Ultrastructural and Morphometrical Evaluation of the Parathyroid Gland in Iron-Lactate-Overloaded Rats

Abstract

Iron lactate was given to Sprague–Dawley rats intravenously at the dosage of 10 mg/kg/day and the early effects on the parathyroid gland were examined ultrastructurally along with the blood level of parathyroid hormone (PTH) after single, 3-day or 6-day administration. Blood levels of electrolytes and other parameters related to osteoclast dynamics were also measured by blood chemistry and histopathology. The plasma parathyroid hormone (PTH) level was elevated in the single and 3-day dosing group but was reduced in the 6-day dosing group. Histopathologically, an increase of osteoclasts in the primary spongiosa was observed in the 3- and 6-day dosing groups. Image analysis of the parathyroid gland revealed that the average area of the storage granule decreased during a experimental period, with the number of storage granules decreasing in the 3- and 6-day dosing group. The chief cells of the parathyroid gland were moderately atrophied in the 6-day dosing group. These results demonstrate that iron lactate immediately promotes discharge of PTH from the storage granules after the treatment and induces an increase of osteoclasts in the primary spongiosa. The findings collectively suggest a pathophysiological mechanism of iron lactate-induced osteopenia in rats.

Introduction

PTH regulates the blood calcium level against hypocalcemia under pathophysiological conditions (Felsenfeld and Llach, 1993). PTH secretion has been reported to increase in experimental osteopenia, and bone resorption and formation simultaneously increase, such as in ovariectomy (Hodgkinson et al., 1978; Meng et al., 1996; Mitalk et al., 1996), immobilization (Okumura et al., 1986), and partial 5/6th nephrectomy (Shimamura and Morrison, 1975). However, we unexpectedly encountered an event in which PTH secretion was suppressed in a study of iron lactate-induced osteopenia, although similar bone lesions were observed in the rat tibia as well as other experimental osteopenia. No remarkable lesions were observed in the parathyroid gland by light microscopy (Matsushima et al., 2001). It is not clear why the change is comparable to that in other experimental models and the morphological features are not accompanied by hyperfunction of the parathyroid gland.

Bourdeau et al. (1987) revealed that aluminum inhibited PTH-release and caused severe cell alterations in porcine parathyroid tissue slices. Ng et al. (2004) reported that magnesium might be involved in the suppression of PTH secretion by investigation of the trace element in a dialysis patient. The cellular activity of the parathyroid gland was suppressed after treatment with ethanol (Chen et al., 1998). However, there have hardly been any reports of iron causing injury to the parathyroid gland and reducing PTH secretion.

The present study was done to ultrastructurally examine acute lesions in the parathyroid gland of iron-lactate-overloaded rats and to elucidate the mechanism of PTH suppression in osteopenia using morphometrical analysis in ultrastructural evaluation.

Materials and Methods

Animals

Thirty-six male Sprague–Dawley rats (Jcl:SD) were obtained at 5 weeks of age from Clea Japan Inc. (Ishibe, Shiga) and acclimated for 1 week before treatment. Groups of 6 rats each received 0 or 10 mg/kg iron lactate by intravenous administration as a single dose or repeated doses once daily for 3 or 6 days. The rats were placed in plastic cages, with 2 animals per cage in an animal room kept under controlled conditions (temperature: 21°C–25°C with a relative humidity of 40%–70% with 15 air changes/hr under a 12-hour light/12-hour dark cycle). All rats were fed pellet diet (CA1, Clea Inc., Japan) and supplied tap water ad libitum throughout the experimental periods. Body weight was measured every day throughout the experimental periods. The body weights ranged from 162 g to 190 g at the beginning of the dosing.

Test Materials

Iron lactate (C₉H₁₆O₉Fe·H₂O, bivalent iron ions; 17.56%, trivalent iron ions; 0.04%, Musashino Chemical; Japan) was prepared daily before use and diluted with physiological saline to produce 0.05 w/v% solution. It was administered at up to 10 mg/kg at the speed of 0.32–0.46 mL/kg/min for 10 minutes once daily via the tail vein through a polyethylene catheter connected to a plastic syringe mounted on an infusion pump (STC-525, Terumo Corp., Japan). Control animals received physiological saline in the same way. The animals were treated humanely during the test period, in accordance with the principles outlined in the Guidelines for Animal Experimentation, published by the Japanese Association for Laboratory Animal Sciences (1987, Exp Anim 36, 285–8) and our laboratories.

Blood Biochemistry

At the end of each treatment, blood was collected from the posterior vena cava of all rats under ether anesthesia. Routine blood chemistry parameters were also examined with an automated analyzer (Hitachi 7170; Japan); glucose, total protein, albumin, total cholesterol, creatinine, urea nitrogen, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase, amylase, triglyceride, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, and iron. PTH was measured with a commercial ELISA kit (Immutopics, Inc., USA) for detecting the full-length biologically active intact form of rat PTH.

Pathology

After blood collection, the animals were perfused through the heart with fixative (2% glutaraldehyde, 2% paraformaldehyde, 0.1 M phosphate buffer, at pH 7.4) under deep ether anesthesia. The tibia was fixed in 10% neutral buffered formalin after the perfusion, and was decalcified with a mixture of 5% formic acid and formalin aqueous solution, and then embedded in paraffin. Paraffin sections were stained with toluidine blue and examined microscopically.

For microscopic and electron microscopic observation, the thyroid-parathyroid glands were excised and postfixed with 2% osmium tetroxide, dehydrated through a graded series of ethanol and embedded in epoxy resin (Epok 812). Semithin-sections of parathyroid gland were stained with Richardson’s solution and examined microscopically. Ultrathin sections of parathyroid gland were stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (JEOL JEM-1010, Japan).

Proximal metaphysis of the tibia and subcellular components of the parathyroid gland was quantitatively analyzed using a computer-digitizing image system consisting of a light microscope with a camera (Fuji HC-2000, Fujifilm, Japan), a flat scanner (CanoScan N676U, Canon, Japan) and software (Image-Pro Plus, Media Cybernetics^®; USA). Osteoclast number was measured for an area of 1 × 4 mm² of primary spongiosa directly below the growth plate-metaphyseal junction.

For ultrastructural evaluation, 10 electron micrographs for an area of 5 × 7 μm² were taken from different regions of the parathyroid glands of each animal from the six groups. The number of storage granules larger than 300 nm in diameter was accurately counted, on the basis of the anatomical information that developing immature storage granules were smaller than 300 nm in diameter according to Setoguti et al. (1995). The contents of lysosomes are usually of a median or low electron density, have the electron-dense residues of various kinds and are occasionally vesicle containing. In the meantime, the storage granules are homogenous granules of moderate to high electron density with a finely particulate texture, usually distributed singly or in groups either near the Golgi area or with in the peripheral membrane and have a clear space between the limiting membrane and dense core (Setoguti et al., 1995). Based on the above findings, the storage granules were distinguished from the lysosomes. Granules containing vesicular components were excluded from the counting. The area of storage granules was estimated. The surface area of the Golgi complex, which was occupied by Golgi cisternae, Golgi vacuoles, and vesicles, and the area of the mitochondria were also measured.

Statistical Analysis

All data were expressed as the mean ± S.D. in the tables. All measured values were analyzed by two-factor ANOVA and p-values were attached to all the results. The significance of the difference between the control group and the treatment group was analyzed by Student’s t-test. The probability level was set at 0.05 or 0.01 for the criteria of significance.

Results

PTH and Blood Biochemistry

There was no clinical sign nor difference in the change of body weight between the treatment group and the control group throughout the experimental period (Table 1).

The plasma PTH level was significantly greater in the single (2.5-fold) and 3-day (2.1-fold) dosing groups than those of the control groups, while it decreased and was less than the control in the 6-day dosing group (−19%). The level of plasma calcium of the treated rats was low throughout the experimental period. The 2-way ANOVA revealed a significant interaction between the dosing groups and dosing periods in the plasma PTH and calcium levels (Tables 2 and 3).

The plasma iron level was significantly lower in the single and 6-day dosing groups than those of the control group (Table 4). The plasma inorganic phosphorus level was significantly lower in the 6-day dosing group than the control group (Table 5). No remarkable changes were observed in the other parameters of the blood chemistry.

Histopathology of the Tibia and Parathyroid Glands

The number of osteoclasts in the primary spongiosa was significantly greater in the 3- and 6-day dosing groups than the control group. The osteoclasts markedly increased in the zone of provisional calcification in the 6-day dosing group. Two-way ANOVA revealed a significant interaction between the dosing groups and dosing periods (Figure 1, Table 6). Atrophy of the parathyroid cells, dilatation of the intercellular and edema of connective tissue was observed in the 6-day dosing groups (Figure 2). No remarkable changes were observed in the secondary spongiosa.

Ultrastructural Evaluation of the Parathyroid Gland

In the control groups, the chief cells were oval or polygonal in shape. The nucleus was irregularly shaped, having a few spots of chromatin area located in the margin, and the nuclear membrane was infolded. The plasma membrane showed interdigitations of the tortuous plasma membranes and the intercellular spaces were narrow. Free ribosomes were diffusely scattered in the cytoplasm. Mitochondria were dispersed throughout the cytoplasm, the cisternae of the rough-surfaced endoplasmic reticulum (rER) were arranged in parallel arrays or randomly distributed in the cytoplasm, and the Golgi complexes were well developed and contained a few coated vesicles and numerous prosecretory granules. Storage granules, 300–620 nm in diameter, were filled with a finely particulate and an electron-dense material, and were scattered in the Golgi area as well as in the cytoplasm (Figure 3). Lipid droplets were occasionally present in the cytoplasm (Isono et al., 1990; Chen et al., 2001).

There were morphological differences between the control and 3- or 6-day iron lactate dosing groups. The chief cells were minimally atrophied and mild extension of the intercellular spaces was diffusely observed in a 3-day dosing group (Figure 4). Vesicles were observed in the vascular endothelium, and enlargement (edematous loosing) of the perivascular space was also evident in the 3-day dosing group. The morphology of the subcellular organelle was similar to that of the control group.

Marked extension of the intercellular spaces, a well-developed tortuous outline and complicate interdigitation were observed in the 6-day dosing group. Additionally, the chief cells of the parathyroid gland were moderately atrophied. The nuclear membrane was similar to that of the control rats, while the nucleoplasm was less than that of the control, and the chromatin was apparently condensed (Figure 5). A moderate amount of vesicles of the vascular endothelium and enlargement (edematous loosening) of the perivascular space were obvious and the cytoplasmic vacuoles were also present within the perivascular peripheral cytoplasm of the chief cell adjacent to the vessel (Figure 6). Some storage granules and prosecretory granules were electron-dense with halos and were situated in the cytoplasm adjacent to the enlarged intercellular space, with larger storage granules sometimes containing floccular material or vesicles without electron-dense granules. Transitional forms between the large storage granules and vacuolar bodies were also observed. The chief cells had developed Golgi complexes associated with many prosecretory granules. The rER was decreased in the cytoplasm of the chief cells (Figure 7). The ultrastructure of the mitochondria was similar to that of the control animals.

Morphometric Analysis of the Parathyroid Gland

The number of storage granules was not different in the single dosing group but was significantly less in the 3- and 6-day dosing groups than those of the control group, and it was the least in the 6-day dosing group. Two-way ANOVA revealed a significant interaction between dosing groups and dosing period. The effect of the experimental period differed between control groups and iron lactate-treatment groups (Table 7). The average area of storage granules was significantly less in each dosing group than those of the control group (Table 8). There was no significant difference in the average area of the Golgi complex and mitochondria in comparison with the control group (Tables 9 and 10).

Discussion

The coupled action of reduction of bone volume and increase of PTH secretion has been observed in other experimental osteopenia as in the present study. However, the osteopenia that was chronically induced in rats fed a diet containing 5% iron lactate was characterized by bone resorption of the tibia without simultaneous PTH secretion at the end of the administration period (Matsushima et al., 2001, 2003). Therefore, we considered that the parathyroid gland was affected only in the early stages by iron lactate even with repeated dosing for long periods.

PTH secretion increased immediately after the intravenous administration of iron lactate but showed a downward tendency in the 6-day treatment. The average area of a storage granule decreased over the experimental periods and the number of storage granules was also decreased in the 3- and 6-day treatment. The storage granules with a wide clear space or halo were sometimes seen near the peripheral cytoplasm for each treatment period. These features are thought to be undischarged remnants in the chief cells of the parathyroid gland. The decrease of storage granules and rER along with the downward tendency of PTH secretion in the 6-day treatment is considered to reflect the beginning of the depression of PTH. Since no remarkable histopathological change was detected in the parathyroid gland on long-term treatment with iron lactate, the only interference to PTH production may have been due to iron lactate.

The increase of the PTH level resulted from the discharge from the storage granule in the early stages of iron lactate treatment. It is likely that a high PTH level induces an increase of osteoclasts in the primary spongiosa and bone lesion is formed in the initial stage of the long-term treatment with iron lactate. Generally, although the activity of osteoclasts was suppressed due to the reduction of PTH secretion (Felsenfeld and Llach, 1993), what remained unclear was the mechanism of iron lactate-induced osteopenia not accompanied by an increase of PTH secretion. The present study revealed that the increase of PTH acted as a trigger of the subsequent bone resorption.

In our previous study that the fed a diet containing 5.0% iron lactate for 3 months, angiopathy was not observed in any organs and tissues. However, the thrombus and angionecrosis of the parenteral site was present in 0.5 w/v% iron lacerate group of the preliminary study (data not shown). It is thought that iron lactate is a compound with the angiotoxicity and is accompanied by the widespread vascular injury. The vesicle of the vascular endothelium and enlargement of the perivascular space (edematous loosening) were ultrastructurally observed in the present study. These were considered to be changes due to the direct stimulation by iron lactate. Atrophy of the chief cells was thought to be the result of a decrease in the storage granules and rER.

The parathyroid gland maintains sustained secretion of PTH, including a release of stored granules, synthesis and degradation of PTH, reutilization of degraded hormone and mobilization of a secondary storage pool. PTH is instantly secreted in response to the decreased blood concentration. Increased PTH levels restore blood calcium to normal concentration by enhancing renal reabsorption and bone resorption of calcium. Furthermore, a relatively small decrement in blood calcium rapidly produces a maximal and sustained rise in PTH levels (Felsenfeld and Llach, 1993). Setoguti et al. (1995) reported that the threshold value of calcium concentration required for the release of PTH from storage granules is on the order of 0.88–0.83 mM Ca²⁺ (8.0–7.5 mg/dl). In the present study, the level of the calcium concentration was 10.9–11.8 mg/dl during the experimental period. Therefore, the rise of the PTH level due to iron lactate-treatment has little effect on the calcium concentration.

There have not yet been reports that the iron ion or oxygen free radicals induce the discharge from the storage granules of the parathyroid gland. However, Nakamichi et al. (2002) has reported that ferrous iron selectively prevented the increase in intracellular free Ca²⁺ concentration following activation of N-methyl-D-aspartate receptor in immature cultured rat cortical neurons in a manner different from various channel inhibitors (MK-801, MgCl₂, and ZnCl₂). Since iron lactate retains bivalent iron ions to high rate, ferrous ion may prevent influx into the chief cells in the parathyroid gland of calcium ion and promote a rapid release of PTH.

The plasma phosphate level decreased in the 6-day iron lactate treatment. Our previous study also showed the low level of inorganic phosphate in the rats fed 50,000 ppm iron lactate (Matsushima et al., 2001). In ultrastructural evaluation, the decrease of rER was observed in the 6-day dosing group. It is thought that the lowered PTH level mainly originates in the inhibition of PTH synthesis from this morphological change. Lopez et al. (2003) also reported that PTH levels clearly declined before a decrease in the blood phosphate level on hyperventilation in dogs. Moallem et al. (1998) have shown that hypophosphatemia rapidly decreases the stability of PTH mRNA messages. Therefore, hypophosphatemia may be one of the factors lowering the PTH level in the iron lactate treatment.

In our previous report, the plasma iron levels in the 4-week dosing group with iron lactate was equivalent to the control group, however, it shifted to the increase in the 13-week dosing group. And an accumulation of excess iron was observed in the liver, kidney, bone marrow and lymph node in the 13-week dosing group with iron lactate. Therefore, the long-term treatment with iron lactate is required for the increase of plasma iron levels (Matsushima et al., 2001, 2003). It is speculated that the combination of transferrin and iron increases in the short-term treatment with iron lactate, and the iron with transferrin is immediately accumulated in the liver and bone marrow and the plasma iron levels decreases.

These results collectively suggest a pathophysiological mechanism of iron lactate-induced osteopenia in rats, which we propose as a unique experimental model.