c-Jun-N-Terminal Kinase Signaling Is Involved in Cyclosporine-Induced Epithelial Phenotypic Changes

Tubular epithelial cells play a central role in the pathogenesis of chronic nephropathies. Previous toxicogenomic studies have demonstrated that cyclosporine- (CsA-) induced epithelial phenotypic changes (EPCs) are reminiscent of an incomplete epithelial to mesenchymal transition (EMT) in a TGF-β-independent manner. Furthermore, we identified endoplasmic reticulum (ER) stress as a potential mechanism that may participate in the modulation of tubular cell plasticity during CsA exposure. Because c-jun-N-terminal kinase (JNK), which is activated during ER stress, is implicated in kidney fibrogenesis, we undertook the current study to identify the role of JNK signaling in EPCs induced by CsA. In primary cultures of human renal epithelial cells, CsA activates JNK signaling, and the treatment with a JNK inhibitor reduces the occurrence of cell shape changes, E-cadherin downregulation, cell migration, and Snail-1 expression. Our results suggest that CsA activates JNK signaling, which, in turn, may participate in the morphological alterations through the regulation of Snail-1 expression

Conséquences rénales de l'activation de la réponse UPR (Unfolded protein response) par des stress toxique et ischémique

Le rein natif et le greffon rénal peuvent être soumis à de multiples agressions conduisant à la détérioration progressive du parenchyme. Ces agressions peuvent être spécifiques (stress toxique, immunologique) et/ou non spécifiques (stress ischémique) et vont engendrer des réponses pouvant entraîner à la fois une diminution de la consommation d énergie, une augmentation des apports afin de maintenir l homéostasie tissulaire et la survie mais aussi une réaction inflammatoire et l apoptose pouvant conduire à la fibrose. Parmi celles-ci, on peut nommer les voies HIF1a, mTOR, le stress du réticulum endoplasmique (RE), l autophagie, l activation de l immunité innée et acquise. La réponse adaptative qui suit le stress du RE, la réponse UPR (Unfolded protein response), est une voie adaptative dont les implications sont actuellement encore peu connues dans le domaine de la pathologie rénale. Celle-ci se compose de trois effecteurs principaux : Perk, Ire1 et ATF6. A l aide de deux modèles de stress toxique (ciclosporine) et ischémique (carence en glucose) sur deux modèles cellulaires distincts (cellulaires endothéliales artérielles et cellules tubulaires rénales), et dans des modèles in vivo, nous avons montré que le stress du RE était impliqué à la fois dans l apparition de modifications phénotypiques endothéliales évocatrices de transition endothélio-mésenchymateuse induites par la ciclosporine et à la fois dans l induction de réponses inflammatoire (régulation de NF- B par Ire1) et angiogénique (régulation distincte de VEGF, bFGF et angiogénine par Perk et Ire1) induites par la carence en glucose. La réponse UPR semble modulée de façon subtile au cours de ces stress car les trois effecteurs n engendrent pas des réponses identiques. Ces travaux apportent ainsi une meilleure compréhension des mécanismes d adaptation au cours de stress variés, montrent que le stress du RE est impliqué dans ces réponses adaptatives et que la réponse peut être différente selon les effecteurs de la réponse UPR. Cette meilleure compréhension pourra permettre de valider des biomarqueurs précoces et des modulateurs de la réponse UPR afin de prévenir la dégradation du parenchyme rénal.Native and grafted kidneys are stressed by multiple specific or non-specific insults leading to progressive structural deterioration. Responses to these insults are adaptive and preserve cell survival but may also promote inflammation, fibrosis and apoptosis. The most important of these adaptive pathways are HIF1a pathway, mTOR pathway, autophagy, unfolded protein response (UPR). The consequences of the UPR in kidney injuries are not well known. The objective of this study is to delineate the mechanisms and consequences of the activation of the UPR in response to toxic (cyclosporine) and ischemic (glucose starvation) stresses in two distinct cellular models (arterial endothelial cells and renal tubular cells). Here, we showed that UPR was engaged in cyclosporine-induced endothelial phenotypic changes, glucose starvation-induced inflammatory and angiogenic responses: NF- B regulation by Ire1; distinct VEGF, bFGF and angiogenin regulation by Perk and Ire1. UPR is subtly modulated since its transducers do not induce identical processes. In conclusion these comprehensive works, we demonstrate the UPR is implicated in stress-induced adaptive pathways with different downstream responses according to the effector. Renal tissue degradation could be prevented by discovering and validating early biomarker and UPR modulators.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF

Rôle du stress du réticulum endoplasmique et de l'autophagie dans la régulation des réponses immune et angiogénique activées par des stress ischémiques et inflammatoires dans l'épithélium rénal humain

Dans le cadre de situations pathologiques, le rein peut être soumis à de multiples agressions toxiques, ischémiques et immunologiques pouvant favoriser la survenue d une maladie rénale chronique et le développement d une insuffisance rénale. En réponse à ces stress, les cellules du parenchyme rénal vont activer des processus biologiques adaptatifs permettant le maintien de la viabilité cellulaire et l homéostasie de l organe. Ces réponses adaptatives peuvent également activer l immunité innée et induire le remodelage tissulaire (fibrogenèse et angiogenèse). Cependant, les mécanismes précis de cette régulation sont mal connus. L objectif de ce travail a été de caractériser les mécanismes de régulation et les conséquences microenvironnementales (inflammation et angiogenèse) de l activation de la réponse UPR (Unfolded Protein Response) et de l autophagie, en réponse à des stress ischémiques et immunologiques. Dans un premier travail, nous avons montré que la réponse UPR est impliquée dans la génération d une réponse inflammatoire induite par un stress métabolique dans des cellules tubulaires rénales. Le stress métabolique, caractérisé par une carence en glucose, induit un stress du RE et active la réponse UPR. Ce stress active le facteur NF-.B et favorise la transcription de cytokines et chimiokines pro-inflammatoires. La voie PERK/eIF2 : - n est pas nécessaire à l activation de l inflammation mais amplifie l expression des cytokines alors que la voie IRE1 - est impliquée dans la génération de cette réponse inflammatoire. De plus, l ischémie aigue active le stress du RE et l inflammation dans les reins de rat. Enfin, à partir de biopsies de déclampage de greffons rénaux, l expression de GRP78, marqueur du stress du RE, et de NF-.B p65/RelA dans les tubules rénaux, est significativement plus élevée en comparaison avec des biopsies de greffons rénaux stables, à distance de la greffe. Dans un second travail, nous avons montré que la réponse UPR régule l angiogenèse dans les cellules tubulaires rénales lors d une carence en glucose. La voie PERK est un régulateur majeur de l expression des facteurs angiogéniques (VEGFA, bFGF et angiogénine). De plus, l expression de l angiogénine est modulée par les voies PERK et IRE1.. Enfin, l ischémie aigue induite chez le rat, active la réponse UPR parallèlement à l augmentation de l expression de VEGFA, bFGF et de l angiogénine. Dans un troisième travail, nous avons mis en évidence un nouveau mécanisme par lequel l interféron. (IFN.) active l autophagie dans les cellules tubulaires rénales. Nous avons montré que l IFN. entraine une déplétion en tryptophane, active la voie GCN2, une kinase eIF2., ce qui conduit à l augmentation du flux autophagique. De plus, la supplémentation entryptophane et l utilisation d ARN interférence dirigés contre GCN2 inhibent l autophagie induite par l IFN. Enfin, l autophagie intervient dans la régulation de la sécrétion de cytokines inflammatoires et de facteurs de croissance en réponse à l IFN.. En conclusion, nous avons caractérisé dans ce travail des mécanismes originaux de régulation d une réponse inflammatoire et angiogénique par la réponse UPR et l autophagie en réponse à des stress ischémiques et immunologiques au sein de l épithélium tubulaire rénal humain.Under pathological conditions, kidney is subjected to multiple toxic, ischemic and immunological failures that promote the occurrence of chronic kidney disease and the development of acute kidney injury. In response to stress, renal parenchymal cells activate biological adaptive processes permitting the maintenance of cell viability and renal homeostasis. These adaptive responses can also activate innate immunity and induce tissue remodeling (fibrogenesis and angiogenesis). However, accurate mechanisms of this regulation are still unclear. The aim of this work was to characterize regulation mechanisms and micro environmental consequences(inflammation and angiogenesis) of the activation of the UPR (Unfolded Protein Response) and autophagy, in response to ischemic and immunological stress. In a first study, we demonstrated that the UPR is involved in the generation of inflammatory response induces by metabolic stress in tubular renal cells. Metabolic stress, characterized by glucose deprivation, induces an ER stress and activates the UPR. This stress activates NF-.B and promotes the transcription of pro inflammatory cytokines and chemokines. The PERK signaling is not required for the induction of inflammation but amplifies cytokine expression whereas IRE1 is involved in the generation of inflammatory response. Moreover, acute ischemia activates ER stress and inflammation in rat kidneys. Finally, from kidney transplant biopsies performed before implantation, the expression of GRP78, an ER stress marker, and NF-.B p65/RelA in renal tubules is significantly increased in comparison with stable human kidney transplant biopsies. In a second study, we showed that the UPR regulates angiogenesis in tubular renal cells during glucose deprivation. The PERK pathway is a major regulator of angiogenic factors expression (VEGFA, bFGF and angiogenin). Furthermore, angiogenin expression is modulated by PERK and IRE1. pathways. Finally, acute ischemia activates the UPR and, in parallel, increases VEGFA, bFGF and angiogenin expression in rat kidneys. In a third work, we identified a novel mechanism by which IFN. activates autophagy in human kidney epithelial cells. We showed that IFN. promotes tryptophan depletion, activates the eIF2. kinase GCN2, and leads to an increase of the autophagic flux. Moreover, tryptophan supplementation and RNA interference directed against GCN2 inhibit IFN.-induced autophagy. Finally,autophagy regulates the secretion of inflammatory cytokines and growth factors in response to IFN..In conclusion, we characterized in this work original mechanisms that regulate inflammatory and angiogenic responses by the UPR and autophagy in response to ischemic and immunological stress in tubular renal human epithelium.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF

Home and Office Blood Pressure Monitoring in Renal Transplant Recipients

Background. Arterial hypertension in renal transplant recipients (RTR) is associated with increased morbid mortality. In the general population, home blood pressure monitoring (HBPM) was found to be superior to office blood pressure (OBP) in identifying true hypertensive patients. The aim of this study was to investigate HBPM for the assessment of blood pressure profile in RTR. Methodology and Principal Findings. We included prospectively 87 stable RTR. Sitting OBP was measured during the outpatient clinic. HBPM was performed by measuring BP every morning and night for 4 days. The accepted limits for the OBP and HBPM, were respectively, 140/90 mmHg and 135/85 mmHg. Patients were classified as “normotensive,” “uncontrolled,” “white-coat hypertensive” and “masked hypertensive”, (OBP below the limit and HBPM above). During the study, 81 patients (55 males, age 48.5 ± 14 years) were available for analysis. The mean OBP and HBP were 138/83 ± 14/10 mmHg and 133/79 ± 14/8 mmHg; 29% of patients were uncontrolled, 28% normotensive, 21% white coat, and 21% masked hypertensive. Age, glycemia, and number of antihypertensive drugs were associated with hypertension. Conclusion and Significance. In RTR, HBPM is well accepted and better define BP profile since there is 42% discrepancy between OBPM and HBPM. Whether this discrepancy is associated with worst outcome in the long term remains to be demonstrated

Population Pharmacokinetics of Tacrolimus in Adult Kidney Transplant Patients: Impact of CYP3A5 Genotype on Starting Dose.

Objectives: The aims of this study were to develop a population pharmacokinetic model of tacrolimus in adult kidney transplant recipients, to use this model to compare cytochrome P450 3A5 (CYP3A5) genotype-based initial dosing of tacrolimus with standard per-kilogram-based dosing, and to predict the best starting dose of tacrolimus based on patient genotype to achieve a trough concentration between 6 and 10 mu g/L by day 5 posttransplantation

Donor CYP3A5 genotype influences tacrolimus disposition on the first day after paediatric liver transplantation

AIM The aim of the present study was to investigate the influence of the cytochrome P450 (CYP) 3A4/5 genotype in paediatric liver transplant recipients and donors, and the contribution of age and gender to tacrolimus disposition on the first day after transplantation. METHODS The contribution of the CYP3A4/5 genotype in paediatric liver transplant recipients and donors to the tacrolimus blood trough concentrations (C0) and the tacrolimus concentration/weight-adjusted dose ratio on day 1 was evaluated in 67 liver-transplanted children: 33 boys and 34 girls, mean age 4.5 years. RESULTS Donor CYP3A5 genotype appears to be significantly associated with tacrolimus disposition on the first day after liver transplantation (P < 0.0002). Other physiological factors, such as recipient age and donor gender may also play a role and lead to significant differences in tacrolimus C0 and tacrolimus concentration/weight-adjusted dose ratio on day 1. However, according to the general linear model, only recipient age appears to be independently associated with tacrolimus disposition on the first day after liver transplantation (P < 0.03). Indeed, there was a faster tacrolimus metabolism in children under 6 years of age (P < 0.02). CONCLUSIONS Donor CYP3A5 genotype, recipient age and, to a lesser extent, donor gender appear to be associated with tacrolimus disposition on day 1 after transplant. This suggests that increasing the starting tacrolimus doses in paediatric patients under 6 years of age who receive a graft from a male extensive metabolizer may enhance the possibility of their tacrolimus levels reaching the therapeutic range sooner

CYP3A4 and CYP3A5 genotyping by Pyrosequencing

BACKGROUND: Human cytochrome P450 3A enzymes, particularly CYP3A4 and CYP3A5, play an important role in drug metabolism. CYP3A expression exhibits substantial interindividual variation, much of which may result from genetic variation. This study describes Pyrosequencing assays for key SNPs in CYP3A4 (CYP3A4*1B, CYP3A4*2, and CYP3A4*3) and CYP3A5 (CYP3A5*3C and CYP3A5*6). METHODS: Genotyping of 95 healthy European and 95 healthy African volunteers was performed using Pyrosequencing. Linkage disequilibrium, haplotype inference, Hardy-Weinberg equilibrium, and tag SNPs were also determined for these samples. RESULTS: CYP3A4*1B allele frequencies were 4% in Europeans and 82% in Africans. The CYP3A4*2 allele was found in neither population sample. CYP3A4*3 had an allele frequency of 2% in Europeans and 0% in Africans. The frequency of CYP3A5*3C was 94% in Europeans and 12% in Africans. No CYP3A5*6 variants were found in the European samples, but this allele had a frequency of 16% in the African samples. Allele frequencies and haplotypes show interethnic variation, highlighting the need to analyze clinically relevant SNPs and haplotypes in a variety of ethnic groups. CONCLUSION: Pyrosequencing is a versatile technique that could improve the efficiency of SNP analysis for pharmacogenomic research with the ultimate goal of pre-screening patients for individual therapy selection

Proteinuria and hematuria after remission induction are associated with outcome in ANCA-associated vasculitis

In anti-neutrophil cytoplasmic antibody (ANCA)- associated vasculitis (AAV), hematuria and proteinuria are biomarkers reflecting kidney involvement at diagnosis. Yet, the prognostic value of their persistence after immunosuppressive induction therapy, reflecting kidney damage or persistent disease, remains uncertain. To study this, our post hoc analysis included participants of five European randomized clinical trials on AAV (MAINRITSAN, MAINRITSAN2, RITUXVAS, MYCYC, IMPROVE). Urine protein-creatinine ratio (UPCR) and hematuria of spot urine samples collected at the end of induction therapy (four-six months after treatment initiation) were correlated with the occurrence of a combined end point of death and/or kidney failure, or relapses during follow-up. Among 571 patients (59% men, median age 60), 60% had anti-proteinase 3-ANCA and 35% had anti- myeloperoxidase-ANCA, while 77% had kidney involvement. After induction therapy, 157/526 (29.8%) had persistent hematuria and 165/481 (34.3%) had UPCR of 0.05 g/mmol or more. After a median follow-up of 28 months (interquartile range 18-42), and adjustment for age, ANCA type, maintenance therapy, serum creatinine and persistent hematuria after induction, a UPCR of 0.05 g/mmol or more after induction was associated with significant risk of death/kidney failure (adjusted Hazard Ratio [HR] 3.06, 95% confidence interval 1.09-8.59) and kidney relapse (adjusted subdistribution HR 2.22, 1.16-4.24). Persistent hematuria was associated with significant kidney relapse (adjusted subdistribution HR 2.16, 1.13-4.11) but not with relapse affecting any organ nor with death/kidney failure. Thus, in this large cohort of patients with AAV, persistent proteinuria after induction therapy was associated with death/kidney failure and kidney relapse, whereas persistent hematuria was an independent predictor of kidney relapse. Hence, these parameters must be considered to assess long-term kidney prognosis of patients with AAV.IP1AImmunopathology of vascular and renal diseases and of organ and celltransplantatio