Role of RPTP-gamma in sensing and transducing acid-base disturbances in the renal proximal tubule

酸碱紊乱发生在广泛的疾病中，并导致动脉（a）和细胞内pH的紊乱，这可能对患者造成破坏性后果。作为对这些紊乱的反应，肾近端小管（PT）-通常处理肾脏分泌的~80%的H+-适当地调节其H+分泌速率（JH）。以前的工作表明，PT不感测PT的基底外侧（BL）侧的pH值。相反，PT JH随着[CO2]BL的增加或[HCO 3 −]BL的减少而显著增加。然而，对于感知Δ[CO2]BL和Δ[HCO 3 −]BL并将其转换为ΔJH的机制知之甚少。重要的线索是，基底外侧CO2诱发的JH增加需要内源性分泌的ANG II结合顶端AT 1A受体，并被靶向包括ErbB 1和ErbB 2的受体酪氨酸激酶亚组的抑制剂阻断。新的数据表明，敲除（KO）通常存在于PT基底膜中的受体蛋白酪氨酸磷酸酶γ（RPTPγ），消除了Δ[CO2]BL和Δ [HCO−]BL产生的ΔJH，并显著降低了整个小鼠在代谢性酸中毒（MAc）期间调节pHa的能力。奇怪的是，RPTPγ的细胞外侧有一个区域--碳酸酐酶样结构域（CALD）--与经典的碳酸酐酶（CAs）约有40%相同。本提案的三个目标是一种多学科方法，在三个整合水平上解决PT如何感知Δ[CO2]BL和Δ[HCO 3 −]BL并将其转化为ΔJH，以及这些过程在全身MAc和呼吸性酸中毒（RAc）期间pHa调节pHa中所起的作用：在分子水平上，我们问（1）RPTPγ的机制是什么？Δ[CO2]和Δ[HCO 3 −]如何控制RPTPγ的二聚化，从而控制其磷酸酶活性？RPTPγ与ErbB 1和ErbB 2相互作用吗？CALD是否缺乏CA活性，如果是，为什么？在细胞水平上，我们要问（2）RPTPγ和假定的下游元件在孤立的PT中是如何起作用的？我们将使用病毒构建体，注射到RPTPγ -/-小鼠的肾脏中，以确定RPTPγ的CALD和磷酸酶结构域的作用。是否需要ErbB 1和ErbB 2来抑制 Δ[CO2]BL和Δ[HCO 3 −]BL信号到ΔJH？[CO2]BL和[HCO 3 −]BL是否通过调节管腔ANG II在心尖AT 1A或其下游发出信号的能力来控制JH？在整个动物水平上，我们问（3）RPTPγ和下游效应物（ErbB 1、ErbB 2、ACE和AT 1A）在对MAc和RAc的全身反应中是必不可少的吗？使用KO小鼠的方法是施加MAc或RAc，并评估动脉血气、尿液化学以及靶向转录和蛋白质谱。我们的工作将对RPTPγ如何感知和转导酸碱状态的变化，下游元件如何在PT中发挥作用以及对整个动物的影响产生重大影响。由于RPTPγ在广泛的细胞类型中表达，与RPTP γ密切相关（主要在CNS星形细胞中），并且由于RPTPγ的CALD与三种孤儿CA相似，因此我们工作的影响将扩展到各种器官系统和细胞类型。它将提供重要的见解临床问题，包括酸碱失衡和与RPTPγ相关的疾病，包括精神分裂症和癌症。

Acid-base disturbances occur in a wide range of diseases, and lead to disturbances in arterial (a) and intracellular pH that can have devastating consequences for the patient. In response to such disturbances, the renal proximal tubule (PT)—which normally handles ~80% of the H+ secreted by the kidney—appropriately adjusts its rate of H+ secretion (JH). Previous work showed that the PT does not sense the pH on the basolateral (BL) side of the PT. Rather, PT JH markedly rises with increases in [CO2]BL or decreases in [HCO3−]BL. Nevertheless, mechanisms for sensing Δ[CO2]BL and Δ[HCO3−]BL and transducing these to ΔJH are poorly understood. Important clues are that the basolateral CO2-evoked increase in JH requires that endogenously secreted ANG II bind to apical AT1A receptors, and is blocked by inhibitors that target a subset of receptor tyrosine kinases that include ErbB1 and ErbB2. New data show that the knockout (KO) of receptor protein tyrosine phosphatase γ (RPTPγ), normally present in the PT basal membrane, eliminates the ΔJH produced by Δ[CO2]BL and [HCO−]BL, and markedly reduces the ability of the whole mouse to regulate pHa during metabolic acidosis (MAc). Curiously, the extracel- lular side of RPTPγ has a region—the carbonic-anhydrase–like domain (CALD)—that is ~40% identical to clas- sical carbonic anhydrases (CAs). The three aims of this proposal are a multidisciplinary approach to address, at three levels of integration, how PTs senses Δ[CO2]BL and Δ[HCO3−]BL and transduce them into ΔJH, and the role played by these processes in pHa regulation of pHa during whole-body MAc and respiratory acidosis (RAc): At the molecular level, we ask (1) what is the mechanism of RPTPγ? How do Δ[CO2] and Δ[ HCO3−] control RPTPγ’s dimerization, which controls its phosphatase activity? Does RPTPγ interact with ErbB1 and ErbB2? And does the CALD lack of CA activity, and if so, why? At the cellular level we ask (2) how do RPTPγ and putative down- stream elements function in isolated PTs? We will use viral constructs, injected into kidneys of RPTPγ –/– mice, to determine the roles of RPTPγ’s CALD and phosphatase domains. Are ErbB1 and ErbB2 required to transduce Δ[CO2]BL and Δ[ HCO3−]BL signals to ΔJH? Do [CO2]BL and [HCO3−]BL control JH by modulating ability of luminal ANG II to signal at or downstream to apical AT1A? At the whole-animal level, we ask (3) are RPTPγ and downstream effectors (ErbB1, ErbB2, ACE, and AT1A) essential in the whole-body responses to MAc and RAc? The approach, using KO mice, is to impose MAc or RAc and assess arterial blood gases, urine chemistry, and targeted transcription and protein profiles. Our work will produce major insights into how RPTPγ senses and transduces changes in acid-base status, how downstream elements function in PTs, and the impact for the whole animal. Because RPTPγ is expressed in a broad range of cell types, is closely related to RPTPζ (mainly in CNS astro- cytes), and because RPTPγ’s CALD is similar to three orphaned CAs, the impact of our work will extend across a diverse range of organ systems and cell types. It will provide important insights clinical problems that include acid-base disturbances and to diseases linked to RPTPγ, which include schizophrenia and cancer.