Elsevier

Bone

Volume 79, October 2015, Pages 252-258
Bone

Original Full Length Article
Indoxyl sulfate exacerbates low bone turnover induced by parathyroidectomy in young adult rats

https://doi.org/10.1016/j.bone.2015.06.010Get rights and content

Highlights

  • Bone abnormalities are often observed in patients with chronic kidney disease (CKD).

  • Low bone turnover model rats were produced by parathyroidectomy (PTX).

  • Rats were fed a diet containing indole to elevate blood level of indoxyl sulfate, an indole metabolite.

  • Indole treatment further decreased the low bone formation observed in PTX rats.

  • The uremic toxin indoxyl sulfate exacerbates low bone turnover by mechanisms other than skeletal resistance to parathyroid hormone.

Abstract

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.

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], [6], [7] and skeletal resistance to parathyroid hormone (PTH) [5], [8], [9]. Analyses of the prevalence of bone turnover abnormalities in 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 protein-bound 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], [20], [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 progression of low-turnover bone disease and reverses the down-regulation 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 indole-supplemented 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.

Section snippets

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

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

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

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.

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