ADAPTATION TO ACIDOSIS/ALKALOSIS IN THE COLLECTING DUCT

该提案的主要目标是了解细胞和 介导对改变的适应性反应的分子机制 肾皮质闰细胞 (ICC) 的酸/碱平衡 收集管（CCD）。 根据生物体的酸/碱状态，CCD 要么秘密 或重吸收HCO3。 HCO3的分泌和重吸收分两次进行 具有相反功能极性的 ICC 亚型。适应酸中毒 似乎涉及转运蛋白的上调和下调 介导 HCO3 分泌细胞的这些相反功能和转化 H 分泌者。细胞外的适应信号 潜在的分子机制尚不清楚。我们的观察 在上一个资助期内表明适应酸中毒 涉及 H-ATP 酶 mRNA 水平与新型结肠的相反变化 H/K-ATP酶亚型和阴离子交换剂 1 与 2 的 mRNA 水平。 其中一些体内变化可以通过以下方法在培养细胞中模拟 改变细胞外 pH 值或增加 NH4 浓度。在 此外，我们证明了适应伴随着显着的 其他基因表达的变化，其中包括新的 EP3 前列腺素受体。 通过目标 (l)，我们将探讨结肠 H,K-ATP 酶在 HCO3 中的作用 CCD 中的传输。通过测量分离的 ICC 中的 mRNA 和蛋白质水平 亚型并通过免疫组织化学进行亚细胞定位，我们 将确定结肠 H,K- 的细胞类型和膜 ATP 酶的存在，以及它如何受酸/碱平衡调节。有目标 (2) 我们将检验以下假设：信号是由于 更近端肾单位段的适应影响 CCD 功能。在 特别是，我们将研究 NH4 慢性变化的影响 浓度和心尖液成分对 H /HCO3 运输的影响 关键转运蛋白的表达。目标 (3a) 的目标是定义角色 新型 pH 调节 EP3 受体适应 酸中毒/碱中毒。我们将确定相关的信号通路 对于这种受体，其酸/碱依赖性的功能后果 酸碱平衡的调节及其调节机制 表达。在目标 (3b) 下，我们将继续系统地搜索 与酸中毒适应有关的其他调节基因。这样的基因 将使用差异显示来识别，及其监管 将通过确定其后果来测试潜力 过度表达和抑制。 预计这些研究获得的新信息将 有助于阐明适应酸中毒/碱中毒的机制 发生在远端肾单位，因此将导致更好的 了解导致肾脏疾病的病因 酸/碱稳态紊乱，如 l 型肾小管性酸中毒 或戏谑综合症。

The main objective of this proposal is to understand the cellular and molecular mechanisms that mediate the adaptive responses to alterations in acid/base balance in intercalated cells (ICCs) of the renal cortical collecting duct (CCD). Depending on the acid/base status of the organism, the CCD either secrets or reabsorbs HCO3. HCO3 secretion and reabsorption take place in two subtypes of ICCs with opposing functional polarity. Adaptation to acidosis seems to involve both up- and down-regulation of the transporters mediating these opposing functions and conversion of HCO3 secreting cells to H+ secretors. The extracellular signals of adaptation and the underlying molecular mechanisms are poorly understood. Our observations during the previous grant period indicate that adaptation to acidosis involves opposite changes in mRNA levels of H-ATPase vs. the novel colonic isoform of H/K-ATPase and in mRNA levels of anion exchangers 1 vs. 2. Several of these in vivo changes can be mimicked in cultured cells by altering extracellular pH or by increasing the concentration of NH4+. In addition, we demonstrated that adaptation is accompanied by significant changes in the expression of other genes among them a novel EP3 prostaglandin receptor. With Aim (l) we will explore the role of the colonic H,K-ATPase in HCO3 transport in the CCD. By measuring mRNA and protein levels in isolated ICC subtypes and by subcellular localization with immunohistochemistry, we will determine in which cell type and in which membrane the colonic H,K- ATPase resides, and how it is regulated by acid/base balance. With Aim (2) we will test the hypothesis that signals generated as a result of adaptation in more proximal nephron segments influence CCD function. In particular, we will examine the effects of chronic changes in NH4+ concentrations and apical fluid composition on H+/HCO3 transport and the expression of key transporters. The goal of Aim (3a) is to define the role of the novel pH-regulated EP3 receptor in adaptation to acidosis/alkalosis. We will determine the signalling pathway associated with this receptor, the functional consequences of its acid/base-dependent regulation and the mechanisms by which acid/base balance regulates its expression. Under Aim (3b) we will continue our systematic search for additional regulatory genes involved in adaptation to acidosis. Such genes will be identified using differential display, and their regulatory potential will be tested by determining the consequences their overexpression and suppression. It is anticipated that the new information acquired by these studies will help to clarify the mechanisms underlying adaptation to acidosis/alkalosis occurring in the distal nephron and consequently will lead to a better understanding of the etiology of renal disorders which result in disturbances of acid/base homeostasis, like type l renal tubular acidosis or Banter's syndrome.