Original Report: Laboratory Investigation

Direct Injection of Calcitriol or Its Analog Improves Abnormal Gene Expression in the Hyperplastic Parathyroid Gland in Uremia
Kazuhiro Shiizaki^a, Masafumi Fukagawa^b, Qunsheng Yuan^f, Ikuji Hatamura^c, Tomoko Nii-Kono^b, Fumie Saji^d, Takashi Shigematsu^d, Tadao Akizawa<sup>e

a</sup>Division of Nephrology, Department of Internal Medicine, Jichi Medical University, Shimotsuke,
^bDivision of Nephrology and Dialysis Center, Kobe University School of Medicine, Kobe,
^cThe First Department of Pathology and
^dDivision of Nephrology and Blood Purification Medicine, Wakayama Medical University, Wakayama, and
^eDepartment of Nephrology, Showa University School of Medicine, Showa, Japan;
^fDivision of Nephrology, Peking Union Medical College Hospital, Beijing, China

Address of Corresponding Author

Am J Nephrol 2008;28:59-66 (DOI: 10.1159/000109240)

 Outline

• Key Words
• Abstract
• Introduction
• Methods
• Animals and Treatments
• Laboratory Measurements
• Isolation of Total RNA and Real-Time Polymerase Chain Reaction
• Immunohistochemical Analysis of Proliferating Cell Nuclear Antigen Expression
• Statistical Analyses
• Results
• Laboratory Data
• Effects of Uremia-Induced SHPT on Various Gene Expression Levels in the PTG
• Effects of DI-CAL and DI-OCT on Various Gene Expression Levels in the PTG
• Comparison of the Effects of DI-CAL and DI-OCT on Various Gene Expression Levels in the PTG
• PCNA Expression in the PTG
• Discussion
• Acknowledgment
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Gene
• Hyperparathyroidism
• Nephrectomy, 5/6
• Polymerase chain reaction, real-time
• Vitamin D
• Vitamin D metabolite

  Abstract

Aims: In this study, we investigated the effects of direct injection (DI) of calcitriol or maxacalcitol into the hyperplastic parathyroid gland (PTG) on altered gene expression related to the advanced status of secondary hyperparathyroidism (SHPT). Methods: Sprague-Dawley rats were 5/6-nephrectomized (uremic) or sham-operated (normal). In each uremic rat, one of the bilateral PTG was treated by DI of calcitriol (PTG_CAL) or maxacalcitol (PTG_OCT), and the other gland was treated with control solution (PTG_CONT). The PTG were evaluated for levels of expression of various mRNA and immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Results: Significant differences in levels of expression of mRNA and PCNA were confirmed between the uremic and normal groups. In PTG_CAL and PTG_OCT, expressions of almost all mRNA and PCNA were significantly improved; both agents were able to normalize the abnormalities of the uremic PTG, in contrast to the baseline and individual PTG_CONT. However, the difference in effect between PTG_CAL and PTG_OCT was only small. Conclusion: Our results suggest that very high concentrations of calcitriol or maxacalcitol in the PTG improve abnormal gene expression and proliferation activity of parathyroid cells, and might explain the better control of SHPT using the DI technique.

Copyright © 2007 S. Karger AG, Basel

 Introduction

In patients with end-stage renal disease, secondary hyperparathyroidism (SHPT) is characterized by hyperplasia of the parathyroid gland (PTG) and high serum levels of parathyroid hormone (PTH). The early stage of SHPT can be controlled by medical treatment with phosphorus binders and vitamin D (VD) supplementation, but advanced disease, characterized by severely hyperplastic PTG, is unresponsive to medical treatments. Little is known about the genetic abnormalities of the parathyroid cell (PTC) in SHPT. Two previous reports showed that most cases of advanced SHPT have a monoclonal neoplasm, assessed by X chromosome inactivation analysis, and one or more chromosomal changes as detected by chromosomal comparative genomic hybridization [1, 2].

Ultrasound-guided direct injection (DI) of calcitriol (CAL) or its analog into the hyperplastic PTG induces a significant decrease in PTH levels in patients with advanced SHPT. Moreover, this decrease is maintained by conventional treatments subsequent to DI, and one of the mechanisms underlying this favorable clinical effect is regression of hyperplasia by induction of PTC apoptosis [3,4,5]. The development of animal models of advanced SHPT and a method of DI into the PTG have made it possible to investigate the cellular effects of these treatments in detail. In advanced SHPT, DI of maxacalcitol (OCT) simultaneously ameliorates the important etiological factors of resistance to medical treatment: marked suppression of PTH synthesis and secretion, upregulation of both the vitamin D receptor (VDR) and the Ca-sensing receptor (CaSR), and induction of PTC apoptosis [6]. We recently showed that these effects were specifically caused by local administration of the agents at high concentrations, thereby enabling their specific interaction with nuclear VD binding sites, and that the improvement in clinical status of SHPT was maintained with subsequent conventional treatment [7, 8].

In this study, we analyzed the differences in the expression of various genes that might directly and/or indirectly be related to PTH synthesis and secretion, and PTC proliferation and sensitivity to medical treatments for SHPT, in order to further explain the mechanisms of the beneficial effects of DI-CAL or DI-OCT therapy.

 Methods

 Animals and Treatments

We used four treatment groups of male Sprague-Dawley rats: (1) normal rats without any treatment (normal group; n = 7), (2) uremic rats without any treatment (basic uremic group; n = 7), (3) uremic rats with PTG_CAL and PTG_CONT (CAL group; n = 8), and (4) uremic rats with PTG_OCT and PTG_CONT (OCT group; n = 8). Uremia was induced by 5/6 nephrectomy at 7 weeks old and subsequent feeding of a high phosphorus diet for 8 weeks, and normal rats received sham operations and were fed a normal diet for the same period.

The bilateral PTG of uremic rats were exposed surgically and each gland was directly injected with 10 µl of CAL (1 µg/ml; DI-CAL), OCT (22-oxacalcitriol; 10 µg/ml; DI-OCT), or control solution (phosphate buffer containing 0.01% polyoxyethylene sorbitan monolaurate and 0.2% ethanol; DI-CONT) using a 30-gauge needle, that is DI-CAL into one PTG (PTG_CAL) and DI-CONT into the other PTG (PTG_CONT) or DI-OCT into one PTG (PTG_OCT) and DI-CONT into the other PTG (PTG_CONT). This procedure has been described in detail elsewhere [6]. The Animal Studies Committee of Wakayama Medical University approved all experimental animal protocols.

 Laboratory Measurements

Serum intact PTH, ionized Ca (Ca²⁺) and P levels, and other data were measured 24 h before and after DI in the CAL and OCT groups, and immediately before PTG removal in the normal and basic uremic groups. Serum intact PTH levels were determined using a two-antibody method using a rat intact PTH ELISA kit (Immutopics, San Clemente, Calif., USA). Hemoglobin, hematocrit and Ca²⁺ levels were measured using an i-STAT Portable Clinical Analyzer (i-STAT, East Windsor, N.J., USA). Other parameters were measured using an automated analyzer (DRI-CHEM3500V, Fuji Film, Tokyo, Japan).

 Isolation of Total RNA and Real-Time Polymerase Chain Reaction

At 24 h after the treatments, the PTG were removed for processing as follows. Total RNA (2 µg) was extracted from each PTG specimen using TRIzol reagent (Life Technologies, Gaithersburg, Md., USA) and reverse-transcribed using oligo (dT) as a primer (Life Technologies) for polymerase chain reaction (PCR), as described previously [3]. The TaqMan® probe and primers for PTH, VDR, CaSR, vitamin D₃-upregulated protein-1 (VDUP-1), cyclin-dependent kinase (Cdk) inhibitor (p21), phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), Snail, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from TaqMan Gene Expression Assays (Perkin-Elmer Applied Biosystems, Foster City, Calif., USA). Their respective set assay IDs were Rn00566882_m1, Rn00566976_m1, Rn00566496_m1, Rn01533885_g1, Rn00589996_m1, Rn00448130_m1, Rn00441533_g1 and Rn99999916_s1. TaqMan PCR reactions and evaluation methods were described in a previous report [8].

We analyzed differences in mRNA expression levels between the PTG of the normal and basic uremic groups, PTG_CAL and PTG_CONT, and PTG_OCT and PTG_CONT, and the ratios of PTG_CAL/PTG_CONT and PTG_OCT/PTG_CONT.

 Immunohistochemical Analysis of Proliferating Cell Nuclear Antigen Expression

PTG samples were fixed in 4% paraformaldehyde phosphate-buffered saline for 8 h and embedded in paraffin. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was performed using a kit (Zymed Laboratories, San Francisco, Calif., USA). Levels of expression in individual glands were determined using light microscopy (BX50, Olympus, Tokyo, Japan). Three independent observers counted PCNA-positive cells in 10 high-power fields with approximately 400 cells per field at ×400 magnification (the ratio of positive cells per 1,000 cells), from which the average was taken.

 Statistical Analyses

Data are expressed as mean ± SD. Significant differences in laboratory data and expression levels of mRNA and PCNA were analyzed using analysis of variance (ANOVA), with post hoc multiple comparisons using Tukey-Kramer's and Dunnett's tests and Student's t test. A p value <0.05 was considered statistically significant.

 Results

 Laboratory Data

Table 1 shows the baseline laboratory data of all groups. Almost all study parameters in the uremic groups (i.e. basic uremic, CAL and OCT groups) were significantly different from those of the normal group. No significant differences were observed in baseline data between uremic groups, or in post-treatment levels of serum intact PTH, Ca²⁺ and P between DI-CAL and DI-OCT.

Table 1. Laboratory data

 Effects of Uremia-Induced SHPT on Various Gene Expression Levels in the PTG

The PTH/GAPDH mRNA ratio was significantly higher, and the other mRNA ratios were significantly lower, in the basic uremic group than in the normal group (fig. 1), confirming that all gene expressions analyzed in this study were significantly abnormal in the PTG of this model of advanced SHPT.

Fig. 1. Gene expression levels in the PTG. a-g Differences in the gene expression levels in the PTG of the normal vs. basic uremic groups, PTGCAL vs. PTGCONT in the same rat (CAL group), and PTGOCT vs. PTGCONT in the same rat (OCT group) are shown. * p < 0.05; ** p < 0.01 compared with the expression level of the basic uremic group.

 Effects of DI-CAL and DI-OCT on Various Gene Expression Levels in the PTG

In PTG_CAL, the PTH/GAPDH mRNA ratio was significantly lower, and the VDR/GAPDH, CaSR/GAPDH, VDUP-1/GAPDH, p21/GAPDH and PHEX/GAPDH mRNA ratios were significantly higher than in the basic uremic group (fig. 1). DI-CAL was not effective for all genes (e.g. Snail); however, all abnormal gene expressions examined in this study were normalized by DI-OCT. These normalizations were confirmed by comparison with mRNA expressions of the respective PTG_CONT in each uremic rat (fig. 1).

 Comparison of the Effects of DI-CAL and DI-OCT on Various Gene Expression Levels in the PTG

Table 2 shows the gene expression ratios of PTG_CAL/PTG_CONT (CAL group) and PTG_OCT/PTG_CONT (OCT group). Ratios of p21/GAPDH and Snail/GAPDH in the OCT group were significantly higher than those in the CAL group. No significant differences were seen in the other ratios. These results also suggest that DI-OCT may normalize the abnormal gene expressions that could not be normalized by DI-CAL.

Table 2. Gene expression ratios of PTGCAL/PTGCONT and PTGOCT/PTGCONT

 PCNA Expression in the PTG

The number of PCNA-positive PTC in the basic uremic group was markedly higher than in the normal group; however, in both the PTG_CAL and PTG_OCT it was significantly decreased compared with the basic uremic group, and numbers were significantly lower than in their respective controls. A significant difference between the CAL and OCT groups was not found (fig. 2).

Fig. 2.a Immunohistochemical staining for PCNA in PTC. Representative micrographs of immunohistochemical staining for PCNA in PTC for the individual treatment groups are shown. Green bars: 50.0 µm. Magnification ×400. b Quantitative analysis of PCNA-positive PTC. * p < 0.01 compared with the expression level in the basic uremic group..

 Discussion

In this study we confirmed from the laboratory data that this animal model produces uremia-induced SHPT, agreeing with a previous study involving a model of advanced SHPT, in which PTH synthesis and secretion, and PTC proliferation activity, were very high, and levels of VDR and CaSR in the PTG were very low [6]. Moreover, it should be noted that significant differences in terms of changes in the levels of serum intact PTH, Ca²⁺ and P between the CAL and OCT groups were not observed, indicating that differences in gene expression levels were independent of these biochemical changes. In our investigation of the effects of DI on the mRNA expression levels of PTG, we compared differences in PTG_CAL and PTG_OCTnot only with those in the basic uremic control group, but also with their own internal controls (i.e. PTG_CONT), because it is known that there is heterogeneity of mRNA expression levels in the PTG (especially under uremic conditions). However, in the consideration of PTG_CONT, the influence of the leakage of the therapeutic agent following DI cannot be disregarded. Our recent report showed that some leakage does enter the systemic circulation, and is taken up by the PTG_CONT [7]. Thus, it is considered that mRNA levels in the PTG_CONT were slightly changed.

The results of the present as well as previous studies indicate that PTH mRNA levels in PTG_OCT are significantly lower, and VDR and CaSR mRNA levels significantly higher in both the basic uremic group and in PTG_CONT in the same rat. Similar normalization in PTG_CAL was also confirmed in this study. In this way, we confirmed that in this model of advanced SHPT both DI-CAL and DI-OCT were effective in suppressing PTH mRNA and in upregulating the VDR and CaSR in the PTG.

Hyperplasia of the PTG is the most important factor in controlling PTH levels and managing SHPT. Previous reports showed a significant relationship between the expression levels of VDR and p21 in the PTG [9, 10], and this was confirmed in the present study, in which PTG levels of p21 mRNA in uremic rats were significantly lower than in normal rats, and levels of expression in PTG_CAL and PTG_OCT were significantly higher than in the basic uremic group. These results indicate that DI-CAL and DI-OCT induced significant improvement in these expression levels. VDUP-1 accelerates apoptosis signal-regulating kinase (ASK)-1-dependent apoptosis through the suppression of thioredoxin (TRX) in target cells [11]. It is a well-known relationship between VD and cell apoptosis in various tissues and cell lines. In particular, induction of PTC apoptosis by VD has been reported. We have shown that both DI-CAL and DI-OCT induce apoptosis in the PTC of uremic patients and rats with advanced SHPT [3, 5, 6]. In contrast, another report showed that CAL upregulated VDUP-1 expression, suppressing proliferation and causing cell cycle arrest at the G1 phase [12]. In this study, PCNA expression levels in PTG with upregulation of VDUP-1 (both PTG_OCT and PTG_CAL) were significantly lower than those in the basic uremic group and control glands (PTG_CONT). These results suggest that one mechanism for the control of PTC proliferation activity by VD might be altered regulation of apoptosis and proliferation by p21 and VDUP-1, although the major regulators of this effect are unclear.

Previous studies reported that both animal models (Hyp and Gy mice) and patients with untreated X-linked hypophosphatemia, which is caused by mutations in the PHEX gene, exhibit elevated serum PTH levels and PTG hyperplasia [13, 14]. Thus, we hypothesize that decreased levels of expression of the PHEX gene and its product might be related to elevated PTH levels and PTG hyperplasia, and that suppression of PHEX expression in the PTG, caused by uremia-induced high P and low Ca and VD levels, might facilitate the progression of SHPT. Both DI-CAL and DI-OCT induced upregulation of PHEX expression levels, and this may be related to the improvements seen in both SHPT symptoms and PTG hyperplasia.

Snail gene expression represses VDR expression and its transcriptional activity in human colon cancer cells [15]. In the present study, however, both VDR and Snail mRNA expressions in the PTG of normal rats were significantly higher than in the uremic rats. Moreover, after DI-OCT both of these abnormally low expression levels significantly increased compared with the basic uremic group and the control glands; however, the same effect was not seen following DI-CAL. These results suggest that Snail expression does not repress VDR expression in the PTG, but that suppressed Snail expression levels might be related to progression of SHPT, in either a VDR-independent and/or VDR-dependent fashion, and that a difference in the regulation of the Snail expression level may exist between CAL and OCT. The difference in the genomic effects of CAL and OCT is unclear. However, a previous study indicated that OCT differs from CAL in terms of its biological effect through interactions of the VDR with specific co-activators [16]. We may therefore presume that one of the mechanisms underlying the difference between the two agents is a specific effect of OCT on the VDR signaling pathway.

With regard to clinical implications, it has been suggested that the indication for DI treatment is a patient who does not have a giant 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 >1,500 pg/ml, respectively) [3]. Another important criterion is that there are no ectopic PTG that cannot be treated under ultrasonographic guidance. We consider that some of the various abnormal gene expressions and increased proliferation activity of PTG in uremia-induced SHPT can be normalized by either DI-CAL or DI-OCT in such patients. We also suggest that some of the favorable clinical and cellular effects of these treatments are based on the normalization of gene expressions. However, there are some differences between the effects of CAL and OCT, and more detailed studies are required in this area.

 Acknowledgment

This study is supported by Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. And the authors are grateful to the Kirin Brewery Co., Ltd., Tokyo, Japan and Chugai Pharmaceutical Co., Ltd., who supported this study by providing the calcitriol and maxacalcitol solutions and their vehicles, respectively.

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  Author Contacts

Kazuhiro Shiizaki, MD, PhD
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@jichi.ac.jp

  Article Information

Part of this study was presented at the Annual Meeting of the American Society of Nephrology held in Philadelphia, Pennsylvania, USA, 2005, and has been punblished as an abstract (J Am Soc Nephrol 16:494A, 2005).

Received: June 28, 2007
Accepted: August 13, 2007
Published online: September 28, 2007
Number of Print Pages : 8
Number of Figures : 2, Number of Tables : 2, Number of References : 16

  Publication Details

American Journal of Nephrology

Vol. 28, No. 1, Year 2008 (Cover Date: November 2007)

Journal Editor: Bakris, G. (Chicago, Ill.)
ISSN: 0250-8095 (print), 1421-9670 (Online)

For additional information: http://www.karger.com/AJN

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.