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NDT Advance Access originally published online on December 21, 2007
Nephrology Dialysis Transplantation 2008 23(5):1529-1536; doi:10.1093/ndt/gfm850
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Highly concentrated calcitriol and its analogues induce apoptosis of parathyroid cells and regression of the hyperplastic gland—study in rats*

Kazuhiro Shiizaki1, Ikuji Hatamura2, Shigeo Negi3, Toshifumi Sakaguchi3, Fumie Saji3, Ikuo Imazeki4, Eiji Kusano1, Takashi Shigematsu3 and Tadao Akizawa5

1 Division of Nephrology, Department of Internal Medicine, Jichi Medical University, Shimotsuke 329-0498 2 The First Department of Pathology, Wakayama Medical University, Wakayama 641-0012 3 Division of Nephrology and Center of Blood Purification Therapy, Wakayama Medical University, Wakayama 641-0012 4 Medical Section, Sendai Branch, Chugai Pharmaceutical Co. Ltd, Sendai 980-0014 5 Department of Nephrology, Showa University School of Medicine, Tokyo 142-0064, Japan

Correspondence and offprint requests to: 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



   Abstract
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 Abstract
Short summary
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Controlling hyperplasia of the parathyroid gland (PTG) is important in the management of secondary hyperparathyroidism (SHPT). Regression of the hyperplastic PTG requires a decrease in the number of parathyroid cells (PTCs), so the present study investigated cell death caused by toxic agents or by clinically usable vitamin D metabolites.

Methods. The PTGs of Sprague–Dawley rats, which had been 5/6-nephrectomized and fed a high-phosphate diet for 12 weeks, were treated with two consecutive direct injections (DI) of calcitriol, maxacalcitol, paricalcitol, doxercalciferol or phosphate-buffered saline containing either 0.01% or 90% ethanol (0.01-ET or 90-ET, respectively). Laboratory data, including serum levels of intact parathyroid hormone (intact-PTH), were obtained before and after the treatments. The PTGs were excised 24 h after the final injection and evaluated for PTC apoptosis using light and electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) method and DNA electrophoresis.

Results. Treatment with any of the vitamin D metabolites and 90-ET significantly decreased the serum intact-PTH level, but only the latter significantly decreased the serum Ca level. Either treatment markedly increased the number of TUNEL-positive PTCs, but not in PTG treated with 0.01-ET. In PTGs treated with DI of any vitamin D metabolites was there ladder formation on DNA electrophoresis, as well as the characteristic morphological features of apoptosis in both the light and electron microscopic studies.

Conclusions. DI of vitamin D metabolites may be effective in controlling not only the PTH level, but also PTG hyperplasia, in advanced SHPT by, at least in part, apoptosis-induced cell death. Our study was performed in rats.

Keywords: cell death; electron microscopy; hyperparathyroidism; renal osteodystrophy; vitamin D



   Short summary
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 Abstract
 Short summary
Introduction
 Methods
 Results
 Discussion
 References
 
Direct injection (DI) of highly concentrated vitamin D metabolites or ethanol into a hyperplastic PTG decreases both the PTH level and the number of PTCs by apoptosis- and necrosis-induced cell death, respectively. These effects were confirmed by the TUNEL method, DNA electrophoresis and light and electron microscopy. Vitamin D metabolites are used clinically in many countries, so uraemic patients with advanced SHPT can benefit from this therapy, with few complications. Our study was performed in rats.



   Introduction
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 Abstract
 Short summary
 Introduction
Methods
 Results
 Discussion
 References
 
Uraemia-induced secondary hyperparathyroidism (SHPT) is characterized not only by a high serum level of parathyroid hormone (PTH), but also by hyperplasia of the parathyroid gland (PTG). 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. In the early stage of SHPT the P binder, calcitriol, and its analogue are effective in suppressing both the serum level of PTH and progression of PTG hyperplasia, but advanced SHPT with a severely hyperplastic PTG is resistant to medical treatment because of the low content of vitamin D and Ca-sensing receptors (VDR and CaSR, respectively) in the parathyroid cells (PTCs) [1,2]. Thus, controlling PTG hyperplasia is most important in the management of SHPT.

It is also well known that maintaining appropriate levels of PTH, Ca and P is required in uraemic patients with SHPT in order to improve their prognosis [3]. Therefore, vitamin D analogues with a low calcaemic action and strong suppression of PTH have been developed and for the past few decades intravenous administration of calcitriol (1,25-dihydroxyvitamin D3: CAL), maxacalcitol (22-oxa-1,25-dihydroxyvitamin D3: OCT), paricalcitol (19Nor-1,25-dihydroxyvitamin D2: 19Nor) or doxercalciferol (1-alpha-hydroxyvitamin D2: DOX) has been used in the management of SHPT in Japan, the United States, Europe and elsewhere, although Japan is the only country where OCT is used. The beneficial clinical effects of these agents in decreasing the serum PTH level with a little increase in the serum Ca and P levels in uraemic patients with SHPT have been reported [4–6].

For cases of advanced SHPT, less invasive treatments using ultrasonography (US), endoscopy and other imaging tools have been developed because the conventional surgical treatment (i.e. parathyroidectomy (PTX)) requires general anaesthesia and hospitalization, and often there are complications. In Japan, US-guided techniques of DI of the PTG have been developed, with subsequent development of subsidiary equipment, such as high-performance US tomography and specific injection needles [7,8], and there are now many reports about this treatment [7–11]. DI of CAL or OCT into the hyperplastic PTG of patients with advanced SHPT that is resistant to other medical treatments, including the intravenous administration of these agents, induces a significant decrease in the serum PTH level without changing the Ca and P levels. Moreover, the decreased level of PTH is subsequently maintained by conventional medical treatment. It is considered that one of the mechanisms underlying the favourable clinical effects of DI is regression of the hyperplastic PTG because of induction of PTC apoptosis [9–11]. The development of animal models of advanced SHPT and a modified technique of DI have made it possible to investigate the cellular effects in detail using molecular biological techniques. DI therapy for advanced SHPT can simultaneously ameliorate some important aetiological factors of the resistance to medical treatments; that is, marked suppression of PTH synthesis and secretion, upregulation of the VDR and CaSR and induction of PTC apoptosis [12]. Control of the PTH level by this treatment enables amelioration of the osteitis fibrosa and high rate of bone turnover caused by SHPT [13]. Thus, DI therapy is expected to have significant effects in patients with advanced SHPT, similar to those of PTX.

However, results to date have been shown in PTGs treated by OCT only. In addition, despite previous reports of a significant increase in the number of PTCs positive for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and a ladder pattern on DNA electrophoresis, which is a characteristic feature of cell apoptosis, it is well known that non-specific TUNEL-positive cells, such as necrotic cells, and DNA fragmentation without apoptosis can be observed [14]. Thus, in the present study we considered it necessary to show the morphological changes by electron microscopy, in addition to the TUNEL method and DNA electrophoresis, to confirm the induction of PTC apoptosis following local administration of CAL and its analogues. As the reference data, we performed DI using highly concentrated ethanol.



   Methods
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 Abstract
 Short summary
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
We performed 5/6 nephrectomies in 7-week-old male Sprague–Dawley rats, under intraperitoneal pentobarbital anaesthesia (50 mg/kg body weight). They were fed a normal diet (0.9% P, 1.12% Ca; protein sources: grain and white fish meal) for 1 week after the procedure and then switched to a high-P and low-Ca (HP-LC) diet (1.2% P, 0.4% Ca; protein sources: grain and milk casein) for 12 weeks [12]. All feed was obtained from Oriental Yeast, Inc. (Chiba, Japan).

DI treatments
Under diethylether inhalation, both PTGs were exposed surgically and we performed DI with 10 µL of CAL (1 µg/mL) (DI-CAL), OCT (10 µg/mL) (DI-OCT), 19Nor (5 µg/mL) (DI-19Nor), DOX (2 µg/mL) (DI-DOX) or phosphate-buffered saline containing 0.01% or 90% ethanol (0.01-ET: DI-CONT or 90-ET: DI-ET) on 2 successive days, using a 30-gauge needle (made exclusively by Tochigi Seikou Co. Inc., Tochigi, Japan). With regard to the concentration of ethanol, 0.01% is the conventional concentration in the vehicle of vitamin D solution and 90% is the clinically proven effective concentration for US-guided percutaneous ethanol injection therapy (PEIT) [7]. Two consecutive injections were given because it reliably induces PTC apoptosis [12]. Immediately after each injection, any leakage from the PTG was flushed with saline. We previously reported that the 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) [12]. PTG of the rat is very dense compared with that of a uraemic patient, so we considered that an injection pressure with a volume exceeding that of the actual gland is required for improved distribution of the injected solution within the tissue. We confirmed this method of DI in our previous study [12]. The Animal Studies Committee of Wakayama Medical University approved all experimental protocols.

Laboratory measurements
Serum levels of intact-PTH, ionized calcium (Ca2+) and P, and other data, were obtained before and 24 h after the final injection. The serum intact-PTH level was measured by a two-antibody method using an appropriate ELISA kit (Immutopics, Inc., CA, USA), which can measure between 1.6 and approximately 2000 pg/mL, so the samples were diluted with the attached 0 pg/mL Standard at 1:10 and analysed together. All standards, controls and test samples were assayed in duplicate and averages were taken. Haemoglobin (Hb), haematocrit (Hct) and Ca2+ levels were measured using an i-STAT Portable Clinical Analyzer (i-STAT Corporation, NJ, USA). Other data were determined using an automated analyser (DRI-CHEM3500V, Fuji Film, Tokyo, Japan).

Light microscopy
PTGs were excised 24 h after the final injection and half of the left side of the PTG was fixed with 4% paraformaldehyde phosphate-buffered saline (PBS) for 8 h and then paraffin-embedded for routine tissue processing. Sections were prepared, stained with haematoxylin and eosin and examined under a light microscope (BX50, Olympus, Tokyo, Japan).

Apoptosis analysis by the TUNEL method
Paraffin-embedded tissue blocks were sectioned, deparaffinized in xylene and alcohol and placed in PBS. The tissue sections were then treated with proteinase K and washed with PBS. An in situ apoptosis detection kit (ApopTag; Serologicals Co., GA, USA) was used for labelling the free 3'-OH terminus. These procedures were automated using VENTANA (Ventana Medical Systems, Inc., AZ, USA). The expression level in individual glands was determined by light microscopy (BX50, Olympus). Three independent observers counted the TUNEL-positive PTCs in 10 high-power fields, with approximately 400 cells per field at x400 magnification (the ratio of the number of TUNEL-positive PTCs per 1000 PTCs = TUNEL-index), after which the average was taken.

Apoptosis analysis by DNA electrophoresis
DNA was extracted from the other side of the PTGs, using a DNA extraction kit (QIAamp; QIAGEN GmbH, Hilden, Germany). DNA electrophoresis was performed at a constant voltage of 100 V in horizontal 2% agarose gels and the DNA bands were visualized by ethidium bromide staining.

Electron microscopy
Apoptosis was also demonstrated by electron microscopy, for which the residue of the left side of the PTG fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) were refixed in 2% osmium tetroxide for 2 h at 4 °C, dehydrated in ethanol at room temperature, and embedded in Quetol 812 (Nisshin EM Co., Tokyo, Japan). Next, 80-nm sections of tissue were contrasted with 4% uranyl acetate for 15 min and subsequently with lead citrate for 5 min at room temperature. Samples were examined with a Hitachi H-300 transmission electron microscope (Hitachi Ltd, Tokyo, Japan).

Statistical analyses
Data are expressed as means ± SD. Laboratory data from all the treatment groups at baseline were analysed by analysis of variance (ANOVA) with post hoc multiple comparisons using Tukey–Kramer's test. The differences in serum levels of intact-PTH, Ca2+ and P between before and after the treatments in individual treatment groups were analysed by Student's paired t-test. The differences in serum intact-PTH, Ca2+ and P ratios and TUNEL indexes among the treatment groups (DI-ET, DI-CAL, DI-OCT, DI-19Nor and DI-DOX relative to DI-CONT) were analysed by ANOVA, with post hoc multiple comparisons using Dunnett's test. A P value <0.05 was considered statistically significant.



   Results
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 Methods
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Discussion
 References
 
Changes in laboratory data
Table 1 shows the baseline laboratory data for all the treatment groups and, in particular, the serum Ca2+, P and intact-PTH levels before and after the treatments. Body weight, Hb, Hct and Ca2+ levels were lower and serum blood urea nitrogen (BUN), creatinine (Cr), P and intact-PTH levels were higher at baseline than previously reported for normal rats [12]. However, significant differences among the treatment groups for any of the parameters were not observed.


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  		Table 1 Laboratory data for all treatment groups

 
The serum intact-PTH level following DI-ET, DI-CAL, DI-OCT, DI-19Nor and DI-DOX was significantly decreased compared with before treatment; however, DI-CONT had no effect. The serum Ca2+ level following DI-ET was significantly decreased compared with before the treatment and there were mild increases and decreases following DI-CAL and DI-OCT, and DI-19-Nor and DI-DOX, respectively; however, these changes in Ca were not significant compared with individual baseline levels. A mild increase in the serum P level following DI-ET and DI-19Nor was observed, but was not significant compared with individual baseline levels.

Morphological changes under light microscopy
Many intact PTCs were observed (i.e. hyperplasia) after DI-CONT (Figure 1A). Following DI-ET the PTG comprised almost uniform eosinophilic tissue lacking nuclei, which was indicative of necrosis (Figure 1B). Among all the vitamin D treatment groups, partial defects of the PTCs and PTCs with various types of nuclear changes, such as nuclear condensation and fragmentation, were observed (Figure 1C).


Figure 1
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  		Fig. 1 Morphological examination by light microscopy. Representative images of parathyroid tissue following DI-CONT (A), DI-ET (B) and treatment with vitamin D metabolites (C) (x200; bars = 100 µm). CONT, control; DI, direct injection; ET, ethanol (see the text for full explanation of agents).

 
Apoptosis analysis by the TUNEL method
Figure 2 shows the PTCs subjected to the TUNEL method. After DI-CONT, few TUNEL-positive PTCs were observed (Figure 2A), but after all of the other treatments there were many TUNEL-positive PTCs (TUNEL-index of DI-CONT, DI-ET, DI-CAL, DI-OCT, DI-19Nor and DI-DOX: 8 ± 9, 814 ± 224, 890 ± 84, 825 ± 114, 785 ± 149 and 765 ± 184, respectively).


Figure 2
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  		Fig. 2 Detection of DNA fragmentation in parathyroid cells by the TUNEL method. Representative findings of in situ detection of DNA fragmentation by the TUNEL method in parathyroid tissues following DI-CONT (A), DI-ET (B) and treatments with vitamin D metabolites (C) (x400; bars = 50.0 µm). See Figure 1 for abbreviations.

 
Apoptosis analysis by DNA electrophoresis
In tissue samples from PTG treated with any of the vitamin D treatments there was a ladder pattern, which indicates the presence of DNA fragmentation, but this was not seen in PTG tissue after DI-CONT or DI-ET (Figure 3).


Figure 3
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  		Fig. 3 Detection of DNA fragmentation using 2% agarose gel electrophoresis. The ladder pattern, indicating DNA fragmentation, can be seen after all treatments using vitamin D metabolites (C), but not with either DI-CONT (A) or DI-ET (B). See Figure 1 for abbreviations.

 
Morphological changes under electron microscopy
The characteristic features of apoptosis, such as chromatin condensation and fragmentation of the nucleus, with an intact cell membrane and cytoplasmic organelles, were observed (Figure 4C–F). In particular, deformities of the mitochondria were not observed. Occasionally, apoptotic bodies surrounded by a cellular membrane were also present (Figure 4E). These findings were observed in PTGs from all the vitamin D treatment groups, but never in the PTGs treated by DI-CONT or DI-ET. In the PTGs treated by DI-CONT, almost all PTCs had an intact nucleus, cytoplasmic organelles and a cell membrane rich in secretory granules, which indicated an intact cell with hyperactive PTH secretion (Figure 4A). In the PTG treated by DI-ET, there were many necrotic PTC, all of which showed significant destruction of the nucleus, cytoplasmic organelles and membrane (Figure 4B).


Figure 4
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  		Fig. 4 Ultrastructure of PTCs by electron microscopy. In the hyperplastic PTG, the PTCs are rich in secretory granules (white arrowheads in A). In the PTG treated with vitamin D metabolites, there are many PTCs with the characteristic features of cell apoptosis, such as condensation (black arrows in C and D) and fragmentation (black arrowheads in F) of the nucleus, apoptosis bodies (white arrow in E), and intact cytoplasmic organelles (black dotted arrows indicate intact mitochondria in D) and membrane. Such PTCs were never observed in PTG treated by DI-CONT or DI-ET. (A) DI-CONT, x5000; (B) DI-ET, x7000; (C) DI-CAL, x7000; (D) DI-OCT, x5000; (E) DI-19Nor, x5000; (F) DI-DOX, x7000. See Figure 1 and text for abbreviations.

 


   Discussion
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 Abstract
 Short summary
 Introduction
 Methods
 Results
 Discussion
References
 
For better control of the serum level of PTH in patients with advanced SHPT, new therapeutic techniques, such as US-guided DI of highly concentrated ethanol or vitamin D into the hyperplastic PTG, have been developed in Japan. Injection of either agent is effective, with few complications, and both induce regression of PTG hyperplasia. Ethanol destroys PTG tissue by necrosis of the PTCs, whereas vitamin D analogues, such as OCT, induce PTC apoptosis with the high concentrations used in the DI technique, an effect that does not occur with conventional intravenous administration [10]. Experiments using 3H-labelled OCT have revealed that the specific effects of DI are related to local administration at high concentration, which enables specific interaction with various nuclear vitamin D binding sites, including the VDR, in PTCs, and is considered to be the mechanism of the beneficial clinical effects of DI-OCT [15]. In the present study, we investigated whether all the vitamin D analogues that have been developed for SHPT treatment and are being used clinically in many countries, might induce PTC apoptosis in the PTG of severely advanced SHPT after DI at high concentrations.

The relationship between vitamin D and cell apoptosis in various tissues and cell lines is known [16–19], but PTC apoptosis induced by vitamin D has been controversial for the past few decades [20–23] because investigators have not been able to show the characteristic features of PTC apoptosis 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 limited PTC uptake of vitamin D related to the significantly decreased content of VDR. However, the DI technique overcomes this limitation and has been shown to induce PTC apoptosis, as indicated by results from the TUNEL method and DNA electrophoresis [10–12,15]. Moreover, it was recently reported that even conventional administration of CAL could induce PTC apoptosis in animals with mild SHPT, as indicated by a significant increase in the number of TUNEL-positive PTCs (perhaps because of some retention of VDR) [24], so in the present study, we used a model of advanced SHPT to focus on the mechanisms of cell death caused by local administration of highly concentrated vitamin D metabolites and ethanol.

First, we confirmed that the PTGs of the rats were severely hyperplastic and that the serum levels of intact-PTH and P were extremely high, with an extremely low serum Ca2+ level. DI-CAL, DI-OCT, DI-19Nor and DI-DOX successfully decreased the serum intact-PTH level without significant changes in the serum Ca2+ and P levels (Table 1), although there were slight changes in both these parameters. The rapid decrease in the serum PTH level immediately after surgical PTX induces ‘hungry bone syndrome’ in patients with advanced SHPT and the most characteristic feature is a marked decrease in the serum Ca level. The same phenomenon is sometimes observed in the patients treated by PEIT (‘chemical PTX’) and in the present study DI-ET similarly induced a significant decrease in the serum Ca2+ level. However, among the vitamin D treatment groups, there were only small differences in the change of the serum Ca2+ level, mainly because of differences in the level of PTH suppression, the strength of the calcaemic action and the administered dose. These findings indicate that DI of vitamin D is considered safer than PEIT, without the rapid decrease in the serum Ca level and associated complications such as tetany. Moreover, small increases in the serum P level were observed with DI-ET and DI-19Nor, because of decreased phosphaturic action as a result of greater suppression of PTH.

DI-CAL, DI-OCT, DI-19Nor and DI-DOX induced a significant increase in the number of TUNEL-positive PTCs and showed the characteristic ladder pattern indicating the presence of DNA fragmentation. However, only a significant increase in the number of TUNEL-positive PTCs was also observed in the PTGs treated by DI-ET (Figure 2B). It is well known that non-specific TUNEL-positive cells (not apoptotic cells) sometimes can be seen; however, in the present study, the TUNEL method was suitable for in situ detection of apoptotic cells, in particular, whether apoptotic cells were PTCs or not and the quantitative changes caused by the target treatment compared with the control treatment (DI-CONT) could be analysed. Thus, in our previous reports we showed both an increase in the number of TUNEL-positive PTCs and the characteristic finding of DNA electrophoresis as evidence of the induction of PTC apoptosis following DI-OCT in uraemic patients [10] and animals [12], and included additional examination using electron microscopy in the present study. Little is known about the ultrastructure of uraemic PTCs, however many PTCs with intact cell membrane and nucleus and rich in secretory granules that varied in size were observed in PTG treated by DI-CONT (Figure 4A). We consider that these granules contain PTH and that this finding suggests hyperactive PTH secretion. In the future, examination using immune-electron microscopy will clarify this hypothesis. Electron microscopic examination also revealed many PTCs with the characteristic features of apoptosis in tissue samples from PTGs treated by the vitamin D injections, but only necrotic, never apoptotic, PTCs in PTGs treated by DI-ET. Moreover, we failed to show DNA fragmentation by DNA electrophoresis in the PTGs treated by DI-ET. The conclusion from these findings is that DI with any of the vitamin D metabolites developed for SHPT treatment (i.e. OCT, CAL, 19Nor and DOX) can induce PTC apoptosis and that the same treatment using a control solution not containing vitamin D or highly concentrated ethanol cannot.

However, some problems still exist; namely, one vitamin D analogue has been shown to induce cell death by neither apoptosis nor necrosis [25], but rather by autophagy, which is characterized by an increase in the number of autophagosomes and vacuoles surrounded by a double membrane, which are sequestered from the cytoplasm or organelles. Subsequently, the autophagosomes fuse with lysosomes to form autolysosomes in which sequestered material is digested. It is considered that autophagy plays an important role in normal protein and organelle turnover caused by the recycling of nutrients and/or removal of damaged organelles (in particular, mitochondria). In the characteristic process of autophagy, damaged organelles are surrounded by autophagosomes, followed by apoptosis-like nuclear changes such as condensation and fragmentation of chromatin. The morphological findings of the completion of autophagy can sometimes look like apoptosis [25]. Thus, detailed morphological evaluation by electron microscopy is required to unequivocally confirm the induction of PTC apoptosis in PTGs treated by DI of vitamin D. In the present study all PTGs treated by DI-CAL, DI-OCT, DI-19Nor and DI-DOX showed the characteristic features of apoptosis (i.e. chromatin condensation and fragmentation, and apoptotic bodies without changes of the cellular membrane and organelles, in particular, mitochondria) in the electron microscopic study and these findings were completely different from the characteristics of autophagy-induced cell death. Thus, the findings of present study are convincing evidence that the regression of PTG hyperplasia following DI of vitamin D 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, including immune-electron microscopic examination for the detection of mitochondrial markers in vacuoles, are required to distinguish these differences more precisely.

With regard to the clinical implications, it has been suggested that the indication for DI treatment with vitamin D is a patient who does not have a gigantic PTG (i.e. volume >2 cm3) 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 [10]. However, the most important criterion for all of DI treatments is that there are no ectopic PTG that cannot be treated under US guidance. Such patients should be treated by ‘surgical PTX’. Clinically, DI with either vitamin D or ethanol can be performed repeatedly to achieve the target PTH level, but the number of injections is limited with the animal model because of the necessites for general anaesthesia and surgical exposure of the gland. Moreover, the rat PTG is very small, even in the uraemic condition, and is very hard compared with the PTG of a uraemic patient. Thus, the mode of diffusion of the solution within the animal PTG, when administered by DI technique, may differ from the case of uraemic patients. However, this model and method have been confirmed as appropriate for the basic examination of this treatment [12,13]. Moreover, the manifestation following DI treatment in this animal model corresponds with the clinical course (i.e. decreased PTH level and regression of PTG hyperplasia maintained by subsequent conventional treatment) [9–11,13]. Improvements in the basic technique and clinical tools of DI treatment will contribute to improvement in the status of this novel treatment in the near future.

In conclusion, the most important factor in the management of SHPT is suppression of PTG hyperplasia. US-guided percutaneous injection therapy using highly concentrated ethanol or vitamin D metabolites achieves this goal by decreasing the number of PTCs by necrosis or apoptosis, respectively. The induction of PTC apoptosis was confirmed by the TUNEL method, DNA electrophoresis and electron microscopy. Thus, DI of any of the vitamin D metabolites that are being used clinically can significantly decrease the serum PTH level and regress PTG hyperplasia by inducing PTC apoptosis without any significant complications. It is expected that uraemic patients with advanced SHPT in many countries will benefit from these novel treatments.



   Acknowledgments
 
This study was supported by Chugai Pharmaceutical Co. Ltd, Tokyo, Japan. The authors thank Kirin Brewery Co. Ltd, Tokyo, Japan and Chugai Pharmaceutical Co. Ltd for providing the calcitriol and maxacalcitol solutions, respectively.

Conflict of interest statement. None declared.



   Notes
 
* Part of this study was presented at the Annual Meeting of the American Society of Nephrology, Philadelphia, PA, USA, 2005, and appeared as an abstract (J Am Soc Nephrol 2005; 16: 494A). Back



   References
Top
 Abstract
 Short summary
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 17. 7.07
Accepted in revised form: 5.11.07


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T. NITTKE, E. KALLAY, T. MANHARDT, and H. S. CROSS
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