Platelet-derived growth factor-DD induces the proliferation and differentiation of rat renal fibroblasts into myofibroblasts

doi:10.3969/j.issn.2095-4344.2015.11.010

Abstract

Abstract:

BACKGROUND: Platelet-derived growth factors can induce proliferation, migration, transformation and extracellular matrix expression of glomerular mesangial cells and renal interstitial cells.

OBJECTIVE: To investigate the effect of activation of platelet-derived growth factor-DD/βR signaling pathway on the proliferation and α-smooth muscle actin expression in rat renal fibroblasts.

METHODS: Normal rat kidney interstitial fibroblasts (NRK-49F) cultured in vitro were divided into the following groups by platelet-derived growth factor-DD concentrations: control, 1 μg/L, 10 μg/L, 50 μg/L, 100 μg/L. And NRK-49Fs were then divided into four groups according to the stimulation time of 50 μg/L platelet-derived growth factor-DD:control, 12 hours, 24 hours, 48 hours. The cell viability of the NRK-49Fs was assessed by Cell Counting Kit-8 after platelet-derived growth factor-DD administration. The mRNA and protein expression levels of platelet-derived growth factor-βR and α-smooth muscle actin in all groups stimulated by different concentrations of platelet-derived growth factor-DD were determined by RT-PCR and western blot, respectively.

RESULTS AND CONCLUSION: Platelet-derived growth factor-DD could facilitate the proliferation rates of the NRK-49Fs at a dose- and time-dependent manner as compared with the control groups. Platelet-derived growth factor-DD could stimulate the mRNA and protein expressions of platelet-derived growth factor-βR and α-smooth muscle actin in a dose-dependent manner. This suggest that the activation of platelet-derived growth facto-DD/βR signaling pathway can obviously promote the proliferation of NRK-49Fs as well as tranformation into myofibroblasts.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: Kidney, Fibrosis, Fibroblasts, Phenotype, Receptors, Platelet-Derived Growth Factor

CLC Number:

R318

Zhao Jia, Sun Jian-ping, Gao Yan-xia, Tang Ni-na, Niu Meng, Cui Meng, Han Xiao-qing, Sui Ai-hua . Platelet-derived growth factor-DD induces the proliferation and differentiation of rat renal fibroblasts into myofibroblasts [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(11): 1688-1693.

Figures/Tables 4

2.1 血小板衍化生长因子DD对NRK-49F增殖的影响与对照组比较，不同质量浓度的血小板衍化生长因子DD均可明显促进NRK-49F的增殖，差异有显著性意义(P < 0.05)。 1 μg/L时即可促进细胞增殖，50 μg/L时，其促NRK-49F增殖作用最强，50 μg/L组与100 μg/L组比较差异无显著性意义(P > 0.05，表2)。此外，与对照组相比，血小板衍化生长因子在作用12 h时即有明显促进NRK-49F增殖的作用，随着时间延长，致观察终点(48 h)细胞增殖效应最大，呈现时间依赖性(P < 0.05，表3)。 2.2 不同质量浓度血小板衍化生长因子DD对血小板衍化生长因子β R、α-平滑肌肌动蛋白mRNA表达的影响血小板衍化生长因子DD主要与血小板衍化生长因子ββR结合，仅少量结合于血小板衍化生长因子αβR与其相应血小板衍化生长因子β R结合后激活信号传导通路，产生生物学效应。α-平滑肌肌动蛋白是活化的肌成纤维细胞的特异性标志，少见于正常肾间质细胞。与对照组比较，各质量浓度组血小板衍化生长因子βR及α-平滑肌肌动蛋白均高于对照组(P < 0.05)；除50 μg/L组与100 μg/L组之间比较差异无显著性意义(P > 0.05)外，其余各处理组间比较血小板衍化生长因子βR及α-平滑肌肌动蛋白表达均不同(P < 0.05)。另外，血小板衍化生长因子DD质量浓度在1 μg/L时即可明显上调血小板衍化生长因子βR及α-平滑肌肌动蛋白的mRNA表达，并可剂量依赖性地上调血小板衍化生长因子βR、α-平滑肌肌动蛋白的mRNA表达，维持该通路的活化状态，随血小板衍化生长因子DD质量浓度增大，表达量逐渐增多(P < 0.05)，50 μg/L与100 μg/L组比较无明显差异(图1A)。RT-PCR电泳图(图1B)呈现与上述一致的结果，随血小板衍化生长因子DD质量浓度增大，血小板衍化生长因子βR及α-平滑肌肌动蛋白mRNA表达量增加，至血小板衍化生长因子DD 50 μg/L时mRNA表达量最大， 50 μg/L与100 μg/L组比较差异无显著性意义。 2.3 血小板衍化生长因子DD对血小板衍化生长因子β R、α-平滑肌肌动蛋白的蛋白表达影响与对照组相比，血小板衍化生长因子DD可剂量依赖性地上调血小板衍化生长因子R、α-平滑肌肌动蛋白的蛋白表达。1 μg/L血小板衍化生长因子DD即可明显上调血小板衍化生长因子βR及 α-平滑肌肌动蛋白的蛋白表达，随血小板衍化生长因子DD质量浓度增大，血小板衍化生长因子βR及α-平滑肌肌动蛋白表达量逐渐增多(图2)，至50 μg/L时血小板衍化生长因子β、α-平滑肌肌动蛋白表达最多，50 μg/L与 100 μg/L组比较无明显差异。"

References

[1] Nicolas P, Oliver B, Alexander V, et al. Origin of renal myofbroblasts in the model of unilateral ureter obstruction in the rat. Histochem Cell Biol.2008:130:141-155.
[2] Chuang PY, Memon MC, He JC. Molecular targets for treatment of kidney fibrosis. J Mol Med.2013;91:549-559.
[3] Wynn TA，Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med.2012; 18(7):1028-1040.
[4] Wynn TA. Cellular and molecular mechanisms of renal fibrosis. J Pathol.2008; 214(2):199-200.
[5] 叶琨,刘伏友,刘映红.肾间质纤维化发生机制的研究进展[J]. 国外医学泌尿系统分册, 2005,1(1):94-98.
[6] Ostendorf T, Eitner F, Floege J. The PDGF family in renal fibrosis. Pediatr Nephrol.2012; 27:1041-1050.
[7] Ina K, Kitamura H, Tatsukawa S, et al. Significance of α-SMA in myofibroblasts emerging in renal tubulointerstitial fibrosis. Histol Histopathol.2011; 26(7):1041-1050.
[8] Zeisberg M, Strutz F, Müller GA. Renal fibrosis: an update. Curr Opin Nephrol Hypertens.2001;10(3):315-320.
[9] Farris AB, Colvin RB. Renal interstitial fibrosis: mechanismsandevaluation. Curr Opin Nephrol Hypertens. Curr Opin Nephrol Hypertens.2012;21(3):289-300
[10] van Roeyen CR, Ostendorf T, Floege J. The platelet-derived growth factor system in renal disease: an emerging role of endogenous inhibitors. Eur J Cell Biol. 2012, 91:542-551.
[11] 张倩倩,孙建平,高延霞,等.单侧输尿管梗阻模型大鼠肾间质纤维化过程中血小板衍生生长因子D的表达[J].中国组织工程研究与临床康复, 2011,15(24):4444-4447.
[12] Ostendorf T, van Roeyen CR, Peterson JD, et al. A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis. J Am Soc Nephrol.2003;14(9):2237-2247.
[13] Hu K, Lin L, Tan X, et al. tPA protects renal interstitial fibroblasts and myofibroblasts from apoptosis.J Am Soc Nephrol.2008;19(3):503-514.
[14] Hinz B. The myofibroblast: paradigm for a mechanically active cell. J Biomech. 2010;43(1):146-155.
[15] Desmoulière A, Chaponnier C, Gabbiani G. Tissue repair, contraction, and the myofibroblast.Wound Repair Regen. 2005;13(1):7-12.
[16] Hinz B, Plan SH, Thannickal VJ, et al. The myofibroblast: one function, multiple origins.Am J Pathol.2007; 170(6): 1807-1816.
[17] Yang J, Dai C, Liu Y. A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition. J Am Soc Nephrol.2005;16(1):68-78.
[18] Badid C, Vincent M, Fouque D. Myofibroblast: a prognostic marker and target cell in progressive renal disease. Ren Fail.2001; 23(3-4):543-549.
[19] Boor P, Konieczny A, Villa L, et al. PDGF-D inhibition by CR002 ameliorates tubulointerstitial fibrosis following experimental glomerulonephritis. Nephrol Dial Transplant. Nephrol Dial Transplant.2007; 24(25):1323-1331.
[20] Floege J, Eitner F, CE A. A new look at platelet-derived growth factor in renal disease. J Am Soc Nephrol.2008; 19(1):12-23.
[21] Jhaveri KD, Flombaum CD, Kroog G. Nephrotoxicities associated with the use of tyrosine kinase inhibitors: a single-center experience and review of the literature. Nephron Clin Pract.2011;117(4):c312-c319.
[22] 吕彦洁,孙建平,高延霞,等.丹参酮ⅡA磺酸钠对大鼠肾间质纤维化的影响及机制探讨[J].山东医药,2014,54(10):30-32.
[23] 孙兴旺,曹灵,于国华,等.丹参酮IIA磺酸钠对纤维化人间充质成纤维细胞体外增及cyclin E蛋白表达的影响[J].第三军医大学学报,2007,29(7):585-587.