Roles of fluid shear stress and retinoic acid in the differentiation of primary cultured human podocytes

• Published in: Experimental cell research Vol. 354; no. 1; pp. 48 - 56
• Main Authors: Yang, Seung Hee, Choi, Jin Woo, Huh, Dongeun, Jo, Hyung Ah, Kim, Sejoong, Lim, Chun Soo, Lee, Jung Chan, Kim, Hee Chan, Kwon, Hyug Moo, Jeong, Chang Wook, Kwak, Cheol, Joo, Kwon Wook, Kim, Yon Su, Kim, Dong Ki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2017
• Subjects: Cell Differentiation - drug effects; Cell Shape - drug effects; Differentiation; Gene Expression Regulation, Developmental - drug effects; Humans; Kidney Glomerulus - growth & development; Kidney Glomerulus - metabolism; Kruppel-Like Transcription Factors - biosynthesis; Kruppel-Like Transcription Factors - genetics; Microfluidics; Nuclear Proteins - biosynthesis; Nuclear Proteins - genetics; Podocyte; Podocytes - drug effects; Podocytes - metabolism; Primary Cell Culture; Retinoic acid; Stress, Mechanical; Synaptophysin - genetics; Tretinoin - administration & dosage; Podocyte; Differentiation; Retinoic acid; Microfluidics
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2017.03.026

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. •Effect of fluid shear stress and retinoic acid on differentiation of human podocytes.•Differentiation protocol of human podocytes using microfluidics device.•Increased expression of KLF15 through proposed differentiation protocol.