Genetic Depletion of Thromboxane A2 Synthase and Thromboxane-Prostanoid Receptor Signaling Attenuates Ischemia/Reperfusion Induced Oxidative Stress, Inflammation, Apoptosis, Autophagy and Pyroptosis in the Kidney

Research Article

Thromb Haemost Res. 2019; 3(2): 1023..

Genetic Depletion of Thromboxane A2 Synthase and Thromboxane-Prostanoid Receptor Signaling Attenuates Ischemia/Reperfusion Induced Oxidative Stress, Inflammation, Apoptosis, Autophagy and Pyroptosis in the Kidney

Chueh TH1, Lin1, Chen KH2,3 and Chien CT1*

¹School of Life Science, National Taiwan Normal University, Taiwan

²Department of Surgery, Far-Eastern Memorial Hospital, Taiwan

³Department of Electrical Engineering, Yuan Ze University, Taiwan

*Corresponding author: Chiang-Ting Chien, School of Life Science, National Taiwan Normal University, No. 88, Section 4, Tingzhou Road, Taipei 11677, Taiwan

Received: April 20, 2019; Accepted: May 14, 2019; Published: May 21, 2019

Abstract

Aims: Enhancement of Thromboxane A2 Synthase (TXAS) activity, Thromboxane A2 (TXA2) release and Thromboxane-Prostanoid receptor (TP) activation leads to severe vasoconstriction and oxidative injury. We explored whether genetic deletion of TXAS/TXA2/TP signaling may reduce renal Ischemia/Reperfusion (I/R) injury in mice.

Methods: Renal hemodynamics and function were evaluated from TXAS+/+TP+/+, TXAS–/–, TP–/– and TXAS–/–TP–/– mice in response to intravenous U46619 (TXA2 mimetic) and I/R injury. We examined renal TXAS and TP expression, BUN and creatinine, Reactive Oxygen Species (ROS) amount, proinflammatory cytokines and pathophysiologic mechanisms including apoptosis, autophagy and pyroptosis under I/R injury.

Results: Renal I/R enhanced the levels of TXAS, TP, nuclear NF-κB, NADPH oxidase gp91, Bax/Bcl-2/Caspase 3/apoptosis, Beclin-1/LC3 II/autophagy, IL- 1β/pyroptosis, renal TXB2 concentration, ROS amount, plasma BUN, creatinine and TXB2 and depressed renal eNOS expression in TXAS+/+TP+/+ mice. All these enhanced parameters were significantly depressed in TXAS–/–, TP–/– and TXAS– /–TP–/– mice. Intravenous U46619 significantly depressed renal microcirculation and enhanced gp91 and Bax/Bcl-2 in TXAS+/+TP+/+ and TXAS–/–, not TP–/– and TXAS–/–TP–/– mice. I/R significantly depressed renal microcirculation in four groups of mice, however, the time for recovery to baseline renal blood flow level was significantly shortened in TXAS–/–, TP–/– and TXAS–/–TP–/– mice vs. TXAS+/+TP+/+ mice. Blocking TXAS/TP signaling also attenuated I/R enhanced pro-inflammatory cytokines profile.

Conclusion: Blockade of TXAS/TXA2/TP signaling confers renal protection against I/R injury through the action of anti-oxidation, anti-inflammation, antiapoptosis, anti-autophagy and anti-pyroptosis.

Keywords: Apoptosis; Autophagy; Pyroptosis; Thromboxane A2 synthase; Thromboxane-prostanoid receptor

Abbreviations

COX-1: Cyclooxygenase-1; COX-2: Cyclooxygenase-2; eNOS: endothelial Nitric Oxide Synthase; ES: E14TG2a Embryonic Stem; I/R: Ischemia/Reperfusion; KO: Knockout; NO: Nitric Oxide; O2-: Superoxide Anion; ROS: Reactive Oxygen Species; TXAS: Thromboxane A2 Synthase; TXAS–/–: TXAS-Knockout; TXA2: Thromboxane A2; TXB2: Thromboxane B2; TP: Thromboxane- Prostanoid Receptor; TP–/–: TP-Knockout; TXAS–/–TP–/–: TXAS and TP Double-Knockout

Introduction

Thromboxane A2 (TXA2) is a member of the prostanoid family of arachidonic acid metabolites generated by the sequential action of phospholipase A2, Cyclooxygenase-1 (COX-1), Cyclooxygenase-2 (COX-2) or TXA2 Synthase (TXAS). The very unstable TXA2 regulates multiple biological processes via its specific thromboxaneprostanoid (TP) receptor to stimulate platelet aggregation and vasoconstriction in vascular and respiratory smooth muscles [1- 4]. The cellular and tissue distribution of TP receptors is closely correlated with that of TXAS [5]. TXA2 [6] as well as isoprostanes [7,8], nonenzymatic free radical-derived products of arachidonic acid, that can activate the TP in vivo [9], is highly elevated in renal, myocardial infarction, atherosclerosis, stroke, bronchial asthma and preeclamptic placenta [1,10-13]. In addition, TXAS and TP are highly expressed within the atherosclerotic lesion areas [14] and during vascular and atherothrombotic diseases [15].

Renal Ischemia/Reperfusion injury (I/R) frequently occurs after infarction, sepsis and organ transplantation and exacerbates kidney dysfunction initiating an inflammatory cascade including Reactive Oxygen Species (ROS), cytokines/chemokines, and leukocytes activation/infiltration [16-18]. Renal TXA2 is elevated in cyclosporine treated kidneys [19], nephritic glomeruli [10,20] and I/R injury [21] reflecting an enhanced TXAS/TXA2/TP signaling to impair the kidney. TXA2 release activates TP via PKC-ζ-mediated NADPH oxidase enhancement [15] and increases O2·- and ONOO-, resulting in endothelial Nitric Oxide Synthase (eNOS) uncoupling in endothelial cells [22]. Anoxia/reoxygenation evokes overproduction of platelet TXB2 and isoprostanes through the action of NADPH oxidase-dependent ROS generation [23]. Targeting the TP or TXAS effectively improves renal function and pathology in humans [21,24]. In animal models, aspirin decreases the severity of renal I/R injury and the development of tubular atrophy [25]. In clinical trials, preoperative low dose aspirin treatment reduced postoperative acute kidney injury, decreased hemodialysis requirements and decreased postoperative hospital stay without increasing bleeding [26]. Some TXAS inhibitors offer an advantage over aspirin in that they may redirect arachidonate metabolism towards PGI2 and other protective eicosanoids [27]. TXAS inhibitors used together with TP receptor antagonists exert a greater anti-platelet effect than low-dose aspirin therapy [28]. However, despite promising results from animal models, the clinical efficacy of TXAS/TP inhibitors is not conclusive and there are even findings suggesting that these agents may inhibit platelet aggregation via other mechanisms besides inhibiting TXA2 activity [3]. Thus, mice with double deletions of TXAS and TP receptors are valuable not only for investigating the phenotype of complete absence of TXA2 activity but also for exploring the pharmacological mechanisms related to these dual inhibitory agents.

In response to oxidative injury, renal cells displayed three types of programmed cell death including apoptosis, autophagy, and pyroptosis [18,29]. We hypothesize these types of programed cell death may be involved in I/R enhancing TXAS/TXA2/TP signalinginduced renal dysfunction. To explore the TXA2 effects and mechanisms on I/R induced autophagy, apoptosis and pyroptosis formation in the damaged kidney is important for alleviating and preventing TXA2-induced renal dysfunction. This study was the first use of four types of targeting gene depleted mice, including TXAS+/+TP+/+, TXAS–/–TP+/+, TXAS+/+TP–/–, TXAS–/–TP–/– mice to explore the effect of blocking TXAS/TXA2/TP signaling on renal I/R injury.

Materials and Methods

Animals

Thromboxane A synthase knockout (TXAS–/–) mice: The TXAS– /– mice are provided by Dr. Shu-Wha Lin’s group from National Taiwan University College of Medicine. A targeting vector is designed to replace the distal portion exon 9 of the endogenous gene with a phosphoglycerate kinase-hypoxanthine phosphoribosyltransferase) cassette. This construct is electroporated into E14TG2a Embryonic Stem (ES) cells. Correctly targeted ES cells are injected into C57BL/6J blastocysts. Chimeric mice are bred with C57BL/6J for 10 generations, the homozygous TXAS–/– mice were obtained by intercrossing the heterozygous TXAS+/– mice.

Thromboxane A2 receptor knockout (TP–/–) mice: The TP–/– mice are generated by Dr. Thomas M. Coffman’s group and also provided by Dr. Su-Wha Lin. A neomycin resistance gene driven by the phosphoglycerate kinase promoter is inserted into a unique SfiI site near the proximal end of exon 2. This insertion targeting vector disrupts the coding sequence of the TP gene in the third transmembrane domain. This construct is electroporated into ES cells. Correctly targeted ES cells are injected into C57BL/6J blastocysts. Chimeric mice are bred with C57BL/6J for 10 generations, the homozygous TP–/– mice are obtained by intercrossing the heterozygous TP+/– mice.

Thromboxane A2 synthase and thromboxane A2 receptor double knockout (TXAS–/–TP–/–) mice: TXAS–/– and TP–/– mouse in the C57BL/6 background are crossbred to generate TXAS–/–TP–/– double knockout mice. Male and female mice with or without targeting gene depletion, including TXAS+/+TP+/+, TXAS–/–TP+/+, TXAS+/+TP–/– and TXAS–/–TP–/– mice (B6 background), have been developed and are provided from National Taiwan University core laboratory. The mice were housed at the Experimental Animal Center of National Taiwan University College of Medicine, with a temperature- and humidityregulated environment (22±2°C, 55±5% RH) and a consistent light cycle (light from 07:00 to 18:00 o’clock). Standard powdered diet containing 58% of carbohydrates, 28.5% of proteins and 13.5% of fat (Laboratory Rodent diet 5001, Young Li Trading Company, LTD, Sijhih City, New Taipei City, Taiwan) and tap water were provided ad libitum. All surgical and experimental procedures are approved by Institutional Animal Care and Use Committee of National Taiwan Normal University and are in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997).

Renal ischemia/reperfusion (I/R)

TXAS+/+TP+/+, TXAS–/–TP+/+, TXAS+/+TP–/– and TXAS–/–TP–/– mice with or without renal I/R (n = 6 in each group) were anesthetized with intraperitoneal urethane (1.2 g/kg). The trachea was exposed via a midline cervical incision and intubated. PE-10 catheters were placed in the left carotid artery for measurement of the heart rate and arterial blood pressure by an ADI system (Power-Lab/16S) with a transducer (P23 1D, Gould-Statham, Quincy, USA), and in the jugular vein for administration of test agents. Renal I/R was induced in the mice as described below. For induction of ischemia, the bilateral renal arteries were clamped 45 min with a small vascular clamp. Sham-operated animals underwent similar operative procedures without occlusion of the renal arteries. Reperfusion was initiated by removal of the clamp for 4 hours. After I/R insults, arterial blood and urine were collected for renal functional determination (Blood Urea Nitrogen (BUN) and creatinine) and plasma and renal TXB2 determination (an enzyme immunoassay, Cayman Chemical, Ann Arbor, MI) [30]. After sacrifice with intravenous KCl, these two kidneys were resected and divided into two parts. One part was stored in 10% neutral buffered formalin for immunohistochemistry and in situ pathologic assay, and another was quickly frozen in liquid nitrogen and stored at –70oC for protein isolation.

Renal microcirculation determination

To examine the in vivo response of renal arterial constriction to U46619 and renal I/R, a full-field laser perfusion imager (MoorFLPI, Moor Instruments Ltd., Devon, UK) was used to continuously quantitate the renal microcirculatory blood flow intensity [18]. In brief, the imager used laser speckle contrast imaging, which would exploit the random speckle pattern generated when tissue was illuminated by laser light. The random speckle pattern changed when blood cells moved within the Region Of Interest (ROI). When there was a high level of movement (fast flow), the changing pattern became more blurred, and the contrast in that region reduced accordingly. The contrast image was processed to obtain a 16-color coded image that correlated with blood flow in the heart. Blue was defined as low flow and red as high flow. The microcirculatory blood flow intensity of each ROI was recorded as Flux with perfusion unit, which was related to the product of average speed and concentration of moving red blood cells in the kidney sample volume. The negative control value was set at 0 perfusion unit (blue color) and the positive value was at 1000 perfusion unit (red color). The perfusion units were realtime analyzed by the MoorFLPI software version 3.0.

In vivo renal ROS detection

We directly measured the renal ROS in response to renal I/R in vivo via an intravenous infusion of a superoxide anion probe, 2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a]- pyrazin- 3-one-hydrochloride (MCLA) (0.2 mg/mL/hour, TCIAce, Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan) and detected by a Chemiluminescence Analyzing System (CLD-110, Tohoku Electronic In. Co., Sendai, Japan) [16]. In brief, after surgery, the rat was maintained on a respirator and a circulating water pad at 37°C during photon detection. For excluding photon emission from sources other than the kidney, the anesthetized animal was housed in a dark box with a shielded plate. Only the kidney was left unshielded and was positioned under a reflector, which reflected the photons from the exposed kidney surface onto the detector area. The MCLA-enhanced chemiluminescent signal from the kidney surface was recorded continuously by the chemiluminescence analyzer. The total ROS value was measured by area under curve from the kidney. The chemiluminescent signal obtained from 0.2 mL saline in 1 mL of MCLA (0.2 mg/ml) or 0.2 ml xanthine (0.75 mg/kg body weight)/ xanthine oxidase (24.8 mU/kg body weight) in 1 ml of MCLA (0.2 mg/ml) was regarded as negative or positive control.

Western blotting

The kidneys were ground to powder in liquid nitrogen and then the powder was lysed in RIPA Buffer (Bio Basic, NY, USA) supplemented with protease inhibitor (Roche, Basel, Switzerland) for 10 minutes at 4°C. Detection of signals was performed by western lightning plus-ECL (PerkinElmer, Waltham, USA). The expression levels of TXAS, TP, NF-κB, gp91, apoptosis-related proteins including Bcl-2, Bax, Caspase 3, autophagy-related proteins Beclin-1 and LC3-II and pyroptosis-related protein IL-1β were analyzed by Western blotting in kidney tissues. The Western blotting method has been described elsewhere [18].

Antibodies raised against TXAS (Cayman), TP (Cayman), NF-κB (R&D Systems), NADPH oxidase (gp91phox; Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology, Inc., Danvers, MA), LC3-II (Cell Signaling Technology, Inc.), Bax (Chemicon), Bcl- 2 (Transduction, Bluegrass-Lexington, KY), caspase-3 (Cell Signaling Technology, Denver, USA) and β-actin (Sigma, Saint Louis, MI) were used. The density of the band with the appropriate molecular mass was determined semi-quantitatively by densitometry using an image analyzing system (Alpha Innotech, San Leandro, CA).

Secondary antibodies included HRP-conjugated goat anti-mouse IgG, HRP-conjugated rabbit anti-goat IgG, and HRP-conjugated goat anti-rabbit IgG (all for 1:10000; all from SouthernBiotech Laboratories, Birmingham, USA). For quantitative comparison of the protein expression levels, intensities of specific bands, corresponding to the proteins of interest were measured using densitometry analysis.

Histologic studies

Tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. We obtained 5-μm sections and stained them with the hematoxylin and eosin. We calculated the mean renal injury score in each mouse with light microscope and averaged the scores for each group. Twenty tubules in each kidney were randomly selected at a × 200 magnification, and the degree of renal damage was scored using the scoring system for renal injury reported by Wagner et al. (2003) [31]. The percentage of tubular injury parameters containing epithelial flattening, tubular atrophy, tubular dilatation, and brush border loss were estimated by a pathologist who was blind to the identity of the specimen using a 4-point scale in ten randomly chosen, nonoverlapping fields. Degree of injury was graded on a scale from 0 to 4: 0 = normal; 1 = mild, involvement of less than 25% of the cortex; 2 = moderate, involvement of 25 to 50% of the cortex; 3 = severe, involvement of 50 to 75% of the cortex; and 4 = extensive damage involving > 75% of the cortex.

Detection of autophagy, apoptosis and pyroptosis in the I/R kidneys

To examine the effect of TXAS or TP knockout on several oxidative stress parameters, we performed LC3-II-related autophagy, Caspase 3/terminal deoxynucleotidyl Transferase-Mediated Nick- End Labeling (TUNEL) apoptosis method [18] and IL-1β-mediated pyroptosis to investigate the presence and extent of three types of programmed cell death in renal I/R injury. The renal sections (5 μm) were prepared, deparaffinized, and stained by the hematoxylin & eosin, LC3-II stain, TUNEL-avidin-biotin-complex methods and IL- 1β stains. A biotinylated secondary antibody (Dako, Botany, NSW, Australia) was then applied followed by streptavidin conjugated to HRP (Dako). The chromogen used was Dako Liquid diaminobenzene (DAB). Twenty high-power (×400) fields were randomly selected for each section, and the value of each oxidative stress was analyzed using a Sonix Image Setup (Sonix Technology Co., Ltd) containing image analyzing software Carl Zeiss AxioVision Rel.4.8.2 (Future Optics & Tech. Co. Ltd., Hangzhou, China). The apoptotic index was calculated as the number of TUNEL-positive nuclei per high-power field (×400).

Statistical analysis

All values were expressed as mean ± Standard Error Mean (SEM). Differences within groups were evaluated by paired t-test. One-way analysis of variance was used for establishing differences among groups. Intergroup comparisons were made by Duncan’s multiplerange test. Differences were regarded as significant if P ‹ 0.05 is attained.

Results

Blocking TXAS/TP signaling reduces I/R induced oxidative stress

The original graph of renal I/R on TXAS, TP, NADPH oxidase gp91 ans eNOS expression by western blotting was displayed in Figure 1A. Renal I/R significantly enhanced TXAS, TP and NADPH oxidase gp91 and depressed eNOS expression as compared to the baseline control in the TXAS+/+TP+/+ kidney (Figure 1B). The increased renal TXAS, TP and gp91 expressions responding to I/R were significantly decreased in the TXAS–/–, TP–/– or TXAS–/–TP–/– mice (Figure 1B). The degree of decreased renal eNOS by I/R was significantly preserved in the TXAS–/–, TP–/– or TXAS–/–TP–/– mice as compared to TXAS+/+TP+/+ mice. We further compared the plasma and kidney TXB2 level before and after renal I/R injury. Renal I/R significantly increased plasma (Figure 1C) and kidney TXB2 concentration (Figure 1D) in the TXAS+/+TP+/+ and TXAS+/+TP–/– mice. However, renal I/R did not significantly increase plasma and kidney TXB2 level in the TXAS–/– and TXAS–/–TP–/– mice. We used immunofluorescence stain to confirm the higher fluorescent intensity of TXAS or TP in the TXAS+/+TP+/+ mice with I/R injury but less expression of TXAS or TP fluorescence in TXAS–/–, TP–/– or TXAS–/–TP–/– mice subjected to I/R injury (Figure 1E). These data implicate blocking TXAS/TP signaling may reduce gp91 mediated oxidative stress in the kidney.