TLR9 Mediates Remote Liver Injury following Severe Renal Ischemia Reperfusion

Ischemia reperfusion injury is a common cause of acute kidney injury and is characterized by tubular damage. Mitochondrial DNA is released upon severe tissue injury and can act as a damage-associated molecular pattern via the innate immune receptor TLR9. Here, we investigated the role of TLR9 in the context of moderate or severe renal ischemia reperfusion injury using wild-type C57BL/6 mice or TLR9KO mice. Moderate renal ischemia induced renal dysfunction but did not decrease animal well-being and was not regulated by TLR9. In contrast, severe renal ischemia decreased animal well-being and survival in wild-type mice after respectively one or five days of reperfusion. TLR9 deficiency improved animal well-being and survival. TLR9 deficiency did not reduce renal inflammation or tubular necrosis. Rather, severe renal ischemia induced hepatic injury as seen by increased plasma ALAT and ASAT levels and focal hepatic necrosis which was prevented by TLR9 deficiency and correlated with reduced circulating mitochondrial DNA levels and plasma LDH. We conclude that TLR9 does not mediate renal dysfunction following either moderate or severe renal ischemia. In contrast, our data indicates that TLR9 is an important mediator of hepatic injury secondary to ischemic acute kidney injury.     

Attenuation of ischemia/reperfusion induced MAP kinases by N-acetyl cysteine,sodium nitroprusside and phosphoramidon

Ischemia followed by reperfusion has a number of clinically significant consequences. A number of pathophysiological processes appear to be involved in ischemia/reperfusion (I/R) injury. The mitogen activated protein kinases (MAPK) are integral components of the parallel MAP kinase cascades activated in response to a variety of cellular stress inducing ischemia/ATP depletion and inflammatory cytokines. Many studies suggest that members of the MAP kinase family in particular Jun N-terminal kinase (JNK) are activated in kidney following ischemia/reperfusion of this tissue. The present study underlines the therapeutic potential of the combination of N-acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphoramidon (P), an endothelin-1 converting enzyme inhibitor in ameliorating the MAPK induced damage during renal ischemia/reperfusion injury. Our previous results showed that 90 min of ischemia followed by reperfusion caused very severe injury and that the untreated animals had 100% mortality after the 3rd day whereas there was improved renal function and 100% survival of animals in the three drug combination treatment group. The present study, mainly on tissue sections, further supports the protection provided by the triple drug therapy. A higher degree of expression of all the three classes of MAPK, i.e. JNK, P³⁸ MAP kinases and P-extracellular signal regulated kinases (ERKs) can be seen in kidneys subjected to ischemia/reperfusion insult. Pretreatment with a combination of N-acetyl cysteine, sodium nitroprusside, and phosphoramidon completely inhibits all three classes of MAPK and ameliorates AP-1 whereas individual or a combination of any two drugs is not as effective.     

Delayed administration of pyroglutamate helix B surface peptide (pHBSP), a novel nonerythropoietic analog of erythropoietin, attenuates acute kidney injury

In preclinical studies, erythropoietin (EPO) reduces ischemia-reperfusion-associated tissue injury (for example, stroke, myocardial infarction, acute kidney injury, hemorrhagic shock and liver ischemia). It has been proposed that the erythropoietic effects of EPO are mediated by the classic EPO receptor homodimer, whereas the tissue-protective effects are mediated by a hetero-complex between the EPO receptor monomer and the β-common receptor (termed "tissue-protective receptor"). Here, we investigate the effects of a novel, selective-ligand of the tissue-protective receptor (pyroglutamate helix B surface peptide [pHBSP]) in a rodent model of acute kidney injury/dysfunction. Administration of pHBSP (10 μg/kg intraperitoneally [i.p.] 6 h into reperfusion) or EPO (1,000 IU/kg i.p. 4 h into reperfusion) to rats subjected to 30 min ischemia and 48 h reperfusion resulted in significant attenuation of renal and tubular dysfunction. Both pHBSP and EPO enhanced the phosphorylation of Akt (activation) and glycogen synthase kinase 3β (inhibition) in the rat kidney after ischemia-reperfusion, resulting in prevention of the activation of nuclear factor-κB (reduction in nuclear translocation of p65). Interestingly, the phosphorylation of endothelial nitric oxide synthase was enhanced by EPO and, to a much lesser extent, by pHBSP, suggesting that the signaling pathways activated by EPO and pHBSP may not be identical.     

Enterocyte viability and mitochondrial function after graded intestinal ischemia and reperfusion in rats

Ischemia/reperfusion of the small intestine can lead to metabolic and structural alterations in the mucosa. Cellular dysfunction occurs when mitochondrial metabolism is compromised, which may ultimately lead to impaired organ function. The aims of this study were to assess the suppression of cellular and mitochondrial oxidative metabolism and involvement of mitochondria in the ischemia/reperfusion injury. The mitochondria were prepared from isolated enterocytes obtained from the small intestine of anesthetized adult rats following different time periods of ischemia and ischemia followed by 5 min reperfusion. Cellular and mitochondrial function were assessed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay. Ischemia of increasing time periods caused a progressive decrease in cellular and mitochondrial MTT reduction in enterocytes and reperfusion showed further decrease of MTT formazan formation. Inclusion of 1 mM succinate, as respiratory subs trate, showed reversal of suppression of mitochondrial function in 30-60 min ischemia whereas 90 min ischemia or short time period ischemia followed by 5 min reperfusion indicated an irreversible damage to mitochondria. This study indicated that mitochondria are a sensitive target of damage due to oxygen deficiency and possibly due to sudden burst of oxygen free radicals. Mitochondria can withstand short periods of ischemia whereas long duration ischemia or reperfusion results in irreversible damage to mitochondrial function. (Mol Cell Biochem 167: 81-87, 1997)     

Early activation of bradykinin B2 receptor aggravates reactive oxygen species generation and renal damage in ischemia/reperfusion injury

The kallikrein/kinin system is beneficial in ischemia/reperfusion injury in heart, controversial in brain, but detrimental in lung, liver, and intestine. We examined the role of the kallikrein/kinin system in acute ischemia/reperfusion renal injury induced by 40 min occlusion of the renal artery followed by reperfusion. Rats were infused with tissue kallikrein protein 5 days before (pretreated group) or after (treated group) ischemia. Two days later, the pretreated group exhibited the worst renal dysfunction, followed by the treated group, then the control group. Kallikrein increased tubular necrosis and inflammatory cell infiltration with generation of more tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Reactive oxygen species (ROS), malondialdehyde, and reduced/oxidized glutathione measurement revealed that the oxidative stress was augmented by kallikrein administration in both ischemic and reperfusion phases. The groups with more ROS generation also had more apoptotic renal cells. The deleterious effects of kallikrein on ischemia/reperfusion injury were reversed by cotreatment with bradykinin B2 receptor (B2R) antagonist, but not B1 receptor antagonist, and were not associated with hemodynamic changes. We conclude that early activation of B2R augmented ROS generation in ischemia/reperfusion renal injury, resulting in subsequent apoptosis, inflammation, and tissue damage. This finding suggests the potential application of B2R antagonists in acute ischemic renal disease associated with bradykinin activation.     

The double-edged role of nitric oxide in brain function and superoxide-mediated injury.

Fetal ischemia or hypoxia can lead to cerebral palsy, mental retardation and epilepsy. We propose that the production of nitric oxide and oxygen radicals by neurons when ischemic or hypoxic brain is reperfused may contribute to cerebral injury. Ischemia will depolarize neuronal membranes causing the synaptic discharge of the excitatory neurotransmitter glutamate, which in turn opens the voltage-dependent, N-methyl-D-aspartic acid-specific glutamate receptor/ionophore, allowing calcium to accumulate in the neuron. Calcium in turn activates an oxygen-dependent neuronal nitric oxide synthetase, which oxidizes arginine to produce nitric oxide (.NO) when oxygen is readmitted to brain by reperfusion. Nitric oxide reacts with the oxygen radical superoxide (O2-), also produced by reperfusion, to form peroxynitrite (ONOO-). Peroxynitrite can diffuse for several micrometers before decomposing to form the powerful and cytotoxic oxidants hydroxyl radical and nitrogen dioxide. The hypothesis is consistent with available evidence on the protective action of glutamate antagonists and of oxygen radical scavengers for limiting cerebral infarction following focal ischemia.     

Attenuation of reperfusion injury by renal ischemic postconditioning: the role of NO

Ischemic postconditioning (Postcond) is defined as rapid intermittent interruptions of blood flow in the early phase of reperfusion and mechanically alters the hydrodynamics of reperfusion. Although Postcond has been demonstrated to attenuate ischemia/reperfusion (I/R) injury in the heart and brain, its roles to renal I/R injury remain to be defined. In the present study, we examined the role of Postcond in I/R injury in a right-nephrectomized rat model. Postcond prevents the renal dysfunction and cell apoptosis induced by I/R and increases nitric oxide (NO) release and renal NO synthase (endothelial, eNOS and inducible, iNOS) expression. In contrast, enhancement of endothelin-1 (ET-1) in the kidney after the reperfusion was markedly suppressed by Postcond. These findings indicate that Postcond can inhibit renal I/R injury. The protective effect of Postcond is closely related to the NO production following the increase in eNOS and iNOS expression and the suppressive effect of ET-1 overproduction.     

Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning

Hepatic ischemia/reperfusion injury has immediate and deleterious effects on the outcome of patients after liver surgery. The precise mechanisms leading to the damage have not been completely elucidated. However, there is substantial evidence that the generation of oxygen free radicals and disturbances of the hepatic microcirculation are involved in this clinical syndrome. Microcirculatory dysfunction of the liver seems to be mediated by sinusoidal endothelial cell damage and by the imbalance of vasoconstrictor and vasodilator molecules, such as endothelin (ET), reactive oxygen species (ROS), and nitric oxide (NO). This may lead to no-reflow phenomenon with release of proinflammatory cytokines, sinusoidal plugging of neutrophils, oxidative stress, and as an ultimate consequence, hypoxic cell injury and parenchymal failure. An inducible potent endogenous mechanism against ischemia/reperfusion injury has been termed ischemic preconditioning. It has been suggested that preconditioning could inhibit the effects of different mediators involved in the microcirculatory dysfunction, including endothelin, tumor necrosis factor-alpha, and oxygen free radicals. In this review, we address the mechanisms of liver microcirculatory dysfunction and how ischemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

Complement-dependent NADPH oxidase enzyme activation in renal ischemia/reperfusion injury

NADPH oxidase plays a central role in mediating oxidative stress during heart, liver, and lung ischemia/reperfusion injury, but limited information is available about NADPH oxidase in renal ischemia/reperfusion injury. Our aim was to investigate the activation of NADPH oxidase in a swine model of renal ischemia/reperfusion damage. We induced renal ischemia/reperfusion in 10 pigs, treating 5 of them with human recombinant C1 inhibitor, and we collected kidney biopsies before ischemia and 15, 30, and 60 min after reperfusion. Ischemia/reperfusion induced a significant increase in NADPH oxidase 4 (NOX-4) expression at the tubular level, an upregulation of NOX-2 expression in infiltrating monocytes and myeloid dendritic cells, and 8-oxo-7,8-dihydro-2′-deoxyguanosine synthesis along with a marked upregulation of NADPH-dependent superoxide generation. This burden of oxidative stress was associated with an increase in tubular and interstitial expression of the myofibroblast marker α-smooth muscle actin (α-SMA). Interestingly, NOX-4 and NOX-2 expression and the overall NADPH oxidase activity as well as α-SMA expression and 8-oxo-7,8-dihydro-2′-deoxyguanosine synthesis were strongly reduced in C1-inhibitor-treated animals. In vitro, when we incubated tubular cells with the anaphylotoxin C3a, we observed an enhanced NADPH oxidase activity and α-SMA protein expression, which were both abolished by NOX-4 silencing. In conclusion, our findings suggest that NADPH oxidase is activated during ischemia/reperfusion in a complement-dependent manner and may play a potential role in the pathogenesis of progressive renal damage in this setting.     

Ischemia and reperfusion--from mechanism to translation

Ischemia and reperfusion-elicited tissue injury contributes to morbidity and mortality in a wide range of pathologies, including myocardial infarction, ischemic stroke, acute kidney injury, trauma, circulatory arrest, sickle cell disease and sleep apnea. Ischemia-reperfusion injury is also a major challenge during organ transplantation and cardiothoracic, vascular and general surgery. An imbalance in metabolic supply and demand within the ischemic organ results in profound tissue hypoxia and microvascular dysfunction. Subsequent reperfusion further enhances the activation of innate and adaptive immune responses and cell death programs. Recent advances in understanding the molecular and immunological consequences of ischemia and reperfusion may lead to innovative therapeutic strategies for treating patients with ischemia and reperfusion-associated tissue inflammation and organ dysfunction.     

Reduction of asymmetric dimethylarginine involved in the cardioprotective effect of losartan in spontaneously hypertensive rats

Previous studies have indicated that nitric oxide synthase (NOS) inhibitors can induce an increase of blood pressure and exacerbate myocardial injury induced by ischemia and reperfusion, whereas angiotensin II receptor antagonists protect the myocardium against injury induced by ischemia and reperfusion. Isolated hearts from male spontaneously hypertensive rats (SHR) or male Wistar-Kyoto rats (WKY) were subjected to 20 min global ischemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dP/dt(max)) were recorded, and serum concentrations of asymmetric dimethylarginine (ADMA) and NO and the release of creatine kinase in coronary effluent were measured. The level of ADMA was significantly increased and the concentration of NO was decreased in SHR. Ischemia and reperfusion significantly inhibited the recovery of cardiac function and increased the release of creatine kinase, and ischemia and reperfusion-induced myocardial injury in SHR was aggravated compared with WKY. Vasodilation responses to acetylcholine of aortic rings were decreased in SHR. Treatment with losartan (30 mg/kg) for 14 days significantly lowered blood pressure, elevated the plasma level of NO, and decreased the plasma concentration of ADMA in SHR. Treatment with losartan significantly improved endothelium-dependent relaxation and cardiac function during ischemia and reperfusion in SHR. Exogenous ADMA also aggravated myocardial injury induced by ischemia and reperfusion in isolated perfused heart of WKY, as shown by increasing creatine kinase release and decreasing cardiac function. The present results suggest that the protective effect of losartan on myocardial injury induced by ischemia and reperfusion is related to the reduction of ADMA levels.     

The β subunit of soluble guanylyl cyclase GUCY1B3 exerts cardioprotective effects against ischemic injury via the PKCε/Akt pathway

The soluble form of guanylyl cyclase (sGC) is the main receptor for the signaling agent nitric oxide (NO), which regulates cardiomyocyte contractile function and attenuates cardiomyocyte hypertrophy. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), a regulator of vascular tone, and cardiac NO-sGC-cGMP signaling modulates cardiac stress responses, including ischemia and reperfusion (IR) injury. Here, we investigated the role of GUCY1B3 (the β subunit of sGC) in cardiomyocyte IR injury and myocardial infarction (MI) in vitro and in vivo. GUCY1B3 was upregulated in neonatal rat ventricular myocytes in response to IR injury, and GUCY1B3 overexpression restored IR-induced cell death and apoptosis. Treatment with specific inhibitors of PKCδ, PKCε, and Akt suggested that the protective effects of GUCY1B3 were mediated by PKCε/Akt signaling. In a mouse model of coronary artery ligation-induced MI, GUCY1B3 silencing aggravated MI-induced cardiac dysfunction and increased infarct size and exacerbated cardiomyocyte apoptosis in association with the inactivation of PKCε and Akt. Our results suggest that GUCY1B3 exerts cardioprotective effects through the modulation of the PKCε/Akt activity and identify a potential mechanism involved in NO-sGC-cGMP signaling in the heart.     

Dexamethasone attenuates neutrophil infiltration in the rat kidney in ischemia/reperfusion injury: the possible role of nitroxyl

Neutrophil infiltration to the tissue, which is one of the important pathogenetic factors in ischemia/reperfusion injury, can be inhibited by glucocorticoids. The purpose of the present study was to clarify the mechanisms by which glucocorticoids inhibit neutrophil infiltration in renal ischemia/reperfusion injury in rats. Pretreatment with dexamethasone significantly attenuated the enhanced neutrophil infiltration and expression of intercellular adhesion molecule-1 induced by renal ischemia/reperfusion. Treatment with nitroxyl anion releaser known as Angeli's salt abolished the beneficial effect of dexamethasone in renal ischemia/reperfusion. Renal dysfunction and tubular damage induced by renal ischemia/reperfusion were not ameliorated by pretreatment with dexamthasone. These results indicate that the attenuation by dexamethasone of neutrophil infiltration and intercellular adhesion molecule-1 expression during renal ischemia/reperfusion may be mediated by the suppressed production of nitroxyl anion. Thus, neutrophil infiltration in renal ischemia/reperfusion injury may be mediated, at least in part, by the enhanced production of nitroxyl anion.     

Overexpression of manganese superoxide dismutase protects against ATP depletion-mediated cell death of proximal tubule cells

We have previously shown that in vivo renal ischemia/reperfusion results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. Current studies were designed to compare the effect of ATP depletion (Antimycin A treatment) on cell death pathways using renal proximal tubular cells and identical cells that overexpress MnSOD. ATP depletion in wild-type cells induced an apoptotic cascade that involved caspase 9 activation; MnSOD overexpressing cells afforded protection against apoptosis. This protection did not appear to involve a cytochrome c-related mechanism, but may be related to altered levels of nitric oxide within the cell. Further studies suggested that nitric oxide was required to protect the renal cells from caspase-mediated cell death. Interestingly, treatment of renal cell extracts with reductants (DTT and ascorbate) enhanced caspase activation. Taken together, these results suggest that cysteine nitrosylation may be playing a role in caspase dysfunction in cells overexpressing MnSOD following ATP depletion.     

Acute treatment with relaxin protects the kidney against ischaemia/reperfusion injury

Although recent preclinical and clinical studies have demonstrated that recombinant human relaxin (rhRLX) may have important therapeutic potential in acute heart failure and chronic kidney diseases, the effects of acute rhRLX administration against renal ischaemia/reperfusion (I/R) injury have never been investigated. Using a rat model of 1‐hr bilateral renal artery occlusion followed by 6‐hr reperfusion, we investigated the effects of rhRLX (5 μg/Kg i.v.) given both at the beginning and after 3 hrs of reperfusion. Acute rhRLX administration attenuated the functional renal injury (increase in serum urea and creatinine), glomerular dysfunction (decrease in creatinine clearance) and tubular dysfunction (increase in urinary excretion of N‐acetyl‐β‐glucosaminidase) evoked by renal I/R. These beneficial effects were accompanied by a significant reduction in local lipid peroxidation, free radical‐induced DNA damage and increase in the expression/activity of the endogenous antioxidant enzymes Mn‐ and CuZn‐superoxide dismutases (SOD). Furthermore, rhRLX administration attenuated the increase in leucocyte activation, as suggested by inhibition of myeloperoxidase activity, intercellular‐adhesion‐molecule‐1 expression, interleukin (IL)‐1β, IL‐18 and tumour necrosis factor‐α production as well as increase in IL‐10 production. Interestingly, the reduced oxidative stress status and neutrophil activation here reported were associated with rhRLX‐induced activation of endothelial nitric oxide synthase and up‐regulation of inducible nitric oxide synthase, possibly secondary to activation of Akt and the extracellular signal‐regulated protein kinase (ERK) 1/2, respectively. Thus, we report herein that rhRLX protects the kidney against I/R injury by a mechanism that involves changes in nitric oxide signalling pathway.     

Prevention of ischemia-reperfusion injury in rat ovarian tissue with the on-off method

Ischemia is defined as cell death caused by insufficient perfusion of the tissue due to reduction in arterial or venous blood flow, depletion of cellular energy storages, and accumulation of toxic metabolites. The positive effects of controlled reperfusion are known and are used clinically. But the positive effects of controlled reperfusion on ovarian tissue have not been seen in the literature yet. The biochemical and histopathological comparative investigation of rat ovaries that were experimentally exposed to ischemia (IG), ischemia-reperfusion (I/R), and ischemia-controlled reperfusion (ICR) was aimed. Forty rats were divided into four groups (10 rats per group). First group: 3 h ischemia by vascular clips on ovarian tissue. Second group: 3 h ischemia + 1 h reperfusion. Third group: 3 h ischemia + 1 h controlled reperfusion (on-off method: controlled reperfusion by opening and closing the clips (on/off) in 10-second intervals, for 5 times for a total of 100 seconds). Fourth group: healthy rats. Biochemical (tGSH, MDA, and DNA damage level and SOD activity) and histopathological analysis were performed. The highest glutathione and superoxide dismutase measurements were found in ischemia/controlled reperfusion group among the ischemia or ischemia/reperfusion groups. Similarly the damage indicators (malondialdehyde, DNA damage level and histopathological damage grade) were the lowest in ischemia/controlled reperfusion group. These results indicate that controlled reperfusion can be helpful in minimizing ischemia-reperfusion injury in ovarian tissue exposed to ischemia for various reasons (ovarian torsion, tumor, etc.).     

Evodiamine alleviates kidney ischemia reperfusion injury in rats: A biochemical and histopathological study

Renal ischemia/reperfusion (I/R) injury resulting in acute renal failure, is a major clinical problem due to its high mortality rate. Renal I/R increases the reactive oxygen species, secretion of inflammatory cytokines, chemokines and other factors. This suggests that initiating the apoptosis process in the presence of oxidative stress may play a role in life-threatening conditions, such as ischemia. Ischemia reperfusion-induced renal damage can result in renal failure and death. Although many treatment procedures have been carried out to reduce or destroy renal I/R damage in experimental models, so far, a routine method of treatment has not yet been found. For this reason, the current study was planned to investigate the possible protective effects of evodiamine on tissue damage caused by ischemia-reperfusion in kidney tissue in rats and an experimental renal I/R model was used for this purpose. Four groups were formed in the study: the control, sham control, ischemia reperfusion (I/R), and evodiamine (10 mg/kg) + I/R groups. The effects of evodiamine against kidney I/R injury were investigated. TAS (total oxidant status), TOS (total oxidant status), interleukin-1β (IL-1β), IL-6, IL-10 and tumor necrosis factor-α levels were determined by enzyme-linked immunosorbent assay. The oxidative stress index was calculated from TAS and TOS levels. In addition, the renal ischemia reperfusion injury was examined histopathologically. The IL-10 and TAS levels in the I/R group decreased when compared with the control and Sham groups, while these levels increased in the evodiamine group. Histopathologic examination revealed that caspase 3 and nuclear factor-κB levels decreased in the evodiamine group compared with the I/R group. The application of evodiamine significantly reduced ischemia reperfusion-induced kidney damage due to its antioxidant, anti-inflammatory and antiapoptotic properties.     

Postischemic anti-inflammatory effects of bradykinin preconditioning

We sought to determine the mechanisms whereby brief administration of bradykinin (bradykinin preconditioning, BK-PC) before prolonged ischemia followed by reperfusion (I/R) prevents postischemic microvascular dysfunction. Intravital videomicroscopic approaches were used to quantify I/R-induced leukocyte/endothelial cell adhesive interactions and microvascular barrier disruption in single postcapillary venules of the rat mesentery. I/R increased the number of rolling, adherent, and emigrated leukocytes and enhanced venular albumin leakage, effects that were prevented by BK-PC. The anti-inflammatory effects of BK-PC were largely prevented by concomitant administration of a B(2)-receptor antagonist but not by coincident B(1) receptor blockade, nitric oxide (NO) synthase inhibition, or cyclooxygenase blockade. However, NO synthase blockade during reperfusion after prolonged ischemia was effective in attenuating the anti-inflammatory effects of BK-PC. Pan protein kinase C (PKC) inhibition antagonized the beneficial effects of BK-PC but only when administered during prolonged ischemia. In contrast, specific inhibition of the conventional PKC isotypes failed to alter the effectiveness of BK-PC. These results indicate that bradykinin can be used to pharmacologically precondition single mesenteric postcapillary venules to resist I/R-induced leukocyte recruitment and microvascular barrier dysfunction by a mechanism that involves B(2) receptor-dependent activation of nonconventional PKC isotypes and subsequent formation of NO.