Bicarbonate Transport by the Maturing Renal Tubule

描述(申请人提供)：代谢性酸中毒是一种常见但可能危及生命的疾病，当血液中积累过多的酸时就会发生。肾脏是主要负责控制血液中酸性物质水平的器官。皮质集合管是肾小管的一个关键部分，它适应酸液的水平并将其泵出体外。长期目标是通过改变细胞对酸和碳酸氢盐的运输来确定细胞如何适应酸碱失衡。兔CCDs通常分泌碳酸氢盐，在pH值为6.8的条件下孵育3h，会分泌酸性物质。新的Hensin蛋白在适应B细胞周围的细胞外基质(ECM)中表达。目的1研究环孢菌素A(CsA)如何通过阻止B-ICs的适应而引起远端肾小管酸中毒。CsA通过抑制亲环素的脯氨酸顺反异构酶活性来抑制Hensin聚合。使用这些功能的特定抑制剂的研究是在培养的ICs和CCDs中进行的，培养的ICs和CCDs在pH 6.8x3h孵育以模拟酸中毒。亲环素的“敲除”将决定在培养的IC中，哪种亲环素是聚合亨氏蛋白的重要因素，以及亲环素的缺陷是否会导致酸中毒。目的2研究Galectin-3在Hensin聚合中的作用。Galectin-3在体外可以促进Hensin的聚合。这一目标将决定哪些细胞表达Galectin-3，以及在酸中毒期间是否有刺激。目的将确定Galectin-3是否与Hensin在适应性CCD的ECM中结合，以及显性负Galectin-3是否阻止Hensin聚合和B-ICs在CCD中的适应性。这一目的将确定表达的亲环素和Galectin-3是否可以在Hensin的体外聚合中协同作用。目的3确定哪些整合素在ICs中表达，哪些与Hensin相互作用。目的4研究Hensin聚合在CCDIC适应代谢性碱中毒中的作用，假设碱中毒通过降解Hensin和允许B-ICs分泌碳酸氢盐来纠正这种异常，从而降低Hensin聚合。这些研究应该表明Hensin和相关蛋白质如何调节肾脏对酸碱失衡的适应。

DESCRIPTION (provided by applicant): Metabolic acidosis is a common but potentially life-threatening disorder that occurs when too much acid accumulates in the blood. The kidney is the organ that is primarily concerned with controlling the level of acid in the blood. The cortical collecting duct (CCD) is a key segment of kidney tubule that adapts to the level of acid and pumps it out of the body. The long term goal is to determine how intercalated cells of the CCD adapt to acid-base disturbances by changing their transport of acid and bicarbonate. Rabbit CCDs, which normally secrete bicarbonate, exposed to 3h incubation at pH 6.8 secrete acid. The novel protein hensin is expressed in the extracellular matrix (ECM) surrounding adapting B-intercalated cells (ICs). Aim 1 examines how Cyclosporin A (CsA) causes a distal renal tubular acidosis by preventing adaptation of B-ICs. CsA inhibits hensin polymerization via an inhibition of the proline cis/trans isomerase activity of cyclophilin. Studies using specific inhibitors of these functions are performed in cultured ICs and CCDs incubated at pH 6.8 x 3 h to simulate acidosis. A "knockdown" of cyclophilin will determine which cyclophilin is important in polymerizing hensin in cultured ICs and whether a defect in this cyclophilin causes acidosis. Aim 2 examines the role of galectin-3 in hensin polymerization. Galectin-3 can help polymerize hensin in vitro. This aim will determine which cells express galectin-3 and whether there is stimulation during acidosis. The aim will determine if galectin-3 associates with hensin in the ECM in the adapting CCD and whether a dominant negative galectin-3 prevents hensin polymerization and adaptation of B-ICs in CCD. This aim will establish whether expressed cyclophilin and galectin-3 can cooperate in the in vitro polymerization of hensin. Aim 3 determines which integrins are expressed in ICs and which interact with hensin. Aim 4 examines the role of hensin polymerization in the adaptation of CCD ICs to metabolic alkalosis, with the hypothesis that alkalosis reduces hensin polymerization by degrading hensin and allowing B-ICs to secrete bicarbonate to correct the abnormality. These studies should show how hensin and associated proteins mediate the kidney's adaptation to acid-base disturbances.