Transcriptional in Chronic Renal Disease Pathogenesis

这些研究的目的是确定许多慢性肾脏疾病（如小管囊肿形成和肾小球硬化）常见的致病过程中的早期和致病事件。我们最近在病毒诱导的慢性肾脏疾病的转基因小鼠模型中确定了发病机制、上皮细胞增殖和凋亡的最早事件。这些致病事件已被证明发生在许多由NF-kB持续激活引起的转基因表达增加中。已知NF- kB调节与肾脏发病有关的许多基因的表达，并被认为是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-kB的失调，进而激活参与发病机制的基因，如控制细胞凋亡的基因。具体目的是：1)研究肾细胞NF-kB激活异常的机制及其对基因表达的影响；2)研究细胞凋亡的机制以及NF-kB在细胞凋亡中的介导作用。为了完成这些研究，我们开发了一个体外系统，使我们能够功能性地测试NF-kB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位移测定和western blotting确定肾小球和小管上皮细胞中NF-kB复合物的组成。持续NF-kB激活的机制将通过NF-kB的特异性抑制剂和关键调节蛋白IkBalpha和IkBbeta的显性突变体来确定。研究NF-kB对细胞凋亡的调控作用及机制。因此，这项建议的长期目标是在分子水平上更好地理解慢性肾脏疾病发病机制中发生的起始事件。了解这些初始致病事件是如何调节的，可能会导致设计更有效的干预策略。

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.