Date Published: July 17, 2017
Publisher: Public Library of Science
Author(s): Jeffrey A. Beamish, Evan Chen, Andrew J. Putnam, Shree Ram Singh.
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.
Acute kidney injury (AKI) is common, costly, and associated with increased mortality [1–3]. Many forms of AKI are reversible and often involve the regeneration of damaged renal tubular epithelium. There is great interest in better understanding factors that influence renal tubular epithelial regeneration to mitigate the short-term and long-term consequences of AKI in terms of patient health and societal costs.
In this study, we report an in vitro model using a biocompatible scaffold system with human RPTECs to explore the role of ECM stiffness in recovery from acute kidney injury. We developed and characterized a versatile PEG-based scaffold platform with well-controlled substrate mechanics, which were independent of ECM conjugation. Using this well-characterized platform, we observed that increasing substrate stiffness promotes spreading and proliferation of RPTECs. We then showed cytoskeletal organization and phosphorylation of FAK, but not YAP shuttling or apoptosis, was correlated with proliferation and that ERK 1/2 activation was required to mediate this effect. We also showed that substrate stiffness contributed to optimal RPTEC epithelialization.