Alveolar macrophages polarity switch via α 2 -adrenoceptor activation ameliorates pulmonary inflammation following kidney ischemia reperfusion

doi:10.21203/rs.3.rs-2594924/v1

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Alveolar macrophages polarity switch via α 2 -adrenoceptor activation ameliorates pulmonary inflammation following kidney ischemia reperfusion

https://doi.org/10.21203/rs.3.rs-2594924/v1

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Purpose: To investigate the anti-inflammatory mechanism of dexmedetomidine (Dex), an α2-adrenoceptor (α2-AR) agonist, on renal ischemia-reperfusion (RIR)-induced acute lung injury (ALI).

Methods: RIR was performed in C57BL/6J mice by bilateral renal pedicles occlusion for 60min and reperfusion for 24h. Mice were pre-treated with or without Dex alone or combined atipamezole (Atip), an α2-AR antagonist. The pulmonary histopathological evaluation, arterial blood gas analysis, cell count and multiple cytokines examination in BALF, global inflammation status assessment in lung tissue and alveolar macrophages phenotype investigation were accomplished. In vitro, the polarity of mice alveolar macrophages (MH-S) treated with serum from normal or RIR mice were indirectly detected by qPCR.

Results: The results indicated that, compared to RIR animal, dexmedetomidine reduced lung injury and significantly promoted macrophage polarization towards an anti-inflammatory M2 phenotype in the pulmonary tissue. Meanwhile, the reduction of inflammatory cell infiltration and pro-inflammatory cytokines levels were observed. In vitro studies confirmed that dexmedetomidine skewed MH-S towards M2 phenotype after stimulation of RIR serum. After administration of the atipamezole, these above effects were abolished.

Conclusion: Dexmedetomidine ameliorates renal ischemia-reperfusion induced ALI through activation of α2-adrenoceptor to skew macrophages towards an anti-inflammatory phenotype to reduce pulmonary global inflammation.

Renal ischemia-reperfusion

Acute lung injury

α2-adrenoceptor

Dexmedetomidine

Macrophage polarization

Acute kidney injury (AKI) is estimated to account for 2 million deaths worldwide annually and becoming a growing global health concern [1,2]. Although the dialytic techniques and therapeutic methods have been greatly improved over the past decades, the morbidity and mortality associated with AKI seems to be no significant improvement [3]. The optimal therapeutic treatments to prevent AKI or promote recovery following AKI are still insufficient. Accumulated studies on AKI animal models induced by the nephrectomy or kidney ischemia demonstrated that AKI was often associated with the pathological damage and dysfunction of extra-renal multiple organs, especially lung, which was one of the important reasons causing high AKI mortality [4]. The clinical statistical data have shown that the AKI mortality of critically ill patients was 44.7-53% [5], and it would rise sharply to 80% when combined with acute lung injury (ALI) [6].

Pulmonary secondary inflammatory response is a predominant cause of AKI-induced ALI. Macrophage recruitment and activation are the early events during immune cells recruitment and cytokines/chemokines networks formation, which maintain the activation and development of inflammation. Macrophages with multifunctional characteristic possess the strong phagocytic capacities being well known. Over the last few decades, more and more in vivo and in vitro experiments proved that the macrophages play a wide range of complex roles in host defense, tissue development, homeostasis maintenance, as well as involving in the processes of tissue injury, repair and fibrosis [7]. Unlike the peripheral blood macrophages, the alveolar macrophages (AMs) are derived from embryonic precursors and have an unique longevity and the self-renewal capacity [8]. AMs account for 90-95% of the alveoli lumen and usually do not move much more in the alveolar lumen under homeostasis. Given that there are several million alveoli and around one to two million AMs in a mouse lung, only about one in every three alveoli can be found [9]. However, AMs can travel between alveoli through the pores of Cohn to maintain homeostasis [10]. A mouse lacking AMs would not be alive due to pulmonary failure and hypoxia [11]. The previous studies indicated that AMs play a critical role in the initiation, maintain and resolution of pulmonary inflammation during ALI process, which is closely associated with the polarity switch of AMs subtypes: M1 macrophages, a pro-inflammatory phenotype and releasing IL-1β, TNF-α, IL-12, iNOS and CCL2, and M2 macrophages, an anti-inflammatory phenotype and secreting high deal of IL-10, IL-1ra, Arg, Fizz1 and CCL22 [12]. Evidences have indicated that the original polarization of macrophage is easily reversible upon environmental changes. Therefore, it is believed as an effective treatment strategy for AKI-induced ALI to orchestrate the macrophage polarity, from M1 to M2 phenotype, to ameliorate pulmonary inflammation as well as promote tissue repair.

Dexmedetomidine (Dex), a potent and highly selective α₂-adrenergic agonist, exhibits sedative, analgesic, amnestic, and sympatholytic properties, and is widely used in critically ill and anesthetic patients. Dex has been found to possess organ protective effects in clinical practice [13], including pulmonary protection [14]. Similarly, our previous studies have proved that Dex is able to protect against ALI after renal ischemia reperfusion [15,16], predominantly due to its ability to anti-inflammation, ameliorate pulmonary microvascular hyper-permeability and anti-apoptosis [17,18]. However, the mechanism of anti-inflammatory effect of Dex remains unclear. The aim of this study is to investigate the hypothesis that dexmedetomidine can modulate macrophage polarity to ameliorate acute pulmonary inflammation when renal ischemia reperfusion occurs.

Animals

C57BL/6J male mice (8- to 12-week-old, weighing 18-22g) were purchased from the laboratory animal center of Army Medical University. Animals were housed in a humidity- and temperature-controlled animal facility in the central laboratory of southwest hospital with a 12-hour light-dark cycle for at least 7 days before induction of renal ischemia reperfusion. Food and water were freely accessible. All the experimental procedures were approved by the Animal Care Committee of Army Medical University and in compliance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Surgical procedure and groups

A renal ischemia reperfusion injury model was performed in mice with bilateral renal pedicles clamped as described previously [15]. Briefly, all mice were anesthetized with 1.5% isoflurane and placed on a heating pad to maintain the temperature at 36 ± 0.1℃. A midline abdominal incision was made. The sterile swabs were used to expose the bilateral renal pedicles. The pedicles were clamped for 60 min with the microvascular clamps followed by reperfusion for 24 hrs. Dex (Orion Pharma, Espoo, Finland) and α₂-adrenergic antagonist Atip (Sigma-Aldrich, St. Louis, MO, USA) were administrated or not used in the following groups. Sham group (Sham): underwent laparotomy without bilateral renal blood flow obstructions. Dex group (Dex): Dex was intraperitoneally injected at 25 μg/kg without laparotomy. Renal ischemia reperfusion injury group (RIR): bilateral renal pedicles were clamped for 60 min and then kidney blood flow was recovered for 24 hrs. Pretreatment with Dex group (Dex + RIR): Dex 25 μg/kg was intraperitoneally injected at 15 min prior to renal ischemia. Combination of the atipamezole group (Atip + Dex + RIR): Atip 250 μg/kg was intraperitoneally injected 10 min prior to Dex pretreatment.

Hematoxylin and eosin (HE) staining

At 24 hrs after RIR, animals were euthanized and perfused with saline through right atrium till the lung tissue turned white. Lungs were harvested and paraffin embedded, then the lung tissues were cut into 5-μm thick sections. Sections were stained with hematoxylin/eosin, then examined under a light microscope. The degree of lung injury was scored on a scale from 0 to 3 using a previously described scoring system: Grade 0, normal pulmonary appearance; Grade 1, mild moderate interstitial congestion and neutrophil leukocyte infiltrations; Grade 2, perivascular edema formation, partial leukocyte infiltration, moderate neutrophil leukocyte infiltration; Grade 3, severe destruction of the lung architecture and massive neutrophil leukocyte infiltration.

Arterial blood gas analysis

Arterial blood samples were obtained for blood gas analysis. A 0.5 ml sample of arterial blood was drawn from the abdominal aorta. pH, partial pressure of oxygen (PaO₂) and partial pressure of carbon dioxide (PaCO₂) were measured at the end of the reperfusion period with a blood gas analyzer (Beckman Coulter, Inc., USA)

Isolation of AMs from murine bronchoalveolar lavage fluid (BALF) and treatment

At 24 hrs after renal ischemia reperfusion, animals were anesthetized with isoflurane (3%, mask inhalation), and a midline cervical incision was made. Gently blunt dissection was used to expose the airway, and a small incision (<2mm) was made on the trachea posterior to the larynx. Inserted a 22G catheter without a needle into the trachea towards the lungs and secured the catheter with a silk braided suture (4-0; non-absorbable) with a square knot. 0.8 ml ice-cold BALF buffer (Ca²⁺ and Mg²⁺ free /PBS + Ethylenediaminetetraacetic acid, EDTA1mM) was slowly perfused into the lungs. Keep the buffer in the lung for 5 seconds and then aspirate the fluid, repeat for 2 more times and pool the lavage fluid. Then euthanize the mouse by an approved protocol. Centrifuge the approximately 3 ml BALF at 300g for 5 min at 4℃. The cell pellet was collected, and the supernatant was stored at -80℃.The collected cells from BALF was resuspended and cell counting was performed with a cell count analyzer (ThermoFisher). Alveolar macrophage ratios were analyzed by flow cytometry, and macrophage polarization-related indicators were detected by immunofluorescence and western-blot.

Protein array

Inflammatory mediators in BALF and lung tissue were assessed respectively with LEGENDplextm mouse macrophage/microglia panel (Biolegend) and mouse inflammation array Q1 (Raybiotech) at 24 hrs after RIR according to the instruction from manufacturer.

Cell culture and treatment

The murine alveolar macrophages cell line (MH-S) were cultured with RPMI 1640 medium which contains 15% fetal serum at 37℃ and 5% CO₂. Cells were incubated with or without Atip 1μM for 3 hrs before pre-administrated with 0.1 μM Dex for 1 hr. Then replace the medium with normal or RIR serum for another 24 hrs.

Flow cytometry

Cells collected from BALF were resuspended in Cell Staining Buffer (Biolegend Cat. No. 420201). Then TruStain FcX™ PLUS (anti-mouse CD16/32) Antibody (Biolegend, Inc. Cat. No. 156603) was used for blocking non-specific binding of immunoglobulin to the Fc receptors. After cell-surface staining with FITC anti-mouse CD11c and PerCP anti-mouse/human CD11b antibody (Biolegend, Inc.; Cat. No. 117305，No. 101229 respectively), flow cytometric analysis was performed with Agilent NovoCyteTM and analyzed by NovoExpressTM.

Immunofluorescence staining and quantification

Cell samples were fixed with 4% paraformaldehyde in PB for 20 min at room temperature. Then, samples were washed with PBS, permeabilized, blocked, and stained with primary antibody and then secondary fluorescence-conjugated antibody. Nuclei were stained by DAPI. Images were visualized with a confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany). The antibodies used were as follows: rabbit polyclonal anti-iNOS (1:100; Abcam), goat polyclonal anti-Arg1 (1:200; Abcam), donkey anti-rabbit secondary antibody, Alexa Fluor 647, donkey anti-goat secondary antibody, Alexa Fluor 488 (1:500; all from Abcam).

Western blot

The lung tissues were harvested at the end of the experiment. Total proteins were extracted from tissue utilizing RIPA lysis buffer. After determining the concentration of protein by bicinchoninic acid assay (Thermo Fisher Scientific), 20 μg of the total protein fractions were separated by 10% SDS-PAGE and electroblotted onto PVDF membranes (Millipore Sigma). The membranes were blocked with 5% bovine serum albumin for 1hr and then probed with primary antibodies against iNOS, Arg1 (1:1000; Abcam, Cambridge, United Kingdom), and GAPDH (1:10000; Bioworld Technology, Bloomington, MN, USA) overnight at 4℃. These blots were then incubated with horseradish peroxidase–conjugated secondary antibodies for 1hr and visualized using an ECL detection system (Millipore Sigma). Quantitative analysis was performed for immunoreactive bands using ImageJ software (National Institutes of Health, Bethesda, MD, USA)

Statistical analysis

All results were expressed as mean ± standard error of the mean. All statistical analyses were performed with SPSS 24.0. The differences in the means among multiple groups were analyzed using one-way analysis of variance followed by post hoc Newman keuls test. Two-group comparisons were tested by the Student's t test. p<0.05 was considered to be of statistical significance.

α₂-adrenoceptor activation exerted by Dex protected against the pulmonary tissue pathological injury and improved the blood gas after renal ischemia reperfusion

HE staining for lung histology was evaluated (Fig.1A). The alveoli were integrated and the exudation in the alveoli was less in the sham or Dex group. In contrast, the alveolar structures in the RIR group were damaged with a significant pulmonary interstitial edema, a large number of red blood cells and inflammatory cells in the alveolar cavity. However, the pulmonary injury was ameliorated by Dex administration (Dex + RIR group), as evidence by the more intact alveolar structure and improved interstitium edema, and a reduction of alveolar infiltration of red cells and inflammatory cells. These protective effects of Dex were abolished by the α₂-adrenoceptor antagonist atipamezole (Atip + Dex + RIR group). All of these changes were corroborated by the histological scores (Fig.1B). The pulmonary injury was further assessed through arterial blood gas analysis (Fig.1C). The arterial partial pressure of oxygen (PaO₂) and CO₂ (PaCO₂) decreased in RIR group compared to the sham or Dex group (p<0.01), which was improved slightly by Dex pre-treatment, but there was no statistical significance. The pH value in the RIR group decreased after the experiment compared with sham or Dex group (p<0.01). Acidosis can be improved significantly with Dex pre-treatment (p<0.01). Besides, atipamezole abolished this effect.

α₂-adrenoceptor activation exerted by Dex reduced BALF cells number after renal ischemia reperfusion and the measurement of alveolar macrophages ratio in BALF

The BALF cell counts were counted by a cell counter (Fig.2A). Compared with Sham group, the BALF cell counts in RIR group significantly increased (p<0.01). Dex pre-treatment significantly attenuated the cells increasing (p<0.01), while Atip reversed the effect of Dex. Flow cytometry was performed to detect CD11c negative and CD11b positive cells to distinguish alveolar macrophages in BALF (Fig.2B). The results showed that AMs predominated in BALF cells of all groups.

Dex improved BALF anti-inflammatory milieu after renal ischemia reperfusion

The expression of inflammatory cytokines in BALF was measured via a cytokine array, which focus on cytokines associated with macrophage polarization (Fig.3). Dex pre-treatment down-regulated M1-associated cytokines TNF-α, IL-1β (p<0.05) and up-regulated M2-associated cytokines IL-10, CCL17. The down-regulation of other M1-associated factors including CXCL1 and IL-12p40 as well as the up-regulation of other M2-associated factors including G-SCF, CCL22 were observed after Dex pre-treatment, although no statistical significance compared to the RIR group. Taken together, renal ischemia caused the pro-inflammatory milieu while Dex attenuated it, which might be associated with the polarity switch of alveolar macrophage from M1 to M2.

Dex reduced various lung tissue inflammatory factors after renal ischemia reperfusion

The expressions of inflammatory factors in lung tissue 24 hrs after reperfusion were measured using a commercial protein array, focusing on the inflammatory cytokines and chemokines (Fig.4A). Dex resulted in remarkable decreases (Fig.4B) in the levels of 15 kinds of cytokines (IL-1α、IL-β、IL-2、IL-6、IL-7、IL-15、TNF-α、CD54、G-CSF、MIP-1γ、TCA-3) and chemokines (CCL5、CCL11、CXCL1、CXCL13) in the premise of comparable reference protein. The protein expressions levels of other 25 inflammatory factors remained unchanged, while their decline trends were observed. These data indicated that pre-treatment of Dex effectively improved the lung global inflammatory microenvironment after renal ischemia reperfusion.

Dex abated alveolar macrophages polarity switch to pro-inflammatory M1 phenotype after RIR through α₂-adrenoceptor dependent way

To determine the effects of Dex on the phenotype changes of alveolar macrophages after renal ischemia reperfusion, the isolated alveolar macrophages were subjected to immunofluorescence detection and Western-Blot assay. The immunofluorescence staining (Fig.5A) and western blot (Fig.5B) were used to detect the phenotype markers of M1 (iNOS) and M2 (Arg1). The immunofluorescence indicated that iNOS positive cells markedly increased and Arg1 positive cells slightly decreased after RIR, while Dex pre-treatment significantly decreased iNOS positive cells (Dex + RIR) and Atip inhibited Dex’s effect (Atip + Dex +RIR). The protein expressions of iNOS and Arg1 detected using western blot showed some similar changes with the immunofluorescence results (Fig.5B, C). Briefly, compared to sham and Dex treatment, RIR significantly increased the iNOS expression (p<0.01) as well as reduced the Arg1 expression (p<0.01), pre-treatment of Dex attenuated the iNOS up-regulated expression (p<0.01) and upregulated Arg1 expression level. Finally, Atip abolished the effects of Dex on iNOS and Arg1 expression (Fig.5C), which was consistent with the results of immunofluorescence.

α₂-adrenoceptor activation exerted by Dex attenuated M1 markers and promoted M2 markers in alveolar macrophages treated by RIR serum at mRNA level

The murine alveolar macrophages cell line (MH-S), after pre-administrated with or without only Dex or combined Atip before RIR serum treatment, was subjected to gene investigation for M1 and M2 markers via quantitative real-time PCR (qPCR). The RIR serum caused significant increase of M1 markers mRNA (iNOS, IL-1β and TNF-α, Fig.6A, B and C) and decrease of M2 markers mRNA (MRC and Arg1, Fig.6D, E) in alveolar macrophages (p<0.01). Dex pre-treatment attenuated the above M1 markers and promoted the above M2 markers (p<0.05 or p<0.01). However, atipamezole almost reversed all effects of Dex (Fig.6).

Our current study indicated that the polarization of alveolar macrophage was promoted to M1 pro-inflammatory phenotype, which might be one of key factors involve in pulmonary inflammation initiation following renal ischemia reperfusion. Dex pre-treatment modulated the polarization switch of alveolar macrophage, from M1 to M2 phenotype with anti-inflammatory properties, to significantly reduce the expression of various pro-inflammatory cytokines at different biological levels in lung, as well as attenuated ALI induced by AKI. The present data partly elucidated the anti-inflammatory and pulmonary protection mechanism of Dex when AKI occurs.

The perioperative ischemia-induced AKI is commonly in the clinical ICU setting, epidemiologically characterized with high morbidity, mortality and medical costs. Lots of experiments demonstrated that AKI is associated with pathological damage and dysfunction of multiple extra-renal organs, including lung, heart, brain, liver and intestine, which can directly or indirectly lead to poor prognosis of AKI [19]. Secondary lung injury following AKI is the common extra-renal complication among AKI induced multiple organ dysfunction. The previous basic and clinical studies explained the possible pathophysiological mechanism of distal organ damage induced by AKI, including leukocyte infiltration, production of soluble factors (such as inflammatory cytokines/chemokines), endothelial damage, oxidative stress, and cell apoptosis [20-22]. A recent study revealed that the local injury could activate macrophages in remote organs, the systemic complications following myocardial infarction, stroke, and sepsis could be improved if regulation of targeted macrophage function [16].

AKI-induced ALI is characterized by increased vascular permeability, interstitial edema, alveolar hemorrhage and red blood cell deposition [6]; consistent with the pulmonary pathological reported in this experiment (Fig.1A). Dex effectively improved the lung pathological score compared to RIR group (Fig. 1B). Arterial blood gas analysis showed that RIR caused acidosis, hypoxemia, and hypocapnia and Dex significantly reversed these statuses (Fig.1C). Low partial pressure of carbon dioxide might be caused by the accelerated respiratory rate due to acidosis. The cell number in alveolar lavage fluid were significantly increasing, accompany with higher pro-inflammatory and decreased anti-inflammatory factors after renal ischemia-reperfusion. Dex pretreatment reduced the cell aggregation and pro-inflammatory factors contents, instead increase anti-inflammatory factors levels (Fig.2, 3). These results implied that Dex could improve alveolar ventilation and oxygenation by adjusting pulmonary inflammatory microenvironment.

As we know, AMs play a key role in the inflammatory initiation, maintenance and resolution, even later repair and reconstruction. Moreover, the AMs subpopulation plays different roles in the process [12]. AMs are divided into three subtypes upon their polarity: steady-state M0 phenotype, pro-inflammatory M1 phenotype, anti-inflammatory and pro-repair M2 phenotype. Some scholars believe that M2 is the default phenotype of colonized macrophages, which is also M0 type [23]. M1 macrophage can engulf the pathogens and cell debris, secrete some chemokines and pro-inflammatory factors and complete antigens presentation to initiate immune responses. While M2 macrophage possesses the opposite abilities, secreting IL-10, TGF-β, etc., to exert immunity suppression effects [24]. iNOS and Arg1 are classic markers for M1 and M2 macrophage respectively. iNOS is not a physiologically expressed protein in macrophages and its expression is commonly stimulated by pro-inflammatory cytokines such as IL-1, TNF-α and IFN-γ. Arg1 is significantly expressed in macrophages after stimulation with IL-4 and IL-13, while Arg1 expression is found in macrophages in a variety of tissues [25,26]. Here, we revealed that Dex pretreatment or Dex combined with RIR decreased M1 phenotype and increased M2 phenotype, indicating that Dex could promote the conversion of macrophages to M2 phenotype both under the physiological and pathological conditions, which may be a vital anti-inflammatory mechanism of Dex and thus to protect lung against RIR-induced injury. In addition, α₂-AR antagonists reversed this effect of Dex, suggesting that Dex works by activating α₂-AR.

The polarity switch in macrophages is orchestrated by a series of complex molecules and signaling pathways. The signaling pathways mentioned in current researches include JNK, Notch, TGF-β, JAK/STAT, TLR/NF-κB and hypoxic dependence. Epigenetic, post-transcriptional and post-translational modifications are also involved in the regulation of macrophage polarity [27,28]. Some other studies have shown that M1 type of macrophages rely on glycolysis to generate energy, while M2 type produces ATP through the tricarboxylic acid cycle [29,30], which corresponds to the pathway of energy provision under stress- and steady-state. α₂-AR is widely distributed in central nervous system and peripheral tissues, such organ protective effects of Dex have been detected in brain [31], heart [32] and lung [33] injury. Likewise, our previous experiments found that excitation of α₂-AR exhibits cytoprotective effects on human renal tubular epithelial cells [15]and pulmonary microvascular Endothelial cells [17]including activating intracellular focal adhesion kinase (FAK) and PI3K/Akt pathway, which increase Akt phosphorylation [18,34]. Moreover, a previous study demonstrated that FAK and PI3K/Akt were involved in the regulation of the polarity switch of glioma infiltrating microglia/macrophages though the accurate mechanism remained unclear [35]. Therefore, it is speculated that α₂-AR/FAK/PI3K/Akt may be a potential signaling pathway of macrophage polarity switch according to Dex’s capacity in the present study. However, some recent studies have pointed out that the activation pathways of macrophages in vivo and in vitro are not consistent, and a considerable part of them belong to private signal pathways, which brings challenges to the exploration of polarization regulation mechanism of macrophages [36].

Then we used tissue chips to detect the changes of lung inflammatory factors (Fig.4). Secondary inflammatory reaction is an important cause of acute lung injury after renal ischemia-reperfusion [37-40]. The results showed that the levels of inflammatory factors generally increased, and notably TNF-α [41], IL-6, IL-1β [42], the key ALI-related factors increased significantly after RIR. Furthermore, some anti-inflammatory factors also increased, which may be associated with the feedback of pro-inflammatory factors. After Dex intervention, it was found that the levels of 15 inflammatory factors decreased, including IL-6, IL-1β, and TNF-α. Other inflammatory factors also trended to reduce, although there was no statistical difference. This result reflects that Dex pretreatment can improve the inflammatory microenvironment of lung tissue after renal ischemia-reperfusion.

We proved that Dex is available to promote the switch of pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype and greatly ameliorate the secondary lung inflammation, thereby attenuating acute lung injury induced by renal ischemia-reperfusion, which might indicate a promising therapeutic strategy in remote organ crosstalk injury.

Authors' contributions

Zhigang Qin, Jieyu Li and Xinhai He contribute equally to this paper, they designed the study, analyzed data, and wrote the manuscript. Zhigang Qin, Ziheng Yang and Xiangfeng Liu performed the experiments, analyzed data, and drafted the manuscript. Yueming Xu and Zhengwei Xue analyzed data, and assisted with writing the manuscript. Bin Yi and Peng Li supervised and contributed to the results’ discussion and manuscript preparation. Jianteng Gu provided ideas,result’ discussion and experimental funding. All authors have reviewed the final version of the manuscript and approved its submission for publishing.

Availability of data and materials

The data underlying this article will be shared on reasonable request to the corresponding author.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81772050) and Excellent Talents Foundation of Army Medical University (XZ2019-505-028), Chongqing, China.

Competing Interests

The author reports no conflicts of interest in this work.

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Alveolar macrophages polarity switch via α 2 -adrenoceptor activation ameliorates pulmonary inflammation following kidney ischemia reperfusion

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Abstract

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Introduction

Materials And Methods

Animals

Surgical procedure and groups

Hematoxylin and eosin (HE) staining

Arterial blood gas analysis

Isolation of AMs from murine bronchoalveolar lavage fluid (BALF) and treatment

Protein array

Cell culture and treatment

Flow cytometry

Immunofluorescence staining and quantification

Western blot

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

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