Exposure of Madin-Darby canine kidney (MDCK) cells to oxalate and calcium oxalate crystals activates nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.
Exposure of Madin-Darby canine kidney (MDCK) cells to oxalate and calcium oxalate crystals activates nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.
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DOI：
10.1016/j.urology.2013.10.038
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发表时间：
2014-02
期刊：
影响因子：
2.1
通讯作者：
Khan SR
中科院分区：
文献类型：
--
作者：
Khan A;Byer K;Khan SR
研究草酸盐（Ox）或草酸钙（CaOx）晶体对Madin-Darby犬肾（MDCK）细胞中烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶活性和活性氧产生的影响。将汇合的Madin-Darby犬肾细胞单层暴露于100、300、500 μmol、1 mmol Ox或33、66、132 μg/cm 2 CaOx晶体15分钟、30分钟、1小时、2小时或3小时。在暴露于Ox和CaOx晶体的特定时间段后，使用标准程序测定乳酸脱氢酶释放、台盼蓝排除、NADPH氧化酶活化和超氧化物产生。通过蛋白质印迹分析确定Nox 4（NADPH氧化酶的膜相关亚基）的产生。暴露于Ox和CaOx晶体导致NADPH氧化酶的时间和浓度依赖性活化。Western印迹分析显示Nox 4的产生增加。超氧化物的产生也改变了时间和浓度依赖性的方式，最大的增加后30分钟暴露于最高浓度的Ox和CaOx晶体。更长时间的暴露不会改变结果或导致活动减少。暴露于较高浓度也导致乳酸脱氢酶释放增加和台盼蓝排斥，表明细胞损伤。结果表明，远端肾小管起源的细胞配备有NADPH氧化酶，其通过暴露于Ox和CaOx晶体而被激活。较高浓度的两者导致细胞损伤，最有可能是通过暴露的细胞产生的活性氧增加。
To investigate nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in Madin-Darby canine kidney (MDCK) cells and the production of reactive oxygen species on exposure to oxalate (Ox) or calcium oxalate (CaOx) crystals. Monolayers of confluent Madin-Darby canine kidney cells were exposed to 100, 300, 500 μmol, 1 mmol Ox or 33, 66, 132 μg/cm2 CaOx crystals for 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours. After specified periods of exposure to Ox and CaOx crystals, lactate dehydrogenase release, trypan blue exclusion, activation of NADPH oxidase, and superoxide production were determined using standard procedures. The production of Nox4, a membrane associated subunit of the NADPH oxidase enzyme, was determined by western blot analysis. Exposure to Ox and CaOx crystals leads to time- and concentration-dependent activation of NADPH oxidase. Western blot analysis showed an increase in the production of Nox4. The production of superoxide also changed in a time- and concentration-dependent manner, with maximum increases after 30-minute exposure to the highest concentrations of Ox and CaOx crystals. Longer exposures did not change the results or resulted in decreased activities. Exposure to higher concentrations also caused increased lactate dehydrogenase release and trypan blue exclusion indicating cell damage. Results indicate that cells of the distal tubular origin are equipped with NADPH oxidase that is activated by exposures to Ox and CaOx crystals. Higher concentrations of both lead to cell injury, most probably through the increased reactive oxygen species production by the exposed cells.
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