The Role of the Calcium Activated Potassium Channel, KCa3.1, in the Pathogenesis

描述(申请人提供)：常染色体显性遗传性多囊肾病(ADPKD)是终末期肾脏疾病的常见原因。ADPKD是由两个基因中的一个突变引起的，这两个基因分别由多囊蛋白1(PC)和2编码。PC1和PC2两个拷贝的丢失与突变细胞内钙流入的减少有关，突变细胞被认为通过促进肾上皮细胞的生长和通过囊性纤维跨膜传导调节因子(CFTR)刺激顶端氯的分泌来介导囊形成和囊扩大。虽然CFTR是由环磷酸腺苷直接调节的，是将氯离子分泌到囊腔的主要通道，但我们有证据表明，钙激活的通道KCa3.1在CFTR刺激的肾上皮细胞氯离子外流中起着关键作用；KCa3.1通过调节K的外流，通过将膜电位设置为更负的值来维持氯-分泌的电化学驱动力。KCa3.1通道在许多细胞的增殖中也起着重要作用。因此，KCa3.1的抑制剂可能具有双重作用，既能抑制肾上皮细胞的增殖，又能抑制CFTR分泌的氯离子。这一应用的重点是研究KCa3.1在PKD发病机制中的作用，并确定KCa3.1是否为减缓疾病进展的可行药物靶点。在特定的目的(SA)1中，我们将测试：(I)是否通过siRNA和过表达脂质磷酸酶肌管蛋白相关蛋白6(MTMR6)来抑制KCa3.1是否抑制MDCK细胞的Cl-分泌；(Ii)KCa3.1是否定位于顶端或基底部；(Iii)DCEBIO是否直接激活KCa3.1，刺激跨MDCK单层的Cl-分泌。在(B)中，我们将确定已知的影响KCa3.1通道活性的基因(MTMR6和核苷二磷酸激酶B[NDPK-B])是否作为修饰物调节CFTR分泌氯离子和体外囊泡生长。在(C)中，我们将把对MDCK细胞中SA1A、B的观察从野生型和PKD-/-细胞扩展到人和小鼠肾小管细胞，并确定PKD1或PKD2突变是否影响KCa3.1的调节、功能或活性。在SA2中，我们将在PKD1和PKD2的小鼠模型中确定KCa3.1与包囊形成的相关性。我们将确定用KCa3.1的特异性抑制剂TRAM-34治疗小鼠是否能阻止PKD1和PKD2模型小鼠的囊性形成和进展为肾功能衰竭。常染色体显性遗传性多囊肾病是终末期肾病的常见病因。随着时间的推移，这些囊变得越来越多，体积越来越大，取代了正常的肾组织，导致肾功能丧失。在这项建议中，我们正在研究一种钾通道，我们有证据表明，钾通道对盐分和水进入囊腔的移动非常重要，这被认为是囊腔随时间扩大的主要机制之一。此外，这一通道也可能在这些细胞的增殖或生长中发挥重要作用，这也是囊性形成的重要因素。研究这一通道的兴奋之处在于，抑制这一通道的药物(KCa3.1)已经存在，并正在进行人体试验，没有任何重大副作用。因此，如果抑制这一通道在ADPKD小鼠模型中显示出希望，我们就可以迅速进入临床来评估患者的治疗。

DESCRIPTION (provided by applicant): Autosomal-dominant polycystic kidney disease (ADPKD) is a common cause of end stage kidney disease. ADPKD is caused by mutations in one of two genes, PKD1 or PKD2, which are encoded by polycystin 1 (PC) and 2 respectively. Loss of both copies of PC1 or PC2 is associated with a decrease in of Ca2+ influx into mutant cells which is thought to mediate cyst formation and cyst enlargement by stimulating the enhanced growth of renal epithelia and the stimulation of apical chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). While the CFTR is directly regulated by cyclic AMP and is the predominant channel that secretes Cl- into the cyst lumen, we have evidence that a Ca2+-activated channel, KCa3.1, plays a critical role in CFTR-stimulated Cl- efflux in renal epithelia; by mediating the outflux of K+, KCa3.1 maintains the electrochemical driving force for Cl- secretion by setting the membrane potential at more negative values. KCa3.1 channels also play an important role in proliferation of a number of cells. Thus, inhibitors of KCa3.1 may serve a dual function to both inhibit the proliferation of renal epithelia and to inhibit Cl- secretion by the CFTR. The focus of this application is to study the role of KCa3.1 in the pathogenesis of PKD and to determine whether KCa3.1 is a viable drug target to slow disease progression. In Specific Aim (SA) 1 we will test: (i) whether inhibiting KCa3.1 by siRNA and by overexpression of a lipid phosphatase, myotubularin related protein 6 (MTMR6), inhibits Cl- secretion by MDCK cells; (ii) whether KCa3.1 is localized apically or basolaterally; (iii) whether direct activation of KCa3.1 by DCEBIO, stimulates Cl- secretion across an MDCK monolayer. In (B) we will determine whether genes known to affect KCa3.1 channel activity (MTMR6 and nucleoside diphosphate kinase B [NDPK-B]) function as modifiers to regulate Cl- secretion by the CFTR and cyst growth in vitro. In (C) we will extend the observations in SA1 A,B in MDCK cells to human and mouse renal tubule cells from wild type and PKD-/- cells and determine whether mutation in PKD1 or PKD2 affects KCa3.1 regulation, function, or activity. In SA2, we will determine the relevance of KCa3.1 to cyst formation in mouse models of PKD1 and PKD2. We will determine whether treatment of mice with TRAM-34, a specific inhibitor of KCa3.1, blocks cyst formation and progression to renal failure in mice models for PKD1 and PKD2. Autosomal-dominant polycystic kidney disease affects is a common cause of end stage kidney disease. Over time, these cysts become more numerous and larger in size and replace normal kidney tissue leading to loss of renal function. In this proposal, we are studying a potassium channel that we have evidence is important for the movement of salt and water into the cyst lumen which is thought to be one of primary mechanism whereby cysts enlarge over time. In addition, this channel may also play an important role in proliferation or growth of these cells, which is also important for cyst formation. The excitement in studying this channel is that drugs that inhibit this channel (KCa3.1) already exists and are in human trials without any major side effects. Thus, if inhibiting this channel shows promise in mouse models of ADPKD, we can rapidly move into the clinic to assess treatment in patients.