Acute kidney injury (AKI) is a major public health concern, which contributes to serious hospital complications, chronic kidney disease (CKD) and even death. Renal ischemia- reperfusion injury (IRI) remains a leading cause of AKI.IRI combines major cell stress, significant burst of free radicals, and strong inflammatory responses leading to extensive cell injury, necrosis, and late interstitial fibrosis. Moreover, IRI- induced AKI releases pro-inflammatory cytokines (e.g. IL-1β, TNF-α, IL-6) that induce a systemic inflammatory response, resulting in pro-inflammatory cells recruitment and remote organ damage. AKI is associated with poor outcomes, particularly when extrarenal complications or distant organ injuries occur.The stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against renal IRI and may be preventively induced using hemin prior to renal insult. This HO-1 induction pathway called hemin preconditioning is largely known in the literature to be effective.We first confirmed that hemin-induced HO-1 improved renal outcomes after IRI (i.e. fewer renal damage, renal inflammation and oxidative stress). We then demonstrated that this protective pathway mitigated AKI-induced ALI, a major extrarenal complication after renal IRI, through modulation of systemic and lung inflammation.Afterwards, we focused on the specific contribution of myeloid HO-1 to renal IRI, which remains poorly characterized. We therefore investigated the contribution of myeloid HO-1 to renal IRI using mice with myeloid-restricted deletion of HO-1 (HO-1M-KO). We observed that myeloid HO-1 appeared to be a critical regulator of the earliest phases of IRI (i.e. higher plasma creatinine, tubular damage, and renal inflammation/oxidative stress in HO-1M-KO mice).As a link between the severity of renal injury and the risk maladaptive repair leading to CKD has been established, we thereby decided to focus on tubular repair and fibrosis deposition upon IRI. We identified that myeloid HO-1 prevented maladaptive repair and subsequent CKD through modulation of cell-cycle and autophagy regulatory proteins.We then showed that hemin-mediated protection requires specific expression of HO-1 within myeloid cells. We therefore identified CD11b+ F4/80lo macrophages as the main protective myeloid source of HO-1 upon renal IRI. Interestingly, we observed this myeloid cell sub- population in the kidney and spleen, suggesting that protective effects might be provided by both tissue-resident and infiltrating/circulating HO-1+ myeloid cells.Based on its promising cytoprotective effects when giving preemptively, we investigated the use of hemin-induced myeloid HO-1 as a strategy to mitigate established AKI. However, due to its chemical structure and oxidative properties, hemin worsened IRI-induced AKI. We thereby identified that hemin had a dual effect on renal IRI, protective or deleterious, depending on the timing of its administration.Altogether, this work suggests that myeloid HO-1 plays a critical role in the modulation of IRI- induced AKI by improving short- and long-term functional outcomes after renal IRI. We conclude that hemin-induced myeloid HO-1 pathway might be an efficient preventive strategy in many renal IRI situations with predictable AKI such as renal transplantation or partial nephrectomy. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Ischemic heart disease is a leading cause of death worldwide. Osteopontin (OPN), a cell-secreted extracellular matrix protein, is suggested to play a cardioprotective role in mouse models of ischemic heart disease. The objective of this study was to examine the role of OPN in modulation of systolic and diastolic functional parameters of the heart following mouse ischemia/reperfusion (I/R) injury. For this, wild-type (WT) and OPN-knockout (KO) mice aged approximately 4 months were subjected to cardiac ischemia for 45 minutes by the ligation of the left anterior descending coronary artery (LAD) followed by reperfusion of LAD by snipping the ligature. Heart function was measured using echocardiography at baseline, 1, 3, 7, 14, and 27 days post-I/R injury. M-mode echocardiographic images were used to calculate % fractional shortening [%FS], % ejection fraction [%EF], end-systolic volume [ESV], and end-diastolic volume [EDV], while pulsed wave Doppler images were used to measure aortic ejection time [AET], isovolumic relaxation time [IVRT], and total systolic time [TST]. Velocity of circumferential fiber shortening (Vcf) was calculated using FS and AET. I/R injury significantly decreased %EF and %FS in both WT and KO groups at all time points (1, 3, 7, 14, and 27 days post-I/R) versus the baseline. However, the decrease in % EF and %FS was significantly greater in KO-I/R group versus WT-I/R at 3, 7, 14 and 27 days post-I/R. I/R-mediated increase in ESV and EDV were significantly greater in KO-MI group versus WT-MI 3 day post-I/R. AET was significantly higher in WT-I/R group 27 days post-I/R versus baseline. However, AET was significantly lower in KO-I/R group 3 and 27 days post-I/R versus WT-I/R. IVRT was significantly higher in KO-I/R group 27 days post-I/R vs baseline. However, IVRT was significantly lower in KO-I/R group 1 day post-I/R vs WT-I/R. TST remained unchanged in WT and KO groups post-I/R versus their respective baseline groups. However, TST was significantly lower in KO-I/R group versus WT-I/R at 3 days post-I/R. Vcf was significantly higher at basal levels in the KO versus WT mice. I/R injury decreased Vcf in both groups versus their baseline at all time-points. These data provide evidence that lack of OPN deteriorates systolic and diastolic functional parameters of the heart following I/R injury, suggesting a cardioprotective role of OPN in myocardial remodeling post-IR.

Background: Myocardial apoptosis plays an important role in myocardial ischemia/reperfusion (I/R) injury. Activation of PI3K/Akt signaling protects the myocardium from I/R injury. This study investigated the role of miR-214 in hypoxia/ reoxygenation (H/R)-induced cell damage in vitro and myocardial I/R injury in vivo. Methods and Results: H9C2 cardiomyoblasts were transfected with lentivirus expressing miR-214 (LmiR-214) or lentivirus expressing scrambled miR-control (LmiR-control) respectively, to establish cell lines of LmiR-214 and LmiR-control. The cells were subjected to hypoxia for 4 h followed by reoxygenation for 24 h. Transfection of LmiR-214 suppresses PTEN expression, significantly increases the levels of Akt phosphorylation, markedly attenuates LDH release, and enhances the viability of the cells subjected to H/R. In vivo transfection of mouse hearts with LmiR-214 significantly attenuates I/R induced cardiac dysfunction and reduces I/Rinduced myocardial infarct size. LmiR-214 transfection significantly attenuates I/Rinduced myocardial apoptosis and caspase-3/7 and caspase-8 activity. Increased expression of miR-214 by transfection of LmiR-214 suppresses PTEN expression, increases the levels of phosphorylated Akt, represses Bim1 expression and induces Bad phosphorylation in the myocardium. In addition, in vitro data shows transfection of miR-214 mimics to H9C2 cells suppresses the expression and translocation of Bim1 from cytosol to mitochondria and induces Bad phosphorylation. Conclusions: Our in vitro and in vivo data suggests that miR-214 protects cells from H/R induced damage and attenuates I/R induced myocardial injury. The mechanisms involve activation of PI3K/Akt signaling by targeting PTEN expression, induction of Bad phosphorylation, and suppression of Bim1 expression, resulting in decreases in I/R-induced myocardial apoptosis.

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 ± 3% in the transgenic hearts as compared to 30 ± 5% in the nontransgenic hearts (p < .05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

Scavenger receptor A (SR-A), also known as the macrophage scavenger receptor and cluster of differentiation 204 (CD204), plays roles in lipid metabolism, atherogenesis, and a number of metabolic processes. However, recent evidence points to important roles for SR-A in infammation, innate immunity, host defense, sepsis, and ischemic injury. Herein, we review the role of SR-A in infammation, innate immunity, host defense, sepsis, cardiac and cerebral ischemic injury, Alzheimer's disease, virus recognition and uptake, bone metabolism, and pulmonary injury. Interestingly, SR-A is reported to be host protective in some disease states, but there is also compelling evidence that SR-A plays a role in the pathophysiology of other diseases. These observations of both harmful and beneficial effects of SR-A are discussed here in the framework of inflammation, innate immunity, and endoplasmic reticulum stress.

Aims: Bifunctional apoptosis regulator (BAR) is an endoplasmic reticulum protein that interacts with both the extrinsic and intrinsic apoptosis pathways. We hypothesize that over-expression of BARΔRING prevents apoptosis and injury following ischaemia/reperfusion (I/R) and attenuates doxorubicin (DOX)-induced cardiotoxicity. Methods and results: We generated a line of transgenic mice that carried a human BARΔRING transgene under the control of the mouse α-myosin heavy chain promoter. The RING domain, which binds ubiquitin conjugating enzymes, was deleted to prevent auto-ubiquitination of BAR and allow accumulation of the BAR protein, which binds apoptosis-regulating proteins. High levels of human BARΔRING transcripts and 42 KDa BARΔRING protein were expressed in the hearts of transgenic mice. When excised hearts were reperfused ex vivo for 45 min as Langendorff preparations after 45 min of global ischaemia, the functional recovery of the hearts, expressed as left ventricular developed pressure x heart rate, was 23 ± 1.7% in the non-transgenic hearts compared with 51.5 ± 4.3% in the transgenic hearts (P < 0.05). For in vivo studies, mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by 4 h of reperfusion. The infarct sizes following I/R injury, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (29 ± 4 vs. 55 ± 4%, P < 0.05). In hearts of mice subjected to cardiac I/R injury, BAR transgenic hearts had significantly fewer in situ oligo-ligation-positive cardiac cells (5.0 ± 0.4 vs. 13.4 ± 0.5%, P < 0.05). Over-expression of BARΔRING also significantly attenuated DOX-induced cardiac dysfunction and apoptosis. Conclusion: Our results demonstrate that over-expression of BARΔRING renders the heart more resistant to I/R injury and DOX-induced cardiotoxicity, and this protection correlates with reduced cardiomyocyte apoptosis.

Free-radical generation is one of the primary causes of myocardial ischemia/reperfusion (I/R) injury. Melatonin is an efficient free-radical scavenger and induces the expression of antioxidant enzymes. We have previously shown that melatonin can prevent free-radical-induced myocardial injury. To date, the mechanism underlying melatonin's cardioprotective effect is not clear. In this study, we assessed the ability of melatonin to protect against I/R injury in mice deficient in glutathione peroxidase 1 (Gpx1). Mice hearts were subjected to 40 min of global ischemia in vitro followed by 45 min of reperfusion. Myocardial I/R injury (expressed as % of recovery of left ventricular developed pressure × heart rate) was exacerbated in mice deficient in Gpx1 (51 ± 3% for Gpx1+/+ mice versus 31 ± 6% for Gpx1-/- mice, P < 0.05). Administration of melatonin for 30 min protected against I/R injury in both Gpx1+/+ mice (72 ± 4.8%) and Gpx1-/- mice (63 ± 4.7%). This protection was accompanied by a significant improvement in left ventricular end-diastolic pressure and a twofold decrease in lactate dehydrogenase (LDH) level released from melatonin-treated hearts. In another set of experiments, mice were subjected to 50 min of ligation of the left descending anterior coronary artery in vivo followed by 4 hr of reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly larger in Gpx1-/- mice than in Gpx1+/+ mice (75 ± 9% versus 54 ± 6%, P < 0.05) and were reduced significantly in melatonin-treated mice (31 ± 3.7% Gpx1-/- mice and 33 ± 6.0% Gpx1+/+ mice). In hearts subjected to 30 min of coronary artery occlusion followed by 3 hr of reperfusion, melatonin-treated hearts had significantly fewer in situ oligo ligation-positive myocytes and less protein nitration. Our results demonstrate that the cardioprotective function of melatonin is independent of Gpx1.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18645号 / 医博第3944号 / 新制||医||1006(附属図書館) / 31559 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 羽賀 博典, 教授 千葉 勉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19226号 / 医博第4025号 / 新制||医||1011(附属図書館) / 32225 / 京都大学大学院医学研究科医学専攻 / (主査)教授 福田 和彦, 教授 木村 剛, 教授 三嶋 理晃 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM