Mechanisms of Transport in Proximal and Distal Tubules

近端和远端肾小管的运输机制

• 批准号: 7868984
• 负责人: William B. Guggino
• 金额: $ 1.64万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-14 至 2011-08-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7868984
• 关键词: Abbreviations; Address; Adult; Affect; Area; Autosomal Dominant Polycystic Kidney; Award; Binding; CLCA2 gene; Calcium; Canis familiaris; Cell membrane; Cell surface; Cilia; Complex; Cyst; Disease; Distal; Endoplasmic Reticulum; Figs - dietary; Genes; Hamsters; Health; Immunoblotting; Immunoprecipitation; Inositol; Integral Membrane Protein; Ions; Kidney; Kidney Failure; Lead; MDCK cell; Mechanics; Mediating; Mutation; Oocytes; Ovary; PKD2 gene; PKD2 protein; Pathogenesis; Pathway interactions; Plasma; Polycystic Kidney Diseases; Proprotein Convertase 1; Proprotein Convertase 2; Proteins; Regulation; Role; Route; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; STIM1 gene; Signal Pathway; Signal Transduction; Thapsigargin; Xenopus oocyte; gene function; kidney cell; mouse model; mutant; public health relevance; receptor; receptor function; tripolyphosphate; water channel

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease arises from mutations in either the PKD1 or the PKD2 gene which encode PC1 or 2. Although it is increasing clear that PC1 is expressed at the plasma membrane and the primary cilium, the cellular localization of PC2 has been controversial. PC2 localizes to the ER, the plasma membrane, and the primary cilium. A popular hypothesis is that PC1 acts as a mechanical receptor and PC2 as a Ca2+ influx channel in the primary cilium to sense renal flow. It has also been suggested that PC2 functions as an intracellular Ca2+ channel. In addition, evidence suggests that PC1 mutants can cause additional mis-localization of PC2 from the cell surface to the ER, which might also contribute even further to the pathogenesis of ADPKD. Therefore, it is important to investigate the role of the ER localized PC2 in regulating intracellular Ca2+ signaling. We propose that PC1 and 2 operate at the ER and at the plasma membrane in concert with the primary cilium to regulate cytosolic Ca2+. Our studies indicate that both PC1 & 2 functionally interact with the IP3 receptor (IP3R) to modulate intracellular Ca2+ signaling. IP3 mediated intracellular Ca2+ signaling is one of the most important intracellular signaling pathways. We also show that a fragment of PC1 which we call PEC inhibits Ca2+ dependent Cl- currents in oocytes suggesting that it inhibits Ca2+ entry. The fragment coimmunoprecipitates with a component of the store operate Ca2+ entry pathway, STIM1. The overall hypothesis is that PC1 & 2 have a precise role in setting the range of intracellular Ca2+ concentrations over which Ca2+ is released from IP3R and simultaneously inhibiting SOCE. We speculate that PC1 inhibition of store-operated Ca2+ entry is necessary to poise renal cells for Ca2+ influx via the primary cilium as a major route of Ca2+ entry. The proposal has three essential parts: Part I. How specifically do the polycystins modulate ER Ca2+ release? Part II. Do the polycystins regulate store-operated Ca2+ entry? Part III. What is the role of IP3R and store-operated calcium entry in tubule and cyst formation? The overall focus of the past 25 years has been on the mechanisms of transport in proximal and distal tubules. During the tenure of this current award, we made fundamental discoveries regarding how ions move through aquaporin water channels and how PC-1 & 2 form a functional complex. We expanded the previous scope into the area of mouse models. The current application takes a new direction into the area of intracellular Ca2+ signaling. PUBLIC HEALTH RELEVANCE Autosomal dominant polycystic kidney disease, a major cause of kidney failure, arises from mutations in more that one gene. When the polycystic kidney genes function abnormally, intracellular calcium is not regulated properly. Thus, understanding normal and abnormal calcium regulation in health and disease, respectively, will ultimately lead to new ways of treating this serious disease.

描述（由申请人提供）：常染色体显性多囊肾病由编码PC 1或2的PKD 1或PKD 2基因突变引起。虽然越来越清楚的是，PC 1的表达在质膜和初级纤毛，PC 2的细胞定位一直有争议。PC 2定位于ER、质膜和初级纤毛。一个流行的假设是，PC 1作为一个机械受体和PC 2作为一个Ca 2+内流通道在初级纤毛感觉肾流量。也有人认为PC 2作为细胞内Ca 2+通道发挥作用。此外，有证据表明，PC 1突变体可导致PC 2从细胞表面到ER的额外错误定位，这也可能进一步促进ADPKD的发病机制。因此，研究ER定位的PC 2在调节细胞内Ca 2+信号中的作用是重要的。我们建议，PC 1和2在ER和质膜与初级纤毛一起调节胞质Ca 2+。我们的研究表明，PC 1和2功能上与IP 3受体（IP 3R）相互作用，以调节细胞内Ca 2+信号。IP 3介导的细胞内Ca ~（2+）信号转导是细胞内最重要的信号转导途径之一。我们还表明，一个片段的PC 1，我们称之为PEC抑制钙依赖性的Cl-电流在卵母细胞中，这表明它抑制Ca 2+进入。该片段与储存操作Ca 2+进入途径STIM 1的组分免疫共沉淀。总的假设是，PC 1和2在设定胞内Ca 2+浓度范围中具有精确的作用，在该范围内Ca 2+从IP 3R释放并同时抑制SOCE。我们推测，PC 1抑制钙池操作的Ca 2+进入是必要的平衡肾细胞的Ca 2+内流通过初级纤毛作为一个主要途径的Ca 2+进入。该提案有三个基本部分：多囊蛋白如何特异性地调节ER Ca 2+释放？第二部分.多囊藻毒素是否调节钙池操作的钙离子内流？第三部分. IP 3R和钙库操纵的钙进入在小管和囊肿形成中的作用是什么？在过去的25年中，总体重点是近端和远端小管的运输机制。在这个奖项的任期内，我们在离子如何通过水通道蛋白水通道以及PC-1和2如何形成功能复合物方面取得了根本性的发现。我们将先前的范围扩展到小鼠模型领域。目前的应用需要一个新的方向进入细胞内Ca 2+信号传导的领域。 公共卫生相关性 常染色体显性遗传性多囊肾病是肾衰竭的主要原因，它是由一个以上基因的突变引起的。当多囊肾基因功能异常时，细胞内钙不能正常调节。因此，分别了解健康和疾病中正常和异常的钙调节，最终将导致治疗这种严重疾病的新方法。