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Indoxyl sulfate exacerbates low bone turnover induced by parathyroidectomy in young adult rats
Bone 79 (2015) 252–258
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Original Full Length Article
Indoxyl sulfate exacerbates low bone turnover induced by parathyroidectomy in young adult rats Junya Hirata a,b,⁎, Kazuya Hirai a, Hirobumi Asai a, Chiho Matsumoto b, Masaki Inada b, Chisato Miyaura b, Hideyuki Yamato a, Mie Watanabe-Akanuma a a b
Safety Research Center, Kureha Corporation, Tokyo 169-8503, Japan Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan
a r t i c l e
i n f o
Article history: Received 1 December 2014 Revised 29 May 2015 Accepted 16 June 2015 Available online 23 June 2015 Edited by Robert Recker Keywords: Indoxyl sulfate Indole Parathyroidectomy Chronic kidney disease Low bone turnover
a b s t r a c t Low-turnover bone disease is one of the bone abnormalities observed in patients with chronic kidney disease (CKD) and is recognized to be associated with low serum parathyroid hormone (PTH) level and skeletal resistance to PTH. Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood as renal dysfunction progresses in CKD patients. A recent in vitro study using an osteoblastic cell culture system suggests that IS has an important role in the pathogenesis of low bone turnover through induction of skeletal resistance to PTH. However, the effects of IS on the progression of low bone turnover have not been elucidated. In the present study, we produced rats with low bone turnover by performing parathyroidectomy (PTX) and fed these rats a diet containing indole, a precursor of IS, to elevate blood IS level from indole metabolism. Bone metabolism was evaluated by measuring histomorphometric parameters of secondary spongiosa of the femur. Histomorphometric analyses revealed significant decreases in both bone formation–related parameters and bone resorption–related parameters in PTX rats. In indole-treated PTX rats, further decreases in bone formation–related parameters were observed. In addition, serum alkaline phosphatase activity, a bone formation marker, and bone mineral density of the tibia tended to decrease in indole-treated PTX rats. These findings strongly suggest that IS exacerbates low bone turnover through inhibition of bone formation by mechanisms unrelated to skeletal resistance to PTH. © 2015 Elsevier Inc. All rights reserved.
Introduction Abnormalities of bone turnover are commonly observed in patients with chronic kidney disease (CKD), and this condition has recently been termed CKD-related mineral and bone disease (CKD-MBD) [1]. The Kidney Disease Improving Global Outcomes (KDIGO) CKD-MBD Work Group analyzed the prevalence of various types of bone disease in patients with CKD between 1983 and 2006 [2]. The group reported that 84% of the patients with CKD at stages 3–5 had some kind of bone abnormality including osteitis fibrosa, which is a high-turnover bone disease (32%), adynamic bone disease, which is a low-turnover bone disease (18%), osteomalacia (8%), and mixed disease (20%). A more recent study reported that low bone turnover accounted for more than 60% in 543 white patients with CKD stage 5 [3]. Low-turnover bone disease, so-called adynamic bone disease, is mainly characterized by an abnormally low bone formation rate [4,5] and the disease is recognized to be associated with low serum PTH level [5–7] and skeletal resistance to parathyroid hormone (PTH) [5,8, 9]. Analyses of the prevalence of bone turnover abnormalities in ⁎ Corresponding author at: Safety Research Center, Kureha Corporation, 3-26-2 Hyakunin-cho, Shinjuku-ku, Tokyo 169-8503, Japan. E-mail address: [email protected] (J. Hirata).
http://dx.doi.org/10.1016/j.bone.2015.06.010 8756-3282/© 2015 Elsevier Inc. All rights reserved.
patients with end-stage renal disease showed that patients with adynamic bone disease had the lowest serum concentration of intact parathyroid hormone (PTH) compared to patients with other bone abnormalities [10,11]. Other studies also reported an association between reduced serum PTH levels and increases in fractures [12] and all-cause mortality [13] in patients on dialysis. Renal dysfunction leads to an accumulation of uremic toxins in CKD patients [14,15], and more than 100 substances have been proposed as uremic toxins as of 2008 [14,16]. Some uremic toxins have a negative impact on many body functions such as cardiovascular systems in CKD patients and CKD model animals [16,17]. Indoxyl sulfate (IS) is one of the organic anion uremic toxins that belongs to the family of proteinbound retention solutes [14]. The metabolic pathway for the synthesis of IS is shown in Fig. 1. In the intestine, dietary tryptophan is metabolized to indole by intestinal bacteria. Indole is absorbed and transported to the liver where it is converted to IS via indoxyl [18]. IS is rapidly excreted into urine in healthy subjects, but accumulates in the blood of patients with impaired renal function [19–21]. Several reports indicate that IS is related to glomerular sclerosis and renal fibrosis, and accelerates the progression of CKD in rats [19,22]. Iwasaki et al. [23] reported that administration of an oral charcoal adsorbent (AST-120), which adsorbs uremic toxins and/or their precursors in the intestine, to partially nephrectomized rats suppresses
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Indole treatment
Fig. 1. Schematic drawing of the metabolic pathway for the synthesis of indoxyl sulfate.
progression of low-turnover bone disease and reverses the downregulation of PTH receptor gene in osteoblasts, which is implicated as a cause of PTH resistance. Subsequently, Nii-Kono et al. [24] showed that IS suppresses PTH-stimulated intracellular cAMP production and PTH receptor expression, and induces oxidative stress in primary cultured osteoblastic cells. Moreover, a clinical study reported a significant negative correlation between IS and bone-specific alkaline phosphatase (r = − 0.34) independent of PTH in 47 hemodialysis patients [25]. These results suggest that IS has an important role in the pathogenesis of low bone turnover through induction of skeletal resistance to PTH. However, the effects of IS on low bone turnover have not been elucidated. In the present study, we fed an indolesupplemented diet to rats with low bone turnover induced by parathyroidectomy (PTX) to increase blood IS level via biological metabolic pathways. Using this model, we examined whether IS exacerbates low bone turnover. Materials and methods Animals Eight-week-old male SD rats (Crl:CD) weighing 280 to 310 g were purchased from Charles River Japan (Kanagawa, Japan). Rats were housed in polycarbonate cages and in an animal room under controlled illumination (12-h light/dark cycle), temperature (22 ± 2 °C), and humidity (55 ± 10%). During 6 days of acclimatization, they were allowed free access to standard powder diet CE-2 (containing approximately 1% calcium; CLEA Japan Inc., Tokyo, Japan) and tap water. All experimental procedures were approved by the Committee of Ethics on Animal Experiments at Kureha Corporation (Tokyo, Japan), and conducted in accordance with the guidelines of Kureha Corporation.
As shown in Fig. 1, indole synthesized in the intestines is metabolized to indoxyl sulfate in the liver. Therefore, we administered indole orally to PTX rats to elevate blood concentration of IS. After confirming the success of PTX, 18 parathyroidectomized rats were selected and divided into 2 groups matched for body weight and serum calcium level: a group fed a normal diet (PTX group, n = 8) and a group fed an indole-supplemented diet (PTX + ID group, n = 10). Rats in the sham group were also divided into 2 groups matched for body weight: a group fed a normal diet (sham group, n = 6) and a group fed an indole-supplemented diet (sham + ID group, n = 6). The sham group and the PTX group were fed a standard powder diet CE-2 (containing approximately 1% calcium; CLEA Japan Inc.) for 4 weeks. Animals in the sham + ID group and PTX + ID group were fed CE-2 containing 0.25% indole (Wako Pure Chemical Industries Ltd., Osaka, Japan) for 4 days followed by CE-2 containing 0.5% indole until the end of 4 weeks. The dose of indole was selected to mimic the serum IS level of patients with CKD and those on dialysis [19], and the administration method was chosen to maintain elevated serum IS levels during the indole treatment period. Body weight and food intake were measured every week during the indole treatment period. Rats were euthanized at the end of the indole treatment, as described below. Blood collection Blood was collected from the cervical vein under isoflurane anesthesia 2 weeks after PTX and every 2 weeks during the indole treatment period. The blood samples were collected into capillary blood collection tubes (CAPIJECT®, Terumo Corporation, Tokyo, Japan) and centrifuged at 3,300g for 2 min. The serum samples were stored at − 80 °C until use for blood biochemistry. Measurement of serum IS concentration Serum IS levels at each blood sampling time during the indole treatment period were measured using liquid chromatography/ electrospray ionization-tandem mass spectrometry (API 4000™ LC/ MS/MS System, Takara Bio Inc., Shiga, Japan) as described by Kikuchi et al. [26]. Blood biochemistry Serum PTH levels at 2 weeks after PTX were determined by Rat Intact PTH ELISA Kit (Immutopics, Inc., California, USA). Serum calcium levels (at 2 weeks after PTX and at each blood sampling time during the indole treatment) and creatinine (S-Cr) levels (at each blood sampling time) were measured by Unicel DxC600 (Beckman Coulter, Inc., California, USA). Both serum alkaline phosphatase activity and tartrateresistant acid phosphatase (TRAP) activity at each blood sampling time during the indole treatment were measured using TRACP & ALP Assay Kit (Takara Bio Inc., Shiga, Japan). Bone histomorphometry
Induction of low bone turnover by parathyroidectomy Rats were divided into 2 groups: a sham-operated group and a parathyroidectomy (PTX) group, that were matched for body weight. Animals in the PTX group (n = 24) underwent PTX using a cautery knife under isoflurane anesthesia. Six animals in the sham group underwent sham operation under isoflurane anesthesia. Two weeks after PTX, body weight, serum PTH, and calcium levels were measured. Serum PTH levels lower than 50 mg/dL and serum calcium levels lower than 7.5 mg/dL were taken as evidence of successful resection of the parathyroid glands.
On day 9 and day 2 before euthanization, all rats were injected subcutaneously with calcein (in 2% NaHCO3 solution) at a dose of 8 mg/kg body weight (Dojindo Laboratories, Kumamoto, Japan) for labelling. At the end of indole treatment, rats were euthanized by exsanguination via the abdominal aorta under isoflurane anesthesia. The right femur was removed and fixed in 10% neutral buffered formalin, and embedded in 4% CMC compound (Leica Microsystems, Tokyo, Japan) without decalcification. The femur was sectioned longitudinally at 4-μm thickness using a cryostat (Leica Microsystems) and stained with toluidine blue to identify cellular components. Histomorphometric analysis of the
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secondary spongiosa at the distal metaphysis of the femur between 1.1 and 2.45 mm distal to the growth plate-epiphyseal junction was performed using an OsteoMeasure Histomorphometry System (Osteometrics, Inc., Georgia, USA), and measurements were made at ×200 magnification. Area of distal epiphyseal cartilage was calculated from the outer perimeter of the epiphyseal cartilage. Total bone surface (BS) in the secondary spongiosa was traced, and osteoid surface (OS/BS, %) was calculated as a percentage of the total BS. Dynamic parameters were determined as follows. Single-labelled surface (sLS) and doublelabelled surface (dLS) as well as BS in the secondary spongiosa were traced. Then, the percentages of single-labelled surface (sLS/BS, %), double-labelled surface (dLS/BS, %), and mineralized surface (MS/BS, %) were calculated as a percentage of the total BS. Mineral apposition rate (MAR, μm/day) was calculated by dividing the distance between double label by the number of days between the two calcein injections. Bone formation rate per bone surface (BFR/BS, μm3/μm2/year) was the product of (dLS + sLS/2) MAR/BS. Percent trabecular osteoblast surface (Ob.S/BS, %), osteoclast surface (Oc.S/BS, %), and eroded surface (ES/BS, %) were determined. The nomenclature, units, and abbreviations used in this study followed the recommendations of the American Society for Bone Mineral Research [27].
Measurement of bone mineral density Right tibial bone samples were collected and all connective tissues were carefully removed. After measuring the bone length, BMD of the right tibia was determined by single-energy X-ray absorptiometry using a bone mineral analyzer (DCS-600EX-IIIR, Aloca Co., Ltd., Tokyo, Japan). The tibial bone was divided into 20 equal parts, and BMD were measured individually. BMD for the entire length of the tibial bone was calculated by averaging the BMD of the 20 parts.
Statistical analysis Numerical data are expressed as means ± SD. Statistical analyses were performed using JMP ver. 10.0.0 (SAS Institute Inc., North Carolina, USA). For data analyses, Bartlett’s test for variance homogeneity was performed. When Bartlett’s test was not significant at 5% level, oneway ANOVA followed by post hoc Tukey–Kramer test (parametric allpairs multiple comparisons) was conducted. When Bartlett’s test was significant or data was expressed in percentage, Kruskal–Wallis’ test followed by post hoc Steel–Dwass test (non-parametric all-pairs multiple comparisons) was performed. P values less than 5% were considered to be statistically significant.
Results Induction of low bone turnover by PTX The body weights of parathyroidectomized rats were 8.5% lower than those of sham-operated rats at 2 weeks after PTX. Serum PTH and Ca levels are shown in Fig. 2. Marked decreases in serum PTH level were observed in 21 of 24 animals in the PTX group (Fig. 2A). The results provided evidence for successful resection of the parathyroid glands in 21 animals. Serum Ca levels in these animals decreased dramatically (Fig. 2B). Effects of indole-supplemented feeding on serum bone turnover markers in PTX rats As shown in Fig. 3A, body weights in the PTX and PTX + ID groups were lower than those in the sham and sham + ID groups throughout the indole treatment period. There was no difference in food intake between the PTX and PTX + ID groups, although a tendency of decreased food intake was observed in the PTX + ID group compared with the sham group on week 4 (Fig. 3B). Body weight and food intake in the sham + ID group were similar to those in the sham group. Mean indole intake during the indole treatment period was 113.7 mg/kg/day in the sham + ID group and 122.7 mg/kg/day in the PTX + ID group. Serum IS levels in indole-treated groups were elevated and the high levels were maintained during the indole treatment period (Fig. 3C). Serum IS levels in the present study were similar to those reported in CKD model rats, which were approximately 4.1 mg/dL in adenineinduced CKD rats [28] and 1.2 mg/dL in 5/6 nephrectomized rats [29]. S-Cr levels were similar in all groups throughout the indole treatment period and the levels on week 4 were 0.33 mg/dL in the sham group, 0.32 mg/dL in the sham + ID group, 0.40 mg/dL in the PTX group, and 0.36 mg/dL in the PTX + ID group. Changes in serum bone turnover markers during the indole treatment period are shown in Fig. 3(D–F). Decreased serum Ca levels were observed after PTX in the PTX group and the low levels were maintained throughout the experiment. Serum Ca levels in the PTX + ID group were similar to those in the PTX + ID group during the indole treatment period (Fig. 3D). Serum ALP activity in the PTX + ID group apparently decreased from week 2 when compared to the sham group and PTX group, although significant differences were not observed (Fig. 3E). On the other hand, there were no differences in serum TRAP activity among all groups during the indole treatment period, with the exception of a lower level in the PTX + ID group compared to the sham group before indole treatment (Fig. 3F). This low baseline level was considered to be incidental because no difference was observed on weeks 2 and 4. In addition, serum TRAP activity was not different between the PTX + ID group and PTX group before indole treatment. Serum PTH levels on week 4 were 384.1 ± 173.1 pg/mL in the sham group, 575.0 ± 270.5 pg/mL in the sham + ID group, 17.0 ± 4.2 pg/mL in the PTX group, and 21.2 ± 10.6 pg/mL in the PTX + ID group. Thus, serum PTH levels were not affected by indole treatment. Decreased serum PTH levels observed after PTX were maintained throughout the experiment. Effects of indole-supplemented feeding on the histomorphometric analysis of the secondary spongiosa in PTX rats
Fig. 2. Serum parathyroid hormone (A) and calcium (B) levels in PTX animals. Data are presented as individual levels (triangles) and mean ± SD (vertical bar with short horizontal line). Dotted line shows upper threshold for successful resection of parathyroid glands. PTH, parathyroid hormone; Ca, calcium; Sham, sham-operated control; PTX, parathyroidectomy.
Low magnification images of distal metaphysis of femur are shown in Fig. 4A. Areas of epiphyseal cartilage in the femur were 2.1 ± 0.2 mm2 in the sham group, 2.1 ± 0.3 mm2 in the sham + ID group, 2.1 ± 0.3 mm2 in the PTX group, and 2.3 ± 0.3 mm2 in the PTX + ID group, with no significant difference among the four groups. Thus, growth impairment did not occur as a result of PTX or indole treatment. Images of calcein staining of the secondary spongiosa in the femur are shown in Fig. 4B. Bone formation represented by the distance between
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Fig. 3. Changes in body weight (A), food intake (B), serum IS level (C), and serum bone turnover markers (D, serum Ca level; E, serum ALP activity; F, serum TRAP activity) in rats. IS, indoxyl sulfate; Ca, calcium; ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase, A, absorbance; Sham, sham-operated control; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID group were fed an indole-containing diet (0.5% w/w) for 4 weeks. Sham group, n = 6; sham + ID group, n = 6; PTX group, n = 8; PTX + ID group, n = 10. Data are expressed as mean ± SD. Statistical analysis was not conducted for serum IS level and serum Ca level because increases in serum IS level in the ID-treated groups and decreses in serum Ca level in the PTX groups were apparent. ⁎P b 0.05, ⁎⁎P b 0.01 vs. sham (Tukey–Kramer test).
calcein labels was suppressed slightly in the sham + ID group and decreased markedly in the PTX group compared to the sham group. Bone formation decreased further in the PTX + ID group, and calcein double-labelling was almost invisible. Fig. 4C shows light micrographs at the same locations of the upper calcein staining images. Light microscopy confirmed that osteoblasts were arranged tightly on the bone surface in the sham group. Osteoblasts in the sham + ID group decreased slightly compared to the sham group. On the other hand, osteoblasts decreased in number and arranged sparsely on the bone surface in the PTX group. The number of osteoblasts in the PTX + ID group decreased further compared to the PTX group. The results of histomorphometric analysis of the secondary spongiosa are summarized in Table 1. The PTX group showed low bone turnover indicated by a significant decrease in OS/BS and significant decreases in dynamic parameters comprising MS/BS, MAR, and BFR/BS, compared to the sham group. Moreover, significant decreases in Oc.S/BS and ES/BS were also observed in the PTX group compared to the sham group. Indole treatment resulted in further significant decreases in MS/BS, BFR/BS, and Ob.S/BS, and a tendency for a decrease in MAR compared to the PTX group. On the other hand, there were no differences in bone resorption–related parameters comprising Oc.S/BS and ES/BS between the PTX and PTX + ID groups. In the sham + ID group, Oc.S/BS tended to decrease when compared to the sham group. Correlation between serum IS levels and histomorphometric parameters for sham groups (sham and sham + ID groups) and PTX groups (PTX and PTX + ID groups) were examined. Significant correlations were observed between IS level and Oc.S/BS or ES/BS in sham and sham + ID groups, and between IS level and MS/BS, MAR, BFR/BS, or Ob.S/BS in PTX and PTX + ID groups (data not shown). Effects of indole-supplemented feeding on BMD in PTX rats The bone length and mean BMD in tibial bone are shown in Table 2. The bone lengths were similar and there were no differences in all of the sham, sham + ID, and PTX groups. Only a slight but significant decrease in bone length was observed in PTX + ID compared to the sham group.
There were no differences in bone length between the PTX and PTX + ID groups. There were no differences in BMD between the sham and PTX groups. On the other hand, BMD in the PTX + ID group apparently decreased when compared to the sham group and PTX group although significant differences were not observed.
Discussion Animal models of CKD with low bone turnover are usually produced by partial nephrectomy combined with PTX [23,30,31]. Moreover, Iwasaki et al. [23] constructed a rat model of renal failure with skeletal resistance to PTH by continuous infusion of a physiological level of 1–34 PTH in addition to PTX and nephrectomy. Thus, models commonly used for the evaluation of bone metabolism represent low bone turnover with skeletal resistance to PTH. In these models, partial nephrectomy is conducted to reproduce the pathophysiology of CKD. As a result, a variety of uremic toxins probably accumulate in the blood of these animals because of renal dysfunction. In the present study, we induced low bone turnover in rats by PTX without partial nephrectomy to avoid the effects of diverse uremic toxins. Since serum PTH in our model rats were almost undetectable, bone abnormalities are caused by PTH depletion and are not related to skeletal resistance to PTH. Our study was designed to examine the effect of IS independent of other uremic toxins by not using a CKD model and by exogenously administering indole to specifically increase blood IS level. We confirmed that serum IS levels in the sham + ID and PTX + ID groups were elevated by indole treatment. On the other hand, the resulting high IS in blood could theoretically impair the renal function of rats causing increased production of various uremic toxins that could affect BMD and other bone metabolic parameters. Indeed, IS has been reported to accelerate the progression of CKD in rats [19,22]. However, the renal function as indicated by serum creatinine was similar among the groups in the present study, indicating that renal function in the model rats were intact and that the observed effects were unlikely to be caused by uremic toxins other than IS.
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Fig. 4. Low magnification images of distal metaphysis of femur (A), calcein staining images (B) and light micrographs (C) of the secondary spongiosa. Sham, sham-operated control; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID group were fed an indole-containing diet (0.5% w/w) for 4 weeks. On days 9 and 2 before euthanization, all rats were injected subcutaneously with calcein at a dose of 8 mg/kg body weight for labelling. The femur was sectioned and stained with toluidine blue. No osteoblasts are observed in the micrograph of PTX + ID rat.
In histomorphometric analysis of the secondary spongiosa, decreases in OS/BS, MS/BS, MAR, and BFR/BS as bone formation–related parameters and Oc.S/BS and ES/BS as bone resorption–related parameters were observed in PTX rats. These findings confirmed that marked decrease or depletion of PTH in blood as a result of PTX induced low bone turnover. We demonstrated that the decreases in bone formation–related parameters observed in PTX rats were further worsened by high IS in blood, while bone resorption–related parameters did not deteriorate. Given the nature of our model, these findings strongly
suggest that IS exacerbates low bone turnover by mechanisms other than skeletal resistance to PTH. In fact, the expression of PTH/PTHrP receptor in the spongiosa of the tibia was not influenced by PTX and/ or indole treatment by quantitative RT-PCR analysis (data not shown). In addition, a significant decrease in Ob.S/BS was observed in the PTX + ID group when compared to the PTX group, but there were no differences in bone resorption–related factors including histomorphometric parameters of Oc.S/BS and ES/BS. These results suggest that IS mainly targets osteoblasts to exacerbate low bone turnover. In the
Table 1 Histomorphometric analysis. Group
Sham Sham + ID PTX PTX + ID
OS/BS
MS/BS
MAR
BFR/BS
Ob.S/BS
Oc.S/BS
ES/BS
(%)
(%)
(μm/day)
(μm3/μm2/year)
(%)
(%)
(%)
10.3 ± 0.5 10.9 ± 0.5 2.0 ± 0.4† 0.9 ± 0.4††,##
25.1 ± 5.3 20.4 ± 4.6 5.1 ± 1.5† 1.9 ± 1.5 ††, #
2.2 ± 0.4 2.0 ± 0.3⁎ 1.0 ± 0.2⁎⁎ 0.7 ± 0.2⁎⁎
201.7 ± 63.3 149.0 ± 46.4 19.3 ± 6.4† 5.6 ± 5.5††,#
13.8 ± 3.5 11.0 ± 2.1 10.4 ± 2.6 5.5 ± 0.8††,##
13.9 ± 2.5 10.5 ± 2.6 7.9 ± 2.6† 8.1 ± 1.8†
10.4 ± 1.6 7.2 ± 2.2 5.1 ± 1.8† 4.2 ± 0.9††
OS/BS, osteoid surface per bone surface; MS/BS, mineralized surface per bone surface; MAR, mineral apposition rate; BFR/BS, bone formation rate per bone surface; Ob.S/BS, osteoblast surface per bone surface; Oc.S/BS, osteoclast surface per bone surface; ES/BS, eroded surface per bone surface; Sham, sham-operated control; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID group were fed an indole-containing diet for 4 weeks. Sham group, n = 6; sham + ID group, n = 6; PTX group, n = 8; PTX + ID group, n = 10. ⁎ P b 0.05. ⁎⁎ P b 0.01 vs. sham group (Tukey–Kramer test). † P b 0.05. †† P b 0.01 vs. sham group (Steel–Dwass test). # P b 0.05. ## P b 0.01 vs. PTX group (Steel–Dwass test).
J. Hirata et al. / Bone 79 (2015) 252–258 Table 2 Bone mineral density of tibial bone. Group
Sham Sham + ID PTX PTX + ID
Bone length
BMD
(mm)
(mg/cm2)
44.9 ± 1.0 44.7 ± 1.2 43.8 ± 1.4 43.4 ± 0.4†
114.5 ± 3.6 115.3 ± 4.8 114.7 ± 4.9 109.7 ± 3.8
BMD, bone mineral density; Sham, sham-operated control; Sham + ID, sham-operated and indole treatment; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID groups were fed an indole-containing diet for 4 weeks. Sham group, n = 5; sham + ID group, n = 6; PTX group, n = 8; PTX + ID group, n = 9. † P b 0.05 vs. sham group (Steel–Dwass test).
sham + ID group, decreases in bone formation–related parameters were not observed in histomorphometric analysis. The difference in the effect on bone formation between sham + ID group and PTX + ID group was considered to be caused by the differences in serum PTH level between these groups. Nii-Kono et al. [24] have suggested that IS is taken by primary cultured osteoblasts via organic anion transporter-3 (OAT-3) present in these cells, where it augments free radical production inside the cells and induces osteoblast dysfunction including decreased expression of PTH receptor. Another study has shown that IS transported via OAT-3 in MC3T3-E1 cells increases free radical production and inhibits osteoblast differentiation [32]. In addition, IS has been shown to increase free radicals in proximal tubular cells [33], in the kidney [34], and in endothelial cells [35]. These findings suggest that IS may act directly on osteoblasts by increasing free radical production after being taken up by osteoblasts via OAT-3. A team in our research group examined the direct effects of IS on bone formation and expression of related genes. In cultures of mouse primary osteoblasts, IS dose-dependently suppressed both mineralization of osteoblasts and mRNA expression of bone formation–related genes such as BMP2, Col1α1, and osteocalcin (Matsumoto et al., unpublished data). Therefore, IS may act directly on osteoblasts and inhibit bone formation. In an in vitro study, Mozar et al. [36] reported that IS inhibited both osteoclast differentiation and bone-resorbing activity induced by receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor. The indole treatment slightly suppressed Oc.S/BS in sham rats in the present study, although there were no significant differences (Table 1). This suggests that IS may act on osteoclasts. On the other hand, indole treatment in PTX rats did not affect the histomorphometric parameters of bone resorption (Table 1) and serum TRAP activity (Fig. 3F) in the present study. It is possible that RANKL expression was reduced accompanying the reduction in number of osteoblasts in our PTX animals, because the animals showed a decrease in Ob.S/BS. Moreover, an analysis of the relationship between IS and biochemical markers of bone turnover in 47 hemodialysis patients reported that serum IS level correlated with bone-specific alkaline phosphatase (r = − 0.34) but did not correlate with serum TRAP 5b level [25]. It may be difficult to confirm the effect of IS on bone resorption under low bone turnover conditions. Serum IS levels reported in CKD model rats were approximately 4.1 mg/dL in adenine-induced CKD rats [28] and 1.2 mg/dL in 5/6 nephrectomized rats [29]. Niwa et al. [19] reported that serum IS level was 1.8 ± 1.5 mg/dL in predialysis CKD patients and 5.3 ± 2.1 mg/dL in patients on dialysis (measured before dialysis session). In our study, the mean serum IS levels in PTX + ID treatment group on weeks 2 and 4 of indole treatment were 5.1 ± 0.9 mg/dL and 3.9 ± 1.2 mg/dL, respectively. Thus, the effects of IS on low bone turnover were observed at serum levels of IS in CKD rat models as well as in CKD patients and patients on dialysis, suggesting that IS may play a role in the progression of low-turnover bone disease associated with CKD. On the other hand, various uremic toxins increase in the blood of uremic patients [14,15], and in vitro studies have shown that some of these uremic toxins such
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as p-cresyl sulfate [37] and phenylacetic acid [38] inhibit osteoblast differentiation. Hence, further studies are required to examine whether other uremic toxins may also contribute to progression of low-turnover bone disease in patients with CKD or on dialysis similar to IS. In conclusion, the present study demonstrated for the first time that IS exacerbates low bone turnover through inhibition of bone formation by mechanisms unrelated to skeletal resistance to PTH in rats. Our data suggest that IS may be one of the uremic toxins that contribute to the progression of low-turnover bone disease in patients with CKD or on dialysis whose serum IS levels are elevated because of renal dysfunction. Further studies are warranted to examine uremic toxins other than IS, which impact bone turnover, with the objective to explore potential therapeutic targets for treating low-turnover disease in these patients. Competing interest statement The authors declare no competing interests relevant to this work. Acknowledgments We thank Rie Takagi, Sayaka Seki, Misaki Miyamoto, and Ayumi Hirano for technical assistance, and Kaori Kikuchi for assistance with measurement of serum IS levels. References [1] S. Moe, T. Drüeke, J. Cunningham, W. Goodman, K. Martin, K. Olgaard, et al., Kidney Disease: Improving Global Outcomes (KDIGO). Definition, evaluation, and classification of renal osteodystrophy: A position statement from Kidney Disease: Improving Global Outcomes (KDIGO), Kidney Int 69 (2006) 1945–1953. [2] Kidney Disease: Improving Global Outcomes(KDIGO)CKD-MBD Work Group, KDIGO clinical practice guideline for the diagnosis, evaluation, prevention and treatment of Chronic Kidney Disease-Mineral and Bone Disorder(CKD-MBD), Kidney Int 76 (Suppl. 113) (2009) S1–S130. [3] H.H. Malluche, H.W. Mawad, M.C. Monier-Faugere, Renal osteodystrophy in the first decade of the new millennium: Analysis of 630 bone biopsies in black and white patients, J Bone Miner Res 26 (2011) 1368–1376. [4] A. Fournier, P. Morinière, M.E. Cohen Solal, B. Boudailliez, J.M. Achard, A. Marie, et al., Adynamic bone disease in uremia: May it be idiopathic? Is it an actual disease? Nephron 58 (1991) 1–12. [5] I.B. Salusky, W.G. Goodman, Adynamic renal osteodystrophy: Is there a problem? J Am Soc Nephrol 12 (2001) 1978–1985. [6] M.M. Couttenye, P.C. D'Haese, W.J. Verschoren, G.J. Behets, I. Schrooten, M.E. De Broe, Low bone turnover in patients with renal failure, Kidney Int Suppl 73 (1999) S70–S76. [7] A.J. Hutchison, R.W. Whitehouse, H.F. Boulton, J.E. Adams, E.B. Mawer, T.J. Freemont, et al., Correlation of bone histology with parathyroid hormone, vitamin D3, and radiology in end-stage renal disease, Kidney Int 44 (1993) 1071–1077. [8] S.G. Massry, R. Stein, J. Garty, A.I. Arieff, J.W. Coburn, A.W. Norman, et al., Skeletal resistance to the calcemic action of parathyroid hormone in uremia: role of 1,25 (OH)2 D3, Kidney Int 9 (1976) 467–474. [9] G. Coen, S. Mazzaferro, P. Ballanti, D. Sardella, S. Chicca, M. Manni, et al., Renal bone disease in 76 patients with varying degrees of predialysis chronic renal failure: a cross-sectional study, Nephrol Dial Transplant 11 (1996) 813–819. [10] D.J. Sherrard, G. Hercz, Y. Pei, N.A. Maloney, C. Greenwood, A. Manuel, et al., The spectrum of bone disease in end-stage renal failure—an evolving disorder, Kidney Int 43 (1993) 436–442. [11] A. Torres, V. Lorenzo, D. Hernández, J.C. Rodríguez, M.T. Concepción, A.P. Rodríguez, et al., Bone disease in predialysis, hemodialysis, and CAPD patients: evidence of a better bone response to PTH, Kidney Int 47 (1995) 1434–1442. [12] M.D. Danese, J. Kim, Q.V. Doan, M. Dylan, R. Griffiths, G.M. Chertow, PTH and the risks for hip, vertebral, and pelvic fractures among patients on dialysis, Am J Kidney Dis 47 (2006) 149–156. [13] M. Naves-Díaz, J. Passlick-Deetjen, A. Guinsburg, C. Marelli, J.L. Fernández-Martín, D. Rodríguez-Puyol, et al., Calcium, phosphorus, PTH and death rates in a large sample of dialysis patients from Latin America. The CORES Study, Nephrol Dial Transplant 26 (2011) 1938–1947. [14] R. Vanholder, R. De Smet, G. Glorieux, A. Argilés, U. Baurmeister, P. Brunet, et al., Review on uremic toxins: Classification, concentration, and interindividual variability, Kidney Int 63 (2003) 1934–1943. [15] R. Vanholder, G. Glorieux, R. De Smet, N. Lameire, European Uremic Toxin Work Group, New insights in uremic toxins, Kidney Int Suppl 63 (2003) S6–S10. [16] R. Vanholder, S. Van Laecke, G. Glorieux, What is new in uremic toxicity? Pediatr Nephrol 23 (2008) 1211–1221. [17] R. Vanholder, U. Baurmeister, P. Brunet, G. Cohen, G. Glorieux, J. Jankowski, et al., A bench to bedside view of uremic toxins, J Am Soc Nephrol 19 (2008) 863–870. [18] L.J. King, D.V. Parke, R.T. Williams, The metabolism of [2-14C] indole in the rat, Biochem J 98 (1966) 266–277.
258
J. Hirata et al. / Bone 79 (2015) 252–258
[19] T. Niwa, M. Ise, Indoxyl sulfate, a circulating uremic toxin, stimulates the progression of glomerular sclerosis, J Lab Clin Med 124 (1994) 96–104. [20] F.C. Barreto, D.V. Barreto, S. Liabeuf, N. Meert, G. Glorieux, M. Temmar, et al., Serum indoxyl sulfate is associated with vascular disease and mortality in chronic kidney disease patients, Clin J Am Soc Nephrol 4 (2009) 1551–1558. [21] K. Atoh, H. Itoh, M. Haneda, Serum indoxyl sulfate levels in patients with diabetic nephropathy: Relation to renal function, Diabetes Res Clin Pract 83 (2009) 220–226. [22] T. Niwa, M. Ise, T. Miyazaki, Progression of glomerular sclerosis in experimental uremic rats by administration of indole, a precursor of indoxyl sulfate, Am J Nephrol 14 (1994) 207–212. [23] Y. Iwasaki, H. Yamato, T. Nii-Kono, A. Fujieda, M. Uchida, A. Hosokawa, et al., Administration of oral charcoal adsorbent (AST-120) suppresses low-turnover bone progression in uraemic rats, Nephrol Dial Transplant 21 (2006) 2768–2774. [24] T. Nii-Kono, Y. Iwasaki, M. Uchida, A. Fujieda, A. Hosokawa, M. Motojima, et al., ndoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells, Kidney Int 71 (2007) 738–743. [25] S. Goto, H. Fujii, Y. Hamada, K. Yoshiya, M. Fukagawa, Association between indoxyl sulfate and skeletal resistance in hemodialysis patients, Ther Apher Dial 14 (2010) 417–423. [26] K. Kikuchi, Y. Itoh, R. Tateoka, A. Ezawa, K. Murakami, T. Niwa, Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography/tandem mass spectrometry, J Chromatogr B Analyt Technol Biomed Life Sci 878 (2010) 2997–3002. [27] A.M. Parfitt, M.K. Drezner, F.H. Glorieux, J.A. Kanis, H. Malluche, P.J. Meunier, et al., Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee, J Bone Miner Res 2 (1987) 595–610. [28] Y. Inami, C. Hamada, T. Seto, Y. Hotta, S. Aruga, J. Inuma, et al., Effect of AST-120 on endothelial dysfunction in adenine-induced uremic rats, Int J Nephrol 164125 (2014). http://dx.doi.org/10.1155/2014/164125. [29] A. Enomoto, M. Takeda, A. Tojo, T. Sekine, S.H. Cha, S. Khamdang, et al., Role of organic anion transporters in the tubular transport of indoxyl sulfate and the induction of its nephrotoxicity, J Am Soc Nephrol 13 (2002) 1711–1720.
[30] Y. Iwasaki-Ishizuka, H. Yamato, T. Nii-Kono, K. Kurokawa, M. Fukagawa, Downregulation of parathyroid hormone receptor gene expression and osteoblastic dysfunction associated with skeletal resistance to parathyroid hormone in a rat model of renal failure with low turnover bone, Nephrol Dial Transplant 20 (2005) 1904–1911. [31] J.C. Ferreira, G.O. Ferrari, K.R. Neves, R.T. Cavallari, W.V. Dominguez, L.M. Dos Reis, et al., Effects of dietary phosphate on adynamic bone disease in rats with chronic kidney disease - role of sclerostin? PLoS One 8 (2013) e79721. [32] Y.H. Kim, K.A. Kwak, H.W. Gil, H.Y. Song, S.Y. Hong, Indoxyl sulfate promotes apoptosis in cultured osteoblast cells, BMC Pharmacol Toxicol 14 (2013) 60. [33] M. Motojima, A. Hosokawa, H. Yamato, T. Muraki, T. Yoshioka, Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-κB and free radical in proximal tubular cells, Kidney Int 63 (2003) 1671–1680. [34] S. Owada, S. Goto, K. Bannai, H. Hayashi, F. Nishijima, T. Niwa, Indoxyl sulfate reduces superoxide scavenging activity in the kidneys of normal and uremic rats, Am J Nephrol 28 (2008) 446–454. [35] L. Dou, N. Jourde-Chiche, V. Faure, C. Cerini, Y. Berland, F. Dignat-George, et al., The uremic solute indoxyl sulfate induces oxidative stress in endothelial cells, J Thromb Haemost 5 (2007) 1302–1308. [36] A. Mozar, L. Louvet, C. Godin, R. Mentaverri, M. Brazier, S. Kamel, et al., Indoxyl sulphate inhibits osteoclast differentiation and function, Nephrol Dial Transplant 27 (2012) 2176–2181. [37] H. Tanaka, Y. Iwasaki, H. Yamato, Y. Mori, H. Komaba, H. Watanabe, et al., p-Cresyl sulfate induces osteoblast dysfunction through activating JNK and p38 MAPK pathways, Bone 56 (2013) 347–354. [38] S. Yano, T. Yamaguchi, I. Kanazawa, N. Ogawa, K. Hayashi, M. Yamauchi, et al., The uraemic toxin phenylacetic acid inhibits osteoblastic proliferation and differentiation: An implication for the pathogenesis of low turnover bone in chronic renal failure, Nephrol Dial Transplant 22 (2007) 3160–3165.
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Bone journal homepage: www.elsevier.com/locate/bone
Original Full Length Article
Indoxyl sulfate exacerbates low bone turnover induced by parathyroidectomy in young adult rats Junya Hirata a,b,⁎, Kazuya Hirai a, Hirobumi Asai a, Chiho Matsumoto b, Masaki Inada b, Chisato Miyaura b, Hideyuki Yamato a, Mie Watanabe-Akanuma a a b
Safety Research Center, Kureha Corporation, Tokyo 169-8503, Japan Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan
a r t i c l e
i n f o
Article history: Received 1 December 2014 Revised 29 May 2015 Accepted 16 June 2015 Available online 23 June 2015 Edited by Robert Recker Keywords: Indoxyl sulfate Indole Parathyroidectomy Chronic kidney disease Low bone turnover
a b s t r a c t Low-turnover bone disease is one of the bone abnormalities observed in patients with chronic kidney disease (CKD) and is recognized to be associated with low serum parathyroid hormone (PTH) level and skeletal resistance to PTH. Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood as renal dysfunction progresses in CKD patients. A recent in vitro study using an osteoblastic cell culture system suggests that IS has an important role in the pathogenesis of low bone turnover through induction of skeletal resistance to PTH. However, the effects of IS on the progression of low bone turnover have not been elucidated. In the present study, we produced rats with low bone turnover by performing parathyroidectomy (PTX) and fed these rats a diet containing indole, a precursor of IS, to elevate blood IS level from indole metabolism. Bone metabolism was evaluated by measuring histomorphometric parameters of secondary spongiosa of the femur. Histomorphometric analyses revealed significant decreases in both bone formation–related parameters and bone resorption–related parameters in PTX rats. In indole-treated PTX rats, further decreases in bone formation–related parameters were observed. In addition, serum alkaline phosphatase activity, a bone formation marker, and bone mineral density of the tibia tended to decrease in indole-treated PTX rats. These findings strongly suggest that IS exacerbates low bone turnover through inhibition of bone formation by mechanisms unrelated to skeletal resistance to PTH. © 2015 Elsevier Inc. All rights reserved.
Introduction Abnormalities of bone turnover are commonly observed in patients with chronic kidney disease (CKD), and this condition has recently been termed CKD-related mineral and bone disease (CKD-MBD) [1]. The Kidney Disease Improving Global Outcomes (KDIGO) CKD-MBD Work Group analyzed the prevalence of various types of bone disease in patients with CKD between 1983 and 2006 [2]. The group reported that 84% of the patients with CKD at stages 3–5 had some kind of bone abnormality including osteitis fibrosa, which is a high-turnover bone disease (32%), adynamic bone disease, which is a low-turnover bone disease (18%), osteomalacia (8%), and mixed disease (20%). A more recent study reported that low bone turnover accounted for more than 60% in 543 white patients with CKD stage 5 [3]. Low-turnover bone disease, so-called adynamic bone disease, is mainly characterized by an abnormally low bone formation rate [4,5] and the disease is recognized to be associated with low serum PTH level [5–7] and skeletal resistance to parathyroid hormone (PTH) [5,8, 9]. Analyses of the prevalence of bone turnover abnormalities in ⁎ Corresponding author at: Safety Research Center, Kureha Corporation, 3-26-2 Hyakunin-cho, Shinjuku-ku, Tokyo 169-8503, Japan. E-mail address: [email protected] (J. Hirata).
http://dx.doi.org/10.1016/j.bone.2015.06.010 8756-3282/© 2015 Elsevier Inc. All rights reserved.
patients with end-stage renal disease showed that patients with adynamic bone disease had the lowest serum concentration of intact parathyroid hormone (PTH) compared to patients with other bone abnormalities [10,11]. Other studies also reported an association between reduced serum PTH levels and increases in fractures [12] and all-cause mortality [13] in patients on dialysis. Renal dysfunction leads to an accumulation of uremic toxins in CKD patients [14,15], and more than 100 substances have been proposed as uremic toxins as of 2008 [14,16]. Some uremic toxins have a negative impact on many body functions such as cardiovascular systems in CKD patients and CKD model animals [16,17]. Indoxyl sulfate (IS) is one of the organic anion uremic toxins that belongs to the family of proteinbound retention solutes [14]. The metabolic pathway for the synthesis of IS is shown in Fig. 1. In the intestine, dietary tryptophan is metabolized to indole by intestinal bacteria. Indole is absorbed and transported to the liver where it is converted to IS via indoxyl [18]. IS is rapidly excreted into urine in healthy subjects, but accumulates in the blood of patients with impaired renal function [19–21]. Several reports indicate that IS is related to glomerular sclerosis and renal fibrosis, and accelerates the progression of CKD in rats [19,22]. Iwasaki et al. [23] reported that administration of an oral charcoal adsorbent (AST-120), which adsorbs uremic toxins and/or their precursors in the intestine, to partially nephrectomized rats suppresses
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Indole treatment
Fig. 1. Schematic drawing of the metabolic pathway for the synthesis of indoxyl sulfate.
progression of low-turnover bone disease and reverses the downregulation of PTH receptor gene in osteoblasts, which is implicated as a cause of PTH resistance. Subsequently, Nii-Kono et al. [24] showed that IS suppresses PTH-stimulated intracellular cAMP production and PTH receptor expression, and induces oxidative stress in primary cultured osteoblastic cells. Moreover, a clinical study reported a significant negative correlation between IS and bone-specific alkaline phosphatase (r = − 0.34) independent of PTH in 47 hemodialysis patients [25]. These results suggest that IS has an important role in the pathogenesis of low bone turnover through induction of skeletal resistance to PTH. However, the effects of IS on low bone turnover have not been elucidated. In the present study, we fed an indolesupplemented diet to rats with low bone turnover induced by parathyroidectomy (PTX) to increase blood IS level via biological metabolic pathways. Using this model, we examined whether IS exacerbates low bone turnover. Materials and methods Animals Eight-week-old male SD rats (Crl:CD) weighing 280 to 310 g were purchased from Charles River Japan (Kanagawa, Japan). Rats were housed in polycarbonate cages and in an animal room under controlled illumination (12-h light/dark cycle), temperature (22 ± 2 °C), and humidity (55 ± 10%). During 6 days of acclimatization, they were allowed free access to standard powder diet CE-2 (containing approximately 1% calcium; CLEA Japan Inc., Tokyo, Japan) and tap water. All experimental procedures were approved by the Committee of Ethics on Animal Experiments at Kureha Corporation (Tokyo, Japan), and conducted in accordance with the guidelines of Kureha Corporation.
As shown in Fig. 1, indole synthesized in the intestines is metabolized to indoxyl sulfate in the liver. Therefore, we administered indole orally to PTX rats to elevate blood concentration of IS. After confirming the success of PTX, 18 parathyroidectomized rats were selected and divided into 2 groups matched for body weight and serum calcium level: a group fed a normal diet (PTX group, n = 8) and a group fed an indole-supplemented diet (PTX + ID group, n = 10). Rats in the sham group were also divided into 2 groups matched for body weight: a group fed a normal diet (sham group, n = 6) and a group fed an indole-supplemented diet (sham + ID group, n = 6). The sham group and the PTX group were fed a standard powder diet CE-2 (containing approximately 1% calcium; CLEA Japan Inc.) for 4 weeks. Animals in the sham + ID group and PTX + ID group were fed CE-2 containing 0.25% indole (Wako Pure Chemical Industries Ltd., Osaka, Japan) for 4 days followed by CE-2 containing 0.5% indole until the end of 4 weeks. The dose of indole was selected to mimic the serum IS level of patients with CKD and those on dialysis [19], and the administration method was chosen to maintain elevated serum IS levels during the indole treatment period. Body weight and food intake were measured every week during the indole treatment period. Rats were euthanized at the end of the indole treatment, as described below. Blood collection Blood was collected from the cervical vein under isoflurane anesthesia 2 weeks after PTX and every 2 weeks during the indole treatment period. The blood samples were collected into capillary blood collection tubes (CAPIJECT®, Terumo Corporation, Tokyo, Japan) and centrifuged at 3,300g for 2 min. The serum samples were stored at − 80 °C until use for blood biochemistry. Measurement of serum IS concentration Serum IS levels at each blood sampling time during the indole treatment period were measured using liquid chromatography/ electrospray ionization-tandem mass spectrometry (API 4000™ LC/ MS/MS System, Takara Bio Inc., Shiga, Japan) as described by Kikuchi et al. [26]. Blood biochemistry Serum PTH levels at 2 weeks after PTX were determined by Rat Intact PTH ELISA Kit (Immutopics, Inc., California, USA). Serum calcium levels (at 2 weeks after PTX and at each blood sampling time during the indole treatment) and creatinine (S-Cr) levels (at each blood sampling time) were measured by Unicel DxC600 (Beckman Coulter, Inc., California, USA). Both serum alkaline phosphatase activity and tartrateresistant acid phosphatase (TRAP) activity at each blood sampling time during the indole treatment were measured using TRACP & ALP Assay Kit (Takara Bio Inc., Shiga, Japan). Bone histomorphometry
Induction of low bone turnover by parathyroidectomy Rats were divided into 2 groups: a sham-operated group and a parathyroidectomy (PTX) group, that were matched for body weight. Animals in the PTX group (n = 24) underwent PTX using a cautery knife under isoflurane anesthesia. Six animals in the sham group underwent sham operation under isoflurane anesthesia. Two weeks after PTX, body weight, serum PTH, and calcium levels were measured. Serum PTH levels lower than 50 mg/dL and serum calcium levels lower than 7.5 mg/dL were taken as evidence of successful resection of the parathyroid glands.
On day 9 and day 2 before euthanization, all rats were injected subcutaneously with calcein (in 2% NaHCO3 solution) at a dose of 8 mg/kg body weight (Dojindo Laboratories, Kumamoto, Japan) for labelling. At the end of indole treatment, rats were euthanized by exsanguination via the abdominal aorta under isoflurane anesthesia. The right femur was removed and fixed in 10% neutral buffered formalin, and embedded in 4% CMC compound (Leica Microsystems, Tokyo, Japan) without decalcification. The femur was sectioned longitudinally at 4-μm thickness using a cryostat (Leica Microsystems) and stained with toluidine blue to identify cellular components. Histomorphometric analysis of the
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secondary spongiosa at the distal metaphysis of the femur between 1.1 and 2.45 mm distal to the growth plate-epiphyseal junction was performed using an OsteoMeasure Histomorphometry System (Osteometrics, Inc., Georgia, USA), and measurements were made at ×200 magnification. Area of distal epiphyseal cartilage was calculated from the outer perimeter of the epiphyseal cartilage. Total bone surface (BS) in the secondary spongiosa was traced, and osteoid surface (OS/BS, %) was calculated as a percentage of the total BS. Dynamic parameters were determined as follows. Single-labelled surface (sLS) and doublelabelled surface (dLS) as well as BS in the secondary spongiosa were traced. Then, the percentages of single-labelled surface (sLS/BS, %), double-labelled surface (dLS/BS, %), and mineralized surface (MS/BS, %) were calculated as a percentage of the total BS. Mineral apposition rate (MAR, μm/day) was calculated by dividing the distance between double label by the number of days between the two calcein injections. Bone formation rate per bone surface (BFR/BS, μm3/μm2/year) was the product of (dLS + sLS/2) MAR/BS. Percent trabecular osteoblast surface (Ob.S/BS, %), osteoclast surface (Oc.S/BS, %), and eroded surface (ES/BS, %) were determined. The nomenclature, units, and abbreviations used in this study followed the recommendations of the American Society for Bone Mineral Research [27].
Measurement of bone mineral density Right tibial bone samples were collected and all connective tissues were carefully removed. After measuring the bone length, BMD of the right tibia was determined by single-energy X-ray absorptiometry using a bone mineral analyzer (DCS-600EX-IIIR, Aloca Co., Ltd., Tokyo, Japan). The tibial bone was divided into 20 equal parts, and BMD were measured individually. BMD for the entire length of the tibial bone was calculated by averaging the BMD of the 20 parts.
Statistical analysis Numerical data are expressed as means ± SD. Statistical analyses were performed using JMP ver. 10.0.0 (SAS Institute Inc., North Carolina, USA). For data analyses, Bartlett’s test for variance homogeneity was performed. When Bartlett’s test was not significant at 5% level, oneway ANOVA followed by post hoc Tukey–Kramer test (parametric allpairs multiple comparisons) was conducted. When Bartlett’s test was significant or data was expressed in percentage, Kruskal–Wallis’ test followed by post hoc Steel–Dwass test (non-parametric all-pairs multiple comparisons) was performed. P values less than 5% were considered to be statistically significant.
Results Induction of low bone turnover by PTX The body weights of parathyroidectomized rats were 8.5% lower than those of sham-operated rats at 2 weeks after PTX. Serum PTH and Ca levels are shown in Fig. 2. Marked decreases in serum PTH level were observed in 21 of 24 animals in the PTX group (Fig. 2A). The results provided evidence for successful resection of the parathyroid glands in 21 animals. Serum Ca levels in these animals decreased dramatically (Fig. 2B). Effects of indole-supplemented feeding on serum bone turnover markers in PTX rats As shown in Fig. 3A, body weights in the PTX and PTX + ID groups were lower than those in the sham and sham + ID groups throughout the indole treatment period. There was no difference in food intake between the PTX and PTX + ID groups, although a tendency of decreased food intake was observed in the PTX + ID group compared with the sham group on week 4 (Fig. 3B). Body weight and food intake in the sham + ID group were similar to those in the sham group. Mean indole intake during the indole treatment period was 113.7 mg/kg/day in the sham + ID group and 122.7 mg/kg/day in the PTX + ID group. Serum IS levels in indole-treated groups were elevated and the high levels were maintained during the indole treatment period (Fig. 3C). Serum IS levels in the present study were similar to those reported in CKD model rats, which were approximately 4.1 mg/dL in adenineinduced CKD rats [28] and 1.2 mg/dL in 5/6 nephrectomized rats [29]. S-Cr levels were similar in all groups throughout the indole treatment period and the levels on week 4 were 0.33 mg/dL in the sham group, 0.32 mg/dL in the sham + ID group, 0.40 mg/dL in the PTX group, and 0.36 mg/dL in the PTX + ID group. Changes in serum bone turnover markers during the indole treatment period are shown in Fig. 3(D–F). Decreased serum Ca levels were observed after PTX in the PTX group and the low levels were maintained throughout the experiment. Serum Ca levels in the PTX + ID group were similar to those in the PTX + ID group during the indole treatment period (Fig. 3D). Serum ALP activity in the PTX + ID group apparently decreased from week 2 when compared to the sham group and PTX group, although significant differences were not observed (Fig. 3E). On the other hand, there were no differences in serum TRAP activity among all groups during the indole treatment period, with the exception of a lower level in the PTX + ID group compared to the sham group before indole treatment (Fig. 3F). This low baseline level was considered to be incidental because no difference was observed on weeks 2 and 4. In addition, serum TRAP activity was not different between the PTX + ID group and PTX group before indole treatment. Serum PTH levels on week 4 were 384.1 ± 173.1 pg/mL in the sham group, 575.0 ± 270.5 pg/mL in the sham + ID group, 17.0 ± 4.2 pg/mL in the PTX group, and 21.2 ± 10.6 pg/mL in the PTX + ID group. Thus, serum PTH levels were not affected by indole treatment. Decreased serum PTH levels observed after PTX were maintained throughout the experiment. Effects of indole-supplemented feeding on the histomorphometric analysis of the secondary spongiosa in PTX rats
Fig. 2. Serum parathyroid hormone (A) and calcium (B) levels in PTX animals. Data are presented as individual levels (triangles) and mean ± SD (vertical bar with short horizontal line). Dotted line shows upper threshold for successful resection of parathyroid glands. PTH, parathyroid hormone; Ca, calcium; Sham, sham-operated control; PTX, parathyroidectomy.
Low magnification images of distal metaphysis of femur are shown in Fig. 4A. Areas of epiphyseal cartilage in the femur were 2.1 ± 0.2 mm2 in the sham group, 2.1 ± 0.3 mm2 in the sham + ID group, 2.1 ± 0.3 mm2 in the PTX group, and 2.3 ± 0.3 mm2 in the PTX + ID group, with no significant difference among the four groups. Thus, growth impairment did not occur as a result of PTX or indole treatment. Images of calcein staining of the secondary spongiosa in the femur are shown in Fig. 4B. Bone formation represented by the distance between
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Fig. 3. Changes in body weight (A), food intake (B), serum IS level (C), and serum bone turnover markers (D, serum Ca level; E, serum ALP activity; F, serum TRAP activity) in rats. IS, indoxyl sulfate; Ca, calcium; ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase, A, absorbance; Sham, sham-operated control; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID group were fed an indole-containing diet (0.5% w/w) for 4 weeks. Sham group, n = 6; sham + ID group, n = 6; PTX group, n = 8; PTX + ID group, n = 10. Data are expressed as mean ± SD. Statistical analysis was not conducted for serum IS level and serum Ca level because increases in serum IS level in the ID-treated groups and decreses in serum Ca level in the PTX groups were apparent. ⁎P b 0.05, ⁎⁎P b 0.01 vs. sham (Tukey–Kramer test).
calcein labels was suppressed slightly in the sham + ID group and decreased markedly in the PTX group compared to the sham group. Bone formation decreased further in the PTX + ID group, and calcein double-labelling was almost invisible. Fig. 4C shows light micrographs at the same locations of the upper calcein staining images. Light microscopy confirmed that osteoblasts were arranged tightly on the bone surface in the sham group. Osteoblasts in the sham + ID group decreased slightly compared to the sham group. On the other hand, osteoblasts decreased in number and arranged sparsely on the bone surface in the PTX group. The number of osteoblasts in the PTX + ID group decreased further compared to the PTX group. The results of histomorphometric analysis of the secondary spongiosa are summarized in Table 1. The PTX group showed low bone turnover indicated by a significant decrease in OS/BS and significant decreases in dynamic parameters comprising MS/BS, MAR, and BFR/BS, compared to the sham group. Moreover, significant decreases in Oc.S/BS and ES/BS were also observed in the PTX group compared to the sham group. Indole treatment resulted in further significant decreases in MS/BS, BFR/BS, and Ob.S/BS, and a tendency for a decrease in MAR compared to the PTX group. On the other hand, there were no differences in bone resorption–related parameters comprising Oc.S/BS and ES/BS between the PTX and PTX + ID groups. In the sham + ID group, Oc.S/BS tended to decrease when compared to the sham group. Correlation between serum IS levels and histomorphometric parameters for sham groups (sham and sham + ID groups) and PTX groups (PTX and PTX + ID groups) were examined. Significant correlations were observed between IS level and Oc.S/BS or ES/BS in sham and sham + ID groups, and between IS level and MS/BS, MAR, BFR/BS, or Ob.S/BS in PTX and PTX + ID groups (data not shown). Effects of indole-supplemented feeding on BMD in PTX rats The bone length and mean BMD in tibial bone are shown in Table 2. The bone lengths were similar and there were no differences in all of the sham, sham + ID, and PTX groups. Only a slight but significant decrease in bone length was observed in PTX + ID compared to the sham group.
There were no differences in bone length between the PTX and PTX + ID groups. There were no differences in BMD between the sham and PTX groups. On the other hand, BMD in the PTX + ID group apparently decreased when compared to the sham group and PTX group although significant differences were not observed.
Discussion Animal models of CKD with low bone turnover are usually produced by partial nephrectomy combined with PTX [23,30,31]. Moreover, Iwasaki et al. [23] constructed a rat model of renal failure with skeletal resistance to PTH by continuous infusion of a physiological level of 1–34 PTH in addition to PTX and nephrectomy. Thus, models commonly used for the evaluation of bone metabolism represent low bone turnover with skeletal resistance to PTH. In these models, partial nephrectomy is conducted to reproduce the pathophysiology of CKD. As a result, a variety of uremic toxins probably accumulate in the blood of these animals because of renal dysfunction. In the present study, we induced low bone turnover in rats by PTX without partial nephrectomy to avoid the effects of diverse uremic toxins. Since serum PTH in our model rats were almost undetectable, bone abnormalities are caused by PTH depletion and are not related to skeletal resistance to PTH. Our study was designed to examine the effect of IS independent of other uremic toxins by not using a CKD model and by exogenously administering indole to specifically increase blood IS level. We confirmed that serum IS levels in the sham + ID and PTX + ID groups were elevated by indole treatment. On the other hand, the resulting high IS in blood could theoretically impair the renal function of rats causing increased production of various uremic toxins that could affect BMD and other bone metabolic parameters. Indeed, IS has been reported to accelerate the progression of CKD in rats [19,22]. However, the renal function as indicated by serum creatinine was similar among the groups in the present study, indicating that renal function in the model rats were intact and that the observed effects were unlikely to be caused by uremic toxins other than IS.
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Fig. 4. Low magnification images of distal metaphysis of femur (A), calcein staining images (B) and light micrographs (C) of the secondary spongiosa. Sham, sham-operated control; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID group were fed an indole-containing diet (0.5% w/w) for 4 weeks. On days 9 and 2 before euthanization, all rats were injected subcutaneously with calcein at a dose of 8 mg/kg body weight for labelling. The femur was sectioned and stained with toluidine blue. No osteoblasts are observed in the micrograph of PTX + ID rat.
In histomorphometric analysis of the secondary spongiosa, decreases in OS/BS, MS/BS, MAR, and BFR/BS as bone formation–related parameters and Oc.S/BS and ES/BS as bone resorption–related parameters were observed in PTX rats. These findings confirmed that marked decrease or depletion of PTH in blood as a result of PTX induced low bone turnover. We demonstrated that the decreases in bone formation–related parameters observed in PTX rats were further worsened by high IS in blood, while bone resorption–related parameters did not deteriorate. Given the nature of our model, these findings strongly
suggest that IS exacerbates low bone turnover by mechanisms other than skeletal resistance to PTH. In fact, the expression of PTH/PTHrP receptor in the spongiosa of the tibia was not influenced by PTX and/ or indole treatment by quantitative RT-PCR analysis (data not shown). In addition, a significant decrease in Ob.S/BS was observed in the PTX + ID group when compared to the PTX group, but there were no differences in bone resorption–related factors including histomorphometric parameters of Oc.S/BS and ES/BS. These results suggest that IS mainly targets osteoblasts to exacerbate low bone turnover. In the
Table 1 Histomorphometric analysis. Group
Sham Sham + ID PTX PTX + ID
OS/BS
MS/BS
MAR
BFR/BS
Ob.S/BS
Oc.S/BS
ES/BS
(%)
(%)
(μm/day)
(μm3/μm2/year)
(%)
(%)
(%)
10.3 ± 0.5 10.9 ± 0.5 2.0 ± 0.4† 0.9 ± 0.4††,##
25.1 ± 5.3 20.4 ± 4.6 5.1 ± 1.5† 1.9 ± 1.5 ††, #
2.2 ± 0.4 2.0 ± 0.3⁎ 1.0 ± 0.2⁎⁎ 0.7 ± 0.2⁎⁎
201.7 ± 63.3 149.0 ± 46.4 19.3 ± 6.4† 5.6 ± 5.5††,#
13.8 ± 3.5 11.0 ± 2.1 10.4 ± 2.6 5.5 ± 0.8††,##
13.9 ± 2.5 10.5 ± 2.6 7.9 ± 2.6† 8.1 ± 1.8†
10.4 ± 1.6 7.2 ± 2.2 5.1 ± 1.8† 4.2 ± 0.9††
OS/BS, osteoid surface per bone surface; MS/BS, mineralized surface per bone surface; MAR, mineral apposition rate; BFR/BS, bone formation rate per bone surface; Ob.S/BS, osteoblast surface per bone surface; Oc.S/BS, osteoclast surface per bone surface; ES/BS, eroded surface per bone surface; Sham, sham-operated control; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID group were fed an indole-containing diet for 4 weeks. Sham group, n = 6; sham + ID group, n = 6; PTX group, n = 8; PTX + ID group, n = 10. ⁎ P b 0.05. ⁎⁎ P b 0.01 vs. sham group (Tukey–Kramer test). † P b 0.05. †† P b 0.01 vs. sham group (Steel–Dwass test). # P b 0.05. ## P b 0.01 vs. PTX group (Steel–Dwass test).
J. Hirata et al. / Bone 79 (2015) 252–258 Table 2 Bone mineral density of tibial bone. Group
Sham Sham + ID PTX PTX + ID
Bone length
BMD
(mm)
(mg/cm2)
44.9 ± 1.0 44.7 ± 1.2 43.8 ± 1.4 43.4 ± 0.4†
114.5 ± 3.6 115.3 ± 4.8 114.7 ± 4.9 109.7 ± 3.8
BMD, bone mineral density; Sham, sham-operated control; Sham + ID, sham-operated and indole treatment; PTX, parathyroidectomy; ID, indole treatment. Sham + ID group and PTX + ID groups were fed an indole-containing diet for 4 weeks. Sham group, n = 5; sham + ID group, n = 6; PTX group, n = 8; PTX + ID group, n = 9. † P b 0.05 vs. sham group (Steel–Dwass test).
sham + ID group, decreases in bone formation–related parameters were not observed in histomorphometric analysis. The difference in the effect on bone formation between sham + ID group and PTX + ID group was considered to be caused by the differences in serum PTH level between these groups. Nii-Kono et al. [24] have suggested that IS is taken by primary cultured osteoblasts via organic anion transporter-3 (OAT-3) present in these cells, where it augments free radical production inside the cells and induces osteoblast dysfunction including decreased expression of PTH receptor. Another study has shown that IS transported via OAT-3 in MC3T3-E1 cells increases free radical production and inhibits osteoblast differentiation [32]. In addition, IS has been shown to increase free radicals in proximal tubular cells [33], in the kidney [34], and in endothelial cells [35]. These findings suggest that IS may act directly on osteoblasts by increasing free radical production after being taken up by osteoblasts via OAT-3. A team in our research group examined the direct effects of IS on bone formation and expression of related genes. In cultures of mouse primary osteoblasts, IS dose-dependently suppressed both mineralization of osteoblasts and mRNA expression of bone formation–related genes such as BMP2, Col1α1, and osteocalcin (Matsumoto et al., unpublished data). Therefore, IS may act directly on osteoblasts and inhibit bone formation. In an in vitro study, Mozar et al. [36] reported that IS inhibited both osteoclast differentiation and bone-resorbing activity induced by receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor. The indole treatment slightly suppressed Oc.S/BS in sham rats in the present study, although there were no significant differences (Table 1). This suggests that IS may act on osteoclasts. On the other hand, indole treatment in PTX rats did not affect the histomorphometric parameters of bone resorption (Table 1) and serum TRAP activity (Fig. 3F) in the present study. It is possible that RANKL expression was reduced accompanying the reduction in number of osteoblasts in our PTX animals, because the animals showed a decrease in Ob.S/BS. Moreover, an analysis of the relationship between IS and biochemical markers of bone turnover in 47 hemodialysis patients reported that serum IS level correlated with bone-specific alkaline phosphatase (r = − 0.34) but did not correlate with serum TRAP 5b level [25]. It may be difficult to confirm the effect of IS on bone resorption under low bone turnover conditions. Serum IS levels reported in CKD model rats were approximately 4.1 mg/dL in adenine-induced CKD rats [28] and 1.2 mg/dL in 5/6 nephrectomized rats [29]. Niwa et al. [19] reported that serum IS level was 1.8 ± 1.5 mg/dL in predialysis CKD patients and 5.3 ± 2.1 mg/dL in patients on dialysis (measured before dialysis session). In our study, the mean serum IS levels in PTX + ID treatment group on weeks 2 and 4 of indole treatment were 5.1 ± 0.9 mg/dL and 3.9 ± 1.2 mg/dL, respectively. Thus, the effects of IS on low bone turnover were observed at serum levels of IS in CKD rat models as well as in CKD patients and patients on dialysis, suggesting that IS may play a role in the progression of low-turnover bone disease associated with CKD. On the other hand, various uremic toxins increase in the blood of uremic patients [14,15], and in vitro studies have shown that some of these uremic toxins such
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as p-cresyl sulfate [37] and phenylacetic acid [38] inhibit osteoblast differentiation. Hence, further studies are required to examine whether other uremic toxins may also contribute to progression of low-turnover bone disease in patients with CKD or on dialysis similar to IS. In conclusion, the present study demonstrated for the first time that IS exacerbates low bone turnover through inhibition of bone formation by mechanisms unrelated to skeletal resistance to PTH in rats. Our data suggest that IS may be one of the uremic toxins that contribute to the progression of low-turnover bone disease in patients with CKD or on dialysis whose serum IS levels are elevated because of renal dysfunction. Further studies are warranted to examine uremic toxins other than IS, which impact bone turnover, with the objective to explore potential therapeutic targets for treating low-turnover disease in these patients. Competing interest statement The authors declare no competing interests relevant to this work. Acknowledgments We thank Rie Takagi, Sayaka Seki, Misaki Miyamoto, and Ayumi Hirano for technical assistance, and Kaori Kikuchi for assistance with measurement of serum IS levels. References [1] S. Moe, T. Drüeke, J. Cunningham, W. Goodman, K. Martin, K. Olgaard, et al., Kidney Disease: Improving Global Outcomes (KDIGO). Definition, evaluation, and classification of renal osteodystrophy: A position statement from Kidney Disease: Improving Global Outcomes (KDIGO), Kidney Int 69 (2006) 1945–1953. [2] Kidney Disease: Improving Global Outcomes(KDIGO)CKD-MBD Work Group, KDIGO clinical practice guideline for the diagnosis, evaluation, prevention and treatment of Chronic Kidney Disease-Mineral and Bone Disorder(CKD-MBD), Kidney Int 76 (Suppl. 113) (2009) S1–S130. [3] H.H. Malluche, H.W. Mawad, M.C. Monier-Faugere, Renal osteodystrophy in the first decade of the new millennium: Analysis of 630 bone biopsies in black and white patients, J Bone Miner Res 26 (2011) 1368–1376. [4] A. Fournier, P. Morinière, M.E. Cohen Solal, B. Boudailliez, J.M. Achard, A. Marie, et al., Adynamic bone disease in uremia: May it be idiopathic? Is it an actual disease? Nephron 58 (1991) 1–12. [5] I.B. Salusky, W.G. Goodman, Adynamic renal osteodystrophy: Is there a problem? J Am Soc Nephrol 12 (2001) 1978–1985. [6] M.M. Couttenye, P.C. D'Haese, W.J. Verschoren, G.J. Behets, I. Schrooten, M.E. De Broe, Low bone turnover in patients with renal failure, Kidney Int Suppl 73 (1999) S70–S76. [7] A.J. Hutchison, R.W. Whitehouse, H.F. Boulton, J.E. Adams, E.B. Mawer, T.J. Freemont, et al., Correlation of bone histology with parathyroid hormone, vitamin D3, and radiology in end-stage renal disease, Kidney Int 44 (1993) 1071–1077. [8] S.G. Massry, R. Stein, J. Garty, A.I. Arieff, J.W. Coburn, A.W. Norman, et al., Skeletal resistance to the calcemic action of parathyroid hormone in uremia: role of 1,25 (OH)2 D3, Kidney Int 9 (1976) 467–474. [9] G. Coen, S. Mazzaferro, P. Ballanti, D. Sardella, S. Chicca, M. Manni, et al., Renal bone disease in 76 patients with varying degrees of predialysis chronic renal failure: a cross-sectional study, Nephrol Dial Transplant 11 (1996) 813–819. [10] D.J. Sherrard, G. Hercz, Y. Pei, N.A. Maloney, C. Greenwood, A. Manuel, et al., The spectrum of bone disease in end-stage renal failure—an evolving disorder, Kidney Int 43 (1993) 436–442. [11] A. Torres, V. Lorenzo, D. Hernández, J.C. Rodríguez, M.T. Concepción, A.P. Rodríguez, et al., Bone disease in predialysis, hemodialysis, and CAPD patients: evidence of a better bone response to PTH, Kidney Int 47 (1995) 1434–1442. [12] M.D. Danese, J. Kim, Q.V. Doan, M. Dylan, R. Griffiths, G.M. Chertow, PTH and the risks for hip, vertebral, and pelvic fractures among patients on dialysis, Am J Kidney Dis 47 (2006) 149–156. [13] M. Naves-Díaz, J. Passlick-Deetjen, A. Guinsburg, C. Marelli, J.L. Fernández-Martín, D. Rodríguez-Puyol, et al., Calcium, phosphorus, PTH and death rates in a large sample of dialysis patients from Latin America. The CORES Study, Nephrol Dial Transplant 26 (2011) 1938–1947. [14] R. Vanholder, R. De Smet, G. Glorieux, A. Argilés, U. Baurmeister, P. Brunet, et al., Review on uremic toxins: Classification, concentration, and interindividual variability, Kidney Int 63 (2003) 1934–1943. [15] R. Vanholder, G. Glorieux, R. De Smet, N. Lameire, European Uremic Toxin Work Group, New insights in uremic toxins, Kidney Int Suppl 63 (2003) S6–S10. [16] R. Vanholder, S. Van Laecke, G. Glorieux, What is new in uremic toxicity? Pediatr Nephrol 23 (2008) 1211–1221. [17] R. Vanholder, U. Baurmeister, P. Brunet, G. Cohen, G. Glorieux, J. Jankowski, et al., A bench to bedside view of uremic toxins, J Am Soc Nephrol 19 (2008) 863–870. [18] L.J. King, D.V. Parke, R.T. Williams, The metabolism of [2-14C] indole in the rat, Biochem J 98 (1966) 266–277.
258
J. Hirata et al. / Bone 79 (2015) 252–258
[19] T. Niwa, M. Ise, Indoxyl sulfate, a circulating uremic toxin, stimulates the progression of glomerular sclerosis, J Lab Clin Med 124 (1994) 96–104. [20] F.C. Barreto, D.V. Barreto, S. Liabeuf, N. Meert, G. Glorieux, M. Temmar, et al., Serum indoxyl sulfate is associated with vascular disease and mortality in chronic kidney disease patients, Clin J Am Soc Nephrol 4 (2009) 1551–1558. [21] K. Atoh, H. Itoh, M. Haneda, Serum indoxyl sulfate levels in patients with diabetic nephropathy: Relation to renal function, Diabetes Res Clin Pract 83 (2009) 220–226. [22] T. Niwa, M. Ise, T. Miyazaki, Progression of glomerular sclerosis in experimental uremic rats by administration of indole, a precursor of indoxyl sulfate, Am J Nephrol 14 (1994) 207–212. [23] Y. Iwasaki, H. Yamato, T. Nii-Kono, A. Fujieda, M. Uchida, A. Hosokawa, et al., Administration of oral charcoal adsorbent (AST-120) suppresses low-turnover bone progression in uraemic rats, Nephrol Dial Transplant 21 (2006) 2768–2774. [24] T. Nii-Kono, Y. Iwasaki, M. Uchida, A. Fujieda, A. Hosokawa, M. Motojima, et al., ndoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells, Kidney Int 71 (2007) 738–743. [25] S. Goto, H. Fujii, Y. Hamada, K. Yoshiya, M. Fukagawa, Association between indoxyl sulfate and skeletal resistance in hemodialysis patients, Ther Apher Dial 14 (2010) 417–423. [26] K. Kikuchi, Y. Itoh, R. Tateoka, A. Ezawa, K. Murakami, T. Niwa, Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography/tandem mass spectrometry, J Chromatogr B Analyt Technol Biomed Life Sci 878 (2010) 2997–3002. [27] A.M. Parfitt, M.K. Drezner, F.H. Glorieux, J.A. Kanis, H. Malluche, P.J. Meunier, et al., Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee, J Bone Miner Res 2 (1987) 595–610. [28] Y. Inami, C. Hamada, T. Seto, Y. Hotta, S. Aruga, J. Inuma, et al., Effect of AST-120 on endothelial dysfunction in adenine-induced uremic rats, Int J Nephrol 164125 (2014). http://dx.doi.org/10.1155/2014/164125. [29] A. Enomoto, M. Takeda, A. Tojo, T. Sekine, S.H. Cha, S. Khamdang, et al., Role of organic anion transporters in the tubular transport of indoxyl sulfate and the induction of its nephrotoxicity, J Am Soc Nephrol 13 (2002) 1711–1720.
[30] Y. Iwasaki-Ishizuka, H. Yamato, T. Nii-Kono, K. Kurokawa, M. Fukagawa, Downregulation of parathyroid hormone receptor gene expression and osteoblastic dysfunction associated with skeletal resistance to parathyroid hormone in a rat model of renal failure with low turnover bone, Nephrol Dial Transplant 20 (2005) 1904–1911. [31] J.C. Ferreira, G.O. Ferrari, K.R. Neves, R.T. Cavallari, W.V. Dominguez, L.M. Dos Reis, et al., Effects of dietary phosphate on adynamic bone disease in rats with chronic kidney disease - role of sclerostin? PLoS One 8 (2013) e79721. [32] Y.H. Kim, K.A. Kwak, H.W. Gil, H.Y. Song, S.Y. Hong, Indoxyl sulfate promotes apoptosis in cultured osteoblast cells, BMC Pharmacol Toxicol 14 (2013) 60. [33] M. Motojima, A. Hosokawa, H. Yamato, T. Muraki, T. Yoshioka, Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-κB and free radical in proximal tubular cells, Kidney Int 63 (2003) 1671–1680. [34] S. Owada, S. Goto, K. Bannai, H. Hayashi, F. Nishijima, T. Niwa, Indoxyl sulfate reduces superoxide scavenging activity in the kidneys of normal and uremic rats, Am J Nephrol 28 (2008) 446–454. [35] L. Dou, N. Jourde-Chiche, V. Faure, C. Cerini, Y. Berland, F. Dignat-George, et al., The uremic solute indoxyl sulfate induces oxidative stress in endothelial cells, J Thromb Haemost 5 (2007) 1302–1308. [36] A. Mozar, L. Louvet, C. Godin, R. Mentaverri, M. Brazier, S. Kamel, et al., Indoxyl sulphate inhibits osteoclast differentiation and function, Nephrol Dial Transplant 27 (2012) 2176–2181. [37] H. Tanaka, Y. Iwasaki, H. Yamato, Y. Mori, H. Komaba, H. Watanabe, et al., p-Cresyl sulfate induces osteoblast dysfunction through activating JNK and p38 MAPK pathways, Bone 56 (2013) 347–354. [38] S. Yano, T. Yamaguchi, I. Kanazawa, N. Ogawa, K. Hayashi, M. Yamauchi, et al., The uraemic toxin phenylacetic acid inhibits osteoblastic proliferation and differentiation: An implication for the pathogenesis of low turnover bone in chronic renal failure, Nephrol Dial Transplant 22 (2007) 3160–3165.