Effect of tocilizumab on ischemia-reperfusion-induced oxido-inflammatory renal damage and dysfunction in rats

Ischemia-reperfusion-induced (I/R) renal damage is a pathogenic process that starts with ischemia, then progresses through oxidative stress and inflammation. Tocilizumab (TCZ), a recombinant human monoclonal antibody produced against the IL-6 receptor, will be tested against renal I/R injury. TCZ is known to lower the levels of proinflammatory cytokines and oxidant mediators while raising the amounts of antioxidant molecules. Our purpose is to evaluate the biochemical and histological effects of TCZ against I/R-induced oxido-inflammatory kidney damage and dysfunction in rats. Animals were divided into 3 groups as renal I/R (RIR), I/R+ TCZ (IRT), and healthy group (HG). TCZ was administered at a dose of 8 mg/kg to the IRT group (n=6) of the animals, and distilled water as a solvent was administered intraperitoneally (ip) to the RIR (n=6) and HG (n=6) groups. Then, two hours of ischemia and six hours of reperfusion were applied to the left kidneys of IRT and RIR animals. TCZ significantly inhibited the increase in the levels of malondialdehyde (MDA), nuclear kappa B (NF-κB), tumour necrosis factor alpha (TNF-α), interleukin 1-β (IL-1β), IL-6, creatinine (Cr) and blood urea nitrogen (BUN) and decrease in total glutathione (tGSH) with I/R in renal tissue. TCZ also attenuated severe histopathological damage due to I/R in renal tissue. TCZ protected renal tissue from I/R-induced oxidative and inflammatory damage. These results indicate that TCZ may be useful in the treatment of renal I/R injury.     

Autophagy activation attenuates renal ischemia-reperfusion injury in rats

Ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI), which is a common clinical complication but lacks effective therapies. This study investigated the role of autophagy in renal I/R injury and explored potential mechanisms in an established rat renal I/R injury model. Forty male Wistar rats were randomly divided into four groups: Sham, I/R, I/R pretreated with 3-methyladenine (3-MA, autophagy inhibitor), or I/R pretreated with rapamycin (autophagy activator). All rats were subjected to clamping of the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. The Sham group underwent the surgical procedure without ischemia. 3-MA and rapamycin were injected 15 min before ischemia. Renal function was indicated by blood urea nitrogen and serum creatinine. Tissue samples from the kidneys were scored histopathologically. Autophagy was indicated by light chain 3 (LC3), Beclin-1, and p62 levels and the number of autophagic vacuoles. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and expression of caspase-3. Autophagy was activated after renal I/R injury. Inhibition of autophagy by 3-MA before I/R aggravated renal injury, with worsened renal function, higher renal tissue injury scores, and more tubular apoptosis. In contrast, rapamycin pretreatment ameliorated renal injury, with improved renal function, lower renal tissue injury scores, and inhibited apoptosis based on fewer TUNEL-positive cells and lower caspase-3 expression. Our results demonstrate that autophagy could be activated during I/R injury and play a protective role in renal I/R injury. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Furthermore, autophagy activator may be a promising therapy for I/R injury and AKI in the future.     

Effect of BQ-123 and nitric oxide inhibition on liver in rats after renal ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury induces an inflammatory response and production of oxygen-derived reactive species which affect many organs including heart, brain, kidney and gastrointestinal tract. The aim of this study was to assess the hepatic changes after renal I/R injury. Male Sprague Dawley rats were subjected to either sham operation or treatment with L-NAME, L-arginine and BQ-123 during 30 min renal ischemia and 2 h reperfusion injury. Hepatic superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) activities, and thiobarbituric acid-reactive substances (TBARS) and nitric oxide (NO) levels were evaluated to show hepatic response to renal I/R injury. Catalase and SOD activities showed significant differences between the control and the other groups after I/R. On the other hand, GSH-Px activity did not show any significant changes between the control and the other experimental groups mentioned under above conditions. Meanwhile, levels of TBARS were not different between the control and the other experimental groups, whereas NO level showed changes between the control and experimental groups except the one to which endothelin receptor antagonist agent (BQ-123) subjected. Experimental period may not be enough to determine the changes in GSH-Px activity and level of TBARS. However, catalase and SOD activities decreased in experimental groups treated by chemical agents. NO level decreased in chemicalagent-applied experimental groups but not in the group to which endothelin receptor antagonist BQ-123 was applied alone.     

Effect of peripheral administration of leptin on the renal sympathetic nerve activity in high-fat diet-related hypertensive rats

A previous study of ours demonstrated that a high-fat diet (FAT) causes body fat accumulation, as well as elevation of plasma leptin level, renal sympathetic nerve activity (RSNA), and blood pressure (BP). In the study reported here, we analyzed the role of leptin in these elevations of the RSNA and BP due to FAT feeding by assessing sympathetic and cardiovascular responses to intravenous (IV) administration of leptin in rats fed either a FAT or a high-carbohydrate diet (CHO). The results showed that baseline body fat, plasma leptin level, RSNA and BP were significantly higher in the FAT group than in the CHO group, and that IV administration of leptin elevated RSNA and plasma leptin levels but lowered BP in the CHO group. However, these effects of leptin were eliminated in the FAT group. These findings suggest that FAT-fed rats which expose basal elevation of plasma leptin levels, RSNA and BP might be hyposensitive to endogenous leptin. Therefore, leptin resistance appeared obviously in FAT-induced hypertension might indicate that leptin is implicated in generating the elevation of RSNA and BP induced by long-term FAT feeding.     

Brief small intestinal ischemia lessens renal ischemia-reperfusion injury in rats

Ischemic preconditioning (IPC) not only reduces local tissue injury caused by subsequent ischemia-reperfusion (IR) but may also have a beneficial effect on IR injury of tissues remote from those undergoing preconditioning. In this study, we investigated the effect of small intestinal IPC on renal IR injury in rats. Renal IR injury was induced by a 45-min renal artery occlusion and reperfusion for 2 or 24 h in rats with a previous contralateral nephrectomy, and ischemic preconditioning was induced by 3 cycles of 8-min ischemia and 5-min reperfusion of the small intestine. We then measured the concentrations of plasma creatinine (Cr) and blood urine nitrogen (BUN) and the level of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT) in the renal cortex. Renal histopathology also was evaluated. Pretreatment with intestinal ischemic preconditioning significantly alleviated renal IR injury, as shown by decreases in the levels of Cr, BUN, and MDA, decreased renal morphologic change, and improved preservation of SOD and CAT activities. These results suggest that remote ischemic preconditioning of the small intestine protects against renal IR injury by inhibition of lipid peroxidation and preservation of antioxidant enzyme activities.     

Effect of aminoguanidine on ischemia-reperfusion induced myocardial injury in rats

Myocardial ischemia-reperfusion (MI/R) has been implicated in the induction of inducible nitric oxide synthase (iNOS) that leads to increase production of nitric oxide (NO). Recently, excessive production of NO has been involved in causing myocardial injury. In our in vivo model, we examined the effects of aminoguanidine (AMG), a known iNOS inhibitor, on percentage infarct size in anaesthetized rats. A total of 14 rats were equally divided into two groups (n = 7 in each group). To produce myocardial necrosis, the left main coronary artery was occluded for 30 min, followed by 120 min of reperfusion, in anesthetized rats. AMG (200 mg kg⁻¹) was given intravenously 10 min before occlusion. The volume of infarct size and the risk zone were determined by planimentry of each tracing and multiplying by the slice thickness. Infarct size was normalized by expressing it as a percentage of the area at risk. Hemodynamic parameters were measured via the left carotid artery. Compared to MI/R group, whereas AMG administration elevated mean arterial blood pressure, statistically reduced the myocardial infarct size (21± 1 and 14± 4%, respectively) and infract size/risk zone (53± 3 and 37± 5%, respectively) in rat model of ischemia-reperfusion. In conclusion, this study indicates that iNOS inhibitor, AMG, show reduction in NO’s side effect in I/R injury.     

Intestinal ischemia-reperfusion injury made leptin decreased

To explore the role and the rule of leptin levels in severe traumatism, an ischemia-reperfusion injury model was established to observe change of leptin levels, and platelet activating factor, noradrenaline, lipopolysaccharide, and endothelin-1 were utilized to induce vascular endothelial cells. Leptin concentrations in serum and supernatant were detected by murine and human leptin radioimmunoassay. The results showed that the first serum leptin level significantly decreased after an injury of 60 min ischemia and 30 min reperfusion versus pre-experimental serum values, and leptin level in serum showed a variational trend to increase as reperfusion time extended; the second, supernatant leptin level significantly decreased after PAF and ET-1 treatments of 6 and 24 h versus the control group. It can be concluded that leptin maybe an inflammatory cytokine to play a protection role in acute inflammation and traumatism.     

Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats

We investigated the effects of estrogen on global myocardial ischemia-reperfusion injury in rats that were ovariectomized (Ovx), sham-operated, or ovariectomized and then given 17beta-estradiol (E(2)beta) supplementation (Ovx+E(2)beta). Hearts were excised, cannulated, perfused with and then immersed in chilled (4 degrees C) cardioplegia solution for 30 min, and then retrogradely perfused with warm (37 degrees C), oxygenated Krebs-Henseleit bicarbonate buffer for 120 min. The coronary flow rate, first derivative of left ventricular pressure, and nitrite production were all significantly lower in Ovx than in sham-operated or Ovx+E(2)beta hearts. However, coronary flow rates or nitrate production were not consistently different throughout the entire reperfusion period. Ca(2+) accumulated more in Ovx rat hearts than in sham-operated or Ovx+E(2)beta hearts, and mitochondrial respiratory function was lower in Ovx hearts than in hearts from the other two groups. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of Ovx rat hearts but not in hearts from either of the other groups. Hematoxylin-basic fuchsin-picric acid-stained sections revealed fewer viable myocytes in hearts from the Ovx group than from the sham or Ovx+E(2)beta group. Transmission electron microscopy demonstrated more severely damaged mitochondria and ultrastructural damage to myocytes in Ovx rat hearts. Our results indicate that estrogen plays a cardioprotective role in global myocardial ischemia-reperfusion injury in female rats.     

The effects of resveratrol administration on lipid oxidation in experimental renal ischemia-reperfusion injury in rats

ABSTRACT

We investigated how resveratrol affects lipid oxidation during experimental renal ischemia-reperfusion injury in rats. We used 48 adult male rats assigned to five groups: group 1, control; group 2, renal ischemia; group 3, renal ischemia + reperfusion; group 4, resveratrol + renal ischemia; group 5, resveratrol + renal ischemia + reperfusion. Plasma and renal tissue malondialdehyde (MDA), and erythrocyte and renal tissue glutathione (GSH) levels were measured and histologic changes in the renal tissue were examined. Ischemia-reperfusion affected the MDA-GSH balance adversely and caused histopathological changes in the renal tissue of the ischemia and ischemia + reperfusion groups. Resveratrol treatment normalized MDA and GSH levels as well as the histopathology that occurred in the renal tissue of the ischemia and ischemia + reperfusion groups.     

Pancreatic damage and regeneration in the course of ischemia-reperfusion induced pancreatitis in rats.

The objective of this study was to assess the biochemical and histological signs of pancreatic damage development and pancreatic recovery in the course of ischemia-reperfusion induced pancreatitis. Acute pancreatitis was induced in rats by limitation of pancreatic blood flow (PBF) in inferior splenic artery for 30 min using microvascular clips, followed by reperfusion. Rats were sacrificed at the time: 1 h, 12 h, 24 h, and 2, 3, 5, 7, 10, 14, 21 and 28 days after ischemia. PBF was measured using laser Doppler flowmeter. Plasma amylase, interleukin 1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, as well as, morphological features of pancreatic damage were examined. Ischemia with reperfusion caused acute necrotizing pancreatitis followed by pancreatic regeneration. After removal of microvascular clips, PBF was reduced and the maximal fall of PBF was observed 24 h after ischemia, then PBF grew reaching the control value at 28th day. Plasma amylase activity was increased between 12th h and 3rd day with maximum at 24 h after ischemia. Also plasma IL-1beta and IL-10 were elevated with maximal value at the first and second day after ischemia, respectively. DNA synthesis was maximally reduced at the first day (by 70%) and from second day the reversion of this tendency was observed with full restoration of pancreatic DNA synthesis within four weeks. Morphological features of pancreatic tissue showed necrosis, strongly pronounced edema and leukocyte infiltration. Maximal intensity of morphological signs of pancreatic damage was observed between first and second day of reperfusion. During pancreatic regeneration between second and tenth day after ischemia the temporary appearance of chronic pancreatitis-like features such as fibrosis, acinar cell loss, formation of tubular complexes and dilatation of ducts was observed. The regeneration was completed within four weeks after pancreatitis development. We conclude that partial and temporary pancreatic ischemia followed by reperfusion causes acute necrotizing pancreatitis with subsequent regeneration within four weeks. Pancreatic repair after necrotizing pancreatitis is connected with the increase in plasma IL-10 concentration and transitory formation of tubular complexes.     

Gastroprotective effect of leptin on gastric mucosal injury induced by ischemia-reperfusion is related to gastric histamine content in rats

Leptin has cytoprotective effect to gastric mucosal injury in rats. We aimed to test the hypothesis that leptin induced histamine is involved in the prevention of ischemia-reperfusion (I/R) induced gastric mucosal injury in rats. At the end of the 30 min celiac artery occlusion and 12h reperfusion process, serum and gastric tissue samples were taken from three group of rats to measure oxidative status, histamine levels and for histological examinations. Leptin decreased ulcer and polymorphonuclear leukocyte (PMNL) index, and serum malondialdehyde (MDA) and protein carbonyl content but increased gastric tissue histamine levels. We concluded that leptin exerts a protective effect on gastric mucosa to I/R induced gastric injury probably through increasing tissue histamine content which, in turn, maintain the gastric mucosal blood flow.     

The protective effect of carvacrol on acetaminophen-induced renal damage in male rats

Molecular Biology Reports - Acetaminophen overdose causes renal injury via oxidative stress and apoptosis induction. Carvacrol has several pharmacological properties such as antioxidant,...     

Effect of leptin on the acute feeding-induced hypothalamic serotonergic stimulation in normal rats

Both hypothalamic serotonin and leptin reduce energy intake and stimulate expenditure. There are evidence that increased serotonin metabolism may be involved in leptin actions. Using microdialysis, we directly assessed the effect of an intracerebroventricular leptin injection on 5-HT release in the lateral hypothalamus of normal rats. When LH microdialysates were collected in the absence of food intake, neither artificial cerebrospinal fluid (CSF) nor 10 microg leptin i.c.v. caused significant variations in 5-HT release, measured for 2 h post-injection, at 20-min periods. When food was ingested after CSF, 5-HT release increased significantly, with a maximal elevation of 51+/-16% above baseline occurring at the 100-120 min post-injection interval. Leptin inhibited food intake (-75% at 0-20 min and -73% at 20-40 min) while it accentuated the food-induced serotonergic activation. At the 0-20 min interval, serotonin release was significantly higher after leptin (42+/-12% above baseline) than after CSF (6+/-5%) and the maximal increase after leptin was of 126+/-53% above baseline (100-120 min, p>0.05 vs. CSF). These observations indicate that leptin probably interacts with the serotonergic-stimulating mechanisms elicited by food intake, intensifying them. The additional serotonergic activation induced by leptin may be significant for the hormone effects on energy balance.     

Complement plays an important role in gastric mucosal damage induced by ischemia-reperfusion in rats.

Ischemia-reperfusion (I/R) of stomach causes gastric mucosal injury. Complement can also cause tissue damage, however its role in gastric I/R injury has not been thoroughly investigated. We evaluated the effect of complement suppression in reducing damage to the gastric epithelium caused by local I/R. Local gastric ischemia was induced by clamping the left gastric artery. The blood-to-lumen clearance of 51Cr-labeled EDTA (51Cr-EDTA) served as an index of epithelial damage. 51Cr-EDTA clearance increased shortly after reperfusion with peak values at 10 min. Intraperitoneal administration of cobra venom factor (CVF; 50 units) prior to I/R, which reduced the serum complement value (CH50) to an undetectable level, remarkably suppressed the 51Cr-EDTA clearance following reperfusion. A monocarboxylic acid derivative of K-76 (K-76 COOH) reduced the CH50 by more than 30% (100 mg/kg) and 60% (200 mg/kg). Rats pretreated with K-76 significantly attenuated the increase in 51Cr-EDTA clearance produced by I/R. These results suggest that complement inhibitor could be used to protect gastric mucosal injury induced by local I/R stress.     

Effect of ischemia-reperfusion on the renal brush-border membrane sodium-dependent phosphate cotransporter NaPi-2

To understand the mechanisms underlying ischemia-reperfusion-induced renal proximal tubule damage, we analyzed the expression of the Na+-dependent phosphate (Na+/Pi) cotransporter NaPi-2 in brush border membranes (BBM) isolated from rats which had been subjected to 30 min renal ischemia and 60 min reperfusion. Na+/Pi cotransport activities of the BBM vesicles were also determined. Ischemia caused a significant decrease (about 40%, P < 0.05) in all forms of NaPi-2 in the BBM, despite a significant increase (31+/-3%, P < 0.05) in the Na+/Pi cotransport activity. After reperfusion, both NaPi-2 expression and Na+/Pi cotransport activity returned to control levels. In contrast with Na+/Pi cotransport, ischemia significantly decreased Na+-dependent glucose cotransport but did not affect Na+-dependent proline cotransport. Reperfusion caused further decreases in both Na+/glucose (by 60%) and Na+/proline (by 33%) cotransport. Levels of NaPi-2 were more reduced in the BBM than in cortex homogenates, suggesting a relocalization of NaPi-2 as a result of ischemia. After reperfusion, NaPi-2 levels returned to control values in both BBM and homogenates. These data indicate that the NaPi-2 protein and BBM Na+/Pi cotransport activity respond uniquely to reversible renal ischemia and reperfusion, and thus may play an important role in maintaining and restoring the structure and function of the proximal tubule.     

Effect of aging on 24-hour pattern of stress hormones and leptin in rats

This work analyzes the 24-hour changes of hypothalamic-pituitary-adrenal (HPA) axis activity and leptin release in aged rats. Three- and 22-month-old male Wistar rats were killed at 6 time intervals during a 24-hour cycle (n=8-10 rats/group). Aging augmented plasma ACTH while it decreased plasma and adrenal gland corticosterone levels. Plasma and adrenal corticosterone levels attained high levels during all the scotophase, concomitantly with the maxima in ACTH levels, whereas in aged rats only a brief plasma corticosterone peak at the early scotophase and no time of day variations of adrenal corticosterone were observed. Aging augmented circulating leptin, with a significant interaction "agextime" in the factorial ANOVA, i.e. only in young rats time of day changes were significant, with the lowest values of leptin at the middle of the light period and higher values at night. When plasma leptin was expressed on body weight basis, the age-related differences became not significant but the daily pattern of plasma leptin found in young rats persisted. Plasma and adrenal corticosterone levels correlated significantly with plasma ACTH only in young rats. Likewise, plasma leptin correlated with plasma corticosterone only in young rats. These changes can be attributed to a disrupting effect of aging on the homeostatic mechanisms modulating HPA activity and leptin release.     

Effect of triethylenepentaminehexaacetic acid on the renal damage in cadmium-treated Syrian hamsters

Cadmium (Cd)-induced nephropathy was treated by triethylene-pentaminehexaacetic acid (TTHA) in male Syrian hamsters. Hamsters injected three times a week with 3 mg/kg body wt CdCl₂ showed proteinuria, urinaryN-acetyl-β-d-inglucosaminidase (NAG), and fractional excretion of sodium (FENa) when compared to saline-injected control. Cd-treated hamsters injected ip with TTHA 10 mg/kg body wt five times a week showed reduction of renal damage, including reductions in urinary protein (from 6.7±2.2 to 4.3±0.5 mg/d) and NAG (0.17±0.06 to 0.04±0.02 U/d). Urinary excretion of Cd was significantly increased (from 87±51.3 to 3052±1485 mg/L) by TTHA administration. Cd concentration in renal cortical tissue was slightly reduced (26.4±3.0 to 21.8±2.7 mg/g. protein). Excretion of malondialdehyde (MDA) was increased only in Cd-injected hamsters (to 2.1±1.6 nM/L), and elevated MDA in renal cortical tissue was not reduced by the administration of TTHA (1041±105 vs 1104±358 nM/g protein). Glutathione (GSH) concentration in the renal cortex was significantly elevated after Cd administration and further increased after TTHA administration (5.5±2.1 to 9.8±2.0 μg/50 mg protein). There were no marked effects on creatinine clearance (Ccr) and hematocrit. Moreover, renal morphological changes were improved significantly by treatment with TTHA. We demonstrated the efficacy of TTHA in the treatment of Cd-induced nephropathy in hamsters. Although the precise mechanism of the TTHA effects on Cd-induced nephropathy has not been elucidated, it might involve GSH reducing the elevated MDA concentration in renal tissue.     

Effect of malotilate on ethanol-induced gastric mucosal damage in capsaicin-pretreated rats

We studied the role of afferent sensory neurons in malotilate-mediated gastric mucosal protection. Intact and capsaicin sensory-denervated rats were used in the experiments. Gross gastric mucosal injury was assessed and evaluated as a main criterion of the gastroprotective effect of the tested substances. Besides malotilate, methyl-prostaglandin E2 was applied alone or in combination with malotilate to compare the effects and the mechanism of action of both substances. The results revealed that both malotilate as well as methyl-prostaglandin E2 exerted a significant protective action on 96% ethanol-induced gastric mucosal damage. However, there were no significant differences between intact and capsaicin-denervated rats. Only the use of 50% ethanol as a milder mucosal irritating agent resulted in significant differences in both groups of animals. We propose that malotilate (like methyl-prostaglandin E2) has a gastroprotective effect on ethanol-induced gastric mucosal injury. This effect is partly dependent on the sensory nervous system and the combination of both above substances has an additive effect.     

Effect of social isolation on 24-h pattern of stress hormones and leptin in rats

This work analyzes the effect of social isolation of growing male rats on 24-h changes of plasma prolactin, growth hormone, ACTH and leptin, and on plasma and adrenal corticosterone concentrations. At 35 days of life, rats were either individually caged or kept in groups (6-8 animals per cage) under a 12:12 h light/dark schedule (lights on at 08:00 h). A significant arrest of body weight gain regardless of unchanged daily food intake was found in isolated rats after 2 weeks of isolation. On the 4th week, rats were killed at 6 time intervals during a 24-h cycle, beginning at 09:00 h. In isolated rats the 24-h pattern of all parameters tested became distorted, as assessed by Cosinor analysis. When analyzed as a main factor in a factorial analysis of variance, isolation decreased plasma prolactin and growth hormone, increased plasma leptin and corticosterone while decreased adrenal corticosterone. Plasma corticosterone levels correlated significantly with plasma ACTH and with adrenal corticosterone levels in group-caged rats only. These changes can be attributed to an effect of mild stress on the endogenous clock that modulates the circadian hormone release.