| Background and objectives:The mechanism of high-turnover renal osteodystrophy (ROD) has not been fully clear to date. The differentiation from bone marrow mesenchymal stem cells (BM-MSC) to osteoblast is the biological foundation of bone transformation, which was enhanced in high-turnover ROD uremic rat. In the present study, we cultured primarily BM-MSC from high-turnover ROD uremic rat in vitro and and identified the characteristics and the signaling pathways involved in its proliferation and differentiation.Method:High-turnover ROD uremic models were made with male Sprague-Dawley rats (body weight180-200g) by5/6nephrectomy fed a high phosphate diet (1.2%phosphorus and1.0%calcium). Male body-weight-matched rats were fed with normal phosphorus diet (0.9%phosphorus and1.0%calcium) as control group. After12weeks feeding, all rats were executed and BM-MSC was isolated from femur and tibia bone and cultured primarily by adherence method. The3rd passage cells were identified for BM-MSC molecular markers (CD90+/CD34-/CD44+/CD45-) by flow cytometry. The cell viability was investigated by MTT assay, and the proliferation level was measured by propidium iodide DNA staining by flow cytometry. The signaling pathway involved was detected by immunohistochemistry and western blot. Then BM-MSC was induced to differentiate to osteoblast in culture medium containing glycerin and dexamethasone phosphate. The differences in the level of cell proliferation, the degree of ERK1/2protein phosphorylation, and the expression of specific osteoblast markers alkaline phosphatase (ALP), osteopontin (OPN), the calcium phosphate deposits (von Kossa staining) and the number of bone nodule (HE staining) were investigated between high-turnover ROD uremic group and the control group.Results:(1) BM-MSC from the high-turnover ROD rats was separated and cultured by adherence method. The3rd passage cells were identified as CD90+/CD34-/CD44+/CD45-cells by flow cytometry. Flow cytometry analysis of DNA showed the proportion of G1phase, S phase and G2/M phase cells in the BM-MSC from uremic group was79.55±3.66%,20.12±1.96%and0.33±0.10%, respectively, while84.62±3.10%,15.13±0.87%and0.25±0.11%in the BM-MSC from control group, respectively. The proportion of S phase cells was significantly higher in uremic group than that in control group (P＜0.05), which were consistent with the results of MTT assays.(2) ERK1/2signaling pathway of the cultured BM-MSC was activated obviously, characterized by p-ERK1/2increased significantly (p＜0.05). Western blot showed that the ERK1/2pathway of control group was activated at the72h point and was maintained behind for24h, whereas the activation in uremic group began at the48h point and was maintained behind for as long as48h. The p-ERK1/2level in uremic group was higher than that in control group at all time points from48h to96h (P＜0.05). Flow cytometry and MTT assay showed that BM-MSC proliferation in both groups decreased significantly after adding of ERK1/2blockers, and the proportion of S phase cells decreased more significantly in uremic group than in control group (P＜0.05).(3) Compared with BM-MSC from control group, BM-MSC from uremic group expressed ALP more significantly1week after differentiation to osteoblast, similarly, for OPN (P＜0.05) and calcium phosphate deposits (P＜0.05) after2weeks, and for the formation of bone nodule after3weeks (P＜0.05).Conclusions:BM-MSC from uremic rat with high-turnover ROD survives in vitro, with an enhanced proliferation and differentiation ability in which the activation of ERK1/2might involed. This study might be valuable for preventing and treating uremia with high-turnover ROD. |
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