REGULATION OF RENAL BICARBONATE AND CHLORIDE ABSORPTION

慢性酸中毒、肾小球高滤过和钾耗竭均导致 近端肾小管Na/H逆向转运蛋白活性增加， Na/3 HCO 3协同转运蛋白，当转运蛋白 在改变的环境之外进行研究（记忆效应）。 的 介导这些适应的细胞内信号目前是未知的。 我们假设，一些不同的细胞外信号发生， 酸中毒、高滤过和缺钾，都集中在一个共同的 细胞途径，影响增加H/HCO 3转运活性， 转录和翻译水平。 我们建立了 培养的近端小管细胞中的慢性酸中毒模型，其中 在酸性介质中长期孵育会产生一种组织特异性蛋白质 Na/H反向转运蛋白活性的合成依赖性增加。 的一个目的 这种授权是为了定义细胞信号机制， 调节H/HCO 3转运蛋白活性;并检查这些作用 介导慢性酸孵育对Na/H影响的途径 反转运蛋白活性 将被检查的细胞信号包括：1） 磷脂酶C/PKC/细胞Ca; 2）腺苷酸环化酶/CAMP/PKA;和3）细胞 [Na]我们还将尝试建立K. 消耗和超滤。 本建议的第二个目的是 研究Na/H逆向转运蛋白的分子机制， Sardet et al，长期受上述决定因素调节。 具体来说，我们将研究蛋白质丰度，相对分布 微粒体和质膜组分之间的蛋白质， 合成/降解、磷酸化程度、mRNA丰度，以及 mRNA转录速率。 最后，我们得到了来自 人Na/H逆向转运蛋白基因的启动子/增强子区。我们将 利用这一点来研究上述调节器如何改变 通过CAT测定的核蛋白和Na/H反向转运蛋白/增强子区域， 迁移率改变和DNA酶保护测定。

Chronic acidosis, glomerular hyperfiltration, and K depletion all lead to increased activities of the proximal tubular Na/H antiporter and Na/3HCO3 cotransporter, effects that persist when the transporters are studied outside the altered environment (memory effect). The intracellular signals mediating these adaptations are presently unknown. We postulate that a number of different extracellular signals occurring acidosis, hyperfiltration, and K deficiency, all converge on a common cellular pathway that effects increased H/HCO3 transporter activities at the transcriptional and translational levels. We have established a model for chronic acidosis in cultured proximal tubule cells, where chronic incubation in acid medium leads to a tissue-specific, protein synthesis-dependent increase in Na/H antiporter activity. One aim of this grant is to define the cell signalling mechanisms that chronically regulate H/HCO3 transporter activities; and to examine the role of these pathways in mediating the effect of chronic acid incubation on Na/H antiporter activity. Cell signals that will be examined include: 1) phospholipase C/PKC/cell Ca; 2) adenyl cyclase/CAMP/PKA; and 3) cell [Na] and Ph. We will also attempt to establish cell culture models for K depletion and hyperfiltration. The second aim of this proposal is to study the molecular mechanisms by which the Na/H antiporter cloned by Sardet et al, is chronically regulated by the above determinants. Specifically, we will examine protein abundance, relative distribution of protein between microsomal and plasma membrane fractions, rates of synthesis/degradation, degree of phosphorylation, abundance of MRNA, and rates of MRNA transcription. Lastly, we have a 1400 bp fragment from the promoter/enhancer region of the human Na/H antiporter gene. We will use this to study how the above regulators modify interactions between nuclear proteins and the Na/H antiporter/enhancer region by CAT assay, mobility shift, and Dnase protection assays.