Ischemia and reperfusion to an organ causes a complex physiological response, which often leads to a pathological condition. In the ischemic state, the restriction of blood flow disturbs the metabolic supply and demand and leads to tissue hypoxia. Though oxygen is restored with blood reperfusion, an increase in reactive oxygen species, inflammatory response, and cell death programs exacerbate the tissue damage [14]. Research on renal IR is abundant; however, the dissonance between animal models and humans is a major pitfall. The extensive renal damage and functional decline, observed immediately in rodent models, are not completely imitated in humans following surgical renal artery occlusion. Furthermore, various biomarker studies and therapeutic interventions in preclinical IR failed to replicate their effectiveness in humans [15]. Moreover, former ‘traditional’ human kidney IR injury prevention drugs were found to be inefficient [16]. Consequently, a thorough understanding of the human pathophysiological response to renal IR and its contribution to AKI is needed.

In this study, we found that kidney injury, in the form of sCr elevation, occurs concomitant with renal clamping in patients undergoing PN. The other circulating metabolites, BUN and serum glucose, were also immediately increased due to renal ischemia, more drastically than was sCr. Although these levels remained elevated after renal reperfusion, they normalized by the first day post operation, probably representing both kidney recovery, as well as compensation by the healthy contra-lateral kidney. In the general population, an AKI incidence is a high-risk factor for CKD development. Although this association is debatable for AKI following PN [17,18], we believe that elucidating the mechanism underlying surgical IR-induced AKI may shed light on the kidney injury and recovery processes.

This study is the first to evaluate the ECS response to renal IR in humans. By self-comparison at multiple time points, the study design offsets the multiple physiological factors known to affect circulating eCBs, and it provides a complete picture of the dynamic eCB changes per individual [19]. The ischemia-induced elevation in AEA levels we observed in non-obese patients provides clinical relevance to ECS involvement reported by numerous preclinical studies. AEA mediates renal hemodynamics by relaxing the vasculature of the renal endothelium [20], dilating afferent and efferent arterioles, and increasing renal blood flow [21]. Furthermore, rodent models of IR-induced injury have displayed decreases in kidney AEA levels [10,12]. Like AEA, the eCB-like compounds, OEA and PEA, were systemically elevated in response to ischemia. These three N-acylethanolamine compounds share common metabolic pathways, suggesting an involvement in their synthetic or degradative enzymes [8]. Indeed, fatty acid amide hydrolase (FAAH), the primary enzyme responsible for N-acylethanolamine degradation, was overexpressed in a mouse model post-renal IR injury [22]. Rodent studies support a protective role of N-acylethanolamine in IR injury. PEA administration attenuated the inflammation, injury, and renal functional decline when administered concurrently with renal ischemia [23]. Moreover, in Chen et al. 2022, increasing renal AEA by FAAH inactivation or transgenic knockout mice was found to mediate renal fibrogenesis observed post-IR injury [22]. Though this study did not measure other N-acylethanolamines, others have demonstrated that a transgenic mouse strain lacking the FAAH enzyme (FAAH^−/−) reveals a significant elevation in the levels of AEA, OEA, and PEA [24]. In light of these pre-clinical studies, our findings may suggest that the overall increase in circulating N-acylethanolamines 10 min following ischemia represents an immediate way for the body to protect itself from the detrimental consequences of artery clamping. However, larger and long-term follow-up studies are needed to determine whether the N-acylethanolamine elevation we observed in human renal ischemia indeed serves as a protective function.

Notably, only the non-obese patients displayed ischemia-induced elevation in N-acylethanolamines, with no response observed in the four patients with BMI > 30. In line with the literature [25], we observed that baseline AEA, OEA, and PEA levels directly correlated with the patients’ BMI. This initial hyper N-acylethanolamine ‘tone’ in the obese patients may be implicated for their lack of response to renal ischemia. Whether this absence of eCB response is connected to the post-operative increases in sCr observed in three out of four of these patients needs further investigation. This research is especially important, since obesity is associated with a higher incidence of RCC [26].

Elucidating the role of N-acylethanolamines in renal ischemia may provide a therapeutic opportunity to mitigate the damage caused by renal IR. Further examination of the functional role of the ECS in this pathological clinical setting, including the expression and localization of the cannabinoid receptors and eCB metabolic enzymes, is needed. ECS homeostasis is altered by the ingestion of cannabis, which contains phytocannabinoids that compete with the canonical receptors [27]. This study supports further investigation of novel ECS-targeted drugs or exploiting phytocannabinoid-induced ECS changes as preventative therapies in various clinical settings that result in renal IR.

Whereas systemic 2-AG levels showed no changes throughout the PN procedure, the levels of this eCB were higher in patients with lower kidney function (significantly correlated with sCr, BUN, and eGFR) and larger tumor masses. The ECS is known to regulate cancer proliferation, migration, and tumorigenesis, with its impact depending on the cancer type and organ [28]; however, its role in RCC is not well established. Early studies found a down-regulation of CB₁R and no expression of CB₂R in clear cell RCC human tissue in comparison with non-cancerous tissue [29]. In contrast, later studies in larger patient cohorts found CB₁R and CB₂R to be overexpressed in the RCC cancer tissue. For both receptors, higher expression levels in the cancer tissue were associated with reduced patient survival [30,31]. In this context, the association we found between tumor size/renal decline and systemic 2-AG, a full agonist of CB₁R and CB₂R, warrants further interest in the ECS mediation of the RCC progression, pointing to the rationale of considering systemic 2-AG as a biomarker for RCC. Moreover, the enhanced levels of systemic 2-AG may in themselves be a source of renal decline, with renal CB₁R overactivity contributing to the pathogenesis of various kidney diseases [9]. Finally, considering the known ECS-modulation of both cancer progression and metabolic disorders, the link between RCC and obesity amidst the observed eCB changes found in this study is worth further investigation.

The limitations of this study include its small population size and the lack of follow-up samples to understand the complete eCB profile changes. Additionally, this study focused on the systemic responses to renal IR, thus providing no data on the eCB changes within the occluded kidney. Since all patients had a functioning contralateral kidney, the question of whether the systemic eCB changes originate in the occluded kidney or they are a function of the compensatory mechanism of the non-affected kidney is unclear.