ROLE OF CYCLIC AMP IN CYST CELL PROLIFERATION

描述：（直接摘自申请）常染色体显性多囊肾病（ADPKD）是一种缓慢进展的肾脏疾病，其特征是源自肾小管的无数上皮囊肿的发展和稳定扩大。小管上皮细胞的增殖是囊肿发生和扩大的重要过程。新的证据表明，ADPKD囊肿上皮细胞可能对环状ANT的有丝分裂作用异常敏感。这个项目是基于我们的新发现，cAMP刺激ADPKD囊肿上皮细胞的体外增殖，而PD98059，一种高度选择性的丝裂原激活的erk通路抑制剂，阻断了核苷酸的这些作用。相比之下，正常人肾皮质细胞（HKC）在体外对cAMP没有增殖反应。本研究的中心目的是验证以下假设：在ADPKD中，细胞内MAP水平升高会加速肾囊肿的扩大。该核苷酸通过激活蛋白激酶A，刺激MAP激酶通路，导致持续的上皮有丝分裂和细胞增殖。以下具体目标将验证这一假设：1)使用来自人ADPKD囊肿和正常人肾皮质（HKC）的上皮细胞，以及cAMP敏感和不敏感的NOCK细胞株来确定增殖对细胞内cAMP浓度的依赖性以及细胞凋亡在整体增殖反应中的作用；2)确定erk1、2、MEK和Raf-I在camp介导的肾上皮细胞增殖增加中的参与程度；3)确定rap - 1和B-Raf在camp介导的肾上皮细胞增殖增加中的作用；4)确定细胞内原位cAMP水平的变化是否会改变遗传性PKD的Han: SPRD大鼠和CD1 pCY小鼠的疾病进展速度。实现这些目标需要应用分子生物学、细胞生物学、生物化学、药理学以及细胞和综合生理学等技术。这些研究将对控制肾囊肿扩大的分子机制产生新的见解，并为设计治疗措施提供框架，以对抗囊肿对肾实质的进行性破坏。

Description: (Taken directly from the application) Autosomal dominant polycystic kidney disease (ADPKD) is a slowly progressive renal disorder characterized by the development and steady enlargement of innumerable epithelial cysts that are derived from renal tubules. Proliferation of tubule epithelial cells is an important process in the generation and enlargement of cysts. New evidence indicates that ADPKD cyst epithelial cells may be unusually sensitive to a mitogenic action of cyclic ANT. This project is based on our new findings that cAMP stimulates the in vitro proliferation of ADPKD cyst epithelial cells and that PD98059, a highly selective inhibitor of the mitogen-activated-ERK-pathway, blocks these effects of the nucleotide. By contrast, cells of normal human kidney cortex (HKC) do not proliferate in response to cAMP in vitro. The central thrust of this project is to test the hypothesis that: In ADPKD, renal cyst enlargement is accelerated by elevated levels of intracellular MAP. This nucleotide, through the activation of protein kinase A, stimulates the MAP kinase pathway leading to sustained epithelial mitogenesis and cellular proliferation. The following Specific Aims will test this hypothesis: 1) Epithelial cells from human ADPKD cysts and normal human kidney cortex (HKC), and cAMP sensitive and insensitive strains of NOCK cells will be used to determine the dependence of proliferation on intracellular concentrations of cAMP and the role of apoptosis in the overall proliferative response; 2) Determine the level of involvement of ERK 1,2, MEK and Raf-I in the cAMP-mediated increase in proliferation of responsive renal epithelial cells; 3) Determine the involvement of Rap-I and B-Raf in cAMP-mediated increase in proliferation of responsive renal epithelial cells; 4) Determine if changes in intracellular cAMP levels in situ alter the rate of disease progression in Han: SPRD rats and CD1 pCY mice with inherited forms of PKD. Achievement of these aims requires application of the techniques of molecular biology, cell biology, biochemistry, pharmacology and cellular and integrative physiology. These studies will yield new insights into the molecular mechanisms governing the enlargement of renal cysts and provide a framework for the design of therapeutic measures to combat the progressive destruction of renal parenchyma by cysts.