The primary cilium, which protrudes from the cell surface, is associated with the pathogenesis of various diseases, including acute kidney injury (AKI). Primary cilium length dynamically changes during the progression of diseases. However, its relevance in disease and the underlying mechanism are largely unknown. In this study, we investigated the role of primary cilia in AKI induced by cisplatin, an effective anticancer drug, and the underlying mechanisms. In addition, we evaluated the usefulness of length alteration and deciliation of primary cilia into the urine for the diagnosis of AKI. Cisplatin induced shortening, elongation, and normalization of the primary cilia in kidney epithelial cells over time. During shortening, primary cilia fragments and ciliary proteins were excreted into the urine. During deciliation, cell proliferation and the expression of cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen were not significantly changed. Shortening and deciliation of primary cilia were observed before significant increases in plasma creatinine and blood urea nitrogen concentration occurred. Pretreatment with Mito-Tempo, a mitochondria-targeted antioxidant, prevented cisplatin-induced primary cilium shortening and inhibited the increases in superoxide formation, lipid peroxidation, blood urea nitrogen, and tissue damage. In contrast, isocitrate dehydrogenase 2 (Idh2) gene deletion, which results in defect of the NADPH-associated mitochondrial antioxidant system, exacerbated cisplatin-induced changes in mice. Taken together, our findings demonstrate that cisplatin induces deciliation into the urine and antioxidant treatment prevents this deciliation, renal dysfunction, and tissue damage after cisplatin injection. These results suggest that cisplatin-induced AKI is associated with primary cilia and urine primary cilia proteins might be a non-invasive biomarker of kidney injury.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) is a major NADPH-producing enzyme which is essential for maintaining the mitochondrial redox balance in cells. We sought to determine whether IDH2 deficiency induces mitochondrial dysfunction and modulates auditory function, and investigated the protective potential of an antioxidant agent against reactive oxygen species (ROS)-induced cochlear damage in Idh2 knockout (Idh2-/-) mice. Idh2 deficiency leads to damages to hair cells and spiral ganglion neurons (SGNs) in the cochlea and ultimately to apoptotic cell death and progressive sensorineural hearing loss in Idh2-/- mice. Loss of IDH2 activity led to decreased levels of NADPH and glutathione causing abnormal ROS accumulation and oxidative damage, which might trigger apoptosis signal in hair cells and SGNs in Idh2-/- mice. We performed ex vivo experiments to determine whether administration of mitochondria-targeted antioxidants might protect or induce recovery of cells from ROS-induced apoptosis in Idh2-deficient mouse cochlea. MitoQ almost completely neutralized the H2O2-induced ototoxicity, as the survival rate of Idh2-/- hair cells were restored to normal levels. In addition, the lack of IDH2 led to the accumulation of mitochondrial ROS and the depolarization of ΔΨm, resulting in hair cell loss. In the present study, we identified that IDH2 is indispensable for the functional maintenance and survival of hair cells and SGNs. Moreover, the hair cell degeneration caused by IDH2 deficiency can be prevented by MitoQ, which suggests that Idh2-/- mice could be a valuable animal model for evaluating the therapeutic effects of various antioxidant candidates to overcome ROS-induced hearing loss.

Cadmium (Cd2+) is toxic to living organisms because it causes the malfunction of essential proteins and induces oxidative stress. NADP+-dependent cytosolic isocitrate dehydrogenase (IDH) provides reducing energy to counteract oxidative stress via oxidative decarboxylation of isocitrate. Intriguingly, the effects of Cd2+ on the activity of IDH are both positive and negative, and to understand the molecular basis, we determined the crystal structure of NADP+-dependent cytosolic IDH in the presence of Cd2+. The structure includes two Cd2+ ions, one coordinated by active site residues and another near a cysteine residue. Cd2+ presumably inactivates IDH due to its high affinity for thiols, leading to a covalent enzyme modification. However, Cd2+ also activates IDH by providing a divalent cation required for catalytic activity. Inactivation of IDH by Cd2+ is less effective when the enzyme is activated with Cd2+ than Mg2+. Although reducing agents cannot restore activity following inactivation by Cd2+, they can maintain IDH activity by chelating Cd2+. Glutathione, a cellular sulphydryl reductant, has a moderate affinity for Cd2+, allowing IDH to be activated with residual Cd2+, unlike dithiothreitol, which has a much higher affinity. In the presence of Cd2+-consuming cellular antioxidants, cells must continually supply reductants to protect against oxidative stress. The ability of IDH to utilise Cd2+ to generate NADPH could allow cells to protect themselves against Cd2+.

This study was undertaken to evaluate chemical characteristics and oxidative stability of tree-borne seed oils. A total of 15 different fatty acids were identified in six tree-borne seed oils, which included seven types of saturated fatty acids, four types of monounsaturated fatty acids, and four types of polyunsaturated fatty acids. Japanese camphor tree (JCT) had a high content of medium-chain fatty acids (97.94 ± 0.04%), in which fatty acid composition was distinct from those of the other five plant seed oils. Overall, contents of tocopherols, a type of fat-soluble vitamin, ranged between 3.82 ± 0.04 mg/100 g and 101.98 ± 1.34 mg/100 g, respectively. Phytosterol contents ranged from 117.77 ± 1.32 mg/100 g to 479.45 ± 4.27 mg/100 g, respectively. Of all tree-borne seed oils, β-sitosterol was the phytosterol at the highest concentration. Contents of unsaponifiables were between 0.13 ± 0.08 and 2.01 ± 0.02, and values of acid, peroxide, and p-anisidine were between 0.79 ± 0.01 and 38.94 ± 0.24 mg KOH/g, 3.53 ± 0.21 and 127.67 ± 1.79 meq/kg, and 2.07 ± 0.51 and 9.67 ± 0.25, respectively. Oxidative stability of tree-borne seed oils was assessed through measurement of oxidation-induction periods. These results should serve as a foundation to identify the potential of tree-borne seed oils in industrial application as well as in providing fundamental data.

The mechanisms underlying the progression to non-alcoholic steatohepatitis (NASH) remain to be elucidated. In the present study, we aimed to identify the proteins involved in the pathogenesis of liver tissue inflammation and to investigate the effects of silibinin, a natural polyphenolic flavonoid, on steatohepatitis. We performed comparative proteomic analysis using methionine and choline-deficient (MCD) diet-induced NASH model mice. Eighteen proteins were identified from the two-dimensional proteomic analysis, which are not only differentially expressed, but also significantly improved, by silibinin treatment. Interestingly, seven of these proteins, including keratin cytoskeletal 8 and 18, peroxiredoxin-4, and protein disulfide isomerase, are known to undergo GlcNAcylation modification, most of which are related to structural and stress-related proteins in NASH model animals. Thus, we primarily focused on how the GlcNAc modification of these proteins is involved in the progression to NASH. Remarkably, silibinin treatment alleviates the severity of hepatic inflammation along with O-GlcNAcylation in steatohepatitis. In particular, the reduction of inflammation by silibinin is due to the inhibition of the O-GlcNAcylation-dependent NF-κB-signaling pathway. Therefore, silibinin is a promising therapeutic agent for hyper-O-GlcNAcylation as well as NASH.

Heart failure is a frequent unfavorable outcome of pathological cardiac hypertrophy. Recent increase in dietary fructose consumption mirrors the rise in prevalence of cardiovascular diseases such as cardiac hypertrophy leading to concerns raised by public health experts. Mitochondria, comprising 30% of cardiomyocyte volume, play a central role in modulating redox-dependent cellular processes such as metabolism and apoptosis. Furthermore, mitochondrial dysfunction is a key cause of pathogenesis of fructose-induced cardiac hypertrophy. Naringin, a major flavanone glycoside in citrus species, has displayed strong antioxidant potential in models of oxidative stress. In this study, we evaluated protective effects of naringin against fructose-induced cardiac hypertrophy and associated mechanisms of action, using in vitro and in vivo models. We found that naringin suppressed mitochondrial ROS production and mitochondrial dysfunction in cardiomyocytes exposed to fructose and consequently reduced cardiomyocyte hypertrophy by regulating AMPK-mTOR signaling axis. Furthermore, naringin counteracted fructose-induced cardiomyocyte apoptosis, and this function of naringin was linked to its ability to inhibit ROS-dependent ATM-mediated p53 signaling. This result was supported by observations in in vivo mouse model of cardiac hypertrophy. These findings indicate a novel role for naringin in protecting against fructose-induced cardiac hypertrophy and suggest unique therapeutic strategies for prevention of cardiovascular diseases.

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells.

Ischemic preconditioning (IPC) by ischemia/reperfusion (I/R) renders resistance to the kidney. Strong IPC triggers kidney fibrosis, which is involved in angiotensin II (AngII) and its type 1 receptor (AT1R) signaling. Here, we investigated the role of AngII/AT1R signal pathway in the resistance of IPC kidneys to subsequent I/R injury. IPC of kidneys was generated by 30 minutes of bilateral renal ischemia and 8 days of reperfusion. Sham-operation was performed to generate control (non-IPC) mice. To examine the roles of AngII and AT1R in IPC kidneys to subsequent I/R, IPC kidneys were subjected to either 30 minutes of bilateral kidney ischemia or sham-operation following treatment with AngII, losartan (AT1R blocker), or AngII plus losartan. IPC kidneys showed fibrotic changes, decreased AngII, and increased AT1R expression. I/R dramatically increased plasma creatinine concentrations in non-IPC mice, but not in IPC mice. AngII treatment in IPC mice resulted in enhanced morphological damage, oxidative stress, and inflammatory responses, with functional impairment, whereas losartan treatment reversed these effects. However, AngII treatment in non-IPC mice did not change I/R-induced injury. AngII abolished the resistance of IPC kidneys to subsequent I/R via the enhancement of oxidative stress and inflammatory responses, suggesting that the AngII/AT1R signaling pathway is associated with outcome in injury-experienced kidney.

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome.

Isocitrate dehydrogenase 2 (IDH2) is a key enzyme that maintains the balance of mitochondrial redox status by generating NADPH as a reducing factor, which is used to reduce oxidized antioxidant proteins and oxidized glutathione. Therefore, the role of IDH2 is crucial in organs that are easily influenced by reactive oxygen species (ROS) or mechanical damage. Humans are constantly exposed to ultraviolet (UV) radiation throughout their lifetime, which can cause various cutaneous diseases, such skin carcinoma, dermatitis, and sunburn. ROS play an important role in the initial step of these diseases; therefore, IDH2 deficient mice (Idh2-/-) could be a useful model to investigate UV-mediated skin damage. When we exposed the dorsal skin of Idh2-/- mice to UVB, pyrimidine dimers and (6-4) photoproducts (6-4PPs), marker of photoproducts generated by UVB, were found in the dermis of the knockout mice. Increased collagen degradation, apoptosis, inflammation, and ROS levels in the dermis were also observed. These results indicated that UVB could reach the dermis by penetrating the epidermis. We then attempted to determine how the epidermis was breached, and observed a decrease in the expression level of ΔNp63, a major protein required for epidermis generation, in the Idh2-/- mice. The mito-TEMPO supplement significantly ameliorates UVB-induced damage in the skin of Idh2-/- mice. In the present study, we provided a role for IDH2 in protection against UVB-induced skin damage and a new connection between IDH2 and ΔNp63.

Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF), secreted by the retinal pigment epithelium (RPE), in pathological angiogenesis and the development of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA) is a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase (IDH), which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS). Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development.

Explosive synaptogenesis and synaptic pruning occur in the hippocampus during the first two weeks of postnatal life, coincident with a heightened susceptibility to seizures in rodents. To determine the temporal correlation between microglial development and age-dependent susceptibility and response to seizures, we quantified developmental changes in basal microglia levels and seizure-induced microglial activation in the hippocampus of Cx3Cr1(GFP /+) transgenic mice.

Acetaminophen (APAP)-induced hepatotoxicity is a major factor in liver failure and its toxicity is associated with the generation of reactive oxygen species (ROS), decreased levels of reduced glutathione (GSH) and overall oxidative stress. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) was demonstrated as an essential enzyme for mitochondria to maintain their antioxidant system by generating NADPH, which is an essential reducing equivalent for GSH turnover in mitochondria. Here, we investigated the role of IDH2 in APAP-induced liver injury with IDH2 deficient (idh2-/-) mice. Hepatotoxicity was promoted through apoptotic cell death following APAP administration in IDH2 deficient hepatocytes compared to that in wild-type hepatocytes. Apoptosis was found to result from the induction of ER stress and mitochondrial dysfunction as shown by the blocking the effect of phenylbutyrate and Mdivi1, respectively. In addition, mito-TEMPO, a scavenger of mitochondrial ROS, was seen to ameliorate APAP-induced hepatotoxicity in idh2-/- mice. In conclusion, IDH2 deficiency leads to a fundamental shortage of GSH that increases susceptibility to ROS generation and oxidative stress. This leads to excessive mitochondrial dysfunction and ER stress induction in response to APAP administration. Our study provides further evidence that IDH2 has a protective role against APAP-induced liver injury and emphasizes the importance of the elaborate linkages and functions of the antioxidant system in liver health.

Lysine-specific histone demethylase 5C (KDM5C) belongs to the jumonji family of demethylases and is specific for the di- and tri-demethylation of lysine 4 residues on histone 3 (H3K4 me2/3). KDM5C is expressed in the brain and skeletal muscles of humans and is associated with various biologically significant processes. KDM5C is known to be associated with X-linked mental retardation and is also involved in the development of cancer. However, the developmental significance of KDM5C has not been explored yet. In the present study, we investigated the physiological roles of KDM5C during Xenopus laevis embryonic development.

Diets high in red meats, particularly meats cooked at high temperature, increase the risk of colon cancer due to a production of heterocyclic aromatic amines (HAAs). Of the identified HAAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most mass abundant colon carcinogen in charred meat or fish. Here, we comprehensively examined sex-dependent colon transcriptome signatures in response to PhIP treatment to identify biological discrepancies. Eight-week-old male and female C57BL/6N mice were intraperitoneally injected with PhIP (10 mg/kg of body weight) and colon tissues were harvested 24 h after PhIP injection, followed by colon transcriptomics analysis. A list of differentially expressed genes (DEGs) was utilized for computational bioinformatic analyses. Specifically, overrepresentation test using the Protein Analysis Through Evolutionary Relationships tool was carried out to annotate sex-dependent changes in transcriptome signatures after PhIP treatment. Additionally, the most significantly affected canonical pathways by PhIP treatment were predicted using the Ingenuity Pathway Analysis. As results, male and female mice presented different metabolic signatures in the colon transcriptome. In the male mice, oxidative phosphorylation in the mitochondrial respiratory chain was the pathway impacted the most; this might be due to a shortage of ATP for DNA repair. On the other hand, the female mice showed concurrent activation of lipolysis and adipogenesis. The present study provides the foundational information for future studies of PhIP effects on underlying sex-dependent mechanisms.

Alcohol-induced hepatic steatosis and inflammation are key drivers of alcohol-induced liver injury, mainly caused by oxidative stress. The roots bark of Ulmus davidiana var. japonica is well known for its substantial antioxidative and antitumorigenic potency. In this study, we examined whether this plant can ameliorate alcohol-induced liver injuries characterized by hepatic steatosis and inflammation through its antioxidative activity. C57BL/6J mice were treated with the root bark extract of Ulmus davidiana var. japonica (RUE; 100 mg of extract/kg bodyweight; oral gavage) and alcohol (1 g/kg of bodyweight; oral gavage) for 5 days. Markers of acute alcohol-induced hepatic steatosis were determined and putative molecular mechanisms responsible for the protection of RUE were investigated. RUE noticeably protected against alcohol-induced hepatic steatosis and inflammation. Reactive oxygen species (ROS), over-produced by alcohol, negatively orchestrated various signaling pathways involved in the lipid metabolism and inflammation. These pathways were restored through the ROS scavenging activity of RUE in the liver. In particular, the expression of lipogenic genes (e.g., SREBP-1, ACC, and FAS) and inflammatory cytokines (e.g., IL-1β, and NF-κB p65) significantly decreased with RUE treatment. Conversely, the expression of fatty acid oxidation-related genes (e.g., SIRT1, AMPKα, and PGC1α) were increased in mice treated with RUE. Thus, the results indicate that RUE counteracts and thus attenuates alcoholic hepatic steatosis onset in mice, possibly by suppressing ROS-mediated steatosis and inflammation.

Mounting evidence shows that ROS regulation by various antioxidants is essential for the expression of enzymes involved in steroidogenesis and maintenance of progesterone production by the corpus luteum (CL). However, the underlying mechanisms of peroxiredoxin 1 (PRDX1), an antioxidant enzyme, in luteal function for progesterone production in mice have not been reported. The aim of this study was to evaluate the functional link between PRDX1 and progesterone production in the CL of Prdx1 knockout (K/O) mice in the functional stage of CL.

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) plays an important role in the formation of NADPH, which is critical for the maintenance of mitochondrial redox balance. Cis-diamminedichloroplatinum II (cisplatin), an effective anticancer drug, induces oxidative stress-related nephrotoxicity, limiting its use. Therefore, we investigated whether IDH2, which is a critical enzyme in the NADPH-associated mitochondrial antioxidant system, is involved in cisplatin nephrotoxicity. Idh2 gene-deleted (Idh2-/-) mice and wild-type (Idh2 +/+ ) littermates were treated with cisplatin, with or without 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito-T), a mitochondria-specific antioxidant. Cisplatin-induced renal functional and morphological impairments were greater in Idh2-/- mice than in Idh2 +/+ mice. Mito-T mitigated those impairments in both Idh2-/- and Idh2 +/+ mice and this mitigation was greater in Idh2-/- than in Idh2 +/+ mice. Cisplatin impaired IDH2 function in the mitochondria, decreasing mitochondrial NADPH and GSH levels and increasing H2O2 generation; protein, lipid, and DNA oxidation; mitochondrial damage; and apoptosis. These cisplatin-induced changes were much more severe in Idh2-/- mice than in Idh2 +/+ mice. Mito-T treatment attenuated cisplatin-induced alterations in both Idh2-/- and Idh2 +/+ mice and this mitigation was greater in Idh2-/- than in Idh2 +/+ mice. Altogether, these data demonstrate that cisplatin induces the impairment of the mitochondrial IDH2-NADPH-GSH antioxidant system and IDH2 deficiency aggravates cisplatin-induced mitochondrial oxidative damage, inducing more severe nephrotoxicity. This suggests that the mitochondrial IDH2-NADPH-GSH antioxidant system is a target for the prevention of cisplatin-induced kidney cell death.

A number of studies have suggested that acrolein-induced lung injury and pulmonary diseases are associated with the depletion of antioxidants and the production of reactive oxygen species. Therefore, compounds that scavenge reactive oxygen species may exert protective effects against acrolein-induced apoptosis. Because hesperetin, a natural flavonoid, has been reported to have an antioxidant activity, we investigated the effect of hesperitin against acrolein-induced apoptosis of lung cells.

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2(-/-)) and wild-type (idh2(+/+)) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.