ADAPTATION TO ACIDOSIS/ALKALOSIS IN THE COLLECTING DUCT

本提案的主要目的是了解细胞和 介导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）。 根据有机体的酸/碱状态，CCD要么秘密地 或者重吸收HCO 3 HCO 3的分泌和重吸收发生在两个 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 介导这些相反的功能和HCO 3分泌细胞的转化 到H+分泌细胞细胞外信号的适应和 对潜在的分子机制知之甚少。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。 这些体内变化中的几种可以在培养的细胞中模拟， 改变细胞外pH或增加NH 4+浓度。在 此外，我们证明，适应伴随着显着的 其他基因表达的变化，其中包括一种新的EP 3 前列腺素受体 目的（1）探讨结肠H，K-ATP酶在HCO_3中的作用 在CCD中运输。通过检测分离的ICC中mRNA和蛋白水平， 亚型和亚细胞定位与免疫组化，我们 将决定结肠H，K- ATP酶的存在，以及它是如何通过酸/碱平衡调节的。有目的（2） 我们将测试的假设，信号产生的结果， 更近端肾单位段的适应影响CCD功能。在 特别是，我们将研究NH 4+的慢性变化的影响 浓度和顶液组成对H+/HCO 3转运的影响， 关键转运蛋白的表达。目标（3a）的目标是定义角色 新的pH调节的EP 3受体在适应 酸中毒/酸中毒我们将确定相关的信号通路 有了这种受体，其酸/碱依赖性的功能后果 调节和酸/碱平衡调节其 表情在目标（3b）下，我们将继续系统地寻找 参与适应酸中毒的其他调节基因。此类基因 将使用差异显示进行鉴定， 将通过确定其后果来测试其潜力。 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/酸中毒的潜在机制 发生在远端肾单位，因此将导致更好的 了解肾脏疾病的病因，导致 酸碱平衡紊乱，如l型肾小管酸中毒 或戏谑综合征

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.