Calcium ions promote primary renal epithelial cell differentiation into cells with bone-associated phenotypes via transforming growth factor-β1-induced epithelial-mesenchymal transition in idiopathic hypercalciuria patients
Calcium ions promote primary renal epithelial cell differentiation into cells with bone-associated phenotypes via transforming growth factor-β1-induced epithelial-mesenchymal transition in idiopathic hypercalciuria patients
复制标题
DOI：
10.3892/mmr.2014.2941
复制
发表时间：
2015-03-01
影响因子：
3.4
通讯作者：
Qin, Baolong
中科院分区：
文献类型：
--
作者：
He, Deng;Wang, Shaogang;Qin, Baolong
本研究旨在确定特发性高钙尿症（IH）肾结石患者中转化生长因子β1（TGF-β1）和钙离子之间的特征和交互作用，以阐明骨相关因子诱导细胞表型变化的潜在机制及其对肾肾结石形成的影响。采用 ELISA 法对 29 例伴 IH 的肾结石患者、29 例无 IH 的肾结石患者和 29 名健康年龄匹配的正常对照的血样进行外周血清 TGF-β 1、骨桥蛋白 (OPN) 和骨形态发生蛋白 2 (BMP2) 的定量。随后使用逆转录定量聚合酶链反应 (RT-qPCR) 和蛋白质印迹分析检测 IH 和 HK-2 人近端肾小管细胞系（对照）的原代肾上皮细胞 (PREC) 中的 BMP2、OPN 和 1,25-二羟基维生素 D-3 受体 (VDR) mRNA 和蛋白质水平。使用 RT-qPCR 分别评估不同浓度的 TGF-β 1（0.5、2.0 和 5.0 ng/ml）、Ca2+（0.5、1.5 和 2.5 mM）或 TGF-β 1 和 Ca2+ 组合刺激后 PREC 和 HK-2 中 BMP2、OPN 和 VDR 的 mRNA 表达水平。 IH患者TGF-β1、BMP2、OPN的表达水平均显着高于对照组。 PRECs 中 BMP2 和 VDR 的 mRNA 和蛋白表达水平显着高于 HK-2 细胞。与TGF-β1和/或Ca2+一起孵育后，PRECs中BMP2、OPN和VDR的mRNA表达水平以剂量依赖性方式增加；然而，随着TGF-β1剂量的增加，HK-2细胞中没有观察到显着差异。在 PREC 和 HK-2 细胞系中与 TGF-β 1 和 Ca2+ 共孵育会产生类似的效果，并且 BMP2、OPN 和 VDR mRNA 的表达以时间依赖性方式增加。总之，本研究结果表明TGF-β1调节PRECs中BMP2、OPN和VDR的表达，但不调节HK-2细胞中的BMP2、OPN和VDR的表达。 TGF-β1和Ca2+共孵育显着增加了PRECs和HK-2细胞中骨相关因子的表达水平，这表明该过程可能是钙结石形成的发病机制的部分原因，并且还与骨形成和TGF-β1诱导的上皮间质转化有关。
The present study aimed to identify the characteristics and cross-talk between transforming growth factor beta 1 (TGF-beta 1) and calcium ions in nephrolithiasis patients with idiopathic hypercalciuria (IH) in order to elucidate the potential mechanisms underlying changes in cell phenotype induced by bone-associated factors and their influence on renal nephrolithiasis formation. Blood samples from a total of 29 nephrolithiasis patients with IH, 29 renal stone patients without IH and 29 healthy age-matched normal controls were subjected to quantification of peripheral serum TGF-beta 1, osteopontin (OPN) and bone morphogenetic protein 2 (BMP2) using ELISA. This was followed by detection of BMP2, OPN and 1,25-dihydroxyvitamin D-3 receptor (VDR) mRNA and protein levels in primary renal epithelial cells (PRECs) of IH and HK-2 human proximal tubular cell lines (control) using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses. The mRNA expression levels of BMP2, OPN and VDR in PRECs and HK-2 were evaluated following stimulation with various concentrations of TGF-beta 1 (0.5, 2.0 and 5.0 ng/ml), Ca2+ (0.5, 1.5 and 2.5 mM) or TGF-beta 1 and Ca2+ combined using RT-qPCR, respectively. TGF-beta 1, BMP2 and OPN expression levels in patients with IH were all significantly higher than those in the control group. The mRNA and protein expression levels of BMP2 and VDR were significantly higher in PRECs than those in HK-2 cells. Following incubation with TGF-beta 1 and/or Ca2+, the mRNA expression levels of BMP2, OPN and VDR in PRECs increased in a dose-dependent manner; however, no significant differences were observed in HK-2 cells with increasing TGF-beta 1 dosage. Co-incubation with TGF-beta 1 and Ca2+ in PRECs and HK-2 cell lines resulted in similar effects and the expression of BMP2, OPN and VDR mRNA increased in a time-dependent manner. In conclusion, the results of the present study demonstrated that TGF-beta 1 regulated the expression of BMP2, OPN and VDR in PRECs, but not in HK-2 cells. Co-incubation with TGF-beta 1 and Ca2+ significantly increased the expression levels of bone-associated factors in PRECs and HK-2 cells, which suggested that this process may be partially responsible for the pathogenesis of calcium stone development, and also associated with bone formation and the TGF-beta 1-induced epithelial to mesenchymal transition.