While multiple complex processes are involved in the pathogenesis of ischemic AKI, tubular cell injury, increased oxidant stress, and inflammation are common denominators.IR-induced renal tubular injury results in increased expression of adhesion molecules, such as vascular and intercellular adhesion molecule1 , and selectins, such as Pselectin and E-selectin.This is followed by the attachment, activation, and transmigration of immune cells into renal tissue, which results in inflammation. Subsequent production of reactive oxygen species and disruption of the nitric oxide pathways lead to further tubular damage, inflammation, and oxidative stress.7 These abnormalities trigger a pathologic cascade of events that ultimately lead to propagation of renal injury and manifest clinically as kidney failure. The endocannabinoids are endogenous, bio-active lipid mediators that exert their effects mainly through specific G protein-coupled receptors: type-1 cannabinoid receptor and type-2 cannabinoid receptor . The most extensively studied ECs are arachidonoyl ethanolamide and 2-arachidonoylglycerol . They are synthesized on demand through distinct cellular pathways and are released in the local micro-environment, leading to autocrine or paracrine downstream effects. Given the abundance of CB1 receptors found in the central nervous system and CB2 receptors on immune cells, the ECs were initially thought to be active only in these systems. However, CB1 and CB2 receptors have been discovered in a multitude of peripheral tissue, including the kidneys.Although not fully understood,heavy duty propagation trays the activation of CB1 receptors in the periphery has been shown to be associated with increased oxidative stress and inflammation, whereas activation of CB2 receptor has been known to have the opposite effect.Furthermore, the ECs and their metabolites can also exert hemodynamic and other effects through CB receptor dependent and -independent pathways.
Given the major role that inflammation and oxidative stress play in IRI and the known involvement of the EC system in these pathways, there has been extensive evaluation of the EC system in pathophysiology of IRI of several organ systems, including the brain, heart, and the liver. Several reports indicate that both the blockade of CB1 receptors and the activation of CB2 receptors protect against IRI in the tissues mentioned.Interestingly, these findings have also been confirmed in nephrotoxic AKI using a murine cisplatin renal tubular injury model.In addition, there are now numerous reports that highlight the involvement of the EC system in renal injury and fibrosis in a variety of settings, including diabetic nephropathy.Despite the preponderance of evidence implicating the EC system and ECs in nonrenal IRI, data on the role of ECs in renal IRI remain sparse.In addition, most of the studies examining the role of the EC system in renal injury focus on the effects of the activation or inhibition of the CB receptors and do not provide data on the tissue level of the endogenous ligands for these receptors, 2-AG and AEA. In this study, we show for the first time that renal IRI leads to a significant increase in renal level of 2-AG, one of the major activators of the EC system. Furthermore, enhancement of renal 2-AG levels using pharmacologic tools improved indices of renal function without changing markers of inflammation or oxidative stress. These results indicate that renal ECs are involved in the pathogenesis of IR-induced AKI, and how modulation of the EC system may impact renal injury and function will need to be studied in further detail.All animals were handled and procedures were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the University of California, Irvine Institutional Animal Care and Use Committee. Male C57BL/6 mice 8–12 weeks old were obtained from Jackson Laboratories . They were housed in the UC Irvine facility under specific pathogen-free conditions, were allowed free access to standard chow and water, and were kept in a 12-h light:12-h dark cycle. In the first set of experiments, 20 mice were divided into two groups: one was sham operated and the other underwent 30 min of bilateral ischemia . For the second set of experiments, 20 animals were divided into two groups: one received the monoacylglycerol lipase inhibitor JZL184 and the other received the correspondent vehicle 2 h before I/R .We used an established mouse model of ‘‘warm’’ renal I/R injury.27 Briefly, mice were anesthetized with ketamine/xylazine and kept on a homoeothermic station to maintain body temperature at 37 C. A warming blanket was used throughout the procedure and for 30 min post procedure . The body temperature of each animal was monitored closely throughout the procedure and afterward using a noninvasive infrared digital temperature device. A midline incision was made and bilateral renal pedicles were exposed.
Using atraumatic Micro Serrefne straight clamps , both renal pedicles were cross-clamped. To maintain fluid balance, mice received 0.7 mL of sterile 0.9% saline by intraperitoneal injection. After 30 min of warm ischemia, clamps were removed initiating renal reperfusion. Sham control animals were subjected to identical operation without clamping. Mice were sacri- fificed at 24 h after reperfusion for serum/kidney sampling under terminal general anesthesia using isoflurane. Blood was taken by intracardiac puncture after accessing the chest cavity from underneath the diaphragm. Briefly, before the opening of the chest for blood collection through cardiac puncture, anesthesia was induced in a chamber with 4–5% isoflurane in 100% O2 and then maintained by continuous administration of 1– 2% isoflurane through nose cone. We made certain that all animals were fully anesthetized prior and during surgery. This method is approved by the University of California Irvine Institutional Animal Care and Use Committee and consistent with the AVMA Guidelines for Euthanasia. Approximately 100 lL of serum was isolated and stored at 80 C. The kidneys were harvested after euthanasia/cardiac puncture. Both kidneys were cut in half along the renal pelvis. One and half kidney was immediately snap-frozen in liquid nitrogen while half of the kidney was fixed immediately. To avoid inter tissue variability and avoid comparing different regions of the kidney, we fixed the same half of the kidney for all animals and compared the same region of the fixed kidney for histopathology evaluation.This is the first study that examines the effect of kidney IRI on renal EC levels and their potential impact on pathophysiology of AKI. We have found that kidney IRI is associated with a significant increase in renal2-AG content. Kidney 2-AG levels correlated positively and significantly with serum BUN and creatinine. Furthermore, enhancement of renal 2-AG levels using the selective MGL inhibitor, JZL184, caused a modest but significant improvement in renal function. Interestingly, the latter findings were not associated with improvement in renal markers of inflammation and oxidative stress, indicating that the improvement in renal function induced by 2-AG may be independent of proinflammatory and oxidative processes.
The findings of our study are unique in several respects. First, while the role of the EC system in AKI has been examined through modulation of CB receptors in animal models of nephrotoxic tubular injury, this is the first investigation examining EC levels and the role of the EC system in ischemic AKI and renal IRI.Therefore, the current findings shed light on how the EC system as a whole may be involved in the changes observed in renal IRI. Furthermore, our study demonstrates that renal IRI is associated with increased kidney 2-AG content. The latter findings are significant because understanding how ECs are mobilized in renal IRI is crucial to their potential exploitation as a therapeutic strategy. It is also intriguing to note that increased tissue 2-AG content has also been reported in IRI in other organ systems such as the liver,vertical cannabis and, just as in the kidney, enhancement of tissue 2-AG levels was associated with improvement of IRI.Therefore, it is noteworthy that the findings described in this study are not exclusive to the kidney, thus reflecting a potential physiopathological role of 2-AG and the EC system in IRI, which needs to be further explored. While increased 2-AG levels have mostly been reported to be associated with reduced tissue injury in IR, the mechanisms through which 2-AG affords protection in IRI remain unclear. Several possibilities have been examined, one of them being a potential anti-inflammatory action of 2-AG mediated by CB2 receptors. However, available data on the impact of 2-AG on inflammation are contradictory, with some studies reporting anti-inflammatory properties, while others noting proinflammatory effects.In our study, we did not find significant changes in the expression of proinflammatory cytokines or adhesion molecules following pharmacological enhancement of renal 2-AG levels. In fact, we observed a trend toward an increased expression of these markers. Indeed, there are recent studies that link increased 2-AG levels with worsening of inflammation.It is possible that increased tissue levels of 2-AG may lead to activation of CB1 receptor, hence causing a trend toward worsening inflammation; however, this mechanism has not been established in renal IRI. Given that indices of renal inflammation and oxidative stress remain unchanged, our findings support the notion that the modest improvement in renal function observed with enhanced 2-AG levels may be related to effects independent of its role in in- flammation and oxidative stress. In this regard, there are several studies indicating that 2-AG has vasodilatory properties through CB1-dependent and -independent mechanisms.For instance, Awumey et al. have demonstrated that 2-AG can cause relaxation of arterial smooth muscle through its metabolite glycerated epoxyeicosatrienoic acid, which can activate potassiumgated calcium channels on vascular smooth muscle cells resulting in hyperpolarization of these cells and vascular relaxation.One possibility is that increased kidney 2- AG levels in renal IRI could cause arterial vasodilatation, which would lead to improved renal perfusion and enhanced glomerular filtration rate, thereby explaining the improvement observed in renal function.
It should also be noted that since we administered JZL184 systemically and most likely increased 2-AG levels in other parts of the body, it is possible that the renoprotective effect observed in our study may be emanating from outside of the kidneys . These possibilities will need to be examined in future studies.First, there is emerging evidence that dysregulation of this system may be involved in diabetic nephropathy, proteinuria, renal fibrosis, and AKI.Second, pharmacological agents are available, which can modulate EC levels and CB receptor activity, thereby providing potential therapeutic strategies. Finally, considering recent reports pointing at synthetic cannabinoid use as a cause of AKI,investigation of the EC system in renal disease may shed light on the mechanism by which these recreational drugs can potentially cause renal injury and help formulate preventive plans in risk population. Several limitations of our study need to be mentioned. The potential role of 2-AG as a mediator of vasodilation and its impact on renal blood flow rate will need to be thoroughly explored in future studies. Furthermore, in our mouse model of AKI, we had a limited supply of plasma, and therefore, systemic levels of ECs in IR AKI remain to be determined. Moreover, given that JZL184 was administered systemically, we cannot rule out inhibition of MGL in other organs that could have had a downstream effect on the kidneys. In addition, despite its specificity for MGL, it is possible that JZL184 may have an impact on other serine hydrolase enzymes that may explain some of the results we are observing in our studies. Furthermore, our evaluation of renal markers of inflammation and oxidative stress pathways was limited to mRNA analysis, and therefore, renal abundance of each protein will need to be determined to complement the mRNA findings. Finally, our study does not address the mechanism/s responsible for increased renal 2-AG levels. We have recently reported that oxidative stress can cause the reversible sulfenylation of MGL and inhibition of its activity, hence leading to decreased 2-AG breakdown and increased 2-AG levels.42 Given that kidney injury is associated with significant oxidative stress, it is possible that MGL inhibition is the mechanism responsible for increased 2-AG levels in renal IRI, however, this possibility will need to be confirmed in future studies. In conclusion, renal IRI is associated with a significant increase in kidney 2-AG content. Further enhancement of renal 2-AG levels using a pharmacologic tool, which inhibits its breakdown, improves indices of renal function and kidney injury, without affecting expression of markers of inflammation and oxidative stress.