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

RPTP-gamma 在肾近曲小管中传感和转导酸碱紊乱中的作用

• 批准号: 9926240
• 负责人: Walter F Boron
• 金额: $ 51.28万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-10 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926240
• 关键词: Acidosis; Acids; Address; Adenoviruses; Animals; Apical; Astrocytes; Bicarbonates; Binding; Blood; Blood gas; Carbon Dioxide; Carbonic Anhydrase II; Cell membrane; Cells; Chemistry; Chronic; Clinical; Data; Dimerization; Disease; ERBB2 gene; Elements; Enzymes; Epidermal Growth Factor Receptor; Evolution; Exhibits; Face; Fluorescence Resonance Energy Transfer; Functional disorder; GTP-Binding Proteins; Genetic Transcription; Goals; Hypertension; Kidney; Knock-out; Knockout Mice; Lead; Ligand Binding Domain; Link; Liquid substance; Malignant Neoplasms; Measures; Membrane; Metabolic; Metabolic acidosis; Metabolism; Molecular; Monitor; Mus; Mutation; Organ; Orphan; Patients; Peptidyl-Dipeptidase A; Phosphoric Monoester Hydrolases; Physicians; Play; Process; Protein Tyrosine Phosphatase; Proteins; Proximal Kidney Tubules; Receptor Protein-Tyrosine Kinases; Recombinant Proteins; Regulation; Respiratory Acidosis; Role; Sampling; Schizophrenia; Shock; Side; Signal Transduction; Structure; Testing; Translating; Urine; Viral; Water; Wild Type Mouse; Work; apical membrane; base; body system; carbonate dehydratase; cell type; extracellular; inhibitor/antagonist; inorganic phosphate; insight; interdisciplinary approach; kidney cell; mutant; prevent; protein profiling; receptor; respiratory; response; sensor; soft drink; virtual

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.

酸碱紊乱发生在多种疾病中，并导致动脉 (a) 和细胞内 pH 值紊乱，这可能会给患者带来毁灭性的后果。为了应对此类干扰，肾近曲小管 (PT)（通常处理肾脏分泌的约 80% H+）会适当调整其 H+ 分泌率 (JH)。先前的研究表明，PT 无法感知 PT 基底外侧 (BL) 侧的 pH 值。相反，PT JH 随着 [CO2]BL 的增加或 [HCO3−]BL 的减少而显着上升。然而，人们对感测 Δ[CO2]BL 和 Δ[HCO3−]BL 并将其转换为 ΔJH 的机制知之甚少。重要线索是，基底外侧 CO2 诱发的 JH 增加需要内源性分泌的 ANG II 与顶端 AT1A 受体结合，并被针对受体酪氨酸激酶子集（包括 ErbB1 和 ErbB2）的抑制剂阻断。新数据表明，通常存在于PT基底膜中的受体蛋白酪氨酸磷酸酶γ（RPTPγ）的敲除（KO）消除了Δ[CO2]BL和[HCO−]BL产生的ΔJH，并显着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)——与经典碳酸酐酶 (CA) 的相似度约为 40%。该提案的三个目标是采用多学科方法，在三个整合水平上解决 PT 如何感知 Δ[CO2]BL 和 Δ[HCO3−]BL 并将其转换为 ΔJH，以及这些过程在全身 MAc 和呼吸性酸中毒 (RAc) 期间 pHa 调节中所发挥的作用：在分子水平上，我们问（1）RPTPγ 的机制是什么？ Δ[CO2] 和 Δ[ HCO3−] 如何控制 RPTPγ 的二聚化，从而控制其磷酸酶活性？ RPTPγ 是否与 ErbB1 和 ErbB2 相互作用？ CALD 是否缺乏 CA 活性？如果是，为什么？在细胞水平上，我们问（2）RPTPγ和假定的下游元件如何在孤立的PT中发挥作用？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。 ErbB1 和 ErbB2 是否需要转导 Δ[CO2]BL 和 Δ[HCO3−]BL 向 ΔJH 发出信号？ [CO2]BL 和 [HCO3−]BL 是否通过调节管腔 ANG II 在顶端 AT1A 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 和下游效应器（ErbB1、ErbB2、ACE 和 AT1A）对于 MAc 和 RAc 的全身反应至关重要吗？该方法使用 KO 小鼠，施加 MAc 或 RAc 并评估动脉血气、尿液化学以及目标转录和蛋白质谱。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要见解。由于 RPTPγ 在多种细胞类型中表达，与 RPTPδ（主要在 CNS 星形胶质细胞中）密切相关，并且由于 RPTPγ 的 CALD 与三个孤儿 CA 相似，因此我们工作的影响将扩展到多种器官系统和细胞类型。它将提供重要的见解，包括酸碱平衡紊乱以及与 RPTPγ 相关的疾病（包括精神分裂症和癌症）等临床问题。