Transferrin-mediated uptake of aluminium by human parathyroid cells results in reduced parathyroid hormone secretion

Karine A. Smans, Patrick C. D'Haese, Glen F. Van Landeghem, Luc J. Andries1, Ludwig V. Lamberts, Geoffrey N. Hendy2 and Marc E. De Broe,

Department of Nephrology, 1 Department of Physiology, University of Antwerp, Antwerp, Belgium, and 2 Calcium Research Laboratory, Royal Victoria Hospital, Montreal, Canada



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. The present study investigates whether aluminium–transferrin (Al–Tf) uptake by Tf receptor-mediated endocytosis induces hypoparathyroidism and thus might contribute to the increasing prevalence of adynamic bone disease (ABD) in the current dialysis population.

Methods and Results. Human parathyroid glands as well as in vitro cultured human parathyroid cells were shown to express Tf receptors. Five-day-old cultures of parathyroid cells were incubated for 48 h in serum-free DMEM/F12 supplemented with 12 µM apo–Tf: 12 µM Tf to which 150 µg/l Al or 150 µg/l Al–citrate (Al–ci) was bound. The amount of Al taken up by the parathyroid cells either as Al–Tf or Al–ci did not differ. However, incubation of cell cultures with Al–Tf showed a significant proportional decrease (mean±SEM, -23.1±4.5%) in iPTH secretion as compared to the reference apo–Tf cultures. Al–ci did not suppress PTH secretion (+3.4±6.5%). The Al uptake after incubation with Al–Tf was found to be dose-dependent. With regard to iPTH secretion, a tendency toward a dose response relationship was observed. Northern blot analysis of parathyroid cells incubated in 12 µM apo–Tf or 12 µM Al–Tf demonstrated that the PTH mRNA synthesis was unaffected by the Tf-mediated uptake of Al. These observations suggest an effect of Al on PTH release rather than on PTH synthesis. Since the cytoskeleton can play an important role in the release of secretory vesicles, the influence of Al on the structure of actin, ß-tubulin and vimentin was investigated by confocal microscopy. Comparison of cultures incubated with apo–Tf and Al–Tf revealed no difference in the organization of these cytoskeletal proteins in relation to the inhibitory effect of Al–Tf on PTH secretion.

Conclusion. In summary, data in the present paper demonstrate that the (i) human parathyroid gland/parathyroid cells exhibit Tf receptors; (ii) Al–Tf complex is taken up by the parathyroid gland in a dose-dependent manner; and (iii) uptake of Al by Tf receptor-mediated endocytosis reduces the secretion of PTH but not its synthesis. These in vitro findings allow us to suggest that Tf receptor-mediated uptake of Al might, besides other factors such as vitamin D, high calcium dialysate or CaCO3 intake, play a role in the development of hypoparathyroidism associated with ABD. The exact mechanism by which Al–Tf suppresses iPTH secretion remains to be elucidated.

Keywords: adynamic bone disease; aluminium-citrate; aluminium-transferrin; human parathyroid cell



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The association between bone aluminium (Al) accumulation and the development of osteomalacia and adynamic bone disease (ABD) is now well recognized [1,2]. Hence, it was expected that with the replacement of Al-containing phosphate binders by CaCO3 these low bone turnover diseases would disappear. In recent years, a dramatic decrease in the prevalence of clinically overt Al-induced osteomalacia has indeed been observed. However, the incidence of adynamic bone disease (ABD) within the dialysis population, which is associated with a relative hypoparathyroidism, has increased rather unexpectedly over the past few years [3]. This observation was reported independently by several research groups; incidences of ABD varied between 20 and 60% [36].

The aetiology of ABD is largely unknown and is most likely to be multifactorial. Aluminium accumulation, diabetes, iron overload, hypothyroidism, corticosteroids, fluoride and acidosis, as well as clinical risk factors such as time on dialysis, age, calcium administration and mode of dialysis have all been associated with the development of ABD [7]. All patients with ABD have a relatively low level of intact PTH in common. It is not yet clear whether ABD is secondary to a decrease in the osteoblastic activity or if it is primarily caused by a direct suppression of the parathyroid gland. With regard to the latter, an inhibitory effect of Al on iPTH secretion was described a decade ago [810]. Aluminium overload is now rarely seen. However, with the introduction of erythropoietin, the present-day dialysis population may have a greater risk of iron (Fe) deficiency [11,12]. As we recently demonstrated, this relative iron depletion results in an increased in vitro binding of Al to transferrin (Tf) [13]. In the in vivo situation this may lead to an alteration in tissue Al distribution [14] due to its preferential deposition in tissues expressing Tf receptors, such as the liver, the osteoblast or the parathyroid gland. Hence, one can reasonably suggest that even the currently observed relatively low serum Al levels within the dialysis population may still have the potential to act as a causal factor in the development of hypoparathyroidism.

Addition of Al salts to bovine parathyroid cells [8,9] and slices of porcine parathyroid glands [10] has previously been reported to result in the suppression of PTH secretion. The extent to which uptake and toxic effects of Al at the level of the parathyroid gland may occur via Tf receptor-mediated endocytosis has not yet been reported. This is in contrast with other tissues where in vitro studies have demonstrated an inhibitory effect of Al–Tf on proliferation of osteoblasts [,16] and hepatocytes [17], and to a lesser extent erythroleukaemia K562 cells [18], as well as an inhibition of haemoglobin synthesis by haematopoietic progenitor cells [19].

The mechanism by which Al interferes with PTH secretion is not yet clear. Morrissey et al. [8] demonstrated that the incorporation of [3H]leucine in total cellular protein, pro-PTH and PTH of bovine parathyroid cells did not change in the presence of high concentrations of Al-chloride, suggesting an effect of the element on PTH release rather than on PTH synthesis. The cytoskeleton, cytosolic and membrane proteins, ATP and a number of secretory granule proteins [20] can all be involved in the release of secretory granules. The interaction of Al with one of these components could therefore possibly influence the release of PTH-containing secretory granules.

The present study investigates: (i) the presence of Tf receptors on human parathyroid cells; (ii) the in vitro effect of both Al–Tf and aluminum-citrate (Al–ci) complexes on secretion and synthesis of PTH by human parathyroid cells; and (iii) the possible involvement of the cytoskeleton proteins actin, ß-tubulin and vimentin in the Al–Tf mediated inhibition of PTH release.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immunohistochemistry
In order to demonstrate the presence of Tf receptors on human parathyroid cells, human parathyroid glands and parathyroid cells were stained for Tf receptor expression.

Parathyroid cells were fixed for 30 min at room temperature in 4% formalin buffered with 0.1 M Na-cacodylate at pH 7.4. Parathyroid glands were frozen in liquid nitrogen. Cryostate sections (6 µm) were fixed with acetone (5 min), washed for 5 min with Tris-buffered saline (TSB; 0.01 M Tris–HCl, pH 7.6, 0.154 M NaCl, 0.4% merthiolate) containing 1% bovine serum albumin (BSA; Boehringer, Mannheim, Germany) and blocked with normal horse serum (1/5 in TSB) for 20 min. The primary mouse anti-human Tf receptor antibody B-D12 (1/100) (Serotec, Oxford, UK) was incubated overnight at room temperature. Following a 5-min wash step with TSB, sections were incubated for 30 min with a biotinylated horse anti-mouse antiserum (Amersham, Arlington Heights, USA).

Both human parathyroid gland and parathyroid cell-bound antibodies were revealed with the vectastain avidin–biotin immunoperoxidase detection system (Vector, Burlingame, USA). Staining was done with 3-amino-9-ethyl-carbozole (AEC; Sigma, St Louis, USA). Preparations were counterstained with 1% methyl green in 0.1 M acetic acid (pH 4.3). Stains of liver tissue were used as a positive control. Experiments done in the absence of the primary antibody served as negative controls.

Aluminium-transferrin and iron-transferrin solutions
Apo–Tf (Sigma) was dissolved in Tris buffer (pH 7.4) supplemented with 24 mM of NaHCO3 to obtain a concentration of 36 µM apo–Tf. Three milliliters of this solution were then incubated with 310 µl of a 20 mg/l Al–ci solution or with 310 µl of a 40 mg/l Fe-ci solution for 1 h at 37°C. The Al–ci and Fe-ci solutions were obtained by diluting, respectively, 1 ml of a 1 g/l Al stock solution (Baker) or 2 ml of a 1 g/L Fe stock solution (Baker) in 50 ml to which 15 mg of citrate (Merck) was added. After incubation with apo–Tf, the samples were ultracentrifuged on a centricon-30 (Amicon) to remove non-Tf bound metal, diluted to 12 µM Tf in DMEM/F12 (Life Technologies, Paisley, UK) and added to the parathyroid cell cultures. Cultures prepared in this way contained 150 µg/l of Al bound at the specific binding sites on Tf. This corresponded with a 23% Tf-saturation as determined by a high performance liquid chromatographic/electrothermal atomic absorption hybrid technique (HPLC/ETAAS); a method we previously described in detail elsewhere [21]. HPLC/ETAAS was also used to check whether all non-Tf bound Al had been removed before adding the Al–Tf solution to the cell culture media.

Cell cultures
Parathyroid cells were isolated from human parathyroid glands of patients with primary or secondary hyperparathyroidism by collagenase digestion. Briefly, parathyroid glands obtained during surgery were placed in cold culture medium and transported to the laboratory. Fat and connective tissues were removed and glands were minced to pieces of ~1–2 mm2. About 2 g of minced tissue was digested in 20 ml of DMEM/F12 containing 0.5 mM MgCl2, 1.0 mM CaCl2, 10% FCS and 2.5 mg/ml collagenase type IA (Sigma) for 1 h at 37°C. The digested pieces were then pushed through a nylon mesh and parathyroid cells were washed with culture medium and collected by centrifugation (1000 r.p.m. for 10 min).

Effect of Al–Tf on PTH secretion
Parathyroid cells (2x106 per well) from 20 patients were grown for 5 days in DMEM/F12 containing 0.5 mM MgCl2, 1.0 mM CaCl2 and 10% FCS in a 24-well plate; allowing the parathyroid cells to adhere to the plates. Subsequently, parathyroid cells were incubated for a further 48 h in serum-free DMEM/F12 containing 0.5 mM MgCl2 and 1.0 mM CaCl2 supplemented with either 12 µM apo–Tf or 12 µM Al–Tf (12 µM apo–Tf to which 150 µg/l Al was bound). In total, parathyroid cells from 20 patients were brought into culture corresponding to 67 culture wells for apo–Tf and Al–Tf each.

Dose-response relationship of Al uptake and iPTH secretion
To study the dose-response relationship of the Tf-mediated Al uptake by parathyroid cells, cultures were incubated with 4, 12 or 36 µM Tf (n=4 for each of the concentrations studied) containing either 84, 252 or 756 µg/l Al. For each of these Al–Tf cultures both the uptake of Al and the iPTH secretion was compared with the cell cultures to which apo–Tf was added at corresponding concentrations.

Effect of Al–ci on PTH secretion
To study the effect of Al–ci on PTH release and compare it with the effect noted for Al–Tf, PTH cells were incubated in the presence of Al–ci, either alone or in the presence of Tf. In the latter case, the binding of Al to Tf had to be avoided when adding Al–ci to the culture medium. Therefore, 100% saturated Fe-Tf was used instead of apo–Tf. Thus, cells were grown for 5 days in DMEM/F12 containing 0.5 mM MgCl2, 1.0 mM CaCl2 and 10% FCS. Following this, they were incubated for a further 48 h in serum-free DMEM/F12 containing 0.5 mM MgCl2, 1.0 mM CaCl2 and 150 µg/l Al–ci (corresponding to the concentration of Al added to the Al–Tf complex; see above) in the presence of 12 µM fully (100%) saturated Fe–Tf. The change in PTH secretion in the presence of Al–ci was studied in parathyroid glands of 13 patients (40 culture wells).

Cultures containing 12 µM apo–Tf supplemented with 3 mM CaCl2 served as controls for the suppressibility of parathyroid glands. About half of the parathyroid glands collected did not respond to Ca and were excluded from the study. In these glands the lack of any effect of Ca on PTH secretion seemed to coincide with a lack of any effect of Al.

The addition of Al–ci to the culture medium might result in a decreased concentration of ionized calcium, which in turn may affect PTH secretion. Therefore, in all cultures studied we checked the concentration of ionized calcium using a Mod-EH-P Fresenius ionometer. Neither the addition of Al–ci nor any other mediator that was added had a significant effect on the ionized calcium levels.

For measurement of the secreted PTH, all culture media were replaced by fresh DMEM/F12, containing the same supplements as before, at the end of a 44 h incubation period, unless stated otherwise. Following another 4 h of incubation, the culture media were collected for measurements of PTH release using the N-tact PTH SP kit from Incstar (Stillwater, Minnesota, USA). This working procedure was used to prevent PTH concentrations increasing to too high levels and to prevent degradation of the molecule during incubation; phenomena that will negatively affect the analytical performance of the PTH measurement. The adherent parathyroid cells were washed with PBS and dissolved in 300 µl 0.5% Triton X-100 in PBS. Samples were then used for protein determination by BCA (Pierce) and direct intracellular Al concentration by atomic absorption spectrometry [22] respectively. In order to correct for variations in cellular content/well and/or possible differences in cell growth the protein content of each cell culture well was also measured. Both PTH released in the culture medium and cellular Al content were expressed in relation to the cellular protein content of each culture well. It needs to be mentioned that Al seemed not to have any effect on cell growth/number. Data from a representative sample indicate that, even after 48 h incubation of PTC without supplements or with Al–ci, apo–Tf, Al–Tf or Fe–Tf, the protein concentrations (247±19, 243±33, 250±19, 255±47 and 227±17 mg protein/l, respectively) did not differ significantly.

Northern blot analysis
Total RNA was extracted from parathyroid cell cultures (107 cells/well in 6-well plates) incubated in either apo–Tf (n=3) or Al–Tf (n=3) as follows: 1 ml of denaturing solution (4 M guanidinium thiocyanate, 0.5% sodium sarcosyl, 25 mM sodium citrate pH 7.0, 0.1 M ß-mercaptoethanol) was added to each culture well and transferred to RNase free tubes. Upon addition of 100 µl 2 M sodium-acetate pH 4.0, 1 ml water-saturated phenol and 200 µl chloroform : isoamyl alcohol (49 : 1), incubation (4°C, 15 min) and centrifugation (10 000 g, 15 min) of the samples, the aqueous phase was transferred to a fresh tube. After addition of 1 ml of 100% isopropanol (-20°C for 30 min), the RNA was pelleted by centrifugation (10 000 g, 10 min for 4°C). The RNA pellet was dissolved in 300 µl of denaturing solution and precipitated again with 100% isopropanol after which it was washed with 75% ethanol, air dried and dissolved in 10 µl of DEPC-water. RNA samples were loaded on a 1% agarose–2.2 M formaldehyde denaturing gel, transferred onto a nylon membrane filter, and analyzed for the presence of PTH [23] and GAPDH [24] mRNA by standard procedures.

Confocal microscopy
The influence of Al on the structure of the cytoskeleton proteins actin, ß-tubulin and vimentin was analyzed by confocal microscopy, according to the method of Van de Water et al. [25].

Human parathyroid cell cultures (5x106 cells/well), were grown on a 22 mm diameter glass coverslip put in a 12-well plate, in the presence of either apo–Tf (n=3) or Al–Tf (n=3), as described above, were washed with Ca and Mg containing PBS (PBS, 1 mM CaCl2, 1 mM MgCl2), and fixed in 2% p-formaldehyde in PEM (80 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.5) for 15 min. Coverslips were washed three times with PBS, treated with 1 mg/ml NaBH4 (10 min) in PBS, and washed again in PBS. Following 10 min of permeabilization in 0.1% Triton X-100 in PBS and washing with PBS, the cells were blocked with 0.2% BSA in PBS (10 min).

For the actin staining, the parathyroid cells were incubated with rhodamine phalloidin (Molecular Probes, Eugene, USA) for 20 min, washed in PBS containing 0.1% Triton X-100 and 0.2% BSA and in PBS without supplements, after which the cells were mounted using the slowfade-light kit from Molecular Probes.

For the ß-tubulin and vimentin stainings, the parathyroid cells were incubated overnight with the mouse Ab TUB 2.1 (Sigma) and a mouse anti-vimentin Ab (Dako) respectively. Upon washing with PBS and blocking with 0.2% BSA in PBS for 10 min the secondary FITC-labelled RAM-F(ab')2 Ab (Dako) was added (60 min). Following another wash step with PBS, the cells were post-fixed with 4% p-formaldehyde in 50 mM cacodylate, pH 7.5 (30 min), quenched with 50 mM NH4Cl in PBS (45 min), washed in PBS, and mounted as before. Samples were stored at -20°C and analyzed within 1 week by confocal microscopy (Bio-Rad MCR-600).

Statistics
For statistical analysis, the paired Student's t-test was used for comparison of PTH secretion and Al-uptake by the various parathyroid glands after addition of Al–Tf vs Al–ci. The unpaired Student's t-test was used for comparison of the percentage decrease in PTH secretion in Al–Tf vs Al–ci containing culture media. Data on dose-response relationship of PTH secretion were analysed by ANOVA followed by the Bonferroni's t-test when more than two groups were considered. Data are expressed as mean±SD, with the exception of data concerning percentage change in iPTH secretion and comparative data on Al-uptake (Figures 2Go and 3Go) and iPTH secretion which are expressed as mean±SEM because of the great biological gland-to-gland variability. A P-value <0.05 was considered significant at a two-tailed level.



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Fig. 2. Mean±SEM Al-uptake by cultured parathyroid gland cells isolated from glands of 13 patients and incubated with (i) 12 µM apo–Tf or 12 µM Al–Tf; (ii) 12 µM Fe–Tf either alone or in the presence of Al–ci (40 culture wells for either culture condition).

 


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Fig. 3. Percentage change (mean±SEM) in PTH secretion of (i) parathyroid cells isolated from glands of 20 patients and incubated with Al–Tf (12 µM) vs apo–Tf (12 µM) (a total of 67 culture wells for each culture condition); (ii) parathyroid cells isolated from glands of 13 patients, incubated with Fe–Tf (12 µM) in the presence of Al–ci vs Fe–Tf (12 µM) alone (a total of 40 culture wells from each culture condition).

 


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 Abstract
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 Materials and methods
 Results
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 References
 
Effect of Al–Tf and Al–ci uptake on PTH suppression
Both human parathyroid glands and in vitro cultured human parathyroid cells were shown to express Tf receptors (Figure 1Go). As expected, the Tf-receptor density in the parathyroid gland was lower than that noted in liver tissue, which was used as a positive control. No signal was noted for the negative control; i.e. experiments done in the absence of the primary antibody.



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Fig. 1. Immunohistological staining for Tf receptors on human parathyroid glands (a) and parathyroid cells (b). Bar represents 50 µm.

 
Comparison of the Al-concentration in parathyroid cells after adding identical concentrations of either Al–Tf complex or Al–ci to parathyroid cell cultures indicated the uptake of both components did not differ significantly (Figure 2Go). Significant (P<0.001) increases from 15.7 ng Al/mg protein to 56.7 ng/mg protein and 15.0 ng/mg protein to 58.4 ng Al/mg protein were noted for Al–Tf and Al–ci uptake, respectively. Some variation in the Al-uptake was noted from gland-to-gland varying from 13 ng Al/mg protein up to 112 ng Al/mg protein in the glands with lowest and highest uptake, respectively. There was no significant difference in Al-uptake when cells were grown in the presence of Al–ci or of Fe–Tf + Al–ci.

The fact that parathyroid cells grown in the absence of any growth factor such as Tf had a low basal PTH release made it impossible to reliably measure the influence of Al–ci alone on PTH release, as can be demonstrated by the following example. While in cultures of this parathyroid gland, the PTH secretion declined from a mean±SEM value of 461±91 pg/mg protein in the presence of apo–Tf (n=4) to 216±14 pg/mg protein in Al–Tf containing cultures (n=4), parathyroid cells grown in the absence of any growth factor such as Tf (n=4), had a basal PTH release of only 146±2 pg/mg protein as compared with 152±6 pg/mg protein in the presence of Al–ci (n=4). Therefore, further experiments made use of the combination of Fe–Tf and Al–ci. By doing so, the binding of Al to transferrin was prevented in the presence of the protein, which acts as a growth factor and allows the initial PTH secretion to increase to basal values comparable to those noted in the presence of apo–Tf. Indeed, the addition of either apo–Tf or Fe–Tf to the cell culture media importantly stimulated PTH secretion (a 3- to 10-fold increase) reaching mean±SEM values of 11.771±3.193 pg PTH/mg protein and 10.667±2.800 pg PTH/mg protein for both compounds, respectively (NS). Both apo–Tf and Fe–Tf stimulated iPTH secretion were found to be suppressible by the addition of CaCl2. The addition of Al–Tf resulted in a distinct decrease in PTH secretion (mean±SEM percentual change: -23.14±4.5%; n=20 parathyroid glands; n=67 culture wells); however, Al–ci had no effect on the PTH secretion (mean±SEM percentual change: +3.42±6.45%, n=13 parathyroid glands, n=40 culture wells; Figure 3Go). As for the Ca-induced PTH suppression, there was a substantial gland-to-gland variation in the percentual change of PTH secretion induced by Al–Tf. In three out of the 20 glands that were incubated with the latter compound, PTH secretion was not decreased while in PTCs of the 17 remaining glands a decrease varying from 9.5% down to even 61.7% was noted. As can be deduced from a representative experiment, PTH suppression induced by adding 12 µM Al–Tf (150 µg/l of Al) to the culture medium was substantially less than when 3 mM CaCl2 was added. While the PTH secretion decreased from 7136±2254 pg/mg protein (n=4) down to 4431±512 pg/mg protein in apo–Tf containing culture media when Al–Tf was added, the addition of Ca (apo–Tf +3 mM CaCl2) resulted in a decrease down to 2075 pg PTH/mg protein.

Dose-response relationship of Al uptake and iPTH secretion
The Tf-mediated uptake of Al by the parathyroid cells was dose-dependent. The incubation of cultures with 4, 12 or 36 µM Al–Tf (n=4) containing either 84, 252, or 756 µg/l Al, respectively, resulted in a cellular Al content of 55±18, 114±20 and 172±26 ng Al/mg protein (P<0.0001), corresponding with a mean±SD percentage change in iPTH secretion of -8±10%, -13±26% and -47±23% vs apo–Tf cultures (P=0.053), respectively.

Al–Tf does not suppress PTH mRNA synthesis in parathyroid cells
Northern blot analysis of mRNA extracted from parathyroid cell cultures incubated either in 12 µM apo–Tf (n=3) or 12 µM Al–Tf (n=3) demonstrated that the mean±SD PTH/GAPDH ratios for apo–Tf and Al–Tf were 1.79±0.12 and 1.86±0.21 (NS), respectively, indicating that the mRNA synthesis of PTH was unaffected by the Al uptake (Figure 5Go).



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Fig. 5. Northern blot analysis of mRNA extracted from parathyroid cells cultures incubated either in 12 µM apo–Tf (n=3) or 12 µM Al–Tf (n=3). Bars represent mean±SD of absorbance ratios of PTH mRNA vs GAPDH mRNA. Statistical analysis was performed with Student's t-test for comparison of treatment with and without Al.

 



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Fig. 4. Dose-response relationship between Al uptake and iPTH secretion after addition of 4, 12 and 36 µM Al–Tf corresponding to Al concentrations of 84, 252 and 756 µg/l respectively.

 

The structural organization of the cytoskeleton is not influenced by Al–Tf
The previous results suggested an Al–Tf -mediated inhibition of PTH release rather than synthesis; therefore, a possible effect of Al on the exocytosis of secretory granules was investigated in more detail. One of the components involved in both constitutive and regulated exocytosis is the cytoskeleton. In this study the cytoskeleton proteins ß-actin, ß-tubulin and vimentin in parathyroid cell cultures incubated with either 12 µM apo–Tf or 12 µM Al–Tf were compared (Figure 6Go). Our results show no difference in the structural organization of either cytoskeleton protein in the presence or absence of Al–Tf, although the PTH secretion was decreased by 30±10%.



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Fig. 6. Organization of the cytoskeleton components ß-actin, ß-tubulin and vimentin in primary cultures derived from human parathyroid glands incubated with either 12 µM apo–Tf or 12 µM Al–Tf as compared by confocal microscopy. Cell cultures contain both parathyroid cells of small and intermediate size and fibroblast-like cells. Magnification: all panels, x400, except upper-left, x800.

 



   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the past, Al has played a major role in the development of the low bone turnover diseases. With the replacement of Al-containing phosphate binders and the introduction of reversed osmosis for water treatment, a decrease in the incidence of low bone turnover diseases could reasonably be expected. Although such an evolution was noted for osteomalacia, the number of patients suffering from ABD associated with a distinct hypoparathyroidism has increased during the last decade [36].

The aetiology of ABD is still unknown. Whether the causal factors of the hypoparathyroidism in ABD are primarily related to either the parathyroid gland or the osteoblast is unknown [26,27].

The introduction of CaCO3 and vitamin D therapy both predisposing to hypercalcaemia might play an important role in the increasing prevalence of ABD [26], although it cannot explain all cases of ABD.

In the past 10 years erythropoietin has been used to improve the patients' anaemic state. This was associated with a substantial increase in the number of patients with a relative iron depletion [11,12], which in turn might enhance the binding of Al to Tf because of: (i) the increased availability of binding sites on Tf; and (ii) the increased affinity between Tf and Al at low Fe-Tf saturations [13]. An additional important consequence of iron depletion is the increased expression of Tf receptors on several tissues, including the osteoblast and possibly the parathyroid gland. All these factors may predispose to the increased Tf receptor-mediated uptake and subsequent toxicity of Al–Tf. These considerations allow the postulation that in the current dialysis population a clinical relevant uptake of Al may still exist in particular tissues despite the relatively lower serum Al concentrations [14,28].

In vitro experiments have indicated that the uptake of Al via Tf receptor mediated endocytosis might have toxic consequences as shown at the level of the osteoblast [15], bone marrow cell [16], hepatocyte [17], erythroleukaemia K562 cell [18], the haematopoietic progenitor cell [19]. To the best of our knowledge, the Tf-mediated uptake of Al by the human parathyroid gland has not been demonstrated before. Results presented in this paper for the first time demonstrate that human parathyroid glands express Tf receptors in vivo as well as in vitro. This makes the uptake of Al through the Al–Tf pathway by the parathyroid cell with possible toxic consequences a feasible process.

As could be expected, the use of human parathyroid glands derived from patients suffering from primary or secondary hyperparathyroidism introduced a great deal of variability both in the amount of PTH secreted per cell and in the suppressibility of the cells. Also, it is of importance that the uptake of Al differed from gland-to-gland. Within one gland, the overall uptake of both Al–Tf and Al–ci turned out to be highly comparable. Whereas the uptake of the Al–Tf complex results in the suppression of PTH secretion, no such an effect could be demonstrated in the presence of the Al–ci compound. These findings suggest that Al–Tf might sequester Al in (an) intracellular compartment(s) different from Al–ci. Therefore, different mechanisms underlying the element's uptake (e.g. Tf-mediated endocytosis vs uptake via ion channels, active transport etc.) must be active at the cellular level.

When studying the effect of non-Tf-bound Al on PTH secretion, the presence of apo–Tf in the culture medium should obviously be avoided. We used a 100% saturated Fe–Tf solution as an alternative growth factor for apo–Tf to stimulate PTH secretion. Addition of Fe–Tf stimulated iPTH secretion up to values comparable to those noted in the presence of apo–Tf. Moreover, apo–Tf and Fe–Tf stimulated iPTH secretion was suppressible by the addition of CaCl2, while no suppression occurred after the addition of Al–ci to Fe–Tf.

The apparent discrepancy between earlier observations by Morrissey et al. [8,9] and Bourdeau et al. [10], who reported a decrease in PTH secretion using Al salts, and our own data might be due to the use of: (i) different species i.e. human instead of bovine or porcine glands; (ii) normal instead of uraemic parathyroid glands; (iii) the amount of Al added; or (iv) the use of different Al salts, i.e. Al-chloride and Al-phosphate [10]. Furthermore in the study by Morrissey et al., the addition of 135 µg/l Al-chloride resulted in a 50% reduction in the PTH secretion of normal bovine parathyroid cells. In the latter study, however, 0.5% of Tf-containing fetal calf serum was added to the culture medium making it difficult to interpret whether only an effect of Al-chloride or of both Al–chloride and Al–Tf was observed. In agreement with our own observation in the human parathyroid gland, a lack of toxicity of Al–ci as opposed to Al–Tf has been shown in some other cell types; for instance the hepatocyte [17] and the haematopoietic progenitor cell [19]. Tf has also been demonstrated to enhance the antiproliferative effect of Al [15] as compared with Al–ci in osteoblasts. Whether the uptake of Al–Tf and Al–ci in these cells was comparable is not clear. Unfortunately in none of these experiments has the intracellular Al content after incubation with Al–Tf been assessed, and/or compared with the element's uptake after the addition of Al–ci.

As shown above, human parathyroid cells express Tf receptors enabling them to take up Al by Tf receptor-mediated endocytosis, resulting in a decreased PTH secretion. To what extent Al affects the Tf receptor density at the level of the parathyroid gland has not been investigated in the present study. However, this might be of interest for future studies on the mechanism(s) underlying the element's toxic effects. Data in the present study indicate that Al–Tf -induced PTH suppression is not due to a decrease in PTH mRNA levels. These results are in agreement with the findings of Bourdeau et al. [10] who also found the PTH suppression in porcine parathyroid glands slices to be reversible. Moreover, the suppression went along with an accumulation of secretory granules in the parathyroid glands [10].

The cytoskeleton plays an important role in the accumulation and release of secretory granules. Furthermore, the observation that in neurons Al intoxication resulted in the accumulation of disordered whorls of neurofilaments in perikarya [29] supports the idea that Al can interact with and affect certain cytoskeleton components. In view of this, the possible involvement of the cytoskeleton in Al–Tf-induced suppression of PTH release was investigated. Comparison of apo–Tf and Al–Tf cultures, however, could not reveal any difference in the organization of either of the analysed proteins. The observation that in neurons Al does have an effect on neurofilaments might be due to the extremely high doses of Al-chloride (1 mM) that these authors added [29]; our experiments were performed at much lower, clinically relevant, concentrations. It is therefore unlikely that in human parathyroid glands changes in either actin, ß-tubulin or vimentin are responsible for the observed PTH suppression by Al–Tf, at least in the concentration range we have used. The mechanism by which Al–Tf suppresses the iPTH secretion therefore remains to be elucidated.

In summary, we have been able to: (i) demonstrate for the first time the presence of Tf receptors at the level of the human parathyroid gland/human parathyroid cell; (ii) present evidence for a dose-dependent Tf-mediated uptake of Al by the parathyroid gland; and (iii) demonstrate an Al–Tf -induced suppression of the PTH secretion, not synthesis.

Although, in vivo data in Al-loaded rats provide evidence that bone is much more susceptible to developing features of Al-toxicity than suppression of the parathyroid glands [30], definite proof of our findings will only be provided by demonstrating an increased Al-content in the parathyroid gland of patients with ABD. These in vitro results allow us to hypothesize that Al uptake via Tf receptor-mediated endocytosis might be linked to the increasing prevalence of ABD by inducing a state of hypoparathyroidism. Our findings become of greater interest in view of the prevalence of patients in the current dialysis population with either relative Fe-depletion on the one hand or ABD on the other. Indeed, Fe-depletion, as observed in erythropoietin-treated patients, results in an increased binding of Al to Tf. Together with the higher expression of Tf receptors in tissues such as the osteoblast or the parathyroid cell, this might lead to a preferential accumulation and increased toxicity of the element in these tissues, possibly leading to the development of hypoparathyroidism-associated ABD even at the relatively low serum Al levels that are observed in the present dialysis population.



   Acknowledgments
 
The authors wish to thank Dr Arnouts, Dr Hostyn, Dr Lonke, Dr Segaert, Dr Somers and Dr Ysebaert who delivered the parathyroid glands and without whom this research would not have been possible. Technical support by Dirk De Weerdt and Erik Snelders is once again appreciated.



   Notes
 
Correspondence and offprint requests to: Dr Marc E. De Broe, University of Antwerp, Department of Nephrology-Hypertension, University Hospital Antwerp, Wilrijkstraat 10, B-2650 Edegem/Antwerpen, Belgium. Back



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 11. 6.99
Revision received 4. 5.00.