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Gene Pathways Analysis of the Effects of Suspension Culture on Primary Human Renal Proximal Tubular Cells

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Abstract

Drug-induced acute kidney injury causes massive morbidity and mortality at exorbitant cost, yet there is currently no effective method for preclinical in vitro testing for nephrotoxicity. Proximal tubule cells are a key target for nephrotoxins, but heretofore, it has been a challenge to maintain their differentiation in vitro. One promising approach is to culture them in suspension, under physiological levels of shear, so as to induce and maintain structural and functional differentiation. Advances in materials, additive manufacturing, and injection molding have reduced the complexity and cost of suspension cultures hardware by orders of magnitude, making it a viable alternative for high throughput screening. This study defines the global transcriptome responses of human renal proximal tubular cells to suspension culture. GSEA/Cytoscape/ClueGO analysis showed near perfect concurrence with GPS-SIGORA analysis in the areas of mineral absorption and ribosome assembly, and defined the nucleic acid and protein mechanisms underlying the transcriptome response to suspension culture. Proximal tubular cells in suspension culture showed increased growth and viability assayed as reducing potential compared to cells cultured under static conditions. Suspension culture of human renal proximal tubular cells now allows investigations in basic cell biology, toxicology, drug screening, and tissue engineering, free of artificial matrices, feeder layers, fetal gene expression, or the need for complex engineering technologies.

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Abbreviations

ABC:

ATP-binding cassette

FDR:

False Discovery Rate

FWER:

Family Wise Error Rate

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

GO:

Gene ontology

GPS:

Gene-Pair Signatures

GSEA:

Gene Set Enrichment Analysis

NES:

Normalized Enrichment Scores

OAT:

Organic anion transporter

OCT:

Organic cation transporter

RFU:

Relative fluorescence units

SIGORA:

Signature Over-Representation Analysis

SLC:

Solute carrier

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Acknowledgements

NASA Grants NNX13AN32G, NNX12AM93G, and NNX10AP01G supported these studies. This material is the result of work supported with resources and the use of facilities at the Durham Veterans Affairs Medical Center and the Veterans Health Administration Office of Research and Development in the Department of Veterans Affairs. Contents do not represent the views of the Department of Veterans Affairs or the United States of America. We thank the Medical College of Georgia for performing gene arrays. We thank David Corcoran Ph.D. and the Duke Genomic Analysis and Bioinformatics Shared Resource for performing Gene Array analysis. We thank Erica Acton and Corey Nislow of the University of British Columbia for performing analysis both by Cytoscape using ClueGO and SIGORA. The authors declare that there is no conflict of interest regarding the publication of this paper.

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Authors and Affiliations

  1. Medicine and Research & Development Service, Durham VA Medical Center, Building 15, Room 210, 508 Fulton Street, Durham, NC, 27705, USA

    Timothy G. Hammond & Patricia L. Allen

  2. Nephrology Division, Department of Internal Medicine, Duke University School of Medicine, Durham, NC, 27705, USA

    Timothy G. Hammond

  3. Space Policy Institute, Elliott School of International Affairs, George Washington University, Washington, DC, 20052, USA

    Timothy G. Hammond & Holly H. Birdsall

  4. Department of Veterans Affairs Office of Research and Development, Washington, DC, 20420, USA

    Holly H. Birdsall

  5. Departments of Otorhinolaryngology, Immunology, and Psychiatry, Baylor College of Medicine, Houston, TX, 77030, USA

    Holly H. Birdsall

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Hammond, T.G., Allen, P.L. & Birdsall, H.H. Gene Pathways Analysis of the Effects of Suspension Culture on Primary Human Renal Proximal Tubular Cells. Microgravity Sci. Technol. 30, 951–963 (2018). https://doi.org/10.1007/s12217-018-9658-x

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