Reduction of hemorrhagic shock–induced acute kidney injury by lower limb ischemic preconditioning in rats

Introduction: During hemorrhagic shock (HS), the kidneys are one of the primary target organs involved. Oxidative stress is shown to be enhanced in different models of acute kidney injury (AKI). Remote ischemic preconditioning (RPC) by brief limb ischemia is considered to be a safe method to protect different organs from further damage. In this study, we investigated the effects of brief hind limb occlusion on protection against AKI and whether this protection is related to a reduction in oxidative stress. Materials and Methods: Twenty one rats were divided into three groups of seven rats. Shamoperated animals underwent surgical procedures, without hemorrhage. HS was induced by bleeding from a femoral arterial catheter to remove 44% of total blood volume. In RPC group, four cycles of lower limb ischemic preconditioning (5 min ischemia followed by 5 min reperfusion) were performed immediately before HS. Three hours later, plasma and renal tissue samples were collected for renal function monitoring and oxidative stress assessment. Results: Compared with the sham group, HS resulted in renal dysfunction, significantly increased blood urea nitrogen (BUN), plasma creatinine (Cr) and renal malondialdehyde (MDA) levels as well as decreased superoxide dismutase (SOD) activity in the kidneys (P0.05). In the RPC group, renal function was significantly improved. Plasma Cr and BUN and renal MDA levels were significantly lower in RPC group comparing to HS group (P0.05). Renal SOD activity was significantly higher in RPC group compared to HS group (P0.05). Conclusion: These results demonstrate that induction of brief periods of lower limb ischemic preconditioning improves kidney function, restores SOD activity and reduces oxidative stress injury caused by hemorrhagic shock. Reduction of shock–induced kidney injury by preconditioning Physiol Pharmacol 19 (2015) 31-37 | 32 HS, compensatory mechanisms maintain blood flow in favor of vital organs such as brain and heart. These events lead to exacerbated renal damage by diminishing oxygen delivery (Hultstrom, 2013). AKI is a serious complication among hospitalized patients. Despite improvements in the management, incidence of AKI is increasing and in some patients leads to chronic kidney disease and death (Mayeur et al., 2011). Most clinical ischemic AKI occurs as a result of renal hypo perfusion which develops in HS (Onen et al., 2003). In recent years, different animal models have been proposed to simulate and study HS conditions (Mayeur et al., 2011; Ronn et al., 2011). The goal is the development of interventions for restoration of tissue survival during ischemia (Mulier et al., 2012). RPC, non-lethal short term episodes of ischemia – reperfusion (IR) in a remote organ, is thought to trigger pathways that confer protection to a target organ against a subsequent lethal prolonged ischemia (Przyklenk et al., 1993). Using transient ischemic upper and lower extremities (hands and feet), instead of more vital organs, we can create protective effects similar to local conditioning models. The limbs are resistant to ischemic damage and are easily accessible (Schmidt et al., 2007). In 2011, Jan et al. showed that bilateral lower limb IR could alleviate lung injury in hemorrhagic shock/resuscitation rats (Jan et al., 2011). Free radicals are normal metabolic products that are continuously generated during consumption of oxygen in the body. Increases in the generation of free radicals that exceed the capacity of antioxidants, results in oxidative stress (Zheng et al., 2008). In different models of HS in rodents, the production of peroxynitrite (ONOO •), a highly reactive product of nitric oxide (NO) and superoxide anion (O2 •), is shown. Peroxynitrite starts oxidative reactions and cause lipid peroxidation which leads to degradation of the cytoskeleton (Wang et al., 2012). MDA, a product of lipid peroxidation, is a well-known oxidative stress biomarker (Moore and Roberts, 1998). In normal conditions, reactive oxygen species (ROS) are eliminated by antioxidant defense system. Among them superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are very important (Scandalios, 2002). The present study was conducted to evaluate the effects of RPC on the improvement of renal ischemic injury following hemorrhagic shock. We then measured the level of the oxidative stress markers in different groups. Materials and methods Twenty one male Wistar rats, weighing 250–300 g, were randomly selected. Rats were housed under standard conditions (12 h light–dark cycle; 20–22°C) and were allowed ad libitum access to food and water. All procedures were approved by the Animal Ethics Committee of Tehran University of Medical Sciences. Experimental protocols There were three groups (n = 7 in each): (i) Sham group (Anesthesia and surgery without induction of HS) (ii) Hemorrhagic shock group (iii) RPC group (RPC plus HS). Rats were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally and maintained with ketamine (20 mg/kg). A sterile cut down was performed in left groin, and cannula was inserted into the left femoral artery. During these experiments, animals were kept warm using a heat pad. In hemorrhagic shock group, experimental animals were bled to 44% of total blood volume (animal weight [g] × 0.03 + 0.7 ml is equal to 50% of total blood volume) using syringes. Sham animals underwent anesthesia and surgery (groin incision and cannulation) without induction of HS. In RPC group, four cycles of lower limb ischemic preconditioning (IP) were performed by applying rubber band tourniquet high around right thigh for 5 min followed by 5 min reperfusion immediately before HS (Ahmadi-Yazdi et al., 2009; Wu et al., 2010). Renal functional assessments After three hours, rats were euthanized and blood samples were collected. Plasma creatinine (Cr) and blood urea nitrogen (BUN) were measured by colorimetric methods (Hitachi 704 auto-analyzer, 33 | Physiol Pharmacol 19 (2015) 31-37 Ahghari et al.