Introduction In the field of heart transplantation and in the context of a global shortage of grafts, new sources of grafts need to be considered. Prior to transplantation, grafts are commonly preserved by being immersed in a solution and kept at 4°C, a method called Cold Static Storage (CSS). Although this method is generally effective, it could cause damage to more fragile organs. Improving graft preservation methods could therefore be the answer to increase the number of available grafts. Objective Our objective is thus to test the effect of different temperatures combined with preservation solutions, especially with LYPS (a new preservation solution developed by our team). We aim to compare those conditions with clinical solutions commonly used for heart preservation by assessing the involvement of different cell death and survival pathways. Method We developed an in-vitro model to simulate the preservation of heart grafts by using cardiomyocytes (both cell lines and primary cells) cultured in different preservation solutions (LYPS, Plegisol, UW, Custodiol and Celsior) and at different temperatures (from 5°C to 25°C). The impact of the preservation method on ischemia-reperfusion injuries was assessed mainly by looking at survival and cell death pathways. Results At the end of conservation, cells preserved in LYPS showed higher viability than other solutions tested (96% for LYPS vs. 68% for Plegisol). In addition, for each solution tested, preservation at 15°C resulted in less cell death than at 4°C. Cells preserved at 15°C in LYPS also showed less commitment to cell death pathways, particularly necrosis, and higher autophagy levels thus suggesting a survival aspect of the latter. Moreover, in those conditions, cells maintained an intact mitochondrial membrane potential (around 80% for LYPS vs. 50% for Plegisol) suggesting less functional alteration. Results were similar in our 2 cellular models, even if the primary cardiomyocyte model needs to be further developed. Conclusion In conclusion, our cellular models are concordant and provides valuable insights to potentially improve graft preservation methods. Interestingly, preservation at 15°C in LYPS provided less cell death and superior maintenance of mitochondrial function.

Introduction Because of the shortage of heart grafts, new strategies are needed to expand the donor pool. We aimed to develop a new dynamic approach to extend graft preservation time, currently limited to 4hours using cold static storage (CSS). Our team also aimed to develop a new preservation solution for organ perfusion, named LYPS. Objective Our objective was thus to determine the optimal perfusion conditions (temperature, perfusion pressure, oxygenation) for cardiac graft preservation and to investigate the underlying mechanisms of cardioprotection. Method An isolated Wistar rat heart graft model was used. Hearts were preserved for 16hours using either static or dynamic preservation in LYPS solution under deep (4°C) or mild (15°C) hypothermia. Dynamic preservation was performed using a perfusion system developed by our team, allowing the control of temperature, perfusion pressure, and oxygenation. After hypothermic preservation, cardiac function was assessed during 1hour of ex-vivo reperfusion and compared with non-preserved control hearts. Implication of cell death and survival pathways were analysed using western blot, immunofluorescence, and enzymatic assays. Results CSS did not allow functional recovery after 16hours of preservation, whereas dynamic preservation enabled successful reanimation. Under deep hypothermia (4°C), optimal conditions consisted of continuous low-pressure perfusion (around 20cmH2O), independent of oxygenation, resulting in 55% functional recovery compared with controls. Under mild hypothermia (15°C), higher perfusion pressures (60–80cmH2O) and oxygenation (21% O2) were required, leading to functional recovery up to 90% of control hearts. Mild hypothermia was associated with higher oxygen and energy substrate consumption during preservation and reduced activation of cell death pathways with enhanced survival signalling. Conclusion In conclusion, optimized dynamic perfusion under deep hypothermia and mild hypothermia allowed a fourfold extension of cardiac preservation time in an ex-vivo rat model. Preservation at 15°C provided superior functional recovery compared with 4°C, likely through reduced cell death and maintenance of energy metabolism.

Abstract Tissue perfusion and oxygenation, key factors of renal ischemia‐reperfusion injury (RIR), can be assessed using contrast‐enhanced ultrasound (CEUS) and photoacoustic (PA) imaging. We hypothesized that early changes in renal perfusion and oxygenation caused by IRI, or protection by mild therapeutic hypothermia (mTH), could be detected and correlate with fibrosis. C57BL6 mice underwent 15‐min unilateral renal ischemia, with or without mTH, followed by 1‐month reperfusion. They were assigned to sham (n = 5), normothermic renal ischemia (IR‐37°C, n = 7), mTH renal ischemia (IR‐34°C, n = 7). The IR‐37°C and IR‐34°C groups had body temperature maintained at 37°C or 34°C during surgery. CEUS and PA were conducted at baseline, 20 min, and 1 month after reperfusion. Histological analysis was performed at 1 month. IRI induced renal atrophy and fibrosis at 1 month, prevented by mTH. CEUS revealed altered perfusion in IR‐37°C at 20 min, sustained at 1 month, compared to Sham, while perfusion was preserved with mTH in IR‐34°C. Oxygenation assessed by PA was not modified. However, oxygenation measured 20 min after IRI correlated with fibrosis at 1 month. CEUS and PA are promising tools for non‐invasive assessment of renal IRI. IRI induced early and sustained perfusion alterations, while early assessment of oxygenation correlated with fibrosis. mTH prevented these alterations.

Introduction New strategies are warranted to expand heart donor pool due to the shortage of available grafts. LYPS preservation solution and its optimal preservation conditions were developed to extend heart graft preservation time, currently limited to 4hours for cold static storage. Objective Our objective was first to compare energy metabolism conservation of currently used cardioplegic solutions (Plegisol, Celsior, Custodiol) with LYPS for static preservation at 4°C. In a second step, we explored energy metabolism between deep (4°C) and midthermia (15°C) during dynamic preservation with LYPS to evaluate the impact of temperature preservation. Method To explore energy metabolism ex-vivo, an isolated Wistar rat heart graft model combined with 31P NMR spectroscopy was used. Hearts were preserved for 4 or 8hours using either static (with Plegisol, Celsior, Custodiol or LYPS at 4°C) or dynamic preservation (LYPS at 4 or 15°C) and placed in a 9.4T bore (Bruker Advance 400) before being reanimated for 1hour with Krebs-Henseleit Buffer at 37°C. Energy metabolism (PCr, ATP, Pi), pHi were quantified during the whole protocol by 31P MRS along with cardiac function during reanimation. Results During 4h of static preservation at 4°C, hearts placed in Celsior solution exhibited higher ATP and PCr pools compared to LYPS, Custodiol and Plegisol, with PCr being noticeable until 2hours of preservation. LYPS maintained higher levels of PCr until 1hour, and a trend in higher ATP levels compared to Plegisol and Custodiol. After reanimation, hearts preserved in LYPS and Celsior showed a significantly similar higher function compared to Plegisol, associated with similar PCr and ATP contents. Interestingly, dynamic perfusion with LYPS, under deep or mild hypothermia, is associated with maintained PCr and ATP throughout the conservation with a trend in higher PCr preservation at 15°C compared to 4°C. No differences were observed for ATP. During reanimation, significantly higher developed pressure, associated with higher PCr and ATP contents were observed with LYPS at 15°C compared to 4°C, suggesting that 15°C midthermia is ideal for dynamic preservation with LYPS. Conclusion Midthermia during dynamic heart preservation with LYPS is preferable, as it ensures better preservation of myocardial energy metabolism, evidenced by a fourfold increase in PCr preservation and associated with superior post-preservation functional recovery compared with deep hypothermia under static or dynamic conditions.

Acute kidney injury (AKI) is a predictor of mortality after a resuscitated cardiac arrest (CA). We aimed to evaluate plasma extracellular cold-induced RNA-binding protein (eCIRP), a pro-inflammatory alarmin released in metabolic stress situations, in predicting AKI after CA. We retrospectively analyzed data of patients prospectively followed-up after their admission to a tertiary hospital following CA. eCIRP levels were assessed using ELISA on frozen plasma samples from admission to day 3. We considered the maximum eCIRP value within the first 24 h (eCIRPmax) for subsequent statistical analyses. We included 153 patients with a median age of 64 years and 91 (59%) were male. The mortality rate was 77%, and AKI occurred in 47% of the patients. Plasma eCIRP was higher in patients who developed AKI (diagnosed after day 1) than in those who did not, regardless of the timing of measurement from admission to day 3, or when using eCIRPmax (Fig. 1A). Plasma eCIRPmax was higher in patients who died than in survivors (Fig. 1B). The AUC of the ROC curve for eCIRPmax in diagnosing AKI was 0.745 (95% CI 0.651–0.840; Fig. 1C). In multiple regression analyses, plasma eCIRPmax remained associated with AKI when accounting for covariates related to patient demographics (Model 1; P < 0.001), admission severity (Model 2; P = 0.018), CA characteristics (Model 3; P < 0.001), or all of them (Model 1 + 2 + 3; P = 0.006). Plasma eCIRP measured in the first 24 h after CA is an independent predictor of AKI and has promising value as a biomarker.

Introduction Due to the shortage of heart grafts, we must improve the quality of graft conservation to increase the pool of donors, in particular by extending the transport time which is currently around 4hours. To do so, we developed a new original preservation solution named LYPS (LYon Preservative Solution), aiming to improved graft protection during preservation. Objective Our objectives were to evaluate the impact of LYPS during hypothermic perfusion on different cell death pathways and their impact on heart functional recovery. Method We used a mimetic cold-ischemia-reperfusion H9C2 cellular model as follows: 20hours of cold preservation, followed by 2hours of warm “reoxygenation” (37°C). In parallel, isolated heart rat graft model was used. Hearts were harvested and submitted to static or dynamic preservation in 3 different solutions (LYPS, Celsior or Plegisol), at deep hypothermia (4°C) and during short (4h) or extended (8h) preservation. Then, cardiac functions were assessed at the time of reperfusion using an ex-vivo retrograde perfusion for 1hour. Cell necrosis, apoptosis, necroptosis and autophagy were evaluated in the two models by flow cytometry and western-blot. Results Flow cytometry in vitro results showed that, after cold hypothermia storage (5°C) and reoxygenation, necrosis (Propidium iodide staining) and apoptosis (annexinV: 10% Control, 9% LYPS vs 76% Plegisol, 85% Celsior) were not significantly different between control and LYPS but they were significantly increased with Celsior and Plegisol. The ex vivo results showed that, for usual static storage time (4h) and extended (8h), there was a significant impact of the solution used, with a better recovery with Lyps and Celsior. This was associated with less LC3II, RIPK3 and cleaved caspase 3 protein expression determined by western blot. Conclusion In conclusion, our data showed that LYPS could offer a cellular protection allowing good preservation of cardiac cells during 20h where commercial solutions failed. Moreover, LYPS appears to provide functional protection in rat ex vivo model, when preservation time was extended, and when perfusion and midthermia were used.

BACKGROUND:In vivo studies have reported cardiovascular benefits of lactate administration after cardiac arrest (CA). However, it remains unclear whether these improvements are due to direct or indirect myocardial effects of lactate. Therefore, we investigated the effects of lactate administration in an ex vivo CA model. METHODS:Isolated rat hearts were subjected to 20 minutes of nonshockable CA followed by 40 minutes of reperfusion. Four groups were analyzed: Control (n = 9), reperfused with standard Krebs buffer; lactate (n = 9), reperfused with Krebs containing 20 mmol/L L-lactate; Lact 5min (n = 5), reperfused with Krebs for 5 minutes and then with Krebs containing 20 mmol/L L-lactate; Lact Hyper (n = 6), reperfused with hypertonic (Na + 163 mmol/L) Krebs containing 20 mmol/L L-lactate. Endpoints included rate-pressure product, left ventricular end-diastolic pressure, coronary flow, and arrhythmia incidence. Troponin and creatine kinase release, mitochondrial calcium retention capacity, and mitochondrial respiration were assessed. RESULTS:Rate-pressure product recovery at the end of reperfusion was 47% ± 4% of baseline in Control versus 45% ± 4%, 40% ± 3%, and 42% ± 3% in Lactate, Lact 5min , and Lact Hyper ( P = ns). Left ventricular end-diastolic pressure and coronary flow were comparable across the groups. Ventricular arrhythmias occurred in 44%, 33%, 40%, and 50% of hearts, respectively ( P = ns). Troponin and creatine kinase levels were similar ( P = ns) between Controls and Lactate-treated hearts. Mitochondrial functions, which were significantly impaired by CA, did not differ significantly between the two groups. CONCLUSIONS:Lactate administration did not improve post-CA myocardial function and was not deleterious. These findings suggest that the in vivo benefits of lactate administration are unlikely due to direct myocardial effects.

Heart transplantation is severely limited by the shortage of suitable donor grafts, partly due to myocardial vulnerability to ischemia–reperfusion injury and the lack of standardized preservation strategies. Current solutions only partially maintain myocardial viability, compromising post-transplant function. To address this issue, we made further improvements to our preservation solution, LYPS (Lyon Preservation Solution), based on mitochondrial metabolic activation and the limitation of membrane depolarization. We first evaluated commonly used extracellular solutions (Celsior and St. Thomas (ST)) on cardiac cell lines (H9C2) exposed to 20 h of cold (4 °C) simulated ischemia followed by 2 h of simulated reperfusion. In parallel, the same three solutions were compared in isolated pig hearts subjected to 20 h of cold static storage followed by reperfusion, with a group directly reperfused with blood at 37 °C serving as the control. Heart function was assessed using a non-working heart preparation, while mitochondrial functions and electrophysiological analysis were evaluated via biopsies and isolated cardiomyocytes. LYPS provided superior protection against cell death and mitochondrial membrane potential loss in vitro, outperformed ST in preserving mitochondrial function, and limited troponin I release by the heart. During reperfusion, LYPS-treated hearts showed improved functional recovery and contractility and better rhythmicity with almost no defibrillation requirements. These effects may involve the modulation of the repolarizing IK1 current. Overall, LYPS effectively preserves myocardial viability and function, representing a promising strategy to enhance graft quality during long-term cold preservation, even through using cold static storage.

Introduction The world of heart transplantation is cruelly impacted by a shortage of grafts. This can be explained by the poor quality of preservation of heart grafts before transplantation and the absence of consensus in the solutions used worldwide. We developed a new original preservation solution named LYPS (for LYon Preservative Solution). Objective Our objective was to compare LYPS to clinical solutions and to determine the impact of temperature on cell death pathways and their impact on heart functional recovery. Method We used a mimetic cold-ischemia-reperfusion H9C2 cellular model (20h preservation at 5-10-15-20 and 25°C, followed by 2h of reoxygenation at 37°C). Cell necrosis, apoptosis, and mitochondrial membrane potential were evaluated by flow cytometry. In parallel, an ex vivo retrograde perfusion rat heart model was used. Hearts were submitted to static preservation in LYPS, Celsior or Plegisol at 4°C and 15°C, then reperfused for 1h where cardiac functions are assessed. A preservation time kinetic was carried out to determine the maximum time at which the rat heart could be resuscitated to regain function (4-8-12-16-20h). Results In vitro results showed that, after 20h of cold hypothermia (5°C) preservation and 2h of reoxygenation, necrosis (2.8% LYPS vs. 79.3%, Plegisol, 61.6%, Celsior and 90% UW, respectively) and apoptosis were significantly reduced in LYPS comparing to other solutions. LYPS was effective in preserving DyM (85% in LYPS vs. 5% in other solutions). Moreover, mid-thermie seemed to be associated with less cell death with all solutions used (for ex: 90% at 4°C vs. 31% at 20°C in Plegisol). Preliminary ex vivo results show that, for short storage times (4h), there is no significant difference impact of the solution used on the cardiac functions but when the storage time is extended to 8 or 12hours, storage in LYPS seems to ensure a better functional recovery in comparison with Plegisol solution. Conclusion Our preliminary data show that LYPS offers a cellular protection that allow a good preservation of cells during 20h where commercial solutions failed. Moreover, LYPS seems to offer better functional protection in ex vivo model, when preservation time is extended.

Abstract Background and Aims Renal 3D photo-acoustic imaging (3D-PAI) and contrast-enhanced ultrasound (CEUS) are promising tools in mice models to assess repeatedly and non-invasively renal ischemia-reperfusion (RIR) consequences and damages, or their improvement by nephroprotective strategies like mild therapeutic hypothermia (mTH), at the early phase of reperfusion as well as in the chronic late phase. Method C57Bl6 mice underwent 15 minutes of unilateral renal vascular clamping, with body temperature at 37°C (RIR-37°C, n=7) or mTH at 34°C (RIR-34°C, n=7), or a sham procedure (Sham, n=5). Renal volume and oxygen saturation (sO2-3D; corresponding to the percentage of oxyhemoglobin over the total hemoglobin content) were measured with 3D-PAI (3D reconstruction of the whole kidney through multi-plane acquisition with an automatized mobile support), and renal perfusion parameters (rBV, mTT, rBF) with CEUS (“destruction-replenishment” model of intravenous microbubbles), performed with VEVO3100 echograph (Fujifilm Visualsonic) 1 week before RIR (basal), 20 minutes after reperfusion, and 1 month after. Renal fibrosis was quantified at 1 month with Masson's trichrome coloration on histological samples. Data were compared with Mann-Whitney test or Wilcoxon test (paired data) as appropriate. Correlation was tested by Spearman test. All animal procedures were approved by the Ethics Committee. Results Sham showed no significant changes during follow-up. RIR-37°C led to renal hypotrophy (volume on 3D-PAI) and fibrosis at 1 month, compared to RIR-34°C (median volume 76 IQR [71-119] vs 143 IQR[108-180] mm3; p=0.03), and those parameters were correlated (R=−0.49; p=0.03). In RIR-37°C, sO2-3D tended to lower at 20 minutes compared to basal values (median sO2-3D 37 IQR [37-44] vs 49 IQR [36-57]%; p=0.08), but not in RIR-34°C (p &gt; 0.99), and these early variations of sO2-3D were correlated with fibrosis at 1 month (R=−0.48; p=0.04). Renal perfusion was altered in RIR-37°C at 20 minutes compared to basal values (median rBV 73 IQR [46-105] vs 100 IQR [89-126] a.u.; p=0.047), and remained altered at 1 month (median rBV 71 IQR [57-83] a.u.; p=0.03), but not in RIR-34°C (p=0.47 at both 20 minutes and 1 month). Renal perfusion early alterations at 20 minutes were correlated with late alterations at 1 month (R=0.63; p=0.005), and tended to correlate with fibrosis (R=−0.41; p=0.08). Conclusion Renal 3D-PAI (on the whole 3D-reconstructed kidney) and CEUS can detect early alterations of renal perfusion and oxygen saturation after RIR, and predict chronic disturbances of perfusion and the onset of fibrosis, as well as the protection conferred by mTH. 3D-PAI can also be used to evaluate non-invasively renal volume as a surrogate of renal fibrosis after RIR.

Background: Myocardial infarction is one of the leading causes of mortality worldwide; hence, there is an urgent need to discover novel cardioprotective strategies. Kynurenic acid (KYNA), a metabolite of the kynurenine pathway, has been previously reported to have cardioprotective effects. However, the mechanisms by which KYNA may be protective are still unclear. The current study addressed this issue by investigating KYNA’s cardioprotective effect in the context of myocardial ischemia/reperfusion. Methods: H9C2 cells and rats were exposed to hypoxia/reoxygenation or myocardial infarction, respectively, in the presence or absence of KYNA. In vitro, cell death was quantified using flow cytometry analysis of propidium iodide staining. In vivo, TTC-Evans Blue staining was performed to evaluate infarct size. Mitochondrial respiratory chain complex activities were measured using spectrophotometry. Protein expression was evaluated by Western blot, and mRNA levels by RT-qPCR. Results: KYNA treatment significantly reduced H9C2-relative cell death as well as infarct size. KYNA did not exhibit any effect on the mitochondrial respiratory chain complex activity. SOD2 mRNA levels were increased by KYNA. A decrease in p62 protein levels together with a trend of increase in PARK2 may mark a stimulation of mitophagy. Additionally, ERK1/2, Akt, and FOXO3α phosphorylation levels were significantly reduced after the KYNA treatment. Altogether, KYNA significantly reduced myocardial ischemia/reperfusion injuries in both in vitro and in vivo models. Conclusion: Here we show that KYNA-mediated cardioprotection was associated with enhanced mitophagy and antioxidant defense. A deeper understanding of KYNA’s cardioprotective mechanisms is necessary to identify promising novel therapeutic targets and their translation into the clinical arena.

It is well known that heart graft deteriorates rapidly during the hypothermic preservation phase before transplantation. Many preservative solutions are used in clinical practice, but there is no consensus on any of them. A new preservation solution named LYon Preservative Solution (LYPS) was developed in our laboratory to improve heart survival during preservation to allow a better functional recovery after transplantation. Our objective is to understand cell death mechanisms that occur during cold ischemia at different temperatures and to test the LYPS compared to the other solutions currently used in clinic (UW, Celsior, and Plégisol). We performed 20 hours of cold to mild-hypothermic (5, 10, 15 and 20 °C) static preservation, followed by 2 hours of warm "reoxygenation" (37 °C) on H9C2 cells. PI, Annexin V and DILC1 flow cytometry staining were used to determined cell necrosis, cell apoptosis and mitochondrial membrane potential respectively at the end of the preservation, and at the end of the reoxygenation. Apoptosis was also quantified by caspase 3 cleavage by Western-Blot. Evaluation of autophagy was made using immunofluorescent labelling of LC3B protein. It appears that LYPS solution protects from necrotic cell death compared to Celsior, UW and Plegisol (8.8% compared to 72.8%; 66.2%; 95% respectively at 5 °C). In addition, LYPS allows a better maintenance of the mitochondrial membrane potential (76% compared to 4.6%; 26%; 3.3%, respectively). This trend is observed at all temperatures studied. Furthermore, we observed that autophagic marker LC3B was stimulated in LYPS compared to other studied solutions. All of these data show that the LYPS solution allows better activation of survival signals, preservation of mitochondrial membrane potential and protection against necrosis. The next step is now to study other cell death pathways such as necroptosis, pyroptosis, and ferroptosis to better understand the mechanisms involved in graft deterioration during preservation in order to find the best preservation conditions and expand the number of potential grafts.

RATIONALE:Mitochondria are key organelles involved in cell survival and death during the acute phenomena of myocardial ischemia-reperfusion (i.e., myocardial infarction). To investigate the functions of isolated mitochondria such as calcium retention capacity, oxidative phosphorylation, and reactive oxygen species (ROS) production, already established methods are based on extramitochondrial measurements of the whole mitochondria population.OBJECTIVE:The aim of this study was to develop a reliable and well-characterized method for multiparametric analysis of isolated single mitochondrion by flow cytometry (FC) in the context of myocardial infarction. The advantage of FC is the possibility to give a simultaneous analysis of morphological parameters (side and forward scatters: SSC and FSC) for each mitochondrion, combined with intramitochondrial measurements of several biological markers, such as ROS production or membrane potential (Δφm), using specific fluorescent probes.METHODS AND RESULTS:For this study, a rat model of ischemia-reperfusion and a protective approach of post-conditioning using low reperfusion pressure was used. Thanks to the use of specific probes (NAO, MTR, TMRM, DilC1, and DHR123) combined with flow cytometry, we propose a method: (i) to identify mitochondrial populations of interest based on quality criteria (NAO/TMRM double staining); (ii) to monitor their morphological criteria, especially during swelling due to calcium overload; and (iii) to compare mitochondrial functions (membrane potential and ROS production) in different experimental groups. Applied to mitochondria from ischemic hearts, these measurements revealed that individual mitochondria are altered and that cardioprotection by low-pressure reperfusion reduces damage, as expected.CONCLUSIONS:Our results highlight FC as a reliable and sensitive method to investigate changes in mitochondrial functions and morphology in pathological conditions that disrupts their activity such as the case in ischemia-reperfusion. This methodological approach can be extended to other pathologies involving mitochondrial dysfunctions. Moreover, FC offers the possibility to work with very small amounts of isolated mitochondria, a factor that may limit the use of classical methods.

Despite the decrease in cardiovascular mortality over the past 3 decades, it still remains the leading cause of death in women. Young women have a lower risk of cardiovascular disease (CVD), but this trend is reversed after menopause. There are many reasons for this difference between men and women, including traditional risk factors such as diabetes, smoking, dyslipidemia, or aging for which women are clearly more impacted than men. Additionally, there are female-specific risk factors, called non-traditional risk factors, that are associated with increased risk of cardiovascular disease in women. These so-called non-traditional risk factors concern women with pre-eclampsia, recurrent pre-eclampsia, gestational diabetes and premature delivery. In addition, there is also an increased risk for women who use contraceptives, who have suffered recurrent miscarriages, premature ovarian failure and early menopause. There are also psychological, social and cultural aspects related to sex. Indeed, lower level of education is more frequently observed in women. Further, numerous pre-clinical animal studies have highlighted some of the cellular mechanisms involved in the differences in the cardiovascular risk in females. These studies have shown a link between high estradiol levels, calcium handling and cardioprotection in young females. In addition, it seems that the mitochondria, which are essential to cardiac function by providing ATP for contraction and play a central role in the management of oxygen and calcium, are also, differentially regulated between males and females. Therefore, it is important to better understand the origin of these differences between men and women in order to improve the diagnosis, prevention and management of CVD in the future.

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.

Ischemia-reperfusion (IR) leads to systemic inflammation. Mild therapeutic hypothermia (mTH) has been suggested to bring protection against IR lesions. However, mTH remains limited to intensive care units (ICU) after resuscitated cardiac arrest (rCA) and is not currently used in nephrology. Extracellular cold-inducible RNA-binding protein (eCIRP) is a pro-inflammatory cytokine that can be secreted during stress situations such as hypoxia. To test if eCIRP plasma level is correlated to renal IR injuries in mice with or without mTH. A 20-minutes bilateral renal ischemia by clamping (or a sham procedure) was conducted on C57BL6 mice with core body temperature maintained at 37 °C (normothermia) or 34 °C (mTH). Plasma eCIRP, IL-6, IL-10 and urea were dosed 2 h and 24 h after reperfusion. Acute tubular necrosis (ATN) was scored (from 0 to 4) on histological exam of kidneys at 24 h. Bilateral renal ischemia in mice was associated with AKI compared to sham as soon as 2 h after reperfusion (median urea 18.7 vs. 11.7 mmol/L, P = 0.02) and more severe 24 h after reperfusion (median urea 60.8 vs. 6.2 mmol/L, P = 0.004), and with ATN 24 h after reperfusion (median histological score 2.23 vs. 0.80, P < 0.001). Plasma eCIRP levels were significantly increased by renal IR 24 h after reperfusion compared to sham (median 123.1 vs. 21.5 pg/mL, P = 0.03). Renal IR also led to a peak of IL-6 secretion 2 h after reperfusion, and of IL-10 secretion 24 h after reperfusion. We showed a correlation between eCIRP and urea levels 2 h after reperfusion (Spearman r = 0.60, P = 0.007) and 24 h after reperfusion (Spearman r = 0.84, P < 0.001), and with ATN score (Spearman r = 0.65, P < 0.001). The use of mTH during renal ischemia was followed by a significantly reduced plasma urea (median 22.7 mmol/L, P = 0.001), ATN score (median 1.45, P = 0.03) and eCIRP elevation (median 60.8 pg/mL, P = 0.03) 24 h after reperfusion, and an inhibition of IL-6 and IL-10 secretion (Fig. 1). Plasma eCIRP increases 24 h after renal IR in mice and is correlated to AKI and ATN. The use of mTH during ischemia alleviates eCIRP elevation in parallel of renal IR injury and inflammation. We aim to measure eCIRP level in ICU after rCA (i.e. global IR) at admission, and at day 1 and 3 to confirm our results in a cohort of 33 patients in order to test if eCIRP is correlated to relevant clinical and biological outcomes, such as AKI and its severity.