Role of C1C5 in Endocytosis and NHE3 Trafficking

登特氏病是一种以低分子量蛋白尿，氨基酸尿， 糖尿、多尿、高磷酸盐尿（Fanconi综合征）以及高钙尿、肾钙质沉着症， 肾结石登特氏病已被证实是由CICN-5基因突变引起的， 其产物是CIC-5，一种在肾小管中以最高丰度表达的电压门控氯离子通道 近端肾单位我们在CICN-5基因敲除小鼠模型中的初步研究表明， 概括了Dent病的特征，包括高钙尿、氨基酸尿、糖尿、低钙尿、高钙尿、 分子量蛋白尿、肾钙化和肾衰竭。拟议的研究将有助于 定义这些再吸收缺陷的潜在分子基础。 具体目标1：其他CIC通道或CIC-5交换如何调节近端细胞中的顶端内吞作用？ 肾小管细胞？凌晨1 CIC-3和/或CIC-4是否有助于细胞内的残余酸化和顶端内吞作用？ ko细胞？18. CIC-3和CIC-4的过表达能否恢复CIC-5 ko细胞的白蛋白摄取？并 在ko细胞中CIC-5的氯/质子交换活性恢复正常白蛋白摄取？具体目标二： 什么是CIC-5的损失和巨蛋白在ko近端小管下调之间的关系 细胞？2A.巨蛋白在ko细胞中的半衰期短于野生型细胞吗？2B.回收行为如何 巨蛋白在KO和WT细胞中的改变？2C.内体pH或CIC-5的缺乏是否调节巨蛋白 动力学？具体目标3。CIC-5的缺失如何导致NHE 3分布/活性的变化 和其他顶端转运蛋白3A.肾近端小管、回肠或空肠中NHE 3活性是否降低 老鼠？3B.近曲小管细胞中NHE 3的稳态水平和动力学是什么？3C.什么 是稳态水平和动力学的NaPi-IIa在KO近端小管细胞？3D，核内体pH，或 缺乏CIC-5，调节NHE 3或NaPi-IIa动力学？ 这项研究的长期目标是了解分子事件如何导致表型 Dent's disease的症状

Dent's disease, is a disorder that is characterized by low molecular weight proteinuria, aminoaciduria, glycosuria, polyuria, hyperphosphaturia (Fanconi syndrome) as well as hypercalciuria, nephrocalcinosis, nephrolithiasis. Dent's disease has been established to be caused by mutations of the CICN-5 gene, the product of which is CIC-5 a voltage-gated chloride channel expressed in highest abundance in renal tubules of the proximal nephron. Our preliminary studies in a CICN-5 knockout mouse model, shows that it recapitulates the features of Dent's disease including hypercalciuria, aminoaciduria, glycosuria, low molecular weight proteinuria, renal calcifications, and renal failure. THE PRPOSED STUDY WILL HELP DEFINE THE UNDERLYING MOLECULAR BASIS OF THESE REABSORPTION DEFECTS. Specific aim 1: How do other CIC channels or CIC-5 exchange regulate apical endocytosis in proximal tubule cells? 1 A. Do CIC-3 and/or CIC-4 contribute to the residual acidification and apical endocytosis in the ko cells? 18. Does the over-expression of CIC-3 and CIC-4 restore albumin uptake in CIC-5 ko cells? Does the chloride/proton exchanger activity of CIC-5 in ko cells restore normal albumin uptake? Specific Aim 2: What is the relationship between loss of CIC-5 and the downregulation of megalin in the ko proximal tubule cells? 2A. Is the half-life of megalin shorter in ko versus wt cells? 2B. How is the recycling behavior of megalin altered in ko versus wt cells? 2C. Does endosomal pH, or lack of CIC-5, regulate megalin dynamics? Specific Aim 3. How does the loss of CIC-5 lead to changes in the distribution/activity of NHE3 and other apical transporters? 3A. Is NHE3 activity decreased in the proximal tubule, ileum or jejunum of ko mice? 3B. What are the steady state levels and dynamics of NHE3 in ko proximal tubules cells? 3C. What are the steady state levels and dynamics of NaPi-lla in ko proximal tubule cells? 3D, Does endosomal pH, or lack of CIC-5, regulate NHE3 or NaPi-lla dynamics? The long term goal of this research is to understand how molecular events cause the phenotypical outcomes of Dent's disease.