[Experimental Cell Research] Roles of fluid shear stress and retinoic acid in the differentiation of
Due to the distinct features that distinguish immortalized podocyte cell lines from their in vivo counterparts, primary cultured human podocytes might be a superior cell model for glomerular disease studies. However, the podocyte de-differentiation that occurs in culture remains an unresolved problem. Here, we present a method to differentiate primary cultured podocytes using retinoic acid (RA) and fluid shear stress (FSS), which mimic the in vivo environment of the glomerulus. RA treatment induced changes in the cell shape of podocytes from a cobblestone-like morphology to an arborized configuration with enhanced mobility. Moreover, the expression of synaptopodin and zonula occludens (ZO)−1 in RA-treated podocytes increased along with Krüppel-like factor 15 (KLF15) expression. Confocal microscopy revealed that RA increased the expression of cytoplasmic synaptopodin, which adopted a filamentous arrangement, and junctional ZO-1 expression, which showed a zipper-like pattern. To elucidate the effect of FSS in addition to RA, the podocytes were cultured in microfluidic devices and assigned to the static, static+RA, FSS, and FSS+RA groups. The FSS+RA group showed increased synaptopodin and ZO-1 expression with prominent spikes on the cell-cell interface. Furthermore, interdigitating processes were only observed in the FSS+RA group. Consistent with these data, the mRNA expression levels of synaptopodin, podocin, WT-1 and ZO-1 were synergistically increased by FSS and RA treatment. Additionally, the heights of the cells were greater in the FSS and FSS+RA groups than in the static groups, suggesting a restoration of the 3D cellular shape. Meanwhile, the expression of KLF15 increased in the RA-treated cells regardless of fluidic condition. Taken together, FSS and RA may contribute through different but additive mechanisms to the differentiation of podocytes. These cells may serve as a useful tool for mechanistic studies and the application of regenerative medicine to the treatment of kidney diseases.
Seung Hee Yanga, b, 1, Jin Woo Choic, 1, Dongeun Huhd, Hyung Ah Joa, Sejoong Kima, e, Chun Soo Lima, b, f, Jung Chan Leeg, h, i, Hee Chan Kimg, h, i, Hyug Moo Kwonj, Chang Wook Jeongk, Cheol Kwakk, Kwon Wook Jooa, b, Yon Su Kima, b, Dong Ki Kima, b, , a Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea b Kidney Research Institute, Seoul National University, Seoul, Republic of Korea c Interdisciplinary Program in Bioengineering Major, Graduate School, Seoul National University, Seoul, Republic of Korea d Department of Bioengineering, University of Pennsylvania, Philadelphia, USA e Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea f Department of Internal Medicine, Seoul National University Boramae Medical Center, Seoul, Republic of Korea g Department of Biomedical Engineering, Seoul National University College of Medicine, Seoul, Republic of Korea h Department of Biomedical Engineering, Seoul National University Hospital, Seoul, Republic of Korea i Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University, Seoul, Republic of Korea j School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea k Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea Received 30 September 2016, Revised 10 March 2017, Accepted 13 March 2017, Available online 16 March 2017 http://dx.doi.org/10.1016/j.yexcr.2017.03.026