Increased long noncoding RNA maternally expressed gene 3 contributes to podocyte injury induced by high glucose through regulation of mitochondrial fission.
ABSTRACT: Excessive mitochondrial fission plays a key role in podocyte injury in diabetic kidney disease (DKD), and long noncoding RNAs (lncRNAs) are important in the development and progression of DKD. However, lncRNA regulation of mitochondrial fission in podocytes is poorly understood. Here, we studied lncRNA maternally expressed gene 3 (Meg3) in mitochondrial fission in vivo and in vitro using human podocytes and Meg3 podocyte-specific knockdown mice. Expression of lncRNA Meg3 in STZ-induced diabetic mice was higher, and correlated with the number of podocytes. Excessive mitochondrial fission of podocytes and renal histopathological and physiological parameters were improved in podocyte-specific Meg3 knockdown diabetic mice. Elongated mitochondria with attenuated podocyte damage, as well as mitochondrial translocation of dynamin-related protein 1 (Drp1), were decreased in Meg3 knockout podocytes. By contrast, increased fragmented mitochondria, podocyte injury, and Drp1 expression and phosphorylation were observed in lncRNA Meg3-overexpressing podocytes. Treatment with Mdivi1 significantly blunted more fragmented mitochondria and reduced podocyte injury in lncRNA Meg3-overexpressing podocytes. Finally, fragmented mitochondria and Drp1 mitochondrial translocation induced by high glucose were reduced following treatment with Mdivi1. Our data show that expression of Meg3 in podocytes in both human cells and diabetic mice was higher, which regulates mitochondrial fission and contributes to podocyte injury through increased Drp1 and its translocation to mitochondria.
Project description:Mitochondria are dynamic organelles frequently undergoing fission and fusion events to maintain their integrity, bioenergetics and spatial distribution, which is fundamental to the processes of cell survival. Disruption in mitochondrial dynamics plays a role in cancer. Therefore, proteins involved in regulating mitochondrial dynamics are potential targets for treatment. mDIVI1 is an inhibitor of the mitochondrial fission protein DRP1, which induces i) mitochondrial oxidative stress and ii) effectively reduces mitochondrial metabolism. We show here that mDIVI1 is able to inhibit 3D tumorsphere forming capacity, cell migration and stemness-related signalling in breast cancer cells, indicating that mDIVI1 can potentially be used for the therapeutic elimination of cancer stem cells (CSCs).
Project description:Several lines of evidence suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy. However, the signaling pathways by which hyperglycemia leads to mitochondrial dysfunction are not fully understood. Here we examined the role of Rho-associated coiled coil-containing protein kinase 1 (ROCK1) on mitochondrial dynamics by generating two diabetic mouse models with targeted deletions of ROCK1 and an inducible podocyte-specific knockin mouse expressing a constitutively active (cA) mutant of ROCK1. Our findings suggest that ROCK1 mediates hyperglycemia-induced mitochondrial fission by promoting dynamin-related protein-1 (Drp1) recruitment to the mitochondria. Deletion of ROCK1 in diabetic mice prevented mitochondrial fission, whereas podocyte-specific cA-ROCK1 mice exhibited increased mitochondrial fission. Importantly, we found that ROCK1 triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. These findings provide insights into the unexpected role of ROCK1 in a signaling cascade that regulates mitochondrial dynamics.
Project description:The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1α, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).
Project description:<h4>Background</h4>Diabetic nephropathy (DN) is one of the principal complications of diabetes and podocyte injury plays an important role in the DN pathogenesis. Wnt/?-catenin signaling overactivation confers podocyte injury and promotes multiple types of renal disease. However, the underlying mechanism of Wnt/?-catenin signaling activation in DN progression has not been fully elucidated. Long noncoding RNA (lncRNA) is a large class of endogenous RNA molecules lacking functional code capacity and which participates in the pathogenesis of human disease, including DN.<h4>Method</h4>A diabetes model was constructed by intraperitoneal injection of Streptozotocin in rats. The MPC5 cells were used to create the in vitro model. Western blot and Quantitative reverse-transcriptase-PCR were used to examine the expression of protein and mRNA. The migrated capacity was analyzed by Transwell migration assay. The cell viability was detected by CCK8.<h4>Results</h4>In the present study, we revealed the association of lncRNA Maternally Expressed Gene 3 (MEG3) with aberrant activation of Wnt/?-catenin signaling and the role of MEG3/Wnt axis in podocyte injury. We found that high glucose (HG) treatment suppressed MEG3 expression in cultured podocytes, activated Wnt/?-catenin signaling and caused podocyte injury as indicated by the downregulation of podocyte-specific markers (podocin and synaptopodin) and the upregulation of snail1 and ?-smooth muscle actin. Overexpression of MEG3 attenuated HG-induced podocyte injury by reducing Wnt/?-catenin activity, repressing cell migration, reactive oxygen species production and increasing the viability of podocytes. Furthermore, we provided evidences that restoration of Wnt/?-catenin signaling by specific agonist impeded the protective effect of MEG3 on podocyte injury. Current results demonstrated that MEG3/Wnt axis plays an important role in fostering podocyte injury and may serve as a potential therapeutic target for the treatment of DN.<h4>Conclusion</h4>lncRNA MEG3 ameliorates podocyte injury in DN via inactivating Wnt/?-catenin signaling.
Project description:Elevated levels of plasma free fatty acid (FFA) and disturbed mitochondrial dynamics play crucial roles in the pathogenesis of diabetic kidney disease (DKD). However, the mechanisms by which FFA leads to mitochondrial damage in glomerular podocytes of DKD and the effects of Berberine (BBR) on podocytes are not fully understood. Methods: Using the db/db diabetic mice model and cultured mouse podocytes, we investigated the molecular mechanism of FFA-induced disturbance of mitochondrial dynamics in podocytes and testified the effects of BBR on regulating mitochondrial dysfunction, podocyte apoptosis and glomerulopathy in the progression of DKD. Results: Intragastric administration of BBR for 8 weeks in db/db mice significantly reversed glucose and lipid metabolism disorders, podocyte damage, basement membrane thickening, mesangial expansion and glomerulosclerosis. BBR strongly inhibited podocyte apoptosis, increased reactive oxygen species (ROS) generation, mitochondrial fragmentation and dysfunction both in vivo and in vitro. Mechanistically, BBR could stabilize mitochondrial morphology in podocytes via abolishing palmitic acid (PA)-induced activation of dynamin-related protein 1 (Drp1). Conclusions: Our study demonstrated for the first time that BBR may have a previously unrecognized role in protecting glomerulus and podocytes via positively regulating Drp1-mediated mitochondrial dynamics. It might serve as a novel therapeutic drug for the treatment of DKD.
Project description:The purpose of our study was to determine the synergistic protective effects of mitochondria-targeted antioxidant SS31 and mitochondria division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Using biochemical methods, we assessed mitochondrial function by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity, mitochondrial ATP, and GTPase Drp1 enzymatic activity in mutant A?PP cells. Using biochemical methods, we also measured cell survival and apoptotic cell death. Amyloid-? (A?) levels were measured using sandwich ELISA, and using real-time quantitative RT-PCR, we assessed mtDNA (mtDNA) copy number in relation to nuclear DNA (nDNA) in all groups of cells. We found significantly reduced levels of A?40 and A?42 in mutant A?PP cells treated with SS31, Mdivi1, and SS31+Mdivi1, and the reduction of A?42 levels were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. The levels of mtDNA copy number and cell survival were significantly increased in SS31, Mdivi1, and SS31+Mdivi1 treated mutant A?PP cells; however, the increased levels of mtDNA copy number and cell survival were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. Mitochondrial dysfunction is significantly reduced in SS31, Mdivi1, and SS31+Mdivi1 treated mutant A?PP cells; however, the reduction is much higher in cells treated with both SS31+Mdvi1. Similarly, GTPase Drp1 activity is reduced in all treatments, but reduced much higher in SS31+Mdivi1 treated cells. These observations strongly suggest that combined treatment of SS31+Mdivi1 is effective than individual treatments of SS31 and Mdivi1. Therefore, we propose that combined treatment of SS31+Mdivi1 is a better therapeutic strategy for AD. Ours is the first study to investigate combined treatment of mitochondria-targeted antioxidant SS31 and mitochondrial division inhibitor 1 in AD neurons.
Project description:Previous studies have shown that mitochondrial dysfunction plays an important role in high- glucose(HG)-induced podocyte injury and thus contributes to the progression of diabetic nephropathy(DN). The histone deacetylase Sirtuin6 (Sirt6) has been revealed to have an essential role in the regulation of mitochondrial function in skeletal muscle and cardiomyocytes. However, its specific role in mitochondrial homeostasis in podocytes is undetermined. Here, we aimeds to explore the physiological function of Sirt6 in podocyte mitochondria and apoptosis under HG conditions and explore the possible mechanism. Herein, we observed that Sirt6-WT-1 colocalization was suppressed in the glomeruli of patients with DN. In addition, diabetic mice exhibited reduced Sirt6 expression and AMP kinase (AMPK) dephosphorylation accompanied by mitochondrial morphological abnormalities. In vitro, podocytes exposed to HG presented with mitochondrial morphological alterations and podocyte apoptosis accompanied by Sirt6 and p-AMPK downregulation. In addition, HG promoted a decrease in mitochondrial number and an increase in mitochondrial superoxide production as well as a decreased mitochondrial membrane potential. ROS production was also increased in HG-treated podocytes. Conversely, all these mitochondrial defects induced by HG were significantly alleviated by Sirt6 plasmid transfection. Sirt6 overexpression simultaneously alleviated HG-induced podocyte apoptosis and oxidative stress, as well as increased AMPK phosphorylation. Increased levels of H3K9ac and H3K56ac induced by HG were attenuated in podocytes transfected with Sirt6 plasmids. Therefore, these results elucidated that Sirt6 protects mitochondria of podocytes and exerts anti-apoptotic effects via activating AMPK pathway. The present findings provide key insights into the pivotal role of mitochondria regulation by SIRT6 in its protective effects on podocytes.
Project description:Myocardial contractile dysfunction is associated with an increase in mitochondrial fission in patients with diabetes. However, whether mitochondrial fission directly promotes diabetes-induced cardiac dysfunction is still unknown. Melatonin exerts a substantial influence on the regulation of mitochondrial fission/fusion. This study investigated whether melatonin protects against diabetes-induced cardiac dysfunction via regulation of mitochondrial fission/fusion and explored its underlying mechanisms. Here, we show that melatonin prevented diabetes-induced cardiac dysfunction by inhibiting dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Melatonin treatment decreased Drp1 expression, inhibited mitochondrial fragmentation, suppressed oxidative stress, reduced cardiomyocyte apoptosis, improved mitochondrial function and cardiac function in streptozotocin (STZ)-induced diabetic mice, but not in SIRT1<sup>-/-</sup> diabetic mice. In high glucose-exposed H9c2 cells, melatonin treatment increased the expression of SIRT1 and PGC-1? and inhibited Drp1-mediated mitochondrial fission and mitochondria-derived superoxide production. In contrast, SIRT1 or PGC-1? siRNA knockdown blunted the inhibitory effects of melatonin on Drp1 expression and mitochondrial fission. These data indicated that melatonin exerted its cardioprotective effects by reducing Drp1-mediated mitochondrial fission in a SIRT1/PGC-1?-dependent manner. Moreover, chromatin immunoprecipitation analysis revealed that PGC-1? directly regulated the expression of Drp1 by binding to its promoter. Inhibition of mitochondrial fission with Drp1 inhibitor mdivi-1 suppressed oxidative stress, alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. These findings show that melatonin attenuates the development of diabetes-induced cardiac dysfunction by preventing mitochondrial fission through SIRT1-PGC1? pathway, which negatively regulates the expression of Drp1 directly. Inhibition of mitochondrial fission may be a potential target for delaying cardiac complications in patients with diabetes.
Project description:Phosphorylation of Dynamin-related protein1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here we established the role of Drp1 Serine 600 (S600) phosphorylation on mitochondrial fission in vivo, and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in progression of diabetic nephropathy (DN). We generated a knockin mouse in which S600 was mutated to alanine (Drp1S600A). We found that diabetic Drp1S600A mice exhibited improved biochemical and histological features of DN along with reduced mitochondrial fission and diminished mitochondrial ROS in vivo. Importantly, we observed that the effect of Drp1S600 phosphorylation on mitochondrial fission in the diabetic milieu was stimulus- but not cell type-dependent. Mechanistically, we showed that mitochondrial fission in high glucose conditions occurs through concomitant binding of phospho-Drp1S600 with mitochondrial fission factor (Mff) and actin-related protein 3 (Arp3), ultimately leading to accumulation of F-actin and Drp1 on the mitochondria. Taken together, these findings establish that a single phosphorylation site in Drp1 can regulate mitochondrial fission and progression of DN in vivo, and highlight the stimulus-specific consequences of Drp1S600 phosphorylation on mitochondrial dynamics.
Project description:Metformin is a widely used antidiabetic drug that exerts cardiovascular protective effects in patients with diabetes. How metformin protects against diabetes-related cardiovascular diseases remains poorly understood. Here, we show that metformin abated the progression of diabetes-accelerated atherosclerosis by inhibiting mitochondrial fission in endothelial cells. Metformin treatments markedly reduced mitochondrial fragmentation, mitigated mitochondrial-derived superoxide release, improved endothelial-dependent vasodilation, inhibited vascular inflammation, and suppressed atherosclerotic lesions in streptozotocin (STZ)-induced diabetic ApoE-/- mice. In high glucose-exposed endothelial cells, metformin treatment and adenoviral overexpression of constitutively active AMPK downregulated mitochondrial superoxide, lowered levels of dynamin-related protein (Drp1) and its translocation into mitochondria, and prevented mitochondrial fragmentation. In contrast, AMPK-?2 deficiency abolished the effects of metformin on Drp1 expression, oxidative stress, and atherosclerosis in diabetic ApoE-/-/AMPK-?2-/- mice, indicating that metformin exerts an antiatherosclerotic action in vivo via the AMPK-mediated blockage of Drp1-mediated mitochondrial fission. Consistently, mitochondrial division inhibitor 1, a potent and selective Drp1 inhibitor, reduced mitochondrial fragmentation, attenuated oxidative stress, ameliorated endothelial dysfunction, inhibited inflammation, and suppressed atherosclerosis in diabetic mice. These findings show that metformin attenuated the development of atherosclerosis by reducing Drp1-mediated mitochondrial fission in an AMPK-dependent manner. Suppression of mitochondrial fission may be a therapeutic approach for treating macrovascular complications in patients with diabetes.