ADAPTIVE CHANGES IN KIDNEY H+-ATPASE IN RENAL DISEASE

这项建议的长期目标是：1)阐明 肾酸升高的细胞和分子机制 慢性给药时的排泄，以及2)调查 远端致病的细胞和分子机制 单侧输尿管梗阻后酸化缺陷。 第一个具体目标是调查适应性的基础 酸负荷时H+分泌增加。我们将研究是否 通过分析H+-ATPase含量在酸负荷过程中的变化 嵌入型细胞特异性亚基异构体的表达 大鼠肾脏液泡H+-ATPase免疫分析及RNA印迹分析 酸负荷。我们研究了H~+-的细胞基础的几个方面。 酸负荷时的ATPase极化。我们将努力孤立和 一种新发现的诱导H+-ATPase的血清因子的特征 慢性酸负荷的两极分化；我们将尝试开发一个模型 用于管状悬浮液或原代培养物的极化 嵌入细胞以允许H+-ATPase分布的改变 在体外检测；我们将检测细胞骨架的变化 在H+-ATPase极化反应中发生组织；我们将 检测Rab低分子量GTP结合蛋白在 肾脏参与H+-ATPase靶向质膜和 调节极化。我们将研究H+水平的变化是否- ATPase调节蛋白(抑制物和激活物)可能在 适应性过程中H~+-ATPase动力学性质的变化 对酸负荷的反应。 第二个具体目标是调查导致 单侧输尿管梗阻的酸化缺陷。我们会 已确定H+-ATPase极化是否存在异常 使用免疫细胞化学，我们将确定H+-ATPase的变化 数量是通过检查嵌入细胞的表达来实现的- 空泡H+-ATPase亚基异构体的免疫分析 RNA印迹；我们研究了H+-ATPase的动力学变化 通过检查H+-水平的变化是否导致缺陷 ATPase调节蛋白(抑制物和激活物)存在于梗阻中。 这些研究将促进我们对生理和心理问题的理解 肾适应中的细胞机制及细胞和 梗阻性肾脏疾病离子转运缺陷的分子基础。

The long term objectives of this proposal are: 1) To elucidate the cellular and molecular mechanisms responsible for increased renal acid excretion during chronic administration, and 2) To investigate the cellular and molecular mechanisms responsible for the distal acidification defect following unilateral ureteral obstruction. The first specific aim is to investigate the basis for adaptational increase in H+ secretion in acid loading. We will examine whether changes in H+-ATPase content occur in acid loading by analyzing the expression of an intercalated cell-specific subunit isoform of the vacuolar H+-ATPase using immunoassays and RNA blots in rat kidneys after acid loading. We study several aspects of the cellular basis for H+ - ATPase polarization in acid loading. We will endeavor to isolate and characterize a newly identified serum factor that induces H+ ATPase polarization in chronic acid loading; we will attempt to develop a model for polarization in tubular suspensions or primary cultures of intercalated cells to allow changes in H+ -ATPase distribution to be examined in vitro; we will examine whether changes in cytoskeletal organization occur in the H+-ATPase polarization response; and we will examine whether the rab low molecular weight GTP-binding proteins in kidney participate in H+-ATPase targeting to plasma membrane and regulated polarization. We will examine whether changes in levels of H+- ATPase regulatory proteins (inhibitor and activator) may have a role in altering kinetic properties of the H+-ATPase during the adaptational response to acid loading. The second specific aims is to investigate the mechanisms responsible for the acidification defect in unilateral ureteral obstruction. We will determined whether abnormalities in H+-ATPase polarization are present using immunocytochemistry; we will determined if changes in H+-ATPase quantity occur by examining the expression of an intercalated cell- specific subunit isoform of the vacuolar H+-ATPase using immunoassays and RNA blots; and we investigate whether kinetic changes in the H+-ATPase contribute to the defect by examining whether changes in level of H+- ATPase regulatory protein (inhibitor and activator) occur in obstruction. These studies will advance our understanding of the physiologic and cellular mechanisms operating in renal adaptation and the cellular and molecular basis for ion transport defects in obstructive renal disease.