Regulation of Proximal Tubule Transport

描述(申请人提供)：肾近端小管(PT)重吸收肾小球过滤的~80%的HCO-3。前人在兔和小鼠离体灌流兔和小鼠的研究表明，HCO3重吸收速率(JHCO3)随[CO2]([CO2]BL)的增加而显著增加或[HCO3]BL的降低，但不受pHBL的改变。因此，PT必须具有感知CO2/HCO-3的机制。顶端Ang II AT1a受体的拮抗剂和ErbB受体家族的特异性抑制剂(如ErbB1)可阻断二氧化碳诱导的JHCO3的增加。我们很感兴趣地了解到一种孤儿受体蛋白酪氨酸磷酸酶(RPTP3)，其胞外配体结合域与碳酸氢酶(CA)非常相似。初步数据表明，敲除RPTP3会消除JHCO3对？[CO2]BL和？[HCO-3]BL的反应。这三个目标是一个多学科的方法，在三个集成水平上解决PT如何感知？[CO2]BL和改变JHCO3：(1)灌流的PTS。RPTP3的作用是什么？[CO2]BL中的海拔高度是否增强了对添加管腔Ang II或Ang-(1-7)的反应？慢性酸中毒是否会增加二氧化碳引起的碳酸氢钠的升高？AT1a缺失和RPTP3缺失的小鼠对慢性酸中毒更敏感吗？(2)PT混悬液中的生物化学。二氧化碳/HCO-3是否在ErbB1上产生独特的依赖RPTP3的磷酸酪氨酸指纹？蛋白质组学方法能识别CO2/RPTP3/ErbB1下游靶点吗？(3)RPTP3分子。是二氧化碳还是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？这项拟议的工作将阐明一种非常新颖的感知二氧化碳和/或HCO-3的机制-独立于pH的变化-并可能对酸中毒、高血压和癌症的临床治疗方法产生重要影响。 与公共卫生相关：人体的酸碱状态主要是通过肾脏近端小管(PT)细胞分泌体内H+(酸)到液体中，最终排出为尿液。PT细胞的酸分泌本身并不直接受到体酸增加的刺激，而是受到体内CO2/HCO-3(苏打水的成分)的比率的刺激，这本身就是酸敏感的。该项目的目标是确定PT及其下游靶标中的二氧化碳感应蛋白，并了解二氧化碳增加增加酸分泌的高度新颖的机制。这项拟议的工作可能会对酸中毒、高血压和癌症的临床治疗方法产生重要影响。

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.