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.

High-Dimensional Mediation Analysis: A New Method Applied to Maternal Smoking, Placental DNA Methylation, and Birth Outcomes.

BACKGROUND: High-dimensional mediation analysis is an extension of unidimensional mediation analysis that includes multiple mediators, and increasingly it is being used to evaluate the indirect omics-layer effects of environmental exposures on health outcomes. Analyses involving high-dimensional mediators raise several statistical issues. Although many methods have recently been developed, no consensus has been reached about the optimal combination of approaches to high-dimensional mediation analyses. OBJECTIVES: We developed and validated a method for high-dimensional mediation analysis (HDMAX2) and applied it to evaluate the causal role of placental DNA methylation in the pathway between exposure to maternal smoking (MS) during pregnancy and gestational age (GA) and birth weight of the baby at birth. METHODS: HDMAX2 combines latent factor regression models for epigenome-wide association studies with max2 tests for mediation and considers CpGs and aggregated mediator regions (AMRs). HDMAX2 was carefully evaluated using simulated data and compared to state-of-the-art multidimensional epigenetic mediation methods. Then, HDMAX2 was applied to data from 470 women of the Etude des Déterminants pré et postnatals du développement de la santé de l'Enfant (EDEN) cohort. RESULTS: HDMAX2 demonstrated increased power in comparison with state-of-the-art multidimensional mediation methods and identified several AMRs not identified in previous mediation analyses of exposure to MS on birth weight and GA. The results provided evidence for a polygenic architecture of the mediation pathway with a posterior estimate of the overall indirect effect of CpGs and AMRs equal to 44.5g lower birth weight representing 32.1% of the total effect [standard deviation (SD)=60.7g]. HDMAX2 also identified AMRs having simultaneous effects both on GA and on birth weight. Among the top hits of both GA and birth weight analyses, regions located in COASY, BLCAP, and ESRP2 also mediated the relationship between GA and birth weight, suggesting reverse causality in the relationship between GA and the methylome. DISCUSSION: HDMAX2 outperformed existing approaches and revealed an unsuspected complexity of the potential causal relationships between exposure to MS and birth weight at the epigenome-wide level. HDMAX2 is applicable to a wide range of tissues and omic layers. https://doi.org/10.1289/EHP11559.

Big data in pharmacogenomics: current applications, perspectives and pitfalls.

The efficiency of new generation sequencing methods and the reduction of their cost has led pharmacogenomics to gradually supplant pharmacogenetics, leading to new applications in personalized medicine along with new perspectives in drug design or identification of drug response factors. The amount of data generated in genomics fits the definition of big data, and need a specific bioinformatics processing following standard steps: data collection, processing, analysis and interpretation. Pitfalls of pharmacogenomics studies are directly related to these steps. This review aims to describe these steps from a pharmacogenomic point of view, focusing on bioinformatics aspects.

New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation.

Human induced pluripotent stem cells (hiPSc) are a very useful solution to create and observe the behavior of specific and usually inaccessible cells, such as human motor neurons. Obtained from a patient biopsy by reprograming dermal fibroblasts (DF), hiPSc present the same properties as embryonic stem cells and can generate any cell type after several weeks of differentiation. Today, there are numerus protocols which aim to control hiPSC differentiation. The principal challenge is to obtain a sufficiently enriched specific cell population to study disease pathophysiology and to provide a good model for further investigation and drug screening. The differentiation process is very costly and time-consuming, because many specific factors and different culture media must be used. In this study, we used Sedimentation Field Flow Fractionation (SdFFF) to prepare enriched populations derived from hiPSc after only 10 days of culture in a classical medium. Based on phenotypic and proteomic characterization, "hyperlayer" elution resulted in a fraction expressing markers of endothelial progenitors while another fraction expressed markers of neural progenitors. The isolation of subpopulations representing various differentiation lineages is of major interest for the production of specialized, cell-enriched fractions and in the preparation of increasingly complex models for the development of new therapeutic tools.