Pharmacological research · 2023
A multi-omics investigation of tacrolimus off-target effects on a proximal tubule cell-line.
СтатьяНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Aouad Hassan, Faucher Quentin, Sauvage François-Ludovic, Pinault Emilie, Barrot Claire-Cécile, Arnion Hélène, Essig Marie, Marquet Pierre
Аннотация
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.
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.