ROLE OF OXIDANT STRESS IN PROGRESSION OF PKD

多囊蛋白-1（PC 1）是ADPKD中最常突变的基因的蛋白产物，是受体/离子通道信号复合物的一部分，被认为参与维持细胞分化。最近，PC 1被证明在肾上皮细胞中具有主要的基于纤毛的机械感觉作用。我们有初步的数据表明，抗氧化反应元件（ARE）调节的基因是PCI介导的信号转导的目标，通过激活NF-E2相关因子-2（Nrf 2）转录因子。Nrf 2/ARE调节的基因产物构成了细胞对内源性氧化剂和外源性毒物的主要防御，保护细胞免受未解决的氧化应激引起的增殖增加、损伤或死亡。我们已经发现，在纯合和杂合Pkd 1缺失小鼠的肾脏中，许多ARE调节基因的表达减少，PC 1的C尾可以在小鼠的肾脏中表达。 本发明的另一个方面是，人ADPKD细胞组成型地发出信号以上调ARE报告基因的活性，人ADPKD细胞响应于氧化应激而显示出增加的增殖和凋亡，并且流体流动诱导ARE调节的基因在纤毛肾上皮细胞中的表达。两者合计，这些观察结果表明，PC 1有一个新的作用，通过上调保护性Nrf 2/ARE基因表达的一般细胞保护。我们的总体假设是，PC 1的缺乏导致抗氧化保护受损，导致PKD相关的细胞增殖和凋亡失调。该提案的主要目标是确定缺乏PC 1的细胞具有降低的抗氧化保护，并且更容易受到氧化剂诱导的损伤、增殖和凋亡。利用来自人ADPKD肾脏和Pkd 1缺失小鼠模型的肾上皮细胞系，我们将确定：（1）PC 1缺陷型肾上皮细胞是否具有减少的ARE调节的基因表达并且更易于氧化剂诱导的损伤;（2）PC 1缺陷型肾上皮细胞是否对氧化剂诱导的增殖和凋亡敏感;以及（3）流体切应力是否保护肾上皮细胞免于氧化剂诱导的增殖、损伤和凋亡。从这些研究中获得的知识将为PKD的病理机制提供新的见解， 治疗PKD的新的治疗途径。

Polycystin-1 (PC1), the protein product of the gene most commonly mutated in ADPKD, is part of a receptor/ion channel signaling complex that is thought to be involved in maintaining cellular differentiation. Recently, PC1 was shown to have a primary cilium-based mechanosensory role in renal epithelial cells. We have preliminary data suggesting that antioxidant response element (ARE)-regulated genes are targets of PCI-mediated signaling through the activation of NF-E2 related factor-2 (Nrf2) transcription factor. Nrf2/ARE-regulated gene products constitute the cell's primary defense against endogenous oxidants and exogenous toxicants, protecting cells from the increased proliferation, damage, or death that can be caused by unresolved oxidant stress. We have found that the expression of a number of ARE-regulated genes is reduced in kidneys from homozygous and heterozygous Pkd1 null mice, that the C-tail of PC1 can constitutively signal to upregulate the activity of an ARE reporter gene, that human ADPKD cells display increased proliferation and apoptosis in response to an oxidant stress, and that fluid flow induces expression of ARE-regulated genes in ciliated renal epithelial cells. Taken together, these observations suggest that PC1 has a novel role in general cytoprotection by upregulating protective Nrf2/ARE gene expression. Our overall hypothesis is that deficiency of PC1 results in impaired antioxidant protection that contributes to the dysregulated cell proliferation and apoptosis associated with PKD. The major goals of this proposal are to establish that cells deficient in PC1 have reduced antioxidant protection and are more susceptible to oxidant-induced damage, proliferation and apoptosis. Utilizing renal epithelial cell lines from human ADPKD kidneys and from a Pkd1 null mouse model, we will determine: (1) if PC1-deficient renal epithelial cells have reduced ARE-regulated gene expression and are more prone to oxidant-induced damage; (2) if PC1-deficient renal epithelial cells are susceptible to oxidant-induced proliferation and apoptosis; and (3) if fluid shear stress protects renal epithelial cells from oxidant-induced proliferation, damage and apoptosis. The knowledge gained from these studies will provide new insights into the pathological mechanism of PKD and will provide new therapeutic avenues for the treatment of PKD.