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

Estimating Extracellular Fluid Volume in Healthy Individuals: Evaluation of Existing Formulae and Development of a New Equation

peer reviewedIntroduction: Several clinical settings require an accurate estimation of the physiologically expected extracellular fluid volume (ECFV). We aimed to analyze the performances of existing ECFV-estimating equations and to develop a new equation. Methods: The performances of 11 ECFV-estimating equations were analyzed in 228 healthy kidney donor candidates (Bichat Hospital, Paris, France) who underwent ECFV measurement using the distribution volume of 51Cr-labeled EDTA (51Cr-EDTA). An equation was developed using a penalized linear modeling approach (elastic net regression) and externally (Tenon Hospital, Paris, France, N = 142) validated. Results: Participants from Bichat (mean age 45.2 ± 12.0 years, 43.0% men) and Tenon (47.8 ± 10.3 years, 29.6% men) hospitals had a mean measured ECFV of 15.4 ± 2.8 l and 15.1 ± 2.1 l, respectively. Available ECFV-estimating formulae have highly variable precision and accuracy. The new equation incorporating body weight, height, sex, and age had better precision and accuracy than all other equations in the external validation cohort, with a median bias of −0.20 (95% CI: −0.35 to −0.05) l versus −2.63 (−2.87 to −2.42) l to −0.57 (− 0.83 to −0.40) l and 0.21 (0.12 to 0.43) l to 2.89 (2.65 to 3.11) l, for underestimating and overestimating equations, respectively, an interquartile range for the bias of 0.88 (0.70 to 1.08) l versus 0.91 (0.71 to 1.20) l to 1.93 (1.67 to 2.25) l, and an accuracy within 10% of 90.9% (83.8 to 94.4) versus 88.0% (81.0 to 92.3) to 8.5% (4.2 to 13.4). These results were consistent across subgroups defined by sex, body mass index (BMI), body surface area (BSA), age, and ethnicity. Conclusion: We developed and validated a new equation to estimate the individual reference value of ECFV, which is easily usable in clinical practice. Further validation in cohorts including individuals of extreme age and corpulence remains needed

Health-Related Quality of Life in Long-Term Survivors of Relapsed Childhood Acute Lymphoblastic Leukemia

BACKGROUND: Relapses occur in about 20% of children with acute lymphoblastic leukemia (ALL). Approximately one-third of these children can be cured. Their risk for late effects is high because of intensified treatment, but their health-related quality of life (HRQOL) was largely unmeasured. Our aim was to compare HRQOL of ALL survivors with the general population, and of relapsed with non-relapsed ALL survivors. METHODOLOGY/PRINCIPAL FINDINGS: As part of the Swiss Childhood Cancer Survivor Study (SCCSS) we sent a questionnaire to all ALL survivors in Switzerland who had been diagnosed between 1976-2003 at age <16 years, survived ≥5 years, and were currently aged ≥16 years. HRQOL was assessed with the Short Form-36 (SF-36), which measures four aspects of physical health and four aspects of mental health. A score of 50 corresponded to the mean of a healthy reference population. We analyzed data from 457 ALL survivors (response: 79%). Sixty-one survivors had suffered a relapse. Compared to the general population, ALL survivors reported similar or higher HRQOL scores on all scales. Survivors with a relapse scored lower in general health perceptions (51.6) compared to those without (55.8;p=0.005), but after adjusting for self-reported late effects, this difference disappeared. CONCLUSION/SIGNIFICANCE: Compared to population norms, ALL survivors reported good HRQOL, even after a relapse. However, relapsed ALL survivors reported poorer general health than non-relapsed. Therefore, we encourage specialists to screen for poor general health in survivors after a relapse and, when appropriate, specifically seek and treat underlying late effects. This will help to improve patients' HRQOL

Evolution à l'âge adulte des vascularites à ANCA à début pédiatrique

LIMOGES-BU Médecine pharmacie (870852108) / SudocSudocFranceF

Immunosuppresseurs et néphropathie chronique d'allogreffe

LIMOGES-BU Médecine pharmacie (870852108) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

Étude protéomique des modifications phénotypiques des cellules rénales exposées aux immunosuppresseurs inhibiteurs de la calcineurine

Les médicaments inhibiteurs de la calcineurine, la cyclosporine (CsA) et le tacrolimus (TAC), sont utilisés depuis plus de 30 ans dans les protocoles d immunosuppression afin de prévenir les rejets du greffon et traiter des maladies auto-immunes. Si ces traitements ont considérablement amélioré la survie du greffon à court terme, ils contribuent à long terme à l apparition d une atteinte rénale appelée néphropathie chronique d allogreffe ainsi qu à une dysfonction chronique du greffon, qui conduit au déclin progressif, irréversible de la fonction rénale et à la perte du greffon, en dehors de tout surdosage médicamenteux. Les mécanismes moléculaires impliqués dans la néphrotoxicité des inhibiteurs de la calcineurine sont nombreux et, bien que largement étudiés, restent mal compris. Notre objectif principal a été de développer et de mettre en application une approche protéomique à large échelle, basée sur la spectrométrie de masse, afin d identifier et de quantifier les modifications d expression protéique induites par la CsA et le TAC sur un modèle de cellules rénales en culture. La méthodologie utilisée est l approche SILAC (ou Stable Isotope Labelling by Amino acids in Cell culture), qui consiste en un marquage métabolique des protéines cellulaires avec des acides aminés alourdis par du carbone isotope 13. Nous montrons que l expression intra-cellulaire et la sécrétion de nombreuses protéines sont modulées par la CsA et que le TAC agit différemment sur ces mêmes protéines. Nos résultats confirment ainsi des observations précédemment décrites et nous conduisent également à formuler de nouvelles hypothèses concernant les mécanismes moléculaires potentiellement impliqués dans la néphrotoxicité induite par les inhibiteurs de la calcineurine. Des études ultérieures seront développées pour tester certaines de ces protéines comme biomarqueurs potentiels de cette néphrotoxicité chez des patients transplantés et traités par CsA ou TAC.LIMOGES-BU Médecine pharmacie (870852108) / SudocSudocFranceF

Modifications du cytosquelette des cellules tubulaires proximales rénales par les inhibiteurs de la calcineurine (rôle dans la dysfonction chronique du greffon)

La ciclosporine et le tacrolimus sont des inhibiteurs de la calcineurine utilisés comme immunosuppresseurs pour prévenir les rejets de greffe. Alors que la survie à court terme des greffons rénaux est bien maitrisée, la survie à long terme est encore compromise par l apparition d une maladie rénale appelée dysfonction chronique du greffon qui conduit au déclin progressif et irréversible de la fonction rénale. Paradoxalement, l utilisation des inhibiteurs de la calcineurine contribue à l apparition des lésions en raison de leur importante néphrotoxicité qui induit, à dose thérapeutique, des modifications phénotypiques perturbant le fonctionnement normal des cellules. Le but de ce travail était d identifier les effets des inhibiteurs de la calcineurine sur l organisation du cytosquelette des cellules du tube proximal et les conséquences sur le phénotype des cellules. Nous montrons ici que la CsA, indépendamment de la voie du NFAT, perturbe le cytosquelette d actine et inhibe le système fibrinolytique des cellules tubulaires proximales. Nous identifions également, pour la première fois au niveau rénal, la cofiline comme une voie de signalisation potentiellement impliquée dans les effets de la ciclosporine. En revanche, les premiers résultats obtenus avec le tacrolimus ne reproduisent pas les effets de la ciclosporine sur le cytosquelette. Ce travail permet de formuler un certain nombre d hypothèses sur les mécanismes physiopathologiques qui participent à la détérioration progressive de la structure et de la fonction des cellules tubulaires proximales après la transplantation rénale et sur la contribution des inhibiteurs de la calcineurine dans ce processus.LIMOGES-BU Médecine pharmacie (870852108) / SudocSudocFranceF

The COVID-19 outbreak and the angiotensin-converting enzyme 2: too little or too much?

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Calcineurin regulation of cytoskeleton organization: a new paradigm to analyse the effects of calcineurin inhibitors on the kidney.

International audienceCalcineurin is a serine/threonine phosphatase originally involved in the immune response but is also known for its role as a central mediator in various non-immunological intracellular signals. The nuclear factor of activated T cell (NFAT) proteins are the most widely described substrates of calcineurin, but ongoing work has uncovered other substrates among which are the cytoskeleton organizing proteins (i.e. cofilin, synaptopodin, WAVE-1). Control over cytoskeletal proteins is of outmost interest because the phenotypic properties of cells are dependent on cytoskeleton architecture integrity, while rearrangements of the cytoskeleton are implicated in both physiological and pathological processes. Previous works investigating the role of calcineurin on the cytoskeleton have focused on neurite elongation, myocyte hypertrophic response and recently in kidney cells structure. Nuclear factor of activated T cell activation is expectedly identified in the signalling pathways for calcineurin-induced cytoskeleton organization, however new NFAT-independent pathways have also been uncovered. The aim of this review is to summarize the current knowledge on the effects of calcineurin on cytoskeletal proteins and related intracellular pathways. These newly described properties of calcineurin on cytoskeletal proteins may explain some of the beneficial or deleterious effects observed in kidney cells associated with the use of the calcineurin inhibitors, cyclosporine and tacrolimus

Reply to: Renal impairment is frequent in chronic hepatitis C patients under triple therapy with telaprevir or boceprevir.

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