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

钙激活钾通道 KCa3.1 在发病机制中的作用

• 批准号: 7484997
• 负责人: EDWARD Y SKOLNIK
• 金额: $ 16.61万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7484997
• 关键词: Adverse effects; Affect; Apical; Autosomal Dominant Polycystic Kidney; Calcium-Activated Potassium Channel; Canis familiaris; Cell Count; Cell Line; Cells; Chloride Ion; Chlorides; Clinic; Cyclic AMP; Cyst; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Disease Progression; Distal; Drug Delivery Systems; End stage renal failure; Epithelial Cells; Epithelium; Figs - dietary; Forskolin; Genes; Growth; Human; In Vitro; Kidney; Kidney Failure; Lipids; Localized; MDCK cell; Measurement; Mediating; Membrane Potentials; Messenger RNA; Movement; Mus; Mutation; Nucleoside diphosphate kinase B; PKD1 gene; Pathogenesis; Patients; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Play; Polycystic Kidney Diseases; Polymerase Chain Reaction; Potassium Channel; Proprotein Convertase 1; Proprotein Convertase 2; Protein Overexpression; Proteins; Regulation; Renal function; Renal tubule structure; Role; Small Interfering RNA; Sodium Chloride; Testing; Thinking; Time; Tissues; Water; Work; cell growth; driving force; follow-up; inhibitor/antagonist; monolayer; mouse model; mutant; myotubularin; polycystic kidney disease 1 protein; renal epithelium; size

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.

描述（由申请方提供）：常染色体显性多囊肾病（ADPKD）是终末期肾病的常见原因。ADPKD是由PKD 1或PKD 2两个基因之一突变引起的，PKD 1或PKD 2分别由多囊蛋白1（PC）和2编码。PC1或PC2两个拷贝的丢失与突变细胞中Ca2+内流的减少相关，这被认为是通过刺激肾上皮细胞的生长增强和通过囊性纤维化跨膜传导调节因子（CFTR）刺激顶端氯化物分泌来介导囊肿形成和囊肿扩大。虽然CFTR直接受环AMP调节，并且是将Cl-分泌到囊肿腔中的主要通道，但我们有证据表明，Ca2+激活通道KCa3.1在肾上皮细胞中CFTR刺激的Cl-流出中起关键作用;通过介导K+的流出，KCa3.1通过将膜电位设置为更负的值来维持Cl-分泌的电化学驱动力。KCa 3.1通道在许多细胞的增殖中也起重要作用。因此，KCa 3.1抑制剂可能具有双重功能，既抑制肾上皮细胞的增殖，又抑制CFTR的Cl-分泌。本申请的重点是研究KCa3.1在PKD发病机制中的作用，并确定KCa3.1是否是减缓疾病进展的可行药物靶点。在特异性目的（SA）1中，我们将测试：（i）通过siRNA和脂质磷酸酶、肌管蛋白相关蛋白6（MTMR 6）的过表达抑制KCa 3.1是否抑制MDCK细胞的Cl-分泌;（ii）KCa 3.1是否位于顶部或基底外侧;（iii）DCEBIO直接激活KCa 3.1是否刺激MDCK单层的Cl-分泌。在（B）中，我们将确定已知影响KCa 3.1通道活性的基因（MTMR 6和核苷二磷酸激酶B [NDPK-B]）是否作为调节剂在体外调节CFTR的Cl-分泌和囊肿生长。在（C）中，我们将在MDCK细胞中SA 1A、B中的观察结果从野生型和PKD-/-细胞扩展到人和小鼠肾小管细胞，并确定PKD 1或PKD 2中的突变是否影响KCa 3.1调节、功能或活性。在SA2中，我们将确定KCa 3.1与PKD 1和PKD 2小鼠模型中囊肿形成的相关性。我们将确定用KCa3.1的特异性抑制剂TRAM-34治疗小鼠是否能阻断PKD 1和PKD 2小鼠模型中囊肿的形成和肾衰竭的进展。常染色体显性多囊肾病是终末期肾病的常见原因。随着时间的推移，这些囊肿变得越来越多，体积越来越大，并取代正常的肾组织，导致肾功能丧失。在这项提案中，我们正在研究一种钾通道，我们有证据表明它对盐和水进入囊肿腔的运动很重要，这被认为是囊肿随时间扩大的主要机制之一。此外，该通道还可能在这些细胞的增殖或生长中发挥重要作用，这对囊肿形成也很重要。研究这一通道的兴奋之处在于，抑制这一通道（KCa3.1）的药物已经存在，并且正在进行人体试验，没有任何重大副作用。因此，如果抑制该通道在ADPKD小鼠模型中显示出希望，我们可以迅速进入临床以评估患者的治疗。