Kansas Interdisciplinary Center for PKD Research

描述(申请人提供)：多囊肾病(PKD)的特征是形成无数充满液体的囊，由于细胞的异常增殖，这些囊的直径可扩大到几厘米。持续增大的囊性病变破坏了正常的肾脏结构，导致肾功能的进行性丧失。CAMP激动剂，包括精氨酸加压素，刺激丝裂原活化蛋白激酶(MAPK)ERK，加速囊壁上皮细胞的增殖。B-Raf是一种MAPK激酶，在cAMP依赖的PKD细胞增殖过程中起重要作用。CAMP增加B-Raf活性，刺激MEK/ERK信号转导和细胞增殖。在初步实验中，我们发现索拉非尼(Bay 43-9006)抑制Raf可降低cAMP诱导的ERK活性和PKD细胞的增殖。与PKD细胞不同，在正常肾小管细胞中，B-Raf被抑制，cAMP不刺激ERK和细胞增殖。因此，体外证据表明，B-Raf是减缓PKD囊性生长的潜在治疗靶点。为了确定B-Raf的激活是否足以诱导肾囊肿的形成和PKD的发展，我们产生了表达由CMV启动子驱动的构建体的转基因小鼠，该构建物包含一个位于野生型B-Raf或最常见的B-Raf激活突变BRAF(V600E)之前的FLOXED-LacZ-多腺苷基化停止序列。这些转基因小鼠将与表达肾脏特异性Cre重组酶的KSP-Cre小鼠杂交，选择性地在收集管中过表达野生型B-Raf或BRAF(V600E)。该项目的长期目标是确定活性B-Raf的肾脏特异性表达是否会诱导肾囊肿和/或肿瘤的形成，从头开始，和/或加速具有遗传型PKD的小鼠的囊性生长速度。我们将使用ARRA刺激计划的竞争性修订资金聘请一名新的全职研究助理，为期12个月，购买研究用品，并支付动物费用。这些研究的完成将提供重要的概念证据，证明B-Raf是参与囊性扩张的异常小管上皮细胞增殖的中心中间体，并将为开发针对人PKD中B-Raf的小分子药物提供强有力的理论基础。 相关性：申请的补充资金将有助于刺激当地和美国经济，因为它们将为新的实验室人员提供短期支出，为购买研究用品提供资金，并为包括兽医和畜牧业工作人员在内的动物护理提供资金。

Description (as provided by applicant): Polycystic kidney disease (PKD) is characterized by the formation of innumerous fluid-filled cysts that can expand to several centimeters in diameter due to aberrant cell proliferation. The insistent enlargement of cysts disrupts the normal renal architecture, leading to progressive loss of renal function. cAMP agonists, including arginine vasopressin, stimulate the mitogen-activated protein kinase (MAPK) ERK and accelerate the proliferation of cyst-lining epithelial cells. B-Raf, a MAPK kinase kinase appears to be an important intermediate in cAMP-dependent proliferation of PKD cells. cAMP increases B-Raf activity leading to stimulation of MEK/ERK signaling and cell proliferation. In preliminary experiments, we found that Raf inhibition with Sorafenib (Bay 43-9006) decreased cAMP-induced ERK activity and PKD cell proliferation. In contrast to PKD cells, in normal tubule cells, B-Raf is repressed and cAMP does not stimulate ERK and cell proliferation. Thus, in vitro evidence suggests that B-Raf is a potential therapeutic target for slowing cyst growth in PKD. To determine if B-Raf activation is sufficient to induce the formation of renal cysts and development of PKD, we generated transgenic mice that express constructs driven by a CMV promoter that contain a floxed-lacZ-polyadenylation-stop sequence placed in front of wild type B-Raf or BRAF(V600E), the most common B-Raf activating mutation. These transgenic mice will be crossed with Ksp-Cre mice, which express kidney specific Cre recombinase, to selectively overexpress wild type B-Raf or BRAF(V600E) in the collecting ducts. The long term goal of this project is to determine if kidney-specific expression of active B-Raf induces renal cyst and/or tumor formation, de novo, and/or accelerates the rate of cyst growth in mice with genetic forms of PKD. We will use Competitive Revision funds from the ARRA stimulus to hire a new full-time Research Assistant for 12 months, purchase research supplies, and pay animal costs. Completion of these studies will provide important "proof of concept" evidence that B-Raf is a central intermediate for aberrant tubule epithelial cell proliferation involved in cyst expansion and will provide a strong rationale for the development of small molecule drugs that target B-Raf in human PKD. RELEVANCE: The requested supplemental funds will help stimulate the local and United States economy as they will provide short-term expenditures for new laboratory personnel, provide funds to purchase research supplies and provide funding for animal care, including veterinary and animal husbandry staff.