前言: 肺泡上皮細胞有許多離子通道，包括上皮鈉離子通道(ENaC)，囊性纖維化跨膜傳導調節因子(CFTR)，鈉鉀幫浦（Na+-K+-ATPase），及鈉鉀氯共同轉換因子(NKCC)等。這些離子通道所產生的滲透壓梯度，具主動回收鹽分與水分的功能，可將肺泡中過多的液體帶回血液循環，稱之為肺泡液清除機制。先前的研究顯示，在腎小管細胞中，With-no-lysine kinase 4 (WNK4)和Ste20-related proline-alanine-rich kinase (SPAK) 調控上皮細胞離子通道之鹽分與水分的平衡扮演ㄧ個重要的角色，但WNK/SPAK如何調控肺泡液體清除機制目前未知。方法:本研究使用SPAK基因剔除小鼠和WNK4D561A/+嵌入小鼠於氧氣暴露誘發急性肺損傷模式中探討SPAK增加NKCC1磷酸化與肺泡液體清除之間的相關性。結果:將小鼠氧氣暴露60小時後計算肺泡白血球浸潤、肺泡灌洗液蛋白質含量、肺泡灌洗液中的乳酸脫氫酶、肺乾溼重比等指數皆明顯增加，並且肺泡液體清除率下降造成嚴重的急性肺損傷。其中氧氣暴露會造成SPAK和NKCC1的磷酸化表現增加，而SPAK基因剔除小鼠中SPAK和NKCC1磷酸化表現量明顯減弱並且原來被抑制的肺泡液體清除率明顯恢復，改善肺損傷。相反的，在WNK4D561A/+(SPAK-NKCC1持續表現)的動物模式中，氧氣暴露造成更嚴重的肺損傷且肺泡液體清除率明顯下降，這樣的現象可以利用NKCC1的抑制劑(furosemide)明顯改善氧氣誘發嚴重的肺損傷，是因為WNK4D561A/+小鼠本身增加肺泡上皮鈉離子通道將水分回收。 結論：在氧氣暴露誘發急性肺損傷的模式中，WNK/SPAK-NKCC1路徑藉由調控肺泡液體清除的機制扮演ㄧ個重要的角色。

Objectives: To investigate mechanisms involved in the regulation of epithelial ion channels and alveolar fluid clearance in hyperoxia-induced lung injury.Subjects: Wild-type, STE20/SPS1-related proline/alanine rich kinase knockout (SPAK–/–), and with-no-lysine kinase 4 knockin (WNK4D561A/+) mice. Interventions: Mice were exposed to room air or 95% hyperoxia for 60 hours.Methods and Main Results: Exposure to hyperoxia for 60 h increased the lung expression of WNK4 and led to SPAK and sodium–potassium–chloride cotransporter (NKCC1) phosphorylation, which resulted in the suppression of AFC and increase of lung edema. WNK4D561A/+ mice at the baseline presented an abundance of epithelium sodium channel (ENaC) and high levels of SPAK and NKCC1 phosphorylation. Compared with the wild-type group, hyperoxia caused greater ENaC expression in WNK4D561A/+ mice, but no significant difference in SPAK and NKCC1 phosphorylation. The functional inactivation of NKCC1 by gene knockout in SPAK–/– mice yielded a lower severity of lung injury and longer animal survival, whereas constitutive expression of WNK4 exacerbated the hyperoxia-induced lung injury. Pharmacological inhibition of NKCC1 by inhaled furosemide improved animal survival in WNK4D561A/+ mice. In contrast, inhibition of ENaC exacerbated the hyperoxia-induced lung injury and animal death.
Conclusions: WNK4 plays a crucial role in the regulation of epithelial ion channels and AFC, mainly via phosphorylation and activation of SPAK and NKCC1.

CONTENS
Title page
Contents………………………………………………...……...II
List of Illustrations……………………………………………..V
List of tables…………………………………………………..IX
Abbreviations………………………………………………..…X
Chinese Abstract……………………………………………...XII
English Abstract……………………………………...……XIV

Contents
Chapter 1 Introduction……………………………………….……………..….…..1
1.1 Acute respiratory distress syndrome………....……………...……….1
1.1.1 Pathogensis of ARDS…………….......……………………...…2
1.1.2 Animal models of acute lung injury……………………………2
1.2 Alveolar fluid clearance……………………………………...…..…..4
1.2.1 Epithelial sodium channel……………….……………………..8
1.2.2 Na+-K+-ATPase………………………………...…………......10
1.2.3 Sodium-potassium-chloride cotranspoter…………………..…10
1.3 WNK and ion transports in kidney…………………..……….…......13
1.4 The mechanisms regulating ion channel activities and fluid
movement in kidney………………………………………..….13
1.5 The regulating mechanism of alveolar epithelial ion channels…..…17
Aim of this thesis……………………………………………………17



Chapter 2
Material and methods………………………………………….….…..19
2.1 Animals and mouse model of hyperoxia-induced lung injury……...19
2.2 Hyperoxia exposure………………………………………………...22
2.3 Pulmonary edema and bronchial–alveolar lavage fluid study……...22
2.4 AFC determination………………………………………………….23
2.5 Histology……………………………………………………….…...25
2.6 Immunoblotting………………………………………………..........25
2.7 Immunofluorescence……………………………………………......26
2.8 Pharmacological modulation of ENaC and NKCC1…………..…....26
2.9 Urine volume and fractional excretion of sodium………………..…27
2.10 Statistical analysis………………………………………………....27
Chapter 3
Results……………………………………………………………….….28
3.1 Hyperoxia activates WNK4 in the lung…………………………......28
3.2 Hyperoxia activates the SPAK pathway in the lung………….…..…38
3.3 Modulation of hyperoxia-induced ALI by SPAK or WNK4 gene
manipulation………………………………………………….……..45
3.4 WNK4 gene manipulation modulates the inflammation cascade…...56

3.5 Effects of pharmacological modulation of ion channels on
hyperoxia-induced ALI……………………………………….…………65
Chapter 4
Discussion……………………………………….………………...…....74
4.1 Summary…………………………………….……………………....74
4.2 SPAK-NKCC1 pathway in lung injury……….…………………......75
4.3 The role of WNK4/SPAK in AFC regulation….………………..…..76
4.4 WNK4-MAPK pathway in lung inflammation……………………..78
4.5 Ion channel regulation and lung inflammation……………….…..…79
Chapter 5
Conclusion and future perspectives……………………………….….82
Chapter 6
References………………………………………………………….…...84
Appendixes…………………………………………………...………...98






List of figures
Figure 1.1 AFC involves ion channels in the lung epithelium…………...7
Figure 1.2 NKCC1 contributes to pulmonary edema………………...…12
Figure 1.3 WNK-SPAK/OSR1 signalling pathway………………...…...15
Figure 1.4 Hypothetical model of the regulation of WNK/SPAK/NKCC……………………………………….18
Figure 2.1 Targeting strategy for generating WNK4D561A/+ knockin mice…………………………………………………………20
Figure 2.2 Targeting strategy for generating SPAK null mice.................21
Figure 2.3 AFC in practice photo………………………….....................24
Figure 3.1 Hyperoxia-induced expression of WNK4 and ENaC in the lungs………………………………………………………...30
Figure 3.2 Hyperoxia-induced expression of WNK4 in the lungs…..….31
Figure 3.3 Hyperoxia-induced expression of ENaC in the lungs…..…...32
Figure 3.4 Expression and activation of SPAK/OSR1 and NKCC1 in WNK4D561A/+ mouse lungs………………………………….33
Figure 3.5 Expression of SPAK/OSR1 in WNK4D561A/+ mouse lungs….34
Figure 3.6 Phosphorylation of SPAK/OSR1 in WNK4D561A/+ mouse lungs……………………………………………………...…35
Figure 3.7 Expression of NKCC1 in WNK4D561A/+ mouse lungs………36
Figure 3.8 Phosphorylation of NKCC1 in WNK4D561A/+ mouse lungs....37
Figure 3.9 Expression and activation of SPAK/OSR1 and NKCC1 in SPAK–/– mouse lungs…………………..……………..…..…39
Figure 3.10 Expression of SPAK/OSR1 in SPAK–/– mouse lungs…....…40
Figure 3.11 Phosphorylation of SPAK/OSR1 in SPAK–/– mouse lungs...41
Figure 3.12 Expression of NKCC1 in SPAK–/– mouse lungs………...…42
Figure 3.13 Phosphorylation of SPAK/OSR1 and NKCC1 in SPAK–/– mouse lungs…………………………………………….…..43
Figure 3.14 Expression of total SPAK/OSR1, p-SPAK/OSR1, total NKCC1, and p-NKCC1 in the lungs, as assessed by immunofluorescence staining………………………………44
Figure 3.15 Animal survival in mice after exposure to hyperoxia……..47
Figure 3.16 Pulmonary edema in mice after exposure to hyperoxia…...48
Figure 3.17 Animal AFC in mice after exposure to hyperoxia…………49
Figure 3.18 Severity of hyperoxia-induced ALI in mice with SPAK or WNK4 gene manipulation………………………………….51
Figure 3.19 Lung tissue micrographs of hyperoxia-induced ALI in mice with SPAK or WNK4 gene manipulation………………......52
Figure 3.20 Neutrophil count of hyperoxia-induced ALI in mice with SPAK or WNK4 gene manipulation………………………..53
Figure 3.21 Lung injury score of hyperoxia-induced ALI in mice with SPAK or WNK4 gene manipulation………………………..54
Figure 3.22 Bronchial–alveolar lavage fluid proteins of hyperoxia-induced ALI in mice with SPAK or WNK4 gene manipulation……………………….………………………..55
Figure 3.23 Activation of the NFB cascade in lung tissues after hyperoxia……………………………………………………58
Figure 3.24 Activation of the p-IKK in lung tissues after hyperoxia….59
Figure 3.25 Expression of the IκB-α in lung tissues after hyperoxia…...60
Figure 3.26 Activation of the p-p65 in lung tissues after hyperoxia…....61
Figure 3.27 Activation of the p-p38 in lung tissues after hyperoxia……62
Figure 3.28 Activation of the p-ERK in lung tissues after hyperoxia…..63
Figure 3.29 Activation of the p-MSK1 in lung tissues after hyperoxia...64
Figure 3.30 Effects of the pharmacological modulation of ion channels on AFC in WT mice………………………………………..…..67
Figure 3.31 Effects of the pharmacological modulation of ion channels on animal survival in WT mice……………………………...…68
Figure 3.32 Effects of the pharmacological modulation of ion channels on AFC in WNK4D561A/+ mice……………………………...…..69
Figure 3.33 Effects of the pharmacological modulation of ion channels on animal survival in WNK4D561A/+ mice…………………...….70
Figure 3.34 Effects of inhaled furosemide on daily urine amount……...72
Figure 3.35 Effects of inhaled furosemide on urinary sodium excretion…………………………………………………….73
Figure 3.36 Proposed mechanism to explain the functional regulation of ENaC and NKCC1 by WNK4 in hyperoxia-induced lung injury………………………………………………………...81
List of tables
Table 1.1 Models of acute lung injury in rodents………………...………3
Table 1.2 Tissue specific anomalies inhuman and mouse with ENaC mutations…………………………………………….………...9
Table 3.1 Summary for survival times by hyperoxia exposure in WT, SPAK-/- and WNK4D561A/+ mice………………………...…….47
Table 3.2 Data for survival times by inhalation in WT and WNKD561A/+ mice…………………………………………………..……....71

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