Transcriptional Programs Driving Shear Stress-Induced Differentiation of Kidney Proximal Tubule Cells in Culture.

Hyun Jung Park,Natalie Rittenhouse,Qidong Ren,Zhenjiang Fan,Joseph D Locker,Youssef Rbaibi,Megan L Gliozzi,Kimberly R Long,Amanda C Poholek,Yulong Bai,Ora A Weisz

doi:10.3389/fphys.2020.587358

Hyun Jung Park, Natalie Rittenhouse + Show 9 more

Open Access

https://doi.org/10.3389/fphys.2020.587358

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Abstract

Cultured cell models are an essential complement to dissecting kidney proximal tubule (PT) function in health and disease but do not fully recapitulate key features of this nephron segment. We recently determined that culture of opossum kidney (OK) cells under continuous orbital shear stress (OSS) significantly augments their morphological and functional resemblance to PTs in vivo. Here we used RNASeq to identify temporal transcriptional changes upon cell culture under static or shear stress conditions. Comparison of gene expression in cells cultured under static or OSS conditions with a database of rat nephron segment gene expression confirms that OK cells cultured under OSS are more similar to the PT in vivo compared with cells maintained under static conditions. Both improved oxygenation and mechanosensitive stimuli contribute to the enhanced differentiation in these cells, and we identified temporal changes in gene expression of known mechanosensitive targets. We observed changes in mRNA and protein levels of membrane trafficking components that may contribute to the enhanced endocytic capacity of cells cultured under OSS. Our data reveal pathways that may be critical for PT differentiation in vivo and validate the utility of this improved cell culture model as a tool to study PT function.

Highlights

Cells lining the kidney proximal tubule (PT) consistently recover ∼70% of water, sodium, chloride, and other solutes entering the tubule lumen as well as essentially all of the glucose and filtered proteins (Alan et al, 2015)
To identify transcriptional changes that lead to the remarkable differentiation we observed in cells exposed to orbital shear stress (OSS), we performed a time course RNASeq study using RNA isolated from opossum kidney (OK) cells exposed to OSS or maintained under static conditions for 0-96 h
To identify transcriptional changes that lead to the remarkable differentiation we observed in cells exposed to OSS, OK cells were plated on permeable supports, and the following day (t = 0h) shifted to OSS for 12, 48, and 96 h or maintained under static conditions over this period (0, 48, and 96 h) before harvesting RNA

Summary

Introduction

Cells lining the kidney proximal tubule (PT) consistently recover ∼70% of water, sodium, chloride, and other solutes entering the tubule lumen as well as essentially all of the glucose and filtered proteins (Alan et al, 2015). Studying PT function in cell culture has been hampered by the paucity of well-differentiated immortalized cell lines or primary cell culture models. In terms of their similarity to PTs in vivo, opossum kidney (OK) cells currently represent the best in vitro model in which to study transport and endocytic functions of this nephron segment. Unlike other PT cell models, OK cells uniquely retain the regulatory cascade that directs sodium dependent phosphate transport in response to parathyroid hormone (Malmström and Murer, 1986). OK cells internalize albumin and other filtered molecules with high efficiency compared to other PT cell lines and have been a useful model in which to study PT endocytosis (Gekle et al, 1995, 1996, 1997, 1998; Raghavan et al, 2014; Long et al, 2020; Ren et al, 2020)

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