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. 2025 Mar 28;63(6):935–943. doi: 10.1097/SHK.0000000000002586

METHANE ADMINISTRATION DURING OXYGENATION MITIGATES ACUTE KIDNEY INJURY IN A PIG MODEL OF 24-H VENO-VENOUS EXTRACORPOREAL MEMBRANE OXYGENATION

Noémi Vida 1, Zoltán Varga 2, Antal Szabó-Biczók 3, Gábor Bari 3, Gyöngyvér Vigyikán 1, Ádám Hodoniczki 1, Ámos Gajda 1, Attila Rutai 1, László Juhász 1, Szabolcs Péter Tallósy 1, Sándor Turkevi-Nagy 4, Anett Bársony 1, Nándor Öveges 2, Andrea Szabó 1, Mihály Boros 1, Gabriella Varga 1, Dániel Érces 1
PMCID: PMC12105969  PMID: 40148266

ABSTRACT

Background: Severe respiratory failure often requires veno-venous extracorporeal membrane oxygenation (v-v ECMO) treatment, a procedure frequently associated with acute kidney injury (AKI). Preclinical studies have demonstrated the anti-inflammatory properties of inhaled methane (CH4). This experimental protocol aimed to investigate whether CH4 gas administration could mitigate the development of AKI in a clinically relevant large animal model of v-v ECMO. Methods: Anesthetized miniature pigs were divided into three groups (n = 6 each). Following cannulation of the right femoral and internal jugular veins, v-v ECMO was initiated and maintained for 24 h, followed by a 6-h post-ECMO observation. The control group underwent cannulation without ECMO, while the v-v ECMO+CH4 group received a 2% CH4-air mixture via the oxygenator. Urine output was recorded, and kidney injury was assessed using plasma and urine neutrophil gelatinase-associated lipocalin levels. Inflammatory activation was evaluated through plasma interleukin-1β (IL-1β) and interleukin-8 (IL-8) levels. Kidney tissue samples were analyzed for histopathological changes, xanthine oxidoreductase and myeloperoxidase activities, and nitrite/nitrate levels. Results: The CH4-treated group exhibited significantly higher post-ECMO renal arterial flow (244.7 ± 70 vs. 96.3 ± 21 mL/min) and increased average urine output (5.75 ± 1.6 vs. 3.25 ± 0.4 mL/h/kg) compared to the v-v ECMO group. CH4 administration reduced urine and plasma neutrophil gelatinase-associated lipocalin levels and demonstrated lower histological damage scores (0.8 ± 0.3 vs. 3.3 ± 0.8). Furthermore, CH4 treatment decreased xanthine oxidoreductase and myeloperoxidase activities and reduced inflammatory mediators, including IL-1β, IL-8, and nitrite/nitrate. Conclusion: CH4 admixture significantly mitigates inflammatory activation and renal injury associated with v-v ECMO. These findings suggest that CH4 may serve as an effective adjunctive means to reduce renal complications of v-v ECMO therapy.

KEYWORDS: Extracorporeal membrane oxygenation, kidney injury, renal artery flow, oxidative stress, methane, inflammation

INTRODUCTION

The use of veno-venous extracorporeal membrane oxygenation (v-v ECMO) has significantly increased during the recent pandemic. This technique is particularly valuable in cases of severe respiratory failure, including acute respiratory distress syndrome (ARDS), where conventional mechanical ventilation fails to meet the patient’s oxygenation requirements (1). Since its introduction in 1965, the successful application of v-v ECMO has been well documented. However, despite significant technological advancements, the procedure remains associated with a range of serious complications. These adverse effects arise from various factors, including prolonged exposure of the blood circulation to synthetic materials, the administration of antithrombotic therapies, and the hemodynamic instability often accompanying the underlying pathological conditions necessitating ECMO support (2,3). Acute kidney injury (AKI) is the most common complication, with reported incidence rates ranging from 26% to 85%, depending on the patient’s physiological status and the underlying disease etiology (2). Notably, approximately 45% of patients with v-v ECMO-associated renal failure require renal replacement therapy, and their mortality rate is 3.7 times higher than that of patients without renal complications. Additionally, the risk of persistent kidney dysfunction after discharge may be as high as 58% (4,5).

The precise mechanisms underlying AKI in this situation are not yet fully understood. However, it is recognized that both activation of systemic inflammatory responses and renal hemodynamic fluctuations play a significant role in its development (6). The artificial ECMO circuit generates biomechanical forces that can lead to hemolysis, exacerbate coagulation, and trigger inflammatory cascades. These processes are accompanied by nitrosative and oxidative stress, all of which contribute to cellular injury. The underlying mechanisms of AKI are not yet fully understood; however, it is noteworthy that in a previous study using a large animal model with veno-venous (v-v) ECMO, early signs of AKI were detected even before significant hemodynamic changes occurred (7).

Over recent decades, several well-known and theoretically promising biological gases, such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), have been investigated in similar contexts due to their recognized anti-inflammatory properties. However, none of these gases has achieved breakthrough success (8). Methane (CH4) has traditionally been regarded as physiologically inert. However, recent studies have unequivocally demonstrated bioactivity and its ability to modulate key inflammatory processes. Moreover, previous research suggests that dissolved or inhaled CH4 can alleviate oxidative and nitrosative stress responses, which are central features of ischemia-reperfusion injuries (9). Exogenous administration of nonasphyxiating concentrations of CH4 has been shown to reduce epithelial permeability and improve local microcirculation in such settings (10). Its anti-inflammatory effects have been repeatedly demonstrated in various animal models of local and systemic inflammation (11,12). Moreover, CH4 administration has been associated with increased renal blood flow in a short-term cardiopulmonary bypass model, suggesting its potential benefits in the context of v-v ECMO (13).

Based on this background, we hypothesized that (1) CH4 gas, when applied via a v-v ECMO oxygenator, would be delivered to the kidneys through systemic circulation, and (2) it would mitigate the AKI-inducing side effects of v-v ECMO treatment. To test this hypothesis, we utilized a clinically relevant 24-h large animal model.

MATERIALS AND METHODS

The experiments were performed on female (n = 10) and castrated male (n = 8) outbred Vietnamese minipigs (46.5 ± 4.5 kg bodyweight) obtained from a local, licensed breeder, in accordance with the National Institutes of Health guidelines on the handling of and care for experimental animals and EU Directive 2010/63 on the protection of animals used for scientific purposes (license number: V./3262/2022).

Animals and anesthesia

The animals were kept in a licensed, conventional hygienic level animal house 7–10 days acclimatization with natural circadian light and free access to water and food. Prior to the experiments, the animals were fasted for 12 h with free access to tap water. At the beginning, anesthesia was induced with an intramuscularly (im) administered mixture of tiletamine-zolazepam (5 mg/kg im; Zoletil, Virbac, Carros, France) and xylazine (2 mg/kg im; Produlab Pharma, Raamsdonksveer, the Netherlands). After endotracheal intubation, mechanical ventilation was started with a tidal volume (VT) of 8–10 mL/kg, the respiratory rate (RR) was adjusted to maintain the end-tidal pressure of carbon dioxide in the 35–45 mmHg range, and the fraction of inspired oxygen (FiO2) was set to keep arterial partial pressure of oxygen (PaO2) between 80 and 100 mmHg. Anesthesia was maintained with a continuous infusion of propofol (6 mg/kg/h intravenously; Fresenius Kabi, Bad Homburg, Germany), midazolam (1.2 mg/kg/h; Torrex Chiesi Pharma, Vienna, Austria) and fentanyl (0.02 mg/kg/h; Richter Gedeon, Budapest, Hungary). Ringer’s lactate infusion was administered at a rate of 10 mL/kg/h. The depth of anesthesia was regularly controlled by monitoring the jaw tone, the absence of the interdigital reflex and the position of the eyeballs (medioventral view).

Surgical preparations

The surgical interventions and the v-v ECMO line (establishment and settings) were described earlier (7). Briefly, the anesthetized animals were placed in supine position on a heating pad to maintain body temperature between 36°C and 37°C. The left jugular vein was cannulated for fluid and drug administration, and the left femoral artery was cannulated for invasive hemodynamic monitoring and to measure cardiac output (CO) by transpulmonary thermodilution (PULSION Medical Systems, Munich, Germany). A urinary catheter was surgically placed in the bladder via the femoral incision. To establish the ECMO circuit, the access cannula was inserted into the right femoral vein and the return cannula was introduced into the right jugular vein (21 Fr, HLS cannulas; Maquet, Rastatt, Germany). The position of the cannulas (access: proximal of the hepatic artery; return: orifice of the superior vena cava) was checked using an X-ray image intensifier (Ziehm SOLO; Ziehm Imaging GmbH, Nuremberg, Germany).

Before setting up the ECMO circuit, CO was directly measured with the PiCCO system via thermodilution, and the flow settings were initially adjusted accordingly at the start of ECMO circulation. Lung-safe ventilation was initiated during ECMO periods (VT = 3–5 mL/kg; positive end-expiratory pressure (PEEP) = 10–15 cmH2O; RR = 4–15 breaths/min). Sweep gas and circuit blood flow rates were refined according to the arterial partial pressure of the carbon dioxide (PaCO2; 35–45 mmHg) and PaO2 (70–100 mmHg) values of the arterial blood gas samples, drawn from the left femoral artery. During ECMO phase, pre- and post-membrane blood samples were also collected for blood gas analysis to ensure proper sweep gas and blood flow settings to provide adequate gas exchange. Transmembrane pressure was monitored, and the blood flow in the circuit was measured with the inline ultrasound flow probe of the pump (Biomedicus 550; Medtronic, Eden Prairie, MN). Mean arterial pressure (MAP) was monitored continuously and kept over 60 mmHg. As a positive inotropic treatment, norepinephrine was administered if necessary (0.05–0.35 μg/kg/h iv; Arterenol; Sanofi-Aventis, Frankfurt am Main, Germany).

The ECMO circulation was stopped after 24 h, the ECMO cannulas were removed, ventilation was set to the pre-ECMO settings. Oxygen and breathing rates were set according to the blood gas values if necessary. After median laparotomy, the right renal artery was dissected free, and a perivascular flow probe was placed around it (Transonic Systems Inc., Ithaca, NY) to measure the renal blood flow. The wound in the abdominal wall was then temporarily closed with clips.

Experimental protocol

Prior to the experiments, the animals underwent a general health check and they were included in the study if no outer injuries, discharge from body orifices, or any signs of inflammation (swelling, edema, epithelial hyperemia) could have been observed. The animals (n = 18) were randomly allocated into three experimental groups (n = 6, each group; the castrated male-female ratio: 0.5 for v-v ECMO, 0.5 for v-v ECMO+CH4 group, and 0.33 for control group). In the v-v ECMO groups, extracorporeal circulation was maintained for 24 h. After 24 h, ECMO was abandoned, the cannulas were removed, and the cannulation entry points were surgically closed. A further 6-h post-ECMO observation followed. For technical reasons, renal artery flow (RAF) was measured in this phase only. In the control group, identical ECMO cannulation was completed, but extracorporeal circulation was not initiated. The same interventions and time frames were applied as in the v-v ECMO groups. In case of the animals of the v-v ECMO and CH4 treatment (v-v ECMO+CH4) group 2% CH4-normoxic air mixture (flow: 1 L/min to maintain at least 60% FiO2) was added to the sweep gas (previous sweep gas flow was reduced accordingly, the total gas flow remained unchanged) through the oxygenator during the ECMO period. To the sweep gas of the animals of the untreated v-v ECMO group artificial air was added at the same rate and considerations as in the v-v ECMO+CH4 group (Fig. 1). No animals were excluded from the study for any reasons.

Fig. 1.

Fig. 1

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Scheme of experimental protocol. Examination of the effects of a 24-h vv-ECMO treatment and vv-ECMO combined with inhaled CH4 treatment. Post-ECMO monitoring was continued for a further 6 h.

During the 30-h total observation time, blood samples were taken in every 60 min for blood gas analysis (Cobas b 123, Roche Ltd., Basel, Switzerland) and every 6 h (at baseline and hours 6, 12, 18, 24 and 30) to measure neutrophil gelatinase-associated lipocalin (NGAL), interleukin-1β (IL-1β), and interleukin-8 (IL-8) values. The urine was collected and measured hourly during the observation period to calculate the average hourly diuresis, and further urine samples were taken for NGAL determination every 6 h (baseline and hours 6, 12, 18, 24, and 30).

At the end of the observation period, kidney tissue biopsies were taken to measure myeloperoxidase (MPO) and xanthine oxidoreductase (XOR) activities, nitrite/nitrate (NOx) levels, and for histological examinations. After removal of tissue samples, the animals were overanesthetized through the jugular vein cannula with a single 120 mg/kg dose of sodium pentobarbital (Sigma-Aldrich Inc, St. Louis, MO).

Hemodynamic measurements

MAP was monitored continuously and registered hourly and transpulmonary thermodilution was used to measure CO hourly during the observation period (PiCCO Plus monitoring system; PULSION Medical Systems; Munich, Germany). Blood flow signals (T206 Animal Research Flowmeter; Transonic Systems Inc., Ithaca, NY) were recorded and registered at every hour during the post-ECMO period with a computerized data acquisition system (SPELL Haemosys; Experimetria, Budapest, Hungary).

Measurement of tissue XOR and MPO activities and NOx content

Tissue samples were harvested immediately after the animals were sacrificed. A circular 1–2.5-cm-thick sample was excised from the distal pole of the left kidney. After saline rinsing, the sample was stored in liquid nitrogen for further analysis. Tissue biopsies kept on ice were homogenized in a phosphate buffer (pH 7.4) containing 50 mM Tris-HCl (Reanal, Budapest, Hungary), 0.1 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL soybean trypsin inhibitor, and 10 μg/mL leupeptin (Sigma-Aldrich GmbH, Germany). The homogenate was centrifuged at 4°C for 20 min at 24,000g, and the supernatant was loaded into centrifugal concentrator tubes (Amicon Centricon-100; 100,000 MW cutoff ultrafilter).

XOR activity was determined in the ultrafiltered, concentrated supernatant with a fluorometric kinetic assay based on the conversion of pterine to isoxanthopterine in the presence (total XOR) or absence (xanthine oxidase activity) of the electron acceptor methylene blue (14).

MPO activity was measured on the pellet of the homogenate (15). Briefly, the pellet was resuspended in a K3PO4 buffer (0.05 M; pH 6.0) containing 0.5% hexa-1,6-bis-decyltriethylammonium bromide. After three repeated freeze-thaw procedures, the material was centrifuged at 4°C for 20 min at 24,000g, and the supernatant was used for MPO determination. Next, 0.15 mL of 3,3′,5,5′-tetramethylbenzidine (dissolved in DMSO; 1.6 mM) and 0.75 mL of hydrogen peroxide (dissolved in K3PO4 buffer; 0.6 mM) were added to 0.1 mL of the sample. The reaction led to the hydrogen peroxide-dependent oxidation of tetramethylbenzidine, which was detected spectrophotometrically at 450 nm (UV-1601 spectrophotometer; Shimadzu, Kyoto, Japan). MPO activity was measured at 37°C; then, the reaction was halted after 5 min with the addition of 0.2 mL of H2SO4 (2 M). The data were referred to protein content.

Tissue samples were assayed for NOx levels with the Griess reaction (16). This assay depends on the enzymatic reduction of nitrate to nitrite, which is then converted into a colored azo compound detected spectrophotometrically at 540 nm. Briefly, the samples were divided in two (200 μL each), and the nitrite concentration was measured in the first aliquot by adding the Griess reagent (550 μL) to the samples. In parallel, 200 μL of samples were mixed with 0.1 M potassium phosphate buffer (pH 7.5 buffer; 300 μL), β-NADPH (1 mM; 20 μL), FAD (0.3 μM; 10 μL), and nitrate reductase (EC 1.7.1.1; 10 mU; Sigma-Aldrich) and then incubated for 30 min at 37°C. The Griess reagent (550 μL) was added to the mixture and incubated for 10 min at room temperature. The absorbances measured were converted to nitrite content or total NOx content using NaNO2 or NaNO3 standard curves, respectively. Total NOx was calculated and expressed in μmol/(mg protein) in the case of tissue samples.

Measurement of IL-1β and IL-8 concentrations

Blood samples (0.4 mL) were taken from the jugular vein (baseline and hours 6, 12, 18, 24, and 30) into precooled EDTA (1 mg/mL)-containing polypropylene tubes, centrifuged at 1,200 g for 10 min at 4°C, and then stored at −70°C until assay. Plasma IL-1β and IL-8 concentrations were determined by means of commercially available enzyme-linked immunosorbent assays (ELISA; Quantikine ultrasensitive ELISA kit for pig IL-1β, IL-8; Biomedica Hungaria Ltd, Budapest, Hungary). The minimum detectable levels of pig IL-1β were <5 pg/mL that of pig IL-8 was <10 pg/mL.

Measurement of NGAL

Four-milliliter blood samples were drawn from the jugular vein into chilled polypropylene tubes containing EDTA (1 mg/mL) and 4-mL urine samples were collected in Eppendorf tubes at baseline and hours 6, 12, 18, 24, and 30. The blood samples were centrifuged at 1,200g for 10 min at 4°C. The plasma samples were then collected and stored at −70°C until assay. The urine samples were kept at −70°C until assay. The plasma and urine concentration of NGAL was measured with a commercially available ELISA kit (Quantikine ELISA for Human Lipocalin-2/NGAL Immunoassay; R&D Systems, Minneapolis, MN).

Kidney histology

Kidney samples were fixed in 4% neutral buffered formalin. Tissue slices were embedded in paraffin using a Thermo Shandon PathCentre tissue processor (Thermo Fisher Scientific, Waltham, MA). The 3-μm-thick sections were cut with a Leica RM2125 rotary microtome (Leica Microsystems Ltd., Wetzlar, Germany). After deparaffinization and rehydration, the sections were routinely stained for hematoxylin-eosin and periodic acid Schiff. The extent of tubular cell edema, apical cytoplasm vacuolization, tubular cell vacuolization, tubular lumen irregularity, loss of brush border, sloughing of tubular cells, tubular dilation, tubular cell necrosis, flattened and simplified tubular epithelium, and denudement of the tubular basal membrane was assessed by a renal pathologist in a blinded way.

To quantify these changes, we used a kidney injury score, based on the extent of the histological changes noted above (0 points = 0%; 1 point = 1%–20%; 2 points = 21%–40%; 3 points = 41%–60%; 4 points = 61%–80%; 5 points = 81%–100%). The values of the tubular cell necrosis and denuded basement membranes were weighted by a factor of two (7).

Measurements of blood CH4 content

To confirm the successful introduction of CH4 into the bloodstream via the oxygenator, blood samples were collected from the jugular vein, femoral artery, and the post-ECMO membrane cannula at the initiation of CH4 administration, as well as at the 12-h and 30-h time points. These samples were analyzed using photoacoustic spectroscopy (PAS) based on a near-infrared laser technique. A volume of 10 mL was extracted from each sampling site and transferred into a 20-mL airtight container equipped with an outlet connected to the spectroscopy apparatus, as previously described (13).

The CH4 concentration was measured from the airspace above the blood sample, with continuous CH4 emission facilitated by magnetic stirring. Measurements were conducted continuously, and the recorded values were corrected for background CH4 levels in the environment. The results were expressed in parts per million (ppm).

Statistical analysis

Data analysis was performed with a statistical software package (SigmaStat for Windows; Jandel Scientific, Erkrath, Germany). Normality of data distribution was analyzed with the Shapiro–Wilk test. Friedman repeated-measures analysis of variance on ranks was applied within groups. Time-dependent differences from the baseline for each group were assessed by Dunn’s method. Differences between groups were analyzed with the Kruskal-Wallis test followed by Dunn’s test. Median values and 75th and 25th percentiles are provided in the figures; P values <0.05 were considered significant. The sample sizes were estimated with PS: Power and Sample Size Calculation 3.1 software (17) and for sample size estimation the differences in the tissue XOR activity were the primary outcome measures.

RESULTS

Changes in systemic hemodynamics and renal function

During both the ECMO and post-ECMO periods, no significant differences in MAP were detected among the experimental groups (data not shown). Similarly, CO remained comparable among groups throughout the v-v ECMO and post-ECMO phases (Fig. 2A).

Fig. 2.

Fig. 2

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Cardiac output (A) during the whole (30-h) observation period and changes in renal artery flow (B) during the 6-h post-ECMO period and differences in average hourly diuresis (C) in the control, v-v ECMO group and v-v ECMO + CH4 groups. In panel A and B, median, 25th and 75th percentiles are shown. The plots in panel C demonstrate the median and the 25th (lower whisker) and 75th (upper whisker) percentiles. xP < 0.05 relative to the control group; #P < 0.05 relative to the v-v ECMO group.

In contrast, RAF values were significantly reduced in the v-v ECMO group starting from the 25th hour, compared to both the control and CH4-treated groups (Fig. 2B). These data specifically pertain to the post-ECMO period.

Hourly diuresis followed a comparable pattern (Fig. 2C); with the v-v ECMO group showing a significant reduction in average urine output relative to the control group. Notably, CH4 administration was associated with a marked improvement in renal function, mitigating the decline in urine output observed in the v-v ECMO group.

Changes in inflammatory mediator levels (XOR, NOx, MPO, IL-1β, IL-8)

XOR (Fig. 3A), MPO (Fig. 3B) activities, and NOx concentrations (Fig. 3C) were measured to assess oxidative and nitrosative stress within kidney tissue. The v-v ECMO group exhibited significantly elevated levels of these inflammatory markers compared to the control group. However, CH4 administration resulted in a marked reduction in XOR and MPO activities, as well as the decrease in NOx levels relative to the v-v ECMO group.

Fig. 3.

Fig. 3

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Differences in xanthine-oxidoreductase (A) and myeloperoxidase (B) enzyme activities and nitrite/nitrate levels (C) in the renal tissue in the control v-v ECMO and v-v ECMO + CH4 groups at hour 30 of the protocol. The plots demonstrate the median (horizontal line in the box), the 25th and 75th percentiles, and the range of data (whiskers). xP < 0.05 relative to the control group; #P < 0.05 relative to the v-v ECMO group.

Plasma concentrations of IL-1β (Fig. 4A) and IL-8 (Fig. 4B) were quantified at 0, 6, 12, 18, 24, and 30 h. In the v-v ECMO group, levels of these proinflammatory cytokines were significantly elevated starting at the 6-h time point. In contrast, CH4 administration significantly reduced IL-1β and IL-8 levels in the treatment group.

Fig. 4.

Fig. 4

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Changes in plasma IL-1β (A) and IL-8 (B) in the control v-v ECMO and v-v ECMO + CH4 groups. The plots demonstrate the median (horizontal line in the box), the 25th and 75th percentiles, and the range of data (whiskers). *P < 0.05 relative to the baseline values (t = 0 h); xP < 0.05 relative to the control group; #P < 0.05 relative to the v-v ECMO group.

Changes in plasma and urine levels of NGAL

The concentration of NGAL was quantified in the urine (Fig. 5A) and the plasma (Fig. 5B) at 0, 6, 12, 18, 24, and 30 h. In the v-v ECMO group, NGAL levels were significantly elevated when compared to both baseline and control group values, indicating renal injury. Following CH4 administration, a significant reduction in NGAL levels was observed from the sixth hour in both the plasma and urine samples.

Fig. 5.

Fig. 5

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Changes in the neutrophil gelatinase-associated lipocalin levels in the urine (A) and plasma (B) in the control v-v ECMO and v-v ECMO + CH4 groups. The plots demonstrate the median (horizontal line in the box), the 25th and 75th percentiles, and the range of data (whiskers). *P < 0.05 relative to the baseline values (t = 0 h); xP < 0.05 relative to the control group; #P < 0.05 relative to the v-v ECMO group.

Kidney histology

In parallel with the functional changes, histological assessments revealed significant differences between the v-v ECMO group and the CH4-treated v-v ECMO group. Renal tissue damage was markedly attenuated by CH4 administration, as evidenced by substantial improvements in six key histological parameters (Fig. 6A). To semiquantitatively evaluate the extent of renal damage and recovery, a scoring system was employed, providing an overall assessment of renal condition and total tissue damage. In the v-v ECMO+CH4 group the score of histological evaluation was significantly lower compared to the nontreated group (Fig. 6B).

Fig. 6.

Fig. 6

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Differences in the components of renal histology score (A) and in the overall renal histology score (B) in the control v-v ECMO and v-v ECMO + CH4 groups. In panel A, occurrence of histological changes in % of high power of fields; median, 25th and 75th percentiles are shown. The plots in panel B demonstrate the median and the 25th (lower whisker) and 75th (upper whisker) percentiles. xP < 0.05 relative to the control group values; #P < 0.05 relative to the v-v ECMO values.

CH4 content in the blood

During transoxygenator CH4 treatment, CH4 levels were significantly elevated in all types of blood samples (postmembrane, arterial and venous) at 12 h. Notably, increased CH4 concentrations were still detectable in arterial and venous blood samples even 6 h after CH4 treatment was discontinued (at 30 h). These findings confirm the efficacy of the CH4 administration protocol via the oxygenator and suggest the potential for CH4 to reach target organs through systemic circulation (Fig. 7).

Fig. 7.

Fig. 7

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Changes in the blood methane content in the venous blood (white dotted box), arterial blood (dotted box with cross), and post-membrane blood (black dotted box) at different stages of the experiments. The plots demonstrate the median (horizontal line in the box), the 25th and 75th percentiles, and the range of data (whiskers). *P < 0.05 relative to the baseline values (t = 0 h).

DISCUSSION

The increasingly frequent use of v-v ECMO in severe ICU cases highlights the critical need to understand and address its associated complications (18). In the present study, we employed a porcine model of v-v ECMO, meticulously replicating all aspects of the experimental setup to align with clinical v-v ECMO treatment conditions. Domestic pigs were chosen due to the anatomical and functional similarities of their immune responses and kidneys to those of humans (19). Notably, a comparative study involving four species (rat, guinea pig, domestic pig, and human) demonstrated that XOR activity in porcine tissues closely parallels human values, whereas rat tissues exhibit activity levels an order of magnitude higher. Furthermore, the therapeutic response to the XOR inhibitor allopurinol is comparable between pigs and humans, and the histopathological alterations observed in renal tissues closely parallel those reported in human patients with ischemic AKI (20,21).

Utilizing this model, we investigated the therapeutic effects of CH4 gas administration via the ECMO oxygenator. Long-term (24-hour) continuous administration of CH4 at low concentrations during v-v ECMO treatment resulted in improved RAF and increased average urine output. These functional benefits were accompanied by reduction in systemic proinflammatory cytokine levels (IL-1β and IL-8), renal oxidative and nitrosative stress enzyme activities (XOR and MPO), and associated marker levels (NOx).

Reduced tissue damage was evidenced by histology and with the changes in the levels of NGAL, an early biomarker for AKI, which reflects renal tubular injury and inflammation (22). Elevated levels of NGAL in serum and urine have been associated with various forms of AKI, including those induced by ischemia or nephrotoxins (23). It has been shown that NGAL levels can rise significantly in response to acute renal injury, often preceding traditional markers such as serum creatinine (24). Therefore, we consider this early elevation in NGAL to be a response to tubular epithelial cell injury, which can be exacerbated by oxidative stress (25). Similar changes were present in our setup from the early phase of v-v ECMO treatment.

The reduced signs of tissue injury after CH4 administration were also associated with improved RAF during the post-ECMO period. In renal afferent arterioles, superoxide can directly increase Ca2+ influx in smooth muscle cells (26), leading to vasoconstriction, which may explain the reduced RAF observed in the v-v ECMO group. Besides, the inflammatory activation triggered by v-v ECMO inevitably stimulates the activity of superoxide-producing enzymes, such as NADPH oxidase and XOR (27).

CH4, traditionally regarded as biologically inert gas, has recently gained attention for its potent therapeutic effects in modulating inflammatory responses (11,28). Its anti-inflammatory properties are mediated through the inhibition of multiple cellular and mitochondrial nitro-oxidative stress pathways, including NF-κB–MAPKs (29), TLR4–NF-κB–NLRP3 (30), PPAR-γ–NF-κB (31), and PI3K–AKT–NF-κB (32) pathways (as reviewed by Poles et al (9)). Additionally, CH4 has been shown to modulate the activity of XOR in various tissues (13). XOR is a major producer of ROS during reoxygenation conditions (33) and in our model, the CH4-induced reduction in XOR activity likely plays a pivotal role in mitigating ECMO-induced kidney injury, as evidenced by the higher average urine output and decreased tubular damage. XOR inhibition has also been shown to suppress NLRP3 inflammasome activation (34,35), potentially leading to reduced release of IL-1β, a key proinflammatory cytokine and a central finding of this study.

The production of NOx, end products of NO metabolism, is also closely tied to inflammatory processes. Elevated NOx levels often reflect increased NO synthase activity, which can be stimulated by proinflammatory cytokines such as IL-1β (36). Furthermore, IL-1β can induce the release of IL-8, a potent chemoattractant (37). CH4 may reduce IL-8 transcriptional upregulation by inhibiting the NF-κB–MAPKs pathway (29,38). In our model, the parallel reduction of IL-1β and NOx following CH4 administration suggests that CH4 treatment contributes to renal protection through these mechanisms.

During v-v ECMO, elevated IL-8 levels were associated with a significant increase in MPO activity in renal tissue, indicative of PMN infiltration and exacerbated oxidative stress. However, CH4 administration mitigated these effects as well, further supporting its role in suppressing ROS-induced IL-1β release and downstream proinflammatory pathways.

Nonetheless, our study is not without limitations such as the relatively small sample size (n = 6 per group) and the short observation period and therefore, the demonstration of pharmacological benefits of CH4 administration has not been accompanied by the translation of these benefits to important clinical outcomes such as mortality. Nevertheless, we aimed to keep the balance between clinical relevance with the logistical and resource constraints inherent in large animal in vivo studies. We anticipated that the renal protective effects of CH4 treatment would be most apparent within the early 30-h timeframe, as ECMO-induced oliguria typically develops within 24–48 h (39). Furthermore, in our previous study, increases in inflammatory mediators were observed as early as 6 h after ECMO initiation (7). Additionally, noninvasive techniques such as Doppler ultrasound to monitor RAF were not utilized, as they require repositioning the animal, posing a risk of accidental decannulation, and are generally less reliable than perivascular flow probes (40).

An important technical consideration is the method of CH4 gas administration. In this protocol a 2% CH4-normoxic air mixture was added to the sweep gas (FiO2 = 0.21), which inevitably reduced the oxygen concentration. However, the flow rate (1 L/min) was carefully selected to ensure the total gas flow necessary for adequate oxygenation and carbon dioxide removal. The lowest total sweep gas flow rate used in this study was 2 L/min (1 L/min pure O2, FiO2 = 1, and 1 L/min 2% CH4-air mixture, FiO2 = 0.21), which still provided a final O2 concentration of approximately 60%. Blood gas monitoring at multiple sites (pre- and postmembrane and left femoral arterial samples) confirmed the absence of hypoxia or hypercapnia with this setup.

It is important to note that the animals in this study were healthy, while human patients requiring ECMO support typically suffer from severe circulatory or pulmonary conditions. Therefore, this O2 concentration might be insufficient in certain clinical cases. However, with the use of specialized gas-mixing equipment, higher O2 concentrations (>98%) can be safely achieved by directly blending 2% CH4 into the sweep gas. The lowest flammability limit of CH4 in O2 is 5% (41), while in this study, the concentration of CH4 in the total sweep gas was ≤1%.

In conclusion, our study demonstrates that CH4 gas administration via the oxygenator during v-v ECMO significantly attenuates AKI in pigs. The results indicate that CH4 not only improves RAF and urine output but also mitigates histological damage, oxidative stress, and proinflammatory cytokine levels—key biomarkers of renal injury.

By integrating this strategy into the therapeutic framework of v-v ECMO, we aimed to target critical pathophysiological mechanisms underlying renal dysfunction. Future studies are essential to further validate its efficacy and organ-specific effects in preclinical, mechanically ventilated critical care models, including AKI secondary to major cardiac surgery, septic shock, or trauma.

While translating these findings to human patients necessitates careful consideration of physiological and species-specific differences, this porcine model provides valuable insights into the therapeutic potential of CH4. Overall, our results underscore the promise of the potential applicability of CH4 as an adjunctive therapeutic agent in managing v-v ECMO-induced AKI with broader implications for improving critical care nephrology outcomes.

ACKNOWLEDGMENTS

The authors thank Virág Molnár, Bence Győrfi, Csilla Mester, Natália Dudás, István Szabó, and Krisztián Daru for their skillful assistance.

The study was supported by EFOP-3.6.2-16-2017-00006, 2024–1.2.3-HU-RIZONT-2024–00033, and NKFI-149858 grants and by the University of Szeged Open Access Fund, Grant ID: 7603.

Authors’ contributions: NV and ZV contributed equally to the paper as first authors. DÉ and GV contributed equally to the paper as last authors.

Footnotes

The authors report no conflicts of interest.

N.V. and Z.V. contributed equally to this study and share first authorship.

G.V. and D.É. have contributed equally to this study and share last authorship.

Contributor Information

Noémi Vida, Email: noemividaa@gmail.com.

Zoltán Varga, Email: vzoli8@gmail.com.

Antal Szabó-Biczók, Email: szba01@gmail.com.

Gábor Bari, Email: drbarigabor@gmail.com.

Gyöngyvér Vigyikán, Email: vigyikangyongyver@gmail.com.

Ádám Hodoniczki, Email: hodiadi2@gmail.com.

Ámos Gajda, Email: amosgajda@gmail.com.

Attila Rutai, Email: rutai.attila@med.u-szeged.hu.

László Juhász, Email: juhasz.laszlo.1@med.u-szeged.hu.

Szabolcs Péter Tallósy, Email: tallosy.szabolcs@med.u-szeged.hu.

Sándor Turkevi-Nagy, Email: tenagysanyi@gmail.com.

Anett Bársony, Email: drbarsonyanett@gmail.com.

Nándor Öveges, Email: nandoroveges@gmail.com.

Andrea Szabó, Email: szabo.andrea.exp@med.u-szeged.hu.

Mihály Boros, Email: boros.mihaly@med.u-szeged.hu.

Gabriella Varga, Email: varga.gabriella.1@med.u-szeged.hu.

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