The WNK1-SPAK/OSR1 signaling pathway in pneumonia-induced lung barrier failure

Abstract

Objective Despite the availability of effective antibiotics and pneumococcal vaccines, lung barrier failure remains a major complication of pneumonia, with high mortality and morbidity rates. Loss of function of the endothelial cystic fibrosis transmembrane conductance regulator (CFTR) has been associated with lung barrier failure in pneumonia. Additionally, pharmacological restoration of CFTR function with ivacaftor has shown barrier-protective properties. The regulatory cascade of CFTR includes lysine-deficient protein kinase 1 (WNK1), which has been suggested as a possible mediator of barrier breakdown. Yet, the mechanisms by which CFTR contributes to lung barrier integrity and its functional interaction with WNK1 remain incompletely understood. To address this, this study has three main objectives: (i) to test whether in vivo WNK1 activation could mimic the effect of ivacaftor-mediated CFTR potentiation; (ii) to investigate the role of WNK1 and its downstream target kinases, SPS/Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), in pneumonia-induced lung barrier disruption, and (iii) to determine whether the regulation of WNK1-SPAK/OSR1 is cell-type specific and differs between airway epithelial and endothelial cells. Methods A murine model (C57BL/6J wild-type) of severe pneumococcal pneumonia was used to study WNK1 activation and its effects on vascular leakage and inflammatory response. Furthermore, a heterozygous knockout of WNK1 (Wnk1+/-) and a homozygous knock-in of SPAK (SpakL502A/L502A) were used to investigate the functional role of the kinases in the murine infection model. To further characterize cell-type specific regulation of WNK1-SPAK/OSR1 signaling, human primary pulmonary microvascular endothelial cells (HPMEC) and primary human pulmonary alveolar epithelial cells (HPAEpiC) were used. Transepithelial/transendothelial electrical resistance (TEER) was used to measure the cellular integrity after pharmacological inhibition of WNK1 or SPAK. In addition, the effect of WNK1 inhibition (WNK-In-11) and nonspecific WNK1 activation (temozolomide) in uninfected or S. pneumoniae-infected conditions and their consequence on cell-specific regulation were investigated by proteomic and phosphoproteomic profiling. Results Pharmacological WNK1 activation prior to S. pneumoniae infection partly prevented severe barrier disruption, improved body temperature, and reduced bacterial burden. Genetic deficiency of WNK1 and SPAK in mice, however, did not affect lung permeability distinctly. Whereas, under in vitro conditions, inhibition of WNK1 decreased cellular resistance HPMEC but not in HPAEpiC. Notably, SPAK inhibition decreased resistance in both cell types. Although no obvious changes in the global proteome were observed, the phosphoproteomic profiling revealed that inhibitory WNK1 phosphorylation is increased in HPMEC but not in HPAEpiC after targeted WNK1 inhibition and S. pneumoniae infection. Conclusion The results suggest an integral role for WNK1-SPAK/OSR1 signaling in lung barrier function. Activation of WNK1 in vivo was shown to have beneficial effects on barrier stability. Furthermore, the signaling was shown to have distinct and cell-type-specific characteristics. In addition to WNK1 and SPAK/OSR1, the regulation of pulmonary permeability is likely to depend on other kinases, including other members of the WNK family.