Rashed Tanvir, Menon Mani, Thamilselvan Sivagnanam
Department of Urology, Vattikuti Urology Institute and Henry Ford Health Sciences Center, Detroit, Mich. 48202, USA.
Am J Nephrol. 2004 Sep-Oct;24(5):557-68. doi: 10.1159/000082043. Epub 2004 Nov 10.
Peroxidation of renal cells is a critical event in the nucleation and formation of calcium oxalate crystals under hyperoxaluric conditions. We previously demonstrated that oxalate-induced peroxidative injury is one of the major mechanisms in promoting crystal attachment to renal epithelial cells.
In this study we have demonstrated that the mechanism of oxalate-induced peroxidative injury is through the induction of TGF-beta1 and glutathione (GSH) redox imbalance in LLC-PK1 cells.
LLC-PK1, renal epithelial cells exposed to oxalate had significantly higher reactive oxygen species (ROS) production; higher TGF-beta1 levels, as measured by ELISA (1.89 +/- 0.035 fold increase) or Western blot (1.65 +/- 0.01 fold increase); increased malondialdehyde formation; increased LDH release, and loss of cell viability. In addition, oxalate exposure significantly decreased GSH content, glutathione reductase, glucose-6-phosphate dehydrogenase activities, and increased oxidized GSH content. Treatment with vitamin E, neutralizing anti-TGF-beta antibody, or diphenylene iodium, an inhibitor of NAD(P)H oxidase, significantly inhibited oxalate-induced ROS production and prevented peroxidative injury and cytolysis. Vitamin E, catalase, or desferoxamine treatment also significantly restored the oxalate-induced cellular GSH redox status toward the control level, and vitamin E treatment significantly attenuated the oxalate-mediated increase in TGF-beta1 protein in cultured LLC-PK1 cells.
This is the first study to demonstrate that the mechanism of oxalate-induced free radical production in renal tubular epithelial cells is through the activation of NAD(P)H oxidase via cytokine TGF-beta1 induction. These results also provide direct evidence that antioxidant therapy might prevent calcium oxalate nucleation and kidney stone formation by preventing oxalate-mediated peroxidative injury and GSH redox imbalance.
在高草酸尿症条件下，肾细胞的过氧化是草酸钙晶体成核和形成的关键事件。我们之前证明，草酸诱导的过氧化损伤是促进晶体附着于肾上皮细胞的主要机制之一。
在本研究中，我们证明了草酸诱导的过氧化损伤机制是通过诱导LLC-PK1细胞中的转化生长因子-β1（TGF-β1）和谷胱甘肽（GSH）氧化还原失衡。
暴露于草酸的肾上皮细胞LLC-PK1产生的活性氧（ROS）显著增加；通过酶联免疫吸附测定（ELISA）（增加1.89±0.035倍）或蛋白质免疫印迹法（Western blot）（增加1.65±0.01倍）测得的TGF-β1水平更高；丙二醛形成增加；乳酸脱氢酶（LDH）释放增加，细胞活力丧失。此外，草酸暴露显著降低了GSH含量、谷胱甘肽还原酶、葡萄糖-6-磷酸脱氢酶活性，并增加了氧化型GSH含量。用维生素E、中和性抗TGF-β抗体或二苯碘嗡（一种NAD(P)H氧化酶抑制剂）处理，可显著抑制草酸诱导的ROS产生，并预防过氧化损伤和细胞溶解。维生素E、过氧化氢酶或去铁胺处理也显著使草酸诱导的细胞GSH氧化还原状态恢复至对照水平，并且维生素E处理显著减弱了草酸介导的培养LLC-PK1细胞中TGF-β1蛋白的增加。
这是第一项证明肾小管上皮细胞中草酸诱导自由基产生的机制是通过细胞因子TGF-β1诱导激活NAD(P)H氧化酶的研究。这些结果还提供了直接证据，即抗氧化治疗可能通过预防草酸介导的过氧化损伤和GSH氧化还原失衡来预防草酸钙成核和肾结石形成。
