The current study is the first to address the renoprotective effect of AEA against HgCl₂-induced AKI evidenced by the improved early (cystatin C/ΚIM-1/IL-18) and late (creatinine/BUN) kidney injury diagnostic markers. The ability of AEA to suppress the IP3/NFATc1 axis offers one mechanism against the HgCl₂-induced cytotoxicity. Besides, the anti-inflammatory potential of AEA was evidenced by the inhibition of the HMGB-1/RAGE hub and its downstream NF-κB p65, which was associated with a decrease in the caspase-1/IL-18 cue, despite the unpredicted decrease of the transcription factor PPAR-γ that was sharply boosted by HgCl₂. Additionally, AEA pinned down its antioxidant capacity by suppressing lipid peroxidation and augmenting the defense molecule GSH. By hampering the heavy metal-mediated decrease of WNT-5A/BCL-2 and increase in caspase-3, AEA promoted cell survival and reduced apoptosis in the kidney.

Our results showed that HgCl₂ boosted the IP3/NFATc1 axis, whereas treatment with AEA has abated it. HgCl₂-induced AKI is known to be mediated partly by disturbing Ca²⁺ homeostasis to increase its intracellular concentration and hence renal cytotoxicity, an effect that was mediated by ER injury⁵, mitochondrial dysfunction, and activation of PLC signal⁶. The current results offer a new mechanism by stimulating the IP3/NFATc1. Indeed, both IP3 and Hg have been shown to stimulate Ca²⁺ release from endogenous stores by activating the IP3R, which in turn activates the Ca²⁺-dependent phosphatase calcineurin^42,43. The latter dephosphorylates/activates NFATc1 resulting in its nuclear accumulation and transcriptional activity^42,44. Of note, previous studies have tethered activated NFATc1 to kidney injury, leading to proteinuria, glomerulosclerosis, and I/R-induced AKI^45–47, verities that brace the present injurious role of IP3/NFATc1 in the HgCl₂-induced AKI. A previous clinical study also showed that the use of cyclosporine A, an inhibitor of calcineurin, ameliorated idiopathic membranous nephropathy⁴⁸, as well as proteinuria⁴⁹. Thus, the inhibition of IP3/NFATc1 in the AEA-treated group offers one mechanism for its renoprotection, which may be attributed to a decrease in Ca²⁺, a notion that can be clarified by an earlier study⁵⁰ using activated murine bone marrow-derived mast cells. These co-authors provided evidence for the important role of the AEA-induced CB2R/GPR55 heterodimer in limiting the rise of intracellular Ca²⁺ to suppress mast cell degranulation that was not CB1R dependent.

The current model depleted WNT-5A to be markedly increased by the AEA administration. WNT-5A is an upstream protein of multiple signaling pathways including the non-canonical WNT cascade to increase intracellular Ca²⁺ partly by stimulating the IP3/NFATc1 axis to trigger cytotoxicity^8,18. However, as proven herein, its beneficial effect is not related to the IP3/NFATc1 cue but may rely on its anti-inflammatory capacity, a notion that concurs with earlier studies pointing to its anti-inflammatory capacity^19,20,51.

Additionally, activated WNT-5A can play a role in reducing cell demise by regulating cell survival and resistance to apoptosis in different cell lines as previously documented^52,53. The current results showed that the AEA-treated group displayed anti-apoptotic and cell survival potentials evidenced by increasing the anti-apoptotic molecule BCL-2 and inhibiting caspase-3, besides augmenting WNT-5A, which was reported to also mediate the formation of BCL-2⁵⁴. The reduction of cystatin C and KIM-1 is another culprit for AEA-mediated cell survival, where cystatin C has been reported to downregulate the expression of BCL-2⁵⁵ and KIM-1, which was boosted after HgCl₂ injury here and hitherto⁵⁶ was expressed in all apoptotic tubules in a chronic cadmium nephrotoxicity model⁵⁷ facilitating apoptotic epithelial cell phagocytosis⁵⁸.

The HgCl₂-mediates injury is associated with the overproduction of reactive oxygen species (ROS) and the overwhelming of the defense system^3,4. Thus, the antioxidant aptitude of AEA verified by curtailing the increased lipid peroxidation and elevating the renal content of the defense molecule GSH proffers a further mechanism for its ability to protect against HgCl₂-induced AKI. The antioxidant effect of AEA may be partly linked to the suppressed IP3/NFATc1 axis, where the HgCl₂-induced oxidative stress⁵⁹ was especially linked to hydrogen peroxide (H₂O₂), which partakes in the generation of IP3⁶⁰. Moreover, it was reported that the transcription factor NFATc1, among others, participates in the transcriptional regulation of NADPH oxidase, one of the crucial oxidative enzymes⁶¹. Moreover, the restored WNT-5A can play a role in the antioxidant profile of AEA, where Lin et al.⁶² recounted that exogenous administration of the antioxidant enzyme superoxide dismutase increased WNT-5A to protect against in a diabetic nephropathy model.

Scarce data regarding the antioxidant effect of AEA are available; in one study AEA suppressed intracellular ROS and augmented GSH in H₂O₂-induced HT22 neuronal cells⁶³. The latter co-workers owed the antioxidant response of AEA to the activation of CB1R, and recently, the selective CB2R agonists 1-phenylisatin and HU-308 have attenuated the cisplatin-induced renal oxidative stress^25,35. Moreover, in an experimental myocardial infarction, Wang et al.⁶⁴ stated that stimulation of CB2R facilitates the antioxidant machinery to induce cellular antioxidant genes that control GSH synthesis and regeneration⁶⁵. Moreover, Gallelli et al.⁶⁶ provided an overview of the role of CB and non-CB receptors for AEA in mediating its antioxidant effect not only CB2R.

In addition to the corrected redox balance, AEA confirmed its anti-inflammatory potential by inhibiting the inflammatory transcription factor NF-κB to support the results of a previous in vitro model⁶⁷. A high degree of complexity characterizes the interaction between ROS and NF-κB, where, in a vicious cycle, the latter triggers the production of the superoxide anion radical responsible for the activation of the ROS/NF-κB cue¹¹. Accordingly, by its antioxidant/anti-inflammatory properties, AEA interjects the ROS/NF-κB vicious cycle, affording further clarification of its ability to suppress IP3/NFATc1 signaling.

Besides the induction of ROS, NF-κB is responsible for the expression of an array of pro-inflammatory mediators, including the renal injury marker IL-18¹², a verity that is reflected herein and associated by inflammatory cell infiltration as shown in the microphotographs of HgCl₂ exposed kidney. Along with the boosted IL-18, HgCl₂ heightened its activator caspase-1 to suggest a possible role for inflammasome signaling in the HgCl₂-induced AKI, a point that warrants investigation. Treatment with AEA, on the other hand, has sharply reduced both IL-18 and its activator, caspase-1, to match the findings of Li et al.¹⁶, who showed that AEA inhibited the NLRP3 inflammasome and its downstream axis caspase-1/IL-18. The endocannabinoid also reduced the renal inflammatory cell infiltration to corroborate the decline in this inflammatory cytokine.

The HMGB-1/RAGE hub was also bolstered by HgCl₂ to be markedly suppressed by AEA. When HMGB-1, an endogenous danger-associated molecular pattern molecule, is released from the nucleus in a ROS-dependent manner⁶⁸, it reactivates NF-κB upon binding to its receptor RAGE in a hostile intercalating loop⁶⁹. Hence, the aptitude of AEA to oppose the inflammatory effect of HgCl₂ and to inhibit the HMGB-1/RAGE cue, documented herein for the first time, pins down the anti-inflammatory capacity of AEA, besides its antioxidant effect. It is worth mentioning that the current antioxidant and earlier reported anti-TNF-α effects of AEA⁷⁰ may be responsible also for the anti-apoptotic effect of AEA since they can inhibit caspase-3 by both intrinsic and extrinsic death pathways, respectively.

The third transcription factor investigated herein is PPAR-γ, which was unexpectedly upregulated in the HgCl₂ group, a response that might be compensatory since the activation of PPAR-γ is well documented to antagonize NF-κB, ROS, and inflammation⁷¹. This assumption could be braced by earlier findings in spontaneously hypertensive rat aorta and mesenteric resistance arteries⁷². On the other hand, the AEA-associated decrease in PPAR-γ can be explained by the ability of the endocannabinoid to quench ROS and inhibit inflammation, as seen here, which no longer necessitates the upregulation of PPAR-γ.

To this end, we have reported the nephroprotective role of AEA, which amended HgCl₂-induced AKI by multiple intersecting mechanisms. AEA reduced inflammation, oxidative stress, and apoptosis through the activation of the WNT-5A/BCL-2 survival pathway and inhibiting the IP-3/NFATc1, HMGB-1/RAGE/NF-κB, and caspase-1/IL-18 cues.