Pre-treatment with isoflurane ameliorates renal ischemic-reperfusion injury in mice

AimsPerioperative renal dysfunction is associated with a high mortality. The aim of this study was to investigate whether isoflurane preconditioning provides a protection against renal ischemic–reperfusion injury and whether hypoxia inducible factor 1α (HIF-1α) is responsible for the protection afforded by isoflurane in mice.Main methodsAdult male C57BL/6 mice received vehicle (PBS), scrambled siRNA or HIF-1α siRNA via hydrodynamic injection through tail vein. Twenty-four hours after injection, they were exposed to 1.5% isoflurane in oxygen enriched air for 2 h while controls without injection were exposed to oxygen enriched air. Twenty-four hours after gas exposure, mice were sacrificed and their kidney were harvested for western blot while other cohorts underwent renal ischemia–reperfusion injury induced by bilateral renal pedicle clamping for 25 min for renal histological or functional analysis 24 h after reperfusion or by unilateral clamping for 40 min for survival rate analysis.Key findingsSurvival rate and the expression of HIF-1α and erythropoietin were significantly increased while apoptosis, renal tubule score, blood plasma creatinine and urea were decreased by isoflurane preconditioning. HIF-1α siRNA but not scrambled siRNA injection abrogated the protective effect of isoflurane preconditioning.SignificanceOur data suggested that isoflurane preconditioning provided a protection against renal ischemic–reperfusion injury which is very likely due to hypoxia inducible factor-1α upregulation.     

Remote Limb Ischemic Preconditioning Protects Rats Against Cerebral Ischemia via HIF-1α/AMPK/HSP70 Pathway

Remote limb ischemic preconditioning (RIPC) is a clinically feasible strategy to protect against ischemia/reperfusion injury, but the knowledge concerning the mechanism underlying RIPC is scarce. This study was performed to examine the effect of RIPC on brain tissue suffering from ischemia challenge and explore its underlying mechanism in a rat model. The animals were divided into four groups: Sham, middle cerebral artery occlusion (MCAO), RIPC, and MCAO+RIPC. We found that previous exposure to RIPC significantly attenuated neurological dysfunction and lessened brain edema in MCAO+RIPC group. Moreover, other important events were observed in MCAO+RIPC group, including substantial decrements in the concentrations of oxidative response indicators [malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), and protein carbonyl], significant reductions in levels of inflammation mediators [myeloperoxidase (MPO), tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), and IL-6], and significant decline in neuronal apoptosis revealed by a smaller number of TUNEL-positive cells. Interestingly, both MCAO and RIPC groups exhibited meaningful elevations in the levels of HIF-1a, HSP70, and AMP-activated protein kinase (AMPK) compared to Sham group, and previous exposure to RIPC further elevated the levels of HIF-1a, HSP70, and AMPK in MCAO+RIPC group. Furthermore, the administration of YC-1 (HIF-1 inhibitor), 8-bAMP (AMPK inhibitor), and Quercetin (HSP70 inhibitor) to MCAO+RIPC rats demonstrated that HIF-1α/AMPK/HSP70 was involved in RIPC-mediated protection against cerebral ischemia.     

Transient ureteral obstruction prevents against kidney ischemia/reperfusion injury via hypoxia-inducible factor (HIF)-2α activation

Although the protective effect of transient ureteral obstruction (UO) prior to ischemia on subsequent renal ischemia/reperfusion (I/R) injury has been documented, the underlying molecular mechanism remains to be understood. We showed in the current study that 24 h of UO led to renal tubular hypoxia in the ipsilateral kidney in mice, with the accumulation of hypoxia-inducible factor (HIF)-2α, which lasted for a week after the release of UO. To address the functions of HIF-2α in UO-mediated protection of renal IRI, we utilized the Mx-Cre/loxP recombination system to knock out target genes. Inactivation of HIF-2α, but not HIF-1α blunted the renal protective effects of UO, as demonstrated by much higher serum creatinine level and severer histological damage. UO failed to prevent postischemic neutrophil infiltration and apoptosis induction in HIF-2α knockout mice, which also diminished the postobstructive up-regulation of the protective molecule, heat shock protein (HSP)-27. The renal protective effects of UO were associated with the improvement of the postischemic recovery of intra-renal microvascular blood flow, which was also dependent on the activation of HIF-2α. Our results demonstrated that UO protected the kidney via activation of HIF-2α, which reduced tubular damages via preservation of adequate renal microvascular perfusion after ischemia. Thus, preconditional HIF-2α activation might serve as a novel therapeutic strategy for the treatment of ischemic acute renal failure.     

缺氧诱导因子(HIFs)在肾癌发生中的作用及其分子机制

肾癌是一种常见的泌尿系统肿瘤,但在临床上对转移性肾癌的治疗手段还非常有限。缺氧是实体肿瘤微环境的一个重要的基本特征。近年来基于分子机制和临床研究的数据表明,低氧诱导因子(hypoxia-inducible factors, HIFs)在肾细胞癌(renal cell carcinoma, RCC)的发生发展中发挥着至关重要的作用。HIF是一类介导细胞适应低氧状况必需的转录激活因子,由α亚基和β亚基组成。研究已经证实,在RCC中HIF-1α和HIF-2α具有相反的作用,前者作为肿瘤抑制因子,而后者作为癌基因发挥功能。本文综述了HIF信号通路在RCC发生发展中的作用及其分子机制,并对靶向HIF信号通路治疗RCC的进行了探讨,以期指导临床肾细胞癌的精准化治疗和进一步的研究。     

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.     

Hypoxia-inducible factor 1 contributes to N-acetylcysteine’s protection in stroke

Stroke is a leading cause of adult morbidity and mortality with very limited treatment options. Evidence from preclinical models of ischemic stroke has demonstrated that the antioxidant N-acetylcysteine (NAC) effectively protects the brain from ischemic injury. Here, we evaluated a new pathway through which NAC exerted its neuroprotection in a transient cerebral ischemia animal model. Our results demonstrated that pretreatment with NAC increased protein levels of hypoxia-inducible factor-1α (HIF-1α), the regulatable subunit of HIF-1, and its target proteins erythropoietin (EPO) and glucose transporter (GLUT)-3, in the ipsilateral hemispheres of rodents subjected to 90 min middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Interestingly, after NAC pretreatment and stroke, the contralateral hemisphere also demonstrated increased levels of HIF-1α, EPO, and GLUT-3, but to a lesser extent. Suppressing HIF-1 activity with two widely used pharmacological inhibitors, YC-1 and 2ME2, and specific knockout of neuronal HIF-1α abolished NAC’s neuroprotective effects. The results also showed that YC-1 and 2ME2 massively enlarged infarcts, indicating that their toxic effect was larger than just abolishing NAC's neuroprotective effects. Furthermore, we determined the mechanism of NAC-mediated HIF-1α induction. We observed that NAC pretreatment upregulated heat-shock protein 90 (Hsp90) expression and increased the interaction of Hsp90 with HIF-1α in ischemic brains. The enhanced association of Hsp90 with HIF-1α increased HIF-1α stability. Moreover, Hsp90 inhibition attenuated NAC-induced HIF-1α protein accumulation and diminished NAC-induced neuroprotection in the MCAO model. These results strongly indicate that HIF-1 plays an important role in NAC-mediated neuroprotection and provide a new molecular mechanism involved in the antioxidant’s neuroprotection in ischemic stroke.     

Effects of YC-1 targeting hypoxia-inducible factor 1 alpha in oesophageal squamous carcinoma cell line Eca109 cells

HIF-1α (hypoxia-inducible factor 1 alpha) is believed to promote oesophageal squamous tumour growth. Thus, an HIF-1α inhibitor is viewed as a therapeutic target in treating oesophageal cancer. Recently, YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] has been widely used as a potential HIF-1α inhibitor and is being developed as a novel anticancer drug. However, little is known about the effects of YC-1 in human oesophageal cancer. In the present study, we aimed to investigate these effects in an esophageal squamous cancer cell line; i.e. Eca109 cells. We found that YC-1 abolished the hypoxia-induced up-regulation of HIF-1α. YC-1 arrested cell growth and inhibited cell migration activities in Eca109 cells. These results suggest that YC-1 may be a chemotherapy candidate against oesophageal squamous cancers.     

Effects of nitric oxide on renal interstitial fibrosis in rats with unilateral ureteral obstruction

AimsIt is well recognized that microvascular injury is a major determinant of renal fibrosis. Mounting evidence shows that nitric oxide (NO) plays an important role in angiogenesis. Therefore, we investigated to the effects of NO on kidney angiogenesis and renal fibrosis.MethodsIn the present study, a unilateral ureteral obstruction (UUO) model was established with l-arginine (l-Arg, 1 g/dl) and N-nitro-l-arginine methyl ester (L-NAME, 5 mg/dl) serving as interference factors. We investigated the alteration of NO concentration with spectrophotometry, peritubular capillary (PTC) density with aminopeptidase P (JG12) immunohistochemical staining, and the expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), hypoxia inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) with immunohistochemical staining and Western blotting at weeks 2, 3 and 4.Key findingsOur findings showed that the expressions of VEGF, eNOS and PTC density were significantly decreased in rats with UUO, which was accompanied by a progressive increase in HIF-1α, TGF-β1 and an area of renal interstitial fibrosis. The administration of l-Arg promoted the synthesis of NO and significantly elevated the expressions of VEGF, eNOS and PTC density with the conspicuous loss of HIF-1α and TGF-β1 expressions and ultimately ameliorated renal fibrosis, which was markedly aggravated by L-NAME administration.SignificanceThese findings demonstrate that NO appears to play an important role in kidney angiogenesis and in slowing the progression of renal interstitial fibrosis, which suggests that NO may serve as a novel therapeutic strategy for preventing renal fibrosis as well as fibrosis in other organs.     

Hypoxia. Cross talk between oxygen sensing and the cell cycle machinery

A fundamental physiological property of mammalian cells is the regulation of proliferation according to O(2) availability. Progression through the cell cycle is inhibited under hypoxic conditions in many, but not all, cell types, and this G1 arrest is dependent on hypoxia-inducible factor (HIF) 1α. Components of the hexameric MCM helicase, which binds to replication origins before the onset of DNA synthesis, are present in large excess in mammalian cells relative to origins, suggesting that they may have additional functions. Screens for HIF-1α interacting proteins revealed that MCM7 binds to the amino-terminal PER-SIM-ARNT (PAS) domain of HIF-1α and stimulates prolyl hydroxylation-dependent ubiquitination and degradation of HIF-1α, whereas MCM3 binds to the carboxyl terminus of HIF-1α and enhances asparaginyl hydroxylation-dependent inhibition of HIF-1α transactivation domain function. Thus MCM proteins inhibit HIF activity via two distinct O(2)-dependent mechanisms. Under prolonged hypoxic conditions, MCM mRNA expression is inhibited in a HIF-1α-dependent manner. Thus HIF and MCM proteins act in a mutually antagonistic manner, providing a novel molecular mechanism for homeostatic regulation of cell proliferation based on the relative levels of these proteins.