Do You Know Oxidative Stress Can Cause Kidney Injury?

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Part Ⅰ：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

1. Introduction

Acute kidney injury (AKI) occurs in various clinical settings, including shock, sepsis, organ transplantation, and vascular surgery [1-3]. AKI (Acute kidney injury) causes damage to distant organs, including the lungs, liver, heart, and brain [4-9]. This distant organ injury following AKI (Acute kidney injury) is recognized as a major risk factor for poor outcomes [5,9]; therefore, appropriate treatment of distant organ injury is important to improve the outcome of patients with AKI (Acute kidney injury). However, the precise mechanisms involved in AKI (Acute kidney injury)-related distant organ injury remain to be defined.

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Cilia are centriole-derived projections from the cell surface that contain a microtubule-based cytoskeleton (axoneme), surrounded by a ciliary membrane. Cilia are characterized by their structure and motility and are defined as either immotile cilia (primary cilia)or motile cilia. Primary cilia are solitary, non-motile, microtubule-based 9 +0 axonemal antenna-like organelles that protrude from the cell membrane and transduce extracellular signals into the cell via the coordination of several signaling pathways [10-12]. Motile cilia, with a multiple, 9 +2 axonemal structure, are mainly found in the respiratory tract and oviduct and function to transport materials [10,13]. Increasing evidence has demonstrated that defects in the formation and function of cilia are associated with diverse human diseases, including polycystic kidney diseases and primary ciliary dyskinesia [14-20]. Furthermore, recent studies have demonstrated that the disruption of cilia occurs under the influence of pathological conditions and that this disruption is involved in mediating cell injury and dysfunction [15-20].

Accumulating evidence has demonstrated that kidney ischemia-reperfusion (IR), a cause of AKI (Acute kidney injury), induces distant organ injury [6-8,21]. Distant organ injury is associated with inflammatory responses, changes in signaling pathways associated with cell death and survival, and oxidative stress [8,22-24]. Oxidative stress is directly linked to the dysfunction of cellular components, which can result in organ disorders, and is recognized as one of the major causes of kidney IR-induced distant organ injury [21,25,26]. We have previously shown that kidney IR-and cisplatin-induced AKI (Acute kidney injury) alters the lengths of primary cilia in kidney epithelial cells via assembly, disassembly, and disruption (deciliation, shedding, or fragmentation) of cilia [17-19]. These processes are associated with reactive oxygen species (ROS)and oxidative stress. depending on the concentration of hydrogen peroxide and degree of oxidative stress; low H2O2 concentration induces the elongation of primary cilia length, whereas high H, O, concentration induces disruption [17-20]. In addition, we found that disrupted primary cilia of injured kidney tubular epithelial cells are excreted into the urine and that this disruption and excretion can be prevented by antioxidant treatment [17-20]. Rodriguez-Ribera et al. also reported that reactive carbonyl compounds such as malondialdehyde, a product of oxidative stress, induce the loss of primary cilia in human kidney proximal tubule cells [27]. Therefore, we hypothesized that AKI (Acute kidney injury)-induced acute lung injury (ALD) and cell injury are associated with cilia and lung cell oxidative stress and that prevention of oxidative stress reduces AKI (Acute kidney injury)-induced lung injury. In the present study, we investigated whether kidney IR-induced AKI (Acute kidney injury) causes the disruption of cilia and cells of the lungs, and if so, whether these disruptions are associated with oxidative B. To this end, we employed pharmacological and genetic approaches, including the use of isocitrate dehydrogenase 2(Idh2)-deleted mice. IDH2 is an enzyme localized in the mitochondria [28]. In the mitochondria, IDH2 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, accompanied by the reduction of NADP to NADPH, which is a critical factor in the thioredoxin and glutathione antioxidant system [29-32]. In addition, we investigated whether the presence of ciliary proteins and fragments in bronchoalveolar lavage fluid (BALF)is indicative of lung injury. We report here, for the first time, that AKI (Acute kidney injury) causes disruption of lung cell cilia and their release into the BALF as well as lung injury, which is exacerbated by IDH2 deletion.

oxidative stress causes kidney injury

2. Materials & methods

Animal preparation.

All experiments were conducted using 8-10-week-old male C57BL/6 mice (Koatech, Pyeongtaek, Gyeonggi-do, Korea), female Idh2 gene-deleted (Idh2~/) mice, and wild-type (dh2+/)mice [28]. The mice were allowed free access to water and standard mouse chow. The animal study was approved by the Institutional Animal Care and Use Committee of Kyungpook National University, Republic of Korea. To induce bilateral renal ischemia, the kidneys were exposed through flank incisions under anesthetization with pentobarbital sodium (60 mg/kg BW), and then, the pedicles of the kidneys were completely clamped for 35 min using microaneurysm clamps. The same procedure, exclusive of kidney pedicle clamping, was performed in the sham operation Body temperature was maintained at 36.5°C-37 ℃C throughout surgical procedures using a temperature-controlled heating device (FHC, Bowdoin, ME, USA).Some mice were administered 2-(2,2,6,6-Tetramethylpiperidin-1-ox-yl-4-arylamino)-2-oxoethyl triphenylphosphonium chloride (Mito--TEMPO,0.7 mg/kg BW;Sigma, St.Louis, MO,USA)at either 17 and 1 h before operation (pretreatment) or 6 h after operation (post-treatment).

At the end of the experiments, the lung and kidney tissues were snap-frozen in liquid nitrogen or perfusion fixed with PLP(4 % paraformaldehyde,75 mM L-lysine,10 mM sodium periodate: Sigma) solution for biochemical and histological studies, respectively. Frozen tissues were stored at -70°C until required.

Collection of BALF.

BALF was collected after euthanizing the mice by overdose injection of pentobarbital sodium. Lung leakages were evaluated by determining the protein concentration and cell number in the BALF. To collect BALF, the bronchus of the left lung was completely clamped using microaneurysm clamps. A needle was inserted into the trachea and 0.8 ml cold phosphate-buffered saline (PBS)was slowly injected into the right lung lobes. BALF was collected by retracting the piston of the syringe once. Total cell number and protein concentration in BALF were analyzed immediately using a hematocytometer and BCA assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA), respectively. BALF was kept at-70°C until required for western blotting analysis.

Kidney function. Blood was collected from mice using a heparinized syringe. The concentration of blood urea nitrogen (BUN) in plasma was determined using a Vitros 250 Chemistry Analyzer(Johnson& Johnson, New Brunswick, NJ, USA).

Periodic acid-Schiff (PAS) and hematoxylin and eosin (H&E)staining.

PLP-fixed kidney and lung tissues were paraffin-embedded, cut into 3-μm thick sections using a microtome （Leica， Bensheim， Ger-many), and mounted on glass slides. Kidney and lung sections were stained with PAS and H&E. The kidney damage score was evaluated blindly by investigators as described previously [3,19].

Western blot analysis.

Western blotting was performed as previously described [3]. The antibodies used for western blotting were as follows: anti-4-hydroxy nominal (4-HNE; Abcam, Cambridge, MA, USA), anti-α-tubulin (Santa Cruz, CA, USA), anti-acetylated α-tubulin (ac-α-tubulin, Sigma), anti-manganese-dependent superoxide dismutase (MnSOD; Calbiochem, San Diego, CA, USA), anti-catalase (Fitzgerald, Concord, MA, USA), anti-ADP-ribosylation factor-like protein 13B (Arl13B, Proteintech, Chicago, IL, USA),anti-isocitrate dehydrogenase 2 (IDH2; Santa Cruz), anti-optic atrophy 1(Opal; BD Bioscience, San Diego, CA), anti-fission 1(Fis1; Sigma), anti-dynamin related protein 1 (Drpl; Cell Signaling Technology, Danvers, MA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase(GAPDH; NOVUS, Littleton, CO, USA).

Immunofluorescent staining.

After deparaffinization, sections were incubated in PBS containing 0.2 % Triton X-100(Sigma)for 1 min and then washed in PBS for 10 min. To expose the antigen epitope, sections were boiled in 10 mm sodium citrate buffer (pH 6.0)for 10 min, cooled for 20 min, and then washed thrice with PBS for 5 min on each wash. The sections were blocked with 3% bovine serum albumin in PBS (blocking buffer)for 30 min and then incubated with anti-Arll3b anti-body at 4°C overnight. After washing, the sections were incubated with FITC-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA)for 60 min and then washed 3 times with PBS for 5 min each. Cell nuclei were stained using 4'-6-diamidino-2-phenylindole (DAPI; Sigma).

Measurement of superoxide in lung tissue.

Superoxide levels were measured using dihydroethidium (DHE; Sigma) as described previously 【33，34】. Briefly，10 μM DHE in 1 ml of pre-warmed（37°C）PBS was added to a 96-well plate containing 20 μl of lung tissue lysate. The plate was read every 10 min for a total of 30 min at excitation/emission filters of 530 nm/620 nm.

Measurement of IL-6.

IL-6 levels in plasma and BALF were determined via ELISA assay using ELISA assay kit according to the manufacturer's instructions (BD Bioscience).

Statistics.

All data were analyzed using GraphPad Prism 7 software (San Diego, CA, USA). The results are expressed as the mean±standard error of the mean (SEM). Statistical analysis was performed using Student's t-test and one-way analysis of variance with Tukey's post hoc procedure. Differences were considered statistically significant when p-values were <0.05.

3. Results

1. Kidney IR causes lung injury.

As expected, significant morphological damage to the kidneys (Fig.1A and B)and increases in plasma BUN(Fig.1C) were observed 4 and 24 h after 35 min of bilateral kidney ischemia. In the lung, an increase in neutrophils and alveolar wall thickening were observed 4 and 24 h after kidney ischemia (Fig. 1D). In addition, the increase in the expression of lymphocyte antigen 6 complex locus G6D (Ly6G, a marker for monocytes, granulocytes, and neutrophils)in lung tissue was observed in a reperfusion time-dependent manner (Fig.1E and F). Interleukin-6 (IL-6)concentration in plasma increased after kidney IR, peaking at 4 h after ischemia (Fig. 1G). The I-6 and protein concentrations as well as total cell number in BALF gradually increased at 4 and 24 h after kidney ischemia (Fig.1H-J). The survival rate at 24 h after 35 min of kidney IR was approximately 92 %(data not shown).

When kidney ischemic time was extended to 45 min, post-ischemic increases in BUN concentration in plasma and total protein concentration and cell number in BALF were greater than those after 35 min of ischemia (Fig.1K-M). These data indicate that lung injury induced by kidney IR injury relies on the degree of kidney injury.

Fig. 1. Histological and functional damage of the lung after kidney IR. C57BL/6 male mice were subjected to 35 (A–M) and 45 (K–M) min of bilateral renal ischemia or sham operation. Kidney, lung, blood, and BALF were collected 4 and 24 h after operation. BALF was collected as described in the Materials and Methods. (A–J) Mice were sacrificed either 4 or 24 h after 35 min of ischemia. (A, B) Kidney sections (3 μm) were stained with PAS, and kidney tubular damage was scored. (C, K) BUN concentrations were measured in plasma. (D) Lung sections (3 μm) were stained with H&E. (E, F) Ly6G expression in lung tissue was analyzed by western blotting. GAPDH was used as a loading control. The densities of the bands were measured using the ImageJ software. (G, H) The IL-6 concentration was measured in plasma and BALF. (I, J, L, M) The protein concentration and total cell number were measured in BALF. (K–M) Mice were sacrificed 4 h after either 35 or 45 min of ischemia. Results are expressed as the means ± SEM (n = 3–6). *p < 0.05 vs. sham. †p < 0.05 vs. 4 h after ischemia or ischemia for 35 min.

2. Kidney IR causes disruption of cilia of lung epithelial cells and these fragments are released into BALF.

Lung tissue sections and BALF-loaded slides were immunostained with anti-ADP-ribosylation factor-like protein 13B(Arl13B, a marker of cilia) antibody. Arll3B positive signals were observed on the luminal part of the alveoli and the terminal bronchioles of the lung (Fig.2A). Loss of Arl13B-positive signal was observed in the lung of kidney IR-subjected mice (Fig.2A). This loss of Arll13B-positive signal gradually increased after kidney ischemia over time (Fig. 2A). In BALF, various lengths of Arl13B-positive particles were observed after kidney ischemia, and the number of Arl13B-positive particles in BALF gradually increased over time after ischemia(Fig.2B and C). The expression of Arl13B, acetylated α-tubulin (ac-α-tubulin, granulocytes, and neutrophils)in lung tissue was observed in a reperfusion time-dependent manner (Fig.1E and F). Interleukin-6 (IL-6)concentration in plasma increased after kidney IR, peaking at 4 h after ischemia (Fig. 1G). The I-6 and protein concentrations as well as total cell number in BALF gradually increased at 4 and 24 h after kidney ischemia (Fig.1H-J). The survival rate at 24 h after 35 min of kidney IR was approximately 92 %(data not shown).

When kidney ischemic time was extended to 45 min, post-ischemic increases in BUN concentration in plasma and total protein concentration and cell number in BALF were greater than those after 35 min of ischemia (Fig.1K-M). These data indicate that lung injury induced by kidney IR injury relies on the degree of kidney injury.

Fig. 2. Disruption in lung cell cilia and their fragments and proteins released into BALF after kidney IR. C57BL/6 male mice were subjected to either 35 min of bilateral renal ischemia or sham operation Lung tissue and BALF were collected 4 and 24 h after the operation as described in the Materials and Methods. (A) Lung sections (5 μm) were subjected to immunofluorescent staining using an anti-Arl13B antibody; green indicates Arl13B-positive. DAPI stain (4′,6-diamidino-2-phenylindole; blue) was used to visualize the nuclei of cells. Arrowheads indicate cilia. (B) BALF (10 μl) was put on a glass slide which was immunofluorescent-stained using an anti-Arl13B antibody; green indicates Arl13B-positive (n = 3). (C) The number of Arl13B-positive particles was counted under a fluorescence microscope (n = 3). (D–G) Arl13B, acetylated-α-tubulin (ac-α-tub), and α-tubulin (α-tub) expressions in BALF were analyzed by western blotting. The densities of the bands were measured using the ImageJ software. Results are expressed as the means ± SEM (n = 3). *p < 0.05 vs. sham. †p < 0.05 vs. 4 h after ischemia. A: Alveoli, TB: Tubule.

3. Kidney IR increases the levels of superoxide and oxidative stress in both lung tissue and BALF.

Kidney IR increased the super-oxide level in lung tissue (Fig.3A), whereas it reduced the expression of IDH2(Fig.3B and C), but not MnSOD and catalase (Fig, 3B.D.E). These findings indicate that kidney IR-induced lung injury is associated with increases in ROS levels and oxidative stress in the lungs. In support of this, the level of 4-hydroxy nominal (4-HNE), a product of lipid peroxidation, in the lung tissue significantly increased 4 and 24 h after kidney ischemia (Fig。3F-D). 4-HNE was observed in almost all areas in lung tissue, including the alveoli and bronchioles(Fig. 3H and D. Furthermore, the expression of 8-hydroxy-2'-deoxyguanosine (8-OhdG), a well-known marker of DNA oxidation as an oxidized derivative of deoxy-guanosine, increased in both the cytosol and nuclei in pulmonary epithelial cells after kidney ischemia (Fig.3J and K. In BALF, 4-HNE expression also increased after kidney ischemia in a reperfusion-time-dependent manner (Fig. 3L, M). Furthermore, when kidney ischemic time was extended to 45 min,post-ischemic increase in 4-HNE expression in the lungs was greater than that after 35 min of kidney ischemia (Fig.3N, O). These results indicate that both mitochondrial DNA and nuclear DNA are oxidatively injured by increased ROS formation and impairments in removal systems, suggesting that the damage to pulmonary epithelial cell cilia is associated with oxidative stress.

Fig. 3. Oxidative stress in lung tissue after kidney IR. C57BL/6 male mice were subjected to 35 (A–O) and 45 (N, O) min of bilateral renal ischemia or sham operation. Lung and kidney were harvested 4 and 24 h after either 35 min or 45 min of ischemia. (A–M) Mice were sacrificed either 4 or 24 h after 35 min of ischemia. (A) The superoxide level in the lung tissue was measured as described in the Materials and Methods (n = 3–5). (B–E) IDH2, catalase, and MnSOD expression in the lung tissue were analyzed by western blotting (n = 4). GAPDH was used as the loading control. (C–E) The densities of the bands were measured using ImageJ software. (F, G) 4-HNE expression in the lung was analyzed by western blotting, and the band density was measured using ImageJ (n = 4). (H–K) Lung sections (3 μm) were immunohistochemically stained using anti-4-HNE and 8-OHdG antibodies and counterstained using hematoxylin; brown indicates 4-HNE- and 8-OHdG-positive (n = 3). (I, K) The intensities of 4-HNE- and 8-OHdG-positive signals were measured using the i-Solution program (n = 3). (L, M) 4-HNE expression in BALF was analyzed by western blotting, and the band density was measured using ImageJ (n = 3). (N, O) Mice were sacrificed 4 h after either 35 min or 45 min of ischemia. 4-HNE expression in the lung was analyzed by western blotting, and the band density was measured using ImageJ (n = 3). Results are expressed as the means ± SEM (n =3–5). *p < 0.05 vs. sham. †p < 0.05 vs. 4 h after ischemia or 35 min of ischemia. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

In cells, mitochondria. a major ROS-producing intracellular organelle normally undergoes fusion and fission to adapt to physiological and pathological conditions. Impairment in these fusion and fission processes can induce cell dysfunction and damage[36]. Previous studies have demonstrated that oxidative stress impairs the normal dynamics of mitochondrial fusion and fission, resulting in mitochondrial dysfunction and cell injury [32,36-38]. We sought to evaluate the mitochondrial dynamics by determining mitochondrial fission- and fusion-regulating protein expressions. The expressions of Fis1 and Drp1, which regulate mitochondrial fission, were significantly increased in the lungs of mice with kidney ischemia (Fig.4A-C). However, the expression of OPA1, which regulates mitochondrial fusion, was not significantly altered by kidney IR (Fig.4A, D). These data indicate that the balance of mitochondrial fusion and fission in lung cells is disrupted as a result of AKI (Acute kidney injury).

Fig. 4. Change in mitochondrial dynamics in the lung after kidney IR. C57BL/6 male mice were subjected to either 35 min of bilateral renal ischemia or sham operation. The lungs were harvested 4 and 24 h after ischemia. (A–D) Drp1, Fis1, and OPA1 expressions in lung tissues were analyzed by western blotting. GAPDH was used as the loading control. The densities of the bands were measured using the ImageJ software. Results are expressed as the means ± SEM (n = 4). *p < 0.05 vs. sham. †p < 0.05 vs. 4 h after ischemia.

4. Mitochondria-specific antioxidant treatment reduces kidney IR-induced lung injury and lung cilia disruption.

To confirm the role of mitochondrial oxidative stress on kidney IR-induced lung injury and lung epithelial cell cilia disruption, we tested whether pretreatment (Fig.5A-D or post-treatment (Fig. 5J-L) with Mito-TEMPO, a mitochondria-specific antioxidant, inhibits kidney IR-induced oxidative stress, I-6 production, and deciliation in the lungs of C57BL/6 male mice. Pretreatment (Fig, 5A-D), but not post-treatment (Fig. 5J-L), with Mito-TEMPO, significantly inhibited increases in BUN and IL-6 concentrations in plasma (Fig.5A and B) as well as IL-6 concentration, total protein concentration, and total cell number in BALF (Fig, 5C-E). The expressions of Arl13B, ac-α-tubulin, and α-tubulin in the BALF of Mito-TEMPO-pre-treated mice were less than those in vehicle-treated mice(Fig.5F-I).In this study, a 6-h post-treatment did not protect the lungs against kidney IR(Fig.5J-L). These results indicate that early ROS production, its accumulation, and oxidative stress may importantly contribute to AKI (Acute kidney injury)-induced lung injury. Superoxide levels and 4-HNE expression in lung tissue(Fig.6A-C)and in BALF(Fig.6D and E)were less in Mito-TEMPO-pretreated mice than in vehicle-treated mice. These results indicate that kidney IR-induced lung injury and lung epithelial cell deciliation are associated with mitochondrial oxidative stress.

Fig. 5. Prevention of kidney IR-induced lung injury and cilia disruption by Mito-TEMPO, a mitochondria-targeted antioxidant. C57BL/6 male mice were subjected to 35 min of bilateral renal ischemia. Some mice were administered with Mito-TEMPO (Mito-T, 0.7 mg/kg BW, i.p.) either 17 and 1 h before ischemia, twice, (A–I) or 6 h after ischemia (J–L). Lung, BALF, and blood were harvested 24 h after ischemia. (A, J) The BUN concentration in plasma was measured. (B, C) IL-6 concentrations in plasma and BALF were measured using the ELISA assay kit. (D, E, K, L) The protein concentration and cell number in BALF were measured. (F–I) Arl13B, acetylated-α-tubulin (ac-α-tub), and α-tubulin (α-tub) expressions in BALF were analyzed by western blotting. The densities of the bands were measured using the ImageJ software. Results are expressed as the means ± SEM (n = 4). *p < 0.05 vs. vehicle.

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