The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model.

The eIF5A hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) was shown to protect from ischemic injuries. We hypothesized that GC7 could be useful for preconditioning kidneys from donors before transplantation. Using a preclinical porcine brain death (BD) donation model, we carried out in vivo evaluation of GC7 pretreatment (3 mg/kg iv, 5 minutes after BD) at the beginning of the 4h-donor management, after which kidneys were collected and cold-stored (18h in University of Wisconsin solution) and 1 was allotransplanted. Groups were defined as following (n = 6 per group): healthy (CTL), untreated BD (Vehicle), and GC7-treated BD (Vehicle + GC7). At the end of 4h-management, GC7 treatment decreased BD-induced markers, as radical oxygen species markers. In addition, GC7 increased expression of mitochondrial protective peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC1α) and antioxidant proteins (superoxyde-dismutase-2, heme oxygenase-1, nuclear factor [erythroid-derived 2]-like 2 [NRF2], and sirtuins). At the end of cold storage, GC7 treatment induced an increase of NRF2 and PGC1α mRNA and a better mitochondrial integrity/homeostasis with a decrease of dynamin- related protein-1 activation and increase of mitofusin-2. Moreover, GC7 treatment significantly improved kidney outcome during 90 days follow-up after transplantation (fewer creatininemia and fibrosis). Overall, GC7 treatment was shown to be protective for kidneys against BD-induced injuries during donor management and subsequently appeared to preserve antioxidant defenses and mitochondria homeostasis; these protective effects being accompanied by a better transplantation outcome.

Influence of Hypoxic Preservation Temperature on Endothelial Cells and Kidney Integrity.

Ischemia-reperfusion (IR) injury is unavoidable during organ transplantation and impacts graft quality. New paradigms are emerging including preservation at higher temperature than "hypothermia" or "cold": although 4°C remains largely used for kidney preservation, recent studies challenged this choice. We and others hypothesized that a higher preservation temperature, closer to physiological regimen, could improve organ quality. For this purpose, we used an in vitro model of endothelial cells exposed to hypoxia-reoxygenation sequence (mimicking IR) and an ex vivo ischemic pig kidneys static storage model. In vitro, 19°C, 27°C, and 32°C provided protection against injuries versus 4°C, by reducing cell death, mitochondrial dysfunction, leukocyte adhesion, and inflammation. However, ex vivo, the benefits of 19°C or 32°C were limited, showing similar levels of tissue preservation damage. Ex vivo 4°C-preserved kidneys displayed a trend towards reduced damage, including apoptosis. Macrophage infiltration, tubulitis, and necrosis were increased in the 19°C and 32°C versus 4°C preserved kidneys. Thus, despite a trend for an advantage of subnormothermia as preservation temperature, our in vitro and ex vivo models bring different insights in terms of preservation temperature effect. This study suggests that temperature optimization for kidney preservation will require thorough investigation, combining the use of complementary relevant models and the design of elaborated preservation solution and new technologies.

Rapid or Slow Time to Brain Death? Impact on Kidney Graft Injuries in an Allotransplantation Porcine Model.

The use of donors deceased after brain death (DBD) with extended criteria in response to the shortage of grafts leads to the removal of more fragile kidneys. These grafts are at greater risk of not being grafted or delayed function. A better knowledge of the pathophysiology of DBDs would improve this situation. There is a difference between the results from animal models of DBD and the clinical data potentially explained by the kinetics of brain death induction. We compared the effect of the induction rate of brain death on the recovery of post-transplant renal function in a pig model of DBD followed by allografts in nephrectomized pigs. Resumption of early function post-transplant was better in the rapidly generated brain death group (RgBD) and graft fibrosis at three months less important. Two groups had identical oxidative stress intensity but a greater response to this oxidative stress by SIRT1, PGC1-α and NRF2 in the RgBD group. Modulation of mechanistic target of rapamycin (mTOR) stimulation by NRF2 would also regulate the survival/apoptosis balance of renal cells. For the first time we have shown that an allostatic response to oxidative stress can explain the impact of the rapidity of brain death induction on the quality of kidney transplants.

Individual and Combined Impact of Oxygen and Oxygen Transporter Supplementation during Kidney Machine Preservation in a Porcine Preclinical Kidney Transplantation Model.

Marginal kidney graft preservation in machine perfusion (MP) is well-established. However, this method requires improvement in order to mitigate oxidative stress during ischemia-reperfusion, by using oxygenation or an O2 carrier with anti-oxidant capacities (hemoglobin of the marine worm; M101). In our preclinical porcine (pig related) model, kidneys were submitted to 1h-warm ischemia, followed by 23 h hypothermic preservation in Waves® MP before auto-transplantation. Four groups were studied: W (MP without 100%-O2), W-O2 (MP with 100%-O2; also called hyperoxia), W-M101 (MP without 100%-O2 + M101 2 g/L), W-O2 + M101 (MP with 100%-O2 + M101 2 g/L) (n = 6/group). Results: Kidneys preserved in the W-M101 group showed lower resistance, compared to our W group. During the first week post-transplantation, W-O2 and W-M101 groups showed a lower blood creatinine and better glomerular filtration rate. KIM-1 and IL-18 blood levels were lower in the W-M101 group, while blood levels of AST and NGAL were lower in groups with 100% O2. Three months after transplantation, fractional excretion of sodium and the proteinuria/creatinuria ratio remained higher in the W group, creatininemia was lower in the W-M101 group, and kidney fibrosis was lower in M101 groups. We concluded that supplementation with M101 associated with or without 100% O2 improved the Waves® MP effect upon kidney recovery and late graft outcome.

Defining the optimal duration for normothermic regional perfusion in the kidney donor: A porcine preclinical study.

Kidneys from donation after circulatory death (DCD) are highly sensitive to ischemia-reperfusion injury and thus require careful reconditioning, such as normothermic regional perfusion (NRP). However, the optimal NRP protocol remains to be characterized. NRP was modeled in a DCD porcine model (30 minutes of cardiac arrest) for 2, 4, or 6 hours compared to a control group (No-NRP); kidneys were machine-preserved and allotransplanted. NRP appeared to permit recovery from warm ischemia, possibly due to an increased expression of HIF1α-dependent survival pathway. At 2 hours, blood levels of ischemic injury biomarkers increased: creatinine, lactate/pyruvate ratio, LDH, AST, NGAL, KIM-1, CD40 ligand, and soluble-tissue-factor. All these markers then decreased with time; however, AST, NGAL, and KIM-1 increased again at 6 hours. Hemoglobin and platelets decreased at 6 hours, after which the procedure became difficult to maintain. Regarding inflammation, active tissue-factor, cleaved PAR-2 and MCP-1 increased by 4-6 hours, but not TNF-α and iNOS. Compared to No-NRP, NRP kidneys showed lower resistance during hypothermic machine perfusion (HMP), likely associated with pe-NRP eNOS activation. Kidneys transplanted after 4 and 6 hours of NRP showed better function and outcome, compared to No-NRP. In conclusion, our results confirm the mechanistic benefits of NRP and highlight 4 hours as its optimal duration, after which injury markers appear.

Decoding cold ischaemia time impact on kidney graft: the kinetics of the unfolded protein response pathways.

The relationship between cold ischaemia time (CIT) and adverse outcome is now acknowledged. However, the underlying mechanisms remain to be defined, which slows the development of adapted therapeutics and diagnostics. We explored the impact of CIT in both preclinical and in vitro models of preservation. We determined that the endoplasmic reticulum (ER) and its stress response (unfolded protein response, UPR) were regulated in close association with CIT; the eIF2α-ATF4 pathway was inhibited early (1-8 h) at the detriment of cell survival, while the ATF6 pathway was activated late (12-24 h) and associated with cell death. The IRE1α-XBP1 branch was activated at reperfusion only if CIT extended beyond 8 h, and had a dual role on cell fate - deleterious through IRE1's RNase activity and beneficial through IRE1α other roles. Finally, the pro-apoptotic factor CHOP was a common target of both ATF6 and IRE1α pathways and was associated with elongated CIT and increased cell death. Microarray analysis of human transplanted kidney confirmed that UPR markers were regulated by CIT and that CHOP was associated with adverse outcome. We show that UPR could be a critical pathway explaining the relationship between CIT and graft outcome, highlighting the potential for UPR-based therapeutics and diagnostics to improve transplantation.

Dynamic transcriptomic analysis of Ischemic Injury in a Porcine Pre-Clinical Model mimicking Donors Deceased after Circulatory Death.

Due to organ shortage, clinicians are prone to consider alternative type of organ donors among them donors deceased after circulatory death (DCD). However, especially using these organs which are more prone to graft dysfunction, there is a need to better understand mechanistic events ocuring during ischemia phase and leading to ischemia/reperfusion injuries (IRI). The aim of this study is to provide a dynamic transcriptomic analysis of preclinical porcine model kidneys subjected to ischemic stress mimicking DCD donor. We compared cortex and corticomedullary junction (CMJ) tissues from porcine kidneys submitted to 60 min warm ischemia (WI) followed by 0, 6 or 24 hours of cold storage in University of Wisconsin solution versus control non-ischemic kidneys (n = 5 per group). 29 cortex genes and 113 CMJ genes were significantly up or down-regulated after WI versus healthy kidneys, and up to 400 genes were regulated after WI followed by 6 or 24 hours of cold storage (p < 0.05). Functionnal enrichment analysis (home selected gene kinetic classification, Gene-ontology-biological processes and Gene-ontology-molecular-function) revealed relevant genes implication during WI and cold storage. We uncovered targets which we will further validate as biomarkers and new therapeutic targets to optimize graft kidney quality before transplantation and improve whole transplantation outcome.

Therapeutic drug monitoring of clomipramine and amitriptyline in a depressed patient with upper digestive tract resection.

We describe a 55-year-old woman with extensive digestive resection and recurrent depressive disorder resistant to oral clomipramine tablets but not to an oral solution of amitriptyline. In the light of this case report, the potential mechanisms of drug resistance after digestive resection are discussed, including the importance of drug monitoring.

[Factors for success in the first year of medical school (PCEM 1) at Paris V University (France)]. / Critères de réussite en première année de médecine (PCEM 1) à Paris V.

OBJECTIVES: The first year of medical school in France (PCEM 1) is very competitive, as the 2004-2005 success rate at the Paris V university was below 20%. Future medical students, as well as the other actors of medical education, are therefore willing to know what are the factors for success in PCEM 1 as well as whether or not the widespread complementary classes are necessary. METHODS: In order to determine these factors, a survey among the first and second year medical students (PCEM 1 and PCEM 2) was carried on. RESULTS AND CONCLUSION: The only two significant success factors found were the good results obtained at the scientific high school graduation exam (Baccalauréat S) and doing the first year for the second time. Neither attending complementary classes nor a short time to go to the faculty seemed to higher the odds for success.

Gamma-observable neighbours for vector quantization.

We define the gamma-observable neighbourhood and use it in soft-competitive learning for vector quantization. Considering a datum v and a set of n units w(i) in a Euclidean space, let v(i) be a point of the segment [vw(i)] whose position depends on gamma a real number between 0 and 1, the gamma-observable neighbours (gamma-ON) of v are the units w(i) for which v(i) is in the Voronoï of w(i), i.e. w(i) is the closest unit to v(i). For gamma = 1, v(i) merges with w(i), all the units are gamma-ON of v, while for gamma = 0, v(i) merges with v, only the closest unit to v is its gamma-ON. The size of the neighbourhood decreases from n to 1 while gamma goes from 1 to 0. For gamma lower or equal to 0.5, the gamma-ON of v are also its natural neighbours, i.e. their Voronoï regions share a common boundary with that of v. We show that this neighbourhood used in Vector Quantization gives faster convergence in terms of number of epochs and similar distortion than the Neural-Gas on several benchmark databases, and we propose the fact that it does not have the dimension selection property could explain these results. We show it also presents a new self-organization property we call 'self-distribution'.