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

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. μs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations

Impact of hypoxia and reoxygenation on the extra/intracellular metabolome and on transporter expression in a human kidney proximal tubular cell line

Introduction Ischemia-reperfusion injury (IRI) induces several perturbations that alter immediate kidney graft function after transplantation and may affect long-term graft outcomes. Given the IRI-dependent metabolic disturbances previously reported, we hypothesized that proximal transporters handling endo/exogenous substrates may be victims of such lesions. Objectives This study aimed to determine the impact of hypoxia/reoxygenation on the human proximal transport system through two semi-targeted omics analyses. Methods Human proximal tubular cells were cultured in hypoxia (6 or 24 h), each followed by 2, 24 or 48-h reoxygenation. We investigated the transcriptomic modulation of transporters. Using semi-targeted LC–MS/MS profiling, we characterized the extra/intracellular metabolome. Statistical modelling was used to identify significant metabolic variations. Results The expression profile of transporters was impacted during hypoxia (y + LAT1 and OCTN2), reoxygenation (MRP2, PEPT1/2, rBAT, and OATP4C1), or in both conditions (P-gp and GLUT1). The P-gp and GLUT1 transcripts increased (FC (fold change) = 2.93 and 4.11, respectively) after 2-h reoxygenation preceded by 24-h hypoxia. We observed a downregulation (FC = 0.42) of y+LAT1 after 24-h hypoxia, and of PEPT2 after 24-h hypoxia followed by 2-h reoxygenation (FC = 0.40). Metabolomics showed that hypoxia altered the energetic pathways. However, intracellular metabolic homeostasis and cellular exchanges were promptly restored after reoxygenation. Conclusion This study provides insight into the transcriptomic response of the tubular transporters to hypoxia/reoxygenation. No correlation was found between the expression of transporters and the metabolic variations observed. Given the complexity of studying the global tubular transport systems, we propose that further studies focus on targeted transporters

Perfusate Metabolomics Content and Expression of Tubular Transporters During Human Kidney Graft Preservation by Hypothermic Machine Perfusion

Background. Ischemia-related injury during the preimplantation period impacts kidney graft outcome. Evaluating these lesions by a noninvasive approach before transplantation could help us to understand graft injury mechanisms and identify potential biomarkers predictive of graft outcomes. This study aims to determine the metabolomic content of graft perfusion fluids and its dependence on preservation time and to explore whether tubular transporters are possibly involved in metabolomics variations. Methods. Kidneys were stored on hypothermic perfusion machines. We evaluated the metabolomic profiles of perfusion fluids (n=35) using liquid chromatography coupled with tandem mass spectrometry and studied the transcriptional expression of tubular transporters on preimplantation biopsies (n=26), both collected at the end of graft perfusion. We used univariate and multivariate analyses to assess the impact of perfusion time on these parameters and their relationship with graft outcome. Results. Seventy-two metabolites were found in preservation fluids at the end of perfusion, of which 40% were already present in the native conservation solution. We observed an increase of 23 metabolites with a longer perfusion time and a decrease of 8. The predictive model for time-dependent variation of metabolomics content showed good performance (R2=76%, Q2=54%, accuracy=41%, and permutation test significant). Perfusion time did not affect the mRNA expression of transporters. We found no correlation between metabolomics and transporters expression. Neither the metabolomics content nor transporter expression was predictive of graft outcome. Conclusions. Our results call for further studies, focusing on both intra- and extratissue metabolome, to investigate whether transporter alterations can explain the variations observed in the preimplantation period

Étude de petits ARNs chez une bactérie : Helicobacter pylori

Helicobacter pylori (H. pylori) est une bactérie Gram-négative colonisant durablement l’estomac d’une personne sur deux à travers le monde. Sa présence est associée avec la plupart des ulcères et cancers gastriques. Bien que les facteurs protéiques impliqués dans la virulence et l’adaptation de la bactérie à l’environnement gastrique ont été largement étudiés, peu d’informations sont disponibles concernant la régulation de l’expression de ses gènes.Les récentes techniques de séquençage à haut-débit ont révolutionné l’analyse des transcriptomes bactériens, et donc la compréhension de la régulation des gènes chez les procaryotes. Mon laboratoire d’accueil s’intéresse plus particulièrement à la régulation par les petits ARNs non codant (sARNnc). Chez H. pylori, l’analyse du transcriptome de la souche26695 a révélé une centaine de sARNnc et l’expression de soixante d’entre eux a été confirmée par Northern Blot.Mon travail s’est inscrit dans l’analyse de ces différents sARNs c’est à dire dans la compréhension du rôle de ces ARNs dans les mécanismes de régulation de la traduction ainsi que dans la physiologie ou la pathogénicité d’H. pylori. Pour cela mon étude s’est portée sur un petit ARN très abondant, le HPnc5490 et d’une famille de sARN nommés IsoA.Des études préliminaires ont montré que le HPnc5490 pourrait être impliqué dans la régulation de la traduction de la protéine TlpB par un mécanisme « trans » antisens. La protéine TlpB étant un récepteur membranaire impliqué dans le pH-tactisme de la bactérie, le sARN HPnc5490 serait lui aussi impliqué dans ce processus physiologique via la régulation de TlpB. Les résultats obtenus lors de cette étude confirment cette hypothèse.Par ailleurs, la famille des IsoA compte 6 membres homologues dans la souche 26695. Ils régulent de manière « cis » antisens la traduction de petits peptides de 30 acides aminés codés par des ARNs messagers nommés aapA. Ce duo aapA/IsoA est très similaire aux cassettes toxine-antitoxine (TA) de type I décrites dans la littérature. Une partie de notre étude a permis de confirmer l’appartenance de ces cassettes aux TA de type I. Une autre partie de notre travail, une étude bioinformatique, nous a permis de montrer la grande conservation de ces cassettes au sein de nombreuses souches d’H. pylori. Une dernière partie nous a permis de monter l’implication de la RNase III dans la maturation des ARNs d’H. pylori.Ce travail a donc permis d’apporter de nouvelles données dans la compréhension de la régulation de l’expression des gènes de H. pylori

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

Structural patterns of the human ABCC4/MRP4 exporter in lipid bilayers rationalize clinically observed polymorphisms

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