Nephroprotective potential of syringic acid in experimental diabetic nephropathy: Focus on oxidative stress and autophagy.

BACKGROUND: Diabetic nephropathy (DN) is a chronic hyperglycemic manifestation of microvascular damage in the kidneys. Widespread research in this area suggests the involvement of perturbed redox homeostasis and autophagy in renal cells phrase- promote the progression of DN. MATERIALS AND METHODS: Reframed sentences-The present study investigates the pharmacological effect of Syringic acid (SYA), in streptozotocin (STZ, 55 mg/kg, i.p) induced diabetic nephropathy model and in high glucose (30 mM) challenged rat renal epithelial cells (NRK 52E) cells with a focus on oxidative stress and autophagy mechanisms. RESULTS: Both in vivo and in vitro experimental data revealed elevated oxidative stress markers along with compromised levels of nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal cellular redox-regulated transcription factor in renal cells upon glycemic stress. Elevated blood glucose also reduced the autophagy process as indicated by low expression of light chain (LC) 3-IIB in diabetic kidney and in NRK 52E cells subjected to excess glucose. SYA (25 and 50 mg/kg, p.o.) administration for 4 weeks to diabetic rats, Reframed sentence-preserved the renal function as evidenced by reduced serum creatinine levels as well as improved urine creatinine and urea levles as compared to non treated diabetic animals. At the molecular level, SYA improved renal expression of Nrf2 and autophagy-related proteins (Atg5, Atg3, and Atg7) in diabetic rats. Similarly, SYA (10 and 20 µM) co-treatment in high glucose-treated NRK 52E cells displayed increased levels of Nrf2 and autophagy induction. CONCLUSION: Results from this study signify the renoprotective effect of SYA and highlight the modulation of oxidative stress and autophagy mechanisms to mitigate diabetic kidney disease.

Melatonin prevents diabetes-induced nephropathy by modulating the AMPK/SIRT1 axis: Focus on autophagy and mitochondrial dysfunction.

Impaired nutrient sensing mechanisms such as AMPK/silent information regulator type 1 (SIRT1) axis and autophagy in renal cells upon chronic diabetic condition accelerate renal injury and upregulating these mechanisms has been reported to prevent renal damage. Melatonin, a neuroendocrine agent, also possess antioxidant and AMPK modulatory effect. In the current study, the protective effect of melatonin against diabetic renal injury was assessed in streptozotocin-induced diabetic nephropathy model and in in vitro model of high-glucose-induced tubular injury. Melatonin (3 and 10 mg/kg) was administered for 28 days after 4 weeks of diabetes induction in Sprague-Dawley rats. For in vitro model, the NRK-52E cells were co-incubated with high glucose and melatonin (25 and 50 µM). Melatonin supplementation abrogated the diabetes-induced renal injury and improved renal function in diabetic rats. Immunoblot analysis of renal tissue lysates revealed improved expression of AMPK, as well as upregulated the expression of nuclear factor erythroid 2-related factor 2, SIRT1, PGC-1α, TFAM and enhanced autophagy upon melatonin treatment in diabetic rats. Likewise, melatonin treatment in high glucose exposed NRK-52E cells improved expression of AMPK, enhanced mitochondrial biogenesis and positively modulated autophagy. However, these effects were repressed upon inhibition of AMPK activity in NRK-52E cells by treatment of Compound-C, suggesting that the protective effects of melatonin were mainly mediated through activation of AMPK. These results suggest that melatonin might mediate the renoprotective effect by upregulating the AMPK/SIRT1 axis, enhancing the autophagy and mitochondrial health in DIabetic Nephropathy.

Renoprotective potential of myo-inositol on diabetic kidney disease: Focus on the role of the PINK1/Parkin pathway and mitophagy receptors.

Recent studies have emphasized the role of mitochondria in renal function as well as in renal injury. Poor mitochondrial quality control mechanisms including mitochondrial fusion, fission and mitophagy are major contributors for progression of diabetic renal injury. The current study is aimed to evaluate the protective role of myo-inositol (MI) against diabetic nephropathy (DN) by utilizing high glucose exposed NRK 52E cell and streptozotocin (STZ) induced DN model. MI supplementation (at doses 37.5 and 75 mg/kg) ameliorated albuminuria and enhanced the renal function as indicated significant improvement in urinary creatinine and urea levels. On the other hand, the western blot analysis of both in vitro and in vivo studies has revealed poor mitophagy in renal cells which was reversed upon myo-inositol treatment. Apart from targeting the canonical PINK1/Parkin pathway, we also focused on the role mitophagy receptors prohibitin (PHB) and NIP3-like protein (NIX). A significant reduction in expression of NIX and PHB2 was observed in renal tissue of diabetic control rats and high glucose exposed NRK 52E cells. Myo-inositol treatment resulted in positive modulation of PINK1/Parkin pathway as well as PHB2 and NIX. Myo-inositol also enhanced the mitochondrial biogenesis in renal tissue of diabetic rat by upregulating Nrf2/SIRT1/PGC-1α axis. The current study thus underlines the renoprotective effect myo-inositol, upregulation of mitophagy proteins and mitochondrial biogenesis upon myo-inositol treatment.

GSK2606414 attenuates PERK/p-eIF2α/ATF4/CHOP axis and augments mitochondrial function to mitigate high glucose induced neurotoxicity in N2A cells.

Neuronal dysfunction and subsequent apoptosis under high glucose conditions during diabetes contribute majorly to the manifestation of diabetic peripheral neuropathy (DPN). PERK (protein kinase RNA (PKR)-like ER kinase) one among the three canonical arms of unfolded protein response (UPR), is believed to play a crucial role in determining the cell fate during endoplasmic reticulum stress (ERS/ER stress) conditions. We evaluated the role of PERK inhibitor GSK2606414 in high glucose (30 â€‹mM) treated neuroblastoma (N2A) cells. High glucose resulted in disruption of ER proteostasis by activation of UPR which is evident through increased (p â€‹< â€‹0.001) expression of GRP78, p-PERK, p-eIF2α, ATF-4 and CHOP when compared to normal cells. It is accompanied with enhanced GRP78 localization in Endoplasmic Reticulum (ER) lumen evident from ER labeling Immunofluorescence (IF) staining. PERK activation resulted in altered mitochondrial function evident by increased mitochondrial superoxide production and compromised mitochondrial homeostasis with decrease in Mfn-2 levels. Additionally, ER stress induced neuronal apoptosis was attenuated by GSK2606414 treatment via inhibiting the PERK-eIF2α-ATF4-CHOP axis that not only curtailed the levels of apoptotic proteins like Bax and caspase 3 but also elevated the levels of anti-apoptotic Bcl-2. Collectively, our findings revealed the neuroprotective potential of GSK2606414 against high glucose induced neurotoxicity in N2A cells.

Indole-3-propionic acid attenuates high glucose induced ER stress response and augments mitochondrial function by modulating PERK-IRE1-ATF4-CHOP signalling in experimental diabetic neuropathy.

OBJECTIVES: We aimed to evaluate the neuroprotective effect of Indole-3-propionic acid (IPA) against streptozotocin (STZ) induced diabetic peripheral neuropathy (DPN) in rats and in high glucose (HG) induced neurotoxicity in neuro2a (N2A) cells. METHODS: Diabetes was induced in male SD rats STZ (55 mg/kg, i.p.) and IPA (10 and 20 mg/kg, p.o.) was administered for two weeks, starting from sixth week after diabetes induction. Neurobehavioral, functional assessments were made, and various molecular studies were performed to evaluate the effect of IPA on HG induced ER stress and mitochondrial dysfunction in sciatic nerves, DRGs and in N2A cells. RESULTS: Diabetic rats and high glucose exposed N2A cells showed marked increase in oxidative damage accompanied by ER stress and mitochondrial dysfunction along with increased apoptotic markers. IPA treatment for two weeks markedly alleviated these changes and attenuated pain behaviour. CONCLUSION: IPA exhibited neuroprotective activity against hyperglycaemic insults.

Carvacrol abates NLRP3 inflammasome activation by augmenting Keap1/Nrf-2/p62 directed autophagy and mitochondrial quality control in neuropathic pain.

Objectives: We aimed to evaluate the effect of carvacrol (CRC), a phenolic monoterpene with high nutritional value on NLRP3 activation against chronic constriction injury (CCI) of sciatic nerve induced neuropathic pain (NP) in rats and in lipopolysacharide (LPS) induced neuroinflammation in neuro2a (N2A) cells. Methods: NP was induced in male SD rats by performing CCI and CRC (30 and 60 mg/kg, p.o) was administered for 14 days. Behavioural and functional parameters were evaluated using standard procedures. Various molecular experimentations were conducted to evaluate the efficacy of CRC against CCI induced neuropathy and in LPS (1 µg/ml) primed and ATP (5 µM) treated N2A cells.Results: CCI resulted in marked development of hyperalgesia and allodynia. Further, CCI rats, LPS and ATP treated N2A cells showed enhanced expression of NLRP3, ASC, Caspase-1 and IL-1ß. In addition, CCI rats exhibited diminished levels of Nrf-2 with an increase in Keap1 expression. Also, CCI animals manifested with compromised mitochondrial function along with decreased autophagy markers and enhanced p62 levels when compared to sham rats. However, CRC administration significantly ameliorated these changes suggesting NLRP3 inhibition by CRC may be attributed to activation of autophagy via Keap1/Nrf-2/p62 forward feedback loop and augmentation of mitochondrial quality control. Intriguingly, pretreatment of CRC (50 and 100 µM) to LPS and ATP treated N2A cells resulted in decreased colocalization of NLRP3 and ASC.Discussion: These findings revealed the neuroprotective potential of CRC against CCI induced NP and delineate the critical role of autophagy and mitochondrial quality control in NLRP3 regulation.

Mitochondrial remodelling-a vicious cycle in diabetic complications.

Diabetes mellitus (DM) is a chronic, metabolic condition characterized by excessive blood glucose that causes perturbations in physiological functioning of almost all the organs of human body. This devastating metabolic disease has its implications in cognitive decline, heart damage, renal, retinal and neuronal complications that severely affects quality of life and associated with decreased life expectancy. Mitochondria possess adaptive mechanisms to meet the cellular energy demand and combat cellular stress. In recent years mitochondrial homeostasis has been point of focus where several mechanisms regulating mitochondrial health and function are evaluated. Mitochondrial dynamics plays crucial role in maintaining healthy mitochondria in cell under physiological as well as stress condition. Mitochondrial dynamics and corresponding regulating mechanisms have been implicated in progression of metabolic disorders including diabetes and its complications. In current review we have discussed about role of mitochondrial dynamics under physiological and pathological conditions. Also, modulation of mitochondrial fission and fusion in diabetic complications are described. The available literature supports mitochondrial remodelling as reliable target for diabetic complications.

SIRT1 Activation by Polydatin Alleviates Oxidative Damage and Elevates Mitochondrial Biogenesis in Experimental Diabetic Neuropathy.

Mitochondrial dysfunction has been implicated as a one of the major factors linked to the development of painful diabetic neuropathy (DN). Several studies have demonstrated that sirtuin (SIRT1) activation recuperates nerve function by activating mitochondrial biogenesis. Polydatin, a resveratrol glycoside, has been explored to improve mitochondrial function via SIRT1 activation. However, the neuroprotective effects of polydatin in DN remain elusive. In this study, polydatin (25 and 50 mg/kg, oral) was administered for last 2 weeks of 8-week study to diabetic Sprague-Dawley rats weighing 250-300 g (post 6-weeks of streptozotocin 55 mg/kg, intraperitoneal). Treatment with polydatin significantly attenuated mechanical and thermal hyperalgesia in diabetic rats. Treated diabetic rats also showed improvement in motor/sensory nerve conduction velocities and nerve blood flow. For in vitro studies, Neuro2a cells were exposed to high-glucose (30 mM) condition to simulate short-term hyperglycemia. Polydatin was evaluated for its role in SIRT1 and Nrf2 activation at a dose of 5, 10, and 20 µM concentrations. Polydatin exposure normalized the mitochondrial superoxides, membrane potentials and improved neurite outgrowth in high-glucose-exposed Neuro2a cells. Increased SIRT1 activation by polydatin resulted in peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) directed mitochondrial biogenesis. SIRT1 activation also facilitated Nrf2-directed antioxidant signaling. Study results inferred that decline in mitochondrial biogenesis and oxidative function in diabetic rats and high-glucose-exposed Neuro2a cells, could be counteracted by polydatin administration, postulated via enhancing SIRT1 and Nrf2 axis.

Chronic hyperglycemia impairs mitochondrial unfolded protein response and precipitates proteotoxicity in experimental diabetic neuropathy: focus on LonP1 mediated mitochondrial regulation.

BACKGROUND: Disturbed mitochondrial homeostasis has been identified to contribute to the pathogenesis of diabetic neuropathy (DN). However, the role of Mitochondrial Lon peptidase 1 (Lonp1) and Heat shock proteins (HSP's) in DN remains elusive. Here we studied the role of these proteins in experimental DN. METHODS: Rats were injected with STZ (55 mg/kg, ip) to induce diabetes. After confirmation of diabetes, animals were maintained for 8 weeks to develop neuropathy. Resveratrol was administered at two dose levels 10 and 20 mg/kg for last 2 weeks. Neuronal PC12 cells was challenged with 30 mM of ß-D glucose to evaluate the molecular changes. RESULTS: Diabetic rats showed reduced expression of various mitochondrial proteases in dorsal root ganglions (DRG). This effect may increase proteotoxicity and diminish electron transport chain (ETC) activity as evident by increased protein oxidation and reduced ETC complexes activities under diabetic condition. In particular, we focused on our efforts to characterize the expression pattern of Lonp1 which was found to be significantly (p < 0.01 vs. control group) under expressed in DRG of diabetic rats. We used Resveratrol to characterize the importance of Lonp1 in regulation of mitochondrial function. High glucose (HG) (30 mM) exposed PC12 cells suggested that Resveratrol treatment attenuated the HG induced mitochondrial damage via induction of mitochondrial proteases. Moreover, siRNA directed against Lonp1 has impaired the activity of Resveratrol in attenuating the HG induced mitochondrial dysfunction. CONCLUSION: These results would signify the importance of modulating mitochondrial proteases for the therapeutic management of DN.

Bardoxolone Methyl Ameliorates Hyperglycemia Induced Mitochondrial Dysfunction by Activating the keap1-Nrf2-ARE Pathway in Experimental Diabetic Neuropathy.

Bardoxolone methyl (Bard), a nuclear factor erythroid 2-related factor 2 (Nrf2) activator regulates multiple oxidative and inflammatory diseases. However, the role of Bard in painful diabetic neuropathy (DN) remains unknown. Bard administration at two dose levels (15 & 30 mg/kg/day) to STZ (55 mg/kg, i.p) induced diabetic rats for last two weeks of eight week study significantly improved motor nerve conduction velocity (61.84 ± 1.9 vs. 38.57 ± 1.08 m/s), sensory nerve conduction velocity (66.86 ± 5.1 vs. 39.43 ± 3.3 m/s), nerve blood flow (86.28 ± 6.4 vs. 56.56 ± 1.62 PU), and intraepidermal nerve fiber density. Additionally, Bard treatment attenuated thermal and mechanical hyperalgesia in diabetic rats. Further molecular investigation on dorsal root ganglions (DRG) tissue isolated from L4-L6 regions of diabetic rats and High glucose (HG) exposed PC12 cells displayed decreased expression and transcriptional activity of Nrf2 which might have resulted in depleted antioxidant enzymes and mitochondrial chaperones. Bard treatment significantly reversed these effects in diabetic rats and also in HG exposed PC12 cells. Moreover, mitochondrial complex activities were diminished in DRG mitochondrial fractions of diabetic rats and mitochondrial isolates of HG exposed PC12 cells and Bard treatment significantly reversed these effects. Furthermore, Bard treatment significantly impeded the impact of hyperglycemic insults on mitochondrial membrane potential, ROS production and mitochondrial oxygen consumption rate (OCR) (Basal respiration, Maximal respiration, ATP production and spare respiratory capacity) in PC12 cells. Collectively our data suggests that Bard treatment to STZ induced diabetic rats robustly reduces DN which may be due to its effect on Keap1-Nrf2-ARE pathway and have contributed to improvement in mitochondrial function.

Adenosine monophosphate-activated protein kinase modulation by berberine attenuates mitochondrial deficits and redox imbalance in experimental diabetic neuropathy.

Adenosine monophosphate-activated protein kinase (AMPK) has been studied for its myriad metabolic and mitochondrial benefits in several chronic diseases. Recent studies have uncovered its therapeutic potential against mitochondrial dysfunction in cultured dorsal root ganglion (DRG) neurons isolated from streptozotocin (STZ) induced diabetic rats. The present study is aimed at evaluating the pharmacological efficacy of berberine (BRB), a natural AMPK activator against experimental diabetic neuropathy (DN) phenotype developed in STZ (55 mg/kg, i.p.) induced diabetic rats and neurotoxicity in high-glucose (30 mM) stimulated neuro 2a (N2A) cells. Diabetic-rats have shown reduced expression of p-AMPK (Thr 172) in sciatic nerves with a consequent reduction in mitochondrial biogenesis and autophagy. BRB (50 & 100 mg/kg, po) administration to diabetic rats for 2-weeks rescued mitochondrial functional deficits and autophagy impairment by increasing the p-AMPK expression. BRB administration also augmented the NEF-2 related factor 2 (Nrf2) mediated endogenous antioxidant defence systems to restrain neuronal damage and neuroinflammation. These effects after BRB administration resulted in enhanced conduction velocity, improved nerve blood flow and attenuated hyperalgesia. Similarly, BRB exposure (5 & 10 µM) to N2A cells attenuated high glucose induced ROS generation, mitochondrial membrane depolarization through the promotion of Nrf2 mediated antioxidant defence systems. BRB exposure augmented peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mediated mitochondrial biogenesis in neuronal cells. Results from this study signify the importance of mitoprotection conferred by BRB in DN and can be used as a preliminary basis for further molecular exploration.