Toxicity of depleted uranium on isolated rat kidney mitochondria

Background

Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in comparison to other organs. Although the oxidative stress and mitochondrial damage induced by DU has been well investigated, the precise mechanism of DU-induced nephrotoxicity has not been thoroughly recognized yet.

Methods

Kidney mitochondria were obtained using differential centrifugation from Wistar rats and mitochondrial toxicity endpoints were then determined in both in vivo and in vitro uranyl acetate (UA) exposure cases.

Results

Single injection of UA (0, 0.5, 1 and 2 mg/kg, i.p.) caused a significant increase in blood urea nitrogen and creatinine levels. Isolated mitochondria from the UA-treated rat kidney showed a marked elevation in oxidative stress accompanied by mitochondrial membrane potential (MMP) collapse as compared to control group. Incubation of isolated kidney mitochondria with UA (50, 100 and 200 μM) manifested that UA can disrupt the electron transfer chain at complex II and III that leads to induction of reactive oxygen species (ROS) formation, lipid peroxidation, and glutathione oxidation. Disturbances in oxidative phosphorylation were also demonstrated through decreased ATP concentration and ATP/ADP ratio in UA-treated mitochondria. In addition, UA induced a significant damage in mitochondrial outer membrane. Moreover, MMP collapse, mitochondrial swelling and cytochrome c release were observed following the UA treatment in isolated mitochondria.

General significance

Both our in vivo and in vitro results showed that UA-induced nephrotoxicity is linked to the impairment of electron transfer chain especially at complex II and III which leads to subsequent oxidative stress.     

The decreased expression of mitofusin-1 and increased fission-1 together with alterations in mitochondrial morphology in the kidney of rats with chronic fluorosis may involve elevated oxidative stress

This study was designed to characterize changes in the expression of mitofusin-1 (Mfn1) and fission-1 (Fis1), as well as in mitochondrial morphology in the kidney of rats subjected to chronic fluorosis and to elucidate whether any mitochondrial injury observed is associated with increased oxidative stress. Sixty Sprague-Dawley (SD) rats were divided randomly into 3 groups of 20 each, i.e., the untreated control group (natural drinking water containing <0.5 mg fluoride/L), the low-fluoride group (drinking water supplemented with 10 mg fluoride/L, prepared with NaF) and the high-fluoride group (50 mg fluoride/L), and treated for 6 months. Thereafter, renal expression of Mfn1 and Fis1 at both the protein and mRNA levels was determined by immunohistochemistry and real-time PCR, respectively. In addition, the malondiadehyde (MDA) was quantitated by the thiobarbituric acid procedure and the total antioxidative capability (T-AOC) by a colorimetric method. The morphology of renal mitochondria was observed under the transmission electron microscope. In the renal tissues of rats with chronic fluorosis, expression of both Mfn1 protein and mRNA was clearly reduced, whereas that of Fis1 was elevated. The level of MDA was increased and the T-AOC lowered. Swollen or fragmented mitochondria in renal cells were observed under the electronic microscope. These findings indicate that chronic fluorosis can lead to the abnormal mitochondrial dynamics and changed morphology in the rat kidney, which in mechanism might be induced by a high level of oxidative stress in the disease.     

Thioacetamide-Induced Fulminant Hepatic Failure Induces Cerebral Mitochondrial Dysfunction by Altering the Electron Transport Chain Complexes

Fulminant hepatic failure (FHF) is an acute form of hepatic encephalopathy resulting from severe inflammatory or necrotic liver damage without any previously established liver damage. This develops as a complication due to viral infections, and drug abuse. FHF also occurs in acute disorders like Reye’s syndrome. Although the exact mechanisms in the etiology of FHF are not understood, elevated levels of brain ammonia have been consistently reported. Such increased ammonia levels are suggested to alter neurotransmission signals and impair cerebral energy metabolism due to mitochondrial dysfunctions. In the present study we have examined the role of cerebral electron transport chain complexes, including complex I, II, III IV, and pyruvate dehydrogenase in the non-synaptic mitochondria isolated from the cortex of the thioacetamide-induced FHF rats. Further, we have examined if the structure of mitochondria is altered. The results of the current study demonstrated a decrease in the activity of the complex I by 31 and 48% at 18 and 24 h respectively after the thioacetamide injection. Similarly, the activity of electron transport chain complex III was inhibited by 35 and 52% respectively, at 18 and 24 h, respectively. The complex II and complex IV, on the other hand, revealed unaltered activity. Further the activity of pyruvate dehydrogenase at 18 and 24 h after the induction of FHF was inhibited by 29 and 43%, respectively. Our results also suggest mitochondrial swelling in FHF induced rats. The inhibition of the respiratory complexes III and I and pyruvate dehydrogenase might lead to the increased production of free radical resulting in oxidative stress and cerebral energy disturbances thereby leading to mitochondrial swelling and further contributing to the pathogenesis of FHF.     

The effects of mesenchymal stem cell mitochondrial transplantation on doxorubicin‐mediated nephrotoxicity in rats

The effect of dysfunctional mitochondria in several cell pathologies has been reported in renal diseases, including diabetic nephropathy and acute kidney injury. Previous studies have reported that mitochondrial transplantation provided surprising results in myocardial and liver ischemia, as well as in Parkinson's disease. We aimed to investigate the beneficial effects of isolated mitochondria transplantation from mesenchymal stem cells (MSCs) in vivo, to mitigate renal damage that arises from doxorubicin‐mediated nephrotoxicity and its action mechanism. In this study, a kidney model of doxorubicin‐mediated nephrotoxicity was used and isolated mitochondria from MSCs were transferred to the renal cortex of rats. The findings showed that the rate of isolated mitochondria from MSCs maintains sufficient membrane integrity, and was associated with a beneficial renal therapeutic effect. Following doxorubicin‐mediated renal injury, isolated mitochondria or vehicle infused into the renal cortex and rats were monitored for five days. This study found that mitochondrial transplantation decreased cellular oxidative stress and promoted regeneration of tubular cells after renal injury (P < .001, P = .009). Moreover, mitochondrial transplantation reduced protein accumulation of tubular cells and reversed renal deficits (P = .01, P < .001). Mitochondrial transplantation increased Bcl‐2 levels, and caspase‐3 levels decreased in injured renal cells (P < .015, P < .001). Our results provide a direct link between mitochondria dysfunction and doxorubicin‐mediated nephrotoxicity and suggest a therapeutic effect of transferring isolated mitochondria obtained from MSCs against renal injury. To our knowledge, this study is the first study in the literature that showed good therapeutic effects of mitochondrial transplantation in a nephrotoxicity model, which is under‐researched.     

Dietary High Vanadium Causes Oxidative Damage-Induced Renal and Hepatic Toxicity in Broilers

The purpose of this study was to investigate the renal and hepatic oxidative damage and toxicity caused by dietary high vanadium in broilers. A total of 420 one-day-old avian broilers were divided into six groups and fed on a corn–soybean basal diet as control diet (vanadium 0.073 mg/kg), and five high vanadium diets (vanadium 5 mg/kg, high vanadium group I; 15 mg/kg, high vanadium group II; 30 mg/kg, high vanadium group III; 45 mg/kg, high vanadium group IV; and 60 mg/kg, high vanadium group V) throughout the experimental period of 42 days. The results showed that the renal and hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, ability to inhibit hydroxy radical, and malondialdehyde (MDA), glutathione, and vanadium contents were not significantly changed in high vanadium group I and II when compared with those of the control groups. However, the SOD and GSH-Px activities, ability to inhibit hydroxy radical, and GSH content were significantly decreased, and the MDA and vanadium contents were markedly increased in high vanadium groups III, IV, and V. At the same time, the lesions were also observed in the kidney and liver of high vanadium groups III, IV, and V. The renal tubular epithelial cells showed granular degeneration and vacuolar degeneration, and hepatocytes showed granular degeneration, vacuolar degeneration, and fatty degeneration. It was concluded that dietary vanadium in the range of 30–60 mg/kg could cause oxidative damage and vanadium accumulation, which induced renal and hepatic toxicity and lesions. The renal and hepatic function was finally impaired in boilers.     

A diet rich in n-3 polyunsaturated fatty acids reduced prostaglandin biosynthesis, ovulation rate, and litter size in mice

Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to > 2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation.     

MitoQ Blunts Mitochondrial and Renal Damage during Cold Preservation of Porcine Kidneys

Cold preservation has greatly facilitated the use of cadaveric kidneys for transplantation but damage occurs during the preservation episode. It is well established that oxidant production increases during cold renal preservation and mitochondria are a key target for injury. Our laboratory has demonstrated that cold storage of renal cells and rat kidneys leads to increased mitochondrial superoxide levels and mitochondrial electron transport chain damage, and that addition of Mitoquinone (MitoQ) to the preservation solutions blunted this injury. In order to better translate animal studies, the inclusion of large animal models is necessary to develop safe preclinical protocols. Therefore, we tested the hypothesis that addition of MitoQ to cold storage solution preserves mitochondrial function by decreasing oxidative stress, leading to less renal tubular damage during cold preservation of porcine kidneys employing a standard criteria donor model. Results showed that cold storage significantly induced oxidative stress (nitrotyrosine), renal tubular damage, and cell death. Using High Resolution Respirometry and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex II/III respiration of the electron transport chain following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. These results suggested that MitoQ decreased oxidative stress, tubular damage and cell death by improving mitochondrial function during cold storage. Therefore this compound should be considered as an integral part of organ preservation solution prior to transplantation.     

The influence of aging on renal response to cadmium in Syrian hamsters

To determine the renal effects of cadmium (Cd) in older animals, we administered subcutaneously a single dose of cadmium, 3.0 mg/kg/BW, to Syrian hamsters aged 16 wk (“young”) and 60 wk (“old”). Marked morphologic changes in the kidney and renal dysfunction were observed, especially in the older animals. The concentration of MDA in the renal cortex was significantly increased only in young hamsters treated with cadmium. Concentrations of glutathione (GSH) in the renal cortex were increased in the old hamsters on d 6. Increased levels of renal MDA after cadmium treatment may induce the production of GSH in the kidney thus preventing renal damage. Aging can increase the susceptibility to the renal effects of cadmium.     

Melatonin attenuates the effects of sub-acute administration of lead on kidneys in rats without altering the lead-induced reduction in nitric oxide

Exposure to lead induces oxidative stress and renal damage. Although most forms of oxidative stress are characterized by simultaneous elevation of nitrogen and oxidative species, lead-induced oxidative stress is unusual in that it is associated with a reduction in nitric oxide (NO) levels in the kidney. The role of NO in kidney injury is controversial; some studies suggest that it is associated with renal injury, whereas others show that it exerts protective effects. Concentration-dependent effects have also been proposed, linking low levels with vasodilatation and high levels with toxicity. The aim of this study was to evaluate the effects of melatonin co-exposure on the lead-induced reduction in renal NO levels. We found that sub-acute intraperitoneal administration of 10 mg/kg/day of lead for 15 days induced toxic levels of lead in the blood and caused renal toxicity (pathological and functional). Under our experimental conditions, lead induced an increase in lipid peroxidation and a decrease in NO. Melatonin co-treatment decreased lead-induced oxidative stress (peroxidation level) and toxic effects on kidneys without altering the lead-induced reduction in renal NO. These results suggest that, in our experimental model, the reduction in renal NO levels by lead exposure is not the only responsible factor for lead-induced kidney damage.     

Effect of methionine dietary supplementation on mitochondrial oxygen radical generation and oxidative DNA damage in rat liver and heart

Methionine restriction without energy restriction increases, like caloric restriction, maximum longevity in rodents. Previous studies have shown that methionine restriction strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative damage to mitochondrial DNA, lowers membrane unsaturation, and decreases five different markers of protein oxidation in rat heart and liver mitochondria. It is unknown whether methionine supplementation in the diet can induce opposite changes, which is also interesting because excessive dietary methionine is hepatotoxic and induces cardiovascular alterations. Because the detailed mechanisms of methionine-related hepatotoxicity and cardiovascular toxicity are poorly understood and today many Western human populations consume levels of dietary protein (and thus, methionine) 2–3.3 fold higher than the average adult requirement, in the present experiment we analyze the effect of a methionine supplemented diet on mitochondrial ROS production and oxidative damage in the rat liver and heart mitochondria. In this investigation male Wistar rats were fed either a L-methionine-supplemented (2.5 g/100 g) diet without changing any other dietary components or a control (0.86 g/100 g) diet for 7 weeks. It was found that methionine supplementation increased mitochondrial ROS generation and percent free radical leak in rat liver mitochondria but not in rat heart. In agreement with these data oxidative damage to mitochondrial DNA increased only in rat liver, but no changes were observed in five different markers of protein oxidation in both organs. The content of mitochondrial respiratory chain complexes and AIF (apoptosis inducing factor) did not change after the dietary supplementation while fatty acid unsaturation decreased. Methionine, S-AdenosylMethionine and S-AdenosylHomocysteine concentration increased in both organs in the supplemented group. These results show that methionine supplementation in the diet specifically increases mitochondrial ROS production and mitochondrial DNA oxidative damage in rat liver mitochondria offering a plausible mechanism for its hepatotoxicity.     

Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.     

Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCβ/p66Shc axis and activation of FOXO-3a

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p⁶⁶Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- β-1 (TGF-β1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CβII (PKCβII), NADPH oxidase, and p⁶⁶Shc, and decreased the activation of p⁶⁶Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCβII/p⁶⁶Shc axis.     

Myoglobin causes oxidative stress,increase of NO production and dysfunction of kidney's mitochondria

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

Taurine plays a beneficial role against cadmium-induced oxidative renal dysfunction

The present study has been carried out to investigate the role of taurine (2-aminoethanesulfonic acid), a conditionally essential amino acid, in ameliorating cadmium-induced renal dysfunctions in mice. Cadmium chloride (CdCl₂) has been selected as the source of cadmium. Intraperitoneal administration of CdCl₂ (at a dose of 4 mg/kg body weight for 3 days) caused significant accumulation of cadmium in renal tissues and lessened kidney weight to body weight ratio. Cadmium administration reduced intracellular ferric reducing/antioxidant power (FRAP) of renal tissues. Levels of serum marker enzymes related to renal damage, creatinine and urea nitrogen (UN) have been elevated due to cadmium toxicity. Cadmium exposure diminished the activities of enzymatic antioxidants, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD) as well as non-enzymatic antioxidant, reduced glutathione (GSH) and total thiols. On the other hand, the levels of oxidized glutathione (GSSG), lipid peroxidation, protein carbonylation, DNA fragmentation, concentration of superoxide radicals and activities of cytochrome P450 enzymes (CYP P450s) have been found to increase due to cadmium intoxication. Treatment with taurine (at a dose of 100 mg/kg body weight for 5 days) before cadmium intoxication prevented the toxin-induced oxidative impairments in renal tissues. The beneficial role of taurine against cadmium-induced renal damage was supported from histological examination of renal segments. Vitamin C, a well-established antioxidant was used as the positive control in the study. Experimental evidence suggests that both taurine and vitamin C provide antioxidant defense against cadmium-induced renal oxidative injury. Combining all, results suggest that taurine protects murine kidneys against cadmium-induced oxidative impairments, probably via its antioxidative property.     

Cardiolipin as an oxidative target in cardiac mitochondria in the aged rat

The aged heart sustains greater injury during ischemia (ISC) and reperfusion (REP) compared to the adult heart. In the Fischer 344 (F344) rat, aging decreases oxidative phosphorylation and complex III activity increasing the production of reactive oxygen species in interfibrillar mitochondria (IFM) located among the myofibrils. In the isolated, perfused 24 month old elderly F344 rat heart 25 min of stop–flow ISC causes additional damage to complex III, further decreasing the rate of oxidative phosphorylation. We did not observe further progressive mitochondrial damage during REP. We next asked if ISC or REP increased oxidative damage within mitochondria of the aged heart. Cardiolipin (CL) is a phospholipid unique to mitochondria consisting predominantly of four linoleic acid residues (C18:2). Following ISC and REP in the aged heart, there is a new CL species containing three oxygen atoms added to one linoleic residue. ISC alone was sufficient to generate this new oxidized molecular species of CL. Based upon oxidative damage to CL, complex III activity, and oxidative phosphorylation, mitochondrial damage thus occurs in the aged heart mainly during ISC, rather than during REP. Mitochondrial damage during ischemia sets the stage for mitochondrial-driven cardiomyocyte injury during reperfusion in the aged heart.     

Mitochondrial damage, cytotoxicity and apoptosis in iron-potentiated alcoholic liver fibrosis: amelioration by taurine

Taurine effectively prevents ischemia-induced apoptosis in the cardiomyocytes and hypothalamic nuclei. The present study explores the influence of taurine on mitochondrial damage, oxidative stress and apoptosis in experimental liver fibrosis. Male albino Wistar rats were divided into six groups and maintained for a period of 60 days as follows: Group I, control; Group II, ethanol treatment [6 g/(kg/day)]; Group III, fibrosis induced by ethanol and iron (0.5% w/w); Group IV, ethanol + iron + taurine (2% w/v); Group V, ethanol + taurine treatment and Group VI, control + taurine treatment. Hepatocytes isolated from ethanol plus iron-treated rats showed decreased cell viability and redox ratio, increased reactive oxygen species formation, lipid peroxidation, DNA fragmentation, and formation of apoptotic bodies. Liver mitochondria showed increased susceptibility to swell, diminished activities of mitochondrial respiratory chain complexes and antioxidants. Taurine administration to fibrotic rats restored mitochondrial function, reduced reactive oxygen species formation, prevented DNA damage, and apoptosis. Thus taurine might contribute to the amelioration of the disease process.     

Rapid pacing of embryoid bodies impairs mitochondrial ATP synthesis by a calcium-dependent mechanism—A model of in vitro differentiated cardiomyocytes to study molecular effects of tachycardia

Tachycardia may cause substantial molecular and ultrastructural alterations in cardiac tissue. The underlying pathophysiology has not been fully explored. The purpose of this study was (I) to validate a three-dimensional in vitro pacing model, (II) to examine the effect of rapid pacing on mitochondrial function in intact cells, and (III) to evaluate the involvement of L-type-channel-mediated calcium influx in alterations of mitochondria in cardiomyocytes during rapid pacing. In vitro differentiated cardiomyocytes from P19 cells that formed embryoid bodies were paced for 24 h with 0.6 and 2.0 Hz. Pacing at 2.0 Hz increased mRNA expression and phosphorylation of ERK1/2 and caused cellular hypertrophy, indicated by increased protein/DNA ratio, and oxidative stress measured as loss of cellular thiols. Rapid pacing additionally provoked structural alterations of mitochondria. All these changes are known to occur in vivo during atrial fibrillation. The structural alterations of mitochondria were accompanied by limitation of ATP production as evidenced by decreased endogenous respiration in combination with decreased ATP levels in intact cells. Inhibition of calcium inward current with verapamil protected against hypertrophic response and oxidative stress. Verapamil ameliorated morphological changes and dysfunction of mitochondria. In conclusion, rapid pacing-dependent changes in calcium inward current via L-type channels mediate both oxidative stress and mitochondrial dysfunction. The in vitro pacing model presented here reflects changes occurring during tachycardia and, thus, allows functional analyses of the signaling pathways involved.     

Ox-LDL aggravates contrast-induced injury of renal tubular epithelial cells

Hypercholesterolemia can aggravate contrast-induced acute kidney injury, and the exacerbation of renal tubular epithelial cell (RTEC) injury is a major cause. However, the exact mechanisms remain obscure. Mitophagy, a type of autophagy, selectively eliminates damaged mitochondria and reduces mitochondrial oxidative stress, which is strongly implicated in cell homeostasis and acute kidney injury. Oxidized low-density lipoprotein (Ox-LDL) is accumulated in hypercholesterolemia and has a cytotoxic effect. This study aimed to determine whether and how ox-LDL exacerbates contrast-induced injury in RTECs and to further explore whether PINK1/Parkin-dependent mitophagy is involved in this process. Iohexol and ox-LDL were used alone or in combination to treat HK-2 cells. Rapamycin pretreatment was utilized to enhance mitophagy. Cell viability, apoptosis, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) were detected by cell counting kit-8, TUNEL staining, JC-1 kit and MitoSOX fluorescence, respectively. The expression of mitophagy-related proteins (including PINK1, Parkin, and so on) and cleaved caspase-3 was confirmed by western blot. Colocalization of MitoTracker-labeled mitochondria and LysoTracker-labeled lysosomes was observed by fluorescence microscopy to evaluate mitophagy. The results of our study showed that ox-LDL aggravated MMP decline, mtROS release and apoptosis in iohexol-treated HK-2 cells, accompanied by a further increased autophagy level. Enhancement of PINK1/Parkin-dependent mitophagy by rapamycin alleviated apoptosis and mitochondrial injury in HK-2 cells in response to iohexol under ox-LDL condition. Therefore, our findings indicate that ox-LDL aggravates contrast-induced injury of RTECs by increasing mitochondrial damage and mitochondrial oxidative stress, which may be associated with the relative insufficiency of PINK1/Parkin-dependent mitophagy.     

Protective effect of gallic acid and gallic acid-loaded Eudragit-RS 100 nanoparticles on cisplatin-induced mitochondrial dysfunction and inflammation in rat kidney

Cisplatin is used as a chemotherapy drug in the treatment of various types of cancer. Mitochondrial dysfunction, oxidative stress and inflammation have been identified as major mechanisms of cisplatin nephrotoxicity. The present study investigated the protective effects of pure gallic acid and nanoparticle gallic acid nanoparticles (nano-gallic acid) on cisplatin induced nephrotoxicity. Nano-gallic acid was prepared by double emulsions-solvent evaporation technique using Eudragit RS 100 polymer and polyvinyl alcohol as carrier. Then, the physicochemical characterization of the nanoparticles was examined. In the present study, renal mitochondria were isolated using different centrifugal methods. Our data indicated that the doses of 50 and 100 mg/kg gallic acid and 10 mg/kg nano-gallic acid significantly decreased mitochondrial reactive oxygen species (ROS) formation, mitochondrial membrane damage (ΔΨm), mitochondrial malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and significantly increased mitochondrial glutathione (GSH), mitochondrial superoxide dismutase (MnSOD), mitochondrial glutathione peroxidase (GPX) and mitochondrial catalase compared to the cisplatin treated group. Histopathological studies also confirmed biochemical tests. Finally, our results confirmed that the pure gallic acid and its nanoparticle improved renal oxidative stress, inflammation and mitochondrial dysfunction in acute nephrotoxicity induced by cisplatin in rat. Nano-gallic acid (10 mg/kg) was selected as the most effective dose. The findings of this study showed the superiority of nano-gallic acid against pure gallic acid. In conclusion, nano-gallic acid-loaded Eudragit-RS 100 as a novel antioxidant can be considered in the treatment of renal complications of cisplatin.