Protective role of heparin in the injury of the liver and kidney on the experimental model of ischemia/reperfusion

Surgical repair of thoracoabdominal aortic aneurysms (TAAAs) are highly complex and challenging interventions. It is not surprising that this operation is associated with a high incidence of serious complications, often leading to respiratory failure, renal failure, neurological deficits or even death.

Multiorgan dysfunction, presumably related to activation of inflammatory pathways and over production of cytokines and other inflammatory mediators, is a major cause of death. The systemic inflammatory response syndrome (SIRS), ischemia-reperfusion injury (I/Ri), and inflammatory pathway activation are considered precursors of morbidity and mortality after open TAAA repair [1–3].

Cytokines such as IL-6 are important mediators of the inflammatory response in ischemia. Plasma IL-6 concentration increased gradually, reached its peak 24 hours after surgery, and correlated with aortic cross-clamp time and morbidity. The observed correlation between plasma IL-6 and aortic cross-clamp time supports the hypothesis that the IL-6 response mainly reflects the impact of ischemia and reperfusion rather than the impact of surgical trauma in elective TAAA surgery. The strong correlation between plasma IL-6 and morbidity in previous reports suggesting that plasma IL-6 is a reliable predictor of outcome in elective TAAA repair [4–6].

Heat shock proteins (HSPs) are cellular stress proteins which have been shown to have an important role for the survival of cells under stress conditions [7]. Zhang et al. [8] pointed out that, HSP70 could respond to a wide variety of stress conditions such as ischemia, and inflammation. It can prevent the irreversible denaturation of proteins [9]. Elevated expression of HSP70 can prevent cell death processes [10]. It has been showed that overexpression of HSP70 attenuate the release of inflammatory factors and interferes with the process of apoptotic cell death [10–12].

Myeloperoxidase (MPO) is one of the obvious indicators for the tissue infiltration of neutrophilic granulocytes. MPO activity increases in response to the I/R injury [13].

Heparin continues to dominate anticoagulation therapy for surgical repair of TAAA and other cardiovascular surgical procedures. Heparin is best recognized for its ability to prevent blood coagulation by catalytically accelerating the interaction of antithrombin III (AT III) with thrombin, as well as with factors XIIa, IXa, Vlla, and Xa, thereby inhibiting the proteases necessary for completion of the coagulation cascade [14].

Beside heparin is known to possess effects independent of its anticoagulant activity, it plays a role in inhibition of leukocyte-mediated damage [15, 16]. Other important and often unrecognized actions of heparin and other glycosaminoglycans are their ability to inhibit complement activation [17] and to exhibit anti-inflammatory effects [18–20].

In this study, we investigated the effect of the degree of anticoagulation on remote organ I/R injury and whether heparin is protective against I/R injury beside its anticoagulant properties.

There was no significant difference with regards to body temperature, mean arterial blood pressure and heart rate among the groups.

Histopathological evaluation of the tissue samples taken from the kidney showed significant intergroup differences in terms of the cytoplasmic vacuole formation, hemorrhage, tubular cell degeneration and tubular dilatation (P = 0.031, P = 0.003, P = 0.007, P = 0.011 respectively).

As depicted in Table 2, heparinized group of the examination compared with other groups showed no hemorrhage according to histopathologic evaluation. Hemorrhage was worst in the ischemic control group followed by sham and high dose-heparinized group. Hemorrhage in the kidney specimens of high dose-heparinized group may be due to high levels of ACT (Table 1).

Examination of the kidney tissue revealed that ischemic control group showed most tubular cell degeneration according to histopathologic evaluation (P = 0.007); whereas there was no degeneration in the high dose-heparinized group.

There was no tubular dilatation in the kidney specimens of sham and heparinized groups while tubular dilatation was worst in the ischemic control group (P = 0.011).

Despite contemporary approaches to organ protection, open abdominal aortic aneurysm (AAA) repair is still associated with significant morbidity and mortality in high-risk patients [21]. Multiple organ failure (i.e., the lung, kidney) and spinal cord injury are related with activation of inflammatory pathways and over production of other inflammatory mediators during the thoracoabdominal aortic surgery [22, 23]. Thoracoabdominal aortic clamping can lead to systemic inflammatory response and ischemia occurs as the blood flow through the major arteries supplying blood to remote organs slows down or stops [24, 25].

Among various factors that has been related with end organ ischemia/reperfusion injury such as distal aortic hypotension after aortic occlusion, duration of ischemia and aneurysm extent and microthrombus formation are most important and avoidable factors [26]. So microthrombus formation associated with blood flow slowing, stopping and inadequate heparin dose can cause multiorgan disorders together with histopathological and functional changes [27, 28]. Especially, when the hematocrit level is higher than 40 mg/dl microthrombus may easily form. It was suggested that microcirculatory disturbances such as higher blood viscosity characterized by hemoconcentration and microthrombus formation were associated with ischemia [29].

Heparin still remains the leading drug in the management of anticoagulation. Not only heparin has anticoagulant activity but also it has direct anti-inflamatory action. The role of heparin as an anti-inflamatory and anticoagulant agent has a wide spectrum of interactions with various enzymes, hormones, biogenic amines, and plasma proteins. There is great variability in plasma concentration of heparin in relation to dose, so the anticoagulant response is not linear. To provide equilibrium between preventing coagulation and in the mean time preventing excessive bleeding is difficult. This difficulty has increased the importance of the identification of the level of anticoagulation. The most popular and predominant test to monitor anticoagulation is ACT, so we thought to investigate the effect of ACT levels on end organ ischemia in an experimental model.

The presence of patients with postoperative organ dysfunction relationships with response of plasma inflammatory mediators after I/Ri. The overexpression and release of inflammatory mediators, cytokines, activation of phospholipase A2 and complement system, effects of adhesion molecules, activation of arachidonic acid system make I/R injury very complicated and difficult to understand [30–32]. Actually, ischemia and reperfusion is a chain reaction resulting in free oxygen radical generation, respiratory burst of activated neutrophils that occurs in response to tissue injury and the autooxidation of cathecolamines [8]. Severin et al. [33] reported that heparin has anti-inflammatory properties beside its anti-coagulant effects. This characteristic of heparin has been attributed to binding certain cytokines, like chemokines which mediate inflammation through their control of leukocyte migration and activation. We investigated the levels of IL-6, MPO and HSP-70 to evaluate the inflammatory response to I/R injury and the effect of heparin on it.

Acute phase proteins, myeloperoxidase and IL-6 are increase with inflammation [5, 34]. HSP-70, a well known cytoprotective agent increases after I/R and its level is directly correlated with the degree of tissue injury and inflammation. In the present study ,the lowest HSP-70 and MPO level in kidney tissue was measured in the sham group and heparinized groups, showed that the inflammation and injury was significantly lower with this treatment. The decrease in HSP-70 in these groups also was an indirect proof of lower tissue injury and better cellular protection. We could not find a significant difference between the groups in terms of the degree of inflammatory response, degree of IL6 kidney tissue.

Cytoplasmic vacuole formation, hemorrhage, tubular cell degeneration and tubular dilatation in the kidney tissue after I/R were decreased after heparin treatment and the histopathologic protective effect of heparin was shown in the present study.

Comparison of immunohistochemical markers and histopathological evaluation of the ischemia- reperfusion injury of the liver tissue in an aortic occlusion model of rats revealed that there was no statistically significant difference. The reason for this outcome for us that; the afferent blood supply to the liver arises from two sources: (i) the hepatic artery, which carries oxygenated blood and accounts for approximately 25% of hepatic blood flow and 50% of total oxygen, and (ii) the portal vein, which drains the splanchnic circulation and accounts for approximately 75% of hepatic blood flow. Because of its protection by a dual blood supply and the capacity for anaerobic metabolism of glycogen stored in the liver, hypoxic damage can occur later in this organ [35].

The half-life of heparin is approximately 30 minutes, 60 minutes and 150 minutes respectively following IV bolus of 25 U/kg, 100 U/kg and 400 U/kg [36]. Our clinical routine in abdominal aortic surgery is to administer bolus of 100 U/kg heparin and to maintain ACT level around 200 seconds. In the present study there was no significant difference between heparin groups (ACT: 200 and ACT: 600) in terms of histopathologic changes or biochemical inflammatory response in the liver and kidney. Also, keeping ACT level around 200 sec during thoracoabdominal surgery seems both to ensure the adequate level of anticoagulation and to avoid the adverse effects such as bleeding.

Many current analysis clearly evidences that the surgical repair of TAAA remains a challenge even in this century. Recent major progress in our understanding of the pathophysiology and operative strategy have decreased the risk of open TAAA repair complications but obviously further investigation is necessary. Heparin is an important anticoagulation agent in thoracoabdominal and abdominal surgical procedures but no beneficial effects were seen with the use of higher levels of heparin in the present study. Taking into account that, bleeding complications will be much less when heparin used in the real-world clinical practice as ACT levels of 200. Furthermore, the results of this investigation indicate that heparin molecule has provide to potential anti-inflammatory property and renal protective action.