Abstract
Background: The endocannabinoid (EC) system is well characterized in the central nervous system but scarcely studied in peripheral organs. In this paper, we newly identify the effect of the EC anandamide (AEA) upon renal proximal tubule cells. Methods: Measurement of lactate dehydrogenase (LDH) release after treatment of primary renal proximal tubule cells (RPTEC) and renal carcinoma cell line (Caki-1) with AEA, arachidonic acid (AA), ethanolamide (EtAm), EC receptor CB1 antagonist (AM251), CB2 receptor antagonist (SR144528), TRPV1 receptor antagonist (capsazepine), degradation enzyme fatty acid amide hydrolase (FAAH) antagonist (URB597), antioxidants GSH-EE; Trolox, GSH depletor BSO, membrane cholesterol depletor (MCD), apoptosis inhibitor zVAD, necroptosis inhibitor Nec-1 or ferroptosis inhibitor Fer-1. Western blot and qRT-PCR analysis plus determination of reactive oxygen species (ROS) via H2-DCFDA were performed. Histology for EC enzymes, N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) and FAAH, as well as the determination of physiological levels of ECs in human and rat renal tissue via liquid chromatography were conducted. Results: AEA both dose- and time-dependently induces cell death in RPTEC and Caki-1 within hours, characterized by cell blebbing, not influenced by blocking the described EC receptors by AM251, SR144528, capsazepine or FAAH by URB597 or MCD. Cell death is mediated via ROS. There is no difference found in the histology of the enzymes FAAH and NAPE-PLD in human renal tissue with interstitial nephritis. Blocking of apoptotic, necroptotic or ferroptotic cell death does not lead to a reduction in LDH release in vitro. Conclusion: The endocannabinoid anandamide induces cell death in renal proximal tubule cell in a time- and dose-dependent manner. This pathway is mediated via ROS and is independent of cannabinoid receptors, membrane cholesterol or FAAH activity.
Highlights
Endocannabinoids (ECs) are endogenous lipids that have been gaining more interest during the last years as the exogenous equivalent is known as Δ9-THC (Δ9-tetrahydrocannabinol), the active psychotropic natural product of Cannabis [1].The EC system is extensively studied in immune system function and the central nervous system, as it is involved in memory processing, regulation of pain, neuroprotection, addiction and appetite [2] [3] [4]
Cells were pretreated with AM251 (CB1 receptor antagonist, 1 μM) from Tocris (Wiesbaden, Germany); SR144528 (CB2 receptor antagonist, 1 μM), Capsazepine (TRPV1 receptor antagonist, 25 μM), Trolox, Necrostatin1 (Nec-1, necroptosis inhibitor, 1 μM), Ferrostatin1 (Fer-1, ferroptosis inhibitor, 60 nM), zVAD, arachidonic acid (25 μM), ethanolamine (25 μM), URB597 (FAAH inhibitor, 10 μM), all from Cayman Chemicals; glutathione reduced ethyl ester, buthionine sulfoximine (GSH depletor, 100 μM), methyl-β-cyclodextrin, ActD (Actinomycin D, 0.4 μg/ml), Tnf alpha (Tumor necrosis factor alpha, 40 ng/ml), TritonX (0.1%); and H2O2 (1 mM), all from Sigma-Aldrich (Deisenhofen, Germany)
To further support the observation of an expression of EC signaling by liquid chromatography, the effect of a>]. The EC anandamide (AEA) within the renal cell lines Caki-1 and renal proximal tubule cells (RPTEC) was considered
Summary
Endocannabinoids (ECs) are endogenous lipids that have been gaining more interest during the last years as the exogenous equivalent is known as Δ9-THC (Δ9-tetrahydrocannabinol), the active psychotropic natural product of Cannabis [1].The EC system is extensively studied in immune system function and the central nervous system, as it is involved in memory processing, regulation of pain, neuroprotection, addiction and appetite [2] [3] [4]. We considered renal disease models resulting in cell loss, like nephritis or ischemic damage during kidney transplantation as well as a renal cell carcinoma (RCC). Understanding those mechanisms resulting in cell loss and the possible involvement of the EC system is an important step towards understanding disease entities. Methods: Measurement of lactate dehydrogenase (LDH) release after treatment of primary renal proximal tubule cells (RPTEC) and renal carcinoma cell line (Caki-1) with AEA, arachidonic acid (AA), ethanolamide (EtAm), EC receptor CB1 antagonist (AM251), CB2 receptor antagonist (SR144528), TRPV1 receptor antagonist (capsazepine), degradation enzyme fatty acid amide hydrolase (FAAH) antagonist (URB597), antioxidants GSH-EE; Trolox, GSH depletor BSO, membrane cholesterol depletor (MCD), apoptosis inhibitor zVAD, necroptosis inhibitor Nec-1 or ferroptosis inhibitor Fer-1. Results: AEA both dose- and time-dependently induces cell death in RPTEC and Caki-1 within hours, characterized by cell blebbing, not influenced by blocking the described EC receptors by AM251, SR144528, capsaze-
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