The injurious effects of hypercapnia on the alveolar epithelium

描述(申请人提供)：在使用“允许性高碳酸血症”策略的呼吸衰竭患者中观察到高二氧化碳水平。我们和其他研究人员最近报告说，二氧化碳浓度升高会对肺泡上皮细胞产生有害影响。具体地说，我们已经报道，高碳酸血症通过抑制肺泡上皮Na，K-ATPase和损害肺泡液清除而导致短期和长期的肺泡上皮功能障碍。因此，这一应用的重点是确定在高二氧化碳状态下导致肺泡液Na，K-ATPase下调和肺泡液清除障碍的具体机制/信号通路。我们建议通过四个相互关联的目的来研究高碳酸血症对肺泡上皮细胞的影响：在特定目的#1的实验中，我们将确定可溶性腺酰环化酶是否参与了高碳酸血症诱导的肺泡上皮细胞Na，K-ATPase的下调；在特定目的2的实验中，我们将确定高碳酸血症诱导的JNK激活是否导致LIM结构域唯一蛋白7(LMO7)的磷酸化和Na，K-ATPase的内吞；在特定目的#3的研究中，我们将确定Na，K-ATPase泛素化是否导致其下调，并建议确定特定的E2和E3泛素连接酶；在与特定目标4相关的研究中，我们将确定高碳酸血症是否会进一步损害轻度呼吸机诱导的肺损伤(VILI)模型，以及SAC、JNK、LMO7和泛素化途径的下调是否会逆转高碳酸血症对啮齿动物肺泡液清除的有害影响。在这笔赠款的第一个周期中，我们已经完成了大部分拟议研究，并就支持这项赠款提案可行性的每一个当前具体目标进行了初步试验。这些实验将为高碳酸血症对肺泡上皮细胞的影响提供新的信息，并阐明导致肺泡上皮功能障碍的机制，这对理解和治疗急性肺损伤患者具有重要意义。 公共卫生相关性：肺水肿和急性呼吸窘迫综合征(ARDS)患者经常暴露于高碳酸血症(血液中高二氧化碳水平)，这对肺泡上皮细胞有有害影响，发病率很高。这项资助计划的重点是确定在高碳酸血症条件下导致肺泡液清除下调和损害的具体机制/信号通路。我们将研究长期高碳酸血症对啮齿动物的影响，特别是测量肺泡液体清除。这项赠款申请中提出的实验所产生的信息将为理解这些疾病的病理生理学以及可能在ARDS患者的治疗中提供新的见解。

DESCRIPTION (provided by applicant): High pCO2 levels are observed in patients with respiratory failure ventilated with the "permissive hypercapnia" strategy. We and other investigators have recently reported that elevated pCO2 levels can have deleterious effects on the alveolar epithelium. Specifically, we have reported that hypercapnia leads to short and long term dysfunction of the alveolar epithelium by inhibiting the alveolar epithelial Na,K-ATPase and impairing alveolar fluid clearance. Therefore, the focus of this application is to determine the specific mechanisms/signaling pathways that lead to alveolar Na,K-ATPase downregulation and impairment in alveolar fluid clearance during hypercapnic conditions. We propose to study the effects of hypercapnia on the alveolar epithelium via four interrelated aims: in experiments pertaining to specific Aim # 1, we will determine whether the soluble adenylyl cyclase participates in the hypercapnia-induced Na,K- ATPase downregulation in the alveolar epithelium; in experiments pertaining to specific Aim #2, we will determine whether hypercapnia-induced JNK activation leads to LIM-domain only protein 7 (LMO7) phosphorylation and Na,K-ATPase endocytosis; in studies pertaining to specific Aim # 3, we will determine whether Na,K-ATPase ubiquitination leads to its downregulation and propose to identify the specific E2 and E3 ubiquitin ligase; and in studies pertaining to specific Aim # 4, we will determine whether hypercapnia further impairs injury in a model of mild ventilator-induced lung injury (VILI) and whether the downregulation of of sAC, JNK, LMO7 and ubiquitination pathway will reverse the deleterious effects of hypercapnia on alveolar fluid clearance in rodents. In the first cycle of this grant, we have completed most of the proposed research and conducted preliminary experiments for each of the current specific aims which support the feasibility of this grant proposal. The proposed experiments will generate novel information on the effects of hypercapnia on the alveolar epithelium and shed light on the mechanisms leading to alveolar epithelial dysfuntion which is of significance to the understanding and treatment of patients with acute lung injury. PUBLIC HEALTH RELEVANCE: Patients with pulmonary edema and acute respiratory distress syndrome (ARDS) are often exposed to hypercapnia (high CO2 levels in the blood) which has deleterious effects on alveolar epithelium and high morbidity rate. This grant proposal focuses on determining the specific mechanisms/signaling pathways that lead to downregulation and impairment in alveolar fluid clearance during hypercapnic conditions. We will study the effects of prolonged hypercapnia on rodents, specifically measuring alveolar fluid clearance. The information generated from the experiments proposed in this grant application will provide new insights to the understanding of the pathophysiology of these diseases and possibly in the treatment of patients with ARDS.