Abstract
Abnormalities of bone turnover are commonly observed in patients with chronic kidney disease (CKD), and the low‐turnover bone disease is considered to be associated with low serum parathyroid hormone (PTH) levels and skeletal resistance to PTH. Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood of patients with CKD. Recently, we have reported that IS exacerbates low bone turnover induced by parathyroidectomy (PTX) in adult rats, and suggested that IS directly induces low bone turnover through the inhibition of bone formation by mechanisms unrelated to skeletal resistance to PTH. To define the direct action of IS in bone turnover, we examined the effects of IS on bone formation and bone resorption in vitro. In cultures of mouse primary osteoblasts, IS suppressed the expression of osterix, osteocalcin, and bone morphogenetic protein 2 (BMP2) mRNA and clearly inhibited the formation of mineralized bone nodules. Therefore, IS directly acts on osteoblastic cells to suppress bone formation. On the other hand, IS suppressed interleukin (IL)‐1‐induced osteoclast formation in cocultures of bone marrow cells and osteoblasts, and IL‐1‐induced bone resorption in calvarial organ cultures. In cultures of osteoblasts, IS suppressed the mRNA expression of RANKL, the receptor activator of NF‐κB ligand, which is a pivotal factor for osteoclast differentiation. Moreover, IS acted on osteoclast precursor, bone marrow‐derived macrophages and RAW264.7 cells, and suppressed RANKL‐dependent differentiation into mature osteoclasts. IS may induce low‐turnover bone disease in patients with CKD by its direct action on both osteoblasts and osteoclast precursors to suppress bone formation and bone resorption.
Highlights
Abnormalities of bone turnover are commonly observed in patients with chronic kidney disease (CKD), and the low-turnover bone disease is considered to be associated with low serum parathyroid hormone (PTH) levels and skeletal resistance to PTH
The primers used for the quantitative PCR (q-PCR) for the mouse RANK ligand (RANKL), osterix, Col1a1, osteocalcin, bone morphogenetic protein 2 (BMP2), Nfatc1, receptor activator of NF-κB (RANK), and tartrate-resistant acid phosphatase (TRAP) genes were constructed from the sequence of respective gene
Using CKD rat models, previous studies have indicated that an increase in Indoxyl sulfate (IS) in the blood is related to glomerular sclerosis, renal fibrosis, and the progression of CKD in rats [18,21]
Summary
Newborn and six-week-old ddy mice were obtained from Japan SLC Inc. (Shizuoka, Japan). Osteoblastic cells were collected from fractions 2-4 and combined, and cultured for 3 days in aMEM with 10% FBS under 5% CO2 in air at 37 °C. Primary osteoblastic cells were cultured for 14 days in a medium containing bone-inducing factors, ascorbic acid. A medium without bone-inducing factors was used. The areas of alizarin-positive cells were defined as mineralized bone nodules. Total RNA was extracted from mouse osteoblasts and from RAW264.7 cells using ISOGEN (Nippon Gene, Tokyo, Japan). The primers used for the q-PCR for the mouse RANKL, osterix, Col1a1, osteocalcin, BMP2, Nfatc, RANK, and TRAP genes were constructed from the sequence of respective gene. The RT-PCR was performed to examine the mRNA expression of organic anion transporters (OATs), OAT1 and OAT3, in mouse osteoblasts, bone marrow macrophages, and RAW264.7 cells. Statistical analyses were performed using IBM SPSS Statistics version 23 software
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