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Updated: Jul 25, 2025
Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells
Published on: June 20, 2018
Improvement of Protein Expression Profile in Three-Dimensional Renal Proximal Tubular Epithelial Cell Spheroids
Naoki Ishiguro1, Etsushi Takahashi2, Hiroshi Arakawa2
1Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Company, Ltd., Kobe, Japan (N.I., A.S., G.M., M.T., R.T., T.K.); R&D Department, Industrial Division, Nikkiso Company, Ltd., Kanazawa, Japan (E.T., F.K., Ma.K., Y.J.); Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (H.A., Mo.K., D.H., Y.N., I.T.); and Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan (S.M., K.O.) naoki.ishiguro@boehringer-ingelheim.com y.jinbo@nikkiso.co.jp.
Abstract:
The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.
Insights
Developing a novel 3D spheroid culture for renal proximal tubular epithelial cells (RPTECs) enhances drug transporter expression, mimicking human kidney tissue for improved toxicity testing.
Area of Science:
- Nephrology
- Pharmacology
- Biotechnology
Background:
- Renal proximal tubule is crucial for kidney function and a key site for drug interactions and toxicity.
- Current in vitro assays for kidney toxicity lack sufficient drug transporter function representation in renal proximal tubular epithelial cells (RPTECs).
Purpose of the Study:
- To establish a simple, reproducible method for culturing RPTECs using three-dimensional (3D) spheroid aggregates.
- To enhance the expression of drug transporters, specifically organic anion transporter 1 (OAT1), in cultured RPTECs.
Main Methods:
- Culturing RPTECs in 3D spheroids, using OAT1 expression as a selection marker.
- Proteome analysis to compare protein expression in 3D spheroids versus conventional 2D cultures and human renal cortices.
- Assessing drug-induced toxicity (cisplatin, adefovir) in the 3D RPTEC spheroid model.
Main Results:
- 3D spheroid culture significantly increased OAT1 protein expression in RPTECs compared to 2D culture, reaching levels similar to human renal cortices.
- Proteome analysis revealed maintained expression of key proximal tubule markers and improved expression of transporter proteins in 3D spheroids.
- The 3D RPTEC spheroids demonstrated transporter-dependent ATP decreases upon exposure to cisplatin and adefovir, indicating functional relevance.
Conclusions:
- The developed 3D RPTEC spheroid culture system is a simple, reproducible, and effective in vitro model.
- This system exhibits enhanced gene and protein expression profiles, closely resembling human kidney cortices.
- The 3D RPTEC spheroids show potential for accurate in vitro evaluation of renal proximal tubular toxicity and drug disposition during drug development.
