Maturation of K transport in the distal nephron

说明(申请人提供)：全身钾含量取决于摄入量和排出量的平衡，后者主要受连接(CNT)和收集管(CNT)和集合管的肾脏K分泌调节。在CNT和皮质集合管中，存在两种顶端K选择性通道：一种是低电导SK通道，一种是高电导Ca/牵张激活的BK通道(或MAXI-K通道)。而SK通道仅限于钠、钾转运的主细胞，而BK通道在酸碱转运的嵌入细胞中密度最高。我们实验室最近发表的和初步的研究提供了令人信服的证据，证明SK通道介导基线K分泌，BK通道参与流刺激的钙依赖性K分泌。此外，我们已经报道，在出生后的发育过程中以及对饮食中钾摄入量的长期变化做出反应时，Ccd中血流刺激的钾分泌速率和BK通道的表达水平是受调节的。根据这一数据，我们假设远端肾单位血流刺激的净K分泌是由嵌入细胞中的BK通道以钙依赖的方式介导的，而在发育过程中和对表观遗传因素的响应中，通道活性的调节是由BKα亚基剪接变体和β亚基的差异表达决定的。这一假说将使用包括功能(体外微灌注、荧光功能成像、电生理学)、生化和分子方法在内的综合方法进行验证，这些方法通常应用于哺乳动物的CCDs。在SA1中，我们将确定短暂的血流诱导的细胞内钙浓度增加和持续刺激钾分泌之间的关系，重点是导致这种反应的钙的来源、时间和来源，以及钙/钙调蛋白激酶在信号转导通路中的作用。在SA2中，我们将研究正常出生后发育过程中通道变异和异构体表达的分子调控，以及对饮食中钾摄入量和代谢性酸碱紊乱的急剧变化的响应；这些研究将在观察到的对流动刺激的钾分泌的影响的背景下进行解释。这项研究的结果将为健康和疾病(例如Bartters综合征)肾脏钾适应的分子生理学提供新的见解，并扩展我们对上皮细胞功能的生物力学调节的新理解。总结：我们最近在肾小管中发现了一个“BK”通道(孔)，它在高尿流率时被激活，这使得细胞受到剪切、拉伸和细胞钙浓度增加的影响。这一应用的目的是为了加深我们对BK通道在健康和疾病条件下在哺乳动物肾脏中的表达、个体发育、定位和调控的理解。

DESCRIPTION (provided by applicant): Total body K content depends on the balance between intake and output, the latter regulated primarily by renal K secretion in the connecting (CNT) and collecting ducts. The magnitude of K secretion in these segments is determined by the electrochemical gradient and the permeability of the apical membrane to K. Two apical K-selective channels have been identified in the CNT and cortical collecting duct (CCD): a low- conductance SK and a high-conductance Ca/stretch-activated BK (or maxi-K) channel. Whereas the SK channel is restricted to Na and K transporting principal cells, the density of BK channels is highest in acid- base transporting intercalated cells. Recent published and preliminary studies by our lab have provided compelling evidence that the SK channel mediates baseline K secretion and that the BK channel participates in flow-stimulated Ca-dependent K secretion. Furthermore, we have reported that the rates of flow-stimulated K secretion and levels of BK channel expression in the CCD are regulated during postnatal development and in response to chronic changes in dietary K intake. Based on this data, we hypothesize that flow-stimulated net K secretion in the distal nephron is mediated by BK channels in intercalated cells in a Ca-dependent manner, and that the regulation of channel activity during development and in response to epigenetic factors is determined by the differential expression of BK alpha subunit splice variants and beta subunits. This hypothesis will be tested using an integrated approach including functional (in vitro microperfusion, fluorescence functional imaging, electrophysiology), biochemical, and molecular approaches, generally applied to mammalian CCDs. In SA1, we will define the relationship between the transient flow-induced increase in cell Ca concentration and sustained stimulation of K secretion, focusing on the, timing and source of the Ca leading to the response, and the role of Ca/calmodulin kinase in the signal transduction pathway. In SA2, we will examine the molecular regulation of channel variant and isoform expression during normal postnatal development, and in response to acute changes in dietary K intake and metabolic acid-base disturbances; these studies will be interpreted in the context of observed effects on flow-stimulated K secretion. The results of this investigation should provide new insight into the molecular physiology underlying renal K adaptation in health and disease (e.g., Bartters syndrome), and extend our emerging understanding of the biomechanical regulation of epithelial cell function. Lay summary: We have recently identified a "BK" channel (pore) in kidney tubules that is activated at high urinary flow rates, which subject the cells to shear, stretch and an increased cell calcium concentration. The goal of this application is to enhance our understanding about the expression, ontogeny, localization, and regulation of BK channels in the mammalian kidney under conditions of health and disease.