Bicarbonate Transport by the Maturing Renal Tubule

描述(申请人提供)：肾脏的集合管有两种主要的细胞类型：主细胞和插层细胞(ICs)，前者负责输送水、盐和钾，后者负责调节酸碱的转运。有两种不同的功能亚型：A-IC通过顶端H+-ATPase和碱侧Cl-/HCO3-交换器(肾脏AE1)分泌质子，而B-IC通过碱侧H+ATPase和顶端Cl-/HCO3-交换器(Pendrin)分泌碳酸氢盐，从而使集合管适应酸碱失衡。代谢性酸中毒将集合管从净HCO3-分泌转变为HCO3-吸收(H+分泌)。HCO3-通量极性的这种逆转与B-IC的主要重塑有关，并依赖于大蛋白Hensin分泌到这些细胞周围的细胞外基质中；我们最近的研究表明，Galectin-3促进Hensin聚合，并在酸中毒时上调。该项目的长期目标是确定Hensin如何调节对代谢性酸中毒的反应。我们将利用一只小鼠，在小鼠中，Hensin的表达从收集的管道细胞中选择性地删除，这会导致kAE1的表达丢失，并导致Al-Awqati实验室产生的远端肾小管酸中毒(DRTA)。在目标1中，我们将通过对野生型和Hensin缺失小鼠的骨髓细胞群进行转录组学分析，研究Hensin如何调节H+/HCO3-转运体的表达。验证将通过RT-qPCR和免疫标记，以及EMSA和荧光素酶报告基因检测在永生化IC细胞系中得到确认。对克隆C中Hensin基因敲除前后的Hensin和整合素a6相关蛋白的蛋白质组学研究将被用来识别Hensin信号通路中的分子。目的2将利用Hensin或Galectin-3缺乏症小鼠的生理学和免疫标记研究，研究Hensin依赖的机制，这些机制介导了对酸中毒和碱中毒的适应。我们将测试Hensin缺乏是否影响悬挂蛋白的调节，如果我们发现Galectin-3缺陷的小鼠有不完整的dRTA，我们将探索Galectin-3促进Hensin聚合的机制。目的3将确定SDF-1/CXCR4通路在酸中毒适应中作为早期反应的作用。SDF-1在酸中毒小鼠的肾脏中上调，我们发现CXCR4受体的阻断阻止了CCDs对体外酸中毒的适应。这些研究的结果可能会改变我们对酸碱生理学的理解，并为迄今尚未解释的远端肾小管酸中毒的原因引出新的机制。

DESCRIPTION (provided by applicant): The collecting duct of the kidney has two major cell types: principal cells which transport water, salt, and potassium, and intercalated cells (ICs) that mediate acid-base transport. There are two functionally distinct subtypes: A-ICs secrete protons via an apical H+ATPase and basolateral Cl-/HCO3- exchanger (kidney AE1), while B-ICs secrete bicarbonate via a basolateral H+ATPase and apical Cl-/HCO3- exchanger (pendrin), thereby enabling the collecting duct to adapt to acid-base disturbances. Metabolic acidosis converts the collecting duct from net HCO3- secretion to HCO3- absorption (H+ secretion). This reversal of polarity of HCO3- flux is associated with major remodeling of the B-ICs and depends on secretion of the large protein hensin into the extracellular matrix surrounding these cells; we have recently shown that galectin-3 facilitates hensin polymerization and is up-regulated during acidosis. The long term goal of this project is to determine how hensin mediates this response to metabolic acidosis. We will make use of a mouse in which hensin expression is selectively deleted from collecting duct cells, which results in a loss of kAE1 expression and the development of distal renal tubular acidosis (dRTA), generated by the Al-Awqati lab. In Aim 1 we will examine how hensin regulates H+/HCO3- transporter expression by performing transcriptome analysis of medullary cell populations from wild-type and hensin-depleted mice. Validation will be confirmed by RT-qPCR and immunolabeling, and EMSA and luciferase reporter gene assays in an immortalized IC cell line. A proteomics study of hensin- and integrin a6-associated proteins in clone C with and without hensin knockdown will be used to identify molecules in the hensin signaling pathway. Aim 2 will examine hensin-dependent mechanisms that mediate adaptation to acidosis and alkalosis, utilizing physiologic and immunolabeling studies in mice with hensin or galectin-3 deficiency. We will test whether hensin deficiency affects pendrin regulation and should we find that galectin-3 deficient mice have an incomplete dRTA, we will explore the mechanisms by which galectin-3 facilitates hensin polymerization. Aim 3 will determine the role of the SDF-1/CXCR4 pathway as an early response in the adaptation to acidosis. SDF-1 is up-regulated in kidneys from acidotic mice, and we are finding that blockade of the CXCR4 receptor prevents adaptation of CCDs to in vitro acidosis. The results of these studies are likely to change our understanding of acid-base physiology and elicit new mechanisms for heretofore unexplained causes of distal renal tubular acidosis.