, Noe¨l Knops1,2, Sante Princiero Berlingerio1
, Celien Lismont3
, Dirk J. Kuypers4
, Elena Levtchenko1,2, Lambert
P. van den Heuvel1,5, Marc Fransen3*
1 Laboratory of Pediatric Nephrology, Department of Growth and Regeneration, University of Leuven,
Leuven, Belgium, 2 Department of Pediatric Nephrology and Solid Organ Transplantation, University
Hospitals Leuven, Leuven, Belgium, 3 Laboratory of Peroxisome Biology and Intracellular Communication,
Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium, 4 Department of
Nephrology and Renal Transplantation and Department of Microbiology, Immunology and Transplantation,
University of Leuven, Leuven, Belgium, 5 Translational Metabolic Laboratory and Department of Pediatric
Nephrology, Radboud University Medical Center, Nijmegen, The Netherlands
Immunology, Medical Research
The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.
Fluorescence was evaluated by using a previously described microscopic setup  or by using a motorized inverted IX-81 microscope, controlled by cellSens Dimension software (version 2) and equipped with (i) a temperature, humidity, and CO2-controlled incubation chamber, (ii) a CoolLED pE-4000 illumination system, (iii) a 100x Super Apochromat oil immersion objective, (iv) the band-pass excitation filters D405/20x and BP470-495 (v) the barrier filter BA510-550 (vi) a dichromatic mirror with a cut-off 505 nm, and (vii) a DP73 high-performance Peltier-cooled digital color camera (Olympus Belgium).
Product Associated Features
High-speed channel switching, software integration into Olympus cellCens and the choice of 256 wavelengths have made the pE-4000 the ideal light source for this ratiometric fluorescence assay.
Year of Publication
Country of Publication
Belgium, The Netherlands