How Acute Kidney Injury Affects Lung Cells?
Mar 11, 2022
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Part Ⅱ：Oxidative stress following acute kidney injury causes disruption of lung cell cilia and their release into the bronchoalveolar lavage fluid and lung injury, which are exacerbated by Idh2 deletion
Yong Kwon Hana, Ji Su Kim, Gwan Beom Leea, Jae Hang Lim, Kwon Moo Park
Acute kidney injury (AKI) induces distant organ injury, which is a serious concern in patients with AKI (Acute kidney injury). Recent studies have demonstrated that distant organ injury is associated with oxidative stress of organs and damage of cilium, an axoneme-based cellular organelle. However, the role of oxidative stress and cilia damage in AKI (Acute kidney injury)-induced lung injury remains to be defined. Here, we investigated whether AKI (Acute kidney injury)-induced lung injury is associated with mitochondrial oxidative stress and cilia disruption in lung cells. AKI (Acute kidney injury) was induced in isocitrate dehydrogenase 2(Idh2, a mitochondrial antioxidant enzyme)-deleted(Idh2-/) and wild-type (Idh2+7)mice by kidney ischemia-reperfusion(IR). A group of mice was treated with Mito-TEMPO, a mitochondria-specific antioxidant. Kidney IR caused lung injuries, including alveolar septal thickening, alveolar damage, and neutrophil accumulation in the lung, and increased protein concentration and total cell number in bronchoalveolar lavage fluid (BALF). In addition, kidney IR caused fragmentation of lung epithelial cell cilia and the release of fragments into BALF, Kidney IR also increased the production of superoxide, lipid peroxidation, and mitochondrial and nuclei DNA oxidation in lungs and decreased IDH2 expression. Lung oxidative stress and injury relied on the degree of kidney injury, Idh2 deletion exacerbated kidney IR-induced lung injuries. Treatment with Mito-TEMPO attenuated kidney IR-induced lung injuries, with greater attenuation in Idh2-7-than Idh2+/+mice. Our data demonstrate that AKI (Acute kidney injury) induces the disruption of cilia and damages cells via oxidative stress in lung epithelial cells, which leads to the release of disrupted ciliary fragments into BALF.
kidney disease: AKI (Acute kidney injury)
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Idh2 deletion aggravates kidney IR-induced lung injury.
Lastly, we investigated the role of IDH2 on kidney IR-induced lung injury. To define the role of IDH2 on lung oxidative stress and lung injury under conditions of no or minimal differences of kidney injury, we induced 35 min of kidney ischemia followed by 4 h of reperfusion in female Idh2-/-and Idh2+/ mice based on two points; 1)female mice are less susceptible to kidney IR injury and 4 h reperfusion causes less severe kidney injury than 24 h reperfusion [3,39,40]. As expected. 35 min of ischemia and 4 h reperfusion increased BUN concentrations in both Idh2~/and Idh2+/ mice without significant difference in BUN between Idh2-/ and Idh2/+ mice (Fig.7A). However, lung injury including increased neutrophil infiltration and alveolar septal thickening-was greater in Idh2-7-mice than Idh2+7+mice(Fig. 7B). Consistent with histological damage, approximately 58.5 % protein concentration, and 37.8% total cell number in BALF of Idh2-/- mice were higher than those in BALF of Idh2+/+ mice(Fig. 7C and D). No significant differences in BUN and lung injury between Idh2-/mice and Idh2+/+ mice were observed after sham operation (Fig. 7A-D). These results indicate that the deficiency of the IDH2 gene, a mitochondrial antioxidant enzyme, worsens kidney IR-induced lung injury.
To further confirm the role of IDH2 and mitochondrial oxidative stress on kidney IR-induced lung injury, we evaluated whether Mito-TEMPO inhibits the increase of protein concentration and cell number in BALF after kidney IR. Mito-TEMPO treatment prevented the increase in protein concentration and total cell number after kidney IR in both Idh2-/mice and Idh2+/+ mice. This prevention was greater in Idh2-/-mice than in Idh2+/ mice(approximately 32.2%in Idh2+/ and 38.6%in Idh2-/- in protein concentration in BALF and 8.4 % in Idh2+/+ and 35.4% in Idh2-7-in total cell number in BALF)(Fig.7C and D).Mito-TEMPO slightly reduced BUN levels in both mice, but this was not statistically significant (Fig.7A). These results indicate that IDH2 plays an important role in kidney IR-induced lung injury.
Fig.7.Greater lung injury after kidney IR in Idh2-/-mice than in Idh2+/+ mice and greater prevention of kidney IR-induced lung injury by Mito-TEMPO in Idh2-/-mice than in Idh2+/mice. Female Idh2-deleted (dh2-7)and wild-type (Idh2+/)littermates were subjected to 35 min of bilateral renal ischemia. Some mice were administered Mito-TEMPO(Mito-T,0.7 mg/kg BW, IP.)17 and 1 h before ischemia, twice. Lung, BALF, and blood were collected 4 h after ischemia. (A) The BUIN in plasma was measured as described in the Materials and Methods (n =4). (B)Perfusion fixed lung tissues were cut into 3-um thick sections, which were then subjected to H&E staining (n= 3). (C. D) The protein concentration and total cell number in BALF were measured as described in the Materials and Methods(n =4). Results are expressed as the means ± SEM (n =3-4).
Kidney IR significantly increased 4-HNE expression in lungs in both Idh2-/-and Idh2+/mice 4 h after kidney ischemia, and this increase was greater in Idh2-/mice than in Idh2+/+ mice (p<0.001)(Fig.8A and B). Mito-TEMPO prevented the increase in 4-HNE expression in both mice(37.5 % in Idh2+/and 28.4 % in Idh2-/)(Fig.8C and D). There were no significant differences in 4-HNE expression between sham-operated Idh2+/+ and Idh2-/mice (Fig. 8A and B). Superoxide levels in the lungs were greater in Idh2-/ mice than in Idh2+/ mice (p<0.001)(Fig.8E).Mito-TEMPOreduced superoxide levels in both mice(p = 0.052 in Idh2+/+ and p= 0.001 in Idh2-/-).This reduction was greater in Idh2-/-mice than in Idh2+/+ mice (approximately 11.6 % in Idh2+/+and 27.2 %in Idh2-7-)(Fig.8E).The levels of 4-HNE in BALF increased in both Idh2-/-mice and Idh2+/+ mice, with a greater increase in Idh2-/mice than inIdh2+/+ mice(p<0.001)(Fig.8F and G). Mito-TEMPO prevented the increase in 4-HNE expression in both mice (14.9 % in Idh2+/+and 45.6 % in Idh2-/)(Fig.8F and G). Next, we determined the levels of Arl13B, ac-α-tubulin, and α-tubulin in BALF. Kidney IR induced increases in Arl13B, ac-α-tubulin, and α-tubulin expression in BALF; these increases were also greater in Idh2-7mice than in Idh2+/+ mice(Fig. 8H-K). Mito-TEMPO inhibited kidney IR-induced increases in the expression of Arll3B, ac-α-tubulin, and α-tubulin in the BALF, and these inhibitions were also greater in the Idh2-/-mice than in the Idh2+/t mice (approximately 16.7% in Idh2+/+ and70.4%inIdh2-/in Arll3B;33.5%in Idh2+/+ and 51.7 %inIdh2-/-in ac-α-tubulin; 39.9 % in Idh2+/+ and 52.5 % in Idh2~/-in α-tubulin)(Fig.8H-K). These results indicate that the deletion of Idh2 augmented kidney IR-induced lung injury by increasing mitochondrial oxidative stress.
Fig.8. Greater lung oxidative stress and cilia damage after kidney IR in Idh2-/- mice than in Idh2+/+ mice and greater prevention of stress and damage by Mito-TEMPO in Idh2-7mice than in Idh27mice. Female Idh2-deleted(Idh2-7-)and wild-type (Idh2+7+)littermates were subjected to either 35 min of bilateral renal ischemia (isch) or sham operation. Some mice were administered either Mito-TEMPO (Mito-T,0.7 mg/kg BW,i.p.)or vehicle 17 and 1h before ischemia, twice. Lung and BALF were collected 4 h after ischemia.(A-D,F,G)4-HNE expression in the lung tissues and BALF was evaluated by Western blot analysis (n =3-4).GAPDHwas used as the loading control. The densities of the bands were measured using the ImageJ software. (E)The superoxide level in the lung tissue was measured (n=4). (F-K)4-HNE, Arl13B, ac-α-tubulin (ac-α-tub)and α-tubulin (α-tub)expressions in BALF were analyzed by western blotting (n =4). The densities of bands were measured using the ImageJ software. Results are expressed as the means ± SEM (n = 4).
4. Discussion
In the present study, we report that kidney IR-induced lung injury is exacerbated by Idh2 deletion and that mitochondrial antioxidant treatment attenuates kidney IR-induced lung injury. In addition, kidney IR induces the disruption of cilia in lung cells via oxidative stress, and the resulting disrupted ciliary fragments and proteins are released into BALF. Importantly, this disruption of cilia is prevented by mitochondria-specific antioxidant treatment. In contrast, Idh2-deletion exacerbates kidney IR-induced lung cell cilia disruption. These data indicate that kidney IR impairs the redox balance in lung cells in an ischemic-time-dependent manner, resulting in oxidative stress of lung tissue and cilia disruption. Moreover, cilia disruption, at least in part, involves AKI (Acute kidney injury)-induced lung injury. To the best of our knowledge, this is the first report to demonstrate that AKI (Acute kidney injury) causes the disruption of cilia in lung cells by oxidative stress, and the resulting disrupted ciliary fragments and proteins are released into BALF. These results indicate that the prevention of cilia disruption could be a novel strategy for the treatment of AKI (Acute kidney injury)-related ALI. Additionally, these data indicate that ciliary proteins and fragments in BALF could be used as indicators of lung injury.
Kidney IR-induced distant organ injury is a complex process in which numerous factors are involved [4,8,41,42]. Several studies have demonstrated that ROS and oxidative stress contribute to distant organ injury following kidney injury [4,8,41]. In lung injury following kidney IR, it has been proposed that neutrophils, which are infiltrated into the lung, function as major ROS-producing cells in the lung following the stimulation of cytokines produced in the injured kidney. This production of ROS subsequently causes additional ROS increases in the lung via the activation of the ROS-producing system and the inhibition of the ROS scavenging system, or both [5,43]. Recently, Hepokoski, M. et al. reported that the extracellular accumulation of kidney mitochondrial DAMPs is caused by AKI (Acute kidney injury), affects lung metabolic pathways, and induces mitochondrial dysfunction [42]. In the present study, we found increased neutrophil infiltration into the lung after kidney IR, depending on kidney ischemic time, i.e., the severity of kidney IR injury. We also found that kidney IR increases superoxide formation, lipid peroxidation, and DNA oxidization in a broad range of lung tissues, including interstitial alveolar septa, alveolar, and airway epithelia. Moreover, we found a decrease in IDH2 expression in the lung tissue after kidney IR. These data indicate that lung cells are exposed to oxidative stress following kidney IR and that this increased oxidative stress, including that by the mitochondria of lung cells, may cause cell and tissue damage and dysfunction. Further, mitochondria-specific antioxidant treatment reduces kidney IR-induced lung injury and inhibits superoxide formation and oxidation of lipids and DNA in the lungs.
kidney disease: oxidative stress on kidney
IDH2 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate in the mitochondria, accompanied by the reduction of NADP to NADPH [31,32,35,44]. NADPH is a source of reducing equivalents for both the thioredoxin and glutathione systems of peroxide detoxification [31,32,35,44,45]. Recently, Park et al. reported that Idh2 deficiency increases susceptibility to acrolein-induced lung toxicity in Lewis lung carcinoma cells and mice through the disruption of mitochondrial redox balance, leading to mitochondrial oxidative stress and apoptosis [31]. In this study, we found that kidney IR reduced IDH2 expression in the lung and that Idh2 deletion in mice exacerbated kidney IR-induced lung injury and oxidative stress. In addition, mitochondrial antioxidant supply reduced lung injury and lung oxidative stress with greater reductions in Idh2-deleted mice than in wild-type littermates. These data indicate that kidney IR-induced lung injury is associated with reduced IDH2 function and a subsequent increase in mitochondrial oxidative stress. Moreover, there may be sex differences in lung injury and lung oxidative stress (note Figs. 1 and 7). However, although we found greater lung injury and oxidative stress and greater protective effect of Mito-TEMPO in Idh2-deficient female mice than in wild-type female mice, at similar post-kidney BUN levels(in turn, under a similar degree of kidney injury), an association of the severity of kidney injury may not be completely ignored because of the dependency of lung injury on kidney injury. This may be answered by the use of lung-specific antioxidant delivery systems or lung-cell-specific conditional knock animals.
The ciliary length in cells is dynamically altered under both physiological and pathophysiological conditions. Recent studies have demonstrated that abnormal alteration of cilia length is associated with the development and progression of various diseases, and cilia are associated with mitochondrial function [17-20,43,46,47]. Under physiological conditions, change in cilia length occurs by either reabsorption or elongation during the normal cell cycle [48-51]. Resorption is a normal process in which cells retract the cilium into the cell during progression from the GO/Gl phase to the S to G2 phases of the cell cycle, and complete cilia reabsorption occurs before mitosis [48-51]. Recent studies have demonstrated that shortening of the cilia length is caused by cell injury [17-20,46,52]. In previous studies, we found that AKI (Acute kidney injury) induces the disruption of cilia in kidney tubular epithelial cells as a result of oxidative stress [18-20]. Furthermore, we found that hepatic IR injury induces the deciliation of primary cilia of tubular cells of distant kidneys owing to oxidative stress in kidney tubule cells and that this deciliation is prevented by antioxidant treatment [20]. Moreover, primary cilia deficiency activates the epithelial to mesenchymal transition, which is critical for the progression of fibrosis [53], indicating that cilia are associated with the progression of post-injury responses. In the present study, we found that kidney IR causes the disruption of lung cell cilia, and these disrupted ciliary fragments are released into BALF. In addition, Idh2 deletion augmented AKI (Acute kidney injury)-induced cilia disruption, whereas Mito-TEMPO treatment prevented kidney IR-induced lung cilia damage, with greater preventive effects in Idh2-/mice than in Idh2+/+mice. Additionally, we found that BALF-containing molecules are highly oxidized and Mito-TEMPO injection reduces the oxidization of BALF contents. Therefore, we speculate that ROS and oxidative stress cause cilia disruption. Supporting this, in previous studies, we found that high concentrations of hydrogen peroxide disrupt cilia in kidney tubule cells [18].
The lung is typically a motile cilia-rich tissue and the dysfunction of respiratory cilia is linked to impaired mucociliary clearance, chest infections, and the progressive destruction of lung architecture [54]. In the bronchiole apical region, ciliated cells have abundant mitochondria to produce ATP for the ciliary motion [13]. Studies have reported that mitochondrial damage in ciliated cells causes dysfunction of both cilia and lung cells, which may lead to an abundance of ROS [55,56]. Several studies have reported that impaired mitochondrial dynamics and mitochondrial damage are associated with lung diseases [56,57]. In this present study, we also found that kidney IR causes an abnormal increase in superoxide generation and mitochondrial fission in the lung. In addition, we found increases in ciliary fragments and proteins in BALF of kidney IR mice. Therefore, we speculated that the deciliation is linked, at least in part, to AKI (Acute kidney injury)-induced ALI. However, we could not distinguish which types of cilia are disrupted and which cells are major ciliary fragment-releasing cells among lung cells owing to the lack of specific markers of primary and motile cilia and histological study limitations. This limitation may be overcome in additional experiments using lung-specific drug delivery systems or lung-specific ciliary genesis-associated gene targeting. However, our data clearly demonstrate that AKI (Acute kidney injury)-related ALI is associated with cilia disruption and oxidative stress of lung cells.
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Acknowledgments
This study was supported by a grant from the National Research Foundation of Korea (NRF)(NRF-202OR1A2C2006903, MIST)funded by the Ministry of Science and ICT(MIST) and a grant from the Korea Health Technology R&D Project (HI15C0001)through the Korea Health Industry Development Institute (KHIDD funded by the Ministry of Health & Welfare, Korea government.
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