Cytoprotective Effects And Mechanism Of Nitro-Oleic Acid In An In Vitro Model Of Ischemia/Reperfusion Injury

Background and ObjectiveAcute kidney injury is a frequent complication which is associated with increased mortality and medical expense. Renal ischemia/reperfusion (I/R) injury is the major cause of clinical acute kidney injury (AKI), which is a frequent and serious problem in transplantation, renal surgery, renal diseases and trauma. This clinical setting usually progresses into end stage renal disease and is associated with other organ injury. Till now, there is still no effective therapy available to treat renal I/R injury or ischemic AKI. Thus, new therapeutic approaches are desperately needed.A number of pathologic processes contribute to renal I/R injury, and oxidative stress has been reported to be the primary factor in the initiation of the pathological response. In ischemic kidney and subsequent reperfusion, generation of supra-physiological levels of reactive oxygen species (ROS) impairs antioxidant enzymes and causes cell damage by lipid peroxidation, DNA breakdown and protein damage, which was confirmed by the accumulation of a sensitive marker of oxidative damage,8-oxodG, in swine renal ischemia/reperfusion injury. NADPH oxidase derived superoxide is central to I/R induced oxidative stress. NADPH oxidase is a multi-subunit complex composed of membrane-associated nox homologs, p22phox subunits and cytosolic subunits, including p47phox, p67phox, p40phox and Rac. Among the Nox homologs, NOX4 is the predominant form in the kidney, and NOX2 is also expressed. The expression of NOX4 and NOX2 has been dramatically upregulated in swine renal ischemia/reperfusion injury. Thus targeting recovery of cellular anti-oxidative defense system and inhibition of excessive oxidative species generation would be promising therapeutic strategies.Although renal epithelial cell necrosis has long been regarded as the pathognomonic lesion of ischemic AKI, the amount of necrosis detected in human renal tissue fails to predict renal function, and accumulating evidences indicate that apoptosis correlates far better with the level of renal dysfunction than necrosis. In renal ischemia/reperfusion injury, Bax is activated and translocates into mitochondrial membrane; meanwhile, the expression of Bcl2 is downregulated, which trigger the mitochondrial apoptotic process. Besides, Peroxisome proliferator-activated receptor y (PPAR y) has emerged as a novel therapeutic target in ischemia/reperfusion injury, and the PPAR y agonists have been reported to control cell apoptosis and contribute to tissue protection.Nitrated unsaturated fatty acids (NO2-FA) are byproducts of nitric oxide (·NO) and NO2- dependent oxidative reactions with unsaturated fatty acids, including nitro-oleic acid (OA-NO2) and nitro-linoleic acid (LNO2). They undergo reversible Michael addition with susceptible nucleophilic amino acids such as cysteine and histidine, thus mediate pluripotent cell signaling actions including PPAR y activation, cardiovascular protection, anti-inflammation and anti-oxidation. NO2-FA could reduce cardiac infarct size and enhance cell vitality of cardiomyocytes after ischemia/reperfusion injury. And we previously showed that OA-NO2 attenuated functional and histological indices in a mouse model of renal I/R injury. However, it is unclear whether NO2-FA can alleviate renal I/R injury via directly regulating cell apoptotic signaling.Based on these backgrounds mentioned above, we tested our hypothesis by establishing the combined oxygen and glucose deprivation/re-oxygenation (OGD/R) model, a widely used in vitro model of I/R injury to explore the protective effect and mechanism of OA-NO2 on cell apoptosis and oxidative stress.Methods1. To explore the protective effect and mechanism of OA-NO2 on oxygen and glucose deprivation/re-oxygenation (OGD/R) triggered apoptosis in renal tubular cells, we subjected the HK-2 cells to oxygen and glucose deprivation (OGD) for 16 h, followed by 0 h,1 h,3 h,8 h and 24 h of re-oxygenation. Cell viability was evaluated by CCK-8 assay; cell apoptosis was analyzed by Annexin V/7-AAD staining. The expression of caspase3, cleaved caspase3, cleaved PARP was evaluated by western blotting. Pretreatment with OA-NO2 (45 min,1.25 μM) or OA(45 min,1.25 μM) was conducted when cell apoptosis reached peak level. And then, cell viability and apoptosis were analyzed by CCK-8 assay, Annexin V/7-AAD staining and Hoechst33342 staining. Pro-apoptotic biomarkers, Bax mitochondrial translocation, cytochrome c and apoptosis-inducing factor (AIF) cytosolic leakage and Akt/Gsk 3β phosphorylation were evaluated by western blotting. Bax activation was visualized by immunocytochemistry. GW9662 and PPAR γ siRNA transfection were employed to examine the involvement of PPAR γ.2. To explore the protective effect and mechanism of OA-NO2 on oxygen and glucose deprivation/re-oxygenation (OGD/R) triggered oxidative stress in renal tubular cells, we subjected the HK-2 cells to oxygen and glucose deprivation (OGD) for 16 h, followed by 0 h,1 h,2 h,4 h,8 h,16 h or 24h. Intracellular ROS production was monitored by DCFH-DA staining. Pretreatment with OA-NO2 (45 min,1.25 μM) or OA (45 min,1.25 μM) was conducted when intracellular ROS generation reached peak level. And then, intracellular ROS production was evaluated by DCFH-DA and DHE staining. Mitochondrial potential loss was analyzed by JC-1 staining. The mRNA and protein expression levels of Nrf2, HO-1, GCLM, SOD1, NOX4, NOX2 and p22phox were measured by real time RT-PCR and western blotting. The overall cellular endogenous anti-oxidative capability was analyzed by ABTS assay. The NADPH oxidase-dependent superoxide generation was assessed by evaluating the consumption of NADPH. Nrf2 siRNA transfection were employed to examine the involvement of Nrf2.Results1. Nitro-oleic acid attenuated OGD/R-triggered apoptosis in renal tubular cells via inhibition of Bax mitochondrial translocation in a PPAR γ/Akt/Gsk 3β-dependent manner1.1 Oxygen and glucose deprivation/re-oxygenation (OGD/R) gradually decreased viability and increased apoptosis in HK-2 cellsHK-2 cells were subjected to combined deprivation of oxygen and glucose for 8 h,16 h or 24 h followed by a re-oxygenation period of 24 h to mimic renal ischemia/reperfusion injury in vitro, and the survival rates were 80.27±0.96%,51.62 ± 1.37% and 29±2.69%, respectively indicated by CKK-8 assay. The results of Annexin V/7-AAD staining and western blotting indicated cell apoptosis reached peak level after 16 h of OGD followed by 3 h of re-oxygenation, which was selected as the standard assay condition for studying the OGD/R-induced apoptosis.1.2 OA-NO2 but not oleic acid (OA) improved cell viability after OGD/R injuryThe CCK-8 assay showed that proliferation was significantly inhibited in HK-2 cells treated with OA-NO2 (2.5 or 5 μM), but was not affected at lower concentrations (0.5,0.75 or 1.25 μM). Pretreatment with OA-NO2 (1.25 μM) for 45 min dramatically increased cell viability from 48.88±3.13% to 61.17±4.90%(P<0.05) after 16h-OGD followed by 3h-re-oxygenation. In contrast, the native fatty acid precursor of OA-NO2, oleic acid (OA), did not show a protective effect.1.3 OA-NO2 protected HK-2 cells against OGD/R-induced apoptosisThe results of Annexin V/7-AAD, Hoechst 33342 staning and western blotting indicated that pretreatment with OA-NO2 decreased cell apoptosis rate, restored the nuclei shape and reduced the expression of cleaved caspase3 and cleaved PARP induced by 16h-OGD and 3h-re-oxygenation.1.4 OA-NO2 inhibited OGD/R-induced Bax activation, mitochondrial translocation and subsequent Mitochondrial Outer Membrane PermeabilizationThe results of western blotting indicated that OGD/R injury markedly promoted the translocation of Bax from cytosol to mitochondria and subsequent mitochondrial cytochrome c and AIF leakage, while OA-NO2 pretreatment inhibited these processes. Besides, OGD/R injury increased active Bax immunoreactivity, which was abrogated by OA-NO2 pretreatment.1.5 OA-NO2 restored Akt and Gsk 3(3 phosphorylation in OGD/R injuryThe results of western blotting indicated that OGD/R injury dramatically decreased the phosphorylation of Akt and Gsk 3(3 (P< 0.001), whereas OA-NO2 normalized the Ser473 phosphorylated Akt and Ser9 phosphorylated Gsk 3(3 levels.1.6 GW9662 and PPAR γ siRNA abolished the protective effects of OA-NO2 against OGD/R induced injuryThe PPAR γ siRNA successfully suppressed PPAR γ protein expression by>55%, and induced a slight reduction in cell proliferation, compared to the control siRNA. Neither GW9662 nor PPAR γ siRNA/control siRNA affected the apoptotic rate of cell under normoxia. However, the protective effects of OA-NO2 were completely abolished after pretreatment with GW9662 for 1 h or infection with PPAR γ siRNA before the OGD/R assay, as indicated by declined cell viability and increased apoptosis. Moreover, GW9662 or PPAR γ siRNA largely eliminated the action of OA-NO2 on phosphorylated Akt and Gsk 3β. Meanwhile, similar profiles were observed in apoptotic biomarkers.1.7 GW9662 and PPAR-γ siRNA eliminated the suppression of OA-NO2 on Bax activationThe immunocytochemistry assay indicated that Bax was activated intensively upon OGD/R induction in cells pre-incubated with GW9662 or transfected with PPAR-γ siRNA even in the presence of OA-NO2 after 16h-OGD followed by 3 h-re-oxy genation.2. Nitro-oleic acid ameliorated OGD/R triggered oxidative stress in renal tubular cells via induction of antioxidants and suppression of NADPH oxidase activation2.1 OA-NO2 but not OA attenuated Oxygen and Glucose Deprivation/Re-oxygenation (OGD/R) induced oxidative stressHK-2 cells were subjected to combined deprivation of oxygen and glucose for 16 h followed by a re-oxygenation period of 0 h,1 h,2 h,4 h,8 h,16 h or 24 h. The intracellular ROS production was monitored by DCFH-DA staining. The levels of ROS elevated remarkably after OGD injury, and reached peak level by 4 h of re-oxygenation. Thus we chose 16 h of OGD without re-oxygenation (H16R0) and 16 h of OGD followed by 4 h re-oxygenation (H16R4) to mimic renal ischemia and ischemia/reperfusion injury in vitro, separately.The results of DCFH-DA, DHE and JC-1 staining indicated that pretreatment with OA-NO2 reduced the fluorescence intensity of DCF/oxidized DHE, and restore the decrease of Δψm after OGD/R injury, while pretreatment with OA had no protective effects.2.2 OA-NO2 upregulated the expressions of anti-oxidative enzymesOA-NO2 upregulated the mRNA and protein expression levels of HO-1 and GCLM in normoxia and after OGD or OGD/R injury, while the expression of SOD1 was only upregulated after OGD or OGD/R injury. The results of ABTS assay indicated that OA-NO2 improved the cellular total antioxidant capacity under normal culture condition and prevent the reduction of Trolox Equivalent Antioxidant Capacity (TEAC) induced by OGD and OGD/R injury.2.3 OA-NO2 increased Nrf2 expression and nuclear accumulationPretreatment with OA-NO2 increased Nrf2 mRNA expression to 1.44±0.04 fold compared to vehicle under normal culture condition, but had no further induction after OGD and OGD/R injury. The protein expression and nuclear translocation of Nrf2 were both increased dramatically by OA-NO2 under normoxia and after OGD/R injury.2.4 Nrf2 siRNA partially eliminated OA-NO2 mediated antioxidants induction and anti-oxidative effectsThe Nrf2 siRNA successfully suppressed Nrf2 mRNA and protein expression levels by 70% and 59%, separately. The OA-NO2 mediated upregulation of mRNA and protein expression levels of HO-1 and GCLM was completely abolished by Nrf2 siRNA. The anti-oxidative effects of OA-NO2 were partially abrogated after transfection with Nrf2 siRNA before the OGD/R assay, indicated by elevated relative DCF fluorescence intensity and decreased Trolox Equivalent Antioxidant Capacity (TEAC). Furthermore, the prevention of mitochondrial membrane potential loss by OA-NO2 pretreatment was also eliminated by Nrf2 siRNA.2.5 OA-NO2 inhibited NADPH oxidase activation in OGD/R injuryNADPH oxidase-dependent superoxide generation, assessed by evaluating the consumption of NADPH, was significantly higher after OGD and OGD/R injury, and pretreatment with OA-NO2 significantly attenuated the elevated NADPH oxidase activity. Moreover, OA-NO2 reduced mRNA and protein expression levels of NOX4, NOX2 and p22 phox after OGD/R injury.2.6 Nrf2 siRNA did not abolish the inhibition of OA-NO2 on NADPH oxidase activation in OGD/R injuryNeither control nor Nrf2 siRNA had effect on the potential of OA-NO2 to inhibit NOX4, NOX2 and p22 phox mRNA expressions after OGD/R injury.Conclusion1. OA-NO2 reduced cell apoptosis after OGD/R injury in HK-2 cells.2. OA-NO2 alleviated intracellular oxidative stress after OGD/R injury in HK-2 cells.3. OA-NO2 attenuated OGD/R-induced apoptosis by inhibiting Bax translocation and activation and the subsequent mitochondria-dependent apoptotic cascade in a PPAR y/Akt/Gsk 3β dependent manner.4. OA-NO2 exerted its anti-oxidative effects via upregulation of Nrf2 dependent HO-1, GCLM and independent SOD1 and suppression of NADPH oxidase activity after OGD/R injury.