Regulation of Proximal Tubule Transport

描述（由申请方提供）：肾近端小管（PT）重吸收约80%的肾小球滤过的HCO-3。先前对离体灌注的兔和小鼠PT的研究表明，HCO-3重吸收率（JHCO 3）随基底外侧[CO 2]（[CO 2]BL）的增加或[HCO-3]BL的减少而显著升高，但不因pHBL的变化而改变。因此，PT必须具有用于感测CO2/HCO-3的机制。CO2诱发的JHCO 3增加被顶端ANG II AT 1a受体的拮抗剂和受体酪氨酸激酶ErbB家族的特异性抑制剂（例如，ErbB1）。我们感兴趣的是了解一个孤儿受体蛋白酪氨酸磷酸酶（RPTP 3）与一个假定的胞外配体结合结构域，强烈类似于碳酸酐酶（CA）。初步数据表明，敲除RPTP 3消除了JHCO 3对两者的反应？[CO2]BL和？[HCO-3]BL.这三个目标是一个多学科的方法来解决，在三个层次的整合，如何PT的感觉？[CO2]BL和改变JHCO 3：（1）灌注PT。RPTP 3的作用是什么？[CO2]BL升高是否会增强对增加的管腔ANG II或ANG-（1-7）的反应？慢性酸中毒是否会增强CO2诱发的JHCO 3增加？AT 1a缺失和RPTP 3缺失小鼠对慢性酸中毒是否更敏感？(2)PT混悬液中的生物化学。CO2/HCO-3是否在ErbB 1上产生独特的RPTP 3依赖性磷酸酪氨酸指纹？蛋白质组学方法能否鉴定CO2/RPTP 3/ErbB 1的下游靶点？(3)RPTP 3分子。是CO2还是HCO 3激活了RPTP 3的磷酸酶活性？RPTP 3在哪里？RPTP 3的CA样结构域的突变能产生CA活性吗？CA抑制剂能结合CA样结构域吗？CA样结构域的晶体结构是什么？拟议的工作将阐明一个非常新颖的机制，用于传感CO2和/或HCO-3-独立于pH值的变化，并可能对酸中毒，高血压和癌症的临床方法具有重要意义。 公共卫生相关性：身体的酸碱状态主要通过分泌身体H+（酸）穿过肾脏的近端小管（PT）细胞进入最终作为尿液排出的液体来维持。PT细胞的酸分泌并不直接受体内酸增加的刺激，而是受体内CO2/HCO-3（苏打水的成分）的比例的刺激，而这本身就是酸敏感的。该项目的目标是确定PT及其下游靶点中的CO2敏感蛋白，并了解CO2增加增加酸分泌的高度新颖的机制。这项工作可能对酸中毒、高血压和癌症的临床治疗有重要意义。

DESCRIPTION (provided by applicant): The renal proximal tubule (PT) reabsorbs ~80% of the HCO-3 filtered at the glomerulus. Previous work on isolated perfused rabbit and mouse PTs shows that the rate of HCO-3 reabsorption (JHCO3) markedly rises with increases in basolateral [CO2] ([CO2]BL) or decreases in [HCO-3]BL, but is not altered by changes in pHBL. Thus, the PT must have a mechanism for sensing CO2/HCO-3. The CO2-evoked increase in JHCO3 is blocked by antagonists of apical ANG II AT1a receptors and by specific inhibitors of the ErbB family of receptor tyrosine kinases (e.g., ErbB1). We were intrigued to learn of an orphaned receptor protein tyrosine phosphatase (RPTP3) with a putative extracellular ligand binding domains that strongly resembles carbonic anhydrase (CA). Preliminary data suggest that knocking out RPTP3 eliminates the JHCO3 response to both ?[CO2]BL and ?[HCO-3]BL. The three aims are a multidisciplinary approach to address, at three levels of integration, how the PT senses ?[CO2]BL and alters JHCO3: (1) Perfused PTs. What is the role of RPTP3? Do elevations in [CO2]BL enhance the response to added luminal ANG II or ANG-(1-7)? Does chronic acidosis enhance the CO2-evoked increase in JHCO3? And are AT1a-null and RPTP3-null mice more sensitive to chronic acidosis? (2) Biochemistry in PT suspensions. Does CO2/HCO-3 produce a unique RPTP3-dependent phosphotyrosine fingerprint on ErbB1? Can a proteomic approach identify downstream - targets of CO2/RPTP3/ErbB1? (3) The RPTP3 molecule. Is it CO2 or HCO3 that activates the phosphatase activity of RPTP3? Where is RPTP3 located? Can mutations in the CA- like domain of RPTP3 generate CA activity? Can a CA inhibitor bind to the CA-like domain? What is the crystal structure of the CA-like domain? The proposed work will illuminate a highly novel mechanism for sensing CO2 and or HCO-3-independent of changes in pH-and could have important implications for clinical approaches to acidosis, hypertension, and cancer. PUBLIC HEALTH RELEVANCE: The acid-base status of the body is predominantly maintained by secretion of bodily H+ (acid) across the proximal tubule (PT) cells of the kidney into fluid that is eventually excreted as urine. Acid secretion by PT cells is not directly stimulated by increased body acid per se-but by the ratio of body CO2/HCO-3 (the components of soda water), which itself is acid-sensitive. The goals of this project are to identify the CO2-sensing protein in the PT and its downstream targets, and to understand the highly novel mechanism by which increased CO2 increases acid secretion. The proposed work could have important implications for clinical approaches to acidosis, hypertension, and cancer.