reduce the dose of gadolinium if possible, and also to monitor the cumulative dose in at-risk patients.It is unclear whether acidosis correction before gadolinium administration and haemodialysis immediately after gadolinium administration can prevent the development of, or treat, NSF, but such actions should be considered in children already on haemodialysis. This underlines the necessity for good communication between radiologists and nephrologists, both for issuing local guidelines and for management of the individual child.
It is often the case that MRI is requested after an inconclusive US examination. In our own experience, more meticulous patient preparation and dedicated rescanning of the patient with US can often give diagnostically adequate results. In patients with CKD not yet on dialysis CT is not a desirable alternative due to ionizing radiation and the nephrotoxicity of iodinated contrast agents. However, CT may provide an option in patients who are already on dialysis. MRI without intravenous contrast agent administration has not yet been validated for vascular anatomy prior to renal transplantation, but several techniques may be valuable, such as 3-D-balanced steady-state free precession imaging and time-of-flight angiography.
Patients with renal impairment may be referred for MRI from non-nephrology units and a crucial question is how to identify them. Our approach would be to include a mandatory tick box on the referral form requiring the referring clinician’s statement about renal function, and also to include a question about any known renal problems on the patient/guardian prescan checklist. This is a pragmatic approach, which will need revision after an initial phase.
Conclusion
We would advocate prudence, and in practice suggest:
Information collection from clinicians and patients/guardians on renal dysfunction and active collaboration with the local nephrology unit.
A case-by-case assessment in terms of the necessity for gadolinium-enhanced MRI, and whether an alternative test (repeat US, noncontrast-enhanced MRI, CT) would be acceptable.
Cumulative dose recording, reduction of the dose of any gadolinium-based contrast agent, and acting accordingly to guidance published in the literature.
Longer follow-up, especially in high-risk children.
GFR measurement or estimation should be part of a systematic prescan investigation in children with known or suspected renal dysfunction; however, a general screening is probably not justified.
To minimize the risks, we should take advantage of the current knowledge, and would suggest using cyclic nonionic compounds as they are more stable in vitro and therefore theoretically less likely to undergo transmetallation.

gadolinium
nephrogenic systemic fibrosis
children
chronic kidney disease