ADAPTATION TO ACIDOSIS/ALKALOSIS IN THE COLLECTING DUCT

这项建议的主要目标是了解细胞和 介导适应性反应的分子机制 肾皮质间质细胞的酸碱平衡 集热管(CCD)。 根据生物体的酸/碱状态，电荷耦合蛋白要么是秘密 或重新吸收HCO3。HCO3的分泌和重吸收发生在两个阶段 具有相反功能极性的ICCs的亚型。对酸中毒的适应 似乎同时涉及对转运蛋白的上调和下调 调节HCO3分泌细胞的这些相反功能和转化 致H秘书。适应的细胞外信号和细胞外信号 其潜在的分子机制还知之甚少。我们的观察 在之前的赠款期间，表明对酸中毒的适应 涉及H-ATPase的mRNA水平与新的结肠相反的变化 H/K-ATPase的异构体和阴离子交换器1与2的mRNA水平。 这些体内变化中的一些可以通过以下方式在培养细胞中模拟 改变胞外pH或增加NH4浓度。在……里面 此外，我们证明了适应伴随着显著的 其他基因表达的变化，其中包括一种新的EP3 前列腺素受体。 我们将与AIM(L)一起探索结肠H，K-ATPase在HCO3中的作用 在电荷耦合器件中进行运输。通过检测分离的ICC中的mRNA和蛋白质水平 亚型和免疫组织化学的亚细胞定位。 将决定在哪种细胞类型和哪种膜上的结肠H，K- ATPase的存在，以及它是如何受酸/碱平衡调节的。目标(2) 我们将检验这样一种假设，即信号产生于 更近端肾单位节段的适应会影响CD功能。在……里面 特别是，我们将研究NH4的慢性变化的影响 浓度和顶端流体成分对H/HCO3传输的影响 关键转运蛋白的表达。目标(3a)的目标是定义角色 新型pH调节的EP3受体在适应 酸中毒/碱中毒。我们将确定相关的信号通路 有了这种受体，它的酸/碱依赖的功能后果 调节和酸/碱平衡调节的机制 表情。根据AIM(3b)，我们将继续系统地寻找 其他调节基因参与了对酸中毒的适应。这样的基因 将使用差异显示进行识别，并对其进行监管 将通过确定其后果来测试其潜力 过度表达和压抑。 预计这些研究获得的新信息将 有助于阐明酸中毒/碱中毒的潜在适应机制 发生在远端肾单位，因此将导致更好的 对肾脏病病因的认识 酸碱平衡紊乱，L样肾小管性酸中毒 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.