Bicarbonate Transport by the Maturing Renal Tubule

描述（由申请人提供）：代谢性酸中毒是一种常见但可能危及生命的疾病，当过多的酸在血液中积聚时发生。肾脏是主要负责控制血液中酸水平的器官。皮质集合管（CCD）是肾小管的一个关键部分，它适应酸的水平并将其泵出体外。长期目标是确定CCD的嵌入细胞如何通过改变其酸和碳酸氢盐的运输来适应酸碱紊乱。正常分泌碳酸氢盐的兔CCD暴露于pH 6.8孵育3小时后分泌酸。新的蛋白hensin表达在细胞外基质（ECM）周围的适应B-嵌入细胞（IC）。目的1探讨环孢素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式/反式异构酶活性来抑制hensin聚合。使用这些功能的特异性抑制剂的研究在培养的IC和CCD中进行，在pH 6.8 × 3 h下孵育以模拟酸中毒。亲环素的“敲低”将决定哪种亲环素在培养的IC中聚合汉辛素中是重要的，以及这种亲环素的缺陷是否会导致酸中毒。目的2研究半乳糖凝集素-3在亨辛聚合中的作用。半乳糖凝集素-3在体外可帮助甘草素。这一目标将确定哪些细胞表达半乳糖凝集素-3以及酸中毒期间是否存在刺激。目的将确定半乳糖凝集素-3是否与适应CCD中ECM中的hensin缔合，以及显性负性半乳糖凝集素-3是否阻止hensin聚合和CCD中B-IC的适应。这一目标将确定表达的亲环素和半乳糖凝集素-3是否可以在汉辛素的体外聚合中合作。目的3确定哪些整合素在IC中表达，哪些整合素与hensin相互作用。目的4研究hensin聚合在CCD IC适应代谢性代谢紊乱中的作用，假设代谢紊乱通过降解hensin和允许B-IC分泌碳酸氢盐来纠正异常而减少hensin聚合。这些研究应该显示汉辛蛋白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.