Dose-dependent deleterious and salutary actions of the Nrf2 inducer dh404 in chronic kidney disease

Oxidative stress and inflammation play a central role in the progression and complications of chronic kidney disease (CKD) and are, in part, due to impairment of the Nrf2 system, which regulates the expression of antioxidant and detoxifying molecules. Natural Nrf2-inducing phytochemicals have been shown to ameliorate kidney disease in experimental animals. However, owing to adverse outcomes a clinical trial of a synthetic Nrf2 activator, bardoxolone methyl (BARD), in CKD patients was terminated. BARD activates Nrf2 via covalent modification of reactive cysteine residues in the Nrf2 repressor molecule, Keap1. In addition to Nrf2, Keap1 suppresses IKKB, the positive regulator of NF-κB. Treatment with a BARD analog, dh404, at 5–20 mg/kg/day in diabetic obese Zucker rats exacerbates, whereas its use at 2 mg/kg/day in 5/6 nephrectomized rats attenuates, CKD progression. We, therefore, hypothesized that deleterious effects of high-dose BARD are mediated by the activation of NF-κB. CKD (5/6 nephrectomized) rats were randomized to receive dh404 (2 or 10 mg/kg/day) or vehicle for 12 weeks. The vehicle-treated group exhibited glomerulosclerosis; interstitial fibrosis and inflammation; activation of NF-κB; upregulation of oxidative, inflammatory, and fibrotic pathways; and suppression of Nrf2 activity and its key target gene products. Treatment with low-dose dh404 restored Nrf2 activity and expression of its target genes, attenuated activation of NF-κB and fibrotic pathways, and reduced glomerulosclerosis, interstitial fibrosis, and inflammation. In contrast, treatment with a high dh404 dosage intensified proteinuria, renal dysfunction, and histological abnormalities; amplified upregulation of NF-κB and fibrotic pathways; and suppressed the Nrf2 system. Thus therapy with BARD analogs exerts a dose-dependent dimorphic impact on CKD progression.     

Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca²⁺ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca²⁺ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca²⁺ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca²⁺ signaling in situ, by analyzing Ca²⁺ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca²⁺ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca²⁺ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca²⁺ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca²⁺ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca²⁺ release events.     

Curcumin Suppresses Gelatinase B Mediated Norepinephrine Induced Stress in H9c2 Cardiomyocytes

Background

Extracellular matrix (ECM) remodeling facilitates biomechanical signals in response to abnormal physiological conditions. This process is witnessed as one of the major effects of the stress imposed by catecholamines, such as epinephrine and norepinephrine (NE), on cardiac muscle cells. Matrix metalloproteinases (MMPs) are the key proteases involved in degradation of the ECM in heart.

Objectives

The present study focuses on studying the effect of curcumin on Gelatinase B (MMP-9), an ECM remodeling regulatory enzyme, in NE-induced cardiac stress. Curcumin, a bioactive polyphenol found in the spice turmeric, has been studied for its multi-fold beneficial properties. This study focuses on investigating the role of curcumin as a cardio-protectant.

Methods

H9c2 cardiomyocytes were subjected to NE and curcumin treatments to study the response in stress conditions. Effect on total collagen content was studied using Picrosirus red staining. Gelatinase B activity was assessed through Gel-Diffusion Assay and Zymographic techniques. RT-PCR, Western Blotting and Immunocytochemistry were performed to study effect on expression of gelatinase B. Further, the effect of curcumin on the localization of NF-κB, known to regulate gelatinase B, was also examined.

Results

Curcumin suppressed the increase in the total collagen content under hypertrophic stress and was found to inhibit the in-gel and in-situ gelatinolytic activity of gelatinase B. Moreover, it was found to suppress the mRNA and protein expression of gelatinase B.

Conclusions

The study provides an evidence for an overall inhibitory effect of curcumin on Gelatinase B in NE-induced hypertrophic stress in H9c2 cardiomyocytes which may contribute in the prevention of ECM remodeling.     

氧化和化学应激的防御性转导通路——Nrf2/ARE

Nrf2/ARE是近年新发现的机体抵抗内外界氧化和化学等刺激的防御性转导通路．生理条件下,NF-E2相关因子2(Nrf2,NF-E2-related factor 2)在细胞质中与Keap1结合处于非活性、易降解的状态.在内外界自由基和化学物质刺激时,Keap1的构象改变或者Nrf2直接被磷酸化,导致Nrf2与Keap1解离而活化．活化的Nrf2进入细胞核,与抗氧化反应元件(ARE)结合,启动ARE下游的Ⅱ相解毒酶、抗氧化蛋白、蛋白酶体/分子伴侣等基因转录和表达以抵抗内外界的有害刺激．MAPK、PI3K/AKT、PKC等信号通路分子广泛参与了Nrf2的活化和核转位过程,但是具体何种通路被激动、何种通路发挥主导作用,取决于刺激物种类、刺激方式和细胞类型．本文就Nrf2分子结构、Nrf2活化机制、Nrf2/ARE调控的下游基因、与Nrf2相关的信号通路分子以及其在肿瘤、炎症、衰老等应用领域的最新进展进行综述．     

Impaired Nuclear Nrf2 Translocation Undermines the Oxidative Stress Response in Friedreich Ataxia

Background

Friedreich ataxia originates from a decrease in mitochondrial frataxin, which causes the death of a subset of neurons. The biochemical hallmarks of the disease include low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression.

Methodology/Principal Findings

We thus investigated signaling pathways involved in antioxidant defense mechanisms. We showed that cultured fibroblasts from patients with Friedreich ataxia exhibited hypersensitivity to oxidative insults because of an impairment in the Nrf2 signaling pathway, which led to faulty induction of antioxidant enzymes. This impairment originated from previously reported actin remodeling by hydrogen peroxide.

Conclusions/Significance

Thus, the defective machinery for ISP synthesis by causing mitochondrial iron dysmetabolism increases hydrogen peroxide production that accounts for the increased susceptibility to oxidative stress.     

A Novel Telomerase Activator Suppresses Lung Damage in a Murine Model of Idiopathic Pulmonary Fibrosis

The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2–4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.     

Genetic Activation of Nrf2 Protects against Fasting-Induced Oxidative Stress in Livers of Mice

Acute fasting causes elevated oxidative stress. The current study investigated the effects of the nuclear factor erythoid 2-related factor 2 (Nrf2), the sensor of oxidative stress in cells, on energy homeostasis and liver pathophysiology during fasting. Feed was removed from mice possessing none (Nrf2-null), normal (wild-type, WT), enhanced (Keap1-knockdown, K1-KD), and maximum (hepatocyte-specific Keap1-knockout, K1-HKO) Nrf2 activity in liver for 24 h. Body weight, blood glucose, and blood lipid profiles were similar among mice with graded Nrf2 activity under either fed or fasted conditions. Fasting reduced liver size in mice expressing Nrf2, but not in Nrf2-null mice. Nrf2-null mice accumulated more non-esterified free fatty acids and triglycerides in liver after fasting than the other genotypes of mice. Fatty acids are mainly catabolized in mitochondria, and Nrf2-null mice had lower mitochondrial content in liver under control feeding conditions, which was further reduced by fasting. In contrast, mitochondrial contents in mice with enhanced Nrf2 activity were not affected by fasting. Oxidative stress, determined by staining of free radicals and quantification of malondialdehyde equivalents, was highest in Nrf2-null and lowest in K1-HKO mice after fasting. The exacerbated oxidative stress in livers of Nrf2-null mice is predicted to lead to damages to mitochondria, and therefore diminished oxidation and increased accumulation of lipids in livers of Nrf2-null mice. In summary, the Nrf2-regulated signaling pathway is critical in protecting mitochondria from oxidative stress during feed deprivation, which ensures efficient utilization of fatty acids in livers of mice.     

The Warburg Effect Suppresses Oxidative Stress Induced Apoptosis in a Yeast Model for Cancer

Background

Otto Warburg observed that cancer cells are often characterized by intense glycolysis in the presence of oxygen and a concomitant decrease in mitochondrial respiration. Research has mainly focused on a possible connection between increased glycolysis and tumor development whereas decreased respiration has largely been left unattended. Therefore, a causal relation between decreased respiration and tumorigenesis has not been demonstrated.

Methodology/Principal Findings

For this purpose, colonies of Saccharomyces cerevisiae, which is suitable for manipulation of mitochondrial respiration and shows mitochondria-mediated cell death, were used as a model. Repression of respiration as well as ROS-scavenging via glutathione inhibited apoptosis and conferred a survival advantage during seeding and early development of this fast proliferating solid cell population. In contrast, enhancement of respiration triggered cell death.

Conclusion/Significance

Thus, the Warburg effect might directly contribute to the initiation of cancer formation - not only by enhanced glycolysis - but also via decreased respiration in the presence of oxygen, which suppresses apoptosis.     

3-Pyridinylboronic Acid Ameliorates Rotenone-Induced Oxidative Stress Through Nrf2 Target Genes in Zebrafish Embryos

Neurochemical Research - Parkinson’s disease (PD) is one of the most common forms of neurodegenerative diseases and research on potential therapeutic agents for PD continues. Rotenone is a...     

低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响

Nrf2可调节多种抗氧化酶的表达,Nrf2的缺失可能影响机体的运动能力,而低氧可提高机体的抗氧化能力并改善运动能力。为了考察低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响,本研究分别在常氧和低氧环境(12%氧浓度)中对野生型大鼠和Nrf2敲除大鼠进行4周的跑台运动。研究显示,低氧运动可提高野生型大鼠的跑台运动力竭时间,Nrf2敲除可缩短大鼠的力竭时间;低氧运动可上调大鼠的Nrf2 m RNA表达量;Nrf2敲除明显抑制HIF-1α蛋白表达,而低氧运动可上调野生型和Nrf2敲除大鼠的HIF-1α蛋白表达;Nrf2敲除大鼠的骨骼肌ROS水平明显升高,并且低氧均可降低野生型和Nrf2敲除大鼠骨骼肌ROS水平。低氧运动可上调Nrf2敲除大鼠的CAT和GSH-PX蛋白表达。苏木精和伊红(HE)染色显示,Nrf2敲除大鼠在力竭跑台运动完成后出现更严重的骨骼肌病理改变,而低氧运动可减轻骨骼肌损伤。本研究认为,Nrf2敲除导致了大鼠骨骼肌中抗氧化酶的抑制及ROS的过量累积,从而造成了骨骼肌损伤并降低了运动能力。此外,低氧可通过上调Nrf2的表达,进而激活HIF-1α及抗氧化酶活性,从而提高运动能力,并防止骨骼肌损伤。     

Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.     

Neuroprotective Effects of the Triterpenoid,CDDO Methyl Amide,a Potent Inducer of Nrf2-Mediated Transcription

The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F₂-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinson''s disease and Huntington''s disease.