Percutaneous maxacalcitol injection therapy regresses hyperplasia of parathyroid and induces apoptosis in uremia

Kidney International, Vol. 64 (2003), pp. 992–1003

Percutaneous maxacalcitol injection therapy regresses hyperplasia of parathyroid and induces apoptosis in uremia KAZUHIRO SHIIZAKI,1 IKUJI HATAMURA,1 SHIGEO NEGI, NOBUHIKO NARUKAWA, MASAHIDE MIZOBUCHI, TOSHIFUMI SAKAGUCHI, AKIRA OOSHIMA, and TADAO AKIZAWA Center of Blood Purification Therapy, Wakayama Medical University, Wakayama, Japan; and The First Department of Pathology, Wakayama Medical University, Wakayama, Japan

Percutaneous maxacalcitol injection therapy regresses hyperplasia of parathyroid and induces apoptosis in uremia. Background. A high level of parathyroid hormone (PTH) is considered to be an indicator of poor prognosis and a poor quality of life of dialysis patients; therefore, an effective and safe therapy for secondary hyperparathyroidism (SHPT) has been developed. Methods. In 20 patients with SHPT resistant to maxacalcitol (OCT) intravenously administered, all detectably enlarged parathyroid glands were treated by percutaneous maxacalcitol injection therapy (PMIT) under ultrasonographic guidance consecutively 6 times, which was followed by OCT that was intravenously administered. The clinical effects of PMIT were evaluated based on the changes in the serum intact-PTH, adjusted Ca, phosphorus, and bone marker levels, and the parathyroid gland volume determined by ultrasonography. Morphologic examination, apoptosis analysis, and PTH mRNA expression level determination by reverse transcription-polymerase chain reaction (RT-PCR) using parathyroid tissues obtained by a biopsy technique were performed. Results. PMIT and subsequent intravenous OCT administrations significantly decreased the serum intact-PTH level and parathyroid gland volume for at least 12 weeks after PMIT without major complications. Parathyroid tissues obtained after PMIT exhibited some partial defects of parathyroid cells, a marked increase in the number of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)– positive cells, the ladder formation determined by DNA electrophoresis, and the decrease in the PTH mRNA expression level. Conclusion. PMIT is effective and safe for the treatment of refractory SHPT, and a locally high level of OCT suppresses PTH secretion and regresses parathyroid hyperplasia, which is involved in the induction of apoptosis of parathyroid cells.

Secondary hyperparathyroidism (SHPT) caused by chronic renal failure results in complications such as renal osteodystrophy. In addition, high levels of parathyroid hormone (PTH) induce anemia resistant to recombinant human erythropoietin treatment, cardiovascular disorders, and ectopic calcification, among others [1, 2]. Recently, new active vitamin D derivatives for controlling SHPT have been developed. However, for severe SHPT resistant to these agents, direct injection of a high concentration of vitamin D into the parathyroid gland was attempted to suppress parathyroid function [3]. The development of ultrasonography has enabled the performance of percutaneous ethanol injection therapy (PEIT) in patients with refractory SHPT, and PEIT is known to be as effective as parathyroidectomy-autotransplantation (PTx-AT) [4]. However, there are some problems with PEIT. Maxacalcitol (22-oxacalcitriol; OCT) is an analog of 1,25-dihydroxy vitamin D3 (1,25-D3) and has been shown to strongly suppress parathyroid function dose dependently and to improve bone disorder caused by SHPT in chronic dialysis patients [5, 6]. Experimentally, it has been proved that OCT suppresses PTH mRNA expression in uremic rats without causing hypercalcemia and prevents the decrease in the vitamin D receptor (VDR) expression level in parathyroid gland as well as that of 1,25-D3 [7–9]. Thus, we developed a direct percutaneous injection therapy of maxacalcitol (PMIT) into parathyroid glands. We applied this for the treatment of refractory SHPT and examined the in vivo effects on parathyroid cells.

1

Kazuhiro Shiizaki and Ikuji Hatamura contributed equally to this work.

Key words: end-stage renal disease (ESRD), secondary hyperparathyroidism, vitamin D analogue, interventional ultrasonography, DNA fragmentation, in vivo effects.

METHODS Patients

Received for publication September 24, 2002 and in revised form February 3, 2003, and March 27, 2003 Accepted for publication April 17, 2003

Twenty patients with end-stage renal disease (ESRD) and undergoing regular hemodialysis [mean duration of hemodialysis therapy, 13.5 ⫾ 4.24 (from 4 to 20) years]

 2003 by the International Society of Nephrology

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Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients Table 1. Background features of patients Demographic data Number of patients Underlying renal disease Age yrs Duration of dialysis yrs Baseline data Serum intact-PTH level pg/mL Serum adjusted Ca level mg/dL Serum phosphorus level mg/dL Serum BAP level IU/L Serum intact-BGP level ng/mL Number of enlarged parathyroid glands Total volume of parathyroid glands cm3 Z-score by DEXA

20 (10 male, 10 female) CGN, 17 Nephrosclerosis, 2 Unknown, 1 55.4 ⫾ 9.76 (from 39 to 72) 13.5 ⫾ 4.24 (from 4 to 20) 1018 ⫾ 450 11.3 ⫾ 0.65 6.73 ⫾ 1.49 233 ⫾ 155 278 ⫾ 182 3.55 ⫾ 1.32 1.98 ⫾ 1.21 ⫺2.11 ⫾ 1.65

Abbreviations are: CGN, chronic glomerulonephritis; BAP, bone alkaline phosphatase; BGP, bone Gla-protein; DEXA, dual energy x-ray absorptiometry. Data are mean ⫾ SD.

participated in our study (Table 1). All of them had a complication of severe SHPT that was resistant to OCT intravenously administered for more than 3 months. The patients underwent dialysis for 240 minutes 3 times a week, and bicarbonate dialysate was used in all cases. The dialysate calcium (Ca) concentration was 1.5 mmol/L for most of the patients; however, 1.25 mmol/L Ca was used for 4 patients with hypercalcemia after PMIT. The types of dialysis membrane used were cellulose acetate for 8 patients, polysulfone for 10 patients, and polymethylmethacrylate for 2 patients. The mean Kt/V was 1.52 ⫾ 0.31. Blood flow was between 150 to 220 mL/min, and dialysate flow was 500 mL/min. This study was approved by the local medical ethics committee, and informed consent was obtained from each patient. Percutaneous maxacalcitol injection therapy (PMIT) and biopsy of parathyroid glands Enlarged parathyroid glands were examined by ultrasonography (SSD 5500; Aloka, Tokyo, Japan) and their sizes were estimated by three-dimensional measurement (␲/6 ⫻ a ⫻ b ⫻ c) [10]. PMIT was performed using the same type of needle developed for PEIT (KM-N type; Hakko, Tokyo, Japan) [4]. Under ultrasonographic guidance, the needle was inserted into the center of parathyroid glands without any anesthesia. If the parathyroid gland was large, we injected various sites in order to saturate the entire parathyroid gland with OCT solution. PMIT was performed consecutively six times a week. The injected volume of OCT solution (10 or 5 ␮g/mL) was almost the same for each gland (mean dose of OCT, 15.8 ⫾ 5.25 ␮g/injection for total glands). The dose and the total volume of parathyroid glands showed a good

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correlation (R2 ⫽ 0.871, P ⬍ 0.01). Table 2 shows the parathyroid gland volume of each patient and the injected dose and volume of OCT per one PMIT. In patients with multiple enlarged parathyroid glands, all detectably enlarged glands were treated by PMIT simultaneously, even if they existed bilaterally. In 13 patients (Table 2), parathyroid gland tissues were obtained by biopsy under ultrasonographic guidance, using the same technique as in PMIT, with an 18-guage needle (Bard MonoptyTM; C.R. Bard, Covington, GA, USA) before and after (i.e., immediately before the sixth PMIT) PMIT under local anesthesia with 1% lidocaine. Therefore, only the sixth PMIT was performed under local anesthesia in these patients for the complete second biopsy. After a series of PMIT, all patients were intravenously administered with OCT at the end of each hemodialysis session. The initial dose was 10 ␮g for each hemodialysis and was changed according to the serum Ca and intactPTH (iPTH) levels measured periodically. Laboratory measurements and radiologic examinations The serum iPTH, adjusted Ca [adjusted Ca (mg/dL), Ca (mg/dL) ⫹ 4 ⫺ albumin (g/dL)] [11], inorganic phosphorus (P) levels, and other biochemical data were obtained before and immediately after (i.e., prior to the first hemodialysis session after a series of PMIT) PMIT in all the patients. Moreover, for 18 patients, these data were also obtained 4 and 12 weeks after PMIT. In order to detect complications of PMIT in the early phase, the levels of Ca and P were measured before each hemodialysis session during the week of PMIT. We also measured serum bone alkaline phosphatase (BAP) and intact bone Gla-protein (iBGP) levels in 18 patients before and 12 weeks after PMIT in order to examine changes in bone metabolism caused by PMIT. Moreover, these laboratory data of 24 weeks after PMIT were obtained from 14 patients that did not require other treatments for insufficient PTH suppression. Serum iPTH levels were measured with the two-antibody method using AllegroTM intact-PTH (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). Serum BAP levels were measured by precipitation with wheat-germ lectin using an Iso-ALP test kit (Roche Diagnostics GmbH, Mannheim, Germany) and iBGP levels by immunoradiometric assay using a BGP-immunoradiometric assay (IRMA) kit (Yuka-Medias, Tokyo, Japan) (upper limit, 150 IU/L; 12.7 ng/mL, respectively). The levels of serum Ca, P, and albumin were determined with an automated analyzer (TBA-200FR; Toshiba, Tokyo, Japan). The individual parathyroid gland volumes were also determined before and 12 weeks after PMIT in 18 patients using ultrasonography. The bone mineral density was measured at the radius one-third the distal portion of the nonarteriovenous fis-

R1 L1

19

1.612 0.320

0.858 0.004 0.151 1.911 0.515 0.148 0.182 0.098 0.030 0.098

0.218 1.206 0.393 0.351 0.076 0.189 0.864 0.334 0.160 0.908 0.065 2.514 0.109 0.009 0.600 2.547 0.135 0.089

0.073 1.938 ND 0.485

NS NS

0.234 0.036 0.131 0.756 0.320 0.207 0.298 0.113 0.123 0.462

0.127 1.418 0.054 0.310 0.029 0.194 0.150 0.124 0.126 1.312 0.061 2.045 0.106 0.013 0.327 0.486 0.074 0.104

0.021 0.569 0.107 0.049

Parathyroid gland volume 12 wks after c cm3

(1.0) (0.1) (0.3) (2.0) (0.6) (0.3) (0.3) (0.1) (0.1) (0.1)

(0.2) (1.4) (0.4) (0.7) (0.15) (0.4) (1.0) (0.5) (0.3) (1.2) (0.1) (2.4) (0.2) (0.1) (0.6) (2.4) (0.1) (0.1)

12.0 (1.6) 3.0 (0.4)

6.25 0.625 1.875 12.5 3.75 3.0 3.0 1.0 1.0 1.0

1.25 8.75 2.5 7.0 1.5 4.0 5.0 2.5 1.5 6.0 0.625 15.0 1.25 0.625 3.0 12.0 0.5 0.5

1.5 (0.2) 13.5 (1.8) NP 10.0 (1.0)

Dose and volume of injected OCT lg cm3/one PMIT

72.0 18.0

37.5 3.75 11.25 75.0 22.5 18.0 18.0 6.0 6.0 6.0

7.5 52.5 15.0 42.0 9.0 24.0 30.0 21.0 9.0 30.0 3.75 90.0 7.5 3.75 18.0 72.0 3.0 3.0

9.0 81.0 NP 60.0

Total injected OCT /6 PMITs lg

20

18

16

14

12

10

8

6

4

2

Patient no. L1 L2 R1 R2 R3 R1 R2d,f L1 R1d,e,f R2 L1 R1 R2 L1 L2d R1 R2d,e,f L1 L2 R1 R2 R3 L1d,e L2 R1 R2 R2 L1 L2 R1 R2 R3 L1 L2 L3 R1 R2 L1d,f L2

Parathyroid gland no.a 0.177 0.197 0.480 0.199 0.052 0.045 3.537 ND 2.517 0.251 1.781 0.276 0.314 0.116 0.736 0.318 2.084 0.340 0.150 0.100 0.265 0.898 1.186 0.315 0.234 0.087 0.230 0.120 0.183 0.083 0.028 0.154 0.008 0.074 0.809 0.281 0.528 1.242 0.021

Parathyroid gland volume before PMITb cm3 0.195 0.171 0.428 0.246 0.084 0.113 2.258 0.046 1.902 0.162 0.954 0.333 0.188 0.235 0.412 0.165 1.314 0.183 0.131 0.005 0.055 0.224 0.186 0.106 0.177 0.044 0.068 0.077 0.054 0.124 0.094 0.206 0.068 0.324 0.823 NS NS NS NS

Parathyroid gland volume 12 wks after c cm3 4.0 (0.4) 6.0 (0.6) 6.0 (0.6) 3.0 (0.3) 1.0 (0.1) 1.0 (0.2) 19.0 (3.8) NP 15.6 (2.6) 2.4 (0.4) 12.0 (2.0) 3.0 (0.4) 3.0 (0.4) 1.5 (0.2) 7.5 (1.0) 2.0 (0.4) 13.0 (2.6) 3.0 (0.6) 2.0 (0.4) 1.0 (0.15) 2.0 (0.3) 6.0 (0.9) 9.0 (1.35) 2.0 (0.3) 3.5 (0.35) 1.0 (0.1) 3.5 (0.35) 2.0 (0.2) 3.0 (0.3) 1.5 (0.2) 0.75 (0.1) 1.5 (0.2) 0.75 (0.1) 1.5 (0.2) 9.0 (1.2) 1.5 (0.2) 3.75 (0.5) 9.0 (1.2) 0.75 (0.1)

Dose and volume of injected OCT lg cm3/one PMIT 24.0 36.0 36.0 18.0 6.0 6.0 114.0 NP 93.6 14.4 72.0 18.0 18.0 9.0 45.0 12.0 84.0 12.0 12.0 6.0 12.0 36.0 54.0 12.0 21.0 6.0 21.0 12.0 18.0 9.0 4.5 9.0 4.5 9.0 54.0 9.0 22.5 54.0 4.5

Total injected OCT /6 PMITs lg

Abbreviations are: PMIT, percutaneous maxacalcitol injection therapy; OCT, maxacalcitol; PG, parathyroid gland; ND, not detected; NP, not treated by PMIT; NS, not studied; R, right; L, left. a Parathyroid gland no. was set as follows: R and L, and the numbers were assigned from the head side b Individual parathyroid gland volumes before PMIT c Individual parathyroid gland volumes 12 weeks after PMIT d Parathyroid gland, which was biopsied both before and after PMIT and examined for morphologic changes and subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays e Parathyroid gland from which DNA was obtained for DNA electrophoresis both before and after PMIT f Parathyroid gland from which mRNA was obtained for reverse transcription-polymerase chain reaction (RT-PCR) analysis of PTH mRNA both before and after PMIT

17

R1 R2 R3 L1d,e L2 R1 R2 R3 L1 L2

R1 L1d,f L2 R1d,e R2 L1 R1 R2 R3 L1d,f R1 R2d,f L1 L2 R1 R2d,e,f L1 L2

R1 L1d,e R1 L1

Parathyroid gland no.a

15

13

11

9

7

5

3

1

Patient no.

Parathyroid gland volume before PMITb cm3

Table 2. Volume of individual parathyroid gland before and 12 weeks after PMIT and OCT injection at indicated dose and volume

994 Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

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Table 3. Primer design and size of fragment

PTH GAPDH

Sense primer

Antisense primer

Size bp

5⬘-ATGATACCTGCAAAAGACAT-3⬘ 5⬘-CGCCTGGTCACCAGGGCTGC-3⬘

5⬘-TTTAGCTTTAGTTAATACA-3⬘ 5⬘-CTTACTCCTTGGAGGCCATGT-3⬘

339 963

Abbreviations are: PTH, parathyroid hormone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pairs.

tula side by dual energy x-ray absorptiometry (DEXA) (DPX-L; GE Lunar, Madison, WI, USA). Technetium99m methoxyisobutylisonitrile imaging (99mTc-MIBI) or thallium-201-technetium-99m subtraction scintigraphy (Tl-Tc) of parathyroid glands was performed for all the patients in order to rule out the existence of ectopic parathyroid gland. Morphologic examination and apoptosis detection Parathyroid gland specimens obtained by needle biopsy (before and after PMIT) were fixed with 4% paraformaldehyde phosphate-buffered saline (PBS) for 8 hours and embedded in paraffin for routine tissue processing. Sections of these specimens were prepared and stained with hematoxylin and eosin. We identified all specimens with parathyroid tissues and then carried out the following studies. The morphologic changes following PMIT were examined under a light microscope (BX50; Olympus, Tokyo, Japan). For the examination of apoptosis induced by PMIT, we used the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method for specimens obtained from 13 patients before and after PMIT. 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. An in situ apoptosis detection kit, TACS XL威 (R & D Systems, Minneapolis, MN, USA) was used for labeling the free 3⬘-OH terminus. The number of TUNEL-positive cells was determined in randomly chosen fields for all tissues under a light microscope. Three independent observers counted the TUNEL-positive cells in 10 high-power fields with approximately 200 cells per field at ⫻400 magnification and then the average was taken. Furthermore, we also performed DNA electrophoresis to detect DNA fragmentation, which is a characteristic feature of cell apoptosis [12]. From the biopsied parathyroid gland specimens (before and after PMIT, in 7 patients, shown in Table 2), DNA was extracted using a DNA extraction kit QIAampTM (QIAgen GmbH, Hilden, Germany). DNA electrophoresis was performed at a constant voltage of 100 V in horizontal 2% agarose gels. DNA bands were visualized by ethidium bromide staining. Isolation of total RNA and reverse transcriptionpolymerase chain reaction (RT-PCR) for PTH Total RNA was prepared from biopsied parathyroid gland specimens (before and after PMIT for 8 patients,

shown in Table 2) using the TRIzol reagent (Life Technologies, Rockville, MD, USA). Total RNA (2 ␮g) was digested with DNase I (RNase-free) for 10 minutes at 37⬚C in the presence of 5 U of RNase inhibitor. After heat inactivation of DNase I, total RNA was reverse transcribed for 50 minutes at 42⬚C using SuperScript II Reverse Transcriptase and oligo (dT) as a primer (Life Technologies). The reaction was terminated by heating samples at 70⬚C. Subsequently, 1 ␮L of the cDNA mixture was suspended in a total volume of 50 ␮L of a solution containing 50 mmol/L KCl, 10 mmol/L Tris-HCl, 1.2 mmol/L MgCl2, 0.5 ␮mol/L of each primer, 200 ␮mol/L of each nucleotide and 1 U of Ampli-Taq GoldTM DNA Polymerase (Roche Molecular Systems, Branchburg, NJ , USA). Amplification was then performed using specific primers (Table 3). For each specific assay, PCR conditions were optimized with respect to the template cDNA per tube, annealing temperature, and number of cycles. For normalization in this assay, the sample RNA was also amplified using primers for the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Amplification products were analyzed on 2% agarose gels containing 0.5 ␮g/mL ethidium bromide and then photographed. PCR products of PTH and GAPDH were quantitated using NIH Image (National Institutes of Health, Bethesda, MD, USA). For each sample, PTH mRNA levels were then normalized by GAPDH mRNA levels. To compile the results of densitometric analysis from independent experiments, PTH/GAPDH mRNA ratios for individual patients (N ⫽ 8) were averaged. Statistical analysis Data were expressed as mean ⫾ SD. The correlation between the total parathyroid gland volume measured by ultrasonography before PMIT and the injected OCT dose for each PMIT was analyzed. Post-hoc multiple comparisons using Dunnett tests were performed to determine the significance of differences in values immediately, 4 weeks, and 12 weeks after PMIT as compared with those before PMIT. Mann-Whitney U test was used to determine the significance of differences in the number of TUNEL-positive cells and the PTH/GAPDH mRNA ratio. Other laboratory data and changes in the parathyroid gland volume were analyzed by Student t test. A P value of less than 0.05 was considered to indicate statistical significance.

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Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

Fig. 1. Changes in serum intact-PTH, adjusted calcium (Ca), and phosphorus (P) levels before and after percutaneous maxacalcitol injection therapy (PMIT) (N ⫽ 20). After PMIT, serum intact-PTH levels significantly decreased, but adjusted serum Ca and P levels did not change significantly. *P ⬍ 0.01 vs. before PMIT.

RESULTS Patient characteristics Table 1 shows background features of our patients before PMIT. The mean serum iPTH level was 1018 ⫾ 450 pg/mL, indicating severe hyperparathyroidism in spite of intravenous OCT administration. The mean serum adjusted Ca and P levels were 11.3 ⫾ 0.65 and 6.73 ⫾ 1.49 mg/dL, respectively. Thus, these patients were diagnosed as having refractory SHPT resistant to OCT intravenously administered. In addition, the high levels of bone markers (the mean serum BAP and iBGP levels; 233 ⫾ 155 IU/L and 278 ⫾ 182 ng/mL, respectively) and the low bone mineral content determined by DEXA (mean Z-score, –2.11 ⫾ 1.65) suggest bone mineral loss due to high bone–turnover disease resulting from SHPT. The mean total volume was 1.98 ⫾ 1.21 cm3, and the mean number of detectably enlarged parathyroid glands was 3.55 ⫾ 1.32. 99mTC-MIBI or Tl-Tc did not detect any ectopic parathyroid glands in all 20 patients.

Fig. 2. Time course changes in serum intact-PTH, adjusted serum calcium (Ca), and phosphorus (P) levels (N ⫽ 18). The decrease in serum intact-PTH levels continued for 12 weeks after percutaneous maxacalcitol injection therapy (PMIT) without significant changes in both adjusted serum Ca and P levels. *P ⬍ 0.01 vs. before PMIT.

Fig. 3. Changes in serum bone alkaline phosphatase (BAP) and intact bone Gla-protein (iBGP) levels before and 12 weeks after percutaneous maxacalcitol injection therapy (PMIT) (N ⫽ 18). Serum iBGP levels significantly decreased 12 weeks after PMIT. *P ⬍ 0.05 vs. before PMIT.

Changes in laboratory data after PMIT Serum iPTH levels significantly decreased after PMIT (385 ⫾ 321 pg/mL; P ⬍ 0.01), and the mean reduction rate was 62.7 ⫾ 20.3%. In contrast, adjusted serum Ca and P levels did not change significantly throughout the course of PMIT (adjusted serum Ca and P levels immediately after PMIT; 11.6 ⫾ 1.29 and 6.22 ⫾ 1.05 mg/dL, respectively) (Fig. 1). We determined the above-mentioned parameters repeatedly in 18 patients and confirmed the continued suppression of parathyroid function for 12 weeks after PMIT (serum iPTH, adjusted serum Ca and P levels before and immediately, 4, and 12 weeks after PMIT, 992 ⫾ 461, 348 ⫾ 246, 560 ⫾ 313, and 459 ⫾ 256 pg/mL, P ⬍ 0.01; 11.3 ⫾ 0.67, 11.7 ⫾ 1.15, 11.1 ⫾ 0.91, and 11.7 ⫾

0.98 mg/dL; and 6.73 ⫾ 1.54, 6.32 ⫾ 1.05, 6.48 ⫾ 1.48, and 6.00 ⫾ 1.20 mg/dL, respectively) (Fig. 2). Moreover, levels of bone markers tended to decrease after PMIT and the subsequent intravenous OCT administrations. In particular, serum iBGP levels significantly decreased (serum BAP and iBGP levels before and 12 weeks after PMIT, 227 ⫾ 150 vs. 177 ⫾ 120 IU/L, 284 ⫾ 191 vs. 212 ⫾ 156 ng/mL, P ⬍ 0.05, respectively) (Fig. 3). Total parathyroid gland volumes also significantly decreased 12 weeks after PMIT compared with those before PMIT (1.98 ⫾ 1.28 vs. 1.26 ⫾ 0.79 cm3, P ⬍ 0.01, respectively) (Fig. 4A). Particularly for more severely swollen parathyroid glands before PMIT, the ratio of parathyroid

Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

Fig. 4. Changes in parathyroid gland volume before and 12 weeks after percutaneous maxacalcitol injection therapy (PMIT) (N ⫽ 18). (A ) Total parathyroid gland volume significantly decreased after PMIT and the subsequent intravenous maxacalcitol (OCT) administrations. (B ) Changes in individual parathyroid gland volumes 12 weeks after PMIT compared with before PMIT for each size are shown. For the larger parathyroid glands before PMIT, the regressive ratios of parathyroid gland volume were higher (PGv ⬍ ␲/6 ⫻ 0.53, ␲/6 ⫻ 0.53 ⱕ PGv ⬍ ␲/6 ⫻ 1.03, ␲/6 ⫻ 1.03 ⱕ PGv ⬍ ␲/6 ⫻ 1.53 and PGv ⱖ ␲/6 ⫻ 1.53 cm3; 8, 40, 9 and 8 glands; 18 patients were examined, respectively). PGv is parathyroid gland volume. *P ⬍ 0.01 vs. before PMIT.

gland volume (PGv) after 12 weeks with respect to that before PMIT was lower than that of mildly swollen parathyroid glands (PGv ⬍ ␲/6 ⫻ 0.53, ␲/6 ⫻ 0.53 ⱕ PGv ⬍ ␲/6 ⫻ 1.03, ␲/6 ⫻ 1.03 ⱕ PGv ⬍ ␲/6 ⫻ 1.53, PGv ⱖ ␲/6 ⫻ 1.53 cm3; 393 ⫾ 315, 96.1 ⫾ 94.6, 62.2 ⫾ 47.8, and 53.2 ⫾ 22.1%, respectively) (Fig. 4B). Table 4 shows the individual laboratory data in all patients and those 24 weeks after PMIT followed by intravenous OCT administrations in 14 patients. Morphologic changes after PMIT Figure 5 a, b, e, f, i, j, m, and n show light microscopy images of biopsied parathyroid gland specimens before and after PMIT. In Figure 5 a, e, i, and m (before PMIT), numerous parathyroid cells are observed, indicating hyperplasia. After PMIT, some partial defects of parathyroid cells were observed, and some of the parathyroid cells were observed to have condensed and fragmented nuclei, which are considered the expressions of cell apoptosis (Fig. 5b). Moreover, Figure 5 f, j, and n show the existence of many parathyroid cells and the abnormal structure of parathyroid tissue. Most of the nuclei in these parathyroid cells were condensed. Analysis of apoptosis induced by PMIT Analysis of parathyroid tissues by the TUNEL method revealed distinct patterns of staining of TUNEL-positive

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cells [brown spots; diaminobenzidine (DAB)] and TUNEL-negative cells (green spots; methyl green). Figure 5 c, d, g, h, k, l, o, and p show apoptotic parathyroid cells stained by the TUNEL method before and after PMIT. In the specimens obtained before PMIT, few or no apoptotic cells were observed (Fig. 5 c, g, k, and o), but after PMIT, a number of apoptotic (TUNEL-positive) parathyroid cells were noted (Fig. 5 d, h, l, and p). Some of the apoptotic cells were scattered in the specimen, but most of them were found in one segment. Furthermore, boundaries between the segment of apoptotic cells and that of non-apoptotic cells were clear. From these findings, the TUNEL method revealed a significant increase in the number of TUNEL-positive cells (expressed per 1000 cells) in parathyroid tissues obtained after PMIT as compared with those before PMIT (rates of TUNELpositive cells before and after PMIT, 1.08 ⫾ 1.66 vs. 619 ⫾ 58.2/1000 cells; P ⬍ 0.01, N ⫽ 13, respectively) (Fig. 6). By DNA electrophoresis of parathyroid tissues obtained after PMIT, a ladder pattern indicating the presence of DNA fragmentation, a characteristic feature of cell apoptosis [12], was observed; this feature was not observed before PMIT (Fig. 7). This change was confirmed in all 7 patients in whom DNA for parathyroid cells were extracted and examined. Changes in PTH mRNA level determined by RT-PCR Figure 8A shows the representative changes in PTH and GAPDH mRNA levels in parathyroid tissues before and after PMIT. The PTH mRNA level after PMIT clearly decreased in comparison with that before PMIT, but the GAPDH mRNA level did not change. Figure 8B shows the comparison of the average of PTH/GAPDH mRNA ratio between before and after PMIT. After PMIT, this ratio significantly decreased compared with that before PMIT; an average of 0.452 ⫾ 0.98-fold decrease in the PTH mRNA level was observed (P ⬍ 0.01, N ⫽ 8). DISCUSSION OCT is a vitamin D analog developed to strongly suppress parathyroid function with low calcemic and phosphatemic actions. Thus, OCT is now benefiting many dialysis patients with SHPT in Japan. However, there are some patients with SHPT resistant to OCT intravenously administered because of its poor suppressive effects on PTH and/or complications such as hypercalcemia. For the treatment of these refractory SHPT, PMIT has been developed. Kitaoka et al [3] reported the efficacy and safety of direct calcitriol injection into parathyroid glands 3 times a week for 2 weeks and showed the necessity of following it up with calcitriol pulse therapy. On the other hand,

10.8 11.2 11.2 11.8 11.7 10.5 10.3 11.0 11.3 10.5 12.9 11.0 12.0 11.1 11.1 10.6 12.0 11.9 11.4 10.7

Beforeb

11.1 12.9 12.2 11.8 10.1 11.5 10.9 10.9 12.2 10.2 12.1 13.4 14.4 10.9 10.3 11.7 12.7 11.6 8.8 12.7

Imc

10.0 12.1 11.5 11.0 10.8 12.9 10.9 11.1 11.8 11.6 13.4 13.3 10.9 11.5 13.3 11.0 11.1 11.6 NS NS

12 wd 10.5 11.2 10.9 10.7 9.6 NS 10.5 10.6 11.4 10.8 NS NS 10.7 10.8 NS 11.0 11.2 10.8 NS NS

24 we

Serum adjusted Ca level mg/dL

7.0 8.5 6.8 7.0 8.5 9.0 6.1 5.4 7.4 5.0 5.9 6.7 6.5 5.4 9.8 7.3 4.1 4.8 7.7 5.6

Beforeb 6.1 6.4 5.4 6.1 7.7 4.9 5.3 6.8 8.1 5.7 6.3 7.0 5.9 6.4 8.8 6.0 6.0 4.9 4.9 5.6

Imc 5.1 6.7 6.2 5.7 6.6 6.6 4.3 5.2 4.4 6.2 6.2 9.2 5.6 5.7 7.9 5.6 6.2 4.6 NS NS

12 wd 6.2 6.5 6.1 4.4 6.2 NS 4.5 5.5 4.7 5.5 NS NS 5.2 5.7 NS 5.3 5.9 4.8 NS NS

24 we

Serum phosphorus level mg/dL

1500 520 1690 890 890 730 956 1320 1500 1300 1700 510 473 1000 1480 474 547 376 1500 1010

Beforeb 480 240 178 440 360 540 211 550 350 310 1100 170 167 148 599 99.5 148 181 1300 122

Imc 510 286 381 520 215 503 402 645 310 530 1090 230 295 269 1070 253 430 331 NS NS

12 wd

Serum intact-PTH level pg/mL

182 247 258 312 306 NS 341 294 203 327 NS NS 238 309 NS 237 279 263 NS NS

24 we 188 93 220 178 650 226 315 298 85 64 302 113 116 408 384 125 81 242 471 104

Beforeb 292 331 47 116 57 239 156 170 183 210 546 84 95 103 186 118 103 141 NS NS

12 wd

214 269 62 140 83 NS 162 112 264 187 NS NS 124 144 NS 84 153 147 NS NS

24 we

Serum BAP level IU/L

510 85 169 120 86 730 312 243 270 220 550 110 91.1 390 536 257 121 320 215 215

Beforeb

130 116 38.4 210 39.2 485 154 228 220 260 597 96.5 127 426 328 115 68.6 178 NS NS

12 wd

101 154 69.3 309 54.1 NS 176 237 240 210 NS NS 113 235 NS 163 88.4 108 NS NS

24 we

Serum intact-BGP level ng/mL

Abbreviations are: PMIT, percutaneous maxacalcitol injection therapy; PTH, parathyroid hormone; BAP, bone alkaline phosphatase; BGP, bone Gla-protein; NS, not studied (because of the impossibility of controlling PTH by subsequent intravenous maxacalcitol (OCT) administrations in patients no. 6, 11, 12, and 15, and because patients no. 19 and 20 dropped out of the study). a Patient no. is the same as that shown in Table 2 b Before PMIT c Immediately after PMIT d 12 weeks after PMIT e 24 weeks after PMIT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Patient no.a

Table 4. Time course of individual data before and after PMIT in each patient

998 Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

we utilized the same therapeutic method but injected OCT consecutively 6 times a week, and found that PMIT could markedly suppress parathyroid function and reduce the parathyroid gland volume without significantly changing serum Ca and P levels. It has been considered that the calcemic and phosphatemic actions of OCT and the elevated serum concentration of OCT applied following PMIT contributed little to these effects because serum Ca and P levels did not significantly change during the period of PMIT, and we confirmed that the serum OCT concentration following PMIT was not markedly elevated, in spite of PMIT with a total OCT dose of 30 ␮g/PMIT, and that serum OCT cleared off rapidly (data not shown). Therefore, it has been considered that the excessive elevation of intra-parathyroid gland OCT concentration by PMIT played the most important role in the expression of these effects. Furthermore, PMIT did not cause aggravated injury of surrounding tissues, which was observed in PEIT, such as laryngeal palsy. PMIT was performed on all detectably enlarged parathyroid glands simultaneously, even if these enlarged parathyroid glands existed bilaterally. All the patients showed tolerance to PMIT. Thus, PMIT was confirmed to clinically improve and to be safe for refractory SHPT. Moreover, these effects were not temporary but continued for at least 12 weeks. Thus, this continued suppression of SHPT, as shown by the significant decrease in the serum iBGP level, might contribute to improving the high bone–turnover disease caused by SHPT. Table 5 shows the clinical outcomes of PMIT. SHPT in 13 of 18 patients could be controlled for more than 48 weeks by following up PMIT with intravenous OCT administrations. One patient (patient no. 9) required PMIT again 48 weeks after the first series of PMIT because of the re-elevation of the serum iPTH level (serum iPTH levels before, 24, and 48 weeks after PMIT, 1500, 203, and 482 pg/mL, respectively). Four patients required PEIT 12 weeks after PMIT because of insufficient suppression of originally severe SHPT. The continuation of high serum Ca level for more than 12 weeks indicates the necessity of discontinuing OCT intravenously administered and treatment by PEIT or PTx-AT instead. These 5 patients had more severely swollen parathyroid glands before PMIT and/or their serum Ca levels increased in the early phase after PMIT. Therefore, it is suggested that PMIT is indicated for patients without a parathyroid gland volume of more than 2 cm3 or severely high levels of P and/or PTH (serum P and iPTH levels more than 9.0 mg/dL and/or 1500 pg/mL, respectively). Regarding the parathyroid gland volume reduction by PMIT and subsequent OCT intravenous administrations, the parathyroid gland volumes 12 weeks after PMIT were contrarily increased in the smallest group with a parathyroid gland volume of less than ␲/6 ⫻ 0.53 cm3 (Fig. 4B). Therefore, it is not necessary for parathyroid glands with a volume

999

of less than ␲/6 ⫻ 0.53 cm3 to be treated by PMIT. However, the most important point is the possibility that all glands can be treated by PMIT in the case without ectopic parathyroid glands. Therefore, it is necessary to rule out the existence of ectopic parathyroid glands by 99m Tc-MIBI or Tl-Tc; in fact, we excluded one patient with ectopic parathyroid gland in the upper mediastinum from this study. Next, the mechanisms underlying the suppression of parathyroid function by PMIT were studied. As one of the mechanisms, OCT was shown to suppress the synthesis and secretion of PTH, as proven by the reduction in the PTH mRNA level [7, 8, 13]. The in vivo effect of the suppression of PTH synthesis and secretion by PMIT was observed in this study (Figs. 1 and 8). It is considered the suppression of PTH gene transcription by OCT administered at a high concentration that may mostly contribute to the early-stage regression of SHPT by PMIT. As an explanation for the continued improvement of SHPT following PMIT and the subsequent intravenous OCT administrations, the reduction in the parathyroid gland volume was suggested to be one of the underlying factors. Morphologic findings in parathyroid tissues obtained after PMIT showed not only some partial defects of parathyroid cells but also condensation and fragmentation of nuclei in many of the extant parathyroid cells. This finding suggests that the decrease in the number of parathyroid cells is partially caused by the induction of apoptosis by PMIT. First, we showed the marked increase in the number of TUNEL-positive parathyroid cells after PMIT (from 1.08 ⫾ 1.66 to 619 ⫾ 58.2/1000 cells) (Figs. 5 d, h, l, p, and 6). Biopsies of the biggest parathyroid gland of each patient were performed (Table 2), and Figure 4B shows the greater reduction in the parathyroid gland volume in bigger parathyroid glands before PMIT. Thus, it is considered that many cells in biopsied parathyroid glands underwent apoptosis induced by PMIT (the mean ratio of parathyroid gland volume 12 weeks after, with respect to that before PMIT in biopsied parathyroid glands was lower than that in nonbiopsied parathyroid glands without less than ␲/6 ⫻ 0.53 cm3; 66.2 and 89.7%, respectively). Parathyroid gland specimens were obtained by needle biopsies from the center of each gland for safety and accuracy. PMIT was performed in various sites of each parathyroid gland, but most of the maxacalcitol was injected into the center and extended to the periphery. Thus, the parathyroid gland specimens were obtained from parts most greatly influenced by PMIT and it is considered these parts contained more TUNELpositive cells than any other part in each parathyroid gland. Thus, it is considered that our result (i.e., the marked increase in the ratio of TUNEL-positive cells with respect to the total number of parathyroid cells) was exaggerated by these factors. For the exclusion of heterogeneous TUNEL-positive cells in the biopsied

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Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

Fig. 5. Light microscopy and detection of DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method in serial sections. (a, e, i, m ) Representative light microscopy images of parathyroid tissue before and (b, f, j, n ) after percutaneous maxacalcitol injection therapy (PMIT) are shown. Before PMIT, a number of hyperplastic parathyroid cells were observed (a, e, i, m), but (b) after PMIT, few parathyroid cells with partial defects and those exhibiting condensation and fragmentation of nuclei, which are considered the expressions of cell apoptosis, were observed. Many parathyroid cells were observed; however, most of their nuclei were condensed (f, j, n). Representative findings of in situ detection of DNA fragmentation in parathyroid tissues by the TUNEL method (c, g, k, o) before and (d, h, l, p) after PMIT are shown. (c, g, k, o) Before PMIT, no or few apoptotic (TUNEL-positive) cells were observed but (d, h, l, p) after PMIT, a number of apoptotic parathyroid cells were noted. The magnification of all figures: 1, low magnification, 2, high magnification, respectively.

specimens, we performed the same TUNEL method on more than 30 parathyroid glands obtained by surgical excisions in patients administered with OCT intravenously immediately before the operations, and confirmed the ratio of TUNEL-positive cell to be similar to the specimens obtained before PMIT (data not shown) or a previous report [14]. Second, it is well known that nonspecific TUNEL-positive cells such as necrotic cells are often observed, so we performed DNA electrophoresis on parathyroid gland specimens before and after PMIT in 7 patients. A ladder pattern indicating the presence of DNA fragmentation, which is characteristic of cell apoptosis [12], was observed only in specimens obtained after PMIT, and this phenomenon was observed in all 7 patients in whom DNA in parathyroid gland was analyzed. Moreover, we investigated the induction of apoptosis in parathyroid glands surgically opened and

directly injected with OCT or its vehicle in uremic rats. The ratios of TUNEL-positive parathyroid cells in rats injected with OCT were significantly higher than those in rats injected with its vehicle, and the ladder pattern was also observed on parathyroid glands injected with OCT (data not shown). Therefore, the induction of apoptosis following PMIT is caused neither by the effects of the vehicle nor mechanical and pressure injury. Previously, Fukagawa et al [10] reported a significant decrease in the parathyroid gland volume following calcitriol oral pulse therapy for 12 weeks and hypothesized that the apoptosis of parathyroid cells is induced by calcitriol at high concentrations. In contrast, Quarles et al [15] failed to observe a decrease in parathyroid gland volume in hemodialysis patients in response to calcitriol pulse therapy for 36 weeks, as assessed by ultrasound and/or magnetic imaging. In animals, a very high

Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

1001

Fig. 5. (Continued)

dose of calcitriol [equivalent to that for a 70 kg human (145.6 ␮g)] intraperitoneally administered did not induce apoptosis of parathyroid cells [16]. Moreover, an in vitro study showed the inhibition of cell apoptosis by calcitriol at high concentrations in hyperplastic parathyroid tissues from patients with SHPT [17]. However, all of these previous studies focused on the induction of apoptosis by calcitriol only. A recent study indicated the possibility that OCT differs from calcitriol in terms of its biologic effect through the interactions of VDR with specific coactivators [18]. Therefore, we presumed that the mechanism underlying the induction of apoptosis by PMIT might be the specific effect of OCT on the VDR signaling pathway of parathyroid cells. Furthermore, reports supporting our hypothesis that there is a relationship between vitamin D and cell apoptosis in various tissues and cell lines have been published recently. In particular, in Wilms’ tumor, gene product (WT1)-expressing renal embryonic cells, Wagner et al [19] suggested that transcriptional activation of VDR by WT1 mediates apoptosis of these cells. In myeloma [20], breast cancer [21], colorectal carcinoma [22], and melanoma cells [23], an obvious relationship between vitamin D and cell apoptosis was reported. In addition, Hewison et al [24] reported that VDR antisense transfection inhibits the induction

of apoptosis in the monoblastoid cell line. Nakagawa et al [25] reported that stimulation of VDR/RXR␣/vitamin D response elements (VDRE) complex formation induces apoptosis of promyelocytic leukemia cells. Guzey et al [26] reported that vitamin D induces decreases in the levels of antiapoptotic proteins such as Bcl-2 in association with enhancement of apoptosis in prostate cancer. Based on these previous reports and our data, we suggest that one of the mechanisms for the regression of SHPT is apoptosis via VDR in parathyroid cells. In our specimens obtained after PMIT, parathyroid cells, which showed immunohistochemical staining of VDR, tended to show TUNEL-positive simultaneously. However, a more indepth study is necessary to elucidate the details of this biologic reaction. The biologic response to OCT that leads to the suppression of PTH synthesis is considered to be due to the control of the gene transcription mediated by a nuclear receptor (VDR) in target cells. However, nodular hyperplasia shows a more marked decrease in the content of VDR on parathyroid cells [27], thus advanced SHPT shows resistance to vitamin D administration. In dialysis patients, bolus oral or intravenous calcitriol administration not only reduced PTH levels, but also improved the response to calcitriol by VDR up-regulation in parathy-

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Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

Fig. 6. Change in ratios of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive parathyroid cells following percutaneous maxacalcitol injection therapy (PMIT). TUNELpositive parathyroid cell ratios significantly increased after PMIT (N ⫽ 13). *P ⬍ 0.01 vs. before PMIT.

Fig. 8. Effect of percutaneous maxacalcitol injection therapy (PMIT) on parathyroid hormone (PTH) mRNA levels. Representative changes in PTH mRNA levels in parathyroid glands following PMIT determined using reverse transcription-polymerase chain reaction (RT-PCR) are shown. (A ) These levels decreased after PMIT without obvious changes in GAPDH mRNA levels. (B ) After PMIT, PTH/GAPDH mRNA ratios obtained by densitometric analysis significantly decreased compared with that before PMIT (N ⫽ 8). *P ⬍ 0.01 vs. before PMIT.

Table 5. Clinical outcomes after PMIT Clinical outcomes

Number of patients (patient no.a)

Good control by i.v. OCTb Re-PMITc PEITd

13 1 (patient no. 9) 4 (patient no. 6, 11, 12, and 15)

Abbreviations are: OCT, maxacalcitol; Re-PMIT, re-percutaneous maxacalcitol injection therapy; PEIT, percutanous ethanol injection therapy. a Patient no. is the same as that shown in Table 2 b These patients were controlled by the subsequent intravenous (i.v.) OCT administrations for more than one year c This patient was treated by the same series of PMIT again immediately one year after the first series d These patients were treated by PEIT due to the insufficient suppression of parathyroid hormone levels and severely elevated serum Ca levels for 12 weeks after PMIT

Fig. 7. DNA fragmentation observed by 2% agarose gel electrophoresis. The ladder pattern indicating the presence of DNA fragmentation, which is a characteristic of cell apoptosis, was observed after percutaneous maxacalcitol injection therapy (PMIT). This feature was not observed before PMIT. This change was observed in all 7 patients in whom DNA for parathyroid cells were extracted and examined.

roid cells [13, 28–30]. Intraperitoneal administration of OCT has also been reported to induce VDR up-regulation in parathyroid cells of uremic rat [9]. In the present study, the number of parathyroid cells immunohistochemically stained by the monoclonal anti-VDR antibody tended to increase, thus it is presumed that VDR up-regulation by PMIT is another factor that contributes to amplifying the effects of PMIT followed by intravenous OCT administrations and to the continued regres-

Shiizaki and Hatamura et al: PMIT regresses SHPT in uremic patients

sion of SHPT for a long period. For the up-regulation of VDR in parathyroid cells following direct OCT injections, we have confirmed the time course of VDR contents in rat parathyroid gland directly injected with OCT by both RT-PCR and immunohistochemical study (data not shown). However, it is considered that more precise studies are required to elucidate the mechanisms underlying the effect of PMIT. CONCLUSION We have clarified that PMIT could reduce not only serum iPTH levels but also the volume of parathyroid glands without major complications. Our results suggest that the suppression of PTH synthesis and secretion, and the decreased number of parathyroid cells due to the induction of apoptosis by OCT at a very high concentration in the parathyroid gland are contributing factors to the regression of SHPT by PMIT. ACKNOWLEDGMENTS Chugai Pharmaceutical Co., Ltd., Tokyo, Japan, supported this study by measuring serum OCT concentrations in patients treated by PMIT and providing OCT and its vehicle for animal experiments. This study was partially supported by a research grant from Renal Osteodystrophy Foundation. The authors thank Dr. Masafumi Kitaoka (Division of Endocrinology and Metabolism, Showa General Hospital, Tokyo, Japan) for the kind training in parathyroid intervention technique under ultrasonography. Reprint requests to Kazuhiro Shiizaki, M.D., Center of Blood Purification Therapy, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan. E-mail: [email protected]

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