NDT Plus 2008 1(Supplement 3):iii42-iii48; doi:10.1093/ndtplus/sfn095
© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Cellular changes following direct vitamin D injection into the uraemia-induced hyperplastic parathyroid gland
Kazuhiro Shiizaki1,
Ikuji Hatamura2,
Shigeo Negi3,
Eiko Nakazawa1,
Ryoko Tozawa1,
Sayoko Izawa1,
Tadao Akizawa4 and
Eiji Kusano1
1 Division of Nephrology, Department of Internal Medicine, Jichi Medical University, Shimotsuke 329-0498
2 The First Department of Pathology
3 Division of Nephrology and Blood Purification Medicine, Wakayama Medical University, Wakayama 641-0012
4 Department of Nephrology, Showa University School of Medicine, Tokyo 142-0064, Japan
Correspondence: Kazuhiro Shiizaki, Division of Nephrology, Department of Internal Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Tel: +81-285-58-7346; Fax: +81-285-44-4869; E-mail: shiizaki{at}jichi.ac.jp
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Abstract
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Background. Hyperplasia of the parathyroid gland (PTG) is associated
not only with excessive secretion of parathyroid hormone (PTH)
but also with changes in the parathyroid cell (PTC) characteristics
(i.e. hyperproliferative activity and low contents of vitamin
D and calcium-sensing receptors). The control of PTG hyperplasia
is most important in the management of secondary hyperparathyroidism
(SHPT), because the advanced stage of hyperplasia is considered
irreversible. For the better control of the PTH level in dialysis
patients with such advanced SHPT, percutaneous vitamin D injection
therapy (PDIT) under ultrasonographic guidance was developed
and various cellular changes caused by this treatment were also
investigated using an animal model.
Methods. The PTGs of Sprague–Dawley rats, which had been 5/6-nephrectomized and fed a high-phosphate diet, were treated with the direct injections of vitamin D agents, and cellular effects focusing the above-mentioned characters were investigated.
Results. An adequacy of the direct injection technique into the rats’ PTGs and the successful effects of this treatment in various biochemical parameters were confirmed. Such characteristics of advanced SHPT were simultaneously improved; in particular, it was confirmed that this treatment may be effective in controlling PTG hyperplasia by, at least in part, apoptosis-induced cell death.
Conclusions. A locally high level of vitamin D strongly may suppress PTH secretion and regress hyperplasia, which is involved in the induction of apoptosis in PTCs, based on the simultaneous improvements of cellular characters of advanced SHPT. The PTH control introduced by this treatment successfully ameliorated osteitis fibrosa (high bone turnover rate).
Key Words: apoptosis • Ca-sensing receptor (CaSR) • parathyroid hyperplasia • percutaneous vitamin D injection therapy (PDIT) • secondary hyperparathyroidism • vitamin D receptor (VDR)
Received for publication February 26, 2008. Accepted for publication March 7, 2008.
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Introduction
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Secondary hyperparathyroidism (SHPT) is a common complication
of chronic kidney disease (CKD) and is characterized not only
by a high serum level of parathyroid hormone (PTH) but also
by hyperplasia of the parathyroid gland (PTG). In CKD, PTH plays
a very important role in bone and mineral metabolisms. The abnormalities
in bone in the setting of CKD include the effects of high levels
of PTH on bone, which results in the high-turnover bone disease
osteitis fibrosa. In addition, the effects of both absolute
and relative lack of PTH lead to a different skeletal abnormality
known as adynamic bone, which is characterized by an extremely
low bone turnover. It was reported that the relationship between
bone fracture and PTH level showed a U-shaped curve, in other
words that both low and high levels of PTH carried high risk
of bone fracture [
1]. Moreover, the incidence of bone fracture
is associated with an increased risk of mortality in the dialysis
population and it is well known that SHPT may contribute to
vascular calcification and potentially thereby worsen the prognosis
in patients with CKD [
2–4]. Thus, it is important to control
the PTH level, not only for appropriate bone and mineral metabolisms
but also for potential improvement of the prognosis in patients
with CKD.
Under uraemic conditions, phosphorus (P) retention and the low levels of calcium (Ca) and active vitamin D play very important roles in the progression of PTG hyperplasia. As medical treatments for SHPT, P binders, as well as calcitriol, and its analogue are effective in suppressing both the serum level of PTH and progression of PTG hyperplasia in the early stages, but advanced SHPT with a severely hyperplastic PTG is resistant to the above-mentioned treatments because of the low contents of vitamin D and Ca-sensing receptors (VDR and CaSR, respectively) in the parathyroid cells (PTCs) [5,6]. Thus, it is most important to pay attention to these factors, which affect resistance to medical treatments for SHPT, in particular PTG hyperplasia, when controlling SHPT.
When SHPT progresses into such an advanced status, it is considered irreversible. Thus, patients with advanced SHPT require the surgical removal of PTG (PTX) or percutanous ethanol injection therapy (PEIT) for the mass reduction of PTG. For the safe control of SHPT in such patients, a novel therapeutic tool, percutaneous direct vitamin D injection therapy (PDIT) was developed. The patients, who satisfied the guideline of the Japanese Society for Parathyroid Intervention [7], were treated by the series of six consecutive daily episodes of PDIT. Both serum intact-PTH levels and the PTG volumes following PDIT were significantly decreased. Thus, we considered that one of the mechanisms of the favourable clinical effect was regression of the hyperplastic PTG (Figure 1) [8–10]. In some patients non-severe complications were observed, such as a mild pain accompanied by the injection and mild subcutaneous haemorrhage. In particular, some problems complicating with PEIT were never observed, e.g. injury of surrounding tissues by ethanol leaking from the PTG, such as transient recurrent or sympathetic nerve palsy and severe pain. Therefore, it is considered that PDIT is a safe and effective treatment for advanced SHPT (Table 1).
The mechanisms for explaining the preferable clinical effects
of this treatment were investigated using molecular and morphological
examinations. At present, there are no available cell lines
of PTC for investigating the cellular effects of the target
agent; thus, the method of primary culture using the removed
PTGs was performed and a relationship was shown between PTC
death and various agents using this method [
11,12]. However,
it is considered that this method is not suitable for the investigation
of cell death in particular, due to many non-physiological effects.
Thus, an animal model and a method of direct injection into
its PTGs were developed and these advantages made it possible
to investigate the
in vivo effects of a highly concentrated
agent for PTC of uraemia-induced SHPT.
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Developments of an animal model of SHPT rat and the direct injection technique into PTGs
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For the animal model of SHPT, we adapted male Sprague–Dawley
rats with 5/6-nephrectomy, which were fed a normal diet (0.9%
P, 1.12% Ca) for 1 week after this procedure and then switched
to a high-P and low-Ca diet (1.2% P, 0.4% Ca) for 8 weeks. At
the completion of these procedures, the body weight, haemoglobin
(Hb) and ionized Ca (Ca
2+) levels were lower and serum blood
urea nitrogen (BUN), creatinine (Cr), P and intact-PTH levels
were higher than those of normal rats. The PTG of this uraemic
rat was severely hyperplastic, and the expression levels of
VDR and CaSR in PTC were significantly decreased [
13]. Moreover,
it was confirmed that the intravenous administration of vitamin
D even at a very high dose failed to decrease the serum PTH
level in these rats [
14]. These results indicated that this
animal was appropriate for uraemia-induced advanced SHPT model,
which were resistant to medical treatments including vitamin
D therapy.
Bilateral PTGs of the rats were exposed surgically and maxacalcitol (OCT: 10 µg/ml; DI-OCT) or its vehicle [phosphate buffer containing 0.01% polyoxyethylene sorbitan monolaurate and 0.2% ethanol (pH 8.0, isotonic solution)] (DI-vehicle) was directly injected using a 30-gauge (G) needle (specially made by Tochigi Seikou Co., Inc., Tochigi, Japan) under a zoom stereo microscope. The needle tip is blind and a one-side hole exists for manoeuvring it to the gland's centre. Immediately after the injection, the solution that had leaked from the PTG was washed away with saline. These procedures were performed under diethyl ether inhalation. The appropriateness of the method was confirmed by the results of direct injection of both Indian ink and [26-3H]-OCT (3H-OCT), and the actual volume of solution injected was similar to the original volume of the PTGs of this rat model (2.47 ± 0.65 µL/PTG; N = 10) [13,15].
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Specific effects of DI-OCT in PTC of uraemia-induced SHPT model rat
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The development of the animal model of advanced SHPT and the
method of direct injection into the PTG have made it possible
to investigate the cellular effects of PDIT in detail. Time
course changes of VDR and CaSR mRNA expressions determined by
reverse transcription-polymerase chain reaction (RT-PCR) and
the immunohistochemical expressions in PTGs following DI-OCT
were investigated. The expression levels of both VDR and CaSR
in PTGs following DI-OCT were significantly higher than those
before injection and at the corresponding time after DI-vehicle
(Figure
2). Moreover, for confirmation of the functional activity
of upregulated CaSR following DI-OCT, the changes in four parameters
calculated from the model of Brown [
16] in the Ca–PTH
response curve were investigated. Not only maximum PTH levels
but also the set point after DI-OCT significantly decreased
compared with those before DI-OCT. The sigmoid curve clearly
shifted to the left and downward following the DI-OCT (Figure
3). However, these findings were not observed after DI-vehicle
[
13]. These results indicate that this treatment can make it
possible to appropriately control both Ca and PTH levels. In
an experimental rat advanced SHPT model, kidney transplantation
normalized serum PTH, Ca, P and urea levels but did not upregulate
VDR and CaSR mRNA expressions in the PTG [
17]; however, DI-OCT
could functionally improve these sensitivity of PTCs.
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Fig. 3 Changes in the Ca–PTH response curve following DI-OCT. The means of following parameters are fitted to the Brown's equation [16]: intact-PTH = (a – d)/(1 + (Ca2+/c)b) + d, where c is the set point, d and a are minimum and maximum PTH levels achieved by hypercalcaemia and hypocalcaemia, respectively, and b is proportional to the slope of the Ca–PTH relationship at the set point.
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The mechanism of PTG regression following PDIT
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In the clinical study, the significant decrease in PTG volume
determined by ultrasonographic examination was confirmed and
one of the important mechanisms explaining the preferable clinical
effects of PDIT was considered to be the regression of PTG hyperplasia.
This required a decrease in the number of PTCs, so the cell
death caused by DI-OCT was investigated. The PTGs of this advanced
SHPT model rats were treated with two consecutive DI-OCT or
DI-vehicle. The PTGs were excised 24 h after the final injection
and evaluated for PTC apoptosis using light and electron microscopy,
TUNEL method and DNA electrophoresis. DI-OCT markedly increased
the number of TUNEL-positive PTCs and there was ladder formation
on DNA electrophoresis (Figure
4), as well as the characteristic
morphological features of apoptosis in both the light and electron
microscopic studies: nuclear pyknosis and fragmentation with
formation of apoptotic bodies, and intact cell membrane and
cytoplasmic organs. These findings were never observed in PTGs
following DI-vehicle. The induction of apoptosis in PTC following
PDIT was also confirmed by analysing the PTG samples obtained
by the biopsy technique before and after PDIT in uraemic patients
[
9,10]. Interestingly, the induction of apoptosis in PTC following
direct injection of all the injectable vitamin D metabolites
such as calcitriol, paricalcitol, and doxercalciferol as well
as OCT, which were developed for the treatment for SHPT and
are clinically used in many countries, was confirmed [
13,
18].
PTC-apoptosis induced by vitamin D has been controversial for
the past few decades [
11,
19–21] because investigators
have not been able to show the characteristic features using
the available cellular and molecular biological techniques.
In particular, it was considered that conventional intravenous
administration of vitamin D, even at very high doses, failed
to induce PTC-apoptosis in advanced SHPT (severely hyperplastic
PTG), principally because of the limited PTC uptake of vitamin
D related to the significantly decreased content of VDR. However,
the direct injection technique overcomes this limitation and
has been shown to induce PTC-apoptosis, as indicated by results
from the TUNEL method, DNA electrophoresis and electron microscopic
examination [
9,10,
13,
15,
18]. Thus, these findings are convincing
evidence that the regression of PTG hyperplasia following PDIT
is related to, at least in part, a decrease in the number of
PTCs because of apoptosis-induced cell death. However, it is
considered that more advanced studies are required to investigate
the detailed mechanism about the induction of PTC-apoptosis
by vitamin D.
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PDIT enables administration of the higher concentrated vitamin D to nuclear vitamin D binding sites of PTC
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These preferable cellular effects were not induced by conventional
administration of vitamin D. Thus, to explain the mechanisms
of the specific cellular changes in PTCs undergoing DI-OCT,
the particular difference in the degree of nuclear localization
of OCT between direct injection and systemic administration
was investigated using a bioimaging analyser system (BAS) and
microautoradiography (mARG) with
3H-OCT. Previous reports showed
the methods of these examinations and the evaluation of the
results in detail [
22,23]. The bilateral PTGs of rat were surgically
exposed, and only the left gland was directly injected with
3H-OCT (DI-
3H-OCT). The time course of the changes in both radioactivity
and localization of
3H-OCT in the bilateral glands was analysed
using a BAS and mARG, respectively. A very high dose of unlabelled
calcitriol was administered intravenously (IV-1,25D
3) prior
to DI-
3H-OCT, as a competitive study. Peak radioactivity levels
in the directly injected and intact PTG occurred immediately
and 1 h, respectively, after DI-
3H-OCT, and the difference was
about 50-fold higher in the treated gland. The latter level
was almost the same as that following intravenous administration
of
3H-OCT at a very high dosage, and it was considered that
this level indicated the limitation of the OCT uptake into the
PTG of this uraemia-induced advanced SHPT model by the conventional
administration. The mARG showed a marked concentration of silver
grains in the nuclei of PTC in the gland treated with DI-
3H-OCT.
However, this concentration was significantly suppressed by
IV-1,25D
3 prior to DI-
3H-OCT. These results indicated that DI-OCT
enables the administration of a highly concentrated drug for
specific binding to nuclear vitamin D binding sites, including
VDR of PTC, which markedly suppresses PTH, improves the response
to Ca and vitamin D and induces apoptosis in PTC [
15].
Moreover, it was recently reported that very high concentrations of both calcitriol and OCT in the PTG improve abnormal gene expression of PTG, which might directly and/or indirectly be related to PTH synthesis and secretion, and PTC proliferation and sensitivity to medical treatments for SHPT. Such normalizations might contribute to the better control of SHPT following PDIT [24].
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Amelioration of skeletal abnormality caused by PTH control based on the specific cellular effects of PDIT
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Next, we examined the maintenance of the above-mentioned cellular
effects by intravenous OCT administrations following DI-OCT,
as well as the histomorphometric alterations in the bone induced
in the control of SHPT on the basis of ameliorating these cellular
characteristics. The same advanced SHPT model rats were divided
into four treatment groups: (1) basic uraemic (at the baseline;
basic uraemic group), (2) one injection of DI-OCT followed by
intravenous OCT administration for 4 weeks (IV-OCT) (DI-OCT
+ IV-OCT group), (3) one injection of DI-vehicle followed by
the same dose of IV-OCT (DI-vehicle + IV-OCT group) and (4)
no treatment for an additional four weeks (uraemic control group).
The effects of these treatments on serum intact-PTH level, PTG
weight, VDR and CaSR expression levels in PTGs and bone histomorphometric
parameters were investigated. In the DI-OCT + IV-OCT group,
the significant decrease in the serum intact-PTH level and the
upregulations of VDR and CaSR expression levels in PTGs were
maintained by the subsequent IV-OCT. A significant decrease
in PTG weight compared with that of the basic uraemic group
was also observed. In the basic uraemic, DI-vehicle + IV-OCT
and uraemic control groups, the findings of severe osteitis
fibrosa such as enhanced bone and osteoid formations, hypomineralization
of bone, bone erosion with active osteoclasts, double labelling
and diffuse and irregular labelling are observed. These findings
indicate pathologic bone features associated with advanced SHPT.
However, in the bone treated by DI-OCT + IV-OCT, an enhanced
bone lamellar structure formation, attenuations of osteoid formation
and bone erosion, and regular labelling were noted, and these
findings were almost the same as the features of the normal
bone (Figure
5) [
14].
Previous reports showed that the conventional administration
of OCT or another vitamin D analogue mediates the effective
prevention of bone disease and ameliorates the established bone
histopathology and bone histomorphometric abnormalities in uraemic
animals and patients [
25–27]. However, the severity of
SHPT was mild in subjects of these previous studies, whose serum
PTH levels were significantly decreased by the conventional
administration of these agents. In general, patients with advanced
SHPT, which is unresponsive to conventional medical treatments,
undergo the PTX. PTX is very effective in decreasing serum PTH
and Ca levels; however, it was reported that this treatment
often induces a low bone turnover rate [
28,29]. A low turnover
and adynamic bone diseases caused by an excessive decrease in
the PTH level, particularly after PTX, could affect the progression
of arterial calcification [
4]. Moreover, PTX can lead to complications,
such as the necessity for general anaesthesia, the hyper- or
hypofunction of autotransplanted PTGs and psychological distress.
DI-OCT can make it possible to appropriately control the serum
PTH level based on the normalizations of above-mentioned aetiological
factors of advanced SHPT. Moreover, these effects successfully
ameliorated osteitis fibrosa (high bone turnover rate). Thus,
it is considered that this novel treatment with the appropriate
following treatments including the control of P and the conventional
administration of vitamin D may contribute to the improvement
of the prognosis of dialysis patients with advanced SHPT.
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Conclusion
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PDIT enables inducement of a significant decrease in PTH levels
in patients with advanced SHPT, who require PTX. Moreover, this
decrease is maintained by conventional treatments subsequent
to PDIT, and one of the mechanisms underlying this favourable
clinical effect is simultaneous amelioration of the important
aetiological factors that relate to the resistance to medical
treatments for SHPT; marked suppression of PTH synthesis and
secretion, upregulation of both the VDR and the CaSR and induction
of PTC-apoptosis. Thus, it is possible for some patients with
very severe SHPT to continue medical treatments and to avoid
PTX or PEIT following the introduction of this novel treatment.
PDIT is a safe and effective treatment for advanced SHPT (Table
1). However, it has been suggested that the indication for PDIT
is a patient who does not have a gigantic PTG (i.e. volume >2
cm
3) nor severely high levels of P and PTH (specifically, serum
P and intact-PTH levels >9.0 mg/dL and >1500 pg/mL, respectively),
and that patients with at least one of these criteria should
be treated by PEIT [
9]. Thus, the clinical indication of PDIT
based on this limitation should be carefully discussed.
Conflict of interest statement. None declared.
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Abstract
Introduction
