Role of TRP Channels on Collecting Duct Calcium Dynamics

描述（由申请方提供）：本提案的总体目标是阐明钙渗透性TRPV通道亚型在调节肾皮质集合管中机械诱导的细胞内Ca、[Ca]i、动力学和Ca重吸收中的作用。机械应力，例如流体剪切应力或低渗应力，在晚期远端小管和皮质集合管（CCD）中变化很大，特别是在病理生理状态（例如，低钠血症、尿崩症、糖尿病、利尿剂给药），这将促成导致改变的Ca平衡的低尿和/或高尿状态。我们已经暗示两个TRPV通道是机械敏感的和中央调节机械刺激的钙离子进入和重吸收小鼠CCD。为实现这一总体目标，提出了以下四个具体目标：1.确定机械调节的Ca内流途径（Ca成像，膜片钳）在小鼠CCD细胞[Ca]i动力学控制中的相对作用和药理学/电生理学特性; 2.鉴定在小鼠CCD细胞中表达的TRPV通道亚型（RT-PCR、北方印迹、免疫荧光、免疫印迹）并确定哪些特异性亚型参与机械调节的[Ca]动力学; 3.描述特异性机械调节TRPV通道在小鼠CCD细胞中Ca重吸收中的相对贡献（过表达/siRNA敲低）并检查通道的调节机制（磷脂酶、蛋白激酶）;和4.评价TRPV亚型的膜定位/运输（共聚焦显微镜/免疫荧光、生物素化-链霉亲和素纯化）以及与辅助蛋白（免疫沉淀/下拉）（包括其他TRP）的相关性对机械调节[Ca]i动力学和Ca重吸收的作用。该研究的结果具有重要的健康相关性，因为它将为肾小管中钙信号传导和重吸收的机制提供新的见解，并为改变的病理生理条件调节肾细胞中钙重吸收的机制提供关键的新理解。

DESCRIPTION (provided by applicant):The overall goal of this proposal is to elucidate the role of Ca-permeable TRPV channel isoforms in regulating mechanically-induced intracellular Ca, [Ca]i, dynamics and Ca reabsorption in renal cortical collecting duct. Mechanical stresses, such as fluid shear stress or hypoosmotic stress, vary widely in late distal tubule and cortical collecting duct (CCD) especially during pathophysiological states (e.g., hyponatremia, diabetes insipidus, diabetes mellitus, diuretic administration), which will contribute to hypocaliuric and/or hypercaliuric states that lead to altered Ca balance. We have implicated two TRPV channels as being mechanosensitive and central to regulating mechano-stimulated Ca entry and reabsorption in mouse CCD. The following four specific aims are proposed to achieve the overall goal: 1. To determine the relative role and pharmacological/ electrophysiological properties of mechanoregulated Ca entry pathways (Ca imaging, patch clamp) in control of [Ca]i dynamics in mouse CCD cells; 2. To identify TRPV channel isoforms expressed in mouse CCD cells (RT-PCR, Northern blot, immunofluorescence, immunoblots) and to determine which specific isoforms participate in mechanoregulated [Ca] dynamics; 3. To delineate the relative contribution of specific mechanoregulated TRPV channels in Ca reabsorption in mouse CCD cells (overexpression/siRNA knockdown) and to examine the mechanism of regulation of the channels (phospholipases, protein kinases); and 4. To evaluate the role of membrane localization/trafficking of TRPV isoforms (confocal microscopy/immunofluorescence, biotinylation-streptavidin purification) and the association with accessory proteins (immunoprecipitation/pull-downs), including other TRPs, on mechanoregulated [Ca]i dynamics and Ca reabsorption. The outcome of the study has important health relatedness as it will provide new insights into the poorly understood mechanisms of calcium signaling and reabsorption in renal tubules and provide a critical new understanding of the mechanism by which altered pathophysiological conditions regulate calcium reabsorption in renal cells.