Beamish Jeffrey A, Chen Evan, Putnam Andrew J
Division of Nephrology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS One. 2017 Jul 17;12(7):e0181085. doi: 10.1371/journal.pone.0181085. eCollection 2017.
Acute kidney injury (AKI) is common and associated with significant morbidity and mortality. Recovery from many forms of AKI involves the proliferation of renal proximal tubular epithelial cells (RPTECs), but the influence of the microenvironment in which this recovery occurs remains poorly understood. Here we report the development of a poly(ethylene glycol) (PEG) hydrogel platform to study the influence of substrate mechanical properties on the proliferation of human RPTECs as a model for recovery from AKI. PEG diacrylate based hydrogels were generated with orthogonal control of mechanics and cell-substrate interactions. Using this platform, we found that increased substrate stiffness promotes RPTEC spreading and proliferation. RPTECs showed similar degrees of apoptosis and Yes-associated protein (YAP) nuclear localization regardless of stiffness, suggesting these were not key mediators of the effect. However, focal adhesion formation, cytoskeletal organization, focal adhesion kinase (FAK) activation, and extracellular signal-regulated kinase (ERK) activation were all enhanced with increasing substrate stiffness. Inhibition of ERK activation substantially attenuated the effect of stiffness on proliferation. In long-term culture, hydrogel stiffness promoted the formation of more complete epithelial monolayers with tight junctions, cell polarity, and an organized basement membrane. These data suggest that increased stiffness potentially may have beneficial consequences for the renal tubular epithelium during recovery from AKI.
急性肾损伤（AKI）很常见，且与显著的发病率和死亡率相关。多种形式的AKI恢复过程涉及肾近端肾小管上皮细胞（RPTECs）的增殖，但对发生这种恢复的微环境的影响仍知之甚少。在此，我们报告了一种聚乙二醇（PEG）水凝胶平台的开发，以研究底物机械性能对人RPTECs增殖的影响，作为AKI恢复的模型。基于聚乙二醇二丙烯酸酯的水凝胶通过对力学和细胞-底物相互作用的正交控制生成。使用该平台，我们发现底物硬度增加会促进RPTECs的铺展和增殖。无论硬度如何，RPTECs显示出相似程度的凋亡和Yes相关蛋白（YAP）核定位，表明这些不是该效应的关键介质。然而，随着底物硬度增加，粘着斑形成、细胞骨架组织、粘着斑激酶（FAK）激活和细胞外信号调节激酶（ERK）激活均增强。抑制ERK激活可显著减弱硬度对增殖的影响。在长期培养中，水凝胶硬度促进形成具有紧密连接、细胞极性和有组织的基底膜的更完整上皮单层。这些数据表明，硬度增加可能对AKI恢复过程中的肾小管上皮具有有益影响。
