Regulation of Proximal Tubule Transport

描述（由申请人提供）：肾近端小管（PT）重新吸收约80%在肾小球滤过的HCO-3。先前对离体灌注兔和小鼠PTs的研究表明，HCO-3重吸收率（JHCO3）随着基底外侧[CO2] （[CO2]BL）的增加或[HCO-3]BL的降低而显著升高，但不受pHBL变化的影响。因此，PT必须具有传感CO2/HCO-3的机制。二氧化碳引起的JHCO3的增加被顶端ANG II AT1a受体拮抗剂和ErbB受体酪氨酸激酶家族的特异性抑制剂（如ErbB1）阻断。我们很感兴趣地了解了一种孤儿受体蛋白酪氨酸磷酸酶（RPTP3），其假定的细胞外配体结合结构域与碳酸酐酶（CA）非常相似。初步数据表明，敲除RPTP3可消除JHCO3对这两种病毒的反应。[CO2]BL和[HCO-3]BL。三个目标是一个多学科的方法来解决，在三个层次的整合，PT如何感觉？[CO2]BL和改变JHCO3:(1)灌注PTs。RPTP3的作用是什么？[CO2]BL升高是否会增强对添加的腔内ANG II或ANG-(1-7)的反应？慢性酸中毒是否会增强co2引起的JHCO3升高？at1a缺失和rptp3缺失小鼠是否对慢性酸中毒更敏感？(2) PT悬液的生物化学。CO2/HCO-3是否在ErbB1上产生独特的rptp3依赖性磷酸酪氨酸指纹？蛋白质组学方法能否识别CO2/RPTP3/ErbB1的下游靶标？3) RPTP3分子。是CO2还是HCO3激活了RPTP3的磷酸酶活性？RPTP3位于哪里？RPTP3的CA样结构域的突变能产生CA活性吗？CA抑制剂能与CA样结构域结合吗？类ca结构域的晶体结构是什么？这项工作将阐明一种高度新颖的传感CO2和/或hco -3的机制，不依赖于ph的变化，并且可能对酸中毒、高血压和癌症的临床方法具有重要意义。

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.