Basic Mineral Metabolism

Biochemical and Cellular Effects of Direct Maxacalcitol Injection into Parathyroid Gland in Uremic Rats

Kazuhiro Shiizaki, Shigeo Negi, Ikuji Hatamura, Toshifumi Sakaguchi, Fumie Saji, Ken Kunimoto, Masahide Mizobuchi, Ikuo Imazeki, Akira Ooshima and Tadao Akizawa
JASN January 2005, 16 (1) 97-108; DOI: https://doi.org/10.1681/ASN.2004030236

Abstract

The most important etiological factors of resistance to medical treatments for secondary hyperparathyroidism are the decreased contents of the vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in parathyroid cells and a severely swollen parathyroid gland (PTG) as a result of hyperplasia. The effects of direct maxacalcitol (OCT) injection into PTG in terms of these factors were investigated in this study. The PTG of Sprague-Dawley rats that were 5/6 nephrectomized and fed a high-phosphate diet were treated by a direct injection of OCT (DI-OCT) or vehicle (DI-vehicle). The changes in serum intact parathyroid hormone (PTH), Ca2+, and phosphorus levels, in VDR and CaSR expression levels in parathyroid cells, and in Ca2+-PTH curves were examined. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA electrophoresis for PTG. DI-OCT markedly decreased serum intact PTH level, and a significant difference in this level between DI-OCT and DI-vehicle was observed. However, serum Ca2+ and phosphorus levels did not changed markedly in both groups. The upregulations of both VDR and CaSR, the clear shift to the left downward in the Ca2+-PTH curve, and the induction of apoptosis after DI-OCT were observed. These findings were not observed in the DI-vehicle-treated rats. Moreover, these effects of DI-OCT were confirmed by the DI-OCT into one PTG and DI-vehicle alone into another PTG in the same rat. DI-OCT may introduce simultaneous VDR and CaSR upregulations and the regression of hyperplastic PTG, and these effects may provide a strategy for strongly suppressing PTH levels in very severe secondary hyperparathyroidism.

Secondary hyperparathyroidism (SHPT) resulting from ESRD causes not only renal osteodystrophy but also cardiovascular disorders because of ectopic calcification in vascular tissues. A low level of parathyroid hormone (PTH) causes adynamic bone disease; easily increases serum calcium (Ca), phosphorus (P), and Ca×P levels; and may contribute to a poor prognosis in patients with ESRD. Thus, the maintenance of appropriate levels of PTH, Ca, and P is required for the improvement of the prognosis and quality of life of these patients (1–3).

SHPT develops in conditions of P retention, low Ca level, and the inactivation of vitamin D (VD) (4–6); therefore, the control of P level and supplementations of Ca and VD are necessary in patients with SHPT as preventive or medical treatment. However, advanced SHPT with severely swollen parathyroid glands (PTG) as a result of hyperplasia is resistant to these medical treatments because of the low contents of VD receptor (VDR) and Ca-sensing receptor (CaSR) in parathyroid cells (PTC) (7,8). When SHPT progresses into such status, it is considered irreversible. Thus, patients with very severe SHPT require parathyroidectomy-autotransplantation (PTx-AT), which may be complicated by some problems, such as the necessity of general anesthesia, the hyper- or hypofunction of autotransplanted PTG, and mental suffering.

The developments of ultrasonography and specific injection needles have enabled parathyroid intervention therapy by which PTH-suppressive agents are injected directly into the PTG. The most commonly used agent is ethanol, and percutaneous ethanol injection therapy is as effective as PTx-AT (9). Moreover, we previously reported the clinical effects and safety of the direct injection of a highly concentrated VD or its analogue into the PTG of patients with very severe SHPT (10,11).

In the present study, we developed a method of direct injection into the PTG of uremic rats to investigate in detail the effects of direct maxacalcitol (OCT) injection (DI-OCT). Our results indicate that DI-OCT may ameliorate the important etiologic factors of resistance to medical treatments for SHPT.

Materials and Methods

Animals

In 7-wk-old male Sprague-Dawley rats, 5/6 nephrectomy (uremic rat = u-rat) or sham operation (sham-operated rat = s-rat) was performed under intraperitoneal pentobarbital anesthesia (50 mg/kg body wt). Rats were fed a normal diet (0.9% P, 1.12% Ca, 1580 IU/kg VD) until 1 wk after these procedures and then switched to a high-P, low-Ca, and low-VD (HP-LC-LD) diet (1.2% P, 0.4% Ca, <300 IU/kg VD) for 8 wk. The feed was obtained from Oriental Yeast, Inc. (Chiba, Japan). The Animal Studies Committee of Wakayama Medical University approved all animal protocols.

Direct Injection into PTG of U-Rats

Bilateral PTG of u-rats were exposed surgically and were injected OCT (10 μg/ml) or its vehicle (phosphate buffer that contained 0.01% polyoxyethylene sorbitan monolaurate and 0.2% ethanol [pH 8.0, isotonic solution]; DI-vehicle) using a 30-G needle (specially made by Tochigi Seikou Co., Inc., Tochigi, Japan) under a zoom stereo microscope (Carton Optical Industries, Ltd., Tokyo, Japan). The needle tip is blind, and a one-side hole exists for maneuvering it to the gland’s center. Immediately after the injection, the solution that had leaked from the PTG was washed with saline and removed. These procedures were performed under diethylether inhalation. Figure 1A shows the image of a direct injection into the PTG, and Figure 1B shows the distribution of the injected solution in the PTG.

Figure 1.
Figure 1.

(A) Direct injection into the parathyroid gland (PTG) of a uremic rat (u-rat). (B) Distribution of directly injected solution into the PTG. The injected solution (Indian ink) is distributed to almost all parts of the PTG.

The volume of injected solution was estimated using 10 u-rats. DI-OCT into one PTG (PTGOCT) and DI-vehicle alone into the other PTG (PTGvehicle) in the same rat were performed. Immediately after these injections, PTG were removed and washed carefully with vehicle. Each PTG was homogenized in 1 ml of vehicle, and the OCT concentration in the supernatant was determined by HPLC with tandem mass spectrometry (12). The mean OCT contents in PTGOCT and in PTGvehicle determined on the basis of OCT concentration in the supernatant were 25.2 ± 6.7 and 0.51 ± 0.42 pg/PTG, respectively. Thus, the mean amount (volume) of the injected OCT solution into one PTG was estimated to be 24.7 ± 6.5 pg (2.47 ± 0.65 μl; because 10 μg/ml OCT was used) by calculating the difference. This value was similar to the original PTG volume of u-rats.

Laboratory Measurements

Serum intact-PTH (iPTH), Ca2+ and P levels, and other data were obtained before (baseline) and 1 and 2 d after DI-OCT or DI-vehicle in u-rats. Serum iPTH and Ca2+ levels were measured by the two-antibody method using a rat iPTH ELISA kit (Immutopics, Inc., San Clemente, CA) and an i-STAT Portable Clinical Analyzer (i-STAT Corporation, East Windsor, NJ), respectively. Other data were determined using an automated analyzer (7070; Hitachi, Tokyo, Japan). In s-rats, the same data were obtained at the completion of the HP-LC-LD diet for 8 wk.

Isolation of Total RNA and Reverse Transcription-PCR for PTH, VDR, and CaSR

Total RNA was prepared from each PTG specimen (before and 6, 12, 24, and 72 h after DI-OCT or DI-vehicle) using the TRIzol reagent (Life Technologies, Gaithersburg, MD). Total RNA (2 μg) was reverse-transcribed using oligo (dT) as a primer (Life Technologies) for reverse transcription-PCR (RT-PCR), as in a previous report (10). Subsequently, 1 μl of the cDNA mixture was suspended in a total volume of 50 μl of a solution that contained 50 mM KCl, 10 mM Tris-HCl, 1.2 mM MgCl2, 0.5 μM of each primer, 200 μM of each nucleotide, and 1 U of Ampli-TaqGold DNA Polymerase (Roche Molecular Systems, Branchburg, NJ). Amplification then was performed using specific primers (Table 1). For each specific assay, PCR conditions were optimized with respect to the template cDNA per tube, annealing temperature, and number of cycles. Amplification products were analyzed on 2% agarose gels that contained 0.5 μg/ml ethidium bromide and photographed. All PCR products were quantified using NIH-Image (National Institutes of Health, Bethesda, MD). For each sample, target mRNA levels then were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level, and then PTH, VDR, and CaSR/GAPDH mRNA ratios for individual samples were averaged.

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Table 1.

Primer design and size of fragmenta

Immunohistochemistry

PTG of u-rats before and 0.5, 1, 3, 5, and 7 d after DI-OCT or DI-vehicle and those of s-rats were fixed with 4% paraformaldehyde PBS for 8 h and paraffin-embedded. Immunohistochemical staining for VDR and CaSR was performed using a mouse monoclonal anti-VDR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a mouse monoclonal anti-CaSR antibody (provided by Dr. Nemeth, NPS Pharmaceutical, Inc., Toronto, Ontario, Canada) as the primary antibodies. The method using the dextran polymer conjugate two-step visualization system developed by Vyberg and Nielsen (Dako Envision System; HRP, Dako, Glostrup, Denmark) was applied (13). The slides were exposed to anti-VDR and CaSR antibodies (1:100 and 1:500 dilutions, respectively) for 1 h each at room temperature. The slides were rinsed and then incubated with a peroxidase-labeled dextran polymer conjugated to goat anti-mouse immunoglobulins against the primary antibody. The immunostaining was visualized using a 3,3-diamino-benzidine substrate chromogen and was observed by light microscopy (BX50; Olympus, Tokyo, Japan). The expression levels were determined in individual glands. Three independent observers counted the VDR-positive PTC in 10 high-power fields with approximately 400 cells per field at ×400 magnification, after which the average was taken. The ratio of CaSR-positive area per total PTG (excluding the part lacking PTC) was estimated for all PTG specimens using NIH image.

Calcium-PTH Response Curves

A previously reported method (14) was modified and applied for the present examination. U-rats were anesthetized with pentobarbital, and the right (before) or left (after) femoral artery was catheterized with 26-G fluoroplastics tubing (NIPRO Co., Tokyo, Japan). A blood sample (0.3 ml) was drawn for the measurement of baseline parameters. Immediately after blood sampling, Ca2+ level was measured, and then the sample was centrifuged. The serum was frozen and stored at −20°C until iPTH level determination. Blood cells and additional heparinizedsaline (10 U/ml) equivalent to the actual volume of serum obtained for the measurement of iPTH level were returned to the rat via a catheter. EGTA disodium salt solution (EGTA-2Na; 0.1 M; 10 ml/kg) was infused intraperitoneally. After EGTA-2Na infusion, blood samples were collected every 7 min, and Ca2+ level was measured immediately. After approximately 30 min, the Ca2+ level decrease to a minimum value (<0.5 mM) was completed and then 0.1 M calcium gluconate solution (gCa) at 10 ml/kg was infused intraperitoneally. After gCa infusion, blood samples were also collected every 5 min. After approximately another 30 min, Ca2+ level was confirmed to increase to a maximum value (>1.6 mM). When the Ca2+ minimum and/or maximum value was not achieved, additional EGTA-2Na and/or gCa solution at 5 ml/kg was reinfused. These procedures were performed before and 2 d after the DI-OCT or DI-vehicle. Four parameters (15) for each sigmoid curve between serum iPTH and Ca2+ levels were estimated by exploratory analysis using graphic and analysis software (Origin 7.0; Light Stone, Tokyo, Japan).

Apoptosis Analysis by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling Method and DNA Electrophoresis

DI-OCT or DI-vehicle was administered one, two, or three times every 24 h. The PTG were excised 24 h after the final injection, fixed, and embedded in paraffin for the above-mentioned processing. Paraffin-embedded tissue blocks were sectioned, deparaffinized in xylene and alcohol, and placed in PBS. The tissue sections then were treated with proteinase K and washed with PBS. The in situ apoptosis detection kit ApopTag (Serologicals Co., Spaulding Drive Norcross, GA) was used for labeling the free 3′-OH terminus. These procedures were automatically stained using VENTANA (Ventana Medical Systems, Inc., Tucson, AZ). The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive PTC was determined by the same evaluation method for VDR-positive PTC.

Furthermore, we also performed DNA electrophoresis to detect DNA fragmentation. From the PTG that were treated by DI-OCT or DI-vehicle at the same time as the TUNEL method, DNA was extracted using the DNA extraction kit QIAamp (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.

Confirmation of DI-OCT Effects Using One of the PTG in the Same Rat as Control

Administration of DI-OCT into the left PTG and DI-vehicle into the right PTG was performed in the same u-rat. The differences in PTH, VDR, and CaSR mRNA levels between PTG 24 h after DI-OCT and DI-vehicle were evaluated by RT-PCR in each rat. For the immunohistochemistry of VDR and CaSR, PTG 1 and 5 d after the treatments were examined. Both TUNEL and DNA electrophoresis were performed to analyze apoptosis using PTG that were injected twice consecutively every 24 h.

Statistical Analyses

Data were expressed as means ± SD. The time course of the changes in all data among the DI-OCT–or DI-vehicle-treated rats were analyzed by ANOVA with post hoc multiple comparisons using Dunnett tests. For the statistical significance of DI-OCT compared with the corresponding times of DI-vehicle and with DI-vehicle into one of the PTG as control, unpaired and paired t test, respectively, were used. Other data were also analyzed by unpaired t test. P < 0.05 was considered statistically significant.

Results

Changes in Laboratory Data

Table 2 shows baseline data of u-rats and s-rats. Serum iPTH levels in u-rats were markedly higher than those in s-rats. All baseline data did not show any significant differences between DI-OCT–and DI-vehicle-treated u-rats except for the body weight.

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Table 2.

Baseline data of sham-operated and uremic ratsa

Figure 2 shows the changes in serum iPTH, Ca2+, and P levels after DI-OCT or DI-vehicle. DI-OCT significantly decreased serum iPTH level relative to the baseline level; however, DI-vehicle did not change this level. Marked differences in serum Ca2+ and P levels between DI-OCT and DI-vehicle were not observed.

Figure 2.
Figure 2.

Changes in serum intact parathyroid hormone (i-PTH; A), ionized calcium (Ca2+; B), and phosphorus (P; C) levels. The level of serum i-PTH decreased after direct maxacalcitol injection (DI-OCT) into PTG; however, this level did not change significantly after direct vehicle injection (DI-vehicle) into PTG. The significant differences in both serum Ca2+ and P levels between DI-OCT and DI-vehicle were not observed. (The numbers of rats that were treated with DI-OCT and DI-vehicle were 12 and 11, respectively; *P < 0.05, **P < 0.01 versus before direct injection; #P < 0.05, ##P < 0.01 versus the corresponding DI-vehicle group.) Solid line, DI-OCT; dotted line, DI-vehicle.

Changes in PTH, VDR, and CaSR mRNA Levels Determined by RT-PCR

Figures 3 through 5⇓⇓ show the representative photographs of PTH, VDR, CaSR, and GAPDH mRNA levels and the time courses of PTH, VDR, and CaSR/GAPDH mRNA ratios in PTG before and after DI-OCT or DI-vehicle. The PTH mRNA levels from 6 to 72 h after DI-OCT clearly decreased in comparison with the original level (Figure 3A). PTH/GAPDH mRNA ratios after DI-OCT were significantly lower than those before injection and corresponding time of DI-vehicle (Figure 3C). The VDR mRNA level 24 h after DI-OCT clearly increased in comparison with those at any other time (Figure 4A). The CaSR mRNA levels 6 to 72 h after DI-OCT clearly increased in comparison with the original level (Figure 5A). The VDR/GAPDH mRNA ratio 24 h after DI-OCT and the CaSR/GAPDH mRNA ratios after DI-OCT were significantly higher than those before injection and corresponding times of DI-vehicle (Figures 4C and 5C).

Figure 3.
Figure 3.

Changes in PTH mRNA expression levels and PTH/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios of PTG before and after DI-OCT (A) or DI-vehicle (B). The decreased level of PTH mRNA expression after DI-OCT was continued for at least 72 h (A). However, GAPDH mRNA expression levels did not change after DI-OCT, and these levels did not change after DI-vehicle at all time points. PTH/GAPDH mRNA ratios after DI-OCT significantly decreased compared with that before DI-OCT and were significantly lower than those after DI-vehicle after 12 to 72 h (C). (The number of rats that were treated with DI-OCT and DI-vehicle was eight at each time; **P < 0.01 versus before direct injection; #P < 0.05, ##P < 0.01 versus the corresponding DI-vehicle group.) Solid line, DI-OCT; dotted line, DI-vehicle.

Figure 4.
Figure 4.

Changes in vitamin D receptor (VDR) mRNA expression levels and VDR/GAPDH mRNA ratios of PTG before and after DI-OCT (A) or DI-vehicle (B). The level of VDR mRNA expressions peaked 24 h after DI-OCT (A). However, GAPDH mRNA expression levels did not change after DI-OCT, and these levels did not change after DI-vehicle at all time points. VDR/GAPDH mRNA ratio 24 h after DI-OCT significantly increased compared with that before DI-OCT and were significantly higher than that in the corresponding DI-vehicle–treated group (C). (The number of rats that were treated with DI-OCT and DI-vehicle was eight at each time; **P < 0.01 versus before direct injection; ##P < 0.01 versus the corresponding DI-vehicle group.) Solid line, DI-OCT; dotted line, DI-vehicle.

Figure 5.
Figure 5.

Changes in calcium-sensing receptor (CaSR) mRNA expression levels and CaSR/GAPDH mRNA ratios of PTG before and after DI-OCT (A) or DI-vehicle (B). The increased level of CaSR mRNA expression after DI-OCT continued for at least 72 h (A). However, GAPDH mRNA expression levels did not change after DI-OCT, and these levels did not change after DI-vehicle at all time points. CaSR/GAPDH mRNA ratios after DI-OCT significantly increased compared with that before DI-OCT and were significantly higher than those in the corresponding DI-vehicle–treated group (C). (The number of rats that were treated with DI-OCT and DI-vehicle was eight at each time; *P < 0.05, **P < 0.01 versus before direct injection; #P < 0.05, ##P < 0.01 versus the corresponding DI-vehicle group.) Solid line, DI-OCT; dotted line, DI-vehicle.

Immunohistochemistry of VDR and CaSR Expressions

At baseline, the numbers of VDR- and CaSR-positive PTC in u-rats were markedly smaller than those in s-rats (Figure 6). However, these numbers were markedly increased after DI-OCT even in u-rats. The numbers of both VDR- and CaSR-positive PTC peaked 3 d after DI-OCT and were maintained for at least 7 d. However, these findings were not observed after DI-vehicle (Figures 7 and 8).

Figure 6.
Figure 6.

Immunohistochemical staining of VDR and CaSR in parathyroid cells (PTC) at baseline. Representative photomicrographs of immunohistochemical staining of VDR (A) and CaSR (B) in PTC at baseline in sham-operated (s-) and uremic (u-) rats are shown. Both VDR and CaSR expression levels in PTC in s-rats were significantly higher than those in u-rats (C). (The numbers of examined s-rats and u-rats both were 10.) Magnification, ×400 in A and B.

Figure 7.

Immunohistochemical staining of VDR (A) and CaSR (B) in PTC after DI-OCT or DI-vehicle. The upregulations of both VDR and CaSR expressions in PTC 1, 3, 5, and 7 d after DI-OCT were confirmed by the immunohistochemical examinations. However, these expressions did not change significantly after DI-vehicle. Magnification, ×400.

Figure 8.
Figure 8.

Changes in VDR (A) and CaSR (B) expression levels in PTC before and after DI-OCT or DI-vehicle. The changes in ratios of VDR-positive cells and CaSR-positive areas after both direct injections are shown. The significantly increased VDR and CaSR expression levels after DI-OCT continued for at least 7 d. However, these expression levels did not change significantly after DI-vehicle. (The numbers of rats that were treated with DI-OCT and DI-vehicle were 10; **P < 0.01 versus before direct injection; ##P < 0.01 versus the corresponding DI-vehicle injection group.) Solid line, DI-OCT; dotted line, DI-vehicle.

Changes in Calcium-PTH Response Curve

The four parameters calculated from the model of Brown (15) before and 2 d after DI-OCT or DI-vehicle are shown in Table 3. Not only maximum PTH levels but also the set point after DI-OCT significantly decreased compared with those before DI-OCT. The sigmoid curve clearly shifted to the left downward after DI-OCT (Figure 9). However, these findings were not observed after DI-vehicle.

Figure 9.
Figure 9.

Changes in calcium-PTH response curve after DI-OCT (A) or DI-vehicle (B). The means of parameters shown in Table 3 are fitted to the Brown’s equation (15): i-PTH = (a − d)/[1 + (Ca2+/c)b] + d, where c is the set point, d and a are minimum and maximum PTH levels achieved by hypercalcemia and hypocalcemia, respectively, and b is proportional to the slope of the Ca-PTH relationship at the set point. After DI-OCT, this curve shifted to the left downward (A). This result suggests that the sensitivity of PTC to Ca improved and that the number of PTC decreased after DI-OCT. These findings were not observed after DI-vehicle (B). Dotted line, before; solid line, after.

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Table 3.

Ca-PTH response curve before and after DI-OCT or DI-vehiclea

Apoptosis Analysis

Figure 10 shows the apoptotic PTC that were subjected to TUNEL after DI-OCT or DI-vehicle. In the specimens after DI-vehicle, some TUNEL-positive PTC were observed (Figure 10B), but in those after DI-OCT, a large number of TUNEL-positive PTC were noted. In particular, repeated DI-OCT more significantly increased the number of TUNEL-positive PTC (Figure 10A). A significant increase in the number of TUNEL-positive PTC after DI-OCT compared with those after DI-vehicle was confirmed (Figure 10C). By the DNA electrophoresis of PTG after two and three consecutive DI-OCT (PTG that were subjected to this repeated DI-OCT contained a large number of TUNEL-positive PTC), a ladder pattern indicating the presence of DNA fragmentation was observed, but this finding was never observed after DI-vehicle (Figure 11).

Figure 10.
Figure 10.

Detections of DNA fragmentation in PTC by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and changes in the ratios of TUNEL-positive PTC after repeated DI-OCT (A) or DI-vehicle (B). Representative findings of in situ detection of DNA fragmentation by the TUNEL method in PTC after DI-OCT (A) and DI-vehicle (B) are shown. After DI-vehicle, some TUNEL-positive PTC were observed (B), but after DI-OCT, a large number of TUNEL-positive PTC were noted (A). (C) Changes in the ratio of TUNEL-positive PTC after DI-OCT or DI-vehicle. After DI-OCT, the ratio of TUNEL-positive PTC significantly increased. Moreover, repeated DI-OCT induced a marked increase in the number of TUNEL-positive PTC compared with that after repeated DI-vehicle. (The numbers of rats that were treated with DI-OCT and DI-vehicle were 10 at each time; *P < 0.05, **P < 0.01 versus that before direct injection; #P < 0.05, ##P < 0.01 versus the corresponding timed DI-vehicle–treated group.) Solid line, DI-OCT; dotted line, DI-vehicle. Magnifications, ×200 (low) and ×400 (high) in A and B.

Figure 11.
Figure 11.

Detection of DNA fragmentation using 2% agarose gel electrophoresis. A ladder pattern indicating the presence of DNA fragmentation, which is characteristic of cell apoptosis, was observed after two or three times of DI-OCT. This feature was never observed after repeated DI-vehicle injections. The numbers of examined rats were >10.

Confirmation of DI-OCT Effects by Its Own Control

Table 4 shows the ratios of the levels of PTGOCT with respect to those of PTGvehicle in the same u-rat. The suppression of PTH mRNA expression, the upregulations of VDR and CaSR, and the induction of apoptosis in only PTGOCT were confirmed by the same techniques; however, these effects were not observed in PTGvehicle.

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Table 4.

Confirmation of the effects of DI-OCT injection into PTG by its own controla

Discussion

Despite recent advances in the treatment for SHPT, several patients have advanced SHPT that is resistant to treatment with a large amount of intravenous VD derivatives, such as OCT or calcitriol (1,25-dihydroxyvitamin D3 [1,25-D3]) (16). The most important etiologic factors of this resistance are markedly decreased VDR and CaSR contents in PTC and severely swollen PTG as a result of hyperplasia. In dialysis patients, these agents not only decrease PTH levels but also improve the response to VD by VDR upregulation in PTC (17–21). A previous study showed that the CaSR mRNA expression level in PTG is dose-dependently increased by 1,25-D3 without any changes in PTH and Ca2+ levels (22). Recently, the genomic relationship between VD and CaSR was also clarified (23). However, it is considered that these upregulations by conventional VD treatment are not sufficient to decrease PTH level to a target level in very severe SHPT. It is possible for PTx-AT and percutaneous ethanol injection therapy to decrease the number of PTC that over-synthesize and secrete PTH. Moreover, we previously reported that percutaneous maxacalcitol injection therapy (PMIT) induces apoptosis in PTC and reduces the ultrasonographic PTG volume (10). However, regarding clinical investigations, more invasive examinations are difficult, and the development of direct injection into rat PTG enables further elucidation of these biochemical and cellular effects.

First, we established the rat model of very severe SHPT and the method of direct injection into the PTG. In u-rats, the levels of VDR and CaSR in PTC are markedly lower than those in s-rats (Figure 6); moreover, the PTH level could not be decreased by intravenous administration of a very high dose of OCT (2.5 μg/kg; data not shown). Thus, it is considered that u-rat is an appropriate model for very severe SHPT. Rat PTG are very small (they weigh from 1 to 3 μg) and are solid; thus, it is very difficult to inject a solution to saturate completely a PTG despite a direct injection under a zoom stereo microscope. Therefore, we designed a specific needle for a direct injection into very small tissue and confirmed the accuracy of injection using Indian ink (Figure 1). The volume of a solution injected into the PTG was almost the same as that of PTG. In the near future, this technique will be available for the in vivo study of a direct injection of various agents and the introduction of target genes into the PTG.

Serum iPTH level significantly decreased without significant changes in serum Ca2+ and P levels immediately after DI-OCT; however, DI-vehicle failed to reduce this level. Thus, it is considered that this decrease was caused by OCT, not by the effect of vehicle and mechanical stress on PTG by direct injection. The suppression of PTH mRNA expression in the PTG was also confirmed by RT-PCR analysis; therefore, we concluded that a highly concentrated OCT in the PTG suppresses the synthesis and secretion of PTH. The subsequent intravenous administration of OCT maintained the reduction in serum iPTH level for 4 wk after DI-OCT in u-rats (data not shown). Moreover, we previously reported the maintenance of this decreased level for >12 wk after PMIT in a clinical investigation (10).

The biologic response to medical treatments such as VD that leads to the suppression of PTH synthesis and PTC proliferation is mediated via VDR and CaSR in target cells. However, nodular hyperplasia shows a more marked decrease in both levels in PTC (7,8); thus, advanced SHPT is resistant to the supplementations of VD and Ca. In an experimental rat advanced SHPT model, kidney transplantation normalized serum PTH, Ca, P, and urea levels but did not upregulate VDR and CaSR mRNA expressions in the PTG (24). However, in the present study, the upregulations of both VDR and CaSR expressions in PTC after DI-OCT were revealed by both RT-PCR analysis and immunohistochemistry despite very severe SHPT. Moreover, the Ca2+-PTH response curve clearly shifted to the left downward after DI-OCT; thus, it is considered that this finding indicates the decreased number and improved Ca sensitivity of PTC after DI-OCT.

The induction of apoptosis in PTC after DI-OCT was also revealed in the present study. We performed repeated DI-OCT to confirm the consistency of the marked increase in the number of TUNEL-positive PTC, because it was considered that apoptosis was induced by OCT via VDR in the cells. Namely, repeated (the second and the third) DI-OCT after the increase in VDR levels in PTC after the first DI-OCT was considered to be more effective than a single DI-OCT. As expected, repeated DI-OCT also revealed a ladder pattern, indicating the presence of DNA fragmentation, which is a characteristic feature of cell apoptosis (25), as determined by DNA electrophoresis. However, these findings were never observed after repeated DI-vehicle or intravenous OCT (data not shown). Therefore, the induction of apoptosis after DI-OCT is caused by the highly concentrated OCT and not by that of the vehicle or mechanical and pressure injury. The induction of apoptosis in PTC by VD is controversial (26–29). However, our previous (10) and present studies revealed that this phenomenon occurs under a specific condition of a highly concentrated OCT in the PTG. Moreover, the relationship between VD and cell apoptosis in various tissues and cell lines was published recently (30–37). On the basis of previous reports and our data, we suggest that one of the mechanisms underlying the regression of SHPT is apoptosis via VDR in PTC. However, the detailed relationship between cell apoptosis and VD remains unclarified; therefore, more precise studies are required to elucidate the mechanisms underlying these effects.

In conclusion, the effects of DI-OCT in PTC were as follows: The suppression of PTH synthesis and secretion, the upregulation of VDR and CaSR expressions, and the induction of apoptosis were confirmed not only by the experiments using vehicle control but also by experiments using one of PTG in the same rat as control (e.g., DI-OCT into one PTG and DI-vehicle alone into the other PTG in the same u-rat). PMIT in very severe SHPT can simultaneously induce the improvement of these etiologic factors of resistance to medical treatments for SHPT; thus, it is possible for some patients with very severe SHPT to continue medical treatments and avoid PTx-AT that may be accompanied by some problems after the introduction of PMIT.

Acknowledgments

This study was supported by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan), which provided Oxarol (OCT solution) and its vehicle, and was partially supported by a research grant from 2003 Wakayama Medical Award for Young Researchers.

We thank Dr. Edward F. Nemeth, (NPS Pharmaceuticals, Inc.) for providing the CaSR monoclonal antibody used in this study.

Footnotes

  • K.S. and S.N. contributed equally to this work.

References

  1. ↵
    Ganesh S, Stack A, Levin N, Hulbert-Shearon T, Port F: Association of elevated serum PO4, Ca × PO4 product, and parathyroid hormone with cardiac mortality risk in chronic hemodialysis patients. J Am Soc Nephrol 12: 2131–2138, 2001
    OpenUrlAbstract/FREE Full Text
  2. London G, Marty C, Marchais S, Guerin A, Metivier F, de Vernejoul M: Arterial calcifications and bone histomorphometry in end-stage renal disease. J Am Soc Nephrol 15: 1943–1951, 2004
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Block G, Klassen P, Lazarus J, Ofsthun N, Lowrie E, Chertow G: Mineral metabolism, mortality, and morbidity in maintenance hemodialysis. J Am Soc Nephrol 15: 2208–2218, 2004
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Reichel H, Szabo A, Uhl J, Pesian S, Schmutz A, Schmidt-Gayk H, Ritz E: Intermittent versus continuous administration of 1,25-dihydroxyvitamin D3 in experimental renal hyperparathyroidism. Kidney Int 44: 1259–1265, 1993
    OpenUrlPubMed
  5. Naveh-Many, Rahamimo R, Livin N, Silver J: Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest 96: 1786–1793, 1995
  6. ↵
    Cozzolino M, Lu Y, Finch J, Slatopolsky E, Dusso AS: p21WAF1 and TGF-α mediate parathyroid growth arrest by vitamin D and high calcium. Kidney Int 60: 2109–2117, 2001
    OpenUrlCrossRefPubMed
  7. ↵
    Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino Y: Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 92: 1436–1443, 1993
  8. ↵
    Gogusev J, Duchambon P, Hory B, Giovannini M, Goureau Y, Sarfati E, Drueke T: Decreased expression of calcium receptor in parathyroid gland tissue of patients with hyperparathyroidism. Kidney Int 51: 328–336, 1997
    OpenUrlCrossRefPubMed
  9. ↵
    Kitaoka M, Fukagawa M, Ogata E, Kurokawa K: Reduction of functioning parathyroid cell mass by ethanol injection in chronic dialysis patients. Kidney Int 46: 1110–1117, 1994
    OpenUrlCrossRefPubMed
  10. ↵
    Shiizaki K, Hatamura I, Negi S, Narukawa N, Mizobuchi M, Sakaguchi T, Ooshima A, Akizawa T: Percutaneous maxacalcitol injection therapy regresses hyperplasia of parathyroid and induces apoptosis in uremia. Kidney Int 64: 992–1003, 2003
    OpenUrlCrossRefPubMed
  11. ↵
    Shiizaki K, Negi S, Mizobuchi M, Hatamura I, Narukawa N, Sakaguchi T, Kitabata Y, Sumikado S, Akizawa T: Effect of percutaneous calcitriol injection therapy on secondary hyperparathyroidism in uraemic patients. Nephrol Dial Transplant 18[Suppl 3]: iii42–iii46, 2003
  12. ↵
    Ishigai M, Yoshinori A, Kumaki K: Determination of 22-oxacalcitriol, a new analog of 1α,25-dihydroxyvitamin D3, in human serum by liquid chromatography-mass spectrometry. J Chromatogr B 706: 261–267, 1998
    OpenUrlCrossRef
  13. ↵
    Vyberg M, Nielsen S: Dextran polymer conjugate two-step visualization system for immunohistochemistry. A comparison of En Vision + with two three-step avidin-biotin techniques. Appl Immunohistochem 6: 3–10, 1998
    OpenUrlCrossRef
  14. ↵
    Imanishi Y, Hall C, Sablosky M, Brown E, Arnold A: A new method for in vivo analysis of parathyroid hormone-calcium set point in mice. J Bone Miner Res 17: 1656–1661, 2002
    OpenUrlCrossRefPubMed
  15. ↵
    Brown E: Four-parameter model of the sigmoid relationship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. J Clin Endocrinol Metab 56: 572–581, 1983
    OpenUrlCrossRefPubMed
  16. ↵
    Malberti F, Marcelli D, Conte F, Limido A, Spotti D, Locatelli F: Parathyroidectomy in patients on renal replacement therapy: An epidemiologic study. J Am Soc Nephrol 12: 1242–1248, 2001
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin K: Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxycholecalciferol in uremic patients. J Clin Invest 74: 2136–2143, 1984
  18. Levine S, Song M: Pharmacokinetics and efficacy of pulse oral versus intravenous calcitriol in hemodialysis patients. J Am Soc Nephrol 7: 488–496, 1996
    OpenUrlAbstract
  19. Brown A, Ritter C, Finch J, Morrissey J, Martin KJ, Murayama E, Nishii Y, Slatopolsky E: The non-calcemic analog of vitamin D, 22-oxacalcitriol (OCT) suppresses parathyroid hormone synthesis and secretion. J Clin Invest 84: 728–732, 1989
  20. Denda M, Finch J, Brown A, Nishii Y, Kubodera N, Slatopolsky E: 1,25-Dihydroxyvitamin D3 and 22-oxacalcitriol prevent the decrease in vitamin D receptor content in the parathyroid glands of uremic rat. Kidney Int 50: 34–39, 1996
    OpenUrlCrossRefPubMed
  21. ↵
    Patel R, Ke Q, Hsu H: Regulation of calcitriol receptor and its mRNA in normal and renal failure rats. Kidney Int 45: 1020–1027, 1994
    OpenUrlPubMed
  22. ↵
    Brown AJ, Zhong M, Finch J, Ritter C, McCracken R, Morrissey J, Slatopolsky E: Rat calcium-sensing receptor is regulated by vitamin D but not by calcium. Am J Physiol (Lond) 270: F454–F460, 1996
    OpenUrl
  23. ↵
    Canaff L, Hendy G: Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. J Biol Chem 277: 30337–30350, 2002
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Lewin E, Garfia B, Recio F, Rodriguez M, Olgaard K: Persistent downregulation of calcium-sensing receptor mRNA in rat parathyroids when severe secondary hyperparathyroidism is reversed by an isogenic kidney transplantation. J Am Soc Nephrol 13: 2110–2116, 2002
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Wyllie A: Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555–556, 1980
    OpenUrlCrossRefPubMed
  26. ↵
    Fukagawa M, Okazaki R, Takano K, Kaname S, Ogata E, Kitaoka M, Harada S, Sekine N, Matsumoto T, Kurokawa K: Regression of parathyroid hypertrophy by calcitriol-pulse therapy in patients on long-term dialysis. N Engl J Med 323: 421–422, 1990
    OpenUrlCrossRefPubMed
  27. Quarles L, Yohay D, Carroll B, Spritzer C, Minda S, Bartholomay D, Lobaugh B: Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int 45: 1710–1721, 1994
    OpenUrlCrossRefPubMed
  28. Jara A, Gonzalez S, Felsenfeld J, Chacon C, Valdivieso A, Jalil R, Chuaqui B: Failure of high dose of calcitriol and hypercalcaemia to induce apoptosis in hyperplastic parathyroid glands of azotaemic rats. Nephrol Dial Transplant 16: 506–512, 2001
    OpenUrlCrossRefPubMed
  29. ↵
    Canalejo A, Almaden Y, Torregrosa V, Gomez-Villamandos J, Ramos B, Campistol J, Felsenfeld A, Rodriguez M: The in vitro effect of calcitriol on parathyroid cell proliferation and apoptosis. J Am Soc Nephrol 11: 1865–1872, 2000
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Wagner D, Wagner N, Sukhatme P, Scholz H: Activation of vitamin D receptor by the Wilms’ tumor gene product mediates apoptosis of renal cells. J Am Soc Nephrol 12: 1188–1196, 2001
    OpenUrlAbstract/FREE Full Text
  31. Puthier D, Bataille R, Barille S, Mellerin M, Harousseau J, Ponzio A, Robillard N, Wijdenes J, Amiot M: Myeloma cell growth arrest, apoptosis, and interleukin-6 receptor modulation induced by EB1089, a vitamin D derivative, alone or in association with dexamethasone. Blood 88: 4659–4666, 1996
    OpenUrlAbstract/FREE Full Text
  32. Wang Q, Yang W, Uytingco M, Christakos S, Wieder R: 1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death. Cancer Res 60: 2040–2048, 2000
    OpenUrlAbstract/FREE Full Text
  33. Diaz G, Paraskeva C, Thomas M, Binderup L, Hague A: Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: Possible implications for prevention and therapy. Cancer Res 60: 2304–2312, 2000
    OpenUrlAbstract/FREE Full Text
  34. Guzey M, Kitada S, Reed JC: Apoptosis induction by 1 alpha,25-dihydroxyvitamin D3 in prostate cancer. Mol Cancer Ther 1: 667–677, 2002
    OpenUrlAbstract/FREE Full Text
  35. Danielson C, Torma H, Vahlquist A, Carlberg C: Positive and negative interaction of 1,25-dihydroxyvitamin D3 and the retinoid CD 437 in the induction of human melanoma cell apoptosis. Int J Cancer 81: 467–470, 1999
    OpenUrlCrossRefPubMed
  36. Hewison M, Dabrowski M, Vadher S, Faulkner L, Cockerill F, Brickell P, O’Riordan J, Katz D: Antisense inhibition of vitamin D receptor expression induces apoptosis in monoblastoid U937 cells. J Immunol 156: 4391–4400, 1996
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Nakagawa K, Sowa Y, Kurobe M, Ozono K, Siu-Caldera ML, Reddy G, Uskokovic M, Okano T: Differential activities of 1α,25-dihydroxy-16-ene-vitamin D(3) analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis. Steroids 66: 327–337, 2001
    OpenUrlCrossRefPubMed
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Biochemical and Cellular Effects of Direct Maxacalcitol Injection into Parathyroid Gland in Uremic Rats
Kazuhiro Shiizaki, Shigeo Negi, Ikuji Hatamura, Toshifumi Sakaguchi, Fumie Saji, Ken Kunimoto, Masahide Mizobuchi, Ikuo Imazeki, Akira Ooshima, Tadao Akizawa
JASN Jan 2005, 16 (1) 97-108; DOI: 10.1681/ASN.2004030236
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