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Kidney Week

Abstract: PO0244

High-Content Imaging of Kidney Cell Function to Elucidate Mechanisms of Antiviral Drug Toxicity

Session Information

  • AKI Mechanisms - 3
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Pearson, Adam, Universitat Zurich, Zurich, ZH, Switzerland
  • Haenni, Dominik, Universitat Zurich, Zurich, ZH, Switzerland
  • Hall, Andrew, Universitat Zurich, Zurich, Switzerland
Background

Globally, millions of people live with HIV and hepatitis B virus (HBV). Toxicity from antiviral drugs is a major cause of kidney disease in these individuals. Tenofovir disoproxil fumarate (TDF) is a first line therapy for HIV and HBV. TDF induces functional proximal tubule (PT) defects for reasons that are unknown, partly due to a lack of appropriate experimental models. Clinically, TDF toxicity is characterized by two major phenotypes: isolated defects in PT solute transport; and severe tubular damage (Fanconi syndrome/acute kidney injury) associated with grossly enlarged mitochondria. The aim of our study was to establish realistic in vitro models of TDF toxicity, to investigate the underlying mechanisms.

Methods

Experiments were performed on monolayers of differentiated human-derived PT cells (RPTEC/TERT1). A high-content image analysis pipeline was established, using automated microscopy and machine learning, to quantify transport function, using dome formation as a readout. Metabolism was evaluated by antibody staining for mitochondrial morphology and autophagy.

Results

We screened numerous treatment regimens and generated phenotypes matching those observed in patients, including transport inhibition and mitochondrial hypertrophy. Further experiments using these models revealed that TDF caused a dose dependent decrease in ATP despite increased glycolysis and mtDNA content. Basal and ATP-linked respiration were decreased but maximal respiration was achieved, suggesting inhibition of complex V (ATP synthase). Metabolomic analysis confirmed that TDF was converted to the active antiviral metabolite Tenofovir diphosphate (TFVpp), a structural analogue of ATP. Using an in vitro assay of complex V activity, we observed a dose dependent inhibition with TFVpp. Metabolomics revealed no major defects in the TCA cycle or beta-oxidation, but clear evidence of oxidative stress.

Conclusion

In summary, we have developed a high-content image analysis pipeline of human-derived PT cells to generate realistic in vitro models of functional TDF toxicity. Metabolic characterization of these revealed a clear phenotype consistent with ATP synthase inhibition, which most likely explains toxicity observed in patients, since PT solute transport is heavily dependent on aerobic respiration. ATP depletion might trigger compensatory mitochondrial biogenesis, leading to hypertrophy.

Funding

  • Government Support - Non-U.S.