Department of Nephrology and Hypertension, Meir Medical Center, Kfar Saba, Israel.
J Steroid Biochem Mol Biol. 2011 Mar;124(1-2):19-24. doi: 10.1016/j.jsbmb.2011.01.001. Epub 2011 Jan 12.
In advanced chronic kidney disease (CKD), hypocalcemia, high levels of advanced glycation end products (AGEs), and parathyroid hormone (PTH) coexist and are considered to play a role in the development of chronic vasculopathies. The aim of the present study was to evaluate the impact of a CKD-like environment on cultured endothelial cell (EC) functions and to assess the impact of calcitriol on the expression of parameters such as endothelial nitric oxide synthase (eNOS), receptor of AGEs (RAGE), interleukin 6 (IL-6) and nuclear factor kappa B (NFκB). Human umbilical vein cord endothelial cells (HUVEC) were grown in medium containing low Ca(2+) concentration stimulated with AGE-HSA and PTH and treated with calcitriol for additional incubation. mRNA expression was established by reverse transcriptase-PCR, protein expression by Western blot analysis, IL-6 secretion by ELISA, NOS activity by conversion of [(14)C]arginine to [(14)C]citrulline and DNA-binding activity of NFκB-p65 assayed colorimetrically in nuclear extracts. The CKD-like environment characterized by the association of low Ca(2+) and high levels of AGEs and PTH, depressed eNOS system activity and enhanced RAGE and IL-6 expression/secretion. DNA-binding activity of nuclear NFκB-p65 was increased and the expression of IκBα decreased. Addition of calcitriol normalized the expression, secretion and activity of eNOS, RAGE and IL-6. The enhanced NFκB activity was also counteracted probably due to the increased IκBα expression. The effect of CKD-like environment on EC may partly explain the increased vasculopathies in CKD patients, in contrast to calcitriol, which suggests a vascular protective action.
在慢性肾脏病（CKD）晚期，低钙血症、晚期糖基化终产物（AGEs）水平升高和甲状旁腺激素（PTH）共存，并被认为在慢性血管病变的发展中起作用。本研究旨在评估 CKD 样环境对培养的内皮细胞（EC）功能的影响，并评估骨化三醇对内皮型一氧化氮合酶（eNOS）、AGEs 受体（RAGE）、白细胞介素 6（IL-6）和核因子 κB（NFκB）等参数表达的影响。将人脐静脉内皮细胞（HUVEC）在含有低钙浓度的培养基中培养，并用 AGE-HSA 和 PTH 刺激，并在额外的孵育中用骨化三醇处理。通过逆转录聚合酶链反应（RT-PCR）确定 mRNA 表达，通过 Western blot 分析确定蛋白质表达，通过 ELISA 测定 IL-6 分泌，通过核提取物中[(14)C]精氨酸转化为[(14)C]瓜氨酸测定 NOS 活性，通过比色法测定核 NFκB-p65 的 DNA 结合活性。由低钙和高水平 AGEs 和 PTH 联合引起的 CKD 样环境特征为，eNOS 系统活性降低，RAGE 和 IL-6 表达/分泌增强。核 NFκB-p65 的 DNA 结合活性增加，IκBα 的表达减少。添加骨化三醇可使 eNOS、RAGE 和 IL-6 的表达、分泌和活性正常化。NFκB 活性的增强也可能由于 IκBα 的表达增加而受到抑制。CKD 样环境对 EC 的影响部分解释了 CKD 患者血管病变增加的原因，而骨化三醇则提示具有血管保护作用。
