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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 2 | Page : 73-84 |
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The roles of AMPK/mTOR autophagy pathway in the acute kidney injury-induced acute lung injury
Si-Heng Shen, Ruo-Lin Wang, Qi Yuan, Lu-Yong Jian, Hua-Hui Guo, He-Sheng Li, Xue-Pin Liu, Ren-Fa Huang
Department of Nephropathy, Shenzhen Hospital (Futian) of Guangzhou University of Chinese Medicine, Shenzhen, China
| Date of Submission | 30-Oct-2022 |
| Date of Decision | 29-Nov-2022 |
| Date of Acceptance | 01-Dec-2022 |
| Date of Web Publication | 20-Apr-2023 |
Correspondence Address:
Dr. Ren-Fa Huang
No. 6001, Beihuan Avenue, Futian District, Shenzhen
China
 Source of Support: None, Conflict of Interest: None  | 57 |
DOI: 10.4103/cjop.CJOP-D-22-00122
Acute kidney injury (AKI) is one of the most challenging clinical problems in kidney disease due to serious complications and high mortality rate, which can lead to acute lung injury (ALI) through inflammatory reactions and oxidative stress. Adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has been reported to be involved in the development of renal ischemia-reperfusion through autophagy and it remains unclear whether AMPK/mTOR pathway has an effect on the AKI-induced ALI. In this study, we aimed to investigate the effects of autophagy-related AMPK/mTOR signaling pathway on inflammatory factors and oxidative stress in an AKI-induced ALI model. The 48 male Sprague–Dawley rats were divided into four groups randomly: (i) sham, (ii) ischemia/reperfusion injury (IRI), (iii) IRI + rapamycin (RA), and (iv) IRI + 3-methyladenine (3-MA). Unilateral flank incisions were made and right kidneys were excised. The left kidney was subjected to 60 min of ischemia followed by 12, 24, 48, and 72 h of reperfusion. The levels of Scr, blood urea nitrogen (BUN), Wet/Dry ratio, indexes of inflammation, and oxidative stress were assayed. Histological examinations were performed. The protein expression of AMPK, mTOR, LC3-II/LC3-I ratio, and Beclin-1, ULK1 was evaluated by western blotting and immunohistochemistry. Compared to the rats from the sham group, IRI rats showed significantly pulmonary damage after AKI with increased Scr, BUN, Wet/Dry ratio, indexes of inflammation, and oxidative stress. The expression of AMPK, LC3-II/LC3-I ratio, Beclin-1, and ULK1 and were increased, while p62 and mTOR were decreased. In addition, RA treatment significantly attenuated lung injury by promoting autophagy through the activation of the AMPK/mTOR pathway, and 3-MA treatment exhibited adverse effects inversely. Therefore, the activation of the AMPK/mTOR pathway after renal IRI induction could significantly attenuate kidney injury and following AKI-induced ALI by inducing autophagy, which alienates inflammation, oxidative stress, and apoptosis.
Keywords: Acute lung injury, AMPK/mTOR pathway, autophagy, inflammation, renal ischemic-reperfusion
How to cite this article:
Shen SH, Wang RL, Yuan Q, Jian LY, Guo HH, Li HS, Liu XP, Huang RF. The roles of AMPK/mTOR autophagy pathway in the acute kidney injury-induced acute lung injury. Chin J Physiol 2023;66:73-84 |
How to cite this URL:
Shen SH, Wang RL, Yuan Q, Jian LY, Guo HH, Li HS, Liu XP, Huang RF. The roles of AMPK/mTOR autophagy pathway in the acute kidney injury-induced acute lung injury. Chin J Physiol [serial online] 2023 [cited 2023 May 29];66:73-84. Available from: https://www.cjphysiology.org/text.asp?2023/66/2/73/374409 |
Si-Heng Shen and Ruo-Lin Wang, contributed equally to this work and share first authorship.
| Introduction | |  |
Acute kidney injury (AKI) is one of the most challenging clinical problems in kidney disease due to serious complications and high mortality rate.[1] Renal ischemia-reperfusion injury (IRI) is the most critical pathophysiological mechanism of AKI.[2] Renal IRI can induce the release of chemokines that act as a major mediator of inflammation, including the expression of pro-inflammatory cytokines and adhesion molecules, and leukocyte infiltration and activation.[3] In addition, significantly increased reactive oxygen species and malonaldehyde (MDA) were observed in renal IRI, which exceeds the scavenging capacity of antioxidant enzymes such as superoxide dismutase (SOD), thus causing apoptosis of renal tubular epithelial cells.[4]
AKI rarely occurs as a single disease but interacts with the injury of extrarenal organs, especially the lung, which is one of the most common extrarenal organs in patients with AKI.[5] Some circulating biochemical mediators, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, have been related with the mediation of lung injury associated with AKI by enhancing lung inflammation, neutrophil recruitment to the lung, and pulmonary capillary leakage.[6],[7] In addition, macrophages target the site of injury by releasing pro-inflammatory DAMP molecules as well as hypoxia-inducible factors from damaged tubular epithelial cells in post-ischemic kidneys.[8] They initiate a pathological cascade and increase the mortality rate, especially 80% when AKI is combined with acute lung injury (ALI).[9]
Autophagy is an important conserved catabolic process that utilizes the activity of lysosomes to degrade cytoplasmic substances, such as cytoplasmic proteins and organelles. Researches have recognized a connection between the autophagy and ischemic kidney injury.[10],[11] Numerous pieces of evidence have also demonstrated that autophagy is significantly involved in ALI. A study by Meng confirmed that the inhibition of autophagy in lipopolysaccharide-induced ALI can significantly increase the levels of inflammation (IL-6 and TNF-α) and oxidative stress (H2O2, O–2, and NO).[12] Another study also demonstrated that improving autophagy activity could attenuate myocardial IRI-induced ALI in diabetic rats, possibly by constraining the inflammation and oxidative stress.[13] These results suggested that ischemia-reperfusion of distal organs can lead to ALI through inflammatory reactions and oxidative stress, and autophagy can mitigate the damage in lung.
The adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway is one of the signaling pathways which tightly associated with autophagy. AMPK can effectively promote the activation of autophagy via suppressing the activity of mTOR.[14] AMPK is a conserved sensor of cellular energy change, which maintains energy balance by reducing ATP-consuming processes while increasing metabolic pathways to conserve ATP when energy is insufficient. For example, fatty acid oxidation, autophagy, mitochondrial synthesis, and oxidative metabolism are enhanced during this process.[15] AMPK activation was found to greatly increase autophagy flux in renal tubular cells, thereby preventing them from apoptosis and maintaining mitochondrial homeostasis.[16] Activation of AMPK greatly reduces proteinuria and alleviates kidney injury by inhibiting the activation of mTOR.[17] mTOR is also a serine/threonine kinase, acting as a central regulator of cell growth and survival. mTORC1 and mTORC2 are two different complexes of mTOR. Among them, mTORC1 is sensitive to rapamycin (RA) inhibition and associates with a variety of signals, such as growth factors, amino acids, cellular energy content, and cellular stress, to regulate cellular activities, including protein synthesis, energy metabolism, and stress response.[18] The increased expression of p-mTOR, suppressed expression of p-AMPK, and reduced autophagy activity was observed in the renal tissue from obese mice. However, normalizing hyperinsulinemia and reducing body weight can activate AMPK and deactivate mTOR, subsequently improving autophagy to protect the kidney.[19] The present study indicates that sirtuin 3 can activate autophagy via the AMPK/mTOR pathway and protect renal function in cecal ligation and puncture-induced AKI.[20] These investigations have proved that the AMPK/mTOR signaling pathway could be reactivated in renal IRI rats.
Accordingly, in this study, we probed the effect of autophagy-related AMPK/mTOR signaling pathway in AKI-induced ALI using a rat model of renal IRI. We hypothesized that the activation of AMPK/mTOR signaling pathway could attenuate acute pulmonary injury caused by AKI through inflammatory factors and oxidative stress. The study findings may be useful for the clinical application for treating AKI-induced ALI.
| Materials and Methods | | |
Experimental drug intervention
In this study, RA was used to promote autophagy and 3-methyladenine (3-MA) was used to inhibit autophagy.[21],[22]
Animals and induction of renal ischemia/reperfusion injury
Experiments were performed in male adult healthy Sprague–Dawley (SD) rats (6-week-old, 200 ± 20 g), which were purchased from the Guangdong Provincial Medical Laboratory Animal Center. They were housed in the Animal Experiment Laboratory Department of Guangzhou University of Chinese Medicine, with 25°C ± 2°C room temperature, 55% ± 5% humidity and 12–12 h light-dark cycle. All rats were unrestricted access to standard diet and water.
The 48 SD rats were randomly divided into four groups: (i) normal saline (NS)-treated sham group (sham, n = 12), (ii) NS-treated IRI group (IRI, n = 12), (iii) RA-treated IRI group (IRI + RA, n = 12), and (iv) 3-MA-treated IRI group (IRI + 3-MA, n = 12). Renal IRI models were induced as described previously.[23] Rats were anesthetized with intraperitoneal 2% pentobarbital sodium (0.3 ml/100g) and placed in a heating pad under a warming light to maintain body temperature between 36°C and 38°C. Additional pentobarbital sodium was given as needed. Unilateral flank incisions were made and the right kidney was removed. After 10 min stabilization period, the left kidney was subjected to 60 min of ischemia using an atraumatic vascular clamp. The kidney showed restoration of renal blood flow within 5 min after removing the clamp. 12 hours before operation and after reperfusion, the rats in the RA treatment group received RA (2 mg/kg, 1 ml) and in the 3-MA treatment group received 3-MA (15 mg/kg, 1 ml) by oral gavage. IRI group received the vehicle NS with an equivalent volume. Rats in the sham group were administered the same volume of NS and received the same surgery without clamping the left renal vessels. The rats in each group (n = 3) were executed at 12 h, 24 h, 48 h, and 72 h after IRI, respectively [Figure 1]. Anesthetizing rats with intraperitoneal 2% pentobarbital sodium (0.3 ml/100g) again before execution, collect the blood from aorta abdominalis. Bronchoalveolar lavage fluid (BALF) was collected by cannulating the trachea with repeated 1 mol/L of phosphate buffer saline (PBS) up to a total volume of 1.0 mL twice. The BALF was centrifuged at 1000 g/min for 15 min and took 500 μL serum. The removed kidneys and lungs were stored in liquid nitrogen for examination. All animal experiments were approved in advance by the Guangzhou University of Chinese Medicine Ethics Committee (Approval number: 20201011002). All methods were carried out in accordance with relevant guidelines and regulations, and the study was carried out in compliance with the ARRIVE guidelines.[24]
Biochemical indexes analysis, ELISA assay and wet/dry ratio
Kidney function was evaluated by the measurement of serum creatinine (Scr) and blood urea nitrogen (BUN) with a Biochemical Autoanalyzer (Roche COBAS C501, Germany). The levels of TNF-α, IL-1β in serum, and BALF were determined by using the corresponding ELISA kits (J&L Biomedical, Shanghai, China). SOD, MDA, and myeloperoxidase (MPO) in serum and BALF were measured using the corresponding kits (J&C Biomedical, Nanjing, China). All experiments were performed according to the manufacturer's instructions. The right part of the lung tissue was weighed and dried in an oven at a constant temperature of 80°C for 48 h to obtain a dehydrate consistency, then weighed again to calculate wet/dry (W/D) ratio. W/D ratio = (wet weight - dry weight)/wet weight × 100%), representing the level of water content of the lung tissues. The catalog numbers: IL-1β (JL20884), TNF-α (JL13202), SOD (A001-3-2), MDA (A003-1-2), MPO (A044-1-2).
Histopathological analysis
Serial sections of kidney were stained with Periodic Acid-Schiff (PAS) for the histopathological analysis. In terms of semi-quantitative score from 0 to 5, histopathological change was scored based on the percentage of injured renal tubules:[25] 0, none; 1, <10%; 2, < 25%; 3, <45%; 4, <75%; and 5, higher than 75%. Histological changes included effacement and loss of proximal tubule brush border, patchy loss of tubule cells, focal areas of proximal tubular dilation and distal tubular casts, as well as areas of cellular regeneration.
The specimens of lung tissue were fixed, sectioned, and stained with Hematoxylin and Eosin (H and E). Quantify damage by lung injury score through microscopic examination. The results were classified into four grades,[26] where Grade 1 represented normal histopathology; Grade 2 indicated only few neutrophil leukocyte infiltration; Grade 3 represented moderate neutrophil leukocyte infiltration perivascular edema formation, and partial destruction of pulmonary architecture; and finally, Grade 4 included dense neutrophil leukocyte infiltration, abscess formation, and complete destruction of pulmonary architecture.
Transmission electron microscopy
The number of autophagosome and ultrastructural changes were detected by the TEM. Tissues were cut into about 1 mm × 1 mm × 1 mm pieces and placed in 2.5% glutaraldehyde PBS buffer at the room temperature for 2 h and 4°C overnight. The samples were washed by 0.1 M PBS three times for 10 min each, post-fixed in 1% osmic acid for 1h and washed by water three times for 10 min each. After that, the samples were stained with uranyl acetated and ehydrated in a graded series of ethanol (50%, 70%, and 90%) and embedded in epoxy resin. The ultrathin sections were prepared and observed with TEM (TECNA-10, Philips, Netherlands).
Western blot analysis
The expression of AMPK, mTOR, unc-51 like autophagy activating kinase 1 (ULK1), Beclin-1, light chain 3 II (LC3-II)/light chain 3 I (LC3-I), and Sequestosome1 (p62) were measured using western blot according to the manufacturer's instructions. After treatment of kidney and lung tissue, the cells were washed three times with cold PBS and lysed. Total cytoplasmic and nuclear protein was collected using cell lysis buffer. The protein concentration was measured using a BCA assay. Equal amounts of protein (25 μg) were separated by 10% SDS-PAGE, followed by transfer to PVDF membranes. The membranes were blocked in 5% BSA at room temperature for 1 h and 1×TBST for 5 min, and then incubated with primary antibodies against AMPK (1:1,000), mTOR (1:1,000), ULK1 (1:1,000), Beclin-1 (1:500), LC3-II/LC3-I (1:500), p62 (1:500), and GAPDH (1:10,000) at 4°C overnight. Anti-GAPDH antibody was selected as an internal reference. Subsequently, the membranes were washed with TBST and incubated with HRP-labeled secondary antibodies (1:5000, Proteintech) at the room temperature for 2 h. Finally, the blots were visualized using ECL (Affinity Biosciences) and the results were analyzed using ImageJ software (National Institutes of Health, USA). The catalog numbers: anti-ULK1 (A00584-1), anti-Beclin-1 (PB9076), anti-LC3-II/LC3-I (BM4827), anti-p62 (PB0458), anti-AMPK (ab32047), and anti-mTOR (ab32028).
Immunohistochemical staining
After the paraffin-embedded sections were dewaxed, we transferred the slides to 100% alcohol, 95% alcohol, 75% alcohol, and ddH2O 2 min twice respectively. After antigen retrieval, put them in hydrogen peroxide (3%) for 10 min to eliminate endogenous peroxidase activity. After washing with PBS three times, the sections of different groups were incubated with anti-AMPK (1:100, abcam, Cambridge, UK), anti-mTOR (1:400, abcam, Cambridge, UK), anti-ULK1 (1:300, Boster, Wuhan, Hubei, China), anti-Beclin-1 (1:200, Boster, Wuhan, Hubei, China), anti-p62 (1:200, Boster, Wuhan, Hubei, China), anti-LC3-II/LC3-I (1:100, Boster, Wuhan, Hubei, China) at 37 ℃ for 1 h. After washing with PBS three times, the secondary antibody (Boster) was added and immunostaining was performed using a DAB horseradish peroxidase color development kit (Boster, Wuhan, Hubei, China), and then, sections were counterstained with hematoxylin and made transparent with xylene. Finally, sections were observed with the PD37 type microscope (Olympus, Japan). Under 20× magnification, pictures were taken in five random fields. The catalog numbers: anti-ULK1 (A00584-1), anti-Beclin-1 (PB9076), anti-LC3-II/LC3-I (BM4827), anti-p62 (PB0458), anti-AMPK (ab32047), and anti-mTOR (ab32028).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
Paraffin-embedded kidney and lung sections were stained using TUNEL assay. This assay was performed following the manufacturer's instructions for a commercially available kit (ROCHE, Shanghai, China, 11684817910) and evaluated by immunofluorescence staining. Five random fields of view were assessed per section. The ratio of TUNEL-positive cells to the total number of cells in ten fields per section (original magnification ×20) was used to represent cell apoptosis.
Statistical analysis
All results are presented mean ± SD and were analyzed using the SPSS 15.0 software. The paired t-test was used to determine intra-group mean values, one-way ANOVA was used for inter-group mean comparison. A value of P < 0.05 was considered statistically significant.
| Results | | |
In this study, renal IRI rat model was used to explore the effect of autophagy-related AMPK/mTOR signaling pathway in AKI-induced ALI. We use RA to promote autophagy and 3-MA to inhibit autophagy. All data were obtained from no less than 3 experiments.
Activation of autophagy in acute kidney injury and acute kidney injury-induced acute lung injury
The production of autophagosomes in tissues can reflect the activation of autophagy. TEM indicated that there was almost no autophagosome in the lung and kidney tissues from sham rats. However, autophagosomes were found in the rats of IRI + RA group. In comparison to the rats in the IRI + RA group, the rats from the IRI + 3-MA group and the IRI group exhibited reduced autophagosomes. We also observed that the renal pathological changes of IRI rats were more severe with deformed nucleus, fell-off epithelial cells, and intra-cellular lipid droplet vacuoles. The lung tissue of IRI rats had more severe pathological changes, with nuclear necrosis, deformation, large intercellular spaces, and lamellar vacuoles [Figure 2]. | Figure 2: The 3-MA group and the IRI group had fewer autophagosome than the RA group. Representative images of autophagosome through TEM in 24 h. (red arrow: autophagosome). 3-MA: 3-methyladenine, IRI: Ischemia/reperfusion injury, TEM: Transmission electron microscopy, RA: Rapamycin.
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We next examined the expression of autophagy-related proteins. Immunochemistry and western blotting analysis revealed significantly increased Beclin-1, ULK1, and LC3-II/LC3-I proteins of IRI group compared to those of sham group. The expression of these three proteins was significantly increased in IRI + RA rats and decreased in IRI + 3-MA rats compared with IRI rats. However, the expression of p62 was lower in IRI + RA group and higher in IRI + 3-MA group compared with IRI group [Figure 3] and [Figure 4]. All of the above results could be observed both in kidney and lung tissues. These results indicated that autophagy was activated after renal IRI insult both in the kidney and lung, and RA could promote autophagy while 3-MA could inhibit autophagy. | Figure 3: Autophagy was promoted by RA and inhibited by 3-MA in immunohistochemical staining. (a) Immunohistochemical images of autophagy proteins in kidney tissue at 24 h. (b) Immunohistochemical images of autophagy proteins in lung tissue at 24 h. p62, Beclin-1, ULK1 and LC3-II/LC3-I are localized in the cytoplasm of cells. 3-MA: 3-methyladenine, RA: Rapamycin.
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 | Figure 4: Autophagy was promoted by RA and inhibited by 3-MA in western blot. (a) Representative western blot demonstrating p62, Beclin-1, ULK1 and LC3-II/LC3-I expression in kidney with GAPDH as loading control. (b) p62, Beclin-1, ULK1 and LC3-II/LC3-I expression in kidney were calculated by relative densitometric values. (c) Representative western blot demonstrating p62, Beclin-1, ULK1 and LC3-II/LC3-I expression in lung with GAPDH as loading control. (d) p62, Beclin-1, ULK1 and LC3-II/LC3-I expression in lung were calculated by relative densitometric values. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. 3-MA: 3-methyladenine, IRI: Ischemia/reperfusion injury, RA: Rapamycin, SD: Standard deviation.
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Autophagy attenuates acute kidney injury and acute kidney injury-induced acute lung injury
To explore the effect of autophagy on renal and pulmonary function, we detected the relative biochemical indicators. In comparison to the rats in the sham group, the levels of BUN and Scr in the IRI group were significantly increased and reached a peak at 24 h after reperfusion. These indexes of kidney injury were decreased in IRI + RA group and enhanced in 3-MA group [Figure 5]. In addition, the semi-quantitative score of tubular damage showed the similar trend in these groups [Figure 6]a. PAS staining indicated that the levels of inflammatory cell infiltration, destruction of the renal tubular structure, and interstitial tissue edema in kidney tissues were enhanced after IRI [Figure 6]b. | Figure 5: Effect of RA and 3-MA treatment on renal function. (a) The level of BUN. (b) The level of serum creatinine. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. 3-MA: 3-methyladenine, IRI: Ischemia/reperfusion injury, RA: Rapamycin, BUN: Blood urea nitrogen, SD: Standard deviation.
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 | Figure 6: Effect of RA and 3-MA treatment on histological changes in kidney tissues. (a) Results of semi-quantitative score of tubular damage. The scores of sham group are 0. (b) Results of PAS staining for kidney sections at 24 h. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. 3-MA: 3-methyladenine, IRI: Ischemia/reperfusion injury, RA: Rapamycin, SD: Standard deviation, PAS: Periodic Acid-Schiff.
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The W/D ratio in 24 h and lung injury score of IRI group rats was also increased compared with sham group rats [Figure 7]. However, the IRI + 3-MA group displayed a significantly higher level of lung injury as compared with the IRI group, and the IRI + RA group led to the opposite results. IRI + RA group showed decreased W/D ratio and lung injury score compared with IRI group [Figure 8]a. Meanwhile, the increased interstitial edema, inflammatory cell infiltration, as well as reduced alveoli, and distorted alveolar structure were observed in the lung tissues of IRI rats by HE staining. Compared with IRI group, inflammatory cell infiltration and distorted alveolar structure were alleviated in IRI + RA group [Figure 8]b. These results showed that the activation of autophagy could attenuate AKI and AKI-induced ALI after renal IRI. | Figure 7: The W/D ratio of lung tissue in 24 h. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. W/D: Wet/dry, IRI: Ischemia/reperfusion injury, SD: Standard deviation.
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 | Figure 8: Effect of RA and 3-MA treatment on histological changes in lung tissues. (a) Results of semi-quantitative score of pulmonary damage. The scores of sham group are 0. (b) Results of haemotoxylin and eosin staining for lung sections at 24 h. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. 3-MA: 3-methyladenine, IRI: Ischemia/reperfusion injury, RA: Rapamycin, SD: Standard deviation.
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Activation of AMPK/mTOR signaling pathway
Then we tested whether AMPK/mTOR signaling pathway was involved in autophagy. Immunohistochemistry and western blot indicated that there were slight AMPK expressions in the sham group rats. However, IRI induced increased AMPK and decreased mTOR protein expression in the renal tubules and alveoli compared to sham group. While, in comparison to the rats from the IRI group, the rats treated with RA showed dramatically decreased mTOR expression and increased AMPK expression in both the kidney and lung tissues. The reverse effects were observed in IRI + 3-MA group. The mTOR expression was higher and AMPK expression was lower in IRI + 3-MA group than IRI group [Figure 9]. These results indicated that AMPK/mTOR signaling pathway was activated after IRI and stronger via autophagy. | Figure 9: AMPK/mTOR pathway was activated after IRI. (a) Representative images of AMPK and mTOR immunohistochemical staining. (b) Representative western blot demonstrating AMPK and mTOR expression with GAPDH as loading control. (c) AMPK and mTOR expression in kidney and lung were calculated by relative densitometric values. AMPK and mTOR are localized in the cytoplasm of cells. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. IRI: Ischemia/reperfusion injury, SD: Standard deviation, AMPK/mTOR: Adenosine monophosphate-activated protein kinase/mammalian target of rapamycin.
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Autophagy attenuates inflammation and oxidative stress
To clarify the specific mechanism of the protective effect of autophagy, we examined the levels of inflammation (IL-1β, TNF-α) and oxidative stress (MDA, MPO, SOD) in serum and BALF. After the renal injury, the levels of IL-1β, TNF-α, MDA, and MPO both from IRI rats were higher than those from sham rats. The autophagy inducer RA significantly decreased the level of these inflammatory cytokines and oxidative stress factors, but the autophagy inhibitor 3-MA enhanced those effects caused by IRI. Meanwhile, IRI rats showed a significantly decreased SOD activity compared to sham rats. We also observed significantly increased SOD in IRI + RA group rats and reduced SOD in IRI + 3-MA group rats [Figure 10]. All of the above results could be observed both in serum and BALF. These results revealed that autophagy protected kidney and lung against damage after AKI by attenuating inflammation and oxidative stress. | Figure 10: RA treatment reduced inflammation and oxidative stress while 3-MA aggravated them. (a) The levels of IL-1β and TNF-α in serum. (b) The levels of IL-1β and TNF-α in BALF. (c) The levels of MDA, MPO and SOD in serum. (d) The levels of MDA, MPO and SOD in BALF. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. 3-MA: 3-methyladenine, IRI: Ischemia/reperfusion injury, RA: Rapamycin, SD: Standard deviation, BALF: Bronchoalveolar lavage fluid.
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Autophagy alleviates apoptosis on renal and lung tissue
Autophagy is usually closely related to apoptosis, so we examined the impact of autophagy on apoptosis. Our results showed, that compared to the rats from the sham group, the rats from the IRI treated group exhibited more TUNEL-positive cells in renal and pulmonary, which peaked 24 h after renal IRI. The proportion of apoptotic cells in the IRI + RA group was lower than that in the IRI group, while the ratio of TUNEL-positive cells was significantly higher in the 3-MA group [Figure 11]. It indicated that promoting autophagy could inhibit apoptosis. | Figure 11: RA treatment inhibited apoptosis while 3-MA treatment promoted apoptosis. (a) The ratio of TUNEL-positive cells (number of apoptotic cells/(number of apoptotic cells + number of non-apoptotic cells) × 100%) of kidney tissue. (b) The ratio of TUNEL-positive cells of lung tissue. (c) Representative TUNEL images from kidney at 24 h. (d) Representative TUNEL images from lung at 24 h. Values are presented as mean ± SD. *P < 0.05, **P < 0.01 versus sham; †P < 0.05, ††P < 0.01 versus IRI at the same time point. 3-MA: 3-methyladenine, IRI: Ischemia/reperfusion injury, RA: Rapamycin, SD: Standard deviation.
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| Discussion | | |
In our study, we have proved that both the inflammatory reaction and oxidative stress are related with the AMPK/mTOR pathway-mediated autophagy activation after renal IRI. Interestingly, improving autophagy by RA could effectively alleviate AKI-induced ALI. These results confirmed our hypothesis that activation of the AMPK/mTOR pathway after renal IRI could significantly attenuate kidney and lung injury by inducing autophagy, and inhibit inflammatory reaction, oxidative stress and apoptosis.
AKI is the leading cause of nephrology consultation and is associated with ischemia, hypoxia, or nephrotoxicity.[27],[28] Recent advances in present studies have confirmed that the physiologic and molecular mechanisms of distant organ interactions in AKI.[29] Moreover, pulmonary dysfunction is a common complication in patients with AKI that contributes to increasing the mortality rate.[9] Studies confirm that RA can attenuate mitochondrial injury and renal tubular cell apoptosis in AKI.[30] It also plays a protective role by inhibiting the level of IL-1β and IL-18 during ALI.[31] In this study, we found that RA could significantly improve the pulmonary and renal functions in AKI-induced ALI.
Autophagy is a conserved multistep pathway that maintains intracellular homeostasis by degrading and recycling damaged organelles and macromolecules. RA is a potent and selective inhibitor of the mTOR protein kinase,[32] which promotes anabolic metabolism and inhibits autophagy induction.[33] mTOR negatively regulates autophagy through phosphorylation of autophagy-related (ATG) protein 13, preventing activation of the autophagy-initiating ATG1-ATG13 kinase complex. Following mTOR inhibition, the activated ULK1 phosphorylates Beclin-1 on Ser 14 to enhance the activity of autophagy, indicating that RA positively regulates autophagy.[34] Previous studies have confirmed that LC3 and p62 serve as autophagosomal markers.[35],[36] LC3-I combines with phosphatidylethanolamine forming LC3-II, which is essential for autophagosome formation.[37] p62 is degraded by binding with LC3-II and contributes to the transport of cargo to the autophagosome.[38] When autophagic flux increases, LC3-II/LC3-I ratio enhances, while p62 quantities decrease.[39] Consistent with previous studies, our present study found that pulmonary and renal autophagy was further increased by RA, attenuating the AKI-induced ALI.
AMPK is one of the mTOR (mTORC1) upstream regulation signals, which can be activated by energy state of the cells.[40] mTORC1 and AMPK are involved in regulating the activity of the ULK1/2 complex.[41] mTORC1 negatively regulates autophagy, and AMPK phosphorylates tuberous sclerosis complex, which inhibits mTORC1.[42] AMPK can also phosphorylate the raptor component of mTORC1 when released from the ULK1 complex, resulting in inactivation of mTORC1.[41] Some studies have proved that AMPK/mTOR signaling pathway could be an essential role in protecting from IRI via activating autophagy.[43],[44] Research has shown that restoring AMPK-dependent suppression of mTOR can alleviate endotoxemia-induced ALI.[45] In our present study, it was similarly found that the AMPK/mTOR signaling pathway was activated after renal IRI. In addition, AKI-induced ALI after renal IRI could be attenuated by promoting autophagy via regulating the AMPK/mTOR pathway.
Researches have indicated that the mechanisms of distant organ communications in AKI were involved in leukocyte activation and infiltration, inflammatory cytokines, and endothelial injury.[29] As the release of inflammatory mediators could cause significant damage to the endothelial monolayer and endothelial vascular leakage.[46] Renal IRI can also lead to neutrophil and monocyte infiltration in the lung alveoli through pulmonary trans-endothelial migration, causing the injury of alveolar and bronchial epithelial cells. The increased pro-inflammatory action on the pulmonary tissues also caused oxidative damage, aggravating the ALI.[47],[48] Autophagy activation could attenuate the excessive inflammatory response and mitigate lung injury of the mice from ALI.[49] Harris et al. have demonstrated that activation of autophagy with RA leads to the degradation of inflammatory cytokines, such as IL-1β in serum, and reduces the secretion of late cytokines.[50] Our present study revealed that the level of inflammatory cytokines (TNF-α and IL-β) and oxidative stress indexes (MDA and MPO) decreased in RA group. However, the expression of SOD was significantly up-regulated. These results indicated that the activated autophagy could attenuate kidney and lung injury via the down-regulation of inflammation and oxidative stress.
There is a close relationship between apoptosis and autophagy, which share common pathways in their response mechanisms.[51] Autophagy and apoptosis may be triggered by common upstream signals, which sometimes leads to a combination of autophagy and apoptosis. In other cases, cells switch between the two responses in a mutually exclusive manner. We used TUNEL to represent the apoptosis in the renal and lung tissues. Our results revealed that autophagy can protect renal and pulmonary cells from severe apoptosis after IRI.
| Conclusion | | |
The present study has revealed that autophagy was associated with the AMPK/mTOR signaling pathway and could attenuate the injury of renal IRI rats. Inflammatory reaction and oxidative stress are the most critical pathophysiological processes involved in AKI-induced ALI and can be alleviated by autophagy. Our results demonstrated the therapeutic effect of the AMPK/mTOR signaling pathway in ALI following AKI by inhibiting the inflammation and oxidative stress through autophagy. Furthermore, the promotion of autophagy can significantly suppress apoptosis following IRI. Therefore, AMPK/mTOR signaling pathway may be a new target for the treatment of AKI-induced ALI.
Data availability
All data included in this study are available upon request by contact with the corresponding author.
Financial support and sponsorship
This work was supported by the grants from the National Natural Science Foundation of China (No. 81460682, 81760805), the Natural Science Foundation of Guangdong Province (No. 2020A1515010566), and the Shenzhen foundation of science and technology research and development (No. JCYJ20190809102413156).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
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