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

Abstract

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.

• Ischemia/reperfusion
• kidney
• oxidative stress
• trimetazidine

Introduction

Renal ischemia reperfusion (I/R) is a common complication of clinical procedures, such as renal transplantation, partial nephrectomy and renal artery operations.1 Renal I/R is a major cause of acute renal failure (ARF), initiating a complex and interrelated sequence of events, resulting in eventual cells death and organ dysfunction.1,2 Increasing evidence has been accumulated over the past decades indicating that production of reactive oxygen species (ROS) play a crucial role in the pathophysiology of renal I/R injury.3,5 The excessive ROS generation induces lipid peroxidation, inactivation of antioxidant enzymes, disruptions of the cellular cytoskeleton and cellular integrity, DNA breakdown and endothelial cell damage.2,4 These factors contribute to cell death and tissue damage.6

Cellular defense against ROS induced by I/R is provided by enzymatic (catalase, superoxide dismutase, and glutathione peroxidase) and non-enzymatic (alpha tocopherol, glutathione (GSH), ascorbic acid, carotene, etc) antioxidant systems.5 Cellular defenses against free radical injury become insufficient during I/R events due to abundant ROS formation. Thus, therapeutic strategies to inhibit oxidative stress have the potential to provide a powerful tool to reduce I/R injury. Several drugs have been showed to protect ischemic organs against I/R damage.7,8 Trimetazidine or (1–2(2,3,4- trimeoxibenzyl)-piperazine) (TMZ), is used as an anti-anginal agent, selectively inhibits long-chain 3-ketoacyl coenzyme A thiolase (the last enzyme involved in β-oxidation) activity.9 Its anti-ischemic effects on the kidney have been experimentally assessed in various models including cell culture10 isolated and perfused kidneys11 and in vivo.12,13 TMZ has different pharmacological properties that could be relevant for the prevention of organ damage from I/R by reduction of intracellular acidosis,14 preservation of ATP production, limitation of inflammatory reaction, and thus of ROS generation.15 However, the exact mechanism of action of TMZ on renal cells is not clear. Several protective pharmacological agents are known to mitigate the I/R-mediated oxidative stress through modulation of numerous signaling components related to cell survival including Akt/eNOS pathway. It was reported that NO plays a crucial role in defending ischemic kidneys against I/R injury.16 Besides, Cau et al.13 reported that HIF-1α accumulation protects the kidney against acute ischemic injury. The present study was undertaken to investigate whether the renoprotective mechanisms of TMZ implicated the Akt/eNOS/HIF-1α signaling pathway to reduce oxidative stress following warm renal I/R injury in rats.

Methods

Experimental model

All animal care and experimental protocols complied with the European Union Regulations (Directive 86/609 EEC) for animal experiments. Adult male Wistar rats (200250g) were housed under standard conditions with a 12 hours light/dark cycle, and were permitted access to water and food ad libitum. Animals were anesthetized by intra peritoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). After maintaining the core body temperature at 37 °C, a midline laparotomy was performed, and both left and right renal arteries and veins were exposed.16 In order to evaluate kidney function, catheters were inserted into (i) the jugular vein to perfuse (Minipuls 3 peristaltic pump, Gilson, France) heated (37 °C) mannitol (10%) and heparin (50 U/I); (ii) the carotid artery to measure arterial pressure (Pression Monitor BP-1; Pression Instruments, Sarasota, FL) and to collect blood samples; (iii) the bladder to collect urine samples. To induce renal ischemia, the renal pedicles were occluded with non traumatic vascular clamps. Reperfusion was initiated by removal of the clamps.

Experimental groups

The rats were randomly divided into 3 groups (n = 6 for each group):

1. I/R group: Both renal pedicles were clamped for 60 min and then subjected to 120 min of reperfusion.16

2. I/R + TMZ group: As I/R group but rats were treated with 3 mg/kg (i.p. injection) of TMZ (Sigma Chemical, St. Louis, MO) 30 min before warm ischemia. TMZ was dissolved in NaCl 9%.

3. Sham group: Animals were subjected to the same surgical procedure described above but were not subjected to renal I/R.

Biochemical determinations

Antioxidant enzymes activities assays

Catalase (CAT) activity was measured as described previously by Claiborne.17 Activity was expressed as μmol H2O2/min/mg of proteins. Superoxide dismutase (SOD) activity was determined by the method of Marklund & Marklund.18 Activity was expressed in international units/mg of proteins. Glutathione peroxidase (GPx) activity was measured according to Flohe & Gunzler.19 Enzyme activity was expressed as µmol of GSH/min/mg of proteins. GSH was measured according to the method of Tietze.20 and was expressed as µg GSH/mg of proteins.

Determination of lipid peroxidation

Lipid peroxidation was determined by measuring the formation of malondialdehyde (MDA) with the thiobarbiturate reaction as previously described.21

Determination of lactate dehydrogenase activity

Cytolysis was assessed in terms of lactate dehydrogenase (LDH) level in plasma, with commercial kits (BioMerieux Vitek, Inc., Durham, NC ).

Renal functional parameters

Renal function was evaluated during the last thirty minutes of reperfusion by calculating the creatinine clearance (μL/min/g kidney weight) using the standard formula:

Creat_p: Creatinine concentration in plasma (μmol/L)

Creat_u: Creatinine concentration in urine (μmol/L)

V: Urine flow (μL/min/g kidney weight)

Serum and urine creatinine concentrations were measured spectrophotometrically at 490 nm by the Jaffé kinetic reaction (BioMerieux Vitek, Inc., Durham, NC).

The formula to calculate the sodium reabsorption rate (%) is:

Na_u: Sodium concentration in urine (mmol/L)

Na_p: Sodium concentration in plasma (mmol/L)

Sodium concentrations in plasma and urine were evaluated by a flame photometer (BT.634, Biotecnica Instruments, Roma, Italy).

Western blotting of p-Akt, eNOS and HO-1

Kidney tissue was homogenized as previously described.16 The supernatants were collected to determine protein concentrations by the Bradford assay. Proteins were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Membranes were immuno-blotted with antibodies against total and phosphorylated Akt (Cell Signaling Technology Inc., Beverly, MA), eNOS, HO-1 and β-actin (Sigma Chemical, St. Louis, MO). The bands were visualized using an enhanced chemiluminescence kit (Bio Rad Laboratories, Hercules, CA). The values were obtained by densitometric scanning and the Quantity One software program (Bio Rad Laboratories, Hercules, CA).

Determination of nitrite and nitrate level

NO production in kidney was determined by tissue accumulation of nitrite and nitrate level as previously reported22 using the Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical Company, Ann Arbor, MI, No.780001).

HIF-1α measurement

Tissue HIF-1α concentration was quantified by using the Trans AM HIF-1α kit (Active Motif, Carlsbad, CA). Results are expressed as μg HIF-1α/mg of protein.

Statistics

Data were expressed as mean values ± SEM (n = 6 for each group). The one-way analysis of variance (ANOVA) test was used for comparison of group data. If the ANOVA was significant, multiple comparisons were carried out using the Newman–Keuls test (Graph Pad Software, Inc., La Jolla, CA, version 4.03, 2005). Statistical significance was defined as p < 0.05.

Results

TMZ treatment reduced oxidative stress and cytolysis

As shown in Figure 1 and 2, renal I/R produced a significant decrease in the CAT (38 ± 9 vs. 81 ± 6 μmol H₂O₂/min/mg of protein, p < 0.05), SOD (11 ± 2 vs. 20 ± 3 U/mg of protein, p < 0.05), and GPx (61 ± 3 vs. 102 ± 5 µmol GSH/min/mg of protein) activities and GSH level (0.93 ± 0.16 vs. 2.19 ± 0.3 µg GSH/mg of protein) versus sham group. Parallely, we noted a significant (p < 0.05) increase in MDA concentration (0.59 ± 0.03 vs. 0.17 ± 0.01 nmol/mg of protein) as compared to sham group (Table 1). TMZ treatment resulted in a significant reduction in MDA concentration (0.33 ± 0.01 nmol/mg of protein) and a rise in GSH level (2.6 ± 0.2 µg GSH/mg of protein), SOD (25 ± 3 U/mg of protein), GPx (95 ± 4 µg GSH/min/mg of protein) and CAT (91 ± 12 μmol/min/mg of protein, p < 0.05) activities compared to I/R group. Interestingly, no statistical differences were obtained between sham and TMZ + I/R groups for all these parameters, except for MDA.

Figure 1. Assessment of catalase (CAT) (a) and superoxide dismutase (b) activities in tissue kidneys. Results are expressed as mean ± SEM for six independent experiments. Note: *p < 0.05 versus Sham group. #p < 0.05 versus I/R group.

Figure 2. Assessment of glutathione peroxidase (GPx) (a) activity and glutathione level (GSH) in tissue kidneys (b). Results are expressed as mean ± SEM for six independent experiments. Note: *p < 0.05 versus Sham group. #p < 0.05 versus I/R group.

Table 1. Evaluation of LDH activity in plasma and tissular MDA concentration in sham, I/R and I/R + TMZ groups.

Parameter	Sham group	I/R group	TMZ + I/R group
MDA (nmol/mg of proteins)	0.17 ± 0.01	0.59 ± 0.03*	0.33 ± 0.01*^#
LDH (U/L)	456 ± 15	1028 ± 30*	845 ± 13*^#

Note: *p < 0.05 versus Sham group, #p < 0.05 versus I/R group, mean ± SEM, n = 6.

TMZ treatment significantly (p < 0.05) improved the integrity of cells as compared to I/R group. Indeed after intraperitoneal injection of TMZ, we found that LDH activity, an index of cytolysis, decreased to 845 ± 13 U/L versus 1028 ± 30 U/L in I/R group (Table 1).

TMZ treatment improved renal function

Renal function was evaluated comparing the variation of creatinine clearance and sodium reabsorption rate among the three groups (Table 2). An important functional recovery was observed subsequently to TMZ administration in comparison to I/R group. We found respectively 105 ± 12 versus 31 ± 11 μL/min/g kidney weight for creatinine clearance (p < 0.05 vs. I/R group) and 95 ± 1% versus 68 ± 5% for the sodium reabsorption rate (p < 0.05 vs. I/R group). Statistical analysis showed a significant difference between sham and TMZ + I/R groups concerning the creatinine clearance (p < 0.05 respectively).

Table 2. Evaluation of Creatinine clearance and sodium reabsorption rate in sham, I/R and I/R + TMZ groups.

Parameter	Sham group	I/R group	TMZ + I/R group
Creatinine clearance (µL/min/g)	484 ± 68	30 ± 12*	105 ± 12*^#
Na^+ reabsorption rate (%)	97 ± 1	68 ± 5*	95 ± 1^#

Note: *p < 0.05 versus Sham group,^#p < 0.05 versus I/R group, mean ± SEM, n = 6.

TMZ increased Akt, eNOS and nitrite and nitrate levels

We evaluated the influence of TMZ on the Akt level. As shown in Figure 3, Western blot analysis revealed that renal I/R markedly increased Akt phosphorylation when compared to sham group (79 ± 2 vs. 41 ± 2, respectively, p < 0.05). This rise was significantly enhanced by TMZ treatment compared to I/R group (153 ± 7, p < 0.05). Furthermore, we assessed the impact of TMZ on the eNOS, one of the main targets of p-Akt (Figure 3). Interestingly, rats undergoing TMZ treatment demonstrated a higher level of eNOS (223 ± 7, p < 0.05) in the kidney compared with its respective sham (30 ± 6) and I/R group (76.5 ± 11). This was concomitant with a significant increase in nitrite and nitrate levels in TMZ + I/R group (18 ± 2 pmol/mg of protein) versus sham (3.7 ± 0.1 pmol/mg of protein) and I/R groups (8 ± 0.1 pmol/mg of protein, p < 0.05; Figure 4).

Figure 3. Representative western blotting of p-Akt. The β-actin was used as a loading control. One representative blot of six independent experiments is shown at the top whereas densitometric analysis is shown at the bottom. Results are expressed as mean ± SEM for six independent experiments. Note: *p < 0.05 versus Sham group. #p < 0.05 versus I/R group.

Figure 4. Representative western blotting of endothelial nitric oxide synthase (eNOS) (a) The β-actin was used as a loading control. One representative blot of six independent experiments is shown at the top whereas densitometric analysis is shown at the bottom. (b) Nitrite/nitrate level. Results are expressed as mean ± SEM for six independent experiments. Note: *p < 0.05 versus Sham group. #p < 0.05 versus I/R group.

TMZ increased HIF 1α and HO-1 levels

Our results showed that TMZ treatment enhanced the stabilization of HIF-1α as judged to I/R and sham groups. We observed 333 ± 48 μg/mg of protein, 177 ± 14 μg/mg of protein and 81 ± 5 μg/mg of protein respectively for TMZ + I/R, I/R and sham groups (p < 0.05, respectively; Figure 5).

Figure 5. Hypoxia-induced factor-1α (HIF-1α) levels. Results are expressed as mean ± SEM for six independent experiments. Note: *p < 0.05 versus Sham group. #p < 0.05 versus I/R group.

As shown in Figure 6, renal I/R resulted in a significant rise (123 ± 16, p < 0.05) in the HO-1 level compared to sham group (55 ± 11). HO-1 level was, significantly (p < 0.05), exacerbated in TMZ treated group (231 ± 16) when compared to I/R group.

Figure 6. Representative western blotting of HO-1. The β-actin was used as a loading control. One representative blot of six independent experiments is shown at the top whereas densitometric analysis is shown at the bottom. Results are expressed as mean ± SEM for six independent experiments. Note: *p < 0.05 versus Sham group. #p < 0.05 versus I/R group.

Discussion

Excessive production of ROS plays a crucial role in producing structural and functional damage of the ischemic kidneys.23 ROS activate a host of pathological processes including DNA damage, protein oxidation and nitrosylation, lipid peroxidation, and induction of apoptosis.24 Indeed, peroxidation of membrane lipids can disrupt membrane fluidity and cell compartmentalization, which can result in cell lysis.4 Numerous studies concerning the renoprotective effects of TMZ have been well documented.12,13,25 It is recognized that TMZ has antioxidant effects and its role in lipid peroxidation inhibition has been well established in different organs.26–28 Hauet et al.11 demonstrated, in isolated perfused pig kidneys, that addition of TMZ to the Euro-Collins (EC) storage solution reduces lipid peroxidation and improves renal tubular function compared to EC solution alone. Sulikowski et al.12 demonstrated that the pretreatment of animals with TMZ markedly attenuated renal dysfunction, morphological alterations and restored the depleted renal antioxidant enzymes. It agrees too with Yoon and his colleagues who reported that pretreatment with TMZ before the onset of ischemia in rat hearts, reduced mitochondrial ROS production.29 Oxidative stress-induced cell injury is not only mediated by increased ROS production, but also by a decreased anti-oxidant defense system. In the present study, oxidative stress was evaluated by detecting tissular MDA and GSH levels and SOD, GPx and CAT activities. Data from our study showed that pretreatment with TMZ reversed the oxidative stress caused by warm renal I/R. We found a significant lower MDA and higher GSH level and SOD, GPx and CAT activities in TMZ + I/R group regarding I/R group, which is in accordance with previous reports showing that TMZ is able to reduce oxidative stress.15,30,31 An increase in the level of antioxidants may make the kidney more resistant to I/R injury.12 The antioxidant properties of TMZ may explain, at least partially, the preservation of cell membranes integrity in TMZ + I/R group, as revealed by the decrease of LDH release. Taken together, these results corroborated the improvement of functional parameters (creatinine clearance and Na⁺ reabsorption rate) in TMZ + I/R group. The antioxidant mechanisms responsible for this renoprotection seem to be more complex. Previous reports demonstrated the activation of Akt/eNOS signaling pathway to mitigate I/R induced oxidative stress. Khan and colleagues32 reported that TMZ is cardioprotective when administered before reperfusion and that this protection appears to be mediated by activation of p38 mitogen-activated protein kinase and Akt signaling pathway. Recently, Wu and coworkers31 demonstrated that TMZ ameliorated H₂O₂-induced impairment of biological functions in endothelial progenitor cells with involvement of antioxidation and Akt/eNOS signaling pathways. In line with previous reports, our results revealed a significant increase in the p-Akt protein levels in TMZ + I/R group.

It was reported that NO plays a crucial role in defending various organs against I/R injury through its antioxidant,28 anti-apoptotic and anti inflammatory properties.33 Napoli and his coworkers34 confirmed that TMZ improves post-ischemic recovery by preserving eNOS expression in isolated working rat hearts. In addition, Jaley and his coworkers.25 showed that the anti-ischemic effects of TMZ seem to be associated with its capacity to generate NO in pig renal I/R injury. In agree with literature, our results revealed an increase of eNOS level following TMZ treatment. Such results corroborated the rise of nitrite and nitrate level in TMZ + I/R group, which was markedly higher than that of I/R group. NO may augment antioxidant protection by decreasing ROS release through inhibition of NADPH oxidase activity.35 According to our findings, elevated NO induced by TMZ might contribute to reducing MDA concentration and increasing GSH level and SOD, CAT and GPx activities following renal I/R.

In order to explore other mechanisms by which TMZ ameliorates kidney tolerance to warm I/R, we assessed the possible involvement of HIF-1α in the renoprotective mechanisms of TMZ. Induction of HIF-1α is an early event in the sequence of cellular changes following the interruption of blood flow and a primary defense mechanism for the adaptive response to ischemia in the kidney.13 Under normoxic conditions, degradation of HIF-1α is mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative alpha-subunits by HIF prolyl hydroxylases (PHD). Several reports have shown that pharmacological up-regulation of HIF-1α represents a novel strategy in the prevention of acute kidney injury.36 Wisel and his coworkers reported that preconditioned rat mesenchymal stem cells with TMZ resulted in a significant protection against H₂O₂ induced loss of cellular viability, membrane damage, and oxygen metabolism. This was accompanied by a significant increase in HIF-1α, pAkt, and Bcl-2 protein levels.37 In the current study, we found that renal I/R up-regulated the level of HIF-1α. Certainly, the content of HIF-1 α in TMZ + I/R group was significantly higher than that in I/R group. Zaouali and his coworkers28 demonstrated that NO could favor the stabilization of HIF-1α in the liver. Guo and his coworkers33 proved that NO can promote binding of HIF-1α to hypoxia response elements in HIF-1α target genes and act as a transcriptional co-activator. In a previous study, we demonstrated that ischemic preconditioning enhanced the stabilization of HIF-1α to protect ischemic kidneys against warm I/R injury. Nevertheless, the treatment with NG-nitro-L-arginine methylester hydrochloride, a non specific NOS inhibitor, abolished this effect and reduced HIF-1α level.16 Based on these findings and our results, it thus seems that TMZ ameliorates kidney tolerance to I/R injury throughout enhancement of NO which induces HIF-1α stabilization.

The next finding of this study shows that TMZ significantly enhanced the expression of HO-1. HO-1 (also known as heat shock protein-32) is the isoform of heme oxygenase that is induced by a variety of cellular stresses, including hypoxia.1 HO-1 catalyzes the rate-limiting oxidation of heme to the biologically active molecules biliverdin and bilirubin, releasing iron and carbon monoxide in the process. These HO-1 enzyme-generated products exhibit free radical scavenging, vasodilating, anti-inflammatory, and anti- apoptotic proprieties.1,38,39 Indeed, it has been demonstrated that HO-1 induction by pharmacologic preconditioning can prevent cold I/R induced injury in renal graft.38 Li Volti and his coworkers39 demonstrated that pharmacologic induction of HO-1 attenuates renal warm I/R injury via inhibiting apoptosis. In the current study, our findings revealed an increase of HO-1 level in TMZ + I/R group as compared to I/R group. Taken together, we can suggest that HO-1 pathway is another mechanism through which TMZ might modulate oxidative stress and so, protect ischemic kidney.

Conclusion

These results suggest that TMZ protects the kidney against warm I/R injury via reducing oxidative stress. These effects are associated, essentially, with an activation of Akt, eNOS, HO-1and stabilization of HIF-1α.

Declaration of interest

The authors declare that they have no conflict of interest. This work was supported by the Tunisian Ministry of Higher Education and Scientific Research.