CellBio  Vol.6 No.4 , December 2017
The Endocannabinoid, Anandamide, Induces Cannabinoid Receptor-Independent Cell Death in Renal Proximal Tubule Cells
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.
Cite this paper: Schlosser, M. , Löser, H. , Siegmund, S. , Montesinos-Rongen, M. , Bindila, L. , Lutz, B. , Barrett, D. , Sarmad, S. , Ortori, C. , Grau, V. , von Brandenstein, M. and Fries, J. (2017) The Endocannabinoid, Anandamide, Induces Cannabinoid Receptor-Independent Cell Death in Renal Proximal Tubule Cells. CellBio, 6, 35-55. doi: 10.4236/cellbio.2017.64004.

[1]   Chen, R., et al. (2013) Delta9-THC-Caused Synaptic and Memory Impairments Are Mediated through COX-2 Signaling. Cell, 155, 1154-1165.

[2]   Marsicano, G. and Lutz, B. (2006) Neuromodulatory Functions of the Endocannabinoid System. Journal of Endocrinological Investigation, 29, 27-46.

[3]   Freund, T.F., Katona, I. and Piomelli, D. (2003) Role of Endogenous Cannabinoids in Synaptic Signaling. Physiological Reviews, 83, 1017-1066.

[4]   Pacher, P., Batkai, S. and Kunos, G. (2006) The Endocannabinoid System as an Emerging Target of Pharmacotherapy. Pharmacological Reviews, 58, 389-462.

[5]   Siegmund, S.V., et al. (2016) Cyclooxygenase-2 Contributes to the Selective Induction of Cell Death by the Endocannabinoid 2-Arachidonoyl Glycerol in Hepatic Stellate Cells. Biochemical and Biophysical Research Communications, 470, 678-684.

[6]   Siegmund, S.V. (2010) Role of the Endocannabinoid System in Alcoholic Liver Disease. Digestive Diseases, 28, 751-755.

[7]   Siegmund, S.V. and Brenner, D.A. (2005) Molecular Pathogenesis of Alcohol-Induced Hepatic Fibrosis. Alcoholism: Clinical and Experimental Research, 29, 102S-109S.

[8]   Wojtalla, A., et al. (2012) The Endocannabinoid N-Arachidonoyl Dopamine (NADA) Selectively Induces Oxidative Stress-Mediated Cell Death in Hepatic Stellate Cells But Not in Hepatocytes. American Journal of Physiology. Gastrointestinal and Liver Physiology, 302, G873-G887.

[9]   Biswas, K.K., et al. (2003) Membrane Cholesterol But Not Putative Receptors Mediates Anandamide-Induced Hepatocyte Apoptosis. Hepatology, 38, 1167-1177.

[10]   Sampaio, L.S., et al. (2015) The Endocannabinoid System in Renal Cells: Regulation of Na(+) Transport by CB1 Receptors through Distinct Cell Signalling Pathways. British Journal of Pharmacology, 172, 4615-4625.

[11]   Tam, J. (2016) The Emerging Role of the Endocannabinoid System in the Pathogenesis and Treatment of Kidney Diseases. Journal of Basic and Clinical Physiology and Pharmacology, 27, 267-276.

[12]   Ritter, J.K., Li, G., Xia, M. and Boini, K. (2016) Anandamide and Its Metabolites: What Are Their Roles in the Kidney? Frontiers in Bioscience, 8, 264-277.

[13]   Vanden Berghe, T., Linkermann, A., Jouan-Lanhouet, S., Walczak, H. and Vandenabeele, P. (2014) Regulated Necrosis: The Expanding Network of Non-Apoptotic Cell Death Pathways. Nature Reviews Molecular Cell Biology, 15, 135-147.

[14]   Sanz, A.B., Santamaria, B., Ruiz-Ortega, M., Egido, J. and Ortiz, A. (2008) Mechanisms of Renal Apoptosis in Health and Disease. Journal of the American Society of Nephrology, 19, 1634-1642.

[15]   Martin-Sanchez, D., et al. (2017) Ferroptosis, But Not Necroptosis, Is Important in Nephrotoxic Folic Acid-Induced AKI. Journal of the American Society of Nephrology, 28, 218-229.

[16]   Linkermann, A., et al. (2012) Rip1 (Receptor-Interacting Protein Kinase 1) Mediates Necroptosis and Contributes to Renal Ischemia/Reperfusion Injury. Kidney International, 81, 751-761.

[17]   Linkermann, A., et al. (2014) Regulated Cell Death in AKI. Journal of the American Society of Nephrology, 25, 2689-2701.

[18]   Degterev, A., et al. (2008) Identification of RIP1 Kinase as a Specific Cellular Target of Necrostatins. Nature Chemical Biology, 4, 313-321.

[19]   Vanden Berghe, T., et al. (2010) Necroptosis, Necrosis and Secondary Necrosis Converge on Similar Cellular Disintegration Features. Cell Death & Differentiation, 17, 922-930.

[20]   Xie, Y., et al. (2016) Ferroptosis: Process and Function. Cell Death & Differentiation, 23, 369-379.

[21]   Siegmund, S.V., Uchinami, H., Osawa, Y., Brenner, D.A. and Schwabe, R.F. (2005) Anandamide Induces Necrosis in Primary Hepatic Stellate Cells. Hepatology, 41, 1085-1095.

[22]   Linkermann, A., et al. (2014) Synchronized Renal Tubular Cell Death Involves Ferroptosis. Proceedings of the National Academy of Sciences, 111, 16836-16841.

[23]   Grau, V., Herbst, B. and Steiniger, B. (1998) Dynamics of Monocytes/Macrophages and T Lymphocytes in Acutely Rejecting Rat Renal Allografts. Cell and Tissue Research, 291, 117-126.

[24]   Von Brandenstein, M., et al. (2012) MicroRNA 15a, Inversely Correlated to PKCalpha, Is a Potential Marker to Differentiate between Benign and Malignant Renal Tumors in Biopsy and Urine Samples. American Journal of Pathology, 180, 1787-1797.

[25]   Bindila, L. and Lutz, B. (2016) Extraction and Simultaneous Quantification of Endocannabinoids and Endocannabinoid-Like Lipids in Biological Tissues. Methods in Molecular Biology, 1412, 9-18.

[26]   Siegmund, S.V., et al. (2007) The Endocannabinoid 2-Arachidonoyl Glycerol Induces Death of Hepatic Stellate Cells via Mitochondrial Reactive Oxygen Species. FASEB J, 21, 2798-2806.

[27]   Von Brandenstein, M., et al. (2015) Vimentin 3, the New Hope, Differentiating RCC versus Oncocytoma. Disease Markers, 2015, Article ID: 368534.

[28]   Von Brandenstein, M.G., et al. (2008) A p38-p65 Transcription Complex Induced by Endothelin-1 Mediates Signal Transduction in Cancer Cells. Biochimica et Biophysica Acta, 1783, 1613-1622.

[29]   Gerstung, M., Roth, T., Dienes, H.P., Licht, C. and Fries, J.W. (2007) Endothelin-1 Induces NF-kappaB via Two Independent Pathways in Human Renal Tubular Epithelial Cells. American Journal of Nephrology, 27, 294-300.

[30]   Kim, J., Carlson, M.E. and Watkins, B.A. (2014) Docosahexaenoyl Ethanolamide Improves Glucose Uptake and Alters Endocannabinoid System Gene Expression in Proliferating and Differentiating C2C12 Myoblasts. Frontiers in Physiology, 5, 100.

[31]   Kim, J. and Watkins, B.A. (2014) Cannabinoid Receptor Antagonists and Fatty Acids Alter Endocannabinoid System Gene Expression and COX Activity. The Journal of Nutritional Biochemistry, 25, 815-823.

[32]   Kim, J., Carlson, M.E., Kuchel, G.A., Newman, J.W. and Watkins, B.A. (2016) Dietary DHA Reduces Downstream Endocannabinoid and Inflammatory Gene Expression and Epididymal Fat Mass While Improving Aspects of Glucose Use in Muscle in C57BL/6J Mice. International Journal of Obesity, 40, 129-137.

[33]   Gao, M., et al. (2016) Ferroptosis Is an Autophagic Cell Death Process. Cell Research, 26, 1021-1032.

[34]   Jenkin, K.A., Verty, A.N., McAinch, A.J. and Hryciw, D.H. (2012) Endocannabinoids and the Renal Proximal Tubule: An Emerging Role in Diabetic Nephropathy. The International Journal of Biochemistry & Cell Biology, 44, 2028-2031.