Osteoblastic differentiation of bone marrow mesenchymal stem cells in uremic rats

https://doi.org/10.1016/j.bbrc.2020.05.096Get rights and content

Highlights

  • Osteoblastic differentiation of mesenchymal stem cell from chronic kidney disease rat.

  • Secondary hyperparathyroidism represents a high turnover bone disease in vivo.

  • Parathyroid hormone little affect differentiation of mesenchymal stem cell in vitro.

  • Chronic kidney disease may affect differentiation potential of mesenchymal stem cells.

Abstract

Severe secondary hyperparathyroidism (SHPT) represents a high turnover bone disease, osteitis fibrosa, but the pathogenesis of osteitis fibrosa remains to be fully elucidated. We examined the characteristics of the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts in uremic rats.

We bred 5/6 nephrectomized (Nx) rats with a high phosphorus (P) diet to induce SHPT (Nx + HP), or Nx (Nx + ND) and normal rats (Nc + ND) fed a standard diet (ND). After 8 weeks, BMSCs were isolated from the femur and serum were analyzed. BMSCs underwent flow cytometric examination for the expression patterns of cell surface markers (CD90+, CD29+, CD45, and CD31).

Serum creatinine (Cre) levels were significantly elevated in the Nx + NP rats compared with the Nc + NP rats. Cre levels in the Nx + HP rats were levels to those in the Nx + ND rats. Serum P and PTH levels were significantly elevated in the Nx + HP rats compared with the Nx + ND rats. Bone morphometrical analysis showed increases in both osteoid volume and eroded surfaces in the Nx + HP but not in the Nx + ND rats. The populations of harvested BMSCs were similar between all three groups. Alp, Runx2, Pth1r and Cyclin D1 mRNA expression in the BMSCs from the Nx + ND rats were significantly suppressed compared with those isolated from the Nc + ND groups. Alizarin red staining tended to be similar to the expression of these mRNA. These results suggest that the BMSCs differentiation into osteoblasts was disturbed in the uremic rats.

Introduction

Chronic kidney disease (CKD) is often complicated by disturbances in mineral metabolism that lead to the development of renal osteopathy, which is characterized by increased bone loss and bone fractures [1]. Serum parathyroid hormone (PTH) is elevated in CKD patients, and severe secondary hyperparathyroidism (SHPT) represents a high turnover bone disease that results in reduced bone density and increased risk of bone fracture [2,3]. The bone and mineral injury is referred to as chronic kidney disease-mineral and bone disorder (CKD-MBD), which frequently affects the quality of life, morbidity and mortality [4].

Bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells that have the ability to differentiate into osteoblasts and other cellular types, such as chondrocytes and adipocytes [5]. Osteogenesis takes place via a multi-step differentiation cascade that is thought to originate from multipotent mesenchymal cells, proceeding through osteoprogenitors and osteoblasts to generate mature bone osteocytes [6]. Whether MSCs proliferate as multipotent stem cells capable of differentiating in multiple mesenchymal lineages, or as committed progenitors of a particular lineage, is likely regulated by numerous growth factors, cytokines and hormones [7].

Many previous studies have shown that CKD with SHPT leads to increased progression of cardiovascular disease, vascular calcification, endothelial dysfunction, disorders of mineral and bone metabolism, including bone fracture, and a higher mortality rate [[8], [9], [10]]. However, it is unclear how the osteogenic differentiation of BMSCs can be achieved in the state of CKD and SHPT. In this study, we examined the characteristics of the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts in uremic rats with SHPT.

Section snippets

Animals

Study protocols were approved by the Showa University Animal Studies Committee in accordance with federal regulations. Eight-week-old male Sprague Dawley rats weighing 225–250 g were randomly assigned to three groups: (1) normal rats fed a standard diet (Nc + ND; n = 7), (2) 5/6 nephrectomized rats fed a standard diet (Nx + ND; n = 7) and (3) the 5/6 nephrectomized rats fed a high phosphorus diet (Nx + HP: n = 12). The standard diet contained 0.6% phosphate and the high phosphorus diet

Renal function and biochemical parameters

The biochemical data of the rats at 18 weeks of age are shown in Fig. 1. Compared with the Nc + ND rats, rats in the Nx + ND and Nx + HP groups showed significantly increased serum creatinine levels. Serum inorganic phosphate was higher level in the Nx + HP rats than that in the Nc + ND or Nx + ND rats. In contrast, serum calcium levels were lower level in the Nx + HP rats than in the Nc + ND or Nx + ND rats. Intact PTH and intact FGF-23 levels showed similar trends.

Bone histology and histomorphometry

Histological examination of

Discussion

The present study demonstrated that osteoblastic differentiation of MSCs from CKD model rats was suppressed compared with MSCs from normal rats. Furthermore, MSCs from SHPT model rats may exhibit even greater suppression of osteoblastic differentiation. In this study, MSCs from all groups of rats were incubated in the same osteogenic medium when osteoblastic differentiation of the MSCs was assessed.

In CKD, there is a broad range of bone turnover phenotypes, which are often categorized

Author contributions

TK, MM, KS, AY, HO, HH, AS and RK: were involved in the study concept. TK, MM and KS: were involved in the formal analysis; TK, MM, AY, HO, AS and RK: acquired funding; TK: investigated the study; TK, MM and RK: applied the methodology for the study; MM and HO: project administration; MM and HO: collection of the resources; MM and HO: supervision and validation of the study; TK: wrote the original manuscript; TK, MM, HO, HH and RK: reviewed and edited the original manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was supported in part by Grant-in-Aid for Young Scientists and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (19K17753, 19H03820, 18K19655, 18K09512, 19K18950, 18K08253, 17K09737), and by Grant for pathophysiological research conference in chronic kidney disease from the Kidney Foundation, Japan (JKFB18-17).

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1

Present address: Department of Internal Medicine, Showa University Northern Yokohama Hospital, 35-1 Chigasakichuo, Yokohama, Kanagawa 224–8503, Japan.

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