目的: 探讨Klotho mRNA及蛋白在高磷诱导的血管平滑肌细胞（vascular smooth muscle cells, VSMCs）钙化中的表达变化。方法: 体外培养大鼠原代VSMCs,利用细胞计数试剂-8（cell counting kit-8, CCK-8）试剂盒检测不同浓度磷刺激条件下 VSMCs的增殖情况。高磷培养基（3 mmol/L）刺激细胞3 d或6 d后,采用茜素红染色评估VSMCs钙化情况,用碱性磷酸酶（alkaline phosphatases, ALP）试剂盒和钙含量试剂盒分别检测细胞内ALP活性和钙含量,实时荧光定量聚合酶链反应（polymerase chain reaction, PCR）和蛋白印迹法检测钙化相关蛋白骨形态发生蛋白2（bone morphogenetic protein 2, BMP-2）、Runt相关转录因子2（Runt related transcription factor 2, Runx2）和 Klotho mRNA及蛋白的表达变化。结果: 培养基磷浓度升高抑制了VSMCs增殖;而高磷刺激VSMCs 6 d后,茜素红染色可见钙化结节明显形成,细胞内钙含量显著增加[(1.52±0.14) mmol/g比(0.03±0.01) mmol/g,P=0.043],成骨相关蛋白BMP-2、Runx2的mRNA和蛋白表达均增加,而Klotho的mRNA和蛋白表达均下降。结论: 高磷可刺激VSMCs钙化形成和表型转换,Klotho可能参与了高磷诱导的VSMCs钙化机制。
Objective: To investigate the changes of Klotho expression in rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphate. Methods: Rat VSMCs was cultured in vitro, the proliferation of VSMCs induced by different concentrations of phosphate was detected by cell counting kit-8 (CCK-8). VSMCs were treated with high phosphate (3 mmol/L) for 3 days or 6 days, and the calcification of VSMCs was evaluated by alizarin red staining. The activity of alkaline phosphatase (ALP) and calcium content in VSMCs were detected by ALP assay kit and calcium colorimetric assay kit, respectively. The expression of mRNA and protein of bone morphogenetic protein 2 (BMP-2), Runt related transcription factor 2 (Runx2) and Klotho were detected by quantitative real-time polymerase chain reaction (qrt-PCR) and Western blotting (WB). Results: Higher-phosphate media inhibited the proliferation of VSMCs. After being incubated in high-phosphate media for 6 days, calcified nodules were observed with alizarin red staining and calcium content was significantly increased; qrt-PCR demonstrated that levels of BMP-2 and Runx2 mRNA and protein were increased and the expressions of Klotho mRNA and protein levels were decreased. Conclusions: Klotho might be involved in the hyperphosphate-induced calcification and phenotype changes in VSMCs.
[1] 陈孜瑾, 陈晓农, 马晓波, 等. 应用腰椎侧位片评价血液透析患者腹主动脉钙化[J]. 中华肾脏病杂志,2012, 28(8):606-610.
[2] 陈孜瑾, 陈晓农, 马晓波, 等. 维持性血液透析患者心脏瓣膜钙化相关危险因素分析[J]. 中国血液净化,2013,12(10):538-542.
[3] Chen XN, Chen ZJ, Ma XB, et al.Aortic artery and cardiac valve calcification are associated with mortality in chinese hemodialysis patients: A 3.5 years follow-up[J]. Chin Med J (Engl),2015,128(20):2764-2771.
[4] Zitt E, Knoll F, Lhotta K.et al.Variable mechanisms of vascular calcification in different segments of the arterial tree in patients with chronic kidney disease[J]. Kidney Int,2014,86(4):858.
[5] Chen Z, Chen X, Xie J, et al.Fibroblast growth factor 23 is a predictor of aortic artery calcification in maintenance hemodialysis patients[J]. Ren Fail,2013,35(5):660-666.
[6] Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing[J]. Nature,1997,390(6655):45-51.
[7] Hu MC, Shi M, Zhang J, et al.Klotho deficiency causes vascular calcification in chronic kidney disease[J]. J Am Soc Nephrol,2011,22(1):124-136.
[8] Lim K, Lu TS, Molostvov G, et al.Vascular Klotho deficiency potentiates the development of human artery calcification and mediates resistance to fibroblast growth factor 23[J]. Circulation,2012,125(18):2243-2255.
[9] Lindberg K, Olauson H, Amin R, et al.Arterial klotho expression and FGF23 effects on vascular calcification and function[J]. PLoS One,2013,8(4):e60658.
[10] Scialla JJ, Lau WL, Reilly MP, et al.Fibroblast growth factor 23 is not associated with and does not induce arterial calcification[J]. Kidney Int,2013,83(6):1159-1168.
[11] Moe SM, Drüeke T, Lameire N, et al.Chronic kidney disease-mineral-bone disorder: a new paradigm[J]. Adv Chronic Kidney Dis,2007,14(1):3-12.
[12] Crouthamel MH, Lau WL, Leaf EM, et al.Sodium-dependent phosphate cotransporters and phosphate-induced calcification of vascular smooth muscle cells: redundant roles for PiT-1 and PiT-2[J]. Arterioscler Thromb Vasc Biol,2013,33(11):2625-2632.
[13] Ohnishi M, Nakatani T, Lanske B, et al.In vivo genetic evidence for suppressing vascular and soft-tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin d levels[J]. Circ Cardiovasc Genet,2009, 2(6):583-590.
[14] Li X, Yang HY, Giachelli CM.BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells[J]. Atherosclerosis,2008,199(2):271-277.
[15] Sage AP, Lu J, Tintut Y, et al.Hyperphosphatemia-induced nanocrystals upregulate the expression of bone morphogenetic protein-2 and osteopontin genes in mouse smooth muscle cells in vitro[J]. Kidney Int,2011,79(4):414-422.
[16] Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts[J]. Cell,1997 May 30,89(5):755-764.
[17] Otto F, Thornell AP, Crompton T, et al.Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development[J]. Cell,1997,89(5):765-771.
[18] Byon CH, Javed A, Dai Q, et al.Oxidative stress induces vascular calcification through modulation of the osteogenic transcription factor Runx2 by AKT signaling[J]. J Biol Chem,2008,283(22):15319-15327.
[19] Otto F, Kanegane H, Mundlos S.Mutations in the RUNX2 gene in patients with cleidocranial dysplasia[J]. Hum Mutat,2002,19(3):209-216.
[20] Tyson KL, Reynolds JL, McNair R, et al. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification[J]. Arterioscler Thromb Vasc Biol,2003,23(3):489-494.
[21] Sun Y, Byon CH, Yuan K, et al.Smooth muscle cell-specific runx2 deficiency inhibits vascular calcification[J]. Circ Res,2012,111(5):543-552.
[22] Prosdocimo DA, Wyler SC, Romani AM, et al.Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia[J]. Am J Physiol Cell Physiol,2010,298(3):C702-C713.
[23] Yao L, Sun YT, Sun W, et al.High phosphorus level leads to aortic calcification via β-catenin in chronic kidney disease[J]. Am J Nephrol,2015,41(1):28-36.
[24] Jimbo R, Kawakami-Mori F, Mu S, et al.Fibroblast growth factor 23 accelerates phosphate-induced vascular calcification in the absence of Klotho deficiency[J]. Kidney Int,2014,85(5):1103-1111.
