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Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells

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Abstract

Objectives

Hepatic ischemia and hypoxia are accompanied by reduced bile flow, biliary sludge and cholestasis. Hepatobiliary transport systems, nuclear receptors and aquaporins were studied after hypoxia and reoxygenation in human hepatic cells.

Methods

Expression of Aquaporin 8 (AQP8), Aquaporin 9 (AQP9), Pregnane X receptor (PXR), Farnesoid X receptor (FXR), Organic anion transporting polypeptide 1 (OATP1), and the Multidrug resistance-associated protein 4 (MRP4) were investigated in induced pluripotent stem cells (iPSCs) derived hepatic cells and the immortalized hepatic line HepG2. HepG2 was subjected to combined oxygen and glucose deprivation for 4 h followed by reoxygenation.

Results

Expression of AQP8 and AQP9 increased during differentiation in iPSC-derived hepatic cells. Hypoxia did not alter mRNA levels of AQP8, but reoxygenation caused a marked increase in AQP8 mRNA expression. While expression of OATP1 had a transient increase during reoxygenation, MRP4 showed a delayed downregulation. Knock-down of FXR did not alter the expression of AQP8, AQP9, MRP4, or OATP1. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation compared to normoxic controls.

Conclusions

Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Expression patterns differed between hepatobiliary transport systems during hypoxia and reoxygenation.

Impact

  • Expression of AQP8 and AQP9 increased during differentiation in induced pluripotent stem cells.

  • Expression of hepatobiliary transporters varies during hypoxia and reoxygenation.

  • Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation.

  • Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells.

  • Hypoxia and reoxygenation may affect aquaporins in hepatic cells and potentially affect bile composition.

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Fig. 1: Expression of aquaporins and hepatobiliary transporters during differentiation.
Fig. 2: Metabolic activity assessed with the MTT assay after 4 h of hypoxia followed by 20 (n = 5), 44 (n = 6) or 68 h (n = 6) of reoxygenation.
Fig. 3: mRNA expression of AQP8, AQP9, PXR, FXR, MRP4 and OATP1 measured with quantitative PCR against endogenous control GAPDH.
Fig. 4: mRNA expression of AQP8, AQP9, FXR, MRP4 and PXR in transfected cells.
Fig. 5: AQP8 proteins levels after hypoxia and rexygenation.

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Funding

N.S.W. has received financial support from University of Oslo. Runar Almaas has received financial support from The Eckbo Foundation.

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Authors and Affiliations

  1. Department of Pediatric Research, Oslo University Hospital, Oslo, Norway

    Niklas Starck Westerberg, Maria Melheim, Sean P. Harrison, Gareth J. Sullivan & Runar Almaas

  2. Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Niklas Starck Westerberg, Monica Atneosen-Åsegg, Maria Eugenia Chollet & Runar Almaas

  3. Department of Haematology, Oslo University Hospital, Oslo, Norway

    Maria Eugenia Chollet

  4. Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway

    Maria Eugenia Chollet

  5. Department of Molecular Medicine, University of Oslo, Oslo, Norway

    Richard Siller

  6. School of Medicine, University of St Andrews, St Andrews, UK

    Gareth J. Sullivan

  7. European Reference Network—Rare Liver, Hamburg, Germany

    Runar Almaas

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  1. Niklas Starck Westerberg
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Contributions

N.S.W. participated in conceptualization and design of the study, acquisition and analysis of data, drafted the initial manuscript, and revised the manuscript. M.A-Å., M.M., M.E.C., S.P.H, R.S. participated in acquisition of data, reviewed and revised the manuscript. G.J.C participated in design of the study, analysis of data, reviewed and revised the manuscript. R.A. participated in conceptualization and design of the study, analysis of data, writing and revision of the manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

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Correspondence to Runar Almaas.

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Westerberg, N.S., Atneosen-Åsegg, M., Melheim, M. et al. Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells. Pediatr Res (2024). https://doi.org/10.1038/s41390-024-03368-0

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  • DOI: https://doi.org/10.1038/s41390-024-03368-0

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