A multi-omics investigation of tacrolimus off-target effects on a proximal tubule cell-line.

INTRODUCTION: Tacrolimus, an immunosuppressive drug prescribed to a majority of organ transplant recipients is nephrotoxic, through still unclear mechanisms. This study on a lineage of proximal tubular cells using a multi-omics approach aims to detect off-target pathways modulated by tacrolimus that can explain its nephrotoxicity. METHODS: LLC-PK1 cells were exposed to 5 µM of tacrolimus for 24 h in order to saturate its therapeutic target FKBP12 and other high-affine FKBPs and favour its binding to less affine targets. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, as well as of the other gluconeogenesis-limiting enzymes FBP1 and FBP2, was measured using RT-qPCR. Cell viability with this concentration of tacrolimus was further checked until 72 h. RESULTS: In our cell model of acute exposure to a high concentration of tacrolimus, different metabolic pathways were impacted including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p < 0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis and acid-base balance control. DISCUSSION: The variations found using a multi-omics pharmacological approach clearly point towards a dysregulation of energy production and decreased gluconeogenesis, a hallmark of chronic kidney disease which may also be an important toxicity pathway of tacrolimus.

Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform.

In the field of quantitative proteomics, the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In the present study, after peptide and protein identification, we compared the widespread quantitation method based on the calculation of MS/MS reporter ion peaks areas ratios (ProteinPilot) to the alternative method based on the calculation of ratios of the sum of peak intensities (jTRAQx [Quant]) and we processed output data with the in-house Customizable iTRAQ Ratios Calculator (CiR-C) algorithm. Quantitation based on peak area ratios displayed no significant linear correlation with Western blot quantitation. In contrast, quantitation based on the sum of peak intensities displayed a significant linear association with Western blot quantitation (non-zero slope; Pearson correlation coefficient test, r = 0.296, P=0.010**) with an average bias of 0.087 ± 0.500 and 95% Limits of Agreement from -0.893 to 1.068. We proposed the Mascot-jTRAQx-CiR-C strategy as a simple yet powerful data processing adjunct to the iTRAQ technology.

Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous detection of 10 illicit drugs in oral fluid collected with FLOQSwabs™ and application to real samples.

According to French law, the roadside testing for drugs of abuse (DOA) should be performed in oral fluid (OF) using an immunological screening kit. If the screening is positive, confirmation has to be done in OF collected by a special swab, called the FLOQSwab™ (FS). Unlike other sampling kits, this device was not designed to collect OF since it does not contain an elution buffer. An analytical method was developed for the simultaneous detection of 10 DOA under control in France: tetrahydrocannabinol (THC) at 1 ng/mL, and cocaine, benzoylecgonine (BZE), morphine, 6-monoacetylmorphine (6-MAM), amphetamine, methamphetamine, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxy-N-methylamphetamine (MDMA) at 10 ng/mL. Samples were eluted using the Quantisal® buffer and extracted by liquid-liquid extraction for THC and by solid-phase extraction for the remaining analytes. Analyses were performed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The validated method made it possible to detect the concentrations required by law and was successfully applied to samples from drivers who screened positive. The main limitations of this kit are the large variability of the collected OF volume and the poor stability of DOA in OF, requiring the use of a conservation buffer.