PHI-1, an Endogenous Inhibitor Protein for Protein Phosphatase-1 and a Pan-Cancer Marker, Regulates Raf-1 Proteostasis.

Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin-proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed ERK1/2 hyperphosphorylation was attributable to an upregulation of Raf-1 expression, rather than an increase in Ras levels, Raf-1 Ser338 phosphorylation, or B-Raf levels. The elevated Raf-1 expression, stemming from PHI-1 knockdown, enhanced EGF-induced ERK1/2 phosphorylation through MEK. Moreover, PHI-1 knockdown significantly contributed to Raf-1 protein stability without affecting Raf-1 mRNA levels. Conversely, ectopic PHI-1 expression suppressed Raf-1 protein levels in a manner that correlated with PHI-1's inhibitory potency. Inhibiting PP1 to mimic PHI-1's function using tautomycin led to a reduction in Raf-1 expression. In summary, our findings highlight that the PHI-1-PP1 signaling axis selectively governs Raf-1 proteostasis and cell survival signals.

Plum-Derived Exosome-like Nanovesicles Induce Differentiation of Osteoblasts and Reduction of Osteoclast Activation.

Osteoblasts and osteoclasts play crucial roles in bone formation and bone resorption. We found that plum-derived exosome-like nanovesicles (PENVs) suppressed osteoclast activation and modulated osteoblast differentiation. PENVs increased the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells and osteoblasts from mouse bone marrow cultures. Notably, PENVs elevated the expression of osteoblastic transcription factors and osteoblast differentiation marker proteins in MC3T3-E1 cells. Higher levels of phosphorylated BMP-2, p38, JNK, and smad1 proteins were detected in PENV-treated MC3T3-E1 cells. Additionally, the number of TRAP-positive cells was significantly decreased in PENV-treated osteoclasts isolated from osteoblasts from mouse bone marrow cultures. Importantly, osteoclastogenesis of marker proteins such as PPAR-gamma, NFATc1, and c-Fos were suppressed by treatment with PENVs (50 µg/mL). Taken together, these results demonstrate that PENVs can be used as therapeutic targets for treating bone-related diseases by improving osteoblast differentiation and inhibiting osteoclast activation for the first time.

RPL27 contributes to colorectal cancer proliferation and stemness via PLK1 signaling.

Although expression of ribosomal protein L27 (RPL27) is upregulated in clinical colorectal cancer (CRC) tissue, to the best of our knowledge, the oncogenic role of RPL27 has not yet been defined. The present study aimed to investigate whether targeting RPL27 could alter CRC progression and determine whether RPL27 gains an extra­ribosomal function during CRC development. Human CRC cell lines HCT116 and HT29 were transfected with RPL27­specific small interfering RNA and proliferation was assessed in vitro and in vivo using proliferation assays, fluorescence­activated cell sorting (FACS) and a xenograft mouse model. Furthermore, RNA sequencing, bioinformatic analysis and western blotting were conducted to explore the underlying mechanisms responsible for RPL27 silencing­induced CRC phenotypical changes. Inhibiting RPL27 expression suppressed CRC cell proliferation and cell cycle progression and induced apoptotic cell death. Targeting RPL27 significantly inhibited growth of human CRC xenografts in nude mice. Notably, polo­like kinase 1 (PLK1), which serves an important role in mitotic cell cycle progression and stemness, was downregulated in both HCT116 and HT29 cells following RPL27 silencing. RPL27 silencing reduced the levels of PLK1 protein and G2/M­associated regulators such as phosphorylated cell division cycle 25C, CDK1 and cyclin B1. Silencing of RPL27 reduced the migration and invasion abilities and sphere­forming capacity of the parental CRC cell population. In terms of phenotypical changes in cancer stem cells (CSCs), RPL27 silencing suppressed the sphere­forming capacity of the isolated CD133+ CSC population, which was accompanied by decreased CD133 and PLK1 levels. Taken together, these findings indicated that RPL27 contributed to the promotion of CRC proliferation and stemness via PLK1 signaling and RPL27 may be a useful target in a next­generation therapeutic strategy for both primary CRC treatment and metastasis prevention.

Pathologic HDAC1/c-Myc signaling axis is responsible for angiotensinogen transcription and hypertension induced by high-fat diet.

High-fat diet (HFD)-induced obesity is a cause of resistant hypertension. We have shown a possible link between histone deacetylases (HDACs) and renal angiotensinogen (Agt) upregulation in the HFD-induced hypertension, whereas the underlying mechanisms remain to be elucidated. Here, using a HDAC1/2 inhibitor romidepsin (FK228) and siRNAs, we determined roles of HDAC1 and HDAC2 in HFD-induced hypertension and found the pathologic signaling axis between HDAC1 and Agt transcription. Treatment with FK228 canceled the increased blood pressure of male C57BL/6 mice induced by HFD. FK228 also blocked upregulation of renal Agt mRNA, protein, angiotensin II (Ang II) or serum Ang II. Activation and nuclear accumulation of both HDAC1 and HDAC2 occurred in the HFD group. The HFD-induced HDAC activation was associated with an increase in deacetylated c-Myc transcription factor. Silencing of HDAC1, HDAC2 or c-Myc in HRPTEpi cells decreased Agt expression. However, only HDAC1 knockdown, but not HDAC2, increased c-Myc acetylation, suggesting selective roles in two enzymes. Chromatin immunoprecipitation assay revealed that HFD induced the binding of HDAC1 and deacetylated c-Myc at the Agt gene promoter. A putative c-Myc binding sequence in the promotor region was necessary for Agt transcription. Inhibition of c-Myc downregulated Agt and Ang II levels in kidney and serum, ameliorating HFD-induced hypertension. Thus, the abnormal HDAC1/2 in the kidney may be responsible for the upregulation of the Agt gene expression and hypertension. The results expose the pathologic HDAC1/c-myc signaling axis in kidney as a promising therapeutic target for obesity-associated resistant hypertension.

The Effect of Apple-Derived Nanovesicles on the Osteoblastogenesis of Osteoblastic MC3T3-E1 Cells.

Osteoporosis is characterized by low bone mass and elevated structural deterioration of the bone tissue, resulting in bone weakness with an increased risk of fracture. Considering biological activities of various phytochemicals extracted from apples, we herein demonstrated the potential antiosteoporotic effects of apple-derived nanovesicles (apple NVs) using osteoblastic MC3T3-E1 cells. Apple NVs significantly stimulated the growth of MC3T3-E1 cells. The cellular alkaline phosphatase (ALP) activity was significantly upregulated in the 5 µg/mL apple NVs-treated group. In addition, the concentrarion of mineralized nodules was significantly increased in the apple NVs-treated groups. Furthermore, apple NVs increased the expression of the genes and proteins associated with osteoblast growth and differentiation, such as Runx2, ALP, OPN, and BMP2/4, which further activated ERK- and JNK-related mitogen-activated protein kinase signaling. These results demonstrate that apple NVs have a potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells through regulating the BMP2/Smad1 pathways.

Deep learning-based diagnosis of stifle joint diseases in dogs.

In this retrospective, analytical study, we developed a deep learning-based diagnostic model that can be applied to canine stifle joint diseases and compared its accuracy with that achieved by veterinarians to verify its potential as a reliable diagnostic method. A total of 2382 radiographs of the canine stifle joint from cooperative animal hospitals were included in a dataset. Stifle joint regions were extracted from the original images using the faster region-based convolutional neural network (R-CNN) model, and the object detection accuracy was evaluated. Four radiographic findings: patellar deviation, drawer sign, osteophyte formation, and joint effusion, were observed in the stifle joint and used to train a residual network (ResNet) classification model. Implant and growth plate groups were analyzed to compare the classification accuracy against the total dataset. All deep learning-based classification models achieved target accuracies exceeding 80%, which is comparable to or slightly less than those achieved by veterinarians. However, in the case of drawer signs, further research is necessary to improve the low sensitivity of the model. When the implant group was excluded, the classification accuracy significantly improved, indicating that the implant acted as a distraction. These results indicate that deep learning-based diagnoses can be expected to become useful diagnostic models in veterinary medicine.

Machine Learning for Antimicrobial Resistance Prediction: Current Practice, Limitations, and Clinical Perspective.

Antimicrobial resistance (AMR) is a global health crisis that poses a great threat to modern medicine. Effective prevention strategies are urgently required to slow the emergence and further dissemination of AMR. Given the availability of data sets encompassing hundreds or thousands of pathogen genomes, machine learning (ML) is increasingly being used to predict resistance to different antibiotics in pathogens based on gene content and genome composition. A key objective of this work is to advocate for the incorporation of ML into front-line settings but also highlight the further refinements that are necessary to safely and confidently incorporate these methods. The question of what to predict is not trivial given the existence of different quantitative and qualitative laboratory measures of AMR. ML models typically treat genes as independent predictors, with no consideration of structural and functional linkages; they also may not be accurate when new mutational variants of known AMR genes emerge. Finally, to have the technology trusted by end users in public health settings, ML models need to be transparent and explainable to ensure that the basis for prediction is clear. We strongly advocate that the next set of AMR-ML studies should focus on the refinement of these limitations to be able to bridge the gap to diagnostic implementation.

Hydrogen sulfide, a gaseous signaling molecule, elongates primary cilia on kidney tubular epithelial cells by activating extracellular signal-regulated kinase.

Primary cilia on kidney tubular cells play crucial roles in maintaining structure and physiological function. Emerging evidence indicates that the absence of primary cilia, and their length, are associated with kidney diseases. The length of primary cilia in kidney tubular epithelial cells depends, at least in part, on oxidative stress and extracellular signal-regulated kinase 1/2 (ERK) activation. Hydrogen sulfide (H2S) is involved in antioxidant systems and the ERK signaling pathway. Therefore, in this study, we investigated the role of H2S in primary cilia elongation and the downstream pathway. In cultured Madin-Darby Canine Kidney cells, the length of primary cilia gradually increased up to 4 days after the cells were grown to confluent monolayers. In addition, the expression of H2S-producing enzyme increased concomitantly with primary cilia length. Treatment with NaHS, an exogenous H2S donor, accelerated the elongation of primary cilia whereas DL-propargylglycine (a cystathionine γ-lyase inhibitor) and hydroxylamine (a cystathionine-ß-synthase inhibitor) delayed their elongation. NaHS treatment increased ERK activation and Sec10 and Arl13b protein expression, both of which are involved in cilia formation and elongation. Treatment with U0126, an ERK inhibitor, delayed elongation of primary cilia and blocked the effect of NaHS-mediated primary cilia elongation and Sec10 and Arl13b upregulation. Finally, we also found that H2S accelerated primary cilia elongation after ischemic kidney injury. These results indicate that H2S lengthens primary cilia through ERK activation and a consequent increase in Sec10 and Arl13b expression, suggesting that H2S and its downstream targets could be novel molecular targets for regulating primary cilia.

Ellagic Acid Prevents Binge Alcohol-Induced Leaky Gut and Liver Injury through Inhibiting Gut Dysbiosis and Oxidative Stress.

Alcoholic liver disease (ALD) is a major liver disease worldwide and can range from simple steatosis or inflammation to fibrosis/cirrhosis, possibly through leaky gut and systemic endotoxemia. Many patients with alcoholic steatohepatitis (ASH) die within 60 days after clinical diagnosis due to the lack of an approved drug, and thus, synthetic and/or dietary agents to prevent ASH and premature deaths are urgently needed. We recently reported that a pharmacologically high dose of pomegranate extract prevented binge alcohol-induced gut leakiness and hepatic inflammation by suppressing oxidative and nitrative stress. Herein, we investigate whether a dietary antioxidant ellagic acid (EA) contained in many fruits, including pomegranate and vegetables, can protect against binge alcohol-induced leaky gut, endotoxemia, and liver inflammation. Pretreatment with a physiologically-relevant dose of EA for 14 days significantly reduced the binge alcohol-induced gut barrier dysfunction, endotoxemia, and inflammatory liver injury in mice by inhibiting gut dysbiosis and the elevated oxidative stress and apoptosis marker proteins. Pretreatment with EA significantly prevented the decreased amounts of gut tight junction/adherent junction proteins and the elevated gut leakiness in alcohol-exposed mice. Taken together, our results suggest that EA could be used as a dietary supplement for alcoholic hepatitis patients.

Inhibition of HDACs (Histone Deacetylases) Ameliorates High-Fat Diet-Induced Hypertension Through Restoration of the MsrA (Methionine Sulfoxide Reductase A)/Hydrogen Sulfide Axis.

[Figure: see text].

IDH2 gene deficiency accelerates unilateral ureteral obstruction-induced kidney inflammation through oxidative stress and activation of macrophages.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) produces NADPH, which is known to inhibit mitochondrial oxidative stress. Ureteral obstruction induces kidney inflammation and fibrosis via oxidative stress. Here, we investigated the role and underlying mechanism of IDH2 in unilateral ureteral obstruction (UUO)-induced kidney inflammation using IDH2 gene deleted mice (IDH2-/-). Eight- to 10-week-old female IDH2-/- mice and wild type (IDH2+/+) littermates were subjected to UUO and kidneys were harvested 5 days after UUO. IDH2 was not detected in the kidneys of IDH2-/- mice, while UUO decreased IDH2 in IDH2+/+ mice. UUO increased the expressions of markers of oxidative stress in both IDH2+/+ and IDH2-/- mice, and these changes were greater in IDH2-/- mice compared to IDH2+/+ mice. Bone marrow-derived macrophages of IDH2-/- mice showed a more migrating phenotype with greater ruffle formation and Rac1 distribution than that of IDH2+/+ mice. Correspondently, UUO-induced infiltration of monocytes/macrophages was greater in IDH2-/- mice compared to IDH2+/+ mice. Taken together, these data demonstrate that IDH2 plays a protective role against UUO-induced inflammation through inhibition of oxidative stress and macrophage infiltration.

Isocitrate dehydrogenase 2 deficiency aggravates prolonged high-fat diet intake-induced hypertension.

The development of hypertension is associated with mitochondrial redox balance disruptions. NADP+-dependent isocitrate dehydrogenase 2 (IDH2) plays an important role in the maintenance of mitochondrial redox balance by producing mitochondrial NADPH, which is an essential cofactor in the reduction of glutathione (from GSSG to GSH) to reduced form of glutathione (GSH). We investigated the association of IDH2 between the development of prolonged high-fat diet (HFD)-induced hypertension. Idh2 gene-deleted (Idh2-/-) male mice and wild-type (Idh2+/+) littermates were fed either HFD or low-fat diet (LFD). Some mice were administrated with Mito-TEMPO, a mitochondria-specific antioxidant. HFD feeding increased blood pressure (BP) in both Idh2-/- mice and Idh2+/+ mice. HFD-induced BP increase was greater in Idh2-/- than Idh2+/+ mice. HFD intake decreased IDH2 activity, NADPH levels, and the GSH/(GSH + GSSG) ratio in the renal mitochondria. However, HFD intake increased mitochondrial ROS levels, along with the accompanying oxidative stress and damage. HFD intake increased angiotensin II receptor 1 type 1 mRNA levels in the kidneys and plasma renin and angiotensin II concentrations. These HFD-induced changes were more prominent in Idh2-/- mice than Idh2+/+ mice. Mito-TEMPO mitigated the HFD-induced changes in both Idh2-/- and Idh2+/+ mice, with greater effects in Idh2-/- mice than Idh2+/+ mice. These results indicate that prolonged HFD intake disrupts the IDH2-NADPH-GSH-associated antioxidant system and activates the renin-angiotensin system in the kidney, leading to increased BP, suggesting that IDH2 is a critical enzyme in the development of hypertension and that the IDH2-associated antioxidant system could serve as a potential hypertension treatment target.

Orexin A-induced inhibition of leptin expression and secretion in adipocytes reducing plasma leptin levels and hypothalamic leptin resistance.

Orexin A (OXA) is a neuropeptide associated with plasma insulin and leptin levels involved in body weight and appetite regulation. However, little is known about the effect of OXA on leptin secretion in adipocytes and its physiological roles. Leptin secretion and expression were analysed in 3T3-L1 adipocytes. Plasma leptin, adiponectin and insulin levels were measured by ELISA assay. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) levels in the hypothalamus were evaluated by western blotting. OXA dose-dependently suppressed leptin secretion from 3T3-L1 adipocytes by inhibiting its gene expression while facilitating adiponectin secretion. The leptin inhibition by OXA was mediated via orexin receptors (OXR1 and OXR2). In addition to the pathway via extracellular signal-regulated kinases, OXA triggered adenylyl cyclase-induced cAMP elevation, which results in protein kinase A-mediated activation of cAMP response element-binding proteins (CREB). Accordingly, CREB inhibition restored the OXA-induced downregulation of leptin gene expression and secretion. Exogenous OXA for 4 weeks decreased fasting plasma leptin levels and increased hypothalamic pSTAT3 levels in high-fat diet-fed mice, regardless of increase in body weight and food intake. These results suggest that high dose of OXA directly inhibits leptin mRNA expression and thus secretion in adipocytes, which may be a peripheral mechanism of OXA for its role in appetite drive during fasting. It may be also critical for lowering basal plasma leptin levels and thus maintaining postprandial hypothalamic leptin sensitivity.

Fragmentation of kidney epithelial cell primary cilia occurs by cisplatin and these cilia fragments are excreted into the urine.

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.

Adaptive Data Boosting Technique for Robust Personalized Speech Emotion in Emotionally-Imbalanced Small-Sample Environments.

Personalized emotion recognition provides an individual training model for each target user in order to mitigate the accuracy problem when using general training models collected from multiple users. Existing personalized speech emotion recognition research has a cold-start problem that requires a large amount of emotionally-balanced data samples from the target user when creating the personalized training model. Such research is difficult to apply in real environments due to the difficulty of collecting numerous target user speech data with emotionally-balanced label samples. Therefore, we propose the Robust Personalized Emotion Recognition Framework with the Adaptive Data Boosting Algorithm to solve the cold-start problem. The proposed framework incrementally provides a customized training model for the target user by reinforcing the dataset by combining the acquired target user speech with speech from other users, followed by applying SMOTE (Synthetic Minority Over-sampling Technique)-based data augmentation. The proposed method proved to be adaptive across a small number of target user datasets and emotionally-imbalanced data environments through iterative experiments using the IEMOCAP (Interactive Emotional Dyadic Motion Capture) database.

Repressive effects of red bean, Phaseolus angularis, extracts on obesity of mouse induced with high-fat diet via downregulation of adipocyte differentiation and modulating lipid metabolism.

Obesity is generally caused by quantitative changes in adipocyte differentiation and fat metabolism. Only a few studies have been determined the effect of red beans extract on obesity and plasma cholesterol concentration. We have been studied the functional activities of red-bean extracts including anti-oxidative effect against DNA and cell damages. Histological study including micro CT analysis showed that the accumulation of fat in hepatocytes and intestines was significantly decreased in red bean extract treated group. In addition, plasma cholesterol and triglyceride levels were decreased in blood samples. In addition, it was confirmed that the red bean extract inhibited the expression of PPARγ, Fabp4 and RETN genes, which regulate total adipocyte differentiation and lipid metabolism. Red bean extract inhibits the expressions of transcription factors associated with adipocyte differentiation in a dose-dependent manner, thereby inhibiting fat accumulation and decreasing blood lipid levels in obese mice induced by high fat diet.

Iss2Image: A Novel Signal-Encoding Technique for CNN-Based Human Activity Recognition.

The most significant barrier to success in human activity recognition is extracting and selecting the right features. In traditional methods, the features are chosen by humans, which requires the user to have expert knowledge or to do a large amount of empirical study. Newly developed deep learning technology can automatically extract and select features. Among the various deep learning methods, convolutional neural networks (CNNs) have the advantages of local dependency and scale invariance and are suitable for temporal data such as accelerometer (ACC) signals. In this paper, we propose an efficient human activity recognition method, namely Iss2Image (Inertial sensor signal to Image), a novel encoding technique for transforming an inertial sensor signal into an image with minimum distortion and a CNN model for image-based activity classification. Iss2Image converts real number values from the X, Y, and Z axes into three color channels to precisely infer correlations among successive sensor signal values in three different dimensions. We experimentally evaluated our method using several well-known datasets and our own dataset collected from a smartphone and smartwatch. The proposed method shows higher accuracy than other state-of-the-art approaches on the tested datasets.

LY3009120, a pan-Raf kinase inhibitor, inhibits adipogenesis of 3T3-L1 cells by controlling the expression and phosphorylation of C/EBP-α, PPAR-γ, STAT­3, FAS, ACC, perilipin A, and AMPK.

Excessive preadipocyte differentiation/adipogenesis is closely linked to the development of obesity. LY3009120 is a pan­Raf kinase inhibitor and is known for its anticancer activities. In the present study, the effect of LY3009120 on 3T3­L1 cell adipogenesis was investigated. The differentiation of 3T3­L1 preadipocytes into adipocytes was measured by Oil Red O staining and AdipoRed assay. Changes of cellular protein expression and phosphorylation levels in differentiating 3T3­L1 preadipocytes in the absence or presence of LY3009120 were determined by western blotting analysis. Cell count assay was used to assess the cytotoxicity of LY3009120 on 3T3­L1 cells. At 0.3 µM, LY3009120 markedly inhibited lipid accumulation and decreased triglyceride content in differentiating 3T3­L1 cells. However, it had minimal effect on the elevated expression and phosphorylation of three Raf kinase isoforms (C­Raf, A­Raf, and B­Raf) observed in the cells. LY3009120 reduced not only the expression of CCAAT/enhancer­binding protein­α (C/EBP­α), peroxisome proliferator­activated receptor­Î³ (PPAR­Î³), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A, but also reduced the phosphorylation of signal transducer and activator of transcription­3 (STAT­3) in differentiating 3T3­L1 cells. LY3009120 also increased the phosphorylation of adenosine 3',5'­cyclic monophosphate (cAMP)­activated protein kinase (AMPK), but did not affect the phosphorylation or expression of liver kinase B1 in these cells. In summary, this is the first report, to the best of our knowledge, demonstrating that LY3009120 has an anti­adipogenic effect on 3T3­L1 cells, which may be mediated through control of the expression and phosphorylation of C/EBP­α, PPAR­Î³, STAT­3, FAS, ACC, perilipin A, and AMPK.

Mitochondrial NADP+-dependent isocitrate dehydrogenase deficiency increases cisplatin-induced oxidative damage in the kidney tubule cells.

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.

Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration.

Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration.