Effect of PERLA®, a new cold-storage solution, on oxidative stress injury and early graft function in rat kidney transplantation model.

BACKGROUND: The composition of organ preservation solutions is crucial for maintaining graft integrity and early graft function after transplantation. The aim of this study is to compare new organ preservation solution PERLA® with the gold standard preservation solution University of Wisconsin (UW) regarding oxidative stress and early graft injury. METHODS: In order to assess oxidative stress after cold storage, kidney grafts have been preserved for 18 h at 4° C in either UW solution or PERLA® solution and then assessed for oxidative stress injury (protocol 1). To assess kidney injuries and oxidative stress after reperfusion, rat kidneys were harvested, stored in cold UW or in PERLA® solutions for 18 h at 4 °C and then transplanted heterotopically for 6 h (protocol 2). PERLA® is a high Na+/low K+ solution including PEG-35 (1 g/L), trimetazidine (1 µM), carvedilol (10 µM) and tacrolimus (5 µM). RESULTS: Our results showed that preservation of kidneys in PERLA® solution significantly attenuates oxidative stress parameters after cold storage and reperfusion. We found a significant decrease in oxidative damage indicators (MDA, CD and CP) and a significant increase in antioxidant indicators (GPx, GSH, CAT, SOD and PSH). Moreover, PERLA® solution decreased kidney injury after reperfusion (creatinine, LDH and uric acid). CONCLUSION: PERLA® solution was more effective than UW storage solution in preserving rat's kidney grafts.

Interleukin 10 (IL-10) gene variants and haplotypes in Tunisian women with polycystic ovary syndrome (PCOS): a case-control study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder that affects women in their child-bearing age, and is associated with insulin resistance and type 2 diabetes. The etiology of PCOS involves multiple factors including genetic, metabolic and immunological factors. Interleukin - 10 (IL-10), as an anti-inflammatory cytokine, plays a critical role in this regard. We investigated the potential role of IL-10 gene variants in the development of PCOS in Tunisian population. METHODS AND RESULTS: 115 cases and 120 controls were recruited in the current case control study. Rotterdam consensus criteria were used to diagnose PCOS patients. Genotyping for IL-10, rs1800896, rs1800871 and rs1800872 variants, was performed by real time PCR. The results obtained showed that the minor allele frequency of rs1800896, rs1800871and rs1800872 were comparable between PCOS cases and control subjects (P = 0.30, P = 0.71, and P = 0.57 respectively). The distribution analysis revealed an unsignificant association of the three tested variants, in all genetic models. Haplotype analysis identified one haplotype CCA with a protective role in PCOS development (P = 0.05; OR (95% CI) = 0.56 (0.32 - 0.99)). This association did not persist after adjustment for multiples covariates (Pc = 0.154). CONCLUSIONS: Our study is the first to show how ethnicity influences the association of IL-10 gene variants with PCOS susceptibility. No allelic nor genetic association were observed between the tested variants and PCOS in Tunisian women, however, a particular IL-10 haplotype with a protective effect for PCOS was identified.

Protective effects of diclofenac on liver graft preservation.

This study aims to evaluate the effect of diclofenac addition to the preservation solution Celsior on liver graft preservation. Liver from Wistar rats were cold flushed in situ, harvested, and then stored in Celsior solution (24 h, 4 °C) supplemented or not with 50 mg/L of diclofenac sodium salt. Reperfusion was performed (120 min, 37 °C) using the isolated perfusion rat liver model. Perfusate samples were collected to evaluate transaminases' activities after cold storage and by the end of reperfusion. To evaluate liver function, bile flow, hepatic clearance of bromosulfophthalein, and vascular resistance were assessed. Diclofenac scavenging property (DPPH assay) as well as oxidative stress parameters (SOD and MPO activities and the concentration of glutathione, conjugated dienes, MDA, and carbonylated proteins) were measured. Transcription factors (PPAR-γ and NF-κB), inflammation (COX-2, IL-6, HMGB-1, and TLR-4), as well as apoptosis markers (Bcl-2 and Bax) were determined by quantitative RT-PCR. Enriching the preservation solution Celsior with diclofenac sodium salt attenuated liver injuries and improved graft function. Oxidative stress, inflammation, and apoptosis were significantly reduced in Celsior + Diclo solution. Also, diclofenac activated PPAR-γ and inhibited NF-κB transcription factors. To decrease graft damage and improve transplant recovery, diclofenac sodium salt may be a promising additive to preservation solution.

Preeclampsia is associated with reduced renin, aldosterone, and PlGF levels, and increased sFlt-1/PlGF ratio, and specific angiotensin-converting enzyme Ins-Del gene variants.

We investigated the association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism with preeclampsia (PE) in Tunisian women. ACE I/D genotyping was done by PCR in 342 pregnant women with PE and 289 healthy pregnant women. The association between ACE I/D and PE and associated features were also evaluated. Decreased active renin concentration, plasma aldosterone concentration, and placental growth factor (PlGF) were observed in PE cases, while soluble fms-like tyrosine kinase-1 (sFlt-1)/PlGF ratio was significantly higher in the PE group. Distribution of ACE I/D alleles and genotypes were comparable between women with PE and control women. A significant difference in the frequency of the I/I genotype was seen between PE cases and control women according to the recessive model, with a trend towards association in the codominant model. Carriers of the I/I genotype had significantly higher infant birth weights compared to the I/D and the D/D genotype carriers. A dose-dependent relationship was also seen in VEGF and PlGF plasma levels and specific ACE I/D genotypes, with the lowest VEGF levels seen in the I/I genotype carriers compared to the D/D genotype carriers. Similarly, the I/I genotype carriers had the lowest PlGF levels compared to I/D and D/D genotype carriers. Furthermore, when studying the linkage between PE features, we found a positive correlation between PAC and PIGF. Our study suggests a role for ACE I/D polymorphism in the pathogenesis of PE, possibly through modulating VEGF and PlGF levels and infant birth weight, and highlights the relationship between PAC and PlGF.

Effect of the Non-steroidal Anti-inflammatory Drug Diclofenac on Ischemia-Reperfusion Injury in Rat Liver: A Nitric Oxide-Dependent Mechanism.

Ischemia/reperfusion injury (IRI) is an inevitable complication of liver surgery and transplantation. The purpose of this study was to examine the beneficial effects of diclofenac on hepatic IRI and the mechanism behind it. Wistar rats' livers were subjected to warm ischemia for 60 min followed by 24 h of reperfusion. Diclofenac was administered intravenously 15 min before ischemia at 10, 20, and 40 mg/kg body weight. To determine the mechanism of diclofenac protection, the NOS inhibitor L-Nitro-arginine methyl ester (L-NAME) was administered intravenously 10 min after diclofenac injection (40 mg/kg). Liver injury was evaluated by aminotransferases (ALT and AST) activities and histopathological analysis. Oxidative stress parameters (SOD, GPX, MPO, GSH, MDA, and PSH) were also determined. Then, eNOS gene transcription and p-eNOS and iNOS protein expressions were evaluated. The transcription factors PPAR-γ and NF-κB in addition to the regulatory protein IκBα were also investigated. Finally, the gene expression levels of inflammatory (COX-2, IL-6, IL-1ß, IL-18, TNF-α, HMGB-1, and TLR-4) and apoptosis (Bcl-2 and Bax) markers were measured. Diclofenac, at the optimal dose of 40 mg/kg, decreased liver injury and maintained histological integrity. It also reduced oxidative stress, inflammation, and apoptosis. Its mechanism of action essentially depended on eNOS activation rather than COX-2 inhibition, since pre-treatment with L-NAME abolished all the protective effects of diclofenac. To our knowledge, this is the first study demonstrating that diclofenac protects rat liver against warm IRI through the induction of NO-dependent pathway. Diclofenac reduced oxidative balance, attenuated the activation of the subsequent pro-inflammatory response and decreased cellular and tissue damage. Therefore, diclofenac could be a promising molecule for the prevention of liver IRI.

A Potential Route to Reduce Ischemia/Reperfusion Injury in Organ Preservation.

The pathophysiological process of ischemia and reperfusion injury (IRI), an inevitable step in organ transplantation, causes important biochemical and structural changes that can result in serious organ damage. IRI is relevant for early graft dysfunction and graft survival. Today, in a global context of organ shortages, most organs come from extended criteria donors (ECDs), which are more sensitive to IRI. The main objective of organ preservation solutions is to protect against IRI through the application of specific, nonphysiological components, under conditions of no blood or oxygen, and then under conditions of metabolic reduction by hypothermia. The composition of hypothermic solutions includes osmotic and oncotic buffering components, and they are intracellular (rich in potassium) or extracellular (rich in sodium). However, above all, they all contain the same type of components intended to protect against IRI, such as glutathione, adenosine and allopurinol. These components have not changed for more than 30 years, even though our knowledge of IRI, and much of the relevant literature, questions their stability or efficacy. In addition, several pharmacological molecules have been the subjects of preclinical studies to optimize this protection. Among them, trimetazidine, tacrolimus and carvedilol have shown the most benefits. In fact, these drugs are already in clinical use, and it is a question of repositioning them for this novel use, without additional risk. This new strategy of including them would allow us to shift from cold storage solutions to cold preservation solutions including multitarget pharmacological components, offering protection against IRI and thus protecting today's more vulnerable organs.

Effect of oleuropein on oxidative stress, inflammation and apoptosis induced by ischemia-reperfusion injury in rat kidney.

AIMS: This study aimed to evaluate the effect of oleuropein (OLE), the main phenolic compound present in olive leaves, on kidney ischemia-reperfusion injury (IRI) and to explore the underlying protective mechanism. MAIN METHODS: Rat kidneys were subjected to 60 min of bilateral warm ischemia followed by 120 min of reperfusion. OLE was administered orally 48 h, 24 h and 30 min prior to ischemia at doses of 10, 50 and 100 mg/kg body weight. The creatinine, urea, uric acid concentrations and lactate dehydrogenase (LDH) activity in plasma were evaluated. Oxidative stress and inflammation parameters were also assessed. Renal expression of AMP-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS), mitogen-activated protein kinases (MAPK), inflammatory proteins and apoptotic proteins were evaluated using Western blot. KEY FINDINGS: Our results showed that OLE at 50 mg/kg reduced kidney IRI as revealed by a significant decrease of plasmatic creatinine, urea, uric acid concentrations and LDH activity. In parallel, OLE up-regulated antioxidant capacities. Moreover, OLE diminished the level of CRP and the expression of cyclooxygenase 2 (COX-2). Finally, OLE enhanced AMPK phosphorylation as well as eNOS expression whereas MAPK, and cleaved caspase-3 implicated in cellular apoptosis were attenuated in the ischemic kidneys. SIGNIFICANCE: In conclusion, this study shows that OLE could be used as therapeutic agent to reduce IRI through its anti-oxidative, anti-inflammatory and anti-apoptotic properties.

The effect of zinc acexamate on oxidative stress, inflammation and mitochondria induced apoptosis in rat model of renal warm ischemia.

AIM: Zinc has proved its efficacy in many models of ischemia reperfusion (I/R) injury. In this study, we used zinc acexamate (ZAC) as an exogenous source of zinc against renal I/R injury and we investigated whether its protective effects are mediated by the decrease of oxidative stress, inflammation, and mitochondria induced-apoptosis. METHODS: Rats were orally pretreated with vehicle or ZAC (10 or 100 mg/kg) 24 h and 30 min prior to 1 h of bilateral renal warm ischemia and 2 h of reperfusion. RESULTS: Our data showed that 10 mg/kg of ZAC, but not 100 mg/kg, improved renal architecture and function. Also, the low dose of ZAC up-regulated antioxidant enzymes activities and glutathione level and decreased lipids and proteins oxidation. Interestingly, the use of ZAC resulted in a significant reduce of pro-inflammatory cytokines (IL-1ß, IL-6 and MCP-1), enhanced mitochondria integrity and decreased expression of the pro-apoptotic protein caspase-9. CONCLUSION: We conclude that renal I/R induced oxidative stress, inflammation and apoptosis and that the use of ZAC at 10 mg/kg, but not 100 mg/kg, protects rat kidneys from I/R injury by down-regulating these processes.

Zinc mitigates renal ischemia-reperfusion injury in rats by modulating oxidative stress, endoplasmic reticulum stress, and autophagy.

Oxidative stress is a major factor involved in the pathogenesis of renal ischemia/reperfusion (I/R). Exogenous zinc (Zn) was suggested as a potent antioxidant; however, the mechanism by which it strengthens the organ resistance against the effects of reactive oxygen species (ROS) is not yet investigated. The present study aims to determine whether acute zinc chloride (ZnCl2 ) administration could attenuate endoplasmic reticulum (ER) stress, autophagy, and inflammation after renal I/R. Rats were subjected to either sham operation (Sham group, n = 6), or 1 hr of bilateral ischemia followed by 2 hr of reperfusion (I/R groups, n = 6), or they received ZnCl2 orally 24 hr and 30 min before ischemia (ZnCl2 group, n = 6). Rats were subjected to 1 hr of bilateral renal ischemia followed by 2 hr of reperfusion (I/R group, n = 6). Our results showed that ZnCl2 enhances renal function and reduces cytolysis (p < 0,05). In addition, it increased significantly the activities of antioxidant enzymes (SOD, CAT, and GPX) and the level of GSH in comparison to I/R (p < 0,05). Interestingly, ZnCl2 treatment resulted in significant decreased ER stress, as reflected by GRP78, ATF-6,p-eIF-2α, XPB-1, and CHOP downregulaion. Rats undergoing ZnCl2 treatment demonstrated a low expression of autophagy parameters (Beclin-1 and LAMP-2), which was correlated with low induction of apoptosis (caspase-9, caspase-3, and p-JNK), and reduction of inflammation (IL-1ß, IL-6, and MCP-1) (p < 0,05). In conclusion, we demonstrated the potential effect of Zn supplementation to modulate ER pathway and autophagic process after I/R.

Thymoquinone protects rat liver after partial hepatectomy under ischaemia/reperfusion through oxidative stress and endoplasmic reticulum stress prevention.

Ischaemia reperfusion (I/R) is associated with liver injury and impaired regeneration during partial hepatectomy (PH). The aim of this study was to investigate the effect of thymoquinone (TQ), the active compound of essential oil obtained from Nigella sativa seeds, on rat liver after PH. Male Wistar rats were divided equally into four groups (n = 6) receiving an oral administration of either vehicle solution (sham and PH groups) or TQ at 30 mg/kg (TQ and TQ + PH groups) for 10 consecutive days. Then, rats underwent PH (70%) with 60 minutes of ischaemia followed by 24 hours of reperfusion (PH and TQ + PH groups). Alanine aminotransferase (ALT) activity and histopathological damage were determined. Also, antioxidant parameters, liver regeneration index, hepatic adenosine triphosphate (ATP) content, endoplasmic reticulum (ER) stress and apoptosis were assessed. In response to PH under I/R, liver damage was significantly alleviated by TQ treatment as evidenced by the decrease in ALT activity (P < .01) and histological findings (P < .001). In parallel, TQ preconditioning increased hepatic antioxidant capacities. Moreover, TQ improved mitochondrial function (ATP, P < .05), attenuated ER stress parameters and repressed the expression of apoptotic effectors. Taken together, our results suggest that TQ preconditioning could be an effective strategy to reduce liver injury after PH under I/R. The protective effects were mediated by the increase of antioxidant capacities and the decrease of ER stress and apoptosis.

Angiotensin IV improves subnormothermic machine perfusion preservation of rat liver graft.

This study aims to determine whether Angiotensin IV (Ang IV) addition to Celsior preservation solution could improve hepatic endothelium function and provide better liver protection during subnormothermic machine preservation (SMP). Two experimental models were used: In the first part of the study, rings isolated from rat hepatic artery were preserved in Celsior solution (6 h, 20 °C) with and without Ang IV (10-9 M), then, endothelium-dependent relaxation (EDR) and the concentration of acetylcholine inducing half-maximal relaxation of pre-contracted rings (EC50) were measured. Also, in order to investigate the implication of nitric oxide (NO) on EDR, the rings of hepatic artery were incubated with L-NG-nitroarginine metyl ester (L-NAME). In the second part of the study, rat livers were subjected to SMP with oxygenated Celsior solution (6 h, 20 °C), supplemented or not with Ang IV (10-9 M) and then perfused (2 h, 37 °C) with Krebs Henseleit solution. We found that Ang IV supplementation to Celsior solution decreased EC50 value and improved EDR of hepatic artery rings, 6h after sub-normothermic preservation. Interestingly, Ang IV amplified the vessel relaxation in a NO-dependent manner. Moreover, liver SMP with Ang IV reduced oxidative stress and cell injury and improved organ function. Ang IV activated pAkt, increased eNOS protein level and decreased apoptosis in the preserved liver grafts. In conclusion, we showed that the use of Ang IV in Celsior solution for sub-normothermic graft preservation insured a better NO-dependent relaxation and improved liver functional recovery.

Olprinone protects the liver from ischemia-reperfusion injury through oxidative stress prevention and protein kinase Akt activation.

Liver ischemia-reperfusion (IR) injury is inevitable in surgical procedures such as hepatic resection and liver transplantation. It represents a leading cause of liver graft dysfunction and primary nonfunction after transplantation. Phosphodiesterase (PDE) inhibitors are emerging as effective drugs able to reduce IR damage. The aim of this study was to investigate the effect of selective PDE-3 inhibitor olprinone (Olp) against liver IR injury. Male Wistar rats were subjected to 1 h of partial warm ischemia (70%) followed by 6 h of reperfusion. Before ischemia, rats were treated with saline (IR group), Olp (Olp group), or Olp with Akt inhibitor LY294002 (Olp plus LY group). After reperfusion, hepatic injury (transaminase activities), mitochondrial damage (glutamate dehydrogenase activity), oxidative stress (malondialdehyde and glutathione concentrations and catalase and superoxide dismutase activities), and protein kinase Akt activation were evaluated. Rat treatment with Olp reduced liver injury, prevented mitochondrial damage, decreased lipid peroxidation, and enhanced antioxidant enzymes. Also, Olp induced a significant activation in protein kinase Akt. Inhibition of Akt with LY294002 abolished all of the protective effects of Olp. In conclusion, Olp treatment may be an effective strategy in reducing liver IR injury through oxidative stress prevention and Akt activation.

Thymoquinone prevents endoplasmic reticulum stress and mitochondria-induced apoptosis in a rat model of partial hepatic warm ischemia reperfusion.

This study was undertaken to evaluate the protective effect of thymoquinone (TQ), the bioactive compound of Nigella sativa seeds, against warm ischemia-reperfusion (I/R) injury in liver. Rats were given an oral administration of a vehicle solution (sham group) or TQ at the appropriate dose (10, 20, 30 and 40mg/kg) for ten days consecutively. Following, they were subjected to 60min of partial hepatic ischemia followed by 24h of reperfusion. .Transaminase activities, histopathological changes, TNFα and antioxidant parameters were evaluated. Also, endoplasmic reticulum (ER) stress, mitochondrial damage and apoptosis were studied. In addition, ERK and P38 phosphorylation was determined by Western blot technique. We found that TQ at 30mg/kg is the effective dose to protect rat liver against I/R injury. Moreover, 30mg/kg of TQ prevented histological damages, inflammation and oxidative stress. Interestingly, it decreased the expression of ER stress parameters including GRP78, CHOP and caspase-12. In parallel, it improved mitochondrial function and attenuated the expression of apoptotic parameters. Furthermore, TQ significantly enhanced ERK and P38 phosphorylation. In conclusion, we demonstrated the potential of TQ to protect the rat liver against I/R injury through the prevention of ER stress and mitochondrial dysfunction. These effects implicate the prevention of oxidative stress.

Nitrite enhances liver graft protection against cold ischemia reperfusion injury through a NOS independent pathway.

INTRODUCTION: Nitrite has been found to protect liver graft from cold preservation injury. However, the cell signaling pathway involved in this protection remains unclear. Here, we attempt to clarify if the NOS pathway by using the NOS inhibitor, L-NAME (L-NG-Nitroarginine methyl ester). ANIMALS AND METHODS: Rat livers were conserved for 24 h at 4°C in (IGL-1) solution enriched or not with nitrite at 50 nM. In a third group, rats were pretreated with 50 mg/kg of L-NAME before their liver procurement and preservation in IGL-1 supplemented with nitrite (50 nM) and L-NAME (1 mM). After 24 h of cold storage, rat livers were ex-vivo perfused at 37°C during 2 h. Control livers were perfused without cold storage. RESULTS: Nitrite effectively protected the rat liver grafts from the onset of cold I/R injury. L-NAME treatment did not abolish the beneficial effects of nitrite. Liver damage, protein oxidation and lipid peroxidation remained at low levels in both nitrite-treated groups when compared to IGL-1 group. Antioxidant enzyme activities and functional parameters were unchanged after NOS inhibition. CONCLUSION: Despite NOS inhibition by L-NAME, nitrite can still provide hepatic protection during cold I/R preservation. This suggests that nitrite acts through a NOS-independent pathway.

Effects of Nitrite Addition to IGL-1 Solution on Rat Liver Preservation.

BACKGROUND The ability of nitrite to provide protection following ischemia/reperfusion (I/R) has been demonstrated, but its mechanism is still poorly understood. This study aimed to determine the optimal nitrite concentration to add into Institut Georges Lopez (IGL-1) storage solution and to assess its effect on antioxidant enzymes and autophagy. MATERIAL AND METHODS Livers from Sprague-Dawley rats were conserved in IGL-1 for 24 hours at 4°C or in IGL-1 enriched with nitrite at 50, 500 and 1,000 nM, respectively, before being perfused ex-vivo at 37 °C for 120 minutes. Sham livers were perfused ex vivo without cold preservation. RESULTS All biological and functional parameters of the preserved livers were significantly impaired as compared to shams. Interestingly, the supplementation of nitrite to IGL-1 protected the liver from I/R injury. Among the doses of nitrite evaluated, the 50 nM was proved efficient: it significantly reduced cytolysis, mitochondrial damage, and lipid peroxidation, and enhanced antioxidant enzyme activity (superoxide dismutase, catalase, and glutathione peroxidase activity) and hepatic function parameters (portal resistance, bile flow, and bromosulfophthalein clearance). In addition, increased levels of the autophagy parameters were observed when 50 nM of nitrite were added to IGL-1 solution, but this effect disappeared completely with higher concentrations of nitrite. CONCLUSIONS It seems that 50 nM of nitrite added to IGL-1 is the optimal concentration able to maintain cell integrity and hepatic function through autophagy induction and oxidative stress prevention.

Effectiveness of a single versus repeated administration of trimetazidine in the protection against warm ischemia/reperfusion injury of rat liver.

BACKGROUND/AIM: The aim of this study was to compare the effects of single and repeated trimetazidine (TMZ) administration against warm hepatic ischemia/reperfusion (I/R) injury and to explore the possible mechanisms affected by TMZ. MATERIALS AND METHODS: Wistar rats were divided into 4 groups (n = 6). Sham: rats were subjected to dissection. I/R: rats were subjected to 60 min of partial hepatic ischemia followed by 24 h of reperfusion. TMZ1: Same as I/R group but rats were pretreated with a single dose of TMZ (10 mg/kg, intraperitoneal injection) 30 min before warm ischemia. TMZ3: Same as I/R but rats were treated with 10 mg/kg TMZ for 3 successive days. RESULTS: TMZ treatment decreased liver injury, lipid peroxidation, and apoptosis. The repeated administration of TMZ conferred more protection than the single dose treatment concerning all studied parameters. In parallel, we noted a significant increase in phosphorylated adenosine monophosphate activated protein kinase (p-AMPK) and endothelial nitric oxide synthase (eNOS) levels in TMZ3 as compared to TMZ1. CONCLUSION: Repeated administration of TMZ for 3 days was more efficient than a single dose of TMZ in protecting the liver against I/R induced apoptosis and lipid peroxidation. These effects implicate AMPK and eNOS activation.

Melatonin modulates endoplasmic reticulum stress and Akt/GSK3-beta signaling pathway in a rat model of renal warm ischemia reperfusion.

Melatonin (Mel) is widely used to attenuate ischemia/reperfusion (I/R) injury in several organs. Nevertheless, the underlying mechanisms remain unclear. This study was conducted to explore the effect of Mel on endoplasmic reticulum (ER) stress, Akt and MAPK cascades after renal warm I/R. Eighteen Wistar rats were randomized into three groups: Sham, I/R, and Mel + I/R. The ischemia period was 60 min followed by 120 min of reperfusion. Mel (10 mg/kg) was administrated 30 min prior to ischemia. The creatinine clearance, MDA, LDH levels, and histopathological changes were evaluated. In addition, Western blot was performed to study ER stress and its downstream apoptosis as well as phosphorylation of Akt, GSK-3ß, VDAC, ERK, and P38. Mel decreased cytolysis and lipid peroxidation and improved renal function and morphology compared to I/R group. Parallely, it significantly reduced the ER stress parameters including GRP 78, p-PERK, XBP 1, ATF 6, CHOP, and JNK. Simultaneously, p-Akt level was significantly enhanced and its target molecules GSK-3ß and VDAC were inhibited. Furthermore, the ERK and P38 phosphorylation were evidently augmented after Mel administration in comparison to I/R group. In conclusion, Mel improves the recovery of renal function by decreasing ER stress and stimulating Akt pathway after renal I/R injury.

Sirtuin 1 in rat orthotopic liver transplantation: an IGL-1 preservation solution approach.

AIM: To investigate the possible involvement of Sirtuin 1 (SIRT1) in rat orthotopic liver transplantation (OLT), when Institute Georges Lopez 1 (IGL-1) preservation solution is enriched with trimetazidine (TMZ). METHODS: Male Sprague-Dawley rats were used as donors and recipients. Livers were stored in IGL-1 preservation solution for 8h at 4 °C, and then underwent OLT according to Kamada's cuff technique without arterialization. In another group, livers were stored in IGL-1 preservation solution supplemented with TMZ, at 10(-6) mol/L, for 8 h at 4 °C and then underwent OLT. Rats were sacrificed 24 h after reperfusion, and liver and plasma samples were collected. Liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity) oxidative stress (malondialdehyde levels), and nicotinamide adenine dinucleotide (NAD(+)), the co-factor necessary for SIRT1 activity, were determined by biochemical methods. SIRT1 and its substrates (ac-FoxO1, ac-p53), the precursor of NAD(+), nicotinamide phosphoribosyltransferase (NAMPT), as well as the phosphorylation of adenosine monophosphate activated protein kinase (AMPK), p-mTOR, p-p70S6K (direct substrate of mTOR), autophagy parameters (beclin-1, LC3B) and MAP kinases (p-p38 and p-ERK) were determined by Western blot. RESULTS: Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preserved in IGL-1 solution alone. In addition, livers preserved in IGL-1 + TMZ presented reduced levels of oxidative stress. This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity, as indicated by decreased acetylation of p53 and FoxO1. The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD(+)/NADH levels. Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of mTOR and its direct substrate (p-p70S6K). As a consequence, autophagy mediators (beclin-1 and LC3B) were over-expressed. Furthermore, MAP kinases were regulated in livers preserved with IGL-1 + TMZ, as they were characterized by enhanced p-ERK and decreased p-p38 protein expression. CONCLUSION: Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT, through SIRT1 up-regulation.

Polyethylene glycol rinse solution: an effective way to prevent ischemia-reperfusion injury.

AIM: To test whether a new rinse solution containing polyethylene glycol 35 (PEG-35) could prevent ischemia-reperfusion injury (IRI) in liver grafts. METHODS: Sprague-Dawley rat livers were stored in University of Wisconsin preservation solution and then washed with different rinse solutions (Ringer's lactate solution and a new rinse solution enriched with PEG-35 at either 1 or 5 g/L) before ex vivo perfusion with Krebs-Heinseleit buffer solution. We assessed the following: liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity), liver function (bile output and vascular resistance), oxidative stress (malondialdehyde), nitric oxide, liver autophagy (Beclin-1 and LCB3) and cytoskeleton integrity (filament and globular actin fraction); as well as levels of metalloproteinases (MMP2 and MMP9), adenosine monophosphate-activated protein kinase (AMPK), heat shock protein 70 (HSP70) and heme oxygenase 1 (HO-1). RESULTS: When we used the PEG-35 rinse solution, reduced hepatic injury and improved liver function were noted after reperfusion. The PEG-35 rinse solution prevented oxidative stress, mitochondrial damage, and liver autophagy. Further, it increased the expression of cytoprotective heat shock proteins such as HO-1 and HSP70, activated AMPK, and contributed to the restoration of cytoskeleton integrity after IRI. CONCLUSION: Using the rinse solution containing PEG-35 was effective for decreasing liver graft vulnerability to IRI.

Effects of trimetazidine on the Akt/eNOS signaling pathway and oxidative stress in an in vivo rat model of renal ischemia-reperfusion.

Renal ischemia reperfusion (I/R) injury, which occurs during renal surgery or transplantation, is the major cause of acute renal failure. Trimetazidine (TMZ), an anti-ischemic drug, protects kidney against the deleterious effects of I/R. However its protective mechanism remains unclear. The aim of this study is to examine the relevance of Akt, endothelial nitric oxide synthase (eNOS), and hypoxia inducible factor-1α (HIF-1α) on TMZ induced protection of kidneys against I/R injury. Wistar rats were subjected to 60 min of warm renal ischemia followed by 120 min of reperfusion, or to intraperitoneal injection of TMZ (3 mg/kg) 30 min before ischemia. In sham operated group renal pedicles were only dissected. Compared to I/R, TMZ treatment decreased lactate dehydrogenase (845 ± 13 vs. 1028 ± 30 U/L). In addition, creatinine clearance and sodium reabsorption rates reached 105 ± 12 versus 31 ± 11 µL/min/g kidney weight and 95 ± 1 versus 68 ± 5%, respectively. Besides, we noted a decrease in malondialdehyde concentration (0.33 ± 0.01 vs. 0.59 ± 0.03 nmol/mg of protein) and an increase in glutathione concentration (2.6 ± 0.2 vs. 0.93 ± 0.16 µg GSH/mg of protein), glutathione peroxidase (95 ± 4 vs. 61 ± 3 µg GSH/min/mg of protein), and superoxide dismutase (25 ± 3 vs. 11 ± 2 U/mg of protein) and catalase (91 ± 12 vs. 38 ± 9 µmol/min/mg of protein) activities. Parallely, we noted a significant increase in p-Akt, eNOS, nitrite and nitrate (18 ± 2 vs. 8 ± 0.1 pomL/mg of protein), HIF-1α (333 ± 48 vs. 177 ± 14 µg/mg of protein) and heme oxygenase-1 (HO-1) levels regarding I/R. TMZ treatment improves renal tolerance to warm I/R. Such protection implicates an activation of Akt/eNOS signaling pathway, HIF-1α stabilization and HO-1 activation.