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Cognitive impairment is one of the major complications of chronic kidney disease (CKD). The present study aims to evaluate the protective effects of carbon monoxide (CO) and hydrogen sulfide (H2S) and their interactions on CKD-induced cognitive deficits by considering the Nrf2/HO-1 signaling pathway. Sixty rats were divided into six experimental groups: sham, five-sixth (5/6) nephrectomy (CKD), CKD + H2S donor (NaHS), CKD + CO-releasing molecule (CORM3), CKD + NaHS and zinc protoporphyrin IX (Znpp), CKD + CORM3 and amino-oxy acetic acid (AOAA). Eleven weeks after 5/6Nx, behavioral tests (Novel object recognition test, Passive avoidance test and Barnes maze test) were performed to evaluate the cognitive level. At the end of the twelfth week, blood urea nitrogen (BUN) and serum creatinine (sCr) levels, as well as the expression levels of nuclear factor-erythroid factor 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), and neuronal loss in the hippocampus and prefrontal cortex were evaluated. CKD caused enhancement of BUN and sCr, reduction of Nrf2 and HO-1 proteins and enhancement of neuronal loss in the hippocampus and prefrontal cortex. In addition, CKD led to cognitive disturbances and memory impairment. CORM3 and NaHS returned all above indices to the levels measured in the control group. However, improving effects of CORM3 on cognitive impairment and Nrf2/HO-1 signaling pathway were prevented by AOAA and decreased H2S level as well as reciprocally improving effects of NaHS on cognitive disturbances and Nrf2/HO-1 signaling pathway were prevented by Znpp and decreased CO level. In conclusion, this study demonstrated that formation of CO and H2S were interdependently improved CKD-induced cognitive dysfunctions, through interaction with Nrf2/HO-1 signaling pathway.

Asthma is one of the most common respiratory diseases in the world. Nevertheless, it is reported that inflammation induced by asthma is not only restricted to the lung and may cause damaging effects on remote organs. Therefore, this study was designed to investigate the beneficial effects of long-term sodium hydrosulfide (NaHS) administration on lung inflammation and oxidative stress markers to protect the kidney during chronic asthma. BALB/c mice were divided into three groups (

In this study, we hypothesized that sepsis induction impairs memory retrieval in rats while transplanted mesenchymal stem cells (MSCs) and MSC-conditioned medium (MSC-CM) application are capable of attenuating those complications. MSCs were obtained from adipose tissue of rats and at the second culture passage; MSCs and MSC–CM were collected. Rats were randomly divided into four experimental groups: sham, CLP, MSC, and MSC-CM. Sepsis was induced by cecal ligation and puncture (CLP) model in the CLP, MSC, and MSC-CM groups. The MSC group received 1 × 106 MSCs/rat (i.p., 2 h after CLP surgery); the MSC-CM rats received the conditioned medium (CM) from 1 × 106 MSCs intraperitoneally 2 h after sepsis induction. Novel object recognition test, sepsis score, and blood pressure measurement were performed 24 h after the treatments. The right hippocampus was taken for western blot analysis. CLP rats showed a significantly higher sepsis score and systolic blood pressure. They also had a significant increase in the phosphorylated form of CAMKII-α, cleaved caspase 3 and Bax/Bcl2 ratio, and a reduction in c-fos protein in the hippocampus tissue samples compared with the sham group. MSC transplantation and MSC-CM administration significantly decreased the mean sepsis score and prevented sepsis-induced attenuation of blood pressure compared with the CLP rats. Animals in the MSC and MSC-CM groups showed a better memory retrieval, attenuation in phosphorylated form of CAMKII-α, cleaved caspase 3 and Bax/Bcl2 ratio, and an increase in c-fos protein expression compared with the CLP group. It seems that CAMKII and c-fos are inversely involved in regulating memory processes in hippocampus. Phosphorylated form of CaMKII-α overexpression may impair the ability of object recognition. Our findings confirmed that MSC-CM application has more advantages compared with transplanted MSCs and may be offered as a promising therapy for inflammatory diseases such as severe sepsis.

Context: Airway remodelling is one of the most refractory problems in asthma. According to the critical roles of oxidative stress and inflammation in airway remodelling, it is supposed that ascorbic acid and calcitriol have beneficial effects. However, a combination of antioxidants may be more effective for asthma therapy. Objective: This study investigated the protective effects of ascorbic acid in combination with calcitriol on airway remodelling in ovalbumin (OVA)-induced chronic asthma. Materials and methods: BALB/c mice were assigned into seven groups: (1) Control; (2) Asthma; (3) Ineffective C (orally 39 mg/kg ascorbic acid); (4) Ineffective D (intraperitoneally 1.5 μg/kg calcitriol); (5) Effective C (orally 130 mg/kg ascorbic acid); (6) Effective D (intraperitoneally 5 μg/kg calcitriol); (7) Combination (orally 39 mg/kg ascorbic acid + intraperitoneally 1.5 μg/kg calcitriol). All animals were sensitized and challenged with OVA except in the control group (normal saline). In all treatment groups, mice were administrated vitamins 30 min before each challenge (three times per week for 8 consecutive weeks). Results: In comparison with the asthma group, co-administration of ineffective doses of ascorbic acid and calcitriol led to the decreased levels of IL-13 (50.5 ± 1.85 vs. 42.13 ± 0.37 pg/mL, p = 0.02) and IgE (58.74 ± 0.43 vs. 45.78 ± 2.05 ng/mL, p = 0.003) as well as the reduction of goblet hyperplasia and subepithelial fibrosis (5 vs. 1 score, p = 0.001 and 5 vs. 2 score, p = 0.001, respectively). Discussion and conclusions: Combination of ascorbic acid with calcitriol in ineffective doses improves airway remodelling due to additive effects possibly through reduction of oxidative stress and inflammation. This study provides a scientific basis for further research and clinical applications of ascorbic acid and calcitriol and can be generalized to the broader pharmacological studies.

Aims This study was designed to investigate the protective effects of centrally administered erythropoietin (EPO) on brain oxidative stress and inflammatory markers to protect the kidneys during hemorrhagic shock (HS). Main methods Animals were assigned into three groups (n = 6). Sham rats were subjected to cannulation of femoral artery and vein as well as stereotaxic surgery. In HS group, 50% of total blood volume was withdrawn and resuscitation was started 2 h later. In EPO group, stereotaxic surgery in lateral ventricle was performed one week before induction of HS for administration of EPO (2 IU) just before resuscitation. Plasma samples, kidney and brain tissues were allocated after a further 3 h in all animals. Key findings There was a significant increase in survival rate in the EPO group (69.3%) compared to the HS group (35.7%). Brain EPO administration significantly attenuated the rises in BUN, plasma Cr and NGAL, brain and renal MDA content and also increased SOD activity in the kidney and brain compared to the HS group. Brain, plasma and kidney TNF-α and IL-6 levels were significantly reduced by EPO compared to HS group. EPO increased the phosphorylation of Akt on Ser473 and eNOS mRNA expression in the kidney tissue compared to the HS group. Significance In conclusion, centrally administered EPO reduced pro-inflammatory and oxidative stress indices in the kidney and reduced apoptosis by activation of the Akt/eNOS signaling pathway. Hence, it can be hypothesized that EPO may play a major role in the central regulation of renal system as a neuromodulator.

Aim Erythropoietin (EPO) is shown to exert protective effects on different tissues in haemorrhagic shock (HS) states. Nitric oxide (NO), as a multifunctional signaling molecule, is implicated in diverse physiologic and pathologic processes. In order to understand the exact mechanism of EPO protection, in this study we evaluated the role of different NOS enzymes in the EPO signaling pathway in male rats. Methods Rats were randomized to five groups: 1) Sham, 2) HS 3) EPO 4) L-NAME, a non-specific NOS inhibitor 5) 1400 W, a specific iNOS inhibitor. HS was induced by withdrawal of 50% of total blood volume. After 2 h, resuscitation was performed with the shed blood and Ringer’s lactate. In group 3, rats were treated with EPO (300 IU/kg, i.v.) over 10 min before HS induction. In the L-NAME and 1400 W groups, L-NAME (10 mg/kg, i.p.) and 1400 W (2 mg/kg, i.p.) were administered 30 min before EPO injection. Blood and kidney tissue samples were obtained 3 h after resuscitation. Results EPO increased the survival rate and significantly improved kidney function and histology compared to the HS group. There were less renal oxidative stress, apoptosis and systemic inflammatory responses in the EPO group. EPO increased eNOS and more abundantly iNOS mRNA expressions. L-NAME and 1400 W significantly abolished all beneficial effects of EPO. Conclusion In this in vivo animal model, we showed that EPO administration prior to HS attenuates renal injury and dysfunction in rats. The protective effects of EPO may be mediated by nitric oxide and the expression of different NOS enzymes, especially iNOS isoform.

The effects of erythropoietin (EPO) alone or in conjunction with ischemic preconditioning (IPC) on nitric oxide synthase as well as comparing their effects on oxidative stress and proinflammatory cytokines are studied. Rats underwent bilateral renal ischemia of 50 min followed by 24 h reperfusion. They were administered EPO (5000 iu/kg i.p.) and/or subjected to IPC and sacrificed after 24 h, then plasma and tissue samples were obtained. Treatment of either EPO or IPC and their combination attenuates oxidative stress, decreases histological damages, inhibits proinflammatory response, and up-regulates iNOS and eNOS gene expression compared to IR group. In addition, EPO+IPC and EPO treatment produced significant up-regulation in iNOS gene expression compared to IPC group. In IPC and EPO+IPC groups, more powerful effect on up-regulation of eNOS gene expression was shown compared to EPO group. Our findings suggest that treatment with EPO or IPC and their combination improve renal function and preserve tubular damage induced by IR injury. These advantageous effects were closely related to reducing oxidative stress, suppressing proinflammatory response and enhancing generation of NO. IPC was more powerful in enhancement of eNOS gene expression compared to EPO that was more effective in increasing of iNOS gene expression.

High-mobility group box 1 (HMGB1), also called amphoterin, HMG1 and p30, is a highly conserved protein between different species that has various functions in nucleus such as stabilization of nucleosome formation, facilitation of deoxyribonucleic acid (DNA) bending and increasing the DNA transcription, replication and repair. It has also been indicated that HMGB1 acts as a potent pro-inflammatory cytokine with increasing concentrations in acute and chronic inflammatory diseases. Asthma is a common chronic respiratory disease associated with high morbidity and mortality rates. One central characteristic in its pathogenesis is airway inflammation. Considering the inflammatory role of HMGB1 and importance of inflammation in asthma pathogenesis, a better understanding of this protein is vital. This review describes the structure, cell surface receptors, signaling pathways and intracellular and extracellular functions of HMGB1, but also focuses on its inflammatory role in asthma. Moreover, this manuscript reviews experimental and clinical studies that investigated the pathologic role of HMGB1.

The main aim of this study was to assay the testicular H2 S levels in the varicocele rat model and then to investigate the protective effects of NaHS on morphometric changes, sperm parameters, oxidative stress and apoptosis markers in rat's testis. D,L-propargylglycine (PAG) was administrated to show the effects of cystathionine γ-lyase enzyme (CSE) inhibition in the varicocele. Rats were assigned to four groups: (a) Sham, (b) varicocele, (c) varicocele + PAG and (d) varicocele + NaHS. Animals in varicocele + NaHS group received 30 µmol/L NaHS in drinking water for 56 days. In the varicocele + PAG group, animals received PAG 19 mg/kg twice a week. Morphometric assessment, oxidative stress markers, testicular H2 S levels, sperm parameters, TUNEL assay and expression of Bax/Bcl2 were evaluated at the end of experiment. Testicular H2 S levels were significantly decreased in varicocele group. NaHS significantly improved sperm parameters, morphometric characteristics and oxidative stress compared to varicocele group. Oxidative stress status deteriorated in the PAG group compared to the varicocele group. This study showed that a low testicular H2 S level might play a critical role in male infertility. Thus, NaHS administration may be a promising treatment strategy for male infertility in varicocele. In addition, CSE may not be the only important enzyme in testicular H2 S production.

Physical exercise is shown to have protective effects on chronic kidney disease (CKD). CKD itself is associated with a reduction in renal hydrogen sulfide (H2S) concentration. This study was designed to investigate whether protective effects of exercise in 5/6 nephrectomized (5/6 NX) rats is associated with H2S levels in the kidney? Twenty four male Wistar rats weighing 250-300 g were assigned into 4 groups: 1- Sham 2- Sham exercise 3-5/6 NX 4-5/6 NX+exercise. To induce CKD, 4 days after removing upper and lower one-third parts of the left kidney, total right nephrectomy was performed. In the Sham groups, anesthesia and surgery were performed like the other groups without removal of the kidney mass. Exercise was performed by treadmill at a speed of 18 m/min for 8 weeks. At the end of the twelfth week, blood and kidney samples were collected to measure renal function (levels of plasma urea and creatinine), oxidative stress markers (renal MDA level and SOD activity), and histological indices. Eight weeks exercise significantly improved serum creatinine, BUN, renal MDA level, SOD activity, renal sympathetic nerve activity (RSNA), hypertension, and renal histology in addition to renal H2S level compared to the 5/6 NX group. The results suggest that regular exercise improves renal oxidative status and ameliorates renal damage, hypertension, and RSNA in 5/6 nephrectomized rats. These improvements by exercise might be associated with the increase in renal H2S level.

Renal ischemia–reperfusion injury (IRI) is a major cause of acute kidney injury. Hydrogen sulfide (H2S) has been known as a novel gaseous signaling molecule. The aim of this study was to investigate whether the efficacy of H2S in protecting against renal IRI is through its antioxidative effect. In this study, rats were randomized into Sham, IR, or sodium hydrosulfide (NaHS, an H2S donor) groups. To establish a model of renal IRI, both renal arteries were occluded for 55 min and then declamped to allow reperfusion for 24 h. Rats in the NaHS group received intraperitoneal injections of 75 μmol/kg NaHS 10 min before the onset of ischemia and immediately after the onset of reperfusion. Sham group underwent laparotomy without cross-clamping of renal pedicles. After reperfusion, plasma and renal tissue samples were collected for functional, histological, and oxidative stress evaluation. The IR group exhibited significant rise in plasma creatinine, blood urea nitrogen (BUN), renal malondialdehyde (MDA) concentration, and significant reduction of renal superoxide dismutase (SOD) activity. Treatment with NaHS reduced the levels of plasma creatinine, BUN, renal MDA concentration, and increased SOD activity in the kidneys. NaHS improved renal histological changes in comparison to IR group. Our data demonstrated that H2S can protect against renal IRI and that its therapeutic effects may be mediated by reducing oxidative stress.

Background To investigate the effect of angiotensin II (Ang II) in the hypothalamic paraventricular nucleus (PVN) on renal ischemia–reperfusion (IR) injury and to assay the role of renal sympathetic nerve activity (RSNA). Methods A cannula was inserted into the right side PVN in Sprague–Dawley rats for microinjection of Ang II (3, 30, and 300 ng); Ang II AT1 receptor antagonist, losartan (0.3 μg); and the superoxide dismutase (SOD) mimetic, tempol (20 nmol) before right side nephrectomy. After 1 wk, renal IR injury was induced by clamping the left renal artery for 45 min, and then reperfusion for 3 or 24 h. The extent of renal damage was determined by evaluation of renal functional indices. RSNA was recorded in all groups. Oxidative stress indices (SOD activity and malondialdehyde levels) were assayed in the PVN. Results Microinjection of pharmacologic doses of Ang II into the PVN exaggerated renal IR injury, increased RSNA and oxidative stress in the PVN dose dependently. The effects of Ang II (3 ng) was prevented by pretreatment with losartan into the PVN. Furthermore, the deleterious effects of Ang II on renal IR injury, RSNA, and oxidative stress were abolished by pretreatment with tempol. Conclusions These results indicate that the PVN is a responsive site for central Ang II increment damage in renal ischemia–reperfusion injury. We suggested the central effects of Ang II in the PVN on renal IR injury are mediated by AT1 receptors and oxidative stress in the PVN, and the peripheral effects by a sympathetic pathway.

Background: Opioids produce reactive oxygen species (ROS) which are highly reactive molecules that damage cells and tissues, and are suggested to contribute to the opioid use disorders. Thus, antioxidant supplementation might improve the disturbance in redox (oxidation-reduction) homeostasis. However, randomized trials on antioxidant therapy have not shown beneficial effects. Objectives: The purpose of this review is to shed lights on the oxidative changes resulting from opioid use and to highlight the unanswered questions regarding oxidative profile in an effort to provide a comprehensive view of different aspects of an efficient antioxidant therapy in clinical settings. Methods: The studies were identified and gathered from the PubMed database over the past 16 years (2000-2016). Our search results were limited to articles in English, both animals and human and in vitro and in vivo studies. A total of 50 full text articles were reviewed and summarized. Results: Opioids elevate the level of ROS and decrease the function of enzymatic antioxidants such as superoxide dismutase, catalase, and glutathione peroxidase. They increase the risk of vitamin deficiency and modify gene expression of target cells through ROS production. The effects of opioids on their target cells are exerted through different way and various mechanisms. Conclusion: Opioids modulate the redox homeostasis; therefore, understanding the profile of oxidative changes in individuals with opioid use disorder could be of significant benefits in the clinical setting, to help with selection of an efficient antioxidant therapy and diminishing oxidative damage.

The present study aimed to analyze and compare the effects of classical and remote ischemic postconditioning (POC) on rat renal ischemia/reperfusion (IR)-induced acute kidney injury. After right nephrectomy, male rats were randomly assigned into four groups ( n = 8). In the IR group, 45 min of left renal artery occlusion was induced followed by 24 h of reperfusion. In the classical POC group, after induction of 45 min ischemia, 4 cycles of 10 s of intermittent ischemia and reperfusion were applied to the kidney before complete restoring of renal blood. In the remote POC group, 4 cycles of 5 min ischemia and reperfusion of left femoral artery were applied after 45 min renal ischemia and right at the time of renal reperfusion. There was a reduction in renal function (increase in blood urea and creatinine) in the IR group. Application of both forms of POC prevented the IR-induced reduction in renal function and histology. There were also significant improvements in kidney oxidative stress status in both POC groups demonstrated by a reduction in malondialdehyde (MDA) formation and preservation of antioxidant levels comparing to the IR group. We concluded that both methods of POC have protective effects on renal function and histology possibly by a reduction in IR-induced oxidative stress.

Airway inflammation and oxidative stress are the two major characteristics of asthma pathogenesis. Therefore, this study evaluated the protective effects of ascorbic acid in combination with calcitriol on the oxidative damages and inflammation in asthma model. All animals, except in the control group, were sensitized and challenged with ovalbumin. One day after the last challenge, samples of bronchoalveolar lavage fluid was collected for the assessment of total white blood cell counts and differential count of white blood cell and plasma was used for the measurement of pro-oxidant/antioxidant balance level. Lung tissue samples were also stored for examining peribronchial inflammatory cell infiltration, phosphorylated nuclear factor-kappa B expression and measurement of malondialdehyde level. Induction of asthma caused significant increases in total white blood cell counts, percentage of neutrophils and eosinophils and a decrease in the percentage of lymphocytes. Moreover, asthma resulted in significant increases of peribronchial inflammatory cell infiltration, phosphorylated nuclear factor-kappa B expression and malondialdehyde level. However, no significant changes were observed in pro-oxidant/antioxidant balance level with the induction of asthma. Co-administration of low doses of ascorbic acid and calcitriol returned all to the levels measured before sensitization and challenge. Combination of low doses of ascorbic acid with calcitriol improves mouse asthma model by a possible additive effects through the decrease of oxidative stress and inflammation.

The present study investigates the role of leukocyte transfer in the induction of kidney damage from mice that have undergone a severe renal ischemia-reperfusion insult into the intact recipient mice. First, Balb/c (inbred) mice were subjected to either sham operation (Sham donors) or bilateral renal IR injury (60 min ischemia-3 h reperfusion, IR donors). Leukocytes were isolated from blood and were transferred to two recipient groups: intact recipient mice received leukocytes from Sham donor group (Sham recipient) or from IR donor group (IR recipient). After 24 h, recipient mice were anesthetized for sample collections. Renal malondialdehyde increased and total glutathione concentration and superoxide dismutase activity decreased significantly in the IR recipient group compared to the Sham recipient group. BUN and plasma creatinine were significantly different between donor groups, but these parameters were not significantly different in the two recipient groups. In the IR donor group, there have been extensive changes in renal tissues comparing to Sham including severe destruction of the tubules, necrosis and tubular obstruction plus tubular flattening. IR recipient kidneys showed significant differences from their corresponding Sham group, demonstrating some degrees of injury including loss of brush borders from proximal tubules, cellular vacuolation and flattening of the tubules. However, less tissue damage was seen in this group comparing to IR donor kidneys. These findings showed that leucocytes transferred from post-ischemic mice induced oxidative stress and consequent damage to native kidneys, suggesting a role of leucocytes in the oxidative processes of reperfusion injury.

Purpose Renal ischemia/reperfusion (I/R) injury is a common clinical problem associated with significant mortality and morbidity. One newly described strategy to reduce this damage is remote perconditioning (RPEC), in which short-time ischemia of a limb during renal ischemia reduces the I/R-induced kidney injury. This study aimed to assess whether RPEC confer protection through changes in pro-inflammatory mediators. Methods Rats were subjected to right nephrectomy and randomized into: sham (no intervention), I/R (subjected to 45-min left renal ischemia) and RPEC group (subjected to four cycles of 5-min I/R of the femoral artery administered during renal ischemia). After 24-h, blood, urine, and kidney samples were collected. Biochemical indicators of renal dysfunction were measured in the cases of Neutrophil gelatinase-associated lipocalin (NGAL), and N-acetyl-B-diglucosaminidase (NAG) activity. Inflammatory cytokines [interleukin (IL)-6 and tumor necrosis factor-alpha, TNF-α] expression in the renal tissues as well as Periodic acid-Schiff stained histological sections were evaluated. Results I/R resulted in renal dysfunction, as evidenced by higher renal NGAL expression and urinary NAG activities. This was accompanied by increased TNF-α and IL-6 expressions as well as histological changes in this group. However, RPEC improved renal histology and function compared with the I/R group. Furthermore, the RPEC group showed decreases in TNF-α and IL-6 expression. Conclusions These results suggest that RPEC reduces the dysfunction and injury associated with I/R of the kidney. This technique reduced the pro-inflammatory cytokine in the kidney. RPEC could be a promising strategy against I/R-induced acute kidney injury partly by down-regulation of inflammatory mediators.

The purpose of this study was to ascertain the onset expression of pro-inflammatory cytokines in the aorta and kidney and establish their correlation with the increase in arterial blood pressure in rats subjected to DOCA-salt treatment. Male Sprague-Dawley rats underwent unilateral nephrectomy and received subcutaneous DOCA (20 mg/rat/week) as well as 1% NaCl and 0.2% KCl for drinking for 2 weeks. Blood pressure and expression of pro-inflammatory cytokines in aorta and kidney were studied weekly during the induction of hypertension. The treated rats exhibited a mild elevation of blood pressure at 1 week and a profound increase at 2 weeks. Quantitative RT-PCR demonstrated a 4.9-fold and a 3.6-fold enhancement in the expression of TNF-alpha and IL-6, respectively, in aorta as early as 1 week. The expression of IL-6 and TNF-alpha in the kidney remained almost unchanged at 1 week but mildly increased at 2 weeks DOCA-salt treatment. This study indicates a robust increase in the expression of IL-6 and TNF-alpha in aorta in DOCA-salt treated rats. This enhancement suggests that the activation of pro-inflammatory cytokines may contribute to onset of the elevation of blood pressure in DOCA-salt hypertension model.

Objective: Reperfusion injury (RI) is associated with high generation of reactive oxygen species (ROS), but the extent of involvement of these agents in the injury remains controversial. The present study aimed to examine the effectiveness of ROS scavengers against hepatic reperfusion injury in the rat. Conclusion: ROS scavengers can reduce superoxide-induced damage and improve the liver function, but they are not able to prevent the structural changes. It shows that ROS are not the sole causative mechanism of hepatic RI and other mechanisms and mediators may be involved Methods: The RI was induced in the liver using an isolated slow-flow, reflow perfused rat liver in both anterograde and retrograde perfusion. The effects of gentisic acid, N-acetyl cysteine, and trolox C on the superoxide production, liver function, and morphological changes were examined using different biochemical and histological assays. Results: The hepatic RI caused a significant (p < 0.05) increase in superoxide production and enzyme releases and a decrease in bile flow in both directions. Histological changes induced by RI include apoptosis, necrosis, pale cytoplasm, cell vacuolation, and attenuation of cell cords. Although the production of superoxide in retrograde direction was significantly less than the anterograde, the extent of the injury in the retrograde was greater than the anterograde direction. Pretreatment of the livers with each of the test compounds significantly reduced the release of lactate dehydrogenase and aspartate aminotransferase and improved bile flow in the liver exposed to hypoxia/reperfusion. However,they failed to protect the liver against the structural alterations induced by RI. Refereed/Peer-reviewed

Different nitric oxide synthase (NOS) isoforms are found in the kidney. Some studies provided evidences that increased endothelial NOS (eNOS) activity leads to restoration of renal function after injury, but activation of inducible NOS (iNOS) aggravates renal failure. In the present study, the beneficial effects of selective iNOS blockade in gentamicin (GM) induced nephrotoxicity have been investigated. Four groups of rats were studied. Untreated control rats received saline. In GM group, GM was injected (IV, 4 mg kg(-1)). In GM + L-NAME group rats received L-NAME (N-omega-L-arginine methyl ester, a non-selective NOS inhibitor) simultaneously with GM (IV, 30 mg kg(-1)). Additional doses of L-NAME were administered 2 and 4 h after GM (IP, 30 mg kg(-1)). In GM + L-NIL group rats were treated by N-imino-ethyl lysine (L-NIL, a selective iNOS inhibitor). First dose (IV, 3 mg kg(-1)) administrated simultaneously with GM. Next doses (IP, 3 mg kg(-1)) were administered 2 and 4 h after GM. In all groups, serum and urine creatinine levels were measured. Creatinine clearance was calculated and considered as an estimation of glomerular filtration rate (GFR). Urine N-acetyl-b-D-glucose aminidase (NAG) activities were also determined. After experiments, kidney sections were histologically studied. Selective iNOS inhibition by L-NIL prevented the GM-induced decrease in GFR and increase in creatinine levels, while complete non-selective NOS inhibition by L-NAME aggravated the GFR reduction, elevation of creatinine levels and enzyme release (P0.05). Histological studies showed that GM-treated kidneys had evidences of tubular damages and these damages were less evident by the administration of L-NIL. In conclusion, selective inhibition of iNOS may prevent GM-induced nephrotoxicity, whereas non-selective inhibition of NOS aggravates it.

Background: It is generally believed that reactive oxygen species (ROS) formation and nitric oxide (NO) generation by the inducible isoform of nitric oxide synthase (iNOS) are the key mediators of ischemia-reperfusion (IR)-induced damage to the kidney. The present study was designed to investigate the effects of ROS and NOS inhibition in prevention of renal IR injury. MnTBAP (Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin), a broad-spectrum reactive species scavenger was administered to inhibit ROS formation and L-Nil (N6-(1-iminoethyl)-L-lysine hydrochloride) was used for iNOS inhibition. Methods: Ischemic acute renal failure (ARF) was induced by 40-min clamping of the renal arteries followed by a 6-hour reperfusion. Rats were administered saline, MnTBAP (10 mg/kg i.v.), L-Nil (3 mg/kg i.v. bolus followed by infusion of 1 mg/kg/h) or co-administration of MnTBAP and L-Nil. Plasma creatinine (Cr) and BUN levels as well as fractional excretion of Na+ (FENa+) and urinary N-acetyl-β-D-glucosaminidase (NAG) activities were measured. Renal damages were evaluated by light microscopy. Results: MnTBAP, L-Nil and their co-administration significantly improved renal functional and histological indices. Co-administration of the mentioned drugs did not demonstrate significant difference with the administration of either drug alone. Conclusion: These results suggest that the significant portion of ROS and iNOS nephrotoxicities in this model of ARF may be mediated by peroxynitrite (ONOO–). These results emphasize the multifactorial nature of ARF and the need for a multidrug therapy in the future.

In recent years, several lines of evidence have implicated reactive species as contributors to renal ischemia/reperfusion injury (I/R). This study was designed to investigate the effect of Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a broad-spectrum reactive species scavenger, in the prevention of renal I/R injury.Experiments were performed on rats anesthetized with pentobarbital. After tracheotomy, the right femoral artery was cannulated and the mean arterial pressure and heart rate were recorded. A midline laparatomy was performed, and the renal arteries were carefully separated from surrounding tissues. After surgery and a stabilization period (60 min), the animals were randomly assigned to four groups: sham-operated; sham+MnTBAP; I/R; I/R+MnTBAP. In I/R groups, the rats were subjected to bilateral renal artery occlusion for 40 min followed by 6 h reperfusion. Other groups underwent the surgery protocol but did not undergo renal artery occlusion, and were maintained under anesthesia for the duration of the experiment. Rats were administered either MnTBAP (10 mg kg(-1), i.v. bolus, 15 min prior to I/R) or saline. Renal function was assessed by plasma creatinine (Cr), blood urea nitrogen (BUN), and aspartate aminotransferase (AST) measurements. The fractional excretion of Na(+) (FE(Na+)) and urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were also measured. Renal section damage was evaluated by light microscopy, and oxidative stress status was evaluated by measurements of plasma and renal vitamin E levels.We found that MnTBAP significantly reduced the I/R-mediated increases in plasma Cr, BUN, AST, FE(Na+), and NAG and improved the renal tissue histology.Our results showed that MnTBAP was effective in preventing the development of I/R-induced renal injury.

SUMMARY 1. The aim of present study was to investigate the effects of magnesium (Mg) on cardiac function and infarct size and to compare it effects with those of adenosine. The mechanism of Mg-mediated cardioprotection was explored by combined use of Mg and a selective adenosine A1 receptor antagonist. 2. Rat isolated hearts were used for Langendorff perfusion. Hearts were either non-preconditioned or preconditioned with Mg (6 mmol/L) or adenosine (1 mmol/L) before 30 min sustained ischaemia followed by 120 min reperfusion. Within each of these protocols, hearts were divided into two groups; one group was exposed to the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 200 nmol/L). Infarct size was measured by the triphenyltetrazolium chloride method. Left ventricular function was assessed by left ventricular developed pressure (LVDP), the product of heart rate × LVDP and coronary flow (CF). 3. The administration of Mg had an anti-infarct effect independent of its effect on postischaemic functional recovery in rats. Both Mg and adenosine equipotently reduced infarct size, but this effect of Mg was not blocked by the simultaneous administration of DPCPX. Cardiac function was improved by both adenosine and Mg and blockade of adenosine A1 receptors attenuated these effects for both agents. 4. In conclusion, the results of the present study indicate that stimulation of adenosine A1 receptors is not responsible for the anti-infarct effect of Mg in ischaemic myocardium in rats, but that the Mg-mediated protection of postischaemic functional recovery in rats is mediated by these receptors.

SUMMARY 1. Gram-negative bacterial lipopolysaccharide (LPS) release and subsequent septic shock is a major cause of death in intensive care units. Lipopolysaccharide has been reported to increase the production of nitric oxide (NO) and the formation of oxygen-derived free radicals (OFR) in different organs. The aim of the present study was to evaluate the role of an inducible form of NO synthase (iNOS) and OFR production in LPS-induced renal impairment. 2. Measurement of vitamin E as the most important fat-soluble anti-oxidant was used as a marker of tissue oxidative stress. Lipopolysaccharide (10 mg/kg), l-iminoethyl lysine (L-Nil; 3 mg/kg, i.p.; a specific inhibitor of iNOS activity) and dimethyl thiourea (DMTU; 500 mg/kg i.p.; a well-known OFR scavenger) were used. Four groups of eight rats were studied. One group received LPS, whereas a second group received LPS + L-Nil. A third group received LPS + DMTU and the fourth group, receiving saline, acted as a control group. To evaluate renal function, plasma creatinine and blood urea nitrogen (BUN) were measured. High-pressure liquid chromatography and ultraviolet detection were used to measure plasma and tissue vitamin E levels. Light microscopy was used to examine histopathological changes in the four groups. 3. Lipopolysaccharide markedly decreased the vitamin E content of renal plasma and tissue (P

Iron overload and enhanced hydroxyl radical (•OH) formation have been implicated as the causative factors of oxidative stress in different organs. Both pro-oxidant and anti-oxidant properties have been reported for nitric oxide (NO) in iron-mediated tissue injury. To determine the contribution of NO to iron-induced renal injury, eight groups of rats (eight in each group) were studied as follows: control (normal saline), L-Arg (L-arginine as a substrate of NO synthase, 400 mg/kg), L-NAME (an inhibitor of NO synthase, 8 mg/kg), Fe (iron dextran, 600 mg/kg), DFO (deferroxamine as a chelator of iron, 150 mg/kg), Fe+L-Arg, Fe+L-NAME, DFO+L-Arg. Twenty-four hours after the injections, blood samples were taken and kidneys removed for biochemical analysis. Plasma creatinine and urea were used to stimulate renal function. Renal tissue and plasma vitamin E levels, the most important endogenous fat soluble antioxidant, were measured by HPLC and UV detection. In this study, renal function was markedly reduced in the Fe group compared to controls (creatinine, 1.02± 0.05 mg/dL versus 0.78±0.04 P

Inhibition of nitric oxide (NO) synthesis has been found to produce learning deficits in spatial tasks. Recent studies also suggest a regulatory effect of endogenous NO on hippocampal serotonin (5-HT) release and have shown that NO-synthase (NOS) inhibitors increased extracellular levels of serotonin (5-HT) in the rat hippocampus. To clarify possible interactions between NO and 5-HT in the hippocampus on learning processes, the effect of selective hippocampal 5-HT depletion on NOS inhibition-induced spatial learning deficits was investigated. Rats received bilateral injections of 5,7-dihydroxytryptamine (5,7-DHT), a 5-HT neurotoxin, or its vehicle in the CA1 region of hippocampus following pretreatment with desipramine. Rats were subjected to 5 days of training in the Morris water maze (MWM); 4 days with the invisible platform to test spatial learning and the 5th day with the visible platform to test motivation and sensorimotor coordination. N ω-nitro- l -arginine methyl ester ( l -NAME), a NOS inhibitor, was administered to either sham-operated or 5,7-DHT-lesioned groups 30 min before training each day. Results showed that l -NAME significantly impaired the ability of rats to locate the hidden platform. This impairment was reversed by co-administration of mole equivalent dose of l -arginine, the NO precursor. Although the 5,7-DHT-induced lesion had no effect by itself on rat performance in the MWM, it attenuated the memory impairment caused by l -NAME. The observed effect suggests an interaction between NO and 5-HT in the hippocampus on spatial memory formation; however, the mechanism of interaction is still unclear and requires further investigation.

Acute renal failure commonly follows reduced renal perfusion or ischemia. Reperfusion is essential for recovery but can itself cause functional and structural injury to the kidney. The separate contributions of ischemia and of reperfusion were examined in the isolated perfused rat kidney. Three groups were studied: brief (5 min) ischemia, 20 min ischemia, and repetitive brief ischemia (4 periods of 5 min) with repetitive intervening reperfusion of 5 min. A control group had no intervention, the three ischemia groups were given a baseline perfusion of 30 min before intervention and all groups were perfused for a total of 80 min. In addition, the effects of exogenous *NO from sodium nitroprusside and xanthine oxidase inhibition by allopurinol were assessed in the repetitive brief ischemia-reperfusion model. Brief ischemia produced minimal morphological injury with near normal functional recovery. Repetitive brief ischemia-reperfusion caused less functional and morphological injury than an equivalent single period of ischemia (20 min) suggesting that intermittent reperfusion is less injurious than ischemia alone over the time course of study. Pretreatment with allopurinol improved renal function after repetitive brief ischemia-reperfusion compared with the allopurinol-untreated repetitive brief ischemia-reperfusion group. Similarly, sodium nitroprusside reduced renal vascular resistance but did not improve the glomerular filtration rate or sodium reabsorption in the repetitive brief ischemia-reperfusion model. Thus, these studies show that the duration of uninterrupted ischemia is more critical than reperfusion in determining the extent of renal ischemia-reperfusion injury and that allopurinol, in particular, counteracts the oxidative stress of reperfusion.

PURPOSE We investigated the impact of sex on the protective effect of postconditioning (POC), a series of brief ischemia-reperfusion (IR) cycles at the reperfusion onset, as a recently described novel approach to attenuate renal IR injury. In this study, the left renal pedicles of uni-nephrectomized male and female rats were clamped for 45 minutes followed by 24 hours of reperfusion as IR groups. Uni-nephrectomized, sham-operated male and female rats served as control groups. Ischemic postconditioning was performed using 4 cycles of 10 seconds of IR of renal pedicle at the end of the ischemia. Twenty-four hours later, BUN (blood urea nitrogen), plasma creatinine (Cr), and renal histological changes, as well as kidney levels of MDA (malondialdehyde) and SOD (superoxide dismutase) as oxidative stress markers were evaluated to detect the protective effect of POC against IR injury in rats. RESULTS Induction of IR resulted in significant reduction in renal function, demonstrated by increase in plasma Cr and BUN, histological changes and oxidative stress in both genders. Application of POC afforded significant protection against these injuries in male rats, namely decreased levels of BUN and Cr, histological improvements and less oxidative damages. However, there were no significant differences in the above-mentioned parameters in female rats. CONCLUSION While POC is shown to be beneficial against renal IR injury in male rats, it did not show any protective effect in female rats.

This study was designed to investigate whether microinjection of angiotensin II (Ang II) into the hypothalamic paraventricular nucleus (PVN) in renal ischemia–reperfusion (IR) injury has any effect on renal oxidative stress and damage through renal sympathetic nerve activity (RSNA). One week before the induction of left renal IR injury, right nephrectomy was performed and a cannula was placed into the right PVN. Rats were then distributed among 4 groups (n = 6); Sham, IR, IR + Ang II, and IR + Ang II + losartan. Renal IR injury was induced by clamping the left renal artery for 45 min followed by 24 h reperfusion. Losartan (0.3 μg) and Ang II (3 ng) were microinjected into the PVN at 20 min and 10 min, respectively, before the induction of IR injury. Ang II increased plasma creatinine, urinary NAG activity, and histological changes, and enhanced RSNA compared with the IR group (p < 0.05). Ang II increased malondialdehyde (MDA) levels and reduced superoxide dismutase (SOD) activity in the kidney compared with IR injury. Losartan caused a reduction in plasma creatinine, urinary NAG activity, histological changes, renal sympathetic nerve activity (RSNA), and renal MDA levels, and increased renal SOD activity compared with the IR group (p < 0.05). These data demonstrated that increased RSNA activity, via microinjection of Ang II into the PVN, exaggerated renal IR injury by inducing oxidative stress in the kidney.

Background/aim: Since the nature of ischemia/reperfusion (IR)-induced tissue damage is multifactorial and complex, in the current study, the effects of multiple treatment strategies via concomitant administration of erythropoietin (EPO) and N-acetylcysteine (NAC) with an ischemic preconditioning (IPC) regimen on renal IR injury were examined.Materials and methods: Thirty male Wistar rats were subjected to bilateral occlusion of the renal pedicles for 50 min followed by reperfusion. EPO (1000 IU/kg) was administered for 3 days, as well as IPC before the IR and NAC (150 mg/kg) administration for 4 days after IR. The animals were randomly allocated into 6 groups (n = 5): sham, IR, EPO+IR, IPC+IR, NAC+IR, and EPO+IPC+NAC+IR. Kidney tissues and blood samples were obtained for oxidative stress, proinflammatory cytokines, and renal functional evaluations.Results: IR caused significant inflammatory response, oxidative stress, and reduced renal function. Treatment with EPO, IPC, and NAC or a combination of two of them attenuated renal dysfunction and reduced the oxidative stress and inflammatory markers. Rats treated with the combination of EPO, IPC, and NAC showed a higher degree of protection compared to the other groups.Conclusion: These results showed that concomitant administration of EPO and IPC along with posttreatment NAC may have additive beneficial effects on kidney IR injury during IR-induced acute renal failure.

Liver ischemia/reperfusion (IR) injury is a complex phenomenon that may cause local as well as remote organ injuries. Reactive oxygen species (ROS) along with many pro- and anti- inflammatory cytokines are implicated in the development of organ injury. The renal functional, histological, oxidative stress and inflammatory indices were studied during a short and a longer period of liver IR. Rats were subjected to either sham operation or 90 min partial liver ischemia followed by 4 or 24 h of reperfusion. Serum ALT, AST, ALK and LDH levels, BUN and creatinine, renal MDA level, SOD and catalase activities were evaluated as well as serum IL-6 and IL-10 concentrations along with renal histological evaluation. Ninety minutes liver ischemia /4 h reperfusion caused an increase in BUN and renal MDA levels and a decrease in SOD and catalase activities. It also caused an increase in serum IL-6 and IL-10 levels. 24 h liver reperfusion resulted in a reduction in BUN levels and lower oxidative damages demonstrated by a decrease in renal MDA levels and an increase in renal SOD and catalase activities comparing to 4 h reperfusion group. Evaluations indicated improvement in histology such as less cytoplasmic vacuolation and lower tubular debris. Serum inflammatory indices (IL-6 and IL-10 levels) were also reduced. This study showed that liver IR damage causes renal injury including functional, inflammatory and oxidative status changes. The remote kidney damage was then improved by continuing reperfusion from 4 to 24 h.

Remote preconditioning (rPeC) is a phenomenon by which short-time intermittent ischaemia-reperfusion (I/R) of a remote organ during ischaemia protects other organs from I/R injury (IRI). The aim of the present study was to investigate the protective effect of rPeC on renal IRI in rats. Rats were subjected to right nephrectomy and randomized as into a sham group (no additional intervention), an I/R group (subjected to 45 min left renal pedicle occlusion) and an rPeC group (subjected to four cycles of 5 min I/R of the left femoral artery administered at the beginning of renal ischaemia). After 24 h, blood, urine and tissue samples were collected. Compared with the sham group, I/R resulted in renal dysfunction, as evidenced by significantly lower creatinine clearance (CCr; 0.52 ± 0.06 vs 0.11 ± 0.02 mL/min, respectively) and higher fractional excretion of sodium (FE(Na) ; 0.80 ± 0.07% vs 2.46 ± 0.20%, respectively). This was accompanied by decreased superoxide dismutase (SOD; 6.9 ± 1.7 vs 26.7 ± 2.7 U/g tissue) and catalase (CAT; 20.2 ± 8.8 vs 32.2 ± 8.7 K/g tissue) activity in the I/R group, as well as decreased levels of reduced glutathione (GSH; 21.7 ± 8.1 vs 81.2 ± 20.2 μmol/g tissue) and increased malondialdehyde levels (MDA; 1.2 to 0.1 vs 0.5 ± 0.2 μmol/100 mg), cyclo-oxygenase (COX)-2 expression and histological damage. In the rPeC group, renal histology and function were significantly improved (CCr 0.32 ± 0.02 mL/min; FE(Na) 1.33 ± 0.12%) compared with the I/R group. Furthermore, compared with the I/R group, the rPeC group exhibited increases in SOD and CAT activity (22.8 ± 3.8 U/g tissue and 21.7 ± 8.6 K/g tissue, respectively), increased GSH levels (74.0 ± 4.9) and decreased MDA levels (1.1 ± 0.3 μmol/100 mg) and COX-2 expression. In conclusion, rPeC appears to exert protective effects against renal IRI. This protection may be a consequence of reductions in lipid peroxidation, intensification of anti-oxidant systems and downregulation of COX-2 expression. A simple approach, rPeC may be a promising strategy for protection against IRI in clinical practice.

Acute hepatic injury causes systematic inflammatory responses which may finally lead to functional disturbances in remote organs. In this study, the effects of an inhibitor of inflammatory cytokines (pentoxifylline, PTX) and a well-known antioxidant, N-acetylcysteine (NAC), were evaluated on renal damage and oxidative stress following liver ischemia reperfusion (IR).Five groups of six male rats were used. Group 1 was sham operated. In group 2, 90 min liver partial ischemia was induced by a clamp around both hepatic artery and portal vein and then followed by 4 h of reperfusion. In groups 3 and 4, PTX or NAC was injected intraperitoneally before the ischemia, while in group 5 both drugs were co-administered. The levels of alanine amino-transferase (ALT), aspartate amino-transferase (AST), blood urea nitrogen (BUN), and creatinine in serum as well as malonyldialdehyde (MDA) and glutathione (GSH) levels and morphological changes in renal tissues were assessed.Significant increase in the serum levels of ALT and AST in IR group is indicative of liver functional damages. Elevated BUN and renal tissue MDA, decreased GSH levels, and morphological damages in IR group demonstrate a significant kidney injury and oxidative stress comparing to sham group. Administration of PTX alone and PTX + NAC prevented the IR-induced increase in renal MDA levels. Administration of both drugs and their co-administration prevented the reduction in renal GSH levels and morphological changes.Pretreatment with PTX and NAC before liver IR may be useful to ameliorate renal oxidative damage by preservation of cellular GSH concentration and a reduction in MDA levels.

Uric acid is considered as an antioxidant in the blood. Despite its proposed protective properties, elevated plasma uric acid has been associated with hypertension in a variety of disorders. The purpose of this study was to investigate the relationship between the increase of arterial blood pressure and the changes in serum uric acid, measured during the gradual development of experimental hypertension in deoxycorticosterone (DOCA)-salt-treated rats. Blood pressure was monitored by tail-cuff method, urinary and plasma uric acid was measured by autoanalyzer during the induction of hypertension in 1-, 2-, 3- and 4-week DOCA-salt-treated Sprague-Dawley rats. Vitamin E (200 mg/kg/day/gavage) was co-administered with DOCA-salt for 4 weeks. From the first week of DOCA-salt treatment, rats exhibited marked increases in blood pressure. DOCA-salt treatment also resulted in a significant increase in serum uric acid and a significant decrease in urinary uric acid at the end of the first week. These changes in serum and urinary uric acid remained until the 4th week of DOCA-salt treatment but blood pressure continued to increase throughout the study. Vitamin E treatment increased urinary excretion of uric acid and decreased blood pressure and serum uric acid in DOCA-salt-treated rats. These data suggest that enhanced serum uric acid may be a contributing factor to the onset of hypertension in DOCA-salt-treated rats. A uricosuric effect is suggested for vitamin E in the treatment of hypertension.

Renal ischemia-reperfusion (IR) induces organ damage in remote organs. The aim of this study was to assess the role of leukocytes in the induction of liver damage after renal IR injury.Inbred mice were subjected to either sham operation or bilateral renal IR injury (60 min ischemia followed by 3 h reperfusion). Mice were then anesthetized for collection of leukocytes by heart puncture. Isolated leukocytes were transferred to two other groups: intact recipient mice that received leukocytes from IR mice and intact recipient mice that received leukocytes from sham-operated control mice. After 24 h, recipient mice were anesthetized and samples were collected.Alanine aminotransferase, aspartate aminotransferase, and hepatic malondialdehyde increased significantly, and hepatic glutathione decreased significantly in intact recipient mice that received leukocytes from IR mice in comparison with intact recipient mice that received leukocytes from sham-operated control mice. Loss of normal liver architecture, cytoplasmic vacuolization, and focal infiltration of leukocytes were seen.These results suggest that leukocytes are one of the possible factors that contribute to liver damage after renal IR injury and this damage is partly due to the induction of oxidative stress.

Background. Cyclosporine (CsA) is known to cause metabolic and distal tubular acidosis. There is some evidence that CsA reduces net HCO 3 ― absorption. The aim of this study was to elucidate whether bicarbonate administration prevented CsA-induced functional or structural nephrotoxicity. Methods. Seven days after uninephrectomy, 20 rats were divided into 4 groups. NaHCO 3 (0.28 mol/L) was added in drinking water for 7 days, whereas control rats received regular tap water. The bicarbonate pretreated rats were administered either CsA (50 mg/kg intraperitoneally) or vehicle daily for a week. At the end of the procedure, animals were placed in metabolic cages for 24 hours after which we measured creatinine clearance (Ccr), urinary total proteins, pH, and N-acetyl-β-D-glucosaminidase (NAG) activity. The kidney was fixed in formaldehyde. Results. Ccr was significantly affected by the administration of CsA. The effects of CsA on serum pH and HCO 3 ― concentration were prevented by pretreatment with NaHCO 3 . However, it did not affect the CsA-induced increased urinary NAG activity or decreased Ccr. There was no protection of CsA-induced changes in renal tissues by NaHCO 3 . Conclusion. Overall NaHCO 3 administration did not prevent CsA-induced changes in Ccr and NAG activity. These data suggested involvement of factors other than acid-base status in CsA-induced nephrotoxicity. However, correction of acidosis should still be considered for patients receiving CsA because acidosis exacerbates tissue damage.

Background Ischemia-reperfusion (IR)-induced nephrotoxicity is associated with proteinuria. There are reports on the involvement of inducible nitric oxide synthase (iNOS) in proteinuria in conjunction with renal disease. This study was designed to investigate the effect of N6-(1-iminoethyl)-L-lysine hydrochloride (L-Nil), a selective inhibitor of iNOS, to prevent proteinuria in IR injury. Methods Ischemia was induced by 40-minute clamping of the renal arteries followed by 6-hour reperfusion. Rats were administered either L-Nil (3 mg/kg intravenous bolus followed by infusion of 1 mg/kg/h) or saline. To monitor glomerular and tubular functional changes before and after treatment, we measured blood urea nitrogen, plasma creatinine, and urinary N-acetyl-β-D-glucosaminidase activity. Total protein (TP), albumin, and low- (LMW) and high-molecular-weight (HMW) protein excretion rates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of urine samples. Kidney ultrastructure was examined through a transmission electron microscope (TEM). Results IR resulted in significant LMW and HMW proteinuria. L-Nil significantly prevented the IR-induced increases in TP, albumin, and α1-microglobulin excretion. TEM showed loss of microvilli of the proximal tubule cells, injured mitochondria, and foamy changes in the structure of nuclear and cytoplasm in IR group. L-Nil reduced IR-mediated renal ultrastructural changes and tubular proteinuria. Discussion This study suggested possible differences in the mechanism(s) of nephrotoxicity induced by iNOS in the glomeruli and tubular cells. The types of proteins excreted in the urine should be considered in the treatment strategy. In conclusion, this study suggested the involvement of iNOS in IR-induced tubular proteinuria.

We assessed whether co-supplementation of vitamins C and E has additive beneficial effects on reducing the kidney damage and attenuation of the arterial pressure elevation compared to administration of either vitamin C or vitamin E alone in deoxycorticosterone acetate-salt-induced hypertension.Forty rats were divided into 4 study groups and 1 sham-operated group. Unilateral nephrectomy was carried out in the study groups and hypertension was induced by deoxycorticosterone injection and 1% sodium chloride and 0.2% potassium chloride added to the drinking water. Vitamins C and E (200 mg/kg/day) or combination of them were administered with DOCA-salt for 4 weeks in 3 study groups. The effects of DOCA and salt and treatment with vitamins were compared in terms of blood pressure, urinary protein excretion, antioxidant activity of the kidneys, and renal histological changes.Four weeks of supplementations of vitamins C, vitamin E, and both in the DOCA-salt-treated rats had comparable significant effects in decreasing systolic blood pressure. Urinary protein excretion and histological damage did not significantly change with the combination therapy of vitamins C and E compared to the vitamin C or E alone. The renal levels of glutathione and ferric reducing/antioxidant power in combination therapy group were similar to the two other treatment groups and were significantly higher than non-treated group.Co-administration of vitamin C and E does not have an additive beneficial effect on reducing the kidney damage and hypertension compared to either vitamin C or E alone in DOCA-salt-induced hypertension.

Objective Cyclosporine (CsA) has been shown to improve long-term survival after organ transplantation. However, CsA therapy is associated with a variety of side effects, among which nephropathy is the major one. Recent studies have suggested increased oxidative stress as a cause of drug nephrotoxicity. Therefore, this study was designed to evaluate the effects of CsA administration on the antioxidant capacity of kidney tissue. Methods Adult male Sprague-Dawley rats were randomly assigned into 2 groups: one group received CsA (25 mg/kg/d, IP for 2 weeks) and a control group (no CsA administration). After 2 weeks, the kidneys of the rats from both groups were removed under anesthesia. A 50 mg fresh kidney tissue sample was homogenized in ice-cold phosphate buffer. Total antioxidant capacity (Ferric Reducing Ability of Plasma [FRAP]) in the homogenates was assayed based on the Benzie spectrophotometric method. Results FRAP in the kidney tissues had been significantly decreased by 2 weeks of CsA administration when compared with control rats (P Conclusions These data suggested that CsA administration may decrease the antioxidant capacity of renal tissues. More studies on the evaluation of the protective effects of antioxidant therapy against CsA nephrotoxicity are underway.

Objective. The clinical use of gentamicin (G) is limited due to its known nephrotoxic actions. Generation of reactive oxygen species has been proposed as a causative factor of cell death in G-induced acute renal failure (ARF). Previous studies using superoxide dismutase (SOD) mimetics have indirectly suggested a role for the superoxide ion in G-induced ARF. In this study, we directly measured the enzyme activities using in situ isolated kidneys seeking to investigate the effects of antioxidant therapy on preservation of endogenous antioxidant levels in ARF. Methods. Thirty-five male Sprague-Dawley rats were randomly assigned to 5 groups: control, Tyrode-perfused; G, gentamicin (200 mg/L) added to the perfusate; G + vitamin E (Vit E; 100 mg/100 g BW, IM); G + vitamin C (Vit C) added to the drinking water for 3 days (200 mg/L) and to the perfusate (100 mg/L); G + Vit E + Vit C. SOD activities were determined in renal tissues based on NAPDH oxidation at 340 nm by spectrophotometry. Results. SOD activity was significantly reduced in the G group compared with the controls (P

Short time pretreatment with oxygen is reported to be protective against subsequent ischemia-reperfusion (IR) injury of heart and spinal cord in some animal models. The purpose of this study was to investigate the effects of pre-exposure to hyperoxic environment on rat renal IR injury for the first time.The effects of 1 h/d pretreatment with oxygen (or=95%) for 5 days on a right nephrectomized rat model of renal IR injury was investigated by comparing creatinine clearance, fractional excretion of sodium, plasma creatinine, blood urea nitrogen, and histological injury scores among three groups: IR (40 min ischemia-24 h reperfusion), sham (no IR), and hyperoxia (5 days intermittent pretreatment with oxygen + IR).Intermittent pretreatment with oxygen resulted in significant improvement of creatine clearance and fractional excretion of sodium (Por= 0.05). Plasma creatinine above 175 micromol/L or blood urea nitrogen beyond 26 mmol/L was significantly less frequent in hyperoxia than IR group (P0.05). Jablonski histological injury score was also significantly lower in hyperoxia compared to IR group (P0.05) and hyperoxic preconditioning significantly reduced the frequency of massive proximal tubular necrosis (12.5% versus 75%, P0.05).The present findings demonstrate that intermittent pre-exposure to hyperoxic environment can reduce subsequent renal IR injury.

There have been many studies in recent years concerning the role of nitric oxide (NO) in acute renal failure (ARF). In this study, the effects of the inhibition or the induction of NO synthase (NOS) on gentamicin-induced ARF was investigated in isolated perfused rat kidneys.Kidneys from male Sprague-Dawley rats were perfused in situ for 90 min. Perfusion was conducted in the presence of inulin (60 mg/dL in perfusion buffer) as a glomerular filtration rate (GFR) marker. Six groups (total: 42 rats) were studied: group 1, controls with no treatment; group 2, L-arginine (2 mM in perfusate); group 3, L-nitro-arginine-methyl ester (L-NAME, 0.1 mM in perfusate); group 4, gentamicin (GM, 0.5 mg/mL in perfusate); group 5, GM + L-arginine (same dose as groups 2 and 4) and; group 6, GM + L-NAME (same dose as groups 3 and 4). Cell injury was assessed by measuring N-acetyl-beta-D-glucosaminidase (NAG), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity in urine.L-arginine prevented, whereas L-NAME enhanced, GM-induced enzyme release and GFR reduction. Histological studies showed that GM-treated kidneys had clear signs of tubular damage and this damage was increased by simultaneous L-NAME and GM administration.This study suggests that NO formation could prevent the GM-induced nephrotoxicity in this ARF model.

Background Hypertension is an important risk factor for the progression of chronic renal disease, which may result in the development of end-stage renal disease. Since reactive oxygen species are implicated in the induction of hypertension, antioxidants have been used to reduce blood pressure and renal impairment in animal models and in human hypertension. However, the available data are not conclusive. Methods To investigate oxidative stress in hypertension, we evaluated renal and serum vitamin E levels as the most effective antioxidant to reduce lipid peroxidation by high-performance liquid chromatography among rats subjected to deoxycorticosterone acetate (DOCA)-salt treatment for 4 weeks. Renal levels of malondialdehyde (MDA) as a marker of cell lipid peroxidation were also assayed in treated rats. Systolic blood pressure (SBP) was measured in conscious rats by the tail-cuff method using a PowerLab/4sp data acquisition system. Results SBP increased significantly in DOCA-salt-treated rats compared to the sham group after 4 weeks of treatment. Serum vitamin E levels were significantly lower and renal MDA concentrations significantly higher in treated compared to sham rats. However, renal vitamin E levels were also significantly higher among treated compared to sham rats. Discussion Decreased plasma vitamin E levels and increased renal MDA levels show systemic and local oxidative stress in DOCA-salt-treated rats. However, the higher renal vitamin E level suggested a local compensatory mechanism. Vitamin E administration might be appropriate, as significant decreases in vitamin E levels were observed in the serum of DOCA-salt-treated rats.

Objective Cyclosporine (CsA) causes tubular dysfunction characterized by polyuria, calcium wasting, distal tubular acidosis, and hyperkalemia. The hypercalciuria induced by CsA administration is associated with an inhibition of calbindin D 28k expression. It has also been shown that chronic metabolic alkalosis increased the expression of Ca 2+ transport proteins accompanied by diminished urine Ca 2+ excretion. The aim of this study, therefore, was to determine the effect of acid-base status on CsA-induced hypercalciuria. Methods Experiments were performed on male Sprague-Dawley rats. Metabolic alkalosis and acidosis were induced respectively by adding 0.28 mol/L NaHCO 3 and 0.28 mol/L NH 4 Cl in the drinking water for 7 days; control rats received regular tap water. Seven days after NaHCO 3 or NH 4 Cl administration, rats were treated with CsA (25 mg/kg, IP) daily for 14 days. To estimate glomerular filtration rate (GFR) over time, animals were placed in metabolic cages. Fractional urinary calcium excretion was determined by standard formula. Results The CsA group showed decreased serum calcium and increased fractional urinary calcium excretion compared with the control group. Creatinine clearance was also significantly reduced. Metabolic alkalosis alone did not affect GFR, but significantly prevented an increase in fractional urinary calcium excretion induced by CsA, whereas chronic metabolic acidosis resulted in the exact opposite effect. Conclusions It is essential for nephrologists to fully understand the mechanisms of CsA-induced renal injury. In this study, metabolic alkalosis reduced CsA-induced hypercalciuria. Further studies are needed to elucidate whether this effect may be achieved pharmacologically by the expression of Ca 2+ transport proteins.

Recent studies suggest that oxygen-derived free radicals are involved in mediating renal reperfusion injury. EPR spectroscopy and spin trapping with the spin traps DMPO and PBN, were used to detect and quantitate the formation of hydroxyl radicals in rat kidney after ischaemia-reperfusion in vivo and in vitro in the isolated rat kidney perfused in the absence of leucocytes. EPR analysis of homogenised kidneys and of venous samples did not detect radical adducts with either spin trap. With PBN, radical adducts were not detected in vitro. When DMPO was used as the spin trap in kidneys perfused without albumin in the perfusate, EPR signals characteristic of hydroxyl and carbon-centred radical adducts were detected during early reperfusion following ischaemia. These studies confirm the generation of hydroxyl radicals during ischaemia-reperfusion in kidney. During reperfusion the total DMPO adduct concentration reached 4.35 +/- 1.05 nmol/g kidney/3 min, p < 0.05. In control kidneys total adduct were present at lower concentration (2.55 +/- 1.1 nmol/g kidney/3 min). Addition of 15 mM dimethylthiourea abolished formation of these adducts following ischaemia-reperfusion but did not prevent a reduction in glomerular filtration rate. These results indicate that significant levels of hydroxyl and carbon-centred radicals are formed in the absence of circulating neutrophils during early renal reperfusion following ischaemia.

The difficulty in direct detection of oxygen-derived free radicals (OFR) in the intact kidney has left uncertain the role of OFR in renal hypoperfusion injury. Salicylate hydroxylation was used as a sensitive method of estimating the extent of production of highly reactive hydroxyl radicals in renal ischaemia-reperfusion injury in the intact rat kidney perfused with recirculating cell-free medium. The reaction products were detected and quantified by HPLC with electrochemical detection. Hydroxyl radicals were detected as 2,5-dihydroxybenzoic acid (2,5-DHBA). Ischaemia for 15 min followed by reperfusion for 15 min caused more than a twofold increase in 2,5-DHBA concentration (to 2279 +/- 225 pg/g tissue weight) compared to controls (933 +/- 103, P0.001). Addition of 15 mM dimethylthiourea (DMTU) before induction of ischaemia prevented this increase. Induction of hypoxia for 15 min with continued perfusion (as a model of low-flow ischaemia) had no significant effect on hydroxyl radical formation. We conclude that significant quantities of hydroxyl radicals form in the absence of circulating leucocytes during reperfusion following ischaemia, but not during hypoxia in the perfused rat kidney.

Renal ischemia/reperfusion (IR) is a widespread phenomenon that occurs in many clinical scenarios when blood supply to the kidneys is interrupted and then restored. However, it is the reperfusion, rather than ischemia, that causes the major damage to the organs (1). Remote ischemic conditioning is a powerful method of protection in which brief nonlethal cycles of IR of the arm or leg are applied before (preconditioning), during (perconditioning) (2), or after (postconditioning) (3) prolonged ischemia of a vital distant organ (4, 5). We have recently evaluated the effects of remote ischemic (application of brief episodes of IR in limb) perconditioning and postconditioning (rPeC and rPoC, respectively) during a sustained episode of renal ischemia and reperfusion. Experiments were performed on male Sprague–Dawley rats weighing 250 to 300 g which were randomly assigned into four groups (n 6). All animals underwent right nephrectomy. In IR group, with the use of a nontraumatic vascular clip, 45 min of left renal artery occlusion was induced followed by 24 hr of reperfusion. In sham group, all of the above surgical procedures were applied except that IR was not induced. In rPeC group, four episodes of 5-min ischemia and 5-min reperfusion of left femoral artery (occlusion and release of an arterial clamp) were applied during renal ischemia and before reperfusion. In rPoC group, the same cycles of intermittent IR of left femoral artery were applied after ischemia and right at the time of restoring the blood to the kidney. At the end of the reperfusion, plasma samples were collected for renal functional monitoring by measuring blood concentrations of creatinine and blood urea nitrogen (BUN) by colorimetric methods using commercially available kits. After 24 hr of reperfusion, both rPeC and rPoC significantly improved renal function and prevented the IRinduced increase in BUN and plasma creatinine. As shown in Figure 1, there were significant differences in BUN levels between sham (18 1.29 mg/dL) and IR group (118.4 5.73 mg/dL), between rPeC (49.66 7.24 mg/dL) and IR group, and between rPoC (65.66 6.90 mg/dL) and IR group. Plasma creatinine was also significantly different between sham (0.73 0.16 mg/ dL) and IR group (3.76 0.57 mg/dL), between rPeC (1.43 0.29 mg/dL) and IR group, and between rPoC (2.16 0.71 mg/ dL) and IR group. These data suggest that rPeC and rPoC have protective effects on renal function. Remote ischemic conditionings as noninvasive and applicable methods may be promising strategies against IR injury in clinical practice, especially during renal transplantation.

Objective To evaluate the role of gender in hepatic oxidative stress response and production of inflammatory cytokines in acute uremia after bilateral nephrectomy. Published studies indicate that the severity of tissue damage in kidney, brain, or heart injury may differ according to gender. We recently demonstrated that acute renal failure after kidney injury or bilateral nephrectomy activates oxidative stress and causes damage to the liver. Methods Male and female rats were subjected to bilateral nephrectomy and euthanized four hours later. Serum and liver tissues were collected and analyzed. To ascertain the role of testosterone and estrogen in hepatic oxidative stress, castration was carried out 15 days before bilateral nephrectomy. In some groups, animals were administrated 17-β-estradiol or vehicle for 2 weeks before bilateral nephrectomy. Results Hepatic oxidative stress was significantly pronounced in male rats as determined by increase in malondialdehyde (MDA) levels and decrease in total glutathione (GSH) contents. An increase in proinflammatory cytokine concentration was seen in male rats, whereas the antiinflammatory cytokine level was more elevated in females. Castration reduced hepatic oxidative stress and proinflammatory cytokine concentration, whereas exogenous estradiol after castration did not have an additional effect on these parameters. Conclusions There is a gender difference with regard to the severity of hepatic oxidative stress and inflammatory response in acute uremia after bilateral nephrectomy, with female rats displaying significant protection relative to male rats. We suggest that sex hormones could play an important role in the severity of remote tissue damage in acute kidney failure.

AIM: To assess the hepatic changes after induction of different periods of renal ischemia. METHODS: Rats were subjected to either sham operation or ischemia (30, 45 and 60 min) followed by 60 min reperfusion. Liver and renal functional indices were measured. Hepatic glutathione (GSH) and ferric reducing antioxidant power levels and the concentration of interleukin (IL)-10 and tumor necrosis factor (TNF-α) were evaluated. Portions of liver and kidney tissues were fixed for histological evaluation. RESULTS: Forty-five minutes renal ischemia followed by 60 min reperfusion caused significant changes in liver structure and a significant reduction in renal function. These rats showed a significant decrease in liver GSH, as well as a significant increase in TNF-α and IL-10 concentrations. These results demonstrated that renal ischemia caused changes in liver histology, function, oxidative stress and inflammatory status, which led to a reduction in hepatic antioxidant capacity. With 30 min ischemia, the magnitude of these changes was less than those with 45 or 60 min ischemia. CONCLUSION: A minimum of 45 min ischemia is needed to study the effects of renal injury on the liver as a remote organ.

Background During renal transplantation, the kidney remains without blood flow for a period of time. The following reperfusion of this ischemic kidney causes functional and structural injury. Formation of oxygen-derived free radicals (OFR) and subsequent lipid peroxidation (LP) has been implicated as the causative factors of these injuries. Vitamin E is known to be the main endogenous antioxidant that stabilizes cell membranes by interfering with LP. The present study was designed to examine the role of ischemic-preconditioning (repeated brief periods of ischemia, IPC) in prevention of renal injury caused by ischemia-reperfusion (IR) in rats. Methods IPC included sequential clamping of the right renal artery for 5 min and release of the clamp for another 5 min for a 3 cycles. IR was induced by 30 min ischemia followed by 10 min reperfusion. Four groups of male rats were used: Control, IPC, IR and IPC-IR. Vitamin E, an endogenous antioxidant and as an index of LP, was measured by HPLC and UV detection in renal venous plasma and tissue. Renal function was assessed by serum creatinine and BUN levels. Renal damage was assessed in sections stained with Haematoxylin and Eosin. Results In the IR group, there was a significant decrease in vitamin E in plasma and tissue compared to a control group (p,0.05). In the IPC-IR group, vitamin E concentration was significantly higher than in the IR group (p,0.01). The results showed that 30 min ischemia in the IR group significantly (p,0.05) reduced renal function demonstrated by an increase in serum creatinine levels as compared with the control group. These results in the IPC group also showed a significant difference with the IR group but no significant difference in serum BUN and creatinine between IR and IPC-IR group were detected. Histological evaluation showed no structural damage in the IPC group and an improvement in the IPC-IR group compared to IR alone. Conclusions In this study, IPC preserved vitamin E levels, but it could not markedly improve renal function in the early phase (1–2 h) of reperfusion. IPC may be a useful method for antioxidant preservation in organ transplantation.