Protection against post-resuscitation renal injury by Artesunate via inhibition of caspase-3/Gasdermin E -mediated pyroptosis

Abstract

Background

Post-resuscitation renal injury is the common complication after cardiac arrest and resuscitation. Artesunate (Art) has been confirmed to protect against regional ischemia–reperfusion injuries in multiple organs. This study aimed to determine the effects of Art on post-resuscitation renal injury and clarify whether the mechanism is related to inhibition of the caspase-3/Gasdermin E pyroptosis pathway.

Methods

Twenty-two swine were divided into a Sham, Cardiopulmonary resuscitation (CPR), and CPR + Art groups. Blood samples were collected to measure serum creatinine and blood urea nitrogen levels. Samples of renal tissue were obtained for histopathological assessment of injury, the apoptosis index, contents of proinflammatory cytokines, kidney injury molecule-1, neutrophil gelatinase-associated lipid carrier, and pyroptosis-related protein expression levels.

Results

Compared with the Sham group, the biomarkers of renal injury were significantly increased in the CPR and CPR + Art groups. Levels of these biomarkers were significantly lower in the CPR + Art group than in the CPR group. Renal pathological damage and cell apoptosis were observed in the CPR and CPR + Art groups after resuscitation and were markedly less severe in the CPR + Art group. The expression levels of caspase-3/Gasdermin E pyroptosis related proteins and proinflammatory cytokines were significantly increased in the CPR and CPR + Art groups compared with the Sham group. Treatment with Art significantly decreased the expression of these indicators of cell pyroptosis.

Conclusions

Art treatment could alleviate post-resuscitation renal injury, and its mechanism may be related to the inhibition of caspase-3/Gasdermin E pyroptosis pathway.

Introduction

Cardiac arrest (CA) remains a major public health burden in terms of high mortality rates and fatal sequelae.1, 2 Although recent years have seen unremitting efforts to improve the success rate of resuscitation and minimize post-resuscitation organ dysfunction, the prognosis of patients with CA remains poor. Systemic ischemia–reperfusion (I/R) resulting from CA and resuscitation can cause post-cardiac arrest syndrome, which is characterized by activation of the immune system and release of inflammatory mediators, leading to multiple organ dysfunction. The kidney is one of the most vulnerable organs after CA and resuscitation, and renal injury post-resuscitation is an independent risk factor for mortality in CA patients and may lead to irreversible loss of renal function.3, 4 A recent study showed that post-resuscitation renal injury occurred in 69.8 % of CA survivors and had a poor outlook.5, 6 Therefore, relieving renal injury after CA and cardiopulmonary resuscitation (CPR) might be a strategy that can delay the progression of post-cardiac arrest syndrome and improve the prognosis.

Artesunate (Art) is the only effective derivative of artemisinin that can be made into a water-soluble preparation and is used mainly in the treatment of malaria worldwide. In recent years, a growing number of studies have demonstrated that Art can improve regional I/R injury of brain, heart, liver, kidney and other organs through its anti-inflammatory, anti-apoptosis, anti-oxidation, anti-pyroptosis effects7, 8, 9 with a low risk of adverse reactions. One study found that Art protects against the fibrosis induced by renal I/R injury in rats.¹⁰ However, the vast majority of these studies have focused on isolated organ I/R injury models. The unique pathophysiology of post-resuscitation renal injury, which characterized by systemic I/R injury and a devastating whole-body inflammatory response, may engage distinct molecular mechanisms. Whether Art is effective in mitigating post-resuscitation renal injury in this specific clinical context, and through what mechanisms, remains an unanswered question. This study aims to investigate the therapeutic potential of Art for post-resuscitation renal injury, as well as its potential mechanisms.

Pyroptosis is a novel mode of inflammatory programmed cell death.¹¹ Recent studies have confirmed that pyroptosis is an important cause of I/R-induced target organ injury,¹² including in the kidney.¹³ However, its role in systemic I/R injury after CA and resuscitation is unclear. Previous studies on organ dysfunction after resuscitation mainly focused on the classical pyroptosis pathway involving caspase-1/GSDMD.¹⁴ Gasdermin E (GSDME) is a newly identified protein that mediates pyroptosis via cleavage of the caspase-3 generating pyroptotic N-terminal fragment of GSDME (GSDME-N). Recent studies have demonstrated that GSDME promotes pyroptosis in various renal diseases, including renal I/R injury,¹⁵ acute kidney injury¹⁶ and obstructive nephropathy.¹⁷ Meanwhile, our previous research found that Art treatment effectively alleviates post-resuscitation intestinal injury by inhibiting caspase-3/GSDME pyroptosis pathway.¹⁸ However, whether GSDME-mediated pyroptosis is involved in post-resuscitation renal injury remains unknown.

In this study, we established a porcine model of CA/CPR in which we hypothesized that Art could reduce post-resuscitation renal injury by inhibiting the caspase-3/GSDME pyroptosis pathway. These findings offer a novel perspective for application of Art in the field of resuscitation and provide an important basis for the prevention and treatment of post-resuscitation renal injury.

Methods

Animals and groups

Twenty-two male swine (age 4–6 months; mean weight 36 ± 2 kg) were sourced from Shanghai Jiagan Biotechnology Inc (Shanghai, China). The animal qualification certificate number was SCXK (Shanghai) 2020-0006. After arrival, the experimental animals continued to be fed under standard conditions and underwent acclimatization to their new environment before being used for the experiments. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011). The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (Approval Number: 2022-027). All methods are reported in accordance with ARRIVE guidelines (Supplementary Table. S1).

The animals were divided into three groups by random number table method: Sham group (n = 6), CPR group (n = 8), and CPR + Art group (n = 8). The Sham group only underwent routine arterial-venous catheterization surgery without induction of CA or performance of CPR, while the CPR and CPR + Art groups were prepared by electrically induced ventricular fibrillation (VF) for 9 min and CPR for 6 min. At 5 min after resuscitation,19, 20 the swine in the CPR + Art group received an intravenous infusion of Art (APExBIO Inc., Houston, TX, USA) at a dose of 4.8 mg/kg (the solution was dissolved in 10 mL of dimethyl sulfoxide and then diluted with 250 mL of phosphate buffer) for 2 h. Animals in the Sham and CPR groups received an intravenous infusion of same amount of solvent. The dosage of Art was selected with reference to previous literature studies.21, 22 The study protocol was developed before the experiment began, which specified the research question, key design features and the primary outcome measure. However, the protocol was not prospectively registered in a public repository. The unregistered protocol is available from the corresponding author upon reasonable request.

Inclusion and exclusion criteria

Inclusion Criteria: All male swine (4–6 months, 36 ± 2 kg) that survived for 24 h following the experimental protocol were included in the final analysis. The protocol consisted of 9 min of electrically induced VF and 6 min of CPR.

Exclusion Criteria: Animals that died during the surgery or within 24 h at post-resuscitation were excluded.

Animal preparation

All swine were fasted for 12 h before experiment. They were anesthetized with an initial intramuscular injection of tiletamine/zolazepam (5 mg/kg) and xylazine (1 mg/kg) followed by an intravenous infusion of propofol (2 mg/kg) and a continuous infusion of propofol (4 mg/kg/h) to maintain anesthesia. After premedication, endotracheal intubation was performed with the assistance of a long straight laryngoscope. The animal was then connected to a ventilator (Monnal T75, Air Liquide Medical Systems, Antony Cedex, France) with the setting of tidal volume (10 mL/kg), peak flow (40 L/min) and oxygen concentration (21 %). End-tidal carbon dioxide (ETCO₂) was maintained by a monitor/defibrillator (M Series, ZOLL Medical Corporation, Chelmsford, MA, USA) in the range of 35–40 mmHg (1 mmHg = 0.133 kPa) with adjustment of the respiratory rate on the ventilator. An 8F pressure monitoring catheter was placed in the right femoral artery with the tip inserted to the thoracic aorta level for monitoring of major arterial blood pressure, right atrial pressure, core temperature, and collection of arteriovenous blood samples. A 7F pressure monitoring catheter was placed in the right femoral vein with the tip inserted to the right atrial level. A 5F pacemaker electrode was inserted into the right ventricle via the right external jugular vein for induction of VF. All catheters were intermittently flushed with the saline containing heparin 5 IU/mL. An iM60 dynamic ECG monitor (iM60, Edan, Shenzhen, China) was used for multi-parameter monitoring in real time. Rectal temperature was monitored and maintained at a normal level of 38.0 ± 0.5 °C using an ultralow temperature system (Blanketrol III, Cincinnati Sub-Zero, Cincinnati, OH, USA).

Experimental procedures

Basic physiological indices were measured for each experimental animal 10 min before electrical stimulation-induced VF. VF was induced using a right ventricular pacemaker electrode with a 1-mA alternating current. Mechanical ventilation was stopped and the fibrillation pacemaker catheter was removed when VF was successfully induced. After 9 min of untreated VF, manual chest compression was initiated and accompanied by ventilation at a ratio of 30:2. At the same time, a PalmCPR chest compression feedback instrument (Sunlife, Shanghai, China) was used to ensure the quality of CPR, which included a compression depth of 5–6 cm and a frequency of 100–120 /min according to international cardiopulmonary resuscitation guidelines. The first dose of epinephrine 20 μg/kg was injected intravenously 2 min after CPR; thereafter, it was administered at 3-min intervals. A 150-J biphasic electrical shock was delivered by the M Series Monitor Defibrillator (M Series, ZOLL Medical Corporation, Chelmsford, MA, USA) at 6 min of CPR and measure whether the return of spontaneous circulation (ROSC) occurred. The ROSC standard criterion was supraventricular spontaneous rhythm with mean arterial pressure >50 mmHg for more than 5 min. If ROSC was not observed, CPR was immediately resumed for 2 min before another electrical shock. This protocol was repeated up to 5 times until ROSC or resuscitation failed. All resuscitation swine were kept under anesthesia and monitored for 4 h. After recovery, the catheter was removed, the wound was sutured, and the swine were transferred to the feeding room for 20 h of observation. During the entire experiment, compound sodium chloride solution (2 mL/kg/h) was continuously infused to maintain fluid balance.²³

Outcome measurements

During CPR, coronary perfusion pressure, duration of CPR, epinephrine dosage, number of shocks, resuscitation success rate, and 24 h survival rate was recorded. Heart rate, mean arterial pressure, and ETCO₂ were recorded at baseline and at 1, 2, 4 and 24 h after ROSC. Venous blood samples and blood gases were also analyzed at these time points for evaluation of renal injury.

Venous blood samples were collected at baseline and 1, 2, 4, and 24 h post-resuscitation. Each sample was centrifuged, after which the supernatant was collected and stored in a cryogenic freezer at –80 °C. Enzyme-linked immunosorbent assay kits (Meixuan Biotechnology Inc, Shanghai, China) were used to measure serum creatinine (Cr) and blood urea nitrogen (BUN) levels. At 24 h after resuscitation, the swine were euthanized, and tissue samples were rapidly dissected from the left upper kidney and stored at –80 °C. Kidney tissue lysates were run on 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. Blots were blocked with 5 % bovine serum albumin and then exposed to the primary antibody in dilutions. Primary antibodies against kidney injury molecule-1 (KIM-1; 1:1000, Proteintech, Rosemont, IL, USA) and neutrophil gelatinase-associated lipocalin (NGAL; 1:1000, Proteintech) were used, followed by incubation with species-specific secondary antibodies (anti-mouse or anti-rabbit; 1:5000, BBI Life Sciences Corporation, Shanghai, China).The proteins were visualized by enhanced chemiluminescence substrates and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

For evaluation of renal histopathological injury, a portion of each left kidney sample obtained at 24 h post-resuscitation for the pathological tissue slices was fixed in paraformaldehyde for 24 h, dehydrated, embedded in paraffin, sliced into 5-μm sections, and stained with hematoxylin-eosin. Six random fields were then photographed at 200 × magnification using an optical microscope (CX31, Olympus, Tokyo, Japan) to determine the severity of renal injury. Renal injury was scored according to criteria adapted from Chan et al.,²⁴ which is based on a combination of the following pathological features: (1) tubular epithelial cell loss, (2) tubular necrosis, (3) accumulation of cellular debris, and (4) tubular cast formation. The scoring was defined by the percentage of affected tubules: Score 0: No damage (normal); Score 1: Mild injury (10–25 % of tubules affected); Score 3: Moderate to severe injury (51–75 % of tubules affected); Score 4: Severe injury (>75 % of tubules affected). The scoring was completed by an experienced pathologist in a blind state.

For evaluation of renal apoptosis, the sections were stained using a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay kit (Boster Biological Technology Co., Wuhan, China) according to the manufacturer’s instructions. Six views were randomly captured at 200 × magnification under the optical microscope to count the numbers of TUNEL-positive cells and total cells, the ratio of which was considered to be the rate of apoptosis.

For the evaluation of caspase-3/GSDME pyroptosis pathway-related proteins, and proinflammatory cytokine in the kidney, tissue specimens were similarly harvested at 24 h post-resuscitation. Protein was extracted from the kidney tissue using RIPA lysate and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was absorbed, and the protein concentration was determined. Equivalent amounts of the protein samples were separated using 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). After blocking the nonspecific binding sites with 5 % nonfat milk at room temperature for 1 h, all membranes were incubated overnight at 4 °C with primary antibodies to caspase-3 (1:1000; Proteintech, Rosemount, United States), cleaved caspase-3 (1:1000, Cell Signaling Technology, Danvers, Massachusetts, United States), GSDME (1:1000; Proteintech, Rosemount, United States), GSDME-N (1:1000; Abcam, Cambridge, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, BBI Life Sciences Corporation, Shanghai, China), and then incubated with the secondary antibody (1:5000; BBI Life Sciences Corporation, Shanghai, China) at room temperature for 1 h. The membranes were washed a further three times and developed using enhanced chemiluminescence substrates. The densities of the protein bands were analyzed using ImageJ software (NIH, Bethesda, United States). Finally, the expression level of the target protein was reflected by the ratio of the gray value for the protein to that of glyceraldehyde-3-phosphate dehydrogenase. High mobility group box 1 (HMGB1), interleukin (IL)-1β, and IL-18 levels were measured in the supernatants of renal homogenates by enzyme-linked immunosorbent assay (Meixuan Biotechnology Inc, Shanghai, China).

Statistical analysis

Sample size was calculated based on our previous investigation to detect a difference with respect to the Cr release (primary outcome) of ≥20 %.²⁵ We calculated a sample size of 6 animals in each group for an α of 0.05 and a power of 80 %. Considering that those animals who did not achieve ROSC or died during the post-resuscitation period, and the guiding principles underpinning the humane use of animals in scientific research (three Rs), 8 animals were used in the CPR and CPR + Art groups, respectively. Meanwhile, Potential confounders related to the order of procedures were minimised through randomization. The order in which animals received treatments and underwent behavioral tests was randomized across experimental days. However, the location of cages within the rack was not systematically controlled. Blinding was applied to outcome assessment and data analysis where possible in our study. Due to the nature of the intervention, the personnel performing the surgeries were aware of the group allocation. All investigators responsible for outcome measurements were blinded to the treatment groups. Continuous variables were are presented as the mean ± standard deviation when normally distributed and as the median (interquartile range) when not normally distributed. Continuous variables were tested for normality using the Kolmogorov–Smirnov test. The resuscitation data were examined for statistical significance using the Student’s t-test. One-way analysis of variance and the Bonferroni post hoc test were used for comparisons among three groups and the least significant difference t-test for comparisons between two groups. The counting data were compared using Fisher’s exact test. All statistical analyses were performed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). A p-value of <0.05 was considered statistically significant.

Results

Baseline characteristics and resuscitation data

Twenty-two swine were used in the study. The three groups were similar at baseline in terms of body weight, heart rate (HR), mean arterial pressure (MAP), ETCO₂, pondus hydrogenii (pH), and lactate (Fig. 1, Fig. 1; Supplementary Table. S2) (all P > 0.05). There was no significant difference in coronary perfusion pressure［(22.1 ± 3.6) mmHg vs. (22.8 ± 4.2) mmHg, P = 0.75］, duration of CPR［(7.50 ± 3.51) min vs. (8.80 ± 4.53) min, P = 0.55］, dose of epinephrine［(1.80 ± 0.81) mg vs. (2.15 ± 0.93) mg, P = 0.56］, or number of shocks［(1.80 ± 1.81) vs. (2.45 ± 2.33), P = 0.55］between the CPR and CPR + Art groups during the establishment of the animal model (Fig. 2, Fig. 2). Following the standard CA/CPR procedure, seven animals in the CPR group and six in the CPR + Art group were successfully resuscitated and alive at 24 h post-resuscitation. There was no significant difference in the resuscitation success rate or the 24 h survival rate between the two groups (Fig. 2E) (P > 0.05). However, compared to the Sham group, both the CPR and CPR + Art groups exhibited significant metabolic acidosis after ROSC. Specifically: The pH (at 1 h and 2 h) and lactate (at 1 h and 2 h) were significantly lower in both groups compared to the sham group. The base excess (BE) remained persistently lower throughout the monitoring period (CPR group: 1 h, 2 h, 4 h; CPR + Art group: 1 h, 2 h). HCO3^– were significantly reduced in the CPR group (1 h, 2 h, 4 h) and the Art group (1 h). It is noteworthy that no statistically significant differences were observed in any blood gas parameters between the CPR and CPR + Art groups at all time points. However, the trend of blood gas analysis parameters in the Art group was better than that in the CPR group. (Fig. 3; Supplementary Table. S3). Compared to the CPR group, the CPR + Art group showed statistically significant differences in HR at 1 h (P = 0.00), 2 h (P = 0.01), 3 h (P = 0.02), and 4 h (P = 0.03) after ROSC, with a more favorable recovery trend in HR observed in the CPR + Art group, which was not significantly different from the Sham group. Regarding MAP, although there was no statistically significant difference between the CPR and CPR + Art groups, the CPR + Art group exhibited more stable blood pressure with less fluctuation, closely resembling the Sham group. In contrast, the CPR group had significantly lower MAP at 1 h (P = 0.02) and 2 h (P = 0.03) after ROSC compared to the Sham group. Furthermore, compared to the Sham group, both groups showed significantly higher neurological deficit scores (NDS) (P < 0.05); however, the CPR + Art group had significantly lower NDS than the CPR group, and this difference was statistically significant (P = 0.00) (Fig. 4).

Fig. 1.

Baseline characteristics in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups. A, Body weight. B, Heart rate. C, Mean arterial pressure. D, End-tidal CO₂. E, pH. F, Lactate. The data are presented as the mean ± standard deviation and were compared among groups by analysis of variance and Bonferroni’s test. CPR, cardiopulmonary resuscitation; Art, Artesunate; pH, pondus hydrogenii.

Fig. 2.

Clinical data in the CPR (n = 8) and CPR + Art (n = 8) groups during the resuscitation period. A, Coronary perfusion pressure at 1, 2, 3, 4, 5, and 6 min during the resuscitation period. B, Duration of CPR. C, Dosage of epinephrine. D, Number of shocks. E, Number of animals with ROSC. The data are presented as the mean ± standard deviation and were compared among the groups using the Student’s t-test. The data of ROSC rates were compared among the groups using the Fisher's exact test. CPR, cardiopulmonary resuscitation; Art, Artesunate; ROSC, return of spontaneous circulation.

Fig. 3.

Blood gas analyses in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups at baseline and at 1, 2, and 4 h during the post-resuscitation period. A, pH. B, PCO2. C, PO2. D, Base excess. E, HCO3^–. F, Lactate. The data are presented as the mean ± standard deviation and were compared among groups by analysis of variance and Bonferroni’s test. *P < 0.05 vs. the Sham group. CPR, cardiopulmonary resuscitation; Art, Artesunate; pH, pondus hydrogenii.

Fig. 4.

Clinical data and NDS in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups at baseline and during the post-resuscitation period. A, Heart rate. B, Mean arterial pressure. C. NDS. The data are presented as the mean ± standard deviation and were compared among the groups by analysis of variance and Bonferroni’s test. *P < 0.05 vs. the Sham group; #P < 0.05 vs. the CPR group. CPR, cardiopulmonary resuscitation; Art, artesunate; BL, baseline; NDS, Neurological Deficit Score.

Assessment of renal function

Compared with the Sham group, those in the CPR and CPR + Art groups showed post-resuscitation renal injury, which manifested as gradual increases in serum Cr and BUN levels and in KIM-1 and NGAL protein concentrations after resuscitation; all differences among the groups were statistically significant (P < 0.05). Serum levels of Cr at 2, 4, and 24 h and of BUN at 4 and 24 h after resuscitation were significantly lower in the CPR + Art group than in the CPR group, as were the protein concentrations of KIM-1 and NGAL (all P < 0.05) (Fig. 5, Fig. 6).

Fig. 5.

Cr and BUN levels in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups at baseline and at 1, 2, 4, and 24 h during the post-resuscitation period. A, Cr. B, BUN. CPR, cardiopulmonary resuscitation; Art, artesunate; Cr, creatinine; BUN, blood urea nitrogen; BL, baseline. *P < 0.05 vs. the Sham group; #P < 0.05 vs. the CPR group. The data are presented as the mean ± standard deviation and were compared among the groups by analysis of variance and Bonferroni’s test.

Fig. 6.

Changes in KIM-1 and NGAL levels at 24 h post-resuscitation in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups. A, representative protein bands of western blots in renal tissue. B, C, KIM-1 and NGAL expression levels in renal tissue. The data are presented as the mean ± standard deviation and were compared among the groups by analysis of variance and Bonferroni’s test. *P < 0.05 vs. the Sham group; #P < 0.05 vs. the CPR group. CPR, cardiopulmonary resuscitation; Art, artesunate; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Histopathological changes in the kidney

Observation by light microscopy after hematoxylin-eosin staining showed that the glomeruli and renal tubules in the Sham group were normal with no exfoliated epithelial cells or abnormalities. However, at 24 h after resuscitation, the CPR group exhibited glomerular edema, focal cellular necrosis, tubular dilation, shedding of tubular epithelial cells, and inflammatory cell infiltration. Renal histopathological alterations in the CPR + Art group were markedly attenuated compared to those in the CPR group (Fig. 7A). Accordingly, the kidney injury score was significantly elevated in both the CPR and CPR + Art groups relative to the Sham group. However, the score in the CPR + Art group remained significantly lower than that in the CPR group (Fig. 7B).

Fig. 7.

Histopathological changes in the kidney at 24 h post-resuscitation in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups. A, Representative photomicrographs of hematoxylin and eosin staining in kidney tissue (200 × magnification). B, Kidney injury score. Tissue samples were measured at 24 h after resuscitation in the Sham, CPR, and CPR + Art groups, respectively. The data are presented as the mean ± standard deviation and were compared among the groups by analysis of variance and Bonferroni’s test. *P < 0.05 vs. the Sham group; #P < 0.05 vs. the CPR group. CPR, cardiopulmonary resuscitation; Art, Artesunate.

Apoptotic changes in kidney tissues

At 24 h after ROSC, pathological analysis showed that the percentages of TUNEL-positive cells in kidney tissues were significantly higher in the CPR and CPR + Art groups than in the sham group. The rate of renal apoptosis was significantly lower in the CPR + Art group than in the CPR group (Fig. 8, Fig. 8).

Fig. 8.

Changes in apoptosis in renal tissues at 24 h post-resuscitation in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups. A, representative photomicrographs of the TUNEL assay results. B, percentage of TUNEL-positive cells. Tissue samples were measured at 24 h after resuscitation in the Sham, CPR, and CPR + Art groups. The data are presented as the mean ± standard deviation and were compared among groups by analysis of variance and Bonferroni’s test. *P < 0.05 vs. the Sham group; #P < 0.05 vs. the CPR group. CPR, cardiopulmonary resuscitation; Art, artesunate; TUNEL, TdT-mediated dUTP-biotin nick end labeling.

Caspase-3 and GSDME in kidney tissues

Caspase-3 and GSDME were measured in kidney tissues at 24 h after resuscitation. The concentrations of caspase-3 protein, the downstream protein GSDME and their activated forms, cleaved caspase-3 and GSDME-N, were significantly higher in the CPR and CPR + Art groups than in the Sham group. However, caspase-3, GSDME, cleaved caspase-3 and GSDME-N protein concentrations were significantly lower in the CPR + Art group than in the CPR group (Fig. 9, Fig. 9).

Fig. 9.

Changes in caspase-3/GSDME-mediated pyroptosis pathway-related proteins at 24 h post-resuscitation in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups. A, representative western blotting images of caspase-3, cleaved caspase-3, GSDME, and GSDME-N in the kidney. B–E, Quantitative analysis of caspase-3, cleaved caspase-3, GSDME, and GSDME-N proteins in the kidney. The data are presented as the mean ± standard deviation and were compared among groups by analysis of variance and Bonferroni’s test. *P < 0.05 vs. the Sham group; #P < 0.05 vs. the CPR group. CPR, cardiopulmonary resuscitation; Art, artesunate; GSDME, Gasdermin E; GSDME-N, N-terminal fragment of GSDME; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Changes in proinflammatory cytokine levels

HMGB1, IL-1β, and IL-18 levels in the kidney tissues were examined at 24 h post-resuscitation (Fig. 10, Fig. 10). The levels of these three proinflammatory cytokines were significantly increased in the post-resuscitation kidney tissues, consistent with the changes in the caspase-3/GSDME-mediated pyroptosis pathway-related proteins. A marked reduction in the levels of HMGB1, IL-1β, and IL-18 was observed following Art treatment.

Fig. 10.

Changes in proinflammatory cytokine levels at 24 h post-resuscitation in the Sham (n = 6), CPR (n = 8), and CPR + Art (n = 8) groups. A, HMGB1. B, IL-1β. C, IL-18. The data are presented as the mean ± standard deviation and were compared among groups by analysis of variance and Bonferroni’s test. *P < 0.05 vs. the Sham group; #P < 0.05 vs. the CPR group. CPR, cardiopulmonary resuscitation; Art, artesunate; HMGB1, high mobility group box 1; IL, interleukin.

Discussion

In this study, we investigated the effects of treatment with Art on post- resuscitation renal injury in a porcine model of CA/CPR. We found that administration of Art markedly alleviated post-resuscitation renal injury when compared with the CPR group during the 24-hour observation period. Art treatment also reduced the expression levels of caspase-3, GSDME, and their activated forms (cleaved caspase-3 and GSDME-N), as well as the levels of proinflammatory cytokines, including HMGB1, IL-1β, and IL-18. These results suggest that Art confers renoprotective effects following resuscitation, which may be partially mediated through suppression of the caspase-3/GSDME-dependent pyroptosis pathway.

CA is a leading cause of mortality worldwide. Most post-resuscitation-related deaths are attributable to post-cardiac arrest syndrome, which is characterized by systemic I/R injury. Previous studies of post-cardiac arrest syndrome have focused predominantly on post-resuscitation myocardial dysfunction and brain injury because of the importance of the heart and brain. Thus far, there has been limited research on post-resuscitation renal injury. In a recent study, the death rate among post-resuscitation patients with renal injury was 69.4 %,²⁶ indicating that renal injury after resuscitation is closely associated with the high mortality of CA. Therefore, there is an urgent need to identify a feasible therapeutic strategy to prevent post-resuscitation renal injury and improve the prognosis. In addition to its significant anti-malarial effect, Art has recently been found to have anti-inflammatory, anti-apoptotic, anti-pyroptosis and anti-oxidant effects on regional I/R injury in the brain, heart, liver, kidney and other organs.7, 8, 9 Although there is no information on the role of Art in renal injury after CA/CPR, Zhang et al.²⁷ found that Art attenuates cisplatin-induced acute kidney injury by suppressing oxidative stress and inflammatory activity. Furthermore, Lei et al. demonstrated that Art relieves cisplatin-induced acute kidney injury by inhibiting macrophagic Mincle-mediated necroptosis and inflammation in tubular epithelial cells.²⁸ Another study showed that Art may protect against renal injury secondary to diabetes.²⁹ Therefore, we hoped to be able to confirm that Art has a protective effect on post-resuscitation renal injury and identify the mechanism.

In view of the urgent need for treatment of post-resuscitation renal injury and the potential therapeutic effectiveness of Art, we conducted this pre-clinical exploration of the ability of Art to alleviate post-resuscitation renal injury. With reference to the Art dosage and route of administration previously reported in the literature21, 22 combined with the earliest possible clinical timing, we opted to inject Art intravenously at a dose of 4.8 mg/kg over 2 h starting 5 min after resuscitation. We found that biomarkers of renal injury, including Cr, BUN, KIM-1, and NGAL, were significantly higher in both the CPR and CPR + Art groups compared to the Sham group within 24 h after resuscitation. Hematoxylin-eosin staining of renal tissue obtained at 24 h post-resuscitation showed that compared to the Sham group, the CPR and CPR + Art groups exhibited varying degrees of renal damage, including glomerular edema, focal cellular necrosis, tubular dilation, epithelial cell shedding, and inflammatory cell infiltration. Furthermore, the percentage of TUNEL-positive cells in renal tissues was significantly higher in the CPR and CPR + Art groups than in the Sham group. Notably, the CPR + Art group showed significant attenuation of renal damage compared to the CPR group. Specifically, serum Cr levels at 2, 4, and 24 h; BUN levels at 4 and 24 h; protein expression of KIM-1 and NGAL; and the percentage of TUNEL-positive cells in renal tissue at 24 h were all significantly lower in the CPR + Art group. Consistent with these findings, histopathological alterations in the kidneys were also markedly milder in the CPR + Art group than in the CPR group. Furthermore, our findings indicate that Art treatment exhibited more stable hemodynamic stability with less fluctuation and the better trend of blood gas analysis parameters as time progresses compared to CPR group, closely resembling the Sham group. These results suggest that Art could protect against post-resuscitation renal injury.

Pyroptosis is a recently identified form of programmed cell death that depends on the activity of specific caspases and is mediated by the Gasdermin proteins, among which Gasdermin D and GSDME are the main executive proteins. Recent studies have confirmed that caspase-1/GSDMD-mediated pyroptosis is a classical pathway and an effective intervention target in various renal injuries.30, 31, 32 GSDME is a newly discovered executive protein in pyroptosis and a focus of considerable research interest.33, 34 A previous study found that cleavage of GSDME is involved in the process of membrane pore formation and lysis of renal tubular epithelial cells and aggravates acute renal tubular injuries.¹⁶ It has also been reported that caspase-3/GSDME-mediated pyroptosis is involved in the progression of various kidney diseases, including local I/R renal injury,¹⁵ chemotherapy drug-induced acute kidney injury,³⁵ and obstructive nephropathy.³⁶ Our previous research has shown that Art can alleviate intestinal injury after resuscitation by inhibiting the GSDME- mediated pyroptosis pathway.¹⁰ Therefore, we performed this study to investigate the phenomenon of pyroptosis mediated by the caspase-3/GSDME pathway and its potential regulation by Art in the kidney after CA/CPR. We found that caspase-3, cleaved caspase-3, GSDME, and GSDME-N protein concentrations in renal tissues were significantly higher in the CPR and CPR + Art groups than in the Sham group, as were the proinflammatory HMGB1, IL-1β, and IL-18 levels. These findings suggest that the caspase-3/GSMDE pathway is activated and promotes pyroptosis in resuscitated renal tissue. Art treatment markedly decreased the levels of the above-mentioned proteins and proinflammatory factors, indicating that Art may exert a protective effect on post-resuscitation renal injury by inhibiting the caspase-3/GSDME-mediated pyroptosis pathway.

Limitations

This study had some limitations. First, urine output was not quantitatively monitored during the experiment. Although we used validated biomarkers and histopathological evaluations, the lack of urine output data limits direct assessment of renal function after resuscitation. Future studies will include continuous urine output monitoring to better characterize post-resuscitation renal dysfunction. Second, only a single dose of Art was tested; thus, the optimal dosing and therapeutic time window remain undetermined. Third, based on previous reports,37, 38 which suggest that sex may influence the efficacy of resuscitation interventions. we only chose the male swine in this study. Consequently, it is unknown whether Art exerts similar renoprotective effects in females. Fourth, renal function was assessed primarily using Cr and BUN. Although these are routine parameters, it is important to acknowledge their limitations, such as hydration status, muscle mass, and diet. The absence of more precise measures of glomerular filtration rate represents another limitation. Future investigations should incorporate more accurate renal function metrics. Fifth, all analyses were performed at 24 h after ROSC. Although this time point effectively captures early acute injury changes, rising trends in Cr and BUN suggest that renal damage may not have peaked or begun to resolve. Thus, the long-term progression and outcome of injury could not be evaluated. Longer observation periods are needed in future work to fully understand injury and recovery dynamics. Sixth, histological scoring was performed by a single pathologist, who was blinded and used predefined criteria, yet the lack of inter-rater reliability assessment may introduce subjectivity. Finally, the relatively small sample size per group may limit statistical power and generalizability. Larger cohorts are required to confirm and extend these findings.

Conclusions

In conclusion, Art treatment significantly alleviated post-resuscitation renal injury in a swine model of CA and resuscitation. The protective effects are related to inhibition of the caspase-3/GSDME-mediated pyroptosis pathway.

Data sharing statement

The data presented in this study are available upon reasonable request from the corresponding author.

CRediT authorship contribution statement

Qianqian Wang: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Peng Shen: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Wang Wu: Writing – review & editing, Writing – original draft, Methodology, Data curation. Jiangang Zhu: Writing – review & editing, Writing – original draft, Methodology, Data curation. Jiefeng Xu: Writing – review & editing, Writing – original draft, Visualization, Supervision, Resources, Project administration, Methodology.

Funding

This work was supported by Zhejiang Provincial Medical Science Foundation (2024KY435), the Provincial Key Clinical Specialty of Zhejiang (2024-ZJZK-002), Jiaxing Science and Technology Plan Project (2024AY30004) and Hospital-level Research Project of The First Hospital of Jiaxing (2023-YB-059).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: