Modulation of energy status and cytotoxicity induced by FK506 and cyclosporin A in a renal epithelial cell line

Abstract

FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC50 was assessed via thiazolyl blue (MTT) assay after incubation for 4-24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC50. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (-25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity.

In vitro Assessment of Renal Toxicity and Inflammatory Events of Two Protein Phosphatase Inhibitors Cantharidin and Nor‐Cantharidin*

“…Incubation was stopped by adding 0.6 M perchloric acid for deproteinization. After scraping the cells, ATP content was determined by high pressure liquid chromatography as previously described (Massicot et al 1997). ATP is expressed as nmol/ml.…”

Oxidative Stress-Mediated Renal Dysfunction by Cyclosporine a in Rats: Attenuation by Trimetazidine

“…[11,29] Moreover, CsA affects mitochondrial function and cell energetics that specifically inhibits mitochondrial energy production and decreases ATP levels characteristic of ischemia in kidney in vitro and ex vivo. [12,13] In addition, CsA increases ROS formation leading to reduction of oxidative phosphorylation and decreased mitochondrial ATP concentration. [30] These alterations could theoretically lead to a classical ischemiareperfusion injury involving free radicals.…”

“…[11] Furthermore, CsA specifically inhibits mitochondrial energy production and decreases ATP levels, events characteristic of ischemia. [12,13] Trimetazidine (TMZ) (1-(2,3,4-trimethoxybenzyl) piperazine) is a novel anti-ischemic agent that exerts cytoprotection by counteracting the metabolic disorder at the level of ischemic cell, independently of any hemodynamic effect. [14] TMZ limits intracellular acidosis, minimizes sodium and calcium accumulation, maintains intracellular ATP levels, and reduces creatinine phosphokinase release.…”

Organ-specific response to inhibition of mitochondrial metabolism by cyclosporine in the rat

“…Furthermore, organ-specific responses to CyA treatment under the same experimental/ clinical conditions in a whole intact animal are, to our knowledge, not available. However, organ-specific metabolic responses to CyA treatment may help to explain why certain organs (e.g., kidney) are at increased risk of CyA toxicity, Previous in vitro studies focused mainly on the effect of CyA on energy production in isolated kidney and liver rat mitochondria [8,11,171. In the last decade, magnetic resonance spectroscopy (MRS) has shown great promise in toxicology, drug discovery, pre-clinical drug development, and hypothesis-driven biomedical research.…”