Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-κB and free radical in proximal tubular cells
Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-κB and free radical in proximal tubular cells
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DOI：
10.1046/j.1523-1755.2003.00906.x
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发表时间：
2003-05-01
影响因子：
19.6
通讯作者：
Yoshioka, T
中科院分区：
文献类型：
--
作者：
Motojima, M;Hosokawa, A;Yoshioka, T
背景尿毒症毒素已被认为是促进慢性肾衰竭的进展。我们已经表明，有机阴离子转运蛋白介导的尿毒症毒素的摄取诱导氧化应激负鼠肾肾小管细胞过度表达转运蛋白。纤溶酶原激活物抑制剂-1（派-1）和核因子-κ B（NF-κ B）是已知促进肾小管间质纤维化的主要因子。本研究探讨尿毒症毒素激活人肾小管上皮细胞（HK-2）中派-1和NF-κ B的信号通路。检测了有机阴离子形式的尿毒症毒素诱导HK-2中氧化应激、派-1基因表达和NF-κ B活化的能力。采用酶联免疫吸附试验（ELISA）和北方印迹法检测派-1的表达。通过荧光素酶报告基因（NKkappaB-luc）测定来估计人派-1启动子活性。通过pNF-κ B-luc报告基因和电泳凝胶迁移率变动分析检测NF-κ B的活化。在测试的有机阴离子物种中，吲哚酚硫酸盐和吲哚乙酸诱导HK-2的自由基产生。非特异性转运蛋白抑制剂（丙磺舒）抑制IS刺激的自由基产生。硫酸吲哚酚和吲哚乙酸剂量依赖性地增加派-1 mRNA和蛋白的表达。荧光素酶报告基因检测显示，硫酸吲哚酚和吲哚乙酸剂量依赖性地激活NF-κ B和派-1启动子。NF-κ B的激活也通过电泳凝胶迁移率变动测定证实。抗氧化剂和NF-κ B抑制剂均能剂量依赖性地抑制硫酸吲哚酚对派-1启动子的激活。尿毒症毒素诱导肾小管细胞产生自由基并激活NF-κ B，NF-κ B反过来上调派-1表达。因此，慢性肾功能衰竭的进展可能是由尿毒症毒素诱导的派-1的上调。
Background. Uremic toxins have been suggested to promote progression of chronic renal failure. We have shown that organic anion transporter-mediated uptake of uremic toxins induces oxidative stress in opossum kidney renal tubular cells overexpressing the transporter. Plasminogen activator inhibitor-1 (PAI-1) and nuclear factor-kappa B (NF-kappaB) are major factors known to promote tubulointerstitial fibrosis. The present study examined the signaling pathway that is activated by uremic toxins to induce PAI-1 and activate NF-kappaB in human renal proximal tubular cells (HK-2).Methods. Uremic toxins in the form of organic anion were examined their ability to induce oxidative stress, PAI-1 gene expression, and NF-kappaB activation in HK-2. PAI-1 expression was measured by enzyme-linked immunosorbent assay (ELISA) and the Northern blotting. Human PAI-1 promoter activity was estimated by luciferase reporter gene (NKkappaB-luc) assay. NF-kappaB activation was measured by the pNFkappaB-luc reporter gene and electrophretic gel mobility shift assay.Results. Among organic anion species tested, indoxyl sulfate and indoleacetic acid induced free radical production in HK-2. A nonspecific transporter inhibitor (probenecid) suppressed the IS-stimulated radical production. Indoxyl sulfate and indoleacetic acid dose dependently increased the expressions of PAI-1 mRNA and protein in these cells. The luciferase reporter gene assay revealed that indoxyl sulfate and indoleacetic acid dose dependently activated NF-kappaB and PAI-1 promoter. Activation of NF-kappaB was also confirmed by an electrophoretic gel mobility shift assay. Both antioxidant and NF-kappaB inhibitors dose dependently inhibited the activation of PAI-1 promoter by indoxyl sulfate.Conclusion. Uremic toxins induce free radical production by renal tubular cells and activate NF-kappaB which, in turn, up-regulates PAI-1 expression. Thus, progression of chronic renal failure may be promoted by PAI-1 up-regulation induced by uremic toxins.