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Renal ischemia reperfusion injury (IRI) is associated with significant morbidity and mortality. Given the importance of microRNAs (miRNAs) in regulating gene expression, we examined expression profiles of miRNAs following renal IRI. Global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805, and miR-194) that are differentially expressed following IRI when compared with sham controls. These miRNAs were also differently expressed following IRI in immunodeficient RAG-2/ common [gamma]-chain double-knockout mice, suggesting that the changes in expression observed are not significantly influenced by lymphocyte infiltration and therefore define a lymphocyte-independent signature of renal IRI. In vitro studies revealed that miR-21 is expressed in proliferating tubular epithelial cells (TEC) and up-regulated by both cell-intrinsic and -extrinsic mechanisms resulting from ischemia and TGF-[beta] signaling, respectively. In vitro, knockdown of miR-21 in TEC resulted in increased cell death, whereas overexpression prevented cell death. However, overexpression of miR-21 alone was not sufficient to prevent TEC death following ischemia. Our findings therefore define a molecular fingerprint of renal injury and suggest miR-21 may play a role in protecting TEC from death. kidney | miR-21 | tubular epithelial cells | programmed cell death protein 4 doi/ 10.1073/pnas.0912701107

In the United States, angioplasty or bypass surgery to remove coronary occlusions is performed on approximately two million patients each year. Although reperfusion is essential for salvaging ischemic myocardium, it also promotes infarction by activating programmed cell death in the formerly ischemic tissue. Reperfusion injury begins when oxidative stress and calcium accumulation by the mitochondria cause activation of the so-called mitochondrial death channels. These channels have become the focus of evolving strategies to protect the heart from infarction. Preclinical and preliminary clinical studies indicate that agents with diverse modes of action can reduce infarct size by 50% or more and significantly preserve myocardial functions. This article reviews the most advanced pharmacological approaches for their ability to reduce infarct size by inhibiting the mitochondrial death pathways.

Previously, we demonstrated gender differences in Na-K-ATPase (NKA) expression and function after renal ischemia-reperfusion (I/R) injury (Sex differences in the alterations of [Na.sup.+], [K.sup.+]-ATPase following ischemia-reperfusion injury in the rat kidney. J Physiol 555: 471-480, 2004). Postischemic membrane destruction causes inhibition of NKA, whereas heat shock protein (HSP) 72 helps to preserve it. We tested the sex differences in postischemic expression of HSP72 and colocalization with NKA. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 (T2), 16 (TI6), and 24 h (T24) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls. Postischemic blood urea nitrogen (BUN), serum creatinine, and renal histology were analyzed. HSP72 mRNA expression was detected by RT-PCR, protein levels by Western blot analysis. Fluorescent immunohistochemistry was performed to evaluate the localization of HSP72 and NKA [[alpha].sub.1]-subunit. Postischemic BUN and creatinine were higher, and renal histology showed more rapid progression in M vs. F (P < 0.05). HSP72 mRNA expression was higher in F vs. M in control and in all I/R groups (P < 0.05). Similar changes were observed in HSP72 protein levels (F vs. M, P < 0.05, control, T2, TI6, T24, respectively). Immunohistochemical localization of HSP72 and NKA [[alpha].sub.1] was similar in control F and M. In postischemic F kidneys, the majority of NKA [[alpha].sub.1] and HSP72 was colocalized on the basolateral membrane of tubular cells, whereas in M prominent staining was observed in the cytosol and apical domain. This study indicates that in female kidneys the higher basal and postischemic levels of HSP72 and different colocalization with NKA might contribute to the gender differences in renal I/R injury. gender

Matrix metalloproteinase-2 (MMP-2) is a central component of the response to injury in the heart. During ischemia, MMP-2 influences ventricular performance and is a determinant of postinfarction remodeling. Elevation of MMP-2 during reperfusion after ischemia suggests that new protein is synthesized, but the molecular regulation of MMP-2 generation during ischemia-reperfusion (I/R) injury has not been studied. Using the MMP-2 promoter linked to a [beta]-galactosidase reporter in transgenic mice, we investigated the transcriptional regulation and cellular sources of MMP-2 in isolated, perfused mouse hearts subjected to acute global I/R injury. I/R injury induced a rapid activation of MMP-2 promoter activity with the appearance of [beta]-galactosidase antigen in cardiomyocytes, fibroblasts, and endothelial cells. Activation of intrinsic MMP-2 transcription and translation was confirmed by real-time PCR and quantitative Western blot analyses. MMP-2 transcription and translation were inhibited by perfusion with 1.0 mM hydroxyl radical scavenger N-(-2-mercaptopropionyl)-glycine. Nuclear extracts demonstrated increased abundance of two activator proteins-1 (AP-1) components JunB and FosB following I/R injury. Immunohistochemical staining localized JunB and FosB proteins to the nuclei of all three cardiac cell types following UR injury, consistent with enhanced nuclear transport of these transcription factors. Chromatin immunoprecipitation (ChIP) of the AP-1 binding site in the intrinsic murine MMP-2 promoter yielded only JunB under control conditions, whereas ChIP following I/R injury recovered both JunB and FosB, consistent with a change in occupancy from JunB homodimers in controls to JunB/FosB heterodimers following I/R injury. We conclude that enhanced MMP-2 transcription and translation following I/R injury are mediated by induction, via oxidant stress, of discrete AP-1 transcription factor components. reperfusion injury; transcription factor; activator protein-1

To examine ischemic tolerance in the absence of [A.sub.1] adenosine receptors ([A.sub.1]ARs), isolated wild-type (WT) and [A.sub.1]AR knockout ([A.sub.1]KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either [A.sub.1]AR deletion and/or ischemia-reperfusion. [A.sub.1]KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by [A.sub.1]AR deletion. After 20 min of ischemia, CF was attenuated in [A.sub.1]KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A1KO hearts (54.4 [+ or -] 5.1 vs. WT 81.1 [+ or -] 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in [A.sub.1]KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of [A.sub.1]AR transcript in [A.sub.1]KO hearts, and the message for [A.sub.2A], [A.sub.2B] and [A.sub.3] adenosine receptors was similar in uninstrumented [A.sub.1]KO and WT hearts. Ischemia-reperfusion increased [A.sub.2B] mRNA expression 2.5-fold in both WT and [A.sub.1]KO hearts without changing [A.sub.1] or [A.sub.3] expression. In WT hearts, ischemia transiently doubled [A.sub.2A] mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in [A.sub.1]KO hearts. Together, these data affirm the cardioprotective role of [A.sub.1]ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts. gene expression; coronary flow; lactate dehydrogenase

Epidemiological studies have linked ambient particulate matter (PM) levels to an increased incidence of adverse cardiovascular events. Yet little is definitively known about the mechanisms accounting for the cardiovascular events associated with PM exposure. The goal of this study was to determine the effects of ultrafine ( myocardial infarction; vascular reactivity; oxidant injury; acetylcholine doi:10.1152/ajpheart.01362.2005

Water accumulation in the heart is important in ischemia-reperfusion injury and operations performed by using cardiopulmonary bypass, with cardiac dysfunction associated with myocardial edema being the principal determinant of clinical outcome. As an initial step in determining the role of aquaporin (AQP) water channels in myocardial edema, we have assessed the myocardial expression of AQPs in humans, rats, and mice. RT-PCR revealed expression of AQP-1, -4, -6, -7, -8, and -11 transcripts in the mouse heart. AQP-1, -6, -7, and -11 mRNAs were found in the rat heart as well as low levels of AQP-4 and -9. Human hearts contained AQP-1, -3, -4, -5, -7, -9, -10, and -11 mRNAs. AQP-1 protein expression was confirmed by Western blot analysis in all three species. AQP-4 protein was detected in the mouse heart but not in the rat or human heart. To determine the potential functional consequences of myocardial AQP expression, water permeability was measured in plasma membrane vesicles from myocardial cells of wild-type versus various AQP knockout mice. Water permeability was reduced by AQP-1 knockout but not by AQP-4 or AQP-8 knockout. With the use of a model of isolated rat heart perfusion, it was found that osmotic and ischemic stresses are not associated with changes in AQP-1 or AQP-4 expression. These studies support a possible functional role of AQP-1 in myocardium but indicate that early adaptations to osmotic and ischemic stress do not involve transcriptional or posttranslational AQP-1 regulation. aquaporin knockout; cardiac myocyte; cardiopulmonary bypass doi:10.1152/ajpheart.00090.2006

Ischemia-reperfusion activates ERK and p38 MAPK in cardiac membranes, but the role of caveolae in MAPK signaling during this stress has not been studied. The purpose of this study was to determine the effect of in vivo myocardial ischemia-reperfusion on the level and distribution of caveolin-1 and -3 and cholesterol as well as MAPK activation in caveolin-enriched fractions. Adult male rats were subjected to in vivo regional myocardial ischemia induced by 25 min of coronary artery occlusion and 10 min (n = 5) or 2 h (n = 4) of reperfusion. Another group of rats served as appropriate nonischemic time controls (n = 4). A discontinuous sucrose density gradient was used to isolate caveolae/lipid rafts from ischemic and nonischemic heart tissue. Caveolin-1 and -3, as well as cholesterol, were enriched in the light fractions. A redistribution of caveolin-3 and a reduction in caveolin-1 and cholesterol levels in the light fractions occurred after 10 min of reperfusion. The ERKs were activated in ischemic zone light and heavy fractions by 10 min of reperfusion, p44 ERK was activated alter 2 h of reperfusion only in the light fractions, whereas p42 ERK phosphorylation was increased in the light and heavy fractions. Although no p38 MAPK activation occurred after 10 min of reperfusion, 2 h of reperfusion caused significant activation of p38 MAPK in nonischemic zone light and heavy fractions. These results show the importance of caveolar membrane/lipid rafts in MAPK signaling and suggest that subcellular compartmentation of p44/p42 ERKs and p38 MAPK may play distinct roles in the response to myocardial ischemia-reperfusion. caveolae; caveolin-3; caveolin-1 doi:10.1152/ajpheart.01354.2005

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels ([K.sub.ATP]); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 [+ or -] 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 [+ or -] 1.9%, 11,12-EET; and 8.4 [+ or -] 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 [+ or -] 2.1%, 11,12-EET; and 9.7 [+ or -] 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 [+ or -] 4.6%) by the [K.sub.ATP] channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 [+ or -] 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12, 12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP [omega]-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 [+ or -] 1.2%, DDMS; and 22.2 [+ or -] 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP [omega]-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP to-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac [K.sub.ATP] channels. cytochrome P-450 epoxygenase; 11,12-epoxyeicosatrienoic acid; 14,15-epoxyeicosatrienoic acid; 20-hydroxyeicosatetraenoic acid; ischemic preconditioning; adenosine 5'-triphosphate-sensitive potassium channels doi:10.1152/ajpheart.00071.2006

Diverse functions have been reported for lipocalin 2. To investigate these functions in vivo, we generated gene-targeted lipocalin 2-deficient mice (Lcn[2.sup.-/-] mice). In vitro studies have suggested that lipocalin 2 is important for cellular apoptosis induced by IL-3 withdrawal, and for the induction of kidney differentiation during embryogenesis. Analysis of Lcn[2.sup.-/-] mice showed normal cell death upon IL-3 withdrawal and normal kidney development. However, we found that Lcn[2.sup.-/-] mice exhibited an increased susceptibility to bacterial infections, in keeping with the proposed function of lipocalin 2 in iron sequestration. Neutrophils isolated from Lcn[2.sup.-/-] mice showed significantly tess bacteriostatic activity compared with WT controls. The bacteriostatic property of the WT neutrophils was abolished by the addition of exogenous iron, indicating that the main function of lipocalin 2 in the antibacterial innate immune response is to limit this essential element. Another important function ascribed to lipocalin 2 has been its protective role against kidney ischemia-reperfusion injury. We analyzed Lcn[2.sup.-/-] mice using a mouse model for severe renal failure and could not detect any significant differences compared with their WT littermates. 24p3 bacteriostatic | kidney ischemia-reperfusion injury | NGAL

Partial bladder outlet obstruction (PBOO) results in cellular damage due to ischemia and reperfusion injury. Our study seeks to establish how early this damage can occur and the role that nitric oxide may play in its pathophysiology. Surgical PBOO (1, 3, and 7 days) were performed on male New Zealand White rabbits. Half of the animals were premedicated for 3 days with [N.sup.G]-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase before obstruction. Bladder weight increased with duration of PBOO but was significantly lower at 3 and 7 days in animals treated with L-NAME compared with their untreated counterparts. Contractile function decreased progressively with PBOO duration. At 1 day postobstruction, bladder contractility was significantly lower in the L-NAME rabbits than in the untreated rabbits. At 3 and 7 days, contractility of the L-NAME bladders was equal or higher than the untreated bladders. The level of hypoxia at 1 day after obstruction was significantly higher in the L-NAME-treated animals than in the untreated controls but equal at 3 and 7 days obstruction. Increased nitrotyrosine was seen by Western blot in all obstructed animals. However, the amount was significantly less in the L-NAME-treated animals at 3 and especially at 7 days. Nerve density decreased progressively after obstruction; however, it decreased to a significantly lesser degree in the L-NAME-treated bladders than in the untreated groups. These results suggest that L-NAME pretreatment enhanced ischemic damage at 1 day after obstruction but protected the bladder from nitric oxide-generated free radical damage at the later time periods by inhibiting the generation of nitrotyrosine. ischemia-reperfusion; rabbit

These experiments were designed to determine whether green tea extract (GTE), which contains polyphenolic free radical scavengers, prevents ischemia-reperfusion injury to the liver. Rats were fed a powdered diet containing 0-0.3% GTE starting 5 days before hepatic warm ischemia and reperfusion. Free radicals in bile were trapped with the spin-trapping reagent [alpha]-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and measured using electron spin resonance spectroscopy. Hepatic ischemia-reperfusion increased transaminase release and caused pathological changes including focal necrosis and hepatic leukocyte infiltration in the liver. Transaminase release was diminished by over 85% and pathological changes were almost totally blocked by 0.1% dietary GTE. Ischemia-reperfusion increased 4-POBN/radical adducts in bile nearly twofold, an effect largely blocked by GTE. Epicatechin, one of the major green tea polyphenols, gave similar protection as GTE. In addition, hepatic ischemia-reperfusion activated NF-KB and increased TNF-[alpha] mRNA and protein expression. These effects were all blocked by GTE. Taken together, these results demonstrate that GTE scavenges free radicals in the liver after ischemia-reoxygenation, thus preventing formation of toxic cytokines. Therefore, GTE could prove to be effective in decreasing hepatic injury in disease states where ischemia-reperfusion occurs. ischemia; free radicals; liver; nuclear factor-kappaB; cytokines

Coronary occlusion and reperfusion produce tachyarrhythmias. We tested the hypothesis that variations in transmural activation after global ischemia and reperfusion were responsible for arrhythmias. We arterially perfused 36 isolated transmural wedges from canine left ventricular free walls. After [is greater than or equal to] 100 min of stabilization, the artery was occluded for 25 min, followed by reperfusion at various flow rates. We recorded 256 channels of fluorescent action potentials on transmural surfaces from preocclusion to [is greater than] 15 min after reperfusion. During endocardial pacing at 300 ms, ischemia of [is greater than or equal to] 570 [+ or -] 165 s (n = 34) produced 1:1 endocardial conduction and then 2:1 and 4:1 block as the wave fronts conducted toward epicardium. Transmural reentry appeared after 535 [+ or -] 146 s of ischemia (n = 31). Further ischemia caused epicardial inactivation and eliminated reentry (n = 24). During reperfusion, tissues progressed through sequences of epicardial inactivation and reappearance of activation with 1:1, 2:1, and 4:1 conduction; both sustained and nonsustained reentry occurred. We conclude that heterogeneous activation responses to endocardial pacing during acute ischemia provide the substrate for initiating reentry, suppressed reentry during further ischemia, and caused reentry during reperfusion. heart; optical mapping; arterial occlusion; tachyarrhythmias; fibrillation