Roles of fluid shear stress and retinoic acid in the differentiation of primary cultured human podocytes.
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.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Kidney Research Institute, Seoul National University, Seoul, Republic of Korea.
Seung Hee
SH
Yang
Interdisciplinary Program in Bioengineering Major, Graduate School, Seoul National University, Seoul, Republic of Korea.
Jin Woo
JW
Choi
Department of Bioengineering, University of Pennsylvania, Philadelphia, USA.
Dongeun
D
Huh
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
Hyung Ah
HA
Jo
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea.
Sejoong
S
Kim
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Kidney Research Institute, Seoul National University, Seoul, Republic of Korea; Department of Internal Medicine, Seoul National University Boramae Medical Center, Seoul, Republic of Korea.
Chun Soo
CS
Lim
Department of Biomedical Engineering, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Biomedical Engineering, Seoul National University Hospital, Seoul, Republic of Korea; Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University, Seoul, Republic of Korea.
Jung Chan
JC
Lee
Department of Biomedical Engineering, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Biomedical Engineering, Seoul National University Hospital, Seoul, Republic of Korea; Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University, Seoul, Republic of Korea.
Hee Chan
HC
Kim
School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.
Hyug Moo
HM
Kwon
Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea.
Chang Wook
CW
Jeong
Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea.
Cheol
C
Kwak
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Kidney Research Institute, Seoul National University, Seoul, Republic of Korea.
Kwon Wook
KW
Joo
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Kidney Research Institute, Seoul National University, Seoul, Republic of Korea.
Yon Su
YS
Kim
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Kidney Research Institute, Seoul National University, Seoul, Republic of Korea. Electronic address: dkkim73@gmail.com.
Dong Ki
DK
Kim
D002454:Cell Differentiation / Q000187:drug effects*; D048430:Cell Shape / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D007678:Kidney Glomerulus / Q000254:growth & development / Q000378:metabolism*; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D050199:Podocytes / Q000187:drug effects / Q000378:metabolism; D061251:Primary Cell Culture; D013314:Stress, Mechanical; D016708:Synaptophysin / Q000235:genetics*; D014212:Tretinoin / Q000008:administration & dosage
C414172:KLF15 protein, human; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; C504260:SYNPR protein, human; D016708:Synaptophysin; D014212:Tretinoin
Differentiation; Microfluidics; Podocyte; Retinoic acid