Introduction
Sepsis-associated acute kidney injury (SA-AKI) is a prevalent sepsis complication strongly linked with adverse outcomes, such as high mortality rates [1,2]. Kidney injury development in sepsis is linked to various factors, including heightened inflammation, harmful oxidative substances, microcirculatory dysfunction, tissue hypoxia, programmed cell death pathway activation, and subsequent renal tissue injury [3–4]. Despite our understanding of SA-AKI, no endorsed standard treatment directly targets these pathophysiological mechanisms. Existing sepsis treatment guidelines emphasize essential conservative measures, such as urgent disease recognition, timely fluid resuscitation, blood purification, and empirical antibiotic therapy [5].
Hyperbaric oxygen therapy (HBOT), a treatment modality that administers 100% oxygen at pressures greater than a 1.4 atmospheric absolute, is widely utilized for decompression sickness, necrotizing fasciitis, chronic wounds, osteomyelitis, and various other diseases [6–9]. Regarding infectious diseases, preclinical studies have reported that HBOT may confer a mortality benefit in sepsis. However, its beneficial effect on SA-AKI remains relatively unexplored. The correlation between HBOT and renal injury has been investigated in diverse animal disease models, including ischemic reperfusion injury, drug-induced renal toxicity, and ureter obstruction kidney injury [10–15]. Although diverse HBOT protocols were adopted as an intervention, these studies have proposed various renoprotective HBOT mechanisms, such as reducing oxidative stress and inflammation, suppressing apoptosis, and promoting autophagy and tissue regeneration [10–15]. Given these proposed mechanisms in various disease conditions, HBOT may offer renoprotective effects in SA-AKI. However, research investigating HBOT’s impact on SA-AKI is limited [16], and the precise mechanisms through which HBOT exerts its renoprotective effects in SA-AKI remain elusive, warranting further investigation.
This study determined whether HBOT exerts a renoprotective effect in an LPS-induced endotoxemia mice model and elucidated the mechanisms behind this effect. We hypothesized that HBOT provides renoprotective benefits in LPS-induced AKI by mitigating renal tubular cell apoptosis, modulating signaling pathways associated with apoptosis, and enhancing antioxidant capacity.
Methods
Ethics statement
Animal care procedures and experiments of this work were conducted per the Animal Care Regulations established by the Committee of Chonnam National University Medical School (No. CNU IACUC-2023009).
Experimental protocol
This experimental study involved 8-week-old male C57BL/6 mice obtained from Samtako Bio in Korea. All mice were pair-housed in cages within the animal care facility at the Chonnam National University Medical School. Mice were allowed 7 days to facilitate acclimation in a temperature-controlled room with a 12-hour light/dark cycle at 23 °C. The mice had ad libitum access to standard chow and water during this period.
Fig. 1 illustrates the experimental procedure timeline. Mice were allocated into four distinct groups: Sham (n = 10), HBOT (n = 10), lipopolysaccharide (LPS; inducing endotoxemia, n = 10), and LPS + HBOT (LPS-induced endotoxemia with HBOT, n = 10). Based on previous studies [17], mice were intraperitoneally injected with LPS (10 μg/kg; Sigma-Aldrich) to induce endotoxemia-associated acute kidney injury (AKI). The Sham and HBOT groups received an equivalent volume of saline. Following administration, mice were randomly assigned into Sham, HBOT, LPS, and LPS + HBOT groups. The HBOT and LPS + HBOT groups initiated HBOT 30 minutes post-LPS administration. All mice were kept in home cages with free food and water access and were euthanized 24 hours post-LPS administration. The entire kidney was harvested from each mouse for measurements.
Hyperbaric oxygen therapy
HBOT was administered with an automatic control hyperbaric mono-chamber designed for animal experimentation (IBEX Medical System). Thirty minutes after LPS administration, mice assigned to the HBOT and LPS + HBOT groups were placed in a cage inside the hyperbaric oxygen chamber. Based on the previous study [18], HBOT sessions were programmed to sustain a constant pressure of 2.5 atmospheres absolute with 100% oxygen for 60 minutes and a 10-minute compression/decompression period. The chamber’s temperature was continuously monitored, kept at 25 °C, and ventilated with a 20 L/min gas flow rate to prevent carbon dioxide accumulation. Concurrently, the control group was exposed to identical normobaric temperature conditions to control potential experimental stress.
Renal functional parameters
Plasma creatinine levels were assessed utilizing the Jaffe method (Olympus 5431; Olympus Optical) calibrated for isotope dilution mass spectrometry. Plasma neutrophil gelatinase-associated lipocalin (NGAL) levels were quantified using a commercially available enzyme-linked immunosorbent assay kit (R&D Systems) per the manufacturer’s protocol. A 1:4,000 dilution was employed for plasma NGAL measurements.
Semiquantitative immunoblotting
Western blot analysis was conducted as previously delineated [19]. Kidney tissues from the left side were sectioned and subjected to homogenization in an ice-cold isolation buffer comprising 0.3-M sucrose, 25-mM imidazole, 1-mM ethylenediaminetetraacetic acid, 8.5-mM leupeptin, and 1-mM phenylmethylsulfonyl fluoride (pH 7.2). Homogenates were centrifuged at 4,000 ×g for 15 minutes at 4 °C to eliminate whole cells, nuclei, and mitochondria. Total protein concentrations were ascertained via a bicinchoninic acid assay kit (Pierce). All samples were normalized to the same final protein concentration, dissolved in a sodium dodecyl sulfate (SDS)-containing sample buffer at 100 °C for 5 minutes, and stored at –20 °C. An initial gel was stained with Coomassie blue to ensure equal protein loading. SDS-polyacrylamide gel electrophoresis was executed on 9% or 12% polyacrylamide gels.
Proteins were electrophoretically transferred to nitrocellulose membranes (Hybond ECL RPN3032D; Amersham Pharmacia Biotec) utilizing a Bio-Rad Mini Protean II apparatus (Bio-Rad). The blots were blocked with 5% milk in PBS-T for 1 hour, incubated with primary antibodies overnight at 4 °C, followed by additional incubation with horseradish peroxidase-conjugated secondary anti-rabbit, anti-mouse, or anti-goat antibodies. Immunoblots were visualized with an enhanced chemiluminescence system, and protein levels were quantified through densitometry. Relative immunoblot signal intensities were assessed through densitometry employing Scion Image for Windows software (2000–2001, version Alpha 4.0.3.2; Scion Corp.) and expressed as fold changes relative to the control. Supplementary Table 1 (available online) presents the primary and secondary antibodies employed for immunoblotting.
Real-time polymerase chain reaction
The polymerase chain reaction (PCR) analysis was executed as previously described [19]. The renal cortex was homogenized in Trizol reagent (Invitrogen). RNA was extracted using chloroform, precipitated with isopropanol, washed with 75% ethanol, and dissolved in distilled water. RNA concentrations were ascertained by measuring absorbance at 260 nm (Ultraspec 2000; Pharmacia Biotech). The messenger RNA (mRNA) expressions of genes associated with inflammation and fibrosis, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP1), and alpha smooth muscle actin (α-SMA), were assessed through real-time PCR utilizing complementary DNA (cDNA) templates. cDNA was synthesized by reverse transcribing 5 µg of total RNA using oligo (dT) priming and SuperScript II Reverse Transcriptase (Invitrogen). Then, cDNA was quantified employing the Smart Cycler II system (Cepheid).
SYBR Green was utilized as the real-time PCR DNA detection dye. Each PCR reaction contained 10-pM forward primers, 10-pM reverse primers, TOPreal qPCR 2x PreMIX (SYBR Green; Enzynomics), 1-µg cDNA, and distilled water to achieve a final volume of 20 µL. Relative mRNA levels were ascertained through real-time PCR, employing a Rotor-Gene TM 3000 Detector system (Corbett Research). Supplementary Table 2 (available online) details the primer sequences used for the cDNA and PCR steps. Data were collected and analyzed using Corbett Research Software. Comparative critical threshold values from quadruplicate measurements were employed to calculate gene expression and normalized to GAPDH as an internal control. Melting curve analysis was conducted to confirm amplification specificity. Supplementary Table 2 (available online) lists the primer sequences used in this study.
Histology
Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 3 µm-thick sections. Then, hematoxylin and eosin (H&E) staining and periodic acid-Schiff (PAS) staining were performed to assess histological morphology. Renal tubular injuries were observed under microscopy and characterized by brush border loss and cytoplasmic degeneration. The qualitative damage score was determined as follows: 0, no damage; 1, mild damage; 2, degenerated cytoplasm with a nucleus; and 3, total necrosis without a nucleus. The quantitative score was assigned as: 0, no damage; 1, 1%–25% damage; 2, 26%–50% damage; and 3, more than 50% damage. The tubular injury score was calculated using the formula: % tubular injury score = [(qualitative score + quantitative score) / 6] × 100.
Statistical analysis
Data distribution was assessed. Normally distributed data are presented as the mean ± standard error of mean or standard deviation and illustrated with bar graphs. Nonnormally distributed data are shown as the median and interquartile range, represented with box and whisker plots. Multiple comparisons between groups were performed using a one-way analysis of variance and the post-hoc Tukey’s honestly significant difference test. Differences with p-values of <0.05 were considered significant.
Results
Table 1 displays changes in rat characteristics, including functional parameters. LPS-induced endotoxemia significantly increased the kidney weight-to-body weight ratio with marked bodyweight reduction compared to the Sham and HBOT groups. HBOT treatment alleviated these changes, and body weight was significantly reduced compared to non-LPS groups. As functional markers, plasma creatinine and NGAL in the LPS group were higher than in the other groups. However, the differences did not reach statistical significance compared to the LPS + HBOT group.
Early single-session hyperbaric oxygen therapy mitigates histopathologic alterations in the kidneys of lipopolysaccharide-induced endotoxemia mice
Twenty-four hours post-exposure, histopathologic changes were more apparent in the kidneys of the LPS group than in the other groups. H&E staining revealed tubular cell degeneration, luminal membrane disruption, and brush border loss in the proximal tubules of the LPS group (Fig. 2). Additionally, PAS staining exhibited glomerular shrinkage and increased basement membrane thickness in the LPS group (Fig. 2). However, these pathological changes were significantly mitigated post-HBOT treatment.
Early single-session hyperbaric oxygen therapy reduces renal apoptosis in lipopolysaccharide-induced endotoxemia
We examined the changes in apoptosis-associated proteins to evaluate HBOT’s effect on apoptosis in renal tubular cells. Immunoblotting indicated a significant increase in the Bax/Bcl-2 and cleaved caspase 3/caspase 3 ratios in the kidney tissue of the LPS group (Fig. 3A). However, while the cleaved caspase 3/caspase 3 ratio was significantly attenuated in HBOT-treated mice, the Bax/Bcl-2 ratio merely indicated a tendency towards reduction. Notably, phosphorylated p53 protein levels were significantly lower in the LPS + HBOT group than in the LPS group (Fig. 3B). Additionally, cytochrome C levels, potent proapoptotic stimulators, were significantly reduced in the LPS + HBOT group than in the LPS group (Fig. 3B). These findings suggest that HBOT intervention inhibits cytochrome C release from the mitochondria, aligning with the mitochondrial membrane stabilization indicated by the decreased Bax-to-Bcl-2 ratio. Collectively, these data suggest that HBOT effectively mitigated renal tubular cell apoptosis.
Effect of hyperbaric oxygen therapy on renal inflammatory mediator genes in lipopolysaccharide-induced endotoxemia
To elucidate the impact of HBOT on renal inflammation induced by LPS endotoxemia, we used quantitative PCR (qPCR) to evaluate specific inflammatory mediator mRNA levels, including IL-6, TNF-α, and MCP1 (Fig. 4). Previous studies have verified that quantifying cytokine gene expression in affected organs via qPCR is an effective method for assessing early inflammatory responses [20,21]. Elevated IL-6, TNF-α, and MCP1 mRNA expression levels were observed in the LPS and LPS + HBOT groups compared to the Sham and HBOT-only groups. Furthermore, there were no discernable differences between the LPS and LPS + HBOT groups. These observations imply that an early single session of HBOT may not substantially influence an inflammatory response.
Effect of hyperbaric oxygen therapy on antioxidant capacity in lipopolysaccharide-induced endotoxemia: catalase, SOD2, GPx4, and HO-1
To evaluate how HBOT impacts antioxidant capacity, we examined changes in antioxidant-associated enzymes, specifically catalase, manganese-dependent superoxide dismutase (SOD2), glutathione peroxidase 4 (GPx4), and heme oxygenase-1 (HO-1) (Fig. 5). Immunoblotting revealed a notable SOD2 decrease in the LPS group relative to the Sham and HBOT-only groups. Interestingly, SOD2 levels in the LPS + HBOT group were considerably higher than in the HBOT and LPS groups. However, there were no significant intergroup differences in GPx4, catalase, and HO-1 levels. These findings suggest early hyperbaric oxygen exposure during LPS-induced endotoxemia may enhance antioxidant defenses by stabilizing SOD2 enzyme levels.
Effect of hyperbaric oxygen therapy on the TGF-β/Smad signaling activation in lipopolysaccharide-induced endotoxemia
We conducted immunoblot assays and qPCR on transforming growth factor beta (TGF-β)/Smad pathway components to investigate the signaling pathways associated with renal fibrosis. Immunoblot results revealed elevated TGF-β protein levels and its downstream effector Smad2/3 in the kidneys of the LPS group relative to the Sham group (Fig. 6). Additionally, the LPS group exhibited a rising trend in Smad4 protein expression. However, HBOT intervention significantly reduced TGF-β and Smad4 expression in the LPS + HBOT group compared to the LPS group. Correspondingly, α-SMA mRNA levels were markedly lower in the LPS + HBOT group compared to the LPS group. These findings imply that HBOT may modulate the TGF-β/Smad signaling pathway, downregulating this pathway in particular during LPS-induced endotoxemia. Interestingly, the phosphorylated Smad2/3 to Smad2/3 ratio was significantly higher in the HBOT group than in the Sham group.
Discussion
This study found that an early single session of HBOT provides renoprotective benefits in LPS-induced AKI by mitigating renal tubular cell apoptosis. This anti-apoptotic effect is mediated by suppressing p53 activation and significantly reducing cytochrome C levels. The observed decrease in TGF-β and downstream Smad4 expression and antioxidant capacity preservation, such as SOD2 following HBOT in LPS-induced endotoxemia, may influence these effects.
Surprisingly, early HBOT significantly mitigated p53 phosphorylation and cytochrome C release in an LPS-induced sepsis rat model, indicating reduced apoptosis. The p53 protein, known for mediating programmed cell death in response to hypoxia, reactive oxygen species (ROS), DNA damage, and other cellular stressors, has been implicated in AKI pathogenesis [22–24]. Previous studies have primarily investigated p53-associated AKI pathogenesis in ischemic reperfusion injuries or drug-induced AKI models [23–25]. However, Sun et al. [26] recently demonstrated that p53 deacetylation alleviates sepsis-associated AKI in sepsis-induced mice models.
Despite HBOT treatment in LPS endotoxemia dramatically reducing phosphorylated p53 levels in the current study, the underlying mechanisms are not fully elucidated. This effect may be due to HBOT ameliorating the conditions stimulating p53 activation [27]. Primarily, HBOT can dissolve an additional large amount of oxygen into the plasma beyond oxygen-hemoglobin’s binding capacity, promoting oxygen diffusion to hypoxic tissue. This feature could alleviate hypoxia, a crucial trigger for p53 activation. Calzavacca et al. [28] demonstrated that renal medullary ischemia and hypoxia progress the first hour after infection, several hours before AKI was detected via oliguria or associated biomarkers. Therefore, early HBOT interventions to resolve kidney ischemia in sepsis may prevent further renal injury and p53 activation. However, this tissue oxygenation elevation is temporary and returns to baseline level soon after HBOT [29]. As such, other underlying mechanisms must be considered to attenuate p53 activation and exert renoprotective effects.
We noted that an early single session of HBOT, administered 30 minutes post-LPS injection in mice, significantly upregulated SOD2 enzymes. Notably, this observation contrasts with the LPS group, which exhibited decreased SOD2 enzyme levels. These results partially corroborate the study by Edremitlioğlu et al. [16], who demonstrated that SOD and catalase levels in renal tissue were significantly decreased after Escherichia coli-induced sepsis. In addition, this study found that HBOT increased these enzyme levels in renal tissue [16]. However, catalase and GPx4 levels did not change in the LPS + HBOT group. One possible explanation for these differences may be the HBOT protocols used, as our study employed a single session rather than multiple sessions. Similarly, other studies have demonstrated that HBOT can enhance renal protection by upregulating antioxidant capacity and reducing oxidant levels in various disease conditions [12,15,30]. Despite the heterogeneity of the study design and injury model diversity, these findings collectively suggest that HBOT may provide renal protection by regulating the balance of antioxidants and oxidants, a benefit that extends to sepsis models.
We observed a significant rise in inflammatory mediator genes, specifically TNF-α, IL-6, and MCP1, 24 hours post-LPS administration compared to the Sham and HBOT groups. Notably, integrating HBOT into the LPS group did not affect these levels. This finding deviates from earlier research, demonstrating that HBOT diminished inflammatory cytokines in a sepsis model [30,31]. For instance, Lin et al. [30] reported a 39% reduction in TNF-α levels following an HBOT session 1 hour after LPS treatment. Comparatively, Luongo et al. [31] reported that HBOT notably decreased TNF-α levels 4 hours post-zymosan administration, maintaining these reduced levels for 24 hours. This discrepancy may stem from differences in the HBOT protocols employed, particularly regarding session frequency. Our protocol incorporated a single HBOT session, contrasting the multiple sessions conducted in the referenced studies [30,31]. Furthermore, Halbach et al. [18] demonstrated a substantial cytokine decline in liver tissue 2 hours following a single 1-hour post-cecal ligation and puncture (CLP) HBOT session. However, these levels rebounded 6 hours post-CLP [18]. This pattern indicates that a single HBOT session may not sustain anti-inflammatory benefits over 24 hours in sepsis models, underscoring the need for further studies to refine HBOT protocols.
The TGF-β/Smad signaling pathway is a predominant mediator in renal fibrosis, frequently activated during AKI [32]. Although the role of TGF-β in AKI progression is still debated, its activation in tubular cells promotes epithelial dedifferentiation, cell cycle arrest, and decreased cell survival [32–36]. Previous studies, such as those by Gewin et al. [34], have demonstrated that TGF-β signaling can induce proapoptotic effects in renal injury models [34–36]. These effects can be attenuated by inhibiting the TGF-β type II receptor or downstream Smad signaling [34,37,38]. In our study, HBOT reduced TGF-β and Smad4 protein expressions and alpha-SMA mRNA expression in an LPS-induced endotoxemia model. This suggests that HBOT can modulate the TGF-β signaling pathway in sepsis, potentially reducing apoptosis in renal tubular cells. However, there was a more substantial increase in the phosphorylated Smad2/3 to Smad2/3 ratio in the HBOT group than in the Sham group. While the underlying cause remains elusive, this rise may be attributed to HBOT elevating ROS production or decreasing antioxidant enzymes.
The pronounced increase in tissue oxygen partial pressure during HBOT often augments ROS production, aligning with the concept of hormesis [39]. An imbalanced redox system can activate latent TGF-β, subsequently triggering the Smad pathway [40]. In fact, our study noted a decrease in antioxidant enzymes within the HBOT group. Conversely, treating the LPS-induced endotoxemia group with HBOT attenuated TGF-β/Smad pathways. This difference could be attributed to the distinct effects HBOT has under physiological and pathophysiological conditions. Furthermore, it may suggest that HBOT’s impact on the TGF-β/Smad pathway may be contingent upon the cellular environment, potentially influencing the pathway’s activation or suppression. This complexity underscores the need for further research to achieve a comprehensive understanding of these interactions and the diverse responses to HBOT.
While our study offers valuable insights, there are several limitations. First, we employed a LPS-induced AKI model. Although this model provides a consistent injury and allows for control over injury severity, its clinical relevance is limited. Second, we primarily focused on gene expression changes to assess renal inflammation. However, it is important to note that gene-level changes may not fully represent the inflammatory status in the tissue. Third, our study did not evaluate survival benefits or long-term kidney changes; a more thorough grasp on HBOT’s impact on sepsis-associated renal injury necessitates further research. Fourth, our study did not include common sepsis treatments that could influence renal damage outcomes, such as antibiotics, vasoactive drugs, or fluid administration. Additionally, we only tested a single-specific HBOT protocol at an early stage of endotoxemia. SA-AKI is a multifaceted condition encompassing several interrelated etiologies. In addition, this injury’s progression continuously shifts the balance between tissue tolerability and damage [1]. Future studies should explore the relationship between HBOT and these treatments with different HBOT protocols to acquire a more comprehensive understanding of potential therapeutic strategies.
In conclusion, our study substantiates that an early single session of HBOT may offer protection in LPS-induced kidney injuries, primarily by mitigating apoptosis. These potential effects are associated with suppressing p53 activation and reducing cytochrome C levels. Additionally, the changes in TGF-β/Smad4 expression and the preservation of antioxidant capacity following HBOT in LPS-induced endotoxemia may contribute to these renoprotective effects. While these findings are encouraging, applying HBOT as an adjunctive treatment for sepsis-associated kidney injuries requires further investigation in more complex clinical scenarios. Our study provides preliminary insights and underscores the need for additional research to uncover the full therapeutic potential of HBOT in treating sepsis.
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