The endocannabinoid, anandamide, induces cannabinoid receptor-independent cell death in renal proximal tubule cells

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, capsaze pine 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

Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells.

Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs

Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells.

Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs

SAA1 and SAA3 levels are elevated during hepatic fibrogenesis.

<p><b>A.</b> Hepatic fibrosis was induced in 8 week old Balb/c mice by bile duct ligation (n = 3 mice per time point) or by treatment with CCl₄ (8 injections of 0.5 μl CCl₄/g body weight, dissolved in olive oil, every 3 days, n = 4 mice/group). Following RNA extraction and reverse transcription, mRNA levels of SAA1 and SAA3 were determined by quantitative real time PCR. Levels are expressed as fold-induction in comparison to sham or oil control mice after normalization to 18s. <b>B.</b> RNA was isolated from quiescent mouse HSCs (18 hours after plating, n = 3 independent isolations) or activated mouse HSCs (5 days after plating, n = 3 independent isolations). mRNA levels of SAA1 and SAA3 were determined by quantitative real time PCR. Levels are expressed as fold-induction in comparison to quiescent HSCs after normalization to 18s.</p

SAA induces apoptosis in HSCs after NF-κB inhibition.

<p>Activated rat HSCs were infected with adenoviruses expressing either GFP or IκBsr. 24 hours later, HSCs were treated with rhSAA (5 μM). <b>A.</b> After 18h or treatment, cell death was determined by LDH activity assay. <b>B.</b> Caspase 3 and PARP cleavage were determined by western blot analysis after 8 hours of rmSAA1 treatment. <b>C.</b> After 8 hours of rhSAA treatment, cells were stained with Annexin V (green) and propidium iodide (PI, red) to visualize apoptosis and necrosis. <b>D.</b> Primary mouse hepatocytes were serum-starved for 12h and treated with rmSAA (5 μM), ActD (0.2 μg/ml) or rmTNFα (30 ng/ml) for 18. Cell death was determined by LDH activity assay (*p<0.05 vs. untreated vehicle control). <b>E.</b> Primary mouse hepatocytes were treated with rmSAA (5 μM) for the indicated times and rmTNFα (30 ng/ml) for 120 min. Phosphorylation of Erk, Akt, p65 and c-Jun was determined by western blot analysis.</p

SAA induces HSC proliferation in an Erk-, JNK- and Akt-dependent manner.

<p><b>A-B.</b> Activated rat HSCs were treated with rhSAA (5 μM) for the indicated times. Phosphorylation of Erk at threonine 202 and tyrosine 204 (<b>A</b>), and Akt at serine 473 (<b>B</b>) was determined by western blot analysis in the absence or presence of MEK inhibitor PD98059 or PI-3 kinase inhibitor LY294002, respectively. <b>C.</b> Serum starved activated rat HSCs were pretreated with PD98059 (5 μM), LY294002 (10 μM) or SP600125 (20 μM) followed by stimulation with rhSAA for 24 hours. 18 hours after stimulation cells were pulsed with 1 uCi/ml 3H-thymidine followed by TCA precipitation and measurement in a scinitillation counter.</p

SAA induces chemokine and MMP 9 expression in HSCs in an NF-κB- and JNK-dependent manner in HSCs.

<p>Activated human HSCs were treated with rhSAA in the prescence or absence of JNK inhibitor SP600125 (20 μM), or after infection with adenoviruses expressing either IκBsr or GFP. <b>A.</b> 24 hours after rhSAA treatment, supernatants were analyzed for MCP-1, IL-8 and RANTES secretion by ELISA. <b>B-C.</b> 6 hours after rhSAA treatment, RNA was extracted and analyzed for mRNA levels of MCP-1, IL-8 and RANTES (<b>B</b>) or MMP9 (<b>C</b>) by quantitiative real time PCR (*p<0.05 vs. SP600125 treatment or IκBsr infection, resp.). <b>D.</b> 24 hours after SAA treatment, proteins were extracted and MMP9 activity was analyzed by zymography.</p

SAA induces IKK and JNK activation in HSCs.

<p><b>A-B.</b> Activated human HSCs were treated with recombinant human SAA (5 μM) for the indicated times or with rhTNFα for 15 minutes. Extracts were subjected to western blot analysis using antibodies that recognize IκBα, phospho-p65 (S536), phospho-c-Jun or actin (<b>A</b>). Results were confirmed by <i>in vitro</i> kinase assay using GST-IκBα, GST-p65, GST-p65 (S536A) and GST-c-Jun substrates (<b>B</b>). <b>C.</b> Activated human HSCs were treated with recombinant human sFasR (10 μM) for the indicated times or with rhTNFα for 15 minutes. Extracts were analyzed by western blot using antibodies against IκBα or GAPDH. <b>D.</b> Activated human HSCs were infected with adenoviruses expressing NF-κB driven luciferase followed by treated with the indicated concentrations of SAA or with rhTNFα (10 ng/ml) for 6h. Lucerifase activity is expressed as fold induction in comparison to untreated control. <b>E.</b> Primary rat HSCs were activated for 2, 4 and 8 days and treated with SAA (5 μM) or rmTNFα (30 ng/ml) for 15 minutes. Extracts were subjected to western blot analysis using antibodies that recognize IκBα, phospho-p65 (S536), phospho-c-Jun or actin. <b>F.</b> Primary rat HSCs were activated for infected with adenoviruses expressing NF-κB driven luciferase at day 3 or day 7 after plating. 18 hours after infection, cells were treated with with SAA (5 μM) or rmTNFα (30 ng/ml) for 15 minutes. NF-κB driven luciferase was determined as described above.</p