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Kidney Epithelial Cells (kidney + epithelial_cell)
Selected AbstractsDetection of environmental androgens: A novel method based on enzyme-linked immunosorbent assay of spiggin, the stickleback (Gasterosteus aculeatus) glue proteinENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2002Ioanna Katsiadaki Abstract We report the development and validation of a novel in vivo biomarker test for waterborne androgens. During breeding, male sticklebacks (Gasterosteus aculeatus) manufacture a glue protein, spiggin, in their kidneys that they use to build their nests. Spiggin production is under the control of androgens. Until now, however, it has only been possible to quantify its production by measurement of the height of kidney epithelial cells. In the present study, we report the development of an enzyme-linked immunosorbent assay (ELISA) for spiggin and demonstrate its application to the measurement of spiggin in the kidneys of female sticklebacks that have been exposed to androgens in water. Results from the ELISA procedure revealed a strong correlation with measurement of kidney epithelial cell height (r2 = 0.93). However, the ELISA was much quicker and had a considerably higher response range (100,000-fold vs fourfold). Clear, graded responses in spiggin production were obtained by exposing intact females to increasing concentrations of 17,-methyltestosterone and 5,-dihydrotestosterone over three-week test periods. The lowest effective concentrations for these two steroids were 100 ng/L and 3 ,g/L, respectively. Female sticklebacks that were exposed to pulp mill effluent also produced spiggin in their kidneys. Possession of an androgen-regulated protein by the female stickleback makes it a unique bioassay organism for detecting androgenic contamination in the aquatic environment. [source] A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprinFEBS JOURNAL, Issue 18 2008Daniel Ambort In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin,Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin,Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unkown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively. [source] Transgenic mice expressing tamoxifen-inducible Cre for somatic gene modification in renal epithelial cellsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 5 2006Irma S. Lantinga-van Leeuwen Abstract Gene inactivation often leads to an embryonic-lethal phenotype. In focal diseases like renal cell carcinomas and polycystic kidney disease, somatic gene inactivation in subsets of cells is likely to occur at later stages. We generated a transgenic mouse line with an inducible form of Cre recombinase for conditional gene modifications in kidney epithelial cells. To this end a 1.4-kb promoter fragment of the kidney-specific cadherin gene (KspCad) was cloned upstream of a tamoxifen-inducible Cre recombinase (CreERT2) encoding sequence. Expression and activity of Cre was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) analysis and by crossbreeding to Z/EG reporter mice. One KspCad-CreERT2 line showed kidney-specific Cre expression and mediated recombination upon tamoxifen treatment in Z/EG reporter mice. No reporter gene expression was detected in untreated animals or in extrarenal tissues upon treatment. Within the kidneys, enhanced green fluorescent protein (EGFP) fluorescence was observed in epithelial cells in several nephronic segments. In addition, the system successfully recombined a floxed Pkd1 gene. genesis 44:225,232, 2006. © 2006 Wiley-Liss, Inc. [source] Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Masato Nagaoka Abstract Rearrangement of cell,cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E-cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E-cadherin-rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E-cadherin-immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E-cadherin-mediated cell adhesion vary with the state of differentiation. J. Cell. Biochem. 103: 296,310, 2008. © 2007 Wiley-Liss, Inc. [source] v-Ha- ras mitogenic signaling through superoxide and derived reactive oxygen speciesMOLECULAR CARCINOGENESIS, Issue 4 2002Ji-Qin Yang Abstract The ras proto-oncogene is frequently mutated in human tumors and functions to constitutively stimulate signal transduction cascades, resulting in unchecked proliferation and malignant transformation. In certain cells, superoxide functions as a signal-transduction messenger, mediating the downstream effects of ras and rac. We demonstrated previously that v-Ha -ras,transfected rat kidney epithelial cells (RECs) overproduced superoxide anion and that this superoxide production was mediated by ras. In the present study, we further demonstrated that v-Ha -ras overexpression transformed immortal nonmalignant RECs into malignant cancer cells; v-Ha -ras,transfected cells formed clones in soft agar, had high plating efficiency, and formed tumors in nude mice. Our data suggest that superoxide radical plays a role in ras-induced transformation; modulation of intracellular superoxide level by overexpression of manganese-containing superoxide dismutase or copper- and zinc-containing superoxide dismutase inhibited ras-induced transformation, as evidenced by in vitro studies of plating efficiency and by in vivo studies of tumor formation in nude mice. Overexpression of catalase (CAT) alone was found to have little effect on tumor cell growth, but overexpression of glutathione peroxidase 1 (GPx1) completely suppressed tumor cell growth in nude mice. This finding suggests that peroxides removed by GPx1, but not by CAT, are also involved in ras-induced transformation. © 2002 Wiley-Liss, Inc. [source] Cytotoxic and antimitotic effects of N -containing Monascus metabolites studied using immortalized human kidney epithelial cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 4-5 2006Anja Knecht Abstract Recently the first Monascus metabolites with a pyridine ring were detected, the monascopyridines A and B. They are formally dehydrogenated derivatives of the red rice pigments rubropunctamine and monascorubramine. Because of their structural similarity, the toxicological effects of these secondary metabolites were studied using immortalized human kidney epithelial cells. The cytotoxicity was determined with the following different endpoint detection methods: metabolic activity, trypan blue exclusion, and electronic cell counting. The compounds led to EC50 values between 11 and 31 ,mol/L but the pigments caused a stronger reduction of the cell viability. Also, the apoptotic potential was examined by measuring caspase 3 activity and detecting apoptotic bodies, but none of the tested compounds induced apoptosis. All four substances caused a rise of the mitotic index to about 9% (100 ,mol/L monascopyridine A and B) and 20% (25 ,mol/L rubropunctamine and monascorubramine). The significant decrease of the ratio of cells in the ana- and telophase to cells in the prometa- and metaphase proved a stop of the mitosis at the meta- to anaphase control point. The compounds caused mitotic arrest and the formation of structural damages like c-mitosis through interaction with the mitotic spindle. These effects point to an aneuploidy inducing potential, which is linked to cancer formation. [source] A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Nicholas Ferrell Abstract We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell,cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO-1 tight junction protein and acetylated ,-tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F-actin and exhibited disassembly of cytosolic F-actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca2+ medium. The transport rate of a fluorescently labeled tracer molecule (FITC-inulin) was measured before and after Ca2+ switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes. Biotechnol. Bioeng. 2010;107:707,716. © 2010 Wiley Periodicals, Inc. [source] Adenosine receptor expression in Escherichia coli -infected and cytokine-stimulated human urinary tract epithelial cellsBJU INTERNATIONAL, Issue 11 2009Susanne Säve OBJECTIVE To assess the expression and regulation of adenosine receptors in unstimulated, uropathogenic Escherichia coli (UPEC)-infected and cytokine-stimulated human urinary tract epithelial cells, and to examine the regulation of interleukin (IL)-6 secretion in response to A2A receptor activation. MATERIALS AND METHODS Human urinary tract epithelial cells (A498, T24 and RT4) were grown in cell culture and stimulated with a mixture of pro-inflammatory cytokines (CM) or UPEC. The expression of adenosine receptors was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry. IL-6 secretion was measured with an enzyme-linked immunosorbent assay. RESULTS RT-PCR analysis showed the presence of transcripts for the A1, A2A and A2B receptor subtypes but not for the A3 receptor in A498 kidney epithelial cells. The expression of A2A receptor mRNA increased in A498 epithelial cells exposed to CM and UPEC, while A1 and A2B receptor transcripts decreased or remained unchanged. Up-regulation of A2A receptors was confirmed at the protein level using Western blot analysis and immunocytochemistry. There was also an increase in A2A receptor mRNA in human bladder epithelial cells (T24 and RT4) and in mouse bladder uroepithelium in response to cytokines and UPEC. IL-6 secretion in UPEC-infected A498 cells was decreased by 38% when exposed to the A2A receptor agonist CGS 21680. CONCLUSION Our data showed a subtype-selective plasticity among adenosine receptors in urinary tract epithelial cells in response to UPEC-infection and cytokines. There was a consistent up-regulation of A2A receptors in kidney and bladder epithelial cells. Functionally, A2A receptor activation reduced UPEC-induced IL-6 secretion. These findings suggest that adenosine might be a previously unrecognized regulator of the mucosal response in urinary tract infection. [source] Association between leptin receptor gene polymorphisms and early-onset prostate cancerBJU INTERNATIONAL, Issue 1 2003Z. Kote-Jarai Significant tissue loss is a consistent feature of ureteric obstruction with, most studies showing increased programmed cell death or apoptosis of kidney epithelial cells. The study by Chuang et al. showed that there is also muscular damage during obstruction, specifically of the ureteric myocytes. More importantly they show for the first time that this induction of cell death is associated with the increased expression of cytochrome c and the caspases, key proteins that drive the induction of apoptosis. Admittedly they do not show whether cytochrome c is released from the mitochondria or that the caspases are truly activated, important events in the cell death pathway, but an increase in their expression does indicate their role in this process. Understanding the pathways leading to tissue loss during ureteric obstruction has important implications in the development of novel treatments for this condition. OBJECTIVE To report a case-control study examining the relationship between polymorphisms in the leptin receptor (OBR) gene and the development of young-onset prostate cancer, because epidemiological studies report that prostate cancer risk is associated with animal fat intake, and thus we investigated if this association occurs via this genetic mechanism. PATIENTS, SUBJECTS AND METHODS The Lys109Arg (OBR1) and Gln223Arg (OBR2) polymorphisms in the coding region of OBR were studied in blood DNA from 271 patients with prostate cancer aged < 56 years at diagnosis and 277 geographically matched control subjects. Cases were collected through the Cancer Research UK/British Prostate Group Familial Prostate Cancer Study. Blood DNA was genotyped using the polymerase chain reaction and a restriction enzyme digest. RESULTS There was no statistically significant association between the OBR genotype and prostate cancer risk; men homozygous for 109Arg genotype had a slightly increased risk for prostate cancer, with a relative risk (95% confidence interval) of 1.36 (0.65,2.85), and those homozygous for the 223Arg allele had some reduction in prostate cancer risk, at 0.82 (0.58,1.26), but neither was statistically significant. CONCLUSION This case-control study showed no significant association between leptin receptor gene polymorphisms and the risk of young-onset prostate cancer, suggesting that genetic variations in OBR are unlikely to have a major role in the development of early-onset prostate cancer in the UK. [source] | |||||||||||||