Pendrin inhibition is associated with protective effect of prone positioning in a ventilator-induced lung injury mouse model

Pendrin inhibition is associated with protective effect of prone positioning in a ventilator-induced lung injury mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pendrin inhibition is associated with protective effect of prone positioning in a ventilator-induced lung injury mouse model Ji Soo Choi, Mi Hwa Shin, Go Eun Oh, Doo Na Song, Wan NamKung, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6561550/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Pendrin (SLC26A4), a transmembrane anion exchanger, is upregulated in inflammatory airway diseases. In this study, we analyzed the role of pendrin expression in a ventilator-induced acute lung injury (VILI) animal model. VILI was induced in the supine or prone position by a high tidal volume (HTV) of 30 mL/kg for 5 hours in pendrin wild-type (WT) and knockout (KO) 129SVEV mice. Pendrin inhibitor (YS-01) was intraperitoneally administered to modulate pendrin signaling. Lung injury parameters were assessed based on bronchoalveolar lavage fluid (BALF) analysis, inflammatory cytokine analysis by ELISA, and histopathological findings. Pendrin expression was determined by western blotting and transmission electron microscopy (TEM) using immunogold labeling methods. The degree of lung injury was significantly attenuated in pendrin-KO mice and pendrin-WT mice with YS-01 compared with pendrin-WT animals after HTV ventilation. Pendrin expression was down-regulated in pendrin-KO mice and pendrin-WT mice with YS-01 compared with pendrin-WT mice with VILI, as determined by western blotting and TEM-immunogold labeling. Prone positioning during ventilation attenuated lung inflammation and pendrin expression. Our results suggest that pendrin is critical in VILI and could be a novel target for modulating VILI. Prone positioning and pendrin inhibition in VILI may be effective in managing these conditions. Biological sciences/Biochemistry/Ion channels Health sciences/Molecular medicine pendrin SLC26A4 ventilator-induced lung injury acute lung injury acute respiratory distress syndrome prone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterized by the rapid onset of hypoxemia with diffuse bilateral pulmonary infiltrates. It has a high mortality rate and poses a significant financial burden on critically ill patients 1 . Mechanical ventilation (MV) is often necessary to manage ALI/ARDS; however, it can cause lung damage and aggravate ALI/ARDS 2 . This condition is known as “ventilator-induced lung injury” (VILI), and occurs due to the inhomogeneous distribution of lung damage and edema in ALI/ARDS 3 . Although a ventilator strategy with low tidal volume improves the prognosis of VILI in patients with ALI/ARDS, some patients are inherently susceptible to VILI 4 , 5 . Prone positioning effectively prevents VILI by reducing uneven lung injury 6 ; however, research on the molecular mechanisms of prone position management is limited 7 . Pendrin (SLC26A4) is a transmembrane anion exchanger protein located on the apical surface of epithelial cells in the kidney, thyroid, lung, and inner ear 8 – 10 . This protein acts as a Cl⁻/anion exchanger that transports Cl⁻ to bases, including iodide (I⁻), bicarbonate (HCO3⁻), hydroxide (OH⁻), and thiocyanate (SCN⁻) 11 . Mutations in pendrin are associated with Pendred syndrome, a recessive genetic disorder characterized by congenital deafness, goiter, or thyroid hormone abnormalities 12 . Pendrin is negligibly expressed in normal airway epithelia 13 . Respiratory diseases characterized by excessive mucus production, including bronchial asthma or chronic obstructive pulmonary disease (COPD), are identified by pendrin overexpression 14 . Pendrin exacerbates airway diseases triggered by viral infections or allergen exposure 15 . An animal model study showed that pendrin knockout mice exhibit significantly decreased lung inflammation caused by Bordetella pertussis infection 16 . Recent research suggests that pendrin plays a role in the pathogenesis of lipopolysaccharide (LPS)-induced ALI, and airway epithelial cells treated with pendrin inhibitor may serve as a therapeutic target for managing ALI/ARDS 17 . Previous in vivo and in vitro studies have identified increased pendrin expression in an ALI/ARDS animal model using LPS induction, and a novel pendrin inhibitor (YS-01) reduces the inflammatory response in LPS-induced ALI, suggesting that pendrin could be a target for ALI/ARDS treatment 18 . Alveolar epithelial cell damage occurs and contributes to the lung inflammatory process in an animal model of lung injury by ventilator stretching 19 ; however, the role of pendrin in VILI has remained unclear and has not been reported. Based on these observations, we hypothesized that pendrin expression is associated with the pathogenesis of VILI-induced ALI/ARDS. Therefore, we constructed a VILI mouse model and observed an increase in pendrin expression. We also assessed whether pendrin-null mice and pendrin inhibitor treatment exhibited lower levels of inflammation during VILI. Furthermore, the protective effects of prone positioning were examined in a VILI mouse model. RESULTS 1. Deletion of the pendrin decreases the inflammatory response in VILI We performed analysis of BALF and histological assessments by comparing pendrin-WT and pendrin-KO mice to evaluate the role of pendrin in VILI. The total cell count in BALF and lung injury score assessed by histological examination was markedly increased in pendrin-WT mice after HTV ventilation compared with those in the non-ventilation control group (Figs. 2 and 3 ). Cell differentiation in pendrin-WT mice with HTV ventilation predominantly comprised macrophages with few neutrophils and lymphocytes (Fig. 2 B) In the group of pendrin-KO mice, the total cell count in BALF after HTV ventilation was significantly lower compared to pendrin-WT mice group (Figs. 2 A and 2 C), and lung injury scores were also decreased compared to pendrin-WT mice group (Fig. 3 ). The group of pendrin-WT mice administered pendrin inhibitor (YS-01) showed reduced total cell count in BALF and lung injury scores compared with those of WT mice after HTV ventilation (Fig. 2 and Fig. 3 ). 2. Prone positioning during ventilation with HTV attenuated the VILI We compared BALF and lung histology results between supine and prone HTV mice to investigate the protective effect of prone positioning during HTV ventilation. In the BALF, the total cell count was significantly decreased in prone HTV mice relative to supine HTV pendrin-WT mice (Fig. 2 ). Assessment of lung histology in prone HTV mice revealed a significantly lower degree of leukocyte infiltration and lung injury scores relative to supine HTV pendrin-WT mice (Fig. 3 ). Furthermore, between the pendrin-WT and pendrin-KO mice groups subjected to HTV in the prone position, the BALF total cell count and lung injury score were lower in the pendrin-KO mice group compared to the pendrin-WT mice group (Fig. 2 and Fig. 3 ). 3. Pendrin deletion and prone positioning suppressed inflammatory cytokine release in the VILI mice model We further investigated the levels of inflammatory cytokines using ELISA to determine the effects of pendrin deletion and prone positioning in a mouse model of VILI. After HTV ventilation, the levels of cytokines, including tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and interleukin 1 beta (IL-1β) were remarkably elevated than those in non-ventilation (Fig. 4 ). In the supine HTV group, pendrin-KO mice and pendrin-WT mice administered with YS-01 showed lower levels of inflammatory cytokines such as TNF-α and MIP-2 (Fig. 4 ). 4. Increased pendrin expression in HTV-ventilated mice To confirm the increase in pendrin expression, we performed western blot analysis and TEM-immunogold labeling for pendrin (SLC26A4). Western blotting showed that pendrin expression was higher in pendrin-WT mice with VILI compared with the control non-ventilation group, pendrin-KO mice, and pendrin-WT mice administered YS-01. Furthermore, when HTV was applied in the prone position, both the WT and KO mice groups showed lower pendrin expression compared to when HTV was applied in the supine position (Fig. 5 ). We conducted TEM-immunogold labeling for pendrin to compare the expression of pendrin in each group at the ultrastructural level. The HTV WT mice in the supine and prone positions revealed increased pendrin expression on the cell membrane and in vesicles compared with that on the apical side of epithelial cells in the control non-ventilation group, pendrin-KO mice, and pendrin-WT mice administered with YS-01 (Fig. 6 A- 6 G). The number of gold particles labeled with pendrin was also higher in pendrin-WT mice with HTV ventilation than in the control non-ventilation group, pendrin-KO mice, and pendrin-WT mice administered with YS-01. Although there was no statistically significant difference between the groups HTV in the supine and prone positions, the number of gold particles labeled with pendrin tended to be lower in the prone position (Fig. 6 H). DISCUSSION In this study, pendrin expression was increased in response to HTV ventilation, and increased pendrin expression was associated with the inflammatory cascade. The modulation of pendrin through pendrin-KO mice and pendrin inhibitor (YS-01) resulted in a decreased inflammatory response in VILI. Furthermore, prone positioning during HTV attenuated lung injury in WT mice and was associated with pendrin expression. The transmembrane anion exchanger protein pendrin (SLC26A4) is associated with various disorders in related organs 20 . Recent research has provided evidence supporting the role of pendrin as a pivotal protein in airway diseases, such as asthma, COPD, and rhinitis 21 . The involvement of pendrin in ALI/ARDS has been suggested in previous studies but its exact mechanism of action remains unclear. Jia et al. 17 showed that the distal airway plays a role in ALI/ARDS, and pendrin in alveolar epithelial cells is involved in the inflammatory process of LPS-induced ALI. Furthermore, they demonstrated that the treatment of LPS-induced ALI mice with methazolamide, a carbonic anhydrase inhibitor, significantly reduced lung inflammation. Lee et al . 18 confirmed that pendrin expression increased in both human BAL samples with pneumonia and an LPS-induced ALI mouse model, and pendrin-null mice presented reduced lung damage by LPS. Moreover, they identified that the pendrin inhibitor (YS-01) could attenuate LPS-induced lung injury by inhibiting the pendrin/OSCN-/NF-κB-mediated pathway, indicating the possibility of developing a novel treatment of ALI. They concluded that airway epithelial cells could be a therapeutic target for developing new drugs and strategies for ALI/ARDS treatment. However, additional research is necessary to explore the molecular mechanism of pendrin upregulation and its therapeutic potential in ALI/ARDS. The clinical outcomes of ALI/ARDS have significantly improved owing to therapeutic strategies such as low tidal volume with low plateau pressure; however, it remains a challenging condition to manage 22 , 23 . Mechanical ventilation is an important treatment option for ALI/ARDS 24 ; notwithstanding, it may paradoxically lead to further lung injury, known as VILI, which can result in poor prognosis 25 . The mechanism of VILI, including conventional barotrauma, volutrauma, and atelectrauma, comprises initiation by induced tensile strain and direct tissue damage to the lungs, leading to increased permeability and disruption of the alveolar–capillary barrier 26 . This mechanical strain interrupts the clearance of edema fluid from the airspaces and triggers the release of inflammatory mediators within the distal lung, inducing end-organ dysfunction by entering the systemic circulation 27 . An increased inflammation response due to mechanical stretching during HTV ventilation, including elevation of TNF-α, IL-1β, and IL-6, has been observed in the BALF of patients with ARDS and in lung injury mice models 28 – 30 . In the present study, we observed increased inflammatory pathology by developing HTV-induced lung injury, consistent with previous studies. Prone positioning during invasive mechanical ventilation improves the outcome in patients with ARDS 31 . The protective mechanism of prone positioning in ARDS includes improvement of hypoxemia by enhancing ventilation-perfusion mismatching and reducing dependent atelectasis 32 . Prone positioning can improve cardiac function and hemodynamics 33 , respiratory mechanics, and prevent VILI 34 . In a previous study, Broccard et al. 35 confirmed a significant reduction in lung injury and homogeneous distribution of VILI in normal dogs with HTV ventilation for 6 h in the prone position. Moreover, a recent study by Park reported that prone positioning could affect VILI at the molecular level by influencing the activation of mitogen protein kinases in rodents exposed to HTV ventilation 36 . Recent research has focused on elucidating the molecular mechanism of the mechanotransduction pathway in VILI and prone positioning 37 . For example, Held et al . 38 reported that mechanical stimuli were found to trigger NF-κB activation, leading to the release of inflammatory cytokines. Notably, they demonstrated the possibility of a therapeutic strategy in which NF-κB activation by mechanical stimuli is inhibited using corticosteroids. Lee et al . 39 reported that NOX4 and Eph/ephrin signaling are implicated in VILI, and a NOX4 inhibitor could potentially serve as a therapeutic intervention for VILI. Furthermore, Park et al . 7 reported that the lung-protective effects of altered EphA2/ephrinA1 signaling through EphA2 antagonism were observed during injurious mechanical ventilation. They also identified that prone positioning exerted a protective effect on VILI. However, the molecular mechanisms underlying VILI and prone positioning remain largely unknown. Our data showed that pendrin expression was elevated in the VILI mouse model, demonstrating an increased response to lung inflammation. Additionally, pendrin deletion exerted a preventive effect in a VILI mouse model, confirmed using a pendrin inhibitor (YS-01) and pendrin-null mice. Furthermore, we demonstrated that prone positioning is also effective in reducing lung inflammatory responses in VILI, and is associated with pendrin expression. Our data suggest that pendrin expression is associated with lung injury due to mechanical stretching, and the modulation of pendrin might be a novel strategy for preventing VILI. Our study has some limitations that warrant further consideration. We could not identify the exact mechanism of pendrin expression or its downstream signaling cascades. However, several signaling cascades have already been extensively explored in our previous study. Nonetheless, our study is the first to investigate the relevance of pendrin and prone positioning in VILI caused by mechanical strain. A novel molecule pendrin inhibitor represents a potential new therapeutic approach for VILI treatment. Additionally, we identified the changes in pendrin expression in response to VILI and prone positioning using TEM-immunogold labeling, as in previous studies 40 . In conclusion, increased pendrin expression in response to HTV ventilation is related to the inflammatory cascade induced by mechanical strain and tissue injury. Our results showed that modulation of pendrin resulted in decreased inflammatory response in VILI, indicating its protective role against lung injury. We showed that prone positioning during HTV attenuated lung injury in WT mice. We identified pendrin as a potential therapeutic target for managing VILI and improving the outcomes of patients with VILI. Further studies are needed to investigate the relationship between pendrin expression and prone position and explore therapeutic strategies for modulating the expression and activity of pendrin. METHODS 1. Experimental animals Pendrin wild-type (WT) and knockout (KO) 129SVEV mice (weight 20–25 g, age 6–8 weeks) were donated by JY Choi at Yonsei University. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Yonsei University College of Medicine (6-2018-0164) and were conducted in accordance with the National Animal Care and Use Committee (IACUC). Animal research in this study was performed in accordance with the ARRIVE 2.0 guidelines. 2. VILI model in mice The mice were subjected to intraperitoneal anesthesia using a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) before performing the tracheostomy. After euthanasia, each mouse was positioned supine and secured on a surgical board. The upper incisors were secured to gently extend the neck, and the exposed trachea was punctured carefully using a 21-gauge needle. The mice were ventilated in the supine or prone position at a high tidal volume (HTV) of 30 mL/kg and a rate of 100 breaths per minute for 5 hours to induce VILI 41 , 42 . Other ventilator settings were 0 cm H 2 O end-expiratory pressure and 0.21 inspired oxygen fraction. Pendrin inhibitor (YS-01) was administered intraperitoneally for 1 hour before inducing VILI in the supine position (Fig. 1 ). At the completion of the experiment, each mouse was humanely euthanized in a carbon dioxide (CO₂) chamber, following institutional and national guidelines for the humane treatment of laboratory animals. 3. Study design and experimental protocol Wild-type (WT) and KO mice were randomly assigned to each HTV ventilation group in the supine and prone positions. The experimental groups were as follows: 1) non-ventilated group, pendrin WT (NVC, WT), n = 10 2) non-ventilated group, pendrin KO (NVC, KO, n = 6 3) supine HTV group, pendrin WT (supine_HTV, WT), n = 10 4) supine HTV group, pendrin KO (supine_HTV, KO), n = 9 5) supine HTV group with YS-01, pendrin WT (1 hour before MV, supine HTV pre-YS-01), n = 5 6) prone HTV group, pendrin WT (prone_HTV, WT), n = 10 7) prone HTV group, pendrin KO (prone_HTV, KO), n = 8 4. Bronchoalveolar lavage fluid (BALF) analysis After mechanical ventilation with HTV for 5 hours, bronchoalveolar lavage (BAL) was performed through a tracheal catheter. BALF was directed into a tracheal catheter, gently retracted using 1 mL of sterile saline, and centrifuged (3000 rpm for 10 min at 4℃). Inflammatory cytokines were analyzed using ELISA. The remaining pellets were resuspended in 100 µL PBS to determine cell count. 5. Histological assessment After BALF collection, the abdominal cavity was opened with midline incision, followed by excision of the lungs for further histological or molecular analysis. The inflated lung with 4% low-melting agarose was fixed in 10% formaldehyde for a day. It was then embedded in paraffin and sectioned into 5-µm thick slices. Subsequently, lung sections were subjected to hematoxylin and eosin staining and analyzed using bright-field microscopy. Histological evaluation of lung injury on each slide was performed using the weighted scoring scale described in the official American Thoracic Society Workshop Report 42 . 6. Transmission electron microscopy Lung injury was assessed to visualize the target protein of pendrin in the tissue using gold-conjugated antibody labeling procedures for localization at the ultrastructural level through transmission electron microscopy (TEM). 7. Statistical analysis All statistical tests were performed using Student’s unpaired two-tailed t-test for comparison of each group using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA). Statistical significance was considered at p < 0.05. Data are presented as the mean ± SEM. The lung injury score is presented as the median ± SE. Declarations Data availability The datasets used and analysed during the current study available from the corresponding author on reasonable request. Acknowledgments Not applicable Author’s contributions Ji Soo Choi: Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review and editing, Mi Hwa Shin: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Go Eun Oh: Data curation, Formal analysis, Investigation, Doo Na Song: Conseptualization, Resources, Wan NamKung: Conseptualization, Resources, Gyoon Hee Han: Conseptualization, Resources, Jae Young Choi: Conseptualization, Resources, Writing – review and editing, Moo Suk Park: Conseptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing – review and editing Additional Information Competing interests The authors declare no competing interests. Ethics approval and consent to participate All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Yonsei University College of Medicine (6-2018-0164) and were conducted in accordance with the National Animal Care and Use Committee (IACUC). Animal research in this study was performed in accordance with the ARRIVE 2.0 guidelines. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Meyer, N. J., Gattinoni, L. & Calfee, C. S. Acute respiratory distress syndrome. The Lancet 398 , 622-637 (2021). Beitler, J. R., Malhotra, A. & Thompson, B. T. Ventilator-induced Lung Injury. Clin Chest Med 37 , 633-646 (2016). Brower, R. G. et al. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 342 , 1301-1308 (2000). Terragni, P. P. et al. 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Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Jul, 2025 Reviews received at journal 14 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers agreed at journal 29 Jun, 2025 Reviewers invited by journal 23 Jun, 2025 Editor assigned by journal 23 Jun, 2025 Editor invited by journal 31 May, 2025 Submission checks completed at journal 08 May, 2025 First submitted to journal 08 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6561550","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":476388610,"identity":"47b0e4e0-795a-4bff-b45b-7aa11e198f13","order_by":0,"name":"Ji Soo Choi","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Soo","lastName":"Choi","suffix":""},{"id":476388611,"identity":"353b6bff-1e08-40a3-bc4c-5f8f902e9c3c","order_by":1,"name":"Mi Hwa Shin","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mi","middleName":"Hwa","lastName":"Shin","suffix":""},{"id":476388612,"identity":"85ced714-cfdd-43e5-95e1-4b0d07630025","order_by":2,"name":"Go Eun Oh","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Go","middleName":"Eun","lastName":"Oh","suffix":""},{"id":476388613,"identity":"98cec1b7-b132-401f-b432-115da6cab943","order_by":3,"name":"Doo Na Song","email":"","orcid":"","institution":"Yonsei University","correspondingAuthor":false,"prefix":"","firstName":"Doo","middleName":"Na","lastName":"Song","suffix":""},{"id":476388614,"identity":"78f27d17-9f87-4cd4-b147-74fe3578fc72","order_by":4,"name":"Wan NamKung","email":"","orcid":"","institution":"Yonsei University","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"","lastName":"NamKung","suffix":""},{"id":476388615,"identity":"70a81b71-172d-41ad-bb0f-d72fdbd9bce0","order_by":5,"name":"Gyoon Hee Han","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gyoon","middleName":"Hee","lastName":"Han","suffix":""},{"id":476388616,"identity":"8ae2e0c5-c806-4271-a74e-ba5fd69aabcb","order_by":6,"name":"Jae Young Choi","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Young","lastName":"Choi","suffix":""},{"id":476388617,"identity":"f7cf13d1-435b-48d2-8889-7377728d8572","order_by":7,"name":"Moo Suk Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYHAC9h8JFQwMBiTpkfhwhlQtkjPbSNFiLnb8gTHvvMOJ2/kPMH74QYwWy9k5Bsm82w4n7pyRwCzZQ4wWg9s5DIdBWjbcYGCQJsphBrfTHzbzzgFqOX+A+TeRWhKMGWc2ALUcSGAjzhagX8wYPhxLN95wI7HNkii/mEunP2NIqLGW3XD+8OEbRIUYNDqagZixgSh3wbTUEad6FIyCUTAKRiYAAHgTNuGzXUMXAAAAAElFTkSuQmCC","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Moo","middleName":"Suk","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2025-04-30 06:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6561550/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6561550/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-24241-y","type":"published","date":"2025-11-20T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85474616,"identity":"cbdc7a0e-6360-451b-95b4-d6f51ae8e92c","added_by":"auto","created_at":"2025-06-26 09:53:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVentilator-induced lung injury model in mice. \u003c/strong\u003ePendrin wild-type (WT) and knockout (KO) mice were tracheostomized and ventilated in the supine or prone position with a high tidal volume (HTV) of 30 mL/kg and a rate of 100 breaths/min for 5 h. The other ventilator settings were 0.21 inspired oxygen fraction and 0 cm H\u003csub\u003e2\u003c/sub\u003eO end-expiratory pressure. Pendrin inhibitor (YS-01) or vesicle was administered intra-peritoneally 1 hour before inducing VILI in the supine position.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6561550/v1/e40690200ccc3b24d71a4946.jpg"},{"id":85474332,"identity":"2b33bb20-e61f-4ae0-ab99-e90261685474","added_by":"auto","created_at":"2025-06-26 09:45:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":268556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeletion of pendrin and prone positioning attenuated ventilator-induced lung injury in a mice model, as demonstrated by bronchoalveolar lavage (BAL) fluid and histologic findings. \u003c/strong\u003ePendrin wild-type (WT) and knockout (KO) mice were subjected to a tracheostomy and ventilation with a supine or prone position at a high tidal volume (HTV) of 30 mL/kg. Pendrin inhibitor (YS-01) was administered intra-peritoneally 1 hour before inducing VILI in the supine position. (\u003cem\u003eA\u003c/em\u003e) Total cell count in BAL fluid. (\u003cem\u003eB\u003c/em\u003e) Cell differential count of BAL fluid. (\u003cem\u003eC\u003c/em\u003e) BAL fluid cytology presented cell differentiation with visual fields. NVC, non-ventilation control group; supine HTV, HTV ventilation in supine position; prone HTV, HTV ventilation in prone position. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 analyzed by Student’s unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6561550/v1/07e3779c6831b64e863854b5.jpg"},{"id":85473185,"identity":"d010875e-5e67-4e7a-9331-05090b396fe0","added_by":"auto","created_at":"2025-06-26 09:37:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":404790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLung histology in pendrin-KO mice after 5-h HTV ventilation showed a significantly lower degree of leukocyte infiltrations and lung injury score. \u003c/strong\u003ePendrin wild-type (WT) and knockout (KO) mice were subjected to a tracheostomy and ventilation with a supine or prone position at a high tidal volume (HTV) of 30 mL/kg. Pendrin inhibitor (YS-01) was administered intra-peritoneally 1 hour before inducing VILI in the supine position. (\u003cem\u003eA\u003c/em\u003e) Lung injury scoring of each group. (\u003cem\u003eB\u003c/em\u003e) Histopathologic image of hematoxylin and eosin (H\u0026amp;E) staining (×400). NVC, non-ventilation control group; supine HTV, HTV ventilation in supine position; prone HTV, HTV ventilation in prone position. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 analyzed by Student’s unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6561550/v1/40ad08a242cf6b02ddd6868f.jpg"},{"id":85473181,"identity":"3f3d16c6-c445-4bf8-863d-7df23bc69587","added_by":"auto","created_at":"2025-06-26 09:37:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":124350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInflammatory cytokines were significantly decreased in the pendrin-KO mice group than those in pendrin-WT mice in the supine HTV group, analyzed using ELISA.\u003c/strong\u003eNVC, non-ventilation control group; supine HTV, HTV ventilation in supine position; prone HTV, HTV ventilation in prone position; HTV, high tidal volume; WT, wild-type; KO, knockout. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 analyzed by Student’s unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6561550/v1/d6eb9757ead5eda9f155ae75.jpg"},{"id":85473187,"identity":"1a1a5881-d629-426a-b465-3d643fa8ea3f","added_by":"auto","created_at":"2025-06-26 09:37:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePendrin expression increases in HTV ventilation mice. \u003c/strong\u003ePendrin wild-type (WT) and knockout (KO) mice were conducted to a tracheostomy and ventilation with a supine or prone position at a high tidal volume (HTV) of 30 mL/kg. Pendrin inhibitor (YS-01) was administered intra-peritoneally 1 hour before inducing VILI in the supine position. (\u003cem\u003eA\u003c/em\u003e) Western blot and (\u003cem\u003eB\u003c/em\u003e) densitometry analyses of pendrin bands. NVC, non-ventilation control group; supine HTV, HTV ventilation in supine position; prone HTV, HTV ventilation in prone position; PDS, pendrin. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 analyzed by Student’s unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6561550/v1/541c0517b11cf0905608ffd2.jpg"},{"id":85474335,"identity":"7653b0aa-f144-44a9-b50b-5692322cbfe8","added_by":"auto","created_at":"2025-06-26 09:45:14","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":893236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of pendrin deletion on VILI as demonstrated by transmission electron micrographic immune-gold labeling. \u003c/strong\u003ePendrin wild-type (WT) and knockout (KO) mice were subjected to tracheostomy and ventilation with a supine or prone position at a high tidal volume (HTV) of 30 mL/kg. (\u003cem\u003eA\u003c/em\u003e) Non-ventilated group, pendrin-WT mice. (\u003cem\u003eB\u003c/em\u003e) Non-ventilated group, pendrin-KO mice. (\u003cem\u003eC\u003c/em\u003e) HTV ventilation in the supine position, pendrin-WT mice. (\u003cem\u003eD\u003c/em\u003e) HTV ventilation in the supine position, pendrin-KO mice. (\u003cem\u003eE\u003c/em\u003e) HTV ventilation with YS-01 before 1hr in the supine position. (\u003cem\u003eF\u003c/em\u003e) HTV ventilation in the prone position, pendrin-WT mice. (\u003cem\u003eG\u003c/em\u003e) HTV ventilation in the prone position, pendrin-KO mice. (\u003cem\u003eH\u003c/em\u003e) Comparisons of gold particle count labeled with pendrin. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 analyzed by Student’s unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6561550/v1/34332ab3fc09e69d898e4aaa.jpg"},{"id":96650134,"identity":"77c38c31-6fab-4a48-86cc-9d292a5213e6","added_by":"auto","created_at":"2025-11-24 16:08:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2985789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6561550/v1/4ed81347-0349-40ce-bac8-1e2fed7bb1d5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pendrin inhibition is associated with protective effect of prone positioning in a ventilator-induced lung injury mouse model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAcute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterized by the rapid onset of hypoxemia with diffuse bilateral pulmonary infiltrates. It has a high mortality rate and poses a significant financial burden on critically ill patients \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Mechanical ventilation (MV) is often necessary to manage ALI/ARDS; however, it can cause lung damage and aggravate ALI/ARDS \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This condition is known as \u0026ldquo;ventilator-induced lung injury\u0026rdquo; (VILI), and occurs due to the inhomogeneous distribution of lung damage and edema in ALI/ARDS \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Although a ventilator strategy with low tidal volume improves the prognosis of VILI in patients with ALI/ARDS, some patients are inherently susceptible to VILI \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Prone positioning effectively prevents VILI by reducing uneven lung injury \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e; however, research on the molecular mechanisms of prone position management is limited \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePendrin (SLC26A4) is a transmembrane anion exchanger protein located on the apical surface of epithelial cells in the kidney, thyroid, lung, and inner ear \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This protein acts as a Cl⁻/anion exchanger that transports Cl⁻ to bases, including iodide (I⁻), bicarbonate (HCO3⁻), hydroxide (OH⁻), and thiocyanate (SCN⁻) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Mutations in pendrin are associated with Pendred syndrome, a recessive genetic disorder characterized by congenital deafness, goiter, or thyroid hormone abnormalities \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Pendrin is negligibly expressed in normal airway epithelia \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Respiratory diseases characterized by excessive mucus production, including bronchial asthma or chronic obstructive pulmonary disease (COPD), are identified by pendrin overexpression \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Pendrin exacerbates airway diseases triggered by viral infections or allergen exposure \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. An animal model study showed that pendrin knockout mice exhibit significantly decreased lung inflammation caused by Bordetella pertussis infection \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent research suggests that pendrin plays a role in the pathogenesis of lipopolysaccharide (LPS)-induced ALI, and airway epithelial cells treated with pendrin inhibitor may serve as a therapeutic target for managing ALI/ARDS \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Previous \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies have identified increased pendrin expression in an ALI/ARDS animal model using LPS induction, and a novel pendrin inhibitor (YS-01) reduces the inflammatory response in LPS-induced ALI, suggesting that pendrin could be a target for ALI/ARDS treatment \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Alveolar epithelial cell damage occurs and contributes to the lung inflammatory process in an animal model of lung injury by ventilator stretching \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e; however, the role of pendrin in VILI has remained unclear and has not been reported.\u003c/p\u003e \u003cp\u003eBased on these observations, we hypothesized that pendrin expression is associated with the pathogenesis of VILI-induced ALI/ARDS. Therefore, we constructed a VILI mouse model and observed an increase in pendrin expression. We also assessed whether pendrin-null mice and pendrin inhibitor treatment exhibited lower levels of inflammation during VILI. Furthermore, the protective effects of prone positioning were examined in a VILI mouse model.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Deletion of the pendrin decreases the inflammatory response in VILI\u003c/h2\u003e \u003cp\u003eWe performed analysis of BALF and histological assessments by comparing pendrin-WT and pendrin-KO mice to evaluate the role of pendrin in VILI. The total cell count in BALF and lung injury score assessed by histological examination was markedly increased in pendrin-WT mice after HTV ventilation compared with those in the non-ventilation control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Cell differentiation in pendrin-WT mice with HTV ventilation predominantly comprised macrophages with few neutrophils and lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB)\u003c/p\u003e \u003cp\u003eIn the group of pendrin-KO mice, the total cell count in BALF after HTV ventilation was significantly lower compared to pendrin-WT mice group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and lung injury scores were also decreased compared to pendrin-WT mice group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The group of pendrin-WT mice administered pendrin inhibitor (YS-01) showed reduced total cell count in BALF and lung injury scores compared with those of WT mice after HTV ventilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. Prone positioning during ventilation with HTV attenuated the VILI\u003c/h3\u003e\n\u003cp\u003eWe compared BALF and lung histology results between supine and prone HTV mice to investigate the protective effect of prone positioning during HTV ventilation. In the BALF, the total cell count was significantly decreased in prone HTV mice relative to supine HTV pendrin-WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Assessment of lung histology in prone HTV mice revealed a significantly lower degree of leukocyte infiltration and lung injury scores relative to supine HTV pendrin-WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, between the pendrin-WT and pendrin-KO mice groups subjected to HTV in the prone position, the BALF total cell count and lung injury score were lower in the pendrin-KO mice group compared to the pendrin-WT mice group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e3. Pendrin deletion and prone positioning suppressed inflammatory cytokine release in the VILI mice model\u003c/h3\u003e\n\u003cp\u003eWe further investigated the levels of inflammatory cytokines using ELISA to determine the effects of pendrin deletion and prone positioning in a mouse model of VILI. After HTV ventilation, the levels of cytokines, including tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and interleukin 1 beta (IL-1β) were remarkably elevated than those in non-ventilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the supine HTV group, pendrin-KO mice and pendrin-WT mice administered with YS-01 showed lower levels of inflammatory cytokines such as TNF-α and MIP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e4. Increased pendrin expression in HTV-ventilated mice\u003c/h3\u003e\n\u003cp\u003eTo confirm the increase in pendrin expression, we performed western blot analysis and TEM-immunogold labeling for pendrin (SLC26A4). Western blotting showed that pendrin expression was higher in pendrin-WT mice with VILI compared with the control non-ventilation group, pendrin-KO mice, and pendrin-WT mice administered YS-01. Furthermore, when HTV was applied in the prone position, both the WT and KO mice groups showed lower pendrin expression compared to when HTV was applied in the supine position (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe conducted TEM-immunogold labeling for pendrin to compare the expression of pendrin in each group at the ultrastructural level. The HTV WT mice in the supine and prone positions revealed increased pendrin expression on the cell membrane and in vesicles compared with that on the apical side of epithelial cells in the control non-ventilation group, pendrin-KO mice, and pendrin-WT mice administered with YS-01 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). The number of gold particles labeled with pendrin was also higher in pendrin-WT mice with HTV ventilation than in the control non-ventilation group, pendrin-KO mice, and pendrin-WT mice administered with YS-01. Although there was no statistically significant difference between the groups HTV in the supine and prone positions, the number of gold particles labeled with pendrin tended to be lower in the prone position (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, pendrin expression was increased in response to HTV ventilation, and increased pendrin expression was associated with the inflammatory cascade. The modulation of pendrin through pendrin-KO mice and pendrin inhibitor (YS-01) resulted in a decreased inflammatory response in VILI. Furthermore, prone positioning during HTV attenuated lung injury in WT mice and was associated with pendrin expression.\u003c/p\u003e \u003cp\u003eThe transmembrane anion exchanger protein pendrin (SLC26A4) is associated with various disorders in related organs \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Recent research has provided evidence supporting the role of pendrin as a pivotal protein in airway diseases, such as asthma, COPD, and rhinitis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The involvement of pendrin in ALI/ARDS has been suggested in previous studies but its exact mechanism of action remains unclear. Jia et al.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e showed that the distal airway plays a role in ALI/ARDS, and pendrin in alveolar epithelial cells is involved in the inflammatory process of LPS-induced ALI. Furthermore, they demonstrated that the treatment of LPS-induced ALI mice with methazolamide, a carbonic anhydrase inhibitor, significantly reduced lung inflammation. Lee \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e confirmed that pendrin expression increased in both human BAL samples with pneumonia and an LPS-induced ALI mouse model, and pendrin-null mice presented reduced lung damage by LPS. Moreover, they identified that the pendrin inhibitor (YS-01) could attenuate LPS-induced lung injury by inhibiting the pendrin/OSCN-/NF-κB-mediated pathway, indicating the possibility of developing a novel treatment of ALI. They concluded that airway epithelial cells could be a therapeutic target for developing new drugs and strategies for ALI/ARDS treatment. However, additional research is necessary to explore the molecular mechanism of pendrin upregulation and its therapeutic potential in ALI/ARDS.\u003c/p\u003e \u003cp\u003eThe clinical outcomes of ALI/ARDS have significantly improved owing to therapeutic strategies such as low tidal volume with low plateau pressure; however, it remains a challenging condition to manage \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Mechanical ventilation is an important treatment option for ALI/ARDS \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e; notwithstanding, it may paradoxically lead to further lung injury, known as VILI, which can result in poor prognosis \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The mechanism of VILI, including conventional barotrauma, volutrauma, and atelectrauma, comprises initiation by induced tensile strain and direct tissue damage to the lungs, leading to increased permeability and disruption of the alveolar–capillary barrier \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This mechanical strain interrupts the clearance of edema fluid from the airspaces and triggers the release of inflammatory mediators within the distal lung, inducing end-organ dysfunction by entering the systemic circulation \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. An increased inflammation response due to mechanical stretching during HTV ventilation, including elevation of TNF-α, IL-1β, and IL-6, has been observed in the BALF of patients with ARDS and in lung injury mice models \u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e–\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In the present study, we observed increased inflammatory pathology by developing HTV-induced lung injury, consistent with previous studies.\u003c/p\u003e \u003cp\u003eProne positioning during invasive mechanical ventilation improves the outcome in patients with ARDS \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The protective mechanism of prone positioning in ARDS includes improvement of hypoxemia by enhancing ventilation-perfusion mismatching and reducing dependent atelectasis \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Prone positioning can improve cardiac function and hemodynamics \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, respiratory mechanics, and prevent VILI \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In a previous study, Broccard et al. \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e confirmed a significant reduction in lung injury and homogeneous distribution of VILI in normal dogs with HTV ventilation for 6 h in the prone position. Moreover, a recent study by Park reported that prone positioning could affect VILI at the molecular level by influencing the activation of mitogen protein kinases in rodents exposed to HTV ventilation \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent research has focused on elucidating the molecular mechanism of the mechanotransduction pathway in VILI and prone positioning \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. For example, Held \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e reported that mechanical stimuli were found to trigger NF-κB activation, leading to the release of inflammatory cytokines. Notably, they demonstrated the possibility of a therapeutic strategy in which NF-κB activation by mechanical stimuli is inhibited using corticosteroids. Lee \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e reported that NOX4 and Eph/ephrin signaling are implicated in VILI, and a NOX4 inhibitor could potentially serve as a therapeutic intervention for VILI. Furthermore, Park \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e reported that the lung-protective effects of altered EphA2/ephrinA1 signaling through EphA2 antagonism were observed during injurious mechanical ventilation. They also identified that prone positioning exerted a protective effect on VILI. However, the molecular mechanisms underlying VILI and prone positioning remain largely unknown.\u003c/p\u003e \u003cp\u003eOur data showed that pendrin expression was elevated in the VILI mouse model, demonstrating an increased response to lung inflammation. Additionally, pendrin deletion exerted a preventive effect in a VILI mouse model, confirmed using a pendrin inhibitor (YS-01) and pendrin-null mice. Furthermore, we demonstrated that prone positioning is also effective in reducing lung inflammatory responses in VILI, and is associated with pendrin expression. Our data suggest that pendrin expression is associated with lung injury due to mechanical stretching, and the modulation of pendrin might be a novel strategy for preventing VILI.\u003c/p\u003e \u003cp\u003eOur study has some limitations that warrant further consideration. We could not identify the exact mechanism of pendrin expression or its downstream signaling cascades. However, several signaling cascades have already been extensively explored in our previous study. Nonetheless, our study is the first to investigate the relevance of pendrin and prone positioning in VILI caused by mechanical strain. A novel molecule pendrin inhibitor represents a potential new therapeutic approach for VILI treatment. Additionally, we identified the changes in pendrin expression in response to VILI and prone positioning using TEM-immunogold labeling, as in previous studies \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, increased pendrin expression in response to HTV ventilation is related to the inflammatory cascade induced by mechanical strain and tissue injury. Our results showed that modulation of pendrin resulted in decreased inflammatory response in VILI, indicating its protective role against lung injury. We showed that prone positioning during HTV attenuated lung injury in WT mice. We identified pendrin as a potential therapeutic target for managing VILI and improving the outcomes of patients with VILI. Further studies are needed to investigate the relationship between pendrin expression and prone position and explore therapeutic strategies for modulating the expression and activity of pendrin.\u003c/p\u003e "},{"header":"METHODS","content":"\u003ch2\u003e1. Experimental animals\u003c/h2\u003e\u003cp\u003ePendrin wild-type (WT) and knockout (KO) 129SVEV mice (weight 20–25 g, age 6–8 weeks) were donated by JY Choi at Yonsei University. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Yonsei University College of Medicine (6-2018-0164) and were conducted in accordance with the National Animal Care and Use Committee (IACUC). Animal research in this study was performed in accordance with the ARRIVE 2.0 guidelines.\u003c/p\u003e\u003ch3\u003e2. VILI model in mice\u003c/h3\u003e\u003cp\u003eThe mice were subjected to intraperitoneal anesthesia using a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) before performing the tracheostomy. After euthanasia, each mouse was positioned supine and secured on a surgical board. The upper incisors were secured to gently extend the neck, and the exposed trachea was punctured carefully using a 21-gauge needle. The mice were ventilated in the supine or prone position at a high tidal volume (HTV) of 30 mL/kg and a rate of 100 breaths per minute for 5 hours to induce VILI \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Other ventilator settings were 0 cm H\u003csub\u003e2\u003c/sub\u003eO end-expiratory pressure and 0.21 inspired oxygen fraction. Pendrin inhibitor (YS-01) was administered intraperitoneally for 1 hour before inducing VILI in the supine position (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At the completion of the experiment, each mouse was humanely euthanized in a carbon dioxide (CO₂) chamber, following institutional and national guidelines for the humane treatment of laboratory animals.\u003c/p\u003e\u003ch3\u003e3. Study design and experimental protocol\u003c/h3\u003e\u003cp\u003eWild-type (WT) and KO mice were randomly assigned to each HTV ventilation group in the supine and prone positions. The experimental groups were as follows:\u003c/p\u003e\u003cp\u003e1) non-ventilated group, pendrin WT (NVC, WT), n = 10\u003c/p\u003e\u003cp\u003e2) non-ventilated group, pendrin KO (NVC, KO, n = 6\u003c/p\u003e\u003cp\u003e3) supine HTV group, pendrin WT (supine_HTV, WT), n = 10\u003c/p\u003e\u003cp\u003e4) supine HTV group, pendrin KO (supine_HTV, KO), n = 9\u003c/p\u003e\u003cp\u003e5) supine HTV group with YS-01, pendrin WT (1 hour before MV, supine HTV pre-YS-01), n = 5\u003c/p\u003e\u003cp\u003e6) prone HTV group, pendrin WT (prone_HTV, WT), n = 10\u003c/p\u003e\u003cp\u003e7) prone HTV group, pendrin KO (prone_HTV, KO), n = 8\u003c/p\u003e\u003ch2\u003e4. Bronchoalveolar lavage fluid (BALF) analysis\u003c/h2\u003e\u003cp\u003eAfter mechanical ventilation with HTV for 5 hours, bronchoalveolar lavage (BAL) was performed through a tracheal catheter. BALF was directed into a tracheal catheter, gently retracted using 1 mL of sterile saline, and centrifuged (3000 rpm for 10 min at 4℃). Inflammatory cytokines were analyzed using ELISA. The remaining pellets were resuspended in 100 µL PBS to determine cell count.\u003c/p\u003e\u003ch2\u003e5. Histological assessment\u003c/h2\u003e\u003cp\u003eAfter BALF collection, the abdominal cavity was opened with midline incision, followed by excision of the lungs for further histological or molecular analysis. The inflated lung with 4% low-melting agarose was fixed in 10% formaldehyde for a day. It was then embedded in paraffin and sectioned into 5-µm thick slices. Subsequently, lung sections were subjected to hematoxylin and eosin staining and analyzed using bright-field microscopy. Histological evaluation of lung injury on each slide was performed using the weighted scoring scale described in the official American Thoracic Society Workshop Report \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003e6. Transmission electron microscopy\u003c/h2\u003e\u003cp\u003eLung injury was assessed to visualize the target protein of pendrin in the tissue using gold-conjugated antibody labeling procedures for localization at the ultrastructural level through transmission electron microscopy (TEM).\u003c/p\u003e\u003ch2\u003e7. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical tests were performed using Student’s unpaired two-tailed t-test for comparison of each group using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA). Statistical significance was considered at p \u0026lt; 0.05. Data are presented as the mean ± SEM. The lung injury score is presented as the median ± SE.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJi Soo Choi: Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review and editing, Mi Hwa Shin: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Go Eun Oh: Data curation, Formal analysis, Investigation, Doo Na Song: Conseptualization, Resources, Wan NamKung: Conseptualization, Resources, Gyoon Hee Han: Conseptualization, Resources, Jae Young Choi: Conseptualization, Resources, Writing – review and editing, Moo Suk Park: Conseptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing – review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were approved by the Institutional Animal Care and Use Committee of the Yonsei University College of Medicine (6-2018-0164) and were conducted in accordance with the National Animal Care and Use Committee (IACUC). Animal research in this study was performed in accordance with the ARRIVE 2.0 guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMeyer, N. J., Gattinoni, L. \u0026amp; Calfee, C. S. Acute respiratory distress syndrome. \u003cem\u003eThe Lancet\u003c/em\u003e \u003cstrong\u003e398\u003c/strong\u003e, 622-637 (2021).\u003c/li\u003e\n\u003cli\u003eBeitler, J. R., Malhotra, A. \u0026amp; Thompson, B. T. Ventilator-induced Lung Injury. \u003cem\u003eClin Chest Med\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 633-646 (2016).\u003c/li\u003e\n\u003cli\u003eBrower, R. 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W.\u003cem\u003e et al.\u003c/em\u003e Angiotensin II acts through the angiotensin 1a receptor to upregulate pendrin. \u003cem\u003eAm J Physiol Renal Physiol\u003c/em\u003e \u003cstrong\u003e301\u003c/strong\u003e, F1314-1325 (2011).\u003c/li\u003e\n\u003cli\u003eVaneker, M.\u003cem\u003e et al.\u003c/em\u003e Mechanical ventilation in healthy mice induces reversible pulmonary and systemic cytokine elevation with preserved alveolar integrity: an in vivo model using clinical relevant ventilation settings. \u003cem\u003eAnesthesiology\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 419-426 (2007).\u003c/li\u003e\n\u003cli\u003eMatute-Bello, G.\u003cem\u003e et al.\u003c/em\u003e An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. \u003cem\u003eAm J Respir Cell Mol Biol\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 725-738 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pendrin, SLC26A4, ventilator-induced lung injury, acute lung injury, acute respiratory distress syndrome, prone","lastPublishedDoi":"10.21203/rs.3.rs-6561550/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6561550/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePendrin (SLC26A4), a transmembrane anion exchanger, is upregulated in inflammatory airway diseases. In this study, we analyzed the role of pendrin expression in a ventilator-induced acute lung injury (VILI) animal model. VILI was induced in the supine or prone position by a high tidal volume (HTV) of 30 mL/kg for 5 hours in pendrin wild-type (WT) and knockout (KO) 129SVEV mice. Pendrin inhibitor (YS-01) was intraperitoneally administered to modulate pendrin signaling. Lung injury parameters were assessed based on bronchoalveolar lavage fluid (BALF) analysis, inflammatory cytokine analysis by ELISA, and histopathological findings. Pendrin expression was determined by western blotting and transmission electron microscopy (TEM) using immunogold labeling methods. The degree of lung injury was significantly attenuated in pendrin-KO mice and pendrin-WT mice with YS-01 compared with pendrin-WT animals after HTV ventilation. Pendrin expression was down-regulated in pendrin-KO mice and pendrin-WT mice with YS-01 compared with pendrin-WT mice with VILI, as determined by western blotting and TEM-immunogold labeling. Prone positioning during ventilation attenuated lung inflammation and pendrin expression. Our results suggest that pendrin is critical in VILI and could be a novel target for modulating VILI. Prone positioning and pendrin inhibition in VILI may be effective in managing these conditions.\u003c/p\u003e","manuscriptTitle":"Pendrin inhibition is associated with protective effect of prone positioning in a ventilator-induced lung injury mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 09:37:09","doi":"10.21203/rs.3.rs-6561550/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-24T17:16:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-14T14:56:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188632718054598310915057674885094894268","date":"2025-07-09T13:49:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160612851882668954387475416550362858066","date":"2025-06-29T10:15:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T02:40:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-24T02:33:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-31T13:39:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-09T01:37:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-09T01:36:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ecd814ef-3658-437b-89a4-0165be73bef5","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50587923,"name":"Biological sciences/Biochemistry/Ion channels"},{"id":50587924,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2025-11-24T16:02:06+00:00","versionOfRecord":{"articleIdentity":"rs-6561550","link":"https://doi.org/10.1038/s41598-025-24241-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-20 15:57:15","publishedOnDateReadable":"November 20th, 2025"},"versionCreatedAt":"2025-06-26 09:37:09","video":"","vorDoi":"10.1038/s41598-025-24241-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24241-y","workflowStages":[]},"version":"v1","identity":"rs-6561550","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6561550","identity":"rs-6561550","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}