Transcriptional in Chronic Renal Disease Pathogenesis

这些研究的目的是确定许多慢性肾脏疾病常见的致病过程中的早期和致病事件，如管状囊肿形成和肾小球硬化。 我们最近在病毒诱导的慢性肾病转基因小鼠模型中确定了发病机制中最早的事件，即上皮细胞增殖和凋亡。 这些致病事件已被证明发生在许多增加的转基因表达所引起的持续激活的NF-κ B。 已知NF-kB调节与肾脏发病机制有关的许多基因的表达，并且已被提出为建立或加速疾病的共同调节点。 因此，我们假设转基因表达诱导NF-κ B的失调，随后激活参与发病机制的基因，如那些控制细胞凋亡。 具体目的是：1）研究NF-κ B激活失调的机制及其对肾细胞基因表达的影响，以及2）研究细胞凋亡的机制和NF-κ B在介导细胞凋亡中的作用。 为了完成这些研究，我们已经开发了一种体外系统，使我们能够功能性地测试NF-κ B在介导上皮细胞功能障碍中的调节作用。 我们将通过电泳迁移率变动分析和蛋白质印迹法确定肾小球和肾小管上皮细胞中NF-κ B复合物的组成。 将使用NF-kB的特异性抑制剂和关键调节蛋白IkB α和IkB β的显性突变体来确定持续NF-kB激活的机制。 细胞凋亡的机制以及NF-κ B的调节作用将被确定。 因此，本提案的长期目标是在分子水平上更好地了解慢性肾脏疾病发病机制中发生的起始事件。 了解这些起始致病事件是如何调节的，可能会导致更有效的干预策略的设计。

The purpose of these studies is to identify early and causative events in pathogenic processes common to many chronic renal diseases, such as tubular cyst formation and glomerulosclerosis. We have recently identified the earliest events in pathogenesis, epithelial cell proliferation and apoptosis, in a transgenic mouse model of virus-induced chronic renal disease. These pathogenic events have been shown to occur in many increased transgene expression caused by a persistent activation of NF-kB. NF- kB is known to regulate the expression of numerous genes implicated in renal pathogenesis, and has been proposed to be a common regulatory point in establishing or accelerating disease. Thus, we hypothesize that transgene expression induces a dysregulation of NF-kB, which subsequently activates genes involved in pathogenesis, such as those controlling apoptosis. The specific aims are 1) to examine the mechanism of dysregulated NF-kB activation and its effect on gene expression in renal cells, and 2) investigate the mechanism(s) of apoptosis and the role of NF-kB in mediating apoptosis. To accomplish these studies, we have developed an in vitro system that will allow us to functionally test the regulatory role of NF-kB in mediating epithelial cell dysfunction. We will define the composition of NF-kB complexes in glomerular and tubular epithelial cells by electrophoretic mobility shift assays and western blotting. The mechanism of persistent NF-kB activation will be determined using specific inhibitors of NF-kB and dominant mutants of the critical regulatory proteins, IkBalpha and IkBbeta. The mechanism of apoptosis will be determined as well as the regulatory role of NF-kB. Thus, the long-term goal of this proposal is to better understand at the molecular level the initiating events that occur in chronic renal disease pathogenesis. An understanding of how these initiating pathogenic events are regulated may lead to the design of more effective interventional strategies.