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      Differential Role of Reactive Oxygen Species in Chemical Hypoxia-Induced Cell Injury in Opossum Kidney Cells and Rabbit Renal Cortical Slices

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          This study was undertaken to evaluate the role of reactive oxygen species (ROS) and lipid peroxidation in chemical hypoxia in opossum kidney (OK) cells and rabbit renal cortical slices. Chemical hypoxia was induced by incubating cells or slices with antimycin A, an inhibitor of mitochondrial electron transport. Exposure of OK cells to chemical hypoxia resulted in a time-dependent cell death and parallel depletion of intracellular ATP. In OK cells subjected to chemical hypoxia, the generation of ROS was increased, and this was prevented by the H<sub>2</sub>O<sub>2</sub> scavenger catalase, but not by the hydroxyl radical scavenger dimethylthiourea (DMTU). Catalase prevented OK cell death induced by chemical hypoxia, but [Cu, Zn]-superoxide dismutase (SOD) and DMTU were not effective. The iron chelators deferoxamine and phenanthroline prevented chemical hypoxia-induced OK cell death, but the potent antioxidants N,N′-diphenyl- p-phenylenediamine (DPPD) and butylated hydroxyanisole (BHA) showed no beneficial effect. Antimycin A in OK cells increased lipid peroxidation, which was prevented by DPPD and phenanthroline. In rabbit renal cortical slices, antimycin A caused an increase in LDH release and lipid peroxidation, and these effects were prevented by ROS scavengers (SOD, catalase, and DMTU), iron chelator (deferoxamine), and antioxidants (DPPD and BHA). However, in primary cultured rabbit proximal tubular cells the antimycin A-induced cell death was not altered by antioxidants. The extent of ATP depletion was similar in renal cortical slices and primary cultured cells treated with antimycin A. These results indicate that chemical hypoxia-induced cell injury is not directly resulted from lipid peroxidation in OK cells, but this cell injury is mediated by lipid peroxidation in rabbit renal cortical slices. This discrepancy may be due to the difference in cell preparation (freshly prepared tubules and cultured cells).

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          A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate.

          A fluorometric microplate assay was established for the detection of respiratory burst activity in phagocytic cells by assessing oxidation of 2',7'-dichlorofluorescin-diacetate (DCFH-DA). This method is based on flow cytometric studies by Bass et al. (J. Immunol. 130 (1983) p. 1910) describing intracellular detection of DCFH oxidation due to the presence of hydrogen peroxides. In the present study we have adapted the assay for use in microtiter plates to determine the amount of extracellular reactive oxidative products. DCFH-DA, granulocytes and stimuli (phorbol myristate acetate, n-formyl-methionyl-leucylphenylalanine, concanavalin A) were added to microtiter plates and after incubation at 37 degrees C, the development of fluorescence intensity was read in a fluorescence concentration analyzer (FCA, Baxter). Calibration of fluorescence units recorded by the FCA was achieved by comparison with defined amounts of fluorescent DCF. The change in measured fluorescence was linear with cell density over the range of 2 x 10(5)-1 x 10(6) cells/well. Cumulative DCF generation in individual wells could be recorded non-destructively at frequent intervals for time course measurements. Results from FCA measurements correlated perfectly with the FACS analysis of the same samples (r = 0.99). In conclusion, this assay can be useful for screening monoclonal antibodies recognizing cell surface structures possibly involved in signal transduction as well as for testing phagocytes for their capacity to release reactive oxidative intermediates.
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            Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

            Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.

              Author and article information

              Nephron Exp Nephrol
              Cardiorenal Medicine
              S. Karger AG
              27 June 2002
              : 10
              : 4
              : 275-284
              Department of Physiology, College of Medicine, Pusan National University, Pusan, Korea
              63702 Exp Nephrol 2002;10:275–284
              © 2002 S. Karger AG, Basel

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              Page count
              Figures: 8, Tables: 2, References: 34, Pages: 10
              Self URI (application/pdf): https://www.karger.com/Article/Pdf/63702
              Original Paper


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