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

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. METHODOLOGY: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. PRINCIPAL FINDINGS: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. CONCLUSIONS: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively

The Current Status of Kidney Cancer Urine Markers - A Systematic Review

BACKGROUND: Renal cell carcinoma is the 9th most common malignant disease in the Western World. Typically, patients develop symptoms in a late stage of the disease and most of them are diagnosed by chance. Up to 30% of the patients at the time of diagnosis had metastatic disease. Therefore, highly specific and sensitive biomarkers for the detection and progression of kidney cancer are of great importance. Here, urine markers can be a major advantage and can have a huge clinical impact on the diagnosis, differentiation and prognosis of kidney cancer. At the moment there are several approaches to improve these conditions. METHODS: Asystematic literature research was performed according to the PRISMA guidelines to identify studies reporting urine markers for kidney cancer between 2012 and 2021. A two-step process for the selection of the studies was initiated. In total 287 studies were considering for the final analysis. In total, 6 studies, which presented potential urinary biomarker were analyzed in depth. RESULTS: The major focus was on urinary markers for the detection, progression and differentiation of renal cell carcinoma. In total, a study population of 1099 patients were investigated in the different studies that were analyzed in depth. The median patient sample size of the different studies was 157 patients. The focus was based on the investigation of different microRNAs and proteins as urinary marker for kidney cancer detection. CONCLUSION: Overall, there are different approaches present for the detection, prognosis and differentiation of kidney cancer in urine but most of the studies are based on a small sample size and need to be validated in a greater collective. Furthermore, the standard should be improved to bring these biomarkers into routine clinical practice

MicroRNAs: Small but amazing, and their association with endothelin

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the expressional regulation of genes by inhibiting gene translation. MicroRNAs are recruited and incorporated into the miRISC, ribonucleoprotein complex, targeting specific mRNAs through mechanisms specific for a miRNA sequence. Here we review the biogenesis, regulation, and monitoring of miRNAs, as well as the current evidence for potential roles of miRNAs in human diseases associated with activation of the endothelin system. These diseases include cancer, kidney disease, cardiovascular diseases, inflammatory diseases, infectious diseases, and blood diseases, that may all be aggravated by aberrant miRNA expression. In this review we will also discuss regulatory mechanisms determining production of miRNA as well as measuring or targeting miRNAs as potential novel approaches for diagnosis and treatment. Targeting miRNAs possibly will allow one to detect diseases or to interfere with the progression of diseases associated with activation of the endothelin system. (c) 2012 Elsevier Inc. All rights reserved

Endothelin-1 induced Mxi-2/Ago2 complex formation resulting in 5P536 downregulation promoting breast cancer development

Increased endothelin-1 decreases PKC alpha (PKCα), resulting in high miRNA 15a levels in kidney tumors. Breast cancer cells treated with ET-1, β-estrogen, Tamoxifen, Tamoxifen + β-estrogen and Tamoxifen + ET-1 were analysed regarding miRNA 15a expression. Significantly increased miRNA 15a levels were found after ET-1, becoming further increased in Tamoxifen + ET-1 treated cells. Our group already showed that miRNA 15a induces MAPK p38 splicing resulting in a truncated product called Mxi-2, whose function has yet to be defined in tumors. We described for the first time in ET-1 induced tumor cells that Mxi-2 builds a complex with Ago2, a miRNA binding protein, which is important for the localization of miRNAs to the 3′UTR of target genes. Furthermore, we show that Mxi-2/Ago2 is important for the interaction with the miRNA 1285 which binds to the 3′end of the tumor suppressor gene p53, being responsible for the downregulation of p53. Tissue arrays from breast cancer patients were performed, analysing Mxi-2, p53 and PKCα. Since the Mxi-2 levels increase in Tamoxifen + ET-1 treated cells, we claim that increasing ET-1 levels in Tamoxifen treated breast cancer patients are responsible for decreasing p53 levels. In summary, ET-1 decreases nuclear PKCα levels, while increasing the amount of miRNA 15a. This causes high levels of Mxi-2, necessary for complex formation with Ago2. The newly identified Mxi-2/Ago2 complex interacting with miRNA 1285 leads to increased 3′UTR p53 interaction, resulting in decreased p53 levels and subsequent tumor progression. This newly identified mechanism is a possible explanation for the development of ET-1 induced tumors

Novel noninvasive marker of regression of clear cell renal cell carcinoma (ccRCC) (Publication with Expression of Concern. See vol. 48, pg. 392, 2022)

Objective: Analyzing protein kinase C (PKC) alpha, iota, and zeta as well as levels of Mxi-2 and Vim3 in regressive clear cell renal carcinomas (ccRCCs) and urine samples. Material and methods: Fresh samples of ccRCCs (predominantly pT1a/b) with different degrees of regression (= 70%) vs normal renal tissue and oncocytomas were studied by Western blot, using antibodies of different PKC isoforms. Urine samples from these tumors were analyzed by ELISA (PKC isoforms, Mxi-2, and Vim3). Results: With increasing degree of regression beyond 10%, nuclear Mxi-2 and Vim3 were highly overexpressed in fresh tumor samples. In urine samples, Vim3 was significantly overexpressed in oncocytoma and downregulated in RCCs with 70% regression. Western blot analysis shows that PKC alpha and iota levels were significantly increased in fresh tumor tissue samples (tumors with >= 30% regression). PKC zeta was expressed in normal kidney and significantly increased in oncocytoma but not found in ccRCCs. In patients' urines, Mxi-2 was significantly reduced (regression > 50%), while PKC isofonn alpha was significantly increased by advanced regression rate. PKC iota in patients' urine was overexpressed in oncocytoma and reduced in all ccRCC urines. Conclusion: Tumor regression in ccRCC tissue shows strong nuclear overexpression of Mxi-2, Vim3, and PKC alpha and iota. In respective urines, PKC alpha was overexpressed; PKC iota was decreased. Mxi-2 and Vim3 decreased with increasing regression rates. These reagents could serve as noninvasive ccRCC markers for regression

Preoperative Stating of Pelvic Lymph Nodes in Prostate Cancer Patients via Endorectal Magnetic Resonance Imaging

Background/Aim: The aim of this study was to evaluate the diagnostic sensitivity, specificity and accuracy of endorectal magnetic resonance imaging (e-MRI), as a preoperative staging modality in the diagnosis of lymph node metastasis (LNM) in patients with prostate cancer (PCa). Patients and Methods: Retrospectively, we analyzed data from N=168 patients who underwent radical prostatectomy (RP) between 2004 and 2013 at two tertiary medical centres. Prior to RP all patients underwent an e-MRI. Inclusion criteria were: PSA levels >20 ng/ml or Gleason score >7. Examinations were performed on a closed 1.0-T system combined with an endorectal body phased-array coil, imaging results were correlated with histopathology. Results: 10.7% (N=18 patients) had histologically-proven LNM. e-MRI was true-positive in N=6 (33.3%) and false-negative N=12 patients (66.6%). N=150 (89.3%) patients without LNM e-MRI were true-negative in 96% and false-positive in 4%. Sensitivity was 96%, specificity was 33%, accuracy was 64.5%. Conclusion: eMRI can be considered a useful preoperative staging modality in diagnosis of LNM

Protein kinase C alpha regulates nuclear pri-microRNA 15a release as part of endothelin signaling

Endothelin-1 induced signaling is characterized by an early induction of a nuclear factor-kappa B p65/mitogen-activated phosphokinase p38 transcription complex via its A-receptor versus a late induction via diacylglycerol, and protein kinase C. A possible interaction between these two pathways and a potential function for protein kinase C in this context has not previously been elucidated. Here we report that in Caki-1 tumor cells, protein kinase C alpha is a part of the transcription complex. With importin alpha 4 and alpha 5 as chaperones, the transcription complex transmigrates into the nucleus. Protein kinase C alpha blocks the nuclear release of primicroRNA 15a by direct binding shown by electrophoretic mobility shift assay and Duolink immune histology. The expression levels of miRNA 15a can be further manipulated by transfection of si-protein kinase C alpha, or an expression vector containing protein kinase C alpha or miRNA 15. The miRNA 15a regulation by protein kinase C alpha is detectable in different malignant human tumor cell lines (renal cell carcinoma, breast carcinoma, and melanoma). Furthermore, all three cell lines harbor both endothelin receptors (ETAR/ETBR). Specific blockage of each receptor leads to major reduction of miRNA 15a expression due to increased nuclear protein kinase C alpha translocation. We conclude that the nuclear binding of pri-microRNA 15a is a novel function of protein kinase C alpha, which plays an important role in endothelin-1 mediated signaling. Since several endothelin-sensitive, malignant tumor cell lines harbor this regulation, it could indicate a more general role in tumor biology. (C) 2011 Elsevier B.V. All rights reserved

Vimentin 3 Expression in Prostate Cancer Cells

Background/Aim: Vimentin3 (Vim3) was recently described as a tumour marker for the direct discrimination between benign and malignant kidney tumours. Here, we examined its expression in prostate cancer (PCa) cell lines and the regulation of its expression by endothelin receptors. Materials and Methods: Prostate cancer cell lines (PC3, DU145, LNCap) were incubated with endothelin 1 (ET-1), BQ123 [endothelin A receptor (ETAR) antagonist], BQ788 [endothelin B receptor (ETBR) antagonist), BQ123+ET-1, BQ788+ET-1 for 24 h and a scratch assay was performed. Cell extracts were analysed by western blotting and qRT-PCR. Results: ET-1 induced Vim3 overexpression. Blocking the ETBR in the different prostate cancer cell lines yielded a higher migration rate, whereby Vim3 expression was significantly increased. Conclusion: Vim3 concentration increases in cell lines without a functional ETBR and may be used as a marker for PCas where ETBR is frequently methylated